4.5.4. Collagenase Inhibition

Collagenase inhibition assay was determined as reported by Andrade et al. [52] with some modifications. The substract N-(3-furyl-acryloyl)-Leu-Gly-Pro-Ala (FALGPA) 1 mM was dissolved in tricine buffer (50 mM, pH 7.5). Collagenase enzyme was prepared in the buffer at 1 U mL⁻¹. First, 10 μL of extract dilutions (0.75 and 1.0 mg mL⁻¹), 45 μL of tricine buffer, and 50 μL of collagenase (1 U mL⁻¹) were added to a 96-well plate and kept at 37 °C for 15 min. The reaction was started by adding 120 μL of FALGPA. The substrate hydrolysis was monitored with a Synergy HT Multi-detection Microplate Reader operated by GEN5^TM (Biotek, Bad Friedrichshall, Germany), operating in kinetic function, at room temperature, for 8 min, at 345 nm. Negative control was performed in the absence of extract, and gallic acid was used as positive control. Results were expressed as percentage of enzyme inhibition in comparison to the untreated control. The assay was performed in triplicate.