3.7. Inhibition of Albumin Denaturation

Anti-denaturation assay was conducted as described by Kumari et al. [20,59,78] with slight modifications. The reaction mixture consisted of 0.5 mL of 5% aqueous solution of human albumin (Albunorm 20, Octapharma (IP) SPRL, 1070 Anderlecht, Belgium) and 0.2 mL of the tested compound, dissolved in DMSO, at different concentrations (20–500 μg/mL). The samples were incubated at 37 °C for 15 min. After that, 2.5 mL phosphate-buffered saline (pH 6.3) was added to each tube and the samples were heated for 30 min to 80 °C and then cooled for 5 min. Turbidity was measured spectrophotometrically at 660 nm (Cary 60 UV-Vis, Agilent Technologies, Santa Clara, CA 95051, USA). For the blank sample, a mixture of 2.5 mL buffer and 0.2 mL DMSO was used instead of the compounds, while the product control test lacked the compounds’ concentration having 0.5 mL serum albumin, and 2.5 mL buffer only. The percentage inhibition of protein denaturation was calculated as follows:

The control represents 100% protein denaturation. Commercially available anti-inflammatory drugs diclofenac and ibuprofen were used for comparison. Their anti-inflammatory effect was determined as a percentage of inhibition of albumin denaturation, following the same protocol as for the novel compounds.