CN103006575B - Application of salvianolic acid A lyophilized powder injection in preparation of drugs for improving neurological function symptoms after cerebral ischemia - Google Patents

Application of salvianolic acid A lyophilized powder injection in preparation of drugs for improving neurological function symptoms after cerebral ischemia Download PDF

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CN103006575B
CN103006575B CN201210487346.XA CN201210487346A CN103006575B CN 103006575 B CN103006575 B CN 103006575B CN 201210487346 A CN201210487346 A CN 201210487346A CN 103006575 B CN103006575 B CN 103006575B
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salvianolic acid
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dried powder
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cerebral
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CN103006575A (en
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钟阳桂
王章伟
吕武清
刘尧奇
蔡元魁
刘地发
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Jiangxi Qingfeng Pharmaceutical Co., Ltd.
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JIANGXI QINGFENG PHARMACEUTICAL CO Ltd
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Abstract

The invention relates to application of salvianolic acid A lyophilized powder injection in preparation of drugs for improving neurological function symptoms after cerebral ischemia. The salvianolic acid A lyophilized powder injection comprises, by weight, 20-60g of salvianolic acid A, 20-60g of filling agent, and an antioxidant accounting for 0.02%-0.1% of the total amount of the finished product.

Description

Salvianolic acid A freeze-dried powder is for the preparation of the purposes of improving the function of nervous system's symptom medicine after cerebral ischemia
Technical field
The present invention relates to a kind of salvianolic acid A freeze-dried powder for the preparation of the purposes of improving the function of nervous system's symptom medicine after cerebral ischemia.
Background technology
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, cerebral tissue impaired and occur series of symptoms.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 that neurocyte, glial cell and contact fiber are due to the degeneration of blood supply disorder generation, the afunction that property is crossed in necrosis or.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), due to the supply of 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.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 uses just effectively in 4.5 hours in apoplexy; But also exist hemorrhage and Ischemia Reperfusion to increase the weight of the danger of brain injury.And comprise that for the neuroprotective drug of ischemic stroke treatment calcium channel blocker is if nimodipine, glutamate receptor antagonists are if dizocilpine (dizocilPine), antioxidant or free radical scavenger are as Edaravone, NO signal transduction pathway regulator lu pei Shandong mile (Iubeluzole) and inflammation inhibitor enlimomab (enlimomab) etc. at present.But the therapeutical effect having in them is imprecise or specificity is not strong, some toxic and side effects compared with 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.Bring into play very important positive role at this field Chinese medicine.
Salvianolic acid A (Salvianolic acid A), have another name called Salvianolic acid A, it is a kind of water-soluble phenolic compounds contained in the dry root and rhizome of labiate Radix Salviae Miltiorrhizae Salviamiltiorrhiza Bunge, salvianolic acid A has obvious pharmacological action to cerebral microvascular thromboembolism, and effect be better than salvianolic acid B (Zhang Hengai. impact and the Mechanism Study [D] of salvianolic acid A on cerebral microvascular thromboembolism rat. Beijing: Beijing Union Medical College graduate school, 2011), salvianolic acid A is to be formed by danshensu and caffeic acid condensation, contain multiple phenolic hydroxyl groups, hydroxyl, the reactive groups such as ester bond, it is unstable to light and heat, the oxidizable one-tenth salvianolic acid C of exposure air and different salvianolic acid C etc., due to the above-mentioned physicochemical property of salvianolic acid A, make salvianolic acid A injection agent, its stability becomes the key technology of making injection.But particularly injection Research Literature data is few for relevant salvianolic acid A preparation, and the instable problem of ubiquity injection, cannot ensure salvianolic acid A pharmacological action.
Summary of the invention
The above-mentioned defect existing for overcoming prior art, the invention provides a kind of salvianolic acid A freeze-dried powder for the preparation of the purposes of improving the function of nervous system's symptom medicine after cerebral ischemia, wherein, the weight proportion of described salvianolic acid A freeze-dried powder is: salvianolic acid A 20g～60g, filler 20g～60g, antioxidant is to make 0.02%～0.1% of total amount;
The preparation method of described salvianolic acid A freeze-dried powder is:
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 DEG C of water temperature lixiviates are got at every turn, stir with 10～50 revs/min of speed simultaneously, or add 3～15 times of amount water boiling and extraction, 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 DEG C), adds ethanol to make to contain alcohol amount 50%～85%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, obtains Radix Salviae Miltiorrhizae extract; Or
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 is also concentrated into without alcohol taste, 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 DEG C of temperature thermal conversions 1～6 hour;
Conversional solution adjust pH to 2.5～4.5, leave standstill, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 1～10mg, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 35～1: 70 with macroporous adsorbent resin ratio, 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 is also concentrated into without alcohol taste,
Aqueous solution is concentrated into the solution of every 1ml containing 1-10mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 5～1: 25 with polyamide ratio, 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 is also concentrated into without alcohol taste 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, point 2～6 extractions, separate organic layer, reclaim under reduced pressure t-butyl methyl ether, makes the extract of every 1ml containing salvianolic acid A 1g～10g, add 1～3 times of amount silica gel, stir, volatilize;
Be added on 5～20 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 4～1: 25, taking 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, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 5～20 times of water gagings dissolvings, microwave vacuum drying, obtains described salvianolic acid A;
Getting described salvianolic acid A 10g～80g injects water 1500～2800ml and is stirred to dissolve, by adjusting PH with base value 4.0～5.0, add described filler to make its dissolving, add again described antioxidant, be stirred to dissolve and mix, then add active carbon 0.5～2g stirring and adsorbing, filter and remove active carbon, inject fill after water and become bottle, send into and in freeze dryer, carry out lyophilization;
Described lyophilization comprises the steps:
A, freezing: be cooled to-40 DEG C～-45 DEG C with 20 DEG C～30 DEG C/h speed, be incubated freezing 10～16 hours;
B, distillation: be evacuated to below 0.3mbar, in 10～14 hours, the salvianolic acid A formulation temperature freezing risen to-23 DEG C～-27 DEG C, maintain-23 DEG C～-27 DEG C vacuum dryings 6～8 hours;
C, dry: continue to heat up, be evenly warming up to 40 DEG C～45 DEG C with 0.5 DEG C～1.0 DEG C/min, maintain 40 DEG C～45 DEG C and be dried after 2～5 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Preferably, its weight proportion is: salvianolic acid A 20g～40g, and filler 20g～40g, antioxidant is to make 0.03%～0.08% of total amount.
Preferably, wherein said filler is selected from any one or a few in mannitol, glucose, lactose, and consumption is 20mg～40mg/2ml～3ml.
Preferably, wherein said antioxidant selects any one or a few in white vitamin C, thiourea, sodium sulfite, sodium pyrosulfite.
Preferably, its weight proportion is: salvianolic acid A 20g, filler mannitol 20g, antioxidant vitamin C 0.8g; Its preparation method is:
Get described salvianolic acid A 20g, inject water 1900ml and be stirred to dissolve, with sodium hydroxide sodium adjust pH 4.0～5.0, add mannitol 20g to make to dissolve, then add 0.8g vitamin C, be stirred to dissolve and mix, add active carbon 0.5g stirring and adsorbing, filter and remove active carbon; Inject water to 2000ml, fill becomes 1000 bottles, lyophilization;
Describedly be frozen in dry comprising the steps:
A, freezing: be cooled to-45 DEG C with 25 DEG C/h speed, be incubated freezing 15 hours;
B, distillation: be evacuated to below 0.3mbar, in 12 hours, the salvianolic acid A formulation temperature freezing risen to-25 DEG C, maintain-25 DEG C of vacuum dryings 8 hours;
C, dry: continue to heat up, be evenly warming up to 40 DEG C with 0.8 DEG C/min, maintain 40 DEG C and be dried after 3 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Preferably, wherein microwave vacuum drying temperature: 20-100 DEG C, return difference temperature 1-5 DEG C, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.
Preferably, wherein microwave vacuum drying temperature: 50-85 DEG C, return difference temperature 2-4 DEG C, more than vacuum-0.07Mpa, microwave power 10-80KW, dry 100-150 minute.
Preferably, wherein microwave vacuum drying temperature: 55-80 DEG C, return difference temperature 2-3 DEG C, more than vacuum-0.07Mpa, microwave power 25-60KW, dry 120-140 minute.
Salvianolic acid A freeze-dried powder principal agent provided by the invention is salvianolic acid A, the present invention is by the extraction to Radix Salviae Miltiorrhizae, transform, purification, drying process, obtain salvianolic acid A raw material: through screening and the optimization of system, first extraction solvent and the extracting method of initiation material salvianolic acid B have relatively been determined, because salvianolic acid B water solublity is better, employing water extraction or low concentration alcohol extraction are determined, again because salvianolic acid B heat stability is poor, determine that employing hot water warm macerating extracts and adds stirring extracting method, or use low-concentration ethanol reflux, extract, make to extract solubility lower than 100 DEG C, keep salvianolic acid B not to be destroyed, optimum solvent consumption and extraction time are determined by orthogonal experiment, obtain 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 feed intake for initiation material, do not need salvianolic acid B to carry out transforming again after purification, be in catalytic conversion reaction of the present invention, reaction raw materials salvianolic acid B does not need high-purity, for example, do not need purity >=50% of salvianolic acid B.It is generally acknowledged that reaction raw materials is more pure better, but, of the present invention experiment showed, in catalytic conversion reaction, the purity height of salvianolic acid B does not affect conversion reaction effect.On the contrary, when salvianolic acid B purity > 50%, not only produce a large amount of impurity, and do not improve conversion ratio.
Moreover the present invention by experiment repeatedly relatively, has first determined that factor that salvianolic acid A productive rate is produced to material impact is as transformed the concentration, pH value, temperature, time etc. of front pressure differential self.On this basis, be studied 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 again, and these factors how synergism exerts an influence to salvianolic acid A productive rate jointly each other, thereby determine that salvianolic acid B transforms the optimum temperature of the needs control of salvianolic acid A, pH value, time etc., and 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 is low good.But, of the present invention experiment showed, in catalytic conversion reaction, the concentration height of salvianolic acid B does not affect conversion reaction effect.On the contrary, salvianolic acid B concentration height not only produces a large amount of impurity, and does not improve conversion ratio.And not more high better containing the concentration of salvianolic acid B in salvianolic acid B aqueous solution, the above concentration conversion ratio of 30mg/ml is low on the contrary, and effect is poorer.Therefore, technique of the present invention, aspect cost-saving and production cycle, has unforeseeable technique effect.In the situation that prior art does not provide the enlightenment of any technology, if those skilled in the art only infer theoretically, be impossible draw the conclusion that sour pellet B is changed into main part of raw material of salvianolic acid A under above-mentioned each conditional parameter and have better changing effect.
What is more important, the present invention passes through performing creative labour, find that zinc chloride can significantly improve the conversion ratio of main part of raw material of salvianolic acid B conversion salvianolic acid A as catalyst, reaching that conversion ratio is highly stable approaches 60%, most cases can exceed 60%, this is all impossible in any one prior art in the past, therefore, has obtained unforeseeable technique effect.
Because salvianolic acid A content is lower, the large raising of salvianolic acid A content after transforming, but also containing a large amount of impurity, therefore, select 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, remove after impurity part, salvianolic acid A content is brought up to 80% from 10% left and right, to 90%, to 93%, to 96%.
Further, in significantly improving conversion ratio, the present invention is by being suitable for the purification step of actual industry application, especially by after the steps such as a series of separation, eluting processing, do not adopt traditional constant pressure and dry, and preferred microwave vacuum drying, microwave vacuum drying method dry from vacuum drying, spraying, thereby thoroughly overcome in the past in dry excess Temperature the defect that drying time is long and large to the destruction of salvianolic acid A; And sublimation drying is long, cost extract high and lyophilization gained cannot be removed dissolvent residual problem completely.
The present invention can also be by directly mixing low concentration with the salvianolic acid B of high concentration, only need be made into suitable initial conversion concentration, can reach equally the object that changes into salvianolic acid A, the preparation technology of this conversion raw material is very simple, and production cost is very suitable for the application in actual industry when reduction.
Salvianolic acid A freeze-dried powder provided by the invention, according to the chemistry of salvianolic acid A and physical characteristic, from affecting the stable additives of medicine, dosage form, container, extraneous as air, light, moisture, the generation chemical reactions such as impurity cause the decomposition of medicament to carry out creationary experimental analysis on the contrary.By screening preparation prescription, repetition test, mannitol, glucose, lactose etc. are selected, make freeze-dried powder, the molding of powder pin is good, and block is loose, hole, color and luster are even, and stability is very high, solve due to air, light, moisture, the medicine that the generation chemical reactions such as impurity cause decomposes.Salvianolic acid A is made to suitable preparation, solved salvianolic acid A and made injection stability problem, by selecting suitable adjuvant and dosage form, ensured salvianolic acid A pharmacological action, provide safe and effective salvianolic acid A injection formulation for clinical.
For these reasons, we by selecting suitable dosage form, have screened different adjuvants according to the physicochemical property of salvianolic acid A, and preferred different preparation technology and technological parameters, has prepared stable, safe, effective, quality controllable salvianolic acid A freeze-dried powder.
Salvianolic acid A in the present invention is adjusted to applicable scope by alkali liquor by pH value, then by adding mannitol etc., makes it be easier to lyophilization, and do not affect the property of medicine.
The present invention is compared with the prior art and shows: the present invention passes through performing creative labour, finally determined lyophilizing speed cooling rate and cooling time, programming rate and temperature when distillation, determine the vacuum sublimation time, clear and definite dry programming rate and temperature, and drying time; The creationary complex relationship of clearly having determined between frozen cooling speed and temperature, distillation programming rate and the dry programming rate of temperature and temperature and time, and suitable numerical range is definite etc., thus just finally obtain stable lyophilized injectable powder.
What is more important, adopts the salvianolic acid A freeze-dried powder that preparation method of the present invention is made can not change the original chemical attribute of salvianolic acid A completely; The salvianolic acid A freeze-dried powder compositions of making has the features such as good water solubility, heat stability is high, solubility is good compared with salvianolic acid A.
On the other hand, the present invention also provides it for preventing and or the purposes for the treatment of ischemic cerebrovascular aspect.
Known through 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 cage wall, push away trunk reaction, the reaction of antenna to stimulation etc.) significantly lower than sham operated rats, illustrate that after 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 to be starkly lower than sham operated rats, the each dosage group of nimodipine group and salvianolic acid A freeze-dried powder is compared with model group, 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 freeze-dried powder, and after its ischemia, the neurobehavioral of 24h approaches sham operated rats level substantially, recovered normally to the each dosage group of 72h salvianolic acid A freeze-dried powder rat neurobehavioral after ischemia, prompting salvianolic acid A freeze-dried powder can improve neurobehavioral after RHRSP cerebral thrombosis ischemia, the effect of nervous symptoms after having protection and improving cerebral ischemia.
Known through histopathology checking experiment Data Comparison, sham operated rats cerebral tissue Bilateral Symmetry, has no pathological changes, and model group Intravascular Thrombus adheres to seriously.With model group comparison, the contraction in length of the each dosage group of Microscopic observation salvianolic acid A freeze-dried powder rat ischemia side middle cerebral artery (MCA) local thrombus, reduces at 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 and dwindles than model control group is remarkable in the each dosage group of salvianolic acid A freeze-dried powder.HE dyes has obvious cerebral tissue softening in visible model group ischemia side cortex MCA blood supply district (centered by the cortex of volume top) infarct, necrocytosis, the phenomenons such as local necrosis tissue comes off, salvianolic acid A freeze-dried powder high dose group rat has no Telatrophy's obscission herein; The HE dyeing of the each dosage group of salvianolic acid A freeze-dried powder 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 the each dosage group of salvianolic acid A freeze-dried powder and model group comparison hypertrophy significantly increases; In addition, the kitchen range shape that HE dyeing display model matched group differs in size is as seen hemorrhage, and high, the middle dosage group of salvianolic acid A freeze-dried powder there is no and finds that there is kitchen range shape bleeding.Result shows that salvianolic acid A freeze-dried powder can obviously improve RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia histopathology state; reduce thrombosis bond area, reduce infarct size; thereby increase in infarcted region and the little blood vessel number of surrouding brain tissue, reduce the necrosis of cerebral hemorrhage phenomenon protection cerebral tissue and come off, thering is the effect of protecting cerebral thrombosis ischemia to cause brain tissue impairment.
Known through 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 freeze-dried powder; under each dosage group rat cerebral tissue microscope, EB hot spot number and cerebral tissue EB detect content and obviously reduce; more all there were significant differences with model group; illustrate that salvianolic acid A freeze-dried powder causes blood-brain barrier permeability to brain tissue impairment and increases inhibited; can protect blood brain barrier, and the further damaged of protection cerebral tissue.。
Known through experimental data contrast, by the comparison of RHRSP cerebral thrombosis rat model group, sham operated rats has no infarcted cerebral constitution; The each dosage group of salvianolic acid A freeze-dried powder cerebral tissue Infarction volume 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 freeze-dried powder basic, normal, high dosage group Infarction volume and brain water content are all lower than nimodipine group.Illustrate that salvianolic acid A freeze-dried powder can accelerate the reparation of focus, reduce ischemic tissue of brain infarction size after RHRSP cerebral thrombosis, alleviate cerebral tissue edema, have and repair and the effect of protection Neurons Against Cerebral Ischemia tissue injury.
Known through experimental data contrast, with the comparison of RHRSP cerebral thrombosis ischemia model group, after ischemia treatment, the each dosage group of salvianolic acid A freeze-dried powder cerebral tissue ischemia penumbra MVD and blood vessel scene are long-pending significantly to be increased to some extent, and after drug withdrawal, the interior numerical value of 48h is more stable.Show that salvianolic acid A freeze-dried powder can increase MVD and the blood vessel field Area Ratio of cerebral tissue ischemia half blanking bar, show that salvianolic acid A freeze-dried powder has the effect that promotes that ischemic tissue of brain Angiogenesis and collateral circulation are set up.
Known through experimental data contrast; RHRSP cerebral thrombosis ischemia model group VEGF Messenger RNA (VEGFmRNA) expression is increased significantly compared with sham operated rats; illustrate after cerebral tissue hypoxic-ischemic can Stimulation of The Brain tissue in the expression increase of VEGF; after prompting cerebral infarction, body self there will be a kind of protective response that resists ischemia injury; the expression of VEGF is increased, after ischemia, occur self " compensatory revascularization ".The each dosage group of salvianolic acid A freeze-dried powder and the comparison of model group group; VEGFmRNA expression significantly raises; show that salvianolic acid A freeze-dried powder can significantly promote ischemic tissue of brain angiogenesis; promote the foundation of collateral circulation compensation; save ischemia half blanking bar; the brain tissue impairment that protection ischemia causes, and there is certain dose-effect relationship.
Known through experimental data contrast; RHRSP cerebral thrombosis ischemia model group rat cerebral tissue's basic fibroblast growth factor (bFGF) protein expression is increased significantly compared with sham operated rats; but with the prolongation of Ischemia Time; have a declining tendency; after prompting cerebral tissue hypoxic-ischemic; body self produces of short duration protection stress, can organize bFGF protein expression to increase by Stimulation of The Brain.The each dosage group of salvianolic acid A freeze-dried powder and model group comparison, the bFGF protein expression of each time period significantly increases; Each time period of each dosage group self relatively shows, bFGF protein expression is continual and steady; Prompting salvianolic acid A freeze-dried powder can increase ischemic tissue of brain bFGF protein expression, has the effect of good neurocyte protection effect and promotion angiogenesis, contributes to the foundation of collateral circulation, saves ischemia half blanking bar.
Known through experimental data contrast, salvianolic acid A freeze-dried powder can alleviate cerebral ischemia-reperfusion injury in rats neurologic impairment symptom, is conducive to its neurobehavioral recovery, and salvianolic acid A freeze-dried powder has the effect that improves function of nervous system after brain tissue impairment.
Known through experimental data contrast, the each dosage group of salvianolic acid A freeze-dried powder is compared with Ischemia-Reperfusion Injury Model group: cerebral infarction volume is than significantly reducing, cerebral index and brain water content all obviously reduce, show that salvianolic acid A freeze-dried powder can reduce rats after cerebral ischemic reperfusion cerebral tissue infarction size, can alleviate the cerebral tissue edema degree after cerebral ischemia reperfusion injury.
Known through experimental data contrast, Level In Rats With Focal Cerebral Ischemia, give various dose salvianolic acid A 10min and begin, each medicine group compared with self administration before ischemia half blanking bar regional cerebral blood flow (rCBF) have obvious rising, and particularly remarkable with high, the middle dosage group of salvianolic acid A freeze-dried powder; The each dosage group of salvianolic acid A freeze-dried powder is compared, and has good dose-effect relationship; In salvianolic acid A freeze-dried powder, the rCBF of dosage group day part is suitable with nimodipine group.Hence one can see that, and salvianolic acid A freeze-dried powder 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.
Known through experimental data contrast, after rat continuous cerebral ischemia 2h recovers to fill with again, respectively organizing rat ischemia half blanking bar rCBF all has increase in various degree.After filling with, nimodipine, the high, medium and low dosage group of salvianolic acid A freeze-dried powder rat rCBF obviously increase again, and each time period rCBF has utmost point significant difference compared with model group.1h after filling with again, nimodipine and salvianolic acid A freeze-dried powder high dose group rCBF return to approach former rCBF 75%, in salvianolic acid A freeze-dried powder dosage group return to be about former rCBF 69%, salvianolic acid A freeze-dried powder low dose group returns to and is about 61% of former rCBF, and to pouring in 3h, each medicine group rCBF maintains in metastable scope again.Result shows that salvianolic acid A freeze-dried powder 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 ischemic cerebrovascular is had to good therapeutical effect.
Known through 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 freeze-dried powder, reduce cerebral tissue lactic acid (LA) content, can significantly improve the energy metabolism of rat cerebral ischemia and ischemia-reperfusion.Salvianolic acid A freeze-dried powder 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, save the brain cell that ischemia half blanking bar after cerebral ischemia is at death's door, further damage is even dead to prevent cerebral tissue, can perform well in treating ischemic cerebrovascular.
Known through experimental data contrast, rat cerebral ischemia 2h fills with after 24h again, and neuronal apoptosis is serious; And after nimodipine and the intervention of various dose salvianolic acid A freeze-dried powder, with model group comparison, in rat cerebral tissue, neuronal apoptosis significantly reduces (P < 0.001 or P < 0.01); Show that salvianolic acid A freeze-dried powder has the effect that suppresses cerebral ischemia rat cerebral tissue neuronal death, suppresses neuronal apoptosis.
Neurotrophic factor is a neure growth histone matter molecule essential with survival, and the integrity of neure growth, growth and function is played to supporting function.Nerve growth factor (NGF), Brain Derived Neurotrophic Factor (BDNF), neurotrophic factor-3 (NT-3) are parts for neurotrophic factor family, can maintain neuronal survival and promote neurocyte differentiation and induction axon growth; Energy neuroprotective unit, promotion neuron reparation and inhibition delayed neuronal death, thereby to anti-cerebral ischemia, protection cerebral ischemia.After Ischemia Reperfusion, in rat cerebral tissue, increase to some extent compared with sham operated rats, after prompting cerebral reperfusion injury, in cerebral tissue, NGF, BDNF express stress protectiveness increase; But in Neurons Against Cerebral Ischemia tissue, NT-3 content obviously reduces.Known through experimental data contrast; with model control group comparison; the each dosage group of salvianolic acid A freeze-dried powder NGF, BDNF, NT-3 protein expression all obviously strengthen; prompting salvianolic acid A freeze-dried powder can strengthen the expression of ischemic injuries cerebral tissue endogenous neurotrophic factor NGF, BDNF, NT-3, reaches the effect of neuro-protective.
After ischemia-reperfusion, 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 experimental data contrast, the each inflammatory cytokine content of rat cerebral tissue that gives the each dosage group of salvianolic acid A freeze-dried powder obviously reduces compared with model group.Prompting salvianolic acid A freeze-dried powder can suppress the expression of ischemic injuries brain tissue inflammation cytokine, suppresses the generation of brain tissue inflammation's reaction, suppresses neuron and the cerebral tissue cascade damage of inflammatory reaction mediation.
Known through 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 comparison, the each dosage group of nimodipine group and salvianolic acid A freeze-dried powder can significantly reduce Ca in cerebral tissue 2+content, rising K +, Mg 2+content, illustrates that salvianolic acid A freeze-dried powder can suppress Ca 2+interior stream alleviates calcium overload, thereby can suppress Ca 2+the neuronal cell apoptosis of overload induction.1
Known through experimental data contrast; salvianolic acid A freeze-dried powder 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 in rising cerebral ischemia/reperfusion injury of rats cerebral tissue, significantly reduces content and the aminoacid exitotoxicity index of cerebral tissue Glutamic Acid (Glu), day red winter propylhomoserin (Asp), glycine (Gly).And dosage increases, effect strengthens.The salvianolic acid A freeze-dried powder showing in conjunction with histopathology pathological examination can obviously alleviate cerebral edema, improves the result of neuronal cell form, shows that salvianolic acid A freeze-dried powder can suppress monoamine neurotransmitter and excessively discharge, and improves 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.
Known through experimental data contrast, the each dosage group of salvianolic acid A freeze-dried powder is compared with ischemia-reperfusion injury model group, lipid peroxide in rat cerebral tissue (LPO) content significantly reduces, and superoxide dismutase (SOD) and glutathion peroxidase (GSH-Px) activity significantly raise; Illustrate that salvianolic acid A freeze-dried powder can increase the generation of the activity of peroxide scavenger enzyme in ischemical reperfusion injury cerebral tissue, inhibition peroxide, thus the damage to neuronal cell and cerebral tissue to antioxidant radical.
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 cerebral tissue ischemia, and just showed increased of endothelial cell apoptosis reaches peak for 24 hours to ischemia.With model group comparison, the apoptosis digital display work of each time period of the each dosage group of salvianolic acid A freeze-dried powder reduces, illustrate that salvianolic acid A freeze-dried powder can suppress cerebral ischemia hindbrain apoptosis of vascular endothelial cell, improve cerebrovascular endothelial cell survival rate, thereby ensure 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 experimental data contrast, after salvianolic acid A freeze-dried powder and nimodipine effect, anoxia and Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial survival rate significantly raise, and salvianolic acid A freeze-dried powder group survival rate is significantly higher than nimodipine group.Prompting salvianolic acid A freeze-dried powder can be protected microvascular endothelial cells oxygen and Hypoxia/Reoxygenation Injury, strengthens its hypoxia-bearing capability, improves the brain microvessel endothelial cells in vitro survival rate of anoxia-induced apoptosis and Hypoxia/Reoxygenation Injury.
Known through experimental data contrast, Hypoxia/Reoxygenation Injury, can cause the each phase apoptosis rate of Brain Microvascular Endothelial significantly to increase.With model group comparison, the each dosage group of salvianolic acid A freeze-dried powder and nimodipine group all can significantly lower cell early, late period and total apoptosis rate, and be certain dose-effect relationship.And salvianolic acid A freeze-dried powder 10 μ mol/L dosage groups are suitable with nimodipine 40 μ mol/L dosage group effects.Prompting salvianolic acid A freeze-dried powder has the effect of the Brain Microvascular Endothelial apoptosis that good anti-hypoxia-reoxygenation injury causes.
After the each dosage group of salvianolic acid A freeze-dried powder 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) compared with model group, and wherein the middle and high dosage group of salvianolic acid A freeze-dried powder rises to the Normal group level that approaches; Each administration group t-PA/PAI and NO/ET ratio also significantly improve compared with model group.Prompting salvianolic acid A freeze-dried powder can improve the secretory function of brain microvessel endothelial cells in vitro after Hypoxia/Reoxygenation Injury.
After Hypoxia/Reoxygenation Injury, Ca in BMVEC born of the same parents 2+concentration significantly increases.Known through experimental data contrast, with model group comparison, each medicine group Ca 2+concentration extremely significantly reduces, the most remarkable with the middle and high dosage group of salvianolic acid A freeze-dried powder effect, and intracellular calcium concentration is significantly lower than nimodipine 40 μ mol/L dosage groups.Prompting salvianolic acid A freeze-dried powder has the Hypoxia/Reoxygenation Injury of inhibition and causes Ca in BMVEC born of the same parents 2+the effect of concentration.
Known through experimental data contrast, impact and the normal saline matched group comparison of salvianolic acid A freeze-dried powder on rat artery thrombus formation time, the thrombus formation time significant prolongation of the high, medium and low dosage group of salvianolic acid A freeze-dried powder; Low dose group (0.5mg/kg) thrombus formation time is suitable with 20mg/kg aspirin group; Show that salvianolic acid A freeze-dried powder can extend thrombus formation time, the formation of prevention of arterial thrombosis, pre-preventing thrombosis and the ischemic cerebrovascular that causes.
Known through experimental data contrast, impact and the comparison of normal saline group that salvianolic acid A freeze-dried powder forms rat suppository, the high, medium and low dosage group of salvianolic acid A freeze-dried powder rat suppository is wet, dry weight all significantly alleviates.And low dose group is suitable with 20mg/kg aspirin group effect.Illustrate that salvianolic acid A freeze-dried powder has the thrombotic effect of good inhibition, can be used for preventing or treat the ischemic cerebrovascular due to thrombosis.
Known through experimental data contrast, impact and the normal saline group comparison of salvianolic acid A freeze-dried powder on rat vein thromboembolism, the high, medium and low dosage group of salvianolic acid A freeze-dried powder rat vein thrombosis is wet, dry weight significantly alleviates, low dose group (0.5mg/kg) and quite (P > 0.05) of 20mg/kg aspirin group effect; Show that salvianolic acid A freeze-dried powder has the effect that suppresses venous thrombosis, can be used for the venous thromboembolism after prophylaxis of acute ischemic cerebrovascular occurs.
Known through experimental data contrast, salvianolic acid A freeze-dried powder is to rats in vitro thrombosis and the comparison of normal saline group, the thrombotic length of the high, medium and low dosage group of salvianolic acid A freeze-dried powder 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 salvianolic acid A freeze-dried powder forms and has good inhibitory action external thrombus.
Known through experimental data contrast, thrombolytic and the normal saline group comparison of salvianolic acid A freeze-dried powder to established thrombosis in body, normal saline group all continues thromboembolism, and without occurring again logical phenomenon; The number of animals that each medicine group continues thromboembolism is few, all has utmost point significant difference compared with normal saline group; Dosage group in salvianolic acid A freeze-dried powder, its vessel open degree is similar to 2000U/kg urokinase group, and its recanalization rate is suitable; The lasting recanalization rate of salvianolic acid A freeze-dried powder high dose group and recanalization rate are all higher than urokinase group, and bolt rate is starkly lower than urokinase group again; Though salvianolic acid A freeze-dried powder low dose group recanalization rate is lower than urokinase group, bolt rate is suitable with urokinase group again for it, and has lower than the urokinase trend of bolt rate again.Illustrate salvianolic acid A freeze-dried powder have good thrombolytic and prevent thrombolytic after the effect of thromboembolism again.
After cerebral thrombosis, rat whole blood viscosity, plasma viscosity significantly increase, and packed cell volume also significantly raises, known through experimental data contrast, the each dosage group of salvianolic acid A freeze-dried powder and model group comparison, its whole blood viscosity and plasma viscosity and packed cell volume all obviously reduce; Prompting salvianolic acid A freeze-dried powder can be accelerated micro-blood flow velocity, and reduction blood viscosity improves hemorheology and the effect that effectively resists cerebral thrombosis.
In sum, salvianolic acid A freeze-dried powder of the present invention has obvious therapeutic effect to ischemic cerebrovascular, and ischemic cerebrovascular described in the present invention comprises the pathological changes that affects cerebral circulation that the various causes of disease form and the damaged infringement of the ischemic rat brain as master taking neuronal death and function of nervous system causing thereof.Mainly comprise any one or more: (1) cerebral thrombosis: 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 by the embolus from heart.(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 cannot not do under the prerequisite of related experiment in advance, learn that in advance salvianolic acid A freeze-dried powder has above-mentioned good purposes.
Brief description of the drawings
Fig. 1. salvianolic acid B reference substance high-efficient liquid phase chromatogram;
Fig. 2. salvianolic acid B high-efficient liquid phase chromatogram in Radix Salviae Miltiorrhizae extract;
Fig. 3. salvianolic acid A reference substance high-efficient liquid phase chromatogram;
Fig. 4. salvianolic acid A high-efficient liquid phase chromatogram in salvianolic acid B catalyzed conversion liquid.
Fig. 5. salvianolic acid A freeze-dried powder freeze-drying curve figure.
Fig. 6. salvianolic acid A freeze-dried powder vacuum curve figure.
Detailed description of the invention
Embodiment 1
Get red rooted salvia, be ground into 6 order granules, add 92 DEG C of water of 7 times of amounts at every turn, warm macerating extracts 3 times, stirs with 25 revs/min of speed simultaneously, and each warm macerating extracts 3 hours, extracting solution is evaporated to relative density 1.20 (60 DEG C), adds ethanol to make to contain alcohol amount 70%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, 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%ZnCl 2as catalyst, 120 DEG C 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, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 50 with macroporous adsorbent resin ratio, 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 is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 5mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 10 with polyamide ratio, 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 is also 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, point 3 extractions, separate organic layer, and 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, be added on 15 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 10, taking 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, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 12 times of water gagings dissolvings, with microwave vacuum drying (60 DEG C of baking temperatures, 3 DEG C of return difference temperature, more than vacuum-0.07Mpa, microwave power 20KW, dry 100 minutes) dry 130 minutes, obtain salvianolic acid A.
