Herbal Medicine: Biomolecular and Clinical Aspects. 2nd edition.

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Benzie IFF, Wachtel-Galor S, editors.

Boca Raton (FL): CRC Press; 2011.

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Chapter 9Ganoderma lucidum (Lingzhi or Reishi)

A Medicinal Mushroom

Sissi Wachtel-Galor, John Yuen, John A. Buswell, and Iris F. F. Benzie.

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9.1. INTRODUCTION

Ganoderma lucidum, an oriental fungus (Figure 9.1), has a long history of use for promoting health and longevity in China, Japan, and other Asian countries. It is a large, dark mushroom with a glossy exterior and a woody texture. The Latin word lucidus means "shiny" or "brilliant" and refers to the varnished appearance of the surface of the mushroom. In China, G. lucidum is called lingzhi, whereas in Japan the name for the Ganodermataceae family is reishi or mannentake.

FIGURE 9.1. (See color insert.

FIGURE 9.1

(See color insert.) The lingzhi mushroom (Ganoderma lucidum). (Courtesy of North American Reishi/Nammex.)

In Chinese, the name lingzhi represents a combination of spiritual potency and essence of immortality, and is regarded as the "herb of spiritual potency," symbolizing success, well-being, divine power, and longevity. Among cultivated mushrooms, G. lucidum is unique in that its pharmaceutical rather than nutritional value is paramount. A variety of commercial G. lucidum products are available in various forms, such as powders, dietary supplements, and tea. These are produced from different parts of the mushroom, including mycelia, spores, and fruit body. The specific applications and attributed health benefits of lingzhi include control of blood glucose levels, modulation of the immune system, hepatoprotection, bacteriostasis, and more. The various beliefs regarding the health benefits of G. lucidum (Figure 9.2) are based largely on anecdotal evidence, traditional use, and cultural mores. However, recent reports provide scientific support to some of the ancient claims of the health benefits of lingzhi.

FIGURE 9.2. Postulated health benefits of lingzhi (Ganoderma lucidum).

FIGURE 9.2

Postulated health benefits of lingzhi (Ganoderma lucidum).

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9.2. HISTORY: LINGZHI AS A MEDICINAL MUSHROOM

Lingzhi has been recognized as a medicinal mushroom for over 2000 years, and its powerful effects have been documented in ancient scripts (Wasser 2005). The proliferation of G. lucidum images in art began in 1400 AD, and they are associated with Taoism (McMeekin 2005). However, G. lucidum images extended beyond religion and appeared in paintings, carvings, furniture, and even women's accessories (Wasser 2005). The first book wholly devoted to the description of herbs and their medicinal value was Shen Nong Ben Cao Jing, written in the Eastern Han dynasty of China (25-220 AD). This book is also known as "Classic of the Materia Medica" or "Shen-nong's Herbal Classics." It describes botanical, zoological, and mineral substances, and was composed in the second century under the pseudonym of Shen-nong ("the holy farmer"; Zhu, 1998). The book, which has been continually updated and extended, describes the beneficial effects of several mushrooms with a reference to the medicinal mushroom G. lucidum (Zhu, 1998; Upton 2000; Sanodiya et al. 2009). In the Supplement to Classic of Materia Medica (502-536 AD) and the Ben Cao Gang Mu by Li Shin-Zhen, which is considered to be the first pharmacopoeia in China (1590 AD; Ming dynasty), the mushroom was attributed with therapeutic properties, such as tonifying effects, enhancing vital energy, strengthening cardiac function, increasing memory, and antiaging effects. According to the State Pharmacopoeia of the People's Republic of China (2000), G. lucidum acts to replenish Qi, ease the mind, and relieve cough and asthma, and it is recommended for dizziness, insomnia, palpitation, and shortness of breath.

Wild lingzhi is rare, and in the years before it was cultivated, only the nobility could afford it. It was believed that the sacred fungus grew in the home of the immortals on the "three aisles of the blest" off the coast of China (McMeekin 2005). However, its reputation as a panacea may have been earned more by virtue of its irregular distribution, rarity, and use by the rich and privileged members of Chinese society than by its actual effects. Nevertheless, the Ganoderma species continue to be a popular traditional medicine in Asia and their use is growing throughout the world (Wachtel-Galor, Buswell et al. 2004; Lindequist, Niedermeyer, and Jülich 2005).

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9.3. TAXONOMY

The family Ganodermataceae describes polypore basidiomycetous fungi having a double-walled basidiospore (Donk 1964). In all, 219 species within the family have been assigned to the genus Ganoderma, of which G. lucidum (W. Curt.: Fr.) P. Karsten is the species type (Moncalvo 2000). Basidiocarps of this genus have a laccate (shiny) surface that is associated with the presence of thickwalled pilocystidia embedded in an extracellular melanin matrix (Moncalvo 2000). Ganoderma species are found all over the world, and different characteristics, such as shape and color (red, black, blue/green, white, yellow, and purple) of the fruit body, host specificity, and geographical origin, are used to identify individual members of the species (Zhao and Zhang 1994; Woo et al. 1999; Upton 2000). Unfortunately, the morphological characteristics are subject to variation resulting from, for example, differences in cultivation in different geographical locations under different climatic conditions and the natural genetic development (e.g., mutation, recombination) of individual species. Consequently, the use of macroscopic characteristics has resulted in a large number of synonyms and a confused, overlapping, and unclear taxonomy for this mushroom. Some taxonomists also consider macromorphological features to be of limited value in the identification of Ganoderma species due to its high phenotypic plasticity (Ryvarden 1994; Zhao and Zhang 1994). More reliable morphological characteristics for Ganoderma species are thought to include spore shape and size, context color and consistency, and the microanatomy of the pilear crust. Chlamydospore production and shape, enzymatic studies and, to a lesser extent, the range and optima of growth temperatures have also been used for differentiating morphologically similar species (Gottlieb, Saidman, and Wright 1998; Moncalvo 2000; Saltarelli et al. 2009). Biochemical, genetic, and molecular approaches have also been used in Ganoderma species taxonomy. Molecular-based methodologies adopted for identifying Ganoderma species include recombinant (rDNA) sequencing (Moncalvo et al. 1995; Gottlieb, Ferref, and Wright 2000), random amplified polymorphic DNA-PCR (RAPD; PCR stands for polymerase chain reaction), internal transcribed spacer (ITS) sequences (Hseu et al. 1996), sequence-related amplified polymorphism (SRAP; Sun et al. 2006), enterobacterial repetitive intergenic consensus (ERIC) elements, and amplified fragment length polymorphism (AFLP; Zheng et al. 2009). Other approaches to the problem of G. lucidum taxonomy include nondestructive nearinfrared (NIR) methods combined with chemometrics (Chen et al. 2008), nuclear magnetic resonance (NMR)-based metabolomics (Wen et al. 2010), and high-performance liquid chromatography (HPLC) for generating chemical fingerprints (Su et al. 2001; Chen et al. 2008; Shi, Zhang et al. 2008; Chen et al. 2010).

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9.4. CULTIVATION, GLOBAL USE, AND MANUFACTURE OF PRODUCTS

Owing to its irregular distribution in the wild and to an increasing demand for G. lucidum as a medicinal herb, attempts were made to cultivate the mushroom (Chang and Buswell 2008). Different members of the Ganoderma genus need different conditions for growth and cultivation (Mayzumi, Okamoto, and Mizuno 1997). Moreover, different types are favored in different geographical regions. For example, in South China, black G. lucidum is popular, whereas red G. lucidum is preferred in Japan. G. lucidum thrives under hot and humid conditions, and many wild varieties are found in the subtropical regions of the Orient. Since the early 1970s, cultivation of G. lucidum has become a major source of the mushroom. Artificial cultivation of G. lucidum has been achieved using substrates such as grain, sawdust, wood logs (Chang and Buswell 1999; Wasser 2005; Boh et al. 2007), and cork residues (Riu, Roig, and Sancho 1997).

Since it takes several months to culture the fruiting body of G. lucidum, mycelia-based and culture broth-based products have assumed greater importance due to demands for increased quality control and year-round production (Sanodiya et al. 2009). The processes and different growth parameters (e.g., temperature, pH) involved in submerged mycelial culture can easily be standardized under controlled conditions, and purification and other downstream processing of active components such as polysaccharides released into the culture medium usually involve relatively simple procedures. Different culture conditions and medium compositions have also been reported to strongly influence mycelial growth and the production of biopolymers (e.g., polysaccharides) that are extruded from the cell (exopolysaccharides [EPSs]; Mayzumi, Okamoto, and Mizuno 1997; Chang and Buswell 1999; Habijanic and Berovic 2000; Fang and Zhong 2002; Boh et al. 2007; Sanodiya et al. 2009). For example, Yang and Liau (1998) reported that polysaccharide production by fermenter-grown mycelia of G. lucidum was optimum at 30°C-35°C and a pH of 4-4.5, and the addition of supplements such as fatty acids was found to accelerate mycelial growth and the production of bioactive components. In a submerged culture of G. lucidum, the optimum pH for cell growth has been shown to be lower than that for EPS formation. A two-stage pH-control strategy, developed to maximize mycelial biomass and EPS production, revealed that culture pH had a significant effect on EPS yield, chemical composition and molecular weight, and mycelial morphology (Kim, Park, and Yun 2006). The productive mycelial morphological form for EPS production was a dispersed pellet (controlled pH shift from 3.0 to 6.0) rather than a compact pellet with a dense core (pH maintained at 4.5) or a featherlike pellet (controlled pH shift from 6.0 to 3.0). Three different polysaccharides were obtained under each pH condition, and their molecular weights and chemical compositions were significantly different (Kim, Park, and Yun 2006). More recently, a novel three-stage light irradiation strategy has been developed in submerged cultures of G. lucidum for the efficient production of polysaccharides and one of the triterpene components, ganoderic acid (Zhang and Tang 2008).

A decade ago, more than 90 brands of G. lucidum products were registered and marketed internationally (Lin 2000). Worldwide consumption is now estimated at several thousand tonnes, and the market is growing rapidly. Although there are no recently published data relating to the total world market value of ganoderma products, in 1995, the total estimated annual market value given by different commercial sources was US$1628 million (Chang and Buswell 1999). Numerous G. lucidum products, prepared from different parts of the mushroom, are currently available on the market (Chang and Buswell 2008). In manufacturing terms, the simplest type consists of intact fruiting bodies ground to powder and then processed to capsule or tablet form. Other "nonextracted" products are prepared from the following three sources: (1) dried and powdered mycelia harvested from submerged liquid cultures grown in fermentation tanks; (2) dried and powdered combinations of substrate, mycelia, and mushroom primordia, following inoculation and incubation of a semisolid medium with fungal mycelia; and (3) intact fungal spores or spores that have been broken by mechanical means or have had the spore walls removed. Although spore preparations have been researched and promoted vigorously in recent years, any added medicinal effects attributable to the removal or breakage of spore walls, which represents an additional and often costly step in the production process, are still controversial. Other products are prepared with materials (e.g., polysaccharides, triterpenes) extracted, usually with hot water or ethanol, from fruiting bodies or mycelia harvested from submerged liquid cultures and then evaporated to dryness and tabulated/encapsulated either separately or integrated together in designated proportions. The adoption of supercritical fluid CO2 extraction technologies has enlarged the spectrum of extracted substances due to the low temperature required during processing. Several other products have been prepared as binary, ternary or more complex mixtures of powdered ganoderma and other mushrooms (e.g., Lentinula edodes, Agaricus brasiliensis, Grifola frondosa, Pleurotus spp., and Flammulina velutipes) and even with other medicinal herbs (e.g., spirulina powder or flower pollen grains).

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9.5. MAJOR BIOACTIVE COMPONENTS

Most mushrooms are composed of around 90% water by weight. The remaining 10% consists of 10-40% protein, 2-8% fat, 3-28% carbohydrate, 3-32% fiber, 8-10% ash, and some vitamins and minerals, with potassium, calcium, phosphorus, magnesium, selenium, iron, zinc, and copper accounting for most of the mineral content (Borchers et al. 1999). In a study of the nonvolatile components of G. lucidum, it was found that the mushroom contains 1.8% ash, 26-28% carbohydrate, 3-5% crude fat, 59% crude fiber, and 7-8% crude protein (Mau, Lin, and Chen 2001).

In addition to these, mushrooms contain a wide variety of bioactive molecules, such as terpenoids, steroids, phenols, nucleotides and their derivatives, glycoproteins, and polysaccharides. Mushroom proteins contain all the essential amino acids and are especially rich in lysine and leucine. The low total fat content and high proportion of polyunsaturated fatty acids relative to the total fatty acids of mushrooms are considered significant contributors to the health value of mushrooms (Chang and Buswell 1996; Borchers et al. 1999; Sanodiya et al. 2009).

Polysaccharides, peptidoglycans, and triterpenes are three major physiologically active constituents in G. lucidum (Boh et al. 2007; Zhou et al. 2007). However, the amount and percentage of each component can be very diverse in natural and commercial products, as exemplified by the data shown in Table 9.1. When 11 randomly selected samples of commercial lingzhi products purchased in Hong Kong shops were evaluated for the two major active components, triterpenes and polysaccharides, it was found that the triterpene content ranged from undetectable to 7.8% and the polysaccharide content varied from 1.1-5.8% (Chang and Buswell 2008). Such variations can occur for several reasons, including differences in the species or strains of mushroom used and differences in production methods.

TABLE 9.1. Comparison of Triterpene and Polysaccharide Contents of 11 Commercial Lingzhi (G. lucidum) Products currently available on the Market.

TABLE 9.1

Comparison of Triterpene and Polysaccharide Contents of 11 Commercial Lingzhi (G. lucidum) Products currently available on the Market.

9.5.1. Polysaccharides and Peptidoglycans

Fungi are remarkable for the variety of high-molecular-weight polysaccharide structures that they produce, and bioactive polyglycans are found in all parts of the mushroom. Polysaccharides represent structurally diverse biological macromolecules with wide-ranging physiochemical properties (Zhou et al. 2007). Various polysaccharides have been extracted from the fruit body, spores, and mycelia of lingzhi; they are produced by fungal mycelia cultured in fermenters and can differ in their sugar and peptide compositions and molecular weight (e.g., ganoderans A, B, and C). G. lucidum polysaccharides (GL-PSs) are reported to exhibit a broad range of bioactivities, including anti-inflammatory, hypoglycemic, antiulcer, antitumorigenic, and immunostimulating effects (Miyazaki and Nishijima 1981; Hikino et al. 1985; Tomoda et al. 1986; Bao et al. 2001; Wachtel-Galor, Buswell et al. 2004). Polysaccharides are normally obtained from the mushroom by extraction with hot water followed by precipitation with ethanol or methanol, but they can also be extracted with water and alkali. Structural analyses of GL-PSs indicate that glucose is their major sugar component (Bao et al. 2001; Wang et al. 2002). However, GL-PSs are heteropolymers and can also contain xylose, mannose, galactose, and fucose in different conformations, including 1-3, 1-4, and 1-6-linked β and α-D (or L)-substitutions (Lee, Lee, and Lee 1999; Bao et al. 2002). Branching conformation and solubility characteristics are said to affect the antitumorigenic properties of these polysaccharides (Bao et al. 2001; Zhang, Zhang, and Chen 2001). The mushroom also consists of a matrix of the polysaccharide chitin, which is largely indigestible by the human body and is partly responsible for the physical hardness of the mushroom (Upton 2000). Numerous refined polysaccharide preparations extracted from G. lucidum are now marketed as over-the-counter treatment for chronic diseases, including cancer and liver disease (Gao et al. 2005).

Various bioactive peptidoglycans have also been isolated from G. lucidum, including G. lucidum proteoglycan (GLPG; with antiviral activity; Li, Liu and Zhao 2005), G. lucidum immunomodulating substance (GLIS; Ji et al. 2007), PGY (a water-soluble glycopeptide fractionated and purified from aqueous extracts of G. lucidum fruit bodies; Wu and Wang 2009), GL-PS peptide (GL-PP; Ho et al. 2007), and F3 (a fucose-containing glycoprotein fraction; Chien et al. 2004).

9.5.2. Triterpenes

Terpenes are a class of naturally occurring compounds whose carbon skeletons are composed of one or more isoprene C5 units. Examples of terpenes are menthol (monoterpene) and β-carotene (tetraterpene). Many are alkenes, although some contain other functional groups, and many are cyclic. These compounds are widely distributed throughout the plant world and are found in prokaryotes as well as eukaryotes. Terpenes have also been found to have anti-inflammatory, antitumorigenic, and hypolipidemic activity. Terpenes in Ginkgo biloba, rosemary (Rosemarinus officinalis), and ginseng (Panax ginseng) are reported to contribute to the health-promoting effects of these herbs (Mahato and Sen 1997; Mashour, Lin, and Frishman 1998; Haralampidis, Trojanowska, and Osbourn 2002).

Triterpenes are a subclass of terpenes and have a basic skeleton of C30. In general, triterpenoids have molecular weights ranging from 400 to 600 kDa and their chemical structure is complex and highly oxidized (Mahato and Sen 1997; Zhou et al. 2007). Many plant species synthesize triterpenes as part of their normal program of growth and development. Some plants contain large quantities of triterpenes in their latex and resins, and these are believed to contribute to disease resistance. Although hundreds of triterpenes have been isolated from various plants and terpenes as a class have been shown to have many potentially beneficial effects, there is only limited application of triterpenes as successful therapeutic agents to date. In general, very little is known about the enzymes and biochemical pathways involved in their biosynthesis.

In G. lucidum, the chemical structure of the triterpenes is based on lanostane, which is a metabolite of lanosterol, the biosynthesis of which is based on cyclization of squalene (Haralampidis, Trojanowska, and Osbourn 2002). Extraction of triterpenes is usually done by means of methanol, ethanol, acetone, chloroform, ether, or a mixture of these solvents. The extracts can be further purified by various separation methods, including normal and reverse-phase HPLC (Chen et al. 1999; Su et al. 2001). The first triterpenes isolated from G. lucidum are the ganoderic acids A and B, which were identified by Kubota et al. (1982). Since then, more than 100 triterpenes with known chemical compositions and molecular configurations have been reported to occur in G. lucidum. Among them, more than 50 were found to be new and unique to this fungus. The vast majority are ganoderic and lucidenic acids, but other triterpenes such as ganoderals, ganoderiols, and ganodermic acids have also been identified (Nishitoba et al. 1984; Sato et al. 1986; Budavari 1989; Gonzalez et al. 1999; Ma et al. 2002; Akihisa et al. 2007; Zhou et al. 2007; Jiang et al. 2008; Chen et al. 2010). Examples of triterpenes are shown in Figure 9.3.

FIGURE 9.3. Chemical structure of lanosterol and three of the many triterpenes isolated from Ganoderma lucidum.

FIGURE 9.3

Chemical structure of lanosterol and three of the many triterpenes isolated from Ganoderma lucidum. (From Kubota, T., Y. Asaka, I. Miura, and H. Mori. 1982. Helv Chim Acta 65:611-9; Nishitoba, T., H. Sato, T. Kasai, H. Kawagishi, and S. Sakamura. (more...)

G. lucidum is clearly rich in triterpenes, and it is this class of compounds that gives the herb its bitter taste and, it is believed, confers on it various health benefits, such as lipid-lowering and antioxidant effects. However, the triterpene content is different in different parts and growing stages of the mushroom. The profile of the different triterpenes in G. lucidum can be used to distinguish this medicinal fungus from other taxonomically related species, and can serve as supporting evidence for classification. The triterpene content can also be used as a measure of quality of different ganoderma samples (Chen et al. 1999; Su et al. 2001)

9.5.3. Other Components

Elemental analysis of log-cultivated fruit bodies of G. lucidum revealed phosphorus, silica, sulfur, potassium, calcium, and magnesium to be their main mineral components. Iron, sodium, zinc, copper, manganese, and strontium were also detected in lower amounts, as were the heavy metals lead, cadmium, and mercury (Chen et al. 1998). Freeze-dried fruit bodies of unidentified Ganoderma spp. collected from the wild were reported to have a mineral content of 10.2%, with potassium, calcium, and magnesium as the major components (Chiu et al. 2000). Significantly, no cadmium or mercury was detected in these samples. G. lucidum can also contain up to 72 μg/g dry weight of selenium (Se; Falandysz 2008) and can biotransform 20-30% of inorganic selenium present in the growth substrate into selenium-containing proteins (Du et al. 2008).

Some attention has been given to the germanium content of Ganoderma spp. Germanium was fifth highest in terms of concentration (489 μg/g) among the minerals detected in G. lucidum fruit bodies collected from the wild (Chiu et al. 2000). This mineral is also present in the order of parts per billion in many plant-based foods, including ginseng, aloe, and garlic (Mino et al. 1980). Although germanium is not an essential element, at low doses, it has been credited with immunopotentiating, antitumor, antioxidant, and antimutagenic activities (Kolesnikova, Tuzova, and Kozlov 1997). However, although the germanium content of G. lucidum has been used to promote G. lucidum-based products, there is no firm evidence linking this element with the specific health benefits associated with the mushroom.

G. lucidum contains some other compounds that may contribute to its reported medicinal effect, such as proteins and lectins. The protein content of dried G. lucidum was found to be around 7-8%, which is lower than that of many other mushrooms (Chang and Buswell 1996; Mau, Lin, and Chen 2001). Bioactive proteins are reported to contribute to the medicinal properties of G. lucidum, including LZ-8, an immunosuppressive protein purified from the mycelia (Van Der Hem et al. 1995); a peptide preparation (GLP) exhibiting hepatoprotective and antioxidant activities (Sun, He, and Xie 2004; Shi, Sun et al. 2008); and a 15-kDa antifungal protein, ganodermin, which is isolated from G. lucidum fruiting bodies (Wang and Ng. 2006).

The carbohydrate and crude fiber content of the dried mushroom was examined and found to be 26-28% and 59%, respectively (Mau, Lin, and Chen 2001). Lectins were also isolated from the fruit body and mycelium of the mushroom (Kawagishi et al. 1997), including a novel 114-kDa hexameric lectin, which was revealed to be a glycoprotein having 9.3% neutral sugar and showing hemagglutinating activity on pronase-treated human erythrocytes (Thakur et al. 2007). Lectins (from the Latin word legere, which means to pick up, choose) are nonenzymatic proteins or glycoproteins that bind carbohydrates. Many species of animals, plants, and microorganisms produce lectins, and they exhibit a wide range of functions. In animals, for example, lectins are involved in a variety of cellular processes and the functioning of the immune system (Wang, Ng, and Ooi 1998).

Other compounds that have been isolated from G. lucidum include enzymes such as metalloprotease, which delays clotting time; ergosterol (provitamin D2); nucleosides; and nucleotides (adenosine and guanosine; Wasser 2005; Paterson 2006). Kim and Nho (2004) also described the isolation and physicochemical properties of a highly specific and effective reversible inhibitor of α-glucosidase, SKG-3, from G. lucidum fruit bodies. Furthermore, G. lucidum spores were reported to contain a mixture of several long-chain fatty acids that may contribute to the antitumor activity of the mushroom (Fukuzawa et al. 2008).

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9.6. THERAPEUTIC APPLICATIONS

The combination of benefit without toxicity represents the desired end result in the development of effective therapeutic interventions. G. lucidum has been used for hundreds of years as a health promotion and treatment strategy; there are now many published studies that are based on animal and cellculture models and on in vitro assessment of the health effects of G. lucidum, and there are also some reports of human trials in the field. However, there is no cohesive body of research, and the objective evaluation of this traditional therapy in terms of human health remains to be clearly established. In Sections 9.6.1 through 9.6.6, studies on the properties of G. lucidum in relation to cancer (which has attracted the most interest), viral and bacterial infection, diabetes, and liver injury are discussed.

9.6.1. Cancer

G. lucidum is a popular supplement taken by healthy individual to boost the immune system and by cancer patients along with conventional therapies. In this section, the scientific studies of G. lucidum on its anticancer properties are summerized.

9.6.1.1. Introduction

Cancer is a worldwide leading cause of death, and despite comprehensive advances in the early diagnosis of the disease and chemotherapy, it remains a major clinical challenge (WHO 2008). As part of searching for new chemopreventive and chemotherapeutic agents, hundreds of plant species, including mushrooms, have been evaluated. This has resulted in the isolation of thousands of bioactive molecules that were shown to have antitumor activity from numerous mushroom species, including Ganoderma species (Wasser and Weis 1999; Borchers et al. 2008). In G. lucidum, a large number of chemical compounds can be extracted from the fruiting body, mycelia, or spores. Many polysaccharides and triterpenes, the two major groups of components in the mushroom, exhibit chemopreventive and/or tumoricidal effects, as proved by numerous studies from in vitro experiments and animal and human in vivo studies (Yuen and Gohel 2005; Zaidman et al. 2005). Tumorimplanted animal models have shown inhibitory effects on angiogenesis and metastasis. However, evidence from well-designed human trials is still scarce.

9.6.1.2. In Vitro Anticancer Activities

Tomasi et al. (2004) tested 58 basidiomycetes mushrooms, of which G. lucidum was shown to be the most effective in killing cancer cells. G. lucidum induced cell-cycle arrest and apoptosis in various human and rodent tumor cells, including murine lymphocytic leukemia L1210 and Lewis lung carcinoma (LLC; Min et al. 2000; Tomasi et al. 2004), mouse reticulocyte sarcoma L-II (Liu et al. 2002), murine sarcoma Meth-A (Min et al. 2000; Gao, Min et al. 2002) and S180 (Gao, Min et al. 2002; Liu et al. 2002), human leukemia HL-60 (Muller et al. 2006; Kim et al. 2007; Fukuzawa et al. 2008; Liu et al. 2009) and U937, K562, Blin-1, Nalm-6, RPMI8226 (Muller et al. 2006; Shang et al. 2009), human hepatoma PLC/PRF/5, KB (Lin et al. 2003), HepG2 (Liu et al. 2009; Weng et al. 2009), Hep3B (Chung et al. 2001), Huh-7 (Lin et al. 2003; Li, Chan et al. 2005), human liver tumor SMMC7721 (Tang et al. 2006), human breast cancer MDA-MB-123 (Jiang et al. 2008; Liu et al. 2009; Zhao et al. 2010), MCF-7 (Jiang, Slivova, and Sliva 2006; Liu et al. 2009; Shang et al. 2009), T-47D (Gao, Min et al. 2002) and MT-1 (Wu et al. 2006; Xie et al. 2009), human prostate cancer PC-3 (Jiang et al. 2004; Evans et al. 2009), human cervix uteri tumor Hela (Liu et al. 2002; Tang et al. 2006; Shang et al. 2009), human ovarian cancer SKOV4 (Shang et al. 2009), human colonic cancer HT-29 (Hong et al. 2004) and SW480 (Xie et al. 2006), human lung carcinoma PG (Cao and Lin 2006; Cao, Lin, and Wang 2007) and 95-D (Tang et al. 2006), human small-cell lung carcinoma NCI-H69 and multidrug-resistant strain VPA (Sadava et al. 2009), lowgrade bladder cancer MTC-11 (Lu et al. 2004), and human uroepithelial HUC-PC (Yuen, Gohel, and Au 2008) cells.

