Preparation of erythrocyte suspension:

Fresh whole blood (3 ml) collected from healthy volunteers into heparinised tubes was centrifuged at 3000 rpm for 10 min.The volume of the dissolved red blood pellets obtained was measured and reconstituted as a 40% v/v
suspension with isotonic buffer solution (10 mM sodium phosphate buffer, pH 7.4).The mixtures were incubated for 1 hr at room temperature (37?C), and afterwards, centrifuged for 3 min at 1300 g. Absorbance (OD) of the haemoglobin content of the supernatant was estimated at 540 nm using Spectronic  (Milton Roy) spectrophotometer.The hypotonic solution (5 ml) containing graded doses of the extracts (100, 200, 400, 600, 800 and 1000 ug/ml) were put into duplicate pairs (per dose) of the centrifuge tubes.Isotonic solution (5 ml) containing graded doses of the extracts (3.9 - 1000 ug/ml) were also put into duplicate pairs (per dose) of the centrifuge tubes.