CN103142472B - A kind of ginkolide B composition and method of making the same - Google Patents
A kind of ginkolide B composition and method of making the same Download PDFInfo
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Abstract
The present invention relates to a kind of ginkolide B composition, described composition is ginkolide B parenteral solution, by weight, described ginkolide B parenteral solution comprises following active constituents of medicine: 20 parts of ginkolide B extracts, PEG-4000 580-630 part, glycerine 770-830 part and ethanol 640-700 part; Described ginkolide B extract extracts and obtains from ginkgo leaf, in described ginkolide B extract, contains ginkolide B and ginkalide A, and the content of described ginkolide B is greater than 99%, and the content of described ginkalide A is less than 0.4%. In ginkolide B composition provided by the invention, purity as ginkolide B in the ginkolide B extract of bulk drug is high, the content of ginkalide A is extremely low, has met the requirement of injection to bulk drug, has improved the bioavilability of bulk drug and patient's drug safety.
Description
Technical field
The invention belongs to medical technical field, be specifically related to a kind of ginkolide B composition and method of making the same.
Background technology
Ginkolide B, English name GinkgolideB; Molecular formula: C20H24O10; Molecular weight: 424.40; StructureFormula is:
Ginkolide B is the platelet activating factor that a kind of activity is strong (PAF) antagonist, can be competitively and PAFReceptors bind, suppresses the effect of PAF; Therefore, allly relate to the illness relevant to PAF, as: thrombosis, brainThe shock that tissue damage, organ-graft refection, acute inflammation, heart allergy, intracellular toxin and IgG cause etc.,Ginkolide B all has certain curative effect.
But up to now, all not having ginkolide B single component is both at home and abroad the preparation listing of raw material. According to ChinaThis medicine of " medicine registration management way " middle chemicals registration classification belongs in chemicals " at home and abroad not to be gone upThe medicine that sell in city " lower " extraction or new effective monomer and the system thereof extracted by fermentation in natural materialsAgent ", belong to chemicals 1.2 classes.
CN101182325A relates to a kind of intravenous administration bilobalide B and extracting method, wherein ginkolide B content>=97%, ginkgoic acid≤2PPM, Bilobalide does not detect, can be directly as intravenous injection or drip-feed bulk drug;Extracting and adopting leaf dry weight 8～25 times of amounts, concentration is 5%-30% ethanol water refluxing extraction several times, isolated by filtration;Filtrate is concentrated into 0.05～0.2 times of amount of original volume, adds adsorbent fully absorption under stirring, filter filterCake extracts several times with 3～10 times of amount alcohol refluxs, merges extract, is concentrated into 0.01～0.2 times of amount of original volume,Leave standstill crystallization, filter to obtain ginkgolides; The methyl alcohol of 10～50 times of amounts several times that are recrystallized for ginkgolides, crystallizationTemperature-10 DEG C～-25 DEG C, filtering for crystallizing, dry, obtains ginkolide B.
CN101412725A discloses a kind of method of extracting separating bilobalide B from ginkgo leaf, and its step is as follows: 1)Get ginkgo leaf, use the ethanol of variable concentrations from high to low to extract, merge extract, reclaim ethanol, after obtaining concentratingExtract; 2) in the extract after concentrated, add ethyl acetate extraction, be concentrated into medicinal extract after reclaiming organic phase; 3)By step 2) the rear upper selective polarity atresia adsorption resin column of gained medicinal extract dilution, with ethanol elution, gained eluent returnsReceive after ethanol, then obtain ginkgolide compound with alcohol crystal; 4) by silica gel in ginkgolide compound crystallizationPost, with the mixed liquor wash-out of n-hexane and ethyl acetate, collects eluent, obtains the eluent of enrichment ginkolide B; 5)Recycling step 4) in solvent in the eluent collected, then use alcohol crystal, obtain ginkolide B monomer.
CN1680392A relates to employing high-speed countercurrent chromatography (HSCCC) and from ginkgo leaf, separates a kind of high-purity ginkgoThe preparation method of lactone. With three dicyandiamide solutions, respectively after twice HSCCC separates, can obtain ginkalide A,Ginkolide B, ginkalide C, bilobalide J and Bilobalide, purity all reaches more than 99%.
At present, medicinal ginkolide B mainly extracts from ginkgo leaf, but because ginkolide B is in ginkgo leafContent extremely low, and ginkolide B and ginkalide A and ginkgoic acid, Bilobalide exist jointly, in ginkgoEster B is very similar again to the structure of ginkalide A, and polarity difference is very little, extracts highly purified silver from ginkgo leafApricot lactone B monomer is difficulty comparatively, particularly the comparatively difficulty that separates with ginkalide A by ginkolide B. Above-mentionedThe ginkolide B extracting in patent and prior art pure not high enough, and ginkolide B is poorly soluble, because ofThis can only serve as oral medication raw material and can not serve as the bulk drug of injection, and the bioavilability of oral administrationLow, can not give full play to the drug effect of ginkolide B, adopt injection can greatly improve its bioavilability, and faceBed is suitable for crowd---ischemic cerebral disease patient, comprises that the Case treatment time window of acute ischemic stroke cerebral infarction is narrow,And need quick acting, general oral formulations is slow with respect to injection onset, and after making injection, administration onset is rapid,In addition, may there is oral drugs dysphagia in ischemic cerebral disease patient, make injection do not exist swallow tiredDifficult problem. But injection is very high to the purity requirement of ginkolide B, ginkolide B of the prior art is formerMaterial medicine is because purity is low cannot be for the preparation of the parenteral solution of ginkolide B.
