CN100434089C - Use of centipeda volatile oil in preparation of tumor growth and proliferation inhibitor - Google Patents
Use of centipeda volatile oil in preparation of tumor growth and proliferation inhibitor Download PDFInfo
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- CN100434089C CN100434089C CNB2006100200268A CN200610020026A CN100434089C CN 100434089 C CN100434089 C CN 100434089C CN B2006100200268 A CNB2006100200268 A CN B2006100200268A CN 200610020026 A CN200610020026 A CN 200610020026A CN 100434089 C CN100434089 C CN 100434089C
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Abstract
The invention discloses an application of centipede benzine in the inhibiting agent of tumour growth and propagation, which is characterized by the following: placing centipede in the distilling container; adding 6-8 times water; heating to distill for 4-6 h; grinding centipede roughly in the ultra-temporary carbon dioxide extractor at 15-25Mpa and 35-45 deg.c; setting the separating pressure at 5.2-7.2 Mpa and 30-35 deg.c for 1-3 h.
Description
Technical field
The present invention relates to centipeda volatile oil preparation method and the application in preparation tumor growth and inhibition of proliferation agent thereof, the particularly application of the prepared volatile oil of Herba Centipedae steam distillation and supercritical carbon dioxide extraction in preparation tumor growth and inhibition of proliferation agent.
Background technology
Herba Centipedae (Centipeda minima (L.) A.Br.etAschers.) is a Compositae Myriogyne minuta platymiscium, has another name called Myriogyne minuta, eritrichium pedumculare.Herba Centipedae beginning is stated from Southern Tang Dynasty's " feeding habits book on Chinese herbal medicine ", and is also on the books in the famous pharmacopeia Compendium of Material Medica of China, " Herba Centipedae can stuffy nose relieving gas, and the sharp seven apertures are told wind-phlegm, and plug breath is from falling ".Herba Centipedae grows in the dark and damp place of 300～1900 meters of height above sea level, and main product is in China Zhejiang, Hubei, Jiangsu, Guangdong and other places.Mainly be distributed in India Plain and Xi Landao abroad.
The Herba Centipedae main component includes volatility oils, steroidal class, main effective ingredient such as terpenoid and flavonoid.Pharmacological research proves that it has pharmacological action widely, and as antiinflammatory, antigen is given birth to biological, effects such as antibiotic antiallergic.
Terpenoid is one of its main chemical compositions, and a lot of terpenoids have the important physical activity, such as, arnicolide C has similar anti-bacillus subtilis effect with D, and the MIC value is 150 mcg/ml.Centipeda volatile oil 0.05 ml/kg and 0.01 ml/kg dosage group all have obvious inhibitory action to the chmice acute inflammation.
About the disclosed patent of Herba Centipedae and the document of delivering are broadly divided into two classes:
The first kind is the research about Herba Centipedae chemical constituent and quality analysis, Bohlmann F, Chen ZL.Newguaianolides from Centipeda minima.KeXue TongBao 1984; 29 (7): 900-3.Liu Yu Yang Yan virtue Wu and Zhen Yan Liu literary composition .RP-HPLC method are measured the content of geraniin in the Herba Centipedae, and the content of geraniin is 3 milligrams/gram in Chinese crude drug 2005,6 (28): the 473-474 report Herba Centipedae.
Second class is about the bioactive research of Herba Centipedae, Herba Centipedae has antiallergic in these reports, the effect of protozoa resisting (as plasmodium and ameba worm etc.) and treatment rhinitis, Sharma P, Sharma J D.A Review of PlantSpecies Assessed in vitro for Antiamoebic Activity or both Antiamoebic andAntiplasmodial Properties.Phytother.Res.200115,1-17. Qin Ren peace, Chen Min. the preliminary experiment report of centipeda volatile oil antiinflammatory action. Guizhou medicine, 2001,25 (10): 909-910.Yu Hongmeng, Wen Sanli. the experimentation of Herba Centipedae treatment of allergic rhinitis. Chinese combination of Chinese and Western medicine hals,Nasen und Ohrenheilkunde magazine, 2001,9 (5): 220-224
In State Intellectual Property Office's coordinate indexing, do not find preparation method, do not find the patent of the application of centipeda volatile oil in preparation tumor growth and inhibition of proliferation agent simultaneously about centipeda volatile oil steam distillation and supercritical carbon dioxide extraction.
