title: The supercritical CO₂ extract from the skin of Bufo bufo gargarizans Cantor blocks hepatitis B virus antigen secretion in HepG2.2.15 cells.
abstract: The skin of Bufo bufo gargarizans Cantor has long been used for the treatment of hepatitis B in China and supercritical carbon dioxide extraction (SC-CO₂) is widely used in extracting active ingredients from natural products. The aim of present study was to assess the anti-hepatitis B virus (HBV) effect of the supercritical CO₂ extract from the skin of Bufo bufo gargarizans Cantor (SCE-BC). Cytotoxicity of SCE-BC was analyzed using an MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] assay in HepG2.2.15 cells. The hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core-related antigen (HBcrAg) concentrations in cell culture medium were determined by chemiluminescent enzyme immunoassay. HBV mRNA in cells was determined using real-time polymerase chain reaction. SCE-BC concentrations below 10(-2) μg/mL had no significant toxicity to HepG2.2.15 cells. SCE-BC at 10(-4) μg/mL effectively inhibited the secretion of HBeAg by 23.36% on day 6. It was more potent than the positive control lamivudine (100 μg/mL) in terms of the inhibition of HBeAg and HBcrAg secretion on day 6. Consistent with the HBV antigen reduction, HBV mRNA expression was markedly inhibited in comparison to the control when HepG2.2.15 cells were treated with SCE-BC. Moreover, SCE-BC had greater inhibitory activity with respect to HBeAg than to HBsAg. Since HBeAg promotes immune tolerance and persistent infection during HBV infection, the present results suggest that immune tolerance induced by HBeAg might be overcome by SCE-BC. Therefore, SCE-BC warrants further investigation.
authors.affiliation: Translational Center for Stem Cell Research, Tongji Hospital, Department of Regenerative Medicine, Tongji University School of Medicine.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Cui
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Inagaki
authors.forename: Dongliang
authors.initials: D
authors.lastname: Wang
authors.forename: Jianjun
authors.initials: J
authors.lastname: Gao
authors.forename: Fanghua
authors.initials: F
authors.lastname: Qi
authors.forename: Bo
authors.initials: B
authors.lastname: Gao
authors.forename: Norihiro
authors.initials: N
authors.lastname: Kokudo
authors.forename: Dingzhi
authors.initials: D
authors.lastname: Fang
authors.forename: Wei
authors.initials: W
authors.lastname: Tang
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D000956:Antigens, Viral / Q000378:metabolism*; D002023:Bufo bufo / Q000378:metabolism*; D002245:Carbon Dioxide; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000235:genetics; D056945:Hep G2 Cells; D006509:Hepatitis B / Q000188:drug therapy*; D006515:Hepatitis B virus / Q000187:drug effects*; D006801:Humans; D007124:Immunoenzyme Techniques; D060888:Real-Time Polymerase Chain Reaction; D012867:Skin / Q000737:chemistry*; D013778:Tetrazolium Salts; D013844:Thiazoles; D014020:Tissue Extracts / Q000494:pharmacology*
chemical_list: D000956:Antigens, Viral; D017931:DNA Primers; D013778:Tetrazolium Salts; D013844:Thiazoles; D014020:Tissue Extracts; D002245:Carbon Dioxide; C022616:thiazolyl blue