3.6.2. Hypotonic Solution-Induced Erythrocyte Haemolysis

Membrane stabilizing activity of the methanolic extract was assessed using hypotonic solution-induced erythrocyte haemolysis. The sample consisted of stock erythrocyte (RBCs) suspension (0.50 mL) mixed with 5 mL of hypotonic solution (50 mM NaCl) in 10 mM sodium phosphate buffered saline (pH 7.4) containing the methanolic extract (1000–7.81 µg/mL) or indomethacin (as positive control). The negative control sample consisted of 0.5 mL of RBCs mixed with hypotonic-buffered saline solution alone. The mixtures were incubated for 10 min at room temperature 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 haemolysis or membrane stabilization was calculated according to modified method described by Shinde et al. [48].

where OD₁ = Optical density of hypotonic-buffered saline solution alone; OD₂ = Optical density of extract in hypotonic solution.

The IC₅₀ value was defined as the concentration of the sample to inhibit 50% RBCs haemolysis under the assay conditions.