CN103091412B

CN103091412B - Method for detecting ginkgolide effective parts

Info

Publication number: CN103091412B
Application number: CN201210555240.9A
Authority: CN
Inventors: 孙毅; 朱永红; 童正兵; 王婕
Original assignee: Chengdu Baiyu Pharmaceutical Co Ltd
Current assignee: Chengdu Baiyu Pharmaceutical Co Ltd
Priority date: 2012-04-23
Filing date: 2012-04-23
Publication date: 2015-02-25
Anticipated expiration: 2032-04-23
Also published as: CN103091412A

Abstract

The invention provides a method for detecting ginkgolide effective parts, relates to the field of plant extraction, particularly relates to a method for extracting and separating ginkgolide, and based on the extraction and separation method, the ginkgolide with unchanged components is obtained. The ginkgolide with fixed components is obtained by the steps of extraction, leaching, column chromatographic separation, crystallization and crystal mixing and contains 25.0-50.0 percent of bilobalide (C15H18O8), 20.0-45.0 percent of ginkgolide A (C20H24O9), 10.0-30.0 percent of ginkgolide B (C20H24O10) and 5.0-15.0 percent of ginkgolide C (C20H24O11), and the total amount of the bilobalide, the ginkgolide A, the ginkgolide B and the ginkgolide C is more than 95 percent.

Description

A kind of ginkgolides active component detection method

Technical field

The present invention relates to field of plant extraction, be specifically related to a kind of ginkgolides active component detection method.

Background technology

From the sixties in last century, many countries adopt the chemical composition of modern separation technology to ginkgo leaf to study, and through pharmacological evaluation and clinical verification, find that many-sided biologically active of ginkgo leaf is relevant with specified chemical composition contained by it.Germany Dr.WillarSchwabe have registered the simple extract of one of ginkgo leaf first, patent (W Schwabe DE 176708 and DE 2117429) has been applied in 1972, name as EGb761, be used for the treatment of cardiovascular and cerebrovascular disease and the nervous system disease, there is significant curative effect, and have no side effect, ginkgolide compound (ginkgolids) has strong platelet activating factor (PAF) antagonism.There are Germany, France and Chinese in the country that ginkgo agent is listed in medicine, and other countries are all with being health food or over-the-counter medications, and the ginkgo health care food that the U.S. develops has obtained FDA approval.

Ginkgolides belongs to terpenoid, is called terpene lactone, is made up of sesquiterpene lactone and diterpenoid-lactone, is the important active component of ginkgo Ye Zhongyi class.Bilobalide (bilobalide) belongs to sesquiterpene lactone, to be separated in 1969 with K.Weinges in 1967 by R.T.Major and to obtain, the unique sesquiterpene lactone compound found from ginkgo leaf at present, Ginkgolide A. B. C, M, J (ginkgolid A, B, C, M, J) are diterpenic lactone, separated from ginkgo leaf first by S.Furukawa in 1932, within 1967, be just separated further by people such as K.Nakanish, M.Maruyama and K.Okabe and determined its chemical constitution.From structure, Bilobalide quasi-molecule skeleton is made up of 15 carbon atoms, has 4 five-membered rings condensed mutually together, wherein has 1 five yuan of carbocyclic ring, and 3 five yuan of lactone carbocyclic rings, five-membered ring are connected with the tert-butyl group rare in 1 natural products.Bilobalide has very strong biologically active; there is the effect promoting nerve growth; the function that can prevent brain cell mitochondrial oxidation from stress cause changes; improve senile memory; prevent the generation of senile dementia; and preventing brain, spinal nerve demyelinization, its neurotrophy, neuroprotection are stronger than ginkgolides.Ginkolide B has the effect such as anti-inflammatory, Hemorrhagic shock, protection cardiovascular and cerebrovascular, treatment acute pancreatitis.And the molecular skeleton of ginkgolide compound is made up of 20 carbon atoms, there are 6 five-membered rings, wherein there are 2 five yuan of carbocyclic rings, 3 lactone ring five membereds, 1 tetrahydrofuran ring, two five yuan of carbocyclic rings link together with the form of volution, and remaining ring connects in the mode condensed, and form the special stereochemical structure of a rigidity verdant shape.In ginkgolides molecule, all there is the tert-butyl group rare in natural products.Ginkgolides comprises Diterpenes and sesquiterpenoids lactone, and diterpenes diterpenoids lactones mainly contains Ginkgolide A. B. C, J, M etc., and half terpene lactone has Bilobalide.

After early 1970s has found PAF; pharmacologist is studied ginkgolides; specifying ginkgoterpene lactone is strong platelet-activating factor antagonist, and immune system, central nervous system, ischemic injuries have protective effect, and have Hemorrhagic shock, antiallergy and antiinflammatory action.Ginkgolide A. B. C, M, J structural difference are that the position that the hydroxy number contained is connected with hydroxyl is different.Ginkgolides is strong platelet-activating factor antagonist, is the key component of special physiological activity in ginkgo leaf.

Ginkalide A structural formula ginkolide B structural formula

Molecular formula: C ₂₀h ₂₄o ₉molecular weight: 408.4 molecular formula: C ₂₀h ₂₄o ₁₀molecular weight: 424.4

Ginkalide C structural formula Bilobalide structural formula

Molecular formula: C ₂₀h ₂₄o ₁₁molecular weight: 440.4 molecular formula: C ₁₅h ₁₈o ₈molecular weight: 326.3

Ginkgolides has powerful specific inhibitory effect to platelet activating factor paf receptor, and wherein the anti-PAF of ginkgolides is the highest.PAF is a kind of endogenous phosphatide that blood platelet and inflammation tissue secretion produce, and is the most effective platelet aggregation found so far, and the Emergence and Development of it and numerous disease is closely related.And ginkgolides is considered to the natural paf receptor antagonists having potential applicability in clinical practice most at present, its antagonism is active closely related with chemical constitution.When R3 in lactone structure is hydroxyl or hydroxy number increases, the antagonistic activity of PAF is weakened, and when R2 is hydroxyl and R3 is H, then active significantly enhancing, wherein the strongest with the antagonism that ginkolide B produces PAF.

Extraction and the purification process of ginkgolides are more, mainly contain: solvent extraction, post extraction method, solvent extraction-post extraction method, super critical extraction and chromatogram or column chromatography purification method etc.These methods all can not isolate the ginkgolides of high-load effectively, and each proportion of composing of ginkgolides is uncertain, therefore, on Clinical practice, drug effect is different, because its content is not high, also there is certain security risk, without complete pharmacological toxicology and clinical testing data, therefore, said method is all under test, is not implemented on drug production process, though have bilobalide injection Patents in China, but its composition is all different from the present invention, through the retrieval of Duo Jia official website of ICH member state, there is no other bilobalide injector launch so far.The mensuration of ginkgolides constituents at present more adopts HPLC-UV method, HPLC-MS and HPLC-ELSD method, these methods only can measure the content of each composition of ginkgolides, owing to lacking the strict control to bad reaction material in its product, the mass property of medicine can not be reflected really, do not form a perfect Drug's control system, although the patent of invention of ginkgolides is a lot, because control method is too simple, ginkgolides proportion of composing is indefinite, all cannot make traditional Chinese medicine, the security of clinical efficacy and application can not be ensured.

Although in China, there are many patents about ginkgolides and report in the countries such as Germany, but technological process of the present invention, Quality Control Technology and clinical indication and other patent of invention have completely different part, the composition of the terpene lactone that especially different separation purifying techniques obtains is different, up to now, there is no 4 kinds of component (ginkalide As, B, C and Bilobalide) the technique report of the extraction ginkgolides active component composition fixed of ratio, also not to 4 kinds of component finger-print control technologys in ginkgolides and the large molecule that may remain, the report of protein inspection method.Bilobalide injection prepared by the present invention has obtained State Food and Drug Administration's authentication code, the accurate word of traditional Chinese medicines: Z20110035, and be current first ginkgo active component injection in the world, product structure is clear, clear and definite.

Summary of the invention

The object of the invention is to provide a kind of ginkgolides active component detection method.The present invention also provides a kind of extraction separation method of ginkgolides, and this extraction separation method can obtain the fixing ginkgolides of component, and the method step is as follows:

A, extraction: pulverize ginkgo leaf, add Solvent Extract methods, in concentrated extracting solution, add anti-oxidant protective agent, with pH adjusting agent adjust pH to 4 ~ 5, concentrated, refrigeration.

Wherein, extraction organic solvent is ethanol, acetone or ethyl acetate, and concentration is 50 ~ 80%v/v, and consumption is 5 ~ 12 times amount, optimum amount 6 ~ 10 times;

Extracting method is: refluxing extraction or decoction are extracted.