Current, chemotherapy of tumors treatment field urgent problem is to seek the low toxicity that makes new advances, efficient cancer therapy drug resource, and this invention just is consistent with it, therefore, has important economic benefit and social benefit.
Summary of the invention
The objective of the invention is at oncotherapy field urgent problem, seek the low toxicity that makes new advances, efficient cancer therapy drug resource.Provide and use combined tumor growth that forms of adding ingredient and the antiblastic dosage form that centipeda volatile oil or itself and pharmacology upward allow; And provide the preparation method of centipeda volatile oil steam distillation and supercritical carbon dioxide extraction.
Tumor growth provided by the invention and inhibition of proliferation agent are the volatile oil of Chinese medicine Herba Centipedae, as the active substance in preparation tumor growth and the antiblastic.
Centipeda volatile oil extracting method provided by the invention is steam distillation or supercritical carbon dioxide extraction method.
Wherein said steam distillation is meant Herba Centipedae is placed distil container, adds 6～8 times of water gagings, adds 4～6 hours extracting method of thermal distillation.
Said supercritical carbon dioxide extraction method is meant Herba Centipedae is ground into coarse powder, put in the supercritical carbon dioxide extraction instrument, be respectively 15～25 MPas and 35～45 ℃, separating pressure and separation temperature with carbon dioxide in extracting pressure and extraction temperature and be respectively under the condition of 5.2～7.2 MPas and 30～35 ℃, extract 1～3 hour extracting method.
The present invention's said vapor extraction gained centipeda volatile oil and supercritical carbon dioxide extraction gained centipeda volatile oil have certain difference in constituent, antitumous effect is different, but are the antineoplastic active substance.
Tumor growth provided by the invention and inhibition of proliferation agent are centipeda volatile oil and the last combined dosage form that forms of adding ingredient that allows of pharmacology.Described inhibitor dosage form is peroral administration solid type preparation: powder, and pill, tablet or capsule, or be para-oral external agent, ointment sticks agent, injection.
Wherein said centipeda volatile oil extracts gained for adopting steam distillation or supercritical carbon dioxide extraction method.Said tumor is medically affiliated all kinds of tumors, comprises benign tumor and malignant tumor.And said malignant tumor comprises: malignant melanoma, malignant lymphoma, Alimentary tumor (gastric cancer, intestinal cancer, hepatocarcinoma, carcinoma of gallbladder, cancer of bile ducts, cancer of pancreas), pulmonary carcinoma, breast carcinoma, carcinoma of testis, ovarian cancer, uterus carcinoma, carcinoma of prostate, maxillary cancer, carcinoma of tongue, oral cancer, laryngocarcinoma, thyroid carcinoma, brain tumor, each sarcoid, osteosarcoma, leukemia, nervous system neoplasms, tumor of bladder, skin carcinoma, skin accessory organ's cancer and metastatic carcinoma of skin.
Tumor growth provided by the invention and inhibition of proliferation agent, low dose of i.e. generation suppresses the effect of kinds of tumor cells growth and has dose dependent, they medicines as the treatment tumor can be applied in the chemotherapy of tumor.
Tumor growth provided by the invention and inhibition of proliferation agent, under the situation of low concentration, has the effect that suppresses tumor cell proliferation, illustrate not only have the effect of killing tumor cell but also the unlimited multiplication capacity that can contain tumor cell, thereby can bring into play the effect of further treating tumor more.
Tumor growth provided by the invention and inhibition of proliferation agent have different cytotoxicities to different human cancer cells with human normal cell, especially to the IC of cancerous cell
50Than Normocellular IC
50Much lower, and both significant differences (p＜0.05).
Tumor growth of the present invention and inhibition of proliferation agent, low toxicity is suitable for people and mammal per os or non-oral administration.
Tumor growth provided by the invention and inhibition of proliferation agent do not have specific restriction aspect dosage form, can be used as peroral administration powder, pill, and tablet, solid type preparation such as capsule also can be used as para-oral external agent, and ointment sticks agent, injection.
Tumor growth provided by the invention and inhibition of proliferation agent can cooperate with the auxiliary material that does not influence pharmacotoxicological effect usually and make per os or para-oral preparation.
Tumor growth provided by the invention and inhibition of proliferation agent can be through aqueous solvent (as distilled water), water soluble preparation (as normal saline), and fatsolvent (as siritch) equal solvent is modulated with conventional method.
