CN108403793B

CN108403793B - A kind of medicine for treating primary dysmenorrhea and preparation method thereof

Info

Publication number: CN108403793B
Application number: CN201810443139.1A
Authority: CN
Inventors: 刘长红; 张艳红; 郭云辉; 梁爽; 刘颖; 袁琳; 庞博; 彭玉博; 王磊
Original assignee: Heilongjiang University of Chinese Medicine
Current assignee: Heilongjiang University of Chinese Medicine
Priority date: 2018-05-10
Filing date: 2018-05-10
Publication date: 2021-06-01
Anticipated expiration: 2038-05-10
Also published as: CN108403793A

Abstract

The invention belongs to the field of traditional Chinese medicine, and discloses a medicine for treating primary dysmenorrhea, the name of the invention is a medicine for treating primary dysmenorrhea and a preparation method thereof, and the medicine comprises the following components by weight: Angelica 1-3 servings, 1-2 servings of white peony root, and 1-2 servings of milk thistle. The invention provides a preparation method of the medicine. The medicine provided by the invention can effectively treat primary dysmenorrhea, has exact curative effect, simple compatibility and low cost; the medicine prepared by the preparation method provided by the invention has high extraction efficiency of active components, The active ingredient content is high, the impurity content is low, the curative effect is reliable, and the safety is high. The invention also provides a method for determining the content of active ingredients by using high performance liquid chromatography and gas chromatography.

Description

Medicine for treating primary dysmenorrhea and preparation method thereof

Technical Field

The invention belongs to the technical field of traditional Chinese medicines, and relates to a medicine for treating primary dysmenorrhea and a preparation method thereof.

Background

Dysmenorrhea refers to the condition of crampy pain in the lower abdomen before and after menstruation or during menstruation, and general discomfort, which seriously affects daily life. It is divided into primary and secondary types. The patient who can not find obvious abnormality of pelvic organs through detailed gynecological clinical examination is called primary dysmenorrhea and also called functional dysmenorrhea. Secondary dysmenorrhea refers to the condition of marked pathological changes of reproductive organs, such as endometriosis, pelvic inflammatory disease, tumor, etc. Primary dysmenorrhea, i.e. functional dysmenorrhea, refers to menstrual pain, which is usually spastic and concentrated in the lower abdomen. Other symptoms comprise headache, hypodynamia, dizziness, nausea, vomiting, diarrhea, lumbocrural pain, which are very common symptoms of young women, and primary dysmenorrhea is not accompanied by obvious pelvic organic diseases.

In the prior art, there are also a number of compound traditional Chinese medicines for treating primary dysmenorrhea, which are listed as follows:

patent documents relating to the treatment of primary dysmenorrhea include:

1. the patent name is a pharmaceutical composition for treating primary dysmenorrhea, and the patent publication number is CN104435519A and the application number is 201410787568.2, and the formula disclosed in the patent is as follows: 5-15 parts of angelica sinensis, 10-20 parts of peach kernel, 5-15 parts of rhizoma cyperi, 5-10 parts of ligusticum wallichii, 5-10 parts of rhizoma corydalis, 2-6 parts of herba lycopi, 2-6 parts of safflower carthamus, 1-5 parts of radix linderae, 2-4 parts of raw pollen typhae and 1-3 parts of liquorice.

2. The patent is named as a pharmaceutical composition for treating cold-congealing and blood-stasis type primary dysmenorrhea, and the patent publication is CN106074830A and application number 201610423799.4, and the formula disclosed in the patent is as follows: 10-30 parts of peach kernel, 3-12 parts of safflower, 3-15 parts of red peony root, 10-20 parts of ligusticum wallichii, 8-20 parts of prepared rehmannia root, 10-20 parts of radix bupleuri, 12-20 parts of medicinal cyathula root, 10-20 parts of cassia twig, 6-20 parts of roasted evodia rutaecarpa, 3-12 parts of angelica, 10-15 parts of white peony root, 6-25 parts of honey-fried licorice root and 10-20 parts of rhizoma corydalis.

3. The patent name is a drug for treating primary dysmenorrhea, and the patent publication number is CN104524085A and the application number is 2014107394165, and the formula disclosed in the patent is as follows: 6 parts of cinnamon, 6 parts of prepared rhizoma cyperi, 8 parts of ligusticum wallichii and 9 parts of rhizoma corydalis.

4. The patent name is a traditional Chinese medicine preparation for treating primary dysmenorrhea, and the patent with the publication number of CN103405606A and the application number of 2013103658206 discloses a formula as follows: 8-12 g of radix bupleuri, 8-12 g of angelica sinensis, 8-12 g of rhizoma cyperi, 5-7 g of trogopterus dung, 5-7 g of pollen typhae, 13-17 g of salvia miltiorrhiza, 13-17 g of radix paeoniae alba and 13-17 g of poria cocos.

5. The patent refers to the field of 'pharmaceutical preparations'. 20-40 parts of motherwort, 20-40 parts of folium artemisiae argyi, 20-40 parts of radix curcumae, 20-40 parts of turmeric, 20-40 parts of hawthorn, 15-25 parts of cortex moutan, 15-25 parts of scutellaria baicalensis, 10-20 parts of trogopterus dung, 10-20 parts of wolfberry fruit, 10-20 parts of codonopsis pilosula and 10-20 parts of semen brassicae.

6. The patent refers to a Chinese medicinal patch for treating primary dysmenorrhea, published as CN106266404A and applied as 201610861934.3, wherein the formula disclosed in the patent is as follows: 5-10 parts of rhizoma cyperi, 5-10 parts of rhizoma corydalis, 4-8 parts of cassia twig, 4-8 parts of cinnamon, 5-8 parts of elecampane and 2-4 parts of angelica sinensis extract.

7. The patent refers to a Chinese medicinal composition for treating dysmenorrhea, published as CN106214769A and applied as 201610886259.X, the formula disclosed in the patent is: 1-5 parts of safflower, 1-5 parts of snow lotus herb, 1-5 parts of jasmine flower and 1-5 parts of peony.

8. The patent is a traditional Chinese medicine plaster for relieving female primary dysmenorrhea, and the patent publication number is CN106176980A, and the application number is 201610544604.1, and the formula disclosed in the patent is as follows: 15-25 parts of corydalis tuber, 15-25 parts of folium artemisiae argyi, 15-25 parts of cinnamon, 15-25 parts of ligusticum wallichii, 1-10 parts of asarum and 10-20 parts of radix linderae.

9. The patent refers to a Chinese medicinal composition for treating primary dysmenorrhea and its preparation and use, and the patent publication is CN106109802A and application number is 201610802757.1, the formula disclosed in the patent is: 1.5-4.5 parts of dragon's blood; 1.5-4.5 parts of pseudo-ginseng; 1.5-4.5 parts of cinnamon; 1.5-4.5 parts of fructus evodiae; 0.5-2.5 parts of herba lycopi; 0.5-2.5 parts of medicinal cyathula root; 0.5-2.5 parts of ligusticum wallichii; 0.5-1.5 parts of clove.

10. The patent is named as the preparation of a medicinal moxibustion strip for treating primary dysmenorrhea, and the patent publication number is CN106074831A and the application number is 201610424128.X, and the patent discloses a formula as follows: peach kernel, bupleurum, safflower, roasted evodia, red peony root, hemlock parsley, prepared rhizome of rehmannia, medicinal cyathula root, cassia twig, white paeony root, angelica, roasted liquorice and rhizoma corydalis.

11. The patent refers to a Chinese medicinal composition containing folium artemisiae argyi for treating primary dysmenorrhea of the type of cold accumulation and blood stasis, with publication number CN105797099A and application number 201610192274.4, wherein the formula disclosed in the patent is: 5-25 parts of folium artemisiae argyi, 6-25 parts of raspberry, 8-20 parts of amethyst, 5-15 parts of cistanche, 5-15 parts of ligusticum wallichii, 9-15 parts of semen cuscutae, 6-12 parts of glossy privet fruit, 3-9 parts of angelica sinensis, 3-9 parts of verbena, 6-12 parts of medlar, 3-10 parts of ginger and 1-10 parts of liquorice.

12. The patent refers to a Chinese medicinal preparation for treating primary dysmenorrhea due to qi stagnation and blood stasis, with publication number CN103721002A and application number 201310755511.X, and the formula disclosed in the patent is: 3-9 parts of begonia leaf, 15-25 parts of rice ball root, 20-25 parts of jasminum grandiflorum, 18-24 parts of aizoon stonecrop root, 30-40 parts of dragon boat flower root, 3-9 parts of gold incense, 12-30 parts of heart-protecting grass, 9-15 parts of murraya paniculata, 9-15 parts of cauda cauliflora and 15-25 parts of fiveleaf akebia fruit.

13. The patent refers to a Chinese medicine for treating primary dysmenorrhea, published under the number CN105616798A and applied under the number 201410618955.3, and the formula disclosed in the patent is: the traditional Chinese medicine comprises 5-15 parts of loosestrife, 10-20 parts of begonia, 3-12 parts of safflower seeds, 2-10 parts of plumbago indica, 5-15 parts of ploughshare, 2-10 parts of centipedes laid on the ground, 5-15 parts of water chestnut, 10-20 parts of weeping forsythia, 5-15 parts of small white chap, 10-20 parts of towel gourd roots, 4-12 parts of golden silk eucommia bark, 5-20 parts of ambergris and 5-15 parts of snakegourd roots.

14. The patent name is a pharmaceutical composition for treating primary dysmenorrhea, and the patent publication number is CN104306470A, and the application number is 201410625394.X, and the formula disclosed in the patent is as follows: 10-50 parts of artemisia scoparia, 10-30 parts of gangrene grass, 20-30 parts of herb of nux vomica, 10-30 parts of hops, 20-30 parts of cupula and 10-20 parts of thalictrum foeniculatum.

15. The patent refers to a Chinese medicinal preparation for treating dysmenorrhea and its preparation, the publication number is CN105582518A, application number is 201610141446.5, the formula disclosed in the patent is: herba Ainsliaeae Rubrinervis, herba Lagotis, herba Artemisiae Anomalae, radix Linderae, Gymnema Sylvestris, radix Adenophorae, herba Siphonostegiae, radix Angelicae sinensis, semen Lactucae, Zingiberis rhizoma, radix seu folium Caulophylli, flos Jasmini sambac, rhizoma Kaempferiae, fructus Leonuri, Notoginseng radix and night.

