CN103163252A - 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 that the chemical composition of ginkgo leaf is studied, and 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, applied for patent (W Schwabe DE 176708 and DE2117429) in 1972, name and be EGb761, be used for the treatment of cardiovascular and cerebrovascular disease and the nervous system disease, has 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 all with being health food or over-the-counter medications, the ginkgo health care food that the U.S. develops has obtained the FDA approval in other countries.
Ginkgolides belongs to terpenoid, is called terpene lactone, is comprised of sesquiterpene lactone and diterpenoid-lactone, is the important active component of ginkgo Ye Zhongyi class.Bilobalide (bilobalide) belongs to sesquiterpene lactone, separated in 1969 with K.Weinges in 1967 by R.T.Major and obtain, 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, separated from ginkgo leaf first by S.Furukawa in 1932, just further separated and determined its chemical constitution in 1967 by people such as K.Nakanish, M.Maruyama and K.Okabe.On structure, Bilobalide quasi-molecule skeleton is comprised of 15 carbon atoms, has 4 five-membered rings that condense mutually together, and 1 five yuan of carbocyclic ring is wherein arranged, 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; has the effect that promotes nerve growth; can prevent the Functional change that the 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, have 6 five-membered rings, 2 five yuan of carbocyclic rings are wherein arranged, 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.All has the tert-butyl group rare in natural products in the ginkgolides molecule.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 been found PAF; the pharmacologist is studied ginkgolides; clear and definite ginkgoterpene lactone is strong platelet-activating factor antagonist, and immune system, central nervous system, ischemic injuries have protective effect, and anti-shock, antiallergy and antiinflammatory action are arranged.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 the 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, and its antagonism is active closely related with chemical constitution.When R3 in lactone structure is hydroxyl or hydroxy number when increasing, the antagonistic activity of PAF is weakened, and when R2 be hydroxyl and R3 when being H, actively significantly strengthen, wherein the strongest to the antagonism that PAF produces with ginkolide B.
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, drug effect is different in clinical use, 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 in China, the bilobalide injection Patents is arranged, but its composition is all different from the present invention, through many official website's retrievals of ICH member state, there is no so far other bilobalide injector launch.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 that lacks 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, the ginkgolides proportion of composing is indefinite, all traditional Chinese medicine can't 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.The bilobalide injection of the present invention preparation 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, add anti-oxidant protective agent in concentrated extracting solution, with pH adjusting agent adjust pH to 4～5, concentrated, refrigeration.
Wherein, the 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
Preferred 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 extraction:
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;
Preferred 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 in order to prevent the ginkgolides oxygenolysis of being heated, and available anti-oxidant protective agent mainly contains neutral amino acid, comprising: at least a in serine, methionine, asparagine, threonine, and preferred methionine.
PH adjusting agent is mainly organic monoacid, comprising: at least a in citric acid, malic acid, sorbic acid, preferably citric acid is used for adjust pH, is to utilize its faintly acid as stabilizing agent, prevents that ginkgolides from opening bad under alkali condition.Reason is that the 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 purpose of steps A refrigeration is in order to make oil and water separation, to remove the middle oil-soluble impurities that anhydrates.
B, extraction: with concentrate first with normal hexane or petroleum ether extraction 2～3 times (preferred isopyknic normal hexane or petroleum ether extraction), water extracts 4～5 times (preferred isopyknic ethyl acetate extraction) with fat-soluble solvent, use again 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), merge the organic phase extract, reduced pressure concentration.
Wherein, be first in order 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 a in ethyl acetate, ethyl formate, acetone, butanone.
The 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 available ethyl acetate extraction ginkgolides, it is separated with gingkgo flavonoids, separate the ginkgolides crude product can further use activated charcoal, silica gel or resin column adsorption chromatography, further remove impurity, then crystallization can obtain purer ginkgolides in aqueous alcohol.
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 the control eluent flow rate is 2～3BV/h; Merge eluent, reduced pressure concentration, drying;
D, crystallization: the dry thing that will cross after post adds in boiling water, and stirring and dissolving is cooling, supernatant is with 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 of amount 30%～50%v/v ethanol heating stirring and dissolving, filters, refrigeration, crystallize out filters, filtrate I is standby, crystal washs with 30%～50%v/v ethanol, and drying under reduced pressure obtains crystal I.
