CONTENTS

Special Lectures

-Anti-HIV activities of Ganoderma Iucidum...........................1

-Biochemical evaluation of Ganoderma Iucidum using in vitro assay systems.............................................................3

-Relationship between the immunomodulating effect and antitumor activity of Ganoderma lucidum.......................................5

-Limitations and prospects of current clinical Ganoderma research...7

-The anti-aging effects of Ganoderma essence........................9

-Ganoderma lucidum: Is it more useful as a functional food than as a pharmaceutical material ?..........................................10

-Molecular methods for Ganoderma lucidum complex systematic........11

Free Papers

-1-1..........................................................................13

The anti-allergic effect of Ganoderma lucidum

-1-2..........................................................................14

Effects of Ganoderma on behavior of HL-60 cell line

-1-3..........................................................................15

Antitumor effects of Ganoderma lucidum

-1-4..........................................................................16

Combined effects of Ganoderma lucidum and taxol on experimental liver cancer

-1-5..........................................................................17

Total utilization of polysaccharides in Ganoderma tsugae

-1-6..........................................................................18

Wound healing and fibroblasts activated effects by using waste from fruiting body of Ganoderma for skin substitute

-2-1..........................................................................19

Effects of Ganoderma lucidum on cellular immuno-competence in γ-ray-irradiated mice

-2-2..........................................................................20

Effective dosage of Ganoderma nutriceuticals in the treatment of various ailments

-2-3..........................................................................21

Phylogenetic relationships in Ganoderma inferred from the nucleotide sequence of the manganese superoxide dismutase gene

-2-4..........................................................................22

Purification and characterization of manganese superoxide dismutase from Ganoderma microsporum

-2-5..........................................................................23

The comparison of manganese superoxide dismutase activity in Ganoderma species

-2-6..........................................................................24

Characterization of the manganese superoxide dismutase gene from Ganoderma by polymerase chain reaction

-2-7..........................................................................25

The specific linhzhi strain (Ganoderma lucidum (Leyss Ex. Fr) Karst on "Lim"- tree in Viet Nam

-2-8..........................................................................26

Taxonomy and biology of the family Ganodermataceae Donk in Viet Nam

-2-9..........................................................................27

Responses of Ganoderma lucidum to minerals and heavy water

-2-10.........................................................................28

Application of Cs-134 techniques for determination of harvest time in cultivations of lingzhi fungus Ganoderma lucidum (Leyss. ex Fr.)Karst.

-2-11.........................................................................29

Morphological evolution in Ganodermataceae-a hypothesis

-2-12.........................................................................30

Antimicrobial activity and physiological sutdy of Ganoderma spp.native to Indonesia

-3-1..........................................................................31

Selection of optimal medium for Korean native Ganoderma spp.

-3-2..........................................................................32

Growth characteristics of ling-zhi compared to selected Ganoderma in the G. lucidum complex

-3-3..........................................................................33

Habitat characterization of basidiocarps and microconidia formation

-3-4..........................................................................34

Cultivation of Ganoderma lucidum in North America

-3-5..........................................................................35

Extensive ecological surveys of a long-spored Ganoderma species in North America including delignification studies with a radioactive-labelled substrate

-3-6..........................................................................36

Isolation of trierpenoids by preparative HPLC from Ganoderma tsugae

-4-1..........................................................................37

The establishment of analytical methods for specific compounds in Ganoderma tsugae

-4-2..........................................................................38

The establishment of analytical method for organogermanium compounds in Ganoderma lucidum

-4-3..........................................................................39

HPLC analysis of hepato-protective triterpenoid patterns in fruiting body of the genus Ganoderma

-4-4..........................................................................40

The enhancement of Ganodermic acid S on the inhibition of prostaglandin E_1 on collagen responses of human platelet

-4-5..........................................................................41

Antioxidant and radical scavenging activities of extracts from Ganoderma tsugae

-4-6..........................................................................42

Protein purification, cloning, and gene expression of fungal immunomodulatory protein, FIP-gts, from Ganoderma tsugae

S

pecial Lecture ────

Anti-HIV Activities of Ganoderma lucidum

Byong Kak Kim, Ha Won Kim* and Eung Chil Choi

Department of Microbial Chemistry, College of Pharmacy

Seoul National Uniuersity, Seoul 151-742, Korea

*Department of Pathology & Laboratory Medicine

Hospital of the Uniuersity of Pennsyluania

Philadelphia, PA 19104, U.S.A.

