CN102988310B - Salvianolic acid A freeze-dried powder is for the preparation of the purposes of protection ischemic tissue of brain damage medicine - Google Patents

Salvianolic acid A freeze-dried powder is for the preparation of the purposes of protection ischemic tissue of brain damage medicine Download PDF

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CN102988310B
CN102988310B CN201210488923.7A CN201210488923A CN102988310B CN 102988310 B CN102988310 B CN 102988310B CN 201210488923 A CN201210488923 A CN 201210488923A CN 102988310 B CN102988310 B CN 102988310B
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salvianolic acid
freeze
dried powder
cerebral
group
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CN102988310A (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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JIANGXI QINGFENG PHARMACEUTICAL CO Ltd
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Abstract

The present invention relates to the purposes of a kind of salvianolic acid A freeze-dried powder for the preparation 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, and antioxidant is make total amount 0.02% ~ 0.1%.

Description

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 the purposes of a kind of salvianolic acid A freeze-dried powder for the preparation of protection ischemic tissue of brain damage medicine.
Background technology
Cerebrovascular, also known as apoplexy, is a kind of nervous system disease caused by blood vessel generation pathological changes making supply brain blood by the various cause of disease.Its Etiological be the reasons such as cerebral arteries system disease damage (as cerebral arteriosclerosis) the cerebral arteries luminal stenosis, vasospasm, the obturation that cause or break, Oligemia or total blockage, brain blood circulation and dysfunction, cerebral tissue impaired and occur series of symptoms.Mainly comprise ischemic and hemorrhagic apoplexy.Wherein (ICVD, also known as Ischemic Stroke) accounts for about 80%.
Ischemic cerebrovascular refers to degeneration that local brain tissue comprises neurocyte, glial cell and contact fiber and occur due to blood supply disorder, necrosis or transient afunction.The cerebral arteries emphraxis that Intravascular Thrombus formation, thromboembolism, angiostenosis cause is the main cause of cerebral infarction.It is various, complicated that ischemia causes cranial nerve cell to damage with dead mechanism of action, after cerebral infarction (i.e. cerebral ischemia), because brain lacks the supply of blood and oxygen, cause cerebral energy metabolic imbalance, produce a series of pathologic damage, as oxidative stress, toxicity of excitatory amino acid, calcium overload, inflammatory reaction etc., thus cause neuronic mortality.It is commonly encountered diseases, frequently-occurring disease clinically, and mortality rate and disability rate are very high, shows one of three large fatal disease having become universally acknowledged.Clinical treatment mainly thrombolytic, the neuron of being at death's door saving ischemic area (Penumbra zone) and promotion damages the recovery of rear function of nervous system.Control ischemic cerebrovascular is current mankind difficult medical problem in the urgent need to address.Current U.S. FDA only have approved the tissue plasminogen activator factor (tPA) for the thromboembolism treatment after apoplexy, but its therapeutic time window is very narrow, only uses in apoplexy 4.5 hours just effectively; But also there is the danger that hemorrhage and Ischemia Reperfusion increases the weight of brain injury.And comprise calcium channel blocker if nimodipine, glutamate receptor antagonists are if dizocilpine (dizocilPine), antioxidant or free radical scavenger are as Edaravone, NO signal transduction pathway regulator lu pei Shandong mile (Iubeluzole) and inflammation inhibitor enlimomab (enlimomab) etc. for the neuroprotective drug of ischemic stroke treatment at present.But the therapeutical effect had in them is imprecise or specificity is strong, some toxic and side effects compared with large, toleration is little, have be also in clinical before or clinical investigation phase, be difficult to play actively impact at control cerebral infarction.Thus, control brain ischemia medicament that is effective, safety and stability is fast developed extremely urgent.Medicine has played very important positive role in this area.
Salvianolic acid A (SalvianolicacidA), 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 SalviamiltiorrhizaBunge, salvianolic acid A has obvious pharmacological action to cerebral microvascular thromboembolism, and effect be better than salvianolic acid B (Zhang Hengai. salvianolic acid A is on the impact of cerebral microvascular thromboembolism rat and Mechanism Study [D]. Beijing: Beijing Union Medical College graduate school, 2011), salvianolic acid A is formed by danshensu and caffeic acid condensation, containing multiple phenolic hydroxyl group, hydroxyl, the reactive groups such as ester bond, it is unstable to light and heat, the oxidizable one-tenth salvianolic acid C of exposure air and different salvianolic acid C etc., due to the above-mentioned physicochemical property of salvianolic acid A, make salvianolic acid A injection agent, its stability becomes the key technology making injection.But relevant salvianolic acid A preparation particularly injection Research Literature data is few, and the instable problem of ubiquity injection, salvianolic acid A pharmacological action cannot be ensured.
Summary of the invention
For overcoming the above-mentioned defect that prior art exists, the invention provides the purposes of a kind of salvianolic acid A freeze-dried powder for the preparation 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 is make total amount 0.02% ~ 0.1%; 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: the extracting solution that step (1) is obtained, adjust pH to 3.5 ~ 6.5, add the catalyst that molar percentage is 0.1% ~ 3.0%, 100 ~ 140 DEG C of heating 1 ~ 6 hour;
(3) purification:
A. by the solution that step (2) obtains, pH to 2.5 ~ 4.5 are adjusted, centrifugal, supernatant is separated, after washing with water, with eluent through nonpolar or low pole macroporous resin column chromatography, efficient Liquid Detection salvianolic acid A, collects the eluent containing salvianolic acid A;
B. the eluent sephadex lh-20 obtained by step a or ODS-C18 or polyamide chromatography post are separated, and with eluent, efficient Liquid Detection salvianolic acid A, collects the eluent containing salvianolic acid A;
C. pH to 2.0 ~ 4.0 adjusted by the eluent obtained by step b, through organic solvent extraction, and Separation of Organic phase;
D. the solution silica gel column chromatography obtained by step c is separated, and with eluent, efficient Liquid Detection salvianolic acid A, collects the eluent containing salvianolic acid A;
(4) dry: the eluent that steps d is obtained, reclaim under reduced pressure eluant, then be dissolved in water, vacuum drying, spraying dry or microwave vacuum drying, obtain described salvianolic acid A;
(5) get described salvianolic acid A 10g ~ 80g to inject and be stirred to dissolve with water 1500 ~ 2800ml, by adjusting PH with base value 4.0 ~ 5.0, adding described filler makes it dissolve, add described antioxidant again, be stirred to dissolve mixing, then add active carbon 0.5 ~ 2g stirring and adsorbing, filter removing active carbon, inject and become bottle with fill after water, send in freeze dryer and carry out lyophilization;
(6) described lyophilization comprises the steps:
A, freezing: to be cooled to-40 DEG C ~-45 DEG C with 20 DEG C ~ 30 DEG C/h speed, to be incubated freezing 10 ~ 16 hours;
B, distillation: be evacuated to below 0.3mbar, rose to-23 DEG C ~-27 DEG C by the salvianolic acid A formulation temperature freezed in 10 ~ 14 hours, maintains-23 DEG C ~-27 DEG C vacuum dryings 6 ~ 8 hours;
C, drying: continue to heat up, be evenly warming up to 40 DEG C ~ 45 DEG C with 0.5 DEG C ~ 1.0 DEG C/min, maintain 40 DEG C ~ 45 DEG C dryings after 2 ~ 5 hours, sample temperature is down to room temperature, adds a cover, obtain salvianolic acid A freeze-dried powder.
Preferably, the purposes of protection blood brain barrier is also comprised.
Preferably, the purposes reducing ischemic tissue of brain infarction size is also comprised.
Preferably, the purposes alleviating ischemic tissue of brain edema is also comprised.
Preferably, its weight proportion is: salvianolic acid A 20g ~ 40g, filler 20g ~ 40g, and antioxidant is make total amount 0.03% ~ 0.08%.
Preferably, wherein said filler be selected from mannitol, glucose, lactose any one or a few, consumption is 20mg ~ 40mg/2ml ~ 3ml.
Preferably, wherein said antioxidant is selected from any one or a few in vitamin C, thiourea, sodium sulfite, sodium pyrosulfite.
Preferably, its weight proportion is: salvianolic acid A 20g, filler mannitol 20g, antioxidant vitamins C0.8g; Its preparation method is:
Get described salvianolic acid A 20g, inject and be stirred to dissolve with water 1900ml, with sodium hydroxide sodium adjust pH 4.0 ~ 5.0, add mannitol 20g and make dissolving, then add 0.8g vitamin C, be stirred to dissolve mixing, add active carbon 0.5g stirring and adsorbing, filter removing active carbon; Inject water to 2000ml, fill becomes 1000 bottles, lyophilization;
Described lyophilization comprises the steps:
A, freezing: to be cooled to-45 DEG C with 25 DEG C/h speed, to be incubated freezing 15 hours;
B, distillation: be evacuated to below 0.3mbar, rose to-25 DEG C by the salvianolic acid A formulation temperature freezed in 12 hours, maintains-25 DEG C of vacuum dryings 8 hours;
C, drying: continue to heat up, be evenly warming up to 40 DEG C with 0.8 DEG C/min, maintain 40 DEG C of dryings after 3 hours, sample temperature is down to room temperature, adds a cover, obtain salvianolic acid A freeze-dried powder.
Preferably, wherein the water extracting method described in step (1) is: get red rooted salvia, is cut into decoction pieces or is ground into diameter to be about 2mm granule, adds 3 ~ 15 times amount water extraction at every turn, extracts 1 ~ 3 time altogether, extract 1 ~ 4 hour at every turn; Extracting solution is evaporated to relative density 1.10 ~ 1.25 (60 DEG C), adding ethanol makes alcohol content 30% ~ 80%, centrifugal, supernatant decompression recycling ethanol is also concentrated into without alcohol taste, described water extraction adopts decoction to extract or 45 ~ 95 DEG C of water temperature lixiviates are got, stir with 10 ~ 50 revs/min of speed simultaneously, obtain Radix Salviae Miltiorrhizae extract.
Preferably, wherein the alcohol extraction method described in step (1) is: get red rooted salvia, is cut into decoction pieces or is ground into diameter to be about 2mm granule, adds 3 ~ 15 times amount 30% ~ 60% alcohol reflux at every turn, extracts 1 ~ 4 hour at every turn, extract 1 ~ 3 time altogether; Decompression recycling ethanol, obtains Radix Salviae Miltiorrhizae extract.
Preferably, in the extracting solution wherein in step (1), salvianolic acid B concentration is 1mg/ml ~ 30mg/ml or is diluted with water to 1mg/ml ~ 30mg/ml.
Preferably, wherein salvianolic acid B concentration is 5mg/ml ~ 20mg/ml or is diluted with water to 5mg/ml ~ 20mg/ml in extracting solution.
Preferably, the catalyst wherein in step (2) is one or more in iron chloride, ruthenium trichloride, aluminum chloride, zinc chloride, 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 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 uses water, 10 ~ 40% ethanol elutions, then uses 20 ~ 60% ethanol elutions, efficient Liquid Detection salvianolic acid A, and collect containing salvianolic acid A part, eluent is concentrated into without alcohol taste.
Preferably, the described eluant wherein in step b is water and the ethanol of water and different proportion, and first uses water, 20 ~ 60% alcoholic solution eluting remove impurity, use 40 ~ 90% alcoholic solution eluting again, efficient Liquid Detection salvianolic acid A, collect containing salvianolic acid A part, eluent is concentrated into without alcohol taste.
Preferably, the organic solvent wherein described in step c is t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate or Ethyl formate.
Preferably, wherein eluant described in 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 wherein temperature of microwave vacuum drying described in step (4): 20-100 DEG C, return difference temperature 1-5 DEG C, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.
Preferably, wherein vacuum drying temperature described in step (4): 50 DEG C ~ 90 DEG C, more than vacuum-0.07Mpa, power: 1 ~ 60KW, dry 2 ~ 20 hours.
Preferably, wherein spray-dired intake air temperature described in step (4): 150 DEG C ~ 350 DEG C, air outlet temperature: 70 DEG C ~ 95 DEG C, 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, obtain salvianolic acid A: through screening and the optimization of system, first the Extraction solvent and extracting method that determine initiation material salvianolic acid B is compared, because salvianolic acid B water solublity is better, determine and adopt water extraction or low concentration alcohol to extract, again because salvianolic acid B heat stability is poor, determine and adopt hot water warm macerating extract and add stirring extracting method, or use low-concentration ethanol reflux, extract, make extraction solubility lower than 100 DEG C, salvianolic acid B is kept not to be destroyed, optimum solvent consumption and extraction time is determined by orthogonal experiment, obtain the salvianolic acid B optimum extraction process adapting to suitability for industrialized production.
The present invention is compared with the prior art and shows: initiation material red rooted salvia extracting directly can carry out the conversion that feeds intake, transform again after not needing that purification is carried out to salvianolic acid B, namely in catalytic conversion reaction of the present invention, reaction raw materials salvianolic acid B does not need high-purity, such as, do not need purity >=50% of salvianolic acid B.It is generally acknowledged that reaction raw materials is more pure better, in catalytic conversion reaction of the present invention, the purity height of salvianolic acid B does not affect conversion reaction effect.On the contrary, experiment proves, not only produces a large amount of impurity, and do not improve conversion ratio during salvianolic acid B purity > 50%.Moreover the present invention comparing by repeatedly testing, first determining and the factor of material impact is produced as transformed the concentration, pH value, temperature, time etc. of front pressure differential self to salvianolic acid A productive rate.On this basis, repeatedly test by paying a large amount of time, material and energy the concentration of temperature, pH value, time, salvianolic acid B and other correlated conditions are studied again, and these factors how synergism has an impact to salvianolic acid A productive rate jointly each other, thus determine the optimum temperature, pH value, time etc. that salvianolic acid B transforms the needs control of salvianolic acid A, and control salvianolic acid B initial concentration at 1mg/ml ~ 30mg/ml, thus salvianolic acid A conversion ratio of the present invention is made more obviously to be better than other conversion conditions.In chemical reaction, the purity of reactant and concentration usually affect the effect of reaction.Generally there is purity requirement to reactant, and think that concentration height specific concentration is low good.But in catalytic conversion reaction of the present invention, the concentration level of salvianolic acid B does not affect conversion reaction effect.On the contrary, experiment proves, salvianolic acid B concentration height not only produces a large amount of impurity, and does not improve conversion ratio.And not more high better containing the concentration of salvianolic acid B in salvianolic acid B aqueous solution, more than 30mg/ml concentration conversion ratio is low on the contrary, and effect is poorer.Therefore, production technology of the present invention, in cost-saving and production cycle, has unforeseeable technique effect.When prior art does not provide the enlightenment of any technology, if those skilled in the art only infer theoretically, be impossible draw, under above-mentioned each conditional parameter, sour for pellet B 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 as catalyst the conversion ratio that salvianolic acid B transforms salvianolic acid A, highly stable the reaching close to 60% of conversion ratio, most cases can more than 60%, this is all impossible in any one prior art in the past, therefore, achieves unforeseeable technique effect.
Because salvianolic acid A content is lower, the large raising of salvianolic acid A content after transforming, but also containing a large amount of impurity, therefore, have selected low pole respectively and nonpolar macroporous adsorption resin carries out crude separation, select polyamide etc. again, solvent extraction, silica gel is separated, and measures various flows part, after removing impurity portion, salvianolic acid A content is brought up to 80% from about 10%, to 90%, to 93%, to 96%.
Further, while significantly improving conversion ratio, the present invention is by being suitable for the purification step of actual industry application, after the steps such as a series of separation, eluting process, do not adopt traditional constant pressure and dry, but adopt vacuum drying, spraying dry, microwave vacuum drying, thus thoroughly overcome in the past that baking temperature is too high, the defect that drying time is long and large to the destruction of salvianolic acid A; And sublimation drying is long, cost is high and the extract of lyophilization gained cannot remove problem of solvent residual completely.
Low-purity can also directly mix with highly purified salvianolic acid B by the present invention, only need be made into suitable initial conversion concentration, the object changing into salvianolic acid A can be reached equally, the preparation technology of this conversion feedstock is very simple, and production cost is very suitable for the application in actual industry while reducing.
Salvianolic acid A freeze-dried powder provided by the invention, according to chemistry and the physical characteristic of salvianolic acid A, from the additives affecting drug substance stable, dosage form, container, the external world are as air, light, moisture, and impurity etc. produce chemical reaction and cause the decomposition of medicament to carry out creationary experimental analysis on the contrary.By screening preparation prescription, repetition test, have selected mannitol, glucose, lactose etc., make freeze-dried powder, the molding of powder pin is good, and block loosens, hole, uniform color, and stability is very high, solve due to air, light, moisture, impurity etc. produce the medicament decomposes that chemical reaction causes.Salvianolic acid A being made suitable preparation, solves salvianolic acid A and make injection stability problem, by selecting suitable adjuvant and dosage form, ensure that salvianolic acid A pharmacological action, provide safe and effective salvianolic acid A injection formulation for clinical.
For these reasons, we are according to the physicochemical property of salvianolic acid A, by selecting suitable dosage form, have screened different adjuvants, and preferred different preparation technologies and technological parameter, prepared stable, safe, effective, quality controllable salvianolic acid A freeze-dried powder.
PH value is adjusted to applicable scope by alkali liquor by the salvianolic acid A in the present invention, then by adding mannitol etc., making it be easier to lyophilization, and not affecting the property of medicine.
The present invention is compared with the prior art and shows: the present invention passes through performing creative labour, finally determine lyophilizing speed cooling rate and cooling time, programming rate and temperature during distillation, determine the vacuum sublimation time, specify that dry programming rate and temperature, and drying time; Creationaryly brightly determine frozen cooling speed and temperature, distil programming rate and the complex relationship between the dry programming rate of temperature and temperature and time, and the determination etc. of suitable numerical range, thus just finally obtain stable lyophilized injectable powder.
What is more important, the salvianolic acid A freeze-dried powder adopting preparation method of the present invention to make can not change the original chemical attribute of salvianolic acid A completely; The salvianolic acid A freeze-dried powder compositions made has the features such as good water solubility, heat stability is high, solubility is good compared with salvianolic acid A.
On the other hand, present invention also offers its for prevent and or treatment ischemic cerebrovascular aspect purposes.
Known through experimental data contrast, the postoperative recovery from anesthesia scoring of easy apoplectic type renovascular hypertension in rats middle cerebral artery occlusion (MCAO), neural behavior scoring (the spontaneous activity of (12h in) after model group is revived, the symmetry of quadruped locomotion, forelimb extensions is creeped the symmetry of action, climb cage wall, push away trunk reaction, antenna is to the reaction etc. stimulated) significantly lower than sham operated rats, illustrate that after cerebral thrombosis ischemia, easily apoplectic type renovascular hypertension in rats (RHRSP) function of nervous system is seriously impaired, and in the 72h continued, neural behavior scoring continues to be starkly lower than sham operated rats, nimodipine group and each dosage group of salvianolic acid A freeze-dried powder are compared with model group, neurobehavioral after rat revives has rising in various degree, the most obvious with nimodipine group and the middle and high dosage group of salvianolic acid A freeze-dried powder, and after its ischemia, the neurobehavioral of 24h is basic close to sham operated rats level, to ischemia, the behavior of 72h salvianolic acid A freeze-dried powder each dosage group rat has recovered normal, and prompting salvianolic acid A freeze-dried powder can improve neurobehavioral after RHRSP cerebral thrombosis ischemia, has protection and the effect of nervous symptoms after improving cerebral ischemia.
Known through histopathology checking experiment Data Comparison, sham operated rats cerebral tissue Bilateral Symmetry, has no pathological changes, and the attachment of model group Intravascular Thrombus is serious.Compare with model group, the contraction in length of Microscopic observation salvianolic acid A freeze-dried powder each dosage group rat ischemia side middle cerebral artery (MCA) local thrombus, reduce at arterial wall bond area, with high, middle dosage group is the most obvious.TTC dyes visible infarcted cerebral constitution in white, and the cerebral tissue scope being dyed to white in salvianolic acid A freeze-dried powder each dosage group is more remarkable than model control group to be reduced.HE dyes in cortex MCA blood supply district, visible model group ischemia side (centered by the cortex of volume top) infarct has obvious cerebral tissue to soften, necrocytosis, the phenomenons such as local necrosis tissue loss, salvianolic acid A freeze-dried powder high dose group rat has no Telatrophy's obscission herein; The HE dyeing display stove of each dosage group of salvianolic acid A freeze-dried powder and model control group is interior, stove Zhou Jun has thin vessels hypertrophy, but each dosage group of salvianolic acid A freeze-dried powder compares hypertrophy thin vessels number with model group significantly increases; In addition, the stove shape that HE dyeing display model matched group differs in size as seen is hemorrhage, and high, the middle dosage group of salvianolic acid A freeze-dried powder there is no and finds that there is stove shape bleeding.Result display salvianolic acid A freeze-dried powder obviously can improve RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia histopathological status; reduce thrombosis bond area, reduce infarct size; increase in infarcted region and the thin vessels number of surrouding brain tissue, reduce cerebral hemorrhage phenomenon thus the necrosis of protection cerebral tissue comes off, there is the effect protecting cerebral thrombosis ischemia to cause brain tissue impairment.
