CN108403793A

CN108403793A - A kind of drug and its preparation method for treating primary dysmenorrhea

Info

Publication number: CN108403793A
Application number: CN201810443139.1A
Authority: CN
Inventors: 刘长红; 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: 2018-08-17
Anticipated expiration: 2038-05-10
Also published as: CN108403793B

Abstract

The invention belongs to tcm field, disclose a kind of drug for treating primary dysmenorrhea, a kind of entitled drug and its preparation method for treating primary dysmenorrhea, the drug by following parts by weight component：1~3 part of Radix Angelicae Sinensis, 1~2 part of Radix Paeoniae Alba, 1~2 part of milk thistle.The present invention provides the preparation method of the drug, drug provided by the invention can effectively treat that primary dysmenorrhea, curative for effect, compatibility is simple, at low cost；Active constituent extraction efficiency in drug made of preparation method provided by the invention is high, active constituent content is high, impurity content is low, curative effect is reliable, safe.The present invention also provides the methods for measuring active component content using high performance liquid chromatography and gas chromatography.

Description

A kind of drug and its preparation method for treating primary dysmenorrhea

Technical field

The invention belongs to technical field of Chinese medicine, are related to a kind of drug and its preparation method for treating primary dysmenorrhea.

Background technology

Dysmenorrhoea means that the menstrual period is front and back or passes through period, lower abdomen spasmic pain occurs, and have general malaise, serious shadow Ring daily life person.It is divided to primary and two kinds secondary.Fail to find that pelvic organ has obviously by detailed gynecological clinic inspection Abnormal person claims primary dysmenorrhea, also referred to as functional dysmenorrhea.Acquired dysmenorrhea, which then refers to reproductive organs, apparent lesion person, such as uterus Endometrium ectopia, pelvic infecton, tumour etc..Primary dysmenorrhea, that is, functional dysmenorrhea refers to painful menstrual periods, is in often spastic, collection In in lower abdomen.Other symptoms include have a headache weak, dizzy, nausea and vomiting, diarrhea, pain in waist and lower extremities, are that young woman is very common Illness, primary dysmenorrhea be not accompanied by apparent pelvic cavity organic disease.

In the prior art, treatment primary dysmenorrhea herbal mixture also has much, and it is as follows now to enumerate part：

(1) patent document for being related to treating primary dysmenorrhea has：

1, patent name is a kind of pharmaceutical composition for treating primary dysmenorrhea, Publication No. CN104435519A, application Number patent for being 201410787568.2, formula disclosed in the patent are：5~15 parts of Radix Angelicae Sinensis, 10~20 parts of peach kernel, rhizoma cyperi 5 ~15 parts, 5~10 parts of Rhizoma Chuanxiong, 5~10 parts of corydalis tuber, 2~6 parts of Herba Lycopi, 2~6 parts of safflower, 1~5 part of the root of three-nerved spicebush, Pollen Typhae 2~4 Part, 1~3 part of Radix Glycyrrhizae.

2, patent name is a kind of pharmaceutical composition for treating cold blood stasis type primary dysmenorrhea, Publication No. CN106074830A, application No. is 201610423799.4 patent, formula disclosed in the patent is：10~30 parts of peach kernel, 3~12 parts of safflower, 3~15 parts of radix paeoniae rubra, 10~20 parts of Rhizoma Chuanxiong, 8~20 parts of radix rehmanniae preparata, 10~20 parts of radix bupleuri, 12~20 parts of radix cyathulae, 10~20 parts of cassia twig, 6~20 parts of evodia rutaecarpa of toast, 3~12 parts of Radix Angelicae Sinensis, 10~15 parts of Radix Paeoniae Alba, 6~25 parts of honey-fried licorice root, corydalis tuber 10~ 20 parts.

3, patent name is a kind of drug for treating primary dysmenorrhea, Publication No. CN104524085A, application number For 2014107394165 patent, formula disclosed in the patent is：6 parts of official osmanthus, 6 parts of rhizoma cyperi processed, 8 parts of Rhizoma Chuanxiong and corydalis tuber 9 Part.

4, patent name is a kind of Chinese medicine preparation for treating primary dysmenorrhea, authorizes Publication No. CN103405606A, Shen Please number patent for being 2013103658206, formula disclosed in the patent is：8~12g of radix bupleuri, 8~12g of Radix Angelicae Sinensis, rhizoma cyperi 8~ 12g, 5~7g of excrementum pteropi, 5~7g of cattail pollen, 13~17g of 13~17g of Radix Salviae Miltiorrhizae, 13~17g of Radix Paeoniae Alba and Poria cocos.

5, patent name is a kind of Chinese medicine for treating primary dysmenorrhea, application publication number CN103566308A, application number For 201310562561.6 patent, formula disclosed in the patent is：20~40 parts of motherwort, 20~40 parts of folium artemisiae argyi, Radix Curcumae 20~40 parts, 20~40 parts of turmeric, 20~40 parts of hawthorn, 15~25 parts of the root bark of tree peony, 15~25 parts of radix scutellariae, 10~20 parts of excrementum pteropi, 10~20 parts of the fruit of Chinese wolfberry, 10~20 parts of Radix Codonopsis, 10~20 parts of semen brassicae.

6, patent name is a kind of Chinese herbs paste for treating primary dysmenorrhea, Publication No. CN106266404A, application number For 201610861934.3 patent, formula disclosed in the patent is：5~10 parts of rhizoma cyperi, 5~10 parts of corydalis tuber, cassia twig 4~ 8 parts, 4~8 parts of Chinese cassia tree, 5~8 parts of root of Aucklandia lappa Decne, 2~4 parts of angelica extract.

7, patent name is a kind of Chinese medicine composition with treatment dysmenorrhoea effect, Publication No. CN106214769A, Shen Please number be 201610886259.X patents, formula disclosed in the patent is：1~5 part of safflower, 1~5 part of lanatechead saussurea herb with flower, Jasmine 1 ~5 parts, 1~5 part of peony.

8, patent name be it is a kind of alleviate female primary dysmenorrhoea Chinese medicine application cream, Publication No. CN106176980A, Application No. is 201610544604.1 patents, formula disclosed in the patent is：15~25 parts of corydalis tuber, 15~25 parts of folium artemisiae argyi, 15~25 parts of Chinese cassia tree, 15~25 parts of Rhizoma Chuanxiong, 1~10 part of asarum, 10~20 parts of the root of three-nerved spicebush.

9, patent name is a kind of Chinese medicine composition and its preparation and use for treating primary dysmenorrhea, Publication No. CN106109802A, application No. is 201610802757.1 patent, formula disclosed in the patent is：Dragon's blood 1.5~4.5 Part；1.5~4.5 parts of Radix Notoginseng；1.5~4.5 parts of Chinese cassia tree；1.5~4.5 parts of evodia rutaecarpa；0.5~2.5 part of Herba Lycopi；Radix cyathulae 0.5~ 2.5 part；0.5~2.5 part of Rhizoma Chuanxiong；0.5~1.5 part of cloves.

10, patent name is a kind of preparation for treating primary dysmenorrhea medicine moxibustion item, Publication No. CN106074831A, application Number patent for being 201610424128.X, formula disclosed in the patent are：Peach kernel, radix bupleuri, safflower, toast evodia rutaecarpa, radix paeoniae rubra, river Rhizome of chuanxiong, radix rehmanniae preparata, radix cyathulae, cassia twig, Radix Paeoniae Alba, Radix Angelicae Sinensis, honey-fried licorice root and corydalis tuber.

11, patent name is a kind of Chinese medicinal formulae of the treatment cold blood stasis type primary dysmenorrhea containing folium artemisiae argyi, publication number For CN105797099A, application No. is 201610192274.4 patent, 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 Herba Cistanches, 5~15 parts of Rhizoma Chuanxiong, 9~15 parts of Semen Cuscutae, the fruit of glossy privet 6~ 1~10 part of 12 parts, 3~9 parts of Radix Angelicae Sinensis, 3~9 parts of Verbena officinalis, 6~12 parts of matrimony vine, 3~10 parts of ginger and Radix Glycyrrhizae.

12, patent name is a kind of Chinese medicine preparation for treating syndrome of qi stagnation and blood stasis primary dysmenorrhea, Publication No. CN103721002A, application No. is the patent of 201310755511.X, formula disclosed in the patent is：3~9 parts of mountain and sea Chinese bush cherry, 15~25 parts of scarlet kadsura root, 20~25 parts of manyflower jasmine herb or flower, 18~24 parts of scabrous doellingeria root, 30~40 parts of Chinese ixora root, 3~9 parts of pilose triumfetta root and leaf, 12~30 parts of Herb of Michel Galingale, 9~15 parts of daphne odera, 9~15 parts of riparian greenbrier root and rhizome, 15~25 parts of fiveleaf akebia fruit.

13, patent name be a kind of Chinese medicine for treating primary dysmenorrhea, Publication No. CN105616798A, application No. is 201410618955.3 patent, formula disclosed in the patent is：Chinese medicinal component be loosestrife 5~15, mountain and sea Chinese bush cherry 10~20, It is kardiseed 3~12, Plumbago indica 2~10, colter point 5~15, cotoneaster 2~10, membranaceous marshmarigold herb 5~15, drop of water 10~20, small White support 5~15, luffa root 10~20, yunnan euonymus root and stem 4~12, ambergris 5~20, Ophiorrhiza japonica 5~15.

14, patent name is a kind of pharmaceutical composition for treating primary dysmenorrhea, Publication No. CN104306470A, application Number it is 201410625394.X patents, formula disclosed in the patent is：10~50 parts of wild pool wormwood artemisia, 10~30 parts of auriculate dichrocephala herb or leaf, snake 10~20 parts of 20~30 parts of marchantia, 10~30 parts of hops, 20~30 parts of copper arhat and deflection evaluation.

15, patent name is a kind of Chinese medicine preparation for treating dysmenorrhoea and preparation, Publication No. CN105582518A, application number For 201610141446.5 patent, formula disclosed in the patent is：Stinkweed, worm lotus, Artemisia anomala, the root of three-nerved spicebush, spoon leaf grass, monkey The sub- bamboo mat spread on the floor for people to sit in ancient China of camphor tree, pheasant, Radix Angelicae Sinensis, seed of garden lettuce, rhizoma zingiberis, ink seven, jasmine, kaempferia galamga, Semen Leonuri, Radix Notoginseng and row night.

