CN103142571B - A kind of salvianolic acid A compositions and prepare medicinal usage - Google Patents
A kind of salvianolic acid A compositions and prepare medicinal usage Download PDFInfo
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Abstract
The present invention relates to the purposes of a kind of salvianolic acid A compositions for the preparation of prevention and therapy ischemic heart medicine, said composition is made up of salvianolic acid A, alkannic acid, rosmarinic acid, salvianolic acid B, salvianolic acid C, wherein said compositions is made up of following weight proportion: salvianolic acid A 90% ~ 99%, alkannic acid 0.1% ~ 3%, rosmarinic acid 0.1% ~ 3%, salvianolic acid B 0.1% ~ 3%, salvianolic acid C 0.1% ~ 5%.
Description
Technical field
The present invention relates to a kind of salvianolic acid A compositions and prepare medicinal usage.
Technical background
Ischemic heart desease (IHD) is the cardiac damage caused because coronary artery circulation change causes imbalance between coronary flow and myocardial demand, modal reason is coronary atherosclerosis, coronary artery thrombosis and coronary vasospasm, accounts for about 90% of ischemic heart desease.Its common ground is the imbalance between supply and demand of oxygen caused by deficiency myocardial blood supply and causes myocardial ischemia, anoxia, energy metabolism of myocardial is abnormal, can not support normal heart action, thus a series of pathophysiological change and symptom occur, cause angina pectoris attacks, even myocardial infarction and threat to life occur.According to the position of coronary artery pathological changes, scope and lesion severity, degree of myocardial ischemia, clinical point of five classes: asymptomatic myocardial ischemia, angina pectoris, myocardial infarction type, ischemic cardiomyopathy type, sudden death type.Comparatively common with angina pectoris, myocardial infarction, sudden cardiac death.And these are called for short coronary heart disease because heart disease that arteria coronaria pathological change causes is referred to as coronary heart disease.Therefore generally said ischemic heart desease mainly refers to coronary heart diseases and angina pectoris.Heart does not have " oxygen warehouse ", relies on myocardial blood flow completely, so once ischemia, can cause anoxia at once.Oxygen is the movable requisite material of myocardial cell, and normal myocardial cells picked-up blood oxygen levels reaches 65 ~ 75%, and other is organized and only absorbs 10 ~ 25%.The direct result of myocardial ischemia is that myocardial cell aerobic metabolism weakens, and production capacity reduces, and energy required when making cardiomotility is under-supply, causes angina pectoris, arrhythmia, cardiac function to decline.Meanwhile, the refuse of metabolism can not effectively be removed in time, easily has a negative impact.Ischemia, anoxia, scarce energy, finally can affect the contractile function of heart.If there is the cardiac muscle of 20% ~ 25% to stop shrinking, usually there will be left chamber function exhaustion; If there is the cardiac muscle of more than 40% not shrink, just have the exhaustion of severe cardiac pump function.If this situation occurs suddenly, just there will be breakneck cardiogenic shock.Acute myocardial infarction is just normal relevant to this situation.Myocardial ischemia also can damage diastolic function.Shrink bad and diastole is bad combines, can cause that complicated substance metabolism is disorderly and myocardial electrical activity is not normal.
Therefore improve cardiovascular function, increase the blood supply oxygen supply of cardiac muscle, reduce the consumption of oxygen, improve myocardial metabolism, allevating angina pectoris, reduce the main purpose that myocardial infarction area is Drug therapy.Current treatment ischemic heart desease mainly comprises Drug therapy and the operative treatment such as intervention (PICA) or bridging.Nitric acid lipid drug is generally used during angina pectoris attacks, as sublingual administration nitroglycerin, rapid-action, but emergency can only be used for, relief of symptoms, fundamentally can not treat myocardial ischemia.Catabasis medication comprises nitrate esters, Statins, ACEI, beta-blocker, calcium ion antagonist, Thrombolytic Drugs (urokinase, streptokinase etc.) and Chinese medicine etc.Energy metabolism impairment be myocardial defect wound make one of reason element, in recent years, playing an antianginal class by optimizing regulation energy metabolism of myocardial, to urge metabolism class medicine also quite concerned, and being expected to becomes new class treatment or an ancillary drug.And it is main with arteria coronaria revascularization, to recover blood perfusion be principle as early as possible ischemic heart desease Primary Care method at present, also there is very important risk: zoopery and clinical research find, along with the recovery of blood fortune, some impaired myocardial cell function and structural deterioration increase the weight of on the contrary, namely there is myocardial ischemia reperfusion injury (myocardialischemiareperfusioninjury, MIRI).Reperfusion injury is after myocardial ischemia, Reperfu-sion early stage, response to oxidative stress causes producing a large amount of oxygen-derived free radicals, superoxide anion in cardiac muscle, accelerate apoptosis of cardiac muscle and necrosis together with the factor such as calcium overload in cell and mitochondrion, such that myocardial infarct size expands, cardiac function worsens rapidly.Clinical manifestation is after the coronary flow imaging of obturation and infarcted region hemoperfusion are rebuild in a period of time, and some patients blood pressure rapid drawdown, cardiac insufficiency, arrhythmia occur and a series of phenomenon that sb.'s illness took a turn for the worse such as even to die suddenly.After as logical again in thrombolysis in myocardial infarction in clinical practice, coronary artery bypass grafting, heart transplantation, all there is reperfusion injury problem after sudden cardiac arrest cardiac resuccitation, after open-heart surgery etc.How to accomplish the blood flow both having ensured to recover ischemic tissue as early as possible, alleviate again the generation even removing reperfusion injury just become another coronary heart disease control in important topic.
World Health Organization (WHO) gives a warning in " global disease burden " report, and cardiovascular system diseases has become the underlying cause of death of global range.Statistical data shows, and the whole world is often close on 2000 ten thousand people and is happened suddenly acute cardiovascular disease, cause death toll account for the whole world dead more than 30%, wherein 43% die from coronary heart disease.In Regional Distribution, the cardiovascular disease death of more than 80% occurs in low middle income country.It is reported, the U.S. about has 45 ~ 500,000 people to die from sudden cardiac death every year, and wherein accounts for more than 80% of sudden death reason with ischemic heart desease (coronary heart disease).Nearly decades, Incidence of CHD increases year by year in China, ranks first at present in all kinds of heart disease.China national statistical data display in 2010, the people of China more than 40% dies from cardiovascular disease.Effective control of coronary heart disease has become the major global public health problem that can not be ignored, the development of this kind of diseases prevention and treatment medicine is listed in the key content of China's Eleventh Five-Year Plan, the research of " 12 " original new drug key special subjects continuously, and will the drug categories of a very long time primary study from now on be become, there are great social meaning and economic worth.
Radix Salviae Miltiorrhizae is the dry root and rhizome of Lamiaceae Salvia platymiscium Radix Salviae Miltiorrhizae SalviamiltiorrhizaBunge, has effect of blood circulation promoting and blood stasis dispelling, removing heat from blood eliminating carbuncle and nourishing blood to tranquillize the mind.Radix Salviae Miltiorrhizae, as the medicine of traditional Chinese medical science tradition blood circulation promoting and blood stasis dispelling, because of determined curative effect, starts to be widely used in clinical departments from the seventies, becomes one of clinical practice medicine the widest at most.Because having pharmacological actions such as improving microcirculation, coronary artery dilating, inhibition thrombosis, anticoagulant, protection ischemic myocardium, the clinical practice in modern age of its preparation is mainly used in ischemic Cardial or cerebral vascular diseases.The Chinese patent medicine of the conventional treatment myocardial ischemia containing Radix Salviae Miltiorrhizae has: Radix Salviae Miltiorrhizae Tabellae, FUFANG DANSHEN PIAN, the heart can relax, arteria coronaria is peaceful; Enumerate coronary artery dilator, increase coronary flow, reduce blood viscosity and blood clotting, anticoagulant, protection ischemic myocardium and improve the pharmacological actions such as microcirculation.Be applicable to the thoracic obstruction patient and angina pectoris acute attack etc. of Blood stasis.
The effective substance of Radix Salviae Miltiorrhizae is water-soluble phenolic compounds wherein, comprise salvianolic acid A-G, rosmarinic acid and methyl ester thereof, danshensu, protocatechualdehyde, protocatechuic acid etc., be feature with antioxidation, anticoagulant and antiinflammatory, there is the pharmacological action of the multiple beneficial such as protection vascular endothelial cell, blood fat reducing, rising HDL, anticoagulant, activation fibrinolytic, the synthesis of suppression Fibrinogen, chelating calcium iron ion simultaneously; Wherein the strongest with salvianolic acid A activity.Salvianolic acid A can improve the rear rat heart muscle H9c2 cell survival rate of damage, inhibited apoptosis, and has very strong antioxidation, obviously can alleviate mitochondrial injury.Therefore salvianolic acid A is by alleviating apoptosis of cardiac muscle, improving antioxidant ability of organism and protecting the mechanism such as myocardial mitochondria to have protective effect to ischemic myocardium.Salvianolic acid A also has significant protective effect to myocardial ischemia reperfusion injury; and effect be better than salvianolic acid B (Lin Zhirong etc. the comparative study [D] of salvianolic acid A and salvianolic acid B resisting myocardial ischemia-reperfusion injury. time treasure traditional Chinese medical science traditional Chinese medicines; 2011,22 (2): 412-424).
But the natural content of salvianolic acid A is extremely low (being about the 0.01-0.06% of red rooted salvia), and make crude drug high cost, separation and purification difficulty is excessive, seriously governs the R and D of medicine, becomes the bottleneck of its industrialization.In addition, in prior art, salvianolic acid B carries out degraded by Ester hydrolysis decarboxylation and chroman ring ring-opening reaction and can change into salvianolic acid A also to have experiment to confirm, but the conversion process of above-mentioned prior art is uncontrollable, conversion byproducts is many, and the productive rate of principal product salvianolic acid A is lower.
Although also there are some patent documentations to propose to attempt to overcome the defect existed in prior art in prior art, but be all only that simple trial heats up, changes pH value or improve concentration transforming front pressure differential self etc., study conversion reaction deeply, all sidedly without any a patent or prior art and all comprise which major influence factors, how synergism has an impact to salvianolic acid A productive rate jointly each other as transformed the concentration, pH value, temperature, time etc. of front pressure differential self more to propose these factors without any a patent or prior art.
On this basis, other inventors or prior art is seldom had to propose to attempt in conversion, adding catalyst to make reaction more abundant, having related to the related content adopting catalyst to carry out transforming in currently available technology exists only in the two parts of patent application documents submitted on April 6th, 2010 same day, these two parts of patent application documents are application number is respectively 2010101436787, the patent application that denomination of invention is " a kind of catalyzed conversion salvianolic acid B prepares the method for salvianolic acid A " and application number are 2010101436876, denomination of invention is the patent application of " a kind of method of preliminary purification salvianolic acid B conversion feedstock ".In the description of these two parts of patent applications, although disclose the technology contents that catalyzed conversion salvianolic acid B prepares salvianolic acid A, but its catalyst is carbamide, the mol ratio of carbamide and salvianolic acid B is (0.4 ~ 0.6): 1, require to consume and add carbamide very high, therefore production cost is very high.Further, in above-mentioned two parts of patent application documents, concern pH value and other correlative factors work in coordination with the impact produced salvianolic acid A productive rate too.Moreover its conversion feedstock is the radix Salviae Miltiorrhizae water extract through co-chromatography preliminary purification, wherein salvianolic acid B >=50%, this all requires higher to the purity of conversion feedstock and purifying technique, and technique is also comparatively complicated, further increases production cost.More crucially, although claim in its application documents " directed conversion ratio >=10% of the salvianolic acid A salvianolic acid B using this method to prepare, even reaches 60% ".But due to carbamide in fact not open loop, dehydration, decarboxylation, only have into ester effect, therefore, its conversion ratio does not reach 60% at all, and the conversion ratio in the specific embodiment of this application file is only up to 53%, and the conversion ratio of multiple embodiment is only 10%, 20% and 30%.Still there is a big difference in the requirement of this and actual industrialization.
Summary of the invention
For overcome prior art exist above-mentioned defect, the invention provides a kind of salvianolic acid A compositions for prevent and or treatment ischemic heart desease aspect purposes.
The invention provides the purposes of a kind of salvianolic acid A compositions for the preparation of prevention and therapy ischemic heart medicine, in wherein said compositions, salvianolic acid A content is greater than 93%, be less than 100%, and also comprise 4 other compositions by following weight proportion: alkannic acid 0.1% ~ 2%, rosmarinic acid 0.1% ~ 2%, salvianolic acid B 0.1% ~ 2%, salvianolic acid C 0.1% ~ 2%, described salvianolic acid A compositions adopts lower preparation method to obtain:
Get red rooted salvia, be cut into decoction pieces or be ground into diameter and be about 1mm ~ 5mm granule, add 3 ~ 15 times amount at every turn, 45 ~ 95 DEG C of water temperature lixiviates are got, stir with 10 ~ 50 revs/min of speed simultaneously, or add 3 ~ 15 times amount water boiling and extraction, extract 1 ~ 3 time altogether, extract 1 ~ 4 hour at every turn; Extracting solution is evaporated to relative density 1.0 ~ 1.25 (60 DEG C), adds ethanol and makes alcohol content 50% ~ 85%, leaves standstill, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, obtains Radix Salviae Miltiorrhizae extract; Or
Get red rooted salvia, be cut into decoction pieces or be ground into diameter and be about 1mm ~ 5mm granule, add 3 ~ 15 times amount 30% ~ 60% alcohol reflux at every turn, extract 1 ~ 4 hour at every turn, extract 1 ~ 3 time altogether; Decompression recycling ethanol is also concentrated into without alcohol taste, obtains Radix Salviae Miltiorrhizae extract;
Above-mentioned Radix Salviae Miltiorrhizae extract is diluted with water to every 1ml containing salvianolic acid B 1 ~ 30mg, and aqueous solution adjusting PH with base to 3.5 ~ 6.5, add with salvianolic acid B molar percentage 0.1 ~ 3% zinc chloride as catalyst, transforms 1 ~ 6 hour at 100 ~ 140 DEG C of heating temperatures;
Conversional solution adjust pH to 2.5 ~ 4.5, leave standstill, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 1 ~ 10mg, be separated through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 35 ~ 1: 70 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 4 ~ 1: 30, use 1 ~ 8 times of cylinder hydrops respectively, 1 ~ 10 times of column volume 10% ~ 40% ethanol elution, removing impurity, use 2 ~ 10 times of column volume 20% ~ 60% ethanol elutions again, HPLC detects, collect 20% ~ 60% 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 1-10mg salvianolic acid A, be separated by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 5 ~ 1: 25 with polyamide ratio, resin column blade diameter length ratio is 1: 4 ~ 1: 25, use 1 ~ 10 times of cylinder hydrops, the remove impurity of 5 ~ 20 times of column volume 20% ~ 60% alcoholic solution eluting respectively, use 4 ~ 15 times of column volume 40% ~ 90% alcoholic solution eluting again, collect 40% ~ 90% alcoholic solution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste aqueous solution;
Aqueous solution concentrates, acid adjustment pH to 2.0 ~ 4.0, by the t-butyl methyl ether of aqueous solution 1-8 times amount, and point 2 ~ 6 extractions, be separated organic layer, reclaim under reduced pressure t-butyl methyl ether, make the extract of every 1ml containing salvianolic acid A 1g ~ 10g, add 1 ~ 3 times amount silica gel, stir, volatilize;
Stirring sample silica gel is added on the dry silicagel column of 5 ~ 20 times amount installed, silicagel column blade diameter length ratio is 1: 4 ~ 1: 25, with pentane-t-butyl methyl ether for eluant, gradient elution, use pentane-t-butyl methyl ether (4: 6) eluting 6 ~ 30 times of column volumes respectively, pentane-t-butyl methyl ether (6: 4) eluting 6 ~ 30 times of column volumes, reclaim under reduced pressure eluant, the salvianolic acid A reclaimed after organic solvent adds 5 ~ 20 times amount water dissolutioies, microwave vacuum drying, obtains described salvianolic acid A compositions.
