色谱

色谱 ›› 2019, Vol. 37 ›› Issue (3): 305-312.DOI: 10.3724/SP.J.1123.2018.11022

• 研究论文 • 上一篇    下一篇

成骨细胞膜色谱/超高效液相色谱-飞行时间质谱法快速筛选六味地黄汤抗骨质疏松活性成分

吴灿1, 许平翠2, 姚伟宣3, 寿旦2, 朱岩1, 王娜妮1,2   

  1. 1. 浙江大学化学系, 浙江 杭州 310007;
    2. 浙江省中医药研究院中药研究中心, 浙江 杭州 310007;
    3. 浙江警察学院刑事科学技术系, 浙江省毒品防控技术研究重点实验室, 浙江 杭州 310053
  • 收稿日期:2018-11-15 出版日期:2019-03-08 发布日期:2015-01-21
  • 通讯作者: 王娜妮.Tel:88849089,E-mail:wnn8511@163.com.
  • 基金资助:

    国家自然科学基金（81603252）；浙江省自然科学基金（LQ17H280002）.

Rapid screening of anti-osteoporosis active ingredients from Liuwei Dihuang Decoction by osteoblast membrane chromatography/ultra-high performance liquid chromatography-time of flight mass spectrometry

WU Can1, XU Pingcui2, YAO Weixuan3, SHOU Dan2, ZHU Yan1, WANG Nani1,2   

  1. 1. Department of Chemistry, Zhejiang University, Hangzhou 310007, China;
    2. Department of Medicine, Zhejiang Academy of Traditional Chinese Medicine, Hangzhou 310007, China;
    3. Department of Criminal Science and Technology, Key Laboratory of Drug Prevention and Control Technology of Zhejiang Province, Zhejiang Police College, Hangzhou 310053, China
  • Received:2018-11-15 Online:2019-03-08 Published:2015-01-21
  • Supported by:

    National Natural Science Foundation of China (No. 81603252); Zhejiang Provincial Natural Science Foundation of China (No. LQ17H280002).

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摘要：

采用成骨细胞建立了细胞膜色谱/超高效液相色谱-飞行时间质谱（CMC/UPLC-TOF/MS）的分析方法。该法可快速筛选中药方剂六味地黄汤中潜在的抗骨质疏松活性成分，通过细胞试验和斑马鱼骨质疏松模型试验验证了筛选结果梓醇的体内外药效作用。以六味地黄汤水提物（90 g/L）为样品，通过CMC/UPLC-TOF/MS分析，快速鉴别细胞膜色谱柱保留成分群，高选择性获取六味地黄汤中16种潜在活性成分。该文以前沿色谱法分析梓醇、丹皮酚、齐墩果酸与细胞膜色谱固定相的亲和强度，选择亲和强度和含量较高的梓醇进行体内外药效验证，发现梓醇在对小鼠成骨细胞有显著促生长作用，能提高骨质疏松斑马鱼头部骨矿化面积。CMC/UPLC-TOF/MS筛选方法能在复杂中药方剂中快速得到抗骨质疏松活性成分，具有操作简便、快速、高效灵敏的优势。

关键词: 超高效液相色谱-飞行时间质谱, 骨质疏松症, 六味地黄汤, 细胞膜色谱, 中药方剂

Abstract:

A method was established for determination of potential anti-osteoporosis ingredients from Liuwei Dihuang Decoction by osteoblast cell membrane chromatography/ultra-performance liquid chromatography/time-of-flight mass spectrometry (CMC/UPLC-TOF/MS). The osteoblasts were used as the source of cell membrane for the preparation of CMC stationary phase. An osteoblast CMC column (10 mm×2 mm) was prepared by coating silica gel (0.05 g) with cell membrane. The active ingredients in the aqueous extract of Liuwei Dihuang Decoction (90 g/L) were first screened by CMC. Water was used as the mobile phase, and the flow rate was 0.20 mL/min. Then, the eluates of the CMC were qualitatively analyzed by UPLC-TOF/MS using a WATERS ACQUITY UPLC BEH C18 column (10 mm×2 mm). Acetonitrile-water was used as the mobile phase at a flow rate of 0.40 mL/min. This method could quickly and selectively identify 16 potential anti-osteoporotic active ingredients from Liuwei Dihuang Decoction. Due to high catalpol content in Liuwei Dihuang Decoction and its good affinity with CMC column, catapol was selected for in vivo and in vitro pharmacological examinations. It was found that catalpol (1-10 μmol/L) could significantly promote the proliferation of osteoblasts in a dose-dependent manner. It also significantly increased the area of bone staining in osteoporosis zebrafish model. The osteoblast CMC/UPLC-TOF/MS method could quickly screen the anti-osteoporosis active ingredients in complex traditional Chinese medicine prescriptions, and had the advantages of simple operation, high efficiency and high sensitivity.

Key words: cell membrane chromatography (CMC), Liuwei Dihuang Decoction, osteoporosis, traditional Chinese medicine prescription, ultra-performance liquid chromatography/time-of-flight mass spectrometry(UPLC-TOF/MS)

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