16, patent name is a kind of Chinese medicine for treating primary dysmenorrhea, Publication No. CN105561017A, application number For 201610107525.4 patent, formula disclosed in the patent is：15 parts of meat fringe grass, 5 parts of dracontomelon duperreanum, 9 parts of delavay neocinnamomum leaf, too 18 parts of the flat certain kind of berries, thousand 12 parts of solution grass, 10 parts of Auricled Hedyotis Herb, 15 parts of amur foxtail, 6 parts of powder Bi.

17, patent name be a kind of Chinese medicine for treating primary dysmenorrhea, Publication No. CN105497389A, application No. is 201610063392.5 patent, formula disclosed in the patent is：8~16 parts of Radix Crossostephii Chinensis, yellow 5~13 parts of camphor tree, the seed of Oriental arborvitae 8 ~16 parts, it is 8~16 parts of Wilson Begonia Rhizome, false 8~16 parts of rhizoma cyperi, 8~16 parts of cloves, 8~16 parts of Ficus fulva Reinw, 5~13 parts of river cassia bark, smelly 5~13 parts of tree peony root, 5~13 parts of Caulis mucunae Macrocarpae, 11~19 parts of radix rehmanniae preparata, 4~8 parts of beechey fig leaf.

18, patent name is a kind of Chinese medicine preparation for treating primary dysmenorrhea and its preparation, Publication No. CN105288092A, application No. is the patent of 201510861876.X, formula disclosed in the patent is：13~18 parts of rhizoma cyperi, 11~16 parts of Radix Angelicae Sinensis, 2 parts of Caulis mucunae Macrocarpae, 20~25 parts of twoleaf swertia herb, 6~9 parts of rhizoma anemarrhenae, 3~6 parts of Radix Glycyrrhizae.

19, patent name is a kind of Chinese medicine composition for treating cold blood stasis type primary dysmenorrhea, Publication No. CN105250617A, application No. is：2015108531658 patent, formula disclosed in the patent are：Salvia trijuga Diels 10~ 15 parts, 13~18 parts of Herba Lycopi, 8~13 parts of Herb of Michel Galingale, 72 parts of bamboo root, 5~10 parts of rhizoma cyperi, 8~12 parts of nude fern herb, Caulis Spatholobi 9~ 8~12 parts of 15 parts, 15~18 parts of wilsonii and Rhizoma Atractylodis Macrocephalae.

20, patent name is a kind of drug for treating primary dysmenorrhea, Publication No. CN105126044A, application number For 2015107110651 patent, formula disclosed in the patent is：10 parts of Caulis Spatholobi, 8 parts of radix boehmeriae, 7 parts of Poria cocos, curcuma zedoary 4 Part, 8 parts of Herba Cistanches, 9 parts of the fruit of Cherokee rose, 10 parts of dindygulen peperomia herb.

21, patent name is a kind of Chinese medicine composition for treating stagnation of the circulation of vital energy blood group primary dysmenorrhea, Publication No. CN104258074A, application No. is 2014105080322 patent, formula disclosed in the patent is：Rhizoma cyperi 13~17 part, month Spend in season 10~15 parts, 8~13 parts of Artemisia anomala, 6~9 parts of the bletilla striata, 8~13 parts of Polygonum multiflower knotweed, 6~9 parts of 5~8 parts of Fructus Aurantii and Radix Astragali.

22, patent name is a kind of traditional Chinese medicine tea for treating primary dysmenorrhea, Publication No. CN104840557A, Shen Please number patent for being 201510291342.8, formula disclosed in the patent is：1~5 part of lilac, 1~5 part of peach blossom, rose Spend 1~5 part, 1~5 part of Flos Hibisci Mutabilis.

23, patent name be a kind of drug for treating primary dysmenorrhea, Publication No. CN104800307A, application No. is 2015102109179 patent, formula disclosed in the patent are：30~50g of safflower, 20~30g of radix paeoniae rubra, 20~30g of cassia twig, 20~30g of folium artemisiae argyi, 10~15g of 20~30g of Radix Angelicae Sinensis, 30~40g of corydalis tuber and the root of Dahurain angelica.

(2) paper for being related to treating primary dysmenorrhea has：

1, in April, 2013, golden red rock exist《Liaoning University of TCM's journal》On publish thesis《Acupoint application of Chinese herb treatment is former The dysmenorrhoea clinical analysis of hair property》, the clinical efficacy of observation acupoint application of Chinese herb treatment primary dysmenorrhea is disclosed herein.2005~ The primary dysmenorrhea patient of outpatient clinic in 2010 100,50 using Chinese medicine applications in three Qihai, Guan Yuan, Shenque acupuncture points, 50 patients do not have to Chinese medicine, are only sticked at corresponding acupuncture point.After 2 courses for the treatment of, acupoint application of Chinese herb treats dysmenorrhoea effective percentage 98%, Dysmenorrhoea effective percentage 32% is controlled without Chinese medicine application.Acupoint application of Chinese herb treatment primary dysmenorrhea is curative for effect, securely and reliably.

2, in April, 2012, Wang Lichun exist《Chinese Medicine guide》On publish thesis《Chinese medicine treats the clinic of primary dysmenorrhea Observation》, therapeutic effect of the observation Chinese medicine treatment to the dysmenorrhoea symptom of primary dysmenorrhea patient is disclosed herein.Choose Mudanjiang City First People's Hospital is divided into cold solidifying uterus type, qi-blood deficiency from 68 primary dysmenorrhea disease patients accepting for medical treatment, by Chinese medical discrimination Type, qi stagnation and blood stasis type, liver-kidney deficiency type and YANG-insufficiency and internal cold type take the oral mode added and subtracted by silt soup to be treated.68 originals Dysmenorrhoea patient is after treating 3 courses for the treatment of for hair property, cures 48 (70.58%), wherein 12 (17.65%) patients treat the 1st Journey is cured, and 25 (36.76%) patients cure in the 2nd course for the treatment of, and 11 (16.18%) example patients cure in the 3rd course for the treatment of；It is good Turn 19 (27.94%), do not cure 1 (1.47%), total effective rate is (98.53%) %.By silt soup for treating primary The dysmenorrhoea symptom effect of dysmenorrhoea patient is ideal, and safety is higher.

3, in March, 2011, Lei Li《Hubei University of Chinese Medicine's journal》On publish thesis《Chinese medicine treats primary dysmenorrhea 120》, be disclosed herein primary dysmenorrhea refer to women before and after menstruation or menstrual period occur abdominal pain, the soreness of waist, lower abdomen pendant pain be in It is spastic or with the discomforts such as weak, dizzy, nausea,vomiting,diarrhea, working and learning are influenced, the organic disease of pelvic cavity is not accompanied by Change person.Adolescent girls and unmarried young woman are more common in, are the common diseases for influencing women and working normally studying and living quality Disease.Its incidence of epidemiological study is 20%~90%.

4, in January, 2014, Li Zhen《Seldom lack disease magazine》On publish thesis《GUIZHI FULING JIAONANG combines medroxyproges-terone acetate Piece treats the clinical research of primary dysmenorrhea》, it is disclosed herein and clinically uses Ramulus cinnamomi Indian buead capsule joint Western medicine peace Palace progesterone piece treats primary dysmenorrhea, and is inquired into its clinical efficacy.In June, 2011 is looked back to during in June, 2013 150 primary dysmenorrhea patient cases that outpatient service is accepted for medical treatment.It is classified as observation group and control group according to randomized blocks, every group 75 Example.Control group patient clinical is treated using Western medicine medroxyproges-terone acetate piece；Observation group patient combines on the basis of control group Ramulus cinnamomi Indian buead capsule is treated.After treatment 3 months, the therapeutic effect of two groups of patients is observed and analyzed.Two groups Conditions of patients is obtained for control, and the total effective rate of wherein observation group's treatment is up to 97.3% (73/75), and conspicuousness is more than control 73.3% (55/75) of group, P<0.05；The pain degree of observation group is (2.2 ± 1.4), and conspicuousness is less than (the 4.2 of control group ± 1.6), P<0.05；The pain time of observation group is (2.7 ± 1.1) h, and conspicuousness is less than (8.9 ± 2.4) h, P of control group< 0.05.Ramulus cinnamomi Indian buead capsule joint Western medicine medroxyproges-terone acetate piece clinically can effectively treat primary dysmenorrhea, and drop The pain degree and pain time of low patient is worth clinical expansion.

5, in April, 2010, Xu Jing《Magazine is controlled outside Chinese and Western》On publish thesis《Oral medicinal herb coordinates moxibustion treatment primary Dysmenorrhoea 42》It is the common disease of gynecological clinic, frequently-occurring disease that dysmenorrhoea, which is disclosed herein, refers generally to abdomen occur through the capable front and back or menstrual period Bitterly, uncomfortable, degree is serious to influence live and work quality.Since two thousand three, moxibustion treatment is coordinated using oral medicinal herb Dysmenorrhoea 42, satisfactory effect.

6, in June, 2009, magnificent qin, Ye Huizhou, Chi Lifang exist《Chinese traditional Chinese medicine academic periodical》On publish thesis《Exempt to decoct Chinese medicine The clinical observation on the therapeutic effect of granule therapy dysmenorrhoea》, evaluation is disclosed herein and exempts to decoct the clinical efficacy of Chinese medicinal granule.By 150 dysmenorrhoeas Patient is divided to actual situation two classes, 5 kinds of card types, doctor trained in Western medicine diagnosis to divide primary dysmenorrhea and acquired dysmenorrhea and according to dysmenorrhoea by tcm diagnosis Degree is divided into 3 degree.Then it gives and exempts to decoct Chinese medicinal granule treatment, 3 menstrual cycles are 1 course for the treatment of, carry out observation of curative effect.Dysmenorrhoea Effective percentage is 93%, and no recurrence is also without side-effects.By the clinical observation to dysmenorrhoea patient, it is medium-sized and primary that syncope due to pathogenic cold coagulates born of the same parents Property dysmenorrhoea curative effect is the most notable.Exempt from decoct Chinese medicinal granule it is curative for effect, for Chinese medicine form improvement, exempt from decoct Chinese medicinal granule popularization answer With offer foundation.

