CN1847237A - Ginkgo leaf extract and production process of separating high purity effective component of the extract - Google Patents
Ginkgo leaf extract and production process of separating high purity effective component of the extract Download PDFInfo
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Abstract
The present invention discloses new process of extracting and separating high purity bilobalide and ginkgetin. By means of macroporous adsorption resin chromatographic separation technology, the water extract of ginkgo material is eluted in macroporous adsorption resin column successively with distilled water, 5-25 % concentration alcohol solution, 0.01-10 % concentration alkali solution, distilled water and 30-95 % concentration alcohol solution, and the elute of alkali solution and the elute of 30-95 % concentration alcohol solution are collected separately, concentrated and purified to obtain bilobalide or ginkgetin. The process has high efficiency, simple operate, low production cost and environment friendship, and the product has high quality and high yield.
Description
Technical field
The present invention relates to a kind of also method of separate drug effective constituent of from plant, extracting, relate in particular to a kind of macroporous adsorbent resin chromatography isolation technique that adopts and from Ginkgo Leaf or ginkgo biloba crude extract, extract and the method for separating high-purity bilobalide and ginkgolic flavone glycoside, belong to biomedicine field.
Background technology
Ginkgo (Ginkgo biloba L.) has another name called Gong Sunshu, is one of Relict Plant in the most ancient secondary era, have the title of gymnosperm " living fossil ", is China's special product.China's gingko resource owning amount accounts for 70% of world's total amount.Mainly containing effective constituent in the Ginkgo Leaf is flavonoid (flavonoids) and terpene lactones compound (terpene lactones), have the expansion coronary vasodilator, improve cerebral circulation, eliminate oxyradical, suppress platelet activation factor effects such as (PA F), be used for the treatment of coronary heart disease, cerebral thrombosis, cerebral ischemia, disordered brain function, brain injury sequela, nervous system disorders and asthma etc. clinically.
What last decade came studies show that bilobalide is that efficient antagonist, the especially Ginkgolide B of PAF is the strongest to the antagonistic action of PAF.Because the generation of PAF and cardiovascular and cerebrovascular diseases development is closely related, also closely related with generations such as asthma, shock, inflammation, stomach ulcer, ephrosis, so bilobalide is considered to have most the natural paf receptor antagonists of potential applicability in clinical practice at present.Recently a kind of viewpoint of Ti Chuing is: because flavones, bilobalide and bilobalide three big composition pharmacological actions in the Folium Ginkgo extract have nothing in common with each other, for the more economical medicine that reasonably uses, allow every kind of effective ingredient all give full play to pharmacological action, should distinguish the different sick administrations respectively of planting, give bilobalide best when treating nervous system disorders such as spinal nerves demyelination disease and senile dementia, and needn't give ginkgolic flavone glycoside and bilobalide; Then mainly use ginkgolic flavone glycoside and bilobalide when the treatment cardiovascular and cerebrovascular diseases, the disease that causes for PAF then only needs bilobalide just can.
The method of main effective constituent bilobalide and ginkgolic flavone glycoside mainly contains macroporous adsorbent resin method, enzyme process, supercritical CO in extraction both at home and abroad, separation Ginkgo Leaf and the extract thereof at present
2Extraction process and high-speed countercurrent chromatography extraction method (High-speed Countercurrent Chromatography, HSCCC) etc.The product that gets but above-mentioned all methods prepare all is the mixture of bilobalide and ginkgolic flavone glycoside, and wherein the content of two kinds of compositions is relatively low, is respectively about 10% and 30%.If will be with two kinds of component separating, then must more step, complex operation, effective constituent kind and quantity loss are all bigger.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, provide a kind of can be quick, simple and direct from Ginkgo Leaf or ginkgo biloba crude extract, extract and the method for separating high-purity bilobalide and ginkgolic flavone glycoside, this ginkgo biloba crude extract can commercially available purchase obtains also can be according to ordinary method (reference: 1, Klaus-Peter Schwabe, Karlsruhe.Extract from Leaves of Ginkgo Biloba for Intravenous Injection or Infusion.U.S.P., 5512286; 2, Li Xingang, Wei Wei, Chen Wei. be rich in the Ginkgo Leaf dry extract preparation technology of bilobalide. Chinese Journal of Pharmaceuticals, 1998,29 (1): 8～9) prepare.
Technical problem to be solved by this invention realizes by following technological approaches:
A kind of method of extracting and separating bilobalide and ginkgolic flavone glycoside from Ginkgo Leaf or ginkgo biloba crude extract may further comprise the steps:
(1), with fresh or dry Ginkgo Leaf water decoction or with the ginkgo biloba crude extract water dissolution, decoction liquor or the aqueous solution are filtered, gained filtrate is all gone up macroporous adsorptive resins after, use distilled water and 5%～25% ethanol successively, the decoction liquor or the aqueous solution filter, wash-out impurity, then colourless to effluent liquid with 0.01%～10% buck wash-out resin column, collect the buck elutriant, be concentrated into dried behind the accent pH 3～5, washing, oven dry gets ginkgolic flavone glycoside;
(2), resin column continues to be washed till effluent liquid pH with distilled water is neutrality, discards water lotion; It is colourless to be eluted to effluent liquid with 30%～95% ethanolic soln again, collects ethanol eluate, and being evaporated to does not have the alcohol flavor, extraction, remove extraction solvent under reduced pressure, the residue dissolve with ethanol adds sorbent material in lysate, whip attachment, filter, filter residue discards, and filtrate decompression reclaims solvent, residual thing is dry under 40～80 ℃ of (more preferably 50～60 ℃) conditions, gets the bilobalide crude product; Or further the bilobalide crude product is adopted that ordinary method is refining to be the bilobalide elaboration.
