CN102716115B - Pharmaceutical composition - Google Patents
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- CN102716115B CN102716115B CN201210241706.8A CN201210241706A CN102716115B CN 102716115 B CN102716115 B CN 102716115B CN 201210241706 A CN201210241706 A CN 201210241706A CN 102716115 B CN102716115 B CN 102716115B
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Abstract
The invention belongs to the technical field of medicines and provides a pharmaceutical composition. The pharmaceutical composition comprises germacrone, furanodiene, curdione, beta-elemene, curcumol, curzerene and borneol. The components of the pharmaceutical composition are definite, and the quality can be controlled. The pharmaceutical composition can be used for preparing vaginal or rectal preparations and has a good effect of treating HR-HPV (high risk human papillomavirus) and/or LR-HPV (low risk human papillomavirus) infection and cervical diseases and can be used widely clinically.
Description
Technical field
The invention belongs to medical technical field, be specifically related to a kind of pharmaceutical composition that contains 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, furanodiene, curdione, beta-elemene, curcumenol, curzerene, Borneolum Syntheticum.
Background technology
Human papillomavirus (HPV) is that a kind of papillary tumor vacuolating virus A that belongs to papovaviridae belongs to, and is spherical DNA viruses, can cause the squamous epithelial cancer propagation of human body skin mucosa.Isolated at present kind more than 130, different hypotypes causes different clinical manifestations, invades different tissue sites.By sending out classification carcinous, can be divided into low risk HPV and high-risk HPV.HPV resistance is strong, can tolerate dry and long-term preservation, and heating or process through formalin can deactivation, so high-temperature sterilization and the sterilization of 2% glutaraldehyde can deactivations.HPV mainly comprises 13 kinds of hypotypes, and the generation that becomes (CIN) with cervical cancer, cervix uteri squamous epithelial cancer tumor is closely related.Cervix uteri persistent infection HPV 8-10 can develop into cervical cancer, and " cervical cancer " is the second largest gynecological cancer after breast carcinoma, in global new cases 460,000/year, Asia accounts for half, 80% occurs in developing country, dies from every year nearly 200,000 people of patient of cervical cancer.China's new cases 130,000/year, die from cervical cancer 4-5 ten thousand/year, M & M all presents increase trend." cervical cancer " is the cancer of preventing and treating that current unique cause of disease is clear and definite, and removing HPV is the determiner of the blocking-up course of disease, prevention cervical cancer.
In recent ten years, the Chinese medicine that the BAOFUKANG SHUAN (main component is Oleum Curcumae and Borneolum Syntheticum) of take is representative is widely used in the treatment that female genital tract HPV infects, and has obtained good curative effect; Therefore, for active component, develop the diseases such as optimum or malignant change that new pharmaceutical composition comes prophylactic treatment HPV to infect with and bring out on this basis, particularly to the HR-HPV to cervical cancer, infect significant.
Rhizoma Curcumae is the dry rhizome of zingiberaceous plant Rhizoma Curcumae Curcuma phaeocaulis Valeton, Guangxi zedoary C.kwangsiensis S.G.Lee et C.F.Liang or RADIX CURCUMAE C.wenyujin Y.H.Chen et C.Ling.The latter practises title " warm Rhizoma Curcumae ".Rhizoma Curcumae is containing volatile oil 1.5%～2%, the highest with curzerenone content, is secondly curcumenol, Curcumenol, curcumadiol etc.In addition still contain Rhizoma Curcumae polysaccharide.Guangxi zedoary, containing volatile oil 1%-1.2%, is planted composition containing more than 20 in oil, and content is up to Camphora, approximately 17.7%, eucalyptol approximately 7.5%, and containing zingiberene, curcumenol, ar-curcumene, fragrant zingiberone, curdione etc.RADIX CURCUMAE containing volatile oil 1.4%-2.0%, is planted main constituent containing more than 20 in oil, is mainly curcumenol, curdione, gima ethylenic, 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-etc., nearly report therefrom separate α-, β-, δ-elemene, wherein beta-elemene is main anti-tumor active ingredient.
The comparison > > (Chinese herbal medicine of 3 kind Rhizoma Curcumae volatile oil chemical compositions of < <, the 36th the 12nd phase of volume, 1785～1787) Rhizoma Curcumae in above-mentioned three places of production is compared, although point out that different cultivars Rhizoma Curcumae has a lot of total compositions, mass fraction difference is larger; The amount significant difference of the composition such as curzerenone, curdione in the Rhizoma Curcumae of the different places of production.And the main component of Rhizoma Curcumae and the notable difference of content, meeting directly affect the curative effect of aromatic turmeric oil preparation.
