CN102988310A - Application of salvianolic acid A lyophilized powder in preparation of medicine for protecting ischemia brain tissue - Google Patents

Application of salvianolic acid A lyophilized powder in preparation of medicine for protecting ischemia brain tissue Download PDF

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CN102988310A
CN102988310A CN2012104889237A CN201210488923A CN102988310A CN 102988310 A CN102988310 A CN 102988310A CN 2012104889237 A CN2012104889237 A CN 2012104889237A CN 201210488923 A CN201210488923 A CN 201210488923A CN 102988310 A CN102988310 A CN 102988310A
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salvianolic acid
freeze
dried powder
cerebral
group
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CN102988310B (en
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杨小玲
曾发林
刘鹏
罗恒真
魏宇清
李志勇
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JIANGXI QINGFENG PHARMACEUTICAL CO Ltd
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陆文萍
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Abstract

The invention relates to application of salvianolic acid A lyophilized powder in preparation of a medicine for protecting an ischemia brain tissue. The salvianolic acid A lyophilized powder comprises the following components by weight: 20 to 60g of salvianolic acid A, 20 to 60g of filling agent, and 0.02 to 0.1% of antioxidant based on the total preparation amount.

Description

The salvianolic acid A freeze-dried powder is for the preparation of the purposes of protection ischemic tissue of brain damage medicine
Technical field
The present invention relates to a kind of salvianolic acid A freeze-dried powder for the preparation of the purposes of protection ischemic tissue of brain damage medicine.
Background technology
Cerebrovascular claims again apoplexy, be by the various causes of disease make the supply brain blood blood vessel generation pathological changes due to a kind of nervous system disease.Its Etiological be the reasons such as cerebral arteries system disease damage (such as cerebral arteriosclerosis) the cerebral arteries luminal stenosis, vasospasm, the obturation that cause or break, Oligemia or total blockage, brain blood circulation and dysfunction, the impaired and series of symptoms that occurs of cerebral tissue.Mainly comprise ischemic and hemorrhagic apoplexy.Wherein (ICVD claims again Ischemic Stroke) account for about 80%.
Ischemic cerebrovascular refers to that local brain tissue comprises neurocyte, glial cell and contact fiber because the afunction of the degeneration that blood supply disorder occurs, necrosis or a property crossed.The cerebral arteries emphraxis that Intravascular Thrombus formation, thromboembolism, angiostenosis cause is the main cause of cerebral infarction.Ischemia causes that the mechanism of action of cranial nerve cell damage and death is various, complicated, behind the cerebral infarction (being cerebral ischemia), because brain lacks the supply of blood and oxygen, cause large brain energy metabolism unbalance, produce a series of pathologic damage, such as oxidative stress, toxicity of excitatory amino acid, calcium overload, inflammatory reaction etc., thereby cause neuronic mortality.It is clinically commonly encountered diseases, frequently-occurring disease, and mortality rate and disability rate are very high, existing one of the three universally acknowledged large deadly diseases that become.Treatment mainly is 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 the promotion damage clinically.The control ischemic cerebrovascular is present human difficult medical problem in the urgent need to address.At present U.S. FDA has only been ratified the thromboembolism treatment after tissue plasmin activation factor (tPA) is used for 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 at present comprise calcium channel blocker such as nimodipine, glutamate receptor antagonists such as dizocilpine (dizocilPine), antioxidant or free radical scavenger such as Edaravone, NO signal transduction pathway regulator lu pei Shandong mile (Iubeluzole) and inflammation inhibitor enlimomab (enlimomab) etc. for the neuroprotective drug of ischemic stroke treatment.But the therapeutical effect that has in them is imprecise or specificity is not strong, and the toxic and side effects that has is large, toleration is little, have also be in clinical before or the clinical research stage, be difficult in control cerebral infarction performance positive impact.Thereby it is extremely urgent to develop quick control brain ischemia medicament effective, safety and stability.Brought 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 the cerebral microvascular thromboembolism, and the effect be better than salvianolic acid B (Zhang Hengai. salvianolic acid A is on impact and the Mechanism Study [D] of 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 a plurality of phenolic hydroxyl groups, hydroxyl, the reactive groups such as ester bond, it is unstable to light and heat, exposure air easily is oxidized to salvianolic acid C and different salvianolic acid C etc., because the above-mentioned physicochemical property of salvianolic acid A, make the 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, can't guarantee the salvianolic acid A pharmacological action.
Summary of the invention
The defects that exists for overcoming prior art, the invention provides a kind of salvianolic acid A freeze-dried powder for the preparation of the purposes of protection ischemic tissue of brain damage medicine, wherein, the weight proportion of described salvianolic acid A freeze-dried powder is: salvianolic acid A 20g～60g, filler 20g～60g, antioxidant make 0.02%～0.1% of total amount; The preparation method of described salvianolic acid A freeze-dried powder is:
(1) extract: Radix Salviae Miltiorrhizae water or alcoholic solution extract and obtain aqueous extract or alcohol extract;
(2) transform: with the extracting solution that step (1) obtains, transfer pH to 3.5～6.5, add molar percentage and be 0.1%～3.0% catalyst, 100～140 ℃ of heating 1～6 hour;
(3) purification:
A. the solution that step (2) is obtained is transferred pH to 2.5～4.5, and is centrifugal, and supernatant separates through nonpolar or low pole macroporous resin column chromatography, after washing with water, uses the eluant eluting, and high performance liquid chromatogram detects salvianolic acid A, collects the eluent that contains salvianolic acid A;
B. the eluent that step a is obtained is used the eluant eluting with sephadex lh-20 or ODS-C18 or the separation of polyamide chromatography post, and high performance liquid chromatogram detects salvianolic acid A, collects the eluent that contains salvianolic acid A;
C. the eluent that step b is obtained is transferred pH to 2.0～4.0, through organic solvent extraction, and the Separation of Organic phase;
D. the solution that step c is obtained separates with silica gel column chromatography, uses the eluant eluting, and high performance liquid chromatogram detects salvianolic acid A, collects the eluent that contains salvianolic acid A;
(4) drying: with the eluent that steps d obtains, the reclaim under reduced pressure eluant is dissolved in water again, and vacuum drying, spray drying or microwave vacuum drying get described salvianolic acid A;
(5) getting described salvianolic acid A 10g～80g injects water 1500～2800ml and stirs and to make dissolving, with adjusting PH with base value 4.0～5.0, add described filler and make its dissolving, add again described antioxidant, stirring makes the dissolving mixing, adds active carbon 0.5～2g stirring and adsorbing again, filters to remove active carbon, fill becomes bottle after injecting water, sends into and carries out lyophilization in the freeze dryer;
(6) described lyophilization comprises the steps:
A, freezing: be cooled to-40 ℃～-45 ℃ with 20 ℃～30 ℃/h speed, be incubated freezing 10～16 hours;
B, distillation: be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 10～14 hours rises to-23 ℃～-27 ℃, keeps-23 ℃～-27 ℃ vacuum dryings 6～8 hours;
C, drying: continue to heat up, evenly be warming up to 40 ℃～45 ℃ with 0.5 ℃～1.0 ℃/min, keep 40 ℃～45 ℃ dryings after 2～5 hours, sample temperature is down to room temperature, add a cover, namely get the salvianolic acid A freeze-dried powder.
Preferably, also comprise the purposes of protecting blood brain barrier.
Preferably, also comprise the purposes that reduces the ischemic tissue of brain infarction size.
Preferably, also comprise the purposes that alleviates the ischemic tissue of brain edema.
Preferably, its weight proportion is: salvianolic acid A 20g～40g, filler 20g～40g, antioxidant make 0.03%～0.08% of total amount.
Preferably, wherein said filler is selected from any one or a few in mannitol, glucose, the lactose, and consumption is 20mg～40mg/2ml～3ml.
Preferably, wherein said antioxidant is selected from any one or a few in vitamin C, thiourea, sodium sulfite, the 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 stirring and make dissolving, with sodium hydroxide sodium adjust pH 4.0～5.0, add mannitol 20g and make dissolving, add again the 0.8g vitamin C, stir and make the dissolving mixing, 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 ℃ with 25 ℃/h speed, be incubated freezing 15 hours;
B, distillation: be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 12 hours rises to-25 ℃, keeps-25 ℃ of vacuum dryings 8 hours;
C, drying: continue to heat up, evenly be warming up to 40 ℃ with 0.8 ℃/min, keep 40 ℃ of dryings after 3 hours, sample temperature is down to room temperature, add a cover, namely get the salvianolic acid A freeze-dried powder.
Preferably, wherein the water extracting method described in the step (1) is: get red rooted salvia, be cut into decoction pieces or be ground into diameter 2mm granule, add 3～15 times of water gagings at every turn and extract, extract altogether 1～3 time, extracted 1～4 hour at every turn; Extracting solution is evaporated to relative density 1.10～1.25 (60 ℃), adding ethanol makes and contains the alcohol amount 30%～80%, centrifugal, the supernatant decompression recycling ethanol also is concentrated into without the alcohol flavor, described water extraction adopts decoction to extract or 45～95 ℃ of water temperature lixiviates are got, stir with 10～50 rev/mins of speed simultaneously, get Radix Salviae Miltiorrhizae extract.
Preferably, wherein the alcohol extraction method described in the step (1) is: get red rooted salvia, be cut into decoction pieces or be ground into diameter 2mm granule, add 3～15 times of amount 30%～60% alcohol reflux at every turn, extracted 1～4 hour at every turn, extract altogether 1～3 time; Decompression recycling ethanol gets Radix Salviae Miltiorrhizae extract.
Preferably, wherein in the extracting solution in the step (1) salvianolic acid B concentration be that 1mg/ml～30mg/ml or thin up are to 1mg/ml～30mg/ml.
Preferably, wherein in the extracting solution salvianolic acid B concentration be that 5mg/ml～20mg/ml or thin up are to 5mg/ml～20mg/ml.
Preferably, wherein the catalyst in the step (2) is one or more in iron chloride, ruthenium trichloride, aluminum chloride, zinc chloride, the Palladous chloride., and the molar percentage of catalyst and salvianolic acid B is 0.1%～3.0%.
Preferably, wherein the molar percentage of catalyst and salvianolic acid B is 0.5%～2.0%.
Preferably, wherein macroporous resin column described in the step a is HPD-80, HPD-100, HPD-100B, HPD-200A, HPD-300, HPD-450, HPD-722, HPD-826, ADS-5, ADS-8, ADS-21 or D101, AB-8; Described eluant is water and the ethanol of water and different proportion, and first water, 10～40% ethanol elutions, uses 20～60% ethanol elutions again, and high performance liquid chromatogram detects salvianolic acid A, collects and contains the salvianolic acid A part, and eluent is concentrated into without the alcohol flavor.
Preferably, wherein the described eluant among the step b is water and the ethanol of water and different proportion, and first water, 20～60% alcoholic solution eluting remove impurity, use again 40～90% alcoholic solution eluting, high performance liquid chromatogram detects salvianolic acid A, collects to contain the salvianolic acid A part, and eluent is concentrated into without the alcohol flavor.
Preferably, wherein the organic solvent described in the step c is t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate or Ethyl formate.
Preferably, wherein eluant described in the steps d is the two-phase solvent of petroleum ether, pentane, normal heptane, ethyl acetate, methyl acetate, Ethyl formate, t-butyl methyl ether composition.
Preferably, the temperature of microwave vacuum drying described in the step (4) wherein: 20-100 ℃, 1-5 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.
Preferably, vacuum drying temperature described in the step (4) wherein: 50 ℃～90 ℃, more than vacuum-0.07Mpa, power: 1～60KW, dry 2～20 hours.
Preferably, spray-dired intake air temperature described in the step (4) wherein: 150 ℃～350 ℃, the air outlet temperature: 70 ℃～95 ℃, spray velocity: 1～300ml/min.
Preferably, wherein pH adjusting agent is phosphoric acid, hydrochloric acid, sulphuric acid or acetic acid.
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, obtained salvianolic acid A: through screening and the optimization of system, extraction solvent and the extracting method of initiation material salvianolic acid B have at first relatively been determined, because the salvianolic acid B water solublity is better, employing water extraction or low concentration alcohol extraction have been determined, again because the salvianolic acid B heat stability is relatively poor, determine the extraction of employing hot water warm macerating and added the stirring extracting method, or use the low-concentration ethanol reflux, extract,, make extraction solubility be lower than 100 ℃, keep salvianolic acid B not to be destroyed, determine optimum solvent consumption and extraction time by orthogonal experiment, obtained adapting to the salvianolic acid B optimum extraction process of suitability for industrialized production.
The present invention is compared with the prior art and shows: initiation material is with the direct extraction of the red rooted salvia conversion that can feed intake, do not need salvianolic acid B is carried out transforming behind the purification again, be in the catalytic conversion reaction of the present invention, the reaction raw materials salvianolic acid B does not need high-purity, does not for example need the purity of salvianolic acid B 〉=50%.It is generally acknowledged that reaction raw materials is more pure better, in the catalytic conversion reaction of the present invention, the purity of salvianolic acid B height is on the not impact of conversion reaction effect.On the contrary, not only produce a large amount of impurity when experiment showed, salvianolic acid B purity＞50%, and do not improve conversion ratio.Moreover, the present invention by experiment repeatedly relatively, the factor of at first having determined the salvianolic acid A productive rate is produced material impact is such as the concentration that transforms front pressure differential self, pH value, temperature, time etc.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 the salvianolic acid A productive rate jointly each other, thereby determined that salvianolic acid B transforms the optimum temperature of the needs control of salvianolic acid A, pH value, time etc., and the salvianolic acid B initial concentration is controlled at 1mg/ml～30mg/ml, thereby so that salvianolic acid A conversion ratio of the present invention more obviously is better than other conversion conditions.In the chemical reaction, the purity of reactant and concentration usually affect the effect of reaction.Generally speaking reactant there is purity requirement, and thinks that the high specific concentration of concentration hangs down.Yet in the catalytic conversion reaction of the present invention, the concentration of salvianolic acid B height is on the not impact of conversion reaction effect.On the contrary, experiment showed, that salvianolic acid B concentration height not only produces a large amount of impurity, and do not improve conversion ratio.And the concentration that contains salvianolic acid B in the salvianolic acid B aqueous solution is not more high better, and the above concentration conversion ratio of 30mg/ml is low on the contrary, and effect is poorer.Therefore, production technology of the present invention has unforeseeable technique effect aspect saving cost and production cycle.Do not provide in prior art in the situation of any technology enlightenment, if only theoretically deduction of those skilled in the art is impossible draw under above-mentioned each conditional parameter the sour B of pellet is changed into the conclusion that salvianolic acid A has better changing effect.
What is more important, the present invention passes through performing creative labour, find that iron chloride, ruthenium trichloride, aluminum chloride, zinc chloride or Palladous chloride. can significantly improve the conversion ratio that salvianolic acid B transforms salvianolic acid A as catalyst, what conversion ratio was highly stable can reach near 60%, most cases can surpass 60%, this all is 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 contain a large amount of impurity, therefore, selected respectively low pole and nonpolar macroporous adsorption resin to carry out crude separation, select again polyamide etc., solvent extraction, silica gel to separate, and various flows part measured, remove the impurity part after, salvianolic acid A content is brought up to 80% from about 10%, to 90%, to 93%, to 96%.
Further, when significantly improving conversion ratio, the purification step that the present invention uses by being suitable for actual industry, especially by after the steps such as a series of separation, eluting processing, do not adopt traditional constant pressure and dry, but adopt vacuum drying, spray drying, microwave vacuum drying, baking temperature was too high in the past thereby thoroughly overcome, drying time the long and defective large to the destruction of salvianolic acid A; And sublimation drying is long, and cost extract high and the lyophilization gained can't be removed the dissolvent residual problem fully.
The present invention can also directly mix low-purity with highly purified salvianolic acid B, only need be made into suitable initial conversion concentration, can reach the purpose that changes into salvianolic acid A equally, the preparation technology of this conversion raw material is very simple, and production cost is very suitable for the application in the actual industry when reducing.
Salvianolic acid A freeze-dried powder provided by the invention, chemistry and physical characteristic according to salvianolic acid A, from affecting the stable additives of medicine, dosage form, container, the external world are such as air, light, moisture, and the generation chemical reactions such as impurity cause the decomposition of medicament to carry out creationary experimental analysis on the contrary.By the screening preparation prescription, repetition test, mannitol, glucose, lactose etc. have been 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, has solved owing to air, light, moisture, and the medicine that the generation chemical reactions such as impurity cause decomposes.Salvianolic acid A is made suitable preparation, solved salvianolic acid A and made the injection stability problem, by selecting suitable adjuvant and dosage form, guaranteed the 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 prepared stable, safe, effective, quality controllable salvianolic acid A freeze-dried powder.
Salvianolic acid A among the present invention is adjusted to suitable scope by alkali liquor with pH value, by adding mannitol etc., makes it be easier to lyophilization, and does not affect the property of medicine again.
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 during distillation are determined the vacuum sublimation time, clear and definite dry programming rate and temperature, and drying time; The creationary complex relationship of clearly having decided between frozen cooling speed and temperature, distillation programming rate and the dry programming rate of temperature and the temperature and time, and suitable numerical range is definite etc., thus just finally obtained stable lyophilized injectable powder.
What is more important, the salvianolic acid A freeze-dried powder that adopts preparation method of the present invention to make can not change the original chemical attribute of salvianolic acid A fully; The salvianolic acid A freeze-dried powder compositions of making is compared the characteristics such as have good water solubility, heat stability is high, solubility is good with salvianolic acid A.
On the other hand, the present invention also provides it to be used for prevention and or the purposes for the treatment of ischemic cerebrovascular aspect.
Through the experimental data contrast as can be known, easily apoplectic type renovascular hypertension in rats middle cerebral artery occlusion (MCAO) postoperative recovery from anesthesia scoring, neural behavior scoring (the spontaneous activity of (in the 12h) after model group is revived, the symmetry of quadruped locomotion, forelimb stretches the symmetry of the action of creeping, climb the cage wall, push away the trunk reaction, antenna is to the reaction that stimulates etc.) significantly be lower than sham operated rats, illustrate that easily apoplectic type renovascular hypertension in rats (RHRSP) function of nervous system is impaired serious behind the cerebral thrombosis ischemia, and in the 72h that continues, neural behavior scoring continues to be starkly lower than sham operated rats; 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 behind its ischemia the neurobehavioral of 24h substantially near the sham operated rats level; Each dosage group rat neurobehavioral of 72h salvianolic acid A freeze-dried powder has recovered normally to the ischemia, and prompting salvianolic acid A freeze-dried powder can improve neurobehavioral behind the RHRSP cerebral thrombosis ischemia, the effect of nervous symptoms after having protection and improving cerebral ischemia.
Through histopathology checking experiment Data Comparison as can be known, sham operated rats cerebral tissue Bilateral Symmetry has no pathological changes, and the model group Intravascular Thrombus adheres to seriously.With model group relatively, the contraction in length of each dosage group rat ischemia side middle cerebral artery (MCA) local thrombus of Microscopic observation salvianolic acid A freeze-dried powder reduces at the arterial wall bond area, with high, middle dosage group is the most obvious.The TTC visible infarcted cerebral constitution that dyes is white in color, and the cerebral tissue scope that is dyed to white in each dosage group of salvianolic acid A freeze-dried powder is dwindled than model control group is remarkable.HE dyes has in visible model group ischemia side cortex MCA blood supply district (centered by the cortex of the volume top) infarct obvious cerebral tissue softening, necrocytosis, the phenomenons such as the 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 each dosage group of salvianolic acid A freeze-dried powder and model control group shows in the kitchen range, kitchen range Zhou Jun has little blood vessel hyperplasia, but the little number of blood vessel of each dosage group of salvianolic acid A freeze-dried powder and model group comparison hypertrophy significantly increases; In addition, the kitchen range shape that as seen HE dyeing display model matched group differs in size is hemorrhage, and the salvianolic acid A freeze-dried powder is high, middle dosage group there is no and finds to have kitchen range shape bleeding.The result shows that the salvianolic acid A freeze-dried powder can obviously improve RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia histopathology state; reduce the thrombosis bond area, reduce infarct size; come off thereby increase the little blood vessel number, the necrosis of minimizing cerebral hemorrhage phenomenon protection cerebral tissue that reach surrouding brain tissue in the infarcted region, have the effect that protection cerebral thrombosis ischemia causes brain tissue impairment.
