CN103044442B - A kind of method of separation and purification GA, GB and bilobalide from Folium Ginkgo extract - Google Patents
A kind of method of separation and purification GA, GB and bilobalide from Folium Ginkgo extract Download PDFInfo
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Abstract
The present invention discloses a kind of method of separation and purification GA, GB and bilobalide from Folium Ginkgo extract, comprises the following steps: 1) extraction into ethyl acetate; 2) low-carbon alcohol-water mixed solvent lixiviate, obtains alcohol leaching extracting solution and insolubles; 3) insolubles silica gel column chromatography is separated to obtain GA and GB; 4) alcohol leaching extracting solution is concentrated into without alcohol, cooling, filters, and carries out crystallization can obtain bilobalide 3 ~ 5 times after mother liquor concentrations is extremely dry with 30 ~ 50% methyl alcohol.The operational path of institute of the present invention separation and purification Ginkgolide A, Ginkgolide B and bilobalide is simple, is applicable to suitability for industrialized production.The purity of separation and purification gained Ginkgolide A, Ginkgolide B and bilobalide is greater than 98%.The rate of transform of Ginkgolide A, Ginkgolide B and bilobalide is greater than 80%, and product yield is high, and production cost is low.
Description
Technical field
The present invention relates to a kind of method of Ginkgolides a and B and bilobalide, be specially a kind of method of separation and purification GA, GB and bilobalide from Folium Ginkgo extract.
Background technology
Folium Ginkgo extract, promoting to have played vital role in the platelet activation of the recycle system, antioxygenation, antisenility function, anti-senile dementia and ophthalmic retina pathology etc., is regarded as the twin spans medicine for the treatment of nervous system disorders and cardiovascular and cerebrovascular diseases.According to incompletely statistics, in August, 2010, State Food and Drug Administration has issued Folium Ginkgo extract medicine and compound formulation produces certification more than 140, and there is family more than 100 in manufacturer.On market, the world today, the annual sales amount of Ginkgo Leaf goods reaches more than 6,000,000,000 dollars, has become global champion's kind of plant amedica, and gingko leaf preparation becomes the leading kind for the treatment of cardiovascular and cerebrovascular diseases.
In Folium Ginkgo extract, diterpenoid-lactone has bilobalide, mainly contains Ginkgolide A. B. C, M, J and bilobalide, is called for short GA, GB, GC, GM, GJ and BB etc.The anti-platelet activating factor (platelet activating factor.PAF) that bilobalide has specificity is active, is natural PAF antagonist, wherein especially the highest with the activity of GB.GB has caused the extensive concern of Inpharm circle, is expected to the kind new medicine becoming the illness such as treatment shock, burn, allergic asthma, apoplexy, transplant rejection, hemodialysis, enters for II and III phase respectively at present clinical in countries such as France, Britain.Bilobalide (BB) is used for the treatment of nervous system disorders, as amyotrophy, flesh contraction, paresis, top layer and depth perception obstacle, headache, dizziness, eyesight, hearing, dysmnesia etc.
The ginkgolide monomer separation purification method of bibliographical information mainly contains extraction using alcohol-extraction into ethyl acetate-column chromatography, extraction using alcohol-extraction into ethyl acetate-reversed phase chromatography separation method, chromatographic separation-recrystallization method, supercritical fluid extraction method etc.
The mandate patent of invention that GA, GB and BB separation purification method is directly related or apply for a patent mainly contains as follows:
(1) Chinese patent (ZL00117758.3) " prepares medicine Ginkgolide A by Ginkgo Leaf or Ginkgo Leaf medicinal extract, the method of B ", this invention relates to one and prepares medicine Ginkgolide A by Ginkgo Leaf or Ginkgo Leaf medicinal extract, the method of B, it is for raw material with Ginkgo Leaf or Ginkgo Leaf medicinal extract, through adding ethanol, or methyl alcohol, or acetone, or butanone, or ethyl acetate organic solvent soaking extraction, leaching liquid after filtration, stratification, the organic phase obtained carries out decon process, the organic phase warp that stratification obtains again concentrates, drying obtains the enriched material of enrichment Ginkgolide A and B, preparative liquid chromatography is adopted to be separated aftertreatment to containing Ginkgolide A with the enriched material of B, obtain GA and GB, it is characterized in that: while adding leaching agent, add muriate salting-out agent, decon process adds sylvite or sodium salt to the organic layer liquid of leaching liquid, the moving phase of the separator column of preparative liquid chromatography is the mixed solution adopting ethyl acetate and sherwood oil, and stationary phase adopts silica gel.The method preparative scale chromatography is separated GA and GB, and production cost is high, and output is little, is not suitable for the shortcomings such as the large production of mass-producing.
