CN1152034C - Process for extracting active anticancer component from vinca flower - Google Patents
Process for extracting active anticancer component from vinca flower Download PDFInfo
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- CN1152034C CN1152034C CNB011319879A CN01131987A CN1152034C CN 1152034 C CN1152034 C CN 1152034C CN B011319879 A CNB011319879 A CN B011319879A CN 01131987 A CN01131987 A CN 01131987A CN 1152034 C CN1152034 C CN 1152034C
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Abstract
The present invention belongs to the technical field of natural medicines, more specifically a novel technology for extracting an anti-cancer active ingredient from madagascar periwinkle herb. In the novel technology, a certain amount of tartaric acid or inorganic acid is added in an extraction solvent, vinblastine and vincristine are separated from the acid, and ursolic acid is separated from acid insoluble substances. The technology is low in cost, and is suitable for industrial production.
Description
Technical field
The invention belongs to natural medicine technical field.Be specifically related to a kind of technology of from Vinca, extracting active ingredient vinealeucoblastine(VLB), vincristine(VCR) and ursolic acid with antitumous effect.
Background technology
Vinca [Catharanthus roseus (L.) G.Don.] is the Apocynaceae per nnial herb, originates in east, non-state.Widely cultivate each province, China south, does trade, flower bed, potted landscape more and view and admire usefulness, can bloom in the anniversary, has another name called everyday spring, new, ligustrum malongense etc. everyday.Plant height 20-60cm, stem branch green or sorrel, class cylindricality, marrow sky; Leaf is to life, oblong or obovate, and long 3-6cm, wide 1.5-2.5cm, the pure garden of tip, pinniform vein, master pulse is clear; The cyme armpit is given birth to or give birth on the top, has colored 2-3, the high pin dish of corolla shape, and 5 split, purplish red, pink, the white or white lobe red heart of pattern; Fruit be a Follicle radish fruit, long garden tubular, and tiny, the black of seed does not have kind of a hair, two ends are cut the particulate state hump on flat (Chen Junyu, Cheng Xuke: middle national flower is through, the cultural press in Shanghai, and 1990, P.484; Xue Congxian; 150 kinds of the perennial root showy flowers of herbaceous plants, Yunnan science and technology press, 1999)
Noble in 1958, RL, Svoboda in 1961, GH successively is separated to vinealeucoblastine(VLB) (Vinblastin from Vinca, VLB) and vincristine(VCR) (Vincristine, VCR), 1967 and Shanghai Pharmaceutical Inst., Chinese Academy of Sciences in 1969 also extract these two kinds of indoles alkaloids with homemade Vinca and change by Hangzhou pharmaceutical factory and produce (Noble RL et al:Ann N.Y.Acad.Sci.1958,76, P.882; Svoboda, GH:J.Am.Pharm.Assoc.Sci.Ed 1958.47.P.834; Svoboda, GH:Lloydia 1961.24.P.173).Vinealeucoblastine(VLB) and vincristine(VCR) are used year surplus in the of 30 in oncotherapy, be still the main medication on the clinical tumor so far.
Sixties Zhou Yunli researcher once was separated to the monoterpenes compound in the waste liquid that extracts vinealeucoblastine(VLB) and vincristine(VCR), existing through being accredited as ursolic acid (Ursolic acid).Ursolic acid (Ursolicacid) is newfound in recent years anticancer active constituent, the growth of tumour cells such as it can not only the strongly inhibited leukemia, fibrosarcoma, mammary cancer, lung cancer, skin carcinoma, melanoma and can induce teratocarcinoma cell to reverse to be normal cell, to be the new focus of modern tumor drug screening.
(17 (7) P.990 for Young, HS et al:Biol.Phar.Bull.1994; Huang, MT et al:Cancer Res.1994,54 (3) P.701; Dong Jing, Sun Yan: Chinese Journal of New Drugs 1997,6 (2) P.101)
Ursolic acid and vinealeucoblastine(VLB), vincristine(VCR) are the dissimilar compounds of two classes, and ursolic acid is α-amyrin type triterpenic acid, and both are indoles alkaloid for the back.Water insoluble and the sherwood oil of ursolic acid, and be dissolved in hot methanol, hot ethanol, dissolve in benzene, chloroform.Vinealeucoblastine(VLB) and vincristine(VCR)
Also water insoluble and be dissolved in methyl alcohol, chloroform, benzene equal solvent.Therefore, with hot methanol, hot ethanol.The benzene equal solvent all can from Vinca, extract ursolic acid, vinealeucoblastine(VLB) and vincristine(VCR).But vinealeucoblastine(VLB) and vincristine(VCR) are different from ursolic acid again, and alkaloid can form salt and water-soluble under acidic conditions, and ursolic acid then can not.
