CN106727503A - A kind of medicine and its quality determining method for treating cardiovascular and cerebrovascular disease - Google Patents
A kind of medicine and its quality determining method for treating cardiovascular and cerebrovascular disease Download PDFInfo
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- CN106727503A CN106727503A CN201710073366.5A CN201710073366A CN106727503A CN 106727503 A CN106727503 A CN 106727503A CN 201710073366 A CN201710073366 A CN 201710073366A CN 106727503 A CN106727503 A CN 106727503A
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Abstract
The invention discloses a kind of medicine and its quality determining method for treating cardiovascular and cerebrovascular disease.The medicine adds pharmaceutically acceptable auxiliary material or complementary composition is made the formulation for pharmaceutically allowing with ginkgo lactone composition as principle active component；Ginkgo lactone composition includes two kinds of active components of ginkalide A and ginkolide B, and the weight ratio of ginkalide A and ginkolide B is 1：2~9.It was found that having very strong synergy between ginkalide A and B, application effect is notable in cardiovascular and cerebrovascular disease is treated.The present invention is determined while realizing ginkgolides formulation components content, the results showed, the GA for being surveyed and GB separating degrees are good, and linear relationship, reappearance, precision, stability, the rate of recovery is preferable.Designed by preparation formulation of the invention and improve its vivo biodistribution availability, improve drug effect.
Description
The application is divisional application, and its female case is the invention of entitled " a kind of ginkgo lactone composition and its application "
Patent application, its Application No. 201310126461.9, applying date is on April 11st, 2013.
Technical field
The present invention relates to a kind of ginkgo lactone composition and its application, specifically being that the ginkgolides based on component structure is consolidated
Body dispersion is prepared and its dynamic quality control method.
Background technology
Ginkgo is one of ancient seeds of China, and ginkgo (Ginkgoaceae) is used as the existing more than one thousand years of China's traditional Chinese medicine
History.With the development of modern Chinese herbal medicine preparation, ginkgolides turns into the important source material medicine for preparing ginkgo agent.Total Terpene Lactones
In main active include ginkalide A (GA), ginkolide B (GB), ginkalide C (GC) and Bilobalide
(BB), wherein in natural ginkgo leaf A contents highest, and it is ginkolide B that pharmacodynamics effect is most strong, and it can selective antagonism
The platelet aggregation induced by platelet activating factor, can effectively prevent the formation of platelet aggregation and thrombus, ginkgolides group
Dividing has good therapeutic action to cardiac and cerebral vascular diseases.
Ginkgo agent is more with ginkolide B as bulk drug mostly at present, ignores the application of ginkgolides component.Only with list
Composition ginkolide B is not very science as the bulk drug of production ginkgo agent.
The content of the invention
It is an object of the invention to provide a kind of ginkgo lactone composition.
Another object of the present invention is to provide a kind of medicine for treating cardiovascular and cerebrovascular disease.
A further object of the present invention is to provide above-mentioned ginkgo lactone composition to prepare the medicine for the treatment of cardiovascular and cerebrovascular disease
Application in thing.
The purpose of the present invention is achieved through the following technical solutions：
A kind of ginkgo lactone composition, it is characterised in that described composition includes two kinds of ginkalide A and ginkolide B
The weight ratio of active component, ginkalide A and ginkolide B is 1：2~9.
Content >=90% of ginkalide A and ginkolide B in above-mentioned composition.
A kind of medicine for treating cardiovascular and cerebrovascular disease, the medicine is with above-mentioned ginkgo lactone composition as main effective
Composition, adds pharmaceutically acceptable auxiliary material or complementary composition is made the formulation for pharmaceutically allowing.
Described formulation is pill, powder, capsule, tablet, granule, injection, compound preparation or solid dispersions；It is preferred that
It is solid dispersions.
Described solid dispersions are with described ginkgo lactone composition as principle active component, with shitosan as carrier
It is made, the weight ratio of the ginkgo lactone composition and carrier is 1：5~15, preferably 1：10.
The preparation method of the solid dispersions is：Shitosan and ginkgo lactone composition are dissolved in ethanol respectively,
After 30~90min of ultrasonic dissolution, the ethanol solution of the ethanol solution of shitosan and ginkgo lactone composition is mixed, continue ultrasound
30~90min of dissolving, mixed liquor is evaporated to obtain solid dispersions after ethanol is volatilized completely.Solid dispersions of the present invention
Preparation method is not limited in above-mentioned preparation method, it would however also be possible to employ other preparation methods of the prior art prepare solid
Dispersion.But the more simple, cost of method operation of the present invention is lower, and prepared solid dispersions can improve ginkgo
The vivo biodistribution availability of lactone composition, can improve drug effect.
Described shitosan is preferably shitosan 3000.
Application of the above-mentioned ginkgo lactone composition in treatment cardiovascular and cerebrovascular diseases medicament is prepared.