CN102058568 discloses the application of 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-in preparation treatment HPV infection medicine; CN102091059 discloses the application of curdione in preparation treatment HPV infection medicine; CN102091064 discloses the application of furanodiene in preparation treatment HPV infection medicine; CN102091065 discloses the application of curcumenol in preparation treatment HPV infection medicine.Mentioned component is all proved to be HPV is infected effectively, but because HPV hypotype is numerous, each hypotype viral phenotype, growth characteristics, pathogenic, the everyways such as the sensitivity of medicine and clinical prognosis be there are differences, thereby single component can not be effective to all hypotypes, so the preparation of single component is unfavorable for clinical use.
Summary of the invention
For solving existing problem in above-mentioned prior art, the invention provides a kind of new pharmaceutical composition and preparation thereof.
The object of this invention is to provide a kind of pharmaceutical composition, said composition has good therapeutical effect to HR-HPV (high-risk human mammilla papillomavirus) and/or LR-HPV (high-risk human mammilla papillomavirus) infection.
The object of this invention is to provide a kind of pharmaceutical composition, said composition is effective to vaginitis.
The object of this invention is to provide a kind of preparation that contains this pharmaceutical composition.
The object of this invention is to provide the application of a kind of preparation that contains this pharmaceutical composition in anti-HPV virus.
The object of this invention is to provide the application of a kind of preparation that contains this pharmaceutical composition in treatment vaginitis.
Particularly, the invention provides:
A pharmaceutical composition, its feature pharmaceutical composition comprises 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, furanodiene, curdione, beta-elemene, curcumenol, curzerene, Borneolum Syntheticum.
Pharmaceutical composition described above, the composition that comprises following weight portion:
3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-5-16 weight portion;
Furanodiene 10-40 weight portion;
Curdione 5-25 weight portion;
Beta-elemene 4-11 weight portion;
Curcumenol 1-10 weight portion;
Curzerene 8-22 weight portion;
Borneolum Syntheticum 30-90 weight portion.
Borneolum Syntheticum described above is selected from natural Broneolum Syntheticum and/or synthetic borneol.
Pharmaceutical composition described above is prepared into vagina administration preparation or rectally preparation.
Described preparation comprises suppository, ointment, capsule, effervescent tablet, gel, lotion, membrane or foam.
Pharmaceutical composition described above prevents and/or treats the application in the medicine of human papilloma virus infection in preparation.
Wherein human papilloma virus infection comprises high-risk human mammilla papillomavirus infection and/or low risk human papilloma virus infection.
Pharmaceutical composition described above prevents and/or treats the application in the medicine of vaginitis, cervical erosion, cervical cancer in preparation.
The present invention compared with prior art has the following advantages and good effect:
1, the bright described pharmaceutical composition definite ingredients of we, quality controllable;
2, pharmaceutical composition of the present invention is not subject to the restriction in the places of origin of raw materials, is applicable to the large production of industry;
3, pharmaceutical composition of the present invention all has good therapeutical effect to HR-HPV and/or LR-HPV, cervical disease etc., is suitable for wide clinical application.
Pharmacological experimental example
Below the invention will be further described for the description by the specific embodiment, but this is not limitation of the present invention, those skilled in the art are according to basic thought of the present invention, can make various modifications or improvement, but only otherwise depart from basic thought of the present invention, all within the scope of the present invention.
HPV nucleic acid amplification (PCR) fluorescence detection reagent kit is purchased from PiJi Biology Engineering Co., Ltd., Shenzhen City.
Oleum Curcumae is purchased from Ji'an, Jiangxi magnificent spice refinery, and according to the requirement of 2010 editions Firsts of Chinese Pharmacopoeia, detecting 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-content is 12.3%.
3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, furanodiene, curdione, beta-elemene, curcumenol, curzerene purchased from Shanghai along vigorous biological company limited.
Borneolum Syntheticum is purchased from Tianjin Li Lang Chemical Industry Science Co., Ltd.
Test example 1: the effect of anti-HPV
Test reagent: HPV nucleic acid amplification (PCR) fluorescence detection reagent kit (comprising DNA extraction liquid 1, DNA extraction liquid 2, PCR reactant liquor, Taq enzyme, UNG).Other reagent are analytical pure.
Trial drug:
Test 1 group: 88mg Oleum Curcumae and 75mg Borneolum Syntheticum.
Test 2 groups: 5mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+40mg furanodiene+8mg curdione+10mg beta-elemene+4mg curcumenol+21mg curzerene+75mg Borneolum Syntheticum.
Test 3 groups: 8mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+32mg furanodiene+15mg curdione+8mg beta-elemene+7mg curcumenol+18mg curzerene+75mg Borneolum Syntheticum.
Test 4 groups: 13mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+36mg furanodiene+12mg curdione+7mg beta-elemene+6mg curcumenol+14mg curzerene+75mg Borneolum Syntheticum.
Test 5 groups: 14mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+28mg furanodiene+20mg curdione+8mg beta-elemene+2mg curcumenol+16mg curzerene+75mg Borneolum Syntheticum.
