CN1887282A - Medicine composition containing bailobalide - Google Patents
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Abstract
The present invention discloses bailobalide injection with mixed solvent comprising common solvents alcohol, glycerin and water normally for injection and citric acid for regulating pH value to 2.5-6.5. The bailobalide injection has certain ratio of bailobalide and high long-term stability.
Description
Technical field
The present invention relates to a kind of preparation of bilobalide-containing, particularly bilobalide injection, the preparation technology of this injection also is provided simultaneously, belong to field of medicinal compositions.
Background technology
Bilobalide is to extract the active substance that obtains in the middle of the Chinese herbal medicine Folium Ginkgo, bilobalide is two terpene compounds, be to comprise 1 oxolane ring, 3 r-lactone group and be attached at the spiral shell [4 that forms on 1 carbon atom by 2 penta carbocyclic rings, 4] nonane system, the C20 cage of the uniqueness that combines with the tert-butyl group of totally 6 penta carbocyclic rings covers molecule.Its cis condensed ring F, A, D and C are fashioned into hemispheric cavity, and this cavity is to be enough to hold 1 cation or positive polar organic molecule.
The known bilobalide of P.Braquet etc. (1988) report has 5 kinds, name (Ginkgolides A, B, C, M, J) with A, B, C, M, J, the basic structure of bilobalide is common, and the difference between the different bilobalides only is number and position different of hydroxyl.Bilobalide M and J are actually the isomer of ginkalide B.Ginkalide A .B.C both had been present in the Cortex Ginkgo, also was present in the Folium Ginkgo, and bilobalide M exists only in the root bark; Bilobalide J is found later, exists only in the Folium Ginkgo.The chemical property torpescence of bilobalide is very stablely (to see Zheng Weiping, the research overview of the brilliant bilobalide of building phoenix.1999 23 the 2nd phases of volume of pharmacy progress, the 82-86 page or leaf).
Extracting method for bilobalide has had detailed bibliographical information, for example, people such as Li Xingang have introduced the laboratory extracting method of bilobalide and (have participated in Li Xin hilllock etc., the laboratory Study on Extraction Method of bilobalide in the Folium Ginkgo, the 1st phase of Chinese Journal of Pharmaceuticals 1998), Chinese patent literature CN1195665A has introduced the extracting method of bilobalide and has contained the preparation of bilobalide, this method is that Folium Ginkgo is extracted with water boil, with adsorbent extraction filtrate is adsorbed, reuse ethanol desorption reclaims ethanol, with the crystallization dissolving of separating out, recrystallization, drying makes bilobalide.Chinese patent literature CN1313287A discloses a kind of production technology of bilobalide.The method of separating ginkgolide monomer in the middle of the total extract of bilobalide, a lot of relevant bibliographical informations are also arranged, for example, people such as You Song have introduced the separation of bilobalide in the bilobalide and structure determination method (referring to You Song etc., the separation of bilobalide and structure determination in the Folium Ginkgo, Chinese pharmaceutical chemistry magazine the 4th phase of nineteen ninety-five).Chinese patent literature CN1287121A discloses the method that is prepared medicine ginkalide A, B by Folium Ginkgo or Folium Ginkgo extractum.Bilobalide can be prepared into it different clinical medicine dosage forms as an effective active substance.Use Folium Ginkgo injections such as Liu Jie treatments acute cerebral infarction obtained certain curative effect [referring to Liu Jie etc., YINXINGYE ZHUSHEYE to acute cerebral infarction after the influence of extremity motor function and TXB-2,6-Keto-PGF-(la)].About the pharmacological action of bilobalide, comparatively detailed bibliographical information (referring to Chen Weijun etc., the chemical constitution of bilobalide and Advance on Pharmacological Activities, 1998 the 9th phases of Chinese Pharmaceutical Journal) has been arranged.Although the research for bilobalide and preparation thereof in the middle of the prior art has had a lot of reports,, never stop for the research of bilobalide, particularly, bibliographical information is arranged constantly for the separation of ginkgolide monomer and the research of derivant thereof.Present research for effective monomer in the middle of the bilobalide, structures such as ginkalide A, B, C have been reported, bilobalide is prepared into injection, a very important technical problem is how to solve its difficult dissolubility, up to the present, in the middle of the bilobalide injector preparation process, also extremely important for the selection of cosolvent, this directly has influence on the stability and the clinical efficacy of product.
According to the lactonic ring structure of bilobalide, generally be easy to expect making open loop with acid or alkali, perhaps use organic solvent dissolution, but its dissolubility in general organic solvent is also very poor, as ethanol, PEG400, glycerol, dissolubility is all very little in the propylene glycol.So at present the bilobalide method for preparing injection has the dissolubility by using aqueous slkali to increase bilobalide to make, CN200510040497.0 for example, CN00115134.7, CN02134223.7.Add under the situation of meglumine, bilobalide is had for example CN02128962.X of good hydrotropy effect.Bilobalide is adopted for example CN200510072466.3 of aqueous solution that the HP-solubilising makes, CN200410013937.9, CN200410041120.2.In addition, contain 5-99% Folium Ginkgo extract, 1-95% poloxamer 188 and pH value regulator and other excipient in the open injection of CN03100334.6.
In these methods, add HP-, poloxamer, reagent such as Polyethylene Glycol, its safety as injection is worth discussion.HP-has potential nephrotoxicity, only uses at present in the antifungal agent Fu Likang azoles injection, does not have as legal injection additives in China.Poloxamer is not because safety issue has as legal injection additives in China yet.Polyethylene Glycol concentration has hemolytic danger when big.So there is not practical possibility substantially.Add alkali or add meglumine and all belong to and allow lactone open loop salify, under neutrality or acid condition, be difficult to be formed with the lactone original shape of drug effect form, even do not have bilobalide.And bilobalide is a chemical constituent of greatest concern in the present Semen Ginkgo, and animal experiment report, bilobalide have the effect that promotes nerve growth, and prevent brain, spinal nerves demyelination, and its neurotrophy, neuroprotective are stronger than bilobalide.Find also that in recent years bilobalide can prevent the changing function that brain cell mitochondrion oxidative stress causes, the seed selection anti-oxidation stress is to improving senile memory, useful effect is played in the generation and the development that prevent alzheimer disease, therefore at present be that content of bilobalide should be 2.6-3.2% to the requirement (specification that German Ministry of Public Health Commission E works out) of Semen Ginkgo extrac in the world.
