CN101947247A - Method for preparing ginkgodipyidamolum injection - Google Patents
Method for preparing ginkgodipyidamolum injection Download PDFInfo
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Abstract
The invention provides a method for preparing ginkgodipyidamolum injection. The method comprises the following steps of: 1) extracting a ginkgo leaf extract from ginkgo leaves; 2) extracting the ginkgo leaf extract once again with over 90 percent ethanol so as to refine the ginkgo leaf extract; 3) adding water into the refined ginkgo leaf extract and dipyridamole for inverse flow and filtering so as to obtain ginkgo leaf extracting solution; and 4) mixing the ginkgo leaf extracting solution, the dipyridamole and auxiliary materials so as to obtain the ginkgodipyidamolum injection, wherein in the ginkgodipyidamolum injection, the weight ratio of the ginkgo leaf extracting solution to the dipyridamole based on total ginkgo flavone-glycoides is 0.9-1.1:0.3-0.5. The method of the invention has the advantages of effectively removing various impurities, enhancing medicament effectiveness, improving the quality stability of the ginkgodipyidamolum injection and reducing safety risk.
Description
Technical field
The present invention relates to a kind of preparation method of injection formulation, specifically, relate to a kind of preparation method of Ginkgo Leaf Extract and Dipyridamole Injection.
Background technology
Semen Ginkgo has another name called Folium Ginkgo, Gong Sunshu, duck's foot tree etc., belongs to gymnosperm, is one of distinctive ancient seeds of China.Contain multiple nutritional components in the Folium Ginkgo, its protein, vitamin C, carotene, vitamin E and mineral element, extremely abundant as calcium, ferrum, fluorine, copper, zinc, chromium, cobalt, boron, selenium equal size; Also contain tens of kinds of active component in the Folium Ginkgo simultaneously, wherein topmost have flavonoid and a terpene lactones chemical compound, these compositions have quite intensive antioxidation, can remove biological intravital superfluous free radical, stop the body lipid peroxidating, suppress functions such as platelet activating factor, blood circulation promoting and brain metabolism.Can be used for treating coronary heart disease, angina pectoris, senile dementia clinically and prevent and treat multiple diseases such as dermatosis, alopecia.
Ginkgo Leaf Extract and Dipyridamole Injection is the Chinese herbal medicine Folium Ginkgo extract; compound formulation with the Western medicine dipyridamole; be the free radical scavenger of generally acknowledging at present, have neuroprotective and the effect of antagonism platelet activating factor, be used for prevention and treatment coronary heart disease, thrombotic disease etc.Wherein the Ginkgo total flavones of Folium Ginkgo extract has dilating coronary blood vessel, cerebrovascular, improves symptom and memory function that cerebral ischemia produces; Double density reaches not can anticoagulant, and high concentration (50 μ g/ml) can suppress platelet and discharge.
At present, domestic have many manufacturer production Ginkgo Leaf Extract and Dipyridamole Injection, but great majority all are to be formed by Folium Ginkgo extract and the direct mixed preparing of dipyridamole, it is in put procedure, especially can separate out the invalid components such as protein, tannin etc. of in the Folium Ginkgo extract preparation process, not removing after the freeze thawing, constant product quality is relatively poor, also can bring risk to the drug safety of human body.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of Ginkgo Leaf Extract and Dipyridamole Injection, this method can reduce the various impurity of Folium Ginkgo extract, improves the quality stability of Ginkgo Leaf Extract and Dipyridamole Injection.
In order to realize purpose of the present invention, the preparation method of Ginkgo Leaf Extract and Dipyridamole Injection of the present invention, it comprises the steps:
1) from Folium Ginkgo, extracts Folium Ginkgo extract;
2) once more Folium Ginkgo extract is extracted with the ethanol of concentration expressed in percentage by volume more than 90%, thus refined ginkgo biloba extract;
3) Folium Ginkgo extract and the dipyridamole after will making with extra care adds water backflow after-filtration, makes ginkgo biloba succi;
4) ginkgo biloba succi, residue dipyridamole and adjuvant are mixed, make Ginkgo Leaf Extract and Dipyridamole Injection; In the described Ginkgo Leaf Extract and Dipyridamole Injection, ginkgo biloba succi is 0.9～1.1: 0.3～0.5 in the weight ratio of Ginkgo total flavones and dipyridamole total amount.
Wherein, step 2) is specially: the ethanol of concentration expressed in percentage by volume more than 90% that adds 8～12 times of weight in the Folium Ginkgo extract, be stirred to dissolving, be concentrated into nothing alcohol flavor after the centrifugalize, add 10 times of purified water that concentrate the back medicine liquid volumes then, leave standstill after the stirring cooling, get supernatant, after centrifugalize, concentrate to receive cream, vacuum drying to water content below 6% (weight), pulverize.
Step 3) is specially: in the Folium Ginkgo extract after refining, add the dipyridamole of extract weight 0.5～2% and the purified water of 10～15 times of weight of extract weight, behind the mix homogeneously, regulate pH value to 3.5～5.5, reflux 0.5～1 hour, be cooled to room temperature, repetition measurement pH to 3.5～5.5 are got supernatant then, with 0.2 μ m membrane filtration, the filtrate ultrafilter membrane below 100000 dalton is again carried out ultrafiltration, and last heat sterilization is made ginkgo biloba succi.
