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

Bio-guided fractionation of the ethanolic extract of the leaves of Alstonia scholaris (Apo-cynaceae) growing in Egypt was carried out to evaluate its antihyperglycemic acti vity in alloxan-induced diabetic rats and its hepatoprotective activity against CCl4-induced hepato-toxicity in rats. The ethyl acetate fraction of the ethanolic extract showed the highest an-tihyperglycemic [(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-β-D-xylopyranosyl (1'''→2")-β-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 bio active 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 μg/mL. This developed HPLC method was shown to be simple, rapid, precise, reproducible, robust, specific, and accurate for quality assessment of the bioactive fraction.