Potential antiviral activities of chrysin against hepatitis B virus

Abstract

Background: Interferon and nucleos(t)ide analogues are current therapeutic treatments for chronic Hepatitis B virus (HBV) infection with the limitations of a functional cure. Chrysin (5, 7-dihydroxyflavone) is a natural flavonoid, known for its antiviral and hepatoprotective activities. However, its anti-HBV activity is unexplored.

Methods: In the present study, the anti-hepatitis B activity of chrysin was investigated using the in vitro experimental cell culture model, HepG2 cells. In silico studies were performed where chrysin and lamivudine (used here as a positive control) were docked with high mobility group box 1 protein (HMGB1). For the in vitro studies, wild type HBV genome construct (pHBV 1.3X) was transiently transfected in HepG2. In culture supernatant samples, HBV surface antigen (HBsAg) and Hepatitis B e antigen (HBeAg) were measured by enzyme-linked immunosorbent assay (ELISA). Secreted HBV DNA and intracellular covalently closed circular DNA (cccDNA) were measured by SYBR green real-time PCR. The 3D crystal structure of HMGB1 (1AAB) protein was developed and docked with the chrysin and lamivudine. In silico drug-likeness, Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties of finest ligands were performed by using SwissADME and admetSAR web servers.

Results: Data showed that chrysin significantly decreases HBeAg, HBsAg secretion, supernatant HBV DNA and cccDNA, in a dose dependent manner. The docking studies demonstrated HMGB1 as an important target for chrysin as compared to lamivudine. Chrysin revealed high binding affinity and formed a firm kissing complex with HMGB1 (∆G = - 5.7 kcal/mol), as compared to lamivudine (∆G = - 4.3 kcal/mol), which might be responsible for its antiviral activity.

Conclusions: The outcome of our study establishes chrysin as a new antiviral against HBV infection. However, using chrysin to treat chronic HBV disease needs further endorsement and optimization by in vivo studies in animal models.

Keywords: CccDNA; Chrysin; HMGB1; Hepatitis B virus; In silico.

Fig. 1

A and B represents optimized two-dimensional molecular geometries of anti-HBV compounds lamivudine (nucleoside analogue used as a reference drug in molecular docking analysis only) and chrysin respectively

Fig. 2

Represents cytotoxicity of chrysin measured by MTT assay. Cells seeded in 96-well plate and were incubated overnight. Cells were treated with different concentrations of chrysin. Untreated wells act as a control. In an ELISA reader, the absorbance of the MTT formazan was determined at 450 nm. The percentage of absorbance (OD) of treated cells to untreated cells was measured as % viability. Data presents mean ± standard deviations (SD) of three independent experiments carried out in triplicate. ^∗p < 0.05, **p < 0.01, compared with control

Fig. 3

Inhibitory effects of chrysin, in a dose dependent manner, on the levels of HBsAg in HepG2 cells transfected with 1.3X pHBV (replication competent) plasmid. Commercial ELISA kit was used for the detection of HBsAg in the culture supernatants after 72 h incubation. The data are presented as mean ± S.D from three independent experiments

Fig. 4

Inhibitory effects of chrysin, in a dose dependent manner, on the levels of HBeAg in HepG2 cells transfected with 1.3X pHBV (replication competent) plasmid. Commercial ELISA kits were used for the detection of HBeAg in the culture supernatants after 72 h incubation. The data represents mean ± S.D from three independent experiments

Fig. 5

Inhibitory effect of chrysin on the secretion of extracellular HBV DNA in HepG2 cells transfected with 1.3X pHBV in a dose dependent manner. Cells were seeded into 24 well plates and were subjected to the tested compound chrysin and subsequently incubated for 72 h. Samples of HepG2 culture supernatant was collected and the HBV DNA quantified by real-time PCR. Untreated cells acted as control. Each experiment was done in triplicate. The data represents mean ± SD from three independent experiments (ns is non-significant, ***p < 0.001 compared with control)

Fig. 6

Inhibitory effects of chrysin on cccDNA in a dose dependent manner after 72 h of treatment. Cells in untreated wells serve as control. Data are presented as mean ± SD of three independent experiments. ^∗p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared with control

Fig. 7

A Ball and socket cartoon model (blue) of the ligand (lamivudine) positive control interacting with the protein (HMGB1), B 2-D representation of various types of interactions (showed in color codes) of the ligand (lamivudine) in ball and socket model (blue) with specific amino acid residues of the protein

Fig. 8

A Ball and socket cartoon model (blue) of the ligand (chrysin) interacting with the protein (HMGB1), B 2-D representation of various types of interactions (showed in color codes) of the ligand (chrysin) in ball and socket model (blue) with specific amino acid residues of the protein