In order to solve validity problem.The invention discloses a kind of prescription and compound method of bilobalide injection, can make ginkgo lactone material and the bilobalide injection thrown, each component and ratio remain unchanged.Because the basic structure of ginkalide A, B, C is common, the difference between the different bilobalides only is number and position different of hydroxyl, but its drug effect (paf receptor antagonists) but is not quite similar; Bilobalide has the effect that promotes nerve growth.In order to keep each component and ratio to remain unchanged, the invention discloses a kind of mixed solvent of forming with the common solvent of injection.Solvent cost is cheap, and processing technology is simple, the more important thing is to have solved safety and validity problem.Can make ginkgo lactone material and the bilobalide injection thrown, each component and ratio remain unchanged.And it is approaching with the component ratio in the Folium Ginkgo.And the material that is surprised to find that pH value and adjusting PH is crucial.
Summary of the invention
Be surprised to find that the mixed solvent of forming with the common solvent of several injection, can make the bilobalide dissolving and can make bilobalide injection.Transfer pH value to 2.5 ~ 6.5 with citric acid.This mixed solvent makes that the ratio of each composition and each composition is consistent with the bilobalide extract of being thrown in the injection.And can in for a long time, keep stable.Once test the material of different pH values and adjusting PH, comprised hydrochloric acid, phosphoric acid, tartaric acid, acetic acid, sulphuric acid, succinic acid, malic acid etc.Put for a long time and can cause that all pH value changes, finally each composition ratio changes.
The purpose of this invention is to provide a kind of preparation that contains bilobalide, particularly bilobalide injection, the preparation technology of this injection also is provided simultaneously.
The preparation of bilobalide of the present invention is to be that active ingredient is prepared from highly purified bilobalide, ginkalide A, and ginkalide B, ginkalide C, the bilobalide sum is greater than more than 80% (weight ratio) of total extract.
The weight ratio of each ingredient of injection of the present invention is:
Bilobalide: ethanol: glycerol: water=(0.001% ~ 0.15%): (1% ~ 80%): (1% ~ 80%): (15% ~ 99%)
The preferred weight ratio of above-mentioned component is:
Bilobalide: ethanol: glycerol: water=(0.05 ~ 0.15): (20 ~ 70): (10 ~ 40): (20 ~ 70)
The preferred weight ratio of above-mentioned component is:
Bilobalide: ethanol: glycerol: water=(0.05 ~ 0.15): (30 ~ 50): (10 ~ 30): (30 ~ 50)
Wherein also need add 10% an amount of citric acid solution and regulate pH value.
Preparation prescription
Prescription (in 1000 dosage units):
Bilobalide 10g
Glycerol 400ml
Ethanol 800ml
Water for injection adds to 2000ml
Make 1000
Preparation method
Get the water for injection of recipe quantity about 80%, the glycerol and the ethanol of prescription full dose are put in the dosing cylinder, add the bilobalide of recipe quantity, after the stirring and dissolving, add to the full amount of water for injection, regulate pH to 4.0 with 10% citric acid solution, stir evenly, after the inspection of semifinished product is qualified, clear and bright with the mocromembrane filtration system filters that 0.65 μ m and the compound anti-pure microporous filter membrane of 0.45 μ m are made filter material, embedding is in 2ml glass bottle, in 100 ℃ of sterilizations 20 minutes, cooling, lamp inspection, decals, promptly.
Prepare the employed ginkgo lactone material of ester injection in the bilobalide Fructus Pruni of the present invention, can extract by known extracting method, perhaps buy, but the ratio of ginkalide A, B, C and bilobalide should be unanimous on the whole with the ratio in the Folium Ginkgo by commercially available.
Content assaying method with the HPLC-ELSD method measure 4 kinds of terpene lactones in the Folium Ginkgo (see Yan Yuzhen, Xie Peishan. pharmaceutical analysis magazine calendar year 2001 21 volumes 3 phase 173-176 pages or leaves)
From the HPLC collection of illustrative plates of the ginkgo lactone material that fed intake and gained finished product as seen: the ratio of each composition of ginkalide A, B, C and bilobalide and each composition does not change.(seeing accompanying drawing one, two)
The preliminarily stabilised test
Imitative commercially available back, place (8～32 ℃ of temperature down in normal temperature laboratory, relative humidity 60%～90%), after sample was investigated once this month, respectively at January, February, March, June, December, sampling in 18 months by investigate (wherein aseptic, haemolysis, zest were only carried out at 0,3 and 18 month) from " the bilobalide injection quality standard (small-volume injection) (draft) " intended.The result is all up to specification, illustrates that bilobalide injection of the present invention is basicly stable.
Bilobalide injection Pharmacodynamic test of active extract data
For the reagent product
Bilobalide injection,, bilobalide injector adjuvant solution (abbreviation blank solution), Breviscapini injection, specification: 5mg/2mL is Gejiu Bio-Pharmaceutical Co., Ltd., Yunnan's product.
1 antiplatelet aggregation test
Platelet rich plasma (platelet-rich plasma, PRP) and platelet poor plasma (platelet-poor plasma, preparation PPP): get healthy rabbits, body weight 2 ~ 3kg, male and female are all used.Get blood from clear-headed rabbit carotid artery and be collected in the plastic centrifuge tube, with 3.8% sodium citrate anticoagulant (blood and anticoagulant volume ratio 9: 1).The centrifugal 10min of 900rpm under the room temperature gets PRP, and sucking-off PRP is in clean plastic tube; Residue blood gets PPP with the centrifugal 10min of 3000rpm again.PPP is used for returning to zero or adjusts the number of platelets of PRP, and platelet count is controlled at 5 * 10 in the process of the test
11Cell/L.
1. bilobalide injection is in external influence to platelet aggregation
Bilobalide injection is standby with the medicinal liquid that normal saline is diluted to variable concentrations with blank solution, Breviscapini injection, get 10 μ L medicinal liquids or blank solution respectively in PRP, 37 ℃ of incubation 10min.Press Born turbidimetry (Born GVR.Aggregation of blood platelets by adenosinediphosphate and its reversal.Nature, 1962,194:927-929), after being PPP zeroing and PRP amplitude modulation, the PAF (final concentration 7.2nmol/L) that adds 5 μ L measures platelet aggregation and writes down maximum platelet aggregation rate in the 5min on platelet aggregation instrument.Platelet aggregation inhibition rate is calculated as follows, and obtains half-inhibition concentration (50%inhibitoryconcentration, the IC of medicine with return law of the straight line
50).
Platelet aggregation inhibition rate (%)=[1-(control tube is assembled percentage rate/delivery tube and assembled percentage rate)] * 100
The result shows that bilobalide injection is concentration dependent and obviously suppresses the inductive platelet aggregation of PAF, its IC
50Be 9.4mg/L, Breviscapini injection IC
50Be 214.8mg/L (table 1,2).Show that bilobalide injection suppresses the inductive platelet aggregation of PAF, significantly is better than Breviscapini injection.