Through the experimental data contrast as can be known, 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; each the microscopically EB of dosage group rat cerebral tissue hot spot number and cerebral tissue EB detect content obviously to be reduced; more all there were significant differences with model group; illustrate that the salvianolic acid A freeze-dried powder increases inhibited to brain tissue impairment hyperamization brain Barrier Permeability; can protect blood brain barrier, and the further damaged of protection cerebral tissue.。
Through the experimental data contrast as can be known, by RHRSP cerebral thrombosis rat model group is compared, sham operated rats has no infarcted cerebral constitution; Each dosage group cerebral tissue Infarction volume of salvianolic acid A freeze-dried powder 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 all are lower than the nimodipine group.Illustrate that the salvianolic acid A freeze-dried powder can accelerate the reparation of focus, reduce ischemic tissue of brain infarction size behind the RHRSP cerebral thrombosis, alleviate the cerebral tissue edema, have and repair and the effect of protection Neurons Against Cerebral Ischemia tissue injury.
Through the experimental data contrast as can be known, compare with RHRSP cerebral thrombosis ischemia model group, after the ischemia treatment, each dosage group cerebral tissue ischemia penumbra MVD of salvianolic acid A freeze-dried powder and blood vessel scene are long-pending significantly to be increased to some extent, and the interior numerical value of 48h is more stable after the drug withdrawal.Show that the 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 the salvianolic acid A freeze-dried powder has the effect that promotes that ischemic tissue of brain Angiogenesis and collateral circulation are set up.
Through the experimental data contrast as can be known; RHRSP cerebral thrombosis ischemia model group VEGF Messenger RNA (VEGFmRNA) expression is increased significantly than sham operated rats; illustrate behind the cerebral tissue hypoxic-ischemic can Stimulation of The Brain tissue in the expression increase of VEGF; a kind of protective response that resists ischemia injury can appear in body self behind the prompting cerebral infarction; the expression of VEGF is increased, self " compensatory revascularization " behind ischemia, occur.Each dosage group of salvianolic acid A freeze-dried powder and model group group are relatively; the VEGFmRNA expression significantly raises; show that the salvianolic acid A freeze-dried powder can significantly promote the ischemic tissue of brain angiogenesis; promote the foundation of collateral circulation compensation; save ischemia half blanking bar; the brain tissue impairment that the protection ischemia causes, and have certain dose-effect relationship.
Through the experimental data contrast as can be known; RHRSP cerebral thrombosis ischemia model group rat cerebral tissue's basic fibroblast growth factor (bFGF) protein expression is increased significantly than sham operated rats; but the prolongation with Ischemia Time; have a declining tendency; behind the prompting cerebral tissue hypoxic-ischemic; body self produces of short duration protection stress, but Stimulation of The Brain organizes the bFGF protein expression to increase.Each dosage group of salvianolic acid A freeze-dried powder and model group compare, and the bFGF protein expression of each time period significantly increases; Each time period of each dosage group self shows that relatively the 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, helps the foundation of collateral circulation, saves ischemia half blanking bar.
Through experimental data contrast as can be known, the salvianolic acid A freeze-dried powder can alleviate cerebral ischemia-reperfusion injury in rats neurologic impairment symptom, is conducive to its neurobehavioral recovery, and the salvianolic acid A freeze-dried powder has the effect that improves function of nervous system behind the brain tissue impairment.
Through the experimental data contrast as can be known, each dosage group of salvianolic acid A freeze-dried powder is compared with the Ischemia-Reperfusion Injury Model group: cerebral infarction volume is than significantly reducing, cerebral index and brain water content all obviously reduce, show that the salvianolic acid A freeze-dried powder can reduce rats after cerebral ischemic reperfusion cerebral tissue infarction size, can alleviate the cerebral tissue edema degree behind the cerebral ischemia reperfusion injury.
Through the experimental data contrast as can be known, Level In Rats With Focal Cerebral Ischemia, give various dose salvianolic acid A 10min and begin, each medicine group than self administration before ischemia half blanking bar regional cerebral blood flow (rCBF) obvious rising is arranged, and high with the salvianolic acid A freeze-dried powder, middle dosage group is particularly remarkable; Each dosage group of salvianolic acid A freeze-dried powder is compared, and good dose-effect relationship is arranged; The rCBF of dosage group day part is suitable with the nimodipine group in the salvianolic acid A freeze-dried powder.Hence one can see that, and the 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.
Through experimental data contrast as can be known, after rat continuous cerebral ischemia 2h recovered to fill with again, each organizes rat ischemia half blanking bar rCBF all in various degree increase.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 compared utmost point significant difference with model group.1h after filling with again, nimodipine and salvianolic acid A freeze-dried powder high dose group rCBF return near former rCBF 75%, in the 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 the 3h, each medicine group rCBF maintains in the metastable scope again.The result shows that the salvianolic acid A freeze-dried powder can obviously improve the cerebral blood flow of ischemia half blanking bar cerebral tissue after the Ischemia and Reperfusion in vivo in Rats, recovering brain cell blood supplies, thereby save ischemia half blanking bar, prevent further damage even dead of cerebral tissue, ischemic cerebrovascular is had good therapeutical effect.
Through the experimental data contrast as can be known, 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.The 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, the brain cell that ischemia half blanking bar after the redemption cerebral ischemia is at death's door, prevent further damage even dead of cerebral tissue, can perform well in treating ischemic cerebrovascular.
Through the experimental data contrast as can be known, after rat cerebral ischemia 2h filled with 24h again, neuronal apoptosis was serious; And after nimodipine and the intervention of various dose salvianolic acid A freeze-dried powder, compare with model group, neuronal apoptosis significantly reduces (P＜0.001 or P＜0.01) in the rat cerebral tissue; Show that the salvianolic acid A freeze-dried powder has the effect that suppresses cerebral ischemia rat cerebral tissue neuronal death, suppresses neuronal apoptosis.
The neurotrophic factor histone matter molecule essential with survival that be neure growth plays supporting function to the integrity of neure growth, growth and function.Nerve growth factor (NGF), Brain Derived Neurotrophic Factor (BDNF), neurotrophic factor-3 (NT-3) are the parts of neurotrophic factor family, can keep neuronal survival and the differentiation of promotion neurocyte and induce axon growth; Energy neuroprotective unit, promotion neuron reparation and inhibition delayed neuronal death, thereby to anti-cerebral ischemia, protection cerebral ischemia.Behind the Ischemia Reperfusion, increase to some extent than sham operated rats in the rat cerebral tissue, behind the prompting cerebral reperfusion injury, NGF, BDNF express in the cerebral tissue stress protectiveness increase; But NT-3 content obviously reduces in the Neurons Against Cerebral Ischemia tissue.Through the experimental data contrast as can be known; compare with model control group; salvianolic acid A freeze-dried powder each dosage group 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.
Behind the ischemia-reperfusion, 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 in the cerebral tissue; Through the experimental data contrast as can be known, each the inflammatory cytokine content of rat cerebral tissue that gives each dosage group of salvianolic acid A freeze-dried powder obviously reduces than 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.
Through the experimental data contrast as can be known, 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; Compare with model group, each dosage group of nimodipine group and salvianolic acid A freeze-dried powder can significantly reduce Ca in the cerebral tissue 2+Content, rising K +, Mg 2+Content illustrates that the salvianolic acid A freeze-dried powder can suppress Ca 2+Interior stream alleviates calcium overload, thereby can suppress Ca 2+The neuronal cell apoptosis that overload is induced.
Through the experimental data contrast as can be known; the 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) in the rising cerebral ischemia/reperfusion injury of rats cerebral tissue, taurine (Tau) content, significantly reduces content and the aminoacid exitotoxicity index of cerebral tissue Glutamic Acid (Glu), aspartic acid (Asp), glycine (Gly).And dosage increases, and effect strengthens.The salvianolic acid A freeze-dried powder that shows in conjunction with the histopathology pathological examination can obviously alleviate cerebral edema, improves the result of neuronal cell form, shows that the salvianolic acid A freeze-dried powder can suppress monoamine neurotransmitter and excessively discharge, and improves the monoamine neurotransmitter disorder; Suppress the accumulation of excitatory amino acid in the 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.
Through the experimental data contrast as can be known, each dosage group of salvianolic acid A freeze-dried powder is compared with the ischemia-reperfusion injury model group, lipid peroxide in the rat cerebral tissue (LPO) content significantly reduces, and superoxide dismutase (SOD) and glutathion peroxidase (GSH-Px) activity significantly raise; Illustrate that the salvianolic acid A freeze-dried powder can increase the generation of the activity of peroxide scavenger enzyme in the ischemical reperfusion injury cerebral tissue, inhibition peroxide, thereby to the damage of antioxidant radical to neuronal cell and cerebral tissue.
Through experimental data contrast as can be known, the cerebrovascular endothelial cell apoptosis number of cerebral thrombosis ischemia model each time period of group is significantly higher than sham operated rats, and 6h behind the cerebral tissue ischemia, endothelial cell apoptosis be showed increased just, reaches the peak to the ischemia in 24 hours.Compare with model group, the apoptosis digital display work of each time period of each dosage group of salvianolic acid A freeze-dried powder reduces, illustrate that the salvianolic acid A freeze-dried powder can suppress cerebral ischemia hindbrain apoptosis of vascular endothelial cell, improve the cerebrovascular endothelial cell survival rate, thereby guarantee the normal of cerebrovascular endothelial cell function, keep the complete of ischemic injuries hindbrain blood vessel structure and function and strengthen ability to anti-cerebral ischemia damnification, the effect of performance treatment ischemic cerebrovascular.
Through the experimental data contrast as can be known, 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 the 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.
Through the experimental data contrast as can be known, Hypoxia/Reoxygenation Injury can cause each phase apoptosis rate of Brain Microvascular Endothelial significantly to increase.With model group relatively, each dosage group of salvianolic acid A 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 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) than model group, and wherein the middle and high dosage group of salvianolic acid A freeze-dried powder rises near the Normal group level; Each administration group t-PA/PAI and NO/ET ratio also significantly improve than model group.Prompting salvianolic acid A freeze-dried powder can improve the secretory function of brain microvessel endothelial cells in vitro behind the Hypoxia/Reoxygenation Injury.
Behind the Hypoxia/Reoxygenation Injury, Ca in the BMVEC born of the same parents 2+Concentration significantly increases.Through the experimental data contrast as can be known, compare each medicine group Ca with model group 2+Concentration extremely significantly reduces, and is the most remarkable with the middle and high dosage group of salvianolic acid A freeze-dried powder effect, and intracellular calcium concentration significantly is 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 the BMVEC born of the same parents 2+The effect of concentration.
Through the experimental data contrast as can be known, the salvianolic acid A freeze-dried powder compares the thrombus formation time significant prolongation of the high, medium and low dosage group of salvianolic acid A freeze-dried powder to impact and the normal saline matched group of rat artery thrombus formation time; Low dose group (0.5mg/kg) thrombus formation time is suitable with 20mg/kg aspirin group; Show that the salvianolic acid A freeze-dried powder can prolong thrombus formation time, the formation of prevention of arterial thrombosis, pre-preventing thrombosis and the ischemic cerebrovascular that causes.
Through the experimental data contrast as can be known, the salvianolic acid A freeze-dried powder compares impact and the normal saline group that rat suppository forms, and 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 the salvianolic acid A freeze-dried powder has the thrombotic effect of good inhibition, can be used for the ischemic cerebrovascular due to prevention or the treatment thrombosis.
Through the experimental data contrast as can be known, the salvianolic acid A freeze-dried powder compares impact and the normal saline group of 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 20mg/kg aspirin group effect suitable (P＞0.05); Show that the salvianolic acid A freeze-dried powder has the effect that suppresses venous thrombosis, can be used for the venous thromboembolism after the prophylaxis of acute ischemic cerebrovascular occurs.
Through the experimental data contrast as can be known, the salvianolic acid A freeze-dried powder compares rats in vitro thrombosis and 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; Formation has preferably inhibitory action to external thrombus to show the salvianolic acid A freeze-dried powder.
Through the experimental data contrast as can be known, the salvianolic acid A freeze-dried powder compares thrombolytic and the normal saline group of established thrombosis in the body, and the normal saline group all continues thromboembolism, and without logical phenomenon occurring again; The number of animals that each medicine group continues thromboembolism is few, and comparing with the normal saline group all has utmost point significant difference; Dosage group in the salvianolic acid A freeze-dried powder, its vessel open degree is similar to 2000U/kg urokinase group, and its recanalization rate is suitable; Salvianolic acid A freeze-dried powder high dose group continues recanalization rate and recanalization rate all is higher than the urokinase group, and the bolt rate is starkly lower than the urokinase group again; Though salvianolic acid A freeze-dried powder low dose group recanalization rate is lower than the urokinase group, bolt rate is suitable with the urokinase group again for it, and has and be lower than the again trend of bolt rate of urokinase.Illustrate the salvianolic acid A freeze-dried powder thrombolytic is arranged preferably and prevents thrombolytic after the again effect of thromboembolism.
Behind the cerebral thrombosis, rat whole blood viscosity, plasma viscosity significantly increase, and packed cell volume also significantly raises, through the experimental data contrast as can be known, each dosage group of salvianolic acid A freeze-dried powder and model group compare, and its whole blood viscosity and plasma viscosity and packed cell volume all obviously reduce; Prompting salvianolic acid A freeze-dried powder can be accelerated little blood flow velocity, and blood viscosity lowering 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 ischemic rat brain infringement as the master take neuronal death and function of nervous system that causes thereof.Mainly comprise any one or more: (1) cerebral thrombosis: by forming embolus and flow into cerebral vessels with blood from the embolus of heart such as artificial valve, atrial fibrillation, ventricle thrombosis, DCM (dilated cardiomyopathy), moving/vein blood vessel inflammation etc., cause consisting of the front circulation of whole brain blood circulation and any position and/or the multiple location thrombosis of metacyclic blood vessels at different levels.(2) cerebral ischemia reperfusion injury (3) cerebral embolism.(4) atherosis or narrow (5) lacunar infarction of cranium arteria carotis externa and brain basilar artery.(6) Transient ischemic attacks (7) vascular dementia.
Medical science and study of pharmacy personnel can't in advance under the prerequisite of not doing related experiment, learn that in advance the salvianolic acid A freeze-dried powder has above-mentioned good purposes.
Description of drawings
Fig. 1. salvianolic acid B reference substance high-efficient liquid phase chromatogram;
Fig. 2. salvianolic acid B high-efficient liquid phase chromatogram in the 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 the 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.
The specific embodiment
Embodiment 1
Get red rooted salvia, be ground into 6 order granules, add 92 ℃ of water of 7 times of amounts at every turn, warm macerating extracts 3 times, stirs with 25 rev/mins of speed simultaneously, and each warm macerating extracted 3 hours; Extracting solution is evaporated to relative density 1.20 (60 ℃), and adding ethanol makes and contains the alcohol amount 70%, leaves standstill, and filters, and decompression filtrate recycling ethanol also is concentrated into without pure flavor; Thin up contains salvianolic acid B 20mg to every 1ml, and aqueous solution is transferred pH to 4.0 with 10% sodium hydroxide, adds 0.5%ZnCl 2As catalyst, 120 ℃ of temperature thermal conversions 4 hours, conversional solution is with 20% phosphoric acid adjust pH to 2.5, centrifugal, supernatant is evaporated to every 1ml and contains salvianolic acid A 3mg, separate through the HPD-100 macroporous resin column chromatography, the salvianolic acid A applied sample amount is 1: 50 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 10, uses respectively 3 times of cylinder hydrops, 5 times of column volume 25% ethanol elutions, removes impurity, use again 4 times of column volume 40% ethanol elutions, HPLC detects, and collects the 40% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol also is concentrated into without the alcohol flavor; Aqueous solution is concentrated into the solution that every ml contains the 5mg salvianolic acid A, separate by polyamide chromatography post, the salvianolic acid A applied sample amount is 1: 10 with the polyamide ratio, the resin column blade diameter length ratio is 1: 8, use respectively 3 times of cylinder hydrops, 12 times of column volume 40% alcoholic solution eluting remove impurity, use 8 times of column volumes, 60% alcoholic solution eluting again, collect the 60% alcoholic solution part contain salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml and contains salvianolic acid A 15mg aqueous solution; Aqueous solution is transferred pH to 2.5 with 20% phosphoric acid, and with the t-butyl methyl ether of 3 times of amounts of aqueous solution, minute 3 extractions separate organic layer, and the reclaim under reduced pressure t-butyl methyl ether is made the extract that every 1ml contains salvianolic acid A 0.5g, adds 1～3 times of amount silica gel, stirs, and volatilizes; Be added on 15 times of dried silicagel columns of amount that installed stirring sample silica gel, the silicagel column blade diameter length ratio is 1: 10, take pentane-t-butyl methyl ether as eluant, gradient elution, use respectively pentane: 10 times of column volumes of t-butyl methyl ether (4: 6) eluting, pentane: 10 times of column volumes of t-butyl methyl ether (6: 4) eluting, the reclaim under reduced pressure eluant, salvianolic acid A behind the recovery organic solvent adds 12 times of water gagings dissolvings, (the microwave vacuum drying temperature: 60 ℃, 3 ℃ of return difference temperature are more than vacuum-0.07Mpa with microwave vacuum drying, microwave power 20KW) 100 minutes, gets salvianolic acid A.
Embodiment 2
Get red rooted salvia, be cut into decoction pieces, add 90 ℃ of water temperature lixiviates of 8 times of amounts at every turn and get 3 times, stir with 20 rev/mins of speed simultaneously, each warm macerating extracted 2.5 hours; Extracting solution is evaporated to relative density 1.15 (60 ℃), and adding ethanol makes and contains the alcohol amount 75%, leaves standstill, and filters, and decompression filtrate recycling ethanol also is concentrated into without pure flavor; Thin up contains salvianolic acid B 15mg to every 1ml, and aqueous solution is transferred pH to 4.0 with 10% potassium hydroxide, adds 0.6%FeCl 3As catalyst, 120 ℃ of temperature thermal conversions 3.5 hours, conversional solution is with 15% hydrochloric acid adjust pH to 2.5, centrifugal, supernatant is evaporated to every 1ml and contains salvianolic acid A 5mg, separate through the HPD-100 macroporous resin column chromatography, the salvianolic acid A applied sample amount is 1: 45 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 8, uses respectively 3.5 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions, removes impurity, use again 5 times of column volume 45% ethanol elutions, HPLC detects, and collects the 45% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol also is concentrated into without the alcohol flavor; Aqueous solution is concentrated into the solution that every ml contains the 5mg salvianolic acid A, separate by polyamide chromatography post, the salvianolic acid A applied sample amount is 1: 9 with the polyamide ratio, the resin column blade diameter length ratio is 1: 7, use respectively 4 times of cylinder hydrops, 10 times of column volume 40% alcoholic solution eluting remove impurity, use 8 times of column volumes, 65% alcoholic solution eluting again, collect the 65% alcoholic solution part contain salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml and contains salvianolic acid A 12mg aqueous solution; Aqueous solution is transferred pH to 2.6 with 15% hydrochloric acid, and with the t-butyl methyl ether of 4 times of amounts, minute 4 extractions separate organic layer, and the reclaim under reduced pressure t-butyl methyl ether is made the extract that every 1ml contains salvianolic acid A 0.8g, adds 2～3 times of amount silica gel, stirs, and volatilizes; Be added on 13 times of dried silicagel columns of amount that installed stirring sample silica gel, the silicagel column blade diameter length ratio is 1: 8, take normal heptane-ethyl acetate as eluant, and gradient elution, use respectively 8 times of column volumes of normal heptane-ethyl acetate (4: 6) eluting, 8 times of column volumes of normal heptane-ethyl acetate (7: 3) eluting, the reclaim under reduced pressure eluant adds 8 times of water gaging dissolvings again, vacuum drying (baking temperature: 65 ℃, more than vacuum-0.07Mpa, power 10KW) 3 hours, gets salvianolic acid A.
