Briefly, for each experiment an aliquot of blood was washed three times with Alsever’s solution composed of 120 mM D-glucose, 30 mM sodium citrate, 7 mM NaCl, and 2 mM citric acid, pH 7.4. Washing was done by low-speed centrifugation (1,000 × g, four minutes) of blood at 4°C (Hermle Z 323 K centrifuge, Lab-Tech Instrumentation, Germany). Washed erythrocytes were diluted in 1% Alsever’s solution (v/v) and combined with different amounts of the aqueous extract (0.03, 0.10, 0.32, 1.00, 3.16, 10.00, 31.60, 100.00, and 316.00 μg protein/mL). Then the samples were incubated at 37°C for 30 minutes (Eppendorf AG 22331Thermomixer, Brinkmann Instruments, Germany). Subsequently, the reaction was stopped by centrifuging for four minutes at 1,000 × g. The A415 of the supernatant fluid containing the hemoglobin released from lysed erythrocytes was measured in a spectrophotometer (Lambda Bio, Perkin Elmer Co., USA). Each experiment was normalized with respect to complete hemolysis, which was measured by diluting the erythrocyte sample in deionized water instead of Alsever’s solution. One hemolytic unit (HU50) was defined as the amount of protein sample required to cause 50% hemolysis.

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