2.4.1. Protein denaturation inhibition assay

The method of Saleem et al. (2020) was used. The reaction mixture was composed of 450 μL of 5% w/v BSA solution and 50 μL of CM extract or standard drug solutions (aspirin and diclofenac sodium) at different concentrations (25–400 µg/mL). The sample mixture was kept for 15 min at 37 °C and then for 5 min at 70 °C. After cooling he samples, 2.5 mL PBS (pH 6.3) was added to the mixture. The absorbance of the test and control samples were measured at 660 nm. The experiment was run three times and the % inhibition in protein denaturation was calculated using the equation

Where, K₀ = absorbance value of control, K_t = absorbance value of CM extract or standards.

The IC₅₀ values of the CM extract, aspirin, and diclofenac sodium were generated in GraphPad Prism-9 (San Diego, CA, USA) using Fit spline analysis.

This assay has been done as described previously (Dadoriya et al. 2020). The reaction mixture was composed of 0.2 mL of fresh hen’s egg albumin, 2.8 mL phosphate buffer saline (pH 6.4) and 2 mL of various concentrations (25–400 µg/mL) of CM extract or standard drugs. All the samples were set aside at 37 °C for 25 min followed by heating for 5 min at 70 °C. The cooled solutions were centrifuged at 3000 rpm for 10 min. Then the absorbance of supernatant solutions was measured at 660 nm. The % of inhibition and IC₅₀ values were calculated as mentioned in BSA denaturation inhibition assay procedure.