CN1911257A - Extractive of ginkgo tree leaves, prepn. method and application thereof - Google Patents
Extractive of ginkgo tree leaves, prepn. method and application thereof Download PDFInfo
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- CN1911257A CN1911257A CN 200510090129 CN200510090129A CN1911257A CN 1911257 A CN1911257 A CN 1911257A CN 200510090129 CN200510090129 CN 200510090129 CN 200510090129 A CN200510090129 A CN 200510090129A CN 1911257 A CN1911257 A CN 1911257A
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Abstract
A gingko leaf extract used for preparing liquid injection or freeze-dried powder injection is prepared from gingko leaves through breaking, thermal reflux extracting with the aqueous solution of alcohol, vacuum volatilizing of solvent, depositing in ammonia water to remove polyphenol, extracting with n-hexane to remove ginkgolic acid, adsorbing by macroreticular resin, separating, refining, ultrafiltration and spray drying.
Description
Technical field
The present invention relates to the production technology of Folium Ginkgo extract and the preparation method of pharmaceutical formulation, particularly relate to the preparation method of gingko leaf extract injection and lyophilized injectable powder.
Background technology
Disturbance of blood circulation such as cardiovascular and cerebrovascular vessel, peripheral blood vessel disease is one of clinical modal disease, and being influences people ' s health and life-prolonging one class principal disease.Folium Ginkgo extract and preparation thereof more and more are subjected to the attention of countries in the world in the effect aspect cardiovascular and cerebrovascular vessel, the peripheral angiopathy.Semen Ginkgo (Ginkgo bilobaL.) for losing lonely plant ancient times, has another name called maidenhair tree.Semen Ginkgo is the rare species of China, also is precious in the world resources of medicinal plant.Especially over nearest 40 years, in succession Semen Ginkgo chemical constituent, pharmacological action are furtherd investigate both at home and abroad, a large amount of pharmacy, pharmacology, drug effect, clinical research show: the main active of Folium Ginkgo extract is Ginkgo total flavones alcohol glycoside, terpene lactones chemical compound, and this two constituents also is an important indicator of passing judgment on the Folium Ginkgo extract quality.Clinical and pharmacological research finds that Folium Ginkgo extract has the cardiac function of enhancing, microcirculation improvement, reduction blood viscosity, antagonism platelet activating factor (PAF), remove interior free yl and tangible fibrinogenolysis, damage after suppressing blood flow and pouring into strengthens heart cell to the tolerance of ischemia etc.In the 60-70 age, especially Germany, the existing a certain amount of Folium Ginkgo extract of France are applied to clinical and have proved its effect abroad, have developed into tens countries such as Europe, Asia, U.S. at present.At present utilize Folium Ginkgo extract to develop many medicine and the health foods that can effectively treat and prevent cardiovascular and cerebrovascular vessel, peripheral blood vessel, nervous system, diseases of urinary system both at home and abroad, Folium Ginkgo extract and preparation thereof have had certain application in clinical and people's life, but level of application is not high, ginkgo agent is used for clinical mainly as ancillary drug, main cause is that the effective ingredient amount is on the low side in the Folium Ginkgo extract quality standard, has influenced curative effect.The current purpose Folium Ginkgo extract (Tebonin that is used for the treatment of commonly used
, Tanakan
, Roekan
Or Egb761) ginkgetin glycosides 24%, terpene lactones 6% in its quality standard, alkylphenol compounds is lower than 10ppm.The preparation that application said extracted thing is done has been the theme of some patent, as European patent EP 431 535, EP 431 536 and US 5,389,370.The nineties, the scientific research personnel of various countries tries one's best and improves the content of effective ingredient flavonol glycosides and Folium Ginkgo terpene lactones in the Folium Ginkgo extract, reduce the content of ginkgoic acid simultaneously as far as possible, ginkgoic acid is the principal element of Folium Ginkgo extract toxic action, because of it can produce serious gastrointestinal side effect, headache, skin irritation and cause edema, clinical practice can cause severe anaphylactic reaction.The theme that United States Patent (USP) (US4,428,965) relates to be the antagonism ginkgoic acid due to desensitization material hypersensitive and method, the Folium Ginkgo extract that the subject matter of Chinese patent CN1159022C also relates to high active constituent content gets preparation method.
