Rapid preparative isolation of erythrocentaurin from Enicostemma littorale by medium-pressure liquid chromatography, its estimation by high-pressure thin-layer chromatography, and its α-amylase inhibitory activity

Abstract

Erythrocentaurin is a relatively simple natural product present among the members of Gentianaceae. A preparative method for the isolation of erythrocentaurin from the ethyl acetate fraction of Enicostemma littorale using medium-pressure liquid chromatography has been reported. The method consisted of a simple step gradient from 10 to 20% ethyl acetate in n-hexane. Using a 70 × 460 mm Si60 column, this method is capable of processing 20 g of material in <3 h (purity ≈ 97%). The recovery of erythrocentaurin was 87.77%. Estimation of erythrocentaurin in extracts and fractions based on high-pressure thin-layer chromatography was carried out on silica gel 60 F(254) plates with toluene/ethyl acetate/formic acid (80:18:2 v/v/v) as the mobile phase. The densitometric analysis was performed at 230 nm. A well-separated compact band of erythrocentaurin appeared at R(f )0.54 ± 0.04. The analytical method showed good linearity in the concentration range of 200-1500 ng/band with a correlation coefficient of 0.99417. The limits of detection and quantification were found to be ≈60 and ≈180 ng/band, respectively. Erythrocentaurin exhibited a concentration-dependent α-amylase inhibition (IC(50) 1.67 ± 0.28 mg/mL). The outcome of the study should be considered for pharmacokinetic and biotransformation studies involving E. littorale.

Keywords: Enicostemma littorale; Erythrocentaurin; High-pressure thin-layer chromatography; Medium-pressure liquid chromatography; alpha-Amylase inhibition.