CN103083302A

CN103083302A - Application of salvianolic acid A composition in preparing medicines for protecting ischemic brain tissue damage

Info

Publication number: CN103083302A
Application number: CN2012104903380A
Authority: CN
Inventors: 李志勇; 刘地发; 曾发林; 钟阳桂; 蒋春红; 杨小玲
Original assignee: 蒋春红
Current assignee: JIANGXI QINGFENG PHARMACEUTICAL CO Ltd
Priority date: 2012-11-20
Filing date: 2012-11-20
Publication date: 2013-05-08
Anticipated expiration: 2032-11-20
Also published as: CN103083302B

Abstract

The invention relates to application of a salvianolic acid A composition in preparing medicines for protecting ischemic brain tissue damage. The composition is prepared from 94-97% of salvianolic acid A, 0.1-1.5% of alkannic acid, 0.1-1.5% of rosmarinic acid, 0.1-1.5% of salvianolic acid B and 0.1-2.0% of salvianolic acid C.

Description

The salvianolic acid A compositions is for the preparation of the purposes of protection ischemic tissue of brain damage medicine

Technical field

The present invention relates to the purposes of a kind of salvianolic acid A compositions for the preparation of protection ischemic tissue of brain damage medicine.

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 (Lubeluzole) 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, the salvianolic acid A structure is as follows:

But, adopt the existing related preparations of cardiovascular and cerebrovascular disease all to pursue simply purity or the content that improves salvianolic acid A in preparation, and ignored, salvianolic acid A is inquired in trial and whether other relevant composition combinations can play equally even better effect.

Yet 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.

Summary of the invention

The invention provides the purposes of a kind of salvianolic acid A compositions for the preparation of protection ischemic tissue of brain damage medicine; in wherein said compositions, salvianolic acid A 94%～97%; alkannic acid 0.1%～1.5%; rosmarinic acid 0.1%～1.5%; salvianolic acid B 0.1%～1.5%; salvianolic acid C 0.1%～2.0%, described salvianolic acid A compositions adopts lower preparation method to obtain:

Get red rooted salvia, be cut into decoction pieces or be ground into diameter 1mm～5mm granule, add 3～15 times of amounts, 45～95 ℃ of water temperature lixiviates are got at every turn, with 10～50 rev/mins of speed, stir simultaneously, or add 3～15 times of water gagings decoction extractions, and extract altogether 1～3 time, extract 1～4 hour at every turn; Extracting solution is evaporated to relative density 1.0～1.25 (60 ℃), adds ethanol to make to measure 50%～85% containing alcohol, standing, filters, and decompression filtrate recycling ethanol also is concentrated into without the alcohol flavor, obtains Radix Salviae Miltiorrhizae extract; Perhaps

Get red rooted salvia, be cut into decoction pieces or be ground into diameter 1mm～5mm 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 also is concentrated into without the alcohol flavor, obtains Radix Salviae Miltiorrhizae extract;

Above-mentioned Radix Salviae Miltiorrhizae extract was diluted with water to every 1ml containing salvianolic acid B 1～30mg, and adjusting PH with base to 3.5 for aqueous solution～6.5 add with salvianolic acid B molar percentage 0.1～3% zinc chloride as catalyst, 100～140 ℃ of temperature thermal conversions 1～6 hour;

Conversional solution adjust pH to 2.5～4.5, standing, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 1～10mg, through the HPD-100 macroporous resin column chromatography, separate, the salvianolic acid A applied sample amount is 1: 35～1: 70 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 4～1: 30, use respectively 1～8 times of cylinder hydrops, 1～10 times of column volume 10%～40% ethanol elution, remove impurity, use again 2～10 times of column volume 20%～60% ethanol elutions, HPLC detects, 20%～60% 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 1-10mg salvianolic acid A, by polyamide chromatography post, separate, the salvianolic acid A applied sample amount is 1: 5～1: 25 with the polyamide ratio, the resin column blade diameter length ratio is 1: 4～1: 25, use respectively 1～10 times of cylinder hydrops, 5～20 times of column volume 20%～60% alcoholic solution eluting remove impurity, use again 4～15 times of column volumes, 40%～90% alcoholic solution eluting, 40%～90% alcoholic solution part that collection contains salvianolic acid A, decompression recycling ethanol also is concentrated into without alcohol flavor aqueous solution;

Aqueous solution is concentrated, acid adjustment pH to 2.0～4.0, the t-butyl methyl ether of doubly measuring with aqueous solution 1-8, minute 2～6 extractions, separate organic layer, the reclaim under reduced pressure t-butyl methyl ether, make the extract of every 1ml containing salvianolic acid A 1g～10g, add 1～3 times of amount silica gel, stir, volatilize;

Stirring sample silica gel, be added on 5～20 times of dry silicagel columns of amount that installed, the silicagel column blade diameter length ratio is 1: 4～1: 25, take pentane-t-butyl methyl ether as eluant, gradient elution, use respectively 6～30 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 6～30 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 5～20 times of water gagings and dissolves, microwave vacuum drying, obtain described salvianolic acid A compositions.

Preferably, also comprise the purposes of protecting blood brain barrier.

Preferably, also comprise the purposes that reduces the ischemic tissue of brain infarction size.

Preferably, also comprise the purposes that alleviates the ischemic tissue of brain edema.

Preferably, wherein said compositions is to be made by following weight proportion: salvianolic acid A 95%～96%, alkannic acid 0.1%～1.2%, rosmarinic acid 0.1%～1.2%, salvianolic acid B 0.1%～1.2%, salvianolic acid C 0.1%～1.5%.

Preferably, microwave vacuum drying temperature: 20-100 ℃ wherein, return difference temperature 1-5 ℃, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.

Preferably, microwave vacuum drying temperature: 50-85 ℃, return difference temperature 2-4 ℃, more than vacuum-0.07Mpa, microwave power 10-80KW, dry 100-150 minute.

Preferably, microwave vacuum drying temperature: 55-80 ℃ wherein, return difference temperature 2-3 ℃, more than vacuum-0.07Mpa, microwave power 25-60KW, dry 120-140 minute.

Preferably, adopt the high effective liquid chromatography for measuring salvianolic acid A, alkannic acid, rosmarinic acid, salvianolic acid B, salvianolic acid C content, condition determination is as follows:

Take octadecylsilane chemically bonded silica as filler;

Detect wavelength 286nm; Flow velocity 1.0ml/min; 30 ℃ of column temperatures;

Theoretical cam curve should be not less than 10000 by salvianolic acid A;

The preparation precision of reference substance solution takes salvianolic acid A, alkannic acid, rosmarinic acid, salvianolic acid B, salvianolic acid C reference substance in right amount to volumetric flask, adds methanol and makes the mixing reference substance solution;

The preparation of need testing solution: precision takes sample 10mg in the 100ml volumetric flask, adds dissolve with methanol and shakes up, and be diluted to scale, obtains;

Eluting be take methanol as mobile phase A, take 0.1～0.5% phosphoric acid as Mobile phase B, by following condition, carries out gradient elution, moves 60 minutes;

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%.

Algoscopy: the accurate absorption mixed reference substance solution and each 10 μ l of need testing solution respectively, and the injection liquid chromatography, measure, and calculates the content of salvianolic acid A, alkannic acid, rosmarinic acid, salvianolic acid B, salvianolic acid C.

The present invention is by the extraction to Radix Salviae Miltiorrhizae, transform, purification, drying process, obtained take salvianolic acid A as main compound: through screening and the optimization of 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 concentration with the salvianolic acid B of high concentration, only need be made into suitable initial conversion concentration and get final product, and can reach the purpose that changes into the salvianolic acid A compositions 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 salvianolic acid A compositions 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 concentration requirement, and think that the high specific concentration of concentration hangs down.In catalytic conversion reaction of the present invention, the concentration 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 zinc chloride can significantly improve as catalyst the conversion ratio that salvianolic acid B transforms the salvianolic acid A compositions, 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 the salvianolic acid A composition levels is lower, the large raising of salvianolic acid A composition levels 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 in the salvianolic acid A compositions is brought up to 80% 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 normal temperature drying, and from vacuum drying, spray drying, microwave vacuum drying method preferred microwave vacuum drying, thereby thoroughly overcome, baking temperature was too high in the past, drying time is long, the large defect to the destruction of salvianolic acid A compositions; And sublimation drying is long, the serious problem of compositions organic solvent residual of the high and lyophilization gained of cost.

