CN103163252B - Determination method for total ginkgolic acid in ginkgolide composition - Google Patents
Determination method for total ginkgolic acid in ginkgolide composition Download PDFInfo
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Abstract
The invention relates to the field of phytoextraction and in particular relates to a determination method for total ginkgolic acid in ginkgolide composition. By adopting the ginkgolide extraction separation method provided by the invention, ginkgolid composition with fixed components can be obtained. In the determination method for the total ginkgolic acid, 20mul of each of a test solution, a reference substance solution and a contrast solution for locating is precisely sucked and injected into a liquid chromatograph, the total peak area of a chromatographic peak corresponding to a total ginkgolic acid reference substance in the test solution is calculated, and the content of the total ginkgolic acid is calculated by adopting a ginkgo new acid reference substance external standard method.
Description
Technical field
The present invention relates to field of plant extraction, be specifically related to the assay method of total ginkgoic acid in a kind of ginkgolides composition.
Background technology
From the sixties in last century, many countries adopt modern separation technology to study the chemical composition of ginkgo leaf, through pharmacological evaluation and clinical verification, find that many-sided biologically active of ginkgo leaf is relevant with its contained specific chemical composition.Germany Dr.Willar Schwabe has registered a kind of simple extract of ginkgo leaf first, in 1972, applied for patent (W Schwabe DE 176708 and DE2117429), 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, sesquiterpene lactone and diterpenoid-lactone, consists of, and is the important active component of ginkgo Ye Zhongyi class.Bilobalide (bilobalide) belongs to sesquiterpene lactone, by R.T.Major in 1967 with K.Weinges in separated obtaining in 1969, a unique sesquiterpene lactone compound of finding from ginkgo leaf at present, Ginkgolide A. B. C, M, J (ginkgolidA, B, C, M, J) are diterpenic lactone, in 1932, by S.Furukawa, separated first from ginkgo leaf, 1967 just by further separated and definite its chemical constitution of the people such as K.Nakanish, M.Maruyama and K.Okabe.From structure, Bilobalide quasi-molecule skeleton is comprised of 15 carbon atoms, has 4 five-membered rings that condense mutually together, wherein has 1 five yuan of carbocyclic ring, and 3 five yuan of lactone carbocyclic rings, are connected with the rare tert-butyl group in 1 natural products on five-membered ring.Bilobalide has very strong biologically active; there is the effect that promotes nerve growth; can prevent the Functional change that brain cell mitochondrial oxidation stress cause; 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 effects such as anti-inflammatory, anti-shock, protection cardiovascular and cerebrovascular, treatment acute pancreatitis.And the molecular skeleton of ginkgolide compound is comprised 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 mode of encircling to condense connects, and forms 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.
From early 1970s, found after PAF; pharmacologist is studied ginkgolides; defining ginkgoterpene lactone is strong platelet-activating factor antagonist, and immune system, central nervous system, ischemic injuries have protective effect, and has anti-shock, antiallergy and antiinflammatory action.The position that the hydroxy number that Ginkgolide A. B. C, M, J structural difference are to contain 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 of finding so far, and the Emergence and Development of it and numerous disease is closely related.And ginkgolides is considered to have most the natural paf receptor antagonists of potential applicability in clinical practice at present, its antagonism is active closely related with chemical constitution.In lactone structure, R3 is hydroxyl or hydroxy number while increasing, the antagonistic activity of PAF is weakened, and when R2 be hydroxyl and R3 while being H, actively significantly strengthen, the antagonism wherein with ginkolide B, PAF being produced is the strongest.
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 be isolated the ginkgolides of high-load effectively, and each proportion of composing of ginkgolides is uncertain, therefore, in clinical use, 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, the retrieval of Duo Jia official website of JingICH member state, there is no other bilobalide injector launch so far.The mensuration of ginkgolides constituents is HPLC-UV method, HPLC-MS and the HPLC-ELSD methods of adopting at present more, these methods only can be measured the content of each composition of ginkgolides, due to the strict control lacking bad reaction material in its product, the mass property that can not reflect really medicine, do not form a perfect drug quality hierarchy of control, although the patent of invention of ginkgolides is a lot, because control method is too simple, ginkgolides proportion of composing is indefinite, all traditional Chinese medicine cannot be made, the security of clinical efficacy and application can not be guaranteed.
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 fixing extraction ginkgolides active component composition of ratio, also not to 4 kinds of component finger-print control technologys and possible residual large molecule in ginkgolides, the report of protein inspection method.Bilobalide injection prepared by the present invention has obtained State Food and Drug Administration's authentication code, and the accurate word of traditional Chinese medicines: Z20110035 is first ginkgo active component injection in the world at present, and product structure is clear, clear and definite.
Summary of the invention
Technical matters solved by the invention is to provide a kind of extraction separation method of ginkgolides, and this extraction separation method can obtain the fixing ginkgolides of component.The method step is as follows:
A, extraction: pulverize ginkgo leaf, add organic solvent extraction, 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 of amounts, 6～10 times of optimum amounts;
Extracting method is: refluxing extraction or decoction are extracted.
