この論文はInternational journal of Medicinal Mushrooms, Vol.3, pp.291-298(2001)に発表されました。

Beneficial Effects of Edible and 　Medicinal Mushrooms on Health Care

Tetsuro Ikekawa

Japanese Association for Integrative Medicine, Association for Popularization of Integrative Medicine and Treatments (NPO, Japan), Sanshin Bldg, 2-15-14, Uchikanda, Chiyoda-ku, Tokyo I O1 -0047, Japan ABSTRACT: Studies on antitumor activities of Basidiomycetes have been in progress at the National Cancer Center Research Institute of Japan since 1966. At present, studies on mushrooms are being conducted worldwide. At the time when we started the studies, it had been said in Japan that hard mushrooms such as Polyporaceae (sarunokoshikake in Japanese) were effective against cancer. We therefore tested such hard mushrooms using a bioassay to measure the so-called "host-mediated" antitumor activities, but the results were not always satisfactory Including Trametes versicolor (L.:Fr.) Lloyd (kawaratake) or Phellinus linteus (Berk. et Curt.) Teng (meshimakobu). However, the aqueous extracts of edible mushrooms showed high growth inhibitory activity against solid Sarcoma 180. Antitumor polysaccharides such as glucans were isolated from the mushrooms, and among them, a β-(1-3)-glucan, which we isolated from Lentinus edodes (Berk.) Sing. (shiitake), was named lentinan and used clinically by a narrow adaptation in Japan. Another popular edible mushroom, Flammulina velutipes (Curt.: Fr.) P. Karst. (enokitake), also has high antitumor activity, and polysaccharides and a low molecular weight protein-bound polysaccharide (EA6) were isolated, It was demonstrated that EA6 was active against tumors when administered orally(p.o.), but not so effective by intraperitoneal (i.p.) injection. It proved to be especially useful by p.o. in combination with surgery and other antitumor agents. By an antitumor screening test we isolated "proflamin" from mycelia of F velutipes, which was highly active against allogeneic and syngeneic tumors when administered p.o in mice. An epidemiological study in Nagano Prefecture, Japan indicated that the cancer death rate among farmers producing F velutipes as a main occupation was remarkably lower than that of other people in the Prefecture. A detailed epidemiological study is ongoing. The cancer prevention effect of Hypsizygus marmoreus (Peck) Bigel.(bunashimeji), one of the most popular edible mushrooms, was investigated. It also has high antitumor activity and a preventive effect in tumor metastasis. Cancer preventive study of edible mushrooms was performed. Control mice were bred on ordinary food and the treated mice with a food containing 5% of dried fruiting body of H, marmoreus. All mice were injected i.d. with a strong carcinogen, methylcholanthrene, and carcinogenesis in the mice was investigated. As a result of the 76-week observation, 21 of 36 of the control mice developed tumors, but only 3 of 36 mice in the treated group had tumors. The mechanism of cancer inhibitory and preventive activities of edible mushrooms was due to immunopotentiation and antioxidant activity. Thus, the intake of mushrooms proved to be effective in cancer prevention and growth inhibition. From ancient times, it has been said in Oriental traditional medicine that medicine and food have the same origin (Ishoku Dougen or Yakushoku Dougen in Japanese). KEY WORDS: Medicinal mushrooms, antitumor, immunomodulator, antitumor olygosaccharides, glucans. ABBREVIATIONS BRM: Biological response modifier; EEM: extracts of edible mushrooms; ILS: increase in life span; i.p.: intraperitoneal administration; p.o.: oral administration; RTA: radical trapping activity; s.c.: subcutaneous administration. The beneficial effects of edible and medicinal mushrooms are now recognized. Studies on mushrooms have been developed worldwide within the discipline life sciences. They have become popular as ordinary foods or supplements. Studies on antitumor activities of Basidiomycetes started at the National Cancer Center Research Institute of Japan 1996; we had started the studies at the dawn of molecular immunology, before the concept of biological response modifiers (BRMS) had been proposed. The late Dr. W. Nakahara, the former President of the National Cancer Center, Japan had intended to apply