Among the above-mentioned preparation method, preferably the Ginkgo Leaf medicinal powder is added the water of 1～100 times of medicinal material amount (W/V) in the step (1), decoct 1～3 time, decoct 5min～3h at every turn; During macroporous adsorptive resins, the macroporous resin consumption is 1: 10～10: 1 with the ratio (V/W) of crude drug amount, more preferably 1: 1～3: 1 on the filtrate; The flow rate of liquid of control filtrate upper prop is 0.5～10 times of resin column volume/h, and the flow rate of liquid that is preferably control filtrate upper prop is 1～3 times of resin column volume/h;
When using distillation washing post in the step (1), preferably use the distilled water wash-out of 3～50 times of resin column volumes, more preferably use the distilled water wash-out of 5～8 times of resin column volumes; The flow velocity of control elutriant is 0.5～10 times of resin column volume/h, is preferably 1～3 times of resin column volume/h;
During with 5%～25% ethanol elution impurity, preferably carry out wash-out with 20% ethanol in the step (1), elution amount is till the effluent liquid clear;
During with 0.01%～10% buck wash-out resin column, preferably carry out wash-out in the step (1) with 0.1～1%NaOH, elution amount be to effluent liquid light or colourless till;
In the step (2), during with distillation washing post, till its elution amount is preferably when being eluted to effluent liquid pH value for neutrality;
During with 30%～95% ethanolic soln wash-out resin column, preferably carry out wash-out in the step (2) with 60% ethanol, elution amount be to effluent liquid light or colourless till;
Step (2) is middle with collected ethanol eluate, being evaporated to does not have when extracting after alcohol is distinguished the flavor of, preferably use the ethyl acetate, ethyl formate, methyl acetate, propyl carbinol, amylalcohol of 1～20 times of weight or extracted with diethyl ether 2～10 times, more preferably use the ethyl acetate extraction 5 times of 4 times of weight;
After removing extraction solvent under reduced pressure in the step (2), residue is preferably used the dissolve with ethanol of 1/10～5 times of weight, the preferred gac that adds 1/100～1/5 times of weight in lysate, whip attachment 15min～5h; Preferred, remove ethyl acetate under reduced pressure after, residue adds the gac of 1/10 times of weight, whip attachment 1～2h with the dissolve with ethanol of 1～3 times of weight in lysate;
When in the step (2) the bilobalide crude product refining being the bilobalide elaboration, as preferably, may further comprise the steps: the bilobalide crude product is dissolved in methyl alcohol, ethanol, propyl alcohol, acetone or the above-mentioned mixing solutions that two or more is formed with arbitrary proportion, add distilled water, centrifugal gluey water liquid, the drying under reduced pressure removed; Dried bilobalide solid is dissolved in methyl alcohol, ethanol, propyl alcohol, acetone or the above-mentioned mixing solutions that two or more is formed with arbitrary proportion, smart filter, filtrate is spent the night being lower than to place under 25 ℃ of (more preferably 10～12 ℃) conditions, separate out white crystals, filter, drying gets the bilobalide elaboration;
Preferred, the bilobalide crude product is dissolved in the acetone of 0.5～10 times of weight, stir the distilled water that slowly adds 2～20 times of weight down; In dried bilobalide solid, add 0.5～5 times of amount (W/V), 10%～60% methyl alcohol,, adopt the smart filter of millipore filtration in 40～80 ℃ of dissolvings; Most preferred, the bilobalide crude product is dissolved in the acetone of 2～3 times of weight, stir the distilled water that slowly adds 8 times of weight down; 30% methyl alcohol that adds equivalent (W/V) in dried bilobalide solid is in 50～60 ℃ of dissolvings.
Macroporous adsorptive resins filler model described in the present invention is optional from macroporous adsorptive resins such as AB-8, X-5, S-8, SIP-1300, MDA, NKA-II, D-101, HPD-100, HPD-400, ADS-17 or BS-55; The material of described macroporous adsorptive resins is the organic polymer of glass, stainless steel or acid-and base-resisting and organic solvent; The blade diameter length ratio of macroporous adsorptive resins is 1: 1～1: 20.
Used alkali can be any one highly basic in the inventive method, and weak base or faintly acid an alkali metal salt etc. for example can be NaOH, KOH, NaHCO
3, Na
2CO
3, NH
4OH etc.
The common trait of ginkgolic flavone glycoside constituents chemical structure is to contain a plurality of weakly acidic phenolic hydroxyl groups, particularly all contains acid the strongest 7 and 4 ' free phenolic hydroxyl group.Therefore, the compound of this class formation all dissolves in weak base
(R
1～R
4Replaced by H or glycosyl respectively)
The gingkgo flavonoids structural formula
The property solution, on macroporous adsorbent resin, just can generate water miscible salt by first wash-out, realize and the separating fully of bilobalide constituents (as follows).3 lactonic rings are arranged in the bilobalide constituents chemical structure, though
It is water-soluble to be insoluble in water
(R
1～R
4Replaced by H or glycosyl respectively)
The easily open loop in alkaline environment of the chemical structure change synoptic diagram of gingkgo flavonoids in acid, alkaline solution, but only just easily generation of ring-opening reaction under system is heated situation, lactonic ring is difficult for open loop at normal temperatures.The elution process of macroporous adsorbent resin of the present invention all is to carry out at normal temperatures, can not make lactone composition chemical structure generation chemical transformation with sig water wash-out flavones ingredient the time.Simultaneously, when Ginkgo Leaf water extract was flowed through macroporous adsorptive resins, phenolic constituent such as a large amount of tannins was also by absorption with macroporous adsorbent resin in the water extract, and distilled water or lower concentration alcohol there is no good wash-out effect to specific examples of such components; During again with higher concentration alcohol wash-out effective constituent, this type of material is also by a large amount of wash-outs, thereby influences quality product.Therefore, the inventive method adopts carries out sig water wash-out operation steps on resin column, effective phenol impurity such as wash-out tannin, thus obtain highly purified bilobalide.By bilobalide and the ginkgolic flavone glycoside that the inventive method preparation get, the two both can use by compatibility, is used for the treatment of cardiovascular and cerebrovascular disease, also can use bilobalide to treat illness due to the PAF such as obstinate asthma separately.So not only make crude drug obtain more economically reasonably utilizing, also will help domestic the 4th generation ginkgo agent research and development.
When prior art is extracted bilobalide and ginkgolic flavone glycoside from Ginkgo Leaf or its extract, use ethanol or acetone and other organic solvent as ginkgo medicinal material extract solvent, remove solvent again under reduced pressure after with an organic solvent extracting the ginkgo medicinal material, raffinate is gone up macroporous adsorbent resin again, on the ginkgo herbal extract behind the macroporous adsorbent resin, elder generation's water and low-concentration ethanol wash-out are removed impurity, use higher concentration ethanol elution effective constituent again, products obtained therefrom is the mixture of flavones and terpene lactones, if both will be separated, then need extremely numerous and diverse technology, prepared high purity ginkgo total flavones is to be cost to lose total terpene lactones fully, and vice versa.
