CN101190258A - Total sesquiterpene lactone extract containing rich parthenolide and preparation method and application thereof - Google Patents
Total sesquiterpene lactone extract containing rich parthenolide and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a total sesquiterpene lactone extract containing rich parthenolide and the preparation method and application thereof. Particularly, the invention relates to a method of extracting sesquiterpene lactone containing rich parithenolide from yulan magnolia leaves and/or flower; yulan magnolia leaves and/or flower are taken as raw materials, crushed, extracted by polarity organic solvent and non-polarity organic solvent so as to get yulan magnolia total lactone; the yulan magnolia total lactone is processed with columnchromatography, and then the invention extract is obtained by collecting necessary components. The invention has definite effective components, the content of which is high, and is suitable for industrialized production. The invention has the anti-tumor virus function. The invention can be prepared into various pharmaceutical preparations with anti-tumor function.
Description
Technical field
The invention belongs to the effective ingredients in plant preparation field, be specifically related to the extraction separation method that from YULAN extraction separation is rich in the total sesquiterpene lactone compounds of parthenolide.The invention still further relates to the application of this extract in pharmacy and antitumor drug.
Background technology
YULAN is one of taxon important in Magnoliaceae (Magnoliaceae) plant, is a class fast-growing woods, special good species with economic forest, forests for water and soil conservation and the town and country landscape ecological forest, and development and exploitation prospect are arranged very much.The dry flower of Magnoliacea plant Flos Magnoliae (Magnolia biondii Pamp.), YULAN (Magnolia denudate desr.) or Flos Magnoliae (Magnolia sprengeri Pamp.) is the medicinal part of Chinese medicine Flos Magnoliae, is used for treating cold symptoms such as headche due to wind-cold, nasal obstruction clinically.Document (Zhao Liqin. Y biondii(Pamp)D.L.Fu. ter penoids and bioactivity research thereof progress. the time precious traditional Chinese medical science traditional Chinese medicines [J] .2005.16 (4): 298-230) record, be rich in ter penoids in the YULAN, be mainly monoterpene and sesquiterpene and containing oxygen derivative thereof.(Chen Xingrong such as Chen Xingrong, Deng. the evergreen magnolia chemical constitution study. Chinese crude drug [J] .2000,13 (3): 159-160) grade is carried out chemical constitution study to evergreen magnolia, therefrom separates and has differentiated three sesquiterpene lactones compounds, is respectively syringaresinol, costunolide and parthenolide.Document (Two newgermacranolides from Magnolia gradiflora.J.Asian Nat Prod Res.2001,3 (2): 95-102) reported isolating sesquiterpene lactones compounds from southern magnolia, comprising lactone compounds such as 2-Alpha-hydroxy-dihydro parthenolide, parthenolide, constuslactones.The document that more than separates parthenolide all obtains when the research plant chemical ingredient, is a basic Separation Research experiment, is not suitable for suitability for industrialized production.And do not have so far yet document open from YULAN specially at the parthenolide extraction and separation process and the research of being rich in the total sesquiterpene lactone compounds extraction and separation process of parthenolide.
Sesquiterpene lactones is that a class has than Johnson ﹠ Johnson and manages active chemical compound, especially aspect anti-bacteria and anti-virus, studies more have arteannuin, costunolide, parthenolide etc.Parthenolide becomes the popular object of study of Chinese scholars in recent years.U.S. Pat 4758433 disclose a kind of from chryanthemum parthenium effective component extracting be used for the treatment of migraine, its main pharmacodynamics composition is a parthenolide.PCT/US00/34469 discloses with parthenolide and has come the anticancer activity.U.S. research worker discovery parthenolide can push away ruins the myelomatosis cell, and the development leukemia medicament is had very great help.
Summary of the invention
The purpose of this invention is to provide a kind of total sesquiterpene lactone extract that is rich in parthenolide and preparation method thereof, the used method of the present invention not only can obtain high-load parthenolide (content that the extract that obtains with this method can make parthenolide more than or equal to 80% less than 100%), and the efficiency of pcr product height, be fit to suitability for industrialized production.
