2.4. Collagenase inhibition assay

Twenty-five microliters of plant extract was dissolved in 50% methanol and mixed with 25 µl of suspended collagenase (Sigma-Aldrich, St. Louis, MO; 26 units/ml) in 0.1 M Tris HCl and 200 µl of 0.5 mg/ml phenylazobenzyloxycarbonyl-Phe-Leu (PZ-pepride; Bachem, Budendorf, Switzerland) solution, and incubated at 37 °C for 30 min. After the incubation, 0.5 ml of 25 mM citric acid and 2.5 ml of ethyl acetate were added to the mixture and agitated. The mixture was centrifuged (10 °C, 1500 rpm, 5 min) and the ethyl acetate layer was collected. Absorbance at 320 nm of the layer was measured, and the inhibition of collagenase activity was calculated as: inhibition (%) = C–[S–B]/C × 100, where S is the absorbance at 320 nm in the reaction in the presence of plant extract and collagenase, C is the absorbance in the presence of plant extract, and B is the absorbance in the absence of collagenase. The assays were conducted in triplicate.