1) refluxing extraction:
50～80%v/v ethanol: extract 75～85 ℃ of temperature, extraction time 2～3 times, each 1～2 hour of time;
50～80%v/v acetone: extract 45～55 ℃ of temperature, extraction time 2～3 times, each 1～2 hour of time;
50～80%v/v ethyl acetate: extract 55～65 ℃ of temperature, extraction time 2～3 times, each 1～2 hour of time;
Vacuum tightness :-0.02～0.08MPa
Preferably extraction conditions is: extract each 1.5 hours 3 times; Concentration of alcohol should be selected 65%v/v; Acetone concentration should be selected 50%v/v; Ethyl acetate concentration should be selected 60%v/v.
2) decoct and extract:
50～80%v/v ethanol: extract 80～90 ℃ of temperature, extraction time 2～3 times, each 1～2 hour of time;
50～80%v/v acetone: extract 50～60 ℃ of temperature, extraction time 2～3 times, each 1～2 hour of time;
50～80%v/v ethyl acetate: extract 60～65 ℃ of temperature, extraction time 2～3 times, each 1～2 hour of time;
Preferably extraction conditions is: extract each 1.5 hours 3 times; Concentration of alcohol should be selected 65%v/v; Acetone concentration should be selected 50%v/v; Ethyl acetate concentration should be selected 60%v/v.
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 in order to prevent the ginkgolides oxygenolysis of being heated: in serine, methionine, asparagine, threonine at least one, preferred methionine.
PH adjusting agent is mainly organic monoacid, comprising: in citric acid, malic acid, sorbic acid at least one, preferably citric acid for adjust pH, is to utilize its faintly acid as stabilizing agent, prevents that ginkgolides from opening bad under alkali condition.Reason is that ginkgolides structure is 5 rings, and stable under solutions of weak acidity, citric acid is weak acid, can prevent ginkgolides open loop under alkali condition.
The object of steps A refrigeration is in order to make oil and water separation, to remove 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), fat-soluble solvent extraction 4～5 times for water (preferred isopyknic ethyl acetate extraction), use again water saturation sec-butyl alcohol (normal butyl alcohol)-ethyl acetate mixed extractant solvent 4～5 times (preferably equal-volume water saturation sec-butyl alcohol-ethyl acetate mixed extractant solvent), merge organic phase extract, reduced pressure concentration.
Wherein, with normal hexane or petroleum ether extraction, be first in order to remove the impurity such as chlorophyll, ginkgolic acid.
With fat-soluble solvent, extract ginkgolides again, available fat-soluble solvent has at least one in ethyl acetate, ethyl formate, acetone, butanone.
Ginkgo terpene lactone is soluble in ethyl acetate, GINKGO BILOBA EXTRACT dissolubility in ethyl acetate is relatively low, in hot water and in aqueous alcohol, solubleness is larger, therefore can be extracted with ethyl acetate ginkgolides, it is separated with gingkgo flavonoids, the separated ginkgolides crude product obtaining can further be used activated charcoal, silica gel or resin column adsorption chromatography, further removes impurity, and then in aqueous alcohol, crystallization can obtain purer ginkgolides.
C, mistake post: extract is crossed polyamide (30～60 order) resin column, uses successively 1～5BV water, 3～5BV20%～40%v/v ethanol, 2～3BV60%～90%v/v ethanol elution, and control eluent flow rate is 2～3BV/h; Merge eluent, reduced pressure concentration, dry;
D, crystallization: the dry thing of crossing after post is added in boiling water, stirring and dissolving, cooling, equal-volume ethyl acetate for supernatant, ethyl formate or acetone extract 4～5 times, combining extraction liquid, reduced pressure concentration, evaporate to dryness, adds 5～8 times of amount 30%～50%v/v ethanol heating stirring and dissolving, filters, refrigeration, crystallize out, filters, filtrate I is standby, 30%～50%v/v ethanol washing for crystal, drying under reduced pressure, obtains crystal I.
It is 10%～30%v/v that filtrate I is concentrated into containing alcohol amount, refrigeration, and crystallize out, filters, and filtrate II is standby; 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, filters, and it is 10～30%v/v that filtrate is concentrated into containing alcohol amount, refrigeration, and crystallize out, filters, and filtrate II I is standby; 30%～50%v/v ethanol washing for crystal, drying under reduced pressure, obtains crystal III.
Filtrate II I is concentrated, 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, it is 10%～30%v/v that collection eluent is concentrated into containing alcohol amount again, refrigeration crystallization, leach crystal, filtrate IV is standby; Crystal washs with 30% ethanol, and drying under reduced pressure obtains crystal IV.
Concentrated filtrate IV, refrigeration, crystallize out, filters, 30%v/v ethanol washing for crystal, drying under reduced pressure, obtains crystal V.
Testing result according to HPLC to remaining ginkgolides in mother liquor, considers whether filtrate IV is carried out to crystallization.
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%.
Adopt the present invention to get the parameter of the ginkgolides that separation method obtains as follows:
A) proterties: off-white color or micro-yellow crystalline powder.Easily molten in ethyl acetate, in methyl alcohol, ethanol, dissolve, 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 is measured 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 is measured macromolecular substances and the polymkeric substance that is greater than 1000 without molecular weight.
H) heavy metal: be less than 10ppm.
I) arsenic salt: be less than 2ppm.
K) undue toxicity: make the solution that contains 0.2mg in every 1ml, meet intravenous injection administration.
