Uv vis 1650

Whole blood was collected from adult mice and centrifuged at 1000 g for 10 min at 4°C. The plasma and buffy coat layers were discarded, and an equal volume of phosphate-buffered saline (PBS) (pH 7.4) was added. After repeating three times, 4% suspension of red blood cells (RBC) was obtained with PBS dilution.
Erythrocytes were hemolyzed following the modified method.[24 (link)] One milliliter of erythrocyte suspension (4%) was mixed with 1 ml of the different concentrations of MPE (2, 4, 6, 8, and 10) μg/ml ethanol extract was prepared and then added 1 ml of H₂O₂ (100 mmol/L in PBS). The blank control consisted of 2 ml of PBS and 1 ml RBC suspension, and the induced control consisted of 1 ml of PBS, 1 ml RBC suspension, and 1 ml of H₂O₂. The mixture was incubated in a shaking water bath at 37°C for varying time intervals (30, 60, 90, 120, and 150 min) and centrifuged at 1000 g for 10 min at 4°C. The RBC-free supernatant solution from each tube was transferred to cuvettes. Absorbance was measured at 415 nm in a spectrophotometer (Shimadzu UV-VIS 1650, Tokyo, Japan). Each sample was measured in triplicate.[25 (link)] The percentage of hemolysis was calculated by the following equation:
Hemolysis percentage (%) = (Abs_[sample]/Abs_{[induced control]}) × 100%.

Erythrocytes were hemolyzed using H₂O₂ and the modified method of Lalitha and Selvam.[22 (link)] One milliliter of erythrocyte suspension (4%) was mixed with 1 mL of FEM at the concentrations (10, 30, 55, 80, and 100 µg/mL) and then added 1 mL of H₂O₂ (100 mmol/L in PBS). The blank control consisted of 2 mL of PBS and 1 mL RBC suspension and the induced control consisted of 1 mL of PBS, 1 mL RBC suspension, and 1 mL of H₂O₂. The mixture was incubated in a shaking water bath at 37°C for 30 min, 60 min, 90 min, 120 min, and 150 min, and then centrifuged at 1000 g for 10 min at 4°C. The RBC-free supernatant solution from each tube was transferred to cuvettes. Absorbance was measured at 415 nm in a spectrophotometer (Shimadzu UV-VIS 1650, Tokyo, Japan). Each sample was measured in triplicate. The percentage of hemolysis was calculated using the equation:
Hemolysis percentage (%) = [Ab_(sample)/Ab_{(induced control)}] × 100%
Percent of hemolysis inhibition
= [(Ab_{(induced control)} − Ab_(sample))/Ab_{(induced control)} − Ab_{(blank control)}] ×100%