The inventive method adopts the water of cheapness and environmental protection as ginkgo solvent extraction medicinal material, and the extracts active ingredients rate of transform is suitable with existing method in the medicinal material, the environmental pollution that the while has avoided the use of organic solvent to cause production cost to rise and caused again.Extracting solution is directly crossed macroporous adsorptive resins after filtering, needing in the prior art to have avoided adopting with loaded down with trivial details steps such as the heating of organic solvent extraction liquid, concentrating under reduced pressure, so the inventive method technology is simple, energy consumption is low, production cost is low.In addition, owing to reduced heating schedule, can avoid heat-labile terpene lactones composition to be damaged in the production technique of the present invention.On the ginkgo medicinal material water extract behind the absorption with macroporous adsorbent resin, the present invention's elder generation's water and low-concentration ethanol wash-out are removed impurity, obtain the weak acid yellow ketones component of higher degree again with the sig water wash-out, obtain the neutral terpene lactones composition of higher degree again with the higher concentration ethanol elution, on macroporous adsorptive resins, just can make ginkgolic flavone glycoside better be separated (product purity reaches 80% and 87% respectively) with Folium Ginkgo terpene lactones through simple single stepping, through the simple purification step product content is reached more than 95% again, and intermediate product content is higher, has avoided traditional extraction process to significant wastage that herb resource caused.
In a word, the inventive method is efficient, and is simple and direct, and production cost is low, little to human body and environmental damage, had by the prepared product of the inventive method that quality is good, the yield advantages of higher.
Embodiment
Further describe the present invention by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
Embodiment 1
In 12kg dry Folium Ginkgo powder, add 120L water, boil 0.5h.Filtration adds 60L in the filter residue more successively and 36L water respectively boils 0.5h, merges No. three times extracting solution.Water is carried the AB-8 macroporous adsorbent resin that 12kg anticipates on the soup (Tianjin Chemical Plant of Nankai Univ. product), and control water carries that column flow rate is 1 times of resin column volume/h on the soup.Behind the whole upper props of liquid to be extracted, resin column is with the distilled water wash-out of 8 times of resin column volumes, and the flow velocity of control elutriant is 3 times of resin column volume/h, and is colourless to effluent liquid, colourless to effluent liquid with 20% ethanol elution again.
Resin column continues colourless to effluent liquid with the 1%NaOH eluant solution, collects the diluted alkaline elutriant, transfers pH to 3 with 6N HCl.Solution spray drying, solid are with an amount of distilled water wash, and drying gets yellow powder shape solid.
Get above-mentioned yellow powder shape solid 2mg, add dehydrated alcohol 1ml and make dissolving, add hydrochloric acid solution (2mol/L) 1ml again, put in the water-bath and refluxed 2 hours, put and be chilled to room temperature, filter, filtrate evaporate to dryness, residue add dehydrated alcohol 0.5ml makes dissolving, as need testing solution; Other gets the Quercetin reference substance, adds dehydrated alcohol and makes the solution that contains 0.5mg among every 1ml, in contrast product solution.Draw each 5 μ l of need testing solution and reference substance solution respectively, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid (6: 3: 1) is developping agent, launches, and takes out, dry, put in the ammonia steam smoked about 5min, in the trial-product chromatogram, with the reference substance chromatogram on position accordingly, show the spot of same color, determine that this yellow powder shape solid contains flavones ingredient.
It is an amount of to get above-mentioned flavones ingredient, measure flavonol glycosides content according to " assay " method in high performance liquid chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005) and " Ginkgo Leaf " item of Pharmacopoeia of People's Republic of China version in 2005, the result shows that content of flavonoids is 83.2% in this yellow powder shape solid.
Resin column is behind the diluted alkaline wash-out, and being washed till effluent liquid pH with distilled water is neutrality, again with 60% ethanol elution, colourless to effluent liquid, collect 60% ethanol eluate, reclaim under reduced pressure is to not having alcohol flavor, raffinate 3L ethyl acetate extraction, coextraction 5 times, the combined ethyl acetate extraction liquid is with 1% (V/W) Na
2SO
4After dehydrating, reclaim ethyl acetate, 60 ℃ of oven dry of residue, porphyrize gets 350g white powder solid.
It is an amount of to get above-mentioned white powder solid, adds acetone and is mixed with the solution that every 1ml contains the 3.0mg sample, as need testing solution.Other gets Ginkgolide A, B, C and bilobalide reference substance, adds acetone and makes the mixing solutions that every 1ml contains 0.5mg, in contrast product solution.Draw each 5 μ l of above-mentioned two kinds of solution, put respectively on the silica gel H thin layer plate of same CMC-Na formulations prepared from solutions with 4% sodium-acetate, with toluene-ethyl acetate-acetone-methyl alcohol (6: 3: 3: 0.1) be developping agent, launching below 15 ℃, take out, dry,, put under the ultraviolet lamp (365nm) and inspect at 140～160 ℃ of about 30min of heating.In the trial-product chromatogram, with the reference substance chromatogram on corresponding position, show the fluorescence spot of same color, so definite this white powder solid bilobalide-containing constituents.
It is an amount of to get above-mentioned bilobalide constituents, measure bilobalide content according to " assay " method in high performance liquid chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005) and " Ginkgo Leaf " item of Pharmacopoeia of People's Republic of China version in 2005, the result shows that the content of ginkgolide compound is 87.6% in this white powder solid.
With the dissolve with ethanol Folium Ginkgo terpene lactones crude product of 1000ml 95%, and add the 100g activity charcoal powder, stir 1h, suction filtration, behind the filtrate recycling ethanol, 60 ℃ of oven dry get white solid 330g.This solid is dissolved in the 1000ml acetone, stirs down and slowly adds 3L distilled water, stir 0.5h slowly, centrifugally remove gluey water liquid, filter residue in 60 ℃ dry 210g dry powder.Adding 210ml content is 30% methyl alcohol in this dry powder, in 50 ℃ of water-baths, make it to dissolve, and the smart filter of millipore filtration, filtrate relays under 4 ℃ of conditions, leaves standstill 8h, separates out white crystals, gets bilobalide elaboration 140g.
It is an amount of to get above-mentioned bilobalide elaboration, adds acetone and is mixed with the solution that every 1ml contains the 3.0mg sample, as need testing solution.Other gets Ginkgolide A, B, C and bilobalide reference substance, adds acetone and makes the mixing solutions that every 1ml contains 0.5mg, in contrast product solution.Draw each 5 μ l of above-mentioned two kinds of solution, put respectively on the silica gel H thin layer plate of same CMC-Na formulations prepared from solutions with 4% sodium-acetate, with toluene-ethyl acetate-acetone-methyl alcohol (6: 3: 3: 0.1) be developping agent, launching below 15 ℃, take out, dry,, put under the ultraviolet lamp (365nm) and inspect at 140～160 ℃ of about 30min of heating.In the trial-product chromatogram, with the reference substance chromatogram on corresponding position, show the fluorescence spot of same color.
