Materials and methods

Cell culture and virus

BHK-21 (ATCC No. CCL-10), African green monkey kidney cell line Vero (ATCC No. CCL-81) and the Rhesus monkey kidney epithelial cell line LLC-MK2 (ATCC No. CCL-7) were cultured in either 10% or 5% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA) in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Gaithersburg, MD) with 100 units/ml of penicillin/streptomycin solution (Pen/Strep, Merck KGaA, Darmstadt, Germany) and maintained at 37 °C with 5% CO2. C6/36 cells (ATCC CRL-1660) were cultured at 28 °C in 10% FBS in minimum essential medium (MEM, Thermo Fisher Scientific, Waltham, MA) with ambient CO2.

DENV 2 (strain 16681) was propagated in C6/36 cells as described previously [13]. Virus titer was determined by plaque assay on LLC-MK2 cells as described previously [13].

Compound preparation

OA (Additional file 1: Fig. 1) (CAS no. 480115; Chengdu Biopurify Phytochemicals Ltd., Sichuan, China) was dissolved in absolute dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO). DMSO was also used as a diluent control. Desired concentrations were obtained by further diluting in culture media from an original stock of 100 mM.

Fig. 1
figure 1

Cytotoxicity and virucidal activity of oroxylin A. A Cell viability of BHK-21 cells after 24 h incubation with OA or vehicle control was assessed by the MTT assay and data was used to calculate the CC50. B Virucidal activity of OA towards DENV was assessed by incubating stock virus with different concentrations of OA or equivalent vehicle control (DMSO) for 1 h, after which virus titer was determined by plaque assay. Data is shown as mean ± S.E.M. Cytotoxicity analysis was undertaken as four independent replicates, while virucidal activity was assessed with three independent biological replicates with duplicate plaque assay

Cytotoxicity and virucidal activity

BHK-21 cells were cultured for 18–20 h to achieve 70–80% confluency, after which cells were treated with OA (0.5–500 µM) or DMSO (0.0005–0.5%) for 24 h before assessing cell viability using the thiazolyl blue tetrazolium bromide dye (MTT) assay (Applichem GmBH, Darmstadt, Germany). The intensity of the dissolved formazan was measured at 570 nm (Beckman Coulter DX880 ST-52, Brea, CA). Experiment was undertaken as four independent replicates.

Assessment of virucidal activity was undertaken as previously described [5, 7, 14] and was performed by direct incubation of stock DENV 2 with OA (10, 50 or 100 µM) or DMSO (0.01, 0.05, 0.1%) or DMEM for 1 h at 37 °C. Viral titer was then determined by plaque assay as described elsewhere [13]. Experiment was undertaken as three independent replicates, with duplicate plaque assay.

Compound activity assays

The treatment conditions were essentially previously described [7]. Briefly BHK-21 cells were treated with selected concentrations of OA (or vehicle control) in three different treatment conditions. In pre-treatment, cells were treated with OA for 2 h prior to DENV 2 (MOI 2) or mock infection and after 2 h incubation cells were washed with PBS and then maintained in complete media under standard conditions for 24 h. In post-treatment, cells were infected with DENV 2 or mock infected for 2 h before washing with PBS and adding complete media containing OA or vehicle control before incubation under standard conditions for 24 h. In the combined pre- and post-infection treatment, cells were both pre-incubated and post infection incubated with compound or vehicle control, but the infection was undertaken in the absence of the compound or vehicle control. At 24 h post-infection, cell pellets were collected for flow cytometry analysis of viral infectivity as previously described [7], and the supernatants of the same conditions were also collected for standard plaque assays to determine infectious viral titer.

Flow cytometry

Flow cytometry was undertaken as previously described [7]. Briefly, cells were collected by trypsinization followed by centrifugation, blocked with 10% goat serum (Thermo Fisher Scientific, Waltham, MA) before fixing with 4% paraformaldehyde (Merck KGaA, Darmstadt, Germany) and subsequently permeabilizing with 0.2% Triton X-100. Cells were incubated with a 1:150 dilution of a pan-specific mouse anti-DENV E protein monoclonal antibody from hybridoma HB114 [15], and subsequently with a 1:40 dilution of a goat anti-mouse IgG conjugated with fluorescein isothiocyanate. Samples were run on a BD FACSCalibur cytometer (Becton Dickinson, BD Biosciences, San Jose, CA), using CELLQuest pro (Version 6.0) software. All experiments were undertaken independently in triplicate.

Western blotting

Mock infected or DENV 2 infected cells treated or untreated with OA as appropriate were collected by trypsinization followed by centrifugation at 5000g at 4 °C for 5 min and were subsequently lysed with 100 µl of radioimmunoprecipitation (RIPA) buffer containing protease inhibitor cocktail (Bio Basic Inc., Markham, Canada) and kept on ice for 30 min with vortexing every 10 min, and then centrifuged at 12,250g at 4 °C for 15 min. Protein concentrations were measured by the Bradford assay. Protein were separated by electrophoresis though 10% SDS polyacrylamide gels before transfer to 0.2 µm nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). Membranes were subsequently blocked with 5% skim milk in TBS/0.05% Tween 20 at room temperature and subsequently incubated with a pan-specific mouse anti-flavivirus E protein monoclonal antibody from hybridoma HB112 [15] at a 1:500 dilution, or a rabbit anti-dengue type 2 NS1 antibody at a 1:2000 dilution (PA5-32207; Thermo Scientific, Waltham, MA), or a rabbit polyclonal anti-DENV 2 NS3 antibody at a 1:8000 dilution (GTX124252; GeneTex Inc., Irvine, CA), or a mouse anti-DENV 2 NS5 monoclonal antibody at a 1:5000 dilution (MA5-17295, Thermo Fisher Scientific, Waltham, MA), or a 1:5000 dilution of mouse anti-GAPDH monoclonal antibody (sc-32233; Santa Cruz Biotechnology Inc., Dallas, TX) overnight at 4 °C. Secondary antibodies were either a horseradish peroxidase (HRP) conjugated goat anti-mouse IgG at a 1:5000 dilution (A4416, Sigma-Aldrich, St.Louis, MO) or a HRP-conjugated goat anti-rabbit IgG at a 1:8000 dilution (31460, Pierce, Rockford, IL) as appropriate, and these were incubated with the membrane for 1 h at room temperature. The signals were developed with the Amersham ECL plus Western Blotting Detection Reagents (GE Healthcare, Chicago, IL) and immediately captured using a visible western blot imaging system (Azure c400, Azure Biosystems, Inc., Dublin, CA).

Data analysis

The CC50 estimation was performed using AAT Bioquest-calculator: (accessed on 23 March 2022) with a four-parameter, background correction (subtract smallest response), and normalization (divide by largest response). Statistical analysis was performed with GraphPad Prism 9 for Windows (GraphPad Software Inc., San Diego, CA); EC50 was calculated using a non-linear fit dose response curve, multiple sample comparison using one way ANOVA comparing to DMSO control. Data is shown as mean ± S.E.M. Significance is denoted by *p ≤ 0.05, **p ≤ 0.01 ***p ≤ 0.001, ****p ≤ 0.0001.

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