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浙江大学学报（医学版）
J Zhejiang Univ (Med Sci)  2017, Vol. 46 Issue (1): 66-73    DOI: 10.3785/j.issn.1008-9292.2017.02.10
    
LC/MS guided approach to discovering nephroprotective substances from Huangkui capsule
MA Tingting1(),WANG Yi2,CHEN Xiaoqian2,ZHAO Xiaoping1,*()
(1) Zhejiang Chinese Medical University, Hangzhou 310053, China
(2) College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
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Abstract  Objective

To discover the nephroprotective substances from Huangkui capsule.

Methods

The components of Huangkui capsule were isolated by preparative liquid chromatography, and the active components were screened by LC/MS and identified. The adriamycine-injured HK-2 cells were treated with various active components with different concentrations, and the malonaldehyde (MDA) content, adenosine triphosphate (ATP) level and mitochondrial oxygen consumption rate were measured to verify the protective activity of the compounds.

Results

Four active components in Huangkui capsule were identified to exert nephroprotective effects. Fifteen flavanoids from these four components were tentatively identified by LC/MS, and hyperin, myricetin, quercetin, rutin and isoquercetin were confirmed. Hyperin, myricetin quercetin and rutin showed dose-dependent protective effects on injured HK-2 cells. Espacially, hyperin significantly reduced MDA content, quercetin and rutin significantly increased ATP level, and myricetin significantly increased mitochondrial oxygen consumption rate.

Conclusion

Hyperin, myricetin, querctein and rutin might be the potential nephroprotective compounds in Huangkui capsule, their effects may be related to the inhibition of lipid peroxidation and the alleviation of mitochondrial damage.

Key words： Abelmoschus/drug effects      Capsules      Epithelial cells/cytology      Kidney tubules, proxima/cytology      Kidney/injuries      Mass spectrometry      Chromatography, liquid      Disease models, animal     
Received: 04 May 2016      Published: 06 July 2017
CLC:  R284.1  
  R285.5  
Corresponding Authors: ZHAO Xiaoping     E-mail: 960144756@qq.com;zhaoxiaoping@zcmu.edu.cn
About author: ZHAO Xiaoping, E-mail: zhaoxiaoping@zcmu.edu.cn
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MA Tingting
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Cite this article:

MA Tingting,WANG Yi,CHEN Xiaoqian,ZHAO Xiaoping. LC/MS guided approach to discovering nephroprotective substances from Huangkui capsule. J Zhejiang Univ (Med Sci), 2017, 46(1): 66-73.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2017.02.10     OR     http://www.zjujournals.com/med/Y2017/V46/I1/66


液相色谱-质谱联用导向的黄葵胶囊肾保护活性物质研究

目的

本研究旨在发现黄葵胶囊肾保护活性物质。

方法

采用组分制备结合液相色谱—质谱联用（LC/MS）导向筛选技术，快速发现黄葵胶囊中具有肾保护活性的物质，在盐酸阿霉素损伤HK-2细胞模型上验证其活性，并检测这些活性物质对损伤HK-2细胞中丙二醛（MDA）含量、ATP含量、线粒体耗氧率的影响。

结果

筛选发现黄葵胶囊中四个组分具有肾保护活性，运用LC/MS分析推测出15个黄酮类化合物，其中鉴定出金丝桃苷、杨梅素、槲皮素、芦丁和异槲皮素五个化合物。验证结果表明，金丝桃苷、杨梅素、槲皮素、芦丁对损伤HK-2细胞的保护作用呈一定的量效关系，均能降低损伤细胞MDA含量、升高ATP含量、提高线粒体耗氧率，其中金丝桃苷能降低MDA含量，槲皮素和芦丁能升高ATP含量，杨梅素能提高线粒体耗氧率。

结论

金丝桃苷、杨梅素、槲皮素、芦丁可能是黄葵胶囊中潜在的肾保护活性物质，其保护作用可能与抗脂质过氧化、减轻线粒体损伤有关。

关键词： 黄葵属/药物作用,  胶囊,  上皮细胞/细胞学,  肾小管, 近端/细胞学,  肾/损伤,  质谱分析法,  色谱法, 液相,  疾病模型, 动物 
Fig 1 Protection effects of total component and components 1-6 on injured HK-2 cells
Fig 2 HPLC-PDA chromatograms of components 3-6 and mixed reference substances
活性成分编号 转速 (tR/min) 化合物 [M-H]-峰 (m/z) HPLC-ESI-MS/MS检测 (m/z)
1 23.1 Floramanoside B 495 333[M-H-Glc]-
2 24.3 杨梅素-3-O-芸香糖苷 625 317 [M-H-Rha-Glc]- 316 [M-2H-Rha-Glc]-
3 24.7 杨梅素-3-O-β-D-半乳糖苷 479 317[M-H-Gal]- 316 [M-2H-Gal]-
4 25.8 杨梅素-3-O-β-D-葡萄糖苷 479 317[M-H-Glc]- 316 [M-2H-Glc]-
5 27.1 槲皮素-3-O-β-D-木糖基 595 463 [M-H-Gal]- 301 [M-H-Xyl-Gal]-
(1→2)-O-β-D-半乳糖苷300 [M-2H-Xyl-Gal]-
6 32.8 Floramanoside C 509 333[M-H-GluA]-
7 34.7 槲皮素-3-O-刺槐糖苷 609 463 [M-H-Rha]- 301[M-H-Rha-Gal]-
8* 36.4 芦丁 609 463 [M-H-Rha]- 301[M-H-Rha-Glc]-
9* 38.9 金丝桃苷 463 301[M-H-Gal]-
10* 42.9 异槲皮素 463 301[M-H-Glc]-
11 46.5 杨梅素-3′-O-β-D-葡萄糖苷 479 317[M-H-Glc]-
12 53.6 棉皮素-8-O-β-D-葡萄糖醛酸 493 317[M-H-GluA]-
13* 56.5 杨梅素 317 317[M-H]- 299[M-H-H2O]-
14 57.7 槲皮素-3′-O-葡萄糖苷 463 301 [M-H-Glc]-
15* 63.9 槲皮素 301 179[M-H-C7H6O2]- 151[M-H-C8H6O3]-
Tab 1 Chromatographic and mass spectral information of compounds in components 3-6
Fig 3 Effects of hyperosin, myricetin, quercetin and rutin on cell morphology in doxorubicin hydrochloried-injured HK-2 cells
Fig 4 Dose-dependent protection of hyperosin, myricetin, quercetin and rutin in doxorubicin hydrochloride-injured HK-2 cells
(n=3, $\overline {x}$±s)
组别 MDA含量
(nmol/mg)		 ATP相对含量
(%)		 线粒体耗氧率Δ
溶剂对照组 5.09±1.25 100.00±9.86 3.08±0.30
模型对照组 11.52±4.79** 50.63±7.32** 1.57±0.26**
阳性对照组 7.55±1.79 85.33±12.67## 2.57±0.53#
金丝桃苷组 5.67±0.46# 73.72±0.16 2.37±0.46
杨梅素组 7.89±1.33 74.32±9.77 2.67±0.44#
槲皮素组 7.14±1.08 87.61±13.83## 2.23±0.32
芦丁组 6.52±0.80 88.29±10.99## 2.26±0.35
Tab 2 Effects of hyperosin, myricetin, quercetin and rutin on MDA content, ATP level and mitochondrial oxygen consumption rate in doxorubicin hydrochloride-injured HK-2 cells
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