CN103130816A - Method of preparing a plurality of active substances from ginkgo leaves - Google Patents
Method of preparing a plurality of active substances from ginkgo leaves Download PDFInfo
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Abstract
The invention discloses a method of preparing a plurality of activation materials from ginkgo leaves, and belongs to a manufacturing method of chemical substances or a manufacturing method of drugs. Strong polarity macroporous adsorption resin or polarity macroporous adsorption resin is adopted for separation, and is adopted to solve the problems that the amount of ginkgolic acid is high when the prior ginkgo extract is prepared, and solve the problems that a large amount of organic solvent is consumed when the prior ginkgo active substances are extracted and prepared, and accordingly the environment is polluted, or that a complex separation technology can not enlarge production, or that extraction efficiency is not high, and use ratio of raw materials is low and the like. The method of preparing the plurality of activation materials from the ginkgo leaves can simultaneously obtain a plurality of active substances such as ginkgo polysaccharide, ginkgol biloba extracts, ginkgol biloba general flavone, ginkgol biloba total lactones, gbilobalide A monosome, gbilobalide B monosome, gbilobalide C monosome and the like. The technical scheme is that the method comprises a raw material pretreatment step, an extraction step, a polysaccharide precipitation step, a macroporous resin separation step, a silica gel dry-column separation step, a reversed phase chromatography purification step, a crystallization step and the like, saves production cost, and improves work efficiency. The process of the method is simple and convenient to conduct. The method can be easily enlarged to the commercial process, and meanwhile greatly increase use ratio of raw materials.
Description
Technical field
The invention belongs to the manufacture method of chemical substance or medicine, be specifically related to take Ginkgo Leaf as raw material, through extraction separation and purification, obtain the method for various active material.
Background technology
Before 200,000,000 years, the Ginkgoaceae plant extensively is present in Asia, non-, Europe, South America and Australia.Almost be dispersed throughout whole earth surface.Due to the tertiary period and earth generation great change in period in the quaternary period, most of Ginkgoaceae plant extinction only has a kind of survival of ginkgo, thereby is called as " living fossil " plant.Ginkgo is the distinctive rare tree of China, and especially on the south the Changjiang river and Taiwan Province, account for more than 70% of world's gingko resource total amount.Along with excavation and the exploitation of the comprehensive values such as medicinal, the health care of effective constituent in Ginkgo Leaf, the systematic study of Ginkgo Leaf has worldwide been started climax, the Action mechanism of its Ginkgo Leaf chemical composition and clinical application become the focus of research.
China has recognized the pharmaceutical use of ginkgo very early, and Ming Dynasty's LI Shi-Zhen is mentioned in Compendium of Material Medica, ginkgo " Dingchuan is coughed, and dwindles just for people's lung channel, beneficial lung qi ".The sixties in 20th century, Germany scientist discovery ginkgolic flavone glycoside can be prevented and treated cardiovascular and cerebrovascular diseases and reducing blood-fat.20 century 70s, German medical science media report, Folium Ginkgo extract energy increase maincenter and peripheral blood vessel volume of blood flow can be used for the heart, brain and peripheral blood vessel ischemic disease.The eighties in 20th century, French scientist Brapuat has found that the Ginkgolide Component of Ginkgo Leaf has the effect of very strong antagonism platelet activation factor (PAF), gingko leaf preparation enters clinical PAF antagonist as first and has carried out the clinical observation of three phases subsequently, and the research and development of gingko leaf preparation has entered a new field.1991, the scholar of Harvard University won the Nobel prize because of the molecular structure of finding Ginkgolide B.So far in same research in the world, have ginkgo only and obtain the Nobel prize twice.Subsequently, more deep, research widely that the purposes of Folium Ginkgo extract obtains, as be used for treating the illnesss such as chronic renal failure, coronary heart disease, sudden deafness, mucosal lesion, schizophrenia, Alzheimer's disease, cerebral thrombosis, and can be antitumor.Further research also shows, Ginkgolide B is used for treatment and the hemodialysis of apoplexy, organ transplant rejection, shock etc. clinically, and its successful is better than the bilobalide mixture, so the purified extract B of Ginkgo Leaf has appearred again in the world market.