Table 1. bilobalide injection is in external influence to the inductive platelet aggregation of PAF
| Medicine dense (mg/L) | Platelet aggregation rate (%) |
| 0 3.125 6.25 12.5 25 50 | 62.3±3.8 55.2±3.7 ** 44.1±4.3 ** 24.5±5.4 ** 17.8±4.1 ** 8.4±2.9 ** |
N=5, x ± s,
*(Student t-check) compared with the blank solution group in P＜0.01
Table 2. Breviscapini injection is in external influence to the inductive platelet aggregation of PAF
| Medicine dense (mg/L) | Platelet aggregation rate (%) |
| 0 31.25 62.5 125 250 500 | 64.2±5.7 57.2±6.2 50.3±4.1 ** 38.1±5.3 ** 25.1±6.0 ** 18.6±4.7 ** |
N=5, x ± s,
*Compare with the blank solution group P＜0.01. and (Student t-check)
2 bilobalide injections are in vivo to the influence of platelet aggregation
30 of healthy rabbits, body weight 2.0 ~ 2.5kg, male and female are usefulness all, is divided into 5 groups at random, and 6 every group, i.e. the Breviscapini injection group of the bilobalide injection group of equal-volume blank solution group, 2.0,4.0,8.0mg/kg and 13.2mg/kg.Above-mentioned each component all with the 5.0mL/kg body weight through the auricular vein drug administration by injection.Get blood before the administration once, after the administration 10,30,60,90 and 120min get blood respectively, prepare PRP and PPP with external test method, observe after the medicine intravenous injection influence to PAF induced platelet aggregation capability.
Experimental result shows, after the bilobalide injection intravenous injection, when dosage is 2.0mg/kg the inductive platelet aggregation of PAF there is not obvious inhibitory action, 4.0mg/kg the time, obviously suppress the platelet aggregation that PAF causes in injection back 30 and 60min, during 8.0mg/kg, in the 10min onset of injection back, reach maximum inhibitory action in 60min, anti-PAF effect continues to 90min; 13.2mg/kg the bilobalide injection of the effect characteristics of the Breviscapini injection of (clinical dosage 40 times) and 8.0mg/kg (clinical dosage 20 times) is similar.Injection back 60min, the anti-PAF action intensity of 8.0mg/kg (clinical dosage 20 times) bilobalide injection is apparently higher than 13.2mg/kg (clinical dosage 40 times) Breviscapini injection group (table 3).
The intravenous injection of table 3. bilobalide injection is to the influence of the inductive platelet aggregation of PAF
| Group | Dosage (mg/ kg) | Clinical dose multiple | Platelet aggregation rate (%) | |||||
| 0min | 10min | 30min | 60min | 90min | 120min | |||
| The blank solution bilobalide injection | Equal-volume 2.0 4.0 8.0 | 5 10 20 | 61.7± 3.1 64.7± 3.7 61.2± 3.2 63.0± | 65.9± 2.7 63.3± 2.0 58.8± 2.5 53.6± | 66.6± 4.5 63.9± 3.1 51.4± 4.1 * 46.9± | 62.0± 2.6 63.5± 3.8 47.7± 3.2 * 31.4± | 64.5± 4.4 61.0± 2.5 57.2± 2.7 49.2± | 62.6± 4.0 62.3± 2.4 59.4± 2.7 59.6± |
| Breviscapini injection | 13. 2 | 40 | 3.6 65.0± 3.8 | 3.2 ** 54.9± 5.7 * | 4.5 ** 49.7± 2.6 ** | 10.1 **# 42.5± 4.9 ** | 4.1 ** 51.6± 3.4 ** | 3.1 61.0± 3.5 |
N=5, x ± s,
*P＜0.05,
*P＜0.01, with the blank solution group or the 0min comparison of equi-time point,
#(Student t-check) compared with Breviscapini injection in P＜0.05.
3 pairs of thrombotic influence tests of electricity irritation rat carotid artery
50 of male SD rats, body weight 190 ~ 230g is divided into 5 groups at random, and 10 every group, i.e. the Breviscapini injection group of the bilobalide injection group of blank solution group (equal-volume), 2.0,4.0,8.0mg/kg and 13.2mg/kg.Above-mentioned each component is all with 0.2mL/100g body weight lumbar injection (ip), 1 time/d, totally 2 times, behind last administration 30min, use method (CharltonPA such as Charlton, Faint RW, Bent F, Bryans J, Chicarelli-Robinson I, Mackie I, MachinS, Bevan P.Evaluation of a low molecular weight modulator of humanplasminogen activator inhibitor-1 activity.Thrombosis and Haemostasis, 1996,75 (5): 808-15) also improved, promptly use 30mg/kg pentobarbital sodium intraperitoneal injection of anesthesia rat, separate left common carotid artery, measure the normal blood flow amount; Medicine again with the 0.2mL/100g body weight through vena femoralis injection, the rearmounted two silvery electrodes of 10min are in the blood vessel two ends, spacing 0.5cm, electrode and blood vessel be lining one insulation film down, pass to 1.5mA unidirectional current continued stimulus 7min, use 5MHz point type ultrasonic probe simultaneously, the blood flow of continuous measurement stimulation location distal end.Beginning to blood flow from stimulation is zero interval, and (occlusion time, OT), this is a thrombus formation time to represent the blood vessel embolism time.If blood vessel is still open in the 60min, then with 60min as the record terminal point.Experimental data Student t-check row statistical procedures.
The result shows, after the bilobalide injection intravenous injection, is the thrombus formation time that dosage correlation obviously prolongs the electricity irritation rat carotid artery; Suitable (table 4) of thrombus formation time of (13.2mg/kg clinical dosage 40 times) Breviscapini injection group and 8.0mg/kg (clinical dosage 20 times) bilobalide injection group.
The intravenous injection of table 4. bilobalide injection is to the thrombotic influence of electricity irritation male rat carotid artery
| Group | Dosage (mg/kg) | The multiple of clinical dosage | Thrombus formation time (OT, min) |
| Blank solution bilobalide injection Breviscapini injection | Equal- | 5 10 20 40 | 18.5±2.9 19.9±2.9 23.3±2.5 **# 35.9±8.8 ** 33.7±11.9 ** |
N=10, x ± s,
*Compare with the blank solution group P＜0.01,
#Compare with the Breviscapini injection group P＜0.01
(Student t-check).
The thrombolytic test of 4 bilobalide injections
The preparation of thrombus model
50 of male SD rats, body weight 240 ~ 280kg is divided into 5 groups at random, and 10 every group, promptly isopyknic blank solution group, 13.2mg/kg Breviscapini injection group, 2.0,4.0 and the bilobalide injection group of 8.0mg/kg.Improve methods such as Charlton, promptly use the pentobarbital sodium ip anesthetized rat of 30mg/kg, separate the rat left common carotid artery, put two silvery electrodes (spacing 0.8cm), ultrasonic probe is put distal end.With the direct current continued stimulus rat carotid artery 5min of electrostimulator with 2mA.With the two-way blood flowmeter continuous probe of ultrasound wave carotid artery flow amount.Be reduced to 50% before stimulating as thrombosis with blood flow.