16. The patent is named as a traditional Chinese medicine for treating primary dysmenorrhea, and the patent publication number is CN105561017A, and the application number is 201610107525.4, and the formula disclosed in the patent is as follows: 15 parts of panicled fameflower herb, 5 parts of Chinese prickly ash, 9 parts of three-ply tendon, 18 parts of Taiping strawberry, 12 parts of club moss, 10 parts of auricularia, 15 parts of alopecurus, and 6 parts of rhizoma dioscoreae hypoglaucae.

17. The patent refers to a Chinese medicine for treating primary dysmenorrhea, published under the number CN105497389A and applied under the number 201610063392.5, and the formula disclosed in the patent is: 8-16 parts of hibiscus roots, 5-13 parts of sassafras, 8-16 parts of platycladi seeds, 8-16 parts of emilia sonchifolia, 8-16 parts of rhizoma cyperi, 8-16 parts of clove, 8-16 parts of ficus microcarpa, 5-13 parts of cinnamomum cassia, 5-13 parts of osmunda japonica roots, 5-13 parts of black sargentgloryvine stems, 11-19 parts of prepared rehmannia roots and 4-8 parts of bupleurum falcatum.

18. The patent refers to a Chinese medicinal preparation for treating primary dysmenorrhea and its preparation, published as CN105288092A and application number 201510861876.X, the formula disclosed in the patent is: 13-18 parts of rhizoma cyperi, 11-16 parts of angelica sinensis, 2 parts of sargentgloryvine stem, 20-25 parts of Japanese milkwort herb, 6-9 parts of rhizoma anemarrhenae and 3-6 parts of liquorice.

19. The patent refers to a Chinese medicinal composition for treating primary dysmenorrhea of the type of cold accumulation and blood stasis, published under the number CN105250617A, filed under the number: 2015108531658, the formulation disclosed therein is: 10-15 parts of three-pair salvia miltiorrhiza, 13-18 parts of herba lycopi, 8-13 parts of heart-protecting grass, 2 parts of rhizoma bambusae, 5-10 parts of rhizoma cyperi, 8-12 parts of herba lycopi, 9-15 parts of caulis spatholobi, 15-18 parts of acanthopanax and 8-12 parts of bighead atractylodes rhizome.

20. The patent name is a drug for treating primary dysmenorrhea, and the patent publication number is CN105126044A and the application number is 2015107110651, and the formula disclosed in the patent is as follows: 10 parts of caulis spatholobi, 8 parts of ramie root, 7 parts of poria cocos, 4 parts of curcuma zedoary, 8 parts of cistanche, 9 parts of cherokee rose fruit and 10 parts of dindyceps.

21. The patent refers to a Chinese medicinal composition for treating primary dysmenorrhea of qi and blood stagnation type, published as CN104258074A and applied for 2014105080322, the formula disclosed in the patent is: 13-17 parts of rhizoma cyperi, 10-15 parts of Chinese rose, 8-13 parts of artemisia anomala, 6-9 parts of bletilla striata, 8-13 parts of tuber fleeceflower stem, 5-8 parts of fructus aurantii and 6-9 parts of astragalus membranaceus.

22. The patent is named as a traditional Chinese medicine tea for treating primary dysmenorrhea, and the patent publication number is CN104840557A, and the application number is 201510291342.8, and the formula disclosed in the patent is as follows: 1-5 parts of clove flower, 1-5 parts of peach flower, 1-5 parts of rose and 1-5 parts of hibiscus flower.

23. The patent name is a drug for treating primary dysmenorrhea, and the patent publication number is CN104800307A and the application number is 2015102109179, and the formula disclosed in the patent is as follows: 30-50 g of safflower, 20-30 g of red peony root, 20-30 g of cassia twig, 20-30 g of folium artemisiae argyi, 20-30 g of angelica, 30-40 g of rhizoma corydalis and 10-15 g of radix angelicae.

(II) the papers related to the treatment of primary dysmenorrhea are:

1. in 2013, 4 months, a paper "clinical analysis of primary dysmenorrhea by acupoint application of traditional Chinese medicine" published by jinhong rock in "university of Liaoning traditional Chinese medicine" published in the paper, and the observation of the clinical efficacy of primary dysmenorrhea by acupoint application of traditional Chinese medicine is disclosed in the paper. 100 cases of primary dysmenorrhea patients are treated in outpatient service in 2005-2010, 50 cases of primary dysmenorrhea patients are pasted on three acupuncture points of Qihai, Guanyuan and Shenque, and 50 cases of primary dysmenorrhea patients are pasted only on corresponding acupuncture points without using traditional Chinese medicines. After 2 treatment courses, the effective rate of the traditional Chinese medicine acupoint application for treating the dysmenorrhea is 98 percent, and the effective rate of the traditional Chinese medicine acupoint application-free treatment for the dysmenorrhea is 32 percent. The Chinese medicine acupoint application has definite curative effect on primary dysmenorrhea, and is safe and reliable.

2. In 4 months 2012, Wang Li Chun published a paper "clinical observation of treating primary dysmenorrhea with traditional Chinese medicine" in the Chinese medical guideline "and the paper discloses observation of the treatment effect of traditional Chinese medicine treatment on dysmenorrhea symptoms of patients with primary dysmenorrhea. 68 cases of primary dysmenorrheal patients who are self-collected by the first people hospital in the city of the peony river are selected, and are classified into a cold accumulation uterus type, a qi-blood deficiency type, a qi stagnation and blood stasis type, a liver and kidney deficiency type and a yang deficiency and internal cold type according to the differentiation of symptoms and signs of the traditional Chinese medicine, and the treatment is carried out by adopting an oral stasis-removing decoction plus or minus decoction. After 3 treatment courses of 68 patients with primary dysmenorrhea, 48 patients (70.58%) were cured, wherein 12 patients (17.65%) were cured in the 1 st treatment course, 25 patients (36.76%) were cured in the 2 nd treatment course, and 11 patients (16.18%) were cured in the 3 rd treatment course; 19 cases (27.94%) were improved, 1 case (1.47%) was not cured, and the total effective rate was (98.53%). The stasis-removing decoction has ideal effect on treating dysmenorrhea symptoms of patients with primary dysmenorrhea and has high safety.

3. In 2011, 3 months, Leli published a paper 120 cases of primary dysmenorrhea treated by traditional Chinese medicines in the university of traditional Chinese medicine of Hubei, and the paper discloses that the primary dysmenorrhea refers to the condition that women have abdominal pain, waist soreness and lower abdomen pain which are spastic before and after menstruation or menstrual period, or have discomfort such as hypodynamia, dizziness, nausea, vomiting, diarrhea and the like, which affect work and study and are not accompanied by pelvic organ degeneration. It is common disease affecting women's normal work, study and quality of life, and is mostly seen in adolescent girls and unmarried young women. Epidemiological studies have seen morbidity rates of 20% to 90%.

4. In 1 month in 2014, Li Zhen published on the journal of rare diseases in the thesis of clinical research on treating primary dysmenorrhea by combining cassia twig and tuckahoe capsules with an angong progesterone tablet, and clinically used traditional Chinese medicines of cassia twig and tuckahoe capsules with western medicines of angong progesterone tablet are disclosed in the thesis to treat primary dysmenorrhea, and the clinical curative effect of the traditional Chinese medicines is discussed. 150 cases of primary dysmenorrhea were reviewed at outpatient clinic visits during the period from 2011 6 months to 2013 6 months. The test pieces were divided into observation groups and control groups by a random grouping method, and 75 cases were added to each group. The control group patients are treated clinically by adopting western medicine of medroxyprogesterone tablets; the patients in the observation group are treated by combining the traditional Chinese medicine cassia twig and poria cocos capsules on the basis of the control group. After 3 months of treatment, the treatment effect of both groups of patients was observed and analyzed. The disease conditions of two groups of patients are controlled, wherein the total effective rate of the observation group is up to 97.3 percent (73/75), the significance is greater than 73.3 percent (55/75) of the control group, and P is less than 0.05; the pain degree of the observation group is (2.2 +/-1.4), the significance is lower than that of the control group (4.2 +/-1.6), and the P is less than 0.05; the pain time of the observation group is (2.7 +/-1.1) h, the significance is lower than that of the control group (8.9 +/-2.4) h, and the P is less than 0.05. The combination of the traditional Chinese medicine cassia twig and poria cocos capsule and the western medicine medroxyprogesterone tablet can effectively treat primary dysmenorrhea clinically, reduce the pain degree and pain time of patients, and is worthy of clinical popularization.

5. In 4 months in 2010, xu jing, published on a journal of external treatment of traditional Chinese medicine and moxibustion 42 cases, the article discloses that dysmenorrhea is a common disease and frequently encountered disease in gynecology clinic, and generally refers to abdominal pain and discomfort before and after menstruation or during menstruation, and the serious degree can affect the quality of life and work. Since 2003, 42 cases of dysmenorrhea were treated by oral administration of traditional Chinese medicines and moxibustion, and the curative effect was satisfactory.

6. In 2009, 6 months, jinyuqin, phyllojiang and chi lifang published a paper "observation of clinical efficacy of decoction-free Chinese medicinal granule for treating dysmenorrhea" in the journal of Chinese traditional medicine and pharmacology, and the text discloses evaluation of clinical efficacy of decoction-free Chinese medicinal granule. 150 cases of dysmenorrhea patients are classified into two types of deficiency and excess, 5 types of syndromes according to the traditional Chinese medical diagnosis, primary dysmenorrhea and secondary dysmenorrhea according to the western medical diagnosis, and 3 degrees according to the degree of dysmenorrhea. Then the decoction-free traditional Chinese medicine particles are used for treatment, the 3-month menstruation period is 1 course of treatment, and the curative effect observation is carried out. The effective rate of the dysmenorrhea is 93%, no recurrence is caused, and no side effect is caused. Clinical observation of patients with dysmenorrhea shows that the curative effects of cold congealing of intercellular type and primary dysmenorrhea are the most remarkable. The decoction-free traditional Chinese medicine granules have definite curative effect and provide basis for improving the dosage form of the traditional Chinese medicine and popularizing and applying the decoction-free traditional Chinese medicine granules.

