This work was supported by the Chongqing Municipal Health and Family Planning Commission Chinese Medicine Science and Technology Project (Project Number: ZY201802297), General project of Chongqing Natural Science Foundation (Project Number: cstc2019jcyj-msxmX065), Chongqing Modern Mountain Area Characteristic High-efficiency Agricultural Technology System Innovation Team Building Plan 2022 [10], and Chongqing Municipal Health Commission Key Discipline Construction Project of Chinese Materia Medica Processing.

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 &#177; 20 g were used in this experiment.
1. Preparation of the extraction
<ol>
	<li>Calculation
	<ol>
		<li>Plan to administer the CR or CRV extract to a treatment group of six Sprague-Dawley rats (10 g/[kg&#8729;day]) for 3 days. Use a CR or CRV extract concentr...

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 technology29,30. 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...

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 &#62;1 per 1,000 women, and the risk is highest during the first 6-12 months and in users older than 40 years4.
Long used in traditional Chinese medicine (TCM), Cyperi rhizoma (CR) is derived from the dried rhizome of the Cyperus rotundus L. of the Cyperaceae family. CR regulates menstrual disorders and relieves depression and pain5. CR is widely used in gynecology and is considered a general medicine to treat women&#39;s diseases6. CR processed with vinegar (CRV) is typically used clinically. Compared with CR, CRV shows enhanced regulation of menstruation and pain relief. Modern studies have shown that CR inhibits cyclooxygenase-2 (COX-2) and the subsequent synthesis of prostaglandins (PGs), thus achieving an anti-inflammatory effect. Meanwhile, CR exhibits an analgesic effect without side effects7, making CR a good choice for dysmenorrhea patients. However, the mechanism underlying the regulation of menstruation and provision of pain relief by CRV is unclear. CR research has mainly focused on changes in its active chemical components and pharmacologic activities, such as its anti-inflammatory, antidepressant, and analgesic effects8,9,10,11,12.
Although the ingredients of TCM are complex, they are absorbed into the blood and must reach a specific blood concentration to be effective13. The scope of screening the active ingredients of TCM can be narrowed by utilizing the strategy of constituent determination in the blood. Blindness can be avoided in studying the chemical components in vitro, and one-sidedness can be avoided in studying the individual constituents14. By comparing the compositions of CR and CRV in the blood, changes in the active ingredients of the processed CR can be detected effectively and quickly. Drug efficacy is the process by which a drug influences the body. Changes in the drug components due to the body&#39;s metabolic response, which may be related to the action mechanism of the drug, can be determined with metabonomics. Metabonomics aims to measure the overall and dynamic metabolic responses, which is consistent with determining the overall efficacy of traditional Chinese medicine15. Furthermore, metabolites are the final product of gene expression, which is most closely related to phenotypes16. Thus, metabonomics may be suitable for exploring the differences in the metabolic pathways between CR and CRV in the treatment of PD. Liquid chromatography-high-resolution tandem mass spectrometry (LC-MS/MS)-based untargeted metabolomics is characterized by high throughput, high sensitivity, and high resolution and can be used to measure many different small molecular components17,18. This method can simultaneously determine the endogenous metabolites and exogenous constituents absorbed into the blood. Metabonomics has been widely used in studies on TCM19, drug toxicology20, health management21, sports22, food23, and other fields.
In this study, the differences in the exogenous constituents absorbed into the blood and the endogenous metabolites were measured between CR-treated and CRV-treated dysmenorrhea model rats using LC-MS/MS-based untargeted metabolomics to reveal the mechanisms of the analgesic effects of CRV.

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			<td>BECKMAN COULTER Microfuge 20</td>
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			<td>Estradiol benzoate</td>
			<td>Shanghai Macklin Biochemical Co., Ltd</td>
			<td>C10042616</td>
			<td></td>
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			<td>formic acid</td>
			<td>Fisher Scientific, Pittsburg, PA, USA</td>
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			<td></td>
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			<td>LC 30A system</td>
			<td>Shimadzu, Kyoto, Japan</td>
			<td>228-45162-46</td>
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			<td>Olive oil</td>
			<td>Shanghai Yuanye Biotechnology Co., Ltd</td>
			<td>H25A11P111909</td>
			<td></td>
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			<td>Oxytocin synthetic</td>
			<td>Zhejiang peptide biology Co., Ltd&#160;</td>
			<td>2019092001</td>
			<td></td>
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			<td>Rat PGF2&#945; ELISA kit</td>
			<td>Shanghai lmai Bioengineering Co., Ltd</td>
			<td>202101</td>
			<td></td>
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			<td>Rat PGFE2 ELISA kit</td>
			<td>Shanghai lmai Bioengineering Co., Ltd</td>
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			<td>SPF Sprague-Dawley rats</td>
			<td>Hunan SJA Laboratory Animal Co., Ltd</td>
			<td>Certificate number SCXK (Hunan) 2019-0004</td>
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			<td>Tecan Infinite 200 PRO&#160;&#160;</td>
			<td>Tecan Austria GmbH, Austria</td>
			<td>1510002987</td>
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			<td>SCIEX, Framingham, MA, USA</td>
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			<td>Fisher Scientific, Pittsburg, PA, USA</td>
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analysis of raw and processed cyperi rhizoma samples using liquid chromatography-tandem mass spectrometry in rats with primary dysmenorrhea

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.

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 PGF2&#945;, PGE...

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

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).

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 ...

Metabonomics measures the overall and the dynamic metabolic responses and is consistent with determining the overall efficacy of traditional Chinese medicine. Changes in the drug components due to the body's metabolic response can be determined with the metabonomics. The scope of screening the active ingredients of TCM can be narrowed by utilizing this technique.One-sidedness can be avoided by studying the individual constituents. This method can be simultaneous, determine the endogenous metabolites and exogenous constituents absorbed into the blood. Metabonomics has been widely used in studies on TCM, drug toxicology, health management, sports, food, and other fields.To begin, determine the required extract of Cyperi rhizoma, or CR, or Cyperi rhizoma processed with vinegar, or CRV, to treat a group of six Sprague Dawley rats for three days. Calculate the volume of CR or CRV to be applied per rat using this equation. To process the CRV, thoroughly mix 100 grams of CR and 20 grams of vinegar, which has more than 5.5 grams of acetic acid per 100 milliliters, and incubate for 12 hours.After the incubation, stir fry the mixture in an iron pan for 10 minutes at 110 to 120 degrees Celsius. Then, remove the mixture, and allow it to cool at room temperature. To prepare the CR extract, soak the CR for two hours with pure water 10 times the CR amount taken, making sure that the medicinal materials are below the liquid level.Next, bring the mixture of water and medicine to boil over high heat, and keep it boiling on low heat for 20 minutes. Then, filter the contents through a 100 mesh filter cloth, and collect the filtrate. Then, concentrate the collected filtrate into an extract with a rotary evaporator to one gram per milliliter.To prepare the CRV extract, perform the steps of soaking, boiling, and concentrating as demonstrated for the CR extraction. For testing the CR and CRV extracts, pipette 500 microliters of the extract to 500 microliters of methanol in 1.5-milliliter microcentrifuge tubes, and vortex for 30 seconds to mix. Centrifuge the samples for 15 minutes at 16, 500 g at four degrees Celsius.Then, remove the supernatant, and transfer it to the sample vial for testing. After testing the samples, perform the principal component analysis, or PCA, and the modeling using the analysis software. Import the standardized data of the metabolites to the software, and then use Autofit to build the analysis model.Finally, use the scores to obtain the score scatter plot of the PCA. To perform the orthogonal partial least square discriminant analysis, or OPLS-DA, import the standardized data on the metabolites and the CR and CRV groups into the software. Then, import the CR and CRV data into their respective groups created.Then, use Autofit to build the analysis model, and use the score to obtain the score scatter plot of the OPLS-DA. Finally, use VIP to obtain the variable significance in the projection or VIP value in the OPLS-DA. To identify the potentially differential metabolites, screen out the metabolites with VIP values greater than one.Then, use the statistical software to calculate the P-value of the screened metabolites by the student's T-test. Next, to identify the differential metabolites, use the annotated metabolites, and screen out the differential metabolites to be matched in the KEGG database. Show the changes in the differential metabolites in the CR and CRV groups by drawing a heat map.To examine the potential metabolic pathways, go to the MetaboAnalyst database. Use Pathways Analysis to upload the different metabolites to obtain the potential metabolic pathways. Upload the different metabolites to the KEGG database to analyze the potential metabolic pathways.The analyzed dysmenorrhea model experiment showed significant differences in the prostaglandin levels. The rats in the model CR and CRV groups displayed substantial writhing reactions following the oxytocin injection. The PCA analysis results demonstrated that the clusters of the CR and CRV compared to the model groups were significantly separated in both the positive and negative ion modes.The OPLS-DA was used to screen for metabolic differences, and the scatter plot results demonstrated that the CR and CRV groups were separated. Univariate statistical analyses were performed to identify metabolic variations. A volcano plot is shown, wherein each point corresponds to a different metabolite.Significant changes were observed in 63 metabolites in the positive mode and 30 in the negative mode. The differential metabolites were determined using the KEGG and HDMB databases, and the accurately matched compounds were listed. The quantitative values of the differential metabolites between the CR and CRV groups were calculated and clustered.The color patches indicate how each metabolite is expressed relative to the others. Compared with the CR group, the levels of four differential metabolites in the CRV group increased while 11 metabolites decreased in the positive ion mode. In the case of negative ion mode, four differential metabolites increased, and seven metabolites decreased.KEGG pathway analysis results showed that the differential metabolites were associated with nine pathways in the positive and negative modes. The more differential metabolites, the better the results, so enough metabolic pathways can be linked and the key metabolic pathways screened will be more accurate.

analysis-raw-processed-cyperi-rhizoma-samples-using-liquid

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

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