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
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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。
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.
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.
引文
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[4] Pharmacopoeia Commission of People’s Republic of China. Pharmacopoeia of People’s Republic of China, Part 1. Beijing: China Medical Science Press, 2015: 705 国家药典委员会. 中华人民共和国药典, 一部. 北京: 中国医药科技出版社, 2015: 705
[5] Bo Y, Sun G X. J Sep Sci, 2017, 41(6): 1182
[6] Du H, Li J J, Fan F, et al. Chinese Journal of Chromatography, 2017, 35(2): 191 杜晖, 李静静, 范菲, 等. 色谱, 2017, 35(2): 191
[7] Xu L, Xu B, Zhao Z Y, et al. J Chromatogr A, 2017, 1516: 125
[8] Ding X, Cao Y, Yuan Y F, et al. Anal Chem, 2016, 88(24): 12081
[9] Wang N N, Xin H L, Zhang Q Y, et al. Talanta, 2017, 162: 10
[10] Wang N N, Zhang Q Y, Xin H L, et al. J Sep Sci, 2017, 40(22): 4311
[11] Wang J H, Lei X, Cheng X R, et al. Alzheimers Res Ther, 2016, 8(10): 57
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[14] Maasumi K, Michael R L, Rapoport A M. Drugs, 2018, 78(9): 1
[15] Filipovic B, Sosic-Jurjevic B, Ajdzanovic V, et al. J Anat, 2017, 230(6): 787
[16] Zhang L B, Man Z T, Li W, et al. Mol Immunol, 2017, 87: 249
[17] Ren H, Liang D, Shen G Y, et al. Menopause, 2016, 23(4): 441
[18] Li S Y, Wang L N, Yang J, et al. J Sep Sci, 2016, 39(11): 2050
[19] Lucie G, Nikzad N, Ian R B, et al. Chem Sci, 2013, 4: 764
[20] Liu C Y, Chen K, Lu Y W, et al. Phytother Res, 2018, 32(6): 1047
[21] Carnovali M, Luzi L, Terruzzi I, et al. Endocrine, 2017, 61(6): 1