The present invention also aims to provide a kind of medicine that contains extract of the present invention, this medicine can be used for antitumor and tumor aid treatment.The medicine that contains extract of the present invention, can be to be main active with extract of the present invention, add that pharmaceutically acceptable pharmaceutical aids makes required pharmaceutical dosage form, also can be that extract of the present invention adds other medicines with antitumor action together as active component, better to reach the purpose of Synergistic treatment tumor.
Preparation contains the medicine of extract of the present invention, can realize by following proposal: it is an amount of to get extract of the present invention, adds the adjuvant in the pharmacy, as disintegrating agent, lubricant, binding agent, stabilizing agent, diluent, excipient etc., by the pharmaceutical methods of routine, make required dosage form.Required dosage form can be an oral formulations, also can be ejection preparation, can also be transdermal absorption formulation.Add other in the present invention and have the antineoplastic active component, then can be made into the pharmaceutical preparation of extract of the present invention and the combination of other active component.
Another object of the present invention is to provide a kind of application of sesquiterpene lactones compounds in preparation treatment antitumor drug of being rich in parthenolide.
The raw material YULAN that the present invention is used, the flower that can be southern magnolia (Magnolia gradiflora) is or/and leaf, and the flower that also can be other plant that contains parthenolide and sesquiterpene lactones in this platymiscium is or/and leaf.
Extract of the present invention is mainly realized by two operations, at first is the preparation of YULAN total sesquiterpene lactone, and the YULAN leaf is pulverized or/and flower is raw material, through solvent extraction and extraction preparation YULAN total sesquiterpene lactone; Next be the YULAN total sesquiterpene lactone that will prepare through column chromatography for separation, the mixed solvent gradient elution is collected required stream part, concentrates, drying promptly gets extract of the present invention.
The extract that contains effective ingredient of the present invention, can realize by following concrete technical scheme: the YULAN leaf soaks 2 times with deionized water or/and flower is ground into coarse powder, and the time was respectively 24 hours, 12 hours.Be soaked in water and remove most of water soluble ingredient in the plant, can avoid the generation of emulsion in the extraction of back like this.
Medicinal residues behind the water logging bubble dry, and it is thin out to be extracted into the alcohol extract primary colors with lower alcohol (as ethanol, methanol, acetone etc.), merges each time extracting solution.Extracting solution reclaim under reduced pressure alcoholic solvent, get extractum shape extract, add 20%～40% ethanol again in the extract, backflow makes the extract dissolving or becomes the suspendible shape, after room temperature cools, with organic solvent (petroleum ether, hexane) extraction is 3 times, each solvent load is about 0.5～1 times of medicinal liquid, merge extremely about 1/4 extract volume of each time extract and concentrating under reduced pressure extract, the ethanol back extraction of extract reuse 80% (can reduce the parthenolide that is fallen by petroleum ether extraction 2 times like this, although its dissolubility in petroleum ether is very little), merge alcohol extraction liquid, be incorporated in the pure water liquid behind organic solvent extraction concentrating under reduced pressure, make and contain the alcohol amount less than 10%, reuse organic solvent (chloroform, ethyl acetate, ether) extraction is thin out to the extract color, needs 3～4 times approximately, merge each time organic solvent extraction liquid, both sesquiterpene lactones crude product of the present invention.
The present invention extracts the method that is rich in parthenolide extract (total sesquiterpene lactone compounds), it is sesquiterpene lactones crude product with gained, separate through silica gel column chromatography, with petroleum ether (hexane)-ethyl acetate 20: 1-1: 20 or petroleum ether (hexane)-acetone 20: 1-1: 20 or chloroform-ethyl acetate (acetone) 40: 1-1: 1 gradient elution, UV, TLC, HPLC follows the tracks of detection, collection contains stream part of parthenolide, merge this stream part, concentrating under reduced pressure, the crude product sesquiterpene lactones that must contain parthenolide, separate through the pressurization reversed-phase column again, use the water-containing organic solvent gradient elution, UV, TLC, HPLC follows the tracks of detection, collect parthenolide content greater than stream part of 75%, reclaim solvent, drying, both must contain parthenolide more than or equal to 80% less than 100% sesquiterpene lactones compounds.Collect parthenolide content greater than stream part of 90%, reclaim solvent, drying, both parthenolide content more than or equal to 90% less than stream part of 100%.Wherein used reverse phase filler can be Sephadex-LH20 (polydextran gel), ODS (reverse phase silica gel), MCI-GEL.The water-containing organic solvent of used gradient elution can be methanol-water 4: 6-10: 0 or alcohol-water 4: 6-9: 1.
