HD-03/ES is a herbal formulation used for the treatment of hepatitis B. However, the molecular mechanism involved in the antihepatitis B (HBV) activity of this drug has not been studied using in vitro models. The effect of HD-03/ES on hepatitis B surface antigen (HBsAg) secretion and its gene expression was studied in transfected human hepatocarcinoma PLC/PRF/5 cells. The anti-HBV activity was tested based on the inhibition of HBsAg secretion into the culture media, as detected by HBsAg-specific antibody-mediated enzyme assay (ELISA) at concentrations ranging from 125 to 1000？μg/mL. The effect of HD-03/ES on HBsAg gene expression was analyzed using semiquantitative multiplex RT-PCR by employing specific primers. The results showed that HD-03/ES suppressed HBsAg production with an IC50 of 380？μg/mL in PLC/PRF/5 cells for a period of 24？h. HD-03/ES downregulated HBsAg gene expression in PLC/PRF/5 cells. In conclusion, HD-03/ES exhibits strong anti-HBV properties by inhibiting the secretion of hepatitis B surface antigen in PLC/PRF/5 cells, and this action is targeted at the transcription level. Thus, HD-03/ES could be beneficial in the treatment of acute and chronic hepatitis B infections. 1. Introduction Hepatitis B virus (HBV) infection is a major health problem throughout the world, affecting more than 350 million people who are carriers of this virus that can cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma [1]. A variety of serological markers appear following the infection with HBV, and first among these is HBsAg (hepatitis B surface antigen), which is observed two to three weeks before the clinical and biological symptoms appear. Prevalence of HBsAg in India varies from 1 to 13 percent with an average of 4.7 percent [2–4]. The molecular diagnosis which detects the HBsAg in the serum samples plays a significant role in the early diagnosis during hepatitis B (HB) infection. PLC/PRF/5 is a continuous human hepatocarcinoma cell line whose genome contains integrated HBV DNA and secretes two of the hepatitis B virus envelope proteins [5]. The cells could secrete HBsAg continuously into the culture medium [6, 7]. These cells are suitable to study the effects of drugs on HBsAg expression and secretion [6]. Since the cells do not produce infectious virion particles, it is safe to handle the cell line with biosafety level 2 containment [8]. Several antivirals are currently available for the treatment of HBV, which include IFN-α, lamivudine, entecavir, telbivudine, and tenofovir. However, interferon therapy has limited efficacy, is
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