Known through experimental data contrast, cerebral thrombosis tissues following MCAO in rats blood-brain barrier disruption, permeability increases, and azovan blue (EB) albumin-binding enters cerebral tissue by open blood brain barrier (BBB).After the administration of salvianolic acid A freeze-dried powder; under each dosage group rat cerebral tissue microscope, EB hot spot number and cerebral tissue EB detect content and obviously reduce; more all there were significant differences with model group; illustrate that salvianolic acid A freeze-dried powder causes blood-brain barrier permeability to brain tissue impairment and increases inhibited; can blood brain barrier be protected, and protect the further damaged of cerebral tissue.。
Known through experimental data contrast, by comparing RHRSP cerebral thrombosis rat model group, sham operated rats has no infarcted cerebral constitution; Salvianolic acid A freeze-dried powder each dosage group cerebral tissue Infarction volume and brain water content are significantly less than model control group, and dosage is larger, and Infarction volume is less, and brain water content is fewer.Salvianolic acid A freeze-dried powder basic, normal, high dosage group Infarction volume and brain water content are all lower than nimodipine group.Illustrate that salvianolic acid A freeze-dried powder can accelerate the reparation of focus, ischemic tissue of brain infarction size after reduction RHRSP cerebral thrombosis, alleviates cerebral tissue edema, has the effect of repairing and protecting Neurons Against Cerebral Ischemia tissue injury.
Known through experimental data contrast, compare with RHRSP cerebral thrombosis ischemia model group, after anti-ischemic therapy, salvianolic acid A freeze-dried powder each dosage group brain tissue ischemia Penumbra zone district MVD and blood vessel scene are long-pending significantly to be increased to some extent, and after drug withdrawal in 48h numerical value more stable.Show that salvianolic acid A freeze-dried powder can increase MVD and the blood vessel field area ratio of brain tissue ischemia Penumbra zone, show that salvianolic acid A freeze-dried powder has the effect promoting that ischemic tissue of brain Angiogenesis and collateral circulation are set up.
Known through experimental data contrast; RHRSP cerebral thrombosis ischemia model group VEGF Messenger RNA (VEGFmRNA) expression comparatively sham operated rats is increased significantly; after brain tissue ischemia anoxia is described, in energy Stimulation of The Brain tissue, the expression of VEGF increases; after prompting cerebral infarction, body self there will be a kind of protective response resisting ischemia injury; the expression of VEGF is increased, occurs after ischemia self " compensatory revascularization ".The each dosage group of salvianolic acid A freeze-dried powder compares with model group group; VEGFmRNA expression significantly raises; show that salvianolic acid A freeze-dried powder can significantly promote ischemic tissue of brain angiogenesis; promote the foundation of collateral circulation compensation; save cerebral ischemic penumbra; the brain tissue impairment that protection ischemia causes, and there is certain dose-effect relationship.
Known through experimental data contrast; RHRSP cerebral thrombosis ischemia model group rat cerebral tissue basic fibroblast growth factor (bFGF) protein expression comparatively sham operated rats is increased significantly; but with the prolongation of Ischemia Time; have a declining tendency; after prompting brain tissue ischemia anoxia; body self produces of short duration protection stress, can bFGF protein expression be organized to increase by Stimulation of The Brain.The each dosage group of salvianolic acid A freeze-dried powder compares with model group, and the bFGF protein expression of each time period significantly increases; Self each time period of each dosage group compares display, and bFGF protein expression is continual and steady; Prompting salvianolic acid A freeze-dried powder can increase ischemic tissue of brain bFGF protein expression, has the effect of good neurocyte protection effect and promotion angiogenesis, contributes to the foundation of collateral circulation, save cerebral ischemic penumbra.
Known through experimental data contrast, salvianolic acid A freeze-dried powder can alleviate cerebral ischemia-reperfusion injury in rats neurologic impairment symptom, is conducive to its neurobehavioral recovery, and salvianolic acid A freeze-dried powder has the effect improving function of nervous system after brain tissue impairment.
Known through experimental data contrast, the each dosage group of salvianolic acid A freeze-dried powder is compared with Ischemia-Reperfusion Injury Model group: cerebral infarction volume than significantly reduces, cerebral index and brain water content all obviously reduce, show that salvianolic acid A freeze-dried powder can reduce rats after cerebral ischemic reperfusion cerebral tissue infarction size, the cerebral tissue edema degree after cerebral ischemia reperfusion injury can be alleviated.
Known through experimental data contrast, Level In Rats With Focal Cerebral Ischemia, give various dose salvianolic acid A 10min to begin, each medicine group comparatively before self administration cerebral ischemic penumbra regional cerebral blood flow (rCBF) have obvious rising, and particularly remarkable with high, the middle dosage group of salvianolic acid A freeze-dried powder; The each dosage group of salvianolic acid A freeze-dried powder is compared, and has good dose-effect relationship; In salvianolic acid A freeze-dried powder, the rCBF of dosage group day part is suitable with nimodipine group.It can thus be appreciated that salvianolic acid A freeze-dried powder has the effect increasing rats with cerebral ischemia brain tissue ischemia Penumbra zone rCBF, be conducive to the cerebral tissue that redemption cerebral ischemic penumbra is at death's door, play the effect for the treatment of cerebral ischemia.
Known through experimental data contrast, after rat continuous cerebral ischemia 2h recovers filling again, each group rat ischemia Penumbra zone rCBF all has increase in various degree.After filling with, nimodipine, salvianolic acid A freeze-dried powder high, medium and low dosage group rat rCBF obviously increase, and each time period rCBF has pole significant difference compared with model group again.1h after filling with again, nimodipine and salvianolic acid A freeze-dried powder high dose group rCBF return to close to former rCBF 75%, in salvianolic acid A freeze-dried powder dosage group return to be about former rCBF 69%, salvianolic acid A freeze-dried powder low dose group returns to and is about 61% of former rCBF, and in Reperfu-sion 3h, each medicine group rCBF maintains in metastable scope.Result shows that salvianolic acid A freeze-dried powder obviously can improve the cerebral blood flow of cerebral ischemic penumbra cerebral tissue after Ischemia and Reperfusion in vivo in Rats, recover brain cell blood to supply, thus redemption cerebral ischemic penumbra, prevent cerebral tissue from damaging further even dead, have good therapeutical effect to ischemic cerebrovascular.
Known through experimental data contrast, salvianolic acid A freeze-dried powder can raise the content of ischemic tissue of brain adenosine triphosphate (ATP), adenosine diphosphate (ADP) (ADP), (AMP) AMP, phosphagen (PC), reduce cerebral tissue lactic acid (LA) content, significantly can improve the energy metabolism of rat cerebral ischemia and ischemia-reperfusion.Salvianolic acid A freeze-dried powder is by improving the energy metabolism of Neurons Against Cerebral Ischemia tissue, increase the supply of cerebral tissue energy matter, strengthen the survival ability of brain cell, save the brain cell that the cerebral ischemic penumbra after cerebral ischemia is at death's door, prevent cerebral tissue from damaging further even dead, can perform well in treating ischemic cerebrovascular.
Known through experimental data contrast, after rat cerebral ischemia 2h fills with 24h again, neuronal apoptosis is serious; And after nimodipine and various dose salvianolic acid A freeze-dried powder intervene, compare with model group, in rat cerebral tissue, neuronal apoptosis significantly reduces (P < 0.001 or P < 0.01); Show that salvianolic acid A freeze-dried powder has the effect suppressing cerebral ischemia rat cerebral tissue neuronal death, suppress neuronal apoptosis.
The neurotrophic factor histone matter molecule that to be neure growth required with survival, 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 parts for neurotrophic factor family, can maintain neuronal survival and promote neural cellular differentiation and inducing axonal growth; Energy neuroprotective unit, promotion neuron reparation and suppression delayed neuronal death, thus to anti-cerebral ischemia, protection cerebral ischemia.After Ischemia Reperfusion, in rat cerebral tissue, comparatively sham operated rats increases to some extent, and after prompting cerebral reperfusion injury, in cerebral tissue, NGF, BDNF express stress protectiveness increase; But NT-3 content obviously reduces in Neurons Against Cerebral Ischemia tissue.Known through experimental data contrast; compare with model control group; the each dosage group NGF of salvianolic acid A freeze-dried powder, 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 neural trophic factors NGF, BDNF, NT-3, reaches the effect of neuro-protective.
After ischemia-reperfusion, in cerebral tissue, inflammatory cytokine interleukin-1 ' beta ' (IL-1 β), interleukin-6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor-alpha (TNF-α), intercellular adhesion molecule-1 (ICAM-1) content all extremely significantly increase; Known through experimental data contrast, each inflammatory cytokine content of rat cerebral tissue giving salvianolic acid A freeze-dried powder each dosage group comparatively model group obviously reduces.Prompting salvianolic acid A freeze-dried powder can suppress the expression of ischemic injuries brain tissue inflammation cytokine, suppresses the generation of brain tissue inflammation's reaction, suppresses neuron and the cerebral tissue cascade damage of inflammatory reaction mediation.
Known through experimental data contrast, cerebral ischemia re-pouring model group compares cerebral tissue Ca with sham operated rats 2+content extremely significantly increases, K +, Mg 2+content significantly reduces; Compare with model group, nimodipine group and each dosage group of salvianolic acid A freeze-dried powder can significantly reduce Ca in cerebral tissue 2+content, rising K +, Mg 2+content, illustrates that salvianolic acid A freeze-dried powder can suppress Ca 2+interior stream alleviates calcium overload, thus can suppress Ca 2+the neuronal apoptosis of overload induction.
Known through experimental data contrast; salvianolic acid A freeze-dried powder can significantly 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), raises γ-aminobutyric acid (GABA) in cerebral ischemia/reperfusion injury of rats cerebral tissue, taurine (Tau) content, significantly reduces cerebral tissue Glutamic Acid (Glu), aspartic acid (Asp), the content of glycine (Gly) and aminoacid exitotoxicity index.And dosage increases, effect strengthens.Salvianolic acid A freeze-dried powder in conjunction with the display of histopathology pathological examination obviously can alleviate cerebral edema, improves the result of neuronal cell morphology, shows that salvianolic acid A freeze-dried powder can suppress monoamine neurotransmitter excessively to discharge, improve monoamine neurotransmitter disorder; Suppress the accumulation of excitatory amino acid in cerebral ischemia re-pouring, stablize the balance of excitatory amino acid-inhibitory aminoacid mediator, alleviate toxicity of excitatory amino acid; Thus suppress the neuronal apoptosis of monoamine neurotransmitter disorder and toxicity of excitatory amino acid induction.
Known through experimental data contrast, the each dosage group of salvianolic acid A freeze-dried powder is compared with ischemia-reperfusion injury model group, in rat cerebral tissue, lipid peroxide (LPO) content significantly reduces, and superoxide dismutase (SOD) and glutathion peroxidase (GSH-Px) activity significantly raise; Illustrate salvianolic acid A freeze-dried powder can increase peroxide scavenger enzyme in ischemical reperfusion injury cerebral tissue activity, suppress the generation of peroxide, thus to the damage of antioxidant radical to neuronal cell and cerebral tissue.
Known through experimental data contrast, the cerebrovascular endothelial cell apoptosis number of cerebral thrombosis ischemia model group each time period is significantly higher than sham operated rats, 6h after brain tissue ischemia, and endothelial cell apoptosis just showed increased, reaches peak in 24 hours to ischemia.Compare with model group, the apoptosis digital display work of each time period of salvianolic acid A freeze-dried powder each dosage group reduces, illustrate that salvianolic acid A freeze-dried powder can suppress cerebral ischemia hindbrain apoptosis of vascular endothelial cell, improve cerebrovascular endothelial cell survival rate, thus ensure the normal of cerebrovascular endothelial cell function, maintain the complete of cerebral vessel structures and function after ischemic injuries and the ability that strengthens anti-cerebral ischemia damnification, play the effect for the treatment of ischemic cerebrovascular.
Known through experimental data contrast, after salvianolic acid A freeze-dried powder and nimodipine effect, anoxia and Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial survival rate significantly raise, and salvianolic acid A freeze-dried powder group survival rate is significantly higher than nimodipine group.Prompting salvianolic acid A freeze-dried powder can protect microvascular endothelial cells oxygen and Hypoxia/Reoxygenation Injury, strengthens its hypoxia-bearing capability, improves the brain microvessel endothelial cells in vitro survival rate of anoxia-induced apoptosis and Hypoxia/Reoxygenation Injury.
Known through experimental data contrast, Hypoxia/Reoxygenation Injury, can cause each phase apoptosis rate of Brain Microvascular Endothelial significantly to increase.Compare with model group, 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 in certain dose-effect relationship.And salvianolic acid A freeze-dried powder 10 μm of ol/L dosage groups are suitable with nimodipine 40 μm of ol/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, Brain Microvascular Endothelial secretory tissue fiber proenzyme activator (t-PA) of Hypoxia/Reoxygenation Injury and the secretory volume of nitric oxide (NO) comparatively model group significantly raise (P < 0.01 or P < 0.001), and wherein the middle and high dosage group of salvianolic acid A freeze-dried powder rises to close to levels found in normal controls; Each administration group t-PA/PAI and NO/ET ratio also comparatively model group significantly improve.Prompting salvianolic acid A freeze-dried powder can improve the secretory function of brain microvessel endothelial cells in vitro after Hypoxia/Reoxygenation Injury.
After Hypoxia/Reoxygenation Injury, Ca in BMVEC born of the same parents 2+concentration significantly increases.Known through experimental data contrast, compare with model group, each medicine group Ca 2+concentration extremely significantly reduces, the most remarkable with salvianolic acid A freeze-dried powder middle and high dosage group effect, and intracellular calcium concentration is significantly lower than nimodipine 40 μm of ol/L dosage groups.Prompting salvianolic acid A freeze-dried powder has suppression Hypoxia/Reoxygenation Injury and causes Ca in BMVEC born of the same parents 2+the effect of concentration.
Known through experimental data contrast, salvianolic acid A freeze-dried powder compares with saline control group the impact of rat artery thrombus formation time, the thrombus formation time significant prolongation of the high, medium and low dosage group of salvianolic acid A freeze-dried powder; Low dose group (0.5mg/kg) thrombus formation time is suitable with 20mg/kg aspirin group; Show that salvianolic acid A freeze-dried powder can extend thrombus formation time, the formation of prevention of arterial thrombosis, pre-preventing thrombosis and the ischemic cerebrovascular caused.
Known through experimental data contrast, salvianolic acid A freeze-dried powder compares with normal saline group the impact that rat suppository is formed, and salvianolic acid A freeze-dried powder high, medium and low dosage group rat suppository is wet, dry weight all significantly alleviates.And low dose group is suitable with 20mg/kg aspirin group effect.Illustrate that salvianolic acid A freeze-dried powder has the effect of good inhibition thrombosis, can be used for preventing or treat the ischemic cerebrovascular caused by thrombosis.
Known through experimental data contrast, salvianolic acid A freeze-dried powder compares with normal saline group the impact of rat vein thromboembolism, salvianolic acid A freeze-dried powder high, medium and low dosage group rat vein thrombosis is wet, dry weight significantly alleviates, and low dose group (0.5mg/kg) and 20mg/kg aspirin group effect are quite (P > 0.05); Show that salvianolic acid A freeze-dried powder has the effect suppressing venous thrombosis, can be used for the venous thromboembolism after preventing acute ischemic cerebrovascular disease to occur.
Known through experimental data contrast, salvianolic acid A freeze-dried powder compares with normal saline group rats in vitro thrombosis, the thrombotic length of salvianolic acid A freeze-dried powder high, medium and low dosage group rats in vitro significantly shortens, thrombosis is wet, dry weight significantly alleviates, and low dose group (0.5mg/kg) is suitable with 20mg/kg aspirin group effect; Show that salvianolic acid A freeze-dried powder has good inhibitory action to ex vivo thrombosis.
Known through experimental data contrast, the thrombolytic of salvianolic acid A freeze-dried powder to thrombosis established in body compares with normal saline group, and normal saline group all continues thromboembolism, and without appearance logical phenomenon again; The number of animals that each medicine group continues thromboembolism is few, all has pole significant difference compared with normal saline group; Dosage group in salvianolic acid A freeze-dried powder, its vessel open degree is similar to 2000U/kg urokinase group, and its recanalization rate is suitable; Salvianolic acid A freeze-dried powder high dose group continues recanalization rate and recanalization rate all higher than urokinase group, and bolt rate is starkly lower than urokinase group again; Though salvianolic acid A freeze-dried powder low dose group recanalization rate is lower than urokinase group, bolt rate and urokinase group are suitable again for it, and have the trend lower than urokinase bolt rate again.Illustrate that salvianolic acid A freeze-dried powder has good thrombolytic and the effect of thromboembolism again after preventing thrombolytic.
After cerebral thrombosis, rat whole blood viscosity, plasma viscosity significantly increase, and packed cell volume also significantly raises, known through experimental data contrast, the each dosage group of salvianolic acid A freeze-dried powder compares with model group, and its whole blood viscosity and plasma viscosity and packed cell volume all obviously reduce; Prompting salvianolic acid A freeze-dried powder can accelerate micro-blood flow velocity, reduction blood viscosity, improves hemorheology and effectively resists the effect of 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 affecting cerebral circulation that the various cause of disease formed and damaging based on the ischemic rat brain of the defect of neuronal death and function of nervous system of causing thereof.Mainly comprise any one or more: (1) cerebral thrombosis: form embolus with blood flow into cerebral vessels by from the embolus of heart is as scorching in artificial valve, atrial fibrillation, ventricular thrombus, DCM (dilated cardiomyopathy), dynamic/vein blood vessel etc., cause forming the front circulation of whole brain blood circulation and any position of metacyclic blood vessel at different levels and/or multiple location thrombosis.(2) cerebral ischemia reperfusion injury (3) cerebral embolism.(4) atherosis or narrow (5) lacunar infarction of cranium arteria carotis externa and basilar artary.(6) Transient ischemic attacks (7) vascular dementia.
Medical science and study of pharmacy personnel in advance under the prerequisite not doing related experiment, cannot learn that salvianolic acid A freeze-dried powder has above-mentioned good purposes in advance.
Accompanying drawing explanation
Fig. 1. salvianolic acid B reference substance high-efficient liquid phase chromatogram;
Fig. 2. salvianolic acid B high-efficient liquid phase chromatogram in Radix Salviae Miltiorrhizae extract;
Fig. 3. salvianolic acid A reference substance high-efficient liquid phase chromatogram;
Fig. 4. salvianolic acid A high-efficient liquid phase chromatogram in salvianolic acid B catalyzed conversion liquid.
Fig. 5. salvianolic acid A freeze-dried powder freeze-drying curve figure.
Fig. 6. salvianolic acid A freeze-dried powder vacuum curve figure.
Detailed description of the invention
Embodiment 1
Get red rooted salvia, be ground into 6 order granules, add 7 times amount, 92 DEG C of water, warm macerating extracts 3 times at every turn, and stir with 25 revs/min of speed, each warm macerating extracts 3 hours simultaneously, extracting solution is evaporated to relative density 1.20 (60 DEG C), adds ethanol and makes alcohol content 70%, leaves standstill, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 20mg, aqueous solution 10% sodium hydroxide adjusts pH to 4.0, adds 0.5%ZnCl 2as catalyst, 120 DEG C of heating temperatures transform 4 hours, conversional solution 20% phosphoric acid adjust pH to 2.5, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 3mg, be separated through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 50 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 10, uses 3 times of cylinder hydrops, 5 times of column volume 25% ethanol elutions respectively, removing impurity, use 4 times of column volume 40% ethanol elutions again, HPLC detects, and collect the 40% ethanol elution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 5mg salvianolic acid A, be separated by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 10 with polyamide ratio, resin column blade diameter length ratio is 1: 8, use 3 times of cylinder hydrops, the remove impurity of 12 times of column volume 40% alcoholic solution eluting respectively, use 8 times of column volume 60% alcoholic solution eluting again, collect the 60% alcoholic solution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 15mg aqueous solution, aqueous solution 20% phosphoric acid adjusts pH to 2.5, and by the t-butyl methyl ether of aqueous solution 3 times amount, point 3 extractions, are separated organic layer, reclaim under reduced pressure t-butyl methyl ether, make the extract of every 1ml containing salvianolic acid A 0.5g, add 1 ~ 3 times amount silica gel, stir, volatilize, stirring sample silica gel is added on the dry silicagel column of 15 times amount installed, silicagel column blade diameter length ratio is 1: 10, with pentane-t-butyl methyl ether for eluant, gradient elution, use pentane respectively: t-butyl methyl ether (4: 6) eluting 10 times of column volumes, pentane: t-butyl methyl ether (6: 4) eluting 10 times of column volumes, reclaim under reduced pressure eluant, the salvianolic acid A reclaimed after organic solvent adds 12 times amount water dissolutioies, by microwave vacuum drying (microwave vacuum drying temperature: 60 DEG C, return difference temperature 3 DEG C, more than vacuum-0.07Mpa, microwave power 20KW) 100 minutes, obtain salvianolic acid A.