When handling the YULAN leaf raw material, once three kinds of method results were compared:
(1) the YULAN leaf is pulverized, the alcohol reflux through 95%, and respectively through petroleum ether, chloroform extraction, emulsion takes place in result easily when extraction, and non-pharmaceutical ingredient content is many in the chloroform extract behind the recovery solvent.
(2) the YULAN leaf is pulverized, and with 95% alcohol reflux, directly uses chloroform extraction behind the recovery solvent through water logging bubble back, the complicated component of extraction, and color is dark, and the chlorophyll content height is unfavorable for follow-up being further purified.
(3) the inventive method, the crude extract paler colour of gained, composition is simple relatively, has removed a lot of interference and invalid composition, and extraction the time does not have the generation of emulsion.
The present invention prepares the method for high-load parthenolide, and wherein used extracting method comprises that reflux, extract,, percolation extract, soak extraction, countercurrent extraction etc. when extracting raw material.
It can be methanol, ethanol, acetone that the present invention extracts used solvent, preferred alcohol, more preferably 80%～95% ethanol.
The present invention prepares the method for high-load parthenolide, and wherein temperature should be controlled less than 60 ℃ during decompression and solvent recovery.Dry used method comprises drying under reduced pressure, lyophilization and spray drying.
The sesquiterpene lactones compounds of parthenolide is rich in the present invention, can be used for making various dosage forms, as oral formulations, ejection preparation, lagging preparation, spray agent etc.
The sesquiterpene lactones compounds of parthenolide is rich in the present invention, has anticancer effect, and cancer comprises breast carcinoma, nasopharyngeal carcinoma, ovarian cancer, colon cancer, hepatocarcinoma, carcinoma of prostate, leukemia etc.
The sesquiterpene lactones compounds that obtains with this method has following characteristics: (1): parthenolide content height, and effective ingredient is clear and definite; (2): the efficiency of pcr product height; (3): process stabilizing is fit to suitability for industrialized production.
The specific embodiment
The present invention will be further described below in conjunction with specific embodiment, be interpreted as, below the purpose of cited embodiment be for the present invention is described, and do not limit the present invention.
Embodiment 1
The preparation of total sesquiterpene lactone: take by weighing leaf of Magnolia gradiflora 10kg, be ground into coarse powder, soak twice with deionized water, soak 24h respectively, 12h, filter, abandon the water extract, filtering residue dries, ethanol percolate extraction with 95% 3 times, solvent load is 8 times of medical material amount, merges ethanol extract, is evaporated to the extractum shape under 60 ℃, add 30% ethanol 3L backflow, make extractum become suspendible shape dissolving, with petroleum ether extraction three times, each petroleum ether consumption is 3000mL, merge petroleum ether extraction liquid, 40 ℃ of following reclaim under reduced pressure petroleum ether solvents are to about 2000mL, the ethanol back extraction petroleum ether twice with 80%, 1000mL at every turn, merge alcohol extraction liquid, be incorporated in the pure water admixing medical solutions, 60 ℃ of concentrating under reduced pressure add 8% ethanol 1L and make concentrated solution become the suspendible shape, reuse chloroform extraction three times, each chloroform consumption is 2000mL, combined chloroform extract, 50 ℃ of following concentrating under reduced pressure, vacuum drying gets total sesquiterpene lactone 210.35g.
Embodiment 2～5
Implement 2～5 and be the total sesquiterpene lactone preparation, 2,3 are respectively the embodiment with methanol, acetone extraction, and all the other are identical with embodiment 1; 4,5 be respectively the embodiment that chloroform is become ethyl acetate, ether, all the other are identical with embodiment 1.