Embodiment 3
Get red rooted salvia, be ground into diameter 2mm granule, add 85 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 2 times, stir with 30 rev/mins of speed simultaneously, each warm macerating extracted 3.5 hours; Extracting solution is evaporated to relative density 1.10 (60 ℃), and adding ethanol makes and contains the alcohol amount 75%, leaves standstill, and filters, and decompression filtrate recycling ethanol also is concentrated into without pure flavor; Thin up contains salvianolic acid B 18mg to every 1ml, and aqueous solution is transferred pH to 5.2 with 10% sodium carbonate, adds 0.6%AlCl 3As catalyst, 123 ℃ of temperature thermal conversions 4.5 hours, conversional solution is with 15% nitric acid adjust pH to 2.8, centrifugal, supernatant is evaporated to every 1ml and contains salvianolic acid A 6mg, separate through the HPD-100B macroporous resin column chromatography, the salvianolic acid A applied sample amount is 1: 40 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 7, uses respectively 4 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions, removes impurity, use again 5 times of column volume 40% ethanol elutions, HPLC detects, and collects the 40% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol also is concentrated into without the alcohol flavor; Aqueous solution is concentrated into the solution that every ml contains the 6mg salvianolic acid A, separate by polyamide chromatography post, the salvianolic acid A applied sample amount is 1: 8 with the polyamide ratio, the resin column blade diameter length ratio is 1: 8, use respectively 4 times of cylinder hydrops, 9 times of column volume 40% alcoholic solution eluting remove impurity, use 7 times of column volumes, 65% alcoholic solution eluting again, collect the 65% alcoholic solution part contain salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml and contains salvianolic acid A 10mg aqueous solution; Aqueous solution is transferred pH to 2.6 with 15% nitric acid, and with the methyl acetate of 4 times of amounts, minute 4 extractions separate organic layer, and the reclaim under reduced pressure methyl acetate is made the extract that every 1ml contains salvianolic acid A 0.7g, adds 3 times of amount silica gel, stirs, and volatilizes; Be added on 10 times of dried silicagel columns of amount that installed stirring sample silica gel, the silicagel column blade diameter length ratio is 1: 7, take petroleum ether, Ethyl formate as eluant, and gradient elution, use respectively 8 times of column volumes of petroleum ether-Ethyl formate (4: 6) eluting, 9 times of column volumes of petroleum ether-Ethyl formate (6: 4) eluting, the reclaim under reduced pressure eluant adds 8 times of water gaging dissolvings again, spray drying (intake air temperature: 170 ℃, 85 ℃ of air outlet temperature, spray velocity: 150ml/min), get salvianolic acid A
Embodiment 4
Get red rooted salvia, be cut into decoction pieces, add 80 ℃ of water temperature lixiviates of 10 times of amounts at every turn and get 3 times, stir with 15 rev/mins of speed simultaneously, each warm macerating extracted 3 hours; Extracting solution is evaporated to relative density 1.18 (60 ℃), and adding ethanol makes and contains the alcohol amount 70%, leaves standstill, and filters, and decompression filtrate recycling ethanol also is concentrated into without pure flavor; Thin up contains salvianolic acid B 20mg to every 1ml, and aqueous solution is transferred pH to 5.4 with 10% sodium bicarbonate, adds 0.8%RuCl 3As catalyst, 128 ℃ of temperature thermal conversions 4.0 hours, conversional solution is with 20% sulphuric acid adjust pH to 2.6, centrifugal, supernatant is evaporated to every 1ml and contains salvianolic acid A 5mg, separate through the HPD-826 macroporous resin column chromatography, the salvianolic acid A applied sample amount is 1: 40 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 7, uses respectively 4 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions, removes impurity, use again 5 times of column volume 40% ethanol elutions, HPLC detects, and collects the 40% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol also is concentrated into without the alcohol flavor; Aqueous solution is concentrated into the solution that every ml contains the 6mg salvianolic acid A, separate by the ODS chromatographic column, the salvianolic acid A applied sample amount is 1: 10 with the ODS ratio, the resin column blade diameter length ratio is 1: 15, use respectively 4 times of cylinder hydrops, 7 times of column volume 35% alcoholic solution eluting remove impurity, use 6 times of column volumes, 60% alcoholic solution eluting again, collect the 60% alcoholic solution part contain salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml and contains salvianolic acid A 12mg aqueous solution; Aqueous solution is transferred pH to 2.8 with 20% sulphuric acid, and with the butyl acetate of 4 times of amounts, minute 4 extractions separate organic layer, and the reclaim under reduced pressure butyl acetate is made the extract that every 1ml contains salvianolic acid A 0.5g, adds 2.5 times of amount silica gel, stirs, and volatilizes; Be added on 9 times of dried silicagel columns of amount that installed stirring sample silica gel, the silicagel column blade diameter length ratio is 1: 8, take pentane, methyl acetate as eluant, gradient elution, use respectively 8 times of column volumes of pentane-methyl acetate (4.5: 5.5) eluting, 8 times of column volumes of pentane-methyl acetate (6.5: 3.5) eluting, the reclaim under reduced pressure eluant, add again 10 times of water gaging dissolvings, (the microwave vacuum drying temperature: 55 ℃, 2 ℃ of return difference temperature are more than vacuum-0.07Mpa with microwave vacuum drying, microwave power 30KW) 120 minutes, gets salvianolic acid A.
Embodiment 5
Get red rooted salvia, be ground into diameter 2mm granule, add 85 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir with 20 rev/mins of speed simultaneously, each warm macerating extracted 2.5 hours; Extracting solution is evaporated to relative density 1.22 (60 ℃), and adding ethanol makes and contains the alcohol amount 70%, leaves standstill, and filters, and decompression filtrate recycling ethanol also is concentrated into without pure flavor; Thin up contains salvianolic acid B 10mg to every 1ml, and aqueous solution is transferred pH to 3.5 with 20% sodium citrate, adds 0.4%PdCl 3As catalyst, 132 ℃ of temperature thermal conversions 3.5 hours, conversional solution is with 20% acetic acid adjust pH to 2.6, centrifugal, supernatant is evaporated to every 1ml and contains salvianolic acid A 5mg, separate through the HPD-826 macroporous resin column chromatography, the salvianolic acid A applied sample amount is 1: 35 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 8, uses respectively 3 times of cylinder hydrops, 4.5 times of column volume 25% ethanol elutions, removes impurity, use again 6 times of column volume 40% ethanol elutions, HPLC detects, and collects the 40% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol also is concentrated into without the alcohol flavor; Aqueous solution is concentrated into the solution that every ml contains the 8mg salvianolic acid A, separate by the ODS chromatographic column, the salvianolic acid A applied sample amount is 1: 12 with the ODS ratio, the resin column blade diameter length ratio is 1: 18, use respectively 4 times of cylinder hydrops, 8 times of column volume 30% alcoholic solution eluting remove impurity, use 5 times of column volumes, 65% alcoholic solution eluting again, collect the 65% alcoholic solution part contain salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml and contains salvianolic acid A 15mg aqueous solution; Aqueous solution is transferred pH to 2.7 with 20% acetic acid, and with the ethyl acetate of 5 times of amounts, minute 5 extractions separate organic layer, and the reclaim under reduced pressure ethyl acetate is made the extract that every 1ml contains salvianolic acid A 0.5g, adds 2 times of amount silica gel, stirs, and volatilizes; Be added on 10 times of dried silicagel columns of amount that installed stirring sample silica gel, the silicagel column blade diameter length ratio is 1: 10, take pentane, methyl acetate as eluant, gradient elution, use respectively 8 times of column volumes of pentane-methyl acetate (4.5: 5.5) eluting, 8 times of column volumes of pentane-methyl acetate (6.5: 3.5) eluting, the reclaim under reduced pressure eluant, add again 12 times of water gaging dissolvings, (the microwave vacuum drying temperature: 65 ℃, 1 ℃ of return difference temperature is more than vacuum-0.07Mpa with microwave vacuum drying, microwave power 30KW) 90 minutes, gets salvianolic acid A.
Embodiment 6
Get red rooted salvia, be ground into diameter 2mm granule, add 88 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir with 22 rev/mins of speed simultaneously, each warm macerating extracted 3.5 hours; Extracting solution is evaporated to relative density 1.16 (60 ℃), and adding ethanol makes and contains the alcohol amount 75%, leaves standstill, and filters, and decompression filtrate recycling ethanol also is concentrated into without pure flavor; Thin up contains salvianolic acid B 13mg to every 1ml, and aqueous solution is transferred pH to 3.6 with 10% sodium hydroxide, adds 0.5%CuCl 2As catalyst, 133 ℃ of temperature thermal conversions 4.5 hours, conversional solution is with 10% hydrochloric acid adjust pH to 2.7, centrifugal, supernatant is evaporated to every 1ml and contains salvianolic acid A 5mg, separate through the HPD-D101 macroporous resin column chromatography, the salvianolic acid A applied sample amount is 1: 40 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 9, uses respectively 4 times of cylinder hydrops, 4 times of column volume 22% ethanol elutions, removes impurity, use again 6 times of column volume 43% ethanol elutions, HPLC detects, and collects the 43% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol also is concentrated into without the alcohol flavor; Aqueous solution is concentrated into the solution that every ml contains the 10mg salvianolic acid A, separate by the ODS chromatographic column, the salvianolic acid A applied sample amount is 1: 15 with the sephadex lh-20 ratio, the resin column blade diameter length ratio is 1: 20, use respectively 4 times of cylinder hydrops, 8 times of column volume 30% alcoholic solution eluting remove impurity, use 5 times of column volumes, 60% alcoholic solution eluting again, collect the 60% alcoholic solution part contain salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml and contains salvianolic acid A 12mg aqueous solution; Aqueous solution is transferred pH to 2.8 with 10% hydrochloric acid, and with the methyl acetate of 5 times of amounts, minute 5 extractions separate organic layer, and the reclaim under reduced pressure methyl acetate is made the extract that every 1ml contains salvianolic acid A 0.5g, adds 3 times of amount silica gel, stirs, and volatilizes; Be added on 12 times of dried silicagel columns of amount that installed stirring sample silica gel, the silicagel column blade diameter length ratio is 1: 10, take pentane, methyl acetate as eluant, and gradient elution, use respectively 6 times of column volumes of pentane-methyl acetate (4: 6) eluting, 7 times of column volumes of pentane-methyl acetate (6: 4) eluting, the reclaim under reduced pressure eluant adds 10 times of water gaging dissolvings again, vacuum drying (baking temperature: 60 ℃, more than vacuum-0.07Mpa, power 15KW) dry 3.5 hours, get salvianolic acid A.
Embodiment 7
Get red rooted salvia, be cut into decoction pieces, add 90 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir with 25 rev/mins of speed simultaneously, each warm macerating extracted 3 hours; Extracting solution is evaporated to relative density 1.12 (60 ℃), and adding ethanol makes and contains the alcohol amount 75%, leaves standstill, and filters, and decompression filtrate recycling ethanol also is concentrated into without pure flavor; Thin up contains salvianolic acid B 20mg to every 1ml, and aqueous solution is transferred pH to 5.0 with 10% sodium carbonate, adds 1.0%ZnCl 2As catalyst, 135 ℃ of temperature thermal conversions 4.5 hours, conversional solution is with 15% sulphuric acid adjust pH to 3.0, centrifugal, supernatant is evaporated to every 1ml and contains salvianolic acid A 5mg, separate through the AB-8 macroporous resin column chromatography, the salvianolic acid A applied sample amount is 1: 36 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 9, uses respectively 3 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions, removes impurity, use again 5 times of column volume 45% ethanol elutions, HPLC detects, and collects the 45% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol also is concentrated into without the alcohol flavor; Aqueous solution is concentrated into the solution that every 1ml contains the 6mg salvianolic acid A, separate by polyamide chromatography post, the salvianolic acid A applied sample amount is 1: 10 with the polyamide ratio, the resin column blade diameter length ratio is 1: 10, use respectively 4 times of cylinder hydrops, 8 times of column volume 45% alcoholic solution eluting remove impurity, use 8 times of column volumes, 65% alcoholic solution eluting again, collect the 65% alcoholic solution part contain salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml and contains salvianolic acid A 10mg aqueous solution; Aqueous solution is transferred pH to 2.7 with 15% sulphuric acid, and with the t-butyl methyl ether of 4 times of amounts, minute 4 extractions separate organic layer, and the reclaim under reduced pressure t-butyl methyl ether is made the extract that every 1ml contains salvianolic acid A 0.6g, adds 3 times of amount silica gel, stirs, and volatilizes; Be added on 10 times of dried silicagel columns of amount that installed stirring sample silica gel, the silicagel column blade diameter length ratio is 1: 8, take pentane, ethyl acetate as eluant, gradient elution, use respectively 8 times of column volumes of pentane-ethyl acetate (4: 6) eluting, 8 times of column volumes of pentane-ethyl acetate (6: 4) eluting, the reclaim under reduced pressure eluant, add again 8 times of water gaging dissolvings, (the microwave vacuum drying temperature: 70 ℃, 4 ℃ of return difference temperature are more than vacuum-0.07Mpa with microwave vacuum drying, microwave power 25KW) 110 minutes, gets salvianolic acid A.
Embodiment 8
Get red rooted salvia, be ground into diameter 2mm granule, add 85 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir with 22 rev/mins of speed simultaneously, each warm macerating extracted 3 hours; Extracting solution is evaporated to relative density 1.14 (60 ℃), and adding ethanol makes and contains the alcohol amount 70%, leaves standstill, and filters, and decompression filtrate recycling ethanol also is concentrated into without pure flavor; Thin up contains salvianolic acid B 25mg to every 1ml, and aqueous solution is transferred pH to 4.2 with 10% sodium citrate, adds 0.4%AlCl 3As catalyst, 133 ℃ of temperature thermal conversions 4.5 hours, conversional solution is with 10% hydrochloric acid adjust pH to 2.8, centrifugal, supernatant is evaporated to every 1ml and contains salvianolic acid A 5mg, separate through the HPD-300 macroporous resin column chromatography, the salvianolic acid A applied sample amount is 1: 45 with the macroporous adsorbent resin ratio, the resin column blade diameter length ratio is 1: 10, uses respectively 5 times of cylinder hydrops, 6 times of column volume 20% ethanol elutions, removes impurity, use again 5 times of column volume 45% ethanol elutions, HPLC detects, and collects the 45% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol also is concentrated into without the alcohol flavor; Aqueous solution is concentrated into the solution that every 1ml contains the 8mg salvianolic acid A, separate by polyamide chromatography post, the salvianolic acid A applied sample amount is 1: 12 with the polyamide ratio, the 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 contain salvianolic acid A, decompression recycling ethanol also is concentrated into every 1ml and contains salvianolic acid A 10mg aqueous solution; Aqueous solution is transferred pH to 2.9 with 15% hydrochloric acid, and with the t-butyl methyl ether of 5 times of amounts, minute 5 extractions separate organic layer, and the reclaim under reduced pressure t-butyl methyl ether is made the extract that every 1ml contains salvianolic acid A 0.6g, adds 2.5 times of amount silica gel, stirs, and volatilizes; Be added on 12 times of dried silicagel columns of amount that installed stirring sample silica gel, the silicagel column blade diameter length ratio is 1: 8, take pentane, t-butyl methyl ether as eluant, gradient elution, use respectively 8 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 8 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, the reclaim under reduced pressure eluant, add again 9 times of water gaging dissolvings, (the microwave vacuum drying temperature: 65 ℃, 5 ℃ of return difference temperature are more than vacuum-0.07Mpa with microwave vacuum drying, microwave power 40KW) 120 minutes, gets salvianolic acid A.
Embodiment 9
Get the salvianolic acid A 80g that embodiment 3 makes, inject water 2800ml stirring and make dissolving, with 10% sodium hydroxide adjust pH 4.5, add mannitol 60g and make dissolving, add again vitamin C 0.5g, stir and make dissolving, mixing, add active carbon 2g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear usefulness 0.22 μ m microporous filter membrane and filter, the filtrate fill is in aseptic cillin bottle, and part is butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot after fill is finished, is sent into freeze dryer, close chamber door, open freeze dryer, be cooled to-40 ℃ with 20 ℃/h speed, be incubated freezing 16 hours; Be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 10 hours rises to-25 ℃, keeps-25 ℃ of vacuum dryings 8 hours; Continue to heat up, evenly be warming up to 41 ℃ with 0.5 ℃/min, keep 41 ℃ of dryings after 3 hours, sample temperature is down to room temperature, add a cover, namely get the salvianolic acid A freeze-dried powder.
Embodiment 10
Get the salvianolic acid A 20g that embodiment 4 makes, inject water 1900ml stirring and make dissolving, with 10% sodium hydroxide adjust pH 4.8, add mannitol 20g and make dissolving, add again vitamin C 0.8g, stir and make dissolving, mixing, add active carbon 0.5g stirring and adsorbing, filter, remove active carbon; Inject water to 2000ml, detect intermediate content, pH value, detect qualified rear usefulness 0.22 μ m microporous filter membrane and filter, the filtrate fill is in aseptic cillin bottle, and part is butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot after fill is finished, is sent into freeze dryer, close chamber door, open freeze dryer, be cooled to-45 ℃ with 25 ℃/h speed, be incubated freezing 15 hours; Be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 12 hours rises to-25 ℃, keeps-25 ℃ of vacuum dryings 8 hours; Continue to heat up, evenly be warming up to 40 ℃ with 0.8 ℃/min, keep 40 ℃ of dryings after 3 hours, sample temperature is down to room temperature, add a cover, namely get the salvianolic acid A freeze-dried powder.
Embodiment 11
Get the salvianolic acid A 10g that embodiment 5 makes, inject water 1500ml stirring and make dissolving, with 10% sodium hydroxide adjust pH 4.6, add mannitol 20g and make dissolving, add again vitamin C 0.8g, stir and make dissolving, mixing, add active carbon 1.2g stirring and adsorbing, filter, remove active carbon; Inject water to 2000ml, detect intermediate content, pH value, detect qualified rear usefulness 0.22 μ m microporous filter membrane and filter, the filtrate fill is in aseptic cillin bottle, and part is butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot after fill is finished, is sent into freeze dryer, close chamber door, open freeze dryer, be cooled to-42 ℃ with 25 ℃/h speed, be incubated freezing 12 hours; Be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 13 hours rises to-23 ℃, keeps-23 ℃ of vacuum dryings 7 hours; Continue to heat up, evenly be warming up to 42 ℃ with 0.8 ℃/min, keep 42 ℃ of dryings after 4 hours, sample temperature is down to room temperature, add a cover, namely get the salvianolic acid A freeze-dried powder.
Embodiment 12
Get the salvianolic acid A 30g that embodiment 6 makes, inject water 2000ml stirring and make dissolving, with 20% sodium carbonate adjust pH 4.2, add mannitol 20g, lactose 10g and make dissolving, add again thiourea 0.5g, stir and make dissolving, mixing, add active carbon 1.0g stirring and adsorbing, filter, remove active carbon; Inject water to 2000ml, detect intermediate content, pH value, detect qualified rear usefulness 0.22 μ m microporous filter membrane and filter, the filtrate fill is in aseptic cillin bottle, and part is butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot after fill is finished, is sent into freeze dryer, close chamber door, open freeze dryer, be cooled to-44 ℃ with 29 ℃/h speed, be incubated freezing 14 hours; Be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 11 hours rises to-26 ℃, keeps-26 ℃ of vacuum dryings 6.5 hours; Continue to heat up, evenly be warming up to 43 ℃ with 0.6 ℃/min, keep 43 ℃ of dryings after 3.5 hours, sample temperature is down to room temperature, add a cover, namely get the salvianolic acid A freeze-dried powder.
Embodiment 13
Get the salvianolic acid A 35g that embodiment 7 makes, inject water 2500ml stirring and make dissolving, with 10% potassium hydroxide adjust pH 4.5, add glucose 30g and make dissolving, add again sodium sulfite 3g, stir and make dissolving, mixing, add active carbon 1.5g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear usefulness 0.22 μ m microporous filter membrane and filter, the filtrate fill is in aseptic cillin bottle, and part is butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot after fill is finished, is sent into freeze dryer, close chamber door, open freeze dryer, be cooled to-43 ℃ with 27 ℃/h speed, be incubated freezing 11 hours; Be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 12.5 hours rises to-24 ℃, keeps-24 ℃ of vacuum dryings 7.5 hours; Continue to heat up, evenly be warming up to 40 ℃ with 0.7 ℃/min, keep 40 ℃ of dryings after 5 hours, sample temperature is down to room temperature, add a cover, namely get the salvianolic acid A freeze-dried powder.
Embodiment 14
Get the salvianolic acid A 40g that embodiment 8 makes, inject water 2700ml stirring and make dissolving, with 10% sodium hydroxide adjust pH 4.3, add glucose 20g, lactose 20g and make dissolving, add again sodium pyrosulfite 4g, stir and make dissolving, mixing, add active carbon 1.5g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear usefulness 0.22 μ m microporous filter membrane and filter, the filtrate fill is in aseptic cillin bottle, and part is butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot after fill is finished, is sent into freeze dryer, close chamber door, open freeze dryer, be cooled to-41 ℃ with 28 ℃/h speed, be incubated freezing 13 hours; Be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 13.5 hours rises to-25.5 ℃, keeps-25.5 ℃ of vacuum dryings 6.5 hours; Continue to heat up, evenly be warming up to 44 ℃ with 0.9 ℃/min, keep 44 ℃ of dryings after 3.5 hours, sample temperature is down to room temperature, add a cover, namely get the salvianolic acid A freeze-dried powder.