Filtrate I is concentrated into that to contain alcohol amount be 10%～30%v/v, refrigeration, and crystallize out filters, and filtrate II is standby; With the washing of 30%～50%v/v ethanol, drying under reduced pressure gets crystal II.
Concentrated filtrate II adds 0.1～0.5% (g/L) charcoal absorption, filters, and it is 10～30%v/v that filtrate being concentrated into contains the alcohol amount, refrigeration, and crystallize out filters, and filtrate II I is standby; Crystal washs with 30%～50%v/v ethanol, and drying under reduced pressure gets 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 again 70%～90%v/v ethanol elution, collect eluent and be concentrated into that to contain the alcohol amount be 10%～30%v/v, the refrigeration crystallization, leach crystal, filtrate IV is standby; Crystal washs with 30% ethanol, and drying under reduced pressure gets crystal IV.
Concentrated filtrate IV, refrigeration, crystallize out filters, and crystal washs with 30%v/v ethanol, and drying under reduced pressure gets crystal V.
According to the testing result of HPLC to remaining ginkgolides in mother liquor, consider whether filtrate IV is carried out crystallization.
E, mixed crystal: crystal I, II, III, IV, V are mixed, pulverize, obtain the 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 little yellow crystalline powder.Easily molten in ethyl acetate, dissolve in methyl alcohol, ethanol, almost insoluble in water.
B) moisture: less than 5.0%.
C) protein: in 595nm wavelength place absorbance less than 0.05.
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: the HPLC method is measured total ginkgoic acid content less than 5ppm.
G) large molecule and polymkeric substance: the large molecule of gel chromatography noresidue and polymkeric substance.The LC-MS method is measured without molecular weight greater than 1000 macromolecular substances and polymkeric substance.
H) heavy metal: less than 10ppm.
I) arsenic salt: less than 2ppm.
K) undue toxicity: make the solution that contains 0.2mg in every 1ml, meet the intravenous injection administration.
L) finger-print: the HPLC method is measured, take Bilobalide reference substance, ginkalide A reference substance, ginkolide B reference substance, ginkalide C reference substance as object of reference, press similarity evaluation, four total peak similarities are greater than 0.95.
M) content: the HPLC method is measured, and presses dry product and calculates, and contains 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%.
Description of drawings
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
Below be the key condition shaker test in ginkgolides preparation method of the present invention.
One, extraction option screening test
Method one: concentrate is first used 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 isopyknic n-hexane extraction 2～3 times, and water is used the equal-volume ethyl acetate extraction 4～5 times again, with the water saturated sec-butyl alcohol-ethyl acetate of equivalent (7: 3) extraction 4～5 times, combining extraction liquid, reduced pressure concentration, drying.
Above two kinds of extraction separation purification method shaker tests are measured respectively the total amount of ginkgolides in two kinds of tests with the HPLC-ELSD method, test findings sees Table 1.
Table 1 extraction option screening test findings
| The 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 the based on very high purity ginkgolides, flavones effectively must be separated with ginkgolides, the separation method that generally adopts 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 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: with extract peracidity alumina column, extract is mixed with the equivalent aluminium oxide, oven dry, the 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 silicagel column, extract is mixed with the equivalent column chromatography silica gel, oven dry, the dry method upper prop, first with petroleum ether-ethyl acetate (2: the 1) wash-out with 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.
Respectively the total amount of ginkgolides in three kinds of tests is measured with the HPLC-ELSD method, test findings sees Table 2.
Table 2 column chromatography test findings
| The 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, and separating effect is better.
Polyamide has suction-operated preferably to flavones, therefore, can effectively ginkgolides be separated effectively with GINKGO BILOBA EXTRACT, investigates crossing post elution processes parameter.
1, the selection of washing volume: the 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 is collected water lotion 5BV from depth to shallow, efflux is limpid, the testing result demonstration, when water volume reached 3BV, the water-solubility impurity in post was substantially clean, inspection does not measure ginkgolides, so select the washing volume of 3BV.Elution volume is seen Fig. 1 to the impact of eluting rate.
