2.10. Phospholipase A2 (PLA2) inhibition

PLA₂ activity was determined using egg yolk as PLA₂ substrate (lecithin) according to Marinetti.²³ Briefly, the assay was carried out by using egg yolk suspension diluted in saline (1:5 w/v). Crude venom sample (200 μg) alone or pre-incubated with different ratios of RMAE (1:0, 1:1, 1:2, 1:3, and 1:4) for 30 min at 37 °C was added to one ml of egg yolk working suspension to a final assay mixture volume of 5 ml with saline and the absorbance was recorded each 5 min for 15 min at 900 nm.

PLA₂ activity was also carried out according to Gutierrez et al.²⁴ Briefly, 200 μg of crude Cc venom was loaded into 3 mm diameter wells of 1% agarose plates containing 4% washed human erythrocytes, 4% egg yolk suspension and 10 mM CaCl₂. The fortified agarose-egg yolk plate was incubated for 20 h at 37 °C. The PLA₂ inhibition was calculated by measuring the zone of clearance around the haloes in the presence and absence of venom/RMAE ratios (1:0, 1:1, 1:2, 1:3, 1:4, and 1:5). PLA₂ activity of venom in the absence of RMAE served as positive control.