CN1911260A - Application of phenolic acids active components from dandelion for inhibiting gynecologic pelvic inflammatory disease - Google Patents
Application of phenolic acids active components from dandelion for inhibiting gynecologic pelvic inflammatory disease Download PDFInfo
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Abstract
An application of the dandelion herb's extract (phenolic acid) in preparing the medicines for preventing and treating the pelvic inflammation of woman is disclosed. Said extract is prepared from dandelion herb through extracting, column chromatography, eluting with alcohol, and refining.
Description
Technical field
The present invention relates to medical technical field, particularly, the present invention relates to a kind of novel medical use with Herba Taraxaci plant phenols acids extract of prevention and treatment gynecological pelvic infection disease.This Herba Taraxaci plant extract that the present invention obtains has the antibiotic and anti-inflammatory efficacy in tangible inside and outside, also has analgesic simultaneously and the analgesia effect, and expection is used for the catch purposes of medicine of prevention and treatment gynecopathy such as pelvic inflammatory.
Background technology
Pelvic inflammatory disease belongs to the common disease occurred frequently of gynecological, and female pelvic cavity genitals inflammation comprises general designation pelvic inflammatory disease such as endometritis, salpingitis, oophoritis, inflammation of pelvic connective tissue and pelvioperitonitis.Be mainly due to the various suppurative bacteriums, inflammation can be confined to a certain position, also can relate to whole internal genitalia, divides acute and chronic two kinds.Inflammation is called adnexitis when being confined to fallopian tube and ovary.Women's pelvic cavity is by fallopian tube, uterus, cervix uteri, vagina and external communicating, easily by the bacterial infection in the external world; Simultaneously because the relation of pelvic tissue anatomical structure also is the place that antibacterial is easily kept, the medicine difficulty is attacked; It is not very thorough adding when many women treat, and disease is shown effect repeatedly, and pathogenic bacteria produces drug resistance, is difficult to cure.Classical symptom: be mainly in the 30-50 middle aged women in year; Common performance is lower abdomen dull pain, the soreness of waist, leucorrhoea grow in quantity, infertile; Symptoms such as acute attack sometimes, appearance heating, belly have an intense pain.Unclean, overfrequency, chaotic sexual life, Chang Yi causes pelvic inflammatory disease.The pelvic cavity complex structure in case pathological changes takes place, will involve and close on organ, as untimely treatment, tends to cause serious consequence.Conventional therapy: (1) acute pelvic inflammatory disease: 1. get fowler's position and have a rest, supply with abundant nutrition and moisture, answer venous transfusion during hyperpyrexia.2. use more heavy dose of antibacterials at pathogenic bacterium.When 3. pelvic cavity has abscess to form, answer incision and drainage.(2) chronic pelvic inflammatory disease: 1. note nutrition, health invigorating.2. acupuncture cv, the middle utmost point, sp 6, come back, shen shu, eight wine with dregs caves.3. physical therapy:, maybe will fry salt and put hot compress hypogastric region in the cloth bag as ultrashort wave diathermy.4. the 2ml of placenta interstitial fluid intramuscular injection, the next day 1 time, 15～20 times is 1 course of treatment.5. posterior fornix seals or the preceding block therapy of sacrum.6. hydrosalpinx and tubo-ovarian cyst are failed to respond to any medical treatment through above-mentioned, can consider excision.7. antibiotic therapy is identical with acute pelvic inflammatory disease.Other therapies: 1. naturopathy: in conjunction with equipment such as microwave therapy apparatus, therapeutic instrument for treating pelvic inflammation, use heat effect and Photochemical effects, in certain temperature range, pathogen kill and inflammatory cell are effectively controlled inflammatory cell invasion and attack and inflammation and are further developed.2. body and mind medical treatment: with pure Chinese medicine compound prescription, the pelvic inflammatory disease blood circulation is quickened in " wash, apply, irritate, obey " four combinations, promotes damaged cell regeneration.Simultaneously, make other inflammation of patient and physical and mental health obtain medical treatment.
Therefore treating gynecological infectious diseases common and occurred frequently all needs to use antibiotic, anti-infective antibiotics.Since the discovery penicillin and after being applied, the mankind day that concerns are tending towards contest with microorganism: antibiotic application, Resistant strain has appearred, continue Application and Development new construction antibiotic, Resistant strain continues to occur, samsara so repeatedly, antibiotic development and Application generation by generation is for the mankind resist microorganism encroach.But long-term clinical application is its toxic and side effects of exposed day by day, particularly broad ectrum antibiotic class also, because misapplication clinically, even abuse, its consequence that causes is troubling: dysbacteriosis in the patient body causes superinfection; Resistant strain constantly occurs, and causes treatment difficulty etc.For rationally utilizing antibiotic, national drug food Surveillance Authority promulgation rules: from July 1st, 2004, exclude the various antibacterials (comprising antibiotic and sulfonamides, quinolones, tuberculosis, antifungal drug) of nonprescription drugs medicine catalogue, all retail pharmacies must write out a prescription and could sell with the medical practitioner in China.This does not have Western medicine anti-infective market and is suspected to have bigger influence.
Clinically, a lot of diseases such as internal medicine, surgery, gynecological all rely on anti-infectives.The Western medicine antibiotics still has irreplaceable advantage in the acute severe infection disease of treatment.But no matter in view of its side effect, for the low-grade infection disease, then there is no need to use antibiotic, if can find the antibiotics succedaneum of " safety, effective, stable ", be social meaning or economic interests, and its value is inestimable.
Herba Taraxaci is feverfew Herba Taraxaci (Taraxacum mongolicum Hand-Mazz) or the dry herb that belongs to several plants together, is heat-clearing and detoxifying herb commonly used, and ancient prescription is put down in writing it and is " key medicine of analgesic removing heat from blood ".Be used for acute mastitis, lung abscess, acute appendicitis, mumps, scrofula, furuncle carbuncle toxin, conjunctival congestion and swelling pain, cold, fever, laryngopharynx swelling and pain, gastritis, enteritis, dysentery, hepatitis, cholecystitis, urinary tract infection, snake bite and insect sting.Among the people, be described as one of heat-clearing and toxic substances removing " eight king kongs ".The Pharmacopoeia of People's Republic of China medication.Herba Taraxaci mainly contains chemical constituents such as triterpenes, phytosterol, sesquiterpene lactones class, Coumarins, flavonoid, phenolic acid compound, carotenoid and fatty acid.Its pharmacological action has anti-inflammation, antioxidation, hepatic cholagogic, immunomodulating and antitumor etc.At present, injection, tablet and the granule etc. that utilize Herba Taraxaci extract to make are widely used in clinical, treat various diseases associated with inflammation and have obtained curative effect preferably.But research and the application to it also only rests on the crude extract preparation at present, it is still quite poor to go deep into exploitation, material base, mechanism research, with " triple effect " (efficient, quick-acting, long-acting), " three is little " (dosage is little, toxicity is little, side effect little), also there is very big distance in the modernized Chinese patent medicine of " three just " (being convenient to preserve, be easy to carry, be convenient to take).
In recent years, the inventor finds under study for action, the Herba Taraxaci plant extract that contains single caffeoyl guinic acid and two caffeoyl guinic acid compounds has the effect that suppresses viral infection, and has applied for national inventing patent " purposes of the preparation of phenolic acid effective kind part and inhibition influenza virus thereof in the Herba Taraxaci " (number of accepting 200610036674.2) thus.Among the present invention, we continue the effective part extract that is rich in phenolic acid compound in this Herba Taraxaci plant has been carried out the pharmacodynamics and the toxicological study of deep antiinflammatory infection direction, cause several animal models such as acute pelvic inflammatory disease model that it is carried out a series of zoopery therapeutic test at adopting colon bacillus or chlamydia trachomatis to be injected into Cavia porcellus uterus or fallopian tube respectively, found that this kind has very outstanding infection, anti-inflammatory activity, also have simultaneously analgesic and analgesic activity, can partly replace antibiotic to use aspect gynecological's pelvic infection disease.
Summary of the invention
The purpose of this invention is to provide and to be rich in the novel medical use of the gynecological infectious diseases such as extract effective site treatment pelvic inflammation of phenolic acid compound in the Herba Taraxaci plant;
Another purpose of the present invention has provided medicine or the pharmaceutical composition that a kind of Herba Taraxaci phenolic acid effective kind part extract is used for the treatment of gynecological's pelvic infection disease.It is characterized by in this extract part except that containing the flavonoid phenoloid and caffeic acid, ferulic acid and chlorogenic acid that luteolin is a basic framework, the two caffeoyl guinic acid class active substances that also contain three structural similarities, their structure is: 3, and the 5-O-dicaffeoyl quinic acid; 3, the 4-O-dicaffeoyl quinic acid; 4, the 5-O-dicaffeoyl quinic acid.