2, the impact of ethanol elution concentration on elute effect: extract is gone up respectively the different polyamide post, absorption 30min, first wash with 3BV, 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, the elution amount difference is little, 30% ethanol elution reaches best eluting rate basically, 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 the different polyamide post, and absorption 30min first with the 3BV washing, then uses 40% ethanol elution, flow velocity 1BV/h.Resolve flow velocity for choosing more excellent ethanol, respectively with 1,2,3,4, the flow velocity of 5BV/h crosses post, wash-out is also collected the 3BV eluent, measures the ginkgolides amount.Elution flow rate and resolution factor have very large relevance, along with flow velocity improves, resolution factor increases, but descends on the contrary during to 3BV/h, this is that speed accelerates to cause ethanol eluate well not exchange with the ginkgolides that adsorbs, thereby can not reach good elute effect.Optimum flow rate 2～3BV/h.The ethanol flow velocity is seen Fig. 2 to the impact of eluting rate.
Three, crystallization conditional filtering test:
Increase to some extent although cross the content of ginkgolides in post, the rear extract of extraction, Flavonoid substances is also effectively separated, and the content of ginkgolides still can not reach the 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 is measured respectively crystal weight, and test findings sees Table 3.
Table 3 30% alcohol crystal test findings
| Solvent adding amount (doubly) | 4 | 6 | 8 | 10 |
| The heating for dissolving situation | Be not dissolved | Dissolve complete | Dissolve complete | Dissolve complete |
| Crystal amount (g) | 3.8 | 4.5 | 4.2 | 2.4 |
Add 5～8 times of amount 30% ethanol during crystallization 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 is measured respectively crystal weight, and test findings sees Table 4.
Table 4 normal hexane-ethyl acetate mixed solvent crystallization test findings
| Solvent adding amount (doubly) | 4 | 6 | 8 | 10 |
| The heating for dissolving situation | Dissolve complete | Dissolve complete | Dissolve complete | Dissolve complete |
| 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 is measured respectively crystal weight, and test findings sees Table 5.
Table 5 10% ethyl acetate crystallization trial result
| Solvent adding amount (doubly) | 4 | 6 | 8 | 10 |
| The heating for dissolving situation | Be not dissolved | Dissolve complete | Dissolve complete | Dissolve complete |
| 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.
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, regulates pH value to 4～5 with citric acid soln, continue to concentrate, low temperature is placed, and filters.First use the equivalent n-hexane extraction, then use the equivalent ethyl acetate extraction, use at last 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, use ethyl acetate extraction, reduced pressure concentration adds ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out filters, and drying gets crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol to 30%, and is standing, and crystallize out filters, and drying gets crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, and is concentrated, lets cool, and crystallize out filters, and drying gets 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, and drying gets crystal IV; Filtrate is concentrated, adds ethanol to 30%, lets cool, and is standing, and crystallize out filters, and drying gets crystal V.Crystal is mixed, get 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%.
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 adds 0.05% methionine stirring and dissolving to without the alcohol flavor, regulates pH value to 4～5 with citric acid soln, continue to concentrate, low temperature is placed, and filters.First use n-hexane extraction, then use ethyl acetate extraction, use at last 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 used ethyl acetate extraction, and reduced pressure concentration adds ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out filters, and drying gets crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, and standing crystallize out filters, and drying gets crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, and is concentrated, adds ethanol, lets cool, and crystallize out filters, and drying gets 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, and drying gets crystal IV; Filtrate is concentrated, adds ethanol to 30%, lets cool, and is standing, and crystallize out filters, and drying gets crystal V.Crystal is mixed, get 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%.
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 adds 0.05% methionine stirring and dissolving to without the alcohol flavor, regulates pH value to 4～5 with citric acid soln, continue to concentrate, low temperature is placed, and filters.First use n-hexane extraction, then use ethyl acetate extraction, use at last 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 used ethyl acetate extraction, and reduced pressure concentration adds ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out filters, and drying gets crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, and standing crystallize out filters, and drying gets crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, and is concentrated, adds ethanol, lets cool, and crystallize out filters, and drying gets crystal III (being mainly ginkalide A and B); Filtrate is concentrated, and upper medical charcoal-silica gel (1: 1) post is used 60% ethanol elution, collects eluent, and is concentrated, lets cool, and crystallize out filters, and drying gets crystal IV; Crystal is mixed, get 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%.