Test 6 groups: 10mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+20mg furanodiene+25mg curdione+11mg beta-elemene+10mg curcumenol+12mg curzerene+75mg Borneolum Syntheticum.
Test method:
HPV infected specimen preparation: make a definite diagnosis HPV from hospital outpatient and infect the specimen of taking (HPV type is 16,52 mixed types, belong to mucosa low risk), focus in 1 centrifuge tube, 30mg weighs, use glass homogenizer homogenate, add 5ml normal saline, be made into the in vitro suspension of 6mg/ml, standby.
Sample treatment: draw 25 μ l sample liquid and 25 μ l specimen suspensions to 15ml centrifuge tube with liquid-transfering gun, jolting is even, and sample and specimen can fully be acted on, and puts in 37 ℃ of water baths and cultivates, in sample treatment specimen, after 1 day, 3 days, 5 days, 7 days, respectively get 1 group and carry out following test.
The extraction of HPV-DNA: take out the specimen suspension through sample treatment from water bath, by test kit step, extract DNA: add 50 μ l normal saline, after mixing, add 100 μ l DNA extraction liquid, jolting is even, the centrifugal 10min of 1200r/min again, abandoning supernatant, add again 25 μ l DNA extraction liquid 2, fully mix 100 ℃ of boiling water boiling 10min, the centrifugal 10min of 1200r/min, supernatant is HPV-DNA template very.
DNA cloning: get the PCR reactant liquor 37.6 μ l in detection kit, Taq archaeal dna polymerase 0.4 μ l, UNG0.03 μ l, in PCR reaction tube, is adding HPV-DNA template 2 μ l, and button strict control lid, is placed in quantitative PCR instrument cocycle amplification.HPV-DNA carries out cyclic amplification through high-temperature denatured, process annealing and extension, and cyclic program is set to: 37 ℃, and 5min; 94 ℃, 1min; 95 ℃, 5sec; 60 ℃, 30sec, circulates 40 times.
Amount standard curve: detect quantitatively with fluorescent probe, according to detection kit requirement, set up the quantitative positive criteria product of HPV-DNA of 4 series concentration, be followed successively by 5 * 10
7/ ml, 5 * 10
6/ ml, 5 * 10
5/ ml, 5 * 10
4/ ml, through PCR detection, (its detection sensitivity is 1 * 10
3/ ml, result is less than this concentration person and is judged to be feminine gender).The natural logrithm of initial copy number of take is abscissa, and circulation threshold is vertical coordinate, and the regression straight line obtaining is standard curve, accordingly the amplification times of sample is carried out quantitatively.
Result of the test: in Table 1.
Table 1: the DNA exercising result that different tests medicine infects HPV
Note :-represent negative.
HPV infected specimen is changed into and from hospital outpatient, is diagnosed as HPV and infects the isolated preparation take (HPV type is the mixed type of HPV1 and HPV12, belong to skin low risk), according to above-mentioned identical condition and method, test, test 2～6 groups of results of cultivating 1 day, 3 days, 5 days, 7 days all negative.
HPV infected specimen is changed into and from hospital outpatient, is diagnosed as HPV and infects the isolated preparation take (HPV type is the mixed type of HPV8 and HPV36, belong to skin high-risk-type), according to above-mentioned identical condition and method, test, test 2～6 groups of results of cultivating 1 day, 3 days, 5 days, 7 days all negative.
HPV infected specimen is changed into and from hospital outpatient, is diagnosed as HPV and infects the isolated preparation take (HPV type is the mixed type of HPV11 and HPV53, belong to mucosa low risk), according to above-mentioned identical condition and method, test, test 2～6 groups of results of cultivating 1 day, 3 days, 5 days, 7 days all negative.
Conclusion (of pressure testing): above-mentioned result of the test shows that the DNA that pharmaceutical composition of the present invention infects different subtype HPV has good inhibitory action.
Test example 2: on the inhibiting impact of human cervical carcinoma cell
Test material
(1) oncocyte: human cervical carcinoma Hela cell system, purchased from Xiangya Medical College, Zhongnan Univ cell centre.
(2) trial drug:
Test 1 group: BAOFUKANG SHUAN, Hainan Bikai Pharmaceutical Co., Ltd produces, every heavy 1.74g, wherein containing Oleum Curcumae 88mg, Borneolum Syntheticum 75mg, all the other compositions are substrate; BAOFUKANG SHUAN is dissolved in the RPMI1640 culture fluid of 44ml serum-free, its concentration is 3.70 * 10
3mg/l (active component), after solution clarification, is the filter filtration sterilization of 0.22 μ m with aperture, and after subpackage, 4 ℃ keep in Dark Place standby.
