ΕΓΚΥΡΗ ΕΠΙΣΤΗΜΟΝΙΚΗ ΠΛΗΡΟΦΟΡΙΑ

Medicinal Properties of Substances Occurring in Higher Basidiomycetes Mushrooms: Current Perspectives

International Journal of Medicinal Mushrooms, Vol 1, 47-50, (1999)

Solomon P. Wasser, Alexander L. Weis

International Center for Cryptogamic Plants and Fungi, Institute of Evolution, University of Haifa, Israel International Myko Biologics, Inc., San Antonio, TX, USA

M.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, Kiev, Ukraine

CARDIOVASCULAR AND HYPERCHOLESTEROLEMIA EFFECTS

The major cause of death in the Western countries is coronary artery disease. A primary risk factor is hypercholesterolelmia, which contributes to hardening of the arteries. In humans, 50% or more of the total cholesterol is derived from de novo synthesis (Rosenfeld, 1989; Steinberg et al., 1989). Clinical intervention studies have demonstrated the therapeutic importance of correcting the hypercholesterolemia. The initial step in lowering cholesterol is a special diet low in fat and saturated fatty acids and rich in crude fibers.

Drug therapy is the next step. The best-known pharmacologic agent that was approved in 1987 is lovastatin (mevinolin) and its analogues (Endo, 1988). This low molecular weight substance is the competitive inhibitor of HMG Co A reductase, the key enzyme in cholesterol metabolism that catalyzes the reduction of HMG CoA into mevalonate.

The best known organisms for potential producers of lovastatin from edible higher Basidiomycetes mushrooms are species of the genus Pleurotus
(Gunde-Cimerman et al;., 1993a,b; Gunde-Cimerman and Cimerman, 1995). The presences of the inhibitor was determined in four species:P. ostreatus, P. cornucopiae, P. eryngii, and P. sapidus. The highest content of lovastatin was found in the fruiting bodies of the P. ostreatus. The appearance of the inhibitor during the development of the fruiting bodies was found in the vegetative mycelium, in the primordial, and in different parts of fruiting bodies of different sizes; less lovastatin was found in stipes when compared with pileus (fig. 5) or in nature stages in the lamellae and basidioiospores (Gund-Cimerman and Cimerman, 1995)

It was shown that lovastatin at the beginning of mushroom growth is unfortunately distributed in small fruit bodies, and there are no substantial differences between the pileus and the stipe. During fruit body growth, the majority of lovastatin is first transferred to the pileus and later the lamellae. Mature fruiting bodies have a diameter of approximately 15 cm and disperse large amounts of basidiospores. These contain less lovastatin in the lamellae when compared with the smaller, 10 cm diameter; less mature mushrooms this transfer is still incomplete (Gunde-Cimerman and cimerman, 1995).

In a series of experiments conducted by Bobek et al. (1991a,b, 1993), it has been found that the addition of 2% to 4% of P. ostreatus to the hyperlipidemic diet efficiently prevented accumulation of C and triacly-glycerols in both sera and livers of animals with exogenous, endogenous, or genetically induced hyperlipidemia. VLDL cholesterol had the dominant role in the reduction of serum C up to 80% induced by the whole mushroom pr its water and 30% of ethanol extracts. The authors attributed this effect to the fiber pulp complex of the oyster mushroom, which limits the resorption of C and gastrointestinal tract, and to an undefined substance which influences, as well, metabolisms outside the phase of resorption. (Bobek et al., 1991a,b,1993). Ryong et al. (1989) have tested alcohol and water extracts of 20 different edible mushrooms in tissue primary culture of cells isolated from atherosclerotic action. Four mushrooms also decreased atherogenic effects by 20% to 40% in sera collected from coronary heart disease patients. In these experiments the effect of dietary fibers was excluded and the efficiency was attributed to an unknown active component (Ryong et al., 1989). Authors suggest that this unknown substance is lovastatin, which can be found in high quantities in the fruiting bodies of various cultured Pleurotus species. Therefore, mature fruiting bodies of P. ostreatus could be recommended for consumption as a natural cholesterol-lowering agent. Lovastatin appears early in the life cycle of the mushroom, in the mycelia from which primordial are being formed.

It is known that Lentinus edodes is able to lower BSC via a factor known as eritadenine (also known as “Lentinacin” or “Lentysine”) was isolated from an 80% ethanol extract of Shiitake mushroom fruiting bodies by absorption on a Amberlite IR-120 (H+) column, followed by elution with 4% NH 4 OH (chibata et al., 1969). A crystalline product had the following properties: mp 261-263° C, C₉H₁₁O₄N₅, MW 253, \ 261 .5 nm (E=14,508)m, Nasapt mp 266 -268 C (decomposition), {a} D + 45.5 (C=1, H2O). Hydrolyses in 6N HCI at 110 C for 72h yielded glycine and new amino acid. Eritadenine apparently reduces serum cholesterol in mice. Its action is not inhibition of cholesterol biosynthesis, but rather the acceleration of excretion of ingested cholesterol and its metabolic decompositions (Makita et al., 1972, cited in Mizuno, 1995a,b).

Apparently, Eritadenine reduces BSC in mice, not by the inhibition of cholesterol biosynthesis but by the acceleration of the excretion of ingested cholesterol and its metabolic decomposition (S. Suzuki and Oshima, 1974, 1976; Higushi et al., 1978; Yagishita et al., 1977, 1978). Eritadenine has been shown to lower blood levels of cholesterol and lipids in animals (Yamamura and Cochran, 1976) Added to the diet of rats, Eritadenine (0.005%) caused a 25% decrease in total cholesterol in as little as one week (Chibata et al., 1969) The cholesterol-lowering activity of this substance is more pronounced in rats fed a high fat diet than those on a low-fat diet (Rokujo et al., 1969). Although feeding studies with humans have indicated a similar effect, further systematic research is needed. S. Suzuki and Ohshima (1974, 1976) have shown that dietary shiitake mushroom lowered BSC levels. Various studies have confirmed (Kimoto et al., 1976;m Kabir and Kmura, 1989) that shiitake mushroom can lower both blood pressure and free cholesterol in plasma, as well as accelerate accumulation of lipids in the liver, by removing them from circulation.

Nucleic acid compound in L. edodes extract have been shown a strong platelet agglutination inhibitive effect (antithrombotic activity). An extract of L. edodes with antithrombotic activity was studied by high-performance liquid chromatography (HPLC). ATP, ADP, UDPG, 5’-GMP, 5’UMP, 5’-CMP, 5’-AMP, uridine, eritadenine, and deoxylentinacin were identified. More antithrombotic activity was shown by 5’-AMP, 5’GMP, eritadenine, and deoxylentinacin (Hokama and Hokama, 1981; Kabir and Kimura, 1989).

Auriculari auricula-judae has shown the following effects and activities in studies on mice and rats: anticoagulation, lowered total cholesterol, triglyceride, and lipid levels (Chen, 1989; Sheng and Chen, 1990); and antiaggregatory activity on blood platelets, which might make it beneficial for coronary heart disease (Agarwal et al., 1982). This mushroom is traditionally used as an immune tonic.

Tremella fuciformis polysaccharides and spore extracts have demonstrated antilipemic activity. T. fuciformis lowered the LDL- cholesterol in rats fed the preparation, which also contained butter sugar, and egg yolks, by 30% over controls (Nakajima, et al., 1989). T. fuciformis polysaccharides prolonged thrombus formation, reduced thrombus size, reduced blood platelet adherence, blood viscosity, and positively influenced other blood coagulation parameters of survival in mice (Sheng and Chen, 1990).