(2) Chinese patent (application number: 200710050245) method of extraction and isolation Ginkgolide B " from the Ginkgo Leaf ", this invention relates to the method for extraction and isolation Ginkgolide B from Ginkgo Leaf, its step is as follows: 1) get Ginkgo Leaf, extract with the ethanol of different concns from high to low, united extraction liquid, reclaim ethanol, obtain the extracting solution after concentrating; 2) in the extracting solution after concentrated, add extraction into ethyl acetate, after reclaiming organic phase, be concentrated into medicinal extract; 3) by step 2) the rear upper selective polarity atresia adsorption resin column of gained medicinal extract dilution, with ethanol elution, after gained elutriant reclaims ethanol, then obtain ginkgolide compound with alcohol crystal; 4) by silicagel column in ginkgolide compound crystallization, with the mixed solution wash-out of normal hexane and ethyl acetate, collect elutriant, obtain the elutriant of enrichment Ginkgolide B; 5) recycling step 4) in solvent in the elutriant collected, then use alcohol crystal, obtain Ginkgolide B.The method steps such as operational path alcohol extracting-extraction-resin purification-crystallization-silicagel column separation-crystallization, operational path is complicated, and only separation obtains GB.
(3) Chinese patent (application number: 201010608847.X) " a kind of method of separating bilobalide B from bilobalide mixture ", this disclosure of the invention a kind of method of separating bilobalide B from bilobalide mixture, the method for extraction agent, adopts fractional-distillation extraction separating bilobalide B from the mixture containing bilobalide homologues such as Ginkgolide A, Ginkgolide B, ginkalide Cs efficiently with ionic liquid or the binary mixed solvent that is made up of ionic liquid water.The method is with bilobalide mixture for raw material, adopts fractional-distillation extraction separating bilobalide B.
(4) Chinese patent (application number: 200410061284.1) " extracting method of Ginkgolides in Ginkgo biloba L. Leaves B and bilobalide ", this disclosure of the invention extracting method of a kind of Ginkgolides in Ginkgo biloba L. Leaves B and bilobalide.By Ginkgo Leaf oven dry, impurity elimination, pulverizing, extract with aqueous ethanolic solution, upper adsorption column decon, is then extracted with ethyl acetate out bilobalide, upper alumina column purifying, and fractional crystallization and recrystallization obtain purer Ginkgolide B and bilobalide.
(5) Chinese patent (application number: 200610014207.X) " preparation method of bilobalide ", a kind of preparation method of bilobalide of this disclosure of the invention, comprise the steps: that (1) gets ginkgo biloba crude extract in order, pulverize, add vinyl acetic monomer and stir extraction, filter, filtrate recovery of acetic acid ethyl ester is to most, drying, obtains bilobalide crude extract; (2) get bilobalide crude extract, pulverize, fully dissolve, acidic alumina column on solution with ethanol, collect effluent liquid, then use ethanol elution, collect elutriant, merge effluent liquid and elutriant, concentrating under reduced pressure, crystallization obtains bilobalide mixed crystal; (3) by a small amount of washing with alcohol of bilobalide mixed crystal, filter, then recrystallization, obtain bilobalide mixed crystal.The operational path of the inventive method is reasonable, and finished product pick-up rate is high, can not destroy effective constituent, can obtain highly purified Ginkgolide A and Ginkgolide B mixed crystal.