Embodiment 6
From the total sesquiterpene lactone 210.35g that last embodiment 1 prepares, get 50g, dissolve with ethanol, 30～60 purpose silica gel mixed samples, other takes by weighing silica gel (200～300 order) 2kg, dry method is packed in the glass column, and column internal diameter * length is 80 * 100cm, and the silica gel mixed sample sample is contained on the clean silica gel, cover clean silica gel of one deck and absorbent cotton on the sample again, with 10: 1 eluting of mobile phase petroleum ether-ethyl acetate, thin out earlier to the eluent color, do not finish 3: 1 eluting of reuse petroleum ether-ethyl acetate when having required composition and getting off, UV, TLC, HPLC follows the tracks of detection, collection contains stream part of parthenolide, merges, and 50 ℃ of following decompression and solvent recoveries are to doing, with few dissolve with methanol of trying one's best, last C
18Reverse phase silica gel post, column internal diameter * length are 60 * 800mm, earlier with 4: 6 eluting of methanol-water, thin out to the eluent color, use 7: 3 system's eluting of methanol-water instead, UV, TLC, HPLC follow the tracks of detection, collect parthenolide content greater than stream part of 70%, merge, 60 ℃ of following decompression and solvent recoveries, lyophilization must be rich in the sesquiterpene lactones compounds 8.4g of parthenolide, HPLC detects, and the content of parthenolide is 80.71%.
Embodiment 7
From the total sesquiterpene lactone 210.35g that last embodiment 1 prepares, get 50g, dissolve with ethanol, 30～60 purpose silica gel mixed samples, other takes by weighing silica gel (200～300 order) 2kg, dry method is packed in the glass column, and column internal diameter * length is 80 * 100cm, and the silica gel mixed sample sample is contained on the clean silica gel, cover clean silica gel of one deck and absorbent cotton on the sample again, with 10: 1 eluting of mobile phase hexane-ethyl acetate, thin out earlier to the eluent color, do not finish 3: 1 eluting of reuse hexane-ethyl acetate when having required composition and getting off, UV, TLC, HPLC follows the tracks of detection, collection contains stream part of parthenolide, merges, and 50 ℃ of following decompression and solvent recoveries are to doing, with few dissolve with methanol of trying one's best, last C
18Reverse phase silica gel post, column internal diameter * length are 60 * 800mm, earlier with 4: 6 eluting of methanol-water, thin out to the eluent color, use 7: 3 system's eluting of methanol-water instead, UV, TLC, HPLC follow the tracks of detection, collect parthenolide content greater than stream part of 75%, merge, 60 ℃ of following decompression and solvent recoveries, lyophilization must be rich in the sesquiterpene lactones compounds of parthenolide, HPLC detects 7.9g, and the content of parthenolide is 84.68%.
Embodiment 8
From the total sesquiterpene lactone 210.35g that last embodiment 1 prepares, get 50g, dissolve with ethanol, 30～60 purpose silica gel mixed samples, other takes by weighing silica gel (200～300 order) 2kg, dry method is packed in the glass column, and column internal diameter * length is 80 * 100cm, and the silica gel mixed sample sample is contained on the clean silica gel, cover clean silica gel of one deck and absorbent cotton on the sample again, with 10: 1 eluting of mobile phase petroleum ether-acetone, thin out earlier to the eluent color, do not finish 4: 1 eluting of reuse petroleum ether-acetone when having required composition and getting off, UV, TLC, HPLC follows the tracks of detection, collection contains stream part of parthenolide, merges, and 50 ℃ of following decompression and solvent recoveries are to doing, with few dissolve with methanol of trying one's best, last C
18Reverse phase silica gel post, column internal diameter * length are 60 * 800mm, earlier with 40: 60 eluting of alcohol-water, thin out to the eluent color, use 68: 32 system's eluting of alcohol-water instead, UV, TLC, HPLC follow the tracks of detection, collect parthenolide content greater than stream part of 83%, merge, 60 ℃ of following decompression and solvent recoveries, lyophilization must be rich in the sesquiterpene lactones compounds 7.3g of parthenolide, HPLC detects, and the content of parthenolide is 89.24%.
Embodiment 9
From the total sesquiterpene lactone 210.35g that last embodiment 1 prepares, get 50g, dissolve with ethanol, 30～60 purpose silica gel mixed samples, other takes by weighing silica gel (200～300 order) 2kg, dry method is packed in the glass column, and column internal diameter * length is 80 * 100cm, and the silica gel mixed sample sample is contained on the clean silica gel, cover clean silica gel of one deck and absorbent cotton on the sample again, with 10: 1 eluting of mobile phase hexane-acetone, thin out earlier to the eluent color, do not finish 4: 1 eluting of reuse hexane-acetone when having required composition and getting off, UV, TLC, HPLC follows the tracks of detection, collection contains stream part of parthenolide, merges, and 50 ℃ of following decompression and solvent recoveries are to doing, with few dissolve with methanol of trying one's best, last C
18Reverse phase silica gel post, column internal diameter * length are 60 * 800mm, earlier with 40: 60 eluting of alcohol-water, thin out to the eluent color, use 68: 32 system's eluting of alcohol-water instead, UV, TLC, HPLC follow the tracks of detection, collect parthenolide content greater than stream part of 88%, merge, 60 ℃ of following decompression and solvent recoveries, lyophilization must be rich in the sesquiterpene lactones compounds 6.7g of parthenolide, HPLC detects, and the content of parthenolide is 92.84%.