Ursolic acid extracts from psyllium (Plantago major L.) or Herba Rabdosiae glaucocalycis { Robdosiaamethytoiles (Benth) Haro} (Chen Dechang, Chemistry for Chinese Traditional Medicine reference substance workbook, Chinese Medicine science and technology press at present; Yu Ruisheng: Botany Gazette 1984,6; 675).Psyllium and Herba Rabdosiae glaucocalycis all are wild plant, collect inconvenience.Find that so far the psyllium that contains ursolic acid only is an a kind of Big Semen Plantaginis.(P.asiatica L.) Plantago depressa Willd (p.depressa L.) etc. is not seen and is contained the ursolic acid report before other common psyllium such as the car.
Summary of the invention
Technical problem to be solved by this invention is to develop extracts the active ingredient with antitumous effect from Vinca.
The present invention utilizes the different qualities of vinealeucoblastine(VLB), vincristine(VCR) and ursolic acid to separate from Vinca.
The invention provides a kind of from Vinca, the extraction and have the technology of antitumous effect active ingredient.
The invention provides a kind of technology of extracting vinealeucoblastine(VLB), vincristine(VCR) and ursolic acid from Vinca, this technology comprises following method:
(1) with Vinca herb siccative crushed material, to mix and stir the back with less water and drop into and to install in advance in the diacolation bucket of benzene, the ratio of crude drug powder and benzene is 1: 5-10, add a cover after lixiviate 1-3 days and begin diacolation, collection benzene percolate;
In above-mentioned benzene percolate, add the 3-10% tartaric acid solution and do the extraction of liquid-liquid anti-phase, vinealeucoblastine(VLB) and vincristine(VCR) are changed in the sour water with the tartrate form, and ursolic acid still stays in acidifying benzene liquid, add the chloroform extraction after benzene liquid is concentrated, filter, behind concentrating under reduced pressure, get the chloroform enriched material again; Then, application of sample is 1/1 wash-out with n-hexane/ethyl acetate on silica gel column chromatography in batches, and elutriant is dried through being evaporated to, and adds chloroform and makes moltenly, uses activated carbon decolorizing, filters, and filtrate is drained, and adds dehydrated alcohol, promptly gets white ursolic acid crystallization; Acid liquid is transferred pH to 6.5 with ammoniacal liquor earlier, adds the chloroform extraction, and the chloroform layer evaporated under reduced pressure is used dissolve with methanol again, and the elimination insolubles is the crude product that contains vinealeucoblastine(VLB) and vincristine(VCR) behind the filtrate evaporate to dryness; Crude product adds a little anhydrous alcohol solution, transfers pH to 3.8 to 4.1 with 5% sulfate anhydrous ethanol again, and placement is spent the night, and its precipitate is vinealeucoblastine(VLB) and leucocristine sulfate mixture; The said mixture adding distil water is dissolved, with the ammoniacal liquor alkalization, add the chloroform extraction, again with behind the anhydrous sodium sulfate dehydration, last alumina column chromatography column chromatography, with benzene-chloroform mixed solvent wash-out, Fractional Collections elutriant, the thin layer chromatography (tlc) method detects, merge same stream part, decompressing and extracting promptly gets vinealeucoblastine(VLB) from column chromatography leading portion elutriant, promptly get vincristine(VCR) in the back segment elutriant; Or
(2) collect the Changchun floral leaf, dry, pulverize, pour in the extracting cylinder, add the paramount charge level 1-2cm of going out of 80% methyl alcohol, in the extracting cylinder chuck, feed steam, make feed temperature rise to solution boiling, boiling steam through condensing reflux to extracting cylinder, refluxing extraction 3hr like this, after centrifugal extraction liquid, extraction liquid through vacuum decompression concentrate methanol extract, reclaim methyl alcohol simultaneously;
In methanol extract, add 1N H
2SO
