Hepatitis B virus (HBV), an important causative pathogen of cirrhosis-related liver failure and hepatocellular carcinoma (HCC), is a public health problem of worldwide concern, and is responsible for one million deaths each year worldwide . China has the biggest HBsAg carrier population with more than one-third of the world's 350-400 million chronic HBV carriers. Though prophylactic vaccines have been widely used since 1970s, eventual elimination of HBV infection remains an unfulfilled goal.
HBV belongs to the group of animal viruses known as the hepadnaviridae. Virus particles are present in large quantities in blood during HBV infection in humans, which consist of a membrane composed of envelope and nucleocapsid proteins containing circular DNA molecule. The envelope protein carries a hepatitis B surface antigen (HBsAg) while the capsid contains the hepatitis B core antigen (HBcAg) and hepatitis B e antigen (HBeAg). Currently, two therapies, conventional interferon alfa (IFNα) and lamivudine (LAM), are widely approved for treatment of chronic hepatitis B (CHB). Traditional Chinese medicines (TCMs) have a similar beneficial effect when compared with IFN or LAM for CHB on antiviral activity as evidenced by the loss of HBeAg and HBV DNA, which endows them with potential as alternative remedies for patients with CHB.
We have previously isolated a polyphonolic compound 1, 2, 4, 6-tetra-O-galloyl-β-D-glucose (1246 TGG) from Phyllanthus emblica L. (Euphorbiaceae), which is a shrub or tree distributed in subtropical and tropical areas of the People's Republic of China, India, Indonesia, and the Malay Peninsula, and has been used in the Southwest of China for treating eczema, wart, diarrhea, and headache after a fever[13, 18]. Acyl glucoses have been shown to be potent antiviral agents against herpes simplex virus (HSV)[3, 9], human immunodeficiency virus (HIV)[2, 10], severe acute respiratory syndrome coronavirus (SARS-CoV)  as well as other viruses. Here, we investigated the anti-HBV activity of 1246TGG by detecting the HBsAg and HBeAg secretion levels in HepG2.2.15 cell culture, a cell line derived by transfection of cloned HBV DNA into human hepatoblastoma cell line HepG2 and used to assay for anti-HBV agents.
The results of cytotoxicity assay are listed in Table 1. On the 10th day (after the third treatment), 1246 TGG at concentrations ranging from 200μg/mL to 12.5μg/mL all induced cytophathic effects to different extents. To confirm whether the cytotoxicity caused by 1246TGG was specific to HBV DNA transfected cells, a parallel experiment was carried out on the HepG2 cell line and similar results were obtained (data not shown). These results indicate that 1246TGG possesses comparatively high cytotoxicity towards both HepG2.2.15 and HepG2 cells.
Table 1. Cytotoxicity of 1246TGG on HepG2.2.15 cells (x, n = 4)
To determine the inhibitory effects of 1246TGG on HBV antigen secretion, cells were treated with 1246TGG at concentrations of 6.25μg/mL and 3.13μg/mL every 3 d during the 10 d treatment period. As shown in Table 2, on the 4th day, HBsAg levels in the media supernatant was significantly reduced in the presence of 6.25μg/mL 1246TGG (P < 0.01), while 3.13μg/mL of 1246TGG also inhibited HBsAg secretion but without a significant difference compared to the untreated group. On the 7th day, both 6.25μg/mL and 3.13μg/mL of 1246TGG led to a reduction of HBsAg level in media supernatant, however, no significant differences were found compared to the untreated group. At the end of the assay, no decrease of HBsAg levels was detected in the presence of either 6.25μg/mL or 3.13μg/mL of 1246TGG. Compared to the untreated group, the relative secretion levels of HBsAg in the presence of 6.25μg/mL 1246TGG were 62%, 84% and 100% on the 4th, 7th and 10th day respectively, and that of 3.13μg/mL 1246TGG were 84%, 87% and 94% respectively.
Similar results were obtained for HBeAg secretion assay. As shown in Table 3, treatment with 1246TGG at a concentration of 6.25μg/mL significantly inhibited HBeAg secretion both on the 4th (P < 0.05) and 7th (P < 0.01) day, while 3.13μg/mL of 1246TGG also reduced HBeAg levels but without significant differences compared to the untreated group. On the 10th day, no reduction of HBeAg levels in the presence of 1246TGG was found. Compared to the untreated group, the relative secretion levels of HBeAg treated with 6.25μg/mL 1246TGG were 67%, 60% and 103% on the 4th, 7th and 10th day respectively, while that of 3.13μg/mL 1246TGG were 82%, 92% and 100% respectively.
Table 2. Effects of 1246TGG on HBsAg secretion in HepG2.2.15 cell culture (x±S.D, n=4)
Table 3. Effects of 1246TGG on HBeAg secretion in HepG2.2.15 cell culture (x±S.D, n=4)