Through the regulation of expression of different signals, tumor cells were arrested by G. lucidum at different points of cell cycle, for example, breast at G0/G1 phase; lung at G1 phase; liver at G1/G2 phase; and bladder, prostate, and leukemia at G2 phase. A selenium-enriched extract of G. lucidum mycelia was shown to induce G1/S phase arrest in human erythroid chronic myeloid leukemia K562 cells (Shang et al. 2009). Another extract induced G0/G1 phase arrest in estrogen-dependent breast MCF-7 cells through the downregulation of estrogen-α receptor and serine/threonine-specific protein kinase Akt/nuclear factor κB (NF-κB) signaling (Jiang, Slivova, and Sliva 2006). In various human cancer cell lines, extracts of G. lucidum were shown to suppress the progression of the G1 phase in cell cycle, and apoptosis was confirmed by using terminal deoxynucleotidyl transferase dUTP nick and labeling (TUNEL) assay (Liu et al. 2009). Many of these activities were accompanied by apoptosis. Cao and Lin (2006) demonstrated that a fraction of GL-PP decreased the antiapoptotic protein Bcl-2 expression and increased the proapoptotic protein Bax expression in human umbilical cord vascular endothelial cells (HUVECs). A triterpene-rich extract from G. lucidum induced progressive apoptosis in the premalignant HUC-PC cell line by increasing the early apoptosis marker annexin-V within 3 hours. Half the cells stained positive for 7-amino-actinomycin D (indicating late apoptosis) after 8 hours. All cells were dead at 24 hours, and this was associated with the downregulation of telomerase (Yuen, Gohel, and Au 2008). Similar apoptotic activities were also demonstrated in other human cancer cells (Fukuzawa et al. 2008). An ethanol extract of G. lucidum decreased cyclooxygenase 2 (COX)-2 enzyme expression and increased nitric oxide synthesis in colon HT-29 cells (Hong et al. 2004). In lung 95-D tumor cells, the pure compound ganoderic acid T caused mitochondrial dysfunction, which resulted from the upregulation of proapoptotic p53 and Bax expression (Tang et al. 2006). Moreover, the use of a combination of G. lucidum and Duschesnea extracts upregulated cytochrome c and Bax translocation to trigger caspase-3 apoptosis in leukemia HL-60 cells (Kim et al. 2007). Activation of caspases-7 and -9 by G. lucidum has been demonstrated in breast MCF-7 and lung H69-SCLC cancer cells, respectively (Hu et al. 2002; Sadava et al. 2009). In hepatoma HepG2 cells, a lucidenic acid-rich G. lucidum extract was shown to suppress phosphorylation of ERK1/2 and Akt signaling, which downregulated their downstream NF-κB and proto-oncoproteins (c-Jun and c-Fos) activities, favoring apoptosis (Weng et al. 2009).

A tumor mass requires a continuous nutrient supply via new blood vessels formed by the process of angiogenesis. Invasive cancer cells spread to distant sites through blood and lymphoid vessels. Therefore, agents that inhibit angiogenesis inhibit tumor growth and spread. The potential antiangiogenic activities of G. lucidum have been demonstrated in ex vivo chick embryo chorioallantoic membrane (CAM) assay (Cao and Lin 2004; Song et al. 2004). Polysaccharide peptide and ethanol extract from G. lucidum has been proved to decrease microvessels around a microfiber filter disc containing an embryo with intact yolks. Using a prostate cancer cell line, two angiogenic factors, known as vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β1, were suppressed by G. lucidum through inhibition of the ras/extracellular signal-regulated kinase (Erk1/2) and Akt signaling pathways (Johnston 2005; Stanley et al. 2005). Similar effects were also observed in a human lung cancer cell lines under hypoxic conditions after exposure to a high dose of GL-PP (Cao and Lin 2006).

Cell adhesion, invasion, and migration are the key factors in determining the aggressiveness of cancer; hence, control of cell motility is effective in avoiding cancer metastasis. A polysaccharide extract of G. lucidum mycelia inhibited the formation of oncogenic ras-induced transformed foci in an R6 embryo fibroblast cell line (Hsiao et al. 2004). Spores and the fruiting body of G. lucidum were shown to inhibit the regulatory proteins phosphatidylinositol and NF-κB in highly invasive breast and prostate cancer cells (Sliva et al. 2002). Cell adhesion, invasion, and colony formation of breast cancer cells were significantly inhibited on exposure to G. lucidum extracts (Sliva 2004). In addition, Lu et al. (2004) demonstrated that water and ethanol extracts of G. lucidum modulated the F/G-actin ratio, which, in turn, reduced the formation of stress fiber and focal adhesion complexes of bladder cancer cells, suggesting the actin remodeling was associated with the inhibition of carcinogen-induced cell migration. Inhibition of mitogen-induced invasion of HepG2 cells was demonstrated in a study by using Matrigel-coated filter inserts assay (Weng et al. 2009).

9.6.1.3. Animal Studies

Rodent studies of possible antitumorigenic effects of G. lucidum can be traced back to the early 1980s. Ten days of intraperitoneal (i.p.) injections of a polysaccharide fraction (GL-1) from the fruit body was reported to inhibit (by 95-98%) the growth of transplanted sarcoma 180 tumor cells in mice (Miyazaki and Nishijima 1981). A complex of polysaccharides and protein from the mushroom was also found to show significant antitumor activity in a similar study conducted by Kim et al. (1980). An inhibition rate of 88% was reported, and there was complete regression of tumor in a third of the test animals. In a study conducted by Hyun, Choi, and Kim (1990), which used a similar protocol but used various extracted polysaccharides, inhibition rates of 52-81% were found. A hot water extract (2 mg/mouse) given i.p. for 3 days resulted in an average 74% inhibition of tumor growth in mice, with 3 out of 10 animals showing complete regression, and an oral administration (daily for 5 weeks) showed 45-63% inhibition (Ohno et al. 1998). Similar inhibitory effects were shown with implanted sarcoma 180 cells after polysaccharide was given orally to mice (Zhang and Lin 1999). A pure β-(1→3) glucan was tested in parallel with crude G. lucidum extracts, which resulted in 90% inhibition of tumor growth (Ohno et al. 1998). A dry powder preparation of the antlered form of G. lucidum (known as deerhorn lingzhi due to its appearance) was shown to inhibit tumor growth and elongate the life span in both allogeneic sarcoma-180-bearing ddY mice and synergenic MM-46 mammary tumor-bearing C3H/He mice (Nonaka et al. 2006).

G. lucidum is a major component of many traditional botanical formulations, such as TBS-101, which was demonstrated to inhibit tumor growth and invasion in PC-3-implanted mice (Evans et al. 2009). Yun (1999) reported that 9 weeks of oral administration of mycelial extract significantly inhibited lung adenoma formation in mice. Oral administration of triterpenoid fractions for 18 consecutive days inhibited Martigel-induced angiogenesis, which significantly reduced tumor weight and the number of tumor cell colonies that had metastasized to the liver in female C57BL/6J strain mice with intrasplenic implantation of Lewis lung cancer cells (Kimura, Taniguchi, and Baba 2002; Wang et al. 2007). In male ICR-nu/nu nude mice injected with hepatoma HepG2 cells, daily oral administration of lucidenic acid-rich extract for 68 days (800 mg/kg dosage) decreased both the number and size of tumors by up to 99%, and also the number of metastatic tumors occurring in liver and lung (Weng et al. 2009). An aqueous extract (administered i.p. at 10, 20, and 40 mg/mouse) of the fruit body significantly increased the life span of mice implanted with Lewis lung carcinoma cells. However, no dose-response effect was seen (Furusawa et al. 1992). An additive effect was seen when G. lucidum was given in combination with cytotoxic antineoplastic drugs, and there was a suggestion of a possible synergistic effect with cisplatin (Furusawa et al. 1992). In another study, G. lucidum was found to also prolong the life span of tumor-transplanted mice by inhibiting metastasis to the lung (Lee et al. 1995). When given 1 week prior to the administration of a carcinogenic agent, a hot water extract of the mycelium/growth medium complex decreased the development of aberrant crypt foci (ACF) and precancerous lesions in the colon (Lu et al. 2001; Lu et al. 2003). No toxicity or side effects were seen in the rats when the extract was administered for 3 months. When tested with mouse colon tumor-implanted chambers, a polysaccharide mixture containing isoflavone aglycons from cultured G. lucidum mycelia was found to inhibit angiogenesis in vivo (Miura et al. 2002).

The chemopreventive activities of the mushroom on prostate cancer were demonstrated by a triterpenoid-rich extract of G. lucidum that suppressed the ventral prostate growth induced by testosterone (Liu et al. 2007a). Ganoderol B was identified as the active principle that was able to bind to an androgen receptor and inhibit 5α-reductase, suppressing androgen-induced LNCaP cell growth and downregulating the prostate-specific antigen (Liu et al. 2007b).

9.6.1.4. Human Studies

In humans, whether the antitumor effect of lingzhi is a direct one or is mediated via effects on the immune system is a key question to address. G. lucidum is one of the eight components of an herbal mixture called "prostate cancer-hope" (known as PC-SEPS), which has been used as an alternative in the treatment of androgen-dependent and -independent prostate cancer (Gao and Zhou 2009). However, only a few clinical trials have used G. lucidum as a single agent on cancer patients (Gao, Zhou et al. 2002; Gao, Zhou et al. 2003; Gao, Sai et al. 2003). Two randomized, controlled trials have been conducted using a GL-PS-rich extract (a patented over-the-counter product, Ganopoly; Gao et al. 2003; Gao and Sai et al. 2003). Gao, Zhou et al. (2003) recruited 134 patients with advanced cancers of different sites and supplemented them with G. lucidum capsules at a dosage of 1800 mg/ day for 12 weeks. Cellular immunity in 80% of these patients was significantly enhanced in terms of elevated plasma interleukin (IL)-2, IL-6, and interferon γ (IFN-γ) levels and natural killer (NK) cell activity. In another study, the same protocol was followed with 68 lung cancer patients (Gao, Sai et al. 2003) in whom immune parameters including total T cells, NK cells, and CD4/CD8 ratio were significantly enhanced in the G. lucidum-treated group. In addition, quality of life in terms of Karnofsky score was improved in about 65% of these patients (Gao, Sai et al. 2003). Ganopoly was also demonstrated to enhance mitogenic activity and NK cells in patients with advanced cancer in a before-and-after comparison study (Gao, Min et al. 2002). These results provide some evidence that the antitumor effects of G. lucidum are mediated via effects on the immune system. However, it must be noted that all studies were conducted by the same research group and that other direct antitumor effects of G. lucidum have not yet been studied on humans in vivo.

9.6.2. Immunomodulation

Agents that enhance the functioning of the host immune system could be expected to enhance health in terms of improved resistance and, thus, removal of malignant or premalignant cells. Many G. lucidum products on the market are labeled or promoted as immunomodulating agents.

There is considerable evidence to support the immunostimulating activities of G. lucidum via induction of cytokines and enhancement of immunological effector (Wang et al. 1997; Zhu and Lin 2006). Different components from G. lucidum were proved to enhance the proliferation and maturation of T and B lymphocytes, splenic mononuclear cells, NK cells, and dendritic cells in culture in vitro and in animal studies in vivo (Bao et al. 2001; Cao and Lin 2002; Zhu, Chen, and Lin 2007; Ma et al. 2008). In normal BALB/c mice, a polysaccharide-rich extract of G. lucidum promoted the proliferation of splenocytes and enhanced the activities of macrophages and NK cells, which resulted in the increase of IL-6 and IFN-γ (Chang et al. 2009). Although a commercial G. lucidum extract did not stimulate proliferation of lymphocytes, it activated the gene expression of IL-1β, IL-6, IL-10, and tumor necrosis factor (TNF)-α (Mao et al. 1999). A polysaccharide fraction (F3) was shown to enhance both adaptive and innate immunities by triggering the production of cytokines IL-1, IL-6, IL-12, IFN-γ, TNF-α, and colony stimulating factors (CSFs) from mouse splenocytes (Chen et al. 2004). It was reported also that TNF-α and IL-6 production were stimulated in human and murine macrophages by G. lucidum mycelia (Kuo et al. 2006). This effect might be due to increased synthesis of nitric oxide (NO) induced by β-D-glucan (Ohno et al. 1998). These polysaccharides were also found to be highly suppressive to tumor cell proliferation in vivo while enhancing the host's immune response (Ooi and Liu 2000).

Wang et al. (1997) found that a polysaccharide-enriched fraction from G. lucidum activated cultured macrophages and T lymphocytes in vitro, which led to an increase of IL-1β, TNF-α, and IL-6 in the culture medium. In another study (Zhang and Lin 1999), incubation of macrophages and T lymphocytes with a polysaccharide resulted in an increase in TNF-α and INF-γ levels in the culture medium. This "conditioned" culture medium was found to inhibit cell growth and induce apoptosis in sarcoma 180 and HL-60 cells (Zhang and Lin 1999). Furthermore, serum-incorporated treatment with a polysaccharide peptide fraction from G. lucidum markedly inhibited the proliferation of human lung carcinoma (PG) cells, whereas the pure fraction by itself did not induce similar effects (Cao and Lin 2004). In addition to polysaccharides, a lanostane triterpenoid, ganoderic acid Me, inhibited tumor growth and metastasis of Lewis lung carcinoma in "T helper 1 responder" C57BL/6 mice by enhancing immune function in terms of IL-2 and IFN-γ expression and NK cell activity (Wang et al. 2007). Zhu and Lin (2006) used cytokine-induced killer (CIK) cells to investigate the interaction between GL-PSs and cytokines, which mediated cell proliferation and antitumor activity. The cytotoxicity of CIK cells was correlated well with the expression of perforin and granzyme B induced by IL-2 and anti-CD3. Results indicated that GL-PSs enhance IL-2 and TNF-α production as well as protein and messenger ribonucleic acid (mRNA) expression of granzyme B and perforin in CIK cells culture, and thus decrease the doses of IL-2 and anti-CD3 without affecting the killing effects on NK-resistant mouse P815 mastocytoma cells and NK-sensitive mouse YAC-1 lymphoma cells (Zhu and Lin 2006).

9.6.3. Lingzhi as an Antioxidant

Consumption of antioxidant-rich plants may help prevent cancer and other chronic diseases (Collins 2005; Benzie and Wachtel-Galor 2009). Antioxidants protect cellular components from oxidative damage, which is likely to decrease risk of mutations and carcinogenesis and also protect immune cells, allowing them to maintain immune surveillance and response. Various components of G. lucidum, in particular polysaccharides and triterpenoids, show antioxidant activity in vitro (Lee et al. 2001; Mau, Lin, and Chen 2002; Shi et al. 2002; Wachtel-Galor, Choi, and Benzie 2005; Yuen and Gohel 2008; Saltarelli et al. 2009; Wu and Wang 2009). As shown in Figure 9.4, antioxidants from lingzhi were found to be absorbed quickly after ingestion, resulting in an increase in the plasma total antioxidant activity of human subjects (Figure 9.4; Wachtel-Galor, Szeto et al. 2004).

FIGURE 9.4. Mean +SEM (standard errors of the mean) change in plasma total antioxidant power (as the ferric reducing ability of plasma [FRAP] value) at 90 minutes postingestion of placebo (vertical lines), 1.

FIGURE 9.4

Mean +SEM (standard errors of the mean) change in plasma total antioxidant power (as the ferric reducing ability of plasma [FRAP] value) at 90 minutes postingestion of placebo (vertical lines), 1.1 g of G. lucidum extract (horizontal lines), and 3.3 g (more...)

Ooi and Liu (2000) reported that protein-bound polysaccharide (PBP) and polysaccharide peptide were able to mimic the endogenous antioxidant superoxide dismutase (SOD) in cancer-bearing animals in vivo. These polysaccharides were also reported to protect the immune cells from oxidative damage (Ooi and Lui 2000). The protective effects of G. lucidum on DNA strand scission induced by a metal-catalyzed Fenton reaction, ultraviolet irradiation, and hydroxyl radical attack were shown in agarose gel electrophoresis in vitro (Lee et al. 2001). Hot water extracts of G. lucidum significantly protected Raji cells from hydrogen peroxide (H2O2)-induced DNA damage (Shi et al. 2002). Hot water extracts protected human lymphocyte DNA only at low (<.001% w/v) concentrations, and caused H2O2-mediated damage at higher concentrations (>.01% w/v) (Wachtel-Galor, Choi, and Benzie 2005). Two antioxidant-enriched extracts from G. lucidum acted oppositely in premalignant HUC-PC cells under carcinogenic attack (Yuen and Gohel 2008). The aqueous extract protected cellular DNA from oxidative damage, whereas the ethanolic extract damaged cellular DNA, with increased H2O2 production and significant cell-killing effects observed. The results suggested that different effects of G. lucidum could be exhibited by different extractable components in bladder chemoprevention. Methanol extracts of G. lucidum were reported to prevent kidney damage (induced by the anticancer drug cisplatin) through restoration of the renal antioxidant defense system (Sheena, Ajith, and Janardhanan 2003). In contrast, a fraction of ganoderma triterpenes (GTS) was found to enhance the intracellular reactive oxygen species (ROS)-producing effect of doxorubicin (DOX) in Hela cells, leading to more DNA damage and apoptosis, whereas such synergism was inhibited by a ROS scavenger (Yue et al. 2008). In an animal study (diabetic rats), nonenzymic and enzymic antioxidants levels increased and lipid peroxidation levels decreased with G. lucidum treatment (Jia et al. 2009). However, a direct link has not been established between the antioxidant properties of G. lucidum and its immunomodulatory and anticancer effects, and whether lingzhi acts as an antioxidant or pro-oxidant may depend on concentration and environment.

9.6.4. Viral and Bacterial Infections

The goal of research in the treatment of viral and bacterial infections is the discovery of agents that specifically inhibit viral and bacterial multiplication without affecting normal cells. The undesired side effects of antibiotics and antivirals and the appearance of resistant and mutant strains make the development of new agents an urgent requirement. This has led researchers to investigate the antibacterial and antiviral activity of medicinal plants and fungi (Wasser and Weis 1999; Zhong and Xiao 2009). Isolation of various water- and methanol-soluble, high-molecular-weight PBPs from G. lucidum showed inhibitory effects on herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and vesicular stomatitis virus (VSV) New Jersey strain in a tissue culture system. Using the plaque reduction method, a significant inhibitory effect was seen at doses that showed no cytotoxicity (Eo et al. 1999; Oh et al. 2000). In addition, there was a marked synergistic effect when PBP from G. lucidum was used in tissue culture in conjunction with antiherpetic agents, acyclovir or vidarabine, and with IFN-α (Kim et al. 2000; Oh et al. 2000). Similar results were shown in HSV-1 and HSV-2 with a GLPG isolated from the mycelia of G. lucidum (Liu et al. 2004; Li, Liu, and Zhao 2005). The cells were treated before, during, and after infection, and viral titer in the supernatant of cell culture 48 hours postinfection was determined. The antiviral effects of the GLPG were more remarkable before viral treatment than after treatment. Although the mechanism was not defined, the authors concluded that GLPG inhibits viral replication by interfering with early events of viral adsorption (Li, Liu, and Zhao 2005).

Some triterpenes from G. lucidum have also been reported to have an inhibitory effect against human immunodeficiency virus (HIV)-1 protease activity, with IC50 values ranging from 20 to more than 1000 μM; however, not all of the examined triterpenes showed anti-HIV activity (El-Mekkawy et al. 1998; Min et al. 1998). In another study, a ganoderic acid isolated from G. lucidum showed inhibitory effects on the replication of hepatitis B virus (HBV) in HepG2215 cells (HepG2- HBV-producing cell line) over 8 days. Production of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) were, respectively, 20% and 44% of controls without ganoderic acid treatment (Li and Wang 2006).

Some small studies in human patients have also reported beneficial effects of lingzhi intake. A dried hot water extract of G. lucidum taken orally (equivalent to 36 or 72 g of dried mushroom per day) was used as the sole treatment for postherpetic (varicella zoster virus) neuralgia in 4 elderly patients. This treatment was reported to dramatically decrease pain and promote the healing of lesions, without any toxicity even at very high doses (Hijikata and Yamada 1998). In another study, a mixture of G. lucidum with other herbs improved recovery time in patients with herpes genitalis (n = 15) and herpes labiallis (n = 13; Hijikata, Yamada, and Yasuhara 2007).

For evaluating the antibacterial effects of the mushroom, several in vitro and in vivo animal studies using G. lucidum were performed. Mice injected with G. lucidum extract (2 mg/mouse) 1 day prior to injection with Escherichia coli showed markedly improved survival rates (>80% compared to 33% in controls; Ohno et al. 1998). In an in vitro study that used the disk assay (Keypour et al. 2008), a chloroform extract of G. lucidum was investigated for its antibacterial effect on gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis) and gram-negative bacteria (E. coli, Pseudomonas aeruginosa). Results showed that the extract had growth-inhibitory effects on two of the gram-positive bacteria with a minimal inhibitory concentration (MIC) of 8 mg/mL for S. aureus and B. subtilis. In another in vitro study, the direct antimicrobial effect of a G. lucidum water extract was examined against 15 species of bacteria alone and in combination with 4 kinds of antibiotics (Yoon et al. 1994). G. lucidum was found to be more effective than antibiotics against E. coli, Micrococcus luteus, S. aureus, B. cereus, Proteus vulgaris, and Salmonella typhi, but less effective against other species tested. The antimicrobial combination of G. lucidum with four commonly used antibiotics (Yoon et al. 1994) resulted in an additive or synergistic effect in most, but not all, instances, with apparent antagonism against cefazolin and ampicillin effects on P. vulgaris.

To date, the antimicrobial components of the tested crude extracts have not been identified, although antimicrobial polysaccharides have been identified in other fungi and plant terpenes have been reported to have antimicrobial activity (Wasser and Weis 1999; Zhong and Xiao 2009). In addition, the bioavailability of putative antimicrobial components of G. lucidum has not been established. Nonetheless, G. lucidum offers a potentially effective therapy. There is also the implication that combination therapy may be more safe and cost effective, as lower amounts of cytotoxic antiviral and antibacterial drugs could be used with a concomitant decrease in the risk of side effects. However, this needs further investigation in terms of in vitro studies and well-designed clinical trials.

9.6.5. Diabetes Mellitus

Components of G. lucidum have been proved to have a hypoglycemic effect in animals. The administration of ganoderans A and B (dose of 100 mg/kg), two polysaccharides isolated from fruit-body water extracts, by i.p. injection to normal and alloxan-induced diabetic mice significantly decreased (by up to 50%) the plasma glucose concentrations, and the hypoglycemic effect was still evident after 24 hours (Hikino et al. 1985). Using a mouse model, ganoderan B was also reported to increase plasma insulin, decrease hepatic glycogen content, and modulate the activity of glucose-metabolizing enzymes in the liver (Hikino et al. 1989). The same group reported that a third polysaccharide (ganoderan C) isolated from G. lucidum also showed significant hypoglycemic effects in mice, and that ganoderan B increased plasma insulin levels in both normal and glucose-loaded mice (Hikino et al. 1989; Tomoda et al. 1986).

In a more recent study, oral administration of G. lucidum hot water extract (0.03 and 0.3 g/kg BW) for 4 weeks was found to lower the serum glucose levels in obese/diabetic (+db/+db) mice, with effects seen after the first week of treatment (Seto et al. 2009). However, the glucose levels were still higher in these animals than in the control animals, and insulin levels were not altered. The extract markedly reduced levels of phosphoenol-pyruvate carboxykinase (PEPCK), which are usually high in obese/diabetic mice. The suggested mechanism, according to the authors, is that of lowering the serum glucose levels through suppression of the hepatic PEPCK gene expression. In another study (Jia et al. 2009), a polysaccharides-rich extract showed beneficial effects in streptozotocin-induced diabetic rats. The diabetic rats were treated with G. lucidum for 30 days. Following the treatment, serum insulin levels increased (compared with the nontreated diabetic group) and glucose levels decreased in a dose-dependent way. Treatment with streptozotocin also elevated levels of lipid peroxidation markers (thiobarbituric acid reactive substances [TBARS]), lipid hydroperoxides, and conjugated dienes); decreased levels of nonenzymic antioxidants (vitamin C, reduced glutathione [GSH] vitamin E); and decreased activities of the antioxidant enzymes, SOD, catalase, and glutathione peroxidase (Gpx). Following treatment with GL-PSs, levels of nonenzymic and enzymic antioxidants increased and lipid peroxidation levels decreased. Therefore, in addition to its glycemic modulation, treatment with G. lucidum helped to decrease oxidative stress (Jia et al. 2009).

In one study reported in the literature, 71 adult patients with confirmed type 2 diabetes mellitus (DM) were supplemented with Ganopoly (polysaccharide fractions extracted from G. lucidum). The patients received either Ganopoly or placebo orally at 1800 mg, three times daily for 12 weeks. Glycosylated hemoglobin (HbA1C) and plasma glucose decreased significantly after 12 weeks, indicating a hypoglycemic effect of the extract (Gao, Lan et al. 2004). Overall, the data from different studies suggest that G. lucidum intake helps in modulating blood glucose levels. However, the studies were performed mostly in animals. More support from well-planned human clinical studies is needed with and without combination with conventional medicines.

9.6.6. Liver and Gastric Injury

Hot water and water-ether extracts of the fruit body of G. lucidum were found to have a potent hepatoprotective effect on liver injury induced by carbon tetrachloride (CCl4) given orally and intraperitoneally to rats (Lin et al. 1995; Kim et al. 1999). The measured markers for liver injury included aspartate and alanine transaminases (AST and ALT) and lactate dehydrogenase (LDH). One active compound of the extract was separated and identified as ganoderenic acid A. This was found to have a potent inhibitory effect on β-glucuronidase, and the authors suggest that this inhibitory effect may have mediated the hepatoprotection seen when this isolated compound was given (Kim et al. 1999). Protection was also reported in a study in which a hot water extract of G. lucidum was given orally to mice 30 minutes before administration of ethanol. The extract was found to have an inhibitory effect against the formation of malondialdehyde (MDA), a degradation product of lipid peroxides, in mouse liver and renal homogenate, with evidence of a dose response seen (Shieh et al. 2001). The MDA effect was also reported by Shi et al. (2008) when the extract was given orally to mice (at 60, 120, and 180 mg/kg/day) for 2 weeks prior to treatment with D-galactosamine, which induced hepatic injury. In addition, pretreatment with G. lucidum maintained normal values of AST, ALT, SOD, and GSH (Shi et al. 2008). Alcohol and CCl4 toxicity is associated with increased oxidative stress and free-radical-associated injury. Therefore, hepatoprotection may also be mediated by the radical-scavenging properties of G. lucidum. Lin et al. (1995) reported that hot water extracts of G. lucidum showed significant radical-scavenging activity against both superoxide and hydroxyl radicals.

Further, G. lucidum methanolic extract was reported to show hepatic protection. The extract was given orally to rats (500 mg/kg/day) for 30 days before hepatic damage was caused by benzo(a) pyrene (Lakshmi et al. 2006). The extract prevented the increase of serum AST, ALT, and alkaline phosphatase (ALP) activities that result from benzo(a)pyrene challenge, and enhanced the levels of GSH, SOD, GpX, CAT, and glutathione S-transferase (GST). Protection of liver injury induced by CCl4 was also observed in mice treated with ganoderic acid (from G. lucidum) at 10 mg and 30 mg/kg/day given by intravenous injection for 7 days (Li and Wang 2006). The medium in which G. lucidum was grown was also proved to have liver-protective effects in an animal study of CCl4- induced liver damage (Liu et al. 1998). Oral administration of the medium in which G. lucidum mycelia were grown (but not the mycelia alone) had marked beneficial effects, as assessed by lower serum AST and ALT activities at 96 hours postinjury. No decrease was seen in the actual damage caused, as transaminase activities at 24 hours were not different from levels in control animals, implying that the mycelium medium may have promoted recovery in some way. The release of a hepatoprotective component from G. lucidum mycelium was also reported by Song et al. (1998). In this study, an extracellular peptidoglycan (a polysaccharide/amino acid complex named WK-003) produced during mycelium fermentation was administered orally to rats for 4 days prior to CCl4 intoxication. The increase in serum ALT levels was significantly decreased (by 70%; P < .01) at 24 hours postinjury compared with untreated, intoxicated rats. The AST levels decreased by 27%; however, this was not statistically significant. These studies of a possible mycelial product with hepatoprotective activity being extruded into the culture medium are of interest because the mycelia of G. lucidum are much easier and less costly to cultivate than the fruit body.

Polysaccharides extracted from G. lucidum and given orally to rats for 28 days were found to ameliorate cirrhosis induced by biliary ligation (Park et al. 1997). In addition, collagen (measured by hydroxyproline) content in the rat liver was lowered and improved liver morphology was found in comparison with control animals. The treatment significantly decreased ligation-induced increases in serum biochemical markers of liver damage (AST, ALT, ALP, and total bilirubin). Similar results were noticed in a study conducted by Wu, Fang, and Lin (2010) in which a decrease in hepatic hydroxyproline content and an improved liver histology were found in mice. In this study, liver fibrosis was induced by the administration of thioacetamide (TAA) for 12 weeks, which was followed by 4 weeks of treatment with G. lucidum extract (0.5 and 1.0 g/kg/day, per oral administration). The RT-QPCR analysis showed the extract treatment decreased mRNA expression of collagen (α1), smooth muscle α actin, and the enzymes metalloproteinase-1 and metalloproteinase-13. In addition, the TAA-induced decrease in total collagenase activity was reversed by the extract treatment, indicating that G. lucidum protection against injury may be related to the enhancement of collagenase activity.