Test 2 groups: 5mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+40mg furanodiene+8mg curdione+10mg beta-elemene+4mg curcumenol+21mg curzerene+75mg Borneolum Syntheticum, according to method described in embodiment 1, prepare suppository, every heavy 1.74g; Suppository is dissolved in the RPMI1640 culture fluid of 44ml serum-free, its concentration is 3.70 * 10
3mg/l (active component), after solution clarification, is the filter filtration sterilization of 0.22 μ m with aperture, and after subpackage, 4 ℃ keep in Dark Place standby.
Test 3 groups: 8mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+32mg furanodiene+15mg curdione+8mg beta-elemene+7mg curcumenol+18mg curzerene+75mg Borneolum Syntheticum, every heavy 1.74g; Suppository is dissolved in the RPMI1640 culture fluid of 44ml serum-free, its concentration is 3.70 * 10
3mg/l (active component), after solution clarification, is the filter filtration sterilization of 0.22 μ m with aperture, and after subpackage, 4 ℃ keep in Dark Place standby.
Test 4 groups: 13mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+36mg furanodiene+12mg curdione+7mg beta-elemene+6mg curcumenol+14mg curzerene+75mg Borneolum Syntheticum, every heavy 1.74g; Suppository is dissolved in the RPMI1640 culture fluid of 44ml serum-free, its concentration is 3.70 * 10
3mg/l (active component), after solution clarification, is the filter filtration sterilization of 0.22 μ m with aperture, and after subpackage, 4 ℃ keep in Dark Place standby.
Test 5 groups: 14mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+28mg furanodiene+20mg curdione+8mg beta-elemene+2mg curcumenol+16mg curzerene+75mg Borneolum Syntheticum, every heavy 1.74g; Suppository bolt is dissolved in the RPMI1640 culture fluid of 44ml serum-free, its concentration is 3.70 * 10
3mg/l (active component), after solution clarification, is the filter filtration sterilization of 0.22 μ m with aperture, and after subpackage, 4 ℃ keep in Dark Place standby.
Test 6 groups: 10mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+20mg furanodiene+25mg curdione+11mg beta-elemene+10mg curcumenol+12mg curzerene+75mg Borneolum Syntheticum, every heavy 1.74g; Suppository is dissolved in the RPMI1640 culture fluid of 44ml serum-free, its concentration is 3.70 * 10
3mg/l (active component), after solution clarification, is the filter filtration sterilization of 0.22 μ m with aperture, and after subpackage, 4 ℃ keep in Dark Place standby.
(3) reagent: super new-born calf serum, Hangzhou Ilex purpurea Hassk.[I.chinensis Sims company product; RPMI1640, U.S. Gibco company product, four formylmerphalan azoles blue (MTT), trypsin are U.S. Sigma company product; Dimethyl sulfoxide (DMSO) is domestic analytical pure.
(4) key instrument: ELX800 type enzyme-linked immunosorbent assay instrument, U.S. BIO-TEK company produces; Epics XL flow cytometer, U.S. Beckmen Coulter product.
Test method
(1) cell culture: human cervical carcinoma Hela cell, with the RPMI1640 culture fluid containing 10% super new-born calf serum, puts 37 ℃, 5% CO
2in incubator, cultivate.Take the logarithm trophophase cell for experiment.
(2) MTT measures: with 1 * 10
5/ ml inoculating cell is in 96 well culture plates, and every hole 100 μ l, are divided into administration group and matched group after adherent 8h, establishes 6 multiple holes for every group.In administration group, add above-mentioned each test group medicine of 20 μ l; Matched group only adds equivalent culture fluid.After continuing to cultivate 48h, with per minute 800, leave heart 10min, absorb culture fluid, add serum-free medium 90 μ l and 10 μ l MTT (5mg/ml), hatch the centrifugal supernatant that goes after 6h for 37 ℃, add DMSO 150 μ l, vibration 10min surveys A570 value by enzyme-linked immunosorbent assay instrument.Repeat 3 plates, every plate all has and only adds the zeroing hole that culture fluid does not add cell.Cell proliferation inhibition rate=(1-experimental group A
570value/control group A
570value) * 100%.
(3) flow cytometry analysis: 0.25% trypsinization collect matched group and test group effect 48h cell each 1 * 10
6individual, add cold PBS washing 2 times, 70% cold ethanol is fixed, 4 ℃ of preservations.Survey timing centrifugal and abandon ethanol, PBS washing 2 times, 37 ℃ of digestion 30min of RNA enzyme (0.1mg/ml), propidium iodide (propidium iodide, PI) examination with computer after dyeing (50mg/ml) 15min, print DNA content distribution prescription figure, automatic Fitting go out cell cycle each time phase ratio
With following formula, calculate proliferation index: PI=(S+G2/M)/(G0/G1+S+G2/M) * 100%.
Statistical disposition
Experimental results is processed through SPSS statistical package, relatively adopts t check between group.