It is an amount of to get above-mentioned bilobalide elaboration, measure bilobalide content according to " assay " method in high performance liquid chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005) and " Ginkgo Leaf " item of Pharmacopoeia of People's Republic of China version in 2005, the result shows that the content of ginkgolide compound is 97.5% in this white powder solid.
Embodiment 2
In 10kg dry Folium Ginkgo powder, add 100L water, boil 1h.Filtration adds 50L in the filter residue more successively and 30L water respectively boils 1h, merges No. three times extracting solution.Water is carried the D-101 macroporous adsorbent resin that 10kg anticipates on the soup (Tianjin Chemical Plant of Nankai Univ. product), and it is 2 times of resin column volume/h that control water is carried the soup flow velocity.Behind the whole upper props of liquid to be extracted, resin column is with the distilled water wash-out of 5 times of resin column volumes, and the flow velocity of control elutriant is 1 times of resin column volume/h, and it is colourless to be eluted to effluent liquid, colourless to effluent liquid with 25% ethanol elution again.
Resin column continues colourless to effluent liquid with the 0.5%NaOH eluant solution, collects the diluted alkaline elutriant, transfers pH to 4 with 6N HCl.Solution spray drying, solid are with an amount of distilled water wash, and drying promptly gets yellow powder shape solid.
It is an amount of to get above-mentioned yellow powder shape solid, add dehydrated alcohol and make the solution that every 1ml contains 0.5mg,, draw above-mentioned solution 6 μ l according to tlc (appendix VIB of Pharmacopoeia of People's Republic of China version in 2005) test, point is on the silica gel g thin-layer plate that contains 4% sodium-acetate, (5: 3: 1: 1) be developping agent, expansion was taken out with ethyl acetate-butanone-formic acid-water, dry, spray is with the aluminum chloride ethanolic soln, and hot blast drying is put under the ultraviolet lamp (365nm) and detected.Show the distinctive yellow-green fluorescence principal spot of flavonoid compound in the trial-product chromatogram, so determine that this yellow powder shape solid contains flavones ingredient.
It is an amount of to get above-mentioned flavones ingredient, measure flavonol glycosides content according to " assay " method in high performance liquid chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005) and " Ginkgo Leaf " item of Pharmacopoeia of People's Republic of China version in 2005, the result shows that content of flavonoids is 80.3% in this yellow powder shape solid.
Resin column is behind the diluted alkaline wash-out, and being washed till effluent liquid pH with distilled water is neutrality, again with 70% ethanol elution, colourless to effluent liquid, collect 70% ethanol eluate, reclaim under reduced pressure is to not having the alcohol flavor, raffinate 2.5L ethyl formate extraction, coextraction 3 times merges the ethyl formate extraction liquid, with 10% (V/W) Na
2SO
4After dehydrating, reclaim ethyl formate, 55 ℃ of oven dry of residue, porphyrize gets 320g white powder solid.
It is an amount of to get above-mentioned white powder solid, adds acetone and makes the solution that every 1ml contains the 3.0mg sample, as need testing solution.Other gets Ginkgolide A, B, C and bilobalide reference substance, adds acetone and makes the mixing solutions that every 1ml contains 0.5mg, 0.5mg, 0.5mg, 1.0mg, in contrast product solution.Test according to tlc (appendix VIB of Pharmacopoeia of People's Republic of China version in 2005), draw each 5 μ l of above-mentioned trial-product and reference substance solution, put respectively on the same silica gel g thin-layer plate for preparing with the carboxymethylcellulose sodium solution that contains 4% sodium-acetate, with toluene-ethyl acetate-acetone-methyl alcohol (10: 5: 5: 0.6) be developping agent, launching below 15 ℃, taking out, drying, about 30 minutes of 140～160 ℃ of heating, put under the ultra-violet lamp (365nm) and inspect.In the trial-product chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.
It is an amount of to get above-mentioned bilobalide constituents, measure bilobalide content according to " assay " method in high performance liquid chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005) and " Ginkgo Leaf " item of Pharmacopoeia of People's Republic of China version in 2005, the result shows that the content of ginkgolide compound is 86.3% in this white powder solid.
With the dissolve with ethanol Folium Ginkgo terpene lactones crude product of 1000ml 95%, and add the 100g activity charcoal powder, stir 1h, suction filtration, behind the filtrate recycling ethanol, 55 ℃ of oven dry get white solid 305g.This solid is dissolved in the 1000ml ethanol, stirs down and slowly adds 3L distilled water, stir 0.5h slowly, centrifugally remove gluey water liquid, filter residue in 60 ℃ dry 195g dry powder.Adding 195ml content is 30% methyl alcohol in this dry powder, in 50 ℃ of water-baths, make it to dissolve, and the smart filter of millipore filtration, filtrate relays under 4 ℃ of conditions, leaves standstill 8h, separates out white crystals, gets bilobalide elaboration 135g.
It is an amount of to get above-mentioned bilobalide elaboration, adds acetone and makes the solution that every 1ml contains the 3.0mg sample, as need testing solution.Other gets Ginkgolide A, B, C and bilobalide reference substance, adds acetone and makes the mixing solutions that every 1ml contains 0.5mg, 0.5mg, 0.5mg, 1.0mg, in contrast product solution.Test according to tlc (appendix VIB of Pharmacopoeia of People's Republic of China version in 2005), draw each 5 μ l of above-mentioned trial-product and reference substance solution, put respectively on the same silica gel g thin-layer plate for preparing with the carboxymethylcellulose sodium solution that contains 4% sodium-acetate, with toluene-ethyl acetate-acetone-methyl alcohol (10: 5: 5: 0.6) be developping agent, launching below 15 ℃, taking out, drying, about 30 minutes of 140～160 ℃ of heating, put under the ultra-violet lamp (365nm) and inspect.In the trial-product chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.
It is an amount of to get above-mentioned bilobalide elaboration, measure bilobalide content according to " assay " method in high performance liquid chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005) and " Ginkgo Leaf " item of Pharmacopoeia of People's Republic of China version in 2005, the result shows that the content of ginkgolide compound is 96.3% in this bilobalide elaboration.