In Ginkgo Leaf, the preparation technology of effective constituent mainly contains with Types Below: 1, steam distillation: after Ginkgo Leaf drying, pulverizing, 100～120 ℃ of water distillations are after distillate is cooling, with non-polar macroporous resin absorption, concentrated, then use ethanol elution, evaporate to dryness namely obtains the powdery crystallization.The method extraction yield is very low; 2, organic extractant solution method: the patent of its extraction process is also more, can be summarized as two kinds of forms: a kind of is the preparation ginkgo biloba crude extract, these class methods be dry Ginkgo Leaf, soak, extraction, filter with organic solvent (such as ethanol or acetone, sherwood oil etc.) after pulverizing, solvent in filtrate gets ginkgo biloba extract after removing with underpressure distillation.The preparation of this method is simple, the yield of product high (approximately 12%～14%), but in product, foreign matter content is higher, usually is brownish black.Another kind is preparation ginkgo leaf extract extract, and this method prepares on the basis at crude extract usually, and further refining, process for purification commonly used has: liquid-liquid extraction (for example using methylene dichloride, chloroform or benzene degreasing), the precipitator method (NH
4The precipitation agent such as OH or PbAc) and diatomite or silica gel adsorption.By after drying, filtration, concentrating under reduced pressure desolventizing, can obtain extract again.Be generally the tawny powder, yield is 1～3%, and these class methods expend a large amount of organic reagents, and the product purity that obtains is not high; 3, supercritical fluid extraction: over past ten years, also more and more extensive with the effective medicinal components that this technology is extracted in natural phant.It and above-mentioned extraction process are compared, has extraction yield high, in no solvent residue toxicity, natural phant, activeconstituents and thermolability composition are difficult for being decomposed and destroy and keep the advantage such as its natural feature, but the further investigation along with supercritical fluid extraction, the deficiency of this technology is needing can not obtain the single component sterling of separation, is roughing out for Ginkgolide A. B. C.
At present, also have manyly about adopting post separation and Extraction, purifying, prepare the method for Semen Ginkgo extrac, but these methods all consume a large amount of organic solvents basically, cause environmental pollution; Or use comparatively complicated isolation technique to be difficult to amplify and produce; Or it is not high to get efficient, and raw material availability is lower.
Summary of the invention
The invention provides a kind of method for preparing the various active material from Ginkgo Leaf, adopt strong polarity or polar macroporous adsorption resin to separate, solve the higher problem of existing preparation Semen Ginkgo extrac ginkgoic acid, and solution has extraction now and preparation ginkgo active substance expends a large amount of organic solvents, causes environmental pollution; Or use comparatively complicated isolation technique to be difficult to amplify and produce; Or it is not high to get efficient, the problem such as raw material availability is lower.And can obtain simultaneously the various active materials such as polysaccharides from ginkgo biloba, Semen Ginkgo extrac, Ginkgo total flavones, Ginkgolides, Ginkgolide A. B. C monomer.
A kind of method for preparing the various active material from Ginkgo Leaf of the present invention comprises:
(1) raw material pre-treatment step; After Ginkgo Leaf dry product raw material pulverizing, add the aqueous solution that contains cellulase and polygalacturonase to carry out the broken wall reaction, oven dry obtains extracting raw material;
(2) extraction step; At the extraction raw material that step (1) obtains, 50～80% extraction using alcohols extract under 60～90 ℃, and solid-liquid ratio 1: 8～1: 20 extracts 2～4 times, obtains extracting solution after merging;
(3) polysaccharide precipitation step; The precipitate and separate step; After the extracting solution that obtains in step (3) is concentrated, add flocculation agent to carry out precipitin reaction, filter and be precipitated the thing polysaccharides from ginkgo biloba; The supernatant liquor that filtration obtains is precipitate and separate liquid;
(4) macroporous resin separating step; With the precipitate and separate liquid that obtains in step (3), through polarity or strong polar macroporous resin isolation, obtain Semen Ginkgo extrac;
(5) the dried post separating step of silica gel; The Semen Ginkgo extrac wet method that obtains in step (4) is splined on the dried post of silica gel, and the mass ratio of Semen Ginkgo extrac and silica gel is 1/50～1/100, utilizes pressure difference with the developping agent descending development under normal pressure, obtains respectively Ginkgo total flavones and Ginkgolides; Developping agent is the mixed solution of methyl alcohol and normal hexane;
(6) reversed phase chromatography purification step; To the Ginkgolides that obtains in step (5), with C
18Filler is stationary phase, take the mixed solution of methyl alcohol and water as moving phase, carries out the mesolow reversed phase chromatography, collects respectively the positive component that is rich in Ginkgolide A, Ginkgolide B, ginkalide C;
(7) crystallisation step; Respectively with three positive component underpressure distillation of reversed phase chromatography to the dried solid substance that obtains, respectively adding concentration is 60%～80% methanol aqueous solution, after fully dissolving under 40 ℃～60 ℃, after filtered while hot, freezing and crystallizing obtains respectively purity greater than 98% Ginkgolide A, Ginkgolide B, ginkalide C.
A kind of described method for preparing the various active material from Ginkgo Leaf, it is characterized in that: in described raw material pre-treatment step, the aqueous solution of described cellulase and polygalacturonase is: the volume of the aqueous solution and the mass ratio of raw material are 0.5～2: 1 (volume mass ratio), the amount of cellulase and the mass ratio of raw material are 0.5～3: 1000 (mass ratioes), and the quality of polygalacturonase and the mass ratio of raw material are 0.5～2: 1000 (mass ratioes); Described broken wall reaction is: 30～60 ℃ of temperature of reaction, 1～3 hour reaction times.