Apart from its effects on chemical-induced liver injury, the effects of lingzhi on gastric injury have also been investigated. Gastric ulcers were induced in rats by acetic acid (Gao, Tang et al. 2004), and treatment with GL-PS fractions of 0.5 and 1.0 g/kg for 14 days significantly accelerated the ulcer healing by 40% and 56%, respectively. Treatment with 1.0 g/kg extract significantly restored mucus and prostaglandin levels compared with the control group.

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9.7. CONCLUDING REMARKS

G. lucidum is a well-known Asian herbal remedy with a long and impressive range of applications. Global consumption of G. lucidum is high, and a large, increasing series of patented and commercially available products that incorporate G. lucidum as an active ingredient are available as food supplements. These include extracts and isolated constituents in various formulations, which are marketed all over the world in the form of capsules, creams, hair tonics, and syrups.

With its growing popularity, many studies on G. lucidum composition, cultivation, and reputed effects are being carried out, and there are data that support its positive health benefits, including anticancer effects; blood glucose regulation; antioxidant, antibacterial, and antiviral effects; and protection against liver and gastric injury. However, most studies have been performed on animals or in cell-culture models. Human experimental studies have often been small, and the results are not always supportive of the in vitro findings. Now, the great wealth of chemical data and anecdotal evidence on the effects of G. lucidum needs to be complemented by reliable experimental and clinical data from well-designed human trials in order to clearly establish if the reported health-related effects are valid and significant. Many challenges are encountered due to a range of factors from dosage to production quality. Strategies for enhancing quality control procedures to define and standardize G. lucidum preparations are needed to determine mechanisms of action and to help characterize the active component(s) of this putative medicinal mushroom.

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ACKNOWLEDGMENTS

The authors thank the Hong Kong Polytechnic University for funding this study.

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Fallbericht: Ling Zhi in elf Tagen erfolgreich gegen Magenkrebs!

Non-Hodgkin-Lymphome gehören zu den häufigsten Krebserkrankungen: allein in Deutschland erkranken pro Jahr etwa 12.500 Menschen. Besonders aggressiv ist das großzellige, diffuse B-Zell-Lymphom, das oft Magen- oder Darmschleimhäute betrifft. Ohne Therapie sterben die Betroffenen oft innerhalb weniger Monate. Aus diesem Grund berichtete eine internationale chirurgische Fachzeitschrift über einen ungewöhnlich positiven Verlauf, an dem Ling Zhi möglicherweise entscheidend mit beteiligt war.
Ein 47-jähriger Mann mit Oberbauchschmerzen wurde endoskopiert. Es zeigte sich ein großes gastrisches Geschwür, das sich nach einer Biopsie als großzelliges gastrisches B-Zellen-Lymphom herausstellte. Bei einer Magenresektion elf Tage später wurde trotz sorgfältiger Probeentnahmen kein Hinweis auf ein großzelliges gastrisches B-Zellen-Lymphom mehr gefunden. Stattdessen wurde festgestellt, dass die gesamte Magenwand von zytotoxischen kleinen T-Lymphozyten durchdrungen war. Die Autoren gehen davon aus, dass die zytotoxischen T-Zellen als Immun-Antwort zur Bekämpfung des B-Zell-Lymphoms gebildet wurden und zur kompletten Regression führten.
Nach Rücksprache teilte der Patient mit, dass er Megadosierungen von Ganoderma lucidum eingenommen hatte - der Grund für die erfolgreiche Immunreaktion?
Quelle: Cheuk W; Chan JK; Nuovo G; Chan MK; Fok M: Regression of gastric large B-Cell lymphoma accompanied by a florid lymphoma-like T-cell reaction: immunomodulatory effect of Ganoderma lucidum (Lingzhi)? Int J Surg Pathol 2007 Apr;15(2):180-6 (ISSN: 1066-8969) Department of Pathology, Queen Elizabeth Hospital, Wylie Road, Kowloon, Hong Kong

TERCÜMESİ:

Olmuş Vak'a Haberi: Ling-Zhi 11 Günde Mide Kanserini Çok İyi Düzeye Getirmiş!

            Non-hodgkin-lymphomları en sık rastlanan kanser hastalıklarındandır. Sadece Almanya'da her yıl yaklaşık 12.500 insan hastalanmaktadır. Bilhassa büyük hücreli difüz B-Cell-Lymphom tipi çok agresiftir ve en çok mide-barsak mukozalarında ortaya çıkmaktadır. Tedavi görmeyen hastalar birkaç ayda kaybedilirler. Bu yüzden önem taşıyan böyle bir konuda uluslar arası bir cerrahi dergisinde olağanüstü olumlu bir gelişmeden söz edilmiş ve sonucun Ling-Zhi (Reishi) etkinliğinden kaynaklandığına vurgu yapılmıştır. 47 yaşında bir adam, üst karın bölgesi ağrılarıyla hastaneye geldiğinde, endoskopi yapılmış ve kendisinde büyük gastrik bir tümör tespit edilince uygulanan biyopsi sonucunda tümörün büyük hücreli gastrik B-cell-lymphomu özelliği taşıdığı anlaşılmıştır. Midesi rezeke edildikten 11. Gün bütün ihtimama rağmen alınan hiçbir örnekte büyük hücreli gastrik B-cell-lymphoma varlığına dair bulgu elde edilememiştir. Bunun yerine bütün mide duvarının sitotoksik etkinliği olan küçük T-lymphositleriyle doldurulmuş olduğu belirlenmiştir. Araştırmacılar, sitotoksik T hücrelerinin B-cell-lymphomuna karşı güçlü bir bağışık cevap olarak ortaya çıktığını ve böylece bölgenin tamamen rejenere edildiğini düşünmüşlerdir. Hasta ile yapılan konuşmalarda hastanın yüksek dozda Ganoderma lucidum ekstraktı aldığı ortaya çıkmıştır. Hiçbir diğer hastada görülmeyen bu tablonun ortaya çıkmasında yani, bu denli üst düzeyde bir bağışık cevap reaksiyonunda sebep nedir? Tabii ki Ganoderma lucidum.Kaynak: Cheuk W; Chan JK; Nuovo G; Chan MK; Fok M: Regression of gastric large B-Cell lymphoma accompanied by a florid lymphoma-like T-cell reaction: immunomodulatory effect of Ganoderma lucidum (Lingzhi)? Int J Surg Pathol 2007 Apr;15(2):180-6 (ISSN: 1066-8969) Department of Pathology, Queen Elizabeth Hospital, Wylie Road, Kowloon, Hong Kong

 

Nutrition and Cancer

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Pharmacological Values of Medicinal Mushrooms for Prostate Cancer Therapy: The Case of Ganoderma Lucidum

Jamal Mahajna a , Nesly Dotan a b , Ben-Zion Zaidman a b , Roumyana D. Petrova c & Solomon P. Wasser d

a Cancer Drug Discovery Program, Migal-Galilee Technology Center , Kiryat Shmona, Israel b The Institute of Evolution and Department of Evolutionary and Environmental Biology, Faculty of Science and Science Education , University of Haifa , Israel

c Institute of Botany, Bulgarian Academy of Sciences , Sofia, Bulgaria

d Institute of Evolution and Department of Evolutionary and Environmental Biology, Faculty of Science and Science Education , University of Haifa , Israel Published online: 30 Dec 2008.

To cite this article: Jamal Mahajna , Nesly Dotan , Ben-Zion Zaidman , Roumyana D. Petrova & Solomon P. Wasser (2008) Pharmacological Values of Medicinal Mushrooms for Prostate Cancer Therapy: The Case of Ganoderma Lucidum , Nutrition and Cancer, 61:1, 16-26

To link to this article: http://dx.doi.org/10.1080/01635580802379323

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Nutrition and Cancer, 61(1), 16-26 Copyright © 2009, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635580802379323

Pharmacological Values of Medicinal Mushrooms for Prostate Cancer Therapy: The Case of Ganoderma Lucidum

Jamal Mahajna

Cancer Drug Discovery Program, Migal-Galilee Technology Center, Kiryat Shmona, Israel Nesly Dotan and Ben-Zion Zaidman

Cancer Drug Discovery Program, Migal-Galilee Technology Center, Kiryat Shmona, Israel and the Institute of Evolution and Department of Evolutionary and Environmental Biology, Faculty of Science and Science Education, University of Haifa, Israel

Roumyana D. Petrova

Institute of Botany, Bulgarian Academy of Sciences, Sofia, Bulgaria

Solomon P. Wasser

Institute of Evolution and Department of Evolutionary and Environmental Biology, Faculty of Science and Science Education, University of Haifa, Israel 

Prostate cancer (PCa) is the most common male malignancy in many Western countries. Primary PCa is hormone dependent and is manageable by hormonal therapy. However, it rapidly develops to hormone-refractory tumors due to the accumulation of muta­tions in the androgen receptor and/or the acquisition of alternative cellular pathways that support proliferation and inhibit apopto- sis of prostate cancer. To date, no effective therapy is available for clinically hormone-insensitive or hormone-refractory stages of prostate cancer.

Whereas prostate cancer is very common in Western coun­tries, its levels are very low in Asia, providing evidence for a potential link between diet, environmental factors, and can­cer incident. Natural products have been used as a source of new pharmaceuticals including anticancer drugs. The medici­nal properties of mushrooms have been well known in Eastern Asia for thousands of years. Of special interest is the impli­cation of several mushrooms in prostate cancer prevention in­cluding the popular mushroom Ganoderma lucidum (Ling Zhi or Reishi) that has been widely used for the general promotion of health and longevity in Asia. Dried powder of G. lucidum was popular as a cancer chemotherapy agent in ancient China. The pharmaceutical activities of G. lucidum substances targeting

Submitted 27 November 2007; accepted in final form 13 April 2008. Address correspondence to Jamal Mahajna, Cancer Drug Dis­covery Program, Migal, P.O. Box 831, Kiryat Shmona 11016, Israel. Phone: +972-4-6953537. Fax: +972-4-6944980. E-mail: jamalm@migal.org.il

signal transduction pathways or molecular targets implicated in prostate carcinogenesis are reviewed.

OVERVIEW

For thousands of years, natural products have played an im­portant role throughout the world in treating and preventing hu­man diseases. Natural product medicines have originated from various source materials including plants, fungi, microorgan­isms, and animals. Such natural bioactive substances possess an enormous structural and chemical diversity, unsurpassable by any synthetic library; they are evolutionally optimized as drug-like molecules and might be considered biologically val­idated. Moreover, these molecules can serve as templates for semisynthetic and fully synthetic modifications.

An analysis of the origin of drugs developed between 1981 and 2002 showed that natural products or natural product- derived drugs comprised 28% of all novel chemical entities (NCEs) launched onto the market (1). In addition, 24% of these NCEs were synthetic or natural mimic compounds based on a study of pharmacophores related to natural products. This com­bined percentage (52% of all NCEs) suggests that natural prod­ucts are important sources for new drugs and are also good lead compounds suitable for further modification during drug devel­opment (2). In the case of anticancer agents, natural products have made significant contributions as either direct treatments or templates for synthetic modification. In this category, there are some 140 anticancer agents available to Western countries and Japan; 62% of them are nonsynthetic agents.

It is estimated that there are approximately 1.5 million species of fungi in the world, of which approximately 82,000 are described (3). About 20,000 of the known species belong to macrofungi, of which about 5,000 are edible and over 2,000 safe (4). Fungi from the Basidiomycota division are of great in­terest due to the large number of biologically active compounds they contain (5-13). Fungal fruiting bodies, mycelium, or the culture broth in which the mycelium has been cultivated are all explored for biological activities. Consequently, approximately 650 species of higher Basidiomycetes have been found to pos­sess antitumor activities (7).

Prostate Cancer

Prostate cancer is the most common male malignancy in many Western countries and the third leading cause of can­cer deaths in men worldwide. The age-standardized incidence rate of prostate cancer is highest in the United States (137 per 100,000 in Black men), lower in European countries (28 and 31 per 100,000 in England and Denmark, respectively), and lowest in Asia (10 and 2.3 per 100,000 in Japan and China, respectively) (15).

The epithelium of the prostate gland is under the hormonal control of androgens, the production of which depends on the hypothalamic-pituitary-testicular axis. The Leydig cells of the testes produce 95% of total androgens, and the adrenal glands produce the remaining 5%. In the prostate, free testosterone diffuses directly into the epithelial or stromal cells where it is converted into the functionally active androgen, dihydrotestos- terone (DHT), by the action of 5-a reductase enzyme system located on the nuclear membrane. Dihydrotestosterone action is mediated by the androgen receptor (AR) (16), which functions to preserve the normal function and structure of the prostate. The androgen receptor is a structurally conserved member of the nu­clear receptor superfamily of ligand-activated transcription fac­tors. Androgen ablation therapy has been shown to produce the most beneficial responses in patients with hormone-responsive prostate cancer (19). Castration, either surgically or chemically, remains the standard treatment option for most patients. Com­bined with antiandrogens that interfere with androgen receptor function, these methods significantly prolong the survival of prostate cancer patients (20). Androgen deprivation therapy is initially effective in repressing primary prostate tumors. How­ever, progression to the clinically hormone-insensitive stage is usually inevitable. To date, no effective therapy is available for hormone-insensitive or hormone-refractory stages of prostate cancer (21).

Prostate cancer has a complex etiology; currently age, ethnic­ity, obesity, and family history are the most consistently reported risk factors associated with the disease. Other potential risk fac­tors, such as environmental (17) and dietary (18) factors, have also been suggested. Conversion of a normal cell into the malig­nant state is often linked to genetic alterations of the cell (22,23). The known tumor-suppressor genes, Retinoblastoma (Rb) and p53, were reported to play an important role in the progression of prostate cancer (24). Both genes appear to be early events in prostatic carcinogenesis (25,26). In addition, mutations or dele­tions of pTEN, a tumor suppressor gene, have been found in 30% of primary prostate cancers and 63% of malignant cases, rank­ing it as one of the most common determinants of prostate tumor progression (27,28,57). The abnormal expression of growth fac­tors and their receptors including the epidermal growth factor (EGF) (29), the transforming growth factor-^ (TGF-£) (30), the transforming growth factor-a (TGF-a) (31), HER-2/neu, and c-erbB-3 oncogenes (32) may also contribute to the growth and development of both local and metastatic prostate cancer. The progression of prostate tumors to a hormone-refractory state is frequently associated with the increased expression of the antiapoptotic gene Bcl-2 (33), and the mutation of pTEN (34).

Androgen Receptor in Prostate Cancer

Like all steroid hormone receptors, the androgen receptor consists of 4 distinct regions in the expressed receptor that have specific functions (35), including the DNA-binding do­main, which is responsible for the interaction with hormone- responsive elements of the target gene promoters (36), and the ligand binding region, which is located at the carboxyl termi­nus of the receptor and contains the activation function 2 (AF2) domain and regulates ligand-dependent receptor function. Fur­thermore, the ligand-binding domain is thought to be important in the function of coactivator proteins (37) and in regulating transcription (38). The androgen receptor remains cytoplasmic until ligand binding occurs, and the dissociation of heat-shock proteins (HSPs) is thought to allow conformational change in the androgen receptor and mediate translocation to the nucleus. In the absence of ligand, the androgen receptor in the cytoplasm is rapidly degraded. In the presence of ligand, the androgen re­ceptor dimerizes, translocates to the nucleus, and initiates gene transcription by binding to specific androgen responsive ele­ments (AREs) found in androgen-responsive promoters (39). During androgen-independent progression, prostate cancer re­lies on various cellular pathways, some involving the androgen receptor and others bypassing it. In the former type of path­ways, a mutated androgen receptor may be activated by various ligands. In addition, deregulated growth factors and cytokines can activate the androgen receptor, usually with the help of androgen-receptor coactivators. Most of these growth factors, including EGF, insulin-like growth factor (IGF) and fibroblast growth factor (FGF), are potent mitogens and are upregulated by androgens, although the exact mechanisms are unknown. In contrast, TGF-j activity is downregulated by androgens (49). In the pathways that bypass the androgen receptor, the loss of pTEN reverses the inhibition of the PI3K/Akt pathway, permit­ting activated Akt to phosphorylate Bad. This activation results in the release of Bcl-2, which eventually leads to cell survival. In addition, androgen-independent cells may overexpress Bcl- 2 (50). Recently, increasing evidence implicated the canoni­cal Wnt signaling pathway in modulating the androgen signal­ing at multiple levels (51,52). j-catenin protein, a particularly critical molecular component of canonical Wnt signaling, is now known to promote androgen signaling through its ability to bind to the androgen receptor protein in a ligand-dependent fash­ion and to enhance the ability of liganded androgen receptor to activate transcription of androgen-regulated genes (53). Further­more, other components of the Wnt signaling pathway also aug­ment androgen receptor function through diverse mechanisms (51-55).

Historical and Contemporary Uses of Medicinal Mushrooms

The medicinal use of fungi dates back thousands of years and is recorded in the histories of both traditional Western medicine (TWM) and traditional Chinese medicine (TCM) (72). Although fungi have often been marginalized in traditional Western medicine and have not been widely incorporated into Western diets, the use of fungi has been central to the cultures of most Asian countries since antiquity (73). In traditional Chi­nese medicine, fungi are used in their entirety, fresh or dried, to treat the patient (and the patient's ailments) as a whole (72). In addition, certain fungi have been used in both traditional Western medicine and in traditional Chinese medicine to target specific conditions. Such is the case with Fomitopsis officinalis (Vill.) Bond. et Singer, called Agaricum by Dioscorides in his collection De Materia Medica (~60 A.D.), where it is described as a powerful panacea, especially suited to treat intestinal ail­ments (73). Other medicinal fungi are described in the "Herbal Classic" of traditional Chinese medicine, a compilation of two ancient bodies of writing collected between 200-264 A.D., the Sien nung Pen ts'ao king (or Pen king) and Ming I pie lu (or Pie lu) (73). A number of ancient medicinal fungi discussed are still in use. These include Ganderma lucidum, Poria cocos (Schwein.) F.A. Wolf, Grifola umbellata (Pers.) Pilat, Calvatia lilacina (Berk et Mont.) Lloyd, and Tremella fuciformis Berk. Special attention is given to "chi" or "Ling chi (zhi)" varieties of G. lucidum, said to promote well-being and immortality (73). In Asia, this fungus has traditionally been used to treat age-related ailments such as coronary disease, hypertension, bronchial prob­lems, and cancer (74).

Fungal Secondary Metabolites

Secondary metabolite production in fungi is a complex pro­cess coupled with morphological development (75). In most cases, the function of secondary metabolites for producing fun­gus is unknown but is inferred from several studies using mu­tants or enzyme inhibitors. These substances have their origins as derivatives from many intermediates in primary metabolism, but most can be classified according to 5 main metabolic sources: 1) amino acid-derived pathways, 2) the shikimic acid pathway for the biosynthesis of aromatic amino acids, 3) the acetate- malonate pathway from acetyl coenzyme A, 4) the mevalonic acid pathway from acetyl coenzyme A that functions in primary metabolism for the synthesis of sterols, and 5) polysaccharides and peptidopolysaccharides. The polyketide and mevalonic acid pathways are most often involved, and they produce a greater variety of compounds than the other pathways (11). Some of these compounds have tremendous importance to humankind in that they display a broad range of useful antibacterial, antivi­ral, and pharmaceutical activities as well as less desirable toxic activities.

Two major groups of secondary metabolites are responsible for toxic activities (the division is rather arbitrary): mycotoxins and mushroom poisons (76). All mycotoxins are low-molecular- weight natural products (i.e., small molecules) produced as sec­ondary metabolites by filamentous microfungi, whereas mush­rooms and other macroscopic fungi produce mushroom poisons. Depending on the definition used, and recognizing that most fun­gal toxins occur in families of chemically related metabolites, some 300 to 400 compounds are now recognized as mycotoxins

  • (77) . The second group of toxic metabolites is mushroom poi­sons. About 300 species of mushrooms are poisonous to humans. These species produce a wide spectrum of poisons that have been divided into the following 7 main categories (in brackets, an ex­ample of producing fungi): amanitin (Amanita phalloides [Vaill. : Fr.] Link, Galerina autumnalis [Peck] A.H. Sm. et Singer), orellanine (Cortinarius orellanus Fr.), gyromitrin (Gyromitra esculenta [Pers.] Fr.), muscarine (Clitocybe dealbata [Sowerby] Gillet), ibotenic acid (Amanita cothurnata G.F. Atk.), psilo- cybin (Psilocybe baeocystis Singer et A.H. Sm., Panaeolus castaneifolius [Murrill] A.H. Sm., Conocybe cyanopus [G.F. Atk.] Kuihner) and coprine (Coprinus atramentarius [Bull.] Fr.)
  • (78) .

Among the fungal secondary metabolites are lectins, lac- tones, terpenoids, alkaloids, antibiotics, and metal chelating agents (6). Fungi also contain a number of enzymes such as lac- case, superoxide dismutase, glucose oxidase, and peroxidases. It has been shown that such an enzyme therapy can also play an important role in cancer treatment preventing oxidative stress and inhibiting cell growth (79).

Anticancer Activity of Fungal Substances

It has been demonstrated that fungal metabolites can be used as inhibitors of molecular targets in malignant cells in order to combat certain cancers. Fungal anticancer substances can be roughly divided into two groups of high- and low- molecular-weight molecules. The major difference between these two groups is their mechanism of action. Most of the high- molecular-weight compounds are polysaccharides or protein- bound polysaccharides (80). It appears that these compounds are capable of interacting nonspecifically with the immune sys­tem to upregulate or downregulate many aspects of the host re­sponse (81). The second group comprises low-molecular-weight secondary compounds that can penetrate the cell membrane and act on specific signal-transduction pathways (11). These include mainly sesquiterpenes (which are the predominant secondary metabolites of Basidiomycetes), triterpenes, steroids and sterols,

and a few polyketides (abundantly produced by Actinomycetes).

Anticancer Activity of Ganoderma Lucidum

Ganoderma is the most popular and intensely investigated genus among the medically active mushrooms. Plenty of its species are famous for their antiviral, antibacterial, antifun- gal, anticancer, and immunostimulating activities and have been used traditionally in the folk medicine of Eastern countries for centuries. These activities were due to the production of various metabolites such as proteins, terpenes, sterols, and so forth.

The genus Ganoderma belongs to the class of Hymeno- mycetes. Within the genus Ganoderma, over 250 taxonomic names have been reported worldwide (82) including G. adsper- sum, G. applanatum, G. australe, G. lucidum, and G. tsugae, to name a few. However, the majority of reports in the literature appear to refer to one species, G. lucidum (83).

Ganoderma lucidum (W. Curti: Fr.) P. Karst. (Ling Zhi or Reishi), an oriental medical mushroom, has been used widely in Asian countries for centuries to prevent or treat different diseases including cancer (Fig. 1). Dried powder of G. lucidum, which was recommended as a cancer chemotherapy agent, is currently used popularly worldwide in the form of dietary supplements.

G. lucidum extracts were reported to possess cytotoxic ac­tivity against various cancer cell lines including leukemia, lymphoma, multiple myeloma (84,112), human hepatoma PLC/PRF/5 and KB, human breast cancer MDA-MB-231 (85), human prostate cancer PC-3 (86), human breast cancer MCF- 7 (87), human cervix uteri tumor HeLa (88), and low-grade bladder cancer MTC-11 (89) cell lines. The cytotoxic effects of

G. lucidum as demonstrated by the studies of Jiang et al. (85,86) and Zhu et al. (88) were concentration dependent. This activity of G. lucidum can be attributed directly to specific compounds from experiments employing isolated and purified molecules. However, the molecular mechanism(s) responsible for the in­hibitory effects have not been fully elucidated.

Ganoderma Lucidum Inhibits Proliferation of Prostate Cancer Cells

Proliferation is the multiplication or reproduction of cells resulting in the rapid expansion of a cell population. Cell pro­liferation is controlled by cell cycle regulatory elements.

 

Hsieh and Wu (92) tested the ability of extracts from indi­vidual herbs containing the herbal mixture PC-SPES, of which Ganoderma lucidum is one of its components, using amounts estimated to be equivalent to that present in the herbal mixture to suppress LNCaP, an androgen-dependent prostate cancer cell line, growth and/or lower prostate-specific androgen (PSA) ex­pression, compared to cells treated with PC-SPES. Treatment of LNCaP cells with 5 microl/ml ethanol extracts of Ganoderma lucidum showed a 63.5% reduction in cell growth and exhibited a similar decrease in cell viability. Additional studies demon­strated the ability of Ganoderma lucidum extracts to also inhibit cell proliferation of AR-independent cancer cell lines such as PC-3 in a dose- and time-dependent manner (86). Growth in­hibition of PC-3 cells by Ganoderma lucidum was mediated by the downregulation of expression of cyclin B and Cdc2 and by the upregulation of p21 expression. The inhibition of cell growth was also demonstrated by cell cycle arrest at the G2/M
phase (86). Liu et al. (62) reported that the LNCaP growth in­hibitory activity of G. lucidum extract is mediated by Ganoderol B isolated from G. lucidum fruiting body extract. Liu et al. (62) reported that Ganoderol B inhibits 5-a reductase activity and thereby causes inhibition of the proliferation of the androgen- dependent LNCaP cell line.

Ganoderma Lucidum Induces Apoptosis in Prostate Cancer Cells

Apoptosis is the predominant mechanism by which can­cer cells die when subjected to chemotherapy or irradiation. However, cancer cells develop resistance to these therapies that may be due, at least in part, to the development of effective antiapoptotic mechanisms (103). Another mechanism allow­ing escape from apoptosis is the activation of survival signal transduction pathways, including Akt-dependent (104) and Akt- independent mechanisms (105). An Akt-independent example includes EGF-induced survival mechanisms (31). When LNCaP cells are treated with PI3K inhibitors and deprived of survival factors, they spontaneously undergo apoptosis. However, treat­ment with EGF or androgen can protect cells from apoptosis, although Akt activity remains inhibited. It was found that EGF can protect LNCaP cells from apoptosis induced via the mi- tochondrial pathway but not from apoptosis induced via the death-receptor pathway (106,107).

A large portion of prostate cancer cells contain deregulated Akt. For example, in LNCaP prostate cancer cell line, Akt is constitutively active as a result of a frame-shift mutation in the pTEN tumor suppressor gene, which encodes a phosphatase that inactivates the lipid products of PI3K. As a result, the lack of the pTEN protein in these cells resulted in a constitutively activated antiapoptotic NF-kB pathway (108). Thus, these cells are less sensitive to anticancer drugs whose mechanism of action is based on the induction of apoptosis (109).

Ganoderma lucidum induced apoptosis of PC-3 cells with a slight decrease in the expression of NF-kB-regulated Bcl-2 and Bcl-xl and the upregulation of the proapoptotic Bax pro­tein, resulting in the enhancement of the ratios of Bax/Bcl-2 and Bax/Bcl-xl (86). Bemis et al. (110) studied the bioactivity of a unique preparation of concentrated soybean isoflavones fermented with G. lucidum mycelia named genistein com­bined polysaccharide (GCP). During fermentation, a concen­trated mixture of aglycone isoflavones was produced due to the hydrolytic cleavage of the sugar moiety from the isoflavone via G. lucidum-derived j -glycosidase. The potential utility of GCP as a prostate cancer chemopreventative agent was ana­lyzed in vitro and in vivo. GCP was reported to significantly suppress LNCaP and PC-3 cell growth, which was associated with apoptosis in LNCaP cells but not in PC-3 cells. GCP in­duced p27 and p53 (LNCaP only) protein expression within 6 h and suppressed phosphorylated Akt in both cell lines. The 2% GCP-supplemented diet significantly slowed LNCaP tumor growth, increasing apoptosis and decreasing proliferation over 4 wk (110). Zaidman et al. (95,139) measured 3 parameters that indicate induction of apoptosis in LNCaP cells: PARP cleav­age, caspase-3 activity, and annexin V-FITC staining of exter­nalized PS membranes. G. lucidum extracts induce apoptosis in LNCaP cell line by triggering the caspase cascade through the extrinsic or death receptor mediated pathway that includes the "initiator" caspase, caspse-8 and the "effector" caspase, caspase-3.

The ability of G. lucidum extracts and compounds to induce apoptosis was also demonstrated in several cell lines such as hu­man leukemia, lymphoma, and multiple myeloma (112), human breast cancer MCF-7 (87), human prostate cancer PC-3 (86), human hepatoma HuH-7 (113), and human colonic carcinoma HT-29 (114) cell lines. One report showed that a mixture of extracts from G. lucidum and the herb Duchesnea chrysantha induces apoptosis in human leukemia HL-60 cells (115). How­ever, in some of these cell lines, it was found that G. lucidum extracts induce apoptosis through the intrinsic or mitochondrial pathway (87,113,115).

In a series of experiments, Sliva et al. (116,117) and Jiang et al. (85) showed that G. lucidum extracts inhibit Akt/NF-^B signaling in prostate cancer PC-3 and breast cancer MDA-MD- 231 cells, resulting in the inhibition of proliferation, apoptosis induction, and decreased motility.

Ganoderma Lucidum Induces Cell Cycle Arrest in Prostate Cancer Cells

Mammalian cell growth and proliferation are mediated via cell cycle progression (119). In each cell division cycle, chro­mosomes are replicated once (DNA synthesis or S-phase) and segregated to create two genetically identical daughter cells (mitosis or M-phase). These events are spaced by intervals of growth and reorganization (gap phases G1 and G2). Progression through the G1 phase of the cell division cycle is a rate-limiting step in mammalian cell proliferation and is governed by nu­merous mitogenic pathways until the restriction point is passed. CDK4 and CDK6 complexed with cyclin D1 are responsible for cell cycle progression through the G1 phase (120), and the CDK2/cyclin E complex functions in the progression of the cell from the late G1 to the early S-phase (121). These complexes lead to the phosphorylation of retinoblastoma gene product, a tumor suppressor gene active in controlling the G1 phase (122). Hyperphosphorylated pRb leads to its release from the E2F fam­ily of transcription factors and induces expression of a number of genes required for S-phase transition (123).

DNA content (as an indicator of cell proliferation) analysis showed that G. lucidum fungal extracts blocked LNCaP cell cy­cle at the transition from the G1 to the S phase (95,139). These results are in accordance with several recent reports showing G1 phase arrest in a variety of cancer cell types caused by the treatment of chemotherapeutic agents (85-88,124). Cell cycle arrest at the G1 phase was reported to be mediated through the downregulation of cyclin D1 (85,87). In addition, other reports have demonstrated a G2 phase arrest caused by G. lucidum fungal extracts that is mediated by the downregula- tion of cyclin B (86,97,118). These data imply the existence of two separate chemical moieties that regulate cyclin D1 and cyclin B and consequently cause cell cycle arrest at G1 and G2, respectively.

Defects in cell cycle are one of the most common features of cancer cells. In prostate cancer, 16-68% of the cases reveal a loss of p27kip1 protein or low-grade expression (125). The function of the p27kip1 protein is regulated mainly by posttrans- lational, ubiquitin-mediated, proteasomal proteolysis (126). The tumor suppressor gene p21 was also reported missing in sev­eral solid tumors. For example, in prostate cancer PC-3, DU 145, and LNCaP cell lines, p21 was expressed at low or un­detectable levels (127). On the other hand, overexpression of cyclin D1 has been documented in a number of human cancers. However, evidence suggests that this event in prostate cancer is quite rare (128). A common polymorphism in the cyclin D1 gene is associated with the production of an alternate transcript of cyclin D1 termed cyclin D1b. It was found that this vari­ant actually stimulates cell-cycle progression in AR-dependent LNCaP cells but had no effect on AR-independent PC-3 cells (129).

Additional studies by Lu et al. (118) and Hu et al. (87) re­ported that the extract of G. lucidum inhibited cell proliferation in vitro and induced G1 cell cycle in prostate and breast can­cer cell lines including MCF7. Later, Zaidman et al. (95,139) has reported that G. lucidum downregulated cyclin D1 expres­sion leading to dephosphorylation of pRb and growth arrest of LNCaP prostate cancer cell line. Interestingly, cyclin D1 is controlled by the Akt and NF-kB pathways (130), which are constitutively active in LNCaP cells and can be inhibited by G. lucidum.

Ganoderma Lucidum Inhibits Angiogenesis in Prostate Cancer

Angiogenesis is a physiological process involving the growth of new blood vessels from preexisting vessels. It is a normal process in growth and development as well as in wound healing. However, this is also a fundamental step in the transition of tumors from a dormant state to a malignant state. Tumors induce angiogenesis by secreting various growth factors such as VEGF and bFGF that induce capillary growth into the tumor and allow it to grow by supplying nutrients and oxygen and removing waste products. Moreover, the new vessels allow tumor cells to escape into the circulation and lodge in other organs (tumor metastases) (135, 136).

G. lucidum extract was reported to inhibit early events in angiogenesis, capillary morphogenesis of the human aortic en­dothelial cells (137). The anti-angiogenic effect of G. lucidum was mediated by the inhibition of constitutively active AP- 1 in prostate cancer cells, resulting in the downregulation of the secretion of VEGF and TGF-j 1 from PC-3 cells. Inhibi­tion of AP-1 activity was mediated by the inhibition of Erk 1/2 phosphorylation and Akt kinases activity in PC-3 cells

  • (137) .

Ganoderma Lucidum Interferes With Androgen Receptor Function

LNCaP cell lines are considered as a laboratory model for hormone-responsive prostate cancer. These cells are sensitive to androgen and contain high levels of mutated androgen recep­tor, a T877A mutation (ACT ^ GCT, Thr ^ Ala) in the LBD

  • (138) . This mutation renders LNCaP cells sensitive not only to androgen but also to antiandrogens, estrogens, and progestins. In contrast, the androgen insensitive cell lines DU 145 and PC-3 do not express the androgen receptor. DHT is an androgen re­ceptor ligand generated from testosterone by the activity of 5-a reductase. Thus, 5-a reductase inhibitors might have activity against prostate diseases including benign prostatic hyperplasia (BPH) and prostate cancer. Fujita et al. (140) examined the in­hibitory effects of methanol extracts of 19 medicinal mushrooms on 5a-reductase activity including Ganoderma lucidum. The ex­tract of Ganoderma lucidum showed the strongest 5-a reductase inhibitory activity, and it significantly inhibited testosterone- induced growth of the ventral prostate in castrated rats (140).

Zaidman et al. (95,139) have reported that the treatment of LNCaP cell lines with G. lucidum organic extract resulted in a concentration-dependent inhibition of androgen receptor transcriptional activity, a concentration-dependent decrease of androgen receptor-regulated PSA, abrogation of nuclear translocation, and DNA binding activity of the androgen receptor (95,139). Furthermore, an active fraction was isolated from G. lucidum that competes with Dihydrotestosterone for receptor binding and consequently interferes selectively with androgen receptor function but not with the glucocorticoid receptor (GR) (95,139). The authors suggested that inter­ference with androgen receptor function might be explained by the ability of G. lucidum to stimulate the assembly of a transcriptionally inactive androgen receptor on DNA as sug­gested by some other antiandrogen compounds (141) possibly through the inhibition of the interaction with coactivators and/or the enhancement of the interaction with corepressors (61).

Liu et al. (62) isolated Ganoderol B from G. lucidum fruiting body extract with 5-a reductase inhibitory activity, which sup­pressed the regrowth of the ventral prostate induced by testos­terone in rats. In addition, Ganoderol B reduced androgen recep­tor levels in treated animals. The downregulation of androgen receptor signaling by Ganoderol B provided an important mech­anism for its antiandrogenic activity. The important implication of this study was that Ganoderol B intervention strategy could be helpful in controlling the morbidity of prostate cancer. It was suggested that Ganoderol B might be useful in prostate cancer therapy through the inhibition of androgen synthesis and the function and level of its receptor (142).

Ganoderma Lucidum Interferes With Prostate Cancer Invasion: The PI3K/Akt/NF-KB Pathway Connection

To invade and metastasize, cancer cells must effectively de­grade extracellular matrix (ECM) components. Plasminogen ac­tivation has been implicated as one of the mechanisms of ECM degradation. Mammalian cells contain two types of plasminogen activators, the urokinase type (uPA) and the tissue type (tPA), of which uPA is primarily involved in ECM degradation and consequently in tumor invasion (69,99,100). The uPA system consists of the serine proteases uPA and plasmin, their serpin inhibitors PAI-1, PAI-2, and a2AP, as well as an uPA cell surface receptor, uPAR (63). uPA is synthesized and released by cells as an inactive, single-chain, proenzyme: pro-uPA. It binds to an uPAR on the cell surface (63,64), which has an accelerating and positioning function for both the uPA and plasminogen activa­tion (64,65). Pro-uPA is cleaved to an active, two-chain protease form by plasmin (66). uPA has restricted substrate specificity, the main function of which is cleaving plasminogen to plas­min (65), which degrades several ECM components and also activates many promatrix metalloproteases (65,67). The plas- minogen activation system is controlled specifically by their serpin inhibitors PAI-1, PAI-2, and a2AP, of which PAI-1 plays a more important role in cancer invasion, and a2AP is the main inhibitor of plasmin (68,100). However, during cancer invasion and metastasis, this degradation system goes out of control, al­lowing cancer cells to cross the normal tissue boundaries. Both the uPA secretion and the presence of receptor bound uPA at the cell surface characterize prostate cancer having an inva­sive phenotype (70), increased metastatic potential, and poor survival.

The expression of uPA is regulated by the transcription fac­tors NF-kB and AP-1. The NF-kB is a dimeric transcription factor belonging to the Rel/NF-KB family of transcription fac­tors (56-60). The major activator of NF-kB is known as the IkB kinase complex (IKK) (58). G. lucidum extract inhibited the constitutively active transcription factors, NF-kB and AP-1, which resulted in the inhibition of the expression of uPA and its receptor uPAR. G. lucidum also suppressed cell adhesion and cell migration of highly invasive prostate and breast can­cer cells, suggesting its potency in reducing tumor invasiveness (71). Furthermore, Jiang et al. (85) reported that Ganoderma lu­cidum downregulated the expression of NF-kB-regulated uPA and uPAR receptor (uPAR), as well as levels of the antiapoptotic genes, Bcl-2 and Bcl-xl, accompanied by increased expression of the proapoptotic Bax protein, resulting in the enhancement of the ratio of Bax/Bcl-2 and Bax/Bcl-xl. Other reports showed that suppressing NF-kB activity by G. lucidum spore and fruit body extracts is mediated by the inhibition of the prosurvival factor Akt in MDA-MB-231 cells (98,116,117).

Clinical Activity of Ganodermalucidum

Ganoderma lucidum shows a very promising effect on prostate cancer in different preclinical systems. An herbal mix­ture called PC-SPES containing G. lucidum was prepared and recommended as an alternative herbal therapy for prostate can­cer (92). Ethanol extracts of the PC-SPES component were ac­tive in inhibiting the growth of LNCaP cells. Treatment with 5 microl/ml of the individual herbal extract suppressed the growth of LNCaP cells in the following order: Dendranthema mori- folium (85.2% reduction), Panax pseudo-ginseng (80.9%), Gly- cyrrhiza uralensis (73%), Rabdosia rubescens (70.8%), Scutel­laria baicalensis (66.5%), Ganoderma lucidum (63.5%), Isatis indigotica (50.0%), and Serenoa repens (14.5%). Analysis of the efficacy of the individual herbs in controlling intracellu- lar/secreted PSA levels and the expression of the androgen receptor and PSA revealed that only Glycyrrhiza uralensis, Scutellaria baicalensis, and Serenoa repens lowered intracel- lular and secreted PSA, whereas the remaining herbs actually increased PSA expression. In addition, no uniform response in AR/PSA was observed in individual herb-treated cells, contrary to PC-SPES, which elicited a coordinated change in AR/PSA. Lack of concordance between changes in prostate cell growth and prostate specific gene expression makes it unlikely that the activity of a single herb can account for the overall effects of PC-SPES (92). The PC-SPES was also tested in prostate cancer patients. de la Taille et al. (93) reported two cases of hormone-refractory prostate cancer patients who showed a fa­vorable response to therapy with the PC-SPES herbal combi­nation, controlling the progression of the disease. The report included two cases of biopsy proven prostate cancer patients with metastatic disease treated with total androgen blockade, progressing to an androgen-independent status. PC-SPES ex­tract decreased the PSA value for both patients from initial values of 100 and 386 ng/ml to 24 and 114 ng/ml after 1 yr and 4 mo, respectively. No gynecomastia or hot flashes were observed in these patients, and the treatment was well tolerated. PC-SPES showed strong estrogenic in vitro and in vivo activ­ity as an alternative tool in the management of prostate cancer patients. These cases suggest that PC-SPES might have some potential activity in hormone-independent prostate cancers. An­other independent study conducted by Small et al. (94) treated 33 patients having androgen-dependent prostate cancer (ADPCa) and 37 patients having androgen-independent (AIPCa) prostate cancer with PC-SPES at a dose of 9 capsules daily. Clinical outcome was assessed with serial serum PSA level measure­ment and imaging studies. As a result, 100% of ADPCa patients experienced a PSA decline of 80%. No patient developed PSA progression. Thirty-one patients (97%) had reductions in testos­terone to the anorchid range. Of the 35 AIPCa patients, 19 (54%) had a 50% PSA reduction, including 8 (50%) of the 16 patients who had received prior ketoconazole therapy. Median time to PSA progression was 16 wk (ranged from 2 to 69+ wk). It was concluded that PC-SPES shows good efficacy in the treatment of both ADPCa and AIPCa with a tolerated toxicity. At this stage, the exact contribution of the Ganoderma lucidum extract to the observed clinical effect is unclear; however, it is reason­able to speculate that a kind of synergy might exist among the different herbs used that resulted in the enhanced clinical effi­cacy of Ganoderma lucidum extract.

Ghafar et al. (111) reported the case of a patient with biopsy proven prostate cancer showing clinical and pathologic evidence of regression following administration of GCP, a concentrated soybean isoflavones fermented with G. lucidum mycelia, named genistein combined polysaccharide (GCP. The patient received GCP for 6 wk prior to radical prostatectomy. The patient's PSA decreased from an initial value of 19.7 to 4.2 ng/ml after 44 days of low-dose GCP. No cancer was identified in the radical prostatectomy specimen and no side-effects were observed in this patient. This case also suggested that GCP, which had shown potent inhibitory effects against prostate cancer cell lines in in vitro studies, may exhibit some potential activity in the treatment and prevention of prostate cancer.

CONCLUSIONS

Ganoderma lucidum is a popular medicinal mushroom that has been used as a home remedy in traditional Chinese medicine (TCM) for the prevention or treatment of a variety of diseases including cancer. Today, G. lucidum is recognized as a dietary supplement recommended in many countries as a cancer thera­peutic. In addition, G. lucidum is considered by some investiga­tors as a therapeutic biofactory that consists of diverse bioactive molecules mediating its biological functions. This raises the need to explore the full potential of this natural product and also to recognize its various active substances capable of combating a variety of diseases including cancer.

Preliminary clinical data based on a number of clinical tri­als that were conducted have shown promising efficacies of G. lucidum extracts or powders in cancer treatment as well as in other indications. Some of the clinical trails were not well de­signed and lacked appropriate controls. We believe that there is a need to explore the full potential of the dietary supplement of G. lucidum to assess its safety and efficacy in well-designed, double-blinded, randomized, placebo-controlled clinical trials using G. lucidum powders or extracts as stand-alone treatment or in combination with other treatments. To achieve this goal, standardization of G. lucidum is an important element. Because the composition and amount of biologically active substances depend on places of production, cultivation conditions, extrac­tion procedures, and the strains of G. lucidum, standardization will help its acceptance as a natural product suitable for cancer treatment.

Ganoderma lucidum extracts exhibited anticancer activity in in vitro systems against a variety of cancer cells including leukemia, lymphoma, breast, prostate, liver, lung, and myeloma cell lines. The anticancer activity of G. lucidum includes the inhibition of proliferation, induction of apoptosis, induction of cell cycle arrest, inhibition of invasive behavior, and suppression tumor angiogenesis in many experimental systems including prostate cancer. Studies have aimed at elucidating the mecha­nism of action revealed that G. lucidum inhibits the function an­drogen receptor and interferes with the PI3K/Akt/NF-KB path­way. The reported activity of G. lucidum is mostly reported using crude extractions. Thus, isolating active fractions and moieties responsible for the reported activity is an obstacle that must be overcome to allow the structural elucidation of active moieties and to define the exact mechanism of action of such substances. Moreover, G. lucidum extracts that exhibited diverse pharma- cologic functions were also shown to contain highly diverse pharmacological moieties (40-48,91). Currently, more than 100 different moieties have been reported from G. lucidum. Thus, the immediate goal must be to identify the different chemi­cal structures mediating the different pharmaceutical activities aimed at improving their potency, selectivity, bioavailability, as well as pharmacokinetics and pharmacodynamics parameters and explore their potential synergy with other pharmaceutical compounds available for combating different diseases including prostate cancer.

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Int J Endocrinol Metab. 2012;10(2):497-502. DOI: 10.5812/ijem.3644

  

An Update on Plant Derived Anti-Androgens

Paul Grant 1[1], Shamin Ramasamy 1

1 Department of Endocrinology, Kings College Hospital, Denmark Hill, London, UK

  

ABSTRACT

Anti-androgens are an assorted group of drugs and compounds that reduce the levels or activity of androgen hormones within the human body. Disease states in which this is relevant include polycystic ovarian syndrome, hirsutism, acne, benign prostatic hyper­plasia, and endocrine related cancers such as carcinoma of the prostate. We provide an overview and discussion of the use of anti-androgen medications in clini­cal practice and explore the increasing recognition of the benefits of plant-derived an- ti-androgens, for example, spearmint tea in the management of PCOS, for which some evidence about efficacy is beginning to emerge. Other agents covered include red reishi, which has been shown to reduce levels 5-alpha reductase, the enzyme that facilitates conversion of testosterone to dihydrotestosterone (DHT); licorice, which has phytoestro­gen effects and reduces testosterone levels; Chinese peony, which promotes the aroma- tization of testosterone into estrogen; green tea, which contains epigallocatechins and also inhibits 5-alpha reductase, thereby reducing the conversion of normal testosterone into the more potent DHT; black cohosh, which has been shown to kill both androgen- responsive and non-responsive human prostate cancer cells; chaste tree, which has a re­duces prolactin from the anterior pituitary; and saw palmetto extract, which is used as an anti-androgen although it shown no difference in comparison to placebo in clinical trials.

Copyright © 2012 Kowsar Corp. All rights reserved.

► Implication for health policy/practice/research/medical education:

There are several endocrine conditions that are a challenge to treat as our current medication regimes are not as effective as we would like them to be. Patients are often keen to try alternative or complementary therapies that may be viewed as more 'natural'. It is impor­tant to recognise that plant derived therapies represent another potential source of therapy - but they still need to be subjected to rigor­ous clinical evaluation.

► Please cite this paper as:

Grant P, Ramasamy S. An Update on Plant Derived Anti-Androgens. Int J Endocrinol Metab. 2012;10(2):497-502. DOI: 10.5812/ijem.3644

 

 

 

 

1. Introduction

An androgen antagonist (anti-androgen) can broadly be defined as any compound that has the biological effect of blocking or suppressing the action of male sex hormones such as testosterone within the human body. This may occur at any point in the hypothalamic- pituitary-gonadal-end-organ axis and could be through a direct effect on gonadotropin production at the level of the pituitary or by competing for binding sites at the re­ceptor level on the normally androgen sensitive tissues in the body. Androgens themselves have a diverse range of effects in both males and females and their dysregu- lation can give rise to a variety of clinical disorders, in­cluding polycystic ovarian syndrome, the most common endocrine disorder in females, which affects up to 7% of the population (1); hirsutism; acne vulgaris; prostatic hy- perplasia; and male pattern baldness.

There are already several medical treatments that act as androgen antagonists and have recognized uses; howev­er, in recent years, there has been an increasing demand for complementary and alternative therapies, and this has included an interest in the development and use of more plant-derived anti-androgen therapies. This is espe­cially relevant as some medications currently in use have
been found to have sub-optimal efficacy in clinical prac­tice, and many patients are keen to try 'natural' or 'alter­native' approaches as opposed to synthetically derived compounds. This review article provides an overview of the conditions, indications, and uses of anti-androgen medications, with a special focus on the renewed inter­est in the ancient area of plant-derived therapies.

•2.  Methodology

In order to obtain a maximum amount of high qual­ity evidence, we undertook a literature search using the PubMed/Medline and Athens databases using the linked keywords androgens, anti-androgens, phytoestrogens, PCOS, prostate cancer, benign prostatic hyperplasia, spearmint, black cohosh, Camellia sinensis, licorice, Chi­nese peony, chaste tree, and saw palmetto. We were inter­ested in both laboratory and clinical studies as well as in systematic reviews and meta-analyses.

•3.  Indications for the Use of Androgen An­tagonists

Anti-androgen medications are of use in a variety of androgen-driven medical and psychological conditions. The most common ones are listed below in Sidebar 1. Anti- androgen therapies in the male can lead to impaired de-

Sidebar 1. Conditions Pertinent for the Use of Anti-Androgens

Disease state

Anti-androgen use

Prostate cancer

Anti-androgens are useful as anti-neo- plastic agents and in palliative, adju­vant or neoadjuvant therapy

Benign prostatic hypertrophy

Prostate enlargement

Male sexual disorders

Hypersexuality, also known as excessive sexual desire or sexual deviation such as paraphilias

Acne vulgaris

To improve skin condition

Androgenic Alopecia

Male pattern baldness

Idiopathic Hirsutism

Excessive female hairiness

Polycystic ovarian syndrome

Regulation of menstrual cycle and re­duction of hirsutism

Gender reassignment therapy

In male to female transsexuals, anti-an- drogens are used to suppress the mas­culinizing effects of androgens

'Chemical castration'

Occasionally anti-androgens are used in registered sex offenders released from prisons to reduce the likelihood of re­peat offending by reducing sexual drive

 

velopment or reversal of secondary sexual characteristics, reduced libido, testicular atrophy, and erectile and sexual dysfunction. There may also be a corresponding change in other androgen dependent characteristics including mus­cle bulk and strength, fat mass, male pattern hair growth, skin appearance, energy levels, mood, concentration, and aggression. These are summarized in Sidebar 2 (2).

Sidebar 2. Androgen Deficiency in the Adult Male

System

Signs & Symptoms of androgen defi­ciency

Circulation/ Central nervous system

Hot flushes Sweats Insomnia Nervousness

Mood & cognition

Irritability & fatigue Reduced sense of well being Reduced motivation Impaired short term memory Depression and low self esteem

Masculinity

Reduced vigor and physical strength

Sexuality

Reduced libido Erectile failure Impaired orgasm

Impaired ejaculation and reduced ejac­ulation volume

Physical features

Decreased muscle mass Abdominal obesity Loss of body hair

Biochemistry

Decreased HDL, Increased LDL Increased total body fat Osteoporosis Reduced red cell volume

 

4. An Overview of Current Anti-Androgen Therapies

•4.1. Cyproterone Acetate

Cyproterone acetate is a synthetically derived steroid that acts as a potent anti-androgen. It also possesses pro­gestational properties and can be used to assist concep­tion in subfertile females.

•4.2.  Spironolactone

Spironolactone, a synthetic 17-spironolactone cortico­steroid, is commonly used as a competitive aldosterone antagonist and acts as a potassium sparing diuretic. It used to treat low-renin hypertension, hypokalemia, and Conn's syndrome. It has recognized anti-androgen ef­fects.

•4.3. Flutamide/Nilutamide/Bicalutamide

Flutamide/nilutamide/bicalutamide are all non-ste­roidal, pure anti-androgens. Bicalutamide is the newest agent and has the fewest side effects.

•4.4.  Ketoconazole

Ketoconazole is a derivative of imidazole that is used as a broad spectrum antifungal agent. Recognized effects are severe liver damage, but there is also an adrenolytic function. Ketoconazole reduces androgen production in the testes and the adrenal glands. It is a relatively weak anti-androgen, but is used with good effect in patients with Cushing's syndrome.

•4.5. Finasteride/Dutasteride

Finasteride/dutasteride are inhibitors of 5-alpha reduc- tase, an enzyme that prevents the conversion of testoster­one into the active form dihydrotestosterone (DHT). They are specific anti-androgens in that they only counteract the effects of testosterone and not other androgens.

•4.6. Plant-Derived Anti-Androgen Therapies

There is an ever-increasing demand for complemen­tary therapies, or those that are perceived as being more natural. The presence of anti-androgenic chemicals in plants, herbs, and foodstuffs provides an alternative to modern synthetic pharmaceuticals. It is also commonly believed that there are fewer adverse effects of such alter­native therapies.

•4.7. Reishi (Ganoderma lucidum)

Red reishi, commonly known as LingZhi in Chinese, is a mushroom thought to have many health benefits. In a re­search study exploring the anti-androgenic effects of 20 species of mushrooms, reishi mushrooms had the stron­gest action in inhibiting testosterone (3). That study found that reishi mushrooms significantly reduced lev­els of 5-alpha reductase, preventing conversion of tes­tosterone into the more potent DHT. High levels of DHT are a risk factor for conditions such as benign prostatatic hypertrophy (BPH), acne, and baldness.

•4.8. Licorice (Glycyrrhiza glabra)

Licorice is a flavorful substance that has been used in food and medicinal remedies for thousands of years. It is also known as "sweet root," licorice root contains a com­pound that is about 50 times sweeter than sugar. It has been used in both Eastern and Western medicine to treat a variety of illnesses ranging from the common cold to liver disease. Licorice affects the endocrine system be­cause it contains isoflavones (phytoestrogens), which are chemicals found in plants that may mimic the effects of estrogen and relieve menopausal symptoms and men­strual disorders. Licorice may also reduce testosterone levels, which can contribute to hirsutism in women.

A small clinical trial published in 2004 by Armanini and colleagues found that licorice root significantly de­creases testosterone levels in healthy female volunteers. Women taking daily licorice root experienced a drop in total testosterone levels after 1 month and testosterone levels returned to normal after discontinuation. It is un­clear as to whether licorice root affects free testosterone levels (4). The endocrine effect is thought to be due to phytoestrogens and other chemicals found in licorice root, including the steroid glycyrrhizin and glycyrrhetic acid, which also have a weak anti-androgen effect (5, 6).

•4.9.  White Peony (Paeonia lactiflora)

Chinese peony is a widely grown ornamental plant with several hundred selected cultivars. Many of the cultivars have double flowers with the stamens modified into addi­tional petals. White peony has been important in tradition­al Chinese medicine and has been shown to affect human androgen levels in vitro. In a 1991 study in the American Jour­nal of Chinese Medicine Takeuchi et al described the effects of paeoniflorin, a compound found in white peony that in­hibited the production of testosterone and promoted the activity of aromatase, which converts testosterone into es­trogen (7). To date, there have been no studies that translate or explore the clinical effects.

•4.10. Green Tea (Camellia sinensis)

In addition to supporting the cardiovascular system and somewhat reducing the risk of cancer and type 2 diabetes (8), green tea may also have an important anti- androgen effect because it contains epigallocatechins, which inhibit the 5-alpha-reductase conversion of nor­mal testosterone into DHT. As previously noted, this anti-androgen mechanism may help to reduce the risk of BPH, acne, and baldness. As yet, no randomized con­trolled trials of green tea for these androgen dependent conditions have been conducted.

•4.11.         Spearmint (Mentha spicata [Labiatae])

Spearmint, usually taken in the form of tea, has been thought for many years to have testosterone reducing properties. It is commonly used in Middle Eastern re­gions as an herbal remedy for hirsutism in females. Its anti-androgenic properties reduce the level of free testos­terone in the blood, while leaving total testosterone and DHEAS unaffected, as demonstrated in a study from Tur­key by Akdogan and colleagues, in which 21 females with hirsutism (12 with polycystic ovary syndrome and 9 with idiopathic hirsutism) drank a cup of herbal tea steeped with M. spicata twice daily for 5 days during the follicular phases of their menstrual cycles. After treatment with the spearmint tea, the patients had significant decreases in free testosterone with increases in luteinizing hor­mone, follicle-stimulating hormone, and estradiol (9). There were no significant decreases in total testosterone or DHEAS levels. This study was followed by a random­ized clinical trial by Grant (10), which showed that drink­ing spearmint tea twice daily for 30 days (vs. chamomile tea, which was used as a control) significantly reduced plasma levels of gonadotropins and androgens in pa­tients with hirsutism associated with polycystic ovarian syndrome. There was a significant change in patients' self-reported dermatology-related quality of life indices, but no objective change on the Ferriman-Gallwey scale. It is possible that sustained daily use of spearmint tea could result in further abatement of hirsutism.