Because the Folium Ginkgo extract that all belongs to the low content effective ingredient that the extracting method of prior art obtains, can not satisfy clinical curative effect requirement, especially be not suitable for ejection preparation, cause Folium Ginkgo extract active constituent content reason on the low side much relations to be arranged with the production technology of Folium Ginkgo extract, though and the preparation of Folium Ginkgo such as tablet, oral liquid, capsule etc. are in wide clinical application, but the shortcoming that oral formulations exists is very obvious, as absorb unhappy, bioavailability is not high, influences treatment of diseases.The gingko leaf extract injection of 99124228 Patent publish for example, employing adds metal ion complex and polysulfones or polyethylene with extract in the preparation of injection be that the mode of the Hollow Fiber Ultrafiltration of material is removed impurity, the shortcoming of the mode of this ultrafiltration remove impurity is that it is to separate the organic macromolecule material, colloid, ultramicron, antibacterial is main, is primarily aimed at macromolecular substances.Only the concentrated etc. of degree of depth the turbid descending clarification and macromolecular substances had effect, also need high temperature sterilize if the extract after the ultrafiltration is applied to injection.
The existing Technology of the Folium Ginkgo extract of having reported has: resin absorption method of purification, hot dipping, microwave extraction method, CO
2Super critical extraction etc.The subject matter that prior art exists: extract not high, the process route of effective ingredient total amount inadequately rationally or industrialized condition does not possess and effective ingredient can not be directly used in injection type etc.
How to obtain the Folium Ginkgo extract of the low impurity high-load effective ingredient of high-purity of suitable injection, become and study focus in the industry.
Summary of the invention
The objective of the invention is to propose a kind of preparation method of high active constituent content Folium Ginkgo extract, extract Ginkgo total flavones＞64% by this method preparation, Ginkgo total lactones＞11%, the procyanidin compounds that has higher antioxidant activity simultaneously contains 7-10%, toxic component ginkgoic acid compounds＜5ppm.The innovative point of this invention is advanced ceramic membrane ultrafitration technology is applied among the preparation technology of extract, not only improved active constituent content greatly, also effectively remove the vegetalitas macromole that causes allergic reaction easily in the vegetable drug, increased the effectiveness and the safety of the preparation of developing.
Another innovative point of the present invention is to provide that another effective ingredient procyanidin compounds gets content assaying method in the extract, has further determined the composition of effective ingredient in the extract, has optimized the ratio of different effective ingredient.
The preparation method that has also proposed gingko leaf extract injection and injectable powder of the present invention, the quality of the pharmaceutical preparations that makes by this method is stable, determined curative effect.
The invention provides a kind of preparation method of Folium Ginkgo extract, it is characterized in that, may further comprise the steps: the alcohol reflux of the 50%-75% concentration that dry Folium Ginkgo adding 4-10 doubly measures at least 1 time, extracting solution is merged filtration, reclaim under reduced pressure is removed ethanol and is obtained concentrated solution; In concentrated solution, add isopyknic water, place, filter or the centrifugal precipitation of removing; Filtrate or supernatant are regulated PH to 8-10, place to make precipitation fully, filter or the centrifugal precipitation (tannin and polymerized polyphenolic class impurity) of removing; In filtrate or supernatant, add acid and regulate PH to 2.5-4.0 (this pH value is the pH value of extracting solution before the adding ammonia), add n-hexane extraction again; Water liquid after the extraction is crossed macroporous adsorptive resins, earlier with the deionized water eluting, abandons water lotion; With the 60%-70% ethanol elution, collect alcohol eluen again, the alcohol solvent is flung in decompression, obtains containing the solution of pure 5-10%; The solution that obtains is through membrane ultrafiltration, and molecular cut off is 0.7-5 ten thousand, and ultrafiltrate is through concentrating under reduced pressure.
Above-mentioned preparation method, wherein the ethanol of Ti Quing is preferably the ethanol of 60% concentration; Water is preferably deionized water; It is 9 that described strong aqua ammonia is transferred pH value; Regulating the acid that pH value adopted is hydrochloric acid or sulphuric acid, preferred hydrochloric acid, and pH value is 3; The concentration of alcohol that described eluting is used is 70%.
In order to reach the raising active constituent content, effectively remove the content of the vegetalitas macromole (as ginkgoic acid etc.) that causes allergic reaction easily in the vegetable drug, the effectiveness and the safety of the preparation of developing have been increased, improve simultaneously in the prior art and to generally acknowledge as impurity and in the present invention as the content of the polyphenol compound (for example proanthocyanidin) of the tannin characteristic of effective ingredient, ultrafiltration described in the extracting method of above-mentioned Folium Ginkgo extract is preferably ceramic membrane ultrafitration, hold back branch and be 1-3 ten thousand in amount, the extract that obtains like this lacks than the refining extract impurity that obtains of other membrane ultrafiltration methods, molecular weight distribution is more concentrated, pharmacological effect is more obvious, and can in preparation injection process, reach the requirement of the injection of pharmacopeia regulation without high temperature sterilize, this ceramic membrane that is we find in experiment sieving is applied to an advantage of the Folium Ginkgo extract of injection, is that other membrane ultrafiltration are irreplaceable.
In a preferred embodiment of the invention, the extracting method of described Folium Ginkgo extract may further comprise the steps:
(1) get dry Folium Ginkgo 20-40 order coarse powder, 60% the alcohol reflux that adds that 4-10 doubly measures 2-4 time merges filtration with extracting solution, and reclaim under reduced pressure is to 1/10 of cumulative volume.
(2) deionized water of adding equal volume in the concentrated solution is placed 12hr in the shady and cool environment, filters or centrifugal removing.
(3) fully stir, add strong aqua ammonia in filtrate that obtains in (2) or the supernatant, regulate PH to 8-10, place 1hr, filtration or the centrifugal precipitated impurities of removing.
(4) add hydrochloric acid in filtrate in (3) or the supernatant and regulate PH to 2.5-4.0, add n-hexane extraction 2-3 time.
(5) the water liquid after step (4) extraction is crossed macroporous adsorptive resins, with the deionized water eluting of 2 times of bed volumes, abandons water lotion earlier; With the 60%-70% ethanol elution of 3 times of bed volumes, collect alcohol eluen again, the alcohol solvent is flung in decompression, obtains containing the solution of pure 5-10%.
(6) the solution ceramic membrane ultrafitration that obtains through step (5), molecular cut off is 1-3 ten thousand, ultrafiltrate promptly obtains Folium Ginkgo extract through concentrating under reduced pressure after the concentrated solution spray drying.
The present invention also provides a kind of Folium Ginkgo extract, this Folium Ginkgo extract adopts said method to make, wherein the gross weight content of effective component of ginkgo leaf is (80%-95% more than 80%, preferred 80%-85%), this extract comprises total flavones, terpene lactones and procyanidin at least, this content of total flavone is (for example 64%-70%) more than 64%, and the content of terpene lactones is (for example 11%-15%) more than 11%, and the content of procyanidin is 7% (for example 7%-10%); Wherein, this Folium Ginkgo extract also comprises ginkgoic acid, and its weight content is no more than 5ppm (for example 2-4ppm).
The present invention also provides a kind of pharmaceutical composition that contains above-mentioned Folium Ginkgo extract, and wherein, said composition is an injection preparation, comprises injection and lyophilized injectable powder.
Above-mentioned injection preparation comprises 1 part of Folium Ginkgo extract, 15-25 part sorbitol or mannitol.
What the present invention also provided total flavones in the Folium Ginkgo extract contains the survey method, and its operating procedure is as follows:
(1) preparation of reference substance solution: it is an amount of that precision takes by weighing dry rutin, in volumetric flask, gets the reference substance stock solution with 70% ethanol standardize solution.
(2) drafting of standard curve: get not commensurability reference substance storing solution to volumetric flask, add quantitative water, pH is acetic acid-sodium acetate buffer solution of 4.5, the liquor alumini chloridi of 0.1mol/L, use 70% ethanol standardize solution then, measure trap, drawing standard curve at the 270nm place.
(3) sample determination: precision takes by weighing an amount of sample,, handles by above-mentioned identical method in volumetric flask with 70% ethanol standardize solution, measures absorption value at the 270nm place, presses anhydride calculates total flavones with rutin amount.Should be 90～110% of labelled amount.
The invention provides the content assaying method of Ginkgo total lactones, its operating procedure is as follows:
(1) preparation of reference substance solution: precision takes by weighing ginkalide A, B, C and bilobalide reference substance place volumetric flask, add the methanol ultrasonic dissolution after, be chilled to room temperature, rare with mobile phase to scale, shake up, be stock solution.Precision is measured 0.5ml, and the 1ml stock solution moves into the 5ml volumetric flask, is diluted to scale with mobile phase, shakes up, in contrast product solution.
(2) preparation of sample solution: it is an amount of that precision takes by weighing extract, place beaker, add that ethanol is ultrasonic to make dissolving, the solution precision is transferred in the separatory funnel, with the water saturation ethyl acetate extraction, totally twice, divide and get in the ethyl acetate stratification evaporating dish water bath method, residue dissolves with small amount of methanol and is transferred in the volumetric flask, with mobile phase dilution and be settled to scale, shake up, standby.
(3) precision is measured each 20 μ l of contrast and need testing solution, injects high performance liquid chromatograph, calculates the content of total lactone in the test sample with the two-point method logarithmic equation.