A kind of described method for preparing the various active material from Ginkgo Leaf is characterized in that: in described polysaccharide precipitation step, described extracting solution simmer down to: the volume mass ratio that extracting solution is concentrated into Ginkgo Leaf dry product raw material is 2～5: 1 (L/kg); Described flocculation agent agent is: a kind of in chitosan, polyphenyl alkene acid amides, polyaerylic acid sodium, polyaerylic acid calcium; The described flocculation agent that adds carries out precipitin reaction, filtration is precipitated the thing Flos Lonicerae polysaccharide, its process is: add flocculation agent in concentrated solution, to add the mass volume ratio of flocculation agent and concentrated solution be 0.05～2: 100 (kg/L), after stirring 10～30min, standing 6～12 hours of normal temperature obtains Flos Lonicerae polysaccharide after filtration is precipitated the thing drying.
A kind of described method for preparing the various active material from Ginkgo Leaf is characterized in that: in described macroporous resin separating step, described polarity or strong polar macroporous resin are: a kind of in ADS-2, ADS-7, HPD-826, HPD-600; Described through polarity or strong polar macroporous resin isolation, its process is: precipitate and separate liquid is loaded on macroporous resin, aqueous ethanolic solution wash-out 2～4 column volumes with pure water or 5%～15%, discard elutriant, use again 40%～60% aqueous ethanolic solution wash-out 2～4 column volumes, collect elutriant, drying obtains Semen Ginkgo extrac.
A kind of described method for preparing the various active material from Ginkgo Leaf, it is characterized in that: in the dried post separating step of described silica gel, the dried post of described silica gel is: be filled with silica gel based filler, the aspect ratio device of 5: 1～20: 1, the silica gel based filler is comprised of silica gel and diatomite, and the mass percent of diatomite and silica gel is 10%～20%; The silica gel particle diameter is 30 μ m～80 μ m, and the silica gel activity is II～III level;
The filling process of the dried post of described silica gel is: silica gel based filler and isopyknic organic solvent are mixed into suspension liquid, pour the hollow PVC post that is plugged with glass wool and two logical pistons into, the silica gel based filler constantly deposits, solvent flows out through glass wool, control the take-off rate of solvent, silica gel based filler surface is covered all the time, until be filled into required height by solvent; Described organic solvent is a kind of in normal hexane, sherwood oil, Skellysolve A.
A kind of described method for preparing the various active material from Ginkgo Leaf is characterized in that: in the dried post separating step of described silica gel, the volume ratio of described developping agent is: methyl alcohol: normal hexane=90: 10～98: 2; The process of described descending development is: developping agent is added from the dried capital of silica gel, utilize the pressure difference descending development under normal pressure, when developer flow to silica gel is done the column bottom, stop adding developping agent; The dried shell of column cutting of silica gel that to be rich in respectively ginkgolic flavone glycoside and bilobalide scrapes, with trichloromethane vibration wash-out 10～20 minutes, repeat wash-out 2～3 times, with the chloroform soln evaporated under reduced pressure that obtains, the solid substance that obtains respectively is Ginkgo total flavones and Ginkgolides; The described dried shell of column of silica gel that is rich in ginkgolic flavone glycoside and bilobalide is both the Rf value R that obtains with reference to silica gel thin-layer experiment under identical developping agent condition
fDetermine.
A kind of described method for preparing the various active material from Ginkgo Leaf is characterized in that: in described reversed phase chromatography purification step: stationary phase is C
18Filler, particle diameter are 20～50 μ m, load into aspect ratio and be the chromatography column of 20: 1～60: 1; In described moving phase, the volume ratio of methyl alcohol and water is 60: 40～90: 10; Described mesolow reversed phase chromatography process is: described Ginkgolides is added from the chromatography capital, and the mass ratio of Ginkgolides and C18 filler is 0.01～0.02: 1; Chromatography column pressure is 5～40bar; Described flow rate of mobile phase is 2～10ml/min, collects respectively the positive component that is rich in Ginkgolide A, Ginkgolide B, ginkalide C.
A kind of described method for preparing the various active material from Ginkgo Leaf is characterized in that: in described crystallisation step, the mass ratio of the volume of described methanol aqueous solution and evaporate to dryness solid substance is 0.5～2: 1 (ml/g); Described freezing and crystallizing temperature is :-20 ℃～4 ℃.
The invention has the advantages that, conception rationally, method is easy, easily promote, the invention provides a kind of method for preparing the various active material from Ginkgo Leaf, adopt strong polarity or polar macroporous adsorption resin to separate, solve the higher problem of existing preparation Semen Ginkgo extrac ginkgoic acid, and solution has extraction now and preparation ginkgo active substance expends a large amount of organic solvents, causes environmental pollution; Or use comparatively complicated isolation technique to be difficult to amplify and produce; Or it is not high to get efficient, the problem such as raw material availability is lower.And can obtain simultaneously the various active materials such as polysaccharides from ginkgo biloba, Semen Ginkgo extrac, Ginkgo total flavones, Ginkgolides, Ginkgolide A. B. C monomer.The project technology comprises raw material pre-treatment step, extraction step, polysaccharide precipitation step, macroporous resin separating step, the dried post separating step of silica gel, reversed phase chromatography purification step and crystallisation step etc., saved production cost, improved production efficiency, and simple process also is easy to be amplified to suitability for industrialized production, can greatly improve raw material availability simultaneously.
Embodiment
Embodiment 1
(1) raw material pre-treatment step:
Getting the Ginkgo Leaf dry product is raw material, after pulverizing and sieving, accurately take 1000g, add the aqueous solution 500ml that contains cellulase 0.50g, polygalacturonase 0.50g, wherein the mass ratio of the volume of the aqueous solution and raw material is 0.5: 1 (volume mass ratio), and the amount of cellulase and the mass ratio of raw material are 0.5: 1000 (mass ratio), the quality of polygalacturonase and the mass ratio of raw material are 0.5: 1000 (mass ratio), be heated to 60 ℃, reacted 1 hour, oven dry obtains extracting raw material;
(2) extraction step
With the extraction raw material of gained in step (1), 80% extraction using alcohol extracts under 60 ℃, and solid-liquid ratio 1: 20 extracts united extraction liquid 2 times;
(3) polysaccharide precipitation step
Obtain concentrated solution 5000ml after the extracting solution that obtains in step (2) is concentrated, concentrated solution is 5: 1 (L/kg) with the volume mass ratio of Japanese Honeysuckle dry product raw material, adding the 0.05kg chitosan is flocculation agent, to add the mass volume ratio of flocculation agent and concentrated solution be 1: 100 (kg/L), after stirring 30min, standing 12 hours of normal temperature filters and is precipitated thing, obtains polysaccharides from ginkgo biloba 71.15g after drying; The precipitation supernatant liquor is precipitate and separate liquid.
(4) macroporous resin separating step
After the precipitate and separate liquid that obtains in step (3) was loaded to the ADS-2 macroporous adsorbent resin, 5% aqueous ethanolic solution wash-out 4 column volumes after abandoning, after 40% aqueous ethanolic solution wash-out 2 column volumes, were collected elutriant, obtain Semen Ginkgo extrac 45.90g after drying;
(5) the dried post separating step of silica gel
Get 4590g, the III level activated silica gel silica gel of particle diameter 50 μ m, the mass ratio of Semen Ginkgo extrac and silica gel is 1/100, and the diatomite (mass percent of diatomite and silica gel is 10%) of interpolation 459g, add isopyknic normal hexane to mix and become suspension liquid, pour the hollow PVC post that is plugged with glass wool and two logical pistons into, the silica gel based filler constantly deposits, solvent flows out through glass wool, control the take-off rate of solvent, silica gel based filler surface is covered by solvent all the time, until filler all changes the PVC post over to, the post aspect ratio is 5: 1;
The Semen Ginkgo extrac 45.90g wet method that obtains is splined on the dried post of silica gel, under condition of normal pressure, adopts methyl alcohol: normal hexane=90: 10 (V/V) solvent is as developping agent, and descending development until solvent is opened up at the bottom of the post, stops the exhibition layer; Experiment obtains with reference to thin layer ginkgolic flavone glycoside and bilobalide R
fValue will be rich in respectively the dried shell of column cutting of the silica gel of ginkgolic flavone glycoside and bilobalide and scrape, and trichloromethane soaks the vibration wash-out 2 times, each 20 minutes, the chloroform soln that obtains in evaporated under reduced pressure, be obtained respectively Ginkgo total flavones 5.56g, Ginkgolides 2.40g.
(6) reversed phase chromatography purification step
Getting particle diameter is 20 μ m, 240g C
18Filler, Ginkgolides and C
18The mass ratio of filler is 0.01: 1, loads into aspect ratio and be the chromatography column of 60: 1; After the Ginkgolides 2.40g loading that obtains, methyl alcohol: water=60: 40 (V/V) elutriant wash-out, chromatography column pressure is 5bar, flow velocity is 2ml/min, collects respectively the positive component that is rich in Ginkgolide A, Ginkgolide B, ginkalide C.
(7) crystallisation step
Respectively with three positive component underpressure distillation of reversed phase chromatography to the dried solid substance that obtains, respectively adding concentration is 60% methanol aqueous solution, add the volume of methanol aqueous solution and the mass ratio of evaporate to dryness solid substance is 0.5: 1 (ml/g), after fully dissolving under 60 ℃, after filtered while hot,-20 ℃ of freezing and crystallizings obtain respectively purity greater than 98% Ginkgolide A 0.27g, Ginkgolide B 0.30g, ginkalide C 0.25g.