Embodiment 2
Get red rooted salvia, be cut into decoction pieces, add the 90 DEG C of water temperature lixiviates of 8 times amount at every turn and get 3 times, stir with 20 revs/min of speed, each warm macerating extracts 2.5 hours simultaneously, extracting solution is evaporated to relative density 1.15 (60 DEG C), adds ethanol and makes alcohol content 75%, leaves standstill, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 15mg, aqueous solution 10% potassium hydroxide adjusts pH to 4.0, adds 0.6%FeCl 3as catalyst, 3.5 hours are transformed at 120 DEG C of heating temperatures, conversional solution 15% hydrochloric acid adjust pH to 2.5, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, be separated through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 45 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 8, use 3.5 times of cylinder hydrops respectively, 4 times of column volume 25% ethanol elutions, removing impurity, use 5 times of column volume 45% ethanol elutions again, HPLC detects, collect the 45% ethanol elution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 5mg salvianolic acid A, be separated by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 9 with polyamide ratio, resin column blade diameter length ratio is 1: 7, use 4 times of cylinder hydrops, the remove impurity of 10 times of column volume 40% alcoholic solution eluting respectively, use 8 times of column volume 65% alcoholic solution eluting again, collect the 65% alcoholic solution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 12mg aqueous solution, aqueous solution 15% hydrochloric acid adjusts pH to 2.6, and by the t-butyl methyl ether of 4 times amount, point 4 extractions, are separated organic layer, reclaim under reduced pressure t-butyl methyl ether, make the extract of every 1ml containing salvianolic acid A 0.8g, add 2 ~ 3 times amount silica gel, stir, volatilize, stirring sample silica gel is added on the dry silicagel column of 13 times amount installed, silicagel column blade diameter length ratio is 1: 8, with normal heptane-ethyl acetate for eluant, and gradient elution, use normal heptane-ethyl acetate (4: 6) eluting 8 times of column volumes respectively, normal heptane-ethyl acetate (7: 3) eluting 8 times of column volumes, reclaim under reduced pressure eluant, then add 8 times amount water dissolutioies, vacuum drying (baking temperature: 65 DEG C, more than vacuum-0.07Mpa, power 10KW) 3 hours, obtain salvianolic acid A.
Embodiment 3
Get red rooted salvia, be ground into diameter and be about 2mm granule, add the 85 DEG C of water temperature lixiviates of 9 times amount at every turn and get 2 times, stir with 30 revs/min of speed, each warm macerating extracts 3.5 hours simultaneously, extracting solution is evaporated to relative density 1.10 (60 DEG C), adds ethanol and makes alcohol content 75%, leaves standstill, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 18mg, aqueous solution 10% sodium carbonate adjusts pH to 5.2, adds 0.6%AlCl 3as catalyst, 4.5 hours are transformed at 123 DEG C of heating temperatures, conversional solution 15% nitric acid adjust pH to 2.8, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 6mg, be separated through HPD-100B macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 40 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 7, use 4 times of cylinder hydrops respectively, 4 times of column volume 25% ethanol elutions, removing impurity, use 5 times of column volume 40% ethanol elutions again, HPLC detects, collect the 40% ethanol elution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 6mg salvianolic acid A, be separated by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 8 with polyamide ratio, resin column blade diameter length ratio is 1: 8, use 4 times of cylinder hydrops, the remove impurity of 9 times of column volume 40% alcoholic solution eluting respectively, use 7 times of column volume 65% alcoholic solution eluting again, collect the 65% alcoholic solution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 10mg aqueous solution, aqueous solution 15% nitric acid adjusts pH to 2.6, and with the methyl acetate of 4 times amount, point 4 extractions, are separated organic layer, reclaim under reduced pressure methyl acetate, make the extract of every 1ml containing salvianolic acid A 0.7g, add 3 times amount silica gel, stir, volatilize, stirring sample silica gel is added on the dry silicagel column of 10 times amount installed, silicagel column blade diameter length ratio is 1: 7, with petroleum ether, Ethyl formate for eluant, gradient elution, use petroleum ether-Ethyl formate (4: 6) eluting 8 times of column volumes respectively, petroleum ether-Ethyl formate (6: 4) eluting 9 times of column volumes, reclaim under reduced pressure eluant, add 8 times amount water dissolutioies again, spraying dry (intake air temperature: 170 DEG C, air outlet temperature 85 DEG C, spray velocity: 150ml/min), obtain salvianolic acid A
Embodiment 4
Get red rooted salvia, be cut into decoction pieces, add the 80 DEG C of water temperature lixiviates of 10 times amount at every turn and get 3 times, stir with 15 revs/min of speed, each warm macerating extracts 3 hours simultaneously; Extracting solution is evaporated to relative density 1.18 (60 DEG C), adds ethanol and makes alcohol content 70%, leaves standstill, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste; Be diluted with water to every 1ml containing salvianolic acid B 20mg, aqueous solution 10% sodium bicarbonate adjusts pH to 5.4, adds 0.8%RuCl 3as catalyst, 4.0 hours are transformed at 128 DEG C of heating temperatures, conversional solution 20% sulphuric acid adjust pH to 2.6, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, be separated through HPD-826 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 40 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 7, uses 4 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions respectively, removing impurity, use 5 times of column volume 40% ethanol elutions again, HPLC detects, and collect the 40% ethanol elution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste; Aqueous solution is concentrated into the solution of every ml containing 6mg salvianolic acid A, be separated by ODS chromatographic column, salvianolic acid A applied sample amount is 1: 10 with ODS ratio, resin column blade diameter length ratio is 1: 15, use 4 times of cylinder hydrops, the remove impurity of 7 times of column volume 35% alcoholic solution eluting respectively, use 6 times of column volume 60% alcoholic solution eluting again, collect the 60% alcoholic solution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 12mg aqueous solution; Aqueous solution 20% sulphuric acid adjusts pH to 2.8, and with the butyl acetate of 4 times amount, point 4 extractions, are separated organic layer, reclaim under reduced pressure butyl acetate, make the extract of every 1ml containing salvianolic acid A 0.5g, add 2.5 times amount silica gel, stir, volatilize; Stirring sample silica gel is added on the dry silicagel column of 9 times amount installed, silicagel column blade diameter length ratio is 1: 8, with pentane, methyl acetate for eluant, gradient elution, use pentane-methyl acetate (4.5: 5.5) eluting 8 times of column volumes respectively, pentane-methyl acetate (6.5: 3.5) eluting 8 times of column volumes, reclaim under reduced pressure eluant, add 10 times amount water dissolutioies again, with microwave vacuum drying (microwave vacuum drying temperature: 55 DEG C, return difference temperature 2 DEG C, more than vacuum-0.07Mpa, microwave power 30KW) 120 minutes, obtain salvianolic acid A.
Embodiment 5
Get red rooted salvia, be ground into diameter and be about 2mm granule, add the 85 DEG C of water temperature lixiviates of 9 times amount at every turn and get 3 times, stir with 20 revs/min of speed, each warm macerating extracts 2.5 hours simultaneously, extracting solution is evaporated to relative density 1.22 (60 DEG C), adds ethanol and makes alcohol content 70%, leaves standstill, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 10mg, aqueous solution 20% sodium citrate adjusts pH to 3.5, adds 0.4%PdCl 3as catalyst, 3.5 hours are transformed at 132 DEG C of heating temperatures, conversional solution 20% acetic acid adjust pH to 2.6, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, be separated through HPD-826 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 35 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 8, use 3 times of cylinder hydrops respectively, 4.5 times of column volume 25% ethanol elutions, removing impurity, use 6 times of column volume 40% ethanol elutions again, HPLC detects, collect the 40% ethanol elution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 8mg salvianolic acid A, be separated by ODS chromatographic column, salvianolic acid A applied sample amount is 1: 12 with ODS ratio, resin column blade diameter length ratio is 1: 18, use 4 times of cylinder hydrops, the remove impurity of 8 times of column volume 30% alcoholic solution eluting respectively, use 5 times of column volume 65% alcoholic solution eluting again, collect the 65% alcoholic solution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 15mg aqueous solution, pH to 2.7 adjusted by aqueous solution 20% acetic acid, and by the ethyl acetate of 5 times amount, point 5 extractions, are separated organic layer, reclaim under reduced pressure ethyl acetate, make the extract of every 1ml containing salvianolic acid A 0.5g, add 2 times amount silica gel, stir, volatilize, stirring sample silica gel is added on the dry silicagel column of 10 times amount installed, silicagel column blade diameter length ratio is 1: 10, with pentane, methyl acetate for eluant, gradient elution, use pentane-methyl acetate (4.5: 5.5) eluting 8 times of column volumes respectively, pentane-methyl acetate (6.5: 3.5) eluting 8 times of column volumes, reclaim under reduced pressure eluant, add 12 times amount water dissolutioies again, with microwave vacuum drying (microwave vacuum drying temperature: 65 DEG C, return difference temperature 1 DEG C, more than vacuum-0.07Mpa, microwave power 30KW) 90 minutes, obtain salvianolic acid A.
Embodiment 6
Get red rooted salvia, be ground into diameter and be about 2mm granule, add the 88 DEG C of water temperature lixiviates of 9 times amount at every turn and get 3 times, stir with 22 revs/min of speed, each warm macerating extracts 3.5 hours simultaneously, extracting solution is evaporated to relative density 1.16 (60 DEG C), adds ethanol and makes alcohol content 75%, leaves standstill, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 13mg, aqueous solution 10% sodium hydroxide adjusts pH to 3.6, adds 0.5%CuCl 2as catalyst, 4.5 hours are transformed at 133 DEG C of heating temperatures, conversional solution 10% hydrochloric acid adjust pH to 2.7, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, be separated through HPD-D101 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 40 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 9, use 4 times of cylinder hydrops respectively, 4 times of column volume 22% ethanol elutions, removing impurity, use 6 times of column volume 43% ethanol elutions again, HPLC detects, collect the 43% ethanol elution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 10mg salvianolic acid A, be separated by ODS chromatographic column, salvianolic acid A applied sample amount is 1: 15 with sephadex lh-20 ratio, resin column blade diameter length ratio is 1: 20, use 4 times of cylinder hydrops, the remove impurity of 8 times of column volume 30% alcoholic solution eluting respectively, use 5 times of column volume 60% alcoholic solution eluting again, collect the 60% alcoholic solution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 12mg aqueous solution, aqueous solution 10% hydrochloric acid adjusts pH to 2.8, and with the methyl acetate of 5 times amount, point 5 extractions, are separated organic layer, reclaim under reduced pressure methyl acetate, make the extract of every 1ml containing salvianolic acid A 0.5g, add 3 times amount silica gel, stir, volatilize, stirring sample silica gel is added on the dry silicagel column of 12 times amount installed, silicagel column blade diameter length ratio is 1: 10, with pentane, methyl acetate for eluant, gradient elution, use pentane-methyl acetate (4: 6) eluting 6 times of column volumes respectively, pentane-methyl acetate (6: 4) eluting 7 times of column volumes, reclaim under reduced pressure eluant, add 10 times amount water dissolutioies again, vacuum drying (baking temperature: 60 DEG C, more than vacuum-0.07Mpa, power 15KW) dry 3.5 hours, obtain salvianolic acid A.
Embodiment 7
Get red rooted salvia, be cut into decoction pieces, add the 90 DEG C of water temperature lixiviates of 9 times amount at every turn and get 3 times, stir with 25 revs/min of speed, each warm macerating extracts 3 hours simultaneously; Extracting solution is evaporated to relative density 1.12 (60 DEG C), adds ethanol and makes alcohol content 75%, leaves standstill, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste; Be diluted with water to every 1ml containing salvianolic acid B 20mg, aqueous solution 10% sodium carbonate adjusts pH to 5.0, adds 1.0%ZnCl 2as catalyst, 4.5 hours are transformed at 135 DEG C of heating temperatures, conversional solution 15% sulphuric acid adjust pH to 3.0, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, be separated through AB-8 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 36 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 9, uses 3 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions respectively, removing impurity, use 5 times of column volume 45% ethanol elutions again, HPLC detects, and collect the 45% ethanol elution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste; Aqueous solution is concentrated into the solution of every 1ml containing 6mg salvianolic acid A, be separated by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 10 with polyamide ratio, resin column blade diameter length ratio is 1: 10, use 4 times of cylinder hydrops, the remove impurity of 8 times of column volume 45% alcoholic solution eluting respectively, use 8 times of column volume 65% alcoholic solution eluting again, collect the 65% alcoholic solution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 10mg aqueous solution; Aqueous solution 15% sulphuric acid adjusts pH to 2.7, and by the t-butyl methyl ether of 4 times amount, point 4 extractions, are separated organic layer, reclaim under reduced pressure t-butyl methyl ether, make the extract of every 1ml containing salvianolic acid A 0.6g, add 3 times amount silica gel, stir, volatilize; Stirring sample silica gel is added on the dry silicagel column of 10 times amount installed, silicagel column blade diameter length ratio is 1: 8, with pentane, ethyl acetate for eluant, gradient elution, use pentane-ethyl acetate (4: 6) eluting 8 times of column volumes respectively, pentane-ethyl acetate (6: 4) eluting 8 times of column volumes, reclaim under reduced pressure eluant, add 8 times amount water dissolutioies again, with microwave vacuum drying (microwave vacuum drying temperature: 70 DEG C, return difference temperature 4 DEG C, more than vacuum-0.07Mpa, microwave power 25KW) 110 minutes, obtain salvianolic acid A.
Embodiment 8
Get red rooted salvia, be ground into diameter and be about 2mm granule, add the 85 DEG C of water temperature lixiviates of 9 times amount at every turn and get 3 times, stir with 22 revs/min of speed, each warm macerating extracts 3 hours simultaneously, extracting solution is evaporated to relative density 1.14 (60 DEG C), adds ethanol and makes alcohol content 70%, leaves standstill, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 25mg, aqueous solution 10% sodium citrate adjusts pH to 4.2, adds 0.4%AlCl 3as catalyst, 4.5 hours are transformed at 133 DEG C of heating temperatures, conversional solution 10% hydrochloric acid adjust pH to 2.8, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, be separated through HPD-300 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 45 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 10, use 5 times of cylinder hydrops respectively, 6 times of column volume 20% ethanol elutions, removing impurity, use 5 times of column volume 45% ethanol elutions again, HPLC detects, collect the 45% ethanol elution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every 1ml containing 8mg salvianolic acid A, be separated by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 12 with polyamide ratio, resin column blade diameter length ratio is 1: 8, use 3 times of cylinder hydrops, the remove impurity of 6 times of column volume 45% alcoholic solution eluting respectively, use 8 times of column volume 55% alcoholic solution eluting again, collect the 55% alcoholic solution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 10mg aqueous solution, aqueous solution 15% hydrochloric acid adjusts pH to 2.9, and by the t-butyl methyl ether of 5 times amount, point 5 extractions, are separated organic layer, reclaim under reduced pressure t-butyl methyl ether, make the extract of every 1ml containing salvianolic acid A 0.6g, add 2.5 times amount silica gel, stir, volatilize, stirring sample silica gel is added on the dry silicagel column of 12 times amount installed, silicagel column blade diameter length ratio is 1: 8, with pentane, t-butyl methyl ether for eluant, gradient elution, use pentane-t-butyl methyl ether (4: 6) eluting 8 times of column volumes respectively, pentane-t-butyl methyl ether (6: 4) eluting 8 times of column volumes, reclaim under reduced pressure eluant, add 9 times amount water dissolutioies again, with microwave vacuum drying (microwave vacuum drying temperature: 65 DEG C, return difference temperature 5 DEG C, more than vacuum-0.07Mpa, microwave power 40KW) 120 minutes, obtain salvianolic acid A.
Embodiment 9
The salvianolic acid A 80g that Example 3 is made, injects and is stirred to dissolve with water 2800ml, with 10% sodium hydroxide adjust pH 4.5, adds mannitol 60g and makes dissolving, add vitamin C 0.5g again, be stirred to dissolve, mixing, add active carbon 2g stirring and adsorbing, filter, removing active carbon; Inject water to 3000ml, detect intermediates content, pH value, detect qualified rear use 0.22 μm of microporous filter membrane to filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make 1000 bottles altogether, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-40 DEG C with 20 DEG C/h speed, be incubated freezing 16 hours; Be evacuated to below 0.3mbar, in 10 hours, the salvianolic acid A formulation temperature freezed risen to-25 DEG C, maintain-25 DEG C of vacuum dryings 8 hours; Continue to heat up, be evenly warming up to 41 DEG C with 0.5 DEG C/min, maintain 41 DEG C of dryings after 3 hours, sample temperature is down to room temperature, adds a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 10
The salvianolic acid A 20g that Example 4 is made, injects and is stirred to dissolve with water 1900ml, with 10% sodium hydroxide adjust pH 4.8, adds mannitol 20g and makes dissolving, add vitamin C 0.8g again, be stirred to dissolve, mixing, add active carbon 0.5g stirring and adsorbing, filter, removing active carbon; Inject water to 2000ml, detect intermediates content, pH value, detect qualified rear use 0.22 μm of microporous filter membrane to filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make 1000 bottles altogether, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-45 DEG C with 25 DEG C/h speed, be incubated freezing 15 hours; Be evacuated to below 0.3mbar, in 12 hours, the salvianolic acid A formulation temperature freezed risen to-25 DEG C, maintain-25 DEG C of vacuum dryings 8 hours; Continue to heat up, be evenly warming up to 40 DEG C with 0.8 DEG C/min, maintain 40 DEG C of dryings after 3 hours, sample temperature is down to room temperature, adds a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 11
The salvianolic acid A 10g that Example 5 is made, injects and is stirred to dissolve with water 1500ml, with 10% sodium hydroxide adjust pH 4.6, adds mannitol 20g and makes dissolving, add vitamin C 0.8g again, be stirred to dissolve, mixing, add active carbon 1.2g stirring and adsorbing, filter, removing active carbon; Inject water to 2000ml, detect intermediates content, pH value, detect qualified rear use 0.22 μm of microporous filter membrane to filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make 1000 bottles altogether, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-42 DEG C with 25 DEG C/h speed, be incubated freezing 12 hours; Be evacuated to below 0.3mbar, in 13 hours, the salvianolic acid A formulation temperature freezed risen to-23 DEG C, maintain-23 DEG C of vacuum dryings 7 hours; Continue to heat up, be evenly warming up to 42 DEG C with 0.8 DEG C/min, maintain 42 DEG C of dryings after 4 hours, sample temperature is down to room temperature, adds a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 12
The salvianolic acid A 30g that Example 6 is made, injects and is stirred to dissolve with water 2000ml, with 20% sodium carbonate adjust pH 4.2, adds mannitol 20g, lactose 10g makes dissolving, add thiourea 0.5g again, be stirred to dissolve, mixing, add active carbon 1.0g stirring and adsorbing, filter, removing active carbon; Inject water to 2000ml, detect intermediates content, pH value, detect qualified rear use 0.22 μm of microporous filter membrane to filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make 1000 bottles altogether, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-44 DEG C with 29 DEG C/h speed, be incubated freezing 14 hours; Be evacuated to below 0.3mbar, in 11 hours, the salvianolic acid A formulation temperature freezed risen to-26 DEG C, maintain-26 DEG C of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 43 DEG C with 0.6 DEG C/min, maintain 43 DEG C of dryings after 3.5 hours, sample temperature is down to room temperature, adds a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 13
The salvianolic acid A 35g that Example 7 is made, injects and is stirred to dissolve with water 2500ml, with 10% potassium hydroxide adjust pH 4.5, adds glucose 30g and makes dissolving, add sodium sulfite 3g again, be stirred to dissolve, mixing, add active carbon 1.5g stirring and adsorbing, filter, removing active carbon; Inject water to 3000ml, detect intermediates content, pH value, detect qualified rear use 0.22 μm of microporous filter membrane to filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make 1000 bottles altogether, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-43 DEG C with 27 DEG C/h speed, be incubated freezing 11 hours; Be evacuated to below 0.3mbar, in 12.5 hours, the salvianolic acid A formulation temperature freezed risen to-24 DEG C, maintain-24 DEG C of vacuum dryings 7.5 hours; Continue to heat up, be evenly warming up to 40 DEG C with 0.7 DEG C/min, maintain 40 DEG C of dryings after 5 hours, sample temperature is down to room temperature, adds a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 14
The salvianolic acid A 40g that Example 8 is made, inject and be stirred to dissolve with water 2700ml, with 10% sodium hydroxide adjust pH 4.3, add glucose 20g, lactose 20g makes dissolving, then add sodium pyrosulfite 4g, be stirred to dissolve, mixing, add active carbon 1.5g stirring and adsorbing, filter, removing active carbon; Inject water to 3000ml, detect intermediates content, pH value, detect qualified rear use 0.22 μm of microporous filter membrane to filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make 1000 bottles altogether, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-41 DEG C with 28 DEG C/h speed, be incubated freezing 13 hours; Be evacuated to below 0.3mbar, in 13.5 hours, the salvianolic acid A formulation temperature freezed risen to-25.5 DEG C, maintain-25.5 DEG C of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 44 DEG C with 0.9 DEG C/min, maintain 44 DEG C of dryings after 3.5 hours, sample temperature is down to room temperature, adds a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 15
The salvianolic acid A 60g that Example 1 is made, injects and is stirred to dissolve with water 2700ml, with 10% sodium hydroxide adjust pH 4.0, adds mannitol 40g and makes dissolving, add Catergen g again, be stirred to dissolve, mixing, add active carbon 2g stirring and adsorbing, filter, removing active carbon; Inject water to 3000ml, detect intermediates content, pH value, detect qualified rear use 0.22 μm of microporous filter membrane to filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make 1000 bottles altogether, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-42.5 DEG C with 24.5 DEG C/h speed, be incubated freezing 11.5 hours; Be evacuated to below 0.3mbar, in 13.5 hours, the salvianolic acid A formulation temperature freezed risen to-24.5 DEG C, maintain-24.5 DEG C of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 42.5 DEG C with 0.55 DEG C/min, maintain 42.5 DEG C of dryings after 2.5 hours, sample temperature is down to room temperature, adds a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 16
The salvianolic acid A 70g that Example 2 is made, injects and is stirred to dissolve with water 2800ml, with 10% sodium hydroxide adjust pH 5.0, adds mannitol 40g and makes dissolving, add Catergen g again, be stirred to dissolve, mixing, add active carbon 2g stirring and adsorbing, filter, removing active carbon; Inject water to 3000ml, detect intermediates content, pH value, detect qualified rear use 0.22 μm of microporous filter membrane to filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make 1000 bottles altogether, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-44.5 DEG C with 28.5 DEG C/h speed, be incubated freezing 11.5 hours; Be evacuated to below 0.3mbar, in 13.5 hours, the salvianolic acid A formulation temperature freezed risen to-24.5 DEG C, maintain-24.5 DEG C of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 43.5 DEG C with 0.75 DEG C/min, maintain 43.5 DEG C of dryings after 3 hours, sample temperature is down to room temperature, adds a cover, obtain salvianolic acid A freeze-dried powder.