•4.12.         Black Cohosh (Actaea racemosa)

Black cohosh (Actaea racemosa) is a plant of the butter­cup family. Extracts from these plants are thought to pos­sess analgesic, sedative, and anti-inflammatory properties. Black cohosh preparations (tinctures or tablets of dried materials) are used to treat symptoms associated with menopause, such as hot flashes, although the efficacy has been questioned (11). The inhibitory effects of black cohosh extracts (Cimicifuga syn. Actaea racemosa L.) on the prolifera­tion of human breast cancer cells has been reported recent­ly (12), and Hostsanka. et al (13) have examined the plant's effects on prostate cancer, another androgen hormone-de­pendent, epidemiologically important tumor. In that study, the inhibitory effect of an isopropanolic extract of black co­hosh (iCR) on cell growth in androgen-sensitive LNCaP and androgen-insensitive PC-3 and DU 145 prostate cancer cells was investigated.

The authors found that regardless of hormone sensitiv­ity, the growth of prostate cancer cells was significantly and dose-dependently down regulated by iCR. At a con­centration between 37.1 and 62.7 pg/ml, iCR caused 50% cell growth inhibition in all cell lines after 72h. Increases in the levels of the apoptosis-related M30 antigen of ap­proximately 1.8-, 5.9-, and 5.3-fold over untreated controls were observed in black cohosh-treated PC-3, DU 145, and LNCaP cells, respectively, with the induction of apoptosis being dose- and time-dependent.

Black cohosh extract was therefore shown to kill both androgen-responsive and non-responsive human pros­tate cancer cells by induction of apoptosis and activa­tion of caspases. This finding suggested that the cells' hormone responsive status was not a major determinant of the response to the iCR, and indicated that the extract may represent a novel therapeutic approach for the treatment of prostate cancer.

4.13. Chaste Tree (Vitex agnus-castus)

Chaste tree (or chasteberry) is a native of the Mediter­ranean region and is traditionally used to correct hor­mone imbalances. In ancient times, it was believed to be an anaphrodisiac, hence the name chaste tree. Clinical studies have demonstrated effectiveness of medications produced from extract of the plant in the management of premenstrual syndrome (PMS) and cyclical mastalgia (14). The mechanism of action is presumed to be via do- paminergic effects resulting in changes of prolactin se­cretion from the anterior pituitary. At low doses, it blocks the activation of D2 receptors in the brain by competitive binding, causing a slight increase in prolactin release. In higher concentrations, the binding activity is sufficient to reduce the release of prolactin (15).

Reduction in prolactin levels affects FSH and estrogen levels in females and testosterone levels in men. There is as yet no information regarding its efficacy in endocrine disease states such as PCOS, however, one small-scale study has demonstrated this prolactin reducing effect in a group of healthy males, and the implication is that it could be of use in mild hyperprolactinemia (16, 17). One could also theorize that it could be refined for use as a male contraceptive, because testosterone reduction should reduce libido and sperm production. This topic is further explored in a review by Grant & Anawalt (18).

4.14. Saw Palmetto (Serenoa repens)

Saw palmetto is a small palm tree native to eastern re­gions of the United States. Its extract is believed to be a highly effective anti-androgen as it contains phytoester- ols. This has been the subject of a great deal of research with regards to the treatment of BPH (19, 20), androgenic alopecia (21), and PCOS (22). However, controlled trials and other convincing research on its efficacy are still lacking. In the context of BPH, there have been 2 reason­ably sized clinical trials that found that saw palmetto ex­tract use showed no difference in comparison to placebo (23, 24). In meta-analyses, it has been shown to be safe and effective in mild to moderate BPH when compared to finasteride, tamsulosin, and placebo (25, 26). However, a more recent meta-analysis showed that it is only supe­rior specifically with regards to the symptom of nocturia (27). Therefore, evidence for its routine use is far from convincing and additional research is necessary to deter­mine its true effectiveness.

5. Discussion

There is clearly a need for a greater variety and more efficacious drugs to treat androgen related disorders such as those outlined above. The fact that there is an increasing emphasis over recent years on the potential for alternative anti-androgen compounds derived from plants is interesting and reflects the fact that from a clini­cal perspective the medications that are used in practice tend not to work very well for the majority of patients, and it can takes several medication changes and dose ad­justments to find a routine that works for an individual patient. The research that has been undertaken, which is summarized in Sidebar 3 , offers hope that alternative treatment options are available and may produce clini­cally effective therapies in the future, once suitably re­fined. Multiple challenges remain, however. Firstly the number of experimental and clinical studies remains relatively small and secondly they are often limited in terms of their quality (lack of adequate numbers to achieve statistical significance, lack of randomized con­trolled trials, and the findings that these compounds only appear to have marginal efficacy when assessed in head-to-head clinical trials). One extremely good review from the Cochrane group examined the use of Serenoa repens in 9 clinical trials. This is a popular herbal medi­cine for BPH and the review found that it was well toler­ated, but was no better than placebo in improving uri­nary symptom scores. Nor did Serenoa repens provide noticeable relief, generally considered to be a decrease of 3 points, in urinary symptoms (27). To date, there have been relatively few high quality long-term randomized studies evaluating standardized preparations of (poten­
tially) clinically relevant doses. Given the frequent use of Serenoa repens and the relatively low quality of existing evidence, a few more well designed, randomized, place­bo-controlled studies that are adequately powered, use validated symptom-scale scores, and have a placebo arm and a minimum follow-up of 1 year, are needed to con­firm, or deny, these findings. The same argument can be made for all of the plant-derived anti-androgens covered in this review. While there may be detectable and signifi­cant biochemical and in vitro changes in androgen relat­ed parameters, we are still not clear as to the benefits in endocrine practice.

6. Conclusions

The essence of endocrinology is to suppress overactiv­ity and stimulate or replace hypofunction in order to re­store normality to the body's hormonal axes. Androgen dysregulation is a feature of several common, and not so common, disease processes. In terms of male sexual dysfunction, the etiology, pathophysiology, and implica­tions are often complex (28). Targeting drug compounds to block the actions and effects of androgens can be chal­lenging and the standard treatments often have variable rates of success.

Alternative medicine is any healing practice that does not fall within the realm of "conventional" medicine. Reports of its efficacy are often anecdotal and based on historical or cultural traditions, rather than on scien­tific evidence. For the most part, the plant derived anti- androgen therapies discussed above used to fall into this category. However, there is now a small amount of data showing that androgen, prolactin, and gonadotropin levels can be biochemically modulated by the active com­pounds contained within these natural sources. Further work is clearly needed before the use of such compounds becomes part of routine practice. There is currently a paucity of high quality data derived from rigorously conducted trials. However, there are a few clinical and pre-clinical studies, although small in number, that have demonstrated that some natural anti-androgens address the underlying pathophysiology and can have effects on endocrine mediated disorders. What is now required are several larger, well-controlled, randomized studies aimed at proving their efficacy. While this group of treat­ments may be slow to find favor and may not be used first line, it does at least appear to be more acceptable to pa­tients because of its perceived more natural origins (29).

Acknowledgments

Many thanks once again to Dr. Derek Lington, as well as Abney and Teal.

Financial Disclosure

None declared.

Funding/Support

None declared.

References

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  • 2. Lee OD. Think androgen deficiency Am]Mens Health. 2011;5(5)377.
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Sidebar 3. Summary of Effects of Plant Derived Anti-Androgens

Plant Derived Anti-Androgen

Clinical/Biological Effects

Reference

Red Reishi (Ganoderma lucidum)

Reduction in 5-alpha-reducatase enzyme activity, reduction in DHT levels

(3)

Licorice (Glycyrrhiza glabra)

Reduction in total testosterone levels (effect on free testosterone levels not clear)

(4-6)

White Peony (Paeonia lactiflora)

Paeoniflorin inhibits the production of testosterone and promotes the ac­tivity of aromatase - the enzyme that converts testosterone into estrogen

(7)

Green Tea (Camellia Sinensis)

Contains chemicals epigallocatechins, which inhibit the enzyme 5-al- pha-reductase, and thereby reduce the conversion of normal testoster­one into the more potent DHT

(8)

Spearmint (Mentha spicata [Labiatae])

Decreases free testosterone, increases LH, FSH and estradiol. Reduction in patient reported measures of hirsutism

(9,10)

Black Cohosh (Actaea racemosa)

Black cohosh extract has been shown to inhibit the proliferation of human breast cancer cells and kill both androgen-responsive and un­responsive human prostate cancer cells by induction of apoptosis and activation of caspases.

(11-13)

Chaste Tree (Vitex agnus-castus)

Clinical studies have demonstrated effectiveness of medications pro­duced from extract of the plant in the management of premenstrual syndrome (PMS) and cyclical breast pain (mastalgia) as well reduction of mild hyperprolactinemia.

(14-17)

Saw Palmetto (Serenoa repens)

Shown to be of efficacy for the treatment of nocturia in the context of BPH only

(19, 20, 25-27)

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  • 6. Tamir S, Eizenberg M, Somjen D, Izrael S, Vaya J. Estrogen-like ac­tivity of glabrene and other constituents isolated from licorice root. J Steroid Biochem Mol Biol. 2001;78(3):291-8.
  • 7. Takeuchi T, Nishii O, Okamura T, Yaginuma T. Effect of paeoni- florin, glycyrrhizin and glycyrrhetic acid on ovarian androgen production. Am J Chin Med. 1991;19(1):73-8.
  • 8. Grant P, Dworakowska D. Tea and Diabetes: the laboratory and the real world. In: Preedy V, editor. Tea in Health & Disease Prevention. 1st ed. Elsevier Academic Press; 2012.
  • 9. Akdogan M, Tamer MN, Cure E, Cure MC, Koroglu BK, Delibas N. Effect of spearmint (Mentha spicata Labiatae) teas on androgen levels in women with hirsutism. Phytother Res. 2007;21(5):444-7.
  • 10. Grant P. Spearmint herbal tea has significant anti-androgen ef­fects in polycystic ovarian syndrome. A randomized controlled trial. Phytother Res. 2010;24(2):186-8.
  • 11. Newton KM, Reed SD, LaCroix AZ, Grothaus LC, Ehrlich K, Guil- tinan J. Treatment of vasomotor symptoms of menopause with black cohosh, multibotanicals, soy, hormone therapy, or place­bo: a randomized trial. Ann Intern Med. 2006;145(12):869-79.
  • 12. Fang ZZ, Nian Y, Li W, Wu JJ, Ge GB, Dong PP, et al. Cycloartane triterpenoids from Cimicifuga yunnanensis induce apoptosis of breast cancer cells (MCF7) via p53-dependent mitochondrial signaling pathway. Phytother Res. 2011;25(1):17-24.
  • 13. Hostanska K, Nisslein T, Freudenstein J, Reichling J, Saller R. Apoptosis of human prostate androgen-dependent and -inde­pendent carcinoma cells induced by an isopropanolic extract of black cohosh involves degradation of cytokeratin (CK) 18. Anti­cancer Res. 2005;25(1A):139-47.
  • 14. Daniele C, Thompson Coon J, Pittler MH, Ernst E. Vitex ag­nus castus: a systematic review of adverse events. Drug Saf. 2005;28(4):319-32.
  • 15. Webster DE, He Y, Chen SN, Pauli GF, Farnsworth NR, Wang ZJ. Opioidergic mechanisms underlying the actions of Vitex agnus- castus L. Biochem Pharmacol. 2011;81(1):170-7.
  • 16. Azadbakht M, Baheddini A, Shorideh S, Naser Zadeh A. Effect of vitex agnus-castus l. leaf and fruit flavonoidal extracts on serum prolactin concentration. J Med Plants. 2005;4(16):56-61.
  • 17. Merz PG, Gorkow C, Schrodter A, Rietbrock S, Sieder C, Loew D, et al. The effects of a special Agnus castus extract (BP1095E1) on prolactin secretion in healthy male subjects. Exp Clin Endocrinol Diabetes. 1996;104(6):447-53.
  • 18. Grant NN, Anawalt BD. Male hormonal contraception: an update on research progress. Treat Endocrinol. 2002;1(4):217-27.
  • 19. Boyle P, Robertson C, Lowe F, Roehrborn C. Updated meta- analysis of clinical trials of Serenoa repens extract in the treat­ment of symptomatic benign prostatic hyperplasia. BJU Int. 2004;93(6):751-6.
  • 20. Wilt T, Ishani A, Mac Donald R. Serenoa repens for benign pros­tatic hyperplasia. Cochrane Database Syst Rev. 2002;(3):CD001423.
  • 21. Murugusundram S. Serenoa Repens: Does It have Any Role in the Management of Androgenetic Alopecia? J Cutan Aesthet Surg. 2009;2(1):31-2.
  • 22. Liepa GU, Sengupta A, Karsies D. Polycystic ovary syndrome (PCOS) and other androgen excess-related conditions: can changes in dietary intake make a difference? Nutr Clin Pract. 2008;23(1):63-71.
  • 23. Bent S, Kane C, Shinohara K, Neuhaus J, Hudes ES, Goldberg H, et al. Saw palmetto for benign prostatic hyperplasia. N Engl J Med. 2006;354(6):557-66.
  • 24. Dedhia RC, McVary KT. Phytotherapy for lower urinary tract symptoms secondary to benign prostatic hyperplasia. J Urol. 2008;179(6):2119-25.
  • 25. Geavlete P, Multescu R, Geavlete B. Serenoa repens extract in the treatment of benign prostatic hyperplasia. Ther Adv Urol. 2011;3(4):193-8.
  • 26. Sosnowska J, Balslev H. American palm ethnomedicine: a meta­analysis. JEthnobiol Ethnomed. 2009;5:43.
  • 27. Tacklind J, MacDonald R, Rutks I, Wilt TJ. Serenoa repens for benign prostatic hyperplasia. Cochrane Database Syst Rev. 2009;(2):CD001423.
  • 28. Grant P, Ramasamy S. The Pituitary Gland & Erectile Dysfunc­tion. In: Grant P, editor. Erectile Dysfunction: Causes, Risk Factors & Management. Nova Publishers; 2012.
  • 29. Magin PJ, Adams J, Heading GS, Pond DC, Smith W. Complementary and alternative medicine therapies in acne, psoriasis, and atopic eczema: results of a qualitative study of patients' experiences and perceptions. J Altern Complement Med. 2006;12(5):451-7.

 


[1] Corresponding author: Paul Grant, Department of Endocrinology Kings College Hospital, Denmark Hill, London, UK. Tel: +44-23099999 , Fax:+020- 32993445, E-mail: drpaul.grant@doctors.org.uk DOI: 10.5812/ijem.3644

Copyright ©2012 Kowsar Corp. All rights reserved.

 

Suppression of growth and invasive behavior of human prostate cancer cells by ProstaCaidTM: Mechanism of activity

JIAHUA JIANG1, ISAAC ELIAZ2 and DANIEL SLIVA1,3,4

1Cancer Research Laboratory, Methodist Research Institute, Indiana University Health, 1800 N Capitol Ave, E504,

23 Indianapolis, IN 46202; Amitabha Medical Clinic and Healing Center, 7064 Corline Court, Sebastopol, CA; Department

of Medicine and 4Indiana University Simon Cancer Center, Indiana University School of Medicine, Indianapolis IN, USA

Received January 17, 2011; Accepted March 2, 2011 DOI: 10.3892/ijo.2011.996

  

Abstract. Since the use of dietary supplements as alternative treatments or adjuvant therapies in cancer treatment is growing, a scientific verification of their biological activity and the detailed mechanisms of their action are necessary for the acceptance of dietary supplements in conventional cancer treatments. In the present study we have evaluated the anti-cancer effects of dietary supplement ProstaCaidTM (PC) which contains mycelium from medicinal mushrooms (Ganoderma lucidum, Coriolus versi­color, Phellinus linteus), saw palmetto berry, pomegranate, pumpkin seed, green tea [40% epigallocatechin-3-gallate (EGCG)], Japanese knotweed (50% resveratrol), extracts of turmeric root (BCM-95®), grape skin, pygeum bark, sarsaparilla root, Scutellaria barbata, eleuthero root, Job's tears, astragalus root, skullcap, dandelion, coptis root, broccoli, and stinging nettle, with purified vitamin C, vitamin D3, selenium, quercetin, citrus bioflavonoid complex, p sitosterolzinc, lycopene, a lipoic acid, boron, berberine and 3.3'-diinodolymethane (DIM). We show that PC treatment resulted in the inhibition of cell proliferation of the highly invasive human hormone refractory (independent) PC-3 prostate cancer cells in a dose- and time- dependent manner with IC50 56.0, 45.6 and 39.0 ^g/ml for 24, 48 and 72 h, respectively. DNA-microarray analysis demon­strated that PC inhibits proliferation through the modulation of expression of CCND1, CDK4, CDKN1A, E2F1, MAPK6 and PCNA genes. In addition, PC also suppresses metastatic behavior of PC-3 by the inhibition of cell adhesion, cell migration and cell invasion, which was associated with the down-regulation of expression of CAV1, IGF2, NR2F1, and PLAU genes and suppressed secretion of the urokinase plasminogen activator (uPA) from PC-3 cells. In conclusion, the dietary supplement

Correspondence to: Dr Daniel Sliva, Cancer Research Laboratory, Methodist Research Institute, 1800 N Capitol Ave, E504, Indianapolis, IN 46202, USA E-mail: dsliva@iuhealth.org

Key words: ProstaCaidTM, plasminogen activator, prostate cancer

PC is a promising natural complex with the potency to inhibit invasive human prostate cancer.

Introduction

Prostate cancer is one of the leading causes of cancer-related death in American men due to its unpredictable hormonal independence and highly metastatic nature (1). Prostate cancers usually progress from androgen-dependent to androgen- independent phenotype with highly metastatic properties (2-4). Thus, the metastasis of prostate cancer remains the primary issue in improving prostate cancer patient survival. Moreover, hormone ablation therapy and chemotherapy for advanced stage prostate cancer seem not to offer more benefit in improving patient survival rate (5,6). Therefore, there is an urgent need for the identification of new therapies with anti-cancer effects in highly metastatic prostate cancers. Recent epidemiologic and experimental studies show that natural agents have potential chemopreventive and chemotherapeutic action for prostate cancer. Natural herbal and phytochemical agents are being recognized as an alternative therapy of prostate cancer patients (7,8).

ProstaCaid (PC) is a dietary supplement consisting of a 33 -ingredient comprehensive polyherbal and nutrient preparation which inhibits aberrant cell proliferation and induces apoptosis in androgen dependent and independent human and mouse prostate cancer cell lines (9). PC contains mycelium from medi­cinal mushrooms (Ganoderma lucidum, Coriolus versicolor and Phellinus linteus), which separately demonstrated anti­cancer properties (7,10-12). Ganoderma lucidum (G. lucidum) (Ling Zhi, Reishi) is a popular medicinal mushroom used as a traditional medicine in China, Korea and Japan for more than 2,000 years to prevent or treat different diseases, including cancer (13,14). The anti-cancer properties of G. lucidum have been attributed to the polysaccharides, which are responsible for the modulation of the immune system, or triterpenes, which demonstrate cytotoxic activity against a variety of cancer cells including breast, prostate, lung, colon, sarcoma, hepatoma and leukemia cells (13-15). G. lucidum has been shown to inhibit proliferation by cell cycle arrest at the G2/M phase and induced apoptosis in human prostate cancer cells by down-regulation of transcription factors NF-kB (16), resulting in modulating the expression of NF-kB-regulated Bcl-2 and Bcl-xl. G. lucidum has also demonstrated anti-invasive and anti-angiogenic properties which were mediated by the suppression of secretion of plasminogen activator (uPA), vascular endothelial growth factor (VEGF), and transforming growth factor-p1 (TGF-p1) from prostate cancer cells, respectively (17,18). Coriolus versicolor (C. versicolor) (Yunzhi) is a medicinal mushroom traditionally used in Asia to treat cancers as well as improve immunomodulatory activities (19). C. versicolor contains biologically active structurally different protein-bound poly- saccharide-K (PSK, Krestin) and polysaccharopeptide (PSP), which have been shown to inhibit the proliferation of various cancer cells including prostate cancer cells (12,20,21). In addition to the anti-cancer properties, extracts of C. versicolor demon­strated strong immunomodulatory effects such as elevated IL-2, natural killer cell activity and T-cell proliferation (11,22). In vivo studies showed that oral administration of C. versicolor extract or PSP to nude mice significantly suppressed the growth of inoculated prostate cancer cells (12,20,23,24). Although the detailed mechanisms of action of C. versicolor on the growth of cancer cells remains to be addressed, recent studies indicate that PSK and PSP of C. versicolor caused cell cycle arrest at the G0/G1 phase, induced apoptosis, and inhibited metastasis of prostate cancer cells (11,12,23,24). Phellinus linteus (P. linteus) was mainly used in Asian countries for the treatment of various human malignancies including prostate cancer (7,25). Although the major biologically active compo­nents in P. linteus are polysaccharides (25), P. linteus also contains a polysaccharide-protein complex (PPC) which stimulated the tumoricidal activities of macrophages and natural killer (NK) cells, and induced the proliferation of B cells in vitro (26). In addition, P. linteus inhibits growth and induces apoptosis of invasive prostate cancer cells in vitro (7,10,27,28), and sensitizes advanced prostate cancer cells to apoptosis in a xenograft model of prostate cancer (10).

In addition, some of the natural compounds in PC demon­strated a direct effect on prostate cancer cells. For example, resveratrol is a natural polyphenol present in various plants which have demonstrated anti-inflammatory, anti-oxidant, anti-invasive, and cardioprotective properties (29-32). Previous studies showed that resveratrol inhibited growth and increased apoptosis in prostate cancer cells (33,34) and a dimethyl ester derivative of resveratrol (Pterostibene) also inhibited MMP-9 and a methylacyl-CoA recemase of prostate cancer cells, two metastatic markers for the invasion and metastasis of prostate cancer cells (35). Vitamin D3 possesses anti-proliferative, anti-invasive, anti-migration, anti-metastasis, and anti- angiogenesis effect on prostate cancer cells (36,37), which are mediated through the arrest of cell cycle and the down-regulation of expression of caveolin and inhibition of MMP-9 activity (38,39). Epigallocatechin-3-gallate (EGCG), a major poly- phenolic component in the green tea, induced cell cycle arrest and apoptosis in androgen-dependent and -independent human prostate cancer cell lines (40-43). Moreover, EGCG inhibited MMP-2 and MMP-9 via suppression of activation of mitogen- activated protein kinase (MAPK) and also inhibited inflammation-triggered MMP-2 activation and invasion in a murine TRAMP model of prostate cancer (44,45). The molecular mechanisms responsible for the anti-invasive activity of EGCG were associated with down-regulation of activation of c-Jun and NF-kB signaling (43,45).

In the present study, we evaluated anti-proliferative and anti-invasive properties of a dietary supplement PC on highly invasive human hormone refractory (independent) prostate cancer cells PC-3. Here, we show that PC inhibits PC-3 prolifer­ation and modulates expression of prostate cancer-related biomarker genes. In addition, PC also suppresses invasive behavior of PC-3 cells by the inhibition of cell adhesion, migration and invasion. Our results demonstrate a novel mechanism of action of PC in the inhibition of growth and invasive behavior of prostate cancer cells.

Materials and methods

Cell culture and reagents. The human prostate cancer cell line PC-3 was obtained from ATCC (Manassas, VA, USA). PC-3 cells were maintained in DMEM/F-12 medium containing penicillin (50 U/ml), streptomycin (50 U/ml), and 10% fetal bovine serum (FBS). Medium and supplements came from Invitrogen (Grand Island, NY, USA). FBS was obtained from Hyclone (Logan, UT, USA). ProstaCaid (PC) a 33-ingredient comprehensive poly-herbal and nutrition preparation containing the following active weight components: Curcuma longa root extract complex with enhanced bioavailability (BCM-95®) 20%, quercetin 15%, Coriolus versicolor, Ganoderma lucidum, Phellinus linteus mushroom mycelium blend 10% [Astragalus membranaceus root extract (5:1), Coix lacryma-jobi seed extract (5:1), Coptis japonica rhizome extract (10:1), Eleutherococcus senticosus root extract (5:1), Scutellaria baicalensis root extract (5:1), Scutellaria barbata root extract (10:1), Similax glabrae extract (5:1), Taraxacum officinale herb (5:1)] herbal blend 9%, Urtica dioica herb extract (5:1) 6%, ß sitostererol 6%, Serenoa repens berry 5%, Brassica oleracea var. italic herb extract (22:1) 4%, Punica granatum fruit (40% Ellagic acid) 4%, Vitis vinifera fruit skin extract (10:1) 4%, Vitamin C 4%, a lipoic acid 3%, 3.3'-diinodlylmethane (DIM) 3%, Cucurbita pepo seed 2%, Prunus africana bark extract (4:1) 2%, Camellia sinensis herb extract (40% EGCG; 95% phenols; 70% catechens) 1.5%, lycopene 0.6%, Zinc 0.4%, Vitamin D3 0.2%, resveratrol 0.2%, berberine 0.1%, boron 0.06%, selenium 0.004%, was supplied by the EcoNugenics, Inc. (Santa Rosa, CA, USA). PC stock solution was prepared by dissolving PC in dimethyl-sulphoxide (DMSO) at a concentration of 25 mg/ ml and stored at 4°C.

Cell proliferation. Cell proliferation was determined by the tetrazolium salt method (MTT method), according to the manufacturer's instructions (Promega, Madison, WI, USA). Briefly, PC-3 cells were cultured in a 96-well plate and treated with PC (0-80 ^g/ml) for 24, 48 and 72 h. At the end of the incubation period, the cells were harvested and absorption was determined with an ELISA plate reader at 570 nm, as previously described (46). Data points represent mean ± SD in the representative experiment of triplicate determinations. Similar results were obtained in two independent experiments.

DNA microarrays. PC-3 cells were treated with PC (0-80 ^g/ml) for 24 h and total RNA isolated with RNAeasy (Qiagen, Valencia, CA). This RNA was used for the evaluation of prostate cancer genes with Oligo GEArray® Human Prostate Cancer Biomarkers Microarray according to the manufacturer's protocol (SABiosciences, Frederick, MD, USA), as previously described (47). The fold change of gene expression was determined by GEArray expression® analysis suite (SABiosciences).

Cell adhesion, migration and invasion assays. Cell adhesion was performed with Cytomatrix Adhesion Strips coated with human fibronectin (Chemicon International, Temecula, CA, USA). Briefly, PC-3 cells were treated with PC (0-80 ^g/ml) for 24 h, harvested, and counted. Cell adhesion was determined after 1.5 h of incubation at 37°C (46). Cell migration of PC-3 cells treated with PC (0-80 ^g/ml) was assessed in Transwell chambers in the Dulbecco's modified Eagle medium: nutrient mixture F-12 (DMEM/F12) medium containing 10% fetal bovine serum (FBS) (46). Invasion of PC-3 cells treated with PC (0-80 ^g/ml) was assessed in Transwell chambers coated with 100 ^l of MatrigelTM (BD Biosciences, Bedford, MA, USA) diluted 1:3 with DMEM/F12, after 24 h of incubation (46).

uPA secretion. DMEM/F12 media from PC-3 cells treated with PC (0-80 ^g/ml) for 24 h were collected and concentrated, and the secretion of uPA was detected by Western blot analysis with anti-uPA antibody (Oncogene Research Products, Cambridge, MA, USA), as described (46). Quantification of uPA secretion was performed by measuring optical densities of autoradiograms with HP-Scanjet 550c and analyzed by UN-SCAN-IT software (Silk Scientific, Orem, UT, USA).

