FAYDALARI: 

Ganoderma lucidum (Reishi) ve Özellikleri

 **Aşağıdaki kaynak Wikipedia'dan alınarak tercüme edilmiştir.

Ganoderma lucidum (Reishi) Asya'da yaklaşık 4000 yıldan beri özel bir ihtimam görmenin mutluluğunu sürdüren bir mantar olup, tradisyonel Çin tıbbında (Ananevi Çin Tıbbı) tarihin tıbbi kullanım sahası olan en eski mantarı olarak bilinmekte ve kullanılmaktadır. Çincede Ling-Zhi yani ruh gücünün bitkisi olarak bilinen ve bazen ölümsüzlük mantarı olarak (1) adlandırılan bu mantar, hiçbir yan etkisi olmamasına karşılık çok geniş spektrumda tıbbi fayda sağlaması bakımından el üstünde tutulmaktadır. Ling-Zhi (Reishi) günümüzde Amerika Herbal Pharmacopoeia ve Therapotic Compendium kapsamına dahil edilmiştir. Reishi biri Kuzey Amerika'da rastlanan, kısa saplı veya sapsız bir formda görüldüğü gibi, ince ve uzun saplı ve şapkası biraz daha küçük bir formda sıcak tropik bölgelerde rastlanan bir mantardır. Günümüzde birçok bölgede bu iki şeklin çeşitli ara formları olarak yaygındır. Morfolojik olarak Reishi (Ling-Zhi) polipor mantarlar sınıfına dahil olup, odunsu yapısıyla karakterizedir. Kenarları beyaz, orta kısmı kırmızı renkli, parlak ve böbreğe benzer bir görüntü hatırlatan meyvası, yaşlandıkça tamamen kırmızı kahverengi (pas rengi) olur ve üzeri kalın bir spor tabakasıyla örtülür. Meyva çok önemli tıbbi özellikleri olan biyolojik aktif polisakkarit kaynağıdır. Buna ek olarak ergosterol, kumarin, mannitol, laktonlar, alkoloidler, doymamış yağ asitleri, çok çeşitli vitamin ve mineraller içerir. Diğer mantarların aksine, taze Reishi meyvasının su içeriği maksimum %25 dolayındadır. 1993 yılı National Audobon Society kaynaklarına göre, doğal olarak yetiştiği bölgelerde yaklaşık 10.000 yaşlı ağaçtan bir veya ikisi bu mantarı üzerinde taşımaktadır. Dolayısıyla doğada çok ender bulunur, kültürünün mümkün olduğu 1980 yılından beri Asya ve Batı ülkelerinin pazarlarında Ling-Zhi veya Reishi preperatları olarak satılmaktadır. Bu preperatların zaman zaman Red Reishi olmayan ve onun sahip olduğu özellikleri tam olarak taşımayan ürünler olduğu da bilinmektedir.

 

Tıbbi Faydaları

Ling-Zhi immunomodulating ve immunotherapotic aktivitelerinin kapsamı içinde antitümör özellikleri ile öne çıkmaktadır. Bilimsel çalışmalarla ortaya konulduğu kadarıyla (2) klinik testler sonucu polisakkaritlerinin, terpenlerinin ve diğer biyoaktif bileşenlerinin giderek daha iyi anlaşılır olması sağlanmıştır. Bu mantarın adaptojenik, antiallerjenik ve antihipertensive özellikler taşıdığı ve bu etkinliğinin içerdiği triterpenlerden kaynaklandığı gösterilmiştir (3). Birçok çalışma Ling-Zhi'nin antiinflamatuar, antiviral, antiparazitik, antifungal, antidiyabetik, antihipotensive ve hepatoprotektif özellikler taşıdığını göstermiştir. Bu arada platelet agregasyonunu inhibe ettiği, tansiyonu düşürdüğü, kolesterol ve kan şekerini düşürdüğü gösterilmiştir. Öyle ki bu özellikleri sayesinde tansiyon düzenleyici, antioksidan, anarjezik etkinliklerinin yanı sıra, böbrek ve sinir sistemini koruyup besleyen (tonik) bir ajan olarak da kabul edilmektedir. Bronşitin önlenmesinde, kardiyovasküler (kalp-damar) hastalıklarının tedavisinde, yüksek trigliseridlere bağlı ve yüksek tansiyona bağlı hastalıkların tedavisinde, hepatitde, alerjilerde, kemoterapi desteği olarak ve hatta HIV (AIDS) tedavi desteği olarak başarıyla kullanılmıştır. Bazı bilimsel çalışmalar, ganoderik asitin karaciğer hasarlarına ve virüs veya toksik bileşiklerle ortaya çıkacak karaciğer bozukluklarına karşı koruyu etki gösterdiğini, dolayısıyla insanda karaciğer hastalıklarında kullanılmaya uygun bir kaynak olduğunu göstermişlerdir. Özellikle www.pubmed.gov kaynaklarında kanserle savaşta tümörleri başarıyla inhibe eden etkinliği konusunda geniş bilgiler vardır. Sonuçlar kanser tipine ve hastalık tablosunun ciddiyetine bağlı olarak farklılık gösterse de, diğer tıbbi tedavilere destek olarak kullanılması yönünde ciddi tavsiyeler vardır. Kemoterapinin yan etkilerini azaltma konusunda, özellikle kemoterapiden evvel alındığı taktirde, çok başarılı sonuçlar elde edildiği fakat kemoterapi sırasında da hatta sonrasında da alınabildiği gösterilmiştir. Saç dökülmesi, ağız yaraları, iştah kaybı ve benzer yan etkiler büyük ölçüde ortadan kalkmaktadır. İnce kesilmiş Reishi dilimleri, kaynar suda ekstrakte edilerek çay şeklinde tüketilir. Kaynama süresi 2 saat olmalıdır. Elde edilen çay, tat bakımından hafif acı fakat rahatça içilebilir özelliktedir. Reishi (Ling-Zhi) ürünleri kapsül, toz şeklinde veya sıvı halde Reishi ekstraktı olarak piyasada yaygındır (4). Ancak toz haline sokulması durumunda sonradan kontamine olabileceği ve kendine özgü bileşiklerini kaybedebileceği unutulmamalıdır. Diğer ticari formlarının hangi katkı maddeleri içerdiğini mutlaka bilmemiz lazımdır. Dolayısıyla uygun kalınlıkta dilimlenmiş ve kurutulmuş halde tüketicinin kullanması tarafımızdan özellikle tavsiye edilmektedir. Şimdiye kadar, kullanılması sırasında ciddi bir yan etki yaptığına dair hiçbir kayıt yoktur. "Shen Nong's Herbal Classic" 2000 yaşında bir Çin tıp kitabıdır. Günümüzde doğunun bitkisel tıbbı (Herbal Medicine) konusunda en eski kitabı olarak kabul edilir. 365 bitki türünü ve hayvan türünü bünyesine almış ve onları üç yarar grubuna ayırmıştır. Superior grubunu oluşturan birinci kategoride, vücudun dengesini düzenleyen ve korunmasını sağlayan ve çok sayıda rahatsızlığa faydası olan bitkiler toplanmıştır ve bunların hiçbirinin ciddi yan etki yapmadığı vurgulanmıştır. Bunların sürekli alınmalarında bir zarar görülmemektedir. İkinci kategori, tonik ve benzeri kullanım alanı olan bitkileri içermektedir ki, uzun süre alınmalarında sakıncalara değinilmektedir. Üçüncü kategoride sadece özel durumlar için ve ancak düşük dozlarda alınabilecek ürünler toplanmıştır. Ling-Zhi, superior grubun en üstüne yerleştirilmiş ve kendisine "Gatzherb" adı verilmiştir (Tanrıların bitkisi).

 

Modern Bilimsel Çalışmalar

               Ling-Zhi ile özellikle Çin, Kore ve Japonya ve Birleşik Devletlerde yapılan çok sayıda çalışma bu mantarın çok geniş bir hastalık ve semptom spektrumunda olumlu etkiler yaptığını göstermiştir. Tüm bu çalışmalarda hala daha tam olarak etkinliğin neden kaynaklandığı çıkartılamamış, çünkü çok sayıda özel bileşiğin izole edilebilmesine rağmen hiçbirinin tek başına alınması durumunda mantarın kendisinin kullanılmasına kıyasla başarı elde edilemediği görülmüştür. Günümüzde Ling-Zhi'nin tanrısal gücü Çin Halk Tıbbı görüş açısından bakılarak anlaşılmalıdır. Batı dünyasında bilim adamları, her bir hastalığa ayrı ayrı yaklaşarak, bu mantarın etkinlik düzeyini araştırmaktadır. Uzakdoğu tıbbında bu mantarla ilgili bulgular 4000 yıllık bir birikimin bize verdiği bilgilere dayanmaktadır. Geleneksel Uzakdoğu görüşü, bir hastalığı aslında vücudun bozulmuş olan dengesinin tıpkı bir buzdağı gibi suyun üstünden görünen kısmı kabul eder ve bu mantarın hastalıkları tedavi etmesinin temelinde, vücudun biyolojik ve psikolojik dengesinin sağlanmasının yattığına inanır. Reishi'nin en önemli özellikleri çok yüksek dozda bile kullanılsa, nerede ise hiç yan etki yapmaması şeklinde gözlemlenerek bir araya getirilmiştir. Kanı temizlemesi, kanın oksijen taşıma yeteneğinin artması, bağışıklık sisteminin güçlendirilmesi ve sinirsel gerginliğin azaltılması en ana faydalardan birkaçıdır. Bu özellikler vücudun dengesinin düzenlenmesi ve normalleştirilmesi ile iç içe olduğundan, dengenin bozulmasından kaynaklanan birçok hastalığı tedavi etmektedir. Ling-Zhi solunum sistemini güçlendirme ve akciğer kapasitesini arttırma ve buna ek olarak akciğer fonksiyonlarının düzenlenmesini mükemmelleştirme etkisi gösterdiğinden astım başta olmak üzere diğer solunum rahatsızlıklarının tedavisinde çok önemlidir. 1970'lerde bu yönde yapılmış ciddi araştırmalar bu sonucu göstermektedir. 2000'den fazla Çinli kronik bronşit hastasına Reishi şurubu içirilmiş, 2 hafta içerisinde %60-%90'nında iştahın arttığı ve hastaların kendisini çok daha iyi hissettiği görülmüştür (Christopher Hobbs, Helps for Health, Jan/February 97). Japonya'da kanserli farelere günlük enjeksiyon uygulandığında, hayvanların %50'sinde 10 gün içerisinde tümörlerin tamamen baskı altına alındığı gösterilmiştir (Ikikava et al., 1968, Japanese Journal of Cancer Research 59:155-157) Antitümör aktivitesinin birçok benzer çalışmada özellikle mantarın polisakkarit bileşenlerinden ileri geldiği gösterilmiş olsa da (Sashaki et al., 1971), insanla yapılan çok geniş kapsamlı çalışmalara olan ihtiyaç hala devam etmektedir.

Diğer tedavilere destek amacıyla kullanılması 4 açıdan önemlidir:

  • a- Kemoterapi veya radyoterapi sırasında olumsuz yan etkilerin (yorgunluk, iştahsızlık, saç dökülmesi, kemik iliği süpresyonuyla enfeksiyon riski) azalmasına yardımcıdır. Jang, 1994 yorgunluğu azalttığını, Miyamato et al., 1985 saç dökülmesini engellediğini, Jeia et al., 1993 kemik iliğini süpresyonunu azalttığını yayınlamışlardır. Shi, 1993 Ling-Zhi'nin diğer glukan biological responce modifiers etkinliği gibi kanser kemoterapisinde ve radyoterapisinde kullanılmasının uygun olduğunu bildirmektedir. Chang, 1994 kullanım dozunun 5-10 gr/gün kuru meyva ağırlığı şeklinde seçilmesi gerektiğini vurgulamıştır.
  • b- Kanser hastalarına verilen diyetin içerisinde kullanılması, yaşam süresini uzattığı ve metastaz olasılığını azalttığı şeklinde tanımlanabilir. Diğer kıyaslanabilir glukan, BRM'lerinin benzeri bir etki ile Ganoderma kullanımının kanser hastalarının yaşam sürelerini uzattığı bildirilmektedir. Ganoderma'nın (Reishi) hayvanlarda metastaz oluşumunu güçlü bir şekilde engellediği Lee, 1984 tarafından gösterilmiştir.
  • c- Kanser hastalarına Reishi verilmesi durumunda yaşam kalitesinin arttığı gözlenmektedir. Ganoderma'nın yanı sıra PSP taşıyan ve yetiştirilmesi daha kolay olan diğer mantarların da bu yönde fayda sağladığı Yao, 1993 tarafından yayınlanmıştır. Ancak Ganoderma (Reishi) verilen hastalarda özellikle terminal devrede bütün şikayetlerin ve ağrıların önemli derecede görülmediği, azaldığı Cupin, 1994 tarafından vurgulanmaktadır.
  • d- Kanser oluşumundan korunma veya tekrar kanser olmayı önlemek amacıyla kullanılması durumunda özellikle Natural Killer (NK) ve sitotoksik limfosit (CTL) hücrelerinin aktive edildiği ve sanki kanser aşısı gibi bir bağışıklık sağladığı bildirilmektedir (Lotzova, 1985). Ganoderma'nın ağız yoluyla alan hastalarda NK ve CTL aktivitesini arttırdığı bilimsel olarak gösterilmiştir (Von et al., 1989). Bu Ganoderma'yı hem kanserden korunma hem tekrarını önleme yönünde rakipsiz kılmaktadır.

** Aşağıdaki kaynak "Shu-Ting Chang Emeritus Professor of Biology and Director of Centre for International Service to Mushroom Biotechnology The Chineese University, 2005"'dan tercümedir.

Ganoderma lucidum:

               Tıbbi mantarların lideri.

Özet: İnsanoğlu kendini daha sağlıklı ve daha mutlu yapacak ve biyolojik fonksiyonlarını arttıracak yeni koşullar ve ürünler arayıp durmaktadır. Ganoderma lucidum (Reishi) Çin ve diğer bazı Asya ülkelerinde yüzyıllar öncesinden beri tanına gelmiş bir mantardır. Ancak, özelikle son 20 yıldır Reishi ürünlerine olan ticari ilgi o denli artmıştır ki, sadece Asya ülkeleri değil, Kuzey Amerika ve Avrupa'da da son derece popüler hale gelmiştir. 2005 verilerine göre dünyada toplam üretim 6000 tondur. Bunun yarısı Çin'den kaynaklanmaktadır. Ganoderma lucidum ürünlerinin ticari cirosunun 2.5 milyar doları geçtiği kabul edilir. Bu artışın en önemli sebebi, bu mantarın tıbbi özelliklerinin giderek daha iyi anlaşılması ve yeni analitik teknikleri ile mantarın sahip olduğu polisakkaritlerin, triterpenlerin, adenozinlerin ve immunomodulator proteinlerin giderek artan sayıda saptanmasıdır. Bu bileşenlerin büyük çoğunluğu antikanser, antitümör özellikleri taşımaktadır ve bu etkinliklerinin temelinde kişinin bağışıklık sistemini güçlendirmenin yattığı düşünülmektedir.

•1.      Giriş:

İnsanoğlu sağlığıyla ilgili çabalarını tarih boyunca sürdürmüştür. Ancak daha çok Asya ülkelerinde ciddiye alınan bitkisel tedavi ve sağlığın korunması uygulamaları yanında, Batı dünyasında birden bire ortaya çıkan ve giderek artan bitkisel tedaviye yönelme, mantarları kullanma ve diğer bitkilerin yanında benzer özellikli besinleri tercih etme davranışı görülmeye başlanmıştır. Bu tip ürünlere vitaminleri, besin destek elemanlarını ekleyebiliriz. Ganoderma lucidum Çincede Ling-Zhi, Japoncada Reishi, Mannentake veya Sachitake ismiyle anılırken, Korecede Young-Zhi adını alır. Basidiomycetes grubunun Polyporaceae ailesindendir. Yaşlı ağaçlarda rastlanan bir mantardır. Ender olarak bitkilerde patojen kabul edenler de vardır. Eskiden beri bilinegeldiği Çin ve Japonya'nın yanı sıra Kuzey Amerika ve Avrupa'da da bu mantarın meyvaları giderek aranır olmuştur. Sebep uluslar arası dikkatin tüketilebilir bir Çin bitkisel ürünü olarak antitümör, immunomodulatör, kardiyovasküler, solunum sistemi, antihepatotoksik ve ağrılara karşı analjezik etkinlikleri şeklinde tanımlayabileceğimiz biyolojik aktivitelerinden kaynaklanmaktadır. Yapısında bulunan bileşenlerin en önemlilerinin triterpenler, polisakkaritler ve adenozinler olduğu kabul edilir. Kim ve Kim, 2002 150 triterpenden daha fazla sayıda triterpen içerdiğini ve Jong ve Birningham, 1992 50'den fazla karsinostatik (kanser durdurucu) polisakkarit taşıdığını bildirmişler ve bunların izole edildiğini söylemişlerdir. Bu bileşenlerin bu mantarın ana etkinliğinin kaynağı olduğu ileri sürülmüştür. Bu mantarla ilgili üretim endüstrisinde, kullanılan tekniklerin bu bileşenlerin korunmasına uygun olarak seçilmesi çok önemlidir. Dolayısıyla ürünün faydalarının, takdim ve yetiştirme şekli ile çok yakından ilgili olduğu söylenebilir. Ganoderma lucidum en üst düzeye yerleşmiş ve binlerce yıldır bilinen tıbbi bir mantar olmanın yanı sıra, çok uzun süre tüketilse bile hiçbir yan etkisi görülmeyen nontoksik bir ürün olduğu vurgulanmaktadır. Son 30 yıldır en güvenilen ve en yoğun araştırılan tıbbi mantar olduğuna inanmaktayız. Dünya ticaret açısından yenilebilir toplam hacminin 30 milyar dolar olduğunu biliyoruz. Bunun içinde yaklaşık 2.5 milyar dolarla Ganoderma'nın yer aldığı toplam tıbbi mantar ürün hacmi 10 milyar doları bulmaktadır.

 

•2.      Geleneksel Kullanım:

Tek başına veya diğer bitkilerle birlikte Ling-Zhi'nin geleneksel Çin tıbbında önemli rol oynadığı bilinmektedir. İntellektüel kapasitenin, hafızanın artarak korunduğu ve yaşamı uzattığı Ling-Zhi uygulamaları bunlara ek olarak, karaciğeri koruyan böbreği koruyan ve tedavi eden, yağlanmayı (kan lipid seviye yüksekliği) önlediği, artrid, astım, mide ülseri, arterioskleroz (damar daralması), lökopeni (lökosit sayı düşüklüğü), diyabet ve anoreksiya hastalıklarında tedavi edici etkinliği geleneksel Çin tıbbında en öne çıkan özellikleridir (Chang and But, 1996; Jong and Birmingham, 1992). Mayzumi et al., 1997 bildirmektedir ki, uzun sürmüş hastalıklardan hızla sağlığa kavuşmak, nörasdeni, aneroksi, kronik hepatit, hiperkolesterolemi, korener kalp hastalıkları, hipertansiyon, karsinomlar ve bronşiyal hastalıklarla mücadelede ve tedavide Ganoderma etkinliği son derece üst düzeyde gözlenmektedir. Bu mantarın kolesterol sentezini inhibe ettiği Kim et al., 2000 tarafından yayınlanmıştır. Günümüzde dünyada milyonlarca kişi besin desteği ve tonik olarak Ganoderma tüketmektedir. Sindirim yeteneklerinin arttırıldığı, enerjilerinin yükseldiği ve daha iyi uyudukları kesindir. Ganoderma, ileride karşılaşılabilecek birçok hastalığa karşı koruma sağlamada, bağışıklık sisteminin dengelenmesinde ve sağlıklı kalp-damar sistemi oluşturulmasında yaygın kullanım bulmaktadır. Ganoderma esaslı ürünlerin çok iyi denetlenmesi ve tüketici tarafından kalitesinin güvenilir olmasına dikkat edilmesi gerekir. Bütün eski Çin tıbbi kayıtlarında Ling-Zhi en üst düzeyde kabul edilir.

 

•3.      Biyolojik Komponentler:

Lindequist, 1995 Reishi'nin biyoaktif komponentlerinin asıl bölümünün triterpenler, polisakkaritler ve adenozinler olduğunu bildirmekte ve farmakolojik aktivitelerin bu ana bileşenlerden ileri geldiği görüşünü ileri sürmektedir. Fungal immunomodulatory proteins (FIP) adı verilen biyoaktif proteinler, nükleik asitler ve diğer bazı bileşikler de Reishi'den izole edilebilmektedirler (Kino et al., 1989; Ko et al., 1995, 1997). Son 20 yılda 200'den fazla bileşik bu mantardan izole edilmiştir. Biz aşağıda, sadece triterpenler ve polisakkaritlerden söz edeceğiz.

 

•a-     Triterpenler-Triterponoidler

Ganoderma lucidum'da ilk kez ganodermerik asit A ve B isimleriyle iki triterpen izole eden ilk araştırmacılar Kubota ve ark., 1982'dir. O günden bu yana 150'den fazla Lanostan tip triterpenoid Ganoderma lucidum'da bulunmuş ve identifize edilmiştir. Biyolojik ve tıbbi aktiviteleri açısından benzerlikleri ve yapısal yakınlıkları dikkate alınırsa, bunların 10 gruba ayrılabildiği söylenmektedir (Kim and Kim, 2002). Triterpenlerin sitotoksik, hepatoprotektif (karaciğeri koruyan) ve hipolipidemik (kanda yağ düzeyini azaltan) özellikler taşıdığı bilinmektedir. Kan damarları içerisinde pıhtı oluşmasını engellemeleri, histamin deşarjını engelleyerek alerjik reaksiyonları baskı altına aldıkları bilinmektedir (Lindequist, 1995; Misuno et al., 1995; Lin, 1996; Kim and Kim, 2002).

Farklı triterpenlerin farklı biyoaktif özellikler taşıdığı gösterilmiştir. Bunlardan birkaçı şu şekilde sıralanabilir:

  • 1. Acılık: Reishi (Ganoderma lucidum) diğer hiçbir mantarda bulunmayan acımsı bir tada sahiptir. Acılığın kültür koşullarıyla veya kullanılan ırkın özellikleri ile değişebileceği görülmekle beraber, sapın şapkadan daha acı olduğu fark edilmiştir. Acılık, mantarın kalitesi hakkında güvenilir bir belirtidir. Koreliler sadece acı ürünleri itibara alırlar. Acılığın ganoderik asit A, C, İ ve J'den, lucidenic asit A, D, İ'den ve lucidon A ve C'den kaynaklandığı düşünülür (Nishitoba, 1996).
  • 2. Sitoksisite: Sitotoksik triterpenler misel ve meyva ekstraktlarında bulunan güçlü antikanser ajanlardır. Ganoderik asit Z, Y, X, W, V ve T tiplerinin invitro koşullarda hepatom hücrelerine (karaciğer kanser hücreleri) öldürücü etki yaptığı gösterilmiştir. Yine meyvalardan elde edilen lanostanoidler güçlü bir şekilde tümör hücrelerini inhibe etmektedirler (Kim and Kim, 2002).
  • 3. Platelet agregat inhibisyonu (Pıhtı oluşmasının engellenmesi): Kandaki plateletler damarlardaki endotelle reaksiyona girmezler. Ancak damar iç yüzeyi (endotel) hasarlarında hızlı bir şekilde yapışır ve küçük topluluklar oluşturarak pıhtı meydana getirirler. Apopleksi tedavisinde platelet agregasyonu engelleyicilerin önemi büyüktür. Ganodermik asit S, platelet agregasyonu üzerine amfipatik etkili olarak gösterilmiştir (Wang et al., 1991; Sue et al., 1999).
  • 4. Antihipertansiyon etkisi: Morigiva et al., 1996 göstermiştir ki, ganoderik asit F antihipertansif olarak çok güçlü bir etkiye sahiptir. Diğer ganoderik asitler B, D, H ve Y buna kıyasla daha zayıf etki gösterirler.
  • 5. Hepatoprotektif aktivite (Karaciğer koruyucu etkinlik): Çin halk tıbbında Ganoderma lucidum meyvalarının kronik hepatit tedavisinde kullanıldığı bilinmektedir. Hirotani et al., 1986 göstermiştir ki, ganoderik asit R ve S güçlü antihepatotoksik bir aktiviteye sahiptir ve bu etkinliği sıçan hepatotsitleri üzerine açıkça gösterilmiştir. Diğer bir hepatoprotektif bileşen olarak ganosporerik asit A bulunmuştur. Özellikle bu mantarın sporlarından eter yardımıyla izole edilmiştir (Chang and Yu, 1991, 1999).
  • 6. Anti HIV: İnsan Immunodeficiency Vırus (HIV) AIDS hastalarından izole edilmektedir (Barre-Sinoussi et al., 1983; Gallo et al., 1983). Ganoderma lucidum'un sıcak su ekstraktında antiHIV aktiviteleri görülmüştür (Hattori et al., 1997; Kim et al., 1997). Bunlara ek olarak El Maccavi et al., 1998 ganoderiol F ve ganodermanuntriol ismini verdikleri antiHIV bileşenlerini Reishi'den elde etmişlerdir. Min et al., 1998 ganoderik asit B, ganodermanondiol ve ganodermanontriol ile ganolucidic asit A ve lucidumol isimli antiHIV bileşikleri elde ettiğini bildirmektedir.
  • 7. Hipoglisemenik etki: Ganoderans A, B ve C güçlü hipoglisemik etkinliği bulunan ve Reishi meyvalarından izole edilen triterpenlere örnektir (Hikuno et al., 1985). Ganoderma ürünleri birlikte alındığı taktirde diyabet ilacı olarak kullanılan bileşiklerin yan etkileri yok edilmektedir. Günümüzde diyabet hastalarını asıl endişelendiren şey, kroner arter hastalıkları ve damar içi plak oluşumu ile hipertansiyon, bağışıklık sisteminin zayıflaması ve göz hastalıkları şeklinde ortaya çıkan rahatsızlıklar endişelendirmektedir. Ganoderma ekstraktının ana bileşenlerinin bu hastalıkların ve bu tabloların ortaya çıkmasını engellediği bulunmuştur.

 

•b-     Polisakkaritler

1984'te Lee et al., Ganoderma lucidum'un suda çözünen bileşenlerinin farede sarkoma 180 ve fibrosarkomları inhibe ettiğini göstermiştir. Farelerde yapılan tüm testlerde Ganoderma lucidum sıcak su ekstraktlarının hayvanın enerjisini ve yaşam süresini uzattığını göstermiştir. Ganoderma lucidum'un antitümör ve antikanser etkinliğinin sahip olduğu polisakkaritlerden kaynaklandığı ve temelde kişinin immun sistemini güçlendirerek etki ettikleri gösterilmiştir (Lieu et al., 1992; Zhu and Morhi, 1993; Chan et al., 1995; Vank et al., 1997). Bağışıklık sistemimizin çeşitli bileşenleri bu polisakkaritlerden olumlu etkilenmektedir. Örneğin, makrofajların aktive edilmesi, natural killer hücrelerinin aktivasyonu ve sitotoksik T hücrelerinin bunlara özgü tümör negrozis faktör reaktif nitrojen ve oksijen radikalleri ile interlökin salgılama yeteneklerinin de kapsam alanına girdiği aktivasyonu sayılabilir (Ooi et al., 2002). Ekstrakte edilebilen bu tip polisakkarit komponentler, makrofaj koloni stimüle edici faktör olarak etkinlik göstermektedirler. Tümör negrosis faktör A düzeyinin arttırılması, direkt olarak antitümör aktivitesinin arttırılması şeklinde mütala edilebilir. Sonuç olarak Ganoderma lucidum bileşenlerinin genel anlamda antitümör aktivitelerinin, belirli stokinlerin (makrofaj koloni stimüle edici faktör ve TNF-A gibi) üst düzeye çıkarılmasından kaynaklandığı söylenebilir. Unutmamalıdır ki, immun cevap makrofaj ve limfositlerin etkinliğinin de aralarında bulunduğu karmaşık bir reaksiyonu ifade eder. Sitotoksik T limfositlerinin ve natural killer hücrelerinin etkinliği ile ortaya çıkan yok etme yeteneği tümörlere, virüs taşıyan hücrelere, parazitlere ve diğer yabancı faktörlere karşı vücudun savunma mekanizmasında çok önemli bir yer tutar.

 

•c-      Fungal Immunomodulator Protein (FIP)

Çok yeni bilimsel çalışmaların diğer mantarların yanı sıra LZ-8 (Ling-Zhi-8) ismiyle Ganoderma lucidum'dan elde edilen yeni bir FIB grubu ortaya çıkarmıştır. Bu protein grubu aminoasit dizileri ve bağışıklık cevap üzerindeki aktiviteleri yönünden benzerlikleri sebebiyle bir araya getirilmiştir (Koo et al., 1995). LZ-8 optimal uyarıcı aktivite yeteneğine sahip ve dalak hücrelerinin hücre oluşturmasında (blast formasyon aktivitesi) uyarıcı bir bileşik olarak tanımlanmaktadır. LZ8 bir lektin değildir, fare dalak hücrelerinde mitojenik aktiviteyi arttırır ve aynı etkiyi insan periferik lökositleri üzerinde de gösterir. Bu özelliklere ek olarak, FIB'ler otoimmun hastalıklarda (otoimmun diyabet) baskılayıcı rol oynamakta ve istenmeyen olumsuz alerjik tepkilerin ortaya çıkmasını engellemektedir.

 

•d-     Steroidler

Ganoderma lucidum yüksek miktarda steroid içermektedir. Özellikle ergesterol ve kolesterol en önemlileridir. Bu mantardan her iki kategoriye girebilen yaklaşık 20 farklı steroid elde edilmiştir (Ha et al., 2000; Ma et al., 2002). Bu steroidlerin antiaterosklerotik (damar sertliğini engelleyici) ve lipid düşürücü etkinliğin temellerini oluşturduğu kabul edilir ve Ganoderma lucidum'un antihiperlepidemik aktivitesinin kaynağıdır (Kimura et al., 1988).

HEALTH FOODS BUSINESS/JANUARY 1992

CONSUMER EDUCATION SERIES

REISHI: ANCIENT MEDICINE IS MODERN HOPE

By Linda McGlasson, Assistant Editor

Western culture has often frowned on mushrooms, even fearing the small innocuous forest growth. The French prize their truffles, but even truffles and other edible fungi and mushrooms are not as highly valued or show as much potential as a species of mushrooms called Ling Zhi or Reishi (Ganoderma lucidum).

The late Hiroshi Hikino, recognized as the world's authority on the chemistry of Oriental medicinal plants, called Reishi one of "the most important elixirs in the Orient."

Relatively rare and undiscovered in the West, Reishi and other mushrooms have been revered as herbal medicines for thousands of years in Japan and China. Emperors of the great Chinese dynasties and Japanese royalty drank teas and concoctions of the mushroom for vitality and long life. The ancient Taoists were constantly searching for the elixir of eternal youth, and Reishi was believed to be among the ingredients.

In modern times, Ganoderma lucidum and its fellow mushrooms have been well-researched in Asian universities. It is currently being studied in China as a sports performance enhancer. Its long History has sparked interest in the West where it is used by herbalists to treat diverse problems such as allergies, chronic Fatigue Syndrome, diabetes, liver diseases and many immune-related diseases.

As little as 20 years ago, Reishi was rare and not widely found in Asia. It grew in the wild, but was extremely hard to cultivate. Now with an increased knowledge of the climates that it thrives in, scientists are able to set up artificial growth conditions with the correct amounts of oxygen and moisture for the spores to grow into the Reishi mushroom.

JUST ANOTHER FUNGUS?

Reishi mushrooms are polypore mushrooms. Mushrooms are the fruiting body and reproductive structure of a higher order fungus organism, much like an apple is the fruit of an apple tree. The actual mushroom "tree" is a fine thread-like network called mycelium. This mycelium is for the most part subterranean, living in soil, logs and other organic litter.

Unlike green plants, which produce many of their own nutrients by photosynthesis, mushrooms primarily get their nutrients from dead organic matter or soil. Mushrooms and their mycelium are nature's original recyclers. Without them, the planet surface would be piled high with dead, decaying material.

Mushrooms rise out of the mycelium when the right nutrients are amassed and the right environmental conditions are present. Mushrooms release spores at maturity. The wind spreads them and when they land on the right spot, the cycle starts over again.

REISHI'S MEDICAL PROPERTIES

In the 16th Century pharmacopedia Pen T'sao Kang Mu, which contains hundreds of natural medicines the Chinese have used for thousands of years, compiler Le Shih-chen described the uses of Reishi. "It positively affects the life energy, or qi of the heart, repairing the chest area and benefiting those with a knotted and tight chest." He wrote that it also increases intellectual capacity and banishes forgetfulness. "Taken over a long period of time, agility of the body will not cease, and the years are lengthened to those of the Immortal Fairies."

In the Orient, Reishi is considered a Fu Zhen herb (immune modulation). Presently, Reishi has various applications including lowering or raising blood pressure, stimulating liver actions, blood cleansing, and acting as an adaptogen in helping the body fight the effects of stress.

Chinese herbalists prize it for its abilities to regenerate the liver. In high doses, and to some degree normal doses, Ganoderma maybe classified as a liver detoxicant and protectant.

In traditional Oriental applications Reishi is also used to treat insomnia, gastric ulcers, neurasthenia, arthritis, nephritis, asthma, bronchitis, hypertension and poisoning. It is also being used in treating neuromuscular disorders -- stress-induced tension, myasthenia gravis and muscular dystrophy -- all with varying degrees of success.

Toxicity studies show no toxic effects on humans. In research, patients are given much higher doses, as high as 10 grams of extract per day, with no ill effects.

ACTIVE INGREDIENTS

The potency of Reishi mushrooms is usually based on its level of triterpenoids. One can determine the level of this by tasting it. The more bitter it is, the higher the level of triterpenoids. Because Reishi is a polypore, (a group of hard, woody, bracket-like mushrooms) it is not eaten, but cut into pieces and made into a tea. In China, the average dose is 3 to 5 grams a day. Other popular forms of delivery are the water/alcohol extracts and powders.

Reishi mushrooms and mushroom extracts are generally analyzed for specific triterpenoids called Ganoderic acids. When buying a Reishi mushroom product, check for the analysis of how much triterpenoids is in the extract or powder.

"There is no standardization yet, either here or in Asia for Reishi. You have to look for high ganoderic acid-A levels, which indicates high levels of other ganoderic acids," said Kenneth Jones, a researcher/writer specializing in the ethnopharmacology of medicinal plants.

One focus for future research is on Reishi spore extracts. In China, it has been used in injectable form in clinical treatments of various ailments with success. One of the things it has successfully treated is low energy, and debilitation following long illness.

OTHER APPLICATIONS

Chinese women take Reishi for beautification of the skin. The results are probably due to the mushroom's hormone-potentiating effects, Jones said.

Reishi is included in many Japanese patents for hair loss formulas, including products used for alopecia. Spore extract injections of Reishi are also being used to treat lupus in China.

The mycelium of Reishi contains high levels of polysaccharides, which have been shown in research to induce the production of interferon. Interferon is a protein produced inside cells to fight viral infection. Polysaccharides are also tumor fighters and help stimulate the immune system.

Reishi is being recognized for its adjunct use as an immune system stimulator when cancer therapy is being used. The use of Reishi as a cancer treatment in the Orient is centuries old. In following the concept of qi tonics, Reishi is used to strengthen the body's resistance to outside forces.

Former heart surgeon Dr. Fukumi Morishige, a leading authority on vitamin C in Japan, reports that when Reishi and vitamin C are combined the results against cancer and other diseases are far better than when Reishi is ingested. This is because the vitamin makes the polysaccharides more accessible to the immune system.

It is also an adaptogen, with properties similar to ginseng. The adenosine in Reishi may explain why the Chinese use it for patients suffering from nervous tension. Adenosine relaxes skeletal muscles, calms the central nervous system and operates against the stimulating action of caffeine.

"Reishi mushrooms are certainly an herb for the 90s and beyond," commented Jeff Chilton, president of North American Reishi. "Considering that Reishi has a history of use that spans 2,000 years and is more highly revered than ginseng in the Orient, one could readily compare its potential to that of ginseng."


Contributing to this article were Terry Willard, Ph.D. and Kenneth Jones, authors of Reishi Mushroom: Herb of Spiritual Potency and Medical Wonder.


Questions, comments, suggestions, and requests for further information are welcome. Send email to:

Dr. Gilbert Ng


This Web page and all linked pages are designed and developed by Dr. Gilbert Ng. Email: gng@hk.super.net

Copyright © 1996. All rights reserved. No part of this document may be reproduced by any means or in any form without permission from the aut

HOW AN ANCIENT HERBAL TREASURE CAN BENEFIT YOUR HEALTH TODAY

World Health Publishing Inc. 330 - 1501 West Broadway Vancouver, B.C. V6J 4Z6

Copyright©2003 by World Health Publishing Inc. First Edition ISBN: 0-9732103-2-X All rights reserved.

No part of this book may be reproduced in any form without the expressed written consent of the publisher, except by a reviewer, who may quote brief passages in connection with a review.

Author: Wong Shu Sing Project Editor: Trevor Lai Editorial Consultant: Brad K. Moy, M.D. Art Director: Catmagic Animation Workshop Cover Designer: Design Square Printer: Leanne Communications Inc.

CADS10.00 Printed in China

Foreword

Reishi (also known as "Ling Zhi" in China) has been known to the Asian world for over 2,000 years. Its miraculous health- enhancing applications have been employed by Oriental cultures for centuries, but have only been introduced to the Western world in the last 30 years.

Reishi is the legendary wizard of all superior medicinal herbs because of its apparent medical efficacy and the absence of unfavorable side-effects and toxins resulting from consumption. Because of its rarity in nature, reishi was reserved primarily for Asian royalty and wealthy individuals until the late 20th century, when cultivation of red reishi mushrooms by the Japanese made the once rare fungi plant more widely available to the general populace around the world.

This book provides an in-depth introduction on the history and the medicinal value of the red reishi species. Reishi mushrooms have been widely used by Chinese Traditional Medicine (TCM) practitioners for ages as an alternative approach to health and treatment modalities. However, such an approach has not been widely endorsed by Western medicine due to the lack of extensive research by those practitioners. A growing number of Western medical doctors and researchers are now working to better understand herbal therapies such as red reishi consumption so that they can help you make informed health care decisions. It has been reported that many people who choose herbal therapy rely mostly on their family and friends for information. We recommend that if you do choose herbal therapy, be sure to

consult a doctor with some basic knowledge in Chinese medicine first.

As many of the medical applications described in this book are, by definition, alternative, they have not been fully researched, approved, nor endorsed by any government or regulatory agency in the Western world. Accordingly, this book should not be substituted for the advice and care of a physician or other licensed health-care professional. Pregnant women, in particular, are urged to consult a physician before commencing any therapy. Ultimately, you must take full responsibility for your own health. The authors and publisher disclaim any responsibility for how you use the information in this book and any of the claims made herein.

Contents

Foreword................................................................................................ 4

Chapter 1 An Introduction to Reishi......................................................... 9

Chapter 2 The Key Types of Reishi.......................................................... 13

Chapter 3 Physical Characteristics and Chemical Composition of Red

Reishi.................................................................................. 17

Chapter 4 Body & Mind: Using Red Reishi to Improve Your Health........... 21

Chapter 5 Health-Enhancing Applications of Red Reishi........................... 23

Chapter 6 How Red Reishi can Improve Your Physical Appearance.......... 31

Chapter 7 On the Road to Better Health with Reishi................................ 33

Chapter 8 The Cultivation of Red Reishi.................................................. 35

Chapter 9 Selecting the Right Reishi Product for You............................... 41

Chapter 10 Reishi Spores & Mycelium.................................................... 47

RESOURCES

Resources You can Use for More Information about Reishi..................... 51

Bibliography.......................................................................................... 53

 

An Introduction to RReishi

In China, for over 2,000 years, the mushroom known as Reishi (Ganoderma lucidum) has been called "God's Herb". Also recognized by its Chinese name, Ling Zhi, Reishi's reputation for being effective in treating a wide range of ailments moved Chinese Emperors throughout the various Dynasties to order servants to search for wild Reishi mushrooms found atop distant mountains, believing that the consumption of Reishi would grant them eternal youth and enhanced health. Because of the mushroom's remote habitat and the scarcity of high-quality specimens, the use of Reishi for medicinal purposes was reserved primarily for royalty and wealthy individuals. It was not until the late 20th century, that this once-rare plant, through diligent cultivation by the Japanese, was it made widely available to the general public.

life

,The original textbook of Oriental medical science, "Herbal Pharmacopoeia", was compiled by the founding father of Chinese medicine, Shen Nong (Han Dynasty, 206 BC ~ 8 AD). In it, the legendary herbalist-emperor documented 365 species of plants and classified them into three categories: superior, average and fair. These classifications were based on two main criteria: its benefits, based on consumption on a continual basis, and side effects. For those plants graded as "superior", the power to harmonize the functions of the body, mind and spirit and the range of ailments they could treat were greater and broader than those of weaker specimens. In addition, they had to have little or no long- term side effects. Among the specimens in this class, Reishi was ranked the highest in this classic medical text, even superior to the well known ginseng.

In the "Compendium of Materia Medica" (Ben Cao Gang Mu), which contains hundreds of natural medicines the Chinese have used for thousands of years, celebrated physician and naturalist Li Shi Zhen (1518~1593) described the benefits of Reishi:

It benefits the life energy, or "qi" of the heart, repairing the chest area and benefiting those with a knotted and tight chest. Taken over a long period of time, agility of the body will not cease, and the years are lengthened to those of the Immortal Fairies.

Over the ages, Reishi has become ingrained in Oriental art and culture because of its prestigious status in Traditional Chinese Medicine (TCM). Since the first Chinese dynasty, paintings, embroideries, buildings, and sculptures of the gods and immortals have depicted Reishi as a symbol of divinity, longevity and good fortune. Depictions of Reishi are displayed throughout the Forbidden City and the Summer Palace in Beijing as a testimony to its value, and the mushroom's distinctive shape was a favorite ornamental design feature used by royalty and the wealthy. Even the traditional scepter of the emperors of China was a stylized Reishi, called a "Ru Yi".

 

Reishi was further immortalized as the ultimate healing substance and spiritual herb of China in the classic Chinese fairy tale, "The White Snake", wherein a mystical heroine stole a magical Reishi plant from the gods to save the life of her human lover. The celestial battle for the Reishi rivaled the battles portrayed in Homer's Iliad and is a story known by virtually every Chinese person.

The Key Types ofReishi

Although there are more than 2000 known species of Reishi, only six kinds have been studied in greater detail to uncover potential health benefits - red, black, blue, white, yellow and purple Reishi. Of these six types, black and red Reishi have demonstrated the most significant health-enhancing effects, and both are therefore widely used in the global health supplement market today. However, red Reishi has been proven to be the most effective in improving one's overall health by enhancing the immune system, many bodily functions, and vital organs.

Black Reishi (Ganoderma sinensis), is fairly common and can be found in most Chinese herbal shops. This species of Ganoderma tends to be unevenly shaped and can measure up to ten inches in diameter, although most mature specimens are about six inches in diameter. The majority of Reishi products that claim to be using "wild" Reishi generally use black Reishi. While it still possesses some value as a moderate herbal

tonic, black Reishi is considered to be inferior to red Reishi because of its lower polysaccharide content.

Wild purple Reishi grows in the Chang Bai Mountains, north of North Korea in Jilin province of China. They are extremely rare and very similar to red Reishi in appearance, but have a significant purple coloration in the heart of the mushroom cap. There has been limited research and testing on this type of Reishi, due in large part to the scarcity of authentic purple Reishi specimens.

It is the very high content of polysaccharides (complex sugars) in red Reishi that makes it particularly potent. However, due to harsh environmental conditions, the instability of wild red Reishi in nature, and its vulnerability to pollution, disease, and insect infestations, the number of high quality red Reishi specimens that can reach full maturity in the wild is rare.

As a result, approximately 30 years ago, scientists and farmers began experimenting with different methods of domestic cultivation for mass production. Since then, Japanese producers in particular,

have earned the reputation of growing the highest quality red Reishi, thanks in part to strict regulations set by the Japanese government to uphold quality and trade standards. The complex cultivation process developed by Japanese scientists and farmers is detailed further in Chapter 5 of this booklet.

Because of the extensive research conducted to investigate the health benefits of red Reishi, and the proliferation of red Reishi products in the marketplace, this booklet focuses primarily on this specific kind of Reishi.

Physical Characteristics and Chemical Composition of Red Reishi

Since mushrooms have become staples of a healthy diet, much information has been gathered in the biological study of fungi. The object of the following section is not to duplicate the in- depth explanation of such studies, but rather to provide a general description of the physical components of red Reishi fungi and its chemical constituents.

The body of a red Reishi mushroom consists of three main parts:

  • a kidney-shaped cap
  • the stem or shaft of the mushroom
  • spores

The stem of the plant draws the nutrients from the wood on which it is growing. In nature, the mushroom flourishes mainly on the dried trunks of dead plum, guercus serrata or pasonia trees, while red Reishi cultivated in Japan is usually cultured by grafting the Reishi fungi onto aged Japanese oak.

Just as "we are what we eat," the quality of a red Reishi mushroom also depends heavily on the nutrients found in its habitat. The quantity and quality of these nutrients determine the size of the mushroom's cap. As the red Reishi approaches maturity,

spores are produced and are eventually released into the air. Because of the hard outer husks of these spores, germination is next to impossible, contributing to the rarity of fully-grown red Reishi mushrooms in the wild.

Red Reishi mushrooms are primarily composed of complex carbohydrates called polysaccharides, triterpeniods, proteins and amino acids. Studies indicate that it is these polysaccharides, the most active element found in red Reishi, that are responsible for

 

 CNiOH

 

Structural analysis of anti-tumor polysaccharides fS-(1->6) and 0-(1->3)-D-glucan

 

strengthening the body's immune system. Medical practitioners in China and Japan have long used herbs like ginseng, brasenia and astragalus - which all share red Reishi's high polysaccharide content - to decrease the side effects of chemotherapy suffered by cancer patients.

In addition, ganoderic acids in red Reishi have also been shown to help alleviate common allergies by inhibiting the chemical mediators of inflammation, including histamine release.

Aside from the exceptionally high polysaccharide content, no exclusive or unique ingredient has yet been identified in red Reishi that can be credited for the mushroom's remarkable health enhancing abilities. It is more likely that the potency of the mushroom lies in the combination of its chemical constituents - a natural illustration of the idea that "the whole is more than the sum of its parts".

 

Body & Mind Using Red Reishi to Improve Yourflealth

Perhaps red Reishi's greatest potential medical benefit is its ability to strengthen the body's immune system. Consumption of red Reishi is therefore considered preventive and immune enhancing, rather than treatment of a specific disease.

Red Reishi is gaining credence as a potential example of what the Royal Medical Society has termed an "adaptogen" - a treatment concept that is said to help the body adapt to stresses of various kinds; these stressors may be temperature, trauma, sleep deprivation, exposure to toxins, radiation, infection, or psychological stress. Like adaptogens, red Reishi shares the properties of causing no side effects with regular consumption; it is effective in dealing with a wide variety of illnesses; and it helps an organism return to a normal and balanced state.

Furthermore, regular consumption of red Reishi can significantly improve the functioning of the immune system. Red Reishi acts as an immune modulator - a substance that regulates, and fine-tunes the immune system. The primary responsibility of the immune system is to detect pathogens such as viruses, bacteria and other microbes that invade the body. Under normal circumstances, an intact immune system is capable of detering most of these organisms before it causes any damage. However, if the immune system is compromised, viruses, bacteria, parasites and fungi can proliferate and cause disease. Antibiotics, anti-viral agents, and anti-fungal medications are the mainstay of current therapy. Even though they can be effective, every treatment carries a potential side effect. In addition, repeated use of these medications can cause microbes to mutate and develop resistance. Consuming red Reishi eliminates these problems.

Red Reishi is also gaining acceptance for its use as an adjunct to combating the unpleasant side effects associated with radiation and chemotherapy for cancer treatment.

Authentic red Reishi products have also demonstrated exceptional anti-stress properties that help improve memory, ease tension, and sharpen concentration. Its potency is particularly noteworthy in the treatment of degenerative diseases that affect primarily the elderly, such as Alzheimer's disease and other forms of dementia.

 

flealth-EnhancingAppHcations ofRed Reishi

Throughout the ages, the consumption of red Reishi has been linked to the treatment of a vast range of diseases, common ailments, and conditions. From asthma to zoster, the applications of red Reishi seem to be related to a multitude of body organs and systems.

Although some product manufacturers use this information as proof that red Reishi can be used to treat these maladies directly, there is no sufficient medical evidence to support this claim to date. However, most of the scientific research that has been conducted appears to strongly support red Reishi's role as a normalizing substance - a nutritional supplement that can yield medical benefits through its normalization and regulation of the body's organs and functions.

The previous chapter provided a general outline of the overall benefits derived from the consumption of red Reishi. The following is a list of specific conditions for which regular consumption of red Reishi and products consisting of authentic red Reishi extract has been shown to have some beneficial effects. Please note that although many of the medical applications listed below have been supported by researchers in both the medical and nutritional science fields, the information below should be treated as observations gathered over time based on individual cases, and

not a guarantee or endorsement of Reishi's effectiveness. For specific case reports or data, please consult a physician with an expertise in Chinese medicine. Research studies and additional information can be obtained through libraries or found on the internet. You may also contact your local health organizations.

Conditions Related to Blood Circulation

It has been thought that red Reishi extract can reduce the coagulation of blood by decreasing platelet aggregation (cells that form clots), similar to aspirin. In addition, it is possible that it is able to transform red blood cells, and make them more malleable to travel through blood vessels at greater ease, reducing the chances of clogging, which could lead to strokes and heart attacks. Improved circulation is extremely important in diabetics, who invariably develop small artery disease.

Heart Conditions

Several cardiovascular benefits have been demonstrated from the regular consumption of red Reishi. These include:

  • correction of some arrhythmias due to stress
  • lowering of "BAD" LDL cholesterol and Triglycerides (another type of blood fat)
  • lowering of blood pressure - demonstrated in published human studies
  • improved blood circulation, with all of the benefits outlined above.

Liver Disorders

The primary function of the liver is to metabolize and detoxify drugs and poisons.

In addition, it is where cholesterol and certain proteins are synthesized. Damage to the liver can cause an accumulation of toxins and by-products in the body that cause jaundice (yellowing of the skin), weight loss, fatigue, loss of appetite, and many other maladies. Research has shown that red Reishi has hepato (liver) protective properties. Regular consumption of red Reishi may indeed rejuvenate the liver cells, allowing it to perform its vital functions.

Digestive Disorders

Red Reishi consumption has also been shown to decrease gastric acid, which, in excess, can lead to the formation of peptic and gastric ulcers. It has also been shown to regulate bowel movements, relieving symptoms of constipation and diarrhea, such as that found in irritable bowel syndrome.

Lungs

 

Red Reishi has shown some benefits in those suffering
from asthma, chronic bronchitis, and common allergies. By blocking the mediators of inflammation, symptoms of asthma can be alleviated, and by boosting the immune system, colds and flu's are reduced, leading to less exacerbations of asthma. Red Reishi products have been used as an anti-tussive to suppress persistent coughing, and it is used as an expectorant to loosen up secretions. It is thought that red Reishi may even enhance the regeneration of tracheal and small airway epithelium - particularly important in patients who smoke and suffer from chronic bronchitis (smokers' cough).

Kidney Regulation

The kidneys are responsible for maintaining the proper balance of chemicals and elements such as sodium, potassium, and other electrolytes in the body. It also filters the blood constantly and maintains a constant acidity (pH) and molecular concentration. Red Reishi may aid in the regulatory functions of the kidney.

Cancer

 

Cancerous cells are marked by their abnormal proliferation of growth. While it is still unclear how normal cells become cancerous, there has been some suggestion that it may be due to a viral origin, or perhaps a change in the body's natural defense (the immune system). Recently it has been thought that certain individuals are genetically predisposed to cancer. Therefore it is not surprising that as one ages and the immune system weakens, one becomes more susceptible to certain forms of cancer.

Regular consumption of red Reishi has been shown to stimulate the production of interferon and interleukins I and II, potent natural anti-cancer substances produced in our body. Furthermore, researchers claim that polysaccharides called Beta- 1,3-D-glucan and Beta-1,6-D-glucan have been shown to possess powerful anti-tumor properties. These polysaccharides can also activate macrophages, the essential immune cells in our body's first line of defense against microbial intruders such as viruses, bacteria and yeast.

Diabetes

Diabetes is a disease of poor blood sugar regulation. There are 2 types of diabetes. Type I is due to a lack of insulin production, which is a hormone secreted by the pancreas - its function is to regulate blood sugar. Type II is usually adult onset, and is due to insulin resistance. This form is more commonly related to obesity, poor nutrition, and lack of exercise. The mainstay of treatment for diabetes is either insulin injections or oral medications, both of which have its inherent side effects. Red Reishi has blood sugar lowering properties, and can possibly help regulate glucose levels

in borderline Type II cases, without the side effects of medications.

Prostate Disease

As men age, there is a normal increase in the size of the prostate, called benign prostatic hyperplasia (BPH). This can lead to a decrease in the normal urinary outflow, frequent night-time urination, and other prostate problems. In addition, advanced age significantly increases the incidence of prostate cancer. The prostate specific antigen (PSA) blood test is increased in both conditions, and it is used to screen, detect, and monitor prostatic disease, including prostate cancer treatment. There is evidence that red Reishi, when combined with other herbs, can significantly reduce Prostate Specific Antigen (PSA) levels. The mechanism is unknown, but it can occur as early as one month after treatment.

HIV Disease

Anti-HIV substances and protease inhibitors (a major class of drugs used to treat HIV/AIDS) have been found in red Reishi mushrooms. There is anecdotal evidence that it lowers the viral load and increases the CD4 counts.

Summary

In conclusion, while medical research and individual case studies have shown red Reishi to be effective in treating numerous conditions and diseases, it is important to emphasize again that the greatest proven benefit of regular red Reishi consumption is the enhancement of your body's overall health.

Through purification, improved blood flow, immune modulation, detoxification, rejuvenation, and revitalization, red Reishi can enhance the body's ability to combat a myriad of diseases, particularly those that occur more frequently with advanced age.

flow Red Reishi can Improve Your PhysicalAppearance

Obsesity can affect one's self confidence and have a negative impact on one's self-image. In addition, it can lead to diabetes, and cardiovascular disease. Studies have found that regular consumption of red Reishi helps the body cleanse itself of accumulated toxins and excess lipids (fat).

In addition to regulating the body's way of handling fat, red Reishi products can also improve the condition of the skin. The anti-aging properties of red Reishi may be related to improved blood flow and increased delivery of nutrients to the skin. It may also help in the reparation process of sun-damaged skin. People who have taken Reishi regularly have noticed a decrease in pimples

Regular consumption of red Reishi also has a calming and stress-reducing effect on the body, and appears to improve sleep patterns, without the sedation commonly seen with medications. It should be clear to the reader that these benefits combined, will improve one's physical appearance.

Regular red Reishi intake combined with a healthy diet and active lifestyle, can effectively facilitate your body's return to a normal, healthy state.

On the Road to Betterflealth with Red Reishi

If we do not actively maintain a healthy lifestyle, our bodies will deteriorate, and we will gradually become accustomed to this state of declining health. We may lose track of weight that we have gained and not notice the decline in our natural well being until a significant problem arises. It is thought that over time, an inactive body accumulates toxins, and this contributes to the deterioration of overall wellness.

Consuming a red Reishi product on a regular basis is thought to clear some of these toxins. In the beginning, a user may experience a "resetting" period, where his or her body will exhibit signs of "cleansing". The excretion of accumulated poisons and an increase in various metabolic activities are normal signs of recovery and they indicate that the regulatory properties of red Reishi are taking effect.

Common symptoms of "recovery" may include dizziness, sore bones, skin irritation, change in bowel movements, including diarrhea and constipation, and pimple-like blemishes appearing on various parts of the body.

The time frame for these symptoms may vary from person to person, but they generally last only a few days - until the body adjusts to a higher state of wellness and becomes accustomed to

the regular intake of red Reishi. Daily consumption of large amounts of water is recommended. It is important to point out again that these physiological reactions are only temporary, unlike side effects from medications.

 

Clogged blood vessels may impede blood flow

 

Steady blood flow of red blood cells

  

The Cultivation of Red Reishi

Research has shown that the health-enhancing properties of Reishi are strongest when the plant is fully mature, a state that is almost impossible to find in the wild due to the unstable environmental conditions that exist in the natural habitat of red Reishi plants.

As a result, the majority of red Reishi products on the market today use mushrooms that have been cultivated in hothouses. Cultivation of red Reishi originated in Gunma-Ken, Japan, where the Mayuzumi family mastered the technique of culturing Reishi on Japanese oak logs.

Wood pulp, wooden box, and natural wood log cultivation are three common methods of cultivating the red Reishi plant. In wood pulp cultivation, wood pulp is placed in a glass bottle and the Reishi fungi are added. After three months, this growing method

 will usually yield small mushrooms of relatively poor quality. The second method, wood box cultivation, involves grafting the fungi into a wooden log that is then placed in a wooden box. Reishi of medium- size and moderate quality are generally produced through this method after six months.

Large red Reishi of superior quality are most commonly grown using the natural wood log method, which is the most complex form of cultivation. The process begins with the culturing of a high quality Reishi fungus strain in test tubes for about 85 days. The germinating fungi are then inserted into holes drilled into selected high grade logs between 26 to 30 years old. These logs are then placed in a greenhouse and buried under nutrient-rich soil for 5 months. During this growth period, the Reishi fungi absorb almost all of the nutrients from the soil. After five years of use, the soil must lay dormant for 2 to 3 years to allow replenishment of nutrients into the soil before it can be used again.

Growing conditions require continued monitoring to ensure a high quality Reishi harvest. Temperature, humidity, carbon dioxide, light intensity and nutrients are all closely monitored to maintain optimum growth conditions. The greenhouse is sprayed with moisture daily to stimulate growth and

 

maintain a humidity of 90 to 95%. This sprayed moisture is sanitized to prevent the Reishi from being eroded by bacteria. Most Japanese companies are required to follow strict agricultural safety guidelines and abstain from the use of exotic pesticides.

The plants are grown for another 100 days until they mature to their full size. Eventually, the mushrooms produce a thick coating of spores and spraying is stopped when the mushrooms release their spores into the air. Finally, the Reishi grow for an additional two weeks before being harvested.

A healthy and mature red Reishi plant is distinguished by its large size, thick cap and overall weight. Although Reishi mushrooms grow larger with age, they are best harvested at the height of maturity when they are fresh and moist inside; older plants may be larger in size, but they are also usually too dry and brittle to be effective.

 

To prepare Reishi for use in health products, the mushrooms are cut into small pieces and boiled for two hours. This process is repeated three times to completely extract the medicinal properties of the red Reishi. The medicinal extract is prepared using a low-temperature vacuum condenser and an air-sprayed drier to transform it from a concentrated liquid form to a powder state for transfer into capsules. Ambient temperature and moisture are strictly controlled during the drying process to maximize product effectiveness.

In summary, the effectiveness of a red Reishi plant in enhancing one's health depends on four main criteria:

  • 1) the quality of the plant's mother fungi;
  • 2) the method of cultivation;
  • 3) the growing conditions;
  • 4) the harvest time.

Thus, it is clear that companies who use a more complex method of Reishi cultivation like the natural wood log method are more likely to satisfy these four criteria, and produce superior red Reishi crops.

 

Selecting the Right Reishi Product for You

As with other natural supplements that serve as preventative measures against degenerative diseases and conditions, red Reishi products are most effective the sooner they are included as part of your regular diet/vitamin program.

Although the impact of poor habits and diet on your health can accumulate substantially over time, the positive benefits of red Reishi are also cumulative. Thus, if you begin using Reishi in your mid-thirties, you may strengthen your body and immune system to better withstand the trials of time as you age.

There are a growing number of product choices every year available to red Reishi consumers, and the variety of brands and product forms can easily be overwhelming. Due to the growing recognition that companies in Japan are producing exceptionally high quality red Reishi products, there is a disturbing trend among some manufacturers who misrepresent their products as being produced in Japan, when they are actually produced in China or North America, where there is little regulation of the red Reishi industry.

A recent study in Choice magazine, published by the Hong Kong Consumer Council*, aimed to help consumers choose the best Reishi product by comparing the effectiveness of active

ingredients in 32 Reishi products available in the marketplace. The study determined that polysaccharides were the most important health-enhancing ingredients in Reishi products. It then compared the potency of the 32 products based on their polysaccharide content level. According to the test results, a certain red Reishi product made in Japan had the highest amount of polysaccharides amongst all the test products at 29.7%, while other Reishi products had as little as a 0.06% polysaccharide content level.

sX * The Consumer Council was established in April 1974 in Hong Kong. The function of the Council is to protect and promote the interests of consumers of goods and services and purchasers of immovable property. Some objectives of the Council are to conduct tests, surveys and research for the evaluation of goods and services to users; promote public awareness of consumer rights and responsibilities through publicity and media campaigns; and publish a monthly magazine "Choice" for the provision of independent and objective consumer information.

The magazine also mentioned a study that compared boiling the red Reishi slices themselves to prepare tonics for consumption versus the commercially-made product. The study team used 15 grams of red Reishi slices and boiled them in 300 cc (about one bowl) of water for an hour. The analysis showed that the amount of polysaccharide extracted was about 0.068 grams, and the study concluded that this boiling method by the consumer is labour intensive and actually more expensive than consuming ready-made industrially processed Reishi products.

What to Look for in Reishi Products

Here are some important factors to consider in your search for the most effective and safest red Reishi product:

The amount of polysaccharides contained in the product

Since the polysaccharides has been identified by researchers as being the most active health-enhancing ingredient in red Reishi, you should choose a product with the highest polysaccharide content for greater potency.

The reputation of the brand name and product history

The art of Reishi cultivation has been developed and refined over the course of thirty years, and many of the leading Reishi product manufacturers were at the forefront of the research and development involved in creating the best cultivation method to yield the highest quality Reishi. You should therefore invest some time in researching a company's website or background carefully before purchasing its products.

The manufacturer's country of origin

While red Reishi cultivation is now possible around the world, companies in Japan are still regarded as producers of premium red Reishi because of strict government regulation of the industry. Some unscrupulous companies have tried to capitalize on this by designing their product packaging to appear to be of Japanese origin - one should be wary of a product that does not list the name and address of its manufacturer.

Tips for Improving Your Diet While Using Red Reishi

In addition to using red Reishi products on a regular basis, you can also greatly improve your overall health by altering your diet. Nutritionists recommend the following:

  • Refrain from using refined sugar. Substitute with pure honey, or fruit sugar instead.
  • Avoid processed foods (e.g. canned foods, instant noodles, soda pop, etc.)
  • Eat a healthy mix of natural foods from each food group to maintain your nutritional balance.

Include all types of vegetables in your diet, especially those with stalks and roots.

  • Drink more water. Try to drink 8 glasses of water a day. This will prevent cellular dehydration and enhance your metabolic functions to rid your body of poisonous waste.

 

Reishi Spores & Mycelium

The effectiveness of using the fruiting body of red Reishi mushrooms in health supplements has made many companies eager to exploit other aspects of the Reishi mushroom for use in health products.

Recently, there has been much debate about the issue of whether supplements prepared by extracting oil from Reishi spores have more medicinal value than traditional red Reishi products. In the past, the oxidation of Reishi oil was shown to create an unpleasant odor and become harmful to human cells. However, current marketers of these Reishi spore oil products claim to have perfected the technology of cracking the spores and preventing the lipid oil collected from being spoiled by oxidation.

The Hong Kong Consumer Council's Choice magazine has thus far declined to comment on the medical efficacy of Reishi

Reishi spore                      Cracked spore

spore byproducts, stating that consumers should wait for more scientific research and clinical studies on these products before making a decision.

Mycelium

Another recent trend in the Reishi industry is the use of mycelium in place of mature Reishi mushroom extract in certain products. Mycelium is the stage between the spore and the mature mushroom in the Reishi life cycle. Reishi mycelium products are typically produced by mixing a small amount of mycelium with sterilized soybeans, rice, or other grains in a water slurry. The resulting mixture is then dried and ground into a powder for use in tablets or capsules.

While mycelium may possess some health-enhancing properties, specialists point out that all the research conducted thus far has centered on the various health benefits provided by the Reishi mushroom itself, and not the mycelium. Therefore, similar to the situation concerning spore oil products, there is
currently insufficient evidence to support the claim that mycelium- based products can yield the same benefits as products made with the red Reishi's fruiting body.

While mycelium use has become prevalent in North America, the lack of time-tested cultivation techniques for the substance has resulted in most mycelium cultivation to take place in greenhouses, using nutrients of less-than-optimum quality. Growing Reishi mycelium in vats of grain slurry is considerably faster and cheaper than taking the Reishi mycelium through the next stage of growth to produce natural mature mushrooms. In contrast, growing mature Reishi mushrooms from mycelium requires 6 to 8 additional months in large, shaded greenhouses equipped with sophisticated sterility, temperature, and humidity controls. Many producers find the additional cost and time necessary to organically cultivate the Reishi to maturity by this method too costly, and opt for the cheaper vat method, which may dilute the mycelium's potency, if any.

Although this cost-cutting practice is widely accepted in America, it is looked upon with disdain in China and Japan; to this day, companies in both countries where Reishi use has the longest history refuse to use mycelium in health products.

Consumers who are interested in using products derived from either Reishi spore oil or mycelium are advised to carefully research these topics, and consult a qualified health care professional before consuming any such products.

Resources You Can Use for More Information about Reishi

Japan Reishi Association

The Japan Reishi Association (JRA) is an international non­profit organization supported by industry leaders dedicated to helping growers and manufacturers maintain the highest standards of product quality and business practices in the Reishi industry worldwide.

The Japan Reishi Association's primary goal is to inform the general public about Reishi, Reishi-related health food products, and product manufacturers. In addition, the JRA is also committed to holding manufacturers and marketers of Reishi products accountable for claims of Japanese origin. To this end, the JRA strives to set and aid in the enforcement of industry standards for product quality and ethical business practices by working together with authentic Reishi product manufacturers in Japan and international government organizations to alert the public about products and manufacturers that fail to meet these standards.

If you have any questions about a Reishi product or manufacturer (such as the authenticity of a specific product you have purchased), you can contact the Japan Reishi Association at info@japan-Reishi.org or by calling their toll-free number at 1-866- J-REISHI (1-866-573-4744).

 

Bibliography

  • 1. Tao J, Feng K.Y. Experimental and clinical studies on inhibitory effect of ganoderma lucidum on platelet aggregation. J Tongji Med Univ 1990; 10(4):240-243
  • 2. Wasser SP, Weis AL. Therapeutic effects of substances occurring in higher Basidiomycetes mushrooms: a modern perspective. Crit Rev Immunol 1999; 19(1):65-96
  • 3. el-Mekkawy S, Meselhy MR, Nakamura N, et al. Anti-HIV-1 and anti-HIV-1 protease substances from Ganoderma lucidum. Phytochemistry 1998;49(6):1661-1657
  • 4. Kim RS, Kim HW, Kim BK. Suppressive effects of Ganoderma lucidum on proliferation of peripheral blood mononuclear cells. Mol Cells 1997;7(1):52-57
  • 5. Wang SY, Hsu ML, Hsu HC, et al. The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and T lymphocytes. Int J Cancer 1997;70(6):699-705
  • 6. Hijikata Y, Yamada S. Effect of Ganoderma lucidum on postherpetic meuralgia. Am J Chin Med 1998;26(3-4):375-381
  • 7. Kim HS, Kacew S, Lee BM. In vitro chemopreventive effects of plant polysaccharides (Aloe barbadensis miller, Lentinus edodes, Ganoderma lucidum and Coriolus versicolor). Carcinogenesis 1999;20(8):1637-1640
  • 8. Komoda Y, Shimizu M, Sonoda Y, et al. Ganoderic acid and its derivatives as cholesterol synthesis inhibitors; Chem Pharm Bull (Tokyo)1989;37(2):531-533.
  • 9. Hikino H, Ishiyama M, Suzuki Y, et al. Mechanisms of hypoglycemic activity of ganoderan B: a glycan of Ganoderma lucidum fruit bodies. Planta Med 1989;55(5):423-428
  • 10. Lee SY, Rhee HM. Cardiovascular effects of mycelium extract of Ganoderma lucidum: inhibition of sympathetic outflow as a mechanism of its hypotensive action. Chem Pharm Bull (Tokyo) 1990;38(5):1359-1364
  • 11. Kim DH, Shim SB, Kim NJ, et al. Beta-glucuronidase-inhibitory activity and hepatoprotective effect of Ganoderma lucidum. Biol Pharm Bull 1999;22(2):162-164
  • 12. Yoon SY, Eo SK, Kim YS, et al. Antimicrobial activity of Ganoderma lucidum extract alone and in combination with some antibiotics. Arch Pharm Res 1994;17(6):438-442.
  • 13. Chen K, Li C. Recent advances in studies on traditional Chinese anti-aging material medica. J Tradit Chin Med 1993;13(3):223-226.
  • 14. van der Hem LG, van der Vliet JA, Bocken CF, et al. Ling Zhi-8; studies of a new immunomodulating agent. Transplanation 1995:60(5):438-443                                                                                                                                                                          15. Thomas Bartram Fellow of the National Institute of Medical Herbalists, Bartram's Encyclopedia of Herbal Medicine, the Definite Guide to the Herbal Treatment of Diseases: 368
  • 16. Linda B. White M.D., Steven Foster. The Herbal Drugstore: 570
  • 17. John E. Smith, Neil J. Rowan, Richard Sullivan. Medicinal Mushrooms and Cancer; (2):15; (3):26; (3):27-32; (7); (8)
  • 18. Terry Willard, PhD. Reishi Mushroom Herb of Spiritual Potency and Medical Wonder; (3); (4); (5)

 

Wild Reishi

Famous Herb Of Royalty For Power, Super Health And Long Life

So effective is Reishi that it is officially listed by the Japanese Government

as a substance to treat cancer.

 ThE MUSHROOM Of IMMORtality ^^auce^Cas^Histories^

  • 39 year-old woman with advanced breast cancer with complications was diagnosed as terminal. She began taking large amounts of Reishi. After 6 months, the cancerous tumors were entirely gone. Exploratory surgery revealed only scar tissue where the tumors had been.
  • 50 year-old woman had surgery for breast cancer. When it later metastasized to her lungs, she was diagnosed as terminal. She began taking large amounts of Reishi. After 6 months, medical tests showed no evidence of cancer in her lungs.
  • 60 year-old man with pancreatic cancer, a typically fast-moving fatal disease, made a complete recovery by taking Reishi daily.
  • 57 year-old man with stomach cancer began taking large amounts of Reishi daily and completely recovered. He was still in remission 5 years later.

Chronic hepatitis, as with many liver diseases, is difficult to treat and often ends in premature death. One study of people with chronic hepatitis showed that Reishi was effective in 71 to 98% of the cases. Reishi contains a special factor, ganodosterone, which is a liver function stimulant and liver protectant. Promising research shows that Reishi helps regenerate the liver. Because of its dramatic healing effects on both the immune system and the liver, Reishi is now used as the herb of choice by many doctors for hepatitis of all types, chronic fatigue, candidiasis, and arthritis.

'Case histories quoted in Willard's Reishi Mushroom: Herb of Spiritual Potency and Medical Wonder, Sylvan Press, Issaquah, Washington, 1990.

 The Best Reishi

 Be Sure Your Reishi Is:

•The correct species of Reishi

(not similar, nonmedicinal species)

•No solvents, no fumigation, no pesticides •100% pure powder in pure vegetable capsules

(avoid tablets and their toxic binders and glues)

Worldwide Reishi Research

•Priceless: Valued more than gold in ancient times •Immunity: Boosts the weakened immune system

(as seen in cancer, candida, chronic fatigue, heart disease, bronchitis, asthma, diabetes, allergies)

•Cancer: Used extensively with all types of cancer

(cancer of the breast, prostate, lung, pancreas, etc.) •Brain: Helps improve memory, concentration,

clarity, helps clear depression, anxiety, insomnia. •Liver: helps regenerate the liver; helps clear

hepatitis and liver diseases •Women: helps clear PMS and menopause symptoms

•Allergies, Candida: Helps clear allergies and fungal overgrowth

 

LITERATURE SEARCH SERVICES

SANTA MONICA, C A 9 0 4 0 3                     Copyright © 2000 Lit. Sí.rch Sirvics

Wild Reishi

Famous Herb Of Royalty For Power, Super Health And Long Life

 The Greatest Herb In History

From ancient times, legends were told of a miraculous herb so powerful that those who could obtain it could overcome the worst, most life-threatening disease. This herb was said to give you the strength and agility ofa young person and to let you live far beyond your normal life span. Even today, the reputation of this herb is greater than almost any other in history, yet most doctors have never even heard of it. It is called Reishi (in Japan) or Ling-zhi (in Chinese). It's scientific name is Ganoderma lucidum. Reishi is classified as "A-one Medicine" in China. In the Orient, Reishi is treasured more than gold.

Master Terminator Of Infection

Reishi is a master eliminator of harmful bacteria such as staph, strep, E. coli and bacillus pneumoniae. In one amazing study, re­searchers gave mice a fatal injection of E. coli. In the control group, which received no Reishi, 100% died. In the group of mice which were given Reishi 48 hours previous to the fatal injection, 85% lived! No wonder Reishi is unrivalled in its long history as the most powerful of all herbs to give you a long, healthy life: it has a dramatic ability to greatly strengthen the immune system.

Head That Cold Off With Reishi

Reishi contains unique, rare polysaccharides which have been proven to increase the RNA and DNA in the bone marrow, where your body makes B cells. Studies show that Reishi strengthens both B cells and T cells. These specialized cells are a critical part of your internal army that fight infection. Studies show Reishi increases the strength and life span of all the white blood cells, the cells that fight infection. What an incredible boost to a sag­ging immune system! Many doctors recommend Reishi at the first sign of any cold, flu or sore throat to help quickly stop the infection. Better yet, take Reishi daily as your best insurance for a super healthy life.

Superb PH Balance: Organic Germanium

A key element in Reishi is its large amount of organic germanium, up to 2,000 ppm, 6 times more than even ginseng. Organogermanium can dramatically increase the oxygen absorbed by your blood. High oxygenation turns an acid body back to alkaline. Candida and cancer thrive only in an acid body. Organogermanium not only upregulates oxygen but also helps clear toxins.

Viruses: The Wrong Kind Of Friends

Typically present with candida or cancer are "bad friends," such as harmful bacteria and viruses. The internal conditions that have allowed the disease to flourish offers a great breeding ground for other undesirables. Research shows that Reishi can stop viruses by helping your body to increase its own production of interferon, a powerful anti-viral substance. In one study, Reishi provided 90% protection to cells even against deadly viruses such as Vaccinia.

Liver Regeneration

Promising research shows that Reishi helps regenerate the liver. In one study, mice were given a lethal dose of the pain-killer, Indomethacin. In the control group, which received no Reishi, 90% of the mice died. In the second group which received Reishi, 100% lived! Reishi helps accelerate the clearance of drugs and chemicals from the body, thus helping the liver to detoxify faster. Reishi is the ultimate Phase I and Phase II liver detoxification tonic.

Beating Chronic Hepatitis

Chronic hepatitis is very difficult to treat. This disease may plague people for years, slowly eroding their health, often leading to premature death. One study of people with chronic hepatitis showed that Reishi was effective in 71 to 98% of the cases. Reishi contains a special factor, ganodosterone, which is a liver function stimulant and liver protectant.

Conquering Depression

Reishi has profound effects on the mind. Reishi is well known for its calming and mood-altering effect. That's why it's a world-class herb to help clear depression. Impaired liver function, poor nutrition and infection are often at the root cause of depression, anxiety and irritability. Depression can also be caused by food allergies. Research shows Reishi's dramatic help in clearing food allergies. Reishi revitalizes the blood and helps normalize IgG, an immunoglobulin responsible for triggering many food allergies.

The Source Is Everything

Most Reishi currently on the market is commercially grown, solvent-extracted and often the wrong species. Poor quality Reishi will not produce the powerful healing benefits as seen in the research. We found the very best results with wild, mountain- grown Reishi from China, unpolluted with chemicals, additives or solvents.

References

Kampo I-yaku Shimbun, Becoming Healthy with Reishi, Toyo-Igaky Sha Co. Ltd., Tokyo, 1988. Lin Zhi-bi, "The Present Status of Pharmacological Studies of Ling Zhi (ganoderma lucidum);" Yao Hseuh Pao 14 (3), 1979.

Subuti Dharmananda, Chinese Herbal Therapies for Immune Disorders, Institute for Traditional Chinese Medicine

and Preventative Health Care, Portland, Oregon, 1988. Toshio Miyazaki and Motohiro Nishijima, "Studies on Fungal Polysaccharides. XXVII. Structural Examination of a Water-

Soluble, Antitumor Polysaccharide of Ganoderma lucidum," Chemical and Pharmaceutical Bulletin (1 981); 29(12): 3611-3616. Tsutomu Furuya, Yumiko Tsuda, Naoichi Koga et at., "lsolation of ganodosterone and ganoderic acids as liver function stimulants," in Chem. Abstr. 109(18): 156253q. Willard, Terry, Reishi Mushroom: Herb Of .Spiritual Potency And Medical Wonder, Sylvan Press: Issaquah, Washington, 1990.

Rev. 1/23/02

 

LITERATURE SEARCH SERVICES

SANTA MONICA, C A 9 0 4 0 3 © Copyright Lit. Se«rch Services 2000

 

 

(Adaptogenlerin Kralı: Kırmızı Reishi)

Red Reishi: King of Adaptogens

San Francisco, CA 94111 June 12, 2007

Global Medicine Hunter® News Release Dr. Meg Jordan, PhD, RN

GLOBAL HEALTH MEDIA

ADAPTOGENS: PROVIDING WHAT IS NEEDED

(San Francisco --) One of nature's great mysteries is the adaptogenic quality of certain plants. Key word - adapt. Imagine a plant that is able to provide you with the right healing nudge, in the right amount, at the right time, in the right direction. Science has not been able to duplicate this confounding yet therapeutic action in any pharmaceutical agent.

Only one in 300 herbs is an adaptogen. Rhodiola rosea, Eleuthero (once known as Siberian ginseng) and Astragalus are high potency adaptogenic herbs that are known in Traditional Chinese Medicine for giving you what you need: enhancing energy you if you're stagnant, calming you if you're irritable and wired.

An adaptogenic botanical for blood pressure will help reduce it for those with high blood pressure, and yet, taken by someone else with low blood pressure, the plant offers a gentle boost. An adaptogen for cholesterol will help boost the good (HDL) without raising the bad (LDL) cholesterol.

Red Reishi is notably Nature's crowning achievement in adaptogen science. Reishi's balancing qualities offer support for both the body's immune and nervous systems. Herbalists use Reishi to support allergies, bronchitis, viral infections and hypertension, Its adaptogenic effect also extends to detoxification, mood and appetite, vigor, and mental alertness.

ADAPTOGEN FOR DETOXIFICATION

But no organ enjoys Reishi's attention more than the liver! With its 300-plus biochemical processes and functions, the liver has the most to gain from regular intake of nature's strongest adaptogen. This proves to be the case in preliminary studies showing Reishi extract as an effective aid in hepatitis B, helping to reduce elevated liver enzymes. (Stengler, 2005)

Reishi is often used by Chinese medicine practitioners for detoxification, which is a primary function of the liver that occurs in two stages. Today's environmental toxins, overabundance of petrochemicals, additives, pesticides, herbicides and xenoestrogens have our poor livers waving white flags! They are working overtime and barely able to perform the metabolic functions the body insists upon for healthy weight management and thousands of other functions. For the average person, consistent support of the liver's filtering action is crucial for overall health and longevity.

ADAPTOGEN FOR STRESS REDUCTION

Besides detoxification, stress reduction is a key aspect to Reishi's adaptogenic qualities. No one escapes the stress of today's busy world. Noise pollution, overwork, traffic, expenses and more add up to powerful and what's worse - continuous-levels of stress.

As people search for effective de-stressing strategies such as meditation, moderate exercise, and a natural, whole foods diet, they still need a natural remedy to help the body ratchet down its chronic, and life-threatening stress level.

Red Reishi is the superior choice. The one-of-a-kind class of polypeptides acts as precursors to neurotransmitters and endorphins, and adjusts the effects of these chemicals through actions known as mediation, downregulation and upregulation-again, in whatever direction is needed, the perfect adaptogenic response.

ADAPTOGENIC TRAINING EFFECT

The effect of ingesting Reishi on a daily basis is akin to training the cardiovascular system through aerobic exercise, or training the musculoskeletal system through lifting weights. Reishi actually trains the body's immune system and nervous system to perform better. Natural medicine experts are beginning to suspect that the body's immune system requires such training, that we're born with an immature system that requires "cooking" through childhood fevers and early slaying of infections such as chickenpox and stomach flu.

That "cooking" action of a fever helps immune modulators and killer cells do their job later in life, as long-term studies have shown that children who don't live in pristine environments have a higher success rate vanquishing certain cancers. Their immune systems took on early struggles with dirt, poverty, filth, bugs and grew robust as a result.

If you were given every imaginable type of immunizations and live in germicidal-scrubbed homes, I'm not suggesting a daily ingestion of dirt, but I do know that Reishi can provide that training ground for your immune system at any age. Reishi can be taken daily without adverse effect to adjust the body's orchestration of powerful stress hormones such as coritsol and norepinephrine, enzymes, neurotransmitters, catecholamines, prostaglandins and a wide range of other compounds.

The end result is less wear and tear on the body's cellular lining, organs, and tissues, and greater support of the body's immune and nervous systems. Your resistance to infections, colds and flu, virus, bacteria, fungus and exotoxic substances is vastly increased. And your energy efficiency, concentration, mood, sleep, and general sense of well-being are significantly enhanced.

Reishi's adaptogenic qualities include:

  • - supports nerve function
  • - scavenges free radicals
  • - tones and improves immune system function
  • - helps detoxify and protect the liver
  • - reduces inflammation
  • - quells allergies

Dr. Meg Jordan, PhD, RN, is a medical anthropologist and behavioral medicine specialist. She teaches at San Francisco State University. mail@megjordan.com

(constjohn@aol.com) St. John Group 1750 Montgomery Street San Francisco, CA 94111 Phone : 415-454-2243 Fax : 415-459-3165

More on Global Medicine Hunter Dr. Meg Jordan

 

 

 

( AIDS ve bu hastalığın Çin Herbal İlaçlarıyla tedavisi)

AIDS

and its treatment by Chinese Herbal Medicine

©1996 by Gunter R. Neeb

 

In the beginning of the eighties, on patients with severe immune deficiencies a new virus was found by showing that most of these patients had antibodies against this virus. Nowadays this virus is known in two types, called Human Immunedeficiency Virus Type 1 and 2 (HIV-1 and HIV-2) of which the first type is more common in the western world.

It belongs to a subgroup of the Retrovirus-Class, the Lentovirus-Group, which lead only slowly to disease after infect their host, such causing a persisiting infection which remains inactive for a long time. The symptomeless stage may take 10 Years or more, which an aproximate maximum of 15 years after which most patients show symptoms of mortal immune-deficiency.

The virus' genom consists of RNA which is copied to a complementary RNA-string by the enzyme Reverse Transcriptase. After the first flu-like acute infection in which a high virustiter can be seen, the virus has killed and infected many CD4-T-Cells and macrophages. The immunic reaction consists of antibodies, CD-4-Helpercells and -inflammative cells, as well as of cytotoxic CD-8-Cells. After this acute phase the blood is effectively frred from the virus with a few circulating CD-4-Cells carrying one copy of the inactive genome of the virus.

Although the peripheral blood contains no viruses during the inactive phase, the virus reproduces itself actively in the lymphatic tissue, brain, and some other regions. this chronic infection gradually destroys the CD-4-T-Cells I

n a not yet completely explained way. After almost all CD-4-T-Cells are destroyed, i.e. if their count is below 200 per µl, the imunedeficiency stage is reached, and the syndrome is called AIDS. Gradually lymphatic folliculary dendritic cells are damaged leading to a loss of lymphatic follicles and thereafter a complete destruction of the lymphatic tissue. A similar process destroys the thymusstroma.

If a new acute infection with HIV-1 or HIV-2 is suspected or early enough detected, several antivirus herbs from Chinese Medicine can be prescribed to secure a low infection rate. Since the toxicity of some is very low, they can be used even as a preventive measure in order to keep the virus from spreading. One promising candidate among them is "zi hua di ding" or viola yedoensis ( Pls. refer to the following table ).

 

Chinese medical herbs affecting various viruses: List 1

Name

Chinese Name

Effects on  Virus       /    toxicity or LD50

Viola yedoyensis

Zi hua di ding

HIV / low toxicity

Arcticum lappa

Niu ban zi

HIV/ low tox.

Andrographis paniculata *

Chuan xi lian

HIV, ECH011 / 13.4g/kg

Lithospermum   rythrorhizon

Zi cao

HIV, jinke,flu, polio, hepatitis / 681.13mg/kg

Altemanthera  philoxeroides 

Lung xin lian zi cao

HIV, flu, encephalitis B. retrovirus, rabies virus / 455.5g/kg

Lonicera japonica

Jin yin hua

HIV, flu (PR8), herpes / orphan (mice) 53g/kg

Coptis chinensis

Huang-lian

HIV, RuS hepatitis B, Newcastle disease / 24.3mg/kg

Epimedium  grandiflorum

Yin-yang-huo

HIV, polio, ECHO 6,9, Coxsackie A9, B4, B5    / 36g/kg

Woodwardia   unigemmata 

Guo ji jue guan zhong

HIV, flu (PR8, jinke 68-1,57-4,NewA1, Lee, C1232, D), Gland. II, polio II,Coxsackie, Herpes simplex, encephalitis / 1.7g/kg (mice)

Prunella vulgaris

Xia ku cao

HIV  /  low toxicity

Senecio  scandens

Qian li guang

HIV / 302.6g/kg***

Hypericum   japonicum

Di er cao

HIV, hepatitis B / no obvious toxicity

Scutellaria   baicalensis

Huang qin

HIV, flu PR8, (Asian A-flu) xiantai, rhinovirus 17, adenovirus 7,  / 3.081g/kg*

Baphicacanthis  folium 

Da qing ye

flu, mumps,encephalitis B / no obvious toxicity

Baphicacanthis  rhizoma, radix 

Ban lan gen

flu, hepatitis A,B,mumps, encephalitis B. herpes /  no obvious toxicity

Bupleuri radix  

Chai hu

flu, smallpox /  4.7g/kg gland (mice)

Ledebouriellae radix 

Fang feng

flu, Columbia SK

Polygonum  cuspidatum

Hu zhang

herpes, ECHO9, ECHO11,adenovirus, hepatitis B., flu, encephalitis B. Coxsackie A,B, polio II /  1363mg/kg*

Forsythiae fructus

Lian qiao

flu (Asian A), nose-17  /  29.37g/kg (mice)

Taraxaci herba

Pu gong ying

ECHO11, herpes / 156.3g/kg

*LD50 is given in the doses of chemical essences of the herb.

**LD50 is given in the doses of alcohol extract, IV injection.

***LD50 is given dependent on the herb produced in China; in some countries this herb is very toxic (Geissman, 1964).

There are 20 herbs listed in Table 1. The antiviral spectrum and toxicities of each herb are given. Thirteen of them have inhibitory effects on the HIV. Some others have antivirus effects on other viruses. The HIV-Virus' ability to avoid immunity response has much in common with some of these viruses:

The permanent and latent infection is also seen in the Eppstein-Barr-Virus and the Herpes Simplex Virus; the antigene variation by mutations can be found as well in Trypanosomes, the influenza virus and some Streptococcus kinds. The induction of an immune-supression shares the HIV-Virus with some Staphylococcus kinds and the Mycobacterium leprae.

From an allopathic view, it may not be proper to list these herbs until they have been tested in vivo against the HIV virus; however, from a TCM view, if certain herbs have been proven effective against a certain virus in clinical trials, the same mechanism may work against another virus in the human body. TCM holds that any herb or drug taken into the body must first affect the body's metabolism so that it can more effectively fight the pathogen. Although the concept of an antivirus is an allopathic approach, TCM's principle of treating febrile infectious diseases (qing re jie du, heat-clearing-and-toxin-eliminating) with herbal remedies can give this approach a new meaning.

The toxicity of these herbs is very low, so they can be used safely for a long period, even over a lifetime, by HIV-infected persons or, as mentioned before as a preventive measure.

The 13 herbs which showed inhibitory effects on HIV in vitro may also contribute to the treatment of various complications of AIDS. These patients are known to be subject of opportunistic infections and some kinds of cancer. Those are caused by organisms which usually can be controlled easily by the immune system, like Pneumocystis carinii causing pneumonia, as well as e.g. Toxoplasma, the Cytomegaly virusand some Mycobacteria like M. tuberculosis and even some normally harmless kinds like M. avium. The functioning of the cellular defese is necessary for an effective control of these microorganisms. Some patients (about 20%) is also affected by organisms, which are usually controlled by the humoral defense system like Haemophilus influenzae.

Therefore most important are probably those herbs which stimulate the growth of the T-lymphocytes as listed below:

Common Name

asparagus (root)

atractylodes, white (rhizome)

coix (seed)

coriolus (sclerotium)

epimedium (leaves)

ganoderma (whole)

lentinus (whole)

ligustrum (fruit)

ginseng (root)

phaseolus, white (bean)

huang-jing (rhizome)

Besides AIDS-Patients are liable to develope tumors like the Karposi-Syndrome, a usually slowly developing proliferation of bloodvessel cells, and to tumors of the B-Cells, the non-Hodgekin-Lymphomes and such of the brain. This might be explainable by a lacking T-Cell response against tumor cells or a chronic stimulation of the B-cells, another feature of HIV-infections.

Since many herbs in Chinese Medicine have been proven to stimulate the immune system or some parts of it, these herbs can also be used in any stage of the HIV infection, i.e. when an opportunistic infection occurs or generally to stimulate the immune system. Pls. refer to list 2 below:

 

Chinese medical herbs affecting the immune system (list 2)

Promoting Lymphoblast Transformation

 

Botanical Name

Angilica sinensis, acutiloba

Astragalus membranaceus

Atractylodes macrocephala

Codonopsis pilosula

Coix lachryma-jobi

Coriolus versicolor

Epimedium grandiflorum

Ganoderma lucidum

Gelatin equi asini

Ligustrum lucidum

Panax ginseng

Phaseolus vulgaris

Polygonatum sibiricum

Polygonum multiflorum

Increasing the Number of White Blood Cells

 

 

Botanical Name

Acanthopanax senticosus

Astragalus membranaceus

Cinnamomum cassia

Codonopsis pilosula

Comus officinalis

Ganoderma lucidum

Gelatin Equi asini

Ligustrum lucidum

Millettia dielsiana

Panax ginseng

Phaseolus vulgaris

Placenta Homines sapientis

Psoralea corylifolia

 

Increasing Phagocytosis of Neutrophilic WBC

 

Botanical Name                                      Common Name

Astragalus membranaceus                    huangqi (root)

Atractylodes macrocephala                   atractylodes,white

Dioscorea opposita                                dioscorea (root)

Glycyrrhiza uralensis                                licorice (root)

Panax ginseng                                         ginseng (root)

Increasing the Number of Mononucleic Macrophages

 

Botanical Name              Common Name

Coriolus versicolor       coriolus (sclerotium)

Glycyrrhiza uralensis   licorice (root)

Lentinus edodes            lentinus (hole)

Promoting Phagocytosis of Mononucleic Macrophages

 

Botanical Name                      Common Name

Acanthopanax senticosus   ginseng, Siberian (root)

Angilica sinensis, acutiloba tang-kuei (root)

Astragalus membranaceus huangqi (root)

Atractylodes macrocephala atractylodes, white (rhizome)

Codonopsis pilosula              codonopsis (root)

Epimedium grandiflorum     epimedium (leaf)

Eucommia ulmoides             eucommia (bark)

Ganoderma lucidum              ganoderma, lingzhi (whole)

Lentinus edodes                     lentinus (whole)

Panax ginseng                        ginseng (root)

Polyporus umbellatus            polyporus (sclerotium)

Psoralea corylifolia                 psoralea (seed)

Rehmannia glutinosa             rehmannia (root)

 

II. Herbs Affecting Non-Specific Humoral Immunities

Inducing the Production of Interferon

 

Botanical Name                      Common Name

Astragalus membranaceus astragalus (root)

Phaseolus vulgaris (PHA)    phaseolus, white (bean)

(Astragalus can also promote the production of interferon by viral stimulations.)

Anti-Complementary Activities

 

Botanical Name         Common Name

Cinnamon cassia     cinnamon (twigs)

Lentinus edodes       lentinus (whole)

(These herbs have triggering effects on the C3 complementary.)

III. Herbs Affecting Specific Humoral Immunities

Those Promoting Hypertrophy of Antigen-Combining Cells in Mice Spleens at the Early Stage of the Immuno-Reaction

 

Botanical Name         Common Name

Angilica sinensis          tang-kuei (root)

Astragalus membranaceus astragalus (root)

Coix lachryma-jobi       coix (seed)

Comus officinalis           cornus (fruit)

Those Promoting Hypertrophy of Antibody-Producing Cells

 

Botanical Name              Common Name

Asparagus cochinchinensis asparagus (root)

Coix lachryma-jobi              coix (seed)

Coriolus versicolor              coriolus (sclerotium)

Epimedium grandiflorum  epimedium (leaves)

Ganoderma lucidum          ganoderma (whole)

Ligustrum lucidum              ligustrum (fruit)

Ophiopogon japonica        ophiopogon (root)

Polygonatum sibiricum     huang-jing (rhizome)

Polyporus umbellatus       polyporus (sclerotium)

Psoralea corylifolia             psoralea (seed)

Those Suppressing Hypertrophy of Antibody-Producing Cells

 

Botanical Name                  Common Name

Glycyrrhiza uralensis         licorice (root)

Those Regulating Hypertrophy of Antibody-Producing Cells

 

Botanical Name                  Common Name

Astragalus membranaceus astragalus (root)

Those Increasing Antibody Production

 

Botanical Name                  Common Name

Astragalus membranaceus huangqi (root)

Coriolus versicolor                coriolus (sclerotium)

Epimedium grandiflorum     epimedium (leaves)

Lentinus edodes                     lentinus (whole)

Panaxginseng                         ginseng (root)

Placenta hominis sapientis  placenta, human

Polygonum multiflorum         he-shou-wu (whole)

Rehmannia glutinosa             rehmannia (root)

Those Suppressing Antibody Production

 

Botanical Name                      Common Name

Angelica sinensis                 tang-kuei (root)

Glycyrrhiza uralensis            licorice (root)

Psoralea corylifolia               psoralea (seed)

Zizyphus jujuba                     jujube (seed)

IV. Those Herbs Affecting the Production of Different Types of Immunoglobulin (Ig)

Herbs Which Affect Immunoglobulin (Ig)

 

Promotive                                          Suppressive

IgG Lentinus edodes                      Psoralea corylifolia

Astragalus membranaceus

IgA Placenta hominis                      Psoralea corylifolia

(serum) Rehmannia glutinosa

IgA Astragalus membranaceus

(secretion) Ganoderma lucidum

Polygoni multiflori

Epimedium grandiflori

IgM Astragalus membranaceus    Psoralea corylifolia

Coriolus versicolor

IgE Astragalus membrananceus

Because of the disruption of the immune system, various allergic reactions can be seen in AIDS and HIV(+) patients, most notably hay fever and allergic sinusitis.

The following list contains herbs which can be used for different types of allergic conditions:

List 3: Herbs affecting Allergic Reactions

Herbs affecting Type-I Allergic Reactions

Those Suppressing the Secretion of Histamine

 

Botanical Name              Common Name

Ganoderma lucidum     ganoderma (sclerotium)

Those Providing Relief from Bronchial Spasms due to Histamine and Acetylecholine

 

Botanical Name              Common Name

Coriolus versicolor          coriolus (sclerotium)

Ganoderma lucidum     ganoderma (sclerotium)

Epimedium grandiflorum epimedium (leaves)

Placenta hominis sapientis placenta, human

Psoralea corylifolia          psoralea (seed)

Those Providing Relief of Gastrointestinal Smooth-Muscle Spasms Caused by Histamine and Acetylecholine

 

Botanical Name                 Common Name

Angelica sinensis              tang-kuei (root)

Cinnamomi cortex             cinnamon (twigs)

Comus officinalis               cornus (fruit)

Glycyrrhiza uralensis         licorice (root)

Those Which Suppress Allergic Shock or Allergic Skin Reactions Caused by Foreign Proteins

 

Botanical Name              Common Name

Glycyrrhiza uralensis     licorice (root)

Panax ginseng                ginseng (root)

Placenta hominis sapientis placenta, human

II. Those Affecting Type-II Allergic Reactions (Cytolytic)

Those Preventing ABO Hemolysis

 

Botanical Name                  Common Name

Glycyrrhiza uralensis          licorice (root)

Those Increasing the Number of Platelets

 

Botanical Name                  Common Name

Glycyrrhiza uralensis         licorice (root)

Placenta hominis sapientis placenta, human

Rehmannia glutinosa        rehmannia (root)

Those Increasing the Number of Red Blood Cells

 

Botanical Name                  Common Name

Acanthopanax senticosus ginseng, Siberian (root)

Codonopsis pilosula          codonopsis (root)

Equi asini                             equine gelatine

Panax ginseng                    ginseng (root)

(These herbs can also affect hemolysis.)

III. Those Affecting Type-III Allergic Reactions

Botanical Name                Common Name

Astragalus membranaceus astragalus (root)

Glyzyrhiza uralensis         licorice (root)

Rehmanniae radix            rehmannia(root)

IV. Those Affecting Type-IV Allergic Reaction (Delayed)

Botanical Name                  Common Name

Angelica sinensis               tang-kuei (root)

Bombyx batryticatus           silkworm (whole)

Glycyrrhiza uralentsis          licorice (root)

As mentioned above some kinds of cancer can occur in HIV positive patients. Since there are many herbs in TCM proven effective against various kinds of cancer, only an excerpt can be listed here, since this would exceed this topic by far. So finally in List 4 are listed only those herbs which may be effective against cancers typical in AIDS patients, namely leukemia and lymphoma. Since some of these herbst are toxic or cytistatic themselves, they should not be used before they are indicated.

List 4: Herbs for cancers in AIDS patients (late stage)

Botanical Name                  Chinese   Name

Polysticticum                          yun zhi

Hedyotis diffusa                     bai hua she she cao

Indigo naturalis                      qing dai

Catharanthi rosei                  chang chun hua

Lysimachiaiae Clethroidis   zhen zhu cai

Botanical Name                  Chinese Name

Camptothecae Acuminatae        xi shu

Sarcandra glabra                           zhong jie feng

Curcuma aromatica/zedoaria     e zhu

Brucea javanica                             ya dan zi

Of these herbs the first on in the list, Polystipticum is one of the most interesting herbs, since its toxicity is very low, while it is useful for leukemia, lymphoma and liver cancer, by inhibiting the synthesis of the DNA and RNA of tumor cells. Besides this it antagonizes the immunosuppression caused by cyclophosphamides and cortisone, while increasing the amount of macrophages, promotes phagocytosis and enhances the rates of blastogenesis and rosette forming, such being a promising agent for both enhancing the immune system and suppressing tumor developement.

At the moment clinical western medicine has not yet found any solutions exept medicaments like AZT, which inhibit the function of the reverse transcriptase and therefore have a toxic effect on human cells as well. Some other proposals are searching to find specific inhibitors of the viral protease or the developement of antibodies for the virus surface or synthezising substances which ought to block the docking of gp120 to the CD4. But they still are far from ready to be used in clinical practice.