Experimental example 1: salvianolic acid A, the research of salvianolic acid B analytical method
1. instrument and reagent
Instrument: Waterse2695 high performance liquid chromatograph, Empower2 chromatographic work station, 2998 diode array detector; Sartoriuscp225D 100,000/electronic balance.
Chromatographic column: YMCC 18chromatographic column (250 × 4.6mm, 5 μm);
Reagent: methanol is chromatographically pure, water is ultra-pure water prepared by Millipore, and other reagent are analytical pure.
Salvianolic acid B reference substance (lot number 111562-201009) all purchased from Nat'l Pharmaceutical & Biological Products Control Institute, for assay; Salvianolic acid A reference substance is self-control, is 99.52% through purity mark content.
2. the preparation of reference substance solution and need testing solution
The preparation of 2.1 reference substance solution: precision takes salvianolic acid B respectively, salvianolic acid A reference substance is about 10mg, puts in 100ml measuring bottle, adds dissolve with methanol and be diluted to scale, shaking up, in contrast product stock solution; Precision draws above-mentioned each 1ml respectively again, puts in same 10ml measuring bottle, adds methanol dilution to scale, shake up, as mixing reference substance solution.
The preparation precision of 2.3 need testing solutions takes embodiment 1 sample (being about equivalent to salvianolic acid A 10mg) and following " in experimental example 2 1.3 salvianolic acid B raw material preparations " obtain sample (being about equivalent to salvianolic acid B 10mg), put in 100ml measuring bottle, add dissolve with methanol and be diluted to scale, shake up, to obtain final product.
3. chromatographic condition and system suitability
Take octadecylsilane chemically bonded silica as filler; Flow velocity 1.0ml/min; Determined wavelength: get mixing reference substance solution, carries out UV scanning, and result has absorption maximum at 286nm wavelength place, therefore determines that determined wavelength is 286nm; Column temperature 30 DEG C; Number of theoretical plate calculates should be not less than 10000 by salvianolic acid A peak.
During 0-10 minute, the ratio of methanol is down to 60% by the ratio that 30% rises to 40%, 0.1-0.5% phosphate aqueous solution by 70%; During 10-30 minute, the ratio of methanol is down to 45% by the ratio that 40% rises to 55%, 0.1-0.5% phosphate aqueous solution by 50%; During 30-60 minute, the ratio of methanol is down to 20% by the ratio that 55% rises to 80%, 0.1-0.5% phosphate aqueous solution by 45%.
Under these conditions, the chromatographic peak retention time of salvianolic acid A, salvianolic acid B reference substance and Radix Salviae Miltiorrhizae extract, salvianolic acid catalyzed conversion liquid is in table 1, and HPLC collection of illustrative plates is shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4.
The chromatographic peak retention time result of each reference substance of table 1.
4, the investigation of linear relationship
The above-mentioned mixing reference substance solution 0.1ml of accurate absorption, 0.2ml, 0.5ml, 1ml, 2ml, 5ml, put respectively in 10ml measuring bottle, add methanol dilution and become following concentration to be about: the series standard solution of 0.001mg/ml, 0.002mg/ml, 0.005mg/ml, 0.01mg/ml, 0.02mg/ml, 0.05mg/ml.The each 10 μ l injection liquid chromatographies of the above-mentioned standard solution of accurate absorption, peak area is calculated by chromatographic condition under " 3. chromatographic condition and system suitability " item, respectively with integrating peak areas value for vertical coordinate, each concentrations control product sample size is abscissa, drawing standard curve.Result shows that mixing reference substance solution becomes good linear relationship, in table 2 in following scope.
Table 2. mixes reference substance solution linear relationship result
5. precision test
Get mixing reference substance solution, measure according to chromatographic condition under " 3. chromatographic condition and system suitability " item, repeat sample introduction 6 times.Result shows that the precision of the method is good, in table 3.
Table 3. Precision test result
6. stability test
Get need testing solution, measure according to chromatographic condition under " 3. chromatographic condition and system suitability " item, sample introduction is once at regular intervals, in result need testing solution each composition in 24 hours peak area without significant change, show having good stability of need testing solution, in table 4.
Table 4. stability test result
7. replica test
Get this salvianolic acid A raw material, add methanol and make need testing solution 6 parts, measure according to chromatographic condition under " 3. chromatographic condition and system suitability " item.Result shows that the method repeatability is good, in table 5.
Table 5. replica test result
8. recovery test
Adopt application of sample recovery test, get the salvianolic acid A raw material 10mg of known content, accurately weighed, parallel 6 parts, a certain amount of reference substance solution is added respectively in the ratio of each constituent content-reference substance (1: 1), by 2.3 below legal system available test sample solutions, measure and calculate average recovery and the RSD of each composition.Result shows that the accuracy of the method is good, in table 6.
Table 6 recovery test result
Experimental example 2: the extraction of salvianolic acid B
The confirmation of 1.1 Extraction solvent and extracting method
Take red rooted salvia 400g, measure content of danshinolic acid B by method under Chinese Pharmacopoeia Radix Salviae Miltiorrhizae item, be divided into 4 parts, test by following testing program:
Experiment 1: get red rooted salvia 100g, add 8 times of water gagings at every turn and extract 1.5 hours at 80 DEG C of warm macerating, warm macerating extracts three times, merge extractive liquid, altogether, and measure salvianolic acid B and calculate extraction ratio, result is as table 7.
Experiment 2: get red rooted salvia 100g, add 8 times of water gagings at every turn and extract 1.5 hours at 80 DEG C of warm macerating, stirs with 10 ~ 50 revs/min of speed simultaneously, and warm macerating stirs extraction three times altogether, merge extractive liquid, and measure salvianolic acid B and calculate extraction ratio, result is as table 7.
Experiment 3: get red rooted salvia 100g, add 8 times amount soak by water 1.5 hours at every turn, decocts three times, merge extractive liquid, altogether, and measure salvianolic acid B and calculate extraction ratio, result is as table 7.
Experiment 4: get red rooted salvia 100g, add 8 times amount 50% alcohol reflux 1.5 hours at every turn, extracts three times, merge extractive liquid, altogether, and measure salvianolic acid B and calculate extraction ratio, result is as table 7.
Table 7. Extraction solvent and extracting method experimental result
Above-mentioned experimental result display, adopt 50% ethanol to do Extraction solvent and have impact to salvianolic acid B extraction ratio, 50% ethanol extraction is better than water extraction, but soak to add with water temperature and stir that to extract difference little, the extraction ratio of two kinds of extracting modes on salvianolic acid B stirred in water extraction process and do not stir has impact, decocting extraction may be because temperature is higher, and extracting salvianolic acid B has impact.
1.2 orthogonal experiment Optimized extraction techniques
1.2.1 the optimizing research of water extraction process: according to above result of the test, water extraction process is using extraction time (A), extraction time (B), quantity of solvent (C), Extracting temperature (D) four as investigation factor, each factor establishes three levels, by L9 (3 4) orthogonal table carries out Orthogonal Experiment and Design (table 8, table 9), inspection target is the extraction ratio of salvianolic acid B.
Table 8. extraction factor water-glass
Table 9. extraction process orthogonal experiments table
Table 10. the results of analysis of variance
F 0.05(2，2)＝19.00F 0.01(2，2)＝99.00
Water extraction the results of analysis of variance shows, each experimental factor is to the extraction rate of transform of salvianolic acid B there are no significant difference, therefore the water extraction condition of salvianolic acid B of the present invention is for adding 3 ~ 15 times of water gagings, at 45 ~ 95 DEG C, and warm macerating extracts 1 ~ 3 time, stir with 10 ~ 50 revs/min of speed simultaneously, each extraction 1 ~ 4 hour or add at every turn 3 ~ 15 times amount decoct extract, each extraction 1 ~ 4 hour, extracts 1 ~ 3 time altogether.
1.2.2 the optimizing research of alcohol extraction technique: according to above result of the test, alcohol extraction technique is using extraction time (A), extraction time (B), quantity of solvent (C), concentration of alcohol (D) four as investigation factor, each factor establishes three levels, by L9 (3 4) orthogonal table carries out Orthogonal Experiment and Design (table 11, table 12), inspection target is the extraction ratio of salvianolic acid B.
Table 11. extraction factor water-glass
Table 12. extraction process orthogonal experiments
Table 13. the results of analysis of variance
F 0.05(2，2)＝19.00F 0.01(2，2)＝99.00
Alcohol reflux extracts the results of analysis of variance and shows, each experimental factor is to the extraction rate of transform of salvianolic acid B there are no significant difference, therefore the extraction conditions of this salvianolic acid B is for adding 3 ~ 15 times amount 30% ~ 60% alcohol reflux 1 ~ 3 time, extracts 1 ~ 4 hour at every turn.
1.3 salvianolic acid B raw material preparations
According to above-mentioned orthogonal experiment Optimization Technology, get red rooted salvia 10kg, add 8 times of water gagings at every turn and extract 1.5 hours at 80 DEG C of warm macerating, stir with 10 ~ 50 revs/min of speed simultaneously, warm macerating stirs extraction 3 times altogether, filters, merging filtrate, extracting solution is evaporated to relative density 1.10 ~ 1.25 (60 DEG C), adding ethanol makes alcohol content 60%, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, vacuum drying, salvianolic acid extract 4.15kg, recording content of danshinolic acid B is 10.25%.
Experimental example 3: salvianolic acid B transforms salvianolic acid A technics comparing
Experiment 1: the salvianolic acid B raw material getting preparation under 1.3 is about 30g, is mixed with 200ml solution, adds 10% sodium hydroxide solution adjust pH to 4.5;
Experiment 2: the salvianolic acid B raw material getting preparation under 1.3 is about 30g, is mixed with 200ml solution, adds 10% sodium hydroxide solution adjust pH to 4.5, add 1.0%ZnCl 2;
Experiment 3: get and be about 6g containing salvianolic acid B (content 51.54%) raw material, adding purified water, to be diluted to salvianolic acid B concentration be 15mg/ml solution, and add carbamide, making it with the mol ratio of salvianolic acid B is 0.5;
Above-mentioned each experimental group is placed in same autoclave, reacts 4.0 hours at 120 DEG C, and cooling calculates salvianolic acid A productive rate.
Table 14. salvianolic acid B transforms salvianolic acid A experimental result
Table 14 result shows, and salvianolic acid B high-purity is on conversion not impact, and do not need to be purified to more than 50% and transform, salvianolic acid B is converted in salvianolic acid A process, and adjust ph, adds 1.0%ZnCl 2, greatly can improve the productive rate of salvianolic acid A.
Experimental example 4: salvianolic acid B transforms salvianolic acid A process optimization
Above-mentioned experimentation proves, salvianolic acid B purity is not affect the factor that salvianolic acid B transforms salvianolic acid A, but adds certain catalysts influence salvianolic acid B conversion salvianolic acid A, and therefore, we all add 1%ZnCl to each experimental group 2as catalyst, other factors on affecting salvianolic acid B and be converted into salvianolic acid A: salvianolic acid B concentration (A), pH (B), temperature (C), time (D) carry out orthogonal test, each factor establishes three levels, by L9 (3 4) orthogonal table carries out EXPERIMENTAL DESIGN (table 15, table 16), inspection target is the productive rate of salvianolic acid A.
Table 15. transforming agent water-glass
Table 16. conversion process orthogonal experiments
Table 17. the results of analysis of variance table
*F 0.05(2，2)＝19.00ΔF 0.01(2，2)＝99.00
The results of analysis of variance shows, and is A to this orthogonal test according to the optimum condition that intuitive analysis optimizes 3b 2c 2d 2, factor A (salvianolic acid B concentration) has certain influence to salvianolic acid A productive rate, analyzes from K value, concentration does not affect the effect that raising salvianolic acid B is converted into salvianolic acid A higher than 30mg/ml, and the impurity formed is more, therefore, before transforming, salvianolic acid B concentration should select 1 ~ 30mg/ml; Factor B (pH), factor C (temperature) have pole significant difference to salvianolic acid A productive rate, should strictly control pH and temperature in prompting salvianolic acid B conversion process, successfully can realize transforming to salvianolic acid A of salvianolic acid B higher yields.
Experimental example 5: catalyst Z nCl 2consumption is on the impact transformed
Salvianolic acid A process optimization condition is transformed by above-mentioned salvianolic acid B, get salvianolic acid B raw material, add purified water 200ml, make the solution that salvianolic acid B concentration is 15mg/ml, regulate pH to be 4.5, conversion temperature is 120 DEG C, and transformation time is 4 hours, press and salvianolic acid B molar percent, add not commensurability catalyst Z nCl respectively 2, the results are shown in Table 18.
Table 18. catalyst Z nCl 2consumption is to transformations affect experimental result
Catalyst Z nCl 2consumption shows transformations affect experimental result, catalyst amount>=0.02% can produce 55.65% conversion ratio, preferred catalyst consumption 0.1% ~ 3.0%, more preferably after 0.5% ~ 2.0%. catalyst amount > 3%, conversion ratio no longer includes and significantly improves.
Experimental example 6: catalyst transforms the catalytic action of salvianolic acid A to salvianolic acid B
Salvianolic acid A process optimization condition is transformed by above-mentioned salvianolic acid B, get salvianolic acid B raw material, add purified water 200ml, make the solution that salvianolic acid B concentration is 15mg/ml, regulate pH to be 4.5, conversion temperature is 120 DEG C, and transformation time is 4 hours, the catalyst adding different cultivars and various dose respectively transforms, and the results are shown in Table 19.
Table 19. different catalysts transforms the impact of red A to red B
Table 19 result shows, and above 4 kinds of catalyst all can as the catalyst in salvianolic acid B conversion reaction, and each catalyst amount and salvianolic acid B molar percentage are 0.5% ~ 3.0%, productive rate >=40% of salvianolic acid A.Conversion ratio is more than 60%.
Experimental example 7: the purification with macroreticular resin of salvianolic acid A
Get salvianolic acid A transforming solution 13 parts, put in the good each model macroporous resin 100g of pretreatment, shaking table dynamic adsorption is after 8 hours, dress post, wash eluting 3 column volumes, 20% ethanol elution, 5 column volumes, 70% ethanol elution, 5 column volumes successively with water, collect 70% ethanol containing salvianolic acid A eluent part, carry out salvianolic acid A assay, eluent evaporate to dryness calculates dry cream amount, the results are shown in Table 20.
The confirmation of table 20. macroporous resin model
Result shows: above 13 kinds of macroporous resins are good to the adsorption effect of salvianolic acid A, and can well remove impurity with the ethanol gradient elution of variable concentrations, obtain the salvianolic acid A eluent that content is greater than 75%.
Experimental example 8: the second time column chromatography purification of salvianolic acid A
Get the salvianolic acid A solution 3 parts after purification with macroreticular resin, put in the good each model resin 100g of pretreatment, shaking table dynamic adsorption is after 8 hours, dress post, wash eluting 3 column volumes, 20% ethanol elution, 5 column volumes, 70% ethanol elution, 5 column volumes successively with water, collect 70% ethanol elution and carry out salvianolic acid A assay, elution fractions evaporate to dryness calculates dry cream amount, the results are shown in Table 21.
The confirmation of table 21. resin model
Result shows: above 3 kinds of resins are good to the adsorption effect of salvianolic acid A, and can well remove impurity with the ethanol gradient elution of variable concentrations, obtain the salvianolic acid A eluent that content is about 90%.
Experimental example 9: the abstraction purification of salvianolic acid A
By 70% ethanol elution decompression recycling ethanol after second time column chromatography purification, adjust pH 2.0 ~ 4.0, use n-butyl alcohol, t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate, Ethyl formate, ethanol extraction 5 times again, Separation of Organic phase, recycling design, dry, obtain salvianolic acid A, measure salvianolic acid A content, the results are shown in Table 22.
The confirmation of table 22. extraction organic solvent
Table 22 result shows: n-butyl alcohol due to polarity large, salvianolic acid A amount is maximum, but its salvianolic acid A content is not improved significantly, ether due to polarity less than normal, water-solubility impurity is few, and salvianolic acid A content is high, but the salvianolic acid A amount obtained 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 comparatively large, and salvianolic acid A content is all improved.
Experimental example 10: the purification by silica gel column chromatography of salvianolic acid A
Get the salvianolic acid A extract 12 parts after abstraction purification, every part, containing salvianolic acid A 10g, adds the silica gel of 20g, stirs, volatilizes; Stirring sample silica gel is added on the dry silicagel column of the 100g installed, respectively with petroleum ether, pentane, normal heptane, ethyl acetate, methyl acetate, Ethyl formate, t-butyl methyl ether composition two-phase solvent for eluant, HPLC or thin layer chromatography detect, collect salvianolic acid A eluent, eluent evaporate to dryness, get dry extract, measure salvianolic acid A content, the results are shown in Table 23.
The confirmation of table 23. eluant
Result shows: during phenolic acid A normal phase silica gel column chromatography, the two-phase solvent adopting petroleum ether, pentane, normal heptane, ethyl acetate, methyl acetate, Ethyl formate, t-butyl methyl ether composition is eluant, gradient elution, can well impurity be removed, obtain highly purified salvianolic acid A eluent.
Experimental example 11: salvianolic acid A drying means is studied
Get the eluent 4 parts after silica gel purification eluting, every part, containing salvianolic acid A 100g, is concentrated into without paste, adopts vacuum drying, lyophilisation, spraying dry, microwave vacuum drying to obtain salvianolic acid A respectively after adding 10 times amount water dissolutioies, this salvianolic acid A is detected, the results are shown in Table 24.
Table 24. salvianolic acid A drying means testing result
Result shows: adopt lyophilisation overlong time, high cost, and organic solvent residual is serious; Vacuum drying, spraying dry microwave vacuum drying process control simple, and treating capacity is large, and baking temperature is low, and the time is short, and gained salvianolic acid A indices is good, therefore the present invention adopts vacuum drying, spraying dry, microwave vacuum drying to prepare salvianolic acid A.
Determine through further testing, microwave vacuum drying optimum range is chosen as temperature: 20-100 DEG C, return difference temperature 1-5 DEG C, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute;
Vacuum drying optimum range is chosen as temperature: 50 DEG C ~ 90 DEG C, more than vacuum-0.07Mpa, power: 1 ~ 60KW, dry 2 ~ 20 hours;
Spraying dry optimum range is chosen as intake air temperature: 150 DEG C ~ 350 DEG C, air outlet temperature: 70 DEG C ~ 95 DEG C, 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 object: the dissolubility increasing composition in injection, filler need be added as supporter during the molding of powder pin, improve mouldability, regulate powder pin admittedly containing thing weight, regulate osmotic pressure, add antioxidant simultaneously and keep principal agent to stablize.
Get salvianolic acid A raw material by table 25 and make freeze-dried powder from different filler and vitamin C.
Table 25. prescription filler screening table
Get salvianolic acid A by table 25 formula, add the water for injection making total amount 90% and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.5, add adjuvant and make dissolving, mixing, adds active carbon 2g stirring and adsorbing, filters, removing active carbon; Inject water to 3000ml, detect intermediates content, pH value, detect qualified rear use 0.22 μm of microporous filter membrane to filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make 1000 bottles altogether, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-42 DEG C with 28 DEG C/h speed, be incubated freezing 12 hours; Be evacuated to below 0.3mbar, in 11 hours, the salvianolic acid A formulation temperature freezed risen to-25 DEG C, maintain-25 DEG C of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 40 DEG C with 0.8 DEG C/min, maintain 40 DEG C of dryings after 5 hours, sample temperature is down to room temperature, adds a cover, observe mouldability, in table 26.
The different filler of table 26 affects result to mouldability
From table 26 experimental result, add not commensurability filler, larger on the impact of mouldability, filler mannitol, glucose, lactose are better to lyophilized formulations effect, lyophilized powder sedimentation is good, porous nickel, therefore as selecting adjuvant, but lower than 20mg mouldability and solubility general.
Experimental example 13: different filler and antioxidant are on the impact of lyophilized formulations type
According to above-mentioned experimental result, mannitol, glucose, lactose is selected to carry out confirmatory study respectively, and research is compared to antioxidant, select vitamin C, thiourea, sodium sulfite, sodium pyrosulfite as antioxidant respectively, experimental program is in table 27, and with do not add antioxidant prescription and compare, experimental result is in table 28.