7,07 month 2005, Song Zhuomin, Xing Shuli existed《Chinese combination of Chinese tradiational and Western medicine magazine》On publish thesis《Tongjingning Granule Treat the clinical observation of primary dysmenorrhea》, visit the effect that Tongjingning granula of TCD treatment primary dysmenorrhea (PD) is disclosed herein Mechanism.PD patient 120 is treated with Tongjingning Granule, and set Western medicine aspirin for treatment 40 compare, while to part Patient carried out in pretherapy and post-treatment corpus luteum, latter stage serum estradiol (E2), progestational hormone (P), midluteal phase, menstrual period Endothelin (ET) With the detection of calcitonin gene-related peptide (CGRP) content.Treatment group's clinical efficacy is better than control group (P<0.01)；To severe, in The aobvious more rate and main and disease improvement rate for spending PD patient are better than control group (P<0.01).Treatment group E2 levels, ET contain after treatment Amount is decreased obviously (P before relatively treating<0.01)；P, CGRP contents then obviously rise (P<0.01).Tongjingning granula of TCD is adjustable Estrogen and progestogen, ET and CGRP；And with adjustment spirit, mood, promote organismic internal environment balance and the advantage of efficacy consolidation.

In the prior art, the herbal mixture for treating primary dysmenorrhea has much, but is substantially the big compound medicine of Chinese medicine, Few is made of 5 to 8 kinds of flavour of a drug raw materials, and more is then made of 20 to 30 kinds of flavour of a drug raw materials, the treatment effect of this big compound medicine Fruit is simultaneously bad, and the effective active composition of drug is also unclear, does not meet the developing direction of modern Chinese herbal medicine pharmacy, currently, urgently Needing one kind effectively to treat, primary dysmenorrhea, curative for effect, compatibility is simple, at low cost, and effective active composition is substantially specific Drug.

The inventor of this patent is the scientific research personnel of a line of Jiamusi institute of Heilongjiang University of Chinese Medicine, by the several years After concentrating on studies, the innovative technology of the obtained present invention.Meanwhile the innovative technology of this patent with Heilongjiang Province and Jiangsu The several pharmacy corporations saved reach Intentionality cooperation agreement, have developed new drug for the innovative technology of this patent and have carried out early period Preparation.

Invention content

The technical problem to be solved by the invention is to provide a kind of effectively treatment primary dysmenorrhea, curative for effect, compatibility is simple Single, drug at low cost；Further, the present invention provides a kind of preparation method of drug that treating primary dysmenorrhea, the preparation method Active constituent extraction efficiency is high in manufactured drug, active component content is high, impurity content is low, curative effect is reliable, safe.

In order to solve the above technical problems, the technical solution adopted by the present invention is：

A kind of drug for treating primary dysmenorrhea, the drug are made of the flavour of a drug raw material of following parts by weight：1~3 weight of Radix Angelicae Sinensis Measure part, 1~2 parts by weight of Radix Paeoniae Alba, 1~2 parts by weight of milk thistle.

The drug is preferably made of the flavour of a drug raw material of following parts by weight：2 parts by weight of Radix Angelicae Sinensis, 1 parts by weight of Radix Paeoniae Alba, milk thistle 1 Parts by weight.

A kind of process for preparing medicine for treating primary dysmenorrhea, the drug are made of the flavour of a drug raw material of following parts by weight：When 1~3 parts by weight, 1~2 parts by weight of Radix Paeoniae Alba, 1~2 parts by weight of milk thistle, preparation method are returned to be：

(1) Radix Angelicae Sinensis, Radix Paeoniae Alba, milk thistle are taken, carries out steam distillation extraction, collection obtains volatile oil, spare；Vapor steams Extracting solution after extracting is evaporated, is filtered, concentration obtains medicinal extract I；The dregs of a decoction after steam distillation extraction are spare；

(2) dregs of a decoction of step (1) are taken, the bionic extraction solvent of 20~40 times of amounts, 36~38 DEG C of heating water baths, stirring is added 2~4h is extracted, 30~40min, 2500~3000r/min of centrifugal speed are centrifuged, collects supernatant, supernatant filtration, concentration obtains Medicinal extract II；Above-mentioned bionic extraction solvent be by 0.5%~0.8% sodium chloride, 0.5%~0.8% pepsin, 0.5%~ 0.8% trypsase and 0.06molL^-1~0.08molL^-1Hydrochloric acid is configured to solution；

(3) medicinal extract I of step (1) is mixed with the medicinal extract II of step (2), is put into ultrasonic-microwave reactor, is added 60%~70% ethanol solution of 20~40 times of amounts, then reacts in ultrasonic-microwave reactor, ultrasonic power is 1200~1400W, microwave power are 360~380W, the reaction time is 40~50min, reaction temperature is 30~40 DEG C, after reaction Liquid filtration, filtrate concentration, obtain medicinal extract III；

(4) medicinal extract III for taking step (3), with purify it is water-dispersed after, cross H-30 type large pore resin absorption columns, be washed with water 6~ 10 column volumes, eluent discard, then wash 4~7 column volumes with 10%~20% methanol, and eluent is discarded, finally used again 60%~70% methanol washes 10~15 column volumes, collects eluent, and filtration obtains filtrate IV；

(5) filtrate IV of step (4), then upper MCI resin columns are taken, 6~10 column volumes are washed with water, eluent discards, then 4~7 column volumes are washed with 10%~20% methanol, eluent discards again, finally washes 10~15 columns with 60%~70% methanol Volume, collects eluent, filtration, and filtrate concentration obtains medicinal extract V；

(6) medicinal extract V of step (5) is taken to merge with the volatile oil of step (1), mixing, freeze-drying to get.

The process for preparing medicine, the drug are preferably made of the flavour of a drug raw material of following parts by weight：2 parts by weight of Radix Angelicae Sinensis, 1 parts by weight of Radix Paeoniae Alba, 1 parts by weight of milk thistle；Its preferred preparation method is：

(2) dregs of a decoction of step (1) are taken, the bionic extraction solvent of 30 times of amounts, 37 DEG C of heating water baths is added, 3h is extracted in stirring, 35min, centrifugal speed 2800r/min are centrifuged, supernatant, supernatant filtration are collected, concentration obtains medicinal extract II；Above-mentioned bionic extraction Solvent is by 0.6% sodium chloride, 0.7% pepsin, 0.6% trypsase and 0.07molL^-1Hydrochloric acid is configured to solution；

(3) medicinal extract I of step (1) is mixed with the medicinal extract II of step (2), is put into ultrasonic-microwave reactor, is added 65% ethanol solution of 30 times of amounts, then reacts in ultrasonic-microwave reactor, ultrasonic power 1300W, microwave work( Rate is 370W, reaction time 45min, reaction temperature are 35 DEG C, and the liquid filtration after reaction, filtrate concentration obtains medicinal extract III；

(4) medicinal extract III for taking step (3), with purify it is water-dispersed after, cross H-30 type large pore resin absorption columns, be washed with water 8 Column volume, eluent discard, then wash 6 column volumes with 15% methanol, and eluent discards again, finally wash 12 columns with 65% methanol Volume, collects eluent, and filtration obtains filtrate IV；

(5) filtrate IV of step (4), then upper MCI resin columns are taken, 8 column volumes are washed with water, eluent discards, then uses 15% methanol washes 6 column volumes, and eluent discards again, finally washes 12 column volumes with 65% methanol, collects eluent, filters, Filtrate concentrates, and obtains medicinal extract V；

The drug uses pharmaceutical methods conventional in pharmacy of Chinese materia medica that oral preparation is made.

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

A kind of detection method of drug that treating primary dysmenorrhea, the drug are made of the flavour of a drug raw material of following parts by weight： 1~3 parts by weight of Radix Angelicae Sinensis, 1~2 parts by weight of Radix Paeoniae Alba, 1~2 parts by weight of milk thistle, preparation method are：

(6) medicinal extract V of step (5) is taken to merge with the volatile oil of step (1), mixing, freeze-drying to get；

Contained using silydianin in hplc simultaneous determination drug, gallic acid, ferulic acid, vanillic acid Amount, steps are as follows：

(1) chromatographic condition：Using C₁₈Chromatographic column, specification：4.6mm × 250mm, 5 μm；Mobile phase：- 1% glacial acetic acid of acetonitrile Solution, gradient elution, eluting order are：From 0min to 15min, acetonitrile is maintained at 10%, and 1% glacial acetic acid solution is maintained at 90%；From 16min to 35min, acetonitrile is from 10% linear rise to 30%, 1% glacial acetic acid solution from 90% linear decline To 70%；From 36min to 40min, acetonitrile is from 30% linear rise to 40%, 1% glacial acetic acid solution under 70% is linear It is down to 60%；From 41min to 50min, acetonitrile is linear from 40% linear rise to 45%, 1% glacial acetic acid solution from 60% Drop to 55%；From 51min to 60min, acetonitrile is from 45% linear rise to 55%, 1% glacial acetic acid solution from 55% line Property drops to 45%；0.5~1.5mLmin of flow velocity^-1；35~40 DEG C of column temperature；255~260nm of Detection wavelength；Sample size 5~20 μL；

(2) preparation of mixed reference substance solution：Silydianin, ferulic acid, gallic acid, vanillic acid reference substance are taken respectively, It is weighed, with methanol dilution at every 1mL 60~70 μ g containing silydianin, 15~20 μ g of ferulic acid, 10~20 μ g of gallic acid respectively, The mixed reference substance solution of 15~20 μ g of vanillic acid, mixing is to get mixed reference substance solution；

(3) preparation of test solution：Sample is taken, it is finely ground, 1.00g is weighed, is set in conical flask with cover, precision plus methanol 15 ~30mL, weighed, at 350~450W, the power of 35~45kHz, 20~30min of ultrasound, methanol supplies loss of weight, and filtration takes Subsequent filtrate is to get test solution；

(4) it measures：Precision draws mixed reference substance solution, each 5~20 μ L of test solution, injects high performance liquid chromatography Instrument is measured.