Preferably, microwave vacuum drying temperature: 20-100 DEG C, return difference temperature 1-5 DEG C, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.
Preferably, microwave vacuum drying temperature: 50-85 DEG C, return difference temperature 2-4 DEG C, more than vacuum-0.07Mpa, microwave power 10-80KW, dry 100-150 minute.
Preferably, microwave vacuum drying temperature: 55-80 DEG C, return difference temperature 2-3 DEG C, more than vacuum-0.07Mpa, microwave power 25-60KW, dry 120-140 minute.
Preferably, the wherein said medicine for prevention and therapy ischemic heart desease can be used for treating one or more of following disease: coronary heart diseases and angina pectoris, ischemic cardiomyopathy, myocardial infarction, myocardial ischemia apoplexy, acute coronary syndrome, coronary stricture, myocardial ischemia reperfusion injury, atherosclerosis.
Preferably, wherein said salvianolic acid A compositions is for improving the hemodynamic purposes of Ischemic Heart.
Preferably, wherein said salvianolic acid A compositions is for improving the purposes of ischemic heart cardiac function.
Preferably, wherein said salvianolic acid A compositions is for increasing the purposes of the coronary flow of ischemic heart.
Preferably, wherein said salvianolic acid A compositions is for reducing the purposes of ischemic heart myocardial infarct size.
Preferably, wherein said salvianolic acid A compositions is for reducing the purposes of myocardial ischemia scope.
Preferably, wherein said salvianolic acid A compositions is for alleviating the purposes of degree of myocardial ischemia.
Preferably, wherein said salvianolic acid A compositions regulates the purposes of Enzyme Activities for the protection of myocardial cell membrane structure.
Preferably, wherein said salvianolic acid A compositions is for suppressing the purposes of ischemic myocardial tissue inflammatory reaction.
Preferably, the purposes that wherein said salvianolic acid A compositions is damaged for the protection of myocardial mitochondria.
Preferably, wherein said salvianolic acid A compositions is for improving the purposes of myocardial metabolism.
Preferably, wherein said salvianolic acid A compositions comprises for improving one or more of following myocardial metabolism for the purposes improving myocardial metabolism: myocardium radical metabolism, fatty acid metabolism, energy metabolism.
Preferably, wherein said salvianolic acid A compositions is for reducing the purposes of myocardial oxygen consumption.
Preferably, wherein said salvianolic acid A compositions is for improving hemorheological purposes.
Preferably, wherein said salvianolic acid A compositions is used for the purposes of inhibition thrombosis.
The present invention is by the extraction to Radix Salviae Miltiorrhizae, transform, purification, drying process, obtain the compound based on 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, but in catalytic conversion reaction of the present invention, the purity height of salvianolic acid B does not affect on conversion reaction effect.On the contrary, not only produce a large amount of impurity, and do not improve conversion ratio during salvianolic acid B purity > 50%, therefore, the present invention achieves unforeseeable technique effect.
In addition, low concentration can also mix with the salvianolic acid B of high concentration by the present invention, only need be made into suitable initial conversion concentration, can reach the object changing into salvianolic acid A compositions equally.Therefore, the preparation technology of this conversion feedstock is very simple, and production cost is also very suitable for the application in actual industry while reducing.
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 compositions 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 concentration requirement to reactant, and think that concentration height specific concentration is low good.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 that the concentration containing salvianolic acid B in salvianolic acid B aqueous solution is not more high better, and more than 30mg/ml concentration conversion ratio is low on the contrary, and effect is poorer.Therefore, the present invention, in cost-saving and production cycle, achieves unforeseeable technique effect, creative.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 zinc chloride can significantly improve as catalyst the conversion ratio that salvianolic acid B transforms salvianolic acid A compositions, 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 composition levels is lower, the large raising of salvianolic acid A composition levels 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, solvent extraction, silica gel to be separated again, and various flows part is measured, after removing impurity portion, salvianolic acid A content in salvianolic acid A compositions 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 normal temperature drying, and from vacuum drying, spraying dry, microwave vacuum drying method preferred microwave vacuum drying, thus thoroughly overcome in the past that baking temperature is too high, drying time is long, the defect large to the destruction of salvianolic acid A compositions; And sublimation drying is long, the high and problem that the compositions organic solvent residual of lyophilization gained is serious of cost.
Contain except salvianolic acid A, also containing a small amount of salvianolic acid B, alkannic acid, rosmarinic acid, salvianolic acid C except main in salvianolic acid A compositions of the present invention; Salvianolic acid A by scavenging free radicals, alleviates mobility and permeability change that membrane lipid peroxidatio causes, stops spilling of the penetrating and enzyme of the exception of ion, thus reduces the damage caused due to myocardial ischemia-reperfusion, have protective effect to myocardial ischemia.The platelet aggregation that salvianolic acid A compositions can dose-dependently suppress adenosine diphosphate (ADP), thrombin, arachidonic acid, collagen or U46619 to induce, reduce cAMP level in platelet, and reduce expression and the Fibrinogen combination of the P-selectin that ADP causes, thus the platelet leukocyte recruitment stoping ADP to cause, preventive and therapeutic effect is formed to atherosclerosis and thromboembolism;
Salvianolic acid B in salvianolic acid A compositions is the principle active component of Radix Salviae Miltiorrhizae and preparation Radix Salviae Miltiorrhizae Injection thereof, XIANGDAN ZHUSHEYE, and the same with salvianolic acid A compositions have protective effect to myocardial ischemia, is formed with preventive and therapeutic effect to atherosclerosis and thromboembolism; Salvianolic acid B effectively can reduce the infarct scope of experimental myocardial infarction rat, increases fibroblast and capillary vessel number in infarct; The left room EDP of Model of Acute Myocardial Ischemia rat can be reduced, raise the maximum rising of left indoor pressure and fall off rate, effect that heavy dose of application is shunk left room and diastolic function is all significantly increased; To Rabbit Myocardium anoxia one reoxygenation injury model, salvianolic acid B has negative chronotropic, increases the effect of creatine kinase, lactate dehydrogenase levels in coronary flow and reduction coronary outflow, can alleviate myocardial cell injury.Salvianolic acid B dose-dependently can reduce H
2o
2caused endotheliocyte LDH leaks outside and MDA generates, and improves NO release, effectively can also suppress H
2o
2there is lower CD54 express and increase neutrophil adhesion rate, show that salvianolic acid B has and alleviate H
2o
2the oxidative damage of caused endotheliocyte, reduces the effect of Surface of Vascular Endothelial Cells Expression of Cell Adhesion Molecules and suppression neutrophil adhesion, and thinks that this is the mechanism of action of salvianolic acid B resisting myocardial ischemia/reperfusion injury; Alkannic acid, rosmarinic acid, salvianolic acid C effect and salvianolic acid A are roughly the same, all have stronger antioxidation, energy scavenging activated oxygen, anti-lipid peroxidation reacts, its action intensity, higher than vitamin C, vitamin E, is one of natural product that known at present antioxidation is the strongest.Rosmarinic acid also contributes to the cell damage preventing free radical from causing, because this reducing cancer and arteriosclerotic risk.
Alkannic acid in salvianolic acid A compositions also has and suppresses the propagation of vascular smooth muscle cell and migration, and prevention of arterial is atherosis, angiostenosis and vascellum endometrial hyperplasia, has vasodilative effect.Uric acid can be suppressed and cross the formation of ultra-oxygen anion free radical, the generation of suppression peroxide, have the effect of antiinflammatory and uric acid resisting.The external effect that metakentrin can be suppressed to discharge.
Salvianolic acid C in salvianolic acid A compositions also by suppressing tubulin polymerization inducing cell mitotic blockade thus apoptosis-induced, has anti-tumour cell proliferative activity.
After the combination of salvianolic acid A in salvianolic acid A compositions, salvianolic acid B, alkannic acid, rosmarinic acid, salvianolic acid C, there is common ischemic myocardial protection effect, reduce the damage caused due to myocardial ischemia-reperfusion.What formed atherosclerosis and thromboembolism has preventive and therapeutic effect, makes it be used alone effect more by force than salvianolic acid A monomeric compound, also has the effect of antiinflammatory and uric acid resisting simultaneously, reduces cancer and arteriosclerotic risk.
On the other hand, present invention also offers its for prevent and or the purposes for the treatment of ischemic heart medicine, and by experimental results demonstrate:
Known through experimental data contrast, model group ischemia-reperfusion 60min, CBF ,+dp/dt
max,-dp/dt
maxobvious decline, LVEDP significantly increases.Compare with model group, the high, medium and low dosage group of positive drug group, salvianolic acid A compositions, salvianolic acid A monomeric compound group These parameters all have clear improvement, and the coronary flow that ischemia-reperfusion can be suppressed to cause reduces, left room diastolic pressure rises, the maximum climbing speed of left indoor pressure declines, the maximum fall off rate of left indoor pressure declines.And salvianolic acid A compositions group high dose group is suitable with positive drug group effect, effect is better than same dosage salvianolic acid A monomeric compound group.Experimental result display salvianolic acid A compositions can alleviate isolated rat heart ischemical reperfusion injury degree, and prompting salvianolic acid A compositions can obviously be improved coronary flow, improve the systolic and diastolic function of isolated heart aortic diastolic function and damaged myocardium, cardioprotection ischemical reperfusion injury.
Through known to the experimental data contrast of rats with myocardial ischemia electrocardio and effect of heart function, following coronary artery occlusion causes rat long-term myocardial infarction and ischemia model Rat Ecg ST section and significantly improves, its HR, arterial pressure, LVSP ,+dp/dt
max,-dp/dt
maxall significantly decline, model group rats myocardium shrinkage function obviously reduces, and after intravenously administrable 7d, each medicine group rat ST section obviously declines; Indices integrated display, with salvianolic acid A compositions middle and high dosage, effect is improved to the electrocardio of long-term rats with myocardial ischemia and parameters of left ventricular function in each medicine group the most obvious, prompting salvianolic acid A compositions can reduce rats with myocardial ischemia ST section rising, improve heart rate, increase its arterial pressure, improve cardiac muscle and to relax contracting power, and effect is better than same dosage salvianolic acid A monomeric compound, the electrocardio of rats with myocardial ischemia and cardiac function are improved significantly.
Known through experimental data contrast; salvianolic acid A compositions can make SOD activity in rats with myocardial ischemia serum increase; suppress the generation of MDA, reduce AST, CK and LDH activity in serum, illustrate that salvianolic acid A compositions can suppress oxygen-derived free radicals to produce; improve the oxidation resistance of cardiac muscular tissue; protecting myocardial cell film, reduces the spilling of enzyme, improves the energy supply of cardiac muscle; reduce ischemia to the degree of injury of cardiac muscle, thus play ischemic myocardial protection effect.
Known through experimental data contrast, there is obvious myocardial ischemia infarction in myocardial infarction and ischemia model group rat, infarction size reaches about 53%; Compared with model group, after various dose salvianolic acid A compositions drug administration by injection 7d, the myocardial infarct size of rat significantly reduces, and presents certain dose-effect relationship.Less compared with salvianolic acid A monomeric compound group rat myocardial infarction model scope with dosage salvianolic acid A compositions.Prompting salvianolic acid A compositions can reduce myocardial infarct size, alleviates treating myocardial ischemia damage and heart infarction degree, has obvious therapeutic action to myocardial ischemia.
Light microscopic and the prompting of Electronic Speculum result, the myocardium markers that salvianolic acid A compositions obviously can improve rats with myocardial ischemia changes, and alleviates myocardium markers damage, can be used for the treatment of ischemic heart desease.And action effect is more obvious than the salvianolic acid A monomeric compound with dosage.
Observation myocardial ischemia in rats-fill with different time myocardial infarct size again, model group rats myocardial ischemia 1.5h, after Reperfu-sion 6h, myocardial infarction is serious, and in recovery perfusion 48h, infarction size progressively increases, and myocardial damage aggravates.Known through experimental data contrast, compare with model group, various dose salvianolic acid A compositions obviously can reduce myocardial infarct size, alleviate myocardial ischemia-reperfusion damage, and myocardial infarct size does not expand with Ischemia Reperfusion time lengthening; There is certain agent effect relationship between each dosage group, and be better than same dosage salvianolic acid A monomeric compound.Prompting salvianolic acid A compositions can reduce the myocardial infarct size of expeirmental myocardial ischemia Reperfu-sion rat, alleviates Myocardial injury degree, has good preventive and therapeutic effect to Myocardial Ischemia Reperfusion Injury.
Known through experimental data contrast, rat is at ischemia-reperfusion 24h, cardiac muscular tissue SOD, GSH-Px, CAT enzyme is lived and is significantly declined, MDA, XOD enzyme is lived and is significantly raised, after prompting myocardial ischemia in rats-Reperfu-sion, Peroxidation Product is piled up serious, and myocardial clearance Peroxidation Product ability significantly declines, and Ischemia Reperfusion causes the generation of a large amount of oxygen-derived free radicals to cause myocardial damage to increase the weight of.Medicine group compares with model group, and cardiac muscular tissue SOD, GSH-Px, CAT enzyme is lived and raised in various degree, and MDA, XOD enzyme is lived and reduced in various degree, and salvianolic acid A combination object height, middle dosage group effect are the most obvious.Prompting salvianolic acid A can strengthen myocardial clearance free radical ability, suppresses oxygen-derived free radicals to produce, strengthens rat heart muscle oxidation resistance, suppress Myocardial Ischemia Reperfusion Injury.
