Studies On The Screening Of Active Extracts Of Liuyueqing And Their Hepatoprotective Effect And Action Mechanism

Studies on the screening of active extracts of Liuyueqing and their hepatoprotective effect and action mechanismAs we all know,hepatic disease,especially viral liver hepatitis,is the most harmful disease to human health.Recently,there are many achievements on the prevention and treatment of viral liver diseases.But the drugs which are used to treat the viral liver hepatitis are few clinically now.And the curative effect of these medicines is not what we wanted.Therefore,it is an important issue for us to develop new drugs to inhibit HBV.Liuyueqing is one of main herbs of compound liuyuexue(CLYX)which can inhibit HBV.Our team has done many experimental researches on CLYX.It has been proved that CLYX can protect markedly against the acute chemical liver injury induced by CCl₄,AP and D-GalN,and can significantly decrease the activities of ALT and AST and increase the contents of cytochrome P450 and cytochrome b5.CLYX also can decrease the activities of ALT and AST as well as the contents of DHBsAg,DHBeAg and DHBV-DNA in serum of Guangxi brown spot duckling infected by DHBV.In vitro,through directly adding CLYX and the serum with CLYX into the medium of HepG2.2.15 cell,the effect of CLYX on HepG2.2.15 cell was observed.CLYX can obviously inhibit the secretion of HBsAg and HBeAg and the synthesis of HBV-DNA,and this inhibition is dose-and time-depedent. That is to say,CLYX has the inhibitive effects on HBV not only in vitro but also in vivo. In the references of researching on Liuyueqing,except for pharmacognosical report of Liuyueqing done in our laboratory,there is no other reports,especially on chemical composition and pharmacology.Based on the results of the experimental research on CLYX,this research will carry on the systemic experiment for Liuyueqing:Firstly,the constituent of Liuyueqing will qualitatively be analyzed.Secondly,the different extracts will be gotten by solid-liquid isolation method.Thirdly,the active extracts of liver protective effect will be screened by these methods,including the studies on the hepatoprotection of the active extracts against chemical liver injury,the effect on immune function in vivo,the inhibitive effect of HBV,and the toxicity of cell in vitro.The mechanism of liver protective effect of the active extracts will be studied by the experiment.【Objective】To screen the active extracts from five Liuyueqing(LYQ)extracts in vivo and in vitro,and to study the effects of these extracts on HBV in vitro and their protective effect and possible mechanism against chemical acute liver injury.【Methods】1.The traditional qualitative analysis method of chemical constituent was used to analyze LYQ extracts.The isolation of LYQ constituents:The several extracts were isolated from LYQ by traditional methods with different solvent,which were petroliem, chloroform,ethyl acetate,n-butyl alcohol,and 60%alchohol.60%alchohol portion was analyzed by qualitative analysis of chemical constituent,UV scanning and high performance liquid chromatography(HPLC).2.The inhibitory effect of 60%alcoholic portion on HBV-DNA in HepG2.2.15 cell:Different concentrations of 60%alcoholic portion were added into culture fluid respectively,then the quantity of HBV-DNA in cell was detected by FQ-PCR subsequently.3.The effect of 60%alcoholic portion on acute liver injury in rats: The models of CCl₄-and D-Gal-induced acute live injuries in rat were used in the study on the protective effect of 60%alcoholic portion.The liver weight index,ALT,AST ALP,Alb, Glo,TP,T.Bil,G-GT and LDH in serum were detected.The hepatic pathological changes were examined.4.The effect of 60%alcoholic portion on acute liver injury in mice:The CCl₄-,AP-and D-GalN-induced acute liver injuries were used to study the protective effect and possible mechanisms of 60%alcoholic portion in mice.The indexes of liver,thymus gland and spleen were detected.The activities of ALT,AST,ALP and T-AOC,and Alb content in serum were determined.The activities of SOD,NO,GSH and GSH-Px as well as the levels of MDA in hepatic tissue were also measured.The hepatic pathological changes were also examined.5.The concentration of tumor necrosis factorα(TNF-α),the activity of serum lysozyme,and the indexes of thymus gland and spleen were determined in immunosuppressed mice induced by cyclophosphamide.6.The effect of LYQ extracts on HepG2.2.15 cell in vitro:The toxicity of LYQ extracts on HepG-2.2.15 cell and cell viability were evaluated by WST-8 method and TC₅₀was calculated.HBsAg and HBeAg in cell culture medium were detected by ELISA method.The inhibition ratio of antigen,IC50 and TI were calculated.7.To measure MTDs of water extract and alcohol extract of LYQ. The protective effect of water extract and alcohol extract of LYQ against acute liver injury: The activities of serum ALT and AST,and the liver weight index were detected in mice with acute liver injury induced by carbon tetrachloride(CCl₄)after the treatment with water extract and alcohol extract of LYQ.