Reverse transcription-polymerase chain reaction (RT-PCR). PC-3 cells were treated with different concentrations of PC (0-80 ^g/ml) for 24 h. The total RNA from PC-3 cells was isolated by RNeasy® mini kit (Qiagen, Valencia, CA, USA) according to instruction of manufacture. RT-PCR was performed as previously described (48). Briefly, PCR for CDK4, CNKN1A and E2F1 was run for 30 cycles at 95°C for denatu- ration for 45 sec, 60°C for annealing for 45 sec and 72°C for extension for 1 min. PCR for CAV1 was run for 38 cycles at 95°C for denaturation for 45 sec, 60°C for annealing for 1 min and 72°C for extension for 1 min. The primer sequences for CDK4 were 5'-TGGTGAGGGTGGGGTGAGG-3' (sense) and 5'-TGGCCACTGTGGGGATCACG-3' (antisense); the primer sequences for CDKN1A were 5'-CCTGCCCTCATG GCCCCTCT-3' (sense) and 5'-TGGGACCCTCACCCCCA CAG-3' (antisense); the primer sequences for E2F1 were 5'-GGC CGTCCTCCCAGCCTGTT-3' (sense) and 5'-CCCACGCGC ACACATGGACT-3' (antisense); the primer sequences for CAV1 were 5'-CGCCCTCTGCTGCCAGAACC-3' (sense) and 5'-GGCCCGTGGCTGGATGAAAA-3' (antisense); and the primer sequences for |-actin were 5'-ACGAGTCCGGCCC CTCCATC-3' (sense) and 5'-GGGGGCACGAAGGCTCA TCA-3' (antisense). The final RT-PCR products (10 ^l) were run on a 1.5% agarose gel containing ethidium bromide and quantified using imager Fluor Chem HD2 (Cell Biosciences, Santa Clara, CA, USA). The results are presented as the ratio of a specific target gene to |-actin.

Statistical analysis. Data are presented as the means ± SD. Statistical comparison between the control group (0 ^g/ml of PC) and groups with different PC doses were carried out using one-way analysis of variance (ANOVA). P<0.05 was considered to be significant.

 

Results and Discussion

Effects of PC on the growth of the highly invasive prostate cancer cells. Chemopreventive and therapeutic studies in Asia have demonstrated the beneficial effects of herbal supplements upon a variety of diseases including cancer (43). Although chemotherapy and hormone therapy demonstrated initial efficacy for metastatic prostate cancer patients, after the long- term anti-androgen treatment, prostate cancer patients lose their responsiveness to treatment and prostate cancers progress to androgen-independent phenotype with highly metastatic properties (2-4). Moreover, some of these chemotherapeutic drugs have undesirable toxic side effects (43,49). Therefore, there is a significant clinical application in the identification of natural complexes demonstrating anti-proliferative and anti- metastatic properties. As recently demonstrated PC suppresses proliferation of a variety of prostate cancer cells and this effect is associated with cycle arrest at G2/M phase and induction of apoptosis (9). However, the effect of PC on invasive behavior of prostate cancer cells was not previously addressed. First, we evaluated if PC inhibits growth of highly invasive androgen independent PC-3 prostate cancer cells. As seen in Fig. 1, the increased concentration of PC (0-80 ^g/ml) markedly suppressed proliferation of PC-3 cells in a dose- and time- dependent manner. The IC50 of PC for 24, 48 and 72 h treatment was 56.0, 45.6 and 39.0 ^g/ml, respectively. Thus, our results are consistent with the recent report by Yan and Katz (9). Although previous studies with mushroom extracts or isolated components of PC demonstrated anti-proliferative and pro- apoptotic effects in prostate cancer cells, the advantage in the use of complex PC is in the low dose of these isolated compo­nents which can be explained by their synergistic or additive effects. For example, resveratrol inhibited proliferation of prostate cancer cells PC-3 at 50 ^M corresponding to 11.4 ^g/ml (50), vitamin D3 at 100 nM corresponding to 38.5 ng/ml (51), and EGCG at 80 ^M corresponding to 36.7 ^g/ml (52), whereas the concentration of resveratrol, vitamin D3 and EGCG in PC corresponds to 1.6 ^g/mg PC, 2 ^g/mg PC, 5.8 ^g/mg PC, respectively. Therefore, the final concentration of resveratrol
corresponds to 128 ng/ml, vitamin D3 to 160 ng/ml, and EGCG to 464 ng/ml at the highest used dose of 80 ^g/ml of PC in our experiments.

 

Effect of PC on the invasive behavior of prostate cancer cells. Tumor invasion and metastasis are multifaceted processes including cell adhesion, proteolytic degradation of tissue barriers, cell migration, invasion, and angiogenesis (43,53). Invasive behavior of prostate cancer cells is associated with their ability to migrate and invade the surround tissues and is mediated through uPA/uPAR complex (43,53,54). To investi­gate if PC has an inhibitory effect on invasive behavior of
highly invasive prostate cancer cells, PC-3 cells were pretreated with PC (0-80 ^g/ml) for 24 h and their adhesion to fibronectin was determined on strips coated with human fibronectin as described in Materials and methods. As seen in Fig. 2A, adhesion of PC-3 cells to fibronectin was markedly suppressed by the PC treatment by 28.3 and 59.9% at 40 and 80 ^g/ml, respectively. The effect of PC on migratory potential of prostate cancer cells was evaluated in PC-3 cells pretreated with PC (0-80 ^g/ml) for 1 h and cell migration was determined after additional 24 h of incubation. As expected, PC significantly decreased the migration rate of PC-3 cells by 29.7 and 58.5% at 40 and 80 ^g/ml, respectively (Fig. 2B). Cell invasion is another key factor involved in cancer progression and meta­stasis (3,43,46). To examine the effect of PC on the invasive ability of PC-3 cells, cell invasion assays were performed in Transwell chambers coated with Matrigel as described in Materials and methods. As seen in Fig. 2C, PC markedly inhibited invasion of PC-3 cells in a dose-response manner by 28.3 and 48.7% at 40 and 80 ^g/ml, respectively. In order to evaluate the molecular mechanism of action of PC on the invasion of prostate cancer cells, conditioned media from PC-3 cells treated with PC (0-80 ^g/ml) were collected and secretion of uPA was determined by Western blot analysis. As expected, PC markedly decreased secretion of uPA from PC-3 cells (Fig. 2D). This observation is consistent with our previous report demonstrating the anti-invasive effect of
G. lucidum in human prostate cancer cells through the mechanisms including uPA/uPAR signaling (18,46). As in the inhibition of prolifer­ation by PC described above, the concentration of G. lucidum in PC was markedly lower (19.5 ^g/mg PC which corresponds to the final concentration of G. lucidum at 1.56 ^g/ml at the highest used dose of 80 ^g/ml of PC), than in the original experi­ments with individual G. lucidum extracts (0.5-2.5 mg/ml) (18).

 

Effect of PC on the gene expression profiles of prostate cancer- related biomarkers in prostate cancer cells. In order to evaluate
whether anti-proliferative and anti-invasive effects of PC are associated with the expression of genes previously identified in prostate cancer, we used cDNA microarray analysis with human prostate cancer biomarker genes. PC-3 cells were treated with PC (0-80 ^g/ml) for 24 h and cDNA microarray analysis performed as described in Materials and methods. As seen in Table I, PC up-regulated the expression of CDKN1A, and down-regulated expression of
CAV1, CCND1, CDK4, E2F1, ELAC2, IGF2, MAPK6, NR2F2 and PLAU genes in the PC-3 cells. Furthermore, we have confirmed the expression of some genes by RT-PCR. PC-3 cells were treated with PC (80 ^g/ml)

for 24 h. Total RNA was isolated and RT-PCR analysis was

performed. Consistent with the DNA microarray data, PC significantly induced the expression of the CDKN1A mRNA and down-regulated the expression of CDK4 mRNA and CAV1 mRNA (Fig. 3). Interestingly, expression of E2F1 mRNA was not changed by the PC treatment. Therefore, PC regulates the cell cycle progression network through binding to cell cycle regulators such as cyclin D1, Rb, and the transcription factor E2F1 (49,55-58). The cell progression is regulated by cyclins, cyclin dependent kinases (Cdks) and Cdk inhibitors such as p15, p16, 21, and p27; cyclin D1 (CCND1) and CDK4 form a complex to accelerate cell cycle progression, while Cdk inhibi­tors slow cell cycle progression (48,55,56,59,60). Therefore, the up-regulation of CDKN1A (p21) and down-regulation of CCND1 and CDK4 genes will cause cell cycle arrest at G1/G0 phase. Up-regulation of p21 induced strong downstream inhibition of CDK4 and cyclin D1 and hypophosphory- lation of Rb, further leading to the inhibition of transcription factor E2F1. Nevertheless, p21 can bind to proliferating cell nuclear antigen (product of PCNA gene) (61) and PCNA regulated the expression of ERK3/MAPK6 (product of MAPK6 gene) which affect cell viability and regulate the cell cycle (62). Thus, the induction of p21 also resulted in the inhibition of PNCA expression, leading to the reduction of ERK3 protein. In addition to the cell cycle regulatory genes, PC treatment also modulated expression of other genes previously identified in prostate cancer CAV-1, IGF2, ELAC2 and PLAU (Table I). For example, Caveolin-1 (product of CAV-1 gene) is a major structural component of caveolae, specialized in plasma membrane invaginations involved in endocytosis, cell adhesion, and signal transduction (63). Further, Caveolin-1 is over- expressed in advanced prostate cancer where it promotes migration, invasion, and angiogenesis in prostate cancer cells (63,64). The precise role of the insulin-like growth factor 2 (IGF2) on progression of tumor remains unclear. However, polymorphism of the IGF2 gene is associated with increased prostate cancer risk (65,66) and an activation of autocrine IGF2 loop is linked to the neoplastic progression (67). uPA (product of PLAU gene) and its receptor (uPAR) are important in cancer adhesion, migration, and invasion. uPA interacts with uPAR, which further form the multi-complex with integrin receptor a3pj or av|3 and regulate the invasive behavior (adhesion, migration and invasion) of cancer cells (18,43,68,69). Although suggested polymorphism of the elaC homolog-2/hereditary prostate cancer (ELAC2/HPC2) gene and prostate cancer risk demonstrated conflicting results in a variety of studies, a recent meta-analysis showed that ELAC2 is associated with prostate cancer risk (70,71).

In summary, our data clearly demonstrate that PC modulates expression of specific genes related to prostate cancer growth and invasiveness, and special ingredients in the PC may contribute to the inhibition of prostate cancer cells through distinct signaling pathways.

In conclusion, ProstaCaid is a novel dietary supplement that contains multiple ingredients which show an anti-proliferation effect on androgen-dependent and -independent prostate cancer cells. Our results show that PC inhibits proliferation and invasive behavior of prostate cancer cells by the modulation of the expression of genes associated with prostate cancer. Our data suggest that PC has multiple targets for its therapeutic effect and the biological activity of PC is mediated by the additive or synergistic effects of its individual ingredients. In summary, PC may have potential clinical application for an alternative prostate cancer therapy.

Acknowledgements

This study was supported by a research grant from EcoNugenics, Inc., Santa Rosa, CA. We would like to thank Barry Wilk for his contribution to this study. One of the authors, I. Eliaz, acknowledges his interest as the formulator and owner of EcoNugenics, Inc.

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Arthritis & Rheumatism (Arthritis Care & Research) Vol. 57, No. 7, October 15, 2007, pp 1143-1150 DOI 10.1002/art.22994

© 2007, American College of Rheumatology

ORIGINAL ARTICLE

Safety and Efficacy of Ganoderma lucidum (Lingzhi) and San Miao San Supplementation in Patients With Rheumatoid Arthritis: A Double­Blind, Randomized, Placebo-Controlled Pilot Trial

EDMUND K. LI,1 LAI-SHAN TAM,1 CHUN KWOK WONG,1 WAI CHING LI,1 CHRISTOPHER W. K. LAM,1 SISSI WACHTEL-GALOR,2 IRIS F. F. BENZIE,3 YI XI BAO,3 PING CHUNG LEUNG,4 and BRIAN TOMLINSON1

Objective. To examine the efficacy of popular Chinese herbs used in a traditional Chinese medicine (TCM) combination of Ganoderma lucidum and San Miao San (SMS), with purported diverse health benefits including antioxidant properties in rheumatoid arthritis (RA).

Methods. We randomly assigned 32 patients with active RA, despite disease-modifying antirheumatic drugs, to TCM and 33 to placebo in addition to their current medications for 24 weeks. The TCM group received G lucidum (4 gm) and SMS (2.4 gm) daily. The primary outcome was the number of patients achieving American College of Rheumatology (ACR) 20% response and secondary outcomes included changes in the ACR components, plasma levels, and ex vivo-induced cytokines and chemokines and oxidative stress markers.

Results. Eighty-nine percent completed the 24-week study. Fifteen percent in the TCM group compared with 9.1% in the placebo group achieved ACR20 (P > 0.05). Pain score and patient's global score improved significantly only in the TCM group. The percentage, absolute counts, and CD4+/CD8+/natural killer/B lymphocytes ratio were unchanged between groups. CD3, CD4, and CD8 lymphocyte counts and markers of inflammation including plasma interleukin-18 (IL-18), interferon-y (IFNy)-inducible protein 10, monocyte chemoattractant protein 1, monokine induced by IFNy, and RANTES were unchanged. However, in an ex vivo experiment, the percentage change of IL-18 was significantly lower in the TCM group. Thirteen patients reported 22 episodes (14 in placebo group and 8 in TCM group) of mild adverse effects. Conclusion. G lucidum and San Miao San may have analgesic effects for patients with active RA, and were generally safe and well tolerated. However, no significant antioxidant, antiinflammatory, or immunomodulating effects could be demonstrated.

KEY WORDS. Rheumatoid arthritis; Ganoderma lucidum; Cytokines.

 

 

 

INTRODUCTION

Ganoderma lucidum or lingzhi, which in Chinese means "herb of spiritual potency," is one form of the mushroom

ClinicalTrials.gov identifier: NCT00432484.

1Edmund K. Li, FRCP(C), Lai-Shan Tam, MD, Chun Kwok Wong, PhD, Wai Ching Li, RN, Christopher W. K. Lam, PhD, Brian Tomlinson, FRCP: Prince of Wales Hospital, The Chi­nese University of Hong Kong, Hong Kong; 2Sissi Wachtel- Galor, PhD: The Hong Kong Polytechnic University, Hong Kong; 3Iris F. F. Benzie, DPhil, Yi Xi Bao, PhD: Chongqing University of Medicine Science, Chongqing, China; 4Ping Chung Leung, DSc: The Institute of Chinese Medicine, Chi­nese University of Hong Kong, Hong Kong.

Address correspondence to Edmund K. Li, FRCP(C), Depart­ment of Medicine & Therapeutics, Prince of Wales Hospital, New Territories, Shatin, Hong Kong. E-mail: edmundli@cuhk. edu.hk.

Submitted for publication December 15, 2006; accepted in revised form April 18, 2007.

Ganoderma lucida, which has been used to treat all forms of ailments, and is the oldest mushroom known to have been used in ancient Chinese medicine. Lingzhi allegedly has multiple health benefits for a broad range of conditions from arthritis to cancers, and has therefore attained an unparalleled reputation in the East as the ultimate herbal substance. From the spores of G lucidum, 6 highly oxygen­ated lanostane-type triterpenes have been isolated called ganoderic acid, which is the active ingredient. G lucidum is widely cultivated nowadays and is sold as raw material or lingzhi extracts in many Asian markets and Western health shops.

In recent years, there has been an increasing number of reports of the biologic effects of G lucidum in the scientific literature. Many suggest that G lucidum has antioxidant properties as a free radical scavenger (1,2), indicating that some of the antioxidant components are well absorbed, resulting in a significant increase in the total antioxidant power in the plasma as assessed by the ferric-reducing/ antioxidant power (FRAP) test (3). Other studies suggest that G lucidum can improve immunologic functions (4,5), with potential bidirectional effects exhibiting both immu- noreactive and immunomodulatory activity. G lucidum was shown to inhibit histamine release from mast cells (6), to reduce production of antibodies and suppress cytokine production, and to up-regulate adhesion molecules (7,8), but it was also found to activate mitogen-activated protein kinase and reduce cytotoxicity, oxidative damage, and ap- optosis in some in vitro studies (7,9). Its growing popular­ity in patients with cancer may be supported by its sup- pressive effects of tumor growth in vitro (10-13).

Despite the large amount of scientific literature on G lucidum, we could find no published studies in the En­glish language literature of its effects on inflammatory arthritic diseases. Clinical studies in general are limited, despite the widespread use of this product. Recently, there has been increased interest in the role of an enzyme, se­cretory phospholipase A2 (sPLA2), as a mediator in the inflammatory pathway in various types of arthritis includ­ing rheumatoid arthritis (RA) (14). This enzyme can serve as a critical modulator of cytokine-driven inflammation. In ex vivo synovial cell cultures, sPLA2 enhances tumor ne­crosis factor a (TNFa) induction of prostaglandin produc­tion, in part via increased expression of cyclooxygenase 2 and cytosolic phospholipase A2. Mononuclear cells from peripheral blood and synovial fluid of patients with RA respond to sPLA2 with enhanced release of TNFa and interleukin-6 (IL-6) (15). Selective inhibition of sPLA2 has yielded variable results in an adjuvant-induced arthritis model in rats (16). Its combined antioxidant, putative an- tiinflammatory properties and our preliminary unpub­lished data using an in vitro test system showing inhibi­tion of the enzyme phospholipase A2 from bee venom and from hog pancreas suggest that G lucidum may have a role in the treatment of patients with RA.

In addition to Glucidum, San Miao San (SMS; translated as Powder of Three Wonderful Drugs) is another herbal remedy that is of relevance and interest in RA. In tradi­tional Chinese medicine (TCM), RA belongs to the cate­gory Bi Zheng, which is defined as a syndrome marked by arthralgia and dyskinesia of the joints and limbs due to attack of the meridians of the limbs by wind, dampness, and heat or cold pathogens. SMS is a traditional Chinese medicinal formula that has been used empirically to treat Bi Zheng for hundreds of years. It is composed of the herbs Rhizoma atractylodis (Cangzhu), Cotex phellodendri (Huangbai), and Radix achyranthes Bidentatae (Niuxi). In recent years, pharmacologic studies have confirmed anti- inflammatory and pain-relieving properties of SMS in an­imal models, along with some preliminary clinical obser­vations from the Chinese literature that reported that SMS had a therapeutic effect in the treatment of children with RA. Furthermore, use of the combination of SMS and G lucidum for the analgesic and antiinflammatory effects was a common practice according to the experts in TCM. They believed that this combination may have additive effects. The goal of this pilot study was to investigate the potential efficacy of a combined standard formulation of G lucidum and SMS as a therapeutic adjunct for patients with active RA.

PATIENTS AND METHODS

Patients.A total of 65 patients who fulfilled the Amer­ican College of Rheumatology (ACR; formerly the Ameri­can Rheumatism Association) 1987 revised criteria for the classification of RA (17) were recruited from the rheuma­tology outpatient clinic at the Prince of Wales Hospital, the teaching hospital of The Chinese University of Hong Kong. Patients taking disease-modifying antirheumatic drugs (DMARDs) including hydroxychloroquine, sulfasalazine, methotrexate, and leflunomide were eligible for inclusion if they were receiving a stable dose for at least 3 months before screening and remained on this regimen throughout the study. Patients receiving stable doses of one nonsteroi- dal antiinflammatory drug or prednisone in dosages up to 10 mg daily were also included. The dosage of methotrex- ate ranged between 12.5 and 17.5 mg/week with folic acid supplementation. All patients were instructed not to make any changes in their background therapies during the study. Intraarticular or pulse corticosteroids were not per­mitted during the study because they may inhibit phos- pholipase activities. Exclusion criteria were as follows:

In this 24-week randomized, double-blind, placebo-con­trolled trial, patients who met the inclusion and exclusion criteria were randomly assigned to receive either G luci- dum with SMS or placebo using a computer-generated list of random numbers in blocks of 5. The list was generated at the Institute of Chinese Medicine, The Chinese Univer­sity of Hong Kong. Study medications were dispensed as sealed packages in consecutive numbers. A research nurse was responsible for dispensing study medications. The investigators, research nurse, and participants were not aware of the treatment assignments throughout the study. Treatment codes were only broken after completion of the study.

Preparation of the extract. G lucidum and SMS were supplied as capsules, containing 4.0 gm of G lucidum extract, 2.4 gm of Rhizoma atractylodis (Cangzhu), 2.4 gm of Cotex phellodendri (Huangbai), and 2.4 gm of Radix achyranthes Bidentatae (Niuxi). Each patient took either 3 capsules twice daily as recommended by the TCM experts or identical-looking placebo. The compound used in this study was prepared in the Institute of Chinese Medicine at The Chinese University of Hong Kong.

The herbal formula was constructed by combining G lucidum 35.7% and SMS, which comprises 3 Chinese herbs, namely, Rhizoma atractylodis 21.4%, Cortex phel­lodendri Chinensis 21.4%, and Radix achyranthes Biden­tatae 21.4%. The herbal preparation was manufactured,
packaged, and labeled by a factory in Hong Kong based on good manufacturing practice standard. The crude herbs were supplied in one batch from reputable suppliers and were kept and stored in a cool and dry place. Before the water extraction procedure, the crude herbs of G lucidum, Rhizoma atractylodes, Cortex phellodendri Chinensis, and Radix achyranthes Bidentatae were cleaned, washed, and cut into fragments no bigger than 6 cm in length. Rhizoma atractylodis was ground into powder below 100 mesh for later use. During the water extraction process, distilled water was added to macerate the herbs for 1 hour, and then the mixture was boiled at 100°C for 1 hour for the first extraction. There were 3 extractions in total. The second extraction involved adding distilled water and boiling at 100°C for 1 hour and the third extraction involved boiling for half an hour. After extraction, the liquid extract was concentrated at -660 mm Hg and at 60°C and was spray dried to produce a powder extract. The dry extract powder was sieved and mixed with Rhizoma atractylodis powder. It was then encapsulated at 500 mg per capsule and pol­ished and packaged in a clean room at 10-20°C and 50% humidity. Placebo capsules, which were identical in ap­pearance, contained starch and a coloring agent.

Assessment of clinical response. All patients were eval­uated at baseline and weeks 4, 8,16, and 24. The following clinical and laboratory variables were assessed at each visit: number of tender joints and swollen joints, patient's global assessment (using a 0-10-cm visual analog scale [VAS]), physician's global assessment (using a 0-10-cm VAS), duration of morning stiffness, plasma C-reactive protein (CRP) level, concentration, and erythrocyte sedi­mentation rate (ESR).

Primary outcome. The primary outcome was the num­ber of patients who achieved the ACR 20% response, which is defined as having a 20% improvement in the 5 components including the number of tender and swollen joints and a 20% improvement in 3 of the 5 remaining core set measures: patient and physician global assessments; pain; disability as assessed by the Health Assessment Questionnaire (HAQ), which is an objective self-adminis­tered questionnaire that measures the functional disability of patients with RA (18); and an acute-phase reactant, ESR or CRP level (19).

Secondary outcomes. Secondary outcomes included changes in the ACR components including tender and swollen joint count, physician's and patient's global as­sessment, HAQ score, and ESR or CRP level. The other laboratory investigations included measurements of the total antioxidant power of plasma by the FRAP assay and plasma ascorbic acid concentration. Both were simulta­neously measured by the FRASC (20), a modified version of the FRAP assay (US patented) (21).

 

The MultiTEST IMK Kit with TruCOUNT tubes (Becton Dickinson, San Jose, CA) and the lyse/no-wash method were used for the assessment of the ratio and absolute counts ofCD4+ (T helper lymphocytes), CD8+ (T suppres­sor lymphocytes and cytotoxic T lymphocytes), natural killer (NK) cells, and B lymphocytes in EDTA whole blood samples using a 4-color FASCalibur flow cytometer (Bec- ton Dickinson). The concentration in EDTA level and the ex vivo levels of induced cytokines and chemokines were measured after treatment with lipopolysaccharide (25 ¡g/ ml) or phytohemagglutinin (5 ¡g/ml) for 24 hours for in­terferon-y (IFNy)-inducible protein 10 (IP-10), monocyte chemoattractant protein 1, monokine induced by IFNy,

RANTES, IL-8, IL-6, and IL-18 at baseline, and were mea­sured by cytometric bead array using flow cytometry or enzyme-linked immunosorbent assay at 8 and 24 weeks after treatment (22).

Assessment of adverse reactions. At each visit, patients were asked if there were any adverse effects. When an adverse event was claimed, the timing relative to the ad­ministration of the drugs was noted. Blood pressure, blood cell count, serum creatinine, and liver function test results were recorded before study entry and at each visit during the study.

Table 2. Changes in the American College of Rheumatology (ACR) core set variables and antioxidant levels after 6 months of

either Ganoderma lucidum plus SMS or placebo*

 

Ganoderma lucidum (n = 28)

Placebo (n

= 30)

Variables

Baseline

6 months

Baseline

6 months

ACR core set Tender joints, median (IQR) Swollen joints, median (IQR) Physician's global (VAS 0-10) Patient's global (VAS 0-10) Pain (VAS 0-10) ESR, median (IQR) mm/hour C-reactive protein, median (IQR) mg/liter HAQ Antioxidant levels FRAP value, ^moles/liter Serum ascorbic acid levels, ^moles/liter

2(1,5) 3 (2, 5) 4.3 ± 1.5 5.7 ± 2.5 4.9 ± 2.3 40 (22, 50) 9.2 (3.0, 22.2) 1.2 ± 0.8

942 ± 213 46.5 ± 16.0

2 (0, 4) 4 (2, 6)

  • 4.8 ± 2.2 4.7 ± 2.6+
  • 3.9 ± 2.5+ 33 (19, 56)

11.3 (3.0, 15.5) 1.3 ± 0.7

960 ± 188 48.1 ± 20.8

  • 2 (2, 3)
  • 3 (2, 6) 4.5 ± 1.7 5.4 ± 2.3 4.8 ± 2.4

44 (18, 76) 9.8 (3.0, 33.0) 1.1 ± 0.8

956 ± 137 51.1 ± 23.1

1 (0, 6) 4 (2, 8) 4.8 ± 2.5 4.8 ± 2.5 4.5 ± 2.3 42 (32, 75) 13.3 (5.1, 20.7) 1.2 ± 0.7

969 ± 212 46.1 ± 18.7

* Values are the mean ± SD unless otherwise indicated. FRAP = ferric-reducing/antioxidant power test; t P < 0.05.

see Table 1 for additional definitions.

Table 3. Percentage change and absolute counts of lymphocyte subsets after treatment with Ganoderma lucidum plus

 

San Miao San and placebo*

 

 

 

Ganoderma lucidum (n = 28)

Placebo (n = 30)

 

% change at

% change at

% change at

% change at

Immune markers

8 weeks

24 weeks

8 weeks

24 weeks

% of T lymphocyte (CD3 + )

-0.59 ± 0.86

-1.19 ± 1.34

0.30 ± 1.50

1.76 ± 1.39

Absolute counts of T cell (CD3+; cells/^l)

5.83 ± 4.05

-2.59 ± 3.59

15.39 ± 4.49

1.45 ± 4.06

% of Ts and CTL (CD8+)

-2.57 ± 1.64

-2.27 ± 2.14

-0.25 ± 1.73

-1.12 ± 1.93

Absolute counts of Ts and CTL (CD8 + ;

3.27 ± 4.46

-4.03 ± 3.67

14.90 ± 5.01

-0.73 ± 4.67

cells/^l)

 

 

 

 

% of Th (CD4+)

0.12 ± 1.73

-0.40 ± 2.13

0.82 ± 1.99

-3.17 ± 2.30

Absolute counts of Th cells (CD4+; cells/^l)

5.96 ± 4.38

-1.34 ± 4.51

14.59 ± 4.63

1.81 ± 3.84

Absolute counts of lymphocytes (cells/^l)

6.64 ± 4.18

-1.33 ± 3.69

13.94 ± 4.14

-0.23 ± 3.74

% of NK cells

1.01 ± 4.92

2.86 ± 6.15

1.39 ± 4.98

4.91 ± 7.90

Absolute counts of NK cells

12.26 ± 7.76

3.36 ± 8.75

17.02 ± 7.65

5.20 ± 8.02

% of B cells

7.64 ± 6.82

9.51 ± 6.77

4.21 ± 4.19

8.89 ± 5.58

Absolute counts of B lymphocytes

13.89 ± 8.26

6.52 ± 7.97

18.35 ± 5.07

8.15 ± 6.49

Ratio of Th/Ts and CTL

3.86 ± 3.79

3.86 ± 3.78

5.21 ± 3.03

5.21 ± 3.04

* Values are the mean ± SEM. % change = [(parameter at week 8 or week 24

- parameters at baseline)/(parameter at baseline)]

X 100. There were no

significant differences in the changes between groups.

Ts = T suppressor lymphocytes; CTL = cytotoxic T lymphocyte; NK =

natural killer cells.

Statistical analysis. Comparisons between the group treated with Glucidum and SMS and the placebo group for demographic and clinical characteristics were performed using chi-square tests, Student's i-test, or Mann-Whitney

U tests where appropriate. Comparisons before and after treatment in each group were assessed using paired t-tests or Wilcoxon's signed rank test as appropriate. All hypoth­eses were 2-tailed, and P values less than 0.05 were con­sidered significant. Analyses were performed using SPSS for Windows, version 10.0 (SPSS, Chicago, IL).

RESULTS

The demographic features of the 65 patients are shown in Table 1. There were no significant differences between the 2 groups. A total of 58 patients (89.2%) completed the 24-week study, with premature termination occurring in 3 patients in the placebo group (2 due to inefficacy as de­fined when patients required increased doses of drugs or a change in the drugs, and 1 due to emigration) and 4 pa­tients in the G lucidum and SMS group due to inefficacy
that occurred at 8 weeks in 3 patients and at 12 weeks in the other patient.

At week 24, the ACR20 response in the placebo group and the G lucidum and SMS group was not significantly different (9.1% and 15.6%, respectively; P > 0.05) and there were no significant differences at earlier time points. Patients in the G lucidum and SMS group who completed the trial had significant improvement in the pain score from week 4, and this was maintained at week 24 (mean ± SD score 4.9 ± 2.3 at baseline, 4.1 ± 2.3 at week 4, 4.1 ± 2.3 at week 16, and 3.9 ± 2.5 at week 24; P < 0.05). In addition, the patient's global assessment also improved significantly at week 4 and was also maintained at week 24 (mean ± SD score 5.7 ± 2.5 at baseline, 5.3 ± 2.5 at week 4, 4.8 ± 2.6 at week 8, 4.7 ± 2.4 at week 16, and 4.7 ± 2.6 at week 24; P < 0.05). Other ACR components remained unchanged in both groups (Table 2). There were no changes in FRAP and serum ascorbic acid levels after treatment in both groups (Table 2).

Table 4. Percentage change in the plasma concentration of cytokines and chemokines after treatment with either Ganoderma

lucidum plus SMS or placebo*

 

Ganoderma lucidum + SMS (n = 28)

Placebo (n

= 30)

Parameters

% change at 8 weeks

% change at 24 weeks

% change at 8 weeks

% change at 24 weeks

Cytokines

IL-18 Chemokines IP-10 MCP-1 MIG

RANTES IL-8

-1.88 (-9.18, 17.11)

-25.33 (-32.55, 7.65) 1.85 (-7.18, 16.89) - 1.95 ( - 32.05, 24.81) 207.36 (0.81, 676.96) 0.00 (-31.68, 108.33)

-3.11 (-14.01, 16.33)

-2.85 (-16.82, 17.95) 1.44 (-20.61, 37.48) 14.48 (-11.90, 43.03) 3.44 (-67.21, 273.33) -2.5 (-32.20, 100.35)

-2.61 (-15.22, 28.8)

1.95 (-29.51, 16.96) 10.20 (-8.04, 59.77) 2.28 (-12.69, 32.83) 236.45 (21.07, 714.68) - 0.0 (-26.46, 66.37)

4.74 (-21.11, 30.28)

5.26 (-17.17, 66.18) 13.26 (-10.94, 52.01) 2.06 (-14.43, 65.58) 18.28 (-66.61, 218.81) 15.70 (-21.71, 94.17)

* Values are the median (interquartile range). % change = [(parameter at week 8 or week 24 - parameters at baseline)/(parameter at baseline)] X 100. There were no significant differences in the changes between groups. IL-18 = interleukin-18; IP-10 = interferon-y-inducible protein 10; MCP-1 = monocyte chemotactic protein 1; MIG = monokine induced by interferon-y; IL-8 = interleukin-8; see Table 1 for additional definitions.

Table 5. Ex vivo

production of IL-6, IL-18, and chemokines upon stimulation with phytohemagglutinin and lipopolysaccharide

for 24 hours*

 

Ganoderma lucidum + SMS (n = 28)

Placebo (n

= 30)

 

% change at 8 weeks

% change at 24 weeks

% change at 8 weeks

% change at 24 weeks

Cytokines IL-6 IL-18 Chemokines IP-10 MCP-1 MIG

RANTES IL-8

-1.64 (-46.86, 116.57) -28.89 (-80.25, 111.01)

-11.60 (-41.29, 89.38) -36.68 (-61.94, 26.24) -42.62 (-89.53, 634.59) 13.86 (-62.12, 242.87) 14.49 (-80.62, 146.49)

-27.67 (-77.68, 20.38) -62.89 (-77.76, 65.87)+

7.06 (-23.93, 94.29)+ -41.95 (-66.12, 16.76) -34.11 (-73.49, 185.26) -11.45 (-74.74, 176.18) -4.88 (-64.36, 57.88)

0.80 (-30.82, 41.31) - -16.99 (-56.38, 88.57) -

-18.18 (-61.51, 29.38) - -2.99 (-63.54, 90.08) - 10.03 (-75.24, 73.66) -

6.7 (-34.76, 105.03) 12.92 (-59.83, 462.77) -

43.53 (-92.42, 55.71) 11.26 (-40.43, 57.95)

41.53 (-83.22, 19.94) 25.90 (-70.94, 46.36) 43.59 (-81.19, -10.46)

0.32 (-40.63, 68.09) 32.09 (-94.56, 86.69)

* Values are the median (interquartile range). % change = [(parameter at week 8 or week 24 - parameters at baseline)/(parameter at baseline)] X 100. IL-6 = interleukin-6; IL-18 = interleukin-18; IP-10 = interferon-y-inducible protein 10; MCP-1 = monocyte chemotactic protein 1; MIG = monokine induced by interferon-y; IL-8 = interleukin-8; see Table 1 for additional definitions. + P < 0.05 by Mann-Whitney U test versus placebo group.

The plasma concentration of cytokines and chemokines showed no significant differences between groups before or after treatments and no significant differences after treatment. The percentage, absolute counts, and ratio of

CD4+/CD8+/NK/B lymphocytes were unchanged be­tween groups. CD3, CD4, and CD8 lymphocyte counts were unchanged (all P > 0.05) (Table 3). However, the plasma levels of IL-18, IP-10, and IL-8 showed a declining trend in patients treated with G lucidum and SMS at week 8 and week 24, although the differences did not reach statistical significance (Table 4). In ex vivo experiments, the percentage change of IL-18 was significantly lower in the G lucidum and SMS group (Table 5).

There were 22 episodes of adverse events reported by 13 patients, with 14 episodes occurring in patients receiving placebo and 8 occurring in the G lucidum and SMS group (Table 6). There were no reports of severe adverse reac­tions in any patients.

DISCUSSION

Despite claims that the extracts are of benefit to many conditions including various types of arthritis, our study is the first clinical trial to examine the effects of a combina­tion of G lucidumand SMS in patients with RA. Our findings indicate that these compounds may have analge-

Table 6. Adverse events in patients treated with either Ganoderma lucidum plus SMS or placebo*

Ganoderma lucidum + SMS Placebo

Adverse events

(n = 32)

(n = 33)

GI upset

4

5

Palpitations

0

3

Irregular period

0

3

Insomnia

1

1

Polyuria

0

1

Headache

1

0

Sweating

2

0

Rash

0

1

Total

8

14

* GI = gastrointestinal;

see Table 1 for additional

definitions.

 

sic effects but do not appear to have any antioxidant or antiinflammatory properties.

The dosage of G lucidum at 4 gm daily was recom­mended by practitioners of TCM, although the effective dose is not really known. Dosages of 0.5-1 gm daily have been recommended for health maintenance, 2-5 gm for chronic health conditions, and up to 15 gm daily for seri­ous illness. The recommended G lucidum dosage in the Pharmacopoeia of the People's Republic of China is 6-12 gm (23). We chose a lower dose in the combination treat­ment based on the suggestion of the local TCM practitioner and in consideration of safety aspects because the patients were taking other medications and it is not known if in­teractions might occur. It is possible that higher doses may have a more beneficial effect. The toxic dose of G lucidum is also not clear but the median lethal dosage has been estimated to be between 10 and 21 gm/kg, and very high dosages (up to 38 gm/kg) have been tested in animal ex­periments (24,25), indicating the extracts are very safe.

For SMS, the recommended dosage was 2.4 gm daily for each component, and higher dosages of up to 10 gm daily have been reported without any adverse effects in the Chinese literature. The data on the appropriate doses of SMS are more uncertain, and for both SMS and G lucidum, there are no useful biologic markers of their activity that can be measured in relation to dose and the active ingre­dients have not been fully characterized. The combination of SMS and G lucidum for the analgesic and antiinflam- matory effects is a common practice according to our Chi­nese medicine colleagues. Combination was chosen based on the advice from a local Chinese medical practitioner, and our preliminary data using an in vitro test system demonstrated that G lucidum significantly inhibits the enzyme phospholipase A2 from bee venom and from hog pancreas. The lack of efficacy in terms of an antiinflam- matory or immunomodulating effect in our study may be due to a number of causes. First, in retrospect, it would be useful to demonstrate in an experimental animal model that G lucidum alone and/or in combination with SMS has an inhibitory effect on phospholipase A2 in the blood and in the synovial fluid at various concentrations. The addi­tion of SMS to G lucidum may not cause any additional inhibitory activity against phospholipase A2. Second, as­suming the compounds do inhibit phospholipase A2, it is also possible that the concentration of the active ingredi­ent of G lucidum or SMS in the blood or synovial fluid at the current dose was insufficient to inactivate phospho- lipase A2 in synovial fluid. It has been shown previously that the concentration of phospholipase A2 in RA synovial fluid exceeded that in plasma (26). It is possible that the concentrations of G lucidum and SMS achieved in the RA synovial tissue and the synovial fluid were insufficient to achieve adequate inhibition of phospholipase A2. Cur­rently, there is no information on the absorption and sys­temic distribution of the active ingredients of G lucidum in humans. Pharmacokinetics and pharmacodynamic assess­ments of G lucidum and SMS components and a dose- finding study of G lucidum would be desirable in future studies. Finally, potential interactions between G lucidum or SMS with other DMARDs should be addressed.

In this study, significant analgesic effects were seen and the mixture was well tolerated. The mechanisms of the analgesic effects are unknown and deserve further study. Despite a lack of clinical antiinflammatory effects in our patients, the elevated ex vivo production of IP-10, a CXC chemokine for activated T cells and NK cells during in­flammatory reactions, is of interest (22). The precise expla­nation for the elevated IP-10 production is unclear but this may reflect an immunoactivation effect of G lucidum. However, an elevated level of the proinflammatory cyto­kine IL-18 (478 pg/ml) was detected in patients with RA before treatment, a level much higher than that in healthy individuals (normal range 83-195 pg/ml) (27). Increased IL-18 has been associated with RA (28), nephrotic syn­drome (29), systemic lupus erythematosus (30), and asthma (31). The significantly lower IL-18 ex vivo produc­tion seen in patients after treatment with G lucidum and SMS may suggest potential beneficial effects for patients with RA. Alternatively, this may imply a reduced induc­tion of IL-18 at the local inflammatory sites in the patients with RA treated with G lucidum and SMS.

Not withstanding the limitations of clinical trials with Chinese herbal medicines, and despite the large number of in vitro studies, we believe this is the first report of G lucidum use in humans with RA. There are 2 reports describing a reduction of herpes zoster pain in 9 patients (32,33), another report on healthy volunteers demonstrat­ing a lack of impairment of hemostatic function (34) de­spite in vitro studies suggesting that G lucidum might impair hemostasis, and one study demonstrating improve­ment of symptoms in patients with neurasthenia (35). Our study represents the first clinical trial that explores the efficacy of these 2 herbs in a combination that is com­monly used in TCM for the treatment of rheumatic disease. The results of this study do not support the clinical im­portance of G lucidum as an antiinflammatory agent via the inhibitory effects of phospholipase A2, and are consis­tent with the recent finding in a much larger double-blind, placebo-controlled study of treatment with a selective in­hibitor of a secretory phospholiase A2 in patients with RA (15). Nonetheless, the significant lowering of the proin­flammatory cytokine IL-18 in patients seen after treatment (36) and the analgesic properties without any demonstra­ble adverse side effects are noteworthy and deserve further study.

AUTHOR CONTRIBUTIONS

Dr. Edmund Li had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Edmund Li, Tam, Tomlinson, Wachtel-Galor, Ben- zie, Leung.

Acquisition of data. Edmund Li, Wai Ching Li, Tam, Wong, Lam. Analysis and interpretation of data. Edmund Li, Tam. Manuscript preparation. Edmund Li, Tomlinson. Statistical analysis. Tam.

Assessment of cytokine and cellular measurement. Wong, Lam, Bao.

REFERENCES

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  • 10. Hu H, Ahn NS, Yang X, Lee YS, Kang KS. Ganoderma lucidum extract induces cell cycle arrest and apoptosis in MCF-7 human breast cancer cell. Int J Cancer 2002;102: 250-3.
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  • 13. Yuen JW, Gohel MD. Anticancer effects of Ganoderma lucidum: a review of scientific evidence. Nutr Cancer 2005; 53:11-7.
  • 14. Bomalaski J, Clark M. Phospholipase A2 and arthritis. Arthri­tis Rheum 1993;36:190-8.
  • 15. Bradley JD, Dmitrienko AA, Kivitz AJ, Gluck OS, Weaver AL, Wiesenhutter C, et al. A randomized, double-blinded, place­bo-controlled trial of LY333013, a selective inhibitor of group II secretory phospholipase A2, in the treatment of rheumatoid arthritis. J Rheumatol 2005;32:417-23.
  • 16. Garcia Pastor P, de Rosa S, de Giulio A, Paya M, Alcaraz M. Modulation of acute and chronic inflammatory processes by cacospongionolide B, a novel inhibitor of human synovial phospholipase A2. Br J Pharmacol 1999;126:301-11.
  • 17. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid ar­thritis. Arthritis Rheum 1988;31:315-24.
  • 18. Koh ET, Seow A, Pong LY, Koh WH, Chan L, Howe HS, et al. Cross cultural adaptation and validation of the Chinese Health Assessment Questionnaire for use in rheumatoid ar­thritis. J Rheumatol 1998;25:1705-8.
  • 19. Felson DT, Anderson JJ, Boers M, Bombardier C, Chernoff M, Fried B, et al. The American College of Rheumatology pre­liminary core set of disease activity measures for rheuma­toid arthritis clinical trials. Arthritis Rheum 1993;36:729-40.
  • 20. Benzie I, Strain JJ. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol 1999;299:15-27.
  • 21. Benzie I, Strain J. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay. Anal Biochem 1996;239:70-6.
  • 22. Tang NL, Chan PK, Wong CK, To KF, Wu AK, Sung YM, et al. Early enhanced expression of interferon-inducible protein-10 (CXCL-10) and other chemokines predicts adverse outcome in severe acute respiratory syndrome. Clin Chem 2005;51:2333- 40.
  • 23. Compiled by the State Pharmacopoeia Commission of P. R. China. Parmacopoeia of the People's Republic of China. Beijing; Chemical Industry Press: 2000.
  • 24. Chiu SW, Wang ZM, Leung TM, Moore D. Nutritional value of Ganoderma extract and assessment of its genotoxicity and antigenotoxicity using comet assays of mouse lymphocytes. Food Chem Toxicol 2000;38:173-8.
  • 25. Chang R. Effective dose of Ganoderma in humans. In: Buchanan PK, Hseu RS, Moncalvo JM, editors. Proceedings of the Contributed Symposium 59A, B of the 5th International Mycological Congress. Taipei; 1994. p. 101-13.
  • 26. Pruzanski W, Vadas P, Stefanski E, Urowitz M. Phospholipase A2 activity in sera and synovial fluids in rheumatoid arthritis and osteoarthritis: its possible role as a proinflammatory en­zyme. J Rheumatol 1985;12:211-6.
  • 27. Wong CK, Ho CY, Li EK, Tam LS, Lam CW. Elevated produc­tion of interleukin-18 is associated with renal disease in pa­tients with systemic lupus erythematosus. Clin Exp Immunol 2002;130:345-51.
  • 28. Sato M, Takemura M, Shinohe R, Koishi H, Morita T, Seishima M. Clinical significance of serum IL-18 determina­tion in rheumatoid arthritis. Rinsho Byori 2004;52:109-14. In Japanese.
  • 29. Matsumoto K, Kanmatsuse K. Elevated interleukin-18 levels in the urine of nephrotic patients. Nephron 2001;88: 334-9.
  • 30. Wong CK, Ho CY, Li EK, Lam CW. Elevation of proinflamma- tory cytokine (IL-18, IL-17, IL-12) and Th2 cytokine (IL-4) concentrations in patients with systemic lupus erythemato- sus. Lupus 2000;9:589-93.
  • 31. Wong CK, Ho CY, Ko FW, Chan CH, Ho AS, Hui DS, et al. Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-y, IL-4, IL-10 and IL-13) in patients with allergic asthma. Clin Exp Immunol 2001;125:177-83.
  • 32. Hijikata Y, Yamada S. Effect of Ganoderma lucidum on post- herpetic neuralgia. Am J Chin Med 1998;26:375-81.
  • 33. 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.
  • 34. Kwok Y, Ng KF, Li CC, Lam CC, Man RY. A prospective, randomized, double-blind, placebo-controlled study of the platelet and global hemostatic effects of Ganoderma lucidum (Ling-Zhi) in healthy volunteers. Anesth Analg 2005;101: 423-6.
  • 35. Tang W, Gao Y, Chen G, Gao H, Dai X, Ye J, et al. A random­ized, double-blind and placebo-controlled study of a Gano- derma lucidum polysaccharide extract in neurasthenia. J Med Food 2005;8:53-8.

36. Xi Bao Y, Kwok Wong C, Kwok Ming Li E, Shan Tam L, Chung Leung P, Bing Yin Y, et al. Immunomodulatory effects of lingzhi and san-miao-san supplementation on patients with rheumatoid arthritis. Immunopharmacol Immunotoxicol 2006;28:197-200.

 

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 ( aşağıdaki yazı bu dergideki makalenin kısmi tercümesidir.)

KANSER OLUŞUMLARIYLA MÜCADELE

Çin'de kronik viral enfeksiyonların sonucu olarak ortaya çıkan kanser vakalarında çok yüksek düzey görülmesi enteresandır. Kanser odağı olarak karaciğer, mide, özofagus ve nazofarinks başta gelmektedir. Buna ek olarak özellikle serviks kanseri kadınlar arasında o denli yükselmiştir ki, kadınların kaybedildiği ikinci kanser tipi halini almıştır. Göreceli olarak yüksek seviyede ortaya çıkan ve Batı ile karşılaştırıldığında anlaşılmaz düzeyde yüksek olan virüs ilişkili kanser tiplerinin sebebi hala meçhuldür. Enfeksiyonun çok yaygın olması, genetik yatkınlık, az beslenme ve sigara belki de bu yayılımın etkeni olabilir. Kronik viral enfeksiyonları kökünden kazıyacak, yeterli bir bağışıklık sisteminin olmaması belki de kanser hücreleri için en önemli çıkış noktasıdır. 2005 yılında sadece yeni kanser vakası artışı %15'tir. Görüldüğü gibi, virüse bağlı kanser vakalarının artışı en önemli halk sağlığı problemlerinden birini oluşturmaktadır ve gerek tanı gerekse tedavi bazlı yeni datalara ihtiyaç vardır.

               Hepatoselüler karsinom (hcc) Çin'de kanserden ölüm sebebi sıralamasında ikinci sıradadır. Ve özellikle erkekler arasında diğer ülkelere kıyasla çok yüksek bir hızla artmaktadır. Hepatit B (hbv) ve C (hcv) hala en önemli viral faktörler olarak görülür ve Hepatit G virüsü veya diğer transfüzyon kökenli virüsler de şüpheliler arasına girebilirler. Çin'de hcc hastalarının neredeyse hepsi zaten hbv hastasıdır. Hbv'nin hcc ile bu sıkı ilişkisi belki de asıl enfeksiyon etkeninin bu virüs olduğunu düşündürür. Neredeyse hcc hastalarının %12'sinde hbv ve hcv koenfeksiyonu vardır. Çok geniş kapsamlı bir analiz göstermiştir ki, hbv ve hcv enfeksiyonları hcc vakaları için bağımsız bir risk faktörü durumundadırlar ve ikisinin bir arada bulunması herhangi birinin tek başına bulunmasından daha yüksek risk oluşturmaktadır.

               Nazofarinks kanseri Eppstein Bar virüsü ile sıkı ilişkili bir olgudur. Zaman zaman Hotgkin linfoma ve gastrik kanserle de ilişkili olduğu görülmektedir. Güney Çin populasyonunda ailesel yoğunlaşma göstererek en yüksek düzeyde bu vakaya rastlanmaktadır. Yıllık yeni vaka ortaya çıkışı erkeklerde 100.000'de 433, kadınlarda 100.000'de 499 düzeyinde olup, örneğin Hong Kong ile karşılaştırıldığında korkutucu olduğu görülmektedir. Çünkü Hong Kong'da nazofarinks kanser vaka düzeyi erkekler için 100.000'de 24, kadınlar için 100.000'de 10'dur.

               Serviks kanseri giderek artan kanser tiplerindendir. Human Papilloma virüsü (hpv) enfeksiyonları ve sigara anal kanserlerle serviks kanserlerinin tetiklenmesinde sinerjit etkili görülmektedir. Batı'da en sık rastlanan etken olarak hpv16 hattı (subtip) görülürken, Çin'de hpv18 ve 59 gibi farklı tipler en sık rastlananlar olarak karşımıza çıkarlar. Hpv16E7 denilen bir protein variantı bireyin humoral immun cevap sistemini çökertse gerektir. Ancak selüler immun cevap yeteneğinde bir değişiklik yapmaz. Ne kadar aşırı sigara tüketimi varsa, o kadar hpv kökenli anal ve serviks kanser olgusu ortaya çıkmaktadır. Diğer yandan, hücresel düzeyde bu virüsü eradike edecek bir immun cevap tetiklenebilirse bu virüslerin kanser başlatması yönündeki güçleri yok edilebilir. Hpv16 Çin'de özofagus kanserinin birinci sebebidir. Ama Batı'da bu yönde bir bulgu yoktur. Buna ek olarak, gerek Batı'da gerek Çin'de hpv ile bağlantılı baş ve boyun kanserleri artış göstermeye başlamıştır. Bir diğer yoğunlaştığı nokta, kadınların göğüs kanserleridir ve düzeyinde hızlı bir yükselme fark edilmektedir. Herhalde belki de, Çin'e özel bir kombinasyon olarak çok yağlı yemenin, hareketsizliğin ve aktivite düşüklüğünün hpv33 virüsü ile ilişkili göğüs kanseri vakalarında artışın sebebi olabileceği düşünülür.

               Kanserin bitkisel ilaçlarla başarılı bir şekilde tedavi edilebilmesi ile ilgili veriler özellikle Çin bitkisel tıbbının öne çıkardığı preperatların belirli karışımlar halinde kullanılmasıyla daha güvenilir olabileceğine işaret etmektedir. Hcc hastası insanlarda bunların kemoterapi ile birlikte verilmeleri de tavsiye edilmektedir. Ancak gerçekten uzun süreli ve çok titiz yapılmış çalışmalarla ilgili veri azdır. Kontrollü klinik çalışmalarda elde edilen ciddi bulgular göstermektedir ki, antikanser tedavilerinde tıbbi mantarlar çok etkilidir. Coriolus versicolor psksı ile desteklenmiş kolorektal kanser tedavisi tek başına uygulanan kemoterapiden çok daha başarılı bulunmaktadır. Tıbbi mantarlarda, vücudun antitümör immünitesini tetikleyen, teşvik eden ve güçlendiren beta glukan grubu polisakkaritlerin bulunduğu bilinmektedir. Bunlar, sinerjik olarak vücudun kendi ürettiği yeni antikorları destekleyerek, etkili olabilmektedirler. Maiteke mantarı ve Ganoderma lucidum Çin tıbbının en önemli mantarlarından olup, immun sistemin her yönünyle desteklenip güçlendirilmesi ve rehabilite edilmesi yoluyla viral enfeksiyonlar ile bunlara bağlı veya bunlardan bağımsız kanser olgularını subrese ettiği bilinen mantarlardır.

 

Semptom Kontrolü

               Kanser hastalarında bazıları kanserle, bazıları da yapılan tedavi sebebiyle ortaya çıkan karmaşık semptomlar görülmektedir. Diyelim ki kanser tedavi edilse bile, hasta uygulanan tedavinin yan etkilerinden uzun süre ızdırap çekmektedir. Bu yan etkiler hastanın yaşam kalitesi üzerine ciddi olumsuz etki yaptıkları gibi, Batı tıbbının geleneksel metodlarıyla hiç de verimli bir sonuç vermezler. Çin tıbbı işte kanser hastalarının semptomlarının yok olmasında tedavinin desteklenmesi yönünde çok faydalı rol oynarlar. Özellikle tükenmişlik, depresyon ve ağrı kapsamında bu hastalarda ortaya çıkan genel semptomlar son derece etkili bir şekilde kontrol altına alındıkları gibi, buna ek olarak mide barsak sisteminin spesifik semptomları ve kemik iliğinin çalışmasının engellenmesine (miyelosüpresyon) ait yan etkiler de ortadan kalkmaktadır. Kemoterapi alan kanser hastaları, genellikle miyelosüpresyon geliştirdiklerinden ve ciddi gastrointestinal (mide barsak) yan etkilere uğradıklarından, kısa sürede dermansız ve bitik bir davranışa girerler ve iştahları kaybolur. Yine kemoterapik ajanların dalak ve böbrek yetersizliği yaptığı, dolayısıyla direkt olarak kan ve genel fizyolojik dengede hızlı bir bozulmaya yol açtıkları bilinmektedir. Unutmamamız gerekir ki böbrek ve dalak diğer organlarla ilgili kominikasyonu ve vücut bütünlüğünü sağlayan en önemli iki organdır. Kemoterapi ilaçları dalağı ve mideyi darmadağın etmektedir. Fiziksel olarak mide ve barsak hasarları şeklinde kendini belli eder. Klasik Çin tıbbını anlamamış bir onkolog, bu tıbbın kullandığı kelimeleri abartılmış ve tedaviden çok kişinin hissiyatına dönük ifadeler taşıdığını ileri sürer. Ancak vücut-beyin ilişkisinin ve bunu donatan sistemin oluşturduğu karmaşık ağın böyle biri tarafından anlaşılmasının güç olacağı açıktır. Depresyon, anksiyete ve benzeri psişik dünya davranışı klasik kemoterapi uygulayıcılarında hiç dikkate alınmaz. Vücut-beyin ağı anlaşılmaya başladıktan sonra akupunkturun, beslenmenin ve bitkisel desteklerin neler yapabilecekleri daha iyi anlaşılır. Ganoderma ve Shiitake mantarlarının da aralarında bulunduğu tıbbi mantarlar, geleneksel Çin tıbbında Qi olarak tanımlanan vücut katmanını destekleyerek ve güçlendirerek, vücudun antitümör aktivitesinin ve bağışıklık profilinin daha etkin bir hale gelmesine yardım ederler. Yapılan en son beş çalışma şunu göstermiştir ki, Çin bitkisel tedavi uygulamaları miyelosüpresyon düzeyini azaltmakta, gastrointestinal yan etkileri yok etmekte ve iştahı arttırmaktadır. Bir çalışmada geç devre gastrik kanseri olan 669 hastaya yapılan uygulama tanımlanmıştır. Hastalardan bir gruba dalağı ve böbreği destekleyen bitkiler günde 2 defa 4-6 hafta süreyle ve kemoterapiyle aynı anda verilmiştir. Diğer bir grup, sadece kemoterapi almıştır. Bitkilerle desteklenmiş kombine tedavi gören hasta grubu bir yandan çok daha yüksek lökosit ve trombosit düzeyine ulaşırken, genel ve gastrointestinal ağırlıklı yan etki ızdırapları neredeyse yok denecek düzeye inmiştir. Kemoterapiyi planlandığı şekilde tamamlayan hasta sayısı kombine tedavide %95 iken, sadece kemoterapi verilenlerde %74 düzeyindedir. Çin'de yayınlanan bu araştırma sonuçları belki de çok geniş kapsamlı bir kitleyi kapsamamakla birlikte, yine de yön gösterici durumundadır.

