CN102973544B - Salvianolic acid A lyophilized powder and application thereof in preparing medicaments - Google Patents

Salvianolic acid A lyophilized powder and application thereof in preparing medicaments Download PDF

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CN102973544B
CN102973544B CN201210487270.0A CN201210487270A CN102973544B CN 102973544 B CN102973544 B CN 102973544B CN 201210487270 A CN201210487270 A CN 201210487270A CN 102973544 B CN102973544 B CN 102973544B
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salvianolic acid
freeze
dried powder
myocardial
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CN102973544A (en
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刘地发
李志勇
程帆
欧阳婷
罗恒真
刘尧奇
刘艳红
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Jiangxi Qingfeng Pharmaceutical Co., Ltd.
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陈飞虹
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Abstract

The invention relates application of salvianolic acid A lyophilized powder in preparing a medicament for preventing and treating ischemic heart disease. The salvianolic acid A lyophilized powder comprises the following components by weight: 10 to 80g of salvianolic acid A, 10 to 80g of filler, and an antioxidant which accounts for 0.01 to 0.2 percent of the total preparation weight. The lyophilization process in the preparation method comprises the following steps of: A, freezing: cooling to a temperature between minus 40 and minus 45 DEG C at a speed of between 20 and 30 DEG C/h, and insulating and freezing for 10 to 16 hours; B, sublimation: vacuumizing to be below 0.3mbar, increasing the temperature of the frozen salvianolic acid A preparation to a temperature between minus 23 and minus 27 DEG C, drying in vacuum at a temperature between minus 23 and minus 27 DEG C for 6 to 8 hours; and C, drying: keeping on increasing temperature, and uniformly increasing the temperature to be between 40 and 45 DEG C at a speed of between 0.5 and 1.0 DEG C/min, drying at a temperature of between 40 and 45 DEG C for 2 to 5 hours, cooling the sample to room temperature, and covering to obtain the salvianolic acid A lyophilized powder.

Description

A kind of salvianolic acid A freeze-dried powder and prepare medicinal usage
Technical field
The present invention relates to a kind of salvianolic acid A freeze-dried powder and prepare medicinal usage.
Background technology
Ischemic heart desease (IHD) is to cause the uneven cardiac damage causing between coronary flow and myocardium demand because coronary artery circulation changes, modal reason is coronary atherosclerosis, coronary artery thrombosis and coronary vasospasm, accounts for 90% left and right of ischemic heart desease.Its common ground is because the imbalance between supply and demand of oxygen due to deficiency myocardial blood supply causes myocardial ischemia, anoxia, energy metabolism of myocardial is undesired, can not support heart normally to work, thereby a series of pathophysiological changes and symptom occur, cause angina pectoris attacks, myocardial infarction even occurs and threat to life.According to the position of coronary artery pathological changes, scope and the pathological changes order of severity, degree of myocardial ischemia, clinical point of five classes: asymptomatic type 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 heart diseases that cause because of arteria coronaria pathological change are referred to as coronary heart disease abbreviation 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 myocardium cellular uptake blood oxygen content 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 essential energy supply deficiency while making cardiomotility causes that angina pectoris, arrhythmia, cardiac function decline.Meanwhile, the refuse of metabolism can not be removed effectively in time, easily has a negative impact.Ischemia, anoxia, lack energy, finally can affect the contractile function of heart.If have 20%～25% cardiac muscle to stop shrinking, conventionally there will be left chamber function exhaustion; If have more than 40% cardiac muscle 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 consumption of myocardium blood supply oxygen supply, reduction oxygen, improve myocardial metabolism, allevating angina pectoris, reducing myocardial infarction area is the main purpose of Drug therapy.Treatment ischemic heart desease mainly comprises the operative treatment such as Drug therapy and intervention (PICA) or bridging at present.When angina pectoris attacks, generally use nitric acid lipid drug, as sublingual administration nitroglycerin, rapid-action, but can only be used for emergency, relief of symptoms, can not fundamentally 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 myocardium damaged wound make one of reason element, in recent years, regulate energy metabolism of myocardial to bring into play the short metabolism class medicine of an antianginal class by optimization also quite concerned, be expected to become new class treatment or an ancillary drug.And it is at present main take arteria coronaria revascularization, recover the ischemic heart desease Primary Care method of blood perfusion as principle as early as possible, also there is very important risk: zoopery and clinical research are found, along with the recovery of blood fortune, some impaired myocardial cell function and structural deterioration increases the weight of on the contrary, there is myocardial ischemia reperfusion injury (myocardial ischemia reperfusion injury, MIRI).Reperfusion injury is after myocardial ischemia, perfusion is early stage again, response to oxidative stress causes producing in cardiac muscle a large amount of oxygen-derived free radicals, superoxide anion, accelerate apoptosis of cardiac muscle and necrosis together with factors such as calcium overloads in cell and mitochondrion, myocardial infarct size expansion, cardiac function are worsened rapidly.Clinical manifestation be inaccessible coronary artery more logical and infarcted region hemoperfusion rebuild after in a period of time, there are blood pressure rapid drawdown, cardiac insufficiency, arrhythmia a series of phenomenons that sb.'s illness took a turn for the worse such as even die suddenly in some patients.All there is reperfusion injury problem after as logical again in thrombolysis in myocardial infarction in clinical practice, after coronary artery bypass grafting, heart transplantation, sudden cardiac arrest cardiac resuccitation, after open-heart surgery etc.How to accomplish both to have guaranteed to recover as early as possible the blood flow of ischemic tissue, alleviate again the generation of even removing reperfusion injury and just become the important topic in another coronary heart disease control.