Therefore Traditional Chinese Medicine might provide some useful alternatives by combining herbs with western medicaments or even use them alone in different stages of HIV-Positive patients and other types of immune deficiency syndromes.

                     

 

 

 

 

 

( GANODERMA Bileşiklerinin Anti-Androgenik etkinliği ve prostat rahatsızlığında kullanılması)

 

Anti-Androgen Effects of Extracts and Compounds from Ganoderma

lucidum

by Jie Liua), Sadaaki Tamuraa), Kenji Kurashikia), Kuniyoshi Shimizua), Kiyoshi Nodab), Fumiko Konishib), Shoichiro Kumamotob), and Ryuichiro Kondo*a)

a) Department of Forest and Forest Products Science, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581, Japan (phone: + 81-92-6422811; fax: + 81-92-6422811; e-mail: ryukondo@agr.kyushu-u.ac.jp) b) Research Laboratories, Chlorella Industry Co., Ltd., Fukuoka 833-0056, Japan

The 30% EtOH extracts of Ganoderma lucidum Fr. Karst (Ganodermateceae) showed weak 5a- reductase inhibitory activity and binding ability to androgen receptor. When LNCaP (lymph-node carcinoma of the prostate) cells were treated with the EtOH extracts, cell proliferation was inhibited. Treatment with the extracts significantly inhibited the testosterone-induced growth of the ventral prostate in castrated rats. These results showed that G. lucidum might be a useful ingredient in the treatment of androgen-induced diseases, such as benign prostatic hyperplasia or prostate cancer. From the 30% EtOH extracts, we isolated ganoderiol F, which showed binding activity to androgen receptor and inhibited LNCaP cell proliferation, as one of the active compounds in the 30% EtOH extracts.

Introduction. - Androgen-mediated diseases such as prostate cancer, hirsutism, acne, androgenic alopecia, and benign prostatic hyperplasia (BPH) have become serious problems among the modern population [1]. Prostate cancer is one of the most frequently diagnosed malignancies and is the second leading cause of cancer death in American men. BPH, in particular, is one of the most common ailments seen in older men; 40% of men 50 to 60 years of age, and 90% of men 80 to 90 years of age have been diagnosed with BPH. The principal prostatic androgen is dihydrotestosterone (DHT), which is formed by the steroid enzyme 5a-reductase from its substrate testosterone [2]. In several androgen target tissues, like the prostate, testosterone is converted to DHT, which is the most potent natural androgen. This process amplifies the androgenic response, perhaps because of the higher affinity of the androgen receptor (AR) for DHT than for testosterone [3]. Both 5a-reductase and DHT perform critical roles, physiologically and pathologically, in man. The plasma level of DHT has been reported to be elevated in patients with either BPH or prostatic cancer.

The AR is a transcriptional activator that, in a multistep process, transduces extracellular signals to target tissues. The AR regulates transcription in response to androgens, and has a key role in the regulation of prostate growth and the maintenance of prostatic function [4]. It is classically understood that, after ligand binding (mainly DHT), the ligand-AR complex with associated proteins translocates into the nucleus, binds to the consensus sequence of androgen response elements [5], and regulates the expression of androgen-responsive genes (ARGs) [6]. Prostate-specific antigen (PSA)

© 2009 Verlag Helvetica Chimica Acta AG, Zürich

is an ARG known to be under the control of the AR, and is a well-accepted marker for the diagnosis and prognosis of prostate cancer. Amplification of the AR can lead to the development of prostatic diseases or androgen-refractory prostate cancer. Therefore, the first step in the process of transcriptional regulation is the binding of DHT to the AR. The androgen antagonist can suppress DHT-induced prostate regrowth.

The inhibition of 5a-reductase and AR has become a pharmacological strategy for the treatment of BPH and prostate cancer as well as other DHT-related disorders such as acne and male pattern baldness [7].

For thousands of years, mushrooms have been known as a source of medicine. In East Asia, the fruiting body of the fungus Ganoderma lucidum has been used medicinally for centuries. It has long been used as a folk medicine to treat various human diseases such as cancer, hypertension, hepatitis, nephritis, and others [8]. Although the inhibitory effects on the proliferation and migration of prostate cancer cells by hot H2O extracts of G. lucidum [9] have been reported, AR binding activity and suppression of androgen-induced prostate cell growth by 30% EtOH extracts of G. lucidum have never been reported. We reported that 99.5% EtOH extracts of G. lucidum showed the anti-androgen effects, which came from 5a-reductase inhibitory activity [10]. The EtOH extracts of G. lucidum also suppressed the testosterone- induced prostate regrowth in animal experiment. Until now, the safety of 99.5% EtOH extracts of G. lucidum had not been guaranteed. G. lucidum is a popular medicinal mushroom, and its dried powder and liquor are currently used worldwide in the form of dietary supplements. As the alcohol content in the liquor is ca. 30%, we changed the solvent for extraction from the 99.5% EtOH to 30% EtOH to insure the safety and the effectiveness of the EtOH extracts of G. lucidum. In this study, we isolated five compounds, 1 - 5. They are identified as ganoderic acid A (1) [11], ganoderic acid F (2) [12], ganodermanontriol (3) [13], ganoderiol A (4) [13], and ganoderiol F (5) [14] (Fig. 1). We also demonstrated the in vitro and in vivo anti-androgenic activity of 30% EtOH extracts of G. lucidum.

Results and Discussion. - 5a-Reductase Inhibitory Activity of 30% EtOH Extracts of G. lucidum. In our previous screening of 19 edible and medicinal mushrooms, we discovered that the fruiting body of G. lucidum showed higher suppressing effect on testosterone-induced prostate regrowth [10]. The 99.5% EtOH extracts of G. lucidum also showed higher 5a-reductase inhibitory activity [15]. The fruiting body of G. lucidum showed a very strong possibility to be used as health supplement. To use the G. lucidum as a health supplement, its safety must be insured. As there are no records on edible 99.5% EtOH extracts of fruiting body of G. lucidum, the safety of 99.5% EtOH extracts cannot be insured. On the other hand, the liquor of the fruiting body of G. lucidum has been used in China for a long time. As the EtOH in the liquor is ca. 30%, we changed the solvent for extraction from the 99.5% EtOH to 30% EtOH to insure the safety and the effectiveness of the EtOH extracts of G. lucidum. With changing the solvent, the components of the extracts were also changed. In this situation, the components of 30% EtOH extracts include more hydrophilic compounds than those of 99.5% EtOH extracts. The changing of compounds in extracts will also affect the bioactivity. These results led us to further investigate the 30% EtOH extracts of G. lucidum. The inhibitory concentration leading to 50% activity loss (IC50) was estimated to be 3000 mg/ml (Table 1). It should be noted that finasteride [16], which is known as a potent steroidal inhibitor, showed an IC50 value of 0.73 mm in our assay system.

Table 1. The Inhibitory Effect of 30% EtOH Extracts, CHCl3, BuOH, H2O Fraction, and Compounds 1 -

5 on 5a-Reductase

3000 mg/ml

0.73 mm

Compound/Fraction

667 mm

333 mm

200 mg/ml

30% EtOH Extract

 

 

 

CHCl3 Fraction

 

 

45

BuOH Fraction

 

 

22

H2O Fraction

 

 

19

Ganoderic acid A ( 1)

22

 

 

Ganoderic acid F (2)

18

 

 

Ganodermanontriol (3)

32

21

 

Ganoderiol A ( 4)

 

6

 

Ganoderiol F (5)

34

23

 

 

Androgen-Receptor-Binding Activity of 30% EtOH Extracts of G. lucidum. We also examined the androgen-receptor-binding activity. The binding-active concentration leading to 50% fluorescence polarization loss (IC50) was estimated to be 49 mg/ml for 30% EtOH extracts of G. lucidum (Table 2). It should be noted that DHTused as a positive control showed an IC50 value of 20 nM in our assay system.

 

The Inhibitory Effect of 30% EtOH Extracts of G. lucidum on the Prostate Cancer Cell. The LNCaP (lymph-node carcinoma of the prostate) human prostate cancer cell line is a well-established and androgen-dependent cell line [17]. LNCaP Cells retain most of the characteristics of human prostatic carcinoma, like the dependence on androgens, the presence of ARs, and the production of acid phosphatase and PSA [18]. For these reasons, the LNCaP cell line becomes an attractive model for in vitro studies on the biology of human prostate cancer [19].

The effect of the 30% EtOH extracts on a prostate cancer cell is shown in Fig. 2. LNCaP Cells were incubated with varying concentrations of the 30% EtOH extracts (from 10 to 200 mg/ml) with or without testosterone or DHT for 3 d. The NR (= 3- amino-7-(dimethylamino)-2-methylphenazine) assay was performed to measure cell viability. In the absence of 30% EtOH extracts, testosterone alone apparently stimulates the LNCaP cells to ca. 150% proliferation on average above the number of cells in an untreated control, and DHT alone apparently stimulates the LNCaP cell number to increase by ca. 200% on average above the number of cells in an untreated control. The cell cytotoxic effect was caused by 30% EtOH extracts from 50 to 200 mg/ ml when cultured without testosterone or DHT. Treating LNCaP cells with the 30% EtOH extracts in the presence of testosterone or DHT resulted in a dose-dependent inhibition of cell growth, which suggested that the inhibition of cell growth in the presence of testosterone or DHT may come from the anti-androgen effect such as 5a- reductase inhibition and the binding to AR, but we cannot deny the cell cytotoxic effect over 50 mg/ml.

Growth Suppression of Rat Prostate by Administration of 30% EtOH Extracts of G. lucidum. Based on the results of 5a-reductase inhibitory activity, the AR-binding experiment, and the cell-proliferation inhibition experiment, we concluded that the 30% EtOH extracts may inhibit the androgen-induced growth of prostate cancer cells in vitro by inhibiting the 5a-reductase activity and binding to the AR. It still cannot be stated with certainty that the 30% EtOH extracts suppresses the growth of the ventral prostate, because some problems remain such as the absorption to the blood, metabolism in the blood, penetration through the cell membrane, etc. To determine whether the 30% EtOH extracts suppresses ventral prostate growth, we designed an

 experiment as follows. In the rats that received testosterone, administration of the 30% EtOH extracts reduced the increased weight of the ventral prostate (Fig. 3 ). Four days after castration, the weights of the rat prostates were markedly reduced, and the
prostate size was recovered by sc injections of testosterone. Administration of the 30% EtOH extracts at the concentration of 1 and 0.1 mg/kg/d showed 26 and 36% prostate growth inhibition, respectively. This result suggested that the growth suppression of the ventral prostate was at least partially the result of the anti-androgen effects, i.e., 5a- reductase inhibition and the binding to the AR.

In our long-term effect assay, the 30% EtOH extracts of G. lucidum also suppressed the prostate growth. The rats were not castrated and fed for 25 weeks. Administration of the 30% EtOH extracts at the concentration of 0.002% in diet food showed 15% prostate growth inhibition. The weight of the testis, fat, spleen, adrenal, kidney, thymus, heart, lung, liver, brain, and hypophysis were not affected by the administration of 30% EtOH extracts of G. lucidum (Table 3). We also tested the serum of the rats. From these results, we determined that the 30% EtOH extracts of G. lucidum specifically suppressed regrowth of the prostate, but not of other organs.

Table 3. The Effect of the 30% EtOH Extracts on Growth of the Rat Prostrate. Each column includes the

mean ± S.D. n = 10.

 

 

Control

30% EtOH Extracts

Inhibitory effect [%]

Body weight

g

685 ± 57

680 ± 52

1

Prostate

mg/100 g body weight

144 ± 24

122 ± 20

15 a)

Testis

left g/kg body weight

2.8 ± 0.3

2.7 ± 0.3

7

 

right g/kg body weight

2.9 ± 0.3

2.9 ± 0.2

4

Adipose

g/kg body weight

14 ± 4

14 ± 3

2

Spleen

g/kg body weight

0.19 ± 0.04

0.17 ± 0.04

7

Adrenal

left g/kg body weight

5.4 ± 0.8

5.0 ± 1.0

8

 

right g/kg body weight

5.6 ± 0.8

5.5 ± 1.0

2

Kidney

left g/kg body weight

3.2 ± 0.6

3.1 ± 0.4

4

 

right g/kg body weight

3.3 ± 0.5

3.1 ± 0.3

5

Thymus

mg/100 g body weight

8 ± 3

10 ± 5

- 22

Heart

g/kg body weight

2.3 ± 0.2

2.4 ± 0.3

- 2

Lung

g/kg body weight

2.7 ± 0.2

2.7 ± 0.3

2

Liver

g/kg body weight

27.8 ± 3.2

26.8 ± 3.9

4

Brain

g/kg body weight

3.8 ± 0.2

3.6 ± 0.3

6

Hypophysis

mg/100 g body weight

29 ± 15

34 ± 18

-16

 

a) p < 0.05 against control.

 5a-Reductase Inhibitory Activity and AR-Binding Activity of Each Fraction of 30% EtOH Extracts of G. lucidum. To determine the active principles of the 30% EtOH extracts of G. lucidum, we carried out 5a-reductase inhibitory activity-guided fractionation. The 30% EtOH extracts was suspended in H2O, and extracted with CHCl3 and H2O-saturated BuOH successively, to give a CHCl3 fraction, a BuOH fraction, and a H2O fraction. The CHCl3 fraction showed the strongest inhibitory activity on 5a-reductase of 45% at 200 mg/ml, while the BuOH fraction and the H2O fraction showed 5a-reductase inhibitory activity of 23 and 19% at 200 mg/ml, respectively (Table 1). We also examined the AR-binding activity. The binding-active concentration leading to 50% fluorescence polarization loss ( IC50) was estimated to be 73, 40, and 174 mg/ml for the CHCl3, BuOH, and H2O fraction, respectively (Table 2).

Growth Suppression of Rat Prostate by Administration of Each Fraction of 30% EtOH Extracts of G. lucidum. We next conducted animal tests of each fraction. Four days after castration, the weights of the rat prostates were markedly reduced. The prostate weights recovered by sc injection of testosterone, but not completely. In the rats that received testosterone only, the prostate weight was 46.6 ± 12.5 mg/100 g of body weight. Administration of CHCl3 fraction at the concentration of 0.01 mg/kg showed higher suppression effects on the prostate. In the rats that received testosterone and CHCl3 fraction, the prostate weights were 34.6 ± 8.6 mg/100 g of body weight (Fig. 4). It should be noted that the body weights were almost the same in four groups. The BuOH and H2O fraction had no effect on the weight of the prostate in the castrated rats that received testosterone at the concentration of 0.01 mg/kg/d. These results suggested that the CHCl3 fraction inhibited prostatic regrowth through the inhibition of 5a-reductase activity and having a direct effect on the AR.

 The Inhibitory Effect of the CHCl3 Fraction on the Prostate Cancer Cells. We performed a cell proliferation experiment to test the results of CHCl3-fraction treatment. Testosterone alone apparently stimulates LNCaP cell numbers by ca. 100% on average above the number of cells in the untreated control, and DHT alone has the same effect. Treatment of LNCaP cells with the CHCl3 fraction resulted in a dose- dependent inhibition. LNCaP Cell growth was 50% lower than that of in the testosterone-stimulated controls (Fig. 5) at higher concentrations of 80 mg/ml CHCl3 fraction in the presence of testosterone. The same inhibition result of LNCaP cell growth was also observed in the DHT assay. When the CHCl3 fraction was added to the cell without androgen, the cell number did not decrease. The inhibition effect on androgen-induced cell proliferation at this concentration was not caused by the cell toxicity. We detected triterpenoids in the CHCl3 fraction by qualitative HPLC analysis. From the results of these experiments, it is reasonable to conclude that the triterpenoids are the principle active compounds in the CHCl3 fraction. At concentrations of 60 to 80 mg/ml, the light proliferation of the LNCaP cells can be observed. In our preliminary experiments, there were many triterpenoids in this fraction. Some of the isolated

triterpenoids showed the increased proliferation effects (data not shown). In the CHCl3 fraction, the inhibitory effect of triterpenoids on cell proliferation is the balance effect between those inhibiting proliferation and those increasing proliferation. In higher concentration, those inhibitory-effect triterpenoids inhibit the cell growth, but in lower concentration, they do not affect the cell growth.

5a-Reductase Inhibitory Activity of Each Isolated Compound. Ganoderic acid A (1), ganoderic acid F (2), ganodermanontriol (3), ganoderiol A (4), and ganoderiol F (5) were isolated from the CHCl3 fraction (Fig. 1). The 5a-reductase inhibitory activity is shown in Table 1. It should be noted that finasteride, which is known as a potent steroidal inhibitor, showed an IC50 value of 0.73 mm in our assay system. All of these compounds exhibited weak 5a-reductase inhibitory activities. Compared with other ganoderma alcohols and ganoderma acids [20], these compounds were not very active inhibitors of 5a-reductase.

AR-Binding Activity of Each Isolated Compound. In the AR competitor assay, ganoderiol F (5) showed the strongest binding activity to androgen receptor (Table 2). In this experiment, we used DHT as the positive control. DHT showed an IC50 value of 0.02 mM in our assay system. Ganoderiol F gave the IC50 value of 25 mM for AR.

The Inhibitory Effect of Ganoderiol F (5) on the Prostate Cancer Cell. Considering the results of 5a-reductase inhibitory activity and AR-binding experiments, ganoderiol F (5) did not show potent inhibitory activity for 5a-reductase activity and exhibited binding activity to the AR. The effect of ganoderiol F (5) on prostate cancer cells is shown in Fig. 6. LNCaP Cells were incubated with varying concentrations of 5 (5 to 20 mM) with or without testosterone or DHT for 3 d. Ganoderiol F (5) is the only compound among the five compounds that showed proliferation inhibition on LNCaP cells (data not shown). Compound 5 showed no inhibition of LNCaP cell growth when cultured without testosterone or DHT. Interestingly, treating LNCaP cells with 5 in the presence of testosterone or DHT resulted in a dose-dependent inhibition of cell growth. These results suggested that the inhibition of cell growth in the presence of testosterone or DHT was not the result of the cytotoxic effect, but came from an anti-androgen effect such as the binding to AR.

 The fungus G. lucidum (Reishi, Mannentake, or Lingzhi) has been used for centuries in East Asia to treat various human diseases such as hepatitis, hepatopathy, hypertension, nephritis, bronchitis, and cancers [21] [22]. Its dried powder was especially popular as a cancer-chemotherapy agent in the Imperial Court of ancient China [23] . Some of the triterpenoids such as ganoderic and lucidic acids, recently isolated from Ganoderma, have demonstrated cytotoxicity against mouse sarcoma and mouse lung-carcinoma cells in vitro [24]. Intraperitoneal administration of H2O-soluble polysaccharides isolated from Ganoderma has been found to inhibit the growth of sarcoma-180 solid tumors in mice [25]. In addition, polysaccharides from Ganoderma also potentiate the production of cytokines which subsequently suppress the proliferation of HL-60 and U937 leukemic cell lines [26].

The use of herbal therapies in alternative medicine has been increasing, and, although the number of cancer patients using herbal dietary supplements is not exactly known, there is evidence of the increasing use of dietary supplements in cancer treatment [27]. G. lucidum is one of the herbs in the herbal mixture PC-SPES, which has shown activity against hormone-refractory disease in two prostate cancer patients [28]. PC-SPES is labeled as a mixture of eight herbs used in Chinese and Western medicine: Chrysanthemum, Isatis, Licorice, Lucid ganoderma, Pseudoginseng, Rabdo- sia Rubescens, Saw Palmetto, and Scute (Scutellaria baicalensis (huangqin), or

Skullcap). PC-SPES is sold in the USA as a nutritional supplement. The name PC- SPES comes from PC for prostate cancer and SPES, Latin for 'hope'. Extracts of PC- SPES have demonstrated estrogenic effects [29] and decreased the growth of hormone­sensitive as well hormone-insensitive prostate cancer cells [30]. Our results suggest that these effects might be related to not only the anticancer effects of G. lucidum but also its anti-androgen effects. Since excessive 5a-reductase activity has been proposed to be a possible contributing factor in prostate-cancer development and progression, the development and progression of prostate cancer may also be affected by diets containing inhibitors of 5a-reductase.

In this experiment, the treatment with 30% EtOH extracts at 1 and 0.1 mg/kg/d significantly inhibited the growth of the ventral prostate induced by testosterone in rats. The yield of ganoderiol F (5) in the 30% EtOH extracts of G. lucidum was determined as 0.1%, leading to the conclusion that 5 was one of the active compounds in the 30% EtOH extracts. However, it should be noted that G. lucidum has a variety of triterpenoids with structures similar to that of 5, so not just one compound such as 5 but also other structurally similar compounds might contribute to the anti-androgenic activity. Since ganoderiol F (5) shows AR-binding activity and inhibits the androgen- induced growth of LNCaP cells, the growth inhibition might be related to the competitive binding to AR with DHT.

The authors are grateful to Mr. Shuhei Kaneko (Fukuoka Prefecture Forest Research and Extension Center) and Mr. Yoshitaro Suimi (Bisoken and Co., Ltd.) for providing mushroom samples.

Experimental Part

General. G. lucidum (BMC9049) was obtained from Bisoken Inc. (Fukuoka, Japan). The mushroom was identified by Mr. S. Kaneko, Fukuoka Prefecture Forest Research and Extension Center. The voucher specimen (BMC9049) was deposited with the herbarium of the Department of Forest and Forest Products Sciences, Kyushu University in Japan. The fruiting body was dried and ground to powder before use. Unless otherwise specified, chemicals were obtained from Sigma Aldrich Japan Co., Ltd. (Tokyo, Japan). Org. solvents were purchased from Wako Pure Chemical Industries Co. (Osaka, Japan). [4- 14C]Testosterone was obtained from Perkin-Elmer Japan Co., Ltd. (Kanagawa, Japan).

30% EtOH Extracts of G. lucidum. Dried and chipped G. lucidum (200 g) was extracted with 30% EtOH at r.t. for 24 h by using blender. The extracts were filtered through ADVANTEC No. 2 filter paper, concentrated under vacuum, and then freeze-dried. The 30% extracts (10 g) were stored at - 20° before assay.

The 30% EtOH extracts (10 g) were suspended in H2O (0.3 l) and extracted with CHCl3 (11 x 5) and H2O-sat. BuOH (2 l x 5) successively. A part of CHCl3 extract (1.8 g) was treated with NaHCO3 soln. to give neutral CHCl3 fraction (0.2 g) and acidic CHCl3 fraction (1.6 g), resp. The extracts were stored at - 20° before assay. Repeated column chromatography (CC) of CHCl3- and BuOH-soluble fractions led to the isolation of compounds 1 - 5. They are identified as ganoderic acid A (1) [11], ganoderic acid F (2) [12], ganodermanontriol (3) [13], ganoderiol A (4) [13], and ganoderiol F (5) [14]. MS, NMR, and optical-rotation data were in agreement with published data (Fig. 1).

Preparation of Rat Microsomes. Rat liver from female SD rats (7-weeks age) was prepared by a method reported by Shimizu et al. with some modifications [31]. From two mature SD female rats, the liver was removed, and minced tissue was homogenized in 4 tissue volumes of medium A (0.32m sucrose, 1 mM dithiothreitol, and 20 mM sodium phosphate, pH 6.5). The resulting supernatant from the centrifugations was further centrifuged at 105,000g for 1 h twice. The washed microsomes were suspended in 1 pellet volume of medium A, and the dispersion of microsomes was achieved using a syringe with 18, 23, and 26 G needless in succession. The microsome suspension was stored at - 80° just before use.

Measurement of 5a-Reductase Inhibitory Activity. A complete reaction mixture included 1 mM dithiothreitol, 20 mM phosphate buffer (pH6.5), 1.9 nCi [4-14C]testosterone, 150 mM testosterone, 167 mm NADPH, and the microsomes (0.6 mg of protein) in a final volume of 0.3 ml. The concentration of testosterone contributed by [4-14C]testosterone was negligible. The sample was added to the soln. at each conc. The incubation was carried out for 10 min at 37°. It was started by the addition of 10 ml microsomes to the pre-heated reaction soln. in a tube. After 10 min, the incubation was terminated by adding 10 mlof3M NaOH. To extract metabolites, 1 ml of Et2O was added, and the tubes were capped and shaken. The org. phase was applied to a silica gel plate (Kieselgel 60 F254), and the plate was developed in AcOEt/hexane (7:3) at r.t. The radioactivity profile was determined with an imaging analyzer (FLA- 5000 RF, Fuji Film Co., Ltd., Tokyo, Japan). The 5a-reductase activity was calculated from the percentage of the extent of the conversion of [4-14C]testosterone to [4-14C]dihydrotestosterone.

Androgen Receptor (AR) Competitor Assay. The ability of each sample to interact with the AR was evaluated by a fluorescence polarization (FP) method. This method enabled to determine the capacity of these competitor chemicals to displace a high-affinity fluorescent ligand (AL Green) from the purified, recombinant ligand-binding domain (LBD) of the human AR at r.t. Conceptually, the binding of a fluorescent molecule to another molecule can be quantified by the change in its speed of rotation. Hence, the AR-fluorescent ligand bound complex (AR-AL Green) will rotate slowly and have a high FP value. Increasing concentration of competing ligand will displace the AL Green from the AR. Free AL Green will then rotate more rapidly and have a low FP value. Because the measured polarization is an average of the free and bound AL Green molecules, it can be used to assess competitive displacement from the AR-LBD. Briefly, all samples were prepared as stock solns. in DMSO. Chemicals were serially diluted, over at least six log order concentrations, in triplicate in 20-ml volumes in assay buffer on a 384-well plate. No final DMSO concentrations exceeded the manufacturer's recommendations, and, therefore, were not anticipated to alter fluorescence. A mixture of AR (25 nM final) and AL Green (1 nM final) was added in 20-ml volumes to the serially diluted test chemicals. The plate was then incubated in the dark for ca. 4 h at r.t. The polarization was then measured on a Beacon 2000 FP instrument using 485-nm excitation and 535-nm emission interference filters in polarization mode. The polarization values (mP) were plotted against increasing concentrations of the test extracts.

The Inhibitory Effect on the Prostate Cancer Cell. The AR-positive human prostate cancer LNCaP cells were obtained from American Type Culture Collection. The cells were maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS) at 37° in a 5% CO2, 95% air-humidified atmosphere incubator. The cells were used between passages 5-30 at a split ratio of 1:4 at each passage. The cells were plated into a 24-well plate with 2 x 105/well density supplemented with 5% steroid-depleted (DCC- stripped) cFBS. After 24 h, the cells were treated with either vehicle control or androgens (testosterone or DHT) for another 3 d. Cell proliferation was determined by the 3-amino-7-(dimethylamino)-2- methylphenazine (NR) method. The NR soln. was prepared at 5 mg/ml and diluted by culture medium to 5 mg/ml. The NR extract soln. was made by using H2O and 50% EtOH (1% AcOH). The culture medium was changed to NR soln. and incubated for 3 h at 37°, then the NR soln. was aspirated, and the cells were washed with PBS twice. NR Extract soln. (500 ml) was added to each well to extract for 20 min at r.t. The absorbance of each well was measured at 540 nm.

Growth Suppression of the Rat Prostate. The assay for growth suppression of the rat prostate was performed as described by Fukuta et al. [32]. The testes of SD rats were removed at four weeks of age under light anesthesia with pentobarbital. After 4 d, testosterone (100 mg/body) was injected sc into the rats once daily for 8 d. There were 6 rats for each group. Each sample suspended in 0.5% methylcellulose was orally administered at different concentrations once daily for 8 d. The flutamide (10 mg/kg body weight) was used as the positive control and suspended in 0.5% methylcellulose, and orally administered once daily for 8 d. After 8 d, rats were anesthetized by pentobarbital. Then, their prostates were removed and their weights were determined.

Another assay was used to test the long-term effect of 30% EtOH extracts of G. lucidum. The 24-to- 27-week-old SD rats were used. 0.002% EtOH extracts in CE-2 was used for 25 weeks. After 25 weeks, rats were anesthetized by pentobarbital. Then, their prostates were removed, and their weights were

 

determined. The weights of the testes, fat, spleen, adrenal, kidney, thymus, heart, lung, liver, brain, and hypophysis were measured.

This experiment was conducted according to the Guidelines for Animal Experiments in the Faculty of Agriculture and the Graduate Course, Kyushu University, and the Law (No. 105) and Notification (No. 6) of the Japanese Government.

Statistics. Results were expressed as means S.E.M. or S.D. Statistical significance of the animal test was determined by Anova and Bonferroni-type multiple t-test, and the statistical significance of cell proliferation was determined by the t-test.

REFERENCES

  • [1] G. Barrtsch, R. S. Rittmaster, H. Klocker, World J. Urol. 2002,19, 413.
  • [2] D. W. Russell, J. D. Wilson, Annu. Rev. Clin. Biochem. 1994, 63, 25.
  • [3] S. Liao, T. Liang, S. Fang, E. Castaned, T. C. Shao, J. Biol. Chem. 1973, 248, 6154.
  • [4] C. A. Heinlein, C. S. Chang, Endocr. Rev. 2004, 25, 276.
  • [5] P. J. Roche, S. A. Hoare, M. G. Parker, Mol. Endocrinol. 1992, 6, 2229.
  • [6] P. S. Nelson, N. Clegg, H. Arnold, C. Ferguson, M. Bonham, J. White, L. Hood, B. Lin, Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11890.
  • [7] B. W. Metcalf, M. A. Levy, D. A. Holt, Trends Pharmacol. Sci. 1989,10, 491.
  • [8] T. Mizuno, G. Y. Wang, J. Zhang, H. Kawagishi, T. Nishitoba, J. Li, Food Rev. Int. 1995, 11, 151.
  • [9] J. H. Jian, V. Slivova, T. Valachovicova, K. Harvey, D. Sliva, Int. J. Oncol. 2004, 24, 1093.
  • [10] R. Fujita, J. Liu, K. Shimizu, F. Konishi, K. Noda, S. Kumamoto, K. Kurashiki, R. Kondo, J. Ethnopharmacol. 2005,102, 107.
  • [11] T. Kubota, Y. Asaka, I. Miura, H. Mori, Helv. Chim. Acta 1982, 65, 611.
  • [12] H. Kohda, W. Tokumot, K. Sakamoto, M. Fujii, Y. Hirai, K. Yamasaki, Y. Komoda, H. Nakamura, S. Ishihara, M. Uchida, Chem. Pharm. Bull. 1985, 33, 1367.
  • [13] A. Fujita, M. Arisawa, M. Saga, T. Hayashi, N. Morita, J. Nat. Prod. 1986, 49, 1122.
  • [14] T. Nishitoba, K. Oda, H. Sato, S. Sakamura, Agric. Biol. Chem. 1988, 52, 367.
  • [15] J. Liu, K. Shimizu, F. Konishi, K. Noda, S. Kumamoto, K. Kurashiki, R. Kondo, Food Chem. 2007,10, 1691.
  • [16] T. Liang, M. A. Cascieri, A. H. Cheung, G. F. Reynolds, G. H. Rasmusson, Endocrinology 1985,117, 571.
  • [17] J. S. Horoszewicz, S. S. Leong, E. Kawinski, J. P. Karr, H. Rosenthal, T. M. Chu, E. A. Mirand, G. P. Murphy, Cancer Res. 1983, 43, 1809.
  • [18] P. Negri-Cesi, A. Poletti, A. Colciago, P. Magni, P. Martini, M. Motta, Prostate 1998, 34, 283.
  • [19] P. Negri-Cesi, M. Motta, J. Steroid Biochem. Mol. Biol. 1994, 51, 89.
  • [20] J. Liu, K. Kurashiki, K. Shimizu, R. Kondo, Biol. Pharm. Bull. 2006, 29, 392.
  • [21] T. K. Yun, Ann. N.Y. Acad. Sci. 1999, 889, 157.
  • [22] S. P. Wasser, A. L. Weis, Crit. Rev. Immunol. 1999, 19, 65.
  • [23] Y. Mizushina, L. Hanashima, T. Yamaguchi, M. Takemura, F. Sugawara, M. Saneyoshi, A. Matsukage, S. Yoshida, K. Sakaguchi, Biochem. Biophys. Res. Commun. 1998, 249, 17.
  • [24] B. S. Min, J. J. Gao, N. Nakamura, M. Hattori, Chem. Pharm. Bull. 2000, 48, 1026.
  • [25] Y. Song, R. Okuda, N. Wada, Agric. Biol. Chem. 1985, 49, 2641.
  • [26] S. Y. Wang, M. L. Hsu, H. C. Hsu, C. H. Tzeng, S. S. Lee, M. S. Shiao, C. K. Ho, Int. J. Cancer 1997, 70, 699.
  • [27] D. M. Eisenberg, R. B. Davis, S. L. Ettner, S. Appel, S. Wilkey, M. Van Rompay, R. C. Kessler, JAMA, J. Am. Med. Assoc. 1998, 280, 1569.
  • [28] A. de la Taille, O. R. Hayek, M. Burchardt, T. Burchardt, A. E. Katz, J. Altern. Complement. Med. 2000, 6, 449.
  • [29] R. S. DiPaola, H. Zhang, G. H. Lambert, R. Meeker, E. Licitra, M. M. Rafi, B. T. Zhu, H. Spaulding, S. Goodin, M. B. Toledano, W. N. Hait, M. A. Gallo, N. Engl. J. Med. 1998, 339, 785.
  • [30] A. de la Taille, O. R. Hayek, R. Buttyan, E. Bagiella, M. Burchardt, A. E. Katz, BJU Int. 1999, 84, 845.
  • [31] K. Shimizu, M. Fukuda, R. Kondo, K. Sakai, Planta Med. 2000, 66, 16.
  • [32] Y. Fukuta, Y. Fukuda, R. Higashino, K. Yoshida, M. Ogishima, H. Tamaki, M. Takei, J. Pharmacol. Exp. Ther. 1999, 290, 1013.

Received January 10, 2008

 

 

(Ganoderma lucidum'dan saflaştırılmış polysaccharitlerin neutrofil hürelerinin yönlenmesi ve fagositoz yeteneklerinin artmasına katkısı)

Signaling mechanisms of enhanced neutrophil phagocytosis and chemotaxis by the polysaccharide purified from Ganoderma lucidum

^ing-Jen Hsu, 2Shiuh-Sheng Lee, 3Sho Tone Lee & [*]4Wan-Wan Lin

'Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan; 2Department of Biochemistry, National Yang-Ming University, Taipei, Taiwan and 3Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan

•1     The polysaccharide from Ganoderma lucidum (PS-G) has been reported to enhance immune responses and to elicit antitumor effects. In our previous study, we found that PS-G efficiently inhibited spontaneously and Fas-enhanced neutrophil apoptosis when cultured in vitro. Since phagocytosis and chemotaxis play essential roles in host defense mediated by neutrophils, it is of great interest to know the effect of PS-G on these two cell functions, and the molecular events leading to these actions.

•2      Using latex beads and heat-inactive Escherichia coli serving as particles for neutrophil engulfment, we found that PS-G is able to enhance phagocytic activity of human primary neutrophils and neutrophilic-phenotype cells differentiated from all trans retinoic acid-treated HL-60 cells.

•3      Chemotactic assay using Boyden chamber also revealed the ability of PS-G to increase neutrophil migration.

•4      Exposure of neutrophils to PS-G time dependently caused increases in protein kinase C (PKC), p38 mitogen-activated protein kinase (MAPK), Hck, and Lyn activities.

•5      Results with specific kinase inhibitors indicate that phagocytic action of PS-G was reduced by the presence of wortmannin (Phosphatidylinositol 3-kinase, PI3K inhibitor), pyrazolpyrimidine 2 (Src- family tyrosine kinase inhibitor), Ro318220 (PKC inhibitor), and SB203580 (p38 MAPK inhibitor), but not by PD98059 (mitogen-activated protein/ERK kinase inhibitor). Moreover, chemotactic action of PS-G requires the activities of PI3K, p38 MAPK, Src tyrosine kinases and PKC.

•6     All these results demonstrate the abilities of PS-G to enhance neutrophil function in phagocytosis and chemotaxis, and further provide evidence to strengthen the beneficial remedy of G. lucidum in human to enhance defense system.

British Journal of Pharmacology (2003) 139, 289-298. doi:10.1038/sj.bjp.0705243 Keywords: PS-G; phagocytosis; chemotaxis; neutrophil; G. lucidum

Abbreviations: ATRA, all trans retinoic acid; Bis X, bisindolylmaleimide X; dBcAMP, dibutyryl cAMP; ERK, extracellular signal-regulated kinase; fMLP, N-formyl-Met-Leu-Phe; GM - CSF, granulocyte - macrophage colony-stimulat­ing factor; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein/ERK kinase; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PP2, pyrazolpyrimidine 2; PS-G, polysaccharide from Ganoderma lucidum

 Introduction

Chinese medical fungus, Ganoderma (G.) lucidum, has been a favorite remedy in oriental medicine for centuries. It was considered to maintain the vitality of human beings and to promote longevity (Shiao et al., 1994). This fungus has been reported to be effective in the treatment of hypertension, hyperglycemia, chronic hepatopathy, and neoplasia. The ethanol-precipitable fraction of hot-water extracts of G. lucidum was shown to possess antitumor activity in vivo (Miyazaki & Nishijima, 1981; Sone et al., 1985; Wang et al., 1993; 1997; Lee et al., 1995) and to reduce tumor metastasis (Hwang et al., 1989; Lee et al., 1995). The major active component was isolated, purified, and named as PS-G (polysaccharide from G. lucidum) (Miyazaki & Nishijima, 1981).

Structurally, PS-G is a branched (1 - 6)-b-D-glucan, which contains a backbone chain of (1 - 3)-linked D-glucose residues, five out of 16 D-glucose residues being substituted at O-6 positions with single D-glucosyl units (Sone et al., 1985). Studies have indicated the antineoplastic action of PS-G and attributed it to the activated host immune response (Won et al., 1989; Furusawa et al., 1992). PS-G was reported to enhance cytotoxic activity of natural killer cells, and to increase tumor necrosis factor-a and interferon-g release, respectively, from macrophages and lymphocytes (Lee et al., 1995; Wang et al., 1997).

Mature human polymorphonuclear neutrophils are the most abundant leukocytes of the blood and are committed to death via apoptosis in vivo and in vitro within 72 h. Although these cells appear to have a very short life span (half-life 12- 18 h), their survival can be extended significantly by certain growth factors, and proinflammatory cytokines such as granulocyte colony-stimulating factor, granulocyte - macrophage colony- stimulating factor (GM-CSF), interleukin-2, and interleukin-8 (Leuenroth et al., 1998; Klein et al., 2000). In our previous study, we found that PS-G efficiently inhibited spontaneously and Fas-enhanced neutrophil apoptosis, and this effect is independent of the action through GM-CSF, TLR4, and N-formyl-Met-Leu-Phe (fMLP) receptors (Hsu et al., 2002). Neutrophils have a broad array of physical and biochemical attributes that distinguish them from other cells. Most of these pathways are adapted for one major purpose-to ingest and destroy invading pathogens. To achieve this purpose, chemo- taxis of neutrophils to inflammatory site following concentra­tion gradient of chemokine is the first step essential for host defense. Phagocytosis is then a process that involves binding and internalization of pathogens by neutrophils. Since neutrophils are greatly involved in inflammation and are the first type of cells to arrive at an inflammatory site, we are interested to elucidate whether PS-G affects the defense functions of neutrophils in terms of phagocytosis and chemotaxis.

Methods

Materials

RPMI 1640, fetal bovine serum, penicillin, and streptomycin were obtained from Gibco BRL (Grand Island, NY, U.S.A.). Ficoll - Hypaque was purchased from Amersham Pharmacia Biotech (Piscataway, NJ, U.S.A.). PD98059, pyrazolpyrimi- dine 2 (PP2), SB203580, Ro318220, and bisindolylmaleimide X (Bis X) were purchased from Calbiochem (San Diego, CA, U.S.A.). Protein kinase C (PKC) activity assay kit was purchased from Upstate Biotechnology (Lake Placid, NY, U.S.A.). [g-32P]ATP (6000 Cimmol"1) and the enhanced chemiluminescence detection agent were purchased from NEN (Boston, MA, U.S.A.). Rabbit polyclonal antibody specific for p38 mitogen-activated protein kinase (MAPK), Lyn, protein A/G beads, and horseradish peroxidase-conju- gated anti-mouse and anti-rabbit antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Mouse monoclonal antibody specific for Hck was purchased from Cell Signaling & Neuroscience (St Louis, MO, U.S.A.). All materials for SDS - PAGE were obtained from Bio-Rad Laboratories (Hercules, CA, U.S.A.). Heat-inactivated fluor- escein-labeled Escherichia coli particles and phalloidin-FITC were purchased from Molecular Probes (Eugene, OR, U.S.A.). Latex beads, wortmannin, fMLP, zymosan, GM-CSF, and all other chemicals were obtained from Sigma (St Louis, MO, U.S.A.).

PS-G purification from cultured G. lucidum

As our previous report (Wang et al., 1997), fruiting bodies of G. lucidum were washed, disintegrated, and extracted with boiling water for 8 - 12 h. Hot-water extract of G. lucidum was fractionated into a polysaccharide fraction (alcohol insoluble) and nonpolysaccharide fraction (alcohol soluble). The crude polysaccharide obtained was then passed through a gel­filtration Sephadex G 50 column (Pharmacia, Upsala, Sweden), and was further purified by anion exchange chromatography with a column of DEAE-cellulose (Miyazaki & Nishijima, 1981). PS-G we isolated was a protein-bound polysaccharide consisting of about 95% polysaccharide and 5% peptides. To rule out possible endotoxin lipopolysacchar- ide contamination of PS-G samples, we determined lipopoly- saccharide content by the chromogenic limulus ameboctye lysate assay. We found that there was no detectable level of endotoxin (<0.10 EU ml-1) in PS-G samples.

Neutrophil preparation

Neutrophils were isolated from citrate anticoagulated venous blood (20-60 ml) obtained from healthy volunteers. Cells were separated from whole blood by centrifugation and the upper plasma layer was removed. Leukocytes were separated from erythrocytes in the cell pellet by differential sedimenta­tion using 1.5% dextran. Granulocytes were then separated from monocytes and lymphocytes by centrifugation through a Ficoll - Hypaque gradient. Granulocytes were harvested from the interface of the gradient, and contaminating erythrocytes were removed by hypotonic water lysis. Neutrophil prepara­tion contained >95% neutrophils, of which >99% viable as determined by trypan blue dye exclusion. Freshly isolated neutrophils were resuspended in RPMI 1640 medium supple­mented with 10% fetal bovine serum and antibiotics (100Uml-1 penicillin and 100mgml-1 streptomycin). Cells were cultured at 37°C in a humidified 5% CO2 atmosphere.

HL-60 cell culture and induction of differentiation

HL-60 cells were cultured at 37°C with 5% CO2 in the following complete medium: 10% FBS, 100Uml-1 penicillin, and 100 mgml-1 streptomycin in RPMI 1640 medium. The cell density was maintained about 1.0 x 106 cells ml-1. HL-60 cells were induced to differentiate by the addition of following agent: 1 mM all trans retinoic acid (ATRA), 200 mM dibutyryl cAMP (dBcAMP), or 1.25% DMSO, for a maximum 6 days, but normally 3 - 4 days sufficed to achieve the neutrophilic phenotype. To ascertain that ATRA produced the desired neutrophilic phenotype in HL-60 cells, cells were assayed for flow cytometric analysis of surface expression of differentia­tion-related antigens. Briefly, HL-60 cells were incubated with or without different inducers of differentiation for 96 h. Cells were then washed, centrifuged and resuspended in staining buffer (50 mM sodium phosphate pH 7.5, 100 mM KCl, 150mM NaCl, 5% glycerol, 0.2% BSA) containing FITC- labeled anti-human CD11b antibody (Ancell Corporation, MN, U.S.A.) for 45 min on ice. After staining, cells were pelleted (400 x g, 5min), washed twice with PBS (pH 7.4), and immediately analyzed using FACScan and the Cellquest program (Becton Dickinson) with appropriate gating para­meters.

Phagocytosis assay

Phagocytosis assay was performed using latex beads or heat- inactivated fluorescein-labeled E. coli particles for engulfment. Briefly, 200 ml aliquots of neutrophils suspended in RPMI medium (106 cells ml-1) were added into the wells of a 96-well plate. Following treatment with the test compound as indicated for 30 min, cells were challenged with FITC - E. coli particles (10 mgml-1) or PE-latex beads (0.025% solids, 2mm diameter) and stood at 37°C for another 15 min. Then the cells were washed three times with PBS (pH 7.4), and suspended in PBS (pH 7.4). Fluorescence derived from FITC - E. coli or PE- latex beads ingested by neutrophils was immediately measured using FACScan and the Cellquest program (Becton Dick­inson). The relative amount of ingested FITC - E. coli or PE- latex beads was calculated by subtraction of the mean fluorescence intensity of neutrophils alone from that of each test samples.

PKC activity assay

PKC activity was assayed by quantification of 32P incorpora­tion from [g-32P]ATP into the substrate, QKRPSQRSKYL peptide. Briefly, cells following incubation in the presence of PS-G for different time periods were washed twice in ice-cold PBS and then lysed in buffer containing 20 mM Tris-HCl, 0.5mM EGTA, 2mM EDTA, 2mM dithiothreitol, 0.5mM phenylmethylsulfonyl fluoride, and 10 mgml-1 leupeptin (pH 7.5). Assays were then performed at 30°C in a total volume of 60 ml containing 20 mM MOPS, pH 7.2, 25 mM b-glycerol phosphate, 1 mM sodium orthovanadate, 1 mM DTT, 1 mM CaCl2, 500 mM QKRPSQRSKYL peptide, 2 mM PKA inhibitor (PKI) peptide, 20 mM compound R24571 (calmodulin inhibi­tor), 0.5mgml~' phosphatidylserine, 0.05mgml~' diacylgly- cerol and 10 mCi [g-32P]ATP. Reactions were initiated by the addition of [g-32P]ATP and terminated after 10min by transfer of the reaction mixture onto a P81 phosphocellulose paper. The papers were then washed with 0.75% phosphoric acid for three times. After washing, acetone was added and washed for two more minutes, removed and the paper stuffed into the scintillation vial containing 5 ml scintillation cocktail. Incor­poration of 32P into the substrate was then quantified by scintillation counter (Beckman).

Immunoblotting

Cells following incubation in the presence of various stimuli for different time periods were washed twice in ice-cold PBS and then lysed in buffer containing 20 mM Tris-HCl (pH 7.5), 0.5mM EGTA, 2mM EDTA, 2mM dithiothreitol, 0.5mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10mgml~' aprotinin, 10mgml~' leupeptin. Samples of equal amounts of protein were subjected to SDS-PAGE on 10% polyacrylamide gels and transferred onto a nitrocellulose membrane, which was then incubated in 150 mM NaCl, 20 mM Tris, and 0.02% Tween (pH 7.4) containing 5% nonfat milk. The membranes were subsequently probed with primary antibody. The antibody - antigen complexes were detected by IgG peroxidase conjugates, followed by use of an enhanced chemiluminescence kit according to the manufacturer's in­struction and exposure to photographic film.

Immunoprecipitation and protein kinase assay

Cells were washed twice with ice-cold PBS, lysed in 1 ml of lysis buffer containing 20 mM Tris, pH 7.5, 137 mM NaCl, 10% glycerol, 1% NP40, 1 mM sodium orthovanadate, 1 mM p-methylsulfonylfluoride, 10 mgml-1 aprotinin, 10 mgml-1 leupeptin, and centrifuged. The supernatant was then im- munoprecipitated with respective specific antibodies against p38 MAPK, Lyn or Hck in the presence of A/G-agarose beads overnight. The beads were washed three times with lysis buffer and two times with kinase buffer (40 mM HEPES, 10 mM MgCl2, 10 mM MnCl2, 200 mM sodium orthovanadate, 2mM dithiothreitol). Then the beads were equally divided into two parts, respectively, for kinase assay and immunoblotting. The kinase reactions were performed by incubating immunopreci- pitated beads with 20 ml of kinase buffer supplemented with 20 mM ATP and 3 mCi of [g-32P]ATP at 30°C for 30min. For p38 MAPK assay, myelin basic protein (MBP, 1 mg per reaction) was added as a substrate, and for tyrosine kinase assay, acid-denatured enolase (12.5 mg per reaction) was used. The reaction mixtures were analyzed by SDS - PAGE followed by autoradiography. The precipitated protein levels of p38 MAPK, Lyn and Hck were assayed by immunoblotting as an internal control.

Neutrophil transmigration assay

The neutrophil transmigration assays were performed in a Boyden chamber using a 3 mm polyvinylpyrrolidone-free polycarbonate membrane. The chemotactic stimuli, fMLP (1 mgmH) or PS-G (100mgmH) diluted in RPMI medium at different concentrations were added to the bottom wells of the chamber. Cells suspended in RPMI medium (5 x 105 cells ml-1) were added to the top wells of the Boyden chamber and allowed to migrate for 30 min at 37°C in a 5% CO2 atmosphere. In some experiments, neutrophils were pretreated with kinase inhibitors or vehicle for 30 min at 37°C and allowed to migrate in the Boyden chamber. Neutrophils that had migrated to the lower surface of the membrane were quantified by crystal violet staining and measuring the absorbance at 550 nm. In some experiments, the membranes were removed from the chambers, washed twice with PBS, and fixed in 4% paraformaldehyde in PBS for 15min at room temperature. Cells were then permeabilized for 5min in 0.1% Triton X-100. After blocking with 5% nonfat milk for 30 min, cells were labeled with phalloidin - FITC (200ngml-1) for 1 h at room temperature. To observe stained cells, membranes were washed, mounted and examined under fluorescence microscope ( x 400 amplification).

Statistical evaluation

Values were expressed as the mean 7 standard error of the means (s.e.m.) of at least three experiments. ANOVA and Dunnetts tests were used to assess the statistical significance of the differences, with 'P' values of less than 0.05 being considered statistically significant.

Results

PS-G dose dependently enhanced neutrophil phagocytic activity

Freshly isolated neutrophils were measured for their ability to phagocytose latex beads. Phagocytosis of latex particles was demonstrated by a progressive fluorescence shift on a logarithmic scale comparing FL2 of latex beads with control group (Figure 1a). When using mean fluorescence intensity as an index of neutrophil phagocytic activity, latex particles phagocytosed by neutrophils treated with vehicle alone increased time dependently (Figure 1b). This basal phagocytic
activity reached a steady-state level of two-fold increase within 60 - 90 min. When neutrophils were 30 min pretreated with PS­G (100 or 300mgml-1) before latex beads exposure, their phagocytic activity was significantly enhanced at 15 and 30 min (Figure 1b). However, with longer challenge with latex particles up to 60 min, the phagocytic index of PS-G-treated neutrophils was equivalent to that of the cells without priming. When examining the concentration-dependent effect of PS-G on phagocytic activity of neutrophils, the results indicated that concentrations higher than 100 mgml-1 of PS-G were required to increase the rates of phagocytosis.

In addition, we also compared the response of PS-G with GM-CSF and zymosan, which are known to effectively enhance phagocytic activity of neutrophils. Cells with GM- CSF (10ngml-1) or zymosan (100mgml-1) pretreatment resulted in increases of phagocytic rate and maximal level.

In addition to latex beads, we also investigated if PS-G affects the engulfment of E. coli particle by neutrophils. Flow cytometric analysis indicated that within 15 min incubation, fluorescein-labeled E. coli particles were phagocytosed in the PS-G primed neutrophils with higher levels than in cells without priming. This effect of PS-G to enhance bacteria uptake by neutrophils displayed the concentration depen­dency, and an increase of about 45% was observed at 100 mgml-1 PS-G treatment (Figure 2a). In contrast to latex particles whose uptake by neutrophils showed the time dependency and reached maximum at 60 - 90 min, when cells challenged with E. coli particles for different time periods, a constant uptake amount was kept within 15 - 60 min incuba­tion (Figure 2b). After that, the amount of E. coli particle uptake by neutrophils was declined. Interestingly, we found that the enhanced phagocytic ability of PS-G-treated neutro- phils only occurred transiently at 15 min, and then returned to control level. Zymosan at 100mgml-1 possessed similar effect as PS-G in bacteria phagocytosis, at least both in the uptake amount and kinetic pattern. In contrast, the ability of GM- CSF (10 ng ml-1) to increase bacteria phagocytosis was greater and more sustained than PS-G and zymosan.

PS-G-enhanced phagocytic activity of differentiated HL-60 cells

 

In addition to primary neutrophils, we also examined the effect of PS-G on another granulocyte system, the differentiating HL-60 promyeloid cells. First, we verified this granulocyte differentiation process by adding the well-known differentia­tion agent and detecting the expression of CD11b (or Mac -1), the a subunit of fi2 integrin, CR3. CD11b is absent on immature promyeloid cells and has been widely used to reflex the initiation of myeloid differentiation into a more mature phenotype (Collins et al., 1978; Testa et al., 1993). In these experiments, cells were incubated for 96 h in culture medium plus 1 mM ATRA, 200 mM dBcAMP, or 1.25% DMSO. They were then stained with anti-CD11b antibody, and flow
cytometric analysis was performed. A representative result of flow cytometric analysis of CD11b expression was shown (Figure 3a, upper panel). Since different stimuli may obtain various degrees of differentiation in HL-60 cells, and the highest percentage of CD11b positive cells was seen in ATRA-treated group, we choose ATRA-treated cells for the following experiments. The kinetic of expressed level of CD11b in the ATRA-treated cells was shown in Figure 3a (bottom panel). Despite the expression of surface marker like CD11b, we further determined whether the differentiated cells were able to engulf latex particles, which is a characte­rized feature of mature neutrophils. Results from Figure 3b demonstrated that HL-60 cells after 4-day differentiation into granulocytes obtained the ability to phagocytose latex beads.

We next examined the effects of PS-G, GM-CSF, or zymosan on the ability of these ATRA-treated cells to engulf latex particles. Results shown in Figure 3c revealed that all these three agents time dependently increased the engulfment of latex particles. However, compared to the more sustained stimulation by GM-CSF and zymosan for 60-120min, the response of PS-G was weak and active for short-period. These results again confirm the stimulating pattern of PS-G, GM- CSF, and zymosan in latex bead phagocytosis by human neutrophils.

Effects of kinase inhibitors on phagocytic stimulation

To dissect the molecular mechanism involved in PS-G-induced phagocytic stimulation, we used several selective inhibitors. The concentrations of these inhibitors used are specific for the kinases as previously reported in neutrophils (Kodama et al., 1999; Klein et al., 2000; Tohru et al., 2000; Nijhuis et al., 2002) and we reported in other cell systems (Chen & Lin, 2001; Huang et al., 2002). As shown in Figure 4a, when latex beads were used, the stimulated effects of PS-G (100 mgml-1) and zymosan (100mgml-1) were unaffected by mitogen-activated protein/ERK kinase (MEK) inhibitor (PD98059, 30 mM), while the presence of phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin, 300 nM) and PKC inhibitor (Ro318220, 10 mM) significantly diminished their effects. In contrast, only wortmannin reversed the enhanced effect of GM-CSF. When PS-G was cotreated with GM-CSF or zymosan, no further enhancement with statistical significance in phagocytosis was seen. These results suggest that PS-G might partially mimic GM-CSF and zymosan action in enhancing phagocytic activity of neutrophils, through activating the PI3K-dependent signaling pathway. Since the concentration of wortmannin employed might be high and probably nonselective, lower concentration of wortmannin was used to exclude this possibility. In addition, we also examined p38 MAPK inhibitor SB203580 within different concentrations between 1 and 10 mM. As shown in Figure 4b, lower concentrations of wortmannin (30 - 300 nM) still retained the inhibitory effect on PS-G action. Similarly inhibition of the p38 MAPK pathway using SB203580 (3 or 10 mM) abolished the potentiating effect of PS-G on neutrophil phagocytosis.

On the other hand, we further used E. coli particle for engulfment to assess and compare the mechanism of PS-G with zymosan and GM-CSF. Similar to the response using latex beads (Figure 4a and b), Ro318220, wortmannin, and SB203580 effectively inhibited the responses of PS-G and zymosan. In addition, we also tested PKC inhibitor Bis X and Src inhibitor PP2. As shown in Figure 4c, treatment with Bis X (10 mM) and PP2 (100 nM) significantly eliminated PS-G and zymosan actions on the engulfment of E. coli particle by neutrophils. Moreover, PS-G in combination with zymosan had no additive priming effect as compared to PS-G or zymosan individually (Figure 4c). This again implies that PS­G, GM-CSF, and zymosan may share similar downstream signaling pathways involved in the priming effect on neutro- phil phagocytosis. In contrast, the effect of GM-CSF was inhibited by PP2, wortmannin and SB203580, but not sensitive to Ro318220.

PS-G-activated PKC, Src-family protein kinases andp38 MAPK

 

Since PKC inhibitors, such as Ro318220 and Bis X, Src family kinase inhibitor PP2 and p38 MAPK inhibitor

SB203580 could diminish the priming effect of PS-G on phagocytosis, PKC, Src family members and p38 MAPK involved in PS-G action were considered. Figure 5a demon­strated that PS-G (100 mgml-1) could time dependently increase PKC activity within 30 min incubation. In human neutrophils, we found that Hck and Lyn are two major tyrosine kinases of Src family and their activities, using in vitro enolase as a substrate, were rapidly and transiently stimulated by PS-G (Figure 5b). p38 MAPK activity was also assessed using in vitro phosphorylation of MBP as an index. As shown in Figure 5c, the activity of p38 MAPK was increased upon PS-G treatment.

PS-G enhanced neutrophil chemotaxis

Results from the above experiments suggest that PS-G could enhance neutrophil phagocytosis. To further ascertain the physiological implication in stimulating neutrophil function, we determined whether PS-G affects neutrophil chemotaxis. Figure 6a showed that PS-G at 100mgml-1 was active in eliciting chemotaxis as examined from fluores­cence microscopy. Moreover, fMLP (100ngml-1) has compar­able efficacy in this effect (Figure 6a and b). These results suggest that PS-G might function as a chemoattractant to recruit neutrophils. In an attempt to elucidate signaling pathways involved in this response of PS-G, we tested several pharmacological inhibitors. As shown in Figure 6b, the presence of wortmannin (100- 300 nM), Ro318220 (10 mM), PP2 (100 nM), and SB203580 (1 -10 mM) significantly abol­ished the effect of PS-G effect. In contrast, the presence of PD98059 (30 mM) did not significantly alter the effect of PS-G.

Discussion

 

Neutrophils are the principal effectors of the initial host response to injury or infection and constitute a significant threat to invading bacterial pathogens (Bharadwaj et al., 2001; Power et al., 2001). Extravasation or the migration of neutrophils from the vascular system to sites of pathogenic exposure is a key event in immune defense (Tani et al., 2001; Ostermann et al., 2002). Neutrophils also carry potent destructive enzymes that can destroy invasive bacteria or damage normal tissues (Shi et al., 2001). Several reagents are known to alter the functions of neutrophils (Bharadwaj et al.,
2001; Marino et al., 2001; Nishimura et al., 2001). Cytokine like GM-CSF or microbial products such as yeast glucan and zymosan might modulate the function of neutrophils (Bober et al., 1995; Kumaratilake et al., 1996; Richardson & Chung, 1997; Bharadwaj et al., 2001; Wanten et al., 2001). In the present study, we demonstrated that the polysaccharide component of Chinese medicine G. lucidum possesses cap­ability for enhancing neutrophil functions in phagocytosis and migration.

To dissect the signaling events involving in PS-G effects on neutrophil phagocytosis and migration, we used pharmacolo­gical inhibitors. Our data ruled out the involvement of extracellular signal-regulated kinase (ERK) signal pathway in phagocytic and chemotactic effects of PS-G, as inhibitor of the upstream activating molecule PD98059 failed to alter the response of PS-G. On the other hand, inhibitors of PKC (Ro318220, Bis X), PI3K (wortmannin), Src tyrosine kinase (PP2), and p38 MAPK (SB203580) attenuated both biological activities of PS-G. Accordingly, our previous study showing PI3K/PKB stimulation by PS-G (Hsu et al., 2002) as well as here for PKC, Src and p38 MAPK activation all together support the requirement of multiple signaling cascades for cell phagocytosis and migration. In this aspect, accumulated evidences have linked the crucial roles of these signaling cascades in granulocyte phagocytosis and migration. Defects in chemotaxis and loss of directionality of neutrophils isolated from PI3Kg (-/-) mice have been demonstrated (Tohru et al., 2000; Hannigan et al., 2002). PKC inhibition has been reported to prevent phagocytosis in human monocytes and PMN (Fallman et al., 1992; Greenberg et al., 1993; Zhou & Brown, 1994). It appears that Src kinases can regulate PKB activation and thus modulate cytokine or chemoattractant-controlled neutro- phil function (Korade-Mirnics & Corey, 2000; Nijhuis et al., 2002). Additionally, several lines of evidence suggested the involvement of p38 MAPK in phagocytosis and chemotaxis in neutrophils (Heuertz et al., 1999; Tohru et al., 2000).

 

b-Glucans have been recently studied for their ability to activate host defense mechanisms against tumors and micro- bial infections. Although heterogeneity of b-glucans derived from a variety of plant, fungal, and bacterial sources have been

in leukocytes have been partially characterized, and delinea­tion for their ligand specificity as well as signal transduction is currently progressing (Ross et al., 1985; Thornton et al., 1996; Liang et al., 1998; Linehan et al., 2000). Analysis of the response of human leukocytes to b-glucans has shown that the integrin CR3 is primarily responsible for the phagocytic responses mediated by b-glucans (Vetvicka et al., 1996; 1997; Xia et al., 1999). In this study, we for the first time demonstrate the ability of b-glucan from G. lucidum to enhance phagocytosis and induce migration of neutrophils. Similar phagocytic results obtained from neutrophilic-pheno- type cells differentiated from HL-60 as freshly isolated neutrophils may also suggest that this cell line provides an excellent tool for determining the function of neutrophils. Moreover, PS-G in combination with zymosan, a yeast b- glucan, which is commonly studied, had no additive priming effect as compared to PS-G or zymosan individually. Accordingly, zymosan has been reported to stimulate neu- trophils by activating PKC and protein tyrosine kinase pathways (Sergeant & McPhail, 1997; Welch & Maridon- neau-Parini, 1997; Wanten et al., 2001). This implies that PS-G appears to share common signaling pathways to accelerate neutrophil functions as zymosan does.

o

In conclusion, we demonstrated that the polysaccharide from G. lucidum might stimulate the functional activity of mature neutrophils. Together with ability to prolong life span of neutrophils by PS-G, it can thus exert a variety of important regulatory controls of neutrophil function during bacterial infections and is a good remedy to promote host immune response.

 identified, they were shown to produce a state of activation in leukocytes and thus activate the innate immune system (Kokoshis et al., 1978; Bohn & Bemiller, 1995; Wakshull et al., 1999). Furthermore, different b-glucan receptors located

This work was supported by the National Science Council of Taiwan (NSC91-2320-B002-210) and Academia Sinica (IBMS- CRC90-T04; IBMS-CRC 92-T03).

References

BHARADWAJ, D., MOLD, C., MARKHAM, E. & DU CLOS, T.W. (2001). Serum amyloid P component binds to Fc gamma receptors and opsonizes particles for phagocytosis. J. Immunol., 166, 6735­6741.

BOBER, L.A., GRACE, M.J., PUGLIESE-SIVO, C., ROJAS-TRIANA, A., WATERS, T., SULLIVAN, L.M. & NARULA, S.K. (1995). The effect of GM-CSF and G-CSF on human neutrophil function. Immuno- pharmacology, 29, 111-119.

BOHN, J.A. & BEMILLER, J.N. (1995). (1 - 3)-D-glucans as biological response modifiers: a review of structure - functional activity relationships. Carbohydrate Polym., 28, 3 - 14.

CHEN, B.C. & LIN, W.W. (2001). PKC- and ERK-dependent activation of IkB kinase by lipopolysaccharide in macrophages: enhancement by P2Y receptor-mediated CaMK activation. Br. J. Pharmacol., 134, 1055- 1065.

COLLINS, S.J., RUSCETTI, F.W., GALLAGHER, R.E. & GALLO, R. (1978). Terminal differentiation of human promyelocytic leukemia cells induced by dimethylsulfoxide and other polar compounds. Proc. Natl. Acad. Sci. U.S.A., 75, 2458-2462.

FALLMAN, M., GULLBERG, M., HELLBERG, C. & ANDERSSON, T. (1992). Complement receptor-mediated phagocytosis is associated with accumulation of phosphatidylcholine-derived diglyceride in human neutrophils. Involvement of phospholipase D and direct evidence for a positive feedback signal of protein kinase. J. Biol. Chem., 267, 2656-2663.

FURUSAWA, E., CHOU, S.C., FURUSAWA, S., HIRAZAMI, A. & DANG, Y. (1992). Anti-tumor activity of Ganoderma lucidum, an edible mushroom, on intraperitoneally implanted Lewis lung carcinoma in synergenic mice. Phytother. Res., 6, 300 - 304.

GREENBERG, S., CHANG P. & SILVERSTEIN, S.C. (1993). Tyrosine phosphorylation is required for Fc receptor-mediated phagocytosis in mouse macrophages. J. Exp. Med., 177, 529- 534.

HANNIGAN, M., ZHAN, L., LI, Z., AI, Y., DIANQING, W. & KUANG, C.H. (2002). Neutrophils lacking phosphoinositide 3-kinase g show loss of directionality during N-formyl-Met- Leu-Phe-induced chemotaxis. Proc. Natl. Acad. Sci. U.S.A., 99, 3603- 3608.

HEUERTZ, R.M., TRICOMI, S.M., EZEKIEL, U.R. & WEBSTER, R.O. (1999). C-reactive protein inhibits chemotactic peptide-induced p38 mitogen-activated protein kinase activity and human neutrophil movement. J. Biol. Chem., 274, 17968- 17974.

HSU, M.J., LEE, S.S. & LIN, W.W. (2002). Polysaccharide purified from Ganoderma lucidum inhibits spontaneous and Fas-mediated apop- tosis in human neutrophils through activation of the phosphatidy- linositol 3 kinase/Akt signaling pathway. J. Leukocyte Biol., 72, 207 - 216.

HUANG, W.C., CHIO, C.C., CHI, K.H., WU, H.M. & LIN, W.W. (2002). Superoxide anion-dependent Raf/MEK/ERK activation by peroxi­some proliferator activated receptor g agonists 15-deoxy-Al2,14- prostaglandin J2, ciglitazone, and GW1929. Exp. Cell Res., 277, 192-200.

HWANG S.F., LIU, K.J., KUAN, Y.H., TUNG, K.S., SU, C.H. & TUNG, T.C. (1989). The inhibitory effect on artificial pulmonary metastasis of murine S-180 sarcoma cells by orally administered Ganoderma lucidum culture broth. J. Chin. Oncol. Soc., 5, 10 - 15.

KLEIN, J.B., RANE, M.J., SCHERZER, J.A., COXON, P.Y., KETTRITZ, R., MATHIESEN, J.M., BURIDI, A. & MCLEISH, K.R. (2000). Granulocyte - macrophage colony-stimulating factor delays neu- trophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways. J. Immunol., 164, 4286-4291.

KODAMA, T., HAZEKI, K., HAZEKI, O., OKADA, T. & UI, M. (1999). Enhancement of chemotactic peptide-induced activation of phos- phoinositide 3-kinase by granulocyte - macrophage colony-stimu­lating factor and its relation to the cytokine-mediated priming of neutrophil superoxide-anion production. Biochem. J., 337, 201 - 209.

KOKOSHIS, P.L., WILLIAMS, D.L., COOK, J.A. & DI LUZIO, N.R. (1978). Increased resistance to Staphylococcus aureus infection and enhancement in serum lysozyme activity by glucan. Science, 199, 1340- 1342.

KORADE-MIRNICS, Z. & COREY, S.J. (2000). Src kinase-mediated signaling in leukocytes. J. Leukocyte Biol., 68, 603 - 613.

KUMARATILAKE, L.M., FERRANTE, A., JAEGER, T. & RZEPCZYK, C. (1996). GM-CSF-induced priming of human neutrophils for enhanced phagocytosis and killing of asexual blood stages of Plasmodium falciparum: synergistic effects of GM-CSF and TNF. Parasite Immunol., 18, 115- 123.

LEE, S.S., WEI, Y.H., CHEN, C.F., WANG, S.Y. & CHEN, K.Y (1995). Antitumor effects of Ganoderma lucidum. J. Chin. Med., 6, 1 - 12.

LEUENROTH, S., LEE, C., GRUTKOSKI, P., KEEPING, H. & SIMMS, H.H. (1998). Interleukin-8-induced suppression of polymorpho- nuclear leukocyte apoptosis is mediated by suppressing CD95 (Fas/ Apo-1) Fas-1 interactions. Surgery, 124, 409-417.

LIANG, J., MELICAN, D., CAFRO, L., PALACE, G, FISETTE, L., ARMSTRONG, R. & PATCHEN, M.L. (1998). Enhanced clearance of a multiple antibiotic resistant Staphylococcus aureus in rats treated with PGG-glucan is associated with increased leukocyte counts and increased neutrophil oxidative burst activity. Int. J. Immunopharmacol., 20, 595 - 614.

LINEHAN, S.A., MARTINEZ-POMARES, L. & GORDON, S. (2000). Macrophage lectins in host defence. Microbes Infect., 2, 279 - 288.

MARINO, F., CATTANEO, S., COSENTINO, M., RASINI, E., DI GRAZIA, L., FIETTA, A.M., LECCHINI, S. & FRIGO G. (2001). Diazepam stimulates migration and phagocytosis of human neutrophils: possible contribution of peripheral-type benzodiaze- pine receptors and intracellular calcium. Pharmacology, 63, 42 - 49.

MIYAZAKI, T. & NISHIJIMA, M. (1981). Studies on fungal poly- saccharides, XXVII. Structural examination of a water-soluble, anti-tumor polysaccharide of Ganoderma lucidum. Chem. Pharm. Bull., 29, 3611 - 3616.

NIJHUIS, E., LAMMERS, J.W., KOENDERMAN, L. & COFFER, P.J. (2002). Src kinases regulate PKB activation and modulate cytokine and chemoattractant-controlled neutrophil functioning. J. Leuko­cyte Biol., 71, 115-124.

NISHIMURA, H., GOGAMI, A., MIYAGAWA, Y., NANBO, A., MURAKAMI, Y., BABA, T. & NAGASAWA S. (2001). Bacterici­dal/permeability-increasing protein promotes complement activa­tion for neutrophil-mediated phagocytosis on bacterial surface. Immunology, 103, 519 - 525.

OSTERMANN, G., WEBER, K.S., ZERNECKE, A., SCHRODER, A. & WEBER, C. (2002). JAM-1 is a ligand of the beta(2) integrin LFA-1 involved in transendothelial migration of leukocytes. Nat. Immu­nol., 3, 151 - 158.

POWER, C., WANG, J.H., SOOKHAI, S., WU, Q.D. & REDMOND, H P. (2001). Proinflammatory effects of bacterial lipoprotein on human neutrophil activation status, function and cytotoxic potential in vitro. Shock, 15, 461 -466.

RICHARDSON, M.D. & CHUNG, I. (1997). GM-CSF-modulated phagocytosis of Trichosporon beigelii by human neutrophils. J. Med. Microbiol., 46, 321 - 325.

ROSS, G.D., CAIN, J.A. & LACHMANN, P.J. (1985). Membrane complement receptor type three (CR3) has lectin-like properties analogous to bovine conglutinin as functions as a receptor for zymosan and rabbit erythrocytes as well as a receptor for iC3b. J. Immunol., 134, 3307-3315.

SERGEANT, S. & MCPHAIL, L.C. (1997). Opsonized zymosan stimulates the redistribution of protein kinase C isoforms in human neutrophils. J. Immunol., 159, 2877 - 2885.

SHI, J., GILBERT, G.E., KOKUBO, Y. & OHASHI, T. (2001). Role of the liver in regulating numbers of circulating neutrophils. Blood, 98, 1226- 1230.

SHIAO, M.S., LEE, K.R., LIN, L.J. & WANG, C.T. (1994). Natural products and biological activities of the Chinese medical fungus, Ganoderma lucidum. In: Food Phytochemicals for Cancer Prevention. II: Teas, Spices, and Herbs. ed. Ho, C.T., Osawa, T., Huang, M.T. & Rosen, R.T., pp. 342 - 354. Washington, DC: American Chemical Society.

SONE, Y., OKUDA, R., WADA, N., KISHIDA, E. & MISAKI, A. (1985). Structures and anti-tumor activities of polysaccharides isolated from fruiting body and the growing culture of mycelium of Ganoderma lucidum. Agric. Biol. Chem., 49, 2641 -2653.

TANI, K., OGUSHI, F., SHIMIZU, T. & SONE, S. (2001). Protease- induced leukocyte chemotaxis and activation: roles in host defense and inflammation. J. Med. Invest., 48, 133 - 141.

TESTA, U., MASCIULLI, R., TRITARELLI, E., PUSTORINO, R., MARIANI, G., MARTUCCI, R., BARBERI, T., CAMAGNA, A., VALTIERI, M. & PESCHLE, C. (1993). Transforming growth factor- b potentiates vitamin D3-induced terminal monocytic differentia­tion of human leukemic cell lines. J. Immunol., 150, 2418 -2430.

THORNTON, B.P., VETVICKA, V., PITMAN, M., GOLDMAN, R.C. & ROSS, G.D. (1996). Analysis of the sugar specificity and molecular location of the beta-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18). J. Immunol., 156, 1235- 1246.

TOHRU, Y., OSAMU, I., HAJIME, N.Y.D.C. & MIKINORI, K. (2000). Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymor­phonuclear leukocytes. FEBS Lett., 467, 253 - 258.

VETVICKA, V., THORMTON, B.P. & ROSS, G.D. (1996). Soluble b- glucan polysaccharide binding to the lectin site of neutrophil or NK cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b- opsonized target cells. J. Clin. Invest., 98, 50-61.

VETVICKA, V., THORMTON, B.P., WIEMAN, T.J. & ROSS, G.D. (1997). Targeting of NK cells to mammary carcinoma via naturally occurring tumor cell-bound iC3b and b -glucan-primed CR3(CD 11b/CD 18). J. Immunol., 159, 599-605.

WAKSHULL, E., BRUNKE-REESE, D., LINDERMUTH, J., FISETTE, L., NATHANS, R.S., CROWLEY, J.J., TUFTS, J.C., ZIMMERMAN, J., MACKIN, W. & ADAMS, D.S. (1999). PGG-glucan, a soluble beta-(1,3)-glucan, enhances the oxidative burst response, micro- bicidal activity, and activates an NF-kappa B-like factor in human PMN: evidence for a glycosphingolipid beta-(1,3)-glucan receptor. Immunopharmacology, 41, 89 - 107.

WANG, S.Y., HSU, M.L., HSU, H.C., TZENG, C.H., LEE, S.S., SHIAO, M.S. & HO, C.K. (1997). The anti-tumor effect of Ganoderma

lucidum is mediated by cytokines released from activated macro­phages and T lymphocytes. Int. J. Cancer, 70, 699-705.

WANG, G., ZHANG J., MIZUNO, T., ZHUANG, C., ITO, H., MAYUZUMI, H., OKAMOTO, H. & LI, J. (1993). Antitumor active polysaccharides from the Chinese mushroom Songshan lingzhi, the fruiting body of Ganoderma tsugae. Biosci. Biotech. Biochem., 57, 894 - 900.

WANTEN, G., VAN EMST-DE VRIES, S., NABER, T. & WILLEMS, P. (2001). Nutritional lipid emulsions modulate cellular signaling and activation of human neutrophils. J. Lipid Res., 42, 428 - 436.

WELCH, H. & MARIDONNEAU-PARINI, I. (1997). Lyn and Fgr are activated in distinct membrane fractions of human granulocytic cells. Oncogene, 15, 2021 - 2029.

WON, S.J., LEE, S.S., KE, YH. & LIN, M.T. (1989). Enhancement of spenic NK cytotoxic activity by extracts of Ganoderma lucidum mycelium in mice. J. Biomed. Lab. Sci., 2, 201 - 213.

XIA, Y., VETVICKA, V., YAN, J., HANIKYROVA, M., MAYADAS, T. & ROSS, G.D. (1999). The b-glucan-binding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b- opsonized target cells. J. Immunol., 162, 2281 -2290.

ZHOU, M.J. & BROWN, E.J. (1994). CR3 (Mac-1, alpha M beta 2, CD11b/CD18) and Fc gamma RIII cooperate in generation of a neutrophil respiratory burst: requirement for Fc gamma RIII and tyrosine phosphorylation. J. Cell Biol., 125, 1407- 1416.

(Received December 6, 2002 Accepted February 17, 2003)

 


[*]Author for correspondence; E-mail: wwl@ha.mc.ntu.edu.tw

 

 

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

 (Ganoderma lucidum-Reishi- meyvelerinden izole edilmiş iki düşük mol. Ağırlıklı polysakkaritin antioksidan aktivitesi)

Characterization and antioxidant activity of two low-molecular-weight polysaccharides purified from the fruiting bodies of Ganoderma lucidum

Wei Liu, Hengyu Wang, Xiubing Pang, Wenbing Yao [1], Xiangdong Gao [2]

School of Life Science and Technology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China

 ABSTRACT

Two low-molecular-weight polysaccharides, GLPl1 and GLPl2, purified from a crude Ganoderma lucidum polysaccharide preparation GLPP were investigated for their physicochemical properties, structure characterization and antioxidant activities. The results indicated that GLPl1 was a glucan with an average molecular weight of 5.2 kDa, while GLPl2 was composed of glucose, galactose and mannose in a ratio of 29:1.8:1.0 with the average molecular weight of 15.4kDa. GLPl1 and GLPl2 had similar structure characteristic which contained linkages such as ^3)-Glcp-(1^, ^4)-Glcp-(1^, ^6)-Glcp- (1^, ^3,6)-Glcp-(1^, and ^4,6)-Glcp-(1^ in the percentage ratio of 21.9:20.3:23.7:24.0:3.7 and 23.0:34.6:7.0:14.1:3.0 in the backbone or branches, respectively. Antioxidant results showed that both GLPL1 and GLPL2 exhibited antioxidant activities while GLPL 1 was more effective in free radicals scav­enging and Fe2+ chelating. Low-molecular-weight polysaccharide seems to play an important role in the exploration of natural antioxidants in food industry and pharmaceuticals.

© 2010 Elsevier B.V. All rights reserved.

 1. Introduction

Ganoderma lucidum (Leyss.:Fr.) Karst, an edible mushroom, has been used in functional food and preventive medicines in the Far East for more than 2000 years and becomes a popular dietary sup­plement ingredient in Western countries, with an annual global market value of over $1.5 billion for G. lucidum extracts [1,2]. G. lucidum is an important economic crop in certain regions of China, from which more than two hundred polysaccharides have been isolated [3-5]. These polysaccharides were reported to have many biological activities, including antitumor [6], immunomodulatory [7], anti-viral [8], antihepatitis [9], antioxidant [10], antihyperten­sion [11] and antidiabetics [12].

Since oxidative stress induced by reactive oxygen species (ROS) might attribute to induce some diseases, the aforementioned chemoprotective effects of G. lucidum were considered associated with its antioxidant properties [13]. In 1995, Lin et al. studied free radical scavenging activity from three different Ganoderma species. The results showed that all the extracts from three Gan- oderma could suppress free radicals generation [14]. In 2001, Lee and others studied the ability of protecting against lipid perox- idation and oxidative DNA damage by an amino-polysaccharide fraction from G. lucidum. The results showed the fraction exhib­ited significantly inhibition to iron-induced lipid peroxidation and oxidative DNA damage [13]. Recently, several polysaccharides from Ganoderma atrum and Ganoderma tsugae were isolated for their antioxidant activities evaluation in vitro by two research groups [10,15]. The results showed these polysaccharides all exhibited good antioxidant properties. In 2009, in vivo antiox- idant activity of G. lucidum polysaccharides was carried out by Jia and others using streptozotocin-induced diabetic rats. The results indicated G. lucidum polysaccharides could significantly and dose-dependently increase nonenzymic/enzymic antioxidants and reduce lipid peroxidation [16]. These previous studies suggested G. lucidum polysaccharides might be a novel resource for natural antioxidants exploration.

Most of these reported polysaccharides from G. lucidum are high molecular weight, composed of P- or a-D-glucose residues in the main chain, sometimes accompanied with other monosaccharide residues in side chains [9]. However, these high-molecular-weight polysaccharides are limited to exhibit their biological activities because of the high molecular weights, high apparent viscosity or poor water-solubility or complex structures and conforma­tions. It is difficult for the polysaccharides with these properties to pass through organizational barriers to enter the interior of the cell or attach to the receptors [17]. Researches on exploring low- molecular-weight bioactive polysaccharides are a timely demand to explore novel applicable products.

As our continuous effort to enhance the profitability of G. lucidum production and processing industries, the present study was conducted to investigate two low-molecular-weight G. lucidum polysaccharides, which were purified from a G. lucidum
polysaccharide preparation (GLPP) obtained from our previous study [5]. Their structural features and physicochemical prop­erties were investigated. In addition, the antioxidant properties of low-molecular-weight polysaccharides were estimated as the scavenging capacities against hydroxyl, superoxide anion radi­cals and hydrogen peroxide, metal chelating ability, and reducing power.

2. Materials and methods

•2.1.    Materials

G. lucidum polysaccharide preparation (GLPP) was obtained from G. lucidum fruiting bodies by hot-water extraction as described previously [5]. Dextrans with different molecular weights were purchased from National Institute for the Control of Pharmaceutical and Biological Products, China. Ascorbic acid, hydrogen peroxide, chloride ferric, ferrous sulfate, and trichlo- racetic acid was purchased from Shanghai Chemical Reagent Company (Shanghai, China). Sodium salicylate, P-nicotinamide adenine dinucleotide (NADH), phenazine methosulfate (PMS), nitroblue tetrazolium chloride (NBT), and ethylenediaminete- traacetic acid disodium salt (EDTA-2Na) were purchased from Sigma-Aldrich (St. Louis, USA). All other chemicals and solvents were analytical grade and used without further purification.

•2.2.    General methods

The specific rotation was determined at 20 ± 1 °C with an automatic polarimeter (Model WZZ-2, China). Total carbohydrate content was determined by the phenol-sulfuric acid method as d- glucose equivalent [18]. Protein was analyzed by the Coomassie Brilliant G-250 method. The FT-IR spectrum was recorded on a Nicolet Impact 410 spectrophotometer with KBr pellets. 13C NMR was accomplished at 30°C in D2O on a Bruker AV-400 NMR spec­trometer.

Preliminary calibration of the column was conducted using dex­trans with different molecular weights. All data provided by the GPC system were collected and analyzed using the Workstation software package.

•2.5.    Monosaccharide composition analysis ofGLPL1 and GLPL2

Monosaccharide composition was analyzed according to a previously described procedure [19]. GLPL1 and GLPL2 were hydrolyzed with 2 M trifluoroacetic acid (TFA) at 100 °C for 8 h and converted to alditol acetates, which were analyzed by gas chro­matography (GC) on a Hewlett-Packard model 6890 instrument equipped with a capillary column of HP-5.5% phenyl methyl silox- ane (30 m x 0.25 mm x 0.25 |im) and a flame-ionization detector (Hewlett-Packard, Agilent Technologies, USA). The temperature of the column was held at 150 °C for 2min, increased to 220°C at a rate of 2 °C/min, and from 220 to 280 °C at a rate of 30 °C/min. d- mannose, D-glucose, D-galactose, D-fucose and D-rhamnose were used as standard sugars.

•2.6.    Methylation analysis ofGLPL1 and GLPL2

The polysaccharides (10 mg) were methylated four times by the method reported by Needs and Selvendran with minor modifica­tions [20]. The methylated products was depolymerized with 90% formic acid at 100 ° C for 6 h and further hydrolyzed with 2 M TFA at 100 °C for 2 h, respectively. The partially methylated residues were reduced and acetylated. The resulting products were analyzed by GC-MS. The GC temperature program was carried out as described previously [7]. Partially methylated alditol acetates were identi­fied by their fragments in EI-MS and by relative retention times in GC, and the molar ratios were estimated from the peak areas and response factors.

•2.7.   Assay for antioxidant activities ofGLPL1 and GLPl2

 •2.3.    Preparation of GLPLI and GLPL2

GLPP (5.0 g) was firstly applied to D301R macroporous adsorp­tion/ion exchange resin column (3.5 cm x 30 cm) to remove pigments and proteins, eluted with distilled water. The elution was then concentrated and loaded to a column (3.5 cm x 30 cm) of DEAE-cellulose-32 (disposed with sodium borate solution), eluted with distilled water for 500 mL at 16 mL/h, then eluted with 0-2 M NaCl aqueous solution for another 500 mL at 16 mL/h. The isola­tion process was monitored by determining the total sugar content using the phenol-sulfuric acid method. The distilled water eluted fraction and the major eluted fraction from the NaCl solution was further purified by gel-filtration chromatography on a column of Sepharcyl S-200 HR. Two single peaks were collected, tested for total carbohydrate by the phenol-sulfuric acid method, lyophilized to give two polysaccharides, named GLPL1 and GLPL2, respectively.

•2.4.    Purity and molecular weight determination

2.7.1. HO' scavenging activity estimation

HO* scavenging activity was measured according to a litera­ture procedure with a few modifications [21]. HO' were generated from FeSO4 and H2O2, and detected by their ability to hydrox- ylate salicylate. The reaction mixture (2.5 mL) contained 0.5 mL FeSO4 (1.5 mM), 0.35 mL H2O2 (6mM), 0.15 mL sodium salicylate (20 mM), and 1 mL of different concentrations of GLPL1 and GLPL2. Ascorbic acid was used as the positive control. After incubation for 1 h at 37 ° C, the absorbance of the hydroxylated salicylate complex was measured at 562 nm. The percentage scavenging effect was calculated as

(Ai - A2)

%HO* scavenged =

Ao

where Ai was the absorbance of the sample or ascorbic acid, and A0 was the absorbance of the solvent control, whereas A2 was the absorbance of the reagent blank without sodium salicylate.

 Purity and molecular weight determination of GLPL1 and GLPL2 was performed using a size-exclusion HPLC chromatogra- phy instrument (Agilent 1100, USA), samples (10 mg) was dissolved in distilled water (1 mL) and passed through a 0.45-|im filter, applied to a gel-filtration chromatographic column of BioSep- SCE-2000 (300 mm x 7.8 mm, Phenomenex, Torrance, Calif., USA), maintained at a temperature of 35 °C, eluted with the mobile phase which is pure water containing 0.7% Na2SO4 and 0.02% NaN3 at a flow rate of 0.5 mL/min and detected by a refractive index detector.

2.7.2. Assay of superoxide radical (O2'-) scavenging

Superoxide radical was generated in the PMS-NADH system containing 1 mL of 10 |M PMS, 1 mL of 100 |M NADH, 50 |L of 600 |M NBT in 0.1 M PBS at pH 7.8, and 1 mL sample or ascorbic acid solution or Tris-HCl buffer (for the control) as described previously [22]. The reaction was initiated by adding PMS. After incubated the mixture at room temperature for 5 min, the reaction was stopped by adding 50 | L 1 M HCl and the absorbance was measured at 560 nm against the blank. The capability of scavenging O2'- was calculated
as

%O2 scavenged =

where A0 was the absorbance of the control reaction (without sam­ple) and Ai was the absorbance of the sample or ascorbic acid reaction.

2.7.3. Fe2+-chelating activity assay

Fe2+-chelating activity of GLPlI and GLPL2 were measured according to a literature procedure with a few modifications [23]. Sample or ethylenediaminetetraacetic acid (EDTA) solution (1 mL) were mixed with 50 |L ferrous chloride (2mM) and 0.2 mL fer- rozine (5 mM), shaken well, allowed to stay still for 10 min at room temperature, and the absorbance of the mixture was determined at 562 nm. EDTA was included as a positive control. The ion-chelating activity was calculated as

(Ai - A2)

chelating rate =

A0

where A0 was the absorbance of the control (without sample) and A1 was the absorbance in the presence of the sample, A2 was the absorbance without ferrozine.

•2.7.4.    Reducing power assay

The reducing power was quantified following a method described earlier [24]. Briefly, 2.5 mL of GLPL1 or GLPL2 or ascor­bic acid solution in phosphate buffer (0.2 M, pH 6.6) was incubated with 2.5 mL potassium ferricyanide (1%, w/v) at 50°C for 20 min. The reaction was terminated by adding 2.5 mL trichloroacetic acid solution (10%, w/v). Then 5 mL distilled water and 1 mL ferric chlo­ride (0.1%, w/v) were added to the reaction mixture. The absorbance was measured at 700 nm. Ascorbic acid was used as the positive control. A higher absorbance of the reaction mixture indicates a stronger reducing power of the sample.

•2.7.5.    H2O2 scavenging activity assay

The H2O2 scavenging capacity was measured according to a reported procedure with minor modifications [25]. H2O2 (1.0 mL, 0.1 mM) and 1.0 mL of GLPL1 or GLPL2 or ascorbic acid were mixed, followed by adding 100 |L 3% ammonium molybdate, 10 mL H2SO4 (2 M) and 7.0 mL KI (1.8 M). The mixture was titrated by Na2S2O3 (5 mM) until the yellow color disappeared. The scavenging activity was calculated as

%H2O2 scavenged = (V°- V1) x 100%

where V0 was the volume of Na2S2O3 solution used to titrate the control mixture without sample, V1 was the volume of Na2S2O3 solution titrate the mixture containing sample.

2.8. Statistical analysis

Measurements were conducted in triplicate. Data were reported as mean ± SD for triplicate determinations. ANOVA and Tukey's tests performed (Minitab for Windows, Version 13, Minitab Inc., PA) to identify differences among means. Statistical significance was declared at P< 0.05.

 white powder, [a]D° = +15.0° (c 0.2, H2O), while GLPL2 appeared as an ivory white powder, [a]D0 = -37.1° (c 0.2, H2O). Small rota­tion values indicated both GLPL1 and GLPL2 contained P-anomeric configuration. Fig. 1 shows that they were eluted as single and sym­metric sharp peaks from gel-permeation chromatography (GPC) profile, which indicated they were homogeneous, respectively. The total carbohydrate contents of GLPL1 and GLPL2 were 99.8% and 99.1%, respectively. Reaction with Coomassie Brilliant G-250 was negative and no absorption at 280 or 260 nm in the UV spec­trum, indicating that GLPL1 and GLPL2 contained no protein or nucleic acid. The average molecular weights of GLPL1 and GLPL2 were determined as 5.2 and 15.4 kDa, respectively. Monosaccharide composition results showed that GLPL1 was composed of glucose, while GLPL2 contained glucose, galactose and mannose in the ratio of 29:1.8:1.0.

3. Results and discussion

3.1. Isolation, purification and composition ofGLPLI and GLPL2

After separation on DEAE-Cellulose-32 column and Sephacryl S-200 HR column, GLPL1 and GLPL2 were obtained with a yield of 61.53% and 30.47% from GLPP, respectively. GLPl1 appeared as a

3.2. Structural characterization of GLPLI and GLPL2

The IR spectra of GLPL1 and GLPL2 were basically indistinguish­able only by some difference in the intensity of bands (Fig. 2). Both IR spectra of GLPL1 and GLPL2 displayed a broad stretching intense characteristic peak at around 3400 cm-1 for the hydroxyl group, and weak C-H stretching band at 2924 cm-1. Three stretching

 peaks at 1043, 1074 and 1156 cm-1 suggest the presence of C-O bonds and pyranose ring in the monosaccharide in GLPL1 and GLPl2 [26]. The characteristic band at 897 cm-1 belonged to the P-anomeric configuration, which was in good agreement with the anomeric signals at around 5105 ppm in the 13C NMR spectrum (Figs. 2 and 3) [27].

Table 1

GC-MS results of methylation analysis of GLPl1 and GLPl2 purified from G. lucidum fruiting bodies.

Polysaccharide

Componentsa

trb

Mole

Linkage

 

 

 

percent (%)

 

GLPl1

2,3,4,6-Me4-Glc

1.00

6.4

Glcp-(1^)

 

2,4,6-Me3-Glc

1.13

21.9

^3)-Glcp-(1^

 

2,3,6-Me3-Glc

1.14

20.3

^4)-Glcp-(1^

 

2,3,4-Me3-Glc

1.17

23.7

^6)-Glcp-(1^

 

2,3-Me2-Glc

1.28

3.7

^4,6)-Glcp-(1^

 

2,4-Me2-Glc

1.30

24.0

^3,6)-Glcp-(1^

GLPl1

2,3,4,6-Me4-Glc

1.00

15.0

Glcp-(1^)

 

2,4,6-Mea-Glc

1.13

23.0

^3)-Glcp-(1^

 

2,3,6-Mea-Glc

1.14

34.6

^4)-Glcp-(1^

 

2,3,4-Mea-Glc

1.17

7.0

^6)-Glcp-(1^

 

2,3,4-Me3-Gal

1.21

3.3

^6)-Galp-(1^

 

2,3-Me2-Glc

1.28

3.0

^4,6)-Glcp-(1^

 

2,4-Me2-Glc

1.29

14.1

^3,6)-Glcp-(1^

 

Other

 

Trace

Terminal

a 2,3,4,6-Tetra-O-Me-Glc = 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucose, etc.

b Retention times ofthe corresponding alditol acetates compared to 2,3,4,6-tetra- O-methyl-D-glucose.

 

 The methylated results of GLPl1 and GLPl2 showed that they were complex polysaccharides. Both methylation results of GLPL1 and GLPL2 showed 2,3,4,6-tetra-O-methyl-glucose, 2,4,6-tri- O-methyl-glucose,2,3,6-tri-O-methyl-glucose,2,3,4-tri-O-methyl- glucose, 2,3-di-O-methyl-glucose, and 2,4-di-O-methyl-glucose were the main methylated sugar derivatives with a molar percent­age of 6.4:21.9:20.3:23.7:3.7:24.0, and 15.0:23.0:34.6:7.0:3.0:14.1, respectively, while GLPL2 contained another 3.3% component of 2,3,4-tri-O-methyl-galactose (Table 1). These results demonstrated that GLPL1 was a glucan consisting of (1 ^ 3)-linkaged glucosyl, (1 ^ 4)-linkaged glucosyl, and(1 ^ 6)-linkaged glucosyl backbones substituted with 24% branching at C-6 or C-3 and 3.7% branching at C-6 or C-4 [28]. Similar backbones were also investigated in GLPL2,
excepted GLPL2 contained a few (1 ^ 6)-linkaged galactosyls in backbones or in branches.

The signals from 13C NMR spectra of GLPL1 and GLPL2 were mainly distributed in the region between 106 and 60ppm, which also indicated GLPL1 and GLPL2 did not contain any uronic acids residues (Fig. 3a, b, A, and B). The 13C NMR spectrum of GLPL1 showed four anomeric signals at around 105 ppm: one at 105.4 ppm that corresponded to nonreducing end units, the one at 105.1 ppm could be assigned to ^3)-P-Glcp-(W and ^3,6)-P-Glcp-(W units while the one at 105.3 ppm was due to ^4)-P-Glcp-(1 ^ unit and ^4,6)-P-Glcp-(W and the other one at 104.9 ppm could be assigned to C-1 of ^6)-P-Glcp-(W unit (Fig. 3A) [26,28,29]. Sim­ilar anomeric signals were also observed in the anomeric region of GLPL2 (Fig. 3B), while there was an extra anomeric signal at 100.5 ppm which belonged to ^6)-a-Galp-(1 ^ unit [26]. The sig­nals at around 85-87 ppm (85.2, 86.9, and 87.5 ppm in GLPL1, 86.5, 86.7, and 87.1 ppm in GLPL2, Fig. 3A and B) were attributed to the C-3 of ^3)-p-Glcp-(W or ^3,6)-P-Glcp-(W units in differ­ent chemical atmospheres, while that at 77.4 ppm in GLPL1 and 77.1 ppm in GLPL2 corresponded to C-3 of the nonreducing end units [28]. The signals at 81.7 ppm in GLPL1 and 81.2 ppm in GLPL2 arose from C-4 of ^4,6)-P-Glcp-(W units, while that at 78.2 ppm in GLPL1 and 78.4 ppm in GLPL2 corresponded to C-4 of ^4)-P- Glcp-(W units [26,30]. Resonances at 68.8 or 69.5 ppm in GLPL1 and 68.5 or 69.1 ppm in GLPL2 arose from ^6)-P-Glcp-(W and ^3,6)-P-Glcp-(W or ^4,6)-P-Glcp-(W units, while the peak at 69.7 ppm in GLPL2 corresponded to the C-6 of ^6)-a-Galp-(W unit [26,28]. Those signals at 62.7 or 62.6 or 63.3 ppm should arise from nonsubstituted C-6. Other signals distributed at 72.2 and 75.7 ppm were attributed to C-2 or C-5 in different units [31]. These NMR data were in good accordance with the results of methylation analysis.

3.3. Antioxidant activity ofGLPLI and GLPL2

3.3.1. Scavenging activity of hydroxyl radical by GLPL1 and GLPL2

 Hydroxyl radical (HO*) can easily cross cell membranes, and can readily react with biomolecules including carbohydrates, proteins, lipids, and DNA in cells, and cause tissue damage or cell death. Thus, removing HO* is important for the protection of living systems [32]. As shown in Fig. 4, the ability of GLPL1 and GLPL2 to scavenge HO* was determined according to the method of Smirnoff and Cumbes [21], and compared with that of ascorbic acid, a known antioxidant compound. GLPL1 and GLPL2 could both scavenge HO*, and the maximum inhibitions of GLPL1 and GLPL2 were 78.3% and 53.6%, respectively. GLPL1 has a greater scavenging capacity than that of GLPL2 in all seven testing concentrations. However, ascorbic acid could exhibit 99.1% hydroxyl radical scavenging activity at a low concentration of 0.63 mg/mL.

•3.3.2.    Scavenging activity ofGLPLI and GLPL2 against superoxide radical

The superoxide radical (O2*-) is a highly toxic species could be generated by numerous biological and photochemical reactions. In addition to directly attack important biological molecules, O2*- may also decompose to form singlet oxygen and hydroxyl radicals, which may increase local oxidative stress and initiate cellular dam­age or lipid peroxidation and pathological incidents such as arthritis and Alzheimer's disease [33]. GLPL1 and GLPL2 were compared to ascorbic acid for their O2scavenging capacity. As shown in Fig. 5, at the test concentrations, both GLPL1 and GLPL2 exhibited scaveng­ing activity on superoxide radicals in a concentration-dependent manner except for the stable ability of GLPL1 at higher concentra­tions (6.0-10.0 mg/mL). As similar observation in hydroxyl radical scavenging assay, GLPL1 exhibited greater scavenging ability than that for GLPL2 while only 15% less compared to that for ascorbic acid at higher concentrations. It has been suggested that the over­all radical scavenging ability was related to the number of hydroxyl or amino groups in a polysaccharide molecule such as chitosan [34]. This might be explained by the factor that lower molecular weight for GLPL1 may provide more active hydroxyl groups than GLPL2 on a per same weight basis.

•3.3.3.    H2O2 scavenging activity of GLPLI and GLPL2

H2O2 is an oxidative agent and is involved in the formation of other ROS molecules such as hydroxyl radical when it reacts with Fe2+ or the superoxide anion radical. Although H2O2 is not very reactive, it is one of the major inducers for cellular aging and may attack many cellular energy-producing systems because of its high penetrability of cell membranes [25,35]. The H2O2 scavenging capacities of GLPL1 and GLPL2 are depicted in Fig. 6. The scavenging activity of GLPL1 and GLPL2 was 50% and 30% at the concentration of 8 mg/mL, respectively. Compared with GLPL1 and GLPL2, ascorbic acid was more effective for scavenging H2O2.