Tumor growth provided by the invention and inhibition of proliferation agent can be gone up the diluent that allows with the pharmacology usually, coloring agent, and tranquilizer, interfacial agent, cosolvent, buffer agent, correctives, spice, preservative agent or sugar-coat agent are combined, are used for per os or non-per os dosage form.
Tumor growth provided by the invention and inhibition of proliferation agent usually can with the diluted composition that allows on the pharmacology and to be molded into branch combined on the dosage form, be used for per os or non-per os dosage form.
Tumor growth provided by the invention and inhibition of proliferation agent per os dosage form are meant tablet, pill, granule, powder or capsule.Non-per os dosage form is meant intravenous injection, intramuscular dose, and agent, rectum, anus or vagina administration are bathed in the subcutaneous injection agent.
Significant advantage of the present invention and technical progress:
Volatile oil with the multiple human growth of tumour cell of inhibition and proliferation function provided by the invention, having overcome traditionally, Chinese medicine all herbal medicine taking dose reaches the unsettled defective of curative effect greatly, with the means of modern biotechnology as invention, using modern science and technology extracts, dosage is little, have significant inhibition human tumor cell growth and proliferation function (the interior and experiment in vitro of body), less to the Normocellular cytotoxicity of the mankind simultaneously, have a good application prospect.
Description of drawings
Table 1 extracts the centipeda volatile oil result of the test for steam distillation.
Table 2 extracts the centipeda volatile oil result of the test for supercritical carbon dioxide extraction method.
Table 3 is that steam distillation and supercritical carbon dioxide extraction method extract centipeda volatile oil quality analysis result.
Table 4 and table 5 be Different Extraction Method gained centipeda volatile oil under variable concentrations to A375 (human melanoma cell), A431 (people's epidermis squamous cell carcinoma), 95D (the high strain cell that shifts of human lung adenocarcinoma), MKN45 (gastric carcinoma cells), BEL-7402 (human liver cancer cell), the effect of LoVo (human colon cancer cell) cell inhibiting.
Table 6 be Different Extraction Method gained centipeda volatile oil under variable concentrations to 293 (HEKC), the cytotoxic effect of LO2 (normal liver cell) cell.
Fig. 1 is vapor distillation extraction method gained centipeda volatile oil suppresses the growth of C57 mouse interior tumor under variable concentrations comparison.
Fig. 2 extracts the gained centipeda volatile oil suppresses the growth of C57 mouse interior tumor under variable concentrations comparison for supercritical carbon dioxide extraction method.
The specific embodiment
Below by embodiment content of the present invention is described further.The cited case does not limit protection scope of the present invention:
Embodiment 1
Get Herba Centipedae and place distil container in right amount, add 6～8 times of water gagings, adopt steam distillation, added thermal distillation 4～6 hours, collect volatile oil.Experimental result is as shown in table 1:
Table 1 steam distillation extracts the centipeda volatile oil result of the test
Above embodiment explanation, steam distillation can extract centipeda volatile oil preferably, and is 6～8 times of amounts in addition, and the heating distillation time is 4～6 hours, and the volatile oil yield is more or less the same.
Embodiment 2
Herba Centipedae was pulverized 20 mesh sieves, placed the supercritical carbon dioxide extraction instrument, with carbon dioxide abstraction, extracting pressure is 15～25 MPas, extraction temperature is that 35～45 ℃, separating pressure are 5.2～7.2 MPas, and separation temperature is 30～40 ℃, and the extraction time is 1～3 hour.Result of the test is as shown in table 2:
Table 2 supercritical carbon dioxide extraction method extracts the centipeda volatile oil result of the test
Above embodiment explanation, supercritical carbon dioxide extraction method can extract centipeda volatile oil preferably, be respectively 15～25 MPas and 35～45 ℃, separating pressure and separation temperature in extracting pressure and extraction temperature and be respectively 5.2～7.2 MPas and 30～40 ℃, the extraction time is 1～3 hour, the volatile oil yield is more or less the same, but yield is apparently higher than steam distillation.
