Qualitative Analysis of Metabolites of Aristolochiae Fructus Aqueous Extract in Rats

WANG Fang; LI Chunying; YI Yan; LIU Suyan; ZHAO Yong; MENG Jing; TIAN Jingzhuo; WANG Lianmei; HAN Jiayin; PAN Chen; ZHANG Yushi; LIU Chenyue; QIN Shasha; WANG Dunfang; XIAN Zhong; TANG Xuan; LIU Meiting; LIANG Aihua

doi:10.13422/j.cnki.syfjx.20230764

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Qualitative Analysis of Metabolites of Aristolochiae Fructus Aqueous Extract in Rats

更新时间：2023-06-02

- Qualitative Analysis of Metabolites of Aristolochiae Fructus Aqueous Extract in Rats
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 13, Pages: 112-121(2023)
- 作者机构：
  
  中国中医科学院中药研究所北京 100700
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20230764
  CLC： R22;R28;R969.1;O657
- Published：05 July 2023，
  
  Published Online：07 April 2023，
  
  Received：06 February 2023，
- 稿件说明：
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王芳,李春英,易艳等.马兜铃水提物在大鼠体内代谢产物的定性分析[J].中国实验方剂学杂志,2023,29(13):112-121.

WANG Fang,LI Chunying,YI Yan,et al.Qualitative Analysis of Metabolites of Aristolochiae Fructus Aqueous Extract in Rats[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(13):112-121.
王芳,李春英,易艳等.马兜铃水提物在大鼠体内代谢产物的定性分析[J].中国实验方剂学杂志,2023,29(13):112-121. DOI： 10.13422/j.cnki.syfjx.20230764.

WANG Fang,LI Chunying,YI Yan,et al.Qualitative Analysis of Metabolites of Aristolochiae Fructus Aqueous Extract in Rats[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(13):112-121. DOI： 10.13422/j.cnki.syfjx.20230764.

摘要

目的

基于超高效液相色谱-四极杆-飞行时间质谱法（UPLC-Q-TOF-MS

），定性分析马兜铃水提物（AFE）和马兜铃酸Ⅰ（AAⅠ）大鼠体内马兜铃酸类物质（AAs）代谢产物的差异。

方法

该实验选取SD大鼠，分别连续灌胃给予AFE（110 g·kg

-1

·d

-1

）和AAⅠ（5 mg·kg

-1

·d

-1

）5 d，收集血清、尿液和粪便。采用ACQUITY UPLC BEH C

色谱柱（2.1 mm×100 mm，1.7 μm），流动相甲醇（含0.01%甲酸+5 mmol·L

-1

乙酸铵，A）-水（含0.01%甲酸+5 mmol·L

-1

乙酸铵，B）梯度洗脱（0~1.0 min，10%B；1.0~7.0 min，10%~75%B；7.0~7.2 min，75%~95%B；7.2~10.2 min，95%B；10.2~10.3 min，95%~10%B；10.3~12.0 min，10%B），流速0.3 mL·min

-1

。电喷雾离子源（ESI）正离子模式采集，扫描范围

100~1 200。结合UNIFI 1.9.4.053系统，采用Pathway-MS

定性分析鉴定生物样品（血清、尿、粪便）中AAs原型和相关代谢产物的种类，平行比较AFE组和AAⅠ组亚急性毒性实验中大鼠体内代谢产物的异同。

结果

与AAⅠ组比较，AFE组血清、尿液与粪便生物样品中分别鉴定出6、10和13个共有代谢产物，以及14、20和30个特有代谢产物。主要AAs成分均遵循去甲基化、硝酸还原和结合等代谢过程。与AAⅠ组的平行比较分析显示，AFE组血清中AAⅠ原型成分和生物样品中多数AAⅠ代谢产物［AAⅠa、马兜铃内酰胺I（ALⅠ）a、7-OHALⅠ及其结合型衍生物等］表达量均明显升高（

0.05，

0.01），AFE组粪便中ALⅠ的代谢产物表达量显著低于AAⅠ组（

0.01），AFE组尿液和粪便中还鉴定出多种AAⅠ组未发现的ALⅠ外排代谢产物。

结论

AFE中AAs成分均显示出与AAⅠ成分相近的体内代谢规律，但马兜铃中多种AAs成分共存可能会影响AAⅠ的代谢，通过增加AAⅠ与ALⅠ代谢外排，从而达到减小不良反应作用。

Abstract

Objective

Based on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS

） technique， we identified qualitatively the metabolites of aristolochic acid（AAs） in rat in order to analyze the metabolic differences between water extract of Aristolochiae fructus（AFE） and Aristolochic acid Ⅰ（AAⅠ）.

Method

SD rats were selected and administered AFE（110 g·kg

-1

·d

-1

） or AAⅠ（5 mg·kg

-1

·d

-1

） by oral for 5 days， respectively. Serum， urine and feces were collected after administration. Through sample pretreatment， ACQUITY UPLC BEH C

column（2.1 mm×100 mm， 1.7 μm） was used with the mobile phase of 0.01% formic acid methanol（A）-0.01% formic acid water（B， containing 5 mmol·L

-1

ammonium acetate） for gradient elution（0-1 min， 10%B； 1-7 min， 10%-75%B； 7-7.2 min， 75%-95%B； 7.2-10.2 min， 95%B； 10.2-10.3 min， 95%-10%B； 10.3-12 min， 10%B） at a flow rate of 0.3 mL·min

-1

. Positive ion mode of electrospray ionization（ESI

） was performed in the scanning range of

100-1 200. In combination with UNIFI 1.9.4.053 system， the Pathway-MS

was used to qualitatively analyze and identify the AAs prototype and related metabolites in biological samples（serum， urine and feces）， and to compare the similarities and differences of metabolites in rats in the subacute toxicity test between AFE group and AAⅠ group.

Result

Compared with AAⅠ group， 6， 10， 13 common metabolites and 14， 20， 30 unique metabolites were identified in biological samples（serum， urine and feces） of AFE group， respectively. Moreover， the main AAs components always followed the metabolic processes of demethylation， nitrate reduction and conjugation. Compared with common metabolites in AAⅠ group， prototype components of AAⅠ in serum and most metabolic derivatives of AAⅠ［AAⅠa， aristolochic lactam Ⅰ（ALⅠ）a， 7-OHALⅠ and its conjugated derivatives］ in biological samples were significantly increased in AFE group（

0.05，

0.01）， except that the metabolic amount of ALⅠ in feces of AFE group was remarkably lowed than that of AAⅠ group（

0.01）. In addition， a variety of special ALⅠ efflux derivatives were also identified in the urine and feces of the AFE group.

Conclusion

Although major AAs components in AFE all show similar metabolic rules as AAⅠ components

in vivo

， the coexistence of multiple AAs components in Aristolochiae Fructus may affect the metabolism of AAⅠ， and achieve the attenuating effect by increasing the metabolic effection of AAⅠ and ALⅠ.

关键词

马兜铃水提物马兜铃酸超高效液相色谱-四极杆-飞行时间质谱法（UPLC-Q-TOF-MSE）生物样品代谢产物定性分析

Keywords

aqueous extract of Aristolochiae Fructusaristolochic acidultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MSE）biological samplesmetabolitesqualitative analysis

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