L) finger-print: HPLC method is measured, take Bilobalide reference substance, ginkalide A reference substance, ginkolide B reference substance, ginkalide C reference substance is object of reference, press similarity evaluation, four total peak similarities are greater than 0.95.
M) content: HPLC method is measured, calculates by dry product, 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%.
Accompanying drawing explanation
The impact of Fig. 1 elution volume on eluting rate.
The impact of Fig. 2 ethanol flow velocity on 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, extraction option screening test
Method one: concentrate is first used to isopyknic n-hexane extraction 2～3 times, water with the butanone-acetone (4: 6) of 8 times of amounts at warm lower extraction 5 times, combining extraction liquid, reduced pressure concentration.
Method two: concentrate is first used to isopyknic n-hexane extraction 2～3 times, and water is again with equal-volume ethyl acetate extraction 4～5 times, with the water saturated sec-butyl alcohol-ethyl acetate of equivalent (7: 3) extraction 4～5 times, combining extraction liquid, reduced pressure concentration, dry.
Above two kinds of extraction separation purification method shaker tests, measure respectively the total amount of ginkgolides in two kinds of tests by HPLC-ELSD method, test findings is in Table 1.
Table 1 extraction option screening test findings
| Investigation project | Method one | Method two |
| Appearance character | Brown ceramic powder | Brown ceramic powder |
| Total lactone content (%) | 14.1 | 10.8 |
The total lactone content of method two gained is all higher, and ethyl acetate and sec-butyl alcohol are the high solvent of security, and therefore, selecting method two is as extracting from purifying process.
Two, chromatography condition shaker test
Owing to still containing a large amount of Ginkgolides materials and other impurity in extract, obtain very high purity ginkgolides, must flavones is effectively separated with ginkgolides, the separation method generally adopting at present comprises polyamide resin column partition method, alumina column chromatography method and silica gel column chromatography, and inventor's comparative study process and result are as follows:
Method one: extract is crossed to polyamide resin column, first use 30% ethanol elution of 2～3 times of amounts, continuing and using 70% ethanol elution, elution speed is 2BV/h, concentrate eluant, evaporate to dryness.
Method two: by extract peracidity alumina column, extract is mixed with equivalent aluminium oxide, dry, dry method upper prop, with the eluent ethyl acetate of 4～6 times of amounts, elution speed is 2BV/h, concentrate eluant, evaporate to dryness.
Method three: extract is crossed to 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 of amounts, elution speed is 2BV/h, use again normal hexane-ethyl acetate (5: 1) wash-out, elution speed is 2BV/h, concentrate eluant, evaporate to dryness.
By HPLC-ELSD method, respectively the total amount of ginkgolides in three kinds of tests is measured, 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 art | Yellow powder |
| Total lactone content (%) | 47.8 | 35.5 | 38.2 |
Therefore the ginkgolides content that adopts polyamide resin column to obtain is higher, separating effect is better.
Polyamide has good suction-operated to flavones, therefore, can effectively ginkgolides and GINKGO BILOBA EXTRACT be carried out effectively separatedly, to crossing post elution processes parameter, investigates.
1, the selection of washing volume: distilled water washing resin post can play good removal of impurities effect, water washing resin post with 5BV, flow velocity 1～2BV/h, the color of outflow from depth to shallow, is collected water lotion 5BV, efflux is limpid, testing result demonstration, 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.Elution volume is shown in Fig. 1 to the impact of eluting rate.
2, the impact of ethanol elution concentration on elute effect: extract is gone up respectively to different polyamide post, absorption 30min, first with 3BV, wash, use respectively 10% again, 30%, 40%, 50%, 70%, 90% ethanol elution, flow velocity 1BV/h, collect respectively ethanol eluate, measure the amount of ginkgolides in each concentration eluent, rising along with concentration of alcohol, elution amount and eluting rate all rise thereupon, but elution amount Slow lifting during to 40% ethanol, during to 90% ethanol, elution amount difference is little, 30% ethanol elution reaches best eluting rate substantially, therefore, adopt 30% ethanol as best wash-out concentration.
3, ethanol is resolved the elute effect impact of flow velocity: extract is gone up respectively to different polyamide post, and absorption 30min, first with 3BV washing, then uses 40% ethanol elution, flow velocity 1BV/h.For choosing preferably ethanol, resolve flow velocity, respectively with 1,2,3,4, the flow velocity of 5BV/h crosses post, wash-out is also collected 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 during to 3BV/h, this is that speed accelerates to cause ethanol eluate well not exchange with the ginkgolides of absorption, thereby can not reach good elute effect.Optimum flow rate 2～3BV/h.Ethanol flow velocity is shown in Fig. 2 to the impact of eluting rate.
Three, crystallization conditional filtering test:
Although cross the content of ginkgolides in post, the rear extract of extraction, increase to some extent, Flavonoid substances also obtains effective separation, 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, add respectively 4,6,8,10 times of amount 30% ethanol, heating for dissolving, low temperature (0～6 ℃) is standing, filters, and drying under reduced pressure, measures respectively crystal weight, 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 |
During crystallization, add 5～8 times of amount 30% ethanol proper, the crystal of separating out is relatively many.