7. In 2005, songzhen and chenchenshuli published a paper "clinical observation of tongjingning granules for treating primary dysmenorrhea" in journal of chinese and western medicine combination ", and the exploring text discloses the action mechanism of the traditional Chinese medicine tongjingning granules for treating Primary Dysmenorrhea (PD). 120 cases of PD patients are treated by the Tongjingning granules, 40 cases of western medicine aspirin treatment are set as a control, and the content of mid-luteal and terminal serum estrogen (E2), progestogen (P), mid-luteal, menstrual Endothelin (ET) and Calcitonin Gene Related Peptide (CGRP) of partial patients before and after treatment is detected. The clinical curative effect of the treatment group is better than that of the control group (P < 0.01); the cure rate and the improvement rate of main symptoms of severe and moderate PD patients are better than those of a control group (P < 0.01). E2 level and ET content in the treatment group are obviously reduced after treatment (P is less than 0.01) compared with those before treatment; p, CGRP content was significantly increased (P < 0.01). The Chinese medicinal granule for treating dysmenorrhea can regulate female progestogen, ET and CGRP; and has the advantages of regulating spirit and emotion, promoting the balance of the environment in the organism and consolidating the curative effect.

In the prior art, a great number of compound traditional Chinese medicines for treating primary dysmenorrhea exist, but the large number of the compound traditional Chinese medicines is basically a large compound traditional Chinese medicine, the small number of the compound traditional Chinese medicines is prepared from 5 to 8 medicinal raw materials, the large number of the compound traditional Chinese medicines is prepared from 20 to 30 medicinal raw materials, the treatment effect of the large compound traditional Chinese medicine is not good, the effective active ingredients of the large compound traditional Chinese medicine are not clear, the development direction of modern traditional Chinese medicine pharmacy is not met, and currently, a medicine which is used for effectively treating primary dysmenorrhea, has definite curative effect, simple compatibility and low cost and has basically definite effective active ingredients is urgently needed.

The inventor of the patent is a scientific research staff at the front line of the Jia Musi college of the university of traditional Chinese medicine in Heilongjiang, and the inventor of the patent obtains the innovative technology of the invention after years of intensive research. Meanwhile, the innovative technology of the patent has reached an intention cooperative agreement with several pharmaceutical enterprises in Heilongjiang province and Jiangsu province, and the prior preparation work is done for developing new drugs by the innovative technology of the patent.

Disclosure of Invention

The invention aims to solve the technical problem of providing a medicament which can effectively treat primary dysmenorrhea, has definite curative effect, simple compatibility and low cost; furthermore, the invention provides a preparation method of the medicine for treating primary dysmenorrhea, and the medicine prepared by the preparation method has high extraction efficiency of active ingredients, high content of the active ingredients, low impurity content, reliable curative effect and high safety.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the medicine for treating primary dysmenorrhea is prepared from the following medicinal raw materials in parts by weight: 1-3 parts of angelica sinensis, 1-2 parts of white peony root and 1-2 parts of silybum marianum.

The medicine is preferably prepared from the following medicinal raw materials in parts by weight: 2 parts of angelica, 1 part of white peony root and 1 part of milk thistle.

A preparation method of a medicine for treating primary dysmenorrhea comprises the following raw materials in parts by weight: 1-3 parts of angelica sinensis, 1-2 parts of white peony root and 1-2 parts of silybum marianum, and the preparation method comprises the following steps:

(1) extracting radix Angelicae sinensis, radix Paeoniae alba, and herba Silybi Mariani by steam distillation, and collecting volatile oil; extracting the extract after steam distillation, filtering and concentrating to obtain an extract I; distilling the extracted residue with steam for use;

(2) taking the dregs of the decoction obtained in the step (1), adding 20-40 times of bionic extraction solvent, heating in a water bath at 36-38 ℃, stirring and extracting for 2-4 h, centrifuging for 30-40 min at the centrifugation speed of 2500-3000 r/min, collecting supernatant, filtering the supernatant, and concentrating to obtain an extract II; the bionic extraction solvent is prepared from 0.5-0.8% of sodium chloride, 0.5-0.8% of pepsin, 0.5-0.8% of trypsin and 0.06 mol.L^-1～0.08mol·L^-1Preparing hydrochloric acid into a solution;

(3) mixing the extract I in the step (1) with the extract II in the step (2), putting the mixture into an ultrasonic-microwave reactor, adding 20-40 times of 60-70% ethanol solution, then reacting in the ultrasonic-microwave reactor, wherein the ultrasonic power is 1200-1400W, the microwave power is 360-380W, the reaction time is 40-50 min, the reaction temperature is 30-40 ℃, filtering the reacted liquid medicine, and concentrating the filtrate to obtain an extract III;

(4) taking the extract III in the step (3), dispersing the extract III by using purified water, passing the extract III through an H-30 type macroporous adsorption resin column, washing 6-10 column volumes by using water, discarding eluent, washing 4-7 column volumes by using 10-20% methanol, discarding eluent, washing 10-15 column volumes by using 60-70% methanol, collecting eluent, and filtering to obtain filtrate IV;

(5) taking the filtrate IV obtained in the step (4), then loading the filtrate on an MCI resin column, washing 6-10 column volumes with water, discarding the eluent, washing 4-7 column volumes with 10-20% methanol, discarding the eluent, finally washing 10-15 column volumes with 60-70% methanol, collecting the eluent, filtering, and concentrating the filtrate to obtain an extract V;

(6) and (3) combining the extract V in the step (5) with the volatile oil in the step (1), uniformly mixing, and freeze-drying to obtain the extract.

The medicine is prepared by the following medicinal raw materials in parts by weight: 2 parts of angelica, 1 part of white peony root and 1 part of silybum marianum; the preferable preparation method comprises the following steps:

(2) adding 30 times of bionic extraction solvent into the residue obtained in the step (1), heating in water bath at 37 deg.C, stirring for 3 hr, centrifuging for 35min at 2800r/min, collecting supernatant, filtering the supernatant, and concentrating to obtain extract II; the bionic solvent is composed of 0.6% sodium chloride, 0.7% pepsin, 0.6% trypsin and 0.07 mol.L^-1Preparing hydrochloric acid into a solution;

(3) mixing the extract I in the step (1) with the extract II in the step (2), putting the mixture into an ultrasonic-microwave reactor, adding 30 times of 65% ethanol solution, then reacting in the ultrasonic-microwave reactor, wherein the ultrasonic power is 1300W, the microwave power is 370W, the reaction time is 45min, the reaction temperature is 35 ℃, filtering the reacted liquid medicine, and concentrating the filtrate to obtain an extract III;

(4) taking the extract III in the step (3), dispersing the extract III by using purified water, passing the extract III through an H-30 type macroporous adsorption resin column, washing 8 column volumes by using water, discarding eluent, washing 6 column volumes by using 15% methanol, discarding eluent, washing 12 column volumes by using 65% methanol, collecting eluent, and filtering to obtain filtrate IV;

(5) taking the filtrate IV of the step (4), then loading on an MCI resin column, washing 8 column volumes with water, discarding the eluent, washing 6 column volumes with 15% methanol, discarding the eluent, finally washing 12 column volumes with 65% methanol, collecting the eluent, filtering, concentrating the filtrate to obtain an extract V;

The medicine is prepared into oral preparation by adopting a conventional pharmaceutical method in traditional Chinese medicine pharmacy.

The oral preparation is tablet, pill, capsule, powder or oral liquid.

A detection method of a medicine for treating primary dysmenorrhea comprises the following raw materials in parts by weight: 1-3 parts of angelica sinensis, 1-2 parts of white peony root and 1-2 parts of silybum marianum, and the preparation method comprises the following steps:

(6) mixing the extract V of step (5) with the volatile oil of step (1), mixing, and freeze drying to obtain the final product;

the method adopts high performance liquid chromatography to simultaneously determine the content of silydianin, gallic acid, ferulic acid and vanillic acid in the medicine, and comprises the following steps:

(1) chromatographic conditions are as follows: by C₁₈Chromatographic column, specification: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-1% glacial acetic acid solution, gradient elution, the elution order is: from 0min to 15min, acetonitrile was maintained at 10%, and 1% glacial acetic acid solution was maintained at 90%; from 16min to 35min, acetonitrile linearly rose from 10% to 30%, and 1% glacial acetic acid solution linearly fell from 90% to 70%; from 36min to 40min, acetonitrile linearly rose from 30% to 40%, and 1% glacial acetic acid solution linearly rose from 70% to 60%; from 41min to 50min, acetonitrile linearly rose from 40% to 45%, and 1% glacial acetic acid solution linearly rose from 60% to 55%; from 51min to 60min, acetonitrile linearly rose from 45% to 55%, and 1% glacial acetic acid solution linearly rose from 55% to 45%; flow rate of 0.5-1.5 mL/min^-1(ii) a The column temperature is 35-40 ℃; detecting the wavelength of 255-260 nm; the sample injection amount is 5-20 mu L;

(2) preparation of mixed control solution: weighing silydianin, ferulic acid, gallic acid and vanillic acid reference substances respectively, diluting with methanol to obtain a mixed reference substance solution containing silydianin 60-70 μ g, ferulic acid 15-20 μ g, gallic acid 10-20 μ g and vanillic acid 15-20 μ g respectively per 1mL, and mixing uniformly to obtain a mixed reference substance solution;

(3) preparation of a test solution: taking a sample, grinding, weighing 1.00g, placing in a conical flask with a plug, precisely adding 15-30 mL of methanol, weighing, carrying out ultrasonic treatment for 20-30 min at power of 35-45 kHz and 350-450W, supplementing methanol to reduce weight, filtering, and taking a subsequent filtrate to obtain a sample solution;

(4) and (3) determination: precisely sucking 5-20 μ L of the mixed reference solution and the sample solution respectively, and injecting into a high performance liquid chromatograph for measurement.

