Prior to screening in all assays, spectra for all extracts were recorded on a Cary 300 UV-visible spectrophotometer to check for interference and shifts in the lambda max.
The assay employed was based on spectrophotometric methods reported in the literature [12 (link)] with some modifications for use in a microplate reader. The assay was performed in 50 mM Tricine buffer (pH 7.5 with 400 mM NaCl and 10 mM CaCl2). Collagenase from Clostridium histolyticum (ChC – EC.3.4.23.3) was dissolved in buffer for use at an initial concentration of 0.8 units/mL according to the supplier's activity data. The synthetic substrate N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA) was dissolved in Tricine buffer to 2 mM. Plant extracts were incubated with the enzyme in buffer for 15 minutes before adding substrate to start the reaction. The final reaction mixture (150 μL total volume) contained Tricine buffer, 0.8 mM FALGPA, 0.1 units ChC and 25 μg test extracts. Negative controls were performed with water. Absorbance at 335 nm was measured immediately after adding substrate and then continuously for 20 minutes using a Cary 50 Microplate Reader in Nunc 96 well microtitre plates. EGCG, 250 μM (0.114 mg/mL) was used as a positive control.
The assay employed was based on spectrophotometric methods reported in the literature [12 (link)] with some modifications for use in a microplate reader. The assay was performed in 50 mM Tricine buffer (pH 7.5 with 400 mM NaCl and 10 mM CaCl2). Collagenase from Clostridium histolyticum (ChC – EC.3.4.23.3) was dissolved in buffer for use at an initial concentration of 0.8 units/mL according to the supplier's activity data. The synthetic substrate N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA) was dissolved in Tricine buffer to 2 mM. Plant extracts were incubated with the enzyme in buffer for 15 minutes before adding substrate to start the reaction. The final reaction mixture (150 μL total volume) contained Tricine buffer, 0.8 mM FALGPA, 0.1 units ChC and 25 μg test extracts. Negative controls were performed with water. Absorbance at 335 nm was measured immediately after adding substrate and then continuously for 20 minutes using a Cary 50 Microplate Reader in Nunc 96 well microtitre plates. EGCG, 250 μM (0.114 mg/mL) was used as a positive control.
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