4Make pH to 3-4, and stir frequently, centrifugal after leaving standstill, get acid non-soluble substance and acid-soluble material, acid-soluble material extracts vinealeucoblastine(VLB) and vincristine(VCR) according to known method, above-mentioned acid non-soluble substance can through silicagel column absorption, be the 10-6/1-3 wash-out with cyclohexane/ethyl acetate with 5 times of amount chloroform extraction ursolic acid again, after elutriant concentrates, its concentrated solution can in dehydrated alcohol, precipitate white ursolic acid crystallization; Or
(3) Vinca herb or leaf or over-ground part extract with hot ethanol, and alcohol concn 70-95% adds organic acid or mineral acid in the ethanol extract of Vinca, make pH reach 3-4, promptly gets acid solution and acid non-soluble substance; Acid solution adds the benzene extraction after transferring alkali with ammoniacal liquor, go up silica gel column chromatography after the extraction liquid evaporated under reduced pressure, with n-hexane/ethyl acetate elutriant wash-out, monitor with thin-layer chromatography simultaneously, add behind the concentrated evaporate to dryness of elutriant segmentation and contain 4% vitriolic dehydrated alcohol, can from leading portion stream part, separate out vinblastine sulfate, from back segment stream part, separate out vincristine sulphate;
Acid non-soluble substance adds methanol extraction, making silica gel chromatographic column after extraction liquid is evaporated to and does separates, use earlier sherwood oil drip washing, again with containing 2% alcoholic acid sherwood oil wash-out, and make thin-layer chromatography and monitor, collection merges the stream part that contains ursolic acid, concentrates evaporate to dryness post-heating dissolve with ethanol and also places under the low temperature, promptly has the ursolic acid crystallization to separate out.
Description of drawings
Fig. 1 Vinca is extracted the technical process of ursolic acid, vinealeucoblastine(VLB) and vincristine(VCR)
Fig. 2 extracts ursolic acid from Vinca benzene percolate
Fig. 3 extracts vinealeucoblastine(VLB) and vincristine(VCR) from Vinca benzene percolate
Fig. 4 extracts ursolic acid, vinealeucoblastine(VLB) and vincristine(VCR) from Vinca methanol eddy liquid
Fig. 5 extracts ursolic acid, vinealeucoblastine(VLB) and vincristine(VCR) from the Vinca ethanolic soln
The thin-layer chromatogram of Fig. 6 ursolic acid standard substance and Vinca ursolic acid
The HPLC color atlas of Fig. 7 ursolic acid standard substance
The HPLC color atlas of Fig. 8 Vinca ursolic acid
The ultraviolet spectrogram of Fig. 9 ursolic acid standard substance
The ultraviolet spectrogram of Figure 10 Vinca ursolic acid
The infrared spectrogram of Figure 11 ursolic acid standard substance
The infrared spectrogram of Figure 12 Vinca ursolic acid
The mass spectrum of Figure 13 ursolic acid standard substance
The mass spectrum of Figure 14 Vinca ursolic acid
Figure 15 ursolic acid standard substance
1The H nuclear magnetic resonance spectrum
Figure 16 Vinca ursolic acid
1The H nuclear magnetic resonance spectrum
Figure 17 ursolic acid standard substance
13The C nuclear magnetic resonance spectrum
Figure 18 Vinca ursolic acid
13The C nuclear magnetic resonance spectrum
Embodiment
As shown in drawings, the invention provides a kind of technology of extracting vinealeucoblastine(VLB), vincristine(VCR) and ursolic acid from Vinca, this technology is:
(1) with Vinca herb siccative crushed material, to mix and stir the back with less water and drop into and to install in advance in the diacolation bucket of benzene, the ratio of crude drug powder and benzene is 1: 5-10.0, add a cover after lixiviate 1-3 days and begin diacolation, collection benzene percolate;
In above-mentioned benzene percolate, add the 3-10% tartaric acid solution and do the extraction of liquid-liquid anti-phase, vinealeucoblastine(VLB) and vincristine(VCR) are changed in the sour water with the tartrate form, and ursolic acid still stays in benzene liquid.By following flow process 1 from acid non-soluble substance (benzene liquid), carry ursolic acid, by following flow process 2 from acid liquid, carry vinealeucoblastine(VLB) and vincristine(VCR) (Fig. 1,2,3).
