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

A new 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 415 nm 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=7.63%) and inter-day precision (CV=13.61%), recovery (97-102.1%), sensitivity (IC50=14.1 nmol/l) and linearity in the range 7.5-120 mmol/l of glycyl-glycine (r2=0.9921). Besides, the method showed good correlation with a HPLC assay already established for the screening of ACE inhibitors (r=0.9935 and 0.9034, respectively, for captopril solutions and for plant extracts). The method involves only inexpensive reagents and apparatus.