CN101829247B - Chinese medicinal compound effective component for treating gynecological diseases and preparation method and application thereof - Google Patents
Chinese medicinal compound effective component for treating gynecological diseases and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a Chinese medicinal compound effective component for treating gynecological diseases and a preparation method and application thereof. The effective component is prepared by obtaining water or ethanol extract of a Chinese medicinal compound consisting of Chinese angelica, Szechuan lovage rhizome, red paeony root, cattail pollen, trogopterus dung, myrrh and corydalis tuber, removing impurity through ethanol precipitation, then injecting the extract into a macroporous absorption resin column, and purifying the extract by gradient elution of ethanol with optimized concentration. A compatible raw material formula is selected according to the Chinese medicinal theory and the gynecological disease pathogenesis; modern analysis instruments and pharmacological experiments show that the Chinese medicinal compound effective component provided by the invention has good activities of resisting uterine smooth muscle spasm, easing pain, resisting inflammation, adjusting estrogen and progestational hormone level, adjusting immunity and the like, can be used for treating multiple gynecological diseases such as primary dysmenorrhea, endometriosis, acute and chronic pelvic inflammation and the like, has clearer components and lower adverse reaction, and is expected to be developed as a new generation medicament for treating the gynecological diseases.
Description
Technical field
The present invention relates to a kind of Chinese medicine compound effective site, be specifically related to a kind of Chinese medicine compound effective site and preparation method thereof and its application in preparation treatment medicine for gynecopathy with the effect of treatment gynaecopathia.
Background technology
Gynaecopathia is women's commonly encountered diseases, a frequently-occurring disease, and wherein many and inflammation and endocrine regulation are closely related.Sickness rate such as chronic pelvic inflammatory disease, chronic cervicitis, vaginitis are higher in the gynecological inflammation; Gynecological's endocrine regulation serve as main performance usually with the disorder of menstruation, whether the eumenorrhea is decided by the height of steroid hormone level, under the orderly fluctuation variation of gonadal hormone, has formed the rule menstruation.
Epidemiological study shows that dysmenorrhea is common disease in the present gynaecologic emmeniopathy, is divided into primary dysmenorrhea and secondary dysmenorrhea.Mainly show as menstrual period or pass through before and after hypogastric region, flank pain appear, sometimes with dizziness or nausea and vomiting, severe patient pale complexion can occur, is in a cold sweat, deadly cold hand and foot, severe pain are fainted phenomenon.At present, TREATMENT OF DYSMENORRHOEA still adopts steroid hormone and prostaglandin antagonists, and the former is accepted by the patient be difficult for more; The latter is because of can only respite pain can not thoroughly curing; And certain side effect is arranged,, can not be regarded as ideal medicine and Therapeutic Method so should not take for a long time.And medicine such as the YUEYUESHU of treatment dysmenorrhea, dysmenorrhea spirit, YIMUCAO CHONGJI etc. on the Chinese medicine market have good efficacy, and toxic and side effects are very little, receive the welcome of extensive patients.
The incidence rate of endometriosis demonstrates ascendant trend in recent years; Its complication performance multiformity; Mainly show as dysmenorrhea that carrying out property increases the weight of, infertile, pelvic cavity tuberosity and enclosed mass clinically, medicines such as progestogen commonly used, danazol, nemestran, the moving agent of gonadotropin releasing hormone and mifepristone in the clinical drug therapy, but these clinical drug effects are unsatisfactory; Particularly used medicine is powerful fertility inhibitor, has significant side effects.Operative treatment has conservative operation and radical surgery.It is thus clear that for endometriosis patient radical surgery is inappropriate, and the long normal Drug therapy that cooperates of conservative operation.
Chronic pelvic inflammatory disease often fails thoroughly to cure for acute pelvic inflammatory disease or disease delay, patient's body constitution more weak due to.This disease is women's commonly encountered diseases, a frequently-occurring disease, and in developing country, the sickness rate of child-bearing period female pelvic inflammation has characteristics such as the course of disease is long, the state of an illness is obstinate, relapse rate height up to 40%.Treatment to the pelvic inflammatory disease patient mainly is an antibiotic therapy.Also have auxiliary treatment such as the traditional tcm therapy and the combined therapy of Chinese and Western medicine in China, curative effect is sure.The common antibiotics of treatment pelvic inflammatory disease has PCs, cephalosporins, lincomycin class, quinolones, Macrolide etc.; The main principle of Chinese traditional treatment pelvic inflammatory disease is blood circulation promoting and blood stasis dispelling, heat-clearing and toxic substances removing.
The measure of such disease of western medicine but is prone to recurrence efficiently and effectively after the drug withdrawal, and medicine has side effect more.Like Progesterone, acetic acid medroxyprogesterone, acetaminophen etc.; These medicines are to the symptom of gynaecopathia and treat; And side effect such as Progesterone and acetic acid medroxyprogesterone can have dizziness, headache, feel sick, distending pain of the breast; Prolonged application can cause atrophy of endometrium, and MBV reduces, and is prone to take place the vagina fungal infection.