•3.3.4.    Ferrous metal ions chelating activity ofGLPL 1 and GLPL2

Various mechanisms including radical scavenging, binding of

transition metal ion catalyst(s), decomposition or reduction of per­oxides, and prevention of continued hydrogen abstraction, and induction of antioxidative enzyme activities have been proposed for the antioxidant activity of a chemical in biological systems [36]. Metal chelating capacity is important since it reduces the concen­tration of transition metals that may act as catalysts to generate the first few radicals and initiate the radical-mediated oxidative chain
reactions in biological or food systems. lon-chelating agents also may inhibit Fenton reaction and hydroperoxide decomposition. In the present study, the two purified polysaccharides GLPL1 and GLPL2 were compared to EDTA for their Fe2+-chelating capacity. GLPL1 showed moderate Fe2+-chelating activity at concentrations above 3.0 mg/mL, but at a level much lower than that of EDTA, while GLPL2 only showed weak chelating activity less than 22% even at a high concentration (Fig. 7). At a concentration of 1.5-10 mg/mL, the chelating ability ranged from 5.2% to 58% for GLPL1, while only from 4.2% to 21% of that for GLPL2. It is suggested that the chelating activ­ity may be related to the conformations of different polysaccharides [37]. The different conformations for GLPL1 and GLPL2 might be another factor to affect the chelating ability except the factor of different molecular weights.

3.3.5. Reducing power of GLPl1 and GLPL2

 

The reducing power assay measures the electron-donating abil­ity of antioxidants using the potassium ferricyanide reduction method. Fig. 8 compares the reducing power of GLPlI, GLPL2 and ascorbic acid. The reducing power of ascorbic acid was 0.805, and this value was comparable to that reported in a previous study [38]. Both GLPL1 and GLPL2 exhibited weak reducing power and slightly increased with the increasing concentration (Fig. 8). The maximum reducing power by GLPL1 was 0.242 and that by GLPL2 was 0.303 at the concentration of 10 mg/mL under the experiment conditions.

4. Conclusion

Two purified polysaccharides, GLPL1 and GLPL2, were obtained from crude G. lucidum preparation GLPP. GLPL1 was glucan while GLPL2 mainly composed of glucose with minor amounts of galactose and mannose. Both GLPL1 and GLPL2 were low- molecular-weight polysaccharides, with average molecular weight of 5.2 and 15.4 kDa, respectively. The results of structural char­acterization experiments showed GLPL1 and GLPL2 had similar glycosidic linkages composition such as ^3)-Glcp-(W, ^4)- Glcp-(W, ^6)-Glcp-(W, ^3,6)-Glcp-(W, and ^4,6)-Glcp-(W except GLPL2 had one more kind of linkage which is (^6)-Galp- (W). In vitro antioxidant activity study indicated GLPL1 exhibit greater capacity in scavenging free radicals and chelating Fe(ll), which may be related to its lower molecular weight. Besides, the antioxidant activity of polysaccharide is usually not influenced by one single factor but combined other factors. Therefore, further research is needed to elucidate the complete structure, conforma­tion and mechanism of antioxidant activity.

Acknowledgments

This research was supported by grants from the National Natural Science Foundation of China (Grant No. 30672479 and 30873201), a specialized Research Fund for the Doctoral Program of Higher Edu­cation (No. 20060316001) and a scholarship from State Scholarship Fund by the China Scholarship Council (No. 2008706001).

 References

 [1] Y. Sone, R. Okuda, N. Wada, E. Kishida, A. Misaki, Agric. Biol. Chem. 49 (1985) 2641-2653.

  • [2] R. Sullivan, J.E. Smith, N.J. Rowan, Perspect. Biol. Med. 49 (2006) 159-170.
  • [3] C.W. Huie, X. Di, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 812 (2004) 241-257.

•[4]   L. Ye, J. Zhang, Y. Yang, S. Zhou, Y. Liu, Q. Tang, X. Du, H. Chen, Y. Pan, Food Chem. 112(2009) 962-966.

  • [5] X. Pang, Z. Chen, X. Gao, W. Liu, M. Slavin, W. Yao, L.L. Yu, J. Food Sci. 72 (2007) S435-S442.
  • [6] H.S. Chen, Y.F. Tsai, S. Lin, C.C. Lin, K.H. Khoo, C.H. Lin, C.H. Wong, Bioorg. Med. Chem. 12(2004) 5595-5601.
  • [7] X. Bao, C. Liu, J. Fang, X. Li, Carbohydr. Res. 332 (2001) 67-74.
  • [8] S.K. Eo, Y.S. Kim, C.K. Lee, S.S. Han, J. Ethnopharmacol. 68 (1999) 175-181.
  • [9] Y.Q. Li, L. Fang, K.C. Zhang, Carbohydr. Polym. 68 (2007) 323-328.
  • [10] Y. Chen, M.Y. Xie, S.P. Nie, C. Li, Y.X. Wang, Food Chem. 107 (2008) 231-241.

•[11]   J.H. Park, H.W. Kim, Y.C. Kim, E.C. Choi, B.K. Kim, Korean J. Food Hyg. 2 (1987) 57-65.

  • [12] S.W. Seto, T.Y. Lam, H.L. Tam, A.L.S. Au, S.W. Chan, J.H. Wu, P.H.F. Yu, G.P.H. Leung, S.M. Ngai,J.H.K. Yeung, P.S. Leung, S.M.Y. Lee, Y.W. Kwan, Phytomedicine 16(2009) 426-436.
  • [13] J.M. Lee, H. Kwon, H. Jeong, J.W. Lee, S.Y. Lee, S.J. Baek, Y.J. Surh, Phytother. Res. 15(2001)245-249.

[1] Corresponding author. Tel.: +86 25 83271218; fax: +86 25 83271218.

[2] Corresponding author. Tel.: +86 25 83271298; fax: +86 25 83271249.

E-mail addresses: wbyao@cpu.edu.cn (W. Yao), xiangdong_gao@yahoo.com.cn (X. Gao).

0141-8130/$ - see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2010.02.006

 

 

 

Çin Tıbbında ilaçlar ve Kanser hastalarında biyomodilasyon

 INTEGRATIVE THERAPIES FOR ONCOLOGY

 

 ABSTRACT

Traditional Chinese Medicine (tcm) is a whole sys­tem containing therapeutic interventions that individu­ally induce biomodulation at the physiologic, chemi­cal, and molecular levels. The theory of tcm proposes a synergy between specific interventions selected as part of a care plan based on tcm diagnostic theory. Combining tcm with the modern practice of oncology seems, in conjunction with biomedical interventions (surgery, radiotherapy, chemotherapy, and pharmaceu­ticals), to have potential advantages through the synergy of biomodulation. Biomodulation approaches are broadly categorized as modification of tumour response and reduction of adverse effects; modulation of immu­nity; prevention of cancer progression; and enhance­ment of symptom control. Although the database of preclinical studies is rapidly expanding, good-quality clinical trials are notably scarce.

Laboratory studies suggest that some herbs increase the effectiveness of conventional chemotherapy with­out increasing toxicity. A healthy immune system is necessary for control of malignant disease, and the immune suppression associated with cancer contrib­utes to its progression. Many Chinese herbs contain glycoproteins and polysaccharides (among them, con­stituents of Coriolus versicolor, Ganoderma lucidum, Grifola frondosa, Astragalus membranaceus, Panax ginseng, and various other medicinal mushrooms) that can modulate metastatic potential and the innate im­mune system. Phytochemicals such as specific poly- saccharides have been shown to boost the innate im­mune system, especially through interaction with Toll­like receptors in mucosa-associated lymphoid tissue. This intervention can potentially improve the effective­ness of new anticancer vaccines. An increase in virus- associated cancers presents a major public health prob­lem that requires novel therapeutic strategies. A number of herbal therapies have both antiviral activity and the ability to promote immunity, possibly inhibiting the ini­tiation and promotion of virus-associated cancers.

The mechanisms learned from basic science should be applied to clinical trials both of specific interventions and of whole-system care plans that safely combine the tcm approach with the conventional biomedical model. In Western medicine, the combination of tcm herbs with drug therapies is controversial, given lack of knowledge concerning whether a drug is favourably enhanced or whether adverse effects occur. Using ini­tial data from the preclinical studies, future clinical research needs to evaluate the combinations, some of which are showing favourable synergy.

KEY WORDS

chinese medicine, herbs, acupuncture, supportive care, immunity, research

1. INTRODUCTION

Biomodulation is the reactive or associative adjustment of the biochemical or cellular status of an organism. Most modulation events describe an interaction in which a molecule (modulating entity) alters the ability of an enzyme to catalyze a specific reaction. In the context of cancer, biomodulation includes the use of a substance to augment the host's antitumour response, including immunotherapy. It encompasses the regula­tion of innate electrophysiologic, chemical, and mo­lecular pathways through relatively low-intensity physi­cal and chemical interventions. In contrast to conven­tional biomedicine-for example, pharmaceuticals- therapies such as herbs or their extracts are a mixture of chemicals administered at relatively low doses over a prolonged period of time. Acupuncture produces low- level electrochemical changes in the soft-tissue fas­cia. In tcm, the practice model includes the use of a diagnostic philosophy derived from cumulative clini­cal observation to target individual imbalances.

Copyright © 2008 Multimed Inc.

Current Oncology-Volume 15, Number 2

 

In Western medicine, the combination of tcm herbs with drug therapies is controversial, because of a lack of knowledge concerning whether the drug is favour­ably enhanced or whether adverse effects occur. Using initial data from preclinical studies, future clinical re­search has to evaluate the combinations, some of which are showing favourable synergy. Both parts of this arti­cle deal with examples of biomodulation and the results of combining tcm with biomedicine. Part one discussed
the broad principles of tcm; part two discusses the po­tential clinical applications of tcm in oncology.

2. ROLES OF TCM IN BIOMODULATION

The goals of cancer treatment should be to increase survival (when possible) and to improve quality of life for patients. Traditional Chinese Medicine is able to support patients being treated with conventional West­ern medicine (surgery, radiotherapy, and chemotherapy) through four approaches:

  • 1. Modification of tumour response and reduction of adverse effects
  • 2. Modulation of immunity
  • 3. Prevention of cancer progression
  • 4. Enhancement of symptom control

Very often, tcm therapy works through more than one approach synergistically.

2.1 Modification of Tumour Response and Reduc­tion of Adverse Effects

2.1.1 Tumour Physiology

Evidence increasingly suggests that tcm can favour­ably modify the tumour response to conventional West­ern cancer treatment. There is a correspondence be­tween the tcm theory of cancer and recent medical research findings. In tcm, the malignant tumour is viewed as being associated with stagnation of qi (en­ergy) and blood. Qi may be viewed as a model for intracellular and intercellular information and poten­tial energy transfer. That definition would correlate with the known abnormalities of signal transduction, cell contact, and electrophysiology of cancer cells 1-3.

Increased fluid content and a stagnant blood sup­ply have been demonstrated in malignant tumours 4-6. The microcirculation within a tumour is very abnor­mal, and there are regions within the tumour where the blood flow is sluggish. In tcm, stagnation of blood is classically associated with tumours. The impaired blood circulation leads to areas of poor oxygenation in the tumour. cancer cells that survive in an environ­ment of low oxygen tension are also found to be more resistant to radiotherapy and to some types of chemo­therapy 78. Interestingly, the use of anticoagulants such as heparin and coumadin (warfarin) as adjunctive treat­ment with chemotherapy has been shown in laboratory studies in animals to prevent the development of blood- borne metastases and in clinical studies to improve the survival of cancer patients 910. Destagnation or de­toxification herbs are used to move the blood and qi within a malignant tumour. Many of these herbs are proving to be anti-angiogenic agents 11.

Herbs from tcm have been extensively investigated in the laboratory and are known to have multiple phar- macologic effects 11-17. Specifying the botanical parts from which the herbal agent is prepared is important, because the active pharmacologic agents depend on their source: "Radix" (Rx) denotes the root; "Cortex" (Cx), the bark or rind; and "Rhizome" (Rh), the rhi­zome. Many examples of anticancer therapeutic multi­plicity are available:

  • Rx Ginseng has antitumour activity and inhibits platelet aggregation and chemotherapy-induced immunosuppression.
  • Glycyrrhizic acid has antitumour activity, acts as an anti-inflammatory by increasing serum corti­sol, and also increases natural killer (nk) cell ac­tivity against cancer cells.
  • Rx Astragalus membranaceus is a powerful stimu­lator of the immune system, has anti-tumour ac­tivity, and inhibits platelet aggregation.
  • Rx Angelica sinensis stimulates the immune sys­tem, has antitumour activity, inhibits platelet ag­gregation, and inhibits vascular permeability.
  • Rh Atractylodis macrocephala has antitumour ac­tivity and acts as an anti-thrombotic and fibrino­lytic agent.
  • Ginkgo biloba has multiple effects, including inhibi­tion of platelet activation factor; stimulation of the immune system, fibrinolysis, and anti-thrombosis; scavenging offree radicals; and dilation ofblood ves­sels to increase perfusion.

As more is learned about the interactive roles of bone marrow, hematopoietic system, and angiogen- esis in the progression of cancer, the foregoing ef­fects of herbs on the hemostatic coagulation system are interesting 11.

The possible usefulness of destagnation herbs was demonstrated in a randomized controlled clinical trial (rct) evaluating the combined-modality treatment of a Chinese herbal destagnation formula and radiotherapy in patients with nasopharyngeal carcinoma 18. In that trial, 90 patients who received combined herbal and radiation treatment were compared with 98 patients who were randomized to receive radiation treatment alone. The ingredients of the herbal formula included Rx Astragalus membranaceus, Rx Paeoniae rubrae, Rx Ligustici chuanxiong, Rx Angelica sinensis, Semen persica, Flos Carthami tinctorii, Rx et Caulis jixueteng, Rx puerariae, Pericarpium citri reticulatae, and Rx Codonopsitis pilosulae. As compared with the group treated with radiation alone, the combined-treatment group showed a statistically significant increase in lo­cal tumour control and overall 5-year survival. The rate of local recurrence in the intervention group was halved from 29% in those receiving radiation alone to 14% in the group receiving destagnation herbs as well. The 5-year disease-free survival was increased from 37% in the control group to 53% in the group receiving des­tagnation herbs.

It is postulated that the tested herbal destagnation formula may have improved tumour microcirculation and increased tumour blood flow, leading to an improvement in the oxygen tension inside the tumour. Improved oxy­gen tension increases the radiosensitivity of the tumour. In other words, the destagnation formula acted as a radiation sensitizer.

In animal experiments, Ginkgo biloba was also shown to increase perfusion and radiosensitivity 19,20 Chinese herbs such as Salviae miltiorrhizae, which inhibits tumour edema caused by free radicals, may also increase tumour perfusion, oxygenation, and re­sponse to radiotherapy 21,22. Other herbs may directly sensitize neoplastic cells to radiotherapy 23.

Some herbs may protect normal tissues from ra­diotherapy. For example, Panax ginseng and Panax quinquefolium water extract (Rh2 ginsenoside) exer­cise radioprotection through mechanisms involving anti- oxidative and immunomodulating properties 24. The subtle balance between anticancer effects and protec­tion of normal tissue-termed "therapeutic gain"-is not currently understood.

The tcm herbs contain a variety of chemicals that may act synergistically to inhibit tumour cell division, to increase tumour cell death (apoptosis), to increase the proportion of immune cells within the tumour, and to increase blood flow through the tumour. These changes are associated with a change in the balance of cytokines and other communicating peptides released by the immune cells, resulting in a reduction in the proliferation of tumour cells and an increase in tumour cell death, and in the minimization of many side ef­fects for normal tissues. This synergy appears to be secondary to induction of apoptosis, anti-angiogenesis, antagonism of the viral genome, and induction of an immune response.

Some herbs can reverse multi-drug resistance 25. Extracts of multiple Chinese herbs traditionally used for anticancer therapy (for example, Anemarrhena asphodeloides, Artemisia argyi, Commiphora myrrha, Duchesnea indica, Gleditsia sinensis, Ligustrum luci- dum, Rheum palmatum, Rubia cordifolia, Salvia chinensis, Scutellaria barbata, Uncaria rhynchophylla, and Vaccaria segetalis) demonstrate growth-inhibitory activity against various cancer cell lines, but limited inhibitory activity against normal cell proliferation 17 Huanglian (Coptidis rhizoma) induces cell-growth ar­rest and apoptosis by upregulation of interferon-^ and tumour necrosis factor a (TNFa) in human breast can­cer cells 26.

Recent meta-analyses confirmed the utility of Chi­nese herbs both to enhance control of particular cancers (particularly viral-induced cancers such as hepatocellu- lar carcinoma and nasopharyngeal cancers) and to re­duce side effects of chemotherapy 27,28. Laboratory stud­ies suggest that some herbs increase the effectiveness of conventional chemotherapy. For example, red ginseng acidic polysaccharide increases the cytotoxicity of paclitaxel 29, and Phellinus linteus enhances the cytotox- icity of doxorubicin 30. A meta-analysis of Astragalus- based Chinese herbs and platinum-based chemotherapy for advanced non-small-cell lung cancer indicates a prom­ising therapeutic gain 31.

Occasionally, herbs alone are associated with tu­mour regression. For example, a 51-year-old woman with pathology-proven squamous cell carcinoma of the lung attained complete regression with the use of a com­bination of herbs as sole treatment (Herba Hedyotis dif- fusae, Rx Ophiopogonis, Herba Taraxaci, Rx Notoginseng, Pseudobulbus cremastrae seu pleiones, Rx Panacis quin- quefolii, Herba Houttuyniae, Bulbus Fritillariae thunber- gii, Rh Pinelliae preparata) 32. This anecdotal report is unusual, but deserves further exploration.

More clinical trials are required to further evaluate the promising role for herbs in potentially improving therapeutic gain.

2.1.2 Hormone Effects

Phytoestrogens that possess either estrogenic or anti- estrogenic activity are found in some botanical supple­ments. Controversy has arisen concerning these substan­ces for patients with hormone-responsive cancers who could theoretically deteriorate as a result of hormone- enhanced cancer progression. Angelica sinensis (dong quai), Glycyrrhiza glabra (liquorice), and the various ginsengs are cited in this category. However, in vitro and in vivo models of estrogenic activity have produced conflicting data. clinically, the substances appear to serve as chemopreventive agents while also being capable of promoting growth in some estrogen receptor-positive cell lines. In addition, they may exert their estrogenic influence through either or both of receptor-dependent and receptor-independent mechanisms 33-35. Other studies demonstrate inhibition of breast cancer cell models, particularly by ginseng and its extracts 36-41. Piersen reviewed the conflicting data in detail 42

Test-tube assays have limitations. They ignore is­sues related to metabolism and cannot address bio- availability. Phytoestrogens usually demonstrate higher activity in vitro than in vivo. In animals, route of ad­ministration and interspecies variability in metabolism produce unreliable results for the human situation. Hu­man studies are confounded by the composition of gut flora, intestinal transit time, the redox potential of the colon, and genetic differences in metabolism. In addi­tion, herbs are not single-entity drugs. Each is a com­plex mixture of hundreds of compounds that may exert their biologic activity alone or in synergy with other compounds, with multiple targets of action 43. Even if a phytoestrogen compound is present, other compounds may counteract its effect.

In vitro studies of Angelica sinensis show weak agonist activity on McF-7 breast cancer cells 44, but human studies do not support an estrogenic mechanism of action. Indeed, although the proposed main utility of Angelica sinensis is to treat symptoms of menopause, a double-blind rct showed no significant reduction in the relief of such symptoms, in endometrial thickness, or in vaginal maturation index 45.

The evidence for proestrogenic activity of the ginsengs against breast cancer is extremely weak and derives from two in vitro studies on MCF-7 cells 33,35. A study of 20(S)-protopanaxadiol, a major gastro­intestinal metabolite of the ginsenosides, suggests that it inhibits estrogen-stimulated gene expression in McF-7 estrogen receptor (ER)-positive breast cancer cells, inhibits xenograft growth, and enhances the cytotox- icity of tamoxifen in an ER-independent fashion 39. The Rh2 ginsenoside extract hypersensitizes multi- drug-resistant breast cancer cells to paclitaxel 37. The tumoricidal effect of cisplatin on MCF-7 cells is not attenuated by American ginseng (Panax quinquefolius L.) 41. Water-extracted compounds of Panax quin­quefolius L. inhibit MCF-7 cell proliferation by inhibit­ing mitogen-activated protein kinase 40. A combina­tion herbal formula of ginseng and Carthami tinctorii inhibits a breast cancer cell line through apoptosis 38.

Laboratory and epidemiologic data indicate that whole ginseng has anti-proliferative activity 37,46,47, and a clinical study reported improvement in quality of life without increase in cancer recurrence 48. An abstract from the Mao clinic (Rochester) presented at the 2007 American Society of Clinical Oncology meeting reported a phase ii rct of North American ginseng (Panax quinquefolium) for cancer patients suffering from fatigue, demonstrating an up to 40% reduction in fatigue in patients on the highest dose level. The cohort included breast cancer patients, and no adverse effects were reported 49. The safety aspects of ginseng in patients with hormone-responsive can­cers are important because of emerging clinical evi­dence for the anti-fatigue and immunogenic properties of this herb.

The pc-spes herbal combination has partial estro- genic activity associated with activity against pros­tate cancer. One study correlated laboratory activity with clinical response 50. On the basis of those find­ings, a U.S. National Cancer Institute (nci) rct was initiated. Unfortunately, the clinical trial was termi­nated when a batch of pc-spes was found to be con­taminated with the hormone diethylstilbestrol and other pharmacologic agents. It is not certain whether the contamination was accidental or the result of deliber­ate adulteration 51. However, there is evidence that the combination was more effective than diethylstilbes- trol alone 52.

Soybeans contain genistein, which is an isoflavone with multiple anticancer effects demonstrated in the laboratory 15. These include the induction of tumour cell death through the process of apoptosis, inhibition of cancer cell proliferation by a decrease in the avail­ability of sex hormones, inhibition of angiogenesis, in­hibition of tyrosine kinase (involved in intracellular sig­nalling from the membrane to the nucleus), and inhibi­tion of platelet aggregation 53-55. Some epidemiology studies suggest that populations with a high soy or tofu content in their diet may have a reduced risk of breast cancer 56-58; other studies have not been able to con­firm this link 59. Animal studies suggest that exposure to soy during early life may alter differentiation of breast cells in a way that protects them against later assault by carcinogenic agents 60. That finding would imply that soy protects against breast cancer only if regularly ingested before menarche. Some reports suggest that phytoestrogens contained within soy may reduce the symptoms ofhot flashes associated with chemotherapy- induced menopause 61; however, most rcts do not sup­port that hypothesis 62,63. Practitioners should be cau­tious in treating patients with ER-positive breast can­cer, especially those on estrogen antagonist therapy. The use of soy isoflavones to promote health in breast cancer survivors remains controversial because of scant scientific data 64,65.

Limited evidence suggests that acupuncture di­rectly influences hormone levels. There is weak evi­dence that it modulates hormones such as melatonin 66 and corticotrophin-releasing factor 67. Its effect is likely to occur via the central nervous system and the pineal and pituitary glands. A nonrandomized human study reported that acupuncture induced melatonin and was associated with improved sleep 66

2.2 Modulation of Immunity

2.2.1 Herbs

Another strategy that tcm uses in cancer therapy is to strengthen the whole body-mind system by enhanc­ing and harmonizing the energy balance between all the organs. This approach may be viewed as correct­ing an imbalance in the body-mind communication network-an intervention that is reflected in an enhan­cement in immunity. This "Fu Zheng treatment" is mediated by the specific group of tcm herbs called Fu Zheng herbs 68-78. There is some limited evidence that improvement in the immunologic function of can­cer patients is associated with an improvement in their survival. In China, Fu Zheng herbs have been reported to increase survival when combined with radiotherapy for patients with nasopharyngeal cancer 79 and when combined with chemotherapy for patients with stom­ach and liver cancer 1,80, but the clinical evidence is weak because of a lack of rcts.

Fu Zheng herbs, including Rx ginseng, Ganoderma, Rx Astragalus membranaceus, Rx Angelica sinensis, Cordyceps sinensis, and Fructus Lycii, have been shown to enhance the body's defence mechanisms. clinical studies, including two randomized trials, have found that cell counts of nk cells and okt4 (immune- enhancing) lymphocytes were increased with the use of Fu Zheng herbs 68-78. These immunocytes are known to attack cancer cells. In a study of gastric cancer pa­tients, increased survival was found in the combined- treatment group (receiving both Fu Zheng herbs and chemotherapy) as compared with the chemotherapy- alone group.

Many of the Fu Zheng herbs are associated with an increase in cytokines, such as interferon and interleukin (il) 81-83. Chinese studies also suggest that healing of normal tissues may be enhanced. Anti- inflammatory constituents may diminish radiation-in­duced ulcers and chemotherapy-induced stomatitis 84,85. These studies still need to be verified in the West, using acceptable standards and quality assurance.

Recently, the concept of immune enhancement gained new ground with the discovery that cytotoxic therapies and cancer both suppress immunity, and that low immune levels may increase the probability of relapse. In addition, an intact innate immune system is necessary for activity of the new cancer vaccines. The interaction of host immunity with the natural his­tory of cancer is suggested by Burnet's immune sur­veillance theory, by the fact that immunodeficiency diseases are associated with an increased risk of can­cer, and by the fact that immune-enhancing therapies in malignant melanoma and renal cell carcinoma have produced antitumour responses. There is evidence that the healthy immune system is necessary for the con­trol of malignant disease and that the immune sup­pression associated with cancer contributes to disease progression.

Natural immune mediators are implicated in re­sistance against tumour development 86. Adaptive im­munity is often suppressed in tumour-bearing hosts, and specially designed agents are required to boost this defence 87. Hormonal manipulation of the host can re­sult in the elevation of immune defences against can­cer. Such manipulation strengthens both the adaptive and natural immune defences of the host, both of which play significant roles. cytokines and hormones boost natural defence mechanisms during febrile reactions, which are now known as the acute-phase response. Hormonal stimulation of immune mechanisms, cou­pled with other immunostimulants, may be employed to good advantage for the combination immunotherapy of cancer.

Many chinese herbs contain glycoproteins and polysaccharides that can modulate metastatic potential and the innate immune system. Metastasis of malig­nant tumours may be a specific receptor-mediated proc­ess in which organ-specific lectins play a role in the adhesion of disseminated tumour cells. Glycoprotein- mediated membrane identity is part of the human leu­cocyte antigen histocompatibility system. The abnormal carbohydrate group on the tumour cell could have formed during malignant transformation. The metastatic tumour cell, with its membrane-associated glycoprotein (often identical with the tumour marker) is recognized by organ-specific lectins as belonging to the organ, and is thereby captured. In vitro experiments show that galactoglycoconjugates can inhibit the adhesion of tu­mour cells to hepatocytes 88.

Immune suppression in cancer contributes to pro­gression and relapse 86,89-97 Multiple strategies for identifying candidate tumour antigens currently exist, and more is now understood about activation and regu­lation of immunity against cancer. Vaccines can target tumour-specific antigens, but adjuvants are required to boost the innate immune response, especially in patients who already have depressed immunity from tumour- derived signalling molecules and the effects of cyto­toxic therapies 98-100.

Phytochemicals such as specific polysaccharides have been shown to boost the innate immune sys­tem, especially through interaction with Toll-like re­ceptors (tlrs) in mucosa-associated lymphoid tis­sue (malt) 101-103. The tlrs evolved to interact with polysaccharides found in the walls of bacteria; they are an essential part of developing and maintaining a competent immune system 104. Polysaccharide extracts and complexes from chinese medicinal herbs and mushrooms may have a potential role in enhancing in­nate immunity. Clinical trials have provided some evi­dence that they can improve survival 105. The polysac­charide complexes and extracts include constituents of Coriolus versicolor (whose extract is called Krestin, psk, or psp) 106-118, Ganoderma lucidum 119-121, Grifola frondosa (maitake MD-fraction) 122-127, Astra­galus membranaceus 128, Panax ginseng 129-132, and various other medicinal mushrooms 133-135.

Molecular mechanisms for the immunobiologic functions may act through various receptors on mac­rophages, monocytes, and nk cells, which activate sec­retion of nuclear factor kb and antitumour cytokines. Interactions may include complement receptor type 3, cd14, mannose, and beta-glucan receptors. There is evidence that polysaccharides derived from Astragalus membranaceus, Acanthopanax senticosus and koreanum, Ganoderma lucidum, and Platycodon grandiflorum 136,137 interact with tlrs (especially tlr4).

Regulatory T cells (Tregs) and myeloid suppres­sor cells inhibit the anticancer activity of nk and T- helper cells and are partly responsible for tumour pro­gression, resistance to chemotherapy, and ineffective antitumour vaccines. Enhancement of innate immu­nity seems to improve anticancer therapies. Tregs are characterized by cd25 and FoxP3 expression. Their normal role is to control the adaptive immune response through cell contact-dependent mechanisms. The in­terplay between Tregs and antigen-responsive T cells is modulated by dendritic cells (dcs): whereas imma­ture myeloid precursors of dcs suppress T-cell activa­tion and induce Treg development, mature monocytes (macrophages) override Treg-mediated suppression. Mature dc macrophages can be activated through the tlr pattern-recognition receptors found on monocytes in the gastrointestinal tract. They then secrete il-6, which renders T-helper and nk cells refractory to the suppressive effect of Tregs 138.

Other studies have shown that elimination of Tregs can significantly improve the outcome of cancer im- munotherapy in preclinical models. For example, Sutmuller et al. 139 showed that therapeutic whole- cell vaccination against melanoma was significantly more effective upon depletion of cd4+cd25+ Tregs with an anti-cD25 monoclonal antibody. Unfortunately, they also showed that Treg depletion with anti-cD25 antibody carries an inherent risk of depleting tumour- specific effector cd4+ (and possibly cd8+) T cells, thus negatively affecting treatment efficacy. Myeloid sup­pressor cells may have additional properties that can compromise anticancer therapies, such as promotion of angiogenesis 140. Specific cytokines also play a role in immune suppression. The cytokines il-13 and il-4 suppress nk T cell immunosurveillance 141.

Tregs that suppress immune responses may limit the efficiency of cancer immunotherapy. Recent find­ings indicate that tlrs directly regulate the suppres­sive activity of Tregs. Linking tlr signalling to the functional control of Tregs may offer new opportuni­ties to improve the outcome of cancer immunotherapy by co-administration of certain tlr ligands 142. Stimu­lation by tlr blocks the generation of dcs from pro­genitor cells and diverts them to mature macrophage monocytes. That effect is achieved by inhibition of granulocyte-macrophage colony-stimulating factor sig­nalling through the induction of SOCS1.

Microbial ligands are able to skew the dichotomy of macrophage versus dc differentiation from com­mon progenitors. in uninflamed tissues, granulocyte- macrophage colony-stimulating factor induces the gen­eration of immature dcs, preparing the host to sense infectious danger. However, during infectious inflam­mation, tlr stimulation drives incoming monocytes to behave more like macrophages than to differentiate into dcs. That mechanism could be of help for the di­rect antimicrobial defence, which is more effectively mediated by macrophage-like cells with a high capac­ity to phagocytise. Pre-existing resident dcs are suffi­cient to perform the task of antigen sampling and trans­duction of information to the adaptive immune system. Thus, tlr stimulation would guide the innate immune system to assure a sufficient supply of phagocytic cells in inflamed tissues 143.

Garay 144 reviewed the potential benefits of tlr agonists when added to chemotherapy tlr2/4 agonists to induce well-controlled TNFa secretion at plasma levels known to make neoangiogenic tumour vessels permeable to the passage of cytotoxic drugs. Moreo­ver, tlr2/4 agonists induce expression of inducible nitric oxide synthase, and nitric oxide is able to indu­ce apoptosis of chemotherapy-resistant tumour cell clo­nes. Finally, tlr2/4 stimulation activates dendritic cell traffic, macrophage production, and cytotoxic T-cell responses.

Breast cancer patients have increased levels of Tregs 145. Vaccine peptides need to be combined with strong adjuvants, such as tlr agonists. A peptide vac­cination strategy that incorporates a tlr agonist could prevent the occurrence of spontaneous breast tumours. Transgenic mice that carry the activated rat epidermal growth factor receptor HER2/neu oncogene were vacci­nated with a synthetic peptide from the rat HER2/neu gene product in combination with a tlr agonist adjuvant. The results show that, to obtain tumour antigen-specific

T-lymphocyte responses and antitumour effects, the function of cd4/cd25 Tregs had to be blocked with anti-cD25 antibody therapy. Mice that were vaccinated using this approach remained tumour-free or were able to control spontaneous tumour growth; they also exhibited long-lasting T-lymphocyte responses. The results suggest that similar strategies should be fol­lowed for conducting clinical studies in patients 146

The polysaccharide beta-glucans stimulate leu­kocyte anti-infective activity and enhance (murine) hematopoietic activity. In a study of human bone mar­row mononuclear cells, PGG-glucan acted on commit­ted myeloid progenitors to enhance activity by direct action independent of il-3 147. Beta-glucans and poly­saccharides are potent stimulators of tlr. Some spe­cific polysaccharides have already been shown to boost the innate immune system through interaction with

TLRs in MALT 101-103.

Polysaccharide extracts and complexes from Chi­nese medicinal herbs and mushrooms seem to have a potential role for enhancing innate immunity. There is some evidence from clinical trials that they can im­prove survival 105. Polysaccharide extracts from Panax ginseng can increase immunity and enhance chemo­therapy 129-131. There is evidence of interaction with tlrs, especially tlr4 136,137,148.

Maitake D-Fraction, a polysaccharide extracted from maitake mushrooms (Grifola frondosa), has been reported to exhibit an antitumour effect through acti­vation of immunocompetent cells, including macro­phages and T cells, with modulation of the balance between T-helper 1 and 2 cells. It can lower the dos­age of mitomycin chemotherapy that is effective in tumour-bearing mice by increasing the proliferation, differentiation, and activation of immunocompetent cells 123.

Further evidence of the potential usefulness of polysaccharides in stimulating an enhanced immune response comes from a study of orally administered beta-glucans (from maitake mushrooms) that demon­strated an enhancement of the antitumour effects of monoclonal antibody-targeted therapies 149. A meta­analysis of another immune-enhancing botanical, As­tragalus, reported an enhancement of the efficacy of platinum-based chemotherapy for lung cancer 31 and psk (Coriolus versicolor) for the enhancement of tegafur for colorectal cancer 108.

Immunosuppression in cancer patients can reduce the efficacy of anticancer vaccines and increase com­plications from opportunistic infections. Polysac- charides (mainly beta-D-glucans alone or linked to pro­teins) from the cell walls of various traditional chi­nese medicinal mushrooms and plants show antitu- mour and anti-infective activities through activation of monocytes, macrophages, and nk cells. A future research strategy should authenticate the source of these polysaccharide extracts and screen them for in­teraction with tlrs in the gastrointestinal tract of animals. Oral agents that boost cell-mediated immunity through malt may be subsequently evalu­ated in human phase i studies for dose-response (cytokine and immune cell assays) and safety.

Optimized, authenticated polysaccharides may play a role in enhancing the potency of anticancer vaccines and other therapeutic modalities. These non-cytokine molecules appear to signal primarily through the tlrs, which are expressed by dendritic cells. In malt, these agonists can induce a host of pro-inflammatory cytokines such as TNFa, il-12, and il-6, as well as cd4+ and cd8+ T cells.

Combining radiation therapy and tlr agonists may reduce the amount of radiation therapy required to eradicate tumours, with the tlr agonists thus acting as immunosensitizers 150,151. Evidence of the poten­tial usefulness of polysaccharides in stimulating an enhanced immune response is strengthened by the study of orally administered beta-glucans (from maitake mushroom) that showed enhancement of the antitumour effects of targeted monoclonal antibodies 149. Ganopoly (a Ganoderma lucidum polysaccharide extract) modu­lated immune function in advanced-stage cancer pa­tients. Treatment for 12 weeks resulted in a significant increase in the mean plasma concentrations of IL-2, il-6, and interferon-y, and a decrease in il-1 and TNFa. Activity of nk cells increased, but no significant change was observed in the levels of cd4+ or cd8+, or in the cd4+/cd8+ ratio 122

Lymphoproliferative neoplasms, such as lympho­mas and leukemias, may be particularly sensitive to changes in cytokine balance. The Memorial Sloan- Kettering Cancer Center in New York has commenced an NCI-sponsored phase i study of beta-glucan and rituximab in pediatric patients with relapsed or pro­gressive cD20-positive lymphoma or leukemia 152

The evidence indicates that a healthy immune sys­tem is necessary for the control of malignant disease and that the immune suppression associated with can­cer contributes to disease progression. Tumours have developed strategies to successfully evade the host immune system, and various molecular and cellular mechanisms responsible for tumour evasion have been identified. Some of these mechanisms target immune antitumour effector cells. Dysfunction and apoptosis of those cells in the tumour-bearing host creates an immune imbalance that cannot be corrected by tar­geted immunotherapies alone. Reversal of existing immune dysfunction and normalization of lymphocyte homeostasis in patients with cancer needs to be a part of future cancer immunotherapy 86. Therapeutic strat­egies are being designed to correct the immune im­balance, to deliver adequate in vivo stimulation, to transfer effector T cells capable of in vivo expansion, and to provide protection for the immune effector cells repopulating the host. Survival of these cells and long- term immune "memory" development in patients with malignancy are necessary for improving the clinical benefits of cancer immunotherapies. Polysaccharides derived from chinese herbs and mushrooms are emerging agents that seem to enhance cytotoxic drugs, radiotherapy, surgery, and the newer targeted thera­pies and vaccines 31,105,153. Rigorous authentication and quality control of these phytoceuticals are neces­sary before clinical studies begin 43.

2.2.2 Acupuncture

Multiple animal and clinical studies have also suggested that acupuncture has a positive immune-modulating effect in cancer patients 154-162. In those studies, acupuncture has been shown to increase T-lymphocyte proliferation and nk cell activity, to activate the com­plement system and heat-stable mitogenic humoral factor, and to increase okt4 cell counts. Inhibition of the growth of transplanted mammary cancer has also been shown in mice with the use of acupuncture. The main acupoints that were used in these studies were those that support blood formation and spleen function. These points include LI4, LI11, St36, Sp6, Sp10, P6, UB20, GB39, and GV14. An increased level of all components (red blood cells, white blood cells, and platelets) was found.

Lu et al. recently reported an exploratory meta- analysis of the clinical trials 163. The trials from the chinese journals suffered from low quality and biases. An analysis of a small set of seven trials did not find statistical significance in publication bias. The hetero­geneity was explained by the varying treatment char­acteristics. The frequency of the acupuncture was once daily, with a median of 16 sessions. Use of acupunc­ture was associated with an increase in leukocytes during chemotherapy, with a weighted mean difference of1221 cells/|L (95% confidence interval: 636 to 1807; p < 0.0001).

2.3 Prevention of Cancer Progression

In china, a high incidence of chronic viral infections results in cancers. cancer sites include liver, stomach, esophagus, and nasopharynx. In addition, cervix can­cer has increased to become the second most common cancer in women. The cause of the relatively higher incidence of virus-associated cancers as compared with the West is unclear. Factors include spread of infec­tion, genetic predisposition, poor diet, and smoking. An inadequate response from the immune system to eradi­cate chronic viral infections and cancer cells is a com­mon determinant. The total number of new cases of cancer was expected to increase by almost 15% by 2005. The increase in virus-associated cancers presents a major public health problem that requires more data directed at developing novel therapeutic strategies based upon local evidence-based remedies.

Hepatocellular carcinoma (hcc) is ranked second in cancer mortality in china, and the disease is now also increasing in frequency in men in many other coun­tries. Hepatitis B (hbv) and C (hcv) viruses remain the major causative factors, and the hepatitis G virus and other transfusion-transmitted viruses cannot be excluded 164. Infection with hbv is nearly ubiquitous in hcc patients in China. The tight association of hbv with hcc strongly suggests the dominant role of hbv infection in causing the cancer. Almost 12% of hcc patients have co-infection with hbv and hcv 165. A meta-analysis concluded that hbv and hcv infections are important independent risk factors for hcc, and that dual infection with hbv and hcv is associated with a higher risk for hcc than is either infection alone, suggesting a synergism between the two 166

Nasopharyngeal cancer is associated with Epstein- Barr virus infection 167,168. It may also be associated with some cases of Hodgkin lymphoma 169 and gastric cancer 170. Its highest incidence is in the Southern Chinese population, with familial aggregation. The annual detection rate is 433 per 100,000 for men and 499 per 100,000 for women in high-risk families, as compared with an average overall annual incidence in Hong Kong of 24 per 100,000 for men and 10 per 100,000 for women 171.

The incidence of cervix cancer is increasing. The combination of human papilloma virus (hpv) infec­tion and cigarette-smoking is synergistic for the in­duction of cervix and anal cancers. In the West, the subtype hpv16 is the most common cause; in China, other types of hpv viruses (for example, hpv18 and 59) are more commonly associated with the disease. The hpv16 E7 variant protein may induce a host hu­moral immune response, but not a special cellular immune response 172. The association is strengthened in smokers. Inducing an appropriate cell-mediated immune response may be key to eradicating the virus and its potential to induce and promote cancer. The hpv16 virus is also a major factor in the development of esophageal cancer in China, but not yet in the West 173,174 In addition, the West and China are both ex­periencing an increase in Hpv-associated head and neck cancers 175,176. Another major concern is a rise in breast cancer in women. Although a high-fat diet, less exercise, and reduced parity are contributing fac­tors, infection with the hpv33 virus appears to play a role in China 177.

Most evidence for successful herbal treatment of cancers comes from case reports or case series that may be biased by selection or spontaneous re­mission 178; however, a meta-analysis of existing rcts provided promising evidence that combining chinese herbal medicine with chemotherapy may ben­efit patients with hcc 28. A major weakness of the data is that high-quality, rigorously controlled trials are lack­ing. Reasonable epidemiologic evidence suggests that Panax ginseng is a non-organ-specific cancer preven­tive, with a dose-response relationship 179. Ginseng extracts and synthetic derivatives should be examined for their preventive effect on various types of human cancers. A case-control study of green tea consump­tion and lung cancer in a population of women living in Shanghai showed an association with reduced risk in non-smoking women 180

Some chinese herbs increase immunity. Although Astragalus, Ligusticum, and Schizandrae have a long history of medicinal use within the tcm system, the West has only recently begun to understand their pharmacologic possibilities and clinical applications. Astragalus has demonstrated a wide range of immu- nopotentiating effects, and it has proven efficacious as an adjunct cancer therapy. Ligusticum and its active components have been investigated for enhancement of the immune system, treatment of ischemic disor­ders, and use as an anti-inflammatory. clinically, the hepatoprotective and antioxidant actions of Schizandrae have proven beneficial in the treatment of chronic vi­ral hepatitis 181. More data are required to determine the clinical effects of Chinese herbs on immunity and to prevent cancer progression 182.

The prevalence of aids is increasing rapidly 183, and research into the effectiveness of chinese herbs on immunity may also help people with that syndrome. The syndrome is a result of infection with hiv, which subsequently leads to significant suppression of immune function. The search for effective therapies to treat aids is of paramount importance. Several chemical anti- hiv agents have been developed; however, besides high cost, adverse effects and limitations are associated with the use of chemotherapy for the treatment of HIv in­fection. Thus, herbal medicines have frequently been used as an alternative medical therapy by HIv-positive individuals and AIDS patients in china. For example, Scutellaria baicalensis georgi and its identified com­ponents (that is, baicalein and baicalin) have been shown to inhibit infectivity and replication of hiv 184 Some preliminary evidence of efficacy has recently been published 185.

Data from controlled clinical trials suggest that medicinal mushrooms may be beneficial as adjunc­tive anticancer therapies 120,186. A rct in colorectal cancer patients receiving curative resection compared adjuvant chemotherapy alone to chemotherapy plus an extract (psk) from the fungus Coriolus versicolor. Both disease-free and overall survival were significantly higher in the group that received the psk 117. Medici­nal mushrooms contain a class of polysaccharides known as beta-glucans that promote antitumour im­munity. They may act synergistically with some of the new therapeutic antibodies and chemotherapy agents and may protect normal marrow 149,187 Maitake mushroom and Ganoderma lucidum are both chinese medicinal mushrooms that are showing pre­liminary evidence that they can suppress viral infec­tions and inhibit cancer progression through modula­tion of the immune system 124,188-190.

In tcm, appropriate nutrition according to specific constitutional and disease patterns is also emphasized. Green tea (Camellia sinensis) and Panax ginseng are two dietary supplements that have been extensively investigated in both laboratory and epidemiologic stud­ies. Both reduce the risk of cancer induction, and they may prevent cancer recurrence 191-193. Green tea contains isoflavones and a powerful antioxidant called epigallocatechin 194 The latter may potentiate the de­struction of cancer cells by promoting apoptosis and inhibiting angiogenesis 195,196. Panax ginseng may induce the differentiation of neoplastic cells into nor­mal tissue 197. Both epigallocatechin and ginseng re­store normal intercellular communication through the gap junctions 3. Both dietary supplements work through novel mechanisms of signalling.

The isoflavones and phytoestrogens in soy appear to reduce the incidence of prostate cancer and may play a role in prevention and as adjunctive therapy to reduce the risk of recurrence 198-202 Cell culture and animal xenograft studies show that treatment with soy is associated with inhibition of prostate-specific anti­gen, deactivation of nuclear factor kb (a nuclear tran­scription factor), induction of apoptosis (programmed cell death), and inhibition of angiogenesis 203-206.

Future goals for cancer prevention in China include public education to reduce the risk of infection, mass immunization, antiviral drugs, and chemotherapy. These are extremely expensive programs, and more educa­tion and research are required before their implemen­tation. However, many Chinese currently have access to traditional herbal remedies within the culture of tcm. Some of the herbs and their derivatives seem to be effective treatments. An opportunity exists to develop, refine, and evaluate the effectiveness of Chinese herbal medicine to prevent the development of cancers sec­ondary to virus infections.

The idea of using Chinese herbs to prevent cancer progression is already being tested in the West. A tcm herb combination may reduce the risk of lung cancer in ex-smokers. An NCI-sponsored study being conducted through the BC Cancer Agency, led by Dr. Stephen Lam, is recruiting participants 45-74 years of age who are ex-smokers to evaluate the efficacy of a herbal combination called "anti-cancer preventive health agent" (acapha) 207. This compound contains Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus, and Dioscorea bulbifera. In Chinese studies, acapha re­duced the risk of esophageal cancer by 50%, by re­versing severe esophageal dysplasia. In addition, a pi­lot study of 20 former heavy smokers with bronchial dysplasia treated with acapha showed that, after 6 months, 50% had complete regression of dysplasia, as compared with only 13% of subjects in the placebo group. Panax quinquefolium (American ginseng) ap­pears to reduce death and increase quality of life in survivors of breast cancer, suggesting that some botanicals may prevent recurrence 208.

2.4 Symptom Control

Cancer patients experience multiple symptoms related either to the cancer itself or to late treatment side ef­fects. Even if the cancer is cured, the survivor may still be suffering from the late treatment side effects, which have an adverse effect on quality of life and are often not effectively managed with conventional West­ern medicine. chinese medicine plays a useful role in supportive care for these symptomatic cancer patients. Symptoms that can be effectively managed include general constitutional symptoms such as fatigue, de­pression, and pain, and specific symptoms such as gastrointestinal side effects and myelosuppression.

cancer patients receiving chemotherapy usually develop myelosuppression (with risk of infection and bleeding) and gastrointestinal side effects (nausea, vomiting, and diarrhea). They easily become fatigued and develop a reduced appetite. In tcm terms, the chemotherapeutic agents are causing Spleen and Kid­ney deficiency, leading to a general decrease in qi and blood. Note that "Spleen" and "Kidney" refer to com­munication systems (resembling the acupuncture me­ridian system) rather than actual organs. Radiotherapy and chemotherapy act as "heat toxins" that damage yin and qi. "Heart fire" is expressed as stomatitis; "de­ficient Spleen qi" is manifest as diarrhea. Chemo­therapy drugs "disturb Spleen and Stomach qi," ex­pressed physically as damage to the lining of the stom­ach and intestines 51.

The conventional oncologist will often find that the language of a tcm practitioner seems quite unu­sual and metaphoric, but that language is consistent within the tcm system of diagnosis and treatment. The physical expressions are only part of the distur­bance in the body-mind network, and they will inevi­tably be accompanied by emotional disorders (such as depression, anxiety, insomnia) and constitutional change (such as fatigue or hyper-excitability and poor concentration). After an evaluation and diagnosis of the disturbance in the body-mind network, appropri­ate combinations of herbs, acupuncture, nutrition, and Qigong may be utilized.

2.4.1 Herbs

Gastrointestinal Toxicity, Depressed Immunity, and Fatigue: Chinese medicine treats the combination of gastrointestinal symptoms, depressed immunity, and fatigue as a single syndrome. Spleen and Stomach qi are supported by appropriate formulas containing Rx ginseng, Poria, and Rh Atractylodis macrocephala 51. Ginger root has been shown in many clinical studies to have antiemetic activity 209-213. It appears par­ticularly to help nausea that may be intransigent to standard antiemetics. Ginger syrup was shown in a rct to be effective 214. Caution should be used in patients on anticoagulants or those with low platelet levels, because the syrup has anticoagulant effects at higher doses.

Depleted yin leads to dry and sore mouth, thirst, constipation, and scanty dark urine. The harmonious relationship between Kidney and Heart is disturbed, leading to insomnia, restlessness, disorientation, pal­pitations, and low back pain. This combination of symptoms is traditionally alleviated with combinations of Rh Anemarrhenae, Cx Phellodendron, and Rx Rehmanniae.

Vitexina (Vigna radiata) is a flavonoid herb with radioprotective effects that may be useful for reducing some side effects of radiotherapy. It treats the heat (yin)-deficiency side effects of anticancer treatment, such as fatigue, restlessness, insomnia, and constipa­tion. This "empty heat" syndrome is characterized through tongue diagnosis, which reveals a red, denuded, and cracked tongue. Because the tongue is the most densely innervated organ in the body, it may reflect the imbalance between yin and yang via the autonomic nervous system, which in turn may influence blood flow and epithelial cell turnover through the local release of neuropeptides and cytokines. A rct of breast cancer patients receiving radiotherapy showed that vitexina prevented the "empty heat" syndrome, reduced weight loss, and protected against a reduction in peripheral lymphocytes and platelets 215.

According to chinese medicine, the weakening of qi is associated with depressed immunity and suscep­tibility to infection and cancer progression. Medicinal mushrooms such as Ganoderma, Cordyceps sinensis, and Shitake strengthen the qi, which is associated with an improved immune profile and antitumour activity. Another herb with potent immune-stimulating proper­ties is Rx Astragalus membranaceus. In Western ter­minology, these patients are fatigued and have de­pressed immunity that renders them more susceptible to infection. High-quality clinical studies are rare, but the Mao clinic (Rochester) recently reported a prom­ising phase ii rct of ginseng (Panax quinquefolia L.) for fatigue in cancer patients. They reported a dose- response and demonstrated a 40% reduction in fatigue for patients taking the highest dose level as compared with those taking placebo 49.

At least five rcts have shown that Chinese herbal treatment can decrease the degree of myelosuppres- sion, reduce gastrointestinal side effects, and increase appetite 39,68-71,73-78,80,216. Importantly, such treat­ment can also increase the probability of completion of scheduled chemotherapy. One randomized trial recruited 669 patients with late-stage gastric cancer 74 One group of patients was treated with herbs that sup­port Spleen and Kidney function ("jian pi yi shen pre­scription") twice daily for 4-6 weeks with concurrent chemotherapy; another group was treated with the same type of chemotherapy alone. The combined- treatment group showed significantly higher leukocyte and platelet counts with fewer general and gastroin­testinal side effects. The percentage of patients com­pleting the scheduled chemotherapy was 95% in the combined-treatment group as compared with 74% in the chemotherapy-alone group (a statistically signifi­cant result at the 0.01 level). Unfortunately, the qual­ity and verification of the data from these studies, which were reported from china, are not at a high enough standard that a definitive meta-analysis can be undertaken at this stage.

A more recent high-quality study was reported from Hong Kong 217. This double-blind placebo-controlled rct used Chinese herbal medicine for the reduction of chemotherapy-induced toxicity from adjuvant therapy for breast or colon cancer. The herbal combination was individualized by the tcm practitioner and then randomized against placebo. The investigators could not show a reduction in hematologic toxicity, but they reported a significant impact on control of nausea.

The role of Chinese herbs in combination with con­ventional Western pharmaceuticals for symptom con­trol is currently unclear. Laboratory data suggest that herbs can be effective modifiers of biochemical path­ways, immunostimulants, and signal transduction modulators. But detrimental interactions and idiosyn­cratic toxicity are certainly a potential possibility.

A Cochrane systematic review of Chinese medi­cinal herbs for chemotherapy side effects in colorectal cancer patients found some merit in the concoction termed "huangqi compounds" (containing Astragal­us) 27. Four relevant trials were reviewed, all of low quality. None of the studies reported common toxic­ity criteria. There appeared to be a significant reduc­tion in the number of patients who experienced nau­sea and vomiting, a decrease in the rate of leucope- nia, and an increase in T-lymphocyte subsets. Although no adverse effects were reported, these are rarely documented in Chinese studies. Another Cochrane systematic review of chinese medicinal herbs used to treat the side effects of chemotherapy in breast cancer patients provided limited evidence, although benefits in bone marrow improvement and quality of life were suggested 218. Future studies using more rig­orous methodology and quality assurance are required.

Chemotherapy and Radiotherapy-Induced Cognitive

Dysfunction: chinese herbal therapies may have a role in improving cognitive function. Because conventional pharmaceutical interventions have produced very limited improvement, opportunities are opening to investigate some natural health products from the chinese pharmacopoeia.

Many patients complain about changes in cogni­tive function during and after chemotherapy. This phe­nomenon has been particularly studied in breast can­cer patients 219,220. Despite the fact that neurocognitive deficits limit productivity and independence for patients, these problems are underreported by patients and underdiagnosed by health care professionals. At least 18% of cancer patients who received standard­dose chemotherapy manifested cognitive deficits on post-treatment neuropsychological testing, and that effect may be sustained 2 years after treatment 221. The patients typically complain of a "foggy brain." The impairments have an impact on tests that require sustained attention and speed of information process­ing. Fatigue and depression are associated disorders. Whether the initial cause of dysfunction is attribut­able to loss of neuronal integrity or is secondary to a metabolic pathology is as yet unknown. There may be a genetic component, such as the e4 allele of Apo- lipoprotein 222.

Because "chemo-brain" is mainly a subjective phenomenon, it is important to develop techniques to objectively measure neurophysiologic or anatomic changes 223-226. Cytokines such as il-1 and interferons may play a role, according to some animal experiments. Chemotherapy may damage the endothelium of blood vessels, resulting in thromboses and micro-infarcts in the central nervous system. currently, the changes that occur in cerebral tissue after anticancer treatments are poorly understood, and no proven interventions are available.

Radiotherapy treatment of the brain can also impair cognitive function. Long-term survivors experience ma­jor cognitive dysfunction that influences their rehabili­tation and quality of life. Radiotherapy can damage the brain, eventually resulting in demyelination that may appear only months later and lead to the development of cognitive impairment. These deficits manifest as any or all of impaired memory; diminished attention; low­ered concentration; or functional, behavioural, and psy­chiatric deficits; and similarities to Alzheimer disease are seen 226-229. Receptors for N-methyl-D-aspartate (nmda) are overactivated, leading to neuronal damage and learning impairment 230.

Interventions that could ameliorate such disabili­ties would be of great benefit to these patients and their caregivers. Ginkgo biloba, from the ginkgo tree, has a long history of use in tcm. Effects of Ginkgo biloba extracts have been postulated to include improvement of memory, increased blood circulation, and benefi­cial effects to patients with Alzheimer disease. The most unique components of the extracts are the ter- pene trilactones-that is, ginkgolides and bilobalide. These structurally complex molecules have been at­tractive targets for total synthesis. Terpene trilactones are believed to be partly responsible for the neuro- modulatory properties of Ginkgo biloba extracts, and several biologic effects of the terpene trilactones have been discovered in recent years, making them attrac­tive pharmacologic tools that could provide insight into the effects of Ginkgo biloba extracts.

Ginkgolides A, B, C, J, K, L, and M and bilobalide are rare terpene trilactones that have been isolated from leaves and root bark of the Chinese Ginkgo biloba tree. The compounds were found to be potent and se­lective antagonists of platelet-activating factor, res­ponsible for their effect of increasing bleeding time. The mean absolute bioavailability for ginkgolides A and B and bilobalide are 80%, 88%, and 79% respec­tively. Much of the given dose is excreted unchanged in urine. Radioactive isotope studies show cerebral availability, particularly in the hippocampus, striatum, and hypothalamus 231-233.

Lipid peroxidation and brain edema are important factors that produce tissue damage in head injury. An investigation of the effect of mexiletine and Gingko biloba extract (EGb 761) on head trauma in rats showed the usefulness of mexiletine and its combination with EGb 761 as a cerebroprotective agent 234. In vivo studies have indicated that systemically administered bilobalide, a sesquiterpene trilactone constituent of Ginkgo biloba leaf extracts, can reduce cerebral edema produced by triethyltin, decrease cortical infarct volume in certain stroke models, and reduce cerebral ischemia. In vitro and ex vivo studies indicate that bilobalide has multiple mechanisms of action that may be associated with neuroprotection, including its preservation of mitochon­drial atp synthesis, its inhibition of apoptotic damage induced by staurosporine or by serum-free medium, its suppression ofhypoxia-induced membrane deterioration in the brain, and its action in increasing the expression of the mitochondrial DNA-encoded cox iii subunit of cytochrome C oxidase and the nd1 subunit of nadh dehydrogenase. Because multiple modes of action may apply to bilobalide, it could be useful in developing therapy for disorders involving cerebral ischemia and neurodegeneration 235-237.

Standardized ginkgo leaf extracts such as Egb76, 120-720 mg daily, have been used in clinical trials for dementia, memory, and circulatory disorders. A common dose is 80 mg or 240 mg (divided into 2 or 3 doses) daily of 50:1 standardized leaf extract by mouth 238. Ginkgo biloba increases vascular perfusion and improves cognitive function 239. It may also pro­tect damaged neurones by maintaining the balance of inhibitory and excitatory amino acids and inhibiting the effect of glutamate on the nmda receptor 240-242. Some rcts and a Cochrane review concluded that its use is promising 243,244; however, other rcts have not confirmed its effectiveness 245. That ineffectiveness may be a function of inadequate dose or purity of the preparation.

When compared with cholinesterase inhibitors, Ginkgo biloba is better tolerated with similar effica­cy 246. An abstract from Wake Forest University de­scribed a phase II (non-controlled) study in brain- irradiated patients and concluded that Ginkgo biloba can improve cognitive dysfunction 247. A dose of 40 mg 3 times daily was administered for 30 weeks. Of 34 patients entered into the study, 18 completed the 30 weeks of treatment; 16 patients went off-study be­cause of tumour progression, treatment toxicity, or choice to discontinue treatment. Brain quality of life as measured by the Functional Assessment of Can­cer Therapy-Brain Subscale was significantly im­proved at 12, 24, and 30 weeks as compared to base­line. Mood improved on the Profile of Mood States scale, confusion and fatigue were reduced, and cog­nitive testing showed improved attention and execu­tive function. A rct to compare donepezil with Ginkgo biloba (Egb761) is currently being initiated at Wake Forest University 248.

Hasegawa 249 reviewed the metabolism of indi­vidual ginsenosides. Ingested ginsenosides are me­tabolized in the large intestine through deglycosylation by colonic bacteria followed by fatty acid esterifica- tion. The resulting metabolites enter the circulation, where they exert their pharmacologic effects. The gin- senosides can inhibit nmda receptor-mediated sig­nals 250-254 A combination of ginseng and Ginkgo biloba was shown to improve cognitive function in nor­mal volunteers 255. Laboratory studies suggest that inhibition of the neurotoxic effects of nmda receptor overactivity by ginseng derivatives may delay or re­verse neurocognitive dysfunction, but as yet, no con­trolled clinical studies have addressed that hypoth­esis. However, one type of ginseng (Panax quinque- folium) has demonstrated encouraging therapeutic effects in a clinical trial involving memory deficien­cy 256. Ginseng derivatives and Ginkgo biloba both inhibit the overstimulation of the nmda receptor by glutamate, which is implicated in neurodegenerative disorders. Ginsenosides may also increase cerebral acetylcholine levels. Although it is not yet known whe­ther radiotherapy specifically interacts with the nmda receptor or acetylcholine levels, the clinical and patho­logic process appears similar to primary and vascular dementia alike 257 Animal and early clinical evidence suggests that Ginkgo biloba and ginseng derivatives could have a neuroprotective effect, reducing cogni­tive impairment.

2.4.2 Acupuncture

Emesis: Acupuncture treatment at acupoint P6 has been shown to increase the antiemetic effect of drugs for perioperative and chemotherapy-induced nausea and vomiting 258,259 Innovative single-blind rcts have since confirmed these results 260-262 and led to a con­sensus statement from the U.S. National Institutes of Health that "acupuncture is a proven effective treat­ment modality for nausea and vomiting" 263. A three- arm rct comparing conventional modern antiemetics (such as the 5-hydroxytryptamine 3 receptor antago­nists), electroacupuncture, and the combination of antiemetic drugs and acupuncture clearly demonstrated that the combination arm was the most effective for preventing nausea and vomiting 264.

Stimulation of P6 may be carried out more conven­iently with a small transcutaneous nerve stimulation (tens) device, such as the ReliefBand (Neurowave Medical, Chicago, IL, U.S.A.). However, a recent rct could not confirm the efficacy of tens in the control of chemotherapy-induced nausea in women with breast cancer 265, despite promising results in patients with motion sickness. Those results may be attributable to the focus on nausea rather than vomiting, to physiologic tolerance, or to maximal control having already been reached by the application of pharmaceutical antiemetics in these patients such that the device provided no ad­vantage over medication alone. A meta-analysis of acu­puncture-point stimulation for chemotherapy-induced nausea or vomiting shows a benefit over and above drug therapy 266; however, the studies did not all use optimal drug therapy, and the pharmaceutical approach may need to be optimized before acupuncture is used in refractory cases. According to the meta-analysis, self-administered acupressure also appears to have a protective effect against acute nausea.

Pain: Pain is a common symptom of cancer. The causes of the pain can be the cancer itself or its treatment. Acupuncture, along with other interventions, has been shown to be effective in managing pain and other symptoms in cancer patients 267. In a retrospective study from the Royal Marsden Hospital in London, England, 183 cancer patients with malignant pain, iatrogenic pain, and radiation-induced chronic ulcers were treated with acupuncture 268-270. An improvement was seen in 82% of the patients, but effectiveness lasted for more than 3 days in only half of the patients. Iatrogenic pain (for example, pain resulting from radiation fibrosis or skin ulceration) and pain caused by secondary muscle spasm responded better than did malignant pain. Furthermore, increased blood flow, with improved healing of skin ulcers, was demonstrated after treatment with acu­puncture. A rct using ear acupuncture showed a profound effect on cancer pain 271, and we obtained encouraging results from a small pilot study of acu­puncture for chemotherapy-induced neuropathic pain 272 A systematic review could not demonstrate the effec­tiveness of acupuncture as an adjunctive analgesic method for cancer patients 273; however, it included only one rct 271, and all the other studies were generally of poor scientific quality. The intensity of stimulation, especially electrostimulation, may be important 274.

We suggest that acupuncture is a useful treatment modality that may best be combined with other treat­ments to improve pain control, resulting in reduced doses of pharmaceutical analgesics. The reduction in medication use has the benefit of reducing the inci­dence and degree of drug-induced side effects. For some patients, a tens device has the advantage of easy self-administration or administration by staff after minimal training.

Acupuncture-like tens (al-tens) devices have been developed to apply low-frequency (4 Hz, for in­stance), high-intensity stimulation that mimics acupunc­ture treatment 275. The goal is to recruit the high-thresh­old type III afferent nerve fibres that are potent releasers of endorphins. Recent meta-analyses (including a Cochrane systematic review) have shown that al-tens is more effective than placebo and improves function more than standard tens in the treatment of chronic pain 276-279. The al-tens devices are very simple machines that patients can learn to operate with less than 60 minutes of training. An acupoint prescription may then be given to the patient, who can administer the appropriate treatments with al-tens at home. The Codetron (EHM Rehabilitation Technologies, Toronto, ON, Canada) is a sophisticated al-tens device that has the advantage of reducing tolerance to its analgesic effect by electronically rotating through a series of ran­dom electrical stimulation patterns and acupoint locations.

Xerostomia: Radiation-induced xerostomia is one of the distressing late side effects seen in patients who receive radiation treatment involving the parotid glands. The condition leads to loss of taste and difficulty in speaking and swallowing for the patients. Recently, acupuncture treatment has been found to increase blood flow to the parotid glands and may stimulate tissue regeneration in glands damaged by radiotherapy 280-282.

A rct involving 38 patients with radiation xeros­tomia was reported from the Karolinska Institute in Sweden 283. Subjects were randomized to either deep acupuncture treatment or superficial acupuncture treat­ment. The superficial group was used as the control, despite previous evidence that superficial acupuncture treatment can have a certain degree of effectiveness and should not be used as a control in acupuncture treat­ment trials. In the Swedish study, more than 50% of the patients in both groups were found to have experienced a greater than a 20% increase in saliva flow rate. In the deep acupuncture group, 68% of patients demonstrated an increase in salivary flow rate. Changes in the control group were smaller and appeared after a longer latency phase. Moreover, patients in the treatment group reported less dryness, less hoarseness, and improved taste.

In another study, 70 patients with xerostomia at­tributable either to Sjogren syndrome or to irradiation were treated with acupuncture 284. A statistically sig­nificant increase in unstimulated and stimulated sali­vary flow rates was found in all patients immediately after acupuncture treatment, and for up to 6 months of follow-up. After a review at 3 years, patients who chose to be treated with additional acupuncture demon­strated a consistently higher median salivary flow rate as compared with patients not choosing to have addi­tional acupuncture.

Despite some limitations in design, both of the fore­going studies provided evidence suggesting that acu­puncture can be effective in treating radiation-induced xerostomia, with minimal side effects. In a prospec­tive single-cohort visual-analogue-assessed study of acupuncture in palliative care patients with xerosto­mia, highly significant alleviation of subjective xeros­tomia was observed 285. Other studies are confirming the clinical use of acupuncture for relief of radiation- induced xerostomia 286

At the Juravinski Cancer Centre in Ontario, Cana­da, a combined phase i and ii study of al-tens in the treatment of radiation-induced xerostomia was carried out 287. The 45 participating patients were randomized into three treatment groups that received Codetron AL­tens stimulation to one of three different sets of acu­puncture points:

  • Group A: CV24, St36, Sp6, LI4
  • Group B: CV24, St36, Sp6, P6
  • Group C: CV24, St5, St6, Sp6, P6

The goal of the study was to determine the opti­mum pattern of stimulation (based on tcm theory) to feed into the design of a placebo-controlled study. The al-tens treatment was administered twice weekly for a total of 12 weeks. Unstimulated and stimulated sali­vary flow rates before, during, and after treatment were measured, and the patients' quality of life was surveyed at a 1-year follow-up. Improvement in xeros­tomia symptoms was noted, with mean score increases of 86 (p < 0.0005) and 77 (p < 0.0001) on the visual analogue scale at 3 and 6 months respectively after treatment completion. For all patients, the increase in mean basal and citric acid-primed whole saliva production at 3 and 6 months after treatment comple­tion was also statistically significant (p < 0.001 and p < 0.0001 respectively). No statistically significant change in the quality-of-life evaluation as compared with baseline was observed.

Those results suggest that Codetron treatment im­proves saliva production and related symptoms in pa­tients suffering from radiation-induced xerostomia. Treatment effects are sustained at least 6 months after completion of treatment. A recent study using func­tional magnetic resonance imaging showed activation of the insula region of the brain, the location associ­ated with gustatory function 288.

Anxiety, Depression, Cognitive Impairment, and Fatigue:

suppression of anxiety by acupuncture is associated with an increase in the pain threshold 289. Acupuncture can also play a role in the treatment of fatigue and malignant cachexia through the modulation of cytokines and hormones 290-295.

Treatment of depression is an important interven­tion in the management of the body-mind network in cancer patients. Conventionally, clinical depression is treated with oral medication such as amitriptyline or the newer serotonin reuptake inhibitor drugs. Stud­ies indicate that acupuncture treatment may be an equally effective alternative modality to drug treat­ment in patients suffering from mild depression. In one study, the side effects profile associated with acu­puncture treatment was shown to be better than that associated with amitriptyline 296. In a single-blind placebo-controlled study of the antidepressant mian- serin, supplementary acupuncture improved the course of depression more than did pharmacologic treatment with the drug alone 297.

A Cochrane review concluded that the evidence is insufficient to determine the efficacy of acupuncture as compared with medication or with wait list control or sham acupuncture in the management of depression 298; however, because pharmaceutical antidepressants are not usually effective until 2 weeks after the start of therapy, their combination with acupuncture may pro­duce more rapid results with reduced side effects.

The role of acupuncture for cognitive impairment caused by chemotherapy or radiotherapy is unclear. An intriguing study in rats showed improvement in cognitive impairment caused by multiple infarcts 299. A recent re­view concluded that some limited evidence supports the use of acupuncture in effectively managing a range of psychoneurologic problems, some of which are similar to those experienced by patients with chemotherapy-as­sociated cognitive dysfunction 300. A phase II study of acupuncture for post-chemotherapy fatigue (average of 2 years) showed a mean improvement of 30% on the Brief Fatigue Inventory 301. That finding met the research group's pre-specified criterion of clinical importance and has prompted initiation of a sham acupuncture rct.

Miscellaneous Symptoms: Other symptoms that may be helped with acupuncture include constipation, tris- mus (post-radiotherapy contracture of the masseter muscle) 302, radiotherapy-associated proctitis 303, hiccups 304, persistent yawning 305, chemotherapy- induced peripheral neuropathy 272, and dysphagia secondary to an esophageal neoplasm 306. Although observational studies by Filshie et al. 307 showed that acupuncture may improve cancer-associated dyspnea, that group's findings were not supported by a later rct using semi-permanent acupuncture studs 308.

Acupuncture can reduce the hot flushes associated with anticancer hormone therapy. Three prospective un­controlled cohort studies have been completed, one in men who had undergone castration for prostate cancer, and two in women who were taking tamoxifen for breast cancer. All demonstrated a reduction in vasomotor symp­toms 309-311. Prolonged stimulation using semi-perma­nent studs or needles, especially at SP6 appears to be associated with more long-term relief of symptoms 270

•3.    CONCLUSIONS

Emerging scientific evidence suggests that tcm can play an important role in the supportive care of can­cer patients. Enough preliminary evidence is avail­able to encourage good-quality clinical trials evaluat­ing the efficacy of integrating tcm into Western can­cer care 312-316. Currently, the evidence for the utility of tcm in cancer care is promising, but prospective rcts for specific clinical scenarios are necessary to obtain reliable and generalizable data.

Appropriate stratification and individualization according to tcm diagnostic criteria is possible within the context of a rct 317. We believe that an evidence- based approach can be integrated into an individual­ized therapeutic plan and that a major role still exists for individual belief systems and psycho-spiritual ex­perience. Assessment and measurement of coping strat­egies, maintenance of function, quality of life, and patient satisfaction are important. We are hopeful that future integration of various models of health such as tcm may lead to further improvements in survival and quality of life for cancer patients.

•4.    ACKNOWLEDGMENTS

We thank christina M. Garchinski for administrative assistance.

5. REFERENCES

  • 1. Coffey DS. Self-organization, complexity and chaos: the new biology for medicine. Nature Med 1998;4:882-5.
  • 2. Cuzick J, Holland R, Barth V, et al . Electropotential measure­ments as a new diagnostic modality for breast cancer. Lancet 1998;352:359-63.
  • 3. Kang K, Kang B, Lee B, et al. Preventive effect of epicatechin and ginsenoside Rb2 on the inhibition of gap junctional intercel­lular communication by TP and H2O2. Cancer Lett 2000; 152:97-106.
  • 4. Sagar SM, Klassen GA, Barclay KD, Aldrich JE. Tumour blood flow: measurement and manipulation for therapeutic gain. Can­cer Treat Rev 1993;19:299-349.
  • 5. Milosevic MF, Fyles AW, Wong R, et al. Interstitial fluid pres­sure in cervical carcinoma: within tumor heterogeneity, and relation to oxygen tension. Cancer 1998;82:2418-26.
  • 6. Baxter LT, Jain RK. Transport of fluid and macromolecules in tumors. I. Role of interstitial pressure and convection. Microvasc Res 1989;37:77-104.
  • 7. Brizel DM, Sibley GS, Prosnitz LR, Scher RL, Dewhirst MW. Tumor hypoxia adversely affects the prognosis of car­cinoma of the head and neck. Int J Radiat Oncol Biol Phys 1997;38:285-9.
  • 8. Fyles AW, Milosevic M, Wong R, et al. Oxygenation predicts radiation response and survival in patients with cervix cancer. Radiother Oncol 1998;48:149-56.
  • 9. Lebeau B, Chastang C, Brechot JM, et al. Subcutaneous heparin treatment increases survival in small cell lung cancer. "Petites Cellules" Group. Cancer 1994;74:38-45.
  • 10. Hejna M, Raderer M, Zielinski CC. Inhibition of metastases by anticoagulants. J Natl Cancer Inst 1999;91:22-36.
  • 11. Yance DR, Sagar SM. Targeting angiogenesis with integrative cancer therapies. Integr Cancer Ther 2006;5:9-29.
  • 12. Wang JZ, Tsumura H, Shimura K, Ito H. Antitumor activity of polysaccharide from a Chinese medicinal herb, Acanthopanax giraldii Harms. Cancer Lett 1992;65:79-84.
  • 13. Tode T, Kikuchi Y, Kita T, Hirata J, Imaizumi E, Nagata I. Inhibitory effects by oral administration of ginsenoside Rh2 on the growth of human ovarian cancer cells in nude mice. J Can­cer Res Clin Oncol 1993;120:24-6.
  • 14. Lau BH, Ruckle HC, Botolazzo T, Lui PD. Chinese medicinal herbs inhibit growth of murine renal cell carcinoma. Cancer Biother 1994;9:153-61.
  • 15. Boik J. Cancer and Natural Medicine: A Textbook of Basic Science and Clinical Research. Rev ed. Princeton, MN: Oregon Medical Press; 1996.
  • 16. Boik J. Emerging trends in cancer research: development of a mechanism-based approach. Protocol J Botanic Med 1996; 2:5-10.
  • 17. Shoemaker M, Hamilton B, Dairkee SH, Cohen I, Campbell MJ. In vitro anticancer activity of twelve Chinese medicinal herbs. Phytother Res 2005;19:649-51.
  • 18. Xu GZ, Cai WM, Qin DX, et al. Chinese herb "destagnation" series i: combination of radiation with destagnation in the treat­ment of nasopharyngeal carcinoma (NPC): a prospective randomized trial on 188 cases. Int J Radiat Oncol Biol Phys 1989;16:297-300.
  • 19. Kleijnen J, Knipschild P. Ginkgo biloba. Lancet 1992;340:1136-9.
  • 20. Ha SW, Yi CJ, Cho CK, Cho MJ, Shin KH, Park CI. Enhance­ment of radiation effect by Ginkgo biloba in C3H mouse fibro­sarcoma. Radiother Oncol 1996;41:163-7.
  • 21. Sagar SM, Singh G, Hodson DI, Whitton AC. Nitric oxide and anti-cancer therapy. Cancer Treat Rev 1995;21:159-81.
  • 22. Kuang P, Tao Y, Tian Y. Radix Salviae miltiorrhizae treatment results in decreased lipid peroxidation in reperfusion injury. J Tradit Chin Med 1996;16:138-42.

2 3. Sun H, Duan S, Yu G. Free radical mechanism in enhancement of radiosensitization by srsbr. J Tradit Chin Med 1994;14:51-5.

  • 24. Lee TK, Johnke RM, Allison RR, O'Brien KF, Dobbs LJ Jr. Radioprotective potential of ginseng. Mutagenesis 2005;20: 237-43.
  • 25. Zhou RF, Liu PX. Study progress in reversing multidrug resist­ance to breast cancer with Chinese herbs [Chinese]. Zhongguo Zhong Yao Za Zhi 2005;30:1797-800.
  • 26. Kang JX, Liu J, Wang J, He C, Li FP. The extract of huanglian, a medicinal herb, induces cell growth arrest and apoptosis by upregulation of interferon-ß and TNF-a in human breast can­cer cells. Carcinogenesis 2005;26:1934-9.
  • 27. Taixiang W, Munro AJ, Guanjian L. Chinese medical herbs for chemotherapy side effects in colorectal cancer patients. Coch­rane Database Syst Rev 2005;(1):14651858.CD004540.pub2.
  • 28. Shu X, McCulloch M, Xiao H, Broffman M, Gao J. Chinese herbal medicine and chemotherapy in the treatment of hepato­cellular carcinoma: a meta-analysis of randomized controlled trials. Integr Cancer Ther 2005;4:219-29.
  • 29. Shin HJ, Kim YS, Kwak YS, Song YB, Kim YS, Park JD. Enhancement of antitumor effects of paclitaxel (Taxol) in com­bination with red ginseng acidic polysaccharide (rgap). Planta Med 2004;70:1033-8.
  • 30. Collins L, Zhu T, Guo J, Xiao ZJ, Chen CY. Phellinus linteus sensitises apoptosis induced by doxorubicin in prostate can­cer. Br J Cancer 2006;95:282-8.
  • 31. McCulloch M, See C, Shu XJ, et al. Astragalus-based Chinese herbs and platinum-based chemotherapy for advanced non- small-cell lung cancer: meta-analysis of randomized trials. J Clin Oncol 2006;24:419-30.
  • 32. Liang HL, Xue CC, Li CG. Regression of squamous cell carci­noma of the lung by Chinese herbal medicine: a case with an 8- year follow-up. Lung Cancer 2004;43:355-60.
  • 33. Lee YJ, Jin YR, Lim WC, et al. Ginsenoside-Rb1 acts as a weak phytoestrogen in MCF-7 human breast cancer cells. Arch Pharm Res 2003;26:58-63.
  • 34. Amato P, Christophe S, Mellon PL. Estrogenic activity of herbs commonly used as remedies for menopausal symptoms. Meno­pause 2002;9:145-50.
  • 35. Chang CJ, Chiu JH, Tseng LM, et al. Modulation of her2 expression by ferulic acid on human breast cancer MCF7 cells. Eur J Clin Invest 2006;36:588-96.
  • 36. Campbell MJ, Hamilton B, Shoemaker M, Tagliaferri M, Cohen I, Tripathy D. Antiproliferative activity of Chinese medicinal herbs on breast cancer cells in vitro. Anticancer Res 2002;22:3843-52.
  • 37. Jia WW, Bu X, Philips D, et al. Rh2, a compound extracted from ginseng, hypersensitizes multidrug-resistant tumor cells to chemotherapy. Can J Physiol Pharmacol 2004;82:431-7.
  • 38. Loo WT, Cheung MN, Chow LW. The inhibitory effect of a herbal formula comprising ginseng and Carthamus tinctorius on breast cancer. Life Sci 2004;76:191-200.
  • 39. Yu Y, Zhou Q, Hang Y, Bu X, Jia W. Antiestrogenic effect of 20S-protopanaxadiol and its synergy with tamoxifen on breast cancer cells. Cancer 2007;109:2374-82.
  • 40. King ML, Murphy LL. American ginseng (Panaxquinquefolius L.) extract alters mitogen-activated protein kinase cell signaling and inhibits proliferation of MCF-7 cells. J Exp Ther Oncol 2007;6:147-55.
  • 41. Aung HH, Mehendale SR, Wang CZ, Xie JT, McEntee E, Yuan CS. Cisplatin's tumoricidal effect on human breast carcinoma MCF-7 cells was not attenuated by American ginseng. Cancer Chemother Pharmacol 2007;59:369-74.
  • 42. Piersen CE. Phytoestrogens in botanical dietary supplements: implications for cancer. Integrat Cancer Ther 2003;2:120-38.
  • 43. Sagar SM. Future directions for research on Silybum marianum for cancer patients. Integr Cancer Ther 2007;6:166-73.
  • 44. Lau CB, Ho TC, Chan TW, Kim SC. Use of dong quai (Angelica sinensis) to treat peri-or postmenopausal symptoms in women with breast cancer: is it appropriate? Menopause 2005;12:734-40.
  • 45. Hirata JD, Swiersz LM, Zell B, Small R, Ettinger B. Does dong quai have estrogenic effects in postmenopausal women? A dou­ble-blind, placebo-controlled trial. Fertil Steril 1997;68:981-6.
  • 46. Duda RB, Zhong Y, Navas V, Li MZ, Toy BR, Alavarez JG. American ginseng and breast cancer therapeutic agents synergistically inhibit MCF-7 breast cancer cell growth. J Surg Oncol 1999;72:230-9.
  • 47. King ML, Adler SR, Murphy LL. Extraction-dependent ef­fects of American ginseng (Panax quinquefolium) on human breast cancer cell proliferation and estrogen receptor activa­tion. Integr Cancer Ther 2006;5:236-43.
  • 48. Cui Y, Shu XO, Gao YT, Cai H, Tao MH, Zheng W. Associa­tion of ginseng use with survival and quality of life among breast cancer patients. Am J Epidemiol 2006;163:645-53.
  • 49. Barton DL, Soori GS, Bauer B, et al. A pilot, multi-dose, pla­cebo-controlled evaluation of American ginseng (Panax quinquefolius) to improve cancer-related fatigue: NCCTG trial N03CA [abstract 9001]. J Clin Oncol 2007;25:18S.
  • 50. DiPaola RS, Zhang H, Lambert GH, et al. Clinical and biologic activity of an estrogenic herbal combination (PC-SPES) in pros­tate cancer. N Engl J Med 1998;339:785-91.
  • 51. Guns ES, Goldenberg SL, Brown PN. Mass spectral analysis of PC-SPES confirms the presence of diethylstilbestrol. Can J Urol 2002;9:1684-8.
  • 52. Oh WK, KantoffPW, Weinberg V, et al. Prospective, multicenter randomized phase ii trial of the herbal supplement: pc-spes, and diethylstilbestrol in patients with androgen-independent pros­tate cancer. J Clin Oncol 2004;22:3705-12.
  • 53. Kim H, Peterson TG, Barnes S. Mechanisms of action of the soy isoflavone genistein: emerging role for its effects via trans­forming growth factor beta signaling pathways. Am J Clin Nutr 1998;68:1418S-25S.
  • 54. Li Y, Bhuiyan M, Sarkar FH. Induction of apoptosis and inhi­bition of c-erbB-2 in MDA-MB-435 cells by genistein. Int J Oncol 1999;15:525-33.
  • 55. Li Y, Upadhyay S, Bhuiyan M, Sarkar FH. Induction of apoptosis in breast cancer cells MDA-MB-231 by genistein. Oncogene 1999;18:3166-72.
  • 56. Wu AH, Ziegler RG, Horn-Ross PL, et al. Tofu and risk of breast cancer inAsian-Americans. Cancer Epidemiol Biomarkers Prev 1996;5:901-6.
  • 57. Witte JS, Ursin G, Siemiatycki J, Thompson WD, Paganini-Hill A, Haile RW. Diet and premenopausal bilateral breast cancer: a case-control study. Breast Cancer Res Treat 1997;42:243-51.
  • 58. Lu LJ, Cree M, Josyula S, Nagamani M, Grady JJ, Anderson KE. Increased urinary excretion of 2-hydroxyestrone but not 16a-hydroxyestrone in premenopausal women during a soya diet containing isoflavones. Cancer Res 2000;60:1299-305.
  • 59. Key TJ, Sharp GB, Appleby PN, et al. Soya foods and breast cancer risk: a prospective study in Hiroshima and Nagasaki, Japan. Br J Cancer 1999;81:1248-56.
  • 60. Hsieh CY, Santell RC, Haslam SZ, Helferich WG. Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo. Cancer Res 1998;58:3833-8.
  • 61. Scambia G, Mango D, Signorile PG, et al. Clinical effects of a standardized soy extract in postmenopausal women: a pilot study. Menopause 2000;7:105-11.
  • 62. Quella SK, Loprinzi CL, Barton DL, et al. Evaluation of soy phytoestrogens for the treatment of hot flashes in breast can­cer survivors: A North Central Cancer Treatment Group Trial. J Clin Oncol 2000;18:1068-74.
  • 63. MacGregor CA, Canney PA, Patterson G, McDonald R, Paul J. A randomised double-blind controlled trial of oral soy sup­plements versus placebo for treatment of menopausal symp­toms in patients with early breast cancer. Eur J Cancer 2005;41:708-14.
  • 64. Hu SA. Risks and benefits of soy isoflavones for breast cancer survivors. Oncol Nurs Forum 2004;31:249-63.
  • 65. Messina M, McCaskill-Stevens W, Lampe JW. Addressing the soy and breast cancer relationship: review, commentary, and workshop proceedings. J Natl Cancer Inst 2006;98:1275-84.
  • 66. Spence DW, Kayumov L, et al. Acupuncture increases noctur­nal melatonin secretion and reduces insomnia and anxiety: a preliminary report. J Neuropsychiatry Clin Neurosci 2004; 16:19-28.
  • 67. Stener-Victorin E, Lundeberg T, Waldenstrom U, Bileviciute- Ljungar I, Janson PO. Effects of electro-acupuncture on corti- cotropin-releasing factor in rats with experimentally-induced polycystic ovaries. Neuropeptides 2001;35:227-31.
  • 68. Ning CH, Wang GM, Zhao TY, Yu GQ, Duan FW. Therapeu­tical effects of jian pi yi shen prescription on the toxicity reac­tions of postoperative chemotherapy in patients with advanced gastric carcinoma. J Tradit Chin Med 1988;8:113-16.
  • 69. Chen JZ. Clinical effect of chemotherapy combined with Chi­nese herbs and western drugs on leukocytes of gastric cancer patients [Chinese]. Zhong Xi Yi Jie He Za Zhi 1990;10:717-19.
  • 70. Yu RC, Guan CF, Zhang JH. Immune function of cancer pa­tients with spleen-deficiency syndrome [Chinese]. Zhong Xi Yi Jie He Za Zhi 1990;10:535-7,516.
  • 71. Hou J, Liu S, Ma Z, et al. Effects of Gynostemma pentaphyllum makino on the immunological function of cancer patients. J Tradit Chin Med 1991;11:47-52.
  • 72. Rao XQ, Yu RC, Zhang JH. Sheng xue tang on immunological functions of cancer patients with spleen-deficiency syndrome [Chinese]. Zhong Xi Yi Jie He Za Zhi 1991;11:218-19.
  • 73. Li NQ. Clinical and experimental study on shen-qi injection with chemotherapy in the treatment of malignant tumor of digestive tract [Chinese]. Zhongguo Zhong Xi Yi Jie He Za Zhi 1992;12:588-92.
  • 74. Yu G, Ren D, Sun G, Zhang D. Clinical and experimental stud­ies of jpys in reducing side-effects of chemotherapy in late- stage gastric cancer. J Tradit Chin Med 1993;13:31-7.
  • 75. Cao GW, Yang WG, Du P. Observation of the effects of lak/il- 2 therapy combining with Lycium barbarum polysaccharides in the treatment of 75 cancer patients [Chinese]. Zhonghua Zhong Liu Za Zhi 1994;16:428-31.
  • 76. Cheng JH. Clinical study on prevention and treatment to chemo­therapy caused nephrotoxicity with jian-pi yi-qi li-shui decoc­tion [Chinese]. Zhongguo Zhong Xi Yi Jie He Za Zhi 1994;14:331-3.
  • 77. Horie Y, Kato K, Kameoka S, Hamano K. Bu ji (hozai) for treatment of postoperative gastric cancer patients. Am J Chin Med 1994;22:309-19.
  • 78. Lin SY, Liu LM, Wu LC. Effects of Shenmai injection on im­mune function in stomach cancer patients after chemotherapy [Chinese]. Zhongguo Zhong Xi Yi Jie He Za Zhi 1995;15:451-3.
  • 79. Pan MJ, Li YH, Chen LF. Long-term curative effect of 150 cases of nasopharyngeal cancer treated with fu zheng sheng jin decoction and radiotherapy [Chinese]. Zhong Xi Yi Jie He Za Zhi 1985;5:83-5.
  • 80. Wang GT. Treatment of operated late gastric carcinoma with prescription of strengthening the patient's resistance and dis­pelling the invading evil in combination with chemotherapy: follow-up study of 158 patients and experimental study in animals [Chinese]. ZhongXi Yi Jie He Za Zhi 1990;10:712-16.
  • 81. Kawakita T, Nakai S, Kumazawa Y, Miura O, Yumioka E, Nomoto K. Induction of interferon after administration of tra­ditional Chinese medicine, xiao-chai-hu-tang (shosaiko-to). Int J Immunopharmacol 1990;12:515-21.
  • 82. Jin R, Wan LL, Mitsuishi T, et al. Effect of shi-ka-ron and Chinese herbs on cytokine production of macrophage in immunocompromised mice. Am J Chin Med 1994;22:255-66.
  • 83. Feng PF, Liu LM, Shen YY. Effect of shenmai injection on sil- 2r, nk and lak cells in patients with advanced carcinoma. Zhongguo Zhong Xi Yi Jie He Za Zhi 1995;15:87-9.
  • 84. Zhu H, Zhang J. Treatment of stomatological complications in 31 cases of acute leukemia with Chinese herbal drugs. J Tradit Chin Med 1993;13:253-6.
  • 85. Zhu BF. Observation on 17 patients with radio-ulcer with com­bined traditional Chinese medicine and Western medicine therapy [Chinese]. Zhongguo Zhong Xi Yi Jie He Za Zhi 1994;14:89-91,68-9.
  • 86. Whiteside LT. immune suppression in cancer: effects on im­mune cells, mechanisms and future therapeutic intervention. Semin Cancer Biol 2006;16:3-15.
  • 87. Berczi I, Chow DA, Baral E, Nagy E. Neuroimmunoregulation and cancer [review]. Int J Oncol 1998;13:1049-60.
  • 88. Beuth J, Ko HL, Schirrmacher V, Uhlenbruck G, Pulverer G. Inhibition of liver tumor cell colonization in two animal tumor models by lectin blocking with D-galactose or arabinogalactan. Clin Exp Metastasis 1988;6:115-20.
  • 89. Kebudi R, Ayan I, Darendeliler E, et al. Immunologic status in children with brain tumors and the effect of therapy. J Neurooncol 1995;24:219-27.
  • 90. Vuckovic-Dekic LJ, Susnjar S, Stanojevic-Bakic N, Rajner L, Frim O. The protective activity of Thymex L against radiotherapeutically-induced cellular immunodepression in lung cancer patients. Neoplasma 1992;39:171-6.
  • 91. Maier H, Daniel V, Heimlich F, Frank C, Opelz G. Cellular immune defect caused by postoperative irradiation in patients with squamous epithelial carcinomas of the upper aerodigestive tract [German]. HNO 1995;43:364-70.
  • 92. Miyazaki K, Mizutani H, Katabuchi H, Fukuma K, Fujisaki S, Okamura H. Activated (hla-dr+) T-lymphocyte subsets in cervical carcinoma and effects of radiotherapy and immuno- therapy with sizofiran on cell-mediated immunity and sur­vival. Gynecol Oncol 1995;56:412-20.
  • 93. Baniyash M. Chronic inflammation, immunosuppression and can­cer: new insights and outlook. Semin Cancer Biol 2006;16:80-8.
  • 94. Sasada T, Kimura M, Yoshida Y, Kanai M, Takabayashi A. cd4+cd25+ regulatory T cells in patients with gastrointestinal malignancies: possible involvement of regulatory T cells in dis­ease progression. Cancer 2003;98:1089-99.
  • 95. Wichmann MW, Meyer G, Adam M, et al. Detrimental immu­nologic effects of preoperative chemoradiotherapy in advanced rectal cancer. Dis Colon Rectum 2003;46:875-87.
  • 96. Bang S, Kim HS, choo YS, Park SW, chung JB, Song SY. Differences in immune cells engaged in cell-mediated immunity after chemotherapy for far advanced pancreatic cancer. Pan­creas 2006;32:29-36.
  • 97. Koukourakis MI, Ktenidou-Kartali S, Bourikas G, Kartalis G, Tsatalas c. Amifostine protects lymphocytes during radio­therapy and stimulates expansion of the CD95/Fas and cd31 expressing T-cells, in breast cancer patients. Cancer Immunol Immunother 2003;52:127-31.
  • 98. Stevenson FK. Update on cancer vaccines. Curr Opin Oncol 2005;17:573-7.
  • 99. Minev BR. Melanoma vaccines. Semin Oncol2002;29:479-93.
  • 100. Hoffmann TK, Bier H, Whiteside TL. Targeting the immune system: novel therapeutic approaches in squamous cell carci­noma of the head and neck. Cancer Immunol Immunother 2004;53:1055-67.
  • 101. Tsan MF. Toll-like receptors, inflammation and cancer. Semin Cancer Biol 2006;16:32-7.
  • 102. Sen G, Khan AQ, Chen Q, Snapper CM. In vivo humoral im­mune responses to the isolated pneumococcal polysaccharides are dependent on the presence of associated tlr ligands. J Immunol 2005;175:3084-91.
  • 103. Rezaei N. Therapeutic targeting of pattern-recognition receptors. Int Immunopharmacol 2006;6:863-9.
  • 104. Heine H, Ulmer AJ. Recognition of bacterial products by Toll­like receptors. Chem Immunol Allergy 2005;86:99-119.
  • 105. Chang R. Bioactive polysaccharides from traditional Chinese medicine herbs as anticancer adjuvants. JAltern Complement Med 2002;8:559-65.
  • 106. Zeng F, Hon CC, Sit WH, et al. Molecular characterization of Coriolus versicolor PSP-induced apoptosis in human prom- yelotic leukemic HL-60 cells using cdna microarray. Int J Oncol 2005;27:513-23.
  • 107. Ito K, Nakazato H, Koike A, et al., for the Study Group of Immunochemotherapy with PSK for colon cancer. Long-term effect of 5-fluorouracil enhanced by intermittent administra­tion of polysaccharide K after curative resection of colon can­cer. A randomized controlled trial for 7-year follow-up. Int J ColorectalDis 2004;19:157-64.
  • 108. Ohwada S, Ikeya T, Yokomori T, et al. Adjuvant immuno- chemotherapy with oral tegafur/uracil plus PSK in patients with

stage II or III colorectal cancer: a randomised controlled study. Br J Cancer 2004;90:1003-10.