Known through experimental data contrast, after ligation dog coronary artery after 60min to ligation during 180min (namely after administration after 45min to medicine during 165min), each medicine group dog epicardial electrogram ST section raises sum (∑-ST) continuous decrease, more all has significant difference with model group; After recovering Reperfu-sion, each medicine group ∑-ST significantly reduces with model group.A certain amount of effect relationship is there is between salvianolic acid A compositions each dosage group, in salvianolic acid A compositions, dosage group ∑-ST fall is suitable with 0.5mg/kg hydrochloride for injection diltiazem group, and salvianolic acid A compositions declines more obvious than the ∑-ST with dosage salvianolic acid A monomeric compound group.Prompting salvianolic acid A compositions can alleviate the degree of myocardial ischemia of experiment ischemia-reperfusion dog, and effect is better than same dosage salvianolic acid A monomeric compound.Point out it to damage by Ischemic myocardium, to ischemic heart desease, there is good therapeutical effect.
Known through experimental data contrast, after ligation dog coronary artery after 60min to ligation during 180min (namely after administration after 45min to medicine during 165min), each medicine group dog epicardial electrogram N-ST still continues to reduce, and more all has significant difference with model group; After recovering Reperfu-sion, each medicine group N-ST significantly reduces with model group.A certain amount of effect relationship is there is between salvianolic acid A compositions each dosage group, in salvianolic acid A compositions, dosage group N-ST slip is suitable with 0.5mg/kg hydrochloride for injection diltiazem group, and salvianolic acid A compositions reduces obviously than the N-ST with dosage salvianolic acid A monomeric compound group.Prompting salvianolic acid A compositions can reduce the myocardial ischemia scope of experiment ischemia-reperfusion dog, and effect is better than same dosage salvianolic acid A monomeric compound.Point out it to damage by Ischemic myocardium, to ischemic heart desease, there is good therapeutical effect.
Known through experimental data contrast, after model group dog ischemia-reperfusion, myocardial infarction is heavier.Salvianolic acid A compositions, hydrochloride for injection diltiazem, salvianolic acid A monomeric compound all can significantly reduce experimental dog myocardial infarct size, and minimum with salvianolic acid A compositions high dose group dog myocardial infarction ratio, in salvianolic acid A compositions, dosage group myocardial infarct size is less than same dosage salvianolic acid A monomeric compound group.Compare with model group; salvianolic acid A combination object height, middle dosage group extremely significantly can reduce the proportion that infarcted myocardium accounts for heart and ventricle; the proportion that salvianolic acid A compositions low dosage dog myocardial infarction district accounts for heart and ventricle also obviously reduces; prompting salvianolic acid A compositions can dose-dependently reduce experimental dog myocardial infarct size; energy Ischemic myocardium-reperfusion injury, is used for the treatment of ischemic heart desease.And effect is better than same dosage salvianolic acid A monomeric compound.
Known through experimental data contrast, ligation dog coronary artery forms myocardial ischemia, 225min (i.e. ligation 60min to 240min) after 45min to administration after administration, each medication therapy groups coronary flow compares with before administration, adds between 25.3% ~ 52.6%; With salvianolic acid A combination object height, middle dosage group, hydrochloride for injection diltiazem group significantly, amplification is respectively 52.6%, 40.2%, 36.7%.In salvianolic acid A compositions, dosage group amplification is higher than same dosage salvianolic acid A monomeric compound group.Prompting salvianolic acid A compositions can promote that collateral circulation is open, increases coronary flow during myocardial ischemia, thus to resisting myocardial ischemia, Ischemic myocardium damages, and points out it in control ischemic heart desease, have certain purposes.
Known through experimental data contrast, after the administration of salvianolic acid A compositions, 15min (i.e. ischemia 30min) significantly can suppress LVEDP rising, suppression ± dp/dt
maxdecline, suppress dog LVW to reduce, reduce TPR, after administration, 45min (namely after ischemia 60min) can significantly suppress CO to decline; In dose dependent; There is the trend being better than same dosage salvianolic acid A monomeric compound.After the administration of prompting salvianolic acid A compositions, myocardial ischemia dog hemodynamic index can be improved, the myocardial contraction of Enhancement test dog, improve myocardial systolic property, reduce Peripheral resistance, suppress cardiac output to reduce, improve the blood-pumping function of heart, improve myocardial blood supply, improve myocardial ischemia and cardiac function, play function of resisting myocardial ischemia.
Known through experimental data contrast, in myocardial infarction and ischemia model group dog serum, CK, LDH, AST, CK-MB significantly raise, and after ligation coronary ischemia is described, dog myocardial mitochondria is destroyed, myocardial cell injury, and myocardium enzyme is released the rising alive of hyperamization clear center creatase.The each dosage group of salvianolic acid A compositions compares with model group, to reduce after dog myocardial ischemia 4h CK, LDH, AST, CK-MB in serum to some extent active, and in dose dependent; Middle dosage salvianolic acid A compositions group ratio is with dosage salvianolic acid A monomeric compound group more remarkable effect.Illustrate that salvianolic acid A compositions can stablize myocardial cell membrane, reduce the spilling of myocardium enzyme during treating myocardial ischemia damage, to ischemic myocardium, there is obvious protective effect.
Known through experimental data contrast, model group dog is after ischemia 4h, serum free fatty acid (FFA), lipid peroxide (LPO) significantly raise, after prompting myocardial ischemia there is obstacle in myocardial fatty acid metabolic, the activity of glucose oxidase Major Enzymes will be suppressed, thus increase myocardial oxygen consumption, expand myocardial infarct size.And each dosage group of salvianolic acid A compositions compares with model group, FFA, LPO content in myocardial ischemia dog serum significantly can be reduced, and in dose-dependence.Prompting salvianolic acid A compositions can significantly suppress FFA level in serum to raise, and improves myocardial fatty acid metabolic, reduces the accumulation of lactic acid; increase the production capacity of unit oxygen consumption, reduce myocardial oxygen consumption, thus improve cardiac function; suppress myocardial infarct size, Ischemic myocardium damages.
Known through experimental data contrast, after model group dog myocardial ischemia 4h, in serum, MDA content significantly raises, SOD, GSH-Px are active significantly to decline, after prompting dog myocardial ischemia, oxygen-derived free radicals generates obviously to be increased, in body, the enzyme of scavenging activated oxygen is lived and is significantly reduced, and oxygen-derived free radicals can affect normal configuration and the function of cell by a series of oxidation reaction, aggravation treating myocardial ischemia damage degree.The each dosage group of salvianolic acid A compositions compares with model group, significantly can reduce the content of MDA in myocardial ischemia dog serum, makes the active obviously enhancing of SOD, GSH-Px in serum simultaneously, and in certain agent effect relationship; Prompting salvianolic acid A compositions is by certain machine-processed anti-lipid peroxidation process, reduce oxygen-derived free radicals to generate, endogenous anti-oxidative enzymatic activity can be improved again and alleviate the damage of oxygen-derived free radicals to cardiac muscle simultaneously, improve the oxidation resistance of ischemic myocardium and play function of resisting myocardial ischemia.And effect is better than same dosage salvianolic acid A monomeric compound.
Known through experimental data contrast, the salvianolic acid A compositions of various dose can reduce myocardial oxygen consumption and coefficient of oxygen utilization to some extent, and wherein salvianolic acid A combination object height, middle dosage group ischemia 120min (i.e. 105min after administration) coefficient of oxygen utilization comparatively reduce nearly 20%, 16% before administration respectively.Illustrate that salvianolic acid A compositions can reduce myocardial oxygen consumption and coefficient of oxygen utilization, improve the equilibrium of supply and demand of myocardium oxygen, improve ventricular function, play the effect of protection ischemic myocardium, control myocardial ischemia.
Known through experimental data contrast, various dose salvianolic acid A compositions can make myocardial ischemia dog blood viscosity, plasma viscosity, packed cell volume and platelet adhesion rate reduce to some extent, obviously reduces fiber protein content in blood plasma; In certain agent effect trend between each dosage group; Prompting salvianolic acid A compositions can reduce blood viscosity, suppresses platelet adhesion to be assembled, and prevention Intravascular Thrombus is formed, and improves myocardial ischemia dog hemorheology, improves microcirculation, can be used for preventing and treating ischemic heart desease.
Known through experimental data contrast, the salvianolic acid A compositions of various dose significantly can alleviate that rat suppository is wet, dry weight, has obvious inhibitory action to thrombosis; In certain dose-effect relationship between each dosage group, and inhibition is better than same dosage salvianolic acid A monomeric compound.Prompting salvianolic acid A compositions has the effect suppressing rat thrombus in vivo to be formed, and points out it to can be used for preventing and treating the ischemic heart desease caused by thrombosis.
Medical science and study of pharmacy personnel in advance under the prerequisite not doing related experiment, cannot learn that salvianolic acid A compositions has above-mentioned good purposes in advance.
Accompanying drawing explanation
Fig. 1. salvianolic acid A hydrogen nuclear magnetic resonance spectrogram;
Fig. 2. salvianolic acid A carbon-13 nmr spectra figure;
Fig. 3. alkannic acid hydrogen nuclear magnetic resonance spectrogram;
Fig. 4. alkannic acid carbon-13 nmr spectra figure;
Fig. 5. rosmarinic acid hydrogen nuclear magnetic resonance spectrogram;
Fig. 6. rosmarinic acid carbon-13 nmr spectra figure;
Fig. 7. salvianolic acid B hydrogen nuclear magnetic resonance spectrogram;
Fig. 8. salvianolic acid B carbon-13 nmr spectra figure;
Fig. 9. salvianolic acid C hydrogen nuclear magnetic resonance spectrogram;
Figure 10. salvianolic acid C carbon-13 nmr spectra figure;
Figure 11. mixing reference substance solution HPLC figure;
Figure 12. alkannic acid reference substance solution HPLC schemes;
Figure 13. rosmarinic acid reference substance solution HPLC schemes;
Figure 14. salvianolic acid B reference substance solution HPLC schemes;
Figure 15. salvianolic acid A reference substance solution HPLC schemes;
Figure 16. salvianolic acid C reference substance solution HPLC schemes;
Figure 17. salvianolic acid A composition sample HPLC schemes;
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, with microwave vacuum drying (baking temperature 50 DEG C, return difference temperature 4 DEG C, more than vacuum-0.07Mpa, microwave power 60KW) 130 minutes, obtain salvianolic acid A compositions.
After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.57%, alkannic acid content 0.663%; Rosmarinic acid contents 1.16%; Content of danshinolic acid B 1.24%; Salvianolic acid C content 1.48%.
Embodiment 2
Get red rooted salvia, be ground into diameter and be about 2mm granule, add 7 times amount, 90 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 1.17 (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 3.5, 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-t-butyl methyl ether (4: 6) eluting 10 times of column volumes respectively, 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 10 times amount water dissolutioies, with microwave vacuum drying (baking temperature 60 DEG C, return difference temperature 2 DEG C, more than vacuum-0.07Mpa, microwave power 40KW) 110 minutes, obtain salvianolic acid A compositions.
After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.63%, alkannic acid content 0.69%; Rosmarinic acid contents 1.12%; Content of danshinolic acid B 1.20%; Salvianolic acid C content 1.48%.
Embodiment 3
Get red rooted salvia, be cut into decoction pieces, add the 85 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 1.18 (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 5.0, add 0.6%ZnCl2 as 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 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, the salvianolic acid A reclaimed after organic solvent adds 8 times amount water dissolutioies, with microwave vacuum drying (baking temperature 50 DEG C, return difference temperature 5 DEG C, more than vacuum-0.07Mpa, microwave power 80KW) 90 minutes, obtain salvianolic acid A compositions.
After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.68%, alkannic acid content 0.68%; State repeatedly fragrant acid content 1.07%; Content of danshinolic acid B 1.19%; Salvianolic acid C content 1.47%.
Embodiment 4
Get red rooted salvia, be ground into diameter 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 1.20 (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%ZnCl
2as 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-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 40 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 7, uses 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 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 by the t-butyl methyl ether 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 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 9 times of column volumes, reclaim under reduced pressure eluant, the salvianolic acid A reclaimed after organic solvent adds 8 times amount water dissolutioies, with microwave vacuum drying (baking temperature 70 DEG C, return difference temperature 1 DEG C, more than vacuum-0.07Mpa, microwave power 20KW) 100 minutes, obtain salvianolic acid A compositions.
After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.72%, alkannic acid content 0.62%; Rosmarinic acid contents 1.10%; Content of danshinolic acid B 1.28%; Salvianolic acid C content 1.46%.
Embodiment 5
Get red rooted salvia, be cut into decoction pieces, add 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 1.19 (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 4.4, add 0.8%ZnCl2 as 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-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 40 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 7, 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: 10 with polyamide 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 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 0.5g, 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: t-butyl methyl ether is 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, the salvianolic acid A reclaimed after organic solvent adds 10 times amount water dissolutioies, with microwave vacuum drying (baking temperature 50 DEG C, return difference temperature 2 DEG C, more than vacuum-0.07Mpa, microwave power 30KW) 110 minutes, obtain salvianolic acid A compositions.
After measured, in described salvianolic acid A compositions, salvianolic acid A content is 96.13%, alkannic acid content 0.55%; Rosmarinic acid contents 1.00%; Content of danshinolic acid B 1.02%; Salvianolic acid C content 1.34%.
Embodiment 6
Get red rooted salvia, be ground into diameter 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 1.25 (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 5.5, adds 0.4%ZnCl
2as 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-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 35 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 8, use 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 polyamide chromatography post, salvianolic acid A applied sample amount is 1: 12 with polyamide ratio, resin column blade diameter length ratio is 1: 18, use 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 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 0.5g, 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, t-butyl methyl ether is 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, the salvianolic acid A reclaimed after organic solvent adds 12 times amount water dissolutioies, with microwave vacuum drying (baking temperature 65 DEG C, return difference temperature 5 DEG C, more than vacuum-0.07Mpa, microwave power 90KW) 80 minutes, obtain salvianolic acid A compositions.
After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.64%, alkannic acid content 0.69%; Rosmarinic acid contents 1.05%; Content of danshinolic acid B 1.11%; Salvianolic acid C content 1.47%.