1) refluxing extraction:

50 ~ 80%v/v ethanol: Extracting temperature 75 ~ 85 DEG C, extraction time 2 ~ 3 times, each 1 ~ 2 hour of time;

50 ~ 80%v/v acetone: Extracting temperature 45 ~ 55 DEG C, extraction time 2 ~ 3 times, each 1 ~ 2 hour of time;

50 ~ 80%v/v ethyl acetate: Extracting temperature 55 ~ 65 DEG C, extraction time 2 ~ 3 times, each 1 ~ 2 hour of time;

Vacuum tightness :-0.02 ~ 0.08MPa

Preferred extraction conditions is: extract 3 times, each 1.5 hours; Concentration of alcohol should select 65%v/v; Acetone concentration should select 50%v/v; Ethyl acetate concentration should select 60%v/v.

2) extraction is decocted:

50 ~ 80%v/v ethanol: Extracting temperature 80 ~ 90 DEG C, extraction time 2 ~ 3 times, each 1 ~ 2 hour of time;

50 ~ 80%v/v acetone: Extracting temperature 50 ~ 60 DEG C, extraction time 2 ~ 3 times, each 1 ~ 2 hour of time;

50 ~ 80%v/v ethyl acetate: Extracting temperature 60 ~ 65 DEG C, extraction time 2 ~ 3 times, each 1 ~ 2 hour of time;

After extract is heated in concentration process, ginkgolides easily decomposes, and need add protective agent and pH adjusting agent.Adding protective agent is that available anti-oxidant protective agent mainly contains neutral amino acid, comprising: at least one in serine, methionine, asparagine, threonine, preferred methionine in order to prevent ginkgolides to be heated oxygenolysis.

PH adjusting agent is organic monoacid mainly, comprising: at least one in citric acid, malic acid, sorbic acid, and preferably citric acid is used for adjust pH, is utilize its faintly acid as stabilizing agent, prevents ginkgolides open loop in the basic conditions.Reason is ginkgolides structure is 5 rings, stablizes under mildly acidic conditions, and citric acid is weak acid, can prevent ginkgolides open loop in the basic conditions.

The object of steps A refrigeration is to make oil and water separation, with except the middle oil-soluble impurities that anhydrates.

B, extraction: by concentrate first with normal hexane or petroleum ether extraction 2 ~ 3 times (preferred isopyknic normal hexane or petroleum ether extraction), aqueous phase fat-soluble solvent extraction 4 ~ 5 times (preferred isopyknic extraction into ethyl acetate), use water saturation sec-butyl alcohol (normal butyl alcohol)-ethyl acetate mixed extractant solvent 4 ~ 5 times (preferred equal-volume water saturation sec-butyl alcohol-ethyl acetate mixed extractant solvent) again, merge organic phase extract, reduced pressure concentration.

Wherein, be first to remove the impurity such as chlorophyll, ginkgolic acid with normal hexane or petroleum ether extraction.

Extract ginkgolides with fat-soluble solvent again, available fat-soluble solvent has at least one in ethyl acetate, ethyl formate, acetone, butanone.

Terpene lactones is soluble in ethyl acetate, GINKGO BILOBA EXTRACT dissolubility in ethyl acetate is relatively low, larger with solubleness in aqueous alcohol in the hot water, therefore can ginkgolides be extracted with ethyl acetate, it is separated with gingkgo flavonoids, the ginkgolides crude product be separated can use activated charcoal, silica gel or resin column adsorption chromatography further, and remove impurity further, then in aqueous alcohol, crystallization can obtain purer ginkgolides.

C, excessively post: extract crosses polyamide (30 ~ 60 order) resin column, and use 1 ~ 5BV water, 3 ~ 5BV, 20% ~ 40%v/v ethanol, 2 ~ 3BV, 60% ~ 90%v/v ethanol elution successively, control eluent flow rate is 2 ~ 3BV/h; Merge eluent, reduced pressure concentration, dry;

D, crystallization: the dry thing after crossing post is added in boiling water, stirring and dissolving, cooling, supernatant equal-volume ethyl acetate, ethyl formate or acetone extract 4 ~ 5 times, combining extraction liquid, reduced pressure concentration, evaporate to dryness, adds 5 ~ 8 times amount 30% ~ 50%v/v ethanol heating stirring and dissolving, filters, refrigeration, crystallize out, filters, filtrate I is for subsequent use, crystal 30% ~ 50%v/v ethanol washing, drying under reduced pressure, obtains crystal I.

It is 10% ~ 30%v/v that filtrate I is concentrated into alcohol content, refrigeration, crystallize out, and filter, filtrate II is for subsequent use; With the washing of 30% ~ 50%v/v ethanol, drying under reduced pressure, obtains crystal II.

Concentrated filtrate II, adds 0.1 ~ 0.5% (g/L) charcoal absorption, and filter, it is 10 ~ 30%v/v that filtrate is concentrated into alcohol content, refrigeration, crystallize out, and filter, filtrate II I is for subsequent use; Crystal 30% ~ 50%v/v ethanol washing, drying under reduced pressure, obtains crystal III.

Filtrate II I concentrates, and crosses activated charcoal-silica gel (volume ratio 1: 1 ~ 1: 3) post, first uses 30% ~ 50%v/v ethanol elution, use 70% ~ 90%v/v ethanol elution again, it is 10% ~ 30%v/v that collection eluent is concentrated into alcohol content, refrigeration crystallization, leach crystal, filtrate IV is for subsequent use; Crystal 30% ethanol washs, and drying under reduced pressure obtains crystal IV.

Concentrated filtrate IV, refrigeration, crystallize out, filters, and crystal 30%v/v ethanol washing, drying under reduced pressure, obtains crystal V.

According to the testing result of HPLC to ginkgolides remaining in mother liquor, consider whether crystallization is carried out to filtrate IV.

E, mixed crystal: crystal I, II, III, IV, V are mixed, pulverize, obtain off-white color crystalline composition, the HPLC content of this crystalline composition active component (Bilobalide, ginkalide A, ginkolide B, ginkalide C sum) is greater than 95%.

The parameter that employing the present invention gets the ginkgolides that separation method obtains is as follows:

A) proterties: off-white color or micro-yellow crystalline powder.Easily molten in ethyl acetate, dissolve in methyl alcohol, ethanol, almost insoluble in water.

B) moisture: be less than 5.0%.

C) protein: absorbance is less than 0.05 at 595nm wavelength place.

D) tannin, resin, oxalates, potassium ion: must not detect.

E) residual solvent: ethanol and ethyl acetate are all less than 0.5%, and normal hexane is less than 0.029%, and caprolactam is less than 0.0015%.

F) total ginkgoic acid: HPLC method measures total ginkgoic acid content and is less than 5ppm.

G) large molecule and polymkeric substance: the large molecule of gel chromatography noresidue and polymkeric substance.LC-MS method mensuration is greater than macromolecular substances and the polymkeric substance of 1000 without molecular weight.

H) heavy metal: be less than 10ppm.

I) arsenic salt: be less than 2ppm.

K) undue toxicity: make the solution containing 0.2mg in every 1ml, meet intravenous injection administration.

L) finger-print: HPLC method measures, with Bilobalide reference substance, ginkalide A reference substance, ginkolide B reference substance, ginkalide C reference substance for object of reference, by similarity evaluation, four total peak similarities are greater than 0.95.

M) content: HPLC method measures, calculates, containing Bilobalide (C by dry product ₁₅h ₁₈o ₈) 25.0% ~ 50.0%, ginkalide A (C ₂₀h ₂₄o ₉) 20.0% ~ 45.0%, ginkolide B (C ₂₀h ₂₄o ₁₀) 10.0% ~ 30.0%, ginkalide C (C ₂₀h ₂₄o ₁₁) 5.0% ~ 15.0%, and Bilobalide, ginkalide A, ginkolide B, ginkalide C total amount are greater than 95%.

Accompanying drawing explanation

Fig. 1 elution volume is on the impact of eluting rate.

Fig. 2 ethanol flow velocity is on the impact of eluting rate.

Fig. 3 ginkgolides LC-MS collection of illustrative plates (molecular weight 400 ~ 1000).

Fig. 4 ginkgolides LC-MS collection of illustrative plates (molecular weight 400 ~ 3000).

Fig. 5 ginkgolides reference fingerprint;

Total coneincone 2: ginkalide C, peak 3: Bilobalide, peak 4: ginkalide A, peak 5: ginkolide B.

Embodiment

It is below the key condition shaker test in ginkgolides preparation method of the present invention.

One, option screening test is extracted

Method one: by concentrate first with isopyknic n-hexane extraction 2 ~ 3 times, aqueous phase with the butanone-acetone (4: 6) of 8 times amount warm lower extraction 5 times, combining extraction liquid, reduced pressure concentration.

Method two: by concentrate first with isopyknic n-hexane extraction 2 ~ 3 times, aqueous phase uses equal-volume extraction into ethyl acetate 4 ~ 5 times again, with water saturated sec-butyl alcohol-ethyl acetate (7: the 3) extraction of equivalent 4 ~ 5 times, combining extraction liquid, reduced pressure concentration, dry.

Above two kinds of extraction separation purification method shaker tests, measure the total amount of ginkgolides in two kinds of tests respectively by HPLC-ELSD method, test findings is in table 1.

