A quality evaluation method for Curcuma wenyujin and its vinegar products

Abstract

The present invention discloses a quality evaluation method of Curcuma aromatica and its vinegar products, quality markers of Curcuma aromatica and its vinegar products for treating dysmenorrhea due to liver depression and qi stagnation, and application in quality control of decoction pieces. The invention comprises: one or more of 32 efficacy-related components selected from zederone, turmeric alcohol ketone, turmeric dione, isocurcumenol, etc. The quality markers are further screened, and one or more selected from isocurcumenol, germacron, and β-elemene can be used as differential quality markers for Curcuma aromatica and its vinegar products for treating dysmenorrhea due to liver depression and qi stagnation. The present invention provides a candidate compound group for treating dysmenorrhea due to liver depression and qi stagnation, and also provides an example for quality control research of Chinese medicine decoction pieces based on efficacy-related substances.

Description

Technical Field

[0001] The invention belongs to the field of quality control of traditional Chinese medicines, and particularly relates to a quality evaluation method for zedoary turmeric and its vinegar products, and quality markers for zedoary turmeric and its vinegar products in treating dysmenorrhea caused by liver depression and qi stagnation. Background Art

[0002] Dysmenorrhea is very common. 50% to 90% of adolescent girls and women of childbearing age will experience dysmenorrhea, which seriously affects women's work and study and reduces the quality of life. According to whether the pelvic organs are diseased, it can be divided into primary dysmenorrhea and secondary dysmenorrhea. Modern medicine believes that the causes of primary dysmenorrhea are mostly excessive oxytocin, prostaglandins, vasopressin and other substances or mental factors such as tension, anxiety, depression or bad living habits, which lead to excessive contraction of the uterine muscle layer and arterial vasoconstriction, resulting in uterine ischemia, hypoxia and pain. Traditional Chinese medicine believes that primary dysmenorrhea is closely related to the liver. The liver is affected by many factors. Mental stimulation, emotional distress and other diseases can cause the liver to lose its ability to vent and the qi to be blocked, which manifests as liver qi stagnation; qi disease and blood will cause blood stasis. Because the liver governs the Chong and Ren meridians, if the blood flow is not smooth, it will cause Chong and Ren disorders, uterine blood stasis, "no smoothness leads to pain", and primary dysmenorrhea and other symptoms. Therefore, in order to achieve the goal of "no pain if the blood is unobstructed", drugs such as Curcuma zedoaria that soothe the liver, promote blood circulation and relieve pain are often used for treatment.

[0003] Curcuma wenyujin is the dried root of Curcuma wenyujin YHChen et C.Ling, Curcuma longa L., Curcuma kwangsiensis SGLee et CFLiang or Curcumaphaeocaulis Val. of the Zingiberaceae family. It belongs to the liver, heart and lung meridians and has the effects of promoting blood circulation and relieving pain, promoting qi and relieving depression. This study takes Curcuma wenyujin as an example. Curcuma wenyujin has multiple pharmacological effects such as antiplatelet aggregation, anticoagulation, anti-inflammatory, analgesic, liver protection and antidepressant. It is traditionally used to treat inflammatory pain diseases such as amenorrhea, irregular menstruation and dysmenorrhea. Sesquiterpenoids and curcumin-based compounds isolated from Curcuma wenyujin, such as curcumadione, germacronone and β-elemene, have been shown to play a role in promoting blood circulation by reducing blood viscosity, reducing platelet aggregation and improving blood rheology. This also provides a clinical basis for the treatment of dysmenorrhea caused by liver depression and qi stagnation with Wen Yu Jin.

[0004] At present, the new edition of the Pharmacopoeia does not include the quality control standard of Curcuma. In the quality control research of Curcuma wenyujin, sensory indicators, thin layer chromatography, gas / liquid chromatography, near-infrared spectroscopy and other technologies are usually used to distinguish the authenticity and quality of Curcuma wenyujin. Its quality control methods, quality evaluation indicators and quality standards are rarely related to its efficacy, resulting in unclear material basis of its efficacy and difficulty in ensuring the quality of Curcuma wenyujin. Based on the above problems, a new concept of quality control of traditional Chinese medicine products is introduced: the concept of quality markers, and a quality marker group of Curcuma wenyujin and its vinegar products for the treatment of liver depression and qi stagnation type dysmenorrhea is established, so as to improve the quality control standards of Curcuma wenyujin and its vinegar products. Summary of the invention

[0005] The purpose of the present invention is to provide a quality evaluation method for Curcuma zedoaria and its vinegar products in view of the problem that the existing quality control of Curcuma zedoaria is not perfect, to track the quality markers of Curcuma zedoaria and its vinegar products in the treatment of dysmenorrhea caused by liver depression and qi stagnation and their application in the quality control of decoction pieces.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A quality evaluation method for Curcuma wenyujin and its vinegar products, combining serum pharmacochemistry and spectrum-effect association to track the blood components of Curcuma wenyujin and its vinegar products and target their quality markers:

[0008] (1) Preparation of Curcuma aromatica and its vinegar extract

[0009] Take pieces of Curcuma and its vinegar products, heat and reflux with 15 times the amount of 90% ethanol, extract twice, each time for 1.5 hours; filter with double-layer gauze, add 10 times the amount of water to the residue, heat and reflux for 1.5 hours, combine the above extracts, and concentrate under reduced pressure at 50℃ to 0.2g·ml -1 , and obtained raw and vinegar Curcuma aromatica extracts, which were used for qualitative analysis of components and subsequent oral administration to rats.

[0010] (2) Animal model construction, drug administration and sample collection

[0011] Construction of liver depression and qi stagnation dysmenorrhea model: Estradiol benzoate and epinephrine hydrochloride were subcutaneously injected on the back of rats in each group for 10 consecutive days. On the first and tenth days, 5 mg kg of estradiol benzoate was subcutaneously injected on the back of each rat. -1 ·d -1 , 2.5 mg kg was injected subcutaneously on the back of each rat on days 2-9 -1 ·d -1 . Epinephrine hydrochloride 0.6 mg kg twice daily -1 ·d -1 The blank control group received subcutaneous injection of saline on the back of rats without comprehensive stimulation.

[0012] Animal dosing: Oral administration started on the 4th day, once a day, for 7 consecutive days. According to the clinical equivalent dose, the dose of ibuprofen group (group B) was 0.06 g kg -1 , the low-dose group of Curcuma longa (CL group) was 0.3 g·kg -1 , the high-dose group of Curcuma wenyujin (CH group) was 0.9 g·kg -1 The rats in each group were gavaged with the corresponding drugs, and the blank control group and the model group were gavaged with the same amount of normal saline.

[0013] Sample collection: intraperitoneal injection of 3% sodium pentobarbital (1 ml kg -1 ) anesthesia. Blood was collected from the abdominal aorta and kept at room temperature for 1 hour at 3000 r / min -1 Centrifuge for 20 minutes, take the supernatant to obtain serum, and use it for the subsequent qualitative analysis of serum components. At the same time, quickly take fresh rat uterus and liver tissues, rinse with saline, dry with filter paper, and weigh. Store part of the uterus and liver tissues in a -80℃ refrigerator for subsequent ELISA and RT-qPCR experiments.