The detection method of the drug, the drug are made of the flavour of a drug raw material of following parts by weight：It is 2 parts by weight of Radix Angelicae Sinensis, white 1 parts by weight of Chinese herbaceous peony, 1 parts by weight of milk thistle；Preparation method is：

Contained using silydianin in hplc simultaneous determination drug, gallic acid, ferulic acid, vanillic acid Amount, preferred steps are as follows：

(1) chromatographic condition：Using C₁₈Chromatographic column, specification：4.6mm × 250mm, 5 μm；Mobile phase：- 1% glacial acetic acid of acetonitrile Solution, gradient elution, eluting order are：From 0min to 15min, acetonitrile is maintained at 10%, and 1% glacial acetic acid solution is maintained at 90%；From 16min to 35min, acetonitrile is from 10% linear rise to 30%, 1% glacial acetic acid solution from 90% linear decline To 70%；From 36min to 40min, acetonitrile is from 30% linear rise to 40%, 1% glacial acetic acid solution under 70% is linear It is down to 60%；From 41min to 50min, acetonitrile is linear from 40% linear rise to 45%, 1% glacial acetic acid solution from 60% Drop to 55%；From 51min to 60min, acetonitrile is from 45% linear rise to 55%, 1% glacial acetic acid solution from 55% line Property drops to 45%；Flow velocity 1.0mLmin^-1；38 DEG C of column temperature；Detection wavelength 257nm；10 μ L of sample size；

(2) preparation of mixed reference substance solution：Silydianin, ferulic acid, gallic acid, vanillic acid reference substance are taken respectively, It is weighed, with methanol dilution at every 1mL 65 μ g containing silydianin, 20 μ g of ferulic acid, 15 μ g of gallic acid respectively, 20 μ g's of vanillic acid Mixed reference substance solution, mixing is to get mixed reference substance solution；

(3) preparation of test solution：Sample is taken, it is finely ground, 1.00g is weighed, is set in conical flask with cover, precision plus methanol 25mL, weighed, at 400W, the power of 40kHz, ultrasonic 25min, methanol supplies loss of weight, and filtration takes subsequent filtrate to get for examination Product solution；

(4) it measures：Precision draws mixed reference substance solution, each 10 μ L of test solution, injects high performance liquid chromatograph, into Row measures.

The content of β-ocimenum, australene, myrtanal is measured using gas chromatography, steps are as follows：

(1) chromatographic condition：Chromatographic column：HP-5 capillary columns, temperature programming：Initial temperature is 40 DEG C, 5min is kept, with 8 ℃·min^-1Rate be warming up to 120 DEG C, keep 8min；Again with 12 DEG C of min^-1Rate be warming up to 180 DEG C, keep 5min； Again with 15 DEG C of min^-1Rate be warming up to 270 DEG C, keep 5min；Injector temperature is 210~230 DEG C；Fid detector；Inspection It is 260~280 DEG C to survey device temperature；Volume flow is 0.5~2.0mLmin^-1；1~10 μ L of sample size, split sampling, split ratio It is 7~9：3~1；

(2) prepared by mixed reference substance solution：Take β-ocimenum, australene, myrtanal reference substance appropriate, it is accurately weighed, It is 0.5~2.0mgmL to add chloroform to be made containing β-ocimenum^-1, containing australene be 0.5~2.0mgmL^-1, contain hill gooseberry 0.5~2.0mgmL of aldehyde^-1Mixed reference substance solution；

(3) prepared by test solution：This product 0.20~1.0g is taken, it is weighed, it is placed in conical flask with cover, trichlorine is added in precision 15~30mL of methane, close plug, weighed weight, 10~20min of ultrasound are let cool, and weighed weight supplies the weight of less loss with chloroform Amount, shakes up, with 0.45 μm of membrane filtration, takes subsequent filtrate to get test solution.

(4) it measures：Precision draws mixed reference substance solution, each 1~10 μ L of test solution, injects gas chromatograph, into Row measures.

The content of β-ocimenum, australene, myrtanal is measured using gas chromatography, preferred steps are as follows：

(1) chromatographic condition：Chromatographic column：HP-5 capillary columns, specification：30m × 0.32mm, 0.25 μm；Temperature programming：Starting Temperature is 40 DEG C, 5min is kept, with 8 DEG C of min^-1Rate be warming up to 120 DEG C, keep 8min；Again with 12 DEG C of min^-1Speed Rate is warming up to 180 DEG C, keeps 5min；Again with 15 DEG C of min^-1Rate be warming up to 270 DEG C, keep 5min；Injector temperature is 220℃；Fid detector；Detector temperature is 270 DEG C；Volume flow is 1.0mLmin^-1；5 μ L of sample size, split sampling, point Stream is than being 8：2；

(2) prepared by mixed reference substance solution：Take β-ocimenum, australene, myrtanal reference substance appropriate, it is accurately weighed, It is 1.055mgmL to add chloroform to be made containing β-ocimenum^-1, containing australene be 1.115mgmL^-1, contain myrtanal 1.225mg·mL^-1Mixed reference substance solution；

(3) prepared by test solution：This product 0.60g is taken, it is weighed, it is placed in conical flask with cover, chloroform is added in precision 20mL, close plug, weighed weight, ultrasonic 15min are let cool, and weighed weight is supplied the weight of less loss with chloroform, shaken up, with 0.45 μm of membrane filtration, takes subsequent filtrate to get test solution.

(4) it measures：Precision draws mixed reference substance solution, each 5 μ L of test solution, injects gas chromatograph, is surveyed It is fixed.

We are a tcm small compound prescription, and side's solution is as follows：Radix Angelicae Sinensis, it is sweet, pungent, warm；Return liver, the heart, the spleen channel；With work of enriching blood Blood, menstruction regulating and pain relieving, the effect of relaxing bowel.Radix Paeoniae Alba, bitter, acid, is slightly cold；Return liver, the spleen channel；There is blood-nourishing to hold back the moon, mends and oiliness, soft liver In slow, sweat and other effects is received in analgesic.Milk thistle, it is bitter, it is cool.Return liver, gallbladder channel；With clearing heat and detoxicating, soothing liver-gallbladder and other effects；For liver Courage is damp and hot, hypochondriac pain, jaundice.Our all medicines share nourishing the liver tonifying spleen, Spleen Bugan, soothing the liver QI invigorating, menstruction regulating and pain relieving, promoting circulation of blood hemostasis, spleen For the foundation of acquired constitution, source of generating QI and blood, insufficiency of vital energy and blood all kinds of diseases and ailments are given birth to, and qi and blood abundance all kinds of diseases and ailments disappear, and spleen, which passes through, secretes various ferment (ferment Be exactly enzyme, be commonly called as catalyst, including liver and gall lipidase etc.) it is main the palm nutrition absorption and qi and blood the generating resource.Liver and gall belong to wood, spleen The stomach pertaining to earth, wood can dredge soil, introduced in our Return liver, gallbladder channel milk thistle, therefore milk thistle has effects that invigorating the spleen tonifying spleen, makes spleen The gentle blood biochemistry of nutrient absorption can be healthily carried out, in addition, Chinese medicine claims not general rule pain, and we have nourishing the liver tonifying spleen, invigorating the spleen The effect of tonifying liver, can promote the transporting of qi and blood, soothing the liver QI invigorating, menstruction regulating and pain relieving, promoting circulation of blood hemostasis.Have to primary dysmenorrhea fine Therapeutic effect, for effect a radical cure primary dysmenorrhea effective prescription.

Technological means, technical characteristic, reached purpose and effect to make the present invention realize are easy to understand, reality below Test or test the technical solution for being used for that the present invention is further explained.

Experiment one：Treat the medicament screening experiment research of primary dysmenorrhea

1 instrument and material

1.1 instrument

The German full-automatic microplate reader of IFP (RT2100C) is purchased from Beijing Bioisystech Co., Ltd of Bo Ou Shides；BL-420E Biological functional system is purchased from Northern Huaihe River Anhui Anhui Zhenghua Biology Instrument Equipment Co., Ltd..

1.2 drugs, reagent

1.2.1 given the test agent G1：Prescription：Radix Angelicae Sinensis 500g, Radix Paeoniae Alba 250g, milk thistle 250g；

Preparation method：(1) it takes Radix Angelicae Sinensis, Radix Paeoniae Alba, milk thistle, is added the bionic extraction solvents of 30 times of amounts, 37 DEG C of heating water baths, Stirring extraction 3h, centrifuges 35min, centrifugal speed 2800r/min, collects supernatant, supernatant filtration, and concentration obtains medicinal extract I；On It is by 0.6% sodium chloride, 0.7% pepsin, 0.6% trypsase and 0.07molL to state bionic extraction solvent^-1Hydrochloric acid is matched Solution is made；

(2) medicinal extract I for taking step (1), with purify it is water-dispersed after, cross H-30 type large pore resin absorption columns, 8 columns are washed with water Volume, eluent discard, then wash 6 column volumes with 15% methanol, and eluent discards again, finally wash 12 cylinders with 65% methanol Product, collects eluent, filtration, and filtrate concentration is freeze-dried to get given the test agent G1.

1.2.2 given the test agent G2：Prescription：Radix Angelicae Sinensis 500g, Radix Paeoniae Alba 250g, milk thistle 250g；

Preparation method：(1) Radix Angelicae Sinensis, Radix Paeoniae Alba, milk thistle are taken, is put into ultrasonic-microwave reactor, 30 times of amounts of addition Then 65% ethanol solution reacts in ultrasonic-microwave reactor, ultrasonic power 1300W, microwave power are 370W, reaction time 45min, reaction temperature are 35 DEG C, and the liquid filtration after reaction, filtrate concentration obtains medicinal extract I；

(2) medicinal extract I for taking step (1), with purify it is water-dispersed after, 8 column volumes, eluent is washed with water in upper MCI resin columns It discards, then 6 column volumes is washed with 15% methanol, eluent discards again, finally washes 12 column volumes with 65% methanol, collects elution Liquid, filtration, filtrate concentration are freeze-dried to get given the test agent G2.