The detection method of the medicine comprises the following steps of: 2 parts of angelica, 1 part of white peony root and 1 part of silybum marianum; the preparation method comprises the following steps:

the method adopts high performance liquid chromatography to simultaneously determine the content of silydianin, gallic acid, ferulic acid and vanillic acid in the medicine, and preferably comprises the following steps:

(1) chromatographic conditions are as follows: by C₁₈Chromatographic column, specification: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-1% glacial acetic acid solution, gradient elution, the elution order is: from 0min to 15min, acetonitrile was maintained at 10%, and 1% glacial acetic acid solution was maintained at 90%; from 16min to 35min, acetonitrile linearly rose from 10% to 30%, and 1% glacial acetic acid solution linearly fell from 90% to 70%; from 36min to 40min, acetonitrile linearly rose from 30% to 40%, and 1% glacial acetic acid solution linearly rose from 70% to 60%; from 41min to 50min, acetonitrile linearly rose from 40% to 45%, and 1% glacial acetic acid solution linearly rose from 60% to 55%; from 51min to 60min, acetonitrile linearly rose from 45% to 55%, and 1% glacial acetic acid solution linearly rose from 55% to 45%; flow rate 1.0 mL/min^-1(ii) a The column temperature is 38 ℃; the detection wavelength is 257 nm; the sample volume is 10 mu L;

(2) preparation of mixed control solution: weighing silydianin, ferulic acid, gallic acid and vanillic acid reference substances respectively, diluting with methanol to obtain mixed reference substance solution containing silydianin 65 μ g, ferulic acid 20 μ g, gallic acid 15 μ g and vanillic acid 20 μ g per 1mL respectively, and mixing well to obtain mixed reference substance solution;

(3) preparation of a test solution: taking a sample, grinding, weighing 1.00g, placing in a conical flask with a plug, adding 25mL of methanol precisely, weighing, performing ultrasonic treatment for 25min under the power of 400W and 40kHz, supplementing methanol to reduce weight, filtering, and taking a subsequent filtrate to obtain a test solution;

(4) and (3) determination: precisely sucking 10 μ L of each of the mixed reference solution and sample solution, injecting into high performance liquid chromatograph, and measuring.

the content of beta-ocimene, alpha-pinene and myrtanal is determined by adopting a gas chromatography, and the method comprises the following steps:

(1) chromatographic conditions are as follows: a chromatographic column: HP-5 capillary column, temperature programming: the initial temperature is 40 deg.C, and the temperature is maintained for 5min at 8 deg.C/min^-1Heating to 120 ℃ at the rate of (1), and keeping for 8 min; then at 12 ℃ under min^-1The temperature is increased to 180 ℃ at the speed of (1) and kept for 5 min; then at 15 ℃ for min^-1The temperature is increased to 270 ℃ at the speed rate, and the temperature is kept for 5 min; the temperature of a sample inlet is 210-230 ℃; a FID detector; the temperature of the detector is 260-280 ℃; the volume flow rate is 0.5-2.0 mL/min^-1(ii) a The sample introduction amount is 1-10 mu L, the split flow sample introduction is carried out, and the split flow ratio is 7-9: 3-1;

(2) preparation of mixed control solution: taking a proper amount of beta-ocimene, alpha-pinene and myrtenal reference substances, precisely weighing, and adding trichloromethane to prepare the product containing 0.5-2.0 mg/mL of beta-ocimene^-1The alpha-pinene content is 0.5-2.0 mg/mL^-1And contains myrtenal 0.5-2.0 mg/mL^-1Mixed reference solution of (1);

(3) preparing a test solution: weighing 0.20-1.0 g of the product, placing the weighed product in a conical flask with a plug, precisely adding 15-30 mL of trichloromethane, sealing the plug, weighing, carrying out ultrasonic treatment for 10-20 min, cooling, weighing, supplementing the lost weight with the trichloromethane, shaking up, filtering with a 0.45 mu m filter membrane, and taking the subsequent filtrate to obtain the test solution.

(4) And (3) determination: precisely sucking 1-10 μ L of each of the mixed reference solution and the sample solution, injecting into a gas chromatograph, and measuring.

the content of beta-ocimene, alpha-pinene and myrtanal is determined by adopting a gas chromatography, and the preferable steps are as follows:

(1) chromatographic conditions are as follows: a chromatographic column: HP-5 capillary column, specification: 30m × 0.32mm, 0.25 μm; temperature programming: the initial temperature is 40 deg.C, and the temperature is maintained for 5min at 8 deg.C/min^-1Heating to 120 ℃ at the rate of (1), and keeping for 8 min; then at 12 ℃ min^-1The temperature is increased to 180 ℃ at the speed of (1) and kept for 5 min; then at 15 ℃ for min^-1The temperature is increased to 270 ℃ at the speed rate, and the temperature is kept for 5 min; the temperature of a sample inlet is 220 ℃; a FID detector; the temperature of the detector is 270 ℃; the volume flow rate is 1.0 mL/min^-1(ii) a The sample introduction amount is 5 mu L, the split sample introduction is carried out, and the split ratio is 8: 2;

(2) preparation of mixed control solution: taking a proper amount of beta-ocimene, alpha-pinene and myrtenal reference substances, precisely weighing, and adding trichloromethane to obtain a product containing 1.055 mg.mL of beta-ocimene^-1The content of alpha-pinene is 1.115 mg/mL^-11.225 mg/mL of myrtenal^-1Mixed reference solution of (1);

(3) preparing a test solution: weighing 0.60g of the product, weighing, placing in a conical flask with a plug, precisely adding 20mL of trichloromethane, sealing, weighing, ultrasonically treating for 15min, cooling, weighing, supplementing the weight loss with trichloromethane, shaking, filtering with a 0.45 μm filter membrane, and collecting the subsequent filtrate to obtain the test solution.

(4) And (3) determination: precisely sucking 5 μ L of each of the mixed reference solution and sample solution, injecting into gas chromatograph, and measuring.

The prescription is a small compound traditional Chinese medicine, and the prescription is as follows: chinese angelica root, sweet, pungent and warm; it enters liver, heart and spleen meridians; has effects of replenishing blood, promoting blood circulation, regulating menstruation, relieving pain, and loosening bowel to relieve constipation. White peony root, bitter, sour, slightly cold; entering liver and spleen meridians; has the effects of nourishing blood, astringing yin, tonifying but not greasy, softening liver, relieving middle energizer, relieving pain, astringing sweat and the like. Silybum marianum, bitter and cool. It enters liver and gallbladder meridians; has the effects of clearing away heat and toxic materials, soothing liver, and promoting function of gallbladder; can be used for treating damp-heat in liver and gallbladder, hypochondriac pain, and jaundice. The traditional Chinese medicines are used together to nourish the liver and tonify the spleen, strengthen the spleen and tonify the liver, sooth the liver and replenish qi, regulate menstruation and relieve pain, promote blood circulation and stop bleeding, the spleen is the acquired root, the qi and blood are generated, the qi and blood are insufficient and are generated for a plurality of diseases, the qi and blood are sufficient and are eliminated for a plurality of diseases, and the spleen mainly absorbs the palm nutrition and generates the qi and the blood by secreting various enzymes (the enzymes are enzymes, commonly called as catalysts, including liver, gall bladder and lipid enzymes and the like. Silybum marianum which belongs to wood and can dredge soil is introduced into the formula, so that the silybum marianum has the effects of tonifying spleen and tonifying spleen, can healthily absorb nutrition and transform qi and blood, is also called as obstruction leading pain in traditional Chinese medicine, has the effects of nourishing liver and tonifying spleen, tonifying spleen and liver, can promote transportation and transformation of qi and blood, soothe liver, tonify qi, regulate menstruation, relieve pain, promote blood circulation and stop bleeding. Has good treatment effect on primary dysmenorrhea, and is an effective prescription for radically treating primary dysmenorrhea.

In order to make the technical means, technical features, achievement objects and effects of the invention easy to understand, the following experiments or experiments are used for further explaining the technical scheme of the invention.

Experiment one: drug screening experimental study for treating primary dysmenorrhea

1 instruments and materials

1.1 instruments

A German IFP full-automatic enzyme labeling apparatus (RT2100C) available from Beijing Boohou Shirdde Biotech, Inc.; BL-420E biological function experimental system, purchase in Anhui Huaihe Zhenghua biological instruments and equipments Limited company of Anhui Huai.

1.2 drugs, reagents

1.2.1 test sample G1: prescription: 500g of angelica, 250g of white peony root and 250g of silybum marianum;

the preparation method comprises the following steps: (1) adding 30 times of bionic extraction solvent into radix Angelicae sinensis, radix Paeoniae alba and herba Silybi Mariani, heating in water bath at 37 deg.C, stirring and extracting for 3 hr, centrifuging for 35min at 2800r/min, collecting supernatant, filtering supernatant, and concentrating to obtain extract I; the bionic solvent is composed of 0.6% sodium chloride, 0.7% pepsin, 0.6% trypsin and 0.07 mol.L^-1Preparing hydrochloric acid into a solution;

(2) and (2) taking the extract I in the step (1), dispersing the extract in purified water, passing through an H-30 type macroporous adsorption resin column, washing 8 column volumes with water, discarding the eluent, washing 6 column volumes with 15% methanol, discarding the eluent, washing 12 column volumes with 65% methanol, collecting the eluent, filtering, concentrating the filtrate, and freeze-drying to obtain a tested sample G1.

1.2.2 test sample G2: prescription: 500g of angelica, 250g of white peony root and 250g of silybum marianum;

the preparation method comprises the following steps: (1) putting angelica, white paeony root and silybum marianum into an ultrasonic-microwave reactor, adding 30 times of 65% ethanol solution, then reacting in the ultrasonic-microwave reactor, wherein the ultrasonic power is 1300W, the microwave power is 370W, the reaction time is 45min, the reaction temperature is 35 ℃, filtering the reacted liquid medicine, and concentrating the filtrate to obtain an extract I;

(2) and (2) dispersing the extract I obtained in the step (1) by using purified water, then loading the dispersed extract I on an MCI resin column, washing 8 column volumes by using water, discarding eluent, washing 6 column volumes by using 15% methanol, discarding eluent, finally washing 12 column volumes by using 65% methanol, collecting eluent, filtering, concentrating filtrate, and freeze-drying to obtain a tested sample G2.