(6) Chinese patent (application number: 200710106040.4) " a kind of preparation method extracting Ginkgolide B from Ginkgo Leaf or Folium Ginkgo extract ", this invention relates to the preparation method extracting Ginkgolide B from Ginkgo Leaf or Folium Ginkgo extract, comprise the following steps: one, the selection of raw material and process: (1), ginkgo biloba extract (total flavones 24%, total lactones 6%, 15-25 mother-in-law U.S. degree); (2), Semen Ginkgo extrac (powder: total flavones 24%, total lactones 6%), after appropriate (1-2 times of volume) dissolved in purified water, stand-by; (3), fresh, dry ginkgo greenery, dry, temperature controls at 70 DEG C, impurity elimination, pulverizing, crosses 60 mesh sieves, with 60% extraction using alcohol 2 times, each 60-90 minute, united extraction liquid, concentrating under reduced pressure, recovery ethanol; Concentrated solution adds the water of 2 times, precipitates 24 hours, centrifugal, gets HP-20 adsorption column on supernatant liquor, first rinses with water, then uses 80% ethanol elution, collect elutriant, concentrated.Two, high-speed counter-current extraction: 1) ginkgo biloba extract or the extracting solution after concentrating are imported in extraction tower, at the bottom of tower, evenly add ethyl acetate carry out convection current solvent extraction, ethyl acetate consumption is generally 1.5 ~ 2.5 times of liquid, and cycle index is 4 ~ 5 times, different tower body, separate collection; 2) ethyl acetate after extraction is reclaimed, obtain thick paste, be concentrated into 20-25 mother-in-law U.S. degree, 65-70 DEG C of vacuum-drying, be crushed to 90-100 order, obtain bilobalide product; Three, column chromatography, recrystallization: the bilobalide 1) will obtained, after 75-85% dissolve with ethanol, by 80-120 object polyamide column, collects effluent liquid; 2) be the macroporous resin column of HP-20 by model again by effluent liquid, then, be colourless with washed with de-ionized water resin to flowing liquid, be the ethanolic soln wash-out of 75-85% again by concentration, when effluent liquid color is close to time colourless, stop wash-out, collect elutriant, be concentrated into the medicinal extract of 20-25 mother-in-law U.S. degree; 3) medicinal extract is placed in less than 4 DEG C environment refrigerations, after leaving standstill, has crystal to separate out gradually, after 3 hours, collect crystallization, suction filtration, then use the crystallization of 75-85% dissolve with ethanol, recrystallization, after twice, recrystallization, by white precipitate vacuum drying to constant weight, obtain Ginkgolide B product, content can reach more than 95%.
(7) Chinese patent (application number: 200710050242.1) method of extraction and isolation Ginkgolide A. B. C, J and bilobalide monomer " from the Ginkgo Leaf ", this invention is by the method for openly a kind of extraction and isolation Ginkgolide A. B. C, J and bilobalide monomer from Ginkgo Leaf, the steps include: 1) get Ginkgo Leaf, extract with the ethanol of different concns from high to low, united extraction liquid, reclaim ethanol, obtain the extracting solution after concentrating; 2) in the extracting solution after concentrated, add extraction into ethyl acetate, after reclaiming organic phase, be concentrated into medicinal extract; 3) by selective polarity atresia adsorption resin column upper after medicinal extract dilution, with ethanol elution, gained elutriant obtains ginkgolide compound with alcohol crystal after reclaiming ethanol; 4) by silicagel column in ginkgolide compound crystallization, with the mixed solution wash-out of normal hexane and ethyl acetate, Fractional Collections elutriant, obtains bilobalide, Ginkgolide A, Ginkgolide B, ginkalide C and bilobalide J successively respectively; 5) reclaim the solvent in the elutriant collected, then obtain bilobalide, Ginkgolide A, Ginkgolide B, ginkalide C and bilobalide J monomer respectively with alcohol crystal.
(8) Chinese patent (application number: 200810024206.2) " a kind of bilobalide B raw material and preparation method thereof ", a kind of bilobalide B raw material of this disclosure of the invention, it is characterized in that this bilobalide B raw material prepares by the following method: get Ginkgo Leaf or Folium Ginkgo extract, digestion in hydrochloric acid soln, digestion liquid adopts organic solvent extraction after filtering, 100 ~ 200 order silica gel combination dryings are added after the extraction liquid obtained is concentrated, adopt silica gel column chromatography to collect the elutriant being rich in Ginkgolide B, obtain bilobalide B raw material through crystallization; Wherein, Ginkgolide B content >=90% in bilobalide B raw material.