Animal studies on Armillriella mellea demonstrated that it decreases heart rate, reduces peripheral and coronary vascular resistance, and increases cerebral blood flow (Chang and But, 1986). AMG-1-a compound isolated from this mushroom exhibits a cerebral-protective effect (Watanabe et al.,1990), and increases coronary oxygen efficiency without altering blood pressure (Y. Zhang et al., 1985)

Grifola frondosa reduced blood pressure in rats without changing plasma HDL levels (Kyoto et al., 1988). Adachi and coworkers (1988) found a blood pressure lowering effect with the powder of G. frondosa fed to hypertensive rats in their normal food. The effect was of rapid onset, short lived and dose dependent. An aqueous extract of G. frondosa also reduced serum cholesterol levels in rats (Mizuno, 1977).

A glycoprotein obtained from submerged cultured mycelial biomass of Trametes sp. showed activity (in animal and in vitro test). Against experimental hypertension and thrombosis. The protein inhibits blood platelet aggregation and is antihyperlipemic and anti arrhythmic. (Ikuzawa, 1985)Trametes versicolor has been shown to lower serum chooesterol in animals (Yagishita et al., 1977). PKS ( beta-glucan protein) from T. versicolor has been used in clinical studies. Tsukagoshi et al (1984) have reported that PSK causes a significant decrease in LDL cholesterol in hyperlipidemia (stage 11a ) patients.

The Volvariella volvacea cardioactive proteins are know to lower blood pressure (Cochran, 1978; Yao et al., 1998).

S.P. Wasser

Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides

Received: 2 May 2002 / Revised: 17 June 2002 / Accepted: 20 June 2002 / Published online: 10 September 2002 © Springer-Verlag 2002

Abstract
The number of mushrooms on Earth is esti­mated at 140,000, yet maybe only 10% (approximately 14,000 named species) are known. Mushrooms comprise a vast and yet largely untapped source of powerful new pharmaceutical products. In particular, and most impor­tantly for modern medicine, they represent an unlimited source of polysaccharides with antitumor and immuno- stimulating properties. Many, if not all, Basidiomycetes mushrooms contain biologically active polysaccharides in fruit bodies, cultured mycelium, culture broth. Data on mushroom polysaccharides have been collected from 651 species and 7 infraspecific taxa from 182 genera of higher Hetero- and Homobasidiomycetes. These poly­saccharides are of different chemical composition, with most belonging to the group of P-glucans; these have P-(1—>3) linkages in the main chain of the glucan and additional P-(1—6) branch points that are needed for their antitumor action. High molecular weight glucans appear to be more effective than those of low molecular weight. Chemical modification is often carried out to im­prove the antitumor activity of polysaccharides and their clinical qualities (mostly water solubility). The main procedures used for chemical improvement are: Smith degradation (oxydo-reducto-hydrolysis), formolysis, and carboxymethylation. Most of the clinical evidence for antitumor activity comes from the commercial polysac­charides lentinan, PSK (krestin), and schizophyllan, but polysaccharides of some other promising medicinal mushroom species also show good results. Their activity is especially beneficial in clinics when used in conjunc­tion with chemotherapy. Mushroom polysaccharides pre­vent oncogenesis, show direct antitumor activity against

S.P. Wasser (S)

Institute of Evolution, University of Haifa, Mt. Carmel, Haifa 31905, Israel e-mail: spwasser@research.haifa.ac.il Tel.: +972-4-8249218, Fax: +972-4-8288197

S.P. Wasser

N.G. Kholodny Institute of Botany,

National Academy of Sciences of Ukraine, Tereshchenkivska str. 2, Kiev 01001, Ukraine

various allogeneic and syngeneic tumors, and prevent tu­mor metastasis. Polysaccharides from mushrooms do not attack cancer cells directly, but produce their antitumor effects by activating different immune responses in the host. The antitumor action of polysaccharides requires an intact T-cell component;
their activity is mediated through a thymus-dependent immune mechanism. Practi­cal application is dependent not only on biological prop­erties, but also on biotechnological availability. The present review analyzes the pecularities of polysaccha­rides derived from fruiting bodies and cultured myceli­um (the two main methods of biotechnological produc­tion today) in selected examples of medicinal mush­rooms.

Introduction

For millennia, mushrooms have been valued by human­kind as an edible and medical resource. A number of bio­active molecules, including antitumor substances, have been identified in many mushroom species. Polysaccha­rides are the best known and most potent mushroom- derived substances with antitumor and immunomodulat- ing properties (Mizuno 1996, 1999a, b, 2002; Lorenzen and Anke 1998; Borchers et al. 1999; Ooi and Liu 1999; Wasser and Weis 1999;
Tzianabos 2000; Reshetnikov et al. 2001). Historically, hot-water-soluble fractions (de­coctions and essences) from medicinal mushrooms, i.e., mostly polysaccharides, were used as medicine in the Far East, where knowledge and practice of mushroom use primarily originated (Hobbs 1995, 2000). Mushrooms such as Ganoderma lucidum (Reishi), Lentinus edodes (Shiitake), Inonotus obliquus (Chaga) and many others have been collected and used for hundreds of years in Korea, China, Japan, and eastern Russia. Those practices still form the basis of modern scientific studies of fungal medical activities, especially in the field of stomach, pros­tate, and lung cancers. It is notable and remarkable how reliable the facts collected by traditional eastern medicine are in the study of medicinal mushrooms (Ying et al.

1987; Hobbs 1995, 2000; Wasser and Weis 1997a, b, 1999; Stamets 2000).

Ikekawa et al. (1969) published one of the first scien­tific reports on antitumor activities of essences obtained from fruiting bodies of mushrooms
belonging to the fami­ly Polyporaceae (Aphyllophoromycetideae) and a few other families, manifested as host-mediated activity against grafted cancer – such
as Sarcoma 180 – in ani­mals (Ikekawa et al. 1982, 1992; Ikekawa 2001). Soon thereafter the first three major drugs were developed from medicinal
mushrooms. All three were polysaccharides, specifically P-glucans: krestin from cultured mycelial biomass of Trametes versicolor (Turkwey Tail),
lentinan from fruiting bodies of L. edodes, and schizophyllan from the liquid cultured broth product of Schizophyllum com­mune (Split
Gill). For almost 40 years, medicinal mush­rooms have been intensively investigated for medicinal effects in in vivo and in vitro model systems, and many
new antitumor and immunomodulating polysaccharides have been identified and put into practical use (Mizuno 1996, 1999a; Wasser and Weis 1999; Ikekawa
2001).

Biologically active polysaccharides are widespread among higher Basidiomycetes mushrooms, and most of them have unique structures in different species.
More­over, different strains of one Basidiomycetes species can produce polysaccharides with different properties. For ex­ample, the proteoglucan krestin
was developed in Japan from the strain Trametes (Coriolus) versicolor CM-101, whereas polysaccharide-peptide (PSP) in China was de­veloped in
submerged culture of the strain Cov-1 of the same species. Both proteoglucans have the same polysac­charide component but with different protein molecules
bound to the polysaccharide (Hiroshi and Takeda 1993).

In the present review, antitumor and immunomodulat- ing polysaccharides from higher Basidiomycetes mush­rooms are analyzed. More attention is given to
their common features than to specific pecularities. The re­view summarizes the general state of knowledge in the area of biodiversity of mushrooms and
their polysaccha­rides; the chemical structure of polysaccharides and its connection with their antitumor activity, including possi­ble ways of chemical
modification; results of experimen­tal testing and clinical use of antitumor or immunostimu- lating polysaccharides; possible mechanisms of their
bio­logical action; and, finally, the difference in polysaccha­ride fraction composition in fruiting bodies and pure cul­ture mycelia in selected examples
of studied medicinal mushrooms.

The vast quantity and diversity of mushrooms with antitumor polysaccharides

The total number of described fungi of all kinds is cur­rently at least 80,060 species; a figure based on the total derived from addition of the numbers of species in each genus given in the latest edition of the Dictionary of the Fungi (Kirk et al. 2001). This figure includes all organ­isms traditionally studied by mycologists: slime molds, chromistan fungi, chytridiaceous fungi, lichen-forming fungi, filamentous fungi, molds, and yeasts.