Embodiment 15
Get the salvianolic acid A 60g that embodiment 1 makes, inject water 2700ml stirring and make dissolving, with 10% sodium hydroxide adjust pH 4.0, add mannitol 40g and make dissolving, add again Catergen g, stir and make dissolving, mixing, add active carbon 2g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear usefulness 0.22 μ m microporous filter membrane and filter, the filtrate fill is in aseptic cillin bottle, and part is butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot after fill is finished, is sent into freeze dryer, close chamber door, open freeze dryer, be cooled to-42.5 ℃ with 24.5 ℃/h speed, be incubated freezing 11.5 hours; Be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 13.5 hours rises to-24.5 ℃, keeps-24.5 ℃ of vacuum dryings 6.5 hours; Continue to heat up, evenly be warming up to 42.5 ℃ with 0.55 ℃/min, keep 42.5 ℃ of dryings after 2.5 hours, sample temperature is down to room temperature, add a cover, namely get the salvianolic acid A freeze-dried powder.
Embodiment 16
Get the salvianolic acid A 70g that embodiment 2 makes, inject water 2800ml stirring and make dissolving, with 10% sodium hydroxide adjust pH 5.0, add mannitol 40g and make dissolving, add again Catergen g, stir and make dissolving, mixing, add active carbon 2g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear usefulness 0.22 μ m microporous filter membrane and filter, the filtrate fill is in aseptic cillin bottle, and part is butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot after fill is finished, is sent into freeze dryer, close chamber door, open freeze dryer, be cooled to-44.5 ℃ with 28.5 ℃/h speed, be incubated freezing 11.5 hours; Be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 13.5 hours rises to-24.5 ℃, keeps-24.5 ℃ of vacuum dryings 6.5 hours; Continue to heat up, evenly be warming up to 43.5 ℃ with 0.75 ℃/min, keep 43.5 ℃ of dryings after 3 hours, sample temperature is down to room temperature, add a cover, namely get the 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 m);
Reagent: methanol is chromatographically pure, and water is the ultra-pure water of Millipore preparation, and other reagent are analytical pure.
Salvianolic acid B reference substance (lot number 111562-201009) is all available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, for assay; The salvianolic acid A reference substance is 99.52% for self-control through purity mark content.
2. the preparation of reference substance solution and need testing solution
2.1 the preparation of reference substance solution: precision takes by weighing salvianolic acid B, the about 10mg of salvianolic acid A reference substance respectively, puts in the 100ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, in contrast the product stock solution; Precision is drawn above-mentioned each 1ml respectively again, puts in the same 10ml measuring bottle, adds methanol and is diluted to scale, shakes up, as mixing reference substance solution.
2.3 the preparation precision of need testing solution takes by weighing embodiment 1 sample (being equivalent to approximately salvianolic acid A 10mg) and following " 1.3 salvianolic acid B raw materials preparation in the experimental example 2 " obtains sample (being equivalent to approximately salvianolic acid B 10mg), put in the 100ml measuring bottle, add dissolve with methanol and be diluted to scale, shake up, and get final product.
3. chromatographic condition and system suitability
Take octadecylsilane chemically bonded silica as filler; Flow velocity 1.0ml/min; Detect wavelength: get the mixing reference substance solution, carry out UV scanning, the result has absorption maximum at 286nm wavelength place, so determine that detecting wavelength is 286nm; 30 ℃ of column temperatures; Number of theoretical plate calculates by the salvianolic acid A peak should be not less than 10000.
In the time of 0-10 minute, the ratio of methanol is down to 60% by 30% ratio that rises to 40%, 0.1-0.5% phosphate aqueous solution by 70%; In the time of 10-30 minute, the ratio of methanol is down to 45% by 40% ratio that rises to 55%, 0.1-0.5% phosphate aqueous solution by 50%; In the time of 30-60 minute, 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 sees Table 1, HPLC collection of illustrative plates and sees Fig. 1, Fig. 2, Fig. 3, Fig. 4.
The chromatographic peak retention time result of each reference substance of table 1.
Figure BSA00000812340600251
4, the investigation of linear relationship
Accurate above-mentioned mixing reference substance solution 0.1ml, 0.2ml, 0.5ml, 1ml, 2ml, the 5ml of drawing, put respectively in the 10ml measuring bottle, 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.Accurate each the 10 μ l injection liquid chromatography of above-mentioned standard solution of drawing, calculate peak area by " 3. chromatographic condition and system suitability " lower chromatographic condition, take the peak area integrated value as vertical coordinate, each concentration reference substance sample size is abscissa, the drawing standard curve respectively.The result shows that mixing reference substance solution becomes good linear relationship in following scope, see Table 2.
Table 2. mixes reference substance solution linear relationship result
Figure BSA00000812340600252
5. precision test
Get the mixing reference substance solution, measure according to " 3. chromatographic condition and system suitability " lower chromatographic condition, repeat sample introduction 6 times.The result shows that the precision of the method is good, sees Table 3.
Table 3. Precision test result
Figure BSA00000812340600253
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, as a result in the need testing solution each composition in 24 hours peak area without significant change, show having good stability of need testing solution, see Table 4.
Table 4. stability test result
Figure BSA00000812340600261
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.The result shows that the method repeatability is good, sees Table 5.
Table 5. replica test result
Figure BSA00000812340600262
8. recovery test
Adopt the application of sample recovery test, get the salvianolic acid A raw material 10mg of known content, accurately weighed, parallel 6 parts, ratio in each constituent content-reference substance (1: 1) adds a certain amount of reference substance solution respectively, by 2.3 below legal system available test sample solutions, measure and calculate average recovery and the RSD of each composition.The result shows that the accuracy of the method is good, sees Table 6.
Table 6 recovery test result
Figure BSA00000812340600263
Figure BSA00000812340600271
Experimental example 2: the extraction of salvianolic acid B
1.1 extract the affirmation of solvent and extracting method
Take by weighing red rooted salvia 400g, press method mensuration content of danshinolic acid B under the 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 extracted 1.5 hours at 80 ℃ of warm macerating, warm macerating extracts three times altogether, and merge extractive liquid, is measured salvianolic acid B and calculated extraction ratio, result such as table 7.
Experiment 2: get red rooted salvia 100g, add 8 times of water gagings at every turn and extracted 1.5 hours at 80 ℃ of warm macerating, stir with 10～50 rev/mins of speed simultaneously, warm macerating stirs and extracts three times altogether, and merge extractive liquid, is measured salvianolic acid B and calculated extraction ratio, result such as table 7.
Experiment 3: get red rooted salvia 100g, add 8 times of water gagings at every turn and decocted 1.5 hours, decoct altogether three times, merge extractive liquid, is measured salvianolic acid B and is calculated extraction ratio, result such 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, is measured salvianolic acid B and is calculated extraction ratio, result such as table 7.
Table 7. extracts solvent and extracting method experimental result
Figure BSA00000812340600272
Above-mentioned experimental result shows, it is influential to the salvianolic acid B extraction ratio to adopt 50% ethanol to make the extraction solvent, 50% ethanol extraction is better than water extraction, but soak with water temperature and to add that stir to extract difference little, two kinds of extracting modes that stir in the water extraction process and do not stir are influential to the extraction ratio of salvianolic acid B, decocting extraction may be because temperature is higher, extracts influential to salvianolic acid B.
1.2 the preferred extraction process of orthogonal experiment
1.2.1 the optimization of water extraction process research: according to above result of the test, water extraction process with extraction time (A), extraction time (B), quantity of solvent (C), extract four of temperature (D) as the investigation factor, each factor is established three levels, presses L9 (3 4) orthogonal table carries out Orthogonal Experiment and Design (table 8, table 9), investigating index is the extraction ratio of salvianolic acid B.
Table 8. extraction factor water-glass
Table 9. extraction process orthogonal experiments table
Figure BSA00000812340600282
Table 10. the results of analysis of variance
Figure BSA00000812340600283
Figure BSA00000812340600291
F 0.05(2，2)＝19.00 F 0.01(2，2)＝99.00
The water extraction the results of analysis of variance shows, each experimental factor is to the extraction rate of transform of salvianolic acid B there are no significant difference, so the water extraction condition of salvianolic acid B of the present invention is for adding 3～15 times of water gagings, extracting 1～3 time at 45～95 ℃ of lower warm macerating, stir with 10～50 rev/mins of speed simultaneously, extracted 1～4 hour at every turn or add 3～15 times of amounts at every turn and decoct extraction, the each extraction 1～4 hour extracted 1～3 time altogether.
1.2.2 the optimization of alcohol extraction technique research: according to above result of the test, alcohol extraction technique with four of extraction times (A), extraction time (B), quantity of solvent (C), concentration of alcohol (D) as the investigation factor, each factor is established three levels, presses L9 (3 4) orthogonal table carries out Orthogonal Experiment and Design (table 11, table 12), investigating index is the extraction ratio of salvianolic acid B.
Table 11. extraction factor water-glass
Figure BSA00000812340600292
Table 12. extraction process orthogonal experiments
Figure BSA00000812340600293
Figure BSA00000812340600301
Table 13. the results of analysis of variance
Figure BSA00000812340600302
F 0.05(2，2)＝19.00 F 0.01(2，2)＝99.00
Alcohol reflux extracts the results of analysis of variance and shows, each experimental factor, extracted 1～4 hour so the extraction conditions of this salvianolic acid B is measured 30%～60% alcohol reflux 1～3 time for adding 3～15 times the extraction rate of transform of salvianolic acid B there are no significant difference at every turn.
1.3 salvianolic acid B raw material preparation
According to above-mentioned orthogonal experiment Optimization Technology, get red rooted salvia 10kg, add 8 times of water gagings at every turn and extracted 1.5 hours at 80 ℃ of warm macerating, stir with 10～50 rev/mins 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 ℃), adding ethanol makes and contains the alcohol amount 60%, filters, and decompression filtrate recycling ethanol also is concentrated into without pure flavor, vacuum drying, salvianolic acid extract 4.15kg, recording content of danshinolic acid B is 10.25%.
Experimental example 3: salvianolic acid B transforms salvianolic acid A technique relatively
Experiment 1: get the about 30g of salvianolic acid B raw material of 1.3 lower preparations, 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 1.3 lower preparations, be mixed with 200ml solution, add 10% sodium hydroxide solution adjust pH to 4.5, add 1.0%ZnCl 2
Experiment 3: get and contain 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 the mol ratio that makes it with salvianolic acid B is 0.5;
Above-mentioned each experimental group places same autoclave, and 120 ℃ of lower reactions 4.0 hours, the salvianolic acid A productive rate was calculated in cooling.
Table 14. salvianolic acid B transforms the salvianolic acid A experimental result
Figure BSA00000812340600311
Table 14 result shows that the salvianolic acid B high-purity does not need to be purified to more than 50% to transform on transforming not impact, and salvianolic acid B is converted in the 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 the salvianolic acid A process optimization
Above-mentioned experimentation proves that salvianolic acid B purity is not to affect the factor that salvianolic acid B transforms salvianolic acid A, transforms salvianolic acid A but add certain catalysts influence salvianolic acid B, 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, and each factor is established three levels, presses L9 (3 4) orthogonal table carries out EXPERIMENTAL DESIGN (table 15, table 16), investigating index is the productive rate of salvianolic acid A.
Table 15. transforming agent water-glass
Figure BSA00000812340600312
Figure BSA00000812340600321
Table 16. conversion process orthogonal experiments
Figure BSA00000812340600322
Table 17. the results of analysis of variance table
Figure BSA00000812340600323
*F 0.05(2，2)＝19.00 ΔF 0.01(2，2)＝99.00
The results of analysis of variance shows that 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 the salvianolic acid A productive rate, analyzes from the K value, concentration is higher than 30mg/ml is converted into salvianolic acid A on improving salvianolic acid B not impact of effect, and the impurity that forms is more, and therefore, salvianolic acid B concentration should be selected 1～30mg/ml before transforming; Factor B (pH), factor C (temperature) have utmost point significant difference to the salvianolic acid A productive rate, should strictly control pH and temperature in the prompting salvianolic acid B conversion process, the transforming to salvianolic acid A of realization salvianolic acid B higher yields that can be successful.
Experimental example 5: catalyst Z nCl 2The impact of consumption on transforming
Transform salvianolic acid A process optimization condition by above-mentioned salvianolic acid B, get the salvianolic acid B raw material, add purified water 200ml, make the solution that salvianolic acid B concentration is 15mg/ml, regulating pH is 4.5, and conversion temperature is 120 ℃, and transformation time is 4 hours, by with the salvianolic acid B molar percentage, add respectively not commensurability catalyst Z nCl 2, the results are shown in Table 18.
Table 18. catalyst Z nCl 2Consumption affects experimental result to transforming
Figure BSA00000812340600331
Catalyst Z nCl 2Consumption affects experimental result to conversion and shows that catalyst amount 〉=0.02% can produce 55.65% conversion ratio, preferred catalyst consumption 0.1%～3.0%, and more preferably behind 0.5%～2.0%. catalyst amount＞3%, conversion ratio no longer is 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 the salvianolic acid B raw material, add purified water 200ml, make the solution that salvianolic acid B concentration is 15mg/ml, regulating pH is 4.5, and conversion temperature is 120 ℃, and transformation time is 4 hours, the catalyst that adds respectively different cultivars and various dose transforms, and the results are shown in Table 19.
Table 19. different catalysts transforms the impact of red A on red B
Figure BSA00000812340600341
Table 19 result shows, more than 4 kinds of catalyst all can be used as catalyst in the salvianolic acid B conversion reaction, and each catalyst amount and salvianolic acid B molar percentage be 0.5%～3.0%, the productive rate of salvianolic acid A 〉=40%.Conversion ratio surpasses 60%.
Experimental example 7: the purification with macroreticular resin of salvianolic acid A
Get salvianolic acid A and transform 13 parts of solution, put among each good model macroporous resin 100g of pretreatment, behind the shaking table dynamic adsorption 8 hours, the dress post, wash successively 3 column volumes of eluting, 5 column volumes of 20% ethanol elution, 5 column volumes of 70% ethanol elution with water, collect 70% ethanol and contain salvianolic acid A eluent part, carry out the salvianolic acid A assay, the eluent evaporate to dryness calculates dried cream amount, the results are shown in Table 20.
The affirmation of table 20. macroporous resin model
Figure BSA00000812340600342
Figure BSA00000812340600351
The 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 content greater than 75% salvianolic acid A eluent.
Experimental example 8: the column chromatography purification second time of salvianolic acid A
Get 3 parts of salvianolic acid A solution behind the purification with macroreticular resin, put among each good model resin 100g of pretreatment, behind the shaking table dynamic adsorption 8 hours, the dress post, wash successively 3 column volumes of eluting, 5 column volumes of 20% ethanol elution, 5 column volumes of 70% ethanol elution with water, collect 70% ethanol elution and carry out the salvianolic acid A assay, part eluent evaporate to dryness calculates dried cream amount, the results are shown in Table 21.
The affirmation of table 21. resin model
Figure BSA00000812340600352
The result shows: above 3 kinds of resins are good to the adsorption effect of salvianolic acid A, and can well remove impurity with the ethanol gradient elution of variable concentrations, obtain content and are about 90% salvianolic acid A eluent.
Experimental example 9: the abstraction purification of salvianolic acid A
With 70% ethanol elution decompression recycling ethanol behind the second time column chromatography purification, adjust pH 2.0～4.0, use again n-butyl alcohol, t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate, Ethyl formate, ethanol extraction 5 times, the Separation of Organic phase, reclaim solvent, drying gets salvianolic acid A, measure salvianolic acid A content, the results are shown in Table 22.
The table 22. extraction affirmation of organic solvent
Figure BSA00000812340600361
Table 22 is the result show: n-butyl alcohol is because polarity is large, the 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 that obtains 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 all is improved.
Experimental example 10: the purification by silica gel column chromatography of salvianolic acid A
Get 12 parts of salvianolic acid A extracts behind the abstraction purification, every part contains salvianolic acid A 10g, adds the silica gel of 20g, stirs, and volatilizes; Be added on the dried silicagel column of the 100g that has installed stirring sample silica gel, the two-phase solvent that forms take petroleum ether, pentane, normal heptane, ethyl acetate, methyl acetate, Ethyl formate, t-butyl methyl ether respectively is as eluant, HPLC or thin layer chromatography detect, collect the salvianolic acid A eluent, the eluent evaporate to dryness, get dry extract, measure salvianolic acid A content, the results are shown in Table 23.
The affirmation of table 23. eluant
Figure BSA00000812340600362
Figure BSA00000812340600371
The result shows: when phenolic acid A uses normal phase silica gel column chromatography, the two-phase solvent that adopts petroleum ether, pentane, normal heptane, ethyl acetate, methyl acetate, Ethyl formate, t-butyl methyl ether to form is eluant, gradient elution, can well remove impurity, obtain highly purified salvianolic acid A eluent.
Experimental example 11: salvianolic acid A drying means research
Get 4 parts of eluents behind the silica gel purification eluting, every part contains salvianolic acid A 100g, is concentrated into without the thick paste shape, adds to adopt respectively vacuum drying, lyophilisation, spray drying, microwave vacuum drying to get salvianolic acid A after 10 times of water gagings dissolve, this salvianolic acid A is detected, the results are shown in Table 24.
Table 24. salvianolic acid A drying means testing result
Figure BSA00000812340600372
The result shows: adopt the lyophilisation overlong time, and high cost, and organic solvent residual is serious; Vacuum drying, spray drying microwave vacuum drying process are controlled simple, and treating capacity is large, and baking temperature is low, and the time is short, and gained salvianolic acid A indices is good, so the present invention adopts vacuum drying, spray drying, microwave vacuum drying to prepare salvianolic acid A.
Through further experiment is definite, the microwave vacuum drying optimum range is chosen as temperature: 20-100 ℃, and 1-5 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute;
The vacuum drying optimum range is chosen as temperature: 50 ℃～90 ℃, and more than vacuum-0.07Mpa, power: 1～60KW, dry 2～20 hours;
The spray drying optimum range is chosen as intake air temperature: 150 ℃～350 ℃, and the air outlet temperature: 70 ℃～95 ℃, spray velocity: 1～300ml/min.
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 purpose: the dissolubility that increases composition in the injection, need add filler as supporter during the molding of powder pin, improve mouldability, regulate the powder pin and admittedly contain thing weight, regulate osmotic pressure, add simultaneously antioxidant and keep principal agent stable.
Get the salvianolic acid A raw material by table 25 and make freeze-dried powder from different filleies and vitamin C.
Table 25. prescription filler screening table
Figure BSA00000812340600381
Get salvianolic acid A by table 25 prescription, add the water for injection stirring of making total amount 90% and make dissolving, with 10% sodium hydroxide adjust pH 4.5, add adjuvant and make dissolving, mixing adds active carbon 2g stirring and adsorbing, filters, and removes active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear usefulness 0.22 μ m microporous filter membrane and filter, the filtrate fill is in aseptic cillin bottle, and part is butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot after fill is finished, is sent into freeze dryer, close chamber door, open freeze dryer, be cooled to-42 ℃ with 28 ℃/h speed, be incubated freezing 12 hours; Be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 11 hours rises to-25 ℃, keeps-25 ℃ of vacuum dryings 6.5 hours; Continue to heat up, evenly be warming up to 40 ℃ with 0.8 ℃/min, keep 40 ℃ of dryings after 5 hours, sample temperature is down to room temperature, add a cover, observe mouldability, see Table 26.
The different filleies of table 26 affect the result to mouldability
Figure BSA00000812340600391
From table 26 experimental result as can be known, add not commensurability filler, larger on the impact of mouldability, filler mannitol, glucose, lactose are better to the lyophilized formulations effect, good, the porous nickel of lyophilized powder sedimentation, therefore as selecting adjuvant, but be lower than the 20mg mouldability and solubility general.
Experimental example 13: different filleies and antioxidant are on the impact of lyophilized formulations type
According to above-mentioned experimental result, select respectively mannitol, glucose, lactose to carry out confirmatory study, and antioxidant compared research, select respectively vitamin C, thiourea, sodium sulfite, sodium pyrosulfite as antioxidant, experimental program sees Table 27, and compare with not adding antioxidant prescription, experimental result sees Table 28.