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, the 2000 ten thousand people acute cardiovascular disease that happens suddenly is often close in the whole world, cause death toll account for the whole world dead more than 30%, wherein 43% die from coronary heart disease.In Regional Distribution, more than 80% cardiovascular disease death occurs in low middle income country.It is reported, the U.S. approximately has 45～500,000 people to die from sudden cardiac death every year, and wherein accounts for sudden death the more than 80% of 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 in 2010 shows, China exceedes 40% people and dies from cardiovascular disease.Effective control of coronary heart disease has become the major global public health problem can not be ignored, the development of this class diseases prevention and treatment medicine is listed in the key content of China's Eleventh Five-Year Plan, the great special item of " 12 " original new drug continuously, and by becoming the drug categories of a very long time primary study from now on, there are great social meaning and economic worth.
Radix Salviae Miltiorrhizae is the dry root and rhizome of Labiatae salvia Radix Salviae Miltiorrhizae Salvia miltiorrhiza Bunge, 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 the widest at most medicine of clinical practice.Because having the pharmacological actions such as the microcirculation of improvement, coronary artery dilating, inhibition thrombosis, anticoagulant, protection ischemic myocardium, the clinical practice in modern age of its preparation is mainly used in ischemic cardiac, cerebrovascular disease.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 etc.; Include coronary artery dilator, increased coronary flow, reduced blood viscosity and blood clotting, anticoagulant, protection ischemic myocardium and improve the pharmacological actions such as microcirculation.Be applicable to thoracic obstruction patient and the 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., take antioxidation, anticoagulant and antiinflammatory as feature, there is protection vascular endotheliocyte, blood fat reducing, rising HDL, anticoagulant, activation fibrinolytic, suppress the pharmacological action of the multiple beneficial such as Fibrinogen is synthetic, 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 have very strong antioxidation, can obviously alleviate mitochondrial injury.Therefore salvianolic acid A can be by alleviating apoptosis of cardiac muscle, improve 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's traditional Chinese medical science traditional Chinese medicines; 2011,22 (2): 412-424).
But the natural content of salvianolic acid A extremely low (being about the 0.01-0.06% of red rooted salvia), makes crude drug high cost, separation and purification difficulty is excessive, is seriously restricting the R and D of medicine, becomes the bottleneck of its industrialization.In addition, in prior art, also have experiment confirmation salvianolic acid B to degrade and can change into salvianolic acid A by ester hydrolysis decarboxylation and chroman ring ring-opening reaction, 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 existing in prior art in prior art, but be only all that simple trial heats up, changes pH value or improves concentration transforming front pressure differential self etc., without any a patent or prior art deeply, study conversion reaction all sidedly and all comprise which major influence factors, how synergism exerts an influence 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, seldom there are other inventors or prior art to propose to attempt adding catalyst in conversion so that reaction is more abundant, in currently available technology, having related to the related content that adopts catalyst to transform exists only in two parts of patent application documents submitting in same day of on April 6th, 2010, these two parts of patent application documents are respectively that application number is 2010101436787, patent application and application number that denomination of invention is " a kind of catalyzed conversion salvianolic acid B is prepared the method for salvianolic acid A " are 2010101436876, denomination of invention is the patent application of " a kind of method that preliminary purification salvianolic acid B transforms raw material ".In the description of these two parts of patent applications, although disclose the technology contents that catalyzed conversion salvianolic acid B is prepared salvianolic acid A, but its catalyst is carbamide, the mol ratio of carbamide and salvianolic acid B is (0.4～0.6): 1, require consume and add carbamide very high, therefore production cost is very high.And, in above-mentioned two parts of patent application documents, do not pay close attention to too the collaborative impact that salvianolic acid A productive rate is produced of pH value and other correlative factors.Moreover it transforms raw material is the radix Salviae Miltiorrhizae water extract through co-chromatography preliminary purification, wherein salvianolic acid B >=50%, this all has relatively high expectations to the purity and the purifying technique that transform raw material, and technique is also comparatively complicated, has further increased production cost.More crucial, although claim " directed conversion ratio >=10% of salvianolic acid A salvianolic acid B that uses this method to prepare, even reaches 60% " in its application documents.But due in fact not open loop, dehydration, decarboxylation of carbamide, 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.
Salvianolic acid A is to be formed by danshensu and caffeic acid condensation, contain the reactive groups such as multiple phenolic hydroxyl groups, hydroxyl, ester bond, it is unstable to light and heat, the easy oxygen of exposure air, due to the above-mentioned physicochemical property of salvianolic acid A, make salvianolic acid A preparation, its stability also guarantees that salvianolic acid A pharmacological action becomes the key technology of preparation.
Summary of the invention
For overcome prior art exist above-mentioned defect, the invention provides a kind of salvianolic acid A freeze-dried powder for prevent and or treatment ischemic heart desease aspect purposes.
