3.5.1. Membrane Stabilization Inhibition Assay

The membrane-stabilizing activity of the samples was assessed using hypotonic solution-induced erythrocyte (RBCs) hemolysis [67]. For the preparation of erythrocyte suspension, whole blood was obtained with heparinized syringes from rats through cardiac puncture. The blood was washed three times with isotonic buffered solution (154 mM NaCl) in 10 mM sodium phosphate buffer (pH 7.4) and immediately centrifuged for 10 min at 3000× g. The test sample consisted of stock erythrocyte suspension (0.5 mL), 5 mL of hypotonic solution (50 mM NaCl), and the C. amblyocarpa EO (7.81–1000 µg mL⁻¹ in ethanol) or indomethacin (as a standard drug). The control sample consisted of 0.5 mL of stock erythrocyte suspension and hypotonic-buffered saline solution alone. The mixtures were incubated for 10 min at room temperature (25 ± 2 °C) and centrifuged for 10 min at 3000× g. In 96-well plates, the absorbance of the supernatant was measured at 540 nm. The percentage inhibition of hemolysis or membrane stabilization were calculated according to the modified method described by Shinde et al. [67] as follows:

where A_control is the absorbance control, and A_treatment is the absorbance treatment.

The IC₅₀ value was defined as the concentration of the EO required to inhibit 50% of the RBC hemolysis under the assay conditions. It was calculated graphically by linear regression of the inhibition values of different concentrations using MS-EXCEL 2016.