Embodiment 7
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 1.23 (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%ZnCl
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-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 40 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 9, uses 4 times of cylinder hydrops, 4 times of column volume 22% ethanol elutions respectively, removing impurity, use 6 times of column volume 43% ethanol elutions again, HPLC detects, and 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 polyamide chromatography post, salvianolic acid A applied sample amount is 1: 15 with polyamide ratio, resin column blade diameter length ratio is 1: 20, use 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 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 0.5g, 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, t-butyl methyl ether is eluant, gradient elution, use pentane-t-butyl methyl ether (4: 6) eluting 6 times of column volumes respectively, pentane-t-butyl methyl ether (6: 4) eluting 7 times of column volumes, reclaim under reduced pressure eluant, the salvianolic acid A reclaimed after organic solvent adds 10 times amount water dissolutioies, with microwave vacuum drying (baking temperature 55 DEG C, return difference temperature 3 DEG C, more than vacuum-0.07Mpa, microwave power 50KW) 120 minutes, obtain salvianolic acid A compositions.
After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.72%, alkannic acid content 0.70%; Rosmarinic acid contents 1.16%; Content of danshinolic acid B 1.24%; Salvianolic acid C content 1.47%.
Embodiment 8
Get red rooted salvia, be cut into decoction pieces, add 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 1.08 (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 3.5, 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 HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 36 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 9, uses 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, t-butyl methyl ether is 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, the salvianolic acid A reclaimed after organic solvent adds 8 times amount water dissolutioies, with microwave vacuum drying (baking temperature 50 DEG C, return difference temperature 2 DEG C, more than vacuum-0.07Mpa, microwave power 60KW) 120 minutes, obtain salvianolic acid A compositions.
After measured, in described salvianolic acid A compositions, salvianolic acid A content is 94.84%, alkannic acid content 0.63%; Rosmarinic acid contents 1.12%; Content of danshinolic acid B 1.25%; Salvianolic acid C content 1.47%.
Experimental example 1: salvianolic acid A compositions analytical method and separation, Structural Identification research
One, salvianolic acid A compositions analytical method research
1. instrument and reagent
Instrument: 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.
Rosmarinic acid reference substance, salvianolic acid B reference substance all purchased from Nat'l Pharmaceutical & Biological Products Control Institute, for assay; Alkannic acid reference substance, salvianolic acid C reference substance are all purchased from Hai Can bio tech ltd, Shanghai, and 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
2.1 the preparation of reference substance solution: precision takes alkannic acid reference substance and is about 10.00mg respectively, rosmarinic acid reference substance is about 10.00mg, salvianolic acid B reference substance is about 10.00mg, salvianolic acid C reference substance is about 10.00mg, salvianolic acid A reference substance is about 10.00mg, put in different 100ml measuring bottle, add dissolve with methanol respectively and be diluted to scale, shake up, accurate absorption alkannic acid reference substance respectively, rosmarinic acid reference substance, salvianolic acid B reference substance, the each 10ml of salvianolic acid C reference substance, put in different 100ml measuring bottle, add dissolve with methanol respectively and be diluted to scale, shake up, product stock solution in contrast.The each 10ml of the above-mentioned stock solution of accurate absorption, puts in same 100ml 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 and is about 10.00mg, puts in 50ml measuring bottle, adds dissolve with methanol and be diluted to scale, in accurate absorption 10ml to 100ml measuring bottle, adds methanol dilution to scale, shakes 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: 286nm; Column temperature 30 DEG C; Number of theoretical plate calculates should be not less than 10000 by salvianolic acid A peak.
When 0 ~ 10 minute, the ratio of methanol rises to 40% by 30%, and the ratio of 0.1 ~ 0.5% phosphate aqueous solution is down to 60% by 70%; When 10 ~ 30 minutes, the ratio of methanol rises to 55% by 40%, and the ratio of 0.1 ~ 0.5% phosphate aqueous solution is down to 45% by 50%; When 30 ~ 60 minutes, the ratio of methanol rises to 80% by 55%, and the ratio of 0.1 ~ 0.5% phosphate aqueous solution is down to 20% by 45%.
Under these conditions, the chromatographic peak retention time of 5 reference substances is in table 1, and Figure 11 ~ 17 are shown in by HPLC collection of illustrative plates.
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 series standard solution.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 compositions, 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 compositions 10mg of known content, accurately weighed, parallel 6 parts, add corresponding reference substance solution by each constituent content equal proportion respectively, by 2.3 below legal system available test sample solutions, measure and calculate average recovery and the RSD of each composition.Result shows that the accuracy of the method is good, in table 6.
Table 6. recovery test result
Two, salvianolic acid A compositions separation, Structural Identification research
Embodiment 1 is obtained above-mentioned salvianolic acid A compositions respectively with CG161M macroporous adsorbent resin, ODS, SephadexLH-20 column chromatography for separation, with water: ethanol gradient elution, the ratio of water is 80 ~ 20%, the ratio of ethanol is 20 ~ 80%, Fractional Collections eluent, merges same composition, crystallization, salvianolic acid A can be obtained, the monomeric compound of alkannic acid, rosmarinic acid, salvianolic acid B, salvianolic acid C, measures described monomeric compound structure, wherein, salvianolic acid A, molecular formula: C
26h
222o
10, molecular weight: 494.45, structure is as follows:
Salvianolic acid A physicochemical property and spectral data, see Fig. 1,2.
Salvianolic acid A (SalvianolicacidA), pale yellow powder, easily easy methanol, water.ESIMSm/z:493 [M-H]
-, 518 [M+Na]
+, molecular formula: C
26h
22o
10, molecular weight: 494.45;
1h (600MHz, CD
3oD) and
13cNMR (150MHz, CD
3oD) data are as follows:
1NMR(CD
3OD，600MHz)δ：2.96(1H，dd，J＝14.4，8.4Hz，H-7``)，3.06(1H，dd，J＝14.4，4.2Hz，H-7``)，5.17(1H，dd，J＝9.0，4.2Hz，H-8``)，6.27(1H，d，J＝16.2Hz，H-8`)，6.54(1H，dd，J＝7.8，1.8Hz，H-6``)，6.63(1H，d，J＝7.8Hz，H-5``)，6.65(1H，d，J＝16.2，Hz，H-7)，6.72(1H，d，J＝8.4Hz，H-4`)，6.74(1H，d，J＝8.4Hz，H-5)，6.76(1H，d，J＝1.8Hz，H-2``)，6.88(1H，dd，J＝8.4，1.8Hz，H-6)，7.05(1H，d，J＝1.8Hz，H-2)，7.11(1H，d，J＝8.4Hz，H-5`)，7.14(1H，d，J＝16.2Hz，H-8)；8.06(1H，d，J＝16.2Hz，H-7`).
13CNMR(CD
3OD，150MHz)δ：36.6(C-7``)，73.3(C-8``)，112.6(C-2)，113.4(C-5)，114.2(C-8`)，114.9(C-4`)，115.1(C-5``)，116.0(C-2``)，118.7(C-5`)，119.6(C-6)，119.3(C-8)，120.6(C-6``)，124.7(C-6`)，127.0(C-1)，127.9(C-1``)，130.0(C-1`)，136.5(C-7)，
143.1(C-3)，143.9(C-3``)，144.8(C-4``)，145.1(C-2`)，145.4(C-4)，146.0(C-7`)，146.9(C-3`)，167.3(C-9`)，172.1(C-9``).
Alkannic acid, its molecular formula: C
27h
22o
12, molecular weight: 538.46, structure is as follows:
Alkannic acid physicochemical property and spectral data, see Fig. 3,4.
Alkannic acid (lithospermicacid), pale yellow powder, easily easy methanol, water.ESIMSm/z:539 [M+H]
+, 537 [M-1]
-, 560 [M+Na]
-, 492 [M-COOH]
-; Molecular formula: C
27h
22o
12, molecular weight 538.45;
1h (600MHz, CD
3oD) and
13cNMR (150MHz, CD
3oD) data are as follows:
1NMR(CD
3OD，600MHz)δ：3.02(1H，dd，J＝14.4，9.6Hz，H-7``)，3.06(1H，dd，J＝14.4，3.0Hz，H-7``)，4.25(1H，dd，J＝5.4Hz，H-8`)，4.97(1H，dd，J＝9.6，3.0Hz，H-8``)，5.88(1H，d，J＝5.4Hz，H-7`)，6.31(1H，d，J＝16.0Hz，H-8)，6.59(1H，dd，J＝8.0，1.8Hz，H-6``)，6.65(1H，d，J＝8.0Hz，H-5``)，6.73(1H，dd，J＝8.4，2.0Hz，H-6`)，6.74(1H，d，J＝1.8Hz，H-2``)，6.74(1H，d，J＝8.4Hz，H-5`)，6.83(1H，d，J＝8.4Hz，2.4Hz，H-5)，6.97(1H，d，J＝2.0Hz，Hz，H-2`)，7.13(1H，d，J＝8.4Hz，H-6)；7.93(1H，d，J＝16.0Hz，H-7).
13CNMR(CD
3OD，150MHz)δ：37.3(C-7``)，59.9(C-8`)，76.7(C-8``)，88.9(C-7`)，112.4(C-2``)，114.6(C-5``)，114.9(C-5`)，115.7(C-8)，116.1(C-2`)，117.2(C-6``)，119.5(C-6)，119.9(C-6`)，123.5(C-1)，129.3(C-2)，129.8(C-1``)，133.4(C-1`)，142.4(C-7)，143.3(C-4)，143.4(C-3``)，144.7(C-4``)，145.1(C-3`)，145.1(C-4`)，147.4(C-3)，167.8(C-9)，176.5(C-9``)，178.5(C-9`).
The molecular formula of rosmarinic acid: C
18h
16o
8, molecular weight: 360.31, structure is as follows:
Rosmarinic acid physicochemical property and spectral data, see Fig. 5,6.
Rosmarinic acid (RosMarinicacid), white powder, easily easy methanol, water.ESIMSm/z:359 [M-H]
-, 383 [M+Na]
+; Molecular formula: C
18h
16o
8, molecular weight 360.31;
1h (600MHz, CD
3oD) and
13cNMR (150MHz, CD
3oD) data are as follows:
1NMR(CD
3OD，600MHz)δ：3.01(1H，dd，J＝14.4，8.4Hz，H-7`)，3.10(1H，dd，J＝14.4，4.2Hz，H-7`)，5.18(1H，dd，J＝8.4，4.2Hz，H-8`)，6.27(1H，d，J＝16.0Hz，H-8)，6.61(1H，dd，J＝7.8，1.8Hz，H-6`)，6.70(1H，d，J＝7.8Hz，H-5`)，6.75(1H，d，J＝1.8Hz，H-2`)，6.78(1H，d，J＝8.4Hz，H-5)，6.95(1H，dd，J＝8.4，2.4Hz，H-6)，7.04(1H，d，J＝1.8Hz，H-2)，7.55(1H，d，J＝16.0Hz，H-7)；
13CNMR(CD
3OD，150MHz)δ：36.6(C-7`)，73.2(C-8`)，113.1(C-8)，113.9(C-2)，114.9(C-5)，115.1(C-5`)，116.2(C-2`)，120.4(C-6)，121.8(C-6`)，126.3(C-1)，127.9(C-1`)，143.9(C-3)，144.8(C-4)，145.5(C-3`)，146.4(C-4`)，148.4(C-7)，167.1(C-9)，172.1(C-9`).
Salvianolic acid B, its molecular formula: C
36h
30o
16, molecular weight: 718.61, structure is as follows:
Salvianolic acid B physicochemical property and spectral data, see Fig. 7,8.
Salvianolic acid B (SalvianolicacidB), pale yellow powder, easily easy methanol, water.ESIMSm/z:717 [M-H]
-, 741 [M+Na]
+; Molecular formula: C
36h
30o
16, molecular weight 718.62;
1h (600MHz, CD
3oD) and
13cNMR (150MHz, CD
3oD) data are as follows:
1NMR(CD
3OD，600MHz)δ：2.80-3.10(2H，m，H-7``)，2.80-3.10(2H，m，H-7```)，4.35(1H，d，J＝4.8Hz，H-8``)，5.15-5.19(1H，m，H-8`)，5.15-5.19(1H，m，H-8```)，5.85(1H，d，J＝4.8Hz，H-7``)，6.20(1H，d，J＝16.2Hz，H-8)，6.31(1H，dd，J＝8.1，2.1Hz，H-6`)，6.52(1H，d，J＝2.4Hz，H-2```)，6.54(1H，d，J＝7.8Hz，H-5``)，6.62(1H，d，J＝2.1Hz，H-2`)，6.66(1H，dd，J＝8.1，2.4Hz，H-6```)，6.70(1H，d，J＝7.8Hz，H-5`)，6.74(1H，d，J＝8.1Hz，H-5```)，6.75(1H，dd，J＝7.8，2.4Hz，H-6``)，6.76(1H，d，J＝2.4Hz，H-2``)，6.83(1H，d，J＝8.4Hz，H-5)，7.16(1H，d，J＝8.4Hz，H-6)，7.52(1H，d，J＝16.2Hz，H-7)；
13CNMR(CD
3OD，150MHz)δ：36.111(C-7```)，36.51(C-7`)，56.58(C-8``)，73.28(C-8)，74.19(C-8```)，86.93(C-7``)，111.99(C-2``)，115.01(C-5``)，115.04(C-5`)，115.13(C-2`)，115.18(C-2```)，115.95(C-8)，116.20(C-6``)，116.97(C-5```)，117.04(C-5)，120.37(C-6)，120.89(C-6```)，123.28(C-1)，125.04(C-2)，127.55(C-1`)，127.85(C-1```)，132.27(C-1``)，142.20(C-7)，143.70(C-3```)，143.72(C-4```)，143.88(C-4``)，144.57(C-3``)，144.73(C-4`)，145.23(C-3`)，145.39(C-3)，147.71(C-4)，166.66(C-9)，170.92(C-9```)，171.17(C-9``)，172.29(C-9`).
Salvianolic acid C, its molecular formula: C
26h
20o
10, molecular weight: 492.43, structure is as follows:
Salvianolic acid C physicochemical property and spectral data, see Fig. 9,10.
Salvianolic acid C (SalvianolicacidC), pale yellow powder, easily easy methanol, water.ESIMSm/z:491 [M-H]
-, 515 [M+Na]
+; Molecular formula: C
26h
20o
10, molecular weight 492.43;
1h (600MHz, CD
3oD) and
13cNMR (150MHz, CD
3oD) data are as follows:
1NMR(CD
3OD，600MHz)δ：3.07(1H，dd，J＝13.8，8.4Hz，H-7``)，3.15(1H，dd，J＝13.8，4.2Hz，H-7``)，5.25(1H，dd，J＝8.4，4.2Hz，H-8``)，6.46(1H，d，J＝15.9Hz，H-8)，6.46(1H，dd，J＝8.1，2.1Hz，H-2``)，6.72(1H，d，J＝8.1Hz，H-3``)，3.74(1H，d，J＝8.4Hz，H-5)，6.80(1H，d，J＝2.4Hz，H-6``)，6.88(1H，d，J＝7.8Hz，H-5`)，7.20(1H，s，，H-8`)，7.36(1H，d，J＝7.8Hz，H-6)，7.37(1H，dd，J＝7.8Hz，2.4Hz，H-6`)，7.40(1H，d，J＝2.4Hz，2.1Hz，H-2`)，7.94(1H，d，J＝15.9Hz，H-7)；
13CNMR(CD
3OD，150MHz)δ：36.2(C-7``)，73.3(C-8``)，97.9(C-8`)，110.4(C-5)，112.0(C-2`)，113.5(C-8)，114.9(C-5``)，115.4(C-5`)，116.3(C-2``)，117.4(C-6`)，118.2(C-1)，120.5(C-6``)，122.0(C-1`)，125.0(C-6)，128.0(C-1``)，131.3(C-2)，143.0(C-3)，
143.8(C-7)，144.0(C-3``)，144.6(C-4)，144.8(C-4``)，145.3(C-2`)，146.7(C-4`)，158.0(C-7`)，167.3(C-9)，172.4(C-9``).
Experimental example 2: the extraction of salvianolic acid B
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 table
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
Table 13. the results of analysis of variance table
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 composition process and compares
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, cooling, calculates salvianolic acid A compositions productive rate.
Table 14. salvianolic acid B transforms salvianolic acid A compositions experimental result
Table 14 result shows, and salvianolic acid B high-purity, on conversion not impact, does not need to be purified to more than 50% and transforms, salvianolic acid B is converted in salvianolic acid A compositions process, adjust ph, adds 1.0%ZnCl2, greatly can improve the productive rate of salvianolic acid A compositions.
Experimental example 4: salvianolic acid B transforms the optimization of salvianolic acid A composition process
Above-mentioned experimentation proves, salvianolic acid B purity is not affect the factor that salvianolic acid B transforms salvianolic acid A compositions, but add certain catalysts influence salvianolic acid B and transform salvianolic acid A compositions, therefore, we all add 1%ZnCl2 as catalyst to each experimental group, other factors on affecting salvianolic acid B and be converted into salvianolic acid A compositions: 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 compositions.
Table 15. transforming agent water-glass
Table 16. conversion process orthogonal experiments table
Table 17. the results of analysis of variance table
*F
0.05(2，2)＝19.00ΔF
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
2factor A (salvianolic acid B concentration) has certain influence to salvianolic acid A compositions productive rate, analyze 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 compositions productive rate, should strictly control pH and temperature in prompting salvianolic acid B conversion process, successfully can realize transforming to salvianolic acid A compositions of salvianolic acid B higher yields.
Experimental example 5: catalyst Z nCl
2consumption is on the impact transformed
Salvianolic acid A composition process optimal conditions is transformed by above-mentioned salvianolic acid B, get salvianolic acid B raw material, add purified water 200l, 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: different catalyst transforms the catalytic action of salvianolic acid A compositions to salvianolic acid B
Salvianolic acid A composition process optimal conditions 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 salvianolic acid A compositions to red B
Table 19 result shows, and above 4 kinds of catalyst all can as the catalyst in salvianolic acid B conversion reaction, but resultant effect is not as good as ZnCl
2the changing effect that catalyst reaches, therefore optimal choice ZnCl of the present invention
2catalyst.
Experimental example 7: the purification with macroreticular resin of salvianolic acid A compositions
Get salvianolic acid A compositions 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 compositions 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: the adsorption effect of above 13 kinds of macroporous resins to salvianolic acid A is good, and can well impurity be removed with the ethanol gradient elution of variable concentrations, obtain the salvianolic acid A compositions eluent that content is greater than 75%, maximum with the HPD-100 amount of getting dry extract, salvianolic acid A composition levels is high, and resin absorption amount is large.
Experimental example 8: the polyamide column chromatography purification of salvianolic acid A compositions
Get the salvianolic acid A composition 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 compositions eluent that content is about 90%; Maximum with the polyamide amount of getting dry extract, content high adsorption capacity is large.
Experimental example 9: the abstraction purification of salvianolic acid A compositions
By 70% ethanol elution decompression recycling ethanol of polyamide 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 compositions extract, 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 of composition is maximum, but its salvianolic acid content is not improved significantly, ether due to polarity less than normal, water-solubility impurity is few, salvianolic acid A content is high, but the salvianolic acid A amount of composition obtained is few, the salvianolic acid A amount of composition that other Extraction solvent t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate, Ethyl formate obtain is larger, salvianolic acid A content is all improved, and obtains that compositions is many, salvianolic acid A content is high with t-butyl methyl ether.
Experimental example 10: the purification by silica gel column chromatography of salvianolic acid A compositions
Get the salvianolic acid A compositions extract 12 parts of 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 compositions 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 the normal phase silica gel column chromatography of salvianolic acid A compositions, 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, impurity can well be removed, obtain highly purified salvianolic acid A compositions eluent, the two-phase solvent adopting pentane-t-butyl methyl ether composition is that eluant effect is best.
Experimental example 11: salvianolic acid A composition dries technique study
Get the salvianolic acid A compositions eluent 4 parts after silica gel column chromatography, every part containing salvianolic acid A compositions 100g, after adding 10 times amount water dissolutioies, employing respectively, vacuum drying, lyophilisation, spraying dry, microwave vacuum drying obtain salvianolic acid A compositions, said composition is detected, the results are shown in Table 24.
Table 24. salvianolic acid A composition dries method testing result
Result shows: salvianolic acid A compositions microwave vacuum drying process controls simple, and treating capacity is large, and baking temperature is low, and the time is short, and extract obtained indices is good, therefore the present invention adopts microwave vacuum drying to prepare salvianolic acid A compositions.
Determine through further testing, microwave vacuum drying optimum range is 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.
The sample that following experiment salvianolic acid A compositions equal Example 3 preparation method obtains, salvianolic acid A monomeric compound, commercially available, salvianolic acid A content more than 99.62%.
Experimental example 12: salvianolic acid A compositions is to the protective effect of SD isolated rat heart reperfusion injury
Male SD rat, body weight (280 ± 20) g; Thered is provided by Beijing Vital River Experimental Animals Technology Co., Ltd., quality certification SCXK (capital) 2007-0006.Open breast rapidly after intraperitoneal anesthesia, mention heart gently, cut off the tissue around superior and inferior vena cava, pulmonary artery, aorta and heart, taken out by heart rapidly together with one section of aorta, aortic root retains the length of about 0.5 ~ 1cm, and standby heart cathetrization is used.Move to immediately get ready in advance containing in oxygen K-H liquid (about 4 DEG C), extruding heart gently makes intraventricular residual blood discharge, and cleaning heart remained blood, hangs on Langendorff perfusion device, heart aorta ascending branch is connected on the sleeve pipe of perfusion, and fixes with cotton thread ligation.Immediately to carry out perfusion containing oxygen K-H liquid, constant temperature (37 ± 0.5) DEG C; Perfusion pressure 70mm mercury column, flow velocity (11.5 ± 0.5) ml/min.K-H liquid composition (g/L): NaCl:6.9025; KCl:0.3517; CaCl
2: 0.2837; Anhydrous MgSO
4: 0.1424; KH
2pO
4: 0.1611; NaHCO
3: 2.0916; Glu:2.0837; Adjust pH 7.4.Cut an osculum at left atrium sidewall, little latex water pocket is inserted left ventricle by atrioventricular orifice.Latex water pocket is full of ultra-pure water with in the conduit be connected, and the other end of conduit, by T joint pressure transducer, thoroughly removes inner size bubble.The water yield of water pocket in ventricle is regulated to make heart ventricle end diastolic pressure maintain 4-6mmHg scope by fine setting injector.Left indoor pressure is tested after stablizing 30min again.With the coronary flow of graduated cylinder record each minute heart; Pressure transducer signal inputs multitrack recording system by bridge amplifier, carries out real time signal aquisition and process.
Experiment divides 7 groups: Normal group: to contain oxygen K-H liquid continous perfusion 120min; Model group: with containing after oxygen K-H liquid perfusion 15min, stop filling with 25min, then recover containing oxygen K-H liquid perfusion 60min; The high, medium and low dosage group of salvianolic acid A compositions (1.0,0.5,0.25mg/L), positive drug group (diltiazem hydrochloride 0.3mg/L), salvianolic acid A compositions monomeric compound group (0.5mg/L): the pastille infusion liquid perfusion of variable concentrations when recovering perfusion, and continue whole refilling process.
Before each group of record stops filling with and after filling with again from 10min, every coronary flow (CBF), left ventricular diastolic pressure (LVEDP), maximum the climbing speed (+dp/dt of left indoor pressure of 10min
max) and maximum the fall off rate (-dp/dt of left indoor pressure
max), n=8.
Table 25. salvianolic acid A compositions is on the hemodynamic impact of isolated rat heart Reperfu-sion 60min
(
n＝8)
Note: compare with Normal group: #P < 0.01, ##P < 0.001; Compare with model group: Δ P < 0.05, * P < 0.01, * * P < 0.001
Compare with Normal group, model group ischemia-reperfusion 60min, CBF ,+dp/dt
max,-dp/dt
maxobvious decline (P < 0.01 or P < 0.001), LVEDP significantly increases (P < 0.001).Compare with model group, the high, medium and low dosage group of positive drug group, salvianolic acid A compositions, salvianolic acid A monomeric compound group These parameters all have clear improvement, and the coronary flow that ischemia-reperfusion can be suppressed to cause reduces, left room diastolic pressure rises, the maximum climbing speed of left indoor pressure declines, the maximum fall off rate of left indoor pressure declines.And salvianolic acid A compositions group high dose group is suitable with positive drug group effect, and effect is better than same dosage salvianolic acid A monomeric compound group.Dose dependent is there is between salvianolic acid A compositions various dose group.Experimental result display salvianolic acid A compositions can alleviate isolated rat heart ischemical reperfusion injury degree, and prompting salvianolic acid A compositions can obviously be improved coronary flow, improve the systolic and diastolic function of isolated heart aortic diastolic function and damaged myocardium, cardioprotection ischemical reperfusion injury.
Experimental example 13: salvianolic acid A compositions is on the impact of rats with myocardial ischemia electrocardio and cardiac function
Male SD rat, (250 ± 20) g, is provided by Beijing Vital River Experimental Animals Technology Co., Ltd., quality certification SCXK (capital) 2007-0006.Animal is divided into model group, positive drug group (hydrochloride for injection diltiazem 1.5mg/kg), the high, medium and low dosage group of salvianolic acid A compositions (2.5,5.0,10.0mg/kg), salvianolic acid A monomeric compound group (5.0mg/kg) at random; Establish sham operated rats simultaneously.
20% urethane intraperitoneal anesthesia rat, fixing.Connect respirator after tracheal intubation, exhale by the frequency of 10 ~ 12ml tidal volume, 80 times/min, continuous positive pressure breathing, inhale: exhale than being 1: 1.Now connect electrocardiograph and measure rat normal ECG.Breast is opened along between left border of sternum the 3rd, 4 ribs, extrude heart, left anterior descending coronary artery is found out between left auricle and pulmonary conus, under ramus descendens anterior arteriae coronariae sinistrae initial part, 2mm sentences circular noinvasive sewing needle 6-0 silk thread threading, rapid ligation ramus descendens anterior arteriae coronariae sinistrae (a sham operated rats threading not ligation), and be with the little plastics pipe pad of groove at ligation position with one, the ligation thereon of two rear line heads, sew up thoracic wall, recover autonomous respiration.Perform the operation and completely at once observe Rat Ecg, with left room antetheca be cyanosis and the II lead electrocardiogram display S-T section back of a bow upwards obviously raise and continue more than 30min for modeling successfully.
Each treated animal is tail vein injection relative medicine (sham operated rats and model group inject isopyknic normal saline) after modeling success, and every day injects twice, continuous 7d.30min after last administration, animal intraperitoneal anesthesia, through common carotid artery intubate to left ventricle, multiple tracks intelligence physiological signal collection analytical system is connected by pressure converter, post surgery stabilization 30min, recorded heart rate (HR), arterial pressure, left ventricular systolic pressure (LVSP), maximum the climbing speed (+dp/dt of left indoor pressure
max) and maximum the fall off rate (-dp/dt of left indoor pressure
max); Insert limb electrode monitoring standard II lead electrocardiogram simultaneously.
Table 26. salvianolic acid A compositions on the impact of rats with myocardial ischemia electrocardio and cardiac function (
n=10)
Note: compare with sham operated rats: #P < 0.01; Compare with model group: * P < 0.05, * * P < 0.01
Experimental result shows, and compare with sham operated rats, following coronary artery occlusion causes rat long-term myocardial infarction and ischemia model Rat Ecg ST section and significantly improves (P < 0.01), its HR, arterial pressure, LVSP ,+dp/dt
max,-dp/dt
maxall significantly decline (P < 0.01), model group rats myocardium shrinkage function obviously reduces.Compare with model group, after intravenously administrable 7d, each medicine group rat ST section obviously declines, every parameters of left ventricular function is improved in varying degrees (P < 0.05 or P < 0.01); And indices integrated display, with salvianolic acid A compositions middle and high dosage, effect is improved to the electrocardio of long-term rats with myocardial ischemia and parameters of left ventricular function in each medicine group the most obvious, in a certain amount of effect relationship between salvianolic acid A compositions each dosage group, prompting salvianolic acid A compositions can reduce rats with myocardial ischemia ST section rising, improve heart rate, increase its arterial pressure, improve cardiac muscle and to relax contracting power, and effect is better than same dosage salvianolic acid A monomeric compound, the electrocardio of rats with myocardial ischemia and cardiac function are improved significantly.
Experimental example 14: salvianolic acid A compositions is on the impact of enzyme work and Radical Metabolism in rats with myocardial ischemia serum
Experimental example 13 respectively organizes rat after electrocardio and parameters of left ventricular function mensuration terminate, abdominal aortic blood, get serum, by the operation of detection kit description, measure superoxide dismutase (SOD), malonaldehyde (MDA) content and aspartate amino transferase (AST) in serum, creatine kinase (CK), lactic acid dehydrogenase (LDH) activity.