Embodiment 3
Steam distillation and supercritical carbon dioxide extraction method gained centipeda volatile oil quality analysis
Respectively vapor distillation and supercritical carbon dioxide extraction gained centipeda volatile oil being made into every milliliter respectively with ether, to contain the crude drug amount be 14.The need testing solution of 6 grams, respectively getting 1 microlitre and inject Thermo Finnigan gas chromatograph-mass spectrometer, is helium in carrier gas, split ratio 55: 1, press 46 kPas before the post, flow rate of carrier gas is 1 milliliter of a per minute, 300 ℃ of injector temperatures, 100 ℃ of column temperatures, 310 ℃ of detector temperatures, ionizing energy is 70 electron-volts, measures under the condition that multiplier electrode is 200 volts, and measurement result is as shown in table 3:
The different preparation method gained of table 3 centipeda volatile oil quality analysis result
Embodiment 3
Centipeda volatile oil is to the toxic action of tumor cell:
Centipeda volatile oil is respectively and adopts steam distillation, supercritical carbon dioxide extraction method, the extract that extraction separation obtains from Herba Centipedae, (Du Shi improves according to the lattice culture medium with DMEM, Dulbecco ' s modified Eagle medium, DMEM) culture fluid is diluted to desired concn.Adopt the quick colorimetric method for determining centipeda volatile oil of tetrazolium bromide (MTT) to A375, A431,95D, MKN45, BEL-7402, the toxic action of LoVo cell.With exponential phase cell (10
6Individual cells/ml is inoculated in 96 well culture plates, and 0.2 milliliter in every hole adds the centipeda volatile oil of setting concentration respectively and handles, parallel 4 holes of every concentration, and matched group adds the culture fluid of equivalent volumes, puts 37 ℃, 5%CO
2And the incubator of saturated humidity was cultivated 1-4 days, experiment stops preceding 4 hours every holes and adds 5 mg/ml MTT, 10 microlitres, cultivate every hole, end back and add 0.04 equivalent dimethyl sulfoxide (DMSO), every hole 150 microlitres, vibrated 10 minutes, treat that the MTT reduzate dissolves fully, with BioRad 550 type microplate reader, with 550 nanometers is the experiment wavelength, 655 nanometers are for to measure its trap with reference to wavelength, calculate the suppression ratio of tumor cell and the suppression ratio of medicine pair cell, and map, determine the half-inhibition concentration (IC of cell with the variable concentrations and the cell inhibiting rate of medicine
50).Experimental result is shown in following table 4 and table 5.
The different centipeda volatile oil of table 4 is to A375, A431, the effect of 95D cell inhibiting
The different centipeda volatile oil of table 5 is to MKN45, BEL-7402, the effect of LoVo cell inhibiting
Above embodiment explanation, centipeda volatile oil all has good killing action to different types of tumor cell, and this inhibition effect also has dose dependent.
Embodiment 4
Centipeda volatile oil is to the toxic action of normal LO2 cell:
The extract of centipeda volatile oil for adopting vapor distillation and supercritical carbon dioxide extraction to obtain from Herba Centipedae is diluted to desired concn with the DMEM culture fluid.Adopt the toxic action of the quick colorimetric method for determining geraniin of tetrazolium bromide (MTT) to the LO2 cell.With exponential phase cell (10
6Cells/ml) be inoculated in 96 well culture plates, 0.2 milliliter in every hole adds the centipeda volatile oil of setting concentration respectively and handles, parallel 4 holes of every concentration, and matched group adds the culture fluid of equivalent volumes, puts 37 ℃, 5%CO
2And the incubator of saturated humidity was cultivated 4 days, experiment stops preceding 4 hours every holes and adds 5 mg/ml MTT, 10 microlitres, cultivate every hole, end back and add 0.04 equivalent dimethyl sulfoxide (DMSO), every hole 150 microlitres, vibrate and treated that the MTT reduzate dissolved fully in 10 minutes, with BioRad 550 type microplate reader, with 550 nanometers is the experiment wavelength, 655 nanometers are for to measure its trap with reference to wavelength, calculate the suppression ratio of tumor cell and the suppression ratio of medicine pair cell, and, determine the half-inhibition concentration (IC of cell with the variable concentrations of medicine and the mapping of cell inhibiting rate
50).Experimental result is as shown in table 6 below.
The different centipeda volatile oil of table 6 is to 293, the cytotoxicity of LO2 cell
It is bigger to human tumor cell's cytotoxicity that the gained centipeda volatile oils are extracted in above embodiment explanation, two kinds of methods, to Normocellular cytotoxicity a little less than, some human tumor cell is had certain targeting inhibitory action.