The aqueous extract from the carpophores of Ganoderma lucidum were separated into high- and low-molecular-weight fractions. After the carpophores were also extracted with methanol, the extract was separated into eight fractions by their charge. Various concentrations of the fractions of both extracts were used for XTT antiviral assay which showed in uitro cytopathic effects of human immunodeficiency virus (HIV-1, Ⅲ b variant) on human T lymphoblastoid cells (CEM-IW). The assay uses a tetrazolium reagent that is metabolically reduced by viable cells to a soluble, colored formazan product which is measured by a colorimeter at 450 nm. With OD values of the product, 50％ inhibitory concentration (IC50)in virus-uninfected cells, 50％ effective concentration (EC50) for protection in virus-infected cells and in uitro therapeutic index (TI, IC50/EC50)were calculated. Virus multiplications were also assayed and compared with those of control by measuring viral reverse transcriptase (RT) activity in the supernatant of Jurkat T lymphocytes that were infected with HIV-1 (HXBC2 strain) in the presence of each fraction. The values of IC50 and EC50 by the treatment of the high-molecular-weight fraction (GL-HMW) were more than 125.0 μg/ml, indicating no toxicity on target cells and no antiviral activity. On the contrary, values of IC50, EC50 and TI by the low-molecular-weight fraction (GL-LMW) were 125.0 μg/ml, 11.0∼11.2 μg/ml and more than 11.1∼11.3 μg/ml, respectively, indicating that this low-molecular-weight fraction had no cytotoxicity on target cells but had very strong inhibitory activity of virus multiplication. The total methanol extract, GLA, showed 43.6∼44.4 μg/ml of IC50 and 14.4∼43.6 μg/ml of EC50 and its TI value was 1.0∼3.1. Thus this extract had very strong antiviral activity. The hexane layer, GLB, showed 21.5∼22.4 μg/ml and 15.2∼21.5 μg/ml of EC50, indicating that this fraction also had strong antiviral activity. The ethyl acetate fraction, GLC, showed 27.1∼29.3 μg/ml of IC50 and 27.1∼29.3 μg/ml of EC50. The water-soluble fraction, GLD, showed more than 125.0 μg/ml of both IC50 and EC50, indicating no cytotoxicity and no antiviral activity. The neutral fraction, GLE, showed 14.8∼15.0 μg/ml of IC50 and 14.8∼15.0 μg/ml of EC50, indicating that this fraction had strong cytotoxicity but had excellent antiviral activity. The acidic fraction, GLF, that includes ganoderic acids and their derivatives more than 125.0 μg/ml of both IC50 and EC50, indicating no cytotoxicity and no antiviral activity. The basic fraction, GLG, showed 22.4∼24.6 μg/ml of IC50 and 22.4∼24.6 μg/ml of EC50, indicating excellent antiviral activity. The amphoteric fraction, GLH, showed more than 125.0 μg/ml of both IC50 and EC50, indicating no cytotoxicity and no antiviral activity. In the RT assay, GLC and GLG showed significant antiviral activity. GLC suppressed viral proliferation to 75％ after three-day culture with Jurkat T lymphocytes at 50 μg/ml when compared with that of control. GLG also inhibited viral proliferation to 66％ after three day-culture at 100 when compared with that of control. These results were in agreenment with those XTT assay, i.e., both GLC and GLG fractions suppressed viral multiplication in both assays where different HIV-1 strains and different T lymphocytes were used. This shows that the non-hexane fraction and the basic fraction of G. lucidum clearly have antiviral components. One of the interesting findings in this study is that the acidic fraction, GLF, to which ganoderic acids and their derivatives belong, did not have any antiviral activity. Among the hot water extractives, only the low-molecular- weight fraction inhibited viral multiplication, Among the methanol extractives, both neutral and basic fractions were found to inhibit viral multiplication, showing that the carpophores of G. lucidum had several components responsible for the inhibition of human immunodeficiency virus multiplication.