  • 109. Koda K, Miyazaki M, Sarashina H, et al. A randomized con­trolled trial of postoperative adjuvant immunochemotherapy for colorectal cancer with oral medicines. Int J Oncol 2003;23:165-72.
  • 110. Tsang KW, Lam CL, Yan C, et al. Coriolus versicolor polysac- charide peptide slows progression of advanced non-small cell lung cancer. Respir Med 2003;97:618-24.
  • 111. Kanazawa M, Mori Y, Yoshihara K, et al. Effect of psk on the maturation of dendritic cells derived from human peripheral blood monocytes. Immunol Lett 2004;91:229-38.
  • 112. Wong CK, Tse PS, Wong EL, Leung PC, Fung KP, Lam CW. Immunomodulatory effects ofyun zhi and danshen capsules in healthy subjects: a randomized, double-blind, placebo-control­led, crossover study. Int Immunopharmacol 2004;4:201-11.
  • 113. Wong CK, Bao YX, Wong EL, Leung PC, Fung KP, Lam CW. Immunomodulatory activities of yunzhi and danshen in post- treatment breast cancer patients. Am J Chin Med 2005;33:381-95.
  • 114. Munemoto Y, Iida Y, Abe J, et al. Significance of postoperative adjuvant immunochemotherapy after curative resection of colorectal cancers: association between host or tumor factors and survival. Int J Oncol 2002;20:403-11.
  • 115. Hayakawa K, Mitsuhashi N, Saito Y, et al. Effect of Krestin as adjuvant treatment following radical radiotherapy in non-small cell lung cancer patients. Cancer Detect Prev 1997; 21:71-7.
  • 116. Ogoshi K, Satou H, Isono K, Mitomi T, Endoh M, Sugita M. Immunotherapy for esophageal cancer. A randomized trial in combination with radiotherapy and radiochemotherapy. Co­operative Study Group for Esophageal Cancer in Japan. Am J Clin Oncol 1995;18:216-22.
  • 117. Mitomi T, Tsuchiya S, Iijima N, et al. Randomized, controlled study on adjuvant immunochemotherapy with PSK in curatively resected colorectal cancer. The Cooperative Study Group of Surgical Adjuvant Immunochemotherapy for Cancer of Colon and Rectum (Kanagawa). Dis Colon Rectum 1992;35:123-30.
  • 118. Nakazato H, Hoike A, Saji S, et al. Efficacy of immuno- chemotherapy as adjuvant treatment after curative resection of gastric cancer; Study Group of Immunochemotherapy with psk for Gastric Cancer. Lancet 1994;343:1122-6.
  • 119. Shao BM, Dai H, Xu W, Lin ZB, Gao XM. Immune receptors for polysaccharides from Ganoderma lucidum. Biochem Biophys Res Commun 2004;323:133-141.
  • 120. Lin ZB. Cellular and molecular mechanisms of immuno­modulation by Ganoderma lucidum. J Pharmacol Sci 2005; 99:144-53.
  • 121. Kuo MC, Weng CY, Ha CL, Wu MJ. Ganoderma lucidum mycelia enhance innate immunity by activating nf-kb. J Ethno- pharmacol 2006;103:217-22.
  • 122. Gao Y, Zhou S, Jiang W, Huang M, Dai X. Effects of ganopoly (a Ganoderma lucidum polysaccharide extract) on the immune functions in advanced-stage cancer patients. Immunol Invest 2003;32:201-15.
  • 123. Kodama N, Asakawa A, Inui A, Masuda Y, Nanba H. Enhance­ment of cytotoxicity of NK cells by D-Fraction, a polysaccha- ride from Grifola frondosa. Oncol Rep 2005;13:497-502.
  • 124. Kodama N, Komuta K, Nanba H. Effect of maitake (Grifola frondosa) D-Fraction on the activation of NK cells in cancer patients. J Med Food 2003;6:371-7.
  • 125. Kodama N, Murata Y, Asakawa A, et al. Maitake D-Fraction enhances antitumor effects and reduces immunosuppression by mitomycin-C in tumor-bearing mice. Nutrition 2005;21:624-9.
  • 126. Kodama N, Komuta K, Nanba H. Can maitake MD-Fraction aid cancer patients? Altern Med Rev 2002;7:236-9.
  • 127. Inoue A, Kodama N, Nanba H. Effect of maitake (Grifola frondosa) D-Fraction on the control of the T lymph node Th- 1/Th-2 proportion. Biol Pharm Bull 2002;25:536-40.
  • 128. Shao BM, Xu W, Dai H, Tu P, Li Z, Gao XM. A study on the immune receptors for polysaccharides from the roots of Astra­galus membranaceus, a Chinese medicinal herb. Biochem Biophys Res Commun 2004;320:1103-11.
  • 129. Shin HJ, Kim YS, Kwak YS, Song YB, Kim YS, Park JD. Enhancement of antitumor effects of paclitaxel (Taxol) in com­bination with red ginseng acidic polysaccharide (rgap). Planta Med 2004;70:1033-8.
  • 130. Shin JY, Song JY, Yun YS, Yang HO, Rhee DK, Pyo S. Immunostimulating effects of acidic polysaccharides extract of Panax ginseng on macrophage function. Immunopharmacol Immunotoxicol 2002;24:469-82.
  • 131. Han SK, Song JY, Yun YS, Yi SY. Ginsan improved Th1 im­mune response inhibited by gamma radiation. Arch Pharm Res 2005;28:343-50.
  • 132. Lim TS, Na K, Choi EM, Chung JY, Hwang JK. Immuno- modulating activities of polysaccharides isolated from Panax ginseng. J Med Food 2004;7:1-6.
  • 133. Ooi VE, Liu F. Immunomodulation and anti-cancer activity of polysaccharide-protein complexes. Curr Med Chem 2000; 7:715-29.
  • 134. Zaidman BZ, Yassin M, Mahajna J, Wasser SP. Medicinal mushroom modulators of molecular targets as cancer therapeu­tics. Appl Microbiol Biotechnol 2005;67:453-68.
  • 135. Lindequist U, Niedermeyer TH, Julich WD. The pharmaco­logical potential ofmushrooms. EvidBased Complement Alternat Med 2005;2:285-99.
  • 136. Han SB, Yoon YD, Ahn HJ, et al. Toll-like receptor-mediated activation of B cells and macrophages by polysaccharide iso­lated from cell culture of Acanthopanax senticosus. IntImmuno- pharmacol 2003;3:1301-12.
  • 137. Schepetkin IA, Quinn MT. Botanical polysaccharides: macro­phage immunomodulation and therapeutic potential. Int Immunopharmacol 2006;6:317-33.
  • 138. Kabelitz D, Wesch D, Oberg HH. Regulation of regulatory T cells: role of dendritic cells and Toll-like receptors. Crit Rev Immunol 2006;26:291-306.
  • 139. Sutmuller RP, van Duivenvoorde LM, van Elsas A, et al. Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of cd25+ regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses. J Exp Med 2001;194:823-32.
  • 140. Yang L, DeBusk LM, Fukuda K, et al. Expansion of myeloid immune suppressor Gr+cD11b+ cells in tumor-bearing host di­rectly promotes tumor angiogenesis. Cancer Cell 2004;6:409-21.
  • 141. Terabe M, Park JM, Berzofsky JA. Role of il-13 in regulation of anti-tumor immunity and tumor growth. Cancer Immunol Immunother 2004;53:79-85.
  • 142. Wang RF. Regulatory T cells and Toll-like receptors in cancer therapy. Cancer Res 2006;66:4987-90.
  • 143. Bartz H, Avalos NM, Baetz A, Heeg K, Dalpke AH. Involve­ment of suppressors of cytokine signaling in Toll-like receptor- mediated block of dendritic cell differentiation. Blood 2006;108:4102-8.
  • 144. Garay RP, Viens P, Bauer J, et al. Cancer relapse under chemo­therapy: why tlr2/4 receptor agonists can help. Eur J Phar­macol 2007;563:1-17.
  • 145. Liyanage UK, Moore TT, Joo HG, et al. Prevalence of regula­tory T cells is increased in peripheral blood and tumor micro­environment of patients with pancreas or breast adenocarci- noma. J Immunol 2002;169:2756-61.
  • 146. Nava-Parada P, Forni G, Knutson KL, Pease LR, Celis E. Pep- tide vaccine given with a Toll-like receptor agonist is effective for the treatment and prevention of spontaneous breast tumors. Cancer Res 2007;67:1326-34.
  • 147. Turnbull JL, Patchen ML, Scadden DT. The polysaccharide, PGG-glucan, enhances human myelopoiesis by direct action in­dependent of and additive to early-acting cytokines. Acta Haematol 1999;102:66-71.
  • 148. Ahn JY, Choi IS, Shim JY, et al. The immunomodulator ginsan induces resistance to experimental sepsis by inhibiting Toll­like receptor-mediated inflammatory signals. Eur J Immunol 2006;36:37-45.
  • 149. Cheung nk, Modak S, Vickers A, Knuckles B. Orally admin­istered beta-glucans enhance anti-tumor effects of monoclonal antibodies. Cancer Immunol Immunother 2002;51:557-64.
  • 150. Koski GK, Czerniecki BJ. Combining innate immunity with radiation therapy for cancer treatment. Clin Cancer Res 2005;11:7-11.
  • 151. Demaria S, Bhardwaj N, McBride WH, Formenti SC. Combin­ing radiotherapy and immunotherapy: a revived partnership. Int J Radiat Oncol Biol Phys 2005;63:655-66.
  • 152. United States, National Institutes of Health, ClinicalTrials.gov. Beta-Glucan and rituximab in treating young patients with re­lapsed or progressive lymphoma or leukemia, or lymphoproli- ferative disorder related to donor stem cell transplantation [Web page]. Bethesda: National Institutes of Health; 2006. [Avail­able at: www.clinicaltrials.gov/ct/gui/show/NCT00087009; cited July 2007]
  • 153. Chan WL, Lam DT, Law HK, et al. Ganoderma lucidum myc­elium and spore extracts as natural adjuvants for immuno- therapy. J Altern Complement Med 2005;11:1047-57.
  • 154. Bianchi M, Jotti E, Sacerdote P, Panerai AE. Traditional acu­puncture increases the content of beta-endorphin in immune cells and influences mitogen induced proliferation. Am J Chin Med 1991;19:101-4.
  • 155. Yuan J, Zhou R. Effect of acupuncture on T-lymphocyte and its subsets from the peripheral blood of patients with malig­nant neoplasm [Chinese]. Zhen Ci Yan Jiu 1993;18:174-7.
  • 156. Wu B, Zhou RX, Zhou MS. Effect of acupuncture on interleukin-2 level and NK cell immunoactivity of peripheral blood of malignant tumor patients [Chinese]. Zhongguo Zhong Xi Yi Jie He Za Zhi 1994;14:537-9.
  • 157. Wu B, Zhou RX, Zhou MS. Effect of acupuncture on immunomodulation in patients with malignant tumors [Chi­nese]. Zhongguo Zhong Xi Yi Jie He Za Zhi 1996;16:139-41.
  • 158. Yang J, Zhao R, Yuan J, et al. The experimental study of prevention and treatment of the side-effects of chemotherapy with acupuncture (comparison among the effect of acupuncture

at different acupoints) [Chinese]. Zhen Ci Yan Jiu 1994; 19:75-8.

  • 159. Liu LJ, Guo CJ, Jiao XM. Effect of acupuncture on immuno­logic function and histopathology of transplanted mammary cancer in mice. Zhongguo Zhong Xi Yi Jie He Za Zhi 1995;15:615-17.
  • 160. Sato T, Yu Y, Guo SY, Kasahara T, Hisamitsu T. Acupuncture stimulation enhances splenic natural killer cell cytotoxicity in rats. Jpn J Physiol 1996;46:131-6.
  • 161. Petti F, Bangrazi A, Liguori A, Reale G, Ippoliti F. Effects of acupuncture on immune response related to opioid-like pep- tides. J Tradit Chin Med 1998;18:55-63.
  • 162. Zhou JQ, Li ZH, Jin PL. A clinical study on acupuncture for prevention and treatment of toxic side-effects during radiotherapy and chemotherapy. J Tradit Chin Med 1999; 19:16-21.
  • 163. L u W, Hu D, Dean-Clower E, et al. Acupuncture for chemotherapy- induced leukopenia: exploratory meta-analysis of random­ized controlled trials. JSoc Integrat Oncol 2007;5:1-10.
  • 164. Tang ZY. Hepatocellular carcinoma. J Gastroenterol Hepatol 2000;15(suppl):G1-7.
  • 165. Gao JD, Shao YF, Xu Y, et al. Tight association of hepatocellu­lar carcinoma with hbv infection in North China. Hepatobiliary PancreatDis Int 2005;4:46-9.
  • 166. Shi J, Zhu L, Liu S, Xie WF. A meta-analysis of case-control studies on the combined effect of hepatitis b and c virus infec­tions in causing hepatocellular carcinoma in China. Br J Cancer 2005;92:607-12.
  • 167. Cheng WM, Chan KH, Chen HL, et al. Assessing the risk of nasopharyngeal carcinoma on the basis of ebv antibody spec­trum. Int J Cancer 2002;97:489-92.
  • 168. Zong Y, Liu K, Zhong B, Chen G, Wu W. Epstein-Barr virus infection of sinonasal lymphoepithelial carcinoma in Guang­zhou. Chin Med J 2001;114:132-6.
  • 169. Jarett RF. Risk factors for Hodgkin's lymphoma by ebv status and significance of detection of ebv genomes in serum of pa­tients with EBv-associated Hodgkin's lymphoma. Leuk Lym­phoma 2003;44(suppl 3):S27-32.
  • 170. Lo YM, Chan WY, Ng EK, et al. Circulating Epstein-Barr virus DNA in the serum of patients with gastric carcinoma. Clin Can­cer Res 2001;7:1856-9.
  • 171. Ng WT, Yau TK, Yung RW, et al. Screening for family members of patients with nasopharyngeal carcinoma. Int J Cancer 2005;113:998-1001.
  • 172. Li H, Zhao M, Qiu X, Ding X, Tan Y, Wu X. Characterization of a new type HPV16 E7 variant isolated from cervical cancer highest incidence area in Hubei province of China. Eksp Onkol 2004;26:48-54.
  • 173. Chang F, Syrjänen S, Shen Q, et al. Evaluation of hpv, cmv, hsv and ebv in esophageal squamous cell carcinomas from a high- incidence area of China. Anticancer Res 2000;20:3935-40.
  • 174. Li T, Lu ZM, Chen KN, et al. Human papillomavirus type 16 is an important infectious factor in the high incidence of esophageal cancer in Anyang area of China. Carcinogenesis 2001;22:929-34.
  • 175. Mellin H, Friesland S, Lewensohn R, Dalianis T, Munck- Wikland E. Human papillomavirus (hpv) dna in tonsillar can­cer: clinical correlates, risk of relapse, and survival. Int J Can­cer 2000;89:300-4.
  • 176. Almadori G, Galli J, Cadoni G, Bussu F, Maurizi M. Human papillomavirus infection and cyclin D1 gene amplification in laryngeal squamous cell carcinoma: biologic function and clini­cal significance. Head Neck 2002;24:597-604.
  • 177. Yu Y, Morimoto T, Sasa M, et al. Human papillomavirus type 33 dna in breast cancer in Chinese. Breast Cancer 2000; 7:33-6.
  • 178. Cheng HM, Tsai MC. Regression of hepatocellular carcinoma spontaneous or herbal medicine related? Am J Chin Med 2004; 32:579-85.
  • 179. Yun TK, Choi SY, Yun HY. Epidemiological study on cancer prevention by ginseng: are all kinds of cancers preventable by ginseng? J Korean Med Sci 2001;16(suppl):S19-27.
  • 180. Zhong L, Goldberg MS, Gao YT, Hanley JA, Parent ME, Jin F. A population-based case-control study of lung cancer and green tea consumption among women living in Shanghai, China. Epi­demiology 2001;12:695-700.
  • 181. Sinclair S. Chinese herbs: a clinical review of Astragalus, Ligusticum, and Schizandrae. Altern Med Rev 1998;3:338-44.
  • 182. Block KI, Mead MN. Immune system effects of echinacea, ginseng, and astragalus: a review. Integr Cancer Ther 2003; 2:247-67.
  • 183. Qian ZH, Vermund SH, Wang N. Risk of hiv/aids in China: subpopulations of special importance. Sex Transm Infect 2005;81:442-7. [Erratum in: Sex Transm Infect2006;82:93]
  • 184. Wu JA, Attele AS, Zhang L, Yuan CS. Anti-Hiv activity of medicinal herbs: usage and potential development. Am J Chin Med 2001;29:69-81.
  • 185. Tani M, Nagase M, Nishiyama T, Yamamoto T, Matusa R. The effects of long-term herbal treatment for pediatric aids. Am J Chin Med 2002;30:51-64.
  • 186. Matsui Y, Uhara J, Satoi S, et al. Improved prognosis of post­operative hepatocellular carcinoma patients when treated with functional foods: a prospective cohort study. J Hepatol 2002; 37:78-86.
  • 187. Lin H, She YH, Cassileth BR, Sirotnak F, Cunningham Rundles S. Maitake ß-glucan MD-Fraction enhances bone marrow colony formation and reduces doxorubicin toxicity in vitro. Int Immunopharmacol 2004;4:91-9.
  • 188. Fullerton SA, Samadi AA, Torterelis DG, et al. Induction of apoptosis in human prostatic cancer cells with beta- glucan (Maitaki mushroom polysaccharide). Molec Urol 2000;4:7-13.
  • 189. Jiang J, Slivova V, Harvey K, Valachovicova T, Sliva D. Ganoderma lucidum suppresses growth of breast cancer cells through the inhibition of Akt/NF-KB signaling. Nutr Cancer 2004;49:209-16.
  • 190. Sliva D. Ganoderma lucidum (Reishi) in cancer treatment. Integr Cancer Ther 2003;2:358-64.
  • 191. Yang CS, Wang ZY. Tea and cancer. J Natl Cancer Inst 1993;85:1038-49.
  • 192. Kaegi E. Unconventional therapies for cancer: 2. Green tea. The Task Force on Alternative Therapies of the Canadian Breast Cancer Research Initiative. CMAJ 1998;158:1033-5.
  • 193. Yun TK, Choi SY. Non-organ specific cancer prevention of ginseng: a prospective study in Korea. Int J Epidemiol 1998;27:359-64.
  • 194. McKenna DJ, Hughes K, Jones K. Green tea monograph. Altern Ther Health Med 2000;6:61-8,70-2,74 passim.
  • 195. Cao Y, Cao R. Angiogenesis inhibited by drinking tea. Nature 1999;398:381.
  • 196. Fujiki H, Suganuma M, Okabe S, et al. Mechanistic findings of green tea as cancer preventive for humans. Proc Soc Exp Biol Med 1999;220:225-8.
  • 197. Lee YN, Lee HY, Chung HY, et al. In vitro induction of differ­entiation by ginsenosides in F9 teratocarcinoma cells. Eur J Cancer 1996;32A:1420-8.
  • 198. Jacobsen BK, Knutsen SF, Fraser GE. Does high soy milk intake reduce prostate cancer incidence? The Adventist Health Study (United States). Cancer Causes Control 1998;9:553-7.
  • 199. Kamat AM, Lamm DL. Chemoprevention of urological cancer. J Urol 1999;161:1748-60.
  • 200. Moyad MA. Soy, disease prevention, and prostate cancer. Semin Urol Oncol 1999;17:97-102.
  • 201. Stephens FO. The rising incidence of breast cancer in women and prostate cancer in men. Dietary influences: a possible pre­ventive role for nature's sex hormone modifiers-the phyto­estrogens (review). Oncol Rep 1999;6:865-70.
  • 202. Adlercreutz H, Mazur W, Bartels P, et al. Phytoestrogens and prostate disease. J Nutr 2000;130:658S-9S.
  • 203. Aronson WJ, Tymchuk CN, ElashoffRM, et al. Decreased growth of human prostate LNCaP tumors in scid mice fed a low-fat, soy protein diet with isoflavones. Nutr Cancer 1999;35:130-6.
  • 204. Davis JN, Kucuk O, Sarkar FH. Genistein inhibits nf-k b acti­vation in prostate cancer cells. Nutr Cancer 1999;35:167-74.
  • 205. Zhou JR, Gugger ET, Tanaka T, Guo Y, Blackburn GL, Clinton SK. Soybean phytochemicals inhibit the growth of transplant­able human prostate carcinoma and tumor angiogenesis in mice. J Nutr 1999;129:1628-35.
  • 206. Davis JN, Muqim N, Bhuiyan M, Kucuk O, Pienta KJ, Sarkar FH. Inhibition of prostate specific antigen by genistein in pros­tate cancer cells. Int J Oncol 2000;16:1091-7.
  • 207. United States, National Institutes of Health, National Cancer Institute. Notes. cam scientist tests lung cancer herb. NCI Can­cer Bull 2006;3(7):7. [Available online at: www.cancer.gov/ NciCancerBulletin/Nci_Cancer_Bulletin_021406.pdf; cited Oc­tober 28, 2007]
  • 208. Cui Y, Shu XO, Gao YT, Cai H, Tao MH, Zheng W. Associa­tion of ginseng use with survival and quality of life among breast cancer patients. Am J Epidemiol 2006;163:645-53.
  • 209. Mowrey DB, Clayson DE. Motion sickness, ginger, and psy- chophysics. Lancet 1982;1:655-7.
  • 210. Grentved A, Brask T, Kambskard J, Hentzer E. Ginger root against seasickness. A controlled trial on the open sea. Acta Otolaryngol 1988;105:45-9.
  • 211. Grentved A, Hentzer E. Vertigo-reducing effects of ginger root. A controlled clinical study. ORL J Otorhinolaryngol Relat Spec 1986;48:282-6.
  • 212. Bone ME, Wilkinson DJ, Young JR, McNeil J, Charlton S. Ginger root-a new antiemetic. The effect of ginger root on postoperative nausea and vomiting after major gynaecological surgery. Anaesthesia 1990;45:669-71.
  • 213. Fischer-Rasmussen W, Kjaer SK, Dahl C, Asping U. Ginger treatment of hyperemesis gravidarum. Eur J Obstet Gynecol Reprod Biol 1991;38:19-24.
  • 214. Keating A, Chez RA. Ginger syrup as an antiemetic in early pregnancy. Altern Ther Health Med 2002;8:89-91.
  • 215. Hien TV, Huong NB, Hung PM, Duc NB. Radioprotective effects of vitexina for breast cancer patients undergoing radio­therapy with cobalt-60. Integrat Cancer Ther 2002;1:38-43.
  • 216. Ling HY, Wang NZ, Zhu HZ. Preliminary study of traditional Chinese medicine-Western medicine treatment of patients with primary liver carcinoma [Chinese]. Zhong Xi Yi Jie He Za Zhi 1989;9:348-9.
  • 217. Mok TS, Yeo W, Johnson PJ, et al. A double-blind placebo- controlled randomized study of Chinese herbal medicine as complementary therapy for reduction of chemotherapy-induced toxicity. Ann Oncol 2007;18:768-74.
  • 218. Zhang M, Liu X, Li J, He L, Tripathy D. Chinese medicinal herbs to treat the side-effects of chemotherapy in breast cancer patients. Cochrane Database Syst Rev 2007;(2):14651858. CD004921.pub2.
  • 219. Brezden CB, Phillips KA, Abdolell M, Bunston T, Tannock IF. Cognitive function in breast cancer patients receiving adjuvant chemotherapy. J Clin Oncol 2000;18:2695-701.
  • 220. Schagen SB, van Dam FS, Muller MJ, Boogerd W, Lindeboom J, Bruning PF. Cognitive deficits after postoperative adjuvant chemotherapy for breast carcinoma. Cancer 1999;85:640-50.
  • 221. Meyers CA, Abbruzzese JL. Cognitive functioning in cancer patients: effect of previous treatment. Neurology 1992;42:434-6.
  • 222. Ahles TA, Saykin AJ, Noll WW, et al. The relationship of apoe genotype to neuropsychological performance in long-term can­cer survivors treated with standard dose chemotherapy. Psychooncology 2003;12:612-19.
  • 223. Tannock IF, Ahles TA, Ganz PA, Van Dam FS. Cognitive im­pairment associated with chemotherapy for cancer: report of a workshop. J Clin Oncol 2004;22:2233-9.
  • 224. Fan HG, Houede-Tchen N, Yi QL, et al. Fatigue, menopausal symptoms, and cognitive function in women after adjuvant chemotherapy for breast cancer: 1- and 2-year follow-up of a prospective controlled study. J Clin Oncol 2005;23:8025-32.
  • 225. Wefel JS, Lenzi R, Theriault R, Buzdar AU, Cruickshank S, Meyers CA. "Chemobrain" in breast carcinoma? A prologue. Cancer 2004;101:466-75.
  • 226. Wefel JS, Kayl AE, Meyers CA. Neuropsychological dys­function associated with cancer and cancer therapies: a con­ceptual review of an emerging target. Br J Cancer 2004; 90:1691-6.
  • 227. Taphoorn MJB, Schiphorst AK, Snoek FJ, et al. Cognitive functions and quality of life in patients with low-grade glio- mas: the impact of radiotherapy. Ann Neurol 1994;36:48-54.
  • 228. Taphoorn MJ, Klein M. Cognitive deficits in adult patients with brain tumours. Lancet Neurol 2004;3:159-68.
  • 229. Taylor BV, Buckner JC, Cascino TL, et al. Effects of radiation and chemotherapy on cognitive function in patients with high­grade glioma. J Clin Oncol 1998;16:2195-201.
  • 230. Danysz W, Parsons CG. The nmda receptor antagonist memantine as a symptomatological and neuroprotective treat­ment for Alzheimer's disease: preclinical evidence. Int J Geriatr Psychiatry 2003;18:S23-32.
  • 231. van Beek TA. Ginkgolides and bilobalide: their physical, chro- matographic and spectroscopic properties. Bioorg Med Chem 2005;13:5001-12.
  • 232. Stramgaard K, Nakanishi K. Chemistry and biology of terpene trilactones from Ginkgo biloba. Angew Chem Int Ed Engl 2004;43:1640-58.
  • 233. Biber A. Pharmacokinetics of Ginkgo biloba extracts. Pharma­copsychiatry 2003;36(suppl 1):S32-7.
  • 234. Menku A, Koc RK, Tayfur V, Saraymen R, Narin F, Akdemir H. Effects of mexiletine, Ginkgo biloba extract (EGb 761), and their combination on experimental head injury. Neurosurg Rev 2003;26:288-91.
  • 235. Defeudis FV. Bilobalide and neuroprotection. Pharmacol Res 2002;46:565-8.
  • 236. DeFeudis FV, Papadopoulos V, Drieu K. Ginkgo biloba ex­tracts and cancer: a research area in its infancy. Fundam Clin Pharmacol 2003;17:405-17.
  • 237. Ahlemeyer B, Krieglstein J. Pharmacological studies support­ing the therapeutic use of Ginkgo biloba extract for Alzheim­er's disease. Pharmacopsychiatry 2003;36(suppl 1):S8-14.
  • 238. Bressler R. Herb-drug interactions: interactions between Ginkgo biloba and prescription medications. Geriatrics 2005;60:30-3.
  • 239. Santos RF, Galduroz JC, Barbieri A, Castiglioni ML, Ytaya LY, Bueno OF. Cognitive performance, SPECT, and blood viscosity in elderly non-demented people using Ginkgo biloba. Pharmacopsychiatry 2003;36:127-33.
  • 240. Hu B, Sun S, Mei G, Chen L, Tong E. Protective effects of Ginkgo biloba extract on rats during cerebral ischemia/ reperfusion. Chin Med J (Engl) 2002;115:1316-20.
  • 241. Wang YS, Xu L, Ma K, Wang S, Wang JJ. Protective effects of Ginkgo biloba extract 761 against glutamate-induced neuro­toxicity in cultured retinal neuron. Chin Med J (Engl) 2005; 118:948-52.
  • 242. Ivic L, Sands TT, Fishkin N, Nakanishi K, Kriegstein AR, Stramgaard K. Terpene trilactones from Ginkgo biloba are an­tagonists of cortical glycine and gabaa receptors. J Biol Chem 2003;278:49279-85.
  • 243. Kanowski S, Hoerr R. Ginkgo biloba extract EGb 761 in de­mentia: intent-to-treat analyses of a 24-week, multi-center, double-blind, placebo-controlled, randomized trial. Pharmacopsychiatry 2003;36:297-303.
  • 244. Birks J, Grimley Evans J. Ginkgo biloba for cognitive impair­ment and dementia. Cochrane Database Syst Rev 2002;(4): 14651858.CD003120.pub2.
  • 245. van Dongen M, van Rossum E, Kessels A, Sielhorst H, Knipschild P. Ginkgo for elderly people with dementia and age-associated memory impairment: a randomized clinical trial. J Clin Epidemiol 2003;56:367-76.
  • 246. Kurz A, Van Baelen B. Ginkgo biloba compared with cholineste- rase inhibitors in the treatment of dementia: a review based on meta-analyses by the Cochrane collaboration. Dement Geriatr Cogn Disord 2004;18:217-26.
  • 247. Shaw EG. A phase ii study of Ginkgo biloba in irradiated brain tumor patients: effects on quality of life and cognitive function [abstract 61]. In: Society for Integrative Oncology (sio). Online program planner for the 2nd International Conference; Novem­ber 10-12, 2005; San Diego, CA [Web tool]. Mt. Royal, NJ: sio; n.d. [Available by key word search at: www.abstracts online.com/viewer/?mkey=%7B52C9E2CA%2D010B% 2D4149%2D9D7C%2DD25107EF113A%7D; cited October 28, 2007]
  • 248. Shaw EG. Phase II studies of donepezil and Ginkgo biloba in irradiated brain tumor patients. Research base protocol CCCWFU 97100. Winston-Salem, NC: Comprehensive Cancer Center, Wake Forest University; 2000. [Available online at: www1. wfubmc.edu/NR/rdonlyres/4A8DC232-84E1-4401-B673- C6C1617A9BD2/0/97100Am4protocol.pdf; cited July 3,2007]
  • 249. Hasegawa H. Proof ofthe mysterious efficacy of ginseng: basic and clinical trials: metabolic activation of ginsenoside: deglycosylation by intestinal bacteria and esterification with fatty acid. J Pharmacol Sci 2004;95:153-7.
  • 250. Kim S, Ahn K, Oh TH, Nah SY, Rhim H. Inhibitory effect of ginsenosides on nmda receptor-mediated signals in rat hip- pocampal neurons. Biochem Biophys Res Commun 2002; 296:247-54.
  • 251. Kim S, Rhim H. Ginsenosides inhibit nmda receptor-mediated epileptic discharges in cultured hippocampal neurons. Arch Pharm Res 2004;27:524-30.
  • 252. Kim S, Kim T, Ahn K, Park WK, Nah SY, Rhim H. Ginsenoside Rg3 antagonizes nmda receptors through a glycine modulatory site in rat cultured hippocampal neurons. Biochem Biophys Res Commun 2004;323:416-24.
  • 253. Radad K, Gille G, Moldzio R, Saito H, Rausch WD. Ginse­nosides Rb1 and Rg1 effects on mesencephalic dopaminergic cells stressed with glutamate. Brain Res 2004;1021:41-53.
  • 254. Bao HY, Zhang J, Yeo SJ, et al. Memory enhancing and neuroprotective effects of selected ginsenosides. Arch Pharm Res 2005;28:335-42.
  • 255. Kennedy DO, Scholey AB, Wesnes KA. Differential, dose dependent changes in cognitive performance following acute administration of a Ginkgo biloba/Panax ginseng combi­nation to healthy young volunteers. Nutr Neurosci 2001;4:399-412.
  • 256. Johnson A, Jiang N, Stuba E. Whole ginseng effects on human response to demands for performance. In: Proceedings of the 3rd International Ginseng Symposium; September 8-10, 1980; Seoul, Korea. Seoul: Korea Ginseng and Tobacco Research In­stitute; 1980: 244.
  • 257. Moretti R, Torre P, Antonello RM, et al. Neuropsychological evaluation of late-onset post-radiotherapy encephalopathy: a comparison with vascular dementia. J Neurol Sci 2005;229- 230:195-200.
  • 258. Dundee JW, Chestnutt WN, Ghaly RG, Lynas AG. Traditional Chinese acupuncture: a potentially useful antiemetic? Br Med J (Clin Res Ed) 1986;293:583-4.
  • 259. Dundee JW, Ghaly RG, Fitzpatrick KT, Abram WP, Lynch GA. Acupuncture prophylaxis of cancer chemotherapy-induced sickness. J R Soc Med 1989;82:268-71.
  • 260. al-Sadi M, Newman B, Julious SA. Acupuncture in the preven­tion of postoperative nausea and vomiting. Anaesthesia 1997;52:658-61.
  • 261. Schlager A, Offer T, Baldissera I. Laser stimulation of acupunc­ture point P6 reduces postoperative vomiting in children un­dergoing strabismus surgery. Br J Anaesth 1998;81:529-32.
  • 262. Lee A, Done ML. The use of nonpharmacologic techniques to prevent postoperative nausea and vomiting: a meta-analysis. Anesth Analg 1999;88:1362-9.
  • 263. nih Consensus Development Panel on Acupuncture. nih con­sensus conference. Acupuncture. JAMA 1998;280:1518-24.
  • 264. Shen J, Wenger N, Glaspy J, et al. Electroacupuncture for con­trol of myeloablative chemotherapy-induced emesis: a randomized controlled trial. JAMA 2000;284:2755-61.
  • 265. Roscoe JA, Matteson SE, Morrow GR, et al. Acustimulation wrist bands are not effective for the control of chemotherapy- induced nausea in women with breast cancer. J Pain Symptom Manage 2005;29:376-84.
  • 266. Ezzo J, Vickers A, Richardson MA, et al. Acupuncture-point stimulation for chemotherapy-induced nausea and vomiting. J Clin Oncol 2005;23:7188-98.
  • 267. Thompson JW, Filshie J. Transcutaneous electrical nerve stimu­lation (tens) and acupuncture. In: Doyle D, Hanks GWC, MacDonald N, eds. Oxford Textbook of Palliative Medicine. 2nd ed. Oxford, U.K.: Oxford University Press; 1998: 421-37.
  • 268. Filshie J, Redman D. Acupuncture and malignant pain prob­lems. Eur J Surg Oncol 1985;11:389-94.
  • 269. Filshie J. Acupuncture for malignant pain. Acupunct Med 1984;2:12-14.
  • 270. Filshie J, Bolton T, Browne D, Ashley S. Acupuncture and self acupuncture for long-term treatment of vasomotor symptoms in cancer patients-audit and treatment algorithm. Acupunct Med 2005;23:171-80.
  • 271. Alimi D, Rubino C, Pichard-Leandri E, Fermand-Brule S, Dubreuil-Lemaire ML, Hill C. Analgesic effect of auricular acupuncture for cancer pain: a randomized, blinded, controlled trial. J Clin Oncol 2003;21:4120-6.
  • 272. Wong R, Sagar S. Acupuncture treatment for chemotherapy- induced peripheral neuropathy-a case series. Acupunct Med 2006;24:87-91.
  • 273. Lee H, Schmidt K, Ernst E. Acupuncture for the relief of cancer- related pain: a systematic review. Eur J Pain 2005;9:437-44.
  • 274. Barlas P, Ting SL, Chesterton LS, Jones PW, Sim J. Effects of intensity of electroacupuncture upon experimental pain in healthy human volunteers: a randomized, double-blind, pla­cebo-controlled study. Pain 2006;122:81-9.
  • 275. Pomeranz B, Niznik G. Codetron: a new electrotherapy device overcomes the habituation problems of conventional tens de­vices. Am JElectromed 1987;2:22-6.
  • 276. Patel M, Gutzwiller F, Paccaud F, Marazzi A. A meta-analysis of acupuncture for chronic pain. Int JEpidemiol 1989;18:900-6.
  • 277. Gadsby JG, Flowerdew MW. Review: transcutaneous electri­cal nerve stimulation reduces pain and improves range of move­ment in chronic low-back pain. Evid Based Med 1997;2:107.
  • 278. Ernst E, White AR. Acupuncture for back pain: a meta-analy- sis of randomized controlled trials. Arch Intern Med 1998;158:2235-41.
  • 279. Ghoname EA, Craig WF, White PF, et al. Percutaneous electri­cal nerve stimulation for low back pain: a randomized crosso­ver study. JAMA 1999;281:818-23.
  • 280. Blom M, Dawidson I, Angmar-Mânsson B. The effect of acu­puncture on salivary flow rates in patients with xerostomia. Oral Surg Oral Med Oral Pathol 1992;73:293-8.
  • 281. Blom M, Lundeberg T, Dawidson I, Angmar-Mânsson B. Ef­fects on local blood flux of acupuncture stimulation used to treat xerostomia in patients suffering from Sjogren's syndrome. J Oral Rehabil 1993;20:541-8.
  • 282. Talal N, Quinn JH, Daniels TE. The clinical effects of elec­trostimulation on salivary function of Sjögrens syndrome pa­tients: a placebo controlled study. Rheumatol Int 1992;12: 43-5.
  • 283. Blom M, Dawidson I, Fernberg JO, Johnson G, Angmar- Mânsson B. Acupuncture treatment of patients with radiation- induced xerostomia. Eur J Cancer B Oral Oncol 1996; 32B:182-90.
  • 284. Blom M, Lundeberg T. Long-term follow-up of patients treated with acupuncture for xerostomia and the influence of addi­tional treatment. Oral Dis 2000;6:15-24.
  • 285. Rydholm M, Strang P. Acupuncture for patients in hospital- based home care suffering from xerostomia. J Palliat Care 1999;15:20-3.
  • 286. Johnstone PA, Peng YP, May BC, Inouye WS, Niemtzow RC. Acupuncture for pilocarpine-resistant xerostomia following radiotherapy for head and neck malignancies. Int J Radiat Oncol Biol Phys 2001;50:353-7.
  • 287. Wong RK, Jones GW, Sagar SM, Babjak AF, Whelan T. A phase i/ii study in the use of acupuncture-like transcutaneous nerve stimulation in the treatment of radiation-induced xerostomia in head-and-neck cancer patients treated with radical radiotherapy. Int J Radiat Oncol Biol Phys 2003; 57:472-80.
  • 288. Deng G, Hou BL, Holodny AI, Vickers AJ, Cassileth BR. Randomized controlled trial of functional magnetic resonance imaging (fMRi) changes associated with acupuncture at a point used to treat xerostomia versus sham acupuncture or gustatory stimulation [abstract]. In: Society for Integrative Oncology (sio). Abstracts of the 3rd International Conference; Novem­ber 9-11, 2006; Boston, MA. Mt. Royal, NJ: sio; 2006. [Available online at: www.integrativeonc.org/images/ 2006%20Conference %20Abstracts%20&%20Posters.pdf; cited October 28, 2007]
  • 289. Widerstrom-Noga E, Dyrehag LE, Borglum-Jensen L, Aslund PG, Wenneberg B, Andersson SA. Pain threshold responses to two different modes of sensory stimulation in patients with orofacial muscular pain: psychological considerations. J Orofac Pain 1998;12:27-34.
  • 290. Lissoni P, Paolorossi F, Tancini G, et al. Is there a role for melatonin in the treatment of neoplastic cachexia? Eur J Can­cer 1996;32A:1340-3.
  • 291. Campbell SS, Murphy PJ. Extraocular circadian phototrans- duction in humans. Science 1998;279:396-9.
  • 292. Glaus A. Fatigue and cachexia in cancer patients. Support Care Cancer 1998;6:77-8.
  • 293. Stone P, Richards M, Hardy J. Fatigue in patients with cancer. Eur J Cancer 1998;34:1670-6.
  • 294. Bonta IL. Acupuncture beyond the endorphin concept? Med Hypotheses 2002;58:221-4.
  • 295. Han JS. Acupuncture and endorphins. Neurosci Lett 2004;361:258-61.
  • 296. Han JS. Electroacupuncture: an alternative to antidepressants for treating affective diseases. Int J Neurosci 1986;29:79-92.
  • 297. Roschke J, Wolf C, Muller MJ, et al. The benefit from whole body acupuncture in major depression. J Affect Disord 2000;57:73-81.
  • 298. Smith CA, Hay PPJ. Acupuncture for depression. Cochrane Database Syst Rev 2005;(2):14651858.CD004046.pub2.
  • 299. Yu J, Liu C, Zhang X, Han J. Acupuncture improved cognitive impairment caused by multi-infarct dementia in rats. Physiol Behav 2005;86:434-41.
  • 300. Johnston MF, Yang C, Hui KK, Xiao B, Li XS, Rusiewicz A. Acupuncture for chemotherapy-associated cognitive dysfunc­tion: a hypothesis-generating literature review to inform clini­cal advice. Integr Cancer Ther 2007;6:36-40.
  • 301. Vickers AJ, Straus DJ, Fearon B, Cassileth BR. Acupuncture for postchemotherapy fatigue: a phase II study. J Clin Oncol 2004;22:1731-5.
  • 302. Ernst E, White AR. Acupuncture as a treatment for temporo­mandibular joint dysfunction: a systematic review of ran­domized trials. Arch Otolaryngol Head Neck Surg 1999; 125:269-72.
  • 303. Zhang, Z. Effect of acupuncture on 44 cases of radiation rectitis following radiation therapy for carcinoma of the cervix uteri. J Tradit Chin Med 1987;7:139-40.
  • 304. Yan LS. Treatment of persistent hiccupping with electro-acu­puncture at "hiccup-relieving" point. J Tradit Chin Med 1988;8:29-30.
  • 305. Wong R, Sagar SM. The treatment of persistent yawning with acupuncture. Acupunct Med 2000;18:124-6.
  • 306. Feng R. Relief of oesophageal carcinomatous obstruction by acupuncture. J Tradit Chin Med 1984;4:3-4.
  • 307. Filshie J, Penn K, Ashley S, Davis CL. Acupuncture for the relief of cancer-related breathlessness. Palliat Med 1996;10:145-50.
  • 308. Vickers AJ, Feinstein MB, Deng GE, Cassileth BR. Acupunc­ture for dyspnea in advanced cancer: a randomized, placebo- controlled pilot trial [ISRCTN89462491]. BMC Palliat Care 2005;4:5.
  • 309. Hammar M, Frisk J, Grimas O, Hook M, Spetz AC, Wyon Y Acupuncture treatment of vasomotor symptoms in men with prostatic carcinoma: a pilot study. J Urol 1999;161:853-6.
  • 310. Tukmachi E. Treatment of hot flushes in breast cancer patients with acupuncture. Acupunct Med 2000;18:22-7.
  • 311. Cumins SM, Brunt AM. Does acupuncture influence the vaso­motor symptoms experienced by breast cancer patients taking tamoxifen? Acupunct Med 2000;19:28-9.
  • 312. Fontanarosa PB, Lundberg GD. Alternative medicine meets science. JAMA 1998;280:1618-19.
  • 313. Sagar SM. Unproven cancer therapies. Ann R Coll Physicians Surg Can 1998;31:160.
  • 314. Sagar SM. Alternative views on alternative therapies. CMAJ 1999;160:1697-9.
  • 315. Sagar SM. Restored Harmony: An Evidence-based Approach for Integrating Traditional Chinese Medicine into Complemen­tary Cancer Care. Hamilton, ON: Dreaming DragonFly Com­munications; 2001.
  • 316. Tagliaferri M, Cohen I, Tripathy D. Complementary and alter­native medicine in early-stage breast cancer. Semin Oncol 2001;28:121-34.
  • 317. Bensoussan A, Talley NJ, Hing M, Menzies R, Guo A, Ngu M. Treatment of irritable bowel syndrome with Chinese herbal me­dicine: a randomized controlled trial. JAMA 1998;280:1585-9.

Correspondence to: Stephen M. Sagar, Juravinski Can­cer Centre, 699 Concession Street, Hamilton, Ontario L8V 5C2.

E-mail: stephen.sagar@hrcc.on.ca

* Departments of Oncology and Medicine, McMaster University; Juravinski Cancer Program, Hamilton Health Sciences Corporation; and The Brain-Body Institute, St. Joseph's Healthcare Sys­tem, Hamilton, Ontario, Canada.

For in-depth information, readers may want to visit these linked sites:

Society for Integrative Oncology (SIO)

Shanghai International Symposium:

Integrative Oncology Theory, Research, and Practice

April 25-26, 2008, Shanghai, China

and

Society for Integrative Oncology

 

 

Printed With Permission Copyright © HealthWorld Online (http://www.healthy.net)

( Çocuklar için Bağışıklık )

Immune Power For Kids

© Leo Galland M.D., F.A.C.N. Director, Foundation for Integrated Medicine; author, The Four Pillars of Healing: How The New Integrated Medicine Can Cure You.

Asthma, allergic rhinitis, chronic otitis media and sinusitis are increasing at frightening rates among children in the United States. There are several measures that parents can take to effectively reverse this trend. These measures are supported by scientific research and have been very effective in my own clinical practice.

The first step is a nutritious diet, which decreases consumption of foods made with added fat and sugar. The National Cancer Institute reports that only one per cent of U.S. children consume a well-balanced diet and only about a third meet the government's food-pyramid targets for fruits, vegetables, grains, meat and dairy. Most surveys over-estimate vegetable intake because they classify french fries and potato chips as vegetables, accounting for about a fourth of alleged vegetable consumption among children. Previous studies from the U.S., Britain and Australia have shown that the eating of fish and tomatoes and the intake of minerals like magnesium and selenium are inversely related to the risk of developing asthma.

Detailed guidelines for feeding children in every age group, along with recipes, are supplied in my first book, Superimmunity for Kids. Recommendations for nutritional supplementation are listed below.

The second step is provision of a hygienic home environment. The three most important areas to control are smoke, dust and humidity. Children exposed to cigarette smoke at home have a higher frequency of asthma, respiratory infection and otitis. House dust can contains surprisingly high levels of lead and toxic waste, tracked in and concentrated from roadside soil, accumulating for years despite routine vacuum cleaning, sometimes exceeding concentrations found at superfund sites. House dust is especially hazardous to toddlers crawling on carpets, because carpet pile is a repository for dust. Excess moisture in the home (a relative humidity of fifty-five per cent or more) encourages the growth of dust mites and of mold. Mites secrete an enzyme which damages the lining of the respiratory tract; children with a high mite exposure are at increased risk for developing asthma. Children who live in homes with visible mildew or moisture are at increased risk for developing repiratory illness and for missing days from school. I describe nine simple steps for parents to take to ensure an environmentally safe home in previous issues of this column.

The third area is regularity of rest and exercise. Exercise of moderate intensity, such as brisk walking or cycling, thirty minutes a day, improves immune function and mood and prevents migraine headache. Most older children, high school students especially, are sleep-de-prived. Sleep deprivation or interrruption reduces natural killer cell activity.

Parents should help their children plan schedules that permit eight to ten hours of sleep a night. Daytime relaxation also has important health benefits. A period of quiet, focused relaxation each day relieves anxiety, improves nighttime sleep, and stimulates immune function of stressed individuals.

Nutritional supplements and herbs can make a substantial contribution to childhood health. Omega-three essential fatty acids, found in fish oils and flax oil, are essential for normal immune regulation and brain function. The past century has witnessed a dramatic decline in omega-three consumption, due to changes in food processing, food choices and animal husbandry practices. Cod liver oil, which can be a rich source of omega-three's, has long been used as a food supplement for children. A recent double-blind placebo-controlled study found that capsules of flax oil, two grams per day, decreased frequency, severity and duration of illness and days missed from school among children suffering from recurrent respiratory infection. I frequently recommend flax oil as a preventive supplement for children and adolescents at a dose to two to six grams per day.

Vitamin E levels in the blood of U.S. chil-dren are mark--edly low-er than those of Japa-nese, German, Austrian or Canadian children, sugge-sting that children in the U.S. may as a group suffer from a mild deficien-cy. Healthy chil-dren with lower vitamin E lev-els have impaired immunity on laboratory tes-ting. The immune defects associated with a relative vitamin E deficiency in "healthy" children are the same deficits associ-ated with in-creased mortali-ty in the elderly.

Children with recurrent respiratory infections have lower blood levels of zinc, iron and vitamin A than do children without recurrent infection. Adequate intakes of zinc and iron can be difficult to obtain from food, even when the diet is better than average. For young children I recommend a preventive daily supplement supplying ten milligrams each of zinc and of iron and twenty-five hundred units of vitamin A; adolescents need twice the dose. Because zinc and iron interfere with each other's absorption and because iron causes oxidation of vitamin E, children who are not doing well with a multivitamin/mineral pill should take separate doses of zinc, iron and vitamin E at different times of the day. Zinc is best absorbed on an empty stomach, but may cause nausea. The second best time for giving zinc is with a high protein meal. Iron is best absorbed with a high protein meal and when given with vitamin C. Vitamin E is best absorbed with food; the optimal immune-boosting dose is a hundred milligrams per day for small children and two hundred milligrams a day for adolescents.

Adolescents and children may sometimes develop repeated infections despite a hugienic environ-ment, a regular schedule of rest and exercise, and a diet of high nutri-ent density, appropriately tailored to one's consti-tutional needs, supplemented with EFAs and antioxidants. There are many addi-tional measures which may be taken to stimulate resis-tance. I recommend these frequently to patients in my medical practice and have been impressed with their safety and efficacy for children and adolescents:

(1) Vitamin C, five hundred milligrams per day, increases the activity of white blood cells.

  • (3) Granular lecithin, one tablespoon a day, has also been shown to improved the activity of white blood cells.
  • (4) The amino acid dimethylglycine (DMG) has been shown to boost antibody responses to immunization in healthy human volunteers. The dose used was one hundred and twenty milligrams per day.
  • (5) Immune stimulating herbs may help children overcome acute viral infection. The safest and best-studied are:

Echinacea species, which grow wild across the American mid-west from Wisconsin to

Texas. All parts of the Echinacea plant have been used for centu-ries by Native Americans to treat wounds and snake bite. Recent studies on its effects reveal marked stimu-lation of many immune functions, including increased activity of phagocytes. Echinacea is very safe.

The two main species, Echinacea angustifolia and Echinacea purpurea, are primarily recommended for acute treat-ment (ten to fourteen days) of colds or the flu. The dose needed is at least 900 mg per day, and I prefer Echi-nacea purpurea root to other preparations. Some people with chronic or recurrent infec-tions benefit from taking Echinacea for prolonged periods, especially, during the winter. It may be taken continuously for eight weeks at a time and should be stopped for a week or two between each eight-week period.

In the treatment of acute respiratory infection, the activi-ty of Echinacea is often enhanced by Chinese herbal mixtures traditionally used for treating fever. My favorite is called Isatis Formula. It is commercially available as an alcohol extraction of the leaves and roots of six plants. The dose is one to three drop-persful three times a day. During heavy flu seasons, over three quarters of my patients taking the Echinacea and Isatis combina-tions have made statements like, "Everyone around me was sick for weeks, taking antibiotics. I usually get sick for three weeks with the flu, but I was better within a few days after starting these herbs."

Astragalus root is a component of many traditional Chinese herbal formulas, generally considered to be a strong tonic and reistance-builder. Contemporary studies reveal that Astragalus can increase natural killer cell activity. I often recommend Astragalus for maintenance therapy of people with chronic or recurrent infectious diseases of any type, because of its high margin of safety.

 

Leo Galland, M.D. has received international recognition as a leader in the field of Nutritional Medicine for the past 20 years. A board- certified internist, Dr. Galland is a Fellow of the American College of Physicians and the American College of Nutrition, an Honorary Professor of the International College of Nutrition, and the author of

 

(6) Mushrooms. Fungal extracts are widely employed in traditional Chinese medicine. Shiitake (Lentinus edodes) and Reishi (Ganoderma lucidum) contain polysaccharides that increase natural killer cell activity and inhibit tumor growth in animals and in humans. Like Astragalus, Shiitake and Reishi are used in contem-porary Chinese medicine as Fu Zheng remedies, which means they "support the normal", stimu-lating health, rather than being used as medication to treat sickness. A dose which stimulates immune responses is 900 mg per day of each. For people with severe allergies, it is advisable to use Reishi alone, as Reishi may inhibit allergic reactivity and Shiitake may increase it.

Archived columns by Leo Galland M.D., F.A.C.N.

 

 

Earth's Bounty:


(Ganoderma, sana faydaları nedir?)

GANODERMA
And why it is good for you.

Modern living moves frequently at a faster pace. Latest technologies echo this fact with innovations on communications, transportation, and daily rituals that promise efficiency and convenience. Unfortunately, whether you are going to work, playing with the kids, or running some errands, you create with toxins everyday. Toxic chemicals come various places: pollution, global warming, saturated fats on your diets, by products from bodily processes, and other environmental. These toxins weaken your body's immune system - your primary sentinel against diseases.

Ganoderma Lucidum, commonly known as Reishi or Lingzhi in Asia, has been proven most effective among a string of herbal because it strengthens the immune system. A strong immune system works as a round-a-clock body armor: it shields you against microbes, infections, and viruses that bring common illnesses. It needs to be benefited with correct and ample amount of nutrients to fulfill its role in total body wellness. To achieve optimum health, you must take care of your immune system through proper nutrition, supplementation and exercise.

Herbal Medicine: Curative or Preventive?

There is a growing clamor for alternative medicine when modern medication is flooded with side effects harmful to human beings. Despite the benefits and minimized side effects, alternative medicine does not turn heads among contemporary health practitioners. Reports however, have it that in more ways than one; these methods sweeten some bitter endings.

Herbal medicine does not replace a prescribed cure on a diagnosis but in fact controls it by preventions. When the body is built initially with a strong armor of protection, it destroys opportunistic parasites, leaving it hale and hearty. Ganoderma's significant results of healing even extend centuries ago in ancient china. "The King of Herbs" became popular when emperors, royalties, and noblemen subscribed to the benefits of Ganoderma and its medicinal properties. It is said to facilitate longevity and youthfulness; increase vitality and vigor; and recently show evidence of anti-cancer activity.


Why Ganoderma is good for you

Ganoderma contains 200 individual ingredients and the combination of these makes the herb potent for various medicinal needs. Here's a closer look on some important element that make Ganoderma "the elixir of immortality."

Polysaccharides
Polysaccharides are carbohydrates made up of many sugar molecules. They enhance the natural ability of the body to heal itself through revitalized cells. Polysaccharides in Ganoderma help to strengthen the immune system to fight off infections brought by certain diseases. It is also scientifically proven to have high anti-tumor activity that prevents the growth of tumors and cancers.

Triterpenes
Triterpenes are simple lipids needed by the body in boosting the digestive system. It reduces cholesterol, toxins, and fats caused by poor diet. Reishi also contains 112 known triterpenes that directly alleviate allergies and pain. Triterpenes are the ones that give the mushroom its bitter taste but beneficial properties.

Adenosines
Adenosines are vital component of chemicals that power the cells. It is like a supercharged battery that revitalizes the metabolism for improved blood circulation and cholesterol level. Ganoderma also helps reduce altitude sickness by oxygenating the blood. This benefit was studied by Asian mountain climbers who ascended mountains of 17,000 feet high with minimal reaction.

Organic Germanium
Organic germanium transport oxygen at a faster rate to nourish body cells. Its presence in Ganoderma regulates blood flow and electrical charges in various body processes. It makes it easy for the body to absorb oxygen and is thought to aid in perking up the metabolism. Organic germanium also facilitates the body's natural response to inhibit free radicals that causes cancer in humans.


Herbal medicine does not replace a prescribed cure on a
Medical diagnosis but in fact controls it by prevention.

Sterols
The vegetative part of Ganoderma (mycelium) is rich in sterols. Sterols make up the core of cell membranes that build hormones in the body. Food and beverages supplemented with sterols may reduce cholesterol and are a promising addition to interventions aimed at lowering heart disease risks.

Ling-zhi 8
Common allergens such as dust, pollen, drugs and food and animal products heightened the body's sensitivities or worse, cause extreme allergies.
Ling-zhi 8, a protein found in the mushroom, has strong anti-allergy properties that minimize the body's reaction to allergens.

Good health is considered to experience chronological loss. But it doesn't have to be that way almost immediately. Prolonged vitality of the body as a whole can be achieved by cautioned food intake and proper supplementation. DXN health products are fortified with Ganoderma extracts beneficial for a well mind and body. These products are a surely natural way to achieve o a healthier you.


HOW GANODERMA WORKS IN YOUR BODY
DXN RG/GL CAPSULES (GANODERMA LUCIDUM)

Our products are all Ganoderma based. 4000 years ago, anoderma/Lingzhi in Chinese, Reoshi in japan,is being used by the emperor of china and his royal family for longevity, vitality and radiant health. The testimonies through the years have brought interest to the medical profession. Please surf the net about Ganoderma. Hundereds of clinical trials were made proving the efficacy of Ganoderma. Based on the traditional Chinese medicine, among 365 herbs, Ganoderma is ranked no.1 and is considered the king of herbs. Ganoderma is excellent for slow aging, gain unlimited energy, improve sex,sharpen mental clarity, reduce stress, and you will look and feel younger. It is 6X more potent than ginseng, ginseng is found in pharmaton, centrum for stress, rogin-ee, red bull, etc. Ganoderma is an excellent antibacterial, antifungal, anti-inflamation agent and skin care. It will significantly improve the functioning of your immune system.
How does Ganoderma works in you? Ganoderma is the only herb that can address the two reasons of why people get sick (toxins and imbalance of the body function). First, Ganoderma will scan your body and cleans the toxins that have accumulated in your body. Second, it will then balance the function of your body. Third, it will nourish every cell in your body. Fourth,Most important of all(forget the 3 but never forget the fourth action), Ganoderma has the raw materials needed in the creation/formation of new cells. As we grow in age, our cell wall hardens making it hard for the nutrients to go inside and for toxins to go out. If your cells are weak, their replications are not the best cells you want. Since RG/GL capsules have the raw materials needed in the creation of new cells, the new cells reproduced are young, healthy and strong cells. This is what you really want. Replication of cells is faster coming from those new born cells replacing the old, unhealthy and weak cells with young, healthy, strong cells. That is why it is you will become young looking and healthy. Secondly, your soldier cells (protecting you from viruses, bacteria, etc) will dramatically increase in numbers allowing you to fight back on enemy viruses and bacteria. That is why it is common to find people using ganoderma to be healthy, away from disease and looking younger everyday. It is also common among unhealthy people to get colds and fever easily when they are near people who carry viruses, bacteria and the likes. The emperors who quarrel just to get hold of this miraculous herb have indeed a relevant reason for doing so. Your immunity increases. Your body is now functioning at its optimum level and there is no better doctor than your own body. Our body is the best doctor! GANODERMA DOES NOT CURE THE DISEASE. It cures your body so that your body can cure disease.

Who needs Ganoderma? Everyone needs ganoderma. From womb to tomb as DR. Lin Siow Jin(Founder) said. Ganoderma does not understand disease, it only understand cells. If a person has liver, Kidny, heart, or any health problem, these sick organs or sick tissues are all made up of cells. Now, Ganoderma's work is to repair and build needed cells. Imagine if every cell in your body is repaired and or replaced with healthy young cells. Your body will naturally become healthy. According to Dr. Nilay Shah MD, there is no other herb present in the world today that has action to every cell in your body except Ganoderma. Garlic and ginseng can help in specific cells but not every cell in your body. It is only Ganoderma that has capacity to repair and build every cell in your body. Ganoderma is incomparable and unparalled. It is indeed the MIRACULOUS KING OF HERBS. It also works better than multi-vitamins coz of its capacity to reproduce NEW CELLS. Use RG/GL capsules (DXN Ganoderma) as protection and aid you in getting the best of your body functions.

 

 

PHYTOTHERAPY RESEARCH

Phytother. Res. 23, 785-790 (2009)

Published online 14 January 2009 in Wiley InterScience

(www.interscience.wiley.com) DOI: 10.1002/ptr.2623

(Çin Tıbbi Bitkisel komplexlerinin ve Citronellolün kemoterapi ve radyoterapi alan kanser hastalarının bağışıklığı üzerine etkisi.)

Effect of Citronellol and the Chinese Medical Herb Complex on Cellular Immunity of Cancer Patients Receiving Chemotherapy/Radiotherapy

Shu-Ru Zhuang12, Su-Lin Chen1, Jih-Hsin Tsai3, Chi-Chou Huang2, Tzu-Chin Wu2, Wen-Shan Liu2, Hsien-Chun Tseng2, Hong-Sen Lee1, Min-Chang Huang4, Guang-Tzuu Shane4, Cheng-Hua Yang4, You-Cheng Shen1, Yeong-Yu Yan1 and Chin-Kun Wang1*

1Graduate Institute of Nutritional Science, College of Health Care and Management, Chung Shan Medical University, 110, Sec. 1, Chien-Kuo North Rd., Taichung, 40201, Taiwan

2Chung Shan Medical University Hospital, 110, Sec. 1, Chien-Kuo North Rd., Taichung, 40201, Taiwan

3Shian De Hospital No. 420, Yichang E, Rd., Taiping City, Taichung County, 41175, Taiwan

4Medigreen Biotechnology Corp. IIF., No. 453, Sec. 2, Wunhua Rd., Banciao City, 22046, Taipei County, Taiwan

Leukopenia and immunity impairment usually occur during cancer therapy. Citronellol, an oil soluble compound derived from the geranium, has anticancer and antiinflammatory properties, as well as promoting wound healing. Ganoderma lucidum, Codonopsis pilosula and Angelicae sinensis are traditional Chinese herbs, all of which have proven immunomodulatory functions in laboratory-based research. This randomized, double­blind, placebo-controlled study examined whether the Chinese medicinal herb complex (CCMH; a mixture of citronellol and extracts of G. lucidum, C. pilosula and A. sinensis) improves the immune cell counts of cancer patients receiving chemotherapy and/or radiotherapy. A total of 105 cancer patients receiving chemotherapy or radiotherapy were enrolled. The quantities of immune cells in the blood of the subjects were determined before and after 6 weeks of cancer treatment, with either CCMH or a placebo. CCMH significantly reduced the depletion of leukocytes (14.2% compared with 28.2%) and neutrophils (11.0% compared with 29.1%). Analysis of the lymphocyte phenotype revealed that the patients receiving the placebo had reduced CD4 lymphocytes and natural killer (NK) cells than the CCMH-treated patients. Treatment with CCMH for patients receiving chemotherapy and/or radiotherapy may improve their immune function, improving their ability to fight off the cancer, as well as any secondary infections that could compromise their treatment and their health. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: citronellol; Ganoderma lucidum; Codonopsis pilosula; Angelicae sinensis; immunity; cancer.

 INTRODUCTION

Malignant tumors have been the major cause of death in Taiwan since 1993. More than 1.3 million new cases of invasive cancer were diagnosed in Taiwan in 2005. Not only the cancer itself but also the therapy for cancer such as chemotherapy, surgery and radiation therapy impair the patient's immunity (Mafune and Tanaka, 2000). Immune system dysfunction, including leukopenia, neutropenia and thrombocytopenia, is attributable to bone marrow suppression, as well as sup­pression of the proliferation or function of lymphocyte subsets. Neutropenia occurs in approximately 80% of all cancer patients who are undergoing treatment for cancer. The exact incidence of neutropenia in cancer patients and the number of neutropenia-related cancer deaths are uncertain, but the mortality reportedly ranges from 5% to 30% among those older than 70 (Balducci and Yates, 2000).

All populations of lymphocytes decrease following chemotherapy. For example, in one study, CD4 lympho­cytes decreased by more than 60%, and levels had not returned to normal even after 18 months (Hakim et al.,

* Correspondence to: Chin-Kun Wang, 110, Sec. 1, Chien-Kuo North Road, Taichung, 40201, Taiwan. E-mail: wck@csmu.edu.tw

1997). A recent study demonstrated that the propor­tion of total T cells was significantly lower in patients after adjuvant systemic therapy than in those with primary breast cancer or in normal healthy donors (Mafune and Tanaka, 2000). The prognosis of cancer patients depends on the method of treatment and the status of the cancer itself, but other factors, including host defenses against cancer, also have an impact on the outcome. In one breast cancer study, higher NK cell activity was predicted to lower cancer recurrence at a 5-year follow-up (Levy et al., 1991). The immune system also may play an important role in the modulation of tumor growth and metastasis (McCoy et al., 2000).

Many patients undergoing cancer therapy also use complementary and alternative medicine (CAM), including vitamins, Chinese medicinal herbs and other biological products (Tagliaferri et al., 2001). These prod­ucts are used to reduce side effects and organ toxicity, to protect and stimulate immunity, or to prevent further cancers or recurrences (Newman et al., 1998; Von Gruenigen et al., 2001). However, the scientific evidence on the efficacy and safety of herbs or nutritional sup­plements is limited. Ganoderma lucidum (also known as lingzhi), Codonopsis pilosula (also known as dang shen) and Angelica sinensis (also known as dong quai) are often used in Chinese medicine, and also cooked as food in Taiwan. Citronellol (3,7-dimethyl-6-octen-1-ol) is an essential oil found in the rose geranium (Pelargonium graveolens), a multi-harvest high value and aromatic plant. This essential oil has strong antioxidant activity (Sun etal, 2005b). This study examined whether a Chinese medicinal herb complex (CCMH; a mixture of citro- nellol and extracts of G. lucidum, C. pilosula and A. sinensis) improves immune function. Understanding the possible effects of CCMH on the immunity of cancer patients receiving chemotherapy and/or radiotherapy could provide an easily available adjuvant therapy for cancer.

Table 1. Ingredients in the CCMH and placebo pills

Component (mg)

CCMH

Placebo

Angelica extract

64.5

0

Codonopsis Pilosulae

27.1

0

Radix extract

 

 

Ganoderma extract

3.0

0

Citronellol powder

273.6

0

Corn starch

129.3

115.6

Magnesium stearate

2.5

2.5

¡¡-cyclodextrin

0

363.7

Caramel

0

18.2

 MATERIALS AND METHODS

 

Participants. This study involved 105 cancer patients (74 women and 31 men) undergoing chemotherapy and/ or radiotherapy, including 60 breast cancer patients, 24 colorectal cancer patients, 14 nasopharyngeal cancer patients and seven lung cancer patients. Patients were assigned randomly to the experimental (75 patients) and control (64 patients) groups. Exclusion criteria for participation in the study included terminally ill patients, renal failure, congestive heart failure, hepatic failure and intolerance or poor compliance to CCMH. During the study, 34 subjects dropped out (16 did not complete the follow-up, 17 withdrew and one had poor compliance). Fifty control subjects and 55 experimen­tal subjects completed the study. The treatment period for each subject was 6 weeks. All cancer patients undergoing chemotherapy or radiotherapy were ran­domly assigned to receive either nine capsules of CCMH (experimental group) or placebo (control group) every day. A physical examination was performed at the beginning of the study. A questionnaire given at the beginning of the study provided information on any concurrent diseases and the patient's medical history (including cancer location and tumor staging).

This randomized, double-blind and placebo-controlled clinical trial was conducted in Chung Shan Medical University Hospital from December 2003 to November 2005. The trial was carried out in accordance with the Declaration of Helsinki and subsequent revisions and approved by the Institutional Review Board of Chung Shan Medical University Hospital, Taichung, Taiwan. Written informed consent was obtained from all patients.

Ingredients of CCMH. Four crude ingredients, G. lucidum extract (3 mg), C. pilosula extract (27.1 mg), A. sinensis extract (64.5 mg) and citronellol powder (273.6 mg) were mixed into a capsule (Table 1). All capsules were made by Sun Ten Pharmaceutical Co. Ltd, and provided by Medigreen Biotechnology Corporation.

Biochemical analysis. Venous blood samples were ob­tained for the complete blood count and the subjects' immunity status analysis before and after 6 weeks of treatment. Red blood cells, hemoglobin, hematocrit, platelets, leukocytes, neutrophils and lymphocytes were evaluated by automated blood analyser. Lymphocytes (CD3, CD4 and CD8), NK cells and B cells were evalu­ated by flow cytometry. Peripheral blood specimens were labeled in Tru-Count (BD Biosciences, San Jose, CA, USA) with antibodies to lymphocytes (CD3, CD4 and CD8), B cells (CD19) and NK cells (CD16/CD56) and incubated at room temperature for 15 min, followed by erythrocyte lysis. Four-color flow cytometric immuno- phenotyping was performed using the FACSCalibur flow cytometer and Multiset software (BD Biosciences). At least 16 500 ungated events were collected in a list mode file and expressed as a percentage of the total lymphocyte population. Both the percentage and abso­lute count were obtained for each subtype. The value obtained at the beginning of the study for each cancer patient is considered the baseline (100%), with the value at week 6 expressed as the percentage of that baseline.

Statistical analysis. Hematological data are presented as the mean ± standard deviation (SD). The other data are presented as the mean percentage of baseline ± SD. The dependent Student's t-test was used to compare the difference in the same group, and the independent t-test to compare the difference between the control and experimental groups. A value of p < 0.05 was considered significant. SPSS version 10.0 software (Chicago, USA) was used for analysis.

RESULTS AND DISCUSSION

CCMH treatment affected hematological indicatory

There was no significant difference in gender, age, height, weight, body mass index (BMI), type of carcinoma, tumor stage and type of therapy between the two groups (Table 2). Red blood cell counts and hemoglobin concentration were significantly decreased in control and experimental groups after treatment (Table 3). Over the study period, the hematocrit concentration decreased greatly in the control group, but the experimental group showed no difference. In the control group, the platelet concentration was 247.10 x 103/mm3 (±69.66) at the beginning of the study but decreased significantly to 218.24 x 103/mm3 (±67.33) at week 6. For the experi­mental group, there was no difference between the baseline value and the value at week 6.

CCMH treatment reduces the loss of leukocytes and neutrophils

Both leukocytes and neutrophils were significantly lower in the control group than in the experimental group after treatment (Fig. 1). The control group had 71.8% (±22.1%) of the baseline number of cells, while the experi­mental group had 85.8% (±31.5%) of the baseline number

Table 2. Demographic characteristics of the patients

 

Control

Experimental

Characteristic

(n = 50)

(n = 55)

Age (years)

35-78

33-84

Men/Women

17/33

14/41

Height (cm)

158.7 ± 7.7

159.0 ± 7.2

Weight (kg)

58.4 ± 10.7

59.8 ± 9.1

BMI (kg/m2)

24.0 ± 4.0

23.7 ± 2.8

Diagnosis of disease

 

 

Breast cancer

27 (54%)

33 (60%)

Colorectal carcinoma

12 (24%)

12 (21.8%)

Nasopharyngeal cancer

7 (14%)

7 (12.7%)

Lung cancer

4 (8%)

3 (5.5%)

Disease stage

 

 

I

4 (8%)

8 (14.5%)

II

20 (40%)

19 (34.5%)

III

12 (24%)

18 (32.7%)

IV

9 (18%)

10 (18.3%)

Unknown

5 (10%)

0 (0%)

 

Data represent mean ± SD or number (%) for each group.

 

 

of cells after treatment. The number of neutrophils decreased significantly in the control group, to 70.9% of baseline after 6 weeks. In the experimental group, neutrophils decreased to 89.0% of baseline after treatment.

Neutropenia-related cancer deaths range from 5% to 30% among those older than 70 (Balducci and Yates, 2000). In another study, the leukocyte count was reduced by 60% at week 2, 24% at week 3, and 57% at week 4, after chemotherapy (Tong et al., 2000). Herein, it was found that both lymphocytes and neutrophils were higher in patients receiving CCMH than in those re­ceiving a placebo, demonstrating that the CCMH sup­plement may maintain leukocyte and neutrophil counts during chemotherapy/radiotherapy.

Experimental group

 CCMH treatment reduces the loss of lymphocytes and NK cells, but not B cells

The changes in overall lymphocyte count, as well as in the counts of specific lymphocyte phenotypes, after 6 weeks of CCMH or placebo treatment are shown in Fig. 2. Lymphocytes were decreased significantly in both the control (23.8%) and experimental (16.4%) groups. The mean percentage of CD3 lymphocytes after treat­ment was 79.5% (±42.8%) of baseline in the control group and 85.9% (±42.2%) of baseline in the experi­mental group. The mean percentage of CD4 lymphocytes decreased significantly to 80.2% of baseline in the con­trol group after intervention, but did not change in the experimental group after treatment. The mean percent­age of CD8 lymphocytes was 88.0% (±52.9%) and 93.0% (±61.8%) of baseline in the control and experimental groups, respectively.

In both the control and experimental groups, B cells were decreased significantly after treatment (Fig. 3A). The mean percentage of B cells decreased significantly to 68.6% (±47.5%) of baseline in the control group and 67.8% (±45.4%) of baseline in the experimental group. NK cells were decreased significantly in the control group but no change was found in the experimental group after treatment (Fig. 3B).

The immunological activity was assessed by count­ing lymphocytes. A low ratio of CD4 to CD8 and NK activity predicts a poor outcome in esophageal cancer patients (Tsutsui et al., 1992). This study demonstrated

Table 3. Hematology of patients

Control group (n = 50)                                                              Experimental group (n = 55)

Baseline                                       Week 6                                         Baseline                                        Week 6

Red blood cells (x106/mm3)

4.27

±

0.60a

4.06

±

0.64b

4.17

±

0.50a

4.05

±

0.53b

Hemoglobin (g/dL)

12.70

±

1.64a

12.06

±

1.45b

12.18

±

1.64a

11.89

±

1.54b

Hematocrit (%)

38.39

±

4.34a

36.55

±

4.54b

37.22

±

4.27a

36.49

±

4.21a

Platelets (x103/mm3)

247.10

±

69.66a

218.24

±

67.33b

254.64

±

97.57a

245.22

±

97.86a

Data are the mean ± SD; data in the same row and group with different superscript letters are significantly different.

Copyright © 2009 John Wiley & Sons, Ltd.                                                                                                                                           Phytother. Res. 23, 785-790 (2009)

DOI: 10.1002/ptr

 

that CCMH treatment could delay or ease the reduction of CD4 lymphocytes and NK cells during cancer treat­ment. The maintenance of NK cells by CCMH was probably due to the action of the G. lucidum poly­saccharide. G. lucidum, a popular home remedy, has been used for more than 4000 years and is known for its beneficial effects on human health (Stamets, 1993).

Preclinical studies have established that the polysaccharide fractions of G. lucidum show immunomodulating effects (Gao et al., 2003; Chen et al., 2006; Lin et al., 2006; Ji et al., 2007). Some studies indicated that the G. lucidum polysaccharide enhances the activity of NK cells, and increases the levels of both tumor necrosis factor a (TNF-a) and interferon-/ (IFN-/) released by
activated macrophages and lymphocytes, respectively (Chen et al., 1995; Wang et al., 1997a).

The maintenance of CD4 lymphocytes is associated with G. lucidum stimulating cytokine gene expression and human T lymphocyte proliferation in humans (Mao et al., 1999), as well as antibody production (Bao et al., 2001). Ferrandina and colleagues (2003) showed that the recovery of CD3, CD4 and CD8 lymphocytes were independent markers of a longer time to progression, and were survival markers in ovarian cancer patients receiving high-dose chemotherapy with peripheral blood stem cell and growth factor support. Enhanced re­covery of CD3, CD4 and CD8 lymphocytes not only increases the patient's immunity against infection but also increases survival.

C. pilosula is another commonly used Chinese herbal medicine. The root of C. pilosula, of the family Campa- nulaceae, is used commonly in traditional Chinese medicine, and is beneficial to the immune, digestive and hematopoietic systems. It also induces saliva pro­duction, and can be used to treat fatigue, thirst and loss of appetite (Wang et al., 1997b). Wang and colleagues (1996) showed that splenocytes in C57BL/6 mice exhibited lower mitogenic responses to concanavalin A (ConA) and lipopolysaccharide (LPS) when given drinking water containing a polysaccharide-enriched fraction prepared from C. pilosula root.

A. sinensis has been used in traditional Chinese medical therapy for a wide variety of diseases for thousands of years (Hardy, 2000). The A. sinensis polysaccharide (ASP) provides many pharmacological effects, including immunoregulation (Sun et al., 2005a), immunostimula­tion (Yang et al., 2006), antitumor activity (Shang et al., 2003; Cao et al., 2006) and promotion of hematopoiesis (Bradley et al., 1999). It also decreases inflammatory cytokines, such as TNF-a, interleukin-1ß and interleukin- 6, in a rat uterine endometritis model (Mikamo et al., 1999).

In this study, it was demonstrated that CCMH decreases leukopenia and neutropenia, as well as NK cell and CD4 lymphocyte counts. In conclusion, treatment with CCMH could have important clinical implications for patients receiving chemotherapy and/or radiotherapy.

Acknowledgements

The authors would like to thank all the cancer patients who participated in this study.

 REFERENCES

Balducci L, Yates J. 2000. General guidelines for the manage­ment of older adults with cancer. Oncology 14: 221-227.

Bao X, Liu C, Fang J, Li X. 2001. Structural and immunological studies of a major polysaccharide from spores of Ganoderma lucidum (Fr.). Carbohydr Res 332: 67-74.

Bradley RR, Cunniff PJ, Pereira BJ Jaber BL. 1999. Hematopoietic effect of Radix Angelica sinensis in a hemodialysis patient. Am J Kidney Dis 34: 349-354.

Cao W, Li XQ, Liu Li. 2006. Structure of an anti-tumor poly­saccharide from Angelica sinensis (Oliv.) Diels. Carbohydr Polym 66: 149-159.

Chen WC, Hau DM, Wang CC, Lin IH, Less SS. 1995. Effects of Ganoderma lucidum and krestin on subset T-cell in spleen of gamma-irradiated mice. Am J Chin Med 23: 289-298.

Chen X, Hu ZP, Yang XX et al. 2006. Monitoring of immune responses to herbal immuno-modulator in patients with advanced colorectal cancer. Int Immunopharmacol 6: 499­508.

Ferrandina G, Pierelli L, Perillo A et al. 2003. Lymphocyte recovery in advanced ovarian cancer patients after high-dose chemo­therapy and peripheral blood stem cell plus growth factor support: clinical implications. Clin Cancer Res 9: 195-200.

Gao Y, Zhou S, Jiang, W, Huang M, Dai X. 2003. Effects of ganopoly (a Ganoderma lucidum polysaccharide extract) on the immune function in advanced-stage cancer patients. Immunol Invest 32: 201-215.

Hakim FT, Cepeda R, Kaimei S et al. 1997. Constraints on CD4 recovery post chemotherapy in adults: thymic insufficiency and apoptotic decline of expanded peripheral CD4 cells. Blood 90: 3789-3798.

Hardy ML. 2000. Herbs of special interest to women. J Am Pharm Assoc 40: 234-342.

Ji Z, Tang Q, Zhang J, Yang Y, Jia W, Pan Y. 2007. Immunomodulation of RAW264.7 macrophages by GLIS, a proteopolysaccharide from Ganoderma lucidum. J Ethno- pharmacol 112: 445-450.

Levy SM, Herberman RB, Lippman M, D'Angelo T, Lee J. 1991. Immunological and psychosocial predictors of disease re­currence in patients with early-stage breast cancer. Behav Med 17: 67-75.

Lin KI, Kao YY, Kuo HK et al. 2006. Reishi polysaccharides induce immunoglobulin production through the TLR4/TLR2- mediated induction of transcription factor Blimp-1. J Biol Chem 281: 24111-24123.

Mafune K, Tanaka Y. 2000. Influence of multimodality therapy

on the cellular immunity of patients with esophageal cancer. Ann Surg Oncol 7: 609-616.

Mao T, Water J, van de Keen CL. 1999. Two mushrooms, Grifola frondosa and Ganoderma lucidum, can stimulate cytokine gene expression and proliferation in human T lymphocytes. Int J Immunopharmacol 15: 13-22.

McCoy JL, Rucker R, Petros JA. 2000. Cell-mediated immunity to tumor-associated antigens is a better predictor of survival in early stage breast cancer than stage, grade, or lymph node status. Breast Cancer Res Treat 60: 227-234.

Mikamo H, Kawazoe K, Sato Y. 1999. Effects of crude herbal ingredients on serum levels of inflammatory cytokines in a rat uterine endometritis model. Curr Ther Res Clin Exp 60: 105-110.

Newman V, Rock CL, Faerber S, Flatt SW, Wright FA, Piecre JP. 1998. Dietary supplement use by women at risk for breast cancer recurrence: the women's healthy eating and living study group. J Am Diet Assoc 98: 285-292.

Shang P, Qian AR, Yang TH et al. 2003. Experimental study of anti-tumor effects of polysaccharides from Angelica sinensis. World J Gastroenterol 9: 1963-1967.

Stamets P. 1993. Growing Gourmet and Medicinal Mushrooms. Ten Speed Press: Berkeley, CA.

Sun Y, Tang J, Gu X, Li D. 2005a. Water-soluble polysaccharides from Angelica sinensis (Oliv.) Diels: Preparation, characteri­zation and bioactivity. Int J Biol Macromol 36: 283-289.

Sun W, Xu Z, Wang C, Ou W, Lin C. 2005b. Study on antioxidant activity of essential oils and its monomer from Pelargo­nium graveolens. Zhong Yao Cai 28: 87-89.

Tagliaferri M, Cohen I, Tripathy D. 2001. Complementary and alternative medicine in early stage breast cancer. Semin Oncol 28: 121-134.

Tong AW, Seamour B, Lawson JM et al. 2000. Cellular immune profile of patients with advanced cancer before and after taxane treatment. Am J Clin Oncol 23: 463-472.

Tsutsui S, Morita M, Kuwano H et al. 1992. Influence of preoperative treatment and surgical operation on immune function of patients with esophageal carcinoma. J Surg Oncol 49: 176-181.

Von Gruenigen VE, White LJ, Kirven MS, Showalter AL, Hopkin MP, Jenison EL. 2001. A comparison of complementary and alternative medicine use by gynecology and gynecologic oncology patients. Int J Gynecol Cancer 11: 205-209.

Wang SY, Hsu ML, Hsu HC et al. 1997a. The anti-tumor effect of Ganoderma lucidium is mediated by cytokines released from

activated macrophages and T lymphocytes. Int J Cancer70: 699-705.

Wang ZT, Du Q, Xu GJ, Wang RJ, Fu DZ, Ng TB. 1997b. Inves­tigations on the protective action of Condonopsis pilosula (Dangshen) extract on experimentally-induced gastric ulcer in rats. Gen Pharmacol 28: 469-473.

Wang ZT, Ng TB, Yeung HW, Xu GJ. 1996. Immunomodulatory effect of a polysaccharide-enriched preparation of Codonopsis pilosula roots. Gen Pharmacol 27: 1347-1350.

 

 

J. Agric. Food Chem. 2009, 57,10565-10572 10565 D0l:10.1021/jf902597w

 

 

 

 

 

A R T I C

 Kükürtlenmiş ve Karboksimetillenmiş G. lucidum Polisakkaridlerinin Antitümör Aktivitesinde artışın gösterilmesi)

Enhancement of Antitumor Activities in Sulfated and Carboxymethylated Polysaccharides of Ganoderma lucidum

Jianguo Wang,^ Lina Zhang,[1]'^ Yonghui Yu,* and Peter C. K. Cheung§

^Department of Chemistry, Wuhan University, Wuhan 430072, People's Republic of China, fop en Laboratory for Oversea Scientists, Center for Medical Research, Wuhan University, Wuhan 430071, People's Republic of China, and ^Department of Biology, The Chinese University of Hong Kong, Hong Kong, People's Republic of China

Two water-soluble derivatives, sulfated and carboxymethylated Ganoderma lucidem polysacchar­ides, coded as S-GL and CM-GL, were prepared using derivatization of water-insoluble polysac­charides (GL-IV-I) extracted from the fruit body of G. lucidem. The degree of substitution (DS) of S-GL and CM-GL was 0.94 and 1.09, respectively. The weight-average molecular mass (Mw) of GL- IV-I, S-GL, and CM-GL was determined with light scattering to be 13.3 x 104, 10.1 x 104, and 6.3 x 104, respectively. S-GL and CM-GL inhibited the in vitro proliferation of Sarcoma 180 (S-180) tumor cells in a dose-dependent manner, with an IC50 value of 26 and 38 ,«g/mL, respectively. They also inhibited the growth of S-180 solid tumors implanted in BALB/c mice, with low toxicity to the animals. Flow cytometric studies revealed that treatment of S-GL and CM-GL with S-180 tumor cells could mediate the cell-cycle arrest in the G2/M phase. The expression of Bax increased, and the expression of Bcl-2 decreased dramatically, as shown by immuno-histochemical staining of S-180 tumor tissue excised from the animals. The sulfated and carboxmethylated groups in the poly- saccharides played an important part in enhancing their antitumor activities, leading to the potential to be developed into antitumor drugs.

KEYWORDS: Polysaccharide conformation; antitumor activities; sulfated derivative; carboxymethy­lated derivative; cell-cycle; apoptosis

 INTRODUCTION

© 2009 American Chemical Society

The global awareness of cancer as one of the leading causes of death in people of various ages and racial backgrounds has led to a great deal of research efforts and clinical studies in the fight against the disease (1). Therefore, searching for anticancer drugs with higher bioactivities and lower toxicity from nature has been an extremely active domain. Nowadays, development of non­invasive treatments of cancer is required because the traditional cancer treatments are often toxic to normal cells and can cause serious side effects (2). Natural products from plants, micro­organisms, and marines are safe, biocompatible, and biodegrad­able and have bioactivities, such as antioxidantion and immuno- stimulatory effects (3 -5). Mushroom, non-starch polysacchar- ides, have been considered as natural antitumor agents, having tumor-specific and immunomodulatory effects. It has been sug­gested that the action of antitumor activities of polysaccharides are not only immunomodulatory but may also result from a direct cytotoxic effect on the tumor cells. It is generally believed that polysaccharides do not induce direct toxicity on tumor cells, and little is known about its exact mechanism (6, 7). To effectively research and develop natural anticancer drugs, it is necessary to elucidate the mechanism of the antitumor activities of polysac- charides with an understanding of the effect of their chemical structure, molecular weight, and chain conformation.

Published on Web 10/28/2009                                                pubs.acs.org/JAFC

 

Ganoderma lucidum polysaccharides, one of the main effica­cious ingredients of G. lucidum (Leyss, ExFr.) Karst (Gl), has been under modern pharmacological research in recent 30 years and has been reported to be effective in inhibiting tumor growth (8). Its fruit body has long been used as a traditional Chinese medicine to promote health and longevity, coded as "elixir of youth" by ancient emperors. The earliest record of "Lingzhi" was in the "Shen Nong's Materia Medica" in the Han Dynasty of China about 2000 years ago. However, the polysac- charides extracted with alkali from G. lucidum are mainly water- insoluble (1 f 3)-/3-D-glucan and less suitable for pharmaceutical study because of its poor water solubility. In the last few decades, much attention has focused on the biological properties of polysaccharides and their chemical derivatives, especially sulfated and carboxymethylated derivatives (9). It has been demonstrated that chemical sulfation and carboxymethylation of water-insolu­ble polysaccharide should not only enhance the water solubility but also change the chain conformation, resulting in the improve­ment of their biological activities (10-12). The objective of this work was to prepare sulfated and carboxymethylated derivatives from water-insoluble polysaccharides extracted from the fruit body of G. lucidum. Moreover, we investigated the effects of these two derivatives on inhibiting cellular proliferation, cell-cycle progression, and apoptosis against Sarcoma 180 (S-180) tumor
to evaluate their bioactivities. This work can provide important insight for the introduction of the charged group to polysacchar­ides upon the enhancing of their antitumor activities and induc­tion of apoptosis.

MATERIALS AND METHODS

Preparation of Derivatives and Characterization. A linear water- insoluble (1 f 3)-3-D-glucan, coded as GL-IV-I, was isolated from the fruit body of G. lucidum by extracting with sodium hydroxide solution (13). The GL-IV-I polysaccharide was sulfated and carboxymethylated sepa­rately, coded as S-GL and CM-GL, respectively, according to the procedures of Yoshida et al. and Bao et al. (14,15). Both of these two derivatives were white scale-like samples. The degree of substitution (DS) in the above two derivatives was determined with element analysis by the following equation: where OD570(contro[) was obtained in the absence of polymers and OD570(samp[es) was obtained in the presence of polymers.

In Vivo Antitumor Activity Assay. A total 32 BALB/c female mice, weighing 20.0 ± 2.0 g, were randomly divided into 4 groups (n = 8) and allowed free access to a standard laboratory diet and water. S-180 cells (about 1.5 x 106) were subcutaneously implanted into the right-hand groin of the mice. 5-Fluorouracil (5-Fu) and the tested polysaccharides were dissolved in 0.9% aqueous sodium chloride solution and were injected intraperitoneally daily (once starting 24 h after tumor inoculation). The same volume of 0.9% aqueous sodium chloride solution was injected intraperitoneally similar to the controlled group. After 8 days of admin­istration, the tumors were removed from the mice and weighed. The tumor weight was compared to those in the controlled group of mice. The inhibition ratio (I) and the enhancement ratio of the body weight ( j) were calculated as follows:

 where Ma is the total atomic weight of the element from the original sugar unit, Wa is the total atomic weight of the element from the substitution group, and Ms is the molecular mass of a substitution group.

Infrared (IR) spectra of the samples were carried out with a Nicolet Fourier transform infrared (FTIR) spectrometer (Spectrum One, Thermo Nicolet Co., Madison, WI) in the range of 4000-400 cm-1 using the KBr disk method. High-resolution 13C nuclear magnetic resonance (NMR) was carried out with a Mercury 500 NMR spectrometer (Varian, Inc., Palo Alto, CA) at room temperature. The concentrations of the GL-IV-I and its derivatives were adjusted to 100 mg/mL for NMR experiments. Me2SO-d6 was used as a solvent for GL-IV-I, and 99.96% D2O was used as a solvent for the derivatives.

Size-exclusion chromatography combined with multi-angle laser light scattering (SEC-LLS, X = 633 nm Optilab, DAWN DSP, Wyatt Technology Co., Santa Barbara, CA) measurements of the samples were carried out to obtain the molecular mass of the samples. A P100 pump (Thermo Separation Products, San Jose, CA) equipped with columns of G4000PWXL (MicroPak, TSK) in 0.9% NaCl aqueous solution and a column of G4000HXL in Me2SO at 25 °C was used as the SEC instrument. A differential refractive index detector (RI-150, Thermo Separation Products, San Jose, CA) was simultaneously connected. The carrier solutions were 0.9% NaCl aqueous solution and Me2SO. The solvents and polysaccharide solutions were purified by a 0.2 ^m filter and degassed before use. The injection volume was 200 iL, with a concentration of 3 mg/ mL for the sample, and the flow rate was 0.5 mL/min in 0.9% NaCl aqueous solution and 0.5 mL/min in Me2SO. Astra software (version 4.90.07) was used for data acquisition and analysis.

Cytotoxicity and Cell Viability Analysis. Sarcoma tumor cell line (kindly provided by Tongji Medical College of the Huazhong Uni­versity of Science and Technology) was used to test the in vitro and in vivo antitumor activities of the sample. Cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 ig/mL streptomycin, and 100 units/mL penicillin. Cell viability of control and treated cells was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in triplicate. Briefly, an amount of 1 x 104 cells was seeded in each well of a 96-well microplate. The cells were permitted to adhere for 24 h and washed with phosphate buffer solution (PBS), and fresh medium was added and then treated with S-GL and CM-GL, dissolved in medium at the final concentration range of 1-500 ig/ mL (16). The treated cells were incubated for 24 h at 37 °C in a humidified atmosphere of 5% carbon dioxide. After treatment, the culture medium was replaced by fresh medium. Then, cells in each well were incubated at 37 0C in 50 iL of MTT (5 mg/mL) for 4 h. After the medium and MTT were removed, 200 iL of Me2SO and 25 iL of glycine buffer (0.1 M glycine and 0.1 MNaClatpH 10.5) were added to each well. Absorbance at 570 nm of the mixture was detected using a microplate ELISA reader (MRXII, DYNEX Technologies, Chantilly, VA). The absorbance of untreated cells was considered as 100%. The results were determined by three independent experiments. The relative cell viability was calculated as

where Wc is the average tumor weight of the control group, Wt is the average tumor weight of the tested group, and Wa and Wb are the body weight of the mice after and before the treatment, respectively.

Cell-Cycle Analysis. The effects of S-GL and CM-GL on S-180 cell- cycle were assessed by flow cytometry. The cells (5 x 104 cells/mL) were incubated on a 6-well plate with polysaccharide samples (at final con­centrations of 0.02 mg/mL) for 24 h. After the incubation, the cells were washed with phosphate-buffered saline (PBS) twice, fixed in 70% cold ethanol, and stored at -20 0C overnight. Prior the analysis, the fixed cells were washed with PBS twice and stained with 50 ig/mL propidium iodide (PI). The stained cells were then transferred to flow tubes by passing through nylon mesh with a pore size of 40 im. Flow cytometric analysis was performed on a flow cytometer (Beckman Coulter, Epics XL MCL). Apoptotic cells were determined by their hypochromic sub-diploid stain­ing profiles. The distribution of cells in the different cell-cycle phases was analyzed from the DNA histogram using Multicycle software (Phoenix Flow Systems, San Diego, CA)

Detection of Apoptosis. Shrinkage of cells and disorganization of chromatin are usually related to apoptosis. Simultaneous staining with Annexin V-FLOUS and PI distinguished between intact cells, early apoptosis, late apoptosis, or cell death (6). Annexin V-FLOUS, a calcium-dependent phospholipid binding protein with a high affinity for phosphatidylserine, was used to detect cell apoptosis. Treated S-180 cells (1.5 x 105) were plated onto a gelatinized culture flask in RPMI 1640 containing 20% fetal bovine serum (FBC) and incubated at 37 0C and 5% CO2 for 24 h. The medium was replaced with 1 mL of RPMI 1640 with 5% FBC and polysaccharide samples (at final concentrations of 0.02 mg/mL) for 24 h and incubated at 37 0C and 5% CO2 for 48 h. Treated and control S-180 cells were labeled with Annexin V-FLOUS (10 ig/mL) and PI (20 ig/mL) prior to harvesting. After labeling, all plates were washed with binding buffer and harvested by scraping. Cells were resuspended in binding buffer at a concentration of 2 x 105 cells/mL before analysis by flow cytometry (FACScan). Data analysis was performed using standard Cell Quest software (Becton-Dickinson).

Immunohistochemistry for Apoptosis-Related Proteins Bax and Bcl-2. The tests of Bax and Bcl-2 immunohistochemistry were performed by the peroxidase-labeled streptavidin-biotin method with microwave antigen retrieval (17-19).Paraffin in the sections were removed by treating with xylene for 15 min twice and then gradually hydrated through a series from 95, 80, to 70% of graded ethanol (2 min each) and distilled water (2 min). Endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide (H2O2) for 10 min at 37 0C, followed by washing for 4 times with PBS (5 min each). Antigen retrieval was carried out in a microwave oven. Briefly, the sections were heated to 95 0C in 0.01 M citrate buffer (pH 6.0) and were maintained at this temperature for 10 min, followed by rinsing in warm tap water. Then, the sections were treated with normal goat serum for 10 min at 37 0C to reduce non-specific staining. Subsequently, the rabbit anti-mouse Bax and Bcl-2 polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as primary antibodies were added in optimal dilution, and the sections were incubated overnight in a humidified chamber at 4 0C. This step and each of the following were

 succeeded by washing for 4 times in PBS for 5 min each. The sections were incubated for 10 min at 37 °C in a humidified chamber with biotinylated goat anti-rabbit IgG (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China; 1:200) as secondary antibodies, followed by exposure to streptavidin horseradish peroxidase (Zhongshan Golden Bridge Biotech­nology Co., Ltd., Beijing, China) for 10 min at 37 °C. The color was developed using a solution containing 0.05% diaminobenzidine (DAB) as chromogen and 0.02% H2O2. After visualization of horseradish-perox- idase activity by color reaction with DAB, the sections were weakly counterstained with hematoxylin, mounted, and examined using a light microscope equipped with a 40 x objective. To confirm immunospecificity, negative controls consisted of sections in which the primary antibody was omitted and replaced by a buffer or non-immune IgG. Formalin-fixed, paraffin-embedded sections of normal human lymph node served as positive controls. Both the number of immuno-reactive cells and the total number of cells (at least 500 cells) were determined to calculate the percentage of Bax and Bcl-2 positive cells by visual inspection of six different fields per section. To assess the apoptosis, ratios of Bcl-2/Bax were determined.

Statistical Analysis. The mean values and standard deviations of the data between the control and the treatment groups were analyzed by Dunnett's test, and the p values for any significant differences between means were set at less than 0.05. The flow cytometric analyses were repeated twice to confirm the findings.

RESULTS AND DISCUSSION

Chemical Structure. The FTIR spectra of the native GL-IV-I and its derivatives are shown in Figure 1. All samples exhibited the characteristic IR absorption of polysaccharides at 1250 and 1650 cm-1.The native GL-IV-I exhibited absorption peaks at 890 cm-1, which is the characteristic absorption peak of the fi configuration of glucan (20). In comparison to GL-IV-I, the characteristic peak of the fi configuration of S-GL at 890 cm-1 disappeared and two new absorption peaks at 813 and 1210 cm-1 appeared for the sulfated derivative, which corresponds to C-S-C symmetrical vibration and S=O asymmetrical stretch­ing (21 ). This indicated that the sulfated derivative had been

 

Table 1. 13C NMR Chemical Shifts for Native Sample GL-IV-I and Its Derivatives

sample

 

 

 

 

 

à (ppm)

 

 

 

 

 

C-1

C-10

C-2

C-2s

C-3

C-30 C-4

C-4s

C-5

C-50

C-6

C-6s

GL-IV-I

103.9

 

73.6

 

86.9

69.2

 

77.1

 

61.6

 

S-GL

103.8

101.3

73.7

79.7

86.9

69.1

76.2

77.0

72.8

61.4

67.9

CM-GL

103.1

 

73.7

82.1

86.4

85.2 69.0

74.5

76.5

74.1

61.5

68.9

 

successfully synthesized from GL-IV-I. For CM-GL, two new absorption peaks appeared at 1603 and 1430 cm-1, assigned to the asymmetrical -COO stretching vibration. The symmetrical -COO stretching and the new peak at 1340 cm-1 were assigned to the -CH2 group (22-25).

The heteronuclear multiple-quantum coherence (HMQC) and double quantum filter (DQF) were described previously (13). The results could confirm that GL-IV-I was a linear (1 f 3)- ^-D-glucan (26). The 13C NMR spectra for the native sample GL-IV-I and its derivatives (S-GL and CM-GL) are shown in Figure 2, and their chemical shifts are listed in Table 1.The13CNMR spectrum of the native GL-IV-I exhibited six signals around 103.9, 86.9, 77.1, 73.6, 69.2, and 61.6 ppm, attributed to C-1, C-3, C-5, C-2, C-4, and C-6, respectively, indicating the characteristic of (1 f 3)- ^-D-glucan (22). In comparison to the native GL-IV-I sample, there were several new signals caused by sulfated groups in S-GL. The peak at 67.9 was assigned to the signal of C-6s; the peak at 79.7 was assigned to the signal of C-2s; and the peak at 76.2 was assigned to the signal of C-4s. The peak at 61.4 was weakened, indicating that C-6 had been substituted by the sulfated group, whereas C-2 and C-4 have been partially substituted. Because

C-2

their strong peaks existed in the NMR spectrum, the peaks at 79.7 for C-2s and 76.2 for C-4s were much weaker than that of C-6s at 67.9. We could conclude that the C-6 position was more active than the C-2 and C-4 positions as a result of the steric hin­drance (27). The downfield shift of the carbon atoms linked with the sulfur groups was about 6-7 ppm (28). Furthermore, a new peak appeared at 101.3, and the native C-1 peak at 103.8 became weaker in NMR spectra of S-GL. This could be explained by the fact that C-2 and C-6 had been substituted, which could influence the adjacent C-1 to split into two peaks. The upfield shift from C-1 to C-10 was about 2.5 ppm, which is somewhat in line with the description by Gorin (28). With the same situation to C-5, the peak at 72.8 is for C-50 of S-GL. In comparison to the native GL- IV-I, a new strong peak at 175.7 for CM-GL could be attributed to the C=O carbon of the carboxymethyl group, indicating the successful carboxymethylation of GL-IV-I. Peaks for C-6s, C-4s, and C-2s were at 69.0, 74.5, and 82.1, and the peak intensity was in the order C-6s > C-4s > C-2s. From 13C NMR results, we could conclude that non-selectivity substitution occurred on the active hydroxyl groups of GL-IV-I and the multi-substitution at C-2, C-4, and C-6 exhibited different reaction activity. The yields, water solubility, and DS of the native polysaccharide and its derivatives are summarized in Table 2. The DS of the derivatives was calculated from the NMR data according to the method reported by Zhou et al. (27). In brief, the total DS of each kind of derivative was the summation of the DS for C-6, C-4, and C-2, and the relative DS values at the C-2, C-4, and C-6 positions were calculated from the relative intensities of peaks C-2, C-2s, C-4, C-4s, C-6, and C-6s. The DS of C-6, C-4, and C-2 calculated from 13C NMR for S-GL and CM-GL were 0.68:0.12:0.04 and 0.62:0.28:0.19, respectively. Reaction activity of the active hydro- xyl groups was in the order of C-6 > C-4 > C-2 on the whole. This could be explained by the fact that the steric hindrance led to the different activities of C-6, C-4, and C-2. The substitution further caused the adjacent C-1, C-3, and C-5 to split into two peaks, with the upfield shift of 2-3 ppm.

Molecular Mass and Chain Conformation. SEC-LLS, as an absolute method, is a convenient method for the determination of the true molecular mass and its distribution. The molecular mass and chain conformation parameters of GL-IV-I and its deriva­tives are summarized in Table 3. The SEC patterns of GL-IV-I in Me2SO and the two derivatives in 0.9% NaCl aqueous solution analyzed by the LLS (detector d11 = 900) at 25 ^ are shown in Figure 3. Obviously, there were single peaks with good symmetry detected by LLS in GL-IV-I and its derivatives. The weight- average molecular mass (Mw) of GL-IV-I, S-GL, and CM-GL were 13.3 x 104, 10.1 x 104, and 6.3 x 104, respectively. The molecular mass of S-GL and CM-GL is lower than that of GL- IV-I because of the chain degradation during the reaction. 

From the SEC chromatogram detected by LLS, we could obtain the Mw and (S2)z1/2 values of numberless fractions. The Mw-(S2)z1/2 relationship from many experimental points in the SEC can give conformation parameters (29). From data of Mw and (S2)z1/2, the power law describing the relationship between Mw and (S2)z1/2 ((S2)z1/2 - Mwa) can be created. Usually, the chain conformation of the polymer in solution can be estimated by the value of exponent (a). Usually, the a value is 0.3 for globular shape, 0.5-0.6 for flexible chain conformation in good solvent, and 0.6-1 for a semi-flexible or stiff chain (30). The log-log plots of (S2)z1/2 - Mwa for GL-IV-I in Me2SO and its derivatives in 0.9% NaCl are shown in Figure 4. The a value for GL-IV-I was 0.47, indicating that the glucans existed as a flexible

Table 3. Experiment Results from LLS, SEC-LLS, and Viscosity for GL-IV-I and Its Derivatives

sample

solvent

 

LLS

 

SEC-LLS

 

 

[n] (cm3 g 1)

Mw (x10-4) (g mol-1)

A2 (x104) (mol mL g-2)

<SV/2 (nm)

Mw (x10-4) (g mol-1)

Mw/Mn

a

GL-IV-I

Me2SO

12.4

3.67

53.5

13.3

1.37

0.47

27.8

S-GL

0.9% NaCl

12.5

2.14

56.6

10.1

1.86

0.61

29.3

CM-GL

0.9% NaCl

5.2

3.56

35.7

6.3

1.62

0.56

39.7

 

Table 4. Results of in Vivo Antitumor Activities and the Enhancement Ratio of the Body Weight of S-GL and CM-GL Compared to 5-Fua sample                                           dose (mg/kg x days)                             inhibition ratio (%)                           enhancement ratio of body weight (%)                                           complete repression

S-GL                                              40 x 8                                           55.6 ± 2.1                                                       48.5 ± 1.5                                              0/8

CM-GL                                           40 x 8                                           51.3 ± 1.3                                                      45.7 ± 2.7                                              0/8

5-Fu                                               40 x 8                                           61.9 ± 3.8                                                      28.2 ± 1.7b                                             0/8

0.9% NaCl                                     40 x 8                                                                                                                  46.4 ± 2.7                                              0/8

 

Figures. Percentage of viability of S-180 cells treated with sulfated (S-GL) and carboxymethylated (CM-GL) derivatives of GL-IV-I at different concentrations. Data are shown as mean ± standard deviation (SD) (n = 3).

chain conformation in Me2SO at 25 °C. The a values for S-GL and CM-GL were 0.61 and 0.56, respectively. This suggested that the chain stiffness of the two derivatives was slightly higher than that of the native glucan. Namely, the derivatives existed as relatively expanded flexible chains in 0.9% NaCl aqueous solu­tion at 25 °C, as a result of the steric hindrance of the substituted groups in S-GL and CM-GL. The introduction of the charged groups, such as sulfated and carboxymethylated groups, im­proved the water solubility and increased the chain stiffness.

Antiproliferative Effect of S-GL and CM-GL on S-180. The antiproliferative effect of S-GL and CM-GL on the S-180 tumor cell line was investigated. Cells were treated at different concen­trations of these two derivatives (0-500 ^g/mL) for 24 h. Figure 5 shows the percentage of viability of S-180 cells in the presence of S-GL and CM-GL with different concentrations. Cells incubated with 0.2% Me2SO were used as a control. The median inhibitory concentration (IC50) values of S-GL and CM-GL tested against S-180 cancer cells were 26 and 38 ^g/mL after 24 h of treatment. Therefore, S-GL and CM-GL could inhibit the in vitro prolifera­tion of S-180 tumor cells in a dose-dependent manner.

