CN106727498A - Treat the composition and its preparation and the method for inspection of cardiovascular and cerebrovascular disease - Google Patents
Treat the composition and its preparation and the method for inspection of cardiovascular and cerebrovascular disease Download PDFInfo
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Abstract
The invention discloses a kind of composition for treating angiocardiopathy and cranial vascular disease and its preparation and the method for inspection, said composition is by the basis of dry weight, according to 1:6~10 weight than Vitexin and mannitol prepare.The Vitexin of present invention treatment cardiovascular and cerebrovascular disease and the blend compositions of mannitol are resisting myocardial ischemia, reduce myocardial oxygen consumption, improve the aspects such as haemodynamics and anti-cerebral ischemia, determined curative effect is notable, work fast, it is stable and controllable for quality, have no adverse reaction, toxic and side effect is low, cost is relatively low, many patients needs can be better met, patient's body and mind pain is solved, with good Social benefit and economic benefit.
Description
The application is the submission of on December 21st, 2012, application number 201210559202.0, entitled " treatment heart and brain blood
The divisional application of the composition of pipe disease and its preparation and the method for inspection ".
Technical field
The present invention relates to a kind of composition for treating angiocardiopathy and cranial vascular disease and its preparation and inspection party
Method, more particularly to the blend compositions comprising Vitexin and mannitol, said composition is resisting myocardial ischemia, is reducing cardiac muscle consumption
The aspect, effect is significant such as oxygen amount, improvement haemodynamics and anti-cerebral ischemia.
Background technology
According to nearest World Health Report, incidence of coronary heart disease and the death rate in recent years in the trend of rapid growth, according to statistics
Just there is 1 to die from angiocardiopathy in every 3 in global Died Of Disease, it is contemplated that will increase to the year two thousand twenty cardiovascular mortality
50%, epidemiological specialist professor MacMahon of Sydney University points out, the year two thousand twenty myocardial infarction will be dead from present with cerebral apoplexy
The the 5th and the 6th of cause rises to the 1st and the 4th.In China, 12 statistics in city, this disease population death rate is 29.6/10
Ten thousand, wherein with Beijing, the city's highest of Tianjin two.20th century the mid-80, Beijing, Shanghai, Guangzhou San Shi this disease population death rates
Respectively 21.7/10 ten thousand, 15.7/10 ten thousand, 4.1/10 ten thousand, 20 the mid-90s in century increased to 62.0/10 ten thousand, 37.4/10 respectively
Ten thousandth, 19.8/10 ten thousand, its trend north is higher than south.Artery sclerosis, thrombus, coronary heart diseases and angina pectoris be case fatality rate frequently-occurring, high,
Disability disease high, heavy burden is caused to people, family and a society, is that global health care and the huge of health resources are born
Load.
According to WHO, it is expected that cardiovascular and cerebrovascular disease at least causes global 12,000,000 people dead every year, it has also become the head of human health
Number enemy.In global 10 big well selling medicines in 2005, cardio-cerebralvascular medicine has accounted for 4., global cardiovascular and cerebrovascular in 2004
Sales amount of medicine is 60,800,000,000 dollars, it is contemplated that will rise to 91,200,000,000 dollars within 2008, and this numeral in 2010 would be possible to alter
It is upgraded to 104,400,000,000 dollars.According to the statistics made by the departments concerned, the scale of China's cardiovascular and cerebrovascular diseases pharmaceutical market there are about 16,000,000,000~18,000,000,000 yuan
RMB, its Chinese and Western medicine respectively accounts for half of the country with Chinese medicine preparation.Within past 10 years, resisting cardiovascular disease Chinese medicine is (especially with Chinese medicine
Based on injection) the conventional clinical treatment agent as domestic hospitals already.Estimated the year two thousand twenty arrives with aging population, China
Coronary heart disease epidemic peak will be welcome with other developing countries, such medicine will continue to show strong growth.
Clinical practice proves that traditional Chinese medicine treatment cardiovascular and cerebrovascular disease has the irreplaceable unique work(of chemical drugs product
Effect, also has Chinese medicine other formulations to be difficult to the advantage for possessing, and wide market is expected to turn into following Chinese medicine manufacturing enterprise research and development
Focus and market competition field the fiercest, are also Development of Traditional Chinese Medicine and the possible important directions of modernization.Have to hold
Recognize, although domestic annual there are some new treatment coronary heart disease application in TCM in clinic, in addition to only a few medicine, mostly
Number Chinese medicines fail to step into all the time the gate of international market, its reason except pharmaceutical research, active drug composition, medicine for process not
It is very clearly outer, more it is primarily due to without the criterion of therapeutical effect of international uniform is fully used, it is difficult to prove Chinese medicine in treatment coronary disease
Efficacy and saferry advantage in terms of disease.Either optimal dose or method of administration, or effective substance, internal mistake
Journey, current research can't provide the explanation of most convincingness.Current Clinical practice frequency Chinese patent drug kind higher includes
Danshen injections, chuanxiong-rhizome azine injecta, shengmai injection, Breviscapini injection, astragalus injection, SHENMAI ZHUSHEYE, Shenfu
Parenteral solution, puerarin injection etc..
No matter at home or abroad hawthorn leaf preparation, suffers from wide model application on treating cardiovascular disease.According to
Solution, in Europe, haw berry, the mixed extract of Ye Hehua achieve curative effect, leaves of Hawthorn certainly for Stable Angina Pectoris
Treatment of the preparation to early stage aged patients with chronic heart failure also obtains significant curative effect.At home, leaves of Hawthorn recorded in《Middle traditional Chinese medicines
Allusion quotation》2005 and 2010 one, hawthorne leaf P.E and preparation Yixintong piece also recorded in《Chinese Pharmacopoeia》2005
And 2010 one.Yixintong piece records Drug Standard of Ministry of Public Health of the Peoples Republic of China Chinese traditional patent formulation as marketing drugs earliest
The 6th page 151 of preparation.Indication：It is promoting blood circulation and removing blood stasis, declare promoting blood circulation.For the obstruction of qi in the chest caused by hemostasis resistance arteries and veins, symptoms include uncomfortable in chest suppress
Shouting pain, palpitation and amnesia, dizziness and tinnitus are taken before gas, the heart；Coronary disease and angina pectoris, hyperlipidemia, cerebral arterial insufficiency are shown in above-mentioned disease
Hou Zhe.Modern study shows that hawthorne leaf P.E has hypotensive, reduces peripheral vascular resistance, coronary artery dilator, increases hat
Arteries and veins flow, reduce myocardial oxygen consumption, cardiac stimulant, increase cardiac output, resist myocardial ischemia, platelet aggregation-against, improve hemorheology
Property, eliminate the pharmacological actions such as oxygen radical, norcholesterol and triglyceride, enhancing hypoxia-bearing capability and diuresis.Clinical practice in
The premature beat that coronary disease and angina pectoris and a variety of causes cause has obvious curative effects, effectively allevating angina pectoris and the symptom such as palpitaition, uncomfortable in chest,
Improve electrocardiogram, reduction blood fat improves HDL, improves microcirculation and lectin from hemolymph, increases cerebral blood flow (CBF), anti peroxidation of lipid, and peace
Entirely, toxic and side effect is had no.
Vitexin is flavone compound C glycosides, and oral formulations, its bioavilability is low, is unfavorable for that this product clinical efficacy is sent out
Wave, still by Vitexin system and mannitol into freeze-dried powder injection composition.Played beneficial to clinical efficacy is maximum, and beneficial to fortune
Defeated, storage, facilitates clinical application.
At hand, coronary heart diseases and angina pectoris, Patients with Stroke are increasing, and have rejuvenation for social population's aging
Such pharmaceutical requirements are increasingly increased by trend, have listed clinical application medicine as patient is using increasing, various adverse reactions
With increase.Clinical expense remains high, and market in urgent need determined curative effect adverse reaction is few, and toxic and side effect is low, and price is relatively cheap
Medicine.Vitexin, sweet dew alcohol composition, stable and controllable for quality, effect is clear in determined curative effect body, and adverse reaction is few, and poison is secondary
Effect is low, and quality-high and inexpensive is the medicine that ordinary people can be afforded to use.This product exploitation listing can better meet many patients to be needed
Will, patient's body and mind pain is solved, with good Social benefit and economic benefit.
The content of the invention
It is an object of the invention to provide a kind of new medicine for being clinically used for treating cardiovascular and cerebrovascular disease, the effect of drugs
Definitely, work fast, toxic and side effect is low.Present invention also offers Vitexin and the preparation side of the blend compositions of mannitol
Method, the method for inspection.
The present invention seeks to be achieved through the following technical solutions：
The composition of cardiovascular and cerebrovascular disease is treated, its active component is the mixing of the Vitexin and mannitol of therapeutically effective amount
Composition.
The composition for treating cardiovascular and cerebrovascular disease is by the basis of dry weight, according to 1:6~10 weight than Vitex negundo var cannabifolia
Element and mannitol are comprised the following steps that come what is prepared：
A, recipe quantity mannitol, the mixing of Vitexin raw material are weighed, plus water for injection stirs, then adds 0.1mol/L's
Sodium hydroxide solution, stirring and dissolving, then PH is adjusted in 8.5 ± 0.5 or so, plus 0.04-0.05% with the hydrochloric acid solution of 1mol/L
Needle-use activated carbon, stir 10-15 minute, micropore titanium filter decompression coarse filtration takes off charcoal, after inspection content, pH is qualified, then passes through
After 0.2-0.25 μm of miillpore filter end-filtration, it is sub-packed in cillin bottle, frozen drying；
B, pre-freeze：The medicine for having dispensed is put into pre-freeze on freeze drying box internal partition to start to+25 DEG C~-40 DEG C；
C, lyophilization：Condenser temperature is dropped to less than -45 DEG C, starts vavuum pump, treat that vacuum reaches a fixed number
After value, butterfly valve is slowly opened, when the vacuum in drying box is that below 0.1mmHg closes refrigerator up to 13.33Pa, by dividing plate
Under heating system slowly heat, the temperature of frozen product is gradually risen to 22-25 DEG C；
D, re-dry：Dried at 25-30 DEG C, loss on drying meets regulation, aseptically adds a cover plug, rolls lid,
Packaging, inspection, storage.