In view of this, special proposition the present invention.
Summary of the invention
The first object of the present invention is to overcome the deficiencies in the prior art, and a kind of ginkolide B composition is provided, shouldIn parenteral solution, the content of ginkolide B extract content high, ginkalide A is extremely low.
The second object of the present invention is to provide a kind of preparation method of above-mentioned ginkolide B composition.
In order to realize object of the present invention, the following technical scheme of special employing:
A kind of ginkolide B composition, described composition is ginkolide B parenteral solution, by weight, described inGinkolide B parenteral solution comprises following active constituents of medicine: 20 parts of ginkolide B extracts, PEG-4000580-630 part, glycerine 770-830 part and ethanol 640-700 part; Described ginkolide B extract is from ginkgo leafMiddle extraction obtains, and contains ginkolide B and ginkalide A, described ginkgolides in described ginkolide B extractThe content of B is greater than 99%, and the content of described ginkalide A is less than 0.4%.
General oral formulations is slow with respect to injection onset, and after making injection, administration onset is rapid. Ischemic brainMay there is oral drugs dysphagia in sick patient, make injection and do not exist the problem of dysphagia. Be used forThe bulk drug of injection is high to purity requirement, and in parenteral solution provided by the invention, bulk drug ginkolide B extractsIn thing, the purity of ginkolide B is high, and the content of ginkalide A is extremely low, has met injection main medicine is livedProperty composition purity high requirement, greatly improved patient's drug safety, in addition, the ginkalide A of trace withGinkolide B collaborative, cumulative, complementation is strong, than single ginkolide B, its bioavilability is moreHigh.
Glycerine can be used as a kind of energy acid, for the people who carries out high strength physical training, can bring to them outstandingPerformance. For body-builders, glycerine can help they body surface and subcutaneous water transport to blood andIn muscle. Glycerine has important effect aspect stabilizing blood sugar and insulin, heavy dose of takes glycerine hardlyCan there is impact to blood sugar and insulin level. A large amount of evidence promptings, if your target is to reduce carbohydrateIntake, glycerine may be a kind of desirable glycogen.
The solubility property of ginkolide B is bad, and the present invention is taking glycerine, ethanol and PEG-4000 as solvent, butGlycerine viscosity is large, and in the present invention, the consumption of glycerine is relatively little, and in addition, ginkolide B is larger in solvent viscosityIn situation, be difficult for dispersing and dissolving, the solubility in PEG-4000 is relatively large, therefore adopts PEG-4000After first dissolving ginkolide B with ethanol, add again glycerine.
Ginkolide B extract provided by the invention is extraction from natural drug ginkgo leaf, separation, refining forming, the content of described ginkolide B is greater than 99%, and the content of ginkalide A is less than 0.4%, ginkolide BContent is high, is made into injection, has greatly improved the bioavilability of ginkolide B, and the matter of this productQuantity research and stability test result show that this product is quality controllable, stable, for this product clinical practice provides good havingThe basis of effect property and security and quality controllability, has improved patient's drug safety widely.
Preferably, described ginkolide B parenteral solution comprises 20 parts of ginkolide B extracts, PEG-4000590-610 part, glycerine 790-810 part and ethanol 650-670 part.
Preferred, described ginkolide B parenteral solution comprises 20 parts of ginkolide B extracts, PEG-4000660 parts of 600 parts, 800 parts of glycerine and ethanol.
Preferably, the pH of described parenteral solution is 3.8-4.8.
Preferably, the content of described ginkolide B is greater than 99.6%, and the content of described ginkalide A is lower than 0.3%.
Preferably, in described ginkolide B extract, do not contain Bilobalide.
Preferably, in described ginkolide B extract, the content of total ginkgoic acid is less than 0.12ppm.
In ginkolide B extract provided by the invention, the kind of impurity is few, content is low, quality controllable, is onePlant the bulk drug of the ejection preparation of function admirable, and the impact of the stability of impurity on ginkolide B is less.