Table 1 extracts option screening test findings

Investigation project	Method one	Method two
			Appearance character	Brown ceramic powder	Brown ceramic powder
Total lactones content (%)	14.1	10.8

Method two gained total lactones content is all higher, and ethyl acetate and sec-butyl alcohol are the high solvent of security, therefore, selecting method two as extraction from purifying process.

Two, chromatography condition shaker test

Owing to still containing a large amount of Ginkgolides material and other impurity in extract, obtain very high purity ginkgolides, flavones effectively must be separated with ginkgolides, the separation method generally adopted at present comprises polyamide resin column partition method, alumina column chromatography method and silica gel column chromatography, inventor's comparative study process and result as follows:

Method one: extract is crossed polyamide resin column, first uses 30% ethanol elution of 2 ~ 3 times amount, continues and use 70% ethanol elution, and elution speed is 2BV/h, concentrate eluant, evaporate to dryness.

Method two: by extract peracidity alumina column, mixed by extract with equivalent aluminium oxide, dry, dry method upper prop, with the eluent ethyl acetate of 4 ~ 6 times amount, elution speed is 2BV/h, concentrate eluant, evaporate to dryness.

Method three: extract is crossed silicagel column, extract is mixed with equivalent column chromatography silica gel, dry, dry method upper prop, first with petroleum ether-ethyl acetate (2: the 1) wash-out by 4 ~ 6 times amount, elution speed is 2BV/h, use n-hexane-ethyl acetate (5: 1) wash-out again, elution speed is 2BV/h, concentrate eluant, evaporate to dryness.

Measure the total amount of ginkgolides in three kinds of tests respectively by HPLC-ELSD method, test findings is in table 2.

Table 2 column chromatography test findings

Investigation project	Method one	Method two	Method three
				Appearance character	Yellow powder	Yellow powder	Yellow powder
Total lactones content (%)	47.8	35.5	38.2

Therefore the ginkgolides content adopting polyamide resin column to obtain is higher, and separating effect is better.

Polyamide has good suction-operated to flavones, therefore, can be effectively effectively separated with GINKGO BILOBA EXTRACT by ginkgolides, investigates crossing post elution processes parameter.

1, the selection of volume is washed: distilled water washing resin post can play good removal of impurities effect, with the water washing resin post of 5BV, flow velocity 1 ~ 2BV/h, the color of outflow from depth to shallow, collects water lotion 5BV, efflux is limpid, testing result shows, and when water volume reaches 3BV, the water-solubility impurity in post is substantially clean, inspection does not measure ginkgolides, so select the washing volume of 3BV.Fig. 1 is shown in the impact of elution volume on eluting rate.

2, the impact of ethanol elution concentration versus elution effect: extract is gone up different polyamide post respectively, absorption 30min, first wash with 3BV, use 10% respectively again, 30%, 40%, 50%, 70%, 90% ethanol elution, flow velocity 1BV/h, collect ethanol eluate respectively, measure the amount of ginkgolides in each concentration eluent, along with the rising of concentration of alcohol, elution amount and eluting rate rise all thereupon, but to 40% ethanol time elution amount Slow lifting, to 90% ethanol time elution amount difference little, the ethanol elution of 30% reaches best eluting rate substantially, therefore, adopt 30% ethanol as best wash-out concentration.

3, the elute effect impact of flow velocity resolved by ethanol: extract is gone up different polyamide post respectively, absorption 30min, first with 3BV washing, then uses 40% ethanol elution, flow velocity 1BV/h.Resolve flow velocity for choosing preferably ethanol, respectively with 1,2,3,4, the flow velocity of 5BV/h crosses post, wash-out also collects 3BV eluent, measures ginkgolides amount.Elution flow rate and resolution factor have very large relevance, along with flow velocity improves, resolution factor increases, but declines on the contrary to during 3BV/h, this is that speed is accelerated to cause ethanol eluate well not exchange with the ginkgolides of absorption, thus can not reach good elute effect.Optimum flow rate 2 ~ 3BV/h.Fig. 2 is shown in the impact of ethanol flow velocity on eluting rate.

Three, crystallization conditional filtering test:

Although in extract, the content of ginkgolides increases to some extent after crossing post, extraction, Flavonoid substances is also effectively separated, and the content of ginkgolides still can not reach injection requirement, needs further crystallization purifying.Ginkgolides is easily molten in ethanol, ethyl acetate equal solvent, and insoluble in water, normal hexane equal solvent, therefore only selects the suitable mixed solvent of polarity as recrystallisation solvent.

(1) 30%v/v alcohol solvent: get and treat crystallization extract 10g, adds 4,6,8,10 times amount 30% ethanol, heating for dissolving respectively, and low temperature (0 ~ 6 DEG C) leaves standstill, and filter, drying under reduced pressure, measures crystal weight respectively, and test findings is in table 3.

Table 3 30% alcohol crystal test findings

Solvent adding amount (doubly)	4	6	8	10
					Heating for dissolving situation	Be not dissolved	Dissolve completely	Dissolve completely	Dissolve completely
Crystal amount (g)	3.8	4.5	4.2	2.4

Add 5 ~ 8 times amount 30% ethanol during crystallization proper, the crystal of precipitation is relatively many.

(2) n-hexane-ethyl acetate (8: 1) solvent: get and treat crystallization extract 10g, add 4,6,8,10 times amount n-hexane-ethyl acetate (8: 1) mixed solvents respectively, heating for dissolving, low temperature (0 ~ 6 DEG C) leaves standstill, filter, drying under reduced pressure, measures crystal weight respectively, and test findings is in table 4.

Table 4 n-hexane-ethyl acetate mixed solvent crystallization test findings

Solvent adding amount (doubly)	4	6	8	10
					Heating for dissolving situation	Dissolve completely	Dissolve completely	Dissolve completely	Dissolve completely
Crystal amount (g)	2.3	3.5	3.8	3.2

N-hexane-ethyl acetate mixed solvent crystallization amount is less than 30% alcohol solvent crystallize out amount.

(3) 10%v/v ethyl acetate solvent: get and treat crystallization extract 10g, adds 4,6,8,10 times amount 10% ethyl acetate heating for dissolving respectively, and low temperature (0 ~ 6 DEG C) leaves standstill, filter, drying under reduced pressure, measures crystal weight respectively, and test findings is in table 5.

Table 5 10% ethyl acetate crystallization trial result

Solvent adding amount (doubly)	4	6	8	10
					Heating for dissolving situation	Be not dissolved	Dissolve completely	Dissolve completely	Dissolve completely
Crystal amount (g)	2.3	3.5	3.3	2.2

10% ethyl acetate solvent crystallization amount is less than 30% alcohol solvent crystallize out amount.

Experimentally result, recrystallisation solvent selects 5 ~ 8 times amount 30% ethanol proper.

Example below for adopting the inventive method to prepare ginkgolides.

Embodiment 1

Ginkgo leaf meal 50kg, adds 65% alcohol heating reflux and extracts 3 times (10,8,6 times amount), each 1.5 hours, merges extract, filter, reduced pressure concentration, adds 0.05% methionine stirring and dissolving, with citric acid soln adjust ph to 4 ~ 5, continue concentrated, low temperature is placed, and filters.First use equivalent n-hexane extraction, use equivalent extraction into ethyl acetate again, finally use water saturation sec-butyl alcohol-ethyl acetate mixed extractant solvent, cross polyamide (30 ~ 60 order) resin column, first wash with water, continue and use 30% ethanol elution, then use 70% ethanol elution, merge eluent, reduced pressure concentration.Add in 2 ~ 3 times amount boiling water, stirring and dissolving, leave standstill, let cool, be extracted with ethyl acetate, reduced pressure concentration, add ethanol heating stirring and dissolving, filter, let cool, crystallize out, filter, dry, obtain crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol to 30%, leaves standstill, crystallize out, filters, dry, obtains crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, stirring and adsorbing, filters, concentrated, lets cool, crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and B); Filtrate is concentrated, crosses medical charcoal-silica gel (1: 1) post, first use 2 times amount 30% ethanol elutions, then use 4 times amount 70% ethanol elutions, collect eluent, concentrated, add ethanol to 30%, let cool, leave standstill, crystallize out, filter, dry, obtain crystal IV; Filtrate concentrates, and adds ethanol to 30%, lets cool, and leaves standstill, crystallize out, filters, dry, obtains crystal V.Crystal is mixed, obtains ginkgolides 91.6g, HPLC content 97.2%, wherein Bilobalide (C ₁₅h ₁₈o ₈) be 42.5%, ginkalide A (C ₂₀h ₂₄o ₉) be 25.4%, ginkolide B (C ₂₀h ₂₄o ₁₀) be 18.7%, ginkalide C (C ₂₀h ₂₄o ₁₁) be 10.6%.

Embodiment 2

Ginkgo leaf meal 200kg, adds 6 times amount 80% alcohol heating reflux and extracts 3 times, each 1.5 hours, merges extract, filter, decompression filtrate recycling ethanol, to without alcohol taste, adds 0.05% methionine stirring and dissolving, with citric acid soln adjust ph to 4 ~ 5, continue concentrated, low temperature is placed, and filters.First use n-hexane extraction, then be extracted with ethyl acetate, finally use water saturation sec-butyl alcohol-ethyl acetate mixed extractant solvent, cross polyamide (30 ~ 60 order) resin column, first use 30% ethanol elution, continue and use 70% ethanol elution, merge eluent, reduced pressure concentration.Add in boiling water, stirring and dissolving, leave standstill and let cool, be extracted with ethyl acetate, reduced pressure concentration, add ethanol heating stirring and dissolving, filter, let cool, crystallize out, filter, dry, obtain crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, leaves standstill crystallize out, filters, dry, obtains crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, stirring and adsorbing, filters, concentrated, adds ethanol, lets cool, crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and B); Filtrate is concentrated, crosses medical charcoal-silica gel (1: 1) post, first use 2 times amount 30% ethanol elutions, then use 4 times amount 70% ethanol elutions, collect eluent, concentrated, add ethanol to 30%, let cool, leave standstill, crystallize out, filter, dry, obtain crystal IV; Filtrate concentrates, and adds ethanol to 30%, lets cool, and leaves standstill, crystallize out, filters, dry, obtains crystal V.Crystal is mixed, obtains ginkgolides 362.8g, HPLC content 96.8%, wherein Bilobalide (C ₁₅h ₁₈o ₈) be 31.2%, ginkalide A (C ₂₀h ₂₄o ₉) be 28.8%, ginkolide B (C ₂₀h ₂₄o ₁₀) be 28.2%, ginkalide C (C ₂₀h ₂₄o ₁₁) be 8.6%.

Embodiment 3

Ginkgo leaf meal 200kg, adds 8 times amount 75% alcohol heating reflux and extracts 3 times, each 1.5 hours, merges extract, filter, decompression filtrate recycling ethanol, to without alcohol taste, adds 0.05% methionine stirring and dissolving, with citric acid soln adjust ph to 4 ~ 5, continue concentrated, low temperature is placed, and filters.First use n-hexane extraction, then be extracted with ethyl acetate, finally use water saturation sec-butyl alcohol-ethyl acetate mixed extractant solvent, cross polyamide (30 ~ 60 order) resin column, first use 30% ethanol elution, continue and use 75% ethanol elution, merge eluent, reduced pressure concentration.Add in boiling water, stirring and dissolving, leave standstill and let cool, be extracted with ethyl acetate, reduced pressure concentration, add ethanol heating stirring and dissolving, filter, let cool, crystallize out, filter, dry, obtain crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, leaves standstill crystallize out, filters, dry, obtains crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, stirring and adsorbing, filters, concentrated, adds ethanol, lets cool, crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and B); Filtrate concentrates, and upper medical charcoal-silica gel (1: 1) post, uses 60% ethanol elution, collects eluent, concentrated, lets cool, crystallize out, filters, dry, obtains crystal IV; Crystal is mixed, obtains ginkgolides 375.5g, HPLC content 97.1%, wherein Bilobalide (C ₁₅h ₁₈o ₈) be 35.8%, ginkalide A (C ₂₀h ₂₄o ₉) be 28.5%, ginkolide B (C ₂₀h ₂₄o ₁₀) be 26.2%, ginkalide C (C ₂₀h ₂₄o ₁₁) be 6.6%.

Embodiment 4

Get ginkgo leaf meal 200kg, add 10 times amount 75% alcohol heating reflux and extract 3 times, each 1.5 hours, merge extract, filter, decompression filtrate recycling ethanol, to without alcohol taste, adds 0.05% methionine stirring and dissolving, with citric acid soln adjust ph to 4 ~ 5, continue concentrated, low temperature is placed, and filters.First use n-hexane extraction, then be extracted with ethyl acetate, finally use water saturation sec-butyl alcohol-ethyl acetate mixed extractant solvent, cross polyamide (30 ~ 60 order) resin column, first use 25% ethanol elution, continue and use 65% ethanol elution, merge eluent, reduced pressure concentration.Add in boiling water, stirring and dissolving, leave standstill and let cool, be extracted with ethyl acetate, reduced pressure concentration, add 50% ethanol heating stirring and dissolving, filter, let cool, crystallize out, filter, dry, obtain crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, leaves standstill crystallize out, filters, dry, obtains crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, stirring and adsorbing, filters, concentrated, lets cool, crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and B); Filtrate concentrates, and upper medical charcoal-silica gel (1: 1) post, uses 60% ethanol elution, collects eluent, concentrated, lets cool, crystallize out, filters, dry, obtains crystal IV; Filtrate concentrates, and lets cool, crystallize out, filters, dry, obtains crystal V; Crystal is mixed, obtains ginkgolides 362.2g, HPLC content 96.5%, wherein Bilobalide (C ₁₅h ₁₈o ₈) be 35.5%, ginkalide A (C ₂₀h ₂₄o ₉) be 26.0%, ginkolide B (C ₂₀h ₂₄o ₁₀) be 26.2%, ginkalide C (C ₂₀h ₂₄o ₁₁) be 8.8%.

Embodiment 5

Ginkgo leaf meal 200kg, adds 8 times amount 60% ethyl acetate heating and refluxing extraction 3 times, each 1.5 hours, merges extract, filter, filtrate decompression reclaims ethyl acetate, adds 0.05% methionine stirring and dissolving, with citric acid soln adjust ph to 4 ~ 5, continue concentrated, low temperature is placed, and filters.First use petroleum ether extraction, aqueous phase is extracted with ethyl acetate again, finally uses water saturation sec-butyl alcohol-ethyl acetate mixed extractant solvent, cross polyamide (30 ~ 60 order) resin column, first use 30% ethanol elution, continue and use 75% ethanol elution, merge eluent, reduced pressure concentration.Add in boiling water, stirring and dissolving, leave standstill and let cool, with acetone extract, be evaporated to dry, add 50% ethanol heating stirring and dissolving, filter, let cool, crystallize out, filter, dry, obtain crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, leaves standstill crystallize out, filters, dry, obtains crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, stirring and adsorbing, filters, concentrated, adds ethanol, lets cool, crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and B); Filtrate concentrates, and upper medical charcoal-silica gel (1: 1) post, uses 60% ethanol elution, collects eluent, concentrated, lets cool, crystallize out, filters, dry, obtains crystal IV; Crystal is mixed, obtains ginkgolides 350.6g, HPLC content 97.4%, wherein Bilobalide (C ₁₅h ₁₈o ₈) be 40.0%, ginkalide A (C ₂₀h ₂₄o ₉) be 22.5%, ginkolide B (C ₂₀h ₂₄o ₁₀) be 27.2%, ginkalide C (C ₂₀h ₂₄o ₁₁) be 10.3%.

Embodiment 6

Ginkgo leaf meal 200kg, adds 8 times amount 50% acetone heating and refluxing extraction 3 times, each 1.5 hours, merges extract, filter, filtrate decompression reclaims acetone, adds 0.05% methionine stirring and dissolving, with citric acid soln adjust ph to 4 ~ 5, continue concentrated, low temperature is placed, and filters.First use petroleum ether extraction, aqueous phase is extracted with ethyl acetate again, finally uses water saturation sec-butyl alcohol-ethyl acetate mixed extractant solvent, cross polyamide (30 ~ 60 order) resin column, first use 30% ethanol elution, continue and use 70% ethanol elution, merge eluent, reduced pressure concentration.Add in boiling water, stirring and dissolving, leave standstill and let cool, be extracted with ethyl acetate, be evaporated to dry, add 30% ethanol heating stirring and dissolving, filter, let cool, crystallize out, filter, dry, obtain crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, leaves standstill crystallize out, filters, dry, obtains crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, stirring and adsorbing, filters, concentrated, adds ethanol, lets cool, crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and B); Filtrate concentrates, and upper medical charcoal-silica gel (1: 1) post, uses 60% ethanol elution, collects eluent, concentrated, lets cool, crystallize out, filters, dry, obtains crystal IV; Filtrate concentrates, and lets cool, crystallize out, filters, dry, obtains crystal V; Crystal is mixed, obtains ginkgolides 343.5g, HPLC content 96.2%, wherein Bilobalide (C ₁₅h ₁₈o ₈) be 38.2%, ginkalide A (C ₂₀h ₂₄o ₉) be 28.3%, ginkolide B (C ₂₀h ₂₄o ₁₀) be 24.2%, ginkalide C (C ₂₀h ₂₄o ₁₁) be 9.3%.