Embodiment 2
(1) raw material pre-treatment step:
Getting the Ginkgo Leaf dry product is raw material, after pulverizing and sieving, accurately take 2000g, add the aqueous solution 4000ml that contains cellulase 6.0g, polygalacturonase 4.0g, wherein the mass ratio of the volume of the aqueous solution and raw material is 2: 1 (volume mass ratio), the amount of cellulase and the mass ratio of raw material are 3: 1000 (mass ratio), the quality of polygalacturonase and the mass ratio of raw material are 2: 1000 (mass ratio), are heated to 30 ℃, react after 3 hours, oven dry obtains extracting raw material;
(2) extraction step
With the extraction raw material of gained in step (1), 50% extraction using alcohol extracts under 90 ℃, and solid-liquid ratio 1: 8 extracts united extraction liquid 4 times;
(3) polysaccharide precipitation step
Obtain concentrated solution 4000ml after the extracting solution that obtains in step (2) is concentrated, concentrated solution is 2: 1 (L/kg) with the volume mass ratio of Japanese Honeysuckle dry product raw material, adding 0.08kg polyphenyl alkene acid amides is flocculation agent, to add the mass volume ratio of flocculation agent and concentrated solution be 2: 100 (kg/L), after stirring 10min, standing 6 hours of normal temperature filters and is precipitated thing, obtains polysaccharides from ginkgo biloba 146.77g after drying; The precipitation supernatant liquor is precipitate and separate liquid.
(4) macroporous resin separating step
After the precipitate and separate liquid that obtains in step (3) was loaded to the ADS-7 macroporous adsorbent resin, 15% aqueous ethanolic solution wash-out 2 column volumes after abandoning, after 60% aqueous ethanolic solution wash-out 4 column volumes, were collected elutriant, obtain Semen Ginkgo extrac 92.70g after drying;
(5) the dried post separating step of silica gel
Get 7416g, the III level activated silica gel silica gel of particle diameter 30 μ m, the mass ratio of Semen Ginkgo extrac and silica gel is 1/80, and the diatomite (mass percent of diatomite and silica gel is 20%) of interpolation 1483.2g, add isopyknic petroleum ether and stirring to be mixed into suspension liquid, pour the hollow PVC post that is plugged with glass wool and two logical pistons into, the silica gel based filler constantly deposits, solvent flows out through glass wool, control the take-off rate of solvent, silica gel based filler surface is covered by solvent all the time, until filler all changes the PVC post over to, the post aspect ratio is 20: 1;
The Semen Ginkgo extrac 92.70g wet method that obtains is splined on the dried post of silica gel, under condition of normal pressure, adopts methyl alcohol: normal hexane=98: 2 (V/V) solvent is as developping agent, and descending development until solvent is opened up at the bottom of the post, stops the exhibition layer; Experiment obtains with reference to thin layer ginkgolic flavone glycoside and bilobalide R
fValue, the dried shell of column cutting of silica gel that will be rich in respectively ginkgolic flavone glycoside and bilobalide scrapes, and trichloromethane soaks the vibration wash-out 3 times, each 10 minutes, the chloroform soln that obtains in evaporated under reduced pressure, is obtained respectively Ginkgo total flavones 11.90g, Ginkgolides 5.10g.
(6) reversed phase chromatography purification step
Getting particle diameter is 50 μ m, 255g C
18Filler, Ginkgolides and C
18The mass ratio of filler is 0.02: 1, loads into aspect ratio and be the chromatography column of 20: 1; After the Ginkgolides 5.10g loading that obtains, methyl alcohol: water=90: 10 (V/V) elutriant wash-out, chromatography column pressure is 20bar, flow velocity is 10ml/min, collects respectively the positive component that is rich in Ginkgolide A, Ginkgolide B, ginkalide C.
(7) crystallisation step
Respectively with three positive component underpressure distillation of reversed phase chromatography to the dried solid substance that obtains, respectively adding concentration is 80% methanol aqueous solution, add the volume of methanol aqueous solution and the mass ratio of evaporate to dryness solid substance is 2: 1 (ml/g), after fully dissolving under 40 ℃, after filtered while hot,-10 ℃ of freezing and crystallizings obtain respectively purity greater than 98% Ginkgolide A 0.55g, Ginkgolide B 0.57g, ginkalide C 0.43g.
Embodiment 3
(1) raw material pre-treatment step:
The extracting honeysuckle dry product is raw material, after pulverizing and sieving, accurately take 1000g, add the aqueous solution 1000ml that contains cellulase 2.0g, polygalacturonase 1.0g, wherein the mass ratio of the volume of the aqueous solution and raw material is 1: 1 (volume mass ratio), the amount of cellulase and the mass ratio of raw material are 2: 1000 (mass ratio), the quality of polygalacturonase and the mass ratio of raw material are 1: 1000 (mass ratio), are heated to 45 ℃, react after 2 hours, oven dry obtains extracting raw material;
(2) extraction step
With the extraction raw material of gained in step (1), 70% extraction using alcohol extracts under 75 ℃, and solid-liquid ratio 1: 12 extracts united extraction liquid 3 times;
(3) polysaccharide precipitation step
Obtain concentrated solution 5000ml after the extracting solution that obtains in step (2) is concentrated, concentrated solution is 4: 1 (L/kg) with the volume mass ratio of Japanese Honeysuckle dry product raw material, adding 0.05kg polyaerylic acid sodium is flocculation agent, to add the mass volume ratio of flocculation agent and concentrated solution be 0.05: 100 (kg/L), after stirring 20min, standing 8 hours of normal temperature filters and is precipitated thing, obtains polysaccharides from ginkgo biloba 69.75g after drying; The precipitation supernatant liquor is precipitate and separate liquid.