A kind of salvianolic acid A freeze-dried powder provided by the invention is for the preparation of the purposes of prevention and treatment ischemic heart medicine, described its weight proportion of salvianolic acid A freeze-dried powder is: salvianolic acid A 10g～80g, filler 10g～80g, antioxidant is to make 0.01%～0.2% of total amount; The preparation method of described salvianolic acid A freeze-dried powder is:
Get red rooted salvia, be cut into decoction pieces or be ground into diameter 1mm～5mm granule, add 3～15 times of amounts, 45～95 ℃ of water temperature lixiviates are got at every turn, stir with 10～50 revs/min of speed simultaneously, or add 3～15 times of amount water boiling and extraction, and extract altogether 1～3 time, extract 1～4 hour at every turn; Extracting solution is evaporated to relative density 1.0～1.25 (60 ℃), adds ethanol to make to contain alcohol amount 50%～85%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, obtains Radix Salviae Miltiorrhizae extract; Or
Get red rooted salvia, be cut into decoction pieces or be ground into diameter 1mm～5mm granule, add 3～15 times of amount 30%～60% alcohol reflux at every turn, extract 1～4 hour at every turn, extract altogether 1～3 time; Decompression recycling ethanol is also concentrated into without alcohol taste, obtains Radix Salviae Miltiorrhizae extract;
Above-mentioned Radix Salviae Miltiorrhizae extract was diluted with water to every 1ml containing salvianolic acid B 1～30mg, and adjusting PH with base to 3.5 for aqueous solution～6.5 add with salvianolic acid B molar percentage 0.1～3% zinc chloride as catalyst, 100～140 ℃ of temperature thermal conversions 1～6 hour;
Conversional solution adjust pH to 2.5～4.5, leave standstill, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 1～10mg, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 35～1: 70 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 4～1: 30, use respectively 1～8 times of cylinder hydrops, 1～10 times of column volume 10%～40% ethanol elution, remove impurity, use again 2～10 times of column volume 20%～60% ethanol elutions, HPLC detects, 20%～60% ethanol elution part that collection contains salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste,
Aqueous solution is concentrated into the solution of every 1ml containing 1-10mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 5～1: 25 with polyamide ratio, resin column blade diameter length ratio is 1: 4～1: 25, use respectively 1～10 times of cylinder hydrops, 5～20 times of column volume 20%～60% alcoholic solution eluting remove impurity, use again 4～15 times of column volumes, 40%～90% alcoholic solution eluting, 40%～90% alcoholic solution part that collection contains salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste aqueous solution;
Aqueous solution is concentrated, acid adjustment pH to 2.0～4.0, the t-butyl methyl ether of doubly measuring with aqueous solution 1-8, point 2～6 extractions, separate organic layer, reclaim under reduced pressure t-butyl methyl ether, makes the extract of every 1ml containing salvianolic acid A 1g～10g, add 1～3 times of amount silica gel, stir, volatilize;
Be added on 5～20 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 4～1: 25, take pentane-t-butyl methyl ether as eluant, gradient elution, use respectively 6～30 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 6～30 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 5～20 times of water gagings dissolvings, microwave vacuum drying, obtains described salvianolic acid A;
Getting described salvianolic acid A 10g～80g injects water 1500～2800ml and is stirred to dissolve, by adjusting PH with base value 4.0～5.0, add described filler to make its dissolving, add again described antioxidant, be stirred to dissolve and mix, then add active carbon 0.5～2g stirring and adsorbing, filter and remove active carbon, inject fill after water and become bottle, send into and in freeze dryer, carry out lyophilization;
Described lyophilization comprises the steps:
A, freezing: be cooled to-40 ℃～-45 ℃ with 20 ℃～30 ℃/h speed, be incubated freezing 10～16 hours;
B, distillation: be evacuated to below 0.3mbar, in 10～14 hours, the salvianolic acid A formulation temperature freezing risen to-23 ℃～-27 ℃, maintain-23 ℃～-27 ℃ vacuum dryings 6～8 hours;
C, dry: continue to heat up, be evenly warming up to 40 ℃～45 ℃ with 0.5 ℃～1.0 ℃/min, maintain 40 ℃～45 ℃ and be dried after 2～5 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Preferably, its weight proportion is: salvianolic acid A 20g～60g, and filler 20g～60g, antioxidant is to make 0.02%～0.1% of total amount.
Preferably, its weight proportion is: salvianolic acid A 20g～40g, and filler 20g～40g, antioxidant is to make 0.03%～0.08% of total amount.
Preferably, wherein said filler is selected from any one or a few in mannitol, glucose, lactose, and consumption is 20mg～40mg/2ml～3ml.
Preferably, wherein said antioxidant is selected from any one or a few in vitamin C, thiourea, sodium sulfite, sodium pyrosulfite.
Preferably, its weight proportion is: salvianolic acid A 20g, filler mannitol 20g, antioxidant vitamin C 0.8g; Its preparation method is:
Get described salvianolic acid A 20g, inject water 1900ml and be stirred to dissolve, with sodium hydroxide sodium adjust pH 4.0～5.0, add mannitol 20g to make to dissolve, then add 0.8g vitamin C, be stirred to dissolve and mix, add active carbon 0.5g stirring and adsorbing, filter and remove active carbon; Inject water to 2000ml, fill becomes 1000 bottles, lyophilization;
Described lyophilization comprises the steps:
A, freezing: be cooled to-45 ℃ with 25 ℃/h speed, be incubated freezing 15 hours;
B, distillation: be evacuated to below 0.3mbar, in 12 hours, the salvianolic acid A formulation temperature freezing risen to-25 ℃, maintain-25 ℃ of vacuum dryings 8 hours;
C, dry: continue to heat up, be evenly warming up to 40 ℃ with 0.8 ℃/min, maintain 40 ℃ and be dried after 3 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Preferably, microwave vacuum drying temperature: 20-100 ℃, return difference temperature 1-5 ℃, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.