1.2.3 given the test agent G3：Prescription：Radix Angelicae Sinensis 500g, Radix Paeoniae Alba 250g, milk thistle 250g；

Preparation method：(1) Radix Angelicae Sinensis, Radix Paeoniae Alba, milk thistle are taken, carries out steam distillation extraction, collection obtains volatile oil, standby With；Extracting solution after steam distillation extraction, filters, and concentration obtains medicinal extract I；The dregs of a decoction after steam distillation extraction are spare；

1.2.4 acetylsalicylic acid tablet is produced by HARBIN PHARMACEUTICAL GROUP CO., LTD. General Pharm. Factory, authentication code：Chinese medicines quasi-word H23021185, rule Lattice：0.3g；

1.2.5 GUIZHI FULING JIAONANG is produced by Kangyuan Pharmaceutical Co., Ltd., Jiangsu Prov, authentication code：Chinese medicines quasi-word Z10950005, specification：Every dress 0.31g；

1.2.6 diethylstilbestrol injection is produced by Tianjin Kingyork Pharmaceutical Co., Ltd., authentication code：Chinese medicines quasi-word H12020536, specification：1ml:0.5mg.

1.2.7 oxytocin injection is produced by Beijing Saisheng Pharmaceutical Co., Ltd., authentication code：Chinese medicines quasi-word H11020363, dosage form：Injection (small-volume injection), specification：1ml:10 units.

1.2.8 prostaglandin F_2αEnzyme-linked immunologic detecting kit is purchased from Tong Wei reagents (Shanghai) Co., Ltd.；Prostaglandin E₂Enzyme-linked immunologic detecting kit is purchased from the Shanghai bio tech ltd Heng Yuan.

1.3 animal for research

SPF grades of SD rats, 190~210g of weight, female, quality certification number P00102013 are big by Heilungkiang traditional Chinese medicine Drug safety assessment center is learned to provide.Adaptable fed 1 week before experiment, temperature are (20 ± 2) DEG C, 12h illumination, humidity 40%, ad lib, single cage raising excludes diet and environment etc. and is possible to the influence generated to experimental animal.

2 methods

The 2.1 pairs of influences of oxytocin induced pain through rat model

Mouse is taken, is randomly divided into 7 groups, every group of 10 rats, respectively：Blank control group, model control group, aspirin Group, GUIZHI FULING JIAONANG group and given the test agent G1, given the test agent G2, given the test agent G3.Diethylstilbestrol injection is subcutaneously injected, even Continuous 14d, one time a day, each 0.8mg.Since the 5th day after injecting diethylstilbestrol, rat is administered, aspirin group is given 0.03g·kg^-1Aspirin, GUIZHI FULING JIAONANG group gives 2.0gkg^-1GUIZHI FULING JIAONANG, given the test agent G1 groups Give 2.0gkg^-1Given the test agent G1, given the test agent G2 groups give 2.0gkg^-1Given the test agent G2, given the test agent G3 groups Give 2.0gkg^-1Given the test agent G3, according to 10mLkg^-1Capacity gastric infusion, blank control group and model comparison Group gives isometric physiological saline respectively, one time a day, continuous 10d.10d last time injection diethylstilbestrols 12h, administration After 2h, oxytocin is injected intraperitoneally, dosage is 2U/, observes the pain reaction of rat in 40min, is son with writhing response The index of palace strong contraction, including writhing incidence, incubation period and number.

Writhing incidence=generation writhing number of animals/10 × 100%.

After observing 40min, rat is put to death, uterus is removed, uterine tissue 0.5g is weighed, adds physiological saline 1.0mL, It is first homogenized, then homogenate is centrifuged, after centrifugation, taken the supernatant of centrifugation, use prostaglandin F_2αEnzyme linked immunological is examined Test agent box, Prostaglandin PGE₂Enzyme-linked immunologic detecting kit carries out assay.

2.2 statistical method

The data result for testing gained carries out statistical procedures using statistics software SPSS11.0.

3 results

The 3.1 pairs of influences of oxytocin induced pain through rat model

Compared with blank control group, model control group rat writhing incidence increases (P ＜ 0.01), writhing shorter latencies (P ＜ 0.01)；Writhing number increases (P ＜ 0.01) in 30min；Uterine tissue PGF_2αContent increases (P ＜ 0.01), PGE₂Content It reduces (P ＜ 0.05), PGF_2α/ PGE2 ratios increase (P ＜ 0.01).Compared with model control group, given the test agent G3 groups, cassia twig Fu Siberian cocklebur Capsules group, aspirin group have apparent suppression to writhing number in the writhing incubation period of rat caused by oxytocin and 30min It makes and uses (P ＜ 0.01), PGF can be reduced_2α/PGE₂Ratio (P ＜ 0.01)；Given the test agent G3 groups, GUIZHI FULING JIAONANG group, A Si Woods group can reduce the PGF in rat endometrium_2αContent (P ＜ 0.05 or 0.01) increases PGE in rat endometrium₂Contain It measures (P ＜ 0.05).Given the test agent G1 groups, given the test agent G2 groups also have certain effect, but compared with model control group, without significantly Sex differernce.It is shown in Table 1, table 2.

Table 1 to rat pain writhing response caused by oxytocin influence (N=10)

Note：Compared with model group^*P ＜ 0.05,^**P ＜ 0.01；Compared with blank control group^#P ＜ 0.05,^##P ＜ 0.01.

Table 2 is to rat uterus tissue PGF_2αAnd PGE₂Influence (N=10)

4 conclusions

It can be seen from the experiment that given the test agent G3 can effectively inhibit the writhing incidence of dysmenorrhea model rat, effectively extend The incubation period of writhing, the writhing number being effectively reduced in 30min.Given the test agent G3 is capable of the uterus muscle of antagonism oxytocin induction The pain caused reaction of spasm reduces PGF in rat endometrium_2αContent, increase endometrium in PGE₂Content.Illustrate by Test agent G3 has good therapeutic effect to experimental primary dysmenorrhea.

Experiment two：Using silydianin, gallic acid, asafoetide in hplc simultaneous determination drug of the present invention The content of acid, vanillic acid

Premenstruum (premenstrua) in gallic acid, Radix Angelicae Sinensis contained in silydianin, Radix Paeoniae Alba contained in milk thistle studies have shown that contain Ferulic acid, vanillic acid drug therapy dysmenorrhoea of the present invention main active, inventor establishes logical by testing repeatedly It crosses high performance liquid chromatography disposably while measuring the detection method of silydianin, gallic acid, ferulic acid, vanilla acid content, Research is as follows.

1 material and reagent

Shimadzu 20-AD high performance liquid chromatographs (Japanese Shimadzu, including quaternary pump, on-line degassing machine, automatic sampling Device, column oven, DAD detectors, 2010 chromatographic work stations)；

Electronic analytical balance (Shanghai Yue Zi Electronic Science and Technology Co., Ltd.s)；

Ultrasonic cleaner (Shenzhen Keepahead Ultrasonic Cleaning Equipment Co., Ltd.).

Silydianin reference substance (the Shanghai bio tech ltd Ya Ji, lot number 102012-20160325)；

Gallic acid reference substance (Shanghai Rui Qi life sciences Co., Ltd, lot number 111202-20160115)；

Macrotin reference substance (bio tech ltd Wei Keqi of Sichuan Province, lot number 121115-20160220)；

Vanillic acid reference substance (Shanghai Tauto Biotechnology Co., Ltd., lot number 110326-20151228)；

Acetonitrile is chromatographically pure；Ethyl alcohol, glacial acetic acid are that analysis is pure；Water is ultra-pure water.

Drug of the present invention：Prescription：Radix Angelicae Sinensis 500g, Radix Paeoniae Alba 250g, milk thistle 250g；

2 methods and result

The preparation of 2.1 solution

2.1.1 the preparation of mixed reference substance solution

Silydianin, ferulic acid, gallic acid, vanillic acid reference substance are taken respectively, it is weighed, with methanol dilution at every 1mL points The not 65 μ g containing silydianin, 20 μ g of ferulic acid, 15 μ g of gallic acid, the mixed reference substance solution of 20 μ g of vanillic acid, mixing to get Mixed reference substance solution.

2.1.2 the preparation of test solution

Sample is taken, it is finely ground, 1.00g is weighed, is set in conical flask with cover, precision plus methanol 25mL are weighed, in 400W, 40kHz Power under, ultrasonic 25min, methanol supplies loss of weight, filtration, take subsequent filtrate to get test solution.

2.1.3 the preparation of negative test solution

Prepared respectively according to recipe quantity scarce milk thistle, Radix Paeoniae Alba, Radix Angelicae Sinensis negative test sample, be made by method provided by the invention Negative test solution.

2.2 chromatographic conditions and system suitability are investigated

Using C₁₈Chromatographic column, specification：4.6mm × 250mm, 5 μm；Mobile phase：- 1% glacial acetic acid solution of acetonitrile, gradient are washed De-, eluting order is：From 0min to 15min, acetonitrile is maintained at 10%, and 1% glacial acetic acid solution is maintained at 90%；From 16min to 35min, acetonitrile is from 10% linear rise to 30%, 1% glacial acetic acid solution from 90% linear decline to 70%；From 36min to 40min, acetonitrile is from 30% linear rise to 40%, 1% glacial acetic acid solution from 70% linear decline to 60%； From 41min to 50min, acetonitrile from 40% linear rise to 45%, 1% glacial acetic acid solution from 60% linear decline to 55%；From 51min to 60min, acetonitrile is from 45% linear rise to 55%, 1% glacial acetic acid solution from 55% linear decline To 45%；Flow velocity 1.0mLmin^-1；38 DEG C of column temperature；Detection wavelength 257nm；10 μ L of sample size.Under above-mentioned chromatographic condition, mix Closing reference substance solution, test solution, negative test solution chromatogram sees Figure of description 1 to attached drawing 5.In test solution Other compositions interfere very little to ingredient to be measured.