In salvianolic acid A compositions of the present invention, except main, contain salvianolic acid A, also containing 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 the damage caused due to cerebral ischemia re-pouring, and cerebral ischemia is had to protective effect.But salvianolic acid A compositions 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 composition for improved middle cerebral artery occlusion model in rats rat function of nervous system defect state; dwindle infarct size, alleviate cerebral edema, reduce 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 compositions is the main effective ingredient of Radix Salviae Miltiorrhizae and preparation Radix Salviae Miltiorrhizae Injection thereof, XIANGDAN ZHUSHEYE, and the same with the salvianolic acid A compositions 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 compositions 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 compositions 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 compositions, 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, also have 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:

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), neural behavior scoring (the spontaneous activity of (in 12h) after model group is revived, the symmetry of quadruped locomotion, forelimb stretches the symmetry of the action of creeping, 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 72h, neural behavior scoring continues obviously low lower than sham operated rats, the nimodipine group is compared with model group with salvianolic acid A monomeric compound group and each dosage group of salvianolic acid A compositions, neurobehavioral after rat revives has rising in various degree, the most obvious with nimodipine group and the middle and high dosage group of salvianolic acid A compositions, and after its ischemia, the neurobehavioral of 24h approaches false dry art group level substantially, recovered normally to each dosage group rat neurobehavioral of 72h salvianolic acid A compositions after ischemia, and recovered soon than salvianolic acid A monomeric compound group and slightly good with the integrality of dosage salvianolic acid A compositions group rat, neurobehavioral has more excellent trend.Prompting salvianolic acid A compositions can be improved 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 each dosage group rat ischemia side middle cerebral artery (MCA) local thrombus of Microscopic observation salvianolic acid A compositions, 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 compositions and dwindles than model control group is remarkable.HE dyes in visible model group ischemia side cortex 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 compositions high dose group rat has no Telatrophy's obscission herein; The HE dyeing of each dosage group of salvianolic acid A compositions 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 dosage group of salvianolic acid A compositions and model group comparison hypertrophy significantly increases; In addition, the kitchen range shape that HE dyeing display model matched group differs in size as seen is hemorrhage, and salvianolic acid A combination object height, middle dosage group there is no and find that there is kitchen range shape bleeding.Result demonstration salvianolic acid A compositions can obviously be improved 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 be 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 compositions; 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 compositions 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 compositions 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 compositions basic, normal, high dosage group Infarction volume and brain water content are all lower than the nimodipine group; And than low with the salvianolic acid A monomeric compound group of dosage.Illustrate that the salvianolic acid A compositions 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 compositions 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 more stable in 7d.Show that the salvianolic acid A compositions can increase MVD and the blood vessel field Area Ratio of cerebral tissue ischemia half blanking bar, prompting salvianolic acid A compositions 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.Each dosage group of salvianolic acid A compositions and model group group are relatively; the VEGFmRNA expression significantly raises; show that the salvianolic acid A compositions can significantly promote the ischemic tissue of brain angiogenesis; promote the foundation of collateral circulation compensation; save ischemia half blanking bar; the brain tissue impairment that the protection ischemia causes, and have certain dose-effect relationship, the trend that is better than same dosage salvianolic acid A monomeric compound is arranged.

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; but the prolongation with Ischemia Time; have a declining tendency; 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 for each dosage group of salvianolic acid A compositions and model group; Each time period of each dosage group self relatively shows, the bFGF protein expression is continual and steady; Prompting salvianolic acid A compositions 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 compositions can alleviate cerebral ischemia-reperfusion injury in rats neurologic impairment symptom, be conducive to its neurobehavioral recovery, the salvianolic acid A compositions has the effect that improves function of nervous system after brain tissue impairment, 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 compositions is compared with the Ischemia-Reperfusion Injury Model group: cerebral infarction volume is than significantly reducing, cerebral index and brain water content all obviously reduce, show that the salvianolic acid A compositions 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, give various dose salvianolic acid A 10min and begin, each medicine group than self administration before ischemia half blanking bar regional cerebral blood flow (rCBF) obvious rising is arranged, and particularly remarkable with salvianolic acid A combination object height, middle dosage group; Each dosage group of salvianolic acid A compositions is compared, and good dose-effect relationship is arranged; In the salvianolic acid A compositions, the rCBF of dosage group day part is suitable with the nimodipine group.Hence one can see that, and the salvianolic acid A compositions has the effect that increases rats with cerebral ischemia cerebral tissue ischemia half blanking bar rCBF, is conducive to save the cerebral tissue that ischemia half blanking bar is at death's door, the effect of performance treatment cerebral ischemia, and the trend that is better than the salvianolic acid A monomeric compound is arranged.

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 model group rat ischemia again, to fill with in 3h blood perfusion extremely unstable again, recovering to fill with again in 1～1.5h, ischemia half blanking bar rCBF rises to top level, but still before ischemia below 50%, cerebral tissue still is low perfusion state, and it is front below 40% after this sharply to drop to again ischemia, cerebral tissue because of fill with again aggravate impaired.And, after filling with, nimodipine, the high, medium and low dosage group of salvianolic acid A compositions rat rCBF obviously increase again, each time period rCBF has compared utmost point significant difference with model group.1h after filling with again, nimodipine and salvianolic acid A compositions high dose group rCBF return to approach former rCBF 76%, the dosage group returns to and is about 70% of former rCBF in the salvianolic acid A compositions, the nimodipine group returns to and is about 69% of former rCBF, 10mg/kg salvianolic acid A monomeric compound group returns to 65% left and right of former rCBF, salvianolic acid A compositions low dose group returns to and is about 62% of former rCBF, and, to pouring in 3h, each medicine group rCBF maintains in metastable scope again.Result shows that the salvianolic acid A compositions 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 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 compositions, reduce cerebral tissue lactic acid (LA) content, can significantly improve the energy metabolism of rat cerebral ischemia and ischemia-reperfusion.The salvianolic acid A compositions 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 24h again, neuronal apoptosis is serious; And, after nimodipine and the intervention of various dose salvianolic acid A compositions, with model group, compare, in rat cerebral tissue, neuronal apoptosis significantly reduces (P<0.001 or P<0.01); And the apoptosis rate with salvianolic acid A combination object height, middle dosage group is minimum; Show that the salvianolic acid A compositions 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 compositions 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 compositions 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 compositions obviously reduces than model group, and salvianolic acid A monomeric compound group and the nimodipine group with dosage is remarkable.Prompting salvianolic acid A compositions can suppress the expression of ischemic injuries brain tissue inflammation cytokine, suppress the generation of brain tissue inflammation's reaction, suppress neuron and the cerebral tissue cascade damage of inflammatory reaction mediation, 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 compositions can significantly reduce Ca in cerebral tissue ²⁺content, rising K ⁺, Mg ²⁺content, and the most remarkable with the salvianolic acid A composition effect.Illustrate that the salvianolic acid A compositions can suppress Ca ²⁺interior stream alleviates calcium overload, thereby can suppress Ca ²⁺the neuronal cell apoptosis that overload is induced.

Known through the experimental data contrast; the salvianolic acid A compositions 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 compositions 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 compositions 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 the neuronal cell apoptosis that monoamine neurotransmitter is disorderly and toxicity of excitatory amino acid is induced, and effect with dosage salvianolic acid A monomeric compound and Buddhist nun not l equal the advantage that has more.