And Chinese medicine is being brought into play certain characteristic and advantage in treatment in the gynaecopathia, has series of advantages such as the animal economy of adjusting function, endocrine regulation system disorders, effect relax, side effect is little.The existing gynaecopathia patent that is directed against is only protected (like CN 136871A Chinese medicine compound compound medicine prescription compatibility; CN1895458A treats important compound extract of gynaecopathia and preparation method thereof for one kind); And the patent of relevant Chinese medicine compound is not studied its effective ingredient and (is treated Chinese medicine compound of gynaecopathia and preparation method thereof like one kind of CN1651033A; The medicine and the application preparation thereof of CN1470273A treatment gynaecopathia; CN1530126A pharmaceutical composition, its preparation method and the application in preparation treatment medicine for gynecopathy thereof); The Chinese medicine gynaecopathia has long history, and its rule of treatment is attacked heresy when sending out and taken stopgap measures supporting vital QI to relieve the primary cause of disease in not sending out; From present clinical gynecopathy therapeutic; Combine with dialectical differential diagnosis of diseases, Chinese medicine manifests its unique treatment advantage further, but the compound medicine of treating gynaecopathia at present exist more use that inconvenience, dosage are big, complicated component, effective site be indeterminate; The material base that plays curative effect in the compound recipe is unclear; And have certain problems such as untoward reaction, and therefore necessaryly develop under the guidance of Chinese medical theory that a kind of active substance is clear and definite, determined curative effect, side effect is little, the medicine of the treatment gynaecopathia of taking convenience.
Summary of the invention
Goal of the invention: technical problem to be solved by this invention is the deficiency that overcomes prior art; Provide that a kind of prescription proportioning is scientific and reasonable, effective ingredient is clear and definite, good effect, side effect are little, the Chinese medicine compound effective site with the effect of treatment gynaecopathia of taking convenience, another object of the present invention provides method for preparing and its application in preparation treatment medicine for gynecopathy of this Chinese medicine compound effective site with the effect of treatment gynaecopathia.
Technical scheme: in order to realize above purpose, the technical scheme that the present invention takes is:
A kind of Chinese medicine compound effective site with the effect of treatment gynaecopathia, it is processed by the raw material of following parts by weight:
Radix Angelicae Sinensis 10-50 part, Rhizoma Chuanxiong 3-20 part, Radix Paeoniae Rubra 8-35 part, Pollen Typhae 10-50 part, Oletum Trogopterori 8-35 part, Myrrha 3-20 part, Rhizoma Corydalis 3-20 part.
As preferred version, the Chinese medicine compound effective site with the effect of treatment gynaecopathia provided by the invention is processed by the raw material of following parts by weight:
Radix Angelicae Sinensis 30-50 part, Rhizoma Chuanxiong 5-15 part, Radix Paeoniae Rubra 15-30 part, Pollen Typhae 30-50 part, Oletum Trogopterori 15-30 part, Myrrha 5-15 part, Rhizoma Corydalis 5-15 part.
As more preferably scheme, the Chinese medicine compound effective site with the effect of treatment gynaecopathia provided by the invention is processed by the raw material of following parts by weight:
Radix Angelicae Sinensis 35-45 part, Rhizoma Chuanxiong 10-15 part, Radix Paeoniae Rubra 15-25 part, Pollen Typhae 30-45 part, Oletum Trogopterori 15-25 part, Myrrha 10-15 part, Rhizoma Corydalis 10-15 part.
The present invention is screening treating the gynaecopathia prescription at all times; Under the guidance of Chinese medical theory; Determination of treatment based on pathogenesis obtained through differentiation of symptoms and signs is carried out the proportioning prescription in conjunction with the pathogeny and the disease symptom of gynaecopathia, confirms with Radix Angelicae Sinensis, Rhizoma Chuanxiong, Radix Paeoniae Rubra; Compatibility promoting blood circulation to disperse blood clots and relieves pain medicine Pollen Typhae, Myrrha, Rhizoma Corydalis and Oletum Trogopterori are formed seven flavor Chinese medicines and are carried out prescription; Perfect square has promoting blood circulation, removing blood stasis and relieving pain, nourishes blood and enrich blood, and the double effects of endocrine regulation is mainly used to the diseases such as dysmenorrhea, endometriosis or chronic pelvic inflammatory disease that gynecological's blood deficiency and blood stasis, endocrine confusion cause; Chinese angelica blood supplementing is invigorated blood circulation in the side, menstruction regulating and pain relieving; The Radix Paeoniae Rubra removing heat from blood and promoting blood circulation has promoting the circulation of blood, removing blood stasis with potent drugs, the clot that looses, relieving spasm to stop pain effect; The Rhizoma Chuanxiong blood circulation promoting and blood stasis dispelling, promoting the circulation of QI to relieve pain, three medicines match, and are association of activity and inertia, and grow and oiliness; Warm and not dry, tonify without causing stagnation, coordination of potent and mild therapies, YIN and YANG balancing; Nutrient blood must be living, the merit that playing altogether enriches blood invigorates blood circulation, and compatibility Pollen Typhae, Myrrha, Rhizoma Corydalis and Oletum Trogopterori are again enriched blood prescription and are not stayed the stasis of blood; Promoting the circulation of blood and non-blood trouble increase the merit of QI invigorating promoting the circulation of QI to relieve pain, blood circulation promoting and blood stasis dispelling, cure mainly the passages through which vital energy circulates coagulation of QI-blood, the gynaecopathia that endocrine regulation causes.The present invention executes prescription under the guidance of controlling at Chinese herb decoction; And according to the physicochemical property characteristics of prescription flavour of a drug; Extract to perfect square removes crudely and store essence through modern separation and purification means, shows according to Modern Analytical Instrument and pharmacological screening result, and the effective site active component of gained is clearer and more definite; Blood circulation promoting and blood stasis dispelling, pain stop and anti-inflammation effect are strong, and untoward reaction is lower.