The different filleies of table 27 and antioxidant Preliminary screening
Figure BSA00000812340600401
Get salvianolic acid A by table 27 prescription, add the water for injection stirring of making total amount 90% and make dissolving, with 10% sodium hydroxide adjust pH 4.5, add filler and antioxidant and make dissolving, mixing adds active carbon 2g stirring and adsorbing, filters, and removes active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear usefulness 0.22 μ m microporous filter membrane and filter, the filtrate fill is in aseptic cillin bottle, and part is butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot after fill is finished, is sent into freeze dryer, close chamber door, open freeze dryer, be cooled to-42 ℃ with 25 ℃/h speed, be incubated freezing 14 hours; Be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 10～14 hours rises to-25 ℃, keeps-23 ℃ of vacuum dryings 7 hours; Continue to heat up, evenly be warming up to 44 ℃ with 0.6 ℃/min, keep 44 ℃ of dryings after 2 hours, sample temperature is down to room temperature, add a cover, observe mouldability, see Table 28.
The different filleies of table 28 and antioxidant are on the result that affects of mouldability and the quality of the pharmaceutical preparations
Figure BSA00000812340600402
Figure BSA00000812340600411
Table 28 experimental result shows, it is better to preparations shaping to add not commensurability filler mannitol, glucose, lactose, and the lyophilized powder sedimentation is good, porous nickel, and consumption selection 20mg～40mg/2ml～3ml mouldability and solubility are all better.Add water-soluble vitamin c, thiourea, sodium sulfite, sodium pyrosulfite on the content of main constituent and other compositions all without affecting, add salvianolic acid A stable content behind the antioxidant, other compositions are unchanged, the preparation effect is more excellent.
Experimental example 14: lyophilisation condition research
3.1 lyophilisation condition is preferred: mainly contain two factors in the freezing conditions: on the impact of mouldability: the temperature of freezing body before freezing speed, the vacuum drying; Impact on freeze drying rate: the heating curve during vacuum freeze-drying.
Following preferred on freeze dryer:
A, the medicinal liquid chilling rate with 20-30 ℃/hour in freeze dryer is cooled to-40 ℃～-45 ℃, be incubated freezing 10～16 hours, be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 10～14 hours rises to-23 ℃～-27 ℃, keeps-23 ℃～-27 ℃ vacuum dryings 6～8 hours;
B, medicinal liquid first chilling rate with 20-30 ℃/hour in freeze dryer is cooled to-25 ℃, be incubated freezing 5～8 hours, be refrigerated to again-40 ℃～-45 ℃, be incubated freezing 5～8 hours, be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 10～14 hours rises to-23 ℃～-27 ℃, keeps-23 ℃～-27 ℃ vacuum dryings 6～8 hours;
C, the medicinal liquid chilling rate with 20-30 ℃/hour in freeze dryer is cooled to-15 ℃, be incubated freezing 5～8 hours, be refrigerated to again-40 ℃～-45 ℃, be incubated freezing 5～8 hours, be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 10～14 hours rises to-23 ℃～-27 ℃, keeps-23 ℃～-27 ℃ vacuum dryings 6～8 hours;
More than three kinds of result of the tests: 1, medicinal liquid directly is refrigerated to-40 ℃～-45 ℃ with 20-30 ℃/hour, and powder body is more even, and without the crack, drying time is short.2, powder body is inhomogeneous, and the crack is arranged, and 3, freeze-drying time is long, powder body is inhomogeneous, and the crack is arranged.Therefore, directly be down to-40 ℃～-45 ℃ with 20-30 ℃/hour chilling rate when selecting medicinal liquid freezing, powder body is more even, without the crack, can save freeze-drying time.
3.2 pilot scale freeze-drying curve
Freeze-drying curve refers to the relation curve of product temperature in the freeze-drying process or shelf temperature time to time change.What, the many factors such as the kind of separation container and appointed condition of the shape of freeze-drying curve and the performance of product, loading amount are relevant.The freeze dryer that we select when middle trial production is 4000 bottles of scales, substantially meets large production requirement.Namely cooled the temperature to-40 ℃～-45 ℃ at 1～4 hour, under this temperature, kept 10～16 hours, at 10～14 hours temperature is risen to again-23 ℃～-27 ℃ and kept 6～8 hours, then at 6～9 hours temperature is risen to 40 ℃～45 ℃ and kept 2～5 hours, freeze-drying curve 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, with above three lot number samples at ambient temperature, by 0 month, 1 month, 2 months, 3 months, 6 months intervals, make regular check on the situation of change of sample property, solubility and clarity, pH value, salvianolic acid A content, other compositions, the results are shown in Table 29.
Table 29 stabilizing member experimental result
Figure BSA00000812340600421
Figure BSA00000812340600431
Table 29 stability experiment observed result shows that lyophilized injectable powder at ambient temperature salvianolic acid A is stable, and other compositions are also without significant change, and the preparation appearance character is good, and the preparation solubility is good, and pH value is stable, the solution clarification.
Below experiment is all got the sample that embodiment 14 obtains with the salvianolic acid A freeze-dried powder.
Experimental example 16: the impact of function of nervous system's symptom behind 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: U.S. Sigma company; SDP-1 type rat heart rate sphygomanometer: China-Japan Friendship Hospital's development; YAG laser device: Wuhan Chinese workers' laser engineering company limited; Japan Olympus BH-5 microscope; Image is processed: the JVCky-F3OB3-CCD coloured image is shot with video-corder the input instrument; Graphical analysis: German KONTRON IBAS2.0 Image analysis system.
Laboratory animal: 2～3 monthly ages, the male SD rat (Beijing Vital River Experimental Animals Technology Co., Ltd. provides) of body weight (100 ± 20) g.
2, experimental technique and result:
2.1 method
2.1.1 the preparation of animal model:
Get the male SD rat of 2～3 monthly ages, body weight (100 ± 20) g and set up the RHRSP model with two narrow renal artery of kidney double fastener method: rat abdominal cavity anesthesia, through the long otch of the about 5cm of xiphoid-process Ventral Midline stringer, cut skin, abdominal muscle, the blunt separation bilateral renal arteries is that the annular silver brain clip of 0.3mm is clamped respectively the bilateral renal arteries initial part with internal diameter.Measuring blood pressure is once surveyed once weekly continuous 12 weeks after the operation before operation.Other gets 10 SD rats that body weight is suitable, surveys its blood pressure as normal control.Reject: in the operation and postoperative death, the 12 weeks after operation blood pressure is lower than the 160mmHg rat, and softening kitchen range that apoplexy symptom and sign, dissection see that left basal ganglia region is little and the rat of subarachnoid hemorrhage are arranged.Blood pressure 〉=160mmHg and be successful model without the RHRSP of apoplexy symptom after 16 weeks.Cause middle cerebral artery occlusion (MCAO) model to cause middle cerebral artery (MCA) thrombosis with photochemical method the RHRSP that is successfully prepared: after 2% pentobarbital sodium is pressed 40mg/kg body weight intraperitoneal injection of anesthesia, through left temporo side approach, at zygomatic arch and 1mm place, front lower place, aquamous part of temporal bone junction, bore the bone window that a diameter is 5mm with dental burr, can know through cerebral dura mater and see left MCA trunk and branch thereof.Dosage injection 2.5% tiger of pressing the 40mg/kg body weight through femoral vein is red, green laser beam (the spot diameter 2mm of the 532nh that occurs with the YAG laser device behind the 5min, intensity of illumination 0.37W) prolonged exposure MCA trunk 15min, as seen this MCA far-end and each branch blood flow interrupt, and see that under operating microscope local white thrombus points out this MCA thrombosis, the success of MCAO model copy.Rat anus temperature remains on (37 ± 0.3) ℃ in the art.With the flushing of penicillin diluent, the conventional intramuscular injection penicillin of postoperative 80,000 U/ only before local wound was sewed up.Reject in the 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
Divide at random 6 groups with animal first: 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 is not done laser irradiation; Model group, the high, medium and low dosage group of salvianolic acid A freeze-dried powder prepare easy apoplectic type renovascular hypertension in rats MCAO model by the model preparation method.And be administered once by the 0.3ml/100g tail vein injection respectively in MCAO model postoperative 30min, after this be administered once continuous three days altogether every day.Sham operated rats, model control group postoperative tail vein injection saline; The high, medium and low dosage group of salvianolic acid A freeze-dried powder postoperative is tail vein injection salvianolic acid A freeze-dried powder 20mg/kg, 10mg/kg, 5mg/kg respectively, nimodipine group postoperative tail vein injection nimodipine 10mg/kg.
2.2 result
18 minutes point systems according to Garcia etc. are marked: the symmetry of spontaneous activity, quadruped locomotion, forelimb stretch the action of creeping symmetry, climb the cage wall, push away the trunk reaction, the reaction of antenna to stimulating.Nothing activity or reaction in the first three items (extremity or trouble limb) are 0 minute, and a little activity is 1 minute, and reaction calibration constant is 2 minutes, and activity normally is 3 minutes; Rear three reactionless be 1 minute, activity calibration constant is 2 minutes, activity normally is 3 minutes.Total points is the highest 18 minutes, minimum 3 minutes.Scoring is once respectively carried out a neuroethology scoring (at every turn all marking) at MCAO postoperative (after being ischemia) 24h, 48h, 72h to rat respectively afterwards after administration on the same day after MCAO postoperative rat anesthesia is revived.Simultaneously each experimental group rat is carried out blind attitude by 5 people that are unfamiliar with this experiment grouping and observe scoring, average at last.And observe simultaneously the rat body state, measure body weight and blood pressure.
Table 30. salvianolic acid A freeze-dried powder on the RHRSP cerebral thrombosis after the impact (x ± s) of neuroethology scoring
Figure BSA00000812340600451
Annotate: compare #P＜0.01, ※ P＜0.05 with sham operated rats; Compare * P＜0.01, ☆ P＜0.05 with model group
Experimental result shows: the neuroethology scoring of (in the 12h) significantly was lower than sham operated rats (P＜0.01) after model group was revived, RHRSP function of nervous system was impaired serious after the cerebral thrombosis ischemia was described, and in the 72h that continues, the neuroethology scoring continues to be starkly lower than sham operated rats (P＜0.05); 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 middle and high dosage group of salvianolic acid A freeze-dried powder the most obviously (P＜0.01), and behind its ischemia the neuroethology scoring of 24h substantially near the sham operated rats level; Each the dosage group rat neuroethology scoring of 72h salvianolic acid A freeze-dried powder has recovered normal to the ischemia, compares difference with model group and still has statistical significance (P＜0.05).Neuroethology was marked after above results suggest, salvianolic acid A freeze-dried powder can improve RHRSP cerebral thrombosis ischemia, the effect of nervous symptoms after prompting salvianolic acid A freeze-dried powder has protection and improves cerebral ischemia.
Experimental example 17: the salvianolic acid A freeze-dried powder is on the impact of RHRSP cerebral thrombosis tissues following MCAO in rats pathological change
The preparation of RHRSP Cerebrovascular embolism model, grouping, medication are with experimental example 16.After the neuroethology scoring finished the last time, the rat broken end was got brain, and observed MCA local thrombus situation under operating microscope.Put into afterwards smooth vessel be built in-20 ℃ freezing, the row crown section.The Mus brain section, every thick about 2mm.The brain sheet is put into rapidly 2%TTC solution, hatch under 37 ℃, the every face of brain sheet is hatched 15min, and normal cerebral tissue peony, and the ischemic infarction cerebral tissue is white in color.Taking-up brain sheet is observed and is taken pictures.Then cerebral tissue is placed 4% paraformaldehyde PBS buffer (pH7.3) fixing, afterwards row dehydration, paraffin embedding are got cerebral tissue and are done paraffin section and do conventional H E staining analysis, and microscopically is observed, taken pictures.The results are shown in Table 31.
Table 31 salvianolic acid A freeze-dried powder is on the impact of RHRSP cerebral thrombosis tissues following MCAO in rats pathological change
Figure BSA00000812340600461
Experimental result shows: sham operated rats cerebral tissue Bilateral Symmetry, have no pathological changes, and the model group Intravascular Thrombus adheres to seriously.With model group relatively, the contraction in length of the left MCA local thrombus of each dosage group rat of Microscopic observation salvianolic acid A freeze-dried powder reduces at the arterial wall bond area, with high, middle dosage group is the most obvious.The TTC visible infarcted cerebral constitution that dyes is white in color, and the cerebral tissue scope that is dyed to white in each dosage group of salvianolic acid A freeze-dried powder is dwindled than model control group is remarkable.HE dyes has in visible model group left cortical MCA blood supply district (centered by the cortex of the volume top) infarct obvious cerebral tissue softening, necrocytosis, the phenomenons such as the 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 each dosage group of salvianolic acid A freeze-dried powder and model control group shows in the kitchen range, kitchen range Zhou Jun has little blood vessel hyperplasia, but the little number of blood vessel of each dosage group of salvianolic acid A freeze-dried powder and model group comparison hypertrophy significantly increases; In addition, the kitchen range shape that as seen HE dyeing display model matched group differs in size is hemorrhage, the salvianolic acid A freeze-dried powder is high, middle dosage group there is no and finds have kitchen range shape bleeding, salvianolic acid A freeze-dried powder low dose group also only to have individual animal to have the kitchen range shape hemorrhage, and hemorrhagic focus is less than model group.Result of the test shows that the salvianolic acid A freeze-dried powder can obviously improve RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia histopathology state; reduce the thrombosis bond area, reduce infarct size; come off thereby increase the little blood vessel number, the necrosis of minimizing cerebral hemorrhage phenomenon protection cerebral tissue that reach surrouding brain tissue in the infarcted region, have the effect that protection cerebral thrombosis ischemia causes brain tissue impairment.
Experimental example 18: the salvianolic acid A freeze-dried powder on the RHRSP cerebral thrombosis after the impact of blood brain barrier (BBB) permeability
1 test method
Animal grouping and the preparation of RHRSP Cerebrovascular embolism model are with experimental example 16 methods.4.5h after the 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; Sham operated rats and model group tail vein injection are with the normal saline of volume.Each is organized rat and gets the cerebral tissue specimen in MCAO postoperative (after being the cerebral thrombosis ischemia) 6h, 12h, 24h execution respectively in batches, and before putting to death 1h tail vein injection 2% azovan blue (Evans Blue, EB) normal saline solution 2ml/kg body weight, after fully circulating, dark numb animal, cut open breast through heart perfusion normal saline flushing blood vessel inner dye, broken end is got brain.
Each group is randomly drawed the cerebral tissue of 3 rats, frozen section immediately, the continuously crown section of the thick about 30um of row, 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 the cerebral tissue, observe BBB open area and degree.Section is not put 4 ℃ of refrigerators and is kept in Dark Place when observing.Each group is got the cerebral tissue of 5 rats at random in addition, and it is heavy to blot behind the surface moisture weighing cutaneous horn; Cerebral tissue is placed homogenizer, add 50% trichloroacetic acid and make tissue homogenate, move into more than the test tube sealing and standing 60min; The centrifugal 10min of 3000rpm/min gets supernatant, and spectrofluorophotometer is surveyed fluorescent value: excitation wavelength 620nm, emission wavelength 680nm; Calculate EB content in the supernatant by standard curve, calculate again every Borneo camphor and organize EB content, react each rat BBB permeability with this.
2 experimental results
2.1 laser scanning co-focusing microscope observed result
EB is bright-coloured bright red fluorescence under the exciting light state.Observe each treated animal cerebral tissue under the laser microscope and present homogeneous, diffusivity EB dip-dye, the line sample profile that sharpness of border, meninges red color visible fluorescence are sketched the contours of without BBB brain district (such as pinus, area postrema and hypophysis cerebri).And in BBB brain district, other except sham operated rats are respectively organized the equal visible light spot of cerebral tissue, and mainly be distributed in thalamus, hypothalamus, cerebellum, Hippocampus etc. locate, spot size differs, fluorescence intensity weakens to periphery gradually from the center of hot spot, obscurity boundary.The hot spot number of each group is obviously different with distribution: the model group hot spot is maximum, and the hot spot of 120 left and right sides diameter 100～200um is arranged approximately, and intensive place in the form of sheets.Each salvianolic acid A freeze-dried powder injecta medicine group number of spots significantly reduces than model group, and each dosage group relatively presents the remarkable dose-difference opposite sex; Wherein more with the low dose group hot spot, hot spot has 50 approximately on same level, but is fused into the less of lamellar; Middle dosage group hot spot is less, is dispersed in distribution; The high dose group hot spot seldom is dispersed in, and fluorescence is very weak.
2.2 the permeability result of the EB of rat cerebral tissue content detection blood brain barrier
Each is organized rat cerebral tissue's specimen and calculates EB content by the fluorescent value that records, and data statistic analysis sees Table 32.
Table 32. salvianolic acid A freeze-dried powder on the RHRSP cerebral thrombosis after the impact of blood-brain barrier permeability
Figure BSA00000812340600481
Annotate: compare #P＜0.01 with sham operated rats; Compare * P＜0.01 with model group
2.3 conclusion
EB is water-soluble dye, enter behind the blood can be absolutely rapidly and albumin bound, can not see through blood brain barrier under normal circumstances.By 2.1 and 2.2 results as seen, sham operated rats BBB brain district, the cerebral tissue microscopically has no EB and oozes out hot spot; And model group rat cerebral tissue microscopically has a large amount of EB to ooze out hot spot, and the cerebral tissue spectrofluorophotometer detects the EB of high-load, there were significant differences (P＜0.01) than sham operated rats, show cerebral thrombosis tissues following MCAO in rats blood-brain barrier disruption, permeability increases, and the EB albumin-binding can enter cerebral tissue by open BBB.Experimental result shows that salvianolic acid A each the microscopically EB of dosage group rat cerebral tissue hot spot number of freeze-dried powder 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 salvianolic acid A freeze-dried powder is on the impact of RHRSP cerebral thrombosis cerebral infarction scope and brain water content
Method by experimental example 16 prepares RHRSP cerebral thrombosis rat model, grouping, administration; After last administration finished, the rat broken end was got brain.Each group is got the brain tissue slice of 5 rats at random, through TTC dyeing, after microscopically is taken pictures, infarct is delimited centered by infarct, processes and calculate the infarct size of every brain sheet with image analysis software.Every all brain sheet infarct size additions of rat are brain infarction area, multiply by the about 2mm of thickness of every brain sheet, are cerebral infarction volume; Get simultaneously all the in kind approximate calculation of brain sheet of corresponding rat and go out corresponding brain cumulative volume; The two ratio calculation cerebral infarction volume percentage ratio.Each group is got in addition 5 rat cerebral tissues and is claimed immediately weight in wet base, puts afterwards in 105 ℃ of baking ovens and dries to constant weight, calculates brain water content.
Table 33. salvianolic acid A freeze-dried powder is on the impact of RHRSP cerebral thrombosis cerebral infarction scope and brain water content (x ± s)
Figure BSA00000812340600491
Annotate: compare #P＜0.01 with sham operated rats; Compare * P＜0.01 with model group
Adopt the SPSS11.5 statistical package that data are carried out statistical procedures.Experimental result shows: sham operated rats has no infarcted cerebral constitution; Each dosage group cerebral tissue Infarction volume of salvianolic acid A freeze-dried powder and brain water content are significantly less than model control group (P＜0.01), and dosage is larger, Infarction volume is less, and brain water content is fewer, and difference has statistical significance (P＜0.01 or p＜0.05).Salvianolic acid A freeze-dried powder basic, normal, high dosage group Infarction volume and brain water content all are lower than the nimodipine group.Illustrate that the salvianolic acid A freeze-dried powder can accelerate the reparation of focus, reduce ischemic tissue of brain infarction size behind the RHRSP cerebral thrombosis, alleviate the cerebral tissue edema, have and repair and the effect of protection Neurons Against Cerebral Ischemia tissue injury.