Be rich in the phenolic acid compound extract in the Herba Taraxaci plant among the present invention and contain phenoloid content more than 20% by colorimetric method for determining.
The female pelvic cavity scope comprises genitals's (uterus, fallopian tube, fallopian tube), pelvic peritoneum and peritubal connective tissue, and the inflammation of Fa Shenging is referred to as pelvic inflammatory disease herein, is one of common gynecological disease.The pathogen of primary disease is mainly the mixed infection that anaerobe and aerobe cause.Route of infection mainly contain through lymphsystem spread, through the blood circulating propagation, prolong the genitals mucosa uply spread, by the adjacent organ direct extension.Sterilization such as childbirth or miscarriage, intrauterine surgical operation is not strict, the bad acute pelvic inflammatory disease that all can cause of menstrual hygiene, and delay then is a chronic pelvic inflammatory disease as the course of disease.If the fallopian tube adhesion obstruction often can cause infertile.Colon bacillus and chlamydia trachomatis are the main pathogenic microbes that causes chronic pelvic inflammatory disease.Adopt colon bacillus or chlamydia trachomatis is injected into the Cavia porcellus uterus respectively or fallopian tube causes the acute pelvic inflammatory disease model, continuous two weeks, measure uterus or oviducal weight and organize bacteria containing amount by the grouping administration every day, pathologic finding is fixed in injection virgin palace or fallopian tube.The result: but this Herba Taraxaci plant extract effective site that is rich in phenolic acid compound alleviates the infiltration and the congestion and edema of tissue injury, inflammatory cell to the pelvic inflammatory disease model dose dependent ground due to two kinds of pathogenic bacterium, the reduce inflammation tissue weight that causes increases, and alleviates the uterine cancer cell bacteria containing amount of colon bacillus pelvic inflammatory disease model.On two kinds of models, the extract effective site group of 200mg/kg Herba Taraxaci and positive control drug JINJI JIAONANG group (1170mg/kg) equivalence (P＞0.05), the extract effective site group of 400mg/kg Herba Taraxaci are better than JINJI JIAONANG group (1170mg/kg) (P＜0.05) aspect the bacteria containing amount alleviating uterus or fallopian tube weight and organize.Conclusion: this Herba Taraxaci plant extract effective site that is rich in phenolic acid compound all has therapeutical effect preferably to the Cavia porcellus pelvic inflammatory disease that colon bacillus or chlamydia trachomatis cause.
In addition, this Herba Taraxaci plant effective part extract that is rich in phenolic acid compound confirms that through big ear rabbit test it has refrigeration function to rabbit fever models due to the bacterial endotoxin.Intravenous injection bacterial endotoxin pyrogenicity is after 1 hour, the fervescence value of Herba Taraxaci plant effective part extract high dose group (20mg/kg), middle dosage group (100mg/kg) and low dose group (50mg/kg) is all significantly less than blank group (P＜0.05 or P＜0.01), and wherein the effect of high dose group (200mg/kg) and positive control drug aspirin (150mg/kg) are suitable; After the pyrogenicity 2 hours, the fervescence value of Herba Taraxaci plant effective part extract high dose group is also significantly less than blank group (P＜0.05), and is still suitable with the effect of positive control drug aspirin; During 3～6 hours, the fervescence value variation tendency of Herba Taraxaci plant effective part extract high dose group is basic and aspirin is similar after the pyrogenicity.
The present invention also finds: Herba Taraxaci phenolic acid compound effective site is inhibited to the caused ICR mice of chemical stimulation pain reaction.Each administration group all suppresses acetic acid induced mice writhing response, wherein dosage group (200mg/kg), heavy dose of group (400mg/kg) and positive control drug aspirin group (250mg/kg) compare with the blank group in the Herba Taraxaci plant effective part extract, group difference all has significance meaning (P＜0.05, P＜0.01 and P＜0.001), the analgesia rate is respectively 47.44%, 62.82% and 78.53%.Experimental result proves: Herba Taraxaci phenolic acid compound effective site can suppress the mice pain reaction that chemical stimulation causes, and its analgesic activity presents certain dose dependent.In addition, Herba Taraxaci plant effective part extract is inhibited equally to the caused ICR mice of thermostimulation pain reaction.(400mg/kg) administration of the heavy dose of group of Herba Taraxaci phenolic acid compound effective site is onset (P＜0.05) after 30 minutes, three dosage groups and blank group are relatively in the time of 60 minutes, pain threshold difference is remarkable (P＜0.05 or P＜0.01) all, and is suitable with positive control drug aspirin (250mg/kg) effect; Percentage rate is improved apparently higher than the aspirin group in the maximum threshold of pain after the administration of heavy dose of group.Experimental result shows: the various dose group of Herba Taraxaci phenolic acid compound effective site all has in various degree analgesic activity to the mice pain reaction due to the thermostimulation.
Consult through document, up to the present, still do not treat the report of gynecological's pelvic infection disease about the Herba Taraxaci plant phenols acids effective part extract that contains two caffeoyl guinic acid compounds.Finish the present invention in view of the above.
Specific embodiments
Plant is in the past discovered and is contained triterpenes, flavonoid, Coumarins, phenolic acids, sesquiterpenoids, phytosterol, carotenoid and long-chain fat acid compounds in the Herba Taraxaci plant.In the concerned countries patent of invention (the patent number of accepting 200610036674.2) that the inventor declares, the inventor suppresses the result according to different extract parts to influenza virus and finds for activity index: its antiviral activity composition concentrates on the high polarity phenolic acids position of plant extract, by process optimization, the inventor adopts water or water-alcohol solvent extraction to be aided with multiple positive and negative phase chromatography means and obtains this effective site.Through spectroscopic datas such as a peacekeeping two dimensional NMR wave spectrum, mass spectrum, infrared, ultraviolets, we find that first this high polarity phenolic acid effective kind part contains two compounds, and a class is four flavonoid phenoloid compositions with luteolin basic framework; A class is exactly traditional phenolic acids active substances such as caffeic acid and chlorogenic acid again, and two caffeoyl guinic acid class compound of phenolic acid of three structural similarities, and their structure is: 3, and the 5-O-dicaffeoyl quinic acid; 3, the 4-O-dicaffeoyl quinic acid; 4, the 5-O-dicaffeoyl quinic acid.
It is emphasized that: among the present invention our reported first from Herba Taraxaci phenolic acid effective kind part extract, separate obtain from this platymiscium, not separating before three dicaffeoyl quinic acid compounds that be, that have physiologically active.It is as follows to derive three dicaffeoyl quinic acid compounds structures through integration analysis such as infrared, mass spectrum, ultraviolet and NMR (Nuclear Magnetic Resonance) spectrum.
| Chemical compound Z 1 | Chemical compound Z 2 | Chemical compound Z 3 |
| 3, the 5-O-dicaffeoyl quinic acid | 3, the 4-O-dicaffeoyl quinic acid | 4, the 5-O-dicaffeoyl quinic acid |
In addition; modern age, pharmacology test studies show that: this type of has the flavone compound of luteolin skeleton and single, double coffee acyl quinic acid compounds, and to have a leukotriene of inhibition synthetic and discharge, suppress multiple important physiologically actives such as histamine release, potent antioxidation, thereby can be used for antiinflammatory, antiviral and treatment anaphylactic disease.(document: Li Zusheng, Zhu Zhian, medical science summary, 2004,10 (4), 249-250; He Lina etc., contemporary Chinese is used pharmaceutical journal, 2000,17 (5), 362-365; Hu C, Kitts DD.Molecular and CellularBiochemistry, 2004,265,107; Zhao Yu etc., CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2006,31 (11), 869-874).What need emphasize a bit is: phenolic acid compound total contents in the Herba Taraxaci effective part extract such as the flavonoid phenoloid of the luteolin skeleton among the present invention and coffee acyl quinic acid must not be less than 20%.The total content of phenolic acid effective site in extract is lower than its infection of Herba Taraxaci extract of 20% and anti-inflammatory activity, and effective site is unobvious as described in the present invention.
Above-mentioned research according to the inventor, this Herba Taraxaci plant extract effective site that is rich in phenolic acid compound all has therapeutical effect preferably to the Cavia porcellus pelvic inflammatory disease that colon bacillus or chlamydia trachomatis cause, simultaneously also have analgesic and analgesic activity, can be used for the treatment of relevant gynecological infectious diseases and inflammation.Thereby may develop the medicine or the pharmaceutical composition that are used for the treatment of gynecological infectious diseases such as pelvic inflammation.This pharmaceutical composition can be made with the routine techniques in the pharmaceutical field, can make various dosage forms, as tablet, granule, capsule, oral liquid, drop pill, injection, transdermal patch, aerosol, suppository etc.These pharmaceutical compositions can be used to prevent and treat the purposes of gynecological infectious diseases such as pelvic inflammation.
Simultaneously, this Herba Taraxaci plant extract effective site that is rich in phenolic acid compound shows the acute and long term toxicity test of ICR mice, SD rat, beagle dog animal, each organizes experimental animal does not all have obvious adverse reaction in the test dose scope, do not have obvious pathology yet and change.Can think that short-term and life-time service do not have obvious toxic-side effects substantially to human body, have safety in utilization preferably.
In order to understand essence of the present invention better, with the form of embodiment this Herba Taraxaci plant extract effective site that is rich in phenolic acid compound is adopted the antibacterial activity in vitro test of " cup-plate method " development test medicine below respectively, the Cavia porcellus pelvic inflammatory disease test that colon bacillus or chlamydia trachomatis are caused, xylol causes the anti-inflammatory activity of mice ear inhibition test, on Carrageenan causes the influence test of rat paw edema, the influence test that the mice capillary permeability that causes Dichlorodiphenyl Acetate increases, influence test to mice granuloma induced by implantation of cotton pellets model, to rabbit fever models refrigeration function test due to the bacterial endotoxin, the caused ICR mice of chemical stimulation pain reaction inhibitory action is tested, to The pharmacological results such as the caused ICR mice of thermostimulation pain reaction inhibitory action tests, its new purposes in pharmaceutical field is described.Embodiment has provided this and has been rich in the part pharmacodynamics activity data of the Herba Taraxaci phenolic acid effective kind part of phenolic acid compound.Mandatory declaration be that embodiments of the invention are to be used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.
Embodiment 1: cup-plate method is studied this antibacterial activity in vitro that is rich in the Herba Taraxaci plant effective part extract of phenolic acid compound
Adopt the antibacterial activity in vitro of " cup-plate method " development test medicine, be to utilize the trial drug that is added in the cup of Oxford to be diffused into inoculation to have in the culture medium of test organisms, thereby suppress the growth of antibacterial, come the antibacterial activity of confirmed test medicine by the diameter that detects the inhibition zone that around the cup of Oxford, forms.
1.1 bacterial strain:
1. staphylococcus aureus 26003-23 is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
2. staphylococcus aureus 26112-6 is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
3. colon bacillus 44113-7 is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
4. colon bacillus 44103-6 is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
5. Pseudomonas aeruginosa 10102-14 is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
6. Bacillus proteus 49027-9 is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
7. klepsiella pneumoniae 46114-8 is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
8. beta hemolytic streptococcus 32210-18 is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
The concentration of trial drug is 5mg/ml, dissolves with sterile saline.
1.2 be subjected to the reagent thing
Herba Taraxaci plant effective part extract (lot number 040120): Haizheng Tianhua Medicine Research Co., Ltd., Zhejiang Prov provides.
The result of the test antibacterial circle diameter is represented with measured value.The results are shown in following table:
| Medicine | Antibacterial circle diameter (mm) | |||||||
| Gold Portugal bacterium-26003 | Gold Portugal bacterium-26112 | Colon bacillus-44113 | Colon bacillus-44103 | Bacillus pyocyaneus-10102 | Bacillus proteus-49027 | Klepsiella pneumoniae | Group B streptococcus-32210 | |
| Herba Taraxaci plant effective part extract | 13.60 | 9.56 | 14.46 | 20.04 | 15.54 | 16.24 | 14.02 | 21.64 |
Assessment and conclusion: this is rich in, and the Herba Taraxaci plant effective part extract of phenolic acid compound is external to have certain antibacterial activity to common pathogen.
Embodiment 2: the model investigation of mice caused by dimethylbenzene xylene ear swelling this be rich in phenolic acid compound Herba Taraxaci plant extract effective site anti-inflammatory activity
2.1 method and step:
2.1.1 the ICR mice, 20 ± 2 grams, complete male, Zhejiang Province medicine inspecting institute animal center provides.Animal divides into groups, numbers and weighs, and 10 every group, totally 5 groups.Herba Taraxaci phenolic acid effective kind part extract, lot number: 040120, Haizheng Tianhua Medicine Research Co., Ltd., Zhejiang Prov provides.
2.1.2 respectively organize medicine with the sterile saline preparation by experimental design concentration.Positive drug dexamethasone acetate tablets agent: lot number 020161 (the celestial jade pendant in Zhejiang pharmaceutical Co. Ltd produces), DEX, 5.0mg/kg; NS group: sterile saline; Reagent group (Herba Taraxaci effective part extract): low dose group is 100mg/kg, and middle dosage group is 200mg/kg, and high dose group is 400mg/kg.
2.1.3 respectively organize mice by corresponding dosage gastric infusion 3 days, 1 time/day, after the last administration 60 minutes, with 30 μ l analytical pure dimethylbenzene (lot numbers 980501, China chemical reagent work of Quzhou Chemical Industry Co. produces) duplicate local inflammation at the mouse right ear exterior feature, left ear is done contrast.Cause scorching back 30 minutes, the dislocation of mice cervical vertebra is put to death, and cuts left and right sides auricle, sweeps away left and right sides auricle with the card punch of diameter 9mm, weighs.Weight difference with same mice left and right sides auricle is represented " swelling degree ", and whether the difference of the positive group of statistical and reagent group and normal saline group mice auricle swelling degree is remarkable.Annotate:
2.2 result:
In each sample: the suppression ratio of (M), high dose group (H) is respectively 48.6%, 54.96% and 72.4% in low dose group (L), the low dose group, and the suppression ratio 72.55% of high dose group suppression ratio and positive drug DEX (5mg/kg) is basic identical.In addition, have good dose-effect relationship between the sample various dose, the results are shown in following table.
Herba Taraxaci plant extract effective site mice ear result of the test (suppression ratio %)
| n＝10 | NS | DEX | Low dose group | Middle dosage group | High dose group |
| Mean standard deviation suppression ratio (%) P value | 31.77 7.02 | 8.72 8.37 72.55％ *** | 16.33 7.61 48.60％ *** | 14.31 8.47 54.96％ ** | 8.77 5.34 72.40％ *** |
* represents difference highly significant (p＜0.01); * * represents difference extremely significantly (p＜0.001).
Basic principle according to anti-inflammatory drug screening: if the inhibitory rate of intumesce of screening of medicaments is more than 30%, and the average of its swelling degree and model group comparing difference remarkable (P＜0.05), illustrate that this medicine has antiphlogistic remarkable result, can further develop.Present embodiment is the result show, the Herba Taraxaci plant extract effective site that is rich in phenolic acid compound all has stronger inhibition activity to this inflammatory model at basic, normal, high dosage.
Embodiment 3: this is rich in the anti-pelvic inflammatory disease activity of the Herba Taraxaci plant extract effective site of phenolic acid compound the model investigation of female Cavia porcellus pelvic inflammatory disease
3.1. test objective: observe of the influence of Herba Taraxaci phenolic acid effective kind part plant extract, inquire into the therapeutical effect of this medicine to pelvic inflammatory disease to female Cavia porcellus pelvic inflammatory disease model.
3.2. test material
3.2.1 be subjected to the reagent thing
Herba Taraxaci phenolic acid effective kind part plant extract, lot number: 040120, Haizheng Tianhua Medicine Research Co., Ltd., Zhejiang Prov provides.Sodium carboxymethyl cellulose, lot number: F20040112, Shanghai chemical reagents corporation of Chinese Medicine group.JINJI JIAONANG, lot number: Z45020293, Lingfeng Pharmaceutical Co., Ltd., Guangxi.
3.2.2 bacterial strain
Colon bacillus 0111 reference culture: provide by last marine mountain hospital laboratory;
Chlamydia trachomatis E serotype: provide by Haizheng Tianhua Medicine Research Co., Ltd., Zhejiang Prov.
3.2.3 animal
Strain: Cavia porcellus; Source: Shanghai City Si Laike laboratory animal Co., Ltd.Body weight: 350～450 grams; Sex: female; Grade: regular grade; The quality certification number: NO 0003147.Grade: cleaning level, the quality certification number: the moving word of doctor 02-64 number.
