Synthesis,Purification And Hypolipidemic Effect Of Total Triterpenoids From Inonotus Hispidus

Inonotus hispidus is a kind of parasitic fungus which lives on deciduous trees like fraxinus and morus,mostly distributes in Beijing Province,Hebei Province,Heilongjiang Province and other northern temperate regions in China.In Xinjiang Province and the northeast region of China,Inonotus hispidus has been used to treat dyspepsia,cancer and diabetes.There are not only a large number of tawny pigments which possess antibacterial,antioxidative and anti-inflammatory activities,but also an important active ingredient named triterpenoids in Inonotus hispidus.In this study,the effect of compound elicitor and its concentration on biosynthesis of total triterpenoids in Inonotos hispidus were investigated.The effect of the optimal elicitor combination on activities of catalase(CAT)and superoxide dismutase(SOD)were also studied.Furthermore,the mycelium induced by optimal elicitor combination and the fruiting body collected from fraxinus were purified.Finally,Finally,the hypolipidemic in vitro effect of total triterpenoids was investigated.The results are as follows:In this study,MeJA,SA,oleic acid and Cu2+ were compounded and added to the culture medium.the combination and concentration of these elicitors were optimized by the accumulation of mycelium and total intracellular triterpenoids.Furthermore,the effect of the optimal elicitor combination on activities of CAT and SOD were also studied.The results showed that the highest values of mycelium and total intracellular triterpenoids were 3.98 g and 85.17 mg/g,with treatment of 2%oleic acid before fermentation(day 0)and 100 ?mol/L MeJA on day 6.Defense enzyme activity determination showed that the activities of CAT and SOD were promoted with treatment of 2%oleic acid and 100 ?mol/L MeJA.Activity of SOD was the highest.On day 2,SOD activity of Inonotus hispidus in the culture medium containing 2%oleic acid was up to 78.70 U/mgprot.Activity of SOD decreased with the increase of days.After day 6,SOD activity of the control and culture medium containing 2%oleic acid were increased again,with treatment of 100 ?mol/L MeJA.The highest values were 59.70 U/mgprot and 68.69 U/mgprot.In this study,the organic solvent extraction and silica gel purification method were used together on fruiting body and mycelium from Inonotus hispidus.The effect of organic solvent,height-to-diameter ratio(height of silica gel:diameter of silica gel),sample mass-to-silica gel mass,and eluent ratio on purity of total triterpenoids were studied.Then,it was compared with other purification methods.The structure of total triterpenoids was analyzed by infrared spectroscopy.The results showed that the optimal parameters were height-to-diameter ratio of 55:22,sample mass-to-silica gel mass of 4:80,acetone and petroleum ether as elution system with ratio of 3:1.Under this condition,the highest purity of total triterpenoids extracted with ethyl acetate from mycelium and fruiting body were 65.35 and 42.32%,which were 4.26 and 4.60 times of total triterpenoids from the crude extract,respectively.This method attained the highest purity comparing with other methods.Infrared spectroscopy analysis showed that there were differences between the purification of mycelium and fruiting bodies in-CH2,-CH3,-S-O,etc,but they both conformed to the characteristic structure of triterpenoids.In this study,the in vitro hypolipidemic ability of total triterpenoids from Inonotus hispidus was investigated by the analysis of in vitro bile salt binding ability,the establishment of HepG2 cell hyperlipidemia model,and the effect of total triterpenoids on the survival rate,lipid content and lipid profile of HepG2 cells.The results showed that after purification,The binding ability of total triterpenoids from mycelium with sodium cholate,sodium glycocholate and sodium taurocholate were the strongest.The highest values were 1.14,1.78 and 2.06 ?mol/100mg,which were 1.56,1.73 and 1.72 times of crude extract from mycelium,respectively.When the concentration of oleic acid was 400?mol/L and building time was 24 h,the lipid content in HepG2 cells was the highest,which was 2.05 times of control.Total triterpenoids could effectively reduce the contents of lipids,total cholesterol(TC),triglycerides(TG)and low-density lipoprotein cholesterol(LDL-C)and improve the content of high-density lipoprotein cholesterol(HDL-C)in HepG2 cells.When the total triterpenoids concentration was 250 ?g/mL,the lipid content decreased from 2.05 times of control to 1.21 times of control,in other words decreased by 40.98%.The intracellular TC content was 0.75 mmol/gprot,and the intracellular TC clearance rate was 41.86%.The intracellular TG content was 0.62 mmol/gprot,and the clearance rate of intracellular TG was 44.44%.The intracellular LDL-C content was 0.89 mmol/gprot,and the intracellular LDL-C clearance rate was 44.02%.The intracellular HDL-C in content was 1.18 mmol/gprot,and the growth rate of intracellular HDL-C reached 57.33%.