Result of the test
1, the impact on Hela cell proliferation, in Table 2
The impact of each test group of table 2 on Hela cell proliferation
| Test group | A 570Value | Suppression ratio (%) |
| Matched group (culture fluid) | 0.384±0.0656 | - |
| Test 1 group | 0.354±0.0727 | 7.81 |
| Test 2 groups | 0.263±0.0513**# | 31.51 |
| Test 3 groups | 0.220±0.0517**# | 42.70 |
| Test 4 groups | 0.193±0.0396**# | 49.73 |
| Test 5 groups | 0.155±0.0283**# | 59.63 |
| Test 6 groups | 0.130±0.0218**# | 66.15 |
Note: with matched group comparison: with matched group * * P < 0.01 relatively, with 1 group of test #P < 0.05 relatively.
Experiment conclusion: presentation of results pharmaceutical composition of the present invention can obviously suppress the propagation of Hela cell, and be better than the combination of Oleum Curcumae and Borneolum Syntheticum.Its suppression ratio reaches and reaches as high as 66.15%.
2, on the Hela impact of cell generation cycle, in Table 3.
Table 3: on the Hela impact of cell generation cycle
Experiment conclusion: flow cytometer testing result shows, pharmaceutical composition effect 48h of the present invention can make Hela cell proliferation index (PI) decline, S phase cell proportion is reduced, and G0/G1 phase cell proportion increases, and more Hela cell is blocked in the G0/G1 phase.
Test example 3: to tumor suppression experiment in the body of mouse cervical cancer model
Test material
(1) oncocyte: human cervical carcinoma Hela cell system.
(2) experimental animal: the female healthy mice of Kunming kind, 2-3 monthly age, body weight (25 ± 3) g.
(3) trial drug:
1 group of administration: 88mg Oleum Curcumae and 75mg Borneolum Syntheticum.
2 groups of administrations: 10mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+20mg furanodiene+25mg curdione+11mg beta-elemene+10mg curcumenol+12mg curzerene+75mg Borneolum Syntheticum.
(4) reagent: super new-born calf serum, Hangzhou Ilex purpurea Hassk.[I.chinensis Sims company product; Trypsin, Beijing Geyuantianrun Biotechnology Co., Ltd..
The foundation of mouse cervical cancer model:
Hela cell is at 37 ℃, 5%CO
2, containing in the DMEM culture fluid of 10% hyclone, cultivate, by the cell of exponential phase 0.25% trypsinization, with aseptic PBS liquid is resuspended, make 5 * 10
7the single cell suspension of/ml, every mice, in the subcutaneous vaccination 0.2ml of right side nape portion cell suspension, inoculates 30 altogether, observes each injection point every day and has or not red and swollen ulceration.
Tumor suppression experiment
30 mices are divided into 3 groups at random, and 10 every group, administration group and blank group are established in experiment.Administration group is respectively at the medicine (preparing with normal saline) of 2 weeks pneumoretroperitoneum injection 100mg/kg dosage of inoculating cell suspension, every day 1 time, continuous 4 weeks; And the isopyknic normal saline of matched group lumbar injection.In experiment, observe gross tumor volume and the speed of growth, experiment is carried out cervical vertebra dislocation after 4 weeks and is put to death mice, weighs in and tumor weight.
Result
1, whole 30 mices all survive in experimentation, and each cell suspension inoculation point has no red and swollen ulceration and bleeding.
2, at the 14th day of cell suspension inoculation, each vaccination was all shown in tumor nodule, diameter > 0.5cm.
3, mice is after giving medicine, tumor tissue under peeling off in Mice Body is carried out to perusal, be nodositas, canescence, quality is partially hard, and there is pseudocapsule on surface, and easy and surrounding tissue is peeled off, there is obvious necrosis region in two most of visible central authorities in medication group tumor transverse section, matched group is rare necrosis region.
4, weigh administration group and control group mice body weight, and tumor weight, the results are shown in Table 4.
Table 4: mice average weight and tumor weight
Note: compare * * P < 0.01, * P < 0.05 with matched group; With 1 group of comparison of administration, #P < 0.05.
Result: the body weight of administration group mice is apparently higher than the mice of matched group, and P < 0.05; Relatively the Mouse Weight of two administration groups is without obvious difference, but tumor heavy be that 2 groups of administrations (giving pharmaceutical composition of the present invention) are significantly lower than 1 group of administration, P < 0.05.Illustrate that Oleum Curcumae+Borneolum Syntheticum and pharmaceutical composition of the present invention are all inhibited to cervical cancer.
Test example 4: In vitro Bactericidal Experiments
Trial drug: 10mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+20mg furanodiene+25mg curdione+11mg beta-elemene+10mg curcumenol+12mg curzerene+75mg Borneolum Syntheticum.
