马兜铃水提物在大鼠体内代谢产物的定性分析  

Qualitative Analysis of Metabolites of Aristolochiae Fructus Aqueous Extract in Rats

作　　者：王芳 李春英[1] 易艳[1] 刘素彦 赵雍[1] 孟晶[1] 田婧卓 王连嵋[1] 韩佳寅[1] 潘辰[1] 张宇实[1] 柳辰玥 秦莎莎 王敦方 鲜中 唐旋 刘美婷 梁爱华[1] WANG Fang;LIChunying;YIYan;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(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 10070,China)

机构地区：[1]中国中医科学院中药研究所,北京100700

出　　处：《中国实验方剂学杂志》2023年第13期112-121,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：中国中医科学院科技创新工程项目(CI2021A04806、CI2021B016、CI2021A04801);国家自然科学基金项目(82174073、82192913);国家“重大新药创制”科技重大专项(2018ZX09101002-003);国家中医药管理局岐黄学者项目。

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-MSE),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 C18色谱柱(2.1 mm×100 mm,1.7μm),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m/z 100~1200。结合UNIFI 1.9.4.053系统,采用Pathway-MSE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(P<0.05,P<0.01),AFE组粪便中ALⅠ的代谢产物表达量显著低于AAⅠ组(P<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。Objective Based on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MSE)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 acidⅠ(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 C18 column(2.1 mm×100 mm,1.7μm)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 m/z 100-1200.In combination with UNIFI 1.9.4.053 system,the Pathway-MSE 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 AAⅠgroup.Result Compared with AAⅠgroup,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 AAⅠgroup,prototype components of AAⅠin serum and most metabolic derivatives of AAⅠ[AAⅠa,aristolochic lactamⅠ(ALⅠ)a,7-OHALⅠand its conjugated derivatives]in biological samples were significantly increased in AFE group(P<0.05,P<0.01),except that the metabolic amount of ALⅠin feces of AFE group was remarkably lowed than that of AAⅠgroup(P<0.01).In addition,a variety

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 

分 类 号：R22[医药卫生—中医基础理论] R28[医药卫生—中医学] R969.1[理学—分析化学] O657[理学—化学]