Embodiment 2
Get red rooted salvia, be cut into decoction pieces, add 85 DEG C of water temperature lixiviates of 8 times of amounts at every turn and get 3 times, stir simultaneously with 20 revs/min of speed, each warm macerating extracts 2.5 hours, extracting solution is evaporated to relative density 1.16 (60 DEG C), adds ethanol to make to contain alcohol amount 75%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 15mg, aqueous solution is adjusted pH to 4.0 with 10% potassium hydroxide, adds 0.6%ZnCl 2as catalyst, 120 DEG C 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, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 45 with macroporous adsorbent resin ratio, 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 is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 5mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 9 with polyamide ratio, 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 is also concentrated into every 1ml containing salvianolic acid A 12mg aqueous solution, aqueous solution is adjusted pH to 2.6 with 15% hydrochloric acid, and by the t-butyl methyl ether of 4 times of amounts, point 4 extractions, separate organic layer, and 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, be added on 13 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 8, taking 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, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 8 times of water gagings dissolvings, with microwave vacuum drying (50 DEG C of baking temperatures, 3 DEG C of return difference temperature, more than vacuum-0.07Mpa, microwave power 20KW) dry 140 minutes, obtain salvianolic acid A.
Embodiment 3
Get red rooted salvia, be ground into diameter 2mm granule, add 85 DEG C of water temperature lixiviates of 9 times of amounts at every turn and get 2 times, stir simultaneously with 30 revs/min of speed, each warm macerating extracts 3.5 hours; Extracting solution is evaporated to relative density 1.10 (60.DEG C), add ethanol to make to contain alcohol amount 75%, leave standstill, filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 18mg, aqueous solution is adjusted pH to 4.2 with 10% sodium carbonate, adds 0.6%ZnCl 2as catalyst, 123 DEG C 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, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 40 with macroporous adsorbent resin ratio, 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 is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 6mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 8 with polyamide ratio, 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 is also 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, point 4 extractions, separate organic layer, and 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, be added on 10 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 7, taking 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, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 8 times of water gagings dissolvings, with microwave vacuum drying (60 DEG C of baking temperatures, 1 DEG C of return difference temperature, more than vacuum-0.07Mpa, microwave power 10KW) dry 110 minutes, obtain salvianolic acid A.
Embodiment 4
Get red rooted salvia, be cut into decoction pieces, add 80 DEG C of water temperature lixiviates of 10 times of amounts at every turn and get 3 times, stir simultaneously with 15 revs/min of speed, each warm macerating extracts 3 hours, extracting solution is evaporated to relative density 1.15 (60 DEG C), adds ethanol to make to contain alcohol amount 70%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 20mg, aqueous solution is adjusted pH to 5.4 with 10% sodium bicarbonate, adds 0.8%ZnCl 2as catalyst, 128 DEG C 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, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 40 with macroporous adsorbent resin ratio, 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 is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 6mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 10 with polyamide ratio, 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 is also 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, point 4 extractions, separate organic layer, and 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, be added on 9 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 8, taking 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, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 10 times of water gagings dissolvings, with microwave vacuum drying (80 DEG C of baking temperatures, 5 DEG C of return difference temperature, more than vacuum-0.07Mpa, microwave power 60KW) dry 100 minutes, obtain salvianolic acid A.
Embodiment 5
Get red rooted salvia, be ground into diameter 2mm granule, add 85 DEG C of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir simultaneously with 20 revs/min of speed, each warm macerating extracts 2.5 hours, extracting solution is evaporated to relative density 1.12 (60 DEG C), adds ethanol to make to contain alcohol amount 70%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, 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 DEG C 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, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 35 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 8, use respectively 3 times of cylinder hydrops, 4.5 times of column volume 25% ethanol elutions, 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 is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 8mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 12 with polyamide ratio, 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 is also 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, point 5 extractions, separate organic layer, and reclaim under reduced pressure t-butyl methyl ether is made the extract of every lml containing salvianolic acid A 0.5g, adds 2 times of amount silica gel, stirs, and volatilizes, be added on 10 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 10, with 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, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 12 times of water gagings dissolvings, with microwave vacuum drying (45 DEG C of baking temperatures, 3 DEG C of return difference temperature, more than vacuum-0.07Mpa, microwave power 60KW) dry 150 minutes, obtain salvianolic acid A.
Embodiment 6
Get red rooted salvia, be ground into diameter 2mm granule, add 88 DEG C of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir simultaneously with 22 revs/min of speed, each warm macerating extracts 3.5 hours, extracting solution is evaporated to relative density 1.23 (60 DEG C), adds ethanol to make to contain alcohol amount 75%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 13mg, aqueous solution is adjusted pH to 5.6 with 10% sodium hydroxide, adds 0.5%ZnCl 2as catalyst, 133 DEG C 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, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 40 with macroporous adsorbent resin ratio, 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 is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 10mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 15 with polyamide ratio, 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 is also 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, point 5 extractions, separate organic layer, and 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, be added on 12 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 10, with pentane, t-butyl methyl ether is eluant, gradient elution, use respectively 6 times of column volumes of E pentane-t-butyl methyl ether (4: 6) eluting, 7 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 10 times of water gagings dissolvings, with microwave vacuum drying (70 DEG C of baking temperatures, 2 DEG C of return difference temperature, more than vacuum-0.07Mpa, microwave power 5KW) dry 120 minutes, , obtain salvianolic acid A.
Embodiment 7
Get red rooted salvia, be cut into decoction pieces, add 90 DEG C of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir simultaneously with 25 revs/min of speed, each warm macerating extracts 3 hours, extracting solution is evaporated to relative density 1.20 (60 DEG C), adds ethanol to make to contain alcohol amount 75%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 20mg, aqueous solution is adjusted pH to 5.0 with 10% sodium carbonate, adds 1.0%ZnCl 2as catalyst, 135 DEG C 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, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 36 with macroporous adsorbent resin ratio, 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 is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every 1ml containing 6mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 10 with polyamide ratio, 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 is also 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, point 4 extractions, separate organic layer, and 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, be added on 10 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 8, with 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, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 8 times of water gagings dissolvings, with microwave vacuum drying (55 DEG C of baking temperatures, 2 DEG C of return difference temperature, more than vacuum-0.07Mpa, microwave power 70KW) dry 90 minutes, obtain salvianolic acid A.
Embodiment 8
Get red rooted salvia, be ground into diameter 2mm granule, add 85 DEG C of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir simultaneously with 2 revs/min of speed, each warm macerating extracts 3 hours, extracting solution is evaporated to relative density 1.14 (60 DEG C), adds ethanol to make to contain alcohol amount 70%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 25mg, aqueous solution is adjusted pH to 4.2 with 10% sodium citrate, adds 0.4%ZnCl 2as catalyst, 133 DEG C of temperature thermal conversions 4.5 hours, 10% hydrochloric acid adjust pH to 2.8 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 45 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 10, use respectively 5 times of cylinder hydrops, 6 times of column volume 20% 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 is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every 1ml containing 8mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 12 with polyamide ratio, resin column blade diameter length ratio is 1: 8, use respectively 3 times of cylinder hydrops, 6 times of column volume 45% alcoholic solution eluting remove impurity, use 8 times of column volumes, 55% alcoholic solution eluting again, collect the 55% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 10mg aqueous solution, aqueous solution is adjusted pH to 2.9 with 15% hydrochloric acid, and by the t-butyl methyl ether of 5 times of amounts, point 5 extractions, separate organic layer, and reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.6g, adds 2.5 times of amount silica gel, stirs, and volatilizes, be added on 12 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 8, with 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, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 9 times of water gagings dissolvings, with microwave vacuum drying (40 DEG C of baking temperatures, 1 DEG C of return difference temperature, more than vacuum-0.07Mpa, microwave power 80KW) dry 130 minutes, obtain salvianolic acid A.
Embodiment 9
Get the salvianolic acid A 80g that embodiment 3 makes, inject water 2800ml and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.5, add mannitol 60g to make to dissolve, add again vitamin C 0.5g, be stirred to dissolve, mix, add active carbon 2g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-40 DEG C with 20 DEG C/h speed, be incubated freezing 16 hours; Be evacuated to below 0.3mbar, in 10 hours, the salvianolic acid A formulation temperature freezing risen to-25 DEG C, maintain-25 DEG C of vacuum dryings 8 hours; Continue to heat up, be evenly warming up to 41 DEG C with 0.5 DEG C/min, maintain 41 DEG C and be dried after 3 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 10
Get the salvianolic acid A 20g that embodiment 4 makes, inject water 1900ml and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.8, add mannitol 20g to make to dissolve, add again vitamin C 0.8g, be stirred to dissolve, mix, add active carbon 0.5g stirring and adsorbing, filter, remove active carbon; Inject water to 2000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-45 DEG C with 25 DEG C/h speed, be incubated freezing 15 hours; Be evacuated to below 0.3mbar, in 12 hours, the salvianolic acid A formulation temperature freezing risen to-25 DEG C, maintain-25 DEG C of vacuum dryings 8 hours; Continue to heat up, be evenly warming up to 40 DEG C with 0.8 DEG C/min, maintain 40 DEG C and be dried after 3 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 11
Get the salvianolic acid A 10g that embodiment 5 makes, inject water 1500ml and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.6, add mannitol 20g to make to dissolve, add again vitamin C 0.8g, be stirred to dissolve, mix, add active carbon 1.2g stirring and adsorbing, filter, remove active carbon; Inject water to 2000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-42 DEG C with 25 DEG C/h speed, be incubated freezing 12 hours; Be evacuated to below 0.3mbar, in 13 hours, the salvianolic acid A formulation temperature freezing risen to-23 DEG C, maintain-23 DEG C of vacuum dryings 7 hours; Continue to heat up, be evenly warming up to 42 DEG C with 0.8 DEG C/min, maintain 42 DEG C and be dried after 4 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 12
Get the salvianolic acid A 30g that embodiment 6 makes, inject water 2000ml and be stirred to dissolve, with 20% sodium carbonate adjust pH 4.2, add mannitol 20g, lactose 10g to make to dissolve, add again thiourea 0.5g, be stirred to dissolve, mix, add active carbon 1.0g stirring and adsorbing, filter, remove active carbon; Inject water to 2000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-44 DEG C with 29 DEG C/h speed, be incubated freezing 14 hours; Be evacuated to below 0.3mbar, in 11 hours, the salvianolic acid A formulation temperature freezing risen to-26 DEG C, maintain-26 DEG C of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 43 DEG C with 0.6 DEG C/min, maintain 43 DEG C and be dried after 3.5 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 13
The salvianolic acid A 35g that embodiment 7 makes, injects water 2500ml and is stirred to dissolve, and with 10% potassium hydroxide adjust pH 4.5, adds glucose 30g to make to dissolve, add again sodium sulfite 3g, be stirred to dissolve, mix, add active carbon 1.5g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-43 DEG C with 27 DEG C/h speed, be incubated freezing 11 hours; Be evacuated to below 0.3mbar, in 12.5 hours, the salvianolic acid A formulation temperature freezing risen to-24 DEG C, maintain 24 DEG C of vacuum dryings 7.5 hours; Continue to heat up, be evenly warming up to 40 DEG C with 0.7 DEG C/min, maintain 40 DEG C and be dried after 5 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 14
Get the salvianolic acid A 40g that embodiment 8 makes, injecting water 2700ml is stirred to dissolve, with 10% sodium hydroxide adjust pH 4.3, add glucose 20g, lactose 20g to make to dissolve, then add sodium pyrosulfite 4g, be stirred to dissolve, mix, add active carbon 1.5g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-41 DEG C with 28 DEG C/h speed, be incubated freezing 13 hours; Be evacuated to below 0.3mbar, in 13.5 hours, the salvianolic acid A formulation temperature freezing risen to-25.5 DEG C, maintain-25.5 DEG C of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 44 DEG C with 0.9 DEG C/min, maintain 44 DEG C and be dried after 3.5 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 15
Get the salvianolic acid A 60g that embodiment 1 makes, inject water 2700ml and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.0, add mannitol 40g to make to dissolve, add again Catergen g, be stirred to dissolve, mix, add active carbon 2g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-42.5 DEG C with 24.5 DEG C/h speed, be incubated freezing 11.5 hours; Be evacuated to below 0.3mbar, in 13.5 hours, the salvianolic acid A formulation temperature freezing risen to-24.5 DEG C, maintain-24.5 DEG C of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 42.5 DEG C with 0.55 DEG C/min, maintain 42.5 DEG C and be dried after 2.5 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 16
Get the salvianolic acid A 70g that embodiment 2 makes, inject water 2800ml and be stirred to dissolve, with 10% sodium hydroxide adjust pH 5.0, add mannitol 40g to make to dissolve, add again Catergen g, be stirred to dissolve, mix, add active carbon 2g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-44.5 DEG C with 28.5 DEG C/h speed, be incubated freezing 11.5 hours; Be evacuated to below 0.3mbar, in 13.5 hours, the salvianolic acid A formulation temperature freezing risen to-24.5 DEG C, maintain-24.5 DEG C of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 43.5 DEG C with 0.75 DEG C/min, maintain 43.5 DEG C and be dried after 3 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Experimental example 1: salvianolic acid A, the research of salvianolic acid B analytical method
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 are m);
Reagent: methanol is chromatographically pure, water is ultra-pure water prepared by Millipore, other reagent are analytical pure.
Salvianolic acid B reference substance (lot number 111562-201009) is all purchased from Nat'l Pharmaceutical & Biological Products Control Institute, for assay; 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
The preparation of 2.1 reference substance solution: precision takes salvianolic acid B, the about 10mg of salvianolic acid A reference substance respectively, puts in 100ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, in contrast product stock solution; Precision is drawn above-mentioned each 1ml respectively again, puts in same 10ml measuring bottle, adds methanol and is diluted to scale, shakes up, as mixing reference substance solution.
The preparation precision of 2.3 need testing solutions takes embodiment 1 sample (being approximately equivalent to salvianolic acid A 10mg) and following " 1.3 salvianolic acid B raw material preparations in experimental example 2 " obtain sample (being approximately equivalent to salvianolic acid B 10mg), put in 100ml measuring bottle, add dissolve with methanol and be diluted to scale, shake up, to obtain final product.
3. chromatographic condition and system suitability
Taking octadecylsilane chemically bonded silica as filler; Flow velocity 1.0ml/min; Detect wavelength: get mixing reference substance solution, carry out UV scanning, result has absorption maximum at 286nm wavelength place, therefore determine that detecting wavelength is 286nm; 30 DEG C of column temperatures; Number of theoretical plate calculates and should be not less than 10000 by salvianolic acid A peak.
0-10 minute time, the ratio of methanol is down to 60% by 30% ratio that rises to 40%, 0.1-0.5% phosphate aqueous solution by 70%; 10-30 minute time, the ratio of methanol is down to 45% by 40% ratio that rises to 55%, 0.1-0.5% phosphate aqueous solution by 50%; 30-60 minute time, 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 salvianolic acid A, salvianolic acid B reference substance and Radix Salviae Miltiorrhizae extract, salvianolic acid catalyzed conversion liquid is in table 1, and HPLC collection of illustrative plates is shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4.
The chromatographic peak retention time result of the each reference substance of table 1.
Figure BSA00000811717500241
4, the investigation of linear relationship
Accurate above-mentioned mixing reference substance solution 0.1ml, 0.2ml, 0.5ml, 1ml, 2ml, the 5ml of drawing, put respectively in 10ml measuring bottle, add methanol and be diluted to following concentration and be about: the series standard solution of 0.001mg/ml, 0.002mg/ml, 0.005mg/ml, 0.01mg/ml, 0.02mg/ml, 0.05mg/ml.The accurate each 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, taking peak area integrated value as vertical coordinate, each concentration reference substance sample size is abscissa, drawing standard curve respectively.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
Figure BSA00000811717500251
5. precision test
Get 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
Figure BSA00000811717500252
6. stability test
Get need testing solution, measure according to " 3. chromatographic condition and system suitability " lower chromatographic condition, at regular intervals sample introduction once, in result 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
Figure BSA00000811717500253
7. replica test
Get this salvianolic acid A raw material, 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
Figure BSA00000811717500261
8. recovery test
Adopt application of sample recovery test, get the salvianolic acid A raw material 10mg of known content, accurately weighed, parallel 6 parts, add a certain amount of reference substance solution in the ratio of each constituent content-reference substance (1: 1) 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
Figure BSA00000811717500262
Experimental example 2: the extraction of salvianolic acid B
1.1 extract the confirmation of solvent and extracting method
Take red rooted salvia 400g, measure content of danshinolic acid B by method under Chinese Pharmacopoeia Radix Salviae Miltiorrhizae item, be divided into 4 parts, test by following testing program:
Experiment 1: get red rooted salvia 100g, add 8 times of water gagings at every turn and extract 1.5 hours at 80 DEG C 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 DEG C of warm macerating, stir simultaneously with 10～50 revs/min of speed, 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,, measures 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,, measures 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 extraction solvent salvianolic acid B extraction ratio is had to impact, 50% ethanol extraction is better than water extraction, but with water temperature soak add stir extract difference little, two kinds of extracting modes that stir in water extraction process and do not stir have impact to the extraction ratio of salvianolic acid B, decocting extraction may be because temperature is higher, and salvianolic acid B is extracted and has impact.