Experimental example 20: the salvianolic acid A freeze-dried powder on RHRSP cerebral thrombosis ischemia after the microvascular impact of rat cerebral tissue
Animal grouping and the preparation of RHRSP Cerebrovascular embolism model are with experimental example 16 methods.After this 4.5h tail intravenously administrable after the model surgery success is administered once, until it is complete to draw materials every day.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; Model group and sham operated rats injection are with the normal saline of volume.Each group is got rat broken end respectively at MCAO postoperative 6h, 1d, 3d, 7d in batches and is got brain, conventional fixing, the dehydration of the neutral PBS buffer of cerebral tissue 4% paraformaldehyde of ischemic focus half penumbra area or same area, paraffin embedding, section, every thick about 5um.SABC method CD31 immunohistochemical staining is by SABC test kit description operation; The DAB colour developing.Replace primary antibodie to make negative control with PBS.Whether do not count blood vessel with the appearance of erythrocyte or tube chamber.All single endotheliocytes of dying brown color or endotheliocyte be bunch all as a vascular counts, and all tube chambers are not greater than 8 erythrocyte sizes, all count with the angiosomes blood vessel of thicker flesh layer.Vascular counts is undertaken by the Weidner method.Under low power (* 40) visual field, find first high vessel density zone, then under high power (* 400) visual field, carry out Microvessel Count, 10 high power fields are chosen in every section at random, get at last its meansigma methods as the microvessel density value (MVD) of this specimen.And adopt the full automatic colour image processing system to carry out graphical analysis, measure blood vessel field Area Ratio (the Positive Objects area/Statistical Fields gross area).
Table 34. is respectively organized long-pending, the immune positive microvessel density of CD31 (MVD) value (n=6) of rat cerebral tissue's blood vessel scene
Figure BSA00000812340600501
Figure BSA00000812340600511
Annotate: compare #P＜0.01 with sham operated rats; Compare * P＜0.01, ☆ P＜0.05 with model group
Table 34 experimental data shows, with sham operated rats relatively, model group ischemia penumbra MVD and blood vessel scene amass that 1d increases to some extent behind ischemia, but 3d, 7d continue significantly to reduce (P＜0.01) behind the ischemia; Compare with model group, after the ischemia treatment, 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 behind ischemia is long-pending significantly to increase (P＜0.05 or P＜0.01) to some extent, and more stable in the 7d.Relatively, difference has statistical significance (P＜0.05) between three dosage groups of salvianolic acid A freeze-dried powder.The result shows that the 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: the salvianolic acid A freeze-dried powder on RHRSP cerebral thrombosis ischemia after the impact of rat cerebral tissue vascular endothelial growth factor expression
Modeling and grouping administration are with experimental example 19.Respectively at rat MCAO postoperative 2h, 24h, 48h, each component is criticized and is got rat abdominal cavity anesthesia, opens breast and exposes heart, and 4% paraformaldehyde that contains 0.1%DEPC through the heart perfusion is liquid-solid fixed, gets rapidly brain, does the thick crown section of about 2mm in forebrain optic chiasma place.Place 4% paraformaldehyde fixative to spend the night, conventional dehydration, paraffin embedding is cut into the thick paraffin section of about 5um continuously, and routine dewaxes to water, detects the expression that VEGF impels ribonucleic acid (VEGFmRNA) with hybridization in situ.Illustrate by VEGF in situ hybridization test kit (Wuhan Boster Biological Technology Co., Ltd.) to operate, carry out optical density (IOD) analysis with microscopic image analysis software.
The impact that table 35. salvianolic acid A freeze-dried powder is expressed RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia tissue VEGF
Figure BSA00000812340600512
Figure BSA00000812340600521
Annotate: compare #P＜0.05 with sham operated rats; Compare #P＜0.01, ☆ P＜0.05 with model group
VEGF (VEGF) is again the blood vessel opsonin, has the effect of short endothelial cell division, can promote the growth of blood vessel and the foundation of side Zhi Xunhuan.Experimental result shows; model group VEGFmRNA expression is increased significantly (P＜0.05) than sham operated rats; illustrate behind the cerebral tissue hypoxic-ischemic can Stimulation of The Brain tissue in the expression increase of VEGF; a kind of protective response that resists ischemia injury can appear in body self behind the prompting cerebral infarction; the expression of VEGF is increased, self " compensatory revascularization " behind ischemia, occur.Each dosage group of salvianolic acid A freeze-dried powder and model group group are relatively; VEGFmRNA expression significantly raise (P＜0.05 or P＜0.01); prompting salvianolic acid A freeze-dried powder can significantly promote the ischemic tissue of brain angiogenesis; promote the foundation of collateral circulation compensation; save ischemia half blanking bar; the brain tissue impairment that the protection ischemia causes, and have certain dose-effect relationship.
Experimental example 22: the 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 20,2h, 24h, 48h after finishing respectively at the rat administration, each component criticizes that to get 4% paraformaldehyde that the rat heart perfusion contains 0.1%DEPC liquid-solid fixed, gets rapidly brain, the crown section of row, paraffin embedding, section.The ABC immunohistochemic al technique method detects cerebral tissue alkalescence fibroblast growth factor (bFGF) protein expression.Operate by bFGF protein immunization group detection kit (Wuhan Boster Biological Technology Co., Ltd.) description, microscopically is observed, and counting is dyed the positive cell number of brown color, and all visuals field of 5 infarcts are selected in each section at random, average.Data are carried out statistical analysis.
Table 36. salvianolic acid A freeze-dried powder on RHRSP cerebral thrombosis ischemia after the bFGF of rat cerebral tissue protein expression impact (x ± s)
Figure BSA00000812340600522
Figure BSA00000812340600531
Annotate: compare #P＜0.05 with sham operated rats; Compare * P＜0.01 with model group
BFGF is the strong neurotrophic factor with various biological activity, can neuroprotective unit to multiple detrimental effects such as ischemia resisting, anoxia, toxicity of excitatory amino acid, calcium overload, free radical and nitric oxide (NO) are synthetic, slow down neuronal apoptosis and necrosis; And can work in coordination with the effect that VEGF promotes angiogenesis in the infarcted region.
Experimental result shows; after ischemia occurs; the bFGF of model group rat cerebral tissue protein expression raises to some extent than sham operated rats; to ischemia 24h significant difference (P＜0.05) is arranged; but the prolongation with Ischemia Time has a declining tendency, behind the prompting cerebral tissue hypoxic-ischemic; body self produces of short duration protection stress, but Stimulation of The Brain organizes the endogenous bFGF protein expression to increase.Nimodipine group, each dosage group of salvianolic acid A freeze-dried powder and model group compare, and the bFGF protein expression of each time period all significantly increases (P＜0.01); Relatively, difference has statistical significance (P＜0.05) between three dosage groups of salvianolic acid A freeze-dried powder; Each time period of each dosage group self shows that relatively the 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 protein expression, has the effect of good neurocyte protection effect and promotion angiogenesis, helps the foundation of collateral circulation, saves ischemia half blanking bar.
Experimental example 23: the salvianolic acid A freeze-dried powder is on the impact of 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 the standby rat brain focal cerebral ischemia-reperfusion injury model of improvement line bolt legal system: male SD rat, pentobarbital sodium intraperitoneal anesthesia.Expose and separation left common carotid, external carotid artery, internal carotid artery and arteria pterygopalatina, ligation external carotid artery distal end, arteriole folder folder closes common carotid artery, the tie wings arteria palatina, cut a kerf in the nearly common carotid artery crotch of external carotid artery, the head end fishing line that to scribble smooth paraffin diameter be 0.3mm is slowly entered the cranium direction to internal carotid artery through left side external carotid artery trunk otch to be advanced, take the common carotid artery crotch as labelling, when advancing 18～20mm to feel slight resistance, namely block middle cerebral artery and cause cerebral ischemia.Fishing line stays about 1cm in order to pouring into usefulness, sterilization, skin suture outward again.Behind continuous ischemia 2h, extract the bolt line, realize ischemia-reperfusion injury model.Sham operated rats except plug wire not, the same model group of all the other operating process.Each group before ischemia 30min and again when beginning perfusion each be administered once through tail vein injection, 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 with volume.
3h, 24h, 48h, 72h respectively carry out neurological deficits score one time after cerebral ischemic reperfusion in rats respectively, and the Bederson improved method is adopted in scoring: carry the about chi of Mus tail built on stilts, observe forelimb flexing situation, stretch to ground such as two forelimb symmetries and count 0 minute; As the flexing that wrist flexing meter 1 minute, elbow flexing meter 2 minutes, shoulder inward turning meter 3 minutes, existing wrist elbow appear in the offside forelimb of performing the operation has again shoulder inward turning meter 4 minutes.Rat is placed on the level land, push away respectively both shoulders to side shifting, check resistance, symmetrical and strong such as the bilateral resistance, be designated as 0 minute; Resistance descender when promoting to the operation offside, according to decline degree difference be divided into gently, in, severe, count respectively 1,2,3 minute.Rat two forelimbs are placed on the wire netting, observe the muscular tension of two forelimbs, bilateral muscular strength equity and strong person count 0 minute, operation offside muscular tension decline degree is divided into gently, in, severe, count respectively 1,2,3 minute.Rat does not stop to count 1 minute to a side person of turn-taking.According to above standards of grading, full marks are 11 minutes, and mark is higher, illustrate that the behavior disorder degree of rat is more serious, neurologic impairment is more serious, and data see Table 37.
Table 37. salvianolic acid A freeze-dried powder is on the impact (x ± s, n=12) of rats after cerebral ischemic reperfusion neurological deficits score
Figure BSA00000812340600541
Annotate: compare * * P＜0.01, * P＜0.05 with model group
Result of the test as can be known, ischemia-reperfusion injury model group function of nervous system serious defect, last till again fill with after 72h, although more again after the perfusion behavioristics's obstacle of 3h, 24h significantly alleviate (P＜0.05), neurologic impairment is serious (neurological deficits score is still greater than 5) still.Each dosage group of nimodipine group and salvianolic acid A freeze-dried powder is compared with model group, from pouring into 3h, neurologic impairment sx↓, neural row are learned damaged mark and are significantly reduced (P＜0.05 or P＜0.01) again, to pouring into 72h again, the neurobehavioral of each medicine group rat recovers substantially.Between 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); Scoring relatively has the trend of reduction between high dose group and the middle dosage group, but difference does not have statistical significance (P＞0.05).
Experimental result shows that the 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 behind the brain tissue impairment.
Experimental example 24: the salvianolic acid A freeze-dried powder is on the impact of rats after cerebral ischemic reperfusion cerebral infarction scope, cerebral index and brain water content
Experimental example 23 is respectively organized rat the last time after the neuroethology scoring, get 6 broken ends for every group and get brain, place immediately-20 ℃ of refrigerator freezing 10min, after removing olfactory bulb, cerebellum and low brain stem, the cerebral tissue continuous coronal section of row 2mm thickness is immersed the brain sheet in the 2%TTC solution, 37 ℃ of dyeing 10min, take out, place fixedly 2h of 4% paraformaldehyde PBS buffer.Normal structure is dyed redness, and infarct is white in color.Fixing brain sheet is arranged sequentially by section, and microscopically is taken pictures and is inputted computer behind the two sides before and after the brain sheet, calculates the approximation of front brain volume and Infarction volume according to each slice thickness, draws the percentage ratio that Infarction volume accounts for front brain volume.After each is organized other 6 rats broken end and gets brain, claim immediately cutaneous horn heavy, dry to constant weight in 105 ℃ of baking ovens, calculate brain water content and cerebral index.
Table 38. salvianolic acid A freeze-dried powder is on the impact (x ± s, n=6) of rats after cerebral ischemic reperfusion cerebral infarction volume ratio, cerebral index, brain water content
Figure BSA00000812340600551
Annotate: compare * p＜0.05, * * p＜0.01 with model group
Experimental data shows that each dosage group of salvianolic acid A freeze-dried powder is compared with the ischemia-reperfusion injury model group: cerebral infarction volume is than significantly reducing (P＜0.01), and cerebral index and brain water content all obviously reduce (P＜0.01 or P＜0.05); Salvianolic acid A 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 nimodipine group compare, and its Infarction volume and cerebral index and brain water content all significantly reduce (P＜0.05 or P＜0.01); Show that the salvianolic acid A freeze-dried powder can reduce rats after cerebral ischemic reperfusion cerebral tissue infarction size, can alleviate the cerebral tissue edema degree behind the cerebral ischemia reperfusion injury.
Experimental example 25: the salvianolic acid A freeze-dried powder is on the impact of rats with cerebral ischemia cerebral tissue ischemia half blanking bar regional cerebral blood flow (rCBF)
1 test method
Adopt the standby rat brain focal ischemia of improvement line bolt legal system model: male SD rat, pentobarbital sodium intraperitoneal anesthesia.Expose and separation left common carotid, external carotid artery, internal carotid artery and arteria pterygopalatina, ligation external carotid artery distal end, arteriole folder folder closes common carotid artery, the tie wings arteria palatina, cut a kerf in the nearly common carotid artery crotch of external carotid artery, the head end fishing line that to scribble smooth paraffin diameter be 0.3mm is slowly entered the cranium direction to internal carotid artery through left side external carotid artery trunk otch to be advanced, take the common carotid artery crotch as labelling, when advancing 18～20mm to feel slight resistance, namely block middle cerebral artery and cause cerebral ischemia.Fishing line stays about 1cm in order to pouring into usefulness, sterilization, skin suture outward again.Sham operated rats except plug wire not, the same model group of all the other operating process.
Model after the 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 the tail vein give relative medicine, sham operated rats and model group give the normal saline with volume.Adopt Laser Doppler Flowmetry to measure rCBF through cranium.Using cyanoacrylate that diameter is close to skull surface as the laser-Doppler probe of 0.5mm fixes, selecting respectively behind the bregma the other 2mm that opens in 1mm center line right side under the stereotaxic instrument is that other to open 6mm be rCBF monitoring point, ischemia center on 2mm center line right side behind ischemia half blanking bar rCBF monitoring point, the bregma, the record blood flow continues to monitor 2h behind the ischemia.Record before the administration and the blood flow after the administration.
2 result of the tests
Behind the middle cerebral artery occlusion in rat, ischemia center cerebral blood flow drop to normal rCBF before the modeling 10%～20% between, ischemia half blanking bar rCBF drop to rCBF before the modeling 30%～40% between.10min after the administration, each ischemia penumbra rCBF of dosage group rat cerebral tissue of nimodipine and salvianolic acid than administration before all existing risings in various degree, 30min after the administration, each medicine group cerebral tissue ischemia penumbra rCBF peaks.Concrete experimental data statistical analysis sees Table 39.
Table 39. salvianolic acid A freeze-dried powder is on the impact (x ± s, n=10) of rats with cerebral ischemia continuous ischemia phase cerebral tissue ischemia half blanking bar rCBF
Figure BSA00000812340600571
Annotate: compare * P＜0.01 with sham operated rats (baseline value); With comparison before the administration, ★ P＜0.01, ☆ p＜0.05
3 interpretations of result
By table 39 data as can be known, after the operation modeling, model control group and each medicine group cerebral tissue half blanking bar rCBF compare with sham operated rats, and utmost point significant difference (p＜0.01) expression modeling success is arranged.10min begins after the administration, each medicine group than self administration before ischemia half blanking bar rCBF obvious rising is arranged, and with nimodipine and salvianolic acid A freeze-dried powder high dose group particularly significantly (p＜0.01); 30min after the administration, each medicine group ischemia half blanking bar rCBF rises to the highest, and respectively organizes self rCBF baseline value before the administration relatively, has raise 15%～31%; Though after this have a declining tendency, but last till ischemia 2h, each medicine group is compared before than model group and self administration, still has remarkable significant difference (p＜0.01 or p＜0.05); Each dosage group of salvianolic acid A freeze-dried powder is compared, and good dose-effect relationship is arranged; The rCBF of dosage group day part is suitable with the nimodipine group in the salvianolic acid A freeze-dried powder.Hence one can see that, and the 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 salvianolic acid A freeze-dried powder is on the impact of rats after cerebral ischemic reperfusion cerebral tissue ischemia half blanking bar rCBF
Respectively organize rat in the experimental example 25 behind continuous ischemia 2h observation ischemia half blanking bar rCBF, extract the bolt line, realize ischemia-reperfusion injury model.After model was set up, each organized the tail intravenously administrable, and dosage is with experimental example 20.Still press experimental example 20 described methods and measure the rear 3h cerebral tissue ischemia half blanking bar rCBF of again filling.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 see Table 40.
Table 40. salvianolic acid A freeze-dried powder is on the impact (x ± s, n=10) of rats after cerebral ischemic reperfusion cerebral tissue ischemia half blanking bar rCBF
Figure BSA00000812340600581
Annotate: compare * P＜0.01 with sham operated rats; With filling is relatively front again, ☆ p＜0.01; Compare ★ P＜0.01 with model group
Experimental result shows, after rat continuous ischemia 2h recovered to fill with again, each organizes rat ischemia half blanking bar rCBF all in various degree increase.The model group rat rises to top level at 60,90 minutes ischemia half blanking bar rCBF, with filling is front than obvious statistical significance (P＜0.01) is arranged again, but also only be about 50% of the front baseline value of ischemia, after this sharply descend again, fill with again in the 3h, the rCBF value before ischemia baseline value 37%～50% between, in the meantime again the perfusion remain incomplete, still be low perfusion phenomena, cerebral tissue can continue impaired.After filling with, nimodipine, the high, medium and low dosage group of salvianolic acid A freeze-dried powder rat rCBF obviously increase again, and each time period rCBF has compared utmost point significant difference (P＜0.01) with model group.1h after filling with again, nimodipine and salvianolic acid A freeze-dried powder high dose group rCBF return near former rCBF 75%, in the 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 the 3h, each medicine group rCBF maintains in the metastable scope again.The experimental result prompting, the salvianolic acid A freeze-dried powder can obviously improve the cerebral blood flow of ischemia half blanking bar cerebral tissue after the Ischemia and Reperfusion in vivo in Rats, recovers the confession of brain cell blood, thereby saves ischemia half blanking bar, prevent further damage even dead of cerebral tissue, ischemic cerebrovascular is had good therapeutical effect.
Experimental example 27: the salvianolic acid A freeze-dried powder is on the impact of rats after cerebral ischemic reperfusion Energy Metabolism of Brain Tissue
Experimental example 26 each treated animal after the cerebral blood flow of having measured again 3h after the perfusion, sacrificed by decapitation, it is freezing to the liquid nitrogen to get brain; Get the cerebral tissue of 100mg corresponding site after one week, pulverize under the freezing state, remove albumen with perchloric acid homogenate, low-temperature centrifugation 10min, supernatant neutralizes with KOH, vortex oscillation, again 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.
Table 41. salvianolic acid A freeze-dried powder is on the impact (x ± s, μ mol/g, n=10) of rats after cerebral ischemic reperfusion Energy Metabolism of Brain Tissue
Figure BSA00000812340600591
Annotate: compare * P＜0.01 with sham operated rats; Compare * * P＜0.01, ☆ P＜0.05 with model group
ATP, ADP, AMP, LA, PC are the human body energy metabolite.Can be found out by table 41 experimental data, model group ATP, ADP, AMP, PC than sham operated rats significantly reduce, LA significantly raise (P＜0.01), rat cerebral ischemia tissues following MCAO in rats energy metabolism serious hindrance is described, ATP exhausts fast, brain-capacity is supplied with significantly and is reduced, anaerobic metabolism product LA piles up serious, even the energy metabolism disorder still exists after implementing to fill with again.And 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, the 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, the brain cell that ischemia half blanking bar after the redemption cerebral ischemia is at death's door, prevent further damage even dead of cerebral tissue, can perform well in treating ischemic cerebrovascular.
Experimental example 28 salvianolic acid A freeze-dried powders are on the impact of rats after cerebral ischemic reperfusion cerebral tissue neuronal apoptosis rate
Prepare rat pattern of ischemia reperfusion, grouping, administration with experimental example 23 methods.After rat cerebral ischemia 2h filled with 24h again, broken end was got brain, fix with 4% paraformaldehyde, and section, paraffin embedding prepares the thick crown section of thick 3 μ m; Adopt eventually transferase mediated dUTP breach end-labelling (TUNEL method) the detection cerebral tissue neurocyte of last deoxyribonucleic, strictly press the operation of test kit description, the DAB colour developing, apoptotic nucleus is brown under the light microscopic.Unduplicated 5 high powers (40 * 10) visual field is chosen in every section at random, and the input computer carries out image analysis, detects apoptotic cell number and the normal cell number of the TUNEL positive, calculates the neuronal apoptosis rate, results averaged.
