Human red blood cell (HRBC) membrane stabilization test

Fresh human blood (10 ml) was collected in heparinized centrifuge tubes and centrifuged at 3000 rpm for 10 min and washed 3× with an equal volume of normal saline solution. The volume of the blood was measured and reconstituted as a 10% v/v suspension with normal saline [32]. The reaction mixture (2 ml) consisted of 1 ml methanolic plant extract and 1 ml of 10% red blood cell suspension. For the control, saline was added instead of plant extract. Aspirin was used as a standard drug (positive control). The samples were incubated at 56 °C for 30 min, centrifuged at 2500 rpm for 5 min and the absorbance of the supernatant measured at 560 nm. The experiment was performed in triplicate. Percent membrane stabilization activity was calculated by the formula given in Inhibition of protein denaturation method section [33], while the percentage of protection was calculated using the following formula:

where A_C and A_S are the absorbance (at 560 nm) of the control and sample, respectively.