Ginkalide A and ginkolide B are all the active constituents of medicine useful to human body, but former as injectionMaterial medicine, wherein the content of main active ginkolide B should be very high, ginkalide A and ginkolide BStructure is very similar, and the two polarity difference is very little, in the ginkolide B extract extracting from ginkgo leaf, and ginkgoThe content of lactone A is higher, obtain the comparatively difficulty of ginkolide B of high-load. The ginkolide B that the present invention extractsIn extract, the content of ginkalide A is very low, effectively ginkalide A has been separated from ginkolide BCome, the ginkolide B content obtaining is very high, and, the kind of impurity and content in ginkolide B extractThe stability of ginkolide B is also had to considerable influence, the dopant species of ginkolide B extract provided by the inventionLess, content is low, has better stability with respect to prior art, can be used as the bulk drug of ejection preparation, greatlyGround has improved patient's drug safety.
The present invention is with reference to total ginkgoic acid Limit Test side under ginkgo biloba p.e item of " Chinese pharmacopoeia " version in 2010Method, shows the result of determination of raw material, and this product is less than 0.12ppm containing the amount of total ginkgoic acid, extracts far below ginkgo leafThing total ginkgoic acid must not be crossed 10/1000000ths limit regulation, and ginkolide B extract provided by the invention is safetyAnd effectively.
In the present invention, the specific rotation of the methanol solution of described ginkolide B extract in the time of 20 DEG C is-57 °～-61 °.
The solubility of ginkolide B under absolute ethyl alcohol is low compared with methyl alcohol, and the concentration that is mixed with 10mg/ml is more difficult,And the optical activity numerical value of 5mg/ml is lower, error increases, thereby to select methyl alcohol be solvent, is mixed with 10mg/mlSolution, in the time of 20 DEG C measure specific rotation be-57 °～-61 °. The specific rotation of ginkolide B of the present invention than " inState's pharmacopeia " in the specific rotation of ginkolide B of regulation be that the scope of-48 ° to-63 ° narrows down a lot, this is describedThe bright ginkolide B extract providing has very high purity.
The present invention also provides a kind of preparation method of described ginkolide B extract, comprising:
(1) under stirring condition, in ethyl acetate, first add diatomite, then add ginkgo biloba p.e, in 78-85 DEG CAdd hot reflux 0.5-1.5 hour, filter, concentrated; Adding diatomaceous amount is the 0.8-1.2 of ginkgo biloba p.e weightDoubly, the liquid-solid ratio of ethyl acetate and ginkgo biloba p.e is 15-20ml/g;
(2) add distilled water removal of impurities again, separate ethyl acetate layer, ethyl acetate layer peracidity alumina column, uses water saturationEluent ethyl acetate, collects eluent;
(3) eluent is evaporated to without ethyl acetate taste, add appropriate ethanol and make it to dissolve, then add water to make containing alcohol amount be60～70%, leave standstill, make to separate out coarse crystallization, filter, get coarse crystallization, dry;
(4) coarse crystallization is used ethanol and re-crystallizing in ethyl acetate successively, obtains finished product ginkolide B extract.
The present invention adopts ethyl acetate from ginkgo biloba p.e, to extract ginkolide B, and ginkgo biloba p.e is added to secondIn acetoacetic ester, can form the material that a large amount of viscosity is large, the technique of not utilizing subsequent technique and clearing out a gathering place, therefore adds siliconAlgae soil contributes to the dispersion of ginkgo biloba p.e in ethyl acetate; In the time extracting in a large number, directly by diatomiteMix with ginkgo biloba p.e, be unfavorable for the dispersion of ginkgo biloba p.e and mix, therefore in extractor, addEnter after ethyl acetate, under stirring condition, first add diatomite, then add ginkgo biloba p.e, ginkgo biloba p.eCan in ethyl acetate, be uniformly dispersed quickly, be conducive to the stripping of ginkolide B in ethyl acetate, improve thickThe content of ginkolide B in product.
In above-mentioned preparation method's step (1), described ginkgo biloba p.e obtains for extracting from ginkgo leaf, carriesAccess method can be with reference to any method of the prior art, and those skilled in the art do not need to pay creationary laborMoving.
The concentrate that step (1) obtains has precipitate, and precipitate affects column chromatography, therefore in step (2), is enteringBefore row column chromatography, prior to the extraction that adds water in concentrate, can make precipitate water-soluble, branch vibration layer, is conducive to removeA large amount of impurity in concentrate, and in removing precipitate, reduce the viscosity of concentrate, be more conducive to follow-upColumn chromatography technique.
From ginkgo leaf extract prepare ginkolide B, preparation method's difference, the purity of the ginkolide B obtaining andWherein the kind of impurity and content difference are huge, and the present invention is by a large amount of experiments, to the extraction system of ginkolide BPreparation Method constantly improves and optimizes, particularly including to solvent and chromatographic column select and to whole extraction roadThe design of line, has finally obtained the preparation method of a highly purified ginkolide B of preparation, and in the ginkgo obtainingIn ester B extract, the kind of impurity is few, and content is low, measures through HPLC-ELSD, and the content of ginkolide B reachesTo more than 99%, the content of ginkalide A is less than 0.4%, can be used as the bulk drug of ejection preparation, greatly carriesHigh patient's drug safety. In addition, the method is simple to operation, manufacturing cycle is short, has greatly reduced from silverIn apricot leaf, prepare the cost of ginkolide B, further reduced the cost of ginkolide B parenteral solution.
