Elastase Inhibition Assay Protocol

The anti-elastase activity was determined using a method by Vostálová et al. [1 (link)]. The reaction mixture contained 100 μL of 0.1 M HEPES buffer (pH 7.5), 10 μL of the test sample (hesperidin, hesperetin, rutinose, or rhamnose)/inhibitor (oleanolic acid in DMSO; 1.46 mg/mL) or solvent DMSO, and 20 μL of elastase enzyme (1 U/mL) except for the blank. All tubes were incubated at room temperature for 5 min, the reaction was initiated by adding 30 μL of the substrate, N-succinyl-Ala-Ala-Ala-p-nitroanilide (4.4 mM). The reaction was monitored as the change in absorbance (A) at 410 nm (∆A/min) using a UV-VIS recording spectrophotometer (Shimadzu (Kyoto, Japan); UV-2401PC) and the percentage inhibition was calculated according to Equation (1):