Known through experimental data contrast, each dosage group of salvianolic acid A compositions 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 compositions 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, 6h after the cerebral tissue ischemia, and endothelial cell apoptosis is showed increased just, to ischemia, within 24 hours, reaches peak.With model group, compare, the apoptosis digital display work of each time period of each dosage group of salvianolic acid A compositions reduces, and is less than salvianolic acid A monomeric compound and the nimodipine group of same dosage.Illustrate that the salvianolic acid A compositions 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 salvianolic acid A compositions and nimodipine effect, anoxia and Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial survival rate significantly raise, and salvianolic acid A compositions group survival rate is significantly higher than the nimodipine group, and also higher than the cell survival rate of the salvianolic acid A monomeric compound group with dosage.Prompting salvianolic acid A compositions 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 compositions, 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 compositions and be certain dose-effect relationship.And salvianolic acid A compositions 10 μ mol/L dosage groups are suitable with nimodipine 40 μ mol/L dosage group effects, with the salvianolic acid A compositions of dosage, be better than the salvianolic acid A monomeric compound.Prompting salvianolic acid A compositions has the effect of the Brain Microvascular Endothelial apoptosis that good anti-hypoxia-reoxygenation injury causes.

After various dose salvianolic acid A compositions, 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 compositions rises to the Normal group level that approaches; Each administration group t-PA/PAI and NO/ET ratio also significantly improve than model group.Experimental result prompting salvianolic acid A compositions can be improved 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 compositions effect, and 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 compositions has the Hypoxia/Reoxygenation Injury of inhibition and causes Ca in BMVEC born of the same parents ²⁺the effect of concentration.

Known through the experimental data contrast, the salvianolic acid A compositions compares impact and the normal saline matched group of rat artery thrombus formation time, the thrombus formation time significant prolongation of the high, medium and low dosage group of salvianolic acid A compositions; Low dose group (0.5mg/kg) thrombus formation time is suitable with 20mg/kg aspirin group; Middle dosage group extends than the salvianolic acid A monomeric compound group thrombus formation time with dosage; Show that the salvianolic acid A compositions 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, the high, medium and low dosage group of salvianolic acid A compositions rat suppository is wet, dry weight all significantly alleviates for the impact that the salvianolic acid A compositions forms rat suppository and normal saline group.And low dose group is suitable with 20mg/kg aspirin group effect.Illustrate that the salvianolic acid A compositions 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 compositions compares impact and the normal saline group of rat vein thromboembolism, the high, medium and low dosage group of salvianolic acid A compositions rat vein thrombosis is wet, dry weight significantly alleviates, low dose group (0.5mg/kg) and 20mg/kg aspirin group effect suitable (P>0.05); Middle dosage group is with the salvianolic acid A monomeric compound successful of dosage.Show that the salvianolic acid A compositions 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 compositions compares rats in vitro thrombosis and normal saline group, the thrombotic length of the high, medium and low dosage group of salvianolic acid A compositions rats in vitro significantly shortens, thrombosis is wet, dry weight significantly alleviates, and low dose group (0.5mg/kg) is suitable with 20mg/kg aspirin group effect; Show that the salvianolic acid A compositions 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 compositions compares impact and the normal saline group of established thrombosis in body, and 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, with the normal saline group, compares utmost point significant difference is all arranged; Dosage group in the salvianolic acid A compositions, the vessel open degree is similar to 5mg/kg salvianolic acid A monomeric compound group and 2000U/kg urokinase group altogether, 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 compositions high dose group and recanalization rate are all higher than the urokinase group, and the bolt rate is starkly lower than the urokinase group again; Though salvianolic acid A compositions low dose group recanalization rate is lower than the urokinase group, bolt rate is suitable with the urokinase group again for it, and has lower than the urokinase trend of bolt rate again.Illustrate the salvianolic acid A compositions thrombolytic is arranged preferably and prevents thrombolytic after the effect of thromboembolism again, 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, relatively, its whole blood viscosity and plasma viscosity and packed cell volume all obviously reduce for each dosage group of salvianolic acid A compositions and model group; Results suggest salvianolic acid A compositions 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 compositions 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 compositions 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 nuclear magnetic resonance, NMR chlorine spectrogram;

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

Fig. 5. the mystery fragrant sour nuclear magnetic resonance, NMR oxygen spectrogram that changes;

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 composition sample HPLC figure;

The specific embodiment

Embodiment 1

Get red rooted salvia, be ground into 6 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 1.20 (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 4.0 with 10% sodium hydroxide, adds 0.5%ZnC1 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 12 times of water gagings and dissolves, with microwave vacuum drying (50 ℃ of baking temperatures, 4 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 60KW) 130 minutes, obtain the salvianolic acid A compositions.

After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.57%, alkannic acid content 0.663%; Rosmarinic acid contents 1.16%; Content of danshinolic acid B 1.24%; Salvianolic acid C content 1.48%.

Embodiment 2

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 1.17 (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 3.5 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 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, with microwave vacuum drying (60 ℃ of baking temperatures, 2 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 40KW) 110 minutes, obtain the salvianolic acid A compositions.

After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.63%, alkannic acid content 0.69%; Rosmarinic acid contents 1.12%; Content of danshinolic acid B 1.20%; Salvianolic acid C content 1.48%.

Embodiment 3

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 1.18 (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 thermal conversions 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 phenolic acid A12mg 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 (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, with microwave vacuum drying (50 ℃ of baking temperatures, 5 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 80KW) 90 minutes, obtain the salvianolic acid A compositions.

After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.68%, alkannic acid content 0.68%; Rosmarinic acid contents 1.07%; Content of danshinolic acid B 1.19%; Salvianolic acid C content 1.47%.

Embodiment 4

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 1.20 (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 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, with microwave vacuum drying (70 ℃ of baking temperatures, 1 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 20KW) 100 minutes, obtain the salvianolic acid A compositions.

After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.72%, alkannic acid content 0.62%; Rosmarinic acid contents 1.10%; Content of danshinolic acid B 1.28%; Salvianolic acid C content 1.46%.

Embodiment 5

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 1.19 (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 4.4 with 10% sodium bicarbonate, adds 0.8%ZnCl ₂as 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, 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: 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: 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 10 times of water gagings and dissolves, with microwave vacuum drying (50 ℃ of baking temperatures, 2 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 30KW) 110 minutes, obtain the salvianolic acid A compositions.

After measured, in described salvianolic acid A compositions, salvianolic acid A content is 96.13%, alkannic acid content 0.55%; Rosmarinic acid contents 1.00%; Content of danshinolic acid B 1.02%; Salvianolic acid C content 1.34%.

Embodiment 6

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 1.25 (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%ZnCl ₂as 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: 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 12 times of water gagings and dissolves, with microwave vacuum drying (65 ℃ of baking temperatures, 5 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 90KW) 80 minutes, obtain the salvianolic acid A compositions.

After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.64%, alkannic acid content 0.69%; The mystery fragrant acid content 1.05% that changes; Content of danshinolic acid B 1.11%; Salvianolic acid C content 1.47%.

Embodiment 7

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 1.23 (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 3.6 with 10% sodium hydroxide, adds 0.5%ZnCl ₂as 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, uses respectively 4 times of cylinder hydrops, 4 times of column volume 22% ethanol elutions, removes impurity, use again 6 times of column volume 43% ethanol elutions, HPLC detects, and collects the 43% 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 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, with microwave vacuum drying (55 ℃ of baking temperatures, 3 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 50KW) 120 minutes, obtain the salvianolic acid A compositions.

After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.72%, alkannic acid content 0.70%; Rosmarinic acid contents 1.16%; Content of danshinolic acid B 1.24%; Salvianolic acid C content 1.47%.

Embodiment 8

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 1.08 (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 3.5 with 10% sodium carbonate, adds 1.0%ZnCl ₂as 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, uses respectively 3 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions, removes impurity, use again 5 times of column volume 45% ethanol elutions, HPLC detects, and collects the 45% 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 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, with microwave vacuum drying (50 ℃ of baking temperatures, 2 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 60KW) 120 minutes, obtain the salvianolic acid A compositions.