(4) macroporous resin separating step
After the precipitate and separate liquid that obtains in step (3) is loaded to the HPD-826 macroporous adsorbent resin, 10% aqueous ethanolic solution wash-out 3 column volumes are after abandoning, after 50% aqueous ethanolic solution wash-out 3 column volumes, collect elutriant, obtain Semen Ginkgo extrac 49.50g after drying;
(5) the dried post separating step of silica gel
Get 2475g, the II level activated silica gel silica gel of particle diameter 80 μ m, the mass ratio of Semen Ginkgo extrac and silica gel is 1/50, and the diatomite (mass percent of diatomite and silica gel is 15%) of interpolation 371.25g, add isopyknic Skellysolve A to mix and become suspension liquid, pour the hollow PVC post that is plugged with glass wool and two logical pistons into, the silica gel based filler constantly deposits, solvent flows out through glass wool, control the take-off rate of solvent, silica gel based filler surface is covered by solvent all the time, until filler all changes the PVC post over to, the post aspect ratio is 15: 1;
The Semen Ginkgo extrac 49.50g wet method that obtains is splined on the dried post of silica gel, under condition of normal pressure, adopts methyl alcohol: normal hexane=95: 5 (V/V) solvent is as developping agent, and descending development until solvent is opened up at the bottom of the post, stops the exhibition layer; Experiment obtains with reference to thin layer ginkgolic flavone glycoside and bilobalide R
fValue will be rich in respectively the dried shell of column cutting of the silica gel of ginkgolic flavone glycoside and bilobalide and scrape, and trichloromethane soaks the vibration wash-out 3 times, each 15 minutes, the chloroform soln that obtains in evaporated under reduced pressure, be obtained respectively Ginkgo total flavones 5.92g, Ginkgolides 2.65g.
(6) reversed phase chromatography purification step
Getting particle diameter is 30 μ m, 176.67g C
18Filler, Ginkgolides and C
18The mass ratio of filler is 0.015: 1, loads into aspect ratio and be the chromatography column of 40: 1; After the Ginkgolides 2.65g loading that obtains, methyl alcohol: water=70: 30 (V/V) elutriant wash-out, chromatography column pressure is 40bar, flow velocity is 5ml/min, collects respectively the positive component that is rich in Ginkgolide A, Ginkgolide B, ginkalide C.
(7) crystallisation step
Respectively with three positive component underpressure distillation of reversed phase chromatography to the dried solid substance that obtains, respectively adding concentration is 70% methanol aqueous solution, add the volume of methanol aqueous solution and the mass ratio of evaporate to dryness solid substance is 1: 1 (ml/g), after fully dissolving under 50 ℃, after filtered while hot, 4 ℃ of freezing and crystallizings obtain respectively purity greater than 98% Ginkgolide A 0.19g, Ginkgolide B 0.23g, ginkalide C 0.17g.
Embodiment 4
(1) raw material pre-treatment step:
Getting the Ginkgo Leaf dry product is raw material, after pulverizing and sieving, accurately take 2000g, add the aqueous solution 4000ml that contains cellulase 8.0g, polygalacturonase 8.0g, wherein the mass ratio of the volume of the aqueous solution and raw material is 2: 1 (volume mass ratio), the amount of cellulase and the mass ratio of raw material are 2: 1000 (mass ratio), the quality of polygalacturonase and the mass ratio of raw material are 2: 1000 (mass ratio), are heated to 60 ℃, react after 1 hour, oven dry obtains extracting raw material;
(2) extraction step
With the extraction raw material of gained in step (1), 80% extraction using alcohol extracts under 70 ℃, and solid-liquid ratio 1: 12 extracts united extraction liquid 3 times;
(3) polysaccharide precipitation step
Obtain concentrated solution 6000ml after the extracting solution that obtains in step (2) is concentrated, concentrated solution is 3: 1 (L/kg) with the volume mass ratio of Japanese Honeysuckle dry product raw material, adding 0.06kg polyaerylic acid calcium is flocculation agent, to add the mass volume ratio of flocculation agent and concentrated solution be 1: 100 (kg/L), after stirring 30min, standing 12 hours of normal temperature filters and is precipitated thing, obtains polysaccharides from ginkgo biloba 143.19g after drying; The precipitation supernatant liquor is precipitate and separate liquid.