Preparation provided by the invention has the method for the Chinese medicine compound effective site of treatment gynaecopathia effect, specifically may further comprise the steps:
(1) gets the Chinese medicine compound of forming by Radix Angelicae Sinensis 10-50 part, Rhizoma Chuanxiong 3-20 part, Radix Paeoniae Rubra 8-35 part, Pollen Typhae 10-50 part, Oletum Trogopterori 8-35 part, Myrrha 3-20 part, Rhizoma Corydalis 3-20 part; The water or the pure decocting that add 5～10 times of amounts of total medical material boil, and merge decoction liquor and get concentrated solution at 20～70 ℃ of concentrating under reduced pressure;
(2) get the concentrated solution adding ethanol that step (1) obtains, make concentration of ethanol reach 70～90%, place deposition, separation of supernatant concentrates, and gets concentrate;
(3) get macroporous adsorptive resins on the concentrate that step (2) obtains, with the ethanol gradient elution of concentration 20% to 70%, eluent reclaims solvent, Chinese medicine compound effective site.
As preferred version, above-described method for preparing with Chinese medicine compound effective site of treatment gynaecopathia effect, concentration of alcohol is 0-80% in the middle decoction of step (1) the solvent alcohol water, decocting number of times is 1～3 time.
As preferred version, above-described method for preparing with Chinese medicine compound effective site of treatment gynaecopathia effect adds ethanol in the step (2); Make concentration of ethanol reach 75～85%, place deposition, the purpose of this operation is to add ethanol to make that concentration of ethanol reaches 75～85% in the solution; Place; Precipitate, can precipitate and remove a large amount of big macromolecular compounds of polarity in the extracting solution, like polysaccharide, tannin, protein and pectin etc.; Help follow-up separation purifying technique, improve purification efficiency.
As preferred version, above-described method for preparing with Chinese medicine compound effective site of treatment gynaecopathia effect, the macroporous adsorbent resin described in the step (3) is D101, D102 or AB-8 type macroporous adsorbent resin.The present invention is through adsorbing the screening with the purification ability to opposed polarity and different model resin; Confirm that model is that the absorption with macroporous adsorbent resin and the purification effect of D101, D102 or AB-8 type carried out; The present invention has simultaneously carried out preferably the eluting power of 5%～95% different concentration ethanol; As index, confirm that concentration is that 20% to 70% alcoholic acid eluate total content is high with the content of total eluate yield and machine acids, flavonoid, lactone and terpene glycoside, machine acids, flavonoid, lactone and terpene glycoside active constituent content are high; And impurity is few; Therefore the ethanol of confirming concentration 20% to 70% is the gradient elution solvent, and as further preferred version, the ethanol of concentration 30% to 40% is best gradient elution solvent.
The Chinese medicine compound effective site that obtains by method for preparing extraction separation provided by the invention; Analyze through HPLC/HPLC-MS; Effective ingredient in the extract is made up of four compounds such as flavonoid, terpene glycoside, lactone, organic acids, and the percentage by weight of four effective constituents in extract is 20～90%.
As preferred version, flavonoid, terpene glycoside, lactone, organic acid four compounds weight percent consist of 1.5%～9.5% machine acids in the extract; 1%～30% flavonoid; 1%～40% lactone; 13%～40% terpene glycosides compound specifically comprises following component in percentage by weight: compositions such as peoniflorin 3.0%～26%, lactone glucoside of Radix Paeoniae 1.2%～4.8%, ferulic acid 0.35%～1.56%, caffeic acid 0.12%～0.48%, vanillic acid 0.64%～2.56%, protocatechuic acid 1%-2%, typhaneoside 1%～4%, isorhamnetin-3-O-neohesperidin 1.49%～5.96%, Quercetin 0.5%-2.1%, senkyunolide I 3%-15% and senkyunolide H 1%-5%.
The application of Chinese medicine compound effective site in preparation treatment medicine for gynecopathy with the effect of treatment gynaecopathia provided by the invention.
As preferred version, described gynaecopathia is dysmenorrhes, endometriosis or chronic pelvic inflammatory disease disease;
As further preferred version, the medicine of processing capsule, granule, tablet, spray, injection, microcapsule, ointment or skin-permeable and control-released plaster dosage form to the Chinese medicine compound effective site with treatment gynaecopathia effect and pharmaceutically acceptable carrier.
Chinese medicine compound effective site handle when processing tablet with the effect of treatment gynaecopathia provided by the invention has Chinese medicine compound and the carrier lactose or the corn starch of treatment gynaecopathia effect; Add magnesium stearate lubricant when needing; Mix homogeneously, tabletting is processed tablet then.Handle had Chinese medicine compound and the carrier lactose or the corn starch mix homogeneously of treatment gynaecopathia effect, granulate, the encapsulated then capsule of processing when the Chinese medicine compound effective site with the effect of treatment gynaecopathia provided by the invention was processed capsule.When the Chinese medicine compound effective site with the effect of treatment gynaecopathia provided by the invention is processed granule, Chinese medicine compound effective site and diluent lactose or corn starch mix homogeneously with the effect of treatment gynaecopathia, granulate, drying is processed granule.
Beneficial effect: the Chinese medicine compound effective site with the effect of treatment gynaecopathia provided by the invention is compared with prior art and is had the following advantages:
(1) Chinese medicine compound with the effect of treatment gynaecopathia provided by the invention is chosen the material combination prescription according to the pathogenesis of Chinese medical theory and gynaecopathia, and the each component proportioning is scientific and reasonable; And according to the character characteristics of each flavour of a drug chemical constituent of prescription; Perfect square is extracted, and extract is removed crudely and store essence through modern separation and purification means, show according to modern analysis method and pharmacological screening result; The effective site active component of gained is clearer and more definite; Have the disorderly effect of good blood circulation promoting and blood stasis dispelling, pain relieving, mitigation gynecological inflammation, endocrine regulation, and rapid-action, treating both the principal and secondary aspects of a disease, be difficult for recurrence, life-time service has no adverse reaction; And the Chinese medicine compound with the effect of treatment gynaecopathia provided by the invention can become the medicine of various dosage forms, convenient clinical taking easily with pharmaceutically acceptable preparing carriers.