(9) Chinese patent (application number: 200910184916.6) " a kind of separation purification method of Ginkgolides a and B monomer ", this invention for starting raw material, comprises Crystallization Separation, purifying and drying with the Ginkgolides of purity >=95%.Described purifying carries out purifying with recrystallization-SCF-CO 2 combination process, monomer GA and GB first Crystallization Separation obtained respectively with dehydrated alcohol by the solid-liquid ratio recrystallization at least three times of 1: 5 ~ 80, obtain GA and GB of purity >=95% monomer; Then extract respectively with monomer GA and GB of SCF-CO 2 method to purity >=95%, extracting pressure 10 ~ 100Mpa, extraction temperature 32 ~ 150 DEG C.Re-extract once, can prepare monomer GA and GB of purity >=99.5%.With monomer gauge in starting raw material, yield >=90%.
Summary of the invention
The defect that the present invention seeks to exist for prior art provides a kind of method of separation and purification GA, GB and bilobalide in Folium Ginkgo extract.
The present invention for achieving the above object, adopts following technical scheme:
A kind of method of the present invention separation and purification GA, GB and bilobalide from Folium Ginkgo extract, comprises the following steps:
1) extraction into ethyl acetate: take Folium Ginkgo extract, add 20 ~ 50 DEG C, the water stirring of Folium Ginkgo extract 5-10 times amount, then extraction into ethyl acetate 3-5 time of Folium Ginkgo extract 5-10 times amount is added, combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, gained acetic acid ethyl ester extract carry out step 2), wherein times amount refers to weight/volume, lower same;
2) low-carbon alcohol-water mixed solvent lixiviate: take acetic acid ethyl ester extract, low-carbon alcohol-the water mixed solvent 20 ~ 50 DEG C adding acetic acid ethyl ester extract 10-20 times amount carries out lixiviate, filter, gained insolubles (II) carries out step 3), and gained alcohol leaching extracting solution (I) carries out step 4);
3) silica gel column chromatography separating purification: by upper for insolubles (II) silicagel column, carry out wash-out with the mixed solvent of sherwood oil or normal hexane or hexanaphthene and ethyl acetate, Fractional Collections elutriant, reclaim the solvent in elutriant, obtain GA and GB respectively;
4) bilobalide crystallization purifying: be concentrated into by alcohol leaching extracting solution (I) and obtain mother liquor without after alcohol, cooling, filtration, carries out crystallization with 30 ~ 50% methyl alcohol after mother liquor concentrations is extremely dry and obtains bilobalide 3 ~ 5 times.
Also comprise step 5): use low-carbon alcohol-water mixed solvent to carry out recrystallization purifying process respectively to Ginkgolide A and Ginkgolide B.
Step 2) low-carbon alcohol is selected from methyl alcohol or ethanol.
Step 2) in low-carbon alcohol-water mixed solvent, low-carbon alcohol content is 30 ~ 70%.
Step 3) boiling range of sherwood oil is 60 ~ 90 DEG C.
Step 3) sherwood oil or normal hexane or hexanaphthene and ethyl acetate mixed solvent in, the volume ratio of sherwood oil or normal hexane or hexanaphthene and ethyl acetate is 2: 8-4: 6.
Step 5) low-carbon alcohol is selected from methyl alcohol or ethanol.
Step 5) in low-carbon alcohol-water mixed solvent, low-carbon alcohol content is 30 ~ 70%.
The present invention has following beneficial effect:
1. the operational path of institute of the present invention separation and purification Ginkgolide A, Ginkgolide B and bilobalide is simple, is applicable to suitability for industrialized production.
2. separation and purification gained Ginkgolide A of the present invention, Ginkgolide B and bilobalide purity be greater than 98%.
3. the rate of transform of Ginkgolide A, Ginkgolide B and bilobalide is greater than 80%, and product yield is high, and production cost is low.
Embodiment
The present invention is from Folium Ginkgo extract (flavones content > 24%; Lactone content > 6%) middle separation and purification Ginkgolide A, Ginkgolide B and bilobalide, the purity > 98% of gained Ginkgolide A, Ginkgolide B and bilobalide.