[0014] (3) Evaluation of the efficacy of Curcuma zedoariae and its vinegar products in treating dysmenorrhea caused by liver depression and qi stagnation

[0015] Writhing reaction: After the last administration of the drug, oxytocin was injected intraperitoneally, and the writhing reaction was observed within 0.5 h, and the writhing latency and number of writhing times were recorded;

[0016] ELISA experiment: Strictly follow the instructions of the kit to determine the PGF in uterine tissue 2α , PGE2, β-EP, Ca 2+ The level of 5-HT in serum.

[0017] RT-qPCR experiment: extract total cell RNA according to the kit instructions, perform reverse transcription according to the reverse transcription kit instructions, design specific primers according to literature reports, use β-actin as the internal reference gene, prepare the corresponding reaction system, and perform quantification.

[0018] (4) Preparation of serum test solution

[0019] Accurately pipette 200μl of serum sample, add 1000μl of methanol, vortex for 1min, fully precipitate protein, centrifuge, take 1000μl of supernatant and place in 2.0ml centrifuge tube, evaporate the solvent in vacuum centrifuge concentrator (30℃), add 200μl of methanol to re-dissolve, vortex for 5min, centrifuge, take 200μl of supernatant, and get it. At the same time, take 10μl from each sample and mix them to prepare quality control (QC) samples, and the steps of protein removal are the same as before.

[0020] (5) Analysis of the composition of drug-containing serum based on UPLC-Q-TOF-MS / MS

[0021] ①Chromatographic conditions

[0022] Chromatographic column: Agilent Zorbax SB-C18 (2.1mm×100mm, 1.8μm). The mobile phase was 0.1% formic acid water (A)-acetonitrile (B), gradient elution (0-5min, 5%-45% B; 5-10min, 45%-55% B; 10-12min, 55%-70% B; 12-25min, 70%-100% B; 25-27min, 100% B; 27-29min, 100%-5% B; 29-31min, 5% B), flow rate 0.3ml·min -1 , column temperature 35°C, injection volume 1 μl.

[0023] ②Mass spectrometry conditions

[0024] The ESI ion source was used, and the full wavelength scanning was performed in positive and negative ion modes. The mass spectrometer detector parameters were set as follows: ion spray voltage 5.5kV / -4.5kV, ion source temperature 550℃, declustering voltage 60V, collision energy 35eV; auxiliary gas was N2, curtain gas 35psi, Gas155 psi, Gas255 psi; MS / MS secondary mass spectrometry mode was used for scanning, the mass number scanning range was 100~2000m / z; the mass spectrum number scanning range was 100~1500m / z; dynamic background subtraction was performed. Data acquisition was performed by TF 1.6 software is available.

[0025] ③UPLC-Q-TOF-MS / MS analysis

[0026] The blood sample solution in (4) is taken and tested by UPLC-Q-TOF-MS / MS under the above-mentioned chromatography / mass spectrometry conditions, and its blood components and metabolites are characterized by matching multiple information such as molecular weight, structural formula, characteristic fragment ions, etc.

[0027] (6) Correlation analysis between blood components and pharmacodynamic indicators

[0028] SPSS statistical software was used to perform spearman correlation analysis on the two variables of blood components and efficacy indicators. The correlation coefficient r was between -1 and 1. When 0.5<|r|<0.8, it indicated that the two variables were moderately correlated; when |r|>0.8, it indicated that the two variables were highly correlated.

[0029] (7) Identification of quality markers

[0030] Preparation of test solutions of raw and vinegar Curcuma azadirachta extracts: Accurately pipette (1) 1.5 ml of the extract, centrifuge at 4°C, take 1.2 ml of the supernatant, elute the sample with an SPE column, take the filtrate, dilute, centrifuge, take the supernatant, filter through a membrane, and obtain.

[0031] Under (5) chromatography / mass spectrometry conditions, by analyzing mass spectrometry data, detecting by UPLC-Q-TOF-MS / MS, and comparing the molecular ion peaks and ion fragment information with references or databases, a total of 32 blood quality marker groups were characterized: zederone, curcolonol, zedoalactone A, zedoalactone C, turmerone, β-elemenone, curcumalactone, curcumolone, turmericdione, 4-epi-curmerol, isoprotocurenol, linoleic acid, furadiene, γ-elemene, δ-elemene, β-serene, α-humulene, β-caryophyllene, aurantium piperonamide acetate, curcumenol, protocurcumenol, 13-hydroxygermacone, (4S,5S)-(+)-germacone 4,5-epoxide, isocurcumenol, germacone, β-elemene, diisobutyl phthalate, curcumabicycloenone, curcumol, curcumadione, neocurcumadione and (4S)-dihydrocurcumabicycloenone.

[0032] The quality evaluation method of Curcuma longifolia and its vinegar product is characterized in that the quality marker is selected from a further optimized group of 25 blood-entering quality markers: zederone, curcolonol, zedoalactone A, zedoalactone C, turmerone, β-elemenone, turmeric alcohol ketone, turmeric dione, 4-epi-curcumol, isoprotocurenol, linoleic acid, furadiene, γ-elemene, δ-elemene, β-serene, α-humulene, β-caryophyllene, aurantium piperonamide acetate, curcumenol, protocurcumenol, 13-hydroxygermacone, (4S, 5S)-(+)-germacone 4,5-epoxide, isocurcumenol, germacone and β-elemene.

[0033] (8) Searching for differential quality markers

[0034] PCA, OPLS-DA and volcano plot analysis were performed using Simca-P14.1 software, and the ion strength of Curcuma wenyujin and its vinegar products were analyzed using T test using SPSS statistical software. And p<0.05 was used as the standard for judging the significance of the difference to find the difference quality markers.

[0035] Preparation of reference solution: Accurately weigh appropriate amounts of isocurcuminol, germacrone, and β-elemene, add methanol to prepare individual reference stock solutions, and accurately take appropriate amounts of each stock solution to prepare a 5 μg / ml mixed reference solution. Under (5) chromatography / mass spectrometry conditions, analyze the mass spectrometry data and detect it by UPLC-Q-TOF-MS / MS to compare the retention time of the reference and the differential mass marker.

[0036] The quality evaluation method of Curcuma wenyujin and its vinegar product is that the difference quality marker components between Curcuma wenyujin and Curcuma wenyujin vinegar are selected from one or more of curcolonol, curdionolide C, tetrahydrocurcumin, aurantium piperonamide acetate, lycium amide, (+)-thujacene, α-curcumene, isocurcumenol, germacronone, β-elemene and linoleic acid.

[0037] In the quality evaluation method of Curcuma longifolia and its vinegar product, the difference quality marker is selected from one or more of isocurcumenol, germacrone, β-elemene, curcolonol, linoleic acid and orange-yellow piperonamide acetate.

[0038] In the quality evaluation method of Curcuma wenyujin and its vinegar product, the difference quality marker is selected from one or more of isocurcumenol, germacrone and β-elemene.

[0039] In the quality evaluation method of Curcuma wenyujin and its vinegar product, the differential quality marker is selected from isocurcumenol.

[0040] Application of the quality markers in the quality evaluation method of Curcuma wenyujin and its vinegar products in the quality control of Curcuma wenyujin and its vinegar products.

[0041] The invention discloses an application of the difference quality markers in the quality evaluation method of Curcuma wenyujin and its vinegar products in the quality control of Curcuma wenyujin vinegar making process.

[0042] The turmeric is raw turmeric, and the processing method is: take turmeric medicinal materials, wash, moisten, cut into thin slices, and dry.

[0043] Beneficial effects of the present invention

[0044] The present invention is based on the research idea of ​​efficacy-related components, and screens out 32 quality markers for treating liver depression and qi stagnation dysmenorrhea with Wenyujin and its vinegar products, including zederone, curcolonol, zedoalactone A, zedoalactone C, turmerone, β-elemenone, curcumalactone, turmeric alcohol ketone, turmeric dione, 4-epi-curcumol, isoprotocurenol, linoleic acid, furadiene, γ-elemene, δ-elemene, β-serene, α-humulene, β-caryophyllene, orange-yellow piperonamide acetate, curcumenol, protocurcumenol, 13-hydroxygermacone, (4S, 5S)-(+)-germacone 4,5-epoxide, isocurcumenol, germacone, β-elemene, diisobutyl phthalate , one or more of curcuma bicycloenone, curcumol, curcuma dione, neocurcuma dione and (4S)-dihydrocurcuma bicycloenone; screen out differential quality markers for treating liver depression and qi stagnation and dysmenorrhea with Wenyu Jin and its vinegar products, a total of 6 including one or more of isocurcumenol, germacron, β-elemene, curcolonol, linoleic acid and orange yellow piperonamide acetate; through reference verification, one or more of isocurcumenol, germacron, β-elemene can be used as differential quality markers for treating liver depression and qi stagnation and dysmenorrhea with Wenyu Jin and its vinegar products. The present invention provides a candidate compound group for treating liver depression and qi stagnation and dysmenorrhea, and also provides an example for the quality control research of Chinese herbal medicine slices based on efficacy-related components. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Effects of Curcuma aromatica and its vinegar product on body weight in rats with liver depression and qi stagnation (n=8).

[0046] Figure 2 Effects of Curcuma zedoariae and its vinegar products on organ indexes in rats with liver depression and qi stagnation (n=8, )

[0047] Figure 3 Effects of Curcuma zedoariae and its vinegar products on the writhing reaction of rats with liver depression and qi stagnation (n=8, )

[0048] Figure 4 Effects of Curcuma zedoariae and its vinegar products on inflammatory and pain factors in rats with liver depression and qi stagnation (n=8, )

[0049] Figure 5 Typical total ion chromatograms of drug-containing serum in positive and negative ion modes.

[0050] Figure 6 To infer the metabolic pathway of isoproterocurenol in serum.

[0051] Figure 7Heat map of correlation analysis between blood components and efficacy indicators of Curcuma aromatica and its vinegar products in the treatment of dysmenorrhea caused by liver depression and qi stagnation.

[0052] Figure 8 Effects of Curcuma wenyujin and its vinegar products on key genes in rats with liver depression and qi stagnation (n=3, )

[0053] Fig. 9 Heat map of correlation analysis between blood components and key genes in the treatment of dysmenorrhea caused by liver depression and qi stagnation in Curcuma zedoaria and its vinegar products.

[0054] Fig.10 Typical total ion currents of Curcuma wenyujin and its vinegar products in positive and negative ion modes.

[0055] Fig.11 Secondary mass spectra and fragmentation patterns of components of Curcuma zedoaria and its vinegar products.

[0056] Fig.12 PCA and volcano plot analysis of Curcuma wenyujin and its vinegar products

[0057] Fig.13 OPLS-DA and S-pot analysis of Curcuma wenyujin and its vinegar products

[0058] Fig.14 Primary mass spectra of isocurenol, germacrone and β-elemene reference substances.

[0059] Fig.15 Secondary mass spectra of reference substances of isocurenol, germacrone and β-elemene.

[0060] Note: * p<0.05, ** p < 0.01, compared with the normal group; # p<0.05, ## p < 0.01, compared with the model group; & p<0.05, && p<0.01, compared with the raw warm turmeric group.

[0061] Specific implementation cases

[0062] The present invention is described in detail below with reference to diagrams and specific implementation operations.

[0063] Example 1

[0064] 1. Pretreatment of experimental samples

[0065] The dried tubers of Curcuma wenyujin Y.H.Chen et C.Ling, a plant of the ginger family, were purchased from Shazhou Village, Taoshan Town, Ruian City, Wenzhou City, Zhejiang Province. The raw and vinegar-curcuma slices were processed according to the processing methods of raw and vinegar-curcuma in the 2020 edition of the Chinese Pharmacopoeia and relevant provincial processing specifications.

[0066] Raw Curcuma Herba Decoction Slices: Take the Curcuma Herba, wash, moisten, cut into thin slices, and dry.

[0067] Vinegar-warmed turmeric slices: Take raw warm turmeric slices, add rice vinegar and mix well, simmer thoroughly, put in a pot and heat over low heat and stir-fry until dry, take out and let cool.

[0068] (20kg of rice vinegar for every 100kg of clean turmeric)

[0069] 2 Preparation of Curcuma aromatica and its vinegar extract

[0070] The raw and vinegar-curcuma slices were heated and refluxed with 15 times the amount of 90% ethanol, and extracted twice, each time for 1.5 hours; the residues were extracted with 10 times the amount of water for 1.5 hours, and the above extracts were combined, filtered, and concentrated under reduced pressure at 50℃ to contain 0.2g·ml of the raw drug. -1 , for subsequent experiments.

[0071] 3. Study on the efficacy of Curcuma zedoariae and its vinegar extract in treating dysmenorrhea caused by liver depression and qi stagnation

[0072] 3.1 Animal grouping, model construction and drug administration

[0073] Animal grouping: Female SPF SD rats (200 ± 20 g), non-pregnant and unmated. The animals were housed in a controlled room with temperature (25 ± 2 °C), humidity (60% ± 5%) and 12-h light / dark cycle, and had free access to standard diet and water. After 1 week of adaptation, the rats were randomly divided into normal control group (NC, n = 10), model group (M, n = 10), ibuprofen (positive drug control) group (B, n = 10), low-dose raw warm turmeric extract group (CL, n = 10); high-dose raw warm turmeric extract group (CH, n = 10), low-dose vinegar warm turmeric extract group (VL, n = 10); high-dose vinegar warm turmeric extract group (VH, n = 10). The experimental protocol was in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Experiment Ethics Committee of Nanjing University School of Medicine (202105A052) (SYXK (su) 2018-0048).

[0074] Construction of liver depression and qi stagnation dysmenorrhea model: Estradiol benzoate and epinephrine hydrochloride were subcutaneously injected on the back of rats in each group for 10 consecutive days. On the first and tenth days, 5 mg kg of estradiol benzoate was subcutaneously injected on the back of each rat. -1 ·d -1, 2.5 mg kg was injected subcutaneously on the back of each rat on days 2-9 -1 ·d -1 . Epinephrine hydrochloride 0.6 mg kg twice daily -1 ·d -1 , with an interval of about 4 hours in between, supplemented by comprehensive stimulation (A: sound stimulation (60dB, (10±5)kHz, intensity level 3), 60min / time, 2 times / day; B: light stimulation (flash light, frequency (2±1)Hz), 60min / time, 2 times / day; C: restraint tube restraint, 60min / time, 2 times / day; D: book clip tail clamp, 30min / time). Rats in the blank control group were subcutaneously injected with normal saline on the back, and no comprehensive stimulation was given.