The preferred dipyridamole that adds Folium Ginkgo extract weight 1% in the step 3).
Because Folium Ginkgo extract is a kind of plant amedica, its composition is comparatively complicated, and the effective ingredient in the Ginkgo Leaf Extract and Dipyridamole Injection is a Ginkgo total flavones, therefore in order to extract its effective ingredient better, reduce various impurity, improve the medicine effectiveness, reduce security risk, the inventor is through repeatedly experimental studies have found that: when the preparation Folium Ginkgo extract, use the ethanol more than 90% that Folium Ginkgo extract is made with extra care, can the lactone of water will be slightly soluble in effectively in the extract, the rutin constituents is removed, thereby increases the quality stability of injection Ginkgo Leaf Extract and Dipyridamole Injection.
In addition, in preparation during Ginkgo Leaf Extract and Dipyridamole Injection,, place after 6 months dipyridamole for a long time and indicate content and promptly drop to below 90% if only add dipyridamole in the step of preparation injection formulation.In order to address this problem, the present invention earlier with Folium Ginkgo extract with make ginkgo biloba succi after the dipyridamole of Folium Ginkgo extract weight 0.5～2% mixes, make the part dipyridamole fully to be dissolved in the extracting solution, again with this ginkgo biloba succi, remaining dipyridamole and appropriate amount of auxiliary materials are hybridly prepared into Ginkgo Leaf Extract and Dipyridamole Injection, thereby can make dipyridamole stability increase in the injection, this may be dipyridamole in reflux course with extract in certain group of some composition combine closely, strengthen its stability, thereby further improved the quality stability of injection.
Step 1) of the present invention is extracted Folium Ginkgo extract from Folium Ginkgo, extract with conventional method to get final product, for example can for: Folium Ginkgo is carried with the purified water water of 6～8 times of weight, extract twice, each reflux, extract, 2 hours is filtered, and is concentrated into 1/3～1/5 of original volume behind the merging filtrate; Concentrated solution is stirred cooling, leaves standstill, get supernatant then, filter, with filtrate through the D101 macroporous resin adsorption, ethanol elution, eluent are concentrated into does not have the alcohol flavor, adds the purified water of 1～2 times of Folium Ginkgo weight then, leave standstill after stirring cooling, get supernatant, filter, filtrate is concentrated receive cream, vacuum drying to water content is below 6% (weight), pulverizes.
Step 4) preparation injection formulation adopts conventional method to get final product among the present invention, being about to ginkgo biloba succi, dipyridamole and adjuvant dissolves fully with water for injection, adjust pH is 3.5～5.5, and intermediate is through after the assay was approved, with fill behind the 0.22 μ m filtering with microporous membrane;
Adjuvant of the present invention is one or more in propylene glycol, glycerol, Tween 80, sodium pyrosulfite, vitamin C, calcium disodium edetate or the PEG400, also can be other adjuvants commonly used in the injection formulation.
Specifically, the invention provides a kind of preparation method that is used for Ginkgo Leaf Extract and Dipyridamole Injection, it comprises the steps:
1) Folium Ginkgo is carried with the purified water water of 6～8 times of weight, extracted twice, each reflux, extract, 2 hours is filtered, and is concentrated into 1/3～1/5 of original volume behind the merging filtrate; Concentrated solution is stirred cooling, leaves standstill, get supernatant then, filter, through the D101 macroporous resin adsorption, ethanol elution, eluent are concentrated into does not have the alcohol flavor with filtrate, the purified water that adds 1～2 times of Folium Ginkgo weight then, leave standstill after stirring cooling, get supernatant, filter, filtrate is concentrated receipts cream, vacuum drying to water content is below 6% (weight), pulverizes, and makes Folium Ginkgo extract;
2) in the Folium Ginkgo extract that step 1) makes, add the ethanol of concentration expressed in percentage by volume more than 90% of 8～12 times of weight, be stirred to dissolving, be concentrated into after the centrifugalize and do not have the alcohol flavor, add 10 times of purified water that concentrate the back medicine liquid volume then, leave standstill after the stirring cooling, get supernatant, after centrifugalize, concentrate and receive cream, vacuum drying to water content below 6% (weight), pulverize, thus refined ginkgo biloba extract;
3) in the Folium Ginkgo extract after refining, add the dipyridamole of extract weight 0.5～2% and the purified water of 10～15 times of weight of extract weight, behind the mix homogeneously, regulate pH value to 3.5～5.5, reflux 0.5～1 hour, be cooled to room temperature, repetition measurement pH to 3.5～5.5 are got supernatant then, with 0.22 μ m membrane filtration, the filtrate ultrafilter membrane below 100000 dalton is again carried out ultrafiltration, and last heat sterilization is made ginkgo biloba succi;
4) ginkgo biloba succi, remaining dipyridamole and adjuvant are dissolved fully with water for injection, adjust pH is 3.5～5.5, and intermediate is through after the assay was approved, with fill behind the 0.22 μ m filtering with microporous membrane, sterilization.
In addition, the present invention also provides the Ginkgo Leaf Extract and Dipyridamole Injection for preparing with said method.
