Studies On Chemical Constitutes And Anti-Tumor Activity Of The Mycelium Of Armillaria Mellea

Armillaria mellea（Vahl.ex Fr.）Quel.is an edible and medicinal fungus.It exists all over the world.Armillaria mellea possesses a lot of different characteristics including variant metabolite and bioactivity.Based on this,Armillaria mellea has an excellent future in the regions of biological medicine and functional food.In order to make the development and utilization of Armillaria mellea sustainable,my study was focused on the mycelium of Armillaria mellea,especially on its chemical constitution and biofunction,and further explored the mechanism of its anti-tumor activity.1.Studies on chemical constitutes and anti-tumor activity of extract from the mycelium of Armillaria melleaClassical extraction methods and HPLC method were used to extract and separate chemical constitutes from the mycelium of Armillaria mellea.We obtain ten protoilludane sesquiterpene aryl esters,including two new compounds and eight known compounds.We performed plane structure analysis on these ten compounds through NMR,and analyzed stereochemical structure and physical properties of the two new compounds.The stereochemical structure were analyzed by NOESY.The physical properties examining experiments were performed by using melting point apparatus,polarimeter,IR and UV.Pharmacological action is ancestor for the function of suppressing hyderactive liver,so we choose L02 and HepG2 cell lines as experiment materials.We detected the effects of the ten protoilludane sesquiterpene aryl esters on cell viability HepG2 and L02 cell lines by MTT.The results showed that compound 2,compound 3,compound 4,compound 7,compound 8,compound 9and compound 10 inhibited the viability of HepG2 cells.It is noteworthy that compound 10 has excellent anti-tumor activity.Acoording to pervious studies,melleolide has been used as antibacterial medicine and there were less researches to defense against tumors.Since we found its excellent activity in inhibiting the viability of HepG2 cells,we chose to study further about its role in defense against tumors.2.The mechanism research of anti-tumor activity of melleolideBase on perious experiments,we chose melleolide treat L02 cells to detect the viability of L02 cells after 12 h,24 h and 48 h.We obtained nontoxic dose of L02 which are treated with melleolide.Its IC₁₀ values were 10.59μg/mL,5.94μg/mL and 6.80μg/mL.At the same time,the results showed that melleolide inhibits the viability of HepG2 cells after 12 h,24 h and 48 h.And IC₅₀ values are 8.12μg/mL,5.56μg/mL and 4.95μg/mL.Above-mentioned results showed that melleolide possessed high efficiency and low toxicity and it was feasible for melleolide as an anti-cancer drug.Then,to find out how melleolide inhibits cell viability,we detected the effect of melleolide on cell proliferation and cell apoptosis by different concentration melleolide after 12 h,24 h and 48 h.In the aspect of cell proliferation,results of Ki67immunofluorescense and Brd U detection revealed that melleolide inhibited HepG2 cell proliferation and inhibition possessed time and dose dependence.In the aspect of cell apoptosis,we employed methods of cell morphology,flow cytometry of apoptosis and western blot.The results told us that melleolide promted apoptosis of HepG2 cell and inhibition possessed time and dose dependence.In sum,these results showed that melleolide inhibits HepG2 cell proliferation and promotes apoptosis of HepG2 cells.We continue to study the molecular mechanism for anti-tumor effect of melleolide by biological information technology and molecular biology methods.We treat HepG2 cell with melleolide and then extract total RNA.The quality of RNA samples was qualified for further experiments.Then,samples were filtered and analyzed for the content of Clean reads,the quality of base,the coverage and distribution of transcriptions.The results indicated the RNA samples were in a great quality for building bank.Whereafter,we obtained the results of changeable m RNA expression to inhibit viability of HepG2 cells in our samples through RNA-seq.By comparing melleolide-treated samples with control ones,we got the following information.First,there were 1105 genes that were up-regulated significantly in melleolide-treated samples and 1269 genes that were down-regulated.Those genes with significant changes are most involved in Breast cancer pathway,Notch signaling pathway,Estrogen signaling pathway and MicroRNA in cancer,through the results from signaling pathway analysis.Via analyzing the results of RNA-seq,we selected Notch signaling pathway with our most interests for further studies.Results showed that melleolide could regulate the expression of a series of Notch signaling pathway downstream target genes,to inhibit tumor cell proliferation and promote cell apoptosis.In summary,our results showed a strong anti-tumor ability of melleolide on hepatoma cells.The relative mechanism was assessed on HepG2 cells and Notch signaling pathway was found involved in this process.Melleolide is a drug with great potential for cancer treatment.