Preferably, microwave vacuum drying temperature: 50-85 ℃, return difference temperature 2-4 ℃, more than vacuum-0.07Mpa, microwave power 10-80KW, dry 100-150 minute.
Preferably, microwave vacuum drying temperature: 55-80 ℃, return difference temperature 2-3 ℃, more than vacuum-0.07Mpa, microwave power 25-60KW, dry 120-140 minute.
Preferably, wherein saidly can be used for treating one or more of following disease for the medicine preventing and treat ischemic heart desease: 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 freeze-dried powder is used for improving the hemodynamic purposes of Ischemic Heart.
Preferably, wherein said salvianolic acid A freeze-dried powder is for improving the purposes of ischemic heart cardiac function.
Preferably, wherein said salvianolic acid A freeze-dried powder is for increasing the purposes of the coronary flow of ischemic heart.
Preferably, wherein said salvianolic acid A freeze-dried powder is for reducing the purposes of ischemic heart myocardial infarct size.
Preferably, wherein said salvianolic acid A freeze-dried powder is for dwindling the purposes of myocardial ischemia scope.
Preferably, wherein said salvianolic acid A freeze-dried powder is for alleviating the purposes of degree of myocardial ischemia.
Preferably, wherein said salvianolic acid A freeze-dried powder regulates the purposes of Enzyme Activities for the protection of myocardial cell membrane structure.
Preferably, wherein said salvianolic acid A freeze-dried powder is for suppressing the purposes of ischemic myocardial tissue inflammatory reaction.
Preferably, wherein said salvianolic acid A freeze-dried powder is for the protection of the purposes of myocardial mitochondria damage.
Preferably, wherein said salvianolic acid A freeze-dried powder is for improving the purposes of myocardial metabolism.
Preferably, wherein said salvianolic acid A freeze-dried powder comprises for improving one or more of following myocardial metabolism for the purposes of improving myocardial metabolism: myocardium radical metabolism, fatty acid metabolism, energy metabolism.
Preferably, wherein said salvianolic acid A freeze-dried powder is for reducing the purposes of myocardial oxygen consumption.
Preferably, wherein said salvianolic acid A freeze-dried powder is used for improving hemorheological purposes.
Preferably, wherein said salvianolic acid A freeze-dried powder is used for suppressing thrombotic purposes.
Salvianolic acid A freeze-dried powder principal agent provided by the invention is salvianolic acid A, the present invention is by the extraction to Radix Salviae Miltiorrhizae, transform, purification, drying process, obtain salvianolic acid A raw material: through screening and the optimization of system, first extraction solvent and the extracting method of initiation material salvianolic acid B have relatively been determined, because salvianolic acid B water solublity is better, employing water extraction or low concentration alcohol extraction are determined, again because salvianolic acid B heat stability is poor, determine that employing hot water warm macerating extracts and adds stirring extracting method, or use low-concentration ethanol reflux, extract, make to extract solubility lower than 100 ℃, keep salvianolic acid B not to be destroyed, optimum solvent consumption and extraction time are determined by orthogonal experiment, obtain adapting to the salvianolic acid B optimum extraction process of suitability for industrialized production.
The present invention is compared with the prior art and shows: the direct extraction of the red rooted salvia conversion that can feed intake for initiation material, do not need salvianolic acid B to carry out transforming again after purification, be in catalytic conversion reaction of the present invention, reaction raw materials salvianolic acid B does not need high-purity, for example, do not need purity >=50% of salvianolic acid B.It is generally acknowledged that reaction raw materials is more pure better, but, of the present invention experiment showed, in catalytic conversion reaction, the purity height of salvianolic acid B does not affect conversion reaction effect.On the contrary, when salvianolic acid B purity > 50%, not only produce a large amount of impurity, and do not improve conversion ratio.
Moreover the present invention by experiment repeatedly relatively, has first determined that factor that salvianolic acid A productive rate is produced to material impact is as transformed the concentration, pH value, temperature, time etc. of front pressure differential self.On this basis, be studied by paying concentration and other correlated conditions that a large amount of time, material and energy tests repeatedly to temperature, pH value, time, salvianolic acid B again, and these factors how synergism exerts an influence to salvianolic acid A productive rate jointly each other, thereby determine that salvianolic acid B transforms the optimum temperature of the needs control of salvianolic acid A, pH value, time etc., and salvianolic acid B initial concentration is controlled to 1mg/ml～30mg/ml, thereby make salvianolic acid A conversion ratio of the present invention more obviously be better than other conversion conditions.In chemical reaction, the purity of reactant usually affects the effect of reacting with concentration.Generally reactant is had to concentration requirement, and think that the high specific concentration of concentration is low good.But, of the present invention experiment showed, in catalytic conversion reaction, the concentration height of salvianolic acid B does not affect conversion reaction effect.On the contrary, salvianolic acid B concentration height not only produces a large amount of impurity, and does not improve conversion ratio.And not more high better containing the concentration of salvianolic acid B in salvianolic acid B aqueous solution, the above concentration conversion ratio of 30mg/ml is low on the contrary, and effect is poorer.Therefore, technique of the present invention, aspect cost-saving and production cycle, has unforeseeable technique effect.In the situation that prior art does not provide the enlightenment of any technology, if those skilled in the art only infer theoretically, be impossible draw the conclusion that sour pellet B is changed into main part of raw material of salvianolic acid A under above-mentioned each conditional parameter and have better changing effect.