(2) method for preparing with Chinese medicine compound effective site of treatment gynaecopathia effect provided by the invention; Confirm optimum extraction and separating technology through experiment screening; Workable, extract, separation and purification efficient is high, and the method for preparing cost is low, environmental protection, can realize industrialized great production.
The specific embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, process conditions and result thereof only are used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
Embodiment 1
A kind of method with Chinese medicine compound effective site of treatment gynaecopathia effect, its preparation method is:
(1) taking by weighing 10 parts of prescription medical material Radix Angelicae Sinensis, 3 parts of Rhizoma Chuanxiongs, 8 parts of Radix Paeoniae Rubra, 10 parts of Pollen Typhaes, 8 parts of Oletum Trogopteroris, 5 parts of Myrrhas, 5 parts of Rhizoma Corydalis in proportion, is 40 orders through being crushed to particle diameter, mixes; With water boiling and extraction 2 times; 10 times of water gagings of totalling medical material decoct 1.5h for the first time, and 8 times of water gagings of totalling medical material decoct 1h for the second time, merge fried liquid twice; Be concentrated into 1g crude drug/ml, get concentrated solution;
(2) using concentration is that concentrated solution to the concentration of alcohol that 95% ethanol pacing rapid (1) obtains is 80%, places 12h, and filtering-depositing is got supernatant, is concentrated into 5kg, gets concentrate;
(3) get D101 macroporous adsorptive resins on the concentrate that step (2) obtains; After leaving standstill 12h, using pure water, concentration respectively is that 10%～95% ethanol carries out gradient elution, collects concentration 40%～50% ethanol elution position; Concentrate, promptly get Chinese medicine compound effective site with treatment gynaecopathia.(lot number 001)
Analyze through HPLC/HPLC-MS, flavonoid, terpene glycoside, lactone, organic acid four compounds weight percent consist of 9% machine acids in the Chinese medicine compound effective site; 28% flavonoid; 32% lactone; 20% terpene glycosides compound.
Embodiment 2
A kind of method with Chinese medicine compound effective site of treatment gynaecopathia effect, its preparation method is:
(1) getting by 30 parts of Radix Angelicae Sinensis, 10 parts of Rhizoma Chuanxiongs, 15 parts of Radix Paeoniae Rubra, 50 parts of Pollen Typhaes, 15 parts of Oletum Trogopteroris, 10 parts of Myrrhas, 10 parts of Chinese medicine compound of forming of Rhizoma Corydalis, mix, is 40 orders through being crushed to particle diameter; Using concentration is 30% alcohol reflux 3 times; Add for the first time 8 times of amount 30% alcohol reflux 2h, add 6 times of amount 30% alcohol reflux 2h for the second time, add 6 times of amount 30% alcohol reflux 1.5h for the third time; Merge extractive liquid, gets concentrated solution 6kg at 50～70 ℃ of concentrating under reduced pressure;
(2) get the concentrated solution adding ethanol that step (1) obtains, make concentration of ethanol reach 70%, place deposition, separation of supernatant concentrates, and gets concentrate;
(3) get D102 type macroporous adsorptive resins on the concentrate that step (2) obtains,, collect 30%～40% ethanol elution position, reclaim solvent, promptly get Chinese medicine compound effective site with treatment gynaecopathia with the ethanol gradient elution of concentration 10% to 95%.(lot number 002)
Analyze through HPLC/HPLC-MS, flavonoid, terpene glycoside, lactone, organic acid four compounds weight percent consist of 7.6% machine acids in the Chinese medicine compound effective site; 26% flavonoid; 31% lactone; 30% terpene glycosides compound.
Get the above effective site 10g for preparing, add medical starch 16g, mix homogeneously adds water and processes soft material; Cross 20 mesh sieves and granulate, drying is processed granule; Incapsulate, every 0.2g perhaps gets effective site 10g corn starch 20g; Add magnesium stearate lubricant 5g, mix homogeneously, tabletting is processed tablet then.
Embodiment 3
A kind of method with Chinese medicine compound effective site of treatment gynaecopathia effect, its preparation method is:
(1) gets by 50 parts of Radix Angelicae Sinensis, 20 parts of Rhizoma Chuanxiongs, 25 parts of Radix Paeoniae Rubra, 50 parts of Pollen Typhaes, 20 parts of Oletum Trogopteroris, 10 parts of Myrrhas, 10 parts of Chinese medicine compound of forming of Rhizoma Corydalis; Mixing, is 40 orders through being crushed to particle diameter, and using concentration is 70% alcohol reflux 2 times; Add for the first time 10 times of amount 70% alcohol reflux 2h; Add for the second time 8 times of amount 70% alcohol reflux 1.5h, merge extractive liquid, gets concentrated solution 7kg at 50～70 ℃ of concentrating under reduced pressure;
(2) get the concentrated solution adding ethanol that step (1) obtains, make concentration of ethanol reach 90%, place deposition, separation of supernatant concentrates, and gets concentrate;
(3) get AB-8 type macroporous adsorptive resins on the concentrate that step (2) obtains; After leaving standstill 12h, carry out gradient elution with pure water, 10%～95% ethanol respectively, collect 40%～60% ethanol elution position; Reclaim solvent, promptly get Chinese medicine compound effective site with treatment gynaecopathia.(lot number 003)
Analyze through HPLC/HPLC-MS, flavonoid, terpene glycoside, lactone, organic acid four compounds weight percent consist of 8% machine acids in the Chinese medicine compound effective site; 23% flavonoid; 33% lactone; 35% terpene glycosides compound.