1.2.3 test sample G3: prescription: 500g of angelica, 250g of white peony root and 250g of silybum marianum;

the preparation method comprises the following steps: (1) extracting radix Angelicae sinensis, radix Paeoniae alba, and herba Silybi Mariani by steam distillation, and collecting volatile oil; extracting the extract after steam distillation, filtering and concentrating to obtain an extract I; distilling the extracted residue with steam for use;

1.2.4 Aspirin tablets, produced by Harbin group pharmaceutical manufacturing, approved article No.: national standard characters H23021185, specification: 0.3 g;

1.2.5 Guizhi Fuling Capsule, manufactured by Jiangsu Kangyuan pharmaceutical industry GmbH, approved article No.: national standard of medicine Z10950005, specification: each granule is filled with 0.31 g;

1.2.6 Diethylstilbestrol injection, manufactured by Tianjin Jinyao pharmaceutical Co., Ltd, approved No.: national standard of medicine H12020536, specification: 0.5mg in 1 ml.

1.2.7 oxytocin injection, manufactured by beijing selagingpharmacy ltd, approved article No.: chinese medicine standard character H11020363, dosage form: injection (small volume injection), specification: 1ml:10 units.

1.2.8 prostaglandin F_2αEnzyme-linked immunoassay kit, purchased from the company of hollow reagents (Shanghai); prostaglandin E₂An enzyme-linked immunoassay kit is purchased from Shanghai Hengyuan Biotech, Inc.

1.3 Experimental animals

The SPF-grade SD rat weighs 190-210 g, is female, has a qualification number of P00102013, and is provided by the drug safety evaluation center of Heilongjiang university of traditional Chinese medicine. The experimental animal is fed adaptively for 1 week before experiment at 20 +/-2 deg.C under 12 hr of light and 40% humidity, and fed freely in single cage to eliminate the influence of diet and environment on experimental animal.

2 method

2.1 Effect on oxytocin model rats

The rats were taken and randomly divided into 7 groups of 10 rats each, which were: blank control group, model control group, aspirin group, cassia twig and poria cocos capsule group, test sample G1, test sample G2 and test sample G3. The injection of diethylstilbestrol was injected subcutaneously for 14 days continuously, 1 time daily, 0.8mg each time. The rats were administered with 0.03 g/kg of aspirin on day 5 after diethylstilbestrol injection^-1The aspirin and the cassia twig and tuckahoe capsule group are administered with 2.0 g/kg^-1The cassia twig and tuckahoe capsule is administered with 2.0 g.kg in a test sample G1 group^-1Test sample G1 (1), test sample G2 group (2.0G/kg)^-1Test sample G2 (1), test sample G3 group (2.0G/kg)^-1The test sample G3 contained in (1) was 10 mL/kg^-1The blank control group and the model control group are respectively given physiological saline with equal volume for 10 days continuously for 1 time per day. 10d, injecting diethylstilbestrol for 12h for the last time, and injecting oxytocin into abdominal cavity 2U/patientThe pain response of the rats within 40min is observed, and the writhing response is taken as the index of strong uterine contraction, including the writhing incidence, the incubation period and the times.

The rate of writhing is the number of writhing animals/10 × 100%.

Observing for 40min, killing rat, stripping uterus, weighing uterus tissue 0.5g, adding physiological saline 1.0mL, homogenizing, centrifuging, collecting supernatant, and treating with prostaglandin F_2αEnzyme-linked immunoassay kit and prostaglandin PGE₂And (4) carrying out content determination by using the enzyme-linked immunoassay kit.

2.2 statistical methods

The data results obtained from the experiment were statistically processed using statistical software SPSS 11.0.

3 results

3.1 Effect on oxytocin-induced dysmenorrhea model rats

Compared with a blank control group, the rate of writhing of rats in the model control group is increased (P is less than 0.01), and the latency period of writhing is shortened (P is less than 0.01); the number of wriggling increases within 30min (P is less than 0.01); uterine tissue PGF_2αIncreased content (P <0.01), PGE₂The content is reduced (P is less than 0.05), PGF_2αThe ratio/PGE 2 increased (P < 0.01). Compared with the model control group, the tested sample group G3, the cassia twig and poria cocos capsule group and the aspirin group have obvious inhibition effects on the writhing latency period of rats caused by oxytocin and the writhing frequency within 30min (P is less than 0.01), and can reduce PGF_2α/PGE₂The ratio (P < 0.01); the tested samples G3 group, the cassia twig tuckahoe capsule group and the aspirin group can reduce PGF in rat endometrium_2αThe content (P is less than 0.05 or 0.01) of the compound can increase PGE in rat endometrium₂The content (P is less than 0.05). The test sample group G1 and the test sample group G2 also had a certain effect, but had no significant difference from the model control group. See tables 1 and 2.

TABLE 1 Effect on oxytocin-induced pain and writhing response in rats: (

n＝10)

Note: comparison with model group^*P＜0.05，^**P is less than 0.01; comparison with blank control group^#P＜0.05，^##P＜0.01。

TABLE 2 PGF for rat uterine tissue_2αAnd PGE₂Influence of (A), (B)

n＝10)

4 conclusion

Experiments show that the test sample G3 can effectively inhibit the writhing incidence rate of a dysmenorrheal model rat, effectively prolong the latency period of writhing, and effectively reduce the writhing frequency within 30 min. Test sample G3 is able to antagonize the painful response of oxytocin-induced uterine muscle spasm and reduce PGF in rat endometrium_2αIncreasing PGE in the endometrium₂The content of (a). The test sample G3 shows that the test sample G3 has good treatment effect on experimental primary dysmenorrhea.

Experiment two: the contents of silydianin, gallic acid, ferulic acid and vanillic acid in the medicine are simultaneously determined by adopting a high performance liquid chromatography

The early-stage research shows that the silydianin contained in the silybum marianum, the gallic acid contained in the white paeony root, the ferulic acid contained in the angelica and the vanillic acid are the main active ingredients of the medicament for treating the dysmenorrheal, and the inventor establishes a detection method for simultaneously measuring the contents of the silydianin, the gallic acid, the ferulic acid and the vanillic acid at one time by a high performance liquid chromatography through repeated experiments, and researches are as follows.

1 materials and reagents

Shimadzu 20-AD high performance liquid chromatograph (Shimadzu, japan Shimadzu, including quaternary pump, on-line degasser, autosampler, column oven, DAD detector, 2010 chromatography workstation);

electronic analytical balance (Shanghai catalpa electronic technologies, Inc.);

an ultrasonic cleaner (Shenzhen Kewei Da ultrasonic equipment Limited).

Silybinin reference (Shanghai Yaji Biotechnology Co., Ltd., batch No. 102012-20160325);

gallic acid control (Shanghai Rui Qi Life sciences Co., Ltd., batch No. 111202-20160115);

cimicifugal element reference (VICKodd Biotechnology, Inc., Sichuan, Lot 121115-20160220);

vanillic acid control (Shanghai Hotan Biotechnology Co., Ltd., batch No. 110326-20151228);

acetonitrile is chromatographically pure; the ethanol and the glacial acetic acid are analytically pure; the water is ultrapure water.

The medicine of the invention is: prescription: 500g of angelica, 250g of white peony root and 250g of silybum marianum;

2 methods and results

2.1 preparation of the solution

2.1.1 preparation of Mixed control solutions

Weighing silydianin, ferulic acid, gallic acid and vanillic acid reference substances respectively, diluting with methanol to obtain mixed reference substance solution containing silydianin 65 μ g, ferulic acid 20 μ g, gallic acid 15 μ g and vanillic acid 20 μ g per 1mL respectively, and mixing well to obtain mixed reference substance solution.

2.1.2 preparation of test solutions

Taking a sample, grinding, weighing 1.00g, placing in a conical flask with a plug, adding 25mL of methanol precisely, weighing, performing ultrasonic treatment at 400W and 40kHz for 25min, supplementing methanol to reduce weight, filtering, and taking a subsequent filtrate to obtain a test solution.

2.1.3 preparation of negative test solutions

Negative test samples of the silybum marianum, the white paeony root and the Chinese angelica are respectively prepared according to the prescription amount, and the negative test sample solution is prepared according to the method provided by the invention.

2.2 chromatographic Condition and System suitability Studies

By C₁₈Chromatographic column, specification: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-1% glacial acetic acid solution, gradient elution,the elution sequence was: from 0min to 15min, acetonitrile was maintained at 10%, and 1% glacial acetic acid solution was maintained at 90%; from 16min to 35min, acetonitrile linearly rose from 10% to 30%, and 1% glacial acetic acid solution linearly fell from 90% to 70%; from 36min to 40min, acetonitrile linearly rose from 30% to 40%, and 1% glacial acetic acid solution linearly rose from 70% to 60%; from 41min to 50min, acetonitrile linearly rose from 40% to 45%, and 1% glacial acetic acid solution linearly rose from 60% to 55%; from 51min to 60min, acetonitrile linearly rose from 45% to 55%, and 1% glacial acetic acid solution linearly rose from 55% to 45%; flow rate 1.0 mL/min^-1(ii) a The column temperature is 38 ℃; the detection wavelength is 257 nm; the amount of the sample was 10. mu.L. Under the chromatographic conditions, the chromatograms of the reference solution, the test solution and the negative test solution are mixed and shown in the attached figures 1 to 5 of the specification. Other components in the test solution have little interference to the component to be tested.