Above-mentioned ginkgo lactone composition or the method for quality control of above-mentioned solid dispersion drug, comprise the following steps：
(1) reference substance solution is prepared：Precision weighs reference substance ginkalide A and ginkolide B respectively, is dissolved with methyl alcohol,
Control series product solution is obtained, ginkalide A and ginkolide B concentration range are respectively in control series product solution：In ginkgo
Ester A10.14~253.5 μ gmL-1；The μ gmL of ginkolide B 12.66~316.5-1；
(2) need testing solution is prepared：Weigh above-mentioned ginkgo lactone composition and be made solution, the solution of gained is filtered,
Subsequent filtrate is taken as need testing solution；Or weigh above-mentioned medicine and be made solution, the solution of gained is filtered, take subsequent filtrate work
It is need testing solution；
Chromatographic condition is：Agilent SB-C18 posts (4.6 × 150mm, 5 μm)；Mobile phase：Methanol-water (28: 72)；Post
35 DEG C of temperature；Flow velocity 1.0mLmin；EISD ELSD, detects parameter：105 DEG C of evaporation mouth temperature, is atomized mouth temperature
70 DEG C of degree, 30 DEG C of drift tube temperature, flow rate of carrier gas:2.8L/min.
The present invention proves that ginkalide A and ginkolide B its pharmacodynamics effect in certain ratio range are excellent by pharmacodynamics
In other ginkgolides composition proportions, therefore quality control is carried out to ginkgo lactone composition.About the quality control of ginkgolides
Be currently limited to the content for controlling ginkgolides overall, and without proportionate relationship between each composition in clear stipulaties ginkgolides component and
Controlled range window.More comprehensively to control the quality of ginkgolides preparation, set up to be matched between composition in ginkgolides part formulation and close
The quality control of system has great importance.
The present invention overcomes the defect of current ginkgo agent quality control, and Chinese Herbs are that multicomponent collaboration plays drug effect,
But this synergy is not again simple adding and fuzzy total composition quality control, it is impossible to reflect Chinese medicine entirety drug effect.
Whether polypharmacy or single are used as medicine, wherein all immanent structure relations comprising Chinese medicine.For Chinese medicine compound prescription, each medicine
There is a certain amount effect proportion relation between taste, inventor is thought from froming the perspective of on microcosmic angle, this compatibility relationship, substantially group
Dose-effect proportion relation between point；And for single component, it is unit curative effect thing to play curative effect with single composition collaboration in component
Matter basis, the meaning of its deeper is each Chinese medicine compound prescription in the presence of the quantified component structure of itself, only it is clear and definite this can
Control quantifies window, can be only achieved optimal curative effect.
The present invention determines the optimum quality control range of ginkgo lactone composition, ginkgo in composition by the test of pesticide effectiveness
The quantization scale of lactone A and ginkolide B is 1:2~1:In the range of 9, its component drug effect can be optimal.Again in clearly silver
On the basis of apricot lactone material base quantitative relationship, for ginkgolides component physics and chemistry and biopharmaceutical properties, carry out rationally
Formulation design and formulation optimization design, be finally reached the dynamic quality control mistake from bulk drug to preparations shaping whole process
Journey.During ginkgolides component dynamic quality control, because ginkgolides UV absorption is weaker, inventor uses HPLC-
ELSD determines ginkalide A and ginkolide B, ginkgolides component structure after carrying out from bulk drug component to preparations shaping simultaneously
Dynamic quality control method.
The specific screening test process of technical scheme is as follows：
The present invention quantifies the preparation of the ginkalide A and ginkolide B of component inner structure proportions：
By the ginkalide A (GA) in ginkgolides component, ginkolide B (GB), ginkalide C (GC) and Bilobalide
(BB), combined according to following ratio：GA:GB:GC:BB(1:1:1:1)、GA:GB:GC(1:1:1)、GA:GB:BB(1:1:1)、
GA:GC:BB(1:1:1)、GB:GC:BB(1:1:1)、GA:GB(2:1)、GA:GC(2:1)、GB:GC(2:1)、GA:GB(1:2)、
GA:GC(1:2)、GB:GC(1:2)、GA:GB(1:4)、GA:GB(1:6)、GA:GB(1:9) group, each ratio combination is provided with 5
Parallel group.
Ginkalide A (GA), ginkolide B (GB), ginkalide C (GC) and Bilobalide (BB) are dissolved separately in
It is respectively prepared in DMSO in 10mg/ml ginkalide As (GA) solution, 10mg/ml ginkolide Bs (GB) solution, 10mg/ml ginkgoes
Ester C (GC) solution and 10mg/ml Bilobalides (BB) solution.
Prepare GA:GB:GC:BB(1:1:1:1) ratio multi-component approach is：The ginkgo that the concentration of 50 μ 1 is 10mg/ml is taken respectively
Lactone A (GA), the concentration of 50 μ 1 are the ginkolide B (GB) of 10mg/ml, the concentration of 50 μ 1 for 10mg/ml ginkalide C (GC) with
And the Bilobalide (BB) that the concentration of 50 μ 1 is 10mg/ml is mixed and made into the mixed liquor that volume is 200 μ 1.
Prepare GA:GB:GC(1:1:1) ratio multi-component approach is：The ginkalide A that the concentration of 50 μ 1 is 10mg/ml is taken respectively
(GA), the concentration of 50 μ 1 is the ginkolide B (GB) and the concentration of 50 μ 1 of 10mg/ml for the ginkalide C (GC) of 10mg/ml mixes
It is made the mixed liquor that volume is 150 μ 1.
Prepare GA:GB:BB(1:1:1) ratio multi-component approach is：The ginkalide A that the concentration of 50 μ 1 is 10mg/ml is taken respectively
(GA), the concentration of 50 μ 1 is the ginkolide B (GB) and the concentration of 50 μ 1 of 10mg/ml for the Bilobalide (BB) of 10mg/ml mixes
It is made the mixed liquor that volume is 150 μ 1.
Prepare GA:GC:BB(1:1:1) ratio multi-component approach is：The ginkalide A that the concentration of 50 μ 1 is 10mg/ml is taken respectively
(GA), the concentration of 50 μ 1 is the ginkalide C (GC) and the concentration of 50 μ 1 of 10mg/ml for the Bilobalide (BB) of 10mg/ml mixes
It is made the mixed liquor that volume is 150 μ 1.