Treat the method for inspection of the composition of cardiovascular and cerebrovascular disease, including procedure below：
(1) this product is taken appropriate, plus ethanol is made every 1ml μ g solution containing main ingredient 20, as need testing solution, separately takes Vitexin
Reference substance is made in the same way of reference substance solution, is tested according to the one annex thin-layered chromatography of version Chinese Pharmacopoeia in 2010, draws for examination
Each 5 μ l of product solution, reference substance solution, put on same PA membrane respectively, are solvent with 36% acetic acid, launch, and take out, and dry in the air
Dry, with 1% aluminum trichloride solution, hot blast drying is put and inspect under uviol lamp 365nm for spray, in test sample chromatogram, with reference substance color
Compose on corresponding position, show the fluorescence spot of same color；
(2) this product is taken in right amount, plus ethanol is made solution of every 1ml containing the μ g of main ingredient 10, according to version Chinese Pharmacopoeia one in 2010
Portion's annex UV-VIS spectrophotometry is determined, and has absorption maximum at 270nm wavelength；
(3) relevant material：This product content is taken, is well mixed, precision weighs the sample for being approximately equivalent to Vitexin 25mg, puts
In 50ml measuring bottles, plus 60% ethanol 10ml makes to be made in every 1ml the solution containing 500 μ g after dissolving of flowing phase dilution, used as confession
Test sample solution；Precision is measured in right amount, plus flowing phase dilution is made the solution containing 10 μ g in every 1ml, used as contrast solution；It is another accurate
Weigh orientin reference substance in right amount, make to dissolve and quantify dilution and be made in every 1ml with mobile phase to contain the solution of 3 μ g as impurity pair
According to product solution, according to the chromatographic condition under (5) assay, precision measures the μ l of reference substance solution 20 injection liquid chromatographs,
Regulation detection sensitivity, makes the 20% of the peak height about full scale of principal component chromatographic peak；Precision measures the μ l of need testing solution 20 again
Injection liquid chromatograph, record chromatogram to 4 times of principal component chromatographic peak retention time, such as aobvious impurity in need testing solution chromatogram
Peak, impure orientin, with calculated by peak area, must not cross 0.6% by external standard method, any molten less than compareing in need testing solution
In the case that the peak that 0.01 times of liquid main peak area is negligible, the summation of all impurity levels must not cross 2.0%；
(4) mannitol：Take this product appropriate, be approximately equivalent to mannitol 0.2g, it is accurately weighed, put in 250ml measuring bottles, adding water makes
Scale is dissolved and be diluted to, is shaken up；Precision measures 10ml, in putting iodine flask, precision increase sodium iodide solution [take sulfuric acid solution (1 →
20) 90ml and sodium periodate solution (2.3 → 1000) 110ml is mixed] 50ml, put and heated 15 minutes in water-bath, let cool, plus
KI test solution 10ml, close plug is placed 5 minutes, is titrated with 0.05mol/L sodium thiosulfate titrating solution, during to nearly terminal, plus is formed sediment
Powder indicator solution 1ml, continues to be titrated to blue disappearance, and the result of titration is corrected with blank test.Per 1ml sodium thiosulfate drop
Determine C6H14O6 of the liquid equivalent to 0.9109mg, the 90.0~110.0% of labelled amount are should be containing mannitol；
(5) Vitexin assay：According to the one annex high effective liquid chromatography for measuring of version Chinese Pharmacopoeia in 2010；
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler；Acetonitrile-water-glacial acetic acid
It is 18：82：1 is mobile phase；Detection wavelength is 270nm.Number of theoretical plate is calculated by Vitexin peak and should be not less than 2500, Vitexin peak
Separating degree with impurity peaks all should meet the requirements；
Determination method：This product content is taken, is well mixed, precision weighs the sample for being approximately equivalent to Vitexin 25mg, puts 50ml
In measuring bottle, plus 60% ethanol makes to dissolve and be diluted to scale, shakes up；It is accurate again to draw during 1ml puts 10ml volumetric flasks, plus mobile phase
Scale is diluted to, is shaken up, as need testing solution.Precision measures 20 μ l injection liquid chromatographs, records chromatogram；Separately take Vitex negundo var cannabifolia
Plain reference substance is appropriate, accurately weighed, is measured in the same method, and by external standard method with calculated by peak area, (C21H20O10) containing Vitexin should be mark
The 90.0~110.0% of the amount of showing.
The Vitexin of present invention treatment cardiovascular and cerebrovascular disease and the blend compositions of mannitol are resisting myocardial ischemia, are reducing the heart
The aspect such as myocardial oxygen consumption, improvement haemodynamics and anti-cerebral ischemia, significantly, work fast, steady quality can for determined curative effect
Control, has no adverse reaction, and toxic and side effect is low, and cost is relatively low, can better meet many patients needs, solves patient's body and mind pain,
With good Social benefit and economic benefit.
Specific embodiment
The present invention is further described below by way of specific embodiment.
Embodiment 1：The preparation of injection Vitexin mannitol
A, weigh Vitexin 30g, mannitol 180g raw materials and uniformly mix, plus 600ml waters for injection, stir, then add
The sodium hydroxide solution 1500ml of 0.1mol/L, stirring and dissolving, then PH is adjusted to 8.5 or so with the hydrochloric acid solution of 1mol/L, plus
Water for injection to 3000ml, the needle-use activated carbon for plus 0.05% is stirred 10 minutes, and micropore titanium filter decompression coarse filtration takes off charcoal, inspection
Look into content, pH it is qualified after, then through 0.22 μm of miillpore filter end-filtration after, be sub-packed in 10ml cillin bottles, it is low per bottled 3ml
Warm freeze-drying.
B, pre-freeze：The medicine for having dispensed is put into pre-freeze on freeze drying box internal partition to start to+25 DEG C~-40 DEG C, 2 hours；-
40 DEG C, 3 hours.
C, lyophilization：Condenser temperature is dropped to less than -45 DEG C, starts vavuum pump, treat that vacuum reaches a fixed number
After value, butterfly valve is slowly opened, when the vacuum in drying box closes refrigerator below up to 13.33Pa (0.1mmHg), by dividing plate
Under heating system slowly heat, the temperature of frozen product is gradually risen to -20 DEG C, then gradually risen to 25 DEG C by -20 DEG C.
Whole lyophilization process about 26 hours.
D, re-dry：Dried about 5 hours at 25 DEG C, loss on drying meets regulation, aseptically adds a cover plug, rolls
Lid, packaging, inspection, storage.
Embodiment 2：The inspection of injection Vitexin mannitol
(1) this product 14.0mg is taken, plus ethanol is made every 1ml μ g solution containing Vitexin 20, as need testing solution；Separately take male
Jing Su reference substances are made in the same way of reference substance solution.According to thin-layered chromatography (one B of annex VI of Chinese Pharmacopoeia version in 2010) experiment, inhale
Each 5 μ l of need testing solution, reference substance solution are taken, are put respectively on same PA membrane, be solvent with 36% acetic acid, launched,
Take out, dry, with 1% aluminum trichloride solution, hot blast drying is put and inspect under uviol lamp (365nm) for spray, in test sample chromatogram,
On position corresponding with reference substance chromatogram, show the fluorescence spot of same color；
(2) this product 7.0mg is taken, plus ethanol is made solution of every 1ml containing the μ g of main ingredient 10, according to UV-VIS spectrophotometry
(one A of annex V of Chinese Pharmacopoeia version in 2010) is determined, and has absorption maximum at 270nm wavelength；
(3) relevant material takes the content under content uniformity, is well mixed, and precision is weighed and (is approximately equivalent to Vitex negundo var cannabifolia in right amount
Plain 25mg), in putting 50ml measuring bottles, plus 60% ethanol 10ml make to be made of flowing phase dilution in every 1ml after dissolving it is molten containing 500 μ g
Liquid, as need testing solution；Precision is measured in right amount, plus flowing phase dilution is made the solution containing 10 μ g in every 1ml, molten as compareing
Liquid.Another precision weighs orientin reference substance in right amount, makes to dissolve and quantify the solution for diluting and being made in every 1ml and contain 3 μ g with mobile phase
As impurity reference substance solution.According to the chromatographic condition under assay, precision measures the μ l of reference substance solution 20 injection liquid phase colors
Spectrometer, adjusts detection sensitivity, makes the 20% of the peak height about full scale of principal component chromatographic peak；Precision measures need testing solution again
20 μ l inject liquid chromatograph, record chromatogram to 4 times of principal component chromatographic peak retention time, as aobvious in need testing solution chromatogram
Impurity peaks, impure orientin, with calculated by peak area, must not cross 0.6% by external standard method, and the summation of all impurity levels must not mistake
2.0%.(any peak less than 0.01 times of contrast solution main peak area is negligible in need testing solution)；
(4) mannitol takes this product 233.3mg, is approximately equivalent to mannitol 0.2g, accurately weighed, in putting 250ml measuring bottles, plus
Water makes to dissolve and be diluted to scale, shakes up；Precision measures 10ml, puts in iodine flask, and precision is increased sodium iodide solution and [takes sulfuric acid solution
(1 → 20) 90ml and sodium periodate solution (2.3 → 1000) 110ml is mixed] 50ml, put and heated 15 minutes in water-bath, put
It is cold, plus KI test solution 10ml, close plug, place 5 minutes, titrated with sodium thiosulfate titrating solution (0.05mol/L), to nearly terminal
When, plus starch indicator solution 1ml, continue to be titrated to blue disappearance, and the result of titration is corrected with blank test.It is thio per 1ml
The C of sodium sulphate titrating solution (0.05mol/L) equivalent to 0.9109mg6H14O6, the 100.1% of labelled amount is should be containing mannitol；
(5) Vitexin assay is determined according to high performance liquid chromatography (one D of annex VI of Chinese Pharmacopoeia version in 2010)；
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler；Acetonitrile-water-ice vinegar
Acid (18：82：1) it is mobile phase；Detection wavelength is 270nm.Number of theoretical plate is calculated by Vitexin peak and should be not less than 2500, Vitexin
Peak all should meet the requirements with the separating degree of impurity peaks；
Determination method：The content under content uniformity is taken, is well mixed, precision is weighed and (is approximately equivalent to Vitexin in right amount
25mg), in putting 50ml measuring bottles, plus 60% ethanol makes to dissolve and be diluted to scale, shakes up；The accurate 1ml that draws puts 10ml capacity again
In bottle, plus mobile phase is diluted to scale, shakes up, as need testing solution.Precision measures 20 μ l injection liquid chromatographs, records color
Spectrogram；The another Vitexin reference substance that takes is appropriate, accurately weighed, is measured in the same method, by external standard method with calculated by peak area, containing Vitexin
(C21H20O10) should be the 90.0~110.0% of labelled amount.