By the term ‘mushrooms’, we generally mean the defi­nition of Chang and Miles (1992): ‘a macrofungus with a distinctive fruiting body which can be either hypogeous or epigeous, large enough to be seen with the naked eye and to be picked by hand’. The number of filamentous fungi that are mushrooms in the sense of this definition deduced from the Dictionary of the Fungi is at least 14,000 and perhaps as many as 22,000 (Hawksworth 2001). However, the real number of such species on Earth is undoubtedly much higher. Two main reasons for the real number being higher are (1) the great number of as yet undescribed spe­cies and (2) the fact that many morphologically defined mushroom ‘species’ prove to be assemblages of several biological species (Hawksworth 2001).

Most new mushrooms are being discovered in the tro­pics, especially those species forming ectomycorrhizas with native trees. In various tropical areas, 22-55% (in some cases up to 73%) of mushroom species have proved to be undescribed (Hawksworth 2001). An analy­sis of the localities from which fungi new to science have been described and catalogued in the Index of Fungi in the 10 years from 1990 to 1999 revealed that about 60% of all newly described fungi are from the tro­pics (Hawksworth 1993, 2001), and this is also the case for mushrooms, although new species continue to be dis­covered in Europe and North America.

Studies of compatability and molecular sequences be­tween mushrooms previously considered to be the same species on morphological grounds revealed ‘cryptic spe­cies’, i.e., populations functioning as separate biological species but covered by a single scientific name. A single morphologically defined species may consist of 20 or more biological species (Hawksworth 2001).

Taking all this into account, recent estimates of the number of fungi on Earth range from 500,000 to 9.9 mil­lion species, of which only 80,060 are named.
A working figure of 1.5 million species is generally accepted, and new data suggests that this is not unreasonable. The number of mushrooms on Earth is estimated at 140,000, of which maybe only 10% are known. Meanwhile, of those ~14,000 species that we know today, about 50% are considered to possess
varying degrees of edibility, more than 2,000 are safe, and about 700 species are known to possess significant pharmacological properties (Chang 1999;
Wasser and Weis 1999; Reshetnikov et al. 2001). Thus, it is clear that mushrooms represent a major and as yet largely untapped source of powerful new pharmaceutical products.

Higher Basidiomycetes mushrooms represent an un­limited source of antitumor or immunostimulating poly­saccharides. All main taxonomic mushroom groups have been investigated for biologically active polysaccha­rides, and most of them possess such substances. At least 651 species and 7 infraspecific taxa representing 182 genera of Hetero- and Homobasidiomycetes mush­rooms contain antitumor or immunostimulating poly­saccharides, as is evident from Table 1
(adapted from

Reshetnikov et al. 2001). Naturally collected or artifi­cially growing fruit bodies, pure culture mycelia, and culture filtrate (culture broth) all contain
biologically active polysaccharides.

Procedures for polysaccharide purification

After two decades of intensive research on medicinal mushrooms, Mizuno and his co-workers in Japan devel­oped reliable procedures for successful
extraction, frac­tionation and purification of polysaccharides from fruit­ing bodies or culture mycelia. In general, this scheme in­volves elimination of
low molecular weight substances from mushroom material using 80% ethanol, followed by three successive extractions with water (100°C, 3 h), 2% ammonium
oxalate (100°C, 6 h), and 5% sodium hydroxide (80°C, 6 h) (Mizuno 1996, 1999a).

The first extraction results in water-soluble polysac­charides, the other two in water-insoluble polysaccha­rides. Polysaccharides extracted are further
purified us­ing a combination of techniques, such as ethanol concen­tration, fractional precipitation, acidic precipitation with acetic acid, ion-exchange
chromatography, gel filtration, and affinity chromatography. Basically, ion-exchange chromatography through DEAE-cellulose columns sepa­rates neutral
polysaccharides from acidic ones. Neutral polysaccharides are then separated into a-glucans (ad­sorbed fraction) and p-glucans (non-absorbed fraction) with
the help of gel filtration and affinity chromatogra­phy. The same procedure with acidic polysaccharides (after elution with 1 M NaCl) yields purified
polysaccha­rides (Mizuno 1999a).

General schemes for fractional preparations of poly­saccharides from mushrooms are shown schematically in Fig. 1. It should be noted that the particular
fractionation procedure scheme depends in each case on the polysac­charide composition of the extracted material.

Structural composition of antitumor polysaccharides in mushrooms

Polysaccharides belong to a structurally diverse class of macromolecules, polymers of monosaccharide residues joined to each other by glycosidic linkages.
It is note­worthy that, in comparison with other biopolymers such as proteins and nucleic acids, polysaccharides offer the highest capacity for carrying
biological information be­cause they have the greatest potential for structural vari­ability. The nucleotides in nucleic acids and the amino acids in
proteins can interconnect in only one way whereas the monosaccharide units in polysaccharides can interconnect at several points to form a wide variety of
branched or linear structures (Sharon and Lis 1993). This enormous potential variability in polysaccharide structure gives the necessary flexibility to the
precise regulatory mechanisms of various cell-cell interactions in higher organisms.

Fig. 1
Fractional preparation of polysaccharides from mushrooms [adapted from Mizuno (1999a) with modification]

Mushroom polysaccharides are present mostly as glu- cans with different types of glycosidic linkages, such as (1^3), (1^6)-p-glucans and (1^3)-a-glucans,
but some are true heteroglycans. The others mostly bind to protein residues as PSP complexes (PSPC; Gorin and Barreto-Berger 1983). The main source of
antitumor polysaccharides appears to be fungal cell walls that con­sist of polysaccharides. However, chitin and chitosan (fungal chitin) have no antitumor
activity (Mizuno et al. 1995b).

p-D-glucan is a polysaccharide yielding exclusively D-glucose upon acid hydrolysis (Mizuno 1996, 1999a). As for structure of schizophyllan tertiary
conformation, active p-D-glucan has a triple-strand right-winding struc­ture (Marchessault et al. 1977). Acidic glucuronoxylo- mannan isolated from the
fruit body of Tremella fucifor- mis was also demonstrated as having a left-handed, three­fold helical backbone conformation (Yui et al. 1995).

Besides the well-known antitumor p-(1^3)-glucans, a wide range of biologically active glucans with other structures have been described. These
polysaccharides have linear or branched molecules in a backbone com­posed of a- or p-linked glucose units, and they contain side chains that are attached
in different ways. Hetero- glucan side chains contain glucuronic acid, xylose, ga­lactose, mannose, arabinose, or ribose as a main compo­nent or in
different combinations.

Glycans, in general, are polysaccharides containing units other than glucose in their backbone. They are clas­sified as galactans, fucans, xylans, and
mannans by the individual sugar components in the backbone. Hetero- glycan side chains contain arabinose, mannose, fucose, galactose, xylose, glucuronic
acid, and glucose as a main component or in different combinations.

A wide range of antitumor or immunostimulating polysaccharides of different chemical structure from higher Basidiomycetes mushrooms has been investigat­ed;
the main types are presented in Table 2.

The number of antitumor active fractions in the fruit bodies of mushrooms is remarkably high. One example can be seen in an analysis of polysaccharides of
fruit bod­ies of Pleurotus pulmonarius (= P. sajor-caju): 16 poly­saccharide fractions from 21 extractions demonstrated different levels
of antitumor activity (Zhuang et al. 1993, Table 3).