In Vivo Antitumor Activities. To investigate the antitumor activities of S-GL and CM-GL, the sample solutions were injected intraperitoneally (40 mg/kg) once daily for 8 days after S-180 tumor cell inoculation for 24 h. The inhibitions of tumor growth in vivo and enhancement ratio of body weight are summarized in Table 4. The native GL-IV-I showed no antitumor activity because of the poor water solubility. However, the inhibition ratios of S-GL and CM-GL were 55.6 ( 2.1 and 51.3 ( 1.3%, respectively. The growth of the S-180 solid tumors was markedly inhibited by S-GL and CM-GL. It is worth noting that the two derivatives exhibited relatively high inhibition on the tumor growth, only slightly lower than that of 5-Fu. Figure 6 shows pictures of the tumors of the control group treated with 0.9% NaCl aqueous solution and the experimental groups treated with S-GL and CM-GL. Although the 5-Fu group exhibited more effective results, its body weight enhancement ratio was 28.2 ( 1.7%, which was much lower than that of S-GL and CM-GL groups (48.5 ± 1.5 and 45.7 ± 2.7%, respectively). Besides, the enhancement ratio of the body weight of mice treated with S-GL and CM-GL groups was almost equal to the control group. Therefore, our findings indicated that S-GL and CM-GL are potential antitumor agents, having high bioactivities and low toxicity. S-GL had a lower IC50 value than that of CM-GL, implying that the introduction of sulfated groups is more con­ducive to the enhancement of the antitumor activity of the polysaccharides.

Cell-Cycle Arrest and Apoptosis Induction by Polysaccharides. Apoptosis is a physiological and crucial process that is regarded as the preferred way to eliminate cancer cells (31, 32). Figure 7 shows the effect of (a) CM-GL and (b) S-GL on the cell-cycle phases (G0/G1, S, and G2/M) of S-180 cells. S-GL and CM-GL induced accumulation of S-180 cells in the sub-G1 peak, indicat­ing apoptotic phenomenon, as well as led to the G2/M phase arrest. In the cell-cycle analysis, a significant increase of the cell population in the G2/M phase was observed from 16.1 ± 1.4% of the control group to 33.5 ± 2.4% for S-GL and 29.7 ± 1.5% for CM-GL, with the concentration of 0.02 mg/mL. These results suggested that the S-GL and CM-GL derivatives could inhibit the growth of S-180 cells through cell-cycle arrest in the G2/M phase and induced the apoptosis. Figure 8 shows representative dot plots of fluorescence intensities of S-180 cells labeled with Annexin V/PI. The percentage of surviving (Annexin V and PI negative, AnVK/PIK) and apoptotic (Annexin V positive and PI negative, AnVC/PIK) cells was indicated in the quadrants. The percentage of apoptosis in control S-180 cells (2.57 ± 0.39%) was significantly (p < 0.05) lower than that of S-GL (17.9 ± 1.98%) and CM-GL (14.1 ± 2.67%). Treatment with S-GL and CM-GL resulted in a significant increase in the percent of apoptosis of S- 180 cells. The percentage of surviving cells in the three groups was inversely related to the percentages of apoptotic cells. In view of the results, S-GL and CM-GL exhibited a significant increase of apoptosis and decrease in the survival of S-180 cells.

The proto-oncogene Bcl-2 protects cells against apopto- sis, whereas Bax promotes cells against apoptosis (33).

 

We investigated alterations in the expression of apoptosis-related proteins Bcl-2 and Bax in S-180 tumor treated by intraperitoneal injection of the S-GL and CM-GL aqueous solution into BALB/c female mice. Figures 9 and 10 show the apoptosis-related proteins Bax and Bcl-2 immunostain in S-180 tumor of the untreated control mice, 5-Fu group, and S-GL and CM-GL treated mice. The results revealed a significant difference in the expression of the proteins in the control and S-GL and CM-GL groups. In the control group, Bax was not detected, whereas Bcl-2 immunostain was strong in the S-180 tumor. However, the mice treated with S-GL and CM-GL exhibited a significantly decreased expression of Bcl-2 and increased expression of Bax. The uniquely high Bax expression with low Bcl-2 immunostain indicated that the poly- saccharide derivatives induced apoptosis of S-180 tumor cells probably by upregulating the expression of Bax to counteract the effect of Bcl-2. The results from the quantitative analysis of the Bax and Bcl-2 immunostain for the S-180 tumor in the control and S-GL and CM-GL groups are summarized in Table 5.The expression levels of the two apoptotic proteins led to a signifi­cantly lower Bcl-2/Bax ratio, suggesting a favorable therapeutic response to S-GL and CM-GL and an increase of the survival rate in mice transplanted with S-180 tumors. Furthermore, a high expression level of Bax accompanied by a low Bcl-2 immuno- reactivity could sustain a low Bcl-2/Bax ratio in favor of apoptosis,

 or alternatively, it could effectively antagonize the anti-apoptotic levels of the Bcl-2. The immuno-histochemical results indicated that both S-GL and CM-GL could induce apoptosis in xenograft S-180 tumor cells by upregulating Bax and downregulating Bcl-2, while S-GL showed a better therapeutic effect than CM-GL.

On the basis of the results mentioned above, the water-soluble derivatives of polysaccharides having charged groups and relatively expanded chain conformation exhibited high antitumor activity. Therefore, the introduction of the sulfated and carboxymethylated groups could improve the water solubility and increase the chain stiffness, leading to the enhancement of the antitumor activities. The relatively extended chain conformation of carboxymethylated /3-glucan from sclerotium of P. tuber-regium and extracellular

samples

Bax (% immunoreactivity)

Bcl-2 (% immunoreactivity)

control group

3.8 ± 1.3

58.5 ± 4.2

5-Fu dose 40 mg/kg

30.3 ± 3.5b

21.8 ± 2.7b

S-GL dose 40 mg/kg

46.5 ± 2.9b

39.6 ± 3.1c

CM-GL dose 40 mg/kg

42.6 ± 3.6b

30.7 ± 2.4b

a Results are expressed as mean ± SD (n = 8). cp < 0.05 (Dunnett's test).

 polysaccharides from Ganoderma tsugae mycelium correlated with their relatively higher antitumor activity. This could be explained by the fact that charged groups and relatively expanded flexible chains of polysaccharides have more opportunities to bind with receptors on the cell than the compact conformation.

ACKNOWLEDGMENT

We thank Professor Zeng of Tongji Medical College of Huaz- hong University of Science and Technology for his kind help.

LITERATURE CITED

  • (1) Zhang, C.; Huang, K. Mechanism of apoptosis induced by a polysaccharide, from the loach Misgurnus anguillicaudatus (MAP) in human hepatocellular carcinoma cells. Toxicol. Aool. Pharmacol. 2006, 210, 236-245.
  • (2) Wasser, S. P. Medicinal mushrooms as a source of anti-tumor and immuno-modulating polysaccharides. Appl. Microbiol. Biol. 2002, 60, 258-274.
  • (3) Yang, X. B.; Zhao, Y.; Yang, Y.; Ruan, Y. Isolation and character­ization of immunostimulatory polysaccharide from an herb tea, Gynostemma pentaphyllum Makino. J. Agric. Food Chem. 2008, 56, 6905-6909.
  • (4) Zhu, J. F.; Wu, M. Characterization and free radical scavenging activity of rapeseed meal polysaccharides WPS-1 and APS-2. J. Agric. Food Chem. 2009, 57, 812-819.
  • (5) Tommonaro, G.; Segura Rodriguez, C. S.; Santillana, M.; Immirzi, B.; De Prisco, R.; Nicolaus, B.; Poli, A. Chemical composition and biotechnological properties of a polysaccharide from the peels and antioxidative content from the pulp of Passiflora liguralis fruits. J. Agric. Food Chem. 2007, 55, 7427-7433.
  • (6) Cheng, Y. L.; Lee, S. C.; Lin, S. Z.; Chang, W. L.; Chen, Y. L.; Tsai, N. M.; Liu, Y. C.; Tzao, C.; Yu, D. S.; Harn, H. J. Anti-proliferative activity of Bupleurum scrozonerifolium in A549 human lung cancer cells in vitro and in vivo. Cancer Lett. 2005, 222, 183-193.
  • (7) Li, G.; Kim, D.-H.; Kim, T.-D.; Park, B.-J.; Park, H.-D.; Park, J.-I.; Na, M.-K.; Kim, H.-C.; Hong, N.-D.; Lim, K.; Hwang, B.-D.; Yoon, W.-H. Protein-bound polysaccharide from Phellinus linteus induces G2/M phase arrest and apoptosis in SW480 human colon cancer cells. Cancer Lett. 2004, 216, 175-181.
  • (8) Cao, L.; Lin, Z. Regulation on maturation and function of dendritic cells by Ganoderma lucidum polysaccharides. Immunol. Lett. 2002, 83, 163-169.
  • (9) Nie, X.; Shi, B.; Ding, Y.; Tao, W. Preparation of a chemically sulfated polysaccharide derived from Grifola frondosa and its poten­tial biological activities. Int. J. Biol. Macromol. 2006, 39, 228-233.
  • (10) Huang, Q.; Zhang, L. Solution properties of (1 f 3)-a-D-glucan and its sulfated derivertives from Poris cocos mycelis via fermentation tank. Biopolymers 2005, 79, 28-38.
  • (11) Zhang, M.; Zhang, L.; Cheung, P. C. K. Molecular mass and chain conformation of carboxymethylated derivatives of ^-D-glucan from sclerotia of Pleurotus tuber-regium. Biopolymers 2003, 68, 150-159.
  • (12) Zhang, M.; Zhang, P. C. K.; Zhang, L.; Chiu, C. M.; Ooi, V. E. C. Carboxymethylated ¿3-glucan from mushroom sclerotium of Pleur- otus tuber-regium as novel water-soluble anti-tumor agent. Carbo- hydr. Polym. 2004, 57, 319-325.
  • (13) Wang, J.; Zhang, L. Structure and chain conformation of five water- soluble derivatives of ^-D-glucan isolated from Ganoderma lucidum. Carbohydr. Res. 2009, 344, 105-112.
  • (14) Yoshida, T.; Yasuda, Y.; Mimura, T.; Kaneko, Y.; Nakashima, H.; Yamamoto, N.; Uryu, T. Synthesis of curdlan sulfates having inhibitory effects in vitro against AIDS viruses HIV-1 and HIV-2. Carbohydr. Res. 1995, 276, 425-436.
  • (15) Bao, X.; Duan, J.; Fang, X.; Fang, J. Chemical modifications of the (1 f 3)-a-D-glucan from spores of Ganoderma lucidum and investi­gation of their physicochemical properties and immunological activity. Carbohydr. Res. 2001, 336, 127-140.
  • (16) Qiu, H.; Tang, W.; Tong, X.; Ding, K.; Zuo, J. Structure elucidation and sulfated derivatives preparation of two a-D-glucans from Gastrodia elata Bl. and their anti-dengue virus bioactivities. Carbo­hydr. Res. 2007, 342, 2230-2236.
  • (17) Lohmann, C. M.; League, A. A.; Clark, W. S.; Lawson, D.; DeRose, P. B.; Cohen, C. Bcl-2:Bax and Bcl-2:Bcl-x ratios by image cyto­metric quantitation of immunohistochemical expression in ovarian carcinoma: Correlation with prognosis. Cytometry 2000, 42, 61-66.
  • (18) Saxena, A.; McMeekin, J. D.; Thomson, D. J. Expression of Bcl-x, Bcl-2, Bax, and Bak in endarterectomy and atherectomy specimens. J. Pathol. 2002,196, 335-342.
  • (19) Xie, X.; Clausen, O. P. F.; Angelis, P. D.; Boysen, M. The prognostic value of spontaneous apoptosis, Bax, Bcl-2, and p53 in oral squa- mous cell carcinoma of the tongue. Cancer Lett. 1999, 86, 913-920.
  • (20) Kiho, T.; Sakushima, M.; Wang, S.; Nagai, K.; Ukai, S. Polysac­charides in fungi. XXVI. Two branched (1,3)-3-D-glucans from hot water extract of yuer. Chem. Pharm. Bull. 1991, 39, 798-880.
  • (21) Zhang, M.; Zhang, L.; Chen, J.; Zeng, F. Solution properties of antitumor sulfated derivatives of (1 f 3)-a-D-glucan from Gano­derma lucidum. Biosci., Biotechnol., Biochem. 2000, 64, 2172-2178.
  • (22) Wang, Y.; Zhang, L.; Li, Y.; Hou, X.; Zeng, F. Correlation of structure to antitumor activities of five derivatives of a ¿3-glucan from Poria cocos sclerotium. Carbohydr. Res. 2004, 339, 2567-2574.
  • (23) Jin, Y.; Zhang, H.; Yin, Y.; Nishinari, K. Comparison of curdlan and its carboxymethylated derivative by means of rheology, DSC, and AFM. Carbohydr. Res. 2006, 341, 90-99.
  • (24) Zhang, L.; Zhang, M.; Chen, J. Solution properties of antitumor carboxymethylated derivertives of (1 f 3)-a-D-glucan from Gano- derma lucidum. Chin. J. Polym. Sci. 2001, 19, 283-289.
  • (25) Wang, Y.; Zhang, L.; Ruan, D. Preparation and structure of five derivertives of (1 f 3)-3-D-glucan isolated from Poria cocos scler­otium. Chin. J. Polvm. Sci. 2004, 22, 137-145.
  • (26) Kato, T.; Okamoto, T.; Tokuya, T.; Takahashi, A. Solution proper­ties and chain flexibility of pullulan in aqueous solutions. Biopolv- mers 1982, 21, 1623-1633.
  • (27) Zhou, J.; Zhang, L.; Deng, H.; Wu, X. Synthesis and characteriza­tion of cellulose derivatives prepared in NaOH/urea aqueous solu­tions. J. Polvm. Sci.. Part A: Polvm. Chem. 2004, 42, 5911-5920.
  • (28) Gorin, P. A. J. Carbon-13 nuclear magnetic resonance spectroscopy of polysaccharides. Adv. Carbohvdr. Chem. Biochem. 1981, 38, 13-104.
  • (29) Picton, L.; Bataille, G.; Muller, G. Analysis of a complex poly- saccharide (gum arabic) by multi-angle laser light scattering coupled on-line to size exclusion chromatography and flow field flow fractionation. Carbohvd. Polvm. 2000, 42, 23-31.
  • (30) Chen, X.; Xu, X.; Zhang, L.; Zeng, F. Chain conformation and anti­tumor activities of phosphorylated (1 f 3)-3-D-glucan from Poria cocos. Carbohvdr. Polvm. 2009, 78, 581-587.
  • (31) Sogawa, K.; Yamada, T.; Sumida, T.; Hamakawa, H.; Kuwabara, H.; Matsuda, M. Induction of apoptosis and inhibition of DNA topoisomerase-I in K-562 cells by a marine microalgal polysacchar- ide. Life Sci. 2000, 66, 227-231.
  • (32) Zhang, C.; Huang, K. Apoptosis induction on HL-60 cells of a novel polysaccharide from the mucus of the loach, Misgurnus anguillicau- datus. J. Ethnopharmacol. 2005, 99, 385-390.
  • (33) Tsujimoto, Y.; Crose, C. M. Analysis of the structure, transcripts, and protein products of Bcl-2, the gene involved in human follicular lymphoma. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 5214-5218.

Received for review July 27, 2009. Revised manuscript received October 7, 2009. Accepted October 7, 2009. We gratefully acknowledge the major grants of the National Natural Science Foundation of China (30530850), the National Natural Science Foundation (20874079), and the High-Technology Research and Development Program of China (2006AA02Z102).

 


[1]To whom correspondence should be addressed. Telephone: +86­27-87219274. Fax: +86-27-68754067. E-mail: lnzhang@public.wh. hb.cn.

 

International Journal of Biological Macromolecules xxx (2010) xxx-xxx

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

 

 

 

 

 

( G.lucidumdan polisakkarit ektraksiyonu ve bunların bağışıklığın arttırılmasındaki rolleri)

Extraction of polysaccharide from Ganoderma lucidum and its immune enhancement activity

Sheng-quan Huanga,b, Zheng-xiang Ning3'*

2

a South China University of Technology, Guangdong Province, Guangzhou 510640, China b Infinitus (China) Co., Ltd., Jiangmen 529156,China

 

 

 

 

 

6

 

 

 

 

 

ARTICLE INFO

Article history:

Received 15 February 2010

Received in revised form 6 March 2010

Accepted 29 March 2010

Available online xxx

7

8 9 10 11 12

Keywords: Polysaccharide Extraction Immunoassay

ABSTRACT

In the present study, in order to maximize the yield of polysaccharides from Ganoderma lucidum, response surface methodology was employed to optimize the ultrasonic/microwave-assisted extraction (UMAE) conditions. The results indicated that the optimal extraction conditions were ultrasonic power of 50W, microwave power of 284 W, extraction time of 701 s and water/solid ratio of 11.6:1, respectively. Using UMAE, the yield of polysaccharides was 115.56% above that of classical hot water extraction (HWE) Q1 and increased by 27.7% than ultrasound-assisted extraction (UAE), which confirmed the great poten­tial application of UMAE technology in the extraction of polysaccharides. The immunological assays results demonstrated that polysaccharides of G. lucidum extracted by ultrasonic/microwave (UMP) could improve the weight of immune organ of immunocompromised mice, restore delayed-type hypersensitiv­ity (DTH) reaction to DFNB, improve hemolysis antibody level and natural killer cell activity at high-dose. However, UMP had no noticeable effects on phagocytosis of monocyte at the tested dosage range.

© 2010 Published by Elsevier B.V.

 

 

 

 

 

is                                                                                            1. Introduction

  • 19 Glossy ganoderma (known as Lingzhi in China) is an oriental fun-
  • 20 gus and a traditional Chinese medicine used for more than 2000
  • 21 years to promote health and longevity.
  • 22 The fruit bodies, cultured mycelia and spores of Ganoderma
  • 23 lucidum were reported to be effective in the treatment of chronic
  • 24 hepatopathy, hypertension, hyperglycemia and neophasia [1-3].
  • 25 There are many different varieties wildly cultured in China, such
  • 26 as G. lucidum, Ganoderma sinense, Coriolus versicolar, Fructifica-
  • 27 tio Amaurodermatis Rudae. The main functional components of G. 2s lucidum include polysaccharides, protein, peptides, amino acid,
  • 29 triterpenes and the polysaccharides have been well known for their
  • 30 immuno-modulatory and anti-tumor functions [4].
  • 31 Extraction of polysaccharides is an important processing for
  • 32 its application, and this has prompted numerous research papers
  • 33 on the extraction technology of polysaccharides from plentiful of
  • 34 plants or fungus in recent years. Dong et al. optimized the hot
  • 35 water extraction (HWE) process of polysaccharides from cultured
  • 36 mycelium of Cordyceps sinensis using Box-Behnken design [5]. Yang
  • 37 et al. employed ultrasonic technology to extract polysaccharides 3s from longan fruit pericarp and obtained the optimal extracted con-

* Corresponding author at: South China University ofTechnology, College of Light Industry and Food Sciences, Guangdong Province, Guangzhou 510640, China. Tel.: +86 20 87112594; fax: +86 20 87112594.

E-mail address: fezhning@scut.edu.cn (Z.-x. Ning).

0141-8130/$ - see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.ijbiomac.2010.03.019

 

ARTICLE IN PRESS

S.-q. Huang, Z.-x. Ning / International Journal of Biological Macromolecules xxx (2010) xxx-xxx

[15]. However, there exists significant inhomogeneous phenom­ena during microwave treatment [12,15] The ultrasonic-assisted extraction is based on the propagation of ultrasonic pressure waves, and resulting cavitation phenomena, while there exists variation in the extraction yield from different plant varieties because of the difference in the structure, rheology or the susceptibility to ultra­sonic shock wave [13]. It is likely that combining ultrasonic with microwave is a complementary technique and may show unpre­dictable advantages. However, there are a few research papers on the UMAE technology, especially for the polysaccharides from G. lucidum.

Recent literatures showed that the polysaccharides isolated from G. lucidum are important functional factors and they could stimulate the proliferation of mouse spleen lymphocytes [16], and to exhibit various other bioactivities including anti-HIV, anti- herpetic, antiviral, immune regulating and anti-tumor properties [17-20]. Bao et al. obtained crude polysaccharides of G. lucidum by hot water extraction and found it exhibited an immune-stimulating activity in mice [18].

In this paper, the objective was to optimize the UMAE condition using response surface methodology (RSM) design and compared with the traditional hot water extraction and the ultrasonic- assisted extraction. Moreover, the immune regulating activities of polysaccharides from G. lucidum were also investigated.

2. Materials and methods

•2.1.  Materials

G. lucidum was provided by Zhejiang Longquan Keda Agricul­tural Limited Company and was identified as artificial cultural G. lucidum by Mao Xiao-lan researcher of Institute of Micro­biology Chinese Academy of Sciences. Dexamethasone sodium phosphate was produced by Hubei Tianyao Pharmaceutical Co., Ltd. Cyclophosphamide was produced byJiangsu Hengrui Medicine Co., Ltd. Propidium Iodide (PI) solution and 2,4-dinitrofluorobenene were obtained from Sigma. RPMI 1640 cell culture was Gbico's product from the United States. YAC-1 cells were purchased from the Cell Center of Sun Yat-sen University. Fluorescent dye CFSE was produced by Invitrogen Corporation of USA. All reagents were of analytical grade. Pachymaran oral solution: 10mL/branch, 16 mg/ml, produced by Hunan Butian Pharmaceutical Co., Ltd.

UMAE apparatus (CW-2000, Shanghai Xintuo Microwave Instrument Co. Ltd. China) was used for ultrasonic/microwave- assisted extraction of polysaccharides. The microwave power could be adjusted in the range of 0-800 W at a frequency of 2450 MHz, and the ultrasonic power was fixed at 50 W with a fixed frequency of 40 kHz, as shown in Fig. 1.

•2.2.  Experimental animals

Male NIH mice of SPF-level (6-8 weeks old, weighing 18-22 g) were provided by the Guangdong Medical Experimental Animal Center. The permit no. is SCXK (Guangdong) 2008-0002 Guangdong Monitoring and Certificate word 2008A021, qualified certificate no.: 0051028. The mice were housed under controlled conditions of 12/12 h light-dark cycle and 50% relative humidity at the tem­perature of 25-30°C

•2.3.  Extraction procedure

2.3.1. Ultrasonic/microwave extraction

Pretreatment: The G. lucidum were ground to pass through 2 mm screen and pretreated to deactivate the endogenous enzymes and remove some soluble materials, including free sugars, amino acids

 and some phenols, and obtained pellet of G. lucidum according to the method of Li et al. [21].

UMAE procedure: The ultrasonic/microwave extraction proce­dure were carried out at the microwave power of 116-284 W, ultrasonic power of 50 W and ultrasonic frequency of 40 kHz. The pellet of G. lucidum powders were dispersed in a 100 mL flask at water to solid ratios (mL/g) of 11.6:1, 15:1, 20:1, 25:1 and 28.4:1. Then the flask was transferred into the chamber of the apparatus connected with condensing tubes. When extraction of polysaccharide was accomplished, the mixture was cooled to room temperature using ice water and centrifuged (4000 x g, 15 min), the supernatant was concentrated in a rotary evaporator under reduced pressure, and then precipitated by the addition of ethanol (12 h, 4 °C) to a final concentration of 85% (v/v), and the precipi­tates were collected by centrifugation (4000 x g, 15 min), washed with cold 100% ethanol, acetone and finally lyophilized to obtain G. lucidum crude polysaccharide.

2.3.2. Experimental design of RSM

RSM was used to find out the optimal ultrasonic/microwave- assisted extraction condition of polysaccharides from G. lucidum. The extraction experiment was carried out according to a cen­tral composite design with three factors and five levels. Three independent variables selected for this paper were microwave power, extraction time and water/solid ratio (shown in Table 1). For each factor, an experimental range was based on the results of preliminary single-factor experiments. Yield of the extracted polysaccharides were the dependent variables. The complete design consisted of 20 experimental points including eight factorial points and six center points. The experiment was carried out in a random order.

Table 1

Independent variables and their levels in the response surface design.

Independent variables

Factor level

 

 

 

 

-1.68

-1

0

1

1.68

Microwave power (W)

116

150

200

250

284

Time (s)

398

480

600

720

802

Water/solid ratio

11.6:1

15:1

20:1

25:1

28.4:1

 

Data from the central composite design were analyzed by mul­tiple regressions to fit the following quadratic polynomial model.

y = bko + bkixi + bkix2 + bkijXi*

j=1

where y is the dependent variable, bto, bki, bUi and b^j are constant regression coefficients of the model, while xi, xj are the independent variables.

•2.3.3.  Traditional hot water extraction

The traditional hot water extraction was carried out in a water bath (HH-2 Guohua Wiring Company, Shanghai, China) at the optimal extraction condition: extraction temperature of 100 °C, extraction time of 5h, and water/solid ratio of 20:1 based on the preliminary three-factor and three-level designed orthogonal opti­mal experiment.

•2.3.4.   Ultrasonic extraction of polysaccharides Ultrasonic-assisted extraction was carried out in a JY92-2D

ultrasonic generator (Xinzhi Bio-technology Institute, Shanghai, China, 20 kHz) with a power of 550 W, extraction time of 80 min, and water/solid ratio of 25:1 based on the preliminary RSM opti­mized experiment.

2.4. Immune enhancement activity of polysaccharides from G. lucidum extracted by ultrasonic/microwave

•2.4.1.  Experimental group

The mice were randomized to six groups: normal control group, model control, pachymaran oral solution, low-dose UMP with 50mg/kg/d, medium-dose UMP with 100mg/kg/d, and high-dose UMP with 200 mg/kg/d. The moisture content of UMP is 22.78%, polysaccharides content is 40.80% and protein content is 2.28%.

•2.4.2.  Carbon clearance test in mice

The test of carbon clearance was carried out according to the method ofYang et al. [22].The mice were injected intraperitoneally with dexamethasone (50 mg/kg) to cause an immunocompromised model on the 27th and 29th days of gastric feed while the nor­mal control group was injected with an equal volume of normal saline (NS). After 31 days of gastric feed, Indian ink according to 0.1 mL/10g body weight was injected into the tail vein of the mice. A total of 20 |L blood was respectively collected through eye orbit after 2min (t1) and 20min (t2), and added to 2mL 0.1% Na2CO3 at once. The absorbance A (595 nm) of blood after 2 and 20 min was measured in an ELISA reader. At the same time, mice were sacri­ficed by cervical dislocation, liver and spleen weights of the mice were measured. The clearance index (k) and the calibrate index (a) were calculated as follows:

2.4.3. Delayed-type hypersensitivity in mice (DTH)

After 26th day of gastric feed, mice were sensitized to 2,4- dinitrofluorobenzene solution (DNFB) by smearing 20 | L 5% DNFB on the abdominal skin of mice by hair removal agent. On the 27th day, mice were intraperitoneally injected with cyclophosphamide (100 mg/kg) to cause an immunocompromised model. On the 30th day, 20 |L 1% DNFB was smeared on the left ear as an attack, while the right ear was sweared with 20 | L acetone solution as control. Twenty-four hours later, the DTH response to DNFB was evaluated by measuring weight difference of left and right ear.

•2.4.4.  Determination of serum hemolysin test

After 27 days of gastric feed, mice were injected intraperi- toneally by 5% sheep red blood cells (SRBC) with 0.25mL/10g body weight. Serum of 20 | L was collected through eye orbit after four more days, then, blended with 2 mL saline of 0.15 M NaCl and centrifuged for 10 min at 2000 rpm. The supernatant of 50 | L was blended with 250 |L, 2.5% SRBC and 250 |L 1:20 guinea pig alexin. A saline solution was used as a control and every sample was controlled at water bath of 37 °C for 30 min, and then termi­nated the reaction in ice water for 10 min. Centrifuge at 2000 rpm for 10 min, obtained 250 | L supernatant and measured with a microplate reader at wavelength of 540 nm. At the same time, thy­mus and spleen weights of the mice were measured and the wet weight (mg) of each 10 g mouse was taken as the spleen index and thymus index, respectively.

•2.4.5.  Determination of natural killer (NK) cell activity

Mice were injected intraperitoneally with dexamethasone (50 mg/kg) on 29th day and 31st day to cause an immunocompro- mised model. After 33 days of gastric feed, mice were sacrificed by cervical dislocation. Spleens were taken out to make spleen cell suspension conventionally. Splenocytes were adjusted to the cell concentration of 2 x 106 cell/mL with RPMI-1640 medium contain­ing 10% fetal bovine serum (FBS).

YAC-1 cell was used as target for NK cell. The cells were adjusted to the concentration of 2 x 106 cell/mL with PBS and stained with 1 |imol/L fluorescein dye CFSE for 10 min. The cells were adjusted to a concentration of 1 x 105 cell/mL with RPMI1640 medium.

Splenocytes prepared from the spleens of mice were used as a source of natural killer cells (NK) effector cells. The effector cells in the 96-well microplates were co-cultured with target cells (100 |L) at the ratio of 50:1. After 2h incubation at 37°C, cells were col­lected into 5 mL centrifuge tube, added 1 mL PBS, then centrifuge at 1200 rpm for 10 min. After abandoning the supernatant and re- suspending the cells with 400 |L PBS, PI solution of 20 |L was added to stain for 5 min, and then to be analyzed by flow cytometry. Taking the CFSE and PI double positive cells as the death cells, the NK cell activity was calculated according to the following formula. The natural mortality was the death rate ofYAC-1 without effector cells, while test group mortality was the death rate cells ofYAC-1 with effector cells.

(test group mortality-natural mortality) (100 - natural mortality)

•2.5.  Analysis ofsamples

The yield of polysaccharides during the extraction was calcu­lated as a percentage of the weight of polysaccharides to pretreated dry powder. The weight of polysaccharides was examined using phenol-sulfuric acid colorimetric method [23].

•2.6.  Statistical analysis

ARTICLE IN PRESS

S.-q. Huang, Z.-x. Ning / International Journal of Biological Macromolecules xxx (2010) xxx-xxx

 

Data analyses were performed using the Statistical Analysis System (SAS, version8.0). Analyses of variance were performed by ANOVA procedure and Duncan's multiple range method of minimum significant differences for the comparison between

ARTICLE IN PRESS

S.-q. Huang, Z.-x. Ning / International Journal of Biological Macromolecules xxx (2010) xxx-xxx

 

 

 

 

 

Table 2

Results of response surface analysis of the variation of polysaccharides (Y) with microwave power (X1), extracted time (X2) and water/solid ratio (X3).

Number

Xi

X2

X3

Y (%)

1

-1

-1

-1

1.99

2

-1

-1

1

2.48

3

-1

1

-1

2.12

4

-1

1

1

2.68

5

1

-1

-1

2.84

6

1

-1

1

2.36

7

1

1

-1

3.08

8

1

1

1

2.69

9

-1.68

0

0

2.48

10

1.68

0

0

2.92

11

0

-1.68

0

2.13

12

0

1.68

0

2.84

13

0

0

-1.68

2.42

14

0

0

1.68

2.83

15

0

0

0

2.97

16

0

0

0

2.92

17

0

0

0

2.89

18

0

0

0

2.81

19

0

0

0

2.91

20

0

0

0

2.86

 

groups. Mean values were considered significantly different when P< 0.05.

•261         3. Result and discussion

  • 262 3.1. Statistical analysis and the model building
  • 263 The experimental data and the process variables for the yield
  • 264 of polysaccharides at different extracted conditions are presented
  • 265 in Table 2. The yield of polysaccharides varied from 1.99% to
  • 266 3.08%. After the response surface regression (RSREG) procedure,
  • 267 the results of F-test (P<0.001) and the determination coefficient
  • 268 (R2 of 94.15%) showed a good fitness with the model and indicat-
  • 269 ing that 94.15% of the dependent variability in the response could
  • 270 be predicted by the model. A low value of coefficient of the variation
  • 271 (CV of 3.98) clearly indicated a high degree of precision and a good
  • 272 reliability of the experimental data. By applying multiple regres-
  • 273 sion analysis on the experimental data, the dependent variable and
  • 274 independent variable are related by the following second-order
  • 275 polynomial equation:
  • 276 Y = 2.89 + 0.18X1 + 0.15X2 + 0.06X3 - 0.08X2 + 0.03X1X2
  • 277 -0.24X1X3 - 0.16X2 + 0.02X2X3 - 0.11X2

278

  • 279 3.2. Influence of process variables on yield
  • 280 During the MAE procedure, microwave energy is used to heat
  • 281 solvents in contact with samples and to partition analytes from
  • 282 sample matrix into the solvent. Generally, the main parameters
  • 283 influencing MAE performance include solvent type, solvent vol-
  • 284 ume, microwave power, exposure time, and temperature [24].
  • 285 Three-dimensional response surfaces and contour plots for the
  • 286 responses are plotted to study the effects of independent vari-
  • 287 ables and their interactions on polysaccharides yield according
  • 288 to the results of regression equations. As shown in Fig. 2, the
  • 289 yield of polysaccharides changed significantly with the linear terms
  • 290 of microwave power and extraction time, the quadratic terms
  • 291 of microwave power, extraction time and water/solid ratio, and
  • 292 the cross terms between microwave power and water/solid ratio.
  • 293 While the linear terms of water/solid ratio had only a slight effect
  • 294 on the yield of polysaccharides. The effect of extraction time on the
  • 295 yield was agreement with the result of Hou and Chen. They found

  

 ARTICLE IN PRESS

S.-q. Huang, Z.-x. Ning / International Journal of Biological Macromolecules xxx (2010) xxx-xxx

Table 3

Effects of polysaccharides UMP on the carbon clearance ability and the DTH of mice.

Groups

Dose (mg/kg/d)

The corrective clearance index a

Degree of ear swelling (mg)

Normal control

-

5.28 ± 0.67ab

14.42 ± 2.84a

Model control

-

4.75 ± 0.34bc

6.75 ± 2.96c

Pachymaran oral

80

5.33 ± 0.64a

10.77 ± 4.04b

UMP low-dose

50

4.92 ± 0.29abc

8.80 ± 3.38bc

UMP medium-dose

100

4.61 ± 0.66c

7.75 ± 4.71bc

UMP high-dose

200

4.94 ± 0.47abc

10.73 ± 4.48b

 

Note: Results are represented as the means ± SD from nine or 10 mice in each group in each group and there are significant differences with the different letters in the same column. Normal control and model control were treated with equal volume of distilled water.

 

 

 

Table 4

Effects of polysaccharides UMP on the generation of serum hemolysin, the thymus and spleen index in mice.

Groups

Dose (mg/kg/d)

OD540nm

Spleen index

Thymus index

Normal control

-

1.5022 ± 0.2682a

48.72 ± 6.69a

15.90 ± 4.82a

Model control

-

0.5262 ± 0.1905c

38.71 ± 7.36bc

11.05 ± 4.23bc

Pachymaran oral

80

1.1332 ± 0.2406b

40.13 ± 4.19bc

14.84 ± 4.02a

UMP low-dose

50

1.0270 ± 0.2930b

36.84 ± 5.35c

9.78 ± 2.96c

UMP medium-dose

100

1.0956 ± 0.2741b

39.62 ± 3.47bc

13.38 ± 3.05ab

UMP high-dose

200

0.9555 ± 0.4071b

43.56 ± 4.60a

14.79 ± 2.03a

 

Note: Results are represented as the means ± SD from 10 or 11 mice in each group in each group and there are significant differences with the different letters in the same column. Normal control and model control were treated with equal volume of distilled water.



 

 

 

tion time as much as possible. However, in some cases, a very high-power microwave decreases the extraction efficiency through degrading the sample. This result has been approved in the researches of Hu et al. and Madej [15,24]. The effect of extrac­tion time on the yield was accordance with the results of Hu et al. They found that the extraction efficiency decreased when irradia­tion time is more than 4min because of its decomposition at long irradiation time [15]. A greater increase in polysaccharides yield from 2.4% to 3.0% was obtained when the microwave power (X1) was increased in the range from 120 to 260 W at a constant value of extraction time (X2) of 600 s, and water/solid ratio (X3) of 20:1. The yields increased from 2.4% to 3.0% when the extraction time varied from 430 to 730 s at a constant value of microwave power (X1) of 240 W, and water/solid ratio (X3) of 20:1.

In the plot of yield against microwave power and water/solid ratio, the higher yield was obtained at higher microwave power and lower water/solid ratio (Fig. 2C and D). As microwave power (X1) was increased in the range from 116 to 284 W, polysaccharides yield increased. At a constant microwave power, the yield changed slightly with the changes in water/solid ratio, the higher yield could be obtained at extraction time of 540 to 780 s (Fig. 2E and F).

•3.3.   Optimization of the UMAE

Based on the experimental data, the optimal extraction condi­tions were microwave powerof284W, extraction time of701 s and water/solid ratio of 11.6:1 using the numerical optimizer, the yield of polysaccharides is 3.27%.

•3.4.   Comparison of UMAE, ultrasonic-assisted and hot water extraction

Compared with UAE, the hot water extraction, the application of UMAE affected positively the yield of polysaccharides obtained. Using a short application of UMAE, the yield of polysaccharides increased to 3.27% at substantially shorter extraction time (12 min), which represents an increase by 27.7% than UAE and by 115.6% than HWE. The extraction time decreased by a factor of 0.15 and 0.04 compared with the UAE and HWE, respectively.

The higher efficiency of the UMAE can be explained by the mechanical of synergistic effects of ultrasonic and microwave on the cell walls resulting in a significantly increased accessibility and extractability of the polysaccharides. This proves that UMAE should be an appropriate and effective extraction technique for polysac- charides from G. lucidum because it gives the maximum extraction values of polysaccharides at the shortest extraction time.

•3.5.   Carbon clearance test and delayed-type hypersensitivity (DTH) determination

Table 3 showed the effects of polysaccharides UMP on the car­bon clearance ability and the DTH of mice. The test of carbon clearance could reflect the phagocytosis function of monocyte. As shown in Table 3, the corrective clearance index of model con­trol group is significantly lower than that of the normal control group (P< 0.05), which showed that the immunocompromised model was successfully built. The corrective clearance index of UCP was no significantly increased comparing with the model control group. This result is disagreement with that of Yang et al. and they found that pollen polysaccharides could increase the ability of lym­phocyte proliferation of tumor-bearing mice in a dose dependent manner [22]. This may be that the monocyte factor has decreased to the normal level after 4 weeks gastric feed.

DTH test could reflect the effects of polysaccharides on the strength of cellular immune function and specific immune responses. Compared with the normal control group, model control group's ear swelling remarkably decreased (P< 0.05) and showed that the immunocompromised model was successfully built. Com­pared with the model control group, the degree of ear swelling of UMP high-dose group significantly increased (P < 0.05), suggesting that it is conducive to the increase of T-cell immune function of immunocompromised mice. There was no significant difference in the degree of ear swelling between of UMP group and the Pachy- maran oral group (P >0.05), and the test results for details are shown in Table 3.

•3.6.  Serum hemolysin and the thymus, spleen index determination

Compared with the normal control group, the amount of hemolysin generation, thymus index and spleen index of the model control group were significantly decreased (P < 0.05), show­ing that the immunocompromised model was successfully built. Serum hemolysin test could reflect the effect of polysaccharides on

Table 5

Effects polysaccharides UMP on NK cells killing activity of mice.

Groups

Dose (mg/kg/d)

NK cells killing activity (%)

Normal control

-

20.78 ± 1.46a

Model control

-

16.45 ± 2.07c

Pachymaran oral

80

19.74 ± 1.45ab

UMP low-dose

50

17.06 ± 1.71c

UMP medium-dose

100

18.31 ± 1.84bc

UMP high-dose

200

20.04 ± 1.85ab

 

Note: Results are represented as the means ± SD from six mice in each group in each group and there are significant differences with the different letters in the same column. Normal control and model control were treated with equal volume ofdistilled water.

 Tumor cell elimination is known to be mediated in part by the cytotoxic activity of Natural killer (NK) cells [22]. As shown in Table 5, compared with the normal control group, NK cells killing activity of the model control group was significantly decreased (P< 0.05), showing that the immunocompromised model was suc­cessfully built. Compared with the model control group, NK cells killing activity of UMP high-dose group was significantly increased (P<0.05), suggesting that high-dose group have positive effect on the increase of NK cells' functions and it indicates UMP could improve the cell immune function in mice. This result was agree­ment with that of Zhang et al. and they found that there was statistical difference in NK cells killing activity only between the high-dose Chinese medicinal fungus water extract and control [27].

These immunological assays results demonstrated that the polysaccharides from G. lucidum could significantly enhance the immune response of immunocompromised mice. The results stated that the UMP could improve the weight of immune organ of immunocompromised mice, restore delayed-type hypersensitivity reaction to DFNB, improve hemolysis antibody level and natural killer cell activity at high-dose, which was related with the purity of crude polysaccharides and higher dosage could obtain the obvious immune activity at lower purity. However, UMP had no noticeable effects on phagocytosis of monocyte at the tested dosage range.

4. Conclusions

We have studied the ultrasonic/microwave-assisted extraction of polysaccharides from G. lucidum and its immune enhancement activity. The main findings can be drawn from the information pre­sented in this paper:

1. The optimal experimental parameters for the ultrasonic/microwave-assisted extraction of polysaccharides from G. lucidum are: microwave power of 284 W, ultrasonic power of 50 W with ultrasonic frequency of 40 kHz, extraction time of 701 s and water/solid ratio of 11.6:1.

  • 2. The yield of polysaccharides extracted by the ultrasonic/microwave-assisted procedures exceeded those of the classical hot water and single ultrasonic extraction procedures. The advantages support the importance and great potential of ultrasonic/microwave technology on the polysaccharides extraction from different tissues of plant materials.
  • 3. These immunological assays results stated that the UMP had no noticeable effects on phagocytosis of monocyte at the tested dosage range. However, it could improve the weight of immune organ of immunocompromised mice, restore delayed- type hypersensitivity reaction to DFNB, improve hemolysis antibody level and natural killer cell activity at high-dose.

Acknowledgements

The authors send great appreciation to all in the Analysis and

Determination Center of South China University ofTechnology who

supported the research work. Thanks are given to Jianhua Gao and

Jian-xu Chen of South China University ofTechnology for beneficial

discussion and diagram of IR spectra.

 

INVITED REVIEW

Fungal(Mantar  kökenli)Metabolitlerin Antiinflammatorik ve bağışıklık düzenleyici özellikleri

Antiinflammatory and Immunomodulating Properties of Fungal Metabolites

Cristina Lull,1 Harry J. Wichers,1 and Huub F. J. Savelkoul2

1 Agrotechnology and Food Innovations, Wageningen University and Research Center,

Bornsesteeg 59, 6708 PD Wageningen, The Netherlands 2Cell Biology and Immunology Group, Wageningen University and Research Center, Marijkeweg 40, 6709 PG Wageningen, The Netherlands

Received 22 December 2004; accepted 25 January 2005

We discuss current information on the ability of extracts and isolated metabolites from mushrooms to modulate immune responses. This can result in a more enhanced innate and acquired disease resistance. The major immunomodulating effects of these active sub­stances derived from mushrooms include mitogenicity and activation of immune effector cells, such as lymphocytes, macrophages, and natural killer cells, resulting in the production of cytokines, including interleukins (ILs), tumor necrosis factor alpha (TNF)-a, and interferon gamma (INF)-y. In particular, the ability of selective mushroom extracts to modulate the differentiation capacity of CD4+ T cells to mature into TH1 and/or TH2 subsets will be discussed. As a consequence these extracts will have profound effects in particular diseases, like chronic autoimmune TH1-mediated or allergic TH2-mediated diseases. Immunosuppressive effects by mushroom components have also been observed. The therapeutic effects of mushrooms, such as anticancer activity, suppression of autoimmune diseases, and allergy have been associated with their immunomodulating effects. However, further studies are needed to determine the molecular mechanisms of the immunomodulating effects of mushrooms metabolites both individually and in complex mixtures, for example, extracts.

INTRODUCTION

The number of different mushroom species on earth is estimated at 140 000, of which may be only 10% are known. Meanwhile, of those approximately 14 000 species that we know today, about 50% are considered to possess varying degrees of edibility, more than 2000 are safe, and about 700 species are known to possess significant phar­macological properties [1, 2, 3, 4]. Mushrooms have long been attracting a great deal of interest in many areas of foods and biopharmaceuticals. They are well known for their nutritional and medicinal values [1, 4, 5, 6, 7, 8, 9]. In accordance to Breene [10] the gross composition of mushrooms is water (90%), and from the dry matter: pro­tein (10%-40%), fat (2%-8%), carbohydrates (3%-28%), fiber (3%-32%), and ash (8%-10%) (the ash percentage

Correspondence and reprint requests to Huub F. J. Savelkoul, Cell Biology and Immunology Group, Wageningen University and Research Center, Marijkeweg 40, 6709 PG Wageningen, The Netherlands; huub.savelkoul@wur.nl

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

is the fraction of dry matter that remains after inciner­ation of the organic material in a sample, and is mainly composed of salts, metals, and so forth). Many species of mushrooms are cultivated worldwide. Global produc­tion increased to about 6.2 million tons in 1997, with a more than 12% increase annually from 1981 to 1997 [11]. Mushroom extracts have been increasingly sold as dietary supplements. The market value of mushroom dietary sup­plement products worldwide is about US$5-6 billion per year [12].

Medicinal mushrooms have an established history of use in traditional oriental therapies. Historically, hot- water-soluble fractions (decoctions and essences) from medicinal mushrooms were used as medicine in the Far East, where knowledge and practice of mushroom use pri­marily originated [4, 13, 14]. Mushrooms such as Gano- derma lucidum (Reishi), Lentinus edodes (Shiitake), Inono- tus obliquus (Chaga), and many others have been collected and used for hundreds of years in Korea, China, Japan, and eastern Russia [4].

© 2005 Hindawi Publishing Corporation

 

Mushroom metabolites are increasingly being utilized to treat a wide variety of diseases, particularly as they can be added to the diet and used orally, without the need to go through phase-I/II/III trials as an ordinary medicine, and they are considered as a safe and use­ful approach for disease treatment. A lot of scientific
investigations have been performed to discover possible functional properties, which could be efficient in possi­ble treatments of diseases like allergic asthma [15, 16, 17], food allergy [18, 19], atopic dermatitis [20], inflamma­tion [21, 22], autoimmune joint inflammation such as rheumatoid arthritis [23], atherosclerosis [24, 25], hy­perglycemia [26], thrombosis [27], human immunodefi­ciency virus (HIV) infection [28, 29], listeriosis [30], tu­berculosis [31], septic shock [32], and cancer [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55].

In the last years many researchers have studied the possibility that extracts and isolated metabolites from mushrooms stimulate or suppress specific components of the immune system. Immunomodulators can be effective agents for treating and preventing diseases and illnesses that stem from certain immunodeficiencies and other de­pressed states of immunity [56]. Synonymous terms for immunomodulators include biological response modi­fiers, immunoaugmentors, or immunorestoratives [57]. Those metabolites which appear to stimulate the human immune response are being sought for the treatment of cancer, immunodeficiency diseases, or for generalized im- munosuppression following drug treatment, for combi­nation therapy with antibiotics, and as adjuvants for vac­cines [58]. Those metabolites that suppress immune re­actions are potentially useful to mitigate autoimmune or certain gastrointestinal tract diseases (eg, Crohn's) [59].

At least 651 species and 7 infraspecific taxa repre­senting 182 genera of hetero- and homobasidiomycetes mushrooms contain antitumor or immunostimulating metabolites [4]. Bioactive metabolites can be isolated from fruiting bodies (Figure 1), pure culture mycelia, and culture filtrate (culture broth). Nowadays many at­tempts are being made to obtain bioactive metabolites from mycelia through submerged fermentation culture. The cultivation of mushrooms to produce fruiting bod­ies is a long-term process requiring from one to several months for the first fruiting bodies to appear. The growth of mushroom cell cultures in submerged conditions in a liquid culture medium accelerates the process, resulting in biomass yield within a few days and allows to obtain stan­dardized nutriceutical substances.

Several major substances with immunomodulatory and/or antitumor activity have been isolated from mush­rooms. These include mainly polysaccharides (in particu­lar ^-D-glucans (Figure 2)), polysaccharopeptides (PSP), polysaccharide proteins, and proteins. Furthermore, other bioactive substances, including triterpenes, lipids, and phenols, have been identified and characterized in mush­rooms with proven medicinal properties. The major im- munomodulating effects of these active substances de­rived from mushrooms include mitogenicity and activa­tion of immune cells, such as hematopoietic stem cells, lymphocytes, macrophages, dendritic cells (DCs) and nat­ural killer (NK) cells, resulting in the production of cy- tokines. The therapeutic effects of mushrooms, such as anticancer activity, suppression of autoimmune diseases, and allergy have been associated in many cases with their immunomodulating effects.

 Whilst it is known that mushroom extracts have im- munomodulatory and/or antitumor activity, the standard approach has been to isolate, characterize, and administer the pure active constituents. However, different compo­nents in a mushroom extract may have synergistic activi­ties [49, 60]. There are several reports of mushrooms con­taining more than one polysaccharide with antitumor ac­tivity. The responses to different polysaccharides are likely to be mediated by different cell surface receptors, which may be present only on specific subsets of cells and may 

trigger distinct downstream responses. A combination of such responses involving different cell subsets could con­ceivably provide greater tumor inhibition than could be induced by a single polysaccharide [49].

EFFECTS OF MUSHROOM METABOLITES ON HEMATOPOIETIC STEM CELLS

Various metabolites, especially carbohydrates isolated from mushrooms, were reported to affect bone mar­row cells (BMCs), and to induce hematopoiesis (Ta­ble 1). Recently, Lin et al [61] reported that Maitake MD-fraction (obtained by further purification of D- fraction), an extract isolated from the fruit body of Grifola frondosa whose active component is an isolated //-glucan, a protein-bound polysaccharide compound, caused direct enhancement of the colony-forming units- granulocytes/macrophages (CFU-GM) response of BMCs progenitors and enhanced recovery of the CFU-GM re­sponse after doxorubicin (DOX) induced hematopoietic suppression. These studies suggest that MD-fraction has the potential to reduce hematopoietic suppression in­duced by chemotherapy.

PG101, a water-soluble extract that consists of protein-bound polysaccharides, isolated from cultured mycelia of Lentinus lepideus [62], is a potent immune modulator that recovers the radiation-damaged bone marrow system very efficiently. In PG101-treated mice, the number of CFU-GM and erythroid burst-forming units (BFU-E) were increased to almost the levels seen in nonirradiated control as early as 8 days after irradi­ation. Radiation is known to result in serious dysregu- lation of cytokine expression. PG101 increased the lev­els of IL-1& IL-6, and granulocyte macrophage-colony- stimulating factor (GM-CSF) over the 24-day period. PG101 significantly reduced the level of TNF-a. TNF-a, which is increased as a consequence of tissue injury and anemia due to radiation, is thought to be a key mediator for the pathogenesis of radiation damage. Thus, PG101 showed great potential as a supplement or a major ther­apeutics in immunocompromised or immunosuppressed individuals whose bone marrow system is damaged [63].

SCG, a ^-(1^3)-D-glucan with ^-(1^6) branches isolated from fruit bodies of Sparassis crispa, enhanced the hematopoietic response in cyclophosphamide- (CY- ) induced leukopenic mice by intraperitoneal routes over a wide range of concentrations. Monocytes and granulo- cytes in the peritoneal cavity, liver, spleen, and bone mar­row recovered faster than in the control group. The ratio of NK cells and y5T cells in the liver, spleen, and peri­toneal cavity was also increased. These results suggest the usefulness of S crispa in cancer immunotherapy [64].

EFFECTS OF MUSHROOM METABOLITES ON THE INNATE IMMUNE SYSTEM

Macrophages

The recognition of microbes by macrophages and neurophilic granulocytes leads to phagocytosis of the mi­crobes and activation of the phagocytes to kill the ingested microbes. Recognition is mediated by toll-like receptors (TLR) that are specific for different components of mi­crobes. TLR-2 binds lipogycans, TLR-4 binds bacterial
lipopolysaccharide (LPS), TLR-5 binds flagellin, and TLR- 9 binds unmethylated CpG nucleotides present in bac­teria. As a consequence of recognition and phagocyto­sis several enzymes are activated, including oxidases and inducible nitric oxide synthase (iNOS), resulting in the production of bacteriocidal reactive oxygen intermediates (ROI) and nitric oxide (NO).

The effects of mushroom extracts and metabolites on macrophages have been extensively studied in vitro and in vivo. Some mushroom metabolites activate macrophages to produce various mediators, even in normal mice. Ac­tivities are summarized in Table 2.

Water extracts of the mycelial culture and fruiting bodies of Agaricus blazei Murill induced TNF-a secretion by macrophages derived from rat bone marrow. Fractions B-4 and B-5 obtained from ethanol precipitation of fruit­ing bodies markedly induced TNF-a secretion. Similar ef­fects were observed in IL-8 secretion by macrophages. Re­garding NO, fraction B-5 induced a significant increase in NO secretion and fractions B-4 and B-6 slightly in­duced NO secretion. Northern blot analysis showed that the increases in cytokine and NO secretion were due to an increase in cytokine mRNAs or NO synthase mRNA [65]. Thus A blazei Murill contains certain components which activate macrophages contributing to the immune response in vitro.

Wang et al [66] reported that after treatment of macrophage cultures with a polysaccharide from fresh fruiting bodies of G lucidum, the levels of IL-1^, TNF-a, and IL-6 were 5.1-, 9.8-, and 29-fold higher than in cul­tures of untreated cells. In addition, the release of INF- y from T lymphocytes was also greatly enhanced in the presence of this polysaccharide. This proinflammatory cy­tokine response is suggested to facilitate the antitumor ac­tivity of this extract.

Grifolan (GRN), an antitumor ^-glucan isolated from G frondosa induced the release of IL-1, IL-6 and TNF-a from macrophages [67, 68]. Ishibashi et al [69] reported that an insoluble as well as a high-molecular-mass solu­ble form of GRN are required for TNF-a production by macrophages.

The effect of Maitake D-fraction was studied by Sanzen et al [70] on the iNOS-mediated NO production in RAW264.7 macrophages with special reference to anti­tumor activity of MD-fraction against human hepatoma- derived huH-1 cells and the data suggested that MD- fraction is a novel inducer for iNOS which contributes at least in part to antitumor activity of MD-fraction.

Kodama et al [30] examined the effects of Maitake D-fraction on the treatment of Listeria-infected mice in combination with vancomycine (VCM). In mice admin­istered with both D-fraction and VCM, macrophages pro­duced 2.7 times as much IL-1^ as that of nontreated con­trol mice. The bactericidal activity of splenic T cells was also enhanced by 2.6 times of that of nontreated control mice. These results suggest a clinical benefit of D-fraction in the case of antibacterial treatment for patients with high risks.

Monocytes/macrophages seem to be the major tar­get cell type responsive to PG101. Jin et al [62] proposed that PG101 interacts with macrophages or related cells and results in the activation of the transcription factor nuclear factor kappa B (NF-kB), which sets off a series of reactions producing a variety of proinflammatory and antiinflammatory cytokines (TNF-a, IL-1^, IL-10, IL-12, GM-CSF, IL-18) in a sequential manner. Inflammatory- cytokine-induced phosphorylation of a degradative mo­tif in IkB triggers IkB proteolysis, liberating NF-kB from the inactive heterodimer and NF-kB transcription which in turn prevents cytokine-induced death of inflamma­tory cells. Despite its significant biological effect on vari­ous cytokines, PG101 remained nontoxic in both rats and human peripheral blood mononuclear cells (hPBMCs) even at a biological concentration approximately 20 times greater. PG101 demonstrates great potential as a thera­peutic immune modulator.

A galactomannan isolated from a polar extract of Morchella esculenta carpophores enhanced macrophage activation. At 3.0 ^g/mL the galactomannan polysaccha­ride (about 2.4% protein) increased NF-KB-directed lu- ciferase expression in THP-1 human monocytic cells to levels of 50% of those achieved by maximal activating concentration (10.0^g/mL) ofLPS [71].

By administering PL, an acidic polysaccharide iso­lated from Phellinus linteus, the production of NO and tumoricidal activity were increased in murine peritoneal macrophages in vivo and in vitro. PL has been claimed to cause the inhibition of tumor growth and metastasis of murine B16F10 melanoma cells [72]. Such properties of PL may be related to its ability to induce the production of the tumoricidal effector molecule NO through protein tyrosine kinase (PTK) and protein kinase C (PKC) [73]. Considering the main role that proinflammatory cytokine production plays in the pathogenesis of septic shock, Kim et al [32] examined how the in vivo administration of PL can modulate circulating cytokine responses in LPS- treated mice. Administration of PL in vivo decreased IL- 2, IFN-y, and TNF-a production in splenocytes and en­hanced spontaneous cell apoptosis in macrophages and lymphocytes stimulated with LPS in vitro. Thus, part of the antiinflammatory effects of PL treatment in vivo may result from the enhanced apoptosis of a portion of the ac­tivated macrophages and lymphocytes. The ability of PL to significantly reduce TNF-a production indicates the potential of the polysaccharides in possible therapeutic strategies that are based on down regulation of TNF-a [32].

The methanol extract of fruit bodies of Cordyceps pruinosa inhibited IL-1^, TNF-a, NO, and prostaglandin E2 (PGE2) in vitro and in vivo. The extract inhib­ited these inflammatory mediators in LPS-stimulated murine macrophage cell line RAW264.7 and primary macrophages, by suppressing gene expression of IL-1^, TNF-a, iNOS, and cyclooxygenase-2 (COX-2) through the inhibition of NF-kB activation. Administration of the extract significantly decreased the plasma level of these inflammatory mediators in LPS-injected mice. These re­sults suggest that the C pruinosa methanol extract sup­presses inflammation through suppression of NF-kB- dependent inflammatory gene expression, suggesting that the C pruinosa extract may be beneficial for treatment of endotoxin shock or sepsis [74]. Also, the methanol extract of fruit bodies of Pleurotus florida showed anti- inflammatory and antiplatelet-aggregating activities but the exact mechanism for these activities is unknown [21].

A fucogalactan, isolated from Sarcodon aspratus, elicited the release of TNF-a and NO in macrophages of mice in vitro. TNF-a production induced with 50 ^g/mL of fucogalactan was significantly higher than that in­duced by lentinan (500 ^g/mL) by approximately4.3-fold. Mizuno et al [75] suggested that the immunomodulat­ing activity of this fucogalactan on TNF-a and NO pro­ductions might contribute to antituor activity in tumor- bearing hosts as well as various immunomodulating ef­fects.

In mice treated with an immunosuppressive carcino­gen, administration of a mushroom-enriched diet con­taining L edodes, G frondosa, and Pleurotus ostreatus re­stored the normal level of the chemotactic activity of macrophages and the capability of lymphocytes to pro­liferate in response to mitogen [76].

Proteins and peptides from mushrooms are also known to activate macrophages. A ubiquitin-like pep­tide isolated from fruiting bodies of the mushroom Agro- cybe cylindracea enhanced NO production in murine peri­toneal macrophages with a potency comparable to that of LPS [77]. Two lectins isolated from the mushroom Tri- choloma mongolicum (TML-1 and TML-2) stimulated the production of nitrite ions and TNF-a by macrophages in normal and tumor-bearing mice [78].

Natural killer cells

Natural killer cells are a class of lymphocytes that rapidly respond to intracellular infections with viruses or bacteria, by killling the infected cells and by producing the macrophage-activating cytokine, IFN-y.

Some mushroom metabolites exhibit stimulating ef­fects on NK cells (Table 3). Innate immunity is in the critical arms of immune surveillance against tumor de­velopment. Moreover, in the innate immune system, NK cells, which do not express T-cell receptors that recognize specific peptides presented on the major histocompatibil- ity complex (MHC), rather than T cells, seem well suited for this role. NK cells can recognize the surface changes that occur on a variety of tumor cells and virally infected cells [79]. NK cells have two relevant functions, related to the natural immune response against pathogens [80]. One is cytotoxicity, mediated by the recognition and lysis of target cells such as virus- and bacteria-infected cells. The second NK cells function is to produce cytokines such as IFN-y, TNF-a, and GM-CSF, that can modulate natural and specific immune responses. Additionally, in­fected or activated DCs and macrophages produce cy­tokines and chemokines such as IFN-a/3, IL-12, IL-15, and IL-18 that stimulate NK cells to rapidly produce other cytokines (including IFN-y, TNF-a, and GM-CSF) and chemokines (such as ATAC/lymphotactin, mig, and MIP- 1a) [81].

Kodama et al [41, 82] monitored levels of NK cell cy­totoxic activity in cancer patients receiving D-fraction. El­evated levels of cytotoxic activity were maintained for one year. To elucidate the mechanisms underlying long-term activation of NK cells during treatment with D-fraction, the authors examined tumor volume and levels of IFN-y and TNF-a in MM46-bearing C3H/HeN mice to which D-fraction was administered for 19 days. D-fraction markedly suppressed tumor growth, corresponding with increases in TNF-a and IFN-y released from spleen cells and a significant increase in TNF-a expressed in NK cells. Furthermore, D-fraction increased macrophage-derived IL-12, which serves to activate NK cells. Thus, NK cells are not only responsible for the early effects of D-fraction on tumor growth, but also for the long-term tumor- suppressive effects of D-fraction through increased IL-12 released from macrophages. D-fraction was capable of en­hancing and maintaining peripheral blood NK cell activ­ity in patients with lung and breast cancer [41]. In ad­dition, Maitake D-fraction, stimulated the natural immu­nity related to the activation of NK cells indirectly through IL-12 produced by macrophages and DCs in normal mice [83]. IFN-y production by splenic NK cells increased sig­nificantly 3 days after D-fraction administration. In a re­cent study, Kodama et al [84] reported the activation of macrophages and DCs in normal mice as well. Therefore, administration of D-fraction to healthy individuals may serve to prevent infection by microorganisms.

Treatment with hot-water extracts of A blazei fruit­ing bodies increased NK activity of spleen cells in naïve BALB/c mice [85]. In meth A-bearing BALB/c mice, the same extracts enhanced the induction of antigen-specific cytotoxic T lymphocytes (TC) and IFN-y production. Up regulation of NK and TC activity is triggered by IL-12- dependent activation [86]. It is not yet clear whether oral administration of Agaricus extracts enhances IL-12 pro­duction in vivo [85].

Ehrlich-carcinoma-bearing mice treated with the n- hexane, dichloromethane, or methanol extracts from A blazei fruiting bodies were able to maintain the NK ac­tivity of spleen cells during the first 10 days after tumor implantation. The NK activity of these groups was sim­ilar to that of normal controls and higher than that of tumor-bearing mice treated with water. The results of NK activity on the 30th day after the injection of tumor cells suggest that none of the three extracts was able to main­tain the lytic activity against Yac-1 target cells. It is pos­sible that after 30 days the production of soluble factors like prostaglandins, TGF-3, or IL-10 by Ehrlich carcinoma cells was enough to prevent the increase of NK activity by the n-hexane extract [87].

Ahn et al [88] investigated the beneficial effects of the consumption of an extract of A blazei Murill Kyowa (ABMK) on immunological status and qualities of life in cancer patients undergoing chemotherapy. They ob­served that NK cell activity was significantly higher in the ABMK-treated group and suggested that ABMK treat­ment might be beneficial for gyneacological cancer pa­tients undergoing chemotherapy.

The medicinal fungus water extract (FWE) consists of equal amounts of Coriolus versicolor, Cordyceps sinen­sis, L edodes, A blazei, and G lucidum. Zhang et al [89] reported that FWE enhanced the phagocytosis of peri­toneal macrophages, promoted NK activity in mice, and suppressed the growth of B-16 melanoma. FWE had sig­nificantly promoted mouse NK activity at the dose of 400 mg/kg, which suggests that FWE may possess the abil­ity to activate NK to directly kill tumor cells, induce NK to secrete cytotoxic agents to elicit the apoptosis of tumor cells, or remove tumor cells by other pathways.

Dendritic cells

DCs are antigen-presenting cells (APC) with a unique ability to induce primary immune response of both helper (Th) and TC [90]. Beside activating naive T cells, DCs can directly activate naive and memory B cells. DCs at differ­ent stages of differentiation can regulate effectors of innate immunity such as NK cells and NK T cells. The induction of tumor immunity can be initiated by the effectors of in­nate immunity and further developed by cells of adaptive immunity, with DCs playing a central regulatory role.

Cao and Lin [91] studied the regulatory effects of Gl- PS, G lucidum polysaccharides (GLPS), on maturation and function of cultured murine bone-marrow-derived DCs in vitro. Gl-PS could promote not only the matura­tion of cultured murine bone-marrow-derived DCs, but also the immune response initiation induced by DCs.

PL induced maturation of bone-marrow-derived DCs and readies them for T-cell-mediated immune responses. PL significantly increased membrane molecules, includ­ing MHC class I, II, CD80, and CD86, and IL-12p70 in DCs. Also, PL markedly reduced the endocytic activ­ity of DCs and augmented their capacity to promote the proliferation of naive allogeneic T cells [92]. PL enhanced
the phenotypic and functional maturation of DCs via TLR-2- and/or TLR-4-mediated NF-kB, ERK, and p38 MAPK signal pathways. It is the first article reporting that a polysaccharide from mushrooms can activate a TLR signaling [93]. Kim et al [94] reported that the admin­istration of PL induced antitumor and immunomodu- lating activities via maturation of CD11c+CD8+ DCs in tumor-bearing mice. The inhibitory effect of PL on the growth of MCA-102 tumor cells was associated with its immunoregulatory properties, including the induction of IL-12 and IFN-y production leading to a TH1 dominant state. Therefore, PL would be useful in preventing tumor growth, and it also has the advantage of having no side effects.

The existence of a strongly immunosuppressive state in cancer-bearing individuals inhibits DCs maturation. Kanazawa et al [95] reported that a protein-bound polysaccharide K (PSK) isolated from the cultured mycelium of C versicolor promoted both the phenotypic and functional maturation of DCs derived from human CD14+ mononuclear cells. PSK has also been reported to resolve the immunosuppressive state of a cancer-bearing host and might be associated with DCs maturation di­rectly [95]. Activities of mushroom metabolites on DCs are summarized in Table 4.

Complement

Activation of complement by either the classical or al­ternative pathway results in the generation of a wide spec­trum of biological activities with the potential to modify immune responses [96, 97]. Particularly, the activation of complement via the alternative pathway is important in natural immunity to bacterial infections [98, 99].

Although there are a few reports concerning the re­lationship between complement-activating and tumor- regressing activity of glucan including lentinan, the pos­itive correlation between the two activities was found by Okuda et al [100]. They observed a correlation between the ability to activate complement via the alternative path­way in vitro and inhibition of tumor growth in vivo. However, the opposite result, no correlation, was found by Hamuro et al [101]. Thus there is no consistent view on the correlation between the two antagonozing activi­ties.

ABP-F and ABP-M, fine particles of A blazei Murill fruiting body and mycelium, respectively, prepared by mechanical disruption, activated the human complement system via the alternative pathway in human serum (Table 5). When particles from fruiting bodies of A blazei Murill (ABP-F) were reacted with human serum, the formation of complement-opsonized ABP, iC3b-ABP-F complexes, and binding of the complexes to human peripheral blood monocytes, were demonstrated in vitro by immunofluo- rescence. Further, the resident human peripheral nucle­ated cells incubated in the presence of iC3b-ABP-F com­plexes inhibited the proliferation of the human tumor cell line TPC-1 in vitro [102].

An alkali extract from cultured mycelium of G lu- cidum activated both classical and alternative pathways of complement [103]. Min et al [104] reported that triter- penoids such as ganoderiol F, ganodermanondiol, and ganodermanontriol from G lucidum had a potent anti- complement activity against the classical pathway with IC50 values of 4.8-4.17 ^M. A clinical study in elderly pa­tients with insomnia and palpitation has shown that tak­ing G lucidum essence for 4-6 weeks increased their serum C3 levels [105].

Also, LELFD, a ^-(1^3)-glucan, obtained from liq­uid-cultured mycelium of G frondosa, could activate the alternative complement pathway [106].

Anticomplementary activity of 61 strains of higher fungi from Korea was screened for immunostimulation [107]. Extracts from 11 of 61 strains, including 5 of G lucidum, 3 of L edodes, 2 of Cordyceps sp, and 1 of Agari- cus campestris, showed higher anticomplementary activity than Krestin from C versicolor. The most potent anticom- plementary activity was found with an extract from L edo­des IY105, that reduced complement capacity by 31.7%.

EFFECTS OF MUSHROOM METABOLITES ON ADAPTIVE IMMUNE SYSTEM

T lymphocytes

T lymphocytes include T-helper (TH) cells and cyto­toxic T (Tc) cells. TH cells interact with B cells and help them to divide, differentiate, and make antibody or inter­act with mononuclear phagocytes and help them destroy intracellular pathogens. TH cells generate their effects by releasing soluble cytokines and/or by direct cell-cell in­teractions. The TC cells destroy target host cells that have been infected by pathogens.

Th cells

CD4+ cells secrete a number of cytokines that are im­portant in the activation of B and other T cells, as well as cells of the innate immune system. Based on the types of cytokines these CD4+ cells produce, they are classified into a number of TH types (0, 1, 2, or 3). TH1 cells pro­duce IL-2, IFN-y, and TNF-3 (LT), and introduce cel­lular immunity to mainly intracellular infections organ­isms. Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13, and activate humoral immunity, mainly directed against extracellular infections. Precursor or TH0 cells produce IL-4 and IFN-y concomitantly. Less is known about the physiological role of TH0 type cells. Thymus-derived reg­ulatory T-cell populations, including naturally occur­ring CD4+CD25+ T cells and inducible IL-10 or TGF-3- producing TR/TH3 cells, develop in the periphery from Th cells depending on the tolerance-inducing micro­environment in which these T cells reside. By blocking ac­tivation of other lymphocytes and APC either directly (by CTLA4-CD28 interaction) or indirectly (by cytokines like IL-10 and TGF-3), these cells ensure self-tolerance mech­anisms. In diseased states, however, the presence and/or

G lucidum

Promoted both the phenotypic and functional maturation of DC derived from human CD14+ mononuclear cells 

 Anticomplement activity

Activation of the alternative complement pathway

 activity of these cells is often reduced leading to enhanced immunopathology, characteristic of chronic inflamma­tory diseases, like auto-immune and allergic diseases.

The downstream immune response is chosen depend­ing on which subtype of T cell is activated, which means that the proportion of the activated sub-types influences phylaxis immunity and antitumor immunity. This control system is also affected by the production of IL-1^, IL-12, and IL-18 by APC [108, 109]. The development of TH1 or Th2 types from naive cells to effector cells is regulated by the presence of specific cytokines in the microenviron­ment at the time of T cell priming. For the TH1 type, IL- 12 is a necessary cytokine of differentiation [110], whereas for the Th2 type, IL-4 and IL-10 are critical [111]. Recent study shows that many immune disorders are attributable to the collapse of the system controlling the proportion of Th1 to Th2 cells [112]. Many diseases such as leprosy, allergy, multiple sclerosis, and responses to immunotoxic agents have pathology associated with aberrant TH1 and TH2 polarization. TH1 cells may cause immunopathol- ogy and organ-specific autoimmune disease if dysregu- lated [113, 114, 115, 116]. Because cytokines produced by TH2 cells, such as IL-4 and IL-5, can activate mast cells and eosinophils and in addition can result in elevated levels of IgE, they have been strongly implicated in atopy and aller­gic inflammation [117]. Restoration of the proper balance between TH1 and TH2 cells is generally considered essen­tial in the treatment of tumors, which are generated when cellular immunity is affected by immunosuppressing fac­tors.

Some mushroom polysaccharides might induce a type 1 immune response, whereas others favor a type 2 polar­ization [49,118]. Borchers et al [49] reported that the lim­ited data available to date do not allow one to determine whether mushroom polysaccharides do so independently of the animal strain or species and disease state investi­gated or whether the nature of their immunomodulatory effects depends on the model to a greater extent than has been appreciated to date.

Lentinan has been described as a T-cell-oriented ad­juvant [119]. The skewing of TH1/TH1 balance to TH1 by lentinan (Table 6) is directed through the distinc­tive production of IL-12 versus IL-6, IL-10, and PGE2 by peritoneal macrophages, depending on intracellular glutathione redox status [120]. Based on the intracellu- lar content of glutathione, two classes of macrophages have been proposed with diverse functional conse­quences: reductive macrophages with high, and oxidative macrophages with low glutathione levels.

Sclerotinia sclerotiorum glucan (SSG) from Sclero- tinia sclerotiorum IFO 9395 induced the development of Th 1 cells via the IL-12 pathway [118].

Inoue et al [121] investigated the antitumor func­tions of D-fraction in relation to its control of the bal­ance between T lymphocyte subsets TH1 and TH2. D- fraction decreased the activation of B cells and potenti­ated the activation of TH cells, resulting in enhanced cel­lular immunity. It also induced the production of IFN- y, IL-12p70, and IL-18 by whole spleen cells and lymph node cells, but suppressed that of IL-4. These results sug­gest that D-fraction establishes TH1 dominance which in­duces cellular immunity in the population that was TH2 dominated due to the presence of this particular car­cinoma [121]. In a later study, Harada et al [122] re­ported that D-fraction induces the differentiation into Th1 cells of CD4+ T cells in tumor-bearing BALB/c mice  

F velutipes S sclerotiorum IFO 9395 L edodes

Gfrondosa V volvacea

Th1 response Th1 response Th1 response

Enhances TH1 dominant response through enhancement of IL-12p70 and IFN-y produced by activated DCs t TH1-specific cytokines (IL-2, IFN-y , LT), TH2-specific cytokine (IL-4), TNF-a, and IL-2R

in which the TH2 response was dominant through en­hancement of IL-12p70 production by DCs, when the ra­tio of CD8a+ DCs to CD8a~ DCs increased. In addition, examination of the tumor rejection effect of D-fraction- stimulated DCs loaded with tumor antigen revealed that tumor growth is inhibited completely by activating CD4+ T cells and CD8+ T cells. Furthermore, the level of TNF-a, which is produced by activated macrophages and NK cells and is cytotoxic for tumor cells, increased by D-fraction- DCs injection, indicating that D-fraction enhanced the protective immunity by DCs loaded with tumor anti­gen through activating macrophages and NK cells. Al­though the action of D-fraction on DCs and its intra- cellular signal transduction pathway remain unclear, D- fraction may be a useful stimulator of DCs, which in­duce the differentiation of CD4+ T cells to TH1 cells [122].

Vvo, a fungal immunomodulatory protein (FIP) pu­rified from the edible mushroom, Volvariella volvacea, in­duced most TH1-specific cytokines (IL-2, IFN-y, and LT) and one TH2-specific cytokine (IL-4) within 4 hours in mouse spleen cells. This result indicates that Vvo princi­pally acts on TH1 cells and to a lesser extent on TH2 cells in the early event of activation. It is known that IL-4 acts on B cells to induce activation and differentiation, lead­ing in particular to the production of IgE. The lower ef­fect of Vvo compared with other FIPs on the prevention of systemic anaphylaxis may be attributed to the elevated expression of IL-4 [123, 124].

Fve, a FIP isolated from the fruiting body of Flam- mulina velutipes, selectively stimulates a TH1 response in hPBMCs [18]. Recently Hsieh et al [18] have char­acterized the immunomodulatory effects of Fve in more detail and investigated the prophylactic use of Fve via the oral route in a murine model of food allergy. They have demonstrated that oral administration of Fve during allergen sensitization could induce a TH1-predominant allergen-specific immune response in mice and protect the mice from systemic anaphylaxis-like symptoms af­ter subsequent oral challenge with the same allergen. It is worth noting that Fve could be administered orally and retain its activity, while most protein drugs cannot. This characteristic greatly promotes the potential of im- munoprophylactic use of Fve [18]. Liu et al [16, 17] have demonstrated the efficacy of local nasal immunother­apy (LNIT) for group 2 allergen of house dust mite Dermatophagoides-pteronyssinus- (Dp2-) induced airway inflammation in mice, using Dp2 peptide and Fve or LZ- 8, a FIP isolated from G lucidum.

B cells

Three polysaccharides isolated from G lucidum, two heteroglycans (PL-1 and PL-4) and one glucan (PL-3) en­hanced the proliferation of T and B lymphocytes in vitro to varying contents and PL-1 exhibited an immune stim­ulating activity in mice [125].

PGL, a complex ^-D-glucan, has a strong effect on suppressing the antibody production [126].

GLIS, a proteoglycan isolated from the fruiting body of G lucidum, is a B-cell stimulating factor. This com­pound stimulated B lymphocyte activation, proliferation, differentiation and production of immunoglobulins. The activation of B cells by GLIS may be associated with the expression of PKC a and PKC y in B cells [127]. GLIS stimulated the proliferation of mouse spleen lympho­cytes, resulting in a threefold to fourfold increase in the percentage of B cells. GLIS also activated mouse spleen lymphocytes, and most of the activated cells were B cells

  • [127] .

PL selectively activates murine B cells but not T cells

  • [128] . Since PL cannot penetrate cells due to its large molecular mass (approximately 15 kD), this selectivity may be caused by the surface binding of this molecule to receptors specifically expressed on B-cells but not on T cells. The B-cell receptor, BCR, consists of surface im­munoglobulin and CD79a-CD79b. Upon BCR ligation, the BCR-associated kinase Lyn phosphorylates CD79a- CD79b. In addition, coreceptors such as CD19 and CD38 positively regulate BCR signaling. Complement receptor CD11b-CR3, or Mac-1, is expressed on the surface of macrophages and NK cells and has been identified as the receptor of ^-glucans [129]. Although PL and ^-glucans show different specificities on B and T cells, they may use the same receptor on B cells. A further complete investi­gation of the membrane receptors of PL should shed light on its selectivity for B cells.

  Evidence that FIPs suppress antibody production came from the result that the proportion of Arthus reaction-positive mice was reduced to 40% by LZ-8 [130]. Fve also suppressed antibody production as demonstrated by its effect in the hind paw edema test but the inhibition was not complete [131]. Activities are summarized in Ta­ble 7.

Figure 3 summarizes the targets for interaction be­tween mushroom ingredients and various components of the adaptive immune system.

RECOGNITION AND RECEPTORS

Evidence for f-glucan receptor binding of immune cells

The innate immune system is the first line of defense against microbial invasion, and must immediately recog­nize and counter infections while the slower, more spe­cific, adaptive response is mounted. The innate cellular response is comprised principally of phagocytic cells and is dependent on germline encoded receptors which rec­ognize conserved microbial structures. The innate im­mune system identifies infectious agents or compounds by means of pattern-recognition receptors (PRR). These receptors recognize pathogen-specific macromolecules called pathogen-associated molecular patterns (PAMP).

Polysacharides cannot penetrate cells due to their large molecular mass, so the first step in the modulation of cellular activity is binding to immune cell receptors. Among all the immunomodulatory metabolites isolated from mushrooms, glucans and in particular f-glucans have been studied profoundly to identify its target recep­tor in immune cells. It has been postulated that glucans are fungal pattern-recognition molecules for the innate immune system [132,133].The mechanisms by which the innate immune system recognizes and responds to fungal cell wall carbohydrate is a very complex and multifacto- rial process [134]. The various activities of f -glucans may reflect the presence of multiple cellular targets or recep­tors [135]. To date several f-glucan receptors have been identified as candidates mediating these activities [136], namely, complement receptor 3 (CR3, aMf2 integrin, or

CD11b/CD18) [137], lactosylceramide [138], scavengers receptors [139], dectin-1 [140], and toll-like receptors TLR-2 and TLR-4 [141].

Dectin-1 is broadly expressed, with highest sur­face expression on populations of myeloid cells (mono- cyte/macrophage and neutrophil lineages) in the blood, bone marrow and spleen. DCs, and a sub-population of T cells , also expressed dectin-1 but at lower levels [142]. It is plausible that the expression of dectin-1, as a T-cell binding receptor, on a subset of T-cells may be part of a novel mechanism for the regulation of the T cell response by specific subsets of T cells as well as by APC [143].

Recently, Kim et al [93] have shown that PL, proteo­glycan isolated from P linteus, could induce the pheno- typic and functional maturation of DCs via TLR-2 and/or TLR-4. Shao et al [141] suggested that TLR-4 is also in­volved in GLPS-mediated macrophage activation. Rat an- timouse TLR-4 monoclonal antibody (AB) inhibited the proliferation of BALB/c mouse B cells under GLPS stim­ulation. Combination of Abs against mouse TLR-4 and immunoglobulin achieved almost complete inhibition of GLPS-induced B-cell proliferation, implying that both membrane Ig abd TLR-4 are required for GLPS-mediated B cell activation.

Lowe et al [134] reported that a f-D-(1^3)- linked glucan polymer composed of seven glucose subunits is the minimum binding ligand for glucan PRR on a hu­man monocyte cell line and indicated that all available monocyte glucan receptors will recognize the basic f- D-(1^3)-glucan structure with approximately the same affinity. However, as the glucan polymer becomes more complex it appears to be preferentially recognized by one glucan receptor versus another.

Additional studies are required to determine which receptor(s) are essential to the expression of the various immunobiological effects ascribed to f-glucans. The in­tracellular events that occur after glucan-receptor bind­ing have not been fully determined. As long as it remains unclear what receptors are involved in and what down­stream events are triggered by the binding of these glu- cans to their target cells, it will be difficult to make further progress in understanding their biological activities.

CONCLUSIONS

The information presented here illustrates the distinct immunomodulatory properties associated with mush­room constituents. The discovery and identification of new safe drugs, without severe side effects, has become an important goal of research in the biomedical science. Medicinal effects have been demonstrated for many tra­ditionally used mushrooms, with large differences in im- munomodulatory properties. The species studied so far represent a vast source of immunomodulating and an­titumor extracts and metabolites. Thus, the biochemi­cal mechanisms that mediate the biological activity are still not clearly understood. Mushroom metabolites are known to stimulate different cells of the immune sys­tem. The major immunopotentiation effects of these active substances include mitogenicity, stimulation of hematopoietic stem cells, activation of alternative comple­ment pathway, and activation of immune cells, such as TH cells, Tc cells, B cells, macrophages, DCs, and NK cells.

Different profiles have been observed in relation to the activated immune cells, for example, GLPS activate mouse B cells and macrophages but not T cells [141], polysaccharides from P linteus can stimulate B cells, T cells, and macrophages [144], while lentinan is a stimu­lator of T cells and macrophages, but not B cells [145]. Some of them might promote a TH1 response and oth­ers a Th2 response [49]. In the particular case of glu- cans, despite the structural and functional similarities of some of them, they differ in their ability to elicit various cellular responses, particularly cytokine expression and production and in their effectiveness against specific tu­mors [5]. The relationship between polysaccharide origin, structure, and their immunomodulation activity remains to be further characterized [125, 146]. 

Mushroom products are obvious immunoenhancers that potentiate the immune system in multiple ways. Mushroom polysaccharides are among the emerging new agents that could directly support or enhance functional autologous hematopoietic stem cell recovery [61]. In pre­ventive medicine, defense against invasion by foreign bod­ies is dependent on enhancing the natural immune sys­tem, including activation of macrophages and NK cells. Macrophages stimulated by mushroom products release several inflammatory cytokines, IL-1, IL-6, IL-8, TNF- a, and NO, all of which directly induce tumoricidal ac­tivity in macrophages. Macrophages produce also IL-10, IL-10, IL-12, GM-CSF, and IL-18. In other cases mush­room extracts inhibit the production of NO, PGE2, IL- 10, and TNF-a in LPS-stimulated macrophages and LPS- administer mice. This antiinflammatory effect occurs by down regulation of iNOS, COX-2, IL-10, and TNF-a gene expression via the suppression of NF-kB activation. Thus, these mushroom extracts might be relevant for clin­ical use for inflammatory diseases, including endotoxemia or sepsis. Some mushroom metabolites like D-fraction
represent an important biological response modifier (BRM) due to the enhancement of NK cells activity in cancer patients. Mushroom polysaccharides induce reg­ulatory effects on maturation and function of DCs and consequently enhance the capacity of DCs to promote the proliferation of naïve allogenic T cells and readies them for T-cell-mediated immune responses. Both classical and alternative pathways of complement have been activated by mushrooms and also anticomplementary activity has been detected in different mushrooms. T and B lympho­cytes are also activated by mushrooms. Some mushroom polysaccharides stimulate the production of antibodies but others as PGL have a strong effect on suppressing the antibody production [126].

The immunomodulating action of mushroom metabolites is specially valuable as a means of pro­phylaxis, a mild and noninvasive form of treatment, prevention of metastatic tumors, and as a cotreatment with chemotherapy [4]. The enhancement or potenti­ation of host defense mechanisms has been recognized as a possible means of inhibiting tumor growth without harming the host, but other alternative mechanisms are possible, like targeting the ras-mediated signaling pathway [147]. Whether certain metabolites enhance or suppress immune responses can depend on a number of factors, including dose, route of administration, and timing of administrations of the compound in question. The type of activity these metabolites exhibit can also depend on their mechanism of action or the site of activity. Taken together, the present data suggest that mushroom extracts or metabolites should be selected and used properly for modulation of immune responses. Due to the differences in activities among various extracts and isolated metabolites, it is imperative to evaluate its biological properties before any suggestions for use of a particular product in clinical practice. For example, D-fraction enhanced rather than suppressed the development of collagen-induced arthritis (CIA) [148]. Administration of D-fraction stimulates immune function of normal and tumor-bearing mice [84]. GLIS from G lucidum has an effect on lymphocytes or purified B cells from tumor-bearing mice markedly stronger than on lymphocytes or purified B cells from normal mice [127]. It has also been reported that an extract from the deep layer of cultivated mycelia of the Cov-1 strain of C versicolor enhances the immune functions in old mice but not in young mice [149].

For some of the mushroom metabolites described, further research is needed to determine whether there are any in vivo benefits comparable to the in vitro effects re­ported. Although it is unlikely that high molecular weight polysaccharidse would be absorbed after oral administra­tion, it is possible that it could exert a therapeutic effect by direct interaction with the mucosal immune system of the gastrointestinal tract. Thus, they could be developed as a preparation for use as a dietary supplement or phar­maceutical.

Some mushroom metabolites, such as the glucans lentinan and schizophyllan, or the polysaccharide-protein PSK, and the PSP, are used clinically for immune therapy [150, 151, 152, 153] and have been developed as phar­maceuticals in Japan and are now commercially available worldwide. PSK was commercialized by Kureha Chem­icals, Japan. After extensive clinical trials, PSK was ap­proved for use in Japan in 1977, and by 1985, it ranked 19th on the list of the world's commercially most suc­cessful drugs [154]. Annual Japanese sales of PSK in 1987 were worth US$357 million [154]. About 10 years af­ter PSK, PSP appeared on the market. Both compounds have been isolated from C versicolor. In addition to clinically tested PSK and PSP, numerous other extract preparations of C versicolor are on the market as neu- traceuticals and traditional medicines. Neutraceutical PSP preparations are sold worldwide in the form of capsules, ground biomass tablets, syrups, food additives, and teas [153].

Quality control of mushrooms poses significant chal­lenges: small differences in genetics, soil, temperature, moisture, and time of harvesting can lead to signifi­cant differences in the concentration of important con­stituents. The cultivation of mushrooms to produce fruit­ing bodies is a long-term process requiring from one to several months for the first fruiting bodies to appear. Nowadays, more research is carried out in relation to sub­merged culture. Submerged culture has potential advan­tages for higher mycelial production in a compact space and for a shorter incubation time with a lesser chance of contamination. Further optimization of the culture medium composition and physicochemical conditions of growth allows regulation of fungal metabolism in order to obtain standardized nutriceutical substances in higher yield. Mycelia formed by growing pure cultures in sub­merged culture is the best technique for obtaining con­sistent and safe mushroom products [3, 12, 155]. Mush­rooms are still far from being thoroughly studied.

ACKNOWLEDGMENT

The authors acknowledge the financial support of the Valencian authorities (Generalitat Valenciana; CTBPDC/2003/014) for Cristina Lull.

REFERENCES

  • [1] Chang R. Functional properties of edible mush­rooms. NutrRev. 1996;54(pt 2):S91-S93.
  • [2] Wasser SP, Weis AL. Therapeutic effects of sub­stances occurring in higher basidiomycetes mush­rooms: a modern perspective. Crit Rev Immunol. 1999;19(1):65-96.
  • [3] Reshetnikov SV, Wasser SP, Tan KK. Higher ba- sidiomycota as a source of antitumor and im- munostimulating polysaccharides. Int J Med Mushr. 2001;3(4):361-394.
  • [4] Wasser SP. Medicinal mushrooms as a source of an­titumor and immunomodulating polysaccharides. Appl Microbiol Biotechnol. 2002;60(3):258-274.
  • [5] Borchers AT, Stern JS, Hackman RM, Keen CL, Gershwin ME. Mushrooms, tumors, and immu­nity. Proc Soc Exp Biol Med. 1999;221(4):281-293.
  • [6] Manzi P, Gambelli L, Marconi S, Vivanti V, Pizzoferrato L. Nutrients in edible mushrooms: an inter-species comparative study. Food Chem. 1999;65:477-482.
  • [7] Mattila P, Suonpaa K, Piironen V. Functional prop­erties of edible mushrooms. Nutrition. 2000;16(7- 8):694-696.
  • [8] Smith JE, Rowan NJ, Sullivan R. Medicinal mush­rooms: a rapidly developing area of biotechnology for cancer therapy and other bioactivities. Biotech- nol Lett. 2002;24(22):1839-1845.
  • [9] Gao Y, Chan E, Zhou S. Immunomodulating ac­tivities ofGanoderma, a mushroom with medicinal properties. Food Rev Int. 2004;20:123-161.
  • [10] Breene WM. Nutritional and medicinal value of specialty mushrooms. J Food Prot. 1990;53(10):883-894.
  • [11] Chang ST. World production of cultivated edible and medicinal mushrooms in 1997 with emphasis on Lentinus edodes (Berk) Sing in China. Int J Med Mushr. 1999;1:291-300.
  • [12] Wasser SP, Nevo E, Sokolov D, Reshetnikov S, Timor-Tismenetsky M. Dietary supplements from medicinal mushrooms: diversity of types and vari­ety of regulations. Int J Med Mushr. 2000;2(1):1-19.
  • [13] Hobbs C, LAc. Medicinal Mushrooms: An Explo­ration of Tradition, Healing and Culture. Santa Cruz, Calif: Botanica Press; 1995.
  • [14] Hobbs C. Medicinal value of Lentinus edodes (Berk) Sing (Agaricomycetideae). A literature review. Int J Med Mushr. 2000;2(4):287-302.
  • [15] Li XM, Huang CK, Zhang TF, et al. The Chinese herbal medicine formula MSSM-002 suppresses allergic airway hyperreactivity and modulates TH1/TH2 responses in a murine model of allergic asthma. J Allergy Clin Immunol. 2000;106(4):660- 668.
  • [16] Liu YH, Kao MC, Lai YL, Tsai JJ. Efficacy of local nasal immunotherapy for Dp2-induced airway in­flammation in mice: using Dp2 peptide and fun­gal immunomodulatory peptide. J Allergy Clin Im­munol. 2003;112(2):301-310.
  • [17] Liu YH, Tsai CF, Kao MC, Lai YL, Tsai JJ. Effec­tiveness of Dp2 nasal therapy for Dp2- induced air­way inflammation in mice: using oral Ganoderma lucidum as an immunomodulator. J Microbiol Im­munol Infect. 2003;36(4):236-242.
  • [18] Hsieh KY, Hsu CI, Lin JY, Tsai CC, Lin RH. Oral ad­ministration of an edible-mushroom-derived pro­tein inhibits the development of food-allergic reac­tions in mice. Clin Exp Allergy. 2003;33(11):1595- 1602.
  • [19] Li XM, Zhang TF, Huang CK, et al. Food allergy herbal formula-1 (FAHF-1) blocks peanut-induced anaphylaxis in a murine model. J Allergy Clin Im­munol. 2001;108(4):639-646.
  • [20] Kuo YC, Huang YL, Chen CC, Lin YS, Chuang KA, Tsai WJ. Cell cycle progression and cytokine gene expression of human peripheral blood mononu- clear cells modulated by Agaricus blazei. J Lab Clin Med. 2002;140(3):176-187.
  • [21] Jose N, Ajith TA, Janardhanan KK. Methanol ex­tract of the oyster mushroom, Pleurotus florida, in­hibits inflammation and platelet aggregation. Phy- totherRes. 2004;18(1):43-46.
  • [22] Kim SH, Song YS, Kim SK, Kim BC, Lim CJ, Park EH. Anti-inflammatory and related phar­macological activities of the n-BuOH subfraction of mushroom Phellinus linteus. J Ethnopharmacol. 2004;93(1):141-146.
  • [23] Kim GY, Kim SH, Hwang SY, et al. Oral ad­ministration of proteoglycan isolated from Phelli- nus linteus in the prevention and treatment of collagen-induced arthritis in mice. Biol Pharm Bull. 2003;26(6):823-831.
  • [24] Bobek P, Galbavy S. Hypocholesterolemic and an- tiatherogenic effect of oyster mushroom (Pleurotus ostreatus) in rabbits. Nahrung. 1999;43(5):339-342.
  • [25] Yamada T, Oinuma T, Niihashi M, et al. Ef­fects of Lentinus edodes mycelia on dietary-induced atherosclerotic involvement in rabbit aorta. J Atheroscler Thromb. 2002;9(3):149-156.
  • [26] Gray AM, Flatt PR. Insulin-releasing and insulin­like activity of Agaricus campestris (mushroom). J Endocrinol. 1998;157(2):259-266.
  • [27] Yoon SJ, Yu MA, Pyun YR, et al. The nontoxic mushroom Auricularia auricula contains a polysac­charide with anticoagulant activity mediated by an- tithrombin. Thromb Res. 2003;112(3):151-158.
  • [28] Nanba H, Kodama N, Schar D, Turner D. Effects of Maitake (Grifola frondosa) glucan in HIV-infected patients. Mycoscience. 2000;41:293-295.
  • [29] Ngai PH, Ng TB. Lentin, a novel and potent an- tifungal protein from Shiitake mushroom with in­hibitory effects on activity of human immunode­ficiency virus-1 reverse transcriptase and prolifer­ation of leukemia cells. Life Sci. 2003;73(26):3363- 3374.
  • [30] Kodama N, Yamada M, Nanba H. Addition of Maitake D-fraction reduces the effective dosage of vancomycin for the treatment of Listeria-infected mice. Jpn J Pharmacol. 2001;87(4):327-332.
  • [31] Markova N, Kussovski V, Drandarska I, Nikolaeva S, Georgieva N, Radoucheva T. Protective activity of lentinan in experimental tuberculosis. Int Im- munopharmacol. 2003;3(10-11):1557-1562.
  • [32] Kim GY, Roh SI, Park SK, et al. Alleviation of ex­perimental septic shock in mice by acidic polysac­charide isolated from the medicinal mushroom

Phellinus linteus. Biol Pharm Bull. 2003;26(10): 1418-1423.