(2) collect the Changchun floral leaf, dry, pulverize, pour in the extracting cylinder, add 80% methyl alcohol to the high 1-2cm of charge level, in the extracting cylinder chuck, feed steam, make feed temperature rise to the solution boiling, boiling steam through condensing reflux to extracting cylinder.Like this refluxing extraction 3hr, after centrifugal extraction liquid.Extraction liquid through vacuum decompression concentrate methanol extract, reclaim methyl alcohol simultaneously.
In methanol extract, add IN H
2SO
4, make pH to 3-4, and stir frequently, centrifugal after leaving standstill, get acid non-soluble substance and acid-soluble material.Natural medicinal ingredients extraction separation and preparation one book (Chinese Medicine science and technology press that acid-soluble material is write by Sun Wenji, 1999) method of being introduced is extracted vinealeucoblastine(VLB) and vincristine(VCR), and does quality test by two ones of Pharmacopoeias of People's Republic of China (2000 editions P.855-856).Above-mentioned acid non-soluble substance can be with 5 times of amount chloroform extraction ursolic acid, again through silicagel column absorption, with cyclohexane/ethyl acetate (10-6/1-3) wash-out, after elutriant concentrates, its concentrated solution can in dehydrated alcohol, precipitate white ursolic acid crystallization (Fig. 4)
(3) Vinca herb or leaf or stem or over-ground part, except that the extraction of available benzene, methyl alcohol equal solvent, also available heat extraction using alcohol, alcohol concn 70-95% is because ursolic acid is dissolved in the hot ethanol, so should get circumfluence method with alcohol extraction.For asking careless three medicines that get, should add 6N HCL in the extracting solution and make pH reach 3-4.Like this, we can from acid solution, extract water-soluble Vinblastine sulphate and vincristine sulphate, and from acid non-soluble substance, carry to such an extent that ursolic acid (Fig. 5) ursolic acid is identified:
To from the acid non-soluble substance of Vinca benzene or methyl alcohol or ethanol extract, separate white crystals, do following evaluation:
1. thin-layer chromatography:
Sample and ursolic acid standard substance (the biological product calibrating of Chinese medicine institute) are distinguished point sample on the thin-layer silicon offset plate, launch with hexanaphthene/chloroform/ethyl acetate (20/5/8) developping agent, aceticanhydride/sulfuric acid (9/1) colour developing, both spots that develop the color are in same position as a result, the Rf of sample * 100 values are 32.2, and the ursolic acid standard substance are 32.8.With behind the silica gel thin-layer plate point sample, with chloroform/acetone (6/4) exhibition layer, the colour developing spot of results sample and ursolic acid standard substance also is in same level attitude in addition, and Rf * 100 values are respectively 59.2 and 58.9; Get the silica gel thin-layer plate of point sample again, with propyl carbinol/chloroform/ethyl acetate (20/5/8) exhibition layer, Rf * 100 values of colour developing back sample are 80.3, and the ursolic acid standard substance are 80.3 also, and both are in full accord; As seen sample crystallization and standard substance are same substance.(Fig. 6)
2. high performance liquid chromatography (HPLC):
Sample (UA-M) is made HPLC to be analyzed and produces with standard ursolic acid (Sigma) company, numbering U653) makes comparisons, HPLC instrument: Beckman Gold System, analytical column: anti-phase C18 post, moving phase: methanol (95/5) result: sample desolventizes (2.98 minutes time of lag) outside the peak, a main peak only occurs, be 9.02 minutes its time of lag; Be 8.90 minutes main peak time of lag of standard ursolic acid, both basically identicals (Fig. 7,8).
3. UV spectrum:
The sample methanol solution is made uv scan (300-190nm), an absorption peak only occurs, its maximum absorption wavelength is 203.3nm, consistent with the ursolic acid standard substance (202.5m).See figure (9,10)
4. infrared spectra:
Sample and ursolic acid standard substance are measured on infrared spectrometer with pellet technique, and both identical vibration absorption peak occurs at 3540cm-1,2952cm-1 and 1715cm-1 as a result.See figure (11,12)
5. mass spectrum:
Sample is done mass spectroscopy, molecule presents the RDA cracking similar with the standard ursolic acid through electron impact, both except that last cracked molecular ion peak (m/e 456) all occurring, all have ursolic acid cleaved fragment proton peak m/e 248, m/e207, m/e189 and m/e133 in addition on mass spectrum.See figure (13,14)
6. nuclear magnetic resonance spectrum:
Sample is made hydrogen (1H) nucleus magnetic resonance and carbon (13C) nmr analysis, instrument: AVANCE 500 Bruke Co., operating frequency: 1H-NMR 500MH2,13C-NMR127MH2.Solvent: DMSO measures temperature: 300K.Reference substance: standard ursolic acid.Result: sample
1H-MMR spectrum and
13C-NM spectrum and standard ursolic acid basically identical, sample
13.4ppm and 2.5ppm abundance in the H-NMR spectrum are higher.The former is the proton peak of water in the sample, and the latter is the resonance signal of solvent DMSO.Therefore sample NMR spectrum is composed no essential difference with the NMR of standard ursolic acid.(Figure 15,16,17,18)
Brief summary:
The comprehensive above susceptible of proof of analyzing: the crystallization of carrying from the acid non-soluble substance of the benzene of Vinca or methyl alcohol or ethanol extract is a ursolic acid.
