Abstract The number of mushrooms on Earth is esti- mated 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 impor- tantly 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 poly- saccharides are of different chemical composition, with most belonging to the group of ��-glucans these have ��-(1���3) linkages in the main chain of the glucan and additional ��-(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 im- prove 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 polysac- charides 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 conjunc- tion with chemotherapy. Mushroom polysaccharides pre- vent oncogenesis, show direct antitumor activity against various allogeneic and syngeneic tumors, and prevent tu- mor 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. Practi- cal application is dependent not only on biological prop- erties, but also on biotechnological availability. The present review analyzes the pecularities of polysaccha- rides derived from fruiting bodies and cultured myceli- um (the two main methods of biotechnological produc- tion today) in selected examples of medicinal mush- rooms. Introduction For millennia, mushrooms have been valued by human- kind as an edible and medical resource. A number of bio- active molecules, including antitumor substances, have been identified in many mushroom species. Polysaccha- rides 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 (de- coctions 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, pros- tate, 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. S.P. Wasser (���) 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 Appl Microbiol Biotechnol (2002) 60:258���274 DOI 10.1007/s00253-002-1076-7 M I N I - R E V I E W 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

259 1987 Hobbs 1995, 2000 Wasser and Weis 1997a, b, 1999 Stamets 2000). Ikekawa et al. (1969) published one of the first scien- tific reports on antitumor activities of essences obtained from fruiting bodies of mushrooms belonging to the fami- ly Polyporaceae (Aphyllophoromycetideae) and a few other families, manifested as host-mediated activity against grafted cancer ��� such as Sarcoma 180 ��� in ani- mals (Ikekawa et al. 1982, 1992 Ikekawa 2001). Soon thereafter the first three major drugs were developed from medicinal mushrooms. All three were polysaccharides, specifically ��-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 com- mune (Split Gill). For almost 40 years, medicinal mush- rooms 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. More- over, different strains of one Basidiomycetes species can produce polysaccharides with different properties. For ex- ample, the proteoglucan krestin was developed in Japan from the strain Trametes (Coriolus) versicolor CM-101, whereas polysaccharide-peptide (PSP) in China was de- veloped in submerged culture of the strain Cov-1 of the same species. Both proteoglucans have the same polysac- charide 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 mush- rooms are analyzed. More attention is given to their common features than to specific pecularities. The re- view summarizes the general state of knowledge in the area of biodiversity of mushrooms and their polysaccha- rides the chemical structure of polysaccharides and its connection with their antitumor activity, including possi- ble ways of chemical modification results of experimen- tal testing and clinical use of antitumor or immunostimu- lating polysaccharides possible mechanisms of their bio- logical action and, finally, the difference in polysaccha- ride fraction composition in fruiting bodies and pure cul- ture 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 cur- rently 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 organ- isms 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 defi- nition 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 spe- cies 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 tro- pics, 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 