  • [33] Kidd PM. The use of mushroom glucans and pro- teoglycans in cancer treatment. Altern Med Rev. 2000;5(1):4-27.
  • [34] Ohno N, Miura NN, Nakajima M, Yadomae T. An­titumor 1,3-0-glucan from cultured fruit body of Sparassis crispa. Biol Pharm Bull. 2000;23(7):866- 872.
  • [35] Ohno N, Furukawa M, Miura NN, Adachi Y, Motoi M, Yadomae T. Antitumor 0-glucan from the cul­tured fruit body of Agaricus blazei. Biol Pharm Bull. 2001;24(7):820-828.
  • [36] Fisher M, Yang LX. Anticancer effects and mechanisms of polysaccharide-K (PSK): implica­tions of cancer immunotherapy. Anticancer Res. 2002;22(3):1737-1754.
  • [37] Mahajan RG, Patil SI, Mohan DR, Shastry P. Pleu­rotus eous mushroom lectin (PEL) with mixed car­bohydrate inhibition and antiproliferative activity on tumor cell lines. J Biochem Mol Biol Biophys. 2002;6(5):341-345.
  • [38] Ng ML, Yap AT. Inhibition of human colon car­cinoma development by lentinan from Shiitake mushrooms (Lentinus edodes). J Altern Complement Med. 2002;8(5):581-589.
  • [39] Gao Y, Zhou S, Jiang W, Huang M, Dai X. Ef­fects of ganopoly (a Ganoderma lucidum polysac- charide extract) on the immune functions in advanced-stage cancer patients. Immunol Invest. 2003;32(3):201-215.
  • [40] Gao YH, Zhou SF. Cancer prevention and treat­ment by Ganoderma, a mushroom with medicinal properties. Food Rev Int. 2003;19:275-325.
  • [41] Kodama N, Komuta K, Nanba H. Effect of Maitake (Grifola frondosa) D-fraction on the activation of NK cells in cancer patients. J Med Food. 2003;6(4):371-377.
  • [42] Lee IS, Nishikawa A. Polyozellus multiplex, a Ko­rean wild mushroom, as a potent chemopre- ventive agent against stomach cancer. Life Sci. 2003;73(25):3225-3234.
  • [43] Lee YL, Kim HJ, Lee MS, et al. Oral administra­tion of Agaricus blazei (H1 strain) inhibited tumor growth in a sarcoma 180 inoculation model. Exp Anim. 2003;52(5):371-375.
  • [44] Monro JA. Treatment of cancer with mushroom products. Arch Environ Health. 2003;58(8):533- 537.
  • [45] Peng YF, Zhang L, Zeng FB, Xu YX. Structure and antitumor activity of extracellular polysaccharides from mycelium. Carbohyd Polym. 2003;54(3):297- 303.
  • [46] Shin KH, Lim SS, Lee S, Lee YS, Jung SH, Cho SY. Anti-tumour and immuno-stimulating activi­ties of the fruiting bodies of Paecilomyces japon­ica, a new type of Cordyceps spp. Phytother Res. 2003;17(7):830-833.
  • [47] Sliva D. Ganoderma lucidum (Reishi) in cancer treatment. Integr Cancer Ther. 2003;2(4):358-364.
  • [48] Tsang KW, Lam CL, Yan C, et al. Coriolus versi­color polysaccharide peptide slows progression of advanced non-small cell lung cancer. Respir Med. 2003;97(6):618-624.
  • [49] Borchers AT, Keen CL, Gershwin ME. Mushrooms, tumors, and immunity: an update. Exp Biol Med (Maywood). 2004;229(5):393-406.
  • [50] Hattori TS, Komatsu N, Shichijo S, Itoh K. Protein- bound polysaccharide K induced apoptosis of the human Burkitt lymphoma cell line, Namalwa. Biomed Pharmacother. 2004;58(4):226-230.
  • [51] Ho JC, Konerding MA, Gaumann A, Groth M, Liu WK. Fungal polysaccharopeptide inhibits tu­mor angiogenesis and tumor growth in mice. Life Sci. 2004;75(11):1343-1356.
  • [52] Jiang J, Slivova V, Harvey K, Valachovicova T, Sliva D. Ganoderma lucidum suppresses growth ofbreast cancer cells through the inhibition of Akt/NF-KB signaling. Nutr Cancer. 2004;49(2):209-216.
  • [53] Jiang J, Slivova V, Valachovicova T, Harvey K, Sliva D. Ganoderma lucidum inhibits proliferation and induces apoptosis in human prostate cancer cells PC-3. Int J Oncol. 2004;24(5):1093-1099.
  • [54] Nakamura T, Matsugo S, Uzuka Y, Matsuo S, Kawagishi H. Fractionation and anti-tumor activity of the mycelia of liquid-cultured Phellinus linteus. Biosci Biotechnol Biochem. 2004;68(4):868-872.
  • [55] Shibata Y, Kurita S, Okugi H, Yamanaka H. Dra­matic remission of hormone refractory prostate cancer achieved with extract of the mushroom, Phellinus linteus. Urol Int. 2004;73(2):188-190.
  • [56] Chirigos MA. Immunomodulators: current and future development and application. Thymus. 1992;19 (suppl 1) :S7-S20.
  • [57] Masihi KN. Immunomodulatory agents for pro­phylaxis and therapy of infections. Int J Antimicrob Agents. 2000;14(3):181-191.
  • [58] Jong SC, Birmingham JM. Medicinal benefits of the mushroom Ganoderma. Adv Appl Microbiol. 1992;37:101-134.
  • [59] Badger AM. Development in industrial microbiol­ogy. In: Saratosa FL, Nash CH, Underkofler LA, eds. Proceedings of the Fortieth General Meeting of the Society for Industrial Microbiology. Arlington, Va; 1983:274.
  • [60] Vickers A. Botanical medicines for the treatment of cancer: rationale, overview of current data, and methodological considerations for phase I and II trials. Cancer Invest. 2002;20(7-8):1069-1079.
  • [61] Lin H, She YH, Cassileth BR, Sirotnak F, Cun­ningham Rundles S. Maitake beta-glucan MD- fraction enhances bone marrow colony formation and reduces doxorubicin toxicity in vitro. Int Im- munopharmacol. 2004;4(1):91-99.
  • [62] Jin M, Jung HJ, Choi JJ, et al. Activation of selec­tive transcription factors and cytokines by water- soluble extract from Lentinus lepideus. Exp Biol Med (Maywood). 2003;228(6):749-758.
  • [63] Jin M, Jeon H, Jung HJ, et al. Enhancement of repopulation and hematopoiesis of bone marrow cells in irradiated mice by oral administration of PG101, a water-soluble extract from Lentinus lep­ideus. Exp Biol Med (Maywood). 2003;228(6):759- 766.
  • [64] Harada T, Miura N, Adachi Y, Nakajima M, Yado- mae T, Ohn N. Effect of SCG, 1,3-ß-D-glucan from Sparassis crispa on the hematopoietic response in cyclophosphamide induced leukopenic mice. Biol Pharm Bull. 2002;25(7):931-939.
  • [65] Sorimachi K, Akimoto K, Ikehara Y, Inafuku K, Okubo A, Yamazaki S. Secretion of TNF-a, IL- 8 and nitric oxide by macrophages activated with Agaricus blazei Murill fractions in vitro. Cell Struct Funct. 2001;26(2):103-108.
  • [66] Wang SY, Hsu ML, Hsu HC, et al. The anti-tumor effect of Ganoderma lucidum is mediated by cy- tokines released from activated macrophages and T lymphocytes. Int J Cancer. 1997;70(6):699-705.
  • [67] Adachi Y, Okazaki M, Ohno N, Yadomae T. En­hancement of cytokine production by macrophages stimulated with (1^3)-ß-D-glucan, grifolan (GRN), isolated from Grifola frondosa. Biol Pharm Bull. 1994;17(12):1554-1560.
  • [68] Okazaki M, Adachi Y, Ohno N, Yadomae T. Structure-activity relationship of (1^3)-beta-D- glucans in the induction of cytokine produc­tion from macrophages, in vitro. Biol Pharm Bull. 1995;18(10):1320-1327.
  • [69] Ishibashi K, Miura NN, Adachi Y, Ohno N, Yado- mae T. Relationship between solubility of grifolan, a fungal 1,3-ß-D-glucan, and production of tu­mor necrosis factor by macrophages in vitro. Biosci Biotechnol Biochem. 2001;65(9):1993-2000.
  • [70] Sanzen I, Imanishi N, Takamatsu N, et al. Nitric oxide-mediated antitumor activity induced by the extract from Grifola frondosa (Maitake mushroom) in a macrophage cell line, RAW264.7. J Exp Clin Cancer Res. 2001;20(4):591-597.
  • [71] Duncan CJ, Pugh N, Pasco DS, Ross SA. Isolation of a galactomannan that enhances macrophage ac­tivation from the edible fungus Morchella esculenta. JAgricFood Chem. 2002;50(20):5683-5685.
  • [72] Han SB, Lee CW, Jeon YJ, et al. The inhibitory effect of polysaccharides isolated from Phellinus linteus on tumor growth and metastasis. Immunopharma- cology. 1999;41(2):157-164.
  • [73] Kim GY, Oh YH, Park YM. Acidic polysac- charide isolated from Phellinus linteus induces nitric oxide-mediated tumoricidal activity of macrophages through protein tyrosine kinase and protein kinase C. Biochem Biophys Res Commun. 2003;309(2):399-407.
  • [74] Kim KM, Kwon YG, Chung HT, et al. Methanol extract of Cordyceps pruinosa inhibits in vitro and in vivo inflammatory mediators by suppress­ing NF-kB activation. Toxicol Appl Pharmacol. 2003;190(1):1-8.
  • [75] Mizuno M, Shiomi Y, Minato K, Kawakami S, Ashida H, Tsuchida H. Fucogalactan iso­lated from Sarcodon aspratus elicits release of tumor necrosis factor-a and nitric oxide from murine macrophages. Immunopharmacology. 2000;46(2):113-121.
  • [76] Kurashige S, Akuzawa Y, Endo F. Effects of Lentinus edodes, Grifola frondosa and Pleuro- tus ostreatus administration on cancer outbreak, and activities of macrophages and lymphocytes in mice treated with a carcinogen, N-butyl-N- butanolnitrosoamine. Immunopharmacol Immuno- toxicol. 1997;19(2):175-183.
  • [77] Ngai PH, Wang HX, Ng TB. Purification and characterization of a ubiquitin-like peptide with macrophage stimulating, antiproliferative and ri- bonuclease activities from the mushroom Agrocybe cylindracea. Peptides. 2003;24(5):639-645.
  • [78] Wang HX, Ng TB, Ooi VE, Liu WK, Chang ST. Actions of lectins from the mushroom Tricholoma mongolicum on macrophages, splenocytes and life­span in sarcoma-bearing mice. Anticancer Res. 1997;17(1A):419-424.
  • [79] Miller JS. Biology of natural killer cells in cancer and infection. Cancer Invest. 2002;20(3):405-419.
  • [80] Sepulveda C, Puente J. Natural killer cells and the innate immune system in infectious pathology. Rev Med Chil. 2000;128(12):1361-1370.
  • [81] Yokoyama WM, Kim S, French AR. The dynamic life of natural killer cells. Annu Rev Immunol. 2004;22:405-429.
  • [82] Kodama N, Komuta K, Sakai N, Nanba H. Effects of D-fraction, a polysaccharide from Grifola frondosa on tumor growth involve activation of NK cells. Biol Pharm Bull. 2002;25(12):1647-1650.
  • [83] Kodama N, Kakuno T, Nanba H. Stimulation of the natural immune system in normal mice by polysaccharide from Maitake mushroom. Myco- science. 2003;44(3):257-261.
  • [84] Kodama N, Murata Y, Nanba H. Administration of a polysaccharide from Grifola frondosa stimu­lates immune function of normal mice. J Med Food. 2004;7(2):141-145.
  • [85] Takimoto H, Wakita D, Kawaguchi K, Kumazawa Y. Potentiation of cytotoxic activity in naive and tumor-bearing mice by oral administration of hot- water extracts from Agaricus brazei fruiting bodies. Biol Pharm Bull. 2004;27(3):404-406.
  • [86] Emtage PC, Clarke D, Gonzalo-Daganzo R, Jung­hans RP. Generating potent Th1/Tc1 T cell adop­tive immunotherapy doses using human IL-12: harnessing the immunomodulatory potential of

IL-12 without the in vivo-associated toxicity [pub­lished correction appears in J Immunother]. J Im- munother. 2003;26(2):97-106. 2003;26(3):290.

  • [87] Kaneno R, Fontanari LM, Santos SA, Di Stasi LC, Rodrigues Filho E, Eira AF. Effects of extracts from Brazilian sun-mushroom (Agaricus blazei) on the NK activity and lymphoproliferative responsive­ness of Ehrlich tumor-bearing mice. Food Chem Toxicol. 2004;42(6):909-916.
  • [88] Ahn WS, Kim DJ, Chae GT, et al. Natural killer cell activity and quality of life were improved by consumption of a mushroom extract, Agari- cus blazei Murill Kyowa, in gynecological cancer patients undergoing chemotherapy. Int J Gynecol Cancer. 2004;14(4):589-594.
  • [89] Zhang W, Wang Y, Hou Y. Effects ofChinese medic­inal fungus water extract on tumor metastasis and some parameters of immune function. Int Im- munopharmacol. 2004;4(3):461-468.
  • [90] Banchereau J, Briere F, Caux C, et al. Immuno- biology of dendritic cells. Annu Rev Immunol. 2000;18:767-811.
  • [91] Cao LZ, Lin ZB. Regulation on maturation and function of dendritic cells by Ganoderma lucidum polysaccharides. Immunol Lett. 2002;83(3):163- 169.
  • [92] Park SK, Kim GY, Lim JY, et al. Acidic polysac- charides isolated from Phellinus linteus induce phenotypic and functional maturation of murine dendritic cells. Biochem Biophys Res Commun. 2003;312(2):449-458.
  • [93] Kim GY, Han MG, Song YS, et al. Proteoglycan isolated from Phellinus linteus induces toll-like re­ceptors 2- and 4-mediated maturation of murine dendritic cells via activation of ERK, p38, and NF- kappaB. Biol Pharm Bull. 2004;27(10):1656-1662.
  • [94] Kim GY, Oh WK, Shin BC, et al. Proteogly- can isolated from Phellinus linteus inhibits tu­mor growth through mechanisms leading to an activation of CD11c+CD8+ DC and type I helper T cell-dominant immune state. FEBS Lett. 2004;576(3):391-400.
  • [95] Kanazawa M, Mori Y, Yoshihara K, et al. Effect of PSK on the maturation of dendritic cells de­rived from human peripheral blood monocytes. Immunol Lett. 2004;91(2-3):229-238.
  • [96] Di Luzio NR. Update on the immunomodulating activities of glucans. Springer Semin Immunopathol. 1985;8(4):387-400.
  • [97] Ross GD, Vetvicka V, Yan J, Xia Y, Vetvick- ova J. Therapeutic intervention with complement and beta-glucan in cancer. Immunopharmacology. 1999;42(1-3):61-74.
  • [98] Alper CA, Abramson N, Johnston RB Jr, Jandl JH, Rosen FS. Increased susceptibility to infection asso­ciated with abnormalities ofcomplement-mediated functions and of the third component of comple­ment (C3). N Engl J Med. 1970;282(7):350-354.

[99] Winkelstein JA, Smith MR, Shin HS. The role of C3 as an opsonin in the early stages of infection. Proc Soc Exp Biol Med. 1975;149(2):397-401.

  • [100] Okuda T, Yoshioka Y, Ikekawa T, Chihara G, Nishioka K. Anticomplementary activity of antitumor polysaccharides. Nature: New Biol. 1972;238(12):59-60.
  • [101] Hamuro J, Hadding U, Bitter-Suermann D. Solid phase activation of alternative pathway of com­plement by beta-1,3-glucans and its possible role for tumor regressing activity. Immunology. 1978;34(4):695-705.
  • [102] Shimizu S, Kitada H, Yokota H, et al. Activation of the alternative complement pathway by Agaricus blazei Murill. Phytomedicine. 2002;9(6):536-545.
  • [103] Lee JW, Chung CH, Jeong H, Lee KH. Effects of al­kali extract of Ganoderma lucidum IY007 on com­plement system. Korean JMycol. 1990;18:137-144.
  • [104] Min BS, Gao JJ, Hattori M, Lee HK, Kim YH. Anticomplement activity of terpenoids from the spores of Ganoderma lucidum. Planta Med. 2001;67(9):811-814.
  • [105] Yang QY, Pai SS. The anti-ageing effects of Gan- oderma essence. Proceedings of the International Meetingon Ganoderma Science. Beijing; 2000:30.
  • [106] Suzuki I, Hashimoto K, Oikawa S, Sato K, Os- awa M, Yadomae T. Antitumor and immunomod- ulating activities of a beta-glucan obtained from liquid-cultured Grifola frondosa. Chem Pharm Bull (Tokyo). 1989;37(2):410-413.
  • [107] Jeong H, Lee JW, Lee KH. Studies on the anticom­plementary activity of Korean higher fungi. Korean J Mycol. 1990;18:145-148.
  • [108] Okamura H, Tsutsi H, Komatsu T, et al. Cloning of a new cytokine that induces IFN-gamma produc­tion by T cells. Nature. 1995;378(6552):88-91.
  • [109] Micallef MJ, Ohtsuki T, Kohno K, et al. Interferon- gamma-inducing factor enhances T helper 1 cy- tokine production by stimulated human T cells: synergism with interleukin-12 for interferon- gamma production. Eur J Immunol. 1996;26(7): 1647-1651.
  • [110] Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O'Garra A, Murphy KM. Development of TH1 CD4+ T cells through IL-12 produced by Liste- ria-induced macrophages. Science. 1993;260(5107): 547-549.
  • [111] Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis. Annu Rev Im­munol. 1996;14:397-440.
  • [112] Cohen PA, Cohen PJ, Rosenberg SA, Mule JJ. CD4+ T-cells from mice immunized to syngeneic sarco­mas recognize distinct, non-shared tumor antigens. Cancer Res. 1994;54(4):1055-1058.
  • [113] O'Garra A, Murphy K. T-cell subsets in autoimmu­nity. Curr Opin Immunol. 1993;5(6):880-886.
  • [114] Powrie F, Coffman RL. Cytokine regulation of T- cell function: potential for therapeutic interven­tion. Immunol Today. 1993;14(6):270-274.
  • [115] Scott B, Liblau R, Degermann S, et al. A role for non-MHC genetic polymorphism in suscep­tibility to spontaneous autoimmunity. Immunity. 1994;1(1):73-83.
  • [116] Liblau RS, Singer SM, McDevitt HO. Th1 and Th2 CD4+ T cells in the pathogenesis of organ- specific autoimmune diseases. Immunol Today. 1995;16(1):34-38.
  • [117] Romagnani S. Regulation of the development of type 2 T-helper cells in allergy. Curr Opin Immunol. 1994;6(6):838-846.
  • [118] Suzuki Y, Adachi Y, Ohno N, Yadomae T. Th1/Th2- balancing immunomodulating activity of gel- forming (1^3)-0-glucans from fungi. Biol Pharm Bull. 2001;24(7):811-819.
  • [119] Chihara G, Hamuro J, Maeda YY, et al. Antitumor and metastasis-inhibitory activities of lentinan as an immunomodulator: an overview. Cancer Detect PrevSuppl. 1987;1:423-443.
  • [120] Murata Y, Shimamura T, Tagami T, Takatsuki F, Hamuro J. The skewing to Th1 induced by lenti- nan is directed through the distinctive cytokine production by macrophages with elevated intracel- lular glutathione content. Int Immunopharmacol. 2002;2(5):673-689.
  • [121] Inoue A, Kodama N, Nanba H. Effect of Maitake (Grifola frondosa) D-fraction on the control of the T lymph node Th-1/Th-2 proportion. Biol Pharm Bull. 2002;25(4):536-540.
  • [122] Harada N, Kodama N, Nanba H. Relationship be­tween dendritic cells and the D-fraction-induced Th-1 dominant response in BALB/c tumor-bearing mice. Cancer Lett. 2003;192(2):181-187.
  • [123] Hsu HC, Hsu CI, Lin RH, Kao CL, Lin JY. Fip-vvo, a new fungal immunomodulatory protein isolated from Volvariella volvacea. Biochem J. 1997;323(pt 2):557-565.
  • [124] She QB, Ng TB, Liu WK. A novel lectin with potent immunomodulatory activity isolated from both fruiting bodies and cultured mycelia ofthe ed­ible mushroom Volvariella volvacea. Biochem Bio- physRes Commun. 1998;247(1):106-111.
  • [125] Bao XF, Wang XS, Dong Q, Fang JN, Li XY. Struc­tural features of immunologically active polysac­charides from Ganoderma lucidum. Phytochemistry. 2002;59(2):175-181.
  • [126] Bao X, Fang J, Li X. Structural characterization and immunomodulating activity of a complex glucan from spores of Ganoderma lucidum. Biosci Biotech- nol Biochem. 2001;65(11):2384-2391.
  • [127] Zhang J, Tang Q, Zimmerman-Kordmann M, Reut- ter W, Fan H. Activation of B lymphocytes by GLIS, a bioactive proteoglycan from Ganoderma lucidum. Life Sci. 2002;71(6):623-638.
  • [128] Kim GY, Park SK, Lee MK, et al. Proteoglycan iso­lated from Phellinus linteus activates murine B lym­phocytes via protein kinase C and protein tyrosine kinase. Int Immunopharmacol. 2003;3(9):1281- 1292.
  • [129] Di Renzo L, Yefenof E, Klein E. The function of human NK cells is enhanced by beta-glucan, a ligand of CR3 (CD11b/CD18). Eur J Immunol. 1991;21(7):1755-1758.
  • [130] Kino K, Sone T, Watanabe J, et al. Immunomodu­lator, LZ-8, prevents antibody production in mice. Int J Immunopharmacol. 1991;13(8):1109-1115.
  • [131] Ko JL, Hsu CI, Lin RH, Kao CL, Lin JY. A new fungal immunomodulatory protein, FIP-fve iso­lated from the edible mushroom, Flammulina ve- lutipes and its complete amino acid sequence. Eur J Biochem. 1995;228(2):244-249.
  • [132] Janeway CA Jr, Medzhitov R. Lipoproteins take their toll on the host. Curr Biol. 1999;9(23):R879- R882.
  • [133] Mueller A, Raptis J, Rice PJ, et al. The influence of glucan polymer structure and solution confor­mation on binding to (1^3)-ß-D-glucan receptors in a human monocyte-like cell line. Glycobiology. 2000;10(4):339-346.
  • [134] Lowe E, Rice P, Ha T, et al. A (1^3)-ß-D-linked heptasaccharide is the unit ligand for glucan pat­tern recognition receptors on human monocytes. Microbes Infect. 2001;3(10):789-797.
  • [135] Kougias P, Wei D, Rice PJ, et al. Normal hu­man fibroblasts express pattern recognition recep­tors for fungal (1^3)-ß-D-glucans. Infect Immun. 2001;69(6):3933-3938.
  • [136] Brown GD, Gordon S. Fungal ß-glucans and mam­malian immunity. Immunity. 2003;19(3):311-315.
  • [137] Ross GD, Cain JA, Myones BL, Newman SL, Lach­mann PJ. Specificity of membrane complement re­ceptor type three (CR3) for beta-glucans. Comple­ment. 1987;4(2):61-74.
  • [138] Zimmerman JW, Lindermuth J, Fish PA, Palace GP, Stevenson TT, DeMong DE. A novel carbohydrate- glycosphingolipid interaction between a ß-(1-3)- glucan immunomodulator, PGG-glucan, and lac- tosylceramide of human leukocytes. J Biol Chem. 1998;273(34):22014-22020.
  • [139] Rice PJ, Kelley JL, Kogan G, et al. Human mono- cyte scavenger receptors are pattern recognition receptors for (1^3)-ß-D-glucans. J Leukoc Biol. 2002;72(1):140-146.
  • [140] Brown GD, Gordon S. Immune recognition. A new receptor for beta-glucans. Nature. 2001;413(6851): 36-37.
  • [141] Shao BM, Dai H, Xu W, Lin ZB, Gao XM. Im­mune receptors for polysaccharides from Gano- derma lucidum. Biochem Biophys Res Commun. 2004;323(1):133-141.

 

  • [142] Herre J, Gordon S, Brown GD. Dectin-1 and its role in the recognition of ß-glucans by macrophages. Mol Immunol. 2004;40(12):869-876.
  • [143] Taylor PR, Brown GD, Reid DM, et al. The ß- glucan receptor, dectin-1, is predominantly ex­pressed on the surface of cells of the mono­cyte/macrophage and neutrophil lineages. J Im­munol. 2002;169(7):3876-3882.
  • [144] Kim HM, Han SB, Oh GT, et al. Stimulation of humoral and cell mediated immunity by polysac­charide from mushroom Phellinus linteus. Int J Im- munopharmacol. 1996;18(5):295-303.
  • [145] Liu M, Li J, Kong F, Lin J, Gao Y. Induction of immunomodulating cytokines by a new polysac- charide-peptide complex from culture mycelia of Lentinus edodes. Immunopharmacology. 1998; 40(3):187-198.
  • [146] Bao X, Liu C, Fang J, Li X. Structural and immuno- logical studies of a major polysaccharide from spores of Ganoderma lucidum (Fr) Karst. Carbo- hydrRes. 2001;332(1):67-74.
  • [147] Hsiao WL, Li YQ, Lee TL, Li N, You MM, Chang ST. Medicinal mushroom extracts inhibit ras-induced cell transformation and the inhibitory effect re­quires the presence of normal cells. Carcinogenesis. 2004;25(7):1177-1183.
  • [148] Shigesue K, Kodama N, Nanba H. Effects of Maitake (Grifola frondosa) polysaccharide on collagen-induced arthritis in mice. Jpn J Pharma­col. 2000;84(3):293-300.
  • [149] Han SN, Wu D, Leka LS, Meydani SN. Effect of mushroom (Coriolus versicolor) extract on the im­mune response of young and old mice. FASEB J. 1996;10(3):3200.
  • [150] Chihara G, Hamuro J, Maeda Y, Arai Y, Fukuoka F. Fractionation and purification of the polysac- charides with marked antitumor activity, especially lentinan, from Lentinus edodes (Berk) Sing (an ed­ible mushroom). Cancer Res. 1970;30(11):2776- 2781.
  • [151] Suga T, Shiio T, Maeda YY, Chihara G. Antitumor activity of lentinan in murine syngeneic and au­tochthonous hosts and its suppressive effect on 3- methylcholanthrene-induced carcinogenesis. Can- cerRes. 1984;44(11):5132-5137.
  • [152] Ooi VE, Liu F. Immunomodulation and anti­cancer activity of polysaccharide-protein com­plexes. CurrMed Chem. 2000;7(7):715-729.
  • [153] Cui J, Chisti Y. Polysaccharopeptides of Coriolus versicolor: physiological activity, uses, and produc­tion. Biotechnol Adv. 2003;21(2):109-122.
  • [154] Yang QY, Jong SC, Li XY, Zhou JX, Chen RT, Xu LZ. Antitumor and immunomodulating activities of the Polysaccharide-Peptide (Psp) of Coriolus ver­sicolor. Eos-Riv Immunol. 1992;12:29-34.
  • [155] Zhu R, Chen X, Lan J. Advance in the study on liquid fermentation for medicinal fungi. Zhong Yao Cai. 2003;26(1):55-57.

 

    

http://www.reishi.com/News/Study%20Fungus%20compounds%20may.

 

 

 

 

 

ONLINE EDITION

JERUSALEM POST

( Mantar  kökenli bileşikler prostat kanserini yavaşlatabilirler)

Study: Fungus compounds may slow prostate cancer

Judy Siegel-Itzkovich , THE JERUSALEM POST                                                                                                                                                                                     Dec. 12, 2007

Compounds found in a common fungus have the potential to slow the progression of prostate cancer, the tumor recently diagnosed in Prime Minister Ehud Olmert and in around 2,250 other Israeli men every year.

The discovery was made recently by researchers at the University of Haifa and announced on Wednesday.

The compounds identified in Ganoderma lucidum were found help suppress some of the mechanisms involved in the progression of prostate cancer. They disrupt the activity of androgen receptors and impede the proliferation of cancerous cells.

Over the past 40 years, much research has examined the medicinal properties of different fungi, which - except for mushrooms - are largely invisible to the naked eye. They live in soil and dead matter, and in asymbiotic relationship with plants, animals or other fungi. They decompose matter and are indispensable in nutrient cycling and exchange. Many fungi contain bioactive compounds such as alkaloids and polyketides that are toxic to animals and humans.

The compounds in Ganoderma lucidum were found to have the ability to fight cancer in a number of ways, but most of the work concentrated on how fungi affect the immune system. In this research - conducted by Dr. Ben-Zion Zaidman under the direction of Prof. Eviatar Nevo and Prof. Solomon Wasser from the university's institute of evolution and Dr. Jamal Mahajna from the Migal Galilee Technology Center - the researchers examined how fungi fight cancer from within cells.

"Up to now, research has been based on enhancing the immune system with high-molecular-weight polysaccharides that act through specific receptors in cell membranes. We concentrated our research on low-molecular-weight secondary metabolites that can penetrate the cells and act at the molecular level from within the cell itself," Zaidman said.

According to the researcher, prostate cancer - one of the most common cancers among men in the West - is controlled by the androgen receptor, especially in the initial stages of development of the disease. Therefore, all medications currently used to treat prostate cancer work to reduce the production of androgens or to interfere with their function via the androgen receptor.

In the first stage of the research, 201 organic extracts from 68 types of fungi were produced with solvents such as ether, ethyl acetate and ethanol. These solvents are used to select molecules that are small enough to act from within human cells. Of the 201 extracts, 11 were found to deter androgen receptor activity by more than 40 percent. Later, 169 extracts were tested for cancer cell growth inhibition. In this study, 14 extracts were found to be active in inhibiting prostate cancer cells.

The active extracts from Ganoderma lucidum were the most effective in inhibiting the function of the androgen receptor and controlling vital development of cancerous cells.

"The results of this research are particularly interesting from a commercial aspect. Potential possibilities exist to establish research and development of bioactive metabolites from Ganoderma lucidum that could yield an

 

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

 
 

ELSEVIER

journal homepage: www.elsevier.com/locate/bmcl

 

 

 

 

 

(Anti -Androjenik osteoclastogenesis inhibitörü olarak Ganoderic Acid DM.)

Ganoderic acid DM: Anti-androgenic osteoclastogenesis inhibitor

Jie Liu a, Jun Shiono a, Kuniyoshi Shimizu a, Akiko Kukita b, Toshio Kukitac, Ryuichiro Kondo a [1]

a Department of Forest and Forest Products Science, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan b Faculty of Medicine, Saga University, Nabeshima 5-1-1, Saga 849-8501, Japan c Faculty of Dental Science, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan

ARTICLE INFO

Prostate cancer is the most common cancer in men in Western countries, with a high incidence of bone metastasis. Ganoderic acid DM, with 5a-reductase inhibitory and androgen receptor (AR) binding activ­ity, isolated from the ethanol extracts of Ganoderma lucidum, can inhibit prostate cancer cell growth and block osteoclastogenesis.

Article history: Received 4 December 2008 Revised 16 February 2009 Accepted 27 February 2009 Available online 4 March 2009

© 2009 Elsevier Ltd. All rights reserved.

 

 Prostate cancer is the most frequently diagnosed male cancer and second leading cause of cancer deaths in North America.1 Androgens play an important role in the proliferation, differentia­tion, maintenance, and function of the prostate.2 Evidence shows that androgens are also involved in the development and progres­sion of prostate cancer.3 Androgens action is mediated by the 5a- reductase and androgen receptor (AR). 5a-Reductase catalyzes testosterone to the active androgen dihydrotestosterone (DHT). Androgen receptor binds to DHT, translocates into nucleus and reg­ulates androgen responsive genes implicated in the development of prostate cancer.4 A unique requirement for prostate cancer is the initial reliance on androgens for growth and to avoid apopto- sis.5 Because of this requirement, standard therapies block the action of androgens or remove the testicular androgens from the patient (endocrine therapy). These therapies include lower testos­terone levels (androgen ablation); treatment with anti-androgens, such as flutamide or bicalutamide, to block DHT binding to the AR (anti-androgen therapy); and maximal androgen blockade (MAB), in which anti-androgen treatment and androgen ablation therapy are combined.

Prostate cancer preferentially metastasizes to bone, resulting in significant disease morbidity prior to a patient's death. It is known that cancer cells spread to bone and use the local cytokine machin­ery to stimulate osteoclasts, resulting in bone resorption and cancer cell growth.6 Osteoclast activities are important for the development of bone metastasis in prostate cancer.7 Because of the facts that increased osteoclastic activity is associated with tumor growth in bone microenvironment, anti-resorptive thera­pies have been used in cancer patients to block the tumor develop­ment in bone.

On the other hand, more and more attentions begin to focus on the phytotherapeutic agents that may be more effective and safer.8 Growing epidemiological data have reported that Asian men are less susceptible to prostate cancer than Europeans and Americans, which may be due to their high dietary intake of phytoestrogens.9,10 Our group previously has obtained a series of triterpenoids from Ganoderma lucidum (Leyss.:Fr.) Karst. (Ganodermataceae), which have a suppressive effect on proliferation of the androgen-depen- dent prostate cancer cell line LNCaP cells11 and estrogen-like activity on proliferation of the estrogen-dependent MCF-7 cells.12 In the present study, we further investigated ganoderic acid DM, the active constituent derived from the fungi, G. lucidum. We report here that ganoderic acid DM has the potential to inhibit 5a-reductase activity, bind to the AR, inhibit the growth of prostate cancer cell, and sup­press the osteoclastic differentiation by using RAW 264 cell.

In our previous screening of 19 edible and medicinal mushrooms, we discovered that the ethanol extracts of the fruiting body of G. luci­dum showed the strongest 5a-reductase inhibitory activity. In addi­tion, the treatment of the ethanol extracts prepared from G. lucidum at 1.5 and 15 mg/kg/day significantly inhibited the growth of the ventral prostate induced by testosterone in rats.13 To clarify the active principles of the ethanol extracts of G. lucidum,5a-reductase inhibitory activity-guided fractionation was carried out. The ethanol extracts were roughly separated into three fractions (Fr. A, B, C). Only Fr. B showed the suppression effect on the ventral prostate growth induced by testosterone in rats. G. lucidum has been reported to produce many bioactive oxygenated triterpenoids. Over 120 species of triterpenoids have been isolated so far from G. lucidum and the genus Ganoderma.14 Considering the results of ourTLC analysis (data not shown), it is likely that most of the triterpenoids were present in Fr. B. Therefore, we focused on the triterpenoids in the ethanol extracts of G. lucidum. Anti-androgenic assay (5a-reductase inhibi­tory activity and androgen receptor-binding)-guided fractionation led to the isolation of one of active triterpenoid from Fr. B, identified as ganoderic acid DM (Fig. 1).

The inhibition of 5a-reductase by ganoderic acid DM was concentration-dependent, as shown in Figure 2. As the concentra­tions of ganoderic acid DM increased, the residual enzyme activity decreased. The 1C50 of ganoderic acid DM was 10.6 iM. It should be noted that finasteride,15 which is known as a potent steroidal inhibitor, showed an 1C50 of 0.73 iM in our assay system. a-Linole- nic acid, a natural compound with 5a-reductase inhibitory activity was also used as a positive control, showed an 1C50 of 116 iM in our assay system. Ganoderic acid DM showed stronger 5a-reduc- tase inhibitory activity than the natural 5a-reductase inhibiter, a-linolenic acid.15 In the history of 5a-reductase inhibitor study, the inverted steroid-based inhibitors have been extremely impor­tant drugs for hormone dependent cancers.16,17 These 5a-reduc- tase inhibitors, the molecules bind in the active site of the enzyme such that the steroid A-ring mimics the A-ring functional­ity of testosterone or some intermediate along the reaction pathway; the potency and selectivity is determined in part by appropriate substitution of the D-ring.18 Ganoderic acid DM has the unsaturated C-26 carboxy (A24,25) at the 17 side chain and C3-carbonyl, which possibly mimic the A-ring of testosterone.

Also, the blocking of DHT from binding to the androgen recep­tors by ganoderic acid DM has been examined. Thus, we directly assessed the ability of ganoderic acid DM to bind to the AR. As shown by the semilog scale relative to the concentration to polar­ization (Fig. 3), the polarization value (mP) was decreased when the concentration of ganoderic acid DM was increased. Fifty percent of the maximal shift of the highest polarization value is represented by 50% of binding to AR-LBD. A higher concentration of ganoderic acid DM (15 iM) than that of DHT (0.018 iM) was required to bind to 50% of AR-LBD.

The LNCaP (lymph node carcinoma of the prostate) human prostate cancer cell line is a well-established androgen-dependent cell line.19 LNCaP cells retain most of the characteristics of human prostatic carcinoma, like the dependence on androgens and the presence of ARs. For these reasons, the LNCaP cell line becomes an attractive model for in vitro studies of the biology of human prostate cancer.20 LNCaP cells were incubated with varying concentrations of ganoderic acid DM (10-50 iM) and with or with­out testosterone or DHT for three days. The NR assay was performed to measure cell viability. Treating LNCaP cells with ganoderic acid DM resulted in dose-dependent inhibition of cell growth (Fig. 4A). In the absence of ganoderic acid DM, testosterone alone apparently stimulates the LNCaP cell proliferation about 150% on average more than the untreated control (Fig. 4B). DHT alone apparently stimulates the LNCaP cell number about 170% on average more than the untreated control (Fig. 4C). Ganoderic acid DM inhibited androgen induced proliferation of LNCaP cells at all concentration range (10-50 iM). Ganoderic acid DM inhibited the androgen induced cell growth in dose-dependent inhibition (Fig. 4B and C).

100

to >

The prostatic carcinoma cell lines PC-3 have been characterized as true prostatic carcinoma cell lines in 1979.21PC-3 has a greatly reduced dependence upon serum for growth when compared to normal prostatic epithelial cells, does not respond to androgens, glucocorticoids, or epidermal or fibroblast growth factors and did not express PSA. The ability of ganoderic acid DM on the other prostate cancer cell line was also evaluated. The PC-3 cells were treated with ganoderic acid DM at concentration range of 1­50 iM. Ganoderic acid DM also inhibited proliferation of PC-3 cell at high concentration (12.5-50 iM) (Fig. 5). Interestingly, a same concentration range of ganoderic acid DM was needed to inhibition of LNCaP cells and PC-3 cell line, suggesting non-selectivity toward prostate cancer cells.

We have already reported the biological activity of ganoderol B.22 Although the structures of ganoderol B and ganoderic acid DM are quite similar but still different, and both of them were isolated from the ethanol extract of G. lucidum, they showed different 5a-reduc- tase inhibitory activity and mechanism on proliferation of PC-3 cell line. Ganoderol B showed no inhibition on proliferation of PC-3 cell line, but ganoderic acid DM showed a dose-dependent manner on PC-3 proliferation. This result suggested that ganoderic acid DM suppressed cell proliferation somewhere in AR signaling pathway other than DHT formation at least in part. Hence, it seems that a little change in the 17ß-chain and C-3 affect the bioactivity of triterpe- noids in a large way.

To investigate whether ganoderic acid DM also affect the differentiation of osteoclasts, we utilized the preosteoclastic cell line RAW 264 cell to study the effect of ganoderic acid DM on the osteoclast-linage. Ganoderic acid DM clearly suppressed osteo- clastogenesis from the RAW 264 cell (Fig. 6). At the concentration of 12.5 iM, ganoderic acid DM inhibited the 40% RANKL-induced RAW 264 cell differentiation to osteoclasts, but not affected the cell number. At the concentration of 25 iM, 72% RAW 264 cells were survived, but no RANKL-induced RAW 264 cell differentiation to osteoclasts were observed. This result suggested that ganoderic acid DM selectively inhibits the osteoclasts forming, at the concen­tration without cytotoxicity. 1t is critical to understand the mechanisms by which ganoderic acid DM inhibits the proliferation of prostate cancer cell. For early prostate cancer, which is androgen-dependent, reducing the levels of circulation androgens to suppress AR/androgen signaling is an effective treatment choice. For example, finasteride, a 5a-reduc- tase inhibitor, which prevents conversion of testosterone to its more active form DHT in the prostate, is an effective drug for early prostate cancer. However, most prostate cancer patients eventually develop androgen-independent tumors that are resistant to this form of therapy. A significant body of evidence has suggested that in many androgen-independent prostate cancer cases, AR is highly expressed and hypersensitive to low, castrated-level of androgens or even can be activated by nonandrogens to induce tumor cell growth. Therefore, the strategy targeting the inactivation of AR
function is particularly attractive to treat hormone refractory prostate tumors. On the basis of these observations, we examined the 5a-reductase inhibitory activity and AR binding activity of ganoderic acid DM. We found that ganoderic acid DM strongly inhibited the 5a-reductase activity and bound to the AR, which suggested that AR signaling pathway was suppressed by this com­pound. Whether this compound inhibits cell proliferation is also a key question. As expected, ganoderic acid DM significantly inhib­ited LNCaP cell growth. Moreover, it also inhibited PC-3 cells proliferation. The fact that ganoderic acid DM inhibited the prolif­eration of both LNCaP and PC-3 cell suggests that this compound have the potential to serve as an effective therapy for androgen- dependent and androgen-independent prostate cancer.

The most common skeletal manifestation of malignancy is focal osteolysis in association with metastases. In order for tumor cells to grow and invade mineralized bone, osteolysis must occur. Osteoclasts appear uniquely adapted to produce the microenviron­ment and the biochemical milieu that are needed to resorb bone.23 The bulk of the evidence suggests that most tumor cells act indi­rectly by co-opting the physiologic mechanisms that normally favor bone resorption. Thus, they release agents such as hormones, eicosanoids, growth factors, and cytokines into the bone microen­vironment, which act on osteoblastic stromal cells to enhance the production of osteoclast activating factors.24,25 Most notable of these is the cell membrane-associated protein termed receptor activator of RANKL, which is a member of the TNF family of cyto­kines. RANKL can then bind to its cognate receptor (RANK) on osteoclast precursors and, enhance the differentiation and fusion of these cells to produce functioning multinucleated osteoclasts.26 Concomitantly, eliminating the forming of osteoclasts means the bone metastasis could be repressed. In this study, we found that ganoderic acid DM inhibited the osteoclasts differentiation.

Altogether our results illustrate the ability of a natural product- derived substance, ganoderic acid DM, to inhibit the proliferation of prostate cancer and osteoclasts differentiation. Thus makes gan- oderic acid DM can be used in therapeutics of prostate cancer by inhibiting the cancer cell proliferation and bone metastases by inhibiting the osteoclast differentiation.

A portion of the ethanol extracts (50 g) was fractionated into three fractions (Fr. A-C) by column chromatography eluting with an n-hexane-EtOAc step-gradient. A part of Fr. B (5 g) was fraction­ated by the preparative reversed phased HPLC and afforded ganod­eric acid DM (150 mg, yield, 0.3% in ethanol extracts of G. lucidum). Ganoderic acid DM was identified by comparing MS, NMR and optical rotation matched with published data (Fig. 1).27

Rat microsomes were prepared by the method previously reported by Liu et al.28

The 5a-reductase inhibitory activity was measured by a method previously reported by Liu et al.28

The ability of ganoderic acid DM to interact with the AR was evaluated using the utilizing a fluorescence polarization (FP) meth­od previously reported by Liu et al.29

Human LNCaP (AR positive, androgen-dependent prostate can­cer cell), PC-3 (AR negative, androgen-independent prostate cancer cell) were obtained from the American Type Culture Collection. The cells were grown in RPMI1640 containing 10% fetal bovine serum. All cell lines were grown at 37 °C in a humidified atmosphere with 5% CO2. The cells were used between passages 5-30 at a split ratio

of 1:3 in each passage. The cells were plated into a 24-well plate with a 1 x 105 cell/well density supplemented with 5% steroid-de­pleted (DCC-stripped) cFBS. Twenty-four hours later, the cells were treated with either vehicle control or androgens (T or DHT) in the presence or absence of each concentration of sample for another three days. Cell proliferation was determined by the 3-amino-7- dimethylamino-2-methyl-phenazine method.

The osteoclast precursor cell line, RAW 264 cell, was cultured in a-MEM containing 10% FBS (6.8 x 103 cells in 150 il/well in 96 well culture plates) for three days in the presence of sRANKL (30 ng/ml) and TNF-a (10 ng/ml) as described by Watanabe et al.30

Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2009.02.119.

References and notes

  • 1. Jemal, A.; Tiwari, R. C.; Murray, T.; Ghafoor, A.; Samuels, A.; Ward, E.; Feuer, E. J.; Thun, M. J. J. Clin. 2004, 54, 8.
  • 2. Niu, Y. J.; Ma, T. X.; Zhang, J.; Xu, Y.; Han, R. F.; Sun, G. Asin.J. Androl. 2003, 51, 19.
  • 3. DePrimo, S. E.; Diehn, M.; Nelson, J. B.; Reiter, R. E.; Matese, J.; Fero, M.; Tibshirani, R.; Brown, P. O.; Brooks, J. D. Genome Biol. 2002, 3. Research00321.
  • 4. Gelmann, E. P. J. Clin. Oncol. 2002, 20, 3001.
  • 5. Denmeade, S. R.; Lin, X. S.; Isaacs, J. T. Prostate 1996, 28, 251.
  • 6. Hofbauer, L. C.; Schoppet, M. JAMA 2004, 292, 490.
  • 7. Roodman, G. D. N. Engl. J. Med. 2004, 64, 9209.
  • 8. Capodice, J. L.; Katz, A. E. World J. Urol. 2006, 24, 378.
  • 9. Denis, L.; Morton, M. S.; Griffiths, K. Eur. Urol. 1999, 35, 377.
  • 10. McCann, M. J.; Gill, G. I.; McGlynn, H.; Rowland, I. R. Nutr. Cancer 2005, 52,1.
  • 11. Liu, J.; Tamura, S.; Kurashiki, K.; Shimizu, K.; Noda, K.; Konishi, F.; Kumamoto, S.; Kondo, R. Chem. biodivers., in press.
  • 12. Shimizu, K.; Miyamoto, I.; Liu, J.; Konishi, F.; Kumamoto, S.; Kondo, R. J. Wood. Sci., in press.
  • 13. Fujita, R.; Liu, J.; Shimizu, K.; Konishi, F.; Noda, K.; Kumamoto, S.; Ueda, C.; Tajili, H.; Kaneko, S.; Suimi, Y.; Kondo, R J. Ethnopharmacol. 2005, 102,107.
  • 14. Shiao, M. S. Chem. Rec. 2003, 3,172.
  • 15. Liu, J.; Kurashiki, K.; Shimizu, K.; Kondo, R Biol. Pharm. Bull. 2006, 29, 392.
  • 16. Zeelin, F. J. Adv. Drug Res. 1992, 22,149.
  • 17. Metcalf, B. W.; Levy, M. A.; Holt, D. A. Trends Pharmacol. Sci. 1989, 10, 491.
  • 18. McDonald, I. A.; Nyce, P. L.; Muench, D. M.; Gates, C. A.; Blohm, T. R.; Laughlin, M. E.; Werntraub, P. M. Bioorg. Med. Chem. Lett. 1994, 4, 847.
  • 19. Horoszewicz,J. S.; Leong, S. S.; Kawinski, E.; Karr,J. P.; Rosenthal, H.; Chu, T. M.; Mirand, E. A.; Murphy, G. P. Cancer Res. 1983, 43,1809.
  • 20. Negri-Cesi, P.; Motta, M. Biochem. Mol. Biol. 1994, 51, 89.
  • 21. Kaighn, M. E.; Narayan, S.; Ohnuki, Y.; Lechner, J. F.; Jones, L. W. Invest. Urol. 1979, 17, 16.
  • 22. Liu, J.; Shimizu, K.; Konishi, F.; Kumamoto, S.; Kondo. R. Bioorg. Med. Chem. 2007, 15, 4966.
  • 23. Eilon, G.; Mundy, G. R Nature 1978, 276, 726.
  • 24. Kong, Y. Y.; Boyle, W. J.; Penninger, J. M. Immunol. Today 2000, 21, 495.
  • 25. Lee, S. K.; Lorenzo, J. A. Endocrinology 1999, 140, 3552.
  • 26. Suda, T.; Takahashi, N.; Udagawa, N.; Jimi, E.; Gillespie, M. T.; Martin, T. J. Endocrinol. Rev. 1999, 20, 345.
  • 27. Wang, F.; Cai, H.; Yang,J. S.; Zhang, Y. M.; Hou, C. Y.; Liu,J. Q.; Zhao, M.J. Yaoxue Xuebao 1997, 32, 447.
  • 28. The assay was conducted with microsomes from rat livers as previously reported.15 Ganoderic acid DM was incubated with microsomes and NADPH for ten minutes, the percentage of inhibitory activity was calculated by the extent [4-14C] testosterone to [4-14C] DHT.
  • 29. The assay was conducted with androgen receptor ligand-binding domain as previously reported.22 Androgen receptor ligand-binding domain (25 nM, 20 ll) was incubated with each concentration of ganoderic acid DM or DHT for 4 h. The polarization was then measured on a beacon 2000 fluorescence polarization instrument using 485 nm excitation and 535 nm emission interference filters in polarization mode.
  • 30. Watanabe, T.; Kukita, T.; Kukita, A.; Wada, N.; Toh, K.; Nagata, K.; Nomiyama, H.; Iijima, T. J. Endocrinology 2004, 180,193.

 


[1] Corresponding author. E-mail address: ryukondo@agr.kyushu-u.ac.jp (R. Kondo).

0960-894X/$ - see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2009.02.119

 

Journal of Pharmacological Sciences ©2008 The Japanese Pharmacological Society

Short Communication

Ganoderic acid Me 'nin Matrix Meltalloproteinaz 2/9 gen expresyonunu düşürerek Tümör yayılmasını inhibe etme özelliği.)

Ganoderic Acid Me Inhibits Tumor Invasion Through Down-Regulating Matrix Metalloproteinases 2 /9 Gene Expression

Nian-Hong Chen1, Jian-Wen Liu1, and Jian-Jiang Zhong2[*]

'State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China

2Key Laboratory of Microbial Metabolism, Ministry of Education, College of Life Science & Biotechnology, Shanghai Jiao Tong University, 800 Dong-Chuan Road, Shanghai 200240, China

Received December 26, 2007; Accepted July 15, 2008

Abstract. The effect of ganoderic acid Me (GA-Me), which was purified from the fermentation mycelia of the traditional Chinese medicinal mushroom Ganoderma lucidum as reported (Tang W, Gu TY, Zhong JJ. Biochem Eng J. 2006;32:205-210), on anti-invasion was investigated. Wound healing assay indicated that GA-Me inhibited cell migration of 95-D, a human highly metastatic lung tumor cell line, in dose- and time-dependent manners. Results of cell aggregation and adhesion assays showed that GA-Me promoted cell homotypic aggregation and inhibited cell adherence to extracellular matrix (ECM). In addition, GA-Me suppressed matrix metallo­proteinases 2 / 9 (MMP2 / 9) gene expressions at both mRNA and protein levels in 95-D cells according to qRT-PCR and Western blotting, respectively. The results demonstrated that GA-Me effectively inhibited tumor invasion, and it might act as a new MMP2 /9 inhibitor for anti- metastasis treatment of carcinoma cells.

Ganoderma lucidum (Fr.) KARST (Polyporaceae), a traditional Chinese medicinal mushroom (1), has been used as a traditional medicine for the prevention and treatment of a variety of diseases, such as hepatitis, hepatopathy, hypertension, nephritis, bronchitis, HIV, platelet aggregation, immunological disorders, and cancers, in Asia for several thousand years (2). It is well documented that the extracts including polysaccharide or triterpene-enriched extract from G. lucidum inhibit cancer proliferation, induce cell cycle arrest, or apoptosis of human and mouse carcinoma cell lines (3, 4).

Interestingly, the anti-angiogenesis, anti-invasion, and anti-metastasis pharmacological functions of G. lucidum were recently found. The extracts of G. lucidum inhibited primary solid-tumor growth in the spleen, liver metastasis, and secondary metastatic tumor growth in the liver in intrasplenic Lewis lung carcinoma (LLC)- implanted mice, inhibited Martigel-induced angiogene- sis (5), induced actin polymerization in bladder cancer cells in vitro (6), inhibited tumor cell adhesion (7), inhibited induced oxidative stress-invasiveness of cancer cells through suppression of interleukin (IL)-8 secretion (8), and suppressed motility and invasiveness of highly invasive breast and prostate cancer cells (9).

It is well recognized that the development of metastasis is a major cause of death in many human cancers. Use of natural products is emerging as an alternative to tradi­tional medicines in treatment of cancer metastasis. For traditional herbal medicines, limited scientific evidence regarding their effectiveness and a lack of mechanistic understanding of their action have led to the fact that they are not being used in mainstream medicine in western countries. What is more, the treatment against metastasis is still far from satisfactory due to the lack of effective drugs. Obviously it is critical to find new effective drugs to fight against metastasis.

Until now, over one hundred ganoderma triterpenes have been identified, and several triterpenes were already purified from G. lucidumin relatively large quantitites (1), which makes bioassay experiments feasible. Ganoderiol F inhibited Martigel-induced angio- genesis (5). A pure lanostane triterpene, ganoderic acid

Me (GA-Me), purified from G. lucidum by our group

  • (I) , could increase the immune function by enhancing the expression of T-helper type 1 (Th1) cytokines (IL-2 and IFN-y), which led to the inhibition of tumor growth and lung metastasis (10). However, the direct anti- metastasis molecular mechanism of GA-Me was still unclear. Therefore, the present study was conducted to examine the anti-invasion /anti-metastasis mechanism of GA-Me, which is the same purified compound as reported by Tangetal. (1). Data are expressed as means ± S.E.M. One- or two-way ANOVA followed by Dunnett's multiple comparison tests were used for statistical analysis using OriginPro 8 (Origin Lab, Inc., Northampton, MD, USA). Our experimental results indicated that GA-Me suppressed invasion of human highly metastatic lung tumor 95-D cells through inhibiting MMP2 / 9 genes expression.

Tumor metastasis consists of numerous processes, including migration, homotypic and heterotypic cell-cell adhesion, cell-matrix interaction, invasion into surround­ing tissues, release from the primary tumor, intravasa- tion, adhesion to vascular walls, extravasation, and formation of new foci. Consequently, multiple factors are involved in the individual steps of metastasis. In normal tissues, cells are tightly associated with each other, so that they are generally not allowed to migrate freely. However, the malignant cells are more loosely associated and can freely detach from the primary tumor and migrate out. Our results indicated that GA-Me could facilitate the adhesion of cancer cells to each other, resulting in aggregate formation (Fig. 1A), which may contribute to prevention of the initial cell release. The cell aggregation assay was done as previously described

  • (II) . Briefly, a single-cell suspension was obtained through standard trypsinization procedures. A total of 2 x 105 95-D cells in 1ml of RPMI medium 1640 (serum-free) with different concentrations of GA-Me was placed in polystyrene microtubes and shaken gently every 5 min for 1 h at 37°C. Figure 1A shows that GA-Me promoted spontaneous cell-cell aggregation in a dose-dependent manner. Following the treatment with increasing concentrations of GA-Me, the cell aggregates grew larger. The percentage of aggregated cells was about 48.4 ± 7.6% when cells were treated with 20 ^m /ml of GA-Me for 1 h.

The wound healing assay was done as previously reported (11). For the wounding assays, confluent monolayers of 95-D cells were cultured in RPMI medium 1640 with 0.01% bovine serum albumin (BSA) in the absence or presence of different concentrations of GA-Me for 24 h after "wounding" of the cell layer with a pipette tip. The distance that the wounded edge of cells had moved was measured. By this assay, we observed that GA-Me effectively inhibited migration of 95-D cells. In Fig. 1B, the cellular motility of 95-D cells was obviously inhibited in a dose- and time-dependent manner by GA-Me. Compared to the control (untreated), GA-Me suppressed the migration capability of 95-D cells, and an increased suppression in cell migration was seen when 95-D cells were treated with a higher concentration of GA-Me (10-20 ¡g /ml, for 24 h). A clear dose-response effect was observed. What is more, at 6-h intervals, GA-Me (10 ¡g /ml) inhibited the cell motility effectively. This is consistent with data showing that G. lucidum extracts inhibited growth and induced actin polymerization in bladder cancer cells (6). In general, cell differentiation is associated with an increased F / G-actin ratio, whereas dedifferentiation and malignant transformation is associated with a decreased F /G-actin ratio (12). Our results imply that GA-Me may inhibit cell migration through inducing actin poly­merization.

The key step of invasion is attachment of cancer cells to extracellular matrix (ECM) components. It is recognized that adhesive interactions of tumor cells with ECM components may play a critical role in the establishment of metastasis. To evaluate the anti- metastatic activity of GA-Me, we assessed the inhibitory effect of GA-Me on the adhesion and cell aggregation of tumor cells. We examined the effects of GA-Me on cancer cell adhesion to ECM proteins (Fig. 1C). The cell adhesion assay was done as previously reported (11). GA-Me inhibited Matrigel®-mediated attachment of 95- D cells. In the cells treated with 10 and 20 ¡g/ml GA-Me for 18 h, the adhesion ratio with respect to the control was 56.8 ± 3.2% and 20.5 ± 6.9%, respectively. The inhibitory effect on cell adhesion might contribute to the inhibition of invasion by GA-Me. The inhibition of tumor adhesion by the water extracts (polysaccharide) of G. lucidum was reported (7), but until now it has been unknown whether the triterpene had the similar effect. The results of this work provided new information about the effects of G. lucidum metabolites on tumor cell adhesion.

In addition, the cell viability was analyzed by both trypan blue dye exclusion assay and flow cytometry. The results showed that the cell viability of 95-D cells at 0, 5, 10, and 20 ¡g/ml of GA-Me was 98.3 ± 2.9%, 96.4 ± 0.6%, 93.1 ± 2.3%, and 92.4 ± 1.8%, respectively (data not shown). Apoptosis of tumor cells was only 2.3% at 20 ¡g /ml of GA-Me for 24 h. These results can be compared to the reported data on curcumin: Exposure of CL1-5 cells, which have high invasive capacities, to curcumin (0, 1, 5, 10, 20 ¡M) influenced the cell viability of CL1-5 cells in a concentration-dependent manner (92.3 ± 6.1% at 10 ¡M and 70.1 ± 7.3% at gelatinases and considered as the major proteolytic enzymes in the degradation of ECM during cancer cell progression and invasion. Interestingly, MMP2 / 9 expression levels are especially high in lung carcinoma and melanoma cells. In 95-D cells, invasive potential has been related to the activity and expression of MMP2 /9 because they can degrade type IV collagen in the reconstituted basement membrane. To determine the effect of GA-Me on the MMP2 /9 protein expression level of 95-D cells, western blot analysis was carried out. It was observed that treatment of cells with GA-Me dose-dependently resulted in down-regulation of MMP2 / 9 protein expression. This suggests that GA-Me inhibited the invasion of tumor cells by efficiently inhibiting the MMP2/9 protein expression (Fig. 2). Interestingly, our data were different from a previous report, where MMP2 was unchanged in the cells treated with either ethanol extracts or water extracts of G. lucidum (at 80 ¡g/ml for 24 h) and the control cells were just treated with water or ethanol (6). The difference may be the result that different extract mixtures have different effects on the anti-metastasis, and different compound(s) may have different even opposite effects. It is unclear what compound(s) in extract mixtures is responsible for the anti-metastasis, which also makes the study of structure-activity relationship difficult. Therefore, the use of a pure triterpene is required to reveal the mechanism of action of the compound(s) responsible for the anti-metastatic effects and to further screen and rationally design structurally similar lead compounds.

It was demonstrated that G-Me suppressed the expression of MMP2 /9 and eventually inhibited cancer cell invasion. This conclusion is in agreement with those in previous studies that inhibitors of MMP2 / 9 signifi­cantly suppressed tumor metastasis in experimental animals (15). To our knowledge, our work is the first report showing that GA-Me effectively inhibited cancer cell invasion through inhibiting MMP2 / 9 gene expres­sion. This is helpful for an in-depth understanding of the anti-invasion /anti-metastasis molecular mechanisms of ganoderic acids.

The anti-metastasis effects of GA-Me were associated with the promotion of cell aggregation and the inhibition of cell invasion, which was evidenced by inhibition of cell adhesion and motility as well as suppression of MMP2 and MMP9 genes expression. The results highlight the potential of GA-Me in the treatment of cancer metastasis as an MMP2 /9 inhibitor. GA-Me could be a new promising candidate as an antimetastatic agent. The further elucidation of the molecular mecha­nisms responsible for the anti-invasive effects of GA-Me requires more studies in the future.

Acknowledgments

Financial support from the Shanghai Science & Technology Commission (project no. 054319933) and the Shanghai Leading Academic Discipline Project (project nos. B203 and B505) is gratefully acknowledged.

References

  • 1 Tang W, Gu TY, Zhong JJ. Separation of targeted ganoderic acids from Ganoderma lucidum by reversed phase liquid chromatography with ultraviolet and mass spectrometry detec­tions. Biochem Eng J. 2006;32:205-210.
  • 2 Russell R, Paterson M. Ganoderma - a therapeutic fungal biofactory. Phytochemistry. 2006;67:1985-2001.
  • 3 Hu HB, Ahn NS, Yang XL, Lee YS, Kang KS. Ganoderma lucidum extract induces cell cycle arrest and apoptosis in MCF-7 human breast cancer cell. Intl J Cancer. 2002;102:250-253.
  • 4 Lu HM, Kyo E, Uesaka T, Katoh O, Watanabe H. A water- soluble extract from cultured medium of Ganoderma lucidum (Rei-shi) mycelia suppresses azoxymethane-induction of colon cancers in male F344 rats. Oncol Rep. 2003;10:375-379.
  • 5 Kimura Y, Taniguchi M, Baba K. Antitumor and antimetastatic effects on liver of triterpenoid fractions of Ganoderma lucidum: mechanism of action and isolation of an active substance. Anticancer Res. 2002;22:3309-3318.
  • 6 Lu QY, Jin YS, Zhang Q, Zhang Z, Heber D, Go VL, et al. Ganoderma lucidum extracts inhibit growth and induce actin polymerization in bladder cancer cells in vitro. Cancer Lett. 2004;216:9-20.
  • 7 Wu QP, Xie YZ, Li SZ, La Pierre DP, Deng Z, Chen Q, et al. Tumor cell adhesion and integrin expression affected by Ganoderma lucidum. Enzyme Microb Tech. 2006;40:32-4126.
  • 8 Thyagarajan A, Jiang J, Hopf A, Adamec J, Sliva D. Inhibition of oxidative stress-induced invasiveness of cancer cells by Ganoderma lucidum is mediated through the suppression of interleukin-8 secretion. Intl J Mol Med. 2006;18:657-664.
  • 9 Sliva D, Labarrere C, Slivova V, Sedlak M, Lloyd FP Jr, Ho NW. Ganoderma lucidum suppresses motility of highly invasive breast and prostate cancer cells. Biochem Biophys Res Commun. 2002;298:603-612.
  • 10 Wang G, Zhao J, Liu JW, Huang YP, Zhong JJ, Tang W. Enhancement of IL-2 and IFN-gamma expression and NK cells activity involved in the anti-tumor effect of ganoderic acid Me in vivo. Int Immunopharmacol. 2007;7:864-870.
  • 11 Zhang J, Shen YL, Liu JW, Wei DZ. Antimetastatic effect of prodigiosin through inhibition of tumor invasion. Biochem Pharmacol. 2005;69:407-414.
  • 12 Rao JY, Hemstreet GP, Hurst RE, Bonner RB, Jones PL, Min KW, et al. Alterations in phenotypic biochemical markers in bladder epithelium during tumorigenesis, Proc Natl Acad Sci USA. 1993;90:8287-8291.
  • 13 Chen HW, Yu SL, Chen JJ, Li HN, Lin YC, Yao PL, et al. Anti- invasive gene expression profile of curcumin in lung adeno- carcinoma based on a high throughput microarray analysis. Mol Pharmacol. 2004;65:99-110.
  • 14 Elkin M, Reich R, Nagler A, Aingorn E, Pines M, Hochberg A, et al. Inhibition of matrix metalloproteinase-2 expression and bladder carcinoma metastasis by halofuginone. Clin Cancer Res. 1999;5:1982-1988.
  • 15 Hidalgo M, Eckhardt SG. Development of matrix metallo- proteinase inhibitors in cancer therapy. J Natl Cancer Inst. 2001;93:178-193.

 


[*]Corresponding author. jjzhong@sjtu.edu.cn Published online in J-STAGE doi: 10.1254/jphs.SC0080019

 

Biogerontology (2009) 10:627-636 DOI 10.1007/s10522-008-9208-9

RESEARCH ARTICLE

( G. lucidum    Kalb mitokonrial enzimlerinin ve solunum zinciri komplexlerinin aktivitesini arttırarak yaşlı farelerde olumlu etki göstermektedir.)

Ganoderma lucidum (Fr.) P. Karst enhances activities of heart mitochondrial enzymes and respiratory chain complexes in the aged rat

N. P. Sudheesh • T. A. Ajith • K. K. Janardhanan

Received: 7 November 2008/Accepted: 16 December 2008 / Published online: 4 January 2009 © Springer Science+Business Media B.V. 2009

Abstract Aging is associated with increased oxi­dative damage at multiple cellular levels, decline in cellular energy production and enhanced free radical status. The effect of the medicinal mushroom, Ganoderma lucidum on the activities of tricarboxylic acid (Krebs) cycle enzymes and mitochondrial com­plexes I-IV of the electron transport chain in aged rats were investigated. The activity of Krebs cycle enzymes, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase as well as mitochondrial complexes I, II, III, and IV were determined in heart of aged male Wistar rats orally administrated with 70% ethanolic extract (50 and 250 mg/kg) of G. lucidum. DL-a- lipoic acid (100 mg/kg) was taken as the positive control. Administration of the G. lucidum, once daily for 15 days, was significantly (P < 0.05) effective to enhance the Krebs cycle dehydrogenases, and mito- chondrial electron transport chain complex IV activities in aged rats. The profound activity of the extract can be correlated to the significant antioxi- dant property of G. lucidum. The results of the study

N. P. Sudheesh • K. K. Janardhanan (El) Department of Microbiology, Amala Cancer Research Centre, Amala Nagar, Thrissur, Kerala 680 555, India e-mail: kkjanardhanan@yahoo.com

T. A. Ajith

Department of Biochemistry, Amala Institute of Medical Sciences, Amala Nagar, Thrissur, Kerala 680 555, India

revealed that G. lucidum is effective to ameliorate the age associated decline of cellular energy status.

Keywords Antioxidant • Reactive oxygen species • Electron transport chain • Krebs cycle • Aging • Ganoderma lucidum

Introduction

Mitochondria are the major intracellular source and target sites of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in animals (Harman 1992; Salvioli et al. 2001; Sastre et al. 2003). The main mechanism responsible for mitochondrial ROS production are the respiratory chain, in particular its complexes I and III in the inner mitochondrial membrane, and mono- amine oxidase in the outer membrane (Beyer 1992; Cadenas and Davies 2000). Normally, ROS are decomposed or their peroxidation products are neu­tralized by natural defense systems mainly consisting of mitochondrial (manganese-containing) superoxide dismutase and glutathione peroxidase (Neubert et al. 1962; Fridovich 1974; Augustin et al. 1997). How­ever, under conditions of increased free radical generation mainly in ischemia-reperfusion, action of some xenobiotics, inflammation, aging and ultraviolet or ionizing irradiation, or conditions of impaired antioxidant defense system, the free radicals may accumulate, leading to a potent damaging effect on the cell and the whole organism (Halliwell and Gutteridge 1990; Mizuno et al. 1998; Schapira 1999; Cadenas and Davies 2000). Thus, oxidative damage is promoted by the attack of free radicals on membrane polyunsaturated fatty acids, proteins and DNA (Halliwell and Chirico 1993; Ritcher et al. 1998). The mitochondrial 'vicious cycle' theory proposes that chronic ROS generation and increases in oxidative stress can be damaging to mtDNA. Much of this damage can be mutagenic giving rise to mtDNA mutations that may accumulate progressively during life which exponentially increases oxidative damage and dysfunction and ultimately culminates in cell death (Hiona and Leeuwenburgh 2008).

Experimental data also supports the decline of mitochondrial function in human aging and degener­ative diseases (Ozawa 1997; Wei 1998). Supplemen­tation with dietary antioxidants has been one of the approaches attempted to test the free radical theory of aging, and, at least, to try to reduce the impact of age related dysfunctions (Beckman and Ames 1998; Miquel 2002). Preservation of mitochondrial function is important for maintaining overall health during aging. In order to protect the mitochondria against respiration-linked oxidative stress, and to preserve the genomic and structural integrity of these energy- producing organelles dietary supplementation with antioxidants, such as vitamins, N-acetyl cysteine, DL-a-lipoic acid and L-carnitine has been suggested (Chow 1991; Arivazhagan et al. 2001; Haripriya et al. 2004). The heart is one of the organs, which highly depends on oxidative energy generated in mitochon­dria, by fatty acid b-oxidation, respiratory electron chain and oxidative phosphorylation (Cadenas and Davies 2000; Marin-Garcia and Goldenthal 2002; Arai et al. 2003). Age-dependent declines of most of the heart mitochondrial enzymes and membrane functions have been reported (Kumaran et al. 2004; Savitha et al. 2005; Savitha and Pannerselvam 2006). Therefore, it seems very likely that age-related changes at the heart mitochondria are important in the decline of physiological function that accompa­nies senescence.

Ganoderma lucidum (Fr.) P. Karst-known as ''Lingzhi'' in Chinese, ''Reishi'' in Japanese, and ''Youngzhi'' in Korean has been traditionally used as a popular folk medicine for the promotion of health in the Orient. As early as 100 BC., Lingzhi was cited in the Shen Nong's Herbal Classic (widely considered as the oldest book on oriental herbal medicine and the foundation of traditional Chinese medicine) for enhancing ''vital energy'' and promoting ''longevity''. This ''mushroom of longevity'' has been deemed as the most exalted medicine in ancient China (Lin et al. 2003). During the past two decades, modern research has revealed that G. lucidum contains a variety of chemical ingredients, including triterpenes, polysac- charides, nucleosides, steroids, fatty acids, alkaloids, proteins, peptides, amino acids, and inorganic ele­ments (Paterson 2006). Among these ingredients, triterpenes and polysaccharides are the major compo­nents and they have attracted considerable attention because of their potentially significant pharmacolog­ical activities. G. lucidum has been used as a remedy for a wide range of ailments and chronic diseases, such as migraine, hypertension, arthritis, bronchitis, asthma, anorexia, gastritis, haemorrhoids, diabetes, hypercholesterolaemia, nephritis, dysmenorrhoea, constipation, lupus erythematosis, hepatitis, and car­diovascular problems (Jong and Birmingham 1992; Shiao et al. 1994). Previous investigations in our laboratory showed that G. lucidum occurring in South India possessed significant antioxidant, antitumor, anti-inflammatory, antinociceptive, nephroprotective and radioprotective properties (Jones and Janardhanan 2000; Sheena et al. 2003a, b; Lakshmi et al. 2003; Pillai et al. 2008). G. lucidum is also reported to possess significant antioxidant and DNA protective effects (Kim and Kim 1999; Lee et al. 2001). However, no reports are available on the effect of G. lucidum on the Krebs cycle enzymes and electron transport chain complexes on the heart mitochondria of aged animals. Hence, we examined the effect of G. lucidum on the activities of Krebs cycle dehydrogen­ases and respiratory chain complexes I-IV in the heart mitochondria of aged rats.

Materials and methods

Chemicals

Rotenone, antimycin-A, 2,6-diclorophenol indophenol sodium salt (DCIP), decyl ubiquinol, coenzyme A, trisodium isocitrate, nicotinamide adenine dinucleotide (NAD), thiamine pyrophosphate, sodium dithio- nate, a-ketoglutarate, bovine serum albumin (BSA), potassium cyanide, nicotinamide adenine dinucleotide reduced (NADH), oxalo acetate and cytochrome C were purchased from Sigma Chemical Company, Saint Louis, MO, USA. DL-a-Lipoic acid was a gift from Garnett McKeen Laboratories Inc., NY, USA and all other chemicals used were of analytical grade.

Preparation of the extract

Fruiting bodies of G. lucidum growing on the Caesalpinia coriaria wild trees in the local area were collected. The type specimen was deposited in the herbarium of Centre for Advanced Studies in Botany, University of Madras, Chennai, India (Herbarium of Madras University Botany Laboratory number HERB. MUBL-3175). The fruiting bodies of the mushroom were cut into small pieces, dried at 40- 50°C for 48 h and powdered. A total of 100 g samples of the powdered materials were extracted with 70% ethanol on a boiling water bath for 48 h. The supernatant was collected and evaporated at low temperature under vacuum using rotary evaporator and finally lyophilized. The yield of the extraction was 5% (w/w). The extract was suspended in distilled water and employed for the experiments.

Animals

Male albino rats of Wistar strain weighing approxi­mately 350 ± 50 g (age more than 24 months) from Small Animal Breeding Centre, Kerala Agricultural University, Mannuthy, Thrissur, Kerala, India were used in the experiment. The animals were maintained under standardized environmental conditions (22-28°C, 60-70% relative humidity, 12 h dark/light cycle) with free access to standard food (Sai Durga Feeds and Foods, Bangalore, India) and water ad libitum. The animals were housed and the experiment was carried out according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) Government of India and by approval of Institutional Animal Ethics Committee.

Effect of Ganoderma lucidum on the mitochondrial dehydrogenases and respiratory complexes in the heart of aged rats

Animals were divided into four groups with six rats in each group. Group 1: control (distilled water), Group 2: DL-a-Lipoic acid (100 mg/kg body weight) and Group 3 and 4: G. lucidum extract (50 and 250 mg/kg body weight). The DL-a-lipoic acid was dissolved in alkaline saline (0.5% NaOH, w/v). The DL-a-lipoic acid, G. lucidum extract and distilled water were administrated orally once daily for 15 days. On completion of 15 days of drug administration, the animals were sacrificed by cervical decapitation. The heart was surgically excised out and kept at - 70°C for further study.

Preparation of mitochondrial fraction

About 10% of the heart tissue homogenate of rats was prepared in 50 mmol/l phosphate buffer (pH 7.0) containing 0.25 mol/l (w/v) sucrose. Homogenate was centrifuged initially at 3,000g for 10 min and the supernatant was subjected to 11,000g for 10 min at 4°C in a cooling centrifuge. The mitochondrial pellets were washed twice with phosphate buffer to remove the sucrose and suspended in phosphate buffer. Mitochondrial fraction was frozen and thawed 3-5 times to release the enzymes (except complex IV which was extracted with 0.5% Tween 80 in phos­phate buffer, v/v). The protein was estimated in the supernatant using the method of Bradford (1976).

Determination of the effect of G. lucidum on the activity of TCA enzymes

Isocitrate dehydrogenase (ICDH) activity was esti­mated according to the method of Fatania et al. (1993) and the activity was expressed as micromoles of NAD? reduced/min/mg protein using extinction coefficient 6.3 mM-1 cm-1. a-Ketoglutarate dehy- drogenase activity (a-KGDH) activity was estimated by the method of Reed and Mukherjee (1969). The activity was expressed as imoles of NAD? (reduced/ min/mg protein) using extinction coefficient 6.3 mM-1 cm- . Succinate dehydrogenase (SDH) activity was estimated by the method of Nulton-Persson and Szweda (2001) with slight modifications. The activity of SDH was calculated using the extinction coefficient of 2,6- diclorophenol indophenol (DCIP) (19.1 mM-1 cm-') and expressed as imoles of DCIP reduced/min/mg protein. Malate dehydrogenase (MDH) activity was estimated by the method of Mehler et al. (1948) and the activity was expressed as imoles of NADH oxidized/ min/mg protein using the extinction coefficient of NADH 6.3 mM-1 cm-1.

Determination of the effect of G. lucidum on the activity of respiratory complexes

Complex I activity was estimated by the method of Janssen et al. (2007). Briefly 1 imol/l antimycin A, 3 mg BSA, 2 mmol/l KCN, 5 mmol/l MgCl2, 65 imol/l ubiquinone, 80 imol/l DCIP and mito­chondrial protein (approximately 40 ig) were mixed with phosphate buffer (25 mmol pH 7.2) in a net volume of 1 ml. Absorbance at 600 nm was moni­tored at 15 s interval for 2 min at room temperature after the addition of NADH (0.2 mmol/l). After 2 min

  • 1 imol/l rotenone was added and the absorbance was measured again for 2 min with an interval of 30 s. The linear difference in absorbance was calculated before and after the addition of rotenone. The activity was expressed as imoles of DCIP reduced/min/mg protein (extinction coefficient of DCIP is 19.1 mM- cm- ).

Determination of complex II

Complex II activity was estimated by the method of Janssen et al. (2007). A volume of 1 ml of the reaction mixture contained 3 mg/ml BSA, 2 mmol/l EDTA,

  • 2 mmol/l KCN, 1 imol/l antimycin A, 1 imol/l rote­none, 20 mmol/l sodium succinate, 65 imol/l decyl ubiquinone, mitochondrial protein (approximately 40 ig) and 50 mmol/l phosphate buffer (pH 7.2). The reaction was initiated with the addition of 60 imol/l DCIP and was monitored spectrophotometrically at 600 nm for 4 min with 15 s interval. The activity was expressed as imoles of DCIP reduced/min/mg protein (extinction coefficient of DCIP is 19.1 mM-1 cm-1).

Determination of complex III

Decyl ubiquinol preparation: 1.3 mmol/l decylubiq- uinone was mixed with a few grains of (approximately half volume) of sodium dithionate and vortexed vigorously. The transparent solution was centrifuged at 12,000g for 10 min. The supernatant containing decylubiquinol was used for the assay.

Complex III activity was estimated by the method of Krahenbuhl et al. (1991). Mitochondrial protein (approximately 20 ig) was mixed with 100 imol/l EDTA, 2 mg BSA, 3 mmol/l, sodium azide, 60 imol/ l ferricytochrome-C, decylubiquinol and phosphate buffer (50 mmol/l, pH 8) in a final volume of 1 ml. The reaction was started by the addition of decyl- ubiquinol and monitored for 2 min at 550 nm and again after the addition of 1 imol/l of antimycin A. The activity was calculated from the linear part of absorption-time curve, which was not less than 30 s. The extinction coefficient of ferricytochrome C (21 mM-1 cm-1) was used for the calculation. Activ­ity of complex III was expressed as imoles of ferricytochrome-C reduced/min/mg protein.

Determination of complex IV

Preparation of ferrocytochrome C solution: 600 imol/l ferricytochrome-C was stirred with a few grains of sodium dithionate in 30 mmol/l phos­phate buffer (pH 7.4) for 10-20 min in the dark. The solution was centrifuged at 12,000g for 10 min. The transparent supernatant containing ferrocytochrome C was used for the complex IV assay.

Complex IV activity was determined by the method of Capaldi et al. (1995). Briefly 1 ml of ferrocytochrome C solution was mixed with approx­imately 10 ig of mitochondrial protein (extract in 0.5% Tween 80 in 30 mmol/l phosphate buffer, pH 7.4) and phosphate buffer in a net volume of 1.3 ml. The reaction was started by the addition of enzyme source and was monitored at 550 nm with an interval of 15 s for 4 min. The difference in absor­bance was calculated from the linear part of the absorption-time curve. Complex (IV) activity was expressed as imoles of ferrocytochrome-C oxidized/ min/mg protein using the extinction coefficient 21 mM-1 cm-1.

Statistical analysis

All data were represented as mean ± SD. Data were statistically analyzed using one-way analysis of variance (ANOVA) (using the Graph Pad Instat software package). The significant difference between the control group, lipoic acid and G. lucidum admin­istered groups were further analyzed by Bonferroni's t-test. P values less than 0.05 were considered as significant.

Results

The effects of administration of G. lucidum and DL-a-lipoic acid on the activities of TCA enzymes in the heart of aged rats are presented in Table 1. It is clearly evident that the administration of G. lucidum (50 and 250 mg/kg) significantly increased the activities of ICDH, a-KGDH, SDH and MDH compared to the control animals. Similarly a positive control of DL-a-lipoic acid (100 mg/kg) increased the activities of TCA enzymes. Among the two doses of G. lucidum studied, 250 mg/kg was more effective in enhancing the TCA enzymes. However, the values are statistically non significant with respect to 50 mg/ kg treated group. In the case of ICDH, G. lucidum (250 mg/kg) treated rats showed significant (P < 0.001) enhancement in the activity, it was around 1.87-fold greater than that of the control. The increase in the activities of a-KGDH, MDH and SDH in the G. lucidum (250 mg/kg) group were approximately 3.69-, 6.54- and 3.07-fold, respec­tively, than that of the aged control rats. Similarly G. lucidum at 50 mg/kg enhanced the activities of

ICDH (1.47-fold), MDH (5.31-fold) and SDH (3.56­fold) than that of the control group. However, the a- KGDH activity in the G. lucidum (50 mg/kg) treated group was non-significantly (P > 0.05) differ from control group. Similarly ICDH, a-KGDH and MDH activities in the DL-a-lipoic acid (100 mg/kg) treated group were enhanced significantly (P < 0.001) than that of the control group except for the activity of SDH which was non significantly altered (P > 0.05). The increase in activities for ICDH, a-KGDH and MDH in the DL-a-lipoic acid treated group was around 2.95-, 2.52- and 5.21-fold with respect to that of the control group.

The effect of G. lucidum and DL-a-lipoic acid on the respiratory complex I, II, III and IV in aged rats is presented in Table 2. Aging also affected the activ­ities of respiratory complexes in the heart of old rats. In the G. lucidum treated groups, the activity of complexes were non-significantly altered (P > 0.05) except for complex IV which was significantly (P < 0.001) enhanced. In the G. lucidum 250 mg/ kg treated group, complex I, II, and IV activities were enhanced around 1.79-, 1.78- and 1.63-fold,

 

Table 1 Effect of ethanolic extract of Ganoderma lucidum on the TCA enzymes

Groups

ICDH

a-KGDH

SDH

MDH

Control

700.85 ± 183.04

42.51 ± 12.01

34.90 ± 10.55

262.95 ± 35.15

DL-a-Lipoic acid (100 mg/kg)

2063.49 ± 105.21***

151.17 ± 34.21***

62.42 ± 25.00ns

1365.12 ± 203.81***

G. lucidum (50 mg/kg)

1030.00 ± 98.04*

88.00 ± 12.50ns

124.16 ± 17.20***

1397.98 ± 443.11***

G. lucidum (250 mg/kg)

1312.00 ± 250.55***

157.07 ± 45.01***

130.69 ± 53.73***

1720.11 ± 166.22***

Values are the mean ± SD, n = 6, *** P < 0.001, * P < 0.05, and ns P > 0.05 (Bonferroni test) with respect to aged control

Units: Isocitrate dehydrogenase (ICDH)-imoles of NAD? reduced/min/mg protein; a-ketoglutarate dehydrogenase (a-KGDH)- imoles of NAD? reduced/min/mg protein; succinate dehydrogenase (SDH)-imoles of DCIP reduced/min/mg protein; malate dehydrogenase (MDH)-imoles of NADH oxidized/min/mg protein

 

Table 2 Effect of ethanolic extract of Ganoderma lucidum

on the respiratory complexes

 

 

Groups

Complex I

Complex II

Complex III

Complex IV

Control

DL-a-Lipoic acid (100 mg/kg) G. lucidum (50 mg/kg) G. lucidum (250 mg/kg)

21.34 ± 4.12 29.91 ± 9.87ns 37.39 ± 12.54ns 38.22 ± 11.75ns

25.75 ± 3.09 71.85 ± 29.18*** 25.62 ± 4.71ns 45.84 ± 14.96ns

14.57 ± 4.28 18.51 ± 6.61ns 8.92 ± 3.58ns 13.55 ± 2.74ns

29.74 ± 11.93 50.02 ± 2.86*** 42.17 ± 2.11* 48.51 ± 6.67***

Values are the mean ± SD, n = 6, *** P < 0.001, * P < 0.05, and ns P > 0.05 (Bonferroni test) with respect to aged control

Units: Complex I-imoles of DCIP reduced/min/mg protein; Complex II-imoles of DCIP reduced/min/mg protein; Complex III- imoles of ferricytochrome C reduced/min/mg protein; Complex IV-imoles of ferrocytochrome C oxidized/min/mg protein

 respectively, than that of the aged control group. In the case of G. lucidum (50 mg/kg) the enhancement in the activities of complex I and IV were approx­imately 1.75 and 1.41, respectively, whereas the complex II and III were non significantly altered. Similarly the DL-a-lipoic acid (100 mg/kg) treatment enhanced the complex activities. The increase was statistically significant (P < 0.001) in the case of complex II and IV whereas it was non-significant (P > 0.05) in complex I and III. The increase in activity was around 1.12-, 2.79-, 1.05- and 1.68-fold for complex I, II, III and IV, respectively, in the case of DL-a-lipoic acid. The effect of the treatment on the body weight of the animals before and after the treatment was determined. There was no significant (P > 0.05) variation in the body weight in the control, DL-a-lipoic acid and G. lucidum (50 and 250 mg/kg) treated groups before and after the experiment (Fig. 1).

The treatment with G. lucidum at 50 and 250 mg/ kg showed an overall enhanced activities of TCA enzymes and respiratory complexes in the heart of aged rats.

Discussion

Results of the study revealed that administration of G. lucidum significantly enhanced the activities of mitochondrial TCA cycle dehydrogenases and ETC.

complexes. Aging affects all types of nucleated cells. However, the degree of age-related changes in cardiac myocytes was found to be high (Coleman et al. 1987). The heart is dependent on molecular oxygen and oxidative phosphorylation to provide high-energy compounds necessary for contraction, but this exposes the myocardium to harmful ROS that are generated continuously as normal by-products of the mitochondrial ETC. The aging heart undergoes significant functional and structural alterations lead­ing to atrophy and a compensatory hypertrophy, followed by myocardial fibrosis (Muscari et al. 1996). In addition, there is an age-related decline in the capacity to withstand stress like ischemia or reper­fusion (Lesnefsky et al. 2001). In its most severe form, cardiac decay results in congestive heart failure during old age. Loss of mitochondrial function and an increase in oxidative stress has been proposed to be one of the key factors in myocardial aging (Hagen et al. 2001). The dose and selection of DL-a-lipoic acid as a reference standard was based on a recent study that reported the improved mitochondrial- supported bioenergetics and the antioxidant activity of DL-a-lipoic acid (100 mg/kg orally) when treated once daily for 28 days (Savitha and Pannerselvam 2006).

Experimental studies demonstrated a significant decrease in activities of the TCA cycle enzymes (Fannin et al. 1999; Kumaran et al. 2004; Savitha et al. 2005; Savitha and Pannerselvam 2006). This
reduction in the activities of specific dehydrogenases might be because of increased ROS production leading to inefficient electron transport resulting in oxidative damage to the mitochondria thereby com­promising their ability to meet cellular energy demands. The inhibition of these enzymes may affect mitochondrial substrate oxidation resulting in reduced rate of transfer of reducing equivalents to molecular oxygen and depletion of energy produc­tion. Several studies suggest that oxidative damage to mitochondrial DNA may be responsible for decrease in activities of ETC. enzyme complexes in aged rats (Filburn et al. 1996) and these changes are more prevalent in post mitotic cells such as central nervous system, heart and skeletal muscle (Hsieh et al. 1994). Progressive increase in the production of free radicals with aging has been correlated with decrease in the number of functional mitochondria per cell, as well as a decline in the production of ATP, protein synthesis and increase in peroxide leakage (Zhang et al. 1990). The reduced activity of a-KGDH could be because of the inefficient electron transport and increased super­oxide production resulting in oxidative damage to mitochondria thus compromising their ability to meet cellular energy demands (Sohal and Weindruch 1996). The accumulation of a-ketoglutarate resulted in the lowering of activities of ICDH (Zhang et al. 1990). The mitochondrial oxidative phosphorylation operates at a lower level in aged rats despite the higher energy demand during aging (Sastre et al. 1996). NADH/NAD ratio has been reported to rise in aged mitochondria due to prolonged metabolic over­load on cells (Zarchin et al. 2002). This in turn may result in diminution in the activities of TCA enzymes by the mechanism of mass action, as observed in the present study. Supplementation of DL-a-lipoic acid increases the activities of these enzymes and this may be attributed to the enhancement of reduced gluta- thione activity in aged rats (Arivazhagan et al. 2001).

G. lucidum at 250 mg/kg was effective to enhance the activities all the complexes except complex III. However, a significant elevation of activity was observed in the complex IV. No subunits of the enzymes of complex II are synthesized by mtDNA (Wallace 1992). Drouet et al. (1999) have suggested that decline in complex II activity with aging could be secondary to a decline in the levels of active enzyme molecules per mitochondrion, or due to accumulation of altered molecules in the organelle.

NADH-ubiquinone oxidoreductase, also known as Complex I, is a multisubunit integral membrane complex of the mitochondrial ETC. which catalyzes electron transfer from NADH to ubiquinone. The vicious cycle theory proposes that progressive accu­mulations of ROS-induced somatic mutations in mtDNA may result in dysfunctions of respiratory chain, leading to increased production of ROS and subsequent accumulation of more mtDNA mutations as one of the reasons for increase in oxidative damage during aging (Linnane et al. 1989; Wei 1992). Complex I is generally reported to be impaired with age in cardiac mitochondria (Castelluccio et al. 1994). According to the mitochondrial theory of aging, the highest frequency of mutations would affect Complex I and secondarily Complex IV, for which respectively seven and three mitochondrial genes are present (Miquel et al. 1980). Thus, mutations may be one of the reasons for decline in the complex I activity during aging. There may be other possible reasons for Complex I defects in aging, such as direct alterations of the proteins (Paradies et al. 1993).

Enhancement in the activity of ICDH, a-KGDH, SDH and MDH was found in the case of G. lucidum (250 mg/kg) treated group which can be attributed to the in vivo antioxidant activity of G. lucidum extract in the heart of aged rats. Our recent study with G. lucidum extract at 50 and 250 mg/kg showed enhanced activities of Glutathione peroxidase (GPx), Glutathione-S-transferase (GST), Catalase (CAT), Superoxide dismutase (SOD) and levels of Reduced glutathione (GSH) and decline of Malonedialdehyde (MDA) level in the heart mitochondria of old aged balb/c mice (unpublished data). Enhanced GSH content in the aged rats by G. lucidum might be correlated to the enhanced TCA enzymes activities as reported earlier (Savitha and Pannerselvam 2006). In the case of G. lucidum (50 and 250 mg/kg) treated groups, the activity of complexes were non-signifi- cantly enhanced (P > 0.05) except for complex IV. But there was an overall increase in the activity of all the complexes in the mean value than that of the old control. This could be attributed to the improvement in the activities of TCA cycle enzymes, which would provide the substrates for the ETC. Additionally G. lucidum could have increased the levels of NADH by increasing the levels of GSH similar to DL-a- lipoic acid as reported earlier (Kumaran et al. 2004).

G. lucidum had been reported to remove the hydroxyl radical, the main factor in the human ageing process (Liu et al. 1997). Preliminary phyto- chemical analysis of the extract found that protein- polysaccharides are the major component present in the aqueous-alcoholic extract (Sheena et al. 2003a). Recent investigation on the bioactivities of polysaccharides showed that water soluble polysac- charide extracted from the fruiting body of G. lucidum was effective in preventing DNA strand breakage induced by hydroxyl radical and UV irradiation (Kim and Kim 1999). Ganoderma poly- saccharide peptide (GLPP) found to possess anti- oxidant activity by scavenging ROS in mice (You and Lin 2003). Recent studies had shown that G. lucidum have capability to improve the learning and memory ability in senescence accelerated mice by increasing the superoxide dismutase, glutathione peroxidase and glutathione reductase in brain and liver (Wang et al. 2004). G. lucidum also demon­strated to be effective against the Alzheimers disease (AD) by attenuating the b-amyloid (Ab) which is the main reason behind AD pathology (Lai et al. 2008). Dai et al. (1987) demonstrated the mitochondrial superoxide dismutase stimulating activity of Ganoderma. Recent reports also indicated the presence of vitamins C and E, as well as b- carotene and essential elements in various Gano­derma species (Huie and Di 2004). Pharmacological treatment using these antioxidant vitamins shown effectiveness in preventing oxidative stress and mitochondrial function during aging in individual cases (Ghosh et al. 1996; Chinnery and Turnbull 2000; Marriage et al. 2004; Navarro et al. 2005). However, there are no reports on the activities of the individual chemical constituents of this mushroom. The presence of these constituents might be respon­sible for the exhibited activity. Therefore, G. lucidum may have beneficial effects on age-associ­ated diseases by increasing the cellular energy status.

In conclusion, results of the study indicate that treatment with ethanolic extract of G. lucidum is capable to protect the decline in the activity of TCA enzymes and respiratory complexes during aging which can be ascribed with the free radical scaveng­ing activity of G. lucidum. However, the exact mechanism is unknown and needs to be further evaluated.

References

Arai T, Nakahara K, Matsuoka H, Sawabe M, Chida K, Matsushita S, Takubo K, Honma N, Nakamura K, Iz- umiyama N, Esaki Y (2003) Age-related mitochondrial DNA deletion in human heart: its relationship with car­diovascular diseases. Aging Clin Exp Res 15:1-5 Arivazhagan P, Ramanathan K, Panneerselvam C (2001) Effect of DL-alpha-lipoic acid on mitochondrial enzymes in aged rats. Chem Biol Interact 138:189-198. doi:10.1016/ S0009-2797(01)00268-X Augustin W, Wiswedel I, Noack H, Reinheckel T, Reichelt O (1997) Role of endogenous and exogenous antioxidants in the defence against functional damage and lipid peroxi- dation in rat liver mitochondria. Mol Cell Biochem 174:199-205. doi:10.1023/A:1006804423627 Beckman KB, Ames BN (1998) The free radical theory of

aging matures. Physiol Rev 78:547-581 Beyer RE (1992) An analysis of the role of coenzyme Q in free radical generation and as an anti oxidant. Biochem Cell Biol 70:390-403 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254. doi:10.1016/0003-2697(76)90527-3 Cadenas E, Davies KJ (2000) Mitochondrial free radical gen­eration, oxidative stress, and aging. Free Radic Biol Med 29:222-230. doi:10.1016/S0891-5849(00)00317-8 Capaldi RA, Marusich MF, Taanman JW (1995) Mammalian cytochrome-c oxidase: characterization of enzyme and immunological detection of subunits in tissue extracts and whole cells. Methods Enzymol 260:117-132. doi:10. 1016/0076-6879(95)60134-1 Castelluccio C, Baracca A, Fato R et al (1994) Mitochondrial activities of rat heart during aging. Mech Ageing Dev 79:73-88. doi:10.1016/0047-6374(94)91583-0 Chinnery PF, Turnbull DM (2000) Mitochondrial DNA mutations in the pathogenesis of human disease. Mol Med 6:425-432. doi: 10.1007/s0089400060425 Chow CK (1991) Vitamin E and oxidative stress. Free Radic Biol Med 11:215-232. doi:10.1016/0891-5849(91) 90174-2

Coleman R, Silbermann M, Gershon D, Reznick AZ (1987) Giant mitochondria in the myocardium of aging and endurance-trained mice. Gerontology 33:34-39 Dai YR, Gao CM, Tian QL, Yin Y (1987) Effect of extracts of some medicinal plants on superoxide dismutase activity in mice. Planta Med 53:309-310. doi:10.1055/s-2006-96 2723

Drouet M, Lauthier F, Charmes JP, Sauvage P, Ratin MH (1999) Age associated changes in mitochondrial parame­ters on peripheral human lymphocytes. Exp Gerontol 34:69-78. doi:10.1016/S0531-5565(99)00058-3 Fannin SW, Lesnefsky EJ, Slabe TJ, Hassan MO, Hoppel CL (1999) Aging selectively decreases oxidative capacity in rat heart interfibrillary mitochondria. Arch Biochem Biophys 372:399-407. doi:10.1006/abbi.1999.1508 Fatania H, Al-Nassar EK, Sidhan V (1993) Purification and partial characterization NADP'-linked isocitrate dehy­drogenase from rat liver cytosol. FEBS Lett 320:57-60

Filburn CR, Edris W, Tamatani M, Hogue B, Kudryashova I, Hansford RD (1996) Mitochondrial electron transport chain activities and DNA deletions in regions of rat brain. Mech Ageing Dev 87:35-46. doi: 10.1016/0047-6374(96)01696-X Fridovich I (1974) Superoxide dismutase. Annu Rev Biochem

44:147-159. doi:10.1146/annurev.bi.44.070175.001051 Ghosh MK, Chattopadhyay DJ, Chatterjee IB (1996) Vitamin C prevents oxidative damage. Free Radic Res 25:173­179. doi: 10.3109/10715769609149922 Hagen TM, Moreau R, Suh JH, Violi F (2001) Mitochondrial decay in the aging rat heart; evidence for improvement by dietary supplementation with acetyl-L-carnitine and/or lipoic acid. FASEB J 15:700-706. doi:10.1096/fj.00-0176com Halliwell B, Chirico S (1993) Lipid peroxidation: its mecha­nism measurement and significance. Am J Clin Nutr 57:715-725

Halliwell B, Gutteridge JMC (1990) Role of free radicals and catalytic metal ions in human diseases: an overview. Methods Enzymol 186:1-85. doi:10.1016/0076-6879(90) 86093-B

Haripriya D, Devi AM, Kokilavani V, Sangeetha P, Pannersel- vam C (2004) Age-dependant alterations in mitochondrial enzymes in cortex, striatum and hippocampus of rat brain- potential role of L-Carnitine. Biogerentology 5:355-361. doi:10.1007/s10522-004-2575-y Harman D (1992) Free radical theory of aging. Mutat Res 275:257-266

Hiona A, Leeuwenburgh C (2008) The role of mitochondrial DNA mutations in aging and sarcopenia: implications for the mitochondrial vicious cycle theory of aging. Exp Gerontol 43:24-33. doi:10.1016/j.exger.2007.10.001 Hsieh RH, Hou JH, Hsu HS, Wei YH (1994) Age dependent respiratory function and mitochondrial DNA deletion in human skeletal muscle mitochondria. Biochem Mol Biol Int 32:1009-1022 Huie CW, Di X (2004) Chromatographic and electrophoretic methods for Lingzhi pharmacologically active compo­nents. J Chromatogr B Anal Technol Biomed Life Sci 812:241-257

Janssen MJA, Trijbels MJ, Sengers ACR, Smeitink MJA, van den Heuvel PL, Wintjes MTL, Stoltenborg-Hogenkamp MJB, Rodenburg TJR (2007) Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts. Clin Chem 53:729-731. doi: 10.1373/clinchem.2006.078873 Jones S, Janardhanan KK (2000) Antioxidant and antitumor activity of Ganoderma lucidum (Curt: Fr.) P. Karst- Reishi (Aphyllophoromycetideae) from South India. Int J Med Mushroom 2:195-200 Jong SC, Birmingham JM (1992) Medicinal benefits of the mushroom Ganoderma. Adv Appl Microbiol 37:101. doi: 10.1016/S0065-2164(08)70253-3 Kim KC, Kim IG (1999) Ganoderma lucidum extract protects DNA from strand breakage caused by hydroxyl radical and UV irradiation. Int J Mol Med 4:273-277 Krahenbuhl S, Chang M, Brass EP, Hoppel CL (1991) Decreased activities of ubiquinol: ferricytochrome c oxi- doreductase (complex III) and ferrocytochrome c: oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobalamin [c lactam]-induced methyl­malonic aciduria. J Biol Chem 266:20998-21003

Kumaran S, Savitha S, Anusuya Devi M, Panneerselvam C (2004) L-Carnitine and DL-a lipoic acid reverses the age related deficit in glutathione redox state in skeletal muscle and heart tissues. Mech Ageing Dev 125:507-512. doi: 10.1016/j.mad.2004.05.004 Lai SW, Yu SM, Yuen HW, So FK, Zee YS, Chang CCR (2008) Antagonizing b-amyloid peptide neurotoxicity of the anti-aging fungus Ganoderma lucidum. Brain Res 1190:215-224. doi:10.1016/j.brainres.2007.10.103 Lakshmi B, Ajith TA, Sheena M, Nidhi G, Janardhanan KK (2003) Antiperoxidative, anti-inflammatory and antimu- tagenic activities of ethanol extract of the mycelium of Ganoderma lucidum occurring in South India. Teratog Carcinog Mutagen s1:85-97 Lee MJ, Kwon H, Jeong H, June Woo Lee WH, Lee YS, Baek JS, Surh JY (2001) Inhibition of lipid peroxidation and oxidative DNA damage by Ganoderma lucidum. Phyther Res 15:245-249. doi:10.1002/ptr.830 Lesnefsky EJ, Moghaddas S, Tandler B, Kerner J, Hoppel CL (2001) Mitochondria dysfunction in cardiac disease: ischemia-reperfusion, aging and heart failure. J Mol Cell Cardiol 33:1065-1089. doi:10.1006/jmcc.2001.1378 Lin SB, Li CH, Lee SS, Kan LS (2003) Triterpene-enriched extracts from Ganoderma lucidum inhibit growth of hepatoma cells via suppressing protein kinase C, activating mitogen-activated protein kinases and G2-phase cell cycle arrest. Life Sci 72:2381. doi:10.1016/S0024-3205(03)00124-3 Linnane AW, Marzuki S, Ozawa T, Tanaka M (1989) Mito- chondrial DNA mutations as an important contributor to ageing and degenerative disease. Lancet 1:642-645. doi: 10.1016/S0140-6736(89)92145-4 Liu F, Ooi VEC, Chang ST (1997) Free radical scavenging activities of mushroom polysaccharide extracts. Life Sci 64:1005-1011. doi:10.1016/S0024-3205(99)00027-2 Marin-Garcia J, Goldenthal MJ (2002) Understanding the impact of mitochondrial defects in cardiovascular disease: a review. J Card Fail 8:347-361. doi:10.1054/jcaf.2002. 127774

Marriage BJ, Clandinin MT, Macdonald IM, Glerum DM (2004) Cofactor treatment improves ATP synthetic capacity in patients with oxidative phosphorylation disorders. Mol Genet Metab 81:263-272. doi:10.1016/j.ymgme.2003. 12.008

Mehler AH, Kornberg A, Grisolia S, Ochoa S (1948) The enzymatic mechanisms of oxidation and reduction between malate or isocitrate and pyruvate. J Biol Chem 174:961-977

Miquel J (2002) Can antioxidant diet supplementation protect against age related mitochondrial damage? Ann NY Acad Sci 959:508-516 Miquel J, Economos AC, Flaming J, Johnson J (1980) Mito- chondrial role in cell aging. Exp Gerontol 15:575-591. doi:10.1016/0531-5565(80)90010-8 Mizuno Y, Yoshino H, Ikebe S, Hattori N, Kobayashi T, Shi- moda-Matsubayashi S, Matsumine H, Kondo T (1998) Mitochondrial dysfunction in Parkinson's disease. Ann Neurol 44:S99-S109. doi:10.1002/ana.410440116 Muscari C, Giaccari A, Giorano E, Clo C, Guarnieri C, Caldarera CM (1996) Role of reactive oxygen species in cardiovascular aging. Mol Cell Biochem 160-161:159-166. doi:10.1007/BF00240046

Navarro A, Gomez C, Sanchez-Pino MJ, Gonzalez H, Bandez MJ, Boveris AD, Boveris A (2005) Vitamin E at high doses improves survival, neurological performance, and brain mitochondrial function in aging male mice. Am J Physiol Regul Integr Comp Physiol 289:R1392-R1399. doi: 10.1152/ajpregu.00834.2004 Neubert D, Wojtczak AB, Lehninger AL (1962) Purification and enzymatic identity of mitochondrial contraction-fac­tors I and II. Proc Natl Acad Sci USA 48:1651-1658. doi: 10.1073/pnas.48.9.1651 Nulton-Persson AC, Szweda LI (2001) Modulation of mito- chondrial function by hydrogen peroxide. J Biol Chem 276:23357-23361. doi:10.1074/jbc.M100320200 Ozawa T (1997) Genetic and functional changes in mito­chondria associated with aging. Physiol Rev 77:425-464 Paradies G, Ruggiero FM, Petrosillo G, Quagliariello E (1993) Age-dependent decrease in the cytochrome c oxidase activity and changes in phospholipids in rat heart mito­chondria. Arch Gerontol Geriatr 16:263-272. doi:10.1016/ 0167-4943(93)90037-I Paterson RRM (2006) Ganoderma-a therapeutic fungal biofac- tory. Phytochemistry 67:1985-2001. doi:10.1016/j.phyto chem.2006.07.004 Pillai GT, Nair CKK, Janardhanan KK (2008) Polysaccharides isolated from Ganoderma lucidum occurring in Southern parts of India, protects radiation induced damages both in vitro and in vivo. Environ Toxicol Pharmacol 26:80-85. doi:10.1016/j.etap.2008.02.004 Reed LJ, Mukherjee BB (1969) a-Ketoglutarate dehydrogenase complex from Escheriachia coli. Methods Enzymol 13:55-61. doi:10.1016/0076-6879(69)13016-5 Ritcher C, Park JW, Ames BN (1998) Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc Natl Acad Sci USA 8:6465-6467 Salvioli S, Bonafe M, Capri M, Monti D, Franceschi C (2001) Mitochondria, aging and longevity-a new perspective. FEBS Lett 492:9-13. doi:10.1016/S0014-5793(01)02199-8 Sastre J, Pallardo FV, Pla R, Pellin A, Juan G, O'Connor JE, Estrela JM, Miquel J, Vina J (1996) Aging of the liver: age- associated mitochondrial damage in intact hepatocytes. Hepatology 24:1199-1205. doi:10.1002/hep.510240536 Sastre J, Pallardo FV, Vina J (2003) The role of mitochondrial oxidative stress in aging. Free Radic Biol Med 35:1-8. doi:10.1016/S0891-5849(03)00184-9 Savitha S, Pannerselvam C (2006) Mitochondrial membrane damage during aging process in rat heart: potential effi­cacy of L-carnitine and DL-a-lipoic acid. Mech Ageing Dev 127:349-355. doi:10.1016/j.mad.2005.12.004

Savitha S, Sivarajan K, Haripriya D, Kokilavani V, Panneer- selvam C (2005) Efficacy of levo carnitine and alpha lipoic acid in ameliorating the decline in mitochondrial enzymes during aging. Clin Nutr 24:794-800. doi: 10.1016/j.clnu.2005.04.005 Schapira AH (1999) Mitochondrial involvement in Parkinson's disease, Huntington's disease, hereditary spastic paraple­gia and Friedreich's ataxia. Biochim Biophys Acta 1410:159-170. doi:10.1016/S0005-2728(98)00164-9 Sheena N, Ajith TA, Janardhanan KK (2003a) Prevention of nephrotoxicity induced by the anticancer drug cisplatin, using Ganoderma lucidum, a medicinal mushroom occurring in South India. Curr Sci 85:478-482 Sheena N, Ajith TA, Janardhanan KK (2003b) Anti- inflam­matory and antinociceptive activities of Ganoderma lucidum occurring in South India. Pharmceutical Biol 41:4301-4304

Shiao MS, Lee KR, Lin LJ, Wang CT (1994) Natural products and biological activities of the Chinese medical fungus, Ganoderma lucidum. In: Ho CT, Osawa T, Huang MT, Rosen RT (eds) Food phytochemicals for cancer preven­tion. Part II: Teas, spices and herbs. American Chemical Society Press, Washington, DC, pp 342-354 Sohal RS, Weindruch R (1996) Oxidative stress, caloric restriction and aging. Science 273:59-63. doi:10.1126/ science.273.5271.59 Wallace DC (1992) Mitochondrial genetics: a paradigm for aging and degenerative diseases? Science 256:628-632. doi:10.1126/science.1533953 Wang M, Chan Y, Wu C, Wong Y, Hosoda K, Yamamoto S (2004) Effects of Ganoderma on aging and learning and memory ability in senescence accelerated mice. Int Congr Ser 1260:399-404. doi:10.1016/S0531-5131(03)01682-0 Wei YH (1992) Mitochondrial DNA alterations as ageing-

associated molecular events. Mutat Res 275:145-155 Wei YH (1998) Oxidative stress and mitochondrial DNA mutations in human aging. Proc Soc Exp Biol Med 217: 53-63

You YH, Lin ZB (2003) Antioxidant effect of Ganoderma

polysaccharide peptide. Yaoxue Xuebao 38:85-88 Zarchin N, Meilin S, Rifkind J, Mayevsky A (2002) Effect of aging on brain energy-metabolism. Comp Biochem Physiol Part A Mol Integr Physiol 132:117-120. doi: 10.1016/S1095-6433(01)00537-2 Zhang Y, Marcillat O, Giulivi C, Ernster L, Davies KJA (1990) The oxidative inactivation of mitochondrial electron transport chain components and ATPase. J Biol Chem 265:16330-16336

 

Exp Oncol 2006 28,1,25-29

 

 

 

(G.lucidum ekstraktının SW 480 İnsan Kolorektal kanser hücrelerinin proliferasyonunu inhibe etme etkinliği.)