Embodiment 3：The Clinical practice of injection Vitexin mannitol
Drip-feed, one time 1 bottle, 1 times a day；With being used after 150ml physiological saline solutions.Detailed directions are to take aseptic note
Needle ejecting tube draws 3~5ml waters for injection, and equipped with Vitexin, the cillin bottle of sweet dew alcohol composition, shaking up makes dissolving for injection, then
Drawn with aseptic needle tubing and injected in 150ml physiological saline, after shaking up, with the method for drip-feed, instilled in Venous Blood pipe.
Embodiment 4：The Clinical practice of injection Vitexin mannitol
Drip-feed, one time 2 bottles, 1 times a day；With being used after 150ml physiological saline solutions.Detailed directions are to take aseptic note
Needle ejecting tube draws 3~5ml waters for injection, and equipped with Vitexin, the cillin bottle of sweet dew alcohol composition, shaking up makes dissolving for injection, then
Drawn with aseptic needle tubing and injected in 150ml physiological saline, after shaking up, with the method for drip-feed, instilled in Venous Blood pipe.
Embodiment 5：The Clinical practice of injection Vitexin mannitol
Drip-feed, one time 2 bottles, 1 times a day；With being used after 250ml physiological saline solutions.Detailed directions are to take aseptic note
Needle ejecting tube draws 3~5ml waters for injection, and equipped with Vitexin, the cillin bottle of sweet dew alcohol composition, shaking up makes dissolving for injection, then
Drawn with aseptic needle tubing and injected in 250ml physiological saline, after shaking up, with the method for drip-feed, instilled in Venous Blood pipe.
Pharmaceutical composition of the present invention has promoting blood circulation and removing blood stasis, a surname's promoting blood circulation.For feeling of oppression and pain in the chest, limbs caused by hemostasis resistance arteries and veins
Numb, hemiplegia；Headstroke, coronary heart diseases and angina pectoris are shown in above-mentioned disease person.The experiment such as drug effect of the present invention, pharmacology, toxicity is referred to
It is following：
Test example 1：Animal angiocarpy drug efficacy study
1st, animal angiocarpy pharmacodynamics result of study
(1) test material
Vitexin, mannitol blend compositions：There is provided by Qixing Medicine Science and Technology Co., Ltd., Hefei, lot number：101109 (freeze
Dry powder pin, is faint yellow loose block, specification：Every bottle of 30mg/), it is dissolved to required concentration with physiological saline (NS) before use；
Kun Ming mice (body weight 18-24g) and Wistar rats (200~320g of body weight), by Medical University Of Anhui's reality
Animal center offer, animal quality certification number are provided：Anhui doctor is real dynamic accurate No. 01, laboratory rearing temperature：22±2℃；Rabbit (body weight
2.0~2.5kg) and mongrel (7.5~12.0kg of body weight)：Medical University Of Anhui's Experimental Animal Center is provided.
(2) test method：
Mouse tracheae folder closes myocardial anoxia model Kunming mouse 50,18~24g of body weight.It is randomly divided into 5 groups：NS pairs
According to group, Vitexin, 3 dosage groups of mannitol blend compositions (12,6,3mgkg-1) and Puerarin (60mgkg-1) sun
Property medicine control group, every group 10, ♀ ♂ half and half.Mouse tail vein injection gives (iv) test medicine back position and is fixed on frog board,
Neck median incision is done, tracheae is separated, and connect ECG.20min presss from both sides pipe of holding one's breath after administration, starts simultaneously at and clocks, and is led with ECG II
Persistently there is a flat line (i.e. electrocardio disappearance) as the mark of dead mouse in connection.The small mousetrap is recorded to hold one's breath II lead electrocardio after pipe
Duration (min).
Model Rats with Acute Myocardial Ischemia rat 60 is only randomly divided into 6 groups：Sham-operation (Sham) group；NS model control groups；
Vitexin, mannitol blend compositions (6,3,1.5mgkg-1) 3 dosage group and Puerarin (30mgkg-1) positive drug
Control group, every group 10, ♀ ♂ half and half.Rat is with 10% chloraldurate (0.3ml100g- 1) intraperitoneal injection (ip) anesthesia, face upward
Clinostatism is fixed, neck median incision, separates right carotid (CCA), and promoting the circulation of qi cannula connects breathing apparatus's (ratio during breathing
It is 1.5: 1,60 min- 1, 70 min of tidal volume- 1), thoracic cavity is opened in the rib of left border of sternum 3~5, heart is exposed, in lung
At arterious cone left border and left auricle of heart lower edge 2mm one 3/8 silk threads are worn through superficial layer of myocardium.Following coronary artery occlusion left anterior descending branch, causes
Myocardial ischemia.Wherein, Sham groups and model group sublingual vein inject (iv) NS, and Sham group threading is not ligatured, and other are respectively received
Reagent group rat is in the corresponding test medicines of 20min iv before coronary ligation.In experimentation, the lead ECG of periodic monitoring II,
Blood is taken through right side CCA intubations after 30min, 3000rpm centrifugation 10min take supernatant LDH and CK vigor to be measured；After experiment terminates
Heart is won immediately, is left and taken left ventricle and is weighed, and its is crosscutting into 6~8, insert in 0.2%NBT dye liquors, 37 DEG C of dyeing
After 20min, separate infarcted myocardium and weigh, to calculate the percentage that infarcted myocardium accounts for left compartment muscle weight in wet base.
Iso induces 50 rats of Model Rats with Acute Myocardial Ischemia (220~240g of body weight) and is randomly divided into model group (iv
NS+sc Iso), Vitexin, mannitol blend compositions (6,3,1.5mgkg-1) (iv Vitexins, mannitol are mixed 3 dosage groups
Polymeric composition+sc Iso) and Puerarin 30mgkg-1Group (iv Puerarin+sc Iso), totally 5 groups, every group 10, ♀ ♂ half and half.
Wherein, (injection dosage is 5mgkg to Iso-1) given in dorsal sc injection when 20min after each group test medicine iv administrations,
There is acute myocardial ischemia to induce, and produce ischemic ECG waveform (II lead, ST sections is raised or forced down).Each group is in injection Iso
30 ', 1 ', 2 ', 5 ', 10 ', 20 ' record an ECG (II lead) respectively after preceding and injection, with the height of ST field offset baselines
The mv numbers of summation Σ ST and the average value of ST field offsets as treating myocardial ischemia damage degree index.Rat is put to death after 20min,
Intact heart muscle tissue is won rapidly, clip apex fixed position cardiac muscular tissue claims weight in wet base；After 80 DEG C are toasted 15 hours, then claim it
Dry weight.Calculating myocardium water content (MWC).MWC=(weight in wet base-dry weight)/weight in wet base × 100%.
Dog acute experimental experimental myocardial infarction healthy adult experimental dog 36,7.5~12.0kg of body weight.It is randomly divided into 6
Group：Sham groups, NS model groups, Vitexin, mannitol blend compositions (4,2,1mgkg-1) 3 dosage group and Puerarins
15mg·kg-1Group, every group 6, ♀ ♂ dual-purposes, ♀ unpregnancies.By dog with 3% Nembutal vein anesthetic after, right lateral position is fixed on
On operating table, promoting the circulation of qi cannula simultaneously connects breathing apparatus's maintenance breathing.The intercostal of left side the 4th opens chest, exposes heart, two-piece crown
At lower 1/3 in shape left anterior descending artery, threading is in case ligation.30 are fixed by infarcted region, periinfarct and normal tissue site
EECG electrode, at once, the moment such as 5,15,30,45,60,90,120min trace electrocardiogram, with ST after coronary ligation
Duan Shenggao total voltage numbers represent degree of myocardial ischemia (Σ ST), with the ST sections of lead number raised more than 2mv, calculating myocardium ischemic model
Enclose (N-ST).20min medications group is through femoral vein injection institute reagent thing before ligation (administration volume is 1ml/kg).Blood is taken during ligation 2h
CK to be measured, LDH activity, and it is rapid in execution after left ventricle injection burnt black ink, taking-up heart, physiological saline is cleaned hemostasis, is cut
Lower left ventricle, filter paper is weighed after blotting.Ischemic region (white) and non-ischemic region (black) are separated, Weight of Ischemic Myocardium is weighed.Again
Ischemic myocardium is cut into several of uniform thickness, is placed in 0.2%NBT, 37 DEG C of dyeing 20min cut infarcted myocardium and (do not dye or ash
Yellow) weigh.Ischemic scope is represented with ischemic myocardium weight/left room weight × 100%；With infarcted myocardium weight/ischemic myocardium weight ×
100% represents infarction size.And calculate heart infarction index, heart infarction index=infarcted region weight/left room weight × 100%.