Ginkgolide monomer separation purification method mainly contains the methods such as extraction into ethyl acetate, macroporous resin partition method, aluminum oxide column chromatography post, polyamide column chromatography method, silica gel column chromatography partition method, reversed phase chromatography separation method, recrystallization method, supercritical fluid extraction method.The present inventor accumulates the research of bilobalide separation and purification for many years, finds can obtain high purity bilobalide A and Ginkgolide B by extraction into ethyl acetate, alcohol lixiviate, silica gel column chromatography, and alcohol vat liquor can obtain bilobalide through crystallization and recrystallization method.Concrete technical scheme of the present invention is:
With Folium Ginkgo extract be raw material by following processing step separation and purification GA, GB and bilobalide: 1) extraction into ethyl acetate; 2) low-carbon alcohol-water mixed solvent lixiviate, obtains alcohol vat liquor (I) and insolubles (II); 3) insolubles (II) silica gel column chromatography is separated to obtain GA and GB; 4) alcohol vat liquor (I) is concentrated into without alcohol, cooling, filters, and carry out crystallization with 30 ~ 50% methyl alcohol after mother liquor concentrations is extremely dry and can obtain bilobalide 3 ~ 5 times, its concrete technology step is as follows:
1) extraction into ethyl acetate: take Folium Ginkgo extract, add the stirring of 20 ~ 50 DEG C, 5-10 times amount (weight/volume) water, then 5-10 times amount (weight/volume) extraction into ethyl acetate is added, extract 3-5 time, combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, and gained acetic acid ethyl ester extract carry out step 2);
2) low-carbon alcohol-water mixed solvent lixiviate: take acetic acid ethyl ester extract, adds 10-20 times amount low-carbon alcohol-water mixed solvent 20 ~ 50 DEG C and carries out lixiviate, and filter, gained insolubles carries out step 3), and gained alcohol leaching extracting solution carries out step 4);
3) silica gel column chromatography separating purification: by upper for insolubles (II) silicagel column, carry out wash-out with the mixed solvent of sherwood oil or normal hexane or hexanaphthene and ethyl acetate, Fractional Collections elutriant, reclaim the solvent in elutriant, obtain GA and GB respectively.
4) bilobalide crystallization purifying: be concentrated into without alcohol, cooling by alcohol vat liquor (I), filters, and carries out crystallization obtain bilobalide 3 ~ 5 times after mother liquor concentrations is extremely dry with 30 ~ 50% methyl alcohol,
Wherein step 2) low-carbon alcohol is selected from methyl alcohol or ethanol.
Wherein step 2) in low-carbon alcohol-water mixed solvent, low-carbon alcohol content is 30 ~ 70%.
Wherein step 3) boiling range of sherwood oil is 60 ~ 90 DEG C.
Wherein step 3) sherwood oil or normal hexane or hexanaphthene and ethyl acetate mixed solvent in, the volume ratio of sherwood oil or normal hexane or hexanaphthene and ethyl acetate is 2: 8-4: 6.
In one embodiment, also step 5 is comprised): use low-carbon alcohol-water mixed solvent to carry out recrystallization purifying process respectively to Ginkgolide A and Ginkgolide B.
Wherein step 5) low-carbon alcohol is selected from methyl alcohol or ethanol.
Wherein step 5) in low-carbon alcohol-water mixed solvent, low-carbon alcohol content is 30 ~ 70%.
Embodiment 1:
Get Folium Ginkgo extract 100g(GA content 2.1%, GB content 1.2%, BB2.5%), add 50 DEG C, 500mL water and stir 30min, then add 500mL extraction into ethyl acetate, extract 5 times, combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, obtains acetic acid ethyl ester extract 15.5g; Add 155mL70% ethanol 50 DEG C of lixiviates 1 hour, filter, filtration cakes torrefaction obtains insolubles 4.4g and alcohol vat liquor 150mL; By insolubles with using 5g100-200 order silica gel mixed sample after 20mL acetic acid ethyl dissolution, then be separated with 200g200-300 object silica gel column chromatography, wash-out is carried out with the mixed solvent 8: 2 of petroleum ether-ethyl acetate, Fractional Collections elutriant, reclaim the solvent in elutriant, obtain GA1.8g(purity 98.2% respectively), GB1.0g(purity 98.5%).Alcohol vat liquor 150mL60 DEG C is evaporated to 40mL, is cooled to 25 DEG C, filters, 70 DEG C, mother liquor be evaporated to dry after, with 30% methyl alcohol periodic crystallisation 5 times, obtain bilobalide 2.3g(purity 98.2%).
