CN103263411A - Ginkgolide composition and application thereof - Google Patents
Ginkgolide composition and application thereof Download PDFInfo
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- CN103263411A CN103263411A CN2013101264619A CN201310126461A CN103263411A CN 103263411 A CN103263411 A CN 103263411A CN 2013101264619 A CN2013101264619 A CN 2013101264619A CN 201310126461 A CN201310126461 A CN 201310126461A CN 103263411 A CN103263411 A CN 103263411A
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Abstract
The present invention discloses a Ginkgolide composition and a method for the preparation thereof. The composition includes two active components, Ginkgolide A and Ginkgolide B, and the weight ratio of Ginkgolide A to Ginkgolide B is 1: (2-9). A strong synergistic effect is found between Ginkgolide A and Ginkgolide B, which has a remarkable application effect on the treatment of cardio-cerebral vascular disease. The invention realizes simultaneous determination for the component content of the Ginkgolides preparation. Experiments show that the Ginkgolide A and Ginkgolide B have the advantages of good separating degree with better linear relation, reproducibility, precision, stability, and recovery rate. The dosage form of the preparations provided by the present invention is beneficial for improving the bioavailability in vivo and drug efficacy.
Description
Technical field
The present invention relates to a kind of bilobalide compositions and application thereof, specifically be based on the preparation of bilobalide solid dispersion and the dynamic mass control method thereof of component structure.
Background technology
Ginkgo is one of ancient seeds of China, and Semen Ginkgo (Ginkgoaceae) is as the history of the existing more than one thousand years of China's Chinese medicine.Along with the development of modern Chinese medicine preparation, bilobalide becomes the important source material medicine of preparation ginkgo agent.Main active in the Ginkgo total lactones comprises ginkalide A (GA), ginkalide B (GB), ginkalide C (GC) and bilobalide (BB), wherein A content is the highest in the natural silver Folium Pruni, and pharmacodynamics effect the strongest be ginkalide B, the platelet aggregation that it can the selectivity antagonism be induced by platelet activating factor, can effectively prevent the formation of platelet aggregation and thrombosis, the bilobalide component has good therapeutical effect to cardiac and cerebral vascular diseases.
Ginkgo agent is many mostly at present is crude drug with the ginkalide B, has ignored the application of bilobalide component.Be science very not with single composition ginkalide B as the crude drug of producing ginkgo agent only.
Summary of the invention
The object of the present invention is to provide a kind of bilobalide compositions.
Another object of the present invention is to provide a kind of medicine that is used for the treatment of cardiovascular and cerebrovascular disease.
Another purpose of the present invention is to provide the application of above-mentioned bilobalide compositions in the medicine of preparation treatment cardiovascular and cerebrovascular disease.
Purpose of the present invention is achieved through the following technical solutions:
A kind of bilobalide compositions is characterized in that described compositions comprises ginkalide A and two kinds of active components of ginkalide B, and the weight ratio of ginkalide A and ginkalide B is 1:2 ~ 9.
The content of ginkalide A and ginkalide B 〉=90% in the above-mentioned composition.
A kind of medicine that is used for the treatment of cardiovascular and cerebrovascular disease, this medicine is main effective ingredient with above-mentioned bilobalide compositions, adds that acceptable accessories or complementary composition make pharmaceutically the dosage form that allows.
Described dosage form is pill, powder, capsule, tablet, granule, injection, compound preparation or solid dispersion; Be preferably solid dispersion.
Described solid dispersion is to be main effective ingredient with described bilobalide compositions, is that carrier is made with the chitosan, and the weight ratio of described bilobalide compositions and carrier is 1:5 ~ 15, is preferably 1:10.
The preparation method of described solid dispersion is: respectively with chitosan and bilobalide composition dissolves in ethanol, behind ultrasonic dissolution 30 ~ 90min, with the alcoholic solution of chitosan and the alcoholic solution mixing of bilobalide compositions, continue ultrasonic dissolution 30 ~ 90min, after mixed liquor is evaporated to ethanol and volatilizes fully solid dispersion.The preparation method of solid dispersion of the present invention is not limited in above-mentioned preparation method, also can adopt other preparation method of the prior art to prepare solid dispersion.But method operation of the present invention is more simple, cost is lower, and prepared solid dispersion can improve the interior bioavailability of body of bilobalide compositions, can improve drug effect.
Described chitosan is preferably chitosan 3000.
The application of above-mentioned bilobalide compositions in preparation treatment cardiovascular and cerebrovascular diseases medicament.
Above-mentioned bilobalide compositions or the method for quality control of above-mentioned solid dispersion medicine may further comprise the steps:
(1) preparation reference substance solution: precision takes by weighing reference substance ginkalide A and ginkalide B respectively, use dissolve with methanol, obtain serial reference substance solution, ginkalide A and ginkalide B concentration range are respectively in the serial reference substance solution: ginkalide A 10.14 ~ 253.5 μ gmL
-1Ginkalide B 12.66 ~ 316.5 μ gmL
-1
(2) preparation need testing solution: take by weighing above-mentioned bilobalide compositions and make solution, with the solution filtration of gained, get subsequent filtrate as need testing solution; Perhaps take by weighing above-mentioned medicine and make solution, with the solution filtration of gained, get subsequent filtrate as need testing solution;
Chromatographic condition is: Agilent SB-C18 post (4.6 * 150 mm, 5 μ m); Mobile phase: methanol-water (28: 72); 35 ℃ of column temperatures; Flow velocity 1.0 mLmin; Evaporative light scattering detector ELSD, detected parameters: 105 ℃ of evaporation mouthful temperature, 70 ℃ of atomizing mouthful temperature, 30 ℃ of drift tube temperatures, flow rate of carrier gas: 2.8 L/min.