3.3 test method
3.3.1 60 of Cavia porcelluss, body weight 350～450 grams, female.Cavia porcellus grouping: (1): sham operated rats, 0.5% cmc soln are pressed 10ml/kg and are irritated stomach; (2) JINJI JIAONANG group 1.17g/kg, by people's every day the highest consumption carry out body weight/dose factor and convert, it is 117mg/ml that 0.5% cmc soln and JINJI JIAONANG are mixed with final concentration; (3) 100mg/kg, it is 10mg/ml that 0.5% cmc soln and Herba Taraxaci phenolic acid effective kind part extract are mixed with final concentration; (4) 0.5% cmc soln and Herba Taraxaci phenolic acid effective kind part extract are mixed with final concentration is 20mg/ml to 200mg/kg; (5) 0.5% cmc soln and Herba Taraxaci phenolic acid effective kind part extract are mixed with final concentration is 40mg/ml to 400mg/kg Herba Taraxaci phenolic acid effective kind part extract, 10 every group; (6) colon bacillus model group: (a) colon bacillus is cultivated: colon bacillus 0111 bacterial strain is provided by middle mountain hospital laboratory, antibacterial culturing is on the SBA flat board, cultivation temperature is 35～37 ℃, picking colony is gone in the normal saline after 24 hours, with 10000g/ minute 4 ℃ after centrifugal 10 minutes, with twice of normal saline flushing and to regulate density to 105～106cfu/ml standby.(setting up spectrophotometer reduced turbidity and antibacterial chessboard counting method relation curve); (b) modelling: with 60 Cavia porcellus random packet, every group 10, perform the operation preceding 3 days by grouping administration administration, after the lumbar injection pentobarbital sodium 20mg/kg anesthesia, aseptic condition is successively opened lower abdomen, expose the uterus, and respectively inject 50 μ l colon bacillus in the bilateral uterus and use liquid and (contain 2～4 * 10
4CFU), successively close abdomen.The conventional raising, successive administration was put to death laboratory animal after 14 days, and anatomical isolation is taken out the reproductive system tissue of Cavia porcellus under aseptic condition, and slide gauge is measured the thickness in uterus, right side.Local organization quantitative culture: get the uterus, left side, add 10 mL of saline volume calculation (w/v), add liquid homogenate, serial dilution quantitative culture, computation organization's bacteria containing amount by 1 gram tissue weight.The uterus, right side is conventional fixing, paraffin section, HE dyeing, tissues observed pathological change under the light microscopic.Change 3 indexs with tissue thickness, clump count and tectology, with matched group comparative evaluation pelvic inflammatory disease model.Pathological observation index and integration standard are: 1. epithelial cell degeneration necrosis: "-" simple columnar epithelium note 0 minute; "+" epithelial cell is flat or come off＜1/3 note 1 minute; Flat or 1/3～2/3 pathological changes note 2 minutes of coming off of " ++ " epithelial cell; +++" holostrome epithelial cell degeneration necrosis note 3 minutes.2. the chronic inflammation cell is invaded profit: "-" no chronic inflammation cell was invaded the profit note 0 minute; "+" decimal be dispersed in or kitchen range and only in proper mucous membrane the note 1 minute; " ++ " is dispersed in or kitchen range but goed deep into flesh layer note 2 minutes; " +++" majority is dispersed in or stratiform is soaked into and involved the holostrome note 3 minutes.3. inner membrance congestion and edema: "-" no inner membrance congestion and edema note 0 minute; The slight congestion and edema note of "+" lamina propria 1 minute; " ++ " obviously congestion and edema remembers 2 fens; " +++" holostrome congestion and edema note 3 minutes.
3.3.2 60 of Cavia porcelluss, body weight 350～450 grams, female.
Cavia porcellus grouping: (1) sham operated rats, 0.5% cmc soln is pressed 10ml/kg and is irritated stomach (2) JINJI JIAONANG group 1.17g/kg, by people's every day the highest consumption carry out body weight/dose factor and convert, it is 117mg/ml (3) 100mg/kg that 0.5% cmc soln and JINJI JIAONANG are mixed with final concentration, and to be mixed with final concentration be 10mg/ml (4) 200mg/kg that 0.5% cmc soln and Herba Taraxaci phenolic acid effective kind part extract are mixed with final concentration is 20mg/ml (5) 400mg/kg Herba Taraxaci phenolic acid effective kind part extract group with 0.5% cmc soln and Herba Taraxaci phenolic acid effective kind part extract.It is 40mg/ml that 0.5% cmc soln and Herba Taraxaci phenolic acid effective kind part extract are mixed with final concentration, 10 every group.(6) chlamydia trachomatis model group: (a) chlamydia is cultivated: chlamydia trachomatis E serotype strain is provided by microorganism teaching and research room of Zhejiang University, directly in the Tissue Culture Flask that forms monolayer McCoy cell, cultivate with the chlamydia culture fluid, discard cell culture fluid, wash 3 times with the PBS that contains cycloheximide, add growth-promoting media (DMEM/FCS, the cycloheximide 1g/ml) cultivation of going down to posterity, the Ultrasonic Pulverization cell, 3000g/ minute centrifugal 10 minutes, collect chlamydia, and transfer to 2*10
7IFU/ml, preceding taking-up is used in-70 ℃ of preservations.(b) modelling: with 60 rat random packet, 10 every group, after the anesthesia of lumbar injection pentobarbital sodium, aseptic condition is successively opened lower abdomen, exposes fallopian tube, injects 50 μ l chlamydia application liquid and (contains 10
5IFU).Successively close abdomen.The conventional raising performed the operation back 24 hours by the grouping drug treatment, puts to death laboratory animal after continuous 14 days.Anatomical isolation is taken out the reproductive system tissue of Cavia porcellus under aseptic condition, and is conventional fixing, paraffin section, HE dyeing, tissues observed pathological change under the light microscopic.Change 3 indexs with swollen tissue rate, total white blood cells and tectology, with matched group comparative evaluation pelvic inflammatory disease model.The modeling fallopian tube is measured: the last administration is after 24 hours, animal is used pentobarbital sodium 20mg/kg intraperitoneal anesthesia, cut open the belly, win the bilateral fallopian tube, use scales/electronic balance weighing after removing fatty tissue, the weight difference of the fallopian tube left and right sides is inflammation swelling degree, obtains swelling rate and suppression ratio, compares between organizing.Swelling rate=(cause scorching oviductus lateralis weight-do not cause scorching oviductus lateralis weight)/do not cause scorching fallopian tube weight * 100%; Suppression ratio=(the average swelling rate of the model group fallopian tube-average swelling rate of administration group the fallopian tube)/average swelling rate of model group fallopian tube * 100%.(c) modeling fallopian tube pathological change: the last administration with pentobarbital sodium 30mg/kg intraperitoneal anesthesia, was cut open the belly animal after 24 hours, win the bilateral fallopian tube, weigh back formalin fixed, paraffin embedding, conventional section, HE dyeing, light microscopic is observed the fallopian tube morphological changes of various tissue components down.Pathological observation index and integration standard are: 1. epithelial cell degeneration necrosis: "-" simple columnar epithelium note 0 minute; "+" epithelial cell is flat or come off＜1/3 note 1 minute; Flat or 1/3～2/3 pathological changes note 2 minutes of coming off of " ++ " epithelial cell; " +++" holostrome epithelial cell degeneration necrosis note 3 minutes.2. the chronic inflammation cell is invaded profit: "-" no chronic inflammation cell was invaded the profit note 0 minute; "+" decimal be dispersed in or kitchen range and only in proper mucous membrane the note 1 minute; " ++ " is dispersed in or kitchen range but goed deep into flesh layer note 2 minutes; " +++" majority is dispersed in or stratiform is soaked into and involved the holostrome note 3 minutes.3. inner membrance congestion and edema: "-" no inner membrance congestion and edema note 0 minute; The slight congestion and edema note of "+" lamina propria 1 minute; " ++ " obviously congestion and edema remembers 2 fens; " +++" holostrome congestion and edema note 3 minutes.
Statistical disposition: each is organized the result and represents the significance that rate of change differs between employing variance test comparison administration front and back and each group with x ± SD.
3.4 result
3.4.1 colon bacillus is caused the influence of Cavia porcellus pelvic inflammatory disease model:
3.4.1.1 Herba Taraxaci phenolic acid effective kind part extract damages the influence of Aconitum carmichaeli Debx. palace diameter to Cavia porcellus:
Herba Taraxaci phenolic acid effective kind part extract is to the influence of uterus diameter (n=10, x ± s)
| Grouping | Uterus diameter (mm) | Uterus weight (g) |
| Sham-operation group ETEC model group dandelion phenolic acid effective kind part extract (400mg/kg) dandelion phenolic acid effective kind part extract (200mg/kg) dandelion phenolic acid effective kind part extract (100mg/kg) JINJI JIAONANG group (1170mg/kg) | 2.1±0.3 3.9±0.4## 2.9±0.3** 3.0±0.4* 3.7±0.4 2.9±0.4** | 0.16±0.03 0.29±0.03## 0.18±0.03** 0.26±0.01 0.29±0.01 0.24±0.04 |
* P＜0.05, * * P＜0.01vs model group #P＜0.05, ##P＜0.01vs sham operated rats
Compare with sham operated rats, colon bacillus model group uterus weight and diameter all significantly increase (P＜0.01), compare with the colon bacillus model group, JINJI JIAONANG group, high dose Herba Taraxaci phenolic acid effective kind part extract group uterus weight and uterus diameter significantly reduce (P＜0.01).Compare with the colon bacillus model group, the Herba Taraxaci phenolic acid effective kind part extract group of 200mg/kg can significantly alleviate the diameter (P＜0.05) in uterus, with the equivalence of JINJI JIAONANG group (P＞0.05).