2.3 examination of the Linear relationship

1.0mL, 2.0mL, 4.0mL, 6.0mL and 8.0mL of the mixed control solution are respectively put into a 10mL measuring flask and diluted to the scale by adding methanol. Respectively taking 10 μ L, injecting into high performance liquid chromatograph, eluting according to the chromatographic conditions provided by the invention, and determining the concentration X (mg. L) by each control peak area Y^-1) Performing linear regression to obtain a regression equation, which is respectively as follows:

regression equation of silidianin: y is 8.5 × 10⁶X-12568.36, r is 0.9998, and the linear range is 13.25-165.26 mg.L^-1。

Regression equation of gallic acid: y is 3.5 × 10⁷X-6254.23, r is 0.9997, and the linear range is 1.96-41.23 mg.L^-1。

Regression equation for ferulic acid: y is 3.5 × 10⁷X-3526.28, r is 0.9998, and the linear range is 2.18-44.25 mg.L^-1。

Regression equation for vanillic acid: y is 3.4 × 10⁷X +2261.88, r is 0.9996, and the linear range is 1.95-48.65 mg.L^-1。

2.4 precision test

A sample (lot No. 20160625) was taken and prepared into a test solution by the method of the present invention, and the sample was repeatedly introduced 5 times under the conditions of the present invention to determine the peak area. The RSD (n is 5) of the peak areas of silydianin, gallic acid, ferulic acid and vanillic acid are respectively 2.1%, 1.6%, 1.8% and 0.9%, which shows that the precision is good.

2.5 stability test

And (3) sampling the same sample solution for 1, 2, 4, 8, 12 and 16 hours respectively, and determining that the RSD of the peak areas of the silydianin, the gallic acid, the ferulic acid and the vanillic acid are respectively 1.6%, 1.4%, 1.3% and 0.9%, which indicates that the sample solution is stable within 16 hours.

2.6 repeatability test

Taking 6 parts of samples, each part is about 1g, precisely weighing, preparing a test solution according to the method provided by the invention, and measuring by sample injection, wherein the average mass fractions of the silydianin, the gallic acid, the ferulic acid and the vanillic acid are 1052.6, 368.5, 454.2 and 565.8 mu g.g.g.g^-1RSD is 1.5%, 1.4%, 1.6%, 1.9%.

2.7 sample application recovery test

6 portions of samples with known content, each portion is 1.0g, precisely weighed, precisely added with the same amount of mixed reference solution, measured according to the conditions provided by the invention, and the recovery rate is calculated, and the result is shown in Table 3. The result shows that the detection method provided by the invention has good accuracy.

TABLE 3 recovery of Silybin, Gallic acid, Ferulic acid, Vanilla acid (n ═ 6)

2.8 measurement of sample content

Samples of 5 batches of 2 batches of samples are taken, a sample solution is prepared according to the method provided by the invention, the peak area is measured according to the chromatographic conditions, the content is calculated according to an external standard method, and the result is shown in table 4.

TABLE 4 weight fractions (μ g) of silydianin, gallic acid, ferulic acid, vanillic acid^-1)

3 conclusion

The invention adopts high performance liquid chromatography to simultaneously determine the content of 4 index components in the medicine, and the detection method has high precision, good repeatability and stability, and can be used for quality detection of the product.

Experiment three: gas chromatography is used for measuring the contents of beta-ocimene, alpha-pinene and myrtenal in the medicine

Earlier researches show that beta-ocimene and alpha-pinene contained in angelica sinensis and myrtenal contained in radix paeoniae alba are main active ingredients of the medicine for treating dysmenorrhea, and the inventor establishes a detection method for simultaneously determining the contents of the beta-ocimene, the alpha-pinene and the myrtenal in the medicine at one time through a gas chromatography through repeated experiments, and researches are as follows.

1 instruments and materials

1.1 instruments

A gas chromatograph; HP-5 capillary column, specification: 30m × 0.32mm, 0.25 μm; electronic analytical balance (shanghai zhuojing electronics technologies ltd); ultrasonic cleaning apparatus (Ningbo Bor ultrasonic Equipment Co., Ltd.).

1.2 materials

Beta-ocimene control (purchased from Guangdong Wengjiang chemical reagent Co., Ltd., lot number: 101205-20160223, content 99.8%), alpha-pinene control (purchased from Shanghai ancient biology Co., Ltd., lot number: 111014-20160325, content 99.9%), and myrtenal control (purchased from Shanghai Shuzo biotechnology Co., Ltd., lot number: 111230-20160219, content 99.9%).

The medicine of the invention is: the medicine of the invention is: prescription: 500g of angelica, 250g of white peony root and 250g of silybum marianum;

Trichloromethane (analytically pure, available from Shanghai Lu chemical Co., Ltd.).

2 method

2.1 chromatographic conditions

A chromatographic column: HP-5 capillary column, specification: 30m × 0.32mm, 0.25 μm; temperature programming: the initial temperature is 40 deg.C, and the temperature is maintained for 5min at 8 deg.C/min^-1Heating to 120 ℃ at the rate of (1), and keeping for 8 min; then at 12 ℃ min^-1OfHeating to 180 deg.C, and maintaining for 5 min; then at 15 ℃ for min^-1The temperature is increased to 270 ℃ at the speed rate, and the temperature is kept for 5 min; the temperature of a sample inlet is 220 ℃; a FID detector; the temperature of the detector is 270 ℃; the volume flow rate is 1.0 mL/min^-1(ii) a The sample introduction amount is 5 mu L, the split sample introduction is carried out, and the split ratio is 8: 2.

2.2 preparation of Mixed control solutions

Taking a proper amount of beta-ocimene, alpha-pinene and myrtenal reference substances, precisely weighing, and adding trichloromethane to obtain a product containing 1.055 mg.mL of beta-ocimene^-1The content of alpha-pinene is 1.115 mg/mL^-11.225 mg/mL of myrtenal^-1Mixed control solution of (4).

2.3 preparation of test solutions

Weighing 0.60g of the product, weighing, placing in a conical flask with a plug, precisely adding 20mL of trichloromethane, sealing, weighing, ultrasonically treating for 15min, cooling, weighing, supplementing the weight loss with trichloromethane, shaking, filtering with a 0.45 μm filter membrane, and collecting the subsequent filtrate to obtain the test solution.

2.4 negative sample solution preparation

By referring to the preparation method of the medicine, the negative samples lacking angelica and white peony root are prepared. Under the chromatographic conditions, respectively and precisely sucking 5 mu L of the mixed reference solution, the test solution and the negative sample solution respectively, injecting the solutions into a gas chromatograph, and recording a gas chromatogram.

The experimental result shows that the separation degrees of beta-ocimene, alpha-pinene and myrtanal and adjacent chromatographic peaks in the chromatogram of the test solution are all larger than 1.5, and a negative sample has no interference. See the attached figures 6, 7 and 8 in the specification.

2.5 Linear test

Accurately sucking appropriate amount of mixed reference solution, and adding chloroform to obtain solutions with concentration of 1.8-180.0 μ g/mL^-1Mixed control solution of (4). In terms of concentration (μ g. mL)^-1) Taking the peak area as the ordinate to perform linear regression to obtain a regression equation, a correlation coefficient and a linear range, wherein the regression equation, the correlation coefficient and the linear range are respectively as follows:

beta-ocimene regression equation: y is 2025.8X-2156.4, r is 0.9998, and the linear range is 1.954 to 592.5 mug·mL^-1；

α -pinene regression equation: y is 1965.2X-1826.4, r is 0.9999, and the linear range is 2.138-698.6 mu g/mL^-1；

Myrtenal regression equation: y is 1842.8X-2157.6, r is 0.9998, and the linear range is 2.568 to 304.6 mu g/mL^-1；

2.6 precision test

Precisely sucking the mixed reference substance solution with the concentration of 10 mu g/mL, and repeatedly injecting the sample for 5 times. The experimental results show that: the RSD value of the beta-ocimene peak area is 0.36%, the RSD value of the alpha-pinene peak area is 0.52% and the RSD value of the myrtenal peak area is 0.62%, which indicates that the precision of the instrument is good.

2.7 stability test

Precisely sucking the same sample solution, and injecting samples for 6 times at 0h, 2h, 4h, 6h, 8h and 12h respectively. The experimental results show that: the RSD of the area of beta-ocimene is 0.96 percent, the RSD of the area of alpha-pinene is 1.22 percent, and the RSD of the area of myrtenal is 1.06 percent, which shows that the test solution is basically stable within 12 hours.

2.8 repeatability test

Meanwhile, 6 parts of sample is taken, a test solution is prepared according to the method provided by the invention, and the content is measured and calculated. The experimental results show that: the RSD value of the beta-ocimene content in the sample is 0.62%, the RSD value of the alpha-pinene content is 0.58% and the RSD value of the myrtenal content in the sample is 0.56%, which shows that the repeatability of the determination method is good.

2.9 recovery test

6 portions of samples were taken, and the concentration of beta-ocimene added to each portion was 55.45. mu.g.mL^-1And the concentration of alpha-pinene is 57.80 mug.mL^-1The concentration of myrtenal is 58.75 mug. multidot.mL^-1The contents of the mixed control solutions (2) were measured by the method (1.0 mL each) to calculate the recovery rate.

The experimental results show that: the average recovery rate of the beta-ocimene is 100.26 percent, and the RSD is 0.95 percent; the average recovery rate of alpha-pinene is 100.40%, and RSD is 0.56%; the average recovery of myrtenal was 99.46% with an RSD of 0.75%. See tables 5, 6, 7.

TABLE 5 beta-Ocimum recovery test results

TABLE 6 results of alpha-pinene recovery test

TABLE 7 myrtenal recovery test results

2.10 sample measurement

5 batches of the medicine are taken, a test solution is prepared according to the method provided by the invention, and the contents of beta-ocimene, alpha-pinene and myrtenal in the test solution are measured and calculated. The results are shown in Table 8.

TABLE 8 measurement results of sample content (. mu.g. g)^-1)

3 conclusion

The method adopts gas chromatography to simultaneously determine the content of 3 index components in the medicine, has high precision, good repeatability and stability, and can be used for quality detection of the product.