After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.84%, alkannic acid content 0.63%; Rosmarinic acid contents 1.12%; Content of danshinolic acid B 1.25%; Salvianolic acid C content 1.47%.

Experimental example 1: salvianolic acid A compositions analytical method and separation, Structural Identification research

One, salvianolic acid A compositions analytical method research

1. instrument and reagent

Instrument: Waters e2695 high performance liquid chromatograph, Empower2 chromatographic work station, 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 are 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 embodiment 1 sample, 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 and get above-mentioned mixing reference substance solution 0.1ml, 0.2ml, 0.5ml, 1ml, 2ml, 5ml, put respectively in the 10ml measuring bottle, add 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

Two, salvianolic acid A compositions separation, Structural Identification research

Embodiment 1 is obtained to above-mentioned salvianolic acid A compositions respectively with CG161M macroporous adsorbent resin, ODS, Sephadex LH-20 column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80～20%, the ratio of ethanol is 20～80%, the Fractional Collections eluent, merge same composition, crystallization, can obtain salvianolic acid A, the monomeric compound of alkannic acid, rosmarinic acid, salvianolic acid B, salvianolic acid C, measure described monomeric compound structure, 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, referring to 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, referring to Fig. 3,4.

Alkannic acid (lithospcrmic acid), pale yellow powder, easily easy methanol, water.ESIMSm/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`).

The molecular formula of rosmarinic acid: C ₁₈h ₁₆o ₈, molecular weight: 360.31, structure is as follows:

Rosmarinic acid physicochemical property and spectral data, referring to 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 ₁₆o 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(CD3OD，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, referring to 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, referring to Fig. 9,10.

Salvianolic acid C (Salvianolic acid C), pale yellow powder, easily easy methanol, water.ESIMSm/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(CD3OD，150MHz)δ：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 2: 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 3: 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% oxygen 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, salvianolic acid B is converted in salvianolic acid A compositions process, regulate pH value, add 1.0%ZnCl2, can greatly improve the productive rate of salvianolic acid A compositions.

Experimental example 4: 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 add certain catalysts influence salvianolic acid B to transform the salvianolic acid A compositions, therefore, we all add 1%ZnCl2 as catalyst to each experimental group, 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, 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 ₂, factor A (salvianolic acid B concentration) has certain influence to salvianolic acid A compositions productive rate, from the K value, analyzes, concentration is converted into the not impact of effect of salvianolic acid A on improving salvianolic acid B higher than 30mg/ml, and the impurity formed is more, therefore, before transforming, salvianolic acid B concentration should be selected 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 5: 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 2001, 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 6: different 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 salvianolic acid A compositions on red B

The demonstration of table 19 result, above 4 kinds of catalyst all can be used as the catalyst in the salvianolic acid B conversion reaction, but resultant effect is not as good as ZnCl ₂the changing effect that catalyst reaches, therefore optimal choice ZnCl of the present invention ₂catalyst.

Experimental example 7: 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, 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%, with the HPD-100 amount of getting dry extract maximum, the salvianolic acid A composition levels is high, and the resin absorption amount is large.

Experimental example 8: the polyamide column chromatography purification 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;

Maximum with the polyamide amount of getting dry extract, the content high adsorption capacity is large.

Experimental example 9: the abstraction purification of salvianolic acid A compositions

The 70% ethanol elution decompression recycling ethanol by the polyamide column chromatography purification, 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 salvianolic acid A compositions extract, 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, 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, salvianolic acid A content all is improved, and obtains with t-butyl methyl ether that compositions is many, salvianolic acid A content is high.

Experimental example 10: the purification by silica gel column chromatography of salvianolic acid A compositions

Get 12 parts of the salvianolic acid A compositions extracts of abstraction purification, every part containing salvianolic acid A 10g, adds the silica gel of 20g, whisks, 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, the two-phase solvent that adopts pentane-t-butyl methyl ether to form is that the eluant effect is best.

Experimental example 11: salvianolic acid A composition dries method research

Get 4 parts of salvianolic acid A compositions eluents after silica gel column chromatography, every part containing salvianolic acid A compositions 100g, employing respectively after adding 10 times of water gagings and dissolving, vacuum drying, lyophilisation, spray drying, microwave vacuum drying 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: the salvianolic acid A compositions is controlled simple with microwave vacuum drying process, treating capacity is large, and baking temperature is low, and the time is short, and extract obtained indices is good, therefore the present invention adopts microwave vacuum drying to prepare the salvianolic acid A compositions.

Through further experiment is definite, the microwave vacuum drying optimum range is temperature: 20-100 ℃, return difference temperature 1-5 ℃, and more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.

Below experiment all gets by the salvianolic acid A compositions sample that embodiment 3 preparation methoies obtain, the salvianolic acid A monomeric compound, and commercially available, salvianolic acid A content is more than 99.62%.

Experimental example 12: the impact of function of nervous system's symptom after salvianolic acid A compositions 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 IBAS2.0 Image analysis system.

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 and result:

2.1 method

2.1.1 the preparation of animal model:

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.

2.1.2 grouping and administration

First by animal, divide 7 groups at random: sham operated rats, model control group, the high, medium and low dosage group of salvianolic acid A compositions, salvianolic acid A monomeric compound group, nimodipine group.The sham operated rats window that only opens seam, do not do laser irradiation; Model group, the high, medium and low dosage group of salvianolic acid A compositions, salvianolic acid A monomeric compound group, nimodipine group prepare easy apoplectic type renovascular hypertension in rats MCAO model by the model preparation method.And by the 0.3ml/100g tail vein injection, be administered once respectively in the postoperative 30min of MCAO model, after this be administered once every day, continuous three days altogether.Sham operated rats, the postoperative tail vein injection saline of model control group; The postoperative tail vein injection salvianolic acid A respectively of the high, medium and low dosage group of salvianolic acid A compositions compositions 20mg/kg, 10mg/kg, 5mg/kg, the postoperative tail vein injection salvianolic acid A of salvianolic acid A monomeric compound group monomeric compound 10mg/kg, the postoperative tail vein injection nimodipine of nimodipine group 10mg/kg.

2.2 result

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, 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) 24h, 48h, 72h 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 25. salvianolic acid A compositions on the RHRSP cerebral thrombosis after the impact (x ± s) of neuroethology scoring

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

Experimental result shows: the neurobehavioral of (in 12h) after model group is revived (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 (P<0.01), after the cerebral thrombosis ischemia is described, RHRSP function of nervous system is impaired serious, and, in lasting 72h, neurobehavioral continues to be starkly lower than sham operated rats (P<0.01); The nimodipine group is compared with model group with salvianolic acid A monomeric compound group and each dosage group of salvianolic acid A compositions, neurobehavioral after rat revives has rising (P<0.05 or P<0.01) in various degree, after salvianolic acid A compositions ischemia, the neurobehavioral of 24h approaches the sham operated rats level substantially, and 48h has recovered normal level; Recovered normally to each dosage group rat neurobehavioral of 72h salvianolic acid A compositions after ischemia, compared difference with model group and still have statistical significance (P<0.01).10mg/kg salvianolic acid A compositions group and 10mg/kg salvianolic acid A monomeric compound group relatively, although the neuroethology scoring does not have significant difference, also have slightly excellent trend; And, in the laboratory observation process, the integrality of the rat of salvianolic acid A compositions group is recovered soon than salvianolic acid A monomeric compound group and is slightly good.Above results suggest, the salvianolic acid A compositions can improve neurobehavioral after RHRSP cerebral thrombosis ischemia, the effect of nervous symptoms after prompting salvianolic acid A compositions has protection and improves cerebral ischemia, and the trend that is better than same dosage salvianolic acid A monomeric compound is arranged.

Experimental example 13: the impact of salvianolic acid A compositions on RHRSP cerebral thrombosis tissues following MCAO in rats pathological change

The preparation of RHRSP Cerebrovascular embolism model, grouping, medication are with experimental example 12.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 26.