(2) normal hexane-ethyl acetate (8: 1) solvent: get and treat crystallization extract 10g, add respectively 4,6,8,10 times of amount normal hexane-ethyl acetate (8: 1) mixed solvents, heating for dissolving, low temperature (0～6 ℃) is standing, filter, drying under reduced pressure, measures respectively crystal weight, and test findings is in Table 4.
Table 4 normal 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 |
Normal 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, add respectively 4,6,8,10 times of amount 10% ethyl acetate heating for dissolving, low temperature (0～6 ℃) is standing, filters, and drying under reduced pressure, measures respectively crystal weight, 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.
According to experimental result, recrystallisation solvent selects 5～8 times of amount 30% ethanol proper.
Below for adopting the inventive method to prepare the example of ginkgolides.
Embodiment 1
Ginkgo leaf meal 50kg, adds 65% alcohol heating reflux and extracts 3 times (10,8,6 times of amounts), each 1.5 hours, merges extract, filter, reduced pressure concentration, adds 0.05% methionine stirring and dissolving, with citric acid soln, regulates pH value to 4～5, continue to concentrate, low temperature is placed, and filters.First use equivalent n-hexane extraction, then with the extraction of equivalent ethyl acetate, 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, use again 70% ethanol elution, merge eluent, reduced pressure concentration.Add in 2～3 times of amount boiling water, stirring and dissolving, standing, let cool, be extracted with ethyl acetate, reduced pressure concentration, adds ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out, filters, dry, obtains crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol to 30%, standing, and crystallize out filters, dry, obtains crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, concentrated, lets cool, and crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and B); Filtrate is concentrated, cross medical charcoal-silica gel (1: 1) post, first with 2 times of amount 30% ethanol elutions, then with 4 times of amount 70% ethanol elutions, collect eluent, concentrated, add ethanol to 30%, let cool, standing, crystallize out, filters, dry, obtains crystal IV; Filtrate is concentrated, adds ethanol to 30%, lets cool, standing, and crystallize out filters, dry, obtains crystal V.Crystal is mixed, obtain 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 of 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, regulates pH value to 4～5, continue to concentrate, 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, standing letting cool, is extracted with ethyl acetate, and reduced pressure concentration adds ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out, filters, dry, obtains crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, and standing crystallize out filters, dry, obtains crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, concentrated, adds ethanol, lets cool, and crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and B); Filtrate is concentrated, cross medical charcoal-silica gel (1: 1) post, first with 2 times of amount 30% ethanol elutions, then with 4 times of amount 70% ethanol elutions, collect eluent, concentrated, add ethanol to 30%, let cool, standing, crystallize out, filters, dry, obtains crystal IV; Filtrate is concentrated, adds ethanol to 30%, lets cool, standing, and crystallize out filters, dry, obtains crystal V.Crystal is mixed, obtain 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 of 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, regulates pH value to 4～5, continue to concentrate, 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, standing letting cool, is extracted with ethyl acetate, and reduced pressure concentration adds ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out, filters, dry, obtains crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, and standing crystallize out filters, dry, obtains crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, concentrated, adds ethanol, lets cool, and crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and B); Filtrate is concentrated, and upper medical charcoal-silica gel (1: 1) post, uses 60% ethanol elution, collects eluent, concentrated, lets cool, and crystallize out, filters, dry, obtains crystal IV; Crystal is mixed, obtain 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 of 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, regulates pH value to 4～5, continue to concentrate, 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, standing letting cool, is extracted with ethyl acetate, and reduced pressure concentration adds 50% ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out, filters, dry, obtains crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, and standing crystallize out filters, dry, obtains crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, concentrated, lets cool, and crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and B); Filtrate is concentrated, and upper medical charcoal-silica gel (1: 1) post, uses 60% ethanol elution, collects eluent, concentrated, lets cool, and crystallize out, filters, dry, obtains crystal IV; Filtrate is concentrated, lets cool, and crystallize out, filters, dry, obtains crystal V; Crystal is mixed, obtain 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 of 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, regulates pH value to 4～5, continue to concentrate, low temperature is placed, and filters.First use petroleum ether extraction, water 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, standing letting cool, with acetone extract, is evaporated to dryly, adds 50% ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out, filters, dry, obtains crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, and standing crystallize out filters, dry, obtains crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, concentrated, adds ethanol, lets cool, and crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and B); Filtrate is concentrated, and upper medical charcoal-silica gel (1: 1) post, uses 60% ethanol elution, collects eluent, concentrated, lets cool, and crystallize out, filters, dry, obtains crystal IV; Crystal is mixed, obtain 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 of 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, regulates pH value to 4～5, continue to concentrate, low temperature is placed, and filters.First use petroleum ether extraction, water 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, standing letting cool, is extracted with ethyl acetate, and is evaporated to dryly, adds 30% ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out, filters, dry, obtains crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, and standing crystallize out filters, dry, obtains crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, concentrated, adds ethanol, lets cool, and crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and B); Filtrate is concentrated, and upper medical charcoal-silica gel (1: 1) post, uses 60% ethanol elution, collects eluent, concentrated, lets cool, and crystallize out, filters, dry, obtains crystal IV; Filtrate is concentrated, lets cool, and crystallize out, filters, dry, obtains crystal V; Crystal is mixed, obtain 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 of amount 70% ethanol and is heated to micro-decoction extraction 3 times of boiling, and each 1.5 hours, merges extract, filter, filtrate decompression reclaims acetone, adds 0.05% methionine stirring and dissolving, with citric acid soln, regulates pH value to 4～5, continue to concentrate, low temperature is placed, and filters.First use petroleum ether extraction, water 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, standing letting cool, is extracted with ethyl acetate, and is evaporated to dryly, adds 30% ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out, filters, dry, obtains crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, and standing crystallize out filters, dry, obtains crystal II (being mainly Bilobalide and Ginkgolides a and B); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, concentrated, adds ethanol, lets cool, and crystallize out, filters, dry, obtains crystal III (being mainly ginkalide A and C); Filtrate is concentrated, and upper medical charcoal-silica gel (1: 1) post, uses 60% ethanol elution, collects eluent, concentrated, lets cool, and crystallize out, filters, dry, obtains crystal IV; Filtrate is concentrated, lets cool, and crystallize out, filters, dry, obtains crystal V; Crystal is mixed, obtain 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, adopt extraction separation and purification method used in the present invention can 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%.