 

Disfonksiyon:

               Çin bitkisel tedavi unsurları zeka, hafıza ve kavrama ile ilgili fonksiyonları üst düzeyde desteklemektedir. Alışılagelmiş farmosötik uygulamalar bu yönde neredeyse hiç veya çok sınırlı yarar sağladıklarından Çin tıbbı bitkilerinin kullanıldığı sağlık ürünlerine doğru bir kapı açılmıştır. Kemoterapi sırasında veya sonrasında birçok hastanın kavrama yeteneği ve hafızası zayıflamaktadır. Bu fenomen özellikle göğüs kanseri hastalarında dikkat çekmektedir. Nörokognitif yetersizlik bu hastalarda bağımsızlığı azalttığından aslında bu problem ciddi olduğu halde ne hastalar bunu yeterince öne çıkarmışlar, ne de sağlıkçılar bununla yeterince ilgilenmişlerdir. Standart doz kemoterapi alan kanser hastalarının hiç olmassa %18'i kemoterapiye bağlı zihinsel yetersizlik göstermekte ve bu durum uygulamadan iki yıl sonrasına kadar sürmektedir. Hastalar örümcek beyinli olmaktan şikayet ederler. Bitkinlik ve depresyon da zaten bununla birlikte ortaya çıkan bozukluklar durumundadır. Acaba nöronal integritasyonun uygulamadan evvelki bazı olgulara mı bağlı olduğu ve bunun metabolik patolojisinin nasıl gerçekleştiği yönündeki bütün bilgilerimiz eksiktir. Araştırıcılar e4 allelli olarak tanımlanan bir apo-lipoproteinden söz etmektedirler.

               Kemobeyin (kemoterapi beyni) esas itibariyle subjektif bir tanım olduğundan, gerçek nörofizyolojik veya anatomik değişikliklerin ortaya çıkarılabilmesi için yeni teknikler geliştirilme zorunluluğu vardır. Kemoterapi kan damarlarının endotelyumunu bozar, bunun sonucu trombozlar ve merkezi sinir sisteminden mikroinfaktlar ortaya çıkar. Bunun sonucu serebral dokuda neler olabildiğini yani antikanser tedavilerinden sonraki beyin dokusunun durumu hala tam anlaşılamamıştır ve maalesef bu yönde çalışma yapıldığına dair bilgi de yoktur.

               Radyoterapi uygulamaları da beyinde uyumsuz kobnitif fonksiyon değişikliğine yol açar. Bu uygulamalardan sonra uzun süre hayatta kalmış hastaların yaşam kalitelerini ve rehabilitasyonlarını etkileyen en önemli olgu hafıza yetersizliği (kognitive disfunction) olmuştur. Radyoterapi beyne hasar verir. Bu hasar demiyelinizasyon sonucu ortaya çıkar ve uygulamadan birkaç hafta veya kısa bir süre sonra hafıza yetersizliği gelişmesine sebep olur. Bu kayıplar belki de bütün hafızamızı etkileyebilir. Psikiyatrik kayıplar, konsantrasyon düşüklüğü, davranış bozukluğu ve hatta allzheimera benzer tablolar gelişebilir. EN-Metil-D-Aspartat (nmda) reseptörleri aşırı aktive olurlar ve nöronal hasara yol açarak öğrenme, algılama yetimizi bozarlar.

GANODERMA LUCIDUM (A LEADER OF MEDICINAL MUSHROOMS): A PROMINENT SOURCE FOR THE HEALTHCARE MARKET IN THE 21ST CENTURY

 

Writer: S.T.Chang / Date :2005-02-02 / hits: 654

Shu-Ting Chang*
Emeritus Professor of Biology, and Director of Centre for International Service to Mushroom Biotechnology The Chinese University of Hong KongAnd Honorary Visiting Professor, Department of Biological SciencesUniversity of Wollongong, Australia

*Address all correspondence to S. T. Chang, 3 Britton Place, McKellar, A.C.T. 2617,Australia

This paper is posted with the permission from Sub-chamber of Edible Fungi of CFNA.

ABSTRACT: It is believed that human beings have constantly searched for new substances that can improve biological functions and make people fitter and healthier. Ganoderma lucidum (Curt.:Fr.) P. Karst, have been recognized for many centuries in China and other parts of Asia. During the last two decades, there has been a sharp increase in the level of commercial interest in G. lucidum products, not only in Asian countries but also in North America and Europe. Current world production of this mushroom is around 6,000 tonnes, half of which comes from China. Latest available estimates put the annual value of G. lucidum products worldwide at more than US$ 2.5 billion. This is expected to increase even further once the attributed medicinal properties are confirmed in human intervention trials. The application of modern analytical techniques has revealed the mushroom to contain numerous bioactive compounds including polysaccharides, triterpenes, adenosines, and immunomodulatory proteins. Many of these compounds have anticancer and antitumor properties that appear to be based on an enhancement of the host immune systems rather than on a direct cytocidal effect. Further expansion of the market of G lucidumproducts will require the introduction of more reproducible protocols for mushroom production and downstream processing to improve quality control and ensure enduring public trust.

I. INTRODUCTION

It is believed that human beings have constantly searched for new substances that can improve biological functions and make people fitter and healthier. Recently, Western society has increasingly turned to plants, mushrooms, herbs and foods as sources of these enhancers.

These products have been called variously: vitamins, dietary supplements, functional foods, phytochemicals, nutraceuticals (Zeisel, 1999) and nutriceuticals (Chang and Buswell, 1996, 2003).

Ganoderma lucidum (Curt.: Fr.) P. Karst. (Lingzhi in Chinese; Reishi, Mannentake, or Sachitake in Japanese; and Youngzi in Korean) (Figure 1) is a species of basidiomycetes, which belongs to Polyporaceae (or Ganodermaceae) of Aphyllophorales. Commonly it is known as a wood-decaying fungus; it causes white rots on a wide variety of trees and can thus be described as a phytopathogenic fungus. Because of its perceived health benefits, its fruiting body has gained wide popularity recently as a dietary supplement, not only in China and Japan but also in North America and other parts of the world. The reason it attracts international attention as a valuable Chinese herb is due to the variety of its biological activities, such as antitumor, immunomodulatory, cardiovascular, respiratory, antihepatotoxic and antinoeiceptive (acting against pain) effects. The diversity in the biological actions of Lingzhi may be attributed to the fact that it is composed of different chemical entities, including alkaloids, amino acids and peptides, inorganic elements, steroids and fatties, and organic acids. The major compounds with significant pharmacological activities appear to be triterpenes, polysaccharides and adenosines. It is interesting that during the last three decades, more than 150 triterpenes (Kim and Kim 2002) and more than 50 carcinostatic polysaccharides (Jong and Birmingham 1992) have been isolated and known to be unique compounds in this mushroom. Therefore, G. lucidum products with different triterpenes and polysaccharides or combinations of these two groups are most likely to result in different pharmacological activities (Leung et al., 2002).

 

Figure1. The fruiting bodies of Ganoderma lucidum grown on short-wood logs buried in the soil.

According to Chinese tradition, Lingzhi is also known as the "miraculous zhi", or "auspicious herb" and it is usually considered to 'symbolize happy augury, and to bespeak good fortune, good health and longevity, even immortality' (Wasson, 1968).

Ganoderma is now consumed worldwide as a health tonic, and as a dietary supplement. Millions of people take it everyday to enhance their energy, to improve their digestion, and to sleep better. Ganoderma is used also both for the prevention, and for the treatment of a number of health problems that require a balanced immunoresponse system, and also a healthy cardiovascular system.

Of particular concern at present is low reproducibility in terms of production methods and the often-poor quality control to which Ganoderma -based products are subject. For various reasons (seasonal variations, different soil conditions, stage of fruiting body development) the fruiting body and hence the product quality are very difficult to control. Moreover, manufacturers ofGanoderma-based products normally rely on several sources to provide the fruiting bodies used to produce the product. These different sources often show considerable variation with respect to both the quality of the mushroom fruiting body and to the processed product. Unfortunately, the perceived future growth of this sector has also resulted in the appearance of an ever-increasing number of less-reputable companies whose activities will inevitably lead to more intensive scrutiny of this sector has also resulted in the appearance of an ever-increasing number of less-reputable companies whose activities will inevitably lead to more intensive scrutiny of the sector as a whole. Therefore, it is of paramount importance for the manufacturing industries involved to develop and adopt acceptable and reproducible protocols both for growing the raw materials and for downstream processing in order to ensure high-quality, standardized and safeGanoderma products. Such practices are essential for earning and maintaining the enduring public trust, which is vital for securing an expanding market in the future. In summering, it can be said that G. Lucidum is one of the earliest medicinal mushrooms that has been considered to be of superior grade, i.e., that it is a non-toxic tonic herb without side effects even when taken for a long period. For the last three decades it has been and will continue to be, we believe, one of the most promising and intensively studied mushrooms for medicinal and tonic purposes.

Mushroom nutriceuticals (dietary supplements) including Ganoderma-based natural healthcare products are likely to be of increasing interest throughout the world. They represent both challenges and opportunities. Who knows? Perhaps a whole new industry, or even industries, will arise having greater economic value than those currently producing mushrooms for food. The world trade in edible mushrooms is currently around US$30 billion, while world trade market value for medicinal mushroom products including Ganoderma's US$2.5 billion is around US$10 billion.

II. TRADITIONAL USES

Lingzhi has played an important role in Chinese traditional medicine, either alone or in combination with other herbal medicines. Uses in ancient folk medicine include treatment for a "tight chest," to improve intellectual capacity and memory, to promote agility, to lengthen life span, and to relieve hepatopathy, nephritis, hyperlipemia, arthritis, asthma, gastric ulcer, arteriosclerosis, leukopenia, diabetes, and anorexia (Chang and But 1996, Jong and Birmingham, 1992). Contemporary traditional Chinese medicine (TCM) using cultivated Ganoderma mushrooms (Mayzumi et al., 1997) includes treatment for neurasthenia, debility from prolonged illness, insomnia, anorexia, dizziness, chronic hepatitis, hepercholesterolemia, mushroom poisoning, coronary heart disease, hepertension, altitude sickness, carcinoma, bronchial cough in the elderly individuals (Ying et al., 1987) and inhibition of cholesterol synthesis (Kim et al., 2000). For a long time in China, Ganoderma has been known as a kind of panacea in the folklore, curing all kinds of diseases (Liu, 1999). All ancient Chinese Materia Medica have treated Lingzhi as of superior grade, which as indicated previously means that it is a non-toxic tonic herb without side effects even when taken for a long period at a high dosage.

III. BIOLOGICAL COMPOUNDS

Recent application of modern analytical techniques has, in number of cases, provided a scientific basis for these earlier empirical observations.Ganoderma has been reported to have multi-beneficial values and concerted medicinal effects on various diseases. The diversity in the beneficial values and medicinal effects may be attributed to the fact that Lingzhi is composed of a vast number of bioactive compounds. The major compounds with significant pharmacological activities appear to be triterpenes, polysaccharides and adenosines (Lindequist 1995) although bioactive proteins, nucleic acids and other substances have also been identified. Of particular interest are a group of fungal immunomodulatory proteins-Fip's, which have been isolated from G. lucidum, Ganoderma tsugae and other mushrooms (Kino et al. 1989; Ko et al. 1995; Ko et al. 1997). Those Fip's have been classified into a distinct family of proteins on the basis of similarities in their amino acid sequences and immunological effects. During the last two decades, more than 200 substances have been isolated from its fruiting bodies, spores, cultivated mycelium and culture broth, and some of their chemical and physical structures are known. However, only some of these triterpenes and polysaccharides are treated in this review paper.

A. Triterpenes/Triterpenoids

Kubota et al. (1982) were the first to isolate triterpene compounds, ganodermeric acids A and B from G. lucidum. Since then, the physiochemical properties of more than 150 lanostane-type tritermponids found inG. lucidum have been identified. These were divided into 10 groups according to their structural similarities and the known biological and medicinal activities (Kim and Kim 2002). These compounds have been isolated from fruiting bodies, spores as well as mycelia. Their chemical structure is based on lanosterol, which is an important intermediate in the biosynthetic pathway for steroids and triterpenes in microorganisms and animals. Triterpenses have cytotoxic, hepatoprotective, and hypolipidemic properties. They influence platelet aggregation, and inhibit angiotensine-converting enzyme and histamine release (Lindequist 1995, Misuno et al. 1995, Lin 1996, Kim and Kim 2002). The different triterpenes appear to have different bioactive properties. Some of these triterpenes have shown the following activities:

1. Bitterness

G. lucidum has a remarkably strong bitterness which, has not been found in any other mushrooms. The bitterness varies in strength depending on the place of production, cultivation conditions, strain used, etc. It has been observed that the stipe has stronger bitterness than the pileus. Such bitterness has also been found recently in cultured mycelia as well as in the culture broth in certain conditions, but the strength is much reduced. Although the bitterness has not found to be related with any pharmacological effects, the bitterness attracts attention as a marker substance for pharmacological evaluation and chemical quality judgement. In point view of marketing, some customers, e.g., Korea, are only interested in theGanoderma products with bitterness. The mushroom produces bitterness during the course of fruiting and the bitterness of fruiting bodies are contributed by substances, such as genoderic acids A. C, I, and J; lucidenic acids A. D. I; and lucidones A and C. These triterpenoids are generally classified in at least two types: one is C30 ganoderic acid type and the other is C27 lucidenic type (Nishitoba 1996).

2. Cytotoxicity

Cytotoxic triterpenses are potential anticancer agents and some cytotoxic compounds are isolated from the mycelial and fruiting body extracts. It is reported that ganoderic acids Z, Y, X, W, V, and T from the mycelia demonstrated cytotoxic activities in vitro on hepatoma cells, and several lanostanoids isolated from fruiting bodies exhibited potent inhibition of tumour cells in vitro (Kim and Kim 2002).

3. Platelet aggregate inhibition

Platelets in the blood are normally nonreactive to intact vascular endothelium. However, they respond quickly when vascular damage occurs by adhesion, aggregation, and coagulation. Inhibitors of platelet aggregation have potential for the treatment of apoplexy. Ganodermic acid S showed amphipathic effect on the platelet aggregation (Wang et al 1991, Su et al. 1999).

4. Antihypertension

Morigiwa et al. (1996) found that ganoderic acid F had the strongest activity of antihypertension and other terpenoids such as ganoderic acids B, D, H, and Y had weaker effects.

5. Hepatoprotective activity

In Chinese folk medicine, the fruiting bodies of G. lucidum have been used for the treatment of chronic hepatitis. Hirotani et al. (1986) found that ganoderic acids R and S from the cultured mycelia showed strong antihepatotoxic activity in the galactosamine-induced cytotoxic test with primary-cultured rat hepatocytes. Another hepatoprotective compound, ganosporeric acid A, was isolated from the ether soluble fraction of the spores of this mushroom by Chen and Yu (1991, 1999).

6. Anti-HIV.

Human immunodeficiency virus (HIV) was isolated as an etiological agent of acquired immunodeficiency disease syndrome (AIDS) (Barre-Sinoussi et al., 1983; Gallo et al., 1983). Anti-HIV activities were reported in water-soluble extract of G. lucidum (Hattori et al., 1997; Kim et al., 1997). Most recently, el-Mekkawy et al., (1998) isolated the anti-HIV compounds and reported them as Ganoderiol F and ganodermanontriol. Min et al. (1998) also isolated anti-HIV components as ganoderic acid b, ganodermanondiol, ganodermanontriol, and ganolucidic acid A, and lucidumol B.

7. Hypoglycenmic effects.

Several triterpenes, e.g., ganoderans A, B, and C, isolated from G. lucidumfruit bodies have been shown to have strong hypoglycemic effect (Hikino et al., 1985). The side effect of the drugs for diabetes with complementaryGanoderma products could be minimised. Actually, the diabetes patients, in the mean time, should worry more about other serious diseases, which could occur concurrently, e.g., coronary artery disease due to a thickening and hardening of medium-size and large arteries with narrowing of the arterial lumen by alherosclerotic plaques, hypertension, weakening of immune systems and eye diseases, etc. It happens that the major components ofGanoderma products are good for preventing these concurrently occurring diseases.

B. Polysaccharide

Some years ago, Lee et al. (1984) reported that water-soluble extracts ofG. lucidum inhibited the growth of Sarcoma 180 and a fibrosarcom in mice. Subsequently, a polysaccharide moiety present predominantly in the ethanol-precipitable fraction of the aqueous extract was found to increase the life-span of tumour-implanted mice, exhibit antimour effects against fibrosarcoma in C3H mice, and inhibit metastasis of the tumour in the lung (He and Li 1989, Furusawa et al. 1992 and Lee et al. 1995). Since these initial reports, more than 50 carcinostatic polysaccarides have been isolated from the basidiocarps and mycelium of G. lucidum (Jong and Birmingham 1992). Many polysaccharides were extracted with hot water, ammonium oxalate solution, alkali solution, dimethyl sulfoxide solution, and supercritical fluid CO2, etc., and separated by various chromatographic methods. Strong antitumour activities were found in various hetero-¥â-D-glucans having a ¥â-(1a3)-D-glucan branch as the active site, such as ¥â-D-glucan, glucurono-¥â-D-glucan, arabinoxylo-¥â-D-glucan, xylo-¥â-D-glucan, manno-¥â-D-glucan, and xylomanno- ¥â-D-glucan, as well as being present in their protein complexes (Mizuno et al., 1995). These compounds have also been ascribed hypoglycemic properties (Lindeqquist 1995, Mizuno et al. 1995, Lin 1996). Recently, a glycoprotein (containing 82.8% carbohydrate and 17.2% protein) obtained from the mycelium culture broth of G. lucidum exhibited increasing the swimming endurance capacity of mice. However, the fruiting body and mycelium of the mushroom did not have any positive response in this regard (Yang et al., 2001). The extracts from different sources (fruiting body, mycelium and culture broth) have different properties with different functions (Table 1). It has been widely reported that the anti-tumour and anti-cancer effects of the polysaccharides are based on the enhancement of the host's immune systems rather than direct cytocidal effects (Lieu et al. 1992, Zhu and Mori 1993, Chen et al. 1995, Wang et al. 1997). The various components of the immune system shown to be affected include activated macrophages, natural killer cells and cytotoxic T cells together with their secretory products such as tumour necrosis factor, reactive nitrogen and oxygen intermediates and interleukins (Ooi et al., 2002). These extractable polysaccharide compounds may increase the expression level of M-CSF (macrophage-colony stimulating factor) in both splenocytes and peritoneal exudate cells of the mouse. The expression level of TNF-a (tumour necrosis factor-a), one of anti-tumour molecules that has direct anti-tumour activity, was also up-regulated. Therefore, the overall anti-tumour effect of G. lucidum compounds may be due to the up -regulation of certain cytokins such as M-CSF and TNF-a. It should be noted that immune responses are complex reactions involving several types of cells such as macrophages and lymphocytes. The killing mediated by cytotoxic T-lymphocytes and natural killer cells represents an important mechanism in immune defence against tumours, virus-infected cells, parasites and other foreign invaders.

Table 1. Bioactive polysaccharides isolated from different sources of G. lucidum

Source

Major active sites or Compounds

Parmocological activity

Ref.

Fruiting body

¥â-(1a3)-D-glucans

Antitumour

Mizuno et al. 1995

Mycelia

¥â-(1a3)-D-glucn with ¥â-(1a6) branches

Anitumour

Mizuno et al. 1995

Culture broth

Glucoprotein

Swimming endurance Capacity

Yang et al. 2001

Spore

Aqueous extract

Myotonia and poly mycositis

Yu et al., 2000

C. Fungal immunomodulatory protein (Fip)

More recently, various mushroom species have been reported as the source of a new group of fungal immunomodulatory proteins (Fip's). These include LZ-8 (Ling Zhi-8) from G. lucidum (Kino et.al 1989), Fip-fve fromFlammulina velutipes (Gr.) Sing. (Ko et. al 1995), Fip-vvo fromVolvariella volvacea (Bull.: Fr.) Sing., (Hsu et al., 1997) and Fip-gts fromGanoderma tsugae Murr. (Lin et al.,1997). Definition of this group of proteins, which have been classified into a distinct family called fungal immunomodulatory proteins (Fip's) by Ko et al., (1995), is based on similarities in their amino acid sequences on components of the immunological responses. LZ-8, isolated and purified from the mycelium ofG. lucidum, showed optimal stimulatory activity of blast-formation towards mouse spleen cells at a concentration of 3.13 mg/ml (Kino et. al. 1989). LZ-8 is not a lectin as it lacks hemagglutination activity toward human red blood cells, but it possesses mitogenic activity toward mouse spleen cell and human peripheral blood leucocytes. In addition, this Fip can suppress autoimmune diabetes in young female non-obese diabetes mice (Kino et al. 1990) and also has a significant effect on cellular immunity as demonstrated by its effect in delaying the process rejection of transplanted allogeneic pancreatic rat islets and allografted mouse skin (van der Heun et al. 1995).

D. Steroids

G. lucidum also contains a high amount of steroids. There are two categories of steroids, ergosterol and cholesterol. Approximately 20 different steroids from both categories have been isolated from this mushroom (Ha et al. 2000; Ma et al. 2002). These steroids appear to account for the anti-atherosclerotic and lipid lowering effects of G. lucidum(Kimura et al. 1988).

IV. CONTEMPORARY USES

There is currently intense industrial interest in the novel class of compounds extractable from either the mycelium or fruiting body of G. lucidum. These compounds mentioned above exhibit either medicinal or tonic qualities and have immense potential as dietary supplements for use in the prevention and treatment of various human diseases. The issue is that should thoseGanoderma products be categorized as health food, nutriceuticals (which are also called dietary supplements or complementary medicines) or pharmaceuticals?

Unfortunately, many people confuse nutriceuticals with functional foods, which are consumed as part of the normal diet and may have been modified or enriched in some way to provide health-giving benefits. A nutriceutical (Chang and Buswell 1996), on the other hand, is a refined or partially refined food extractive, which is consumed in the form of capsules or tablets as a dietary supplement or complementary medicine (not a food), and which has potential therapeutic applications. A regular intake can enhance the immune response of the human body, thereby increasing resistance to disease and, in some cases, cause regression of a disease state. AsGanoderma nutriceuticals are extracted from a very well known mushroom species, there is again little likelihood of toxicity and overdose. Although some degree of control by the regulatory agencies is both necessary and desirable, accessibility would be comparable with over-the-counter medicines. Nor are nutriceuticals to be confused with pharmaceuticals, which are almost invariably a defined chemical preparation, the specifications of which are listed in a pharmacopoeia. Pharmaceuticals possess medicinal properties, and are mainly used therapeutically for the treatment of specific medical conditions. Because many pharmaceuticals have a high potency, there is often a considerable potential for toxicity and, for this and other reasons (e.g. purity and sterility), their production and usage is subject to close control by regulatory agencies. There is a wide range of availability from over-the-counter medicines to strictly controlled dangerous drugs. The main features of functional foods, nutriceuticals, and pharmaceuticals were discussed in details by Chang and Buswell (1996).

Because the Ganoderma products are hot water extractable complex compounds rather than a single chemical component, they should be considered as a kind of nutriceutiacls or dietary supplements- not as functional food or as pharmaceutical. Scientific evidence is increasingly suggesting that the various compounds occurring in Ganoderma products generate an immense immune-response enhancement, through concerted effects. However, it should be emphasized that good and honest products obtained through an acceptable protocol for growing the materials and processing the products are of paramount importance in earning enduring public credibility and securing an expanding market in the future.

V. MARKET VALUE OF G. LUCIDUM PRODUCTS

It has been estimated (Chang and Buswell, 1999) that total world market value for Ganoderma-based natural healthcare products in 1995 was US$1,628.4 million. This figure was roughly estimated by different commercial sources to be US$ 350 million in China, US$215 million in Taiwan, US$600 million in Korea, US$300 million in Japan, US$ 91.2 million in Malaysia , US$60 million in Hong Kong, US$2.2 million in Singapore and US$10 million in other countries.

The current world production of G. lucidum is around 6,000 tonnes, more than half of which comes from China (Rai, 2003). Estimation (Lai, et al., 2004) of world production of this mushroom was about 5,000 tonnes in 2002, with 3,800 tonnes were produced in China. It should be noted that such huge demand cannot be met from wild collections, though it is available in the wild in the tropics. Moreover, it is not only a question of assured supply but also of genuine G. lucidum true-to-type, nature, stage of maturity of the fruiting bodies, and free from unwanted microbes, insects and chemicals, which is demanded by modern herbal industry as well as by the consumer. Therefore, many countries have developed a great variety of cultivation methods for this mushroom. World trade market value in this mushroom in 2003 is around US$2.5 billion.

VI. A PROTOCOL FOR QUALITY GANODERMANUTRICEUTICALS

It is apparent that Ganoderma-based products can serve as superior dietary supplements or nutriceuticals. The problem is that the products are so diverse, and there is currently a lack of standard protocols for guaranteeing a reproducible high-quality product. There are more than 100 brands of Ganoderma products in the world. The ingredients of the products can be either extracts from fruiting body or mycelium, by hot water or alcohol; or powders grounded from fruiting body or mycelium. The extracts or powders can be processed into different forms for administration, such as tablet, capsule, granule, and small packages. The most popular form is the capsule. There is an alarming concern that some of these products have no enduring public credibility. Consequently, there is serious need for improved quality control, which is essential both to increase and maintain consumer confidence, and to meet current and future standards set by the regulatory authorities.

It is essential that Ganoderma products be of good quality and free from potentially harmful substances. The following five guidelines are suggested as a protocol for obtaining quality Ganoderma products (nutriceuticals):

(1) GLP (Good Laboratory Practice): A known mushroom strain must be used; the source and nature of the strain culture should be properly maintained and preserved without contamination and degeneration.

(2) GAP (Good Agriculture Practice): Growth conditions must be known; the substrate should be free of heavy metals and have consistent levels of ingredients; the water should be free of contamination. The environmental conditions should include unpolluted air, a good sanitary growth area, and optimal temperature and humidity for growth and fruiting of strain used. Finally, fruiting bodies should be harvested at optimal maturity and free of moulds and insects.

(3) GMP (Good Manufacturing Practice): The parameters for processing must be known and maintained. The temperature, duration, and percentage of solvates used in extraction should be constantly monitored.

(4) GPP (Good Production Practice): The following tests must be conducted; laboratory tests should determine dosages effective for a particular health problem; a chemical analysis of the products to determine organic components and heavy metal content; microbial analysis to determine if the type and level of microorganisms present is within safe limits; and standardization of the formulation of the products.

(5) GCP (Good Clinical Practice): Medical practitioners must conduct high quality clinical trials including double-blind study.

VII. CONCLUSION

Although Lingzhi (Ganoderma lucidum) has been used for more than 2000 years in China as a herbal remedy, the general public still remains cautious about the therapeutic effects of these medicinal mushroom products. Recent experimental studies have helped substantiate the longstanding traditional claims and uses of lingzhi. However, the problem is that these products are so diverse, because the bioactive composition of different sources of fruiting bodies is highly variable depending on the species, the growing environment, the cultivation methods, as well as the extraction procedures. There are currently no regulations assuring standard protocols for guaranteeing reproducible high product quality. If these Lingzhi products can be manufactured according to the proposed protocol in Section VI, thenGanoderma-based products should be able to serve as superior dietary supplements or mushroom nutriceuticals. Obviously, there is serious need for improved quality control. This improved quality control is essential both to increase and maintain consumer confidence, and to meet current and future standards set by the regulatory authorities.

Because the active ingredient of G. lucidum products is not single, chemically defined compound as used in conventional drugs treatments, these products do not fulfil the requirement to be classified as pharmaceuticals. Their fruiting bodies are usually thick, corky and tough, and in this form, they are not suitable as dietary food. Therefore, G. lucidum products should be considered as a type of nutriceutical or as a dietary supplement as described by Chang and Buswell (1996 and 2003). However, it should be emphasised again that good quality and trustworthy products are of paramount importance in earning enduring public credibility and securing an expanding market in the future.