Individual standards of Palmatine hydrochloride (Sigma Aldrich, USA) (Cat # 361615), Cordifolioside A (Chem faces, China) (Cat # CFN95040), Magnoflorine (Sigma Aldrich, USA) (Cat. # 361615), Withaferine A (Natural remedies, India) (Cat # W003), Withanoside IV (Natural remedies, India) (Cat # W006),) Withanoside V (Natural remedies, India) (Cat. # W007), Withanone (Natural remedies, India) (Cat. # W005) Rosmarinic acid (Sigma Aldrich, USA) (Cat. # R4033), Palmaitine hydrochloride (Sigma Aldrich, USA) (Cat # 361615), Ursolic acid (Tokyo chemical industries, India) (Cat. # 102067769) and Betulinic acid (Natural remedies, India) (Cat. # B2836) were dissolved in methanol to prepare 1000 ppm standard solution. 0.05 mL of 1000 ppm standard stock solution was taken to prepare 50 ppm of standard working solution.
Purified Omicron Spike (S) proteins and purified human ACE-2 protein were procured from Sino Biological (Beijing, China) (Cat # 10108-H08H-B). Coronil (Internal Batch No. CHIH/CORA/0122/2269) was obtained from Divya Pharmacy (Haridwar, India). Interactions between ACE-2 and seven different types of SARS-CoV-2 (Omicron) spike (S) proteins, namely, SARS-CoV-2(BA.4/BA.5) Spike RBD Protein (His Tag) (Cat # 40592-V08H130), SARS-CoV-2 BQ1.1 (Omicron) Spike RBD Protein (His Tag) (Cat # 40592-V08H143), SARS-CoV-2 XBB (Omicron) Spike RBD Protein (His Tag) (Cat # 40592-V08H144), SARS-CoV-2 BA.2.75.2 (Omicron) Spike RBD Protein (His Tag) (Cat # 40592-V08H141), SARS-CoV-2 (BA.4.6/BF.7) Spike RBD Protein (His Tag) (Cat # 40592-V08H140), SARS-CoV-2 JN.1 (omicron) Spike RBD Protein (aa319-529), His Tag (HPLC-verified) (Cat # 40592-V08H155), SARS-CoV-2 JN.1 (omicron) (Spike ECD His Tag) (Cat # 40589-V08H59) were studied. Bovine serum albumin (BSA) was purchased from Himedia (Thane, India) (Cat # TC194-500G). 3.3′,5,5′-Tetramethylbenzidine (TMB) was purchased from BD Bioscience, San Diego, USA (Cat # 555,214,) and Peroxidase Streptavidin was procured from Jackson ImmunoResearch (Cat # 016-030-084).
Compositional Analysis of Coronil
0.5 gm powdered Coronil tablet sample was dissolved in 10 mL methanol: water (80:20) solution and sonicated for 30 min. The solution was centrifuged at 10000 rpm for 5 min and filtered using 0.45 µm nylon filters. The resulting filtered solution was used for the analysis. Compositional analysis of Coronil was performed by Prominence-XR UHPLC system (Shimadzu, Japan) equipped with Quaternary pump (NexeraXR LC-20AD XR), DAD detector (SPD-M20 A), Auto-sampler (Nexera XR SIL-20 AC XR), Degassing unit (DGU-20A 5R) and Column oven (CTO-10 AS VP). Separation was achieved using a Shimadzu Shim pack GIST-HP C18 (3µm, 3 X 100 mm) column subjected to binary gradient elution. The two solvents used for the analysis were water containing 0.1% orthophosphoric acid (pH 2.5 adjusted with diethyl amine (solvent A)) and Acetonitrile (solvent B). Gradient programming of the solvent system was done initially at 5% B for 0–10 min, 5–15% B from 10–20 min, 15–25% B from 20–40 min, 25–65% B from 40–60 min, 65–90% B from 60–65 min, 90 − 5%B from 65-66min, 5% B from 66–70 min with a flow rate of 0.7 ml/min. 10 µl of standard and test solution were injected and column temperature was maintained at 30°C. Wavelengths were set at 227 nm (for Withaferine A, Withanoside IV, Withanoside V, Withanone, Codifolioside A and Magnoflorine), 325 nm (for Rosmarinic acid and Palmatine) and 210 nm for Ursolic acid and Betulinic acid).
ACE2 binding inhibition assay
ACE2 binding inhibition assay was performed based on the published protocol with slight modifications [29]. Briefly, 100 µl of Spike protein (3 or 6 µg/ml) was coated in the 15 mM Sodium Carbonate, 35 mM Sodium Hydrogen Carbonate, and 7.7 mM Sodium Azide, pH 9.6 in Nunc Maxisorp plates (Denmark) (Cat # 442404.) for 16 hr at 4 oC. The plate was washed thrice with washing buffer (0.5% Tween 20 in PBS). 200 µl of the blocking buffer was added (2% BSA in the PBST) to each well followed by incubation at 37 oC for 1.5 hr. After washing thrice with washing buffer (0.5% Tween 20 in PBS), 100 µl of biotinylated ACE2 (2 µg/ml) was added to each well with different concentrations of Coronil (3.0, 10, 30, 100, and 300 µg/ml) and the plate was incubated at 37 oC for 1 hr. The wells were washed three times with washing buffer (0.1% Tween 20 in PBS) and 100 µl of Streptavidin-HRP solution (0.1 µg/ml) was added to each well and the plate was further incubated at 37 oC for 1 hr. This was followed by five washings with wash buffer (0.1% Tween 20 in PBS). Finally, 200 µl of TMB was added as substrate and the plate was further allowed for color development for up to 30 minutes at 37 0C. The reaction was stopped by adding 50 µl of 2N H2SO4 and absorbance was taken at 450 nm using Perkin Elmer Envision plate reader.
Statistical Analysis
All the statistical analyses were performed using GraphPad Prism software version 9.0 and the data was represented as mean ± SD. Multiple comparisons were done using one-way ANOVA (nonparametric) with a post hoc Tukey’s test. Each experiment was performed in atleast 3 replicates.