基于UV-vis/HPLC-CAD法的不同地区牛膝糖含量测定及其道地性分析

Genuineness Study of Niuxi(Achyranthis Bidentatae Radix)Based on Different Carbohydrate Contents Analysis by UV-Vis/HPLC-CAD

目的对不同地区牛膝药材D-木糖、果糖、D-葡萄糖、蔗糖、蔗果三糖和总多糖含量进行分析,为牛膝道地性研究提供参考依据。方法苯酚硫酸法结合紫外分光光度(UV-Vis)法测定不同地区牛膝中总多糖含量;采用高效液相-电雾检测器(HPLC-CAD)法测定其D-木糖、果糖、D-葡萄糖、蔗糖、蔗果三糖含量,色谱柱为Asahipak NH2P-504E,流动相为乙腈-水,梯度洗脱,流速为1 mL/min,柱温为40℃。采用单因素方差分析(ANOVA)、聚类热图和主成分分析(PCA)研究不同产地牛膝中糖含量变化趋势。结果测定方法线性关系良好,相关系数为0.9993~0.9998,加样回收率为97.34%~103.55%,相对标准偏差为0.86%~2.28%。ANOVA分析结果显示河南牛膝中总多糖、蔗糖和蔗果三糖平均含量均显著高于内蒙古和河北样品(P<0.05),而木糖含量则显著低于内蒙古和河北样品(P<0.05)。不同地区牛膝糖含量指标聚类热图和PCA分析结果均显示,河南牛膝样品可单独聚为一个分支,内蒙古和河北牛膝样品可聚为另一个分支。结论研究建立的牛膝单糖、寡糖和总多糖含量测定与多元统计分析相结合的方法可将河南牛膝样品与内蒙古、河北牛膝样品进行区分,为怀牛膝的道地性研究和品质评价提供依据。

Objective The differences of monosaccharide,oligosaccharide and total polysaccharide contents of Niuxi(Achyran⁃this Bidentatae Radix)in different habitats were compared to study the genuine connotation of Niuxi(Achyranthis Bidentatae Ra⁃dix).Methods The total polysaccharide content in Niuxi(Achyranthis Bidentatae Radix)from different regions was determined by ultraviolet spectrophotometry(UV-Vis)combined with phenol-sulfuric acid method.The contents of D-xylose,fructose,D-glucose,sucrose and kestose were determined by HPLC-CAD method,with Asahipak NH2P-504E column(4.6 mm×250 mm,3.5μm),mobile phase consisted of acetonitrile-water(gradient elution)at the flow rate of 1 mL/min and column temper⁃ature of 40℃.The genuine character was explored based on several kinds of sugar content of Niuxi(Achyranthis Bidentatae Ra⁃dix)from different areas by multivariate statistical analysis.Results The linear relationship of the determination method was good,with correlation coefficients ranging from 0.9993 to 0.9998.The sample recovery rate was 97.34%-103.55%,and the relative standard deviation was 0.86%-2.28%.The ANOVA analysis results showed that the average content of total polysac⁃charides,sucrose and fructose in Henan Niuxi(Achyranthis Bidentatae Radix)was significantly higher than that in Inner Mongo⁃lia and Hebei samples(P<0.05),while the xylose content was significantly lower than that in Inner Mongolia and Hebei sam⁃ples(P<0.05).The clustering heat map and PCA analysis results of the sugar content indicators of Niuxi(Achyranthis Bidenta⁃tae Radix)in different regions showed that the Henan Niuxi(Achyranthis Bidentatae Radix)samples can be clustered into one branch separately,while the Inner Mongolia and Hebei Niuxi(Achyranthis Bidentatae Radix)samples can be clustered into an⁃other branch Conclusion A method to determine the contents of monosaccharide,oligosaccharide and total polysaccharide in Niuxi(Achyranthis Bidentatae Radix)combined with multivariate statistical analysis was established,which could distinguish genuine producing areas of Niuxi(Achyranthis Bidentatae Radix)from the samples of the other areas.The research can provide a new re⁃search strategic basis for the genuine study and quality evaluation of Niuxi(Achyranthis Bidentatae Radix).