Research on Ganoderma

Constituents of Camphor Lingzhi (Ganoderma comphoratum) and their Activity

Faculty Advisor: Professor Cheng Cheng Yung

Graduate student, Graduate School of Chemistry, National Taiwan Normal University: Cheng Yi Hua

Degree: M.S.

Synopsis

First described in 1990, Ganoderma comphoratum, Zang & Su, is also known as Pyroformes sp., Camphor Mushroom, etc., and is parasitic on the inner wall of hollow heartwood in Cinnamomum comphora (L) Prcsl. The fruiting body has a strong camphor odor and is plate- or bell-shaped. The fruiting body is annual, stalkless, and is corky to woody in texture. The cap is semicircular with a diameter of 10~20 x 3~8 cm and a thickness of 2~2.5 cm. The surface is brown to blackish brown in color, has no obvious folds, and has a shiny luster. The edge is flat and blunt. The flesh has two layers; the upper layer is the color of wood, while the lower layer is ivory and 1~1.5 cm thick. The basidiospore is egg-shaped with a two-layer wall. The outer wall is transparent and the inner wall is golden brown with separate or connected spine-like protrusions. Spore diameter is 14~19 x 7.8~14.4 microns. A member of the Polyporaceae, this fungus has dense, fine pores. There are approximately 4~5 pores per square millimeter. The tubule openings are small and deep red in color.

Cinnamomum comphora (L) Prcsl has a strong odor, and is a powerful insect repellant. While most fungi cannot grow on camphorwood, Ganoderma comphoratum Zang & Su is uniquely able to do so, and is consequently popularly considered to be a natural treasure. Ganoderma comphoratum Zang & Su is a superior detoxifying agent that can be used to treat food poisoning, diarrhea, vomiting, and pesticide poisoning. A piece of camphor Lingzhi about the size of a kernel of corn is usually eaten after boiling, or chewed until powdered and then swallowed with water. Many persons currently use camphor Lingzhi to treat cancer and other chronic diseases, and anecdotal reports suggest that it is very effective. Very little camphor Lingzhi is produced and prices are extremely high; 1 kg of camphor Lingzhi mixed with some camphor bark costs approximately US,430 and prices are currently rising. But because of camphor Lingzhi's possible effectiveness, people continue to purchase and use it. To enhance effectiveness, small quantities of Ganoderma Comphoratum Zang & Su are sometimes added to Lingzhi products packaged in gelatin capsules. Unfortunately, no scholarly papers on the constituents or pharmacology of Ganoderma Comphoratum Zang & Su have yet been published in academic journals.

If methanol reflux extraction is performed on the ground fruiting body of Ganoderma Comphoratum Zang & Su, chromatographic analysis using a silica gel column and HPLC analysis reveal that the extract contains 13 constituents. Spectrographic analysis shows that these constituents have the following structures:

• Compounds A:4(-Methylergost-8,24(28)-dien-3,11-dion-26-oic acid.

• Compounds B:4(-Methylergost-8,24(28)-dien-3,7,11-trion-26-oicacid.

• Compounds:7(-Hydroxy-4(-methylergost-8,24(28)-dien-3,11-dion-26-oic acid.

• Compounds D:14(-Hydroxy-4(-methylergost-8,24(28)-dien-3,7,11-trion-26-oic acid.

• Compounds E:4(-Methylergost-8,14,24(28)-trien-3,11-dion-26-oic acid.

• Compounds F:7(-Hydroxy-4(-methylergost-8,14,24(28)-trien-3,11-dion-26-oic acid.

• Compounds G:Methyl-3(,12(-dihydroxy-4(-methylergost-8,24(28)-dien-7,11-dion-26-oate.

• Compounds H:2,2',5,5'-tetramethoxy-3,4,3',4'-di-methylenedioxy-6,6'-dimethyl biphenyl.

• Compounds I:3(-Hydroxy-24-methylenelanost-7,9(11)-dien-21-oic acid.

