采用1-苯基-3-甲基-5-吡唑啉酮(1-phenyl-3-methyl-5-pyrazolone,PMP)试剂柱前衍生结合高效液相色谱法同时测定地参多糖中的8种单糖组分。样品经石油醚脱脂、超声提取、Sevage试剂除蛋白,无水乙醇沉淀制得多糖溶液,然后加入2 mol/L 三氟乙酸,在110 ℃下水解5 h,得到单糖溶液,最后加入0.5 mol/L PMP-甲醇溶液在70 ℃水浴中衍生1 h,进行测定。结果表明,地参多糖主要由甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖、半乳糖、阿拉伯糖和岩藻糖组成,在各自质量浓度范围内线性关系良好(r≥0.999 5),平均回收率为82.58%～108.71%,检出限为0.16～0.85 μg/mL,各单糖组分平均物质的量比为:1.00:0.60:0.54:5.15:266.53:890.11:4.12:1.05,其中半乳糖的平均质量分数为120.446 mg/g、葡萄糖的平均质量分数为35.306 mg/g,为地参中单糖主要成分。主成分分析表明,地参单糖含量的高低在我国地域上呈现出北方＞南方＞西南的趋势。该方法操作简便,灵敏度高,衍生物质稳定性好,重复性好,实用可靠,适用于地参多糖中单糖成分分析和含量测定,可为地参药材的质量控制提供参考。

A method for the simultaneous determination of the eight monosaccharide components of Lycopus lucidus polysaccharides, utilizing pre-column derivatization of 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent and high performance liquid chromatography (HPLC), is reported. The sample was degreased by petroleum ether and extracted by treatment with ultrasound waves, proteins were removed by treatment with the Sevage reagent, and a polysaccharide solution was prepared by precipitation with anhydrous ethanol. The polysaccharide solution was then treated with 2 mol/L trifluoroacetic acid (TFA) for 5 h at 110 ℃ to obtain the corresponding monosaccharide solution by hydrolysis. Finally, 0.5 mol/L of a methanolic solution of PMP was added to the monosaccharide solution kept in a water bath maintained at 70 ℃ for 1 h. All eight monosaccharides exhibited a linear relationship (r≥0.999 5) within their respective mass concentration ranges; the average recovery rate was 82.58%-108.71%, and the detection limit was 0.16-0.85 μg/mL. The polysaccharide extracted from Lycopus lucidus was mainly composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose, and phycosaccharide. Each of these monosaccharide components was present in an average ratio of 1.00:0.60:0.54:5.15:266.53:890.11:4.12:1.05. The average content of galactose and glucose, the major monosaccharide components in Lycopus lucidus polysaccharides, was 120.446 and 35.306 mg/g, respectively. Principal component analysis revealed that the monosaccharide content in Lycopus lucidus polysaccharides showed a trend of northern>southern>southwestern with respect to the different provinces in China. The method reported in this study is simple, sensitive, reproducible, practical, and reliable, and the derivatized substances can exhibit a high degree of stability. Therefore, this method is suitable for the identification and analysis of monosaccharide components in Lycopus lucidus polysaccharides and represents a reference standard for quality control of Lycopus lucidus polysaccharides used for medicinal purposes.