Prepare GB:GC:BB(1:1:1) ratio multi-component approach is：The ginkolide B that the concentration of 50 μ 1 is 10mg/ml is taken respectively
(GB), the concentration of 50 μ 1 is the ginkalide C (GC) and the concentration of 50 μ 1 of 10mg/ml for the Bilobalide (BB) of 10mg/ml mixes
It is made the mixed liquor that volume is 150 μ 1.
Prepare GA:GB(2:1) ratio multi-component approach is：Take respectively the concentration of 100 μ 1 be 10mg/ml ginkalide A (GA),
The concentration of 50 μ 1 is mixed and made into the mixed liquor that volume is 150 μ 1 for the ginkolide B (GB) of 10mg/ml.
Prepare GA:GC(2:1) ratio multi-component approach is：Take respectively the concentration of 100 μ 1 be 10mg/ml ginkalide A (GA),
The concentration of 50 μ 1 is mixed and made into the mixed liquor that volume is 150 μ 1 for the ginkalide C (GC) of 10mg/ml.
Prepare GB:GC(2:1) ratio multi-component approach is：Take respectively the concentration of 100 μ 1 be 10mg/ml ginkolide B (GB),
The concentration of 50 μ 1 is mixed and made into the mixed liquor that volume is 150 μ 1 for the ginkalide C (GC) of 10mg/ml.
Prepare GA:GB(1:2) ratio multi-component approach is：Take respectively the concentration of 50 μ 1 be 10mg/ml ginkalide A (GA),
The concentration of 100 μ 1 is mixed and made into the mixed liquor that volume is 150 μ 1 for the ginkolide B (GB) of 10mg/ml.
Prepare GA:GC(1:2) ratio multi-component approach is：Take respectively the concentration of 50 μ 1 be 10mg/ml ginkalide A (GA),
The concentration of 100 μ 1 is mixed and made into the mixed liquor that volume is 150 μ 1 for the ginkalide C (GC) of 10mg/ml.
Prepare GB:GC(1:2) ratio multi-component approach is：Take respectively the concentration of 50 μ 1 be 10mg/ml ginkolide B (GB),
The concentration of 100 μ 1 is mixed and made into the mixed liquor that volume is 150 μ 1 for the ginkalide C (GC) of 10mg/ml.
Prepare GA:GB(1:4) ratio multi-component approach is：Take respectively the concentration of 50 μ 1 be 10mg/ml ginkalide A (GA),
The concentration of 200 μ 1 is mixed and made into the mixed liquor that volume is 250 μ 1 for the ginkolide B (GB) of 10mg/ml.
Prepare GA:GB(1:6) ratio multi-component approach is：Take respectively the concentration of 50 μ 1 be 10mg/ml ginkalide A (GA),
The concentration of 300 μ 1 is mixed and made into the mixed liquor that volume is 350 μ 1 for the ginkolide B (GB) of 10mg/ml.
Prepare GA:GB(1:9) ratio multi-component approach is：Take respectively the concentration of 50 μ 1 be 10mg/ml ginkalide A (GA),
The concentration of 450 μ 1 is mixed and made into the mixed liquor that volume is 500 μ 1 for the ginkolide B (GB) of 10mg/ml.
The external anticoagulation influence of ginkgolides component of above-mentioned different proportion proportioning：
By selecting PT, APTT, TT and FIB for pharmacological effect index, compare the ginkgolides component confrontation of different ratio
Hemoglutination influences, and selects optimal proportion component.First, with disposably volunteer blood sample 70ml is extracted with Syringe needle for taking samples of blood, dispense
In 26 anti-freezing PV pipes, (3000rpm, 15min) is then centrifuged for, obtains supernatant blood plasma 50ml.Blood plasma is divided into 75 groups, each
Part μ l containing blood plasma 400.50 μ l are prepared 1 is taken respectively:1:1:1 GA:GB:GC:BB；1:1:1 GA:GB:GC；1:1:1
GA:GB:BB；1:1:1 GA:GC:BB；1:1:1 GB:GC:BB；2:1 GA:GB；2:1 GA:GC；2:1 GB:GC；1:2
GA:GB；1:2 GA:GC；1:2 GB:GC；1:4 GA:GB；1:6 GA:GB；1:9 GA:The different ratio ginkgo of GB
Lactone component mixed liquor is added in the blood plasma of 400 μ 1, and being vortexed, it is to be measured to mix.Prothrombin time TT, the part blood coagulation of activation
The measure of movable enzyme time APTT, thrombin time TT and plasmase FIB is by analysis section of the hospitals of traditional Chinese and western medicine of Jiangsu Province
Determine.1 is the results are shown in Table, it is more excellent with ginkalide A and ginkolide B proportioning drug effect in ginkgolides component, and its controllable quantization
Window is 1:2~1:When 9, drug effect is preferable.
The different proportion of table 1 proportioning the external blood coagulation influence of ginkgolides component compare (N=5)
Tab1.The comparison between of the effect of different proportion
ginkgolides component on coagulant system in vitro
Note：Compare * P with control group<0.05,**P<0.01.