The invention has the advantages that, the preparation method of Ginkgo Leaf Extract and Dipyridamole Injection of the present invention, Folium Ginkgo extract is made with extra care with the ethanol more than 90%, can remove various impurity effectively, improve the medicine effectiveness, and then can improve the quality stability of Ginkgo Leaf Extract and Dipyridamole Injection, reduce security risk; And, by adjusting the adding mode of dipyridamole, promptly earlier Folium Ginkgo extract and part dipyridamole are mixed and made into ginkgo biloba succi, be hybridly prepared into Ginkgo Leaf Extract and Dipyridamole Injection by ginkgo biloba succi, remaining dipyridamole and appropriate amount of auxiliary materials again, can make dipyridamole stability increase in the injection, further improve the quality stability of injection.
The specific embodiment
Below further specify the present invention by specific embodiment, but be not used for limiting the scope of the invention.
Embodiment 1
1, extract Folium Ginkgo extract: Folium Ginkgo is carried with the purified water water of 6 times of weight, extracted twice, each reflux, extract, 2 hours is filtered, and is concentrated into 1/3 of original volume behind the merging filtrate; Concentrated solution is stirred cooling, leaves standstill, get supernatant then, filter, with filtrate through the D101 macroporous resin adsorption, ethanol elution, eluent are concentrated into does not have the alcohol flavor, adds the purified water of 2 times of Folium Ginkgo weight then, leave standstill after stirring cooling, get supernatant, filter, filtrate is concentrated receive cream, vacuum drying to water content is below 6% (weight), pulverizes;
2, refined ginkgo biloba extract: 90% (volume) ethanol that in the Folium Ginkgo extract that step 1 obtains, adds 8 times of weight, be stirred to dissolving, be concentrated into after the centrifugalize and do not have the alcohol flavor, add 10 times of purified water that concentrate the back medicine liquid volume then, leave standstill after stirring cooling, get supernatant, after centrifugalize, concentrate and receive cream, vacuum drying to water content below 6% (weight), pulverize;
3, preparation ginkgo biloba succi: the purified water that in Folium Ginkgo extract, adds 15 times of amounts of extract, after being heated to mix homogeneously, the dipyridamole that adds Folium Ginkgo extract weight 1%, regulate pH value 4.59, continue to be heated to boiling, refluxed 1 hour, stop heating, be cooled to room temperature, repetition measurement pH to 4.55; Get supernatant then, with 0.22 μ m filter membrane plate-and-frame filtration, filtrate is carried out ultrafiltration with 100000 daltonian filter membranes, with heat sterilization in the liquid suction sterilization tank after the above-mentioned ultrafiltration, when temperature rises 115 ℃, pick up counting, sterilization is 35 minutes under 115 ± 2 ℃ of conditions, make ginkgo biloba succi after the cooling, standby;
4, preparation Ginkgo Leaf Extract and Dipyridamole Injection preparation:
1) prescription is formed: the prescription of 1000 Ginkgo Leaf Extract and Dipyridamole Injection is composed as follows:
Specification 5ml 10ml
Ginkgo biloba succi: by Ginkgo total flavones 5g by Ginkgo total flavones 10g
Dipyridamole: 2g 4g
Tween 80: 10g 20g
Vitamin C: 25g 50g
Propylene glycol: 500ml 1000ml
Water for injection is extremely: 5000ml 10000ml.
2) preparation process
Dipyridamole with propylene glycol and proper amount of water for injection dissolving surplus makes solution A, and is standby; With an amount of water for injection dissolving Tween 80 and vitamin C, make solution B, standby; After solution A, solution B and proper amount of water for injection mixing, add the active carbon of 0.1% (g/ml) of moistening, insulation is 30 minutes under 80 ℃ temperature, with 0.45 μ m microporous filter membrane decarbonization filtering;
The ginkgo biloba succi that filtrate and step 2 are made mixes, and is 4.52 with 6mol/L sodium hydroxide adjust pH, and with 0.22 μ m microporous filter membrane fine straining, filtrate is added water for injection to recipe quantity; Intermediate detect pH value (4.55) and content qualified after, through twin-stage 0.22 μ m microporous filter membrane fine straining embedding in ampoule bottle, the ampoule bottle 5min that under temperature more than 300 ℃, sterilizes in advance; The 30min (5ml specification) that under 115 ℃ of temperature, sterilizes then, perhaps at 115 ℃ of sterilization 45min (10ml specification), qualified on inspection back packing warehouse-in.