The preferred extraction process of 1.2 orthogonal experiment
1.2.1 the optimization of water extraction process research: according to above result of the test, water extraction process using extraction time (A), extraction time (B), quantity of solvent (C), extract four of temperature (D) as investigation factor, each factor is established three levels, by 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
Figure BSA00000811717500272
Table 9. extraction process orthogonal experiments table
Figure BSA00000811717500281
Table 10. the results of analysis of variance
Figure BSA00000811717500282
F 0.05(2，2)＝19.00F 0.01(2，2)＝99.00
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, the warm macerating extraction 1～3 time for adding 3～15 times of water gagings, at 45～95 DEG C of the water extraction condition of receiving salvianolic acid B of the present invention, stir with 10～50 revs/min of speed simultaneously, extract 1～4 hour at every turn or add 3～15 times of amounts at every turn and decoct extraction, each extraction 1～4 hour, extracts 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 using four of extraction times (A), extraction time (B), quantity of solvent (C), concentration of alcohol (D) as investigation factor, each factor is established three levels, by 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
Figure BSA00000811717500291
Table 12. extraction process orthogonal experiments
Figure BSA00000811717500292
Table 13. the results of analysis of variance
F 0.05(2，2)19.00F 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 preparations
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 DEG C of warm macerating, stir with 10～50 revs/min of speed simultaneously, warm macerating stirs and extracts 3 times altogether, filters merging filtrate, extracting solution is evaporated to relative density 1.10～1.25 (60 DEG C), add ethanol to make to contain alcohol amount 60%, filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, vacuum drying, salvianolic acid extract 4.15kg, recording content of danshinolic acid B is 10.25%.
Experimental example 3: salvianolic acid B transforms the comparison of salvianolic acid A technique
Experiment 1: get the about 30g of salvianolic acid B raw material of preparation under 1.3, be mixed with 200ml solution, add 10% sodium hydroxide solution adjust pH to 4.5;
Experiment 2: get the about 30g of salvianolic acid B raw material of preparation under 1.3, be mixed with 200ml solution, add 10% sodium hydroxide solution adjust pH to 4.5, add 1.0%ZnCl2;
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, 1202 times reactions 4.0 hours, cooling, calculates salvianolic acid A productive rate.
Table 14. salvianolic acid B transforms salvianolic acid A experimental result
Figure BSA00000811717500302
Figure BSA00000811717500311
The demonstration of table 14 result, salvianolic acid B high-purity affects not transforming, does not need to be purified to more than 50% to transform, and salvianolic acid B is converted in salvianolic acid A process, regulates pH value, adds 1.0%ZnCl 2, can greatly improve the productive rate of salvianolic acid A.
Experimental example 4: salvianolic acid B transforms salvianolic acid A process optimization
Above-mentioned experimentation proves, salvianolic acid B purity is not to affect salvianolic acid B to transform the factor of salvianolic acid A, but adds certain catalysts influence salvianolic acid B to transform salvianolic acid A, and therefore, we all add 1%ZnCl to each experimental group 2as catalyst, be converted into other factors of salvianolic acid A 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, by L9 (3 4) orthogonal table carries out EXPERIMENTAL DESIGN (table 15, table 16), the productive rate that to investigate index be salvianolic acid A.
Table 15. transforming agent water-glass
Figure BSA00000811717500312
Table 16. conversion process orthogonal experiments
Figure BSA00000811717500313
Table 17. the results of analysis of variance table
Figure BSA00000811717500322
*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 productive rate, analyzes from K value, concentration is converted into salvianolic acid A on improving salvianolic acid B effect higher than 30mg/ml does not affect, and the impurity forming is more, therefore, before transforming, salvianolic acid B concentration should be selected 1～30mg/ml; (C (temperature) has utmost point significant difference to salvianolic acid A productive rate for factor B (pH), factor, strictly pellet pH processed and temperature in prompting salvianolic acid B conversion process, can successfully realize transforming to salvianolic acid A of salvianolic acid B higher yields.
Experimental example 5: catalyst Z nCl 2consumption is on the impact transforming
Transform salvianolic acid A process optimization condition by above-mentioned salvianolic acid B, get 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 DEG C, and transformation time is 4 hours, by with 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
Figure BSA00000811717500331
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%, more preferably, after 0.5%～2.0%. catalyst amount > 3%, conversion ratio is no longer significantly improved.
Experimental example 6: catalyst transforms the catalytic action of salvianolic acid A to salvianolic acid B
Transform salvianolic acid A process optimization condition by above-mentioned salvianolic acid B, get 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 DEG C, and transformation time is 4 hours, add respectively the catalyst of different cultivars and various dose to transform, the results are shown in Table 19.
Table 19. different catalysts transforms the impact of red A on red B
Figure BSA00000811717500332
Table 19 result shows, above 4 kinds of catalyst all can be used as the catalyst in salvianolic acid B conversion reaction, and each catalyst amount and salvianolic acid B molar percentage be 0.5%～3.0%, productive rate >=40% of salvianolic acid A.Conversion ratio exceedes 60%, but Comprehensive Assessment adopts ZnCl 2optimum.
Experimental example 7: the purification with macroreticular resin of salvianolic acid A
Get salvianolic acid A and transform 13 parts of solution, put in each model macroporous resin 100g that pretreatment is good, after shaking table dynamic adsorption 8 hours, 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 eluent part, carry out salvianolic acid A assay, eluent evaporate to dryness calculates dry cream amount, the results are shown in Table 20.
The confirmation of table 20. macroporous resin model
Figure BSA00000811717500351
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 eluent that content is greater than 75%, with the HPD-100 amount of getting dry extract maximum, content is high, and resin absorption amount is large.Experimental example 8: the polyamide column chromatography purification of salvianolic acid A
Get 3 parts of salvianolic acid A solution after purification with macroreticular resin, put in each model resin 100g that pretreatment is good, after shaking table dynamic adsorption 8 hours, 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 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
Figure BSA00000811717500352
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, obtains content and is about 90% salvianolic acid A eluent; Maximum with the polyamide amount of getting dry extract, content high adsorption capacity is large.
Experimental example 9: the abstraction purification of salvianolic acid A
By 70% ethanol elution decompression recycling ethanol after column chromatography purification for the second time, adjust pH 2.0～4.0, use again n-butyl alcohol, t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate, Ethyl formate, ethanol extraction 5 times, Separation of Organic phase, reclaim solvent, dry, obtain salvianolic acid A, measure salvianolic acid A content, the results are shown in Table 22.
The confirmation of organic solvent for table 22. extraction
Figure BSA00000811717500353
Figure BSA00000811717500361
Table 22 result shows: n-butyl alcohol is because polarity is large, salvianolic acid A amount is maximum, but its salvianolic acid A content is not improved significantly, ether is because polarity is less than normal, water-solubility impurity is few, and salvianolic acid A content is high, but the salvianolic acid A amount obtaining is few, the salvianolic acid A amount that other extraction solvent t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate, Ethyl formate obtain is larger, and salvianolic acid A content is all improved; Obtain with t-butyl methyl ether that extract is many, salvianolic acid A content is high.
Experimental example 10: the purification by silica gel column chromatography of salvianolic acid A
Get 12 parts of salvianolic acid A extracts after abstraction purification, every part containing salvianolic acid A 10g, adds the silica gel of 20g, stirs, and volatilizes; Be added on the dry silicagel column of the 100g having installed stirring sample silica gel, the two-phase solvent forming taking petroleum ether, pentane, normal heptane, ethyl acetate, methyl acetate, Ethyl formate, t-butyl methyl ether is respectively as eluant, HPLC or thin layer chromatography detect, collect salvianolic acid A eluent, 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
Figure BSA00000811717500362
Figure BSA00000811717500371
Result shows: when salvianolic acid A is used normal phase silica gel column chromatography, adopting the two-phase solvent of pentane-t-butyl methyl ether composition is eluant, and gradient elution, can well remove impurity, obtains highly purified salvianolic acid A eluent.，
Experimental example 11: salvianolic acid A drying means research
Get 4 parts of eluents after silica gel purification eluting, every part containing salvianolic acid A 100g, is concentrated into without thick paste shape, adds after 10 times of water gagings dissolve, to adopt respectively that vacuum drying, lyophilisation, spraying are dry, microwave vacuum drying obtains salvianolic acid A, this salvianolic acid A is detected, the results are shown in Table 24.
Table 24. salvianolic acid A drying means testing result
Result shows: adopt lyophilisation overlong time, and high cost, and organic solvent residual is serious; The vacuum drying time is slightly long, and Drying Time of Vertical Spray Dryer is short but instantaneous temperature is higher; Microwave vacuum drying baking temperature is low, and the time is short, and gained salvianolic acid A indices is good.
Through further experiment is definite, microwave vacuum drying optimum range is chosen as temperature: 20-1002, and return difference temperature 1-5 DEG C, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.Experimental example 12: the Preliminary screening of salvianolic acid A freeze-dried powder adjuvant
Freeze-dried powder generally need add appropriate amount of auxiliary materials, adjuvant mainly comprises filler and antioxidant, be mainly used in following object: the dissolubility that increases composition in injection, when the molding of powder pin, need add filler as supporter, improve mouldability, regulate powder pin admittedly containing thing weight, regulate osmotic pressure, add antioxidant to keep principal agent stable simultaneously.
Get salvianolic acid A raw material by table 25 and make freeze-dried powder from different filleies and vitamin C.
Table 25. filler of writing out a prescription is sieved and washed table
Figure BSA00000811717500381
Get salvianolic acid A by table 25 formula, add the water for injection of making total amount 90% and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.5, add adjuvant to make to dissolve, mix, add active carbon 2g stirring and adsorbing, filtration, removes active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-42 DEG C with 28 DEG C/h speed, be incubated freezing 12 hours; Be evacuated to below 0.3mbar, in 11 hours, the salvianolic acid A formulation temperature freezing risen to-25 DEG C, maintain-25 DEG C of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 40 DEG C with 0.8 DEG C/min, maintain 40 DEG C and be dried after 5 hours, sample temperature is down to room temperature, add a cover, observe mouldability, in table 26.
The different filleies of table 26 affect result to mouldability
Figure BSA00000811717500382
Figure BSA00000811717500391
From table 26 experimental result, add not commensurability filler, larger on the impact of mouldability, filler mannitol, glucose, lactose are better to lyophilized formulations effect, lyophilized powder sedimentation is good, porous nickel, therefore as selecting adjuvant, but general lower than 20mg mouldability and solubility.
Experimental example 13: different filleies and the antioxidant impact on lyophilized formulations type
According to above-mentioned experimental result, select respectively mannitol, glucose, lactose to carry out confirmatory study, and antioxidant is compared to research, select respectively vitamin C, thiourea, sodium sulfite, sodium pyrosulfite as antioxidant, experimental program is in table 27, and compare with not adding antioxidant prescription, experimental result is in table 28.
The different filleies of table 27 and antioxidant Preliminary screening
Figure BSA00000811717500392
Figure BSA00000811717500401
Get salvianolic acid A by table 27 formula, add the water for injection of making total amount 90% and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.5, add filler and antioxidant to make dissolving, mix, add active carbon 2g stirring and adsorbing, filtration, removes active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-42 DEG C with 25 DEG C/h speed, be incubated freezing 14 hours; Be evacuated to below 0.3mbar, in 10～14 hours, the salvianolic acid A formulation temperature freezing risen to-25 DEG C, maintain-23 DEG C of vacuum dryings 7 hours; Continue to heat up, be evenly warming up to 44 DEG C with 0.6 DEG C/min, maintain 44 DEG C and be dried after 2 hours, sample temperature is down to room temperature, add a cover, observe mouldability, in table 28.
The different filleies of table 28 and the antioxidant result that affects on profiled member and the quality of the pharmaceutical preparations
Figure BSA00000811717500402
Figure BSA00000811717500411
Table 28 experimental result shows, adds not commensurability filler mannitol, glucose, lactose better to preparations shaping, and lyophilized powder sedimentation is good, porous nickel, consumption select 20mg～40mg/2ml～3ml mouldability and solubility all better.Add content on main constituent of water-soluble vitamin c, thiourea, sodium sulfite, sodium pyrosulfite and other compositions all without impact, add salvianolic acid A stable content after antioxidant, other compositions are unchanged, and preparation effect is more excellent.
Experimental example 14: lyophilisation condition research
3.1 lyophilisation conditions are preferred: in freezing conditions, mainly contain two factors: the impact on mouldability: the temperature of freezing body before freezing speed, vacuum drying; Impact on freeze drying rate: heating curve when vacuum freeze-drying.
Preferably following on freeze dryer:
A, medicinal liquid are cooled to-40 DEG C～-45 DEG C with the chilling rate of 20-30 DEG C/h in freeze dryer, be incubated freezing 10-16 hour, be evacuated to below 0.3mbar, in 10～14 hours, the salvianolic acid A formulation temperature freezing is risen to 23 DEG C～-27 DEG C, maintain-23 DEG C～-27 DEG C vacuum dryings 6～8 hours;
B, medicinal liquid are first cooled to-25 DEG C with the chilling rate of 20-30 DEG C/h in freeze dryer, be incubated freezing 5～8 hours, be refrigerated to again-40 DEG C～-45 DEG C, be incubated freezing 5～8 hours, be evacuated to below 0.3mbar, in 10～14 hours, the salvianolic acid A formulation temperature freezing is risen to-23 DEG C～-27 DEG C, maintain-23 DEG C～-27 DEG C vacuum dryings 6～8 hours;
C, medicinal liquid are cooled to-15 DEG C with the chilling rate of 20-30 DEG C/h in freeze dryer, be incubated freezing 5～8 hours, be refrigerated to again-40 DEG C～-45 DEG C, be incubated freezing 5～8 hours, be evacuated to below 0.3mbar, in 10～14 hours, the salvianolic acid A formulation temperature freezing is risen to-23 DEG C～-27 DEG C, maintain-23 DEG C～-27 DEG C vacuum dryings 6～8 hours;
Above three kinds of result of the tests: 1, medicinal liquid is directly refrigerated to-40 DEG C～-45 DEG C with 20-30 DEG C/h, and powder body is more even, and without crack, drying time is short.2, powder body is inhomogeneous, has crack, and 3, freeze-drying time is long, powder body is inhomogeneous, has crack.Therefore, while selecting medicinal liquid freezing, be directly down to-40 DEG C～45 DEG C with the chilling rate of 20-30 DEG C/h, powder body is more even, without crack, can save freeze-drying time.
3.2 pilot scale freeze-drying curves
Freeze-drying curve refers to the relation curve of product temperature in freeze-drying process or shelf temperature time to time change.The number of the shape of freeze-drying curve and the performance of product, loading amount, the many factors such as kind and appointed condition of separation container are relevant.The freeze dryer that we select in the time of middle trial production is 4000 bottles of scales, substantially meets large production requirement.Cooled the temperature to-40 DEG C～-45 DEG C at 1～4 hour, at this temperature, keep 10～16 hours, again 10～14 hours by temperature rise to-23 DEG C～-27 DEG C and keep 6～8 hours, then at 6～9 hours by temperature rise to 40 DEG C～45 DEG C and keep 2～5 hours, freeze-drying curve is as shown in Figure 5 and Figure 6.
Experimental example 15: preparation stabilization Journal of Sex Research
Get embodiment 10,11,12 samples, by the requirement of stability of drug products experimentation relevant technologies, by above three lot number samples at ambient temperature, by 0 month, 1 month, 2 months, 3 months, 6 months intervals, the situation of change of making regular check on sample property, solubility and clarity, pH value, salvianolic acid A content, other compositions, the results are shown in Table 29.
Table 29 stability experiment result
Figure BSA00000811717500421
The demonstration of table 29 stability experiment observed result, lyophilized injectable powder at ambient temperature salvianolic acid A is stable, and other compositions are also without significant change, and preparation appearance character is good, and preparation solubility is good, and pH value is stable, solution clarification.