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 adds 0.05% methionine stirring and dissolving to without the alcohol flavor, regulates pH value to 4～5 with citric acid soln, continue to concentrate, low temperature is placed, and filters.First use n-hexane extraction, then use ethyl acetate extraction, use at last 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 used ethyl acetate extraction, and reduced pressure concentration adds 50% ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out filters, and drying gets crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, and standing crystallize out filters, and drying gets crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, and is concentrated, lets cool, and crystallize out filters, and drying gets crystal III (being mainly ginkalide A and B); Filtrate is concentrated, and upper medical charcoal-silica gel (1: 1) post is used 60% ethanol elution, collects eluent, and is concentrated, lets cool, and crystallize out filters, and drying gets crystal IV; Filtrate is concentrated, lets cool, and crystallize out filters, and drying gets crystal V; Crystal is mixed, get 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%.
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, regulates pH value to 4～5 with citric acid soln, continue to concentrate, low temperature is placed, and filters.First use petroleum ether extraction, water is used ethyl acetate extraction again, uses at last 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 used acetone extract, is evaporated to driedly, adds 50% ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out filters, and drying gets crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, and standing crystallize out filters, and drying gets crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, and is concentrated, adds ethanol, lets cool, and crystallize out filters, and drying gets crystal III (being mainly ginkalide A and B); Filtrate is concentrated, and upper medical charcoal-silica gel (1: 1) post is used 60% ethanol elution, collects eluent, and is concentrated, lets cool, and crystallize out filters, and drying gets crystal IV; Crystal is mixed, get 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%.
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, regulates pH value to 4～5 with citric acid soln, continue to concentrate, low temperature is placed, and filters.First use petroleum ether extraction, water is used ethyl acetate extraction again, uses at last 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 used ethyl acetate extraction, is evaporated to driedly, adds 30% ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out filters, and drying gets crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, and standing crystallize out filters, and drying gets crystal II (being mainly Ginkgolide A. B. C); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, and is concentrated, adds ethanol, lets cool, and crystallize out filters, and drying gets crystal III (being mainly ginkalide A and B); Filtrate is concentrated, and upper medical charcoal-silica gel (1: 1) post is used 60% ethanol elution, collects eluent, and is concentrated, lets cool, and crystallize out filters, and drying gets crystal IV; Filtrate is concentrated, lets cool, and crystallize out filters, and drying gets crystal V; Crystal is mixed, get 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%.
Ginkgo leaf meal 200kg adds 8 times of amount 70% ethanol and is heated to little 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, regulates pH value to 4～5 with citric acid soln, continue to concentrate, low temperature is placed, and filters.First use petroleum ether extraction, water is used ethyl acetate extraction again, uses at last 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 used ethyl acetate extraction, is evaporated to driedly, adds 30% ethanol heating stirring and dissolving, filters, and lets cool, and crystallize out filters, and drying gets crystal I (being mainly Bilobalide and ginkolide B); Filtrate is continued concentrated, adds ethanol, and standing crystallize out filters, and drying gets crystal II (being mainly Bilobalide and Ginkgolides a and B); Filtrate adds medical charcoal, and stirring and adsorbing is filtered, and is concentrated, adds ethanol, lets cool, and crystallize out filters, and drying gets crystal III (being mainly ginkalide A and C); Filtrate is concentrated, and upper medical charcoal-silica gel (1: 1) post is used 60% ethanol elution, collects eluent, and is concentrated, lets cool, and crystallize out filters, and drying gets crystal IV; Filtrate is concentrated, lets cool, and crystallize out filters, and drying gets crystal V; Crystal is mixed, get 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, contain 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 little yellow crystalline powder.
Easily molten in ethyl acetate, dissolve in methyl alcohol, ethanol, almost insoluble in water.
B) moisture: 60 ℃ of drying under reduced pressure less loss weight are less than 5.0%.
C) protein: in 595nm wavelength place absorbance less than 0.05.
Get approximately 24mg of ginkgolides of the present invention, after adding ethanol 2ml dissolving, be diluted with water to 50ml, as need testing solution.Measure according to Coomassie brilliant blue method (Bradford method), take corresponding reagent as blank, in 595nm wavelength place absorbance 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, placed 10 minutes, do not occur muddy or precipitation.