The different filler of table 27 and antioxidant Preliminary screening
Get salvianolic acid A by table 27 formula, add the water for injection making total amount 90% and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.5, add filler and antioxidant makes dissolving, mixing, adds active carbon 2g stirring and adsorbing, filters, removing active carbon; Inject water to 3000ml, detect intermediates content, pH value, detect qualified rear use 0.22 μm of microporous filter membrane to filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make 1000 bottles altogether, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-42 DEG C with 25 DEG C/h speed, be incubated freezing 14 hours; Be evacuated to below 0.3mbar, in 10 ~ 14 hours, the salvianolic acid A formulation temperature freezed risen to-25 DEG C, maintain-23 DEG C of vacuum dryings 7 hours; Continue to heat up, be evenly warming up to 44 DEG C with 0.6 DEG C/min, maintain 44 DEG C of dryings after 2 hours, sample temperature is down to room temperature, adds a cover, observe mouldability, in table 28.
The different filler of table 28 and antioxidant affect result to mouldability and the quality of the pharmaceutical preparations
Table 28 experimental result shows, and add not commensurability filler mannitol, glucose, lactose are better to preparations shaping, lyophilized powder sedimentation is good, porous nickel, consumption select 20mg ~ 40mg/2ml ~ 3ml mouldability and solubility all better.Add water-soluble vitamin c, thiourea, sodium sulfite, sodium pyrosulfite to the content of main constituent and other compositions all without impact, add salvianolic acid A stable content after antioxidant, other compositions are unchanged, and preparation effect is more excellent.
Experimental example 14: lyophilisation condition is studied
3.1 lyophilisation conditions are preferred: mainly contain two factors in freezing conditions: the impact on mouldability: the temperature of freezing body before freezing speed, vacuum drying; Impact on freeze drying rate: heating curve during vacuum freeze-drying.
Preferably following on freeze dryer:
A, medicinal liquid are cooled to-40 DEG C ~-45 DEG C with the chilling rate of 20-30 DEG C/h in freeze dryer, be incubated freezing 10 ~ 16 hours, be evacuated to below 0.3mbar, in 10 ~ 14 hours, the salvianolic acid A formulation temperature freezed is risen to-23 DEG C ~-27 DEG C, maintain-23 DEG C ~-27 DEG C vacuum dryings 6 ~ 8 hours;
B, medicinal liquid are first cooled to-25 DEG C with the chilling rate of 20-30 DEG C/h in freeze dryer, be incubated freezing 5 ~ 8 hours, be refrigerated to-40 DEG C ~-45 DEG C again, be incubated freezing 5 ~ 8 hours, be evacuated to below 0.3mbar, in 10 ~ 14 hours, the salvianolic acid A formulation temperature freezed is risen to-23 DEG C ~-27 DEG C, maintain-23 DEG C ~-27 DEG C vacuum dryings 6 ~ 8 hours;
C, medicinal liquid are cooled to-15 DEG C with the chilling rate of 20-30 DEG C/h in freeze dryer, be incubated freezing 5 ~ 8 hours, be refrigerated to-40 DEG C ~-45 DEG C again, be incubated freezing 5 ~ 8 hours, be evacuated to below 0.3mbar, in 10 ~ 14 hours, the salvianolic acid A formulation temperature freezed is risen to-23 DEG C ~-27 DEG C, maintain-23 DEG C ~-27 DEG C vacuum dryings 6 ~ 8 hours;
Above three kinds of result of the tests: 1, medicinal liquid is directly refrigerated to-40 DEG C ~-45 DEG C with 20-30 DEG C/h, powder body is comparatively even, and without crack, drying time is short.2, powder body is uneven, has crack, and 3, freeze-drying time is long, powder body is uneven, has crack.Therefore, be directly down to-40 DEG C ~-45 DEG C with the chilling rate of 20-30 DEG C/h when selecting medicinal liquid freezing, powder body is comparatively even, without crack, can save freeze-drying time.
3.2 pilot scale freeze-drying curves
Freeze-drying curve refers to the relation curve of product temperature or shelf temperature time to time change in freeze-drying process.The number of the shape of freeze-drying curve and the performance of product, loading amount, the many factors such as kind and appointed condition of separation container are relevant.The freeze dryer that we select when scale up test is 4000 bottles of scales, substantially meets large production requirement.Namely-40 DEG C ~-45 DEG C were cooled the temperature at 1 ~ 4 hour, keep 10 ~ 16 hours at such a temperature, at 10 ~ 14 hours temperature risen to-23 DEG C ~-27 DEG C again and keep 6 ~ 8 hours, then at 6 ~ 9 hours temperature risen to 40 DEG C ~ 45 DEG C and keep 2 ~ 5 hours, freeze-drying curve as shown in Figure 5 and Figure 6.
Experimental example 15: preparation stabilization Journal of Sex Research
Example 10,11,12 sample, by the requirement of stability of drug products experimentation relevant technologies, by above three sample lots 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
Table 29 stability experiment observed result shows, and lyophilized injectable powder at ambient temperature salvianolic acid A is stablized, and other compositions are also without significant change, and formulation aesthetics character is good, and preparation solubility is good, and pH value is stablized, and solution is clarified.
The sample that the equal Example 14 of following experiment salvianolic acid A freeze-dried powder obtains.
Experimental example 16: the impact of function of nervous system's symptom after salvianolic acid A freeze-dried powder commute apoplectic type renovascular hypertension in rats (RHRSP) cerebral thrombosis
1, experiment material:
Main agents and instrument: tiger red (rosebengal): the true Bioisystech Co., Ltd in Shanghai; TTC: Sigma Co., USA; SDP-1 type rat heart rate sphygomanometer: China-Japan Friendship Hospital develops; YAG laser device: Wuhan Chinese workers' laser engineering company limited; Japan OlympusBH-5 microscope; Image procossing: JVCky-F3OB3-CCD coloured image shoots with video-corder input instrument; Graphical analysis: German KONTRONIBAS2.0 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 2 ~ 3 monthly ages, the male SD rat of body weight (100 ± 20) g sets up RHRSP model with two narrow renal artery of kidney double fastener method: rat abdominal cavity is anaesthetized, the long otch of 5cm is about through the stringer of xiphoid-process Ventral Midline, cut skin, abdominal muscle, blunt separation bilateral renal arteries, clamps bilateral renal arteries initial part respectively with the annular silver brain clip that internal diameter is 0.3mm.In operation consent measuring blood pressure once, Post operation is surveyed weekly once, continuous 12 weeks.Separately get the SD rat that 10 body weight are suitable, survey its blood pressure as normal control.Reject: in operation and postoperative death, 12 weeks after operation blood pressure, lower than 160mmHg rat, has apoplexy symptom and sign, a rat that the softening stove that left basal ganglia region is little and subarachnoid hemorrhage are shown in dissection.Blood pressure >=160mmHg after 16 weeks and RHRSP without apoplexy symptom is successful model.Middle cerebral artery occlusion (MCAO) model is caused to cause middle cerebral artery (MCA) thrombosis the RHRSP photochemical method be successfully prepared: 2% pentobarbital sodium is by after 40mg/kg body weight intraperitoneal injection of anesthesia, through left temporo side approach, at zygomatic arch and 1mm place, front lower place, aquamous part of temporal bone junction, bore by dental burr the bone window that a diameter is 5mm, can know through cerebral dura mater and see left MCA trunk and branch thereof.Dosage injection 2.5% tiger of pressing 40mg/kg body weight through femoral vein is red, with green laser beam (the spot diameter 2mm of the 532nh of YAG laser device generation after 5min, intensity of illumination 0.37W) prolonged exposure MCA trunk 15min, this MCA far-end visible and each branch blood flow ceases, and under operating microscope, see that local white thrombus points out the success of this MCA thrombosis, MCAO model copy.In art, rat anus temperature remains on (37 ± 0.3) DEG C.Local wound is rinsed with penicillin diluent before sewing up, and postoperative conventional intramuscular injection penicillin 80,000 U/ only.Reject in art and postoperative 1d death and rat that under hemorrhage many or operating microscope, whether not clear and definite blood supply is interrupted.
2.1.2 grouping and administration
First divide 6 groups at random by animal: the high, medium and low dosage group of sham operated rats, model control group, salvianolic acid A freeze-dried powder, nimodipine group.Sham operated rats only opens seam window, does not make laser and irradiates; Model group, salvianolic acid A freeze-dried powder high, medium and low dosage group prepare easy apoplectic type renovascular hypertension in rats MCAO model by model preparation method.And be administered once by 0.3ml/100g tail vein injection respectively in the postoperative 30min of MCAO model, be after this administered once every day, continuous three days altogether.The postoperative tail vein injection saline of sham operated rats, model control group; Salvianolic acid A freeze-dried powder high, medium and low dosage group postoperative tail vein injection salvianolic acid A respectively freeze-dried powder 20mg/kg, 10mg/kg, 5mg/kg, nimodipine group postoperative tail vein injection nimodipine 10mg/kg.
2.2 result
Mark according to 18 points of point systems of Garcia etc.: the symmetry of spontaneous activity, quadruped locomotion, forelimb extensions creep action symmetry, climb cage wall, push away trunk reaction, antenna to stimulate reaction.In first three items, nothing activity or reaction (extremity or suffering limb) are 0 point, and a little activity is 1 point, and reaction calibration constant is 2 points, and activity is normally 3 points; Rear three reactionless be 1 point, activity calibration constant is 2 points, and activity is normally 3 points.Total score is the highest 18 points, minimum 3 points.Mark once after the postoperative rat anesthesia of MCAO is revived, at MCAO postoperative (namely after ischemia) 24h, 48h, 72h, a Neurological deficits (at every turn all marking after administration on the same day) is respectively carried out to rat respectively afterwards.Blind state is carried out to each experimental group rat by 5 people being unfamiliar with this experiment grouping simultaneously and observe scoring, finally average.And observe rat body state simultaneously, measure body weight and blood pressure.
Table 30. salvianolic acid A freeze-dried powder is on the impact (x ± s) of Neurological deficits after RHRSP cerebral thrombosis
Note: compare with sham operated rats, #P < 0.01, ※ P < 0.05; Compare with model group, * P < 0.01, ☆ P < 0.05
Experimental result shows: after model group is revived, the Neurological deficits of (in 12h) is significantly lower than sham operated rats (P < 0.01), after cerebral thrombosis ischemia is described, RHRSP function of nervous system is seriously impaired, and in the 72h continued, Neurological deficits continues to be starkly lower than sham operated rats (P < 0.05); Nimodipine group and each dosage group of salvianolic acid A freeze-dried powder are compared with model group, Neurological deficits 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 obvious (P < 0.01), and after its ischemia, the Neurological deficits of 24h is basic close to sham operated rats level; To ischemia, 72h salvianolic acid A freeze-dried powder each dosage group rat neurological deficit score has recovered normal, and difference still has statistical significance (P < 0.05) compared with model group.The prompting of above result, salvianolic acid A freeze-dried powder can improve Neurological deficits after RHRSP cerebral thrombosis ischemia, and prompting salvianolic acid A freeze-dried powder has protection and the effect of nervous symptoms after improving cerebral ischemia.
Experimental example 17: 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 Neurological deficits terminates the last time, rat broken end gets brain, and under operating microscope, observe MCA local thrombus situation.Put into afterwards smooth vessel be built in-20 DEG C freezing, row coronal section.Mus brain section, the thick about 2mm of every sheet.Brain sheet is put into rapidly 2%TTC solution, hatches at 37 DEG C, 15min is hatched in the every face of brain sheet, and normal cerebral tissue peony, and ischemic infarction cerebral tissue is in white.Take out brain sheet observe and take pictures.Then cerebral tissue is placed in 4% paraformaldehyde PBS buffer (pH7.3) to fix, row dehydration afterwards, paraffin embedding, get cerebral tissue and do paraffin section and do conventional H E staining analysis, basis of microscopic observation, to take 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
Experimental result shows: sham operated rats cerebral tissue Bilateral Symmetry, has no pathological changes, and the attachment of model group Intravascular Thrombus is serious.Compare with model group, the contraction in length of the left MCA local thrombus of Microscopic observation salvianolic acid A freeze-dried powder each dosage group rat, reduce at arterial wall bond area, with high, middle dosage group is the most obvious.TTC dyes visible infarcted cerebral constitution in white, and the cerebral tissue scope being dyed to white in salvianolic acid A freeze-dried powder each dosage group is more remarkable than model control group to be reduced.HE dyes in visible model group left cortical MCA blood supply district (centered by the cortex of volume top) infarct has obvious cerebral tissue to soften, necrocytosis, the phenomenons such as local necrosis tissue loss, salvianolic acid A freeze-dried powder high dose group rat has no Telatrophy's obscission herein, salvianolic acid A freeze-dried powder low, middle dosage group necrocytosis is less, accidental tissue loss phenomenon; The HE dyeing display stove of each dosage group of salvianolic acid A freeze-dried powder and model control group is interior, stove Zhou Jun has thin vessels hypertrophy, but each dosage group of salvianolic acid A freeze-dried powder compares hypertrophy thin vessels number with model group significantly increases; In addition, the stove shape that HE dyeing display model matched group differs in size as seen is hemorrhage, high, the middle dosage group of salvianolic acid A freeze-dried powder there is no and finds that there is stove shape bleeding, and salvianolic acid A freeze-dried powder low dose group also only has individual animal to have stove shape hemorrhage, and hemorrhagic focus is less than model group.Result of the test display salvianolic acid A freeze-dried powder obviously can improve RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia histopathological status; reduce thrombosis bond area, reduce infarct size; increase in infarcted region and the thin vessels number of surrouding brain tissue, reduce cerebral hemorrhage phenomenon thus the necrosis of protection cerebral tissue comes off, there is the effect protecting cerebral thrombosis ischemia to cause brain tissue impairment.
Experimental example 18: salvianolic acid A freeze-dried powder is on the impact of blood brain barrier (BBB) permeability after RHRSP cerebral thrombosis
1 test method
Animal grouping and the preparation of RHRSP Cerebrovascular embolism model are with experimental example 16 method.4.5h after model surgery success, each group is tail vein injection salvianolic acid A freeze-dried powder 40mg/kg, 20mg/kg, 10mg/kg respectively, nimodipine 20mg/kg; The normal saline of sham operated rats and model group tail vein injection same volume.Each group of rat is put to death in MCAO postoperative (namely after cerebral thrombosis ischemia) 6h, 12h, 24h respectively in batches and gets cerebral tissue specimen, and in execution before 1h tail vein injection 2% azovan blue (EvansBlue, EB) normal saline solution 2ml/kg body weight, after fully circulating, dark numb animal, cut open breast through heart perfusion normal saline flushing Ink vessel transfusing dyestuff, broken end gets brain.
The each group of cerebral tissue randomly drawing 3 rats, frozen section immediately, the coronal section of the thick about 30um of consecutive row, with 70% glycerol PBS buffer (pH8.5 ~ 9.0) mounting, that under Bio-RadRadiance2100 laser scanning co-focusing microscope, observes EB in cerebral tissue oozes out situation, observes BBB open area and degree.When not observing, section is put 4 DEG C of refrigerators and is kept in Dark Place.The each group of cerebral tissue separately getting 5 rats at random, weighs cutaneous horn weight after blotting surface moisture; Cerebral tissue is placed in homogenizer, adds 50% trichloroacetic acid and make tissue homogenate, move into more than test tube sealing and standing 60min; The centrifugal 10min of 3000rpm/min, gets supernatant, and spectrofluorophotometer surveys fluorescent value: excitation wavelength 620nm, emission wavelength 680nm; Calculate EB content in supernatant by standard curve, then calculate every Borneo camphor and organize EB content, react each rat BBB permeability with this.
2 experimental results
2.1 laser scanning co-focusing microscope observed results
EB is bright-coloured bright red fluorescence under exciting light state.Observe each treated animal cerebral tissue under laser microscope and present homogeneous, diffusivity EB dip-dye without BBB brain district (as pinus, area postrema and hypophysis cerebri), sharpness of border, the line sample profile that meninges red color visible fluorescence is sketched the contours of.And in BBB brain district, other except sham operated rats respectively organize the equal visible light spot of cerebral tissue, and be mainly distributed in the places such as thalamus, hypothalamus, cerebellum, Hippocampus, spot size differs, and fluorescence intensity weakens gradually from the center of hot spot to periphery, obscurity boundary.The hot spot number of each group and distribution are obviously different: model group hot spot is maximum, and about have the hot spot of about 120 diameter about 100 ~ 200um, intensive place in the form of sheets.Each salvianolic acid A freeze-dried powder injecta medicine group number of spots comparatively model group significantly reduces, and each dosage group compares, and presents remarkable dose variability; Wherein more with low dose group hot spot, on same level, hot spot about has 50, but is fused into the less of lamellar; Middle dosage group hot spot is less, is dispersed in distribution; High dose group hot spot is seldom dispersed in, and fluorescence is very weak.
The permeability result of 2.2 rat cerebral tissue EB content detection blood brain barrier
Ge Zu rat cerebral tissue specimen calculates EB content by the fluorescent value recorded, and data statistic analysis is in table 32.
Table 32. salvianolic acid A freeze-dried powder is on the impact of blood-brain barrier permeability after RHRSP cerebral thrombosis
Note: compare with sham operated rats, #P < 0.01; Compare with model group, * P < 0.01
2.3 conclusion
EB is water-soluble dye, and entering after blood can absolutely rapidly and albumin bound, under normal circumstances can not through blood brain barrier.From 2.1 and 2.2 results, sham operated rats BBB brain district, has no EB under cerebral tissue microscope and oozes out hot spot; And under model group rats cerebral tissue microscope, have a large amount of EB to ooze out hot spot, and cerebral tissue spectrofluorophotometer detects the EB of high-load, comparatively there were significant differences (P < 0.01) for sham operated rats, show cerebral thrombosis tissues following MCAO in rats blood-brain barrier disruption, permeability increases, and EB albumin-binding enters cerebral tissue by open BBB.Under experimental result display salvianolic acid A freeze-dried powder each dosage group rat cerebral tissue microscope, EB hot spot number and cerebral tissue EB detect content and obviously reduce; more all there were significant differences (P < 0.01) with model group; prompting salvianolic acid A freeze-dried powder increases inhibited to blood-brain barrier permeability following brain injury; can blood brain barrier be protected, and protect the further damaged of cerebral tissue.
Experimental example 19: salvianolic acid A freeze-dried powder is on the impact of RHRSP cerebral thrombosis cerebral infarction scope and brain water content
Empirically the method for example 16 prepares RHRSP cerebral thrombosis rat model, grouping, administration; After last administration terminates, rat broken end gets brain.The each group of brain tissue slice getting 5 rats at random, through TTC dyeing, after taking pictures under the microscope centered by infarct, delimit infarct, calculate with image analysis software process the infarct size often opening brain sheet.Every rat all brain sheets infarct size is added, and is brain infarction area, is multiplied by the thickness often opening brain sheet and is about 2mm, be cerebral infarction volume; Get the in kind approximate calculation of corresponding rat all brains sheet simultaneously and go out corresponding brain cumulative volume; The two ratio calculation cerebral infarction volume percentage ratio.Separately get 5 rat cerebral tissues for each group and claim weight in wet base immediately, rearmounted 105 DEG C of baking ovens in dry to constant weight, calculate brain water content.
Table 33. salvianolic acid A freeze-dried powder is on the impact (x ± s) of RHRSP cerebral thrombosis cerebral infarction scope and brain water content
Note: compare with sham operated rats, #P < 0.01; Compare with model group, * P < 0.01
SPSS11.5 statistical package is adopted to carry out statistical procedures to data.Experimental result shows: sham operated rats has no infarcted cerebral constitution; Salvianolic acid A freeze-dried powder each dosage group cerebral tissue Infarction volume and brain water content are significantly less than model control group (P < 0.01), and dosage is larger, Infarction volume is less, brain water content is fewer, and difference has statistical significance (P < 0.01 or p < 0.05).Salvianolic acid A freeze-dried powder basic, normal, high dosage group Infarction volume and brain water content are all lower than nimodipine group.Illustrate that salvianolic acid A freeze-dried powder can accelerate the reparation of focus, ischemic tissue of brain infarction size after reduction RHRSP cerebral thrombosis, alleviates cerebral tissue edema, has the effect of repairing and protecting Neurons Against Cerebral Ischemia tissue injury.
Experimental example 20: salvianolic acid A freeze-dried powder is on the microvascular impact of rat cerebral tissue after RHRSP cerebral thrombosis ischemia
Animal grouping and the preparation of RHRSP Cerebrovascular embolism model are with experimental example 16 method.4.5h tail intravenously administrable after model surgery success, is after this administered once, until it is complete to draw materials every day.The each dosage component of salvianolic acid A freeze-dried powder other tail vein injection salvianolic acid A freeze-dried powder 20mg/kg, 10mg/kg, 5mg/kg, nimodipine group injection nimodipine 10mg/kg; The normal saline of model group and sham operated rats injection same volume.Get rat broken end respectively at MCAO postoperative 6h, 1d, 3d, 7d for each group in batches and get brain, cerebral tissue 4% paraformaldehyde of ischemic focus Penumbra zone district or same area neutral PBS buffer routine is fixing, dehydration, paraffin embedding, section, the thick about 5um of every sheet.SABC method CD31 immunohistochemical staining, by immunohistochemical kit description operation; DAB develops the color.Primary antibodie is replaced to make negative control with PBS.Whether blood vessel is not counted with the appearance of erythrocyte or tube chamber.All dye brown color single endotheliocyte or endotheliocyte bunch all as a vascular counts, all tube chambers are greater than 8 erythrocyte sizes, all do not count with the angiosomes blood vessel of thicker muscle layer.Vascular counts is undertaken by Weidner method.First under low power (× 40) visual field, find high vessel density region, then under high power (× 400) visual field, Microvessel Count is carried out, often open section random selecting 10 high power fields, finally get the Microvascular density angle value (MVD) of its meansigma methods as this specimen.And adopt full automatic colour image processing system to carry out graphical analysis, measure blood vessel field area ratio (Positive Objects area/Statistical Fields gross area).