The different proportion ginkgolides component that gives of the present invention influences on the Mouse tail artery bleeding time, and its method is such as
Under：
160 mouse are taken, male and female half and half are randomly divided into 16 groups, and every group 10, experiment is divided into:Blank control group, GA:GB:
GC:BB(1:1:1:1)、GA:GB:GC(1:1:1)、GA:GB:BB(1:1:1)、GA:GC:BB(1:1:1)、GB:GC:BB(1:1:
1)、GA:GB(2:1)、GA:GC(2:1)、GB:GC(2:1)、GA:GB(1:2)、GA:GC(1:2)GB:GC(1:2)、GA:GB(1:
4)、GA:GB(1:And GA 6):GB(1:9) group.By 50 μ l/g be administered, daily gastric infusion once, continuous gavage 7 days.Finally
Docking experiment is carried out after single administration after 30min.Mouse is fixed in fixator, at the 1.5mm of mouse tail end
Cut with operating scissors, record the mouse tail continuous bleeding time.2 are the results are shown in Table, with ginkalide A and silver in ginkgolides component
Apricot lactone B proportioning drug effects are more excellent, and its controllable quantization window is 1:2~1:When 9, drug effect is preferable.
Table 2 compare different proportion ginkgolides component to Mouse tail artery bleeding influence (N=5)
Note：Compare * p with control group<0.05,**p<0.01
Ginkgo lactone composition solid dispersions preparation method of the present invention is as follows：
Shitosan and solid ginkgo lactone composition are dissolved in ethanol respectively, after ultrasonic dissolution, by the second of shitosan
The ethanol solution of alcoholic solution and ginkgo lactone composition is mixed, and continues ultrasonic dissolution, mixed liquor then is evaporated into ethanol complete
Solid dispersions are obtained after volatilizing.
Detailed step is：
Ginkgo lactone composition-chitosan solid dispersion is prepared using solvent method, respectively by shitosan and solid ginkgo
(the weight ratio of ginkalide A and ginkolide B is 1 to lactone composition：2~9) be dissolved in ethanol, ultrasonic dissolution 30~
After 90min, the two is mixed, continue 30~90min of ultrasonic dissolution.Mixed liquor is placed in Rotary Evaporators, treats that ethanol is complete
After volatilizing, solid dispersion powder is taken out.Optimization ginkgo lactone composition is 1 with shitosan weight ratio：5~15, most preferably
1:10, it is 1 in ginkgo lactone composition and shitosan weight ratio:Its accumulation dissolution rate is optimal when 10.
The method of quality control of ginkgo lactone composition of the present invention or solid dispersion drug is specific as follows：
Present invention experiment employs HPLC-ELSD methods and determines ginkalide A and silver in ginkgo lactone composition preparation simultaneously
The content of apricot lactone B, this method is simple and easy to apply, has and determines silver in good precision, reappearance and reliability, 25min simultaneously
Apricot lactone component content, can be used for the quality control of ginkgolides part formulation.
Chromatographic condition is：Agilent SB-C18 posts (4.6 × 150mm, 5 μm)；Mobile phase：Methanol-water (28: 72)；Post
35 DEG C of temperature；Flow velocity 1.0mLmin；EISD ELSD, detects parameter：105 DEG C of evaporation mouth temperature, is atomized mouth temperature
70 DEG C of degree, 30 DEG C of drift tube temperature, flow rate of carrier gas:2.8L/min.Record chromatogram, reference substance is capable of achieving baseline separation, surveys
It is fixed noiseless.
Reference substance is：Ginkalide A and ginkolide B.
The preparation method of reference substance solution is：
Precision weighs reference substance respectively：(1) 5.07mg ginkalide As, (2) 6.33mg ginkolide Bs put 10mL browns appearance
In measuring bottle, plus methyl alcohol dissolving and constant volume, prepare ginkalide A and ginkolide B mass concentration is respectively 507 and 633 μ g/mL's
Mixing reference substance stock solution.It is accurate respectively to draw mixing reference substance stock solution, again with methanol dilution, extension rate is respectively 2,5,
10th, 20,50, obtain control series product solution.Concentration is respectively：Ginkalide A：253.5th, 101.4,50.7,25.35 and
10.14μg/mL；Ginkolide B：316.5th, 126.6,63.3,31.65 and 12.66 μ g/mL.
The preparation method of need testing solution is：Weigh above-mentioned ginkgo lactone composition and be made solution, by the solution of gained
Filtration, takes subsequent filtrate as need testing solution；Or weigh above-mentioned medicine and be made solution, the solution of gained is filtered, take continuous
Filtrate is used as need testing solution.
The present invention devises the ginkgolides quality of the pharmaceutical preparations control method based on component structure.
The inventive method progressive compared with existing ginkgolides quality of the pharmaceutical preparations control method is as follows：
Inventor is based on traditional Chinese medicine basic theory, and network analysis is carried out to each composition of ginkgolides component, finds in ginkgo
There is very strong synergy between ester A and B.Significantly, this method overcomes mesh to application effect in cardiovascular and cerebrovascular disease is treated
The defect of preceding ginkgolides quality of the pharmaceutical preparations control, is the multidimensional component of final preparation shaping from bulk drug to formulation process
The dynamic control process of structure, the optimization effective substance of ginkgo lactone composition preparation for treating cardiovascular and cerebrovascular disease is ginkgo
Lactone A and ginkolide B, preferably controllable quality window are 1: 2~1: 9.Using HPLC-ELSD methods, ginkgolides system is realized
Determined while agent constituent content, the results showed, the GA for being surveyed and GB separating degrees are good, linear relationship, reappearance, precision,
Stability, the rate of recovery is preferable.Because ginkgolides component generally there are solubility, this is likely to cause biological utilisation
One of low big reason of degree, is designed by preparation formulation of the invention and improves its vivo biodistribution availability, improves drug effect.This
Outward, the reasonability of ginkgolides component quantization scale is further illustrated by the test of pesticide effectiveness.
Brief description of the drawings
Equilbrium solubility of Fig. 1 ginkgo lactone compositions in different pH value under the conditions of 37 DEG C.
