Inhibitory activities of Ranunculus japonicus Thunb. ethanol extract against hepatitis B virus

Abstract

The chronic hepatitis B virus (HBV) infection is a worldwide public health problem, which cannot be cured by current therapeutics due to the persistence of viral CCC DNA in the infected hepatocytes. Screening from medicinal herbs for anti-HBV activities showed that the ethanol extract from Ranunculus japonicus Thunb. could decrease the production of HBV e antigen (HBeAg). Further study showed that the extract had no effect on core protein expression but significantly reduced the efficiency of viral capsid assembly. The levels of viral pgRNA and total core DNA were not affected significantly. However, the ratio of RC DNA/SS DNA decreased, indicating that the conversion of RC DNA from SS DNA was delayed by the extract.More interestingly, though similar levels of RC DNA were accumulated, the CCC DNA level and its formation efficiency were reduced significantly, which was also consistent with the decreased level of HBeAg, indicating that R. japonicus Thunb. extract could inhibit the CCC DNA formation. Together, this study found that R. japonicus Thunb. extract could inhibit HBV replication at multiple steps, especially showed significant inhibitory effects on capsid assembly and CCC DNA formation.

Antiviral activities of Polygonum perfoliatum L. extract and related phenolic acid constituents against hepatitis B virus

“…The total ethanol extract (TEE) of Polygonum perfoliatum L. was prepared as described previously. 26 Briefly, dried Polygonum perfoliatum L. was powdered and extracted with 70% ethanol for 12 h at room temperature. The samples were then filtered and the solid residue was retrained and re-extracted an additional three times in the same manner.…”

“…The effects of Polygonum perfoliatum L. TEE, four fractions (PEE, EAE, CE, and WE), and the phenolic acids (protocatechuic acid, gallic acid, and ethyl caffeate) on cell viability were measured by the Cell Counting Kit-8(CCK-8) assays according to the manufacturer's instructions (EpiZyme) and as described previously. 26 Briefly, cells were cultured in 96-well plates for 24 h and then were treated with the abovementioned extracts and compounds at indicated concentrations for 3 or 5 days. The cells were then washed with phosphatebuffered saline (PBS) and incubated with 10 µl CCK-8 in a 100 µl culture medium at 37°C for 1 h. The optical density was measured at an excitation wavelength of 450 nm using a microplate reader.…”

“…HBc assembly and pgRNA packaging were analyzed as previously described. 26 Briefly, viral intact NCs in the cytoplasmic lysate were first separated by native agarose gel electrophoresis and then transferred to a nitrocellulose membrane, which was further baked in a vacuum oven at 80°C for 2 h. The membrane was then incubated in a "releasing buffer" (0.2 M NaOH and 1.5 M NaCl) for 10-20 s and then in a neutralization buffer (0.2 M Tris-HCl, pH 7.5, and 1.5 M NaCl) for 5 min with shaking. The viral pgRNA in the NCs was detected using a digoxigenin (DIG)-labeled HBV plus-strand-specific riboprobe according to the manufacturer's introductions (DIG Northern Starter Kit) and the NCs in the same membrane were subsequently detected with an anti-HBc polyclonal antibody.…”

“…HBV replicative core DNA in NCs and CCC DNA was isolated as previously described. 26,27 Briefly, for isolation of core DNA, cell lysate was incubated twice with MNase (150 U/ml) and CaCl 2 (5 mM) at 37°C for 45 min, and the NCs in the cytoplasmic lysate were collected by PEG-8000 precipitation and treated with proteinase K (0.6 μg/μl)/SDS (0.5%) to release the viral core DNA. The released DNA was then purified by phenol-chloroform extraction and ethanol precipitation.…”

“…The DNA was further treated with heat denaturation and EcoR I linearization to confirm the nature of CCC DNA, as described previously. 26…”