Embodiment 10
From the total sesquiterpene lactone 210.35g that last embodiment 1 prepares, get 50g, dissolve with ethanol, 30～60 purpose silica gel mixed samples, other takes by weighing silica gel (200～300 order) 2kg, dry method is packed in the glass column, column internal diameter * length is 80 * 100cm, the silica gel mixed sample sample is contained on the clean silica gel, cover clean silica gel of one deck and absorbent cotton on the sample again, earlier with 30: 1 eluting of mobile phase chloroform-ethyl acetate, thin out to the eluent color, do not finish when having required composition and getting off, 9: 1 eluting of reuse chloroform-ethyl acetate, UV, TLC, HPLC follows the tracks of detection, collection contains stream part of parthenolide, merge, 50 ℃ of following decompression and solvent recoveries are to doing, with trying one's best few dissolve with methanol, last MCI-GEL reversed-phase column, column internal diameter * length is 60 * 800mm, and is with 40: 60 eluting of methanol-water, thin out to the eluent color earlier, use 70: 30 system's eluting of methanol-water instead, UV, TLC, HPLC follows the tracks of detection, collects required stream part, 60 ℃ of following decompression and solvent recoveries, lyophilization, must be rich in the sesquiterpene lactones compounds 6.5g of parthenolide, HPLC detects, and the content of parthenolide is 95.69%.
Embodiment 11
From the total sesquiterpene lactone 210.35g that last embodiment 1 prepares, get 50g, dissolve with ethanol, 30～60 purpose silica gel mixed samples, other takes by weighing silica gel (200～300 order) 2kg, dry method is packed in the glass column, column internal diameter * length is 80 * 100cm, the silica gel mixed sample sample is contained on the clean silica gel, cover clean silica gel of one deck and absorbent cotton on the sample again, earlier with 30: 1 eluting of mobile phase chloroform-acetone, thin out to the eluent color, do not finish when having required composition and getting off, 9: 1 eluting of reuse chloroform-acetone, UV, TLC, HPLC follows the tracks of detection, collection contains stream part of parthenolide, merge, 50 ℃ of following decompression and solvent recoveries are to doing, with trying one's best few dissolve with methanol, last MCI-GEL reversed-phase column, column internal diameter * length is 60 * 800mm, and is with 40: 60 eluting of methanol-water, thin out to the eluent color earlier, use 70: 30 system's eluting of methanol-water instead, UV, TLC, HPLC follows the tracks of detection, collects required stream part, 60 ℃ of following decompression and solvent recoveries, lyophilization, must be rich in the sesquiterpene lactones compounds 6.1g of parthenolide, HPLC detects, and the content of parthenolide is 97.82%.
Embodiment 12
From the total sesquiterpene lactone 210.35g that last embodiment 1 prepares, get 50g, dissolve with ethanol, 30～60 purpose silica gel mixed samples, other takes by weighing silica gel (200～300 order) 2kg, dry method is packed in the glass column, column internal diameter * length is 80 * 100cm, the silica gel mixed sample sample is contained on the clean silica gel, cover clean silica gel of one deck and absorbent cotton on the sample again, earlier with 10: 1 eluting of mobile phase petroleum ether-acetone, thin out to the eluent color, do not finish when having required composition and getting off, 4: 1 eluting of reuse petroleum ether-acetone, UV, TLC, HPLC follows the tracks of detection, collection contains stream part of parthenolide, merge, 50 ℃ of following decompression and solvent recoveries are to doing, with few dissolve with methanol of trying one's best, last Sephadex-LH20 post, column internal diameter * length is 60 * 800mm, methanol-water 4: 6,1: 1,6: 4,7: 3,8: 2 gradient elutions, UV, TLC, HPLC follows the tracks of detection, collects required stream part, 60 ℃ of following decompression and solvent recoveries, lyophilization, must be rich in the sesquiterpene lactones compounds 5.1g of parthenolide, HPLC detects, and the content of parthenolide is 99.24%.