The impact of table 26. salvianolic acid A compositions 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 compositions, 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 compositions 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 compositions high dose group rat has no Telatrophy's obscission herein, other each administration group necrocytosiss are less, the accidental obscission of organizing; The HE of each administration group and model control group dyeing shows in kitchen range, kitchen range Zhou Jun has little blood vessel hyperplasia, but the little number of blood vessel of each dosage group of each administration composition and model group comparison hypertrophy significantly increases, and with salvianolic acid A combine object height, middle dosage group is particularly remarkable; In addition, the kitchen range shape that HE dyeing display model matched group differs in size as seen is hemorrhage, salvianolic acid A combination object height, middle dosage group there is no and find that there is kitchen range shape bleeding, salvianolic acid A compositions low dose group and salvianolic acid A monomeric compound group and nimodipine group also only have indivedual animals to have the kitchen range shape hemorrhage, and hemorrhagic focus is less than model group.Result of the test demonstration salvianolic acid A compositions can obviously be improved 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 is better than salvianolic acid A watch box compound.

Experimental example 14: the salvianolic acid A compositions on the RHRSP cerebral thrombosis after the impact of blood brain barrier (BBB) permeability

1 test method

Animal grouping and the preparation of RHRSP Cerebrovascular embolism model are with experimental example 12 methods.4.5h after the model surgery success, each group is tail vein injection salvianolic acid A compositions 40mg/kg, 20mg/kg, 10mg/kg respectively, salvianolic acid A monomeric compound 10mg/kg, nimodipine 20mg/kg; Sham operated rats and model group tail vein injection are with the normal saline of volume.Each is organized rat and puts to death and get the cerebral tissue specimen in MCAO postoperative (after being the cerebral thrombosis ischemia) 6h, 12h, 24h in batches respectively, 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 brain.

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.The hot spot number of each group and distribution are obviously different: the model group hot spot is maximum, and the hot spot of 120 left and right diameter 100～200um is approximately arranged, and intensive place in the form of sheets.Each salvianolic acid A composition medicine group number of spots significantly reduces than model group, and the salvianolic acid A compositions is fewer than the salvianolic acid A monomeric compound group hot spot of same dosage; Each dosage group of salvianolic acid A compositions relatively, presents the remarkable dose-difference opposite sex; Wherein more with the low dose group hot spot, on same level, hot spot approximately has 50, but is fused into the less of lamellar; Middle dosage group hot spot is less, is dispersed in distribution; The high dose group hot spot seldom is dispersed in, and fluorescence is very weak.

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 27.

Table 27. salvianolic acid A compositions on the RHRSP cerebral thrombosis after the impact of blood-brain barrier permeability

Annotate: compare #P<0.01 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, there were significant differences (P<0.01), 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 compositions 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 compositions 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 15: the impact of salvianolic acid A compositions on RHRSP cerebral thrombosis cerebral infarction scope and brain water content

Method by experimental example 12 prepares RHRSP cerebral thrombosis rat model, grouping, administration; After last 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 28. salvianolic acid A compositions on RHRSP cerebral thrombosis cerebral infarction scope and brain water content

Adopt the SPSS11.5 statistical package 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 compositions 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 compositions basic, normal, high dosage group Infarction volume and brain water content are all lower than the nimodipine group.10mg/kg salvianolic acid A compositions 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 compositions 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 16: the salvianolic acid A compositions on RHRSP cerebral thrombosis ischemia after the microvascular impact of rat cerebral tissue

Animal grouping and the preparation of RHRSP Cerebrovascular embolism model are with experimental example 12 methods.4.5h tail intravenously administrable after the model surgery success, after this be administered once, until it is complete to draw materials every day.Each dosage group of salvianolic acid A compositions is tail vein injection salvianolic acid A compositions 20mg/kg, 10mg/kg, 5mg/kg respectively, salvianolic acid A monomeric compound group injection salvianolic acid A monomeric compound 10mg/kg, nimodipine group injection nimodipine 10mg/kg; Model group and sham operated rats injection are with the normal saline of volume.Each group is got rat broken end respectively at the postoperative 6h of MCAO, 1d, 3d, 7d in batches and is got brain, conventional fixing, the dehydration of the neutral PBS buffer of cerebral tissue 4% paraformaldehyde of ischemic focus half penumbra area or same area, 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 full automatic colour image place type system to carry out graphical analysis, measure blood vessel field Area Ratio (Positive Objects area/Statistical Fields gross area).

Table 29 respectively organizes that rat cerebral tissue's blood vessel scene is long-pending, the immune positive microvessel density of CD31 (MVD) value (n=6)

Table 29 experimental data shows, with sham operated rats, compares, and model group ischemia penumbra MVD and blood vessel scene are amassed 1d after ischemia to be increased to some extent, but after ischemia, 3d, 7d continue significantly to reduce (P<0.01); With model group, compare, after the ischemia treatment, each dosage group of nimodipine group and salvianolic acid A compositions and salvianolic acid monomeric compound group 6h, 1d, 3d, 7d cerebral tissue ischemia penumbra MVD and blood vessel scene after ischemia is long-pending significantly increases (P<0.05 or P<0.01) to some extent, and more stable in 7d.Three kinds of different pharmaceuticals relatively, the most remarkable with the salvianolic acid A composition effect.Between three dosage groups of salvianolic acid A compositions, relatively, difference has statistical significance (P<0.05).Result shows that the salvianolic acid A compositions can increase MVD and the blood vessel field Area Ratio of cerebral tissue ischemia half blanking bar, and prompting salvianolic acid A compositions 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 17: the salvianolic acid A compositions on RHRSP cerebral thrombosis ischemia after the impact of rat cerebral tissue vascular endothelial growth factor expression

Modeling and grouping administration are with experimental example 12.Respectively at the postoperative 2h of rat MCAO, 24h, 48h, 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 30. salvianolic acid A compositions 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 VEGF the expression increase; 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.Relatively, the VEGFmRNA expression significantly raises (P<0.05 or P<0.01), and is better than same dosage salvianolic acid A monomeric compound group for each dosage group of salvianolic acid A compositions and model group group.Prompting salvianolic acid A compositions can significantly promote the ischemic tissue of brain angiogenesis, promotes the foundation of collateral circulation compensation, saves ischemia half blanking bar, the brain tissue impairment that the protection ischemia causes, and have certain dose-effect relationship.

Experimental example 18: the salvianolic acid A compositions on RHRSP cerebral thrombosis ischemia after the impact of rat cerebral tissue basic fibroblast growth factor protein expression

With experimental example 17, respectively at the postoperative 2h of rat, 24h, 48h, each component criticizes that to get 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 31. salvianolic acid A compositions to 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, Bai Youji and 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 24h, significant difference (P<0.05) is arranged; but the prolongation with Ischemia Time, have a declining tendency, after prompting cerebral tissue hypoxic-ischemic; body self produces of short duration 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 compositions and salvianolic acid A monomeric compound group and model group; With salvianolic acid A composition effect optimum.Between three dosage groups of salvianolic acid A compositions, 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 compositions 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 19: the impact of salvianolic acid A compositions 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 compositions (10mg/kg, 5mg/kg, 2.5mg/kg) group, salvianolic acid A monomeric compound group (10mg/kg), nimodipine matched group (10mg/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 30min and each is administered once through tail vein injection when perfusion starts again before ischemia, after this each injection every day once, continues to and draws materials.Sham operated rats and ischemia-reperfusion injury model matched group give the normal saline of same volume.

After cerebral ischemic reperfusion in rats, 3h, 24h, 48h, 72h 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 32.