Get the above effective site 15g for preparing, add medical starch 30g, mix homogeneously adds water and processes soft material; Cross 20 mesh sieves and granulate, drying, granulate is processed granule; Perhaps get effective site 15g corn starch 30g, add magnesium stearate lubricant 6g, mix homogeneously, tabletting is processed tablet then.
Embodiment 4
A kind of method with Chinese medicine compound effective site of treatment gynaecopathia effect, its preparation method is:
(1) gets by 30 parts of Radix Angelicae Sinensis, 5 parts of Rhizoma Chuanxiongs, 15 parts of Radix Paeoniae Rubra, 30 parts of Pollen Typhaes, 15 parts of Oletum Trogopteroris, 5 parts of Myrrhas, 5 parts of Chinese medicine compound of forming of Rhizoma Corydalis; Mixing, is 40 orders through being crushed to particle diameter, water reflux, extract, 2 times; Add for the first time 10 times of water gaging backflow 3h; For the second time add 8 times of water gaging backflow 2h, merge extractive liquid, gets concentrated solution 6kg at 50～70 ℃ of concentrating under reduced pressure;
(2) get the concentrated solution adding ethanol that step (1) obtains, make concentration of ethanol reach 80%, place deposition, separation of supernatant concentrates, and gets concentrate;
(3) get D101 type macroporous adsorptive resins on the concentrate that step (2) obtains; After leaving standstill 12h, carry out gradient elution with pure water, 10%～95% ethanol respectively, collect 20%～60% ethanol elution position; Reclaim solvent, promptly get Chinese medicine compound effective site with treatment gynaecopathia.(lot number 004)
Analyze through HPLC/HPLC-MS, flavonoid, terpene glycoside, lactone, organic acid four compounds weight percent consist of 6% machine acids in the Chinese medicine compound effective site; 20% flavonoid; 21% lactone; 30% terpene glycosides compound.
Embodiment 5
A kind of method with Chinese medicine compound effective site of treatment gynaecopathia effect, its preparation method is:
(1) gets by 40 parts of Radix Angelicae Sinensis, 10 parts of Rhizoma Chuanxiongs, 30 parts of Radix Paeoniae Rubra, 30 parts of Pollen Typhaes, 20 parts of Oletum Trogopteroris, 10 parts of Myrrhas, 10 parts of Chinese medicine compound of forming of Rhizoma Corydalis; Mixing, is 40 orders through being crushed to particle diameter, with concentration 30% alcohol reflux 2 times; Add for the first time 10 times of amount 30% alcohol reflux 3h; Add for the second time 8 times of amount 30% alcohol reflux 2h, merge extractive liquid, gets concentrated solution 7.5kg at 50～70 ℃ of concentrating under reduced pressure;
(2) get the concentrated solution adding ethanol that step (1) obtains, make concentration of ethanol reach 90%, place deposition, separation of supernatant concentrates, and gets concentrate;
(3) get D102 type macroporous adsorptive resins on the concentrate that step (2) obtains; After leaving standstill 12h, carry out gradient elution with pure water, 10%～95% ethanol respectively, collect 30%～70% ethanol elution position; Reclaim solvent, promptly get Chinese medicine compound effective site with treatment gynaecopathia.(lot number 005)
Analyze through HPLC/HPLC-MS, flavonoid, terpene glycoside, lactone, organic acid four compounds weight percent consist of 7% machine acids in the Chinese medicine compound effective site; 21% flavonoid; 28% lactone; 26% terpene glycosides compound.
The test of embodiment 6 antalgic and inflammation relievings
1, rat primary dysmenorrhea test
(1) preparation of rat primary dysmenorrhea model and experiment
Select that the KM kind is female, the female mice of body weight 18-22g for use, random packet is united with estrogen and oxytocin and to be duplicated the primary dysmenorrhea model.With 0.01mgg
-1D
-1Give the continuous sc estradiol benzoate of mice 8d, make the Mouse Uterus synchronization, the 8th day, ip oxytocin 0.01mLg
-1, make rat primary dysmenorrhea animal model.
Detect index: writhing response.
Medication: the large and small dose groups that mice is divided at random lot number 001 effective site that model control group, positive drug matched group (celecoxib), embodiment 1 prepare; The large and small dose groups of lot number 002 effective site that embodiment 2 prepares; The large and small dose groups of lot number 003 effective site that embodiment 3 prepares.Normal control group, model control group give the isometric(al) normal saline, and the positive drug matched group gives celecoxib 0.0411mgg
-1D
-1, effective site administration 37.0g crude drug/kg, 18.5g crude drug/kg are large and small dose groups, each organizes 10 every group of mices.Duplicate the primary dysmenorrhea model as stated above.From the 6th day, the morning every day lumbar injection estradiol benzoate, afternoon, the normal control group gave normal saline 0.4mL/ only, all the other each groups are irritated stomach relative medicines.Continuous 3d, 60min after the last administration, ip oxytocin 0.2mL/ only write down interior mouse writhing number of times of injection back 20min and writhing response suppression ratio.
(2) experimental result and analysis
Show that by table 1 experimental result Chinese medicine compound effective site provided by the invention all has the effect (P＜0.05 or P＜0.01) of significant inhibition mouse writhing reaction under large and small dosage; Suppression ratio is all more than 50%, and the result shows that Chinese medicine compound effective site provided by the invention has good analgesic activity, is better than the positive control drug celecoxib.