• Compounds J:3(,15(-Dihydroxy-24-methylenelanost-7,9(11)-dien-21-oic acid.

• Compounds K:24-Methylenedihydrolanosterol.

• Compounds L:9(E)-Octadecenoic acid.

• Compounds M:9,12(E,E)-Octadecadienoic acid.

• Indicates new compounds

Structure and Functional Mechanisms of an Immunomodulatory Protein

Faculty Advisor: Professor Ling Jung Yao

Graduate student, Graduate School of Biochemistry, National Taiwan University College of Medicine: Ling Wen Hui

Degree: M.S.

Synopsis

Lingzhi has been an auspicious symbol for the Chinese since ancient times, and many records and legends concerning Lingzhi have been passed down to us from long ago. Physicians have long used Lingzhi to treat a variety of disorders, and it is considered a valuable medicine with moist tonic, strengthening, and corrective properties. Lingzhi's actual functional mechanisms still remain to be clarified, however.

This laboratory has purified an immunity-regulating protein with a molecular weight of 13K we have named FIP-gts (fungal immunomodulatory protein gts) from the mycelia of G. tsugae. This protein can promote the proliferation of human peripheral lymphocytes and mouse spleen cells, and maximum human peripheral lymphocyte proliferation is achieved at a concentration of 5(g/ml. RT-PCR has been used to verify that FIP-gts can stimulate the production of the cytokines IL-2, IL-4, TNF-, and IFN-, and can promote the expression of ICAM-1 (intracellular adhesion molecule-1).

Following hydrolysis using a protein hydrolysis enzyme, an automatic protein sequencer was used to analyze the amino acid sequence of FIP-gts. It was found that the sequence of the 110 amino acids of FIP-gts is very similar to those of the variable region of the immunoglobulin heavy chain (IgVH). Garnier analysis of FIP-gts' secondary structure showed that it possesses two helices, seven sheets, and one turn. Gel filtration analysis indicated that it has a molecular weight of 26K, but SDS-PAGE analysis yielded a weight of 13K. This suggests that FIP-gts may exist in the form of a homodimer; glutaraldehyde can be used to verify that FIP-gts may form a homodimer. We have discovered through the use of PCR technology that many other species of Lingzhi other than G. tsugae possess an identical nucleic acid sequence, but certain not all fungi contain this immunity-strengthening protein.

We used genetic engineering techniques to embed the cDNA for FIP-gts in a PGEX-2T matrix, which was then inserted in E. coli for expression. We employed a column with Glutathione-Sepharose 4B affinity to purify a fusion protein containing GST and recombinant FIP-gts. This yielded pure FIP-gts after thrombin decomposition, and the ability of the resulting FIP-gts to stimulate human lymphocyte proliferation was identical of that of FIP-gts purified from mycelia.

To determine the chemical structure of FIP-gts and its relationship with the protein's physiological activity, we designed several primers with which to construct different proteins with deletion mutations; these were ¡µN1-3 (with the first three amino acids deleted), ¡µN1-6, ¡µN1-13, ¡µN1-27, ¡µN106-110, ¡µN101-110, ¡µN1-13/¡µN101-110, and ¡µN1-27/¡µN101-110. We used site-directed mutagenesis and helical wheel program predictions to verify that FIP-gts forms a helix A at the 13th amino acid from the nitrogen end. If this helix is deleted, the hydrophobic moment of the wild type falls from 0.43 to 0. This causes the molecule to lose its physiological activity and cease to form a homodimer, remaining in monomeric form. If the fifth leucine, the seventh phenylalanine, and the ninth leucine from the nitrogen end (¡µ5L/7F/9L) are also removed, the hydrophobic moment will drop to 0.1. Apart from losing its helix A structure, the molecule also loses its physiological activity. We consequently can assert that the main physiological function of FIP-gts depends on the helix A near the nitrogen end, which enables it to bind to lymphocyte T cells and produce the cascade of signals that stimulate the secretion of various hormone, and bring about the function of immunomodulation.