[0075] Animal dosing: Oral administration started on the 4th day, once a day, for 7 consecutive days. According to the clinical equivalent dose, the dose of ibuprofen group (group B) was 0.06 g kg -1 , the low-dose group of Wenyujin was 0.3 g·kg -1 , the high-dose group of Wenyujin was 0.9 g·kg -1 The rats in each group were gavaged with the corresponding drugs, and the blank control group and the model group were gavaged with the same amount of normal saline.

[0076] 3.2 Animal sample collection

[0077] After the last dose, oxytocin was injected intraperitoneally, and the writhing reaction was observed within 0.5 h. Afterwards, 3% sodium pentobarbital (1 ml kg -1 ) anesthesia. Blood was collected from the abdominal aorta and kept at room temperature for 1 h at 3 000 r / min -1 Centrifuge for 20 minutes, take the supernatant to obtain serum, and use it for the subsequent qualitative analysis of serum components. At the same time, quickly take fresh rat uterus and liver tissues, rinse with saline, dry with filter paper, and weigh. Store part of the uterus and liver tissues in a -80℃ refrigerator for subsequent experiments.

[0078] 3.3 Results of the efficacy evaluation of Curcuma zedoariae and its vinegar products in treating dysmenorrhea due to liver depression and qi stagnation

[0079] 3.3.1 Results of body surface characteristics of animals with liver depression and qi stagnation

[0080] Results of the body surface characteristics of animals with liver depression and qi stagnation are as follows Figure 1As shown. During the experiment, the normal control group rats had shiny fur, rapid activity, bright red eyes, no ecchymosis or petechiae on the ears, normal appetite, formed stools, and steady weight gain. The model group rats had yellow and frizzy fur, fatigue and irritability, reduced spontaneous activity, dark red eyes, purple tongue, reduced appetite, loose and unformed stools, and gradually lost weight. The tails of very few rats were extremely swollen or broken, which was consistent with the surface characteristics of the animal model of Qi stagnation and blood stasis syndrome, indicating that the rat model was successful. The rats in the other drug-treated groups showed varying degrees of the above symptoms after modeling; compared with the model group, the status of each drug-treated group improved after treatment, and the weight increased slowly. Among them, the ibuprofen group, the high-dose group of Curcuma wenyujin and its vinegar products significantly improved the status of the rats.

[0081] 3.3.2 Results of organ coefficients in rats with liver depression and qi stagnation and dysmenorrhea

[0082] The results of organ coefficients of rats with liver depression and qi stagnation and dysmenorrhea are as follows Figure 2 As shown. Compared with the blank group, the organ index of the liver and uterus of the model rats increased significantly (P<0.01), which may be related to liver and uterine congestion or edema. After administration, the ibuprofen group, the high-dose group of Wenyujin and its vinegar products had a significant improvement effect on the organ index of the liver and uterus (P<0.05, P<0.01), and the high-dose group of vinegar Wenyujin had a significant improvement effect on the liver and uterus than the raw Wenyujin group (P<0.05, P<0.01). There was no significant difference in the low-dose group.

[0083] 3.3.3 Results of writhing reaction in rats with liver depression and qi stagnation and dysmenorrhea

[0084] Results of writhing reaction in rats with liver depression and qi stagnation and dysmenorrhea Figure 3 Compared with the model group, the ibuprofen group, Curcuma zedoariae and its high-dose vinegar product could significantly prolong the writhing latency and reduce the writhing times of rats with liver depression and qi stagnation and dysmenorrhea (P<0.05, P<0.01), and the writhing reaction inhibition rates were 40%, 20%, and 27%, respectively. It can be seen that vinegar preparation is beneficial to enhance the analgesic effect of Curcuma zedoariae.

[0085] 3.3.4 ELISA results of rats with liver depression and qi stagnation and dysmenorrhea

[0086] 0.1 g of uterine tissue was weighed in each group, and 0.9 ml of washing solution (0.15% Tween 20 in PBS) was added and mixed evenly using a Thermo Fisher cryogenic tissue homogenizer at 12,000 r / min. -1 Centrifuge for 10 min and collect the supernatant; PGF 2α , PGE2, β-EP and 5-HT were determined by competitive method, Ca 2+The biochemical microplate method was used for the determination. The parameters were set strictly according to the instructions of the kit, and the operation was performed strictly according to the instructions of the kit to make a standard curve, and the levels of PGF2α, PGE2, β-EP, and Ca2+ in uterine tissue, as well as the level of 5-HT in serum were determined in turn.

[0087] ELISA results of rats with dysmenorrhea caused by liver depression and qi stagnation Figure 4 Compared with the blank group, the PGF 2α , Ca 2+ , 5-HT contents increased significantly, PGE2, β-EP contents decreased significantly (P<0.01); compared with the model group, the positive drug ibuprofen group significantly improved all indicators of the primary dysmenorrhea model rats with liver depression and qi stagnation (P<0.01); PGF 2α , Ca 2+ , 5-HT contents were significantly decreased, and PGE2 and β-EP contents were significantly increased (P<0.05, P<0.01); the low-dose vinegar turmeric group significantly reduced PGF 2α The content of PGE2 and β-EP was increased (P<0.05); among them, the low-dose group of raw turmeric had no obvious regulatory effect; compared with the raw turmeric group, the high-dose group of vinegar turmeric had a significantly better regulatory effect than the raw turmeric group (P<0.05, P<0.01). In summary, vinegar processing is beneficial to enhance the inhibitory effect of turmeric on uterine damage.

[0088] 4 UPLC-Q-TOF-MS / MS analysis of drug-containing serum

[0089] 4.1 Chromatographic conditions

[0090] Chromatographic column: Agilent Zorbax SB-C18 (2.1mm×100mm, 1.8μm). The mobile phase was 0.1% formic acid water (A)-acetonitrile (B), gradient elution (0-5min, 5%-45% B; 5-10min, 45%-55% B; 10-12min, 55%-70% B; 12-25min, 70%-100% B; 25-27min, 100% B; 27-29min, 100%-5% B; 29-31min, 5% B), flow rate 0.3ml·min-1, column temperature 35℃, injection volume 1μl.

[0091] 4.2 Mass spectrometry conditions

[0092] The ESI ion source was used, and the full wavelength scanning was performed in positive and negative ion modes. The mass spectrometer detector parameters were set as follows: ion spray voltage 5.5kV / -4.5kV, ion source temperature 550℃, declustering voltage 60V, collision energy 35eV; auxiliary gas was N2, curtain gas 35psi, Gas1 55psi, Gas2 55psi; MS / MS secondary mass spectrometry mode was used for scanning, the mass number scanning range was 100~2000m / z; the mass spectrum number scanning range was 100~1500m / z; dynamic background subtraction was performed. Data acquisition was performed by TF 1.6 software is available.

[0093] 4.3 Test solution

[0094] Accurately pipette 200μl of the above serum sample, add 1000μl of methanol, vortex for 1min, fully precipitate the protein, centrifuge, take 1000μl of the supernatant and place it in a 2.0ml centrifuge tube, evaporate the solvent in a vacuum centrifuge concentrator (30°C), add 200μl of methanol to re-dissolve, vortex for 5min, centrifuge, take 200μl of the supernatant, and get it. At the same time, take 10μl from each sample and mix them to prepare the quality control (QC) sample, and the steps of protein removal are the same as before.