Table 1 Chinese medicine compound effective site is turned round the inhibitory action of body to the primary dysmenorrhea model mice
Compare with model group,
*P＜0.05;
*P＜0.01
The inhibitory action of 2, the mice isolated uterine being shunk
(1) the mice isolated uterine shrinks experiment
Get the unpregnancy female mice, body weight 18-22g; Lumbar injection estradiol benzoate 1mgkg for three days on end before the experiment
-1D
-1, with the synchronization uterus and increase the sensitivity of uterus to oxytocin.Took out Mouse Uterus on 4th, place Locke ' s solution to clean, tie cervix uteri end and both sides ovary end respectively with fine rule, an end is fixed in the bath bottom, and the other end is connected on the tonotransducer.Place 20ml Locke ' s liquid thermostatic bath, temperature is (37 ± 0.1) ℃, continues oxygen supply, and pH 7.3, adds the load (10mN) of 1g through adjusting nut, and balance 40min treats that uterine contraction is stable, and the spontaneous rhythm and pace of moving things begins to experimentize after recovering.
Write down 5-10min normal contraction curve in advance; Continuing adds oxytocin injection 10 μ l, and final concentration is 5ul/L, adds behind the effect 15min respectively to receive reagent thing (the Chinese medicine compound effective site that embodiment 1 to 3 prepares again; Lot number 001,002 and 003); The initial concentration that each sample is added by the reagent thing is 10mg/ml, and the final concentration that receives the reagent thing in the 1L bath is 0.01mg/ml, observes medicine causes the contraction of hysterospasm property to oxytocin inhibitory action; Adopt before the administration of T method of inspection statistics and the contraction frequency and the shrinkage amplitude in uterus after the administration, calculate and suppress uterotonic percentage rate (IR%).Parameter * 100 before computing formula: IR (%)=(parameter after the preceding parameter-administration of administration)/administration.
(2) date processing and statistics
Each organizes data, and all (relatively t check is in twos adopted in the expression of mean ± SD), interclass correlation index, and there is significance P＜0.05 for difference with mean ± standard deviation.
(3) the mice isolated uterine is shunk experimental result, concrete experimental result is as shown in table 2:
1. to the influence of mice isolated uterine smooth muscle contraction frequency
By finding out in the table 2 that Chinese medicine compound effective site provided by the invention all has significant inhibition active (P＜0.01 or P＜0.05) to uterus smooth muscle contraction frequency.
2. to the influence of mice isolated uterine smooth muscle contraction amplitude
By finding out in the table 2 that Chinese medicine compound effective site provided by the invention has significant inhibition active (P＜0.01) to mice isolated uterine smooth muscle contraction amplitude.
3. to the influence of mice isolated uterine smooth muscle muscular tension
By finding out in the table 2 that Chinese medicine compound effective site provided by the invention has significant inhibition active (P＜0.05 or P＜0.01) to mice isolated uterine smooth muscle muscular tension.
3 evaluation indexes that the analysis-by-synthesis isolated uterine shrinks show that Chinese medicine compound effective site provided by the invention presents remarkable activity to suppressing uterine contraction.Its ED
50Be 128.0 μ g/ml, 95% credibility interval is 104.2 μ g/ml～165.9 μ g/ml.
Table 2 Chinese medicine compound effective site is to the result that influences of mice isolated uterine contraction
3, mice formaldehyde causes scorching test
(1) formaldehyde causes scorching Preparation of model
Get 80 mices and be divided into 8 groups at random, 10 every group, matched group, model group, the large and small dose groups of embodiment 1 lot number, 001 effective site; The large and small dose groups of embodiment 2 lot numbers, 002 effective site; The large and small dose groups of embodiment 3 lot numbers, 003 effective site.Appearance body normal saline such as matched group, model control group give, compound extract group dosage is respectively 37.0gKg
-1D
-1, the dose of making a living.At right back sufficient lift injected 2.5% formaldehyde normal saline 20 μ L, behind the 4h, with the mice sacrificed by decapitation, cut foot from knee joint afterwards behind the administration 1h, weigh, sufficient difference about comparing is represented the inflammation degree with this.
(2) anti-mice foot swelling experimental result and analysis, concrete experimental result is as shown in table 3:
Can know by table 3; The heavy dose of Chinese medicine compound effective site all has significant inhibition formaldehyde induced mice foot swelling effect (P＜0.05); Suppression ratio can reach more than 50%, and effect is better than positive drug, shows that the Chinese medicine compound effective site of being invented can significantly suppress the formaldehyde-caused edema of subcutaneous injection.
The anti-foot swelling experimental result of table 3 Chinese medicine compound effective site
Compare with model group,
*P＜0.05;
*P＜0.01
(3) PARA FORMALDEHYDE PRILLS(91,95) causes PGE in the mice foot swelling tissue
2The influence of content
With the right foot of the mice of cutting, be soaked in 1h in the 1mL normal saline after the peeling, with the centrifugal 10min of 2000r/min, draw supernatant 0.2mL, add 0.5mol/L NaOH-CH
3OH solution 1mL, isomerization is 20 minutes in 50 ℃ of water-baths, adds methanol and is diluted to 10mL, measures its absorption value in the 278nm place, and calculates PGE by following formula
2Content:
PGE
2Content (μ g/g)=(E278 * 13.13 * Vt * D)/W
Calculating each group difference [annotates: Vt=PGE
2The soak cumulative volume, D=extension rate, W=right lower extremity weight (g), the absorption value at E278=278nm place].Concrete experimental result is seen table 4.
Table 4 Chinese medicine compound effective site PARA FORMALDEHYDE PRILLS(91,95) causes PGE in the mice foot swelling tissue
2The influence of content
Compare with model group,
*P＜0.05;
*P＜0.01
Can know that by table 4 the Chinese medicine compound effective site of being invented is to causing contained PGE in the scorching tissue
2Inhibitory action with highly significant shows the synthetic and release of the inflammatory factor that can reduce in the inflammation tissue, PGE
2Content is low more, and its antiinflammatory action is remarkable more.