Table 42 salvianolic acid A freeze-dried powder is on the impact of rats after cerebral ischemic reperfusion cerebral tissue neuronal apoptosis rate
Figure BSA00000812340600601
Compare #P＜0.001 with sham operated rats; Compare * * P＜0.001, * P＜0.01 with model group
Neuronal apoptosis is the principal mode of cerebral ischemia and transient ischemia/reperfusion damage delayed neuronal death, can determine the brain tissue impairment degree that ischemic cerebrovascular is final.Experimental result shows that after rat cerebral ischemia 2h filled with 24h again, neuronal apoptosis was serious; And after nimodipine and the intervention of various dose salvianolic acid A freeze-dried powder, compare with model group, neuronal apoptosis significantly reduces (P＜0.001 or P＜0.01) in the rat cerebral tissue; Show that the salvianolic acid A freeze-dried powder has the inhibition neuronal apoptosis, suppresses the effect that cerebral ischemia causes rat cerebral tissue's neuronal death.
Experimental example 29 salvianolic acid A freeze-dried powders are on the impact of rats after cerebral ischemic reperfusion cerebral tissue neurotrophic factor
Prepare rats after cerebral ischemic reperfusion model, grouping and administration with experimental example 28 methods.Ischemia Reperfusion 24h is by aortic cannulation, normal saline flushing, broken end is got brain after the perfusion of 4% paraformaldehyde, cerebral tissue is fixed, dehydration, paraffin embedding, section, thick about 5 μ m, Immunohistochemical Method detect neurotrophic factor nerve growth factor (NGF), Brain Derived Neurotrophic Factor (BDNF), neurotrophic factor-3 (NT-3) protein expression in the 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.Observe under the light microscopic, Cai Tu also uses the image analysis system analysis, measures its average gray value.Average gray is higher, and the intensity of expression positive cell is more weak.
Table 43 salvianolic acid A freeze-dried powder is on the impact of rat cerebral tissue's neurotrophic factor protein expression (gray value)
Figure BSA00000812340600611
Compare with sham operated rats, #P＜0.05,
Figure BSA00000812340600612
Compare * P＜0.05, △ P＜0.01 with model control group.
The neurotrophic factor histone matter molecule essential with survival that be neure growth plays supporting function to the integrity of neure growth, growth and function.NGF, BDNF, NT-3 are the parts of neurotrophic factor family, can keep neuronal survival and the differentiation of promotion neurocyte and induce axon growth; Energy neuroprotective unit, promotion neuron reparation and inhibition delayed neuronal death, thereby to anti-cerebral ischemia, protection cerebral ischemia.Experimental result shows, behind the Ischemia Reperfusion, increases to some extent than sham operated rats in the rat cerebral tissue, and behind the prompting cerebral reperfusion injury, NGF, BDNF express in the cerebral tissue stress protectiveness increase; But NT-3 content obviously reduces in the Neurons Against Cerebral Ischemia tissue.Compare with model control group; salvianolic acid A freeze-dried powder each dosage group 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 salvianolic acid A freeze-dried powders are on the impact of rats after cerebral ischemic reperfusion inflammatory cytokine
Prepare rats after cerebral ischemic reperfusion model, grouping and administration with experimental example 28 methods.Break end rapidly behind the Ischemia Reperfusion 24h and get brain, get ischemia side cerebral tissue and make 10% tissue homogenate, the centrifugal 10min of low temperature 2000r/min, get supernatant, use the content of each inflammatory cytokine of ELISA kit measurement: interleukin-1 ' beta ' (IL-1 β), interleukin-6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor (TNF-α), intercellular adhesion molecule-1 (ICAM-1).
Table 44 salvianolic acid A freeze-dried powder is on the impact of rats after cerebral ischemic reperfusion inflammatory cytokine
Compare #P＜0.001 with sham operated rats; Compare Δ P＜0.01, * P＜0.05 with model group.
Experimental result shows that behind the ischemia-reperfusion, inflammatory cytokine IL-1 β, IL-6, IL-8, TNF-α, ICAM-1 content all extremely significantly increase (P＜0.001) in the cerebral tissue; Each the inflammatory cytokine content of rat cerebral tissue that gives each dosage group of salvianolic acid A freeze-dried powder obviously reduces than 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: the salvianolic acid A freeze-dried powder is to rats after cerebral ischemic reperfusion cerebral tissue Ca 2+, K +, Mg 2+The impact of content
Get experimental example 24 and dry cerebral tissue to constant weight, change conical flask over to after weighing, in (200 ± 10) ℃ digestion, add the deionized water dissolving residue with analytical pure nitric acid and perchloric acid, change color comparison tube and standardize solution over to after, survey Ca in the cerebral tissue with atomic spectrophotometer 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 BSA00000812340600631
Annotate: compare * P＜0.01 with sham operated rats; Compare * P＜0.05, * * P＜0.01 with model group
Ca in the cell 2+Overload is one of encephaloclastic important mechanism of mediation Secondary cases in the During Ischemia, is the last common pathway that ischemia, anoxia produce irreversible neuronal damage.Ca 2+Overload can be by exciting activator protein enzyme as the second message,second messenger, activate phospholipase, activating a series of enzyme reactions such as endonuclease cell is produced infringement, and cell death inducing causes acute cell death and Delayed onset neuronal necrosis.K +, Mg 2+Be Ca 2+Antagonist.K +As the requisite cation of neurocyte polarized state, can suppress calcium ion in flow to and alleviate calcium overload; Mg 2+Can activate plurality of enzymes system in the body, in cerebral tissue, participate in the multiple important metabolic activity of cell, conduction, ion transport, the synthetic all many-sides of energy metabolism that reach of albumen affect the nerves, the spasm of energy alleviating vascular smooth muscle, improve microcirculation, improve hypoxic-ischemic behind the brain injury, calcium overload in the retardance after craniocerebral injury neurocyte.By experimental data as can be known, cerebral ischemia re-pouring model group and sham operated rats compare cerebral tissue Ca 2+Content extremely significantly increases, K +, Mg 2+Content significantly reduces; Compare with model group, each dosage group of nimodipine group and salvianolic acid A freeze-dried powder can significantly reduce Ca in the cerebral tissue 2+Content, rising K +, Mg 2+Content illustrates that the salvianolic acid A freeze-dried powder can suppress Ca 2+Interior stream alleviates calcium overload, thereby can suppress Ca 2+The neuronal cell apoptosis that overload is induced.
Experimental example 32: the salvianolic acid A freeze-dried powder is on the impact of rats after cerebral ischemic reperfusion cerebral tissue neurotransmitter
1 experimental technique
1.1 grouping administration and model preparation
Male SD rat (250 ± 20) g, be divided at random 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) group, nimodipine matched group (10mg/kg).Adopt re-perfusion model (as described in experimental example 23) behind the standby middle cerebral artery occlusion in rat 2h of line bolt legal system, sham operated rats except plug wire not, the same model group of all the other operating process.Each group before ischemia 30min and again when beginning perfusion each be administered once through tail vein injection, sham operated rats and cerebral ischemia re-pouring model group give the normal saline with volume.After filling with 2h again, each group is got 4 broken ends and is got brain and do histopathology section, and HE dyeing is observed histopathology and changed.
1.2 the mensuration of rat cerebral tissue's monoamine neurotransmitters
Each organizes rat after ischemia 2h pours into 2h again, respectively get 8, broken end is got brain fast, separate cerebral cortex and striatum, weigh, add n-butyl alcohol homogenate, the centrifuging and taking supernatant is used fluorescent spectrophotometer assay 5-hydroxy tryptamine (5-HT), norepinephrine (NE), dopamine (DA) content after carrying.Data are carried out statistical analysis, the results are shown in Table 46.
1.3 the mensuration of rat cerebral tissue's content of amino acids neurotransmitter
Each organizes rat after ischemia 2h pours into 2h again; respectively get 8; broken end is got brain fast; broken end is got brain; sharp separation ischemia side cerebral cortex in ice bath; weigh; put in the homogenizer; make homogenate with sulfosalicylic acid; centrifugal, get supernatant, dilution; behind the OPA derivative reaction, detect the content of amino acid neurotransmitter glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), γ-aminobutyric acid (GABA), taurine (Tau) in brain tissue homogenate's supernatant with high performance liquid chromatography.Mobile phase is a certain proportion of potassium phosphate buffer and methanol, tetrahydrofuran solution, and pH is about 6.6, flow velocity 1ml/min.Calculate excitatory toxicity index (EI): EI=[Glu] [Gly]/[GABA].Data are carried out statistical analysis, the results are shown in Table 47.
2 experimental results
2.1 histopathology observed result
Sham operated rats structure of neurons, form normally, matter edema continuously; Ischemia-reperfusion group neuronal cell volume dwindles, distortion, karyopycnosis, and nervous tissue is loose, and neuron and perivascular space increase, and a matter cerebral edema is obvious; Each dosage group of salvianolic acid A freeze-dried powder is compared with model group, and cerebral edema obviously alleviates, and the neuronal cell form is obviously improved, and neuron peripheral clearance is obviously dwindled; Especially high with the salvianolic acid A freeze-dried powder, middle dosage group is the most remarkable, its neuronal cell is more complete, the form normal.
2.2 the salvianolic acid A freeze-dried powder is on the impact of monoamine neurotransmitter in the rats after cerebral ischemic reperfusion cerebral tissue
Compare 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).Compare 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) with the cerebral ischemia re-pouring model group.
Table 46. salvianolic acid A freeze-dried powder is on the impact (x ± s, ng/mg, n=8) of monoamine neurotransmitter in the rats after cerebral ischemic reperfusion cerebral tissue
Figure BSA00000812340600641
Annotate: compare * P＜0.01 with sham operated rats; Compare * P＜0.05, * * P＜0.01 with model group
2.3 the salvianolic acid A freeze-dried powder is on the impact of amino acid neurotransmitter in the rats after cerebral ischemic reperfusion cerebral tissue
Compare with sham operated rats, the content of Glu, Asp, Gly, GABA, Tau all has remarkable increase (P＜0.01 or P＜0.05) in the cerebral ischemia re-pouring model group rat cerebral cortex; Compare with model group, Glu, Asp, Gly all significantly reduce (P＜0.01 or P＜0.05) in each dosage group cerebral tissue of nimodipine group and salvianolic acid A freeze-dried powder, Tau content significantly raise (P＜0.01 or P＜0.05) in each dosage group cerebral tissue of nimodipine group and salvianolic acid A freeze-dried powder, nimodipine group and salvianolic acid A freeze-dried powder are high, GABA content significantly raise (P＜0.01) in the middle dosage group cerebral tissue, the GABA content of salvianolic acid A freeze-dried powder low dose group slightly raises than model group, but difference does not have statistical significance.Cerebral ischemia re-pouring model group EI value has extremely significantly than sham operated rats and increases (P＜0.01), and the EI value of each dosage group of salvianolic acid A freeze-dried powder and nimodipine group obviously reduces (P＜0.01 or P＜0.05) than model group.
Table 47. salvianolic acid A freeze-dried powder is on the impact (x ± s, n=8) of amino acid neurotransmitter in the rats after cerebral ischemic reperfusion cerebral tissue
Annotate: compare * P＜0.01, ※ P＜0.05 with sham operated rats; Compare * P＜0.05, * * P＜0.01 with model group
3 conclusions
Monoamine neurotransmitter disorder and toxicity of excitatory amino acid play an important role in ischemic brain injury, acute cerebral ischemia cell death, reperfusion injury and delayed neuronal death.Monoamine neurotransmitters significantly raises in extracellular fluid during cerebral ischemia, and reduces in brain essence.In cerebral tissue ischemia equivalent damage situation, monoamine neurotransmitter generation metabolism disorder in the brain, NE, DA and 5-HT content obviously reduce, and cerebral ischemia is heavier, and cerebral tissue NE, DA, 5-HT content are just lower.Glu and Asp are the important excitatory neurotransmitters of brain, and GABA and Gly are the important inhibitory neurotransmitters of brain, and Tau has protective effect as a kind of neuromodulator in cerebral ischemia.During cerebral ischemia in the brain amino acid neurotransmitters excited-to suppress unbalance is one of key factor of causing cerebral ischemia.This experimental result shows that the 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, GABA in the rising cerebral ischemia/reperfusion injury of rats cerebral tissue, Tau content, significantly reduces content and the aminoacid exitotoxicity index of Glu in the cerebral tissue, Asp, Gly.And dosage increases, and effect strengthens.The salvianolic acid A freeze-dried powder that shows in conjunction with the histopathology pathological examination can obviously alleviate cerebral edema, improves the result of neuronal cell form, shows that the salvianolic acid A freeze-dried powder can suppress monoamine neurotransmitter and excessively discharge, and improves the monoamine neurotransmitter disorder; Suppress the accumulation of excitatory amino acid in the 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 salvianolic acid A freeze-dried powder is on the impact of LPO, SOD, GSH-Px in the rats after cerebral ischemic reperfusion cerebral tissue
Each organizes 10 remaining rats after 32 lower detections of experimental example, broken end is got brain, weigh, make brain tissue homogenate's liquid of 10% with 4 ℃ of normal saline, centrifugal, remove supernatant, measure the activity of lipid peroxide contents (LPO), superoxide dismutase (SOD) and glutathion peroxidase (GSH-Px).The result carries out statistical analysis.
Table 48. salvianolic acid A freeze-dried powder is on the impact (x ± s, n=10) of LPO, SOD, GSH-Px in the rats after cerebral ischemic reperfusion cerebral tissue
Figure BSA00000812340600661
Figure BSA00000812340600671
Annotate: compare * P＜0.01 with sham operated rats; Compare * P＜0.05, * * P＜0.01 with model group
LPO content significantly raises than sham operated rats in the cerebral ischemia re-pouring model group rat cerebral tissue, and SOD and GSH-Px activity significantly reduce (P＜0.01) than sham operated rats.The brain tissue oxygen free radical sharply increased when Cerebral ischemia and reperfusion was described, the integrity of infringement membrane structure and function causes lipid peroxidation, produces a large amount of lipid peroxide; And the free radical scavenging enzymatic activity significantly lowers; Therefore the dynamic equilibrium heavy damage of free-radical generating and removing.Free radical toxicity chain reaction meeting accelerator nerve units apoptosis, increase the weight of cerebral ischemia.Experimental result shows that each dosage group of salvianolic acid A freeze-dried powder is compared with model group, and LPO content significantly reduces in the rat cerebral tissue, active significantly raise (P＜0.01 or P＜0.05) of SOD and GSH-Px; Illustrate that the salvianolic acid A freeze-dried powder can increase the generation of the activity of peroxide scavenger enzyme in the ischemical reperfusion injury cerebral tissue, inhibition peroxide, thereby to the damage of antioxidant radical to neuronal cell and cerebral tissue.
Experimental example 34: the 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 is pressed 0.3ml/100g immediately after model preparation operation administration volume tail vein injection administration, after this every day the tail intravenously administrable once, continue to and draw materials.Sham operated rats, model control group postoperative tail vein injection saline; The high, medium and low dosage group of salvianolic acid A freeze-dried powder postoperative is tail vein injection salvianolic acid A freeze-dried powder 40mg/kg, 20mg/kg, 10mg/kg respectively; Nimodipine group injection nimodipine 20mg/kg.Each group is got rat respectively at MCAO postoperative 6h, 12h, 24h, 48h, 72h time point and is poured into 4% paraformaldehyde neutral buffered solution through heart, gets brain.Conventional fixing, the dehydration paraffin embedding of cerebral tissue is cut into the thick crown section of 5um continuously.TUNEL method (the dUTP breach end-labelling of deoxyribose nucleotides terminal transferase mediation) detects vascular endothelial cell.Press the operation of TUNEL cell apoptosis detection kit (Wuhan doctor's moral biological reagent company) description, DAB colour developing, observed result under the optical microscope.Nucleus the brown yellow granule the occurs positive apoptosis endotheliocyte of person.Each specimen random observation volume top and Striatum and Basal ganglia under * 400 times of mirrors have blood vessel but nonoverlapping 10 visuals field, calculate its TUNEL positive vessels endotheliocyte sum, i.e. apoptosis cell.Data are carried out statistical analysis and are processed.
Table 49. salvianolic acid A freeze-dried powder is on the impact of RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia apoptosis of vascular endothelial cell (x ± s)
Figure BSA00000812340600681
Annotate: compare #P＜0.01 with sham operated rats; Compare * P＜0.01 with model group
Experimental result shows that the cerebrovascular endothelial cell apoptosis number of each time period of model group is significantly higher than sham operated rats (P＜0.01), and 6h behind the cerebral tissue ischemia, endothelial cell apoptosis be showed increased just, reaches the peak to the ischemia in 24 hours.Compare with model group, the apoptosis digital display work of each time period of 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 the cerebrovascular endothelial cell survival rate, thereby guarantee the normal of cerebrovascular endothelial cell function, keep the complete of ischemic injuries hindbrain blood vessel structure and function and strengthen ability to anti-cerebral ischemia damnification, the effect of performance treatment ischemic cerebrovascular.
Experimental example 35: the salvianolic acid A freeze-dried powder is on the impact of anoxia and Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC) survival rate
The SD rat of the about 100g of male body weight behind the broken end, is got brain behind the alcohol disinfecting, peel off pia mater encephali and macroscopic trunk, remove cerebral white matter and cerebellum, thereafter, shred cerebral tissue, homogenate is crossed successively 90,180 mesh filter screens and is filtered, blood capillary section on flushing and the collection filter screen adds the 0.1%II collagenase, 37 ℃ of digestion 15min, the centrifugal 5min of 1500r/min, repeat 3 times, be inoculated in after precipitation suspends with culture fluid and continue in the culture bottle to cultivate, change liquid 1 time every 2d.Treat that cell grows up to the fusion state, identify that through Morphological Identification and 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, make single cell suspension after the digestion, 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 the cell inoculation, second day changes culture fluid into PBS liquid, is positioned over (37 ℃, 95%N2,5%CO2) effect 4h in the anoxia tank, causes BMVEC anoxia-induced apoptosis (simulated ischemia); Change again common culture fluid into and normally cultivate 12h, cause BMVEC Hypoxia/Reoxygenation Injury (simulated ischemia-fill with again).Each dosage group of nimodipine group 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, detect the survival rate of respectively organizing BMVEC with mtt assay respectively.
Table 50 salvianolic acid A freeze-dried powder is on the impact (x ± s, n=6) of the BMVEC survival rate of anoxia and Hypoxia/Reoxygenation Injury
Figure BSA00000812340600691
Compare #P＜0.05, * P＜0.01 with model control group; With comparison before the reoxygenation, ☆ P＜0.01; Compare with the nimodipine group, △ P＜0.05,
Figure BSA00000812340600692
Experimental result shows that after the anoxia 4h damage, the BMVEC survival rate is about 25.45% only, cell injury occurred after anoxia is described; The trend that 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 rising, but significant difference do not shown; The BMVEC survival rate is significantly higher than model control group (P＜0.05) behind salvianolic acid A freeze-dried powder 40,50, the 60 μ mol/L dosage group anoxia-induced apoptosis 4h.Behind the Hypoxia/Reoxygenation Injury, the BMVEC survival rate only is about 13.22%, with comparison before the reoxygenation, significantly reduces (p＜0.01), illustrates that hypoxia/reoxygenation has aggravated cell injury; After salvianolic acid A freeze-dried powder and nimodipine effect, the 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 its hypoxia-bearing capability, improves the brain microvessel endothelial cells in vitro survival rate of anoxia-induced apoptosis and Hypoxia/Reoxygenation Injury.
Experimental example 36 salvianolic acid A freeze-dried powders are on the impact of 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 to be processed, and cultivates corresponding experimental period by normal method.Each porocyte of trypsinization, centrifugal collecting cell adopts phosphatidyl in conjunction with albumen-Fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) dyeing, uses the cells were tested by flow cytometry cell in early days and the apoptosis rate in late period.
