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Medicina

使用液相色谱-串联质谱分析原发性痛经大鼠的原始和加工的Cyperi Rhizoma样品

Published: December 23, 2022
doi:
10.3791/64691
, , , , ,
1Chongqing Engineering Research Center of Pharmaceutical Evaluation of Chinese Medicine,Chongqing Academy of Chinese Materia Medica

Summary

在这里，使用超高效液相色谱-高分辨率串联质谱（UPLC-MS/MS）对原发性痛经大鼠的原始和加工的Cyperi rhizoma（CR）样品进行了比较分析。检查CR处理的大鼠和醋处理的CR（CRV）大鼠之间代谢物和样品成分的血液水平变化。

Abstract

根瘤（CR）广泛用于妇科，是我国治疗妇女疾病的全科药物。由于用醋加工后CR的镇痛作用增强，因此临床上一般采用醋处理的CR（CRV）。然而，醋加工增强镇痛作用的机制尚不清楚。本研究采用超高压液相色谱串联质谱（UPLC-MS/MS）技术检测CR处理和CRV处理痛经大鼠外源成分和代谢物血液水平的变化。结果显示，这些大鼠血液中的15种成分和两种代谢物含量不同。其中，CRV组的（-）-肉豆蔻醇和[（1R，2S，3R，4R）-3-羟基-1，4，7，7-四甲基双环[2.2.1]庚-2-基]乙酸水平明显高于CR组。CRV降低具有促炎、血小板聚集和血管收缩活性的2系列前列腺素和4系列白三烯的水平，并通过调节花生四烯酸和亚油酸代谢以及不饱和脂肪酸的生物合成提供镇痛作用。本研究表明，醋加工增强了CR的镇痛作用，有助于我们了解CRV的作用机制。

Introduction

原发性痛经（PD）是临床妇科中最普遍的疾病。它的特征是月经前或月经期间背痛、肿胀、腹痛或不适，生殖系统中没有盆腔病变1.一份关于其患病率的报告显示，85.7%的学生患有PD2。低剂量口服避孕药是标准疗法，但其不良副作用，如深静脉血栓形成，引起了越来越多的关注3。口服避孕药使用者深静脉血栓形成的患病率为每 1，000 名妇女 >1 例，在最初 6-12 个月和 40 岁以上的使用者中风险最高4。

长期用于传统中药（TCM），Cyperi rhizoma（CR）来源于Cyperaceae家族Cyperus rotundus L.的干燥根茎。CR调节月经紊乱，缓解抑郁和疼痛5.CR广泛用于妇科，被认为是治疗女性疾病的普通药物6。用醋（CRV）加工的CR通常用于临床。与CR相比，CRV显示出对月经和疼痛缓解的增强调节。现代研究表明，CR抑制环氧合酶-2（COX-2）和随后前列腺素（PGs）的合成，从而达到抗炎作用。同时，CR表现出无副作用的镇痛作用7，使CR成为痛经患者的不错选择。然而，CRV调节月经和缓解疼痛的机制尚不清楚。CR研究主要集中在其活性化学成分和药理活性的变化，如其抗炎，抗抑郁和镇痛作用8，9，10，11，12。

虽然中医的成分很复杂，但它们被吸收到血液中，必须达到特定的血液浓度才能有效13。通过利用血液中成分测定的策略，可以缩小中药活性成分的筛选范围。在体外研究化学成分时可以避免盲目，在研究单个成分时可以避免片面性14。通过比较血液中CR和CRV的组成，可以有效，快速地检测处理后的CR活性成分的变化。药物功效是药物影响身体的过程。由于身体的代谢反应引起的药物成分的变化，可能与药物的作用机制有关，可以用代谢组学来确定。代谢组学旨在测量整体和动态代谢反应，这与确定中药的整体疗效一致15。此外，代谢物是基因表达的最终产物，与表型16最密切相关。因此，代谢组学可能适用于探索CR和CRV在PD治疗中的代谢途径的差异。 基于液相色谱-高分辨率串联质谱（LC-MS/MS）的非靶向代谢组学具有高通量、高灵敏度和高分辨率的特点，可用于测量许多不同的小分子成分17，18.该方法可以同时测定吸收到血液中的内源性代谢物和外源性成分。代谢组学已广泛应用于中医19、药物毒理学20、健康管理21、运动22、食品23等领域。