Table 2
Chemical structure of antitumor and immunostimulat- ing polysaccharides of higher Basidiomycetes

Mannogalactoglucan

Polysaccharide	Species	References
Glucans
α-(1→3)-glucan	Armillariella tabescens	Kiho et al. 1992a
Linear α-(1→3)-glucan	Amanita muscaria	Kiho et al. 1994
Agrocybe	aegerita	Yoshida et al. 1996
α-(1→4)-; β-(1→6)-glucan	Agaricus blazei	Fujimiya et al. 1998b
α-(1→6)-; α-(1→4)- glucan	Agaricus blazei	Mizuno et al. 1990a
β-(1→6)-glucan	Lyophyllum decastes	Ukawa et al. 2000
	Armillariella tabescens	Kiho et al. 1992a
β-(1→6)-; β-(1→3)-glucan	Agaricus blazei	Mizuno et al. 1990a
Grifola frondosa	Nanba et al. 1987
β-(1→6)-; α-(1→ 3)-glucan	Agaricus blazei	Mizuno et al. 1990a
β-(1→3)-glucuronoglucan	Ganoderma lucidum	Saito et al. 1989
Mannoxyloglucan	Grifola frondosa	Mizuno et al. 1986
Galactoxyloglucan	Hericium erinaceus	Mizuno 1999b
Xyloglucan	Grifola frondosa	Mizuno et al. 1986
Polyporus confluens	Mizuno et al. 1992
Pleurotus pulmonarius	Zhuang et al. 1993
Xylogalactoglucan	Inonotus obliquus	Mizuno et al. 1999a
Pleurotus pulmonarius	Gutiérrez et al. 1996
	Pleurotus cornucopiae	Kim et al. 1994
	Ganoderma lucidum	Cho et al. 1999
	Agaricus blazei
Galactomannoglucan	Flammulina velutipes	Ikekawa et al. 1982
	Hohenbuehelia serotina	Mizuno et al. 1994
Leucopaxillus giganteus	Mizuno et al. 1995a
Arabinoglucan	Ganoderma tsugae	Zhang et al. 1994b
Riboglucan	Agaricus blazei	Cho et al. 1999
Glycans
Arabinogalactan	Pleurotus citrinopileatus	Zhang et al. 1994a
Glucogalactan	Ganoderma tsugae	Wang et al. 1993
Fucogalactan	Sarcodon aspratus	Mizuno 2000
α-(1→6)-mannofucogalactan	Fomitella fraxinea	Cho et al. 1998
Fucomannogalactan	Dictyophora indusiata	Hara et al. 1991
Mannogalactan	Pleurotus pulmonarius	Zhuang et al. 1993
Mannogalactofucan	Grifola frondosa	Zhuang et al. 1994a
Xylan	Hericium erinaceus	Mizuno 1999b
Glucoxylan	Hericium erinaceus	Mizuno 1999b
Pleurotus pulmonarius	Zhuang et al. 1993
Mannoglucoxylan	Hericium erinaceus	Mizuno 1999b
α-(1→3)-mannan	Dictyophora indusiata	Ukai et al. 1983
Glucomannan	Agaricus blazei	Hikichi et al. 1999
β-(1→2)-; β-(1→3)-glucomannan	Agaricus blazei	Tsuchida et al. 2001
	Mizuno et al. 1999b
Galactoglucomannan	Lentinus edodes	Fujii et al. 1979

The most antitumor-active water-soluble fractions from P. pulmonarius are Fi_o-a protein-containing xyloglu- can with Man:Gal:Xyl:Glc in
the polysaccharide at a mo­lar ratio 2:12:42:42, and FA-2 protein-containing manno- galactan consisting of Xyl:Man:Gal (9:35:56 molar ratio). The most
antitumor-active water-insoluble polysaccha­rides are FII-1 protein-containing xylan; FIII-1a protein- containing glucoxylan consisting of Glc:Xyl (40:44
molar ratio), and FIII-2a protein-containing xyloglucan consist­ing of Xyl:Glc (36:62 molar ratio).

Correlation of structure and antitumor activities of mushroom polysaccharides

Polysaccharides with antitumor action differ greatly in their chemical composition and configuration, as well as their physical properties. Antitumor
activity is exhibited by a wide range of glycans extending from homopoly­mers to highly complex heteropolymers (Ooi and Liu

1999. . Differences in activity can be correlated with solubility in water, size of the molecules, branching rate and form. Although it is difficult to
      correlate the struc­ture and antitumor activity of complex polysaccharides, some relationships can be inferred.

Table 3
Structure and antitumor activity of Pleurotus pulmonarius fruit bodies polysaccharides against Sarcoma 180 in mice (after Zhuang et al. 1993). FI-FA Water-soluble, FII-FIII water-insoluble polysaccharides

Polysaccharid	MW x103	Protein (%)	Total sugar (%)	Component sugar (molar %)				Tumor inhibition ratio at 3 weeks (%)
				Glc	Xyl	Man	Gal
FIo-a	278	24.1	75.6	43.7	42.3	1.9	11.8	84.8
FIo-a-a	420	23.5	69.5	24.1	72.5	2.7	0.7	53.1
FIo-a-P	68	26.3	67.0	53.5	27.2	1.6	17.7	49.8
FIo-b-a	10	42.1	52.6	56.0	40.7	3.3	–	59.4
FIo-b-P	24	6.9	84.6	–	16.2	–	83.8	31.7
FA-1	11	27.5	67.7	71.6	5.5	–	22.9	48.7
FA-2	115	16.2	76.1	–	9.4	34.6	56.0	74.6
FA-3	10	75.3	22.5	50.0	14.9	13.1	22.0	34.5
FII-1	19	20.5	62.2	5.2	91.2	–	3.6	90.8
FII-2	17	44.1	50.5	9.2	86.2	–	4.6	8.0
FII-3	13	49.0	50.1	2.9	80.5	–	16.6	8.4
FIII-1a	87	70.5	15.4	39.8	43.7	7.8	8.7	76.9
FIII-1b	24	96.8	3.0	–	97.9	–	2.1	51.6
FIII-2	627	2.8	69.6	33.9	40.3	–	1.9	84.5
FIII-2a	700	2.5	68.8	62.2	35.5	–	2.3	100.0
FIII-2b	190	4.5	74.8	30.9	69.1	–	–	84.6

It is obvious that structural features such as P-(1— 3) linkages in the main chain of the glucan and additional P-(1——6) branch points are needed for
antitumor action. P-glucans containing mainly (1—6) linkages have less activity. High molecular weight glucans appear to be more effective than those of
low molecular weight (Mizuno 1996, 1999a, b). However, obvious variations in antitumor polysaccharides have also been noted. Antitu­mor polysaccharides may
have other chemical structures, such as hetero-P-glucans (Mizuno et al. 1995b), hetero- glycan (Gao et al. 1996b), P-glucan-protein (Kawagishi

et al. 1990), a-manno-P-glucan (Mizuno et al. 1995b), a-glucan-protein (Mizuno et al. 1995b) and heteroglycan- protein complexes (Zhuang et al. 1993;
Mizuno et al.

1996).

A triple-helical tertiary conformation of medicinal mushroom P-(1— 3)-glucans is known to be important for their immune-stimulating activity. When lentinan
was denatured with dimethyl sulfoxide, urea, or sodium hydroxide, tertiary structure was lost while primary structure was not affected, but tumor
inhibition proper­ties were lowered with progressive denaturation (Maeda et al. 1988). The same results, which confirm the corre­lation between antitumor
activity and triple helix struc­ture, were obtained upon investigation of schizophyllan (Yanaki et al. 1983, 1986).

Mushroom P-(1—3)-glucans exhibit a variety of bio­logical and immuno-pharmacological activities, and many of these activities, such as macrophage nitrogen
oxide synthesis, and limulus factor G activation, are de­pendent on the triple-helix conformation, while others are independent of this conformation, e.g.,
synthesis of interferon-y and colony stimulating factor (Yadomae

1999. , thus indicating that the a-(1—3)-mannan back­bone structure is of more importance than the tertiary structure of the molecule.

Unlike P-(1— 3)-glucans with medicinal properties that are strongly dependent on high molecular weight, ranging from 500 to 2,000 kDa (Mizuno 1996),
a-(1—3)- glucuronoxylomannans, which are characteristic of Jelly mushrooms, are not strongly dependent on molecular weight. Thus, Gao and co-workers
(1996a) reported that acidic hydrolysate fractions of T. fuciformis fruit bodies contain glucuronoxylomannans with molecular weights of from 53 to
1 kDa that induce human monocytes to pro­duce interleukin-6 as efficiently as non-hydrolyzed het­eropolysaccharide. This indicates that the activity may be
due to the common structure of the a-(1—3)-mannan backbone; differences in molecular weight had no obvi­ous influence on the activity of the heteroglycans
(Gao et al. 1996b).