3.4.1.2 Herba Taraxaci phenolic acid effective kind part extract is to the influence of Cavia porcellus uterine cancer cell bacteria containing amount:
| Grouping | Organize bacteria containing amount (CFU/ml) |
| Sham-operation group ETEC model group JINJI JIAONANG group (1170mg/kg) 100mg/kg dandelion phenolic acid effective kind part extract 200mg/kg 400mg/kg | (7.0±3.3)×10 0 (1.1±0.6)×10 4##** (1.2±0.4)×10 2##** (2.0±0.5)×10 2##** (1.3±0.6)×10 2##** (5.3±2.0)×10 1##** |
* P＜0.05, * * P＜0.01vs model group #P＜0.05, ##P＜0.01vs sham operated rats
Compare with sham operated rats, the uterine cancer cell bacteria containing amount is respectively organized in colon bacillus model group group and medication significantly increases (P＜0.01), compare with the colon bacillus model group, each group of JINJI JIAONANG group and Herba Taraxaci phenolic acid effective kind part extract all can significantly alleviate uterine cancer cell bacteria containing amount (P＜0.01).Wherein, the Herba Taraxaci phenolic acid effective kind part extract group equivalence (P＞0.05) of JINJI JIAONANG group and 20mg/kg
Herba Taraxaci phenolic acid effective kind part extract is learned influence to the Cavia porcellus uterus shape:
| Grouping | The epithelial cell degeneration necrosis | Cell infiltration | Congestion and edema |
| Sham-operation group ETEC model group dandelion phenolic acid effective kind part extract 400mg/kg dandelion phenolic acid effective kind part extract 200mg/kg dandelion phenolic acid effective kind part extract 100mg/kg JINJI JIAONANG group 1170mg/kg | 0±0 1.8±0.4 0.8±0.4** 0.9±0.3* 1.3±0.5 1.0±0.5* | 0±0 2.1±0.3 0.9±0.7** 1.2±0.6* 1.8±0.6 1.1±0.6* | 0±0 1.3±0.5 0.6±0.5** 0.7±0.5* 0.9±0.6 0.7±0.5* |
* P＜0.05, * * P＜0.01vs model group #P＜0.05, ##P＜0.01vs sham operated rats
Compare with sham operated rats, colon bacillus model group uterine epithelial cell degeneration necrosis, cell infiltration and tube wall congestion and edema be (P＜0.01) obviously, compare with the colon bacillus model group, high dose Herba Taraxaci phenolic acid effective kind part extract group can significantly alleviate the uterine epithelial cell degeneration necrosis, cell infiltration and tube wall congestion and edema (P＜0.01), the Herba Taraxaci phenolic acid effective kind part extract group of 200mg/kg can significantly alleviate the uterine epithelial cell degeneration necrosis, cell infiltration and tube wall congestion and edema be (P＜0.05) obviously, with the equivalence of JINJI JIAONANG group (P＞0.05).
3.4.2 chlamydia trachomatis is caused the influence of Cavia porcellus pelvic inflammatory disease model:
3.4.2.1 Herba Taraxaci phenolic acid effective kind part extract is to the influence of Cavia porcellus fallopian tube swelling rate:
Herba Taraxaci phenolic acid effective kind part extract is to the influence of fallopian tube swelling rate (n=10, x ± s)
| Grouping | Swelling rate (%) | Suppression ratio (%) |
| Sham-operation group chlamydia trachomatis model group dandelion phenolic acid effective kind part extract 400mg/kg dandelion phenolic acid effective kind part extract 200mg/kg dandelion phenolic acid effective kind part extract 100mg/kg JINJI JIAONANG group 1170mg/kg | 1.9±0.9 45.6±7.9## 20.3±4.8**## 25.3±8.4*## 29.7±5.7## 26.7±4.7*## | 55.5 44.6 34.9 41.5 |
* P＜0.05, * * P＜0.01vs model group #P＜0.05, ##P＜0.01vs sham operated rats
Compare with sham operated rats, chlamydia trachomatis model group fallopian tube swelling rate significantly increases (P＜0.01), compare with the chlamydia trachomatis model group, high dose Herba Taraxaci phenolic acid effective kind part extract group fallopian tube swelling rate significantly reduces (P＜0.01), the Herba Taraxaci phenolic acid effective kind part extract group of 200mg/kg can significantly alleviate fallopian tube swelling rate (P＜0.05), with the equivalence of JINJI JIAONANG group (P＞0.05).
3.4.2.2 Herba Taraxaci phenolic acid effective kind part extract is to the influence of Cavia porcellus fallopian tube pathological change:
Herba Taraxaci phenolic acid effective kind part extract thing is to the influence of fallopian tube pathological change (n=10, x ± s)
| Grouping | The epithelial cell degeneration necrosis | Cell infiltration | Congestion and edema |
| Sham-operation group chlamydia trachomatis model group dandelion phenolic acid effective kind part extract 400mg/kg dandelion phenolic acid effective kind part extract 200mg/kg dandelion phenolic acid effective kind part extract 100mg/kg JINJI JIAONANG group 1170mg/kg | 0±0 1.6±0.5 0.6±0.5** 0.7±0.5* 0.8±0.6 0.7±0.5* | 0±0 2.2±0.4 0.9±0.7** 1.0±0.5* 1.8±0.6 1.0±0.7* | 0±0 1.1±0.6 0.5±0.5** 0.7±0.5* 0.9±0.6 0.6±0.5* |
* P＜0.05, * * P＜0.01vs model group #P＜0.05, ##P＜0.01vs sham operated rats
Compare with sham operated rats, the necrosis of chlamydia trachomatis model group uterine tubal epithelium cytopathy, cell infiltration and tube wall congestion and edema be (P＜0.01) obviously, compare with the chlamydia trachomatis model group, high dose Herba Taraxaci phenolic acid effective kind part extract group can significantly alleviate the necrosis of uterine tubal epithelium cytopathy, cell infiltration and tube wall congestion and edema (P＜0.01), the Herba Taraxaci phenolic acid effective kind part extract group of 200mg/kg can significantly alleviate the necrosis of uterine tubal epithelium cytopathy, cell infiltration and tube wall congestion and edema be (P＜0.05) obviously, with the equivalence of JINJI JIAONANG group (P＞0.05).
Assessment and conclusion: the female pelvic cavity scope comprises genitals's (uterus, fallopian tube, fallopian tube), pelvic peritoneum and peritubal connective tissue, and the inflammation of Fa Shenging is referred to as pelvic inflammatory disease herein, is one of common gynecological disease.The pathogen of primary disease is mainly the mixed infection that anaerobe and aerobe cause.Route of infection mainly contain through lymphsystem spread, through the blood circulating propagation, prolong the genitals mucosa uply spread, by the adjacent organ direct extension.Sterilization such as childbirth or miscarriage, intrauterine surgical operation is not strict, the bad acute pelvic inflammatory disease that all can cause of menstrual hygiene, and delay then is a chronic pelvic inflammatory disease as the course of disease.If the fallopian tube adhesion obstruction often can cause infertile.Colon bacillus and chlamydia trachomatis are the main pathogenic microbes that causes chronic pelvic inflammatory disease.Herba Taraxaci phenolic acid effective kind part extract and JINJI JIAONANG all can obviously alleviate the inflammation of uterus that colon bacillus causes, wherein, and the Herba Taraxaci phenolic acid effective kind part extract curative effect and the JINJI JIAONANG equivalence of high dose.Herba Taraxaci phenolic acid effective kind part extract and JINJI JIAONANG have certain curative effect equally to the salpingitis tubal that chlamydia trachomatis causes.
Conclusion: but Herba Taraxaci phenolic acid effective kind part extract dose dependent alleviates metritis and salpingitis that colon bacillus, chlamydia trachomatis cause, except that the growth of direct minimizing antibacterial, also with to alleviate tissue inflammation reaction's degree relevant.
Embodiment 4: Herba Taraxaci phenolic acid effective kind part extract causes the refrigeration function test of rabbit fever models to endotoxin
4.1 purpose: utilize endotoxin to cause the refrigeration function that rabbit fever models is observed Herba Taraxaci phenolic acid effective kind part extract.
4.2. material:
4.2.1 medicine and reagent
Herba Taraxaci phenolic acid effective kind part extract (lot number 040120): Haizheng Tianhua Medicine Research Co., Ltd., Zhejiang Prov provides.Face the suspension that is mixed with desired concn with 0.5% sodium carboxymethyl cellulose (CMC-Na) solution with preceding.Aspirin (Aspirin): the Central-South Pharmaceutical Co in Hunan produces lot number 060308.Compound method is with Herba Taraxaci phenolic acid effective kind part extract.Sodium carboxymethyl cellulose (CMC-Na): China Medicine (Group) Shanghai Chemical Reagent Co., produces, lot number 20030626.
