2.5. Hyaluronidase Assay

The enzymatic assay of Echis carinatus hyaluronidase enzyme was performed by using method of Pukrittayakamee et al. [21] with slight modification. Briefly, the assay mixture contained acetate buffer (0.2 M sodium acetate–acetic acid, pH 5.0, containing 0.15 M NaCl), 50 μg of hyaluronic acid (0.5 mg/mL in buffer), and enzymes in a final volume of 1.0 mL. The mixture was incubated for 15 minutes at 37°C, and the reaction was quenched by the addition of 2 mL of 2.5% CTAB in 2% NaOH. The absorbance was read at 400 nm (within ten minutes) against a control solution containing 1 mL of the same buffer and 2 mL of 2.5% CTAB in 2% NaOH. Percentage of the remaining hyaluronic acid was considered as turbidity-reducing activity taking absorbance of sample with no enzyme added as 100%. One unit was defined as the amount of enzyme that induced 50% turbidity reduction. Kinetics data to optimize the hyaluronidase assay were also obtained [22, 23]. To measure medicinal plants neutralizing potential, (0.8 mg) extracts were preincubated with venom for 15 min at 37°C.