Activation of mushroom polysaccharides by chemical modification

Different approaches to improving antitumor activity of mushroom polysaccharides by chemical modification have been described in the literature. The most
success­ful schemes for chemical improvement of mushroom polysaccharides have been developed for Ganoderma lucidum, Grifola frondosa and Leucopaxillus giganteus (= Tricholoma gigantea). These schemes include two main procedures: modification of mushroom polysaccha­rides by
Smith degradation (oxydo-reducto-hydrolysis) and activation by the method of formolysis (Mizuno 1996, 1999a; Mizuno et al. 1996). Five polyaldehydes and
ten polyalcohols were prepared by the Smith degra­dation method from five polysaccharide fractions previ­ously obtained from G. frondosa liquid
culture myceli­um. For this reason, original polysaccharide solutions were first oxidised to polyaldehydes by 0.1 M NaIO₄ in darkness, then
converted into polyalcohols by reduction of NaBH₄ in alkaline medium adjusted to pH 8 with 2 M NaOH, and hydrolysed by 1 M H₂SO ₄ at room tempera­ture (Zhuang et al. 1994b). Chemical activation of mush­room polysaccharides by the method of formolysis in­volves degradation
of polysaccharides by formic acid in 99% HCOOH solution; the reaction solution is then pre­cipitated with 99% EtOH, and one-half of the precipitate is
lyophilized after dialysis, while the other part is dis­solved in hot water and additional fractions obtained by alcohol precipitation (Zhuang et al.
1994b). Four for- mylated polysaccharides and four formolysis products of polysaccharides were prepared by this method from four polysaccharide fractions
obtained from G. frondosa liquid culture mycelium. Although two of the original polysaccharides had no activity, their polyaldehyde poly- ol,
formylated, and formolysis derivatives showed signif­icant activity. Polyaldehyde, and polyol-polysaccharides prepared from a polysaccharide with low
antitumor ac­tivity showed activity higher than the original polysac­charide (Zhuang et al. 1994b). As all original polysac­charide fractions showing
elevated activity levels by chemical modification were p-glucan or xyloglucan, it was suggested that the sugar chain was changed or elimi­nated upon
treatment, resulting in improved solubility and activity (Mizuno 1999a).

Carboxymethylation is the other chemical method used to transform p-glucans into a water-soluble form. For example, whole fruit bodies of Pleurotus ostreatus or their stipes homogenate were treated with 0.15 M so­dium hydroxide solution at 95°C for 2 h. The residue collected was
washed with water until neutral, then sus­pended in 0.06% sodium chlorite solution, adjusted to pH 4.5 with acetic acid, and stirred for 6 h at 50°C. The
polysaccharide obtained was p-(1^3)-linked glu- can, with every fourth glucopyranosyl residue substi­tuted at 0-6 with single D-glucopyranosyl groups. The
heterogeneous etherification of the particulate glucan with monochloroacetic acid (C₂H₃ClO₂) in alkaline medium gave the
sodium salt of the water-soluble O-(carboxymethyl) glucan derivative (Kuniak et al. 1993; Karacsonyi and Kuniak 1994). Carboxymethylated glucan from P. ostreatus (pleuran) exhibited immuno­modulatory effects, especially increased phagocytic activity (Paulik et al. 1996).

In a similar manner, a water-insoluble, alkali-soluble linear a-(1^3)-glucan obtained from fruiting bodies of Amanita muscaria and Agrocybe aegerita had little or no antitumor effect, while their carboxymethylated products showed potent antitumor activity (Kiho et al. 1994;
Yoshida et al. 1996).

Chemical modification of branched mushroom poly­saccharides resulting in side-chain reduction can be de­veloped not only by Smith degradation but also by
enzy­matic reactions. A novel linear polysaccharide compris­ing a-(1^4)-bonded a-D-glucose units of a molecular weight of 500-10,000 kDa was developed
after succes­sive enzymatic treatments of submerged culture broth with amylase, cellulase, and protease (Kosuna 1998).

Linear low molecular weight a-(1^4)-glucans ob­tained after enzymatic reduction of side chains and pro­tein component (active hexose correlated compounds –
AHCC) were demonstrated as having immunomodulato­ry and anticancer properties (Ghoneum et al. 1995; Matsushita et al. 1998). In 1992, a trial was done in
Japan to evaluate the preventive effect of AHCC against recurrence of hepatocellular carcinoma following surgi­cal resection (Kidd 2000).

Sulfated homo- and heteropolysaccharides possessing antiviral activity are widespread in algae, especially in sea algae (Schaeffer and Krylov 2000), but do
not natu­rally occur in higher Basidiomycetes mushrooms. Chem­ically sulfated schizophyllans with different sulfur con­tent were obtained from
p-(1^3)-glucan produced by

Schizophyllum commune
(Itoh et al. 1990; Hirata et al. 1994). It was suggested that the sulfur content in schiz- ophyllan is more important in inhibiting growth of hu­man
immunodeficiency virus (HIV) than the molecular weight or the nature of the sugar component (Itoh et al. 1990; Hobbs 1995). The medicinal tests indicate
that sul- fated schizophyllan with a sulfur content of 5% can be useful as an anti-HIV agent for treatment of HIV-infect­ed hemophiliacs (Hirata et al.
1994; Hobbs 1995).

It is important to realize that chemical modification is necessary in many cases to improve not only the antitu­mor activity of mushroom polysaccharides,
but also their clinical qualities, most importantly water solubility and the ability to permeate stomach walls after oral inges­tion.

Testing antitumor and immunomodulating activity of mushroom polysaccharides

Initial data on the antitumor activity of mushroom ex­tracts was circumstantial and in no way solid and reli­able. However, many indirect data that were
properly collected and processed gave good evidence for the ben­eficial effects of mushrooms on human health. A good example is an epidemiological study in
Nagano Prefec­ture, Japan, where activity was monitored for several de­cades. Researchers demonstrated that the cancer death rate of farmers whose main
occupation was producing Flammulina velutipes (a well known medicinal mush­room in Japan) was remarkably lower than that of the general population
in the Prefecture (Ikekawa 1995, 2001). Another similar observation in Brazil brought about extensive studies – and popularity – of Agaricus blazei (see below).

I would like to emphasize the principal points of anti­tumor and immunomodulating effects of mushroom poly­saccharides. Most important among them are: (1)
preven­tion of oncogenesis by oral consumption of mushrooms or their preparations; (2) direct antitumor activity against various allogeneic and syngeneic
tumors; (3) immunopo- tentiation activity against tumors in combination with chemotherapy; (4) preventive effect on tumor metastasis.

A good example of preventive effect is given by Japa­nese research on their popular edible and medicinal mushroom, Hypsizygus marmoreus (Ikekawa
2001). Control mice were bred on an ordinary diet and treated mice with a diet containing 5% dried fruit body of H. marmoreus. All mice were i.p.
injected with a strong carcinogen, methyl-cholanthrene, and carcinogenesis of the mice was investigated. At the end of the 76-week ob­servation, 21 of the
36 control mice developed tumors, but only 3 of 36 mice in the treated group had tumors. The authors concluded that the mechanism of cancer- inhibitory and
cancer-preventing activities of edible mushrooms was due to immunopotentiation (Ikekawa 2001).

It is well known from clinical practice that mushroom polysaccharides work best in conjunction with other

forms of ‘tough’ chemotherapy and surgery, which is, unfortunately, very invasive and has a lot of negative side effects. Lentinan has been studied best in
this re­spect, both in animal models and in human clinical prac­tice. In one study, 275 patients with advanced or recur­rent gastric cancer were given one
of two kinds of che­motherapy (mitomycin C with 5-fluorouracil or tegafur) either alone or with lentinan injections. The best results were obtained when
lentinan was administered prior to chemotherapy and in patients with a primary lesion who had undergone no previous chemotherapy. The results were
evaluated on the basis of prolongation of life, re­gression of tumors or lesions, and the improvement of immune responses (Hamuro and Chihara 1985; Hobbs
1995; Wasser and Weis 1997a).

