The Study Of New Medical Material Of Shelf Fungus By Fermentation And The Molecular Mechanism Of Hispolon From Shelf Fungus For Its Antitumor Effect

Shelf fungus are a large group of terrestrial fungi in the family of Polypores.Some of them(Phellinus lintenus,Fomes fomentarious,Phellinus chrysoloma)are traditional medicinal mushrooms in China,which have been widely used for the treatment of various diseases,including gastroenteric disorder,peptic ulcers,lymphatic disease and various cancers.However,there is little information available concerning their active components,functional localization,or mechanism.Moreover,It becomes more difficult to obtain the fruiting body of these mushrooms because their habitat is being destroyed.Therefore,it's very important to protect these mushrooms and search for substitutes of the natural products of the fungus,Which has been a major area for drug development from traditional Chinese medicine.In the last decades,there are still two problems for drug development from traditional medicinal mushrooms:1.It becomes more difficult to obtain the fruiting body of these medicinal mushrooms;2.The function mechanism of these medicinal mushrooms is unclear.In the present study,Fomes fomentarious L8 was cultivated by submerged culture, and the optimization of submerged culture conditions and nutritional requirements of mycelial biomass from F.fomentarius was studied.Therefore,the substitutes of the natural products of the fungus were obtained,which might be a model for study other Shelf fungus.Next,we evaluated the anticancer effect of these medicinal mushrooms and found that hispolon,a polyphenol compound,existed in some of these mushrooms. Furthermore,the molecular mechanism of hispolon for its anticancer effect was studied.We found that hispolon induced apoptosis in human gastric cancer cells through a ROS-mediated mitochondrial pathway.To our best knowledge,the mechanism of apoptosis in this study has not been demontrasted.The main results of this study are as follows:1.The strain L8,originally isolated in this laboratory from the fruiting body of Fomes fomentarius,was identified by morphologic,physiological and ITS-5.8S rDNA sequencing analysis.The sequence of the ribosomal RNA gene from the isolated strain was identified as that of F.fomentarius(GeneBank accession no.DQ 513402).Then, the optimization of submerged culture conditions and nutritional requirements of mycelial biomass from F.fomentarius was studied using single factor experiment and orthogonal matrix method.We obtained an optimized culture requirements and conditions in a 15 L autocontrol bioreactor with working volume 9 L.The optimal medium for mycelial biomass was as follows:5%glucose,1%silkworm chrysalis, 0.5%yeast extract,0.05%KH₂PO₄,and 0.05%MgSO₄;and the optimal culture conditions was:temperature 25℃,initial pH 5.0,agitation rate 100 rpm/min,aeration rate 1.0 vvm,inoculate volume 10%(v/v),and working time 6 days.Under the optimal culture condition,the maximum mycelial biomass reached 18.11±1.08 g/L,which is about 2.3 times higher than that at the basal medium.2.Next,we investigated the in vivo effect of the mycelium of L8 on ICR mice bearing implanted S₁₈₀cells.We found that L8 could effectively inhibit the solid tumor growth,with the mean growth inhibitory 23.5%,compared with the control group (p＜0.05).Further study indicated that the mean weight of thymus in L8 group is significantly higher than the control group(p＜0.05);but there is no statistical difference between control group and L8 group on the mean weight of spleen.In contrast,both the mean weight of thymus and spleen in cisplatin group(positive control,1.25 mg/kg) were lower than the control group,although the mean growth inhibitory reached 64.7% in cisplatin group,compared with the control group.Another observation was that the mean weight of ICR mice bearing implanted S₁₈₀cells became higher after L8 treatment.Taken together,these results suggest that the mycelium of L8 could be useful for the treatment of cancer. 3.we used some human cancer cell lines(gastric cancer,liver cancer,colon cancer, oral cancer)as a screening model,and evaluated the anticancer effect of 10 Shelf fungus,which were collected from the mountain of Changbai Shan,Jilin,China.Then we analysed the active components of these medicinal mushrooms and found that hispolon,a polyphenol compound,exsisted in some of these mushrooms.Further study indicated that hispolon showed a dose-dependent inhibition of some human cancer cells proliferation,and it is more toxic towards human gastric cancer cells(SGC-7901), with IC₅₀lower than 10μg/mL after treatment for 72 h.4.Next,we investigated the effects of hispolon on human gastric cancer cells and further examined the cell death mechanism by some kinds of methods in cancer cell biology and molecular pharmacology(such as fluorescent observation,flow cytometry, and Western-blot).We found that hispolon induced apoptosis in human gastric cancer cells through a ROS-mediated mitochondrial pathway,which was associated with a significant increase in the levels of intracellular ROS.Investigating the mechanism by which gastric cancer cells undergo apoptosis in response to hispolon treatment,we found that hispolon abolished the glutathione antioxidant system through depletion of cellular GSH and inhibition of GPX enzyme activity resulted in severe ROS accumulation.Excessive ROS caused oxidative damage to the mitochondria membranes and impaired the membrane integrity,leading to cytochrome c release, caspase activation and apoptosis.In contrast,there is no obvious cell death or ROS increase in human normal gastric cells after hispolon treatment.The degrees of ROS accumulation and cell death induced by hispolon were dependent on endogenous ROS generation.The heightened basal level of ROS in gastric cancer cells seem responsible for their high sensitivity to hispolon.Because gastric cancer cells depend on GSH to impair the active ROS output,abolishment of the GSH antioxidant system by hispolon would severely affect these cells,leading to oxidative damage and cell death.In contrast,it is less likely to induce such severe ROS stress in normal gastric cells,due to their low basal ROS output.5.We found that Low doses of hispolon hispolon dramatically enhanced the cytotoxic effects of other chemotherapeutic agents(such as MMC,5-FU and DOX) commonly used in the treatment of gastric cancer.Further study indicated that the combination of hispolon and MMC,5-FU,or DOX synergistically caused severe ROS accumulation in SGC-7901 cells,leading to massive cell death.Taken together,these findings will contribute to the search for substitutes of the natural products of these medicinal mushrooms,and exploring the molecular mechanism of active components from these medicinal mushrooms.Moreover,this study could be useful for the drug development from traditional Chinese medicinal mushrooms.