Embodiment 7

Ginkgo leaf meal 200kg, adds 8 times amount 70% ethanol and is heated to micro-decoction extraction 3 times of boiling, each 1.5 hours, merge extract, filter, filtrate decompression reclaims acetone, adds 0.05% methionine stirring and dissolving, with citric acid soln adjust ph to 4 ~ 5, continue concentrated, low temperature is placed, and filters.First use petroleum ether extraction, aqueous phase is extracted with ethyl acetate again, finally uses water saturation sec-butyl alcohol-ethyl acetate mixed extractant solvent, cross polyamide (30 ~ 60 order) resin column, first use 30% ethanol elution, continue and use 70% ethanol elution, merge eluent, reduced pressure concentration.Add in boiling water, stirring and dissolving, leave standstill and let cool, be extracted with ethyl acetate, be evaporated to dry, add 30% ethanol heating stirring and dissolving, filter, let cool, crystallize out, filter, dry, obtain crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, leaves standstill crystallize out, filters, dry, obtains crystal II (being mainly Bilobalide and Ginkgolides a and B); Filtrate adds medical charcoal, stirring and adsorbing, filters, concentrated, adds ethanol, lets cool, crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and C); Filtrate concentrates, and upper medical charcoal-silica gel (1: 1) post, uses 60% ethanol elution, collects eluent, concentrated, lets cool, crystallize out, filters, dry, obtains crystal IV; Filtrate concentrates, and lets cool, crystallize out, filters, dry, obtains crystal V; Crystal is mixed, obtains ginkgolides 362.6g, HPLC content 97.4%, wherein Bilobalide (C ₁₅h ₁₈o ₈) be 36.5%, ginkalide A (C ₂₀h ₂₄o ₉) be 25.3%, ginkolide B (C ₂₀h ₂₄o ₁₀) be 28.2%, ginkalide C (C ₂₀h ₂₄o ₁₁) be 7.4%.

To sum up, extraction separation and purification method used in the present invention is adopted can to obtain the relatively-stationary ginkgolides of purity higher composition, wherein, containing Bilobalide (C ₁₅h ₁₈o ₈) 25.0% ~ 50.0%, ginkalide A (C ₂₀h ₂₄o ₉) 20.0% ~ 45.0%, ginkolide B (C ₂₀h ₂₄o ₁₀) 10.0% ~ 30.0%, ginkalide C (C ₂₀h ₂₄o ₁₁) 5.0% ~ 15.0%, and Bilobalide, ginkalide A, ginkolide B, ginkalide C total amount are greater than 95%.

The invention provides the detection method of ginkgolides active component, subitem detection method and the testing result of concrete ginkgolides of the present invention are as follows:

A) proterties: off-white color or micro-yellow crystalline powder.

Easily molten in ethyl acetate, dissolve in methyl alcohol, ethanol, almost insoluble in water.

B) moisture: 60 DEG C of drying under reduced pressure less loss weight are less than 5.0%.

C) protein: absorbance is less than 0.05 at 595nm wavelength place.

Get ginkgolides of the present invention and be about 24mg, after adding ethanol 2ml dissolving, be diluted with water to 50ml, as need testing solution.Measure according to Coomassie Brilliant Blue (Bradford method), with corresponding reagent for blank, at 595nm wavelength place, absorbance is less than 0.05.

D) tannin, resin, oxalates, potassium ion

Adoptable detection method has:

Tannin: get protein check item need testing solution 1ml, add spirit of vinegar 1, then add gelatin sodium chloride test solution 5, shake up, places 10 minutes, does not occur muddiness or precipitation.

Resin: get protein check item need testing solution 5ml, add hydrochloric acid 1, places 30 minutes, separates out without resinoid.

Oxalates: get protein check item need testing solution 2ml, with watery hydrochloric acid adjust ph to 1 ~ 2, filter, filtrate ammoniacal liquor adjust ph is 5 ~ 6, adds 3% calcium chloride solution 3, places 10 minutes, does not occur muddiness or precipitation.

Potassium ion: get protein check item need testing solution 2ml, put in 10ml nessler colorimetric tube, add alkaline formaldehyde solution 0.6ml, 3%EDTA solution 2,3% sodium tetraphenylborate solution 0.5ml, be diluted with water to 10ml, the accurate Klorvess Liquid 0.8ml of another label taking, with method test, the turbidity of need testing solution is not higher than contrast solution.

Result: do not detect tannin, resin, oxalates, potassium ion.

E) residual solvent

(1) ethanol, ethyl acetate and normal hexane: be all less than 0.5% containing ethanol and ethyl acetate, normal hexane is less than 0.029%.

(2) resin residue amount: be less than 0.0015% containing caprolactam.

F) total ginkgoic acid: be less than 5ppm containing total ginkgoic acid.

G) large molecule and polymkeric substance: the large molecule of gel chromatography noresidue and polymkeric substance.LC-MS method measures, and result is greater than large molecule and the polymkeric substance of 1000 without molecular weight.

Assay method:

(1) exclusion chromatography chromatographic column: Phenomenex BioSep-SEC-S2000,300 × 7.8mm, 5um, mobile phase: 0.71% (including the sodium azide of 0.02%) metabisulfite solution, column temperature: 35 DEG C, detector temperature: 35 DEG C, flow velocity: 0.5ml/min.Result: the large molecule of noresidue and polymkeric substance.

(2) HPLC-MS coupling method mobile phase: methanol-water (90: 10), chromatographic column: Agilent RX-C ₁₈(2.1 × 50mm) column temperature: 25 DEG C, flow velocity: 0.3ml/min.Result: result is greater than large molecule and the polymkeric substance of 1000 without molecular weight.

H) heavy metal: be less than 10ppm.

I) arsenic salt: be less than 2ppm.

The preparation of need testing solution: get ginkgolides of the present invention and be about 25mg, with sodium chloride injection makes solution in every 1ml containing 0.2mg after dissolving with ethanol 2ml.

Inspection technique: get body weight 17 ~ 20g mouse 5, injects mouse tail vein need testing solution 0.5ml, and 48 hours without dead.

L) finger-print: HPLC method measures, and records the chromatogram of 60 minutes.By similarity evaluation, four total peak similarities are greater than 0.95.

M) content: HPLC method measures, calculates, containing Bilobalide (C by dry product ₁₅h ₁₈o ₈) should be 25.0% ~ 50.0%, ginkalide A (C ₂₀h ₂₄o ₉) should be 20.0% ~ 45.0%, ginkolide B (C ₂₀h ₂₄o ₁₀) should be 10.0% ~ 30.0%, ginkalide C (C ₂₀h ₂₄o ₁₁) should be 5.0% ~ 15.0%, and Bilobalide, ginkalide A, ginkolide B, ginkalide C total amount are greater than 95%.

L) finger-print is identical with the detection method of m) assay employing, and condition is as follows: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol-tetrahydrofuran-water (25: 10: 65) for mobile phase; By evaporative light-scattering detector, drift tube temperature: 105 DEG C; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 DEG C; Number of theoretical plate calculates should be not less than 2500 by Bilobalide peak.The degree of separation at Bilobalide peak and ginkalide C peak should be greater than 1.5.

N) pyrogen test: body temperature raises lower than 0.6 DEG C.

The preparation of need testing solution: precision takes ginkgolides 20mg of the present invention, adds 2ml ethanol and makes dissolving, then adds in 0.9% sodium chloride injection 100ml.

Inspection technique: get rabbit 3, measures after its normal body temperature in 15 minutes, injects 5ml slowly inject need testing solution from ear vein by the every 1kg of rabbit body weight, body temperature 1 time is measured every 30 minutes, survey 6 times altogether, body temperature raises all should lower than 0.6 DEG C, and 3 rabbit body temperatures raise summations lower than 1.3 DEG C.

Inventor has carried out large molecule and polymer determination research to foregoing invention content and has illustrated, for proving technique effect of the present invention.Following test is used for further illustrating and explaining the present invention, but does not limit the present invention.

(1) test apparatus and reagent

Agilent 1200 type high performance liquid chromatograph, UV-detector, differential refraction detector.

Phenomenex BioSep-SEC-S2000 gel chromatographic columns.

Dextran reference substance D2000 (blue dextran 2000), middle inspection institute, lot number 140646-2000-01

Glucose control product (D0), content: 99.5%, lot number 086K0166, SIGMA.

Ultrapure water Millipore-Q ultrapure water system obtains.

All the other reagent is pure for analyzing.

(2) selection of mobile phase

Choose 0.71% (including the sodium azide of 0.02%) metabisulfite solution as mobile phase.

(3) selection of detecting device

Select common detector differential refraction detector, this detecting device all has good response for the material that there is refraction coefficient difference.

(4) quasi-definite chromatographic condition

Chromatographic column: Phenomenex BioSep-SEC-S2000,300 × 7.8mm, 5 μm

Mobile phase: 0.71% (including the sodium azide of 0.02%) metabisulfite solution

Column temperature: 35 DEG C, detector temperature: 35 DEG C, flow velocity: 0.5ml/min

(5) molecular weight of each composition of ginkgolides

Ginkgolides	Ginkalide A	Ginkolide B	Ginkalide C	Bilobalide	Bilobalide J
						Molecular formula	C ₂₀H ₂₄O ₉	C ₂₀H ₂₄O ₁₀	C ₂₀H ₂₄O ₁₁	C ₁₅H ₁₈O ₈	C ₂₀H ₂₄O ₁₀
Molecular weight	408.4	424.4	440.4	326.3	424.4

(6) methodological study

1. dextran and glucose control product are added the solution that mobile phase makes 10mg/ml respectively, the each 20 μ l of accurate absorption reference substance solution respectively, injecting chromatograph, record chromatogram, result dextran goes out peak in retention time 9.816 ', and glucose goes out peak in retention time 18.712 ', shows to adopt exclusion chromatography, the material that molecular weight is large first goes out peak, goes out peak after the material that molecular weight is little.