Use technology of the present invention can from Vinca, extract vinealeucoblastine(VLB), vincristine(VCR) and three anticancer compositions of ursolic acid, reduce production costs greatly, but large-scale industrial production.
Embodiment one
With Vinca herb siccative crushed material, add 0.3% water and mix and stir the back and drop into and to install in advance in the diacolation bucket of benzene, the ratio of crude drug powder and benzene is 1: 8, adds a cover lixiviate and begins diacolation after 3 days, collection benzene percolate;
In above-mentioned benzene percolate, add 6% tartaric acid solution and do the extraction of liquid-liquid anti-phase, vinealeucoblastine(VLB) and vincristine(VCR) are changed in the sour water with the tartrate form, and ursolic acid still stays in benzene liquid.
Ursolic acid extracts: add the chloroform extraction after above-mentioned benzene liquid is concentrated, filter, get the chloroform enriched material again behind concentrating under reduced pressure.Then, application of sample is on silica gel column chromatography in batches, and with n-hexane/ethyl acetate (1/1) wash-out, elutriant is dried through being evaporated to, and adds chloroform and makes moltenly, use activated carbon decolorizing, and filtrate is drained in filtration, adds dehydrated alcohol, promptly gets white ursolic acid crystallization.(Fig. 1,2)
The extraction of vinealeucoblastine(VLB) and vincristine(VCR): from above-mentioned acid liquid can by Fig. 3 extract vinealeucoblastine(VLB) and vincristine(VCR), concrete steps are as follows:
1. acid liquid is put in the separating funnel, the chloroform that adds 0.4 times earlier, add about 14% ammoniacal liquor adjusting pH to 6.5 then, chloroform layer is emitted in jolting behind the standing demix, add the chloroform extraction three times of 0.2 times of volume in water layer, chloroform extracted solution anhydrates after leaving standstill, evaporated under reduced pressure adds small amount of acetone then and takes out pine, gets always weak alkaloid.
2. total weak alkaloid is added 1.5 times of dissolvings of methyl alcohol, after spending the night, elimination crystalloid impurity, methyl alcohol filtrate, evaporated under reduced pressure is taken out pine, after add 0.5 times of melt into dope of dehydrated alcohol, add 5% ethanol solution of sulfuric acid then to pH3.8, placement is spent the night, and separates out the mixing vinblastine sulfate.Leach, 1.5 times of dissolvings of adding distil water move in the separating funnel, add 1.5 times of amount chloroforms earlier, add ammoniacal liquor then and are adjusted to pH7.5, and jolting carries free alkali into chloroform, and chloroform extracted solution adds anhydrous sodium sulfate drying, leach, and evaporated under reduced pressure gets the free weak base of purifying.
3. with the free weak base of above-mentioned purifying, in vacuum drier after the drying, by 1 gram with 30 ratios that restrain aluminum oxide, chromatographic separation on alumina column is with benzene-chloroform mixed solvent (proportion 1.30) wash-out that heavily steams, Fractional Collections, check with the thin layer chromatography (tlc) method, merge quite stream part.With column chromatography leading portion elutriant pressure reducing and steaming solvent, drain, put in the vacuum drier of P2O5 and drained 12 hours, weigh, add 2 times of dissolvings of dehydrated alcohol, add 5%H2SO4 again, make ethanol solution to pH3.8, close plug placing 48 hours below 25 ℃, is separated out the vinblastine sulfate crystallization, leach, after a small amount of dehydrated alcohol is washed, dry in the dislocation vacuum drier immediately, be the Vinblastine sulphate crude product.