The impact (x ± s, n=12) of table 32. salvianolic acid A compositions on the rats after cerebral ischemic reperfusion neurological deficits score

Annotate: compare * * P<0.01, * P<0.05 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 72h, although more again after perfusion behavioristics's obstacle of 3h, 24h 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 compositions is compared with model group, from pouring into again 3h, the neurologic impairment sx↓, neural row is learned damaged mark and is significantly reduced (P<0.05 or P<0.01), to pouring into 72h again, the neurobehavioral of each medicine group rat recovers substantially.Between each dosage group of salvianolic acid A compositions: low dose group and the scoring of middle and high dosage group relatively have statistical significance (P<0.05); Between high dose group and middle dosage group, scoring relatively has the trend of reduction, but difference does not have statistical significance (P>0.05).Each time period neurologic defect symptom of salvianolic acid A compositions group rat will be lighter than the salvianolic acid A monomeric compound group of same dosage.

Experimental result shows that the salvianolic acid A compositions can alleviate rats after cerebral ischemic reperfusion neurologic impairment symptom, be conducive to its neurobehavioral recovery, prompting salvianolic acid A compositions has the effect that improves nervous symptoms after brain tissue impairment, and action effect is more obvious than salvianolic acid A monomeric compound.

Experimental example 20: the impact of salvianolic acid A compositions on rats after cerebral ischemic reperfusion cerebral infarction scope, cerebral index and brain water content

Experimental example 19 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 33. salvianolic acid A compositions 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 compositions is compared with the ischemia-reperfusion injury model group: cerebral infarction volume is than significantly reducing (P<0.01), and cerebral index and brain water content all obviously reduce (P<0.01 or P<0.05); Salvianolic acid A compositions 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 or P<0.01) for the middle and high dosage group of salvianolic acid A compositions and nimodipine group, and lower than same dosage salvianolic acid A monomeric compound group; Show that the salvianolic acid A compositions 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 21: the impact of salvianolic acid A compositions on rats with cerebral ischemia cerebral tissue ischemia half blanking bar regional cerebral blood flow (rCBF)

1 test method

Adopt the standby rat brain of improvement line bolt legal system with kitchen range 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 an example mouthful 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.

Model after success is divided into nimodipine group (10mg/kg), salvianolic acid A monomeric compound group (10mg/kg), the high, medium and low dosage group of salvianolic acid A compositions (10mg/kg, 5mg/kg, 2.5mg/kg) at random.Each treated animal after model success immediately the tail vein give relative medicine, sham operated rats and model group give the normal saline of same volume.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.Record before administration and administration after blood flow.

2 result of the tests

After middle cerebral artery occlusion in rat, 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%～40% between.10min after administration, the ischemia penumbra rCBF of each medicine group rat cerebral tissue than administration before all existing risings in various degree, 30min after administration, each medicine group cerebral tissue ischemia penumbra rCBF peaks.Concrete experimental data statistical analysis is in Table 34.

The impact (x ± s, n=10) of table 34. salvianolic acid A compositions 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 comparison before administration, ★ P<0.01, ☆ p<0.05

3 interpretations of result

From table 34 data, after the operation modeling, model control group and each medicine group cerebral tissue half blanking bar rCBF compare with sham operated rats, have utmost point significant difference (p<0.01) to mean the modeling success.After administration, 10min begins, each medicine group than self administration before ischemia half blanking bar rCBF obvious rising is arranged, and with nimodipine and salvianolic acid A compositions high dose group particularly significantly (p<0.01); To 30min after administration, each medicine group ischemia half blanking bar rCBF rises to the highest, and respectively organizes self rCBF baseline value before administration relatively, has raise 15%～30%; Though after this have a declining tendency, but last till ischemia 2h, each medicine group is compared before than model group and self administration, still has remarkable significant difference (p<0.01 or p<0.05); Each dosage group of salvianolic acid A compositions is compared, and good dose-effect relationship is arranged; In the salvianolic acid A compositions, the rCBF of dosage group day part is suitable with the nimodipine group, and higher than same dosage salvianolic acid A monomeric compound.Hence one can see that, and the salvianolic acid A compositions 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, the effect of performance treatment cerebral ischemia.

Experimental example 22: the impact of salvianolic acid A compositions on rats after cerebral ischemic reperfusion cerebral tissue ischemia half blanking bar rCBF

Respectively organize rat in experimental example 21 after continuous ischemia 2h observation ischemia half blanking bar rCBF, extract the bolt line, realize ischemia-reperfusion injury model.After model is set up, each organizes the tail intravenously administrable, and dosage is with experimental example 16.Still press the described method of experimental example 16 and measure the rear 3h cerebral tissue ischemia half blanking bar rCBF of filling again.Respectively organize rat and fill with again rear different time sections 10min, 30min, 60min, 90min, 120min, 180min cerebral tissue ischemia half blanking bar rCBF.Data are carried out statistical analysis.Concrete data are in Table 35.

The impact (x ± s, n=10) of table 35. salvianolic acid A compositions on rats after cerebral ischemic reperfusion cerebral tissue ischemia half blanking bar rCBF

Annotate: compare * P<0.01 with sham operated rats; With filling is relatively front again, ☆ p<0.01; Compare ★ 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 model group rat rises to top level at 60,90 minutes ischemia half blanking bar rCBF, with filling is front than obvious statistical significance (P<0.01) is arranged again, but also only be about 50% of the front baseline value of ischemia, after this sharply descend again, fill with again in 3h, the rCBF value before ischemia baseline value 37%～50% between, in the meantime again the perfusion remain incomplete, still be low perfusion phenomena, cerebral tissue can continue impaired.After filling with, nimodipine, the high, medium and low dosage group of salvianolic acid A compositions rat rCBF obviously increase again, and each time period rCBF has compared utmost point significant difference (P<0.01) with model group.1h after filling with again, salvianolic acid A compositions high dose group rCBF return to approach former rCBF 76%, in the salvianolic acid A compositions dosage group return to be about former rCBF 70%, the nimodipine group return to be about former rCBF 69%, 10mg/kg salvianolic acid A monomeric compound group returns to 65% left and right of former rCBF, salvianolic acid A compositions low dose group returns to and is about 62% of former rCBF; Each dosage group of salvianolic acid A compositions relatively, is dose dependent; And, to pouring in 3h, each medicine group rCBF maintains in metastable scope again.The experimental result prompting, the salvianolic acid A compositions can obviously be improved 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 is better than the salvianolic acid A monomeric compound, ischemic cerebrovascular is had to good therapeutical effect.

Experimental example 23: the impact of salvianolic acid A compositions on the rats after cerebral ischemic reperfusion Energy Metabolism of Brain Tissue

Experimental example 22 each treated animals are after the cerebral blood flow of having measured again 3h 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 36. salvianolic acid A compositions 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 36 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 raise (P<0.01), LA significantly reduces, result shows can the raise content of cerebral tissue ATP, ADP, AMP, PC of salvianolic acid A compositions, reduce cerebral tissue LA content, can significantly improve the energy metabolism of rat cerebral ischemia and ischemia-reperfusion.Prompting salvianolic acid A compositions 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 24 salvianolic acid A compositionss on rats after cerebral ischemic reperfusion cerebral tissue neuronal apoptosis rate

Prepare rat pattern of ischemia reperfusion, grouping, administration with experimental example 19 methods.After rat cerebral ischemia 2h fills with 24h 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 whole art deoxyribonucleic 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 37 salvianolic acid A compositions 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 24h again, neuronal apoptosis is serious; And after nimodipine, salvianolic acid A monomeric compound and the intervention of various dose salvianolic acid A compositions, 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 freeze-dried powder; Show that the salvianolic acid A compositions has the inhibition neuronal apoptosis, suppresses the effect that cerebral ischemia causes rat cerebral tissue's neuronal death, effect is better than salvianolic acid A monomeric compound and nimodipine.