The experiment of experimental dog haemodynamics is from healthy experimental dog 30,7.5~12.0kg of body weight.It is randomly divided into 5 groups：
NS control groups, Vitexin, mannitol blend compositions (4,2,1mgkg-1) 3 dosage group and Puerarin 15mgkg-1Group,
Every group 6, ♀ ♂ dual-purposes, female unpregnancy.With 3% Nembutal vein anesthetic, take dorsal position and fix.Neck median incision, cruelly
Reveal and tracheostomize, trachea intubation；Right external jugular vein is separated in case coronary sinus vein intubation carries out the oxygen content of blood and determines use；Point
From left lower extremity femoral artery, connect BL-420E biological functional systems and trace blood pressure；Right lower extremity femoral artery is separated in case blood sampling is carried out
Arterial oxygen content is determined；Left lower extremity femoral vein is separated, slow constant speed is input into physiological saline (about 1ml/min) to supplement loss
Body fluid；The subcutaneous fixed needle electrode of four limbs, traces the lead ECG of standard limb II, to calculate heart rate；Then animal takes the forelimb right side and crouches
Position, anterior pectorial region unhairing, Yu Zuosi, five intercostals cut skin along the 4th rib lower edge, and blunt separation muscle after exposure pleura, meets people
Work lung ventilator, fully hemostasis after cut pleura, lift pericardium cut and make pericardium bed, careful separation LCA and
Preplaced line is stayed after aortic root, according to coronary artery and the thickness of sustainer, suitable doppler ultrasound blood is selected and connect
Stream instrument MP100 probes (2mm, 12mm)；Left ventricular cannulation (heparin-saline is full of in pipe) inserts left through left ventricle apex
Intra-ventricle, connects BL-420E biological functional systems measurement left indoor pressure, left room EDP；From right jugular vein intubation to
Coronary sinus vein (injection heparin-saline anti-freezing in cardiac catheter)；After aforesaid operations terminate, intravenous injection heparin carries out whole body
Test tube of hepari (5mg/kg).After animal stablizes 30 minutes, observe and recording blood pressure, ECG, coronary flow and sustainer stream
Measuring, and take blood from femoral artery and coronary sinus vein carries out oxygen content of blood measure, used as the normal value before administration.Then according to difference
Group and dose intravenous drug administration by injection (administration volume is 1ml/kg), in after administration 5,15,30,45,60,90 and the 120min time-divisions
Not Ce Ding coronary artery and ABF, blood pressure and ECG etc., and in 30min after administration, 60min takes a blood sample survey hat respectively
Shape venous sinus and the femoral artery oxygen content of blood.After experiment terminates, take out heart and weigh.According to result, by blood pressure, heart rate, left interior
Pressure, coronary blood flow, ABF and the oxygen content of blood carry out statistical procedures, and calculate heartbeat according to formula and go out
Amount, cardiac index, SI, left room work done, 100 grams of MBFs, coronary resistance, total peripheral resistance, cardiac muscles per minute
Oxygen demand, myocardium coefficient of oxygen utilization and venous oxygen consumption index etc..
Two-level index computing formula is as follows：
(1) body surface area=(body weight kg)2/3×0.11
(2) cardiac output=cardiac output (ml/min) ÷ hearts rate (beat/min)
(3) cardiac index=cardiac output (ml/min) ÷ body surface areas (m2)
(4) SI=cardiac index (L/min/m2) ÷ hearts rate (beat/min) × 103
(5) left room work done=cardiac index × 1.052 × ÷ of (blood pressure -5) × 13.6 103
(6) it is per minute per 100g MBFs=cardiac weight × 100 of CF (ml/min) ÷ 1/3
(7) coronary resistance=blood pressure (mmHg) ÷ is per minute per 100g MBFs
(8) total peripheral resistance=blood pressure (mmHg) × 79.92 ÷ cardiac outputs (L/min)
(9) MCO=(arterial blood oxygen amount-coronary sinus vein blood oxygen amount) × CF (ml/min) × 10-2
(10) myocardium coefficient of oxygen utilization=(arterial blood oxygen amount-coronary sinus vein blood oxygen amount) ÷ arterial bloods oxygen amount × 100
(11) venous oxygen consumption index=heart rate (beat/min) × blood pressure (mmHg) × 10-2
(12) CBF=flow velocity (cm/sec) × 3.14 × radius (cm)2×60(sec)
(3) result of the test：
Vitexin, mannitol blend compositions are as shown in table 1 to the influence result that tracheae folder closes the mouse electrocardio duration,
After tracheae folder is closed, NS control group mices are died off in 9.26 ± 1.14min, and Vitexin, mannitol blend compositions 12,
6,3mg kg-1The time of 3 dosage group mouse electrocardio disappearances is considerably longer than NS control groups, can at most extend about 32.94%,
Difference has highly significant (P<0.01), effect is better than positive drug.Prompting Vitexin, mannitol blend compositions are remarkably improved
The ability of mouse heart resist oxygen lack.
The Vitexin of table 1, mannitol blend compositions to tracheae folder close the mouse electrocardio duration influence (N=
10)
Compare with NS control groups,*P<0.05,**P<0.01
The influence that Vitexin, mannitol blend compositions are damaged to coronary ligation myocardial ischemia in rats
Influence to myocardial infarct size shows the size of myocardial infarct size with NBT decoration methods, as a result as shown in table 2,
Model group rats coronary ligation 30min, its myocardial infarct size is up to 54.02% ± 14.32%, and Vitexin, mannitol are mixed
3 dosage groups of polymeric composition can in various degree reduce the infarction size of cardiac muscle, wherein 6mgkg-1Group rat myocardial infarction model model
Only 33.47% ± 6.30% is enclosed, 38.04% is reduced compared with model group, difference has highly significant.Curative effect is better than positive drug.
Prompting, Vitexin, mannitol blend compositions have the effect for reducing Acute Myocardial Ischemia Rats myocardial infarct size.
The Vitexin of table 2, mannitol blend compositions to coronary ligation infarcted myocardium of rat weight influence (N=
10)
Compare with Sham groups,△△P<0.001；Compare with model group,**P<0.01
Influence result to LDH and CK vigor in blood plasma is as shown in table 3, compares with Sham groups, in model group rats blood plasma
The vigor of LDH and CK is significantly raised, and shows that coronary ligation has damaged the myocardial cell membrane of rat, LDH and CK from cell at home and abroad
Leakage, reduces its activity in cell, and activity is significantly raised in blood plasma.Vitexin, mannitol blend compositions 6,3mg
kg-1The exception that dosage group can suppress LDH vigor in rat plasma increases, meanwhile, Vitexin 6mgkg-1Dosage group is to CK vigor
Abnormal rising also significantly inhibit effect.
The Vitexin of table 3, mannitol blend compositions to LDH and CK vigor in coronary ligation rat plasma influence (
N=10)
Compare with Sham groups,△△P<0.01；Compare with model group,*P<0.05,**P<0.01
Vitexin, mannitol blend compositions induce Iso the influence of rats with myocardial ischemia treating myocardial ischemia damage
The influence result of exception ECG (ST sections) is as shown in the table during to ischemic, and ST sections of model group rats ECG is in sc Iso
2min is to start substantially to raise afterwards, raises more notable during 5min, and is continued for 20min.Vitexin, mannitol mixing group
Compound 6,3,1.5mg kg-13 dosage groups can be obviously prolonged ST sections of exception and raise the time for starting occur, and and model group
Compare, the ST sections of abnormal order of severity raised can be significantly reduced.Prompting, Vitexin, mannitol blend compositions can obviously improve
Abnormal ECG changes caused by sc Iso.
The Vitexin of table 4, mannitol blend compositions induce Iso the shadow of Acute Myocardial Ischemia in Rats exception ECG (ST sections)
Ring (N=10)
Compare with before medication,#P<0.05,##P<0.01, compare with model group, * P<0.05,**P<0.01
Influence to myocardial water content as can be seen from Table 5, Vitexin, mannitol blend compositions 6mg kg-1Dosage
Group can significantly reduce myocardial water content, suppress myocardial edema.
The Vitexin of table 5, mannitol blend compositions induce Iso the influence of rats with myocardial ischemia myocardial water contentn
=10)
Compare with model group group,*P<0.05,**P<0.01
The influence of Vitexin, mannitol blend compositions to coronary ligation dog Damage of Myocardial Ischemia
To the influence result of myocardial ischemia and the infarct order of severity as shown in table 6-8, cardiac muscle stalk is shown with NBT decoration methods
The size of dead scope, as a result finds, after model group dog coronary ligation 120min, its myocardial ischemia scope up to 56.50% ±
4.70%, infarction size is up to 20.18% ± 12.17%.And Vitexin, mannitol blend compositions 4,2,1mgkg-13 dosage
Group can to some extent reduce the ischemic scope and infarction size of cardiac muscle, and wherein Vitexin, mannitol blend compositions are heavy dose of
Group myocardial ischemia scope is 41.29% ± 6.44%, and inhibiting rate is up to 26.92%；Myocardial infarct size is 7.17% ± 3.51%,
Inhibiting rate is up to 64.47%；Compare with model group, difference be respectively provided with conspicuousness (**P<0.01,*P<0.05).Meanwhile, Vitexin 4,
2,1mg kg-13 dosage groups also reduce heart infarction index to some extent, and maximum reduces percentage up to 74.53%.Prompting, it is male
Jing Su, mannitol blend compositions can substantially mitigate ischemic order of severity during coronary ligation cause dog acute myocardial ischemia.
The Vitexin of table 6, mannitol blend compositions to coronary ligation dog myocardial ischemia scope influence (N=6)
Compare with Sham groups,△△P<0.001；Compare with model group, * P<0.05, * * P<0.01
The Vitexin of table 7 to coronary ligation dog myocardial infarction scope influence (N=6)
Compare with Sham groups,△△P<0.01；Compare with model group, * P<0.05
The Vitexin of table 8, mannitol blend compositions to coronary ligation dog heart infarction index influence (N=6)
Compare with Sham groups,△△P<0.01；Compare with model group, * P<0.05, * * P<0.01
To degree of myocardial ischemia (Σ-ST) and the influence result such as table 9 of myocardial ischemia scope (N-ST), shown in 10, use
Epicardial lead infarcted region, ischemic region and normal area place 30 electrodes, measure model group dog coronary ligation at once until
120min, its degree of myocardial ischemia is serious, and Σ-ST values are all remarkably higher than Sham groups (p<0.01)；Myocardial ischemia scope N-ST
(p is significantly increased compared with Sham groups<0.01), show that coronary ligation makes dog occur in that severe myocardial ischemia situation.And Vitexin, sweet dew
Alcohol blend compositions 4,2mg kg-1Dosage group can be obviously reduced myocardial ischemia scope, and can reduce after coronary ligation 45min and
Degree of myocardial ischemia during 60min, difference has conspicuousness, and curative effect is suitable with Puerarin.It is Vitexin, sweet in other times point
Dew alcohol blend compositions 4,2mg kg-1Dosage group is compared with model group, though difference is not statistically significant, to degree of myocardial ischemia
Also reduction trend has been shown.Prompting, Vitexin, mannitol blend compositions can substantially mitigate cardiac muscle during acute myocardial ischemia
Degree of ischemia and scope.
The Vitexin of table 9, mannitol blend compositions to coronary ligation dog degree of myocardial ischemia (Σ-ST) influence (n
=6)
Compare with Sham groups,△△P<0.01；Compare with model group, * P<0.05
The influence of the Vitexin of table 10, mannitol blend compositions to coronary ligation dog myocardial ischemia scope (N-ST)
Compare with Sham groups,△△P<0.01；Compare with model group, * P<0.05, * * P<0.01
Influence result to LDH and CK vigor in blood plasma is as shown in table 11, the vigor of LDH and CK in model group dog plasma
Significantly raise, Vitexin, the kg of mannitol blend compositions 4,2,1mg-13 dosage groups by comparison, can significantly inhibit blood
LDH vigor increases in slurry, meanwhile, Vitexin, mannitol blend compositions 4,2mg kg-1Dosage group can also reduce CK in blood plasma
Activity level.
The Vitexin of table 11, mannitol blend compositions to LDH and CK vigor in coronary ligation dog plasma influence ( n
=6)
Compare with Sham groups,△△P<0.01；Compare with model group,*P<0.05,**P<0.01
The influence of Vitexin, mannitol blend compositions to cardiac hemodynamics of dogs and MCO
To blood pressure, the influence of heart rate is preceding with administration and NS control groups compare, each dose of Vitexin, mannitol blend compositions
Amount group has no significant effect (P to anaesthetized dog blood pressure, heart rate>0.05).
Influence to dog left indoor pressure is compared with before administration, Vitexin, mannitol blend compositions 4,2mg kg-1Dosage
Downward trend is pressed with group rat left chamber, but there is no significant difference；And compare with NS control groups, Vitexin 2mgkg-1Dosage group
15~45min periods interior energy substantially reduces dog left indoor pressure upon administration, and curative effect is better than positive control drug.
To dog coronary flow, the influence Vitexin of cardiac output, mannitol blend compositions 4mgkg-1 dosage group dogs
Coronary flow and cardiac output are compared with before medication and NS control groups substantially increase, Vitexin, mannitol blend compositions 2mg
Kg-1 dosage groups coronary flow upon administration 15min when substantially increase compared with NS control groups, and be continued for 45min, its heart is defeated
Obvious increase before output and medication.Prompting, Vitexin, mannitol blend compositions can increase dog coronary flow and heart output
Amount.
Dog coronary resistance, the influence Vitexin of total peripheral resistance, mannitol blend compositions 4mg kg-1Dosage group is administered
Coronary resistance is can obviously reduce during 45min afterwards, and is continued for 90min, total peripheral resistance also goes out in 60min after medication
Now it is decreased obviously, and is continued for 120min；Compare with NS control groups, Vitexin 4,2,1mg kg-13 dosage groups may not be used
Coronary resistance, Vitexin, mannitol blend compositions 4,2mg kg are reduced with degree-1Dosage group can also after medication 120min
When reduce total peripheral resistance, difference is respectively provided with conspicuousness.
To dog cardiac output, the influence Vitexin of SI, mannitol blend compositions 4mgkg-1Dosage group is administered
Dog cardiac output and SI can substantially be increased during 120min afterwards, compared with NS control groups with before medication, be respectively provided with significance difference
It is different；Vitexin, mannitol blend compositions 2mgkg-1Cardiac output and SI are relatively used during 60min after dosage group administration
When substantially increasing before medicine, and being continued for 120min.
To dog cardiac index, the influence Vitexin of left room work done, mannitol blend compositions 2mgkg-1Dosage group is administered
After cardiac index is persistently raised, and be significantly increased left room work done in 45~60min time periods, and Vitexin, mannitol
Blend compositions 4mgkg-1Dosage group cardiac index compared with before medication and NS control groups more have a rising, but to left room work done without
Significantly affect (P>0.05).
Dog hectogram MBF per minute, the influence of venous oxygen consumption index are compared with NS control groups, it is Vitexin, sweet
Dew alcohol blend compositions 4,2,1mgkg-1Can to some extent increase every 100 grams of MBFs after 3 dosage group administrations,
But (P is had no significant effect to venous oxygen consumption index>0.05).
Influence to dog MCO, myocardium coefficient of oxygen utilization removes Vitexin, mannitol blend compositions 1mg kg-1Agent
Amount group cardiac muscle coefficient of oxygen utilization decreases outward compared with NS control groups, and remaining each dosage group is to Myocardium in Anaesthetized Dogs oxygen demand, myocardium oxygen profit
(P is had no significant effect with rate>0.05).
Test example 2：Animal cerebrovascular drug efficacy study
(1) test material
Vitexin, mannitol blend compositions：There is provided by Qixing Medicine Science and Technology Co., Ltd., Hefei, lot number：101109 (freeze
Dry powder pin, is faint yellow loose block, specification：Every bottle of 30mg/), it is dissolved to required concentration with physiological saline (NS) before use；
Kunming mouse and ICR kind mouse, respectively by Anhui Province's Experimental Animal Center (cleaning grade, 18~22g of body weight, the animal quality certification
Number：Scxk (Anhui) 2005-001) and Zhejiang Province's Experimental Animal Center (cleaning grade, 18~22g of body weight, animal quality certification number：scxk
(Zhejiang) 2003-001) provide；SD rats and Wistar rats, respectively by Zhejiang Province's Experimental Animal Center (cleaning grade, body weight 180
~250g, animal quality certification number：Scxk (Zhejiang) 2003-001) and Chinese military medicine academy of sciences Experimental Animal Center (cleaning grade,
240~280g of body weight, animal quality certification number：Scxk (army) 2002-001) provide.
(2) test method
Mouse broken end causes cerebral anoxia model：Kunming mouse, is randomly divided into NS control groups, by reagent Vitexin, mannitol
3 dosage groups of blend compositions and positive control drug Nimodipine (Nim) and each one group of ginkgo biloba p.e (EGB), every group small
Mouse ♀ ♂ half and half.Each group mouse distinguishes gavage (ig) NS or corresponding by reagents (0.1ml/10g body weight), once a day, continuously gives
Medicine 5 days, last dose 2h quickly breaks end at the ear line of mouse two, record action of dehiscing after each mouse broken end to breathe number of times and
Duration (sec).
Press from both sides pipe of holding one's breath and cause anoxia in mice model：Ibid, after last dose 2h, 10% is hydrated for ICR kind mouse, packet and administration
Chloral 300mg.kg-1Intraperitoneal injection (ip) is anaesthetized, median incision, separates tracheae, connects ECG monitor monitor ECG
(ECG) pipe of holding one's breath, is then pressed from both sides, the immediate record mouse ECG duration, is cardiac arrest index with ECG disappearances.
Cerebral ischemia animal model induced by middle cerebral artery occlusion in rats：Using the focal of line bolt improved method manufacture middle cerebral artery occlusion (MCAO)
Cerebral ischemia reperfusion model.After rat is anaesthetized with 10% chloraldurate (300mg/kg, ip), by a 5cm long, about 250 μm of left sides of diameter
Right nylon wire enters the propulsion of cranium direction through left side external carotid artery trunk otch is slow to internal carotid, is with arteria carotis communis crotch
Mark, when 18~20mm of propulsion feels light resistance, you can blocking arteria cerebri media.After blocking 2h, nylon wire is extracted, lacked
Blood Reperfu- sion 24h.In operation technique and anaesthesia process, rat incandescent lamp is heated and maintains body temperature, and anus temperature maintains 36.5~
37.5℃.Sham groups in addition to not plug wire, the same model group of remaining surgical procedure.
Wistar rats, are randomly divided into sham-operation (Sham) group, model (NS controls) group, by reagent Vitexin, mannitol
3 dosage groups of blend compositions and each one group of positive control drug Nim and EGB, every group of ♀ ♂ half and half.Each group rat distinguish ig NS or
Corresponding medicine (1.0ml/100g body weight), once a day, successive administration 5 days, the above-mentioned MCAO experiments of last ig administration 2h rows.
22h is administered once after MCAO is filled again, after 2h, carries out scoring (0 point, impassivity defective symptom of abnormal neuron symptom；1 point, before the right side
Limb can not be stretched completely；2 points, to right rotation；3 points, topple over to the right；4 points, without spontaneous activity or stupor).Then broken end takes
Blood, quickly takes brain, and after weighing, coronal cutting brain tissue is put into 2%TTC solution in 37 DEG C of lucifuges dyeing (normal groups into 5
Knit and dye red, it is white that inactive infarction tissue refuses dye) after 30min, taken pictures with digital camera and brain piece and be input into calculating
In machine, with the pale infarct in image analysis software (Image J) respectively the digital pictures section of each 5 brain pieces of mouse of survey calculation
After tissue area and complete brain piece area, multiplied by with brain piece thickness, the percentage that cerebral infarction volume accounts for whole Brain tissue volume is calculated
Than.
Then 5 brain pieces of each mouse are dried to constant weight (i.e. dry weight) in 100 DEG C of baking ovens, calculates its brain water content.Brain
Tissue water content=(weight in wet base-dry weight)/weight in wet base × 100%
After the centrifugal blood that will be taken, Serum LDH activity is determined at 490nm by kit specification method respectively, press
Thiobarbituricacidα- method, Serum MDA (MDA) content is determined with TMP as standard items at 535nm.
Reperfusion following ischemia model：Built using Pulsinell-Brierley tetra- blood vessel blocking (4-VO) method of improvement
Vertical cerebral ischemia, reperfusion model.SD rats are anaesthetized with 10% chloraldurate (300mg/kg, ip), and prone position is fixed, and are drawn
Straight cervical vertebra, neck is horizontal, to row median incision at axis under occipital bone, blunt separation muscle, exposure the
The foramen transversarium of one cervical vertebra both sides, during the coagulation pin of a diameter of 0.5mm inserted into the foramen alare of both sides respectively, burns both sides vertebral artery,
Make its permanent closure, sew up the incision.Again through throat portion median incision, bilateral common carotid arteries, the l before arteria carotis communis bifurcated are separated
Surgical thread is placed in lower section at cm, and line end is placed in vitro, sews up the incision, and is steam again after rat is clear-headed and raised.After 24h, rat is again
Secondary anesthesia, after determining electroencephalogram, removes the suture of neck, lifts preset surgical thread, pulls out the arteria carotis communis that both sides are separate,
Rapid folder being pressed from both sides with arteriole and closing bilateral common carotid arteries, it is model Success Flag to be flattened with brain wave.Blocking arteria carotis communis 30min,
Then bilateral arterial folder is unclamped simultaneously, surgical thread is taken out, and is carried out brain vessel blood and is filled 120min again.The operation behaviour of Sham group animals
Make step identical with ischemia group, but not coagulation bilateral vertebral artery, not press from both sides and close bilateral common carotid arteries, remaining treatment is dynamic with cerebral ischemia group
Thing.
Electroencephalogram (EEG) assay method：After rat both sides vertebral artery blocking 24h, anaesthetize again, in l mm after bregma, arrow
The a diameter about aperture of 2mm is bored on skull with cranial drill at shape seam left side 2mm, single needle electrode is put through skull aperture
In Epidural cavity, reference electrode puts ear skin, and the shielded conductor that electrode is drawn is connected with BE Light electroencephalographs, records ischemic
Before, ischemic when, fill the EEG of 5min, l5min, 30min, 60min and 120min again.Global ischemia/reperfusion is calculated by following equation
The change percentage in arid of rat EEG potential amplitudes.
EEG amplitude × 100% before EEG amplitudes/ischemic after EEG potential amplitudes change percentage in arid=Reperfu- sion
After experiment terminates, after rat broken end takes blood, full brain is quickly removed, be divided into two, weigh rearmounted electric heating of left half brain is permanent
Warm baking oven (100 DEG C) is dried, until calculating brain water content after constant weight.
Right half brain puts fixed in 10% formalin, HE dyeing Hou Hang histopathologic examinations and scored (without, without~
Slightly, slightly, moderate and severe morphological changes of various tissue components remember 0,0.5,1,2 and 3 points respectively).
After by taken centrifugal blood, according to preceding method detection Serum LDH activity and MDA contents.
Rat is grouped and is administered with intraluminal middle cerebral artery occlusion in rats constriction experiment.
Rat carotid artery-venous bypass thrombus experiment：SD rats, are randomly divided into NS control groups, by reagent Vitexin, sweet
Dew 3 dosage groups of alcohol blend compositions and each one group of positive control drug Nim and EGB, every group 10, ♀ ♂ half and half.The water of rat 10%
Chloralization is closed, neck median incision separates LCC and right vena jugularis externa.It is the poly- second of 2mm in a 15cm long, external diameter
A 5cm silk thread long is inserted in alkene pipe and full of 50U/ml heparin-saline solution, one section of insertion of the polyethylene pipe is right
In vena jugularis externa, after ligation is fixed, in another section of insertion LCC and tighten, then open artery clamp and open blood flow
After 15min, take out 6h during the thrombus in lower polyethylene pipe claims to be put into 85 DEG C of baking boxs after its weight in wet base and claim its dry weight again, subtract silk thread weight
Amount obtains wet weight of thrombus and dry weight respectively.
Each group rat distinguishes ig NS or corresponding by reagents (1.0ml/100g body weight), once a day, successive administration 5 days,
Above-mentioned experiment is carried out after last dose 2h.
Rat platelet aggregation is tested：72 rats are randomly divided into NS control groups, by reagent Vitexin, mannitol mixing group
3 dosage groups of compound and each one group of positive control drug Nim and EGB, every group 8, ♀ ♂ half and half.Each group rat distinguishes ig NS or phase
The by reagent (1.0ml/100g body weight) answered, once a day, successive administration 5 days carries out following platelet aggregations after last dose 2h
Collection experiment.
After rat yellow Jackets ip anesthesia, abdominal cavity is opened, separate abdominal aortic blood, carried out with 3.8% sodium citrate
Anti-freezing is processed, and amount for taking blood and anti-coagulants ratio are that 9: 1,1 000r/min is centrifuged 5min, suction out supernatant platelet-rich blood plasma
(PRP), the 000r/min of remaining blood 3 centrifugations 15min, prepares platelet poor plasma (PPP).Blood platelet in PRP is counted, is used
PPP adjusts its platelet count 2.5 × 108Individual .ml-1。
By in PRP immigration silication cuvettes, 37 DEG C of warm bath 5min survey its absorbance (A) at 600nm, and PRP is adjusted with PPP
A values between 0.6~0.7, the PRP that will mix up is moved into another silication cuvette, and addition is surveyed at 600nm by Born methods
A values before ADP, are subsequently added 100 μ l ADP (4 μm of ol/ml of final concentration), then determine 30sec, 1,2min after addition ADP respectively
A values during with 5min, calculate platelet aggregation rate.
A value × 100% before platelet aggregation rate=(adding A values-add A values after ADP before ADP)/addition ADP.
Statistical procedures：Experimental result is with mean ± standard deviationRepresent, entered using SPSS11.5 statistical softwares
Compare between row statistical procedures, one-way analysis of variance, group and checked with t.
(3) result of the test
The influence of Vitexin, mannitol blend compositions to ischemic brain damage
On being dehisced after mouse broken end, snorting influence Vitexin, mannitol blend compositions can be obviously prolonged mouse broken end
After dehisce breathing time.
The influence Vitexin of mouse ECG times, mannitol blend compositions are closed to tracheae folder and causes mouse to pressing from both sides pipe of holding one's breath
The ECG times have obvious extension to act on；Nim has the trend of extension, but no significant difference as EGB.
The influence of Vitexin, mannitol blend compositions to focal cerebral ischemia in rats reperfusion injury
Influence MCAO to abnormal neuron symptom fills rat and occurs in that obvious abnormal neuron symptom again, as EGB,
Vitexin, mannitol blend compositions can significantly reduce the scoring of abnormal neuron symptom.
Influence MCAO to encephaledema and brain tissue infarct volume percentage fills the obvious encephaledema of rat generation and brain again
Infraction, as EGB or Nim, Vitexin, mannitol blend compositions can significantly mitigate encephaledema and reduce brain tissue infraction body
Long-pending percentage, and imitate relation in a certain amount.
MCAO is filled again rat blood serum LDH activity and MDA contents influence rat MCAO fill again can make serum in LDH live
Property and MDA contents are significantly raised.As EGB and Nim, Vitexin, mannitol blend compositions can substantially suppress MCAO and fill again
The rising of LDH activity and MDA contents in rat blood serum；
The influence that Vitexin, mannitol blend compositions are damaged to reperfusion following ischemia
The GBI caused by the vascular ligation of influence four to EEG can force down rat EEG to flatten, and Reperfu- sion can make greatly
Mouse EEG has recovered, but compares with Sham, then still without recovering completely when filling 120min.As EGB and Nim, Vitexin,
Mannitol blend compositions recover have obvious facilitation to global ischemia/reperfusion rat EEG.
Influence global ischemia/reperfusion rat to brain water content there occurs obvious encephaledema, male as EGB and Nim
Jing Su, mannitol blend compositions can obviously reduce the brain water content of global ischemia/reperfusion rat.
Influence to Serum LDH and MDA is compared with Sham, and NS control rats Serum LDH activity and MDA contents have bright
Aobvious rising, as EGB and Nim, Vitexin, mannitol blend compositions can significantly inhibit Serum LDH activity and MDA contents
Rising.
Result Sham groups rat cerebral tissue of histopathologic examination nerve cell is normal, and nuclear membrane understands, kernel is obvious；God
Normal, the light dye of endochylema through spongiocyte form, cell membrane and nuclear membrane understand.Capillary and thin vessels tube chamber are narrower or real in one
Property bar rope.There is obvious pathology and sexually revise in NS model group rats brain tissue, nerve cell endochylema and karyon are concentrated, and dyeing adds
Deep, cell volume reduces, and aixs cylinder and dendron are in irregular shape, and the gap around nerve cell and capillary, thin vessels is broadening,
The loose oedema of interstitial, Deiter's cells swelling.The brain tissue nerve cell of Vitexin, each dosage group of mannitol blend compositions
Concentration and deep dye have mitigation in various degree compared with NS model groups, and Deiter's cells swelling and interstitial porousness are also lighter than NS
Model group.
The influence that Vitexin, mannitol blend compositions are formed to rat suppository：
Rat carotid artery-venous loop can cause the formation of thrombus, as EGB, Vitexin, mannitol blend compositions
Can obviously reduce the weight in wet base of thrombus.
The influence of the platelet aggregation that Vitexin, mannitol blend compositions are induced ADP
Compare with Normal group, 100mg.kg-1EGB can significantly suppress add ADP after 2min and 5min when blood platelet
Aggregation, 5mg.kg-1Nim is to platelet aggregation also has significant suppression during 5min after addition ADP.Vitexin, mannitol mixing group
Platelet aggregation has obvious inhibitory action when compound is to 2min and 5min.
Test example 3：Animal acute toxicity test is studied
(1) test material
Vitexin, mannitol blend compositions：There is provided by Qixing Medicine Science and Technology Co., Ltd., Hefei, lot number：101109 (freeze
Dry powder pin, is faint yellow loose block, specification：Every bottle of 30mg/), it is dissolved to required concentration with physiological saline (NS) before use；
Beagle dogs, regular grade is provided by Chengdu up to large bio tech ltd.Credit number：SCXK (river) 2006-24, by four
The management of laboratory animal committee of river province signs and issues,
(2) test method
Test sample is made into the solutions for administration of 30mg/ml, be designed as 2.0 successively according to 50% incremental method administered volume,
3.0th, 4.5 a series of, 6.8,10.1, dosage groups of 15.2ml/kg, No. 1 dog gives 4.5ml/kg dosage during administration, according to animal
Reaction determines No. 2 dosages of dog, then determines No. three dosages of dog according to the reaction of No. 2 dogs, the like, every
An animal is given every a dosage, minimum lethal dose (MLD) and highest lethal dose is measured, is then given again with dosage between the two
One animal, this dosage is ALD, and the dosage for not finding poisoning symptom is maximal tolerance dose.Maximum can give
20ml/kg, such as not dead yet, then 20ml/kg is maximum dosage-feeding.
(3) result of the test
No. 1 animal (7.6kg)：Vitexin solution 4.5ml (135mg)/kg is given, administration process and administration terminate rear animal
Reaction is not shown any abnormalities.
No. 2 animals (7.5kg)：Vitexin solution 10.1ml (303mg)/kg is given, administration terminates rear 3min animals vomiting
Once, other abnormal responses are had no.
No. 3 animals (6.7kg)：Vitexin solution 6.8ml (204mg)/kg is given, administration process and administration do not go out after terminating
Incumbent what abnormal response；
No. 4 animals (6.6kg)：Vitexin solution 20ml (600mg)/kg is given, administration terminates rear animal to be occurred vomitting immediately
Reaction is told, then intermittent vomiting, recovered after 24h normal.
Do not occur any abnormal response in 14 day observation period of all animals, observation terminates rear cut open inspection, the heart, liver, spleen, lung,
The main organs such as kidney have no the visible exception of naked eyes.
Test example 4：Long-term toxicity test for animals is studied
This research is observed 15 days by continuous 6 months intravenously administrables and recovery of being discontinued, with regard to Vitexin, mannitol hybrid combining
Thing is observed the effect of beasle dog long term toxicity.Result shows to exist without animal dead, administration group during being administered 6 months
5~30 minutes Activities quantitatives are reduced after administration, are partly reposed, with heavy dose group animal as very.Blank control group is respectively given by animal
Give after physiological saline activity freely.Each administration group and control group beasle dog in addition to above-mentioned symptom, sign outward appearance, behavioral activity and
The aspect Non Apparent Abnormality such as diet, stool and urine is normal, and hair is glossy, and ordinary circumstance is good, and body weight increases naturally.Each group animal
Being showed no eye, ear and oral cavity etc. has abnormal secretion.
Hematological examination result shows：Before administration, administration 2 months, 4 months, 6 months and be discontinued convalescence when each dosage group
Hematological indices compare that there was no significant difference with NS control groups.During this medicine taking prompting and be discontinued recover during Vitexin, sweet
To hematology indices, the influence without significant adverse dew alcohol blend compositions occurs.
Blood biochemical analysis inspection result shows：The heavy dose of group of Vitexin, mannitol blend compositions is total during medication 2 months
Protein value and Vitexin, mannitol blend compositions are big at 6 months, the GPT of middle dose group is significantly reduced compared with control group,
But the liver function of the same period other indexs compare that there was no significant difference with control group, this prompting Vitexin, mannitol blend compositions
Long term administration is on each parameter of the liver function of beasle dog substantially without obvious influence.
During medication 2 months, small dose group urea nitrogen is significantly raised compared with NS control groups, but is used during medication 4 months and 6 months
When each dosage group urea nitrogen of medicine and control group indifference, this prompting 2 months the change of small dose group urea nitrogen and medicine without
Close, belong to transient change.Medication 4 months, 6 months when Vitexin, the heavy dose of group beasle dog creatinine of mannitol blend compositions
(CREA) value is significantly raised compared with control group, and creatinine (CREA) is one of Primary Reference index index for reflection renal function,
Therefore prompting Vitexin, mannitol blend compositions heavy dose have a certain impact to beasle dog renal function.Remaining each administration group
Difference that the blood biochemical analysis index of dog compares that there are no significant with NS control groups.The comprehensive biochemical index of whole blood can be seen that,
The Vitexin of each dosage group, the long term administration of mannitol blend compositions influence on the liver function of beasle dog without obvious, but for a long time
Administration may have a certain impact to renal function, it is proposed that note observing the change of renal function during clinical test.
Routine urianlysis shows：Each administration group routine urinalysis is compared with NS control groups when 4 months, 6 months and convalescence is administered
There was no significant difference between group, and this prompting Vitexin, mannitol blend compositions give beasle dog after 6 months to the routine urinalysis of animal
Have no significant effect.
ECG is checked and shown：Before administration, administration 2 months, 4 months, 6 months and convalescence when each administration group ECG items refer to
Difference that mark compares that there are no significant with NS control groups.This prompting ECG inspection result shows the Vitexin of large, medium and small dosage, sweet dew
Alcohol blend compositions long-term prescription does not produce obvious adverse effect to the ECG of beasle dog.
Animal becomes celestial and shows no obvious abnormalities；Organ coefficient inspection result show medication 4 months, 6 months and during convalescence it is each
Dosage group beasle dog organ coefficient compares that there was no significant difference with NS control groups.
The main parenchyma denaturation from internal organs of histopathologic examination, meronecrosis, inflammation, bleeding and congestion of blood vessel expansion
, the histopathological indications such as oedema aspect observed.Result shows：Administration 4,6 months and be discontinued 15 days convalescences when,
The beasle dog heart, liver, spleen, lung, kidney, adrenal gland, thymus gland, the first of the large, medium and small each dosage group of Vitexin, mannitol blend compositions
Shape gland, brain (brain, cerebellum, brain stem) spinal cord, oesophagus, Stomach duodenum, gall-bladder, salivary gland, pancreas, parathyroid gland, mammary gland,
Bone marrow of sternum, testis (and epididymis), prostate, uterus, ovary, brain, hypophysis, optic nerve, ischium lymph node, administration part etc. are
Have no obvious lesion.Without significant difference (P ＞ 0.05) between medication each group and NS control groups.
In sum, the large, medium and small dose intravenous of Vitexin, mannitol blend compositions be administered 6 months it is general to Beagle dog
Behaviouristics, body weight, food ration etc. have no significant effect, and the large, medium and small dosage group hematology of Vitexin, mannitol blend compositions is each
The Non Apparent Abnormality change of item index, blood biochemical analysis inspection prompting Vitexin, mannitol blend compositions heavy dose may be contrasted
The renal function of lattice dog has a certain impact, routine urianlysis discovery without exception, and organ coefficient inspection is showed no obvious influence, disease
Reason histological examination there are no obvious pathology infringement and change, and ECG is checked and do not produced obvious adverse effect；It is Vitexin, sweet
Dew each dosage group of alcohol blend compositions has no the generation of overt toxicity effect when observing convalescence of being discontinued, and also has no new toxicity
The generation of reaction.This prompting Vitexin, mannitol blend compositions clinic long-term prescription are safe comparings.
Test example 5：Animal anaphylaxis, excitant, hemolytic experimental study
Animal sensitivity test result
(1) sample and source
Vitexin, mannitol blend compositions：There is provided by Qixing Medicine Science and Technology Co., Ltd., Hefei, lot number：101109 (freeze
Dry powder pin, is faint yellow loose block, specification：Every bottle of 30mg/), it is dissolved to required concentration with physiological saline (NS) before use；
0.9% physiological saline (NS)：Produced by Shanghai Hua Yuan Pharmacy stock Co., Ltd, lot number：100705076；5% fresh albumen：
Prepared with fresh egg.
(2) animal
Cavy, 32, body weight 300-350g, ♂ are provided, laboratory rearing by Medical University Of Anhui's Experimental Animal Center
Temperature：23 ± 3 DEG C, airconditioning control, relative humidity：50~70%.
(3) method
Cavy 32, body weight 300-350g, ♂ are randomly divided into four groups, NS control groups, positive controls (egg white) and male
(low dosage is that clinical people intends using dosage 0.5mg/kg for Jing Su, mannitol blend compositions high and low dose group：Sensitization 0.5mg/kg,
Attack 1.0mg/kg；High dose is 5 times of low dosage：Sensitization 2.5mg/kg, attacks 5.0mg/kg), every group of 8 cavys, each group
By sterile working, intraperitoneal injection sterile injection physiological saline, Vitexin, high and low dose of mannitol blend compositions the next day respectively
Amount and 5% fresh albumen 0.5ml/, totally 5 times.In the 14th day after last sensitizing injection, cut by forelimb lateral vein skin
Open, injecting normal saline parenteral solution, Vitexin, mannitol blend compositions high and low dose and 5% fresh albumen solution 1.0ml/
Only, injection time is 1min, is attacked, and symptoms of allergic is observed according to table 12, is examined 30 minutes, then proceedes to raise
Support observation 3 hours.
The symptoms of allergic of table 12
| 0：Normally | 7：It is short of breath | 14：Instability of gait |
| 1：It is unpeaceful | 8：Urination | 15：Jump |
| 2：Piloerection | 9：Defecation | 16：Pant |
| 3：Shake | 10：Shed tears | 17：Spasm |
| 4：Scratch nose | 11：Expiratory dyspnea | 18：Horizontal turn |
| 5：Sneeze | 12：Rale | 19：Cheyne-stokes respiration |
| 6：Cough | 13：Purpura | 20：It is dead |
As a result criterion：Judge that allergic reaction is strong and weak according to the evaluation criterion of table 13."-" is asymptomatic；"+" for it is uneasy,
Tremble, scratch nose, urinate, be short of breath；" ++ " is in addition to above-mentioned symptom, to have difficulty in breathing and dyskinesia；" +++ " removes above-mentioned disease
Outside shape, respiratory failure can recover up to spasm；" ++++" it is death.
The cavy allergic reaction series of table 13
(4) result of the test
Vitexin, mannitol blend compositions are can be seen that from the result of the test of table 14 give guinea pig intraperitoneal injection sensitization, vein
Injection is attacked, to cavy without allergic reaction.Vitexin, mannitol blend compositions whole body active hypersensitive test are qualified.
The symptom that the 14th day cavy of table 14 produces after exciting
(5) conclusion
When iv Vitexins, mannitol blend compositions is excited within the 14th day without allergic symptom, result of the test shows cavy
Vitexin, mannitol blend compositions intravenously administrable have no obvious sensitization.
Animal irritation test result
(1) experimental animal
Healthy rabbits 2.2~2.5kg, ♀ ♂ half and half, is provided, laboratory rearing by Medical University Of Anhui's Experimental Animal Center
Temperature：23 ± 3 DEG C, airconditioning control, relative humidity：50%~70%.
(2) test method
Take healthy rabbits 8, ♀ ♂ half and half after weighing, are randomly divided into two groups, every group 4, quiet in fixed ear edge respectively
(concentration is 30mg100ml for arteries and veins instillation Vitexin, mannitol blend compositions-1；5ml.kg-1) and equivalent NS (5.0mlkg-1) drip-feed speed be 1ml.min-1, daily with position be injected intravenously 1 time, dosage it is identical (be intended to during injection local unhairing and
Sterilization), continuous 4 days, be discontinued the 2nd day, put to death animal, take immediately rabbit ear polupus put it is fixed in 10% formalin after row tissue disease
Reason histological examination, histologic lesion's situation at 1.0,2.0,3.0 and 4.0cm is spaced at record auricular vein entry point to proximal part
(first visually observe and whether there is congested, oedema and necrosis, then carry out pathological examination).
(3) result of the test
After drip-feed Vitexin, mannitol blend compositions and NS, freely, feed and stool and urine are normal for rabbit activity,
Hair is glossy, has no obvious abnormal response；Outside injection site skin is shown in that point-like forms a scab, non-show, swollen, heat, pain and part
Thicken, fester, the abnormal response such as tubercle.
Pathology results are as shown in the table, Vitexin, mannitol blend compositions medication group and NS control group rabbit ear edge
Vein blood vessel tube wall is complete, and without thrombosis in tube chamber, interstitial has no that bleeding, oedema and cell infiltration (are shown in Table around blood vessel
15)。
The Vitexin of table 15, mannitol blend compositions (30mg100ml-1) drip-feed is to the stimulation of rabbit vein
Note：–：Normally.
(4) conclusion
Rabbit vein instillation Vitexin, mannitol blend compositions have no obvious vein irritating reaction.
Animal sensitivity test result
(1) experimental animal
Healthy rabbits 2.3kg, ♂, are provided by Medical University Of Anhui's Experimental Animal Center, laboratory rearing temperature：23±3
DEG C, airconditioning control；Relative humidity：50%~70%.
(2) test method
It is prepared by 2% rabbit red cell suspension：Healthy male rabbit one is taken, heart extracting blood 10ml is placed in beaker, is put into
Bead is shaken gently for, and fibrin is removed out after several minutes, takes out defibrinated blood, plus normal saline
(2000rpm, 5min) is centrifuged, and removes supernatant, and the blood cell of precipitation adds physiological saline again, cleans, then is centrifuged, and so repeatedly three
It is secondary, by the capacity of blood cell, 2% suspension is made into physiological saline.
After taking 7 numberings of clean tube, the rabbit erythrocyte suspensions of 2.5ml 2% are separately added into, are separately added into shown according to the form below
The Vitexin of respective volume, mannitol blend compositions (30mg.100ml-1) and NS or double distilled waters, put in 37 DEG C of incubators, divide
Not Guan Cha 0.5,1,2,3h hemolytic reaction situations.
(3) result of the test
Result is as shown in table 16 below, and Vitexin, the mannitol mixing group of different volumes are added in 2% rabbit erythrocyte suspension
0.5 after compound, 1,2, the 3h generations without hemolytic reaction.
The Vitexin of table 16, mannitol blend compositions (30mg.100ml-1) normal saline solution hemolytic test
Note：+：Haemolysis and/or agglutinating reaction ,-：Not haemolysis also not aggegation
(4) conclusion
Vitexin, mannitol blend compositions have no obvious hemolytic reaction.
Claims (5)
1. a kind of composition for treating cardiovascular and cerebrovascular disease, it is characterised in that its active component is the Vitexin of therapeutically effective amount
With the blend compositions of mannitol, the composition do not contain mannose.
2. it is according to claim 1 treatment cardiovascular and cerebrovascular disease composition, it is characterised in that the Vitexin and sweet dew
The weight ratio of alcohol is 1:6~10.
3. the composition for the treatment of cardiovascular and cerebrovascular disease according to claim 1, it is characterised in that the regulation composition to
PH is 8.5 ± 0.5.
4. a kind of preparation method of the composition of the as claimed in claim 1 treatment cardiovascular and cerebrovascular disease, it is characterised in that described
Pharmaceutical composition be by the basis of dry weight, according to 1:6~10 weight than Vitexin and mannitol prepare, tool
Body step is as follows：
A, recipe quantity mannitol, the mixing of Vitexin raw material are weighed, plus water for injection stirs, then adds the hydrogen-oxygen of 0.1mol/L
Change sodium solution, stirring and dissolving, then PH be adjusted to 8.5 ± 0.5 or so with the hydrochloric acid solution of 1mol/L, plus 0.04-0.05% pin
With activated carbon, stir 10-15 minutes, micropore titanium filter decompression coarse filtration takes off charcoal, after checking that content, pH are qualified, then through 0.2-
After 0.25 μm of miillpore filter end-filtration, it is sub-packed in cillin bottle, frozen drying；
B, pre-freeze：The medicine for having dispensed is put into pre-freeze on freeze drying box internal partition to start to+25 DEG C~-40 DEG C；
C, lyophilization：Condenser temperature is dropped to less than -45 DEG C, starts vavuum pump, treat that vacuum reaches certain numerical value
Afterwards, slowly open butterfly valve, when the vacuum in drying box is that below 0.1mmHg closes refrigerator up to 13.33Pa, by every
Heating system under plate is slowly heated, and the temperature of frozen product is gradually risen to 22-25 DEG C；
D, re-dry：Dried at 25-30 DEG C, loss on drying meets regulation, aseptically adds a cover plug, rolls lid, packed,
Inspection, storage.
5. a kind of method of inspection of the composition of the as claimed in claim 1 treatment cardiovascular and cerebrovascular disease, it is characterised in that including
Procedure below：
(1) this product is taken appropriate, plus ethanol is made every 1ml μ g solution containing main ingredient 20, as need testing solution, separately takes Vitexin control
Product are made in the same way of reference substance solution, are tested according to the one annex thin-layered chromatography of version Chinese Pharmacopoeia in 2010, draw test sample molten
Each 5 μ l of liquid, reference substance solution, put on same PA membrane respectively, are solvent with 36% acetic acid, launch, and take out, and dry,
With 1% aluminum trichloride solution, hot blast drying is put and inspect under uviol lamp 365nm for spray, in test sample chromatogram, with reference substance chromatogram
On corresponding position, show the fluorescence spot of same color；
(2) this product is taken in right amount, plus ethanol is made solution of every 1ml containing the μ g of main ingredient 10, it is attached according to version Chinese Pharmacopoeia one in 2010
Record UV-VIS spectrophotometry is determined, and has absorption maximum at 270nm wavelength；
(3) relevant material：This product content is taken, is well mixed, precision weighs the sample for being approximately equivalent to Vitexin 25mg, puts 50ml
In measuring bottle, plus 60% ethanol 10ml makes to be made in every 1ml the solution containing 500 μ g after dissolving of flowing phase dilution, used as test sample
Solution；Precision is measured in right amount, plus flowing phase dilution is made the solution containing 10 μ g in every 1ml, used as contrast solution；Another precision is weighed
Orientin reference substance is appropriate, makes to dissolve and quantify dilution and be made in every 1ml with mobile phase to contain the solution of 3 μ g as impurity reference substance
Solution, according to the chromatographic condition under (5) assay, precision measures the μ l of reference substance solution 20 injection liquid chromatographs, regulation
Detection sensitivity, makes the 20% of the peak height about full scale of principal component chromatographic peak；Precision measures the μ l of need testing solution 20 injections again
Liquid chromatograph, record chromatogram to 4 times of principal component chromatographic peak retention time, such as aobvious impurity peaks in need testing solution chromatogram,
Impure orientin, with calculated by peak area, must not cross 0.6% by external standard method, any less than contrast solution master in need testing solution
In the case that the peak that 0.01 times of peak area is negligible, the summation of all impurity levels must not cross 2.0%；
(4) mannitol：Take this product appropriate, be approximately equivalent to mannitol 0.2g, it is accurately weighed, put in 250ml measuring bottles, adding water makes dissolving
And scale is diluted to, shake up；Precision measures 10ml, puts in iodine flask, and precision is increased sodium iodide solution and [takes sulfuric acid solution (1 → 20)
90ml and sodium periodate solution (2.3 → 1000) 110ml is mixed] 50ml, put and heated 15 minutes in water-bath, let cool, plus iodine
Change potassium test solution 10ml, close plug is placed 5 minutes, titrated with 0.05mol/L sodium thiosulfate titrating solution, during to nearly terminal, plus starch
Indicator solution 1ml, continues to be titrated to blue disappearance, and the result of titration is corrected with blank test；Per the titration of 1ml sodium thiosulfate
C6H14O6 of the liquid equivalent to 0.9109mg, the 90.0~110.0% of labelled amount are should be containing mannitol；
(5) Vitexin assay：According to the one annex high effective liquid chromatography for measuring of version Chinese Pharmacopoeia in 2010；
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler；Acetonitrile-water-glacial acetic acid is
18：82：1 is mobile phase；Detection wavelength is 270nm；Number of theoretical plate is calculated by Vitexin peak and should be not less than 2500, Vitexin peak with
The separating degree of impurity peaks all should meet the requirements；
Determination method：This product content is taken, is well mixed, precision weighs the sample for being approximately equivalent to Vitexin 25mg, puts 50ml measuring bottles
In, plus 60% ethanol makes to dissolve and be diluted to scale, shakes up；It is accurate again to draw during 1ml puts 10ml volumetric flasks, plus flowing phase dilution
To scale, shake up, as need testing solution；Precision measures 20 μ l injection liquid chromatographs, records chromatogram；Separately take Vitexin pair
It is appropriate according to product, it is accurately weighed, it is measured in the same method, by external standard method with calculated by peak area, (C21H20O10) containing Vitexin should be labelled amount
90.0~110.0%.
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| CN1442137A (en) * | 2003-04-09 | 2003-09-17 | 吴梅春 | Crataegus leaf total flarone powder injection for treating heart and brain blood vessel disease and its preparation method |
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