In above-mentioned preparation method's step (4), coarse crystallization is used ethyl alcohol recrystallization 3 above, ethyl acetate weights successivelyCrystallization is more than 1 time, and in the finished product obtaining, the content of ginkolide B is greater than 99.6%, and the content of ginkalide A is lowIn 0.3%.
In described step (1), preferred, add diatomaceous amount be ginkgo biloba p.e weight 0.9-1.1 doubly;The liquid-solid ratio of ethyl acetate and ginkgo biloba p.e is 16-18ml/g. The consumption of diatomite, ginkgo leaf and ethyl acetateWithin the scope of this, be conducive to improve the yield of ginkolide B extract and improve containing of ginkolide B in crude productAmount.
In described step (1), preferred, it is 3-5ml/g that filtrate is concentrated into liquid-solid ratio.
In described step (2), preferred, the blade diameter length ratio of acidic alumina column is 1:4～1:10, and aluminium oxide is200～300 orders, the weight ratio of applied sample amount and aluminium oxide is 1:7～10, elution speed is 1/4～1/2BV/h; More excellentChoosing, the blade diameter length ratio of described acidic alumina column is 1:4～1:7, the weight ratio of applied sample amount and aluminium oxide is 1:8.
By above-mentioned preferred technical scheme, the present invention obtains in ginkolide B extract not containing Bilobalide, alwaysThe content of ginkgoic acid is less than 0.12ppm.
The present invention also provides a kind of preparation method of foregoing ginkolide B composition, comprises the following steps:(1) take recipe quantity PEG-4000, etc. the ethanol of quality, stir, add in the ginkgo of recipe quantityEster B extract, is heated to 50～60 DEG C, and stirring and dissolving adds the glycerine of recipe quantity, with containing 9-11% hydrochloric acidEthanolic solution adjust pH to 3.8～4.8, add the full dose of ethanol to recipe quantity;
(2) add the active carbon of dosing amount 0.1-0.3%g/ml, stir 25-35 minute, coarse filtration takes off charcoal;
(3) cross 0.22 μ m filter membrane, obtain smart filtrate;
(4) embedding;
(5) 118-125 DEG C of pressure sterilizing 13-18 minute, inspection, packaging, warehouse-in.
Grope in process at prescription, find that pH value has a significant impact the stability of parenteral solution, when pH value 3.8～Between 4.8 time, parenteral solution is more stable.
Compared with prior art, beneficial effect of the present invention is:
In ginkolide B composition provided by the invention, the purity of its component ginkolide B extract is very high,In this ginkolide B extract, the content of ginkalide A is very low, and in addition, the kind of impurity is few, content is low,Meet the requirement of injection to bulk drug, improved the bioavilability of bulk drug and patient's drug safety.Wherein the preparation method of ginkolide B extract is simple to operation, and particularly diatomaceous employing, is conducive to follow-upThe carrying out of column chromatography technique, improved the efficiency of preparation, reduced the cost of preparation.
Detailed description of the invention
The method for chromatographic determination of ginkgolides and Bilobalide adopts HPLC-ELSD chromatographic process: use octadecyl siliconAlkane bonded silica gel is filler; Taking methyl alcohol/oxolane (2:1): water=32:68 as mobile phase, evaporative light-scattering inspectionSurvey device and detect (reference conditions: Alltech3300: 60 DEG C of drift tube temperatures, air velocity 1.5L/min, gainBe 8; Agilent380-LC: 30 DEG C of atomization temperatures, 50 DEG C of drift tube temperatures, carrier gas flux 1.5SLM, PMT increasesBenefit is 8). Integration method: area external standard method.
The assay of total ginkgoic acid is with reference to total ginkgo under ginkgo biloba p.e item of " Chinese pharmacopoeia " version in 2010Acid Limit Test method: taking octadecylsilane chemically bonded silica as filler, with methyl alcohol-oxolane-water (22:10:68) be mobile phase; By EISD detection (reference conditions: Alltech3300: drift tube temperatureSpend 60 DEG C, air velocity 1.5L/min, gain is 8; Agilent380-LC: 30 DEG C of atomization temperatures, drift tubeTemperature 50 C, carrier gas flux 1.5SLM, PMT gain is 8). Integration method: area external standard method.
The assay method of specific rotation is with reference to " Chinese pharmacopoeia " version annex VIE in 2010, and reference conditions: WZZ-3 certainlyMoving polarimeter solvent: methanol solution, concentration: 10mg/ml, temperature: 20 DEG C.
Embodiment 1
The preparation of ginkolide B extract:
Under stirring condition, in 750ml ethyl acetate, first add diatomite 40g, then add ginkgo biloba p.e 50g,Add hot reflux 0.5 hour in 85 DEG C, filter, concentrated, it is 5ml:1g that filtrate is concentrated into liquid-solid ratio; ; Toward concentrateIn add the distilled water removal of impurities of approximately 1/10 volume, separate ethyl acetate layer, ethyl acetate layer peracidity alumina column,The blade diameter length ratio of acidic alumina column is 1:4, and aluminium oxide is 200 orders, and the weight ratio of applied sample amount and aluminium oxide is 1:7,Use water saturation eluent ethyl acetate, elution speed is 1/4BV/h, collects eluent; Eluent is evaporated to nothingEthyl acetate taste, add appropriate ethanol make it dissolve, then add water to make containing alcohol amount be 60%, leave standstill, make to separate out coarse crystallization,Filter, get coarse crystallization, dry; Coarse crystallization is used ethanol and re-crystallizing in ethyl acetate successively, obtains finished product ginkolide BExtract. Sample detection ginkolide B content is 99.0%, and the content of ginkalide A is less than 0.4%. Finished productSpecific rotation is-61 °.
Embodiment 2
The preparation of ginkolide B extract:
Under stirring condition, in 1000ml ethyl acetate, first add diatomite 40g, then add ginkgo biloba p.e50g, adds hot reflux 1.5 hours in 79 DEG C, filters, and concentrated, it is 4ml:1g that filtrate is concentrated into liquid-solid ratio; Toward denseThe distilled water removal of impurities that adds approximately 1/9 volume in contracting liquid, separates ethyl acetate layer, ethyl acetate layer peracidity alumina column,The blade diameter length ratio of acidic alumina column is 1:4, and aluminium oxide is 200 orders, and the weight ratio of applied sample amount and aluminium oxide is 1:7,Use water saturation eluent ethyl acetate, elution speed is 1/4BV/h, collects eluent; Eluent is evaporated to nothingEthyl acetate taste, add appropriate ethanol make it dissolve, then add water to make containing alcohol amount be 60%, leave standstill, make to separate out coarse crystallization,Filter, get coarse crystallization, dry; Coarse crystallization is used ethyl alcohol recrystallization 3 times and re-crystallizing in ethyl acetate 1 time successively,Finished product ginkolide B extract. Sample detection ginkolide B content is 99.6%, and the content of ginkalide A is less than0.3%. The specific rotation of finished product is-59.3 °.
Embodiment 3
The preparation of ginkolide B extract:
Under stirring condition, in 850ml ethyl acetate, first add diatomite 60g, then add ginkgo biloba p.e 50g,Add hot reflux 1 hour in 80 DEG C, filter, concentrated, it is 3ml:1g that filtrate is concentrated into liquid-solid ratio; In concentrateThe distilled water removal of impurities that adds approximately 1/11 volume, separates ethyl acetate layer, ethyl acetate layer peracidity alumina column, acidThe blade diameter length ratio of property alumina column is 1:10, and aluminium oxide is 300 orders, and the weight ratio of applied sample amount and aluminium oxide is 1:10,Use water saturation eluent ethyl acetate, elution speed is 1/2BV/h, collects eluent; Eluent is evaporated to nothingEthyl acetate taste, add appropriate ethanol make it dissolve, then add water to make containing alcohol amount be 70%, leave standstill, make to separate out coarse crystallization,Filter, get coarse crystallization, dry; Coarse crystallization is used ethyl alcohol recrystallization 3 times and re-crystallizing in ethyl acetate 1 time successively,Finished product ginkolide B extract. Sample detection ginkolide B content is 99.7%, and Bilobalide does not detect, ginkgoThe content of lactone A is 0.3%, and the content of total ginkgoic acid is 0.12ppm. The specific rotation of finished product is-57.8 °.
Embodiment 4
The preparation of ginkolide B extract:
Under stirring condition, in 900ml ethyl acetate, first add diatomite 45g, then add ginkgo biloba p.e 50g,Add hot reflux 1 hour in 79 DEG C, filter, concentrated, it is 3.5ml:1g that filtrate is concentrated into liquid-solid ratio; In concentrateThe distilled water removal of impurities that adds approximately 1/10 volume, separates ethyl acetate layer, ethyl acetate layer peracidity alumina column, acidThe blade diameter length ratio of property alumina column is 1:7, and aluminium oxide is 300 orders, and the weight ratio of applied sample amount and aluminium oxide is 1:8,Use water saturation eluent ethyl acetate, elution speed is 1/2BV/h, collects eluent; Eluent is evaporated to nothingEthyl acetate taste, add appropriate ethanol make it dissolve, then add water to make containing alcohol amount be 70%, leave standstill, make to separate out coarse crystallization,Filter, get coarse crystallization, dry; Coarse crystallization is used ethyl alcohol recrystallization 3 times and re-crystallizing in ethyl acetate 1 time successively,Finished product ginkolide B extract. Sample detection ginkolide B content is 99.6%, and Bilobalide does not detect, ginkgoThe content of lactone A is 0.2%, and the content of total ginkgoic acid is 0.12ppm. The specific rotation of finished product is-58.4 °.