Embodiment 13
From the total sesquiterpene lactone 210.35g that last embodiment 1 prepares, get 50g, dissolve with ethanol, 30～60 purpose silica gel mixed samples, other takes by weighing silica gel (200～300 order) 2kg, dry method is packed in the glass column, column internal diameter * length is 80 * 100cm, the silica gel mixed sample sample is contained on the clean silica gel, cover clean silica gel of one deck and absorbent cotton on the sample again, earlier with 10: 1 eluting of mobile phase petroleum ether-ethyl acetate, thin out to the eluent color, do not finish when having required composition and getting off, 3: 1 eluting of reuse petroleum ether-ethyl acetate, UV, TLC, HPLC follows the tracks of detection, collection contains stream part of parthenolide, merge, 50 ℃ of following decompression and solvent recoveries are to doing, with few dissolve with methanol of trying one's best, last Sephadex-LH20 post, column internal diameter * length is 60 * 800mm, alcohol-water 4: 6,1: 1,6: 4,7: 3,8: 2 gradient elutions, UV, TLC, HPLC follows the tracks of detection, collects required stream part, 60 ℃ of following decompression and solvent recoveries, lyophilization, must be rich in the sesquiterpene lactones compounds 4.9g of parthenolide, HPLC detects, and the content of parthenolide is 98.86%.
Embodiment 14
From the total sesquiterpene lactone 210.35g that last embodiment 1 prepares, get 50g, dissolve with ethanol, 30～60 purpose silica gel mixed samples, other takes by weighing silica gel (200～300 order) 2kg, dry method is packed in the glass column, and column internal diameter * length is 80 * 100cm, and the silica gel mixed sample sample is contained on the clean silica gel, cover clean silica gel of one deck and absorbent cotton on the sample again, with 30: 1 eluting of mobile phase chloroform-ethyl acetate, thin out earlier to the eluent color, do not finish 9: 1 eluting of reuse chloroform-ethyl acetate when having required composition and getting off, UV, TLC, HPLC follows the tracks of detection, collection contains stream part of parthenolide, merges, and 50 ℃ of following decompression and solvent recoveries are to doing, with few dissolve with methanol of trying one's best, last C
8The reverse phase silica gel post, column internal diameter * length is 60 * 800mm, earlier with 4: 6 eluting of methanol-water, distributes to thin out, the no required one-tenth of eluent color, use 7: 3 isocratic elutions of methanol-water again instead, UV, TLC, HPLC follow the tracks of detection, collect required stream part, 60 ℃ of following decompression and solvent recoveries, lyophilization, must be rich in the sesquiterpene lactones compounds 6.5g of parthenolide, HPLC detects, and the content of parthenolide is 98.54%.
Embodiment 15
From the total sesquiterpene lactone 210.35g that last embodiment 1 prepares, get 50g, dissolve with ethanol, 30～60 purpose silica gel mixed samples, other takes by weighing silica gel (200～300 order) 2kg, dry method is packed in the glass column, and column internal diameter * length is 80 * 100cm, and the silica gel mixed sample sample is contained on the clean silica gel, cover clean silica gel of one deck and absorbent cotton on the sample again, with 30: 1 eluting of mobile phase chloroform-acetone, thin out earlier to the eluent color, do not finish 10: 1 eluting of reuse chloroform-acetone when having required composition and getting off, UV, TLC, HPLC follows the tracks of detection, collection contains stream part of parthenolide, merges, and 50 ℃ of following decompression and solvent recoveries are to doing, with few dissolve with methanol of trying one's best, last C
8The reverse phase silica gel post, column internal diameter * length is 60 * 800mm, earlier with 4: 6 eluting of alcohol-water, distributes to thin out, the no required one-tenth of eluent color, use 68: 32 isocratic elutions of ethanol alcohol-water again instead, UV, TLC, HPLC follow the tracks of detection, collect required stream part, 60 ℃ of following decompression and solvent recoveries, lyophilization, must be rich in the sesquiterpene lactones compounds 6.1g of parthenolide, HPLC detects, and the content of parthenolide is 97.67%.