immunological methods or ideas to cancer chemotherapy fields, and it is my belief that, having spent his academic life at the Rockefeller Institute, New York, he had taken an interest in cancer immunology. At the time we started the studies he was leading the work of the Chemotherapy Division of NCC; I carried out the mushroom studies under his leadership, and he had advocated as "host-mediated" activities those we call BRMS today. At the time it had been said in Japan that hard mushrooms such as Polyporaceae (sarunokoshikake in Japanese) were effective against cancer. We therefore tested such hard mushrooms by a bioassay to measure the so-called "host-mediated" antitumor activity. The bioassay was performed with subcutaneous (s.c.) transplantation of an ascites type of Sarcoma 180 and after a 5-week observation of tumor growth an inhibition ratio was determined by measurement of the solid tumor weight as reported in many articles on mushroom polysaccharide studies. Today it is a popular and established system, but then it was a new trial in cancer chemotherapy. The studies on hard mushrooms were conducted by us and Prof. S. Shibata's institution, the University of Tokyo. Many mushrooms Including Trametes versicolor (L.: Fr.) Lloyd (kawaratake) or Phellinus linteus (Berk. et Curt.) Teng (meshimakobu) were tested by the aforementioned antitumor bioassay, but the result was not necessarily satisfactory (Ikekawa et al., 1968; Shibata et al.,1968). We then directed our attention to edible mushrooms, particularly Japanese mushrooms. The aqueous extracts of the Japanese edible mushrooms showed high growth inhibitory activity against the solid Sarcoma 180 (Table I ). No substantial difference was found in the antitumor activity of edible mushrooms, that Is, Lentinus edodes (Berk.) Sing. (shiitake), Flalmmulina velutipes (Curt.: Fr.) P. Karst. (enokitake), and Pleurotus ostreatus (Jacq. : Fr.) Kumm. (hiratake) (Ikekawa et al., 1969). Activity of the Agaricus series was not high (Ikekawa et al., 1969; Ikekawa et al., unpublished data). Another edible mushroom, Hypsizygus marmoreus (Peck) Bigel. (bunashimeji), showed a very high inhibition ratio in our later experiment (Table 2) (Ikekawa et al., 1989, 1992). Although an extract of Trametes versicolor was approved as an anticancer drug in Japan and that of P Linteus in Korea, they are not useful for obtaining an estimation, and not very active in oral administration (p.o.) experiments (Ikekawa et al., 1968, 1969, 1989, 1992; Shibata et al., 1968; Chihara et al., 1970; Sasaki et al., 1971; Yoshioka et al., 1972, 1973, 1975; Ohkuma et al., 1982; Chihara, 1988; Yoo et al., 1995, 1996; Han et al., 1999). Many antitumor polysaccharides such as glucans were isolated from the edible mushrooms, and among them, a P-(1-3)-glucan that we isolated from L.edodes (Ikekawa et al., 1969) was named lentinan and used clinically as an injection in a narrow adaptation in Japan (Chihara et al., 1 970; Chihara, 1985). No substantial difference was found between the activity of p-(1-3)-glucans isolated from F velutipes or P. ostreatus and that of the glucan. It can be concluded that those antitumor polysaccharides are more effective than polysaccharides of the nonedible mushrooms such as Ganoderma lucidum (Curt.: Fr.) P. Karst., Phellinus linteus, and so on (Ikekawa et al., 1968, 1969, 1989, 1992; Chihara et al., 1970; Shibata et al., 1968; Sasaki et al., 1971 ; Yoshioka et al., 1972; Yoshioka et al., 1973, 1975; Ohkuma et al., 1982; Chihara, 1988; Yoo et al., 1995, 1996; Han et al., l999) . Because the aforementioned results were concerned mainly with intraperitoneal (i.p.) administration, it was important whether or not substances of mushrooms are active by p.o. It was thus necessary to measure quantitatively the immunopotentiation activity against tumors by p.o. sample administration. An experimental model of a combination treatment consisting of cryosurgery and mushroom extracts was useful to analyze immune responses by p.o. administration. One week after the tumor implantation of Sarcoma 180, the solid tumor was destroyed by cryosurgery and mushroom extracts were administered i.p. or p.o. for 7 days. Thereafter the ascites cells of the same tumor were implantated i.p., and an increase in life span (ILS) was