Extract of Scutellaria baicalensis inhibits dengue virus replication

Abstract

Background: Scutellaria baicalensis (S. baicalensis) is one of the traditional Chinese medicinal herbs that have been shown to possess many health benefits. In the present study, we evaluated the in vitro antiviral activity of aqueous extract of the roots of S. baicalensis against all the four dengue virus (DENV) serotypes.

Methods: Aqueous extract of S. baicalensis was prepared by microwave energy steam evaporation method (MEGHE™), and the anti-dengue virus replication activity was evaluated using the foci forming unit reduction assay (FFURA) in Vero cells. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to determine the actual dengue virus RNA copy number. The presence of baicalein, a flavonoid known to inhibit dengue virus replication was determined by mass spectrometry.

Results: The IC(50) values for the S. baicalensis extract on Vero cells following DENV adsorption ranged from 86.59 to 95.19 μg/mL for the different DENV serotypes. The IC(50) values decreased to 56.02 to 77.41 μg/mL when cells were treated with the extract at the time of virus adsorption for the different DENV serotypes. The extract showed potent direct virucidal activity against extracellular infectious virus particles with IC(50) that ranged from 74.33 to 95.83 μg/mL for all DENV serotypes. Weak prophylactic effects with IC(50) values that ranged from 269.9 to 369.8 μg/mL were noticed when the cells were pre-treated 2 hours prior to virus inoculation. The concentration of baicalein in the S. baicalensis extract was ~1% (1.03 μg/gm dried extract).

Conclusions: Our study demonstrates the in vitro anti-dengue virus replication property of S. baicalensis against all the four DENV serotypes investigated. The extract reduced DENV infectivity and replication in Vero cells. The extract was rich in baicalein, and could be considered for potential development of anti-DENV therapeutics.

Figure 1

Cytotoxicity effects of S. baicalensis extract, AHPE-XA-09, on Vero cells. Data are presented as percentage of cell viability from triplicate assay.

Figure 2

Prophylactic effects of S.baicalensis extract against DENVs. Foci forming unit reduction assay (FFURA) was performed to evaluate prophylactic activity of the plant extract on DENVs in vitro replication (A) and the respective DENVs RNA production levels were quantified by qRT-PCR for all four DENV serotypes (B). Data from triplicate assays were plotted using Graph Pad Prism Version 5 (Graph Pad Software Inc., San Diego, CA).

Figure 3

Anti-adsorption activity of S.baicalensis extract against DENVs. Foci forming unit reduction assay (FFURA) was performed to evaluate the effect of plant extract on DENVs adsorption and attachment to the Vero cells (A) and the respective DENVs RNA production levels were quantified by qRT-PCR for all 4 DENV serotypes (B). Data from triplicate assays were plotted using Graph Pad Prism Version 5 (Graph Pad Software Inc., San Diego, CA).

Figure 4

Effects of S.baicalensis extract against DENVs intracellular replication. Foci forming unit reduction assay (FFURA) was performed to determine the antiviral activity of the plant extract on DENVs after adsorption to the Vero cells (A) and the respective DENVs RNA production levels were quantified by qRT-PCR for all 4 DENV serotypes (B). Data from triplicate assays were plotted using Graph Pad Prism Version 5 (Graph Pad Software Inc., San Diego, CA).

Figure 5

Direct virucidal activity of S. baicalensis extract against DENVs. Foci forming unit reduction assay (FFURA) was performed to evaluate the direct virucidal effects of the plant extract on DENVs (A) and the respective DENVs RNA production levels were quantified by qRT-PCR for all 4 DENV serotypes (B). Data from triplicate assays were plotted using Graph Pad Prism Version 5 (Graph Pad Software Inc., San Diego, CA).

Figure 6

Standard curve of the pure baicalein and LC/MS/MS chromatogram of AHPE-XA-09. Pure baicalein standard linear regression curve was plotted from the area under the curve based on the different intensity height determined from the chromatogram of the different concentrations of pure baicalein. The intensity of baicalein in the AHPE-XA-09 was extrapolated from the plot (indicated by a dot line) (A). LC/MS/MS chromatogram shows the intensity and retention time of baicalein in the AHPE-XA-09 (B).