加压毛细管电色谱法高效分离分析葛根多糖中的单糖

High-efficiency separation and analysis of monosaccharides in Pueraria polysaccharides by pressurized capillary electrochromatography

葛根多糖具有抗氧化、抗肿瘤等众多生物活性,对葛根多糖进行单糖组成分析对其活性研究具有重要意义。该研究利用响应面分析法考察了超声辅助提取法中液料比、超声温度、超声时间和超声功率对葛根多糖提取率的交互影响,并拟合数据得到多元二次回归方程。同时建立了柱前衍生加压毛细管电色谱检测糖类的方法,对分离8种中性单糖的色谱条件进行了探索与优化,并将此方法应用于两种葛根实际样品的单糖组成测定。响应面分析结果表明,4个试验因素中,超声温度对两种葛根多糖提取率的影响程度最大,其次为液料比,超声时间和超声功率影响程度较小。结合软件预测分析得到的最佳条件及设备实际情况,确定葛根多糖的最佳提取工艺条件为:超声温度90℃,粉葛多糖液料比20 mL/g,柴葛多糖液料比40 mL/g,超声时间30 min,超声功率180 W。优化后的色谱分离条件为:采用Halo-2.7μm核壳型C18填料毛细管色谱柱,以乙腈-50 mmol/L pH 4.1的醋酸铵水溶液(18∶82,v/v)为流动相,在250 nm波长下检测,施加电压-20 kV。在此条件下可以实现24 min内对葡萄糖等8种中性单糖衍生物的快速分离,相比传统液相色谱方法大大提升了分离检测速度和分离柱效。方法学考察表明此方法具有较好的线性关系和良好的重复性。对实际样品分离鉴定表明,粉葛多糖主要由葡萄糖、甘露糖、鼠李糖和岩藻糖组成,4种单糖物质的量之比为1.00∶0.16∶0.14∶0.07;柴葛多糖主要由葡萄糖和甘露糖组成,2种单糖物质的量之比为1.00∶0.70。该研究为单糖化合物快速高效分离检测提供了新方法,并为葛根多糖单糖组成分析提供了参考。

Pueraria polysaccharides have been proven to possess biological activities such as bacteriostasis,anti-oxidative,anti-tumor,and immunity boosting activities.The variation in the structure,composition,and amount of monosaccharides in these polysaccharides may lead to different spatial structures and biological activities.Therefore,extraction of Pueraria polysaccharides and determination of the monosaccharide composition are of great significance for activity analysis and quality control.Direct detection of saccharides is difficult because they are strongly polar and do not show absorption in the ultraviolet region.At present,the commonly used methods for saccharide detection are liquid chromatography-ultraviolet detection,gas chromatography-ultraviolet detection,and mass spectrometry.Pressurized capillary electrochromatography is a high-efficiency microseparation technology.In this study,two kinds of Pueraria polysaccharides were extracted by an ultrasonic-assisted method,and response surface methodology was performed to explore the conditions for ultrasonic-assisted extraction of polysaccharides from Pueraria.The interaction effects of four factors,the liquid-solid ratio,ultrasonic extraction time,ultrasonic extraction temperature,and ultrasonic power,on the extraction rate of the polysaccharides were analyzed.By combining the optimal conditions predicted by the software and the actual equipment conditions,the optimal extraction conditions for Pueraria polysaccharides were chosen as follows:ultrasonic extraction temperature,90℃;liquid-solid ratio of Pueraria thomsonii Benth,20 mL/g;liquid-solid ratio of Pueraria lobata Ohwi,40 mL/g;ultrasonic extraction time,30 min;ultrasonic power,180 W.Through data fitting,the multiple quadratic regression equation of the four factors on the extraction rate of Pueraria polysaccharides was established.A novel method based on pressurized capillary electrochromatography for the separation and analysis of eight neutral monosaccharides has been established.The monosaccharides were derivatized by the 1-phenyl-3-methyl-5-pyrazolone pre-column derivatization method.The separation conditions for these monosaccharides were explored,and the buffer concentration,buffer pH,applied voltage,type of chromatographic column,and mobile phase ratio were optimized.Finally,the established pressurized capillary electrochromatography-ultraviolet detection method was applied to the detection and identification of two kinds of actual Pueraria polysaccharide samples.The results of response surface analysis showed that among the four experimental factors,ultrasonic extraction temperature had the greatest influence on the extraction rate of polysaccharides from the two kinds of Pueraria,followed by the liquid-solid ratio;the influence of the ultrasonic extraction time and ultrasonic power was relatively weak.The experimental conditions were determined as follows:the separation of eight neutral monosaccharide derivatives could be realized within 24 min on a Halo-2.7μm core-shell C18 capillary column with acetonitrile-50 mmol/L ammonium acetate aqueous solution(18∶82,v/v,pH 4.1)as the mobile phase,by detection at 250 nm under an applied voltage of-20 kV.The separation and detection speeds and the column efficiency achieved with this method were much better than those obtained with the traditional liquid chromatography method.The results show that the proposed method has a good linear relationship and good repeatability.The separation and identification results for the actual samples showed that the polysaccharides of Pueraria thomsonii Benth were mainly composed of glucose,mannose,rhamnose,and fucose in the molar ratio 1.00∶0.16∶0.14∶0.07.The polysaccharides of Pueraria lobata Ohwi were mainly composed of glucose and mannose in the molar ratio 1.00∶0.70.This study provides a novel method for the rapid and efficient separation and detection of neutral monosaccharides,and serves as a reference for analyzing the monosaccharide composition of Pueraria polysaccharides.