Its pharmacodynamics effect in certain ratio range is better than other bilobalide composition proportion by pharmacodynamics proof ginkalide A and ginkalide B in the present invention, so the bilobalide compositions is carried out quality control.The quality control of relevant bilobalide only limits to control the overall content of bilobalide at present, does not clearly stipulate in the bilobalide component proportionate relationship and controlled range window between each composition and have.For controlling the quality of bilobalide preparation more comprehensively, set up in the bilobalide component preparation that the quality control of proportion relation has great importance between composition.
The present invention overcomes the defective of present ginkgo agent quality control, and the Chinese medicine curative effect is the collaborative performance of multicomponent drug effect, but this synergism be not again simply add and, fuzzy total composition quality control can not reflect the whole drug effect of Chinese medicine.No matter be compound recipe medication or single is used as medicine, wherein all comprise the immanent structure relation of Chinese medicine.For Chinese medicine compound, there is certain dose-effect proportion relation between each flavour of a drug, the inventor thinks from microcosmic angle, this compatibility relationship comes down to the dose-effect proportion relation between component; And for single component, working in coordination with the performance curative effect with single composition in the component is unit curative effect material base, but its more the meaning of deep layer be the quantized sets separation structure that there is self in each Chinese medicine compound, have only clear and definite this controlled quantification window, just can reach optimum curative effect.
The present invention determines the optimum quality span of control of bilobalide compositions by the test of pesticide effectiveness, and the quantization scale of ginkalide A and ginkalide B is in 1:2～1:9 scope in the compositions, and it is optimum that its component drug effect can reach.Again on the basis of clear and definite bilobalide material base quantitative relationship, at bilobalide component physics and chemistry and biopharmaceutics character, carry out the design of rational dosage form design and formulation optimization, finally reach the dynamic mass control procedure of whole process from the crude drug to the preparations shaping.In bilobalide component dynamic mass control procedure, since the bilobalide uv absorption a little less than, the inventor adopts HPLC-ELSD to measure ginkalide A and ginkalide B simultaneously, carry out from the crude drug component to preparations shaping after the dynamic mass control method of bilobalide component structure.
The concrete screening test process of technical scheme of the present invention is as follows:
The present invention quantizes the ginkalide A of component inner structure ratio proportioning and the preparation of ginkalide B:
With the ginkalide A in the bilobalide component (GA), ginkalide B (GB), ginkalide C (GC) and bilobalide (BB), according to following ratio combination: 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 groups.
Ginkalide A (GA), ginkalide B (GB), ginkalide C (GC) and bilobalide (BB) be dissolved in respectively make 10mg/ml ginkalide A (GA) solution, 10mg/ml ginkalide B (GB) solution, 10mg/ml ginkalide C (GC) solution and 10mg/ml bilobalide (BB) solution among the DMSO respectively.
Preparation GA:GB:GC:BB (1:1:1:1) ratio component method is: get the bilobalide (BB) that ginkalide A (GA) that 50 μ, 1 concentration is 10mg/ml, ginkalide B (GB) that 50 μ, 1 concentration is 10mg/ml, ginkalide C (GC) that 50 μ, 1 concentration is 10mg/ml and 50 μ, 1 concentration is 10mg/ml respectively and be mixed and made into the mixed liquor that volume is 200 μ 1.
Preparation GA:GB:GC (1:1:1) ratio component method is: get the ginkalide C (GC) that ginkalide A (GA) that 50 μ, 1 concentration is 10mg/ml, ginkalide B (GB) that 50 μ, 1 concentration is 10mg/ml and 50 μ, 1 concentration is 10mg/ml respectively and be mixed and made into the mixed liquor that volume is 150 μ 1.
Preparation GA:GB:BB (1:1:1) ratio component method is: get the bilobalide (BB) that ginkalide A (GA) that 50 μ, 1 concentration is 10mg/ml, ginkalide B (GB) that 50 μ, 1 concentration is 10mg/ml and 50 μ, 1 concentration is 10mg/ml respectively and be mixed and made into the mixed liquor that volume is 150 μ 1.
Preparation GA:GC:BB (1:1:1) ratio component method is: get the bilobalide (BB) that ginkalide A (GA) that 50 μ, 1 concentration is 10mg/ml, ginkalide C (GC) that 50 μ, 1 concentration is 10mg/ml and 50 μ, 1 concentration is 10mg/ml respectively and be mixed and made into the mixed liquor that volume is 150 μ 1.
Preparation GB:GC:BB (1:1:1) ratio component method is: get the bilobalide (BB) that ginkalide B (GB) that 50 μ, 1 concentration is 10mg/ml, ginkalide C (GC) that 50 μ, 1 concentration is 10mg/ml and 50 μ, 1 concentration is 10mg/ml respectively and be mixed and made into the mixed liquor that volume is 150 μ 1.