The investigation of 2.3 linear relationships

Mixed reference substance solution 1.0mL, 2.0mL, 4.0mL, 6.0mL, 8.0mL are taken respectively, are respectively placed in 10mL measuring bottles, Add methanol dilution to scale.Each 10 μ L are respectively taken respectively, are injected high performance liquid chromatograph, are washed by chromatographic condition provided by the invention It is de-, with each reference substance peak area Y to its concentration X (mgL^-1) linear regression is carried out, regression equation is obtained, it is as follows respectively：

The regression equation of silydianin：Y=8.5 × 10⁶X-12568.36, r=0.9998, the range of linearity be 13.25~ 165.26mg·L^-1。

The regression equation of gallic acid：Y=3.5 × 10⁷X-6254.23, r=0.9997, the range of linearity be 1.96~ 41.23mg·L^-1。

The regression equation of ferulic acid：Y=3.5 × 10⁷X-3526.28, r=0.9998, the range of linearity be 2.18~ 44.25mg·L^-1。

The regression equation of vanillic acid：Y=3.4 × 10⁷X+2261.88, r=0.9996, the range of linearity be 1.95~ 48.65mg·L^-1。

2.4 precision test

Sample (lot number 20160625) is taken, test solution is prepared by method provided by the invention, is provided by the present invention Condition repeat sample introduction 5 times, measure peak area.The RSD (n=5) of silydianin, gallic acid, ferulic acid, vanillic acid peak area Respectively 2.1%, 1.6%, 1.8%, 0.9%, show that precision is good.

2.5 stability test

Take same sample solution respectively 1,2,4,8,12,16h sample introduction measure, silydianin, gallic acid, ferulic acid, The RSD of vanillic acid peak area is respectively 1.6%, 1.4%, 1.3%, 0.9%, shows that test solution is stablized in 16h.

2.6 repetitive test

6 parts of sample is taken, every part of about 1g is accurately weighed, is prepared into test solution by method provided by the invention, sample introduction is surveyed It is fixed, silydianin, gallic acid, ferulic acid, vanillic acid average mass fraction be respectively 1052.6,368.5,454.2, 565.8μg·g^-1, RSD 1.5%, 1.4%, 1.6%, 1.9%.

2.7 sample recovery rates are tested

6 parts, every part of 1.0g of the sample of known content is taken, accurately weighed, same amount of mixed reference substance solution is added in precision, It is measured by condition provided by the invention, calculates the rate of recovery, the results are shown in Table 3.The result shows that detection method provided by the invention is accurate It spends.

The sample recovery rate (n=6) of 3 silydianin of table, gallic acid, ferulic acid, vanillic acid

2.8 sample sizes measure

It takes 5 batches, sample, 2 parts of every batch of to prepare test solution by method provided by the invention, is surveyed according to above-mentioned chromatographic condition It is fixed, peak area is recorded, and content is calculated by external standard method, the results are shown in Table 4.

Mass fraction (the μ gg of 4 silydianin of table, gallic acid, ferulic acid, vanillic acid^-1)

3 conclusions

The present invention uses the content of 4 kinds of index ingredients in hplc simultaneous determination drug of the present invention, this hair Bright detection method precision is high, and reproducible, stability can be used for the quality testing of this product.

Experiment three：Gas chromatography measures the content of β-ocimenum, australene, myrtanal in drug of the present invention

Premenstruum (premenstrua), myrtanal contained in Radix Paeoniae Alba was this hair studies have shown that β-ocimenum, australene contained in Radix Angelicae Sinensis The main active of bright drug therapy dysmenorrhoea, inventor establish disposable by gas chromatography by testing repeatedly The detection method of the content of β-ocimenum, australene, myrtanal in drug of the present invention is measured simultaneously, and research is as follows.

1 instrument and material

1.1 instrument

Gas chromatograph；HP-5 capillary columns, specification：30m × 0.32mm, 0.25 μm；Electronic analytical balance (the tall and erect essence in Shanghai Electronic Science and Technology Co., Ltd.)；Ultrasonic washing instrument (Ningbo Bo Er ultrasonic equipments Co., Ltd).

1.2 material

β-ocimenum reference substance (is purchased from Guangdong Weng Jiang chemical reagent Co., Ltd, lot number：101205-20160223, content 99.8%), australene reference substance is (purchased from the biological Co., Ltd in ancient of Shanghai, lot number：111014-20160325, content 99.9%), myrtanal reference substance (is purchased from Shanghai Rong Chuan Bioisystech Co., Ltd, lot number：111230-20160219, content 99.9%).

Drug of the present invention：Drug of the present invention：Prescription：Radix Angelicae Sinensis 500g, Radix Paeoniae Alba 250g, milk thistle 250g；

Chloroform (analysis is pure, is purchased from Shanghai Lv Shi Chemical Co., Ltd.s).

2 methods

2.1 chromatographic condition

Chromatographic column：HP-5 capillary columns, specification：30m × 0.32mm, 0.25 μm；Temperature programming：Initial temperature is 40 DEG C, 5min is kept, with 8 DEG C of min^-1Rate be warming up to 120 DEG C, keep 8min；Again with 12 DEG C of min^-1Rate be warming up to 180 DEG C, keep 5min；Again with 15 DEG C of min^-1Rate be warming up to 270 DEG C, keep 5min；Injector temperature is 220 DEG C；FID is examined Survey device；Detector temperature is 270 DEG C；Volume flow is 1.0mLmin^-1；5 μ L of sample size, split sampling, split ratio 8：2.

It is prepared by 2.2 mixed reference substance solutions

Take β-ocimenum, australene, myrtanal reference substance appropriate, it is accurately weighed, add chloroform to be made containing β-sweet basil Alkene is 1.055mgmL^-1, containing australene be 1.115mgmL^-1, 1.225mgmL containing myrtanal^-1Mixing reference substance it is molten Liquid.

It is prepared by 2.3 test solutions

This product 0.60g is taken, it is weighed, it is placed in conical flask with cover, precision is added chloroform 20mL, close plug, weighed weight, Ultrasonic 15min, lets cool, and weighed weight is supplied the weight of less loss with chloroform, shaken up, and with 0.45 μm of membrane filtration, takes continuous filter Liquid is to get test solution.

It is prepared by 2.4 negative sample solution

With reference to the preparation method of drug of the present invention, scarce Radix Angelicae Sinensis and the negative sample of Radix Paeoniae Alba is made.Under above-mentioned chromatographic condition, respectively Precision draws mixed reference substance solution, test solution and each 5 μ L of negative sample solution, injects gas chromatograph, records gas phase Chromatogram.

The experimental results showed that β-ocimenum, australene, myrtanal divide with adjacent chromatographic peak in test solution chromatography 1.5 are all higher than from degree, and negative sample is noiseless.See Figure of description 6, attached drawing 7, attached drawing 8.

2.5 linear test

Accurate absorption mixed reference substance solution is appropriate respectively, and it is respectively 1.8~180.0 μ g to add chloroform that concentration is made mL^-1Mixed reference substance solution.With concentration (μ gmL^-1) it is abscissa, linear regression is carried out by ordinate of peak area, is obtained Regression equation, related coefficient and the range of linearity are as follows respectively：

β-ocimenum regression equation：Y=2025.8X-2156.4, r=0.9998,1.954~592.5 μ of the range of linearity g·mL^-1；

Australene regression equation：Y=1965.2X-1826.4, r=0.9999,2.138~698.6 μ g of the range of linearity mL^-1；

Myrtanal regression equation：Y=1842.8X-2157.6, r=0.9998,2.568~304.6 μ of the range of linearity g·mL^-1；

2.6 precision test

Precision draws the mixed reference substance solution of a concentration of 10 μ g/mL, repeats sample introduction 5 times.Experimental result is shown：β-sweet basil The RSD values of alkene peak area are 0.36%, the RSD values of australene peak area are 0.52%, the RSD values of myrtanal peak area are 0.62%, show that instrument precision is good.

2.7 stability test

Precision draws same test solution, respectively at 0h, 2h, 4h, 6h, 8h, 12h sample introduction 6 times.Experimental result is shown： The RSD of β-ocimenum area is 0.96%, the RSD of australene area is 1.22%, the RSD of myrtanal peak area is 1.06%, show that test solution is basicly stable in 12h.

2.8 repetitive test

6 parts of samples are taken simultaneously, test solution is prepared according to method provided by the invention, measures and calculate content.Experiment knot Fruit shows：β-ocimenum content RSD values are 0.62% in sample, australene content RSD values are 0.58%, myrtanal content RSD values are 0.56%, show that the repeatability of assay method is good.

2.9 recovery test

Take 6 parts of sample, a concentration of 55.45 μ gmL of every part of addition β-ocimenum^-1, australene a concentration of 57.80 μ g·mL^-1, myrtanal a concentration of 58.75 μ gmL^-1Each 1.0mL of mixed reference substance solution, measure its content in accordance with the law, count Calculate the rate of recovery.

Experimental result is shown：The average recovery rate of β-ocimenum is 100.26%, RSD 0.95%；Australene is averaged The rate of recovery is 100.40%, RSD 0.56%；The average recovery rate of myrtanal is 99.46%, RSD 0.75%.Be shown in Table 5, Table 6, table 7.

5 β of table-ocimenum recovery test result

6 australene recovery test result of table

7 myrtanal recovery test result of table

2.10 sample measures

Drug 5 batches of the present invention is taken, test solution is prepared according to method provided by the invention, measure and calculates wherein β-sweet basil Alkene, australene, myrtanal content.It the results are shown in Table 8.

8 sample size measurement result (μ gg of table^-1)

3 conclusions

The present invention measures the content of 3 kinds of index ingredients in drug of the present invention using gas chromatography simultaneously, of the invention Detection method precision is high, and reproducible, stability can be used for the quality testing of this product.