[0095] 4.4UPLC-Q-TOF-MS / MS analysis

[0096] Take the test solution in 4.3 and detect it by UPLC-Q-TOF-MS / MS under the chromatographic conditions in 4.1 and the mass spectrometric conditions in 4.2. Use Peakview software and Metabolite Pilot metabolite analysis software to identify and analyze the components in the serum of Curcuma aromatica. The specific process is as follows. First, the above mass spectrometry data were imported into Peakview software to characterize the blood components, and then the names, molecular formulas, structural formulas and secondary mass spectrometry data of the characterized chemical components were imported into the Compound Library module in MetabolitePilot software as the compounds to be analyzed; then, by consulting the data, a common metabolic pathway library existing in the blood was summarized and established, with a total of 80 metabolic pathways (phase I metabolism & phase II metabolism), and the names and molecular formula additions and subtractions of these metabolic pathways were imported into the Biotransformation module in the software; then, in the Processing Methods module, Predicted metabolites were checked, and the other parameters were default to establish the analysis method; finally, the serum mass spectrometry file to be analyzed was imported into the software for serum component identification, and the overall scores of Mass defect, Isotope pattern MS / MS and Massaccuracy were combined, and the compounds with a score rate ≥75% were identified as metabolites.

[0097] 4.5 UPLC-Q-TOF-MS / MS results

[0098] The positive and negative total ion currents of the serum containing Curcuma wenyujin are as follows Figure 5 As shown. MetabolitePilot software was used to identify and analyze the blood-entering components and metabolites of Curcuma wenyujin. A total of 66 components were identified in the drug-containing serum of rats in the Curcuma wenyujin administration group, including 31 prototype components and 35 metabolites. The relative ion intensities of the 66 blood-entering components and their metabolites were analyzed by T test. The specific information of these compounds is shown in Table 1. As can be seen from Table 1, the metabolic pathways of the sesquiterpenoid components in Curcuma wenyujin in rats with liver depression and qi stagnation and dysmenorrhea mainly include internal hydrolysis, hydrogenation, oxidation, deoxygenation, desaturation, ketone formation, demethylation or desaturation or carboxylic acid formation and other one-phase metabolism. Figure 6 The main metabolic transformation pathways of isoproterocurenols are shown.

[0099]

[0100]

[0101]

[0102] 5. Correlation analysis results between blood components and pharmacodynamic indicators

[0103] The peak intensity of the above 66 blood components was used as variable X; organ coefficient, twisting reaction, and related biochemical factor index data were used as variable Y, and spearman correlation analysis was performed using SPSS statistical software. The correlation coefficient r was between -1 and 1. When 0.5<|r|<0.8, it indicated that the two variables were moderately correlated; when |r|>0.8, it indicated that the two variables were highly correlated.

[0104] The results of the correlation analysis between blood components and pharmacodynamic indicators are shown in Table 2 and Figure 7 According to the order of the absolute value of the correlation coefficient from large to small, the main efficacy indicators (writhing times, PGF 2α , Ca 2+ , 5-HT, β-EP)|r|>0.5, for different efficacy indicators, the blood components with strong correlation are also different. For example, for the representative indicator of inflammatory response, PGF 2α The negatively correlated blood component is orange-yellow piperonamide acetate, which lost one molecule of C9H 10 O and the oxidized metabolite M2, while the positively correlated component entering the blood is the metabolite M1 after linoleic acid lost one molecule of O and hydrogenated, which is speculated to have anti-inflammatory effects. 2+The negatively correlated blood component is orange-yellow piperonamide acetate, which lost one molecule of C9H 10 O and the oxidized metabolite M2, indicating that the higher the content of these compounds in the blood, the higher the Ca 2+ The lower the content, the more likely it is to inhibit Ca 2+ It flows into the uterine smooth muscle cells to play an antispasmodic effect. For factors such as the number of writhing times and β-EP, which are representative indicators of pain response, the negatively correlated blood-entering component is the loss of one molecule of H2O from furanadiene; the positively correlated blood-entering components are the metabolites of Zederone, turmeric alcohol, turmeric dione, 4-epi-curcumol, isocurcumol and Curcolonol, indicating that the higher the content of these compounds in the blood, the lower the number of writhing times, the lower the 5-HT content, and the higher the β-EP content, which is speculated to have analgesic effects. Further analysis of these highly correlated blood-entering components found that most of them were metabolites. Compared with the prototype components, metabolites play a more important role in the treatment process of the drug. Nine blood-entering components that meet the condition of 0.5<|r|<1 were selected as potential quality markers for Wenyu Jin and its vinegar products in the treatment of dysmenorrhea caused by liver depression and qi stagnation, including furadiene, aurantium piperonamide acetate, zederone, turmeric alcohol ketone, turmericdione, 4-epi-curcumol, isoproterocurenol, curcolonol and linoleic acid.

[0105] Table 2 Ranking of correlation coefficients between blood components and some efficacy indicators of raw and vinegar-warmed turmeric in the treatment of dysmenorrhea caused by liver depression and qi stagnation

[0106]

[0107]

[0108] Example 2

[0109] 1. Pretreatment of experimental samples

[0110] The treatment method is the same as that in Example 1 (1. Pretreatment of experimental samples).

[0111] 2 Preparation of Curcuma aromatica and its vinegar extract

[0112] The preparation method is the same as that of the 2-warm turmeric and its vinegar product extract in Example 1.

[0113] 3. Study on the efficacy of Curcuma zedoariae and its vinegar extract in treating dysmenorrhea caused by liver depression and qi stagnation

[0114] 3.1 Animal model construction and drug administration

[0115] The animal model construction and drug administration were the same as those in 3.1 Animal model construction and drug administration in Example 1.

[0116] 3.2 Animal sample collection

[0117] Animal sample collection is the same as 3.2 Animal sample collection in Example 1.

[0118] 3.3 Results of the efficacy evaluation of Curcuma zedoariae and its vinegar products in treating dysmenorrhea due to liver depression and qi stagnation

[0119] Total RNA was extracted from cells according to the kit instructions, and reverse transcription was performed according to the reverse transcription kit instructions. Specific primers were designed according to the literature reports (Table 3). β-actin was used as the internal reference gene, and the following reaction system was prepared using the AceQ qPCR SYBR Green Master Mix kit: 2×qPCR Mix (10 μl), 10 μM gene primer (2.0 μl), reverse transcription product (2.0 μl), ddH2O (6.0 μl), ROX1 (0.4 μl). Quantification was performed using the Applied Biosystems QuantStudio6-Flex instrument according to the following RT-qPCR program: pre-denaturation at 95°C for 10 min, followed by 40 cycles of 95°C, 15s→60°C, 60s, and finally the temperature was raised from 75°C to 95°C at a rate of 1°C every 20s. -ΔΔCt The relative expression levels of genes were calculated, and each gene was repeated 3 times.

[0120] RT-qPCR results of rats with dysmenorrhea caused by liver depression and qi stagnation Figure 8 The results showed that compared with the normal group, the expression levels of 5-HTR2A, IP3R, COX-2, PKC, cALM, and ERK genes in the uterine tissue of rats with liver depression and qi stagnation type dysmenorrhea were significantly increased ( * P<0.05, ** P<0.01, *** P<0.001); compared with the model group, the expression levels of 5-HTR2A, IP3R, COX-2, PKC, cALM, and ERK genes in the uterine tissues of rats in each drug-treated group were significantly decreased ( * P<0.05, ** P<0.01, *** P<0.001); Compared with the raw warm turmeric group, the vinegar warm turmeric group had a significantly stronger regulation on 5-HTR2A and COX-2 genes ( & P<0.05). Curcuma wenyujin can treat dysmenorrhea of ​​liver depression and qi stagnation type by down-regulating the expression of 5-HTR2A, COX-2, ERK2 and other genes, and its effect of soothing liver and relieving pain is enhanced after being prepared with vinegar.