The impact of experimental example 25 salvianolic acid A compositionss on rats after cerebral ischemic reperfusion cerebral tissue neurotrophic factor

Prepare rats after cerebral ischemic reperfusion model, grouping administration with experimental example 24 methods.Ischemia Reperfusion 24h 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 38 salvianolic acid A compositions on rat cerebral tissue's neurotrophic factor protein expression (gray value)

With sham operated rats, compare, #P<0.05, p<0.01; 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 compositions 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 compositions 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 26 salvianolic acid A compositionss on the rats after cerebral ischemic reperfusion inflammatory cytokine

Prepare rats after cerebral ischemic reperfusion model, grouping administration with experimental example 24 methods.Break end and get brain rapidly after Ischemia Reperfusion 24h, 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 39 salvianolic acid A compositions 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 rat cerebral tissue that gives each dosage group of salvianolic acid A compositions obviously reduces than model group, and salvianolic acid A monomeric compound group and the nimodipine group with dosage is remarkable.Prompting salvianolic acid A compositions can suppress the expression of ischemic injuries brain tissue inflammation cytokine, suppress the generation of brain tissue inflammation's reaction, suppress neuron and the cerebral tissue cascade damage of inflammatory reaction mediation, effect is better than salvianolic acid A monomeric compound and nimodipine.

Experimental example 27: the salvianolic acid A compositions is to rats after cerebral ischemic reperfusion cerebral tissue Ca ²⁺, K ⁺, Mg ²⁺the impact of content

Get experimental example 20 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 40. salvianolic acid A compositions 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; With model group, compare, each dosage group of nimodipine group, salvianolic acid A monomeric compound group and salvianolic acid A can significantly reduce Ca in cerebral tissue ²⁺content, rising K ⁺, Mg ²⁺content, and the most remarkable with the salvianolic acid A composition effect.Illustrate that the salvianolic acid A compositions can suppress Ca ²⁺interior stream alleviates calcium overload, thereby can suppress Ca ²⁺the neuronal cell apoptosis that overload is induced.

Experimental example 28: the impact of salvianolic acid A compositions 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 compositions (10mg/kg, 5mg/kg, 2.5mg/kg) group, salvianolic acid A monomeric compound group (10mg/kg), nimodipine matched group (10mg/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 30min and each is administered once through tail vein injection when perfusion starts again before ischemia, sham operated rats and cerebral ischemia re-pouring model group give the normal saline of same volume.After filling with 2h 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 2h 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 41.

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

Each organizes rat after ischemia 2h pours into 2h again; respectively get 8; broken end is got brain fast; broken end is got brain; 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 that a certain proportion of potassium phosphate buffer and methanol, tetrahydrochysene furan ring 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 42.

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 salvianolic acid A combination object height, middle dosage group, its neuronal cell is more complete, the form normal.

2.2 the impact of salvianolic acid A compositions 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).Compare each administration group rat cerebral cortex, striatum NE, DA, 5-HT content obviously raise (P<0.01 or P<0.05) with the cerebral ischemia re-pouring model group.

The impact (x ± s, ng/mg, n=8) of table 41. salvianolic acid A compositions on monoamine neurotransmitter in the rats after cerebral ischemic reperfusion cerebral tissue

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

With sham operated rats, compare, in cerebral ischemia re-pouring model group rat cerebral cortex, the content of Glu, Asp, Gly, GABA, Tau all has remarkable increase (P<0.01 or P<0.05); 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 compositions 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) 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 or P<0.05) than model group, and salvianolic acid A combines object height, middle dosage group is the most remarkable.。

The impact (x ± s, n=8) of table 42. salvianolic acid A compositions on amino acid neurotransmitter in the rats after cerebral ischemic reperfusion cerebral tissue

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

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 compositions 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 compositions 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 compositions 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 the neuronal cell apoptosis that monoamine neurotransmitter is disorderly and toxicity of excitatory amino acid is induced.

Experimental example 29: the impact of salvianolic acid A compositions on LPO, SOD, GSH-Px in the rats after cerebral ischemic reperfusion cerebral tissue

10 remaining rats after each group detects under 28 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 contents (LPO), superoxide dismutase (SOD) and glutathion peroxidase (GSH-Px).

Result is carried out statistical analysis.

Table 43. salvianolic acid A compositions on LPO, SOD, GSH-Px in the rats after cerebral ischemic reperfusion cerebral tissue impact (x ± s, n=10)

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 compositions is compared with model group, and in rat cerebral tissue, LPO content significantly reduces, 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 or P<0.05), 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 compositions 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 that is better than the salvianolic acid A monomeric compound.

Experimental example 30: the salvianolic acid A compositions is protected RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia vascular endothelial cell

Prepare RHRSP Cerebrovascular embolism model, grouping with experimental example 12 methods.The administration volume tail vein injection administration that each group is pressed immediately 0.3ml/100g after model preparation operation, after this every day the tail intravenously administrable once, continue to and draw materials.Sham operated rats, the postoperative tail vein injection saline of model control group; The postoperative tail vein injection salvianolic acid A respectively of the high, medium and low dosage group of salvianolic acid A compositions compositions 40mg/kg, 20mg/kg, 10mg/kg; Salvianolic acid A monomeric compound group injection salvianolic acid A monomeric compound 20mg/kg, nimodipine group injection nimodipine 20mg/kg.Each group is got rat respectively at the postoperative 6h of MCAO, 12h, 24h, 48h, 72h time point and is poured into 4% paraformaldehyde neutral buffered solution through heart, gets 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 that deoxyribose nucleotides art end is transferase mediated) 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 44. salvianolic acid A compositions on RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia apoptosis of vascular endothelial cell

Experimental result shows, the cerebrovascular endothelial cell apoptosis number of each time period of model group is significantly higher than sham operated rats (P<0.01), 6h after the cerebral tissue ischemia, and endothelial cell apoptosis is showed increased just, to ischemia, within 24 hours, reaches peak.With model group, compare, the apoptosis digital display work of each time period of each dosage group of salvianolic acid A compositions reduces (P<0.01), and is less than salvianolic acid A monomeric compound and the nimodipine group of same dosage.Prompting salvianolic acid A compositions 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 31: the impact of salvianolic acid A compositions 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 compositions (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).Cell is by after planting, and 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 compositions 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 45 salvianolic acid A compositions 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<0.01; With the nimodipine group, compare, △ P<0.05,

p<0.01

Experimental result shows, after anoxia 4h damage, the BMVEC survival rate is only 25.45% 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 compositions 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 compositions 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 13.22% left and right, with comparison before reoxygenation, significantly reduces (p<0.01), illustrates that hypoxia/reoxygenation has aggravated cell injury; After each drug effect, the BMVEC survival rate significantly raises (P<0.01), with salvianolic acid A compositions 30,40,50,60 μ mol/L dosage group best results; And salvianolic acid A compositions 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 compositions 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 compositions 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 32 salvianolic acid A compositionss on Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC) apoptosis

Method by experimental example 31: 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 compositions (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 31.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 46 salvianolic acid A compositions causes the impact (x ± s, n=6) of BMVEC apoptosis on Hypoxia/Reoxygenation Injury

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

p<0.05

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 compositions and be certain dose-effect relationship.And salvianolic acid A compositions 10 μ mol/L dosage groups are suitable with nimodipine 40 μ mol/L dosage group effects, with the salvianolic acid A compositions of dosage, be better than the salvianolic acid A monomeric compound.Prompting salvianolic acid A compositions has the effect of the BMVEC apoptosis that good anti-hypoxia-reoxygenation injury causes.

Experimental example 33 salvianolic acid A compositionss affect Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC) secretory function

As described in experimental example 31, 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 compositions on BMVEC secretory tissue fiber proenzyme activator (t-PA), tissue plasminogen activator's inhibitor (PAI), nitric oxide (NO), Endothelin (ET).

Table 47 salvianolic acid A compositions is on Hypoxia/Reoxygenation Injury (BMVEC) secretory function impact (x ± s, n=6)

With Normal group, compare, #P<0.0l, p<0.05; 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 compositions rises to the Normal group level that approaches; Each administration group t-PA/PAI and NO/ET ratio ratio also significantly improve than model group.Prompting salvianolic acid A compositions can be improved 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.