Embodiment 3
Toward the commercially available Ginkgo Leaf crude extract of 1kg (Zhejiang Kang Enbei pharmaceutical Co. Ltd product, trade name is " a Ginkgo Leaf crude extract ", and it is about 24% that this Ginkgo Leaf crude extract contains the ginkgo total flavones, gingko total terpene lactones about 6%) in add 100L water, stirring makes dissolving, filters.Water is carried the S-8 macroporous adsorbent resin that 50kg anticipates on the soup (Tianjin Chemical Plant of Nankai Univ. product), and control water carries that column flow rate is 1 times of resin column volume/h on the soup.Behind the whole upper props of liquid to be extracted, resin column is with the distilled water wash-out of 5 times of resin column volumes, and the flow velocity of control elutriant is 0.5 times of resin column volume/h, and is colourless to effluent liquid, colourless to effluent liquid with 18% ethanol elution again.
Resin column continues colourless to effluent liquid with the 0.5%KOH eluant solution, collects the diluted alkaline elutriant, transfers pH to 5 with 6N HCl.Solution spray drying, solid are with an amount of distilled water wash, and drying gets yellow powder shape solid.
Get above-mentioned yellow powder shape solid 2mg, add dehydrated alcohol 1ml and make dissolving, add hydrochloric acid solution (2mol/L) 1ml again, put in the water-bath and refluxed 2 hours, put and be chilled to room temperature, filter, filtrate evaporate to dryness, residue add dehydrated alcohol 0.5ml makes dissolving, as need testing solution.Other gets the Quercetin reference substance, adds dehydrated alcohol and makes the solution that contains 0.5mg among every 1ml, in contrast product solution.Draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with toluene-ethyl acetate-formic acid (6: 3: 1) is developping agent, launches, and takes out, dry, put in the ammonia steam smoked about 5min, in the trial-product chromatogram, with the reference substance chromatogram on position accordingly, the spot that shows same color is so determine that this yellow powder shape solid contains flavones ingredient.
Get above-mentioned flavones ingredient 100mg, place the 250ml flask, add ethanol and the 20ml distilled water of 50ml, supersound process 5min, concentrated hydrochloric acid and the zeolite of adding 8ml refluxed 2.25 hours, were cooled to room temperature.Solution quantitatively is transferred in the volumetric flask of 100ml, is diluted to scale with distilled water, mixing filters, as need testing solution.In addition prepare the 0.6mg/ml Quercetin with methyl alcohol, 0.6mg/ml kaempferide and 0.2mg/ml Isorhamnetol solution, the solution that is diluted to 1: 5 and 1: 10 with methyl alcohol is product solution in contrast.Measure flavonol glycosides content according to high performance liquid chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005), the result shows that content of flavonoids is 90.2% in this yellow powder shape solid.
Resin column is behind the diluted alkaline wash-out, and being washed till effluent liquid pH with distilled water is neutrality, again with 90% ethanol elution, colourless to effluent liquid, collect 90% ethanol eluate, reclaim under reduced pressure is to not having alcohol flavor, raffinate 3L extracted with diethyl ether, coextraction 3 times merges ether extraction liquid, with 10% (V/W) Na
2SO
4After dehydrating, reclaim ether, 50 ℃ of oven dry of residue, porphyrize gets 650g white powder solid.
It is an amount of to get above-mentioned white powder solid, adds acetone and is mixed with the solution that every 1ml contains the 3.0mg sample, as need testing solution.Other gets Ginkgolide A, B, C and bilobalide reference substance, adds acetone and makes the mixing solutions that every 1ml contains 0.5mg, in contrast product solution.Draw each 5 μ l of above-mentioned two kinds of solution, put respectively on the silica gel H thin layer plate of same CMC-Na formulations prepared from solutions with 4% sodium-acetate, with toluene-ethyl acetate-acetone-methyl alcohol (6: 3: 3: 0.1) be developping agent, launching below 15 ℃, take out, dry,, put under the ultraviolet lamp (365nm) and inspect at 140～160 ℃ of about 30min of heating.In the trial-product chromatogram, with the reference substance chromatogram on corresponding position, show the fluorescence spot of same color, so definite this white powder solid bilobalide-containing constituents.
It is an amount of to get above-mentioned bilobalide constituents, measure bilobalide content according to " assay " method in high performance liquid chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005) and " Ginkgo Leaf " item of Pharmacopoeia of People's Republic of China version in 2005, the result shows that the content of ginkgolide compound is 92.6% in this white powder solid.
With the dissolve with ethanol Folium Ginkgo terpene lactones crude product of 2000ml 95%, and add the 200g activity charcoal powder, stir 1h, suction filtration, behind the filtrate recycling ethanol, 60 ℃ of oven dry get white solid 630g.This solid is dissolved in the 2000ml acetone, stirs down and slowly adds 6L distilled water, stir 0.5h slowly, centrifugally remove gluey water liquid, filter residue in 60 ℃ dry 610g dry powder.Adding 610ml content is 30% ethanol in this dry powder, in 50 ℃ of water-baths, make it to dissolve, and the smart filter of millipore filtration, filtrate relays under 10 ℃ of conditions, leaves standstill 8h, separates out white crystals, gets bilobalide elaboration 605g.
It is an amount of to get above-mentioned bilobalide elaboration, adds acetone and is mixed with the solution that every 1ml contains the 3.0mg sample, as need testing solution.Other gets Ginkgolide A, B, C and bilobalide reference substance, adds acetone and makes the mixing solutions that every 1ml contains 0.5mg, in contrast product solution.Draw each 5 μ l of above-mentioned two kinds of solution, put respectively on the silica gel H thin layer plate of same CMC-Na formulations prepared from solutions with 4% sodium-acetate, with toluene-ethyl acetate-acetone-methyl alcohol (6: 3: 3: 0.1) be developping agent, launching below 15 ℃, take out, dry,, put under the ultraviolet lamp (365nm) and inspect at 140～160 ℃ of about 30min of heating.In the trial-product chromatogram, with the reference substance chromatogram on corresponding position, show the fluorescence spot of same color.
It is an amount of to get above-mentioned bilobalide elaboration, measure bilobalide content according to " assay " method in high performance liquid chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005) and " Ginkgo Leaf " item of Pharmacopoeia of People's Republic of China version in 2005, the result shows that the content of ginkgolide compound is 98.3% in this white powder solid.
Embodiment 4
In the fresh Ginkgo Leaf of 100kg, add 200L water, boil 1h.Filtration adds 100L in the filter residue more successively and 60L water respectively boils 1h, merges No. three times extracting solution.Water is carried the NKA-II macroporous adsorbent resin that 20kg anticipates on the soup (Tianjin Chemical Plant of Nankai Univ. product), and it is 1 times of resin column volume/h that control water is carried the soup flow velocity.Behind the whole upper props of liquid to be extracted, resin column is with the distilled water wash-out of 15 times of resin column volumes, and the flow velocity of control elutriant is 3 times of resin column volume/h, and it is colourless to be eluted to effluent liquid, colourless to effluent liquid with 23% ethanol elution again.