Preparation GA:GB (2:1) ratio component method is: get the ginkalide A (GA) that 100 μ, 1 concentration is 10mg/ml respectively, the ginkalide B (GB) that 50 μ, 1 concentration is 10mg/ml is mixed and made into the mixed liquor that volume is 150 μ 1.
Preparation GA:GC (2:1) ratio component method is: get the ginkalide A (GA) that 100 μ, 1 concentration is 10mg/ml respectively, the ginkalide C (GC) that 50 μ, 1 concentration is 10mg/ml is mixed and made into the mixed liquor that volume is 150 μ 1.
Preparation GB:GC (2:1) ratio component method is: get the ginkalide B (GB) that 100 μ, 1 concentration is 10mg/ml respectively, the ginkalide C (GC) that 50 μ, 1 concentration is 10mg/ml is mixed and made into the mixed liquor that volume is 150 μ 1.
Preparation GA:GB (1:2) ratio component method is: get the ginkalide A (GA) that 50 μ, 1 concentration is 10mg/ml respectively, the ginkalide B (GB) that 100 μ, 1 concentration is 10mg/ml is mixed and made into the mixed liquor that volume is 150 μ 1.
Preparation GA:GC (1:2) ratio component method is: get the ginkalide A (GA) that 50 μ, 1 concentration is 10mg/ml respectively, the ginkalide C (GC) that 100 μ, 1 concentration is 10mg/ml is mixed and made into the mixed liquor that volume is 150 μ 1.
Preparation GB:GC (1:2) ratio component method is: get the ginkalide B (GB) that 50 μ, 1 concentration is 10mg/ml respectively, the ginkalide C (GC) that 100 μ, 1 concentration is 10mg/ml is mixed and made into the mixed liquor that volume is 150 μ 1.
Preparation GA:GB (1:4) ratio component method is: get the ginkalide A (GA) that 50 μ, 1 concentration is 10mg/ml respectively, the ginkalide B (GB) that 200 μ, 1 concentration is 10mg/ml is mixed and made into the mixed liquor that volume is 250 μ 1.
Preparation GA:GB (1:6) ratio component method is: get the ginkalide A (GA) that 50 μ, 1 concentration is 10mg/ml respectively, the ginkalide B (GB) that 300 μ, 1 concentration is 10mg/ml is mixed and made into the mixed liquor that volume is 350 μ 1.
Preparation GA:GB (1:9) ratio component method is: get the ginkalide A (GA) that 50 μ, 1 concentration is 10mg/ml respectively, the ginkalide B (GB) that 450 μ, 1 concentration is 10mg/ml is mixed and made into the mixed liquor that volume is 500 μ 1.
The external anticoagulation influence of the bilobalide component of above-mentioned different proportion proportioning:
Be the pharmacological effect index by selecting PT, APTT, TT and FIB, the bilobalide component of more different proportionings influences blood coagulation resisting function, selection optimal proportion component.At first, extract volunteer blood sample 70 ml with disposable with Syringe needle for taking samples of blood, be divided in 26 anticoagulant PV pipes, centrifugal then (3000rpm 15min), obtains supernatant blood plasma 50 ml.Blood plasma is divided into 75 groups, and each part contains blood plasma 400 μ l.Get the GA:GB:GC:BB of the 1:1:1:1 that 50 μ l prepare respectively; The GA:GB:GC of 1:1:1; The GA:GB:BB of 1:1:1; The GA:GC:BB of 1:1:1; The GB:GC:BB of 1:1:1; The GA:GB of 2:1; The GA:GC of 2:1; The GB:GC of 2:1; The GA:GB of 1:2; The GA:GC of 1:2; The GB:GC of 1:2; The GA:GB of 1:4; The GA:GB of 1:6; The different proportioning bilobalide component mixed liquors of the GA:GB of 1:9 join in the blood plasma of 400 μ 1, and the vortex mixing is to be measured.Partial thromboplastin time APTT, the thrombin time TT of prothrombin time TT, activation and the mensuration of brinase FIB are measured by analysis section of the Jiangsu Province hospitals of traditional Chinese and western medicine.The results are shown in Table 1, more excellent with ginkalide A and ginkalide B proportioning drug effect in the bilobalide component, and its controlled quantification window is when being 1:2 ~ 1:9, drug effect is better.
The external blood coagulation influence comparison of the bilobalide component of table 1 different proportion proportioning (
+ s, n=5)
Tab1．The?comparison?between?of?the?effect?of?different?proportion
ginkgolides?component?on?coagulant?system?in?vitro
Annotate: compare * P＜0.05, * * P＜0.01. with matched group
The different proportion bilobalide component that gives of the present invention is to mouse tail arterial hemorrhage time effects, and its method is as follows:
Get 160 mices, male and female half and half, be divided into 16 groups at random, every group 10, experiment is divided into: blank 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 (1:6) and GA:GB (1:9) group.By 50 μ l/g administrations, every day gastric infusion once, continuous irrigation stomach 7 days.The experiment of docking behind the 30min after the last administration.Mice is fixed in the holder, cuts off the record mouse tail continuous bleeding time with operating scissors at the terminal 1.5mm of distance mouse tail place.The results are shown in Table 2, more excellent with ginkalide A and ginkalide B proportioning drug effect in the bilobalide component, and its controlled quantification window is when being 1:2 ~ 1:9, drug effect is better.