LogP value of Fig. 2 ginkgo lactone compositions in n-octyl alcohol-buffer solution under the conditions of 37 DEG C.
Fig. 3 prepares ginkgolides component-shitosan (1 by carrier of different molecular weight shitosan:5) solid dispersions accumulation
Stripping curve
(a) shitosan 3000- ginkgo lactone composition solid dispersions, ginkgolides:chitosan 5000(1:5)
SD (b) shitosan 5000- ginkgo lactone composition solid dispersions, the 000- ginkgo lactone compositions solid of (c) shitosan 50 point
A prose style free from parallelism, the 000- ginkgo lactone composition solid dispersions of (d) shitosan 100, ginkgolides:chitosan>100 000
(1:5) SD (e) shitosans are more than 100 000- ginkgo lactone composition solid dispersions, ginkgolides (f) ginkgolides groups
Compound raw material .Data are presented as the mean ± SEM (n=3).
The Dissolution profiles of GA in Fig. 4 different proportions ginkgo lactone composition-chitosan solid dispersion.
(a) ginkgo lactone composition-shitosan 3000 (1:5) solid dispersions, (b) ginkgo lactone composition-shitosan
3000(1:10) solid dispersions, (c) ginkgo lactone composition-shitosan 3000 (1:15) solid dispersions, (d) ginkgolides
Composition material medicine .Data are presented as mean ± SEM (n=3).
The Dissolution profiles of GB in Fig. 5 different proportions ginkgo lactone composition-chitosan solid dispersion.
(a) ginkgo lactone composition-shitosan 3000 (1:5) solid dispersions, (b) ginkgo lactone composition-shitosan
3000(1:10) solid dispersions, (c) ginkgo lactone composition-shitosan 3000 (1:15) solid dispersions, (d) ginkgolides
Composition material medicine .Data are presented as mean ± SEM (n=3).Fig. 6 high doses ginkgo lactone composition is former
Material medicine group, low dosage ginkgo lactone composition bulk drug group, control group, ISO model groups, positive drug group and high dose ginkgolides
Composition solid dispersion group, the SOD activity of low dosage ginkgo lactone composition solid dispersions group.
Fig. 7 high dose ginkgo lactone composition bulk drugs group, low dosage ginkgo lactone composition bulk drug group, control group,
ISO model groups, positive drug group and high dose ginkgo lactone composition solid dispersions group, low dosage ginkgo lactone composition solid
The MDA activity of dispersion group.
Fig. 8 high dose ginkgo lactone composition bulk drugs group, low dosage ginkgo lactone composition bulk drug group, control group,
ISO model groups, positive drug group and high dose ginkgo lactone composition solid dispersions group, low dosage ginkgo lactone composition solid
The LDH activity of dispersion group.
Fig. 9 is the chromatogram for mixing reference substance.
Specific embodiment
The measure of the ginkgo lactone composition of embodiment 1 equilbrium solubility in water and in the PBS of difference pH：
Prepare need testing solution：Prepare pH be respectively 2.0,2.5,5.0,5.8,6.5,7.0,7.4, the phosphoric acid of 7.8-8.0
Salt buffer salting liquid, takes excessive ginkalide A：Ginkolide B weight ratio is 1：2 ginkgo lactone composition equivalent is divided into 10 parts
It is respectively placed in tool plug teat glass, then a tool plug teat glass adds water as blank thereto, to other tool plug glass
The different phosphate buffered saline of pH value is separately added into glass test tube, ultrasound is not redissolved to medicine and is made ginkgolides combination
Thing saturated solution, temperature keeps (37 ± 1) DEG C in being then placed in constant temperature oscillator, shakes 24h.Saturated solution is micro- with 0.45 μm
Hole membrane filtration, discards just filtrate, takes subsequent filtrate as need testing solution.
Prepare reference substance solution：Precision weighs reference substance respectively：(1) 5.07mg ginkalide As, in (2) 6.33mg ginkgoes
Ester B, in putting 10mL brown volumetric flasks, plus methyl alcohol dissolving and constant volume, prepare ginkalide A and ginkolide B mass concentration difference
It is the mixing reference substance stock solution of 507 and 633 μ g/mL.Accurate respectively to draw mixing reference substance stock solution, again with methanol dilutes,
Extension rate is respectively 2,5,10,20,50, obtains control series product solution.Concentration is respectively：Ginkalide A：253.5、
101.4th, 50.7,25.35 and 10.14 μ g/mL；Ginkolide B：316.5th, 126.6,63.3,31.65 and 12.66 μ g/mL.
Chromatographic condition is：Agilent SB-C18 posts (4.6 × 150mm, 5 μm)；Mobile phase：Methanol-water (28: 72)；Post
35 DEG C of temperature；Flow velocity 1.0mLmin；EISD ELSD, detects parameter：105 DEG C of evaporation mouth temperature, is atomized mouth temperature
70 DEG C of degree, 30 DEG C of drift tube temperature, flow rate of carrier gas:2.8L/min.Record chromatogram, reference substance is capable of achieving baseline separation, surveys
It is fixed noiseless.
The test sample of above-mentioned preparation and the sample size of reference substance are 10 μ L；Record time 30min.
2 kinds of active ingredients in ginkgo lactone composition are measured, its equilbrium solubility is calculated, 1 and Fig. 1 is the results are shown in Table
(equilbrium solubility of the ginkgo lactone composition in different pH value under the conditions of 37 DEG C).
Equilbrium solubility of the ginkgo lactone composition of table 1 in different pH value under the conditions of 37 DEG C
As a result：Ginkgo lactone composition dissolubility is poor.Therefore, when considering to prepare ginkgo lactone composition preparation, it is necessary to
Improve based on its solubility, to improve its bioavilability.