Below experiment all gets with salvianolic acid A freeze-dried powder the sample that embodiment 14 obtains.Experimental example 16: the impact of function of nervous system's symptom after salvianolic acid A freeze-dried powder 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: Sigma company of the U.S.; 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 processing: JVCky-F3OB3-CCD coloured image is shot with video-corder 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 two narrow renal artery of kidney double fastener method of male SD rat of 2～3 monthly ages, body weight (100 ± 20) g and set up RH RSP model: rat abdominal cavity anesthesia, through the long otch of the about 5cm of xiphoid-process Ventral Midline stringer, cut skin, abdominal muscle, blunt separation bilateral renal arteries, the annular silver brain clip that is 0.3mm with internal diameter is clamped respectively bilateral renal arteries initial part.Before operation, measuring blood pressure once, is surveyed once weekly continuous 12 weeks after operation.Separately get 10 S that body weight is suitable) rat, survey its blood pressure as normal control.Reject: in operation and postoperative death, 12 weeks after operation blood pressure is lower than 160rmmHg rat, have 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.Blood pressure >=160mmHg and be successful model without the RHRSP of apoplexy symptom after 16 weeks.Cause middle cerebral artery occlusion (MCAO) model to cause middle cerebral artery (MCA) thrombosis with photochemical method the RHRSP being successfully prepared: 2% pentobarbital sodium by 40mg/kg body weight intraperitoneal injection of anesthesia after, warp/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, can know and see left MCA trunk and branch thereof through cerebral dura mater.Dosage injection 2.5% tiger of pressing 40mg/kg body weight through femoral vein is red, green laser beam (the spot diameter 2mm of the 532nm occurring by 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 under operating microscope, see that local white thrombus points out this MCA thrombosis, the success of MCAO model copy.In art, rat anus temperature remains on (37 ± 0.3) DEG C.Local wound is doubted and is closed the front penicillin diluent flushing of using, 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 divide 6 groups at random by animal: sham operated rats, model control group, the high, medium and low dosage group of salvianolic acid A freeze-dried powder, nimodipine group.The sham operated rats window that only opens seam, does not do laser irradiation; Model group, the high, medium and low dosage group of salvianolic acid A freeze-dried powder are prepared easy apoplectic type renovascular hypertension in rats MCAO model by model preparation method.And be administered once by 0.3ml/l00g tail vein injection respectively in the postoperative 30min of MCAO model, be after this 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 freeze-dried powder freeze-dried powder 20mg/kg, 10mg/kg, 5mg/kg, the postoperative tail vein injection nimodipine of nimodipine group 10mg/kg.
2.2 result
18 points of point systems according to Garcia etc. are marked: the symmetry of spontaneous activity, quadruped locomotion, forelimb stretch the action of creeping symmetry, climb cage wall, push away trunk reaction, antenna to the reaction stimulating.In first three items, be 0 point without movable or reaction (extremity or suffering limb), a little activity is 1 point, and reaction calibration constant is 2 points, and activity is normally 3 points; Rear three reactionless be 1 point, activity calibration constant is 2 points, activity is normally 3 points.Total score is the highest 18 points, minimum 3 points.After the postoperative rat anesthesia of MCAO is revived, scoring once, is respectively carried out a neuroethology scoring (at every turn all marking 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 rat body state simultaneously, measure body weight and blood pressure.
Table 30 salvianolic acid A freeze-dried powder on RHRSP cerebral thrombosis after the impact (x ± s) of neuroethology scoring
Figure BSA00000811717500441
Figure BSA00000811717500451
Note: with sham operated rats comparison, #P < 0.01, ※ P < 0.05; With model group comparison, * P < 0.01, ☆ P < 0.05
Experimental result shows: after model group is revived, the neuroethology of (in 12h) is marked significantly lower than sham operated rats (P < 0.01), after cerebral thrombosis ischemia is described, RHRSP function of nervous system is impaired serious, and in lasting 72h, neuroethology scoring continues to be starkly lower than sham operated rats (P < 0.05); The each dosage group of nimodipine group and salvianolic acid A freeze-dried powder is compared with model group, neuroethology scoring after rat revives has rising in various degree, with nimodipine group and the most obviously (P < 0.01) of the middle and high dosage group of salvianolic acid A freeze-dried powder, and after its ischemia, the neuroethology scoring of 24h approaches sham operated rats level substantially; Recovered normal to the each dosage group of 72h salvianolic acid A freeze-dried powder rat neuroethology scoring after ischemia, difference still has statistical significance (P < 0.05) compared with model group.Above results suggest, after salvianolic acid A freeze-dried powder can improve RHRSP cerebral thrombosis ischemia, neuroethology is marked, the effect of nervous symptoms after prompting salvianolic acid A freeze-dried powder has protection and improves cerebral ischemia.
Experimental example 17: the impact of salvianolic acid A freeze-dried powder on RHRSP cerebral thrombosis tissues following MCAO in rats pathological change
The preparation of RHRSP Cerebrovascular embolism model, grouping, medication are with experimental example 16.After neuroethology scoring finishes the last time, rat broken end is got brain, and under operating microscope, observes MCA local thrombus situation.Put into afterwards smooth vessel be built in-20 DEG C freezing, row crown section.Mus brain section, every thick about 2mm.Brain sheet is put into rapidly to 2%TTC solution, hatch at 37 DEG C, the every face of brain sheet is hatched 15min, and normal cerebral tissue peony, and ischemic infarction cerebral tissue is white in color.Taking out brain sheet observes and takes pictures.Then cerebral tissue is placed in to 4% paraformaldehyde PBS buffer (pH7.3) fixing, row dehydration afterwards, paraffin embedding, get cerebral tissue and do paraffin section and do conventional H E staining analysis, micro-Microscopic observation, takes pictures.The results are shown in Table 31.
The impact of table 31 salvianolic acid A freeze-dried powder on RHRSP cerebral thrombosis tissues following MCAO in rats pathological change
Figure BSA00000811717500452
Figure BSA00000811717500461
Real cold junction fruit shows: sham operated rats cerebral tissue Bilateral Symmetry, have no pathological changes, and model group Intravascular Thrombus adheres to seriously.With model group comparison, the contraction in length of the left MCA local thrombus of the each dosage group of Microscopic observation salvianolic acid A freeze-dried powder rat, reduces at 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 and dwindles than model control group is remarkable in the each dosage group of salvianolic acid A freeze-dried powder.HE dyes has obvious cerebral tissue softening in visible model group left cortical MCA blood supply district (centered by the cortex of volume top) infarct, necrocytosis, the phenomenons such as local necrosis tissue comes off, salvianolic acid A freeze-dried powder high dose group rat has no Telatrophy's obscission herein, low, the middle dosage group of salvianolic acid A freeze-dried powder necrocytosis is less, the accidental obscission of organizing; The HE dyeing of the each dosage group of salvianolic acid A freeze-dried powder 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 the each dosage group of salvianolic acid A freeze-dried powder and model group comparison hypertrophy significantly increases; In addition, the kitchen range shape that HE dyeing display model matched group differs in size is as seen hemorrhage, high, the middle dosage group of salvianolic acid A freeze-dried powder there is no and finds that there is kitchen range shape bleeding, and salvianolic acid A freeze-dried powder low dose group also only has indivedual animals to have kitchen range shape hemorrhage, and hemorrhagic focus is less than model group.Result of the test shows that salvianolic acid A freeze-dried powder can obviously improve RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia histopathology state; reduce thrombosis bond area, reduce infarct size; thereby increase in infarcted region and the little blood vessel number of surrouding brain tissue, reduce the necrosis of cerebral hemorrhage phenomenon protection cerebral tissue and come off, thering is the effect of protecting cerebral thrombosis ischemia to cause brain and knitting damage.
Experimental example 18: salvianolic acid A freeze-dried powder to RHRSP cerebral thrombosis after shadow noon of blood brain barrier (BBB) permeability
1 test method
Animal grouping and RHRSP Cerebrovascular embolism model are prepared with experimental example 16 methods.4.5h after model surgery success, each group is tail vein injection salvianolic acid A freeze-dried powder 40mg/kg, 20mg/kg, 10mg/kg respectively, nimodipine 20mg/kg; The normal saline of sham operated rats and model group tail vein injection same volume.Each group rat is put to death and gets cerebral tissue specimen in MCAO postoperative (after being cerebral thrombosis ischemia) 6h, 12h, 24h in batches respectively, and in put to death before 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.
Randomly draw the cerebral tissue of 3 rats for each group, frozen section immediately, the crown section of the thick about 30um of row continuously, with 70% glycerol PBS buffer (pH8.5～9.0) mounting, under Bio-Rad Radiance2100 laser scanning co-focusing microscope, observe the situation of oozing out of EB in cerebral tissue, observe BBB open area and degree.While observation, section is put 4 DEG C of refrigerators and is kept in Dark Place.Each group is separately got the cerebral tissue of 5 rats at random, weighs cutaneous horn weight after blotting surface moisture; Cerebral tissue is placed in to homogenizer, adds 50% trichloroacetic acid to make tissue homogenate, move into test tube more than standing 60min when close; The centrifugal 10min of 3000rpm/min, gets 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, react each rat BBB permeability with this.
2 experimental results
Its focusing microscope observed result of 2.1 laser scannings
EB is bright-coloured bright red fluorescence under exciting light state.Under laser microscope, observe each treated animal cerebral tissue WuBBBNao district (as pinus, area postrema and hypophysis cerebri) 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 BBBNao district, the equal visible light spot of other each group cerebral tissue except sham operated rats, and be mainly distributed in thalamus, hypothalamus, cerebellum, Hippocampus etc. and locate, spot size/differing, 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: model group hot spot is maximum, approximately has the hot spot of 120 left and right diameter 100～200um, and intensive place in the form of sheets.Each salvianolic acid A freeze-dried powder injecta medicine group number of spots significantly reduces compared with model group, and the comparison of each dosage group, presents the remarkable dose-difference opposite sex; Wherein more with 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; High dose group hot spot is seldom dispersed in, and fluorescence is very weak.
The permeability result of 2.2 EB of rat cerebral tissue content detection blood brain barrier
Ge Zu rat cerebral tissue specimen calculates EB content by the fluorescent value recording, and data statistic analysis is in table 32.
Table 32 salvianolic acid A freeze-dried powder faces the impact of bent brain Barrier Permeability after thrombosis on RHRSP
Figure BSA00000811717500471
Figure BSA00000811717500481
Note: with sham operated rats comparison, #P < 0.01; With model group comparison, * P < 0.01
2.3 conclusion
EB is water-soluble dye, enter after blood can be absolutely rapidly and albumin bound, can not see through under normal circumstances blood brain barrier.From 2.1 and 2.2 results, sham operated rats BBBNao district, has no EB under cerebral tissue microscope and oozes out hot spot; And under model group rat cerebral tissue microscope, have a large amount of EB to ooze out hot spot, and cerebral tissue spectrofluorophotometer detects the EB of high-load, compared with 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 EB albumin-binding can enter cerebral tissue by open BBB.Experimental result shows under salvianolic acid A freeze-dried powder each dosage group rat cerebral tissue microscope that EB hot spot number and cerebral tissue EB detect content and obviously reduce; more all there were significant differences (P < 0.01) with model group; prompting salvianolic acid A freeze-dried powder increases inhibited to blood-brain barrier permeability following brain injury; can protect blood brain barrier, and the further damaged of protection cerebral tissue.
Experimental example 19: the impact of salvianolic acid A freeze-dried powder on RHRSP cerebral thrombosis cerebral infarction scope and brain water content
Prepare RHRSP cerebral thrombosis rat model, grouping, administration by the method for experimental example 16; After last administration finishes, rat broken end is got brain.Get at random the brain tissue slice of 5 rats for each group, through TTC dyeing, after taking pictures under the microscope centered by infarct, infarct delimited, process and calculate the infarct size of every brain sheet by image analysis software.Every all brain sheets of rat infarct size is added, and is 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.Separately get 5 rat cerebral tissues for each group and claim immediately weight in wet base, put afterwards in 105 DEG C of baking ovens and dry to constant weight, calculate brain water content.
The impact of the red phenolase A of table 33 freeze-dried powder on RHRSP cerebral thrombosis cerebral infarction scope and brain water content (x ± s)
Figure BSA00000811717500491
Note: with sham operated rats comparison, #P < 0.01; With model group comparison, * P < 0.01 adopts SPSS11.5 statistical package to carry out statistical procedures to data.Experimental result shows: sham operated rats has no infarcted cerebral constitution; The each dosage group of salvianolic acid A freeze-dried powder cerebral tissue Infarction volume and brain water content are significantly less than model control group (P < 0.01), and dosage is larger, Infarction volume is less, brain water content is fewer, and difference has statistical significance (P < 0.01 or p < 0.05).Salvianolic acid A freeze-dried powder basic, normal, high dosage group Infarction volume and brain water content are all lower than nimodipine group.Illustrate that salvianolic acid A freeze-dried powder can accelerate the reparation of focus, reduce ischemic tissue of brain infarction size after RHRSP cerebral thrombosis, alleviate cerebral tissue edema, have and repair and the effect of protection Neurons Against Cerebral Ischemia tissue injury.
Experimental example 20: salvianolic acid A freeze-dried powder on RHRSP cerebral thrombosis ischemia after the microvascular impact of rat cerebral tissue
Animal grouping and RHRSP Cerebrovascular embolism model are prepared with experimental example 16 methods.4.5h tail intravenously administrable after model surgery success, is after this administered once, until it is complete to draw materials every day.The each dosage group of salvianolic acid A freeze-dried powder is tail vein injection salvianolic acid A freeze-dried powder 20mg/kg, 10mg/kg, 5mg/kg respectively, nimodipine group injection nimodipine 10mg/kg; The normal saline of model group and sham operated rats injection same volume.Get rat broken end respectively at the postoperative 6h of MCAO, 1d, 3d, 7d for each group in batches and get 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; DAB colour developing.With PBS replace primary antibodie make negative control.Whether do not count blood vessel with the appearance of erythrocyte or tube chamber.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 Weidner method.First under low power (× 40) visual field, find high vessel density region, then under high power (× 400) visual field, carry out Microvessel Count, 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 processing system to carry out graphical analysis, measure blood vessel field Area Ratio (Positive Objects area/Statistical Fields gross area).
Biao34Ge Zu rat cerebral tissue blood vessel scene is long-pending, the immune positive microvessel density of CD31 (MVD) value (n=6)
Note: with sham operated rats comparison, #p < 0.01; With model group comparison, * P < 0.01, ☆ P < 0.05
The demonstration of table 34 experimental data, with sham operated rats comparison, 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 comparison, after ischemia treatment, the each dosage group of nimodipine group and salvianolic acid A freeze-dried powder 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.Between three dosage groups of salvianolic acid A freeze-dried powder, relatively, difference has statistical significance (P < 0.05).Result shows that salvianolic acid A freeze-dried powder can increase MVD and the blood vessel field Area Ratio of cerebral tissue ischemia half blanking bar, and prompting salvianolic acid A freeze-dried powder can promote the effect that Angiogenesis and collateral circulation are set up.
Experimental example 21: salvianolic acid A freeze-dried powder to RHRSP cerebral thrombosis ischemia after rat cerebral tissue's vascular endothelial growth factor expression shadow noon
Modeling and grouping administration are with experimental example 19.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 heart perfusion, gets rapidly brain, does in forebrain optic chiasma place the crown section that about 2mm is thick.Be placed in 4% paraformaldehyde fixative and spend the night, conventional dehydration, paraffin embedding, is cut into the paraffin section that about 5um is thick continuously, and routine dewaxes to water, impels the expression of ribonucleic acid (VEGFmRNA) with hybridization in situ detection VEGF.Illustrate and operate by VEGF in situ hybridization test kit (Wuhan Boster Biological Technology Co., Ltd.), with microscopic image analysis software carry out optical density (IOD) analyze.
Table 35 salvianolic acid A freeze-dried powder is to RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia tissue VEGF
The impact of expressing
Figure BSA00000811717500511
Note: with sham operated rats comparison, #P < 0.05; With model group comparison, #P < 0.01, ☆ P < 0.05
VEGF (VEGF), is again 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) compared with sham operated rats; illustrate after cerebral tissue hypoxic-ischemic can Stimulation of The Brain tissue in the expression increase of VEGF; after prompting cerebral infarction, body self there will be a kind of protective response that resists ischemia injury; the expression of VEGF is increased, after ischemia, occur self " compensatory revascularization ".The each dosage group of salvianolic acid A freeze-dried powder and the comparison of model group group; VEGFmRNA expression significantly raise (P < 0.05 or P < 0.01); prompting salvianolic acid A freeze-dried powder can significantly promote ischemic tissue of brain angiogenesis; promote the foundation of collateral circulation compensation; save ischemia half blanking bar; the brain tissue impairment that protection ischemia causes, and there is certain dose-effect relationship.
Experimental example 22: salvianolic acid A freeze-dried powder on RHRSP cerebral thrombosis ischemia after the impact of rat cerebral tissue basic fibroblast growth factor protein expression
With experimental example 20, finish rear 2h, 24h, 48h respectively at rat administration, 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.ABC immunohistochemic al technique method detects the expression of cerebral tissue alkalescence fibroblast growth factor (bFGF) albumen.By bF, (GF protein immunization group detection kit (Wuhan Boster Biological Technology Co., Ltd.) description operates wet micro-Microscopic observation, counting is dyed the positive cell number of brown color, the all visuals field of 5 infarcts are selected in each section at random, average.Data are carried out statistical analysis.
Table 36 salvianolic acid A freeze-dried powder to RHRSP cerebral thrombosis ischemia after the bFGF of rat cerebral tissue
Protein expression impact (x ± s)
Figure BSA00000811717500521
Note: with sham operated rats comparison, #P < 0.05; With relatively * P < 0.01 of model group
BFGF is the strong neurotrophic factor with various biological activity, can neuroprotective unit to multiple detrimental effects such as ischemia resisting, anoxia, toxicity of excitatory amino acid, calcium overload, free radical and NO are synthetic, slow down neuronal apoptosis and necrosis; And can work in coordination with the effect of angiogenesis in VEGF promotion infarcted region.
