Study on the Chemical Constituents and anti-HBV Activity of Hypericum japonica

The most common cause of chronic viral hepatitis worldwide is from the Hepatitis B virus (HBV). Screening the safe and effective anti-HBV compounds in traditional Chinese medicine provides a large opportunity for the development of anti-HBV drugs. We found the ethanol extract of H. japonica had the besr inhibitory effects on HBeAg secretion from 20 species of medicinal plants In this study, silica gelSephadex LH-20. and a thin layer of chromatography were used to screen and isolate active anti-hepatitis B virus compounds from Hypericum japonica that was extracted with 60% ethanol. Four compounds were separated from the ethyl acetate extract of H•japonica and their structures were identified by spectral chemical and structural analyses. In addition, using HepG2 2.2.15 cell line with the cytotoxicity and inhibitory effecting on the secretion of HBeAg and HBsAg of the 4 isolated compounds were examined by MTT and enzyme-linked immunosorbent assays. The four isolated compounds were determined and 1,3,6,7-tetrahydroxy-9H-xanthen-9one (Compound 4) exhibited the highest activity in inhibiting secretion of HBeAg in HepG2 2.2.15cells. The results indicated a 67.66% of inhibition rate, 40.25 μg /mL of IC50 value and TI>2. We conducted a qualitatively evaluated research using duck hepatitis B virus model in vivo, the results indicated that ethanol extracts of H. japonica had anti-HBV effects and almost no sign of disease rebound after drug withdrawing.But the compound 4 has no direct inhibitory effect on HBV-DNA in duck experiment.


Introduction
The Hepatitis B virus (HBV) was discovered in 1966, but even after 50 years, it is still a major global public health problem [1], especially in Asian and African countries.Approximately 350-400 million people are affected with HBV in the world, and up to 1 million suffer from are HBV-related complications every year, including viral hepatitis and hepatocellular carcinoma(HCC) [2].According to a national serological survey in 2006, China has one of the highest carrier rates of the Hepatitis B surface antigen (HBsAg), reaching 7.18% of the general population [2][3][4][5][6][7].Currently, a great progress is being made in the treatment of the HBV with the development of effective and well-tolerated nucleotide analogues (NAs).However, the treatments of HBV, relying on some limited available antiviral drugs, are not successful in most of the cases.There are still some issues with the progress.First, there are still no highly effective anti-HBV drugs to remove the HBV-DNA from chronic infections completely.Second, the clinical anti-HBV drugs have some side effects.
However, anti-HBV drugs of constituents from traditional Chinese herbs have some unique advantages such as high efficiency, low toxicity, and multi-target points.Therefore, screening safe and effective anti-HBV constituents in herbs is an important and necessary aspect within the research of anti-HBV drugs.
We screened out four herbs within this study: (Hypericum japonica, Fagopyrum dibotrys (D.Don) Hara, Areca catechu L and Parmelia saxatile ( L. ) Ach)with anti-HBV activity from twenty kinds herbs.The result indicated that Hypericum japonica has the potential to suppress the secretion of HBeAg effectively in a dose-dependent method.In addition, its inhibition rate was the highest out of the four herbs that were screened.

Materials and Methods
In this Duck-Model, Beijing ducks were infected with duck hepatitis B virus (DHBV) used to evaluate anti-HBV activity in this study.HepG2 2.2.15 cell line was used in this study to screen the ethanol extracts from these plants using in vitro assays.The cytotoxicity was examined by MTT assay, and the effects on the secretion of HBeAg and HBsAg was examined by enzyme-linked immunosorbent assay.

Screening of the herbs with anti-HBV activity
Within this study, the "Records of Li Folk Medicine," was used to identify 20 species of medicinal plants (table 1) and were selected to screen the herds with anti-HBV activity in our study.HepG2 2.2.15 cell line was used to screen the ethanol extracts from these plants using in vitro assays [8][9][10][11].The cytotoxicity was examined by MTT assay, and the effects on the secretion of HBeAg and HBsAg were examined by enzyme-linked immunosorbent assay(ELISA).
The data indicated that H.japonica could suppress the secretion of HBeAg effectively in the dose-dependent method and its inhibition rate was 67.66%, the IC50 value was 40.25 µg /mL.

The inhibitory effects of Ethanol extract of H. japonica on HBsAg and HBeAg
In addition, the inhibitory effects of H.japonica on HBeAg and HBsAg had better results compared with 3-TC and Kushenin..The results indicated that the ethanol extract of H. japonica did not have any inhibition on HBsAg; whereas, the ethanol extract of H. japonica had obvious inhibitory effects on HBeAg secretion, and the highest non-toxic concentration of inhibition rate was 69.06%, which was higher than that of Kushenin.The IC50 value was 14.27µg/mL and the therapeutic index (TI) > 2 (table 6); therefore, the ethanol extract of H. japonica had the further research value.The H.japonica ethanol extracts were acquired by trichloromethane, ethyl acetate, n-butanol, and water, respectively, to obtain their different fractions.The four fractions from H.japonica ethanol extracts were tested for anti-HBV activity by MTT and ELISA methods, respectively.Results indicated it had the highest inhibition rate of ethyl acetate fraction on the secretion of HBeAg in HepG2 2.2.15 cell line(table 3).This fraction showed 63.46% of inhibition rate,111.07µg/mL of IC50 value and TI>2(table 6).Consequently, it is necessary to isolate further the ethyl acetate fraction of H. japonica, which has the highest inhibition rate on the secretion of HBeAg in HepG2 2.2.15 cell line.------------------------------------------ Note：----，toxic to cells and not tested,**P<0.01,*P<0.05,ascompared with the no drug control group.

3.4The inhibitory effects of different parts of ethyl acetate fraction on HBeAg
The next step was to isolate the ethyl acetate fraction, as well as obtain 3 parts (YZ1,YZ2 and YZ3) with the active anti-hepatitis B virus by the way of silica gel chromatography.Looking at Table 4, we see that, YZ2 is of the highest inhibition rate on the secretion of HBeAg in HepG2 2.2.15 cell line.This part indicated that it had 71.57%% of inhibition rate, 85.21 µg/mL of IC50 value and TI>2(table 6).Note：----，toxic to cells and not tested,**P<0.01,*P<0.05,ascompared with the no drug control group.

Table 1 .
Inhibitory effects of 20 species of herbs on HBsAg and

Table 2 .
The inhibitory effects of H. japonica, 3-TC and Kushenin on HBsAg and HBeAg Secretion in HepG2 2.2.15 concentrations for 7 days.The HBsAg and HBeAg in the supernatants were quantified using specific ELISA kits.Data are presented as mean ±S.D. of three experiments.**P< 0.01, *P< 0.05, as compared with the no drug control group.

Table 3 .
The inhibitory effects of different fractions from H.

Table 4 .
The different inhibition parts of ethyl acetate fraction from H. japonicaon on HBeAg secretion in HepG2 2.2.15

Table 5 .
The inhibitory effect of the four compounds of the ethyl acetate fraction from H. japonica on HBeAg secretion in

Table 6 .
Evaluation of anti-HBV efficacy of the active site and isolated compound of H. japonica Extract IC 50 (μg/mL) TC 50 (μg/mL) TI