Development and Application of a Rapid and Efficient CZE Method Coupled with Correction Factors for Determination of Monosaccharide Composition of Acidic Hetero-polysaccharides from Ephedra sinica

摘要

Introduction - Ephedrine alkaloids cannot account for all the effects of Ephedra sinica and the polysaccharides are also demonstrated to be one of the main bioactive constituents of E. sinica. However, no work has been reported on the analysis of monosaccharide composition of purified polysaccharides isolated from the stem of E. sinica.Objective - To develop a rapid and efficient capillary zone electrophoresis (CZE) method based on pre-column derivatisation with 1-phenyl-3-methyl-5-pyrazolone for the simultaneous determination of neutral and acidic sugars of purified polysaccharides from E. sinica.Methodology - Three polysaccharides (ESP-A3, ESP-A4 and ESP-B4) were isolated and purified by ion exchange and gel-filtration chromatography from the stem of E. sinica. The effects of background electrolyte pH and concentration, applied voltage and temperature on the separation were investigated. Meanwhile, factors affecting the hydrolysis of ESP-B4 with sulphuric acid were investigated by changing the hydrolysis time, acid concentration and hydrolytic temperature to achieve complete hydrolysis. The standard curves coupled with correction factors were used to calculate molar ratios.Results - The optimal CZE method coupled with correction factors was successfully applied to the determination of molar ratios of three purified polysaccharides and their corresponding partial acid hydrolysis products. ESP-A3, ESP-A4 and ESP-B4 were all typical acidic hetero-polysaccharides and consisted of xylose, arabinose, glucose, rhamnose, mannose, galactose, glucuronic acid and galacturonic acid, and their corresponding molar ratios were 6.8:7.5:1.0:14.0:13.7:22.3:10.2:3.8 for ESP-A3, 1.2:4.1:1.0:5.1:1.6:17.3:3.1:2.2 for ESP-A4, and 1.0:4.5:1.0:2.0:1.0:5.5:1.5:50.0 for ESP-B4.Conclusion - The results provided scientific evidence for the further study of the structure and bioactivity of complex acidic E. sinica polysaccharides