Experimental result shows; after ischemia occurs; the bFGF of model group rat cerebral tissue protein expression raises to some extent compared with sham operated rats; there is significant difference (P < 0.05) to ischemia 24h; but with the prolongation of Ischemia Time, have a declining tendency, after prompting cerebral tissue hypoxic-ischemic; body self produces of short duration protection stress, can organize endogenous bFGF protein expression to increase by Stimulation of The Brain.Nimodipine group, the each dosage group of salvianolic acid A freeze-dried powder and model group comparison, the bFGF protein expression of each time period all significantly increases (P < 0.01); Between three dosage groups of salvianolic acid A freeze-dried powder, relatively, difference has statistical significance (P < 0.05); Each time period of each dosage group self relatively shows, bFGF protein expression is continual and steady; Prompting salvianolic acid A freeze-dried powder can increase former of ischemic tissue of brain and secondary bFGF egg is expressed certainly, has good neurocyte protection effect and the effect that promotes angiogenesis, contributes to the foundation of collateral circulation, saves ischemia half blanking bar.
Experimental example 23: the impact of salvianolic acid A freeze-dried powder on rats after cerebral ischemic reperfusion nervous symptoms
Male SD/ rat (250 ± 20) g, be divided at random 6 groups: sham operated rats, ischemia-reperfusion injury model matched group, the high, medium and low dosage of salvianolic acid A freeze-dried powder (10mg/kg, 5mg/kg, 2.5mg/kg) group, nimodipine matched group (10mg/kg).Adopt improvement line bolt legal system for rat brain focal cerebral ischemia-reperfusion injury 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, 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 slowly enter the propelling of cranium direction to internal carotid artery through left side external carotid artery trunk otch, taking 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 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 of 30min and being respectively 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 Bederson improved method: carry Mus tail built on stilts approximately one chi, observe forelimb flexing situation, stretch to ground count 0 point as two forelimb symmetries; As the offside forelimb of performing the operation occurs that the flexing of 1 point of wrist flexing meter, 2 points of elbow flexing meters, 3 points of shoulder inward turning meters, existing wrist elbow has again 4 points of shoulder inward turning meters.Rat is placed on level land, pushes away respectively both shoulders to side shifting, check resistance, as symmetrical in bilateral resistance and strong, be designated as 0 point; As organized power descender in the time that operation offside promotes, according to decline degree difference be divided into gently, in, severe, count respectively 1,2,3 point.Rat two forelimbs are placed on wire netting, observe the muscular tension of two forelimbs, bilateral muscular strength equity and strong person count 0 point, operation offside muscular tension decline degree is divided into gently, in, severe, count respectively 1,2,3 point.Rat does not stop, to a side person of turn-taking, to count 1 point.According to above standards of grading, full marks are 11 points, and mark is higher, illustrate that the behavior disorder degree of rat is more serious, neurologic impairment is more serious.Concrete data are in table 37.
The impact of table 37 salvianolic acid A freeze-dried powder on rats after cerebral ischemic reperfusion neurological deficits score
(x±s，n＝12)
Figure BSA00000811717500541
Note: with model group comparison, * * P < 0.01, * P < 0.05
Result of the test is known, ischemia-reperfusion injury model group function of nervous system serious defect, last till and fill with again rear 72h, although after perfusion, behavioristics's obstacle of 3h, 24h significantly alleviates (P < 0.05) more again, still serious (neurological deficits score is still greater than 5) of neurologic impairment.Nimodipine group with the each dosage group of salvianolic acid A freeze-dried powder compared with model group, from pouring into again 3h, neurologic impairment symptom alleviates, 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 the each dosage group of salvianolic acid A freeze-dried powder: 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).
Experimental result shows that salvianolic acid A freeze-dried powder can alleviate rats after cerebral ischemic reperfusion neurologic impairment symptom, is conducive to its neurobehavioral recovery, and prompting salvianolic acid A freeze-dried powder has the effect that improves nervous symptoms after brain tissue impairment.
Experimental example 24: the shadow noon of salvianolic acid A freeze-dried powder to rats after cerebral ischemic reperfusion cerebral infarction scope, cerebral index and brain water content
The each group of experimental example 23 rat is the last time after neuroethology scoring, get 6 broken ends for every group and get brain, be placed in immediately-20 (refrigerator freezing 10min, remove after olfactory bulb, cerebellum and low brain stem, the cerebral tissue continuous coronal section of row 2mm thickness, immerses brain sheet in 2%TTC solution, 37 DEG C of dyeing 10min, take out, be placed in 4% paraformaldehyde PBS buffer and fix 2h.Normal structure is dyed redness, and infarct is white in color.Fixing brain sheet is arranged by section order, 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.
Other 6 rats broken end of each group is got after brain, claims immediately cutaneous horn weight, in 105 DEG C of baking ovens, dries to constant weight, calculates brain water content and cerebral index.
Table 38 salvianolic acid A freeze-dried powder to rats after cerebral ischemic reperfusion cerebral infarction volume ratio,
The impact (x ± s, n=6) of cerebral index, brain water content
Figure BSA00000811717500551
Note: with relatively * p < 0.05 of model group, * * p < 0.01
Experimental data shows, the each dosage group of salvianolic acid A freeze-dried powder is compared with ischemia-reperfusion injury model group: cerebral infarction volume is than aobvious remarkable minimizing (P < 0.01), and cerebral index and brain water content all obviously reduce (P < 0.01 or P < 0.05); Salvianolic acid A freeze-dried powder low dose group and nimodipine group be no difference of science of statistics relatively; The middle and high dosage group of salvianolic acid A freeze-dried powder and the comparison of nimodipine group, its Infarction volume and cerebral index and brain water content all significantly reduce (P < 0.05 or P < 0.01); Show that salvianolic acid A freeze-dried powder can reduce rats after cerebral ischemic reperfusion cerebral tissue infarction size, can alleviate the cerebral tissue edema degree after cerebral ischemia reperfusion injury.
Experimental example 25: the impact of salvianolic acid A freeze-dried powder on rats with cerebral ischemia cerebral tissue ischemia half blanking bar regional cerebral blood flow (rCBF)
1 test method
Adopt improvement line bolt legal system for rat brain focal ischemia model: male SD rat, pentobarbital sodium intraperitoneal anesthesia.Expose and separate left carotid, external carotid artery, internal carotid artery and arteria pterygopalatina, ligation external carotid artery distal end, arteriole folder folder closes common carotid artery, tie wings arteria palatina, cut all 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 slowly enter the propelling of cranium direction to internal carotid artery through left side external carotid artery trunk otch, taking 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), the high, medium and low dosage group of salvianolic acid A freeze-dried powder (10mg/kg, 5mg/kg, 2.5mg/kg) at random.Each treated animal after model success immediately 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 use cyanoacrylate is 0.5mm by diameter is close to skull surface and is fixed, under stereotaxic instrument, selecting respectively after bregma the other 2mm that opens in 1mm center line right side 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, after ischemia, continue to monitor 2h.Blood flow before recording administration and after administration.
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 each dosage group of nimodipine and the salvianolic acid ischemia penumbra rCBF of rat cerebral tissue compared with administration before all existing risings in various degree, 30min after administration, each medicine group cerebral tissue ischemia penumbra rCBF peaks.Specific experiment data statistical analysis is in table 39.
Table 39 salvianolic acid A freeze-dried powder is to rats with cerebral ischemia continuous ischemia phase cerebral tissue ischemia half blanking bar
The impact (x ± s, n=10) of rCBF
Figure BSA00000811717500561
Note: with relatively * P < 0.01 of sham operated rats (baseline value); With comparison before administration, ★ P < 0.01, ☆ p < 0.05
3 interpretations of result
From table 39 data, after operation modeling, model control group and each medicine group cerebral tissue half blanking bar rCBF, compared with sham operated rats, have utmost point significant difference (p < 0.01) to represent modeling success.After administration, 10min begins, each medicine group compared with self administration before ischemia half blanking bar rCBF have obvious rising, and with particularly significantly (p < 0.01) of nimodipine and salvianolic acid A freeze-dried powder high dose group; To 30min after administration, each medicine group ischemia half blanking bar rCBF rises to the highest, with each group before administration self rCBF baseline value comparison, has raise 15%～30%; Though after this have a declining tendency, but last till ischemia 2h, each medicine group is compared before compared with model group and self administration, still has remarkable significant difference (p < 0.01 or p < 0.05); The each dosage group of salvianolic acid A freeze-dried powder is compared, and has good dose-effect relationship; In salvianolic acid A freeze-dried powder, the rCBF of dosage group day part is suitable with nimodipine group.Hence one can see that, and salvianolic acid A freeze-dried powder 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 26: the impact of salvianolic acid A freeze-dried powder on rats after cerebral ischemic reperfusion cerebral tissue ischemia half blanking bar rCBF
In experimental example 25, each group rat, after continuous ischemia 2h observation ischemia half blanking bar rCBF, is extracted bolt line, realizes ischemia-reperfusion injury model.Model is respectively organized tail intravenously administrable after setting up, and dosage is with experimental example 20.Still the method for pressing described in experimental example 20 measure and fill with again after 3h cerebral tissue ischemia half blanking bar rCBF.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 40.
Table 40 salvianolic acid A freeze-dried powder is to rats after cerebral ischemic reperfusion cerebral tissue ischemia half blanking bar
The impact (x ± s, n=10) of rCBF
Figure BSA00000811717500571
Note: with sham operated rats comparison, * p < 0.01; With fill with again front comparison, ☆ p < 0.01; With model group comparison, ★ P < 0.01
Experimental result shows, recovers after filling again at rat continuous ischemia 2h, and respectively organizing rat ischemia half blanking bar rCBF all has increase in various degree.Model group rat rises to top level at 60,90 minutes ischemia half blanking bar rCBF, with fill with again front than there being obvious statistical significance (P < 0.01), but be also only about 50% of the front baseline value of ischemia, after this sharply decline again, fill with again in 3h, rCBF value before ischemia baseline value 37%～50% between, in the time of this phase, pour into again and 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 freeze-dried powder rat rCBF obviously increase again, and each time period rCBF has utmost point significant difference (P < 0.01) compared with model group.1h after filling with again, nimodipine and salvianolic acid A freeze-dried powder high dose group rCBF return to approach former rCBF 75%, in salvianolic acid A freeze-dried powder dosage group return to be about former rCBF 69%, salvianolic acid A freeze-dried powder low dose group returns to and is about 61% of former rCBF, is dose dependent; And to pouring in 3h, each medicine group rCBF maintains in metastable scope again.Experimental result prompting, salvianolic acid A freeze-dried powder can obviously improve the cerebral blood flow of ischemia half blanking bar cerebral tissue after Ischemia and Reperfusion in vivo in Rats, recovers the confession of brain cell blood, thereby saves ischemia half blanking bar, further damage is even dead to prevent cerebral tissue, and ischemic cerebrovascular is had to good therapeutical effect.
Experimental example 27: the impact of salvianolic acid A freeze-dried powder on rats after cerebral ischemic reperfusion Energy Metabolism of Brain Tissue
The each treated animal of experimental example 26 is after having measured the cerebral blood flow of 3h after perfusion again, and sacrificed by decapitation, gets brain to freezing in liquid nitrogen; After one week, get the cerebral tissue of 100mg corresponding site, under freezing state, pulverize, with perchloric acid homogenate except albumen, 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 of table 41 salvianolic acid A freeze-dried powder on rats after cerebral ischemic reperfusion Energy Metabolism of Brain Tissue
(x±s，μmol/g，n＝10)
Figure BSA00000811717500581
Note: with sham operated rats comparison, * P < 0.01; With model group comparison, * * P < 0.01, ☆ P < 0.05
ATP, ADP, AMP, LA, PC are human body energy metabolite.Can be found out by table 41 experimental data, model group ATP, ADP, AMP, PC compared with 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 Power supply significantly reduces, anaerobic metabolism product LA piles up serious, even energy metabolism disorder still exists after implementing to fill with again.And the each dosage group of salvianolic acid A freeze-dried powder and nimodipine group are 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 freeze-dried powder, reduce cerebral tissue LA content, can significantly improve the energy metabolism of rat cerebral ischemia and ischemia-reperfusion.Prompting salvianolic acid A freeze-dried powder 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, save the brain cell that ischemia half blanking bar after cerebral ischemia is at death's door, further damage is even dead to prevent cerebral tissue, can perform well in treating ischemic cerebrovascular.
The impact of the red larva of a tapeworm or the cercaria of a schistosome acid of experimental example 28 A freeze-dried powder on rats after cerebral ischemic reperfusion cerebral tissue neuronal apoptosis rate
Prepare rat pattern of ischemia reperfusion, grouping, administration with experimental example 23 methods.Fill with after 24h at rat cerebral ischemia 2h, broken end is got brain again, fixes with 4% paraformaldehyde, cuts into slices, and paraffin embedding, prepares the thick crown section of thick 3 μ m; Adopt the transferase mediated dUTP breach end-labelling (TUNEL method) of last deoxyribonucleic eventually to detect cerebral tissue neurocyte, strictly wash bright book operation by test kit, 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 input computer carries out image analysis, detects apoptotic cell number and the normal cell number of the TUNEL positive, calculates neuronal apoptosis rate, results averaged.
The impact of table 42 salvianolic acid A freeze-dried powder on rats after cerebral ischemic reperfusion cerebral tissue neuronal apoptosis rate
Figure BSA00000811717500591
Figure BSA00000811717500601
With relatively #P < 0.001 of sham operated rats; With model group comparison, * * P < 0.001, * P < 0.01
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.Experimental result demonstration, rat cerebral ischemia 2h fills with after 24h again, and neuronal apoptosis is serious; And after nimodipine and the intervention of various dose salvianolic acid A freeze-dried powder, with model group comparison, in rat cerebral tissue, neuronal apoptosis significantly reduces (P < 0.001 or P < 0.01); Show that salvianolic acid A freeze-dried powder has the neuronal apoptosis of inhibition, suppresses cerebral ischemia and cause the dead effect of rat cerebral tissue's nerve.
Experimental example 29 impacts of salvianolic acid A freeze-dried powder on rats after cerebral ischemic reperfusion cerebral tissue neurotrophic factor
Prepare rats after cerebral ischemic reperfusion model, grouping administration with experimental example 28 methods.After Ischemia Reperfusion 24h through 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.Light Microscopic observation, Cai Tu also uses image analysis system analysis, measures its average gray value.Average gray is higher, represents that the intensity of positive cell is more weak.The impact of table 43 salvianolic acid A freeze-dried powder on rat cerebral tissue's neurotrophic factor protein expression (gray value)
Figure BSA00000811717500602
With sham operated rats comparison, #P < 0.05,
Figure BSA00000811717500603
p < 0.01; With model control group comparison, * P < 0.05, AP < 0.01.
Neurotrophic factor is a neure growth histone matter molecule essential with survival, and the integrity of neure growth, growth and function is played to supporting function.NGF, BDNF, NT-3 are parts for neurotrophic factor family, can maintain neuronal survival and promote neurocyte differentiation and induction 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, in rat cerebral tissue, increases to some extent compared with sham operated rats, and after prompting cerebral reperfusion injury, in cerebral tissue, NGF, BDNF express stress protectiveness increase; But in Neurons Against Cerebral Ischemia tissue, NT-3 content obviously reduces.With model control group comparison; the each dosage group of salvianolic acid A freeze-dried powder NGF, BDNF, NT-3 protein expression all obviously strengthen (P < 0.05 or P < 0.01); prompting salvianolic acid A freeze-dried powder can strengthen the expression of ischemic injuries cerebral tissue endogenous neurotrophic factor NGF, BDNF, NT-3, reaches the effect of neuro-protective.
Experimental example 30 impacts of salvianolic acid A freeze-dried powder on rats after cerebral ischemic reperfusion inflammatory cytokine
Prepare rats after cerebral ischemic reperfusion model, grouping administration with experimental example 28 methods.After Ischemia Reperfusion 24h, broken end is got brain rapidly, get scarce side cerebral tissue and make 10% tissue homogenate, the centrifugal 10min of low temperature 2000r/min, get supernatant, the content by the each inflammatory cytokine of ELISA kit measurement: interleukin-1 ' beta ' (IL-1 β), interleukin-6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor (TNF-α), intercellular adhesion molecule-1 (ICAM-1).
The impact of table 44 salvianolic acid A freeze-dried powder on rats after cerebral ischemic reperfusion inflammatory cytokine
Figure BSA00000811717500611
With sham operated rats comparison, #P < 0.001; With model group comparison, Δ P < 0.01, * P < 0.05.
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); The each inflammatory cytokine content of rat cerebral tissue that gives the each dosage group of salvianolic acid A freeze-dried powder obviously reduces compared with model group.Prompting salvianolic acid A freeze-dried powder can suppress the expression of ischemic injuries brain tissue inflammation cytokine, suppresses the generation of brain tissue inflammation's reaction, suppresses neuron and the cerebral tissue cascade damage of inflammatory reaction mediation.
