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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.
Preparation of erythrocyte suspension:
Fresh whole blood (3 ml) collected from healthy volunteers into heparinised tubes was centrifuged at 3000 rpm for 10 min. A volume of normal saline equivalent to that
of the supernatant was used to dissolve the red blood pellets. 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 buffer solution contained 0.2 g of NaH2PO4, 1.15 g of Na2HPO4 and 9 g of NaCl in 1 litre of distilled water. The reconstituted red blood cells (resuspended supernatant) were used as such.
Hypotonicity induced haemolysis:
Samples of the extract used in this test were dissolved in distilled water (hypotonic solution). The hypotonic solution (5 ml) containing graded doses of the extracts (100, 200, 400, 600, 800 and 1000 μg/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 μg/ml) were also put into duplicate pairs (per dose) of the centrifuge tubes. Control tubes contained 5 ml of the vehicle (distilled water) and 5 ml of (3.9 – 1000 μg/ml) indomethacin respectively. Erythrocyte suspension (0.1 ml) was added to each of the tubes and mixed gently. 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 percentage heamolysis was calculated by assuming the heamolysis produced in the presence of distilled water as 100%. The percent inhibition of haemolysis by the extract was calculated thus:
% Inhibition of haemolysis= 1-((OD2-OD1)/ (OD3-OD1))*100
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