Embodiment 2:
Get Folium Ginkgo extract 100g(GA content 2.1%, GB content 1.2%, BB2.5%), add 30 DEG C, 700mL water and stir 30min, then add 1000mL extraction into ethyl acetate, extract 3 times, combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, obtains acetic acid ethyl ester extract 16.5g; Add 330mL70% ethanol 20 DEG C of lixiviates 1 hour, filter, filtration cakes torrefaction obtains insolubles 4.6g and alcohol vat liquor 310mL; By insolubles with using 5g100-200 order silica gel mixed sample after 20mL acetic acid ethyl dissolution, then be separated with 200g200-300 object silica gel column chromatography, wash-out is carried out with the mixed solvent 3: 7 of petroleum ether-ethyl acetate, Fractional Collections elutriant, reclaim the solvent in elutriant, obtain GA1.7g(purity 98.1% respectively), GB0.98g(purity 98.3%).Alcohol vat liquor 310mL60 DEG C is evaporated to 100mL, is cooled to 25 DEG C, filters, 70 DEG C, mother liquor be evaporated to dry after, with 40% methyl alcohol periodic crystallisation 4 times, obtain bilobalide 2.2g(purity 98.4%).
Embodiment 3:
Get Folium Ginkgo extract 100g(GA content 2.1%, GB content 1.2%, BB2.5%), add 20 DEG C, 1000mL water and stir 30min, then add 1000mL extraction into ethyl acetate, extract 4 times, combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, obtains acetic acid ethyl ester extract 16.0g; Add 320mL30% ethanol 50 DEG C of lixiviates 1 hour, filter, filtration cakes torrefaction obtains insolubles 4.8g and alcohol vat liquor 300mL; By insolubles with using 5g100-200 order silica gel mixed sample after 20mL acetic acid ethyl dissolution, then be separated with 200g200-300 object silica gel column chromatography, wash-out is carried out with the mixed solvent 4: 6 of petroleum ether-ethyl acetate, Fractional Collections elutriant, reclaim the solvent in elutriant, obtain GA1.75g(purity 98.0% respectively), GB1.05g(purity 98.1%).Alcohol vat liquor 300mL60 DEG C is evaporated to 70mL, is cooled to 25 DEG C, filters, 70 DEG C, mother liquor be evaporated to dry after, with 40% methyl alcohol periodic crystallisation 5 times, obtain bilobalide 2.2g(purity 98.1%).
Embodiment 4:
Get Folium Ginkgo extract 100g(GA content 2.1%, GB content 1.2%, BB2.5%), add 50 DEG C, 500mL water and stir 30min, then add 500mL extraction into ethyl acetate, extract 5 times, combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, obtains acetic acid ethyl ester extract 15.5g; Add 155mL70% methyl alcohol 50 DEG C of lixiviates 1 hour, filter, filtration cakes torrefaction obtains insolubles 4.4g and alcohol vat liquor 150mL; By insolubles with using 5g100-200 order silica gel mixed sample after 20mL acetic acid ethyl dissolution, then be separated with 200g200-300 object silica gel column chromatography, wash-out is carried out with the mixed solvent 8: 2 of n-hexane-ethyl acetate, Fractional Collections elutriant, reclaim the solvent in elutriant, obtain GA1.83g(purity 98.4% respectively), GB1.02g(purity 98.1%).Alcohol vat liquor 150mL60 DEG C is evaporated to 45mL, is cooled to 25 DEG C, filters, 70 DEG C, mother liquor be evaporated to dry after, with 40% methyl alcohol periodic crystallisation 4 times, obtain bilobalide 2.3g(purity 98.0%).