Embodiment 2
1, extracts Folium Ginkgo extract: identical with embodiment 1;
2, refined ginkgo biloba extract: 90% (volume) ethanol that in the Folium Ginkgo extract that step 1 obtains, adds 12 times of weight, be stirred to dissolving, be concentrated into after the centrifugalize and do not have the alcohol flavor, add 10 times of purified water that concentrate the back medicine liquid volume then, leave standstill after stirring cooling, get supernatant, after centrifugalize, concentrate and receive cream, vacuum drying to water content below 6% (weight), pulverize;
3, preparation ginkgo biloba succi: the purified water that in Folium Ginkgo extract, adds 10 times of amounts of extract, after being heated to mix homogeneously, the dipyridamole that adds Folium Ginkgo extract weight 0.5%, regulate pH value to 4.51, continue to be heated to boiling, refluxed 1 hour, stop heating, be cooled to room temperature, repetition measurement pH to 4.46; Get supernatant then, with 0.22 μ m filter membrane plate-and-frame filtration, filtrate is carried out ultrafiltration with 100000 daltonian filter membranes, with heat sterilization in the liquid suction sterilization tank after the above-mentioned ultrafiltration, when temperature rises to 115 ℃, pick up counting, sterilization is 35 minutes under 115 ± 2 ℃ of conditions, make ginkgo biloba succi after the cooling, standby;
4, preparation Ginkgo Leaf Extract and Dipyridamole Injection preparation:
1) prescription is formed: identical with embodiment 1;
2) preparation process
Dipyridamole with propylene glycol and proper amount of water for injection dissolving surplus makes solution A, and is standby; With an amount of water for injection dissolving Tween 80 and vitamin C, make solution B, standby; After solution A, solution B and proper amount of water for injection mixing, add the active carbon of 0.1% (g/ml) of moistening, insulation is 30 minutes under 80 ℃ temperature, with 0.45 μ m microporous filter membrane decarbonization filtering;
The ginkgo biloba succi that filtrate and step 2 are made mixes, and is 4.56 with 6mol/L sodium hydroxide adjust pH, and with 0.22 μ m microporous filter membrane fine straining, filtrate is added water for injection to recipe quantity; Intermediate detect pH value (4.52) and content qualified after, through twin-stage 0.22 μ m microporous filter membrane fine straining embedding in ampoule bottle, the ampoule bottle 5min that under temperature more than 300 ℃, sterilizes in advance; The 30min (5ml specification) that under 115 ℃ of temperature, sterilizes then, perhaps at 115 ℃ of sterilization 45min (10ml specification), qualified on inspection back packing warehouse-in.
Embodiment 3
1, extracts Folium Ginkgo extract: identical with embodiment 1;
2, refined ginkgo biloba extract: use dehydrated alcohol, all the other steps are identical with embodiment 1;
3, preparation ginkgo biloba succi: the purified water that in Folium Ginkgo extract, adds 10 times of amounts of extract, after being heated to mix homogeneously, the dipyridamole that adds Folium Ginkgo extract weight 2%, regulate pH value to 4.78, continue to be heated to boiling, refluxed 0.5 hour, stop heating, be cooled to room temperature, repetition measurement pH to 4.65; Get supernatant then, with 0.22 μ m filter membrane plate-and-frame filtration, filtrate is carried out ultrafiltration with 100000 daltonian filter membranes, with heat sterilization in the liquid suction sterilization tank after the above-mentioned ultrafiltration, when temperature rises to 115 ℃, pick up counting, sterilization is 40 minutes under 115 ± 2 ℃ of conditions, make ginkgo biloba succi after the cooling, standby;
4, preparation Ginkgo Leaf Extract and Dipyridamole Injection preparation:
1) prescription is formed: identical with embodiment 1;
2) preparation process
Dipyridamole with propylene glycol and proper amount of water for injection dissolving surplus makes solution A, and is standby; With an amount of water for injection dissolving Tween 80 and vitamin C, make solution B, standby; After solution A, solution B and proper amount of water for injection mixing, add the active carbon of 0.1% (g/ml) of moistening, insulation is 30 minutes under 80 ℃ temperature, with 0.45 μ m microporous filter membrane decarbonization filtering;
The ginkgo biloba succi that filtrate and step 2 are made mixes, and is 4.55 with 6mol/L sodium hydroxide adjust pH, and with 0.22 μ m microporous filter membrane fine straining, filtrate is added water for injection to recipe quantity; Intermediate detect pH value (4.48) and content qualified after, through twin-stage 0.22 μ m microporous filter membrane fine straining embedding in ampoule bottle, the ampoule bottle 5min that under temperature more than 300 ℃, sterilizes in advance; The 30min (5ml specification) that under 115 ℃ of temperature, sterilizes then, perhaps at 115 ℃ of sterilization 45min (10ml specification), qualified on inspection back packing warehouse-in.
Comparative example 1 routine fashion prepares Ginkgo Leaf Extract and Dipyridamole Injection
1, extracts Folium Ginkgo extract: identical with embodiment 1;
2, preparation ginkgo biloba succi: the Folium Ginkgo extract that step 1 is made is without purification step, the purified water that directly adds 15 times of amounts of extract, after being heated to mix homogeneously, regulate pH value to 4.86, continue to be heated to boiling, refluxed 1 hour, stop heating, be cooled to room temperature, repetition measurement pH to 4.59; Get supernatant then, with 0.22 μ m filter membrane plate-and-frame filtration, filtrate is carried out ultrafiltration with 100000 daltonian filter membranes, with heat sterilization in the liquid suction sterilization tank after the above-mentioned ultrafiltration, when temperature rises to 115 ℃, pick up counting, sterilization is 35 minutes under 115 ± 2 ℃ of conditions, make ginkgo biloba succi after the cooling, standby;
3, preparation Ginkgo Leaf Extract and Dipyridamole Injection preparation: prescription composition and preparation process are all identical with embodiment 1.
Comparative example 2
1, extracts Folium Ginkgo extract: identical with embodiment 1;
2, refined ginkgo biloba extract: identical with embodiment 1;
3, preparation ginkgo biloba succi:
The purified water that adds 10 times of amounts of extract in the Folium Ginkgo extract after refining, be heated to mix homogeneously after, regulate pH value to 4.56, continue to be heated to boiling, refluxed 0.5 hour, stop to heat, be cooled to room temperature, repetition measurement pH to 4.57; Get supernatant then, with 0.22 μ m filter membrane plate-and-frame filtration, filtrate is carried out ultrafiltration with 100000 daltonian filter membranes, with heat sterilization in the liquid suction sterilization tank after the above-mentioned ultrafiltration, when temperature rises to 115 ℃, pick up counting, sterilization is 30 minutes under 115 ± 2 ℃ of conditions, make ginkgo biloba succi after the cooling, standby;
4, preparation Ginkgo Leaf Extract and Dipyridamole Injection preparation: prescription composition and preparation process are all identical with embodiment 1.