Special Lecture ────

Biochemical Evaluation of Ganoderma Lucidum Using In Vitro Assay Systems

Research Institute for Traditional Sino-Japanese Medicines, Toyama Medical and Pharmaceutical University, Japan

Masao Hattori

For the purpose of evaluating the biological activities of the fruiting bodies of Ganoderma lucidum, we investigated the extracts and their constituents using the cultured myocardial cell enzymatic systems.

Effects of Ganoderma lucidum extracts on the cultured myocardial cells

The heart cell in culture is a useful system for studying the actions of cardioactive agents. Their actions can be detected sensitively as changes in beating rhythm, contraction force and other parameters. We developed a new monitoring system of myocardial cell motion using a microcomputer-driven image analyzing method and applied to various cardioactive agents and crude drug extracts used for treatment of heart failure in traditional system of medicine.

Hexane and methanol extracts of the fruiting bodies of G. lucidum significantly reduced the beating rate, the latter being stronger in potency. Both extracts, as well as the water extract, tended to increase the latter being stronger in potency. Both extracts, as well as the water extract, tended to increase the beating amplitude. Of the constituents isolated from the extracts, ganoderic acid S1, portensterol and ganodermanontriol significantly increased the beating amplitude. Ganodermanontriol increased the beating amplitude most effectively but it significantly reduced the beating rate.

Effects of Ganoderma lucidum extracts on glucosyltransferase from a cariogenic bacterium, Streptococcus mutans

Streptococcus mutans produces glucosyltransferase. This enzyme catalyzes the formation of sticky glucans in the presence of sucrose, and S. mutans cells are adhered to smooth surfaces on the teeth. The adhered bacteria cause tooth decay by the action of organic acids produced. For the purpose of developing anti-dental caries agents, we examined various crude drug extracts for inhibition of the adherence of S. mutans cells to smooth surfaces and found that the methanol extract appreciably inhibited the adherence of the cells. In addition, the methanol extract of G. lucidum showed potent inhibition against glucosyltransferase and the ethylacetate fraction showed the most potent inhibition against this enzyme. Of the constituents isolated, ganoderic acid S1, ganodermanontriol and ganoderic acid C2 strongly inhibited this enzyme.

Effects of Ganoderma lucidum extracts on HIV-protease

AIDS(acquired immunodeficiency syndrome) is caused by human innunodeficiency virus (HIV). This virus has some specific enzymes, such as reverse transcriptase, RNase H, integrase and protease, which are necessary for its replication. Therefore, these enzymes are targets for development of anti-AIDS agents. Especially, the protease (aspartic acid protease) is responsible for the cleavage of the viral polyproteins at the specific amino acid sequences to give functional proteins or enzymes. We screened various plant extracts for their inhibitory effects on recombinant HIV protease and found that methanol extract of G. lucidum appreciably inhibited the enzyme activity. However, the extract did not inhibit reverse transcriptase.

Effects of Ganoderma lucidum extracts on HCV-protease

Hepatitis C is a quite serious problem in the world because it leads the viral carriers to hepatic cancer at high frequency. This disease is caused by infection of hepatitis C virus (HCV), which is an RNA virus and produces viral specific protease (serine protease) to cleave the viral polyproteins during the maturation processes. In the course of developing anti-HCV agents , we investigated the effect of the methanolic extract of G. lucidum on recombinant HCV-proteaes, The methanol extract showed appreciable inhibition against this enzymes but the water extract did not show any inhibition.

In conclusion, through in vitro bioassays, we found that Ganoderma extract had cardioactive effects on the beating of cultured myocardial cells and the inhibitory effects on the dental plaque formation by S. mutans, as well as on the viral specific enzymes produced by HIV and HCV. Although in vitro bioassay systems introduced here have some limitations, these systems are convenient tools for screening a great number of plant extracts and their constituents available in small quantity.

Special Lecture ────

RELATIONSHIP BETWEEN THE IMMUNOMODULATING EFFECT AND ANTITUMOR ACTIVITY OF GANODERMA LUCIDUM

Wang, S. Y., Hsu, M.L., Lee, S.S., Hsu, H.C., Shiao, M.S., Ho, C.K.

