CN103044442A - Method for separating and purifying GA, GB and bilobalide from folium ginkgo extractive - Google Patents
Method for separating and purifying GA, GB and bilobalide from folium ginkgo extractive Download PDFInfo
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Abstract
The invention discloses a method for separating and purifying GA (Ginkgolide A), GB (Ginkgolide B) and bilobalide from a folium ginkg extractive. The method comprises the following steps that 1), ethyl acetate is adopted for extraction; 2), a lower alcohol-water mixed solvent is adopted for leaching; an alcohol leaching extraction solution and an undissolved substance are obtained; 3), the undissolved substance is subjected to silica gel column chromatography separation to form GA and GB; and 4), the alcohol leaching extraction solution is concentrated till no alcohol exists, cooled and filtered; a mother solution is concentrated to dryness, and then subjected to crystallization with 30-35% of methanol for 3-5 times; and bilobalide is obtained. The method for separating and purifying GA, GB and bilobalide is simple in process route and is suitable for industrial production. The purity of GA, GB and bilobalide obtained by separation and purification is greater than 98%; transfer rates of GA, GB and bilobalide are greater than 80%; the yield is high; the production cost is low.
Description
Technical field
The present invention relates to the method for a kind of Ginkgolides a and B and bilobalide, be specially a kind of from Folium Ginkgo extract the method for separation and purification GA, GB and bilobalide.
Background technology
Folium Ginkgo extract has been brought into play vital role at aspects such as promoting the platelet activation of the recycle system, antioxygenation, antisenility function, anti-ageing year dementia and eye retinopathy, is regarded as treating the twin spans medicine of nervous system disorders and cardiovascular and cerebrovascular diseases.According to incompletely statistics, in August, 2010, State Food and Drug Administration has issued the Folium Ginkgo extract medicine and compound formulation is produced more than 140 of certifications, and there is family more than 100 in manufacturer.The annual sales amount of Ginkgo Leaf goods reaches more than 6,000,000,000 dollars on the market, the world today, 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.
Diterpenoid-lactone has bilobalide in the Folium Ginkgo extract, mainly contains Ginkgolide A. B. C, M, J and bilobalide, is called for short GA, GB, GC, GM, GJ and BB etc.Bilobalide has anti-platelet activating factor (the platelet activating factor.PAF) activity of specificity, is natural PAF antagonist, and wherein the activity with GB is the highest especially.GB has caused the extensive concern of Inpharm circle, is expected to become a kind new medicine of the illnesss such as treatment shock, burn, allergic asthma, apoplexy, transplant rejection, hemodialysis, at present the countries such as France, Britain entered respectively II and the III phase clinical.Bilobalide (BB) is used for the treatment of nervous system disorders, such 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-ethyl acetate extraction-column chromatography, extraction using alcohol-ethyl acetate extraction-reversed phase chromatography separation method, chromatographic separation-recrystallization method, supercritical liq extraction process etc.
It is as follows that the mandate patent of invention that GA, GB and BB separation purification method are directly related or apply for a patent mainly contains:
(1) Chinese patent (ZL00117758.3) " prepares the medicine Ginkgolide A by Ginkgo Leaf or Ginkgo Leaf medicinal extract; the method for B ", this invention relates to and a kind ofly prepares the medicine Ginkgolide A by Ginkgo Leaf or Ginkgo Leaf medicinal extract, the method of B, it is as raw material take 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, standing demix, the organic phase that obtains is carried out decon and is processed, the organic phase that obtains of standing demix is through concentrated again, drying obtains the enriched material of enrichment Ginkgolide A and B, adopt preparative liquid chromatography that the enriched material that contains Ginkgolide A and B is separated aftertreatment, obtain GA and GB, it is characterized in that: when adding leaching agent, add the muriate agent of saltouing; The decon process is organic layer liquid adding sylvite or the sodium salt to leaching liquid; The moving phase of the separator column of preparative liquid chromatography is to adopt the mixed solution of ethyl acetate and sherwood oil, and stationary phase adopts silica gel.The method is separated GA and GB with preparative scale chromatography, 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) " from Ginkgo Leaf, extract the method for separating bilobalide B ", this invention relates to the method for extracting separating bilobalide 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, Recycled ethanol, the extracting solution after obtaining concentrating; 2) add ethyl acetate extraction in the extracting solution after concentrated, be concentrated into medicinal extract after reclaiming organic phase; 3) with step 2) the rear upper selective polarity atresia adsorption resin column of gained medicinal extract dilution, use ethanol elution, behind the gained elutriant Recycled ethanol, obtain ginkgolide compound with alcohol crystal again; 4) with silicagel column in the 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, use again alcohol crystal, obtain Ginkgolide B.The method operational path alcohol extracting-extraction-resin purification-crystallization-steps such as silicagel column separation-crystallization, operational path is complicated, and only separation obtains GB.