[0121] Table 3 RT-QPCR key genes and their primer sequences

[0122]

[0123] Note: “F” stands for forward primer, and “R” stands for reverse primer.

[0124] 4 UPLC-Q-TOF-MS / MS analysis of drug-containing serum

[0125] The conditions, solution preparation and other methods and analysis results were the same as in Example 1 (UPLC-Q-TOF-MS / MS analysis of drug-containing serum).

[0126] 5. Correlation analysis results between blood components and pharmacodynamic indicators

[0127] The peak intensity of the above 66 blood components was used as variable X; 5-HTR2A, IP3R, COX-2, PKC, cALM, ERK index data was used as variable Y, and spearman correlation analysis was performed using SPSS statistical software. The correlation coefficient r was between -1 and 1. When 0.5<|r|<0.8, it indicated that the two variables were moderately correlated; when |r|>0.8, it indicated that the two variables were highly correlated.

[0128] The results of the correlation analysis between blood components and key gene indicators are shown in Table 4 and Fig. 9 As shown. According to the order of |r| from large to small, 32 blood-entering components with |r|>0.5 related to the main efficacy indicators (5-HTR2A, IP3R, COX-2, PKC, cALM, ERK) were screened out. For different efficacy indicators, the blood-entering components with strong correlation are also different. For example, the representative gene 5-HTR2A, which is closely related to neurotransmitters, is negatively correlated with the blood-entering components such as the desaturated metabolite M2 of turmeric dione and the prototype component Zedoalactone A; the negatively correlated blood-entering components linoleic acid and its metabolites M1 and M2 are speculated to exert analgesic effects by regulating the 5-HTR signaling pathway. And Ca 2+ The representative gene IP3R that is closely related to Ca is negatively correlated with furanodiene, etc., indicating that the higher the content of these compounds in the blood, the lower the expression of IP3R gene, which inhibits Ca 2+ Influx, it is speculated that it may regulate Ca 2+Signaling pathways are used to relax uterine smooth muscle and exert antispasmodic effects. COX-2, a representative gene closely related to PGs, is negatively correlated with the metabolite M1 of curcuminol losing a molecule of H2O and the prototype component Zedoalactone A, etc.; the positively correlated metabolite M3 of isoprotocurcose internal hydrolysis is speculated to exert anti-inflammatory effects by regulating arachidonic acid metabolism. ERK, a representative gene closely related to inflammatory response, is negatively correlated with the metabolite M2 of Curcolonol in the body and the prototype component Zederone, etc.; the positively correlated metabolites M2 and M3 of 4-epi-curcumol in the body and the prototype component Germacone, etc., speculated to exert anti-inflammatory effects by regulating the MAPK / ERK signaling pathway. The 32 blood-entering components that meet the condition of 0.5<|r|<1 were selected as potential quality markers for Curcuma wenyujin and its vinegar products in the treatment of dysmenorrhea caused by liver depression and qi stagnation, including zederone, curcolonol, zedoalactone A, zedoalactone C, turmerone, β-elemenone, curcumalactone, curcumolone, turmericdione, 4-epi-curmerol, isoprotocurenol, linoleic acid, furadiene, γ-elemene, δ-elemene, β-serene, α-humulene, β-caryophyllene, aurantium piperonamide acetate, curcumenol, protocurcumenol, 13-hydroxygermacone, (4S,5S)-(+)-germacone 4,5-epoxide, isocurcumenol, germacone, β-elemene, diisobutyl phthalate, curcumabicycloenone, curcumol, curcumadione, neocurcumadione and (4S)-dihydrocurcumabicycloenone.

[0129] Table 4 Correlation coefficients between blood components and key genes in the treatment of dysmenorrhea caused by liver depression and qi stagnation with raw and vinegar-warmed turmeric

[0130]

[0131]

[0132]

[0133] Example 3

[0134] 1 Characterization of quality marker groups

[0135] 1.1 Chromatographic conditions

[0136] The conditions were set the same as those in 3.1 chromatographic conditions in Example 1.

[0137] 1.2 Mass spectrometry conditions

[0138] The conditions were set the same as those in 3.2 mass spectrometry conditions in Example 1.

[0139] 1.3 Test solution

[0140] Accurately pipette 1.5 ml of the extract in Example 1 (2), centrifuge at 4°C, 13,000 rpm for 10 min, and take 1.2 ml of the supernatant for later use. Prepare the SPE column eluent (5%, 25%, 50%, 75%, 100% methanol). First, activate the SPE column according to the methanol concentration from high to low (2 ml for each gradient). Inject 1 ml of the centrifuged extract onto the activated SPE column, and elute the sample according to the methanol concentration from low to high (1 ml 5%, 2 ml 25%, 2 ml 50%, 2 ml 75%, 2 ml 100%). Take the filtrate, dilute it 1-fold with methanol, centrifuge at 4°C, 13,000 rpm for 10 min, take the supernatant, filter the membrane, and you are done.

[0141] 1.4 UPLC-Q-TOF-MS / MS analysis and results

[0142] Take the test solution in 1.3 and detect it by UPLC-Q-TOF-MS / MS under the chromatographic conditions in 1.1 and the mass spectrometry conditions in 1.2. According to the establishment of the chemical component database of Curcuma, the primary and secondary mass spectra of the chemical components of the original mass spectrometry data were extracted using Peakview software. The error value between different samples was set to ≤10ppm, and the retention time error range was set to ±0.2min. The secondary fragments were compared with the database, and the compounds were matched according to various information such as molecular weight, chemical structure, characteristic fragment ions, etc., and the chemical components in the extract of Curcuma wenyu were analyzed. The total positive and negative ion currents of Curcuma wenyu and its vinegar products are shown in Figure 2. Fig.10 As shown, by matching with Peakview software, 42 compounds (32 of which enter the blood) were obtained in positive and negative ion modes, as shown in Table 5. As can be seen from Table 5, these components are mainly sesquiterpenes (33), curcumin (1) and other components (8).

[0143] 1.5 Characterization of quality marker groups

[0144] These mass markers were characterized by analyzing mass spectrometry data and comparing molecular ion peaks and ion fragment information with references or databases. Fig.11 As shown. The mass spectrometry fragmentation behavior of compounds of the same type also has the same characteristics. Taking the sesquiterpenoid components furanodiene and germacrone as examples, the secondary fragmentation pathways were preliminarily analyzed. Compound 20 in positive ion mode m / z 216[M+H] +, observing the secondary mass spectrometry ion fragments, it is speculated that m / z 216 loses 1 O atom and 1 C atom to form m / z 189, m / z 216 loses -C4H7 group to form m / z 161, m / z 216 loses 1 -OH and propylene to form m / z 157, m / z 216 loses 1 2-methylbutenyl to form m / z 147, m / z 216 loses 1 2-methyl-2-butenyl to form m / z 149, and then m / z 149 loses 1 -CH2 to form m / z 135, characterizing compound 20 as furadiene. Compound 38 in positive ion mode m / z 219 [M+H] + , observing the secondary mass spectrometry ion fragments, it was inferred that m / z201 was formed by the loss of 1 molecule of H2O from m / z219, m / z191 was formed by the loss of CO group from m / z219, and m / z163 was formed by the loss of C4H8 group, thus compound 38 was characterized as germacrone.