Table 2 relatively different proportion bilobalide component to the influence of mouse tail arterial hemorrhage (
)
Annotate: compare * p＜0.05, * * p＜0.01 with matched group
Bilobalide composition solid dispersion preparation method of the present invention is as follows:
Respectively with chitosan and solid bilobalide composition dissolves in ethanol, behind the ultrasonic dissolution, with the alcoholic solution of chitosan and the alcoholic solution mixing of bilobalide compositions, continue ultrasonic dissolution, after then mixed liquor being evaporated to ethanol and volatilizing fully solid dispersion.
Detailed steps is:
Adopt solvent method to prepare bilobalide compositions-chitosan solid dispersion, respectively chitosan and solid bilobalide compositions (weight ratio of ginkalide A and ginkalide B is 1:2 ~ 9) are dissolved in the ethanol, behind ultrasonic dissolution 30 ~ 90min, with the two mixing, continue ultrasonic dissolution 30 ~ 90min.Mixed liquor is placed Rotary Evaporators, treat that ethanol volatilizes fully after, take out the solid dispersion powder.Optimizing the bilobalide compositions is 1:5 ~ 15 with the chitosan weight ratio, most preferably is 1:10, and it accumulates dissolution the best when bilobalide compositions and chitosan weight ratio are 1:10.
The method of quality control of bilobalide compositions of the present invention or solid dispersion medicine is specific as follows:
The present invention tests the content that has adopted the HPLC-ELSD method to measure ginkalide A and ginkalide B in the bilobalide composite preparation simultaneously, this method is simple, the precision that tool is good, repeatability and reliability, measure the bilobalide constituent content in 25 min simultaneously, can be used for the quality control of bilobalide component preparation.
Chromatographic condition is: Agilent SB-C18 post (4.6 * 150 mm, 5 μ m); Mobile phase: methanol-water (28: 72); 35 ℃ of column temperatures; Flow velocity 1.0 mLmin; Evaporative light scattering detector ELSD, detected parameters: 105 ℃ of evaporation mouthful temperature, 70 ℃ of atomizing mouthful temperature, 30 ℃ of drift tube temperatures, flow rate of carrier gas: 2.8 L/min.The record chromatogram, reference substance all can be realized baseline separation, measures all noiseless.
Reference substance is: ginkalide A and ginkalide B.
The preparation method of reference substance solution is:
Precision takes by weighing reference substance respectively: (1) 5.07 mg ginkalide A, (2) 6.33 mg ginkalide B, put in the brown volumetric flask of 10 mL, add dissolve with methanol and standardize solution, preparation ginkalide A and ginkalide B mass concentration are respectively the mixing reference substance stock solution of 507 and 633 μ g/mL.The accurate absorption mixed the reference substance stock solution respectively, the dilution of reuse methanol, and extension rate is respectively 2,5,10,20,50, obtains serial reference substance solution.Concentration is respectively: ginkalide A: 253.5,101.4,50.7,25.35 and 10.14 μ g/mL; Ginkalide B: 316.5,126.6,63.3,31.65 and 12.66 μ g/mL.
The preparation method of need testing solution is: take by weighing above-mentioned bilobalide compositions and make solution, with the solution filtration of gained, get subsequent filtrate as need testing solution; Perhaps take by weighing above-mentioned medicine and make solution, with the solution filtration of gained, get subsequent filtrate as need testing solution.
The present invention has designed the bilobalide quality of the pharmaceutical preparations control method based on component structure.
It is as follows that the inventive method and existing bilobalide quality of the pharmaceutical preparations control method are compared progressive:
The inventor carries out systematic analysis based on the Chinese medicine rationale to each composition of bilobalide component, finds to have very strong synergism between ginkalide A and the B.Effect is remarkable in the treatment cardiovascular and cerebrovascular disease, this method has overcome the defective of present bilobalide quality of the pharmaceutical preparations control, be to the formulation preparation process from crude drug, the dynamic control process of the multidimensional component structure of final preparations shaping, the optimization effective substance of bilobalide composite preparation treatment cardiovascular and cerebrovascular disease is ginkalide A and ginkalide B, and preferred controlled quality window is 1: 2～1: 9.Adopt the HPLC-ELSD method, measure when having realized bilobalide formulation components content, the results showed that the GA that surveys and GB separating degree are good, linear relationship, repeatability, precision, stability, the response rate are all better.Because generally there is solubility in the bilobalide component, this is likely and causes one of low big reason of bioavailability, improves bioavailability in its body by preparation formulation design of the present invention, improves drug effect.In addition, further specify the reasonability of bilobalide component quantization scale by the test of pesticide effectiveness.
Description of drawings
Fig. 1 bilobalide compositions is the equilbrium solubility in the different pH value under 37 ℃ of conditions.
Fig. 2 bilobalide compositions is the LogP value in n-octyl alcohol-buffer under 37 ℃ of conditions.
Fig. 3 is preparing carriers bilobalide component-chitosan (1:5) solid dispersion accumulation stripping curve with the different molecular weight chitosan
(a) chitosan 3000-bilobalide composition solid dispersion, ginkgolides:chitosan 5000 (1:5) SD (b) chitosan 5000-bilobalide composition solid dispersion, (c) chitosan 50 000-bilobalide composition solid dispersions, (d) chitosan 100 000-bilobalide composition solid dispersions, ginkgolides:chitosan〉100 000 (1:5) SD (e) chitosan is greater than 100 000-bilobalide composition solid dispersions, ginkgolides (f) bilobalide composition material. Data are presented as the mean ± SEM (n=3).