Metastasis is a very serious and important problem in cancer therapy. A preventive effect of mushroom ex­tracts on cancer metastasis has been studied by
many groups, especially at the National Cancer Center Re­search Institute of Japan. In a successful series of experi­ments, Lewis lung carcinoma was s.c.
transplanted into the foot pads of mice and EA6 or EA6-PII (polysaccha­rides from Flammulina velutipes) were p.o. administered for a period of 10
days. The life span of the group treated with EA6-PII was significantly increased (Ikekawa

1999. . Further study was carried out using Meth-A fibro­sarcoma: 7 days after the tumor was s.c. transplanted in­to the abdomen of female BALB/c mice,
      the solid tumor of each mouse was surgically dissected out, and 7 days after the surgery, a second challenge with the same tu­mor, Meth-A
      fibrosarcoma was made s.c. into the other side of the abdomen of the mouse and the re-challenged tumor growth was observed. The results indicated
      that pre-treatment with EA6 slightly inhibited growth of the rechallenged tumor, but post-treatment was remarkably effective for tumor growth
      inhibition at a dose of 10 mg/kg (Ikekawa 2001).

Specific preparations from particular medicinal mush­rooms have undergone a lot of testing in animal model ex­periments and in clinics. After isolation of
lentinan from Lentinus edodes by Chihara in 1969, most of the experi­mental antitumor testing was performed with this polysac­charide. Its
‘father’, Chihara himself, was one of the first researchers to report the antitumor properties of lentinan. Originally, its effect was tested by using
Sarcoma 180 transplanted in CD-1/ICD mice (Chihara et al. 1969, 1970). Later, lentinan showed prominent antitumor ac­tivity not only against allogenic
tumors, but also against various synergic and autochtonous tumors (Hamuro and Chihara 1985). Injections of lentinan into mice produced either an 80%
reduction in tumor size or complete regres­sion in most of the animals tested (Chihara 1981). A num­ber of clinical tests followed. Among the first of
these was a follow-up, randomized control study on Phase 3 patients with advanced and recurrent stomach cancers (Wasser and Weis 1999; Ikekawa 2001).
Lentinan therapy showed very good results in prolonging the life span of patients and had no toxic side effects. Similar results were obtained in pa­tients
with colorectal and breast cancers. Since then, lenti- nan became a widely used medicine and dietary supple­ment in Japan, other Far East countries, and
later in the United States and Europe.

PSK (commercial name krestin) has remarkable im- mune-enhancing activity and a broad antineoplastic scope. It has been shown to prolong the survival time
of radiated mice, stimulate phagocytotic activity of macro­phages, and improve the functions of the reticuloendo­thelial system (Zhu 1987). With regard to
its antitumor properties, it acts directly on tumor cells, as well as indi­rectly in the host to boost cellular immunity (Hobbs 1995; Stamets 2000). It has
shown antitumor activity in animals with adenosarcoma, fibrosarcoma, mastocyto­ma, plasmacytoma, melanoma, sarcoma, carcinoma, and mammary, colon, and lung
cancer (Sugimachi et al.

1997. . An intriguing feature of this compound is that in­jection of PSK at one tumor site has been shown to in­hibit tumor growth at other sites, thus
      helping to prevent metastasis. PSK has been used both orally and intrave­nously in clinical medicine. It has been shown to be effective against
      many types of cancer (Hobbs 1995; Stamets 2000), but seldom with satisfactory results if administered alone.

The polysaccharide schizophyllan shows antitumor ac­tivity against both the solid and ascite forms of Sarcoma 180, as well as against the solid form only
of sarcoma 37, Erlich sarcoma, Yoshida sarcoma and Lewis lung carci­noma (Hobbs 1995). Schizophyllan has also increased cellular immunity by restoring
suppressed killer-cell activity to normal levels in mice with tumors (Borchers et al. 1999). Best results against radiation damage were found when
schizophyllan was administered shortly after or at the same time as radiation, and schizophyllan re­stored mitosis of bone marrow cells previously
sup­pressed by anticancer drugs (Zhu 1987). Human clinical studies proved the beneficial activity of treatment with schizophyllan for patients with
recurrent and inoperable gastric cancer, stage 2 cervical cancer, and advanced cer­vical carcinoma (Hobbs 1995).

Mechanisms of antitumor and immunomodulating action by mushroom polysaccharides

Mushroom polysaccharides exert their antitumor action mostly via activation of the immune response of the host organism. These substances are regarded as
biological response modifiers (BRMs; Wasser and Weis 1999). This basically means that: (1) they cause no harm and place no additional stress on the body;
(2) they help the body to adapt to various environmental and biological stress­es; and (3) they exert a nonspecific action on the body, supporting some or
all of the major systems, including nervous, hormonal, and immune systems, as well as reg­ulatory functions (Brekhman 1980).

Fig. 2
Possible pathways of lentinan action (after Chihara 1981)

The immunomodulating action of mushroom polysac­charides is especially valuable as a prophylactic, a mild and non-invasive form of treatment, and in the
preven­tion of metastatic tumors, etc., as described above. Poly­

saccharides from mushrooms do not attack cancer cells directly, but produce their antitumor effects by activating different immune responses in the host.
This has been verified in many experiments, such as the loss of the antitumor effect of polysaccharides in neonatal thymec- tomized mice or after
administration of anti-lymphocyte serum (Ooi and Liu 1999). Such results suggest that the antitumor action of polysaccharides requires an intact T-cell
component and that the activity is mediated through a thymus-dependent immune mechanism. Also, the antitumor activity of lentinan and other
polysaccha­rides is inhibited by pretreatment with antimacrophage agents (such as carrageenan). Thus, the various effects of polysaccharides are thought to
be due to potentiation of the response of precursor T cells and macrophages to cytokines produced by lymphocytes after specific recog­nition of tumor cells
(Hamuro and Chihara 1985). In addition, the induction of a marked increase in the amounts of CSF, IL-1, and IL-3 by polysaccharides re­sults in maturation,
differentiation, and proliferation of the immunocompetent cells for host defense mechanisms (Hamuro and Chihara 1985). Mushroom polysaccharides are known
to stimulate natural killer cells, T-cells, B-cells, and macrophage-dependent immune system responses.

Lentinan is known to be able to restore the suppressed activity of helper T-cells in the tumor-bearing host to their normal state, leading to complete
restoration of hu­moral immune responses (Ooi and Liu 1999). The same effect is true for PSK, while it has no substantial effect on immune responses of the
host under normal condi­tions.

Infiltration of eosinophils, neutrophils, and granu­locytes around target tissues is also accelerated by lenti- nan. It activates secretion of active
oxygen and produc­tion of cytokines in peritoneal macrophages. Lentinan also increases peritoneal macrophage cytotoxicity against metastatic tumors; it can
activate the normal and alternative pathways of the complement system and can split C3 into C3a and C3b, enhancing macrophage acti­vation (Aoki 1984;
Wasser and Weis 1997a; Hobbs 2000).

Lentinan’s immune-activating ability may be linked with its modulation of hormonal factors, which are known to play a role in tumor growth. Aoki (1984)
showed that the antitumor activity of lentinan is strongly reduced by administration of thyroxin or hydrocortisone. Lentinan can also restore
tumor-specific antigen-directed delayed-type hypersensitivity reaction.

Schizophyllan activates macrophages (in vitro and in vivo), which results in augmentation of T-cell activities and increases sensitivity of cytotoxic LAK
and NK cells to IL-2 (Mizuno 1996). Although structurally related to lentinan, schizophyllan does not directly activate T-cells (Hobbs 1995). Possible
pathways of such actions for lentinan have been summarized in Chihara (1981) and Hamuro and Chihara 1985), and reviewed by Wasser and Weis (1999), and
those for p-D-glucan BRMs (Mizuno

1999. are shown in Figs. 2 and 3.