Method: adopt trace continuously doubling dilution measure the minimal inhibitory concentration (MIC) of pharmaceutical composition of the present invention to the mark bacterial strain of colon bacillus, staphylococcus aureus, Pseudomonas aeruginosa, staphylococcus epidermidis, proteus vulgaris, streptococcus faecalis, gonococcus and colon bacillus, staphylococcus aureus, Pseudomonas aeruginosa, staphylococcus epidermidis, proteus vulgaris, streptococcus faecalis, gonococcus clinical separation strain, adopt dull and stereotyped infection protocol to measure pharmaceutical composition of the present invention to the minimal bactericidal concentration of above-mentioned antibacterial (MBC).Colon bacillus, staphylococcus aureus, Pseudomonas aeruginosa, staphylococcus epidermidis, proteus vulgaris, vagina Gartner bacterium are inoculated in to MH meat soup, put 37 ℃ of common incubators and cultivate 24h; Streptococcus faecalis is inoculated in TPY meat soup (anaerobe nutrient broth), puts 37 ℃ of anaerobic culture boxes and cultivates 48h; Gonococcus is inoculated in the MH meat soup of antiperspirant 5% calf serum, puts 5%CO
237 ℃ of cultivation 48h of cultivation property.
Table 5 pharmaceutical composition of the present invention is to the MIC of reference culture and MBC
As seen from the above table, pharmaceutical composition of the present invention all has obvious inhibition and deactivation to testing the mark bacterial strain of selected colon bacillus, staphylococcus aureus, Pseudomonas aeruginosa, staphylococcus epidermidis, proteus vulgaris, streptococcus faecalis, gonococcus with the clinical separated Reference Strains of vagina Gartner bacterium, its MBC is MIC 2～4 times.
MIC, the MIC of table 6 pharmaceutical composition of the present invention to 405 strain clinical isolates strains
50, MIC
99and MBC (mg compositions/ml)
| Bacterial strain | Strain number | MIC | MIC 50 | MIC 99 | MBC |
| Staphylococcus aureus | 100 | 0.39～1.56 | 1.0526 | 1.3084 | 0.78～6.25 |
| Escherichia coli | 120 | 6.25～50 | 18.9163 | 23.1657 | 25～100 |
| Staphylococcus epidermidis | 50 | 0.39～3.13 | 0.7232 | 1.5016 | 1.56～6.25 |
| Bacillus proteus | 100 | 12.5～50 | 26.125 | 34.0726 | 25～100 |
| Gonococcus | 24 | 0.195～0.39 | 0.2438 | 0.2751 | 0.195～0.78 |
| Vagina Gartner bacterium | 11 | 0.39～1.56 | 0.7800 | 0.9146 | 0.85～1.65 |
As seen from the above table, pharmaceutical composition of the present invention all has certain inhibition and deactivation to testing 405 selected strain clinical isolates strains, its MBC is MIC 2～4 times.
Test example 5: In Vitro Anti mycologic test
Trial drug: 10mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+20mg furanodiene+25mg curdione+11mg beta-elemene+10mg curcumenol+12mg curzerene+75mg Borneolum Syntheticum.
Method: adopt the trace MIC of doubling dilution mensuration pharmaceutical composition of the present invention to candidiasis continuously, adopt dull and stereotyped infection protocol to measure the MBC of pharmaceutical composition of the present invention to candidiasis.Adopt the trace MIC of doubling dilution mensuration pharmaceutical composition of the present invention to mycete continuously, adopt dull and stereotyped infection protocol to measure the MBC of pharmaceutical composition of the present invention to mycete.
Table 7 pharmaceutical composition of the present invention is to the MIC of test strain and MBC
As seen from the above table, pharmaceutical composition of the present invention all has obvious inhibition and deactivation to testing mark bacterial strain or the clinical separated Reference Strains of selected Candida albicans, Candida parapsilosis, monilia guilliermondii, candida parakrusei, Oidium tropicale, penicillium, Aspergillus flavus, its MBC is MIC 2～4 times.
Table 8 pharmaceutical composition of the present invention is to the MIC of Candida albicans clinical isolates strain, MIC
50, MIC
99and MBC (mg compositions/ml)
| Strain number | MIC | MIC 50 | MIC 99 | MBC |
| 50 | 1.56～6.25 | 0.0991 | 0.1243 | 6.25～12.5 |
As seen from the above table, to discoloration test, 50 selected strain Candida albicans clinical isolates strains have certain inhibition and deactivation to pharmaceutical composition of the present invention.
Test example 6: anti-trichomonal vaginitis test
Trial drug: 10mg 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-+20mg furanodiene+25mg curdione+11mg beta-elemene+10mg curcumenol+12mg curzerene+75mg Borneolum Syntheticum.
Method: adopt extracorporeal culture-ing to measure the minimum parasite killing concentration of pharmaceutical composition of the present invention to trichomonal vaginitis.Trichomonal vaginitis is at 37 ℃ of cultivation 48h of CPLM culture medium of improvement, and infusorian motion is active, well-grown, and natural mortality rate < 2%, and test is 1.8～2.5 * 10 with trichomonal vaginitis liquid containing worm concentration
3/ ml.