determined. Polysaccharides and a protein-bound polysaccharide with low molecular weight (EA6) were isolated from Flammulina velutipes. In the combination study using these substances, both P-(1-3)-glucan (EA3) and EA6 showed a high synergistic effect by i.p. administration. By p.o. administration, however, simple polysaccharides including P-( 1-3)-glucans were not effective and only EA6 showed a high synergistic activity (Ohkuma et al., 1982, 1983; Ikekawa et al., l989). Thus it was concluded that antitumor substances showing p.o. activity were different from simple polysaccharides with high molecular weight and rather low molecular weight protein-bound polysaccharides were important. These experimental results were obtained using allogeneic tumors. The next problem was thus whether the extract of mushrooms was active against syngeneic tumors by p.o. administration. EA6 isolated from fruiting bodies of F velutipes showed 34% or 39% of ILS against Lewis lung carcinoma at a dose of 5 or 10 mg/kg by p.o. administration (Ikekawa et al., 1989; Ikekawa et al., unpublished data). It showed 22% of ILS at 10mg/kg and EA6-PII, the main component of EA6, showed 36% or 42(~o of ILS at a dose of 5 or 20 mg/kg against B-16 melanoma by p.o. as shown in Table 3 (Ikekawa et al., 1989; Ikekawa et al., unpublished data). Metastasis is a very serious and important problem in cancer therapy. We therefore investigated a preventive effect of the mushroom extracts on cancer metastasis. First, Lewis lung carcinoma was transplanted s.c. in footpads of mice and EA6 or EA6-PII was administered p.o. for 10 days. Eight days after the tumor transplantation, legs of each mice with the tumor were surgically dissected out from their femurs and ILS was observed. The treated group with EA6-PII had significantly increased life span (Fig. 1) (Ikekawa et al., unpublished data). A further study was carried out using Meth-A fibrosarcoma. Seven days after the tumor was trans planted s.c. into the abdomen of female BALB/C mice, the solid tumor of each mice was surgically dissected out, and 7 days after the surgery, the second challenge with the same tumor. Meth-A fibrosarcoma, was implanted s.c. into another side of the abdomens of mice and the tumor growth rechallenged was observed. As shown in Fig. 2, pretreatment with EA6 before the surgery slightly inhibited growth of the rechallenged tumor, but post-treatment was remarkably effective for tumor growth inhibition, and the tumors disappeared in two out of seven mice post-administration and in three out of six mice pre- and post-administration at a dose of 10 mg/kg of EA6 (Ikekawa et al., unpublished data). Thus it was illustrated that p.o. administration of EA6 isolated from F velutipes was effective for inhibition of reccurrence or metastasis of tumors after surgery. Moreover, it proved to be useful in combination with not only surgery, but also antitumor agents, especially by p.o. By an antitumor screening test using syngeneic tumors we isolated "proflamin" from mycelia of F velutipes, which was highly active p.o. against allogeneic and syngeneic tumors in mice (Ikekawa et al., 1985). Thereafter, another cultivated edible mushroom, Hypsizygus marmoreus, has been supplied to the Japanese market and become one of the most popular mushrooms in Japan. We investigated the mushroom extract and obtained a high inhibition ratio for the solid Sarcoma 180, that is, 100% at a dose of 30 mg/kg, and the preventive effect on tumor metastasis and immunomodulation activity of the mushroom were also investigated (Ikekawa et al., 1992; Saitoh et al., 1997). A cancer prevention study of edible mushrooms was performed using this mushroom. The control mice were bred with an ordinary feed and the treated mice with a feed adding 5 % of dried fruiting body of H. marmoreus to the ordinary feed. All mice were injected i.d. with a strong carcinogen, methylcholanthrene, and carcinogenesis of the mice was investigated. On the 1 6th week the first tumor was confirmed in an injected side of the control mice. As a result of the 76-week observation, the tumors developed in 21 of 36 mice of the control group. However, only 3 of 36 mice in the treated group had tumors as shown in Fig. 3 (Ikekawa et al., 1992). Thus, the intake of mushrooms proved to be effective