Show that by above antalgic and inflammation relieving result of the test Chinese medicine compound effective site provided by the invention has good analgesia and antiphlogistic effects, and onset is rapid, does not see untoward reaction, can be used for treating the women's chronic pelvic inflammation disease.
Embodiment 7 improves hemorheology and endocrine regulation hormone test
1, the preparation of stasis syndrome SD rat model and experiment
Normal group is conventional raises, and model group, treatment group rat place 0 ℃～1 ℃ frozen water 5min, every day 1 time, continuous seven days.The beginning in the 1st day in modeling is organized in the treatment of Chinese medicine compound effective site, irritates stomach by each sample of large and small dosage Chinese medicine compound, every day 1 time, and logotype 7 days, normal group, model group give the distilled water with volume.In the 8th day subcutaneous injection 0.1% epinephrine 0.1ml/100g, totally 2 times, twice blanking time 4h.Stop eating afterwards, Yu Cichen adopts full-automatic rheology to survey appearance soon and measures hemorheology index; Adopt the method for radioimmunity to measure hormonal readiness in the serum.
2, to the influence of rat acute blood stasis model WBV
Visible by table 5, to compare with model group, Chinese medicine compound effective site provided by the invention all obviously reduces WBV effect (P＜0.01 or P＜0.05) in high and low dose.Concrete experimental result is as shown in table 5.
Table 5 Chinese medicine compound effective site is to the influence of whole blood viscosity
Compare with model group,
*P＜0.05,
*P＜0.01
3, to the influence of stasis syndrome rat model plasma viscosity, erythrocyte sedimentation rate, packed cell volume
Compare with model group, Chinese medicine compound effective site provided by the invention can obviously reduce blood plasma viscosity, erythrocyte sedimentation rate, packed cell volume (P＜0.01 or P＜0.05), but fibrinogen content is not seen significant change (P＞0.05).High dose group has the obvious effect (P＜0.05) that improves erythrocyte sedimentation rate, packed cell volume, and concrete experimental result is as shown in table 6.
Table 6 Chinese medicine compound effective site is to the influence of rat plasma viscosity, erythrocyte sedimentation rate and packed cell volume
#P＜0.05 is compared with matched group;
*P＜0.05,
*P＜0.01 is compared with model group
4, to the influence of E2 and P content in the stasis syndrome rat model serum
Compare with model group, Chinese medicine compound effective site high and low dose group provided by the invention all obviously or significantly reduces E in the serum
2Rising (P＜0.001 or P＜0.01); And progesterone content is all presented rising effect (P＜0.05, P＜0.01), concrete experimental result is as shown in table 7.
Table 7 Chinese medicine compound effective site is to the influence of endocrine hormone level in the cold blood stasis type rat blood serum
| Group | Dosage (g/Kg) | E 2(ng/L) | P(nmol/L) |
| The blank group | The equal-volume normal saline | 168.43±35.69 | 384.98±17.16 |
| Model group | The equal-volume normal saline | 237.50±41.17## | 254.26±62.35### |
| Embodiment 1 | 37.0 | 117.40±12.78 ** | 343.98±48.27 * |
| 18.5 | 104.60±42.65 * | 339.94±23.96 * | |
| Embodiment 2 | 37.0 | 117.57±16.91 *** | 352.89±79.14 * |
| 18.5 | 102.83±31.81 *** | 389.78±152.00 * | |
| Embodiment 3 | 37.0 | 111.17±15.63 *** | 348.73±27.56 *** |
| 18.5 | 106.57±11.06 *** | 342.13±44.66 *** |
##P＜0.01 is compared with matched group;
*P＜0.05,
*P＜0.01,
* *P＜0.001 is compared with model group
Show that by above experimental result Chinese medicine compound effective site provided by the invention has good blood circulation promoting and blood stasis dispelling; Improve blood circulation; Endocrine regulation is chaotic, recovers endocrine and acts on normally, thereby can be used to treat the gynaecopathias such as dysmenorrhea or endometriosis due to the blood stasis.
Embodiment 8 regulates the immunologic function test
1, to mice spleen T lymphocyte transformation experiment (mtt assay)
Each treated animal continuous irrigation stomach 30 days, animal, the aseptic spleen of getting are put to death in the cervical vertebra dislocation; Place the plate that fills an amount of aseptic Hank ' s liquid, gently spleen is ground, process single cell suspension; 200 eye mesh screens filter; Use Hank ' s liquid washing 2 times, each centrifugal 10 minutes living cell counting numbers (should more than 95%), the adjustment cell concentration is 3 * 106/ml.Divide two holes to add in 24 well culture plates cell suspension, every hole 1ml, a hole adds 75 μ l ConA liquid (being equivalent to 715 μ g/ml), and another hole compares.Put 37 ℃, 5%CO
2Cultivated 72 hours in the incubator.Cultivate and finish preceding 4 hours, supernatant 0.7ml is inhaled in every hole gently, adds the RPMI1640 culture fluid that 0.7ml does not contain calf serum, adds MTT (5mg/ml) 50 μ l/ holes simultaneously, continues to cultivate 4 hours.After cultivating end, every hole adds 1ml acid isopropyl alcohol, and the piping and druming mixing dissolves purple crystal fully.Lysate is moved in the 2ml cuvette; Measure OD value (OD) in the 570nm wavelength; Experimental result shows that Chinese medicine compound effective site provided by the invention has the lymphopoietic effect of good promotion spleen T, thereby can play the effect that improves immunity.