Embodiment 5
Centipeda volatile oil suppresses the comparison of C57 mouse interior tumor growth activity:
The extract of centipeda volatile oil for adopting vapor distillation and supercritical carbon dioxide extraction to obtain from Herba Centipedae, two of male lotus tumor C57 mices peel off the solid tumor of mice, shred and grind with sour phosphorus buffer (pH 7.4), the filtration of 80 eye mesh screens.It is standby to be made into cell suspending liquid.Male C57 is by the body weight random packet, and tumor cell suspension (1 * 10 is injected in the forelimb oxter
6Individual/milliliter), in simultaneously different medicine abdominal cavity injection every day of inoculation, negative control is a normal saline.With the positive contrast of cancer therapy drug, administration 8 days, put to death the back, peels off the solid tumor of mice and weigh.With compare the calculating suppression ratio.The variable concentrations of medicine to the experimental result of the influence of the suppression ratio of tumor as shown in Figure 1 and Figure 2.
Above embodiment explanation, centipeda volatile oil also can suppress growth of tumor in vivo.
Claims (6)
1, the application of centipeda volatile oil in preparation tumor growth and inhibition of proliferation agent, it is characterized in that, adopt the centipeda volatile oil of steam distillation or supercritical carbon dioxide extraction method preparation, as the active substance in preparation tumor growth and the antiblastic;
Said steam distillation is meant Herba Centipedae is placed distil container, adds 6～8 times of water gagings, adds 4～6 hours extracting method of thermal distillation;
Said supercritical carbon dioxide extraction method is meant Herba Centipedae is ground into coarse powder, put in the supercritical carbon dioxide extraction instrument, is that 35～45 ℃, separating pressure are that 5.2～7.2 MPas and separation temperature are under 30～35 ℃ the condition with carbon dioxide in extracting pressure 15～25 MPas and extraction temperature, extracts 1～3 hour extracting method.
2, as the application of the said centipeda volatile oil of claim 1 in preparation tumor growth and inhibition of proliferation agent, it is characterized in that, centipeda volatile oil or itself and pharmacology go up combined tumor growth and the antiblastic of being prepared into of adding ingredient that allows, and described inhibitor dosage form is peroral administration solid type preparation.
3, as the application of the said centipeda volatile oil of claim 2 in preparation tumor growth and inhibition of proliferation agent, it is characterized in that, centipeda volatile oil or itself and pharmacology go up combined tumor growth and the antiblastic of being prepared into of adding ingredient that allows, described peroral administration solid type preparation is a powder, pill, tablet or capsule.
4, as the application of the said centipeda volatile oil of claim 1 in preparation tumor growth and inhibition of proliferation agent, it is characterized in that, centipeda volatile oil or itself and pharmacology go up combined tumor growth and the antiblastic of being prepared into of adding ingredient that allows, and described inhibitor dosage form is para-oral external agent.
5, as the application of the said centipeda volatile oil of claim 4 in preparation tumor growth and inhibition of proliferation agent, it is characterized in that, centipeda volatile oil or itself and pharmacology go up combined tumor growth and the antiblastic of being prepared into of adding ingredient that allows, and described para-oral external agent is ointment, stick agent.
6, as the application of the said centipeda volatile oil of claim 1 in preparation tumor growth and inhibition of proliferation agent, it is characterized in that, centipeda volatile oil or itself and pharmacology go up combined tumor growth and the antiblastic of being prepared into of adding ingredient that allows, and described inhibitor dosage form is an injection.
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| CN101732383B (en) * | 2009-12-11 | 2012-01-11 | 暨南大学 | Total sesquiterpene lactone extract of centipeda minima, preparation method and application thereof |
| CN103858764B (en) * | 2014-03-19 | 2015-07-08 | 钦州市林业科学研究所 | Centipeda minima tissue culture rapid propagation medium and centipeda minima one-time seedling formation tissue culture rapid propagation method |
| KR102298839B1 (en) * | 2019-01-04 | 2021-09-07 | 주식회사 디네이쳐 | Method of selective extracting natural material |
| CN110194808B (en) * | 2019-06-12 | 2021-08-31 | 北京果素堂生物科技有限公司 | Plant refined polysaccharide and application thereof in preparation of whitening cosmetics |
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| 鹅不食草挥发油对急性肺损伤大鼠支气管上皮细胞CD54表达的影响. 覃仁安.中华中医药杂志，第8期. 2005 |
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