What is more important, the present invention passes through performing creative labour, find that zinc chloride can significantly improve the conversion ratio of main part of raw material of salvianolic acid B conversion salvianolic acid A as catalyst, reaching that conversion ratio is highly stable approaches 60%, most cases can exceed 60%, this is all impossible in any one prior art in the past, therefore, has obtained unforeseeable technique effect.
Because salvianolic acid A content is lower, the large raising of salvianolic acid A content after transforming, but also containing a large amount of impurity, therefore, select respectively low pole and nonpolar macroporous adsorption resin to carry out crude separation, select again polyamide, solvent extraction, silica gel to separate, and various flows part is measured, remove after impurity part, salvianolic acid A content is brought up to 80% from 10% left and right, to 90%, to 93%, to 96%.
Further, in significantly improving conversion ratio, the present invention is by being suitable for the purification step of actual industry application, especially by after the steps such as a series of separation, eluting processing, do not adopt traditional constant pressure and dry, and preferred microwave vacuum drying, microwave vacuum drying method dry from vacuum drying, spraying, thereby thoroughly overcome, baking temperature was too high in the past, the defect that drying time is long and large to the destruction of salvianolic acid A; And sublimation drying is long, cost extract high and lyophilization gained cannot be removed dissolvent residual problem completely.
The present invention can also be by directly mixing low concentration with the salvianolic acid B of high concentration, only need be made into suitable initial conversion concentration, can reach equally the object that changes into salvianolic acid A, the preparation technology of this conversion raw material is very simple, is very suitable for the application in actual industry when being produced into written or printed documents reduction.
Salvianolic acid A freeze-dried powder provided by the invention, according to the chemistry of salvianolic acid A and physical characteristic, from affecting the stable additives of medicine, dosage form, container, extraneous as air, light, moisture, the generation chemical reactions such as impurity cause the decomposition of medicament to carry out creationary experimental analysis on the contrary.By screening preparation prescription, repetition test, mannitol, glucose, lactose etc. are selected, make freeze-dried powder, the molding of powder pin is good, and block is loose, hole, color and luster are even, and stability is very high, solve due to air, light, moisture, the medicine that the generation chemical reactions such as impurity cause decomposes.Salvianolic acid A is made to suitable preparation, solved salvianolic acid A and made injection stability problem, by selecting suitable adjuvant and dosage form, guaranteed salvianolic acid A pharmacological action, provide safe and effective salvianolic acid A injection formulation for clinical.
For these reasons, we by selecting suitable dosage form, have screened different adjuvants according to the physicochemical property of salvianolic acid A, and preferred different preparation technology and technological parameters, has prepared stable, safe, effective, quality controllable salvianolic acid A freeze-dried powder.
Salvianolic acid A in the present invention is adjusted to applicable scope by alkali liquor by pH value, then by adding mannitol etc., makes it be easier to lyophilization, and do not affect the property of medicine.
The present invention is compared with the prior art and shows: the present invention passes through performing creative labour, finally determined lyophilizing speed cooling rate and cooling time, programming rate and temperature when distillation, determine the vacuum sublimation time, clear and definite dry programming rate and temperature, and drying time; The creationary complex relationship of clearly having determined between frozen cooling speed and temperature, distillation programming rate and the dry programming rate of temperature and temperature and time, and suitable numerical range is definite etc., thus just finally obtain stable lyophilized injectable powder.
What is more important, adopts the salvianolic acid A freeze-dried powder that preparation method of the present invention is made can not change the original chemical attribute of salvianolic acid A completely; The salvianolic acid A freeze-dried powder of making has the features such as good water solubility, heat stability is high, solubility is good compared with salvianolic acid A.
On the other hand, the present invention also provide 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 maxobviously decline, LVEDP significantly increases.With model group comparison, positive drug group, the high, medium and low dosage group of salvianolic acid A freeze-dried powder These parameters all have clear improvement, and can suppress the maximum climbing speed decline of coronary flow minimizing, the rising of left chamber diastolic pressure, left indoor pressure, the maximum fall off rate decline of left indoor pressure that ischemia-reperfusion causes.And salvianolic acid A freeze-dried powder group high dose group is suitable with positive drug group effect.Experimental result shows that salvianolic acid A freeze-dried powder can alleviate isolated rat heart ischemical reperfusion injury degree, and prompting salvianolic acid A freeze-dried powder can obviously improve coronary flow, improve systolic and diastolic function, the cardioprotection ischemical reperfusion injury of isolated heart aorta diastolic function and damaged myocardium.
Known through the experimental data contrast to rats with myocardial ischemia electrocardio and effect of heart function, following coronary artery occlusion causes the long-term myocardial infarction and ischemia model Rat Ecg of rat ST section and significantly improves, its HR, arterial pressure, LVSP ,+dp/dt max,-dp/dt maxall significantly decline, model group rat heart muscle contractile function obviously reduces, and after intravenously administrable 7d, each medicine group rat ST section obviously declines; Indices integrated display, in each medicine group with the middle and high dosage of salvianolic acid A freeze-dried powder the electrocardio to long-term rats with myocardial ischemia and parameters of left ventricular function to improve effect the most obvious, prompting salvianolic acid A freeze-dried powder can reduce rats with myocardial ischemia ST section rising, improve heart rate, increase its arterial pressure, improve the cardiac muscle contracting power of relaxing, electrocardio and cardiac function to rats with myocardial ischemia improve significantly.