Experimental example 31: salvianolic acid A freeze-dried powder is to rats after cerebral ischemic reperfusion cerebral tissue Ca 2+, K +, Mg 2+the shadow noon of content
Get experimental example 24 and dry the cerebral tissue to constant weight, after weighing, proceed to conical flask, use analytical pure nitric acid and perchloric acid in (20010) DEG C digestion, add deionized water dissolving residue, proceed to after color comparison tube standardize solution, with Ca in atomic spectrophotometer survey cerebral tissue 2+, K +, Mg 2+content.Data are carried out statistical analysis.
Table 45 salvianolic acid A freeze-dried powder is to rats after cerebral ischemic reperfusion cerebral tissue
Ca 2+, K +, Mg 2+the impact of content (x ± s)
Figure BSA00000811717500621
Note: with sham operated rats comparison, * P < 0.01; With model group comparison, * P < 0.05, * * P < 0.01
Ca in cell 2+overload is in During Ischemia, to mediate one of encephaloclastic important mechanism of Secondary cases, is the last common pathway that ischemia, anoxia produce irreversible neuronal damage.Ca 2+overload can be by exciting as second message,second messenger's activator protein enzyme, activate phospholipase, activate a series of enzyme reactions such as endonuclease produces infringement to cell, and cell death inducing, causes acute cell death and Delayed onset neuronal necrosis.K +, Mg 2+be Ca 2+antagonist.K +doing, is 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, in cerebral tissue, participate in the multiple important metabolic activity of cell, synthetic and all many-sides of energy metabolism of conduction, ion transport, 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 comparison, each dose group of nimodipine group and salvianolic acid A freeze-dried powder can significantly reduce Ca in cerebral tissue 2+content, rising K +, Mg 2+content, illustrates that salvianolic acid A freeze-dried powder can suppress Ca 2+interior stream alleviates calcium overload, thereby can suppress Ca 2+the neuronal cell apoptosis of overload induction.
Experimental example 32: the impact of salvianolic acid A freeze-dried powder on rats after cerebral ischemic reperfusion cerebral tissue neurotransmitter
1 experimental technique
1.1 grouping administrations and model preparation
Male SD rat (250 ± 20) g, be divided at random 6 groups, every group 30: sham operated rats, cerebral ischemia re-pouring model control group, the high, medium and low dosage of salvianolic acid A freeze-dried powder (10mg/kg, 5mg/kg, 2.5mg/kg) are thin, nimodipine matched group (10mg/kg).Re-perfusion model (as described in experimental example 23) after adopting line bolt legal system for middle cerebral artery occlusion in rat 2h, sham operated rats except plug wire not, the same model group of all the other operating process.Each group before ischemia 30min and again perfusion start time be respectively administered once through tail vein injection, sham operated rats and cerebral ischemia re-pouring model group give the normal saline of same volume.Fill with after 2h again, get 4 broken ends for each group and get brain and do histopathology section, HE dyeing, observes histopathology and changes.
The mensuration of 1.2 rat cerebral tissue's monoamine neurotransmitters
Each group rat is poured into after 2h at ischemia 2h again, respectively get 8, broken end is got brain fast, separate cerebral cortex and striatum, weigh, add n-butyl alcohol homogenate, centrifuging and taking supernatant is used fluorescent spectrophotometer assay 5-hydroxy tryptamine (5-HT), norepinephrine (NE), dopamine (DA) content after carrying.Data are carried out statistical analysis, the results are shown in Table 46.
The mensuration of 1.3 rat cerebral tissue's content of amino acids neurotransmitter
Each group rat is poured into after 2h at ischemia 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 OPA derivative reaction, detect the content of amino acid neurotransmitter glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), γ-aminobutyric acid (GABA), taurine (Tau) in brain tissue homogenate's supernatant by high performance liquid chromatography.Mobile phase is a certain proportion of potassium phosphate buffer and methanol, tetrahydrofuran solution, and pH is about 6.6, flow velocity 1ml/min.Calculate excitatory toxicity index (EI): EI=[Glu] [Gly]/[GABA].Data are carried out statistical analysis, the results are shown in Table 47.
2 experimental results
2.1 histopathology observed results
The thin structure of neurons of sham-operation, form be normal, without interstitial edema; Ischemia-reperfusion group neuronal cell volume dwindles, distortion, karyopycnosis, and nervous tissue is loose, and neuron and perivascular space increase, and interstitial cerebral edema is obvious; The each dosage group of salvianolic acid A freeze-dried powder is compared with model group, and cerebral edema obviously alleviates, and neural tuple born of the same parents' form is obviously improved, and neuron peripheral clearance is obviously dwindled; Especially the most remarkable with high, the middle dosage group of salvianolic acid A freeze-dried powder, its neuronal cell is more complete, form normal.
The impact of 2.2 salvianolic acid A freeze-dried powders on monoamine neurotransmitter in rats after cerebral ischemic reperfusion cerebral tissue
Compared with sham operated rats, cerebral ischemia re-pouring group rat cerebral cortex and striatum NE, DA, 5-HT content all significantly reduce (P < 0.01).Compared with cerebral ischemia re-pouring model group, the each dosage group of salvianolic acid A freeze-dried powder and nimodipine group cerebral cortex, striatum NE, DA, 5-HT content obviously raise (P < 0.01 or P < 0.05).
Table 46 salvianolic acid A freeze-dried powder is in rats after cerebral ischemic reperfusion cerebral tissue
The impact (x ± s, ng/mg, n=8) of monoamine neurotransmitter
Figure BSA00000811717500641
Note: with sham operated rats comparison, * P < 0.01; With model group comparison, * P < 0.05, * * P < 0.01
2.3 impacts of red sour A freeze-dried powder on amino acid neurotransmitter in rats after cerebral ischemic reperfusion cerebral tissue
Compared with sham operated rats, 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), compared with model group, Glu in nimodipine group and the each dosage group of salvianolic acid A freeze-dried powder cerebral tissue, Asp, Gly all significantly reduces (P < 0.01 or P < 0.05), Tau content significantly raise (P < 0.01 or P < 0.05) in nimodipine group and the each dosage group of salvianolic acid A freeze-dried powder cerebral tissue, nimodipine group and salvianolic acid A freeze-dried powder are high, GABA content significantly raise (P < 0.01) in middle dosage group cerebral tissue, the GABA content of salvianolic acid A freeze-dried powder low dose group slightly raises compared with model group, but difference does not have statistical significance.Cerebral ischemia re-pouring model group EI value has extremely significantly and increases (P < 0.01) compared with sham operated rats, and the EI value of the each dosage group of salvianolic acid A freeze-dried powder and nimodipine group obviously reduces (P < 0.01 or P < 0.05) compared with model group.
The impact (x ± s, n=8) of table 47 salvianolic acid A freeze-dried powder on amino acid neurotransmitter in rats after cerebral ischemic reperfusion cerebral tissue
Figure BSA00000811717500651
Note: with sham operated rats comparison, * P < 0.01, ※ P < 0.05; With model group comparison, * P < 0.05, * * P < 0.01
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.When cerebral ischemia, monoamine neurotransmitters can be shown rising in extracellular fluid, and reduces in brain essence.In cerebral tissue ischemia equivalent damage situation, monoamines nerve enters matter generation metabolism disorder in brain, and 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.Amino acid neurotransmitters excitement in brain when cerebral ischemia-suppress is unbalance is one of key factor causing cerebral ischemia.This experimental result shows that salvianolic acid A freeze-dried powder can obviously improve the cerebral cortex of cerebral ischemia-reperfusion injury in rats and the content of striatum monoamine transmitters, the emerging poison exponent of going through of GABA, Tau content, the content that significantly reduces Glu in cerebral tissue, Asp, Gly and aminoacid in rising cerebral ischemia/reperfusion injury of rats cerebral tissue.And dosage increases, effect strengthens.The salvianolic acid A freeze-dried powder showing in conjunction with histopathology pathological examination can obviously alleviate cerebral edema, improves the result of neuronal cell form, shows that salvianolic acid A freeze-dried powder can suppress monoamine neurotransmitter and excessively discharge, and improves 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 33: the impact of salvianolic acid A freeze-dried powder on LPO, SOD, GSH-Px in rats after cerebral ischemic reperfusion cerebral tissue
Each group under 32 of experimental examples, detect after 10 remaining rats, broken end is got brain, weigh, make brain tissue homogenate's liquid of 10% with 4 DEG C 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.
The impact (x ± s, n=10) of table 48 salvianolic acid A freeze-dried powder on LPO, SOD, GSH-Px in rats after cerebral ischemic reperfusion cerebral tissue
Figure BSA00000811717500661
Note: with sham operated rats comparison, * P < 0.01; With model group comparison, * P < 0.05, * * P < 0.01
In cerebral ischemia re-pouring model group rat cerebral tissue, LPO content significantly raises compared with sham operated rats, and SOD and GSH-Px activity significantly reduce (P < 0.01) compared with sham operated rats.When Cerebral ischemia and reperfusion is described, 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 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 demonstration, the each dosage group of salvianolic acid A freeze-dried powder is compared with model group, and in rat cerebral tissue, LPO content significantly reduces, active significantly raise (P < 0.01 or the P < 0.05) of SOD and GSH-Px; Illustrate that salvianolic acid A freeze-dried powder can increase the generation of the activity of peroxide scavenger enzyme in ischemical reperfusion injury cerebral tissue, inhibition peroxide, thus the damage to neuronal cell and cerebral tissue to antioxidant radical.
Experimental example 34: salvianolic acid A freeze-dried powder is protected RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia vascular endothelial cell
Prepare RHRSP Cerebrovascular embolism model, grouping with experimental example 16 methods.Each group of administration volume tail vein injection administration of pressing immediately 0.3ml/100g after model preparation operation, after this every day 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 freeze-dried powder freeze-dried powder 40mg/kg, 20mg/kg, 10mg/kg; Nimodipine group injection nimodipine 20mg/kg.Get rat through heart perfusion 4% paraformaldehyde neutral buffered solution respectively at the postoperative 6h of MCAO, 12h, 24h, 48h, 72h time point, get brain for each group.Conventional fixing, the dehydration paraffin embedding of cerebral tissue, is cut into the thick crown section of 5um continuously.TUNEL method (the dUTP breach art end labelling method 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 occurs brown yellow granule 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 statistical analysis processing.
The impact of table 49 salvianolic acid A freeze-dried powder on RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia apoptosis of vascular endothelial cell (x ± s)
Figure BSA00000811717500671
Note: with sham operated rats comparison, #P < 0.01; With model group comparison, * P < 0.01
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 cerebral tissue ischemia, just showed increased of endothelial cell apoptosis reaches peak for 24 hours to ischemia.With model group comparison, the apoptosis digital display work of each time period of the each dosage group of salvianolic acid A freeze-dried powder reduces (P < 0.01), prompting salvianolic acid A freeze-dried powder can suppress cerebral ischemia hindbrain apoptosis of vascular endothelial cell, improve cerebrovascular endothelial cell survival rate, thereby ensure the normal of cerebrovascular endothelial cell function, maintain the complete of ischemic injuries hindbrain blood vessel structure and function and strengthen the ability to anti-cerebral ischemia damnification, the effect of performance treatment ischemic cerebrovascular.
Experimental example 35: the shadow noon of salvianolic acid A freeze-dried powder to 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, gets brain after alcohol disinfecting, peel off pia mater encephali and macroscopic trunk, remove cerebral white matter and cerebellum, thereafter, shred cerebral tissue, homogenate, crosses successively 90,180 mesh filter screens and filters, rinse and collect the blood capillary section on filter screen, add 0.1%II collagenase, 37 DEG C of digestion 15min, the centrifugal 5mi n of 1500r/mi n, repeat 3 times, precipitation is inoculated in culture bottle and continues to cultivate after suspending with culture fluid, changes liquid 1 time every 2d.Treat that cell grows up to fusion state, through Morphological Identification and the qualification of VIII factor SABC, be defined as Brain Microvascular Endothelial (BMVEC), purity is greater than 98%.The cultivation of going down to posterity.Get third generation BMVEC, after digestion, make single cell suspension, be inoculated in 96 orifice plates.Experiment divides 8 groups: model group, 6 dosage groups of salvianolic acid A freeze-dried powder (final concentration is respectively 10,20,30,40,50,60 μ mol/L) and nimodipine group (final concentration is 30 μ mol/L).After cell inoculation, second day, change culture fluid into PBS liquid, be positioned over (37 DEG C, 95%N in 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).The each dosage group of nimodipine group and salvianolic acid A freeze-dried powder all adds respectively the medicine of respective concentration when 4h, anoxia and reoxygenation before modeling.After anoxia-induced apoptosis 4h, anoxia 4h-reoxygenation 12h damage, recognize with MTT the survival rate that detects each group of BMVEC respectively.
The impact (x ± s, n=6) of the BMVEC survival rate of table 50 salvianolic acid A freeze-dried powder on anoxia and Hypoxia/Reoxygenation Injury
Figure BSA00000811717500681
With model control group comparison, #P < 0.05, * P < 0.01; With comparison before reoxygenation, ☆ P < 0.01; With the comparison of nimodipine group ,/P < 0.05,
Figure BSA00000811717500692
p < 0.01
Experimental result shows, after anoxia 4h damage, BMVEC survival rate is only 25.45% left and right, occurred cell injury after anoxia is described; The survival rate of nimodipine group 30 μ mol/L and salvianolic acid A freeze-dried powder 10,20,30 μ mol/L processed group and model group relatively have the trend of rising, but do not show significant difference; After salvianolic acid A freeze-dried powder 40,50,60 μ mol/L dosage group anoxia-induced apoptosis 4h, BMVEC survival rate is significantly higher than model control group (P < 0.05).After Hypoxia/Reoxygenation Injury, 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 salvianolic acid A freeze-dried powder and nimodipine effect, BMVEC survival rate significantly raises (P < 0.01), with salvianolic acid A freeze-dried powder 30,40,50,60 μ mol/L dosage group best results; And salvianolic acid A freeze-dried powder 20,30,40,50,60 μ mol/L dosage group survival rates are significantly higher than nimodipine 30 μ mol/L groups (P < 0.05, or P < 0.01).Prompting salvianolic acid A freeze-dried powder can be protected microvascular endothelial cells oxygen and Hypoxia/Reoxygenation Injury, strengthens tool hypoxia-bearing capability, improves the brain microvessel endothelial cells in vitro survival rate of anoxia-induced apoptosis and Hypoxia/Reoxygenation Injury.
The impact of experimental example 36 salvianolic acid A freeze-dried powders on Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC) apoptosis
Method by experimental example 35: third generation BMVEC is inoculated in 96 orifice plates, experiment divides 6 groups: Normal group, model group, the high, medium and low dosage group of salvianolic acid A freeze-dried powder ( final concentration 40,20,10 μ mol/L), nimodipine group (final concentration 40 μ mol/L).Modeling and medication are with experimental example 35.Normal group does not carry out hypoxia/reoxygenation processing, cultivates corresponding experimental period by normal method.The each porocyte of trypsinization, centrifugal collecting cell, adopts phosphatidyl in conjunction with albumen-Fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) dyeing, uses cells were tested by flow cytometry cell in early days and the apoptosis rate in late period.
Table 51 salvianolic acid A lyophilizing dry powder causes the impact (x ± s, n=6) of BMVEC apoptosis for Hypoxia/Reoxygenation Injury
Figure BSA00000811717500693
Figure BSA00000811717500701
With Normal group comparison, #P < 0.01; With model group comparison, * P < 0.01, * * P < 0.001; With the comparison of nimodipine group, Δ P < 0.01,
Figure BSA00000811717500702
p < 0.05
Apoptosis especially plays an important role at ischemia injury in transient ischemia/reperfusion damage.The apoptosis of vascular endothelial cell causes the broken words of vascular integrity while understanding, and 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.Shown that by experimental result Hypoxia/Reoxygenation Injury can cause the each phase apoptosis rate of BMVEC significantly to increase.With model group comparison, the each dosage group of salvianolic acid A freeze-dried powder and nimodipine group all can significantly lower cell early, late period and total apoptosis rate (P < 0.01), and be certain dose-effect relationship.And salvianolic acid A freeze-dried powder 10 μ mol/L, dosage group is suitable with nimodipine 40 μ mol/L dosage group effects.Prompting salvianolic acid A freeze-dried powder has the effect of the BMVEC apoptosis that good anti-hypoxia-reoxygenation injury causes.
Experimental example 37 salvianolic acid A freeze-dried powders affect Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC) secretory function
As described in experimental example 35, third generation BMVEC is inoculated in 96 orifice plates to preparation BMVEC hypoxia/reoxygenation model.Grouping and administration are with experimental example 36.Collecting cell supernatant, ELISA method detects the impact of salvianolic acid A freeze-dried powder on BMVEC secretory tissue fiber proenzyme activator (t-PA), tissue plasminogen activator's inhibitor (PAI), nitric oxide (NO), Endothelin (ET).