The ginkalide A of embodiment 2:Ginkolide B weight ratio is 1：The survey of 2 ginkgo lactone composition Determination of oil-water partition coefficient
It is fixed
Take during appropriate ginkgo lactone composition is dissolved in by water saturated n-octyl alcohol, be made the just pungent of ginkgo lactone composition
Alcohol saturated solution, precision is drawn the solution and is respectively placed in 10 10mL cillin bottles (2mL/ bottles), thereto in a cillin bottle
The water of 2ml n-octyl alcohol saturations is added as blank, it is molten to the phosphate-buffered that 2mL difference pH value is separately added into other cillin bottles
Liquid, is put into 37 DEG C of Air oscillators, and shaking 24h is until balance, removes a layer water phase, with 6000rmin-1Centrifugation 10min, presses
Method and chromatographic condition according to the preparation reference substance solution described in embodiment 1 prepare reference substance solution and determine n-octyl alcohol before distribution
Middle ginkgo lactone composition concentration C0With the ginkgo lactone composition concentration C in water phase after distribution, apparent oil is calculated according to formula
Water partition coefficient.Computing formula is as follows：
Papp=(C0V0-CV)/CV
P in above formulaappIt is apparent partition coefficients；C0Initial concentration (the μ gmL for being index components in n-octyl alcohol-1)；V0It is water saturated n-octyl alcohol volume (i.e. 2mL)；C measures concentration (the μ g of index components in water phase when being partition equilibrium
mL-1)；V is water phase volume (i.e. 2mL).The results are shown in Table 2 and Fig. 2 (ginkgo lactone composition n-octyl alcohol-bufferings under the conditions of 37 DEG C
LogP values in liquid).
LogP value of the ginkgo lactone composition of table 2 in n-octyl alcohol-buffer solution under the conditions of 37 DEG C
As a result：LogP values in n-octyl alcohol-buffer solution of the ginkgo lactone composition under different pH all more than 3, explanation
Its permeability is good.In conjunction with the embodiments 1 and embodiment 2, can obtain ginkgo lactone composition can according to Biopharmaceutics Classification
It is classified as the 3rd class component, i.e. low-solubility, high osmosis medicine.Formulation design should be improved for the property.
Embodiment 3 is based on the preparation of the ginkgolides solid dispersions of preferred composition
Method：Select shitosan as solid dispersion carrier using solvent method, ginkalide A is prepared first:In ginkgo
Ester B part by weight is 1：2 ginkgo lactone composition and 3000,5000,50000,100000 and the shitosan more than 100000
Ratio is 1:5, compare different molecular weight influences on ginkgo lactone composition solubility, preferably the chitosan-loaded of optimum weight
Body.Further optimize the part by weight (1 of ginkgo lactone composition and preferred chitin carrier:5、1:10、1:15) silver is prepared
Apricot lactone solid dispersions, the prescription of ginkgolides solid dispersions is optimized with dissolution rate as inspection target.Result is shown in Fig. 3-5.
As a result：Shitosan 3000 can at utmost improve ginkgo lactone composition compared with other molecular weight chitosans
Solubility, and the solid dispersions of different proportion ginkgo lactone composition and shitosan are investigated, find ginkgo lactone composition
With shitosan part by weight 1:10 is optimal prescription, and the accumulation dissolution rate of ginkgolides component can reach 90% in 20min.
The sign of the ginkgo lactone composition solid dispersion preparation of embodiment 4
Method：Investigated using SOD, MDA and LDH biochemical indicator and compare ginkgo lactone composition bulk drug high dose group and low
Dosage group, ginkgo lactone composition solid dispersions (GSD) high dose group and low dose group, the drug effect of positive drug control group are poor
It is different.
Ginkgo lactone composition bulk drug：Ginkalide A and ginkolide B are by weight 1：2 mixing.
Ginkgo lactone composition solid dispersions (GSD)：Respectively by shitosan 3000 and ginkgo lactone composition (in ginkgo
Ester A and ginkolide B weight ratio are 1：2) it is dissolved in ethanol, after ultrasonic dissolution 45min, by the ethanol solution and silver of shitosan
The ethanol solution of apricot lactone composition is mixed, and continues ultrasonic dissolution 45min, and mixed liquor is evaporated to consolidate after ethanol is volatilized completely
Body dispersion.Wherein described ginkgo lactone composition is 1 with the weight ratio of shitosan 3000:10.
Positive control drug：Pu Nuonaier.
ISO modeling methods：Give rats by intraperitoneal injection isoproterenol hydrochloride injection (Isoprenaline within continuous 5 days
Hydrochloride injection, ISO) ISO 10mg/kg modes modeling method, can safely obtain acute myocardial ischemia
Rat model.
Blank control group：Normal diet, after the 5th day, continuous gavage physiological saline 7 days.
ISO model groups：Modeling according to the method described above, normal diet.
Positive drug control group：Modeling according to the method described above, continuous gavage 2mg/kg Pu Nuonaier 7 days.
High dose ginkgo lactone composition solid dispersions group (High dose GSD)：Modeling according to the method described above, continuously
Gavage high dose (50mg/kg is equivalent to ginkgo lactone composition raw material dose) ginkgolides solid dispersions 7 days.
Low dosage ginkgo lactone composition solid dispersions group (Low dose GSD)：Modeling according to the method described above, continuously
Gavage low dosage (10mg/kg is equivalent to ginkgo lactone composition raw material dose) ginkgolides solid dispersions 7 days.
High dose ginkgo lactone composition bulk drug group (High dose GK)：Modeling according to the method described above, continuous gavage
High dose 50mg/kg ginkgo lactone compositions bulk drug 7 days.