The dissolution curve of GA in Fig. 4 different proportion bilobalide compositions-chitosan solid dispersion.
(a) bilobalide compositions-chitosan 3000 (1:5) solid dispersion, (b) bilobalide compositions-chitosan 3000 (1:10) solid dispersion, (c) bilobalide compositions-chitosan 3000 (1:15) solid dispersion, (d) bilobalide composition material medicine. Data are presented as mean ± SEM (n=3).
The dissolution curve of GB in Fig. 5 different proportion bilobalide compositions-chitosan solid dispersion.
(a) bilobalide compositions-chitosan 3000 (1:5) solid dispersion, (b) bilobalide compositions-chitosan 3000 (1:10) solid dispersion, (c) bilobalide compositions-chitosan 3000 (1:15) solid dispersion, (d) bilobalide composition material medicine. Data are presented as mean ± SEM (n=3).
The SOD activity of Fig. 6 high dose bilobalide composition material medicine group, low dosage bilobalide composition material medicine group, matched group, ISO model group, positive drug group and high dose bilobalide composition solid dispersion group, low dosage bilobalide composition solid dispersion group.
The MDA activity of Fig. 7 high dose bilobalide composition material medicine group, low dosage bilobalide composition material medicine group, matched group, ISO model group, positive drug group and high dose bilobalide composition solid dispersion group, low dosage bilobalide composition solid dispersion group.
The LDH activity of Fig. 8 high dose bilobalide composition material medicine group, low dosage bilobalide composition material medicine group, matched group, ISO model group, positive drug group and high dose bilobalide composition solid dispersion group, low dosage bilobalide composition solid dispersion group.
Fig. 9 is for mixing the chromatogram of reference substance.
The specific embodiment
The mensuration of embodiment 1 bilobalide compositions equilbrium solubility in water and among the PBS of different pH:
The preparation need testing solution: preparation pH is respectively 2.0,2.5,5.0,5.8,6.5,7.0,7.4,7.8-8.0 phosphate buffered saline, get excessive ginkalide A: the ginkalide B weight ratio is that the bilobalide compositions equivalent of 1:2 is divided into 10 parts and places tool plug teat glass respectively, add water as blank to one of them tool plug teat glass then, in other tool plug teat glasses, add the different phosphate buffered saline of pH value respectively, ultrasonic no longer the dissolving to medicine made bilobalide compositions saturated solution, put into the constant temperature oscillator temperature then and keep (37 ± 1) ℃, jolting 24 h.Saturated solution with 0.45 μ m filtering with microporous membrane, is discarded filtrate just, get subsequent filtrate as need testing solution.
The preparation reference substance solution: precision takes by weighing reference substance respectively: (1) 5.07 mg ginkalide A, (2) 6.33 mg ginkalide B, put in the brown volumetric flask of 10 mL, add dissolve with methanol and standardize solution, preparation ginkalide A and ginkalide B mass concentration are respectively the mixing reference substance stock solution of 507 and 633 μ g/mL.The accurate absorption mixed the reference substance stock solution respectively, the dilution of reuse methanol, and extension rate is respectively 2,5,10,20,50, obtains serial reference substance solution.Concentration is respectively: ginkalide A: 253.5,101.4,50.7,25.35 and 10.14 μ g/mL; Ginkalide B: 316.5,126.6,63.3,31.65 and 12.66 μ g/mL.
Chromatographic condition is: Agilent SB-C18 post (4.6 * 150 mm, 5 μ m); Mobile phase: methanol-water (28: 72); 35 ℃ of column temperatures; Flow velocity 1.0 mLmin; Evaporative light scattering detector ELSD, detected parameters: 105 ℃ of evaporation mouthful temperature, 70 ℃ of atomizing mouthful temperature, 30 ℃ of drift tube temperatures, flow rate of carrier gas: 2.8 L/min.The record chromatogram, reference substance all can be realized baseline separation, measures all noiseless.
The test sample of above-mentioned preparation and the sample size of reference substance are 10 μ L; Writing times 30 min.
2 kinds of effective ingredient in the bilobalide compositions are measured, calculated its equilbrium solubility, the results are shown in Table 1 and Fig. 1 (the bilobalide compositions is the equilbrium solubility in the different pH value under 37 ℃ of conditions).
Table 1 bilobalide compositions is the equilbrium solubility in the different pH value under 37 ℃ of conditions
The result: the bilobalide composition dissolves is poor.Therefore, when considering preparation bilobalide composite preparation, it is main must improving its dissolubility, to improve its bioavailability.