Department of Medical Research, Veterans General Hospital-Taipei and National Yang-Ming University, Taipei, Taiwan, Republic of China.

Ganoderma(G.)lucidum, an oriental medical fungus, has been widely used as a remedy for promotion of health and longevity in China and other Asian countries (Shiao et al., 1994). The fruiting bodies and cultured mycelia of G. lucidum were reported to be effective in the treatment of many diseases, such as chronic hepatopathy, hypertension, hyperglycemia, and neoplasia. This medical fungus has attracted great attention due to the fact that its poly-saccharide fractions have antitumor activity (Miyazaki and Nishijima, 1981; Sone et al., 1985). Using animal models, many investigators had demonstrated that administration of crude or partially purified polysaccharides of G. lucidum (PS-G)could significantly inhibit the growth of locally implanted S 180 sarcoma (Sone et al., 1985)and reduce the tumor metastasis in mice (Hwang et al., 1989). In addition, an ethanol precipitable fraction of the aqueous G. lucidum extract (crude PS-G) was found to increase the life span of tumor-implanted mice when administered alone or in combination with cytotoxic antitumor drugs (Furusawa et al., 1992). The mechanism of the antitumor effect of PS-G is so far uncertain. Some investigators have suggested that the in vivo tumoricidal activity of PS-G may be related to some extent to the activation of host immune responses (Won et al., 1989). However, substantial and convincing evidence has not been well documented.

The presnt study was to ascertain the immunomodulating and antitumor effects of G. lucidum. PS-G from fresh fruiting bodies of G. lucidum were isolated and used to potentiate the cytokine production by human monocytes-macrophages and T lymphocytes. Our results had shown that the levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 in macrophage cultures treated with PS-G(100 μg/ml)were 4.1-, 9.8-, and 29-fold higher than those of the untreated controls, respectively, In addition, the release of interferon (IFN)-γ from T lymphocytes was also greatly promoted in the presence of PS-G (25-100 μg/ml). Furthermore, these cytokine-containing mononuclear cell conditioned media(PSG0MNC-CM)were found to be cytotoxic to the HL-60 and U937 leukemic cells with respective inhibition rates of 70％ and 75％ at a concentration of 20％9vol.vol). DNA labeling and flow cytometric analysis revealed that only few (2.3±0.8％)apoptotic cells were seen in the control cultures, while PSG-MNC-CM treatment induced a significant increase in the apoptotic population in both the HL-60(38.3±4.5％)and the U937(44.5±3.8％)cell lines. In addition, about 40-45％ of the treated leukemic cells were triggered to differentiate into mature monocytic cells expressing both CD 14 and CD68 surface antigens. However, PS-G alone had no such effects even at a higher dose of 400 μg/mlSince untreated macrophages and T lymphocytes produced little or no cytokine and normal MNC-CM did not suppress leukemic cell growth, it was suggestive that the antitumor activity of PSG-MNC-CM was derived from the elevated levels of cytokines. Antibody neutralization studies further revealed that the tumoritoxic cytokines in the PSG-MNC-CM were mainly of TNF-α and IFN-γ,and these two cytokines acted synergistically on the inhibition of leukemic cell growth.

REFERENCE

FURESAWA, E., et al., Antitumor activity of Ganoderma lucidum on intraperitoneally implanted lewis lung carcinoma in synergenic mice. Phytother. Res., 6, 300-3041992).

HWANG, S.F., et al., The inhibitory effect on artificial pulomonary metastasis of murine S-180 sarcoma cells by orally adimnistered Ganderma lucidum culture broth. J. Chinese Oncol. Soc., 5, 10-15(1989).

MIYAZAKI, T. and NISHIJIMA, M., Studies on fungal polysaccharides, XXVII. Structural examination of a water-soluble, antitumor polysaccharide of Ganoderma lucidum. Chem. Pharm. Bull., 29, 3611-3616(1981).

SHIAO, M.S., et al., In: Food Phytochemicals for Cancer Prevention Ⅱ:Teas, Spices, and Herbs, pp.342-354, Am. Chem. Soc., Washington DC(1994).