Embodiment 16
Below by of the effect of pharmacological evaluation explanation effective ingredient of the present invention at anticancer aspect.
(1), external pharmacological action
Cell strain: leukemia cell line K562, nasopharyngeal carcinoma cell KB, ovarian cancer cell SKOV3, hepatoma carcinoma cell BEL-7402, colon cancer cell (HT-29), cervical cancer cell Hela, prostate gland cancer cell PC-3.
To be in exponential phase leukaemia pearl K562, nasopharyngeal carcinoma cell, ovarian cancer cell, hepatoma carcinoma cell, colon cancer cell, prostate gland cancer cell strain and LAK cell (matched group) be made into 2 * 10 with the CM-1640 culture medium
5/ ml cell suspension adds in the 96 hole circle floor cells culture plates, and every hole 0.2ml adds extract of the present invention (earlier with the DMSO dissolving) more respectively, and make its concentration be: 0.5,1.0,2.5,5.0,10.0 μ g/ml, 37 ℃, 5%CO are put in each test concentrations 5 hole
2Cultivated 72 hours under the saturated humidity condition, record extinction (A) value at enzyme connection detector 570nm wavelength with mtt assay.Calculate the suppression ratio of extract of the present invention to each cell strain.
Table 1 variable concentrations extract of the present invention is to the increment suppression ratio (%) of tumor cell and LAK cell
Compare with the LAK cell:
*P＜0.01
From last table result as can be known, extract of the present invention is when 0.5～10 μ g/ml, deratization bone marrow cell carcinoma, human cervical carcinoma cell do not produce outside the inhibitory action, all the other all have certain inhibitory action, the most suitable during wherein with 5 μ g/ml concentration, up terraced again enrichment degree is not obvious to the inhibitory action contribution of cancerous cell, nonsignificance.The LAK cytotoxicity experiment is shown, when 0.5～5 μ g/ml, the LAK cell is not produced inhibitory action, when 1.0～2.5 μ g/ml, also have certain increment effect, the strongest to the LAK cyto-inhibition when 10.0 μ g/ml.
(2), the interior anti-tumor effect of body to transplanted tumor
Animal: BALB/c nude mouse
JEG-3: colon cancer cell line (HT-29), Os Mus myelocytome SP20
Test solution: PBS titer
Get 80 of BALB/C mice, be divided into 8 groups at random, be respectively two groups of matched groups, lotus HT29 cancerous cell group three groups of (injecting the high, medium and low dosage group of extract of the present invention respectively), three groups of lotus SP20 cancerous cell groups (giving extract of the present invention high, medium and low dosage equally).First winding kind HT29 colon cancer cell line abdominal cavity is simultaneously injected the 0.4mlPBS titer every day, promptly distinguishes lumbar injection extract 0.25,0.5 of the present invention, 1.0mg/kg.d for the 3rd to five group behind inoculation HT29 cell suspension
-1, second group is lumbar injection 0.4mlPBS titer every day behind inoculation SP20 cell suspension, promptly distinguishes lumbar injection extract 0.25,0.5 of the present invention, 1.0mg/kg.d for the 6th to eight group behind inoculation SP20 cell suspension
-1, dissect survival mice on the 14th day in the inoculation back, and win tumor, calculate gross tumor volume
(the maximum major diameter of a, b is maximum minor axis), and calculate tumour inhibiting rate, the tumour inhibiting rate computing formula is
The results are shown in Table 2 and table 3
Table 2 extract of the present invention is to transplanting the tumor-inhibiting action of HT29 cell strain in the mice body
Compare with matched group:
*P＜0.01
Table 3 extract of the present invention is to transplanting the tumor-inhibiting action of SP20 cell strain in the mice body
Compare with matched group:
*P＜0.01
From table 2,3 as can be known, in the tumor bearing nude mice body, extract of the present invention all has the effect that suppresses tumor growth to colon cancer and Mus bone marrow cancer, after 2 weeks of administration, the gross tumor volume of treatment group mice is significantly less than matched group (P＜0.01), to the dose-dependence that is suppressed to of tumor, high dose group of the present invention is respectively 71.48%, 65.49% to the suppression ratio of lotus colon cancer cell tumor and lotus mouse myeloma cell line.