2. get ginkgolides of the present invention and be about 10mg, add ethanol 2ml to dissolve, add dextran reference substance solution (10mg/ml) 1ml, mixing, accurate absorption 10 μ l, injecting chromatograph, record chromatogram, result detects dextran in retention time 9.698 ', bilobalide injection becomes swarming all after 18min, to go out peak, show molecular weight at 180 ~ 450 appearance times at about 18min, molecular weight 5000 ~ 2000000 appearance time is at about 9min, and it is feasible for adopting exclusion chromatography to detect macromolecular substances.

In order to verify not containing large molecule and polymkeric substance in this product again, therefore carry out LC-MS test again.

Chromatographic condition: methanol-water (90: 10) is mobile phase, Agilent RX-C ₁₈(2.1 × 50mm) chromatographic column, column temperature 25 DEG C of flow velocity 0.3ml/min.

The preparation of need testing solution: precision takes ginkgolides 10mg of the present invention and puts in 10ml measuring bottle, adds appropriate 1% acetic acid and makes dissolving, add mobile phase and be diluted to scale, shake up, as need testing solution.

LC-MS coupling is tested: according to the test method determined, get each 10 μ l of need testing solution respectively, test respectively in 400 ~ 1000 and 400-3000 molecular weight ranges, record chromatogram.Test findings is in table 6.

Table 6 LC-MS coupling molecular weight determination

[M+Na] ⁺	M
		419.1、431.5、447.4、463.3、475.7、532.2、588.8、701.8	396.1、408.5、424.4、440.3、452.7、509.2、678.8

From LC-MS coupling molecular weight determination, detect ginkalide A (molecular weight 408.5) respectively, ginkolide B (molecular weight 424.4), ginkalide C (molecular weight 440.4), completely the same with the effective ingredient of ginkgolides of the present invention, because test molecule weight range is between 400-3000, Bilobalide is not tested, the material that molecular weight is greater than more than 700 is not detected in ginkgolides of the present invention, the material of other different molecular weight may be the existence of other impurity, through LC-MS, the molecular weight of different components in ginkgolides is tested, illustrate in this product not containing large molecule or polymkeric substance.Fig. 3 ~ 4 are shown in by ginkgolides LC-MS collection of illustrative plates.

Embodiment 8

Ginkgolides quality control---total ginkgoic acid inspection

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol-1% glacial acetic acid (90: 10) for mobile phase; Flow velocity 1.0ml/min; Determined wavelength is 310nm.Number of theoretical plate should be not less than 4000 by gingko neo-acid peak.

The preparation of reference substance solution: get gingko eo-acid reference substance appropriate, accurately weighed, add methyl alcohol and make the solution in contrast product solution of every 1ml containing 5 μ g; Separately get total ginkgoic acid reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 100 μ g, as location contrast solution.

The preparation of need testing solution: get ginkgolides 5g of the present invention, accurately weighed, put in flask, add normal hexane 50ml, add hot reflux 2 hours, take out, let cool, filter, residue uses a small amount of n-hexane again, merging filtrate and cleansing solution, put evaporate to dryness in water-bath, residue adds methyl alcohol and dissolves and be diluted to 2ml, shakes up, as need testing solution.

Determination method: each 20 μ l of accurate absorption need testing solution, reference substance solution and location contrast solution, injection liquid chromatography, calculate the total peak area of chromatographic peak corresponding to total ginkgoic acid reference substance in need testing solution, calculate total ginkgoic acid content with gingko eo-acid reference substance external standard method, total ginkgoic acid is less than 5ppm.

Inventor is studied and explanation foregoing invention content, for proving technique effect of the present invention.Following test is used for further illustrating and explaining the present invention, but does not limit the present invention.

A, method one

The preparation of need testing solution: get ginkgolides 5g of the present invention, accurately weighed, put in flask, precision adds sherwood oil (60 ~ 90 DEG C) 50ml, refluxes 2 hours, takes out, let cool, filter, residue uses a small amount of petroleum ether 1 time again, merging filtrate and cleansing solution, put evaporate to dryness in water-bath, residue adds methyl alcohol and dissolves and be diluted to 2ml, shakes up, as need testing solution (1).

The preparation of blank sample solution: get sherwood oil (60 ~ 90 DEG C) 50ml, put in conical flask, reflux 2 hours, put evaporate to dryness in water-bath, residue adds methyl alcohol and dissolves and be diluted to 2ml, shakes up, as blank solution (1).

B, method two (normal hexane replacement sherwood oil)

The preparation of need testing solution: get ginkgolides 5g of the present invention, accurately weighed, put in flask, precision adds normal hexane 50ml, refluxes 2 hours, takes out, let cool, filter, residue uses a small amount of n-hexane 1 time again, merging filtrate and cleansing solution, put evaporate to dryness in water-bath, residue adds methyl alcohol and dissolves and be diluted to 2ml, shakes up, as need testing solution (2).

The preparation of blank sample solution: get normal hexane 50ml, puts in flask, and reflux 2 hours, put evaporate to dryness in water-bath, residue adds methyl alcohol and dissolves and be diluted to 2ml, shakes up, as blank solution (2).

Determination method: accurate absorption need testing solution and each 20 μ l of blank solution, injection liquid chromatography, record chromatogram.Test findings is in table 7.

Table 7 two kinds of method testing result tables

Test findings shows: employing method one (sherwood oil) prepares sample, detects chromatographic peak in blank solution, and the chromatographic peak area that its peak area and need testing solution detect is basically identical, illustrates that blank test has interference; And adopting method two (normal hexane) to prepare sample, blank solution and need testing solution all do not detect chromatographic peak, therefore inventor intends adopting method two to do application of sample recovery test, carry out the feasibility of verification method two with this.

The application of sample recovery test of c, total ginkgolic acid

The preparation of need testing solution: get ginkgolides 5g of the present invention, accurately weighed, put in flask, precision adds the total ginkgoic acid reference substance solution 0.2ml that concentration is 1.032mg/ml, then precision adds normal hexane 50ml, refluxes 2 hours, let cool, filter, residue uses a small amount of n-hexane again, merging filtrate and cleansing solution, put water bath method, residue adds methyl alcohol and dissolves and be diluted to 2ml, shakes up, as need testing solution.

The preparation of reference substance solution: precision measures the total ginkgoic acid reference substance solution 0.2ml that concentration is 1.032mg/ml, puts in 2ml measuring bottle, adds methanol dilution to scale, shake up, in contrast product solution.

Determination method: accurate absorption need testing solution and each 20 μ l of reference substance solution, injection liquid chromatography, record chromatogram.

Result: need testing solution can detect total ginkgoic acid chromatographic peak on the position corresponding to total ginkgoic acid reference substance chromatogram, and from peak area, need testing solution is consistent with reference substance solution peak area, illustrates that the recovery is better.Test findings is in table 8.

Table 8 average recovery test findings

D, reappearance are tested

The preparation of reference substance solution: get gingko eo-acid reference substance appropriate, accurately weighed, add methyl alcohol and make the solution in contrast product solution of every 1ml containing 5 μ g.Separately get total ginkgoic acid reference substance appropriate, accurately weighed, add methyl alcohol and make the solution of every 1ml containing 100 μ g, as location contrast solution.

Prepared by need testing solution: get ginkgolides 5g of the present invention, accurately weighed, nominal gets 6 parts, put respectively in flask, add normal hexane 50ml, reflux 2 hours, let cool, filter, a small amount of n-hexane of residue, merging filtrate and cleansing solution, put evaporate to dryness in water-bath, residue adds methyl alcohol and dissolves and be diluted to 2ml, shakes up, as need testing solution.

Determination method: each 20 μ l of accurate absorption need testing solution, reference substance solution and location contrast solution, injection liquid chromatography, record chromatogram.Test findings is in table 9.

Table 9 reproducible test results

Numbering

1#

2#

3#

4#

5#

6#

Total ginkgoic acid check result

Do not detect

Test findings shows, without ginkgolic acid in ginkgolides of the present invention.

E, recovery test

Prepared by need testing solution: get ginkgolides 5g of the present invention, accurately weighed, nominal gets 3 parts, put respectively in flask, adding concentration is respectively 3.04 μ g/ml gingko eo-acid reference substance solution 1.6ml, 2.0ml, 2.4ml, then adds normal hexane 50ml respectively, reflux 2 hours, let cool, filter, the a small amount of n-hexane of residue, merging filtrate and cleansing solution, put evaporate to dryness in water-bath, and residue adds methyl alcohol and dissolves and be diluted to 2ml, shake up, as need testing solution.

The preparation of reference substance solution: with reappearance test item.

Determination method: accurate absorption need testing solution, each 20 μ l of reference substance solution, injection liquid chromatography, record chromatogram.Each concentration determination three times, totally 9 times.Calculate the recovery, RSD value.Test findings is in table 10.