 

GANODERMA LUCIDUM EXTRACT INHIBITS PROLIFERATION OF SW 480 HUMAN COLORECTAL CANCER CELLS

J.T. Xie1C.Z. Wang1S. Wicks12, J.J. Yin 5, J. Kong3, J. Li3, Y.C. Li3, C.S. Yuan 1

Tang Center for Herbal Medicine Research, the Pritzker School of Medicine,

University of Chicago, Chicago, USA 2Department of Anesthesia & Critical Care, the Pritzker School of Medicine, University of Chicago, Chicago, USA 3Section of Gastroenterology, Department of Medicine, The Pritzker School of Medicine,

University of Chicago, Chicago, USA 4Committee on Clinical Pharmacology and Pharmacogenomics, the Pritzker School of Medicine,

University of Chicago, Chicago, USA 5Center for Food Safety and Applied Nutrition, FDA, College Park, USA

 

Aim: Ganoderma lucidum is a commonly used Chinese herb and an important ingredient in traditional Chinese medicine herbal formula­tions for immune dysfunction related illnesses. The effects of this medicinal mushroom on human colorectal cancer cells have not yet been evaluated. In this study, we investigated the effects of Ganoderma lucidum extract using SW480 human colorectal cancer cell line. Materials and Methods: Two different fractions of Ganoderma lucidum extract, i.e., a fraction containing mainly polysaccharides (GLE-1), and a triterpenoid fraction without polysaccharides (GLE-2) were analyzed. Their antiproliferative activity was evaluated by cell proliferation assay and 3H-thymidine incorporation assay. Scavenging effects of DPPH radical were assessed using ESR-spectroscopy. Results: Our data showed that both GLE-1 and GLE-2 significantly inhibited the proliferation of SW 480 cells. The inhibitory effect of GLE-2 was much stronger than that of GLE-1. GLE-1 inhibited DNA synthesis in the cells and reduced the formation of DPPH radicals. Conclusion: Ganoderma lucidum extract inhibits proliferation of human colorectal cancer cells and possesses antioxidant properties. Key Words: Ganoderma lucidum, colorectal cancer, SW 480, proliferation, reactive oxygen species, DPPH radicals.

 Colorectal cancer is the second leading cause of cancer related death in the United States, and the sec­ond most prevalent cancer worldwide [1]. Half of the patients diagnosed with colorectal cancer eventually die from the disease; and only less than ten percent of patients with the metastatic cancer survive more than five years after diagnosis [1]. Although in its early stages the cancer can be cured by surgical resection alone, very often surgery is combined with adjuvant ra­dio- and chemotherapy. Patients treated for advanced stage colorectal cancer with adjuvant chemotherapy show a 40% reduced risk of relapse [2]. Recently, controlled clinical trials support a multimodal, multi- disciplinary approach to treating both early stage and advanced colorectal cancers [3, 4].

While advances continue to be made in developing effective treatment strategies for colorectal cancer pa­tients, chemotherapies are still limited by severe side effects and dose-limiting toxicity. The drug-related adverse events not only worsen patients' quality of life, but can also lead to their refusal to continue the poten­tially curative chemotherapy [5]. Patients with cancer often resort to complementary and alternative medical means to treat the side effects of chemotherapy [6].

Received: December 22, 2005. "Correspondence author: Fax: (773) 834-0601;

E-mail: cyuan@airway.uchicago.edu Abbreviations used: DMSO - dimethyl sulfoxide; DPPH - 2,2-Di- phenyl-1-picrylhydrazyl; ESR - electron spin resonance; GLE - Ganoderma lucidum extract; ROS - reactive oxygen species.

Ganoderma lucidum (or "Lingzhi" in Chinese) has long been recognized by Chinese medical profession­als as a valuable herbal medicine in treating a number of different illnesses [7]. Shizhen Li, a highly influential doctor in Chinese medicine from the Ming Dynasty (1368-1644 A.D.), recorded the effectiveness of Li ngzhi in his famous book, Ban Chao Gang Moo ("Comprehen­sive Pharmacopoeia"), and stated that taking Lingzhi over the long-term would build a strong, healthy body and assure longevity. Ganoderma lucidum is often an important ingredient in traditional Chinese medicine herbal formulations for immune dysfunction related ill­nesses [7, 8].

Currently, there are several Ganoderma lucidum herbal products available. SunRecome is a commonly used Ganoderma lucidum product manufactured by Shanghai Green Valley Pharmaceuticals in China. Previous studies showed that this herbal product could attenuate chemotherapy-induced nausea and vomiting in clinical trials [9] and animal studies [10]. Although human observational data in Chinese lit­erature suggest that Ganoderma lucidum possesses an anticancer property [9], and the active anti tumor constituents are thought to be on the polysaccharides [11], the effects of this medicinal mushroom on human colorectal cancer cells has not been evaluated. In the present study, we investigated the antiproliferative effects of two fractions of SunRecome, i.e., a fraction containing mainly polysaccharides, and a triterpenoid fraction without polysaccharides, using SW 480 human
colorectal cancer cell line. Potential antioxidant activity of Ganoderma lucidum was also evaluated.

MATERIALS AND METHODS

Ganoderma lucidum extract (GLE) and test solution preparation. SunRecome, or Ganoderma lucidum extract (GLE), from one lot, was obtained from Shanghai Green Valley Pharmaceuticals, China. The extract was analyzed by Shanghai Allsuccess Com­modity Inspection Co., who reported that the extract contained 1.89% terpenoids and 15.8% polysaccha­rides using spectrophotometric methods. Fig. 1 shows the chemical structure of terpenoids in Ganoderma lucidum. The extract was also analyzed by Applied Consumer Services (Hialeah Gardens, USA) to confirm that it was free of contamination such as microorgan­isms, pesticide residues, and toxic elements.

Compounds

R1

R2

R3

Ganoderic acid A

0

H,

S>H

Ganoderic acid B

^OH

'""H

H,

0

Ganoderic acid C1

0

H,

0

Ganoderic acid C2

^OM

H,

-"on

Ganoderic acid G

^OH >H

^OH

St

0

Ganoderic acid y*

0

H,

"'■OH

*Structure of side chain:

 

^^^COOH

 

Fig. 1. General structure of terpenoids in Ganoderma lucidum

Two different solutions were used to prepare GLE-1 (a fraction containing mainly polysaccharides) and GLE-2 (a fraction containing triterpenoids without polysac­charides). To prepare GLE-1, the extract powder was dissolved in 5% ethanol-water solution and placed in a 4 °C refrigerator over night. The following day the sealed sample was heated in a water bath at 80 °C for 30 min. When the sample temperature cool down to room tem­perature, the solution was filtered with a Millex 0.2 |m nylon membrane syringe filter (Millipore Co., Bedford, USA). To prepare GLE-2, the extract powder was dis­solved in DMSO in room temperature, and then filtered with a Millex 0.2 |m nylon syringe filter. To ensure that the cell growth was not affected by the DMSO, we studied the effect of DMSO alone on cell proliferation as a vehicle.

Cell culture. Human colorectal SW480 cancer cell line (ATCC, Manassas, USA) was maintained in Leibovitz's L-15 medium with 10% L-glutamine (Gibco, USA), supple­mented with 10% fetal bovine serum and penicillin-strep­tomycin (50 units/ml, Invitrogen, USA), in a humidified incubator (5% CO2 in air at 37 °C) with medium change every 2-3 days. When the cells reach 70-80% confluency, they were trypsinized, harvested, and seeded into a new tissue culture dish (100 mm in diameter).

Cell proliferation assay. On selected days after removal of incubation medium, SW480 cell monolayer was washed twice with phosphate buffered saline (PBS). To examine the antiproliferation effect of the extract, the cells were seeded in a 24-well plate at about 10,000 cells/well with regular medium and allowed to adhere for 24 h. After adhesion of the cells, the culture medium was changed prior to the addition of the test herbal extracts. The cells were incubated with testing material at various concentrations for 72 h. Control cultures were incubated in culture medium alone. At the end of treatments, the cells were detached using trypsin and counted using a Coulter Counter (Counlter Electronics, Hialeah, USA) [12, 13]. All assays were performed at least three times. The inhibition of SW480 cell proliferation was calculated as follows: Cell proliferation (%) = 100 x (each cell num­ber in experimental well/total cell number in the control well).

3H-thymidine incorporation assay. SW 480 cells were seeded in a 24-well plate and allowed to adhere for 24 h. The cells were incubated with GLE-1 at various concentrations in 300 |l media containing 1 A/ml ^-thy­midine (specific activity: 1.48 TBq /mmol 40.0 Ci/mmol) in each well for 72 h. Control cultures were incubated in the medium with 3H-thymidine only. After washing with PBS and 10% TCA (trichloroacetic acid), 0.5 ml NaOH (0.2 M) was added to each well and agitated for 5 min. Finally, the cell lysates were transferred into vials and 5 ml of 30% liquid scintillation was added and mixed in the vials. The radioactivity was measured using Liquid Scintillation Analyzer (Tri-Carb1500, Packard) [14].

Measurement of free radical scavenging ability of GLE-1 in a cell-free system. Antioxidant activity of Ganoderma lucidum was evaluated on the basis of its scavenging effect of DPPH radical using ESR spectroscopy [15]. Conventional ESR spectra were obtained with a Varian E-109 X-band spectrometer. ESR signals were recorded with 10 mW for DPPH, and 100 kHz field modulations of 2G. All measurements were performed at room temperature.

The DPPH stable radical scavenging activity of GLE-1 was estimated by ESR according to the method of Yama- guchi et al. [16]. The reaction mixture contained 50 |l test sample and 50 |l DPPH solution (0.5 mM). The proportion of ethanol/water in the reaction mixture was 5/95. ESR signaling was recorded at room temperature. Time depen­dence of the scavenging effect was determined by adding GLE-1 to the reaction mixture at 5 mg/ml for 2-15 min after mixing sample. Scavenging effect was determined by comparison with a control group. The dose dependency of the scavenging effect was also determined after GLE-1 addetion to the reaction mixture for 15 min.

GLE-2 was diluted in DMSO. Since DMSO pos­sesses antioxidant activity, we were unable to evalu­
ate the free radical scavenging ability of GLE-2 in the cell-free system.

Statistical analysis. Data are expressed as mean ± standard error (S.E.). Statistical analysis was performed using ANOVA in combination with Student's f-test. Dif­ferences were considered significant if P < 0.05.

RESULTS

Inhibitory effect of GLE-1 on SW480 cells. The

inhibitory activity of GLE-1 on the proliferation of SW 480 human colorectal cancer cells at 72 h is shown in Fig. 2, a. GLE-1 decreased cell proliferation significantly in a concentration related manner. Compared to con­trol group (100%), GLE-1 reduced cell proliferation by 3.8 ± 4.5% at 0.05 mg/ml, 8.9 ± 5.2% at 0.25 mg/ml (P < 0.05), and 10.2 ± 5.0% at 1 mg/ml (P < 0.05).

Vehicle 0,05 0,25 Concentration of GLE-2 (mg/ml) Fig. 2. Antiproliferative effects of Ganoderma lucidum extract on SW 480 colorectal cancer cells after 72 h treatment. (a) Effects of GLE-1. (b) Effects of GLE-2. * P < 0.05; ** P < 0.01.

Inhibitory effect of GLE-2 on SW480cells. The inhibitory activity of GLE-2 on the proliferation of SW 480 cells at 72 h is shown in Fig. 2, b. GLE-2 decreased the cell proliferation significantly in a concentration dependent manner. Compared to the control group, GLE-2 reduced cell proliferation by 18.1 ± 3.1% at 0.05 mg/ml (P < 0.05), 40.2 ± 3.7% at 0.25 mg/ml (P < 0.01), and 76.7 ± 1Effects of GLE-1 on DNA synthesis by measur­ing 3H-thymidineincorporation. As shown in Fig. 4, c, treatment with GLE-1 for 72 h at 0.5 and 1 mg/ml suppressed cellular incorporation of 3H-thymidine significantly by 70.0 ± 0.6% and 73.0 ± 1.5% ompared with the control (both P < 0.01), respectively.

Free radical scavenging ability of GLE-1 in cell­free system. The radical scavenging activities of GLE-1 in a cell free system are shown in Fig. 5. Addi­tion of GLE-1 to the DPPH system at a concentration of 5mg/ml remarkably reduced the free radical ESR signal after 2 min. After 15 min, the ESR signal of DPPH was totally eliminated. This time-related effect is shown in Fig. 5, a. The concentration-related scaveng­ing effect of GLE-1 for 15 min is shown in Fig 5, b.

DISCUSSION

Ganoderma lucidum is a commonly used herbal medicine in many Oriental countries. The anticancer effects of polysaccharides extracted from another species of genus Ganoderma were initially reported
in 1971 [17]. Recently, some researchers observed that triterpenoids in Ganoderma lucidum also are ac­tive antitumor constituents [18, 19].

In this study, two different solutions were used in the preparation of GLE-1 and GLE-2. Although both of the extracts showed inhibitory effects on the proliferation of SW 480 human colorectal cancer cells, the percent­age of inhibition was different, with much stronger inhibition using GLE-2. GLE-1 was prepared with 5% ethanol-water solution, which presumably dissolved mostly polysaccharides and a small percentage of ter­penoids, while GLE-2 was prepared with DMSO, which presumably dissolved mostly terpenoids without any polysaccharides [11]. Thus, it seems that terpenoids in the GLE exert significant antiproliferative effects on the colorectal cancer cells in our experiments. In order to explore the possible mechanisms of Ganoderma lucidum's anticancer activity, we evaluated the effect of the extract on the incorporation of 3H-thymidine in SW480 cells by using 3H-thymidine labeling assay. Our data suggest that the extract directly inhibits the synthesis of DNA in the cells [14].

Many herbal medicines possess antioxidant prop­erties [20]. Antioxidants are compounds that protect cells against the damaging effects of reactive oxygen species (ROS). Some ROS, such as superoxide and hydrogen peroxide, are normally produced in cells as by-products of biochemical reactions or as signaling molecules. When ROS-generating reactions are acti­vated excessively, pathological quantities of ROS are released to create an imbalance between antioxidants and ROS, resulting in cellular damage. Oxidative stress was linked with the pathogenesis of many human diseases including cancer, aging, and atheroscle­rosis [21]. Herbal antioxidants may protect against these diseases by contributing to the total antioxidant defense system of the human body [20, 22]. We ob­served the antioxidant effect of Ganoderma lucidum extract in our ESR study. The relationship between the observed antioxidant activity and the antiproliferation effect of SW480 cells remain to be elucidated. In future investigations, we will also use a panel of cell lines to provide us with the opportunity to evaluate whether the effects of a medicinal herb can be generalized across the spectrum of human cancers.

In addition to its possible anticancer effect, Gano- derma lucidum may also help in decrease chemo­therapy-induced side effects. We have demonstrated that the extract of Ganoderma lucidum attenuated cisplatin-induced nausea and vomiting in a rat model [10]. Cancer patients are known to use herbal prod­ucts more often than the general population, and thus, well-investigated herbal therapies can offer patients with a practical alternative [6]. Moreover, if synergistic effects between herbal medicines and chemotherapy agents can be identified, reduction of chemotherapy dose in combination with herbs can further decrease dose-related drug toxicity. The identification of non­toxic anticancer herbal medicines remains an essential step in advancing the treatment of cancer. Data ob­tained from our studies has the potential to advance treatment regimens, and improve the quality of life of patients suffering from colorectal cancer.

ACKNOWLEDGEMENTS

This work was supported in part by the NIH/NCCAM grants AT002176 and AT002445.

REFERENCES

  • 1. Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, Feuer EJ, Thun MJ. Cancer statistics, 2004. CA Cancer J Clin 2004; 54: 8-29.
  • 2. Moertel CG, Fleming TR, Macdonald JS, Haller DG, Laurie JA, Tangen CM, Ungerleider JS, Emerson WA, Tormey DC, Glick JH, Veeder MH, Mailliard JA. Fluorouracil plus levamisole as effective adjuvant therapy after resection of stage III colon carcinoma: a final report. Ann Intern Med 1995; 122: 321-6.
  • 3. Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers B, Ross R, Kabbinavar F. Bevaci- zumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004; 350: 2335-42.
  • 4. Moehler M, Teufel A, Galle PR. New chemotherapeutic strategies in colorectal cancer. Recent Results Cancer Res 2005; 165: 250-9.
  • 5. Schnell FM. Chemotherapy-induced nausea and vomit­ing: the importance of acute antiemetic control. Oncologist 2003; 8: 187-98.
  • 6. Ott MJ. Complementary and alternative therapies in cancer symptom management. Cancer Pract 2002; 10: 162-6.
  • 7. Bensky D, Gamble A, Stoger E. Chinese Herbal Medi­cine: Materia Medica. Eastland Press, Seattle, 2004.
  • 8. Hijikata Y, Yasuhara A, Sahashi Y. Effect of an herbal formula containing Ganoderma lucidum on reduction of herpes zoster pain: a pilot clinical trial. Am J Chin Med 2005; 33: 517-23.
  • 9. Cheng J, Ren W. Anti-cancer research of SunRecome and its clinical application: a review. Chin J Med 2003; 3: 689-93.
  • 10. Wang CZ, Basila D, Aung HH, Mehendale SR, Chang WT, McEntee E, Guan X, Yuan CS. Effects of Gano­derma lucidum extract on chemotherapy-induced nausea and vomiting in a rat model. Am J Chin Med 2005; 33: 807-15.
  • 11. Gao Y, Tang W, Dai X, Gao H, Chen G, Ye J, Chan E, Koh HL, Li X, Zhou S. Effects of water-soluble Ganoderma lucidum polysaccharides on the immune functions of patients with advanced lung cancer. J Med Food 2005; 8: 159-68.
  • 12. Chang JS, Chiang LC, Hsu FF, Lin CC. Chemopreven- tion against hepatocellular carcinoma of Cornus officinalis in vitro. Am J Chin Med 2004; 32: 717-25.
  • 13. Garmanchouk LV, Pyaskovskaya ON, Yanish YV, Shlyakhovenko VA, Dasyukevich OI, Solyanik GI. Influence of aconitine-containing herbal extract BC1 on proliferative and electrokinetic characteristics of endothelial cells. Exp Oncol 2005; 27: 262-6.
  • 14. Gieni RS, Li Y, HayGlass KT. Comparison of [3H]thymidine incorporation with MTT- and MTS-based bioassays for human and murine IL-2 and IL-4 analysis. Tetrazolium assays provide markedly enhanced sensitivity. J Immunol Meth 1995; 187: 85-93.
  • 15. Kumar SS, Devasagayam TP, Bhushan B, Verma NC. Scavenging of reactive oxygen species by chlorophyllin: an ESR study. Free Radic Res 2001; 35: 563-74.
  • 16. Yamaguchi F, Ariga T, Yoshimura Y, Nakazawa H. Anti­oxidative and anti-glycation activity of garcinol from Garcinia indica fruit rind. J Agric Food Chem 2000; 48: 80-185.
  • 17. Sasaki T, Arai Y, Ikekawa T, Chihara G, Fukuoka F. Antitu­mor polysaccharides from some Polyporaceae, Ganoderma applana- tum and Phellinus linteus. Chem Pharm Bull 1971; 19: 821-6.
  • 18. Sliva D. Ganoderma lucidum (Reishi) in cancer treat­ment. Integr Cancer Ther 2003; 2: 358-64.
  • 19. Li CH, Chen PY, Chang UM, Kan LS, Fang WH, Tsai KS, Lin SB.Ganoderic acid X, a lanostanoid triterpene, inhibits topoisomerases and induces apoptosis of cancer cells. Life Sci 2005; 77: 252-65.
  • 20. Cadenas E, Packer L. Handbook of Antioxidants. Marcel Dekker, Inc., New York, 2002.
  • 21. Sauer H, Wartenberg M, Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and dif­ferentiation. Cell Physiol Biochem 2001; 11: 173-86.
  • 22. Chang WT, Dao J, Shao ZH. Hawthorn: potential roles in cardiovascular disease. Am J Chin Med 2005; 33: 1-10.

 

 

Journal of Hematology & Oncology

Open Access

Research

İnsan monositik lösemi hücrelerinin  immune stimulatorik(bağışıklık olaylarını tetikleyen)dendritik hücrelere dönüşmesini sağlayan Ganoderma lucidum Polisakkaritleri.

 

Ganoderma lucidum polysaccharides can induce human monocytic leukemia cells into dendritic cells with immuno-stimulatory function

Wing Keung Chan, Christopher Ching Hang Cheung, Helen Ka Wai Law, Yu Lung Lau and Godfrey Chi Fung Chan*

Address: Department of Paediatrics & Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, PR China

Email: Wing Keung Chan - wingkc@graduate.hku.hk; Christopher Ching Hang Cheung - comcc.chris@gmail.com; Helen Ka Wai Law - hkwlaw@hkucc.hku.hk; Yu Lung Lau - lauylung@hkucc.hku.hk; Godfrey Chi Fung Chan* - gcfchan@hkucc.hku.hk * Corresponding author

Published: 21 July 2008

Journal of Hematology & Oncology 2008, 1:9 doi:I0.II86/l 756-8722-1 -9

This article is available from: http://www.jh00nline.0rg/c0ntent/I/I/9

© 2008 Chan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativec0mm0ns.0rg/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background: Previous studies demonstrated Ganoderma lucidum polysaccharides (GL-PS), a form of bioactive P-glucan can stimulate the maturation of monocyte-derived dendritic cells (DC). The question of how leukemic cells especially in monocytic lineage respond to GL-PS stimuli remains unclear.

Results: In this study, we used in vitro culture model with leukemic monocytic cell-lines THP-I and U937 as monocytic effectors cells for proliferation responses and DCs induction. We treated the THP-I and U937 cells with purified GL-PS (I00 |g/mL) or GL-PS with GM-CSF/IL-4. GL-PS alone induced proliferative response on both THP-I and U937 cells but only THP-I transformed into typical DC morphology when stimulated with GL-PS plus GM-CSF/IL-4. The transformed THP-I DCs had significant increase expression of HLA-DR, CD40, CD80 and CD86 though not as high as the extent of normal monocyte-derived DCs. They had similar antigen-uptake ability as the normal monocyte-derived DCs positive control. However, their potency in inducing allogeneic T cell proliferation was also less than that of normal monocyte-derived DCs.

Conclusion: Our findings suggested that GL-PS could induce selected monocytic leukemic cell differentiation into DCs with immuno-stimulatory function. The possible clinical impact of using this commonly used medicinal mushroom in patients with monocytic leukemia (AML-M4 and M5) deserved further investigation.

 Background

In both Western and Oriental societies, cancer patients commonly take complementary and alternative medicine while they underwent conventional anti-cancer therapy [1-3]. Among different kinds of alternative medicine, herbal medicine is the most popular form taken by patients in United Kingdom [4]. In our community, more than 42% of our pediatric cancer patients took herbal medicine when they received conventional chemotherapy [5]. Among them, the commonest herb being used is the extracts derived from Ganoderma lucidum.

Ganoderma lucidum (GL) is a traditional Chinese medicine known by the layman as the "herb of immortality". It was

 

used as a health tonic to promote longevity for more than two thousands years. It has two major groups of bioactive components: polysaccharides and triterpenes. In recent decade, they have been extensively studied because of its potential immunomodulating and anti-tumor effects as demonstrated in both in vitro and in vivo models [6]. So far, currently available data suggested that GL polysaccha- rides exert anti-cancer functions indirectly by activation of host's immune responses whereas GL triterpenes can kill cancer cells directly via its direct cytotoxic effect [7]. GL polysaccharides are purified from the mushroom myc­elium and they contain branched P-glucan.

Dendritic cells (DCs) are the most potent antigen present­ing cells and have unique ability in linking innate and adaptive immunity. Due to the scarcity of circulating DCs, the current protocol to study DCs biology and differentia­tion is mainly through differentiation of monocytes to DCs with the cytokines GM-CSF and IL-4. Recently, DCs can be induced from acute myeloid leukemic cells (AML) and this raised the possibility of using DCs derived from autologous leukemic cells for therapeutic uses [8]. Several AML cell-lines including monocytic THP-1, KG-1 and CD34+ MUTZ3 cell-lines have been used as cellular mod­els to study the differentiation of leukemic cells and DCs biology. However, the differentiation protocols differed greatly. For example, mature DCs could only be derived from THP-1 and KG-1 by adding GM-CSF and IL-4 together with ionomycin and TNF-a [8]. Interestingly, all study agreed that there is impaired response of leukemic DC to LPS directed DCs maturation [9]. This suggested that these leukemic DCs are somehow defective in response to maturation stimuli.

We and other groups demonstrated GL mycelium polysac- charides have the ability to stimulate the maturation of human DCs [10-12]. While most reports advocating the immunomodulating role of GL on normal monocytic cells, our data provided a novel observation that GL polysaccharides may also enhance monocytic leukemic cells proliferation and induce dendritic cells differentia­tion from monocytic leukemic blasts. The awareness of such phenomenon may help us to design specific treat­ment approach for monocytic leukemia.

Results

Cell proliferation response of THP-1 after GL polysaccharides stimulation

To relay the GL-PS has effect on leukemic cells, we evalu­ated the effect of GL polysaccharides (GL-PS) on the acute myeloid leukemia (AML) cell-lines THP-1 and U937 by cell proliferation assay. GL-PS alone at the dose of 100 |g/ mL could stimulate the growth of both THP-1 and U937 cells. The average increases after the three-day exposure in THP-1 and U937 cells were 1.53-fold and 1.16-fold higher than the untreated control, respectively (Fig. 1A). Vincris- tine, a chemotherapeutic drug, was used as control to show the cells were responding. The cell cycle analysis with PI staining showed that GL-PS did not induce S phase arrest during the three-day treatments (Fig. 1B). By checking the expression of proliferating cell nuclear anti­gen (PCNA), which is an S-phase marker, both THP-1 and U937 cells showed increases in PCNA expression after GL- PS treatment (Fig. 1C).

Induction of DCs-like morphology and phenotype in GL-PS treated THP-1 cells

Under GL-PS treatment (100 |g/mL was used in all exper­iments onwards), we observed DC-like morphology in THP-1 cell culture. Since reports suggest that THP-1 can be induced into DC by a combination of cytokines [8,13], we hypothesized that GL-PS might also induce or enhance the differentiation of THP-1 cells into THP-1 DCs. We cul­tured these cells in the presence of GM-CSF/IL-4 with or without GL-PS. We used Mo-DCs as positive control and untreated THP-1 cells as negative control. THP-1 treated with GL-PS plus GM-CSF/IL-4 yielded atypical large adherent and elongated cells with multiple cytoplasmic spikes (white arrow) comparing to the round floating THP-1 cells and typical Mo-DCs with multiple satellite­like cytoplasmic protrusions (Fig. 2A). Under the forward and side scatter analysis of flow cytometry (lower panel), we found that the THP-1 DCs derived with GL-PS (GL-PS THP-1 DCs) had larger size as if the monocytes when dif­ferentiated into DCs. For U937 cells, we did not observe similar morphological changes (data not shown).

Phenotypic maturation of THP-1 DCs derived with GL-PS

We then checked the surface expression of antigen presen­tation molecules and costimulation molecules, which Mo-DCs normally express. In Fig. 2B, we found that the THP-1 expressed relatively low levels of CD11c, HLA-DR, CD40, CD80 and CD86 when compared with normal Mo-DCs. Challenge of THP-1 with GM-CSF/IL-4 and GL- PS demonstrated increase in all markers when it was com­pared with the untreated THP-1 cells. To check the exper­imental consistency, we then normalized the fluorescence intensity from four experiments using the CD marker expression in THP-1 cells alone as the 100% (Fig. 2C). GL- PS alone could induce significant increase in CD 11c, HLA-DR and CD40 when compared with the negative control THP-1 cells alone. But together with cytokine, the GL-PS THP-1 DCs showed significant increase in all five CD marker expressions, suggesting phenotypic maturity. The GM-CSF/IL-4 alone did not always increase the matu­ration marker expression in THP-1. To show the specifi­city of DC differentiation in THP-1 cells, we repeated the experiments on U937 cells. However, there was no signif­icant increase in all DCs maturation markers (data not shown).

 

sentative experiments. *p < 0.05; ** p < 0.01; ***p < 0.001 versus that of THP-1 cells.

Loss of cell proliferation response after added with cytokines

To show the differentiation commitment of the GL-PS treated THP-1 cells in the presence of GM-CSF/IL-4, we added GM-CSF/IL-4 to the THP-1 cells, which had been treated with GL-PS for three days. We monitored the cell growth for two more days (Day 4 and 5) by XTT prolifer­ation assays (Fig. 3A). Adding GM-CSF/IL-4 induced sig­nificant increase in proliferation in GL-PS treated THP-1 on Day 4. However, the proliferation became static after Day 5. We confirmed the cell proliferation results with cell counting using trypan blue exclusion assay. As shown in Fig. 3B, we recorded significant increase in cell number when either GM-CSF/IL-4 or GL-PS was added to the THP- 1 cells on Day 5. When both GL-PS and GM-CSF/IL-4 were added, the cell number retained similar to the nega­tive untreated THP-1. The decrease was not due to the cell death as indicated by the trypan blue staining (data not shown).

Upregulated endocytotic activity of THP-1 DCs derived with GL-PS

We examined the endocytotic activity of the THP-1 DCs using fluorescent labeled FITC-dextran as antigens and incubated them at 37°C. We used PBS as negative control as well as parallel experiments at 4°C to serve as the back­ground fluorescence. We found that the THP-1, THP-1 DCs with GM-CSF/IL-4 and THP-1 with GL-PS showed similar antigen uptake ability (Fig. 4A). For the GL-PS THP-1 DCs, we unexpectedly found that there was an increase in antigen uptake signals from the FITC-dextran after normalizing with the negative control (Fig. 4B). In order to rule out the possibility of upregulation of man- nose receptor, which could account for the uptake of FITC-dextran, we determined the expression level of man- nose-receptor and we found no change was observed (data not shown).

Low IL-12 and IL-10 production in THP-1 DCs derived with GL-PS

We detected low amount of IL-12 production from THP-1 cells, THP-1 DCs with GM-CSF/IL-4, THP-1 with GL-PS and GL-PS THP-1 DCs (Fig. 5A). Though THP-1 DCs with or without GL-PS had relatively higher IL-12 production, the amount was not significant. In contrast, GL-PS THP-1 DCs showed significant increase in IL-10 production (Fig. 5B).

Decrease in T cell proliferation in allogeneic mixed lymphocyte reaction

We determined the outcome of those THP-1 DCs when they were co-cultured with normal CD3+ T cells, with immature and mature Mo-DCs as positive control. Signif­icant increase in T cell proliferation was induced from immature to mature DCs (Fig. 6A, left). Interestingly, we found that there was a suppression of T cell proliferation in the co-culture of GL-PS THP-1 DCs when compared with other THP-1 cells or THP-1 DCs (Fig. 6A, left). When compared with THP-1 cells alone, the GL-PS THP-1 DCs showed significant decrease in T cell proliferation (Fig. 6A, right). We then examined that the suppression of T cell proliferation was not due to the induction of apopto- sis (data not shown). The suppression did not focus on the CD4+ helper T cells and CD8+ cytotoxic T cells indi­cated by no significant change in CD4/CD8+ ratio (Fig. 6B).

Discussion

We demonstrated herein that purified immunomodula- tory GL polysaccharides, which have been widely used as adjuvant therapy for anti-tumor purposes, could induce both monocytic leukemic cell proliferation and abnormal cellular differentiation in the form of immunoregulatory DCs. Interestingly, such proliferative stimulation was not found in other non-monocytic lymphoid and myeloid leukemic cell lines tested (data not shown), suggesting such effect was lineage specific. We also explored the pos­sibility of using GL-PS to induce DCs from autologous blast cells in order to reduce leukemic cell burden.

We found that even among monocytic leukemic cells THP-1 and U937, there was a differential response to GL- PS. GL-PS only enhanced proliferation of U937, an AML M5 cell-line but could not induce its differentiation into DCs as in THP-1. Contrary to our findings, GL polysaccha- rides were reported to have to ability in inhibiting the growth of U937 cells, but it was under the influence of a conditioned medium primed by GL polysaccharides-stim- ulated human blood mononuclear cells [14]. This finding in fact suggested such inhibition required possible soluble factors secreted by the primed monocytes. In a recent report by Muller et al [15], GL was also demonstrated to be anti-proliferative in leukemic cells rather than inducing cell proliferation as shown in our study. This is mainly related to the choice of purified components being used. In our study, purified GL polysaccharides were used whereas purified GL triterpenes such as ganoderic acids were used in Muller et al. study. From the review of litera­ture (see Table I), GL polysaccharides have consistently been shown to have immunological potency and can sup­press cancer cell growth mainly by activating host's immune responses. In contrast, the triterpenes exert direct cytotoxic effect mainly through induction of cell cycle arrests and apoptosis in cancer cells including human leukemia, lymphoma and multiple myeloma (HL60, U937, K562, Blin-1, Nalm-6 and RPMI8226) [14,15]; breast cancer cells MDA-MB-231; and umbilical cord vas­cular endothelial cells HUVEC [16,17]. These data high­lighted the importance of the choice of GL components selected for the study. Standardization of polysaccharides

 

and triterpenes contents in the GL products has to be con­sidered if we would like to extend these in vitro data into clinical study.

The GL-PS THP-1 DCs so generated morphologically resembled DCs with upregulated CD 11c, HLA-DR, and costimulation molecules CD40, 80 and 86 (Fig. 2). Although the expression levels of these molecules were relatively low when compared with those on normal monocyte-derived DCs, they showed similar DCs func­tion of stimulating allogeneic T cells proliferation responses. They were however immunoregulatory with the evidence of immature uptake of antigens, the IL-10 production as well as low potency in stimulating alloge­neic T cell proliferation (Fig. 4, 5, 6). The suppressed T cell proliferation was believed related to their IL-10 produc­tion. IL-10 is an immunosuppressive cytokine and renders T cell stop proliferation even under the challenge of allo­geneic differences [18]. This is also a mechanism for leukemic cells to escape from immune surveillance by dysregulation of immune systems via secretion of IL-10.

Previous studies demonstrated that GL polysaccharides could induce IL-1 release through the toll-like receptor (TLR)-4 signaling pathways in murine macrophages [19]. This raised a question that whether other TLRs ligands could account for the abnormal cellular responses on monocytic leukemic cells. To test this hypothesis, we also explored the effect of LPS (a ligand of TLR-4) and zymosan (a ligand of TLR-2 and dectin-1) on THP-1 DCs (data not shown). We found that LPS induced more sig­nificant cell adhesion to the culture plates and caused more cell death during cell harvesting. This phenomenon was also reported in previous studies but LPS could not induce the maturation of the leukemic DCs [8,9]. For the zymosan treated THP-1, there was no effect in expression of DC maturation markers; dextran-based endocytosis and IL-10, IL-12 productions. But we recognized that the zymosan treated THP-1 DCs with GM-CSF/IL-4 also had decrease potency in stimulating T cells proliferation. This implied that the leukemic cells THP-1 might respond to TLR ligands in different environments such as during infections and lead to abnormal changes.

Conclusion

In summary, we found that GL polysaccharides could induce proliferation of monocytic leukemic cells. Together with GM-CSF/IL-4, GL-PS could induce THP-1 cells to become DCs with significant upregulation of anti­gen presentation and costimulation molecules expres­sion. The immuno-potent nature was shown by the evidences that they retained ability to uptake antigens with IL-10 productions and decrease in immunostimual- tory potential for T cell proliferation. Differential response of monocytic leukemic cells to GL-PS was observed. Our findings thus suggested that GL polysaccharides or other TLR ligands might have clinical impact on patients with monocytic leukemia. Whether GL-PS could induce DCs differentiation from autologous blast cells to help cancer patients to reduce cancer cell burden require further in vivo study for verification.

Materials and methods

Source and preparation of polysaccharides

Purified Ganoderma lucidum polysaccharides (GL-PS) was kindly provided by Prof. Lin ZB (Department of Pharma­cology, Peking University Health Science Center, School of Basic Medical Sciences, Beijing, China). It is a polysac­charide peptide from GL mycelium with molecular weight of 584,900 and 17 amino acids. The ratio of polysaccha­rides to peptides is 93.51%: 6.49%. The polysaccharides consist of glucose, galactose, arabinose, xylose and man- nose          with                  molar               ratios            of

0.793:0.964:2.944:0.167:0.384:7.94 and linked by P-gly- cosidic linkages [20]. Endotoxin levels in GL-PS were con­stantly measured by using endotoxin-specific kinetic chromogenic Limulus Ameobyocyte Lysate (LAL) assay kit (Pyrochrome®, Associates of Cape Cod, Inc, East Fal­mouth, MA) with glucan inhibition buffer (Glucashield®, Associates of Cape Cod) to reconstitute the reagents according to the manufacturer's instructions. Standard curves were generated using Control Standard Endotoxin (CSE) and for better comparison, the LAL reactivity of P- glucan sample was also compared with that of lipopoly- saccharide (LPS; Sigma). The endotoxin level of GL-PS was equivalent to 0.01% of 1 ng lipopolysaccharide, LPS, E. coli derived, suggesting negligible.

Cell culture of leukemic cells

Leukemic cells, THP-1 and U937 were purchased from ATCC (Manassas, VA). It was characterized as AML M5. Cells were cultured in medium consisting of 90% RPMI 1640, 10% FBS, 100 U/mL penicillin, 100 mg/mL strepto­mycin (Invitrogen, Life Technologies, CA) and main­tained at 37°C in a humidified atmosphere with 5% CO2.

Generation of leukemic DCs in vitro

The generation of leukemic DCs was modified from that for normal Mo-DCs as previously described [11,21]. Leukemic cells THP-1 at the density of 1 x 105 per well were cultured with/without GL-PS (100 |g/mL) in the presence of GM-CSF (40 ng/mL; Novartis Pharma A6, Basle, Switzerland) and IL-4 (40 ng/mL; R&D Systems Inc, Minneapolis, MN) at 37°C under 5% CO2. On Day 3, 90% of the medium was replaced with fresh medium and cytokines. THP-1 DCs were then harvested on Day 5 and washed for further assays. For normal monocyte-derived DCs, mononuclear cells were isolated from buffy coat of healthy adult donors (Red Cross, Hong Kong SAR, China) by Ficoll-Paque Plus density gradient (Amersham Bio­sciences, Uppsala, Sweden). Monocytes were then iso­lated from PBMCs by positive selection using anti-CD14- conjugated magnetic microbeads (Miltenyi Biotec, Ber­gisch Gladbach, Germany). The isolated cells were cul­tured at a density of 1 x 106 cell/mL in RPMI 1640 medium supplemented with 10% FBS, 50 IU/mL penicil­lin and 50 IU/mL streptomycin (Invitrogen) with GM-CSF and IL-4 at 37°C under 5% CO2 for five days. CD3+T cells were isolated with the same method except using anti- CD3-conjugated magnetic microbeads (Miltenyi Biotec.). The purity of isolated monocytes was consistently > 85% while that of T cells was consistently > 9 8% as determined by Coulter Epics Flow Cytometer (Coulter Corporation, Miami, FL). Based on flow cytometry analysis, the imma­ture DCs on Day 5 were 98.3% CD11c+CD1a+ and 99.8% lineage negative (CD3-, CD 14-, CD 16-, CD19-, CD20-, CD56-).

Cell proliferation assay

The effects of GL-PS on cell proliferation were measured using the Cell Proliferation Kit II XTT assay kit (Roche Molecular Biochemicals, Mannheim, Germany) accord­ing to the manufacturer's instructions. Briefly, 5 x 104 cells per well were grown in flat-bottom 96-well plates in a final volume of 100 | L culture medium overnight. Cells were then exposed to GL-PS at different concentrations (1 Ig/mL to 1 mg/mL) for 24, 48 and 72 h. After the fixed time of incubation, 50 |L of the XTT labeling mixture was added to each well, and incubated for 4 h at 37°C in a humidified atmosphere with 5% CO2. The formation of formazan dyes in XTT labeling mixture by metabolically active cells was detected spectrophotometrically at 450 nm. The cell proliferation was calculated from the OD and expressed as percentage of negative control. To confirm the cell proliferation by increase in cell number, trypan blue exclusion assay was performed with trypan blue stain (Invitrogen). A minimum of 300 cells were counted under hemocytometer.

Cell cycle analysis

GL-PS-treated leukemic cells were harvested, washed with PBS, fixed with ice-cold 70% ethanol and stored at 4°C. When for assay, the cell suspensions were incubated with RNase A (100 |g/mL; Sigma) and propidium iodide (4 Ig/mL; Sigma) in PBS. Cell cycle phases were then ana­lyzed with Coulter Epics Flow Cytometer (Beckman Coul­ter, Inc., Fullerton, CA). The percentage of G1, S and G2/ M were determined with Cylchred Version 1.0.2 (Cardiff University, Wales, UK). For the expression of proliferating cell nuclear antigen (PCNA), we stained the cells with Flu­orescein isothiocyanate (FITC) conjugated PCNA anti­body (BD PharMingen, San Diego, CA) together with isotype control FITC-IgGK (BD PharMingen). The cell were analyzed with the flow cytometer and the data were analyzed with WINMDI version 2.8 flow cytometry anal­ysis software (Purdue University, West Lafayette, IN).

Flow cytometry analysis of DCs

On Day 5, DCs were harvested, washed and labeled with fluorochrome-conjugated antibodies. After labeling, the cell suspension was washed and resuspended in 300 |L of 1% paraformaldehyde for flow cytometry. Fluorescein isothiocyanate (FITC), Phycoerythrin (PE) and Phyco- erthrin-cyanin 5.1 (PC5)-conjugated isotype controls and CD14-PE, CD40-FITC, CD80-FITC, CD86-FITC, CD11c- PE and HLA-DR-PC5 antibodies were purchased from BD PharMingen. Flow cytometric analysis was performed with Coulter Epics Flow cytometer (Beckman Coulter) and analyzed with WINMDI software (Purdue Univer­sity). To reduce inter-experimental variation, the mean fluorescence intensities for different CD markers were normalized with that of RPMI treated negative control as relative fluorescence intensity.

FITC-dextran endocytosis assay

Leukemic cell-derived and monocyte-derived DCs were harvested and resuspended in RPMI with 10% FBS. FITC- dextran (molecular weight 40 kDa; Sigma) was added at a final concentration of 1 mg/mL. Cells were then incu­bated at 37°C or 4°C for 1 h. Thereafter, the cells were washed four times with cold PBS and then analyzed with flow cytometer.

ELISA assay for cytokines

The supernatants from DCs cultures were collected after harvesting the cells and stored at -80°C until assayed for cytokines. The levels of IL-12p70 and IL-10 were then measured in duplicate with human Duoset® ELISA Kit (R&D Systems Inc.). The detection ranges for IL-12 and IL- 10 were 31.25-2000 pg/mL and 62.5-4000 pg/mL, respectively.

Allogeneic mixed lymphocyte reaction

The leukemic cell-derived and monocyte-derived DCs were irradiated with a gamma-irradiator (Gammacell 1000 Elite, MDS Nordion Inc., Canada) at 30 Gy and co- cultured at the ratio of 1:10 with 1 x 105 allogeneic responder CD3+T cells in flat-bottom 96-well microtiter plates. Bromodeoxyuridine (BrdU) was added into the wells 16 h before the end of five-day culture. Cell prolifer­ation during the last 16 h of the five-day culture was quan­tified by the Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Molecular Biochemicals).

Statistical analysis

Comparisons between means were based on nonparamet- ric Student's t test (2-tailed). For more than two groups, we compared the means with one-way ANOVA. The dif­ference was statistically significant when p < 0.05.

Competing interests

The authors declare that they have no competing interests. Authors' contributions

WKC designed, performed experiments and wrote up the manuscript; CCC performed experiments, KWL designed the experiments and revised manuscript, YLL provided materials and revised manuscript; GCFC designed, ana­lyzed the data and wrote up the manuscript.

Acknowledgements

This study was supported by the Committee on Research and Conference Grants (CRCG), The University of Hong Kong, Ho-Tung SK Charitable Fund, and Pau K.W. Charitable Fund. We thank Prof. Lin ZB for providing the purified polysaccharides.

References

  • 1. Copeland DR, Silberberg Y, Pfefferbaum B: Attitudes and prac­tices of families of children in treatment for cancer. A cross- cultural study. Am J Pediatr Hematol Oncol 1983, 5(1):65-7l.
  • 2. Kelly KM, Jacobson JS, Kennedy DD, Braudt SM, Mallick M, Weiner MA: Use of unconventional therapies by children with cancer at an urban medical center. J Pediatr Hematol Oncol 2000, 22(5):412-416.
  • 3. Sparber A, Wootton JC: Surveys of complementary and alter­native medicine: Part II. Use of alternative and complemen­tary cancer therapies. J Altern Complement Med 2001, 7(3):28l-287.
  • 4. Ramsay NA, Kenny MW, Davies G, Patel JP: Complimentary and alternative medicine use among patients starting warfarin. Br J Haematol 2005, 1 30(5): 777-780.
  • 5. Chan G, Mullen P, Ha S, Wong G, Lee T, YL L: Use of alternative medical treatments in paediatric oncology patients in Hong Kong. Annual Scientific Meeting of the Paediactric Society of Hong Kong 1998.
  • 6. Lin ZB, Zhang HN: Anti-tumor and immunoregulatory activi­ties of Ganoderma lucidum and its possible mechanisms.

Acta Pharmacol Sin 2004, 25(11): 1387-1 395.

  • 7. Wasser SP, Weis AL: Therapeutic effects of substances occur­ring in higher Basidiomycetes mushrooms: a modern per­spective. Crit Rev Immunol 1999, 19(1):65-96.
  • 8. Berges C, Naujokat C, Tinapp S, Wieczorek H, Hoh A, Sadeghi M, Opelz G, Daniel V: A cell line model for the differentiation of human dendritic cells. Biochem Biophys Res Commun 2005, 333(3):896-907.
  • 9. Kim KD, Choi SC, Noh YW, Kim JW, Paik SG, Yang Y, Kim KI, Lim JS: Impaired responses of leukemic dendritic cells derived from a human myeloid cell line to LPS stimulation. Exp Mol Med 2006, 38(1):72-84.
  • 10. Cao LZ, Lin ZB: Regulation on maturation and function of den­dritic cells by Ganoderma lucidum polysaccharides. Immunol­ogy letters 2002, 83(3): 163-169.
  • 11. Chan WK, Lam DT, Law HK, Wong WT, Koo MW, Lau AS, Lau YL, Chan GC: Ganoderma lucidum mycelium and spore extracts as natural adjuvants for immunotherapy. J Altern Complement Med 2005, 11(6): 1047-1057.
  • 12. Lin YL, Liang YC, Lee SS, Chiang BL: Polysaccharide purified from Ganoderma lucidum induced activation and maturation of human monocyte-derived dendritic cells by the NF-kappaB and p38 mitogen-activated protein kinase pathways. Journal of leukocyte biology 2005, 78(2):533-543.
  • 13. Masterson AJ, Sombroek CC, De Gruijl TD, Graus YM, Vliet HJ van der, Lougheed SM, Eertwegh AJ van den, Pinedo HM, Scheper RJ: MUTZ-3, a human cell line model for the cytokine-induced differentiation of dendritic cells from CD34+ precursors. Blood 2002, 100(2):701-703.
  • 14. Lieu CW, Lee SS, Wang SY: The effect of Ganoderma lucidum on induction of differentiation in leukemic U937 cells. Antican­cer Res 1992, 12(4): 121 1-1215.
  • 15. Muller CI, Kumagai T, O'Kelly J, Seeram NP, Heber D, Koeffler HP: Ganoderma lucidum causes apoptosis in leukemia, lym- phoma and multiple myeloma cells. Leuk Res 2006, 30(7):84l-848.
  • 16. Lu QY, Sartippour MR, Brooks MN, Zhang Q, Hardy M, Go VL, Li FP, Heber D: Ganoderma lucidum spore extract inhibits endothelial and breast cancer cells in vitro. Oncol Rep 2004, 12(3):659-662.
  • 17. Sliva D, Sedlak M, Slivova V, Valachovicova T, Lloyd FP Jr, Ho NW: Biologic activity of spores and dried powder from Gano­derma lucidum for the inhibition of highly invasive human breast and prostate cancer cells. J Altern Complement Med 2003, 9(4):49l-497.
  • 18. Buggins AG, Milojkovic D, Arno MJ, Lea NC, Mufti GJ, Thomas NS, Hirst WJ: Microenvironment produced by acute myeloid leukemia cells prevents T cell activation and proliferation by inhibition of NF-kappaB, c-Myc, and pRb pathways. J Immunol 2001, 167(10):602l-6030.
  • 19. Hsu HY, Hua KF, Lin CC, Lin CH, Hsu J, Wong CH: Extract of Rei- shi polysaccharides induces cytokine expression via TLR4- modulated protein kinase signaling pathways. J Immunol 2004, 1 73(10):5989-5999.
  • 20. Shao BM, Dai H, Xu W, Lin ZB, Gao XM: Immune receptors for polysaccharides from Ganoderma lucidum. Biochem Biophys Res Commun 2004, 323(1): 133-141.
  • 21. Liu E, Law HK, Lau YL: BCG promotes cord blood monocyte- derived dendritic cell maturation with nuclear Rel-B up-reg­ulation and cytosolic I kappa B alpha and beta degradation. Pediatric research 2003, 54(1): 105-112.

Publish with BioMedCentral and every scientist can read your work free of charge

"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK

Your research papers will be:

  • available free of charge to the entire biomedical community
  • peer reviewed and published immediately upon acceptance
  • cited in PubMed and archived on PubMed Central
  • yours - you keep the copyright

Submit your manuscript here:               i } BioMedcentral

http://www.biomedcentral.com/info/publishing_adv.asp   ^

Lipids in Health and Disease BioMed central

Open Access

Research

( G.lucidum'un(Reishi) Kolesterol düşürücü etkinliği)

Cholesterol-lowering properties of Ganoderma lucidum in vitro, ex vivo, and in hamsters and minipigs

A Berger*1,2, D Rein1,3, E Kratky1, I Monnard1, H Hajjaj1,4, I Meirim1, C Piguet-Welsch1, J Hauser1,5, K Mace1 and P Niederberger1

Address: Nestlé Research Center, Lausanne 26, 1000, Switzerland, 2Paradigm Genetics, Research Triangle Park, NC 27709-4528, USA, 3BASF Plant Science Holding GmbH, Agricultural Center, BPH-Li 555, Limburgerhof, 67114, Germany, 4Univerity des Sciences et de Technologie de Lille, B.P. 179, Villeneuve d'Ascq Cedex, 59653, France and 5University of Lausanne, Institut de Biologie Cellulaire et de Morphologie, 1015, Lausanne, Switzerland

Email: A Berger* - aberger@paragen.com; D Rein - dietrich.rein@basf-ag.de; E Kratky - elenanm@earthlink.com; I Monnard - irina.monnard@rdls.nestle.com; H Hajjaj - H_hajjaj@yahoo.com; I Meirim - Isabelle.Meirim@rdls.nestle.com; C Piguet- Welsch - Cristal.Piguet-Welsch@rdls.nestle.com; J Hauser - jonas.hauser@ibcm.unil.ch; K Mace - catherine.mace@rdls.nestle.com; P Niederberger - peter.niederberger@rdls.nestle.com * Corresponding author

Published: 18 February 2004                                                                                         Received: 22 January 2004

,, ,, , ~ ,„„. „„                                                                                                          Accepted: 18 February 2004

Lipids in Health and Disease 2004, 3:2                                                                                        v                             '

This article is available from: http://www.lipidworld.eom/content/3/l/2

© 2004 Berger et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

Abstract

Introduction: There has been renewed interest in mushroom medicinal properties. We studied cholesterol lowering properties of Ganoderma lucidum (Gl), a renowned medicinal species.

Results: Organic fractions containing oxygenated lanosterol derivatives inhibited cholesterol synthesis in T9A4 hepatocytes. In hamsters, 5% Gl did not effect LDL; but decreased total cholesterol (TC) 9.8%, and HDL 11.2%. Gl (2.5 and 5%) had effects on several fecal neutral sterols and bile acids. Both Gl doses reduced hepatic microsomal ex-vivo HMG-CoA reductase activity. In minipigs, 2.5 Gl decreased TC, LDL- and HDL cholesterol 20, 27, and 18%, respectively (P < 0.05); increased fecal cholestanol and coprostanol; and decreased cholate.

Conclusions: Overall, Gl has potential to reduce LDL cholesterol in vivo through various mechanisms. Next steps are to: fully characterize bioactive components in lipid soluble/insoluble fractions; evaluate bioactivity of isolated fractions; and examine human cholesterol lowering properties. Innovative new cholesterol-lowering foods and medicines containing Gl are envisioned.

Background

In Kampo Chinese folk medicine, mushrooms have been known to have medicinal properties since AD1200 [1].

In recent years, there has been interest in the cholesterol lowering properties of mushrooms, including Ganoderma lucidum (Reishi-, Longevity-, or Phantom mushrooms, Biladi Top, Young-zhi, The King Of Herbs, Ling Zhi in

Chinese, Saru-no-koshikake and Mannendake in Japa­nese) [2,3], Pleurotus ostreatus (Oyster mushroom) [4-8], Volvariella volvacea (Straw mushroom) [9], Agaricus bis- porus (champignon) [10], Agaricus campestris [11], Auricu- laria auricula (Tree-ear), Tremella fuciformis (White-jelly leaf) [12,13], Grifola frondosa (Maitake mushroom)

  • [14.15] , Lentinus erodes (Shiitake) and isolated fractions
  • [14.16] , and Polyporus confluens (Ningyotake) [17]. In an earlier work, Kaneda and Tokuda [18] studied cholesterol lowering properties of ether-, water- and ethanol extracts from caps and stems from Lentinus edodes, Auricularia pol­ytrichia (Jews-ear), Flammulina velutipes, and Agaricus bis- porus. The majority of these studies were performed in rats. The cholesterol lowering properties of Cordyceps sinensis were studied in humans [19].

Our focus is Gl, an important medicinal fungus belonging to the Ganodermataceae family that has been studied for its many interesting health promoting properties, includ­ing anti-tumor, anti-inflammatory, and anti-platelet aggregation [20-27]http://kyotan.com/lectures/lectures. Indeed, entire books, symposiums, organizations (e.g., the Ganoderma International Research Institute, New York) and therapies have been devoted to Gl. As further testament to its importance, in ancient Chinese times, a Reishi Goddess (Reishi senshi) was even worshipped to bestow health, life and eternal youth.

As described, Gl has been occasionally studied for its cho­lesterol lowering- and hypotensive properties in the rat [2] and rabbit [28], but not in more physiological cholesterol models [29] such as minipigs. Gl can supposedly lower cholesterol in humans, but the work was not peer- reviewed nor adequately described [24]http://www.gano therapyusa.com/DXN/docs/whatis.htm.

Like humans, minipigs are omnivours, and their lipid and steroid metabolism, and digestive and cardiovascular physiology closely resembles that of humans [30-32]; whereas in contrast to humans, rodents carry most of the cholesterol in HDL fractions unless they are fed high satu­rated fat and cholesterol rich diets, which has the effect of shutting down LDL receptors [29].

The components in Gl that may lower cholesterol are not known, but may include ganoderan-type glucans [22,33,34], hetero-P-glucans, glucan-protein complexes (xyloglucans, uronic acid-P-glucans), other fibers, lectins [25], terpenoid triterpenes [35-38], ergostane sterols [39], and highly oxygenated ganoderic acid-type, lanostanoid triterpenes [38-42]. Gl fibrous components could affect cholesterol absorption and bile acid recycling, whereas lipophilic components could affect cholesterol synthesis.

Gl may affect cholesterol synthesis at the committed 3- hydroxy-3-methylglutaryl coenzyme A reductase (HMG- CoA reductase) rate-limiting step; or at the latter lanos- terol 14a-methyl demethylase:cytochrome P-450 demethylase (P-45014DM) step [43,44], catalyzing the rate limiting step in lanosterol-cholesterol conversion. In non-Gl mushroom species, inhibition of squalene syn- thetase by zaragozic acid fungal metabolites has also been reported in primates [45].

Herein, we tested the effects of Gl on cholesterol metabo­lism in hepatic T9A4 human cells, a hamster small animal model, and a minipig larger animal model having differ­ent lipoprotein cholesterol distribution than the hamster model. Animal models were fed cholesterol-containing diets described in Tables 1, 2.

Results

Active components in Gl and in vitro activity

Organic and aqueous Gl phases did not contain HPLC- detectable lovastatin. The organic extracted phase strongly

Table 1: Proximate analysis of Nafag 924 test diets for hamsters[1]

Weight %

Component                                                Control                                                        2.5% Gl                                                        5.0% Gl

Carbohydrate (by difference)

31.8

31.0

30.3

Starch

23.6

23.0

22.5

Crude Protein

15.6

15.2

14.8

Water

8.0

7.8

7.6

Crude fat

4.4

4.3

4.2

Ash

4.7

4.6

4.5

Crude fiber

3.5

3.4

3.3

Essential amino acids

3.4

3.3

3.2

Gl extract

0.0

2.5

5.0

Vitamin mix (includes choline)

2.4

2.3

2.3

Minerals (Ca, P, Mg, K, Na)

2.3

2.2

2.2

Trace elements

0.3

0.3

0.3

  

inhibited cholesterol biosynthesis (ID50 = 1.3 |g/mL, rel­ative to 0.4 for lovastatin), while the aqueous phase was ineffective (ID50 > 330). Various highly oxygenated lanos- tanoid triterpenes, and 32-methyl- and 26-oxo sterols were found in the organic phase, and likely contributed to inhibition of cholesterol synthesis. A 20% EtOAc/hexane fraction contained ganoderal A; and a 50% EtOAc/hexane contained ganoderols-A and B, and Y ganoderic acid.

Body and organ weights, and food intake of hamsters

Body weights ranged from 68.7-70.8 and 83.2-86.4 g for the experimental groups on D1 and D18, respectively, without significant differences relative to control, on D1, D18, or D18 minus D1. D18 liver and cecum relative weights (g organ/100 g body wt) were 2.77-2.84 and 0.52-0.56 for the various groups, respectively, without significant differences relative to control. Daily food intake was 7.1-7.8 g food/d averaged over D1-16; there were no significant differences relative to control.

Cholesterol and triacylglycerol in hamsters

Starting D1 TC levels did not differ among the groups, whereas there were differences in D1 TAG (Table 3). Gl at 2.5 and 5.0% reduced D18 TAG (likely due to D1 TAG dif­ferential starting values). Gl at 2.5% did not reduce D18

TC, LDL or HDL. With 5.0% Gl, there was a statistical trend (P < 0.10) to reduce TC and HDL; LDL was not affected. Similarly to the higher dose of Gl, lovastatin decreased D18 TC and HDL, but not LDL. LDL/HDL ratio was not statistically significantly different for any dietary treatments relative to control.

Fecal bile acids and neutral sterols in hamsters

Gl (2.5%) increased fecal total bile acids and chenodeox- ycholate (Table 4). Both Gl doses increased coprostanol 3- one, whereas, 5% Gl decreased cholestanol. Lovastatin had no significant effects on bile acids or neutral sterols examined.

Ex vivo hepatic HMG-CoA reductase activity in hamsters

Lovastatin did not affect de-phosphorylated activity, and phosphorylated activity not examined (Table 5). In absence of NaF (inhibitor of phosphatase) and in pres­ence of 2.5 and 5 % Gl, 3-hydroxy-3-methylglutaryl-CoA reductase activity in hamster hepatic microsomes (pmol/ min/g liver) was reduced 2.1- and and 1.5 fold, respec­tively, relative to the control. In presence of NaF, 2.5% and 5% Gl reduced HMG-CoA reductase 3.5- and 1.9­fold, respectively, relative to control.

Table 3: Plasma cholesterol and triacylglycerol in hamsters treated with G. lucidum and lovastatin (mmol/L)

Group

TC

TC

TAG

TAG

VLDL

LDL

HDL

LDL/HDL

 

DI

DI8

DI

DI8

DI8

DI8

DI8

DI8

Control

3.57

3.48

I.I0

I.08

0.22

0.5I

2.75

0.I9

Lovastatin

3.39

3.I6a

I.07

0.98

0.I8a*

0.48

2.50a

0.I9

Gl (2.5%)

3.46

3.40

0.64a

0.90a

0.I8

0.53

2.69

0.20

Gl (5%)

3.34

3.I4a*

0.89a*

0.92a

0.20

0.49

2.44a*

0.20

 

TC, total cholesterol; TAG, triacylglycerol; VLDL, LDL, HDL, very low-, low-, and high-density lipoproteins; D, day; LOVA, 20 mg/kg diet. Values represent mean of 6 animals. 'Significantly different from the control (P < 0.05 or *P < 0.10), students, unpaired, l-tailed,t-test, equal variances.

Table 4: Fecal bile acids and neutral sterols in hamsters treated with G. lucidum and lovastatin (nmol/g dry feces/d)

Bile acids                                                                                                     Neutral Sterols

Group                 C LC DC CDC UDC Total BA COP-ol COP-3-one CHOL erol CHOL anol Total NS

Control

15.0

I4.3

I0.7

5.I

I.7

46.7

I I.5

ND

7.2

I8.4

37. I

Lovastatin

I5.0

I2.8

II.6

6.2

I.6

47.I

I2.0

ND

6.7

I8.9

37.6

Gl (2.5%)

I6.9

I6.2

I4.3

a.5 8.

I.9

57.8a

II.7

4.2a

7.7

I6.9

40.6

Gl (5%)

I2.2

I3.9

I2.0

6.4

I.4

45.8

I2.0

7.8a

7.0

I4.I a

4I.0

 

Feces were collected quantitatively at the conclusion of the experiment (Dl8). C, cholate; LC, lithocholate; DC, deoxycholate; CDC, chenodeoxycholate, UDC, Ursodeoxycholate; COP, coprostan; CHOL, cholest; BA, bile acids; NS, neutral sterols. ND, not detected. Values represent mean of 6 determinations. aSignificantly different from the control (P < 0.05), students unpaired, 2-tailed t-test, equal variances. No statistical trends (P < 0.10) existed in the data set.

Table 5: HMG-CoA reductase activity in hamster hepatic microsomes (pmol [14C]mevalonolactone/min/mg microsomal protein)

Group                                                                                 Activity-NaF                                                                       Total activity+NaF

Control                                                                                12.36                                                                                  9.30

Lovastatin                                                                          13.34                                                                                    ND

Gl (2.5%)                                                                              5.86a                                                                                 2.66a

Gl (5%)                                                                                 8.08a                                                                                 4.86a

Refer to the text for methodologic details. [l4C]mevalonate was converted to [l4C]mevalonolactone with 10 M HCl before the TLC step. Values above were corrected for recovery (15-63%) with [3H]mevalonate. G. lucidum, but not lovastatin, was found to inhibit ex vivo synthesis whether: recovery was accounted for; results were expressed per gram liver weight; [l4C]mevalonolactone was first extracted into organic solvent and then applied to TLC plates [79] rather than applying aqueous extracts directly to TLC plates [55] as reported above (data not shown); and whether or not endogenous phosphatase activity was inhibited with 50 mM NaF (shown above). Values represent mean of 6 determinations. aSignificantly different from control, students unpaired, 2-tailed t-test, equal variances (P < 0.05). ND, not determined.

Fractional cholesterol synthesis rate in hamsters

In hamsters, 24 h FSR values (Atom% enrichment D17- 18) were 1.68 ± 0.20, 1.91 ± 0.16, 1.75 ± 0.36, and 2.29 ± 0.05 (mean of n = 6, ± 1 SEM) for control, lovastatin, 2.5%-, and 5% Gl, respectively. Values were not statisti­cally significantly different from control.

Body weights of minipigs

Minipig body weights increased equivalently with Gl and lovastatin from 19.0-26.9 kg over D1-28. Similar weights per age were previously reported for experimentally-fed Gottingen minipigs [46].

Cholesterol and triacylglycerol in minipigs

The experimental diet increased TC 27-30% from D1-14 (Table 6). In the Gl-fed group, TC significantly decreased 12.5% from D14-21, but not further from D21-29; the decrease in TC from D14-29 was 20% (P < 0.01). Lovasta­tin did not significantly decrease TC during D14-21 (P > 0.13), D21-29, nor D14-29; but TC did decrease >10% in two pigs from D14-21.

There were no significant differences in TAG and VLDL with Gl or lovastatin (Table 6). VLDL was however a minor lipoprotein pool. Lovastatin had not significant effects on LDL nor HDL; Gl decreased LDL 26% and HDL 16% (P < 0.01; D14 vs 29). Gl did not affect statistically significantly affect LDL/HDL since both individual parameters decreased from D14-29.

Fecal bile acids and neutral sterols in minipigs

The high cholesterol-fat diet decreased chenodeoxycho- late; and increased coprostanol, coprostan 3-one, and cholesterol from D1-14 (P < 0.05 or < 0.10; Table 7). Gl trended to increase cholestanol (D14 vs 29; P < 0.10).

Discussion

Active components in Gl and in vitro activity

As described, lovastatin was not detected in our Gl mush­room preparations. By contrast, statin-like compounds have been found in oyster mushrooms [47] and Chrysospo- rium pannorum [48].

Table 6: Plasma cholesterol and triacylglycerol in minipigs treated with G. lucidum and lovastatin (mmol/L)

Group

TC

TC

TC

TC

TAG

TAG

TAG

TAG

VLDL

VLDL

LDL

LDL

HDL

HDL

LDL/HDL

LDL/HDL

 

Dl

Dl4

D2l

D29

Dl

Dl4

D2l

D29

Dl4

D29

Dl4

D29

Dl4

D29

Dl4

D29

Gl (2.5%)

2.47a

3.2lbc

2.81

2.58

0.53

0.57

0.80

0.69

0.07

0.09

l .45c

l.08

l.69c

l.42

0.88

0.79

Lovastatin

2.36a

3.00

2.44

2.81

0.50

0.60

0.59

0.7l

0.l0

0.09

l .40

l.29

l.50

l.43

0.95

0.9l

Lovastatin was administered at 80 mg/d. Between DI-I4, all pigs received a high cholesterol and fat control diet; from DI5-29, pigs received either G. lucidum (Gl) extract or lovastatin. The same statistical conclusions were reached if all 10 pigs were compared between DI-14. Pigs were randomly selected to receive either GI or lovastatin before study commencement. Student's, paired, I-tailed, t-test, was utilized for statistical comparisons. Statistically significant changes (P < 0.05, I-tailed testing) in cholesterol parameters are indicated as follows: aDI vs. I4; bD 14 vs. 2I; cDI4 vs. 29. Abbreviations: refer to Table 3. There was a slight trend for 2.5% Gl to reduce LDL/HDL ratio between DI4-29 (P < 0.II, I -tailed testing).

 

Table 7: Fecal bile acids and neutral sterols in minipigs treated with G. lucidum and lovastatin (nmol/g dry feces)

 

 

Bile acids

 

 

 

Neutral Sterols

 

Group

NCT

C

CDC

COP-ol

COP-3-one

CHOL erol

CHOL anol

All pigs (Dl)

l .98

0.86

0.66a

2.4la

0.l0a

l.73a*

0.96

All pigs (Dl4)

l .98

l .33

0.l6

3.75

0.l7

2.96

0.9l

Gl (Dl4)

l.98

l.6l

0. l4

3.61

0.l8

3.37

0.87b*

Gl (D29)

l.98

0.8l

0. l6

4.44

0.l5

3.l5

l .22

Lovastatin (Dl4)

l.98

0.99

0. l9

3.92

0.l5

2.44

0.96

Lovastatin (D29)

l.98

l.23

0. l8

3.28

0.l6

2.34

l.l4

 

Feces were collected on Dl, 14 and 29. A quantitative fecal collection was not possible, hence results are expressed per gram of feces. Abbreviations: refer to Table 4, except, NCT, 23- Nor p cholanate 5a,7a,l2a triol. A paired, 2-tailed students t-test, equal variances, evaluated effects of the high cholesterol and fat diet, between Dl-14 (10 minipigs); and the effects of Gl or lovastatin between Dl5-29 (5 minipigs/group), indicated as follows: aDl vs 14, for all minipigs combined (P < 0.00l).bDl4 vs 29 (P < 0.05). *0.05 < P < 0.1, to indicate statistical trends.

 

We did however detect oxygenated lanosterol molecules such as 32-methyl- and 26-oxo sterols, ganoderols-A and

B, Y ganoderic acid, and ganoderals-A and B in the organic layer. The organic layer strongly inhibited cholesterol bio­synthesis from acetate. Similar or identical oxygenated lanosteroids had been previously reported in Gl [38-42], and found to inhibit conversion of 24,25-dihydrolanos- terol to cholesterol at the lanosterol 14 a-demethylase step [49-51], and also indirectly to inhibit HMG-CoA reductase activity [51]. The fact that the aqueous phase from Gl was ineffective at inhibiting cholesterol synthesis (ID50 > 330) suggests that hydrophilic molecules such as glucans and fibers in Gl do not affect conversion of acetate to cholesterol. Such molecules may however affect choles­terol absorption and bile acid recycling.

Ex vivo hepatic HMG-CoA reductase and fractional cholesterol synthesis rate in hamsters

The observed inhibition of ex-vivo HMG-CoA reductase activity in hamsters treated with Gl has similarly been observed with Gl in rats [51], and with pure lanosterol analogs [44,52]. Our lack of effect with lovastatin (4.3 | mol/kg body wt) contrasts results with the related statin, simvastatin, where 10, 30, and 60 |mol/kg body wt/d increased ex-vivo hepatic HMG-CoA reductase activity 2-,
17-, and 50-fold, respectively [53]. Lovastatin could have different effects on HMG-CoA reductase and other enzymes than simvastatin, and was not however exam­ined in the above study.

Lanosterol analogs such as those found in Gl are known to inhibit translation of HMG-CoA reductase mRNA, and may also accelerate protein degradation [44,52]. Gl may also affect cholesterol biosynthesis at latter biosynthetic steps such as the conversion of lanosterol [51], which could in turn, indirectly inhibit HMG-CoA reductase activity, as reported for statins in minipigs [53]. Indeed, it was reported that repression of the lanosterol 14 a- demethylase step can result in accumulation of 3 P- hydroxy-lanost-8-en-32-al, a known translational down- regulator of HMG-CoA reductase [54].

If Gl had direct physical effects on HMG-CoA reductase activity, this implies that even after the 16 h fast employed in hamsters, Gl components were still bound to the enzyme during the assay procedure [55]. After the 16 h fast, lovastatin could have been removed from the enzyme accounting for the lack of observed effects of lovastatin on ex-vivo HMG-CoA reductase activity. Due to removal of the drug, other statins have even been found to increase ex-vivo HMG-CoA reductase activity [56]. Hepatic ex-vivo HMG-CoA reductase activity and whole body cholesterol FSR are entirely different types of measurements. It is not clear why Gl and lovastatin did not influence cholesterol FSR in hamsters. In principle, the low saturated fat-choles­terol condition employed via use of a chow diet, should have led to a high endogenous rate of cholesterol synthe­sis, one that could be inhibited by Gl and lovastatin. It is conceivable that the Gl and lovastatin became decom­posed in the dietary mixture. To test this hypothesis, we re-extracted Gl and lovastatin from stored diets after cul­mination of the experiments, and found no differences in bioactive components analyzed, compared to the original starting materials (before addition to the diets; data not shown).

Cholesterol and triacylglycerol in hamsters and minipigs

Hamsters were fed a low-cholesterol chow-based diet with no added exogenous cholesterol or saturated fat. Under these conditions, there was not sufficient cholesterol to redistribute cholesterol from the HDL to LDL pool [29]. This is why in hamsters, 5% Gl and lovastatin reduced D18 TC and HDL, but not LDL [57,58].

Using the same types of diet, lovastatin was similarly found to preferentially reduce HDL in hamsters; and only when dietary saturated fat was added, were both LDL and HDL reduced [57].

Another factor contributing to the lack of strong effects in hamsters, and the total lack of effect in minipigs may be that the dose of lovastatin was insufficient. In hamsters, the employed dose of 2 mg lovastatin/100 g diet is ca. 4.3 |mol lovastatin/kg body wt. Himber et al. [57] treated hamsters with 25 |mol lovastatin/kg body wt, which low­ered HDL; or 50 |mol, which lowered LDL and HDL [57]. Morand et al. [53] found that 20-200 |mol simvastatin/ kg body wt was sufficient to reduce LDL. Ma et al. [59] reduced lipoproteins in hamsters with 100 mg lovastatin/ 100 g diet. In minipigs, we utilized a dose of 80 mg lovas- tatin/minipig/d, which may also have been on the low side. A dose of 24-42 mg was sufficient to lower lipopro­teins in Hyde Park minipigs [60]. Nevertheless, our partic­ular species, strain, and location of minipigs may have responded less aggressively to lovastatin (M. Huff, Per­sonal Communication, December 2000). In Göttingen minipigs, a dose of 80 mg simvastatin lowered LDL, whereas 240 mg lowered LDL and HDL [53]; simvastatin is likely more effective in minipigs than lovastatin at a similar dietary weight percent [61,62].

The reduction in TAG with Gl was likely due to lower D1 TAG values in the Gl groups relative to control. TAG reductions in hamster models typically occur under con­ditions of higher saturated fat intake [6,63]. In the only other peer-reviewed study examining cholesterol lowering properties of Gl in a small animal model, 5 dietary wt% dried Reishi mushroom powder was found to decrease TC in SHR rats; effects on VLDL, LDL and HDL were not stud­ied [2]. In minipigs, with the high fat-cholesterol feeding conditions employed, a Gl-induced inhibition of choles­terol synthesis should result in less availability of hepatic cholesterol for lipoprotein synthesis. In turn, this has the potential effect of reducing plasma VLDL cholesterol secretion, reducing LDL direct secretion; and possibly reducing VLDL-LDL conversion [64,65]. In the present work, we did not observe differences in TAG or VLDL in pigs fed either Gl or lovastatin, however this effect could have been missed since the VLDL pool represented only a small lipoprotein pool and/or there was efficient VLDL- LDL conversion. The reductions in both LDL and HDL with Gl is consistent with that seen with higher statin doses [53].

Fecal bile acids and neutral sterols in hamsters and minipigs

In hamsters, Gl increased fecal total bile acids and cheno- deoxycholate, whereas both doses, increased coprostanol 3-one; the 5% dose decreased cholestanol for unclear rea­sons. An increase in fecal chenodeoxycholate likely indi­cates production or recycling of chenodeoxycholate was enhanced.

Plasma levels of cholestanol are positively associated with cholesterol absorption [66]; whereas decreased fecal cholestanol may indicate plasma cholestanol was increased and cholesterol absorption was enhanced. In minipigs, Gl tended to increase fecal cholestanol, the opposite pattern to that of hamsters fed 5% Gl. Coprosta- nol and coprostanol 3-one are the bacterial products of cholesterol, which are increased when fecal cholesterol is increased, or when gut flora are altered [67]. Since fecal cholesterol and coprostanol levels were not changed by either dose of Gl, it is not obvious why coprostanol 3-one accumulated.

Bile salts are now known to possess many different func­tions acting as detergents, activators of protein kinase C and phosphatidylinositol-3 kinase; and being important gene regulators [68,69]. Chenodeoxycholate, deoxycho- late, and their glycine and taurine conjugates can lead to farnesoid X receptor/retinoid X receptor (FXR/RXR)- induced activation of intestinal bile acid binding protein transcription (I-BABP), and suppression of CYP7a RNA and protein levels (FXR prevents liver X receptor (LXRa)- induced transactivation of CYP7a). CYP7a regulates the committed step in classical bile acid synthesis. Overall, an increased fecal level of chenodeoxycholate would mean less chenodeoxycholate is available to activate FXR. Less activation of FXR would lead to less bile acid recycling and less inhibition of bile acid synthesis, more hepatic choles­terol converted to bile acids, and a lowering of plasma cholesterol.

Overall, it is likely that fibrous and/or lipophilic sterol- like molecules in Gl altered the absorption and recycling of bile acids and neutral sterols, leading to altered fecal accumulation. Monitoring plasma levels of neutral sterols and bile acids, and quantifying conjugated and de-conju­gated bile acids, should help to clarify the potential importance of the observed trends.

Comparing in vitro, ex vivo, and in vivo results

In the present work, the in vitro experiments were per­formed with fractionated Gl extracts, whereas the ex-vivo and in vivo work utilized intact Gl. Intact Gl contains fibrous components, which may have affected bile acid and neutral sterol absorption and recycling. Fibrous com­ponents could also impair the uptake of lipophilic com­ponents, such as those inhibiting in vitro cholesterol synthesis. An additional complexity is that lipophilic components such as ergostane sterols [39] could also affect bile acid and neutral sterol levels. Thus, it is difficult to directly compare our in vitro and in vivo results. Feeding fractionated and intact mushrooms should help to unravel the in vivo bioactive components, as has been accomplished for oyster mushrooms [70].

Conclusions and key findings

In summary, GI was found to have cholesterol lowering potential in vitro, ex-vitro, and in two animal models, with some differences between the two animal models. It is possible that oxygenated lanosterol derivatives in Gl (partly characterized in the present work) contributed to this cholesterol lowering by decreasing cholesterol syn­thesis (changes in in vitro and ex-vivo, but not whole body, cholesterol synthesis were apparent in the present work). Fibrous components and glucans in Gl were likely respon­sible for the observed alterations in fecal neutral sterols and bile acids in both animal species, ultimately affecting cholesterol absorption and bile acid recycling and con­tributing to cholesterol lowering. Next steps are to exam­ine the cholesterol lowering properties of various doses of intact and fractionated, chemically characterized, Gl com­ponents in a placebo-controlled clinical trial. Animal experimentation should also utilize fractionated materi­als, and ideally, elucidate mechanisms of action of each bioactive component. Positive cholesterol-lowering results in such studies will pave the way for adding Gl to new cholesterol-lowering foods and medicines, alone, and in combination with other established cholesterol- lowering ingredients and drugs.

Materials and methods Materials

Gl was from Fermenta SA, Payerne, Switzerland. Mush­rooms were cultivated on a defined formula of sawdust, wheat straw and millet grain. Substrate was sterilized at 90°C for 48 h, then incubated with Gl seed material from Mycotec Sarl (Cernier, Neuchatel). Cultivation was with controlled temperature, light, humidity and carbon diox­ide concentration. Human hepatic T9A4 cells [71] were grown in LCM serum-free media under 3.5% CO2 at 37°C. Lovastatin was purchased as 20 mg Mevacor tablets (MSD Chibropharm GmbH, Haar, Germany). HMG-CoA reductase, DL-3-Glutaryl-3- [14C]-HMG-CoA (2216 MBq/ mmol), R-[5-3H] mevalonic acid ammonium salt (1443 MBq/mmol), and [1-14C] acetic acid sodium salt (2070 MBq/mmol) were from Amersham (Upsala, Sweden). a- 3-HMG-CoA (cold) and liquid scintillation cocktail were from Sigma (Buchs, Switzerland). LCM cell medium was from Biofluids (Rockville, MD). 5P-cholesteane-3a-ol, 5- a-cholestane and 2,3-nor-5P-cholanicacid-3a,7a,12a- triol were from Steraloids, Inc. (Newport, Rhode Island); other steroid standards were from Sigma, and Calbiochem (La Jolla, California). Methanolic HCl and Sylon HTP were from Supelco (Buchs, Switzerland). The Cobas Bio autosampler was from Hoffmann-La Roche (Basel, Swit­zerland) and reagents were from Roche Diagnostics (Rotkreuz, Switzerland). Total Cholesterol Kit 352 and Triacylglycerol Kit 336 were from Sigma. Deuterium was from Cambridge Isotope Laboratories (Andover, MA). Zn catalyst was from Biochemical laboratories (University

Bloomington, IN). Silica gel thin layer chromatography (TLC) plates were from Merck Eurolab (Dietikon, Switzer­land). Coomassie Plus-200 protein assay reagents and bovine serum albumin fraction V were from Pierce (Rock­ford, Illinois). All other chemicals were from Sigma.

Preparation of Gl for in vitro testing

Fruiting bodies from Gl (20 g) were dried, milled and macerated in 0.4 L MeOH/H2O (4:1, v/v) at room temper­ature for 3d. The mixture was then filtered, evaporated, re- dissolved in H2O, acidified to pH 3 with 3 M HCl, extracted 3 x with 150 mL ethyl acetate, and the organic phase evaporated under vacuum at 30°C, re-dissolved in 10 mL MeOH, and dried with Na2SO4, for HPLC analyses and in vitro testing.

Chemical analysis of Gl

The presence of lovastatin in Gl was determined by HPLC with a Nucleosil 100-5 C18 column (250 x 4 mm; Mach- erey-Nagel, Oensingen, Switzerland) and a Lichrospher 100 RP-18 post column (Merck, Glattbrugg, Switzerland). Solvent A was H3PO4/H2O (1:2000, by vol); solvent B was acetonitrile. Separation was initiated with a linear gradi­ent of 95% A, 5% B, reaching 50% A, 50% B in 45 min, 30% A, 70% B in 46 min, 10% A, 90% B in 48 min, and 0% A, 100% B in 50 min; the run was continued isocrati- cally 4 min. Initial conditions were maintained 6 min for re-equilibration; the flow rate was 1 mL/min. The detector was a G1315 A, series 1100 detector (Hewlett Packard, Meyrin, Switzerland); absorbance was measured at 254 nm. After selective extraction and purification with differ­ent adsorbents and solvents, ganoderols and ganoderic acids were detected by mass spectroscopy and NMR (details to be published separately).

In vitro activity of Gl extracts

Human hepatic T9A4 cells were grown in LCM serum-free media under 3.5% CO2 at 37°C. Cells were seeded in 24- well plates and at confluence, incubated with 1 mM 14C- acetate (1 mCi/mmol) for 20 h ± mushroom extracts. Lip­ids were extracted from cells by incubating 2 x with 1.5 mL hexane/isopropanol (3:2, by vol) for 30 min at room temperature. Combined organic extracts were dried under N2, re-dissolved in hexane, and separated by TLC with hexane/diethyl ether/acetic acid (75:25:1, by vol). Choles­terol synthesis was determined by measuring incorpora­tion of 14C from acetate to cholesterol. Radioactivity was assessed with an instant imager and expressed as percent of control.

Administration of Gl and lovastatin to hamsters

Male Golden Syrian hamsters (Harlan, UK), 3-4 wks, 40­60 g, were housed individually in Macrolon Type 3 cages with 12 h alternating periods of light and darkness. Dur­ing 3 wks preceding treatment, hamsters were fed Nafag 924 hamster complete diet (# 3132/20, Eberle Nafag AG, Gossau, Switzerland; Table 1). Following body weight randomization, groups consisted of 6 hamsters/group receiving either: Nafag diet (control), Nafag mixed with 2 mg lovastatin /100 g diet (powdered in liquid N2); or Nafag mixed with 2.5 or 5.0% dried Gl. Hamsters were fed experimental diets for 17 d. Lovastatin is an inhibitor of HMG-CoA reductase [72], and was used as a positive con­trol. Dietary intake was recorded daily, body weights weekly. Feces were collected on D15-18. Hamsters were injected subcutaneously with 250 |L D2O on D17 and killed under anesthesia with isoflurane on D18. Follow­ing a 16 h fast, D1 (0.5 mL) and D18 blood (>3 mL) were obtained from the retro-orbital cavity and cardiac vein, respectively, and transferred to EDTA tubes. Plasma was prepared by centrifugation at 1500 g, 15 min, at 4°C. Plasma, and hepatic and cecum tissues were stored at - 80°C. Animal procedures were authorized by Service Vétérinaire du Canton de Vaud, Switzerland, protocol 1247.

Administration of Gl and lovastatin to minipigs

Nine female and one male Göttingen minipig(s) (Jörg Farm in Bern Switzerland; Minipig-Primärzucht, Auswill, Switzerland) aged 6-12 mo (18-20 kg), with white (7) and black (3 minipigs) colorations, were housed in a 30 m2 box with normal light/dark cycle, and kept at room temperature. Females were chosen because they have fewer age-related lipid modifications and higher lipid concentrations than males [73]. One male was acciden­tally provided in the delivery, however its total cholesterol (TC), lipoproteins, bile acids and neutral sterols were sim­ilar to that of other minipigs. Minipigs were randomly dis­tributed by weight into two separately housed groups, marked with a plastic label in the ear, and fed twice daily for 11 d with powdered commercial pig chow (Diet 574, Minipig-Primärzucht). During a subsequent 4 d adapta­tion period, minipigs were fed an acclimatization mixture of chow and increasing amounts of powdered hypercholesterolemic control diet (custom diet 2604, Kliba, Kaiseraugst, Switzerland; Table 2) from 0% to 100%, in steps of 25%, designed after Burnett et al. [64,74], that was consistent with Göttingen minipig nutri­tional needs [75]. During the following 2 wks (D15-29), groups were fed control hypercholesterolemic diet pre- mixed with 2.5% Gl extract; or hypercholesterolemic diet plus 80 mg lovastatin/pig/d (in four 20 mg tablets) [53], hand fed to each minipig, mornings, in half an apple. For acclimatization, on D12-14, minipigs received a half apple without lovastatin. The study was blinded in that the diets were coded, and the mushroom extract was referred to as "Nestlé Special Fiber." Food intake was 3.5% of body wt/d (based on group average wt), readjusted weekly, to provide sufficient, but not excessive, calories [64,65,75]. Diets were distributed at 0700 and 15h00, and spread linearly on a clean cement surface to facilitate indi­vidual consummation. Distilled water was provided ad libitum. Toys and human contact were provided to avoid boredom. Fasting 16 h blood samples (10 mL; 20 mL on D29) were collected in EDTA tubes on D1, 15, 22, 28, and 29 from anterior vena cava. Plasma was prepared by cen- trifugation as described for hamsters, and stored at -80°C. Blood collection began at 0800 following injection of the intra-muscular relaxant Dormicum® (Hoffmann La Roche, Basel, Switzerland), then the tranqulizer Stresnil® (Janssen Pharmaceuticals, Beerse, Belgium). After blood sampling on D1, 15 and 29, minipigs were isolated for 2 h maxi­mum for individual fecal collections. Some minipigs did not defecate during this period, whereas others defecated again following return to their groups. Hence, the morn­ing fecal collection was qualitative. Feces were stored at - 40°C under N2. Body weight was recorded weekly, and food intake recorded each morning. Minipigs were donated to the University of Geneva at the study's conclu­sion. Animal procedures were authorized by Service Vétérinaire du Canton de Geneve, Switzerland, protocol 1315, authorization 31.1.1014/1719/1.

Cholesterol and triacylglycerol measurements in hamsters and minipigs

Plasma total cholesterol and triacylglycerol were meas­ured using commercial kits and a Roche Cobas Bio autosampler. Plasma lipoproteins were separated by size- exclusion HPLC as previously described [63].

Fractional cholesterol synthesis rate measurements in hamsters and minipigs

Measurements of water- and cholesterol deuterium enrichment were performed with a Finnigan Thermoquest Delta XL plus Isotopic Ratio Mass Spectrometer (Bremen, Germany) as previously described [76,77]. Fractional syn­thesis rate (FSR) of free cholesterol was calculated from a plasma sample collected 24 h after deuterium oxide sub­cutaneous injection as follows: FSR (in % pool/d) = 100 x (cholesterol enrichment/(water enrichment x 0.478)). Due to technical reasons, there were insufficient values in the minipig experiments to reach interpretable conclusions.

Fecal bile acids and neutral sterol measurements in hamsters and minipigs

Fecal neutral sterols and bile acids were extracted from lyopholized feces, deconjugated, derivatized with Sylon HTP and analyzed by gas chromatography as previously described with internal standards: 5-a-cholestane for neu­tral sterols; 2,3-nor-5P-cholanic acid-3a,7a,12a-triol for bile acids [63].

Hepatic ex-vivo HMG-CoA reductase measurements in hamsters

Freshly excised liver (300 mg) was collected after 16 h fast of hamsters, minced with scissors, and homogenized with 0.4 mL buffer (50 mM KH2PO4, 0.1 M sucrose, 50 mM KCl, 50 mM NaCl, 30 mM EDTA, and 2 mM dithiothrei- tol, ± 50 mM NaF) with a Potter-Elvehjem S homogenizer with 400 rpm/5 strokes, on ice, after Conde et al. [55]. NaF inhibits dephosphorylation of HMG-CoA reductase by inactivating phosphoprotein phosphatases, yielding total phosphorylated HMG-CoA reductase activity. After washing homogenizer with 0.2 mL buffer, homogenate was centrifuged at 10000 g, 15 min, at 4°C. Supernatant was decanted, 0.4 mL cold buffer added, and the tube vor- texed and re-centrifuged. Pooled post-mitochondrial supernatants were spun in 1.5 mL ultracentrifuge tubes at 150000 rpm, 10 min, at 4°C in a Sorvall Discovery M 150 micro ultracentrifuge (Kendro Laboratory Products SA, Carouge-Geneva, Switzerland), and microsomal fractions stored at -80°C. Microsomal protein (200 |g, 10-18 |L) was pre-incubated 10 min at 37°C in an agitating bath, then incubated 15 min with 50 |L substrate solution (buffer plus 90 mM glucose-6-phosphate, 72 mM EDTA, 9 mM NADP, 6.2 nmol cold HMG-CoA (0.12 mM), 1.3 nmol [14C]HMG-CoA (0.0025 MBq), 0.3 IU glucoses- phosphate dehydrogenase, and 0.024 MBq [3H]meval- onic acid as recovery standard). After 15 min, reaction was terminated with 25 |L 10 M HCL, then incubated 30 min at 37°C for mevalonate-mevalonolactone conversion. Following centrifugation at 1000 g, 1 min, at 4°C to remove denatured protein, supernatant was applied to activated (1 h, 105°C) TLC plates, developed in fresh ben­zene-acetone (1:1, by vol), the mevalonolactone region scraped (based on migration of cold standards and X-ray film visualization; Rf 0.42-0.5), and radioactivity meas­ured in 10 mL scintillation cocktail.

Statistics

Differences between groups were tested by unpaired/ paired, one-tailed/two-tailed, student t-tests, equal vari­ances, as appropriate for different measurements. Statistical significance was evaluated at P < 0.05 unless stated otherwise.

List of abbreviations

D, day; FPLC, fast protein liquid chromatography; GC, gas chromatography; Gl or G. lucidum, Ganoderma lucidum; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HPLC, high pressure liquid chromatography; TLC, thin layer chromatography; TAG, triacylglycerol; TC, total cholesterol.

Authors' contributions

AB wrote and compiled the majority of the manuscript, was responsible for minipig studies, and served as project leader for animal cholesterol research. DR developed methods for cholesterol lipoprotein measurements, and was responsible for hamster studies. EK, I. Monnard, and JH assisted in both animal studies, and developed meth­ods for neutral sterols, bile acids and ex vivo measure­ments. HH developed methods to chemically analyze Gl.I. Meirim and CPW developed methods for cholesterol syn­thetic rates. KM was responsible for in vitro biological test­ing of Gl extracts. P.Niederberger served as overall project leader.

Acknowledgements

The authors would like to thank P. Weber of Champitec for providing Gl; D. Isler of Kliba for preparation of the custom minipig diets; P. Bidaut and P. Bonfils of the University of Geneva for housing and feeding the minipigs, and assistance with the experimental protocol; J.-L. Sanchez-Garcia or assistance with minipig blood draws; M. Gyger for submitting the veterinary protocols; The Animal Care Facility of the Nestle Research Center for assistance with the hamster experiments; M. Huff of The University of Western Ontario, for helpful discussions concerning minipig diets and lov­astatin doses; Ellegaard Gottingen Minipigs ApS, Dalmose, Denmark, for providing information on Gottingen minipigs; and finally D. Mutch, B. Ger­man, J.-R. Neeser, and O. Ballevre for dynamic discussions concerning ani­mal models for cholesterol research.

References

  • 1. Borchers AT, Stern JS, Hackman RM, Keen CL, Gershwin ME: Mush­rooms, tumors, and immunity. Proc Soc Exp Biol Med 1999, 221:281-293.

•2.       Kabir Y, Kimura S, Tamura T: Dietary effect of Ganoderma luci­dum mushroom on blood pressure and lipid levels in sponta­neously hypertensive rats (SHR). J Nutr Sci Vitaminol (Tokyo) 1988, 34:433-438.

  • 3. Li Khva Ren, Vasil'ev AV, Orekhov AN, Tertov VV, Tutel'ian VA: [Anti-atherosclerotic properties of higher mushrooms (a clinico-experimental investigation)]. Vopr Pitan 1989:16-19.
  • 4. Opletal L, Jahodar L, Chobot V, Zdansky P, Lukes J, Bratova M, Soli- chova D, Blunden G, Dacke CG, Patel AV: Evidence for the anti- hyperlipidaemic activity of the edible fungus Pleurotus ostreatus. Br J Biomed Sci 1997, 54:240-243.
  • 5. Bobek P, Ozdin L, Galbavy S: Dose- and time-dependent hypoc- holesterolemic effect of oyster mushroom (Pleurotus ostreatus) in rats. Nutrition 1998, 14:282-286.

•6.       Bobek P, Galbavy S: Hypocholesterolemic and antiatherogenic effect of oyster mushroom (Pleurotus ostreatus) in rabbits. Nahrung 1999, 43:339-342.

  • 7. Cheung PC, Lee MY: Fractionation and characterization of mushroom dietary fiber (nonstarch polysaccharides) as potential nutraceuticals from sclerotia of Pleurotus tuber- regium (Fries) singer. J Agric Food Chem 2000, 48:3148-3151.
  • 8. Gunde-Cimerman N, Plemenitas A: Hypocholesterolemic activ­ity of the genus Pleurotus (Fr.) Karst. (Agaricales s.l., Basidiomycetes). Int J Medicinal Mushrooms 200l, 3:A9l.
  • 9. Cheung PC: Plasma and hepatic cholesterol levels and fecal neutral sterol excretion are altered in hamsters fed straw mushroom diets. J Nutr 1998, 128:1512-1516.

•10.    Fukushima M, Nakano M, Morii Y, Ohashi T, Fujiwara Y, Sonoyama K: Hepatic LDL receptor mRNA in rats is increased by dietary mushroom (Agaricus bisporus) fiber and sugar beet fiber. J Nutr 2000, 130:2151-2156.

•11.    Beynen AC, Fielmich AM, Lemmens AG, Terpstra AH: Farm-grown mushrooms (Agaricus campestris) in the diet of rats do not affect plasma and liver cholesterol concentrations. Nahrung l996, 40:343-345.

  • 12. Cheung PCK: The hypocholesterolemic effect of extracellular polysaccharide from the submerged fermentation of mushroom. Nutr Res 1996, 16:1953-1957.

•13.     Cheung PCK: The hypocholesterolemic effect of two edible mushroom: Auricularia auricula (Tree-ear) and Tremella fuci- formis (White-jelly leaf) in hypercholesterolemic rats. Nutr Res 1996, 16:1721-1725.

•14.    Kabir Y, Yamaguchi M, Kimura S: Effect of shiitake (Lentinus edo- des) and maitake (Grifola frondosa) mushrooms on blood pressure and plasma lipids of spontaneously hypertensive rats. J Nutr Sci Vitaminol (Tokyo) 1987, 33:341-346.