The bacterial endotoxin working standard: 120EU/ props up, and Nat'l Pharmaceutical ﹠ Biological Products Control Institute produces, lot number 2006-5.Face with preceding and be mixed with 50EU/ml solution with the apyrogeneity normal saline.Sodium chloride injection: Hangzhou Minsheng Pharmaceutical Group Co produces, lot number 40604263.
4.2.2 animal
White big ear rabbit, the male and female dual-purpose, 1.8～2.2 kilograms of body weight are provided by Zhejiang University zoopery center.
4.2.3 instrument
BT-AIICN series electronic clinical thermometer: Fuda, Shenzhen health Industrial Co., Ltd. produces.
4.3. method
4.3.1 endotoxin cause rabbit fever models [Xu Shuyun, Bian Rulian, Chen Xiu chief editor. pharmacological experimental methodology (second edition) Beijing: People's Health Publisher, 1991:729-731]
Test was carried out adaptability to rabbit in preceding continuous 3 days and is surveyed the anus temperature, measured 30 minutes at interval every day 2 times.The 4th day, with the anus temperature is that 32 rabbit of 38.0～39.6 ℃ are divided into blank group (CMC-Na) at random, positive controls (aspirin 150mg/kg), Herba Taraxaci phenolic acid effective kind part extract small dose group (50mg/kg), the heavy dose of group of dosage group (100mg/kg) and Herba Taraxaci phenolic acid effective kind part extract (200mg/kg) in the Herba Taraxaci plant effective part extract.Each is organized all by 6ml/kg volume gastric infusion, every day 1 time, totally 4 days.Last administration beginning in preceding 2 hours fasting, and measure the anus temperature 2 times, 30 minutes at interval, average as the basal body temperature before the pyrogenicity.After the last administration 1 hour, in auricular vein bacterial injection endotoxin 50EU/kg, the anus temperature of measuring each rabbit in 1,2,3,4,5 and 6 hour respectively in injection back as pyrogenicity after body temperature, with the difference of body temperature after the pyrogenicity and basal body temperature as pyrogenicity after intensification value (if negative value counts 0).
4.3.2 statistical procedures
(x ± s) expression, the significance test of group difference adopts the T-Test in the SPSS-13.0 statistical software to carry out to experimental data with mean ± standard deviation.
4.4. result
By following table as seen: intravenous injection bacterial endotoxin pyrogenicity is after 1 hour, the fervescence value of each dosage group of Herba Taraxaci phenolic acid effective kind part extract is all significantly less than blank group (P＜0.05 or P＜0.01), and wherein the effect of high dose group is suitable with resistive contrast medicine aspirin; After the pyrogenicity 2 hours, the fervescence value of Herba Taraxaci phenolic acid effective kind part extract high dose group is also significantly less than blank group (P＜0.05), and is still suitable with the effect of positive control drug aspirin; During 3～6 hours, the fervescence value variation tendency of Herba Taraxaci phenolic acid effective kind part extract high dose group is basic and aspirin is similar after the pyrogenicity.
The influence that Herba Taraxaci phenolic acid effective kind part extract raises to rabbit body temperature due to the endotoxin (x ± s)
| Group | Dosage (mg/ kg) | Number of animals (only) | Basal body temperature (℃) | Fervescence value behind the injection endotoxin (Δ T, ℃) | ||
| 1 hour | 2 hours | 3 hours | ||||
| The CMC-Na aspirin | 150 | 8 6 | 38.43±0.24 38.71±0.36 | 1.37±0.41 0.74±0.31** | 1.38±0.44 0.91±0.35* | 0.98±0.44 0.54±0.34 |
| Herba Taraxaci phenolic acid effective kind part extract | 50 100 200 | 6 6 6 | 38.31±0.29 38.68±0.26 39.06±0.32 | 0.83±0.20* 0.93±0.27* 0.78±0.16** | 1.38±0.27 1.37±0.24 0.91±0.34* | 0.99±0.21 1.08±0.33 0.43±0.54 |
The influence that Herba Taraxaci phenolic acid effective kind part extract raises to rabbit body temperature due to the endotoxin (x ± s) (continuous table)
| Group | Dosage (mg/ kg) | Number of animals (only) | Basal body temperature (℃) | Fervescence value behind the injection endotoxin (Δ T, ℃) | ||
| 4 hours | 5 hours | 6 hours | ||||
| The CMC-Na aspirin | 150 | 8 6 | 38.43±0.24 38.71±0.36 | 0.62±0.31 0.22±0.20* | 0.63±0.32 0.15±0.20** | 0.34±0.34 0.10±0.10 |
| Herba Taraxaci phenolic acid effective kind part extract | 50 100 200 | 6 6 6 | 38.31±0.29 38.68±0.26 39.06±0.32 | 0.74±0.34 0.61±0.45 0.53±0.62 | 0.56±0.33 0.32±0.24 0.13±0.16** | 0.43±0.35 0.28±0.19 0.13±0.16 |
Compare with the CMC-Na group: * P＜0.05, * * P＜0.01.
4.5. conclusion
Herba Taraxaci phenolic acid effective kind part extract has refrigeration function to rabbit fever models due to the bacterial endotoxin.
Embodiment 5: Herba Taraxaci phenolic acid effective kind part extract is to the analgesic activity test of mice chemical stimulation method and thermostimulation method model
5.1 purpose: adopt mouse writhing method and mice hot plate method to observe the analgesic activity of Herba Taraxaci phenolic acid effective kind part extract.
5.2. material:
5.2.1 medicine and reagent
Herba Taraxaci phenolic acid effective kind part extract (lot number 040120): Haizheng Tianhua Medicine Research Co., Ltd., Zhejiang Prov provides.Face the suspension that is mixed with desired concn with 0.5% sodium carboxymethyl cellulose (CMC-Na) solution with preceding.Aspirin (Aspirin): the Central-South Pharmaceutical Co in Hunan produces lot number 060308.Compound method is with Herba Taraxaci phenolic acid effective kind part extract.Sodium carboxymethyl cellulose (CMC-Na): China Medicine (Group) Shanghai Chemical Reagent Co., produces, lot number 20030626.Glacial acetic acid: analytical pure, Hangzhou chemical reagent company limited is produced, lot number 20060330.
5.2.2 animal: the ICR mice, the male and female dual-purpose, body weight 18～22 grams are available from Shanghai Slac Experimental Animal Co., Ltd..
5.2.3 instrument: JJ2000 type precise electronic balance: the two outstanding test instrunment in Changshu factory produces.Stopwatch: stopwatch factory in Shanghai produces.Hot plate apparatus: self-control.
5.3. method
5.3.1 mouse writhing method (chemical stimulation method) [Xu Shuyun, Bian Rulian, Chen Xiu chief editor. pharmacological experimental methodology (second edition) Beijing: People's Health Publisher, 1991:695-701]
Get 50 ICR mices, male and female half and half, be divided into 5 groups at random, every group 10, be blank group (CMC-Na), positive controls (aspirin 250mg/kg), Herba Taraxaci phenolic acid effective kind part extract small dose group (100mg/kg), dosage group (200mg/kg) in the Herba Taraxaci phenolic acid effective kind part extract, the heavy dose of group of Herba Taraxaci phenolic acid effective kind part extract (400mg/kg).Each is organized all by 10ml/kg volume gastric infusion, every day 1 time, totally 4 days.After the last administration 1 hour, every mouse peritoneal is injected 0.7% acetum 0.2ml, write down and respectively organize mice in 20 minutes and turn round the body number of times by what acetic acid caused.
Calculate the analgesia rate by following formula:
Analgesia rate=(matched group is on average turned round body number of times-administration group and on average turned round the body number of times)/matched group is on average turned round body number of times * 100%
5.3.2 mice hot plate method (thermostimulation method) [Xu Shuyun, Bian Rulian, Chen Xiu chief editor. pharmacological experimental methodology (second edition) Beijing: People's Health Publisher, 1991:695-701]
The temperature of hot plate apparatus is controlled at 55 ℃ ± 0.5 ℃, gets female ICR mice and put on the hot plate, the record mice is from putting into hot plate to the metapedes required time occurring licking, with this as this Mus pain threshold.30 minutes at interval, redeterminate pain threshold, get twice meansigma methods as this Mus administration before pain threshold.All pain thresholds were less than 5 seconds or give it up greater than 30 seconds and happiness jumping person Yi Pen.Be divided into 5 groups at random, 10 every group with selecting 50 qualified mices.Each corresponding dosage of organizing gastric infusion is identical with hot plate method, but is the single medication.After administration 30 minutes, 60 minutes, 90 minutes, 120 minutes, measured each Mus pain threshold in 150 minutes and 240 minutes, if still painless reaction in the mice 60 seconds, immediately with its taking-up, pain threshold was by 60 seconds.