Table 51 salvianolic acid A freeze-dried powder causes the impact (x ± s, n=6) of BMVEC apoptosis on Hypoxia/Reoxygenation Injury
Figure BSA00000812340600701
Compare #P＜0.01 with Normal group; Compare * P＜0.01, * * P＜0.001 with model group; Compare with the nimodipine group, Δ P＜0.01,
Figure BSA00000812340600702
Apoptosis especially plays an important role in the transient ischemia/reperfusion damage at ischemia injury.The apoptosis of vascular endothelial cell can cause the broken words of vascular integrity, and the angiolysis damage is the basis of cerebrovascular; The apoptosis of brain microvessel endothelial cells in vitro can affect the integrity of blood brain barrier and the secretory function of vascular endothelial cell, and then increases the weight of ischemic brain injury.Show that by experimental result Hypoxia/Reoxygenation Injury can cause each phase apoptosis rate of BMVEC significantly to increase.With model group relatively, 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 groups are 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 preparation BMVEC hypoxia/reoxygenation model.Grouping and administration are with experimental example 36.Collecting cell supernatant, ELISA method detect the salvianolic acid A freeze-dried powder to the impact of 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 BSA00000812340600711
Compare with Normal group, #P＜0.01,
Figure BSA00000812340600712
Compare * P＜0.01, △ P＜0.05, * * P＜0.001 with model group
Behind the Hypoxia/Reoxygenation Injury, compare with normal group, BMVEC secretion t-PA and NO obviously reduce, and ET raises, and t-PA/PAI and NO/ET ratio also significantly descend.The t-PA of each 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) than model group, and wherein the middle and high dosage group of salvianolic acid A freeze-dried powder rises near the Normal group level; Each administration group t-PA/PAI and NO/ET ratio ratio also significantly improve than model group.Prompting salvianolic acid A freeze-dried powder can improve the secretory function of brain microvessel endothelial cells in vitro behind the 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+Concentration as influencing factor
As described in experimental example 35, third generation BMVEC is inoculated in 96 orifice plates preparation BMVEC hypoxia/reoxygenation model.Grouping and administration are with experimental example 36.Each porocyte of trypsinization behind the centrifugal collecting cell, adds Fluo-3 (a kind of calcium fluorescent probe, final concentration are 5 μ mol/L), hatches 45min for 37 ℃, and the cells were tested by flow cytometry fluorescence intensity is used in PBS liquid flushing 3 times.Almost there is not fluorescence when existing with the free ligand form because of Fluo-3, and and Ca 2+Strengthen in conjunction with rear fluorescence, can be according to fluorescence intensity change-detection Cytoplasmic Ca 2+Concentration change.
Table 53 salvianolic acid A freeze-dried powder is to Ca in the Hypoxia/Reoxygenation Injury BMVEC 2+Concentration as influencing factor (x ± s, n=6)
Figure BSA00000812340600721
Compare #P＜0.01 with Normal group; Compare * P＜0.01 with model group; Compare with the nimodipine group,
Figure BSA00000812340600722
The experimental result demonstration, behind the Hypoxia/Reoxygenation Injury, Ca in the BMVEC born of the same parents 2+Concentration significantly increases (P＜0.01).Compare each medicine group Ca with model group 2+Concentration extremely significantly reduces, and is the most remarkable with the middle and high dosage group of salvianolic acid A freeze-dried powder effect, and intracellular calcium concentration significantly is 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 the BMVEC born of the same parents 2+The effect of concentration.
Experimental example 39: the salvianolic acid A freeze-dried powder is on the impact of rat artery thrombus formation time
Get the SD rat, male and female half and half, body weight are 250～300g, are 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 organizes tail intravenously administrable 1 time every day (matched group gives the isometric(al) normal saline), continuously behind the 7d, 30min after the last administration, pentobarbital sodium (40mg/kg) anesthetized rat, dorsal position is fixed on the Mus plate, the cervical region median incision, separate the about 15mm of right carotid, the stimulating electrode and the temperature sensor probe that thrombus in vivo are formed analyzer place on the common carotid artery, stimulating electrode is positioned at proximal part, with 2mA galvanism blood vessel 7min, makes the tunica intima damage, record is because of the time of thrombosis blocking blood flow in the arterial lumen, i.e. thrombus formation time (OT).Experimental data statistical result sees Table 54.
Table 54. salvianolic acid A freeze-dried powder is on the impact of rat artery thrombus formation time
Figure BSA00000812340600731
Annotate: compare * P＜0.01 with the normal saline group
Experimental result shows, compares the thrombus formation time significant prolongation (P＜0.01) of salvianolic acid A freeze-dried powder 12.5,2.5,0.5mg/kg dosage group with the normal saline matched group; Low dose group thrombus formation time and 20mg/kg aspirin group be (P＞0.05) quite; Thrombus formation time has significant difference (P＜0.05) between the high, medium and low dosage group.Show that the salvianolic acid A freeze-dried powder can prolong thrombus formation time, the formation of prevention of arterial thrombosis, pre-preventing thrombosis and the ischemic cerebrovascular that causes.
Experimental example 40: the salvianolic acid A freeze-dried powder is to thrombotic inhibitory action
The SD rat, male and female half and half, grouping, administration are with experimental example 39.30min pentobarbital sodium anesthesia after the last administration, dorsal position is fixed, and right common carotid artery and left external jugular vein are isolated in operation, connect with three sections polyethylene tubes.Put into the long 5cm operation silk thread of having weighed in the polyethylene tube stage casing.(5u/mL) is full of polyethylene tube with heparin-saline solution.One end of pipe inserts left external jugular vein, and the other end links to each other with right common carotid artery.Open bulldog clamp, blood returns left jugular vein by the right carotid polyethylene tube of flowing through.Herba Clinopodii in behind the open blood flow 15min takes out rapidly silk thread, and filter paper sucks supernatant blood, weighs, and gross weight subtracts line and heavily namely gets wet weight of thrombus; Then put the interior freeze-day with constant temperature of 60 ℃ of baking ovens to constant weight, weigh after the cooling, be the thrombosis dry weight.Experimental data sees Table 55.
The inhibitory action that table 55. salvianolic acid A freeze-dried powder forms rat suppository
Figure BSA00000812340600732
Figure BSA00000812340600741
Annotate: compare * P＜0.01 with the normal saline group
Experimental data shows, compares with the normal saline group, and salvianolic acid A freeze-dried powder 12.5,2.5,0.5mg/kg dosage group rat suppository wet, dry weight all significantly alleviates (P＜0.01), have preferably dose-effect relationship between each dosage group.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 prevention or treat ischemic cerebrovascular due to the thrombosis.
Experimental example 41: the salvianolic acid A freeze-dried powder is on the impact of rat vein thromboembolism
Male SD rat, body weight (200 ± 20) g divides into groups with experimental example 40 pentobarbital sodium (40mg/kg) anesthetized rat, along abdominal part medisection rat stomach wall, open the abdominal cavity, separate postcava, and in left renal vein lower end level place ligation postcava, close the abdominal cavity, closed the abdominal cavity, behind the 1h, the tail vein injection administration; Reopen the abdominal cavity after 3h, folder closes blood vessel in 2cm place, ligation below, simultaneously ligation vein side shoot, and blood vessel is cut in stringer open, exhausts the tube chamber inner blood, and removal of thromboses, filter paper sop up residual blood, take by weighing immediately wet weight of thrombus; After putting afterwards the interior freeze-day with constant temperature of 60 ℃ of baking ovens, claim the thrombosis dry weight.Test data sees Table 56.
Table 56. salvianolic acid A freeze-dried powder is on the impact of rat vein thromboembolism
Figure BSA00000812340600742
Annotate: compare * P＜0.01 with the normal saline group
Experimental data shows, compare with the normal saline group, salvianolic acid A freeze-dried powder 12.5,2.5,0.5mg/kg dosage group rat vein thrombosis wet, dry weight significantly alleviates (P＜0.01), and low dose group and 20mg/kg aspirin group effect be (P＞0.05) quite; Show that the salvianolic acid A freeze-dried powder has the effect that suppresses venous thrombosis, can be used for the venous thromboembolism after the prophylaxis of acute ischemic cerebrovascular occurs.
Experimental example 42: the salvianolic acid A freeze-dried powder is to the thrombotic inhibitory action of rats in vitro
The SD rat, 250～300g, male and female half and half; Divide at random physiology 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, each organize every day tail vein injection to relative medicine 1 time, continuous 7 days; 30min after the last administration, pentobarbital sodium anesthesia, open the abdominal cavity along ventrimeson, the about 1.5ml of abdominal aortic blood injects the silication sebific duct, with the docking of silica gel tube two ends circlewise, the silica gel sheath seal of tube is fixed rapidly, puts 37 ℃ of constant temperature rotation 15min on the extracorporeal thrombosis forming device, removal of thromboses is measured thrombosis length, is claimed its weight in wet base; Survey its dry weight in 60 ℃ of calorstats after the oven dry.Experimental data sees Table 57.
Table 57. salvianolic acid A freeze-dried powder is to the thrombotic inhibitory action of rats in vitro (x ± s)
Figure BSA00000812340600751
Annotate: compare * P＜0.01 with the normal saline group
Experimental data shows, compare with the 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; Formation has preferably inhibitory action to external thrombus to show the salvianolic acid A freeze-dried powder.
Experimental example 43: the salvianolic acid A freeze-dried powder is to the thrombolytic experimental study of established thrombosis in the 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.Reduce to 50% before stimulating with blood flow after stimulate finishing and be considered as thrombosis.Animal is divided 5 groups at random, 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 20min after forming thrombosis, all through the disposable injection relative medicine of femoral vein, revascularization situation in the 1h after the observation administration; If revascularization in this section period then continues to observe vessel open state 1h.The Flow of carotid artery of every animal to be stimulating front blood flow as baseline, with 〉=50% or≤25% stimulate before the blood flow person be judged to be continue to lead to again or continue after again thromboembolism; After logical again in the 1h, each treated animal blood flow is divided into 〉=and 50%, 25%～50% and≤25% baseline values.According to blood flow, the carotid artery vascular degree of opening is divided three kinds of states, is respectively 1. to continue thromboembolism without logical again; 2. logical interlocking with thromboembolism occurs again; 3. continuous openness after leading to again, nothing is thromboembolism again.Experimental result sees Table 58.
Thrombolytic effect (n=10) in the table 58. salvianolic acid A lyophilized powder needle body
Figure BSA00000812340600761
Annotate: 1. logical again number of animals/animal number appears in 1h after recanalization rate=administration
2. the number of animals that appearance is led to again in the 1h after again thromboembolism number of animals/administration that the bolt rate=1h occurs after leading to again again
3. compare * P＜0.01 with the normal saline group; Compare #P＜0.05 with the urokinase group
The result shows: the normal saline group all continues thromboembolism, and without logical phenomenon occurring again; The number of animals that each medicine group continues thromboembolism is few, and comparing with the normal saline group all has utmost point significant difference (P＜0.01); Dosage in the 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; Salvianolic acid A freeze-dried powder high dose (10mg/kg) group continues recanalization rate and recanalization rate all is higher than the urokinase group, and the 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 the group recanalization rate is lower than the urokinase group, and bolt rate is suitable with the urokinase group again for it, and has and be lower than the again trend of bolt rate of urokinase.The again effect of thromboembolism after the above results prompting salvianolic acid A freeze-dried powder has preferably thrombolytic and prevents thrombolytic.
Experimental example 44 salvianolic acid A freeze-dried powders are on the impact of cerebral thrombosis hemorheology of rat
Adopt the method for carotid artery injection self thrombosis to prepare Cerebrovascular embolism model: after the intraperitoneal anesthesia of SD rat pentobarbital sodium, the neck median incision, separate the total tremulous pulse of right side strength (CCA), internal carotid artery (ICA), external carotid artery (ECA), ligation ECA far-end and arteria pterygopalatina, arteriole folder folder closes CCA and ICA, cut an osculum at the ECA near-end, unclamp CCA arteriole folder, extract arterial blood 0.5ml, the sodium citrate anticoagulant, centrifugal, get platelet poor plasma and add a small amount of erythrocyte and thrombinogen and calcium chloride mixing, the preparation diameter is about the thrombosis of 0.35mm, shreds the about 2mm of a trifle; Folder closes CCA, by ECA embolus is injected ICA, and ligation ECA unclamps CCA and arteria pterygopalatina place vascular clamp, sews up the incision.Experiment minutes 6 groups: sham operated rats, model control group, salvianolic acid A freeze-dried powder high, medium and low (10,5,2.5mg/kg) group, nimodipine group (10mg/kg).30min behind the successful surgery, after this each treated animal tail vein relative medicine injects once continuous 7 days every day; Sham operated rats and model group are injected isopyknic normal saline.The second day (fasting 12h) that administration finishes, intraperitoneal anesthesia, abdominal aortic blood, anticoagulant heparin carries out Determination of Blood Rheology.
Table 59 salvianolic acid A freeze-dried powder is on the impact of cerebral thrombosis hemorheology of rat
Figure BSA00000812340600771
Compare #p＜0.01 with sham operated rats; Compare * P＜0.01 with model control group,
Figure BSA00000812340600772
Experimental result shows that behind the cerebral thrombosis, rat whole blood viscosity, plasma viscosity significantly increase, and packed cell volume is significantly rising (P＜0.01) also; Each dosage group of salvianolic acid A freeze-dried powder and model group compare, and its whole blood viscosity and plasma viscosity and packed cell volume all obviously reduce; Prompting salvianolic acid A freeze-dried powder can be accelerated little blood flow velocity, and blood viscosity lowering improves hemorheology and the effect that effectively resists cerebral thrombosis.

Claims (22)

1. the salvianolic acid A freeze-dried powder is for the preparation of the purposes of protection ischemic tissue of brain damage medicine, and wherein, the weight proportion of described salvianolic acid A freeze-dried powder is: salvianolic acid A 20g～60g, and filler 20g～60g, antioxidant make 0.02%～0.1% of total amount;
The preparation method of described salvianolic acid A freeze-dried powder is:
(1) extract: Radix Salviae Miltiorrhizae water or alcoholic solution extract and obtain aqueous extract or alcohol extract;
(2) transform: with the extracting solution that step (1) obtains, transfer pH to 3.5～6.5, add molar percentage and be 0.1%～3.0% catalyst, 100～140 ℃ of heating 1～6 hour;
(3) purification:
A. the solution that step (2) is obtained is transferred pH to 2.5～4.5, and is centrifugal, and supernatant separates through nonpolar or low pole macroporous resin column chromatography, after washing with water, uses the eluant eluting, and high performance liquid chromatogram detects salvianolic acid A, collects the eluent that contains salvianolic acid A;
B. the eluent that step a is obtained is used the eluant eluting with sephadex lh-20 or ODS-C18 or the separation of polyamide chromatography post, and high performance liquid chromatogram detects salvianolic acid A, collects the eluent that contains salvianolic acid A;
C. the eluent that step b is obtained is transferred pH to 2.0～4.0, through organic solvent extraction, and the Separation of Organic phase;
D. the solution that step c is obtained separates with silica gel column chromatography, uses the eluant eluting, and high performance liquid chromatogram detects salvianolic acid A, collects the eluent that contains salvianolic acid A;
(4) drying: with the eluent that steps d obtains, the reclaim under reduced pressure eluant is dissolved in water again, and vacuum drying, spray drying or microwave vacuum drying get described salvianolic acid A;
(5) getting described salvianolic acid A 10g～80g injects water 1500～2800ml and stirs and to make dissolving, with adjusting PH with base value 4.0～5.0, add described filler and make its dissolving, add again described antioxidant, stirring makes the dissolving mixing, adds active carbon 0.5～2g stirring and adsorbing again, filters to remove active carbon, fill becomes bottle after injecting water, sends into and carries out lyophilization in the freeze dryer;
(6) described lyophilization comprises the steps:
A, freezing: be cooled to-40 ℃～-45 ℃ with 20 ℃～30 ℃/h speed, be incubated freezing 10～16 hours;
B, distillation: be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 10～14 hours rises to-23 ℃～-27 ℃, keeps-23 ℃～-27 ℃ vacuum dryings 6～8 hours;
C, drying: continue to heat up, evenly be warming up to 40 ℃～45 ℃ with 0.5 ℃～1.0 ℃/min, keep 40 ℃～45 ℃ dryings after 2～5 hours, sample temperature is down to room temperature, add a cover, namely get the salvianolic acid A freeze-dried powder.
2. purposes according to claim 1 also comprises the purposes of protecting blood brain barrier.
3. purposes according to claim 1 also comprises the purposes that reduces the ischemic tissue of brain infarction size.
4. purposes according to claim 1 also comprises the purposes that alleviates the ischemic tissue of brain edema.
5. each described purposes according to claim 1～4, its weight proportion is: salvianolic acid A 20g～40g, filler 20g～40g, antioxidant make 0.03%～0.08% of total amount.
6. each described purposes according to claim 1～4, wherein said filler is selected from any one or a few in mannitol, glucose, the lactose, and consumption is 20mg～40mg/2ml～3ml.
7. each described purposes according to claim 1～4, wherein said antioxidant is selected from any one or a few in vitamin C, thiourea, sodium sulfite, the sodium pyrosulfite.
8. purposes according to claim 7, 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 stirring and make dissolving, with sodium hydroxide sodium adjust pH 4.0～5.0, add mannitol 20g and make dissolving, add again the 0.8g vitamin C, stir and make the dissolving mixing, 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 ℃ with 25 ℃/h speed, be incubated freezing 15 hours;
B, distillation: be evacuated to below the 0.3mbar, the salvianolic acid A formulation temperature that will freeze in 12 hours rises to-25 ℃, keeps-25 ℃ of vacuum dryings 8 hours;
C, drying: continue to heat up, evenly be warming up to 40 ℃ with 0.8 ℃/min, keep 40 ℃ of dryings after 3 hours, sample temperature is down to room temperature, add a cover, namely get the salvianolic acid A freeze-dried powder.
9. purposes according to claim 1, wherein the water extracting method described in the step (1) is: get red rooted salvia, be cut into decoction pieces or be ground into diameter 2mm granule, add 3～15 times of water gagings at every turn and extract, extract altogether 1～3 time, extracted 1～4 hour at every turn; Extracting solution is evaporated to relative density 1.10～1.25 (60 ℃), adding ethanol makes and contains the alcohol amount 30%～80%, centrifugal, the supernatant decompression recycling ethanol also is concentrated into without the alcohol flavor, described water extraction adopts decoction to extract or 45～95 ℃ of water temperature lixiviates are got, stir with 10～50 rev/mins of speed simultaneously, get Radix Salviae Miltiorrhizae extract.
10. purposes according to claim 1, wherein the alcohol extraction method described in the step (1) is: get red rooted salvia, be cut into decoction pieces or be ground into diameter 2mm granule, add 3～15 times of amount 30%～60% alcohol reflux at every turn, the each extraction 1～4 hour extracted 1～3 time altogether; Decompression recycling ethanol gets Radix Salviae Miltiorrhizae extract.
11. purposes according to claim 1, wherein salvianolic acid B concentration is that 1mg/ml～30mg/ml or thin up are to 1mg/ml～30mg/ml in the extracting solution in the step (1).
12. purposes according to claim 11, wherein salvianolic acid B concentration is that 5mg/ml～20mg/ml or thin up are to 5mg/ml～20mg/ml in the extracting solution.
13. purposes according to claim 1, wherein the catalyst in the step (2) is one or more in iron chloride, ruthenium trichloride, aluminum chloride, zinc chloride, the Palladous chloride., and the molar percentage of catalyst and salvianolic acid B is 0.1%～3.0%.
14. purposes according to claim 13, wherein the molar percentage of catalyst and salvianolic acid B is 0.5%～2.0%.
15. purposes according to claim 1, wherein macroporous resin column described in the step a is HPD-80, HPD-100, HPD-100B, HPD-200A, HPD-300, HPD-450, HPD-722, HPD-826, ADS-5, ADS-8, ADS-21 or D101, AB-8; Described eluant is water and the ethanol of water and different proportion, and first water, 10～40% ethanol elutions, uses 20～60% ethanol elutions again, and high performance liquid chromatogram detects salvianolic acid A, collects and contains the salvianolic acid A part, and eluent is concentrated into without the alcohol flavor.
16. purposes according to claim 1, wherein the described eluant among the step b is water and the ethanol of water and different proportion, and first water, 20～60% alcoholic solution eluting remove impurity, use again 40～90% alcoholic solution eluting, high performance liquid chromatogram detects salvianolic acid A, collection contains the salvianolic acid A part, and eluent is concentrated into without the alcohol flavor.
17. purposes according to claim 1, wherein the organic solvent described in the step c is t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate or Ethyl formate.
18. purposes according to claim 1, wherein eluant described in the steps d is the two-phase solvent of petroleum ether, pentane, normal heptane, ethyl acetate, methyl acetate, Ethyl formate, t-butyl methyl ether composition.
19. purposes according to claim 1, the wherein temperature of microwave vacuum drying described in the step (4): 20-100 ℃, 1-5 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.
20. purposes according to claim 1, wherein vacuum drying temperature described in the step (4): 50 ℃～90 ℃, more than vacuum-0.07Mpa, power: 1～60KW, dry 2～20 hours.
21. purposes according to claim 1, wherein spray-dired intake air temperature described in the step (4): 150 ℃～350 ℃, the air outlet temperature: 70 ℃～95 ℃, spray velocity: 1～300ml/min.
22. purposes according to claim 1, wherein pH adjusting agent is phosphoric acid, hydrochloric acid, sulphuric acid or acetic acid.
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