Resin: get protein check item need testing solution 5ml, add 1 of hydrochloric acid, placed 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 is regulated the 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 the 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 the 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: contain ethanol and ethyl acetate all less than 0.5%, normal hexane is less than 0.029%.
(2) resin residue amount: contain caprolactam less than 0.0015%.
F) total ginkgoic acid: contain total ginkgoic acid less than 5ppm.
G) large molecule and polymkeric substance: the large molecule of gel chromatography noresidue and polymkeric substance.The LC-MS method is measured, result without molecular weight greater than 1000 large molecule and polymkeric substance.
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 temperatures: 25 ℃, flow velocity: 0.3ml/min.Result: result without molecular weight greater than 1000 large molecule and polymkeric substance.
H) heavy metal: less than 10ppm.
I) arsenic salt: less than 2ppm.
K) undue toxicity: make the solution that contains 0.2mg in every 1ml, meet the intravenous injection administration.
The preparation of need testing solution: get approximately 25mg of ginkgolides of the present invention, make with adding the chlorination sodium injection after ethanol 2ml dissolving the solution that contains 0.2mg in every 1ml.
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: the 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: the HPLC method is measured, and presses dry product and calculates, and contains 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; Take methyl alcohol-tetrahydrofuran-water (25: 10: 65) as mobile phase; Use evaporative light-scattering detector, drift tube temperature: 105 ℃; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 ℃; Number of theoretical plate should be not less than 2500 by Bilobalide peak calculating.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 dissolving, 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, measured body temperature 1 time every 30 minutes, survey altogether 6 times, hyperthermia all should be lower than 0.6 ℃, and 3 rabbit body temperature rising summations are lower than 1.3 ℃.
The inventor has carried out large molecule and polymer determination research and explanation to the foregoing invention content, is used 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
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 with 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 the common detector differential refraction detector, this detecting device all has good response for the material that has refraction coefficient difference.
(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 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, dextran is at retention time 9.816 ' go out peak as a result, and glucose shows the employing exclusion chromatography in retention time 18.712 ' go out the peak, the material that molecular weight is large first goes out the peak, goes out the peak after the little material of molecular weight.
2. get approximately 10mg of ginkgolides of the present invention, add ethanol 2ml dissolving, add dextran reference substance solution (10mg/ml) 1ml, mixing, 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 to go out later on the peak at 18min, show molecular weight at 180～450 appearance times in about 18min, molecular weight 5000～2000000 appearance times are in the 9min left and right, it is feasible adopting exclusion chromatography to detect macromolecular substances.
Do not contain large molecule and polymkeric substance in this product in order to verify again, therefore carried out again the LC-MS test.
Chromatographic condition: methanol-water (90: 10) is mobile phase, Agilent RX-C
18(2.1 * 50mm) chromatographic columns, 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 the 10ml measuring bottle, adds appropriate 1% acetic acid and makes dissolving, adds mobile phase and is diluted to scale, shakes up, as need testing solution.
LC-MS coupling test: according to the test method of determining, get respectively each 10 μ l of need testing solution, 400～1000 and the 400-3000 molecular weight ranges in test respectively, record chromatogram.Test findings sees 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, due to the test molecule weight range between 400-3000, Bilobalide is not tested, do not detect molecular weight in ginkgolides of the present invention greater than the material more than 700, 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 and do not contain large molecule or polymkeric substance in this product.Ginkgolides LC-MS collection of illustrative plates is seen Fig. 3～4.
Embodiment 8
Ginkgolides quality control---total ginkgoic acid inspection
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol-1% glacial acetic acid (90: 10) as mobile phase; Flow velocity 1.0ml/min; The detection wavelength is 310nm.Number of theoretical plate should be not less than 4000 by the gingko neo-acid peak.
The preparation of reference substance solution: get gingko eo-acid reference substance appropriate, accurately weighed, add methyl alcohol and make solution that every 1ml contains 5 μ g product solution in contrast; Separately get the total ginkgoic acid reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that every 1ml contains 100 μ g, as the 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, added 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: precision is drawn need testing solution, reference substance solution and is located with each 20 μ l of contrast solution, the injection liquid chromatography, calculate in need testing solution the total peak area with the corresponding chromatographic peak of total ginkgoic acid reference substance, calculate total ginkgoic acid content with gingko eo-acid reference substance external standard method, total ginkgoic acid is less than 5ppm.