(3) Chinese patent (application number: 201010608847.X) " a kind of from the bilobalide mixture separating bilobalide B method ", this disclosure of the Invention a kind of from the bilobalide mixture method of separating bilobalide B, the binary mixed solvent that the method forms take ionic liquid or by ionic liquid water adopts efficiently separating bilobalide B from the mixture that contains the bilobalide homologues such as Ginkgolide A, Ginkgolide B, ginkalide C of fractional-distillation extraction as extraction agent.The method is take the bilobalide mixture as 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 the extracting method of a kind of Ginkgolides in Ginkgo biloba L. Leaves B and bilobalide.With Ginkgo Leaf oven dry, impurity elimination, pulverizing, extract with aqueous ethanolic solution, then upper adsorption column decon goes out bilobalide with ethyl acetate extraction, upper alumina column purifying, fractional crystallization and recrystallization obtain purer Ginkgolide B and bilobalide.
(5) Chinese patent (application number: 200610014207.X) " preparation method of bilobalide ", this disclosure of the Invention a kind of preparation method of bilobalide, comprise the steps: that in order (1) get ginkgo biloba crude extract, pulverize, add vinyl acetic monomer and stir extraction, filter, filtrate recovery of acetic acid ethyl ester is to most, drying gets the bilobalide crude extract; (2) get the bilobalide crude extract, pulverize, fully dissolve with ethanol, acidic alumina column on the solution is collected effluent liquid, uses ethanol elution again, collects elutriant, merges effluent liquid and elutriant, concentrating under reduced pressure, and crystallization gets the bilobalide mixed crystal; (3) with a small amount of washing with alcohol of bilobalide mixed crystal, filter, recrystallization namely gets the bilobalide mixed crystal again.The operational path of the inventive method is reasonable, and the 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 who from Ginkgo Leaf or Folium Ginkgo extract, extracts Ginkgolide B ", this invention relates to the preparation method who extracts Ginkgolide B from Ginkgo Leaf or Folium Ginkgo extract, comprise the following steps: one, selection and the processing of raw material: (1), ginkgo biloba extract (total flavones 24%, total lactone 6%, 15-25 mother-in-law U.S. degree); (2), Semen Ginkgo extrac (powder: total flavones 24%, total lactone 6%), after an amount of (1-2 times of volume) dissolved in purified water, stand-by; (3), fresh, dry ginkgo greenery, oven dry, temperature is controlled at 70 ℃, 60 mesh sieves are crossed in impurity elimination, pulverizing, with 60% extraction using alcohol 2 times, 60-90 minute at every turn, united extraction liquid, concentrating under reduced pressure, Recycled ethanol; Concentrated solution adds 2 times water, precipitates 24 hours, and is centrifugal, gets HP-20 adsorption column on the supernatant liquor, and then first water flushing uses 80% ethanol elution, collects elutriant, concentrated.Two, high-speed counter-current extraction: 1) in the extracting solution importing extraction tower with ginkgo biloba extract or after concentrating, evenly add ethyl acetate at the bottom of the tower and carry out the convection current solvent extraction, the ethyl acetate consumption is generally 1.5～2.5 times of liquid, and cycle index is 4～5 times, different tower bodies, separate collection; 2) ethyl acetate after will extracting reclaims, and obtains thick paste, is concentrated into 20-25 mother-in-law U.S. degree, 65-70 ℃ of vacuum-drying, is crushed to the 90-100 order, obtains the bilobalide product; Three, column chromatography, recrystallization: 1) with the bilobalide that obtains, behind the 75-85% dissolve with ethanol, by 80-120 purpose polyamide column, collect effluent liquid; 2) be the macroporous resin column of HP-20 again by model with 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 with concentration, when the effluent liquid color when 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 the refrigeration of environment below 4 ℃, after leaving standstill, have gradually crystal to separate out, after 3 hours, collect crystallization, suction filtration is used the crystallization of 75-85% dissolve with ethanol, recrystallization again, behind twice in the recrystallization, the white precipitate vacuum drying to constant weight, is got the Ginkgolide B product, content can reach more than 95%.