Below further specify the present invention by stability test.
Experimental example 1
The Ginkgo Leaf Extract and Dipyridamole Injection that embodiment 1～3 and comparative example 1～2 are prepared is placed on respectively below-20 ℃ and carries out freezing-thawing test, investigate appearance character, pH, Ginkgo total flavones content and the dipyridamole content of each Ginkgo Leaf Extract and Dipyridamole Injection respectively, the result is shown in table 1～2.
Table 1 Ginkgo Leaf Extract and Dipyridamole Injection freezing-thawing test result
The Ginkgo Leaf Extract and Dipyridamole Injection of table 2 embodiment 1 freezes the result of the test after preceding and the thawing
According to table 1, table 2 as can be seen: after Folium Ginkgo extract is through the ethanol refinement treatment more than 90% (volume), prepared Ginkgo Leaf Extract and Dipyridamole Injection is more stable, sample is more stable through the freezing thawing back properties of samples that freezes again, macroscopic white point do not occur; Particularly the Ginkgo Leaf Extract and Dipyridamole Injection of embodiment 1 method preparation is more stable through the freezing thawing back properties of samples that freezes again, and significant change, its quality stability the best all do not take place for its content, pH, particulate matter, color etc.
Experimental example 2
The Ginkgo Leaf Extract and Dipyridamole Injection that embodiment 1 and comparative example 1～2 are prepared is placed under relative humidity 75% ± 5%, 40 ℃ ± 2 ℃ condition respectively and was carried out accelerated test in 6 months, and once respectively at sampling at 0,1,2,3,6 the end of month, investigate appearance character, pH, Ginkgo total flavones content and the dipyridamole content of Ginkgo Leaf Extract and Dipyridamole Injection respectively, the result is as shown in table 3.
Table 3 Ginkgo Leaf Extract and Dipyridamole Injection accelerated test result
According to table 2 as can be seen: through accelerated test, the Ginkgo Leaf Extract and Dipyridamole Injection of comparative example 1 and the preparation of comparative example 2 methods, its dipyridamole content presents downward trend, is defective during March to acceleration; The Ginkgo Leaf Extract and Dipyridamole Injection of embodiment 1 method preparation through 6 months accelerated tests after, significant change, quality stability the best all do not take place in its each component content, pH, particulate matter, color etc.
In order to prove the safety of the Ginkgo Leaf Extract and Dipyridamole Injection that the present invention is prepared, we entrust Shanxi Hua Kang novel technique exploitation service centre that the Ginkgo Leaf Extract and Dipyridamole Injection of using the present invention's preparation is carried out main with local, that the whole body administration is relevant specific safety experimentation such as anaphylaxis, hemolytic and local irritation.
Experimental example 4 sensitivity tests
1. test objective
The anaphylaxis that after the administration of investigation this product whole body animal is caused is for data for clinical drug use provides foundation.
2. be subjected to the reagent thing
Tried thing: the Ginkgo Leaf Extract and Dipyridamole Injection of embodiment 1 preparation, specification: 10ml;
Negative control: 0.9% sodium chloride injection, specification: 250ml, Shangdong Hualu Pharmaceutical Co., Ltd.;
Positive control: human albumin's (20%), specification: 50ml:10g, Guangdong Weilun Biological Pharmaceutical Co. Ltd..
3. animal subject
18 of Cavia porcelluss, body weight 300～400g, male and female half and half, prosperous animal cultivation field, Haidian, Beijing provides, the quality certification number: SCXK (capital) 2006-0006.
4. dosage design:
The clinical using dosage of this test dose design considerations Ginkgo Leaf Extract and Dipyridamole Injection (adult's dosage is a 10～25ml, with the glucose injection or the 0.9% sodium chloride injection 500ml dilution that add 5%, 2 times on the one).
5. test method
Cavia porcellus is divided into 3 groups at random by body weight, sex, 6 every group.Negative control group gives 0.5ml 0.9% sodium chloride injection sensitization, and positive controls gives 0.5ml 1% human albumin's sensitization, the Ginkgo Leaf Extract and Dipyridamole Injection sensitization that prepared with 1ml embodiment 1 by the reagent group.
Lumbar injection medicinal liquid next day that each being organized is injected 3 times altogether; Then every group of Cavia porcellus is further divided into 2 groups, wherein 1 group Cavia porcellus quick vein double-injected administration in 14 days behind first time lumbar injection medicinal liquid; The quick vein double-injected administration in the 21st day behind first time lumbar injection medicinal liquid of another group.
Cavia porcellus symptoms of allergic after the observation intravenous injection in 30 minutes, and press symptom shown in the table 4 and observe the reaction symptom of every animal and appearance, the extinction time of symptom in detail, the longest prolongation observed to 3h.