  • 15. Kubo K, Nanba H: The effect of maitake mushrooms on liver and serum lipids. Altern Ther Health Med l996, 2:62-66.
  • 16. Sugiyama K, Yamakawa A, Saeki S: Correlation of suppressed linoleic acid metabolism with the hypocholesterolemic action of eritadenine in rats. Lipids 1997, 32:859-866.

•17.     Sugiyama K, Kawagishi H, Tanaka A, Saeki S, Yoshida S, Sakamoto H, Ishiguro Y: Isolation of plasma cholesterol-lowering compo­nents from ningyotake (Polyporus confluens) mushroom. J Nutr Sci Vitaminol (Tokyo) 1992, 38:335-342.

  • 18. Kaneda T, Tokuda S: Effect of various mushroom preparations on cholesterol levels in rats. J Nutr 1966, 90:371-376.
  • 19. Shao G: [Treatment of hyperlipidemia with cultivated Cordyceps - a double-blind, randomized placebo control trial]. Zhong Xi Yi Jie He Za Zhi (Chin J Integ Med) 1985, 5:642-665.

•20.    Tao J, Feng KY: Experimental and clinical studies on inhibitory effect of Ganoderma lucidum on platelet aggregation. J Tongji Med Univ 1990, 10:240-243.

  • 21. Chang ST: Ganoderma - the leader in production and technol­ogy of mushroom neutraceuticals. In: Recent advances in Gano­derma lucidum research Edited by: Kim B-K, Kim YS. Seoul, Korea: The Pharmaceutical Society of Korea; 1995:43-52.
  • 22. Chen AW, Miles PG: Biomedical research and the application of the mushroom nutriceuticals from Ganoderma lucidum. In: Mushroom Biology and Mushroom Products Edited by: Royse DJ. University Park, PA: The Pennsylvania State University; l996:l6l-l75.
  • 23. Mizuno T: Oriental medicinal tradition of Ganoderma lucidum (Reishi) in China. In: Ganoderma lucidum Edited by: Mizuno T, Kim B- K. Seoul, Korea: II-Yang Pharm. Co. Ltd; l996:l0l-l06.
  • 24. Sun-Soo T: Effective dosage of the extract of Ganoderma luci­dum in the treatment of various ailments. In: Mushroom Biology and Mushroom Products Edited by: Royse DJ. University Park, PA: The Pennsylvania State University; l996:l77-l85.
  • 25. Wasser SP, Weis AL: Therapeutic effects of substances occur­ring in higher Basidiomycetes mushrooms: a modern perspective. Crit Rev Immunol 1999, 19:65-96.
  • 26. Chang ST, Buswell JA: Medicinal Mushrooms - A Prominent Source of Nutriceuticals for the 21st century. Current Topics in Nutraceutical Research 2003, 1:257-280.
  • 27. Shiao MS: Natural products of the medicinal fungus Gano­derma lucidum: occurrence, biological activities, and phar­macological functions. Chem Rec 2003, 3:172-180.

•28.     Lee SY, Rhee HM: Cardiovascular effects of mycelium extract of Ganoderma lucidum: inhibition of sympathetic outflow as a mechanism of its hypotensive action. Chem Pharm Bull (Tokyo) 1990, 38:1359-1364.

  • 29. Rein D, Monnard I, German JB, Berger A: Screening of cholesterol absorption and synthesis inhibiting food ingredients in ham­ster models. FASEB J 2000, I4:A250.
  • 30. Cevallos WH, Holmes WL, Myers RN, Smink RD: Swine in athero­sclerosis research - Development of an experimental animal model and study of the effect of dietary fats on cholesterol metabolism. Atherosclerosis 1979, 34:303-317.
  • 31. Clarkson T, Shively C, Weingand K: Animal Models of diet- Induced Atherosclerosis. In: Use of Animal Models for Research in Human Nutrition Volume 6. Edited by: Beynen A, West C. Basel: Karger; l988:56-82.
  • 32. Fadden K, Hill MJ, Latymer E, Low G, Owen RW: Steroid metabo­lism along the gastrointestinal tract of the cannulated pig. Eur J Cancer Prev 1999, 8:35-40.

•33.     Hikino H, Ishiyama M, Suzuki Y, Konno C: Mechanisms of hypogly­cemic activity of ganoderan B: a glycan of Ganoderma luci- dum fruit bodies. Planta Med l989, 55:423-428.

•34.     Bao X, Duan J, Fang X, Fang J: Chemical modifications of the (I- ->3)-alpha-D-glucan from spores of Ganoderma lucidum and investigation of their physicochemical properties and immu­nological activity. Carbohydr Res 2001, 336:127-140.

  • 35. Zhu M, Chang Q, Wong LK, Chong FS, Li RC: Triterpene antioxi­dants from Ganoderma lucidum. Phytother Res 1999, 13:529-53 1.
  • 36. Wu TS, Shi LS, Kuo SC: Cytotoxicity of Ganoderma lucidum trit- erpenes. J Nat Prod 200', 64:' '2'-''22.
  • 37. Gao JJ, Min BS, Ahn EM, Nakamura N, Lee HK, Hattori M: New trit- erpene aldehydes, lucialdehydes A-C, from Ganoderma luci­dum and their cytotoxicity against murine and human tumor cells. Chem Pharm Bull (Tokyo) 2002, 50:837-840.
  • 38. Luo J, Zhao YY, Li ZB: A new lanostane-type triterpene from the fruiting bodies of Ganoderma lucidum. J Asian Nat Prod Res 2002, 4: '29-' 34.
  • 39. Ma J, Ye Q, Hua Y, Zhang D, Cooper R, Chang MN, Chang JY, Sun HH: New lanostanoids from the mushroom Ganoderma lucidum. J Nat Prod 2002, 65:72-75.
  • 40. Komoda Y, Shimizu M, Sonoda Y, Sato Y: Ganoderic acid and its derivatives as cholesterol synthesis inhibitors. Chem Pharm Bull '989, 37:53' -533.

4'. Mizushina Y, Takahashi N, Hanashima L, Koshino H, Esumi Y, Uzawa J, Sugawara F, Sakaguchi K: Lucidenic acid O and lactone, new terpene inhibitors of eukaryotic DNA polymerases from a basidiomycete, Ganoderma lucidum. Bioorg Med Chem '999, 7:2047-2052.

•42.     Ha TB, Gerhauser C, Zhang WD, Ho-Chong-Line N, Fouraste I: New lanostanoids from Ganoderma lucidum that induce NAD(P)H:quinone oxidoreductase in cultured murine hepatoma cells. Planta Med 2000, 66:68' -684.

  • 43. Walker KA, Kertesz DJ, Rotstein DM, Swinney DC, Berry PW, So OY, Webb AS, Watson DM, Mak AY, Burton PM et al.: Selective inhibition of mammalian lanosterol 14 alpha-demethylase: a possible strategy for cholesterol lowering. J Med Chem '993, 36:2235-2237.
  • 44. Frye LL, Leonard DA: Lanosterol analogs: dual-action inhibitors of cholesterol biosynthesis. Crit Rev Biochem Mol Biol '999, 34:' 23-' 40.
  • 45. Bergstrom JD, Dufresne C, Bills GF, Nallin-Omstead M, Byrne K: Dis­covery, biosynthesis, and mechanism of action of the zarago- zic acids: potent inhibitors of squalene synthase. Annu Rev Microbiol '995, 49:607-639.
  • 46. Bollen P, Ellegaard L: The Gottingen minipig in pharmacology and toxicology. Pharmacol Toxicol '997, 80(Suppl 2):3-4.

•47.     Gunde-Cimerman N, Cimerman A: Pleurotus fruiting bodies con­tain the inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase-lovastatin. Exp Mycol '995, 19: '-6.

  • 48. Ogawa H, Hasumi K, Sakai K, Murakawa S, Endo A: Pannorin, a new 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor produced by Chrysosporium pannorum. J Antibiot (Tokyo) '99', 44:762-767.
  • 49. Morisaki M, Sonoda Y, Makino T, Ogihara N, Ikekawa N, Sato Y: Inhibitory effect of 15-oxygenated sterols on cholesterol syn­thesis from 24,25-dihydrolanosterol. J Biochem (Tokyo) '986, 99:597-600.
  • 50. Aoyama Y, Yoshida Y, Sonoda Y, Sato Y: 7-Oxo-24,25-dihydro- lanosterol: a novel lanosterol 14 alpha-demethylase (P- 45014dm) inhibitor which blocks electron transfer to the oxy- ferro intermediate. Biochim Biophys Acta '987, 922:270-277.

5'. Sonoda Y, Obi N, Onoda M, Sakakibara Y, Sato Y: Effects of 32-oxy- genated lanosterol derivatives on 3-hydroxy-3-methylglu- taryl coenzyme A reductase activity and cholesterol biosynthesis from 24,25-dihydrolanosterol. Chem Pharm Bull (Tokyo) '992, 40:2796-2799.

  • 52. Trzaskos JM, Magolda RL, Favata MF, Fischer RT, Johnson PR, Chen HW, Ko SS, Leonard DA, Gaylor JL: Modulation of 3-hydroxy-3- methylglutaryl-CoA reductase by 15 alpha-fluorolanost-7- en-3 beta-ol. A mechanism-based inhibitor of cholesterol biosynthesis. J Biol Chem '993, 268:2259 '-22599.
  • 53. Morand OH, Aebi JD, Dehmlow H, Ji YH, Gains N, Lengsfeld H, Him- ber J: Ro 48-8.071, a new 2,3-oxidosqualene:lanosterol cyclase inhibitor lowering plasma cholesterol in hamsters, squirrel monkeys, and minipigs: comparison to simvastatin. J Lipid Res '997, 38:373-390.
  • 54. Ness GC, Gertz KR, Holland RC: Regulation of hepatic lanos­terol 14 alpha-demethylase gene expression by dietary cho­lesterol and cholesterol-lowering agents. Arch Biochem Biophys 200', 395:233-238.
  • 55. Conde K, Roy S, Freake HC, Newton RS, Fernandez ML: Atorvasta- tin and simvastatin have distinct effects on hydroxy methyl- glutaryl-CoA reductase activity and mRNA abundance in the guinea pig. Lipids '999, 34: '327-'332.
  • 56. Del Puppo M, Rauli S, Galli Kienle M: Inhibition of cholesterol syn­thesis and hepatic 3-hydroxy-3-methylglutaryl-CoA reduct­ase in rats by simvastatin and pravastatin. Lipids '995, 30: '057-' 06'.
  • 57. Himber J, Missano B, Rudling M, Hennes U, Kempen HJ: Effects of stigmastanyl-phosphocholine (Ro 16-6532) and lovastatin on lipid and lipoprotein levels and lipoprotein metabolism in the hamster on different diets. J Lipid Res ' 995, 36:' 567-'585.
  • 58. Krause BR, Princen HM: Lack of predictability of classical ani­mal models for hypolipidemic activity: a good time for mice? Atherosclerosis '998, 140:'5-24.
  • 59. Ma PT, Gil G, Sudhof TC, Bilheimer DW, Goldstein JL, Brown MS: Mevinolin, an inhibitor of cholesterol synthesis, induces mRNA for low density lipoprotein receptor in livers of ham­sters and rabbits. Proc Natl Acad Sci U S A '986, 83:8370-8374.
  • 60. Huff MW, Telford DE: Regulation of low density lipoprotein apoprotein B metabolism by lovastatin and cholestyramine in miniature pigs: effects on LDL composition and synthesis of LDL subfractions. Metabolism '989, 38:256-264.

6'. Tikkanen MJ: Statins: within-group comparisons, statin escape and combination therapy. Curr Opin Lipidol '996, 7:385-388.

  • 62. Knopp RH: Drug treatment of lipid disorders. N Engl J Med '999, 341:498-5' '.
  • 63. Berger A, Gremaud G, Baumgartner M, Rein D, Monnard I, Kratky E, Geiger W, Burri J, Dionisi F, Allan M, Lambelet P: Cholesterol-low­ering properties of amaranth grain and oil in hamsters. Int J Vitam Nutr Res 2003, 73:39-47.
  • 64. Burnett JR, Wilcox LJ, Telford DE, Kleinstiver SJ, Barrett PH, Newton RS, Huff MW, Barrett P: Inhibition of HMG-CoA reductase by atorvastatin decreases both VLDL and LDL apolipoprotein B production in miniature pigs. Arterioscler Thromb Vasc Biol '997, 17:2589-2600.
  • 65. Burnett JR, Wilcox LJ, Telford DE, Kleinstiver SJ, Barrett PH, Newton RS, Huff MW: The magnitude of decrease in hepatic very low density lipoprotein apolipoprotein B secretion is determined by the extent of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition in miniature pigs. Endocrinology '999, 140:5293-5302.
  • 66. Miettinen TA, Strandberg TE, Gylling H: Noncholesterol sterols and cholesterol lowering by long-term simvastatin treat­ment in coronary patients: relation to basal serum cholestanol. Arterioscler Thromb Vasc Biol 2000, 20:' 340-'346.
  • 67. Collinder E, Cardona ME, Kozakova H, Norin E, Stern S, Midtvedt T: Biochemical intestinal parameters in pigs reared outdoors and indoors, and in germ-free pigs. J Vet Med A Physiol Pathol Clin Med 2002, 49:203-209.
  • 68. Edwards PA, Kast HR, Anisfeld AM: BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis. J Lipid Res 2002, 43:2- '2.
  • 69. Davis RA, Miyake JH, Hui TY, Spann NJ: Regulation of cholesterol- 7alpha-hydroxylase: BAREly missing a SHP. J Lipid Res 2002, 43:533-543.

•70.     Bobek P, Ozdin L, Kuniak L: The effect of oyster mushroom (Pleurotus ostreatus), its ethanolic extract and extraction res­idues on cholesterol levels in serum, lipoproteins and liver of rat. Nahrung '995, 39:98-99.

7'. Pfeifer AM, Cole KE, Smoot DT, Weston A, Groopman JD, Shields PG, Vignaud JM, Juillerat M, Lipsky MM, Trump BF et al.: Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens. Proc Natl Acad Sci U S A '993, 90:5' 23-5 '27.

  • 72. Askenazi M, Driggers EM, Holtzman DA, Norman TC, Iverson S, Zim­mer DP, Boers ME, Blomquist PR, Martinez EJ, Monreal AW et al.: Integrating transcriptional and metabolite profiles to direct the engineering of lovastatin-producing fungal strains. Nat Biotechnol 2003, 21: '50-'56.
  • 73. Berlin E, Khan MA, Henderson GR, Kliman PG: Influence of age and sex on composition and lipid fluidity in miniature swine plasma lipoproteins. Atherosclerosis '985, 54: '87-203.
  • 74. Burnett JR, Wilcox LJ, Telford DE, Kleinstiver SJ, Barrett PH, Newton RS, Huff MW: Inhibition of ACAT by avasimibe decreases both VLDL and LDL apolipoprotein B production in miniature pigs. J Lipid Res ' 999, 40: '3'7-'328.
  • 75. Ritskes-Hoitinga J, Bollen PJA: Nutrition of (Göttingen) minipigs: facts, assumptions and mysteries. Pharmacol Toxicol 1997, 80(Supl II):5-9.
  • 76. Gremaud G, Piguet C, Baumgartner M, Pouteau E, Decarli B, Berger
  • A, Fay LB: Simultaneous assessment of cholesterol absorption and synthesis in humans using on-line gas chromatography/ combustion and gas chromatography/pyrolysis/isotope-ratio mass spectrometry. Rapid Commun Mass Spectrom 2001, 15:1207-1213.
  • 77. Gremaud G, Dalan E, Piguet C, Baumgartner M, Ballabeni P, Decarli
  • B, Leser ME, Berger A, Fay LB: Effects of non-esterified stanols in a liquid emulsion on cholesterol absorption and synthesis in hypercholesterolemic men. Eur J Nutr 2002, 41:54-60.
  • 78. Nutrient Requirements for Swine - 10th Revised Edition. Washington, D. C.: National Research Council/National Academy of Science (NRC/NAS); 1998.
  • 79. Shapiro JD, Nordstrom LJ, Mitschelen JJ, Rodwell VW, Schimke RT: Micro assay for 3-hydroxy-3-methylglutaryl-CoA reductase in rat liver and in L-cell fibroblast. Biochim Biophys Acta 1974, 370:369-377.

Publish with BioMecl Central and every scientist can read your work free of charge

"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK

Your research papers will be:

  • available free of charge to the entire biomedical community
  • peer reviewed and published immediately upon acceptance
  • cited in PubMed and archived on PubMed Central
  • yours - you keep the copyright

Submit your manuscript here:           i } BioMedcentral

http://www.biomedcentral.com/info/publishing_adv.asp        ^

 


[1] Nafag 924 hamster complete diet was from Eberle Nafag AG, Gossau, Switzerland. The detailed ingredients in the diet are not known. Metabolizable energy was estimated to be 3111 kcal/kg diet. The lovastatin diet was identical to the control diet, but contained 2 mg/100 g diet lovastatin. Gl, G. lucidum.

were from Kliba. Proximate analysis of minipig test diets (wt%) was: carbohydrate as nitrogen free extract 46.6, crude protein '5.9, water 11.1,

crude fat '6.8, ash (including vitamins) 5.', and crude fiber 4.6. Total- and digestible energy of the diet were estimated to be 3985 and 3776 kcal/kg

diet, respectively. 2Centravo Schweinefett B 90, Centravo AG, Zurich, Switzerland. Contained 98% fat, 5% free fatty acids, 9% polyunsaturated fatty

acids, protected with antioxidants. 3Migros Genossenschafts-Bund SA, Zurich, Switzerland. 82% fat, 0.5% protein, 0.5% carbohydrate. 4Per '00 g diet, contained ' .06% arginine, 0.75% lysine, 0.26% methionine, 0.56% methionine + cystine, 0.20% tryptophan, and 0.57% threonine [78]. 5Per '00

g diet, contained 0.80% calcium, 0.70% phosphorus, 0.'9% sodium, 0.63% potassium, 0.20% magnesium, and 0.26% chloride. 6Vitacel LC 200 Cellulose, J. Rettenmaier & Söhne (JRS), GMBH + Co, Rosenberg, Holzmühle ', Germany. 0.3% sulfate ash, pH 5.0-7.5, 300 ^M fiber length.

[7]Champitec, Payerne, Switzerland. Prepared as described in the text. Pigs that did not receive the Gl extract, received the control diet plus 80 mg

lovastatin/pig/d in half an apple. 8Fluka 26740, Fluka Holding AG, Buchs, Switzerland. 97% pure. 9Roche Vitamins Ltd, Basel, Switzerland. In mg/'00 g

diet, contained 0.4 vitamin A (800 IU), 2.0 vitamin D3 (80 IU), '0.5 vitamin E, 0.3 vitamin K3, '.2 vitamin Bh 0.8 vitamin B2, 3.0 nicotinic acid, 2.0

pantothenic acid, 0.' folic acid, 0.7 vitamin B6, 0.0034 vitamin B'2, 0.02 biotin, 65.' choline, and 2.0 vitamin C. '0In mg/'00 g diet, contained '.' copper (mg/kg), 7.6 zinc, '' .0 iron, 0.05 iodine, 5.0 manganese, and 0.03 selenium. Gl, G. lucidum.

 

PHYTOTHERAPY RESEARCH Phytother. Res. (2010) Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ptr.3202

(Ganoderma lucidum'un Lymphoma DA-! Hücreleri üzerine sitotoksik aktivitesi)

Cytotoxic Action of Ganoderma lucidum on Interleukin-3 Dependent Lymphoma DA-1 Cells: Involvement of Apoptosis Proteins

Eva Calviño,1 Lucía Pajuelo,1 Jon A. Ochoa de Eribe Casas,2 José Luis Manjón,2 M. Cristina Tejedor,1 Angel Herráez,1 Manuel Díez Alonso3 and José C. Diez1*

departamento de Bioquímica y Biología Molecular, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain

2Departamento de Biología Vegetal, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain 3Servicio de Cirugía, Hospital Universitario Príncipe de Asturias, 28806 Alcalá de Henares, Madrid, Spain

Aqueous extracts and a semipurified fraction obtained by methanol extraction and column chromatography were isolated from Ganoderma lucidum [Ganoderma lucidum (Curtis) P. Karst.; Ganodermataceae Donk] and their effects on interleukin 3-dependent lymphoma cells (DA-1) were studied. Cell viability was reduced by the action of unboiled aqueous extract and by the methanol-extracted column-chromatography semipurified fraction, producing DNA fragmentation in DA-1 cells. Treatments with aqueous extracts showed increments of Bax after 13 h, increments of p53 and Mdm2 after 19 h and a reduction of these three proteins after 24 h. The methanol-extracted semipurified fraction also induced increments of p53 and Mdm2 factors at 19 h with a reduction after 24 h. The methanol-extracted column-chromatography semipurified fraction from Ganoderma lucidum produced minor changes in the level of Akt after treatments for 19 h in DA-1 cells with a slight reduc­tion in the levels of NFkB-p65 factor. Both the unboiled aqueous extract and the methanol-extracted column- chromatography semipurified fraction produced cleavage of inactive caspase 3, as a clear indication of induction of apoptosis by compounds present in Ganoderma lucidum. Copyright © 2010 John Wiley & Sons, Ltd.

Keywords: Bax; Bcl-2; Ganoderma lucidum (Curtis) P. Karst., Ganodermataceae Donk; lymphoma; p53.

INTRODUCTION

In recent times, the search for new more effective thera­peutic agents with more selective action on tumor cells has been conducted. The fungus Ganoderma lucidum (common names: Reishi, Lingzhi) is a well known mush­room in traditional Chinese medicine. Extracts from different parts (mycelia, spores and fruiting bodies) from Ganoderma lucidum have been used as a source of active compounds against tumor processes (Hu et al., 2002; Liu et al., 2002; Hong et al., 2004; Jiang et al., 2004a, 2004b; Lu et al., 2004; Sliva, 2004). Among the different compounds present in Ganoderma lucidum, nucleo- sides, proteins, polysaccharides, fatty acids, sterols and triterpenes have been described as molecules which could be responsible for the antitumor activity (Yeung et al., 2004). Ganoderma lucidum extracts inhibit the growth of human prostate and bladder cancer cell lines (Jiang et al., 2004b; Lu et al., 2004) and cell proliferation inducing apoptosis in human colon carcinoma and breast cancer cell lines (Hu et al., 2002; Hong et al., 2004; Jiang et al., 2004a). Polysaccharides present in Gano- derma lucidum may be responsible for the growth delay of sarcoma cells (Cao and Lin, 2004). In part, their

* Correspondence to: José C. Diez, Departamento de Bioquímica y Biología Molecular, Campus Universitario, Universidad de Alcalá, 28871 Alcalá de Henares (Madrid) Spain. E-mail: josecarlos.diez@uah.es

Copyright © 2010 John Wiley & Sons, Ltd.

antitumor activity may be a consequence of their immunomodulatory properties (Wilasrusmee et al., 2002; Williamson, 2001; Lin, 2005). Also, triterpenes present in Ganoderma lucidum inhibit the growth of hepatoma cells by altering intracellular phosphoryla­tion pathways (Lin et al., 2003).

We have previously studied the apoptotic action of two antitumor compounds, etoposide and hydroxyurea, on DA-1 lymphoma cells (Olmos et al., 2005a, 2005b) as a model for cytotoxicity by inducing apoptosis. These cells, derived from a murine lymphoma, could be sensi­tive to the action of antitumor and toxic compounds of therapeutic interest. Additionally, this model shows the advantage of being modulated by interleukin 3 (Gottlieb et al., 1994, 1996; Gottlieb and Oren, 1998). Thus, antiproliferative and apoptotic effects of antitu­mor compounds can be studied in this cell model. We have demonstrated the possible differential involve­ment of Bax and Bcl-2 factors in the apoptotic death of these cells after treatment with etoposide or hydroxy- urea (Olmos et al., 2005a, 2005b). Thus, the present work studied the cytotoxicity of different Ganoderma lucidum extracts, prepared as described by other authors (Sliva et al., 2002; Stanley et al., 2005) on these interleukin 3 dependent lymphoma cells. Aqueous extracts and a methanol-extracted semipurified fraction from Gano- derma lucidum demonstrated the ability to reduce cell viability in DA-1 cells and also induced DNA fragmen­tation. Changes in protein expression of apoptosis factors were also observed after treatments with these extracts showing activation of apoptosis cascades.

Received 03 September 2009 Revised 28 January 2010 Accepted 29 March 2010

Some authors have previously shown the activation of intracellular kinase cascades by Ganoderma extracts in induced neuronal differentiation in rat PC12 cells (Cheung et al., 2000). Jiang et al. (2004a) also showed inhibition of Akt/NF-kB signalling by Ganoderma lucidum in breast cancer cells. Thus, interest was focused on activation and phosphorylation of intracellular kinases which are altered in other cellular model systems (Jiang et al., 2004a; Cheung et al., 2000). Changes in expression and phosphorylation of Akt have been studied after treatment of DA-1 cells with aqueous extracts and the methanol-extracted semipurified frac­tion from Ganoderma lucidum.

MATERIALS AND METHODS

The combined extracts were evaporated to dryness in a rotary evaporator, and dissolved in absolute methanol. To purify this extract, it was adsorbed on 0.2 g of silica gel to be applied onto a silica gel column (1 x 15.5 cm) and subsequently eluted with 3 :1 : 1 butanol: acetic acid: water. Column fractions (0.2 mL) with an Rf near 0.74 were combined, the solvent was evaporated and the final sample (1.6 mg) was resuspended in 0.01 mL DMSO to obtain the extract 3 (E3).

Aqueous extracts E1 and E2 prepared as described by other authors were used. It is highly probable that sugars are present in these extracts. Several poly- saccharides present in G. lucidum have been shown to have immunomodulatory and antitumor activities (Cao and Lin, 2004; Sliva et al., 2002; Stanley et al., 2005). With respect to the E3 fraction, isolated on column chromatography from a methanol extract of G. lucidum, based on the solvents used it is possible that compounds similar to terpenoids could be present in this fraction (Li et al., 2005; Lin et al., 2003; Min et al., 2000).

Cell treatments. Cultured DA-1 cells were incubated at concentrations of 0.5 x 106 cells/mL at 37°C in an atmo­sphere with 5% CO2 in the presence of IL-3 conditioned medium. DA-1 cells were grown in the presence of IL-3 conditioned medium alone or supplemented with either Ganoderma lucidum extracts or 100 |im etoposide as a positive control for toxicity and apoptosis induction.

The E1 and E2 extracts and E3 fraction were added separately to 1 mL aliquots of cell culture (0.5 x 106 cell/ mL) and allowed to act for 13, 19 and 24 h.

The lyophyllized E1 extract (14.9 mg dried weight) was dissolved in 0.9 mL of sterile distilled water and 60 |L was added per mL of cell culture.

The lyophyllized E2 extract (3 mg dried weight) was dissolved in 0.9 mL of sterile distilled water and 60 |L was added per mL of cell culture.

Fraction 3 (E3) (2.08 mg) dissolved in 13 |L of DMSO was added per mL of cell culture (0.5 x 106 cell/ mL).

DMSO (13 |L) was also added to the cells as a nega­tive control and no toxic effect was observed on cell culture (i.e. no reduction of cell viability, no DNA frag­mentation, etc.).

Cell viability, permeability of treated cells to propidium iodide and flow cytometry analyses. The cell viability of DA-1 cells incubated in the presence of IL-3 and treated with Ganoderma lucidum extracts was determined by flow cytometry by measuring the impermeability to propidium iodide. 2.5 x 10[1] treated cells were collected, washed in PBS, pelleted at 1200 rpm for 5 min, resus­pended in 500 |L of PBS and stained with propidium iodide at 5 |ig/mL final concentration and, analysed by flow cytometry either by FACScan or FACScalibur (Becton Dickinson, San José, CA, USA). For each sample the acquisition was finished at 10000 counts. Data analysis was done using the program Win-MDI (Windows Multiple Document Interface for Flow Cytometry, version 2.8, available from Scripps Research Institute, FACS Core Facility, http://facs.scripps.edu/ software.html) (Trotter, 2004). Cell fragments were dis­criminated from the non viable cells on FSC/FL-2-H dot plots, where FL-2-H corresponds to propidium iodide fluorescence.

Cytometric analysis of cell populations with subdiploid DNA and cell cycle. Apoptotic cells were counted on the basis of DNA content per cell after permeabiliza- tion of cells with NP40. After treatments with 100 ¡¡m etoposide or G. lucidum extracts, 2.5 x 105 cells were collected and washed with PBS. The pellet was resus- pended in 475 ¡¡L of a solution containing 0.5 mg/mL RNase, 0.1% of NP-40 in PBS and incubated for 30 min in order to extract low molecular weight DNA from cell nuclei. The remnant DNA in cells was stained with 0.05 mg/mL propidium iodide, immediately before mea­suring the fluorescence in the cytometer.

Cells with hypodiploid DNA (apoptotic cells) were distinguished from those containing diploid DNA (non apoptotic cells) on the basis of a different fluorescence intensity of propidium iodide in the flow cytometer. Cell cycle progress was studied in the same samples. Histo­grams of the untreated cells were used to define the positions of the different peaks for the G1 and G2/M phases in the cell cycle.

Western blot. 5 x 106 cells treated with the extracts for 13, 19 or 24 h were collected by centrifugation at 1200 rpm for 5 min, resuspended in 200 ¡L of lysis buffer (50 mm Tris/HCl pH 8.0, 150 mm NaCl, 5 mm EDTA, 0.5% NP-40, 1 mm PMSF) and incubated for 20 min at 4°C. The cells were sonicated for 20 s (duty cycle 100%, output control 50%) in a Branson Cell Disruptor B15 Sonifier and then centrifuged (14000 rpm, 5 min, 4°C) and the supernatants were analysed by elec­trophoresis and blotting. Proteins (20 ¡g/well) were loaded on a 10% polyacrylamide gel with SDS, electro- phoretically separated and transferred to nitrocellulose membranes as described (Towbin et al., 1979). Mem­branes were blocked with 5% powder milk in TTBS (50 mm Tris pH 7.2, 140 mm NaCl, 0.06% Tween 20). Afterwards, they were washed with TTBS and incu­bated with antibodies specific against several apoptosis factors diluted in TTBS containing milk.

All the following antibodies were obtained from Santa Cruz Biotechnology, CA, USA, and used at the dilution indicated: Anti-Bax mouse monoclonal IgG2b (B-9; sc-7480; 200 ¡g/mL) 1 : 100; anti-Bcl-2 mouse monoclonal IgGi antibody (C-2; sc7382; 200 ¡g/mL) 1 : 200; anti-MDM2 (SMP14) 1 : 100; anti-Akt1/2/3 (H-136) sc-8312 antibodies, 1 : 400; anti-pAkt1/2/3 (Ser 437) sc-7985-R antibodies 1 : 400; anti Erk1/2 sc-154 anti­bodies 1 : 3000; anti p-Erk1/2 (Tyr204) sc-7383 antibodies 1 : 200; anti-NFkB p50 and p105 (E-10) sc-8414 antibod­ies 1 : 200; anti-NFkB p65 (F-6) sc-8008 antibodies 1 : 200.

Anti-p53 mouse monoclonal IgG antibody (Ab-1, 0P03; 100 ¡g/mL) was purchased from Calbiochem (Oncogene Research Products, MA, USA) and diluted 1 : 50.

Monoclonal anti-P-actin antibodies were purchased from Sigma A5441 and used at a dilution of 1 : 5000.

Polyclonal caspase 3 antibodies were purchased from Cell Signalling Technology (Reference number 9662) and used at a dilution of 1 : 1000.

After incubation of the nitrocellulose membranes with specific antibodies overnight at 4°C the bands were revealed using goat anti-mouse horseradish peroxidase- conjugated polyclonal antibodies (1 : 2000) from Promega (Madison, WI) in an enhanced chemilumines- cence (ECL) detection kit (Amersham). The intensities of the bands were corrected with respect to the intensity of actin band in the blot, as a control for constitutive expression, and quantified relative to the intensity of the band in control cells without any treatment, which were considered as 100%.

All the experiments were repeated three or four times. The mean and SEM were graphically represented. In order to determine the significance of the effect, Stu­dent's t-test was used. The asterisk * indicates p < 0.05.

RESULTS

Cell viability analyses of DA-1 cells

The cell viability of DA-1 lymphoma cells was deter­mined after treatment with Ganoderma lucidum extracts obtained as described in Methods. As can be observed, etoposide (Fig. 1) at concentrations of 100 ^m induced a decrease in cell viability, with values of 70%, 58% and 51% viability in cells treated with etoposide for 13, 19 and 24 h. Also, different extracts obtained from Ganoderma lucidum were used: Both E1 and E3 showed an effective reduction in cell viability with a reduction of up to 70% and 36% at 24 h of treatment, respectively (Fig. 1). On the contrary, the E2 extract showed not so clear action on viability. In the case of E1 the effects were clearly visible after 19 h. E3 showed an even more effective action than E1 extract (Fig. 1). Thus, the analyses of cell viability clearly demonstrate the action of these extracts (E1 and E3) on cell mem­brane permeability which allows the entrance of prop- idium iodide.

 

Cell death induction in DA-1 cells

Previously, it was shown that either of two antitumor compounds (hydroxyurea or etoposide) induces apop- tosis in DA-1 cells (Olmos et al., 2005a, 2005b), as tested by permeability alteration analyses, DNA fragmenta­tion and annexin-FITC studies. The apoptotic action of these compounds can be related to changes in cell cycle. Thus, we studied the effects on cell cycle as well as on the presence of subdiploid DNA. Figure 3 shows the results obtained for treatments of DA-1 cells in the presence of the extracts obtained from Ganoderma lucidum. As can be observed, E1 extract showed a level of cell death induction (as tested by production of subdiploid DNA) similar to that of 100 |im etoposide. At 24 h, the induction of subdiploid DNA was even higher in the case of E1 than etoposide. E3 fraction also showed an important induction of subdiploid DNA in DA-1 cells, an effect that was more clearly visible at 24 h. On the contrary, E2 extract showed a very limited induction of cell death in DA-1 cells even at 24 h. Figure 4 shows representative diagrams of DNA content of control and E1 and E3 treated DA-1 cells. The effects of E1 extract are clearly visible at 24 h (with an impor­tant peak corresponding to subdiploid DNA) although they were already apparent at 13 h. The methanol- extracted semipurified E3 fraction also induced DNA fragmentation (see the panels on the right side of Fig. 4). In this case, it is possible to observe a prominent peak with high fluorescence in FL2-H at 13 and 24 h, that might indicate an arrest in S phase of the cell cycle as a consequence of the treatment.

Analyses of expression of apoptosis related factors

Since some factors such as p53, Bax, Bcl-2 and Mdm-2 are involved in apoptosis processes, the levels of these proteins (Figs 5-8) were analysed by western blot using specific antibodies.

The effect of the E2 extract is shown in Fig. 6. At 13 h, the treated DA-1 cells showed increments in Bax and Mdm2, while Bcl-2 showed a reduction. In the case of Mdm2, a high level of this protein was observed with treatments for 19 h.

Variations in the expression of some other factors such as the kinases Akt and NFkB were also checked (Figs 5-8). Akt was increased after 19 or 24 h treatment with E1 extract (Figs 5 and 8) while NFkB was reduced at 13 h of E1 treatment (Fig. 5).

In the case of E2 treatments, minor variations in Akt and slight increments of pAkt were observed as time passed (Figs 6 and 8) and NFkB showed values of 80% of control at all time points (Fig. 6).

After treatment of DA-1 cells with the E3 fraction, the levels of Akt remained close to controls after 13 h, with a slight increase at 19 h and then a decrease. Its

phosphorylated form p-Akt remained nearly constant through the treatment, although lower than in untreated cells (61%, 60% and 65% at 13, 19 and 24 h, respec­tively) (Figs 7 and 8). NFkB was reduced by E3 treat­ment (Fig. 7).

To further confirm an apoptosis effect induced by either of these extracts uncleaved caspase 3 was quan­titated in DA-1 treated cells in comparison with untreated cells (Fig. 9). As can be observed, both E1 extract and semipurified E3 fraction produced a clear reduction of inactive caspase 3 in western blot analyses (Fig. 9), an effect clearly visible at 24 h. To the contrary, no effect was manifested by treatment with E2.

  

DISCUSSION

Antitumor compounds can exert toxic effects on differ­ent kinds of cells and induce apoptosis in tumor cells. Ganoderma lucidum is a mushroom with components that have been claimed to show antitumor properties.

Some authors have shown inhibition of the growth of solid tumors of sarcoma by using aqueous extracts from G. lucidum (Sone et al., 1985). For instance, Xie et al. (2006) observed significant changes in the induction of cell death in breast cancer and lymphoma cells alter treatment with a polysaccharide extract from G. lucidum. In contrast, Sliva et al. (2002) or Jiang et al. (2004a) in breast and prostate cancer cells, Cao and Lin (2004) in endothelial cells and Müller et al. (2006) in different lymphoma cell lines did not observe significant changes in cell viability alter treatment with different extracts from G. lucidum.

On the basis of previous studies (Gottlieb et al., 1994, 1996; Gottlieb and Oren, 1998; Olmos et al, 2005a, 2005b), the activity of aqueous and alcohol Ganoderma lucidum extracts were studied on DA-1 lymphoma cells. A reduction in viability was observed after treatment with an unboiled aqueous extract (E1) that was obtained similar to that described by others (Sliva et al., 2002; Stanley et al., 2005) (Figs 1 and 2). Thus, aqueous extrac­tion from Ganoderma lucidum renders some com­pounds with toxicity on DA-1 lymphoma cells (Figs 1 and 2). The E3 fraction also showed toxic effects on lymphoma cells reducing cell viability up to 36% (Figs 1 and 2). These results are in accordance with those results described by other authors (Lin, 2005) that showed inhibition of cell growth in liver cancer cells (Huh-7) after treatment with alcohol extracts from G. lucidum.

The results shown in this work (Figs 3 and 4) indicate that the E1 extract and E3 fraction induced fragmenta­tion of DNA, measured as subdiploid DNA. These data are similar to those described by Jiang et al. (2004b) who showed programmed cell death induced by G. lucidum in prostate cancer PC-3 cells and by Cao and Lin (2004) who described the apoptosis induction in endothelial cells from umbilical cord by a polysaccharidic extract from G. lucidum. Müller et al. (2006) also described apoptosis in lymphoma cells induced by a methanol extract from G. lucidum containing C2 ganoderic acid. The data are also in accordance to that described by Hu et al. (2002) who showed inhibition of the proliferation of breast cancer MCF-7 in a time-dependent way and induction of apoptosis after treatment with an alcohol extract from G. lucidum. The results may also be com­pared with those described by Tang et al. (2006) who studied the action of ganoderic acid T in lung cancer 95-D cells finding induction of apoptosis in a time- dependent way. Li et al. (2005) also observed induction of apoptosis in human cancer HuH-7 cells by triterpenes from Ganoderma amboinense, in particular by ganod- eric acid X with inhibition of topoisomerases, which can be correlated to the presence of cytotoxic compounds in Ganoderma sp.

The present work checked whether the observed cell death may be mediated by changes in apoptosis factors. p53 is known to play an essential role in apoptosis induced by some antitumor compounds (Lowe et al., 1994; Newcomb, 1995). In some cell types, p53 is actually required for an apoptotic response (Messmer and Brune, 1997; Palacios et al., 2000). p53 has been identi­fied in many cases as an initiator of apoptotic signals (Merino and Cordero-Campaña, 1998) being a regula­tor of cell cycle progression and a mediator of apoptosis in response to DNA damage. Tang et al. (2006) observed an increase in p53 levels in lung cancer 95-D cells after treatment with ganoderic acid T assuming that this acti­vation of p53 could be stimulated by DNA damage induced by this compound (Li et al, 2005). Cerebrosides isolated from G. lucidum inhibited DNA polymerases and induced DNA damage (Mizushina et al., 1998). In the present study, treatment of DA-1 cells with E1 or E3 fraction produced increments in p53 levels after 19 and 24 h (Figs 5-7), particularly for e3. Thus, this protein might be involved in cell death induced in DA-1 lym­phoma cells by G. lucidum (Figs 5-7). In contrast, the modulation of p53 by E2 extract was quite modest.

p53 regulates the expression of Mdm2 and Bcl-2 (Barak et al., 1993) both of which inhibit the apoptosis mediated by p53 (Chiu et al., 1994; Chen et al., 1996). In the case of Mdm2, the pattern of expression showed a reduction for E1 and E3 treatments at 13 h. One common factor between E1 and E3 appears to be the up/down-regulation of p53/Mdm2, whereas this was not the case for the E2 extract.

p53 could also regulate the expression of bax (Miyas- hita and Reed, 1995), which promotes cell death (Allen et al., 1998). A reduction of Bcl-2 levels was observed in DA-1 cells after treatment for 13 h with E1 or E2 extracts or E3 fraction from Ganoderma lucidum (Figs 5 and 7). Cao and Lin (2004) also observed a reduction of the levels of Bcl-2 and an increment of the levels of Bax, in HUVEC cells treated with polysaccharides from G. lucidum. Tang et al. (2006) found that Bax expression increased in lung cancer 95-D cells after treatment with ganoderic acid T maintaining the levels of Bcl-2. These authors suggested apoptosis induction by ganoderic acid T via alteration of the Bax/Bcl-2 ratio. Jiang et al. (2004b) found that an aqueous extract containing polysaccharides and triterpenes from G. lucidum reduced the expression of Bcl-2 in prostate cancer PC-3 cells and increased the levels of Bax, cor­relating changes in Bax/Bcl-2 ratio with apoptosis induction (Raisova et al., 2001). The increase of Bax in cells treated for 13 h with Ganoderma lucidum E1 or E2 extract could suggest a role of Bax in the apoptosis induced by these extracts (Figs 5 and 6). In the case of the E3 fraction (Fig. 7), Bax disminished slightly with respect to untreated cells while Bcl-2 increased, showing low values for the Bax/Bcl-2 ratio which could be char­acteristic of resistant cells.

NF-kB is a factor related to cell growth, survival, angiogenesis, adhesion and migration (Mayo and Baldwin, 2000; Shibata et al, 2002). Jiang et al. (2004a) studied the effect of an aqueous extract containing poly- saccharides and triterpenes from G. lucidum in MDA- MB-231 breast cancer cells showing inhibition of NF-kB. A similar result has been described in PC-3 prostate cancer cells (Jiang et al., 2004b). In cultures of rat corti­cal neurons exposed to hypoxia, Zhao et al. (2004) also observed a blockage in the activation of NF-kB as a consequence of a polysaccharidic extract from G. lucidum. From the results an inhibition of NF-kB can be inferred, since E1, E2 or E3 reduced the levels of NF-kB in DA-1 cells (Figs 5-7).

Jiang et al. (2004a) studied the implication of Akt on the inhibitory effect of an aqueous extract containing polysaccharides and triterpenes from G. lucidum on NF-kB in MDA-MB-231 cells finding an inhibition of Akt expression in a dose- and time-dependent way. Although they did not find a reduction in phosphoryla- tion of Akt (pAkt) in Thr308, they observed a reduction in the levels of pAkt Ser473. Stanley et al. (2005) carried out a similar study in PC-3 cells, finding no changes either in Akt expression or in its phosphorylation in Thr308. However, the levels of pAkt in Ser473 disminished in a dose-dependent response. The results show that independent treatments with any of the extracts or E3 fraction from G. lucidum slightly increased the levels of Akt at 19 h. In the case of the E1 extract and E3 fraction (Figs 5, 7 and 8) the levels of Akt decreased at 24 h in accordance with Jiang et al. (2004a, 2004b) who carried out longer treatments (up to 96 h). The E1 extract treat­ment also produced a very slight reduction of pAkt with treatment for 24 h which could correlate with that shown by Jiang et al. (2004a, 2004b). Stanley et al. (2005) also showed a reduction of the level of p-Akt-Ser473 with treatments for 24 h with different doses of an aqueous extract from Ganoderma lucidum containing polysac- charides and triterpenes. The E2 extract induced a reduction of Akt and pAkt Ser473 at 13 h (Figs 6 and 8) with an increase of Akt at longer times. The correlation of the effects of E1, E2 extracts or E3 fraction on cell viability and DNA fragmentation in DA-1 cells (Figs 1 and 3) with the involvement of Akt expression and its phosphorylation on the cytotoxic action of Ganoderma lucidum in DA-1 cells can not yet be clearly established.

Finally, apoptosis induction by either the E1 extract or E3 fraction on DA-1 cells was clearly confirmed since caspase 3 activation was stimulated (Fig. 9). Cleavage of inactive caspase 3 demonstrates the role of the apopto- sis mechanism in the cytotoxicity induced in DA-1 cells by the E1 extract or E3 fraction from Ganoderma lucidum.

Acknowledgements

This work was supported in part by grants from UAH PI2004/025, CAM-UAH 2005-040 and CCG06-UAH/SAL-0672. E. Calviño was supported by a 'Miguel de Cervantes' fellowship from the Universi­dad de Alcalá. We also want to thank Isabel Trabado (C.A.I. Medic­ina-Biología, Unidad de Cultivos, Universidad de Alcalá) for technical assistance in cytometric analyses.

Ethics

This work accomplishes all the ethical institutional or national requirements.

Conflict of Interest

The authors declare no conflict of interest with this work.

REFERENCES

Allen RT, Cluck MW, Agrawal DK. 1998. Mechanisms controlling Barak Y, Juven T, Haffner R, Oren M. 1993. Mdm2 expression

cellular suicide: role of Bcl-2 and caspases. Cell Mol Life Sci        is induced by wild type p53 activity. EMBO J 12: 461 -

54: 427-445.                                                                                                                                                            468.

 

Cao QZ, Lin ZB. 2004. Antitumor and anti-angiogenic activity of Ganoderma lucidum polysaccharides peptide. Acta Phar­macol Sin 25: 833-838.

Chang S-T, Miles P. 2004. Mushrooms: Cultivation, Nutritional Value, Medicinal Effect, and Environmental Impact. CRC Press: Boca Raton.

Chen J, Wu X, Lin J, Levine AJ. 1996. Mdm-2 inhibits the G1 arrest and apoptosis functions of the p53 tumor suppressor protein. Mol Cell Biol 16: 2445-2452.

Cheung WMW, Hui WS, Chu PWK, Chiu SW, Ip NY. 2000. Gano­derma extract activates MAP kinases and induces the neu­ronal differentiation of rat pheochromocytoma PC12 cells. FEBS Lett 486: 291-296.

Chiou SK, Rao L, White E. 1994. Bcl-2 blocks p53-dependent apoptosis. Mol Cell Biol 14: 2556-2563.

Gottlieb E, Haffner R, von Ruden T, Wagner EF, Oren M. 1994. Down-regulation of wild-type p53 activity interferes with apoptosis of IL-3-dependent hematopoietic cells following IL-3 withdrawal. EMBO J 13: 1368-1374.

Gottlieb E, Lindner S, Oren M. 1996. Relationship of sequence- specific transactivation and p53-regulated apoptosis in interleukin 3-dependent hematopoietic cells. Cell Growth Diff 7: 301-310.

Gottlieb E, Oren M. 1998. p53 facilitates pRb cleavage in IL-3- deprived cells: novel pro-apoptotic activity of p53. EMBO J 17: 3587-3596.

Hong KJ, Dunn DM, Shen CL, Pence BC. 2004. Effects of Gano­derma lucidum on apoptotic and anti-inflammatory func­tion in HT-29 human colonic carcinoma cells. Phytother Res 18: 768-770.

Hu H, Ahn NS, Yang X, Lee YS, Kang KS. 2002. Ganoderma lucidum extract induces cell cycle arrest and apoptosis in MCF-7 human breast cancer cell. Int J Cancer 102: 250-253.

Jiang J, Slivova V, Harvey K, Valachovicova T, Sliva D. 2004a. Ganoderma lucidum suppresses growth of breast cancer cells through the inhibition of Akt/NF-kappaB signaling. Nutr Cancer 49: 209-216.

Jiang J, Slivova V, Valachovicova T, Harvey K, Sliva D. 2004b. Ganoderma lucidum inhibits proliferation and induces apoptosis in human prostate cancer cells PC-3. Int J Oncol 24: 1093-1099.

Li CH, Chen PY, Chang UM et al. 2005. Ganoderic acid X, a lanostanoid triterpene, inhibits topoisomerases and induces apoptosis of cancer cells. Life Sci 77: 252-265.

Lin SB, Li CH, Lee SS, Kan LS. 2003. Triterpene-enriched extracts from. Ganoderma lucidum inhibit growth of hepatoma cells via suppressing protein kinase C, activating mitogen- activated protein kinases and G2-phase cell cycle arrest. Life Sci 72: 2381-2390.

Lin ZB. 2005. Cellular and molecular mechanisms of immuno­modulation by Ganoderma lucidum. J Pharmacol Sci 99: 144-153.

Liu X, Yuan J-P, Chung Ch-K, Chen X-J. 2002. Antitumor activity of the sporoderm-broken germinating spores of Gano­derma lucidum. Cancer Lett 182: 155-161.

Lowe S, Bodis S, McClatchey A et al. 1994. p53 status and the efficacy of cancer therapy in vivo. Science 266: 807-810.

Lu QY, Jin YS, Zhang Q et al. 2004. Ganoderma lucidum extracts inhibit growth and induce actin polymerization in bladder cancer cells in vitro. Cancer Lett 216: 9-20.

Mayo MW, Baldwin AS. 2000. The transcription factor NF- kappaB: control of oncogenesis and cancer therapy resis­tance. Biochim Biophys Acta 1470: M55-M62.

Merino JJ, Cordero-Campaña MI. 1998. Molecular bases of the programmed cell death process: implications of tumor sup­pressor protein p53 and other proteins in the control of cell cycle. Mechanisms of apoptotic action. Invest Clin 39: 323-358.

Messmer UK, Brüne B. 1997. Attenuation of p53 expression and Bax down-regulation during phorbol ester mediated inhibi­tion of apoptosis. Br J Pharmacol 121: 625-634.

Min BS, Gao JJ, Nakamura N, Hattori M. 2000. Triterpenes from the spores of Ganoderma lucidum and their cytotoxicity against meth-A and LLC tumor cells. Chem Pharm Bull 48: 1026-1033.

Miyashita T, Reed JC. 1995. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80: 293-299.

Mizushina Y, Hanashima L, Yamaguchi T, Takemura M, Sugawara F, Saneyoshi M. 1998. A mushroom fruiting body-inducing substance inhibits activities of replicative DNA polymerases. Biochem Biophys Res Commun 249: 17-22.

Müller CI, Kumagai T, O'Kelly J, Seeram NP, Heber D, Koeffler HP. 2006. Ganoderma lucidum causes apoptosis in leuke­mia, lymphoma and multiple myeloma cells. Leuk Res 30: 841-848.

Newcomb EW. 1995. P53 gene mutations in lymphoid diseases and their possible relevance to drug resistance. Leuk Lym­phoma 17: 211-221.

Olmos G, Prieto A, Herraez A, Tejedor MC, Alvarez-Mon M, Diez JC. 2005a. Involvement of Bax, Bcl-2 and caspase -3 in hydroxyurea- or etoposide-induced apoptosis of mouse interleukin-3-dependent lymphoma cells. Anticancer Res 25: 999-1008.

Olmos G, Prieto A, Herraez A, Tejedor MC, Alvarez-Mon M, Diez JC. 2005b. Quantitation of apoptosis induction by etoposide or hydroxyurea in mouse interleukin 3-dependent lym­phoma cells. In Vivo 19: 455-464.

Palacios C, Gutierrez del Arroyo A, Silva A, Collins MK. 2000. The role of p53 in death of IL-3-dependent cells in response to cytotoxic drugs. Oncogene 19: 3556-3559.

Raisova M, Hossini AM, Eberle J et al. 2001. Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. J Invest Dermatol 117: 333-340.

Shibata A, Nagaya T, Imai T, Funahashi H, Nakao A, Seo H. 2002. Inhibition of NF-kappaB activity decreases the VEGF mRNA expression in MDA-MB-231 breast cancer cells. Breast Cancer Res Treat 73: 237-243.

Sliva D. 2004. Cellular and physiological effects of Ganoderma lucidum (Reishi). Mini Rev Med Chem 4: 873-879.

Sliva D, Labarrere C, Slivova V, Sedlak M, Lloyd Jr FP, Ho NW. 2002. Ganoderma lucidum suppresses motility of highly invasive breast and prostate cancer cells. Biochem Biophys Res Commun 298: 603-612.

Sone Y, Okuda R, Wada N, Kishida E, Misaki A. 1985. Structures and antitumor activities of the polysaccharides isolated from fruiting body and the growing culture of mycelium of Ganoderma lucidum. Agric Biol Chem 49: 2641-2653.

Stanley G, Harvey K, Slivova V, Jiang J, Sliva D. 2005. Gano- derma lucidum suppresses angiogenesis through the inhibition of secretion of VEGF and TGF-beta1 from prostate cancer cells. Biochem Biophys Res Commun 330: 46-52.

Tang W, Liu JW, Zhao WM, Wei DZ, Zhong JJ. 2006. Ganoderic acid T from Ganoderma lucidum mycelia induces mito­chondria mediated apoptosis in lung cancer cells. Life Sci 80: 205-211.

Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350-4354.

Trotter J. 2004. WinMDI (Windows Multiple Document Interface for Flow Cytometry), version 2.8. Available from http://facs. scripps.edu/software.html

Wilasrusmee C, Kittur S, Siddiqui J, Bruch D, Wilasrumee S, Kittur DS. 2002. In vitro immunomodulatory effects of ten commonly used herbs on murine lymphocytes. J Altern Complement Med 8: 467-475.

Williamson EM. 2001. Synergy and other interactions in phyto- medicines. Phytomedicine 8: 401-409.

Xie JT, Wang CZ, Wicks S et al. 2006. Ganoderma lucidum extract inhibits proliferation of SW 480 human colorectal cancer cells. Exp Oncol 28: 25-29.

Yeung WH, Lu QY, Zhang Q, Go VLW. 2004. Chemical and bio­chemical basis of the potential anti-tumor properties of Ganoderma lucidum. Curr Top Nutraceut Res 2: 67-77.

Zhao HB, Lin SQ, Liu JH, Lin ZB. 2004. Polysaccharide extract isolated from Ganoderma lucidum protects rat cerebral cor­tical neurons from hypoxia/reoxygenation injury. J Pharma­col Sci 95: 294-298.

 

Cell lines. Two cell lines were used: Mouse interleukin

3 dependent lymphoma DA-1 cells were kept at 37°C and 5% CO2, in Iscove's MDM medium containing 10% fetal calf serum, 2 mm l-glutamine and 2.5 x 10-5 m ß-mercaptoethanol.

The WEHI-3B cell line was employed and applied as a source of conditioned medium containing interleukin 3, which is required for the survival of DA-1 cells. The supernatant containing the medium with secreted IL-3 was obtained by centrifugation at 1200 x g for 5 min and subsequent filtration. The DA-1 cells were grown in the presence of 5% of this conditioned medium.

Source of Ganoderma lucidum. The Ganoderma lucidum [Ganoderma lucidum (Curtis) P. Karst.; Gano- dermataceae Donk] strain was isolated from the fruit body tissue (context) of a wood-inhabiting filamentous parasitic fungi growing in a natural habitat on Quercus ilex roots in Caceres (Spain). A small part of the tissue context developed Ganoderma lucidum mycelium that was cultured and subcultured in a malt extract solid medium (MA). In order to produce fruiting bodies it was necessary to cultivate the fungus on lignocellulosic solid medium substrate (Chang and Miles, 2004), ade­quately hydrated and autoclaved. Once the lignocellu- losic medium was inoculated with G. lucidum mycelia, the substrate was incubated until a proper mycelial colonization. Subsequently, the bulk mycelium devel­oped fruit bodies that reached maturity in 1-2 months.

Extracts of Ganoderma lucidum. Fruiting bodies of G. lucidum were resuspended in sterile water to a concen­tration of 50 mg/mL (Sliva et al., 2002; Stanley et al., 2005), homogenized and then centrifuged at 14000 rpm (in a Beckman J2.21 centrifuge using JA-20.1 rotor) for

 


[1] min, at room temperature, to obtain a supernatant called extract 1 (E1). The pellet was resuspended in the same volume of sterile water, boiled for 5 min, sonicated for 20 s at 50% intensity, centrifuged at 14000 rpm for 5 min and the supernatant obtained was called extract 2 (E2). Afterwards, these extracts were lyophyllized and disolved in 1/10 the initial volume, giving ten-fold con­centrated extracts.

Methanol extracts of G. lucidum were also obtained. Fruiting bodies (2 g) were disrupted with liquid nitro­gen and the resulting material was resuspended at 50 mg/mL concentration in 10% methanol and extracted three times with 10% methanol for 24 h with agitation.

 

Polishing

Full-length article

( Sitokinle indüklenmiş Killer hücreleri üzerine G.lucidum polysakkaridlerinin etkisi)

Effects of Ganoderma lucidum polysaccharides on proliferation and cytotoxicity of cytokine-induced killer cells

Xiao-ling ZHU, Zhi-bin LIN1

Department of Pharmacology, School of Basic Medical Science, Peking University Health Science Center, Beijing 100083, China

Key words

 

 

 

 

Ganoderma lucidum; polysaccharides; cell proliferation; cytotoxicity; immunomodula­tion

1 Correspondence to Prof Zhi-bin LIN. Phn/Fax 86-10-8280-1686. E-mail linzb@public3.bta.net.cn

Received 2004-11-23 Accepted 2005-03-14

doi: 10.1111/j.1745-7254.2005.00171.x

Aim: To study the effects (and the mechanisms thereof) of Ganoderma lucidum polysaccharides (Gl-PS) on the proliferation and the anti-tumor activity of cytokine- induced killer (CIK) cells, and to make use of CIK cells as a means to investigate the interactions between Gl-PS and cytokines. Methods: CIK cells were prepared by using the standard protocol as a positive control. Experimental groups also underwent the standard protocol, except that Gl-PS (400 mg/L or 100 mg/L) was added and the dose of anti-CD3 and interleukin-2 they received was reduced by 50% and 75%, respectively. For negative controls, Gl-PS in the experimental protocol was replaced with soluble starch or methylcellulose (400 mg/L or 100 mg/L). CIK cell proliferation, cytotoxicity, and phenotype were determined by using the Trypan blue exclusion method, MTT assay, and flow cytometry. Results: By synergizing cytokines, Gl-PS (400 mg/L or 100 mg/L) could decrease the amount of cytokine in lymphokine activated killer (LAK) cells and CIK cells culture, but had no significant effect on the proliferation, cytotoxicity, or phenotype of LAK cells, or CIK cells induced by cytokines at higher doses alone, in which CIK cells expanded about 80-fold and the main effectors, CD3+NK1.1+ cells, expanded by more than 15%. The cytotoxicity of CIK cells in experimental groups was 79.3%± 4.7%, 76.9%±6.8% versus the positive control 80.7%±6.8% against P815 (P>0.05) and 88.9%±5.5%, 84.7%±7.9% versus the positive control 89.8%±4.5% against YAC-1 (P>0.05). The activity of Gl-PS could mostly be blocked by anti-CR3. Conclusion: Gl-PS was shown to be a promising biological response modifier and immune potentiator. The effect of Gl-PS on CIK cells is possibly mediated prima­rily through complement receptor type 3.

 Introduction

The fungus Ganoderma lucidum (Leyss, ex Fr) Karst (known in China as "lingzhi" and in Japan as "reishi") has a long history of use in traditional Chinese medicine. Polysac­charides from Ganoderma lucidum have been reported to have promising immune modulating effects; for example, they have been shown to promote the function of the mononuclear phagocyte system, antigen-presenting cells and cytotoxic T lymphocytes (CTL) induced by dendritic cells[1-3], to enhance lymphocyte proliferation and antibody production[4-6], to potentiate cytokine production by splenocytes and macroph­ages^1, and to inhibit spontaneous and Fas-mediated

1130

apoptosis in neutrophils'81. The antitumor effect of G/-PS is reportedly due to its effects on the immune system, and there is reportedly no associated toxicity in humans[9].

Over the past 20 years or so, investigators have at­tempted to develop adoptive cellular immunotherapy for can­cer treatment. Cytokine-induced killer (CIK) cells have been shown to generate effector cells with higher proliferative capacity, increased cytotoxicity and fewer side effects than lymphokine activated killer (LAK) cells'10111. Some reports have demonstrated that G/-PS can enhance the cytotoxicity of CTL and natural killer (NK) cells'11, but little is known about the effects of G/-PS on the development and cytotoxic

©2005 CPS and SIMM

activity of CIK cells. The present study was undertaken to elucidate the effects (and mechanisms thereof) of Gl-PS on LAK cells and CIK cells induction and anti-tumor activity.

Materials and methods

Animals and drugs Male C57BL/6j mice 6-8-week-old were purchased from the Department of Experimental Animals, Health Science Center, Peking University, Beijing, China. Gl-PS were isolated from Ganoderma lucidum (Leyss, ex Fr) Karst by placing the fungus in boiling water, followed by ethanol precipitation, dialysis, and protein depletion using the Sevag method. The isolated molecule was a polysaccharide pep- tide with a molecular weight of 584 900, and a ratio of polysac­charides to peptides of 93.61%:6.49%. The polysaccharides included D-rhamnose, D-xylose, D-fructose, D-galactose, D- mannose and D-glucose with a molar ratio of 0.793:0.964: 2.944:0.167:0.389:7.94, linked together with P-glycosidic linkages. The peptides contained 16 kinds of amino acids[2]. Gl-PS was isolated as a water-soluble powder that was dis­solved in serum-free RPMI-1640 medium (Gibco BRL, NY, USA), then filtered through a 0.22 ^m filter and stored at 4 °C. This solution was further diluted to the required concen­trations before assay.

Isolation and culture of LAK cells LAK cells were gen­erated as previously described[9]. Briefly, mice were killed by cervical dislocation. Suspensions of spleen single cells were pooled in serum-free RPMI-1640 medium by filtering the suspension through mesh with the aid of a glass homog- enizer to exert gentle pressure on the spleen fragments. Eryth- rocytes were lysed with an ammonium chloride solution [0.15 mol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L edetic acid (EDTA), pH 7.2]. Obtained cells were washed twice with phosphate-buffered saline (PBS) and resuspended in the complete RPMI-1640 medium [RPMI-1640 medium supple­mented with 10% inactivated fetal calf serum (FCS), 2 mmol/L Z-glutamine, 25 mmol/L NaHCO3, 1 mmol/L sodium pyruvate, 25 mmol/L ^-2-hydroxyethylpiperazine-^'-2-ethanesulfonic acid (HEPES), 100 kU/L penicillin G, and 100 mg/L strepto­mycin] . Adherent cells were removed via adherence to glass surfaces. Nonadherent mononuclear cells were obtained and the percentage of viable cells was >95%. Murine recombi­nant interleukin-2 (rIL-2) (300 kU/L) was added on d 0. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 at a concentration of 1*109 cells/L. Cell density was determined every 4 d, and cells were subcultured in fresh complete medium containing 150 kU/L IL-2. LAK cells were cultured in complete medium with 300 kU/L IL-2 for the posi­tive control, with 75 kU/L IL-2 plus 400 mg/L or 100 mg/L Gl- PS for experimental groups 1 and 2, respectively, and with 75 kU/L IL-2 plus 400 or 100 mg/L soluble starch (SS) or methyl- cellulose (MC) as negative controls 1-4, respectively.

Isolation and culture of CIK cells CIK cells were gener­ated as previously described[10]. Nonadherent splenocytes were suspended in complete RPMI-1640 medium at a con­centration of 1*109 cells/L. Murine y-interferon (IFN-y; 1000 kU/L) was added on d 0. After 24 h of incubation, 50 mg/L monoclonal antibodies (mAb) against CD3 (dilution of su­pernatant from hybridoma 145-2C11), 300 kU/L IL-2, and 100 kU/L murine rIL-1 were added. Fresh IL-2 (150 kU/L) and fresh medium were added every 3 d to maintain a cell density of 1.5x109-2.0x109 cells/L for 14-21 d. All the cytokines were purchased from PeproTech EC, London, UK. CIK cells were prepared by using the standard protocol described ear­lier for the positive control. Experimental groups 1 and 2 were prepared according to the standard protocol except that Gl-PS (400 or 100 mg/L) was added, and the dose of anti- CD3 and IL-2 was decreased by 50% and 75%, respectively. For negative control groups 1-4, Gl-PS in the experimental protocol was replaced with SS or MC (400 or 100 mg/L).

LAK and CIK cell proliferation assay Cell density was assessed every 3 d, and cells were subcultured in fresh com­plete medium containing IL-2 as described previously[10]. Viable cell numbers were determined by the Trypan blue dye exclusion method at each time point. LAK and CIK cell proliferation was analyzed by cell growth curves.

Preparation of target cells P815 (NK-resistant) and YAC- 1 (NK-sensitive) cells proliferating in a logarithmic manner were used as target cells for the cytotoxicity assay. Cells (2*108 cells/L) were maintained in complete RPMI-1640 medium as described earlier. On the day of testing, cells were washed once with PBS and re-suspended in a complete medium at a concentration of 2*108 cells/L.

Cytotoxicity assay The MTT colorimetric assay was used for testing cytotoxic activity in vitro[l2 LAK cells or CIK cells were used as effectors, and were coincubated with P815 or YAC-1 cells as targets for 4 h at effector:target ratios of 20:1. Cells were plated in triplicate in 96-well microculture plates (Corning Costar, MA, USA). MTT (Sigma, St Louis, MO, USA) was dissolved at 5 g/L in PBS and filtered through a 0.22 ^m filter. MTT solution (20 ^L) was added to each well, and the microplates were further incubated at 37 °C for 4 h. The 96-well microplates were centrifuged at 200*g for 15 min, then supernatants were discarded, and 150 ^L of Me2SO was added into each well. The tray was gently shaken thoroughly for 10 min to dissolve the dark blue crystals of formazan that could be measured spectrophotometrically. Data are expressed as mean absorbance value (optical density, OD) of samples±SD. Percentage-specific cytotox- icity (% C) was calculated as follows: % C={1-[(OD of effectors+targets)-ftD of effectors]/ftD of targets}x100%.

Flow cytometric assays Effector cells were harvested and washed twice with ice cold FACScan buffer (PBS con­taining 2% FCS and 0.1% sodium azide). To block nonspe­cific antibody binding, 20% mixed mouse and rat serum was used and then cells were stained with mAb against CD3 coupled to fluorescein isothiocyanate (FITC) and/or mAb against NK1.1 coupled to phycoerythrin (PE) (antibodies from Santa Cruz, CA, USA or Pharmingen, CA, USA) for 45 min at 4 °C in the dark. The stained cells were washed twice and fixed with 1% paraformaldehyde in FACScan buffer and then analyzed by using a FACScan flow cytometer (Becton Dickinson, NJ, USA). Dead cells and debris were gated out.

Antibody block experiment CIK cells were induced by using the standard protocol described earlier for the posi­tive control, except that Gl-PS (400 or 100 mg/L) was added and the dose of anti-CD3 and IL-2 was decreased by 50% and 75% (experimental groups 1 and 2, respectively) or Gl-PS in the experimental group protocol was replaced by SS (400 mg/L or 100 mg/L) or MC (400 mg/L or 100 mg/L) as negative controls 1-4, respectively. Anti-CR3 (eBioscience, San Diego, CA, USA) or its isotype immunoglobulin G (IgG) was added 30 min prior to Gl-PS in the experimental group protocol, in anti-CR3 groups 1 or 2 and isotype antibody groups 1 or 2. Cells were harvested on d 7. The proliferation and cytotoxicity of different groups were determined by MTT colorimetric assay.

Statistical analysis Data were analyzed using general linear models (2-way ANOVA) in SPSS statistical software (version 10.0) with a significance level of a=0.05. Compari­sons between means were made using the least significant difference multiple comparisons t- test of Dunnett's t-test. Results are presented as the mean±SD. Values of P<0.05 were considered to be statistically significant.

Results

Effects of Gl-PS on LAK cell proliferation Nonadherent splenocytes were incubated at 1x109 cells/L in complete me­dium with IL-2 alone or IL-2 at different concentrations plus Gl-PS at different concentrations. Cells were harvested after 4 d to generate LAK cells. Statistical tests of the between- subject effects showed that there were significant differences according to univariate analyses of variance (two-way ANOVA). The interaction between IL-2 and Gl-PS was significant, which suggests a synergy between IL-2 and Gl-PS. The effect of IL-2 was bigger than that of Gl-PS. For the factor "IL-2", there was no statistically significant differ­ence between neither the 150 kU/L nor the 75 kU/L level when compared with the 300 kU/L level (P>0.05). For the factor "Gl-PS ", the 400 mg/L and 100 mg/L levels were sig­nificantly different from the 0 mg/L level (P<0.01). The com­bination of 75 kU/L IL-2 and 400 mg/L or 100 mg/L Gl-PS was optimal for the least usage of IL-2 (75 kU/L), and LAK cell proliferation induced by the combination was not less than that of the positive control, in which only IL-2 (300 kU/L) was added (Table 1). Compared with only 75 kU/L IL-2 in the LAK cell culture, the effects of SS or MC in different concen­trations plus 75 kU/L IL-2 were not significant, therefore SS and MC were chosen as negative controls (data not shown).

Cells in different groups were harvested and viable cell numbers were determined on d 1, 4, 8, and 12. The difference in cell numbers across groups was not significant on d 1. Following 4 d of culture, the number of viable cells of the positive control and experimental groups increased 2-fold in comparison to the negative controls, but the difference be­tween experimental groups and the positive control was not significant at that time. The number of viable cells in the positive control and experimental groups increased mark­edly compared with negative controls on d 8. However, at that time, there was no significant difference between the

Effects of Gl-PS on CIK cell proliferation With respect to the synergistic interaction of Gl-PS (400 mg/L or 100 mg/L)

and IL-2 (75 kU/L), experimental groups 1 and 2 were the same as the positive control except for the addition of 400 or 100 mg/L Gl-PS and a reduction in the doses of anti-CD3 and IL-2 by 50% and 75%, respectively. For negative controls 1­4, Gl-PS was replaced with SS or MC (400 mg/L or 100 mg/L) in the protocol used in the experimental groups. We ob­served that the proliferation of CIK cells in the experimental groups was also similar to that of the positive control. The reduction of cell numbers in the negative controls was sig­nificant compared with the positive control on d 6 to d 21. The number of CIK cells in the positive control and experi­mental groups peaked at d 21 and was expanded about 80­fold relative to the beginning culture. Their increase in ac­tivity was much larger than that observed in LAK cells (LAK proliferation peaked at d 4-8 and the cell number increased

  • 2- fold relative to the initial culture). This suggests that 400 mg/L or 100 mg/L Gl-PS decreased the usage of IL-2 and anti-CD3 by 75% and 50%, respectively, and that the influ­ence on CIK cell proliferation was almost equal to that of cytokines alone at higher doses (Figure 2).

Effects of Gl-PS on cytotoxicity of LAK Compared with induction by 300 kU/L IL-2 alone, no significant difference in the cytotoxicity of LAK cells induced by 75 kU/L IL-2 plus 400 mg/L or 100 mg/L Gl-PS was observed, but cytotoxicity induced by 75 kU/L IL-2 plus SS or MC instead of Gl-PS in the negative controls was reduced. This suggests that a certain concentration of Gl-PS increases the cytotoxicity of LAK cells (Figure 3).

Effects of Gl-PS on cytotoxicity of CIK cells Compared with the standard protocol for preparing CIK cells, the cyto- toxicity of CIK cells in the experimental groups was not sig­nificantly different, but was higher than that in the negative control groups. We found that CIK cells in every group achieved higher cytotoxicity than LAK cells. This suggests that 400 mg/L or 100 mg/L Gl-PS decreases the usage of IL-2 and anti-CD3 by 75% and 50%, respectively, but has no influence on CIK cell cytotoxicity induced by cytokines alone at higher doses (Figure 4).

Immunophenotype of effector cells in the presence of Gl-PSFlow cytometric assays showed that LAK cells repre­sented a heterogeneous population, in which the major ef­fector cells generally had an NK1.1+ cell surface phenotype. Compared with LAK cells in the positive control groups, the phenotype of LAK cells in the experimental groups was not significantly different. Most CIK cells expressed CD3, a sur-

 

face marker of T cells. Treatment with 400 mg/L or 100 mg/L Gl-PS in the experimental groups did not alter the phenotype of CIK cells (Table 2).

Effect of anti-CR3 on proliferation and cytotoxicity of CIK cells induced by stimuli containing Gl-PS CIK cell proliferation and cytotoxicity were reduced to <40% of that in the experimental groups by the addition of anti-CR3 30 min prior to the addition of Gl-PS in CIK cell culture, but not by nonspecific isotype IgG (Figures 5, 6).

Discussion

Polysaccharides isolated from Ganoderma are high- molecular-weight polysaccharides linked together by P-gly- cosidic linkages. These polysaccharides exhibit immuno­logical activity, for example promoting cellular immunity and humoral immunity'1-8'13151. Biological and immunopharma- cological activities depend on the conformation of polysac- charides, molecular mass, solubility in water and triple- helical structure. Soluble starch is an a-(1,4)-bonding glu­cose polymer, and methylcellulose links by uniform P-(1,4) glycosidic linkages'161. Because they lack a helical structure, neither of these compounds have the immunological activity, so they served as negative controls. Flow cytometry indi­cated that LAK and CIK cells populations were hetero­geneous. Effector cells in LAK cell culture generally have the CD3"NK1.1+ cell surface phenotype'17]; however, most CIK cells express T-cell markers. Earlier studies have shown that cytotoxicity correlates with the expansion of CD3+NK1.1+ lymphocytes, which are the main effector cells in CIK cell cultures'181. The present study demonstrated that the opti­mal combination of Gl-PS (400 mg/L or 100 mg/L) and certain cytokines did not alter the phenotype of LAK cells or CIK cells, and had no effect on the percentage of effector cells. For this reason, the cytotoxicity of LAK cells and CIK cells in the experimental groups was similar to that of the positive control, which contained cytokines alone at higher doses.

Table 2. Influences of Gl-PS on the immunophenotype of murine LAK cells and CIK cells. n=6. Mean±SD.

Group

CD3+/%

NK1.1+/%

CD3+NK1.1+/%

LAK cells

 

 

 

 

Group 1

62.2±7.3

11.

3±4.3

3.8±0.9

Group 2

58.6±9.3

10.

8±4.5

3.7±1.0

Group 3

57.5±7.2

10.

. 4±5.0

3.4±1.1

CIK cells

 

 

 

 

Group 4

96.8±4.5

20.

3±6.3

18.3±5.4

Group 5

95.4±4.6

19

. 1±5.8

16.9±5.1

Group 6

95.1±3.2

18.

2±6.4

15.7±4.5

 

 

CIK cells with enhanced cytotoxicity and a higher prolif- erative capacity relative to LAK cells were described first by Schmidt-Wolf and colleagues in 1991. They reported that treatment of tumor cells from bone marrow with CIK cells effectively reduced the tumor cell burden, allowing animals to survive'101. As demonstrated here, LAK and CIK cells

possess cytotoxic effects against both NK-sensitive and NK- press both the Fas receptor and FasL. Fas-sensitive and resistant tumor cells, and CIK cells are more cytotoxic than previously activated T cells are eliminated in the early stages LAK cells. Previous studies have shown that CIK cells ex- of CIK cell culture through either IFN-y, activation-induced

cell death or both. To generate CIK cells, splenocytes are stimulated with IFN-y 24 h prior to anti-CD3 and IL-2 treatment. This combination of cytokines might select for a population of Fas-resistant cells'191. In the present study, the CIK cell growth curve decreased in the initial stages, then increased. The initial decrease might be related to the downregulation of cell proliferation caused by IFN-y. In the present study, a synergistic interaction between IL-2 (75­300 kU/L) and Gl-PS (400 mg/L or 100 mg/L) enhanced LAK cell development and cytotoxicity. In combination with 1000 kU/L IFN-y, 75 kU/L IL-2, 25 mg/L anti-CD3 and 100 kU/L IL-1, Gl-PS (400 mg/L or 100 mg/L) decreased the dose of IL-2 and anti-CD3 by 75% and 50% respectively, but did not influence CIK cell proliferation and cytotoxicity. These results show that only intermediate concentrations (not too high or too low) of Gl-PS enhance immunocyte development; and the results also suggest that a certain concentration of Gl-PS could be an immune potentiator to reduce the dose of cytokine inducing LAK and CIK cells. Previous studies have shown that Gl-PS can stimulate splenocytes and macroph­ages to produce cytokines, including IL-1, IL-2, IFN-y and tumor necrosis factor'5'20-231. Whether Gl-PS exerts its activ­ity by promoting the release of cytokines by the precursors of CIK cells is currently under investigation.

ß-Glucans are known to bind to receptors, for example CR3. The binding site of CR3 has a broad specificity for certain polysaccharides containing glucose and mannose, as well as ^-acetyl-D-glucosamine'241. Gl-PS is a hetero- glycan that contains primarily glucose'21, and CR3 is ex­pressed mostly by activated lymphocytes, NK cells, mac­rophages and granulocytes'251. Our results demonstrated that the activity of CIK cells mediated by Gl-PS was drasti­cally inhibited (60%-70%) by prior treatment of murine splenocytes with anti-CR3. These results correspond with those of Xia et al, who found that ß-Glucans bound to CR3 on macrophages, neutrophils, and NK cells in humans and mice, and cytotoxicity mediated by ß-glucans can be blocked by anti-CR3 added before or after ß-glucan treatment'251. It has been confirmed that anti-CR3 had no effect on the cyto- toxic activity of CIK cells'261. These results suggest that the immune-modulating effect of Gl-PS on CIK cells is mediated primarily through CR3. Our study provides useful data for the use of Gl-PS in combination with other immuno- activators.

Acknowledgements

This research was supported by the Research Fund of Shanghai Green Valley Holdings Co. The Gl-PS was kindly provided by Prof Shu-qian LIN and Associate Prof Sai-zhen WANG from the Fuzhou Institute of Green Valley Bio-Pharm Technology.

References

  • 1 Lin ZB, Zhang HN. Anti-tumor and immunoregulatory activi­ties of Ganoderma lucidum and its possible mechanisms. Acta Pharmacol Sin 2004; 25: 1387-95.
  • 2 Cao LZ, Lin ZB. Regulation on maturation and function of dendritic cells by Ganoderma lucidum polysaccharides. Immunol Lett 2002; 83: 163-9.
  • 3 Cao LZ, Lin ZB. Regulatory effect of Ganoderma lucidum polysaccharides on cytotoxic T-lymphocytes induced by den­dritic cells in vitro. Acta Pharmacol Sin 2003; 24: 321-6.
  • 4 Zhang J, Tang Q, Zimmerman-Kordmann M, Reutter W, Fan H. Activation of B lymphocytes by GLIS, a bioactive proteoglycan from Ganoderma lucidum. Life Sci 2002; 71: 623-38.
  • 5 Bao XF, Zhen Y, Ruan L, Fang JN. Purification, characterization, and modification of T lymphocyte-stimulating polysaccharide from spores of Ganoderma lucidum. Chem Pharm Bull (Tokyo) 2002; 50: 623-9.
  • 6 Wasser SP, Weis AL. Therapeutic effects of substances occurring in higher Basidiomycetes mushrooms: a modern perspective. Crit Rev Immunol 1999; 19: 65-96.
  • 7 Lin ZB, Zhang QH. Effect of Ganoderma lucidum polysaccha­ride B on TNF-a and IFN-y production and their mRNA expression. J Beijing Med Univ 1999; 31: 179-83.
  • 8 Hsu MJ, Lee SS, Lin WW. Polysaccharide purified from Ganoderma lucidum inhibits spontaneous and Fas-mediated apoptosis in human neutrophils through activation of the phosphatidylinositol 3 kinase/Akt signaling pathway. J Leukoc Biol 2002; 72: 207-16.
  • 9 Lin ZB. Progress of studies on the anti-tumor activity and immunomodulating effect of Ganoderma. J Beijing Med Univ 2002; 34: 493-8.
  • 10 Schmidt-Wolf IG, Negrin RS, Kiem HP, Blume KG, Weissman IL. Use of a SCID mouse/human lymphoma model to evaluate cytokine-induced killer cells with potent anti-tumor cell activity. J Exp Med 1991; 174: 139-49.
  • 11 Lu PH, Negrin RS. A novel population of expended human CD3+CD56+ cells derived from T cells with potent in vivo antitu­mor activity in mice with severe combined immunodeficiency. J Immunol 1994; 153: 1687-96.
  • 12 Ferrari M, Fornasiero MC, Isetta AM. MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. J Immunol Methods 1990; 131: 165-72.
  • 13 Zakany J, Chihara G, Fachet J. Effect of Lentinan on tumor growth in murine allogeneic and syngeneic host. Int J Cancer 1980; 25: 371-6.
  • 14 Zakany J, Chihara G, Fachet J. Effect of Lentinan on the pro­duction of migration inhibitory factor induced by syngeneic tu­mor in mice. Int J Cancer 1980; 26: 783-8.

1 5 Bao XF, Wang XS, Dong Q, Fang JN, Li XY. Structural features of immunologically active polysaccharides from Ganoderma lucidum. Phytochemistry 2002; 59: 175-81.

16 Chen HL. Studies of the conformation and activity of polysaccharides. Shanghai: Shanghai Medical University Press; 1997.

17 Schmidt-Wolf IG, Lefterova P, Johnston V, Scheffold C, Csipai M, Mehta BA, et al. Sensitivity of multidrug-resistant tumor cell lines to immunologic effector cells. Cell Immunol 1996; 169: 85-90.

  • 1 8 Schmidt-Wolf IG, Lefterova P, Mehta BA, Fernandez LP, Huhn

D, Blume KG, et al. Phenotypic characterization and identifica­tion of effector cells involved in tumor recognition of cytokine- induced killer cells. Exp Hematol 1993; 21: 1673-9.

  • 19 Verneris MR, Kornacker M, Mailänder V, Negrin RS. Resistance of ex vivo expanded CD3+CD56+ T cells to Fas-mediated apoptosis. Cancer Immunol Immunother 2000; 49: 335-45.
  • 20 Wang YY, Khoo KH, Chen ST, Lin CC, Wong CH, Lin CH. Studies on the immuno-modulating and antitumor activities of Ganoderma lucidum (Reishi) polysaccharides: functional and proteomic analyses of a fucose-containing glycoprotein fraction responsible for the activities. Bioorg Med Chem 2002; 10: 1057­62.
  • 2 1 Ma L, Lin ZB, Li RZ, He YQ. Effects of Ganoderma polysac­

charides on IL-2 production by mouse splenocytes in vitro.

J Beijing Med Univ 1991; 23: 412-6.

  • 22 Lei LS, Lin ZB. Effects of Ganoderma polysaccharides on T cell subpopulations and production of interleukin 2 in mixed lymphocyte response. Acta Pharmacol Sin 1992; 27: 331-5.
  • 23 Zhang QH, Lin ZB. The antitumor activity of Ganoderma lucidum (Curt Fr ) P Karst (Ling Zhi) (Aphyllophoromycetideae) polysac­charides is related to tumor necrosis factor-a and interferon-y. Int J Med Mushroom 1999; 1: 207-15.
  • 24 Thornton BP, Vetvicka V, Pitman M, Goldman RC, Ross GD. Analysis of the sugar specificity and molecular location of the beta-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18). J Immunol 1996; 156: 1235-46.

2 5 Xia Y, Vetvicka V, Yan J, Hanikyrova M, Mayadas T, Ross GD. The beta-glucan-binding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized tar­get cells. J Immunol 1999; 162: 2281-90.

26 Schmidt-Wolf IG, Lefterova P, Johnston V, Huhn D, Blume KG, Negrin RS. Propagation of large numbers of T cells with natural killer cell markers. Br J Haematol 1994; 87: 453-8.

 

 

Laborstudie: Ganoderma gegen Eierstockkrebs!

Es gibt viele Studien, die sich mit den krebshemmenden Eigenschaften von Ganoderma lucidum (häufig auch als Ling Zhi bezeichnet) befassen. Eine aktuelle Laboruntersuchung hat sich erstmalig speziell mit Eierstockkrebszellen beschäftigt. Diese Krebsart (auch epitheliales Ovarialkarzinom oder abgekürzt EOC genannt) wird häufig leider erst entdeckt, wenn die Erkrankung schon weit fortgeschritten ist. Der Tumor bildet schnell Metastasen, die sich vor allem in der Bauchhöhle ausbreiten. Üblicherweise wird Eierstockkrebs mit Cisplatin behandelt. Allerdings ist die Entwicklung von Resistenzen häufig: die Krebszellen reagieren dann nicht mehr auf die Chemotherapie.
In dieser Studie wurde jetzt untersucht, ob Ganoderma lucidum die Aktivität der Eierstockkrebszellen reguliert. Dabei wurde festgestellt, dass Ganoderma lucidum bei mehreren EOC-Krebszelllinien für einen starken Rückgang der Zellzahlen sorgte. Dieser Effekt war dosisabhängig, d.h. je mehr Ganoderma-lucidum-Extrakt, desto weniger Krebszellen zeigten sich unter dem Mikroskop - unabhängig davon, ob die Krebszellen Resistenzen gegenüber Chemotherapeutika entwickelt hatten oder nicht.
Die Wirkung von Cisplatin auf die Krebszellen wurde durch Ganoderma lucidum verstärkt, die Apoptose (Zell-Selbstmord) wurde häufiger.
Die Autoren zeigen sich zufrieden mit den Ergebnissen. Sie gehen davon aus, dass Ganoderma lucidum auf mehreren Ebenen Anti-Tumor-Effekte auf Eierstockkrebszellen ausübt und außerdem die Empfindlichkeit der Krebszellen auf Cisplatin erhöhen kann.
Quelle: Zhao S, Ye G, Fu G, Cheng JX, Yang BB, Peng C: Ganoderma lucidum exerts anti-tumor effects on ovarian cancer cells and enhances their sensitivity to cisplatin. Int J Oncol. 2011 May;38(5):1319-27. doi: 10.3892/ijo.2011.965. Epub 2011 Mar 8.

 

TERCÜMESİ:

Yumurtalık kanserine karşı GANODERMA (REISHI)

            Ganoderma'nın (Sık sık Ling-Zhi olarak da adlandırılır.) kanseri  önlemede veya tedavisinde katkı sağlayıcı etkilerinden sık sık yayın yapılmakta ve söz edilmektedir. Güncel bir laboratuar araştırması, bu mantarın yumurtalık kanser hücreleri üzerine etkisini ilk kez ortaya çıkarmaya yönelik gerçekleştirilmiştir. EOC (Epitelial Ovarial Carsinom) adıyla da bilinen bu kanser türü, maalesef vücutta gecikmiş devrelerde fark edilir. Tümör çok hızlı metastaz oluşturma yeteneğindedir ve bunlar özellikle karın boşluğunda yaygındırlar. Günümüz tedavi uygulamalarında bu kansere karşı cisplatin kullanılmaktadır. Ancak çok sık dirençlilik geliştiğinden, bir süre sonra kemoterapiye cevap alınamaz olur.

            Bu çalışmada, Ganoderma lucidum'un yumurtalık kanser hücrelerinin aktivitesini hangi düzeyde ve ne yönde etkilediği araştırılmıştır. Ortaya çıkan sonuçlar göstermektedir ki, EOC kanser hücre hatlarının çoğunda Ganoderma lucidum etkinliği kanser hücresi sayısının üst düzeyde indirgenmesi şeklinde kendini göstermektedir. Bu etkinliğin doza bağlı olduğu ve kemoterapi dirençli veya dirençsiz kanser hücresi farklılığı gözetmeksizin, ne kadar yüksek doz kullanılırsa, kanser hücresi sayısında o kadar azalma görüldüğü bulunmuştur. Ek olarak, cisplatinin kanser hücrelerine etkisinin daha üst düzeylere çıkartıldığı ve Ganoderma lucidum'un bunun yanı sıra apoptoz düzeyini de yükselttiği bulunmuştur.

            Araştırmacılar sonuçlardan memnundurlar. Öyle ki, Ganoderma lucidum'un yumurtalık kanseri hücreleri üzerine çok farklı yönlerde ve düzeylerde antitümör etkinliği gösterdiğini ve kanser hücrelerinin cisplatine hassasiyetinin olumlu yönde arttırıldığını ifade etmektedirler.

Kaynak: Zhao S, Ye G, Fu G, Cheng JX, Yang BB, Peng C: Ganoderma lucidum exerts anti-tumor effects on ovarian cancer cells and enhances their sensitivity to cisplatin. Int J Oncol. 2011 May;38(5):1319-27. doi: 10.3892/ijo.2011.965. Epub 2011 Mar 8.

 

Ling Zhi: ein natürliches Anti-Aging-Mittel!

Das Stichwort Anti-Aging geistert seit Jahren durch die Presse. Hormone und Nahrungsergänzungsmittel sollen den Alterungsprozess aufhalten, für jugendliches Aussehen und geistige Frische sorgen. Eine indische Untersuchung könnte in dieser Szene für Aufruhr sorgen: Ausgerechnet ein Medizinalpilz zeigt spannende Ergebnisse in der „Behandlung" alternder Mäuse. Genauer gesagt: Es ging um Ganoderma lucidum und um alternde Mitochondrien. 
Mitochondrien haben zwar nur die Größe von Bakterien, sie sind aber für alle Lebewesen als Zellkraftwerke oder Energiegeneratoren wichtig. Jede Körperzelle enthält viele Hundert Mitochondrien, die für die Aufrechterhaltung des zellulären Energiestoffwechsels sorgen. Allerdings ist das Erbmaterial der Mitochondrien durch hochreaktive Sauerstoffverbindungen wie Wasserstoffperoxid besonders gefährdet. Sie werden während des Energiestoffwechsels in den Mitochondrien gebildet, ein Abfallprodukt der Stoffwechselaktivität sozusagen. Diese hochreaktiven Sauerstoffverbindungen bewirken Informationsverluste der DNA, so dass die Leistungsfähigkeit der Mitochondrien mit zunehmendem Lebensalter sinkt: der Alterungsprozess läuft.
Ein Gegenspieler der Alterung ist das reduzierte Glutathion. Es kann freie Radikale „entschärfen" und so dafür sorgen, dass die hochreaktiven Sauerstoffverbindungen weniger Schaden anrichten. 
In der indischen Studie wurden alternde Mäuse mit Ganoderma lucidum gefüttert. Das Resultat kann sich sehen lassen: Das reduzierte Glutathion erhöhte sich, während die Wasserstoffperoxidspiegel sanken. 
Das Resultat passt gut zu den Erfahrungen aus Asien: Nicht umsonst heißt der Pilz dort „Pilz der Unsterblichkeit".
Quelle: 
Sudheesh NP, Ajith TA, Ramnath V, Janardhanan KK.: Therapeutic potential of Ganoderma lucidum (Fr.) P. Karst. against the declined antioxidant status in the mitochondria of post-mitotic tissues of aged mice. Clin Nutr.  2009 Dec 29. doi:10.1016/j.clnu.2009.12.003

 

TERCÜMESİ:

Ling-Zhi: Doğal Bir Gençlik İksiri

Basında yıllardan beri anti-Aging (yaşlanmayı önleyen, genç tutan) kelimesi sık sık göze çarpmaktadır. Hormonlar ve besin takviyesi şeklinde kullanılan yiyecekler, yaşlanma olayını durdurur veya hiç olmazsa yavaşlatır şeklinde bahsedilen faktörler olarak tanıtılmaktadırlar. Genç görünüm sağladıkları ve ruhu genç tuttukları belirtilir. Hindistan'da yapılan bir araştırma bu tabloda bir sansasyon yaratacak sonuçlardan bahsedilmektedir. Yaşlanmakta olan farelerle yapılan bu araştırmada, yaşlılığın tedavisi yönünde tıbbi bir mantarın harikalar yarattığından söz edilmektedir. Daha açık ifadeyle, çalışma sonuçları Ganoderma lucidum'un yaşlanmakta olan mitokondriler üzerine etkisinden söz etmektedir.

Mitokondriler, bakteri büyüklüğünde hücre içi organeller olup, tüm canlıların hücresel enerji reaktörleri, enerji jeneratörleri olarak görev yaparlar. Vücudumuzun her hücresi, hücrelerin ihtiyacı olan enerjinin üretilmesi için çalışan birkaç yüz mitokondri taşımaktadır. Diğer taraftan, mitokondrilerin kendilerine özgü DNA'ları (Kalıtım materyali) enerji üretimi sırasında ortaya çıkan hidrojen peroksit gibi yüksek reaktif (okside edici) bileşikler sebebiyle tehlike yaşamaktadır. Yani faaliyeti sırasında ortaya çıkan atıklardan muzdariptir. Bu tip okside edici bileşikler, yavaş yavaş da olsa DNA'nın tahrip olmasına ve ileri yaşlarda mitokondrilerin başarı düzeylerinin düşmesine sebep olur. Yani yaşlanma süreci sürer gider.

Bilindiği gibi yaşlılığın önlenmesinde, yavaşlatılmasında elimizdeki en güçlü bileşik indirgenmiş GLUTATION'dur. Bu bileşik serbest radikalleri yani moleküler oksijen gibi aktif bölgeleri yok eder, sayıca azaltır, böylece verebilecekleri hasar düzeyini düşürür. Sözü edilen çalışmada yaşlanmakta olan fareler, Ganoderma lucidum ile beslenmişlerdir. Sonuç, aman Allah dedirtecek kadar muhteşemdir. Ganoderma verilen farelerde, indirgenmiş Glutation düzeyi önemli ölçüde artmış ve bunun aksine hidrojen peroksit düzeyi müthiş azalmıştır. Bu sonuç, yüzyıllardan beri Asya'dan yükselen sese ve tecrübeye tamamen uymaktadır. Yani Ling-Zhi ismini yani, ölümsüzlük mantarı adını Asyalılar boş yere takmamışlardır.

Kaynak: 
Sudheesh NP, Ajith TA, Ramnath V, Janardhanan KK.: Therapeutic potential of Ganoderma lucidum (Fr.) P. Karst. against the declined antioxidant status in the mitochondria of post-mitotic tissues of aged mice. Clin Nutr.  2009 Dec 29. doi:10.1016/j.clnu.2009.12.003

 

Hoffnung für Parkinson-Patienten

Ganoderma lucidum, der Medizinalpilz Ling Zhi also, könnte durch seine entzündungshemmenden und immunmodulierenden Eigenschaften zur Behandlung der Parkinsonkrankheit beitragen. Zu diesem Ergebnis kommen die Pekinger Wissenschaftler nach einer Reihe von Laborversuchen.  
Die genaue Entstehung der Parkinson-Krankheit ist noch nicht endgültig geklärt. Sicher ist jedoch die Beteiligung entzündlicher Reaktionen. Diese zeigen sich als Mikroglia-Aktivierung. Mikrogliazellen sind für die aktive Immunabwehr im Zentralnervensystem zuständig. Sie haben also eine ähnliche Aufgabe wie Makrophagen, die in anderen Geweben Eindringlinge „fressen". 
Eine Parkinsonerkrankung lässt sich im Tiermodell durch bestimmte Gifte künstlich auslösen. Genau wie die echte Parkinson-Krankheit aktiviert das Gift die Mikroglia, die wie im Krankheitsfall für eine Ausschüttung von Zytokinen und Stickoxid sorgt. 
Dies änderte sich, wenn Ling Zhi hinzu kam. Dieser Medizinalpilz wirkte im Labormodell der Entzündung entgegen. Ganoderma lucidum könnte die Gehirnzellen vor den Schäden durch die Entzündung schützen.  Die Autoren hoffen angesichts ihrer Studienergebnisse, dass es bald bessere Behandlungsmöglichkeiten der Parkinson-Krankheit gibt.
Quelle: Zhang R, Xu S, Cai Y, Zhou M et al.: Ganoderma lucidum Protects Dopaminergic Neuron Degeneration Through Inhibition of Migriglial Activation. eCAM 2009;Page 1 of 9, doi:10.1093/ecam/nep075

TERCÜMESİ:

Parkinson Hastaları İçin Ümit

            Ling-Zhi olarak bilinen tıbbi mantar yani Ganoderma lucidum, enflamasyon önleyici ve immunomodüle edici özelliklerin sayesinde Parkinson hastalarının tedavisinde kullanılabilir kabul edilmektedir. Bu sonuca bir dizi laboratuvar araştırmasından sonra ulaşan Pekin'li bilim adamları aşağıdaki bulguları elde etmişlerdir.

            Her ne kadar Parkinson hastalığının ortaya çıkması henüz tam olarak aydınlatılamamış ve anlaşılamamış ise de, ortaya çıkmasında enflamasyon reaksiyonlarının kesinlikle etken olduğu bilinmektedir. Bu reaksiyonların en önemli bulgularından biri mikroglia aktivasyonudur. Mikroglia hücreleri Merkezi Sinir Sisteminde aktif bağışıklık cevabı fonksiyonu olan hücrelerdir. Yani makrofajlara benzer bir görevi sinir dokusu içinde gerçekleştirirler. Dokuya giren yabancıları yerler. Parkinson hastalığını belirli zehirler kullanarak, hayvan modellerinde oluşturmak mümkündür. Bu zehirler, tıpkı gerçek Parkinson hastalarında olduğu gibi mikrogliaları aktive ederler, aktive olmuş mikroglialar tıpkı gerçek hastalıkta olduğu gibi sitokinler ve azotoksitler salgılamaya başlarlar. Bu olay işin içine Ling-Zhi girince, tamamen değişmektedir. Bu tıbbi mantar, enflamasyona karşı üst düzeyde etkili olduğundan, bu yolla beyin hücrelerinin hasar görmesini engellemekte ve Parkinson'a karşı güçlü bir koruma sağlamaktadır. Araştırmacılar bu sonuçları gördükten sonra, Parkinson hastalığına yönelik tedavilerin çok kısa sürede ortaya çıkacaklarına inanmaktadırlar.

 Kaynak:Zhang R, Xu S, Cai Y, Zhou M et al.: Ganoderma lucidum Protects Dopaminergic Neuron Degeneration Through Inhibition of Migriglial Activation. eCAM 2009;Page 1 of 9, doi:10.1093/ecam/nep075

 

Wenn es nur noch tröpfelt... Heilpilze bei Prostatabeschwerden

Gute Nachrichten für Männer, die Schwierigkeiten mit dem Wasserlassen haben: In einer randomisierten Doppelblindstudie aus Japan zeigte sich Ling Zhi (Ganoderma lucidum) als wirkungsvoll bei Männern mit Miktionsstörungen.
Die Männer nahmen 12 Wochen lang täglich 6mg Ganoderma-Extrakt oder Plazebo ein. Der IPSS (internationale Prostata-Symptom-Score) besserte sich durch Ling Zhi stärker als unter Plazebo, Nebenwirkungen traten nicht auf.
Quelle: Noguchi M, Kakuma T, Tomiyasu K, Yamada A et al: Randomized clinical trial of an ethanol extract of Ganoderma lucidum in men with lower urinary tract symptoms. J Androl. 2007 Dec 20.

 

TERCÜMESİ:

Sadece Damlıyor Olsa Bile...! Mantarlar Prostat Problemlerinde Yardımcı Olmaktadırlar

            Erkeklere güzel haber, artık işemek problem yaratmayacak:

            Çift kontrollü rastgele seçilmiş deneklerle yapılan bir Japon araştırmasında Ling-Zhi (Ganoderma lucidum) miksiyon güçlüğü çeken erkeklerde çok başarılı sonuçlar vermiştir. Denekler 12 hafta boyunca ya günlük 6 mg Ganoderma ekstraktı (öz suyu) almışlar, ya da kontrol olarak klasik tedavi görmüşlerdir. IPSS (International Prostat Symptom Score) düzeyi Ling-Zhi kullanan hastalarda kontrollere göre çok daha olumlu değişiklikler göstermiş ve hiç bir yan etkiye rastlanmamıştır.

Kaynak: Noguchi M, Kakuma T, Tomiyasu K, Yamada A et al: Randomized clinical trial of an ethanol extract of Ganoderma lucidum in men with lower urinary tract symptoms. J Androl. 2007 Dec 20.