The inventor is studied and illustrates the foregoing invention content, is used 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 ℃) 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, refluxed 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, refluxed 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, the injection liquid chromatography records chromatogram.Test findings sees Table 7.
Two kinds of method testing result tables of table 7
Test findings shows: employing method one (sherwood oil) preparation sample, detect 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) preparation sample, blank solution and need testing solution all do not detect chromatographic peak, therefore the inventor intends adopting method two to do the application of sample recovery test, come 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, 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 the 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, the injection liquid chromatography records chromatogram.
Result: need testing solution with total ginkgoic acid reference substance chromatogram corresponding position on can detect the total ginkgoic acid chromatographic peak, from peak area, need testing solution is consistent with the reference substance solution peak area, illustrates that the recovery is better.Test findings sees 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 solution that every 1ml contains 5 μ g product solution in contrast.Separately get the total ginkgoic acid reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that every 1ml contains 100 μ g, as the location contrast solution.
The 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, refluxed 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 need testing solution, reference substance solution and is located with each 20 μ l of contrast solution, and the injection liquid chromatography records chromatogram.Test findings sees Table 9.
Table 9 reproducible test results
| Numbering | 1# | 2# | 3# | 4# | 5# | 6# |
| The 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
The 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, refluxed 2 hours, and let cool, filter, residue washs with a small amount of normal hexane, merging filtrate and cleansing solution are 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 need testing solution, each 20 μ l of reference substance solution of drawing, the injection liquid chromatography records chromatogram.Each concentration determination three times, totally 9 times.Calculate recovery rate, RSD value.Test findings sees Table 10.
Table 10 recovery test is table as a result
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; Take methyl alcohol-tetrahydrofuran-water (25: 10: 65) as mobile phase; Use evaporative light-scattering detector, drift tube temperature: 105 ℃; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 ℃; Number of theoretical plate should be not less than 2500 by Bilobalide peak calculating.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 the mixed solution that every 1ml contains respectively 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 and add methyl alcohol 1ml dissolving in the 10ml measuring bottle, 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 the 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.
The inventor is studied and illustrates the foregoing invention content, is used 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.
In the ginkgolides finger-print, wherein peak 2 is that ginkalide C, peak 3 are ginkolide B for Bilobalide, peak 4 for ginkalide A, peak 5, and in this product, 4 characteristic peaks of active component all can be corresponding one by one in finger-print.The ginkgolides reference fingerprint is seen Fig. 5.
At first adopting Chinese Pharmacopoeia Commission's finger-print designated software in 2004---similarity evaluation A version generates reference fingerprints to the 10 batches of ginkgolides respectively, and test sample finger-print and the reference fingerprint of different batches calculated similarity with similarity software.Test findings sees 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.
Ginkgolides quality control---residual solvent is measured
(1) ethanol, ethyl acetate and normal hexane
The preparation of need testing solution: get approximately 0.1g of ginkgolides of the present invention, accurately weighed, in the top set empty bottle, precision adds DMF 5ml to make dissolving, and sealing is 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 approximately contains the solution of 30 μ g, precision measures 5ml, in the top set empty bottle, sealing, product solution in contrast.
Determination method: take 6% cyanogen propyl group phenyl-94% dimethyl polysiloxane (or polarity is close) as immobile liquid, initial temperature is 50 ℃, keeps 3 minutes, is warming up to 160 ℃ with the speed of 40 ℃ of per minutes, keeps 3 minutes; 200 ℃ of injector temperatures; Detector temperature is 250 ℃; Head space bottle equilibrium temperature is 80 ℃, and equilibration time is 30 minutes.Get the 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, press external standard method with calculated by peak area.
Contain ethanol and ethyl acetate 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 the solution that every 1ml approximately contains 0.1mg, shakes up, as inner mark solution; It is appropriate that precision takes caprolactam, adds inner mark solution and make the solution that every 1ml approximately contains caprolactam 37.5 μ g, in contrast product solution.
The preparation of need testing solution: get approximately 2.5g of ginkgolides of the present invention, accurately weighed, put in conical flask, add normal hexane 25ml, refluxed 2 hours, and took out, let cool, filter, with a small amount of normal hexane washing, merging filtrate and cleansing solution are in 60 ℃ of water bath methods, residue adds inner mark solution 1ml makes dissolving, as need testing solution.