Get an amount of bilobalide composition dissolves in by water saturated n-octyl alcohol, make the n-octyl alcohol saturated solution of bilobalide compositions, accurate this solution of drawing places 10 10 mL cillin bottles (2mL/ bottle) respectively, in one of them cillin bottle, add the saturated water of 2ml n-octyl alcohol as blank, add the phosphate buffered solution of the different pH value of 2 mL in other cillin bottles respectively, put into 37 ℃ of Air oscillators, jolting 24 h are until balance, take off a layer water, with 6000 rmin
-1Centrifugal 10 min prepare reference substance solution and measure bilobalide composition concentration C in the preceding n-octyl alcohol of distribution according to embodiment 1 described method and the chromatographic condition for preparing reference substance solution
0Bilobalide composition concentration C with distributing the back aqueous phase calculates apparent profit partition coefficient according to formula.Computing formula is as follows:
P
app?=?(C
0V
0?-?CV)?/CV
P in the following formula
AppBe the apparent oil water partition coefficient; C
0Be initial concentration (the μ gmL of index components in n-octyl alcohol
-1); V
0Be water saturated n-octyl alcohol volume (being 2mL); When being partition equilibrium, C records concentration (the μ gmL of index components at aqueous phase
-1); V is water volume (being 2mL).The results are shown in Table 2 and Fig. 2 (the bilobalide compositions is the LogP value in n-octyl alcohol-buffer under 37 ℃ of conditions).
Table 2 bilobalide compositions is the logP value in n-octyl alcohol-buffer under 37 ℃ of conditions
The result: the LogP value in the n-octyl alcohol-buffer of bilobalide compositions under different pH illustrates its permeate well all more than 3.In conjunction with the embodiments 1 and embodiment 2, can obtain the bilobalide compositions and can be classified as the 3rd class component according to the biopharmaceutics classification, be i.e. low-solubility, high osmosis medicine.The preparation design should improve at this character.
Method: adopt solvent method to select chitosan as the solid dispersion carrier, at first prepare ginkalide A: the ginkalide B part by weight is that bilobalide compositions and 3000,5000,50000, the 100000 chitosan ratios that reach greater than 100000 of 1:2 are 1:5, relatively different molecular weight is to the influence of bilobalide composition dissolves degree, the chitin carrier of preferred optimum weight.Further optimizing part by weight (1:5,1:10,1:15) the preparation bilobalide solid dispersion of bilobalide compositions and preferred chitin carrier again, is the prescription of investigating index optimization bilobalide solid dispersion with the dissolution.The results are shown in Figure 3-5.
Result: compare the dissolubility that chitosan 3000 can at utmost improve the bilobalide compositions with other molecular weight chitosans, and the solid dispersion of investigation different proportion bilobalide compositions and chitosan, find that bilobalide compositions and chitosan part by weight 1:10 are optimum prescription, the accumulation dissolution of bilobalide component can reach 90% in the 20min.
The sign of embodiment 4 bilobalide composition solid dispersion formulations
Method: the drug effect difference of utilizing SOD, MDA and LDH biochemical indicator investigation comparison bilobalide composition material medicine high dose group and low dose group, bilobalide composition solid dispersion (GSD) high dose group and low dose group, positive drug control group.
Bilobalide composition material medicine: ginkalide A and ginkalide B mix by weight 1:2.
Bilobalide composition solid dispersion (GSD): respectively chitosan 3000 and bilobalide compositions (ginkalide A and ginkalide B weight ratio are 1:2) are dissolved in the ethanol, behind the ultrasonic dissolution 45min, with the alcoholic solution of chitosan and the alcoholic solution mixing of bilobalide compositions, continue ultrasonic dissolution 45min, after mixed liquor is evaporated to ethanol and volatilizes fully solid dispersion.The weight ratio of wherein said bilobalide compositions and chitosan 3000 is 1:10.
Positive control drug: Pu Nuonaier.
The ISO modeling method: continuous 5 days to rats by intraperitoneal injection isoproterenol hydrochloride inj (Isoprenaline Hydrochloride injection, ISO) modeling method of ISO 10mg/kg mode can obtain the rat model of acute myocardial ischemia safely.
The blank group: normal diet, after the 5th day, continuous irrigation stomach normal saline 7 days.
ISO model group: modeling according to the method described above, normal diet.
Positive drug control group: modeling according to the method described above, continuous irrigation stomach 2mg/kg Pu Nuonaier 7 days.
High dose bilobalide composition solid dispersion group (High dose GSD): modeling according to the method described above, continuous irrigation stomach high dose (50mg/kg is equivalent to bilobalide composition material dose) bilobalide solid dispersion 7 days.
Low dosage bilobalide composition solid dispersion group (Low dose GSD): modeling according to the method described above, continuous irrigation stomach low dosage (10mg/kg is equivalent to bilobalide composition material dose) bilobalide solid dispersion 7 days.
High dose bilobalide composition material medicine group (High dose GK): modeling according to the method described above, continuous irrigation stomach high dose 50mg/kg bilobalide composition material medicine 7 days.
Low dosage bilobalide composition material medicine group (Low dose GK): modeling according to the method described above, continuous irrigation stomach low dosage 10mg/kg bilobalide composition material medicine 7 days.
Prepared bilobalide composition solid dispersion release unit, in order to prove that the said preparation technology has effectively improved its drug effect, this problem is at first set up ISO rat heart muscle ischemia model, by SOD, MDA and LDH biochemical indicator, by than higher dosage bilobalide composition material medicine group, low dosage bilobalide composition material medicine group, the blank group, the ISO model group, positive drug group and high dose bilobalide composition solid dispersion group, the drug effect difference of low dosage bilobalide composition solid dispersion group, experimental result shows, bilobalide has concentration dependent for the drug action of rats with myocardial ischemia, and its solid dispersion pharmacodynamics effect obviously is better than the pharmacological effect of bilobalide composition material medicine.Rat to injection ISO modeling myocardial ischemia, bilobalide composition solid dispersion group (GSD) back and the ISO model group of administration high and low dose group compare, it is measured the SOD value and obviously raises, the MDA value obviously reduces, the LDH value also obviously descends, the bilobalide composition material medicine group of its intensity of variation and high dose and low dosage relatively also obviously is better than its end value.The results are shown in Figure 6-8.