Table 27. salvianolic acid A compositions is lived and metabolism of free radical to enzyme in rats with myocardial ischemia serum
(
n＝10)
Note: compare with sham operated rats: #P < 0.01; Compare with model group: * P < 0.05, * * P < 0.01
Produce a large amount of oxygen-derived free radicals during myocardial ischemia, cause damage to cardiac muscle, SOD, MDA are the objective indicators of reflection oxygen free radical injury; Extensively be present in the myocardium enzyme AST in myocardial cell, degree of injury that the activity of LDH, CK can reflect myocardial ischemia.Compare with sham operated rats, in myocardial infarction and ischemia model group rat blood serum, SOD obviously reduces, and MDA, AST, LDH, CK significantly raise (P < 0.01); Compare with model group, after each Drug therapy, in administration group rat blood serum, SOD has rising in various degree, and MDA, AST, LDH, CK all obviously reduce (P < 0.05 or P < 0.01); And it is particularly remarkable with salvianolic acid A combination object height, middle dosage group and positive drug group drug effect.Experimental result is pointed out; salvianolic acid A compositions can make SOD activity in rats with myocardial ischemia serum increase; suppress the generation of MDA, reduce AST, CK and LDH activity in serum, illustrate that salvianolic acid A compositions can suppress oxygen-derived free radicals to produce; improve the oxidation resistance of cardiac muscular tissue; protecting myocardial cell film, reduces the spilling of enzyme, improves the energy supply of cardiac muscle; reduce ischemia to the degree of injury of cardiac muscle, thus play ischemic myocardial protection effect.
Experimental example 15: salvianolic acid A compositions is on the impact of rats with myocardial ischemia myocardial infarct size
Get experimental example 14 blood sampling terminate after rat, core immediately dirty, normal saline clean, cut off right atrium, isolate left ventricle, after sucking excessive moisture, claim left ventricle weight in wet base.Below ligature, parallel coronary sulcus direction is cut into 5 of equal thickness, put into 1%TTC dye liquor, 37 DEG C of constant temperature dyeing 10min, non-infarct is dyed to redness, and infarct is not colored in canescence, and infarct is weighed, calculate infarct and heavily account for the heavy percentage ratio of left ventricle, i.e. myocardial infarct size.
Table 28. salvianolic acid A compositions on the impact of rats with myocardial ischemia myocardial infarct size (
n=10)
Note: compare with sham operated rats: #P < 0.01; Compare with model group: * * P < 0.01
There is obvious myocardial ischemia infarction in myocardial infarction and ischemia model group rat, infarction size reaches about 53%; Compared with model group, after various dose salvianolic acid A compositions drug administration by injection 7d, the myocardial infarct size of rat significantly reduces (P < 0.01), and presents certain dose-effect relationship.Less compared with salvianolic acid A monomeric compound group rat myocardial infarction model scope with dosage salvianolic acid A compositions.Prompting salvianolic acid A compositions can reduce myocardial infarct size, alleviates treating myocardial ischemia damage and heart infarction degree, has obvious therapeutic action to myocardial ischemia.
Experimental example 16: salvianolic acid A compositions is on rats with myocardial ischemia myocardium markers histology and Ultrastructural impact
Animal model and grouping administration are with experimental example 13.Administration terminates latter second day, and sacrifice of animal is cored dirty, and normal saline flushing, cuts left ventricle, each group of left ventricle of getting Some Animals at random, in 10% formaldehyde, through fixing, rinse, after dehydration, dimethylbenzene soaks into, paraffin embedding, section, and HE dyes, mounting.The microstructure of light Microscopic observation cardiac muscle.Get the left ventricle of random residual animal, fixing in 3% glutaraldehyde solution, through dehydration at different levels, soak into expoxy propane, epoxy resin Epon812 embeds, 65 DEG C of polymerizations, section, electron microscopic observation Myocardial ultrastructure.
Result shows, light Microscopic observation sham operated rats animal cardiac muscle marshalling, and heart band is clear, and only have extremely indivedual slight hypertrophy of flesh core, cardiac interstitium has no cell infiltration, without hemorrhagic necrosis.Model group compares with sham operated rats, cardiac muscle fiber obvious tumefaction, degeneration, arrangement disorder, and cardiac muscle cross striation major part disappears, the obvious hypertrophy of flesh core, spatium intermusculare fibroplasia is more, a large amount of cell infiltration, part specimen cardiac tissue necrosis, muscle glycogen is dissolved sample, has obvious stove shape myocardial infarction.Various dose salvianolic acid A compositions group, positive drug (hydrochloride for injection diltiazem) group and salvianolic acid A monomeric compound group compare with model group; pathological changes all obviously alleviates; cardiac muscle fiber mild swelling; arrange still orderly; band is more clear, hypertrophy that flesh core is slight, cardiac interstitium is without hemorrhage; have a small amount of inflammatory cell infiltration do not waited, and salvianolic acid A compositions low dose group and salvianolic acid A monomeric compound group only individual animal cardiac muscle are slight degeneration; Paired observation between each treatment group, salvianolic acid A combination object height, middle dosage and positive drug group cardiac muscle swelling and inflammatory infiltration the gentliest, have more obvious protective effect to ischemic myocardial tissue.
Observe visible sham operated rats animal cardiac muscle cell Z line under transmission electron microscope regular, each muscle segment is clear, and myofilament is evenly distributed, it is closely neat to arrange, and triplet is obvious, and structure of mitochondria is complete, and ridge is many, clear and arrangement is orderly.Model group compares with sham operated rats, sarcostyle arrangement disorder, and visible downright bad muscle fiber, mitochondrion obviously increases, vacuolar degeneration, ridge rareness or fall into disarray, and unintelligible, and muscle segment shortens, structure disturbance, myofilament fracture, downright bad or dissolving; Nuclear membrane structure is discontinuous, and pathological change is fairly obvious.Salvianolic acid A compositions low dose group and salvianolic acid A monomeric compound group structure of mitochondria substantially complete, partial mitochondrial vacuolar degeneration, myofilament arrangement is still neat, and part ridge is smudgy, and Ultrastructural change is obviously lighter than model group; Salvianolic acid A combination object height, middle dosage group, positive drug group myocardial cell Z line fundamental rule, substantially complete, the few partial mitochondrial swelling of structure of mitochondria, ridge is substantially clear, and myofilament arrangement is substantially neat, nuclear structures is complete, and ultrastructure lesions showed is obviously improved.
Light microscopic and the prompting of Electronic Speculum result, the myocardium markers that salvianolic acid A compositions obviously can improve rats with myocardial ischemia changes, and alleviates myocardium markers damage, can be used for the treatment of ischemic heart desease.And action effect is more obvious than the salvianolic acid A monomeric compound with dosage.
Experimental example 17: salvianolic acid A compositions is on the impact of Myocardial Ischemia Reperfusion Injury SD rat myocardial infarction model scope
Male SD rat, (280 ± 20) g, is divided into 7 groups at random: sham operated rats, model group, hydrochloride for injection diltiazem group (1.5mg/kg), the high, medium and low dosage group of salvianolic acid A compositions (10.0,5.0,2.5mg/kg), salvianolic acid A monomeric compound group (5.0mg/kg).
20% urethane intraperitoneal anesthesia rat, fixing.Connect respirator after tracheal intubation, exhale by the frequency of 10 ~ 12ml tidal volume, 80 times/min, continuous positive pressure breathing, inhale: exhale than being 1: 1.Now connect electrocardiograph and measure rat normal ECG.Breast is opened along between left border of sternum the 3rd, 4 ribs, extrude heart, left anterior descending coronary artery is found out between left auricle and pulmonary conus, under ramus descendens anterior arteriae coronariae sinistrae initial part, 2mm sentences circular noinvasive sewing needle 6-0 silk thread threading, rapid ligation ramus descendens anterior arteriae coronariae sinistrae (a sham operated rats threading not ligation), and be with the little plastics pipe pad of groove at ligation position with one, the ligation thereon of two rear line heads, recording ecg.With left room antetheca be cyanosis and the II lead electrocardiogram display S-T section back of a bow upwards obviously raise and continue more than 20min for modeling successfully; Myocardial ischemia, after 90 minutes, unclamps line, takes out pipe pad, realizes Reperfu-sion.Close breast, sew up, recover autonomous respiration.Ligation tail vein injection administration at once (sham operated rats and model group give isometric normal saline), be after this administered once every 2h, altogether administration 4 times; In batches after filling with 6h, 24h, 48h again anesthetized animal (fill with 24 again, 48h treated animal within second day, begin after operation 4 administrations on the same day terminate still every day drug administration by injection twice, until draw materials), fixing, cut open breast and core dirty, be separated left ventricle, after sucking excessive moisture, claim left ventricle weight in wet base.Below ligature, crosscut becomes 5 of equal thickness, puts into 1%TTC dye liquor, 37 DEG C of constant temperature dyeing 10min, non-infarct is dyed to redness, and infarct is not colored in canescence, infarct is weighed, and calculates infarct and heavily accounts for the heavy percentage ratio of left ventricle, i.e. myocardial infarct size.
Table 29. salvianolic acid A compositions on the impact of myocardial ischemia in rats-fill with again different time myocardial infarct size (
n=6)
Note: compare with sham operated rats: #P < 0.01; Compare with model group: * * P < 0.01
Model group rats myocardial ischemia 1.5h, after Reperfu-sion 6h, myocardial infarction is serious, and in recovery perfusion 48h, infarction size progressively increases, and myocardial damage aggravates.Compare with model group, various dose salvianolic acid A compositions obviously can reduce myocardial infarct size (P < 0.01), alleviate myocardial ischemia-reperfusion damage, and myocardial infarct size does not expand with Ischemia Reperfusion time lengthening; There is certain agent effect relationship between each dosage group, and be better than same dosage salvianolic acid A monomeric compound.Prompting salvianolic acid A compositions can reduce the myocardial infarct size of expeirmental myocardial ischemia Reperfu-sion rat, alleviates Myocardial injury degree, has good preventive and therapeutic effect to Myocardial Ischemia Reperfusion Injury.
Experimental example 18: salvianolic acid A compositions is on the impact of Myocardial Ischemia Reperfusion Injury SD metabolism of free radicals in rats
Modeling and medication are with experimental example 17.After recovery Reperfu-sion 24h, Animal Anesthesia, core dirty, cut off atrium, retain ventricle, ice normal saline flushing, blot surface moisture, weigh, make tissue homogenate, centrifugal (3000rpm, 10min), get supernatant, according to the operation of test kit description, detect malonaldehyde (MDA) content in tissue, superoxide dismutase (SOD), xanthine oxidase (XOD), glutathion peroxidase (GSH-Px), catalase (CAT) activity.
Table 30. salvianolic acid A compositions in rat tissue Radical Metabolism relevant enzyme live impact (
n=10)
Note: compare with sham operated rats: #P < 0.01; Compare with model group: * P < 0.05, * * P < 0.01
Experimental result shows, compare with sham operated rats, model group rats is at ischemia-reperfusion 24h, cardiac muscular tissue SOD, GSH-Px, CAT enzyme is lived and is significantly declined, MDA, XOD enzyme is lived and is significantly raised (P < 0.01), and after prompting myocardial ischemia in rats-Reperfu-sion, Peroxidation Product is piled up serious, myocardial clearance Peroxidation Product ability significantly declines, and Ischemia Reperfusion causes the generation of a large amount of oxygen-derived free radicals to cause myocardial damage to increase the weight of.Each medicine group compares with model group, cardiac muscular tissue SOD, GSH-Px, CAT enzyme is lived and is raised in various degree, MDA, XOD enzyme is lived and is reduced (P < 0.05 or P < 0.01) in various degree, and salvianolic acid A combination object height, middle dosage group effect are the most obvious.Prompting salvianolic acid A can strengthen myocardial clearance free radical ability, suppresses oxygen-derived free radicals to produce, strengthens rat heart muscle oxidation resistance, suppress Myocardial Ischemia Reperfusion Injury.
Experimental example 19: salvianolic acid A compositions is on the impact of dog Myocardial Ischemia Reperfusion Injury degree of myocardial ischemia
Healthy dogs, (17.5 ± 2.5) kg, is divided into 6 groups immediately: be respectively model group, hydrochloride for injection diltiazem group (0.5mg/kg), the high, medium and low dosage group of salvianolic acid A compositions (6,3,1.5mg/kg), salvianolic acid A monomeric compound group (3mg/kg).Pentobarbital sodium 30mg/kg intravenous injection is anaesthetized, fixing, and tracheal intubation connects respirator, is separated left common carotid, and intubate to be connected with polygraph recording blood pressure through pressure transducer, and be separated right side external jugular vein, along left side, the 4th intercostal opens breast, makees pericardium bed.Be separated aortic arch, place electromagnetic flowmeter probe, measure cardiac output.After opening breast, intubate is to right ventricle, records center venous pressure.Be separated M-LAD, the standby ligation use of lead-in wire, seam is put 16 epicardial leads of leading and is connected ecg amplifier, record epicardial electrocardiogram.Apex of the heart intubate, Bonding pressure transducer, measures intraventricular pressure.After record indices is stable, adopt two step ligation method ligation arteria coronaria left anterior descending branches.2min before ligation first, gives 2.5mg/kg lignocaine prevention arrhythmia.After Complete Ligation, stablize 15min, femoral vein constant speed injects administration, and administration volume 1ml/kg, 10min injection is complete.Model group and sham operated rats such as to give at the normal saline of capacity.After ischemia 3h, solution bursts at the seams bolt, realizes Reperfu-sion.Respectively before ligation, ligation 15min (namely before administration), 30,60,120,180min, the change of filling with 30min again, filling with 60min record epicardial electrocardiogram ST section again, blood oxygen.Myocardial ischemia journey is represented with total mV number (∑-ST) that the ST section of each mapping point of each moment raises.