Determination method: take polyglycol (PEG-20M) (or polarity is close) as immobile liquid; Initial temperature is 100 ℃, keeps 2 minutes, is warming up to 160 ℃ with the speed of 40 ℃ of per minutes, keeps 3 minutes, then is warming up to 220 ℃ with the speed of 40 ℃, keeps 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.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 in reference substance solution and interior mark peak area by internal standard method.
Caprolactam does not detect.
Embodiment 11
Ginkgolides quality control---assay
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol-tetrahydrofuran-water (25: 10: 65) as mobile phase; Use evaporative light-scattering detector, drift tube temperature: 105 ℃; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 ℃; Number of theoretical plate should be not less than 2500 by Bilobalide peak calculating.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 the mixed solution that every 1ml contains respectively 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 and add methyl alcohol 1ml dissolving in the 10ml measuring bottle, 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, calculate respectively the content of Bilobalide, ginkalide A, ginkolide B and ginkalide C with external standard two-point method logarithmic equation.
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, add the chlorination sodium injection and make the solution that contains 0.2mg in every 1ml.
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, measured body temperature 1 time every 30 minutes, 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:
The preparation method is:
A) preparation: mixed ethanol and glycerine, add ginkgolides, dissolving, add ethanol or water for injection to full dose, regulate pH value to 3.2～3.8 with 5～10% citric acid solns or 1～10% hydrochloric acid solution;
B) filtration sterilization;
C) embedding;
D) sterilization.
(1) protein: get bilobalide injection 2ml, add water and make 50ml, as need testing solution.Take approximately 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 the 1ml need testing solution, shakes up, and places 3min.Do blank with method, under the 595nm wavelength, measure absorbance, the 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, placed 10 minutes, do not occur muddy or precipitation.
(3) resin: get protein check item need testing solution 5ml, add 1 of hydrochloric acid, placed 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 is regulated the 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 the 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 the 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 (press embodiment 14 preparation) 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 is for the test sample pipe, manages negative control tube No. 3, manages positive control tube, No. 5 pipe test sample control tube No. 4.By adding successively 2% red cell suspension, 0.9% sodium chloride solution, distilled water shown in table 12, put immediately after mixing in the constant temperature oven of 37 ℃ ± 0.5 ℃ and carry out incubation.
Table 12 haemolysis and agglutination test addition
| The 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 |
Be clear and bright redness as the solution in test tube, it is residual or have a small amount of red blood cell residual that the bottom is acellular, and showing has haemolysis to occur; All sink as red blood cell, the supernatant achromatism and clarity, though or supernatant coloured clear and bright, 1, No. 2 pipe and manage the visual inspection no significant differences for No. 5 shows without haemolysis to occur.Observe after 3 hours 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; Take methyl alcohol-tetrahydrofuran-water (25: 10: 65) as mobile phase; Use evaporative light-scattering detector, drift tube temperature: 105 ℃; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 ℃; Number of theoretical plate should be not less than 2500 by Bilobalide peak calculating.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 the mixed solution that every 1ml contains respectively 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 the injection liquid chromatography records the chromatogram of 60 minutes.
Press similarity evaluation, test sample finger-print and reference fingerprint through similarity greater than 0.95.
Embodiment 17
Bilobalide injection quality control---assay
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take methyl alcohol-tetrahydrofuran-water (25: 10: 65) as mobile phase; Use evaporative light-scattering detector, drift tube temperature: 105 ℃; Flow rate of carrier gas: 3.00L/min; Column temperature: 40 ℃; Number of theoretical plate should be not less than 2500 by Bilobalide peak calculating.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 the mixed solution that every 1ml contains respectively 0.15mg, 0.12mg, 0.1mg, 0.1mg, shake up, in contrast product solution.
The preparation of need testing solution: precision measures bilobalide injection (pressing embodiment 14 preparations) 1ml, adds phosphate buffer solution (pH6.5) 14ml, shakes up, upper Extrelut-20 post, adsorbed 15 minutes, and with ethyl acetate 100ml wash-out, collected eluent, evaporate to dryness in water-bath, residue dissolves with mobile phase and is transferred in the 10ml measuring bottle, adds mobile phase and is diluted to scale, shakes up, filter with 0.45 μ m miillpore 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, calculate respectively the content of Bilobalide, ginkalide A, ginkolide B and ginkalide C 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, preferred 4.25～5.75mg.