The assay of ginkalide A and ginkalide B in embodiment 5 bilobalide composition material medicines and the bilobalide composition solid dispersion
1. instrument and reagent
The Waters high performance liquid chromatograph comprises 600 quaternary gradient pumps, 717 automatic samplers, ELSD2000E detector.
Reference substance: ginkalide A (lot number 110862) is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute; Ginkalide B (lot number 110863) purity is equal〉98%.
Test sample: bilobalide composition material medicine (ginkalide A: the ginkalide B weight ratio is 1:2); Bilobalide composition solid dispersion (carrier is chitosan 3000, and the weight ratio of bilobalide compositions and chitosan 3000 is 1:10, and ginkalide A and ginkalide B weight ratio are 1:2 in the bilobalide compositions).
Methanol (chromatographically pure, Hanbon Sci. ﹠ Tech. Co., Ltd.), Milli-Q ultra-pure water.All the other reagent are analytical pure.
2. chromatographic condition
Chromatographic column: Agilent SB-C18 post (4.6 * 150 mm, 5 μ m); Mobile phase: methanol-water (28: 72); 35 ℃ of column temperatures; Flow velocity 1.0 mL/min; Evaporative light scattering detector ELSD, detected parameters: 105 ℃ of evaporation mouthful temperature, 70 ℃ of atomizing mouthful temperature, 30 ℃ of drift tube temperatures, flow rate of carrier gas: 2.8 L/min.Writing times 30 min.
3. experimental technique and result
3.1 the preparation of need testing solution
Take by weighing 1.32 mg bilobalide composition material medicines and 2.47 mg bilobalide composition solid dispersions, put respectively in the 25mL measuring bottle, with dissolve with methanol and standardize solution, sample filters, get subsequent filtrate, namely get need testing solution, be used for measuring the bilobalide composition levels.
3.2 the preparation of reference substance solution: prepare reference substance solution according to embodiment 1 described method.
3. content assaying method
Accurate reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid respectively, measure, and the results are shown in Table 3-5.In preparation process design and preparation process, the change of minor swing appears in bilobalide compositions structure proportion, and the part material loss is arranged, but its compositions structure proportioning value control further specifies essence of the present invention in the controlled range window.
The content (mg/g) of ginkalide A and ginkalide B in the table 3 bilobalide composition solid dispersion
Annotate: the testing result of bilobalide composition solid dispersion is the result of calculation behind the removal chitosan.
Material loss amount proportion (%) behind table 4 preparations shaping
Bilobalide compositions structure proportion value before and after table 5 preparation
1. linear relationship
Precision takes by weighing reference substance 5.07 mg ginkalide As respectively, 6.33 mg ginkalide B, put in the brown volumetric flask of 10 mL, add dissolve with methanol and standardize solution, preparation ginkalide A and ginkalide B mass concentration are respectively the conduct mixing reference substance stock solution of 507 and 633 μ g/mL.The accurate absorption mixed the reference substance stock solution respectively, the dilution of reuse methanol, and extension rate is respectively 2,5,10,20,50, obtains serial reference substance solution.Concentration is respectively: ginkalide A: 253.5,101.4,50.7,25.35 and 10.14 μ g/mL; Ginkalide B: 316.5,126.6,63.3,31.65 and 12.66 μ g/mL; Each concentration reference substance solution is sample introduction 10 μ L respectively.Measure peak area with HPLC-ELSD, lnM makes linear regression with the sample size logarithm of peak area logarithm lnA, gets regression equation, sees Table 6.
Table 6 linear relationship experimental result
2. precision test
3 variable concentrations reference substances of accurate absorption, containing GA and GB concentration respectively is 50.7,63.3; 25.3,31.6; 12.7,15.8 μ gmL
-1, to press the described chromatographic condition of embodiment 1 and measure, RSD is in a few days calculated in every kind of concentration determination 5 times; METHOD FOR CONTINUOUS DETERMINATION 5 d calculate RSD in the daytime.The result: its in a few days RSD be respectively 0.96%, 1.53%, 0.78%; In the daytime RSD is respectively 2.81%, 1.79%, 0.75%.
3. repeated experiment
Get 6 parts in bilobalide composition material medicine sample, prepare need testing solution and analysis according to embodiment 5 described test sample preparation methoies and chromatographic condition, the RSD of ginkalide A and ginkalide B chromatographic peak area is respectively 2.25% and 2.92% as a result.
4. stability experiment
Get " replica test " item bilobalide composition material medicine need testing solution down, respectively at 0,2,4,8,12,24 h measure, and calculate the RSD of peak area, are respectively 2.14 % and 2.72%.