(7) Chinese patent (application number: 200710050242.1) " from Ginkgo Leaf, extract the method for separating bilobalide A, B, C, J and bilobalide monomer ", this invention will disclose a kind of method of extracting separating bilobalide 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, Recycled ethanol, the extracting solution after obtaining concentrating; 2) add ethyl acetate extraction in the extracting solution after concentrated, be concentrated into medicinal extract after reclaiming organic phase; 3) with upper selective polarity atresia adsorption resin column after the medicinal extract dilution, use ethanol elution, behind the gained elutriant Recycled ethanol, get ginkgolide compound with alcohol crystal; 4) with silicagel column in the ginkgolide compound crystallization, with the mixed solution wash-out of normal hexane and ethyl acetate, the Fractional Collections elutriant obtains respectively bilobalide, Ginkgolide A, Ginkgolide B, ginkalide C and bilobalide J successively; 5) solvent in the elutriant of recovery collection obtains respectively bilobalide, Ginkgolide A, Ginkgolide B, ginkalide C and bilobalide J monomer with alcohol crystal again.
(8) Chinese patent (application number: 200810024206.2) " a kind of bilobalide B raw material and preparation method thereof ", this disclosure of the Invention a kind of bilobalide B raw material, 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, after filtering, adopts digestion liquid organic solvent extraction, add 100～200 order silica gel combination dryings after the extraction liquid that obtains is concentrated, adopt silica gel column chromatography to collect the elutriant that is rich in Ginkgolide B, obtain bilobalide B raw material through crystallization; Wherein, Ginkgolide B content 〉=90% in the bilobalide B raw material.
(9) (application number: 200910184916.6) " a kind of separation purification method of Ginkgolides a and B monomer ", this invention comprises Crystallization Separation, purifying and drying take the Ginkgolides of purity 〉=95% as starting raw material to Chinese patent.Described purifying is to carry out purifying with recrystallization-SCF-CO 2 combination process, the monomer GA that at first Crystallization Separation is obtained and GB respectively with dehydrated alcohol by 1: 5～80 solid-liquid ratio recrystallization at least three times, obtain GA and the GB of purity 〉=95% monomer; Then extract respectively extracting pressure 10～100Mpa, 32～150 ℃ of extraction temperature with monomer GA and the GB of SCF-CO 2 method to purity 〉=95%.Re-extract once can prepare monomer GA and the GB of purity 〉=99.5%.In amount of monomer in the starting raw material, yield 〉=90%.
Summary of the invention
The present invention seeks to provide for the defective that prior art exists the method for separation and purification GA, GB and bilobalide in a kind of Folium Ginkgo extract.