Biao34.Ge Zu rat cerebral tissue blood vessel scene is long-pending, CD31 immuno positive microvessel density (MVD) value (n=6)
Note: compare with sham operated rats, #P < 0.01; Compare with model group, * P < 0.01, ☆ P < 0.05
Table 34 experimental data shows, and compare with sham operated rats, model group ischemia penumbra MVD and blood vessel scene amass 1d after ischemia to be increased to some extent, but after ischemia, 3d, 7d continue significantly to reduce (P < 0.01); Compare with model group, after anti-ischemic therapy, nimodipine group and each dosage group of salvianolic acid A freeze-dried powder 6h, 1d, 3d, 7d brain tissue ischemia Penumbra zone district MVD and blood vessel scene after ischemia are amassed and are significantly increased to some extent (P < 0.05 or P < 0.01), and more stable in 7d.Compare between salvianolic acid A freeze-dried powder three dosage groups, difference has statistical significance (P < 0.05).Result shows that salvianolic acid A freeze-dried powder can increase MVD and the blood vessel field area ratio of brain tissue ischemia Penumbra zone, and prompting salvianolic acid A freeze-dried powder can promote the effect that Angiogenesis and collateral circulation are set up.
Experimental example 21: salvianolic acid A freeze-dried powder is on rat cerebral tissue's vascular endothelial growth factor expression impact after RHRSP cerebral thrombosis ischemia
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, liquid-solid fixed containing 4% paraformaldehyde of 0.1%DEPC through heart perfusion, gets brain rapidly, makes the thick coronal section of about 2mm in forebrain optic chiasma place.Be placed in 4% paraformaldehyde fixative to spend the night, conventional dehydration, paraffin embedding, is cut into the paraffin section that about 5um is thick continuously, and conventional dewaxing, to water, detects with hybridization in situ the expression that VEGF impels ribonucleic acid (VEGFmRNA).Illustrate by VEGF in situ hybridization test kit (Wuhan Boster Biological Technology Co., Ltd.) and 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
Note: compare with sham operated rats, #P < 0.05; Compare with model group, #P < 0.01, ☆ P < 0.05
VEGF (VEGF), is again blood vessel opsonin, has the effect of short endothelial cell division, can promote the growth of blood vessel and the foundation of Doppler flow mapping.Experimental result shows; model group VEGFmRNA expression comparatively sham operated rats is increased significantly (P < 0.05); after brain tissue ischemia anoxia is described, in energy Stimulation of The Brain tissue, the expression of VEGF increases; after prompting cerebral infarction, body self there will be a kind of protective response resisting ischemia injury; the expression of VEGF is increased, occurs after ischemia self " compensatory revascularization ".The each dosage group of salvianolic acid A freeze-dried powder compares with model group group; VEGFmRNA expression significantly raises (P < 0.05 or P < 0.01); prompting salvianolic acid A freeze-dried powder can significantly promote ischemic tissue of brain angiogenesis; promote the foundation of collateral circulation compensation; save cerebral ischemic penumbra; the brain tissue impairment that protection ischemia causes, and there is certain dose-effect relationship.
Experimental example 22: salvianolic acid A freeze-dried powder is on rat cerebral tissue's basic fibroblast growth factor protein expression impact after RHRSP cerebral thrombosis ischemia
With experimental example 20, terminate rear 2h, 24h, 48h respectively at rat administration, each component criticizes that to get rat heart perfusion liquid-solid fixed containing 4% paraformaldehyde of 0.1%DEPC, gets brain rapidly, row coronal section, paraffin embedding, section.ABC immunohistochemic al technique method detects the expression of cerebral tissue basic fibroblast growth factor (bFGF) albumen.Operate by bFGF protein immunization group detection kit (Wuhan Boster Biological Technology Co., Ltd.) description, basis of microscopic observation, counting dyes the positive cell number of brown color, and all visuals field of 5 infarcts are selected in each section at random, average.Data carry out statistical analysis.
Table 36. salvianolic acid A freeze-dried powder is on rat cerebral tissue bFGF protein expression impact (x ± s) after RHRSP cerebral thrombosis ischemia
Note: compare with sham operated rats, #P < 0.05; * P < 0.01 is compared with model group
BFGF is the strong neurotrophic factor with various biological activity, neuronal apoptosis and necrosis, to multiple detrimental effects such as ischemia resisting, anoxia, toxicity of excitatory amino acid, calcium overload, free radical and nitric oxide (NO) synthesis, slow down in energy neuroprotective unit; And the effect that VEGF promotes angiogenesis in infarcted region can be worked in coordination with.
Experimental result shows; after ischemia occurs; model group rats cerebral tissue bFGF protein expression comparatively sham operated rats raises to some extent; significant difference (P < 0.05) is had to ischemia 24h; but with the prolongation of Ischemia Time, have a declining tendency, after prompting brain tissue ischemia anoxia; body self produces of short duration protection stress, can endogenous bFGF protein expression be organized to increase by Stimulation of The Brain.The each dosage group of nimodipine group, salvianolic acid A freeze-dried powder compares with model group, and the bFGF protein expression of each time period all significantly increases (P < 0.01); Compare between salvianolic acid A freeze-dried powder three dosage groups, difference has statistical significance (P < 0.05); Self each time period of each dosage group compares display, and 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, contributes to the foundation of collateral circulation, save cerebral ischemic penumbra.
Experimental example 23: 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 into 6 groups at random: 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).Reforming Longa method is adopted to prepare rat brain focal cerebral ischemia-reperfusion injury model: male SD rat, pentobarbital sodium intraperitoneal anesthesia.Expose and be separated 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, tie wings arteria palatina, a kerf is cut in the nearly common carotid artery crotch of external carotid artery, head end is scribbled fishing line that smooth paraffin diameter is about 0.3mm slowly to enter cranium direction to internal carotid artery through left side external carotid artery trunk otch and advance, with common carotid artery crotch for labelling, when advancing 18 ~ 20mm to feel light resistance, namely block middle cerebral artery and cause cerebral ischemia.Stay about 1cm in order to Reperfu-sion use outside fishing line, sterilization, skin suture.After continuous ischemia 2h, extract bolt line, realize ischemia-reperfusion injury model.Sham operated rats except not plug wire, the same model group of all the other operating process.Respectively be administered once through tail vein injection when each group 30min and Reperfu-sion start before ischemia, after this respectively inject once every day, continue to and draw materials.Sham operated rats and ischemia-reperfusion injury model matched group give the normal saline of same volume.
After cerebral ischemic reperfusion in rats, 3h, 24h, 48h, 72h respectively carry out a neurological deficits score respectively, and scoring adopts Bederson improved method: carry Mus tail and leave ground about one chi, observe forelimb flexing situation, stretch to ground count 0 point as two forelimb symmetry; As offside forelimb of performing the operation occur wrist flexing meter 1 point, elbow flexing meter 2 points, shoulder inward turning meter 3 points, existing wrist elbow flexing have again shoulder inward turning meter 4 points.Rat is placed on level land, pushes away both shoulders respectively and move to offside, check resistance, as symmetrical in bilateral resistance and strong, be designated as 0 point; Resistance descender during as promoted to operation offside, be divided into gently according to decline degree difference, in, severe, count 1,2,3 point respectively.Rat two forelimb is placed on wire netting, observes the muscular tension of two forelimbs, bilateral muscular strength equity and strong person counts 0 point, operation offside muscular tension decline degree is divided into gently, in, severe, count 1,2,3 point respectively.Rat does not stop, to the side person of turn-taking, to count 1 point.According to above standards of grading, full marks are 11 points, and mark is higher, and illustrate that the behavior disorder degree of rat is more serious, neurologic impairment is more serious, data are in 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
Note: compare with model group, * * P < 0.01, * P < 0.05
Result of the test is known, ischemia-reperfusion injury model group function of nervous system serious defect, last till 72h after filling with again, although significantly alleviate (P < 0.05) compared with behavioristics's obstacle of 3h, 24h after Reperfu-sion, neurologic impairment is comparatively serious (neurological deficits score is still greater than 5) still.Nimodipine group and each dosage group of salvianolic acid A freeze-dried powder are compared with model group, from Reperfu-sion 3h, neurologic impairment symptom alleviates, neural row is learned defect mark and is significantly reduced (P < 0.05 or P < 0.01), to Reperfu-sion 72h, the neurobehavioral of each medicine group rat recovers substantially.Between salvianolic acid A freeze-dried powder each dosage group: low dose group and middle and high dosage group mark to compare statistical significance (P < 0.05); To mark between high dose group with middle dosage group the trend comparing and have reduction, but difference does not have statistical significance (P > 0.05).
Experimental result shows that salvianolic acid A freeze-dried powder can alleviate rats after cerebral ischemic reperfusion neurologic impairment symptom, is conducive to its neurobehavioral recovery, and prompting salvianolic acid A freeze-dried powder has the effect improving nervous symptoms after brain tissue impairment.
Experimental example 24: salvianolic acid A freeze-dried powder is on the impact of rats after cerebral ischemic reperfusion Brain stem injury, cerebral index and brain water content
Experimental example 23 respectively group rat the last time after Neurological deficits, often organize and get 6 broken ends and get brain, be placed in-20 DEG C of refrigerator freezing 10min immediately, after removing olfactory bulb, cerebellum and low brain stem, the cerebral tissue continuous coronal section of row 2mm thickness, immerses brain sheet in 2%TTC solution, 37 DEG C of dyeing 10min, take out, be placed in 4% paraformaldehyde PBS buffer and fix 2h.Normal structure dyes redness, and infarct is in white.By fixing brain sheet by the arrangement of section order, input computer behind two sides before and after brain sheet of taking pictures under microscope, calculate the approximation of front brain volume and Infarction volume according to each slice thickness, show that Infarction volume accounts for the percentage ratio of front brain volume.After each group of other 6 rats broken end gets brain, claim cutaneous horn weight immediately, dry to constant weight in 105 DEG C 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
Note: compare * p < 0.05, * * p < 0.01 with model group
Experimental data shows, the each dosage group of salvianolic acid A freeze-dried powder is compared with ischemia-reperfusion injury model group: cerebral infarction volume reduces (P < 0.01) than significantly, 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 compares no difference of science of statistics with nimodipine group; The middle and high dosage group of salvianolic acid A freeze-dried powder compares with nimodipine group, and its Infarction volume and cerebral index and brain water content all significantly reduce (P < 0.05 or P < 0.01); Show that salvianolic acid A freeze-dried powder can reduce rats after cerebral ischemic reperfusion cerebral tissue infarction size, the cerebral tissue edema degree after cerebral ischemia reperfusion injury can be alleviated.
Experimental example 25: salvianolic acid A freeze-dried powder is on the impact of rats with cerebral ischemia brain tissue ischemia Penumbra zone regional cerebral blood flow (rCBF)
1 test method
Reforming Longa method is adopted to prepare rat brain focal ischemia model: male SD rat, pentobarbital sodium intraperitoneal anesthesia.Expose and be separated 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, tie wings arteria palatina, a kerf is cut in the nearly common carotid artery crotch of external carotid artery, head end is scribbled fishing line that smooth paraffin diameter is about 0.3mm slowly to enter cranium direction to internal carotid artery through left side external carotid artery trunk otch and advance, with common carotid artery crotch for labelling, when advancing 18 ~ 20mm to feel light resistance, namely block middle cerebral artery and cause cerebral ischemia.Stay about 1cm in order to Reperfu-sion use outside fishing line, sterilization, skin suture.Sham operated rats except not plug wire, the same model group of all the other operating process.
Model after success is divided into the high, medium and low dosage group (10mg/kg, 5mg/kg, 2.5mg/kg) of nimodipine group (10mg/kg), salvianolic acid A freeze-dried powder at random.Each treated animal model success after immediately tail vein give relative medicine, sham operated rats and model group give the normal saline of same volume.Laser Doppler Flowmetry is adopted to measure rCBF through cranium.Use cyanoacrylate that the laser Doppler probe that diameter is 0.5mm is close to skull surface to fix, after selecting bregma respectively under stereotaxic instrument, on the right side of 1mm center line, the other 2mm that opens is that other on the right side of 2mm center line after cerebral ischemic penumbra rCBF monitoring point, bregma to open 6mm be central ischemic zone rCBF monitoring point, record blood flow, continues to monitor 2h after ischemia.Blood flow before record administration and after administration.
2 result of the tests
After middle cerebral artery occlusion in rat, central ischemic zone cerebral blood flow drop to normal rCBF before modeling 10% ~ 20% between, cerebral ischemic penumbra rCBF drop to rCBF before modeling 30% ~ 40% between.10min after administration, nimodipine and salvianolic acid each dosage group rat cerebral tissue ischemia penumbra rCBF are compared with rising in various degree all existing before administration, and 30min after administration, each medicine group brain tissue ischemia Penumbra zone district rCBF peaks.Specific experiment data statistical analysis is in 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 brain tissue ischemia Penumbra zone rCBF
Note: compare * P < 0.01 with sham operated rats (baseline value); Compare with before administration, ★ P < 0.01, ☆ p < 0.05
3 interpretations of result
From table 39 data, after operation modeling, model control group and each medicine group cerebral tissue Penumbra zone rCBF, compared with sham operated rats, have pole significant difference (p < 0.01) to represent modeling success.After administration, 10min begins, each medicine group comparatively before self administration cerebral ischemic penumbra rCBF have obvious rising, and with nimodipine and salvianolic acid A freeze-dried powder high dose group particularly significantly (p < 0.01); 30min after administration, each medicine group cerebral ischemic penumbra rCBF rises to the highest, and respectively organizes self rCBF baseline value before administration and compares, and increases 15% ~ 31%; Though after this have a declining tendency, but last till ischemia 2h, each medicine group is comparatively compared before model group and self administration, still has remarkable significant difference (p < 0.01 or p < 0.05); The each dosage group of salvianolic acid A freeze-dried powder is compared, and has good dose-effect relationship; In salvianolic acid A freeze-dried powder, the rCBF of dosage group day part is suitable with nimodipine group.It can thus be appreciated that salvianolic acid A freeze-dried powder has the effect increasing rats with cerebral ischemia brain tissue ischemia Penumbra zone rCBF, thus be conducive to saving the cerebral tissue that cerebral ischemic penumbra is at death's door, play the effect for the treatment of cerebral ischemia.
Experimental example 26: salvianolic acid A freeze-dried powder is on the impact of rats after cerebral ischemic reperfusion brain tissue ischemia Penumbra zone rCBF
In experimental example 25, each group rat is after continuous ischemia 2h observes cerebral ischemic penumbra rCBF, extracts bolt line, realizes ischemia-reperfusion injury model.After model is set up, each group tail intravenously administrable, dosage is with experimental example 20.Still empirically method described in example 20 measures and fills with rear 3h brain tissue ischemia Penumbra zone rCBF again.Respectively organize rat and fill with rear different time sections 10min, 30min, 60min, 90min, 120min, 180min brain tissue ischemia Penumbra zone rCBF again.Data carry out statistical analysis.Concrete data are in table 40.
Table 40. salvianolic acid A freeze-dried powder is on the impact (x ± s, n=10) of rats after cerebral ischemic reperfusion brain tissue ischemia Penumbra zone rCBF
Note: compare with sham operated rats, * P < 0.01; With fill with again before compare, ☆ p < 0.01; Compare with model group, ★ P < 0.01
Experimental result shows, and after rat continuous ischemia 2h recovers filling again, each group rat ischemia Penumbra zone rCBF all has increase in various degree.Model group rats rises to top level at 60,90 minutes cerebral ischemic penumbra rCBF, with fill with again before than there being obvious statistical significance (P < 0.01), but before being also only about ischemia baseline value 50%, after this sharply decline again, fill with again in 3h, rCBF value before ischemia baseline value 37% ~ 50% between, Reperfu-sion remains incomplete in the meantime, still in Low perfusion phenomenon, cerebral tissue can continue impaired.After filling with, nimodipine, salvianolic acid A freeze-dried powder high, medium and low dosage group rat rCBF obviously increase, and each time period rCBF has pole significant difference (P < 0.01) compared with model group again.1h after filling with again, nimodipine and salvianolic acid A freeze-dried powder high dose group rCBF return to close to former rCBF 75%, in salvianolic acid A freeze-dried powder dosage group return to be about former rCBF 69%, salvianolic acid A freeze-dried powder low dose group returns to and is about 61% of former rCBF, in dose dependent; And in Reperfu-sion 3h, each medicine group rCBF maintains in metastable scope.Experimental result is pointed out, salvianolic acid A freeze-dried powder obviously can improve the cerebral blood flow of cerebral ischemic penumbra cerebral tissue after Ischemia and Reperfusion in vivo in Rats, recovers the confession of brain cell blood, thus saves cerebral ischemic penumbra, prevent cerebral tissue from damaging further even dead, have good therapeutical effect to ischemic cerebrovascular.
Experimental example 27: salvianolic acid A freeze-dried powder is on the impact of rats after cerebral ischemic reperfusion Energy Metabolism of Brain Tissue
After the cerebral blood flow of each treated animal of experimental example 26 3h after having measured Reperfu-sion, sacrificed by decapitation, gets in brain to liquid nitrogen freezing; The cerebral tissue of 100mg corresponding site is got after one week, pulverize under freezing state, with perchloric acid homogenate removing protein, low-temperature centrifugation 10min, supernatant KOH neutralizes, vortex oscillation, then low-temperature centrifugation 10min, get supernatant, high effective liquid chromatography for measuring cerebral tissue adenosine triphosphate (ATP), adenosine diphosphate (ADP) (ADP), AMP (AMP), lactic acid (LA), phosphagen (PC) content.
Table 41. salvianolic acid A freeze-dried powder is on the impact (x ± s, μm ol/g, n=10) of rats after cerebral ischemic reperfusion Energy Metabolism of Brain Tissue
Note: compare with sham operated rats, * P < 0.01; Compare with model group, * * P < 0.01, ☆ P < 0.05
ATP, ADP, AMP, LA, PC are human body energy metabolite.As can be seen from table 41 experimental data, comparatively sham operated rats significantly reduces, LA significantly raises (P < 0.01) for model group ATP, ADP, AMP, PC, rat cerebral ischemia tissues following MCAO in rats energy metabolism serious hindrance is described, ATP exhausts fast, brain Power supply significantly reduces, anaerobic metabolism product LA piles up serious, still exists even if implement to fill with rear energy metabolism disorder 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 raises (P < 0.01), LA significantly reduces, result display salvianolic acid A freeze-dried powder can raise the content of cerebral tissue ATP, ADP, AMP, PC, reduce cerebral tissue LA content, significantly can improve the energy metabolism of rat cerebral ischemia and ischemia-reperfusion.Prompting salvianolic acid A freeze-dried powder is by improving the energy metabolism of Neurons Against Cerebral Ischemia tissue, increase the supply of cerebral tissue energy matter, strengthen the survival ability of brain cell, save the brain cell that the cerebral ischemic penumbra after cerebral ischemia is at death's door, prevent cerebral tissue from damaging further even dead, can perform well in treating ischemic cerebrovascular.
Experimental example 28 salvianolic acid A freeze-dried powder is on the impact of rats after cerebral ischemic reperfusion cerebral tissue neuronal apoptosis rate
Rat pattern of ischemia reperfusion, grouping, administration is prepared with experimental example 23 method.After rat cerebral ischemia 2h fills with 24h again, broken end gets brain, fixes with 4% paraformaldehyde, cuts into slices, paraffin embedding, prepares thick 3 μm of thick coronal section; Adopt the dUTP Nick End labelling method (TUNEL method) that last deoxyribonucleic is transferase mediated eventually to detect cerebral tissue neurocyte, strictly press the operation of test kit description, DAB develops the color, and under light microscopic, apoptotic nucleus is brown.Often open section unduplicated 5 high power (40 × 10) visuals field of random selecting, input computer carries out image analysis, detects apoptotic cell number and the normal cell number of the TUNEL positive, calculates neuronal apoptosis rate, results averaged.
Table 42 salvianolic acid A freeze-dried powder is on the impact of rats after cerebral ischemic reperfusion cerebral tissue neuronal apoptosis rate
#P < 0.001 is compared with sham operated rats; Compare with model group, * * P < 0.001, * P < 0.01
Neuronal apoptosis is the principal mode of cerebral ischemia and transient ischemia/reperfusion damage delayed neuronal death, can determine the brain tissue impairment degree that ischemic cerebrovascular is final.Experimental result shows, and after rat cerebral ischemia 2h fills with 24h again, neuronal apoptosis is serious; And after nimodipine and various dose salvianolic acid A freeze-dried powder intervene, compare with model group, in rat cerebral tissue, neuronal apoptosis significantly reduces (P < 0.001 or P < 0.01); Show that salvianolic acid A freeze-dried powder has suppression neuronal apoptosis, suppresses cerebral ischemia to cause the effect of rat cerebral tissue's neuronal death.