With column chromatography back segment elutriant through the chromatoplate inspection, merge same stream part, decompression boils off solvent, drain, put the vacuum drier drying, weigh, add 2 times of dissolvings of dehydrated alcohol, add the 4%H2SO4 ethanol solution to pH3.8, jam-pack was being placed 48 hours below 25 ℃, promptly separated out the vitriol crude product of vincristine(VCR).
4. get the Vinblastine sulphate crude product, adding distil water and methyl alcohol make 60 ℃ of heating molten entirely, filter, container is washed with methyl alcohol, and filtrate merges, reclaim under reduced pressure methyl alcohol, heat is to the dehydrated alcohol adding of boiling in advance with 150 times of amounts immediately, and white needle is separated out in placement very soon after shaking up rapidly, leach drying, promptly get the Vinblastine sulphate elaboration.
5. get the vincristine sulphate crude product in addition, add the small amount of methanol dissolving, filter, add 30 times of amount dehydrated alcohols behind the concentrating under reduced pressure, shake up, placement is spent the night, and leaches the crystallization of separating out, and puts moisture eliminator and drains, and promptly gets the pure product of vincristine sulphate.
Embodiment two
Collect the Changchun floral leaf, dry, pulverize, cross 80 mesh sieve holes and pour in the extracting cylinder, processing industry methyl alcohol is paramount to go out charge level 2cm, feeds steam in the extracting cylinder chuck, makes feed temperature rise to the solution boiling, boiling steam through condensing reflux to extracting cylinder.Each refluxing extraction 3hr repeats 2 times, must extraction liquid after centrifugal.Extraction liquid gets methanol extract at 60 ± 5 ℃ of following concentrating under reduced pressure, reclaims methyl alcohol simultaneously.
In methanol extract, add IN H
2SO
4, make pH to 3.5, and stir frequently, centrifugal after leaving standstill, get acid non-soluble substance and acid-soluble material.Above-mentioned acid non-soluble substance can repeatedly extract ursolic acid with 5 times of amount chloroforms, concentrated extract, divide and pull on sample to silica gel column chromatography, use cyclohexane/ethyl acetate (10/3) wash-out then, elutriant through be evaporated to do after, add in the dehydrated alcohol and at room temperature leave standstill, can separate out white ursolic acid crystallization (Fig. 4)
Above-mentioned acid-soluble material is regulated pH to 6.5 with chlorine water, uses the chloroform extracting, and concentrating under reduced pressure reclaims chloroform and takes out the loose total alkaloids that gets.
Total alkaloids adds the methyl alcohol placement and spends the night, filter methanol solution, reclaim under reduced pressure methyl alcohol is also taken out pine, get the purifying total alkaloids, the purifying total alkaloids adds dehydrated alcohol and 5% sulfate anhydrous dissolve with ethanol solution, placement is placed filtration on the 2nd after waiting to separate out crystallization again, and crystallization mainly is Vinblastine sulphate and vincristine(VCR) mixture.
Above crystallization is water-soluble, ammoniacal liquor alkalization, chloroform extraction, chloroform solution anhydrous sodium sulfate dehydration, concentrate, take out pine, drying is splined on alumina chromatographic column, with benzene, chloroform mixed solution (1: 2) wash-out, Fractional Collections effluent liquid, leading portion contains vinealeucoblastine(VLB), and back segment contains vincristine(VCR).The back segment effluent liquid adds 4% sulfate anhydrous ethanolic soln behind evaporate to dryness, promptly get the vincristine sulphate finished product, and the leading portion effluent liquid adds 4% sulfate anhydrous ethanolic soln behind evaporate to dryness, make its vitriol, is the Vinblastine sulphate finished product.