Selected examples of important medicinal mushrooms with antitumor polysaccharides in fruit bodies and cultured mycelium

Fig. 3
Possible immune mech­anism: P-D-glucan biological response modifier (BRM)

(after Mizuno 2002)

One of the utmost important edible and medicinal bio­technological species, known as

A. blazei

was revaluated by our group. Analysis of data on cultivated mushroom originating from Brazil, and study of type material of

A. blazei

Murrill shows dramatic differences between

them. On the basis of existing differences the correct name for widely cultivated mushroom was proposed as new for science species Agaricus brasiliensis S. Wasser et al. A. blazei is the North American endemic not culti­vated species known from only three localities –
one in Florida and two in South Carolina (Wasser et al. 2002).

Agaricus blazei
mushroom (the Royal Sun Agaricus, ABM, Himematsutake, Cogmelo de Dues) is one of the more newly discovered medicinal mushrooms. This deli­cious
edible mushroom is native to a very small area of the mountains of Brazil, near the town of Sao Paulo. Epi­demiologists studying the native population in
this area found that they had a very low incidence of a variety of illnesses, including cancers as well as viral and bacteria- induced diseases, and a
disproportionately high number enjoyed longevity. Eventually this was correlated with constant consumption of A. blazei mushroom in their nor­mal
diet. During the 1980s and 1990s, A. blazei was dem­onstrated to be an immune system stimulant, promoting the body’s natural defense mechanisms to
fight a variety of infectious agents and conditions, including cancer. The immunostimulating activity and antitumor action of A. blazei extracts
were investigated in different laboratory models, including Sarcoma 180 and (Meth-A) fibrosarco­ma tumor-bearing mice (Kawagishi et al. 1989, 1990; Mizuno
et al. 1990b, 1998; Mizuno 2002; Itoh et al. 1994; Ebina and Fujimiya 1998; Fujimiya et al. 1998a, 2000; Stamets 2000). Of the 17 polysaccharide fractions
obtained from A. blazei fruit bodies (Mizuno et al. 1990a; Mizuno 2002), 7 were demonstrated to have antitumor activity. Analyses of
physico-chemical properties of water-soluble polysaccharide fractions having high anti­tumor activity showed that their main components were P-(1 ——6)-;
P-(1—3)-glucan, acidic P-(1——6)-; a-(1—4)- glucan, and acidic P-(1 ——6)-; a-(1—3)-glucan (Mizuno et al. 1990a). A. blazei was the first mushroom
described to contain anti tumor glucan with a P-(1— 6)-linked back­bone, unlike the well-known P-(1— 3)-glucans. The anti­tumor proteoglucan HM3-G from A. blazei fruit bodies, which mediated natural killer cell activation and apopto- sis, has a molecular mass of 380 kDa and consists of more than
90% glucose, the main component being a-(1—4)-glucan with P-(1— 6)-branching, at a ratio of approximately 4:1 (Fujimiya et al. 1998b). It is interest­ing
to note that a low molecular weight fraction, LM-3, with an average weight of 20 kDa, composed of a-(1—4)-glucan with P-(1— 6)-branching, also
demon­strated tumor-specific cytocidal and immunopotentiating effects (Fujimiya et al. 1999), whereas pure glucan ob­tained from antitumor
p-(1—-6)-glucan-protein complex, isolated from water-insoluble residue of A. blazei fruiting bodies, did not exhibit strong activity (Kawagishi et
al. 1990). Three immunostimulating heteroglucans (AG-2, -3, and -6) were extracted with 0.9% sodium chloride and hot water from fruiting bodies of A. blazei from among the six polysaccharides obtained (Cho et al. 1999). AG-2 and AG-3 were composed of glucose, galactose and mannose in the
molar ratios 74.0:15.3:10.7 and 63.6:17.6:12.7, respectively; and AG-6 was composed of glucose and ribose at a molar ratio of 81.4:12.6.

A xyloglucan (Xyl:Glc, molar ratio =2:10) containing 9% protein obtained by fractionation and purification of A. blazei extract showed significant
activity against Sarcoma 180 in mice (Mizuno 2002).

Not only fruit bodies but also cultured mycelia of A. blazei are a source of antitumor polysaccharides. An antitumor organic substance called ATOM
was devel­oped from A. blazei (Iwade strain 101), which is a PSPC (Ito et al. 1997). Another PSPC, 0041, was obtained from submerged culture
mycelium; the main components of this polysaccharide are glucose and mannose (Hikichi et al. 1999). A new antitumor polysaccharide active against Sarcoma
180 was recently separated from liquid cultured mycelium of A. blazei: p-(1—>2)-; p-(1—-3)-glu- comannan (Tsuchida et al. 2001). This
polysaccharide appears to be completely different from the antitumor polysaccharides from fruiting bodies of A. blazei (Mizuno et al. 1999b).

A liquid medium filtrate separated from mycelium after submerged cultivation of A. blazei contained man- nan-protein complex (AB-FP) with a
molecular weight of 10⁵-10⁷ Da and a small amount of glucose, galactose, and ribose. The yield of AB-FP was 575 mg/1 liquid medium
filtrate, and it possesses significant antitumor activity (Mizuno 2002).

Thus, antitumor polysaccharides investigated in A. blazei fruit body, culture mycelia, or produced extra­cellularly in a culture medium have
different chemical structures. Polysaccharides from fruit bodies represented glucans with different types of glucose unit connections or heteroglucans;
culture mycelia contained glucoman- nans, and mannan-protein complex was produced in a culture medium under submerged cultivation.

Ganoderma tsugae
is the other medicinal mushroom in which polysaccharides have been well investigated in both the fruit body and mycelia. Seven glycans with strong
antitumor activities were obtained from 14 water- soluble and 15 water-insoluble fractions extracted from G. tsugae fruit bodies (Wang et al.
1993). Water-soluble fractions were protein-containing glucogalactans associ­ated with mannose and fucose, and water-insoluble frac­tions represented
protein-containing p-(1—3)-glucans with different protein content.

Sixteen water-soluble polysaccharides were extracted from G. tsugae mycelium and examined for antitumor effects on Sarcoma 180 in mice (Zhang et
al. 1994b). The three active polysaccharides obtained were: a gly- can-protein complex containing 9.3% protein, with a heteropolysaccharide composed of
mannose and xylose; a glucan-protein complex containing 25.8% protein; and a glycan-protein with glucose as the main component, and associated with
arabinose, mannose, xylose, and galactose. Comparison of active water-soluble polysac­charides obtained from fruit body and mycelium showed that those from
the fruiting body were glucogalactan- protein complexes, but those of the mycelium were homoglucan-protein complexes or a heteroglycan com­posed of mannose
and xylose.

Grifola frondosa
is one of the most popular medicinal mushrooms. Fruit bodies of this mushroom contain p-(1——3)-; p-(1—6)-glucan, acidic p-D-glucan (Mizuno et al. 1986;
Jong and Birmingham 1990; Wasser and Weis 1999), and p-(1——6)-; p-(1—3)-glucan (Nanba et al. 1987) in the water-soluble polysaccharide fraction.
Water-insoluble fractions include an acidic xyloglucan with a Glc:Xyl molar ratio of 100:82 and 16.5% glucu­ronic acid; an acidic heteroglycan containing
3.8% pro­tein, component sugars Glc:Xyl:Man:Fuc (100:58:34:14); and three acidic glycoproteins with molecular masses of 20-100 kDa. The major component
sugar is glucose, while fucose, xylose, mannose and galactose are minor components (Mizuno et al. 1986). Thus, all polysaccha­rides detected in G. frondosa fruit bodies are p-glucans with different chain conformation, heteroglucans, or glu- coproteins.

