ABSTRACT
OBJECTIVE： To study in vitro metabolism pathway of effective component of Bletilla striata as Militarine in liver microsomes and kinetics characteristics of enzyme-catalyzed reactions. METHODS： The in vitro incubation system of rat and human liver microsomes was established， and incubation reaction of Militarine was performed. UPLC-QTOF-MS was used to identify the structure of its metabolites in combination with UNIFI database and references. Using puerarin as internal standard， UPLC-Triple Quad-MS was used to quantitatively analyze metabolic transformation of Militarine in rat liver microsomes. The kinetic parameters （vmax， km， CLint） of Militarine enzyme-catalyzed reactions with/without reducing coenzyme Ⅱ （NADPH） were calculated by fitting the curves with GraphPad Prism 5.0 software. RESULTS： After incubation in rat and human liver microsomes， Militarine produced a chemical formula C21H29O11， which was presumed to be a metabolite of Militarine ester bond hydrolysis. The kinetic study of enzyme-catalyzed reactions showed that vmax of Militarine enzyme-catalyzed reactions with/without NADPH were 1.955， 2.129 nmol/（h·mg）； km were 8.601， 9.854 nmol/mL； CLint were 0.227 3， 0.216 1 mL/（h·mg）； there was no significant difference between with NADPH and without NADPH. CONCLUSIONS： The main metabolic pathway of Militarine in liver microsomes is the hydrolysis of C1 and C4 ester bonds. Its metabolism does not depend on the pathway of cytochrome P450 enzymes initiated by NADPH.
ABSTRACT
Objective:To develop a Q-TRAP LC-MS/MS method for the content determination of militarine, protocatechuic acid and caffeic acid in Bletilla Striata. Methods:A Supelco Discovery C-18 (150 mm × 2. 1 mm, 3 μm) column was used and the mobile phase was acetonitrile-water containing 0. 1% formic acid (v/v) with gradient elution. The flow rate was 0. 25 ml·min-1. Bletilla Striata contrast medicinal materials and Bletilla Striata samples were detected by AB Sciex 4000 Q-MRM scan mode. Results: The content of militarine, protocatechuic acid and caffeic acid in the control herb of Bletilla Striata was 1. 167 5, 0. 062 6 mg·g-1 and 0. 001 0 mg·g-1 , respectively, and that in Bletilla Striata samples was 0. 708 8, 0. 001 1 mg·g-1 and 0. 000 4 mg·g-1 , respec-tively(n=3). Conclusion:The method is simple, accurate and reproducible. It can be used to quantitatively analyze militarine, pro-tocatechuic acid and caffeic acid in Bletilla Striata.
ABSTRACT
Objective:To study the polar chemical constituents of Blettila striata(Thunb.) Reichb.f. Methods:The constituents in N-butanol moiety were separated and purified by column chromatography with silica gel and SephadexLH-20, and were identified by spectral analysis. Results:Five compounds were identified as: militarine(1);7-hydroxy-4-methoxy-phenanthrene-2-O-?-D-glucoside(2);4-methoxyphenanthrene-2,7-O-?-D-diglucoside(3);7-hydroxy-2,4-dimethoxyphenanthrene-3-O-glucoside(4);3′-hydroxy-5-methoxybibenzyl-3-O-?-glucopyranoside(5). Conclusion:Compound 1,5 were isolated from this plant for the first time.