【Results】1.There were saponin,cardiac glycosides,lactone,coumarin,phenols, organic acid,amino acid,polypeptide,protein,carbonhydrates and glycoside in the liuyueqing.The result of thin-layer chromatography(TLC)contrast showed that there were not indigo and indirubin in the extract of LYQ.Different solvent were used to separate the alcohol extract of LYQ into five extracts,which were petroliem portion,chloroform portion, ethyl acetate portion,n-butyl alcohol portion,and 60%alchohol portion.The results of qualitative analysis on 60%alchohol portion showed that there was a strong absorption on 209nm wavelength and there were saponin,cardiac glycosides,lactone,coumarin and triterpenoids in it.2.60%alchohol portion could inhibit the replication of HBA-DNA in HepG2.2.15 cell and decrease the copies of HBV-DNA and could increase circulation.3. In CCl₄-and D-Gal-induced acute liver injuries of rat,60%alchohol portion could lower the weight of enlarged liver and could decrease the activities of ALT,AST,ALP,G-GT and LDH in serum as well as the content of T.Bil,and improve the content of Alb and Glo and TP.4.In acute liver injuries of mice induced by CCl₄ and AP and D-GalN,the weight of enlarged liver was decreased by the treatment with 60%alchohol portion,which could increase the weight of thymus gland.60%alchohol portion could decrease the activities of ALT and.AST and ALP and could increase the content of Alb and activities of T-AOC.60% alchohol portion also increased the activities of SOD,GSH,GSH-Px in hepatic tissue and decreased the content of MDA and NO.All results indicated that 60%alchohol portion could protect markedly against the acute liver injury.5.In five extracts of LYQ,the high and low dosage of petroliem and chloroform portion could lowered the enlarged liver index as well as the high dosage of 60%alchohol portion in CCl₄-induced acute liver injury in mice,and the activities of ALT and AST in serum were decreased in acute liver injuried mice induced by CCl₄.The high and low dosage of petroliem and chloroform and 60% alchohol portion could obviously decrease ALT activity,and AST activity was significantly decreased by the high dosage of chloroform and 60%alchohol portion.The rest of portion had no effect on activities of ALT and AST.In cyclophosphamide immunosuppressed mice, five portions could improve the weight of thymus gland and the activities of lysozyme,but they could not improve the weight of spleen.The level of TNF-αwas obviously improved by ethyl acetate and n-butyl alcohol and 60%alchohol portion.6.The effect of extracts of LYQ on HepG2.2.15 cell in vitro:â‘ Chloroform and ethyl acetate and n-butyl alcohol portion showed some toxicity,but the inhibition effect of 60%alchohol portion on cell was increased with the increase in concentration.â‘¡The results of measuring HBsAg and HBeAg in cell culture medium showed that petroliem and 60%alchohol portion could inhibit the expressions of HBsAg and HBeAg in HepG2.2.15 cell(TI＞2)and 60%alchohol portion was active extract.7.The MTDs of aqueous and alcohol extracts of LYQ were respectively 153.0g and 369.0g crude materials/kg body weight,which were 46 and 110.8 times of clinical dose.The high and low dosage of aqueous extract of LYQ and the high dosage of alcohol extract of LYQ could significantly reduce the activities of ALT and AST in serum.The low dosage of aqueous extract of LYQ could obviously decrease the activities of ALT,but could not decrease the activities of AST.The middle and low dosage of alcohol extract of LYQ could not decrease the activities of ALT and AST.【Conclusion】1.There are many kinds of chemical constituents in LYQ,such as saponin, lactone,coumarin,phenols,amino acid and so on,whose water extract and alcohol extract have hepatoprotective efffcts against acute liver injury.2.Five extracts,which are petroliem,chloroform,ethyl acetate,n-butyl alcohol,and 60%alchohol portions are gotten by solid-liquid isolation method.60%alchohol portion has a strong absorption on 209nm wavelength and possibly contains saponin,lactone,coumarin and triterpenoids.HBV is inhibited by 60%alchohol portion in vitro,which can inhibit the replication of HBV-DNA and the expressions of HBsAg and HBeAg in HepG2.2.15 cell.In vivo,it can enhance immunity function,and obviously increase the contents of lysozyme and TNF-αin serum.It can also protect markedly against the acute chemical liver injury,which decreases the activities of ALT,AST and ALP in serum and the content of MDA and NO in hepatic tissue, and increases the content of Alb and activities of T-AOC in serum and the activities of SOD, GSH,GSH-Px in hepatic tissue.Its possible mechanisms of hepatoprotection are related with the inhibition of lipid peroxidation.3.In addition,petroliem portion can enhance immunity function,protect liver and inhibit the expressions of HBsAg and HBeAg in HepG2.2.15 cell.4.Chloroform,ethyl acetate,n-butyl alcohol portions have the effects of enhancing immunity function and are toxic to HepG2.2.15 cell.Chloroform portion can also protect against the acute liver injury.