The mensuration of thrombolysis activity
To stimulate end back to the 50% required time that blood flow is reduced to before stimulating to be decided to be thrombus formation time.Form 20min behind the thrombosis, above-mentioned each component is all through the disposable injection of femoral vein, observes after the administration revascularization situation in the 1h; This section is in the period, if blood vessel is logical again, then thinks and leads to failure again.If logical again, then continue to observe vessel open state 1h.With 〉=50% or≤25% stimulation before the blood flow person be judged to be continue again logical or continue after thromboembolism again; In the logical again back 1h, the carotid artery flow amount of every animal with blood flow before stimulating be baseline can be divided into 〉=50%,＞25% to＜50% and≤25%.The carotid artery vascular degree of opening be respectively (1) continue thromboembolism (persistent occlusion, PO): do not have again logical; (2) logically again occur with thromboembolism is staggered again (cyclic reflow, CR); (3) logical again back continuous openness (persistentpatency, PP): do not have thromboembolism again after logical again.
The result shows that the vascular embolization of 2.0mg/kg bilobalide injection group is similar to the blank solution group, all do not have again and again logical, 4.0, the recanalization rate of 8.0mg/kg bilobalide injection group and Breviscapini injection group is respectively 30,60 and 60%; 4.0mg/kg the blood vessel of bilobalide injection group bolt rate again is 66.7%, the rate of bolt again of 8.0mg/kg (clinical dosage 20 times) bilobalide injection group and 13.2mg/kg (clinical dosage 40 times) Breviscapini injection group is suitable, is 50% (table 5).In the logical again back 1h, the vessel open state shows as, and the blank solution group all continues thromboembolism; 13.2mg/kg the bilobalide injection group of the vessel open state score value of the Breviscapini injection group of (clinical dosage 40 times) and 8.0mg/kg (clinical dosage 20 times) is (table 6) quite.
Table 5. bilobalide injection is to the thrombolytic effect of arterial thrombus
| Medicine | Dosage (mg/kg) | The multiple of clinical dosage | Thrombus formation time (min) | The blood flow time at zero point (min) | Recanalization rate (logical again number/sum) | Bolt rate (bolt number/sum more again |
| The blank solution bilobalide injection | Equal-volume 2.0 4.0 8.0 | 5 10 20 | 8.2±.4 8.9±3.0 7.6±4.2 8.3±3.6 | 21.4±4.3 19.6±3.2 19.4±3.7 20.3±4.2 | 0/10 0/10 3/10 * 6/10 * | 0/0 0/0 2/3 * 3/6 * |
| Breviscapini injection | 13.2 | 40 | 8.1±2.7 | 21.7±4.6 | 6/10 * | 3/6 * |
N=10, x ± s,
*(X is compared with blank solution in P＜0.05
2Test).
1h tremulous pulse open state behind the disposable quiet notes bilobalide injection thrombolytic of table 6.
| Medicine | Dosage (mg/kg) | Clinical dose multiple | Vessel open state score value | ||
| Continue thromboembolism | Thromboembolism is with logical staggered again | Continue again logical | |||
| Blank solution bilobalide injection Breviscapini injection | Equal-volume 2.0 4.0 8.0 13.2 | 5 10 20 40 | 10 10 7 4 4 | 0 0 2 3 3 | 0 0 1 3 3 |
The n=10 male rat
5 bilobalide injections are to mouse anti ischemia, hypoxia endurance test
50 of ICR mices, body weight 18～22g, male and female half and half are divided into 5 groups at random, and 10 every group, the bilobalide injection group of promptly isopyknic blank solution group, 2.0,4.0,8.0mg/kg and the Breviscapini injection group of 13.2mg/kg.Each treated animal is pressed 0.1mL/10g body weight ip, and 1 time/d, continuous 2d.30min after the last administration, slowly inject above-mentioned each component through the tail vein respectively again, behind the 10min mice is pursued only broken end, by dehiscing to breathe dwell time as anti-cerebral ischemia, anoxia enduring index behind the stopwatch record mice broken end, data are with statistical procedures between Student t-check row group immediately.
The result shows, the bilobalide injection of high, middle dosage and Breviscapini injection all obviously prolong the normal mouse broken end dehisces the time of breathing, the time of breathing of the Breviscapini injection group of 13.2mg/kg (clinical dosage 40 times) and 8.0mg/kg (clinical dosage 20 times) bilobalide injection group suitable (table 7).
The intravenous injection of table 7. bilobalide injection is to the dehisce influence of the time of breathing of mice broken end
| Group | Dosage (mg/kg) | The multiple of clinical dosage | (s) dehisces the time of breathing |
| Blank solution | Equal-volume | 17.8±3.6 |
| The bilobalide injection Breviscapini injection | 2.0 4.0 8.0 13.2 | 5 10 20 40 | 18.0±4.2 20.8±1.9 *# 23.3±2.1 ** 24.4±3.2 ** |
N=10, x ± s,
*P＜0.05,
*Compare with the blank solution group P＜0.01,
#P＜0.05 and breviscapine, the injection group is (Student t-check) relatively.
6 pairs of pallasiomy cerebral ischemia reperfusion injury improve test
Model: 40 of healthy pallasiomys, body weight 50～70g, male and female dual-purpose.2% pentobarbital sodium (50mgkg
-1) the ip anesthetized animal, the cervical region median incision separates common carotid artery, close bilateral common carotid arteries 10min with not damaged bulldog clamp folder, pine folder back direct-view revascularization (perfusion again) causes the cerebral ischemia reperfusion injury model, the sham operated rats row is operated equally, does not close common carotid artery but do not press from both sides.The skin suture otch is put back to old terms and is continued to raise 5 days (d).Keep about 37 ℃ of anus temperature in the operation process.
Grouping: animal is divided into 5 groups at random, 8 every group, is respectively: sham operated rats, ischemia model group, the middle and high dosage group of bilobalide injection and Breviscapini injection positive controls.Preceding 2 days of moulding, ip solvent (sham operated rats and ischemia model group), bilobalide injection 4.0,8.0mg/kg and Breviscapini injection 13.2mg/kg, press from both sides and close preceding each given the test agent of 5min vena femoralis injection of tremulous pulse 1 time at every day 1 time respectively, 1 time/d of ip between flush phase again, totally 5 times.