HPLC analysis shows that several types of Ganoderma fruiting bodies contain triterpenoids with liver-protecting effects

Faculty Advisor: Professor Su Ching Hua

Graduate student, Taipei Medical College: Yang Yi Chen

Degree: M.S.

Synopsis

The more than 50 triterpenoids found in Ganoderma are similar in form and cannot be identified using simple methods. This study used high-speed HPLC and TLC methods to analyze 64 Lingzhi samples originated from CCRCs and agricultural test stations in different nations. The scientific names of these Lingzhi species are: G.neo.Japonicum, G.formosanum, G.australe, G.calidophilum,G.mastoporum, G.weberianum, G.pfeifferi, G.resinaceum, G.lucidum, G.subamboinense var. laevisporum, G.boniense¡A G.tropicum, G.fornicatum, G.tsugae, G.curtisii, G.lobatum, G.mirabile¡AG.oerstedii. Taking ganoderic acid B and C2 as standard in an alcohol extract, it was inferred there were 18 types of Lingzhi. These 18 types conform with those used in morphological and hybrid incubation experiments at the plant pathology department of the Taiwan Agriculture Research Institute. In addition, since ganoderic acid B and C2 are purified triterpenoids with known liver-protecting efficacy, HPLC analysis was used to determine the ganoderic acid B and C2 content of each type of Lingzhi. Taking mice with acute liver malfunctions induced with carbon tetrachloride as a model, GOT, GPT, and tissue sections were used to determine the efficacy of the triterpenoids in each type of Lingzhi at easing acute liver malfunction. The results showed that when identical doses of extract containing triterpenoids (30 mg/kg of mouse weight) were given to mice, after three doses G. Tropicum proved best at eliminating the liver malfunction.

Suppression of the Platelet Coagulation Mechanism by Oxidized Triterpenes from Lingzhi

Faculty Advisor: Professor Wang Chang Te

Graduate student, Graduate School of Biology, National Tsinghua University: Su Chen Yi

Degree: Ph.D.

Synopsis

To determine the degree to which oxidized triterpenes can hydrolyze the cell membrane of platelets, we tested the hydrophobicity of different kinds of oxidized triterpenes using different solvent systems, and found that GAS is most hydrophobic. It was also found that the degree of hydrophobicity of the triterpenes is correlated with replaced bases and three-dimensional carbon structure. We compared the effect of eight types of oxidized triterpenes on platelet coagulation induced by ADP-fibrinogen and collagen, and discovered the chemical structure of each preparation, which cause them to have different degrees of suppression or promotion with regard to the effects of the two types of activator. In addition, we also observed that there were differences in platelet coagulation induced by monomeric or fibrous collagen from different sources. Because GAS content is highest in red Lingzhi, we first used GAS to investigate suppression of platelet coagulation caused by collagen from different sources. The results showed that GAS suppressed coagulation activated by collagen from two sources at different initial rates. GAS displayed greater suppressing action as concentration increased when coagulation was induced with human placental collagen, but not so when coagulation was induced with collagen from the skin of cattle.

This research also revealed that GAS promotes the production of cAMP within platelets induced by PGE1 and strengthens the protein phosphorylation of cAMP-dependent protein kinase receptors (250, 60, 50, 39, 30, 24, 22 kDa) stimulated by PGE1. G AS 7.5 (m suppressed the platelet coagulation of 47kDa and 20kDa protein phosphorylation by 10~20% when platelet coagulation was induced with human placental collagen fibers, and suppressed serotonin secretion by 35%. GAS is also able to strengthen the suppressing effect of PGE1 on collagen activation; this may be because GAS increases the synthesis of cAMP under stimulation by PGE1, which strengthens the protein phosphorylation of PKA receptor, or may be due to other suppression pathways connected with cAMP. These findings have given us a deeper understanding of the structure and function of various oxidized triterpenes. Light was also shed on mechanisms by which GAS affects platelet activation by human placental collagen fibers and strengthens the suppression of collagen activation by PGE1. It was shown that GAS is an effective anticoagulant at low concentrations, and clarified the role of GAS in enhancing the suppression of embolism formation by prostaglandin and other drugs. The above results indicate that Lingzhi is likely to be highly effective at preventing and treating embolisms and vascular disease.