The present invention adopts following technical scheme for achieving the above object:
The method of the present invention separation and purification GA, GB and bilobalide from a kind of Folium Ginkgo extract may further comprise the steps:
1) ethyl acetate extraction: take by weighing Folium Ginkgo extract, add 20 ~ 50 ℃ of stirrings of water that Folium Ginkgo extract 5-10 doubly measures, then add ethyl acetate extraction 3-5 time that Folium Ginkgo extract 5-10 doubly measures, the combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, the gained acetic acid ethyl ester extract carry out step 2), wherein doubly amount refers to weight/volume, and is lower same;
2) low-carbon alcohol-water mixed solvent lixiviate: take by weighing acetic acid ethyl ester extract, add 20 ~ 50 ℃ of low-carbon alcohol-water mixed solvents that acetic acid ethyl ester extract 10-20 doubly measures and carry 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: with the upper silicagel column of insolubles (II), carry out wash-out with the mixed solvent of sherwood oil or normal hexane or hexanaphthene and ethyl acetate, the Fractional Collections elutriant, the solvent in the recovery elutriant obtains respectively GA and GB;
4) bilobalide crystallization purifying: alcohol leaching extracting solution (I) is concentrated into without alcohol, cooling, obtains mother liquor after filtering, mother liquor is concentrated into carries out crystallization with 30 ~ 50% methyl alcohol after doing and get bilobalide 3 ~ 5 times.
Also comprise step 5): use low-carbon alcohol-water mixed solvent that Ginkgolide A and Ginkgolide B are carried out respectively the recrystallization purifying processing.
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 ℃.
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.
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 fit to suitability for industrialized production.
Separation and purification gained Ginkgolide A of the present invention, Ginkgolide B and bilobalide purity 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%) separation and purification Ginkgolide A, Ginkgolide B and bilobalide in, the purity of gained Ginkgolide A, Ginkgolide B and bilobalide＞98%.
The ginkgolide monomer separation purification method mainly contains the methods such as ethyl acetate extraction, 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 liq extraction process.The inventor to the research accumulation of bilobalide separation and purification, finds can obtain high purity ginkgo lactone A and Ginkgolide B by ethyl acetate extraction, pure lixiviate, silica gel column chromatography for many years, and pure vat liquor can obtain bilobalide through crystallization and recrystallization method.Concrete technical scheme of the present invention is:
Take Folium Ginkgo extract as raw material by following processing step separation and purification GA, GB and bilobalide: 1) ethyl acetate extraction; 2) low-carbon alcohol-water mixed solvent lixiviate gets pure vat liquor (I) and insolubles (II); 3) insolubles (II) silica gel column chromatography separates to get GA and GB; 4) pure vat liquor (I) is concentrated into without alcohol, cooling, filters, and carries out crystallization with 30 ~ 50% methyl alcohol after mother liquor is concentrated into and does and can get bilobalide 3 ~ 5 times, and its concrete technology step is as follows:
1) ethyl acetate extraction: take by weighing Folium Ginkgo extract, add 5-10 and doubly measure 20 ~ 50 ℃ of stirrings of (weight/volume) water, then add 5-10 and doubly measure (weight/volume) ethyl acetate extraction, extract 3-5 time, the combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, and the gained acetic acid ethyl ester extract carry out step 2);
2) low-carbon alcohol-water mixed solvent lixiviate: take by weighing acetic acid ethyl ester extract, adding 10-20 doubly measures 20 ~ 50 ℃ of low-carbon alcohol-water mixed solvents and carries out lixiviate, filters, and the gained insolubles carries out step 3), and gained alcohol leaching extracting solution carries out step 4);
3) silica gel column chromatography separating purification: with the upper silicagel column of insolubles (II), carry out wash-out with the mixed solvent of sherwood oil or normal hexane or hexanaphthene and ethyl acetate, the Fractional Collections elutriant, the solvent in the recovery elutriant obtains respectively GA and GB.
4) bilobalide crystallization purifying: pure vat liquor (I) is concentrated into without pure, cooling, filters, carry out crystallization with 30 ~ 50% methyl alcohol after mother liquor is concentrated into and does and get bilobalide 3 ~ 5 times,
Step 2 wherein) low-carbon alcohol is selected from methyl alcohol or ethanol.
Step 2 wherein) in low-carbon alcohol-water mixed solvent, low-carbon alcohol content is 30 ~ 70%.