2, to the experimental result that influences of mice tranquillization splenocyte
Can find out by table 8 experimental result; Compare with blank group (normal saline); Chinese medicine compound effective site provided by the invention can obviously strengthen mouse lymphocyte transformation function (P＜0.01 or P＜0.05 or P＜0.001); Have the breeder reaction that promotes the tranquillization chrotoplast, improve the effect of immunity.
Table 8 Chinese medicine compound effective site is to the influence of mice tranquillization splenocyte
Compare with blank control group,
*P＜0.05,
*P＜0.01,
* *P＜0.001.
3, to the inductive activation spleen of ConA T lymphopoiesis and the inductive IFN-γ test of ConA
Experimental result shows that Chinese medicine compound effective site provided by the invention is compared the proliferation function (P＜0.01) with the inductive activating T cell of obvious inhibition ConA and had the inductive IFN-γ of an obvious inhibition ConA inflammatory factor with the DMSO matched group when 200 μ g/ml generation (P＜0.01) shows that further Chinese medicine compound effective site provided by the invention has the activity of good adjusting immunologic function.
The present invention is according to the Chinese medical theory determination of treatment based on pathogenesis obtained through differentiation of symptoms and signs; Pathogeny according to gynaecopathia; Carry out scientific composition, and adopt preferred method for extraction and purification to prepare Chinese medicine compound effective site, show that through experimental result the Chinese medicine compound effective site with the effect of treatment gynaecopathia provided by the invention has good anti-uterine smooth muscle spasm, analgesia, antiinflammatory, adjusting estrogen, progesterone level, adjusting immunologic function isoreactivity; Can be used for treating various gynecological diseases; Comprise gynaecopathias such as primary dysmenorrhea, endometriosis, urgent chronic pelvic inflammatory disease, and owing to removed a large amount of inert matters, effective ingredient is clearer in the effective site; Content is higher; Clinically medication still less, safer, untoward reaction is lower, the Chinese medicine compound with the effect of treatment gynaecopathia therefore provided by the invention is expected to develop the medicine that becomes a new generation's control gynaecopathia.
Claims (5)
1. one kind has the Chinese medicine compound effective site of treating the gynaecopathia effect, it is characterized in that it is processed by following method:
(1) gets the Chinese medicine compound of forming by Radix Angelicae Sinensis 10-50 part, Rhizoma Chuanxiong 3-20 part, Radix Paeoniae Rubra 8-35 part, Pollen Typhae 10-50 part, Oletum Trogopterori 8-35 part, Myrrha 3-20 part, Rhizoma Corydalis 3-20 part; Add the water or the pure water extraction of 5～10 times of amounts of total medical material, merge extractive liquid, gets concentrated solution at 20～70 ℃ of concentrating under reduced pressure;
(2) get the concentrated solution adding ethanol that step (1) obtains, make concentration of ethanol reach 70～90%, place deposition, separation of supernatant concentrates, and gets concentrate;
(3) get macroporous adsorptive resins on the concentrate that step (2) obtains; Ethanol gradient elution with concentration 20% to 70%; Eluent reclaims solvent, gets Chinese medicine compound effective site, includes the organic acid of percentage by weight 1.5%～9.5% in the described Chinese medicine compound effective site; 1%～30% flavonoid; 1%～40% lactone; 13%～40% terpene glycosides compound.
2. one kind prepares the described method with Chinese medicine compound effective site of treatment gynaecopathia effect of claim 1, it is characterized in that may further comprise the steps:
(1) gets the Chinese medicine compound of forming by Radix Angelicae Sinensis 10-50 part, Rhizoma Chuanxiong 3-20 part, Radix Paeoniae Rubra 8-35 part, Pollen Typhae 10-50 part, Oletum Trogopterori 8-35 part, Myrrha 3-20 part, Rhizoma Corydalis 3-20 part; Add the water or the pure water extraction of 5～10 times of amounts of total medical material, merge extractive liquid, gets concentrated solution at 20～70 ℃ of concentrating under reduced pressure;
(2) get the concentrated solution adding ethanol that step (1) obtains, make concentration of ethanol reach 70～90%, place deposition, separation of supernatant concentrates, and gets concentrate;
(3) get macroporous adsorptive resins on the concentrate that step (2) obtains; Ethanol gradient elution with concentration 20% to 70%; Eluent reclaims solvent, gets Chinese medicine compound effective site, includes the organic acid of percentage by weight 1.5%～9.5% in the described Chinese medicine compound effective site; 1%～30% flavonoid; 1%～40% lactone; 13%～40% terpene glycosides compound.
3. the method for preparing with Chinese medicine compound effective site of treatment gynaecopathia effect according to claim 2 is characterized in that the macroporous adsorbent resin described in the step (3) is D101, D102 or AB-8 type macroporous adsorbent resin.
4. the described application of Chinese medicine compound effective site in preparation treatment dysmenorrhes, endometriosis or chronic pelvic inflammatory disease disease medicament of claim 1 with the effect of treatment gynaecopathia.
5. the application of Chinese medicine compound effective site in preparation treatment dysmenorrhes, endometriosis or chronic pelvic inflammatory disease disease medicament with treatment gynaecopathia effect according to claim 4 is characterized in that processing Chinese medicine compound effective site and pharmaceutically acceptable carrier the medicine of capsule, granule, spray, injection, microcapsule, tablet, ointment or skin-permeable and control-released plaster dosage form.
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| 宿树兰等.少腹逐瘀汤对小鼠离体子宫收缩模型的生物效应及物质基础评价.《中国药科大学学报》.2007,第38卷(第06期),544-548. * |
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