Detect index
(1). record electroencephalogram (EEG): reference literature (Sun F, Liu TP.Tetrandrine vsnicardipine in cerebral ischemia-reperfusion damages in gerbils.ActaPharmacol Sin.1995,16 (2): it is subcutaneous 145) to insert the pallasiomy forebrain with single needle electrode, reference electrode places occipitalia subcutaneous, another termination Japanese photoelectricity VC-11 type memory oscilloscope, before the record ischemia, intravenous injection 5min, ischemia 10min, pour into 10 again, 30 and the EEG of 60min, and EEG signals imported Japanese photoelectricity QC-111J type stack Nogata analyser, write down its rectangular histogram, with the v number of x axle as EEG current potential amplitude, with EEG before the ischemia is 100%, is calculated as follows EEG current potential amplitude %.
EEG current potential amplitude (%)=ischemia or amplitude * 100 before the perfusion back amplitude ÷ ischemia again
(2). the Ca of brain cortical tissue
2+, Na
+, water content measures: after cerebral ischemia 10min pours into 5d again, take off neck and put to death animal, with reference to Young method (Young W, Rappaport H, Chalif DJ, FlammES.Regional brain sodium, potassium and water changes in the ratmiddle cerebral artery occlusion of ischemia.Stroke 1987; 18:751) get right side temporo cortex of parietal lobe, inhale the mark of dehematizing with filter paper, put in the crucible of weighing in advance, accurately take by weighing weight in wet base, rearmounted 100 ℃ of baking boxs baking 5h causes constant weight, takes by weighing dry weight again, is calculated as follows the water content of tissue:
Water content (%)=(weight in wet base-dry weight) ÷ weight in wet base * 100
Afterwards through digestion, ashing, dilution process, with atomic absorption spectrophotometer cerebral cortex Ca
2+, Na
+Content (Bradbury, MWB, Kleeman, CR, Bagdoyan, H, Berberian A.The calcium and magnesium content of skeletal muscle, brain, andcerebrospinal fluid as determined by atomic absorption flamephotometry.J Lab Clin Med 1968; 71:884).
Experimental result shows that after folder closed bilateral common carotid arteries, ischemia-reperfusion treated animal EEG current potential amplitude reduced immediately, continue low putting down, comparing difference with Sham-operated control group has significance (P＜0.01), pours into the after-potential amplitude again and recovers slowly, during 60min, only return to 40.5% before the ischemia.After giving bilobalide injection 4.0,8.0mg/kg Breviscapini injection 13.2mg/kg, all can promote the recovery of EEG current potential amplitude, return to 49.5%, 58.1% and 70.8% before the ischemia when pouring into 60min more respectively, apparently higher than ischemia-reperfusion group, P＜0.05 (table 8).
Table 8 bilobalide injection drug administration by injection is to the influence of pallasiomy cerebral ischemia reperfusion damage EEG
| Group | Dosage (mg/kg) | Clinical dose multiple | EEG current potential amplitude (%) | ||||
| Intravenous injection 5 (min) | Ischemia 10 (min) | Perfusion again | |||||
| 10 (min) | 30 | 60 | |||||
| Sham-operation group ischemia-reperfusion group bilobalide injection Breviscapini injection | Equal-volume equal-volume 4.0 8.0 13.2 | 10 20 40 | 105.8± 11.0 95.8± 13.8 106.5± 8.0 99.0± 9.5 102.3± 14.5 | 107.1±7.6 ** 18.9±7.1 20.3±2.1 20.4±4.6 19.6±5.0 | 106.2± 11.4 ** 23.5±7.9 26.0±9.7 33.0±7.2 * 29.6±12.5 | 109.8±12.0 ** 28.9±6.0 36.7±11.2 46.4±9.4 ** 46.9±17.0 * | 113± 14.7 ** 40.5±7.1 49.5± 9.0 * 58.1± 11.5 ** 70.8± 15.1 ** |
X ± s, n=8.
*P＜0.05,
*(Studentt-check) compared with ischemia-reperfusion group in P＜0.01.
After ischemia 10min poured into 5d again, ischemia-reperfusion group animal brain cortex water, sodium and calcium content were apparently higher than Sham-operated control group (P＜0.01).Bilobalide injection 8.0mg/kg (clinical dosage 20 times) can alleviate the cerebral edema that ischemical reperfusion injury causes, with ischemia-reperfusion group relatively, difference has significance; Accumulation has minimizing trend to calcium sodium, but does not reach the remarkable meaning of statistics.Breviscapini injection 13.2mg/kg (clinical dosage 40 times) is not obvious to the cerebral edema influence, but can alleviate calcium sodium accumulation (table 9).
Table 9 bilobalide drug administration by injection is irritated 5d cerebral cortex H again to pallasiomy cerebral ischemia 10min
2O, Na
+, Ca
2+The influence of content
| Group | Dosage (mg/kg) | The multiple of clinical dosage | H 2O (％) | Na + (μmol·g -1Dry weight) | Ca 2+ (μmol·g -1Dry weight) |
| Sham operated rats ischemia-reperfusion group bilobalide injection | Equal-volume equal-volume 4.0 | 10 | 76.6±0.5 ** 77.4±0.4 77.3±0.6 | 365.2±62.1 * 462.6±94.4 489.2±51.6 | 3.59±0.82 * 5.08±1.45 4.22±2.18 |
| Breviscapini injection | 8.0 13.2 | 20 40 | 76.8±0.4 * 77.5±0.5 | 395.2±61.7 378.1±37.0 * | 3.89±1.40 3.61±1.22 * |
X ± s, n=8.
*P＜0.05,
*(Studentt-check) compared with ischemia-reperfusion group in P＜0.01.
7 pairs of focal cerebral ischemia in rats are improved test
80 of male SD rats, body weight 310 ± 17g, be divided into 6 groups at random, be respectively: sham operated rats, ischemia model group (all giving the equal-volume solvent), bilobalide injection high dose group (8.0mg/kg), middle dosage group (4.0mg/kg), low dose group (2.0mg/kg) and Breviscapini injection positive controls (13.2mg/kg).Experiment preceding 2 day every day ip.1 time, 5min administration 1 time before ischemia in the 3rd day.The reference literature method (Xu Shuyun, Bian Rulian, old repairing. the pharmacological experiment methodology, the third edition, the People's Health Publisher, 2002, pp.1066), adopt internal carotid artery line bolt legal system to be equipped with intraluminal middle cerebral artery occlusion in rats obturation (MCAO) model.With 10% chloral hydrate (300mg/kg) ip anesthetized animal, the 0.28mm diameter nylon line of silication is inserted anterior cerebral artery (with the common carotid artery crotch is labelling, is about 17mm) through common carotid artery cause the focal cerebral ischemia model.The sham operated rats row is operated equally, but not inaccessible middle cerebral artery.Keep about 37 ℃ of anus temperature in the operation process.Postoperative 24h carries out behavior scoring to the nervous symptoms that animal occurs, and standard is as follows:
0 is no abnormal
1 perpendicular hair, slight motion is low
2 action are inharmonious, and flexion posture rotatablely moves
3 hemiplegias can not be stood or walk
4 spasm, lethargy
5 death
24h behind the MCAO takes off neck and puts to death animal, takes out full brain, does crown section, hatches 25min with 37 ℃ of lucifuges of 1%TTC solution, separates pale district (infarct) and non-pale district (normal district), weighs, and is calculated as follows percentage ratio:
Infraction percentage ratio (%)=infarct weight ÷ (infarct weight+normally distinguish weight) * 100
The result shows, ip bilobalide injection 4.0mg/kg (clinical dosage 10 times) and 8.0mg/kg (clinical dosage 20 times) and Breviscapini injection 13.2mg/kg (clinical dosage 40 times) all can obviously dwindle the infarction size of MCAO rat, the brain section of each group of test is seen Fig. 1, compare with the ischemia model group, difference has significance (P＜0.05,0.01); Bilobalide injection and Breviscapini injection all can reduce apoplexy scoring and the animal dead rate (table 10) of MCAO rat.