Table 31. salvianolic acid A compositions is on the impact of dog Myocardial Ischemia Reperfusion Injury degree of myocardial ischemia (∑-ST)
(
mV，n＝6)
Note: compare with sham operated rats: #P < 0.001; Compare with model group: * P < 0.05, * * P < 0.01
Experimental result shows, and after ligation dog coronary artery, 5min starts, and each group dog epicardial electrogram ST section is raised sum (∑-ST) and obviously raised; After model group dog to ligation, 15min has been elevated to a metastable level, and after realizing Reperfu-sion, although ∑-ST comparatively declines between ischemic stage, significantly increase (P < 0.001) with sham operated rats, prompting dog cardiac muscle is still in ischemic state.In the 15min of each medicine group before ligation and after ligation (namely before administration), respectively organize ∑-ST and compare with model group and there is no diversity (P > 0.05); And 30min begins (i.e. 15min after administration) after ligation, each medicine group ∑-ST starts to decline, degree of ischemia alleviates, but except salvianolic acid A compositions high dose group to compare with model group have notable difference except (P < 0.05), other medicines group declines does not have statistical significance; But to ligation after 60min to ligation during 180min (namely after administration after 45min to medicine during 165min), each medicine group ∑-ST still continuous decrease, and more all have significant difference (P < 0.05 or P < 0.01) with model group; After recovering Reperfu-sion, each medicine group ∑-ST significantly reduces with model group.There is a certain amount of effect relationship between salvianolic acid A compositions each dosage group, in salvianolic acid A compositions, dosage group ∑-ST fall is suitable with 0.5mg/kg hydrochloride for injection diltiazem group, and has slightly excellent trend.Salvianolic acid A compositions declines more obvious than the ∑-ST with dosage salvianolic acid A monomeric compound group.Prompting salvianolic acid A compositions can alleviate the degree of myocardial ischemia of experiment ischemia-reperfusion dog, and effect is better than same dosage salvianolic acid A monomeric compound.Point out it to damage by Ischemic myocardium, to ischemic heart desease, there is good therapeutical effect.
Experimental example 20: salvianolic acid A compositions is on the impact of dog Myocardial Ischemia Reperfusion Injury myocardial ischemia scope
Modeling, grouping administration are with described in experimental example 19.Record the change of the epicardial electrocardiogram ST section of each treated animal each time period, represent myocardial ischemia scope with mapping point sum (N-ST) of each moment ST section rising >=2mV.
Table 32. salvianolic acid A compositions is on the impact of dog Myocardial Ischemia Reperfusion Injury myocardial ischemia scope (N-ST)
(
n＝6)
Note: compare with sham operated rats: #P < 0.001; Compare with model group: * P < 0.05, * * P < 0.01
Experimental result shows, and after ligation dog coronary artery, each group dog epicardial electrogram N-ST obviously increases; 15min after model group dog to ligation, N-ST has been increased to a metastable level, and ischemia scope is basicly stable, and after realizing Reperfu-sion, although N-ST comparatively reduces during ligation, but still pole is significantly higher than sham operated rats (P < 0.001).In the 15min of each medicine group before ligation and after ligation (namely before administration), each group N-ST compares with model group does not have diversity; And 30min begins (i.e. 15min after administration) after ligation, each medicine group N-ST starts the trend having minimizing, but except salvianolic acid A compositions high dose group slip to compare with model group have notable difference except (P < 0.05), other medicines group no difference of science of statistics; But to ligation after 60min to ligation during 180min (namely after administration after 45min to medicine during 165min), each medicine group N-ST still continues to reduce, and more all has significant difference (P < 0.05 or P < 0.01) with model group; After recovering Reperfu-sion, each medicine group N-ST significantly reduces with model group.A certain amount of effect relationship is there is between salvianolic acid A compositions each dosage group, in salvianolic acid A compositions, dosage group N-ST slip is suitable with 0.5mg/kg hydrochloride for injection diltiazem group, and salvianolic acid A compositions reduces obviously than the N-ST with dosage salvianolic acid A monomeric compound group.Prompting salvianolic acid A compositions can reduce the myocardial ischemia scope of experiment ischemia-reperfusion dog, and effect is better than same dosage salvianolic acid A monomeric compound.Point out it to damage by Ischemic myocardium, to ischemic heart desease, there is good therapeutical effect.
Experimental example 21: salvianolic acid A compositions is on the impact of dog Myocardial Ischemia Reperfusion Injury myocardial infarct size
Modeling, grouping administration are with described in experimental example 19.Each group of dog ischemia 180min, after Reperfu-sion 60min, cores dirty, normal saline flushing, weighs and weighs whole-heartedly.Cut off trunk and atrium, satisfactory room is heavy, and be on average cut into five from the apex of the heart to ligation point, dye in 0.5% nitro blue tetrazolium (N-TB) dye liquor, 15min, infarcted region is not painted, and skipper is dyed in non-infarcted region.Be separated infarcted region and non-infarcted region and weigh, accounting for heavy, infarcted myocardium whole-heartedly with infarcted myocardium and account for the heavy percentage calculation infarction size of ventricle.
Table 33. salvianolic acid A compositions on the impact of dog Myocardial Ischemia Reperfusion Injury myocardial infarct size (
n=6)
Note: compare with sham operated rats: #P < 0.01; Compare with model group: * P < 0.05, * * P < 0.01
Experimental result shows, and after model group dog ischemia-reperfusion, myocardial infarction is heavier.Salvianolic acid A compositions, hydrochloride for injection diltiazem, salvianolic acid A monomeric compound all can significantly reduce experimental dog myocardial infarct size, and minimum with salvianolic acid A compositions high dose group dog myocardial infarction ratio, in salvianolic acid A compositions, dosage group myocardial infarct size is less than same dosage salvianolic acid A monomeric compound group.Compare with model group; salvianolic acid A combination object height, middle dosage group extremely significantly can reduce the proportion (P < 0.01) that infarcted myocardium accounts for heart and ventricle; the proportion that salvianolic acid A compositions low dosage dog myocardial infarction district accounts for heart and ventricle also obviously reduces (P < 0.05); prompting salvianolic acid A compositions can dose-dependently reduce experimental dog myocardial infarct size; energy Ischemic myocardium-reperfusion injury, is used for the treatment of ischemic heart desease.And effect is better than same dosage salvianolic acid A monomeric compound.
Experimental example 22: salvianolic acid A compositions is on the impact of dogs with acute myocardial ischemia coronary flow
Modeling, grouping, administration time and dosage are with method described in experimental example 19.After animal opens breast, be separated LCA, place electromagnetic flowmeter probe, different time sections heart coronary flow (CBF) in ischemic stage 240min before measuring ligation, after ligation.Experiment terminates rear execution animal, using 1/3rd cardiac weights as LC drain district, calculates the blood flow of every 100g cardiac muscle: MBF=(CBF/ cardiac weight) × 100 × 3.
Table 34. salvianolic acid A compositions is on the impact of Myocardial Ischemia Reperfusion Injury dog coronary flow
(
n＝6)
Note: after administration, blood flow amplification compares with model group amplification: * P < 0.05, * * P < 0.01
Experimental result shows, and each time period coronary flow of sham operated rats dog does not have significant change; From each time period coronary flow of model group dog with compare before ligation and respectively organize comparative result before the coronary flow before dog administration and ligation, after ligation dog coronary artery forms myocardial ischemia, coronary flow has short time compensatory to increase, and increasing degree is about 10%; After each dosage of salvianolic acid A compositions, hydrochloride for injection diltiazem, each administration of salvianolic acid A monomeric compound treatment group dog each time period coronary flow with compare the increase had in various degree before administration, each medicine group is 15min (i.e. ligation 30min) upon administration, coronary flow amplification is between 13% ~ 24%, except salvianolic acid A high dose group amplification has except notable difference, other each medicine groups increases are also not obvious; And 225min (i.e. ligation 60min to 240min) after 45min to administration after administration, each medication therapy groups coronary flow compares with before administration, adds between 25.3% ~ 52.6%; With salvianolic acid A combination object height, middle dosage group, hydrochloride for injection diltiazem group significantly, amplification is respectively 52.6%, 40.2%, 36.7%.In salvianolic acid A compositions, dosage group amplification is higher than same dosage salvianolic acid A monomeric compound group.Prompting salvianolic acid A compositions can promote that collateral circulation is open, increases coronary flow during myocardial ischemia, thus to resisting myocardial ischemia, Ischemic myocardium damages, and points out it in control ischemic heart desease, have certain purposes.
Experimental example 23: salvianolic acid A compositions is on myocardial ischemia dog cardiac function and hemodynamic impact
Modeling, grouping administration empirically method described in example 19; Aortic root places electromagnetic flowmeter probe, measures cardiac output (CO); Apex of the heart intubate is to left ventricle, and Bonding pressure transducer, measures left room pressure, left indoor pressure maximum collapse and diastole rate of change (± dp/dtmax); Recording ecg (ECG); Coronary ligation Post operation stablizes 15min administration; Polygraph record, measurement calculate the change of each leading indicator of different time in dog ischemia 240min: CO, left ventricular end diastolic presssure (LVEDP), ± dp/dt
max, the acting of left room (LVW), total peripheral resistance (TPR).
Table 35. salvianolic acid A compositions on the impact of myocardial ischemia dog CO (
l/min, n=6)
Note: compare with before self ischemia, #P < 0.05, ##P < 0.01; Compare with model group, * P < 0.05, * * P < 0.01
Table 36. salvianolic acid A compositions on the impact of myocardial ischemia dog LVEDP (
mmHg, n=6)
Note: compare with before self ischemia, #P < 0.05, ##P < 0.01; Compare with model group, * P < 0.05, * * P < 0.01
Table 37. salvianolic acid A compositions is to myocardial ischemia dog+dp/dt
maximpact (
mmHg/s, n=6)
Note: compare with before self ischemia, #P < 0.05, ##P < 0.01; Compare with model group, * P < 0.05, * * P < 0.01
Table 38. salvianolic acid A compositions is to myocardial ischemia dog-dp/dt
maximpact (
mmHg/s, n=6)
Note: compare with before self ischemia, #P < 0.05, ##P < 0.01; Compare with model group, * P < 0.05, * * P < 0.01
Table 39. salvianolic acid A compositions on the impact of myocardial ischemia dog left room acting (LVW) (
n=6)
Note: compare with before self ischemia, #P < 0.05, ##P < 0.01; Compare with model group, * P < 0.05, * * P < 0.01
Table 40. salvianolic acid A compositions on the impact of myocardial ischemia dog total peripheral resistance (TPR) (
n=6)
Note: compare with before self ischemia, #P < 0.05, ##P < 0.01; Compare with model group, * P < 0.05, * * P < 0.01
Experimental result show, after ligation coronary ischemia 30min, dog cardiac output (CO) with significantly decline before ischemia, LVEDP significantly raises, ± dp/dt
maxremarkable decline, left room acting (LVW) reduce, total peripheral resistance (TPR) enlarges markedly (P < 0.05 or P < 0.01), after dog coronary ligation is described, myocardial function is obviously impaired, myocardial contraction and diastolic function decline, hypokinemia, cardiac pumping function reduces, and not, the impaired cardiac function that causes of myocardial ischemia-anoxemia declines blood oxygen supply.Compare with model group: after the administration of high dose salvianolic acid A compositions, 15min (i.e. ischemia 30min) significantly can suppress LVEDP rising, suppression ± dp/dt
maxdecline, suppress dog LVW to reduce, reduce TPR, after administration, 45min (namely after ischemia 60min) can significantly suppress CO to decline; After the administration of middle dosage salvianolic acid A compositions, 15min (after ischemia 30min) can raise dog ± dp/dt
max, TPR is declined, after administration, 45min (namely after ischemia 60min) significantly can suppress dog LVW to reduce, reduce LVEDP, and increase 105min (i.e. ischemia 120min) dog CO after administration, its action effect is suitable with hydrochloride for injection diltiazem; After the administration of salvianolic acid A compositions low dose group, 105min can obviously suppress dog CO to decline, and after administration, 45min can reduce dog TPR, rising ± dp/dt
maxand increase LVW, and upon administration after 105min (i.e. ischemia 120min) can obviously reduce dog LVEDP, and have the trend being better than same dosage salvianolic acid A monomeric compound.After the administration of result prompting salvianolic acid A compositions, myocardial ischemia dog hemodynamic index can be improved, the myocardial contraction of Enhancement test dog, improve myocardial systolic property, reduce Peripheral resistance, suppress cardiac output to reduce, improve the blood-pumping function of heart, improve myocardial blood supply, improve myocardial ischemia and cardiac function, play function of resisting myocardial ischemia.
Experimental example 24: salvianolic acid A compositions is on the impact of myocardial ischemia dog myocardium enzyme
Modeling, grouping administration are with method described in experimental example 19.Administration after coronary ligation post surgery stabilization 15min, after ligation, 240min (i.e. 4h after ischemia) gets blood from femoral vein respectively, detects creatine kinase (CK), lactic acid dehydrogenase (LDH), aspartate aminotransferase (AST), creatine kinase isozyme (CK-MB) in serum.
Table 41. salvianolic acid A compositions on the impact of myocardial ischemia dog myocardium enzyme (
u/L, n=6)
Note: compare with model group, * P < 0.01, * * P < 0.001
In serum, CK, LDH, AST, CK-MB are the coherent detection myocardial zymetology as Hypoxicichemic myocardial lesion commonly used clinically, can reflecting myocardium degree of injury.Wherein CK-MB is Cardiac-specific enzyme, the most responsive in myocardial damage.Experimental result shows, and compare with sham operated rats, in myocardial infarction and ischemia model group dog serum, CK, LDH, AST, CK-MB significantly raise, after ligation coronary ischemia is described, dog myocardial mitochondria is destroyed, myocardial cell injury, and myocardium enzyme is released the rising alive of hyperamization clear center creatase.The each dosage group of salvianolic acid A compositions compares with model group, CK, LDH, AST, CK-MB activity (P < 0.01 or P < 0.001) in serum to be reduced after dog myocardial ischemia 4h to some extent, and in dose dependent; Middle dosage salvianolic acid A compositions group ratio is with dosage salvianolic acid A monomeric compound group more remarkable effect.Experimental result prompting salvianolic acid A compositions can stablize myocardial cell membrane, reduces the spilling of myocardium enzyme during treating myocardial ischemia damage, has obvious protective effect to ischemic myocardium.
Experimental example 25: salvianolic acid A compositions is on the impact of myocardial ischemia dog fatty acid metabolism and radical metabolism
Modeling, grouping administration are with method described in experimental example 19.Administration after coronary ligation post surgery stabilization 15min, after ligation, 240min (i.e. 4h after ischemia) gets blood from femoral vein respectively, detects serum free fatty acid (FFA), lipid peroxide (LPO), malonaldehyde (MDA) content, superoxide dismutase (SOD), glutathion peroxidase (GSH-Px).