analy- sis 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 tro- pics (Hawksworth 1993, 2001), and this is also the case for mushrooms, although new species continue to be dis- covered in Europe and North America. Studies of compatability and molecular sequences be- tween mushrooms previously considered to be the same species on morphological grounds revealed ���cryptic spe- cies���, 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 mil- lion 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 un- limited source of antitumor or immunostimulating poly- saccharides. All main taxonomic mushroom groups have been investigated for biologically active polysaccha- rides, and most of them possess such substances. At least 651 species and 7 infraspecific taxa representing 182 genera of Hetero- and Homobasidiomycetes mush- rooms contain antitumor or immunostimulating poly- saccharides, as is evident from Table 1 (adapted from

260 Table 1 Higher Basidiomycetes mushrooms containing antitumor or immunostimulating polysaccharides Taxa (number of species studied) Activity against: Source Sarcoma Ehrlich 180 solid solid cancer cancer Heterobasidiomycetes Auriculariales ��� Auricularia (3) 70���90 60���80 Ohtsuka et al. 1973 Ukai et al. 1982 Song et al. 1998 Dacrymycetales ��� Calocera (1) Dacrymyces (1) 60���90 60 Ohtsuka et al. 1973 Tremellales ��� Exidia (1) Guepinia (1) Holtermannia (1) Phlogiotis (1) 60���100 70���100 Ohtsuka et al. 1973 Protodaedalea (1) Pseudohydnum (1) Tremella (2) Tremellodon (1) Gao et al. 1997 Homobasidiomycetes Aphyllophoromycetideae Cantharellaceae ��� Cantharellus (5) Craterellus (2) 60���100 60���90 Ohtsuka et al. 1973 Clavariaceae ��� Clavaria (4) Clavariadeiphus (2) Clavulinopsis (4) Lentaria (1) 60���90 60���100 Ohtsuka et al. 1973 Clavulinaceae ��� Clavulina (1) 70���90 80 Ohtsuka et al. 1973 Sparassidaceae ��� Sparassis (1) 100 100 Ohtsuka et al. 1973 Ohno et al. 2000 Yadomae and Ohno 2000 Ramariaceae ��� Ramaria (5) 60���80 60���70 Ohtsuka et al. 1973 Hydnaceae ��� Hydnum (1) 70 90 Ohtsuka et al. 1973 Chung et al. 1982 Hericiaceae ��� Echinodontium (2) Hericium (2) Laxitextum (1) 70���90 60���80 Ohtsuka et al. 1973 Mizuno 1999b Corticiaceae ��� Aleurodiscus (1) Cotylidia (2) Laxitextum (1) Lopharia (1) 60���100 60���100 Ohtsuka et al. 1973 Merulius (2) Phlebia (2) Sarcodontia (1) Sistotrema (1) Steccherinum (1) Stereum (13) Coniophoraceae ��� Serpula (1) 70 60 Ohtsuka et al. 1973 Thelephoraceae Bankera (1) Calodon (4) Hydnellum (2) Polyozellus (1) 60���100 70���100 Ohtsuka et al. 1973 Song et al. 1998 Sarcodon (2) Thelephora (1) Mizuno 2000 Hymenochaetaceae ��� Coltricia (4) Cryptoderma (6) Cyclomyces (1) 60���100 90���100 Ohtsuka et al. 1973 Fuscoporia (1) Hymenochaete (4) Hymenostilbe (1) Inonotus (6) Onnia (1) Kim et al. 1996 Phellinus (6) Pyrrhoderma (1) Han et al. 1999 Mizuno 2000 Fistulinaceae ��� Fistulina (2) 80 90 Ohtsuka et al. 1973 Ueno et al. 1978 Ganodermataceae ��� Ganoderma (7) 70���100 70���100 Ohtsuka et al. 1973 Nakashima et al. 1979 Miyazaki and Nishijima 1981 Ukai et al. 1983 Zhang and Lin 1999 Polyporaceae ��� Amauroderma (1) Coriolellus (1) Coriolus (8) Cymatoderma (2) 70���90 70���100 Ohtsuka et al. 1973 Cystidiophorus (1) Daedalea (1) Daedaleopsis (3) Dendropolyporus (1) Ito et al. 1976 Favolus (3) Fomes (2) Fomitella (1) Fomitopsis (5) Gloeophyllum (1) Ohtsuka et al. 1977 Gloeoporus (1) Gloeostereum (1) Grifola (2) Hirschioporus (3) Ischnoderma (1) Fujii et al. 1979 Laetiporus (2) Laricifomes (1) Lenzites (1) Meripilus (1) Microporus (2) Liou and Lin 1979 Oxyporus (1) Phaeolus (1) Piptoporus (1) Polyporus (10) Poria (1) Min et al. 1980 Porodisculus (1) Pycnoporus (1) Rigidoporus (2) Trachyderma (1) Nakajima et al. 1980 Trametes (8) Trichaptum (1) Tyromyces (5) Kanayma et al. 1986 Mizuno et al. 1992 Gasiorowski et al. 1993 Cho et al. 1996 Nanba 1998 Fullerton et al. 2000 Schizophyllaceae ��� Schizophyllum (1) 70 ��� Ohtsuka et al. 1973 Okamura et al. 1986