Experimental example 29 salvianolic acid A freeze-dried powder is on the impact of rats after cerebral ischemic reperfusion cerebral tissue neurotrophic factor
Rats after cerebral ischemic reperfusion model, grouping also administration is prepared with experimental example 28 method.Through aortic cannulation after Ischemia Reperfusion 24h, normal saline flushing, after 4% paraformaldehyde perfusion, broken end gets brain, cerebral tissue is fixed, dehydration, paraffin embedding, section, thick about 5 μm, Immunohistochemical Method detects neurotrophic factor nerve growth factor (NGF), Brain Derived Neurotrophic Factor (BDNF), neurotrophic factor-3 (NT-3) protein expression in rat cerebral tissue; Replace primary antibodie for negative control with PBS; With the endochylema of cell, after birth, aixs cylinder is brown or brown yellow granule as immuno positive.Light Microscopic observation, Cai Tu also uses image analysis system analysis, measures its average gray value.Average gray is higher, represents that the intensity of positive cell is more weak.
Table 43 salvianolic acid A freeze-dried powder is on the impact of rat cerebral tissue's neurotrophic factor protein expression (gray value)
Compare with sham operated rats, #P < 0.05, compare with model control group, * P < 0.05, △ P < 0.01.
The neurotrophic factor histone matter molecule that to be neure growth required with survival, plays supporting function to the integrity of neure growth, growth and function.NGF, BDNF, NT-3 are parts for neurotrophic factor family, can maintain neuronal survival and promote neural cellular differentiation and inducing axonal growth; Energy neuroprotective unit, promotion neuron reparation and suppression delayed neuronal death, thus to anti-cerebral ischemia, protection cerebral ischemia.Experimental result shows, and after Ischemia Reperfusion, in rat cerebral tissue, comparatively sham operated rats increases to some extent, and after prompting cerebral reperfusion injury, in cerebral tissue, NGF, BDNF express stress protectiveness increase; But NT-3 content obviously reduces in Neurons Against Cerebral Ischemia tissue.Compare with model control group; the each dosage group NGF of salvianolic acid A freeze-dried powder, 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 neural trophic factors NGF, BDNF, NT-3, reaches the effect of neuro-protective.
Experimental example 30 salvianolic acid A freeze-dried powder is on the impact of rats after cerebral ischemic reperfusion inflammatory cytokine
Rats after cerebral ischemic reperfusion model, grouping also administration is prepared with experimental example 28 method.After Ischemia Reperfusion 24h, broken end gets brain rapidly, get ischemia side cerebral tissue and make 10% tissue homogenate, the centrifugal 10min of low temperature 2000r/min, get supernatant, measure the content of each inflammatory cytokine by ELISA kit: 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 with sham operated rats, #P < 0.001; Compare with model group, Δ P < 0.01, * P < 0.05.
Experimental result shows, and after ischemia-reperfusion, in cerebral tissue, inflammatory cytokine IL-1 β, IL-6, IL-8, TNF-α, ICAM-1 content all extremely significantly increases (P < 0.001); The each inflammatory cytokine content of rat cerebral tissue giving salvianolic acid A freeze-dried powder each dosage group comparatively model group obviously reduces.Prompting salvianolic acid A freeze-dried powder can suppress the expression of ischemic injuries brain tissue inflammation cytokine, suppresses the generation of brain tissue inflammation's reaction, suppresses neuron and the cerebral tissue cascade damage of inflammatory reaction mediation.
Experimental example 31: salvianolic acid A freeze-dried powder is to rats after cerebral ischemic reperfusion cerebral tissue Ca 2+, K +, Mg 2+the impact of content
Get experimental example 24 and dry cerebral tissue to constant weight, after weighing, proceed to conical flask, with analytical pure nitric acid and perchloric acid in (200 ± 10) DEG C digestion, add deionized water dissolving residue, proceed to color comparison tube and after standardize solution, survey Ca in cerebral tissue with atomic spectrophotometer 2+, K +, Mg 2+content.Data carry 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 (x ± s) of content
Note: compare with sham operated rats, * P < 0.01; Compare with model group, * P < 0.05, * * P < 0.01
Ca in cell 2+overload is one of important mechanism mediating subsequent brain damage in During Ischemia, is ischemia, anoxia produces the last common pathway of irreversible neuronal damage.Ca 2+overload by exciting a series of enzyme reaction such as activator protein enzyme, activation phospholipase, activation endonuclease as second message,second messenger to produce infringement to cell, cell death inducing, causes acute cell death and Dalayed neuronal necrosis.K +, Mg 2+be Ca 2+antagonist.K +as the requisite cation of neurocyte polarized state, can suppress flow in calcium ion and alleviate calcium overload; Mg 2+multiple enzyme system in body can be activated, the multiple important metabolic activity of cell is participated in cerebral tissue, affect the nerves conduction, ion transport, albumen synthesis and all many-sides of energy metabolism, the spasm of energy alleviating vascular smooth muscle, improve microcirculation, improve hypoxic-ischemic after brain injury, calcium overload in retardance after craniocerebral injury neurocyte.From experimental data, cerebral ischemia re-pouring model group compares cerebral tissue Ca with sham operated rats 2+content extremely significantly increases, K +, Mg 2+content significantly reduces; Compare with model group, nimodipine group and each dosage group of salvianolic acid A freeze-dried powder can significantly reduce Ca in cerebral tissue 2+content, rising K +, Mg 2+content, illustrates that salvianolic acid A freeze-dried powder can suppress Ca 2+interior stream alleviates calcium overload, thus can suppress Ca 2+the neuronal apoptosis of overload induction.
Experimental example 32: 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 administrations and model preparation
Male SD rat (250 ± 20) g, be divided into 6 groups at random, often organize 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 line brush to prepare re-perfusion model (as described in experimental example 23) after middle cerebral artery occlusion in rat 2h, sham operated rats except not plug wire, the same model group of all the other operating process.Respectively be administered once through tail vein injection when each group before ischemia, 30min and Reperfu-sion start, sham operated rats and cerebral ischemia re-pouring model group give the normal saline of same volume.After filling with 2h again, each group get 4 broken ends get brain do histopathology section, HE dye, observe histopathology change.
The mensuration of 1.2 rat cerebral tissue's monoamine neurotransmitters
Each group of rat is after ischemia 2h Reperfu-sion 2h, respectively get 8, quick broken end gets brain, be separated cerebral cortex and striatum, weigh, add n-butyl alcohol homogenate, centrifuging and taking supernatant, with fluorescent spectrophotometer assay 5-hydroxy tryptamine (5-HT), norepinephrine (NE), dopamine (DA) content after carrying.Data carry out statistical analysis, the results are shown in Table 46.
The mensuration of 1.3 rat cerebral tissue's content of amino acids neurotransmitter
Each group of rat is after ischemia 2h Reperfu-sion 2h; respectively get 8; quick broken end gets brain; broken end gets brain; sharp separation ischemia side cerebral cortex in ice bath; weigh; put in homogenizer; homogenate is made with sulfosalicylic acid; centrifugal, get supernatant, dilution; after OPA derivative reaction, detect the content of amino acid neurotransmitter glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), γ-aminobutyric acid (GABA), taurine (Tau) in brain tissue homogenate's supernatant by high performance liquid chromatography.Mobile phase is a certain proportion of potassium phosphate buffer and methanol, tetrahydrofuran solution, and pH is about 6.6, flow velocity 1ml/min.Calculate excitatory toxicity index (EI): EI=[Glu] [Gly]/[GABA].Data carry out statistical analysis, the results are shown in Table 47.
2 experimental results
2.1 histopathology observed results
Sham operated rats structure of neurons, form be normal, without interstitial edema; Ischemia-reperfusion group neuronal cell volume reduces, be out of shape, karyopycnosis, and nervous tissue loosens, and neuron and perivascular space increase, and interstitial cerebral edema is obvious; The each dosage group of salvianolic acid A freeze-dried powder is compared with model group, and cerebral edema obviously alleviates, and neuronal cell morphology obviously improves, and neuron peripheral clearance obviously reduces; Especially the most remarkable with high, the middle dosage group of salvianolic acid A freeze-dried powder, its neuronal cell is more complete, and form is normal.
2.2 salvianolic acid A freeze-dried powders are on the impact of monoamine neurotransmitter in rats after cerebral ischemic reperfusion cerebral tissue
Compared with sham operated rats, cerebral ischemia re-pouring group rat cerebral cortex and striatum NE, DA, 5-HT content all significantly reduce (P < 0.01).Compared with cerebral ischemia re-pouring model group, each dosage group of salvianolic acid A freeze-dried powder and nimodipine group cerebral cortex, striatum NE, DA, 5-HT content obviously raise (P < 0.01 or P < 0.05).
Table 46. salvianolic acid A freeze-dried powder is on the impact (x ± s, ng/mg, n=8) of monoamine neurotransmitter in rats after cerebral ischemic reperfusion cerebral tissue
Note: compare with sham operated rats, * P < 0.01; Compare with model group, * P < 0.05, * * P < 0.01
2.3 salvianolic acid A freeze-dried powders are on the impact of amino acid neurotransmitter in rats after cerebral ischemic reperfusion cerebral tissue
Compared with sham operated rats, in cerebral ischemia re-pouring model group rats cerebral cortex, the content of Glu, Asp, Gly, GABA, Tau all has remarkable increase (P < 0.01 or P < 0.05), compared with model group, Glu in nimodipine group and salvianolic acid A freeze-dried powder each dosage group cerebral tissue, Asp, Gly all significantly reduces (P < 0.01 or P < 0.05), in nimodipine group and salvianolic acid A freeze-dried powder each dosage group cerebral tissue, Tau content significantly raises (P < 0.01 or P < 0.05), nimodipine group and salvianolic acid A freeze-dried powder high, in middle dosage group cerebral tissue, GABA content significantly raises (P < 0.01), the GABA content of salvianolic acid A freeze-dried powder low dose group comparatively model group slightly raises, but difference does not have statistical significance.Cerebral ischemia re-pouring model group EI value comparatively sham operated rats has and extremely significantly increases (P < 0.01), and the EI value of each dosage group of salvianolic acid A freeze-dried powder and nimodipine group comparatively model group obviously reduce (P < 0.01 or P < 0.05).
Table 47. salvianolic acid A freeze-dried powder is on the impact (x ± s, n=8) of amino acid neurotransmitter in rats after cerebral ischemic reperfusion cerebral tissue
Note: compare with sham operated rats, * P < 0.01, ※ P < 0.05; Compare with model group, * P < 0.05, * * P < 0.01
3 conclusions
Monoamine neurotransmitter disorder and toxicity of excitatory amino acid play an important role in ischemic brain injury, acute cerebral ischemia cell death, reperfusion injury and delayed neuronal death.During cerebral ischemia, monoamine neurotransmitters significantly raises in extracellular fluid, and reduces in brain essence.In brain tissue ischemia equivalent damage situation, monoamine neurotransmitter generation metabolism disorder in brain, NE, DA and 5-HT content obviously reduces, and cerebral ischemia is heavier, and cerebral tissue NE, DA, 5-HT content is lower.Glu and Asp is the important excitatory neurotransmitter of brain, GABA and Gly is the important inhibitory neurotransmitter of brain, and Tau, as a kind of neuromodulator, has protective effect in cerebral ischemia.During cerebral ischemia in brain amino acid neurotransmitters excited-to suppress unbalance be one of key factor causing cerebral ischemia.This experimental result shows that salvianolic acid A freeze-dried powder can significantly improve the cerebral cortex of cerebral ischemia-reperfusion injury in rats and the content of striatum monoamine transmitters, raises GABA, Tau content in cerebral ischemia/reperfusion injury of rats cerebral tissue, the content significantly reducing Glu, Asp, Gly in cerebral tissue and aminoacid exitotoxicity index.And dosage increases, effect strengthens.Salvianolic acid A freeze-dried powder in conjunction with the display of histopathology pathological examination obviously can alleviate cerebral edema, improves the result of neuronal cell morphology, shows that salvianolic acid A freeze-dried powder can suppress monoamine neurotransmitter excessively to discharge, improve monoamine neurotransmitter disorder; Suppress the accumulation of excitatory amino acid in cerebral ischemia re-pouring, stablize the balance of excitatory amino acid-inhibitory aminoacid mediator, alleviate toxicity of excitatory amino acid; Thus suppress the neuronal apoptosis of monoamine neurotransmitter disorder and toxicity of excitatory amino acid induction.
Experimental example 33: salvianolic acid A freeze-dried powder is on the impact of LPO, SOD, GSH-Px in rats after cerebral ischemic reperfusion cerebral tissue
Each group is detected rear 10 remaining rats under experimental example 32, broken end gets brain, weigh, brain tissue homogenate's liquid of 10% is made with 4 DEG C of normal saline, centrifugal, remove supernatant, measure the activity of lipid peroxide contents (LPO), superoxide dismutase (SOD) and glutathion peroxidase (GSH-Px).Result 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 rats after cerebral ischemic reperfusion cerebral tissue
Note: compare with sham operated rats, * P < 0.01; Compare with model group, * P < 0.05, * * P < 0.01
In cerebral ischemia re-pouring model group rats cerebral tissue LPO content comparatively sham operated rats significantly raise, SOD and GSH-Px activity comparatively sham operated rats significantly reduces (P < 0.01).When Cerebral ischemia and reperfusion is described, brain tissue oxygen free radical sharply increases, the integrity of infringement membrane structure and function, causes lipid peroxidation, produces a large amount of lipid peroxide; And free radical scavenging enzymatic activity significantly lowers; Therefore the dynamic equilibrium heavy damage of free radical generation and removing.Free radical toxicity chain reaction is understood accelerator nerve unit apoptosis, is increased the weight of cerebral ischemia.Experimental result shows, and each dosage group of salvianolic acid A freeze-dried powder is compared with model group, and in rat cerebral tissue, LPO content significantly reduces, SOD and GSH-Px is active significantly raises (P < 0.01 or P < 0.05); Illustrate salvianolic acid A freeze-dried powder can increase peroxide scavenger enzyme in ischemical reperfusion injury cerebral tissue activity, suppress the generation of peroxide, thus to the damage of antioxidant radical to neuronal cell and cerebral tissue.
Experimental example 34: salvianolic acid A freeze-dried powder is protected RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia vascular endothelial cell
RHRSP Cerebrovascular embolism model, grouping is prepared with experimental example 16 method.Prepare Post operation immediately by the administration volume tail vein injection administration of 0.3ml/100g at model for each group, after this every day tail intravenously administrable once, continue to and draw materials.The postoperative tail vein injection saline of sham operated rats, model control group; Salvianolic acid A freeze-dried powder high, medium and low dosage group postoperative tail vein injection salvianolic acid A respectively freeze-dried powder 40mg/kg, 20mg/kg, 10mg/kg; Nimodipine group injection nimodipine 20mg/kg.Get rat through heart perfusion 4% paraformaldehyde neutral buffer respectively at postoperative 6h, 12h, 24h, 48h, 72h time point of MCAO, get brain for each group.Cerebral tissue routine is fixing, dehydration paraffin embedding, is cut into the thick coronal section of 5um continuously.TUNEL method (the dUTP Nick End labelling method of deoxyribose nucleotides terminal transferase mediation) detects vascular endothelial cell.By the operation of TUNEL cell apoptosis detection kit (Wuhan doctor's moral biological reagent company) description, DAB develops the color, observed result under optical microscope.Nucleus occurs brown yellow granule, and person is positive apoptosis endotheliocyte.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 carry out statistical analysis process.
Table 49. salvianolic acid A freeze-dried powder is on the impact (x ± s) of RHRSP cerebral thrombosis Neurons Against Cerebral Ischemia apoptosis of vascular endothelial cell
Note: compare with sham operated rats, #P < 0.01; Compare with model group, * P < 0.01
Experimental result shows, the cerebrovascular endothelial cell apoptosis number of model group each time period is significantly higher than sham operated rats (P < 0.01), 6h after brain tissue ischemia, endothelial cell apoptosis just showed increased, reaches peak in 24 hours to ischemia.Compare with model group, the apoptosis digital display work of each time period of salvianolic acid A freeze-dried powder each dosage group reduces (P < 0.01), prompting salvianolic acid A freeze-dried powder can suppress cerebral ischemia hindbrain apoptosis of vascular endothelial cell, improve cerebrovascular endothelial cell survival rate, thus ensure the normal of cerebrovascular endothelial cell function, maintain the complete of cerebral vessel structures and function after ischemic injuries and the ability that strengthens anti-cerebral ischemia damnification, play the effect for the treatment of ischemic cerebrovascular.
Experimental example 35: salvianolic acid A freeze-dried powder is on the impact of anoxia and Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC) survival rate
Male Body Weight is about the SD rat of 100g, after broken end, gets brain after alcohol disinfecting, peel off pia mater encephali and macroscopic trunk, remove cerebral white matter and cerebellum, thereafter, shred cerebral tissue, homogenate, cross 90,180 mesh filter screens successively and filter, rinse and collect the blood capillary section on filter screen, adding 0.1%II collagenase, 37 DEG C of digestion 15min, the centrifugal 5min of 1500r/min, repeat 3 times, precipitation culture fluid is inoculated in after suspending in culture bottle and continues to cultivate, and changes liquid 1 time every 2d.Treat that cell grows up to Fusion Strain, through Morphological Identification and the qualification of VIII factor SABC, be defined as Brain Microvascular Endothelial (BMVEC), purity is greater than 98%.Carry out Secondary Culture.Get third generation BMVEC, after digestion, make single cell suspension, be inoculated in 96 orifice plates.Experiment divides 8 groups: model group, salvianolic acid A freeze-dried powder 6 dosage groups (final concentration is respectively 10,20,30,40,50,60 μm of ol/L) and nimodipine group (final concentration is 30 μm of ol/L).After cell inoculation, second day, culture fluid is changed into PBS liquid, be positioned over (37 DEG C, 95%N2,5%CO2) in anoxic tank and act on 4h, cause BMVEC anoxia-induced apoptosis (simulated ischemia); Change Nostoc commune Vanch liquid again into and normally cultivate 12h, cause BMVEC Hypoxia/Reoxygenation Injury (simulated ischemia-fill with again).Nimodipine group and each dosage group of salvianolic acid A freeze-dried powder all add the medicine of respective concentration respectively before modeling when 4h, anoxia and reoxygenation.Detect the survival rate of each group of BMVEC respectively with mtt assay after anoxia-induced apoptosis 4h, anoxia 4h-reoxygenation 12h damage.
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
Compare with model control group, #P < 0.05, * P < 0.01; Compare with before reoxygenation, ☆ P < 0.01; Compare with nimodipine group, △ P < 0.05,
Experimental result shows, and after anoxia 4h damage, BMVEC survival rate is only about 25.45%, has occurred cell injury after anoxia is described; The survival rate of nimodipine group 30 μm of ol/L and salvianolic acid A freeze-dried powder 10,20,30 μm of ol/L processed group compares the trend having rising with model group, but does not show significant difference; After salvianolic acid A freeze-dried powder 40,50,60 μm of ol/L dosage group anoxia-induced apoptosis 4h, BMVEC survival rate is significantly higher than model control group (P < 0.05).After Hypoxia/Reoxygenation Injury, BMVEC survival rate is only about 13.22%, compares with before reoxygenation, significantly reduces (p < 0.01), illustrates that hypoxia/reoxygenation exacerbates cell injury; After salvianolic acid A freeze-dried powder and nimodipine effect, BMVEC survival rate significantly raises (P < 0.01), with salvianolic acid A freeze-dried powder 30,40,50,60 μm of ol/L dosage group best results; And salvianolic acid A freeze-dried powder 20,30,40,50,60 μm of ol/L dosage group survival rates are significantly higher than nimodipine 30 μm of ol/L groups (P < 0.05, or P < 0.01).Prompting salvianolic acid A freeze-dried powder can protect 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 powder is on the impact of Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC) apoptosis
The empirically method of 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 μm of ol/L), nimodipine group (final concentration 40 μm of ol/L).Modeling and medication are with experimental example 35.Normal group does not carry out hypoxia/reoxygenation process, cultivates corresponding experimental period by normal method.The each porocyte of trypsinization, centrifugal collecting cell, adopts phosphatidyl associated proteins-Fluorescein isothiocyanate/propidium iodide (AnnexinV-FITC/PI) dyeing, the apoptosis rate in and late period early stage with cells were tested by flow cytometry cell.
Table 51 salvianolic acid A freeze-dried powder causes the impact (x ± s, n=6) of BMVEC apoptosis to Hypoxia/Reoxygenation Injury
Compare with Normal group, #P < 0.01; Compare with model group, * P < 0.01, * * P < 0.001; Compare with nimodipine group, Δ P < 0.01,
Apoptosis especially plays an important role at ischemia injury in transient ischemia/reperfusion damage.The apoptosis of vascular endothelial cell can cause the broken words of vascular integrity, and angiolysis damage is the basis of cerebrovascular; The apoptosis of brain microvessel endothelial cells in vitro can affect the integrity of blood brain barrier and the secretory function of vascular endothelial cell, and then increases the weight of ischemic brain injury.Shown by experimental result, Hypoxia/Reoxygenation Injury, each phase apoptosis rate of BMVEC can be caused significantly to increase.Compare with model group, 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 in certain dose-effect relationship.And salvianolic acid A freeze-dried powder 10 μm of ol/L dosage groups are suitable with nimodipine 40 μm of ol/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 powder affects 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 detects 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)
Compare with Normal group, #P < 0.01, compare with model group, * P < 0.01, △ P < 0.05, * * P < 0.001
After Hypoxia/Reoxygenation Injury, compare with normal group, BMVEC secretes t-PA and NO and obviously reduces, and ET raises, t-PA/PAI and NO/ET ratio also significantly declines.T-PA and the NO secretory volume of each dosage group of salvianolic acid A freeze-dried powder and nimodipine group comparatively model group significantly raises (P < 0.01 or P < 0.001), and wherein the middle and high dosage group of salvianolic acid A freeze-dried powder rises to close to levels found in normal controls; Each administration group t-PA/PAI and NO/ET ratio ratio also comparatively model group significantly improve.Prompting salvianolic acid A freeze-dried powder can improve the secretory function of brain microvessel endothelial cells in vitro after Hypoxia/Reoxygenation Injury.