Embodiment 5
The preparation of ginkolide B extract:
Under stirring condition, in 900ml ethyl acetate, first add diatomite 55g, then add ginkgo biloba p.e 50g,Add hot reflux 1 hour in 78 DEG C, filter, concentrated, it is 3.5ml:1g that filtrate is concentrated into liquid-solid ratio; In concentrateThe distilled water removal of impurities that adds approximately 1/10 volume, separates ethyl acetate layer, ethyl acetate layer peracidity alumina column, acidThe blade diameter length ratio of property alumina column is 1:4, and aluminium oxide is 300 orders, and the weight ratio of applied sample amount and aluminium oxide is 1:8,Use water saturation eluent ethyl acetate, elution speed is 1/2BV/h, collects eluent; Eluent is evaporated to nothingEthyl acetate taste, add appropriate ethanol make it dissolve, then add water to make containing alcohol amount be 65%, leave standstill, make to separate out coarse crystallization,Filter, get coarse crystallization, dry; Coarse crystallization is used ethyl alcohol recrystallization 3 times and re-crystallizing in ethyl acetate 1 time successively,Finished product ginkolide B extract. Sample detection ginkolide B content is 99.7%, and Bilobalide does not detect, ginkgoThe content of lactone A is 0.2%, and the content of total ginkgoic acid is 0.10ppm. The specific rotation of finished product is-57 °～-61 °. BecomeThe specific rotation of product is-57 °.
Embodiment 6
The preparation of ginkolide B parenteral solution:
Take PEG-4000 580g, etc. the ethanol of quality, stir, the ginkgo that adds embodiment 1 to prepareLactone B20g, is heated to 50 DEG C, and stirring and dissolving, adds glycerine 770g, with the ethanolic solution tune containing 9% hydrochloric acidPH value to 4.6, adds ethanol 60g, stirs; Add again the active carbon of dosing amount 0.1% (g/ml), stir 25Minute, coarse filtration takes off charcoal; Cross 0.22 μ m filter membrane, obtain smart filtrate; Embedding, every bottled amount 2mL; 118 DEG C of hot pressing are gone outBacterium 18 minutes, inspection, packaging, warehouse-in.
Embodiment 7
The preparation of ginkolide B parenteral solution:
Take PEG-4000 630g, etc. the ethanol of quality, stir, the ginkgo that adds embodiment 2 to prepareLactone B20g, is heated to 55 DEG C, and stirring and dissolving, adds glycerine 830g, with the ethanolic solution tune containing 11% hydrochloric acidPH value to 4.2, adds ethanol 70g, stirs; Add again the active carbon of dosing amount 0.3% (g/ml), stir 35Minute, coarse filtration takes off charcoal; Cross 0.22 μ m filter membrane, obtain smart filtrate; Embedding, every bottled amount 2mL; 125 DEG C of hot pressing are gone outBacterium 13 minutes, inspection, packaging, warehouse-in.
Embodiment 8
The preparation of ginkolide B parenteral solution:
Take PEG-4000 590g, etc. the ethanol of quality, stir, the ginkgo that adds embodiment 3 to prepareLactone B20g, is heated to 60 DEG C, and stirring and dissolving, adds glycerine 790g, with the ethanolic solution tune containing 10% hydrochloric acidPH value to 4.0, adds ethanol 60g, stirs; Add again the active carbon of dosing amount 0.1% (g/ml), stir 30Minute, coarse filtration takes off charcoal; Cross 0.22 μ m filter membrane, obtain smart filtrate; Embedding, every bottled amount 2mL; 121 DEG C of hot pressing are gone outBacterium 15 minutes, inspection, packaging, warehouse-in.
Embodiment 9
The preparation of ginkolide B parenteral solution:
Take PEG-4000 610g, etc. the ethanol of quality, stir, the ginkgo that adds embodiment 4 to prepareLactone B20g, is heated to 50 DEG C, and stirring and dissolving, adds glycerine 810g, with the ethanolic solution tune containing 10% hydrochloric acidPH value to 3.8, adds ethanol 60g, stirs; Add again the active carbon of dosing amount 0.1% (g/ml), stir 30Minute, coarse filtration takes off charcoal; Cross 0.22 μ m filter membrane, obtain smart filtrate; Embedding, every bottled amount 2mL; 121 DEG C of hot pressing are gone outBacterium 15 minutes, inspection, packaging, warehouse-in.
Embodiment 10
The preparation of ginkolide B parenteral solution:
Take PEG-4000 600g, etc. the ethanol of quality, stir, the ginkgo that adds embodiment 5 to prepareLactone B20g, is heated to 60 DEG C, and stirring and dissolving, adds glycerine 800g, with the ethanolic solution tune containing 10% hydrochloric acidPH value to 4.8, adds ethanol 60g, stirs; Add again the active carbon of dosing amount 0.1% (g/ml), stir 30Minute, coarse filtration takes off charcoal; Cross 0.22 μ m filter membrane, obtain smart filtrate; Embedding, every bottled amount 2mL; 121 DEG C of hot pressing are gone outBacterium 15 minutes, inspection, packaging, warehouse-in.
