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 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 was used as the dosage. As the average weight of an adult person is 60 kg, the adult dose is 0.1667 g/kg. According to the weight conversion algorithm24, as the dose conversion coefficient between humans and rats is 6.3, the dosage for rats is 1.05 g/kg. The drug dosage was increased by 10 times to 10.5 g/kg for rats. The dosage was set as 10 g/kg for the convenience of calculation and the actual experiment. For example, by the calculation, if a total of 36 g of CR or CRV is needed, the Chinese herbal medicine should be prepared at least twice. Thus, 200 g of CR was required-100 g of CR was used as the CR, and 100 g of CR was processed into CRV.</li>
		<li>Calculate the volume of CR or CRV to be applied per rat using equation (1): 
		V = 10 g/(kg&#8729;day) &#215; 200 g/(1 g/mL) = 2 mL&#160; &#160;(1)</li>
	</ol>
	</li>
	<li>Processing of the CRV
	<ol>
		<li>Mix 100 g of CR and 20 g of vinegar (&#62;5.5 g acetic acid/100 mL) thoroughly and incubate for 12 h. 
		NOTE: To ensure that the interior of the CR was moistened by vinegar after 12 h, the CR and vinegar were mixed, stirred well, and then stirred again until the inner portion was moist.</li>
		<li>Stir-fry the mixture in an iron pan for 10 min at 110-120 &#176;C. Then, take out the mixture, and let it cool at room temperature. 
		NOTE: To prevent the CR from scorching, it is necessary to stir continuously while heating. If the processed CRV is too wet, it can be dried at 60 &#176;C. When the surface of the CR is sepia, the stir-frying can be stopped.</li>
	</ol>
	</li>
	<li>Extraction
	<ol>
		<li>CR extract
		<ol>
			<li>Add 10 times (the amount of CR) pure water to the CR, and soak for 2 h. Make sure that the medicinal materials are below the liquid level when soaking. 
			NOTE: The CR only needs to be cut in half before extraction. The purpose of soaking is to extract the active constituents more effectively. The process of soaking is essential.</li>
			<li>Bring the mixture of water and medicine to the boil over a high heat, and keep it boiling over a low heat for 20 min. Filter with a filter cloth (100 mesh), and collect the filtrate. 
			NOTE: When decocting, a high heat was used before boiling, and a low heat was used to maintain the boiling.</li>
			<li>Repeat step 1.3.1.2 once, and combine the filtrates.</li>
			<li>Concentrate the extract with a rotary evaporator to 1 g/mL (based on the original medicine, the concentration temperature must be below 60 &#176;C). 
			NOTE: The active components in CR are volatile, so the concentration temperature should not be higher than 60 &#176;C.</li>
		</ol>
		</li>
		<li>CRV extract
		<ol>
			<li>Perform the same steps (1.3.1.1-1.3.1.3) as for the CR extraction method.</li>
		</ol>
		</li>
		<li>CR extract for testing
		<ol>
			<li>Pipette 500 &#181;L of the CR extract and 500 &#181;L of methanol into a 1.5 mL microcentrifuge tube, and vortex for 30 s to mix. 
			NOTE: A mixture of methanol and water extracts the active components better. The extract should not be directly filtered for testing.</li>
			<li>Centrifuge each sample for 15 min at 1,6502 &#215; g at 4 &#176;C. Filter the supernatant, and then transfer it to the sample vial for testing. 
			NOTE: After the high-speed centrifugation of the mixture of methanol and extract, the supernatant can be directly transferred to the sample bottle for determination without filtration. Due to the heat generated by the centrifugation process, it is preferable to use a cryogenic centrifuge.</li>
		</ol>
		</li>
		<li>CRV extract for testing
		<ol>
			<li>Perform steps 1.3.3.1-1.3.3.2 to prepare the CRV extract for testing.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Animals
<ol>
	<li>Calculation
	<ol>
		<li>Take 50 mg of estradiol benzoate, and add it to 50 mL of olive oil to prepare a 1 mg/mL solution. Take 50 mg of oxytocin, and add it to 50 mL of normal saline to prepare a 1 mg/mL solution. 
		NOTE: The dose of the intraperitoneal injection of estradiol benzoate and oxytocin is 10 mg/(kg&#8729;day). Estradiol benzoate is dissolved in olive oil, and oxytocin is dissolved in normal saline. Estradiol benzoate is not easy to dissolve in olive oil and can be treated with ultrasound to accelerate dissolution. Both the estradiol benzoate and oxytocin solutions must be prepared daily.</li>
		<li>Calculate the volume of estradiol benzoate solution to be applied per rat (i.e., V = 10 mg/[kg&#8729;day] &#215; 200 g/[1 g/mL] = 2 mL). Calculate the volume of oxytocin solution to be applied per rat (i.e., V = 10 mg/[kg&#8729;day] &#215; 200 g/[1 g/mL] = 2 mL).</li>
	</ol>
	</li>
	<li>Animal grouping and administration 
	NOTE: Ten days were assigned for administration25,26. During treatment, the rats had unrestricted access to standard chow and water. Within 30 min of oxytocin administration, each rat&#39;s writhing activity was tracked. The PD rat model was developed successfully, as evidenced by the model rats&#39; twisting responses, which included uterine contraction, one limb rotation, hind limb extension, a hollow trunk, and abdominal contraction26.
	<ol>
		<li>Assign 24 female Sprague-Dawley rats (SD rats, 8-10 weeks of age, weighing 200 g &#177; 20 g) into four groups at random-control, model, CR, and CRV-and feed them for 7 days.</li>
		<li>Animal administration
		<ol>
			<li>Intraperitoneally inject rats in the model, CR, and CRV groups with 2 mL of estradiol benzoate solution every day. Intraperitoneally inject the rats in the control group with 2 mL of normal saline.</li>
			<li>From day 8, complete step 2.2.2.1. Then, administer intragastrically 2 mL of CRV extract to the rats in the CRV group, 2 mL of CR extract to the rats in the CR group, and 2 mL of normal saline to the rats in the control and model groups.</li>
			<li>On day 10, complete step 2.2.2.2. Then, intraperitoneally inject the rats in the model, CR, and CRV groups with 2 mL of oxytocin solution and the rats in the control group with 2 mL of normal saline.</li>
		</ol>
		</li>
		<li>Record the writhing times of the rats within 30 min of the oxytocin injection.</li>
	</ol>
	</li>
	<li>Sample collection
	<ol>
		<li>Collect abdominal aorta blood samples, excise the uterus rapidly and completely, and carefully separate the connective tissue and fat adhering to the uterine wall. 
		NOTE: Blood was collected as close to within 1 h after the final dose.</li>
		<li>Use microcentrifuge tubes to preserve and transfer the uterine tissue to liquid nitrogen. Store the tissue samples at &#8722;80 &#176;C.</li>
		<li>Centrifuge the blood samples for 10 min at 4,125 &#215; g. Remove the serum-containing supernatant, and centrifuge at 16,502 &#215; g for 10 min at 4 &#176;C. Centrifuge the serum, and then keep it in the tube at &#8722;80 &#176;C for storage. 
		NOTE: The blood samples must be left at room temperature for 1 h for reprocessing.</li>
	</ol>
	</li>
	<li>Sample processing
	<ol>
		<li>Serum samples
		<ol>
			<li>Put 400 &#181;L of methanol and 200 &#181;L of serum into a microcentrifuge tube, and vortex for 30 s to mix. Centrifuge each sample for 15 min at 16,502 &#215; g at 4 &#176;C. Fill sample bottles with the supernatant after collection and filtration. Mix all the supernatants from each sample with the same volume to prepare quality control samples for testing.</li>
		</ol>
		</li>
		<li>Uterine tissue samples
		<ol>
			<li>Take 100 mg of uterine tissue from the ipsilateral segment and grind it with a ninefold volume of normal saline. Centrifuge the homogenate for 10 min at 4,125 &#215; g, and collect the supernatant. Place the supernatant in a refrigerator at 4 &#176;C for testing or at &#8722;80 &#176;C if not to be tested on the same day.</li>
			<li>Place normal saline, tissue, and steel balls in a 2 mL microcentrifuge tube, put the microcentrifuge tube into liquid nitrogen for 3-5 s, and then put the tissue into a tissue grinder for grinding.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Sample testing
	<ol>
		<li>Use an enzyme-linked immunosorbent assay (ELISA) to measure the PGF2&#945; and PGE2 content in the uterine tissues of the rat samples. 
		NOTE: The rat PGE2 ELISA kit was used to measure the PGE2 content, and the rat PGF2&#945; ELISA kit was used to measure the PGF2&#945; level. The detailed steps can be found in the manufacturer&#39;s instructions. The details of the kit are given in the Table of Materials.</li>
		<li>Assess the serum sample, the CR extract, and the CRV extract using UPLC-MS/MS.
		<ol>
			<li>For UPLC, use a C18 column (2.6 &#181;m, 2.1 mm x 100 mm) and a binary gradient method with mobile phase A containing 0.1% formic acid and mobile phase B containing acetonitrile. Set the elution gradient as follows: from 0 min to 1 min, 15% B; from 1 min to 8.5 min, 15% to 85% B; from 8.5 min to 11.5 min, 85% B; from 11.5 min to 11.6 min, 99% to 1% B; and from 11.6 min to 15 min, 15% B. Set the flow rate to 0.35 mL/min and the injection volume to 2 &#181;L.</li>
			<li>For MS, set the temperature to 600 &#176;C, the curtain gas (CUR) flow rate to 0.17 MPa, and both the sheath and auxiliary gas flow rates to 0.38 MPa. Set the ion spray voltage of the positive ion mode and negative ion mode to 5.5 kV and &#8722;4.5 kV, respectively, the declustering potential (DP) voltage to 80 V or &#8722;80 V, the collision energy (CE) to 40 eV or &#8722;25 eV, and the collision energy superposition (CES) to 35 eV &#177; 15 eV.</li>
			<li>Perform the test following the manufacturer&#39;s protocol using UPLC-MS/MS. Perform MS for a mass range of 50-1,000 m/z.</li>
			<li>Obtain the UPLC-MS/MS results using the matching workstation program in conjunction with the detection mode&#39;s information-dependent acquisition, multiple mass defect filter, and dynamic background deduction. Use the quality control samples of pooled serum to test the repeatability and stability of the UPLC-MS/MS equipment to verify the conventional approach. Before the research samples, inject the quality control samples for four successive runs, and then inject them after every five serum samples.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Data processing
<ol>
	<li>Data preparation
	<ol>
		<li>Use conversion software to convert the raw data to the mzXML format. Normalize the total ion current (TIC) data of each sample. 
		NOTE: An internal R-based application (www.lims2.com) built on XCMS was used to analyze the information for the integration, alignment, extraction, and peak detection27.</li>
		<li>Perform metabolite annotation using a proprietary MS2 database (www.lims2.com). Set the annotation threshold to 0.328. 
		NOTE: The endogenous metabolites were identified in each group.</li>
	</ol>
	</li>
	<li>Principal component analysis and orthogonal partial least square discriminant analysis
	<ol>
		<li>Use analysis software to perform the principal component analysis (PCA) and the modeling. Import the standardized data of the metabolites to the analysis software. Then, use autofit to build the analysis model. Finally, use score to obtain the score scatter plot of the PCA. 
		NOTE: The clustering of each group was obtained with the score scatter plot of the PCA. PCA is an unsupervised analysis mode that mainly groups the samples through dimension reduction without intervention.</li>
		<li>Use the analysis software to perform the orthogonal partial least square discriminant analysis (OPLS-DA).
		<ol>
			<li>Import the standardized data on the metabolites in the CR and CRV groups into the analysis software.</li>
			<li>Import the data from the CR group into the created CR group, and import the data from the CRV group into the created CRV group. 
			NOTE: OPLS-DA is a supervised analysis mode, and grouping of the samples is necessary.</li>
			<li>Then, use autofit to build the analysis model, and use score to obtain the score scatter plot of the OPLS-DA. Finally, use VIP to obtain the variable significance in the projection (VIP) value in the OPLS-DA. 
			NOTE: The variable significance in the projection (VIP) values of the metabolites in the CR and CRV groups were obtained through the OPLS-DA.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Identification of the potential differential metabolites
	<ol>
		<li>Screen out the metabolites with VIP values greater than 1 in step 3.2.2.3.</li>
		<li>Use statistical software to calculate the P value of the metabolites that were screened out in step 3.3.1 by the Student&#39;s t-test. 
		NOTE: Significant differences in the potential differential metabolites in the CR and CRV groups were determined by a Student&#39;s t-test. The potential differential metabolites were those with a Student&#39;s t-test p-value &#60; 0.05 and a variable significance in the projection (VIP) greater than 1. The representation was accomplished using a volcanic plot.</li>
	</ol>
	</li>
	<li>Identification of the differential metabolites
	<ol>
		<li>Screen out the potential differential metabolites in step 3.3. Use the results of step 3.1.2 to identify these differential metabolites. 
		NOTE: A small number of potential differential metabolites were identified, and they became the candidate differential metabolites.</li>
		<li>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 heatmap. 
		NOTE: A small number of candidate differential metabolites were matched, and they became the differential metabolites.</li>
	</ol>
	</li>
	<li>Examination of the potential metabolic pathways
	<ol>
		<li>Upload the different metabolites to the Metaboanalyst (www.metaboanalyst.ca) database.</li>
		<li>Use Pathways Analysis to obtain the potential metabolic pathways. 
		NOTE: The potential metabolic pathways were obtained in the CR and CRV groups. The P value and impact value were two very important indicators in the selection of the critical pathways. The P value was more important than the impact value. Significance was defined by a P value &#60; 0.05; bigger impact values were associated with better correlations.</li>
		<li>Analyze the potential metabolic pathways by uploading the different metabolites to the KEGG (http://www.kegg.jp/kegg/pathway.html) database. 
		NOTE: The relationship between the function of the metabolic pathway and PD should also be considered. The critical metabolic pathways were obtained.</li>
	</ol>
	</li>
	<li>Identification of the constituents absorbed into the blood
	<ol>
		<li>Identify the chemical constituents in the CR and CRV extracts using the in-house MS2 database (www.lims2.com), the Human Metabolome Database (HMDB), and the Massbank and Chemspider databases.</li>
		<li>Determine the constituents absorbed into the blood in the CR and CRV groups, and compare the constituents between the CR and CRV groups. 
		NOTE: The constituents absorbed into the blood must be detected in the CR or CRV groups but cannot be detected in the control group.</li>
	</ol>
	</li>
	<li>Statistical analysis
	<ol>
		<li>Analyze the data using univariate analysis (UVA) and multivariate statistical methods, including the analysis of variance (ANOVA) and the Student&#39;s t-test. 
		NOTE: The experimental information was presented using statistical software as mean &#177; standard deviation (&#177;SD). P &#60; 0.01 was considered a highly significant difference, and P &#60; 0.05 represented a significant difference28.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile&#160;</td>
			<td>Fisher Scientific, Pittsburg, PA, USA</td>
			<td>197164</td>
			<td></td>
		</tr>
		<tr>
			<td>BECKMAN COULTER Microfuge 20</td>
			<td>Beckman&#160;Coulter, Inc.</td>
			<td>MRZ15K047</td>
			<td></td>
		</tr>
		<tr>
			<td>Estradiol benzoate</td>
			<td>Shanghai Macklin Biochemical Co., Ltd</td>
			<td>C10042616</td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid</td>
			<td>Fisher Scientific, Pittsburg, PA, USA</td>
			<td>177799</td>
			<td></td>
		</tr>
		<tr>
			<td>LC 30A system</td>
			<td>Shimadzu, Kyoto, Japan</td>
			<td>228-45162-46</td>
			<td></td>
		</tr>
		<tr>
			<td>Olive oil</td>
			<td>Shanghai Yuanye Biotechnology Co., Ltd</td>
			<td>H25A11P111909</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxytocin synthetic</td>
			<td>Zhejiang peptide biology Co., Ltd&#160;</td>
			<td>2019092001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat PGF2&#945; ELISA kit</td>
			<td>Shanghai lmai Bioengineering Co., Ltd</td>
			<td>202101</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat PGFE2 ELISA kit</td>
			<td>Shanghai lmai Bioengineering Co., Ltd</td>
			<td>EDL202006217</td>
			<td></td>
		</tr>
		<tr>
			<td>SPF Sprague-Dawley rats</td>
			<td>Hunan SJA Laboratory Animal Co., Ltd</td>
			<td>Certificate number SCXK (Hunan) 2019-0004</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan Infinite 200 PRO&#160;&#160;</td>
			<td>Tecan Austria GmbH, Austria</td>
			<td>1510002987</td>
			<td></td>
		</tr>
		<tr>
			<td>Triple TOF 4600 system</td>
			<td>SCIEX, Framingham, MA, USA</td>
			<td>BK20641402</td>
			<td></td>
		</tr>
		<tr>
			<td>water</td>
			<td>Fisher Scientific, Pittsburg, PA, USA</td>
			<td>152720</td>
			<td></td>
		</tr>
	</tbody>
</table>

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...

Watch this Scientific Journal Video about Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea at JoVE.com

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 ...

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

Research

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Medicine

1.4K Views.  Chongqing Academy of Chinese Materia Medica. 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).

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

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson&#039;s Disease

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Predicting Prognosis in Acute Heart Failure Using Phase Angle & BIVA Z-Scores

Clinical Application of Phase Angle and BIVA Z-Score Analyses in Patients Admitted to an Emergency Department with Acute Heart Failure

Acute heart failure is characterized by neurohormonal activation, which leads to sodium and water retention and causes alterations in body composition, such as increased body fluid congestion or systemic congestion. This condition is one of the most common reasons for hospital admission and has been associated with poor outcomes. The phase angle indirectly measures intracellular status, cellular integrity, vitality, and the distribution of spaces between intracellular and extracellular body water. This parameter has been found to be a predictor of health status and an indicator of survival and other clinical outcomes. In addition, phase angle values of &lt;4.8&#176; upon admission were associated with higher mortality in patients with acute heart failure. However, low phase angle values may be due to alterations-such as the shifting of fluids from an intracellular body water (ICW) compartment to an ECW (extracellular body water) compartment and a concurrent decrease in body-cell mass (which can reflect malnutrition)-that are present in heart failure. Thus, a low phase angle may be due to overhydration and/or malnutrition. BIVA provides additional information about the body-cell mass and congestion status with a graphical vector (R-Xc graph). In addition, a BIVA Z-score analysis (the number of standard deviations from the mean value of the reference group) that has the same pattern as that of the ellipses for the percentiles on the original R-Xc graph can be used to detect changes in soft-tissue mass or tissue hydration and can help researchers compare changes in different study populations. This protocol explains how to obtain and interpret phase angle values and BIVA Z-score analyses, their clinical applicability, and their usefulness as a predictive marker for the prognosis of a 90-day event in patients admitted to an emergency department with acute heart failure.

Author Spotlight: Unveiling Prognostic Indicators in Heart Failure - The Role of Phase Angle and Bioelectrical Impedance Analysis 

In this protocol, we explain how to obtain and interpret phase angle values and bioelectrical impedance vectorial analysis (BIVA) Z-score obtained by bioelectrical impedance in patients with acute heart failure admitted to the Emergency Department and their clinical applicability as a predictive marker for the prognosis of a 90-day event.

Acute heart failure is characterized by neurohormonal activation, which leads to sodium and water retention and causes alterations in body composition, ...

Developing an Exercise Program for Rheumatoid Arthritis Patients

Evaluation of Changes in Hydration and Body Cell Mass with Bioelectrical Impedance Analysis after Exercise Program for Rheumatoid Arthritis Patients

Rheumatoid arthritis (RA) is a debilitating disease that can result in complications such as rheumatoid cachexia. While physical exercise has shown benefits for RA patients, its impact on hydration and body cell mass remains uncertain. The presence of pain, inflammation, and joint changes often restrict activity and make traditional body composition assessments unreliable due to altered hydration levels. Bioelectrical impedance is a commonly used method for estimating body composition, but it has limitations since it was primarily developed for the general population and does not consider changes in body composition. On the other hand, bioelectrical impedance vectorial analysis (BIVA) offers a more comprehensive approach. BIVA involves graphically interpreting resistance (R) and reactance (Xc), adjusted for height, to provide valuable information about hydration status and the integrity of the cell mass.
Twelve women with RA were included in this study. At the beginning of the study, hydration and body cell mass measurements were obtained using the BIVA method. Subsequently, the patients participated in a six-month dynamic exercise program encompassing cardiovascular capacity, strength, and coordination training. To evaluate changes in hydration and body cell mass, the differences in the R and Xc parameters, adjusted for height, were compared using BIVA confidence software. The results showed notable changes: resistance decreased after the exercise program, while reactance increased. BIVA, as a classification method, can effectively categorize patients into dehydration, overhydration, normal, athlete, thin, cachectic, and obese categories. This makes it a valuable tool for assessing RA patients, as it provides information independent of body weight or prediction equations. Overall, the implementation of BIVA in this study shed light on the effects of the exercise program on hydration and body cell mass in RA patients. Its advantages lie in its ability to provide comprehensive information and overcome the limitations of traditional body composition assessment methods.

Author Spotlight: Implementation of BIVA for Analyzing Disease Risk Factors in Patients with Low Body Cell Mass 

This protocol assesses alterations in hydration and body cell mass status using bioelectrical impedance vectorial analysis following a dynamic exercise program designed for patients with rheumatoid arthritis. The dynamic exercise program itself is detailed, highlighting its components focused on cardiovascular capacity, strength, and coordination. The protocol details steps, instruments, and limitations.

Rheumatoid arthritis (RA) is a debilitating disease that can result in complications such as rheumatoid cachexia. While physical exercise has shown ...

Multicolor Immunofluorescence of the Nasal Tissue in an Allergic Rhinitis Rat Model

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), affecting approximately 10%-20% of the world's population. While immunofluorescence (IF) staining has long been a standard technique for detecting disease-specific protein expression, conventional IF techniques are limited in their ability to detect the expression levels of three or more proteins in the same sample. Consequently, multicolor IF techniques have been developed in recent years, which allow the simultaneous labeling of multiple targets in cells or tissues.
This protocol provides a comprehensive overview of the process for establishing a rat model of AR, obtaining nasal mucosal samples, and the technical procedures for multicolor immunofluorescence. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose, with behavioral observations scoring &#8805;5 points. Hematoxylin and eosin (H&amp;E) staining revealed increased inflammatory cell counts and disrupted nasal mucosal integrity in the AR group. Multicolor immunofluorescence (mIF) demonstrated increased expression of ROR&#947;t and TICAM-1, while Foxp3 expression decreased in the nasal mucosa tissue of AR rats.

Author Spotlight: Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence

This protocol describes a multicolor immunofluorescence technique for evaluating the rat model of allergic rhinitis.

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), ...

Non-Invasive Biomarker Discovery for Tear Proteomics Using LC-MS

A Protein Suspension-Trapping Sample Preparation for Tear Proteomics by Liquid Chromatography-Tandem Mass Spectrometry

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for several ocular diseases and conditions. The suspension trapping column has been reported to be an efficient and user-friendly sample preparation workflow for the broad application of downstream proteomic analysis. Yet, this strategy has not been well-studied in the analysis of human tear proteome. The present protocol describes an integrated workflow from clinical human tear samples to purified peptides for non-invasive tear protein biomarker research using mass spectrometry, which provides insights into disease biomarkers and monitoring when combined with bioinformatics analysis. A protein suspension trapping sample preparation was applied and demonstrated the discovery of tear proteome with fast, reproducible, and user-friendly procedures, as a universal, optimized sample preparation for human tear fluid analysis. In particular, the suspension trapping procedure outperformed in-solution sample preparation in terms of peptide recovery, protein identification, and shorter sample preparation time.

Author Spotlight: Advancing Tear Fluid Analysis Using a Standardized Protocol for Proteomics Research 

This protocol describes a method for collecting tear samples using Schirmer strips and an integrated quantitative workflow for discovering non-invasive tear protein biomarkers. The suspension trapping sample preparation workflow enables fast and robust tear sample preparation and mass spectrometric analysis, resulting in higher peptide recovery yields and protein identification than standard in-solution procedures.

Tear fluid is one of the easily accessible biofluids that can be collected non-invasively. Tear proteomics has the potential to discover biomarkers for ...