Experimental example 38 salvianolic acid A freeze-dried powder is to Hypoxia/Reoxygenation Injury Brain Microvascular Endothelial (BMVEC) interior Ca 2+the impact of concentration
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.The each porocyte of trypsinization, after centrifugal collecting cell, add Fluo-3 (a kind of calcium fluorescent probe, final concentration is 5 μm of ol/L), hatch 45min for 37 DEG C, PBS liquid rinses 3 times, uses cells were tested by flow cytometry fluorescence intensity.Because almost there is no fluorescence when Fluo-3 exists with free ligand form, and and Ca 2+in conjunction with rear Fluorescence Increasing, Cytoplasmic Ca can be detected according to fluorescence intensity change 2+concentration change.
Table 53 salvianolic acid A freeze-dried powder is to Ca in Hypoxia/Reoxygenation Injury BMVEC 2+the impact (x ± s, n=6) of concentration
Compare with Normal group, #P < 0.01; Compare with model group, * P < 0.01; Compare with nimodipine group,
Experimental result shows, after Hypoxia/Reoxygenation Injury, and Ca in BMVEC born of the same parents 2+concentration significantly increases (P < 0.01).Compare with model group, each medicine group Ca 2+concentration extremely significantly reduces, the most remarkable with salvianolic acid A freeze-dried powder middle and high dosage group effect, and intracellular calcium concentration is significantly lower than nimodipine 40 μm of ol/L dosage groups (P < 0.05).Prompting salvianolic acid A freeze-dried powder has suppression Hypoxia/Reoxygenation Injury and causes Ca in BMVEC born of the same parents 2+the effect of concentration.
Experimental example 39: salvianolic acid A freeze-dried powder is on the impact of rat artery thrombus formation time
Get SD rat, male and female half and half, body weight is 250 ~ 300g, is divided into 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 at random.Each group of tail intravenously administrable 1 time every day (matched group gives isometric(al) normal saline), after continuous 7d, 30min after last administration, pentobarbital sodium (40mg/kg) anesthetized rat, dorsal position is fixed on Mus plate, cervical region median incision, be separated right carotid and be about 15mm, the stimulating electrode and the temperature sensor probe that thrombus in vivo are formed analyzer are placed on common carotid artery, stimulating electrode is positioned at proximal part, with 2mA galvanism blood vessel 7min, make blood vessel internal membrane damage, record the time because of thrombosis blocking blood flow in arterial lumen, i.e. thrombus formation time (OT).Experimental data statistical result is in table 54.
Table 54. salvianolic acid A freeze-dried powder is on the impact of rat artery thrombus formation time
Note: compare with normal saline group, * P < 0.01
Experimental result shows, and compares with saline control group, salvianolic acid A freeze-dried powder 12.5,2.5, the thrombus formation time significant prolongation (P < 0.01) of 0.5mg/kg dosage group; Low dose group thrombus formation time and 20mg/kg aspirin group are quite (P > 0.05); Between high, medium and low dosage group, thrombus formation time has significant difference (P < 0.05).Show that salvianolic acid A freeze-dried powder can extend thrombus formation time, the formation of prevention of arterial thrombosis, pre-preventing thrombosis and the ischemic cerebrovascular caused.
Experimental example 40: salvianolic acid A freeze-dried powder is to thrombotic inhibitory action
SD rat, male and female half and half, grouping, administration are with experimental example 39.30min pentobarbital sodium anesthesia after last administration, dorsal position is fixed, and right common carotid artery and left external jugular vein are isolated in operation, connect with three sections of polyethylene tubes.Put into a long 5cm to have weighed operation silk thread in polyethylene tube stage casing.Polyethylene tube is full of with heparin-saline solution (5u/mL).Left external jugular vein is inserted in one end of pipe, and the other end is connected with right common carotid artery.Open bulldog clamp, blood flows through polyethylene tube by right carotid and returns left jugular vein.Herba Clinopodii in after open blood flow 15min, take out silk thread rapidly, filter paper sucks supernatant blood, weighs, and gross weight subtracts line and heavily namely obtains wet weight of thrombus; Then put freeze-day with constant temperature in 60 DEG C of baking ovens, to constant weight, to weigh after cooling, be thrombosis dry weight.Experimental data is in table 55.
The inhibitory action that table 55. salvianolic acid A freeze-dried powder is formed rat suppository
Note: compare with normal saline group, * P < 0.01
Experimental data shows, compare with normal saline group, salvianolic acid A freeze-dried powder 12.5,2.5, wet, the dry weight of 0.5mg/kg dosage group rat suppository all significantly alleviates (P < 0.01), there is good dose-effect relationship between each dosage group.And low dose group and 20mg/kg aspirin group effect are quite (P > 0.05).Prompting salvianolic acid A freeze-dried powder has the effect of good inhibition thrombosis, can be used for preventing or treat the ischemic cerebrovascular caused by thrombosis.
Experimental example 41: salvianolic acid A freeze-dried powder is on the impact of rat vein thromboembolism
Male SD rat, body weight (200 ± 20) g, divide into groups same experimental example 40, pentobarbital sodium (40mg/kg) anesthetized rat, along abdominal part medisection rat stomach wall, open abdominal cavity, be separated postcava, and in left renal vein lower end level place ligation postcava, close abdominal cavity, close abdominal cavity, after 1h, tail vein injection administration; Reopen abdominal cavity after 3h, below ligation, 2cm place folder closes blood vessel, the vein of ligation simultaneously side shoot, and blood vessel is cut in stringer open, and exhaust tube chamber inner blood, removal of thromboses, filter paper sops up residual blood, takes wet weight of thrombus immediately; Rearmounted 60 DEG C of baking ovens in after freeze-day with constant temperature, claim thrombosis dry weight.Test data is in table 56.
Table 56. salvianolic acid A freeze-dried powder is on the impact of rat vein thromboembolism
Note: compare with normal saline group, * P < 0.01
Experimental data shows, compare with normal saline group, salvianolic acid A freeze-dried powder 12.5,2.5, wet, the dry weight of 0.5mg/kg dosage group rat vein thrombosis significantly alleviates (P < 0.01), low dose group and 20mg/kg aspirin group effect suitable (P > 0.05); Show that salvianolic acid A freeze-dried powder has the effect suppressing venous thrombosis, can be used for the venous thromboembolism after preventing acute ischemic cerebrovascular disease to occur.
Experimental example 42: salvianolic acid A freeze-dried powder is to the thrombotic inhibitory action of rats in vitro
SD rat, 250 ~ 300g, male and female half and half; Random point of normal saline group, aspirin group (20mg/kg) and the high, medium and low dosage of salvianolic acid A (12.5,2.5,0.5mg/kg) group, each group tail vein injection every day is given and relative medicine 1 time, continuous 7 days; 30min after last administration, pentobarbital sodium is anaesthetized, abdominal cavity is opened along ventrimeson, abdominal aortic blood is about 1.5ml and injects silication sebific duct, rapidly silica gel tube two ends are butted into ring-type, the silica gel sheath seal of tube is fixed, and puts 37 DEG C of constant temperature on extracorporeal thrombosis forming device and rotates 15min, removal of thromboses, measures thrombosis length, claims its weight in wet base; Its dry weight is surveyed after drying in 60 DEG C of calorstats.Experimental data is in table 57.
Table 57. salvianolic acid A freeze-dried powder is to the thrombotic inhibitory action of rats in vitro (x ± s)
Note: compare with normal saline group, * P < 0.01
Experimental data shows, compare with 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 shortens (P < 0.01), thrombosis is wet, dry weight significantly alleviates (P < 0.01), and low dose group and 20mg/kg aspirin group effect are quite (P > 0.05); Show that salvianolic acid A freeze-dried powder has good inhibitory action to ex vivo thrombosis.
Experimental example 43: salvianolic acid A freeze-dried powder is to the thrombolytic experimental study of thrombosis established in body
SD male rat (250 ± 20) g, pentobarbital sodium intraperitoneal anesthesia rat, is separated left common carotid artery with the unidirectional current continued stimulus rat artery 7 minutes of 2mA, with blood flowmeter continuous probe Flow of carotid artery.Terminate rear blood flow and reduce to 50% before stimulation to stimulate and be considered as thrombosis.Animal divides 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 of 20min after formation thrombosis, all through the disposable injection relative medicine of femoral vein, revascularization situation in 1h after observation administration; If revascularization in this period, then continue to observe vessel open state 1h.The Flow of carotid artery of every animal to stimulate front blood flow for baseline, before stimulating with >=50% or≤25% blood flow person be judged to continue to lead to again or continue after thromboembolism again; After logical again in 1h, each treated animal blood flow is divided into >=and 50%, 25% ~ 50% and≤25% baseline values.According to blood flow, carotid artery vascular degree of opening divides three kinds of states, is respectively and 1. continues thromboembolism without leading to again; 2. logical interlocking with thromboembolism occurs again; 3. continuous openness after leading to again, nothing thromboembolism again.Experimental result is in table 58.
Thrombolytic effect (n=10) in table 58. salvianolic acid A lyophilized powder needle body
Note: 1. after recanalization rate=administration, number of animals/animal number logical again appears in 1h
2. after the number of animals/administration of thromboembolism again that bolt rate=logical rear 1h occurs again again, 1h is interior there is number of animals logical again
3. compare with normal saline group, * P < 0.01; Compare with urokinase group, #P < 0.05
Result shows: normal saline group all continues thromboembolism, and without appearance logical phenomenon again; The number of animals that each medicine group continues thromboembolism is few, all has pole significant difference (P < 0.01) compared with normal saline group; Dosage (5mg/kg) group in salvianolic acid A freeze-dried powder, its vessel open degree is similar to 2000U/kg urokinase group, and its recanalization rate is suitable; The lasting recanalization rate of salvianolic acid A freeze-dried powder high dose (10mg/kg) group and recanalization rate are all higher than urokinase group, and bolt rate is starkly lower than urokinase group (P < 0.05) again; Salvianolic acid A freeze-dried powder low dosage (2.5mg/kg) is though organize recanalization rate lower than urokinase group, and bolt rate and urokinase group are suitable again for it, and has the trend lower than urokinase bolt rate again.The above results prompting salvianolic acid A freeze-dried powder has good thrombolytic and the effect of thromboembolism again after preventing thrombolytic.
Experimental example 44 salvianolic acid A freeze-dried powder is on the impact of cerebral thrombosis hemorheology of rat
The method adopting carotid artery to inject self thrombosis prepares Cerebrovascular embolism model: after the intraperitoneal anesthesia of SD rat pentobarbital sodium, neck median incision, be separated the right side total tremulous pulse of strength (CCA), internal carotid artery (ICA), external carotid artery (ECA), ligation ECA far-end and arteria pterygopalatina, arteriole folder folder closes CCA and ICA, an osculum is cut at ECA near-end, unclamp CCA arteriole folder, extract arterial blood 0.5ml, sodium citrate anticoagulant, centrifugal, get platelet poor plasma and add a small amount of erythrocyte and thrombinogen and calcium chloride mixing, prepare the thrombosis that diameter is about 0.35mm, shred, one little saving 2mm, folder closes CCA, by ECA, embolus is injected ICA, ligation ECA, unclamps CCA and arteria pterygopalatina place vascular clamp, sew up the incision.Experiment points 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 after successful surgery, each treated animal tail vein relative medicine, after this injects once, continuous 7 days every day; Sham operated rats and model group inject isopyknic normal saline.Second day (fasting 12h) that administration terminates, 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
Compare with sham operated rats, #p < 0.01; * P < 0.01 is compared with model control group,
Experimental result shows, and after cerebral thrombosis, rat whole blood viscosity, plasma viscosity significantly increase, and packed cell volume also significantly raises (P < 0.01); The each dosage group of salvianolic acid A freeze-dried powder compares with model group, and its whole blood viscosity and plasma viscosity and packed cell volume all obviously reduce; Prompting salvianolic acid A freeze-dried powder can accelerate micro-blood flow velocity, reduction blood viscosity, improves hemorheology and effectively resists the effect of cerebral thrombosis.

Claims (21)

1. the preparation method of salvianolic acid A freeze-dried powder, described salvianolic acid A freeze-dried powder is for the preparation 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 is make total amount 0.02% ~ 0.1%;
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: the extracting solution that step (1) is obtained, adjust pH to 3.5 ~ 5.2, adding with the molar percentage of salvianolic acid B is the catalyst of 0.1% ~ 3.0%, 100 ~ 140 DEG C of heating 1 ~ 6 hour, wherein said catalyst was one or more in iron chloride, ruthenium trichloride, aluminum chloride, zinc chloride, Palladous chloride.;
(3) purification:
A. by the solution that step (2) obtains, pH to 2.5 ~ 4.5 are adjusted, centrifugal, supernatant is separated, after washing with water, with eluent through nonpolar or low pole macroporous resin column chromatography, efficient Liquid Detection salvianolic acid A, collects the eluent containing salvianolic acid A;
B. the eluent sephadex lh-20 obtained by step a or ODS-C18 or polyamide chromatography post are separated, and with eluent, efficient Liquid Detection salvianolic acid A, collects the eluent containing salvianolic acid A;
C. pH to 2.0 ~ 4.0 adjusted by the eluent obtained by step b, through organic solvent extraction, and Separation of Organic phase;
D. the solution silica gel column chromatography obtained by step c is separated, and with eluent, efficient Liquid Detection salvianolic acid A, collects the eluent containing salvianolic acid A;
(4) dry: the eluent that steps d is obtained, reclaim under reduced pressure eluant, then be dissolved in water, vacuum drying, spraying dry or microwave vacuum drying, obtain described salvianolic acid A;
(5) get described salvianolic acid A 10g ~ 80g to inject and be stirred to dissolve with water 1500 ~ 2800ml, by adjusting PH with base value 4.0 ~ 5.0, adding described filler makes it dissolve, add described antioxidant again, be stirred to dissolve mixing, then add active carbon 0.5 ~ 2g stirring and adsorbing, filter removing active carbon, inject and become bottle with fill after water, send in freeze dryer and carry out lyophilization;
(6) described lyophilization comprises the steps:
A, freezing: to be cooled to-40 DEG C ~-45 DEG C with 20 DEG C ~ 30 DEG C/h speed, to be incubated freezing 10 ~ 16 hours;
B, distillation: be evacuated to below 0.3mbar, rose to-23 DEG C ~-27 DEG C by the salvianolic acid A formulation temperature freezed in 10 ~ 14 hours, maintains-23 DEG C ~-27 DEG C vacuum dryings 6 ~ 8 hours;
C, drying: continue to heat up, be evenly warming up to 40 DEG C ~ 45 DEG C with 0.5 DEG C ~ 1.0 DEG C/min, maintain 40 DEG C ~ 45 DEG C dryings after 2 ~ 5 hours, sample temperature is down to room temperature, adds a cover, obtain salvianolic acid A freeze-dried powder.
2. preparation method according to claim 1, also comprises the purposes of protection blood brain barrier.
3. preparation method according to claim 1, also comprises the purposes reducing ischemic tissue of brain infarction size.
4. preparation method according to claim 1, also comprises the purposes alleviating ischemic tissue of brain edema.
5. the preparation method according to any one of Claims 1 to 4, its weight proportion is: salvianolic acid A 20g ~ 40g, filler 20g ~ 40g, and antioxidant is make total amount 0.03% ~ 0.08%.
6. the preparation method according to any one of Claims 1 to 4, wherein said filler be selected from mannitol, glucose, lactose any one or a few, consumption is 20mg ~ 40mg/2ml ~ 3ml.
7. the preparation method according to any one of Claims 1 to 4, wherein said antioxidant be selected from vitamin C, thiourea, sodium sulfite, sodium pyrosulfite any one or a few.
8. preparation method according to claim 7, its weight proportion is: salvianolic acid A 20g, filler mannitol 20g, antioxidant vitamins C0.8g; Its preparation method is:
Get described salvianolic acid A 20g, inject and be stirred to dissolve with water 1900ml, with sodium hydroxide sodium adjust pH 4.0 ~ 5.0, add mannitol 20g and make dissolving, then add 0.8g vitamin C, be stirred to dissolve mixing, add active carbon 0.5g stirring and adsorbing, filter removing active carbon; Inject water to 2000ml, fill becomes 1000 bottles, lyophilization;
Described lyophilization comprises the steps:
A, freezing: to be cooled to-45 DEG C with 25 DEG C/h speed, to be incubated freezing 15 hours;
B, distillation: be evacuated to below 0.3mbar, rose to-25 DEG C by the salvianolic acid A formulation temperature freezed in 12 hours, maintains-25 DEG C of vacuum dryings 8 hours;
C, drying: continue to heat up, be evenly warming up to 40 DEG C with 0.8 DEG C/min, maintain 40 DEG C of dryings after 3 hours, sample temperature is down to room temperature, adds a cover, obtain salvianolic acid A freeze-dried powder.
9. preparation method according to claim 1, wherein the water extracting method described in step (1) is: get red rooted salvia, is cut into decoction pieces or is ground into diameter 2mm granule, add 3 ~ 15 times amount water extraction at every turn, extract 1 ~ 3 time altogether, extract 1 ~ 4 hour at every turn; Extracting solution is evaporated to, and " relative density is determined as 1.10 ~ 1.25 at 60 DEG C; add ethanol and make alcohol content 30% ~ 80%; be centrifugal; supernatant decompression recycling ethanol is also concentrated into without alcohol taste; described water extraction adopts decoction to extract or 45 ~ 95 DEG C of water temperature lixiviates are got; stir with 10 ~ 50 revs/min of speed simultaneously, obtain Radix Salviae Miltiorrhizae extract.
10. preparation method according to claim 1, wherein the alcohol extraction method described in step (1) is: get red rooted salvia, be cut into decoction pieces or be ground into diameter 2mm granule, add 3 ~ 15 times amount 30% ~ 60% alcohol reflux at every turn, each extraction 1 ~ 4 hour, extracts 1 ~ 3 time altogether; Decompression recycling ethanol, obtains Radix Salviae Miltiorrhizae extract.
11. preparation methoies according to claim 1, in the extracting solution wherein in step (1), salvianolic acid B concentration is 1mg/ml ~ 30mg/ml or is diluted with water to 1mg/ml ~ 30mg/ml.
12. preparation methoies according to claim 11, wherein in extracting solution, salvianolic acid B concentration is 5mg/ml ~ 20mg/ml or is diluted with water to 5mg/ml ~ 20mg/ml.
13. preparation methoies according to claim 1, wherein in step (2), the molar percentage of catalyst and salvianolic acid B is 0.5% ~ 2.0%.
14. preparation methoies according to claim 1, wherein macroporous resin column described in step a is HPD-80, HPD-100, HPD-100B, HPD-200A, HPD-300, HPD-450, HPD-722, HPD-826, ADS-5, ADS-8, ADS-21 or D101, AB-8; Described eluant is water and the ethanol of water and different proportion, and first uses water, 10 ~ 40% ethanol elutions, then uses 20 ~ 60% ethanol elutions, efficient Liquid Detection salvianolic acid A, and collect containing salvianolic acid A part, eluent is concentrated into without alcohol taste.
15. preparation methoies according to claim 1, described eluant wherein in step b is water and the ethanol of water and different proportion, and first use water, 20 ~ 60% alcoholic solution eluting remove impurity, use 40 ~ 90% alcoholic solution eluting again, efficient Liquid Detection salvianolic acid A, collect containing salvianolic acid A part, eluent is concentrated into without alcohol taste.
16. preparation methoies according to claim 1, the organic solvent wherein described in step c is t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate or Ethyl formate.
17. preparation methoies according to claim 1, wherein eluant described in steps d is the two-phase solvent of petroleum ether, pentane, normal heptane, ethyl acetate, methyl acetate, Ethyl formate, t-butyl methyl ether composition.
18. preparation methoies according to claim 1, the wherein temperature of microwave vacuum drying described in step (4): 20-100 DEG C, return difference temperature 1-5 DEG C, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.
19. preparation methoies according to claim 1, wherein vacuum drying temperature described in step (4): 50 DEG C ~ 90 DEG C, more than vacuum-0.07Mpa, power: 1 ~ 60KW, dry 2 ~ 20 hours.
20. preparation methoies according to claim 1, wherein spray-dired intake air temperature described in step (4): 150 DEG C ~ 350 DEG C, air outlet temperature: 70 DEG C ~ 95 DEG C, spray velocity: 1 ~ 300ml/min.
21. preparation methoies according to claim 1, wherein pH adjusting agent is phosphoric acid, hydrochloric acid, sulphuric acid or acetic acid.
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