Table 42. salvianolic acid A compositions is on the impact of myocardial ischemia dog fatty acid metabolism and radical metabolism
(
n＝6)
Note: compare with model group, * P < 0.05, * * P < 0.01
A series of myocardial metabolism appearance disorder in various degree can be caused during myocardial ischemia, thus cause cardiac insufficiency.The main reason for the treatment of myocardial ischemia damage makes myocardial damage even downright bad owing to causing Power supply obstacle after myocardial ischemia.Free fatty is the main substrate of normal heart energy supply.And cardiac muscle is under ischemia, because fatty acid oxidation energy supply will consume more oxygen than glucose metabolism energy supply, myocardial ischemia phase free fatty acid levels and oxygenation efficiency rising can suppress the oxidation of glucose, increase the gathering of lactic acid in cell, the efficiency that heart therefore can be made to do work reduces, and produces adverse influence to cardiac muscle.Experimental result shows, compare with sham operated rats, model group dog is after ischemia 4h, serum free fatty acid (FFA), lipid peroxide (LPO) significantly raise (P < 0.01), after prompting myocardial ischemia there is obstacle in myocardial fatty acid metabolic, the activity of glucose oxidase Major Enzymes will be suppressed, thus increase myocardial oxygen consumption, expand myocardial infarct size.And each dosage group of salvianolic acid A compositions compares with model group, FFA, LPO content in myocardial ischemia dog serum (P < 0.05 or P < 0.01) significantly can be reduced, and in dose-dependence.Prompting salvianolic acid A compositions can significantly suppress FFA level in serum to raise, and improves myocardial fatty acid metabolic, reduces the accumulation of lactic acid; increase the production capacity of unit oxygen consumption, reduce myocardial oxygen consumption, thus improve cardiac function; suppress myocardial infarct size, Ischemic myocardium damages.
Compare with sham operated rats, after model group dog myocardial ischemia 4h, in serum, MDA content significantly raises, SOD, GSH-Px are active significantly declines (P < 0.01), after prompting dog myocardial ischemia, oxygen-derived free radicals generates obviously to be increased, in body, the enzyme of scavenging activated oxygen is lived and is significantly reduced, and oxygen-derived free radicals can affect normal configuration and the function of cell by a series of oxidation reaction, aggravation treating myocardial ischemia damage degree.The each dosage group of salvianolic acid A compositions compares with model group, significantly can reduce the content of MDA in myocardial ischemia dog serum, make the active obviously enhancing (P < 0.05 or P < 0.01) of SOD, GSH-Px in serum simultaneously, and in certain agent effect relationship; Prompting salvianolic acid A compositions is by certain machine-processed anti-lipid peroxidation process, reduce oxygen-derived free radicals to generate, endogenous anti-oxidative enzymatic activity can be improved again and alleviate the damage of oxygen-derived free radicals to cardiac muscle simultaneously, improve the oxidation resistance of ischemic myocardium and play function of resisting myocardial ischemia.And effect is better than same dosage salvianolic acid A monomeric compound.
Experimental example 26: salvianolic acid A compositions is on the impact of myocardial ischemia dog myocardial oxygen consumption
Prepare myocardial ischemia dog: modeling, grouping, dosage and time are with method described in experimental example 19.Through external jugular vein intubate to coronary sinus vein, in carotid artery intubate, with Oximetry instrument respectively before ligation arteria coronaria and after ischemia 15,30,60,120,240min measures the Coronary vein of each animal, the oxygen content of tremulous pulse, calculating myocardium oxygen consumption and coefficient of oxygen utilization.Myocardial oxygen consumption=(carotid artery blood oxygen amount-coronary sinus vein blood oxygen amount) × CBF; Cardiac muscle coefficient of oxygen utilization=(carotid artery blood oxygen amount-coronary sinus vein blood oxygen amount)/carotid artery blood oxygen amount × 100%.
Table 43. salvianolic acid A compositions on the impact (ml/min) of myocardial ischemia dog myocardial oxygen consumption (
n=6)
Note: compare with model group, * P < 0.05, * * P < 0.01
Table 44. salvianolic acid A compositions on myocardial ischemia dog cardiac muscle coefficient of oxygen utilization impact (%) (
n=6)
Note: compare with model group, * P < 0.05, * * P < 0.01
Oxygen metabolism is lacked of proper care, and oxygen supply and aerobic imbalance are the main causes of myocardial ischemia.Experimental result shows, model group dog is after ligation arteria coronaria causes myocardial ischemia, myocardial oxygen consumption and coefficient of oxygen utilization there is no significant change with comparing before ischemia, but known in conjunction with the experiment about the acting of myocardial ischemia left room aforementioned in this explanation, after myocardial ischemia, the acting of left room reduces, and myocardial oxygen consumption and coefficient of oxygen utilization unchanged, therefore, the oxygen consumption of unit acting increases, and heart mechanical efficiency significantly reduces.Compare with model group, the salvianolic acid A compositions of various dose can reduce myocardial oxygen consumption and coefficient of oxygen utilization to some extent, and wherein salvianolic acid A combination object height, middle dosage group ischemia 120min (i.e. 105min after administration) coefficient of oxygen utilization comparatively reduce nearly 20%, 16% before administration respectively.In conjunction with in this explanation about salvianolic acid A compositions is to the experimental result of the parameters of left ventricular function such as myocardial ischemia dog coronary flow and ventricle acting; prompting salvianolic acid A compositions can reduce myocardial oxygen consumption and coefficient of oxygen utilization; improve the equilibrium of supply and demand of myocardium oxygen; improve ventricular function; play the effect of protection ischemic myocardium, control myocardial ischemia.
Experimental example 27: salvianolic acid A compositions is on the hemorheological impact of myocardial ischemia dog
Prepare myocardial ischemia dog: modeling, grouping, dosage and time are with method described in experimental example 19.Draw blood after ischemia 240min respectively, carry out the mensuration of the hemorheology indexs such as whole blood viscosity, plasma viscosity, plasma fibrinogen content, packed cell volume, platelet adhesion rate.
Table 45. salvianolic acid A compositions on the impact of myocardial ischemia dog whole blood viscosity, plasma viscosity (
n=6)
Note: compare with model group, * P < 0.05
Compare with sham operated rats, ligation arteria coronaria causes the whole blood viscosity of myocardial ischemia dog, plasma viscosity obviously raises, plasma fibrinogen content obviously rises, and packed cell volume and platelet adhesion rate obviously increase (P < 0.05); Compare with model group, in experiment, various dose salvianolic acid A compositions can make myocardial ischemia dog blood viscosity, plasma viscosity, packed cell volume and platelet adhesion rate reduce to some extent, obviously reduces fiber protein content in blood plasma; In certain agent effect trend between each dosage group; Prompting salvianolic acid A compositions can reduce blood viscosity, suppresses platelet adhesion to be assembled, and prevention Intravascular Thrombus is formed, and improves myocardial ischemia dog hemorheology, improves microcirculation, can be used for preventing and treating ischemic heart desease.
Experimental example 28: the impact that salvianolic acid A compositions is formed rat thrombus in vivo
SD rat, (280 ± 20) g, random point 6 groups: normal saline group matched group, the high, medium and low dosage of salvianolic acid A compositions (20,10,5mg/kg) group, aspirin group (10mg/kg), salvianolic acid A monomeric compound group (10mg/kg).Each group of tail vein injection administration every day 1 time (matched group gives isometric(al) normal saline), continuous 7d, 1h after last administration, pentobarbital sodium (40mg/kg) anesthetized rat, dorsal position is fixed, 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.
Impact that table 46. salvianolic acid A compositions is formed rat thrombus in vivo (
n=6)
Note: compare with normal saline group, * P < 0.05, * * P < 0.01
Compare with normal saline group, 60,30,15mg/kg salvianolic acid A compositions significantly can alleviate that rat suppository is wet, dry weight (P < 0.05 or P < 0.01), has obvious inhibitory action to thrombosis; In certain dose-effect relationship between each dosage group, and inhibition is better than same dosage salvianolic acid A monomeric compound.Prompting salvianolic acid A compositions has the effect suppressing rat thrombus in vivo to be formed, and points out it to can be used for preventing and treating the ischemic heart desease caused by thrombosis.
Claims (20)
1. the preparation method of a salvianolic acid A compositions, described salvianolic acid A compositions is for the preparation of prevention and therapy ischemic heart medicine, in wherein said compositions, salvianolic acid A content is greater than 93%, be less than 100%, and also comprise 4 other compositions by following weight proportion: alkannic acid 0.55% ~ 2%, rosmarinic acid 1% ~ 2%, salvianolic acid B 0.1% ~ 2%, salvianolic acid C 0.1% ~ 2%, described salvianolic acid A compositions adopts lower preparation method to obtain:
Get red rooted salvia, be cut into decoction pieces or be ground into diameter 1mm ~ 5mm granule, add 3 ~ 15 times amount at every turn, 45 ~ 95 DEG C of water temperature lixiviates are got, stir with 10 ~ 50 revs/min of speed simultaneously, or add 3 ~ 15 times amount water boiling and extraction, extract 1 ~ 3 time altogether, extract 1 ~ 4 hour at every turn; Extracting solution is evaporated to relative density and is determined as 1.0 ~ 1.25 at 60 DEG C, adds ethanol and makes alcohol content 50% ~ 85%, leaves standstill, and filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, obtains Radix Salviae Miltiorrhizae extract; Or
Get red rooted salvia, be cut into decoction pieces or be ground into diameter 1mm ~ 5mm granule, add 3 ~ 15 times amount 30% ~ 60% alcohol reflux at every turn, extract 1 ~ 4 hour at every turn, extract 1 ~ 3 time altogether; Decompression recycling ethanol is also concentrated into without alcohol taste, obtains Radix Salviae Miltiorrhizae extract;
Above-mentioned Radix Salviae Miltiorrhizae extract is diluted with water to every 1ml containing salvianolic acid B 1 ~ 30mg, and aqueous solution adjusting PH with base to 3.5 ~ 6.5, add with salvianolic acid B molar percentage 0.1 ~ 3% zinc chloride as catalyst, transforms 1 ~ 6 hour at 100 ~ 140 DEG C of heating temperatures;
Conversional solution adjust pH to 2.5 ~ 4.5, leave standstill, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 1 ~ 10mg, be separated through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 35 ~ 1: 70 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 4 ~ 1: 30, use 1 ~ 8 times of cylinder hydrops respectively, 1 ~ 10 times of column volume 10% ~ 40% ethanol elution, removing impurity, use 2 ~ 10 times of column volume 20% ~ 60% ethanol elutions again, HPLC detects, collect 20% ~ 60% 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 1-10mg salvianolic acid A, be separated by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 5 ~ 1: 25 with polyamide ratio, resin column blade diameter length ratio is 1: 4 ~ 1: 25, use 1 ~ 10 times of cylinder hydrops, the remove impurity of 5 ~ 20 times of column volume 20% ~ 60% alcoholic solution eluting respectively, use 4 ~ 15 times of column volume 40% ~ 90% alcoholic solution eluting again, collect 40% ~ 90% alcoholic solution part containing salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste aqueous solution;
Aqueous solution concentrates, acid adjustment pH to 2.0 ~ 4.0, by the t-butyl methyl ether of aqueous solution 1-8 times amount, and point 2 ~ 6 extractions, be separated organic layer, reclaim under reduced pressure t-butyl methyl ether, make the extract of every 1ml containing salvianolic acid A 1g ~ 10g, add 1 ~ 3 times amount silica gel, stir, volatilize;
Stirring sample silica gel is added on the dry silicagel column of 5 ~ 20 times amount installed, silicagel column blade diameter length ratio is 1: 4 ~ 1: 25, with pentane-t-butyl methyl ether for eluant, gradient elution, use pentane-t-butyl methyl ether eluting 6 ~ 30 times column volume of by volume 4: 6 respectively, pentane-t-butyl methyl ether eluting 6 ~ 30 times column volume of volume ratio 6: 4, reclaim under reduced pressure eluant, the salvianolic acid A reclaimed after organic solvent adds 5 ~ 20 times amount water dissolutioies, microwave vacuum drying, obtains described salvianolic acid A compositions.
2. preparation method according to claim 1, is characterized in that microwave vacuum drying temperature: 20-100 DEG C, return difference temperature 1-5 DEG C, more than vacuum-0.07MPa, microwave power 1-100KW, dry 10-200 minute.
3. preparation method according to claim 2, is characterized in that microwave vacuum drying temperature: 50-85 DEG C, return difference temperature 2-4 DEG C, more than vacuum-0.07MPa, microwave power 10-80KW, dry 100-150 minute.
4. preparation method according to claim 3, is characterized in that microwave vacuum drying temperature: 55-80 DEG C, return difference temperature 2-3 DEG C, more than vacuum-0.07MPa, microwave power 25-60KW, dry 120-140 minute.
5. preparation method according to claim 1, the wherein said disease for prevention and therapy ischemic heart desease comprise following one or more: coronary heart disease, myocardial ischemia apoplexy, acute coronary syndrome, coronary stricture, myocardial ischemia reperfusion injury, atherosclerosis.
6., according to the preparation method of claim 1 or 5, wherein said salvianolic acid A compositions is for improving the hemodynamic purposes of Ischemic Heart.
7. preparation method according to claim 6, wherein said salvianolic acid A compositions is for improving the purposes of ischemic heart cardiac function.
8. preparation method according to claim 6, wherein said salvianolic acid A compositions is for increasing the purposes of the coronary flow of ischemic heart.
9., according to the preparation method of claim 1 or 5, wherein said salvianolic acid A compositions is for reducing the purposes of ischemic heart myocardial infarct size.
10. preparation method according to claim 9, wherein said salvianolic acid A compositions is for reducing the purposes of myocardial ischemia scope.
11. preparation methoies according to claim 9, wherein said salvianolic acid A compositions is for alleviating the purposes of degree of myocardial ischemia.
12. preparation methoies according to claim 9, wherein said salvianolic acid A compositions regulates the purposes of Enzyme Activities for the protection of myocardial cell membrane structure.
13. preparation methoies according to claim 9, wherein said salvianolic acid A compositions is for suppressing the purposes of ischemic myocardial tissue inflammatory reaction.
14. preparation methoies according to claim 9, the purposes that wherein said salvianolic acid A compositions is damaged for the protection of myocardial mitochondria.
15. according to the preparation method of claim 1 or 5, and wherein said salvianolic acid A compositions is for improving the purposes of myocardial metabolism.
16. preparation methoies according to claim 15, wherein said salvianolic acid A compositions comprises for improving one or more of following myocardial metabolism for the purposes improving myocardial metabolism: myocardium radical metabolism, fatty acid metabolism, energy metabolism.
17. according to the preparation method of claim 1 or 5, and wherein said salvianolic acid A compositions is for reducing the purposes of myocardial oxygen consumption.
18. according to the preparation method of claim 1 or 5, and wherein said salvianolic acid A compositions is for improving hemorheological purposes.
19. according to the preparation method of claim 1 or 5, and wherein said salvianolic acid A compositions is used for the purposes of inhibition thrombosis.
20. preparation methoies according to claim 5, the wherein said wherein said disease for prevention and therapy ischemic heart desease comprise following one or more: angina pectoris, ischemic cardiomyopathy, myocardial infarction.
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