SONE, Y., et al., Structures and antitumor activites of polysaccharides isolated from fruiting body and the growing culture of mycelium of Ganoderma lucidum. Agric. Biol. Chem., 49, 2641-2653(1985).

Special Lecture ────

Limitations and Prospects of Current Clinical Ganoderma Research

Raymond Y. Chang, MD FACP

Department of Medicine, Memorial Sloan-kettering Cancer Center and Cornell Medical College, New York NY 10021 USA.

Ganoderma's efficacy as a folk medicine is well recognized in Asia and some of its pharmacological properties have been explored and systematically studied and reported in the past twenty years. There is no doubt that Ganoderma is a superior nutriceutical with many potential applications and proven efficacy but without significant toxicity or side-effects. Its recent potential applications in the area of cancer and AIDS treatment especially deserves attention from researchers. But despite these recent advances, there remains serious limitations in current Ganoderma research and development in three general areas:

Ⅰ. PHARMACOGNOSY: Ganoderma is a crude drug and its pharmacologic and pharmacokinetic study falls in the realm of pharmacognosy. In this general area, the pharmacology of its bioactive components and their pharmacokinetics have yet to be fully characterized. For example, we do not understand why Ganoderma is as efficacious as it is although we know its major bioactive components: polysaccharides and triterpenoids etc. Many other lesser nutriceuticals (eg algae, licorice) also contain similar polysaccharides and triterpenoids, so why is Ganoderma unique ? Another example is related to polysaccharides in Ganoderma - often promoted as a major bioactive fraction of the funrus: is it absorbed by the gastrointestinal tract, and what happens to such polysaccharides after the absorption? How about the pharmacokinetics of triterpenoids, adenosine and other bioactive fractions in Ganoderma must involve solving some of these basic pharmacologic and phamacokinetic issues.

Ⅱ. CLINICAL: The efficacy of Ganoderma has been reported for many clinical conditions ranging from alopecia to urticaria and include major human disease categories of allergic diseases, cancer, cardiovascular diseases, diabetes, immune disorders, infectious diseases, and inflammatory diseases. Yet there has not been one single well-designed double-blind controlled trial of Ganoderma in any of these conditions to confirm its efficacy, and its response to treatment is freguently unpredictable and can vary widely from person to person.

Ⅲ. INDUSTRIAL: In order to foster research in addressing some of the above issues, there is an urgent need to have reliable standardized Ganoderma products. Standard species grown under standard conditions with standard bioactive composition processed in standard manners with standard labelling all verifiable by undiased and internationally acceptable standards such as GMP, GLP, GCP and ISO9001 will be the first step towards advancing the research of Ganoderma and further promoting its use for the healthful benefit of all mankind.

Special Lecture ────

THE ANTI-AGING EFFECTS OF GANODERMA ESSENCE

YANG QING-YAO^1 and WANG MIN-MING^2

1.Shanghai Teachers University, Shanghai 200234, China

2.Shanghai Institute of Pharmaceutical Industry, Shanghai 200040, China

Abstract

The comparative observation of the symptoms of weakness and debility of 37 cases of patients about tiredness, palpitation, insomnia, bad memory, etc after taking Ganoderma essence from 4 to 6 weeks. The result showed that the integral values of the weakness and debility were lowered from 10.3 points before taking the medicine to 5.8 points after taking the medicine and the rate of lowering was 56.3％; lymphoid transformation from, 25732 cpm raised to 32657 cpm; The contents of IL-2, IgG and complement C_3 raised from 20.3 (IU/ml), 9.3(g/L)and 0.9(g/L) to 27.7(IU/ml), 13.2(g/L)and 1.2(g/L) respectively. P value was less than 0.05.

We have further observed and determined the effect of Ganoderma essence on eliminating the liver poisoning of mice after injecting CCI_4. The result showed that in the mice of Ganoderma essence group (120 mg/kg for 7 days), their abnormal change and damaged and dead number of liver cells were obviously decreased. The GPT and GOT values of the serum of the control group of CCI_4 were lowered from 164.5 and 103.1 units to 32.4 and 86.0 units respectively (P<0.01 and P<0.05) It suggests that Ganoderma essence has the function of improving symptoms of weakness and debility and of protecting the liver and eliminating the poison.