Subitem detection method and the testing result of ginkgolides of the present invention are as follows:
A) proterties: off-white color or micro-yellow crystalline powder.
Easily molten in ethyl acetate, in methyl alcohol, ethanol, dissolve, almost insoluble in water.
B) moisture: 60 ℃ 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 the about 24mg of ginkgolides of the present invention, add after ethanol 2ml dissolving, be diluted with water to 50ml, as need testing solution.According to Coomassie brilliant blue method (Bradford method), measure, take corresponding reagent as 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 1 of spirit of vinegar, then add 5 of gelatin sodium chloride test solutions, shake up, place 10 minutes, do not occur muddy or precipitation.
Resin: get protein check item need testing solution 5ml, add 1 of hydrochloric acid, place 30 minutes, separate out without resinoid.
Oxalates: get protein check item need testing solution 2ml, regulate pH value to 1～2 with watery hydrochloric acid, filter, it is 5～6 that filtrate regulates pH value with ammoniacal liquor, adds 3 of 3% calcium chloride solutions, places 10 minutes, does not occur muddy or precipitation.
Potassium ion: get protein check item need testing solution 2ml, put in 10ml nessler colorimetric tube, add alkaline formaldehyde solution 0.6ml, 2 of 3%EDTA solution, 3% sodium tetraphenylborate solution 0.5ml, be diluted with water to 10ml, the another accurate Klorvess Liquid 0.8ml of 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: containing ethanol and ethyl acetate, be all less than 0.5%, 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 is measured, large molecule and polymkeric substance that result is greater than 1000 without molecular weight.
Assay method:
(1) exclusion chromatography chromatographic column: Phenomenex BioSep-SEC-S2000,300 * 7.8mm, 5um, mobile phase: 0.71% (including 0.02% sodium azide) metabisulfite solution, column temperature: 35 ℃, detector temperature: 35 ℃, 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 ℃, flow velocity: 0.3ml/min.Result: large molecule and polymkeric substance that result is greater than 1000 without molecular weight.
H) heavy metal: be less than 10ppm.
I) arsenic salt: be less than 2ppm.
K) undue toxicity: make the solution that contains 0.2mg in every 1ml, meet intravenous injection administration.
The preparation of need testing solution: get the about 25mg of ginkgolides of the present invention, make with sodium chloride injection the solution that contains 0.2mg in every 1ml after dissolving with ethanol 2ml.
Inspection technique: get 5 of body weight 17～20g mouse, inject mouse tail vein need testing solution 0.5ml, 48 hours without dead.
L) finger-print: HPLC method is measured, and records the chromatogram of 60 minutes.Press similarity evaluation, four total peak similarities are greater than 0.95.
M) content: HPLC method is measured, calculates by dry product, containing Bilobalide (C
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 and m) detection method that adopts of assay is identical, and condition is as follows: take octadecylsilane chemically bonded silica as filling agent; Methyl alcohol-tetrahydrofuran-the water (25: 10: 65) of take is mobile phase; By evaporative light-scattering detector, drift tube temperature: 105 ℃; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 ℃; Number of theoretical plate calculates and 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: hyperthermia is lower than 0.6 ℃.
The preparation of need testing solution: precision takes ginkgolides 20mg of the present invention, adds 2ml ethanol to make to dissolve, then adds in 0.9% sodium chloride injection 100ml.
Inspection technique: get 3 of rabbit, measure after its normal body temperature in 15 minutes, 5ml slowly injects need testing solution from ear vein by the every 1kg injection of rabbit body weight, every 30 minutes, measure body temperature 1 time, survey altogether 6 times, hyperthermia all should be lower than 0.6 ℃, and 3 rabbit body temperature rising summations are lower than 1.3 ℃.
Inventor has carried out large molecule and polymer determination research and explanation to foregoing invention content, for proving technique effect of the present invention.Following test is used for further illustrating and explains the present invention, but does not limit the present invention.
(1) test apparatus and reagent
Agilent1200 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 reference substance (D0), content: 99.5%, lot number 086K0166, SIGMA.
Ultrapure water makes by Millipore-Q ultrapure water system.
All the other reagent are pure for analyzing.
(2) selection of mobile phase
Choose 0.71% (including 0.02% sodium azide) metabisulfite solution as mobile phase.
