N succinyl l ala 3 p nitroanilide

The elastase inhibitory assay was conducted using the modified Cannell method [25 (link)]. N-succinyl-(L-Ala)₃-p-nitroanilide (Sigma Chemical Co., USA) was used as a substrate and dissolved in buffer solution to a concentration of 2.9 mM. Briefly, 15 μl of 0.5 U/mL elastase (Sigma Chemical Co.) dissolved in buffer (0.1 M Tris-HCl; pH 8.0), 100 μl of same buffer, 20 μl of substrate, and 15 μl of sample were mixed and incubated at 37°C for 30 min. One hundred μg/ml of ursolic acid was used as the positive control. To evaluate elastase inhibitory activity, absorbance was measured at 420 nm using a micro-plate reader (Tecan Sunrise, Tecan, Switzerland).

Ascorbic acid (AA), bovine serum albumin (BSA), Folin-Ciocalteu reagent, 2,2′-diphenyl-1-picrylhydrazyl (DPPH), sodium nitrite, Bradford reagent, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), gallic acid, NADH, nitroblue tetrazolium, phenazine methosulfate, CHAPS, dithiothreitol, N-succinyl-(L-Ala)₃-p-nitroanilide, elastase, hyaluronidase, hyaluronic acid (HA), epigallocatechin gallate (EGCG), apigenin, and Griess reagent were from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Ac-WEHD-methyl-coumarin amide (Ac-WEHD-MCA) was from Peptide Institute Inc. (Osaka, Japan). Cell lysis buffer [25 mM Tris-phosphate (pH 7.8), 2 mM 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 2 mM dithiothreitol, 10% glycerol, 1% Triton X-100] was obtained from Promega Korea (Seoul, Korea). All other chemicals used in this work were of the highest grade commercially available.