(4) macroporous resin separating step
After the precipitate and separate liquid that obtains in step (3) was loaded to the HPD-600 macroporous adsorbent resin, pure water wash-out 4 column volumes after abandoning, after 50% aqueous ethanolic solution wash-out 3 column volumes, were collected elutriant, obtain Semen Ginkgo extrac 94.45g after drying;
(5) the dried post separating step of silica gel
Get 9445g, the II level activated silica gel silica gel of particle diameter 80 μ m, the mass ratio of Semen Ginkgo extrac and silica gel is 1/100, and the diatomite (mass percent of diatomite and silica gel is 10%) of interpolation 944.5g, add isopyknic normal hexane to mix and become suspension liquid, pour the hollow PVC post that is plugged with glass wool and two logical pistons into, the silica gel based filler constantly deposits, solvent flows out through glass wool, control the take-off rate of solvent, silica gel based filler surface is covered by solvent all the time, until filler all changes the PVC post over to, the post aspect ratio is 20: 1;
The Semen Ginkgo extrac 94.45g wet method that obtains is splined on the dried post of silica gel, under condition of normal pressure, adopts methyl alcohol: normal hexane=94: 6 (V/V) solvent is as developping agent, and descending development until solvent is opened up at the bottom of the post, stops the exhibition layer; Experiment obtains with reference to thin layer ginkgolic flavone glycoside and bilobalide R
fValue, the dried shell of column cutting of silica gel that will be rich in respectively ginkgolic flavone glycoside and bilobalide scrapes, and trichloromethane soaks the vibration wash-out 3 times, each 20 minutes, the chloroform soln that obtains in evaporated under reduced pressure, is obtained respectively Ginkgo total flavones 12.16g, Ginkgolides 5.30g.
(6) reversed phase chromatography purification step
Getting particle diameter is 20 μ m, 530g C
18Filler, Ginkgolides and C
18The mass ratio of filler is 0.01: 1, loads into aspect ratio and be the chromatography column of 60: 1; After the Ginkgolides 5.30g loading that obtains, methyl alcohol: water=80: 20 (V/V) elutriant wash-out, chromatography column pressure is 20bar, flow velocity is 8ml/min, collects respectively the positive component that is rich in Ginkgolide A, Ginkgolide B, ginkalide C.
(7) crystallisation step
Respectively with three positive component underpressure distillation of reversed phase chromatography to the dried solid substance that obtains, respectively adding concentration is 70% methanol aqueous solution, add the volume of methanol aqueous solution and the mass ratio of evaporate to dryness solid substance is 0.5: 1 (ml/g), after fully dissolving under 60 ℃, after filtered while hot,-20 ℃ of freezing and crystallizings obtain respectively purity greater than 98% Ginkgolide A 0.58g, Ginkgolide B 0.55g, ginkalide C 0.41g.
Claims (8)
1. method for preparing the various active material from Ginkgo Leaf comprises:
(1) raw material pre-treatment step; After Ginkgo Leaf dry product raw material pulverizing, add the aqueous solution that contains cellulase and polygalacturonase to carry out the broken wall reaction, oven dry obtains extracting raw material;
(2) extraction step; At the extraction raw material that step (1) obtains, 50～80% extraction using alcohols extract under 60～90 ℃, and solid-liquid ratio 1: 8～1: 20 extracts 2～4 times, obtains extracting solution after merging;
(3) polysaccharide precipitation step; The precipitate and separate step; After the extracting solution that obtains in step (3) is concentrated, add flocculation agent to carry out precipitin reaction, filter and be precipitated the thing polysaccharides from ginkgo biloba; The supernatant liquor that filtration obtains is precipitate and separate liquid;
(4) macroporous resin separating step; With the precipitate and separate liquid that obtains in step (3), through polarity or strong polar macroporous resin isolation, obtain Semen Ginkgo extrac;
(5) the dried post separating step of silica gel; The Semen Ginkgo extrac wet method that obtains in step (4) is splined on the dried post of silica gel, and the mass ratio of Semen Ginkgo extrac and silica gel is 1/50～1/100, utilizes pressure difference with the developping agent descending development under normal pressure, obtains respectively Ginkgo total flavones and Ginkgolides; Developping agent is the mixed solution of methyl alcohol and normal hexane;
(6) reversed phase chromatography purification step; To the Ginkgolides that obtains in step (5), take the C18 filler as stationary phase, take the mixed solution of methyl alcohol and water as moving phase, carry out the mesolow reversed phase chromatography, collect respectively the positive component that is rich in Ginkgolide A, Ginkgolide B, ginkalide C;
(7) crystallisation step; Respectively with three positive component underpressure distillation of reversed phase chromatography to the dried solid substance that obtains, respectively adding concentration is 60%～80% methanol aqueous solution, after fully dissolving under 40 ℃～60 ℃, after filtered while hot, freezing and crystallizing obtains respectively purity greater than 98% Ginkgolide A, Ginkgolide B, ginkalide C.