(3) selection of detecting device
Select common detector differential refraction detector, this detecting device is for existing the material of refraction coefficient difference all to have good response.
(4) quasi-definite chromatographic condition
Chromatographic column: Phenomenex BioSep-SEC-S2000,300 * 7.8mm, 5 μ m
Mobile phase: 0.71% (including 0.02% sodium azide) metabisulfite solution
Column temperature: 35 ℃, detector temperature: 35 ℃, 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 reference substance are added respectively to the solution that mobile phase is made 10mg/ml, precision is drawn each 20 μ l of reference substance solution respectively, injecting chromatograph, record chromatogram, result dextran is at retention time 9.816 ' go out peak, and glucose, in retention time 18.712 ' go out peak, shows to adopt exclusion chromatography, the material that molecular weight is large first goes out peak, after the little material of molecular weight, goes out peak.
2. get the about 10mg of ginkgolides of the present invention, adding ethanol 2ml dissolves, add dextran reference substance solution (10mg/ml) 1ml, mix, the accurate 10 μ l that draw, injecting chromatograph, record chromatogram, result is at retention time 9.698 ' detect dextran, bilobalide injection becomes swarming all after 18min, to go out peak, show molecular weight at 180～450 appearance times in about 18min, molecular weight 5000～2000000 appearance times are in 9min left and right, it is feasible adopting exclusion chromatography to detect macromolecular substances.
In order to verify again in this product not containing large molecule and polymkeric substance, therefore carried out again LC-MS test.
Chromatographic condition: methanol-water (90: 10) is mobile phase, Agilent RX-C
18(2.1 * 50mm) chromatographic column, 25 ℃ of flow velocity 0.3ml/min of column temperature.
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 to dissolve, and adds mobile phase and is diluted to scale, shakes up, as need testing solution.
LC-MS coupling test: according to definite test method, get respectively each 10 μ l of need testing solution, 400～1000 and 400-3000 molecular weight ranges in test respectively, record chromatogram.Test findings is in Table 6.
Table 6 LC-MS coupling molecular weight determination result
| [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 result, detect respectively ginkalide A (molecular weight 408.5), ginkolide B (molecular weight 424.4), ginkalide C (molecular weight 440.4), in full accord with the effective ingredient of ginkgolides of the present invention, because test molecule weight range is between 400-3000, Bilobalide is not tested, in ginkgolides of the present invention, do not detect molecular weight and be greater than more than 700 materials, the material of other different molecular weight may be the existence of other impurity, through LC-MS to ginkgolides in the molecular weight test of different components, 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; Methyl alcohol-1% glacial acetic acid (90: 10) of take is mobile phase; Flow velocity 1.0ml/min; Detection 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 every 1ml containing the solution of 5 μ g product solution in contrast; Separately get total ginkgoic acid reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 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 is again with a small amount of normal hexane washing, 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: accurate absorption need testing solution, reference substance solution and each 20 μ l of contrast solution for location, injection liquid chromatography, calculate in need testing solution the total peak area with the corresponding chromatographic peak of total ginkgoic acid reference substance, with gingko eo-acid reference substance external standard method, calculate total ginkgoic acid content, total ginkgoic acid is less than 5ppm.
Inventor is studied and illustrates foregoing invention content, for proving technique effect of the present invention.Following test is used for further illustrating and explains 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 ℃) 50ml, refluxes 2 hours, takes out, let cool, filter, residue is used a small amount of petroleum ether 1 time, merging filtrate and cleansing solution again, 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 ℃) 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 is again with a small amount of normal hexane washing 1 time, 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, put in 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 (2).
Determination method: accurate need testing solution and each 20 μ l of blank solution of drawing, injection liquid chromatography, records chromatogram.Test findings is in Table 7.
Two kinds of method testing result tables of table 7
Test findings shows: employing method one (sherwood oil) is prepared 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, with this, carry out the feasibility of verification method two.
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, it is the total ginkgoic acid reference substance solution 0.2ml of 1.032mg/ml that precision adds concentration, then precision adds normal hexane 50ml, refluxes 2 hours, let cool, filter, residue is again with a small amount of normal hexane washing, 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 methyl alcohol and is diluted to scale, shakes up, in contrast product solution.
Determination method: accurate need testing solution and each 20 μ l of reference substance solution of drawing, injection liquid chromatography, records chromatogram.
Result: need testing solution with the corresponding position of total ginkgoic acid reference substance chromatogram on can detect total ginkgoic acid chromatographic peak, 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 test
The preparation of reference substance solution: get gingko eo-acid reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 5 μ g product solution in contrast.Separately get total ginkgoic acid reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 100 μ g, as location contrast solution.
Need testing solution preparation: get ginkgolides 5g of the present invention, accurately weighed, nominal is got 6 parts, put respectively in flask, add normal hexane 50ml, reflux 2 hours, let cool, filter, residue washs with a small amount of normal hexane, 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: precision is drawn each 20 μ l of contrast solution for need testing solution, reference substance solution and location, and injection liquid chromatography, records 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 | Do not detect | Do not detect | Do not detect | Do not detect | Do not detect |
Test findings shows, in ginkgolides of the present invention without ginkgolic acid.