Step 3 wherein) boiling range of sherwood oil is 60 ~ 90 ℃.
Step 3 wherein) 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.
In one embodiment, also comprise step 5): use low-carbon alcohol-water mixed solvent that Ginkgolide A and Ginkgolide B are carried out respectively the recrystallization purifying processing.
Step 5 wherein) low-carbon alcohol is selected from methyl alcohol or ethanol.
Step 5 wherein) 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 ℃ in 500mL water and stir 30min, then add the 500mL ethyl acetate extraction, extract 5 times, the combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, gets acetic acid ethyl ester extract 15.5g; Add 50 ℃ of lixiviates of 155mL70% ethanol 1 hour, filter, filtration cakes torrefaction gets insolubles 4.4g and pure vat liquor 150mL; To use 5g100-200 order silica gel mixed sample behind the insolubles usefulness 20mL acetic acid ethyl dissolution, then separate with 200g200-300 purpose silica gel column chromatography, mixed solvent with petroleum ether-ethyl acetate carries out wash-out at 8: 2, the Fractional Collections elutriant, reclaim the solvent in the elutriant, obtain respectively GA1.8g(purity 98.2%), GB1.0g(purity 98.5%).150mL60 ℃ of vat liquor of alcohol is evaporated to 40mL, is cooled to 25 ℃, filters, and after 70 ℃ in mother liquor is evaporated to and does, with 30% methyl alcohol periodic crystallisation 5 times, gets 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 ℃ in 700mL water and stir 30min, then add the 1000mL ethyl acetate extraction, extract 3 times, the combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, gets acetic acid ethyl ester extract 16.5g; Add 20 ℃ of lixiviates of 330mL70% ethanol 1 hour, filter, filtration cakes torrefaction gets insolubles 4.6g and pure vat liquor 310mL; To use 5g100-200 order silica gel mixed sample behind the insolubles usefulness 20mL acetic acid ethyl dissolution, then separate with 200g200-300 purpose silica gel column chromatography, mixed solvent with petroleum ether-ethyl acetate carries out wash-out at 3: 7, the Fractional Collections elutriant, reclaim the solvent in the elutriant, obtain respectively GA1.7g(purity 98.1%), GB0.98g(purity 98.3%).310mL60 ℃ of vat liquor of alcohol is evaporated to 100mL, is cooled to 25 ℃, filters, and after 70 ℃ in mother liquor is evaporated to and does, with 40% methyl alcohol periodic crystallisation 4 times, gets 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 ℃ in 1000mL water and stir 30min, then add the 1000mL ethyl acetate extraction, extract 4 times, the combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, gets acetic acid ethyl ester extract 16.0g; Add 50 ℃ of lixiviates of 320mL30% ethanol 1 hour, filter, filtration cakes torrefaction gets insolubles 4.8g and pure vat liquor 300mL; To use 5g100-200 order silica gel mixed sample behind the insolubles usefulness 20mL acetic acid ethyl dissolution, then separate with 200g200-300 purpose silica gel column chromatography, mixed solvent with petroleum ether-ethyl acetate carries out wash-out at 4: 6, the Fractional Collections elutriant, reclaim the solvent in the elutriant, obtain respectively GA1.75g(purity 98.0%), GB1.05g(purity 98.1%).300mL60 ℃ of vat liquor of alcohol is evaporated to 70mL, is cooled to 25 ℃, filters, and after 70 ℃ in mother liquor is evaporated to and does, with 40% methyl alcohol periodic crystallisation 5 times, gets 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 ℃ in 500mL water and stir 30min, then add the 500mL ethyl acetate extraction, extract 5 times, the combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, gets acetic acid ethyl ester extract 15.5g; Add 50 ℃ of lixiviates of 155mL70% methyl alcohol 1 hour, filter, filtration cakes torrefaction gets insolubles 4.4g and pure vat liquor 150mL; To use 5g100-200 order silica gel mixed sample behind the insolubles usefulness 20mL acetic acid