Result shows, pharmaceutical composition of the present invention is external effective to killing trichomonal vaginitis.
Specific embodiment
Embodiment 1
Prescription: 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-15g, furanodiene 39g, curdione 24g, beta-elemene 9g, curcumenol 8g, Borneolum Syntheticum 75g.
Dosage form: suppository.
Preparation method: the 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-of recipe quantity, furanodiene, curdione, beta-elemene, curcumenol and 75g polyoxyethylene sorbitan monoleate (Tween-80) are mixed, and appropriate dissolve with ethanol for Borneolum Syntheticum 75g, mixes with above-mentioned solution.
The Myrj 45 1491g of recipe quantity, puts heating in water-bath and makes fusing, adds above-mentioned medicinal liquid, stirs evenly, and fill, makes 1000 of suppositorys.
Embodiment 2-8
By following formula preparation suppository, preparation method is with embodiment 1.
| Embodiment | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)- | 5.5 | 6 | 9 | 10 | 11 | 13 | 14 |
| Furanodiene | 37 | 33 | 30 | 28 | 25 | 15 | 11 |
| Curdione | 6 | 8 | 9 | 15 | 18 | 20 | 23 |
| Beta-elemene | 10.5 | 10 | 9.5 | 8 | 6.5 | 5 | 4.5 |
| Curcumenol | 1.5 | 2 | 4 | 6 | 8 | 9 | 9.5 |
| Curzerene | 8.5 | 9 | 10 | 15 | 14 | 16 | 21 |
| Borneolum Syntheticum | 30 | 40 | 50 | 60 | 70 | 80 | 90 |
| Polyoxyethylene sorbitan monoleate | 55 | 60 | 65 | 70 | 75 | 80 | 85 |
| Myrj 45 | 1563 | 1574 | 1467 | 1572 | 1583 | 1543 | 1548 |
Embodiment 9
Prescription: 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-6.5g, furanodiene 32g, curdione 21g, beta-elemene 9.5g, curcumenol 2.5g, Borneolum Syntheticum 35g.
Dosage form: effervescent tablet.
Preparation method:
1) get beta-schardinger dextrin-, add water appropriate by 25ml/g, stir and make it entirely molten, heating, obtains beta-schardinger dextrin-solution if desired; The 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-of recipe quantity, furanodiene, curdione, beta-elemene, curcumenol, Borneolum Syntheticum, with slowly adding in beta-schardinger dextrin-solution after dehydrated alcohol dilution, continue to be stirred to into homogeneous phase, in refrigerator, place and spend the night, sucking filtration, by a small amount of petroleum ether 3 times, freezing, white powder.
2) above-mentioned white powder and 35g Borneolum Syntheticum, citric acid 150g and lactose 160g are mixed; Separately get sodium bicarbonate 120g and lactose 160g mixes, granulate respectively with 5%PVP ethanol solution as binding agent, 50 ℃ dry, and granulate, adds PEG6000 appropriate, mixes tabletting.
Embodiment 10
Prescription: 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-7.5g, furanodiene 31g, curdione 19g, beta-elemene 5.5g, curcumenol 3.5g, curzerene 11g, Borneolum Syntheticum 45g.
Dosage form: capsule
Preparation method:
1) capsule leather is standby: press gelatin: glycerol: water=10: the weight portion mixture of 3.7: 9.5, get gelatin, and add suitable quantity of water to make gelatin imbibition.Separately get glycerol and remaining water and put in glue pot and be heated to 70 ℃ 2, mix homogeneously, adds the gelatin of expansion, stir, and melting, insulation, vacuum suction is removed bubble, considers, and adds the PEG400 of approximately 3% weight portion, mixes, and is then incubated 50 ℃, standby.
2) content preparation: get 300g PEG400 and be heated to 80 ℃, add Borneolum Syntheticum 45g, add until completely dissolved 150g Macrogol 4000 to be stirred to dissolving, when temperature is down to 40～50 ℃, the 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-, furanodiene, curdione, beta-elemene, curcumenol, the curzerene that add recipe quantity, stir, be cooled to 25～30 ℃, cross colloid mill mill 2 times, each 5min, make content, under heat-retaining condition, make 1000 soft capsules.
Embodiment 11
Prescription: 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-8.5g, furanodiene 29g, curdione 21g, beta-elemene 4.5g, curcumenol 4.5g, curzerene 22g, Borneolum Syntheticum 55g.
Dosage form: membrane.