本研究采用基于LC-MS/MS的非靶向代谢组学，测定CR处理和CRV治疗痛经模型大鼠血液中吸收的外源成分和内源性代谢物的差异，揭示CRV镇痛作用的机制。

Protocol

所有与动物有关的实验均经重庆中医药研究院实验伦理委员会批准进行。本实验使用了24只8-10周龄，体重200克±20克的雌性Sprague Dawley大鼠（SD）。 1. 提取的准备 计算计划将CR或CRV提取物施用于六只Sprague-Dawley大鼠（10g / [kg∙day]）的治疗组3天。使用浓度为 1 g/mL 的 CR 或 CRV 提取物（1 mL 提取物来自 1 g 草药）。注意：CR的剂量为6-10克。在这项研究中?…

Representative Results

痛经模型实验分析对照组30分钟内无扭动反应，因为这些大鼠未腹腔注射催产素和苯甲酸雌二醇引起疼痛。模型组、CR组和CRV组的大鼠在注射催产素后表现出大量的扭动反应。这些结果证明了苯甲酸雌二醇和催产素联合用药诱导痛经的疗效。模型组和对照组之间PGF 2α、PGE 2和PGF2α/PGE2水平的差异显著（P < 0.001，P < 0.05），证明了 模型的有效性（…

Discussion

由于中药种类繁多，性质各异，这些中药材有时在临床上不起作用，这可能是由于中药加工和煎煮不当造成的。随着当代科学技术的使用，中医的机制变得越来越明显29，30.本研究表明，CR和CRV均对PD模型大鼠具有治疗效果，且CRV的治疗效果更为显著。CRV的作用机制可能与醋加工可能影响被吸收到血液中的CR成分有关，并且可能与亚油酸代谢和不饱和脂肪…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到了重庆市卫生和计划生育委员会中医药科技项目（项目编号：ZY201802297）、重庆市自然科学基金面上项目（项目编号：cstc2019jcyj-msxmX065）、重庆市现代山区特色高效农业科技体系创新团队建设规划2022[10]、重庆市卫生健康委员会中药重点学科建设项目的支持。 医药加工。

Materials

Acetonitrile  Fisher Scientific, Pittsburg, PA, USA 197164
BECKMAN COULTER Microfuge 20 Beckman Coulter, Inc. MRZ15K047
Estradiol benzoate Shanghai Macklin Biochemical Co., Ltd C10042616
formic acid Fisher Scientific, Pittsburg, PA, USA 177799
LC 30A system Shimadzu, Kyoto, Japan 228-45162-46
Olive oil Shanghai Yuanye Biotechnology Co., Ltd H25A11P111909
Oxytocin synthetic Zhejiang peptide biology Co., Ltd  2019092001
Rat PGF2α ELISA kit Shanghai lmai Bioengineering Co., Ltd 202101
Rat PGFE2 ELISA kit Shanghai lmai Bioengineering Co., Ltd EDL202006217
SPF Sprague-Dawley rats Hunan SJA Laboratory Animal Co., Ltd Certificate number SCXK (Hunan) 2019-0004
Tecan Infinite 200 PRO   Tecan Austria GmbH, Austria 1510002987
Triple TOF 4600 system SCIEX, Framingham, MA, USA BK20641402
water Fisher Scientific, Pittsburg, PA, USA 152720
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Tags

MetabonomicsTraditional Chinese MedicineCyperi RhizomaRaw And Processed SamplesLiquid Chromatography-tandem Mass SpectrometryPrimary DysmenorrheaEndogenous MetabolitesExogenous ConstituentsPrincipal Component Analysis
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Chen, Y., Li, N., Wang, D., Fan, J., Chu, R., Li, S. Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea. J. Vis. Exp. (190), e64691, doi:10.3791/64691 (2022).
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