生物学杂志

生物学杂志 ›› 2020, Vol. 37 ›› Issue (3): 110-.doi: 10.3969/j.issn.2095-1736.2020.03.110

• 技术方法 • 上一篇    下一篇

柱前衍生化-HPCE法测定霍山石斛中游离氨基酸

  

  1. 1. 皖西学院 生物与制药工程学院， 六安 237012;2. 安徽省中药资源保护与持续利用工程实验室院， 六安 237012
  • 出版日期:2020-06-18 发布日期:2020-06-10
  • 作者简介:陈乃东，博士，教授，硕士生导师，研究方向为中药药效物质基础、质量标准及质量控制，E-mail: 2004cnd@163.com
  • 基金资助:
    国家自然科学基金(81573536，81274021)；安徽省自然基金面上项目 (1608085MH221);中国博士基金特别资助( 第9批) (2016T90559)；中国博士后研究面上项目(2014M551791)；国家级大学生创新创业项目（006040217104，006040217105）

Simultaneous quantification of free amino acids in Dendrobium huoshanense by a precolumn derivatization-HPCE technology

  1. 1. College of Biotechnology and Pharmaceutical Engineering, West Anhui University, Lu′an 237012; 2. Anhui Engineering Laboratory for Conservation and Sustainable Utilization of Traditional Chinese Medicine Resource, Lu′an 237012, China
  • Online:2020-06-18 Published:2020-06-10

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摘要： 游离氨基酸作为中药炮制与食品加工过程中参与米拉德反应的底物，是影响中药炮制中组分变化和食品风味的关键物质。霍山石斛因其鲜品难以保存，临床以其加热炮制成干燥品的茎入药，开展霍山石斛游离氨基酸研究对于揭示霍山石斛炮制机制、霍山石斛资源综合利用具有重要意义。采用柱前衍生化-高效毛细管电泳法（HPCE）对霍山石斛中12种游离氨基酸进行定性定量分析，结果显示霍山石斛中游离氨基酸最佳毛细管电泳分离条件为：检测波长254 nm、pH 11的60 mmol/L硼砂+20 mmol/L磷酸氢二钠缓冲液、分离电压10 kV、毛细管有效长度50 cm。在该条件下，12种氨基酸可在60 min内实现基线分离，在最优分离条件下，构建了标准曲线并进行了方法学考察，测得霍山石斛根、茎、叶中总游离氨基酸含量分别为1.8958、 2.0736和2.5954 mg/g，各氨基酸线性相关系数R2均大于0.99，加样回收率为90.17%~110.25%，RSD均小于5%（n=3）。方法准确、可靠、重现性较好，可用于霍山石斛中游离氨基酸的定性定量分析。

关键词: 高效毛细管电泳, 柱前衍生化, 游离氨基酸, 霍山石斛

Abstract: As the substrates of Maillard reaction, free amino acids were the key matters which may greatly affect the chemical compositions in traditional Chinese medicines and the flavored constituents in food. Dendrobium husohansense, a rare and endangered medicinal plant, has long been used in clinical as its processed dry stems due to the great difficulty in the preservation of its fresh stems. Thus, it is essential to assay the free amino acids for the investigation of the processing mechanism of D. huoshanese. In this study, we established an efficient high performance capillary electrophoresis (HPCE) method to qualify and quantify the free amino acids in D. huoshanense. The results showed that the optimal HPCE method was as follows: detection wavelength at 254 nm, separation voltage of 10 kV with effective length of uncoated standard fused silica capillary 55 cm. The running buffer was 60 mmol/L Na2B4O7-Na2HPO4 with pH value of 11.0. Under these conditions, all the 12 investigated amino acids could be separated in 60 min with the R2 values of all the calibrations for the 12 amino acids more than 0.99 and recovery within 90.17%-110.25% (RSD≤5%, n=3). The total contents of total amino acids in the root, stem and leaf in D. huoshanense were 1.8958, 2.0736 and 2.5954 mg/g, respectively. These conformed the established method in our experiments is accurate, reliable and reproducible, and can be used to qualify and quantify the free amino acids in D. huoshanense.

Key words: high performance capillary electrophoresis (HPCE), precolumn derivatization, free amino acids, Dendrobium huoshanense

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