Table 10 bilobalide injection drug administration by injection is to scoring of rat MCAO 24h neurological handicap and infraction percentage ratio
| Group | Dosage (mg/kg) | The multiple of clinical dosage | The example number | Mortality rate (%) | Behavior scoring | The example number | Infarction size (%) |
| Sham operated rats ischemia model group bilobalide injection Breviscapini injection | Equal-volume equal-volume 2.0 4.0 8.0 13.2 | 5 10 20 40 | 10 15 15 14 15 10 | 0 53.3 46.7 40.0 26.7 * 20.0 * | 0 3.8±1.5 3.6±1.5 3.0±1.9 2.6±1.7 * 2.0± 1.8 ** | 10 11 12 12 12 9 | 0 30.7±6.4 27.1±6.4 22.8±9.0 * 21.6± 8.2 ** 21.5± 4.7 ** |
X ± s. behavior scoring and infraction percentage ratio adopt Student t-check:
*P＜0.05,
*Compare with the ischemia model group P＜0.01; Mortality rate adopts X
2-check:
*Compare with the ischemia model group P＜0.05.
8 increase the test of mouse peritoneal capillary permeability
Mice is divided into 5 groups at random, and 10 every group, i.e. the Breviscapini injection group of blank solution group, 2.0,4.0,8.0mg/kg bilobalide group and 13.2mg/kg.Each treated animal is all first with 0.1mL/10g body weight lumbar injection, and 1 time/d, totally 2 times, 30min after the last administration, continue with tail vein injection, behind the 10min, 0.5% azovan blue with the also timely lumbar injection 0.7% acetic acid 0.2mL/ of 0.2mL/ tail vein injection only again, put to death animal behind the 15min, intraperitoneal injection of saline 5mL gently rubs the back and extracts peritoneal fluid, and is centrifugal, supernatant is measured its OD value in the 590nm place, t check between the data line group.
The result shows that bilobalide injection and Breviscapini injection high, middle dosage all can promote H
+Stimulate increase (table 11) that causes the mouse peritoneal capillary permeability.
The intravenous injection of table 11. bilobalide injection is to the influence of mouse peritoneal capillary permeability
| Group | Dosage (mg/kg) | The multiple of clinical dosage | The OD value |
| Blank solution bilobalide injection Breviscapini injection | Equal-volume 2.0 4.0 8.0 13.2 | 5 10 20 40 | 0.33±0.03 0.36±0.05 0.41±0.08 *# 0.52±0.13 ** 0.51±0.13 ** |
N=10, x ± s,
*P＜0.05,
*Compare with the blank solution group P＜0.01,
#(Student t-check) compared with the Breviscapini injection group in P＜0.05.
1.8 former being commissioned to train supported the mouse cortex protecting neuron from acute
It is foster that mouse cortex neurocyte former is commissioned to train: get pregnant 14-17d ICR in age tire Mus cerebral cortex, after D-Hanks liquid is washed 2 times, be cut into rotten shape, add 0.125% trypsinization 10min, stop digestion with the DMEM culture fluid that contains 10% calf serum, blow and beat gently, cell is disperseed with suction pipe, 200 order wire-mesh screens filter, and cell filtrate is with 800r.min
-1Centrifugal 5min abandons supernatant, with DMEM culture fluid (containing 10% calf serum, benzylpenicillin 100U/mL, streptomycin 100U/mL) suspension cell again, cell concentration is adjusted into 1.5 * 10
6/ mL is inoculated in the poly-D-lysine bag by in 24 holes of crossing and 96 orifice plates, long-pending 400L and the 100L of being respectively of inoculum.In 37 ℃ of 5%CO2 incubators, cultivate.Change liquid weekly 2 times, behind 3～5d, add cytosine arabinoside 10mol/L and cultivate 24h, to suppress non-neurocyte proliferation.
The nerve cell damage of glutamic acid to cultivating: added given the test agent (using the DMSO hydrotropy) or blank solvent on the 8th day in cultivating, inhale behind the 24h and remove culture fluid, wash 1 time with DMEM, add glutamic acid (final concentration 500umol/L) and handle 1h, suction removes to contain the culture fluid of glutamic acid, wash 1 time with DMEM, add the DMEM that contains calf serum again and continue to cultivate 24h.
The MTT trace is colorimetry automatically: glutamic acid is handled back 24h, in 96 orifice plates, add MTT (final concentration 0.5mg/mL), continue to cultivate 4h, add three liquid dissolving bluish violet crystallization (Formazan) again, inferior daily EL340 type microplate reader is measured the trap of 570nm, by formula calculate the relative survival rate (relative survival rate %=well OD value or Glu damage OD value ÷ blank hole, hole OD value * 100) of cell, the result carries out the t-check.
The LDH determination of activity: glutamic acid is handled back 24h, measure in the 24 orifice plate culture fluid is the LDH activity of extracellular fluid, afterwards with cell cryopreservation in-30 ℃ of refrigerators, to destroy cell, measure LDH activity total in the culture fluid next day once more, the degree of injury of the big or small reacting cells of twice difference, the more little damage of difference is heavy more, it is many more to illustrate that LDH leaks into extracellular fluid, and the result carries out the t-check.
In the Mechanism Study of ischemic brain injury, excitatory amino acid is being brought into play important effect in the ischemic brain injury pathological process, to cause the death of neurocyte after studies show that glutamic acid and former cerebral cortex neurocyte of being commissioned to train foster contacting certain hour, LDH leaks in the extracellular fluid.This experimental result shows: bilobalide injection and Breviscapini injection all can reduce the leakage of the neurocyte LDH that glutamic acid causes, and both compare there was no significant difference, and prompting has the certain protection effect to cell membrane damage due to the glutamic acid; But to improving the relative survival rate influence not obvious (table 12) of glutamic acid damage back cell.