ethyl dissolution, then separate with 200g200-300 purpose silica gel column chromatography, mixed solvent with normal hexane-ethyl acetate carries out wash-out at 8: 2, the Fractional Collections elutriant, reclaim the solvent in the elutriant, obtain respectively GA1.83g(purity 98.4%), GB1.02g(purity 98.1%).150mL60 ℃ of vat liquor of alcohol is evaporated to 45mL, is cooled to 25 ℃, filters, and after 70 ℃ in mother liquor is evaporated to and does, with 40% methyl alcohol periodic crystallisation 4 times, gets 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 ℃ in 700mL water and stir 30min, then add the 1000mL ethyl acetate extraction, extract 3 times, the combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, gets acetic acid ethyl ester extract 16.5g; Add 20 ℃ of lixiviates of 330mL70% methyl alcohol 1 hour, filter, filtration cakes torrefaction gets insolubles 4.6g and pure vat liquor 320mL; To use 5g100-200 order silica gel mixed sample behind the insolubles usefulness 20mL acetic acid ethyl dissolution, then separate with 200g200-300 purpose silica gel column chromatography, mixed solvent with hexanaphthene-ethyl acetate carries out wash-out at 4: 6, the Fractional Collections elutriant, reclaim the solvent in the elutriant, obtain respectively GA1.78g(purity 98.6%), GB0.95g(purity 98.6%).320mL60 ℃ of vat liquor of alcohol is evaporated to 85mL, is cooled to 25 ℃, filters, and after 70 ℃ in mother liquor is evaporated to and does, with 30% methyl alcohol periodic crystallisation 4 times, gets 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 ℃ in 1000mL water and stir 30min, then add the 1000mL ethyl acetate extraction, extract 5 times, the combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, gets acetic acid ethyl ester extract 16.0g; Add 50 ℃ of lixiviates of 320mL30% methyl alcohol 1 hour, filter, filtration cakes torrefaction gets insolubles 4.8g and pure vat liquor 300mL; To use 5g100-200 order silica gel mixed sample behind the insolubles usefulness 20mL acetic acid ethyl dissolution, then separate with 200g200-300 purpose silica gel column chromatography, mixed solvent with normal hexane-ethyl acetate carries out wash-out at 4: 6, the Fractional Collections elutriant, reclaim the solvent in the elutriant, obtain respectively GA1.82g(purity 98.3%), GB0.95g(purity 98.4%).300mL60 ℃ of vat liquor of alcohol is evaporated to 80mL, is cooled to 25 ℃, filters, and after 70 ℃ in mother liquor is evaporated to and does, with 50% methyl alcohol periodic crystallisation 3 times, gets 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 ℃ in 60000mL water and stir 30min, then add the 60000mL ethyl acetate extraction, extract the combined ethyl acetate extraction liquid 3 times, acetic acid ethyl acetate extract is concentrated, dry, gets acetic acid ethyl ester extract 156.0g; Add 30 ℃ of lixiviates of 3000mL50% methyl alcohol 1 hour, filter, filtration cakes torrefaction gets insolubles 51.3g and pure vat liquor 2850mL; To use 60g100-200 order silica gel mixed sample behind the insolubles usefulness 200mL acetic acid ethyl dissolution, then separate with 3000g200-300 purpose silica gel column chromatography, mixed solvent with normal hexane-ethyl acetate carries out wash-out at 2: 8, the Fractional Collections elutriant, reclaim the solvent in the elutriant, obtain respectively GA and GB.GA gets crystal 18.5g(purity 99.2% with the further crystallization of 50% methyl alcohol), GB gets crystal 9.5g(purity 99.3% with the further crystallization of 30% ethanol).2850mL60 ℃ of vat liquor of alcohol is evaporated to 1000mL, is cooled to 25 ℃, filters, and after 70 ℃ in mother liquor is evaporated to and does, with 50% methyl alcohol periodic crystallisation 3 times, gets 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 ℃ in 60000mL water and stir 30min, then add the 60000mL ethyl acetate extraction, extract the combined ethyl acetate extraction liquid 