In contrast to fruit body polysaccharide composition, no p-glucan has been detected among antitumor active fractions obtained from culture mycelium (grown
on Whatman filter paper soaked with liquid nutrient medium) that was collected before initiation of fruit bodies (Mizuno and Zhuang 1995). In the
water-soluble fractions, a fu- cogalactomannan-protein complex, a glucogalactoman- nan, a mannogalactofucan, and a galactoglucomannofu- can-protein complex
were found. In water-insoluble frac­tions, a mannofucoglucoxylan, a mannoglucofucoxylan- protein complex, a mannofucoglucoxylan-protein com­plex, and a
glucomannofucoxylan-protein complex were found (Zhuang et al. 1994a). Thus, polysaccharides from G. frondosa are heteromannans, heterofucans, and
hetero- xylans, or their complexes with protein, i.e., types of poly­saccharide that were not found in fruit bodies of this mushroom.

It must be stated that the polysaccharide structure in cultured mycelia may depend on the composition of the nutrient medium used for cultivation. Thus,
Ohno and co­workers (1985, 1986) concluded that the antitumor glucan grifolan extracted from cultured mycelium of G. frondosa is a p-(1——3)-,
p-(1—6)-glucan, the same as in the fruit body of the mushroom. In this experiment, a pure culture was growing in liquid medium in stationary culture or
with shaking. The mycelium obtained was additionally cultivated for 3 days in a buffer composed of glucose (5%) and citric acid, pH 4.5. Antitumor active
p-(1——3)-, p-(1—6)-glucans were obtained both by extraction of my­celium grown on a nutrient medium and by alcohol pre­cipitation of buffer supernatant
(Adachi et al. 2002).

Table 4
Number of polysaccharide fractions obtained from different Basidiomycetes

Species	Fruit body	Culture mycelium	References
Agaricus blazei	17		Mizuno et al. 1990a
Hericium erinaceus	15		Mizuno 1999b
Grifola frondosa	29	28	Mizuno et al. 1986; Cun et al. 1994; Zhuang et al. 1994a
Hohenbuehelia serotina	20		Ma et al. 1991
Pleurotus pulmonarius	21		Zhuang et al. 1993
Pleurotus citrinopileatus	21		Zhang et al. 1994a
Leucopaxillus giganteus	24		Mizuno et al. 1995a
Lyophyllum decastes	11		Ukawa et al. 2000
Inonotus obliquus	21	8	Mizuno et al. 1999a
Ganoderma tsugae	29	16a	Wang et al. 1993; Zhang et al. 1994b

^a
Number of fractions of water-soluble polysaccharides only

The number of polysaccharides extracted from fruit­ing body or cultured mycelium of the same species is strongly dependent on the method of fractionation
used but, in general, the total amount of polysaccharides in fruiting bodies is higher (Table 4).

The number of fractions indicated in Table 4 includes, in some cases, not only finally purified polysaccharides but also some intermediate fractions that
were tested for antitumor activity.

The proportion of biologically active polysaccharide fractions in fruit body and culture mycelium is very high. Thus, 20 of 29 polysaccharide fractions
obtained from G. frondosa fruit body exhibited different levels of antitumor activity (Mizuno et al. 1986), and 24 of 28 polysaccharide fractions
obtained from culture mycelium of this mush­room showed antitumor activity (Zhuang et al. 1994a).

The total number of polysaccharides extracted from the fruit body is higher, in general, than that obtained from culture mycelium. For example, the total
of both water-soluble and water-insoluble polysaccharides ob­tained from I. obliquus sclerotium is 2-3 times higher than that extracted from
cultured mycelium (Table 5).

Conclusions

Higher Basidiomycetes mushrooms are still far from be­ing thoroughly studied; even the inventory of known species is incomplete, comprising maybe only 10%
of the true number of species existing (Hawksworth 2001; Kirk et al. 2001). The number of mushrooms with known pharmacological qualities is much lower
still. Nevertheless, the species studied so far represeent a vast source of anticancer and immunostimulating polysaccha­rides. Many, if not all,
Basidiomycetes mushrooms con­tain biologically active polysaccharides. Of the 651 spe­cies and 7 infraspecific taxa from 182 genera of higher Hetero- and
Homobasidiomycetes, the overwhelming majority have been demonstrated to possess pharmaco­logically active polysaccharides in their fruit bodies, cul­ture
mycelia, or culture broth (Reshetnikov et al. 2001).

Mushroom polysaccharides are of different chemical composition, mainly belonging to the group of P-glucans

Table 5
Yield of polysaccharide fractions from sclerotia and cul­ture mycelium of Inonotus obliquus (after Mizuno et al. 1999b)

Water-soluble polysaccharides, Water-insoluble polysaccharides, g/kg dry weight g/kg dry weight

Sclerotium

FIS-I	164.5	FII	2.64
FIS-II	12.0	FIII-1	42.48
		FIII-2	87.84
Mycelium
FI	53.9	FII	43.15
		FIII-1	4.6
		FIII-2	21.1

(Mizuno 1999a, 2000). The antitumor polysaccharides from various mushrooms are characterized by their mo­lecular weight, degree of branching, and higher
(tertiary) structure. It is evident that such structural features as P-(1——3) linkages in the main chain of the glucan and additional P-(1——6) branch
points are needed for antitu­mor action. The P-glucans containing mainly (1—6) linkages have less activity. High molecular weight glucans appear to be more
effective than those of low molecular weight (Mizuno 1996, 1999a, b). Unlike P-(1—3)-glucans, a-(1—3)-glucuronoxylomannans,

which are characteristic of jelly mushrooms, are not strongly dependent on molecular weight.

Different approaches exist to improve the antitumor activity of mushroom polysaccharides by chemical modi­fication, which is also necessary to improve
their clinical qualities, water solubility and ability to permeate stomach walls after oral ingestion. Two main procedures for chemical improvement are:
modification of mushroom polysaccharides by Smith degradation (oxydo-reducto- hydrolysis) and activation by the method of formolysis. The most successful
schemes for such methods have been developed for Ganoderma lucidum, Grifola frondosa and Leucopaxillus giganteus (= Tricholoma gigantea). Car- boxymethylation is another chemical method that trans­forms P-glucans into a water-soluble form.

A large body of experimental and clinical evidence demonstrates the beneficial results of mushroom poly­

saccharides for the following purposes: (1) prevention of oncogenesis by oral consumption of mushrooms or their preparations; (2) direct antitumor activity
against various allogeneic and syngeneic tumors; (3) immunopotentia- tion activity against tumors in conjunction with chemo­therapy; (4) preventive effects
on tumor metastasis. Most of the clinical evidence comes from the commercial polysaccharides lentinan, PSK (krestin), and schizophyl- lan, but there are
also impressive new data for polysac­charides from Phellinus linteus, Flammulina velutipes, Hypsizygus marmoreus, A. blazei and
others.

The biochemical mechanisms that mediate the biolog­ical activity of polysaccharides are still not clearly un­derstood. Polysaccharides from mushrooms do
not attack cancer cells directly, but produce their antitumor effects by activating different immune responses in the host. The antitumor action of
polysaccharides requires an in­tact T-cell component; their activity is mediated through a thymus-dependent immune mechanism (Borchers et al. 1999).
Mushroom polysaccharides are known to stimu­late natural killer cells, T-cells, B-cells, and macrophage- dependent immune system responses. The immunomod-
ulating action of mushroom polysaccharides is especially valuable as a means of prophylaxis, a mild and non-inva- sive form of treatment, prevention of
metastatic tumors, and as a co-treatment with chemotherapy.

A wide range of biologically active polysaccharides is found among higher Basidiomycetes mushrooms, and their practical application is dependent not only
on their unique properties but also on biotechnological availabili­ty. Isolation and purification of polysaccharides from mushroom material is relatively
simple and straight­forward, and can be carried out with minimal effort (Mizuno 1996, 1999a). Mycelia formed by growing pure cultures in submerged
conditions are of constant composition, and submerged culture is the best technique for obtaining consistent and safe mushroom products (Wasser et al.
2000; Reshetnikov et al. 2001).

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