Resin column continues colourless to effluent liquid with the 1%KOH eluant solution, collects the diluted alkaline elutriant, transfers pH to 3 with 6N HCl.Solution spray drying, solid are with an amount of distilled water wash, and drying promptly gets yellow powder shape solid.
It is an amount of to get above-mentioned yellow powder shape solid, add dehydrated alcohol and make the solution that every 1ml contains 0.5mg,, draw above-mentioned solution 6 μ l according to tlc (appendix VIB of Pharmacopoeia of People's Republic of China version in 2005) test, point is on the silica gel g thin-layer plate that contains 4% sodium-acetate, (5: 3: 1: 1) be developping agent, expansion was taken out with ethyl acetate-butanone-formic acid-water, dry, spray is with the aluminum chloride ethanolic soln, and hot blast drying is put under the ultraviolet lamp (365nm) and detected.Show the distinctive yellow-green fluorescence principal spot of flavonoid compound in the trial-product chromatogram, so determine that this yellow powder shape solid contains flavones ingredient.
It is an amount of to get above-mentioned flavones ingredient, measure flavonol glycosides content according to " assay " method in high performance liquid chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005) and " Ginkgo Leaf " item of Pharmacopoeia of People's Republic of China version in 2005, the result shows that content of flavonoids is 82.3% in this yellow powder shape solid.
Resin column is behind the diluted alkaline wash-out, and being washed till effluent liquid pH with distilled water is neutrality, again with 40% ethanol elution, colourless to effluent liquid, collect 40% ethanol eluate, reclaim under reduced pressure is to not having the alcohol flavor, raffinate 5.0L methylethylketone extraction, coextraction 3 times merges the methylethylketone extraction liquid, with 5% (V/W) Na
2SO
4After dehydrating, reclaim ethyl acetate, 60 ℃ of oven dry of residue, porphyrize gets 603g white powder solid.
It is an amount of to get above-mentioned white powder solid, adds acetone and makes the solution that every 1ml contains the 3.0mg sample, as need testing solution.Other gets Ginkgolide A, B, C and bilobalide reference substance, adds acetone and makes the mixing solutions that every 1ml contains 0.5mg, 0.5mg, 0.5mg, 1.0mg, in contrast product solution.Test according to tlc (appendix VIB of Pharmacopoeia of People's Republic of China version in 2005), draw each 5 μ l of above-mentioned trial-product and reference substance solution, put respectively on the same silica gel g thin-layer plate for preparing with the carboxymethylcellulose sodium solution that contains 4% sodium-acetate, with toluene-ethyl acetate-acetone-methyl alcohol (10: 5: 5: 0.6) be developping agent, launching below 15 ℃, taking out, drying, about 30 minutes of 140～160 ℃ of heating, put under the ultra-violet lamp (365nm) and inspect.In the trial-product chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.
It is an amount of to get above-mentioned bilobalide constituents, measure bilobalide content according to " assay " method in high performance liquid chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005) and " Ginkgo Leaf " item of Pharmacopoeia of People's Republic of China version in 2005, the result shows that the content of ginkgolide compound is 85.7% in this white powder solid.
With the dissolve with ethanol Folium Ginkgo terpene lactones crude product of 2000ml 95%, and add the 200g activity charcoal powder, stir 1h, suction filtration, behind the filtrate recycling ethanol, 60 ℃ of oven dry get white solid 585g.This solid is dissolved in the 2000ml propyl alcohol, stirs down and slowly adds 6L distilled water, stir 0.5h slowly, centrifugally remove gluey water liquid, filter residue in 60 ℃ dry 574g dry powder.Adding 600ml content is 30% acetone in this dry powder, in 50 ℃ of water-baths, make it to dissolve, and the smart filter of millipore filtration, filtrate relays under 4 ℃ of conditions, leaves standstill 8h, separates out white crystals, gets bilobalide elaboration 565g.
It is an amount of to get above-mentioned bilobalide elaboration, adds acetone and makes the solution that every 1ml contains the 3.0mg sample, as need testing solution.Other gets Ginkgolide A, B, C and bilobalide reference substance, adds acetone and makes the mixing solutions that every 1ml contains 0.5mg, 0.5mg, 0.5mg, 1.0mg, in contrast product solution.Test according to tlc (appendix VIB of Pharmacopoeia of People's Republic of China version in 2005), draw each 5 μ l of above-mentioned trial-product and reference substance solution, put respectively on the same silica gel g thin-layer plate for preparing with the carboxymethylcellulose sodium solution that contains 4% sodium-acetate, with toluene-ethyl acetate-acetone-methyl alcohol (10: 5: 5: 0.6) be developping agent, launching below 15 ℃, taking out, drying, about 30 minutes of 140～160 ℃ of heating, put under the ultra-violet lamp (365nm) and inspect.In the trial-product chromatogram, with the corresponding position of reference substance chromatogram on, show the fluorescence spot of same color.
It is an amount of to get above-mentioned bilobalide elaboration, measure bilobalide content according to " assay " method in high performance liquid chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005) and " Ginkgo Leaf " item of Pharmacopoeia of People's Republic of China version in 2005, the result shows that the content of ginkgolide compound is 95.6% in this bilobalide elaboration.
Test example 1 Different Extraction Method is to the influence test of Ginkgo total flavones, the gingko total terpene lactones rate of transform in the Ginkgo Leaf medicinal material
One, test method
Precision takes by weighing 9 parts of Ginkgo Leaf medicinal powders, every part of 10g.3 parts are adopted 25% extraction using alcohols, and every part adds 10 times of weight 25% ethanol and soaked 1 hour in 60 ℃ of temperature, extracts altogether 3 times, filters merging filtrate; 3 parts are adopted 60% acetone to extract with reference to last method; 3 parts of employings add 10 times of weight water in addition, boil 1 hour, carry altogether 3 times, filter merging filtrate.
Two, measuring method
Content according to efficient form and aspect chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005) and Pharmacopoeia of People's Republic of China " Ginkgo Leaf " Chinese medicinal materials " assay " above-mentioned 3 kinds of method extracting solution kind Ginkgo total flavones of mensuration of version in 2005 and total terpene lactones.
With the cubage content of total flavone of Quercetin, kaempferol, Isorhamnetol, with the total terpene lactones content of the cubage of Ginkgolide A, B, C and bilobalide:
Measurement result sees Table 1.