Low dosage ginkgo lactone composition bulk drug group (Low dose GK)：Modeling according to the method described above, continuous gavage is low
Dosage 10mg/kg ginkgo lactone compositions bulk drug 7 days.
Ginkgo lactone composition solid dispersions drug delivery unit is prepared for, in order to prove that said preparation technology effectively increases it
Drug effect, this problem initially sets up ISO myocardial ischemia in rats models, by SOD, MDA and LDH biochemical indicator, by relatively high dose
Amount ginkgo lactone composition bulk drug group, low dosage ginkgo lactone composition bulk drug group, blank control group, ISO model groups, sun
Property medicine group and high dose ginkgo lactone composition solid dispersions group, the medicine of low dosage ginkgo lactone composition solid dispersions group
Effect difference, test result indicate that, ginkgolides has concentration dependent for the drug action of rats with myocardial ischemia, and it is solid
Body dispersion pharmacodynamics effect is substantially better than the pharmacological effect of ginkgo lactone composition bulk drug.To injection ISO modeling myocardial ischemias
Rat, be administered high and low dose group ginkgo lactone composition solid dispersions group (GSD) compare with ISO model groups afterwards, its survey
Determine SOD values significantly raised, MDA values are substantially reduced, LDH values are also decreased obviously, the silver of its intensity of variation and high dose and low dosage
Apricot lactone composition bulk drug group compares, and is also significantly better than its end value.Result is shown in Fig. 6-8.
Ginkalide A and silver in the ginkgo lactone composition bulk drug of embodiment 5 and ginkgo lactone composition solid dispersions
The assay of apricot lactone B
1. instrument and reagent
Waters high performance liquid chromatographs, including 600 quaternary gradient pumps, 717 automatic samplers, ELSD2000E detectors.
Reference substance：Ginkalide A (lot number 110862) is purchased from Nat'l Pharmaceutical ＆ Biological Products Control Institute；Ginkolide B (lot number
110863) purity is equal>98%.
Test sample：Ginkgo lactone composition bulk drug (ginkalide A:Ginkolide B weight ratio is 1：2)；Ginkgolides
(carrier is shitosan 3000 to composition solid dispersion, and ginkgo lactone composition is 1 with the weight ratio of shitosan 3000:10, silver
Ginkalide A and ginkolide B weight ratio are 1 in apricot lactone composition：2).
Methyl alcohol (chromatographically pure, Hanbon Sci. ＆ Tech. Co., Ltd.), Milli-Q ultra-pure waters.Remaining reagent is pure for analysis.
2. chromatographic condition
Chromatographic column：Agilent SB-C18 posts (4.6 × 150mm, 5 μm)；Mobile phase：Methanol-water (28: 72)；Column temperature 35
℃；Flow velocity 1.0mL/min；EISD ELSD, detects parameter：105 DEG C of evaporation mouth temperature, atomization mouth temperature 70
DEG C, 30 DEG C of drift tube temperature, flow rate of carrier gas:2.8L/min.Record time 30min.
3. experimental technique and result
The preparation of 3.1 need testing solutions
1.32mg ginkgo lactone compositions bulk drug and 2.47mg ginkgo lactone composition solid dispersions are weighed, is put respectively
In 25mL measuring bottles, with methyl alcohol dissolving and constant volume, sample filtration takes subsequent filtrate, need testing solution is obtained final product, for determining ginkgolides
Composition levels.
The preparation of 3.2 reference substance solutions：Reference substance solution is prepared according to the method described in embodiment 1.
3. content assaying method
It is accurate respectively to draw reference substance solution and each 10 μ l of need testing solution, liquid chromatograph is injected, it is measured, as a result
It is shown in Table 3-5.In preparation process design and preparation process, there is the variation of minor swing in ginkgo lactone composition structure proportion, and
And there is part material to be lost, but its composition structure matches somebody with somebody Ratio control in controlled range window, further illustrates of the invention
Essence.
The content (mg/g) of ginkalide A and ginkolide B in the ginkgo lactone composition solid dispersions of table 3
Note：The testing result of ginkgo lactone composition solid dispersions is to remove the result of calculation after shitosan.
Material loss amount proportion (%) after the preparations shaping of table 4
Ginkgo lactone composition structure proportion value before and after the preparation of table 5
The content assaying method of embodiment 6 is investigated
1. linear relationship
Precision weighs reference substance 5.07mg ginkalide As respectively, 6.33mg ginkolide Bs, in putting 10mL brown volumetric flasks,
Plus methyl alcohol dissolves simultaneously constant volume, the conduct that preparation ginkalide A and ginkolide B mass concentration are respectively 507 and 633 μ g/mL is mixed
Close reference substance stock solution.It is accurate respectively to draw mixing reference substance stock solution, again with methanol dilution, extension rate is respectively 2,5,
10th, 20,50, obtain control series product solution.Concentration is respectively：Ginkalide A：253.5th, 101.4,50.7,25.35 and
10.14μg/mL；Ginkolide B：316.5th, 126.6,63.3,31.65 and 12.66 μ g/mL；Each concentrations control product solution difference
The μ L of sample introduction 10.Peak area is determined with HPLC-ELSD, linear regression is made to sample size logarithm lnM with peak area logarithm lnA, must returned
Return equation, be shown in Table 6.
