2.8. Anti-Inflammatory Assay

The human red blood cell (RBC) membrane stabilization assay was applied to investigate the anti-inflammatory activity of the O. persica leaf extract and the phytofabricated AgNPs as described by Vane and Botting [24]. The blood samples were obtained from ten healthy volunteers. The samples were blended with an equal volume of a sterilized Alsever’s solution (containing 0.5% citric acid, 0.8% sodium citrate, 0.42% sodium chloride, and 2% dextrose). To separate the packed cells, the preserved blood samples were centrifuged at 4000 rpm for 15 min. The obtained packed cells were washed with 0.85% isosaline (pH 7.2), and a cell suspension (10% v/v) was prepared in isosaline. This resulting human RBC suspension was applied for the determination of the anti-inflammatory activity. Accordingly, 1 mL amounts of the O. persica leaf extract solutions, the suspensions of the phytofabricated AgNPs, and the diclofenac sodium solutions (as a standard drug) at various concentrations (50, 100, 150, 200, 250, and 300 μg mL⁻¹) were separately blended with 2 mL of 0.36% hypo saline, 1 mL of a 0.15 mol L⁻¹ phosphate buffer (pH 7.4), and 0.5 mL of the human RBC suspension. Distilled water (2 mL) was considered as the control instead of the hypo saline. The resulting mixtures were incubated at 37 °C for 30 min and then centrifuged at 4000 rpm for 15 min. The supernatants were evacuated, and their hemoglobin value was spectrophotometrically measured at 560 nm. The protection (%) or the human RBC membrane stabilization (%) was determined using the following equation: