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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

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).

Streszczenie

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.

Wprowadzenie

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 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'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'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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Protokół

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 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.
    2. Calculate the volume of CR or CRV to be applied per rat using equation (1):
      V = 10 g/(kg∙day) × 200 g/(1 g/mL) = 2 mL   (1)
  2. Processing of the CRV
    1. Mix 100 g of CR and 20 g of vinegar (>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.
    2. Stir-fry the mixture in an iron pan for 10 min at 110-120 °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 °C. When the surface of the CR is sepia, the stir-frying can be stopped.
  3. Extraction
    1. CR extract
      1. 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.
      2. 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.
      3. Repeat step 1.3.1.2 once, and combine the filtrates.
      4. Concentrate the extract with a rotary evaporator to 1 g/mL (based on the original medicine, the concentration temperature must be below 60 °C).
        NOTE: The active components in CR are volatile, so the concentration temperature should not be higher than 60 °C.
    2. CRV extract
      1. Perform the same steps (1.3.1.1-1.3.1.3) as for the CR extraction method.
    3. CR extract for testing
      1. Pipette 500 µL of the CR extract and 500 µL 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.
      2. Centrifuge each sample for 15 min at 1,6502 × g at 4 °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.
    4. CRV extract for testing
      1. Perform steps 1.3.3.1-1.3.3.2 to prepare the CRV extract for testing.

2. Animals

  1. Calculation
    1. 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∙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.
    2. Calculate the volume of estradiol benzoate solution to be applied per rat (i.e., V = 10 mg/[kg∙day] × 200 g/[1 g/mL] = 2 mL). Calculate the volume of oxytocin solution to be applied per rat (i.e., V = 10 mg/[kg∙day] × 200 g/[1 g/mL] = 2 mL).
  2. 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's writhing activity was tracked. The PD rat model was developed successfully, as evidenced by the model rats' twisting responses, which included uterine contraction, one limb rotation, hind limb extension, a hollow trunk, and abdominal contraction26.
    1. Assign 24 female Sprague-Dawley rats (SD rats, 8-10 weeks of age, weighing 200 g ± 20 g) into four groups at random-control, model, CR, and CRV-and feed them for 7 days.
    2. Animal administration
      1. 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.
      2. 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.
      3. 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.
    3. Record the writhing times of the rats within 30 min of the oxytocin injection.
  3. Sample collection
    1. 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.
    2. Use microcentrifuge tubes to preserve and transfer the uterine tissue to liquid nitrogen. Store the tissue samples at −80 °C.
    3. Centrifuge the blood samples for 10 min at 4,125 × g. Remove the serum-containing supernatant, and centrifuge at 16,502 × g for 10 min at 4 °C. Centrifuge the serum, and then keep it in the tube at −80 °C for storage.
      NOTE: The blood samples must be left at room temperature for 1 h for reprocessing.
  4. Sample processing
    1. Serum samples
      1. Put 400 µL of methanol and 200 µL of serum into a microcentrifuge tube, and vortex for 30 s to mix. Centrifuge each sample for 15 min at 16,502 × g at 4 °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.
    2. Uterine tissue samples
      1. 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 × g, and collect the supernatant. Place the supernatant in a refrigerator at 4 °C for testing or at −80 °C if not to be tested on the same day.
      2. 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.
  5. Sample testing
    1. Use an enzyme-linked immunosorbent assay (ELISA) to measure the PGF2α 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 PGF2α ELISA kit was used to measure the PGF2α level. The detailed steps can be found in the manufacturer's instructions. The details of the kit are given in the Table of Materials.
    2. Assess the serum sample, the CR extract, and the CRV extract using UPLC-MS/MS.
      1. For UPLC, use a C18 column (2.6 µm, 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 µL.
      2. For MS, set the temperature to 600 °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 −4.5 kV, respectively, the declustering potential (DP) voltage to 80 V or −80 V, the collision energy (CE) to 40 eV or −25 eV, and the collision energy superposition (CES) to 35 eV ± 15 eV.
      3. Perform the test following the manufacturer's protocol using UPLC-MS/MS. Perform MS for a mass range of 50-1,000 m/z.
      4. Obtain the UPLC-MS/MS results using the matching workstation program in conjunction with the detection mode'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.

3. Data processing

  1. Data preparation
    1. 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.
    2. 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.
  2. Principal component analysis and orthogonal partial least square discriminant analysis
    1. 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.
    2. Use the analysis software to perform the orthogonal partial least square discriminant analysis (OPLS-DA).
      1. Import the standardized data on the metabolites in the CR and CRV groups into the analysis software.
      2. 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.
      3. 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.
  3. Identification of the potential differential metabolites
    1. Screen out the metabolites with VIP values greater than 1 in step 3.2.2.3.
    2. Use statistical software to calculate the P value of the metabolites that were screened out in step 3.3.1 by the Student's t-test.
      NOTE: Significant differences in the potential differential metabolites in the CR and CRV groups were determined by a Student's t-test. The potential differential metabolites were those with a Student's t-test p-value < 0.05 and a variable significance in the projection (VIP) greater than 1. The representation was accomplished using a volcanic plot.
  4. Identification of the differential metabolites
    1. 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.
    2. 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.
  5. Examination of the potential metabolic pathways
    1. Upload the different metabolites to the Metaboanalyst (www.metaboanalyst.ca) database.
    2. 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 < 0.05; bigger impact values were associated with better correlations.
    3. 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.
  6. Identification of the constituents absorbed into the blood
    1. 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.
    2. 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.
  7. Statistical analysis
    1. Analyze the data using univariate analysis (UVA) and multivariate statistical methods, including the analysis of variance (ANOVA) and the Student's t-test.
      NOTE: The experimental information was presented using statistical software as mean ± standard deviation (±SD). P < 0.01 was considered a highly significant difference, and P < 0.05 represented a significant difference28.

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Wyniki

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 PGF2α, PGE...

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Dyskusje

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...

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Ujawnienia

The authors declare that they have no competing financial interests.

Podziękowania

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.

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Materiały

NameCompanyCatalog NumberComments
Acetonitrile Fisher Scientific, Pittsburg, PA, USA197164
BECKMAN COULTER Microfuge 20Beckman Coulter, Inc.MRZ15K047
Estradiol benzoateShanghai Macklin Biochemical Co., LtdC10042616
formic acidFisher Scientific, Pittsburg, PA, USA177799
LC 30A systemShimadzu, Kyoto, Japan228-45162-46
Olive oilShanghai Yuanye Biotechnology Co., LtdH25A11P111909
Oxytocin syntheticZhejiang peptide biology Co., Ltd 2019092001
Rat PGF2α ELISA kitShanghai lmai Bioengineering Co., Ltd202101
Rat PGFE2 ELISA kitShanghai lmai Bioengineering Co., LtdEDL202006217
SPF Sprague-Dawley ratsHunan SJA Laboratory Animal Co., LtdCertificate number SCXK (Hunan) 2019-0004
Tecan Infinite 200 PRO  Tecan Austria GmbH, Austria1510002987
Triple TOF 4600 systemSCIEX, Framingham, MA, USABK20641402
waterFisher Scientific, Pittsburg, PA, USA152720

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