Known through experimental data contrast; salvianolic acid A freeze-dried powder 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 freeze-dried powder can suppress oxygen-derived free radicals and produce; improve the oxidation resistance of cardiac muscular tissue; protecting myocardial cell film, reduces overflowing of enzyme, improves myocardium energy supply; reduce ischemia to myocardium degree of injury, thereby play ischemic myocardial protection effect.
Known through experimental data contrast, there is obvious myocardial ischemia infarction in myocardial infarction and ischemia model group rat, and infarction size reaches 53% left and right; Compared with model group, after various dose salvianolic acid A freeze-dried powder drug administration by injection 7d, the myocardial infarct size of rat significantly reduces, and presents certain dose-effect relationship.Prompting salvianolic acid A freeze-dried powder can dwindle myocardial infarct size, alleviates treating myocardial ischemia damage and heart infarction degree, and myocardial ischemia is had to obvious therapeutic action.
Light microscopic and Electronic Speculum results suggest, salvianolic acid A freeze-dried powder can obviously improve the myocardium pathological change of rats with myocardial ischemia, alleviates myocardium pathology damage, can be used for the treatment of ischemic heart desease.
Observation myocardial ischemia in rats-fill with again different time myocardial infarct size, model group myocardial ischemia in rats 1.5h, then after pouring into 6h, myocardial infarction is serious, and in recovery perfusion 48h, infarction size progressively increases, myocardial damage aggravation.Known through experimental data contrast, with model group comparison, various dose salvianolic acid A freeze-dried powder can obviously dwindle myocardial infarct size, alleviate myocardial ischemia-reperfusion damage, and myocardial infarct size does not expand with Ischemia Reperfusion time lengthening; Between each dosage group, there is certain agent effect relationship.Prompting salvianolic acid A freeze-dried powder can dwindle expeirmental myocardial ischemia and pour into the myocardial infarct size of rat again, alleviates Myocardial injury degree, and Myocardial Ischemia Reperfusion Injury is had to good preventive and therapeutic effect.
Known through experimental data contrast, rat is at ischemia-reperfusion 24h, the SOD of cardiac muscular tissue, GSH-Px, CAT enzyme are lived and are significantly declined, MDA, XOD enzyme are lived and are significantly raise, prompting myocardial ischemia in rats-after pouring into again, Peroxidation Product is piled up serious, and myocardial clearance Peroxidation Product ability significantly declines, and Ischemia Reperfusion causes that a large amount of oxygen-derived free radicals generations cause myocardial damage to increase the weight of.Medicine group and model group comparison, the SOD of cardiac muscular tissue, GSH-Px, CAT enzyme are lived and are raise in various degree, and MDA, XOD enzyme are lived and are reduced in various degree, and high, the middle dosage group of salvianolic acid A freeze-dried powder effect is the most obvious.Prompting salvianolic acid A can strengthen myocardial clearance free radical ability, suppresses oxygen-derived free radicals and produces, and strengthens rat heart muscle oxidation resistance, suppresses Myocardial Ischemia Reperfusion Injury.
Known through experimental data contrast, after ligation dog coronary artery, 60min is to (be after administration 45min to during 165min after medicine) during 180min after ligation, each medicine group dog epicardial electrogram ST section is raised sum (∑-ST) continuous decrease, more all has significant difference with model group; Recover after perfusion again, each medicine group ∑-ST and model group significantly reduce.Between the each dosage group of salvianolic acid A freeze-dried powder, have a certain amount of effect relationship, in salvianolic acid A freeze-dried powder, dosage group ∑-ST fall is suitable with 0.5mg/kg hydrochloride for injection diltiazem group.Prompting salvianolic acid A freeze-dried powder can alleviate the degree of myocardial ischemia of experiment ischemia-reperfusion dog.Point out it to damage by Ischemic myocardium, ischemic heart desease is had to good therapeutical effect.
Known through experimental data contrast, after ligation dog coronary artery, 60min is to (be after administration 45min to during 165min after medicine) during 180min after ligation, each medicine group dog epicardial electrogram N-ST still continues to reduce, and more all has significant difference with model group; Recover after perfusion again, each medicine group N-ST and model group significantly reduce.Between the each dosage group of salvianolic acid A freeze-dried powder, have a certain amount of effect relationship, in salvianolic acid A freeze-dried powder, dosage group N-ST slip is suitable with 0.5mg/kg hydrochloride for injection diltiazem group.Prompting salvianolic acid A freeze-dried powder can reduce the myocardial ischemia scope of experiment ischemia-reperfusion dog.Point out it to damage by Ischemic myocardium, ischemic heart desease is had to good therapeutical effect.
Known through experimental data contrast, after model group dog ischemia-reperfusion, myocardial infarction is heavier.Salvianolic acid A freeze-dried powder, hydrochloride for injection diltiazem all can significantly reduce experimental dog myocardial infarct size, and with salvianolic acid A freeze-dried powder high dose group dog myocardial infarction ratio minimum.With model group comparison; high, the middle dosage group of salvianolic acid A freeze-dried powder can extremely significantly reduce infarcted myocardium and account for the proportion of heart and ventricle; the proportion that salvianolic acid A freeze-dried powder low dosage dog myocardial infarction district accounts for heart and ventricle also obviously reduces; prompting salvianolic acid A freeze-dried powder energy dose dependent ground dwindles experimental dog myocardial infarct size; energy Ischemic myocardium-reperfusion injury, is used for the treatment of ischemic heart desease.