The invention provides the method for measuring total flavonoids in the Folium Ginkgo extract, concrete steps are as follows:
(1) preparation of reference substance solution: it is an amount of that precision takes by weighing Quercetin, with standardize solution behind the dissolve with methanol in volumetric flask, product solution in contrast.
(2) it is an amount of that precision takes by weighing exsiccant ginkgo leaf standard extract, puts in the round bottom ground flask, uses dissolve with methanol, adds 25% hydrochloric acid again, mixing was put on the boiling water bath back hydrolysis 1 hour, put and was chilled to room temperature, quantitatively was transferred in the volumetric flask,, shake up to scale with methanol constant volume, as need testing solution.
(3) accurate respectively above-mentioned reference substance solution and each 10 μ l of need testing solution of drawing inject chromatograph of liquid under above-mentioned chromatographic condition, measure, and calculate the content of total flavonoids with following formula.
Total flavonoids (mg)=(Q * 2.51+K * 2.64 * 1.05+I * 2.39 * 0.95) * R * extension rate/1000
Q=Quercetin peak area
K=kaempferide peak area
I=isorhamnetin peak area
The invention provides the method for measuring ginkgoic acid in the Folium Ginkgo extract, concrete steps are as follows:
(1) the preparation precision of reference substance solution takes by weighing the ginkgoic acid reference substance and arrives suitable concentration concentration, product solution in contrast with methanol constant volume in right amount.
(2) it is an amount of that the preparation precision of sample solution takes by weighing extract, puts in the tool plug test tube, adds dehydrated alcohol, ultrasonicly makes dissolving, be cooled to room temperature after, add an amount of normal hexane and saturated sodium sulfate (regulating pH value to 3) and an amount of kieselguhr with sulphuric acid.Vibration is left standstill, treat layering after, the accurate upper organic phase of drawing is removed extract under protection of nitrogen gas in another test tube, residue is dissolved in the methanol, as need testing solution.
(3) adopt chromatograph of liquid to measure, the record chromatogram calculates the content of total ginkgolic acids and must not cross 5/1000000ths.
The pharmacology pharmacodynamic experiment
The experimentation of the anti-myocardial ischemia of 1 Folium Ginkgo extract
Mice is closed the influence of trachea electrocardio extinction time
Get 60 qualified after testing mices, be divided into six groups at random, every group 10,1.5% concentration pentobarbital sodium lumbar injection 30mg/kg anesthesia, facing upward the position is fixed on the mice dissecting table, the cervical region cropping, operation separates trachea, insert the mice extremity with ECG electrode, gather mark II lead electrocardiogram, the mouse tail vein injection administration, first group of injection isometric(al) normal saline, second group of tail vein injection Ginaton injection 17.5mg/kg, the three～five group respectively through mouse tail vein injection Folium Ginkgo 20mg/kg, 10mg/kg, 5mg/kg irritates stomach Folium Ginkgo extract 10mg/kg for the 6th group, closed mice trachea (small artery is clamped the mice trachea) behind the injectable drug in 30 minutes, write down every group every mice and close the time that trachea to mice electrocardio disappears, the testing data significance test is handled with t check between group, and the result shows that Folium Ginkgo extract holds one's breath behind the pipe to the little mousetrap, electrocardiogram extinction time and normal saline group be the tool notable difference relatively, can obviously prolong electrocardio extinction time P＜0.05.The Ginaton injection has the electrocardio of prolongation extinction time P＜0.05 equally.The Folium Ginkgo extract group of Isodose compares, and difference is not remarkable.Irritate stomach Folium Ginkgo extract 10mg/kg group mice and normal saline group relatively, difference is not remarkable.
2 Folium Ginkgo extract are to the protective effect of ischemic myocardium
Folium Ginkgo extract brings out the influence of rat heart muscle ischemia ECG to pituitrin
Get 60 of Wistar rats, male and female half and half, body weight 250-300g, average weight 271.3 ± 18.0g is divided into six groups at random, 10 every group.First group of rat is by tail vein injection isometric(al) normal saline; Second group of rat injection Ginaton injection 8.5mg/kg; The three～five group of rat is by tail vein injection Folium Ginkgo extract 10mg/kg, 5mg/kg and 2.5mg/kg, the 6th group of rat oral gavage Folium Ginkgo extract 5mg/kg, injection is 2 days continuously, after the last administration 30 minutes, by tail vein injection pituitrin 0.6 μ/kg, record V3 marks the II lead electrocardiogram and is annotating the 15th of pituitrin respectively, 30,45 seconds, 1,1.5,2, electrocardiogram changed in 5 minutes, analyze ST, the T ripple changes, and the gained testing data is checked with t, the difference that compares administration group and normal saline group, after the result shows the rat intravenous injection pituitrin, the ST section, the T ripple has obviously and increases, and after giving Folium Ginkgo extract, the ST section of raising has obvious recovery, the T ripple is descending manner and recovers, at injection of pituitrin after 2 minutes, and the ST section, it is normal that the T ripple is tending towards substantially, administration group and matched group compare, no significant difference.