Preparation method: get polyvinyl alcohol 800g and soak in 800ml water 24 hours, dissolve in water-bath at 80 ℃ of temperature; By the 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-of recipe quantity, furanodiene, curdione, beta-elemene, curcumenol, curzerene, Borneolum Syntheticum, be dissolved in 1000ml ethanol (W/V=75%), and above-mentioned poly-vinyl alcohol solution is mixed, and stirs; In above-mentioned mixed liquor, add antioxidant sodium pyrosulfite 10g, wetting agent glycerol 80g, plasticizer three triacetin 80g, stir; Deviate from according to a conventional method bubble, apply after film forming, dry, cut, obtain 1000 membrane products.
Embodiment 12
Prescription: 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-9.5g, furanodiene 26g, curdione 23g, beta-elemene 4.2g, curcumenol 5.5g, curzerene 19g, Borneolum Syntheticum 65g.
Dosage form: gel
Preparation method: get carbomer 20g and swell in propylene glycol 760g, standing to the completely rear adjusting of swelling pH value to 4～7, and it is standby to add propylene glycol 600g to make substrate; By the 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-of recipe quantity, furanodiene, curdione, beta-elemene, curcumenol, curzerene, Borneolum Syntheticum is dissolved in 120g PEG400 and obtains mixed solution, stirs evenly, and mixed liquor is mixed homogeneously with above-mentioned substrate, and subpackage, obtains.
Embodiment 13
Prescription: 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-10.5g, furanodiene 23g, curdione 24.5g, beta-elemene 4.6g, curcumenol 6.5g, curzerene 14g, Borneolum Syntheticum 70g.
Dosage form: lotion
Preparation method: get Borneolum Syntheticum 70g, add appropriate ethanol and make its dissolving; By the 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-of recipe quantity, furanodiene, curdione, beta-elemene, curcumenol, curzerene and azone 20g, 2500g Tween 80 mix homogeneously, add ethanol to 2800g, after stirring, add water for injection to 10kg, stir evenly subpackage, obtain concentrated solution, 10 times of uses of thin up during use.
Embodiment 14
Prescription: 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-12.5g, furanodiene 17g, curdione 18.5g, beta-elemene 6.3g, curcumenol 7.5g, curzerene 16g, Borneolum Syntheticum 80g.
Dosage form: ointment
Preparation method: at 80～100 ℃ of temperature controls after fusing, 115～120 ℃ of sterilizings of temperature control, are cooled to 70～80 ℃ by sterilized vaseline by vaseline 800g; By lanoline 40g in 115～120 ℃ of sterilizings of temperature control; By 55～65 ℃ of extremely fusings of dimethyl sulfoxine 25g temperature control; Vaseline is put into material-compound tank, put into a half and stir; By Borneolum Syntheticum, dimethyl sulfoxine and lanoline congruent melting, filter and drop in material-compound tank and stir again; Then by the 3,7-Cyclodecadien-1-one, 3,7-dimethyl-10-(1-methylethylidene)-, (E,E)-of recipe quantity, furanodiene, curdione, beta-elemene, curcumenol, curzerene add in material-compound tank, stir.
Claims (7)
1. a pharmaceutical composition, is characterized in that the effective ingredient of this pharmaceutical composition is:
2. pharmaceutical composition according to claim 1, wherein said Borneolum Syntheticum is selected from natural Broneolum Syntheticum and/or synthetic borneol.
3. pharmaceutical composition according to claim 1, is characterized in that, this pharmaceutical composition is vagina administration preparation or rectally preparation.
4. pharmaceutical composition according to claim 3, wherein preparation comprises suppository, ointment, capsule, effervescent tablet, gel, lotion, membrane or foam.
5. pharmaceutical composition according to claim 1 prevents and/or treats the application in the medicine of human papilloma virus infection in preparation.
6. application according to claim 5, wherein human papilloma virus infection comprises high-risk human mammilla papillomavirus infection and/or low risk human papilloma virus infection.
7. pharmaceutical composition according to claim 1 prevents and/or treats the application in the medicine of vaginitis, cervical erosion, cervical cancer in preparation.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101244237A (en) * | 2007-05-24 | 2008-08-20 | 海南碧凯药业有限公司 | Suppository and preparation thereof |
| CN101422585A (en) * | 2008-07-28 | 2009-05-06 | 海南碧凯药业有限公司 | Pharmaceutical use of medicine composition containing zedoary turmeric oil |
| CN101791392A (en) * | 2010-04-22 | 2010-08-04 | 海南碧凯药业有限公司 | Application of medicinal composition |
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| CN101244237A (en) * | 2007-05-24 | 2008-08-20 | 海南碧凯药业有限公司 | Suppository and preparation thereof |
| CN101422585A (en) * | 2008-07-28 | 2009-05-06 | 海南碧凯药业有限公司 | Pharmaceutical use of medicine composition containing zedoary turmeric oil |
| CN101791392A (en) * | 2010-04-22 | 2010-08-04 | 海南碧凯药业有限公司 | Application of medicinal composition |
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