Known through experimental data contrast, ligation dog coronary artery forms myocardial ischemia, after administration, 45min, to 225min (being ligation 60min to 240min) after administration, relatively, has increased between 25.3%～52.6% before each medication therapy groups coronary flow and administration; The most remarkable with high, the middle dosage group of salvianolic acid A freeze-dried powder, hydrochloride for injection diltiazem group, amplification is respectively 52.6%, 40.2%, 36.7%.Prompting salvianolic acid A freeze-dried powder can promote collateral circulation open, the coronary flow while increasing myocardial ischemia, thus to resisting myocardial ischemia, Ischemic myocardium damage, points out it aspect control ischemic heart desease, having certain purposes.
Known through experimental data contrast, 15min after the administration of salvianolic acid A freeze-dried powder (being ischemia 30min) can significantly suppress LVEDP rising, inhibition ± dp/dt maxdecline, suppress dog LVW and reduce, reduce TPR, 45min after administration (after being ischemia 60min) can significantly suppress CO and decline; Be dose dependent; After the administration of prompting salvianolic acid A freeze-dried powder, can improve myocardial ischemia dog hemodynamic index, the myocardial contraction of Enhancement test dog, improve myocardium systolic and diastolic function, reduce Peripheral resistance, suppress the blood-pumping function that cardiac output reduces, improves heart, improve myocardial blood supply, improve myocardial ischemia and cardiac function, performance 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 illustrate after ligation coronary ischemia, and dog myocardial mitochondria is destroyed, myocardial cell injury, and myocardium enzyme is released the rising alive of the clear center of hyperamization creatase.The each dosage group of salvianolic acid A freeze-dried powder and model group comparison, can reduce to some extent after dog myocardial ischemia 4h CK, LDH, AST, CK-MB activity in serum, and be dose dependent; Illustrate that salvianolic acid A freeze-dried powder can stablize myocardial cell membrane, the overflowing of myocardium enzyme when minimizing treating myocardial ischemia damage, has obvious protective effect to ischemic myocardium.
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 Acids metabolism, will suppress the activity of glucose oxidase Major Enzymes, thereby increase myocardial oxygen consumption, expands dog myocardial infarct size.And the each dosage group of salvianolic acid A freeze-dried powder and model group comparison can significantly reduce FFA in myocardial ischemia dog serum, LPO content, and be dose-dependence.Prompting salvianolic acid A freeze-dried powder can significantly suppress FFA level in serum and raise, and improves Myocardial Fatty Acids metabolism, reduces the accumulation of lactic acid; the production capacity that increases unit oxygen consumption, reduces myocardial oxygen consumption, thereby improves cardiac function; suppress myocardial infarct size, Ischemic myocardium damage.
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, remove the enzyme significantly reduction alive of oxygen-derived free radicals, and oxygen-derived free radicals can affect by a series of oxidation reactions normal configuration and the function of cell, aggravation treating myocardial ischemia damage degree.The each dosage group of salvianolic acid A freeze-dried powder and model group comparison, can significantly reduce the content of MDA in myocardial ischemia dog serum, makes that SOD in serum, GSH-Px are active obviously to be strengthened simultaneously, and be certain agent effect relationship; Prompting salvianolic acid A freeze-dried powder can suppress lipid peroxidation process by certain mechanism, reducing oxygen-derived free radicals generates, simultaneously can improve endogenous activities of antioxidant enzymes again and alleviate oxygen-derived free radicals to myocardium damage, improving the oxidation resistance of ischemic myocardium and bring into play function of resisting myocardial ischemia.
Known through experimental data contrast, the salvianolic acid A freeze-dried powder of various dose can reduce myocardial oxygen consumption and coefficient of oxygen utilization to some extent, and wherein high, the middle dosage group of salvianolic acid A freeze-dried powder ischemia 120min (being 105min after administration) coefficient of oxygen utilization has reduced respectively nearly 20%, 16% before compared with administration.Illustrate that salvianolic acid A freeze-dried powder can reduce myocardial oxygen consumption and coefficient of oxygen utilization, improve the equilibrium of supply and demand of myocardium oxygen, improve ventricular function, the effect of performance protection ischemic myocardium, control myocardial ischemia.
Known through experimental data contrast, various dose salvianolic acid A freeze-dried powder 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; Between each dosage group, be certain agent effect trend; Prompting salvianolic acid A freeze-dried powder can reduce blood viscosity, suppresses platelet adhesion and assembles, and prevention Intravascular Thrombus forms, 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 freeze-dried powder of various dose can significantly alleviate that rat suppository is wet, dry weight, and thrombosis is had to obvious inhibitory action; Between each dosage group, be certain dose-effect relationship.Prompting salvianolic acid A freeze-dried powder has and suppresses the effect that rat thrombus in vivo forms, and points out it to can be used for preventing and treating the ischemic heart desease due to thrombosis.
Medical science and study of pharmacy personnel cannot not do under the prerequisite of related experiment in advance, learn that in advance salvianolic acid A freeze-dried powder has above-mentioned good purposes.
Accompanying drawing explanation
Fig. 1. salvianolic acid B reference substance high-efficient liquid phase chromatogram;
Fig. 2. salvianolic acid B high-efficient liquid phase chromatogram in Radix Salviae Miltiorrhizae extract;
Fig. 3. salvianolic acid A reference substance high-efficient liquid phase chromatogram;
Fig. 4. salvianolic acid A high-efficient liquid phase chromatogram in salvianolic acid B catalyzed conversion liquid.