[0145]

[0146]

[0147]

[0148] 2. Search for differential quality markers

[0149] 2.1 PCA analysis and results

[0150] In order to analyze and explore the differences between Curcuma wenyujin and its vinegar products, MarkerView1.2.1 software was used for peak detection and calibration, and the parameters were set as follows: retention time error limit 0.2 minutes; mass range 100-2000Da; mass error limit 0.01Da. The peak intensity threshold was 10. Minimum RT: 2min; minimum spectral peak width: 25ppm; minimum RT peak width: 6 scans; noise threshold: 100; RT error: 0.50min; mass error: 10ppm; maximum number of peaks: 8000. After the total ion flow of each sample was normalized, the raw data was converted into a three-dimensional matrix containing peak name, retention time, sample number, and normalized peak area. The three-dimensional matrix was imported into Simca-P14.1 software for PCA analysis. The PCA analysis showed obvious grouping and clear boundaries ( Fig.12 ).

[0151] 2.2 Volcano map analysis and results

[0152] Using the volcano plot ( Fig.12) analyzed the data set. In the positive ion mode, there were 136 significantly upregulated components and 134 significantly downregulated components. In the negative ion mode, there were 368 significantly upregulated components and 137 significantly downregulated components, which verified that there were differences between Curcuma wenyujin and its vinegar products.

[0153] 2.3OPLS-DA analysis and results

[0154] The three-dimensional matrix was then imported into Simca-P14.1 software for OPLS-DA analysis. The OPLS-DA analysis showed obvious grouping with clear boundaries ( Fig.13 ). The extended statistical analysis method S-pot of OPLS-DA was used to determine the potential differential markers for distinguishing Curcuma wenyujin and its vinegar products. In the S-pot diagram, each point represents an ion TR-m / z pair. The farther the "point" is from the center, the greater the contribution of the ion to the identification of the two groups of samples. Therefore, the ions at both ends of the S curve have the largest contribution rate and are potential differential markers for the difference between the two groups of samples ( Fig.13 ).

[0155] 2.4T test analysis and results

[0156] In order to further obtain the differential components in Wenyujin and its vinegar products, the relative contents of the components in Wenyujin and its vinegar products were analyzed by T test. The results showed that there were 11 components that changed significantly after Wenyujin vinegar processing (p < 0.05), as shown in Table 6, and the change information of other compounds is shown in Table 5. Further analysis found that there were 2 compounds with significantly increased contents after Wenyujin vinegar processing, including tetrahydrocurcumin and curcolonol, and the percentage of increase in their contents was as high as 91.67% and 61.32%; there were 9 compounds with significantly decreased contents after Wenyujin processing, including curdionolide C, orange yellow piperonamide acetate, lycium amide, (+)-thujacene, α-curcumene, isocurcumenol, germacrone, β-elemene and linoleic acid, among which the percentage of germacrone content decreased the most, reaching 60.74%.

[0157] Based on the quality marker groups of Example 1 and Example 2, it was found that a total of 25 blood-entering components that meet the condition of 0.7<|r|<1 are potential quality marker groups for Curcuma zedoariae and its vinegar products in the treatment of liver depression and qi stagnation dysmenorrhea, including:

[0158] Zederone, curcolonol, zedoalactone A, zedoalactone C, turmerone, β-elemenone, curcuminone, turmericdione, 4-epi-curcumol, isoprotocurenol, linoleic acid, furadiene, γ-elemene, δ-elemene, β-serene, α-humulene, β-caryophyllene aurantiamide acetate (aurantiamide), curcumenol, protocurcumenol, 13-hydroxygermacone, (4S,5S)-(+)-germacone 4,5-epoxide, isocurcumenol, germacone and β-elemene.

[0159] Combining the 11 differential components of Example 3 and the above 25 quality marker groups, it was found that 6 components, namely Curcolonol, piperonamide acetate, isocurcumol, germacrone, β-elemene and linoleic acid, can be used as differential quality markers for entering the blood.

[0160] Table 6 Eleven different components in Curcuma wenyujin and its vinegar products

[0161]

[0162] △ p<0.05, △△ p<0.01, △△△ p<0.001. +: enter the blood, -: not enter the blood.

[0163] 3. Identification and verification of differential quality markers

[0164] 3.1 Chromatographic conditions

[0165] The conditions were set the same as those in 3.1 chromatographic conditions in Example 1.

[0166] 3.2 Mass spectrometry conditions

[0167] The conditions were set the same as those in 3.2 mass spectrometry conditions in Example 1.

[0168] 3.3 Reference solution

[0169] The representative active ingredients of Curcuma wenyujin, isocurcumenol, germacron, and β-elemene, were selected for verification. An appropriate amount of reference substance was accurately weighed, and methanol was added to prepare a single reference substance stock solution. An appropriate amount of each stock solution was accurately taken to prepare a 5 μg / ml mixed reference substance solution.

[0170] 3.4 UPLC-Q-TOF-MS / MS analysis and results

[0171] Take the reference solution in 3.3 and detect it by UPLC-Q-TOF-MS / MS under the chromatographic conditions in 3.1 and the mass spectrometry conditions in 3.2. Verify the characterization results of the mass marker by comparing the retention time of the mass marker with the retention time of the reference, the primary mass spectrum, and the secondary mass spectrum ( Fig.14 and Fig.15 ). After comparison with the reference substance, compounds 37, 38, and 40 were verified to be isocurcumenol, germacrone, and β-elemene.

[0172] Based on the potential quality marker groups in Example 1 and Example 2, and the differential quality markers in Example 3, isocurcumenol, germacrone, and β-elemene can be used as differential quality markers for Curcuma aromatica and its vinegar products in the treatment of dysmenorrhea caused by liver depression and qi stagnation.