3 times, acetic acid ethyl acetate extract is concentrated, dry, gets acetic acid ethyl ester extract 146.0g; Add 30 ℃ of lixiviates of 2500mL50% ethanol 1 hour, filter, filtration cakes torrefaction gets insolubles 48.5g and pure vat liquor 2400mL; To use 60g100-200 order silica gel mixed sample behind the insolubles usefulness 200mL acetic acid ethyl dissolution, then separate with 3000g200-300 purpose silica gel column chromatography, mixed solvent with petroleum ether-ethyl acetate carries out wash-out at 2: 8, the Fractional Collections elutriant, reclaim the solvent in the elutriant, obtain respectively GA and GB.GA gets crystal 18.0g(purity 99.5% with the further crystallization of 50% ethanol), GB gets crystal 9.8g(purity 99.4% with the further crystallization of 50% methyl alcohol).2400mL60 ℃ of vat liquor of alcohol is evaporated to 800mL, is cooled to 25 ℃, filters, and after 70 ℃ in mother liquor is evaporated to and does, with 30% methyl alcohol periodic crystallisation 3 times, gets bilobalide 23.3g(purity 98.1%).
As previously mentioned, according to the disclosed content in front, one of skill in the art can use the present invention to greatest extent.Should be pointed out that the present invention aim to provide a kind of from Folium Ginkgo extract the method for separation and purification Ginkgolide A, Ginkgolide B and bilobalide.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 (8)
1. the method for separation and purification GA, GB and bilobalide from a Folium Ginkgo extract is characterized in that may further comprise the steps:
1) ethyl acetate extraction: take by weighing Folium Ginkgo extract, add 20 ~ 50 ℃ of stirrings of water that Folium Ginkgo extract 5-10 doubly measures, then add ethyl acetate extraction 3-5 time that Folium Ginkgo extract 5-10 doubly measures, the combined ethyl acetate extraction liquid, acetic acid ethyl acetate extract is concentrated, dry, the gained acetic acid ethyl ester extract carry out step 2), wherein doubly amount refers to weight/volume, and is lower same;
2) low-carbon alcohol-water mixed solvent lixiviate: take by weighing acetic acid ethyl ester extract, add 20 ~ 50 ℃ of low-carbon alcohol-water mixed solvents that acetic acid ethyl ester extract 10-20 doubly measures and carry 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: with the upper silicagel column of insolubles (II), carry out wash-out with the mixed solvent of sherwood oil or normal hexane or hexanaphthene and ethyl acetate, the Fractional Collections elutriant, the solvent in the recovery elutriant obtains respectively GA and GB;
4) bilobalide crystallization purifying: alcohol leaching extracting solution (I) is concentrated into without alcohol, cooling, obtains mother liquor after filtering, mother liquor is concentrated into carries out crystallization with 30 ~ 50% methyl alcohol after doing and get bilobalide 3 ~ 5 times.
2. method according to claim 1 also comprises step 5): use low-carbon alcohol-water mixed solvent that Ginkgolide A and Ginkgolide B are carried out respectively the recrystallization purifying processing.
3. method according to claim 1 and 2, wherein step 2) low-carbon alcohol is selected from methyl alcohol or ethanol.
4. according to claim 1 ~ 3 each described method, wherein step 2) in low-carbon alcohol-water mixed solvent, low-carbon alcohol content is 30 ~ 70%.
5. according to claim 1 ~ 4 each described method, wherein step 3) boiling range of sherwood oil is 60 ~ 90 ℃.
6. according to claim 1 ~ 5 each described method, 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.
7. according to claim 2 ~ 6 each described method, wherein step 5) low-carbon alcohol is selected from methyl alcohol or ethanol.
8. according to claim 2 ~ 7 each described method, wherein step 5) in low-carbon alcohol-water mixed solvent, low-carbon alcohol content is 30 ~ 70%.
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