Key words: Ganoderma essence, Linzhi, weakness and debility, symptoms, protecte the liver, eliminate toxin.

Special Lecture ────

Ganoderma Iucidum: Is it more useful as a functional food than as a Pharmaceutical material?

Tsutomu Tamura

Pharmaceutical Society of Japan

Ganoderma lucidum(G.L., Man-nen Take-Reisi)has long been syudied by many investigators in Japan focusing on its constituents and their physiological activities^1,2,3).The findings of these previous studies may be that the main physiologically active substance of G.L. is a group of polysacharides with the structure of β-1,3 glucan. Some of β-1,3 glucans with determined structures have already been used as antitumor drugs, such as Krestin (R), Lentinan (R), Sonifilan (R), etc. The purpose of the previous studies on these G.L. constituents, however, was not to develop foods but to develop drugs, and the majority of the studies completed in 1980th.

In this situation, focusing on the function of the basidiomycete to sprout out edible mushrooms, we carried out the present study for the prupose of developing the functional fermented food, biomedical food (BMF) making the best use of characteristics of the basidiomycete. Essentially, we aimed to use a whole fermented material as a food, and did not attempt to do anything else. We report the one of such fermented foods, BMF-1, in this article.

Special Lecture ────

Molecular methods for Ganoderma lucidum complex systematic

Ruey-Shyang Hseu, Jean-Marc Moncalvo, Huei-Fang Wang,

and Hsi-Hua Wang

Department of Agricultural Chemistry National Taiwan University Taipei, Taiwan, ROC

The genus Ganoderma was established by Karsten (1991) for the laccate and stipitate white rot fungus polyporus lucidus W. Curt. Ganoderma is a cosmopolitan genus of wood decaying polypore fungi of economic importance. Several species cause seyere diseases in plantations or in forests. In the Orient Ganoderma is merely regarded as an herb of longevity. The fungus has been used in folk medicine for hundreds of years and strains are commercially cultivated for preparation of health products. Isolates used in pharmaceutical and medicinal studies, and consequently commercially cultivated isolates, largely refer to G. Iucidum. However, as used in the pharmaceutical literature, this name encompasses several laccate Ganoderma species that might differ in their bioactive compounds.

The G. lucidum species complex includes G. tsugae Murr., G. valesiacum Boud., G. oregonense Murr., G. resinaceum Boud., G. pfeifferi Bres., G. oerstedii (Fr.) Torr., G. ahmadii Stey, and several other taxa that are restricted to tropical areas. The use of traditional taxonomic methods was inconclusive in systematic of the group, and these methods are useless to characterize individual strains. However, an accurate identification system and a phylogenetically-based classification of Ganoderma taxa, together with development of genetic markers for individual strains, would have practical implications in epidemiology studies, and pharmacology.

We examined 45 isolates representing several genera, subgenera, sections and species of Ganodermataceae to infer natural relationships in the group. Phylogenetic characters were produced from nucleotide sequence data from the internal transcribed spacer regions (ITS 1 and ITS 2) of the ribosomal gene (rDNA) and from divergent region D2 of the large ribosomal subunit gene (LSU-D2). The ITS dataset provided phylogenetic information at lower taxonomic levels while the LSU dataset was more useful at higher levels. Results of parsimony analysis support Ganoderma, and Amauroderma as distinct genera. The results indicated that subgenus Elfvingia is monophyletic, while sections Ganoderma and Phaeonema are not. A species concept based on monophyly and potential evidence of genetic isolation is proposed, and taxonomy of the G. lucidum complex is revised. Collections named G. lucidum in North America and in Asia are not conspecific with European G. lucidum. The sister group of European G. lucidum is an Argentinean taxon labelled G. oerstedii.

Parsimony analysis of nucleotide sequences produced from the internal transcribed spacers(ITS)of the ribosomal gene (rDNA) distinguished six phyla in G. lucidum complex Each phylum represented one or more putative species. Some isolates shared identical ITS sequence, but all isolates were differentiated by genetic fingerprinting using random amplified polymorphic DNA (RAPDs). This suggested that ITS sequence can be used to identify isolates of the G. lucidum complex, whilst RAPDs can be used to differentiate between isolates having identical ITS sequences.

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