Experiment four: comparative experiment for determining content of active ingredient in tested medicine prepared under different preparation methods

1 test drugs prepared under different preparation methods

1.1 test sample G1: prescription: 500g of angelica, 250g of white peony root and 250g of silybum marianum;

the preparation method comprises the following steps: (1) adding 30 times of bionic extraction solvent into radix Angelicae sinensis, radix Paeoniae alba and herba Silybi Mariani, heating in water bath at 37 deg.C, stirring and extracting for 3 hr, centrifuging for 35min at 2800r/min, collecting supernatantFiltering the supernatant, and concentrating to obtain extract I; the bionic solvent is composed of 0.6% sodium chloride, 0.7% pepsin, 0.6% trypsin and 0.07 mol.L^-1Preparing hydrochloric acid into a solution;

1.2 test sample G2: prescription: 500g of angelica, 250g of white peony root and 250g of silybum marianum;

1.3 test sample G3: prescription: 500g of angelica, 250g of white peony root and 250g of silybum marianum;

(2) adding 30 times of bionic extraction solvent into the residue obtained in the step (1), heating in water bath at 37 deg.C, stirring for 3 hr, centrifuging for 35min at 2800r/min, collecting supernatant, filtering the supernatant, and concentrating to obtain extract II; the bionic solvent is composed of 0.6% sodium chloride, 0.7% pepsin, 0.6% trypsin and 0.07 mol.L^-1Preparation of hydrochloric acidPreparing a solution;

2 measurement of

The content of active ingredients in the medicine is determined by adopting the high performance liquid chromatography and the gas chromatography provided by the invention.

3 results of measurement

The results are shown in Table 9.

TABLE 9 content of active ingredient (. mu.g. g. in test drugs prepared by different preparation methods^-1)

4 conclusion

The experimental results show that the tested drug G3 prepared by the preparation method provided by the invention contains silydianin, gallic acid, ferulic acid, vanillic acid, beta-ocimene, alpha-pinene and myrtenal which are obviously higher than those of the drugs prepared by other preparation methods, which may be the reason that the curative effect of the tested drug G3 (the drug of the invention) in treating dysmenorrhea is better than that of other drugs.

Experiment five: observation of curative effect of the medicine for treating primary dysmenorrhea

The medicine can be used for treating 18 cases of primary dysmenorrhea, and can not be used for large-scale clinical trial research due to the limitation of technical rules, and only small samples of 18 volunteers who are admitted to subsidiary hospitals of the Jia Mus college of the traditional Chinese medicine university in Heilongjiang are used for preliminary clinical trial research. 18 patients signed the book of informed consent and voluntarily received clinical trials. The study was as follows.

1 data and method

1.1 general data

18 patients with dysmenorrhea were gynecological outpatients of subsidiary hospitals of the Jia Mus college of Heilongjiang traditional Chinese medicine university, aged 23-32 years and had a course of disease of 3 months-3 years.

1.2 diagnostic criteria

The diagnosis standard of the Chinese medicine gynecology is referred to for drafting: the women have periodic lower abdominal pain as main symptoms and other discomforts in the menstrual period or 1 week before and after the menstrual period, so that the study, the work and the life are influenced, and no organic lesions of pelvic reproductive organs are caused by gynecological examination.

1.3 case inclusion criteria

(1) The diagnosis standard of primary dysmenorrheal in accordance with traditional Chinese medicine gynecology is met; (2) differentiation of syndromes according to qi stagnation and blood stasis type or congealing cold and blood stasis type; (3) the age is between 20 and 38 years old; (4) the menstrual cycle regularity; (5) no sedative, analgesic, and hormonal drugs were taken 2 weeks prior to this trial; (6) the total score of dysmenorrhea symptom score is more than or equal to 8; (7) signing the informed consent.

1.4 exclusion criteria

(1) The gynecological diseases such as pelvic inflammation, hysteromyoma and endometriosis; (2) less than 15 years of age or greater than 40 years of age; (3) pregnant or lactating women; (4) there are cardiovascular and cerebrovascular diseases, and serious diseases of liver and kidney systems.

2 method of treatment

The medicine of the invention: prescription: 500g of angelica, 250g of white peony root and 250g of silybum marianum;

(6) and (3) combining the extract V in the step (5) with the volatile oil in the step (1), uniformly mixing, adding a proper amount of starch and dextrin, granulating, freeze-drying, filling into hard capsules, and preparing into 1000 hard capsules.

The medicine is taken 2 capsules at a time, twice a day, respectively taken in the morning and at the evening, 4 capsules at a time in a week before menstruation, respectively taken in the morning and at the evening, and is not stopped during the menstrual period; one menstrual cycle is a course of treatment, and 3 courses of treatment are taken.

3 standard of therapeutic effect

3.1 cure: after the medicine is taken, the integral is recovered to 0 point, the abdominal pain and other symptoms in the menstrual period disappear, and the disease does not recur after the medicine is stopped for 3 months;

3.2, significant effect: the score after treatment is reduced to below 1/2 of the score before treatment, the abdominal pain is obviously relieved, other symptoms are improved, and the patient can insist on working without taking analgesic;

3.3 effective: the score after treatment is reduced to 1/2-3/4 of the score before treatment, the abdominal pain is relieved, other symptoms are improved, and the patient can keep working after taking the analgesic;

3.4 invalid: abdominal pain and other symptoms were unchanged.

4 results

4.1 clinical efficacy

18 patients with primary dysmenorrhea were observed, 12 cases (66.66%) were cured, 4 cases (22.22%) were significantly effective, 2 cases (11.12%) were effective, and 0 case (0%) was ineffective. The total effective rate is 100%.

The treatment period is as follows: shortest 15d and longest 60 d.

4.2 toxic side effects

Adverse reactions such as allergy, poisoning and the like do not appear in the observation period.

5 conclusion

The medicament for treating dysmenorrheal has the advantages of remarkable curative effect, small side effect and difficult relapse.

Description of the drawings:

FIG. 1 is a high performance liquid chromatogram of a mixed control solution, wherein: the peak 1 is silydianin, and the peak 2 is ferulic acid; peak 3 is gallic acid; peak No. 4 is vanillic acid.

FIG. 2 is a high performance liquid chromatogram of a test solution, wherein: the peak 1 is silydianin, and the peak 2 is ferulic acid; peak 3 is gallic acid; peak No. 4 is vanillic acid.

FIG. 3 is a high performance liquid chromatogram of a negative test solution of Silybum marianum, wherein: the peak No. 2 is ferulic acid; peak 3 is gallic acid; peak No. 4 is vanillic acid.

FIG. 4 is a high performance liquid chromatogram of a negative test solution lacking white peony root, wherein: the peak 1 is silydianin, and the peak 2 is ferulic acid; peak No. 4 is vanillic acid.

FIG. 5 is a high performance liquid chromatogram of a negative test solution lacking Angelica sinensis, wherein: the 1 st peak is silydianin, the 3 rd peak is gallic acid

FIG. 6 is a gas chromatogram of a mixed control solution, in which peak 1 is β -ocimene, peak 2 is α -pinene, and peak 3 is myrtanal.

FIG. 7 is a gas chromatogram of the sample solution, in which peak No. 1 is β -ocimene, peak No. 2 is α -pinene, and peak No. 3 is myrtanal.

FIG. 8 is a gas chromatogram of a negative sample solution.

Detailed Description

Example 1:

prescription: 500g of angelica, 250g of white peony root and 250g of silybum marianum;

(4) and (3) determination: precisely sucking 10 μ L of each of the mixed reference solution and the sample solution, and injecting into a high performance liquid chromatograph for determination;

(5) and (3) measuring results: each gram of the product contains silydianin 1035.3 μ g^-1Gallic acid 388.6 μ g^-1492.6 mug g of ferulic acid^-1Vanillic acid 614.8 mug g^-1。

(5) And (3) measuring results: the product contains beta-ocimene 241.62 μ g/g per gram^-1Alpha-pinene 322.46. mu.g.g^-1Myrtacenal 180.55 μ g^-1。

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims

1. a medicine for the treatment of primary dysmenorrhea, the medicine is made from the medicinal-flavor raw materials of the following parts by weight: 1-3 parts by weight of Angelica sinensis, 1-2 parts by weight of Radix Paeoniae Alba, 1-2 parts by weight of Silybum marianum, it is characterized in that It is, its preparation method is:

(1) get Angelica sinensis, Radix Paeoniae Alba, Silybum marianum, carry out steam distillation extraction, collect and obtain volatile oil, for subsequent use; extract after steam distillation extraction, filter, concentrate, obtain extract I; medicine after steam distillation extraction slag spare;

(2) Take the medicinal residues of step (1), add 20-40 times the amount of bionic extraction solvent, heat in a water bath at 36-38°C, stir and extract for 2-4 hours, centrifuge for 30-40 minutes, centrifugal speed 2500-3000 r/min, collect The supernatant is filtered and concentrated to obtain extract II; the above-mentioned bionic extraction solvent is composed of 0.5%-0.8% sodium chloride, 0.5%-0.8% pepsin, 0.5%-0.8% trypsin and 0.06mol ·L ^-1 ～0.08mol·L ^-1 hydrochloric acid is prepared into a solution;

(3) Mix the extract I of step (1) with the extract II of step (2), put it into an ultrasonic-microwave reactor, add 20 to 40 times the amount of 60% to 70% ethanol solution, and then put it in an ultrasonic-microwave reactor. The reaction is carried out in an ultrasonic-microwave reactor. The ultrasonic power is 1200-1400W, the microwave power is 360-380W, the reaction time is 40-50min, and the reaction temperature is 30-40℃. paste III;

(4) Take the extract III of step (3), disperse it with purified water, pass through H-30 macroporous adsorption resin column, wash with water for 6-10 column volumes, discard the eluent, and then use 10%～ Wash with 20% methanol for 4 to 7 column volumes, discard the eluent, and finally wash with 60% to 70% methanol for 10 to 15 column volumes, collect the eluent, and filter to obtain filtrate IV;

(5) Take the filtrate IV of step (4), put it on the MCI resin column, wash with water for 6-10 column volumes, discard the eluent, wash with 10%-20% methanol for 4-7 column volumes, wash The liquid is removed and then discarded, and finally washed with 60%-70% methanol for 10-15 column volumes, the eluent is collected, filtered, and the filtrate is concentrated to obtain extract V;

(6) Combine the extract V of step (5) with the volatile oil of step (1), mix well, freeze-dry, and then obtain.

