Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Summary

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

Abstract

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Introduction

Primary dysmenorrhea (PD) is the most prevalent condition in clinical gynecology. It is characterized by backache, swelling, abdominal pain, or discomfort before or during menstruation without pelvic pathology in the reproductive system¹. A report on its prevalence showed that 85.7% of students suffer from PD². Low-dose oral contraceptives are the standard therapy, but their adverse side effects, such as deep vein thrombosis, have drawn increasing attention³. The prevalence of deep vein thrombosis among oral contraceptive users is >1 per 1,000 women, and the risk is highe....

Protocol

All animal-related experiments were conducted with approval from the Experiment Ethics Committee of Chongqing Institute of TCM. Twenty-four female Sprague Dawley rats (SD) that were 8-10 weeks old and weighed 200 g ± 20 g were used in this experiment.

1. Preparation of the extraction

1. Calculation
   1. Plan to administer the CR or CRV extract to a treatment group of six Sprague-Dawley rats (10 g/[kg∙day]) for 3 days. Use a CR or CRV extract concentration of 1 g/mL (1 mL of the extract is obtained from 1 g of herbs).
      NOTE: The dosage of CR is 6-10 g. In this study, the maximum dose of 10 g....

Results

Analysis of the dysmenorrhea model experiment
There was no writhing response within 30 min in the control group because these rats were not intraperitoneally injected with oxytocin and estradiol benzoate to cause pain. The rats in the model, CR, and CRV groups displayed substantial writhing reactions following the oxytocin injection. These outcomes demonstrate the efficacy of the estradiol benzoate and oxytocin combination for inducing dysmenorrhea. The differences in the PGF_2α, PGE.......

Discussion

Due to the wide variety and different nature of TCMs, these herbs sometimes do not work in clinical practice, and this may be due to the inappropriate processing and decocting of TCMs. The mechanisms of TCM are becoming more apparent with the use of contemporary science and technology^29,30. This study shows that both CR and CRV have therapeutic effects in PD model rats and that the therapeutic effect of CRV is more substantial. The mechanism of action of CRV coul.......

Materials

Name	Company	Catalog Number	Comments
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