Table 10 recovery test result table

Test findings shows, the recovery is better.

Embodiment 9

Ginkgolides quality control---finger-print inspection

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; With methyl alcohol-tetrahydrofuran-water (25: 10: 65) for mobile phase; By evaporative light-scattering detector, drift tube temperature: 105 DEG C; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 DEG C; Number of theoretical plate calculates should be not less than 2500 by Bilobalide peak.The degree of separation at Bilobalide peak and ginkalide C peak should be greater than 1.5.

The preparation of object of reference solution: precision takes Bilobalide reference substance respectively, ginkalide A reference substance, ginkolide B reference substance, ginkalide C reference substance are appropriate, add methyl alcohol and make every 1ml respectively containing the mixed solution of 0.15mg, 0.12mg, 0.1mg, 0.1mg, shake up, as object of reference solution.

The preparation of test sample solution: get ginkgolides 6mg of the present invention, accurately weighed, put in 10ml measuring bottle and add methyl alcohol 1ml and dissolve, add mobile phase and be diluted to scale, shake up, as need testing solution.

Determination method: accurate absorption object of reference solution and each 20 μ l of need testing solution respectively, injection liquid chromatography, records the chromatogram of 60 minutes.

By similarity evaluation, test sample finger-print and reference fingerprint similarity are greater than 0.95.

In ginkgolides finger-print, wherein peak 2 is ginkalide C, peak 3 is Bilobalide, peak 4 is ginkalide A, peak 5 is ginkolide B, and in this product, active component 4 characteristic peaks all can one_to_one corresponding in finger-print.Ginkgolides reference fingerprint is shown in Fig. 5.

First Chinese Pharmacopoeia Commission's finger-print designated software in 2004 is adopted---similarity evaluation A version generates reference fingerprint to 10 batches of ginkgolides respectively, and the test sample finger-print of different batches and reference fingerprint similarity software are carried out calculating similarity.Test findings is in table 11.

Table 11 10 batches of ginkgolides similarity result

Lot number	100401	100402	100403	100404	110101
						Similarity	0.992	0.997	0.991	0.996	0.982
Lot number	110102	110103	110601	110602	110603
						Similarity	0.999	0.997	0.992	0.993	0.989

The similarity of 10 batches of ginkgolides finger-prints is all greater than 0.95.

Embodiment 10

Ginkgolides quality control---residual solvent measures

(1) ethanol, ethyl acetate and normal hexane

The preparation of need testing solution: get ginkgolides of the present invention and be about 0.1g, accurately weighed, in top set empty bottle, precision adds DMF 5ml makes dissolving, and sealing, as need testing solution.

The preparation of reference substance solution: get ethanol, ethyl acetate and normal hexane appropriate, accurately weighed, quantitatively dilute with DMF and make each solution about containing 30 μ g in every 1ml, precision measures 5ml, in top set empty bottle, sealing, product solution in contrast.

Determination method: with 6% cyanogen propyl group phenyl-94% dimethyl polysiloxane (or polarity is close) for immobile liquid, initial temperature is 50 DEG C, maintains 3 minutes, with the ramp to 160 DEG C of 40 DEG C per minute, maintains 3 minutes; Injector temperature 200 DEG C; Detector temperature is 250 DEG C; Ml headspace bottle equilibrium temperature is 80 DEG C, and equilibration time is 30 minutes.Get reference substance solution headspace sampling, the peak-to-peak degree of separation of each composition should meet the requirements; Get need testing solution and reference substance solution headspace sampling respectively again, record chromatogram, by external standard method with calculated by peak area.

All be less than 0.5% containing ethanol and ethyl acetate, normal hexane is less than 0.029%.

(2) resin residue amount

The preparation of reference substance solution: get DMA appropriate, accurately weighed, make every 1ml about containing the solution of 0.1mg with water, shake up, as inner mark solution; It is appropriate that precision takes caprolactam, adds inner mark solution and make every 1ml about containing the solution of caprolactam 37.5 μ g, product solution in contrast.

The preparation of need testing solution: get ginkgolides of the present invention and be about 2.5g, accurately weighed, put in conical flask, add normal hexane 25ml, reflux 2 hours, take out, let cool, filter, use a small amount of n-hexane, merging filtrate and cleansing solution, in 60 DEG C of water bath methods, residue adds inner mark solution 1ml makes dissolving, as need testing solution.

Determination method: with polyglycol (PEG-20M) (or polarity is close) for immobile liquid; Initial temperature is 100 DEG C, maintains 2 minutes, with the ramp to 160 DEG C of 40 DEG C per minute, maintains 3 minutes, then with the ramp to 220 DEG C of 40 DEG C, maintains 7 minutes; Injector temperature is 240 DEG C; Detector temperature is 260 DEG C.Precision measures reference substance solution and each 1 μ l of need testing solution, inject gas chromatograph, record chromatogram.By internal standard method with calculated by peak area, in need testing solution, the ratio of caprolactam peak area and interior mark peak area is less than the ratio of caprolactam peak area and interior mark peak area in reference substance solution.

Caprolactam does not detect.

Embodiment 11

Ginkgolides quality control---assay

The preparation of reference substance solution: precision takes Bilobalide reference substance respectively, ginkalide A reference substance, ginkolide B reference substance, ginkalide C reference substance are appropriate, add methyl alcohol and make every 1ml respectively containing the mixed solution of 0.15mg, 0.12mg, 0.1mg, 0.1mg, shake up, in contrast product solution.

The preparation of need testing solution: get ginkgolides 6mg of the present invention, accurately weighed, put in 10ml measuring bottle and add methyl alcohol 1ml and dissolve, add mobile phase and be diluted to scale, shake up, as need testing solution.

Determination method: precision measures reference substance solution 10 μ l, 20 μ l and need testing solution 10 ~ 20 μ l respectively, inject high performance liquid chromatograph, record chromatogram, calculates the content of Bilobalide, ginkalide A, ginkolide B and ginkalide C respectively with external standard two-point method logarithmic equation.

Calculate by dry product, Bilobalide (C ₁₅h ₁₈o ₈) be 42.5%, ginkalide A (C ₂₀h ₂₄o ₉) be 25.4%, ginkolide B (C ₂₀h ₂₄o ₁₀) be 18.7%, ginkalide C (C ₂₀h ₂₄o ₁₁) be 10.6%, and Bilobalide, ginkalide A, ginkolide B, ginkalide C total amount 97.2%.

Embodiment 12

Ginkgolides quality control---abnormal toxicity tests

The preparation of need testing solution: get ginkgolides of the present invention, makes the solution containing 0.2mg in every 1ml with sodium chloride injection.

Inspection technique: get body weight 17 ~ 20g mouse 5, injects mouse tail vein need testing solution 0.5ml respectively, without dead in 48 hours.

Embodiment 13

Ginkgolides quality control---pyrogen test

The preparation of need testing solution: get ginkgolides 10mg of the present invention, joins in 0.9% sodium chloride injection 50ml, shakes up.

Inspection technique: get rabbit 3, measures after its normal body temperature in 15 minutes, injects 5ml slowly inject need testing solution from ear vein by the every 1kg of rabbit body weight, body temperature 1 time is measured every 30 minutes, survey 6 times altogether, body temperature raises all lower than 0.6 DEG C, and 3 rabbit body temperatures raise summation lower than 1.3 DEG C.

Embodiment 14

Bilobalide injection quality control---Related substances separation

Injection formula:

Preparation method is:

A) prepare: mixed ethanol and glycerine, add ginkgolides, dissolving, add ethanol or water for injection to full dose, with 5 ~ 10% citric acid solns or 1 ~ 10% hydrochloric acid solution adjust ph to 3.2 ~ 3.8;

B) filtration sterilization;

C) embedding;

D) sterilizing.

(1) protein: get bilobalide injection 2ml, add water and make 50ml, as need testing solution.Take Coomassie brilliant G-250 and be about 50mg, be dissolved in 25ml ethanol, then add the phosphoric acid 50ml of 85% (w/v), be diluted with water to 500ml, shake up, filter, precision measures filtrate 5ml and puts in test tube, add 1ml need testing solution again, shake up, place 3min.Do blank with method, under 595nm wavelength, measure absorbance, need testing solution absorbance is less than 0.05.

(2) tannin: get protein check item need testing solution 1ml and add spirit of vinegar 1, then add gelatin sodium chloride test solution 5, shake up, place 10 minutes, do not occur muddiness or precipitation.

(3) resin: get protein check item need testing solution 5ml, add hydrochloric acid 1, places 30 minutes, separates out without resinoid.

(4) oxalates: get protein check item need testing solution 2ml, with watery hydrochloric acid adjust ph to 1 ~ 2, filter, filtrate ammoniacal liquor adjust ph is 5 ~ 6, adds 3% calcium chloride solution 3, places 10 minutes, does not occur muddiness or precipitation.

(5) potassium ion: get protein check item need testing solution 2ml, put in 10ml nessler colorimetric tube, add alkaline formaldehyde solution 0.6ml, 3%EDTA solution 2,3% sodium tetraphenylborate solution 0.5ml, be diluted with water to 10ml, the accurate Klorvess Liquid 0.8ml of another label taking, with method test, turbidity is lower than contrast solution.