Table 1 Different Extraction Method is to the influence of Ginkgo total flavones, the gingko total terpene lactones rate of transform in the Ginkgo Leaf medicinal material
| Batch | Diluted Alcohol method (%) | Rare acetone method (%) | Water extraction method (%) | |||
| Total flavones | Total terpene lactones | Total flavones | Total terpene lactones | Total flavones | Total terpene lactones | |
| 1 | 89.6 | 85.1 | 87.1 | 89.3 | 86.2 | 87.2 |
| 2 | 87.3 | 86.4 | 83.7 | 90.1 | 89.1 | 85.3 |
| 3 | 88.4 | 89.2 | 85.4 | 88.6 | 89.4 | 84.5 |
As shown in Table 1, adopt water extraction of the present invention to compare extracts active ingredients rate of transform there was no significant difference in the medicinal material, illustrate and adopt water extraction of the present invention also the effective constituent in the medicinal material fully can be proposed with traditional 25% ethanol or 60% acetone method.
The different enriching methods of test example 2 macroporous adsorbent resins are to the influence test of Ginkgo total flavones, gingko total terpene lactones content in the intermediate
One, test method
1, test group: will test on the prepared ginkgo medicinal material water extract of example 1 behind the AB-8 absorption with macroporous adsorbent resin, elder generation's water and 25% ethanol elution are removed impurity, obtain the weak acid yellow ketones component with the 1.0%NaOH wash-out again, obtain neutral terpene lactones composition with 60～70% ethanol elutions again.
2, control group: on the herbal extract behind the AB-8 macroporous adsorbent resin, it is colourless that elder generation's water is eluted to effluent liquid, it is colourless to effluent liquid to remove impurity with 20% ethanol elution again, uses 60～70% ethanol elution effective constituents then, and the gained intermediate product is the mixture of flavones and terpene lactones.
Two, measuring method
Content according to Ginkgo total flavones and total terpene lactones in efficient form and aspect chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005) and Pharmacopoeia of People's Republic of China " Ginkgo Leaf " Chinese medicinal materials " assay " item above-mentioned test group of mensuration of version in 2005 and the control group gained intermediate.
With the cubage content of total flavone of Quercetin, kaempferol, Isorhamnetol, with the total terpene lactones content of the cubage of Ginkgolide A, B, C and bilobalide.
Three, measurement result
Measurement result sees Table 2.
The different enriching methods of table 2 macroporous adsorbent resin are to the influence of Ginkgo total flavones, gingko total terpene lactones content in the intermediate
| Group | Control group (mixture, %) | Test group (%) | ||
| Total flavones | Total terpene lactones | Total flavones | Total terpene lactones | |
| 1 | 31.6 | 9.2 | 83.6 | 89.2 |
| 2 | 28.3 | 6.4 | 81.9 | 91.3 |
| 3 | 28.4 | 8.9 | 84.2 | 87.5 |
As shown in Table 2, adopt macroporous adsorbent resin diluted alkaline wash-out concentration method of the present invention to compare with the control group method that active constituent content significantly improves in the intermediate product, product can make content all reach more than 95% through simple purification again.Test example 3 different preparation methods are to the influence test of Ginkgo total flavones elaboration, gingko total terpene lactones elaboration yield
One, test method
1, test group: will test in the example 2 test group through the preparation-obtained Ginkgo total flavones crude product of macroporous adsorbent resin enrichment with distilled water wash 3 times, dry, heating is dissolved in an amount of methyl alcohol, filter, filtrate is spent the night in 10～12 ℃ of placements, the crystallization that filtration is separated out in 60 ℃ of dryings, gets the Ginkgo total flavones elaboration.
To test test group in the example 2 through the embodiment 1 method purifying of the preparation-obtained gingko total terpene lactones crude product of macroporous adsorbent resin enrichment, obtain the gingko total terpene lactones elaboration according to the present invention.
2, control group: will test in the example 2 control group effective constituent intermediate product (total flavones and total terpene lactones mixture) in the preparation-obtained Ginkgo Leaf of macroporous adsorbent resin enrichment and be dissolved in water, with an amount of ethyl acetate extraction 3 times.Polymeric amide chromatographic column on the water, washing again with 10% ethanol elution, is collected 95% ethanol elution part, reclaim under reduced pressure is to doing, and heating is dissolved in an amount of methyl alcohol, filters, and filtrate is spent the night in 10～12 ℃ of placements, the crystallization that filtration is separated out in 60 ℃ of dryings, gets the Ginkgo total flavones elaboration.
The acetic acid ethyl acetate extract reclaim under reduced pressure is to doing, add an amount of dissolve with ethanol, be 15% with distilled water diluting ethanol content to the solution again, filter polyamide column on the filtrate, wash post with 5% ethanol again, collect elutriant, reclaim under reduced pressure is to there not being the alcohol flavor, with ethyl acetate extraction, according to the present invention's embodiment 1 method purifying, obtain the gingko total terpene lactones elaboration again.
Two, measuring method
Measure Ginkgo total flavones, gingko total terpene lactones content according to efficient form and aspect chromatography (appendix VID of Pharmacopoeia of People's Republic of China version in 2005), calculate the medicinal material effective component yield according to following formula.
Three, measurement result
The different preparation methods of table 3 are to the influence of Ginkgo total flavones elaboration, gingko total terpene lactones elaboration yield
| Group | Control group (%) | Test group (%) | ||
| Total flavones | Total terpene lactones | Total flavones | Total terpene lactones | |
| 1 | 0.60 | 0.42 | 1.06 | 0.62 |
| 2 | 0.71 | 0.39 | 1.21 | 0.73 |
| 3 | 0.63 | 0.46 | 1.09 | 0.67 |
As shown in Table 3, adopt preparation method's gained Ginkgo total flavones of the present invention and gingko total terpene lactones elaboration yield, so adopt the inventive method that the Ginkgo Leaf herb resource is more made full use of all apparently higher than the method that control group adopted.
