2.6. In vitro anti-inflammatory activity assay

The anti-inflammatory activity of CEO was evaluated using albumin denaturation (AD) method described by Alam and Singh, (Alam and Singh, 2020) and proteinase inhibitory method described by Gunathilake et al. (Gunathilake et al., 2018), with slight modifications.

In the egg albumin method, the reaction mixture, 2.8 mL of phosphate-buffered saline (PBS, pH 6.4), 0.2 mL of fresh hen egg albumin, 2 mL of different CEO dilution (5, 10, 50, 200, and 1000 μg/mL), and Ibuprofen (standard drug) were added in separate test tubes. The reaction mixtures were incubated for 15 min at 37 ± 2 °C, subjected to heating in oven at 70 °C for 10 min, and then allowed to cool; the absorbance of the reaction mixture was measured at 660 nm by using a UV–Vis spectrophotometer; PBS solution was used as the blank. The experiment was repeated thrice, and the percentage inhibition of AD was measured using the following equation:

In the proteinase inhibitory assay, 0.06 mg trypsin, 1 mL of tris-HCl buffer (20 mM), 1 mL of different CEO dilution (5, 10, 50, and 200 μg/mL), and Ibuprofen (standard drug) were added in separate test tubes and mixed thoroughly. The reaction mixtures were incubated at 37 °C for 15 min. The mixtures were again incubated at 37 °C for 20 min after adding 1 mL of casein (0.8% w/v). Then, 2 mL of 70% per chloric acid was added to each mixture, and the mixture was centrifuged at 3000 rpm for 5 min. Supernatant of the centrifuged samples was separated, and absorbance of each sample was measured at 210 nm. The mixture containing all the reagents except the sample and standard solution was used as the control. The experiment was repeated thrice, and the percentage of protein denaturation was calculated using the following equation: