CN103142517A

CN103142517A - Salvianolic acid A dropping pill and application thereof to medicine preparation

Info

Publication number: CN103142517A
Application number: CN2012104872518A
Authority: CN
Inventors: 刘鹏; 崔刚; 刘尧奇; 刘艳红; 钟阳桂; 刘恩位
Original assignee: 楚健
Current assignee: Jiangxi Qingfeng Pharmaceutical Co., Ltd.
Priority date: 2012-11-20
Filing date: 2012-11-20
Publication date: 2013-06-12
Anticipated expiration: 2032-11-20
Also published as: CN103142517B

Abstract

The invention relates to a salvianolic acid A dropping pill. The salvianolic acid A dropping pill contains by weight 5%-60% of a salvianolic acid A composition and 95%-40% of a substrate. The salvianolic acid A composition comprises salvianolic acid A more than 93% and less than 100%, and other 4 ingredients including 0.1%-2% of alkannic acid, 0.1%-2% of rosmarinic acid, 0.1%-2% of salvianolic acid B and 0.1%-2% of salvianolic acid C. The substrate is one or more selected from PEG 4000, PEG 6000, PEG 8000, gelatin, stearic acid, glycerol monostearate, insect wax and hydrogenated vegetable oil. The invention also discloses application of the salvianolic acid A dropping pill to preparation of medicine for preventing and / or treating ischemic cerebrovascular disease.

Description

A kind of salvianolic acid A drop pill and prepare medicinal usage

Technical field

The present invention relates to a kind of salvianolic acid A drop pill and prepare medicinal usage.

Technical background

Cerebrovascular claims again apoplexy, is to make to supply a kind of nervous system disease due to the blood vessel generation pathological changes of brain blood by the various causes of disease.Its Etiological be the reasons such as cerebral arteries system disease damage (as cerebral arteriosclerosis) the cerebral arteries luminal stenosis, vasospasm, the obturation that cause or break, Oligemia or total blockage, brain blood circulation and dysfunction, the impaired and series of symptoms that occurs of cerebral tissue.Mainly comprise ischemic and hemorrhagic apoplexy.Wherein (ICVD claims again Ischemic Stroke) account for 80% left and right.

Ischemic cerebrovascular refers to that local brain tissue comprises the afunction of degeneration, necrosis or a property crossed that neurocyte, glial cell and contact fiber occur due to blood supply disorder.The cerebral arteries emphraxis that Intravascular Thrombus formation, thromboembolism, angiostenosis cause is the main cause of cerebral infarction.Ischemia cause cranial nerve cell damage and dead mechanism of action various, complicated, after cerebral infarction (being cerebral ischemia), supply due to brain shortage blood and oxygen, cause large brain energy metabolism unbalance, produce a series of pathologic damage, as oxidative stress, toxicity of excitatory amino acid, calcium overload, inflammatory reaction etc., thereby cause neuronic mortality.It is commonly encountered diseases, frequently-occurring disease clinically, and mortality rate and disability rate are very high, existing one of the three universally acknowledged large lethal diseases that become.Treatment is mainly the recovery of function of nervous system after thrombolytic, the neuron of being at death's door of saving ischemic area (half blanking bar) and promotion damage clinically.The control ischemic cerebrovascular is current mankind difficult medical problem in the urgent need to address.At present U.S. FDA has only been ratified tissue plasmin activation factor (tPA) for the thromboembolism treatment after apoplexy, but its therapeutic time window is very narrow, only in apoplexy, uses just effectively in 4.5 hours; But also exist hemorrhage and Ischemia Reperfusion to increase the weight of the danger of brain injury.And at present for the neuroprotective drug of ischemic stroke treatment comprise calcium channel blocker as nimodipine, glutamate receptor antagonists as dizocilpine (DizocilPine), antioxidant or free radical scavenger as Edaravone, NO signal transduction pathway regulator lu pei Shandong mile (Lubeluzo1e) and inflammation inhibitor enlimomab (Enlimomab) etc.But the therapeutical effect had in them is imprecise or specificity is not strong, some toxic and side effects than large, toleration is little, have also in clinical before or the clinical research stage, being difficult to actively affects in the performance of control cerebral infarction.Thereby, develop quick control brain ischemia medicament effective, safety and stability extremely urgent.Brought into play very important positive role at this field Chinese medicine.

Red sage formulation is the base therapy medicine of China's cardiovascular and cerebrovascular disease, and because of determined curative effect, Radix Salviae Miltiorrhizae has become China's consumption maximum, sales volume is the highest, preparation factory is maximum, one of Chinese medicine that Clinical Dosage Form is the most complete.The Radix Salviae Miltiorrhizae active chemical mainly contains two large classes: fat-soluble tanshinone compound and water-soluble phenolic compounds.Research shows: the salvianolic acid class is at anti-hepar damnification, atherosclerosis and apoptosis and improve the aspects such as memory dysfunction significant activity is arranged.Wherein the strongest with salvianolic acid A (Salvianolic acid A) antioxidant activity again.

But the natural content of salvianolic acid A extremely low (0.01-0.06% that is about red rooted salvia), make the crude drug high cost, the separation and purification difficulty is excessive, is seriously restricting the R and D of medicine, becomes the bottleneck of its industrialization.In addition, in prior art, also have experiment confirmation salvianolic acid B to degrade and can change into salvianolic acid A by ester hydrolysis decarboxylation and chroman ring ring-opening reaction, but the conversion process of above-mentioned prior art is uncontrollable, conversion byproducts is many, and the productive rate of principal product salvianolic acid A is lower.

Although also there are some patent documentations to propose to attempt to overcome the defect existed in prior art in prior art, but be only all that simple trial heats up, changes pH value or improves concentration that transforms front pressure differential self etc., without any a patent or prior art deeply, study conversion reaction all sidedly and all comprise which major influence factors, how synergism exerts an influence to the salvianolic acid A productive rate jointly each other as the concentration that transforms front pressure differential self, pH value, temperature, time etc. more without any a patent or prior art, to propose these factors.

On this basis, seldom there are other inventors or prior art to propose to attempt adding catalyst in conversion so that reaction is more abundant, having related to the related content that adopts catalyst to be transformed in currently available technology exists only in two parts of patent application documents submitting in same day of on April 6th, 2010, these two parts of patent application documents are respectively that application number is 2010101436787, patent application and application number that denomination of invention is " a kind of catalyzed conversion salvianolic acid B prepares the method for salvianolic acid A " are 2010101436876, the patent application that denomination of invention is " a kind of method that preliminary purification salvianolic acid B transforms raw material ".In the description of these two parts of patent applications, although disclose the technology contents that the catalyzed conversion salvianolic acid B prepares salvianolic acid A, its catalyst is carbamide, the mol ratio of carbamide and salvianolic acid B is (0.4～0.6): 1, require consume and add carbamide very high, so production cost is very high.And, in above-mentioned two parts of patent application documents, do not pay close attention to too the collaborative impact that the salvianolic acid A productive rate is produced of pH value and other correlative factors.Moreover it transforms raw material be the radix Salviae Miltiorrhizae water extract through the co-chromatography preliminary purification, salvianolic acid B >=50% wherein, this all has relatively high expectations to purity and purifying technique of transforming raw material, and technique is complexity comparatively also, has further increased production cost.More crucial, although claim " the directed conversion ratio of the salvianolic acid A salvianolic acid B that uses this method to prepare >=10%, even reach 60% " in its application documents.But because in fact carbamide do not have open loop, dehydration, decarboxylation, the ester effect is only arranged into, and therefore, its conversion ratio does not reach 60% at all, and the conversion ratio in the specific embodiment of this application file is only up to 53%, and the conversion ratio of a plurality of embodiment is only 10%, 20% and 30%.Still there is a big difference in the requirement of this and actual industrialization.

In addition, salvianolic acid A has obvious pharmacological action to the cerebral microvascular thromboembolism, and the effect be better than salvianolic acid B (Zhang Hengai. impact and the Mechanism Study [D] of salvianolic acid A on cerebral microvascular thromboembolism rat. Beijing: Beijing Union Medical College graduate school, 2011), but salvianolic acid A is to be formed by danshensu and caffeic acid condensation, contain a plurality of phenolic hydroxyl groups, hydroxyl, the reactive groups such as ester bond, it is unstable to light and heat, the easy oxygen of exposure air, above-mentioned physicochemical property due to salvianolic acid A, make the salvianolic acid preparation, its stability also guarantees that the salvianolic acid A pharmacological action becomes the key technology of preparation.

Summary of the invention

The above-mentioned defect existed for overcoming prior art, the invention provides a kind of salvianolic acid A drop pill, further, also provides it for prevention and or the purposes for the treatment of ischemic cerebrovascular aspect.

The invention provides a kind of salvianolic acid A drop pill, described salvianolic acid A drop pill comprises that each composition of wherein said drop pill is pressed column weight amount proportioning and made containing salvianolic acid A compositions, substrate: containing salvianolic acid A compositions 5%～60%, and substrate 95%～40%; Wherein saidly containing salvianolic acid A content in the salvianolic acid A compositions, be greater than 93%, be less than 100%, also comprise 4 other compositions: alkannic acid 0.1%～2%, rosmarinic acid 0.1%～2%, salvianolic acid B 0.1%～2%, salvianolic acid C 0.1%～2%, wherein said substrate is any one or a few in Macrogol 4000, polyethylene glycol 6000, PEG 8000, gelatin, stearic acid, glyceryl monostearate, insect wax and hydrogenated vegetable oil.

Preferably, each composition of wherein said drop pill is pressed column weight amount proportioning and is made: containing salvianolic acid A compositions 10%～40%, substrate 90%～60%, wherein saidly containing salvianolic acid A content in the salvianolic acid A compositions, be greater than 94%, be less than 99%, also comprise 4 other compositions: alkannic acid 0.1%～1.5%, rosmarinic acid 0.1%～1.5%, salvianolic acid B 0.1%～1.5%, salvianolic acid C 0.1%～1.5%.

On the other hand, the present invention also provides a kind of preparation method of salvianolic acid A drop pill, comprises the steps:

(1) extract: Radix Salviae Miltiorrhizae water or alcoholic solution extract and obtain aqueous extract or alcohol extract; Wherein in extracting solution, salvianolic acid B concentration is 1mg/ml～30mg/ml or is diluted with water to 1mg/ml～30mg/ml;

(2) transform: the extracting solution that step (1) is obtained, adjust pH to 3.5～6.5, add the catalyst that molar percentage is 0.1%～3.0%, 100～140 ℃ of heating 1～6 hour;

(3) purification:

A. solution step (2) obtained, adjust pH to 2.5～4.5, centrifugal, and supernatant separates through nonpolar or low pole macroporous resin column chromatography, after washing with water, uses the eluant eluting, and high performance liquid chromatogram detects salvianolic acid A, collects the eluent that contains salvianolic acid A;

B. sephadex lh-20 or ODS-C18 or the separation of polyamide chromatography post for eluent step a obtained, use the eluant eluting, and high performance liquid chromatogram detects salvianolic acid A, collects the eluent that contains salvianolic acid A;

C. eluent step b obtained is adjusted pH to 2.0～4.0, through organic solvent extraction, and the Separation of Organic phase;

D. solution step c obtained separates with silica gel column chromatography, uses the eluant eluting, and high performance liquid chromatogram detects salvianolic acid A, collects the eluent that contains salvianolic acid A;

(4) drying: the eluent that steps d is obtained, the reclaim under reduced pressure eluant, then be dissolved in water, vacuum drying, microwave vacuum drying or spray drying, obtain the salvianolic acid A compositions;

(5) drop pill preparation: get the salvianolic acid A compositions that step (4) obtains and mix homogeneously with substrate, splash in condensed fluid and become ball, be able to the drop pill of the unit dose of salvianolic acid A meter.

Preferably, wherein the water extracting method described in step (1) is: get red rooted salvia, be cut into decoction pieces or be ground into diameter 2mm granule, add 3～15 times of water gagings at every turn and extract, extract altogether 1～3 time, extract 1～4 hour at every turn; Extracting solution is evaporated to relative density 1.10～1.25 (60 ℃), add ethanol to make containing the alcohol amount 30%～80%, centrifugal, the supernatant decompression recycling ethanol also is concentrated into without the alcohol flavor, described water extraction adopts decoction to extract or 45～95 ℃ of water temperature lixiviates are got, stir with 10～50 rev/mins of speed simultaneously, obtain Radix Salviae Miltiorrhizae extract.

Preferably, wherein the alcohol extraction method described in step (1) is: get red rooted salvia, be cut into decoction pieces or be ground into diameter 2mm granule, add 3～15 times of amount 30%～60% alcohol reflux at every turn, extract 1～4 hour at every turn, extract altogether 1～3 time; Decompression recycling ethanol, obtain Radix Salviae Miltiorrhizae extract.

Preferably, wherein in the extracting solution in step (1) salvianolic acid B concentration be 5mg/ml～20mg/ml or be diluted with water to 5mg/ml～20mg/ml.

Preferably, it is characterized in that the catalyst in step (2) is one or more in iron chloride, ruthenium trichloride, aluminum chloride, zinc chloride, Palladous chloride..

Preferably, the molar percentage that it is characterized in that catalyst and salvianolic acid B is 0.5%～2.0%.

Preferably, wherein macroporous resin column described in step a is HPD-80, HPD-100, HPD-100B, HPD-200A, HPD-300, HPD-450, HPD-722, HPD-826, ADS-5, ADS-8, ADS-21 or D101, AB-8; The water that described eluant is water and different proportion and ethanol, and first water, 10～40% ethanol elutions, then use 20～60% ethanol elutions, high performance liquid chromatogram detects salvianolic acid A, collects and contains the salvianolic acid A part, and eluent is concentrated into without the alcohol flavor.

Preferably, water and ethanol that wherein the described eluant in step b is water and different proportion, and first water, 20～60% alcoholic solution eluting remove impurity, use again 40～90% alcoholic solution eluting, high performance liquid chromatogram detects salvianolic acid A, collects and contains the salvianolic acid A part, and eluent is concentrated into without the alcohol flavor.

Preferably, wherein the organic solvent described in step c is t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate or Ethyl formate.

Preferably, the two-phase solvent that wherein eluant described in steps d is petroleum ether, pentane, normal heptane, ethyl acetate, methyl acetate, Ethyl formate, t-butyl methyl ether composition.

Preferably, the temperature of microwave vacuum drying: 20-100 ℃ described in step (4) wherein, return difference temperature 1-5 ℃, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.

Preferably, vacuum drying temperature described in step (4) wherein: 50 ℃～90 ℃, more than vacuum-0.07Mpa, power: 1～60KW, dry 2～20 hours.

Preferably, spray-dired intake air temperature described in step (4) wherein: 150 ℃～350 ℃, the air outlet temperature: 70 ℃～95 ℃, spray velocity: 1～300ml/min.

Preferably, the unit dose of salvianolic acid A of wherein take in step (5) is 1mg～100mg, and preferred, unit dose is 5mg～80mg.

Preferably, wherein pH adjusting agent is phosphoric acid, hydrochloric acid, sulphuric acid or acetic acid.

On the one hand, the present invention also provides described salvianolic acid A drop pill for the preparation of the purposes that prevents and/or treats the ischemic cerebrovascular medicine again.

Preferably, wherein said ischemic cerebrovascular mainly comprises any one or a few in atherosis or narrow, the lacunar infarction, Transient ischemic attacks, vascular dementia of cerebral thrombosis, cerebral ischemia reperfusion injury, cerebral embolism, cranium arteria carotis externa and brain basilar artery.

Preferably, described salvianolic acid A drop pill is for improving the purposes of the function of nervous system's symptom after cerebral ischemia.

Preferably, wherein said salvianolic acid A drop pill is for the protection of the purposes of ischemic tissue of brain damage.

Preferably, wherein said salvianolic acid A drop pill is for the protection of the purposes of blood brain barrier.

Preferably, wherein said salvianolic acid A drop pill is for reducing the purposes of ischemic tissue of brain infarction size.

Preferably, wherein said salvianolic acid A drop pill is for alleviating the purposes of ischemic tissue of brain edema.

Preferably, wherein said salvianolic acid A drop pill is for saving the purposes of ischemia half blanking bar.

Preferably, wherein said salvianolic acid A drop pill is for the purposes of the foundation that promotes the new life of cerebral vessels and collateral circulation.

Preferably, wherein said salvianolic acid A drop pill is for increasing the purposes of cerebral tissue microvessel density.

Preferably, wherein said salvianolic acid A drop pill is for promoting the purposes of vascular endothelial growth factor expression.

Preferably, wherein said salvianolic acid A drop pill is for promoting the purposes of basic fibroblast growth factor protein expression.

Preferably, wherein said salvianolic acid A drop pill is for increasing ischemia half blanking bar cerebral blood flow purposes.

Preferably, wherein said salvianolic acid A drop pill is for improving the Energy Metabolism of Brain Tissue purposes.

Preferably, wherein said salvianolic acid A drop pill is organized neuronal damage or dead purposes for suppressing Neurons Against Cerebral Ischemia.

Preferably, wherein said salvianolic acid A drop pill is for suppressing the purposes of cerebral tissue neuronal apoptosis.

Preferably, wherein said salvianolic acid A drop pill is for strengthening the purposes of cerebral tissue endogenous neurotrophic factor expression.

Preferably, wherein said salvianolic acid A drop pill is for suppressing the purposes of brain tissue inflammation's damage.

Preferably, wherein said salvianolic acid A drop pill is for suppressing Ca in the cerebral tissue neurocyte ²⁺the purposes of overload.

Preferably, wherein said salvianolic acid A drop pill is for improving the purposes of monoamine neurotransmitter disorder in cerebral tissue.

Preferably, wherein said salvianolic acid A drop pill is for suppressing the purposes of cerebral tissue toxicity of excitatory amino acid.

Preferably, wherein said salvianolic acid A drop pill is for suppressing the purposes of brain tissue oxygen radical damage.

Preferably, wherein said salvianolic acid A drop pill is for the protection of the purposes of cerebrovascular endothelial cell.

Preferably, wherein said salvianolic acid A drop pill is for the protection of the purposes of Injury of Cerebral Microvascular Endothelial Cells.

Preferably, wherein said salvianolic acid A drop pill is for preventing and/or treating the purposes of cerebral thrombosis.

Preferably, wherein said salvianolic acid A drop pill is for suppressing thrombotic purposes.

Preferably, wherein said salvianolic acid A drop pill is for thrombolytic purposes.

Preferably, wherein said salvianolic acid A drop pill is for improving hemorheological purposes.

The present invention is by the extraction to Radix Salviae Miltiorrhizae, transform, purification, drying process, screening and optimization through system, at first relatively extraction solvent and the extracting method of initiation material salvianolic acid B have been determined, because the salvianolic acid B water solublity is better, employing water extraction or low concentration alcohol extraction have been determined, again because the salvianolic acid B heat stability is poor, determined that employing hot water warm macerating extracts and adds the stirring extracting method, or use the low-concentration ethanol reflux, extract, make to extract solubility lower than 100 ℃, keep salvianolic acid B not to be destroyed, optimum solvent consumption and extraction time have been determined by orthogonal experiment, obtained adapting to the salvianolic acid B optimum extraction process of suitability for industrialized production.

The present invention is compared with the prior art and shows: the direct extraction of the red rooted salvia conversion that can be fed intake for initiation material, do not need salvianolic acid B is carried out being transformed after purification again, be in catalytic conversion reaction of the present invention, the reaction raw materials salvianolic acid B does not need high-purity, does not for example need the purity of salvianolic acid B >=50%.It is generally acknowledged that reaction raw materials is more pure better, yet, in catalytic conversion reaction of the present invention, the purity of salvianolic acid B height is on the not impact of conversion reaction effect.On the contrary, not only produce a large amount of impurity during salvianolic acid B purity>50%, and do not improve conversion ratio, therefore, the present invention has obtained unforeseeable technique effect.

In addition, the present invention can also mix low-purity with highly purified salvianolic acid B, only need be made into suitable initial conversion concentration and get final product, and can reach the purpose that changes into salvianolic acid A equally.Therefore, the preparation technology of this conversion raw material is very simple, and production cost also is very suitable for the application in actual industry when reducing.

Moreover the present invention by experiment repeatedly relatively, has at first determined that factor that the salvianolic acid A productive rate is produced to material impact is as the concentration that transforms front pressure differential self, pH value, temperature, time etc.On this basis, by paying concentration and other correlated conditions that a large amount of time, material and energy tests repeatedly to temperature, pH value, time, salvianolic acid B, be studied again, and these factors how synergism exerts an influence to the salvianolic acid A productive rate jointly each other, thereby determined that salvianolic acid B transforms the optimum temperature of the needs control of salvianolic acid A, pH value, time etc., and the salvianolic acid B initial concentration is controlled to 1mg/ml～30mg/ml, thereby make salvianolic acid A conversion ratio of the present invention more obviously be better than other conversion conditions.In chemical reaction, the purity of reactant usually affects the effect of reacting with concentration.Generally reactant is had to purity requirement, and think that the high specific concentration of concentration hangs down.In catalytic conversion reaction of the present invention, the purity of salvianolic acid B height is on the not impact of conversion reaction effect.On the contrary, it is not more high better to experimental results show that containing the concentration of salvianolic acid B in the salvianolic acid B aqueous solution, and the above concentration conversion ratio of 30mg/ml is low on the contrary, and effect is poorer.Therefore, the present invention, aspect cost-saving and production cycle, has obtained unforeseeable technique effect, creative.In the situation that prior art does not provide any technology enlightenment, if only deduction theoretically of those skilled in the art is impossible draw under above-mentioned each conditional parameter the sour B of pellet is changed into to the conclusion that salvianolic acid A has better changing effect.

What is more important, the present invention passes through performing creative labour, find that iron chloride, ruthenium trichloride, aluminum chloride, zinc chloride or Palladous chloride. can significantly improve as catalyst the conversion ratio that salvianolic acid B transforms salvianolic acid A, reaching that conversion ratio is highly stable approaches 60%, most cases can surpass 60%, this is all impossible in any one prior art in the past, therefore, has obtained unforeseeable technique effect.

Because salvianolic acid A content is lower, the large raising of salvianolic acid A content after transforming, but also containing a large amount of impurity, therefore, selected respectively low pole and nonpolar macroporous adsorption resin to carry out crude separation, select again polyamide, solvent extraction, silica gel to separate, and various flows part is measured, after removing the impurity part, salvianolic acid A content is brought up to 78% from 10% left and right, to 90%, to 93%, to 96%.

Further, when significantly improving conversion ratio, the present invention is by being suitable for the purification step of actual industry application, especially by after the steps such as a series of separation, eluting processing, do not adopt traditional constant pressure and dry, but adopt vacuum drying, spray drying, microwave vacuum drying, thereby thoroughly overcome, baking temperature was too high in the past, drying time long and to the destruction of salvianolic acid A large defect; And sublimation drying is long, the high and extract lyophilization gained of cost can't be removed the dissolvent residual problem fully.

Salvianolic acid A drop pill provided by the invention, chemistry and physical characteristic according to salvianolic acid A, from affecting the stable substrate of medicine, dosage form, extraneous as air, light, moisture, the generation chemical reactions such as impurity cause the decomposition of medicament to carry out creationary experimental analysis on the contrary.By the screening preparation prescription, to substrate Macrogol 4000 commonly used, polyethylene glycol 6000, PEG 8000, gelatin and stearic acid, glyceryl monostearate, insect wax, the hydrogenated vegetable wet goods is filled a prescription, grope into the ball condition, investigated chilling temperature, drip distance, cooling column length, drop pill speed is on becoming the impact of ball, investigated respectively the shaped degree that becomes ball, the ball method of double differences is different, dissolve scattered time limit, the salvianolic acid A changes of contents, pass through repetition test, make the salvianolic acid A drop pill, drop pill is taken and is carried taking convenience, clinical practice is must answering property good, and preparation stability is very high, solved due to air, light, moisture, the medicine that the generation chemical reactions such as impurity cause decomposes.The salvianolic acid A compositions is made to suitable preparation, solved the salvianolic acid A stability problem, by selecting suitable substrate and dosage form, guaranteed the salvianolic acid A pharmacological action, for clinical, provide safe and effective salvianolic acid A drop pill.

Salvianolic acid A drop pill of the present invention, except containing the main component salvianolic acid A, also contains a small amount of salvianolic acid B, alkannic acid, rosmarinic acid, salvianolic acid C; Salvianolic acid A is by removing free radical, alleviates mobility and the permeability that the membrane lipid peroxidating causes and changes, and stops the spilling of abnormal penetrating and enzyme of ion, thereby reduced cerebral ischemia re-pouring and the damage that causes, and cerebral ischemia is had to protective effect.But salvianolic acid A dose dependent ground suppresses the platelet aggregation that adenosine diphosphate (ADP), thrombin, arachidonic acid, collagen or U46619 induce, reduce cAMP level in platelet, and the expression and the Fibrinogen combination that reduce the P-selectin that ADP causes, thereby the platelet leukocyte recruitment that stops ADP to cause, be formed with preventive and therapeutic effect to atherosclerosis and thromboembolism; Salvianolic acid A improves middle cerebral artery occlusion model in rats rat function of nervous system defect state, dwindles infarct size, alleviates cerebral edema, reduces the related brain areas neure damage degree, cerebral ischemia is had to protective effect, to the protective effect of learning memory injury.

Salvianolic acid B in the salvianolic acid A drop pill is the main effective ingredient of Radix Salviae Miltiorrhizae and preparation Radix Salviae Miltiorrhizae Injection thereof, XIANGDAN ZHUSHEYE, and the same with salvianolic acid A have protective effect to cerebral ischemia, and atherosclerosis and thromboembolism are formed with to preventive and therapeutic effect; Cerebral ischemia is had to protective effect; alkannic acid, rosmarinic acid, salvianolic acid C effect and salvianolic acid A are roughly the same; stronger antioxidation is all arranged; can remove oxygen-derived free radicals; suppress lipid peroxidation; its action intensity is higher than vitamin C, vitamin E; it is one of natural product that at present known antioxidation is the strongest; the aggreation that obviously suppresses ADP, arachidonic acid, collagen-induced Rabbit Blood Platelets; and suppress thrombotic effect, and can extend the time-to-live of animal under anoxia condition.Can obviously improve function of nervous system's defect of cerebral reperfusion injury rat, improve behavior disorder, obviously dwindle brain infarction area, obviously improve FeCl ₃due to the animal nerve function damage that causes of rat cerebral ischemia, make the improvement of its obstacle, and can dwindle brain infarction area; Rosmarinic acid also contributes to prevent that the cell that free radical causes is impaired, has therefore reduced cancer and arteriosclerotic risk.

Alkannic acid in the salvianolic acid A drop pill suppresses propagation and the migration of vascular smooth muscle cell in addition, and prevention of arterial is atherosis, angiostenosis and vascellum endometrial hyperplasia, has vasodilative effect.Can suppress uric acid and cross the formation of ultra-oxygen anion free radical, the generation of inhibition peroxide, the effect of antiinflammatory and uric acid resisting is arranged.The external effect that can suppress metakentrin release.

Thereby the salvianolic acid C in the salvianolic acid A drop pill is also apoptosis-induced by suppressing the retardance of tubulin polymerization inducing cell mitosis, has anti-tumour cell proliferative activity.

There is common cerebrovascular is had protective effect, atherosclerosis and thromboembolism are formed with preventive and therapeutic effect, cerebral ischemia is had to protective effect after salvianolic acid A in the salvianolic acid A drop pill, salvianolic acid B, alkannic acid, rosmarinic acid, salvianolic acid C combination; use effect is stronger separately than salvianolic acid A monomeric compound to make it, have in addition simultaneously antiinflammatory and uric acid resisting effect, reduced cancer and arteriosclerotic risk.

On the other hand, the present invention also provide its for prevention and or the purposes for the treatment of ischemic cerebrovascular aspect, and by experimental results demonstrate:

Salvianolic acid A is by removing free radical, alleviates mobility and the permeability that the membrane lipid peroxidating causes and changes, and stops the spilling of abnormal penetrating and enzyme of ion, thereby reduced the damage caused due to cerebral ischemia re-pouring, and cerebral ischemia is had to protective effect.But salvianolic acid A dose dependent ground suppresses the platelet aggregation that adenosine diphosphate (ADP), thrombin, arachidonic acid, collagen or U46619 induce, reduce cAMP level in platelet, and the expression and the Fibrinogen combination that reduce the P-selectin that ADP causes, thereby the platelet leukocyte recruitment that stops ADP to cause, be formed with preventive and therapeutic effect to atherosclerosis and thromboembolism; Salvianolic acid A improves middle cerebral artery occlusion model in rats rat function of nervous system defect state, dwindles infarct size, alleviates cerebral edema, reduces the related brain areas neure damage degree, cerebral ischemia is had to protective effect, to the protective effect of learning memory injury.

Salvianolic acid B is the main effective ingredient of Radix Salviae Miltiorrhizae and preparation Radix Salviae Miltiorrhizae Injection thereof, XIANGDAN ZHUSHEYE, and the same with salvianolic acid A have protective effect to cerebral ischemia, and atherosclerosis and thromboembolism are formed with to preventive and therapeutic effect; Cerebral ischemia is had to protective effect; alkannic acid, rosmarinic acid, salvianolic acid C effect and salvianolic acid A are roughly the same; stronger antioxidation is all arranged; can remove oxygen-derived free radicals; suppress lipid peroxidation; its action intensity is higher than vitamin C, vitamin E; it is one of natural product that at present known antioxidation is the strongest; the aggreation that obviously suppresses ADP, arachidonic acid, collagen-induced Rabbit Blood Platelets; and suppress thrombotic effect, and can extend the time-to-live of animal under anoxia condition.Can obviously improve function of nervous system's defect of cerebral reperfusion injury rat, improve behavior disorder, obviously dwindle brain infarction area, obviously improve FeCl ₃due to the animal nerve function damage that causes of rat cerebral ischemia, make the improvement of its obstacle, and can dwindle brain infarction area; Rosmarinic acid also contributes to prevent that the cell that free radical causes is impaired, has therefore reduced cancer and arteriosclerotic risk.

Alkannic acid suppresses propagation and the migration of vascular smooth muscle cell in addition, and prevention of arterial is atherosis, angiostenosis and vascellum endometrial hyperplasia, has vasodilative effect.Can suppress uric acid and cross the formation of ultra-oxygen anion free radical, the generation of inhibition peroxide, the effect of antiinflammatory and uric acid resisting is arranged.The external effect that can suppress metakentrin release.

Thereby salvianolic acid C is also apoptosis-induced by suppressing the retardance of tubulin polymerization inducing cell mitosis, has anti-tumour cell proliferative activity.

There is common cerebrovascular is had protective effect, atherosclerosis and thromboembolism are formed with preventive and therapeutic effect, cerebral ischemia is had to protective effect after salvianolic acid A, salvianolic acid B, alkannic acid, rosmarinic acid, salvianolic acid C combination; use effect is stronger separately than salvianolic acid A monomeric compound to make it, have in addition simultaneously antiinflammatory and uric acid resisting effect, reduced cancer and arteriosclerotic risk.

Known through the experimental data contrast, the easily postoperative recovery from anesthesia scoring of apoplectic type renovascular hypertension in rats middle cerebral artery occlusion (MCAO), model group revive after (12h in) neural behavior scoring (symmetry of spontaneous activity, quadruped locomotion, forelimb stretch the action of creeping symmetry, climb the cage wall, push away the trunk reaction, antenna is to the reaction that stimulates etc.) significantly lower than sham operated rats, illustrate that after the cerebral thrombosis ischemia, easily apoplectic type renovascular hypertension in rats (RHRSP) function of nervous system is impaired serious, and, in lasting 14d, neural behavior scoring continues to be starkly lower than sham operated rats; Compare high, the middle dosage group of the salvianolic acid A drop pill behavioristics's scoring after rat ischemia 3d that can obviously raise in experiment with model group; After ischemia 7d, each dosage group rat neuroethology of nimodipine group and salvianolic acid A monomeric compound group and salvianolic acid A drop pill all obviously raises, and the most obvious with the middle and high dosage group of salvianolic acid A drop pill, and after its ischemia, the neurobehavioral of 14d approaches the sham operated rats level substantially; And with the integrality of dosage salvianolic acid A drop pill group rat, than salvianolic acid A monomeric compound group, recover soon and slightly good, neurobehavioral has more excellent trend.Prompting salvianolic acid A drop pill can improve neurobehavioral after RHRSP cerebral thrombosis ischemia, the effect of nervous symptoms after thering is protection and improving cerebral ischemia, and effect is better than nimodipine, and the trend that is better than same dosage salvianolic acid A monomeric compound is arranged.

Known through histopathology checking experiment Data Comparison, sham operated rats cerebral tissue Bilateral Symmetry, have no pathological changes, and the model group Intravascular Thrombus adheres to seriously.With model group relatively, the contraction in length of the left MCA local thrombus of each dosage group rat of Microscopic observation salvianolic acid A drop pill, reduce at the arterial wall bond area, with high, middle dosage group is the most obvious.The TTC visible infarcted cerebral constitution that dyes is white in color, and is dyed to white cerebral tissue scope in each dosage group of salvianolic acid A drop pill and dwindles than model control group is remarkable.HE dyes in visible model group left cortical MCA blood supply district (centered by the cortex of volume top) infarct has obvious cerebral tissue softening, necrocytosis, the phenomenons such as the local necrosis tissue comes off, salvianolic acid A drop pill high dose group rat is rare Telatrophy obscission herein, other each administration group necrocytosiss and the tissue degree varies that comes off, than model group, all significantly reduce, each dosage group of salvianolic acid A drop pill relatively is the dose-difference opposite sex; The HE dyeing of each administration group and model control group shows in kitchen range, kitchen range Zhou Jun has little blood vessel hyperplasia, but the little number of blood vessel of each administration group hypertrophy increases than model group is remarkable, and particularly remarkable with high, the middle dosage group of salvianolic acid A drop pill and salvianolic acid A monomeric compound group; In addition, the kitchen range shape that HE dyeing display model matched group differs in size as seen is hemorrhage, high, the middle dosage group of salvianolic acid A drop pill is accidental kitchen range shape bleeding, salvianolic acid A drop pill low dose group and salvianolic acid A monomeric compound group and nimodipine group also only have the minority animal to have the kitchen range shape hemorrhage, and hemorrhagic focus is less than model group.Result of the test shows that the salvianolic acid A drop pill can obviously improve RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia histopathology state; reduce the thrombosis bond area, reduce infarct size; thereby increasing the little blood vessel number, the necrosis of minimizing cerebral hemorrhage phenomenon protection cerebral tissue that reach surrouding brain tissue in infarcted region comes off; there is protection cerebral thrombosis ischemia and cause the effect of brain tissue impairment, and action effect there is the trend that is better than the salvianolic acid A monomeric compound.

Known through the experimental data contrast, cerebral thrombosis tissues following MCAO in rats blood-brain barrier disruption, permeability increases, and azovan blue (EB) albumin-binding can enter cerebral tissue by open blood brain barrier (BBB).After the administration of salvianolic acid A drop pill; under each dosage group rat cerebral tissue microscope, EB hot spot number and cerebral tissue EB detect content and obviously reduce; and the salvianolic acid A monomeric compound group that is less than same dosage; with model group, more all there were significant differences; illustrate that the salvianolic acid A drop pill increases inhibited to brain tissue impairment hyperamization brain Barrier Permeability; can protect blood brain barrier, and the further damaged of protection cerebral tissue, action effect is better than the salvianolic acid A monomeric compound.

Known through the experimental data contrast, by RHRSP cerebral thrombosis rat model group is compared, sham operated rats has no infarcted cerebral constitution; Each dosage group cerebral tissue Infarction volume of salvianolic acid A drop pill and brain water content are significantly less than model control group, and dosage is larger, and Infarction volume is less, and brain water content is fewer.Salvianolic acid A drop pill middle and high dosage group Infarction volume and brain water content are all lower than 30mg/kg nimodipine group; And 30mg/kg salvianolic acid A drop pill group rat cerebral infarction volume and brain water content are all low than the salvianolic acid A monomeric compound group of same dosage.Illustrate that the salvianolic acid A drop pill can accelerate the reparation of focus, reduce ischemic tissue of brain infarction size after the RHRSP cerebral thrombosis, alleviate the cerebral tissue edema, have and repair and the effect of protection Neurons Against Cerebral Ischemia tissue injury, be better than salvianolic acid A monomeric compound and nimodipine.

Known through the experimental data contrast, with RHRSP cerebral thrombosis ischemia model group, compare, after the ischemia treatment, each dosage group cerebral tissue ischemia penumbra MVD of salvianolic acid A drop pill and blood vessel scene are long-pending significantly to be increased to some extent, than nimodipine and more obvious with dosage salvianolic acid A monomeric compound, and, at ischemia 7d to 14d after this, maintain a metastable level.Show that the salvianolic acid A drop pill can increase MVD and the blood vessel field Area Ratio of cerebral tissue ischemia half blanking bar, prompting salvianolic acid A drop pill has the effect that promotes that ischemic tissue of brain Angiogenesis and collateral circulation are set up, and effect is better than salvianolic acid A monomeric compound and nimodipine.

Known through the experimental data contrast; RHRSP cerebral thrombosis ischemia model group VEGF Messenger RNA (VEGFmRNA) expression is increased significantly than sham operated rats; illustrate after the cerebral tissue hypoxic-ischemic can Stimulation of The Brain tissue in the expression increase of VEGF; after the prompting cerebral infarction, body self there will be a kind of protective response that resists ischemia injury; the expression of VEGF is increased, self " compensatory revascularization " occur after ischemia.To ischemia 7d, 14d, in experiment, relatively, the VEGFmRNA expression significantly raises, and the salvianolic acid A drop pill has the trend that is better than same dosage salvianolic acid A monomeric compound for each dosage group of salvianolic acid A drop pill and model group.Prompting salvianolic acid A drop pill can significantly promote the ischemic tissue of brain angiogenesis, promotes the foundation of collateral circulation compensation, saves ischemia half blanking bar, and, to anti-cerebral ischemia, and there is certain dose-effect relationship in the brain tissue impairment that the protection ischemia causes.

Known through the experimental data contrast; RHRSP cerebral thrombosis ischemia model group rat cerebral tissue's basic fibroblast growth factor (bFGF) protein expression is increased significantly than sham operated rats; to ischemia 7d, 14d, significant difference is arranged; after prompting cerebral tissue hypoxic-ischemic; body self produces of short duration protection stress, but Stimulation of The Brain organizes the bFGF protein expression to increase.Relatively, the bFGF protein expression of each time period significantly increases, with salvianolic acid A drop pill effect optimum for each dosage group of salvianolic acid A drop pill and salvianolic acid A monomeric compound group and nimodipine group and model group; Each time period of each dosage group of salvianolic acid A drop pill self relatively shows, the bFGF protein expression is continual and steady; Prompting salvianolic acid A drop pill can increase former of ischemic tissue of brain and secondary bFGF protein expression; effect with good neurocyte protection effect and promotion angiogenesis; contribute to the foundation of collateral circulation; save ischemia half blanking bar, and action effect is better than salvianolic acid A monomeric compound and nimodipine.

Known through the experimental data contrast; the salvianolic acid A drop pill can alleviate cerebral ischemia-reperfusion injury in rats neurologic impairment symptom; be conducive to its neurobehavioral recovery; the salvianolic acid A drop pill has the effect that improves function of nervous system after brain tissue impairment; energy prevention and protection cerebral ischemia reperfusion cause the damage of rat brain function of nervous system, and action effect is more obvious than salvianolic acid A monomeric compound.

Known through the experimental data contrast, each dosage group of salvianolic acid A drop pill is compared with the Ischemia-Reperfusion Injury Model group: cerebral infarction volume ratio, cerebral index and brain water content all obviously reduce, and salvianolic acid A drop pill low dose group and nimodipine group be no difference of science of statistics relatively; Relatively, its Infarction volume and cerebral index and brain water content all significantly reduce for the middle and high dosage group of salvianolic acid A drop pill and nimodipine group, and lower than same dosage salvianolic acid A monomeric compound group; Show that the salvianolic acid A drop pill can reduce rats after cerebral ischemic reperfusion cerebral tissue infarction size, can alleviate the cerebral tissue edema degree after cerebral ischemia reperfusion injury, effect is better than salvianolic acid A monomeric compound and nimodipine.

Known through the experimental data contrast, Level In Rats With Focal Cerebral Ischemia, after ischemia 10min, in experiment, each dosage group of salvianolic acid A drop pill, nimodipine group, salvianolic acid A monomeric compound group rise to some extent than model group ischemia half blanking bar rCBF, after ischemia, 30min begins, and each medication therapy groups ischemia half blanking bar rCBF except salvianolic acid A drop pill low dose group and model group relatively, have raise 12%～30%, after ischemia, in 1～2h, each medicine group ischemia half blanking bar rCBF is all apparently higher than model group.And particularly remarkable with high, the middle dosage group of salvianolic acid A drop pill; Each dosage group of salvianolic acid A drop pill is compared, and good dose-effect relationship is arranged; In the salvianolic acid A drop pill, the rCBF of dosage group day part is higher than the nimodipine group, and also slightly is better than same dosage salvianolic acid A monomeric compound.Hence one can see that, and the salvianolic acid A drop pill has the effect that increases rats with cerebral ischemia cerebral tissue ischemia half blanking bar rCBF, thereby is conducive to save the cerebral tissue that ischemia half blanking bar is at death's door, protection ischemia half blanking bar, the effect of performance treatment cerebral ischemia.

Known through the experimental data contrast, after rat continuous ischemia 2h recovers to fill with again, each organizes rat ischemia half blanking bar rCBF all increase in various degree.But focal cerebral ischemia is filled with the recovery of model group rat again and is filled with rear ischemia half blanking bar rCBF again and recover to fill with front than being increased significantly again, but still before ischemia below 50% of baseline value, fill with again in 7d, drop between ischemia front 40%～45%, perfusion remains very incomplete more in the meantime, and it is further impaired that lasting low perfusion will increase the weight of cerebral tissue.And, after filling with, nimodipine, the high, medium and low dosage group of salvianolic acid A drop pill rat rCBF obviously increase again, each time period rCBF compares with model group that there were significant differences; 7d after filling with again, each medication therapy groups rCBF return to former rCBF 57%～67% between; Each dosage group of salvianolic acid A drop pill relatively, is certain dose-effect trend; The salvianolic acid A drop pill has the salvianolic acid A monomeric compound that is better than same dosage and the trend of nimodipine.The experimental result prompting, the salvianolic acid A drop pill can obviously improve the cerebral blood flow of ischemia half blanking bar cerebral tissue after Ischemia and Reperfusion in vivo in Rats, recovering brain cell blood supplies, thereby save ischemia half blanking bar, further damage is even dead to prevent cerebral tissue, and effect has the trend that is better than the salvianolic acid A monomeric compound, ischemic cerebrovascular is had to good therapeutical effect.

Known through the experimental data contrast, can the raise content of ischemic tissue of brain adenosine triphosphate (ATP), adenosine diphosphate (ADP) (ADP), (AMP) AMP, phosphagen (PC) of salvianolic acid A drop pill, reduce cerebral tissue lactic acid (LA) content, can significantly improve the energy metabolism of rat cerebral ischemia and ischemia-reperfusion.The salvianolic acid A drop pill can be by improving the energy metabolism of Neurons Against Cerebral Ischemia tissue, increase the supply of cerebral tissue energy matter, strengthen the survival ability of brain cell, the brain cell that ischemia half blanking bar after the redemption cerebral ischemia is at death's door, further damage is even dead to prevent cerebral tissue, and action effect is obvious than salvianolic acid A monomeric compound and nimodipine, can perform well in treating ischemic cerebrovascular.

Known through the experimental data contrast, after rat cerebral ischemia 2h fills with 72h again, neuronal apoptosis is serious; And, after nimodipine, salvianolic acid A monomeric compound and the intervention of various dose salvianolic acid A drop pill, with model group, compare, in rat cerebral tissue, neuronal apoptosis significantly reduces (P<0.001 or P<0.01); And the apoptosis rate with high, the middle dosage group of salvianolic acid A drop pill is minimum; Show that the salvianolic acid A drop pill has the effect that suppresses cerebral ischemia rat cerebral tissue neuronal death, suppresses neuronal apoptosis, effect is better than salvianolic acid A monomeric compound and nimodipine.

The neurotrophic factor histone matter molecule essential with survival that be neure growth, play supporting function to the integrity of neure growth, growth and function.Nerve growth factor (NGF), Brain Derived Neurotrophic Factor (BDNF), neurotrophic factor-3 (NT-3) are the parts of neurotrophic factor family, can maintain neuronal survival and promote the neurocyte differentiation and induce axon growth; Energy neuroprotective unit, promotion neuron reparation and inhibition delayed neuronal death, thereby to anti-cerebral ischemia, protection cerebral ischemia.After Cerebral Ischemia-reperfusion, in rat cerebral tissue, than sham operated rats, increase to some extent, after the prompting cerebral reperfusion injury, in cerebral tissue, NGF, BDNF express stress protectiveness increase; But in the Neurons Against Cerebral Ischemia tissue, NT-3 content obviously reduces.Known through the experimental data contrast, with model control group, compare, each dosage group of salvianolic acid A drop pill NGF, BDNF, NT-3 protein expression all obviously strengthen, and the trend that is better than same dosage salvianolic acid A monomeric compound and nimodipine is arranged.Prompting salvianolic acid A drop pill can strengthen the expression of ischemic injuries cerebral tissue endogenous neurotrophic factor NGF, BDNF, NT-3, for neuronal survival provides condition, and reaches the effect of neuro-protective.

After cerebral ischemic reperfusion in rats, in cerebral tissue, inflammatory cytokine interleukin-1 ' beta ' (IL-1 β), interleukin-6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor-alpha (TNF-α), intercellular adhesion molecule-1 (ICAM-1) content all extremely significantly increase; Known through the experimental data contrast, each inflammatory cytokine content of rat cerebral tissue that gives each dosage group of salvianolic acid A drop pill obviously reduces than model group, and salvianolic acid A monomeric compound group and the nimodipine group with dosage is remarkable.Prompting salvianolic acid A drop pill can suppress the expression of ischemic injuries brain tissue inflammation cytokine, suppresses the generation of brain tissue inflammation's reaction, suppresses neuron and the cerebral tissue cascade damage of inflammatory reaction mediation, and effect is better than salvianolic acid A monomeric compound and nimodipine.

Ca in cell ²⁺overload is one of encephaloclastic important mechanism of mediation Secondary cases in During Ischemia, is the last common pathway that ischemia, anoxia produce irreversible neuronal damage.Ca ²⁺overload can be by exciting activator protein enzyme as the second message,second messenger, activate phospholipase, activate a series of enzyme reactions such as endonuclease produces infringement to cell, and cell death inducing, cause acute cell death and Delayed onset neuronal necrosis.K ⁺, Mg ²⁺be Ca ²⁺antagonist.K ⁺as the requisite cation of neurocyte polarized state, can suppress calcium ion in flow to and alleviate calcium overload; Mg ²⁺can activate plurality of enzymes system in body, participate in the multiple important metabolic activity of cell in cerebral tissue, conduction, ion transport, the synthetic all many-sides of energy metabolism that reach of albumen affect the nerves, the spasm of energy alleviating vascular smooth muscle, improve microcirculation, improve hypoxic-ischemic after brain injury, calcium overload in retardance after craniocerebral injury neurocyte.Known through the experimental data contrast, cerebral ischemia re-pouring model group and sham operated rats be cerebral tissue Ca relatively ²⁺content extremely significantly increases, K ⁺, Mg ²⁺content significantly reduces; With model group, compare, each dosage group of nimodipine group, salvianolic acid A monomeric compound group and salvianolic acid A drop pill can significantly reduce Ca in cerebral tissue ²⁺content, rising K ⁺, Mg ²⁺content, and the most remarkable with salvianolic acid A drop pill effect.Illustrate that the salvianolic acid A drop pill can suppress Ca ²⁺interior stream alleviates calcium overload, thereby can suppress Ca ²⁺the neuronal cell apoptosis that overload is induced, the damage of protection cerebral ischemic neuron.

Known through the experimental data contrast; the salvianolic acid A drop pill can obviously improve the content of the cerebral cortex of cerebral ischemia-reperfusion injury in rats and striatum monoamine transmitters 5-hydroxy tryptamine (5-HT), norepinephrine (NE), dopamine (DA), γ-aminobutyric acid (GABA), taurine (Tau) content, the content that significantly reduces cerebral tissue Glutamic Acid (Glu), aspartic acid (Asp), glycine (Gly) and aminoacid exitotoxicity index in rising cerebral ischemia/reperfusion injury of rats cerebral tissue.And dosage increases, effect strengthens.The salvianolic acid A drop pill shown in conjunction with the histopathology pathological examination can obviously alleviate cerebral edema, improve the neuronal cell form, dwindle the result of neuron peripheral clearance, show that the salvianolic acid A drop pill can suppress monoamine neurotransmitter and excessively discharge, improve the monoamine neurotransmitter disorder; Suppress the accumulation of excitatory amino acid in cerebral ischemia re-pouring, stablize the balance of excitatory amino acid-inhibitory aminoacid mediator, alleviate toxicity of excitatory amino acid; Thereby suppress the neuronal cell apoptosis that monoamine neurotransmitter is disorderly and toxicity of excitatory amino acid is induced, and effect has more advantage with dosage salvianolic acid A monomeric compound and nimodipine.

Known through experimental data contrast, each dosage group of salvianolic acid A drop pill is compared with the ischemia-reperfusion injury model group, and lipid peroxide in rat cerebral tissue (LPO) content significantly reduces, and lower than the salvianolic acid A monomeric compound group of same dosage; Superoxide dismutase (SOD) and glutathion peroxidase (GSH-Px) activity significantly raise, and the trend higher than same dosage salvianolic acid A monomeric compound group is arranged, and effect is also more obvious than the nimodipine group; Illustrate that the salvianolic acid A drop pill can increase the generation of the activity of peroxide scavenger enzyme in the ischemical reperfusion injury cerebral tissue, inhibition peroxide, thereby the damage to antioxidant radical to neuronal cell and cerebral tissue, its antioxidation has the trend of the salvianolic acid A monomeric compound that is better than same dosage.

Known through experimental data contrast, the cerebrovascular endothelial cell apoptosis number of cerebral thrombosis ischemia model each time period of group is significantly higher than sham operated rats, and cerebral tissue is to 14d after ischemia, and endothelial cell apoptosis still exists.With model group, compare, the apoptosis digital display work of each time period of each dosage group of salvianolic acid A drop pill reduces, and middle dosage group apoptosis number is less than salvianolic acid A monomeric compound and the nimodipine group of same dosage.Illustrate that the salvianolic acid A drop pill can suppress cerebral ischemia hindbrain apoptosis of vascular endothelial cell, improve the cerebrovascular endothelial cell survival rate, thereby guarantee the normal of cerebrovascular endothelial cell function, maintain the complete of ischemic injuries hindbrain blood vessel structure and function and strengthen the ability to anti-cerebral ischemia damnification, the effect of performance treatment ischemic cerebrovascular.

Known through the experimental data contrast, after doses salvianolic acid A drop pill and nimodipine effect, anoxia and Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial survival rate significantly raise, and salvianolic acid A drop pill 20,30,40,50,60 μ mol/L dosage group survival rates are significantly higher than nimodipine 30 μ mol/L groups, and also higher than the cell survival rate of the salvianolic acid A monomeric compound group with dosage.Prompting salvianolic acid A drop pill can be protected microvascular endothelial cells oxygen and Hypoxia/Reoxygenation Injury; strengthen its hypoxia-bearing capability; improve the brain microvessel endothelial cells in vitro survival rate of anoxia-induced apoptosis and Hypoxia/Reoxygenation Injury, and be better than nimodipine and salvianolic acid A monomeric compound.

Known through the experimental data contrast, Hypoxia/Reoxygenation Injury, can cause each phase apoptosis rate of Brain Microvascular Endothelial significantly to increase.With model group relatively, each dosage group of salvianolic acid A drop pill, salvianolic acid A monomeric compound group and nimodipine group all can significantly lower cell early, late period and total apoptosis rate; Between each dosage group of salvianolic acid A drop pill and be certain dose-effect relationship.And salvianolic acid A drop pill 10 μ mol/L dosage groups are suitable with nimodipine 40 μ mol/L dosage group effects, with the salvianolic acid A drop pill of dosage, be better than the salvianolic acid A monomeric compound.Prompting salvianolic acid A drop pill has the effect of the Brain Microvascular Endothelial apoptosis that good anti-hypoxia-reoxygenation injury causes.

After various dose salvianolic acid A drop pill, salvianolic acid A monomeric compound and the effect of nimodipine group, the Brain Microvascular Endothelial secretory tissue fiber proenzyme activator (t-PA) of Hypoxia/Reoxygenation Injury and the secretory volume of nitric oxide (NO) significantly raise (P<0.01 or P<0.001) than model group, and wherein the middle and high dosage group of salvianolic acid A drop pill effect is especially remarkable; Each administration group t-PA/PAI and NO/ET ratio also significantly improve than model group.Experimental result prompting salvianolic acid A drop pill can improve the secretory function of brain microvessel endothelial cells in vitro after Hypoxia/Reoxygenation Injury, and action effect significantly is better than nimodipine, and the trend that is better than same dosage salvianolic acid A monomeric compound is arranged.

After Hypoxia/Reoxygenation Injury, Ca in BMVEC born of the same parents ²⁺concentration significantly increases.Known through the experimental data contrast, compare each medicine group Ca with model group ²⁺concentration extremely significantly reduces, the most remarkable with the middle and high dosage group of salvianolic acid A drop pill effect, and its intracellular calcium concentration is significantly lower than nimodipine 40 μ mol/L dosage groups, and is better than the salvianolic acid A monomeric compound of same dosage.Prompting salvianolic acid A drop pill has the Hypoxia/Reoxygenation Injury of inhibition and causes Ca in BMVEC born of the same parents ²⁺the effect of concentration, and the protection hypoxia/reoxygenation is to the damage of BMVEC.

Known through the experimental data contrast, the salvianolic acid A drop pill compares impact and the normal saline matched group of rat artery thrombus formation time, the thrombus formation time significant prolongation of salvianolic acid A drop pill 60,30,15mg/kg dosage group; Middle dosage group thrombus formation time is suitable with 30mg/kg aspirin group, and extends than the salvianolic acid A monomeric compound group thrombus formation time with dosage; Show that the salvianolic acid A drop pill can extend thrombus formation time, the formation of prevention of arterial thrombosis, pre-preventing thrombosis and the ischemic cerebrovascular that causes.

Known through the experimental data contrast, relatively, salvianolic acid A drop pill 60,30,15mg/kg dosage group rat suppository wet, dry weight all significantly alleviates for the impact that the salvianolic acid A drop pill forms rat suppository and normal saline group.And middle dosage group is suitable with 30mg/kg aspirin group effect.Illustrate that the salvianolic acid A drop pill has the thrombotic effect of good inhibition, can be for prevention or treatment the ischemic cerebrovascular due to thrombosis, and than salvianolic acid A monomeric compound successful.

Known through the experimental data contrast, the salvianolic acid A drop pill compares impact and the normal saline group of rat vein thromboembolism, salvianolic acid A drop pill 60,30,15mg/kg dosage group rat vein thrombosis wet, dry weight significantly alleviates, middle dosage group and 30mg/kg aspirin group effect be (P>0.05) quite, and with the salvianolic acid A monomeric compound successful of dosage.Show that the salvianolic acid A drop pill has the effect that suppresses venous thrombosis, can be used for the venous thromboembolism after the prophylaxis of acute ischemic cerebrovascular occurs.

Known through the experimental data contrast, the salvianolic acid A drop pill compares rats in vitro thrombosis and normal saline group, salvianolic acid A drop pill 60,30, the thrombotic length of 15mg/kg dosage group rats in vitro significantly shorten, thrombosis is wet, dry weight significantly alleviates, and middle dosage group is suitable with 30mg/kg aspirin group effect; Show that the salvianolic acid A drop pill forms and has inhibitory action preferably external thrombus, and the trend that is better than same dosage salvianolic acid A monomeric compound is arranged.

Known through the experimental data contrast, the salvianolic acid A drop pill compares impact and the normal saline group of established thrombosis in the rat body, and the normal saline group all continues thromboembolism, and without logical phenomenon occurring again; In experiment, the number of animals of the lasting thromboembolism of each medicine group is few, with the normal saline group, compares utmost point significant difference is all arranged; Dosage in the salvianolic acid A drop pill (40mg/kg) group, its vessel open degree is similar to 40mg/kg salvianolic acid A monomeric compound group and 3500U/kg urokinase group, its recanalization rate is suitable, but its vessel open state has more stable trend than salvianolic acid A monomeric compound and urokinase group; The lasting recanalization rate of salvianolic acid A drop pill high dose (80mg/kg) group and recanalization rate are all higher than the urokinase group, and the bolt rate is starkly lower than urokinase group (P<0.05) again; Salvianolic acid A drop pill low dosage (20mg/kg) though the group recanalization rate lower than the urokinase group, its again the bolt rate lower than urokinase bolt rate again.The effect of thromboembolism again after the above results prompting salvianolic acid A drop pill has preferably thrombolytic and prevents thrombolytic, and effect is more stable.。

After cerebral thrombosis, rat whole blood viscosity, plasma viscosity significantly increase, and packed cell volume also significantly raises, known through the experimental data contrast, in experiment, relatively, its whole blood viscosity and plasma viscosity and packed cell volume all obviously reduce for each dosage group of salvianolic acid A drop pill and model group; Results suggest salvianolic acid A drop pill can be accelerated micro-blood flow velocity, reduces blood viscosity, improves hemorheology and the effect that effectively resists cerebral thrombosis, and, than nimodipine group more remarkable effect, the trend that is better than same dosage salvianolic acid A monomeric compound is also arranged.

In sum, salvianolic acid A drop pill of the present invention has obvious therapeutic effect to ischemic cerebrovascular, and ischemic cerebrovascular described in the present invention comprises that the pathological changes that affects cerebral circulation that the various causes of disease form and the damaged of neuronal death and function of nervous system of take caused thereof are main ischemic rat brain infringement.Mainly comprise any one or more: (1) cerebral thrombosis: by the embolus from heart, as artificial valve, atrial fibrillation, ventricle thrombosis, DCM (dilated cardiomyopathy), moving/vein blood vessel inflammation etc. form embolus and flow into cerebral vessels with blood, cause forming the front circulation of whole brain blood circulation and any position and/or the multiple location thrombosis of metacyclic blood vessels at different levels.(2) cerebral ischemia reperfusion injury (3) cerebral embolism.(4) atherosis or narrow (5) lacunar infarction of cranium arteria carotis externa and brain basilar artery.(6) Transient ischemic attacks (7) vascular dementia.

Medical science and study of pharmacy personnel can't, in advance under the prerequisite of not doing related experiment, learn that the salvianolic acid A drop pill has above-mentioned good purposes in advance.

The accompanying drawing explanation

Fig. 1. salvianolic acid A hydrogen nuclear magnetic resonance spectrogram;

Fig. 2. salvianolic acid A carbon-13 nmr spectra figure;

Fig. 3. alkannic acid hydrogen nuclear magnetic resonance spectrogram;

Fig. 4. alkannic acid carbon-13 nmr spectra figure;

Fig. 5. rosmarinic acid hydrogen nuclear magnetic resonance spectrogram;

Fig. 6. rosmarinic acid carbon-13 nmr spectra figure;

Fig. 7. salvianolic acid B hydrogen nuclear magnetic resonance spectrogram;

Fig. 8. salvianolic acid B carbon-13 nmr spectra figure;

Fig. 9. salvianolic acid C hydrogen nuclear magnetic resonance spectrogram;

Figure 10. salvianolic acid C carbon-13 nmr spectra figure;

Figure 11. mix reference substance solution HPLC figure;

Figure 12. alkannic acid reference substance solution HPLC figure;

Figure 13. rosmarinic acid reference substance solution HPLC figure;

Figure 14. salvianolic acid B reference substance solution HPLC figure;

Figure 15. salvianolic acid A reference substance solution HPLC figure;

Figure 16. salvianolic acid C reference substance solution HPLC figure;

Figure 17. salvianolic acid A compositions HPLC figure;

The specific embodiment

Embodiment 1

Get red rooted salvia, be ground into 4 order granules, add 92 ℃ of water of 7 times of amounts at every turn, warm macerating extracts 3 times, with 25 rev/mins of speed, stirs simultaneously, and each warm macerating extracts 3 hours; Extracting solution is evaporated to relative density 1.0～1.30 (60 ℃), adds ethanol to make to measure 70% containing alcohol, standing, filters, and decompression filtrate recycling ethanol also is concentrated into without the alcohol flavor; Be diluted with water to every 1ml containing salvianolic acid B 20mg, aqueous solution is adjusted pH to 5.5 with 10% sodium hydroxide, adds 0.5%ZnCl ₂as catalyst, 120 ℃ of temperature thermal conversions 4 hours, 20% phosphoric acid adjust pH to 2.5 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 3mg, through the HPD-100 macroporous resin column chromatography, separate, the salvianolic acid A applied sample amount is 1: 50 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 10, uses respectively 3 times of cylinder hydrops, 5 times of column volume 25% ethanol elutions, removes impurity, use again 4 times of column volume 40% ethanol elutions, HPLC detects, and collects the 40% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol also is concentrated into without the alcohol flavor; Aqueous solution is concentrated into the solution of every ml containing the 5mg salvianolic acid A, by polyamide chromatography post, separate, the salvianolic acid A applied sample amount is 1: 10 with the polyamide ratio, the resin column blade diameter length ratio is 1: 8, use respectively 3 times of cylinder hydrops, 12 times of column volume 40% alcoholic solution eluting remove impurity, use 8 times of column volumes, 60% alcoholic solution eluting again, collect the 60% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml containing salvianolic acid A 15mg aqueous solution; Aqueous solution is adjusted pH to 2.5 with 20% phosphoric acid, and by the t-butyl methyl ether of 3 times of amounts of aqueous solution, minute 3 extractions, separate organic layer, and the reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.5g, adds 1～3 times of amount silica gel, stirs, and volatilizes; Stirring sample silica gel, be added on 15 times of dry silicagel columns of amount that installed, the silicagel column blade diameter length ratio is 1: 10, take pentane-t-butyl methyl ether as eluant, gradient elution, use respectively pentane: 10 times of column volumes of t-butyl methyl ether (4: 6) eluting, pentane: 10 times of column volumes of t-butyl methyl ether (6: 4) eluting, the reclaim under reduced pressure eluant, salvianolic acid A after the recovery organic solvent adds 10 times of water gagings and dissolves, and microwave vacuum drying, obtain the salvianolic acid A compositions.

After measured, in the salvianolic acid A compositions, salvianolic acid A content is 93.77%, alkannic acid content 0.75%; Rosmarinic acid contents 1.26%; Content of danshinolic acid B 1.35%; Salvianolic acid C content 1.48%.

Embodiment 2

Get red rooted salvia, be ground into diameter 2mm granule, add 95 ℃ of water of 9 times of amounts at every turn, warm macerating extracts 3 times, with 30 rev/mins of speed, stirs simultaneously, and each warm macerating extracts 2.5 hours, extracting solution is evaporated to relative density 1.0～1.30 (60 ℃), adds ethanol to make to measure 70% containing alcohol, standing, filters, and decompression filtrate recycling ethanol also is concentrated into without the alcohol flavor, be diluted with water to every 1ml containing salvianolic acid B 15mg, aqueous solution is adjusted pH to 4.5 with 10% sodium hydroxide, adds 0.5%ZnCl ₂as catalyst, 120 ℃ of temperature (0.01MPa～0.20MPa pressure) thermal conversion 4 hours, 15% phosphoric acid adjust pH to 2.8 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, through the HPD-100 macroporous resin column chromatography, separate, the salvianolic acid A applied sample amount is 1: 45 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 9, use respectively 2 times of cylinder hydrops, 5 times of column volume 28% ethanol elutions, remove impurity, use again 5 times of column volume 40% ethanol elutions, HPLC detects, the 40% ethanol elution part that collection contains salvianolic acid A, decompression recycling ethanol also is concentrated into without the alcohol flavor, aqueous solution is concentrated into the solution of every 1ml containing the 5mg salvianolic acid A, by polyamide chromatography post, separate, the salvianolic acid A applied sample amount is 1: 10 with the polyamide ratio, the resin column blade diameter length ratio is 1: 8, use respectively 3 times of cylinder hydrops, 10 times of column volume 40% alcoholic solution eluting remove impurity, use 7 times of column volumes, 60% alcoholic solution eluting again, collect the 60% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml containing salvianolic acid A 12mg aqueous solution, aqueous solution is adjusted pH to 2.6 with 15% phosphoric acid, and by the t-butyl methyl ether of 4 times of amounts of aqueous solution, minute 4 extractions, separate organic layer, and the reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.5g, adds 2 times of amount silica gel, stirs, and volatilizes, stirring sample silica gel, be added on 12 times of dry silicagel columns of amount that installed, the silicagel column blade diameter length ratio is 1: 10, take pentane-t-butyl methyl ether as eluant, gradient elution, use respectively 10 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 10 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, the reclaim under reduced pressure eluant, salvianolic acid A after the recovery organic solvent adds 10 times of water gagings and dissolves, and microwave vacuum drying, obtain the salvianolic acid A compositions.

After measured, in the salvianolic acid A compositions, salvianolic acid A content is 94.78%, alkannic acid content 0.73%; Rosmarinic acid contents 1.23%; Content of danshinolic acid B 1.22%; Salvianolic acid C content 1.35%.

Embodiment 3

Get red rooted salvia, be ground into diameter 2mm granule, add 90 ℃ of water of 7 times of amounts at every turn, warm macerating extracts 3 times, with 25 rev/mins of speed, stirs simultaneously, and each warm macerating extracts 3 hours, extracting solution is evaporated to relative density 1.0～1.30 (60 ℃), adds ethanol to make to measure 70% containing alcohol, standing, filters, and decompression filtrate recycling ethanol also is concentrated into without the alcohol flavor, be diluted with water to every 1ml containing salvianolic acid B 20mg, aqueous solution is adjusted pH to 5.5 with 10% sodium hydroxide, adds 0.5%ZnCl ₂as catalyst, 120 ℃ of temperature (0.01MPa～0.20MPa pressure) thermal conversion 4 hours, 20% phosphoric acid adjust pH to 2.5 for conversional solution, centrifugal, supernatant is evaporated to every 1m containing salvianolic acid A 3mg, through the HPD-100 macroporous resin column chromatography, separate, the salvianolic acid A applied sample amount is 1: 50 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 10, use respectively 3 times of cylinder hydrops, 5 times of column volume 25% ethanol elutions, remove impurity, use again 4 times of column volume 40% ethanol elutions, HPLC detects, the 40% ethanol elution part that collection contains salvianolic acid A, decompression recycling ethanol also is concentrated into without the alcohol flavor, aqueous solution is concentrated into the solution of every ml containing the 5mg salvianolic acid A, by polyamide chromatography post, separate, the salvianolic acid A applied sample amount is 1: 10 with the polyamide ratio, the resin column blade diameter length ratio is 1: 8, use respectively 3 times of cylinder hydrops, 12 times of column volume 40% alcoholic solution eluting remove impurity, use 8 times of column volumes, 60% alcoholic solution eluting again, collect the 60% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml containing salvianolic acid A 15mg aqueous solution, aqueous solution is adjusted pH to 2.5 with 20% phosphoric acid, and by the t-butyl methyl ether of 3 times of amounts of aqueous solution, minute 3 extractions, separate organic layer, and the reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.5g, adds 3 times of amount silica gel, stirs, and volatilizes, stirring sample silica gel, be added on 15 times of dry silicagel columns of amount that installed, the silicagel column blade diameter length ratio is 1: 10, take pentane-t-butyl methyl ether as eluant, gradient elution, use respectively 10 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 10 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, the reclaim under reduced pressure eluant, salvianolic acid A after the recovery organic solvent adds 10 times of water gagings and dissolves, and vacuum drying, obtain the salvianolic acid A compositions.

After measured, in the salvianolic acid A compositions, salvianolic acid A content is 95.28%, alkannic acid content 0.75%; Rosmarinic acid contents 1.12%; Content of danshinolic acid B 1.26%; Salvianolic acid C content 1.40%.

Embodiment 4

Get red rooted salvia, be cut into decoction pieces, add 85 ℃ of water temperature lixiviates of 8 times of amounts at every turn and get 3 times, stir with 20 rev/mins of speed simultaneously, each warm macerating extracts 2.5 hours, extracting solution is evaporated to relative density 1.0～1.25 (60 ℃), adds ethanol to make to measure 75% containing alcohol, standing, filters, and decompression filtrate recycling ethanol also is concentrated into without the alcohol flavor, be diluted with water to every 1ml containing salvianolic acid B 15mg, aqueous solution is adjusted pH to 5.0 with 10% potassium hydroxide, adds 0.6%ZnCl ₂as catalyst, 120 ℃ of temperature (0.01MPa～0.20MPa pressure) thermal conversion 3.5 hours, 15% hydrochloric acid adjust pH to 2.5 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, through the HPD-100 macroporous resin column chromatography, separate, the salvianolic acid A applied sample amount is 1: 45 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 8, use respectively 3.5 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions, remove impurity, use again 5 times of column volume 45% ethanol elutions, HPLC detects, the 45% ethanol elution part that collection contains salvianolic acid A, decompression recycling ethanol also is concentrated into without the alcohol flavor, aqueous solution is concentrated into the solution of every ml containing the 5mg salvianolic acid A, by polyamide chromatography post, separate, the salvianolic acid A applied sample amount is 1: 9 with the polyamide ratio, the resin column blade diameter length ratio is 1: 7, use respectively 4 times of cylinder hydrops, 10 times of column volume 40% alcoholic solution eluting remove impurity, use 8 times of column volumes, 65% alcoholic solution eluting again, collect the 65% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml containing salvianolic acid A 12mg aqueous solution, aqueous solution is adjusted pH to 2.6 with 15% hydrochloric acid, and by the t-butyl methyl ether of 4 times of amounts, minute 4 extractions, separate organic layer, and the reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.8g, adds 2～3 times of amount silica gel, stirs, and volatilizes, stirring sample silica gel, be added on 13 times of dry silicagel columns of amount that installed, the silicagel column blade diameter length ratio is 1: 8, take the pentane t-butyl methyl ether as eluant, gradient elution, use respectively 8 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 8 times of column volumes of pentane-t-butyl methyl ether (7: 3) eluting, the reclaim under reduced pressure eluant, salvianolic acid A after the recovery organic solvent adds 8 times of water gagings and dissolves, and spray drying, obtain the salvianolic acid A compositions.

After measured, in the salvianolic acid A compositions, salvianolic acid A content is 94.68%, alkannic acid content 0.66%; Rosmarinic acid contents 1.12%; Content of danshinolic acid B 1.25%; Salvianolic acid C content 1.39%.

Embodiment 5

Get red rooted salvia, be ground into diameter 2mm granule, add 85 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 2 times, stir with 30 rev/mins of speed simultaneously, each warm macerating extracts 3.5 hours; Extracting solution is evaporated to relative density 1.0～1.25 (60 ℃), adds ethanol to make to measure 75% containing alcohol, standing, filters, and decompression filtrate recycling ethanol also is concentrated into without the alcohol flavor; Be diluted with water to every 1ml containing salvianolic acid B 18mg, aqueous solution is adjusted pH to 5.2 with 10% sodium carbonate, adds 0.6%ZnCl ₂as catalyst, 123 ℃ of temperature thermal conversions 4.5 hours, 15% nitric acid adjust pH to 2.8 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 6mg, through the HPD-100 macroporous resin column chromatography, separate, the salvianolic acid A applied sample amount is 1: 40 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 7, uses respectively 4 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions, removes impurity, use again 5 times of column volume 40% ethanol elutions, HPLC detects, and collects the 40% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol also is concentrated into without the alcohol flavor; Aqueous solution is concentrated into the solution of every ml containing the 6mg salvianolic acid A, by polyamide chromatography post, separate, the salvianolic acid A applied sample amount is 1: 8 with the polyamide ratio, the resin column blade diameter length ratio is 1: 8, use respectively 4 times of cylinder hydrops, 9 times of column volume 40% alcoholic solution eluting remove impurity, use 7 times of column volumes, 65% alcoholic solution eluting again, collect the 65% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml containing salvianolic acid A 10mg aqueous solution; Aqueous solution is adjusted pH to 2.6 with 15% nitric acid, and by the t-butyl methyl ether of 4 times of amounts, minute 4 extractions, separate organic layer, and the reclaim under reduced pressure methyl acetate is made the extract of every 1ml containing salvianolic acid A 0.7g, adds 3 times of amount silica gel, stirs, and volatilizes; Stirring sample silica gel, be added on 10 times of dry silicagel columns of amount that installed, the silicagel column blade diameter length ratio is 1: 7, take pentane-t-butyl methyl ether as eluant, gradient elution, use respectively 8 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 9 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, the reclaim under reduced pressure eluant, salvianolic acid A after the recovery organic solvent adds 8 times of water gagings and dissolves, and microwave vacuum drying, obtain the salvianolic acid A compositions.

After measured, in the salvianolic acid A compositions, salvianolic acid A content is 94.72%, alkannic acid content 0.55%; Rosmarinic acid contents 1.22%; Content of danshinolic acid B 1.27%; Salvianolic acid C content 1.47%.

Embodiment 6

Get red rooted salvia, be cut into decoction pieces, add 80 ℃ of water temperature lixiviates of 10 times of amounts at every turn and get 3 times, stir with 15 rev/mins of speed simultaneously, each warm macerating extracts 3 hours, extracting solution is evaporated to relative density 1.0～1.25 (60 ℃), adds ethanol to make to measure 70% containing alcohol, standing, filters, and decompression filtrate recycling ethanol also is concentrated into without the alcohol flavor, be diluted with water to every 1ml containing salvianolic acid B 20mg, aqueous solution is adjusted pH to 5.4 with 10% sodium bicarbonate, adds 0.8%FeCl ₃as catalyst, 128 ℃ of temperature thermal conversions 4.0 hours, 20% sulphuric acid adjust pH to 2.6 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, through the HPD-100 macroporous resin column chromatography, separate, the salvianolic acid A applied sample amount is 1: 40 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 7, use respectively 4 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions, remove impurity, use again 5 times of column volume 40% ethanol elutions, HPLC detects, the 40% ethanol elution part that collection contains salvianolic acid A, decompression recycling ethanol also is concentrated into without the alcohol flavor, aqueous solution is concentrated into the solution of every ml containing the 6mg salvianolic acid A, by polyamide chromatography post, separate, the salvianolic acid A applied sample amount is 1: 10 with the polyamide ratio, the resin column blade diameter length ratio is 1: 15, use respectively 4 times of cylinder hydrops, 7 times of column volume 35% alcoholic solution eluting remove impurity, use 6 times of column volumes, 60% alcoholic solution eluting again, collect the 60% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml containing salvianolic acid A 12mg aqueous solution, aqueous solution is adjusted pH to 2.8 with 20% sulphuric acid, and by the t-butyl methyl ether of 4 times of amounts, minute 4 extractions, separate organic layer, and the reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.5g, adds 2.5 times of amount silica gel, stirs, and volatilizes, stirring sample silica gel, be added on 9 times of dry silicagel columns of amount that installed, the silicagel column blade diameter length ratio is 1: 8, take pentane: t-butyl methyl ether is eluant, gradient elution, use respectively 8 times of column volumes of pentane-t-butyl methyl ether (4.5: 5.5) eluting, 8 times of column volumes of pentane-t-butyl methyl ether (6.5: 3.5) eluting, the reclaim under reduced pressure eluant, salvianolic acid A after the recovery organic solvent adds 10 times of water gagings and dissolves, and microwave vacuum drying, obtain the salvianolic acid A compositions.

After measured, in the salvianolic acid A compositions, salvianolic acid A content is 95.53%, alkannic acid content 0.52%; Rosmarinic acid contents 1.01%; Content of danshinolic acid B 1.19%; Salvianolic acid C content 1.36%.

Embodiment 7

Get red rooted salvia, be ground into diameter 2mm granule, add 85 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir with 20 rev/mins of speed simultaneously, each warm macerating extracts 2.5 hours, extracting solution is evaporated to relative density 1.0～1.25 (60 ℃), adds ethanol to make to measure 70% containing alcohol, standing, filters, and decompression filtrate recycling ethanol also is concentrated into without the alcohol flavor, be diluted with water to every 1ml containing salvianolic acid B 10mg, aqueous solution is adjusted pH to 5.5 with 20% sodium citrate, adds 0.4%AlCl ₃as catalyst, 132 ℃ of temperature thermal conversions 3.5 hours, 20% acetic acid adjust pH to 2.6 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, through the HPD-100 macroporous resin column chromatography, separate, the salvianolic acid A applied sample amount is 1: 35 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 8, use respectively 3 times of cylinder hydrops, 4.5 column volume 25% ethanol elution doubly, remove impurity, use again 6 times of column volume 40% ethanol elutions, HPLC detects, the 40% ethanol elution part that collection contains salvianolic acid A, decompression recycling ethanol also is concentrated into without the alcohol flavor, aqueous solution is concentrated into the solution of every ml containing the 8mg salvianolic acid A, by polyamide chromatography post, separate, the salvianolic acid A applied sample amount is 1: 12 with the polyamide ratio, the resin column blade diameter length ratio is 1: 18, use respectively 4 times of cylinder hydrops, 8 times of column volume 30% alcoholic solution eluting remove impurity, use 5 times of column volumes, 65% alcoholic solution eluting again, collect the 65% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml containing salvianolic acid A 15mg aqueous solution, aqueous solution is adjusted pH to 2.7 with 20% acetic acid, and by the t-butyl methyl ether of 5 times of amounts, minute 5 extractions, separate organic layer, and the reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.5g, adds 2 times of amount silica gel, stirs, and volatilizes, stirring sample silica gel, be added on 10 times of dry silicagel columns of amount that installed, the silicagel column blade diameter length ratio is 1: 10, take pentane, t-butyl methyl ether is eluant, gradient elution, use respectively 8 times of column volumes of pentane-t-butyl methyl ether (4.5: 5.5) eluting, 8 times of column volumes of pentane-t-butyl methyl ether (6.5: 3.5) eluting, the reclaim under reduced pressure eluant, salvianolic acid A after the recovery organic solvent adds 12 times of water gagings and dissolves, and spray drying, obtain the salvianolic acid A compositions.

After measured, in the salvianolic acid A compositions, salvianolic acid A content is 95.14%, alkannic acid content 0.61%; Rosmarinic acid contents 1.07%; Content of danshinolic acid B 1.16%; Salvianolic acid C content 1.37%.

Embodiment 8

Get red rooted salvia, be ground into diameter 2mm granule, add 88 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir with 22 rev/mins of speed simultaneously, each warm macerating extracts 3.5 hours, extracting solution is evaporated to relative density 1.0～1.23 (60 ℃), adds ethanol to make to measure 75% containing alcohol, standing, filters, and decompression filtrate recycling ethanol also is concentrated into without the alcohol flavor, be diluted with water to every 1ml containing salvianolic acid B 13mg, aqueous solution is adjusted pH to 5.6 with 10% sodium hydroxide, adds 0.5%RuCl ₃as catalyst, 133 ℃ of temperature thermal conversions 4.5 hours, 10% hydrochloric acid adjust pH to 2.7 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, through the HPD-100 macroporous resin column chromatography, separate, the salvianolic acid A applied sample amount is 1: 40 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 9, use respectively 4 times of cylinder hydrops, 4 times of column volume 22% ethanol elutions, remove impurity, use again 6 times of column volume 43% ethanol elutions, HPLC detects, the 43% ethanol elution part that collection contains salvianolic acid A, decompression recycling ethanol also is concentrated into without the alcohol flavor, aqueous solution is concentrated into the solution of every ml containing the 10mg salvianolic acid A, by polyamide chromatography post, separate, the salvianolic acid A applied sample amount is 1: 15 with the polyamide ratio, the resin column blade diameter length ratio is 1: 20, use respectively 4 times of cylinder hydrops, 8 times of column volume 30% alcoholic solution eluting remove impurity, use 5 times of column volumes, 60% alcoholic solution eluting again, collect the 60% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml containing salvianolic acid A 12mg aqueous solution, aqueous solution is adjusted pH to 2.8 with 10% hydrochloric acid, and by the t-butyl methyl ether of 5 times of amounts, minute 5 extractions, separate organic layer, and the reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.5g, adds 3 times of amount silica gel, stirs, and volatilizes, stirring sample silica gel, be added on 12 times of dry silicagel columns of amount that installed, the silicagel column blade diameter length ratio is 1: 10, take pentane, t-butyl methyl ether is eluant, gradient elution, use respectively 6 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 7 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, the reclaim under reduced pressure eluant, salvianolic acid A after the recovery organic solvent adds 10 times of water gagings and dissolves, and vacuum drying, obtain the salvianolic acid A compositions.

After measured, in the salvianolic acid A compositions, salvianolic acid A content is 94.72%, alkannic acid content 0.69%; Rosmarinic acid contents 1.13%; Content of danshinolic acid B 1.28; Salvianolic acid C content 1.40%.

Embodiment 9

Get red rooted salvia, be cut into decoction pieces, add 90 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir with 25 rev/mins of speed simultaneously, each warm macerating extracts 3 hours, extracting solution is evaporated to relative density 1.0～1.20 (60 ℃), adds ethanol to make to measure 75% containing alcohol, standing, filters, and decompression filtrate recycling ethanol also is concentrated into without the alcohol flavor, be diluted with water to every 1ml containing salvianolic acid B 20mg, aqueous solution is adjusted pH to 5.0 with 10% sodium carbonate, adds 1.0%PdCl ₃as catalyst, 135 ℃ of temperature thermal conversions 4.5 hours, 15% sulphuric acid adjust pH to 3.0 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, through the HPD-100 macroporous resin column chromatography, separate, the salvianolic acid A applied sample amount is 1: 36 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 9, use respectively 3 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions, remove impurity, use again 5 times of column volume 45% ethanol elutions, HPLC detects, the 45% ethanol elution part that collection contains salvianolic acid A, decompression recycling ethanol also is concentrated into without the alcohol flavor, aqueous solution is concentrated into the solution of every 1ml containing the 6mg salvianolic acid A, by polyamide chromatography post, separate, the salvianolic acid A applied sample amount is 1: 10 with the polyamide ratio, the resin column blade diameter length ratio is 1: 10, use respectively 4 times of cylinder hydrops, 8 times of column volume 45% alcoholic solution eluting remove impurity, use 8 times of column volumes, 65% alcoholic solution eluting again, collect the 65% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml containing salvianolic acid A 10mg aqueous solution, aqueous solution is adjusted pH to 2.7 with 15% sulphuric acid, and by the t-butyl methyl ether of 4 times of amounts, minute 4 extractions, separate organic layer, and the reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.6g, adds 3 times of amount silica gel, stirs, and volatilizes, stirring sample silica gel, be added on 10 times of dry silicagel columns of amount that installed, the silicagel column blade diameter length ratio is 1: 8, take pentane, t-butyl methyl ether is eluant, gradient elution, use respectively 8 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 8 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, the reclaim under reduced pressure eluant, salvianolic acid A after the recovery organic solvent adds 8 times of water gagings and dissolves, and microwave vacuum drying, obtain the salvianolic acid A compositions.

After measured, in the salvianolic acid A compositions, salvianolic acid A content is 95.14%, alkannic acid content 0.55%; Rosmarinic acid contents 1.18%; Content of danshinolic acid B 1.22%; Salvianolic acid C content 1.32%.

Embodiment 10

Taking polyethylene glycol (6000) 950g, be heated to 80 ℃ and make melting, get the salvianolic acid A compositions 50g that embodiment 3 makes, add in the Polyethylene Glycol (6000) of melting, mix homogeneously, speed with 15 of per minutes splashes in simethicone liquid coolant (8 ℃), obtains the salvianolic acid A drop pill.

Embodiment 11

Taking polyethylene glycol (6000) 450g, Polyethylene Glycol (4000) 450g, be heated to 75 ℃ and make melting, get the salvianolic acid A compositions 100g that embodiment 5 makes, add in the Polyethylene Glycol (6000), Polyethylene Glycol (4000) of melting, mix homogeneously, speed with 30 of per minutes splashes in simethicone liquid coolant (5 ℃), obtains the salvianolic acid A drop pill.

Embodiment 12

Taking polyethylene glycol (8000) 400g, Polyethylene Glycol (4000) 400g, be heated to 80 ℃ and make melting, get the salvianolic acid A compositions 200g that embodiment 7 makes, add in the Polyethylene Glycol (6000), Polyethylene Glycol (4000) of melting, mix homogeneously, speed with 10 of per minutes splashes in liquid Paraffin liquid coolant (15 ℃), obtains the salvianolic acid A drop pill.

Embodiment 13

Taking polyethylene glycol (6000) 350g, Polyethylene Glycol (4000) 350g, be heated to 80 ℃ and make melting, get the salvianolic acid A compositions 300g that embodiment 5 makes, add in the Polyethylene Glycol (6000), Polyethylene Glycol (4000) of melting, mix homogeneously, speed with 12 of per minutes splashes in liquid Paraffin liquid coolant (10 ℃), obtains the salvianolic acid A drop pill.

Embodiment 14

Get glyceryl monostearate 420g, hydrogenated vegetable oil 300g, be heated to 78 ℃ and make melting, get the salvianolic acid A compositions 280g that embodiment 1 makes, add in the glyceryl monostearate, hydrogenated vegetable oil of melting, mix homogeneously, speed with 18 of per minutes splashes in simethicone liquid coolant (10 ℃), obtains the salvianolic acid A drop pill.

Embodiment 15

Taking polyethylene glycol (6000) 430g, Polyethylene Glycol (4000) 330g, be heated to 75 ℃ and make melting, get the salvianolic acid A compositions 240g that embodiment 8 makes, add in the Polyethylene Glycol (6000), Polyethylene Glycol (4000) of melting, mix homogeneously, speed with 26 of per minutes splashes in simethicone liquid coolant (8 ℃), obtains the salvianolic acid A drop pill.

Embodiment 16

Get glyceryl monostearate 520g, hydrogenated vegetable oil 300g, be heated to 78 ℃ and make melting, get the salvianolic acid A compositions 180g that embodiment 6 makes, add in the glyceryl monostearate, hydrogenated vegetable oil of melting, mix homogeneously, speed with 15 of per minutes splashes in simethicone liquid coolant (12 ℃), obtains the salvianolic acid A drop pill.

Embodiment 17

Taking polyethylene glycol (6000) 450g, Polyethylene Glycol (4000) 400g, be heated to 70 ℃ and make melting, get the salvianolic acid A compositions 150g that embodiment 9 makes, add in the Polyethylene Glycol (6000), Polyethylene Glycol (4000) of melting, mix homogeneously, speed with 30 of per minutes splashes in simethicone liquid coolant (5 ℃), obtains the salvianolic acid A drop pill.

Embodiment 18

Get insect wax 450g, hydrogenated vegetable oil 300g, be heated to 75 ℃ and make melting, get the salvianolic acid A compositions 180g that embodiment 1 makes, add in the insect wax, hydrogenated vegetable oil of melting, mix homogeneously, speed with 25 of per minutes splashes in simethicone liquid coolant (10 ℃), obtains the salvianolic acid A drop pill.

Embodiment 19

Taking polyethylene glycol (6000) 520g, Polyethylene Glycol (8000) 400g, be heated to 80 ℃ and make melting, get the salvianolic acid A compositions 80g that embodiment 2 makes, add in the Polyethylene Glycol (6000), Polyethylene Glycol (8000) of melting, mix homogeneously, speed with 18 of per minutes splashes in liquid Paraffin liquid coolant (9 ℃), obtains the salvianolic acid A drop pill.

Embodiment 20

Get stearic acid 500g, hydrogenated vegetable oil 300g, be heated to 76 ℃ and make melting, get the salvianolic acid A compositions 200g that embodiment 8 makes, add in the stearic acid g, hydrogenated vegetable oil of melting, mix homogeneously, speed with 20 of per minutes splashes in simethicone liquid coolant (9 ℃), obtains the salvianolic acid A drop pill.

Embodiment 21

Taking polyethylene glycol (6000) 500g, insect wax 250g, be heated to 80 ℃ and make melting, get the salvianolic acid A compositions 250g that embodiment 4 makes, add in the ethylene glycol (6000), insect wax of melting, mix homogeneously, speed with 16 of per minutes splashes in simethicone liquid coolant (9 ℃), obtains the salvianolic acid A drop pill.

Embodiment 22

Taking polyethylene glycol (6000) 500g, Polyethylene Glycol (4000) 200g, be heated to 80 ℃ and make melting, get the salvianolic acid A compositions 300g that embodiment 6 makes, add in the ethylene glycol (6000), Polyethylene Glycol (4000) of melting, mix homogeneously, speed with 25 of per minutes splashes in liquid Paraffin liquid coolant (9 ℃), obtains the salvianolic acid A drop pill.

Embodiment 23

Taking polyethylene glycol (6000) 350g, Polyethylene Glycol (4000) 350g, be heated to 80 ℃ and make melting, get commercially available salvianolic acid A (content 99.12%) 300g, add in the Polyethylene Glycol (6000), Polyethylene Glycol (4000) of melting, mix homogeneously, speed with 12 of per minutes splashes in liquid Paraffin liquid coolant (10 ℃), obtains the salvianolic acid A drop pill.

Experimental example 1: salvianolic acid A compositions analytical method research

1. instrument and reagent

Instrument: Waters 2695 high performance liquid chromatographs, Empower 2 chromatographic work stations, 2998 diode array detector; Sartorius cp225D 100,000/electronic balance.

Chromatographic column: YMC C ₁₈chromatographic column (250 * 4.6mm, 5 μ m);

Reagent: methanol is chromatographically pure, and water is ultra-pure water prepared by Millipore, and other reagent are analytical pure.

Rosmarinic acid reference substance, salvianolic acid B reference substance are all purchased from Nat'l Pharmaceutical & Biological Products Control Institute, for assay; Alkannic acid reference substance salvianolic acid C reference substance is all purchased from the bright bio tech ltd in sea, Shanghai, and the salvianolic acid A reference substance, for self-control, is 99.52% through purity mark content.

2. the preparation of reference substance solution and need testing solution

2.1 the preparation of reference substance solution: precision takes the about 10.00mg of alkannic acid reference substance respectively, the about 10.00mg of rosmarinic acid reference substance, the about 10.00mg of salvianolic acid B reference substance, the about 10.00mg of salvianolic acid C reference substance, the about 10.00mg of salvianolic acid A reference substance, put in different 100ml measuring bottles, add respectively dissolve with methanol and be diluted to scale, shake up, precision is drawn the alkannic acid reference substance respectively, the rosmarinic acid reference substance, the salvianolic acid B reference substance, each 10ml of salvianolic acid C reference substance, put in different 100ml measuring bottles, add respectively dissolve with methanol and be diluted to scale, shake up, product stock solution in contrast.Accurate each 10ml of above-mentioned stock solution that draws, put in same 100ml measuring bottle, adds methanol and be diluted to scale, shakes up, as mixing reference substance solution.

2.3 the preparation precision of need testing solution takes the about 10.00mg of sample of the present embodiment 1, puts in the 50ml measuring bottle, adds dissolve with methanol and is diluted to scale, the accurate absorption in 10ml to 100ml measuring bottle, add methanol and be diluted to scale, shakes up, and obtains.

3. chromatographic condition and system suitability

Take octadecylsilane chemically bonded silica as filler; Flow velocity 1.0ml/min; Detect wavelength: 286nm; 30 ℃ of column temperatures; Number of theoretical plate calculates and should be not less than 10000 by the salvianolic acid A peak.

In the time of 0～10 minute, the ratio of methanol is down to 60% by 30% ratio that rises to 40%, 0.1～0.5% phosphate aqueous solution by 70%; In the time of 10～30 minutes, the ratio of methanol is down to 45% by 40% ratio that rises to 55%, 0.1～0.5% phosphate aqueous solution by 50%; In the time of 30～60 minutes, the ratio of methanol is down to 20% by 55% ratio that rises to 80%, 0.1～0.5% phosphate aqueous solution by 45%.

Under these conditions, the chromatographic peak retention time of 5 reference substances is shown in Figure 11～17 in Table 1, HPLC collection of illustrative plates.

The chromatographic peak retention time result of each reference substance of table 1.

4. the investigation of linear relationship

Accurate above-mentioned mixing reference substance solution 0.1ml, 0.2ml, 0.5ml, 1ml, 2ml, the 5ml of drawing, put respectively in the 10ml measuring bottle, adds methanol and be diluted to series standard solution.Accurate each the 10 μ l injection liquid chromatographies of above-mentioned standard solution of drawing, calculate peak area by " 3. chromatographic condition and system suitability " lower chromatographic condition, take the peak area integrated value respectively as vertical coordinate, and each concentration reference substance sample size is abscissa, the drawing standard curve.Result shows to mix reference substance solution and become good linear relationship in following scope, in Table 2.

Table 2. mixes reference substance solution linear relationship result

5. precision test

Get the mixing reference substance solution, measure according to " 3. chromatographic condition and system suitability " lower chromatographic condition, repeat sample introduction 6 times.Result shows that the precision of the method is good, in Table 3.

Table 3. Precision test result

6. stability test

Get need testing solution, according to " 3. chromatographic condition and system suitability " lower chromatographic condition, measure, at regular intervals sample introduction once, as a result in need testing solution each composition in 24 hours peak area without significant change, show having good stability of need testing solution, in Table 4.

Table 4. stability test result

7. replica test

Get this salvianolic acid A compositions, add methanol and make 6 parts of need testing solutions, measure according to " 3. chromatographic condition and system suitability " lower chromatographic condition.Result shows that the method repeatability is good, in Table 5.

Table 5. replica test result

8. recovery test

Adopt the application of sample recovery test, get the salvianolic acid A compositions 10mg of known content, accurately weighed, parallel 6 parts, add corresponding reference substance solution by each constituent content equal proportion respectively, by 2.3 below legal system available test sample solutions, measure and calculate average recovery and the RSD of each composition.Result shows that the accuracy of the method is good, in Table 6.

Table 6. recovery test result

Experimental example 2: the component separating in the salvianolic acid A compositions and evaluation

By embodiment 1 salvianolic acid A compositions respectively with CG161M macroporous adsorbent resin, ODS, SephadexLH-20 column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, the ratio of ethanol is 20～80%, and the Fractional Collections eluent merges same composition, crystallization, can obtain salvianolic acid A, alkannic acid, rosmarinic acid, salvianolic acid B, salvianolic acid C monomeric compound, carry out respectively Structural Identification, wherein:

Salvianolic acid A, molecular formula: C ₂₆h ₂₂₂o ₁₀, molecular weight: 494.45, structure is as follows:

Salvianolic acid A physicochemical property and spectral data, be shown in Fig. 1,2.

Salvianolic acid A (Salvianolic acid A), pale yellow powder, easily easy methanol, water.ESIMS m/z:493[M-H] ^-, 518[M+Na] ⁺, molecular formula: C ₂₆h ₂₂o ₁₀, molecular weight: 494.45; ¹h (600MHz, CD ₃oD) and ¹³c NMR (150MHz, CD ₃oD) data are as follows:

¹NMR(CD ₃OD，600MHz)δ：2.96(1H，dd，J＝14.4，8.4Hz，H-7``)，3.06(1H，dd，J＝14.4，4.2Hz，H-7``)，5.17(1H，dd，J＝9.0，4.2Hz，H-8``)，6.27(1H，d，J＝16.2Hz，H-8`)，6.54(1H，dd，J＝7.8，1.8Hz，H-6``)，6.63(1H，d，J＝7.8Hz，H-5``)，6.65(1H，d，J＝16.2，Hz，H-7)，6.72(1H，d，J＝8.4Hz，H-4`)，6.74(1H，d，J＝8.4Hz，H-5)，6.76(1H，d，J＝1.8Hz，H-2``)，6.88(1H，dd，J＝8.4，1.8Hz，H-6)，7.05(1H，d，J＝1.8Hz，H-2)，7.11(1H，d，J＝8.4Hz，H-5`)，7.14(1H，d，J＝16.2Hz，H-8)；8.06(1H，d，J＝16.2Hz，H-7`).

¹³CNMR(CD ₃OD，150MHz)δ：36.6(C-7``)，73.3(C-8``)，112.6(C-2)，113.4(C-5)，114.2(C-8`)，114.9(C-4`)，115.1(C-5``)，116.0(C-2``)，118.7(C-5`)，119.6(C-6)，119.3(C-8)，120.6(C-6``)，124.7(C-6`)，127.0(C-1)，127.9(C-1``)，130.0(C-1`)，136.5(C-7)，143.1(C-3)，143.9(C-3``)，144.8(C-4``)，145.1(C-2`)，145.4(C-4)，146.0(C-7`)，146.9(C-3`)，167.3(C-9`)，172.1(C-9``).

Alkannic acid, its molecular formula: C ₂₇h ₂₂o ₁₂, molecular weight: 538.46, structure is as follows:

Alkannic acid physicochemical property and spectral data, be shown in Fig. 3,4.

Alkannic acid (lithospermic acid), pale yellow powder, easily easy methanol, water.ESIMS m/z:539[M+H] ⁺, 537[M-1] ^-, 560[M+Na] ^-, 492[M-COOH] ^-; Molecular formula: C ₂₇h ₂₂o ₁₂, molecular weight 538.45; ¹h (600MHz, CD ₃oD) and ¹³c NMR (150MHz, CD ₃oD) data are as follows:

¹NMR(CD ₃OD，600MHz)δ：3.02(1H，dd，J＝14.4，9.6Hz，H-7``)，3.06(1H，dd，J＝14.4，3.0Hz，H-7``)，4.25(1H，dd，J＝5.4Hz，H-8`)，4.97(1H，dd，J＝9.6，3.0Hz，H-8``)，5.88(1H，d，J＝5.4Hz，H-7`)，6.31(1H，d，J＝16.0Hz，H-8)，6.59(1H，dd，J＝8.0，1.8Hz，H-6``)，6.65(1H，d，J＝8.0Hz，H-5``)，6.73(1H，dd，J＝8.4，2.0Hz，H-6`)，6.74(1H，d，J＝1.8Hz，H-2``)，6.74(1H，d，J＝8.4Hz，H-5`)，6.83(1H，d，J＝8.4Hz，2.4Hz，H-5)，6.97(1H，d，J＝2.0Hz，Hz，H-2`)，7.13(1H，d，J＝8.4Hz，H-6)；7.93(1H，d，J＝16.0Hz，H-7).

¹³CNMR(CD ₃OD，150MHz)δ：37.3(C-7``)，59.9(C-8`)，76.7(C-8``)，88.9(C-7`)，112.4(C-2``)，114.6(C-5``)，114.9(C-5`)，115.7(C-8)，116.1(C-2`)，117.2(C-6``)，119.5(C-6)，119.9(C-6`)，123.5(C-1)，129.3(C-2)，129.8(C-1``)，133.4(C-1`)，142.4(C-7)，143.3(C-4)，143.4(C-3``)，144.7(C-4``)，145.1(C-3`)，145.1(C-4`)，147.4(C-3)，167.8(C-9)，176.5(C-9``)，178.5(C-9`).

Rosmarinic acid, the molecular formula of rosmarinic acid: C ₁₈h ₁₆0 ₈, molecular weight: 360.31, structure is as follows:

Rosmarinic acid physicochemical property and spectral data, be shown in Fig. 5,6.

Rosmarinic acid (Rosmarinic acid), white powder, easily easy methanol, water.ESIMS m/z:359[M-H] ^-, 383[M+Na] ⁺; Molecular formula: C ₁₈h ₁₆0 ₈, molecular weight 360.31; ¹h (600MHz, CD ₃oD) and ¹³c NMR (150MHz, CD ₃oD) data are as follows:

¹NMR(CD ₃OD，600MHz)δ：3.01(1H，dd，J＝14.4，8.4Hz，H-7`)，3.10(1H，dd，J＝14.4，4.2Hz，H-7`)，5.18(1H，dd，J＝8.4，4.2Hz，H-8`)，6.27(1H，d，J＝16.0Hz，H-8)，6.61(1H，dd，J＝7.8，1.8Hz，H-6`)，6.70(1H，d，J＝7.8Hz，H-5`)，6.75(1H，d，J＝1.8Hz，H-2`)，6.78(1H，d，J＝8.4Hz，H-5)，6.95(1H，dd，J＝8.4，2.4Hz，H-6)，7.04(1H，d，J＝1.8Hz，H-2)，7.55(1H，d，J＝16.0Hz，H-7)；

¹³CNMR(CD ₃OD，150MHz)δ：36.6(C-7`)，73.2(C-8`)，113.1(C-8)，113.9(C-2)，114.9(C-5)，115.1(C-5`)，116.2(C-2`)，120.4(C-6)，121.8(C-6`)，126.3(C-1)，127.9(C-1`)，143.9(C-3)，144.8(C-4)，145.5(C-3`)，146.4(C-4`)，148.4(C-7)，167.1(C-9)，172.1(C-9`).

Salvianolic acid B, its molecular formula: C ₃₆h ₃₀o ₁₆, molecular weight: 718.61, structure is as follows:

Salvianolic acid B physicochemical property and spectral data, be shown in Fig. 7,8.

Salvianolic acid B (Salvianolic acid B), pale yellow powder, easily easy methanol, water.ESIMS m/z:717[M-H] ^-, 741[M+Na] ⁺; Molecular formula: C ₃₆h ₃₀o ₁₆, molecular weight 718.62; ¹h (600MHz, CD ₃oD) and ¹³c NMR (150MHz, CD ₃oD) data are as follows:

¹NMR(CD ₃OD，600MHz)δ：2.80-3.10(2H，m，H-7``)，2.80-3.10(2H，m，H-7```)，4.35(1H，d，J＝4.8Hz，H-8``)，5.15-5.19(1H，m，H-8`)，5.15-5.19(1H，m，H-8```)，5.85(1H，d，J＝4.8Hz，H-7``)，6.20(1H，d，J＝16.2Hz，H-8)，6.31(1H，dd，J＝8.1，2.1Hz，H-6`)，6.52(1H，d，J＝2.4Hz，H-2```)，6.54(1H，d，J＝7.8Hz，H-5``)，6.62(1H，d，J＝2.1Hz，H-2`)，6.66(1H，dd，J＝8.1，2.4Hz，H-6```)，6.70(1H，d，J＝7.8Hz，H-5`)，6.74(1H，d，J＝8.1Hz，H-5```)，6.75(1H，dd，J＝7.8，2.4Hz，H-6```)，6.76(1H，d，J＝2.4Hz，H-2``)，6.83(1H，d，J＝8.4Hz，H-5)，7.16(1H，d，J＝8.4Hz，H-6)，7.52(1H，d，J＝16.2Hz，H-7)；

¹³CNMR(CD ₃OD，150MHz)δ：36.111(C-7```)，36.51(C-7`)，56.58(C-8``)，73.28(C-8)，74.19(C-8```)，86.93(C-7``)，111.99(C-2``)，115.01(C-5``)，115.04(C-5`)，115.13(C-2`)，115.18(C-2```)，115.95(C-8)，116.20(C-6``)，116.97(C-5```)，117.04(C-5)，120.37(C-6)，120.89(C-6```)，123.28(C-1)，125.04(C-2)，127.55(C-1`)，127.85(C-1```)，132.27(C-1``)，142.20(C-7)，143.70(C-3```)，143.72(C-4```)，143.88(C-4``)，144.57(C-3`)，144.73(C-4`)，145.23(C-3`)，145.39(C-3)，147.71(C-4)，166.66(C-9)，170.92(C-9```)，171.17(C-9``)，172.29(C-9`)

Salvianolic acid C, its molecular formula: C ₂₆h ₂₀o ₁₀, molecular weight: 492.43, structure is as follows:

Salvianolic acid C physicochemical property and spectral data, be shown in Fig. 9,10.

Salvianolic acid C (Salvianolic acid C), pale yellow powder, easily easy methanol, water.ESIMS m/z:491[M-H] ^-, 515[M+Na] ⁺; Molecular formula: C ₂₆h ₂₀o ₁₀, molecular weight 492.43; ¹h (600MHz, CD ₃oD) and ¹³c NMR (150MHz, CD ₃oD) data are as follows:

¹NMR(CD ₃OD，600MHz)δ：3.07(1H，dd，J＝13.8，8.4Hz，H-7``)，3.15(1H，dd，J＝13.8，4.2Hz，H-7``)，5.25(1H，dd，J＝8.4，4.2Hz，H-8``)，6.46(1H，d，J＝15.9Hz，H-8)，6.46(1H，dd，J＝8.1，2.1Hz，H-2``)，6.72(1H，d，J＝8.1Hz，H-3``)，3.74(1H，d，J＝8.4Hz，H-5)，6.80(1H，d，J＝2.4Hz，H-6``)，6.88(1H，d，J＝7.8Hz，H-5`)，7.20(1H，s，，H-8`)，7.36(1H，d，J＝7.8Hz，H-6)，7.37(1H，dd，J＝7.8Hz，2.4Hz，H-6`)，7.40(1H，d，J＝2.4Hz，2.1Hz，H-2`)，7.94(1H，d，J＝15.9Hz，H-7)；

¹³CNMR(CD ₃OD，150MHz)δδ：36.2(C-7``)，73.3(C-8``)，97.9(C-8`)，110.4(C-5)，112.0(C-2`)，113.5(C-8)，114.9(C-5``)，115.4(C-5`)，116.3(C-2``)，117.4(C-6`)，118.2(C-1)，120.5(C-6``)，122.0(C-1`)，125.0(C-6)，128.0(C-1``)，131.3(C-2)，143.0(C-3)，143.8(C-7)，144.0(C-3``)，144.6(C-4)，144.8(C-4``)，145.3(C-2`)，146.7(C-4`)，158.0(C-7`)，167.3(C-9)，172.4(C-9``).

Experimental example 3: the extraction of salvianolic acid B

1.1 extract the confirmation of solvent and extracting method

Take red rooted salvia 400g, the method under Chinese Pharmacopoeia Radix Salviae Miltiorrhizae item of pressing is measured content of danshinolic acid B, is divided into 4 parts, by following testing program, is tested:

Experiment 1: get red rooted salvia 100g, add 8 times of water gagings at every turn and extract 1.5 hours at 80 ℃ of warm macerating, warm macerating extracts three times altogether, and merge extractive liquid, is measured salvianolic acid B and calculates extraction ratio, and result is as table 7.

Experiment 2: get red rooted salvia 100g, add 8 times of water gagings at every turn and extract 1.5 hours at 80 ℃ of warm macerating, stir with 10～50 rev/mins of speed simultaneously, warm macerating stirs and extracts three times altogether, and merge extractive liquid, is measured salvianolic acid B and calculates extraction ratio, and result is as table 7.

Experiment 3: get red rooted salvia 100g, add 8 times of water gagings at every turn and decoct 1.5 hours, decoct altogether three times, merge extractive liquid,, measure salvianolic acid B and calculate extraction ratio, and result is as table 7.

Experiment 4: get red rooted salvia 100g, add 8 times of amount 50% alcohol reflux 1.5 hours at every turn, extract altogether three times, merge extractive liquid,, measure salvianolic acid B and calculate extraction ratio, and result is as table 7.

Table 7. extracts solvent and extracting method experimental result

Above-mentioned experimental result shows, adopt 50% ethanol to make the extraction solvent influential to the salvianolic acid B extraction ratio, 50% ethanol extraction is better than water extraction, but soak and add that stir to extract difference little with water temperature, two kinds of extracting modes that stir in the water extraction process and do not stir are influential to the extraction ratio of salvianolic acid B, decocting extraction may be because temperature is higher, to salvianolic acid B, extracts influential.

1.2 the preferred extraction process of orthogonal experiment

1.2.1 the optimization of water extraction process research: according to above result of the test, water extraction process is usingd extraction time (A), extraction time (B), quantity of solvent (C), extract four of temperature (D) as the investigation factor, each factor is established three levels, presses L9 (3 ⁴) orthogonal table carries out Orthogonal Experiment and Design (table 8, table 9), the extraction ratio that to investigate index be salvianolic acid B.

Table 8. extraction factor water-glass

Table 9. extraction process orthogonal experiments table

Table 10. the results of analysis of variance table

F _0.05(2，2)＝19.00 F _0.01(2，2)＝99.00

The water extraction the results of analysis of variance shows, each experimental factor is to the extraction rate of transform of salvianolic acid B there are no significant difference, therefore the water extraction condition of salvianolic acid B of the present invention is for adding 3～15 times of water gagings, extracting 1～3 time at 45～95 ℃ of lower warm macerating, with 10～50 rev/mins of speed, stir simultaneously, extract 1～4 hour at every turn or add 3～15 times of amounts at every turn and decoct extraction, the each extraction 1～4 hour, extract 1～3 time altogether.

1.2.2 the optimization of alcohol extraction technique research: according to above result of the test, alcohol extraction technique is usingd extraction time (A), four of extraction times (B), quantity of solvent (C), concentration of alcohol (D) be as the investigation factor, each factor is established three levels, presses L9 (3 ⁴) orthogonal table carries out Orthogonal Experiment and Design (table 11, table 12), the extraction ratio that to investigate index be salvianolic acid B.

Table 11. extraction factor water-glass

Table 12. extraction process orthogonal experiments table

Table 13. the results of analysis of variance table

F _0.05(2，2)＝19.00 F _0.01(2，2)＝99.00

Alcohol reflux extracts the results of analysis of variance and shows, each experimental factor, to the extraction rate of transform of salvianolic acid B there are no significant difference, therefore the extraction conditions of this salvianolic acid B is measured 30%～60% alcohol reflux 1～3 time for adding 3～15 times, extracts 1～4 hour at every turn.

1.3 salvianolic acid B raw material preparation

According to above-mentioned orthogonal experiment Optimization Technology, get red rooted salvia 10kg, add 8 times of water gagings at every turn and extract 1.5 hours at 80 ℃ of warm macerating, with 10～50 rev/mins of speed, stir simultaneously, warm macerating stirs and extracts 3 times altogether, filters merging filtrate, extracting solution is evaporated to relative density 1.10～1.25 (60 ℃), add ethanol to make to measure 60% containing alcohol, filter, decompression filtrate recycling ethanol also is concentrated into without the alcohol flavor, vacuum drying, salvianolic acid extract 4.15kg, recording content of danshinolic acid B is 10.25%.

Experimental example 4: salvianolic acid B transforms the salvianolic acid A composition process relatively

Experiment 1: get the about 30g of salvianolic acid B raw material of preparation under 1.3, be mixed with 200ml solution, add 10% sodium hydroxide solution adjust pH to 4.5;

Experiment 2: get the about 30g of salvianolic acid B raw material of preparation under 1.3, be mixed with 200ml solution, add 10% sodium hydroxide solution adjust pH to 4.5, add 1.0%ZnCl ₂;

Experiment 3: get containing the about 6g of salvianolic acid B (content 51.54%) raw material, adding purified water, to be diluted to salvianolic acid B concentration be 15mg/ml solution, adds carbamide, and making it with the mol ratio of salvianolic acid B is 0.5;

Above-mentioned each experimental group is placed in same autoclave, and under 120 ℃, reaction is 4.0 hours, cooling, calculates salvianolic acid A compositions productive rate.

Table 14. salvianolic acid B transforms salvianolic acid A compositions experimental result

The demonstration of table 14 result, the salvianolic acid B high-purity, on transforming not impact, does not need to be purified to more than 50% to be transformed, and salvianolic acid B is converted in salvianolic acid A compositions process, regulates pH value, adds 1.0%ZnCl ₂, can greatly improve the productive rate of salvianolic acid A compositions.

Experimental example 5: salvianolic acid B transforms the optimization of salvianolic acid A composition process

Above-mentioned experimentation proves, salvianolic acid B purity is not to affect the factor that salvianolic acid B transforms the salvianolic acid A compositions, but adds certain catalysts influence salvianolic acid B to transform the salvianolic acid A compositions, and therefore, we all add 1%ZnCl to each experimental group ₂as catalyst, be converted into other factors of salvianolic acid A compositions to affecting salvianolic acid B: salvianolic acid B concentration (A), pH (B), temperature (C), time (D) are carried out orthogonal test, and each factor is established three levels, presses L9 (3 ⁴) orthogonal table carries out EXPERIMENTAL DESIGN (table 15, table 16), the productive rate that to investigate index be the salvianolic acid A compositions.

Table 15. transforming agent water-glass

Table 16. conversion process orthogonal experiments table

Table 17. the results of analysis of variance table

^*F _0.05(2，2)＝19.00 △F _0.01(2，2)＝99.00

The results of analysis of variance demonstration, the optimum condition that this orthogonal test is optimized according to intuitive analysis is A ₃b ₂c ₂d ₂factor A (salvianolic acid B concentration) has certain influence to salvianolic acid A compositions productive rate, from the K value, analyze, concentration is converted into salvianolic acid A to improving salvianolic acid B effect higher than 30mg/ml has no significant effect, and the impurity formed is more, therefore, transform front salvianolic acid B concentration and should select 1～30mg/ml; Factor B (pH), factor C (temperature) have utmost point significant difference to salvianolic acid A compositions productive rate, in prompting salvianolic acid B conversion process, should strictly control pH and temperature, can success realize transforming to the salvianolic acid A compositions of salvianolic acid B higher yields.

Experimental example 6: catalyst Z nCl ₂the impact of consumption on transforming

Transform salvianolic acid A composition process optimal conditions by above-mentioned salvianolic acid B, get the salvianolic acid B raw material, add purified water 200ml, make the solution that salvianolic acid B concentration is 15mg/ml, regulating pH is 4.5, and conversion temperature is 120 ℃, and transformation time is 4 hours, by with the salvianolic acid B molar percentage, add respectively not commensurability catalyst Z nCl ₂, the results are shown in Table 18.

Table 18. catalyst Z nCl ₂consumption affects experimental result to transforming

Catalyst Z nCl ₂consumption affects experimental result to conversion and shows, catalyst amount>=0.02% can produce 55.65% conversion ratio, preferred catalyst consumption 0.1%～3.0%, and more preferably, after 0.5%～2.0%. catalyst amount>3%, conversion ratio no longer is significantly improved.

Experimental example 7: catalyst transforms the catalytic action of salvianolic acid A compositions to salvianolic acid B

Transform salvianolic acid A composition process optimal conditions by above-mentioned salvianolic acid B, get the salvianolic acid B raw material, add purified water 200ml, make the solution that salvianolic acid B concentration is 15mg/ml, regulating pH is 4.5, and conversion temperature is 120 ℃, and transformation time is 4 hours, add respectively the catalyst of different cultivars and various dose to be transformed, the results are shown in Table 19.

Table 19. different catalysts transforms the impact of red A compositions on red B

Table 19 result shows, above 4 kinds of catalyst all can be used as the catalyst in the salvianolic acid B conversion reaction, and each catalyst amount and salvianolic acid B molar percentage be 0.5%～3.0%, the productive rate of salvianolic acid A compositions >=40%.Conversion ratio surpasses 60%.

Experimental example 8: the purification with macroreticular resin of salvianolic acid A compositions

Get the salvianolic acid A compositions and transform 13 parts of solution, put in each model macroporous resin 100g that pretreatment is good, after shaking table dynamic adsorption 8 hours, the dress post, wash successively 3 column volumes of eluting, 5 column volumes of 20% ethanol elution, 5 column volumes of 70% ethanol elution with water, collect 70% ethanol containing salvianolic acid A compositions eluent part, carry out the salvianolic acid A assay, the eluent evaporate to dryness calculates dry cream amount, the results are shown in Table 20.

The confirmation of table 20. macroporous resin model

Result shows: above 13 kinds of macroporous resins are good to the adsorption effect of salvianolic acid A compositions, and can well remove impurity with the ethanol gradient elution of variable concentrations, obtain the salvianolic acid A compositions eluent that content is greater than 75%.

Experimental example 9: the column chromatography purification for the second time of salvianolic acid A compositions

Get 3 parts of salvianolic acid A composition solutions after purification with macroreticular resin, put in each model resin 100g that pretreatment is good, after shaking table dynamic adsorption 8 hours, the dress post, wash successively 3 column volumes of eluting, 5 column volumes of 20% ethanol elution, 5 column volumes of 70% ethanol elution with water, collect 70% ethanol elution and carry out the salvianolic acid A assay, part eluent evaporate to dryness calculates dry cream amount, the results are shown in Table 21.

The confirmation of table 21. resin model

Result shows: above 3 kinds of resins are good to the adsorption effect of salvianolic acid A, and can well remove impurity with the ethanol gradient elution of variable concentrations, obtain content and are about 90% salvianolic acid A compositions eluent.

Experimental example 10: the abstraction purification of salvianolic acid A compositions

By 70% ethanol elution decompression recycling ethanol after post layer column purification for the second time, adjust pH 2.0～4.0, use again n-butyl alcohol, t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate, Ethyl formate, ethanol extraction 5 times, the Separation of Organic phase, reclaim solvent, drying, obtain the salvianolic acid A compositions, measure salvianolic acid A content, the results are shown in Table 22.

The confirmation of organic solvent for table 22. extraction

Table 22 result shows: n-butyl alcohol is because polarity is large, the salvianolic acid A amount of composition is maximum, but its salvianolic acid content is not improved significantly, ether is because polarity is less than normal, water-solubility impurity is few, and salvianolic acid A content is high, but the salvianolic acid A amount of composition obtained is few, the salvianolic acid A amount of composition that other extraction solvent t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate, Ethyl formate obtain is larger, and salvianolic acid A content all is improved.

Experimental example 11: the purification by silica gel column chromatography of salvianolic acid A compositions

Get 12 parts of salvianolic acid A compositions extracts after abstraction purification, every part containing salvianolic acid A 10g, adds the silica gel of 20g, stirs, and volatilizes; Stirring sample silica gel, be added on the dry silicagel column of the 100g installed, the two-phase solvent that petroleum ether, pentane, normal heptane, ethyl acetate, methyl acetate, Ethyl formate, t-butyl methyl ether form of take respectively is eluant, HPLC or thin layer chromatography detect, collect salvianolic acid A compositions eluent, the eluent evaporate to dryness, get dry extract, measure salvianolic acid A content, the results are shown in Table 23.

The confirmation of table 23. eluant

Result shows: when the salvianolic acid A compositions is used normal phase silica gel column chromatography, the two-phase solvent that adopts petroleum ether, pentane, normal heptane, ethyl acetate, methyl acetate, Ethyl formate, t-butyl methyl ether to form is eluant, gradient elution, can well remove impurity, obtain highly purified salvianolic acid A compositions eluent.

Experimental example 12: salvianolic acid A composition dries method research

Get 4 parts of salvianolic acid A compositions eluents after purification by silica gel column chromatography, every part containing salvianolic acid A 100g, be concentrated into without the thick paste shape, adding after 10 times of water gagings dissolve adopts respectively vacuum drying, lyophilisation, spray drying, microwave vacuum drying to obtain the salvianolic acid A compositions, said composition is detected, be the results are shown in Table 24.

Table 24. salvianolic acid A composition dries method testing result

Result shows: adopt the lyophilisation overlong time, and high cost, and organic solvent residual is serious; And that vacuum drying, spray drying microwave vacuum drying process are controlled is simple, treating capacity is large, and baking temperature is low, and the time is short, and gained salvianolic acid A compositions indices is good, therefore the present invention adopts vacuum drying, spray drying or microwave vacuum drying.

Experimental example 13: the salvianolic acid A drop pill becomes the Preliminary screening of ball condition

Drop pill is that medicine evenly splashes in condensed fluid the drop pill that becomes ball and make after mixing with substrate, due to the many factors that affects into ball, relates to into ball substrate, crude drug, liquid coolant and temperature and drips speed etc.; At first we use Macrogol 4000, polyethylene glycol 6000 mixed in equal amounts, get 750g in 80 ℃ of meltings, with the 250g salvianolic acid A, mix homogeneously, take dimethicone as coolant, respectively on the coolant temperature that affects into ball, drip apart from, drip speed, cooling column progress row optimization experiment, scheme with the results are shown in Table 25,26.

Table 25. factor level

Table 26. result of the test

Experimental example 14: the Preliminary screening of salvianolic acid A drop pill substrate

The substrate Main Function of drop pill is to make it molding for dispersion medicine filling weight or volume, substrate Macrogol 4000 commonly used, polyethylene glycol 6000, PEG 8000, gelatin and stearic acid, glyceryl monostearate, insect wax, hydrogenated vegetable wet goods; According to the base parameter of above-mentioned one-tenth ball, we get the salvianolic acid A compositions, press table 27 substrate and form, and dripping becomes drop pill respectively.

Table 27. prescription Screening matrix table

Get the salvianolic acid A compositions by table 27 formula, substrate is placed in to melting on water-bath, add the salviol acid A compositions of regulation ratio, stir mix homogeneously.Take dimethicone as coolant, dripping, cooling under set point of temperature, the coolant of filtering drop pill remained on surface, obtain sample.Investigate respectively gravimetry, roundness, color and luster, the ball method of double differences different, leach time, red sour phenol A assay and other component contents and measure; The results are shown in Table 28.

Table 28. different substrates affects result to mouldability

From table 28 experimental result, but different prescriptions and different proportion adjuvant mix all drippings with salvianolic acid A becomes ball, its shaped degree, appearance character, weight differential, dissolve scattered time limit are all up to specification, salvianolic acid A and other stable components, illustrate that the preparation process is on the not impact of salvianolic acid A compositions.

Experimental example 15: preparation stabilization Journal of Sex Research

Get embodiment 11,12,13 samples, by the requirement of stability of drug products experimentation relevant technologies, by above three lot number samples at ambient temperature, by 0 month, 1 month, 2 months, 3 months, 6 months intervals, make regular check on sample property, dissolve scattered time limit, weight differential, salvianolic acid A changes of contents situation, the results are shown in Table 29.

29. stability experiment result

Table 29 stability experiment observed result shows, the front and back not variation of salvianolic acid A content in 6 months at ambient temperature of salvianolic acid A drop pill, and appearance character, dissolve scattered time limit also, in acceptability limit, illustrates that the salvianolic acid A drop pill is stable.

Below experiment all gets with the salvianolic acid A drop pill sample that embodiment 13 preparation methoies obtain, and salvianolic acid A monomeric compound group is got embodiment 23 samples.

Experimental example 16: the impact of function of nervous system's symptom after salvianolic acid A drop pill commute apoplectic type renovascular hypertension in rats (RHRSP) cerebral thrombosis

1, experiment material:

Main agents and instrument: tiger red (rose bengal): the true Bioisystech Co., Ltd in Shanghai; TTC: U.S. Sigma company; SDP-1 type rat heart rate sphygomanometer: China-Japan Friendship Hospital's development; YAG laser device: Wuhan Chinese workers' laser engineering company limited; Japan Olympus BH-5 microscope; Image is processed: JVCky-F3OB 3-CCD coloured image is shot with video-corder the input instrument; Graphical analysis: German KONTRON IBAS 2.0 Image analysis systems.

Laboratory animal: 2～3 monthly ages, the male SD rat (Beijing Vital River Experimental Animals Technology Co., Ltd. provides) of body weight (100 ± 20) g.

2, experimental technique

Get the male SD rat of 2～3 monthly ages, body weight (100 ± 20) g and set up the RHRSP model with two narrow renal artery of kidney double fastener method: rat abdominal cavity anesthesia, through the long otch of the about 5cm of xiphoid-process Ventral Midline stringer, cut skin, abdominal muscle, the blunt separation bilateral renal arteries, the annular silver brain clip that is 0.3mm with internal diameter is clamped respectively the bilateral renal arteries initial part.Before operation, measuring blood pressure once, is surveyed once weekly continuous 12 weeks after operation.Separately get 10 SD rats that body weight is suitable, survey its blood pressure as normal control.Reject: in operation and postoperative death, the 12 weeks after operation blood pressure is lower than the 160mmHg rat, and softening kitchen range that apoplexy symptom and sign, dissection be shown in that left basal ganglia region is little and the rat of subarachnoid hemorrhage are arranged.Blood pressure >=160mmHg and be successful model without the RHRSP of apoplexy symptom after 16 weeks.With photochemical method, cause middle cerebral artery occlusion (MCAO) model to cause middle cerebral artery (MCA) thrombosis the RHRSP be successfully prepared: after 2% pentobarbital sodium is pressed 40mg/kg body weight intraperitoneal injection of anesthesia, through left temporo side approach, at zygomatic arch and 1mm place, front lower place, aquamous part of temporal bone junction, bore by dental burr the bone window that a diameter is 5mm, through cerebral dura mater, can know and see left MCA trunk and branch thereof.Dosage injection 2.5% tiger of pressing the 40mg/kg body weight through femoral vein is red, green laser beam (the spot diameter 2mm of the 532nm occurred by the YAG laser device after 5min, intensity of illumination 0.37W) prolonged exposure MCA trunk 15min, visible this MCA far-end and each branch blood flow interrupt, and see that under operating microscope local white thrombus points out this MCA thrombosis, the success of MCAO model copy.In art, rat anus temperature remains on (37 ± 0.3) ℃.Local wound is rinsed with the penicillin diluent before sewing up, and postoperative conventional intramuscular injection penicillin 80,000 U/ only.Reject in art and postoperative 1d death and hemorrhage many or operating microscope under the not clear and definite blood supply rat of whether interrupting.

Animal is divided 7 groups at random: sham operated rats, model control group, the high, medium and low dosage group of salvianolic acid A drop pill, salvianolic acid A monomeric compound group, nimodipine group.Wherein the sham operated rats window that only opens seam, do not do laser irradiation.And naturally revive rear gastric infusion once in the postoperative animal of MCAO, after this be administered once every day, altogether continuous 14d.Sham operated rats, model control group gavage are with the normal saline of volume; The high, medium and low dosage group of salvianolic acid A drop pill gavage 60mg/kg, 30mg/kg, 15mg/kg, salvianolic acid A monomeric compound group gavage 30mg/kg, nimodipine group gavage 30mg/kg.

3 experimental results

18 minutes point systems according to Garcia etc. are marked: the symmetry of spontaneous activity, quadruped locomotion, forelimb stretch the action of creeping symmetry, climb the cage wall, push away the trunk reaction, the reaction of antenna to stimulating.In first three items, without movable or reaction (extremity or suffering limb), be 0 minute, a little activity is 1 minute, and reaction calibration constant is 2 minutes, and activity is normally 3 minutes; Rear three reactionless be 1 minute, activity calibration constant is 2 minutes, activity is normally 3 minutes.Total points is the highest 18 minutes, minimum 3 minutes.After the postoperative rat anesthesia of MCAO is revived, before gastric infusion, scoring once, is respectively carried out a neuroethology scoring (at every turn all being marked after administration on the same day) at MCAO postoperative (after being ischemia) 3d, 7d, 14d to rat respectively afterwards.Each experimental group rat is carried out to blind state by 5 people that are unfamiliar with this experiment grouping simultaneously and observe scoring, finally average.And observe the rat body state simultaneously, measure body weight and blood pressure.

Table 30. salvianolic acid A drop pill on the RHRSP cerebral thrombosis after the impact (x ± s) of neuroethology scoring

Annotate: compare #P<0.01 with sham operated rats; Compare * P<0.05, ☆ P<0.01 with model group

Experimental result shows: the neuroethology scoring after each treated animal modeling is revived is significantly lower than sham operated rats (P<0.01), the cerebral thrombosis ischemia be described after RHRSP function of nervous system impaired seriously.Model group neuroethology scoring after cerebral ischemia 3d, 7d, 14d all is starkly lower than sham operated rats (P<0.01); Ischemia 3d, except salvianolic acid A drop pill high, middle dosage group behavior scoring and model group relatively are increased significantly (P<0.05), though other each medicine group has in various degree and raises than model group, difference is not remarkable; Ischemia 7d, each medicine group all is increased significantly (P<0.01 or P<0.05) than the model group behavior scoring, the most remarkable with salvianolic acid A drop pill high dose group; To the scoring of high, the middle dosage group of ischemia 14d salvianolic acid A drop pill rat behavior, followed the sham-operation component not go out difference, each medicine group is apparently higher than model group (P<0.01 or P<0.05).Each time period behavior scoring of salvianolic acid A drop pill low dose group is suitable with the nimodipine group.30mg/kg salvianolic acid A drop pill group and 30mg/kg salvianolic acid A monomeric compound group are relatively, although the neuroethology scoring does not have significant difference, but slightly excellent trend is also arranged, and in the laboratory observation process, the integrality of rat is also good than salvianolic acid A monomeric compound group.Above results suggest, after the salvianolic acid A drop pill can improve RHRSP cerebral thrombosis ischemia, neuroethology is marked, the effect of nerve injury symptom after prompting salvianolic acid A drop pill has protection and improves cerebral ischemia, and the trend that is better than same dosage salvianolic acid A monomeric compound is arranged.

Experimental example 17: the impact of salvianolic acid A drop pill on RHRSP cerebral thrombosis tissues following MCAO in rats pathological change

The preparation of RHRSP Cerebrovascular embolism model, grouping, medication are with experimental example 16.After the neuroethology scoring finishes the last time, the rat broken end is got brain, and observes MCA local thrombus situation under operating microscope.Put into afterwards smooth vessel be built in-20 ℃ freezing, the row crown section.The Mus brain section, every thick about 2mm.The brain sheet is put into rapidly to 2%TTC solution, hatch under 37 ℃, the every face of brain sheet is hatched 15min, and normal cerebral tissue peony, and the ischemic infarction cerebral tissue is white in color.Taking out the brain sheet observes and takes pictures.Then cerebral tissue is placed in to 4% paraformaldehyde PBS buffer (pH7.3) fixing, row dehydration afterwards, paraffin embedding, get cerebral tissue and do paraffin section and do conventional H E staining analysis, micro-Microscopic observation, takes pictures.The results are shown in Table 31.

The impact of table 31. salvianolic acid A drop pill on RHRSP cerebral thrombosis tissues following MCAO in rats pathological change

Experimental result shows: sham operated rats cerebral tissue Bilateral Symmetry, have no pathological changes, and the model group Intravascular Thrombus adheres to seriously.With model group relatively, the contraction in length of the left MCA local thrombus of each dosage group rat of Microscopic observation salvianolic acid A drop pill, reduce at the arterial wall bond area, with high, middle dosage group is the most obvious.The TTC visible infarcted cerebral constitution that dyes is white in color, and is dyed to white cerebral tissue scope in each dosage group of salvianolic acid A drop pill and dwindles than model control group is remarkable.HE dyes in visible model group left cortical MCA blood supply district (centered by the cortex of volume top) infarct has obvious cerebral tissue softening, necrocytosis, the phenomenons such as the local necrosis tissue comes off, salvianolic acid A drop pill high dose group rat is rare Telatrophy obscission herein, other each administration group necrocytosiss and the tissue degree varies that comes off, than model group, all significantly reduce, each dosage group of salvianolic acid A drop pill relatively is the dose-difference opposite sex; The HE dyeing of each administration group and model control group shows in kitchen range, kitchen range Zhou Jun has little blood vessel hyperplasia, but the little number of blood vessel of each administration group hypertrophy increases than model group is remarkable, and particularly remarkable with high, the middle dosage group of salvianolic acid A drop pill and salvianolic acid A monomeric compound group; In addition, the kitchen range shape that HE dyeing display model matched group differs in size as seen is hemorrhage, high, the middle dosage group of salvianolic acid A drop pill is accidental kitchen range shape bleeding, salvianolic acid A drop pill low dose group and salvianolic acid A monomeric compound group and nimodipine group also only have the minority animal to have the kitchen range shape hemorrhage, and hemorrhagic focus is less than model group.Result of the test shows that the salvianolic acid A drop pill can obviously improve RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia histopathology state; reduce the thrombosis bond area, reduce infarct size; thereby increasing the little blood vessel number, the necrosis of minimizing cerebral hemorrhage phenomenon protection cerebral tissue that reach surrouding brain tissue in infarcted region comes off; there is protection cerebral thrombosis ischemia and cause the effect of brain tissue impairment, and action effect there is the trend that is better than the salvianolic acid A monomeric compound.

Experimental example 18: the salvianolic acid A drop pill on the RHRSP cerebral thrombosis after the impact of blood brain barrier (BBB) permeability

1 test method

The preparation of RHRSP Cerebrovascular embolism model and animal grouping, administration are with experimental example 16 methods.Each is organized rat and puts to death in MCAO art ischemia 3d, 7d, 14d respectively in batches, and before putting to death 1h tail vein injection 2% azovan blue (Evans Blue, EB) normal saline solution 2ml/kg body weight, after fully circulating, dark numb animal, cut open breast through heart perfusion normal saline flushing blood vessel inner dye, broken end is got the cerebral tissue specimen.

Each group is randomly drawed the cerebral tissue of 3 rats, frozen section immediately, the crown section of the thick about 30um of row continuously, with 70% glycerol PBS buffer (pH8.5～9.0) mounting, observe the situation of oozing out of EB in cerebral tissue under Bio-Rad Radiance2100 laser scanning co-focusing microscope, observe BBB open area and degree.While observing, section is not put 4 ℃ of refrigerators and is kept in Dark Place.Each group is separately got the cerebral tissue of 5 rats at random, blots weighing cutaneous horn weight after surface moisture; Cerebral tissue is placed in to homogenizer, adds 50% trichloroacetic acid to make tissue homogenate, more than moving into test tube sealing and standing 60min; The centrifugal 10min of 3000rpm/min, get supernatant, and spectrofluorophotometer is surveyed fluorescent value: excitation wavelength 620nm, emission wavelength 680nm; Calculate EB content in supernatant by standard curve, then calculate every Borneo camphor and organize EB content, with this, react each rat BBB permeability.

2 experimental results

2.1 laser scanning co-focusing microscope observed result

EB is bright-coloured bright red fluorescence under the exciting light state.Observe each treated animal cerebral tissue WuBBBNao district (as pinus, area postrema and hypophysis cerebri) under laser microscope and present homogeneous, diffusivity EB dip-dye, sharpness of border, the line sample profile that meninges red color visible fluorescence is sketched the contours of.And the BBBNao district, other except sham operated rats are respectively organized the equal visible light spot of cerebral tissue, and mainly be distributed in thalamus, hypothalamus, cerebellum, Hippocampus etc. locate, spot size differs, fluorescence intensity weakens to periphery gradually from the center of hot spot, obscurity boundary.Hot spot number of each group and distribute obviously different: the model group hot spot is maximum, and ischemia 3d approximately has the hot spot of 120 left and right diameter 100～200um, and to ischemia 7d, the hot spot number approximately has 150 left and right, and it is little that the hot spot number of ischemia 14d and 7d organize difference; The intensive place of hot spot in the form of sheets.Each salvianolic acid A drop pill group number of spots significantly reduces than model group, and the salvianolic acid A drop pill is fewer than the salvianolic acid A monomeric compound group hot spot of same dosage; Each dosage group of salvianolic acid A drop pill relatively, is the obvious dose-difference opposite sex; Wherein more with the low dose group hot spot, ischemia 14d, on same level, hot spot approximately has 80, but is fused into the less of lamellar; Middle dosage group hot spot is few than low dose group; The high dose group hot spot is minimum and be dispersed in distribution, and fluorescence also obviously weakens.

2.2 the permeability result of the EB of rat cerebral tissue content detection blood brain barrier

Each is organized rat cerebral tissue's specimen and calculates EB content by the fluorescent value recorded, and data statistic analysis is in Table 32.

Table 32. salvianolic acid A drop pill on the RHRSP cerebral thrombosis after the impact of blood-brain barrier permeability

Annotate: compare #P<0.001 with sham operated rats; Compare * P<0.01 with model group

2.3 conclusion

EB is water-soluble dye, enter after blood can be absolutely rapidly and albumin bound, can not see through blood brain barrier under normal circumstances.From 2.1 and 2.2 results, sham operated rats BBBNao district, have no EB under the cerebral tissue microscope and ooze out hot spot; And under model group rat cerebral tissue microscope, have a large amount of EB to ooze out hot spot, and the cerebral tissue spectrofluorophotometer detects the EB of high-load, than sham operated rats, utmost point significant difference (P<0.001) is arranged, show cerebral thrombosis tissues following MCAO in rats blood-brain barrier disruption, permeability increases, and the EB albumin-binding can enter cerebral tissue by open BBB.Experimental result shows under each dosage group rat cerebral tissue microscope of salvianolic acid A drop pill that EB hot spot number and cerebral tissue EB detect content and obviously reduce, with model group, more all there were significant differences (P<0.01), and will be less than the salvianolic acid A monomeric compound group of same dosage.Prompting salvianolic acid A drop pill increases inhibited to blood-brain barrier permeability following brain injury, can protect blood brain barrier, and the further damaged of protection cerebral tissue is done by effect and is better than the salvianolic acid A monomeric compound.

Experimental example 19: the impact of salvianolic acid A drop pill on RHRSP cerebral thrombosis cerebral infarction scope and brain water content

Method by experimental example 16 prepares RHRSP cerebral thrombosis rat model, grouping, administration; Second day after administration finishes, the rat broken end is got brain.Each group is got the brain tissue slice of 5 rats at random, through TTC dyeing, after taking pictures under the microscope centered by infarct, infarct is delimited, and processes and calculate the infarct size of every brain sheet by image analysis software.The addition of every all brain sheets of rat infarct size, be brain infarction area, is multiplied by the about 2mm of thickness of every brain sheet, is cerebral infarction volume; Get the in kind approximate calculation of all brain sheets of corresponding rat simultaneously and go out corresponding brain cumulative volume; The two ratio calculation cerebral infarction volume percentage ratio.Each group is separately got 5 rat cerebral tissues and is claimed immediately weight in wet base, puts afterwards in 105 ℃ of baking ovens and dries to constant weight, calculates brain water content.

The impact (x ± s) of table 33. salvianolic acid A drop pill on RHRSP cerebral thrombosis cerebral infarction scope and brain water content

Annotate: compare #P<0.01 with sham operated rats; Compare * P<0.01 with model group

Adopt SPSS 11.5 statistical packages to carry out statistical procedures to data.Experimental result shows: sham operated rats has no infarcted cerebral constitution; Each dosage group cerebral tissue Infarction volume of salvianolic acid A drop pill and brain water content are significantly less than model control group (P<0.01), and dosage is larger, and Infarction volume is less, and brain water content is fewer, and difference has statistical significance (P<0.01 or p<0.05).Salvianolic acid A drop pill basic, normal, high dosage group Infarction volume and brain water content are all lower than 30mg/kg nimodipine group.30mg/kg salvianolic acid A drop pill group rat cerebral infarction volume and brain water content are all low than the salvianolic acid A monomeric compound group of same dosage.Illustrate that the salvianolic acid A drop pill can more effectively accelerate the reparation of focus, reduce ischemic tissue of brain infarction size after the RHRSP cerebral thrombosis, alleviate the cerebral tissue edema, have and repair and the effect of protection Neurons Against Cerebral Ischemia tissue injury.

Experimental example 20: the salvianolic acid A drop pill on RHRSP cerebral thrombosis ischemia after the microvascular impact of rat cerebral tissue

RHRSP Cerebrovascular embolism model preparation and animal grouping, dosage be with experimental example 16 methods, every day gavage once, continue to and draw materials.Each group respectively at the cerebral ischemia of MCAO art after 3d, 7d, 14d get the rat broken end in batches and get brain, the cerebral tissue of ischemic focus half penumbra area or same area with the neutral PBS buffer of 4% paraformaldehyde conventionally fix, dehydration, paraffin embedding, section, every thick about 5um.SABC method CD31 immunohistochemical staining, by SABC test kit description operation; The DAB colour developing.With PBS, replace primary antibodie to make negative control.With the appearance of erythrocyte or tube chamber, whether do not count blood vessel.All single endotheliocytes of dying brown color or endotheliocyte are bunch all as a vascular counts, and all tube chambers are greater than 8 erythrocyte sizes, all do not count with the angiosomes blood vessel of thicker flesh layer.Vascular counts is undertaken by the Weidner method.First under low power (* 40) visual field, find high vessel density zone, then carry out Microvessel Count under high power (* 400) visual field, 10 high power fields are chosen in every section at random, finally get the microvessel density value (MVD) of its meansigma methods as this specimen.And adopt the full automatic colour image processing system to carry out graphical analysis, measure blood vessel field Area Ratio (Positive Objects area/Statistical Fields gross area).

Table 34. respectively organizes that rat cerebral tissue's blood vessel scene is long-pending, the immune positive microvessel density of CD31 (MVD) value (n=6)

Annotate: with sham operated rats, compare,

#P<0.01; Compare * P<0.01, ☆ P<0.05 with model group

Table 34 experimental data shows, with sham operated rats, compares, and 3d after the model group ischemia, long-pending obviously reduce (P<0.05) of ischemia penumbra MVD and blood vessel scene, after ischemia, 7d, 14d still continue reduction (P<0.05 or P<0.01); With model group, compare, after the ischemia treatment, each dosage group of nimodipine group and salvianolic acid A drop pill and salvianolic acid monomeric compound group be 3d cerebral tissue ischemia penumbra MVD and long-pending increase to some extent (P<0.05 or P<0.01) of blood vessel scene after ischemia, and, at ischemia 7d to 14d after this, maintain a metastable level.Three kinds of different pharmaceuticals relatively, the most remarkable with salvianolic acid A drop pill effect.Relatively, with the high dose group best results, be the dose-difference opposite sex (P<0.05) between three dosage groups of salvianolic acid A drop pill.Result shows that the salvianolic acid A drop pill can increase MVD and the blood vessel field Area Ratio of cerebral tissue ischemia half blanking bar, and prompting salvianolic acid A drop pill can promote the effect that Angiogenesis and collateral circulation are set up, and effect is better than salvianolic acid A monomeric compound and nimodipine.

Experimental example 21: the salvianolic acid A drop pill on RHRSP cerebral thrombosis ischemia after the impact of rat cerebral tissue vascular endothelial growth factor expression

Modeling and grouping, dosage be with experimental example 16, every day gavage once, continue to and draw materials.Respectively at the postoperative 3d of rat cerebral ischemia, 7d, 14d, each component is criticized and is got rat abdominal cavity anesthesia, opens breast and exposes heart, liquid-solid fixed containing 4% paraformaldehyde of 0.1%DEPC through the heart perfusion, gets rapidly brain, in forebrain optic chiasma place, does the crown section that about 2mm is thick.Be placed in 4% paraformaldehyde fixative and spend the night, conventional dehydration, paraffin embedding, be cut into the paraffin section that about 5um is thick continuously, and routine dewaxes to water, detects the expression of VEGF Messenger RNA (VEGFmRNA) with hybridization in situ.Illustrate and operated by VEGF in situ hybridization test kit (Wuhan Boster Biological Technology Co., Ltd.), with microscopic image analysis software, carry out optical density (IOD) analysis.

The impact that table 35. salvianolic acid A drop pill is expressed RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia tissue VEGF

Annotate: compare #P<0.05 with sham operated rats; Compare * P<0.01, ☆ P<0.05 with model group

VEGF (VEGF), be again the blood vessel opsonin, has the effect of short endothelial cell division, can promote the growth of blood vessel and the foundation of side Zhi Xunhuan.Experimental result shows; model group VEGFmRNA expression is increased significantly (P<0.05) than sham operated rats; illustrate after the cerebral tissue hypoxic-ischemic can Stimulation of The Brain tissue in the expression increase of VEGF; after the prompting cerebral infarction, body self there will be a kind of protective response that resists ischemia injury; the expression of VEGF is increased, self physiological reaction of " compensatory revascularization " occurs after ischemia.And during this period in medication therapy groups, to ischemia 3d, high, the middle dosage group of salvianolic acid A drop pill, salvianolic acid A monomeric compound group VEGFmRNA expression are than model group obviously raise (P<0.05), salvianolic acid A drop pill low dose group and nimodipine group are expressed and are increased than model group VEGFmRNA, but there was no significant difference; To ischemia 7d, 14d, each medication therapy groups VEGFmRNA expresses all to be had in various degree and increases (P<0.05 or P<0.01) than model group; And the salvianolic acid A drop pill has the trend that is better than same dosage salvianolic acid A monomeric compound.Prompting salvianolic acid A drop pill can significantly promote the ischemic tissue of brain angiogenesis, promotes the foundation of collateral circulation compensation, saves ischemia half blanking bar, and, to anti-cerebral ischemia, and there is certain dose-effect relationship in the brain tissue impairment that the protection ischemia causes.

Experimental example 22: the salvianolic acid A drop pill on RHRSP cerebral thrombosis ischemia after the impact of rat cerebral tissue basic fibroblast growth factor protein expression

Modeling and grouping, medication be with experimental example 20, and respectively at 3d, 7d, 14d after rat cerebral ischemia, each component criticizes that to get the rat heart perfusion liquid-solid fixed containing 4% paraformaldehyde of 0.1%DEPC, gets rapidly brain, the crown section of row, paraffin embedding, section.The ABC immunohistochemic al technique method detects the expression of cerebral tissue alkalescence fibroblast growth factor (bFGF) albumen.By bFGF protein immunization group detection kit (Wuhan Boster Biological Technology Co., Ltd.) description, operated, micro-Microscopic observation, counting is dyed the positive cell number of brown color, and all visuals field of 5 infarcts are selected in each section at random, average.Data are carried out statistical analysis.

Table 36. salvianolic acid A drop pill on RHRSP cerebral thrombosis ischemia after the bFGF of rat cerebral tissue protein expression impact (x ± s)

Annotate: compare #P<0.05 with sham operated rats; Compare * P<0.01 with model group

BFGF is the strong neurotrophic factor with various biological activity, can neuroprotective unit to multiple detrimental effects such as ischemia resisting, anoxia, toxicity of excitatory amino acid, calcium overload, free radical and nitric oxide (NO) are synthetic, slow down neuronal apoptosis and necrosis; And can work in coordination with the effect that VEGF promotes angiogenesis in infarcted region.

Experimental result shows; after ischemia occurs; the bFGF of model group rat cerebral tissue protein expression raises to some extent than sham operated rats; to ischemia 7d, 14d, significant difference (P<0.05) is arranged; after prompting cerebral tissue hypoxic-ischemic; body self produces a kind of protection stress, but Stimulation of The Brain organizes the endogenous bFGF protein expression to increase.Relatively, the bFGF protein expression of each time period all significantly increases (P<0.01) for nimodipine group, each dosage group of salvianolic acid A drop pill and salvianolic acid A monomeric compound group and model group; With salvianolic acid A drop pill effect optimum.Between three dosage groups of salvianolic acid A drop pill, relatively, difference has statistical significance (P<0.05); Each time period of each dosage group self relatively shows, the bFGF protein expression is continual and steady; Prompting salvianolic acid A drop pill can increase former of ischemic tissue of brain and secondary bFGF protein expression; effect with good neurocyte protection effect and promotion angiogenesis; contribute to the foundation of collateral circulation; save ischemia half blanking bar, and action effect is better than salvianolic acid A monomeric compound and nimodipine.

Experimental example 23: the impact of salvianolic acid A drop pill on the rats after cerebral ischemic reperfusion nervous symptoms

Male SD rat (250 ± 20) g, be divided at random 7 groups: sham operated rats, ischemia-reperfusion injury model matched group, the high, medium and low dosage of salvianolic acid A drop pill (60mg/kg, 30mg/kg, 15mg/kg) group, salvianolic acid A monomeric compound group (30mg/kg), nimodipine matched group (30mg/kg).Adopt the standby rat brain focal cerebral ischemia-reperfusion injury model of improvement line bolt legal system: male SD rat, pentobarbital sodium intraperitoneal anesthesia.Expose and separate left carotid, external carotid artery, internal carotid artery and arteria pterygopalatina, ligation external carotid artery distal end, arteriole folder folder closes common carotid artery, the tie wings arteria palatina, cut a kerf in the nearly common carotid artery crotch of external carotid artery, head end is scribbled to the fishing line that smooth paraffin diameter is 0.3mm and through left side external carotid artery trunk otch, slowly to internal carotid artery, enter the propelling of cranium direction, take the common carotid artery crotch as labelling, while advancing 18～20mm to feel slight resistance, block middle cerebral artery and cause cerebral ischemia.Fishing line stays about 1cm in order to pouring into use, sterilization, skin suture outward again.After continuous ischemia 2h, extract the bolt line, realize ischemia-reperfusion injury model.Sham operated rats except plug wire not, the same model group of all the other operating process.Each group starts gastric infusion in first 7 days in modeling, once a day.Sham operated rats and ischemia-reperfusion injury model matched group are filled with the normal saline with same volume.

After cerebral ischemic reperfusion in rats, 1d, 3d, 7d respectively carry out neurological deficits score one time respectively, and scoring adopts the Bederson improved method: carry an approximately chi of Mus tail built on stilts, observe forelimb flexing situation, stretch to ground as two forelimb symmetries and count 0 minute; As the offside forelimb of performing the operation occurs that the flexing of wrist flexing meter 1 minute, elbow flexing meter 2 minutes, shoulder inward turning meter 3 minutes, existing wrist elbow has again shoulder inward turning meter 4 minutes.Rat is placed on level land, pushes away respectively both shoulders to side shifting, check resistance, as symmetrical as the bilateral resistance and strong, be designated as 0 minute; Resistance descender when promoted to the operation offside, according to decline degree difference be divided into gently, in, severe, count respectively 1,2,3 minute.Rat two forelimbs are placed on wire netting, observe the muscular tension of two forelimbs, bilateral muscular strength equity and strong person count 0 minute, operation offside muscular tension decline degree is divided into gently, in, severe, count respectively 1,2,3 minute.Rat does not stop, to a side person of turn-taking, to count 1 minute.According to above standards of grading, full marks are 11 minutes, and mark is higher, illustrate that the behavior disorder degree of rat is more serious, neurologic impairment is more serious.Concrete data are in Table 37.

The impact (x ± s, n=12) of table 37. salvianolic acid A drop pill on the rats after cerebral ischemic reperfusion neurological deficits score

Annotate: compare * P<0.05, * * P<0.01 with model group

Result of the test is known, ischemia-reperfusion injury model group function of nervous system serious defect, last till again fill with after 7d, although more again after perfusion behavioristics's obstacle of 1d significantly alleviate (P<0.05), neurologic impairment is serious (neurological deficits score still is greater than 5) still.Each dosage group of nimodipine group and salvianolic acid A drop pill is compared with model group, from pouring into again 1d, the neurologic impairment sx↓, neural row is learned damaged mark and is significantly reduced (P<0.05 or P<0.01), to pouring into 7d again, the neurobehavioral of each medication therapy groups rat all extremely significantly improves (P<0.01) than model control group.Between each dosage group of salvianolic acid A drop pill: low dose group and high dose group scoring relatively have statistical significance (P<0.05); High dose group has the trend that is better than middle dosage group effect, middle dosage group effect obvious than low dose group, but difference does not have statistical significance (P>0.05).Each time period neurologic defect symptom of salvianolic acid A drop pill group rat will be lighter than the salvianolic acid A monomeric compound group of same dosage.

Experimental result shows that the salvianolic acid A drop pill can alleviate rats after cerebral ischemic reperfusion neurologic impairment symptom; be conducive to its neurobehavioral recovery; prompting salvianolic acid A drop pill has the effect that improves nervous symptoms after brain tissue impairment; energy prevention and protection cerebral ischemia reperfusion cause the damage of rat brain function of nervous system, and action effect is more obvious than salvianolic acid A monomeric compound.

Experimental example 24: the impact of salvianolic acid A drop pill on rats after cerebral ischemic reperfusion cerebral infarction scope, cerebral index and brain water content

Experimental example 23 is respectively organized rat the last time after neuroethology scoring, get 6 broken ends for every group and get brain, be placed in immediately-20 ℃ of refrigerator freezing 10min, after removing olfactory bulb, cerebellum and low brain stem, the cerebral tissue continuous coronal section of row 2mm thickness, immerse the brain sheet in 2%TTC solution, 37 ℃ of dyeing 10min, take out, be placed in fixedly 2h of 4% paraformaldehyde PBS buffer.Normal structure is dyed redness, and infarct is white in color.Fixing brain sheet is sequentially arranged by section, before and after the brain sheet of taking pictures under microscope, behind two sides, inputted computer, calculate the approximation of front brain volume and Infarction volume according to each slice thickness, show that Infarction volume accounts for the percentage ratio of front brain volume.

Each claims the cutaneous horn weight after organizing other 6 rats broken end and getting brain immediately, in 105 ℃ of baking ovens, dries to constant weight, calculates brain water content and cerebral index.

The impact (x ± s, n=6) of table 38. salvianolic acid A drop pill on rats after cerebral ischemic reperfusion cerebral infarction volume ratio, cerebral index, brain water content

Annotate: with model group, compare * p<0.05, * * p<0.01

The experimental data demonstration, each dosage group of salvianolic acid A drop pill is compared with the ischemia-reperfusion injury model group: cerebral infarction volume ratio, cerebral index and brain water content all obviously reduce (P<0.01 or P<0.05); Salvianolic acid A drop pill low dose group and nimodipine group be no difference of science of statistics relatively; Relatively, its Infarction volume and cerebral index and brain water content all significantly reduce (P<0.05) for the middle and high dosage group of salvianolic acid A drop pill and nimodipine group, and lower than same dosage salvianolic acid A monomeric compound group; Show that the salvianolic acid A drop pill can reduce rats after cerebral ischemic reperfusion cerebral tissue infarction size, can alleviate the cerebral tissue edema degree after cerebral ischemia reperfusion injury, effect is better than salvianolic acid A monomeric compound and nimodipine.

Experimental example 25: the impact of salvianolic acid A drop pill on rats with cerebral ischemia cerebral tissue ischemia half blanking bar regional cerebral blood flow (rCBF)

1 test method

Animal is divided into sham operated rats, nimodipine group (30mg/kg), salvianolic acid A monomeric compound group (30mg/kg), the high, medium and low dosage group of salvianolic acid A drop pill (60mg/kg, 30mg/kg, 15mg/kg) at random.With the relative medicine gavage once sham operated rats and model group give the normal saline of same volume every day for each treated animal.Successive administration 7d, and 30min after administration the last time, adopt the standby rat brain of improvement line bolt legal system focal ischemia model: male SD rat, pentobarbital sodium intraperitoneal anesthesia.Expose and separate left carotid, external carotid artery, internal carotid artery and arteria pterygopalatina, ligation external carotid artery distal end, arteriole folder folder closes common carotid artery, the tie wings arteria palatina, cut a kerf in the nearly common carotid artery crotch of external carotid artery, head end is scribbled to the fishing line that smooth paraffin diameter is 0.3mm and through left side external carotid artery trunk otch, slowly to internal carotid artery, enter the propelling of cranium direction, take the common carotid artery crotch as labelling, while advancing 18～20mm to feel slight resistance, block middle cerebral artery and cause cerebral ischemia.Fishing line stays about 1cm in order to pouring into use, sterilization, skin suture outward again.Sham operated rats except plug wire not, the same model group of all the other operating process.

Adopt Laser Doppler Flowmetry to measure rCBF through cranium.The laser-Doppler probe that the use cyanoacrylate is 0.5mm by diameter is close to skull surface and is fixed, selecting respectively after bregma the other 2mm that opens in 1mm center line right side under stereotaxic instrument is that after ischemia half blanking bar rCBF monitoring point, bregma other to open 6mm be rCBF monitoring point, ischemia center on 2mm center line right side, record blood flow, continue to monitor 2h after ischemia.

2 result of the tests

After middle cerebral artery occlusion in rat, model group ischemia center cerebral blood flow drop to normal rCBF before modeling 10%～20% between, ischemia half blanking bar rCBF drop to rCBF before modeling 30%～35% between (take sham operated rats as with reference to).After ischemia 30min, the ischemia penumbra rCBF of each medicine group rat cerebral tissue is than all existing risings in various degree of model group.Concrete experimental data statistical analysis is in Table 39.

The impact (x ± s, n=10) of table 39. salvianolic acid A drop pill on rats with cerebral ischemia continuous ischemia phase cerebral tissue ischemia half blanking bar rCBF

Annotate: with sham operated rats (baseline value), compare * P<0.01; With model group, compare,

3 interpretations of result

From table 39 data, after the operation modeling, model control group is compared with sham operated rats, cerebral tissue half blanking bar rCBF extremely significantly descend (p<0.01).After ischemia 10min, each medicine group rises to some extent than model group ischemia half blanking bar rCBF, but there is no statistical significance; After ischemia, 30min begins, and each medication therapy groups ischemia half blanking bar rCBF except salvianolic acid A drop pill low dose group and model group relatively, have raise 12%～30%, obvious difference (p<0.05); After ischemia, in 1～2h, each medicine group ischemia half blanking bar rCBF is all apparently higher than model group (p<0.05).Each dosage group of salvianolic acid A drop pill is compared, and is certain dose-effect trend; In the salvianolic acid A drop pill, the rCBF of dosage group day part is higher than the nimodipine group, and also slightly is better than same dosage salvianolic acid A monomeric compound.Hence one can see that, and the salvianolic acid A drop pill has the effect that increases rats with cerebral ischemia cerebral tissue ischemia half blanking bar rCBF, thereby is conducive to save the cerebral tissue that ischemia half blanking bar is at death's door, protection ischemia half blanking bar, the effect of performance treatment cerebral ischemia.

Experimental example 26: the impact of salvianolic acid A drop pill on rats after cerebral ischemic reperfusion cerebral tissue ischemia half blanking bar rCBF

Respectively organize rat in experimental example 25 after continuous ischemia 2h observation ischemia half blanking bar rCBF, extract the bolt line, realize ischemia-reperfusion injury model.After setting up re-perfusion model, still press experimental example 21 medications, every day gastric infusion once.Press the described method of experimental example 25 and measure filling rear 3h, 1d, 3d, 7d cerebral tissue ischemia half blanking bar rCBF.Data are carried out statistical analysis.Concrete data are in Table 40.

The impact (x ± s, n=10) of table 40. salvianolic acid A drop pill on rats after cerebral ischemic reperfusion cerebral tissue ischemia half blanking bar rCBF

Annotate: compare * P<0.01 with sham operated rats; Compare ☆ P<0.05, ★ P<0.01 with model group

The experimental result demonstration, after rat continuous ischemia 2h recovers to fill with again, each organizes rat ischemia half blanking bar rCBF all increase in various degree.The recovery of model group rat is filled with rear ischemia half blanking bar rCBF again and is recovered to fill with front than being increased significantly (P<0.01) again, but also only be about front below 50% of baseline value of ischemia, fill with again in 7d, the rCBF value before ischemia baseline value 40%～45% between, perfusion remains very incomplete more in the meantime, and it is further impaired that lasting low perfusion will increase the weight of cerebral tissue.After filling with, nimodipine, the high, medium and low dosage group of salvianolic acid A drop pill, salvianolic acid A monomeric compound group rat rCBF obviously increase again, and each time period rCBF compares with model group that all there were significant differences (P<0.05 or P<0.01).7d after filling with again, each medication therapy groups rCBF return to former rCBF 57%～67% between; Each dosage group of salvianolic acid A drop pill relatively, is certain dose-effect trend; The salvianolic acid A drop pill has the salvianolic acid A monomeric compound that is better than same dosage and the trend of nimodipine.The experimental result prompting, the salvianolic acid A drop pill can obviously improve the cerebral blood flow of ischemia half blanking bar cerebral tissue after Ischemia and Reperfusion in vivo in Rats, recovering brain cell blood supplies, thereby save ischemia half blanking bar, further damage is even dead to prevent cerebral tissue, and effect has the trend that is better than the salvianolic acid A monomeric compound, ischemic cerebrovascular is had to good therapeutical effect.

Experimental example 27: the impact of salvianolic acid A drop pill on the rats after cerebral ischemic reperfusion Energy Metabolism of Brain Tissue

Experimental example 26 each treated animals are after the cerebral blood flow of having measured again 7d after perfusion, and sacrificed by decapitation, get brain to freezing in liquid nitrogen; Get the cerebral tissue of 100mg corresponding site after one week, under freezing state, pulverize, remove albumen with perchloric acid homogenate, low-temperature centrifugation 10min, supernatant neutralizes with KOH, vortex oscillation, then low-temperature centrifugation 10min, get supernatant, high effective liquid chromatography for measuring cerebral tissue adenosine triphosphate (ATP), adenosine diphosphate (ADP) (ADP), AMP (AMP), lactic acid (LA), phosphagen (PC) content.

The impact (x ± s, μ mol/g, n=10) of table 41. salvianolic acid A drop pill on the rats after cerebral ischemic reperfusion Energy Metabolism of Brain Tissue

Annotate: compare * P<0.01 with sham operated rats; Compare * * P<0.01, ☆ P<0.05 with model group

ATP, ADP, AMP, LA, PC are the human body energy metabolite.By table 41 experimental data, can be found out, model group ATP, ADP, AMP, PC than sham operated rats significantly reduce, LA significantly raise (P<0.01), rat cerebral ischemia tissues following MCAO in rats energy metabolism serious hindrance is described, ATP exhausts fast, brain-capacity is supplied with significantly and is reduced, anaerobic metabolism product LA piles up serious, even after implementing to fill with, the energy metabolism disorder still exists again.And each administration group is compared with model group, cerebral tissue ATP content extremely significantly raises, LA significantly reduces (P<0.01), result shows can the raise content of cerebral tissue ATP, ADP, AMP, PC of salvianolic acid A drop pill, reduce cerebral tissue LA content, can significantly improve the energy metabolism of rat cerebral ischemia and ischemia-reperfusion.Prompting salvianolic acid A drop pill can be by improving the energy metabolism of Neurons Against Cerebral Ischemia tissue, increase the supply of cerebral tissue energy matter, strengthen the survival ability of brain cell, the brain cell that ischemia half blanking bar after the redemption cerebral ischemia is at death's door, further damage is even dead to prevent cerebral tissue, and action effect is obvious than salvianolic acid A monomeric compound and nimodipine, can perform well in treating ischemic cerebrovascular.

The impact of experimental example 28 salvianolic acid A drop pill on rats after cerebral ischemic reperfusion cerebral tissue neuronal apoptosis rate

Prepare rat pattern of ischemia reperfusion, grouping, administration with experimental example 23 methods.After rat cerebral ischemia 2h fills with 7d again, broken end is got brain, with 4% paraformaldehyde, fix, and section, paraffin embedding, prepare the thick crown section of thick 3 μ m; Adopt the transferase mediated dUTP breach end-labelling (TUNEL method) of last deoxyribonucleic eventually to detect the cerebral tissue neurocyte, strictly press the operation of test kit description, the DAB colour developing, under light microscopic, apoptotic nucleus is brown.Unduplicated 5 high powers (40 * 10) visual field is chosen in every section at random, and the input computer carries out image analysis, detects apoptotic cell number and the normal cell number of the TUNEL positive, calculates the neuronal apoptosis rate, results averaged.

The impact of table 42. salvianolic acid A drop pill on rats after cerebral ischemic reperfusion cerebral tissue neuronal apoptosis rate

Compare #P<0.001 with sham operated rats; Compare * * P<0.001, * P<0.01 with model group

Neuronal apoptosis is the principal mode of cerebral ischemia and transient ischemia/reperfusion damage delayed neuronal death, can determine the brain tissue impairment degree that ischemic cerebrovascular is final.The experimental result demonstration, after rat cerebral ischemia 2h fills with 72h again, neuronal apoptosis is serious; And after nimodipine, salvianolic acid A monomeric compound and the intervention of various dose salvianolic acid A drop pill, with model group, compare, in rat cerebral tissue, neuronal apoptosis significantly reduces (P<0.001 or P<0.01), and minimum with the apoptosis rate of high, the middle dosage group of salvianolic acid A drop pill; Show that the salvianolic acid A drop pill has the effect that suppresses cerebral ischemia rat cerebral tissue neuronal death, suppresses neuronal apoptosis, effect is better than salvianolic acid A monomeric compound and nimodipine.

The impact of experimental example 29 salvianolic acid A drop pill on rats after cerebral ischemic reperfusion cerebral tissue neurotrophic factor

Prepare rats after cerebral ischemic reperfusion model, grouping administration with experimental example 28 methods.Ischemia Reperfusion 7d is by aortic cannulation, normal saline flushing, after 4% paraformaldehyde perfusion, broken end is got brain, cerebral tissue is fixed, dehydration, paraffin embedding, section, thick approximately 5 μ m, Immunohistochemical Method detects neurotrophic factor nerve growth factor (NGF), Brain Derived Neurotrophic Factor (BDNF), neurotrophic factor-3 (NT-3) protein expression in rat cerebral tissue; Replace the negative contrast of primary antibodie with PBS; With endochylema, after birth, the aixs cylinder of cell be brown or brown yellow granule into immunity positive.Under light microscopic, observe, Cai Tu also uses the image analysis system analysis, measures its average gray value.Average gray is higher, means that the intensity of positive cell is more weak.

The impact of table 43. salvianolic acid A drop pill on rat cerebral tissue's neurotrophic factor protein expression (gray value)

With sham operated rats, compare, #P<0.05,

compare * P<0.05, △ P<0.01 with model control group.

The neurotrophic factor histone matter molecule essential with survival that be neure growth, play supporting function to the integrity of neure growth, growth and function.NGF, BDNF, NT-3 are the parts of neurotrophic factor family, can maintain neuronal survival and promote the neurocyte differentiation and induce axon growth; Energy neuroprotective unit, promotion neuron reparation and inhibition delayed neuronal death, thereby to anti-cerebral ischemia, protection cerebral ischemia.Experimental result shows, after Ischemia Reperfusion, rat cerebral tissue in, NGF, BDNF express and increase to some extent than sham operated rats, and after the prompting cerebral reperfusion injury, in cerebral tissue, express stress the protectiveness increase for NGF, BDNF; But in the Neurons Against Cerebral Ischemia tissue, NT-3 content obviously reduces.With model control group, compare, each dosage group of salvianolic acid A drop pill NGF, BDNF, NT-3 protein expression all obviously strengthen (P<0.05 or P<0.01), and the trend that is better than same dosage salvianolic acid A monomeric compound and nimodipine is arranged.Prompting salvianolic acid A drop pill can strengthen the expression of ischemic injuries cerebral tissue endogenous neurotrophic factor NGF, BDNF, NT-3, for neuronal survival provides condition, and reaches the effect of neuro-protective.

The impact of experimental example 30 salvianolic acid A drop pill on the rats after cerebral ischemic reperfusion inflammatory cytokine

Prepare rats after cerebral ischemic reperfusion model, grouping administration with experimental example 28 methods.Break end and get brain rapidly after Ischemia Reperfusion 7d, get ischemia side cerebral tissue and make 10% tissue homogenate, the centrifugal 10min of low temperature 2000r/min, get supernatant, use the content of each inflammatory cytokine of ELISA kit measurement: interleukin-1 ' beta ' (IL-1 β), interleukin-6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor (TNF-α), intercellular adhesion molecule-1 (ICAM-1).

The impact of table 44. salvianolic acid A drop pill on the rats after cerebral ischemic reperfusion inflammatory cytokine

Compare #P<0.001 with sham operated rats; Compare △ P<0.01, * P<0.05 with model group.

The experimental result demonstration, after ischemia-reperfusion, in cerebral tissue, inflammatory cytokine IL-1 β, IL-6, IL-8, TNF-α, ICAM-1 content all extremely significantly increase (P<0.001); Each inflammatory cytokine content of the rat cerebral tissue of each medication therapy groups obviously reduces (P<0.05 or P<0.01) than model group; Between each dosage group of salvianolic acid A drop pill, dose-effect relationship is obvious, and salvianolic acid A monomeric compound group and the nimodipine group with dosage is remarkable.Prompting salvianolic acid A drop pill can suppress the expression of ischemic injuries brain tissue inflammation cytokine, suppresses the generation of brain tissue inflammation's reaction, suppresses neuron and the cerebral tissue cascade damage of inflammatory reaction mediation, and effect is better than salvianolic acid A monomeric compound and nimodipine.

Experimental example 31: the salvianolic acid A drop pill is to rats after cerebral ischemic reperfusion cerebral tissue Ca ²⁺, K ⁺, Mg ²⁺the impact of content

Get experimental example 24 and dry the cerebral tissue to constant weight, after weighing, proceed to conical flask, use analytical pure nitric acid and perchloric acid in (200 ± 10) ℃ digestion, add the deionized water dissolving residue, after proceeding to color comparison tube standardize solution, with atomic spectrophotometer, survey Ca in cerebral tissue ²⁺, K ⁺, Mg ²⁺content.Data are carried out statistical analysis.

Table 45. salvianolic acid A drop pill is to rats after cerebral ischemic reperfusion cerebral tissue Ca ²⁺, K ⁺, Mg ²⁺the impact of content (x ± s)

Annotate: compare * P<0.01 with sham operated rats; Compare * P<0.05, * * P<0.01 with model group

Ca in cell ²⁺overload is one of encephaloclastic important mechanism of mediation Secondary cases in During Ischemia, is the last common pathway that ischemia, anoxia produce irreversible neuronal damage.Ca ²⁺overload can be by exciting activator protein enzyme as the second message,second messenger, activate phospholipase, activate a series of enzyme reactions such as endonuclease produces infringement to cell, and cell death inducing, cause acute cell death and Delayed onset neuronal necrosis.K ⁺, Mg ²⁺be Ca ²⁺antagonist.K ⁺as the requisite cation of neurocyte polarized state, can suppress calcium ion in flow to and alleviate calcium overload; Mg ²⁺can activate plurality of enzymes system in body, participate in the multiple important metabolic activity of cell in cerebral tissue, conduction, ion transport, the synthetic all many-sides of energy metabolism that reach of albumen affect the nerves, the spasm of energy alleviating vascular smooth muscle, improve microcirculation, improve hypoxic-ischemic after brain injury, calcium overload in retardance after craniocerebral injury neurocyte.From experimental data, cerebral ischemia re-pouring model group and sham operated rats be cerebral tissue Ca relatively ²⁺content extremely significantly increases, K ⁺, Mg ²⁺content significantly reduces (P<0.01); With model group, compare, each dosage group of nimodipine group, salvianolic acid A monomeric compound group and salvianolic acid A drop pill can significantly reduce Ca in cerebral tissue ²⁺content, rising K ⁺, Mg ²⁺content (P<0.05 or P<0.01), and the most remarkable with salvianolic acid A drop pill effect.Illustrate that the salvianolic acid A drop pill can suppress Ca ²⁺interior stream alleviates calcium overload, thereby can suppress Ca ²⁺the neuronal cell apoptosis that overload is induced, the damage of protection cerebral ischemic neuron.

Experimental example 32: the impact of salvianolic acid A drop pill on rats after cerebral ischemic reperfusion cerebral tissue neurotransmitter

1 experimental technique

1.1 grouping administration and model preparation

Male SD rat (250 ± 20) g, be divided at random 7 groups, every group 30: sham operated rats, cerebral ischemia re-pouring model control group, the high, medium and low dosage of salvianolic acid A drop pill (60mg/kg, 30mg/kg, 15mg/kg) group, salvianolic acid A monomeric compound group (30mg/kg), nimodipine matched group (30mg/kg).Adopt re-perfusion model (as described in experimental example 19) after the standby middle cerebral artery occlusion in rat 2h of line bolt legal system, sham operated rats except plug wire not, the same model group of all the other operating process.Each group 7d before operation starts gastric infusion, and once a day, sham operated rats and cerebral ischemia re-pouring model group give the normal saline of same volume.After filling with 7d again, each group is got 4 broken ends and is got brain and do histopathology section, and HE dyeing, observe histopathology and change.

1.2 the mensuration of rat cerebral tissue's monoamine neurotransmitters

Each organizes rat after ischemia 2h pours into 7d again, respectively get 8, broken end is got brain fast, separate cerebral cortex and striatum, weigh, add n-butyl alcohol homogenate, the centrifuging and taking supernatant is used fluorescent spectrophotometer assay 5-hydroxy tryptamine (5-HT), norepinephrine (NE), dopamine (DA) content after carrying.Data are carried out statistical analysis, the results are shown in Table 46.

1.3 the mensuration of rat cerebral tissue's content of amino acids neurotransmitter

Each organizes rat after ischemia 2h pours into 7d again; respectively get 8; broken end is got brain fast; sharp separation ischemia side cerebral cortex in ice bath; weigh; put in homogenizer; make homogenate with sulfosalicylic acid; centrifugal; get supernatant; dilution, after the OPA derivative reaction, detect the content of amino acid neurotransmitter glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), γ-aminobutyric acid (GABA), taurine (Tau) in brain tissue homogenate's supernatant by high performance liquid chromatography.Mobile phase is a certain proportion of potassium phosphate buffer and methanol, tetrahydrofuran solution, and pH is about 6.6, flow velocity 1ml/min.Calculate excitatory toxicity index (EI): EI=[Glu] [Gly]/[GABA].Data are carried out statistical analysis, the results are shown in Table 47.

2 experimental results

2.1 histopathology observed result

Sham operated rats structure of neurons, form be normal, without the interstitial edema; Ischemia-reperfusion group neuronal cell volume dwindles, distortion, karyopycnosis, and nervous tissue is loose, and neuron and perivascular space increase, and the interstitial cerebral edema is obvious; Each administration group is compared with model group, and cerebral edema obviously alleviates, and the neuronal cell form is obviously improved, and neuron peripheral clearance is obviously dwindled; Especially the most remarkable with high, the middle dosage group of salvianolic acid A drop pill, its neuronal cell is more complete, the form normal.

2.2 the impact of salvianolic acid A drop pill on monoamine neurotransmitter in the rats after cerebral ischemic reperfusion cerebral tissue

With sham operated rats, compare, cerebral ischemia re-pouring group rat cerebral cortex and striatum NE, DA, 5-HT content all significantly reduce (P<0.01).With the cerebral ischemia re-pouring model group, compare, each administration group rat cerebral cortex, striatum NE, DA, 5-HT content all have rising, except the rising of nimodipine group and salvianolic acid A drop pill low dose group rat striatum NE content does not have significant difference than model group, other each medication therapy groups all relatively has statistical significance (P<0.01 or P<0.05) than model group.

Table 46. salvianolic acid A drop pill is in the rats after cerebral ischemic reperfusion cerebral tissue

The impact of monoamine neurotransmitter (x ± s, ng/mg, n=8)

2.3 the impact of salvianolic acid A drop pill on amino acid neurotransmitter in the rats after cerebral ischemic reperfusion cerebral tissue

With sham operated rats, compare, after cerebral ischemia re-pouring 7d, in the model group rat cerebral cortex, the content of Glu, Asp, Gly all has remarkable increase (P<0.01); With model group, compare, in each administration group cerebral tissue, Glu, Asp, Gly all significantly reduce (P<0.01 or P<0.05), Tau content significantly raise (P<0.01 or P<0.05); Except the GABA content of salvianolic acid A drop pill low dose group and nimodipine group slightly raises than model group, difference does not have outside statistical significance, GABA content significantly raise (P<0.01 or P<0.05) in other each administration group cerebral tissue.Cerebral ischemia re-pouring model group EI value has extremely significantly and increases (P<0.01) than sham operated rats, and the EI value of each administration group and nimodipine group obviously reduces (P<0.01) than model group.

The impact (x ± s, n=8) of table 47. salvianolic acid A drop pill on amino acid neurotransmitter in the rats after cerebral ischemic reperfusion cerebral tissue

3 conclusions

Monoamine neurotransmitter disorder and toxicity of excitatory amino acid play an important role in ischemic brain injury, acute cerebral ischemia cell death, reperfusion injury and delayed neuronal death.During cerebral ischemia, monoamine neurotransmitters significantly raises in extracellular fluid, and reduces in brain essence.In cerebral tissue ischemia equivalent damage situation, monoamine neurotransmitter generation metabolism disorder in brain, NE, DA and 5-HT content obviously reduce, and cerebral ischemia is heavier, and cerebral tissue NE, DA, 5-HT content are just lower.Glu and Asp are the important excitatory neurotransmitters of brain, and GABA and Gly are the important inhibitory neurotransmitters of brain, and Tau, as a kind of neuromodulator, has protective effect in cerebral ischemia.During cerebral ischemia in brain amino acid neurotransmitters excited-to suppress unbalance be one of key factor of causing cerebral ischemia.This experimental result shows that the salvianolic acid A drop pill can obviously improve the cerebral cortex of cerebral ischemia-reperfusion injury in rats and the content of striatum monoamine transmitters, GABA, Tau content, the content that significantly reduces Glu in cerebral tissue, Asp, Gly and aminoacid exitotoxicity index in rising cerebral ischemia/reperfusion injury of rats cerebral tissue.And dosage increases, effect strengthens, and effect is obvious with dosage salvianolic acid A monomeric compound and nimodipine.The salvianolic acid A drop pill shown in conjunction with the histopathology pathological examination can obviously alleviate cerebral edema, improves the result of neuronal cell form, shows that the salvianolic acid A drop pill can suppress monoamine neurotransmitter and excessively discharge, and improves the monoamine neurotransmitter disorder; Suppress the accumulation of excitatory amino acid in cerebral ischemia re-pouring, stablize the balance of excitatory amino acid-inhibitory aminoacid mediator, alleviate toxicity of excitatory amino acid; Thereby suppress cerebral hypoxia ischemia and cause the neuronal cell apoptosis that monoamine neurotransmitter is disorderly and toxicity of excitatory amino acid is induced.

Experimental example 33: the impact of salvianolic acid A drop pill on LPO, SOD, GSH-Px in the rats after cerebral ischemic reperfusion cerebral tissue

10 remaining rats after each group detects under 32 of experimental examples, broken end is got brain, weigh, make brain tissue homogenate's liquid of 10% with 4 ℃ of normal saline, centrifugal, remove supernatant, measure the activity of lipid peroxide (LPO) content, superoxide dismutase (SOD) and glutathion peroxidase (GSH-Px).Result is carried out statistical analysis.

The impact (x ± s, n=10) of table 48. salvianolic acid A drop pill on LPO, SOD, GSH-Px in the rats after cerebral ischemic reperfusion cerebral tissue

In cerebral ischemia re-pouring model group rat cerebral tissue, LPO content significantly raises than sham operated rats, and SOD and GSH-Px activity significantly reduce (P<0.01) than sham operated rats.When Cerebral ischemia and reperfusion is described, the brain tissue oxygen free radical sharply increases, and the integrity of infringement membrane structure and function causes lipid peroxidation, produces a large amount of lipid peroxide; And the free radical scavenging enzymatic activity significantly lowers; Therefore the dynamic equilibrium heavy damage of free-radical generating and removing.Free radical toxicity chain reaction meeting accelerator nerve units apoptosis, increase the weight of cerebral ischemia.Experimental result shows, each dosage group of salvianolic acid A drop pill is compared with model group, and in rat cerebral tissue, LPO content significantly reduces (P<0.01 or P<0.05), and lower than the salvianolic acid A monomeric compound group of same dosage; SOD and GSH-Px are active significantly to raise (P<0.01), and the trend higher than same dosage salvianolic acid A monomeric compound group is arranged, and effect is obvious than the nimodipine group; Illustrate that the salvianolic acid A drop pill can increase the generation of the activity of peroxide scavenger enzyme in the ischemical reperfusion injury cerebral tissue, inhibition peroxide, thereby the damage of oxygen-derived free radicals to neuronal cell and cerebral tissue during to anti-cerebral ischemia, its antioxidation has the trend that is better than the salvianolic acid A monomeric compound.

Experimental example 34: the salvianolic acid A drop pill is protected RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia vascular endothelial cell

Prepare RHRSP Cerebrovascular embolism model, grouping with experimental example 16 methods.Each treated animal 3d before operation starts gastric infusion, once a day, after the 4th day gastric infusion 1h, starts the modeling of performing the operation.Postoperative still every day gavage once, continue to and draw materials.The high, medium and low dosage group of salvianolic acid A drop pill gavage salvianolic acid A drop pill 80mg/kg, 40mg/kg, 20mg/kg; Salvianolic acid A monomeric compound group gavage salvianolic acid A monomeric compound 40mg/kg, nimodipine group gavage nimodipine 40mg/kg.Sham operated rats, model control group gavage are with the volume normal saline; Each group respectively at cerebral ischemia after 3d, 7d, 14d get rat and pour into 4% paraformaldehyde neutral buffered solution through heart, get brain.Conventional fixing, the dehydration paraffin embedding of cerebral tissue, be cut into the thick crown section of 5um continuously.TUNEL method (the dUTP breach end-labelling of deoxyribose nucleotides terminal transferase mediation) detects vascular endothelial cell.Press the operation of TUNEL cell apoptosis detection kit (Wuhan doctor's moral biological reagent company) description, DAB colour developing, observed result under optical microscope.Nucleus the brown yellow granule the occurs positive apoptosis endotheliocyte of person.Each specimen random observation volume top and Striatum and Basal ganglia under * 400 times of mirrors have blood vessel but nonoverlapping 10 visuals field, calculate its TUNEL positive vessels endotheliocyte sum, i.e. apoptosis cell.Data are carried out the statistical analysis processing.

The impact (x ± s) of table 49. salvianolic acid A drop pill on RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia apoptosis of vascular endothelial cell

Annotate: compare #P<0.001 with sham operated rats; Compare * P<0.05, * * P<0.01 with model group

Experimental result shows, the cerebrovascular endothelial cell apoptosis number utmost point of each time period of model group is significantly higher than sham operated rats (P<0.001), 7d after the cerebral tissue ischemia, endothelial cell apoptosis than ischemia after 3d obviously reduce (P<0.05), but still far above sham operated rats.To 14d after ischemia, endothelial cell apoptosis still exists, and is not lighter than the apoptosis of 7d after ischemia.With model group relatively, the apoptosis digital display of each time period of each medication therapy groups work reduces (P<0.05 or P<0.01), and in the salvianolic acid A drop pill, dosage group apoptosis number is less than salvianolic acid A monomeric compound and the nimodipine group of same dosage.Prompting salvianolic acid A drop pill can suppress cerebral ischemia hindbrain apoptosis of vascular endothelial cell, improve the cerebrovascular endothelial cell survival rate, thereby guarantee the normal of cerebrovascular endothelial cell function, maintain the complete of ischemic injuries hindbrain blood vessel structure and function and strengthen the ability to anti-cerebral ischemia damnification, the effect of performance treatment ischemic cerebrovascular.

Experimental example 35: the impact of salvianolic acid A drop pill on anoxia and Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC) survival rate

The SD rat of the about 100g of male body weight, after broken end, get brain after alcohol disinfecting, peel off pia mater encephali and macroscopic trunk, remove cerebral white matter and cerebellum, thereafter, shred cerebral tissue, homogenate, cross successively 90,180 mesh filter screens and filter, rinse and collect the blood capillary section on filter screen, add the 0.1%II collagenase, 37 ℃ of digestion 15min, the centrifugal 5min of 1500r/min, repeat 3 times, precipitation is inoculated in culture bottle and continues to cultivate after suspending with culture fluid, every 2d, changes liquid 1 time.Treat that cell grows up to the fusion state, identify through Morphological Identification and VIII factor SABC, be defined as Brain Microvascular Endothelial (BMVEC), purity is greater than 98%.The cultivation of being gone down to posterity.Get third generation BMVEC, make single cell suspension after digestion, be inoculated in 96 orifice plates.Experiment divides 8 groups: model group, 6 dosage groups of salvianolic acid A drop pill (final concentration is respectively 10,20,30,40,50,60 μ mol/L), salvianolic acid A monomeric compound group (final concentration 30 μ mol/L) and nimodipine group (final concentration is 30 μ mol/L).After the cell inoculation, second day, change culture fluid into PBS liquid, is positioned over (37 ℃, 95%N in the anoxia tank ₂, 5%CO ₂) effect 4h, cause BMVEC anoxia-induced apoptosis (simulated ischemia); Change again common culture fluid into and normally cultivate 12h, cause BMVEC Hypoxia/Reoxygenation Injury (simulated ischemia-fill with again).Each dosage group of nimodipine group, salvianolic acid A monomeric compound group and salvianolic acid A drop pill all adds respectively the medicine of respective concentration when 4h, anoxia and reoxygenation before modeling.Detect with mtt assay the survival rate of respectively organizing BMVEC respectively after anoxia-induced apoptosis 4h, anoxia 4h-reoxygenation 12h damage.

The impact (x ± s, n=6) of table 50. salvianolic acid A drop pill on the BMVEC survival rate of anoxia and Hypoxia/Reoxygenation Injury

Compare #P<0.05, * P<0.01 with model control group; With comparison before reoxygenation, △ P<0.05, ☆ P<0.01

Experimental result shows, after anoxia 4h damage, the BMVEC survival rate is only 27.58% left and right, cell injury occurred after anoxia is described; The survival rate of nimodipine group 30 μ mol/L and salvianolic acid A monomeric compound 30 μ mol/L and salvianolic acid A drop pill 10,20,30 μ mol/L processed group and the trend that model group relatively has rising, but significant difference do not shown; After salvianolic acid A drop pill 40,50,60 μ mol/L dosage group anoxia-induced apoptosis 4h, the BMVEC survival rate is significantly higher than model control group (P<0.05).After Hypoxia/Reoxygenation Injury, the BMVEC survival rate is only 14.88% left and right, with comparison before reoxygenation, significantly reduces (p<0.01), illustrates that hypoxia/reoxygenation has aggravated cell injury; After each drug effect, after Hypoxia/Reoxygenation Injury, the BMVEC survival rate significantly raises (P<0.01), with salvianolic acid A drop pill 30,40,50,60 μ mol/L dosage group best results; And salvianolic acid A drop pill 20,30,40,50,60 μ mol/L dosage group survival rates are significantly higher than nimodipine 30 μ mol/L group (P<0.05, or P<0.01), salvianolic acid A drop pill 30 μ mol/L dosage groups are also higher than the cell survival rate of the salvianolic acid A monomeric compound group with dosage.Prompting salvianolic acid A drop pill can be protected microvascular endothelial cells oxygen and Hypoxia/Reoxygenation Injury; strengthen its hypoxia-bearing capability; improve the brain microvessel endothelial cells in vitro survival rate of anoxia-induced apoptosis and Hypoxia/Reoxygenation Injury, and be better than nimodipine and salvianolic acid A monomeric compound.

