The anti‑hepatitis B virus activity of sea buckthorn is attributed to quercetin, kaempferol and isorhamnetin

Abstract

The present study assessed the in vitro anti-hepatitis B virus (HBV) effects of cold-adapted sea buckthorn (Hippophae rhamnoides). Sea buckthorn leaf ethanol extracts subjected to chloroform (SB-Chl), ethyl acetate (SB-Eac), n-butanol (SB-But) and aqueous (SB-Aqu) fractionation were first examined (MTT assay) for their toxic effects on HepG2 cells. While SB-Chl (IC₅₀, 32.58 µg/ml) exhibited high cytotoxicity, SB-Eac, SB-But SB-Aqu were non-toxic at up to 150 µg/ml. High performance liquid chromatography analysis led to the identification of the anti-HBV active flavonols, quercetin (93.09 µg/g), kaempferol (44.19 µg/g) and isorhamnetin (138.75 µg/g) in the extract. The analysis of the anti-HBV effects of SB-Eac, SB-But and SB-Aqu (50 µg/ml, each) on HepG2.2.15 cells revealed the marked inhibition of HBsAg and HBeAg expression levels. At the concentration of 10 µg/ml, quercetin and kaempferol exerted potent inhibitory effects on HBsAg (60.5 and 62.3%, respectively) and HBeAg synthesis (64.4 and 60.2%, respectively), as compared to isorhamnetin (30.5 and 28.4%, respectively). The HBV-polymerase inhibitor drug, lamivudine (2 µM), inhibited HBsAg and HBeAg expression by 87.4 and 83.5%, respectively. The data were in good agreement with a previous in vitro and in silico molecular docking analysis performed by the authors where quercetin, kaempferol and lamivudine had formed stable complexes with HBV-polymerase binding-pocket amino acids. On the whole, to the best of our knowledge, the present study provides the first report of the anti-HBV therapeutic potential of sea buckthorn, attributed to quercetin, kaempferol and isorhamnetin.

Keywords: anti-hepatitis B virus; hepatitis B virus; isorhamnetin; kaempferol; quercetin; sea buckthorn.

Figure 1

Sea buckthorn (Hippophae rhamnoides L.) whole plant collected from Ladakh, India (upper panel) and the chemical structures of its bioactive flavonols: Quecrcetin, isorhamnetin and kaempherol (lower panel).

Figure 2

High performance liquid chromatography chromatograms of standard flavonols: quercetin, kaempferol and isorhamnetin (upper panel) and sea buckthorn extract (lower panel).

Figure 3

Cell viability or cytotoxicity assay of HepG2 cells treated with various concentrations of the sea buckthorn SB-But, SB-Chl, SB-Eac and SB-Aqu fractions, as well as DMSO (0.1%). The untreated control served as a negative control. Data are presented as the mean ± SEM (n=3); ^***P<0.001 vs. UT. UT, untreated control; SB-But, n-butanol fraction; SB-Chl, chloroform fraction; SB-Eac, ethyl acetate fraction; SB-Aqu, aqueous fraction.

Figure 4

The anti-HBV assay showing concentration-dependent (left panel) and time-dependent (right panel) inhibitions of HBV surface or ‘s’ antigen (HBsAg) by sea buckthorn SB-But, SB-Eac and SB-Aqu fractions in HepG2.2.15 cells. Lamivudine (2 µM) served as a reference or positive control, while DMSO (0.1%) functioned as the untreated control. Data are presented as the mean ± SEM (n=3); ^**P<0.01 and ^***P<0.001 vs. UT. UT, untreated control; SB-But, n-butanol fraction; SB-Eac, ethyl acetate fraction; SB-Aqu, aqueous fraction; LAM, lamivudine.

Figure 5

The anti-HBV assay showing time-course inhibitions of HBV surface or ‘s’ antigen (HBsAg) by sea buckthorn-derived isorhamnetin (5, 10 and 20 µg/ml), quercetin (10 µg/ml) and kaempferol (10 µg/ml) in HepG2.2.15 cells. Lamivudine (2 µM) served as a reference or positive control, while DMSO (0.1%) functioned as the untreated control. Data are presented as the mean ± SEM (n=3); ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. UT. UT, untreated control; IRM, isorhamnetin; QRC, quercetin; LAM, lamivudine.

Figure 6

The anti-HBV assay showing time-course inhibitions of HBV pre-core or ‘e’ antigen (HBeAg) by the sea buckthorn SB-But, SB-Eac and SB-Aqu fractions (50 µg/ml, each), as well as isorhamnetin (10 µg/ml), quercetin (10 µg/ml) and kaempferol (10 µg/ml) in HepG2.2.15 cells. Lamivudine (2 µM) served as a reference or positive control, while DMSO (0.1%) functioned as the untreated control. Data are presented as the mean ± SEM (n=3); ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. UT. UT, untreated control; SB-But, n-butanol fraction; SB-Eac, ethyl acetate fraction; SB-Aqu, aqueous fraction; IRM, isorhamnetin; QRC, quercetin; LAM, lamivudine.