Effect Of Total Saponins Of Semenlitchi Against Duck Hepatitis B Virus

Objective:1.To extract total saponins of semen litchi and establish the method for determination of content of total saponins of semen litchi.2.To observe the effect of total saponins of semen litchi on the treatment of duck hepatitis B.Methods:1.The method of ethanol reflux extraction was used.Total saponins of semen litchi was extracted by using 50%ethanol.Petroleum ether was used to remove the fat-soluble components and water-saturated butanol extraction method was used to purified the total saponins of semen litchi.With ginsenoside Rg1 as standard,standard curve was prepared.And then the content of total saponins of semen litchi was measured by using vanillin-glacial acetic acid-Perchloric acid colorimetric method.2.One-day-old Guangxi ducks were intraperitoneally injected with duck hepatitis B virus DNA strong positive serum,0.25ml each duck,to establish the animal model of duck hepatitis B virus.60 Guangxi ducks successfully infected with duck hepatitis B virus were randomly divided into five groups:model control group,positive control drug acyclovir group(ACV group)and the high,middle and low-dose total saponins of litchi groups.Each group had 12 ducks.The model control group were given 2mL/kg · d double distilled water,the remaining 4 drug treatment group were given high-dose total saponins of semen litchi(0.64g/kg · d),middle-dose total saponins of semen litchi(0.32 g/kg · d),low-dose total saponins of semen litchi(0.16 g/kg · d)and the positive control drug acyclovir(ACV)for treatment.Ducks in each group were administered with drugs continuously for two weeks.The changes of DHBV DNA,DHBsAg and DHBeAg OD values,ALT and AST activity were observed before treatment(T0),7 days(T7)and 14 days(T14)after the medication and 5 days after stopping therapy(P5)in the serum of ducks.Finally all duck were killed and their liver was taken for pathological examination.3.The statistical analysis of data:Data of duck hepatitis B virus DNA,DHBsAg and ALT which meet the sphericity test wre analyzed by analysis of variance for repeated measurement data.Data of duck hepatitis B virus DNA,DHBeAg and AST which did not meet the sphericity test wre analyzed The data for effect of therapeutic drugs on duck liver histopathology belonged to ranked data.Wilcoxon rank sum test was used for statistical analysis.All data were analyzed by using SPSS 16.0 for windows statistical software.Results:1.The relative standard deviation(RSD)of precision test and stability test were 1.73%and 1.10%,respectively.The average recovery rate was 98.47%and the RSD of recovery rate was 1.90%.193.42 g total saponins powder of semen litchi was obtained after 3 kg semen litchi coarse powder extracted and purified(1g total saponins of semen litchi is equivalent to 15.51 g crude drug).The average yield of total saponins of semen litchi was 6.45%and the average purity was 49.06%.2.The effect of total saponins of semen litchi on the treatment of duck hepatitis B.(1)After ducks were given drugs for treatment,serum DHBV DNA of ducks in the ACV group,high-dose and middle-dose total saponins of semen litchi group was significantly lower than that of the model group.The differences between the three treatment groups and the model control group were statistically significant(P<0.01).It was suggested that ACV,high and middle dose total saponins of semen litchi could effectively inhibit the replication of duck hepatitis B virus.However,compared with the model control group,The duck hepatitis B virus DNA of low-dose total saponins of semen litchi group did not decline obviously.The difference was not statistically significant(P>0.05).It was suggested that we were still unable to conclude that low-dose total saponins of semen litchi inhibit the replication of duck hepatitis B virus.The experimental results also showed that the effect of total saponins of semen litchi against duck hepatitis B virus had a certain relationship with dose size of total saponins of semen litchi.(2)After ducks were given drugs for treatment,compared with the control group,the serum DHBsAg and DHBeAg in the duck of each drug treatment group had no obvious changes.The differences of DHBsAg and DHBeAg OD values between each drug group and the model control group had no statistical significance(P>0.05).So we could not think that total saponins of semen litchi could reduce DHBsAg and DHBeAg activity in serum of duck.(3)After ducks were given drugs for treatment,compared with the control group,the activity of AST and ALT in duck serum did not reduce significantly.There was no significant differences in AST and ALT activity between each drug treatment group and the model control group(P>0.05).It still could not believe that total saponins of semen litchi had effect on reducing AST and ALT activity in duck serum.(4)Five days after stopping treatment,the duck livers were taken to make pathological sections for the pathological examination.Examination results found that ACV,high and middle dose of total saponins of semen litchi had obvious protective effect on duck liver damage caused by duck hepatitis B virus(P<0.05).While low dose of total saponins of semen litchi group compared with the model control group,the difference was not statistically significant(P>0.05).It could not indicate that low dose of total saponins of semen litchi has a protective effect on duck liver damage caused by duck hepatitis B virus.Conclusions:1.Vanillin-glacial acetic acid-Perchloric acid colorimetric method is an economic,simple,accurate analysis method whose precision and stability are good.This method can be used for the determination of content of total saponins of semen litchi.The yield and purity of total saponins of semen litchi are good.2.Total saponins of semen litchi have significant effect on anti-duck hepatitis B virus,and its antiviral effect on hepatitis B virus in vivo has a certain relationship with dose size and time;This experiment did not find that the total saponins of semen litchi could inhibit the expression of DHBsAg and DHBeAg and reduce AST and ALT activity.However,this study finds that total saponins of semen litchi can improve liver injury caused by duck hepatitis B virus.