title: A non-michaelian behavior of the in vitro metabolism of the pentacyclic triterpene alfa and beta amyrins by employing rat liver microsomes.
abstract: Pharmacological studies employing alpha and beta amyrin have demonstrated potential application in several biological activities suggesting their application as promising drugs. In the early drug development, metabolism studies may give important parameters regarding the efficacy and safety of the drug candidate. Therefore, the aim of this work was to determine the enzymatic kinetic parameters of these pentacyclic triterpenes. Chromatographic analyzes were performed using a Shimadzu GC-MS system. The resolution of amyrins was achieved with a DB5-MS column of 0.25 μM film thickness, 30.0 cm length and 0.25 mm diameter. At this condition, the retention times of beta- and alpha-amyrin were 21.3 and 20.2 min, respectively. The proposed method showed to be linear over the concentration range of 0.16-42.18 μM for beta amyrin and 0.11-28.12 μM for alpha amyrin. The lowest concentration quantified by the validated method was 0.16 μM for beta and 0.11 μM for alpha amyrin. The stability study showed that amyrins were stable at room temperature for 12h and at 37°C for 1h. The absolute recovery of the amyrin isomers from the rat microsome was 54.3-59.2%. The enzymatic kinetics presented sigmoidal plots. It was observed a Vmax=0.698 ± 0.022 μmol/mg protein/min, S50=4.4 μM and Hill coefficient of 2.7 ± 0.17 for alpha amyrin and a Vmax=0.775 ± 0.034 μmol/mg protein/min, S50=7.0 μM and Hill coefficient of 2.5 ± 0.21 for beta amyrin. The obtained results give the first clues regarding amyrin metabolism and suggests a more detailed study conducted employing isolated CYP isoforms.
authors.affiliation: Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, São Paulo, Brazil.
authors.forename: Fernanda de Lima
authors.initials: Fde L
authors.lastname: Moreira
authors.forename: Gustavo Henrique Bianco
authors.initials: GH
authors.lastname: de Souza
authors.forename: Ivanildes Vasconcelos
authors.initials: IV
authors.lastname: Rodrigues
authors.forename: Norberto Peporine
authors.initials: NP
authors.lastname: Lopes
authors.forename: Anderson Rodrigo Moraes
authors.initials: AR
authors.lastname: de Oliveira
mesh_terms: D000818:Animals; D055808:Drug Discovery / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D007536:Isomerism; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D053978:Pentacyclic Triterpenes / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D053978:Pentacyclic Triterpenes; D009828:Oleanolic Acid; C036380:beta-amyrin
keywords: Amyrins; Atypical enzymatic kinetics; Hill profile; In vitro metabolism; Natural product