Experimental example 1
This experimental example has been prepared ginkolide B with reference to the method for following documents, and adopts the test side in the present inventionMethod, has measured containing of ginkolide B in ginkolide B extract, Bilobalide, ginkalide A, total ginkgoic acidAmount, the results are shown in Table 1.
The ginkolide B that sample 1 is prepared for the method for the embodiment 1 with reference to patent application CN101182325A extractsThing;
The ginkolide B that sample 2 is prepared for the method for the embodiment 1 with reference to patent application CN101412725A extractsThing;
The ginkolide B extract that sample 3 is prepared for the method for the embodiment 1 with reference to patent application CN1680392A;
The ginkolide B that sample 4 is prepared for the method for the embodiment 1 with reference to patent application CN101302222A extractsThing;
The ginkolide B that sample 5 is prepared for the method for the embodiment 1 with reference to patent application CN102627656A extractsThing;
Table 1
| Ginkolide B | Bilobalide | Ginkalide A | Total ginkgoic acid | |
| Sample 1 | 97.8% | Do not detect | 1.1% | 2ppm |
| Sample 2 | 98.3% | 0.5% | 0.5% | 8ppm |
| Sample 3 | 98.7% | 0.3% | 0.3% | 10ppm |
| Sample 4 | 91.5% | 1.7% | 2.5% | 15ppm |
| Sample 5 | 98.6% | 0.5% | 0.3% | 7ppm |
The measurement result of table 1 shows, compared with the prior art of above-mentioned patent application, in ginkgo prepared by the present inventionIn ester B extract, ginkolide B content is high, the content of ginkalide A is low, and the content of impurity is few, particularlyThe content of Bilobalide, total ginkgoic acid is very low, can be used as the bulk drug of injection, has greatly improved patient'sDrug safety.
Experimental example 2
This experimental example has been tested the stability of ginkolide B extract prepared by the present invention.
Sample 1 is embodiment 1 product, and sample 2 is embodiment 2 products; Sample 3 is embodiment 5 products;
Sample 4 is that the HPLC purity that adopts the method for patent CN101182325A embodiment 1 to obtain is 97.8%Ginkolide B;
The HPLC purity that sample 5 obtains for the method for employing patent CN1680392A embodiment 1 is 99.0%Ginkolide B;
Sample is respectively got 1g, and this experiment is according to 2005 editions second annex XIXC medicine stability test of Chinese pharmacopoeiaGuideline is carried out, and result is as follows:
Table 2, accelerated test result
Table 3, long-term test results
In ginkolide B extract, ginkalide A, Bilobalide and total ginkgoic acid are steady to ginkolide BQualitatively all have a certain impact, the kind of ginkalide A and impurity in apricot lactone B extract provided by the inventionLess with content, the content of ginkolide B is higher, and these materials are less to the stability influence of ginkolide B,Investigate by accelerated test and experiment for long-term stability, result shows that ginkolide B extract of the present invention is with respect to existingThere is technology to there is better stability, accelerate, long term test purity content is little.
Experimental example 3
The pH of parenteral solution and the relation of stability are investigated
The sample 1 ginkolide B parenteral solution for preparing according to the method for embodiment 6, difference is, its pH is3.0；
The sample 2 ginkolide B parenteral solution for preparing according to the method for embodiment 6, difference is, its pH is3.8；
The sample 3 ginkolide B parenteral solution for preparing according to the method for embodiment 7, difference is, its pH is4.8；
The sample 4 ginkolide B parenteral solution for preparing according to the method for embodiment 7, difference is, its pH is4.0；
This experiment is carried out according to 2005 editions second annex XIXC medicine stability test guideline of Chinese pharmacopoeia,Result is as follows:
Table 2, accelerated test result
Table 3, long-term test results
The result of this experimental example shows, in the time that pH is between 3.8-4.8, and ginkolide B injection provided by the inventionLiquid is more stable. In addition, from accelerated test and the long term test of sample 2,3, in ginkgo provided by the inventionEster B parenteral solution has extraordinary stability.
