Validation of a colorimetric assay for the in vitro screening of inhibitors of angiotensin-converting enzyme (ACE) from plant extracts.

Abstract

Anew method for the in vitro screening of plant extracts with potential angiotensin-converting enzyme (ACE) inhibitory activity is proposed. The method is based on the cleavage of the substrate hippuryl-glycyl-glycine by ACE and subsequent reaction with trinitrobenzenesulfonic acid to form 2,4,6-trinitrophenyl-glycyl-glycine, whose absorbance is determined at 415nm in a microtitre plate reader. Rabbit lung dehydrated by acetone was employed as an enzyme source. Validation of the method showed satisfactory intra-day (CV ¼ 7.63%) and inter-day precision (CV ¼ 13.61%), recovery (97–102.1%), sensitivity (IC50 ¼ 14.1 nmol/l) and linearity in the range 7.5–120 mmol/l of glycyl-glycine (r2 ¼ 0:9921). Besides, the method showed good correlation with a HPLC assay already established for the screening of ACE inhibitors (r ¼ 0:9935 and 0:9034; respectively, for captopril solutions and for plant extracts). The method involves only inexpensive reagents and apparatus.