Experiment four：Measure the comparative experiments of active component content in the test medicine prepared under different preparation methods

The test medicine prepared under 1 different preparation methods

1.1 given the test agent G1：Prescription：Radix Angelicae Sinensis 500g, Radix Paeoniae Alba 250g, milk thistle 250g；

1.2 given the test agent G2：Prescription：Radix Angelicae Sinensis 500g, Radix Paeoniae Alba 250g, milk thistle 250g；

1.3 given the test agent G3：Prescription：Radix Angelicae Sinensis 500g, Radix Paeoniae Alba 250g, milk thistle 250g；

2 measure

Using the content of active constituent in high performance liquid chromatography provided by the invention and Drugs by GC.

3 measurement results

Measurement result is shown in Table 9.

Active component content (μ gg in the test medicine prepared under the different preparation methods of table 9^-1)

4 conclusions

By experimental result as it can be seen that the test medicine G3 being prepared using preparation method provided by the invention, is contained Silydianin, gallic acid, ferulic acid, vanillic acid, β-ocimenum, australene, myrtanal content be apparently higher than other systems The drug of gained is prepared under Preparation Method, this is also likely to be that test medicine G3 (drug of the present invention) is better than it the effect for the treatment of dysmenorrhoea The reason of his drug.

Experiment five：The effect of drug therapy primary dysmenorrhea of the present invention, is observed

Using drug therapy primary dysmenorrhea of the present invention totally 18, because of the limitation of technical regulation, drug of the present invention can not also To do large-scale clinical experimental study, 18 only accepted for medical treatment with affiliated hospital of Jiamusi institute of Heilongjiang University of Chinese Medicine are voluntarily The small sample of person has done preliminary clinical experimental study.18 patients endorsed《Informed consent form》, voluntarily receive clinical examination It tests.Research is as follows.

1 data and method

1.1 general information

18 dysmenorrhoea patients are affiliated hospital of Jiamusi institute of Heilongjiang University of Chinese Medicine outpatients of gynecology, age 23 ~32 years old, the course of disease 3 months to 3 years.

1.2 diagnostic criteria

Reference《Gynecology of Chinese Medicine》Diagnostic criteria draft：Women in 1 week, occurs with week before and after menstrual period or menstrual period Phase property hypogastralgia is primary symptom, with other discomforts, and study, work and life is affected, through gynecologial examination, pelvic cavity genitals Official is without organic disease.

1.3 case inclusion criterias

(1) meet《Gynecology of Chinese Medicine》The diagnostic criteria of primary dysmenorrhea；(2) dialectical category qi stagnation and blood stasis type or cold blood stasis Type；(3) age is between 20~38 years old；(4) menstrual cycle rule；(5) 2 Zhou Qianwei of this experiment take sedative, anodyne and Hormone medicine；(6) dysmenorrhoea symptom score total score >=8 point；(7) informed consent form is signed.

1.4 exclusion criteria

(1) gynecological diseases such as pelvic infecton, fibroid, endometriosis are suffered from；(2) age is less than 15 years old or big In 40 years old；(3) gestation or women breast-feeding their children；(4) there is cardiovascular and cerebrovascular disease, there is liver kidney system serious disease.

2 therapies

Give drug of the present invention：Prescription：Radix Angelicae Sinensis 500g, Radix Paeoniae Alba 250g, milk thistle 250g；

(6) medicinal extract V of step (5) is taken to merge with the volatile oil of step (1), suitable starch, dextrin is added in mixing, system Grain, freeze-drying are packed into hard capsule, hard capsule 1000 are made.

Usually give drug of the present invention 2 tablets each time, it is twice daily, oral in the morning and evening respectively, give every time 4 menstruation previous week Grain, oral in the morning and evening respectively, the menstrual period is not discontinued；One menstrual cycle is as a treatment course, 3 courses for the treatment of of taking medicine.

3 criterions of therapeutical effect

3.1 curing：Integral restores to 0 point after medication, and menstrual period abdominal pain and other symptoms disappear, and are discontinued 3 menstrual cycles It does not recur；

3.2 effective：Post treatment integral is down to the 1/2 of prior treatment integral hereinafter, abdominal pain is substantially reduced, remaining symptom improves, Refuse to obey that anodyne can adhere to work；

3.3 effectively：Post treatment integral is down to the 1/2~3/4 of prior treatment integral, and abdominal pain mitigates, remaining symptom improves, and clothes stop Pain medicine can adhere to work；

3.4 invalid：Abdominal pain and other symptoms are without change.

4 results

4.1 clinical efficacy

18 primary dysmenorrhea patients are observed, are cured 12 (66.66%), it is effective 4 (22.22%), effective 2 (11.12%), invalid 0 (0%).Total effective rate is 100%.

Treatment cycle：Most short 15d, longest 60d.

4.2 toxic side effect

Do not occur the adverse reactions such as allergy, poisoning in observation period.

5 conclusions

Drug therapy dysmenorrhoea of the present invention has significant in efficacy, Small side effects, advantage uneasy to recur.

Description of the drawings：

Fig. 1 is the high-efficient liquid phase chromatogram of mixed reference substance solution, wherein：No. 1 peak is silydianin, and No. 2 peaks are asafoetide Acid；No. 3 peaks are gallic acid；No. 4 peaks are vanillic acid.

Fig. 2 is the high-efficient liquid phase chromatogram of test solution, wherein：No. 1 peak is silydianin, and No. 2 peaks are ferulic acid；3 Number peak is gallic acid；No. 4 peaks are vanillic acid.

Fig. 3 is the high-efficient liquid phase chromatogram for the negative test solution for lacking milk thistle, wherein：No. 2 peaks are ferulic acid；No. 3 Peak is gallic acid；No. 4 peaks are vanillic acid.

Fig. 4 is the high-efficient liquid phase chromatogram for the negative test solution for lacking Radix Paeoniae Alba, wherein：No. 1 peak is silydianin, No. 2 Peak is ferulic acid；No. 4 peaks are vanillic acid.

Fig. 5 is the high-efficient liquid phase chromatogram for the negative test solution for lacking Radix Angelicae Sinensis, wherein：No. 1 peak is silydianin, No. 3 Peak is gallic acid

Fig. 6 is mixed reference substance solution gas chromatogram, wherein No. 1 peak is β-ocimenum, No. 2 peaks are australene, No. 3 peaks For myrtanal.

Fig. 7 is test solution gas chromatogram, wherein No. 1 peak is β-ocimenum, No. 2 peaks are australene, and No. 3 peaks are peach Golden ma's aldehyde.

Fig. 8 is negative sample solution gas chromatogram.

Specific implementation mode

Embodiment 1：

Prescription：Radix Angelicae Sinensis 500g, Radix Paeoniae Alba 250g, milk thistle 250g；

(4) it measures：Precision draws mixed reference substance solution, each 10 μ L of test solution, injects high performance liquid chromatograph, into Row measures；

(5) measurement result：Every gram of 1035.3 μ gg containing silydianin of this product^-1, 388.6 μ gg of gallic acid^-1, asafoetide 492.6 μ gg of acid^-1, 614.8 μ gg of vanillic acid^-1。

(5) measurement result：Every gram of 241.62 μ gg containing β-ocimenum of this product^-1, 322.46 μ gg of australene^-1, hill gooseberry 180.55 μ gg of aldehyde^-1。

The above is only a preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Claims

1. a kind of drug for treating primary dysmenorrhea, which is characterized in that the drug is made of the flavour of a drug raw material of following parts by weight：When Return 1~3 parts by weight, 1~2 parts by weight of Radix Paeoniae Alba, 1~2 parts by weight of milk thistle.

2. drug according to claim 1, which is characterized in that the drug is made of the flavour of a drug raw material of following parts by weight：When Return 2 parts by weight, 1 parts by weight of Radix Paeoniae Alba, 1 parts by weight of milk thistle.

3. a kind of process for preparing medicine for treating primary dysmenorrhea, which is made of the flavour of a drug raw material of following parts by weight：Radix Angelicae Sinensis 1 ~3 parts by weight, 1~2 parts by weight of Radix Paeoniae Alba, 1~2 parts by weight of milk thistle, which is characterized in that preparation method is：

(1) Radix Angelicae Sinensis, Radix Paeoniae Alba, milk thistle are taken, carries out steam distillation extraction, collection obtains volatile oil, spare；Steam distillation carries Rear extracting solution is taken, is filtered, concentration obtains medicinal extract I；The dregs of a decoction after steam distillation extraction are spare；

(2) dregs of a decoction of step (1) are taken, the bionic extraction solvent of 20~40 times of amounts, 36~38 DEG C of heating water baths, stirring extraction is added 2~4h centrifuges 30~40min, 2500~3000r/min of centrifugal speed, collects supernatant, supernatant filtration, and concentration obtains medicinal extract Ⅱ；Above-mentioned bionic extraction solvent is by 0.5%~0.8% sodium chloride, 0.5%~0.8% pepsin, 0.5%~0.8% pancreas Protease and 0.06molL^-1~0.08molL^-1Hydrochloric acid is configured to solution；

(3) medicinal extract I of step (1) is mixed with the medicinal extract II of step (2), is put into ultrasonic-microwave reactor, addition 20~ 40 times amount 60%~70% ethanol solutions, then reacted in ultrasonic-microwave reactor, ultrasonic power be 1200~ 1400W, microwave power are 360~380W, the reaction time is 40~50min, reaction temperature is 30~40 DEG C, the liquid after reaction Filtration, filtrate concentration, obtains medicinal extract III；

(4) medicinal extract III for taking step (3), with purify it is water-dispersed after, cross H-30 type large pore resin absorption columns, be washed with water 6~10 Column volume, eluent discard, then wash 4~7 column volumes with 10%~20% methanol, and eluent discards again, finally with 60%~ 70% methanol washes 10~15 column volumes, collects eluent, and filtration obtains filtrate IV；

(5) filtrate IV of step (4), then upper MCI resin columns are taken, 6~10 column volumes are washed with water, eluent discards, then uses 10%~20% methanol washes 4~7 column volumes, and eluent discards again, finally washes 10~15 cylinders with 60%~70% methanol Product, collects eluent, filtration, and filtrate concentration obtains medicinal extract V；

4. process for preparing medicine according to claim 3, which is made of the flavour of a drug raw material of following parts by weight：Radix Angelicae Sinensis 2 Parts by weight, 1 parts by weight of Radix Paeoniae Alba, 1 parts by weight of milk thistle；It is characterized in that, preparation method is：