Claims

1. A quality evaluation method for Curcuma wenyujin and its vinegar products, characterized in that: The steps are: combining serum pharmacochemistry and spectrum-effect association to track the blood components of Curcuma wenyujin and its vinegar products and targeting their quality markers: (1) Preparation of extracts of Curcuma zedoaria and its vinegar products Take pieces of Curcuma wenyujin and its vinegar products, heat and reflux with 15 times the amount of 90% ethanol, extract twice, each time for 1.5 h; filter with double-layer gauze, add 10 times the amount of water to the residue, heat and reflux for 1.5 h, combine the extracts, and concentrate under reduced pressure at 50℃ to 0.2 g•ml -1 , and obtained the raw and vinegar Curcuma Radix extracts, which were used for qualitative analysis of components and subsequent oral administration of rats; (2) Animal model construction, drug administration and sample collection The rat model of dysmenorrhea caused by liver depression and qi stagnation was established by subcutaneous injection of estradiol benzoate and epinephrine hydrochloride on the back of each group of rats for 10 consecutive days. On the first and tenth days, each rat was subcutaneously injected with estradiol benzoate 5 mg kg -1 ·d -1 , 2.5 mg kg was injected subcutaneously on the back of each rat on days 2-9 -1 ·d -1 , inject epinephrine hydrochloride 0.6 mg kg twice a day -1 ·d -1 , with an interval of 4 hours in between, supplemented with comprehensive stimulation, and the blank control group rats were subcutaneously injected with normal saline on the back without comprehensive stimulation; (3) Evaluation of the efficacy of Curcuma zedoariae and its vinegar products in treating dysmenorrhea due to liver depression and qi stagnation Writhing reaction: After the last administration of oxytocin, the writhing reaction was observed within 0.5 h, and the writhing latency and number of writhing times were recorded. ELISA experiment: determination of PGF2α, PGE2, β-EP and Ca in uterine tissue 2+ The content of levels, and the content of 5-HT levels in serum; RT-qPCR experiment: Design specific primers according to literature reports, use β-actin as the internal reference gene, prepare the corresponding reaction system, and perform quantification; (4) Preparation of serum test solution Accurately pipette 200µl of serum sample, add 1000µl of methanol, vortex for 1min to fully precipitate protein, centrifuge, take 1000µl of supernatant and place in a 2.0ml centrifuge tube, evaporate the solvent in a vacuum centrifuge concentrator at 30℃, add 200µl of methanol to re-dissolve, vortex for 5 min, centrifuge, take 200µl of supernatant, and obtain; at the same time, take 10µl from each sample and mix them to prepare quality control QC samples, and the steps of protein removal are the same as before; (5) Analysis of the composition of drug-containing serum based on UPLC-Q-TOF-MS / MS ①Chromatographic conditions Chromatographic column: Agilent Zorbax SB-C18, specification: 2.1 mm×100 mm, 1.8 µm; mobile phase: 0.1% formic acid water-acetonitrile, 0.1% formic acid water as A, acetonitrile as B, gradient elution, concentration: 0-5 min, 5%-45% B; 5-10 min, 45%-55% B; 10-12 min, 55%-70% B; 12-25 min, 70%-100% B; 25-27 min, 100% B; 27-29 min, 100% ~5% B; 29-31 min, 5% B, flow rate 0.3 ml•min -1 , column temperature 35 °C, injection volume 1 μl; ②Mass spectrometry conditions The ESI ion source was used, and the full wavelength scanning was performed in positive and negative ion modes. The parameters of the mass spectrometer detector were set as follows: ion spray voltage 5.5 kV / -4.5 kV, ion source temperature 550 °C, declustering voltage 60 V, collision energy 35 eV; auxiliary gas was N2, curtain gas 35 psi, Gas 155 psi, Gas 255 psi; MS / MS secondary mass spectrometry mode was used for scanning, and the mass number scanning range was 100-2000 m / z; the mass spectrum number scanning range was 100-1500 m / z; dynamic background subtraction was performed, and data acquisition was obtained through Analyst® TF 1.6 software; ③UPLC-Q-TOF-MS / MS analysis Take the blood test solution in (4) above, and detect it by UPLC-Q-TOF-MS / MS under the above-mentioned chromatography / mass spectrometry conditions, and characterize its blood components and metabolites by matching multiple information such as molecular weight, structural formula, characteristic fragment ions, etc.; (6) Correlation analysis between blood components and pharmacodynamic indicators SPSS statistical software was used to perform spearman correlation analysis on the two variables of blood components and efficacy indicators. The correlation coefficient r was between -1 and 1. When 0.5<|r|<0.8, it indicated that the two variables were moderately correlated; when |r|>0.8, it indicated that the two variables were highly correlated. The correlation analysis of blood components and pharmacodynamic indicators, the efficacy indicators were the number of writhing times, PGF2α, and Ca 2+ , 5-HT and β-EP. Nine blood-entering components that meet the condition of 0.5<|r|<1 are used as potential quality markers for Wenyu Jin and its vinegar products in the treatment of liver depression and qi stagnation and dysmenorrhea, including furadiene, orange yellow piperonamide acetate, zederone, curcumolone, turmeric dione, 4-epi-curcumol, isoproterocurenol, Curcolonol and linoleic acid. Correlation analysis between blood-entering components and key gene indicators, the efficacy indicators are 5-HTR2A, IP3R, COX-2, PKC, cALM and ERK. 32 blood-entering components that meet the condition of 0.5<|r|<1 are used as potential quality markers for Wenyu Jin and its vinegar products in the treatment of liver depression and qi stagnation and dysmenorrhea, including zederone, curcolonol, zedoalactone A, zedoalactone C, turmerone, β-elemenone, curcumalactone, curcumolone, turmericdione, 4-epi-curmerol, isoprotocurenol, linoleic acid, furadiene, γ-elemene, δ-elemene, β-serene, α-humulene, β-caryophyllene, aurantium piperonamide acetate, curcumenol, protocurcumenol, 13-hydroxygermacone, (4S,5S)-(+)-germacone 4,5-epoxide, isocurcumenol, germacone, β-elemene, diisobutyl phthalate, curcuma bicycloenone, curcumol, curcuma dione, neocurcuma dione, and (4S)-dihydrocurcuma bicycloenone; (7) Identification of quality markers Preparation of test solution of raw and vinegar Curcuma acuminata extract: accurately pipette (1) 1.5 ml of extract, centrifuge at 4 °C, take 1.2 ml of supernatant, elute the sample with SPE column, take the filtrate, dilute, centrifuge, take the supernatant, filter through membrane, and obtain; Under the conditions of (5) chromatography / mass spectrometry, UPLC-Q-TOF-MS / MS was used for detection, and the relative contents of the components in Curcuma and its vinegar products were analyzed by T-test. Eleven differential components were found in Curcuma and its vinegar products, including curcolonol, curdionolide C, tetrahydrocurcumin, aurantium piperonamide acetate, lycium amide, (+)-thujacene, α-curcumene, isocurcumenol, germacronone, β-elemene and linoleic acid.

2. The quality evaluation method of Curcuma aromatica and its vinegar product according to claim 1, characterized in that: A total of 25 blood-entering components that met the condition of 0.7<|r|<1 were used as potential differential quality markers for Wenyu Jin and its vinegar products in the treatment of liver depression and qi stagnation dysmenorrhea, including 25 blood-entering differential quality marker groups: zederone, curcolonol, zedoalactone A, zedoalactone C, turmerone, β-elemenone, curcumolone, turmerone, 4-epi-curcumol, isoprotocurenol, linoleic acid, furadiene, γ-elemene, δ-elemene, β-serene, α-humulene, β-caryophyllene, aurantium piperonamide acetate, curcumenol, protocurcumenol, 13-hydroxygermacone, (4S,5S)-(+)-germacone 4,5-epoxide, isocurcumenol, germacone and β-elemene.

3. The quality evaluation method of Curcuma aromatica and its vinegar product according to claim 2, characterized in that: Based on the above 11 differential components and the differential quality markers in the 25 blood-entering components, the differential quality markers include isocurcumenol, germacrone, β-elemene, curcolonol, linoleic acid and aurantium piperonamide acetate.

4. The quality evaluation method of Curcuma aromatica and its vinegar product according to claim 3, characterized in that: Differential quality markers included isocurenol, germacrone, and β-elemene.

5. Application of the quality marker in the quality evaluation method of Curcuma aromatica and its vinegar products according to claim 1 or 2 in the quality control of Curcuma aromatica and its vinegar products.

6. Application of the differential quality markers in the quality evaluation method of Curcuma aromatica and its vinegar products according to any one of claims 3 to 4 in the quality control of Curcuma aromatica vinegar making process.

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