E, recovery test
Need testing solution preparation: get ginkgolides 5g of the present invention, accurately weighed, nominal is got 3 parts, put respectively in flask, adding respectively concentration is 3.04 μ g/ml gingko eo-acid reference substance solution 1.6ml, 2.0ml, 2.4ml, then adds respectively normal hexane 50ml, reflux 2 hours, let cool, filter, residue washs with a small amount of normal hexane, 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.
Determination method: accurate need testing solution, each 20 μ l of reference substance solution of drawing, injection liquid chromatography, records chromatogram.Each concentration determination three times, totally 9 times.Calculate recovery rate, 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; Methyl alcohol-tetrahydrofuran-the water (25: 10: 65) of take is mobile phase; By evaporative light-scattering detector, drift tube temperature: 105 ℃; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 ℃; Number of theoretical plate calculates and 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: it is appropriate that precision takes Bilobalide reference substance, ginkalide A reference substance, ginkolide B reference substance, ginkalide C reference substance respectively, 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 dissolving, add mobile phase and be diluted to scale, shake up, as need testing solution.
Determination method: precision is drawn object of reference solution and each 20 μ l of need testing solution respectively, and injection liquid chromatography, records the chromatogram of 60 minutes.
Press similarity evaluation, test sample finger-print and reference fingerprint similarity are greater than 0.95.
Inventor is studied and illustrates foregoing invention content, for proving technique effect of the present invention.Following test is used for further illustrating and explains the present invention, but does not limit the present invention.
In ginkgolides finger-print, wherein peak 2 is that ginkalide C, peak 3 are that Bilobalide, peak 4 are that ginkalide A, peak 5 are ginkolide B, and in this product, 4 characteristic peaks of active component all can be corresponding one by one in finger-print.Ginkgolides reference fingerprint is shown in Fig. 5.
First adopting Chinese Pharmacopoeia Commission's finger-print designated software in 2004---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 are calculated to similarity with similarity software.Test findings is in Table 11.
10 batches of ginkgolides similarity result of table 11
| 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 is measured
(1) ethanol, ethyl acetate and normal hexane
The preparation of need testing solution: get the about 0.1g of ginkgolides of the present invention, accurately weighed, in top set empty bottle, precision adds DMF 5ml to make to dissolve, and sealing, as need testing solution.
The preparation of reference substance solution: get ethanol, ethyl acetate and normal hexane appropriate, accurately weighed, with DMF quantitatively dilution make that in every 1ml, each is approximately containing the solution of 30 μ g, precision measures 5ml, in top set empty bottle, sealing, in contrast product solution.
Determination method: 6% cyanogen propyl group phenyl-94% dimethyl polysiloxane (or polarity is close) of take is immobile liquid, and initial temperature is 50 ℃, maintains 3 minutes, is warming up to 160 ℃ with the speed of 40 ℃ of per minutes, maintains 3 minutes; 200 ℃ of injector temperatures; Detector temperature is 250 ℃; Head space bottle equilibrium temperature is 80 ℃, 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 again need testing solution and reference substance solution headspace sampling respectively, record chromatogram, by external standard method with calculated by peak area.
Containing ethanol and ethyl acetate, be all less than 0.5%, normal hexane is less than 0.029%.
(2) resin residue amount
The preparation of reference substance solution: get DMA appropriate, accurately weighed, water is made every 1ml approximately containing the solution of 0.1mg, shakes up, as inner mark solution; It is appropriate that precision takes caprolactam, adds inner mark solution and make every 1ml approximately containing the solution of caprolactam 37.5 μ g, in contrast product solution.
The preparation of need testing solution: get the about 2.5g of ginkgolides of the present invention, accurately weighed, put in conical flask, add normal hexane 25ml, reflux 2 hours, take out, let cool, filter, with a small amount of normal hexane washing, merging filtrate and cleansing solution, in 60 ℃ of water bath methods, residue adds inner mark solution 1ml to be made to dissolve, as need testing solution.
Determination method: the polyglycol (PEG-20M) (or polarity is close) of take is immobile liquid; Initial temperature is 100 ℃, maintains 2 minutes, with the speed of 40 ℃ of per minutes, is warming up to 160 ℃, maintains 3 minutes, then is warming up to 220 ℃ with the speed of 40 ℃, maintains 7 minutes; Injector temperature is 240 ℃; Detector temperature is 260 ℃.Precision measures reference substance solution and each 1 μ l of need testing solution, and inject gas chromatograph, records 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
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Methyl alcohol-tetrahydrofuran-the water (25: 10: 65) of take is mobile phase; By evaporative light-scattering detector, drift tube temperature: 105 ℃; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 ℃; Number of theoretical plate calculates and 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 reference substance solution: it is appropriate that precision takes Bilobalide reference substance, ginkalide A reference substance, ginkolide B reference substance, ginkalide C reference substance respectively, 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 dissolving, 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, with external standard two-point method logarithmic equation, calculate respectively the content of Bilobalide, ginkalide A, ginkolide B and ginkalide C.
Press dry product and calculate, 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, make the solution that contains 0.2mg in every 1ml with sodium chloride injection.
Inspection technique: get 5 of body weight 17～20g mouse, inject respectively mouse tail vein need testing solution 0.5ml, in 48 hours, nothing is dead.
Embodiment 13
Ginkgolides quality control---pyrogen test
The preparation of need testing solution: get ginkgolides 10mg of the present invention, join in 0.9% sodium chloride injection 50ml, shake up.