Embodiment 5:
Get Folium Ginkgo extract 100g(GA content 2.1%, GB content 1.2%, BB2.5%), add 30 DEG C, 700mL water and stir 30min, then add 1000mL extraction into ethyl acetate, extract 3 times, combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, obtains acetic acid ethyl ester extract 16.5g; Add 330mL70% methyl alcohol 20 DEG C of lixiviates 1 hour, filter, filtration cakes torrefaction obtains insolubles 4.6g and alcohol vat liquor 320mL; By insolubles with using 5g100-200 order silica gel mixed sample after 20mL acetic acid ethyl dissolution, then be separated with 200g200-300 object silica gel column chromatography, wash-out is carried out with the mixed solvent 4: 6 of cyclohexane-ethyl acetate, Fractional Collections elutriant, reclaim the solvent in elutriant, obtain GA1.78g(purity 98.6% respectively), GB0.95g(purity 98.6%).Alcohol vat liquor 320mL60 DEG C is evaporated to 85mL, is cooled to 25 DEG C, filters, 70 DEG C, mother liquor be evaporated to dry after, with 30% methyl alcohol periodic crystallisation 4 times, obtain bilobalide 2.2g(purity 98.4%).
Embodiment 6:
Get Folium Ginkgo extract 100g(GA content 2.1%, GB content 1.2%, BB2.5%), add 30 DEG C, 1000mL water and stir 30min, then add 1000mL extraction into ethyl acetate, extract 5 times, combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, obtains acetic acid ethyl ester extract 16.0g; Add 320mL30% methyl alcohol 50 DEG C of lixiviates 1 hour, filter, filtration cakes torrefaction obtains insolubles 4.8g and alcohol vat liquor 300mL; By insolubles with using 5g100-200 order silica gel mixed sample after 20mL acetic acid ethyl dissolution, then be separated with 200g200-300 object silica gel column chromatography, wash-out is carried out with the mixed solvent 4: 6 of n-hexane-ethyl acetate, Fractional Collections elutriant, reclaim the solvent in elutriant, obtain GA1.82g(purity 98.3% respectively), GB0.95g(purity 98.4%).Alcohol vat liquor 300mL60 DEG C is evaporated to 80mL, is cooled to 25 DEG C, filters, 70 DEG C, mother liquor be evaporated to dry after, with 50% methyl alcohol periodic crystallisation 3 times, obtain bilobalide 2.1g(purity 98.4%).
Embodiment 7:
Get Folium Ginkgo extract 1000g(GA content 2.1%, GB content 1.2%, BB2.5%), add 30 DEG C, 60000mL water and stir 30min, then add 60000mL extraction into ethyl acetate, extract 3 times, combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, obtains acetic acid ethyl ester extract 156.0g; Add 3000mL50% methyl alcohol 30 DEG C of lixiviates 1 hour, filter, filtration cakes torrefaction obtains insolubles 51.3g and alcohol vat liquor 2850mL; By insolubles with using 60g100-200 order silica gel mixed sample after 200mL acetic acid ethyl dissolution, then be separated with 3000g200-300 object silica gel column chromatography, wash-out is carried out with the mixed solvent 2: 8 of n-hexane-ethyl acetate, Fractional Collections elutriant, reclaim the solvent in elutriant, obtain GA and GB respectively.The GA further crystallization of 50% methyl alcohol obtains crystal 18.5g(purity 99.2%), the GB further crystallization of 30% ethanol obtains crystal 9.5g(purity 99.3%).Alcohol vat liquor 2850mL60 DEG C is evaporated to 1000mL, is cooled to 25 DEG C, filters, 70 DEG C, mother liquor be evaporated to dry after, with 50% methyl alcohol periodic crystallisation 3 times, obtain bilobalide 22.3g(purity 98.5%).
Embodiment 8:
Get Folium Ginkgo extract 1000g(GA content 2.1%, GB content 1.2%, BB2.5%), add 30 DEG C, 60000mL water and stir 30min, then add 60000mL extraction into ethyl acetate, extract 3 times, combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, obtains acetic acid ethyl ester extract 146.0g; Add 2500mL50% ethanol 30 DEG C of lixiviates 1 hour, filter, filtration cakes torrefaction obtains insolubles 48.5g and alcohol vat liquor 2400mL; By insolubles with using 60g100-200 order silica gel mixed sample after 200mL acetic acid ethyl dissolution, then be separated with 3000g200-300 object silica gel column chromatography, wash-out is carried out with the mixed solvent 2: 8 of petroleum ether-ethyl acetate, Fractional Collections elutriant, reclaim the solvent in elutriant, obtain GA and GB respectively.The GA further crystallization of 50% ethanol obtains crystal 18.0g(purity 99.5%), the GB further crystallization of 50% methyl alcohol obtains crystal 9.8g(purity 99.4%).Alcohol vat liquor 2400mL60 DEG C is evaporated to 800mL, is cooled to 25 DEG C, filters, 70 DEG C, mother liquor be evaporated to dry after, with 30% methyl alcohol periodic crystallisation 3 times, obtain bilobalide 23.3g(purity 98.1%).
