Amentoflavone

A specific SARS-CoV-2 Spike-ACE2 Interaction Inhibitor Screening Assay Kit (Item No. 502050; Cayman Chemical^®, Ann Arbor, MI, USA) was used to assess the inhibitory potential of the recombinant SARS-CoV-2 Spro S1 RBD interaction with recombinant ACE-2. Amentoflavone, kaempferitrin, quercetin, luteolin, quercetin-7-O-glucoside (Quercimeritrin) and myricetin were obtained commercially (Sigma-Aldrich St. Louis, MO, USA; analytical grade 93–99%) and evaluated each at concentrations of 0.25; 0.5; 1; 2; 4, and 8 µg/mL. The test was prepared according to the manufacturer’s guidelines and read at 450 nm (Synergy™ H1, Biotek/AGILENT TECHNOLOGIES, Santa Clara, CA, USA).

Amentoflavone was purchased from Sigma-Aldrich (St. Louis, MO, USA), and was tested for BRD4 tandem (BD1 + BD2) binding by means of AlphaScreen [58 (link)], using an H4 peptide (1–21) K5/8/12/16 Ac. Experimental work was performed by the Reaction Biology Corp., providing 2-mg samples to obtain duplicate dose-response curves beginning at a 100-μM concentration, following a three-fold dilution. The positive control for the test was the JQ-1 compound. The half maximal inhibitory concentration (IC₅₀) values were obtained from the curves, and the Hill slope for Amentoflavone was calculated.

Reference compound quercetin-3-O-α-l-rhamnoside (98% purity) was purchased from LGC standard, whereas amentoflavone (98% purity) was purchased from Sigma-Aldrich. The reference compounds were prepared by dissolving them in methanol at a final concentration of 1000 mg/L. The standard solutions were diluted to obtain calibration curves with five points in the concentration range of 0.025–0.5 and 0.05–0.5 mg/L, respectively.

This experimental study was conducted at the
Department of Applied Chemistry at Dong-Eui
University (Republic of Korea). In this study,
Amentoflavone was obtained from Sigma-Aldrich
(St. Louis, MO, USA). Dulbecco’s modified Eagle’s
medium (DMEM), trypsin-EDTA, penicillin/
streptomycin/ amphotericin (10000 U/ml, 10000 g/
ml, and 2500 g/ml, respectively), and fetal bovine
serum (FBS) were obtained from Gibco BRL, Life
Technologies (NY, USA). A549 (ATCC # CRL-6323)
and WI38 (ATCC # CRL-75) cells were purchased from
ATCC. 3-(4 (link),5 (link)-dimethyl-2-yl)-2,5- diphenyl tetrazolium
bromide (MTT) reagent, agarose, and other materials
were purchased from Sigma Chemical Co. (St. Louis,
MO, USA).

Alcian blue, aluminium chloride, Folin-Ciocalteu reagent, ethylene diamine tetra acetic acid (EDTA), 5,5-dithio-bis-(2-nitrobenzoic acid (DTNB), gallic acid, thiobarbituric acid, trichloroacetic acid, toluene, ethyl acetate, formic acid, formaldehyde, sodium hydroxide, rutin, phosphoric acid, pyrogallol, haematoxylin, eosin were purchased from HiMedia Laboratories Pvt Ltd, Mumbai, India, and HPTLC plates, tris-HCl buffer, ethanol, amentoflavone were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA).