








key: cord-352988-9ey3ir5e
authors: Xiang, Yang-fei; Ju, Huai-qiang; Li, Shen; Zhang, Ying-jun; Yang, Chong-ren; Wang, Yi-fei
title: Effects of 1,2,4,6-tetra-O-galloyl-β-D-glucose from P. emblica on HBsAg and HBeAg secretion in HepG2.2.15 cell culture
date: 2010-10-08
journal: Virol Sin
DOI: 10.1007/s12250-010-3144-y
sha: 
doc_id: 352988
cord_uid: 9ey3ir5e

A polyphenolic compound, 1,2,4,6-tetra-O-galloyl-β-D-glucose (1246TGG), was isolated from the traditional Chinese medicine Phyllanthus emblica L. (Euphorbiaceae) and assayed for its potential as an anti-hepatitis B virus (HBV) agent. The cytotoxicity of 1246TGG on HepG2.2.15 as well as HepG2 cells was determined by observing cytopathic effects, and the effects of 1246TGG on secretion of HBsAg and HBeAg in HepG2.2.15 cells were assayed by enzyme immunoassay. Results indicates that treatment with 1246TGG (6.25 μg/mL, 3.13 μg/mL), reduced both HBsAg and HBeAg levels in culture supernatant, yet the inhibitory effects tend to decline with the assay time. This study provides a basis for further investigation of the anti-HBV activity and possible mechanism of action of 1246TGG.

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 [7] . China has the biggest HBsAg carrier population with more than one-third of the world's 350-400 million chronic HBV carriers [11] . Though and hepatitis B e antigen (HBeAg) [12] . Currently, two therapies, conventional interferon alfa (IFNα) and 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) [15] 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 [4] .

Compound 1246TGG was isolated and its structure was identified by the State Key Laboratory of Phytochemistry and Plant Resources in West China in the Kunming Institute of Botany, Chinese Academy of Sciences, using procedures as described in a previous paper [18] . Briefly, the ethanol extract of the fresh leaves and branches of P. emblica was suspended into water and then extracted with diethyl ether. The diethyl ether layer was partitioned between hexane and methanol, and the methanol layer was further chromatographed successively over Sephadex LH-20, silica gel, MCI-gel CHP 20P and Chromatorex ODS to obtain the desired compound (purity > 95%) in the form of a pale amorphous powder. Its structure was identified by comparison of the physical and spectral data with literature values and the 1H-1H COSY spectrum (Fig. 1) . The isolated compound was then dissolved in dimethyl sulfoxide (DMSO) before use.

The final concentration of DMSO was less than 0.2%.

HepG2 and HepG2. only 5% FBS was added. Cells were cultured at 37°C in a humid atmosphere with 5% CO 2 .

The cytotoxicity assay was performed by observing cytopathic effect (CPE). HepG2 or HepG2. day. Cytopathic effects were classified into five levels as follows: >75%, between 75% and 50%, between 50% and 20%, <25% and no cytopathic effect. The assay was performed in four parallel wells.

Concentrations without cytotoxicity were used for HBsAg and HBeAg inhibition assay.

For HBsAg and HBeAg secretion assay, HepG2.2.15 cells were seeded onto 24-well tissue culture plates (Corning) 3×10 4 cells/well and incubated at 37℃ in a humid atmosphere with 5% CO 2 for 24 h before the test. Similarly, 1246TGG at two concentrations (6.25 µg/mL and 3.13µg/mL) were diluted in maintenance media and added every 3 d during the 10 d treatment period, namely, on the 1st, 4th and 7th day. Before the second treatment of 1246TGG (on the 4th and 7th day) and at the end of the treatment period (on the 10th day), culture media of each compound concentration was collected and stored at -20℃. HBsAg and HBeAg levels in culture media were measured using an enzyme immunoassay kit (InTec) according to the manufacturer's instructions and absorbance at 450nm was measured using an ELISA reader (Bio-Rad). The assay was performed in four parallel wells.

Results were expressed as  or ±S.D. of four parallel wells. Statistical calculations were carried out with the SPSS 13.0 for Windows software package (Statistica). One-Way ANOVA was used for statistical analyses; P values < 0.05 were considered to be significant.

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 

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 

Polyphenols, especially flavonoid, phenolic acids and other derivates might be potential antiviral agents [14] . Among these, galloyl glucoses, with various number of galloyl groups in the glucose core structure, penta-O-galloyl-β-D-glucose (PGG), was found to be efficient in inhibiting the NS3 protease of HCV [1] .

PGG also decreased extracellular HBV in a dosedependent manner in HepG2.2.15 cell culture [8] . In this study, 1246TGG was isolated from traditional Chinese medicine P. emblica and its activity in affecting HBV antigen secretion was reported for the first time.

1246TGG showed cytotoxicity towards HepG 

Antiviral compounds from traditional Chinese medicines Galla Chinese as inhibitors of HCV NS3 protease

Inhibitory effects of Egyptian folk medicines on human immunodeficiency virus (HIV) reverse transcriptase

Inhibition of herpes simplex virus infection by tannins and related compounds

A cell culture assay for compounds which inhibit hepatitis B virus replication

Human hepatic cell uptake of resveratrol: involvement of both passive diffusion and carrier-mediated process

Resveratrol in human hepatoma HepG2 cells: metabolism and inducibility of detoxifying enzymes

Hepatitis B virus infection

In vitro antiviral activity of 1,2,3,4,6-penta-O-galloyl-beta-Dglucose against hepatitis B virus

Studies on antiviral activity of several hydrolyzable tannins

Sulfated pentagalloyl glucose (Y-ART-3) inhibits HIV replication and cytopathic effects in vitro, and reduces HIV infection in hu-PBL-SCID mice

Management of chronic hepatitis B: experience from China

The hepatitis B virus

Ethnopharmacology of Phyllanthus emblica L

Current status of natural products from plants as anti-herpes simplex virus 1 agents

Respiratory syncytial virus infection induces matrix metalloproteinase-9 expression in epithelial cells

Anti-cancer, anti-diabetic and other pharmacologic and biological activities of penta-galloyl-glucose

Contemporary clinical research of traditional Chinese medicines for chronic hepatitis B in China: an analytical review

Phyllanemblinins A-F, new ellagitannins from Phyllanthus emblica