5. average recovery test
Precision takes by weighing 9 parts in same bilobalide composition material medicine sample, every part of about 15 mg add respectively in the 25mL measuring bottle, be divided into 3 groups, (content of ginkalide A is about 1.40,1.75,3.50 mg respectively in the reference substance solution of adding in each measuring bottle to add a certain amount of reference substance solution respectively in 3 measuring bottles of every group, the content of ginkalide B is about 2.87,3.51 respectively, 7.08mg), with dissolve with methanol and standardize solution, sample filters, get subsequent filtrate, namely get need testing solution.Measure according to embodiment 5 described chromatographic conditions, sample introduction 20 μ L analyze, and calculate the average average recovery of each reference substance, and ginkalide A 102.8% as a result, and RSD is 2.54%; Ginkalide B is that 100.1%, RSD is 2.59%.
To sum up, the method based on the quality control of bilobalide component solid dispersion that the present invention sets up has certain effect experiment basis, can more effective, more fully control the quality of bilobalide component preparation.
Claims (10)
1. a bilobalide compositions is characterized in that described compositions comprises ginkalide A and two kinds of active components of ginkalide B, and the weight ratio of ginkalide A and ginkalide B is 1:2 ~ 9.
2. bilobalide compositions according to claim 1 is characterized in that content 〉=90% of ginkalide A and ginkalide B in the compositions.
3. medicine that is used for the treatment of cardiovascular and cerebrovascular disease, it is characterized in that this medicine is main effective ingredient with any described bilobalide compositions in the claim 1 ~ 2, add that acceptable accessories or complementary composition make pharmaceutically the dosage form that allows.
4. medicine according to claim 3 is characterized in that described dosage form is pill, powder, capsule, tablet, granule, injection, compound preparation or solid dispersion.
5. want 4 described medicines according to right, it is characterized in that described dosage form is solid dispersion.
6. want 5 described medicines according to right, it is characterized in that described solid dispersion is is main effective ingredient with described bilobalide compositions, be that carrier is made with the chitosan, the weight ratio of described bilobalide compositions and carrier is 1:5 ~ 15, is preferably 1:10.
7. want 6 described medicines according to right, the preparation method that it is characterized in that described solid dispersion is: respectively with chitosan and bilobalide composition dissolves in ethanol, behind ultrasonic dissolution 30 ~ 90min, with the alcoholic solution of chitosan and the alcoholic solution mixing of bilobalide compositions, continue ultrasonic dissolution 30 ~ 90min, after mixed liquor is evaporated to ethanol and volatilizes fully solid dispersion.
8. want 6 or 7 described medicines according to right, it is characterized in that described chitosan is chitosan 3000.
9. the application of the described bilobalide compositions of claim 1 in preparation treatment cardiovascular and cerebrovascular diseases medicament.
10. the method for quality control of the described bilobalide compositions of claim 1 or the described medicine of claim 3 is characterized in that may further comprise the steps:
(1) preparation reference substance solution: precision takes by weighing reference substance ginkalide A and ginkalide B respectively, use dissolve with methanol, obtain serial reference substance solution, ginkalide A and ginkalide B concentration range are respectively in the serial reference substance solution: ginkalide A 10.14 ~ 253.5 μ gmL
-1Ginkalide B 12.66 ~ 316.5 μ gmL
-1
(2) preparation need testing solution: take by weighing the solution that the described bilobalide compositions of claim 1 is made, with the solution filtration of gained, get subsequent filtrate as need testing solution; Perhaps take by weighing the described medicine of claim 3 and make solution, with the solution filtration of gained, get subsequent filtrate as need testing solution;
Chromatographic condition is: Agilent SB-C18 post (4.6 * 150 mm, 5 μ m); Mobile phase: methanol-water (28: 72); 35 ℃ of column temperatures; Flow velocity 1.0 mLmin; Evaporative light scattering detector ELSD, detected parameters: 105 ℃ of evaporation mouthful temperature, 70 ℃ of atomizing mouthful temperature, 30 ℃ of drift tube temperatures, flow rate of carrier gas: 2.8 L/min.
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| CN103638017A (en) * | 2013-12-11 | 2014-03-19 | 江西本草天工科技有限责任公司 | Composition of ginkgolide A and ginkgolide B and application of composition |
| CN104140465A (en) * | 2013-12-27 | 2014-11-12 | 江苏康缘药业股份有限公司 | Monoclonal antibody and application thereof, kit and Dot-ELISA method for detecting content of water-soluble proteins in ginkgo leaf |
| CN109883987A (en) * | 2019-01-16 | 2019-06-14 | 江苏康缘药业股份有限公司 | A kind of content assaying method of ginkalide A or ginkolide B |
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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 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103638017A (en) * | 2013-12-11 | 2014-03-19 | 江西本草天工科技有限责任公司 | Composition of ginkgolide A and ginkgolide B and application of composition |
| CN104140465A (en) * | 2013-12-27 | 2014-11-12 | 江苏康缘药业股份有限公司 | Monoclonal antibody and application thereof, kit and Dot-ELISA method for detecting content of water-soluble proteins in ginkgo leaf |
| CN104140465B (en) * | 2013-12-27 | 2016-08-10 | 江苏康缘药业股份有限公司 | Monoclonal antibody and application, test kit, the Dot-ELISA method of detection Folium Ginkgo water-soluble protein content |
| CN109883987A (en) * | 2019-01-16 | 2019-06-14 | 江苏康缘药业股份有限公司 | A kind of content assaying method of ginkalide A or ginkolide B |
| CN109883987B (en) * | 2019-01-16 | 2021-07-27 | 江苏康缘药业股份有限公司 | Method for measuring content of ginkgolide A or ginkgolide B |
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