Table 12. bilobalide injection is supported the influence of the former damage of cortex neural to former being commissioned to train of glutamate induction mice
| Medicine dense (g/ L) | The example number | Relative cell survival rate % | The example number | LDH (U/L) difference | ||
| Bilobalide | Breviscapine | Bilobalide | Breviscapine | |||
| Contrast 0 0.1 1 10 100 1000 | 5 5 5 5 5 5 | 76.2±8.3 68.7±9.0 71.6±12.8 72.9±12.4 76.5±13.7 80.2±14.6 | 76.2±8.3 66.2±6.1 73.2±13.2 72.5±16.7 77.0±16.3 80.1±20.3 | 4 40.5 * 4 4 4 4 19.2 * 4 16.8 * 4 14.3 ** | 189.2± 134.7±12.5 152.5±22.6 161.7±21.6 164.6± 169.1± 177.6± | 189.2± 40.5 * 134.7±12.5 134.4±28.6 154.1±35.9 165.3± 21.4 * 180.5± 20.4 ** 201.2± 25.8 ** |
X ± s, P＞0.01 and 0 concentration ratio be (Student t-check); (Student t-check) compared with Breviscapini injection in P＞0.05.
In sum, bilobalide injection has remarkable ischemia and the anoxia functions that improves cerebral tissue, bilobalide injection have significantly promote blood circulation, blood circulation promoting and blood stasis dispelling and promote the effect of the absorption of blood stasis material.In most of pharmacodynamics test project, bilobalide injection 8.0mg/kg (clinical dosage 20 times) effect and Breviscapini injection 13.2mg/kg (clinical dosage 40 times) are quite, but the powerful antagonism PAF of the former selectivity, the encephaledema effect is stronger, and the latter alleviates calcium, the sodium cumulative function is more obvious.This test provides foundation comparatively reliably for bilobalide injection as preventing and treating apoplexy and periphery thrombotic disease.
Description of drawings
The assay HPLC figure of Fig. 1 bilobalide extract, wherein peak 1 is that ginkalide C, peak 2 are that bilobalide, peak 3 are that ginkalide A, peak 4 are ginkalide B.
The assay HPLC figure of Fig. 2 bilobalide injection, wherein peak 1 is that ginkalide C, peak 2 are that bilobalide, peak 3 are that ginkalide A, peak 4 are ginkalide B.
The specific embodiment
Embodiment one
Prescription (in 1000 dosage units):
Bilobalide 10g
Glycerol 1000ml
Ethanol 2500ml
Water for injection adds to 5000ml
Make 1000
Preparation method
Get the water for injection of recipe quantity about 80%, the glycerol and the ethanol of prescription full dose are put in the dosing cylinder, add the bilobalide of recipe quantity, after the stirring and dissolving, add to the full amount of water for injection, regulate pH to 2.5 with 10% citric acid solution, stir evenly, after the inspection of semifinished product is qualified, clear and bright with the mocromembrane filtration system filters that 0.65 μ m and the compound anti-pure microporous filter membrane of 0.45 μ m are made filter material, embedding is in 5ml glass bottle, in 100 ℃ of sterilizations 20 minutes, cooling, lamp inspection, decals, promptly.
Embodiment two
Prescription (in 1000 dosage units):
Bilobalide 10g
Glycerol 400ml
Ethanol 800ml
Water for injection adds to 2000ml
Make 1000
Preparation method
Get the water for injection of recipe quantity about 80%, the glycerol and the ethanol of prescription full dose are put in the dosing cylinder, add the bilobalide of recipe quantity, after the stirring and dissolving, add to the full amount of water for injection, regulate pH to 4.0 with 10% citric acid solution, stir evenly, after the inspection of semifinished product is qualified, clear and bright with the mocromembrane filtration system filters that 0.65 μ m and the compound anti-pure microporous filter membrane of 0.45 μ m are made filter material, embedding is in 2ml glass bottle, in 100 ℃ of sterilizations 20 minutes, cooling, lamp inspection, decals, promptly.
Embodiment three
Prescription (in 1000 dosage units):
Bilobalide 15g
Glycerol 50ml
Ethanol 100ml
Water for injection adds to 1000ml
Make 1000
Preparation method
Get the water for injection of recipe quantity about 80%, the glycerol and the ethanol of prescription full dose are put in the dosing cylinder, add the bilobalide of recipe quantity, after the stirring and dissolving, add to the full amount of water for injection, regulate pH to 3.5, stir evenly with 10% citric acid solution, after the inspection of semifinished product is qualified, clear and bright with the mocromembrane filtration system filters that 0.65 μ m and the compound anti-pure microporous filter membrane of 0.45 μ m are made filter material, embedding in 2ml glass bottle, every bottle of 1ml.In 100 ℃ of sterilizations 20 minutes, cooling, lamp inspection, decals, promptly.
Embodiment four
Prescription (in 1000 dosage units):
Bilobalide 15g
Glycerol 800ml
Ethanol 100ml
Water for injection adds to 1000ml
Make 1000
Preparation method
Get the water for injection of recipe quantity about 80%, the glycerol and the ethanol of prescription full dose are put in the dosing cylinder, add the bilobalide of recipe quantity, after the stirring and dissolving, add to the full amount of water for injection, regulate pH to 3.5, stir evenly with 10% citric acid solution, after the inspection of semifinished product is qualified, clear and bright with the mocromembrane filtration system filters that 0.65 μ m and the compound anti-pure microporous filter membrane of 0.45 μ m are made filter material, embedding in 2ml glass bottle, every bottle of 1ml.In 100 ℃ of sterilizations 20 minutes, cooling, lamp inspection, decals, promptly.
Embodiment five
Prescription (in 1000 dosage units):
Bilobalide 15g
Glycerol 50ml
Ethanol 900ml
Water for injection adds to 1000ml
Make 1000
Preparation method
Get the water for injection of recipe quantity about 80%, the glycerol and the ethanol of prescription full dose are put in the dosing cylinder, add the bilobalide of recipe quantity, after the stirring and dissolving, add to the full amount of water for injection, regulate pH to 5, stir evenly with 10% citric acid solution, after the inspection of semifinished product is qualified, clear and bright with the mocromembrane filtration system filters that 0.65 μ m and the compound anti-pure microporous filter membrane of 0.45 μ m are made filter material, embedding in 2ml glass bottle, every bottle of 1ml.In 100 ℃ of sterilizations 20 minutes, cooling, lamp inspection, decals, promptly.