(2) dregs of a decoction of step (1) are taken, the bionic extraction solvent of 30 times of amounts, 37 DEG C of heating water baths, stirring extraction 3h, centrifugation is added 35min, centrifugal speed 2800r/min, collect supernatant, supernatant filtration, and concentration obtains medicinal extract II；Above-mentioned bionic extraction solvent It is by 0.6% sodium chloride, 0.7% pepsin, 0.6% trypsase and 0.07molL^-1Hydrochloric acid is configured to solution；

(3) medicinal extract I of step (1) is mixed with the medicinal extract II of step (2), is put into ultrasonic-microwave reactor, be added 30 times 65% ethanol solution of amount, then reacts in ultrasonic-microwave reactor, ultrasonic power 1300W, microwave power are 370W, reaction time 45min, reaction temperature are 35 DEG C, and the liquid filtration after reaction, filtrate concentration obtains medicinal extract III；

(4) medicinal extract III for taking step (3), with purify it is water-dispersed after, cross H-30 type large pore resin absorption columns, 8 cylinders are washed with water Product, eluent discards, then washes 6 column volumes with 15% methanol, and eluent discards again, finally washes 12 cylinders with 65% methanol Product, collects eluent, and filtration obtains filtrate IV；

(5) it takes the filtrate IV of step (4), then upper MCI resin columns, 8 column volumes is washed with water, eluent discards, then with 15% first Alcohol washes 6 column volumes, and eluent discards again, finally washes 12 column volumes with 65% methanol, collects eluent, filters, and filtrate is dense Contracting, obtains medicinal extract V；

5. process for preparing medicine according to claim 3 or 4, which is characterized in that the drug uses in pharmacy of Chinese materia medica often Oral preparation is made in the pharmaceutical methods of rule.

6. process for preparing medicine according to claim 5, which is characterized in that the oral preparation is tablet, pill, capsule Agent, powder or oral solution.

7. a kind of detection method of drug that treating primary dysmenorrhea, the drug are made of the flavour of a drug raw material of following parts by weight：When 1~3 parts by weight, 1~2 parts by weight of Radix Paeoniae Alba, 1~2 parts by weight of milk thistle, preparation method are returned to be：

It is characterized in that, using silydianin, gallic acid, ferulic acid, vanilla in hplc simultaneous determination drug The content of acid, steps are as follows：

(1) chromatographic condition：Using C₁₈Chromatographic column, specification：4.6mm × 250mm, 5 μm；Mobile phase：- 1% glacial acetic acid solution of acetonitrile, Gradient elution, eluting order are：From 0min to 15min, acetonitrile is maintained at 10%, and 1% glacial acetic acid solution is maintained at 90%；From 16min to 35min, acetonitrile is from 10% linear rise to 30%, 1% glacial acetic acid solution from 90% linear decline to 70%； From 36min to 40min, acetonitrile from 30% linear rise to 40%, 1% glacial acetic acid solution from 70% linear decline to 60%；From 41min to 50min, acetonitrile is from 40% linear rise to 45%, 1% glacial acetic acid solution from 60% linear decline To 55%；From 51min to 60min, acetonitrile is from 45% linear rise to 55%, 1% glacial acetic acid solution under 55% is linear It is down to 45%；0.5~1.5mLmin of flow velocity^-1；35~40 DEG C of column temperature；255~260nm of Detection wavelength；5~20 μ L of sample size；

(2) preparation of mixed reference substance solution：Silydianin, ferulic acid, gallic acid, vanillic acid reference substance are taken respectively, it is weighed, With methanol dilution at every 1mL 60~70 μ g containing silydianin, 15~20 μ g of ferulic acid, 10~20 μ g of gallic acid respectively, vanilla The mixed reference substance solution of 15~20 μ g of acid, mixing is to get mixed reference substance solution；

(3) preparation of test solution：Sample is taken, it is finely ground, 1.00g is weighed, is set in conical flask with cover, accurate plus methanol 15~ 30mL, weighed, at 350~450W, the power of 35~45kHz, 20~30min of ultrasound, methanol supplies loss of weight, and filtration takes continuous Filtrate is to get test solution；

(4) it measures：Precision draws mixed reference substance solution, each 5~20 μ L of test solution, injects high performance liquid chromatograph, into Row measures.

8. the detection method of drug as claimed in claim 7, which is made of the flavour of a drug raw material of following parts by weight：Radix Angelicae Sinensis 2 Parts by weight, 1 parts by weight of Radix Paeoniae Alba, 1 parts by weight of milk thistle；Preparation method is：

(1) chromatographic condition：Using C₁₈Chromatographic column, specification：4.6mm × 250mm, 5 μm；Mobile phase：- 1% glacial acetic acid solution of acetonitrile, Gradient elution, eluting order are：From 0min to 15min, acetonitrile is maintained at 10%, and 1% glacial acetic acid solution is maintained at 90%；From 16min to 35min, acetonitrile is from 10% linear rise to 30%, 1% glacial acetic acid solution from 90% linear decline to 70%； From 36min to 40min, acetonitrile from 30% linear rise to 40%, 1% glacial acetic acid solution from 70% linear decline to 60%；From 41min to 50min, acetonitrile is from 40% linear rise to 45%, 1% glacial acetic acid solution from 60% linear decline To 55%；From 51min to 60min, acetonitrile is from 45% linear rise to 55%, 1% glacial acetic acid solution under 55% is linear It is down to 45%；Flow velocity 1.0mLmin^-1；38 DEG C of column temperature；Detection wavelength 257nm；10 μ L of sample size；

(2) preparation of mixed reference substance solution：Silydianin, ferulic acid, gallic acid, vanillic acid reference substance are taken respectively, it is weighed, With methanol dilution at every 1mL 65 μ g containing silydianin, 20 μ g of ferulic acid, 15 μ g of gallic acid respectively, the mixing of 20 μ g of vanillic acid Reference substance solution, mixing is to get mixed reference substance solution；

(3) preparation of test solution：Sample is taken, it is finely ground, 1.00g is weighed, is set in conical flask with cover, precision plus methanol 25mL, Weighed, at 400W, the power of 40kHz, ultrasonic 25min, methanol supplies loss of weight, and filtration takes subsequent filtrate molten to get test sample Liquid；

(4) it measures：Precision draws mixed reference substance solution, each 10 μ L of test solution, injects high performance liquid chromatograph, is surveyed It is fixed.

9. a kind of detection method of drug that treating primary dysmenorrhea, the drug are made of the flavour of a drug raw material of following parts by weight：When 1~3 parts by weight, 1~2 parts by weight of Radix Paeoniae Alba, 1~2 parts by weight of milk thistle, preparation method are returned to be：

It is characterized in that, measuring the content of β-ocimenum, australene, myrtanal using gas chromatography, steps are as follows：

(1) chromatographic condition：Chromatographic column：HP-5 capillary columns, temperature programming：Initial temperature is 40 DEG C, 5min is kept, with 8 DEG C min^-1Rate be warming up to 120 DEG C, keep 8min；Again with 12 DEG C of min^-1Rate be warming up to 180 DEG C, keep 5min；Again with 15℃·min^-1Rate be warming up to 270 DEG C, keep 5min；Injector temperature is 210~230 DEG C；Fid detector；Detector Temperature is 260~280 DEG C；Volume flow is 0.5~2.0mLmin^-1；1~10 μ L of sample size, split sampling, split ratio 7 ~9：3~1；

(2) prepared by mixed reference substance solution：Take β-ocimenum, australene, myrtanal reference substance appropriate, it is accurately weighed, add three It is 0.5~2.0mgmL that chloromethanes, which is made containing β-ocimenum,^-1, containing australene be 0.5~2.0mgmL^-1, contain myrtanal 0.5~2.0mgmL^-1Mixed reference substance solution；

(3) prepared by test solution：This product 0.20~1.0g is taken, it is weighed, it is placed in conical flask with cover, chloroform is added in precision 15~30mL, close plug, weighed weight, 10~20min of ultrasound are let cool, and weighed weight supplies the weight of less loss with chloroform, It shakes up, with 0.45 μm of membrane filtration, takes subsequent filtrate to get test solution；

(4) it measures：Precision draws mixed reference substance solution, each 1~10 μ L of test solution, injects gas chromatograph, is surveyed It is fixed.

10. the detection method of drug as claimed in claim 9, which is made of the flavour of a drug raw material of following parts by weight：Radix Angelicae Sinensis 2 Parts by weight, 1 parts by weight of Radix Paeoniae Alba, 1 parts by weight of milk thistle；Preparation method is：

(1) chromatographic condition：Chromatographic column：HP-5 capillary columns, specification：30m × 0.32mm, 0.25 μm；Temperature programming：Initial temperature It is 40 DEG C, 5min is kept, with 8 DEG C of min^-1Rate be warming up to 120 DEG C, keep 8min；Again with 12 DEG C of min^-1Rate liter Temperature keeps 5min to 180 DEG C；Again with 15 DEG C of min^-1Rate be warming up to 270 DEG C, keep 5min；Injector temperature is 220 ℃；Fid detector；Detector temperature is 270 DEG C；Volume flow is 1.0mLmin^-1；5 μ L of sample size, split sampling, shunting Than being 8：2；

(2) prepared by mixed reference substance solution：Take β-ocimenum, australene, myrtanal reference substance appropriate, it is accurately weighed, add three It is 1.055mgmL that chloromethanes, which is made containing β-ocimenum,^-1, containing australene be 1.115mgmL^-1, contain myrtanal 1.225mg·mL^-1Mixed reference substance solution；

(3) prepared by test solution：This product 0.60g is taken, it is weighed, it is placed in conical flask with cover, chloroform 20mL is added in precision, Close plug, weighed weight, ultrasonic 15min are let cool, and weighed weight is supplied the weight of less loss with chloroform, shaken up, with 0.45 μm Membrane filtration takes subsequent filtrate to get test solution；

(4) it measures：Precision draws mixed reference substance solution, each 5 μ L of test solution, injects gas chromatograph, is measured.