Development and Validation of a High-Performance Liquid Chromatography Method for Standardization of the Bioactive Ethyl Acetate Fraction of Alstonia scholaris (Linn.) R. Br. Growing in Egypt

Abstract

Bio-guided fractionation of the ethanolic extract of the leaves of Alstonia scholaris (Apocynaceae) growing in Egypt was carried out to evaluate its antihyperglycemic activity in alloxan-induced diabetic rats and its hepatoprotective activity against CCl4-induced hepatotoxicity in rats. The ethyl acetate fraction of the ethanolic extract showed the highest antihyperglycemic [(133.6 +/- 4.2) mg/mL, relative to metformin with (92.3 +/- 2.7) mg/mL] and hepatoprotective [(37.9 +/- 1.4) U/L, relative to silymarin with (29.7 +/- 0.8) U/L] activities. Four compounds were isolated from this fraction, and identified by spectroscopic techniques and by comparison with reported data: caffeic acid and isoquercitrin for the first time from this plant, in addition to quercetin 3-O-beta-D-xylopyranosyl (1''' --> 2")-beta-D-galactopyranoside (major compound) and chlorogenic acid. A validated reversed phase-high-performance liquid chromatography (RP-HPLC) method was developed for the standardization of the bioactive ethyl acetate fraction. The calibration curve showed good linearity (r2 > 0.999) within tested ranges. The relative standard deviation of the method was less than 3% for intra- (0.4-2.0%) and inter-day (1.9-2.8%) assays. Mean recovery of the method was within the range of 98.5-102.5%. The minimum detectable concentration of the analyte (LOD) was found to be 0.04 microg/mL. This developed HPLC method was shown to be simple, rapid, precise, reproducible, robust, specific, and accurate for quality assessment of the bioactive fraction.