in cancer prevention and growth inhibition. The cancer preventive effect of the edible mushrooms was thought to be due to the immunomodulation activity. As the effect might also be attributable to antioxidant activities, a study was done. Mice were bred using a diet to which was added 10% of the aqueous extract of H. marmoreus to the ordinary diet in the treated group and mice of the control group were bred using the ordinary diet for 26 days. Then the blood plasma of each mouse was isolated and a radical trapping activity (RTA) was measured. RTA of the mouse plasma of the treated group increased significantly in an alkoxyl radical experiment, compared with that of the control group, and in a peroxy radical experiment RTA of the treated mice indicated an increasing tendency (Matsuzawa et al., 1997, 1998). When the amount of lipid peroxide in the plasma was also measured as a thiobarbituric acid reacting substance in this experiment, the lipid peroxide level of the mice plasma decreased 1 3~1~oin the treated group (Matsuzawa et al., 1997). It was reported by Hiramatsu et al. (1989) that the o t e aqueous extract of several Japanese edible mushrooms such as F velutipes had higher RTA than an aqueous extract of lemon. An antioxidant protein was also isolated from L. edodes (Kawagishi,1996). It can be concluded that cancer preventive and antitumor effects of the edible mushrooms are due to not only immunopotentiation activity, but also to antioxidant activity. In Japan Agaricus blazei that originated in Brazil is sold as a supplement, but its antitumor activity was not established in our tests (Ikekawa, 2000). Furthermore, when mice were treated with the extract of A. blazei, the amount of lipid peroxide in plasma increased, and contents of β-glucan in the mushroom were very low as shown in Table 4 (Ikekawa, 2000). It was shown in an epidemiological study in Nagano Prefecture, Japan that the cancer death rate among farmers producing F velutipes as a main occupation was remarkably lower than that of other people in the Prefecture (Table 5) (Ikekawa et al., 1 989). It was suggested that quantity and frequency of intake of the mushroom were related to the lowered cancer death rate. This epidemiological investigation was performed for 15 years (1972-1986), and now a more detailed epidemiological investigation is ongoing. It is a case control study, which is being conducted by the NCC Research Institute of Japan, Nagano Agricultural Technology Institute and Hospitals belonging to Agricultural Cooperatives in proportion to a cohort study of the Japanese Minister of Health and Welfare. Progress in life sciences, for example, molecular genetics, is astonishing and could not be imagined at the end of World War II, but so-called "life style related" diseases are increasing now. An important goal of modern medicine at present is to conquer these. For these reasons a development of integrative medicine is anticipated, which will combine both Oriental and Western medicine or both traditional and modern medicine. To maintain individual health, thinking of integrative or holistic medicine is necessary and important. In the course of our mushroom research I remembered an old world saying of Oriental medicine that is "Ishoku Dougen" or "Yakushoku Dougen" in Japanese, meaning that medicine and food have the same origin. There is a legend in Oriental traditional medicine from ancient times that human beings discovered that intake of some special foods had been effective for treatments or prevention of some disease. Some food was found to be a functional food as said in the present days. A supplement (named EEM) made from the edible mushrooms was developed as a result of these considerations on the basis of our research and recently supplied in Japan. Its clinical effectiveness was demonstrated by H. Ueno, who showed that natural killer cell activity was augmented in 15 out of 1 8 cases by administration of EEM for 3 months (Ueno et al., unpublished data). The quantity of the edible mushrooms consumed in our country is now increasing, because scientific evidence of the useful function of the mushrooms is reported, and I believe that it contributes to prevention or treatment of "life-style related diseases" and the edible mushrooms are functional foods.