Table 52 salvianolic acid A freeze-dried powder is on Hypoxia/Reoxygenation Injury (BMVEC) secretory function impact (x ± s, n=6)
Figure BSA00000811717500711
With Normal group comparison, #P < 0.01,
Figure BSA00000811717500712
p < 0.05; With model group comparison, * P < 0.01, AP < 0.05, * * P < 0.001
After Hypoxia/Reoxygenation Injury, with normal group comparison, BMVEC secretion t-PA and NO obviously reduce, and ET raises, and t-PA/PAINO/ET ratio also significantly declines.The t-PA of the each dosage group of salvianolic acid A freeze-dried powder and nimodipine group and NO secretory volume significantly raise (P < 0.01 or P < 0.001) compared with model group, and wherein the middle and high dosage group of salvianolic acid A freeze-dried powder rises to the Normal group level that approaches; Each administration group t-PA/PAI and NO/ET ratio ratio also significantly improve compared with model group.Prompting salvianolic acid A freeze-dried powder can improve the secretory function of brain micro blood vessel endothelium group born of the same parents after Hypoxia/Reoxygenation Injury.
Experimental example 38 salvianolic acid A freeze-dried powders are to the interior Ca of Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC) 2+the impact of concentration
As described in experimental example 35, third generation BMVEC is inoculated in 96 orifice plates to preparation BMVEC hypoxia/reoxygenation model.Grouping and administration are with experimental example 36.The each porocyte of trypsinization, after centrifugal collecting cell, adds Fluo-3 (a kind of calcium fluorescent probe, final concentration is 5 μ mol/L), hatches 45min for 37 DEG C, and PBS liquid rinses 3 times, though use cells were tested by flow cytometry fluorescence degree.While existence with free ligand form because of Fluo-3, almost there is no fluorescence, and and Ca 2+strengthen in conjunction with rear fluorescence, can detect Cytoplasmic Ca according to fluorescence intensity change 2+concentration change.
Table 53 salvianolic acid A freeze-dried powder is to Ca in Hypoxia/Reoxygenation Injury BMVEC 2+the impact (x ± s, n=6) of concentration
Figure BSA00000811717500721
With Normal group comparison, #P < 001; With model group comparison, * P < 0.0l; With the comparison of nimodipine group,
Figure BSA00000811717500722
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).With model group comparison, each medicine group Ca 2+concentration extremely significantly reduces, the most remarkable with the middle and high dosage group of salvianolic acid A freeze-dried powder effect, and intracellular calcium concentration is significantly lower than nimodipine 40 μ mol/L dosage groups (P < 0.05).Prompting salvianolic acid A freeze-dried powder has the Hypoxia/Reoxygenation Injury of inhibition and causes Ca in BMVEC born of the same parents 2+the effect of concentration.
Experimental example 39: the impact of salvianolic acid A freeze-dried powder on rat artery thrombus formation time
Get SD rat, male and female half and half, body weight is 250～300g, is divided at random matched group, aspirin group (20mg/kg) and the high, medium and low dosage of salvianolic acid A (12.5,2.5,0.5mg/kg) group.Each group tail intravenously administrable 1 time every day (matched group gives isometric(al) normal saline), continuously after 7d, 30min after art time administration, pentobarbital sodium (40mg/kg) anesthetized rat, dorsal position is fixed on Mus plate, cervical region median incision, separate the about 15mm of right carotid, the stimulating electrode and the temperature sensor probe that thrombus in vivo are formed to analyzer are placed on common carotid artery, stimulating electrode is positioned at proximal part, with 2mA galvanism blood vessel 7min, make tunica intima damage, record is because of the time of thrombosis blocking blood flow in arterial lumen, be thrombus formation time (OT).Experimental data statistical result is in table 54.
The impact of table 54 salvianolic acid A freeze-dried powder on rat artery thrombus formation time
Figure BSA00000811717500723
Note: with the comparison of normal saline group, * P < 0.01
Experimental result shows, with the comparison of normal saline matched group, and the thrombus formation time significant prolongation (P < 0.01) of salvianolic acid A freeze-dried powder 12.5,2.5,0.5mg/kg dosage group; Low dose group thrombus formation time and 20mg/kg aspirin group be (P > 0.05) quite; Between high, medium and low dosage group, thrombus formation time has significant difference (P < 0.05).Show that salvianolic acid A freeze-dried powder can extend thrombus formation time, the formation of prevention of arterial thrombosis, pre-preventing thrombosis and the ischemic cerebrovascular that causes.
Experimental example 40: salvianolic acid A freeze-dried powder is to thrombotic inhibitory action
SD rat, male and female half and half, grouping, administration are with experimental example 39.30min pentobarbital sodium anesthesia after Rhizoma Atractylodis Macrocephalae time administration, dorsal position is fixed, and right common carotid artery and left external jugular vein are isolated in operation, with three sections of polyethylene tubes connections.Put into the long 5cm operation silk thread of having weighed in polyethylene tube stage casing.Be full of polyethylene tube with heparin-saline solution (5u/mL).Left external jugular vein is inserted in one end of pipe, 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, takes 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 DEG C of baking ovens freeze-day with constant temperature to constant weight, weigh after cooling, be thrombosis dry weight.Experimental data is in table 55.
The inhibitory action that table 55 salvianolic acid A freeze-dried powder forms rat suppository
Figure BSA00000811717500731
Note: with the comparison of normal saline group, * P < 0.01
Experimental data shows, with the comparison of normal saline group, salvianolic acid A freeze-dried powder 12.5,2.5,0.5mg/kg dosage group rat suppository are wet, dry weight all significantly alleviates (P < 0.01), between each dosage group, have good dose-effect relationship.And low dose group and 20mg/kg aspirin group effect be (P > 0.05) quite.Prompting salvianolic acid A freeze-dried powder has the thrombotic effect of good inhibition, can be used for preventing or treat the ischemic cerebrovascular due to thrombosis.
Experimental example 41: the impact of salvianolic acid A freeze-dried powder on rat vein thromboembolism
Male SD rat, body weight (200 ± 20) g, divides into groups with experimental example 39, pentobarbital sodium (40mg/kg) anesthetized rat, along the bright rat stomach wall of opening in abdominal part center, open abdominal cavity, separate postcava, and in left renal vein lower end level place ligation postcava, close abdominal cavity, close abdominal cavity, after 1h, 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 tube chamber inner blood, removal of thromboses, filter paper sops up residual blood, takes immediately wet weight of thrombus; Put afterwards after the interior freeze-day with constant temperature of 60 DEG C of baking ovens, claim thrombosis dry weight.Test data is in table 56.
The impact of table 56 salvianolic acid A freeze-dried powder on rat vein thromboembolism
Figure BSA00000811717500741
Note: with the comparison of normal saline group, * P < 0.01
Experimental data shows, with the comparison of normal saline group, salvianolic acid A freeze-dried powder 12.5,2.5,0.5mg/kg dosage group rat vein thrombosis are wet, dry weight significantly alleviates (P < 0.01), low dose group and 20mg/kg aspirin group effect suitable (P > 0.05); Show that salvianolic acid A freeze-dried powder has the effect that suppresses venous thrombosis, can be used for the venous thromboembolism after prophylaxis of acute ischemic cerebrovascular occurs.
Experimental example 42: salvianolic acid A freeze-dried powder is to the thrombotic inhibitory action of rats in vitro
SD rat, 250～300g, male and female half and half; Random point of normal saline group, aspirin group (20mg/kg) and the high, medium and low dosage of salvianolic acid A (12.5,2.5,0.5mg/kg) group, respectively organize every day tail vein injection to relative medicine 1 time, continuous 7 days; 30min after last administration, pentobarbital sodium anesthesia, open abdominal cavity along ventrimeson, the about 1.5ml of abdominal aortic blood injects silication sebific duct, rapidly silica gel tube two ends are docked circlewise, the silica gel sheath seal of tube is fixed, and puts 37 DEG C of constant temperature rotation 15min on extracorporeal thrombosis forming device, removal of thromboses, measures thrombosis length, claims its weight in wet base; After drying in 60 DEG C of calorstats, survey its dry weight.Experimental data is in table 57.
Table 57 salvianolic acid A freeze-dried powder is to the thrombotic inhibitory action of rats in vitro (x ± s)
Note: with the comparison of normal saline group, * P < 0.0l
Experimental data shows, with the comparison of normal saline group, salvianolic acid A freeze-dried powder 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 salvianolic acid A freeze-dried powder forms and has good inhibitory action external thrombus.
Experimental example 43: the thrombolytic experimental study of salvianolic acid A freeze-dried powder to established thrombosis in body
SD male rat (250 ± 20) g, pentobarbital sodium intraperitoneal anesthesia rat, separates 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.Finish rear blood flow and reduce to 50% before stimulation and be considered as thrombosis to stimulate.Points 5 groups at random of animals, normal saline group, urokinase (2000U/kg) and the high, medium and low dosage of salvianolic acid A (10,5,2.5mg/kg) group; Each group of 20min after formation thrombosis, all through the disposable injection relative medicine of femoral vein, revascularization situation in 1h after observation administration; If in this period: revascularization, continues to observe vessel open state 1h.The Flow of carotid artery of every animal to be to stimulate front blood flow as baseline, with >=50% or≤25% stimulate before blood flow person be judged to be to 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, 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 thromboembolism again.Experimental result is in table 58.
Thrombolytic effect (n=10) in table 58 salvianolic acid A lyophilized powder needle body
Figure BSA00000811717500752
Figure BSA00000811717500761
Note: 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 bolt rate=logical rear 1h occurs again again, in 1h, there is again logical number of animals
3. with the comparison of normal saline group, * P < 0.0l; With the comparison of urokinase group, #P < 0.05
Result shows: normal saline group all continues thromboembolism, and without occurring again logical phenomenon; The number of animals that each medicine group continues thromboembolism is few, all has utmost point significant difference (P < 0.01) compared with normal saline group; Dosage in salvianolic acid A freeze-dried powder (5mg/kg) group, its vessel open degree is similar to 2000U/kg urokinase group, and its recanalization rate is suitable; The lasting recanalization rate of salvianolic acid A freeze-dried powder high dose (10mg/kg) group and recanalization rate are all higher than urokinase group, and bolt rate is starkly lower than urokinase group (P < 0.05) again; Salvianolic acid A freeze-dried powder low dosage (2.5mg/kg) is though organize recanalization rate lower than urokinase group, and bolt rate is suitable with 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 freeze-dried powder has good thrombolytic and prevents thrombolytic.
Experimental example 44 impacts of salvianolic acid A freeze-dried powder on 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, neck median incision, separate the total tremulous pulse of right side strength (CCA), internal carotid artery (ICA), external carotid artery (ECA), ligation ECA far-end and arteria pterygopalatina, arteriole folder folder closes CCA and ICA, cut an osculum at ECA near-end, unclamp CCA arteriole folder, extract arterial blood 0.5ml, 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, unclamps CCA and arteria pterygopalatina place vascular clamp, sews up the incision.Experiment points 6 groups: sham operated rats, model control group, salvianolic acid A freezes in powder pin high, medium and low (10,5,2.5mg/kg) group, nimodipine group (10mg/kg).30min after successful surgery, each treated animal tail vein relative medicine, after this injects once continuous 7 days every day; Sham operated rats and model group are injected isopyknic normal saline.Second day (the fasting 12h) that administration finishes, intraperitoneal anesthesia, abdominal aortic blood, anticoagulant heparin, carries out Determination of Blood Rheology.
The impact of table 59 salvianolic acid A freeze-dried powder on cerebral thrombosis hemorheology of rat
Figure BSA00000811717500771
With sham operated rats comparison, #p < 0.01; With relatively * P < 0.01 of model control group,
Figure BSA00000811717500772
p < 0.05.
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; The each dosage group of salvianolic acid A freeze-dried powder and model group comparison, its whole blood viscosity and plasma viscosity and packed cell volume all obviously reduce; Prompting salvianolic acid A freeze-dried powder can be accelerated micro-blood flow velocity, and reduction blood viscosity improves hemorheology and the effect that effectively resists cerebral thrombosis.

Claims (8)

1. the preparation method of salvianolic acid A freeze-dried powder, described salvianolic acid A freeze-dried powder is for the preparation of the purposes of improving the function of nervous system's symptom medicine after cerebral ischemia, wherein, the weight proportion of described salvianolic acid A freeze-dried powder is: salvianolic acid A 20g～60g, filler 20g～60g, antioxidant is to make 0.02%～0.1% of total amount;
The preparation method of described salvianolic acid A freeze-dried powder is:
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 DEG C of water temperature lixiviates are got at every turn, stir with 10～50 revs/min of speed simultaneously, or add 3～15 times of amount water boiling and extraction, and extract altogether 1～3 time, extract 1～4 hour at every turn; Extracting solution is evaporated to relative density and is determined as 1.0～1.25 at 60 DEG C, adds ethanol to make to contain alcohol amount 50%～85%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, obtains Radix Salviae Miltiorrhizae extract; Or
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 is also concentrated into without alcohol taste, 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 DEG C of temperature thermal conversions 1～6 hour;
Conversional solution adjust pH to 2.5～4.5, leave standstill, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 1～10mg, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 35～1: 70 with macroporous adsorbent resin ratio, 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 is also concentrated into without alcohol taste,
Aqueous solution is concentrated into the solution of every 1ml containing 1-10mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 5～1: 25 with polyamide ratio, 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 is also concentrated into without alcohol taste 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, point 2～6 extractions, separate organic layer, reclaim under reduced pressure t-butyl methyl ether, makes the extract of every 1ml containing salvianolic acid A 1g～10g, add 1～3 times of amount silica gel, stir, volatilize;
Be added on 5～20 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 4～1: 25, taking 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, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 5～20 times of water gagings dissolvings, microwave vacuum drying, obtains described salvianolic acid A;
Getting described salvianolic acid A 10g～80g injects water 1500～2800ml and is stirred to dissolve, by adjusting PH with base value 4.0～5.0, add described filler to make its dissolving, add again described antioxidant, be stirred to dissolve and mix, then add active carbon 0.5～2g stirring and adsorbing, filter and remove active carbon, inject fill after water and become bottle, send into and in freeze dryer, carry out lyophilization;
Described lyophilization comprises the steps:
A, freezing: be cooled to-40 DEG C～-45 DEG C with 20 DEG C～30 DEG C/h speed, be incubated freezing 10～16 hours;
B, distillation: be evacuated to below 0.3mbar, in 10～14 hours, the salvianolic acid A formulation temperature freezing risen to-23 DEG C～-27 DEG C, maintain-23 DEG C～-27 DEG C vacuum dryings 6～8 hours;
C, dry: continue to heat up, be evenly warming up to 40 DEG C～45 DEG C with 0.5 DEG C～1.0 DEG C/min, maintain 40 DEG C～45 DEG C and be dried after 2～5 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
2. preparation method according to claim 1, its weight proportion is: salvianolic acid A 20g～40g, filler 20g～40g, antioxidant is to make 0.03%～0.08% of total amount.
3. preparation method according to claim 1, wherein said filler is selected from any one or a few in mannitol, glucose, lactose, and consumption is 20mg～40mg/2ml～3ml.
4. preparation method according to claim 1, wherein said antioxidant is selected from any one or a few in vitamin C, thiourea, sodium sulfite, sodium pyrosulfite.
5. preparation method according to claim 4, its weight proportion is: salvianolic acid A 20g, filler mannitol 20g, antioxidant vitamin C 0.8g; Its preparation method is:
Get described salvianolic acid A 20g, inject water 1900ml and be stirred to dissolve, with sodium hydroxide sodium adjust pH 4.0～5.0, add mannitol 20g to make to dissolve, then add 0.8g vitamin C, be stirred to dissolve and mix, add active carbon 0.5g stirring and adsorbing, filter and remove active carbon; Inject water to 2000ml, fill becomes 1000 bottles, lyophilization;
Described lyophilization comprises the steps:
A, freezing: be cooled to-45 DEG C with 25 DEG C/h speed, be incubated freezing 15 hours;
B, distillation: be evacuated to below 0.3mbar, in 12 hours, the salvianolic acid A formulation temperature freezing risen to-25 DEG C, maintain-25 DEG C of vacuum dryings 8 hours;
C, dry: continue to heat up, be evenly warming up to 40 DEG C with 0.8 DEG C/min, maintain 40 DEG C and be dried after 3 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
6. preparation method according to claim 1, is characterized in that microwave vacuum drying temperature: 20-100 DEG C, return difference temperature 1-5 DEG C, and more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.
7. preparation method according to claim 6, is characterized in that microwave vacuum drying temperature: 50-85 DEG C, return difference temperature 2-4 DEG C, and more than vacuum-0.07Mpa, microwave power 10-80KW, dry 100-150 minute.
8. preparation method according to claim 7, is characterized in that microwave vacuum drying temperature: 55-80 DEG C, return difference temperature 2-3 DEG C, and more than vacuum-0.07Mpa, microwave power 25-60KW, dry 120-140 minute.
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