Inspection technique: get 3 of rabbit, measure after its normal body temperature in 15 minutes, 5ml slowly injects need testing solution from ear vein by the every 1kg injection of rabbit body weight, every 30 minutes, measure body temperature 1 time, survey altogether 6 times, hyperthermia is all lower than 0.6 ℃, and 3 rabbit body temperature rising summations are lower than 1.3 ℃.
Embodiment 14
Bilobalide injection quality control---related substance inspection
Injection formula:
Preparation method is:
A) preparation: 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, regulate pH value 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 the about 50mg of Coomassie brilliant blue G-250, 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, then adds 1ml need testing solution, shakes up, and places 3min.With method, do blank, 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 1 of spirit of vinegar, then add 5 of gelatin sodium chloride test solutions, shake up, place 10 minutes, do not occur muddy or precipitation.
(3) resin: get protein check item need testing solution 5ml, add 1 of hydrochloric acid, place 30 minutes, separate out without resinoid.
(4) oxalates: get protein check item need testing solution 2ml, regulate pH value to 1～2 with watery hydrochloric acid, filter, it is 5～6 that filtrate regulates pH value with ammoniacal liquor, adds 3 of 3% calcium chloride solutions, places 10 minutes, does not occur muddy 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, 2 of 3%EDTA solution, 3% sodium tetraphenylborate solution 0.5ml, be diluted with water to 10ml, the another accurate Klorvess Liquid 0.8ml of 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 (by embodiment 14 preparations) 6ml, join in 0.9% sodium chloride injection 100ml and shake up.
Inspection technique: get 5 of clean glass test tubees, numbering, 1, No. 2 pipe, for test sample pipe, is managed negative control tube for No. 3, manages positive control tube for No. 4, No. 5 pipe test sample control tube.By adding successively 2% red cell suspension, 0.9% sodium chloride solution, distilled water shown in table 12, after mixing, put immediately in the constant temperature oven of 37 ℃ ± 0.5 ℃ and carry out incubation.
Table 12 haemolysis and agglutination test addition
| Test tube numbering | 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 |
As the solution in test tube is clear and bright redness, it is residual or have a small amount of red blood cell residual that bottom is acellular, shows to 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 manage visual inspection no significant differences for No. 5, shows to occur without haemolysis.After 3 hours, observe and do not produce haemolysis and aggregation.
Embodiment 16
Bilobalide injection quality control---finger-print inspection
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Methyl alcohol-tetrahydrofuran-the water (25: 10: 65) of take is mobile phase; By evaporative light-scattering detector, drift tube temperature: 105 ℃; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 ℃; Number of theoretical plate calculates and 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: it is appropriate that precision takes Bilobalide reference substance, ginkalide A reference substance, ginkolide B reference substance, ginkalide C reference substance respectively, 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 need testing solution: get the need testing solution under [assay] item.
Determination method: precision is drawn object of reference solution and each 20 μ l of need testing solution respectively, and injection liquid chromatography, records the chromatogram of 60 minutes.
Press similarity evaluation, test sample finger-print and reference fingerprint are greater than 0.95 through similarity.
Embodiment 17
Bilobalide injection quality control---assay
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Methyl alcohol-tetrahydrofuran-the water (25: 10: 65) of take is mobile phase; By evaporative light-scattering detector, drift tube temperature: 105 ℃; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 ℃; Number of theoretical plate calculates and 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 reference substance solution: it is appropriate that precision takes Bilobalide reference substance, ginkalide A reference substance, ginkolide B reference substance, ginkalide C reference substance respectively, 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: precision measures bilobalide injection (by embodiment 14 preparations) 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 dissolves with mobile phase and is transferred in 10ml measuring bottle, adds mobile phase and is diluted to scale, shakes up, with 0.45 μ m miillpore filter, filter, as need testing solution.
Determination method: precision is drawn reference substance solution 10 μ l, 20 μ l respectively, need testing solution 15 μ l, inject high performance liquid chromatograph, record chromatogram, with external standard two-point method logarithmic equation, calculate respectively the content of Bilobalide, ginkalide A, ginkolide B and ginkalide C.
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, preferred 4.25～5.75mg.
Claims (2)
1. the assay method of total ginkgoic acid in ginkgolides composition, is characterised in that,
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent, 90: 10 methyl alcohol-1% glacial acetic acid of take are mobile phase, flow velocity 1.0ml/min, detection 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 every 1ml containing the solution of 5 μ g product solution in contrast, separately get total ginkgoic acid reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 100 μ g, as location contrast solution;
The preparation of need testing solution: get by ginkgo leaf and extract the ginkgolides composition 5g obtaining, accurately weighed, put in flask, add normal hexane 50ml, add hot reflux 2 hours, take out, let cool, filter, residue is again with a small amount of normal hexane washing, 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: accurate absorption need testing solution, reference substance solution and each 20 μ l of contrast solution for location, injection liquid chromatography, calculate in need testing solution the total peak area with the corresponding chromatographic peak of total ginkgoic acid reference substance, with gingko eo-acid reference substance external standard method, calculate total ginkgoic acid content.
2. according to the assay method of claim 1, it is characterized in that the total ginkgoic acid content recording is less than 5ppm.
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