As previously mentioned, according to content disclosed above, one of skill in the art can apply the present invention to greatest extent.It should be pointed out that the present invention aims to provide a kind of method of separation and purification Ginkgolide A, Ginkgolide B and bilobalide from Folium Ginkgo extract.The following examples can help the present invention of those skilled in the art's comprehend, but do not limit the present invention in any way.
Claims (4)
1. the method for separation and purification Ginkgolide A, Ginkgolide B and bilobalide from Folium Ginkgo extract, is characterized in that comprising the following steps:
1) extraction into ethyl acetate: take Folium Ginkgo extract, add 20 ~ 50 DEG C, the water stirring of Folium Ginkgo extract 5 ~ 10 times amount, then the extraction into ethyl acetate 3 ~ 5 times of Folium Ginkgo extract 5 ~ 10 times amount is added, combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, gained acetic acid ethyl ester extract carry out step 2), wherein times amount refers to weight/volume, lower same;
2) the mixed solvent lixiviate of methanol-water or alcohol-water: take acetic acid ethyl ester extract, the methanol-water or the ethanol-water mixed solvent 20 ~ 50 DEG C that add acetic acid ethyl ester extract 10 ~ 20 times amount carry out lixiviate, filter, gained insolubles (II) carry out step 3), gained alcohol leaching extracting solution (I) carry out step 4), wherein in methanol-water or ethanol-water mixed solvent, the content of methyl alcohol or ethanol is respectively 30% ~ 70%;
3) silica gel column chromatography separating purification: by upper for insolubles (II) silicagel column, wash-out is carried out with the mixed solvent of sherwood oil or normal hexane or hexanaphthene and ethyl acetate, Fractional Collections elutriant, reclaims the solvent in elutriant, obtains Ginkgolide A and Ginkgolide B respectively;
4) bilobalide crystallization purifying: be concentrated into by alcohol leaching extracting solution (I) and obtain mother liquor without after alcohol, cooling, filtration, carries out crystallization with 30 ~ 50% methyl alcohol after mother liquor concentrations is extremely dry and obtains bilobalide 3 ~ 5 times.
2. method according to claim 1, it is characterized in that, also comprise step 5): use the mixed solvent of methanol-water or alcohol-water to carry out recrystallization purifying process respectively to Ginkgolide A and Ginkgolide B, wherein in methanol-water or ethanol-water mixed solvent, the content of methyl alcohol or ethanol is 30%-70%.
3. method according to claim 1 and 2, is characterized in that, wherein step 3) the boiling range 60-90 DEG C of sherwood oil.
4. method according to claim 1 and 2, is characterized in that, wherein step 3) in the mixed solvent of sherwood oil or normal hexane or hexanaphthene and ethyl acetate, the volume ratio of sherwood oil or normal hexane or hexanaphthene and ethyl acetate is 2: 8-4: 6.
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| CN103735543A (en) * | 2013-12-26 | 2014-04-23 | 哈尔滨誉衡药业股份有限公司 | Traditional Chinese medicine composition |
| CN104693214A (en) * | 2014-12-22 | 2015-06-10 | 徐州技源药业有限公司 | Separation and purification method for bilobalide monomers in ginkgo leaf extract |
| CN105061458B (en) * | 2015-09-01 | 2017-05-17 | 合肥工业大学 | Preparation method of nontoxic high-purity ginkgo total lactones and monomers thereof |
| CN113801133B (en) * | 2021-10-28 | 2022-08-19 | 江苏得乐康生物科技有限公司 | Preparation method of ginkgolide and monomer contained in ginkgolide |
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