Study On Anti-hepatitis B Effects And Its Mechanisms Of Total Flavones From Desmodium Multiflorum Dc.

Desmodium multiflorum is a leguminosae herb,distributed in Guangxi,Guangdong,Jiangxi,Fujian and Yunnan provinces in southern China.The main function of the whole plant is to clear hot detoxify,improve digestion and relieve pain.Its main chemical constituents are composed of organic acids,steroid,terpenoids,flavones and phenols.Moreover,in China's Guangxi Zhuang autonomous region,Desmodium multiflorum has been used in folk medicine for liver protection and hepatitis.In the previous study of our research group,the ethanol extract of Desmodium multiflorum was extracted by the system solvent separation method and sequential assayed for anti-HBV activities in vitro.Previous study showed the ethylaeetate fraction was found to contain the highest level of anti-HBV activity and further indicated to probably contain many flavones.However,it has little pharmacological study about total flavones from Desmodium multiflorum,and has not been reported regarding anti-HBV effect yet.So,this issue is to study the antiviral and liver-protective effects of total flavones from Desmodium multiflorum.The research will provide scientific theory for development of new anti-HBV drugs and for further development and utilization of Desmodium multiflorum.Chapter 1 Extraction and confirmation of anti-HBV effective part of Desmodium multiflorumExperiment 1 Extraction and purification of total flavones from Desmodium multiflorumObjective:To study the extraction conditions and purification process parameters of total flavones from herb Desmodium multiflorum with alcohol extraction method and AB-8 macroporous resin adsorption.Methods:The total flavones in Desmodium multiflorum were determined through ultraviolet spectrophotometry with rutin as reference standard.The effects of 4 process parameters including ethanol concentration,liquid-to-material ratio,time of extraction and frequency of extraction on extraction efficiency were investigated by orthogonal array design.The factors affecting separation such as sample concentration,sample volume,elute concentration,elute rate and elute volume were investigated by detecting content and purity of total flavones product.Results:The optimum extraction conditions were:ethanol 60%,liquid-to-material ratio 12:1,3times for extraction,1 hour for each time.The optimum purification technological conditions were:sample concentration were 0.4g/mL(amount of crude drug),sample volume were 2 times of bed volume(BV),70%alcohol as desorption solvent with 4 times of BV,elute rate were 2mL/min.Under these conditions,the purity of total flavones was 61.47%.Conclusion:The optimized extraction and purification process is simple and feasible.Experiment 2 Confirmation of anti-HBV effective part of Desmodium multiflorumObjective:To compare the inhibitory effect of alcohol extract of Desmodium multiflorum(AEDM)and total flavones from Desmodium multiflorum(TFDM)on the anti-HBV effect in vitro.Methods:Cell counting kit-8(CCK-8)method was used to observe the toxic effects of AEDM and TFDM on HepG2.2.15 cells.The inhibitory effect of anti-HBsAg and HBeAg secreted by HepG2.2.15 cells was detected by ELISA analysis.Results:The median toxic concentration(TC50)of AEDM and TFDM was respectively 621.83?g/mL and 310.91?g/mL.The 50%inhibition concentration(IC50)of AEDM on the secretion of HBsAg and HBeAg was 179.60?g/mL and 168.59?g/mL,and the therapeutic index(TI)was 3.46 and 3.69.The IC50 of TFDM on the secretion of HBsAg and HBeAg was 56.25?g/mL ? 39.70?g/mL,and TI was 5.53 and 7.83.Conclusion:The anti-HBV antigen effect of TFDM is better than that of AEDM in vitro.The anti-HBV antigen effect in vitro is positively correlated with the content of flavone in Desmodium multiflorum.Chapter 2 Inhibitory effect of TFDM on the replication of the HBV DNA and HBV cccDNA in vitroObjective:To observe the inhibitory effect of TFDM on the replication of the HBV DNA and HBV cccDNA in vitro.Methods:HepG2.2.15 was used to control group,3TC group(100?g/mL),TFDM groups with different concentration(100?g/mL,50?g/mL,and 25?g/mL).All groups were given the appropriate culture medium;medium was changed once every 2 days.At day 6,fluorescence quantitative PCR(FQ-PCR)was used to detect extracellular HBV DNA,intracellular HBV DNA and intracellular cccDNA.Results:Compared with the control cells,every-dosage group of TFDM could significantly inhibit the viral load of HBV DNA(P<0.01)and cccDNA(P<0.05 or P<0.01).The effect of inhibiting showed an obvious dose-effect relationship.Conclusion:Our research indicates that TFDM can interfere with the replication cycle of HBV,including the formation of cccDNA in HepG2.2.15 cells.Chapter 3 Therapeutic effect of TFDM on DHBV hepatitisObjective:To study the effect of TFDM on DHBV