The linear relationship experimental result of table 6
2. precision test
Precision draws 3 various concentrations reference substances, is respectively 50.7,63.3 containing GA and GB concentration；25.3,31.6；12.7,
15.8μg·mL-1, chromatographic condition as described in embodiment 1 determines, and every kind of concentration mensuration 5 times calculates in a few days RSD；METHOD FOR CONTINUOUS DETERMINATION
5d, calculates RSD in the daytime.As a result：Its in a few days RSD be respectively 0.96%, 1.53%, 0.78%；In the daytime RSD be respectively 2.81%,
1.79%th, 0.75%.
3. repeated experiment
6 parts of ginkgo lactone composition bulk drug sample is taken, according to test sample preparation method and chromatostrip described in embodiment 5
Part prepares need testing solution and analyzes, and as a result the RSD of ginkalide A and ginkolide B chromatographic peak area is respectively 2.25% He
2.92%.
4. stability experiment
Ginkgo lactone composition bulk drug need testing solution under " replica test " item is taken, respectively at 0,2,4,8,12,24h
It is measured, calculates the RSD, respectively 2.14% and 2.72% of peak area.5. average recovery experiment
Precision weighs same 9 parts of ginkgo lactone composition bulk drug sample, and every part of about 15mg is separately added into 25mL measuring bottles,
3 groups are divided into, (reference substance added in each measuring bottle is molten to be separately added into a certain amount of reference substance solution in every group of 3 measuring bottles
In liquid the content of ginkalide A respectively may be about 1.40,1.75,3.50mg, the content of ginkolide B respectively may be about 2.87,3.51,
7.08mg), with methyl alcohol dissolving and constant volume, sample filtration takes subsequent filtrate, obtains final product need testing solution.According to the color described in embodiment 5
Spectral condition is determined, and the μ L of sample introduction 20 are analyzed, and calculates the mean sample recovery rate of each reference substance, as a result ginkalide A
102.8%, RSD are 2.54%；Ginkolide B is that 100.1%, RSD is 2.59%.
To sum up, the method for the quality control based on ginkgolides constituent solid dispersion that the present invention is set up, with
Fixed effect experiment basis, can more effectively, more fully control the quality of ginkgolides part formulation.
Claims (10)
1. a kind of medicine for treating cardiovascular and cerebrovascular disease, it is characterised in that the medicine is based on ginkgo lactone composition
Active ingredient is wanted, pharmaceutically acceptable auxiliary material is added or complementary composition is made the formulation for pharmaceutically allowing；
The ginkgo lactone composition includes two kinds of active components of ginkalide A and ginkolide B, the ginkalide A and institute
The weight ratio for stating ginkolide B is 1：2~9.
2. medicine according to claim 1, it is characterised in that ginkalide A and ginkgo in the ginkgo lactone composition
Content >=90% of lactone B.
3. medicine according to claim 1, it is characterised in that the formulation is pill, powder, capsule, tablet, particle
Agent, injection, compound preparation or solid dispersions.
4. medicine according to claim 3, it is characterised in that the formulation is solid dispersions.
5. medicine according to claim 4, it is characterised in that the solid dispersions are with the ginkgo lactone composition
It is principle active component, is made up with shitosan of carrier.
6. medicine according to claim 5, it is characterised in that the weight of the ginkgo lactone composition and the shitosan
Than being 1：5~15.
7. medicine according to claim 6, it is characterised in that the weight of the ginkgo lactone composition and the shitosan
Than being 1：10.
8. medicine according to claim 5, it is characterised in that the preparation method of the solid dispersions is：Respectively by shell
Glycan and ginkgo lactone composition are dissolved in ethanol, after 30~90min of ultrasonic dissolution, by the ethanol solution of shitosan and ginkgo
The ethanol solution of lactone composition is mixed, and continues 30~90min of ultrasonic dissolution, and mixed liquor is evaporated to be obtained after ethanol is volatilized completely
Solid dispersions.
9. according to any described medicine in claim 5-8, it is characterised in that the shitosan is shitosan 3000.
10. the quality determining method of the medicine described in a kind of claim 1, it is characterised in that comprise the following steps：
(1) reference substance solution is prepared：Precision weighs reference substance ginkalide A and ginkolide B respectively, is dissolved with methyl alcohol, obtains
Control series product solution, ginkalide A and ginkolide B concentration range are respectively in control series product solution：Ginkalide A
10.14~253.5 μ gmL-1；The μ gmL of ginkolide B 12.66~316.5-1；
(2) need testing solution is prepared：The solution that the ginkgo lactone composition described in claim 1 is made is weighed, by the molten of gained
Liquid is filtered, and takes subsequent filtrate as need testing solution；Or the medicine weighed described in claim 1 is made solution, by the molten of gained
Liquid is filtered, and takes subsequent filtrate as need testing solution；
Chromatographic condition is：Agilent SB-C18 posts (4.6 × 150mm, 5 μm)；Mobile phase：Methanol-water (28: 72)；Column temperature 35
℃；Flow velocity 1.0mLmin；EISD ELSD, detects parameter：105 DEG C of evaporation mouth temperature, atomization mouth temperature 70
DEG C, 30 DEG C of drift tube temperature, flow rate of carrier gas:2.8L/min.
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| CN101249085A (en) * | 2008-03-31 | 2008-08-27 | 广州艾格生物科技有限公司 | Bilobalide A, B compound injection and preparation method and application thereof |
| CN102204879A (en) * | 2010-03-29 | 2011-10-05 | 刘全胜 | Preparation method of ginkgolide B solid dispersion |
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| CN101249085A (en) * | 2008-03-31 | 2008-08-27 | 广州艾格生物科技有限公司 | Bilobalide A, B compound injection and preparation method and application thereof |
| CN102204879A (en) * | 2010-03-29 | 2011-10-05 | 刘全胜 | Preparation method of ginkgolide B solid dispersion |
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