3 Folium Ginkgo extract are to the influence of acute myocardial infarction rat myocardium block area
The Wistar rat, male and female half and half, body weight 240-290g is divided into seven groups at random, and 10 every group, distribute unusual and pathologic Q ripple person before ligation, occurs and dead Mus in 24 hours removing arteria coronaria after the operation at any time, guaranteeing at last has 70 rats to be for experiment.
During test respectively by quiet injecting normal saline of tail or Folium Ginkgo extract, successive administration two days, after the last administration 30 minutes, lumbar injection 1.5% pentobarbital sodium 30mg/kg faced upward the position and is fixed on the operating-table, get median incision, cut skin, along the breastbone center left, pure property separating muscle, expose the 3rd, 4 ribs, the subsides left border of sternum cuts off, and opens otch with small-sized machine for chest-opening expansion heart is exposed well, mentions pericardium gently with the ophthalmology tweezer, carefully cut off, mention left auricle with mosquito forceps, export 3.0mm place, below and 5mm place, dual ligation branch of coronary artery in branch of coronary artery apart from arteria coronaria with the ophthalmology suture needle, send heart back to thoracic cavity rapidly then, sew up and close breast, the sacrificed by decapitation rat is got blood, survey FFA in its serum, CPK content sees Table 6.Win heart, in physiological salt liquid, wash quiet surplus blood, cut off atrium and trunk along coronary sulcus, satisfactory chamber weight on torsion balance is cut into ventricle the 4-5 sheet then, and (37 ℃ of constant temperatures dye down in the nitro blue tetrazolium dyeing liquor, and (normal myocardium was dyed skipper in 15 minutes to put N-BT, infarcted myocardium is not painted), cut off coloured part with shears or scalpel, remaining is infarcted myocardium, calculates the percentage rate that infarct accounts for the heart area.Administration group and normal saline group are compared, with the significance of statistical calculations difference.The result shows that gingko leaf preparation can protect damaged myocardium, no matter is myocardial infarction weight or myocardial infarction area, with the normal saline group significant difference P＜0.01 is arranged more all.
4 Folium Ginkgo extract are to the influence of platelet aggregation and thrombus in vivo
Folium Ginkgo extract is to the accumulative influence of rabbit platelet
Get 12 hours rabbit of fasting, with pentobarbital sodium 30mg/kg ear vein injecting anesthetic, operation separates common carotid artery, clip inserts the polyethylene tube blood-letting to being equipped with in advance in the 3.8% liquor sodii citratis silication test tube, the ratio of blood and anticoagulant is 9: 1, with blood and anticoagulant mixing gently, centrifugal 4 minutes of 1000rpm, draw the liquid of upper strata ecru, obtain platelet rich plasma (PRP), with the centrifugal again 3000rpm of remaining blood 10 minutes, draw supernatant, obtain platelet poor plasma (PPP).
Connect platelet aggregation instrument and monitor power supply, made it to give heat 30 minutes, regulate simultaneously and assemble instrument constant temperature knob, make it constant temperature at 37 ± 0.5 ℃, earlier PPP was given warm 3-5 minute, observe pointer between 70-90, must not surpass 100Abs, press " E " key, " Z " key lamp is bright, takes out the PPP pipe, and the PRP pipe is placed instrument connection, add stirring rod, press " STIRRER " key, hear the stirring sound that rustles after, press " E " key, machine begins to print laboratory report figure, after printing finishes, add Folium Ginkgo extract 20 μ g earlier, 10 μ g, 5 μ g and Ginaton injection 10 μ g add ADP liquid 200 μ l (20 μ g) after 1 minute, press " START " key, promptly print empirical curve and data after 5 minutes.
After each organizes off-test, according to A in the data (max) maximum agglutination rate, calculate and suppress percentage rate (%), administration group and normal saline group are relatively and carry out t and check.The results are shown in following table
The table Folium Ginkgo extract is to the accumulative influence of Sanguis Leporis seu oryctolagi plate
| Group | Dosage (μ g/ pipe) | Anticoagulant percentage rate (%) (X ± SD) |
| Normal saline Ginaton injection Folium Ginkgo extract Folium Ginkgo extract Folium Ginkgo extract | - 10 20 10 5 | 52.08±2.46 19.51±6.93 *** 18.87±4.28 *** 38.69±4.43 *** 39.22±3.66 *** |
Annotate: sample number n=10
*Compare P＜0.01 with the normal saline matched group
* *Compare P＜0.001 with the normal saline matched group
Show that by last watch test data Folium Ginkgo extract is assembled rabbit platelet very obvious suppression effect P＜0.001.