hepatitis.Methods:One-day-old ducklings were infected with DHBV.Strong positive ducklings were detected by PCR assay after infection 7 days,and were randomly divided into 5 groups,with each consisting of 10 ducklings:model group,3TC group(50mg/kg),TFDM high-dose group(300mg/kg),TFDM medium-dose group(150mg/kg),TFDM low-dose group(75mg/kg).Additional 10 DHBV negative ducklings were serves as normal control.Drugs were administered orally daily for 14 days.The blood was drawn from jugular vein of all ducks before treatment(TO),after the medication for 7 days(T7),14 days(T14)and withdrawal of the drug for 3 days(P3).The level of DHBV DNA,DHBsAg,DHBeAg,ALT,AST,IL-2 and IFN-? were detected.Results:TFDM could significantly decrease the level of HBV DNA,DHBsAg and DHBeAg at T14 and P3,and without virus rebounding after drug withdrawal.Meanwhile,TFDM could significantly reduce the level of ALT and AST at T7,T14 and P3,significantly increase the content of IL-2 and IFN-y at T14.Conclusion:TFDM has therapeutics effect on DHBV hepatitis,and its mechanism may be contributed to antiviral,hepatic-protective,descending transaminase function and immune adjustment.Chapter 4 Protective Effects of TFDM on Immunological Liver Injury Induced by Concanavalin A in MiceExperiment 1 Effects of TFDM on immunological liver injury induced by Concanavalin A in MiceObjective:To study the effect of TFDM on immunological liver injury and its mechanism.Methods:72 mice were randomly divided into six groups:normal,model,positive control drug bifendate group(200mg/kg),TFDM high-dose group(800mg/kg),TFDM medium-dose group(400mg/kg),TFDM low-dose group(200mg/kg).After intragastric administration of corresponding drugs for 10d,mice were injected by vena caudal is with concanavalin A(Con A)20mg/kg,the control group received saline alone.Animals were sacrificed at the 8h.The index of liver was tested.ALT,AST,SOD,MDA and NO in liver tissue were examined.At the same time,the percentage of T lymphocyte subsets CD4+?CD8+cells in plasma were detected by a flow cytometer.The serum cytokines including IL-2,IFN-y,IL-4 and IL-10 were measured by using ELISA kit.Results:Compared with model group,TFDM could significantly reduce the swelling of the liver and spleen,increase thymic weight,decrease the content of ALT,AST,MDA,NO,and increase the activities of SOD in the liver.Meanwhile,TFDM could increase the percentage of CD4+cell and CD8+cell reduce the level of IL-2,IFN-y,IL-4 and IL-10.Conclusion:TFDM has protective effect on immunological liver injury in mice.Its mechanism may be closely related to anti-oxidative effect,regulation of T-cell subsets and decreasing inflammatory cytokines.Experiment 2 Acute toxicity study of TFDM in MiceObjective:To evaluate the safety of TFDM,and to explore the maximum oral administration dosage.Methods:20 mice were randomly divided into two groups:control group and TFDM group.TFDM group was treated with TFDM with gastric infusion at maximum concentration an volume,while control group was given equal dose of distilled water.The behavior,appearance,body weight,food intake and excrement,the general changes of major organs and death were observed in both groups within two weeks after gastric infusion,and the maximum administration dosage in mice was converted.Results:After administering of TFDM,no obvious abnormality behavior was found in all mice.Body weight in TFDM group showed normal growth and development.No death within 2 weeks in both groups.The maximum administration dosage is as 1157 times as clinical human adult dose.Conclusion:Toxicity of TFDM is very low with oral administration.Chapter 5 Effects of TFDM on the the transcription and translation of some antiviral protein in HepG2.2.15 cellObjective:To observe the effect of TFDM on the the transcription and translation of some antiviral protein in HepG2.2.15cell and to explore the plausible mechanism of TFDM on anti-HBV effect.Methods:HepG2.2.15 was used to control group,TFDM groups with different concentration(100?g/mL,50?g/mL and 25?g/mL).Appropriate culture medium was changed once every 2 days,and cells were collected at 6d after drugs intervention.The mRNA levels of signal transducers and activators of transcription 1(STAT1),signal transducers and activators of transcription 2(STAT2),2',5'-Oligoadenylate synthetase(OAS1),RAN-dependent protein kinase(PKR)and myxovirus resistance protein A(MxA)were quantified by real-time PCR.The protein levels of STAT1,STAT2,OAS1,PKR and MxA was detected by immunohistochemical and Western Blotting.Results:TFDM could increase the intracellular STAT2,PKR and MxA mRNA level(P<0.05)and enhance PKR and MxA protein level(P<0.01).Conclusion:TFDM could induce the transcription and translation of PKR and MxA in JAK-STAT signaling pathway.The anti-HBV effect of TFDM might be contributed to JAK-STAT signaling pathway.