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.
Serra
C P
CP
Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
Côrtes
S F
SF
Lombardi
J A
JA
Braga de Oliveira
A
A
Braga
F C
FC
D000806:Angiotensin-Converting Enzyme Inhibitors / Q000737:chemistry*; D000818:Animals; D001938:Brazil; D003124:Colorimetry; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008519:Medicine, Traditional; D009842:Oligopeptides / Q000737:chemistry; D008517:Phytotherapy; D010936:Plant Extracts / Q000737:chemistry*; D010946:Plants, Medicinal; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014302:Trinitrobenzenesulfonic Acid / Q000737:chemistry
D000806:Angiotensin-Converting Enzyme Inhibitors; D009842:Oligopeptides; D010936:Plant Extracts; C029275:hippuryl-glycyl-glycine; D014302:Trinitrobenzenesulfonic Acid