Claims (13)
1, a kind of method of extracting and separating bilobalide and ginkgolic flavone glycoside may further comprise the steps:
(1), with fresh or dry Ginkgo Leaf water decoction or with the ginkgo biloba crude extract water dissolution, the decoction liquor or the aqueous solution filter, after gained filtrate is all gone up macroporous adsorptive resins, use distilled water and 5%～25% ethanol elution impurity successively, then colourless to effluent liquid with 0.01%～10% buck wash-out resin column, collect the buck elutriant, be concentrated into dried behind accent pH3～5, washing, oven dry gets ginkgolic flavone glycoside;
(2), to continue to be washed to effluent liquid pH value be neutrality to macroporous adsorptive resins, discards water lotion; Colourless with 30%～95% ethanolic soln wash-out macroporous adsorptive resins again to effluent liquid, collect this ethanol eluate, be evaporated to and do not have the alcohol flavor, extraction, remove extraction solvent under reduced pressure, residue adds sorbent material with methyl alcohol, ethanol, propyl alcohol, propyl carbinol, amylalcohol, acetone, methylethylketone or the above-mentioned mixing solutions dissolving back that two or more is formed with arbitrary proportion, whip attachment, filter, filter residue discards, filtrate decompression reclaims solvent, and residual thing is dry in 40～80 ℃ of environment, gets the bilobalide crude product; Or further the bilobalide crude product is adopted that ordinary method is refining to be elaboration.
2, according to the method for claim 1, it is characterized in that in the step (1) the Ginkgo Leaf medicinal powder being added the water of 1～100 times of medicinal material amount (W/V), decoct 1-3 time, decoct 5min～3h at every turn.
3,, it is characterized in that described macroporous adsorptive resins is AB-8, X-5, S-8, SIP-1300, MDA, NKA-II, D-101, HPD-100, HPD-400, ADS-17 or BS-55 macroporous adsorptive resins according to the method for claim 1.
4, according to the method for claim 1, when it is characterized in that filtrate is gone up macroporous adsorptive resins in the step (1), the macroporous resin consumption is 1: 10～10: 1 with the ratio (V/W) of crude drug Ginkgo Leaf consumption, and control filtrate flow rate of liquid is 0.5～10 times of resin column volume/h.
5, according to the method for claim 4, it is characterized in that the macroporous resin consumption and the ratio (V/W) of crude drug Ginkgo Leaf consumption are 1: 1～3: 1, control filtrate flow rate of liquid is 1～3 times of resin column volume/h.
6, according to the method for claim 1, when it is characterized in that using in the step (1) distillation washing post, with the distilled water wash-out macroporous adsorptive resins of 3～50 times of resin column volumes, the flow velocity of control elutriant is 0.5～10 times of resin column volume/h.
7, according to the method for claim 6, it is characterized in that distilled water wash-out with 5～8 times of resin column volumes, the flow velocity of control elutriant is 1～3 times of resin column volume/h.
8,, it is characterized in that in the step (1) that with 20% ethanol elution impurity elution amount is till the effluent liquid clear according to the method for claim 1; Then carry out wash-out with 0.1～1%NaOH, elution amount be to effluent liquid light or colourless till.
9, according to the method for claim 1, when it is characterized in that in the step (2) with distilled water wash-out macroporous adsorptive resins, its elution amount be neutrality to effluent liquid pH value till; Then with 60% ethanol elution macroporous adsorptive resins, elution amount be to effluent liquid light or colourless till; Collected ethanol eluate is evaporated to not to be had when extracting behind the alcohol flavor, with the ethyl acetate of 1～20 times of weight, ethyl formate, methyl acetate, propyl carbinol, amylalcohol or extracted with diethyl ether 2～10 times; After removing the extraction organic solvent under reduced pressure, residue is pure or low charcoal ketone or the mutual mixing solutions dissolving of being formed with arbitrary proportion with the low charcoal of 1/10～5 times of weight, the gac that in lysate, adds 1/100～1/5 times of weight, whip attachment 15min～5h, filter, filter residue discards, and filtrate decompression reclaims solvent, residual thing is dry in 50～60 ℃ of environment, gets the bilobalide crude product.
10,, it is characterized in that collected ethanol eluate is evaporated to and do not have alcohol flavor back with the ethyl acetate extraction of 4 times of weight 5 times according to the method for claim 9; After removing ethyl acetate under reduced pressure, residue adds the gac of 1/10 times of weight, whip attachment 1～2h with the dissolve with ethanol of 1～3 times of weight in lysate.
11,, when it is characterized in that in the step (2) that the bilobalide crude product refining is elaboration, may further comprise the steps according to the method for claim 1:
The bilobalide crude product is dissolved in the mixing solutions that methyl alcohol, ethanol, propyl alcohol, acetone or above-mentioned two or more solvents are formed with arbitrary proportion, adds distilled water, centrifugal gluey water liquid, the drying under reduced pressure removed; Dried bilobalide solid is dissolved in methyl alcohol, ethanol, propyl alcohol, acetone or the above-mentioned mixing solutions that two or more is formed with arbitrary proportion, smart filter, filtrate is spent the night placing below 25 ℃, separate out white crystals, filter, drying gets the bilobalide elaboration.
12, according to the method for claim 11, it is characterized in that the bilobalide crude product is dissolved in the acetone of 0.5～10 times of weight, stir the distilled water that slowly adds 2～20 times of weight down, centrifugal gluey water liquid, the drying under reduced pressure removed; Add in dried bilobalide solid in 0.5～5 times of amount (W/V), 10%～60% methyl alcohol, in 40～80 ℃ of dissolvings, adopt the smart filter of millipore filtration, filtrate is spent the night 10～12 ℃ of placements.
13, according to the method for claim 12, it is characterized in that the bilobalide crude product is dissolved in the acetone of 2～3 times of weight, stir the distilled water that slowly adds 8 times of weight down; In dried bilobalide solid, add in 30% methyl alcohol of equivalent (W/V), in 50～60 ℃ of dissolvings.
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| CN104387356A (en) * | 2013-11-15 | 2015-03-04 | 广西莱吉生物工程有限公司 | Method for extracting flavone from folium ginkgo |
| CN103664981A (en) * | 2013-12-02 | 2014-03-26 | 沃太能源南通有限公司 | Method for extracting lactone from ginkgo nuts |
| CN103664981B (en) * | 2013-12-02 | 2015-12-09 | 沃太能源南通有限公司 | The extracting method of lactone in a kind of gingko |
| CN106176838A (en) * | 2016-07-18 | 2016-12-07 | 湖南绿蔓生物科技股份有限公司 | A kind of production method of Folium Ginkgo extract |
| CN106176838B (en) * | 2016-07-18 | 2019-10-25 | 湖南绿蔓生物科技股份有限公司 | A kind of production method of ginkgo biloba p.e |
| CN106214708A (en) * | 2016-08-31 | 2016-12-14 | 重庆万物春生制药有限公司 | A kind of extracting method of Semen Ginkgo |
| CN106800562A (en) * | 2016-12-14 | 2017-06-06 | 浙江科技学院 | A kind of ginkgolides preparation method for removing ginkgoic acid |
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