Claims (10)
1. total sesquiterpene lactone extract that is rich in parthenolide is characterized in that it being to be prepared by following two operations: at first be the preparation of total sesquiterpene lactone, with the YULAN leaf or/and flower is the feedstock production total sesquiterpene lactone; Next is a process for refining, and the YULAN total sesquiterpene lactone for preparing through column chromatography for separation, is collected required composition, concentrates, dry both the total sesquiterpene lactone extract of parthenolide is rich in the present invention.
2. total sesquiterpene lactone extract as claimed in claim 1, wherein said operation comprises the steps:
1 〉: the preparation of total sesquiterpene lactone:
(1): take by weighing the YULAN coarse powder, soak 2 times with deionized water, be respectively 24h, 12h, filter, remove the water soak, filtering residue dries, and is standby;
(2): get above-mentioned filtering residue, thin out with lower alcohol extraction to the extracting solution color, filter, merge extractive liquid,, concentrating under reduced pressure reclaims lower alcohol to the thick paste shape, makes toward wherein adding ethanol that to contain the alcohol amount be 20%～40%, the dissolving that refluxes, room temperature cools;
(3): dissolve with ethanol liquid is thin out to the extract color with petroleum ether extraction, merges petroleum ether extraction liquid, is evaporated to 1/4 of original volume, ethanol back extraction with 80% 2 times, merge 80% alcohol extraction liquid, be incorporated in the aquiferous ethanol layer, decompression recycling ethanol is less than 10% to containing the alcohol amount;
(4): contain alcohol and be lower than 10% alcohol-water solution reuse organic solvent extracting to be taken to the extract color thin out, merge organic solvent extraction liquid, concentrating under reduced pressure, drying, the total sesquiterpene lactone compounds;
2 〉: the exquisite total sesquiterpene lactone extract that is rich in parthenolide that separates of column chromatography:
(5): with silica gel chromatographic column on the above-mentioned total sesquiterpene lactone compounds, with the mixed organic solvents gradient elution, UV, TLC, HPLC follow the tracks of detection, collection contains stream part of parthenolide, merge this part stream part, reclaim solvent to doing, must be rich in the total sesquiterpene lactone compounds of parthenolide;
(6): with the above-mentioned total sesquiterpene lactone compounds few dissolve with methanol of trying one's best that is rich in parthenolide, last reversed phase column chromatography separates, with the water-containing organic solvent gradient elution, UV, TLC follow the tracks of detection, collect parthenolide content greater than stream part of 75%, merge this part stream part, reclaim solvent, drying promptly gets extract of the present invention, wherein the parthenolide mass fraction more than or equal to 80% less than 100%.
3. total sesquiterpene lactone extract as claimed in claim 2, used lower alcohol solvent can be any in methanol, ethanol, the acetone when wherein extracting.
4. total sesquiterpene lactone extract as claimed in claim 3, described lower alcohol are 80%～95% ethanol.
5. total sesquiterpene lactone extract as claimed in claim 2, wherein extraction contains alcohol to be lower than 10% the used organic solvent of alcohol-water solution is to be selected from chloroform, ethyl acetate, the ether any.
6. total sesquiterpene lactone extract as claimed in claim 2, wherein the used mixed flow of gradient elution silicagel column is mutually for being selected from petroleum ether: ethyl acetate=20: 1～1: 20, hexane: ethyl acetate=20: 1～1: 20, petroleum ether: acetone=20: 1～1: 20, hexane: acetone=20: 1～1: 20, chloroform: ethyl acetate=40: 1～1: 1, chloroform: any of acetone=40: 1～in 1: 1.
7. total sesquiterpene lactone extract as claimed in claim 2, the used reverse phase filler of wherein said reversed-phase column can be among Sephadex-LH20, MCI-GEL, the ODS any.
8. total sesquiterpene lactone extract as claimed in claim 2, wherein the used water-containing organic solvent of gradient elution reversed-phase column can be a methanol: water=4: 6～10: 0 or ethanol: any in the water 4: 6～9: 1.
9. medicine that contains claim 1 or 2 described total sesquiterpene lactone compounds.
10. total sesquiterpene lactone class extract according to claim 1 and 2 has application in the antitumor action medicine in preparation.
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