2. medicine according to claim 1, this medicine is made of the medicinal raw material of following weight parts: Angelica 2 weight parts, white peony root 1 weight part, Silybum marianum 1 weight part; It is characterized in that, its preparation method is:

(2) Take the medicinal residues of step (1), add 30 times the amount of bionic extraction solvent, heat in a water bath at 37°C, stir and extract for 3 hours, centrifuge for 35 minutes at a centrifugal speed of 2800 r/min, collect the supernatant, and filter the supernatant, Concentrate to obtain extract II; the above bionic extraction solvent is prepared into a solution by 0.6% sodium chloride, 0.7% pepsin, 0.6% trypsin and 0.07mol·L ^-1 hydrochloric acid;

(3) Mix the extract I of step (1) with the extract II of step (2), put it into the ultrasonic-microwave reactor, add 30 times the amount of 65% ethanol solution, and then put it in the ultrasonic-microwave reactor In the middle reaction, the ultrasonic power is 1300W, the microwave power is 370W, the reaction time is 45min, and the reaction temperature is 35°C, the reacted medicinal solution is filtered, and the filtrate is concentrated to obtain the extract III;

(4) Take the extract III of step (3), disperse it with purified water, pass through a H-30 macroporous adsorption resin column, wash with water for 8 column volumes, discard the eluent, and then wash with 15% methanol for 6 12 column volumes, the eluent was discarded, and finally washed with 65% methanol for 12 column volumes, the eluent was collected and filtered to obtain filtrate IV;

(5) Take the filtrate IV of step (4), put it on the MCI resin column, wash with water for 8 column volumes, discard the eluent, wash with 15% methanol for 6 column volumes, discard the eluent, and finally Wash with 65% methanol for 12 column volumes, collect the eluent, filter, and concentrate the filtrate to obtain extract V;

3. medicine according to claim 1 and 2 is characterized in that, this medicine adopts conventional pharmaceutical method in traditional Chinese medicine pharmacy to make oral preparation.

4. The medicine according to claim 3, wherein the oral preparation is tablet, pill, capsule, powder or oral liquid.

5. A detection method for a medicine for the treatment of primary dysmenorrhea, the medicine is made from the following medicinal-flavor raw materials in parts by weight: 1-3 parts by weight of Angelica sinensis, 1-2 parts by weight of white peony root, 1-2 parts by weight of milk thistle , its preparation method is:

(6) combine the extract V of step (5) with the volatile oil of step (1), mix well, freeze-dry, and get final product;

It is characterized in that, adopting high performance liquid chromatography to simultaneously measure the content of silymarin, gallic acid, ferulic acid and vanillic acid in the medicine, and the steps are as follows:

(1) Chromatographic conditions: C ₁₈ chromatographic column, size: 4.6mm×250mm, 5μm; mobile phase: acetonitrile-1% glacial acetic acid solution, gradient elution, elution sequence: from 0 min to 15 min, acetonitrile was kept at 10%, 1% glacial acetic acid solution kept at 90%; from 16min to 35min, acetonitrile linearly increased from 10% to 30%, 1% glacial acetic acid solution decreased linearly from 90% to 70%; from 36min to 40min , acetonitrile increased linearly from 30% to 40%, 1% glacial acetic acid solution decreased linearly from 70% to 60%; from 41 min to 50 min, acetonitrile linearly increased from 40% to 45%, 1% glacial acetic acid solution from 60% Linearly decreased to 55%; from 51 min to 60 min, acetonitrile increased linearly from 45% to 55%, and 1% glacial acetic acid solution decreased linearly from 55% to 45%; flow rate 0.5～1.5mL·min ^-1 ; column temperature 35 ～40℃; detection wavelength 255～260nm; injection volume 5～20μL;

(2) Preparation of mixed reference solution: take silymarin, ferulic acid, gallic acid, and vanillic acid reference substances respectively, weigh, and dilute with methanol to obtain 60-70 μg of silymarin and 15 μg of ferulic acid per 1 mL, respectively. ～20μg, 10～20μg of gallic acid, 15～20μg of vanillic acid mixed reference solution, mix well to obtain mixed reference solution;

(3) Preparation of the test solution: take the sample, grind it finely, weigh 1.00 g, put it in a stoppered conical flask, add 15-30 mL of methanol precisely, weigh it, and weigh it at a power of 350-450W and 35-45kHz. , ultrasonic for 20-30min, methanol to make up for weight loss, filtration, take the filtrate to get the test solution;

(4) Determination: Precisely draw 5-20 μL of the mixed reference solution and the test solution, and inject them into a high-performance liquid chromatograph for measurement.

6. the detection method of medicine as claimed in claim 5, this medicine is made of the medicinal raw material of following weight part: Angelica 2 weight parts, white peony root 1 weight part, Silybum marianum 1 weight part; Its preparation method is:

(5) Take the filtrate IV of step (4), put it on the MCI resin column, wash with water for 8 column volumes, discard the eluent, wash with 15% methanol for 6 column volumes, discard the eluent, and finally Wash with 65% methanol for 12 column volumes, collect the eluate, filter, and concentrate the filtrate to obtain extract V;

(1) Chromatographic conditions: C ₁₈ chromatographic column, size: 4.6mm×250mm, 5μm; mobile phase: acetonitrile-1% glacial acetic acid solution, gradient elution, elution sequence: from 0 min to 15 min, acetonitrile was kept at 10%, 1% glacial acetic acid solution kept at 90%; from 16min to 35min, acetonitrile linearly increased from 10% to 30%, 1% glacial acetic acid solution decreased linearly from 90% to 70%; from 36min to 40min , acetonitrile increased linearly from 30% to 40%, 1% glacial acetic acid solution decreased linearly from 70% to 60%; from 41 min to 50 min, acetonitrile linearly increased from 40% to 45%, 1% glacial acetic acid solution from 60% Linearly decreased to 55%; from 51 min to 60 min, acetonitrile increased linearly from 45% to 55%, and 1% glacial acetic acid solution decreased linearly from 55% to 45%; flow rate 1.0 mL·min ^-1 ; column temperature 38°C; Detection wavelength 257nm; injection volume 10μL;

(2) Preparation of mixed reference solution: take silymarin, ferulic acid, gallic acid, and vanillic acid reference substances respectively, weigh them, and dilute with methanol to obtain 65 μg of silymarin, 20 μg of ferulic acid, 20 μg of gallic acid per 1 mL, respectively. Acid 15μg, vanillic acid 20μg mixed reference solution, mix well, that is, mixed reference solution;

(3) Preparation of the test solution: take the sample, grind it finely, weigh 1.00 g, put it in a conical flask with a stopper, add 25 mL of methanol precisely, weigh it, under the power of 400 W, 40 kHz, ultrasonicate for 25 min, and make up with methanol. Lose weight, filter, and take the filtrate to obtain the test solution;

(4) Determination: Precisely draw 10 μL each of the mixed reference solution and the test solution, and inject them into a high-performance liquid chromatograph for determination.

7. A detection method for a medicine for the treatment of primary dysmenorrhea, the medicine is made from the medicinal-flavor raw materials of the following parts by weight: 1-3 parts by weight of Angelica sinensis, 1-2 parts by weight of white peony root, 1-2 parts by weight of milk thistle , its preparation method is:

It is characterized in that, adopting gas chromatography to measure the content of β-ocimene, α-pinene and myrtanal, the steps are as follows:

(1) Chromatographic conditions: chromatographic column: HP-5 capillary column, temperature program: the initial temperature is 40 °C, maintained for 5 min, heated to 120 °C at a rate of 8 °C·min ^-1 , and maintained for 8 min; then at 12 °C· The temperature was raised to 180°C at a rate of min ^-1 and held for 5 minutes; then heated to 270°C at a rate of 15°C·min ^-1 and held for 5 minutes; the inlet temperature was 210-230°C; the FID detector; the detector temperature was 260 ～280℃; volume flow rate is 0.5～2.0mL·min ^-1 ; injection volume is 1～10μL, split injection, split ratio is 7～9:3～1;

(2) Preparation of mixed reference solution: take an appropriate amount of β-ocimene, α-pinene, and myrtle aldehyde reference substances, accurately weigh them, and add chloroform to make β-ocimene containing 0.5-2.0 mg·mL ^-1 . A mixed reference solution containing 0.5-2.0 mg·mL-1 of α-pinene and 0.5-2.0 mg·mL ^-1 ^of myrtanal;

(3) Preparation of the test solution: take 0.20-1.0 g of this product, weigh it, put it in a conical flask with a stopper, add 15-30 mL of chloroform precisely, seal it tightly, weigh it, ultrasonicate it for 10-20 min, Allow to cool, weigh the weight, supplement the lost weight with chloroform, shake well, filter with a 0.45 μm filter membrane, and take the filtrate to obtain the test solution;

(4) Determination: Precisely draw 1-10 μL each of the mixed reference solution and the test solution, and inject them into a gas chromatograph for measurement.

8. the detection method of medicine as claimed in claim 7, this medicine is made of the medicinal raw material of following weight parts: Angelica 2 weight parts, white peony root 1 weight part, silybum 1 weight part; Its preparation method is:

(1) Chromatographic conditions: chromatographic column: HP-5 capillary column, specification: 30m×0.32mm, 0.25μm; temperature program: the initial temperature is 40°C, maintained for 5min, and heated to 120 at a rate of 8°C·min ^-1 ℃, hold for 8min; then raise the temperature to 180℃ at the rate of 12℃·min ^-1 and hold for 5min; then raise the temperature to 270℃ at the rate of 15℃·min ^-1 and hold for 5min; the injection port temperature is 220℃; FID Detector; the detector temperature is 270°C; the volume flow is 1.0mL·min ^-1 ; the injection volume is 5μL, the split injection is performed, and the split ratio is 8:2;

(2) Preparation of mixed reference solution: take appropriate amount of β-ocimene, α-pinene, and myrtle aldehyde reference substance, accurately weigh, add chloroform to make β-ocimene containing 1.055mg·mL ^-1 , a mixed reference solution containing α-pinene at 1.115 mg·mL ^-1 and myrtanal at 1.225 mg·mL ^-1 ;

(3) Preparation of the test solution: take 0.60g of this product, weigh it, put it in a conical flask with a stopper, accurately add 20mL of chloroform, close the stopper, weigh it, ultrasonicate for 15 minutes, let it cool, and weigh it. , supplement the lost weight with chloroform, shake well, filter with a 0.45 μm filter membrane, and take the subsequent filtrate to obtain the test solution;

(4) Determination: Precisely draw 5 μL each of the mixed reference solution and the test solution, and inject them into a gas chromatograph for measurement.