The impact of experimental example 34 salvianolic acid A compositionss on the interior Ca2+ concentration of Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC)

As described in experimental example 31, third generation BMVEC is inoculated in 96 orifice plates to preparation BMVEC hypoxia/reoxygenation model.Grouping and administration are with experimental example 32.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 48 salvianolic acid A compositions 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,

p<0.05 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, the most remarkable with the middle and high dosage group of salvianolic acid A compositions effect, and intracellular calcium concentration is significantly 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 compositions has and suppresses preferably Hypoxia/Reoxygenation Injury and cause Ca in BMVEC born of the same parents ²⁺the effect of concentration.

Experimental example 35: the impact of salvianolic acid A compositions 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 (2.5mg/kg), aspirin group (20mg/kg) and the high, medium and low dosage of salvianolic acid A compositions (12.5,2.5,0.5mg/kg) group.Each organizes tail intravenously administrable 1 time every day (matched group gives the isometric(al) normal saline), continuously after 7d, 30min 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 the 2mA choked flow, stimulates 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 49.

The impact of table 49. salvianolic acid A compositions on the rat artery thrombus formation time

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

Experimental result shows, compares the thrombus formation time significant prolongation (P<0.01) of salvianolic acid A compositions 12.5,2.5,0.5mg/kg dosage group with the normal saline matched group; Low dose group thrombus formation time and 20mg/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 compositions can extend thrombus formation time, the formation of prevention of arterial thrombosis, pre-preventing thrombosis and the ischemic cerebrovascular that causes.

Experimental example 36: the salvianolic acid A compositions is to thrombotic inhibitory action

The SD rat, male and female half and half, grouping, administration are with experimental example 35.30min 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 (5 μ/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 50.

The inhibitory action that table 50. salvianolic acid A compositions forms rat suppository

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

Experimental data shows, with the normal saline group, compares, and salvianolic acid A compositions 12.5,2.5,0.5mg/kg dosage group rat suppository wet, dry weight all significantly alleviates (P<0.01), between each dosage group, have dose-effect relationship preferably.And low dose group and 20mg/kg aspirin group effect be (P>0.05) quite.Prompting salvianolic acid A compositions has the thrombotic effect of good inhibition, can or treat the ischemic cerebrovascular due to thrombosis for prevention, and than salvianolic acid A monomeric compound successful.

Experimental example 37: the impact of salvianolic acid A compositions on the rat vein thromboembolism

Male SD rat, body weight (200 ± 20) g, divide into groups with experimental example 35, pentobarbital sodium (40mg/kg) anesthetized rat, 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, after 1h, the tail vein injection administration; 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 51.

The impact of table 51. salvianolic acid A compositions on the rat vein thromboembolism

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

Experimental data shows, with the normal saline group, compare, salvianolic acid A compositions 12.5,2.5,0.5mg/kg dosage group rat vein thrombosis wet, in the representation work, alleviate (P<0.01), and low dose group and 20mg/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 compositions has the effect that suppresses venous thrombosis, can be used for the venous thromboembolism after the prophylaxis of acute ischemic cerebrovascular occurs.

Experimental example 38: the salvianolic acid A compositions 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 (2.5mg/kg), aspirin group (20mg/kg) and the high, medium and low dosage of salvianolic acid A compositions (12.5,2.5,0.5mg/kg) group, each is organized the every day tail vein injection and gives and relative medicine 1 time, continuous 7 days; 30min after art time 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 52.

Table 52. salvianolic acid A compositions 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 compositions 12.5,2.5, the thrombotic length of 0.5mg/kg dosage group rats in vitro significantly shorten (P<0.01), thrombosis is wet, dry weight significantly alleviates (P<0.01), and low dose group and 20mg/kg aspirin group effect be (P>0.05) quite; Show that the salvianolic acid A compositions 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 39: the thrombolytic experimental study of salvianolic acid A compositions 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 (2000U/kg) group, salvianolic acid A monomeric compound (5mg/kg) group and the high, medium and low dosage of salvianolic acid A compositions (10,5,2.5mg/kg) group; Each group is 20min after forming thrombosis, all through the disposable injection relative medicine of femoral vein, and revascularization situation in 1h after the observation administration; If revascularization in this period, continue to observe vessel open state 1h.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; After logical again in 1h, 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 53.

Thrombolytic effect (n=10) in table 53. salvianolic acid A composition

Annotate: 1. after recanalization rate=administration, logical number of animals/animal number appears in 1h again

2. after the number of animals/administration of thromboembolism again that the bolt rate=logical rear 1h occurs again again in 1h, 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 about thing 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 compositions (5mg/kg) group, its vessel open degree 5mg/kg salvianolic acid A monomeric compound group and similar to 2000U/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 compositions high dose (10mg/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 compositions low dosage (2.5mg/kg) though the group recanalization rate lower than the urokinase group, bolt rate is suitable with the urokinase group again for it, and has lower than the urokinase trend of bolt rate again.The effect of thromboembolism again after the above results prompting salvianolic acid A compositions has preferably thrombolytic and prevents thrombolytic, and effect is more stable.

Experimental example 40: the impact of salvianolic acid A compositions 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, decoct 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 of one trifle, folder closes CCA, by ECA, embolus is injected to ICA, and ligation ECA, unclamp CCA and arteria pterygopalatina place vascular clamp, sews up the incision.Experiment minutes 7 groups: sham operated rats, model control group, salvianolic acid A compositions high, medium and low (10,5,2.5mg/kg) group, salvianolic acid A monomeric compound group (5mg/kg), nimodipine group (10mg/kg).30min after successful surgery, each treated animal tail vein relative medicine, after this inject once continuous 7 days every day; Sham operated rats and model group are injected isopyknic normal saline.The second day (fasting 12h) that administration finishes, intraperitoneal anesthesia, abdominal aortic blood, anticoagulant heparin, carry out Determination of Blood Rheology.

The impact of table 54 salvianolic acid A compositions on the cerebral thrombosis hemorheology of rat

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

p<0.05.

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 compositions 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 compositions 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. the salvianolic acid A compositions is for the preparation of the purposes of protection ischemic tissue of brain damage medicine; in wherein said compositions, salvianolic acid A 94%～97%; alkannic acid 0.1%～1.5%; rosmarinic acid 0.1%～1.5%; salvianolic acid B 0.1%～1.5%; salvianolic acid C 0.1%～2.0%, described salvianolic acid A compositions adopts lower preparation method to obtain:

2. purposes according to claim 1, also comprise the purposes of protecting blood brain barrier.

3. purposes according to claim 1, also comprise the purposes that reduces the ischemic tissue of brain infarction size.

4. purposes according to claim 1, also comprise the purposes that alleviates the ischemic tissue of brain edema.

5. according to the described purposes of claim 1～4 any one, wherein said compositions is to be made by following weight proportion: salvianolic acid A 95%～96%, alkannic acid 0.1%～1.2%, rosmarinic acid 0.1%～1.2%, salvianolic acid B 0.1%～1.2%, salvianolic acid C 0.1%～1.5%.

6. according to the described purposes of claim 1～4 any one, microwave vacuum drying temperature: 20-100 ℃ wherein, return difference temperature 1-5 ℃, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.

7. purposes according to claim 6, microwave vacuum drying temperature: 50-85 ℃ wherein, return difference temperature 2-4 ℃, more than vacuum-0.07Mpa, microwave power 10-80KW, dry 100-150 minute.

8. purposes according to claim 7, microwave vacuum drying temperature: 55-80 ℃ wherein, return difference temperature 2-3 ℃, more than vacuum-0.07Mpa, microwave power 25-60KW, dry 120-140 minute.

9. according to the described purposes of claim 1～8 any one, it is characterized in that adopting the high effective liquid chromatography for measuring salvianolic acid A, alkannic acid, rosmarinic acid, salvianolic acid B, salvianolic acid C content, condition determination is as follows:

The 18 ethyl group silane group silica gel of take are filler;

Theoretical cam curve should be not less than 10000 by salvianolic acid A;