The present invention proposes the preparation method of Folium Ginkgo extract freeze-dried powder, may further comprise the steps:
Dosing: the sorbitol that accurately takes by weighing recipe quantity adds 5% ethanol 4500ml, stirs to make dissolving fully, adds the Folium Ginkgo extract of recipe quantity, stirring makes dissolving fully, and is an amount of with 20%NaOH, transfers pH to be about 5.5, add 5% alcoholic solution at last to prescribed volume, stir and make mix homogeneously.
Filter: filter through 0.45 μ m and the pressurization of 0.2 μ m microporous filter membrane respectively.
Fill: with filterable medicinal liquid by every 5.1-5.2ml sterile filling in the ampoule bottle of having handled.
Preparing ginkgo biloba leaf extract injection of the present invention may further comprise the steps:
Dosing: it is an amount of that the mannitol that accurately takes by weighing recipe quantity adds 5% ethanol, stirring makes dissolving fully, the Folium Ginkgo extract that adds recipe quantity, stirring makes dissolving fully, add the disodium hydrogen phosphate of recipe quantity again, an amount of with 20%NaOH, transfer pH to be about 6.0, add 5% alcoholic solution at last to prescribed volume, stir and make mix homogeneously.
Filter: filter through 0.45 μ m and the pressurization of 0.2 μ m microporous filter membrane respectively.
Fill: the medicinal liquid after will filtering by every 3.1ml fill in the cillin bottle of having handled.
Lyophilizing: our temperature of eutectic point by measuring it of the above-mentioned solution of this product is-23 ℃.So we are being lower than temperature of eutectic point-30 ℃ pre-freeze 5 hours, can this product is fully freezing fully.In placing the vacuum freeze-drying machine, the freeze dryer vacuum degree control is about 0.1～0.3 millibar, and condenser temperature is-40 ℃.Baffle temperature kept 15 hours for-30 ℃ of maintenances are warming up to-10 ℃ after 5 hours, temperature was risen to 30 ℃ again and kept 8 hours.The gland outlet.
The gingko leaf extract injection that the present invention proposes and the preparation method of injectable powder, the extraction process route of Folium Ginkgo extract is more reasonable, and extraction, remove impurity, purification, extraction, separation and subtractive process meet big production requirement; Production cost is low; The effective component extraction rate height (Folium Ginkgo extract total flavonoid glycoside＞64% that this production technology obtains, terpene lactones＞11%, procyanidin 〉=7%, ginkgoic acid constituents＜5ppm reaches the requirement of Chinese medicine injection with raw material); The condition that possesses suitability for industrialized production.The preparation method of gingko leaf extract injection of the present invention and injectable powder studies show that through stability test stable preparation process is feasible, has increased dosage form, has satisfied clinical needs.Safety testing shows that gingko leaf extract injection of the present invention and injectable powder all do not have irritated reaction and haemolysis, and intravenous injection is to the blood vessel nonirritant.Has effect widely in the cardiovascular disease field.
The present invention also has been a large amount of contrast experiments, total effective ingredient all is lower than 80% in the Folium Ginkgo extract that the preparation method that proof is put down in writing with existing document obtains, not clearly record of content to procyanidin, if reached allergin ginkgoic acid constituents＜5ppm, then the lead ion in the product promptly exceeds standard, and can't be used for the composition of injection preparation at all; And the present invention adopts the ethanol extraction of garbled concentration, the series remove impurity, and last macroporous adsorbent resin is refining, and ceramic membrane filter, total flavonoid glycoside in the extract that obtains＞64%, terpene lactones＞11%, procyanidin 〉=7%, ginkgoic acid constituents＜5ppm reaches the requirement of Chinese medicine injection with raw material fully.
The specific embodiment
Embodiment 1: the preparation of Folium Ginkgo extract
The dry Folium Ginkgo of tested number was pulverized 40 mesh sieve, obtain medicinal material coarse powder, 60% ethanol that adds 8 times of amounts, reflux 1.5hr, leach extracting solution, 60% ethanol that adds equal volume carries out the 2nd time and extracts, merge 2 times extracting liquid filtering, reclaim under reduced pressure is flung to solvent, to the concentrated solution volume be 1/10 (being that solid contents is 20～30% concentrated solution) of cumulative volume, add the deionized water of equal volume, place 12hr in the shady and cool environment, fully separate out insoluble matter, filter or the centrifugal precipitation of removing; Under well-beaten condition, in filtrate or supernatant, add strong aqua ammonia, regulate PH to 9, place 1hr in the shady and cool environment, filter or the centrifugal precipitation of removing; Add sulphuric acid adjusting PH to 3 (this pH value adds the pH value of the preceding extracting solution of ammonia) in filtrate or supernatant, the n-hexane extraction of V/3 volume extracts 3 times; Water is flung to solvent and is promptly got ginkgo biloba crude extract.Sample on the D101 type macroporous adsorbent resin, 1.5kg (Folium Ginkgo medical material)/kg (resin); Earlier with the deionized water eluting of 2 times of bed volumes, abandon water lotion behind the last sample; With 70% ethanol elution of 3 times of bed volumes, collect alcohol eluen again, solvent is flung in decompression, passes through ceramic membrane ultrafitration again, and molecular cut off is 1-3 ten thousand, obtains Folium Ginkgo extract.
Embodiment 2: the preparation of gingko leaf extract injection
Take by weighing the 200g sorbitol and add 5% ethanol 4500ml, stir and to make dissolving fully, add the 10g Folium Ginkgo extract, stir and make dissolving fully, an amount of with 20%NaOH, transfer pH to be about 5.5, add 5% alcoholic solution at last to prescribed volume, stir and make mix homogeneously.
Filter: filter through 0.45 μ m and the pressurization of 0.2 μ m microporous filter membrane respectively.
Fill: will filter medicinal liquid and cut open in the bottle in the peace of having handled by every 5.1-5.2ml sterile filling.
Embodiment 3: the preparation of ginkgo leaf extract powder injection
Taking by weighing 150g mannitol, to add 5% ethanol an amount of, stirring makes dissolving fully, add the 10g Folium Ginkgo extract, stirring makes dissolving fully, add the 26g disodium hydrogen phosphate again, an amount of with 20%NaOH, transfer pH to be about 6.0, add 5% alcoholic solution at last to prescribed volume, stir and make mix homogeneously.
Filter: filter through 0.45 μ m and the pressurization of 0.2 μ m microporous filter membrane respectively.
Fill: the medicinal liquid after will filtering by every 3.1ml fill in the cillin bottle of having handled.
Lyophilizing: the above-mentioned solution temperature of eutectic point of this product is-23 ℃.Be lower than temperature of eutectic point-30 ℃ pre-freeze 5 hours, can this product is fully freezing fully.In placing the vacuum freeze-drying machine, the freeze dryer vacuum degree control is about 0.1～0.3 millibar, and condenser temperature is-40 ℃.Baffle temperature kept 15 hours for-30 ℃ of maintenances are warming up to-10 ℃ after 5 hours, temperature was risen to 30 ℃ again and kept 8 hours.The gland outlet.
Embodiment 4: the gingko leaf extract injection prescription
Folium Ginkgo extract 8.0-12.0g
Sorbitol 300-500g
5% ethanol adds to 5000ml
Make 1000 altogether
(1) sorbitol that takes by weighing recipe quantity adds 5% ethanol 4500ml, stirs to make dissolving fully, adds the Folium Ginkgo extract of recipe quantity, stirring makes dissolving fully, and is an amount of with 20%NaOH, transfers pH to be about 5.0-6.0, add 5% alcoholic solution at last to prescribed volume, stir and make mix homogeneously.
(2) filter: filter through 0.45 μ m and the pressurization of 0.2 μ m microporous filter membrane respectively.
(3) fill: the medicinal liquid after will filtering cuts open in the bottle in the peace of having handled by every 5.1-5.2ml sterile filling.
Embodiment 5: Folium Ginkgo extract injection powder injection prescription:
Folium Ginkgo extract 8.0-12.0g
Mannitol 120-180g
Disodium hydrogen phosphate 20-30g
5% ethanol adds to 3000ml
Make 1000 bottles altogether
(1) it is an amount of that the mannitol that takes by weighing recipe quantity adds 5% ethanol, stirring makes dissolving fully, the Folium Ginkgo extract that adds recipe quantity, stirring makes dissolving fully, add the disodium hydrogen phosphate of recipe quantity again, an amount of with 20%NaOH, transfer pH to be about 5.5-7.0, add 5% alcoholic solution at last to prescribed volume, stir and make mix homogeneously.
(2) filter: filter through 0.45 μ m and the pressurization of 0.2 μ m microporous filter membrane respectively.
(3) fill: the medicinal liquid after will filtering by every 3.1ml fill in the cillin bottle of having handled.
(4) lyophilizing: be lower than temperature of eutectic point pre-freeze certain hour, this product is fully freezing fully.In placing the vacuum freeze-drying machine, the freeze dryer vacuum degree control is about 0.1～0.5 millibar, and condenser temperature is-30--50 ℃.Baffle temperature for-20--30 ℃ keep being warming up to after 4-6 hour-5--10 ℃ kept 12-18 hour, temperature risen to 25-30 ℃ again and kept the gland outlet 8 hours.
Claims (10)
1. the preparation method of a Folium Ginkgo extract is characterized in that, may further comprise the steps:
(1) dry Folium Ginkgo adds the alcohol reflux at least 1 time of the 50%-75% concentration that 4-10 doubly measures, and extracting solution is merged filter, and reclaim under reduced pressure is removed ethanol and obtained concentrated solution;
(2) in concentrated solution, add isopyknic water, place, filter or the centrifugal precipitation of removing;
(3) filtrate or supernatant are regulated PH to 8-10, place to make precipitation fully, filter or the centrifugal precipitation of removing;
(4) in filtrate or supernatant, add acid and regulate PH to 2.5-4.0, add n-hexane extraction again;
(5) the water liquid after the extraction is crossed macroporous adsorptive resins, earlier with the deionized water eluting, abandons water lotion; With the 60%-70% ethanol elution, collect alcohol eluen again, the alcohol solvent is flung in decompression, obtains containing the solution of pure 5-10%;
(6) the solution process membrane ultrafiltration that obtains through step (5), molecular cut off is 0.7-5 ten thousand, ultrafiltrate is through concentrating under reduced pressure.
2. the preparation method of Folium Ginkgo extract as claimed in claim 1 is characterized in that, the mixed liquid of water alcohol described in the step (1) is the ethanol of 60% concentration; Water in the step (2) is deionized water; Transferring pH value with strong aqua ammonia in the step (3) is 9; Acid described in the step (4) is hydrochloric acid, and pH value is 3; The concentration of alcohol that eluting described in the step (5) is used is 70%.
3. the extracting method of Folium Ginkgo extract as claimed in claim 1 is characterized in that, the ultrafiltration described in the step (6) is a ceramic membrane ultrafitration, and molecular cut off is 1-3 ten thousand.
4. the extracting method of Folium Ginkgo extract as claimed in claim 1 is characterized in that, may further comprise the steps:
(1) get dry Folium Ginkgo 20-40 order coarse powder, 60% the alcohol reflux that adds that 4-10 doubly measures 2-4 time merges filtration with extracting solution, and reclaim under reduced pressure is to 1/10 of cumulative volume;
(2) deionized water of adding equal volume in the concentrated solution is placed 12hr in the shady and cool environment, filters or centrifugal removing;
(3) fully stir, add strong aqua ammonia in filtrate that obtains in (2) or the supernatant, regulate PH to 8-10, place 1hr, filtration or the centrifugal precipitated impurities of removing;
(4) add hydrochloric acid in filtrate in (3) or the supernatant and regulate PH to 2.5-4.0, add n-hexane extraction 2-3 time;
(5) the water liquid after step (4) extraction is crossed macroporous adsorptive resins, with the deionized water eluting of 2 times of bed volumes, abandons water lotion earlier; With the 60%-70% ethanol elution of 3 times of bed volumes, collect alcohol eluen again, the alcohol solvent is flung in decompression, obtains containing the solution of pure 5-10%;
(6) the solution ceramic membrane ultrafitration that obtains through step (5), molecular cut off is 1-3 ten thousand, ultrafiltrate promptly obtains Folium Ginkgo extract through concentrating under reduced pressure after the concentrated solution spray drying.
5. a Folium Ginkgo extract is characterized in that, this Folium Ginkgo extract adopts the described method of claim 1 to make, and wherein the gross weight content of effective component of ginkgo leaf is more than 80%, and this extract comprises total flavones, terpene lactones and procyanidin at least.
6. Folium Ginkgo extract as claimed in claim 5 is characterized in that, described content of total flavone is more than 64%, and the content of terpene lactones is more than 11%, and the content of procyanidin is 7%.
7. Folium Ginkgo extract as claimed in claim 5 is characterized in that described Folium Ginkgo extract also comprises ginkgoic acid, and its weight content is no more than 5ppm.
8. pharmaceutical composition that contains each described Folium Ginkgo extract of claim 5-7, wherein, said composition is an injection preparation.
9. Pharmaceutical composition as claimed in claim 8, wherein, described injection preparation is injection and lyophilized injectable powder.
10. Pharmaceutical composition as claimed in claim 8, wherein, described injection preparation comprises 1 part of Folium Ginkgo extract, 15-25 part sorbitol or mannitol.
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