Metabolic Regulation And Bioactivity Of Betulinic Acid From Inonotus Obliquus

As a kind of extremely precious medicinal fungus,Inonotus obliquus contains a variety of triterpenoids,such as betulinic acid(BA),and has a variety of biological activities,such as anti-cancer,anti-HIV,anti-inflammatory,anti-viral,anti-oxidation and so on.However,in the natural environment,Inonotus obliquus grows quite slowly because it grows in an extremely cold environment.In the shake flask culture of Inonotus obliquus,the contents of triterpenoids and BA are not very high.Consequently,it is necessary to find a more effective way to obtain triterpenoids and BA in Inonotus obliquus.This dissertation mainly discussed the effects of different stimulators,including fatty acids,fungal elicitors,methyl jasmonate(MeJA)and farnesol,on the fermentation and the expression level of key enzyme genes related to the biosynthesis of BA in the submerged Inonotus obliquus.It also investigated the influence of co-cultivation with engineeringed S.cerevisiae strain ZJUQH311 on the fermentation of Inonotus obliquus.In addition,this study explored the inhibitory effects and mechanism of BA on human hepatocellular carcinoma HepG2 cells.1.The results of the submerged Inonotus obliquus induced by oleic acid and fungal elicitors showed that the optimal addition amount of oleic acid and fungal elicitors were 1.0 g/L and 45 mg/L,respectively.These stimulators could increase the content of BA in Inonotus obliquus by up-regulating the expression level of the key enzyme genes associated with BA synthesis.2.The study on the fermentation of Inonotus obliquus induced by MeJA showed that the optimal addition amounts of MeJA were 50 ?M and 10 ?M for the highest contents of triterpenoids and BA.The optimum addition time was the eighth day of the whole fermentation period.The optimal fermentation period was 12-14 days.10 ?M's addition of MeJA increased the expression levels of HMGR and SQS by 98%and 53%respectively.3.The study on the fermentation of Inonotus obliquus induced by farnesol showed that 50 ?M and 100 ?M were the best addition amounts of farnesol for total triterpenoids and BA in the submerged Inonotus obliquus.4.The results of mixed culture fermentation showed that the addition of engineering yeast ZJUQH311 in the broken state could increase the content of BA in Inonotus obliquus.5.The cell experiments were conduted by MTT assay and flow cytometry.The results showed that BA could reduce the viability rate of HepG2 cells significantly.The IC50 of BA for HepG2 cells was 52 ?g/mL,26 ?g/mL,and 17.5 ?g/mL respectively at 24 h,48 h,and 72 h.BA could induce the apoptosis of HepG2 cells,mainly for early apoptosis.BA could arrest HepG2 cells in S phase and reduce the mitochondrial membrane potential of HepG2 cells,indicating that the BA-induced apoptosis of HepG2 cells was related to the mitochondrial pathway.This study provided some novel ideas for the improvement of triterpenoids and BA production in the submerged Inonotus obliquus,and made initial contributions to the explanation of the mechanism of BA on the apoptosis of HepG2 cells.