Embodiment three:
Vinca herb or leaf or stem or over-ground part, except that the extraction of available benzene, methyl alcohol equal solvent, also available heat extraction using alcohol, alcohol concn 95% is because ursolic acid is dissolved in the hot ethanol, so should get circumfluence method with alcohol extraction.For asking careless three medicines that get, should add 6N HCL in the extracting solution and make pH reach 3.0.Like this, we both can from acid solution, extract water-soluble Vinblastine sulphate and vincristine sulphate, can from acid non-soluble substance, carry again ursolic acid (Fig. 5), concrete scheme is as follows:
In the acid-soluble material of Vinca ethanol extract, transfer pH to 7.0 with ammoniacal liquor, make alkaloids such as vinealeucoblastine(VLB) and vincristine(VCR) change free alkali into, and be dissolved in benzene from salt; Benzene liquid is splined on silicagel column after concentrating evaporate to dryness, with n-hexane/ethyl acetate (3: 1), the elutriant wash-out, collect the leading portion elutriant, after concentrating evaporate to dryness, add and contain 4% vitriolic dehydrated alcohol and promptly get vinblastine sulfate, the same back segment elutriant of collecting adds 4% sulfate anhydrous ethanolic soln after concentrating evaporate to dryness, promptly get vincristine sulphate.
Other gets the acid non-soluble substance of Vinca ethanol extract, adds 5 times of calorimetric benzene extraction, and benzene liquid is dried through being evaporated to, and is splined on silica gel chromatographic column then and makes chromatographic separation.Earlier with the flushing of lower boiling sherwood oil, till no longer including wax and washing out, to remove lipoid impurity.And then continue flushing with containing 2% alcoholic acid sherwood oil, at this moment the similar Oleanolic Acid of ursolic acid and ursolic acid is by wash-out slowly.Elutriant is evaporate to dryness behind low temperature (60 ± 2 ℃ °) vacuum concentration, add methyl alcohol and heated and boiled, filtered while hot, because Oleanolic Acid is soluble in methyl alcohol, be present in the filtrate after the filtration, and ursolic acid is slightly soluble in hot methanol, so still stay in filter residue, add dehydrated alcohol after getting the filter residue evaporate to dryness, put room temperature following 24 hours, can separate out the ursolic acid crystallization.
Claims (1)
1. technology of extracting vinealeucoblastine(VLB), vincristine(VCR) and ursolic acid from Vinca is characterized in that this technology comprises following method:
(1) with Vinca herb siccative crushed material, to mix and stir the back with less water and drop into and to install in advance in the diacolation bucket of benzene, the ratio of crude drug powder and benzene is 1: 5-10, add a cover after lixiviate 1-3 days and begin diacolation, collection benzene percolate;
In above-mentioned benzene percolate, add the 3-10% tartaric acid solution and do the extraction of liquid-liquid anti-phase, vinealeucoblastine(VLB) and vincristine(VCR) are changed in the sour water with the tartrate form, and ursolic acid still stays in acidifying benzene liquid, add the chloroform extraction after benzene liquid is concentrated, filter, behind concentrating under reduced pressure, get the chloroform enriched material again; Then, application of sample is 1/1 wash-out with n-hexane/ethyl acetate on silica gel column chromatography in batches, and elutriant is dried through being evaporated to, and adds chloroform and makes moltenly, uses activated carbon decolorizing, filters, and filtrate is drained, and adds dehydrated alcohol, promptly gets white ursolic acid crystallization; Acid liquid is transferred pH to 6.5 with ammoniacal liquor earlier, adds the chloroform extraction, and the chloroform layer evaporated under reduced pressure is used dissolve with methanol again, and the elimination insolubles is the crude product that contains vinealeucoblastine(VLB) and vincristine(VCR) behind the filtrate evaporate to dryness; Crude product adds a little anhydrous alcohol solution, transfers pH to 3.8 to 4.1 with 5% sulfate anhydrous ethanol again, and placement is spent the night, and its precipitate is vinealeucoblastine(VLB) and leucocristine sulfate mixture; The said mixture adding distil water is dissolved, with the ammoniacal liquor alkalization, add the chloroform extraction, again with behind the anhydrous sodium sulfate dehydration, last alumina column chromatography column chromatography, with benzene-chloroform mixed solvent wash-out, Fractional Collections elutriant, the thin layer chromatography (tlc) method detects, merge same stream part, decompressing and extracting promptly gets vinealeucoblastine(VLB) from column chromatography leading portion elutriant, promptly get vincristine(VCR) in the back segment elutriant; Or