Claims (2)
1. the assay method of total ginkgoic acid in the ginkgolides composition, be characterised in that,
Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent, take 90: 10 methyl alcohol-1% glacial acetic acid as mobile phase, flow velocity 1.0ml/min, the detection wavelength is 310nm, number of theoretical plate should be not less than 4000 by the gingko neo-acid peak;
The preparation of reference substance solution: get gingko eo-acid reference substance appropriate, accurately weighed, add methyl alcohol and make solution that every 1ml contains 5 μ g product solution in contrast, separately get the total ginkgoic acid reference substance appropriate, accurately weighed, add methyl alcohol and make the solution that every 1ml contains 100 μ g, as the location contrast solution;
The preparation of need testing solution: get by ginkgo leaf and extract the ginkgolides composition 5g that obtains, accurately weighed, put in flask, add normal hexane 50ml, added 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: precision is drawn need testing solution, reference substance solution and is located with each 20 μ l of contrast solution, the injection liquid chromatography, calculate in need testing solution the total peak area with the corresponding chromatographic peak of total ginkgoic acid reference substance, calculate total ginkgoic acid content with gingko eo-acid reference substance external standard method.
2. according to claim 1 assay method, the total ginkgoic acid content that it is characterized in that recording is less than 5ppm.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107315058A (en) * | 2017-05-04 | 2017-11-03 | 黑龙江珍宝岛药业股份有限公司 | A kind of method of total ginkgoic acid in detection ginkgo biloba succi |
| CN111487345A (en) * | 2020-04-28 | 2020-08-04 | 劲牌持正堂药业有限公司 | Method for rapidly detecting total ginkgoic acid in sample in production process of ginkgo leaf extract |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6030621A (en) * | 1998-03-19 | 2000-02-29 | De Long; Xie | Ginkgo biloba composition, method to prepare the same and uses thereof |
| CN1853674A (en) * | 2005-02-07 | 2006-11-01 | 贵阳云岩西创药物科技开发有限公司 | Quality controlling method of Xingdan injection |
| CN101757049A (en) * | 2009-03-13 | 2010-06-30 | 北京瑞伊人科技发展有限公司 | Ginkgo leaf extractive and method for extracting total ginkgoic acid in preparation thereof |
| CN101757050A (en) * | 2010-02-23 | 2010-06-30 | 广州汉方现代中药研究开发有限公司 | Method for measuring total ginkgolic acid in ginkgo biloba extract |
| CN102283870A (en) * | 2011-09-09 | 2011-12-21 | 黑龙江省珍宝岛制药有限公司 | High-purity folium ginkgo composition, preparation including same and preparation method thereof |
-
2012
- 2012-04-23 CN CN201310111707.5A patent/CN103163252B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6030621A (en) * | 1998-03-19 | 2000-02-29 | De Long; Xie | Ginkgo biloba composition, method to prepare the same and uses thereof |
| CN1853674A (en) * | 2005-02-07 | 2006-11-01 | 贵阳云岩西创药物科技开发有限公司 | Quality controlling method of Xingdan injection |
| CN101757049A (en) * | 2009-03-13 | 2010-06-30 | 北京瑞伊人科技发展有限公司 | Ginkgo leaf extractive and method for extracting total ginkgoic acid in preparation thereof |
| CN101757050A (en) * | 2010-02-23 | 2010-06-30 | 广州汉方现代中药研究开发有限公司 | Method for measuring total ginkgolic acid in ginkgo biloba extract |
| CN102283870A (en) * | 2011-09-09 | 2011-12-21 | 黑龙江省珍宝岛制药有限公司 | High-purity folium ginkgo composition, preparation including same and preparation method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107315058A (en) * | 2017-05-04 | 2017-11-03 | 黑龙江珍宝岛药业股份有限公司 | A kind of method of total ginkgoic acid in detection ginkgo biloba succi |
| CN111487345A (en) * | 2020-04-28 | 2020-08-04 | 劲牌持正堂药业有限公司 | Method for rapidly detecting total ginkgoic acid in sample in production process of ginkgo leaf extract |
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