Calculate the threshold of pain by following formula and improve percentage rate:
Percentage rate=(the preceding average pain threshold of average pain threshold-administration after the administration)/preceding average pain threshold of administration * 100% is improved in the threshold of pain
5.3.3 statistical procedures
(x ± s) expression, the significance test of group difference adopts the T-Test in the SPSS-13.0 statistical software to carry out to experimental data with mean ± standard deviation.
5.4. result
5.4.1 Herba Taraxaci phenolic acid effective kind part extract is to the influence of chemical stimulation induced mice pain reaction
By table 5.4.1 as seen: each administration group all suppresses acetic acid induced mice writhing response, wherein dosage group, heavy dose of group and positive control drug aspirin group and blank group compare in the Herba Taraxaci phenolic acid effective kind part extract, group difference all has significance meaning (P＜0.05, P＜0.01 and P＜0.001), the analgesia rate is respectively 47.44%, 62.82% and 78.53%.Experimental result proves: Herba Taraxaci phenolic acid effective kind part extract can suppress the mice pain reaction that chemical stimulation causes, its analgesic activity presents certain dose dependent.
The influence of table 5.4.1 Herba Taraxaci phenolic acid effective kind part extract Dichlorodiphenyl Acetate induced mice writhing response (x ± s, n=10)
| Group | Dosage (mg/kg) | Turn round the body number of times (inferior/20min) | Analgesia rate (%) |
| CMC-Na | 31.20±16.84 | - | |
| Aspirin | 250 | 6.70±6.34*** | 78.53 |
| Herba Taraxaci plant effective part extract | 100 200 400 | 26.30±7.70 16.40±9.13* 11.60±11.48** | 15.71 47.44 62.82 |
Compare with the CMC-Na group: * P＜0.05, * * P＜0.01, * * * P＜0.001
5.4.2 Herba Taraxaci phenolic acid effective kind part extract is to the influence of thermostimulation induced mice pain reaction
Data show among the table 5.4.2: the administration of the heavy dose of group of Herba Taraxaci phenolic acid effective kind part extract is onset (P＜0.05) after 30 minutes, three dosage groups and blank group are relatively in the time of 60 minutes, pain threshold difference is remarkable (P＜0.05 or P＜0.01) all, and is suitable with the effect of positive control drug aspirin; Percentage rate is improved apparently higher than the aspirin group in the maximum threshold of pain after the administration of heavy dose of group.Experimental result shows: the various dose group of Herba Taraxaci plant effective part extract all has in various degree analgesic activity to the mice pain reaction due to the thermostimulation.
Table 5.4.2 Herba Taraxaci phenolic acid effective kind part extract to the influence of hot plate method mice pain threshold (x ± s, n=10)
| Group | Dosage (mg/kg) | Pain threshold (second) before the administration | Percentage rate (%) is improved in the pain threshold after the administration (s) and the threshold of pain | ||
| 30 minutes | 60 minutes | 90 minutes | |||
| CMC-Na aspirin dandelion plant effective part extract | 250 100 200 400 | 19.56±3.37 17.44±2.59 18.10±3.28 18.46±3.94 17.91±3.1 | 17.86±9.68 (0) 25.39±18.63 (45.58) 23.99±13.06 (32.54) 26.75±18.51 (44.91) 33.89±16.85* (89.22) | 16.27±2.84 (0) 23.94±15.09 (32.27) 23.96±9.76* (32.38) 24.58±6.67** (33.15) 23.54±9.93* (31.43) | 11.09±2.46 (0) 24.59±13.96** (41.00) 21.33±19.59 (17.85) 21.49±9.83** (16.14) 18.50±5.95** (3.29) |
Table 5.4.2 (continuing)
| Group | Dosage (mg/kg) | Pain threshold (s) before the administration | Percentage rate (%) is improved in the pain threshold after the administration (s) and the threshold of pain | ||
| 120 minutes | 150 minutes | 240 minutes | |||
| CMC-Na aspirin dandelion plant effective part extract | 250 100 200 400 | 19.56±3.37 17.44±2.59 18.10±3.28 18.46±3.94 17.91±3.1 | 12.42±5.84 (0) 12.84±3.89 (0) 23.34±19.06 (28.95) 17.14±5.25 (0) 16.16±4.30 (0) | 11.08±2.68 (0) 11.99±4.21 (0) 18.16±8.90* (0.33) 20.65±9.54* (11.86) 18.87±5.14*** (5.36) | 12.65±3.80 (0) 15.98±16.03 (0) 14.03±4.64 (0) 20.98±14.77 (13.65) 13.44±9.47 (0) |
Compare with the CMC-Na group: * P＜0.05, * * P＜0.01, * * * P＜0.001; Bracket inner digital is that percentage rate is improved in the threshold of pain.
5.5. conclusion
Herba Taraxaci phenolic acid effective kind part extract is inhibited to chemical stimulation and the caused mice pain reaction of thermostimulation.
Embodiment 6: Herba Taraxaci phenolic acid effective kind part extract on Carrageenan causes the influence of rat paw edema
6.1 method: 40 of male SD rats, body weight 180 ± 20 grams, be divided into 5 groups at random: the heavy dose of group of dosage group (TME 100mg/kg) and Herba Taraxaci phenolic acid effective kind part extract (TME 200mg/kg) in normal control group (NS10ml/kg), positive drug Dexamethasone group (DEX 2.5mg/kg), Herba Taraxaci phenolic acid effective kind part extract small dose group (TME 50mg/kg), the Herba Taraxaci phenolic acid effective kind part extract, 8 every group.Measurement causes scorching preceding rat normal foot sole of the foot volume 2 times, gets its meansigma methods.Every animal is irritated stomach and gives 5 ml physiological salines capable water load.After 30 minutes, gastric infusion 1ml/100g, after the administration 60 minutes, left back sufficient plantar aponeurosis injection 1% carrageenin down caused inflammation for 0.1 milliliter, and right back sufficient same area is injected 0.1 ml physiological saline and is compared.Cause scorching back 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours and measure the rat paw volume respectively.
The result:
The influence of the inductive rat paw edema of Herba Taraxaci phenolic acid effective kind part extract on Carrageenan sees Table 6.2.Herba Taraxaci phenolic acid effective kind part extract 50,100 and 200mg/kg dosage under, 1～6 hour rat paw edema on Carrageenan injection back has significant resistancing action, and its suppression ratio is respectively 24.4-39.3%, 34.1～44.3% and 41.5～56.7%.And the positive drug dexamethasone has produced 39.0～54.1% suppression ratio under 2.5mg/kg dosage.
Table 6.2 Herba Taraxaci phenolic acid effective kind part on Carrageenan causes the influence (x ± s) of rat paw edema
| Contrast | Dosage (mg/kg) | Toes swelling (ml) (suppressing %) | |||||
| 1 hour | 2 hours | 3 hours | 4 hours | 5 hours | 6 hours | ||
| Blank group DEX TME | - 2.5 50 100 200 | 0.41±0.03 0.25±0.02 *** (39.0) 0.31±0.02 * (24.4) 0.27±0.02 ** (34.1) 0.24±0.02 *** (41.5) | 0.65±0.03 0.32±0.03 *** (50.8) 0.42±0.02 *** (35.4) 0.36±0.02 *** (44.6) 0.31±0.03 *** (52.3) | 0.66±0.03 0.32±0.02 *** (51.5) 0.44±0.03 *** (33.3) 0.37±0.04 *** (43.9) 0.31±0.04 *** (53.0) | 0.60±0.03 0.30±0.02 ***(50.0) 0.40±0.02 ***(33.3) 0.35±0.02 ***(41.7) 0.28±0.03 ***(53.3) | 0.58±0.04 0.27±0.02 *** (53.4) 0.38±0.02 *** (34.5) 0.33±0.02 *** (43.1) 0.27±0.02 *** (53.4) | 0.61±0.05 0.28±0.03 *** (54.1) 0.37±0.04 *** (39.3) 0.34±0.03 *** (44.3) 0.27±0.04 *** (56.7) |
Numeric representation method: mean+SD, * P＜0.05, * * P＜0.01, * * * P＜0.001 (with the contrast of blank group)
Embodiment 7: Herba Taraxaci phenolic acid effective kind part extract Dichlorodiphenyl Acetate causes the influence that the mice capillary permeability increases
7.1 method: 50 of male ICR mouses, body weight 20 ± 2 grams, be divided into 5 groups at random: the heavy dose of group of dosage group (TME 100mg/kg) and Herba Taraxaci phenolic acid effective kind part extract (TME 200mg/kg) in normal control group (NS10ml/kg), positive drug Dexamethasone group (DEX 2.5mg/kg), Herba Taraxaci phenolic acid effective kind part extract small dose group (TME 50mg/kg), the Herba Taraxaci phenolic acid effective kind part extract, 10 every group.Administration every day 1 time, successive administration 7 days.After the last administration 1 hour, tail vein injection 0.2% Azo-Blue 0.1ml/10g body weight, lumbar injection 0.6% acetic acid 0.2ml/ is only immediately.Cause scorching back 60 minutes and put to death mice, cut off the abdominal cavity, with 5ml normal saline flushing abdominal cavity for several times, collect cleaning mixture, centrifugal 5 minutes of 4500rpm gets supernatant and measures absorbance (OD value) with ultraviolet spectrophotometer outside 590nm.Calculate the average and the standard deviation of each treated animal peritoneal exudate OD value, whether the difference of statistics matched group and administration group is remarkable.
