王小燕 郭常润 常军民 陈丽

摘 要 目的：建立懷牛膝多糖的柱前衍生化-高效液相色谱（HPLC）指纹图谱，测定其主要单糖成分的含量，为怀牛膝饮片的质量评价提供参考。方法：以10批不同生产厂家的怀牛膝饮片为样品，采用水提醇沉法提取其多糖后，以三氟乙酸进行水解，再采用1-苯基-3-甲基-5-吡唑啉酮衍生化处理后，进行HPLC分析。色谱柱为Hanbon Hedera C18，柱温为30 ℃，流速为1.2 mL/min，流动相为乙腈-0.02 mol/L乙酸铵溶液（梯度洗脱），检测波长为250 nm，进样量为20 μL。采用《中药色谱指纹图谱相似度评价系统》（2012A版）建立10批怀牛膝多糖的HPLC指纹图谱并进行相似度评价，通过与对照品比对进行共有峰指认，并采用SPSS 25.0软件进行聚类分析。采用HPLC法对指认的单糖成分进行含量测定。结果：10批怀牛膝多糖样品的相似度均大于0.95;共标定了9个共有峰，并指认了其中5个共有峰，分别为无水葡萄糖（峰1）、甘露糖（峰2）、鼠李糖（峰4）、半乳糖醛酸（峰5）、阿拉伯糖（峰7）。聚类分析结果显示，编号为S1的样品单独为一类，编号为S2、S5、S8和S9的样品聚为一类，编号为S3、S4、S6、S7和S10的样品聚为一类。含量测定结果显示，10批样品中无水葡萄糖的含量为6.17～17.55 mg/g、甘露糖的含量为3.31～7.66 mg/g、鼠李糖的含量为38.80～73.97 mg/g、半乳糖醛酸的含量为2.49～8.95 mg/g、阿拉伯糖的含量为11.30～28.58 mg/g。结论：本研究建立的柱前衍生化-HPLC指纹图谱及单糖组分的含量测定方法可为怀牛膝饮片质量评价提供参考。

关键词 怀牛膝多糖;单糖;柱前衍生化;高效液相色谱法;指纹图谱;含量测定

中图分类号 R284 文献标志码 A 文章编号 1001-0408（2021）03-0294-07

DOI 10.6039/j.issn.1001-0408.2021.03.08

ABSTRACT   OBJECTIVE： To establish pre-column derivatization-HPLC fingerprint of polysaccharide from Achyranthes bidentata，and to determine the contents of monosaccharide components，so as to provide reference for quality evaluation of A. bidentata decoction pieces.  METHODS： Taking 10 batches of A. bidentata decoction pieces from different manufacturers as samples， the polysaccharides were extracted by water extraction and alcohol precipitation， hydrolyzed by trifluoroacetic acid， and then derivatized by 1-phenyl-3-methyl-5-pyrazolone for HPLC analysis. The determination was performed on Hanbon Hedera C18 column with column temperature of 30 ℃ at the flow rate of 1.2 mL/min. The mobile phase consisted of acetonitrile-0.02 mol/L ammonium acetate solution （gradient elution）. The detection wavelength was set at 250 nm， and sample size was 20 μL. HPLC fingerprint was established and similarity evaluation was performed for 10 batches of A. bidentata polysaccharide by using TCM Chromatogramic Fingerprint Similarity Evaluation System （2012A edition）. The common peak was identified by comparing with the reference substance，and cluster analysis was performed by using SPSS 25.0 software. The contents of identified monosaccharides were determined by HPLC. RESULTS： The similarity of 10 batches of A. bidentata polysaccharide were all higher than 0.95. Nine common peaks were fixed and a total of 5 common peaks were identified， namely anhydrous glucose （peak 1）， mannose （peak 2）， rhamnose （peak 4）， galacturonic acid （peak 5） and arabinose （peak 7）. Results of cluster analysis showed that S1 sample was classified into one category; S2， S5， S8 and S9 samples were clustered into one category; S3， S4， S6， S7 and S10 samples were clustered into one category. Results of content determination showed that the contents of anhydrous glucose in 10 batches of samples ranged from 6.17 to 17.55 mg/g; those of mannose ranged from 3.31 to 7.66 mg/g; those of rhamnose ranged from 38.80 to 73.97 mg/g; those of galacturonic acid ranged from 2.49 to 8.95 mg/g; those of arabinose ranged from 11.30 to 28.58 mg/g. CONCLUSIONS： Established pre-column derivatization HPLC fingerprints and content determination method can provide reference for quality evaluation of A. bidentata decoction pieces.

KEYWORDS   Achyranthes bidentata polysaccharide; Monosaccharide; Pre-column derivatization; HPLC; Fingerprint; Contnet determination

怀牛膝Achyranthes bidentata BI.为常用的大宗中药材之一，最早出自《神农本草经》，以根入药，主要含有甾酮类、皂苷类、多糖类等化合物[1-2]。……