Claims (9)
1. a ginkolide B composition, it is characterized in that, described composition is ginkolide B parenteral solution, by weight, described ginkolide B parenteral solution comprises following active constituents of medicine: 20 parts of ginkolide B extracts, PEG-4000 580-630 part, glycerine 770-830 part and ethanol 640-700 part; Described ginkolide B extract extracts and obtains from ginkgo leaf, in described ginkolide B extract, contains ginkolide B and ginkalide A, and the content of described ginkolide B is greater than 99%, and the content of described ginkalide A is less than 0.4%;
The extracting method of described ginkolide B extract, comprising:
(1) under stirring condition, in ethyl acetate, first add diatomite, then add ginkgo biloba p.e, add hot reflux 0.5-1.5 hour in 78-85 DEG C, filter, concentrated; Add diatomaceous amount be ginkgo biloba p.e weight 0.8-1.2 doubly, the liquid-solid ratio of ethyl acetate and ginkgo biloba p.e is 15-20ml/g;
(2) add distilled water removal of impurities again, separate ethyl acetate layer, ethyl acetate layer peracidity alumina column, uses water saturation eluent ethyl acetate, collects eluent;
(3) eluent is evaporated to without ethyl acetate taste, add appropriate ethanol make it dissolve, then add water to make containing alcohol amount be 60～70%, leave standstill, make to separate out coarse crystallization, filter, get coarse crystallization, dry;
(4) coarse crystallization is used ethanol and re-crystallizing in ethyl acetate successively, obtains finished product ginkolide B extract.
2. composition according to claim 1, is characterized in that, described ginkolide B parenteral solution comprises 20 parts of ginkolide Bs, PEG-4000 590-610 part, glycerine 790-810 part and ethanol 650-670 part.
3. composition according to claim 2, is characterized in that, described ginkolide B parenteral solution comprises 660 parts of 20 parts of ginkolide Bs, 600 parts of PEG-4000s, 800 parts of glycerine and ethanol.
4. composition according to claim 1, is characterized in that, the pH of described ginkolide B parenteral solution is 3.8-4.8.
5. composition according to claim 1, is characterized in that, the content of described ginkolide B is greater than 99.6%, and the content of described ginkalide A is lower than 0.3%.
6. composition according to claim 1, is characterized in that, does not contain Bilobalide in described ginkolide B extract.
7. ginkolide B composition according to claim 1, is characterized in that, in described ginkolide B extract, the content of total ginkgoic acid is less than 0.12ppm.
8. according to the composition described in claim 1 or 6 or 7, it is characterized in that, the specific rotation of the methanol solution of described ginkolide B extract in the time of 20 DEG C is-57 °～-61 °.
9. a preparation method for the ginkolide B composition described in claim 1-8 any one, is characterized in that, comprises the following steps:
(1) take recipe quantity PEG-4000, etc. the ethanol of quality, stir, add the ginkolide B extract of recipe quantity, be heated to 50～60 DEG C, stirring and dissolving, add the glycerine of recipe quantity, with ethanolic solution adjust pH to 3.8～4.8 containing 9-11% hydrochloric acid, add the full dose of ethanol to recipe quantity;
(2) add the active carbon of dosing amount 0.1-0.3%g/ml, stir 25-35 minute, coarse filtration takes off charcoal;
(3) cross 0.22 μ m filter membrane, obtain smart filtrate;
(4) embedding;
(5) 118-125 DEG C of pressure sterilizing 13-18 minute, inspection, packaging, warehouse-in;
The extracting method of the ginkolide B extract described in step (1), comprising:
(1) under stirring condition, in ethyl acetate, first add diatomite, then add ginkgo biloba p.e, add hot reflux 0.5-1.5 hour in 78-85 DEG C, filter, concentrated; Add diatomaceous amount be ginkgo biloba p.e weight 0.8-1.2 doubly, the liquid-solid ratio of ethyl acetate and ginkgo biloba p.e is 15-20ml/g;
(2) add distilled water removal of impurities again, separate ethyl acetate layer, ethyl acetate layer peracidity alumina column, uses water saturation eluent ethyl acetate, collects eluent;
(3) eluent is evaporated to without ethyl acetate taste, add appropriate ethanol make it dissolve, then add water to make containing alcohol amount be 60～70%, leave standstill, make to separate out coarse crystallization, filter, get coarse crystallization, dry;
(4) coarse crystallization is used ethanol and re-crystallizing in ethyl acetate successively, obtains finished product ginkolide B extract.
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| CN101244031A (en) * | 2008-03-31 | 2008-08-20 | 广州艾格生物科技有限公司 | Bilobalide A injection, preparation method and application thereof |
| CN101249085A (en) * | 2008-03-31 | 2008-08-27 | 广州艾格生物科技有限公司 | Bilobalide A, B compound injection and preparation method and application thereof |
| CN101301267A (en) * | 2008-05-21 | 2008-11-12 | 南京海陵中药制药工艺技术研究有限公司 | Bilobalide B injection and preparation thereof |
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| CN101244031A (en) * | 2008-03-31 | 2008-08-20 | 广州艾格生物科技有限公司 | Bilobalide A injection, preparation method and application thereof |
| CN101249085A (en) * | 2008-03-31 | 2008-08-27 | 广州艾格生物科技有限公司 | Bilobalide A, B compound injection and preparation method and application thereof |
| CN101301267A (en) * | 2008-05-21 | 2008-11-12 | 南京海陵中药制药工艺技术研究有限公司 | Bilobalide B injection and preparation thereof |
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