The result: Herba Taraxaci phenolic acid effective kind part extract Dichlorodiphenyl Acetate causes the result that influences that the mice capillary permeability increases and is: 50,100 and 200mg/kg dosage under, the mice capillary permeability that Herba Taraxaci phenolic acid effective kind part extract has significantly suppressed acetic-acid induced increases, and its suppression ratio is respectively 23.78%, 33.81% and 42.4%.The positive drug dexamethasone has been brought into play similar inhibitory action under 2.5mg/kg dosage, its suppression ratio is 34.98%.
Embodiment 8: Herba Taraxaci phenolic acid effective kind part extract is to the influence of mice granuloma induced by implantation of cotton pellets model
8.1 method: 50 of male ICR mouses, body weight 20 ± 2 grams, be divided into 5 groups at random: the heavy dose of group of dosage group (TME 100mg/kg) and Herba Taraxaci phenolic acid effective kind part extract (TME 200mg/kg) in normal control group (NS10ml/kg), positive drug Dexamethasone group (DEX 2.5mg/kg), Herba Taraxaci phenolic acid effective kind part extract small dose group (TME 50mg/kg), the Herba Taraxaci phenolic acid effective kind part extract, 10 every group.Used etherization in 1 hour after the first administration, dorsal position is fixed.Respectively make a transverse incision that is about 8-10mm at left and right groin with scalpel, the reuse mosquito forceps expands subcutaneous tissue, and every side is implanted a sterilization cotton balls of handling through penicillin and streptomycin and (weigh 10 ± 1mg), and posterior tubercle sewed up 2 pins.Every day, gastric infusion was 1 time, successive administration 7 days.Animal is put to death in cervical vertebra dislocation in the 8th day, takes out cotton balls, picks most fatty tissue, puts 60 ℃ of baking boxs and dries to constant weight, and it is heavy to deduct former cotton balls, is granuloma weight.Respectively organize granuloma weight (granuloma is represented with the mg/100g body weight), and calculate the resistance rate.
8.2 result
Herba Taraxaci phenolic acid effective kind part extract sees Table 8.1 to the influence of cotton balls inducing mouse granuloma model.The result shows: under 50mg/kg, 100mg/kg and 200mg/kg dosage, Herba Taraxaci phenolic acid effective kind part extract all has remarkable resistancing action (P＜0.01 or P＜0.001) to cotton balls inducing mouse granulation tissue hyperplasia, its suppression ratio is respectively 29.4%, 34.2% and 43.6%, has obvious dose-effect relationship.Positive drug dexamethasone (DEX) also has significant inhibitory effect (P＜0.001) to this model, and its suppression ratio is 48.8%.
The influence that table 8.1 Herba Taraxaci phenolic acid effective kind part extract forms cotton balls inducing mouse granulation tissue
| Group | Dosage (mg/kg) | The animal number of elements | Granulation weight (mg/100g body weight) | Suppression ratio (%) |
| Blank DEX TME | 10ml/kg 5mg/kg 25mg/kg 50mg/kg 100mg/kg | 10 10 10 10 10 | 68.36±21.83 34.98±15.46 *** 48.26±19.48 ** 44.99±17.81 *** 38.58±11.22 *** | - 48.8 29.4 34.2 43.6 |
Numeric representation method: mean+SD, * P＜0.05, * * P＜0.01, * * * P＜0.001 (with the contrast of blank group)
Claims (8)
1. one kind is that the Herba Taraxaci plant extract inside and outside of main effective site is antibiotic and suppress the medical usage of acute and chronic inflammation with phenolic acid compound;
2. one kind is the Herba Taraxaci plant extract treatment bacillus coli of main effective site or the medical usage of the pelvic inflammatory disease that chlamydia trachomatis infection causes with phenolic acid compound;
3. one kind is that the Herba Taraxaci plant extract of main effective site is used for analgesic and the analgesic medical usage with phenolic acid compound;
4. according to the purposes of claim 1～3, it is characterized by the Herba Taraxaci phenolic acid effective kind part that contains the caffeoyl guinic acid compounds and be used for prevention and treat gynecopathy such as pelvic inflammatory catching;
5. one kind is the pharmaceutical composition that the Herba Taraxaci plant extract of main effective site is used to suppress antibacterial, treatment pelvic inflammatory disease with phenolic acid compound, and it contains Herba Taraxaci plant extract and pharmaceutically acceptable auxiliaries as the treatment effective dose of active substance.
6. according to the pharmaceutical composition of claim 5, wherein the content (with the colorimetry test) as phenolic acid compound in the Herba Taraxaci plant extract effective site of the treatment effective dose of active component must not be less than 20%.
7. according to the content of claim 1～6, Herba Taraxaci plant extract phenolic acid effective kind part among the present invention, it is characterized by in this extract except that containing the flavonoid phenoloid and caffeic acid, ferulic acid and chlorogenic acid that luteolin is a basic framework, the two caffeoyl guinic acid class compound of phenolic acid active substances that also contain three structural similarities, they are: 3, and the 5-O-dicaffeoyl quinic acid; 3, the 4-O-dicaffeoyl quinic acid; 4, the 5-O-dicaffeoyl quinic acid.
8. according to the pharmaceutical composition of claim 5～6, it can be externally-applied liniment, injection, aerosol, suppository, membrane, tablet, paster agent, capsule, drop pill, oral liquid or ointment, can also adopt known controlled release of modern pharmaceutical circle or slow release formulation.
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| CNA2006100529985A CN1911260A (en) | 2006-08-18 | 2006-08-18 | Application of phenolic acids active components from dandelion for inhibiting gynecologic pelvic inflammatory disease |
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| CNA2006100529985A CN1911260A (en) | 2006-08-18 | 2006-08-18 | Application of phenolic acids active components from dandelion for inhibiting gynecologic pelvic inflammatory disease |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102973545A (en) * | 2012-12-11 | 2013-03-20 | 中国药科大学 | Abirritation application of dicaffeoylquinic acid type capsaicin receptor antagonist |
| CN103316118A (en) * | 2013-07-02 | 2013-09-25 | 通化斯威药业股份有限公司 | Pure vegetable-fruit essence capable of diminishing inflammation, resisting bacteria and preventing infection, and preparation method thereof |
| CN105696196A (en) * | 2016-04-05 | 2016-06-22 | 东华大学 | Method for preparing dandelion phenol activity antibacterial agent and chitosan composite nanofiber felt |
| CN110251499A (en) * | 2019-06-10 | 2019-09-20 | 陕西理工大学 | Dandelion extract and its purposes in preparation prevention and treatment woman vagina disease medicament |
-
2006
- 2006-08-18 CN CNA2006100529985A patent/CN1911260A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102973545A (en) * | 2012-12-11 | 2013-03-20 | 中国药科大学 | Abirritation application of dicaffeoylquinic acid type capsaicin receptor antagonist |
| CN103316118A (en) * | 2013-07-02 | 2013-09-25 | 通化斯威药业股份有限公司 | Pure vegetable-fruit essence capable of diminishing inflammation, resisting bacteria and preventing infection, and preparation method thereof |
| CN103316118B (en) * | 2013-07-02 | 2015-04-08 | 通化斯威药业股份有限公司 | Pure vegetable-fruit essence capable of diminishing inflammation, resisting bacteria and preventing infection, and preparation method thereof |
| CN105696196A (en) * | 2016-04-05 | 2016-06-22 | 东华大学 | Method for preparing dandelion phenol activity antibacterial agent and chitosan composite nanofiber felt |
| CN110251499A (en) * | 2019-06-10 | 2019-09-20 | 陕西理工大学 | Dandelion extract and its purposes in preparation prevention and treatment woman vagina disease medicament |
| CN110251499B (en) * | 2019-06-10 | 2022-11-01 | 陕西理工大学 | Dandelion extract and application thereof in preparing medicine for preventing and treating woman vaginal diseases |
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