2. a kind of method for preparing the various active material from Ginkgo Leaf as claimed in claim 1, it is characterized in that: in described raw material pre-treatment step, the aqueous solution of described cellulase and polygalacturonase is: the volume of the aqueous solution and the mass ratio of raw material are 0.5～2: 1 (volume mass ratio), the amount of cellulase and the mass ratio of raw material are 0.5～3: 1000 (mass ratioes), and the quality of polygalacturonase and the mass ratio of raw material are 0.5～2: 1000 (mass ratioes); Described broken wall reaction is: 30～60 ℃ of temperature of reaction, 1～3 hour reaction times.
3. a kind of method for preparing the various active material from Ginkgo Leaf as claimed in claim 1, it is characterized in that: in described polysaccharide precipitation step, described extracting solution simmer down to: the volume mass ratio that extracting solution is concentrated into Ginkgo Leaf dry product raw material is 2～5: 1 (L/kg); Described flocculation agent agent is: a kind of in chitosan, polyphenyl alkene acid amides, polyaerylic acid sodium, polyaerylic acid calcium; The described flocculation agent that adds carries out precipitin reaction, filtration is precipitated the thing Flos Lonicerae polysaccharide, its process is: add flocculation agent in concentrated solution, to add the mass volume ratio of flocculation agent and concentrated solution be 0.05～2: 100 (kg/L), after stirring 10～30min, standing 6～12 hours of normal temperature obtains Flos Lonicerae polysaccharide after filtration is precipitated the thing drying.
4. a kind of method for preparing the various active material from Ginkgo Leaf as claimed in claim 1, it is characterized in that: in described macroporous resin separating step, described polarity or strong polar macroporous resin are: a kind of in ADS-2, ADS-7, HPD-826, HPD-600; Described through polarity or strong polar macroporous resin isolation, its process is: precipitate and separate liquid is loaded on macroporous resin, aqueous ethanolic solution wash-out 2～4 column volumes with pure water or 5%～15%, discard elutriant, use again 40%～60% aqueous ethanolic solution wash-out 2～4 column volumes, collect elutriant, drying obtains Semen Ginkgo extrac.
5. a kind of method for preparing the various active material from Ginkgo Leaf as claimed in claim 1, it is characterized in that: in the dried post separating step of described silica gel, the dried post of described silica gel is: be filled with silica gel based filler, the aspect ratio device of 5: 1～20: 1, the silica gel based filler is comprised of silica gel and diatomite, and the mass percent of diatomite and silica gel is 10%～20%; The silica gel particle diameter is 30 μ m～80 μ m, and the silica gel activity is II～III level;
The filling process of the dried post of described silica gel is: silica gel based filler and isopyknic organic solvent are mixed into suspension liquid, pour the hollow PVC post that is plugged with glass wool and two logical pistons into, the silica gel based filler constantly deposits, solvent flows out through glass wool, control the take-off rate of solvent, silica gel based filler surface is covered all the time, until be filled into required height by solvent; Described organic solvent is a kind of in normal hexane, sherwood oil, Skellysolve A.
6. a kind of method for preparing the various active material from Ginkgo Leaf as claimed in claim 1, it is characterized in that: in the dried post separating step of described silica gel, the volume ratio of described developping agent is: methyl alcohol: normal hexane=90: 10～98: 2; The process of described descending development is: developping agent is added from the dried capital of silica gel, utilize the pressure difference descending development under normal pressure, when developer flow to silica gel is done the column bottom, stop adding developping agent; The dried shell of column cutting of silica gel that to be rich in respectively ginkgolic flavone glycoside and bilobalide scrapes, with trichloromethane vibration wash-out 10～20 minutes, repeat wash-out 2～3 times, with the chloroform soln evaporated under reduced pressure that obtains, the solid substance that obtains respectively is Ginkgo total flavones and Ginkgolides; The described dried shell of column of silica gel that is rich in ginkgolic flavone glycoside and bilobalide is both the Rf value R that obtains with reference to silica gel thin-layer experiment under identical developping agent condition
fDetermine.
7. a kind of method for preparing the various active material from Ginkgo Leaf as claimed in claim 1, it is characterized in that: in described reversed phase chromatography purification step: stationary phase is C
18Filler, particle diameter are 20～50 μ m, load into aspect ratio and be the chromatography column of 20: 1～60: 1; In described moving phase, the volume ratio of methyl alcohol and water is 60: 40～90: 10; Described mesolow reversed phase chromatography process is: described Ginkgolides is added from the chromatography capital, and the mass ratio of Ginkgolides and C18 filler is 0.01～0.02: 1; Chromatography column pressure is 5～40bar; Described flow rate of mobile phase is 2～10ml/min, collects respectively the positive component that is rich in Ginkgolide A, Ginkgolide B, ginkalide C.
8. a kind of method for preparing the various active material from Ginkgo Leaf as claimed in claim 1, it is characterized in that: in described crystallisation step, the mass ratio of the volume of described methanol aqueous solution and evaporate to dryness solid substance is 0.5～2: 1 (ml/g); Described freezing and crystallizing temperature is :-20 ℃～4 ℃.
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