Table 4 symptoms of allergic
| Sequence number | Symptom | Sequence number | Symptom | Sequence number | Symptom |
| 0 | Normally | 7 | Rapid breathing | 14 | Instability of gait |
| 1 | Restless | 8 | Urinate | 15 | Jump |
| 2 | Perpendicular hair | 9 | Defecation | 16 | Pant |
| 3 | Tremble | 10 | Shed tears | 17 | Spasm |
| 4 | Scratch nose | 11 | Dyspnea | 18 | Rotation |
| 5 | Sneeze | 12 | Wheezing sound | 19 | Cheyne-Stokes respiration |
| 6 | Cough | 13 | Purpura | 20 | Dead |
Judge the anaphylaxis occurrence degree, calculate the anaphylaxis incidence rate according to the evaluation criterion shown in the table 5, comprehensively judge according to anaphylaxis incidence rate and anaphylaxis occurrence degree.The result is as shown in table 6.
Table 5 whole body sensitization evaluation criterion
| Symptom generation number | Intensity | Conclusion |
| 0 | - | The anaphylaxis feminine gender |
| 1-4 | + | Anaphylaxis is weak positive |
| 5-10 | ++ | The anaphylaxis positive |
| 11-19 | +++ | The anaphylaxis strong positive |
| 20 | ++++ | The extremely strong positive of anaphylaxis |
The anaphylactic reaction result of the test of table 6 Ginkgo Leaf Extract and Dipyridamole Injection
6. result of the test
As can be seen from Table 6: anaphylactic reaction does not all take place respectively at attacking in 14 days and the 21st day behind the 1st lumbar injection in the two group Cavia porcelluss that are subjected to the reagent group; And the positive controls Cavia porcellus occurs dyspnea in injecting in back 2 minutes, and tic is fallen down, and is then dead; Cavia porcellus death all occurs in injects in back 1～5 minute.In addition, anaphylaxis does not all take place in 6 Cavia porcelluss of negative control group.The result shows: under this experimental condition, the Ginkgo Leaf Extract and Dipyridamole Injection of the inventive method preparation is not had sensitization to trying Cavia porcellus.
The test of experimental example 5 hemolytics
1. test objective
Observe Ginkgo Leaf Extract and Dipyridamole Injection man rabbit erythrocyte is had or not haemolysis and agglutination, for data for clinical drug use provides foundation.
2. be subjected to the reagent thing
Tried thing: the Ginkgo Leaf Extract and Dipyridamole Injection of embodiment 1 preparation, specification: 10ml;
Negative control: 0.9% sodium chloride injection, specification: 250ml, Shangdong Hualu Pharmaceutical Co., Ltd. provides;
3. animal subject
1 of white rabbit, body weight 3.0kg, male, prosperous animal cultivation field, Haidian, Beijing provides, the quality certification number: SCXK (capital) 2006-0006.
4. dosage design:
The clinical using dosage of this test dose design considerations Ginkgo Leaf Extract and Dipyridamole Injection (adult's dosage is a 10～25ml, adds in the glucose injection or 0.9% sodium chloride injection of 500ml 5% 2 times on the one).
5. test method
1) preparation of 2% red cell suspension
Rabbit is got blood 20ml, puts into the conical flask that contains bead and vibrates 10 minutes, defibrinates, add the about 10 times of amounts of 0.9% sodium chloride injection, the centrifugal 15min of 2500 commentaries on classics/min, abandoning supernatant adds 0.9% sodium chloride injection again and shakes up gently, centrifugal, abandoning supernatant till not taking on a red color to supernatant several times so repeatedly, is pressed the gained red cell volume then, be mixed with 2% red cell suspension with 0.9% sodium chloride injection, standby.
2) test procedure
Get 7 in test tube, by 1～7 numbering.The 1-5 test tube adds the Ginkgo Leaf Extract and Dipyridamole Injection of 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml embodiment 1 preparation respectively, and adds 0.9% sodium chloride injection respectively to 2.5ml; Add 0.9% sodium chloride injection 2.5ml, distilled water 2.5ml (complete hemolysis contrast) in No. 6, No. 7 test tubes respectively.Last every pipe all adds 2.5ml2% rabbit erythrocyte suspension, shakes up gently, puts in 37 ℃ of water-baths, writes down the haemolysis and the coagulation situation of 15min, 30min, 45min, 1h, 2h, 3h, each test tube of 4h respectively.The result is as shown in table 7.
Haemolysis criterion as a result is:
Complete hemolysis: solution clarification, red, that the pipe end, do not have erythrocyte is residual;
Part haemolysis: solution clarification, red or brown, manage the end and still have a small amount of erythrocyte residual;
Haemolysis not: erythrocyte all sinks, upper strata liquid achromatism and clarity;
Coagulation: though can not disperse after red cell agglutination, the jolting appears in haemolysis not; Or drug precipitation appears.
The hemolytic test result of table 7 Ginkgo Leaf Extract and Dipyridamole Injection
Annotate: "-" be haemolysis not; "+" part haemolysis; " ++ " complete hemolysis
6. result of the test
As can be seen from Table 7: Ginkgo Leaf Extract and Dipyridamole Injection 1-5 pipe does not all cause haemolysis and agglutination in 4h.The result shows: under this experiment condition, the Ginkgo Leaf Extract and Dipyridamole Injection of the inventive method preparation does not have haemolysis and agglutination to rabbit erythrocyte.