The impact of experimental example 36 salvianolic acid A drop pill on Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC) apoptosis

Method by experimental example 35: third generation BMVEC is inoculated in 96 orifice plates, experiment divides 7 groups: Normal group, model group, the high, medium and low dosage group of salvianolic acid A drop pill (final concentration 40,20,10 μ mol/L), salvianolic acid A monomeric compound group (final concentration 20 μ mol/L), nimodipine group (final concentration 40 μ mol/L).Modeling and medication are with experimental example 35.Normal group does not carry out the hypoxia/reoxygenation processing, by normal method, cultivates corresponding experimental period.Each porocyte of trypsinization, centrifugal collecting cell, adopt phosphatidyl in conjunction with albumen-Fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) dyeing, uses the cells were tested by flow cytometry cell in early days and the apoptosis rate in late period.

Table 51. salvianolic acid A drop pill causes the impact (x ± s, n=6) of BMVEC apoptosis on Hypoxia/Reoxygenation Injury

Compare #P<0.001 with Normal group; Compare * P<0.01, * * P<0.001 with model group; With the nimodipine group, compare, △ P<0.01,

Apoptosis especially plays an important role at ischemia injury in the transient ischemia/reperfusion damage.The apoptosis of vascular endothelial cell can cause the destruction of vascular integrity, and the angiolysis damage is the basis of cerebrovascular; The apoptosis of brain microvessel endothelial cells in vitro can affect the integrity of blood brain barrier and the secretory function of vascular endothelial cell, and then increases the weight of ischemic brain injury.By experimental result, shown, Hypoxia/Reoxygenation Injury, can cause each phase apoptosis rate of BMVEC significantly to increase.With model group relatively, each medicine group all can significantly lower cell early, late period and total apoptosis rate (P<0.01); Between each dosage group of salvianolic acid A drop pill and be certain dose-effect relationship.And salvianolic acid A drop pill 10 μ mol/L dosage groups are suitable with nimodipine 40 μ mol/L dosage group effects, with the salvianolic acid A drop pill of dosage, be better than the salvianolic acid A monomeric compound.Prompting salvianolic acid A drop pill has the effect of the BMVEC apoptosis that good anti-hypoxia-reoxygenation injury causes.

Experimental example 37 salvianolic acid A drop pill affect Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC) secretory function

As described in experimental example 35, third generation BMVEC is inoculated in 96 orifice plates to preparation BMVEC hypoxia/reoxygenation model.Grouping and administration are with experimental example 32.The collecting cell supernatant, the ELISA method detects the impact of salvianolic acid A drop pill on BMVEC secretory tissue fiber proenzyme activator (t-PA), tissue plasminogen activator's inhibitor (PAI), nitric oxide (NO), Endothelin (ET).

Table 52. salvianolic acid A drop pill is on Hypoxia/Reoxygenation Injury BMVEC secretory function impact (x ± s, n=6)

With Normal group, compare, #P<0.01,

compare * P<0.01, △ P<0.05, * * P<0.001 with model group

After Hypoxia/Reoxygenation Injury, with normal group, compare, BMVEC secretion t-PA and NO obviously reduce, and ET raises, and t-PA/PAI and NO/ET ratio also significantly descend.The t-PA of each medicine group and NO secretory volume significantly raise (P<0.01 or P<0.001) than model group, and wherein the middle and high dosage group of salvianolic acid A drop pill effect is especially remarkable; Each administration group t-PA/PAI and NO/ET ratio also significantly improve than model group.Prompting salvianolic acid A drop pill can improve the secretory function of brain microvessel endothelial cells in vitro after Hypoxia/Reoxygenation Injury, and action effect significantly is better than same dosage nimodipine, and the trend that is better than same dosage salvianolic acid A monomeric compound is arranged.

The impact of experimental example 38 salvianolic acid A drop pill on the interior Ca2+ concentration of Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC)

As described in experimental example 35, third generation BMVEC is inoculated in 96 orifice plates to preparation BMVEC hypoxia/reoxygenation model.Grouping and administration are with experimental example 36.Each porocyte of trypsinization, after centrifugal collecting cell, add Fluo-3 (a kind of calcium fluorescent probe, final concentration is 5 μ mol/L), hatches 45min for 37 ℃, and PBS liquid rinses 3 times, uses the cells were tested by flow cytometry fluorescence intensity.Almost there is no fluorescence while existing with the free ligand form because of Fluo-3, and and Ca ²⁺strengthen in conjunction with rear fluorescence, can detect Cytoplasmic Ca according to fluorescence intensity change ²⁺concentration change.

Table 53. salvianolic acid A drop pill is to Ca in Hypoxia/Reoxygenation Injury BMVEC ²⁺the impact of concentration (x ± s, n=6)

Compare #P<0.01 with Normal group; Compare * P<0.01 with model group; With the nimodipine group, compare,

The experimental result demonstration, after Hypoxia/Reoxygenation Injury, Ca in BMVEC born of the same parents ²⁺concentration significantly increases (P<0.01).Compare each medicine group Ca with model group ²⁺concentration extremely significantly reduces (P<0.01), and the middle and high dosage group of salvianolic acid A drop pill intracellular calcium concentration is starkly lower than nimodipine 40 μ mol/L dosage groups (P<0.05), and is better than the salvianolic acid A monomeric compound of same dosage.Prompting salvianolic acid A drop pill can suppress Hypoxia/Reoxygenation Injury and cause Ca in BMVEC born of the same parents ²⁺concentration raises, and the protection hypoxia/reoxygenation is to the damage of BMVEC.

Experimental example 39: the impact of salvianolic acid A drop pill on the rat artery thrombus formation time

Get the SD rat, male and female half and half, body weight is 250～300g, is divided at random matched group, salvianolic acid A monomeric compound group (30mg/kg), aspirin group (30mg/kg) and the high, medium and low dosage of salvianolic acid A drop pill (60,30,15mg/kg) group.Each organizes gastric infusion 1 time (matched group gives the isometric(al) normal saline), continuously after 10d, 1h after the last administration, pentobarbital sodium (40mg/kg) anesthetized rat, dorsal position is fixed on the Mus plate, the cervical region median incision, separate the about 15mm of right carotid, the stimulating electrode and the temperature sensor probe that thrombus in vivo are formed to analyzer are placed on common carotid artery, stimulating electrode is positioned at proximal part, with 2mA galvanism blood vessel 7min, makes the tunica intima damage, record is because of the time of thrombosis blocking blood flow in arterial lumen, i.e. thrombus formation time (OT).Experimental data statistical result is in Table 54.

The impact of table 54. salvianolic acid A drop pill on the rat artery thrombus formation time

Annotate: compare #P<0.05, * P<0.01 with the normal saline group

Experimental result shows, compares the thrombus formation time significant prolongation (P<0.05 or P<0.01) of salvianolic acid A drop pill 60,30,15mg/kg dosage group with the normal saline matched group; Middle dosage group thrombus formation time and 30mg/kg aspirin group be (P>0.05) quite; Middle dosage group extends than the salvianolic acid A monomeric compound group thrombus formation time with dosage; Between high, medium and low dosage group, thrombus formation time has significant difference (P<0.05).Show that the salvianolic acid A drop pill can extend thrombus formation time, the formation of prevention of arterial thrombosis, pre-preventing thrombosis and the ischemic cerebrovascular that causes.

Experimental example 40: the salvianolic acid A drop pill is to thrombotic inhibitory action

The SD rat, male and female half and half, grouping, administration are with experimental example 39.1h pentobarbital sodium anesthesia after the last administration, dorsal position is fixed, and right common carotid artery and left external jugular vein are isolated in operation, with three sections polyethylene tubes, connect.Put into the long 5cm operation silk thread of having weighed in the polyethylene tube stage casing.Be full of polyethylene tube with heparin-saline solution (5u/mL).One end of pipe inserts left external jugular vein, and the other end is connected with right common carotid artery.Open bulldog clamp, blood returns to left jugular vein by the right carotid polyethylene tube of flowing through.Herba Clinopodii in after open blood flow 15min, take out rapidly silk thread, and filter paper sucks supernatant blood, weighs, and gross weight subtracts line and heavily obtains wet weight of thrombus; Then put in 60 ℃ of baking ovens freeze-day with constant temperature to constant weight, weigh after cooling, be the thrombosis dry weight.Experimental data is in Table 55.

The inhibitory action that table 55. salvianolic acid A drop pill forms rat suppository

Annotate: compare #<0.05, * P<0.01 with the normal saline group

Experimental data shows, with the normal saline group, compares, and salvianolic acid A drop pill 60,30,15mg/kg dosage group rat suppository wet, dry weight all significantly alleviates (P<0.05 or P<0.01), between each dosage group, have dose-effect relationship preferably.And middle dosage group and 30mg/kg aspirin group effect be (P>0.05) quite.Prompting salvianolic acid A drop pill has the thrombotic effect of good inhibition artery-vein bypass, can or treat the ischemic cerebrovascular due to thrombosis for prevention, and than salvianolic acid A monomeric compound successful.

Experimental example 41: the impact of salvianolic acid A drop pill on the rat vein thromboembolism

Male SD rat, body weight (200 ± 20) g, the grouping administration is with experimental example 40,1h pentobarbital sodium (40mg/kg) anesthetized rat after the last administration, along abdominal part medisection rat stomach wall, open abdominal cavity, separate postcava, and, in left renal vein lower end level place ligation postcava, close abdominal cavity, closed abdominal cavity; Reopen abdominal cavity after 3h, in 2cm place, ligation below, folder closes blood vessel, the vein of ligation simultaneously side shoot, and blood vessel is cut in stringer open, exhausts the tube chamber inner blood, removal of thromboses, filter paper sops up residual blood, takes immediately wet weight of thrombus; After putting afterwards the interior freeze-day with constant temperature of 60 ℃ of baking ovens, claim the thrombosis dry weight.Test data is in Table 56.

The impact of table 56. salvianolic acid A drop pill on the rat vein thromboembolism

Annotate: compare #<0.05, * P<0.01 with the normal saline group

Experimental data shows, with the normal saline group, compare, salvianolic acid A drop pill 60,30,15mg/kg dosage group rat vein thrombosis wet, dry weight significantly alleviates (P<0.05 or P<0.01), and middle dosage group and 30mg/kg aspirin group effect be (P>0.05) quite; Middle dosage group is with the salvianolic acid A monomeric compound successful of dosage.Show that the salvianolic acid A drop pill has the effect that suppresses venous thrombosis, can be used for the venous thromboembolism after the prophylaxis of acute ischemic cerebrovascular occurs.

Experimental example 42: the salvianolic acid A drop pill is to the thrombotic inhibitory action of rats in vitro

The SD rat, 250～300g, male and female half and half; Random minute physiology saline group, salvianolic acid A monomeric compound group (30mg/kg), aspirin group (30mg/kg) and the high, medium and low dosage of salvianolic acid A drop pill (60,30,15mg/kg) group, each is organized the every day tail vein injection and gives and relative medicine 1 time, continuous 7 days; 30min after the last administration, pentobarbital sodium anesthesia, open abdominal cavity along ventrimeson, the about 1.5ml of abdominal aortic blood injects the silication sebific duct, by the docking of silica gel tube two ends circlewise, the silica gel sheath seal of tube is fixed rapidly, puts 37 ℃ of constant temperature rotation 15min on extracorporeal thrombosis forming device, removal of thromboses, measure thrombosis length, claim its weight in wet base; Survey its dry weight after drying in 60 ℃ of calorstats.Experimental data is in Table 57.

Table 57. salvianolic acid A drop pill is to the thrombotic inhibitory action of rats in vitro (x ± s)

Annotate: compare * P<0.01 with the normal saline group

Experimental data shows, with the normal saline group, compare, salvianolic acid A drop pill 60,30, the thrombotic length of 15mg/kg dosage group rats in vitro significantly shorten (P<0.05 or P<0.01), thrombosis is wet, dry weight significantly alleviates (P<0.01), and middle dosage group and 30mg/kg aspirin group effect be (P>0.05) quite; Experiment shows that the salvianolic acid A drop pill forms and has inhibitory action preferably external thrombus, and the trend that is better than same dosage salvianolic acid A monomeric compound is arranged.

Experimental example 43: the thrombolytic experimental study of salvianolic acid A drop pill to established thrombosis in body

SD male rat (250 ± 20) g, pentobarbital sodium intraperitoneal anesthesia rat, separate left common carotid artery with the unidirectional current continued stimulus rat artery of 2mA 7 minutes, with blood flowmeter continuous probe Flow of carotid artery.Reduce to 50% before stimulating with blood flow after stimulate finishing and be considered as thrombosis.Minutes 6 groups at random of animals, normal saline group, urokinase (3500U/kg) group, salvianolic acid A monomeric compound (40mg/kg) group and the high, medium and low dosage of salvianolic acid A drop pill (80,40,20mg/kg) group; Each group 7d before forming thrombosis starts continuous gastric infusion, and once a day, after thrombosis, the 7d same time of every day is surveyed a blood flow, observes the revascularization situation.The Flow of carotid artery of every animal be take stimulates front blood flow as baseline, with >=50% or≤25% stimulate before the blood flow person be judged to be continue to lead to again or continue after thromboembolism again; Each treated animal blood flow is divided into≤and 50%, 25%～50% and≤25% baseline values.According to blood flow, the carotid artery vascular degree of opening is divided three kinds of states, is respectively and 1. continues thromboembolism without logical again; 2. logical and thromboembolism is staggered again occurs; 3. continuous openness after leading to again, nothing is thromboembolism again.Experimental result is in Table 58.

Thrombolytic effect (n=10) in table 58. salvianolic acid A drop pill body

Annotate: 1. logical number of animals/animal number appears in recanalization rate=7d again

2. in the number of animals/7d of thromboembolism again that bolt rate=7d occurs again, logical number of animals appears again

3. compare * P<0.01 with the normal saline group; Compare #P<0.05 with the urokinase group

Result shows: the normal saline group all continues thromboembolism, and without logical phenomenon occurring again; The number of animals that each medicine group continues thromboembolism is few, compares with the normal saline group and utmost point significant difference is all arranged (P<0.01); Dosage in the salvianolic acid A drop pill (40mg/kg) group, its vessel open degree is similar to 40mg/kg salvianolic acid A monomeric compound group and 3500U/kg urokinase group, its recanalization rate is suitable, but its vessel open state has more stable trend than salvianolic acid A monomeric compound and urokinase group; The lasting recanalization rate of salvianolic acid A drop pill high dose (80mg/kg) group and recanalization rate are all higher than the urokinase group, and the bolt rate is starkly lower than urokinase group (P<0.05) again; Salvianolic acid A drop pill low dosage (20mg/kg) though the group recanalization rate lower than the urokinase group, its again the bolt rate lower than urokinase bolt rate again.The effect of thromboembolism again after the above results prompting salvianolic acid A drop pill has preferably thrombolytic and prevents thrombolytic, and effect is more stable.

The impact of experimental example 44 salvianolic acid A drop pill on the cerebral thrombosis hemorheology of rat

Adopt the method for carotid artery injection self thrombosis to prepare Cerebrovascular embolism model: after the intraperitoneal anesthesia of SD rat pentobarbital sodium, the neck median incision, separate the total tremulous pulse of right side strength (CCA), internal carotid artery (ICA), external carotid artery (ECA), ligation ECA far-end and arteria pterygopalatina, arteriole folder folder closes CCA and ICA, cut an osculum at the ECA near-end, unclamp CCA arteriole folder, extract arterial blood 0.5ml, the sodium citrate anticoagulant, centrifugal, getting platelet poor plasma adds a small amount of erythrocyte and thrombinogen and calcium chloride to mix, prepare the thrombosis that diameter is about 0.35mm, shred, the about 2mm folder of one trifle closes CCA, by ECA, embolus is injected to ICA, ligation ECA, unclamp CCA and arteria pterygopalatina place vascular clamp, sew up the incision.Experiment minutes 7 groups: sham operated rats, model control group, salvianolic acid A drop pill high, medium and low (60,30,15mg/kg) group, salvianolic acid A monomeric compound group (30mg/kg), nimodipine group (30mg/kg).Before modeling, 7d starts gastric infusion, once a day, and continuous 14d; Sham operated rats and the isopyknic normal saline of model group gavage.The second day (fasting 12h) that administration finishes, intraperitoneal anesthesia, abdominal aortic blood, anticoagulant heparin, carry out Determination of Blood Rheology.

Table 59. salvianolic acid A drop pill drips rheol impact (x ± s, n=8) to the cerebral thrombosis rat serum

Compare #p<0.01 with sham operated rats; Compare * P<0.01 with model control group,

The experimental result demonstration, after cerebral thrombosis, rat whole blood viscosity, plasma viscosity significantly increase, and packed cell volume is significantly rising (P<0.01) also; Each dosage group of salvianolic acid A drop pill and model group relatively, than nimodipine group more remarkable effect, and have the trend that is better than same dosage salvianolic acid A monomeric compound, and its whole blood viscosity and plasma viscosity and packed cell volume all obviously reduce; Prompting salvianolic acid A drop pill can be accelerated micro-blood flow velocity, and the reduction blood viscosity, improve hemorheology and effectively to resist the effect of cerebral thrombosis comparatively remarkable.

Claims

1. a salvianolic acid A drop pill is characterized in that: described salvianolic acid A drop pill comprises that each composition of wherein said drop pill is pressed column weight amount proportioning and made containing salvianolic acid A compositions, substrate: containing salvianolic acid A compositions 5%～60%, and substrate 95%～40%; Wherein saidly containing salvianolic acid A content in the salvianolic acid A compositions, be greater than 93%, be less than 100%, also comprise 4 other compositions: alkannic acid 0.1%～2%, rosmarinic acid 0.1%～2%, salvianolic acid B 0.1%～2%, salvianolic acid C 0.1%～2%, wherein said substrate is any one or a few in Macrogol 4000, polyethylene glycol 6000, PEG 8000, gelatin, stearic acid, glyceryl monostearate, insect wax and hydrogenated vegetable oil.

2. a kind of salvianolic acid A drop pill according to claim 1, each composition of wherein said drop pill is pressed column weight amount proportioning and is made: containing salvianolic acid A compositions 10%～40%, substrate 90%～60%, wherein saidly containing salvianolic acid A content in the salvianolic acid A compositions, be greater than 94%, be less than 99%, also comprise 4 other compositions: alkannic acid 0.1%～1.5%, rosmarinic acid 0.1%～1.5%, salvianolic acid B 0.1%～1.5%, salvianolic acid C 0.1%～1.5%.

3. the preparation method of the described a kind of salvianolic acid A drop pill of claim 1, comprise the steps:

(3) purification:

(5) drop pill preparation: get the salvianolic acid A compositions that step (4) obtains, mix homogeneously with substrate, splash in condensed fluid and become ball, be able to the drop pill of the unit dose of salvianolic acid A meter.

4. preparation method according to claim 3, wherein the water extracting method described in step (1) is: get red rooted salvia, be cut into decoction pieces or be ground into diameter 2mm granule, add 3～15 times of water gagings at every turn and extract, extract altogether 1～3 time, extract 1～4 hour at every turn; Extracting solution is evaporated to relative density 1.10～1.25 (60 ℃), add ethanol to make containing the alcohol amount 30%～80%, centrifugal, the supernatant decompression recycling ethanol also is concentrated into without the alcohol flavor, described water extraction adopts decoction to extract or 45～95 ℃ of water temperature lixiviates are got, stir with 10～50 rev/mins of speed simultaneously, obtain Radix Salviae Miltiorrhizae extract.

5. preparation method according to claim 3, wherein the alcohol extraction method described in step (1) is: get red rooted salvia, be cut into decoction pieces or be ground into diameter 2mm granule, add 3～15 times of amount 30%～60% alcohol reflux at every turn, the each extraction 1～4 hour, extract 1～3 time altogether; Decompression recycling ethanol, obtain Radix Salviae Miltiorrhizae extract.

6. preparation method according to claim 3, wherein in the extracting solution in step (1), salvianolic acid B concentration is 5mg/ml～20mg/ml or is diluted with water to 5mg/ml～20mg/ml.

7. preparation method according to claim 3, is characterized in that the catalyst in step (2) is one or more in iron chloride, ruthenium trichloride, aluminum chloride, zinc chloride, Palladous chloride..

8. preparation method according to claim 3, the molar percentage that it is characterized in that catalyst and salvianolic acid B is 0.5%～2.0%.

9. preparation method according to claim 3, wherein macroporous resin column described in step a is HPD-80, HPD-100, HPD-100B, HPD-200A, HPD-300, HPD-450, HPD-722, HPD-826, ADS-5, ADS-8, ADS-21 or D101, AB-8; The water that described eluant is water and different proportion and ethanol, and first water, 10～40% ethanol elutions, then use 20～60% ethanol elutions, high performance liquid chromatogram detects salvianolic acid A, collects and contains the salvianolic acid A part, and eluent is concentrated into without the alcohol flavor.

10. preparation method according to claim 3, water and ethanol that wherein the described eluant in step b is water and different proportion, and first water, 20～60% alcoholic solution eluting remove impurity, use again 40～90% alcoholic solution eluting, high performance liquid chromatogram detects salvianolic acid A, collection contains the salvianolic acid A part, and eluent is concentrated into without the alcohol flavor.

11. preparation method according to claim 3, wherein the organic solvent described in step c is t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate or Ethyl formate.

12. preparation method according to claim 3, the two-phase solvent that wherein eluant described in steps d is petroleum ether, pentane, normal heptane, ethyl acetate, methyl acetate, Ethyl formate, t-butyl methyl ether composition.

13. preparation method according to claim 3, the temperature of microwave vacuum drying: 20-100 ℃ described in step (4) wherein, return difference temperature 1-5 ℃, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.

14. preparation method according to claim 3, the temperature of microwave vacuum drying: 20-100 ℃ described in step (4) wherein, return difference temperature 1-5 ℃, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.

15. preparation method according to claim 3, wherein vacuum drying temperature described in step (4): 50 ℃～90 ℃, more than vacuum-0.07Mpa, power: 1～60KW, dry 2～20 hours.

16. preparation method according to claim 3, wherein spray-dired intake air temperature described in step (4): 150 ℃～350 ℃, the air outlet temperature: 70 ℃～95 ℃, spray velocity: 1～300ml/min.

17. preparation method according to claim 3, the unit dose of salvianolic acid A of wherein take in step (5) is 1mg～100mg, and preferred, unit dose is 5mg～80mg.

18. preparation method according to claim 3, wherein pH adjusting agent is phosphoric acid, hydrochloric acid, sulphuric acid or acetic acid.

19. the described salvianolic acid A drop pill of claim 1 or 2 any one is for the preparation of the purposes that prevents and/or treats the ischemic cerebrovascular medicine.

20., according to the purposes of claim 19, wherein said ischemic cerebrovascular mainly comprises any one or a few in atherosis or narrow, the lacunar infarction, Transient ischemic attacks, vascular dementia of cerebral thrombosis, cerebral ischemia reperfusion injury, cerebral embolism, cranium arteria carotis externa and brain basilar artery.

21., according to the purposes of claim 19 or 20, wherein said salvianolic acid A drop pill is for improving the purposes of the function of nervous system's symptom after cerebral ischemia.

22., according to the purposes of claim 19 or 20, wherein said salvianolic acid A drop pill is for the protection of the purposes of ischemic tissue of brain damage.

23., according to the purposes of claim 22, wherein said salvianolic acid A drop pill is for the protection of the purposes of blood brain barrier.

24., according to the purposes of claim 22, wherein said salvianolic acid A drop pill is for reducing the purposes of ischemic tissue of brain infarction size.

25., according to the purposes of claim 22, wherein said salvianolic acid A drop pill is for alleviating the purposes of ischemic tissue of brain edema.

26., according to the purposes of claim 19 or 20, wherein said salvianolic acid A drop pill is for saving the purposes of ischemia half blanking bar.

27., according to the purposes of claim 26, wherein said salvianolic acid A drop pill is for the purposes of the foundation that promotes the new life of cerebral vessels and collateral circulation.

28., according to the purposes of claim 27, wherein said salvianolic acid A drop pill is for increasing the purposes of cerebral tissue microvessel density.

29., according to the purposes of claim 27, wherein said salvianolic acid A drop pill is for promoting the purposes of vascular endothelial growth factor expression.

30., according to the purposes of claim 27, wherein said salvianolic acid A drop pill is for promoting the purposes of basic fibroblast growth factor protein expression.

31., according to the purposes of claim 26, wherein said salvianolic acid A drop pill is for increasing ischemia half blanking bar cerebral blood flow purposes.

32., according to the purposes of claim 26, wherein said salvianolic acid A drop pill is for improving the Energy Metabolism of Brain Tissue purposes.

33., according to the purposes of claim 19 or 20, wherein said salvianolic acid A drop pill is organized neuronal damage or dead purposes for suppressing Neurons Against Cerebral Ischemia.

34., according to the purposes of claim 33, wherein said salvianolic acid A drop pill is for suppressing the purposes of cerebral tissue neuronal apoptosis.

35., according to the purposes of claim 33, wherein said salvianolic acid A drop pill is for strengthening the purposes of cerebral tissue endogenous neurotrophic factor expression.

36., according to the purposes of claim 33, wherein said salvianolic acid A drop pill is for suppressing the purposes of brain tissue inflammation's damage.

37., according to the purposes of claim 33, wherein said salvianolic acid A drop pill is for suppressing Ca in the cerebral tissue neurocyte ²⁺the purposes of overload.

38., according to the purposes of claim 33, wherein said salvianolic acid A drop pill is for improving the purposes of monoamine neurotransmitter disorder in cerebral tissue.

39., according to the purposes of claim 33, wherein said salvianolic acid A drop pill is for suppressing the purposes of cerebral tissue toxicity of excitatory amino acid.

40., according to the purposes of claim 33, wherein said salvianolic acid A drop pill is for suppressing the purposes of brain tissue oxygen radical damage.

41., according to the purposes of claim 19 or 20, wherein said salvianolic acid A drop pill is for the protection of the purposes of cerebrovascular endothelial cell.

42., according to the purposes of claim 41, wherein said salvianolic acid A drop pill is for the protection of the purposes of Injury of Cerebral Microvascular Endothelial Cells.

43., according to the purposes of claim 19 or 20, wherein said salvianolic acid A drop pill is for preventing and/or treating the purposes of cerebral thrombosis.

44., according to the purposes of claim 43, wherein said salvianolic acid A drop pill is for suppressing thrombotic purposes.

45., according to the purposes of claim 43, wherein said salvianolic acid A drop pill is for thrombolytic purposes.

46., according to the purposes of claim 43, wherein said salvianolic acid A drop pill is for improving hemorheological purposes.