(2) collect the Changchun floral leaf, dry, pulverize, pour in the extracting cylinder, add the paramount charge level 1-2cm of going out of 80% methyl alcohol, in the extracting cylinder chuck, feed steam, make feed temperature rise to solution boiling, boiling steam through condensing reflux to extracting cylinder, refluxing extraction 3hr like this, after centrifugal extraction liquid, extraction liquid through vacuum decompression concentrate methanol extract, reclaim methyl alcohol simultaneously;
In methanol extract, add 1NH
2SO
4Make pH to 3-4, and stir frequently, centrifugal after leaving standstill, get acid non-soluble substance and acid-soluble material, acid-soluble material extracts vinealeucoblastine(VLB) and vincristine(VCR) according to known method, above-mentioned acid non-soluble substance can through silicagel column absorption, be the 10-6/1-3 wash-out with cyclohexane/ethyl acetate with 5 times of amount chloroform extraction ursolic acid again, after elutriant concentrates, its concentrated solution can in dehydrated alcohol, precipitate white ursolic acid crystallization; Or
(3) Vinca herb or leaf or over-ground part extract with hot ethanol, and alcohol concn 70-95% adds organic acid or mineral acid in the ethanol extract of Vinca, make pH reach 3-4, promptly gets acid solution and acid non-soluble substance; Acid solution adds the benzene extraction after transferring alkali with ammoniacal liquor, go up silica gel column chromatography after the extraction liquid evaporated under reduced pressure, with n-hexane/ethyl acetate elutriant wash-out, monitor with thin-layer chromatography simultaneously, add behind the concentrated evaporate to dryness of elutriant segmentation and contain 4% vitriolic dehydrated alcohol, can from leading portion stream part, separate out vinblastine sulfate, from back segment stream part, separate out vincristine sulphate;
Acid non-soluble substance adds methanol extraction, making silica gel chromatographic column after extraction liquid is evaporated to and does separates, use earlier sherwood oil drip washing, again with containing 2% alcoholic acid sherwood oil wash-out, and make thin-layer chromatography and monitor, collection merges the stream part that contains ursolic acid, concentrates evaporate to dryness post-heating dissolve with ethanol and also places under the low temperature, promptly has the ursolic acid crystallization to separate out.
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| CNB011319879A CN1152034C (en) | 2001-10-24 | 2001-10-24 | Process for extracting active anticancer component from vinca flower |
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| CNB011319879A CN1152034C (en) | 2001-10-24 | 2001-10-24 | Process for extracting active anticancer component from vinca flower |
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| CN1365978A CN1365978A (en) | 2002-08-28 |
| CN1152034C true CN1152034C (en) | 2004-06-02 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100374446C (en) * | 2005-12-19 | 2008-03-12 | 东北林业大学 | A method for separating vindoline, vinblastine and vincristine from herba Catharanthi rosei |
| CN101416988B (en) * | 2008-12-07 | 2011-12-21 | 中国热带农业科学院热带生物技术研究所 | New use of rosebay hornworm which eats madagascar periwinkle leaves and stool extract thereof in preparing anti-tumor medicine |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100336817C (en) * | 2005-07-12 | 2007-09-12 | 东北林业大学 | Method of chromatography preparing high purity catharanthine sulphate by continuous medium pressure column |
| CN100364996C (en) * | 2005-07-13 | 2008-01-30 | 东北林业大学 | Purification method of catharanthine and vincristine |
| CN100526327C (en) * | 2006-09-05 | 2009-08-12 | 中国科学院昆明植物研究所 | Process for producing ursolic acid |
| CN102372731A (en) * | 2011-11-24 | 2012-03-14 | 广州瑞尔医药科技有限公司 | Preparation method for vinblastine |
| CN104031074A (en) * | 2014-06-30 | 2014-09-10 | 倪建平 | Production method of vincristine |
| CN104262362B (en) * | 2014-09-01 | 2017-02-15 | 海南希源化工科技有限公司 | Vinblastine extraction and purification method |
| CN107880064A (en) * | 2017-10-09 | 2018-04-06 | 广州普星药业有限公司 | A kind of method of semi-synthetic vincristine sulphate |
-
2001
- 2001-10-24 CN CNB011319879A patent/CN1152034C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100374446C (en) * | 2005-12-19 | 2008-03-12 | 东北林业大学 | A method for separating vindoline, vinblastine and vincristine from herba Catharanthi rosei |
| CN101416988B (en) * | 2008-12-07 | 2011-12-21 | 中国热带农业科学院热带生物技术研究所 | New use of rosebay hornworm which eats madagascar periwinkle leaves and stool extract thereof in preparing anti-tumor medicine |
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| CN1365978A (en) | 2002-08-28 |
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