The test of embodiment 6 local irritations
1. test objective
Observing the Ginkgo Leaf Extract and Dipyridamole Injection vein slowly injects to the zest effect after the injection of rabbit auricular vein, for data for clinical drug use provides foundation.
2. be subjected to the reagent thing
Tried thing: the Ginkgo Leaf Extract and Dipyridamole Injection of embodiment 1 preparation, specification: 10ml;
Negative control: 0.9% sodium chloride injection, specification: 250ml, Shangdong Hualu Pharmaceutical Co., Ltd. provides;
3. animal subject
3 of white rabbit, body weight 1.7～3.0kg, male and female half and half, prosperous animal cultivation field, Haidian, Beijing provides, the quality certification number: SCXK (capital) 2006-0006.
4. dosage design:
The clinical using dosage of this test dose design considerations Ginkgo Leaf Extract and Dipyridamole Injection (adult's dosage is a 10～25ml, adds in the glucose injection or 0.9% sodium chloride injection of 500ml 5% 2 times on the one).
5. test method
Get 3 of rabbit, consubstantiality left and right sides own control is taked in intravenous injection, and the right side is the administration group, gives embodiment the Ginkgo Leaf Extract and Dipyridamole Injection of 1 preparation; The left side is 0.9% sodium chloride injection matched group.All take to carry out with the concentration (10ml/kg) after 10 times of the stock solution dilutions, once-a-day, continuous 5 days.48h observes after the last administration.
6. perusal result
Observe the injection site rabbit ear vein and have or not hyperemia, hydropic degeneration, scleroma and downright bad phenomenon, write down lesion degree and the rabbit number that vascular tissue changes occurs, the congestion of blood vessel and edema degree are divided into 0, I, II, III degree, 0 degree is no change, the I degree is slight the variation, the II degree is significant change, and the III degree is serious the variation.The result is as shown in table 8.
Table 8 Ginkgo Leaf Extract and Dipyridamole Injection rabbit auricular vein is injected back perusal result
As can be seen from Table 8: 48h after the last administration, perusal administration group and matched group do not have obvious difference.
7. the result of histopathologic examination
Observe changes such as injection site rabbit ear vein vasodilation hyperemia, thrombosis, edema and inflammatory cell infiltration, every content press lesion degree divide normal, light, in, heavy, respectively with-,+, ++, +++represent.
As a result, 48h after the last administration, the administration group is not seen scleroma, downright bad phenomenon apart from the visible inflammatory cell infiltration of injection site 1cm place rabbit ear edge vein, slight hyperemia, edema etc.; Compare with the 1cm place, obviously alleviate apart from injection site 5cm place Sanguis Leporis seu oryctolagi pipe inflammatory cell infiltration, hyperemia, edema; Matched group auricular vein histopathology presents similar variation, and comparing with the administration group does not have significant difference.
The result shows: under this experimental condition, the Ginkgo Leaf Extract and Dipyridamole Injection intravenously administrable of the inventive method preparation does not have tangible blood vessel irritation to the rabbit auricular vein.
Though above with a general description of the specific embodiments, the present invention is described in detail, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. the preparation method of a Ginkgo Leaf Extract and Dipyridamole Injection, it comprises the steps:
1) from Folium Ginkgo, extracts Folium Ginkgo extract;
2) once more Folium Ginkgo extract is extracted with the ethanol of concentration expressed in percentage by volume more than 90%, thus refined ginkgo biloba extract;
3) Folium Ginkgo extract and the dipyridamole after will making with extra care adds water backflow after-filtration, makes ginkgo biloba succi;
4) ginkgo biloba succi, residue dipyridamole and adjuvant are mixed, make Ginkgo Leaf Extract and Dipyridamole Injection; In the described Ginkgo Leaf Extract and Dipyridamole Injection, ginkgo biloba succi is 0.9～1.1: 0.3～0.5 in the weight ratio of Ginkgo total flavones and dipyridamole total amount.
2. preparation method according to claim 1, it is characterized in that, step 2) be specially: add the ethanol of concentration expressed in percentage by volume more than 90% of 8～12 times of weight in the Folium Ginkgo extract, be stirred to dissolving, being concentrated into after the centrifugalize does not have the alcohol flavor, add 10 times of purified water that concentrate the back medicine liquid volume then, leave standstill after stirring cooling, get supernatant, after centrifugalize, concentrate and receive cream, vacuum drying to water content below 6%, pulverize.
3. preparation method according to claim 1 and 2, it is characterized in that, step 3) is specially: in the Folium Ginkgo extract after refining, add the dipyridamole of extract weight 0.5～2% and the purified water of 10～15 times of weight of extract weight, behind the mix homogeneously, regulate pH value to 3.5～5.5, reflux 0.5～1 hour, be cooled to room temperature, repetition measurement pH to 3.5～5.5 are got supernatant then, with 0.2 μ m membrane filtration, the filtrate ultrafilter membrane below 100000 dalton is again carried out ultrafiltration, and last heat sterilization is made ginkgo biloba succi.
4. preparation method according to claim 3 is characterized in that, adds the dipyridamole of Folium Ginkgo extract weight 1% in the step 3).