Embodiment 15

Bilobalide injection quality control---haemolysis checks with cohesion

The preparation of need testing solution: get bilobalide injection (preparing by embodiment 14) 6ml, join in 0.9% sodium chloride injection 100ml and shake up.

Inspection technique: get 5, cleaned glass test tube, numbering, 1, No. 2 pipe is test sample pipe, and No. 3 pipes are negative control pipe, and No. 4 pipes are positive control pipe, No. 5 pipe test sample control tube.By adding 2% red cell suspension, 0.9% sodium chloride solution, distilled water shown in table 12 successively, putting immediately after mixing in the constant temperature oven of 37 DEG C ± 0.5 DEG C and carrying out incubation.

Table 12 haemolysis and agglutination test addition

Test tube is numbered	1	2	3	4	5
						2% red cell suspension/ml	2.5	2.5	2.5	2.5
0.9% sodium chloride solution/ml	2.2	2.2	2.5		4.7
						Distilled water/ml				2.5
Need testing solution/ml	0.3	0.3			0.3

If the solution in test tube is clear and bright redness, bottom is acellular to be remained or has a small amount of red blood cell to remain, and shows have haemolysis to occur; As red blood cell all sinks, supernatant achromatism and clarity, though or supernatant coloured clear and bright, 1, No. 2 pipe and No. 5 pipe visual inspection no significant differences, then show to occur without haemolysis.Observe after 3 hours and do not produce haemolysis and aggregation.

Embodiment 16

Bilobalide injection quality control---finger-print inspection

The preparation of need testing solution: get the need testing solution under [assay] item.

By similarity evaluation, test sample finger-print and reference fingerprint are greater than 0.95 through similarity.

Embodiment 17

Bilobalide injection quality control---assay

The preparation of need testing solution: precision measures bilobalide injection (preparing by embodiment 14) 1ml, adds phosphate buffered solution (pH6.5) 14ml, shakes up, upper Extrelut-20 post, adsorb 15 minutes, with ethyl acetate 100ml wash-out, collect eluent, evaporate to dryness in water-bath, residue mobile phase dissolves and is transferred in 10ml measuring bottle, adds mobile phase and is diluted to scale, shake up, filter, as need testing solution with 0.45 μm of miillpore filter.

Determination method: accurate absorption reference substance solution 10 μ l, 20 μ l respectively, need testing solution 15 μ l, inject high performance liquid chromatograph, record chromatogram, calculates the content of Bilobalide, ginkalide A, ginkolide B and ginkalide C respectively with external standard two-point method logarithmic equation.

Every 1ml composition containing ginkgo terpene lactone 5.15mg in bilobalide injection.

In bilobalide injection, every 1ml composition containing ginkgo terpene lactone is with Bilobalide (C ₁₅h ₁₈o ₈), ginkalide A (C ₂₀h ₂₄o ₉), ginkolide B (C ₂₀h ₂₄o ₁₀) and ginkalide C (C ₂₀h ₂₄o ₁₁) total amount count 1-10mg, preferably 4.25 ~ 5.75mg.

Claims

1. the detection method of ginkgolides active component, it comprises following subitem and detects:

A) proterties: off-white color or micro-yellow crystalline powder;

B) moisture: 60 DEG C of drying under reduced pressure less loss weight are less than 5%;

C) protein: absorbance is less than 0.05 at 595nm wavelength place;

D) detection of tannin, resin, oxalates, potassium ion:

Tannin: get protein check item need testing solution 1ml, add spirit of vinegar 1, then add gelatin sodium chloride test solution 5, shake up, places 10 minutes, does not occur muddiness or precipitation;

Resin: get protein check item need testing solution 5ml, add hydrochloric acid 1, places 30 minutes, separates out without resinoid; Oxalates: get protein check item need testing solution 2ml, with watery hydrochloric acid adjust ph to 1 ~ 2, filter, filtrate ammoniacal liquor adjust ph is 5 ~ 6, adds 3% calcium chloride solution 3, places 10 minutes, does not occur muddiness or precipitation;

Potassium ion: get protein check item need testing solution 2ml, put in 10ml nessler colorimetric tube, add alkaline formaldehyde solution 0.6ml, 3%EDTA solution 2,3% sodium tetraphenylborate solution 0.5ml, be diluted with water to 10ml, the accurate Klorvess Liquid 0.8ml of another label taking, with method test, the turbidity of need testing solution is not higher than contrast solution;

E) residual solvent

(1) ethanol, ethyl acetate and normal hexane: be all less than 0.5% containing ethanol and ethyl acetate, normal hexane is less than 0.029%;

(2) resin residue amount: be less than 0.0015% containing caprolactam;

F) total ginkgoic acid: be less than 5ppm containing total ginkgoic acid;

G) large molecule and polymkeric substance: gel chromatography, chromatographic column Phenomenex BioSep-SEC-S2000,300 × 7.8mm, 5 μm, mobile phase: include Sodium azide 0.71% metabisulfite solution of 0.02%, column temperature 35 DEG C, detector temperature: 35 DEG C, flow velocity 0.5ml/min, the large molecule of noresidue and polymkeric substance; HPLC-MS coupling method measures, mobile phase: the Agilent Rx-C of 90:10 methanol-water, chromatographic column: 2.1 × 50mm ₁₈, column temperature: 25 DEG C, flow velocity: 0.3ml/min, result is greater than large molecule and the polymkeric substance of 1000 without molecular weight;

H) heavy metal: be less than 10ppm;

I) arsenic salt: be less than 2ppm;

J) undue toxicity: make the solution containing 0.2mg in every 1ml, meet intravenous injection administration;

K) finger-print: HPLC method measures, and take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as the methyl alcohol-tetrahydrofuran-water of 25:10:65 be mobile phase; Use evaporative light detecting device, drift tube temperature: 105 DEG C; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 DEG C; Number of theoretical plate calculates should be not less than 2500 by Bilobalide peak, and the degree of separation at Bilobalide peak and ginkalide C peak should be greater than 1.5, records the chromatogram of 60 minutes, and by similarity evaluation, four total peak similarities are greater than 0.95;

L) content: HPLC method measures, and take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as the methyl alcohol-tetrahydrofuran-water of 25:10:65 be mobile phase; Use evaporative light detecting device, drift tube temperature: 105 DEG C; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 DEG C; Number of theoretical plate calculates should be not less than 2500 by Bilobalide peak, and the degree of separation at Bilobalide peak and ginkalide C peak should be greater than 1.5, calculates, containing Bilobalide (C by dry product ₁₅h ₁₈o ₈) 25.0% ~ 50.0%, ginkalide A (C ₂₀h ₂₄o ₉) 20.0% ~ 45.0%, ginkolide B (C ₂₀h ₂₄o ₁₀) 10.0% ~ 30.0%, ginkalide C (C ₂₀h ₂₄o ₁₁) 5.0% ~ 15.0%, and Bilobalide, ginkalide A, ginkolide B, ginkalide C total amount are greater than 95%;

M) pyrogen test: body temperature raises lower than 0.6 DEG C.

2. the detection method of ginkgolides active component according to claim 1, wherein a), c) item detection is as follows

A) proterties: off-white color or micro-yellow crystalline powder;

Easily molten in ethyl acetate, dissolve in methyl alcohol, ethanol, almost insoluble in water;

C) protein: absorbance is less than 0.05 at 595nm wavelength place;

Get ginkgolides 24mg, after adding ethanol 2ml dissolving, be diluted with water to 50ml, as need testing solution, measure according to Coomassie Brilliant Blue, with corresponding reagent for blank, at 595nm wavelength place, absorbance is less than 0.05.

3. the detection method of ginkgolides active component according to claim 2, k) finger-print of wherein itemizing is identical with the detection method of l) assay employing, and condition is as follows: take octadecylsilane chemically bonded silica as filling agent; Take volume ratio as 25:10:65 methyl alcohol-tetrahydrofuran-water be mobile phase; By evaporative light-scattering detector, drift tube temperature: 105 DEG C; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 DEG C; Number of theoretical plate calculates should be not less than 2500 by Bilobalide peak, and the degree of separation at Bilobalide peak and ginkalide C peak should be greater than 1.5.

4. the detection method of ginkgolides active component according to claim 3, wherein itemizing g) greatly, the assay method of molecule and polymkeric substance is as follows:

(1) exclusion chromatography chromatographic column: Phenomenex BioSep-SEC-S2000,300 × 7.8mm, 5um, mobile phase: include sodium azide 0.71% metabisulfite solution of 0.02%, column temperature: 35 DEG C, detector temperature: 35 DEG C, flow velocity: 0.5ml/min; Result: the large molecule of noresidue and polymkeric substance;

(2) HPLC-MS coupling method mobile phase: the Agilent RX-C of 90:10 methanol-water, chromatographic column: 2.1 × 50mm ₁₈, column temperature: 25 DEG C, flow velocity: 0.3ml/min.