Research on the Antioxidant Properties

Faculty Advisor: Professor Ling Jung Yao

Graduate student, Graduate School of Food Science, National Chungshing University: Wu Jun Yi

Degree: M.S.

Synopsis

This study compared the antioxidant properties of different types of Lingzhi, and investigated their antioxidant characteristics. Of the various types of Lingzhi in use (Ganoderma tsugae, G. formosanum, G. gibbasum, G. lucidum, Trametes versicolor), a methanol extract from G. tsugae exhibited the best antioxidant action. After different solvents were used to make extracts of G. tsugae, including n-hexane, ethyl acetate, acetone, ethanol, methanol, water, and boiling water, it was found that a methanol extract displays the strongest antioxidant properties; while its antioxidant action was superior to that of an equivalent concentration of £\-tocopherol, it was weaker than that of butylated hydroxyanisole (BHA). 

 Research on antioxidant characteristics showed that a 400ppm methanol extract of G. tsugae can suppress the auto-oxidation of linoleic acid by 93.27%. The resstoring strength of the G. tsugae extract increased as its concentration increased. In addition, a methanol extract of G. tsugae also exhibits a strong metal chelating ability, and a 400ppm extract was able to chelate 89.71% of Fe2+. On the other hand, a methanol extract of G. tsugae proved significantly weaker at eliminating free radicals. At a concentration of 200ppm, the methanol extract was able to eliminate only 41.93% of £\¡ßdiphenyl-£]-picrylhydrazyl free radicals. When 4mg were added, the extract was able to eliminate 53.48% of hydroxyl free radicals. This indicates that the antioxidant properties of G. tsugae do not derive solely from a single mechanism, but are rather the combined result of several characteristics.

G. tsugae methanol extract also displays antioxidant action against the lipid peroxidation system, which employs the different oxidation factor promoter linoleic acid. When 400ppm G. tsugae methanol extract was used to test antioxidant action against the auto-oxidation and Fe2+/H2O2-promoted oxidation systems, it was found that a respective 93.3% and 57.4% of lipid peroxidation was suppressed. When 300ppm G. tsugae methanol extract was used to test antioxidant action against the Fe3+-promoted oxidation system, it was found that 67.6% of oxidation was suppressed. It was discovered, however, that the G. tsugae methanol extract is a weaker antioxidant against the Fe2+ and Fe3+/ H2O2/ascorbic acid promoted oxidation systems than against other systems; even at the high concentration of 1,000ppm, G. tsugae methanol extract was able only to suppress a respective 29.6% and 19.3% of lipid peroxidation.

Furthermore, G. tsugae methanol extract displayed excellent antioxidant action against the membrane lipid peroxidation system of rat liver microsomes. A 200ppm methanol extract was able to achieve 92.1% suppression of NADPH-dependent lipid peroxidation. While G. tsugae methanol extract was less effective against the Fenton reaction-promoted oxidation system, it still achieved 79.2% suppression. A G. tsugae methanol extract of only 60ppm concentration achieved 94.4% suppression of lipid peroxidation by the Fe2+-promoted oxidation system. These results indicate that a methanol extract of G. tsugae potentially has the ability to protect against lipid peroxidation induced by enzymes, free radicals, and free metal ions.

To summarize the results of this study, it was found that a methanol extract of G. tsugae is an excellent antioxidant. Apart from dual type I and Type II antioxidant mechanisms, it is able to prevent oxidation damage to in vitro biological membrane lipid systems. This suggests that G. tsugae extracts may also play an antioxidant role in vivo, and prevent damage from active oxygen and free radicals.