5. preparation method according to claim 1 is characterized in that step 1) is specially: Folium Ginkgo is carried with the purified water water of 6～8 times of weight, extracted twice, each reflux, extract, 2 hours is filtered, and is concentrated into 1/3～1/5 of original volume behind the merging filtrate; Concentrated solution is stirred cooling, leaves standstill, get supernatant then, filter, with filtrate through the D101 macroporous resin adsorption, ethanol elution, eluent are concentrated into does not have the alcohol flavor, adds the purified water of 1～2 times of Folium Ginkgo weight then, leave standstill after stirring cooling, get supernatant, filter, filtrate is concentrated receive cream, vacuum drying to water content is below 6%, pulverizes.
6. preparation method according to claim 1, it is characterized in that step 4) is specially: ginkgo biloba succi, dipyridamole and adjuvant are dissolved fully with water for injection, and adjust pH is 3.5～5.5, intermediate is through after the assay was approved, with fill behind the 0.22 μ m filtering with microporous membrane.
7. according to claim 1 or 6 described preparation methoies, it is characterized in that adjuvant described in the step 4) is one or more in propylene glycol, glycerol, Tween 80, sodium pyrosulfite, vitamin C, calcium disodium edetate or the PEG400.
8. preparation method according to claim 1 is characterized in that it comprises the steps:
1) Folium Ginkgo is carried with the purified water water of 6～8 times of weight, extracted twice, each reflux, extract, 2 hours is filtered, and is concentrated into 1/3～1/5 of original volume behind the merging filtrate; Concentrated solution is stirred cooling, leaves standstill, get supernatant then, filter, through the D101 macroporous resin adsorption, ethanol elution, eluent are concentrated into does not have the alcohol flavor with filtrate, the purified water that adds 1～2 times of Folium Ginkgo weight then, leave standstill after stirring cooling, get supernatant, filter, filtrate is concentrated receipts cream, vacuum drying to water content is below 6%, pulverizes, and makes Folium Ginkgo extract;
2) in the Folium Ginkgo extract that step 1) makes, add the ethanol of concentration expressed in percentage by volume more than 90% of 8～12 times of weight, be stirred to dissolving, be concentrated into after the centrifugalize and do not have the alcohol flavor, add 10 times of purified water that concentrate the back medicine liquid volume then, leave standstill after the stirring cooling, get supernatant, after centrifugalize, concentrate and receive cream, vacuum drying to water content below 6%, pulverize, thus refined ginkgo biloba extract;
3) in the Folium Ginkgo extract after refining, add the dipyridamole of extract weight 0.5～2% and the purified water of 10～15 times of weight of extract weight, behind the mix homogeneously, regulate pH value to 3.5～5.5, reflux 0.5～1 hour, be cooled to room temperature, repetition measurement pH to 3.5～5.5 are got supernatant then, with 0.2 μ m membrane filtration, the filtrate ultrafilter membrane below 100000 dalton is again carried out ultrafiltration, and last heat sterilization is made ginkgo biloba succi;
4) ginkgo biloba succi, remaining dipyridamole and adjuvant are dissolved fully with water for injection, adjust pH is 3.5～5.5, and intermediate is through after the assay was approved, with fill behind the 0.22 μ m filtering with microporous membrane, sterilization.
9. with any Ginkgo Leaf Extract and Dipyridamole Injection that described preparation method makes of claim 1-8.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102793730A (en) * | 2012-08-31 | 2012-11-28 | 山西普德药业股份有限公司 | Ginkgo-damole medicinal composition and preparation method thereof |
| CN104523771A (en) * | 2015-01-13 | 2015-04-22 | 中国药科大学 | Ginkgo leaf total flavones and preparing method thereof |
| CN106974945A (en) * | 2017-04-11 | 2017-07-25 | 珠海亿邦医药有限公司 | A kind of Ginkgo Damo injection and preparation method thereof |
| CN107184616A (en) * | 2017-05-07 | 2017-09-22 | 珠海亿邦医药有限公司 | A kind of preparation method of Ginkgo Damo injection |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1939357A (en) * | 2005-09-27 | 2007-04-04 | 巴里莫尔制药（通化）有限公司 | Preparation of Ginkgo Damo injection |
-
2010
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1939357A (en) * | 2005-09-27 | 2007-04-04 | 巴里莫尔制药（通化）有限公司 | Preparation of Ginkgo Damo injection |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102793730A (en) * | 2012-08-31 | 2012-11-28 | 山西普德药业股份有限公司 | Ginkgo-damole medicinal composition and preparation method thereof |
| CN102793730B (en) * | 2012-08-31 | 2013-11-06 | 山西普德药业股份有限公司 | Ginkgo-damole medicinal composition and preparation method thereof |
| CN104523771A (en) * | 2015-01-13 | 2015-04-22 | 中国药科大学 | Ginkgo leaf total flavones and preparing method thereof |
| CN104523771B (en) * | 2015-01-13 | 2018-01-09 | 中国药科大学 | A kind of ginkgo leaf total flavonoid and preparation method thereof |
| CN106974945A (en) * | 2017-04-11 | 2017-07-25 | 珠海亿邦医药有限公司 | A kind of Ginkgo Damo injection and preparation method thereof |
| CN106974945B (en) * | 2017-04-11 | 2018-04-10 | 珠海亿邦医药有限公司 | A kind of Ginkgo Damo injection and preparation method thereof |
| CN107184616A (en) * | 2017-05-07 | 2017-09-22 | 珠海亿邦医药有限公司 | A kind of preparation method of Ginkgo Damo injection |
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