Embodiment six
Prescription (in 1000 dosage units):
Bilobalide 5g
Glycerol 200ml
Ethanol 500ml
Water for injection adds to 1000ml
Make 1000
Preparation method
Get the water for injection of recipe quantity about 80%, the glycerol and the ethanol of prescription full dose are put in the dosing cylinder, add the bilobalide of recipe quantity, after the stirring and dissolving, add to the full amount of water for injection, regulate pH to 4, stir evenly with 10% citric acid solution, after the inspection of semifinished product is qualified, clear and bright with the mocromembrane filtration system filters that 0.65 μ m and the compound anti-pure microporous filter membrane of 0.45 μ m are made filter material, embedding in 2ml glass bottle, every bottle of 1ml.In 100 ℃ of sterilizations 20 minutes, cooling, lamp inspection, decals, promptly.
Embodiment seven
Prescription (in 1000 dosage units):
Bilobalide 10g
Glycerol 1000ml
Ethanol 2500ml
Water for injection adds to 10000ml
Make 1000
Preparation method
Get the water for injection of recipe quantity about 80%, the glycerol and the ethanol of prescription full dose are put in the dosing cylinder, add the bilobalide of recipe quantity, after the stirring and dissolving, add to the full amount of water for injection, regulate pH to 3.5 with 10% citric acid solution, stir evenly, after the inspection of semifinished product is qualified, clear and bright with the mocromembrane filtration system filters that 0.65 μ m and the compound anti-pure microporous filter membrane of 0.45 μ m are made filter material, embedding is in 10ml glass bottle, in 100 ℃ of sterilizations 20 minutes, cooling, lamp inspection, decals, promptly.
Embodiment seven
Prescription (in 1000 dosage units):
Bilobalide 10g
Glycerol 1000ml
Ethanol 2500ml
Water for injection adds to 100000ml
Make 1000 bottles
Preparation method
Get the water for injection of recipe quantity about 80%, the glycerol and the ethanol of prescription full dose are put in the dosing cylinder, add the bilobalide of recipe quantity, after the stirring and dissolving, add to the full amount of water for injection, regulate pH to 4.0 with 10% citric acid solution, stir evenly, after the inspection of semifinished product is qualified, clear and bright with the mocromembrane filtration system filters that 0.65 μ m and the compound anti-pure microporous filter membrane of 0.45 μ m are made filter material, embedding is in 100ml glass bottle, in 120 ℃ of sterilizations 30 minutes, cooling, lamp inspection, decals, promptly.
Claims (11)
1. the pharmaceutical composition of a bilobalide is characterized in that, this pharmaceutical composition contains the mixed solvent of forming with ethanol, and the pH value of this pharmaceutical composition is 2.5-6.5.
2. the pharmaceutical composition of a kind of bilobalide according to claim 1 is characterized in that, the mixed solvent of this pharmaceutical composition contains ethanol, glycerol, water.
3. the pharmaceutical composition of a kind of bilobalide according to claim 2 is characterized in that, bilobalide in the mixed solvent of this pharmaceutical composition: ethanol: glycerol: water=0.001%-0.15%: 1%-80%: 1%-80%: 15%-99%.
4. the pharmaceutical composition of a kind of bilobalide according to claim 2 is characterized in that, bilobalide in the mixed solvent of this pharmaceutical composition: ethanol: glycerol: water=0.05%-0.15%: 20%-70%: 5%-40%: 20%-70%.
5. the pharmaceutical composition of a kind of bilobalide according to claim 2 is characterized in that, bilobalide in the mixed solvent of this pharmaceutical composition: ethanol: glycerol: water=0.05%-0.15%: 30%-50%: 10%-30%: 30%-50%.
6. the preparation of drug combination method of a kind of bilobalide according to claim 2 is characterized in that, the pH value of regulating this pharmaceutical composition is 3.5-5.0.
7. the preparation of drug combination method of a kind of bilobalide according to claim 1 is characterized in that, the pH value regulator of this pharmaceutical composition is a citric acid.
8. the preparation of drug combination method of a kind of bilobalide according to claim 1, it is characterized in that, get the water for injection of recipe quantity about 80%, the glycerol and the ethanol of prescription full dose are put in the dosing cylinder, add the bilobalide of recipe quantity, after the stirring and dissolving, add to the full amount of water for injection, regulate pH value, stir evenly with 10% citric acid solution, after the inspection of semifinished product is qualified, the mocromembrane filtration system filters of making filter material with 0.65 μ m and 0.45 μ m composite microporous filter film is clear and bright, and embedding is in 100 ℃ of sterilizations, cooling, lamp inspection, decals, promptly.
9. the pharmaceutical composition of a kind of bilobalide according to claim 1 is characterized in that, its dosage form is to be fit to the injection that the prison bed is used.
10. the pharmaceutical composition of a kind of bilobalide according to claim 9 is characterized in that, its dosage form also comprises transfusion, freeze-dried powder.
11. the pharmaceutical composition according to each described a kind of bilobalide among the claim 1-10 is characterized in that, this pharmaceutical composition is in the purposes of preventing and treating on apoplexy and the periphery thrombotic disease.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102626383A (en) * | 2012-04-23 | 2012-08-08 | 成都百裕科技制药有限公司 | Bilobalide injection and preparation method thereof |
| CN103877017A (en) * | 2014-04-18 | 2014-06-25 | 夏中宁 | Bilobalide B injection and preparation method thereof |
| CN106580872A (en) * | 2015-10-16 | 2017-04-26 | 上海现代药物制剂工程研究中心有限公司 | Ginkgolides B injection and preparation method thereof |
| CN109925310A (en) * | 2013-12-10 | 2019-06-25 | 成都百裕制药股份有限公司 | Blood-pressure drug containing therapeutically effective amount ginkgolides |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1188124C (en) * | 2000-03-24 | 2005-02-09 | 中国科学院上海药物研究所 | Process for preparing ginkgolide injection |
| CN1232251C (en) * | 2004-01-14 | 2005-12-21 | 江苏康缘药业股份有限公司 | Gingko lactone powder injection and its preparation method |
-
2006
- 2006-07-26 CN CNB2006101036256A patent/CN100464747C/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102626383A (en) * | 2012-04-23 | 2012-08-08 | 成都百裕科技制药有限公司 | Bilobalide injection and preparation method thereof |
| WO2013159411A1 (en) * | 2012-04-23 | 2013-10-31 | 成都百裕科技制药有限公司 | Ginkgolide injection and preparation method thereof |
| CN109925310A (en) * | 2013-12-10 | 2019-06-25 | 成都百裕制药股份有限公司 | Blood-pressure drug containing therapeutically effective amount ginkgolides |
| CN103877017A (en) * | 2014-04-18 | 2014-06-25 | 夏中宁 | Bilobalide B injection and preparation method thereof |
| CN103877017B (en) * | 2014-04-18 | 2016-02-03 | 夏中宁 | A kind of Ginkgolide B injection and preparation method thereof |
| CN106580872A (en) * | 2015-10-16 | 2017-04-26 | 上海现代药物制剂工程研究中心有限公司 | Ginkgolides B injection and preparation method thereof |
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