Fig. 5. salvianolic acid A freeze-dried powder freeze-drying curve figure.
Fig. 6. salvianolic acid A freeze-dried powder vacuum curve figure.
The specific embodiment
Embodiment 1
Get red rooted salvia, be ground into 6 order granules, add 92 ℃ of water of 7 times of amounts at every turn, warm macerating extracts 3 times, stirs with 25 revs/min of speed simultaneously, and each warm macerating extracts 3 hours, extracting solution is evaporated to relative density 1.20 (60 ℃), adds ethanol to make to contain alcohol amount 70%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 20mg, aqueous solution is adjusted pH to 4.0 with 10% sodium hydroxide, adds 0.5%ZnCl 2as catalyst, 120 ℃ of temperature thermal conversions 4 hours, 20% phosphoric acid adjust pH to 2.5 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 3mg, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 50 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 10, uses respectively 3 times of cylinder hydrops, 5 times of column volume 25% ethanol elutions, removes impurity, use again 4 times of column volume 40% ethanol elutions, HPLC detects, and collects the 40% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 5mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 10 with polyamide ratio, resin column blade diameter length ratio is 1: 8, use respectively 3 times of cylinder hydrops, 12 times of column volume 40% alcoholic solution eluting remove impurity, use 8 times of column volumes, 60% alcoholic solution eluting again, collect the 60% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 15mg aqueous solution, aqueous solution is adjusted pH to 2.5 with 20% phosphoric acid, and by the t-butyl methyl ether of 3 times of amounts of aqueous solution, point 3 extractions, separate organic layer, and reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.5g, adds 1～3 times of amount silica gel, stirs, and volatilizes, be added on 15 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 10, take pentane-t-butyl methyl ether as eluant, gradient elution, use respectively pentane: 10 times of column volumes of t-butyl methyl ether (4: 6) eluting, pentane: 10 times of column volumes of t-butyl methyl ether (6: 4) eluting, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 12 times of water gagings dissolvings, with microwave vacuum drying (60 ℃ of baking temperatures, 3 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 20KW, dry 100 minutes) dry 130 minutes, obtain salvianolic acid A.
Embodiment 2
Get red rooted salvia, be cut into decoction pieces, add 85 ℃ of water temperature lixiviates of 8 times of amounts at every turn and get 3 times, stir simultaneously with 20 revs/min of speed, each warm macerating extracts 2.5 hours, extracting solution is evaporated to relative density 1.16 (60 ℃), adds ethanol to make to contain alcohol amount 75%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 15mg, aqueous solution is adjusted pH to 4.0 with 10% potassium hydroxide, adds 0.6%ZnCl 2as catalyst, 120 ℃ of temperature thermal conversions 3.5 hours, 15% hydrochloric acid adjust pH to 2.5 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 45 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 8, use respectively 3.5 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions, remove impurity, use again 5 times of column volume 45% ethanol elutions, HPLC detects, the 45% ethanol elution part that collection contains salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 5mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 9 with polyamide ratio, resin column blade diameter length ratio is 1: 7, use respectively 4 times of cylinder hydrops, 10 times of column volume 40% alcoholic solution eluting remove impurity, use 8 times of column volumes, 65% alcoholic solution eluting again, collect the 65% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 12mg aqueous solution, aqueous solution is adjusted pH to 2.6 with 15% hydrochloric acid, and by the t-butyl methyl ether of 4 times of amounts, point 4 extractions, separate organic layer, and reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.8g, adds 2～3 times of amount silica gel, stirs, and volatilizes, be added on 13 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 8, take pentane-t-butyl methyl ether as eluant, gradient elution, use respectively 8 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 8 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 8 times of water gagings dissolvings, with microwave vacuum drying (50 ℃ of baking temperatures, 3 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 20KW) dry 140 minutes, obtain salvianolic acid A.
Embodiment 3
Get red rooted salvia, be ground into diameter 2mm granule, add 85 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 2 times, stir simultaneously with 30 revs/min of speed, each warm macerating extracts 3.5 hours, extracting solution is evaporated to relative density 1.10 (60 ℃), adds ethanol to make to contain alcohol amount 75%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 18mg, aqueous solution is adjusted pH to 4.2 with 10% sodium carbonate, adds 0.6%ZnCl 2as catalyst, 123 ℃ of temperature thermal conversions 4.5 hours, 15% nitric acid adjust pH to 2.8 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 6mg, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 40 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 7, uses respectively 4 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions, removes impurity, use again 5 times of column volume 40% ethanol elutions, HPLC detects, and collects the 40% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 6mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 8 with polyamide ratio, resin column blade diameter length ratio is 1: 8, use respectively 4 times of cylinder hydrops, 9 times of column volume 40% alcoholic solution eluting remove impurity, use 7 times of column volumes, 65% alcoholic solution eluting again, collect the 65% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 10mg aqueous solution, aqueous solution is adjusted pH to 2.6 with 15% nitric acid, and by the t-butyl methyl ether of 4 times of amounts, point 4 extractions, separate organic layer, and reclaim under reduced pressure methyl acetate is made the extract of every 1ml containing salvianolic acid A 0.7g, adds 3 times of amount silica gel, stirs, and volatilizes, be added on 10 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 7, take pentane-t-butyl methyl ether as eluant, gradient elution, use respectively 8 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 9 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 8 times of water gagings dissolvings, with microwave vacuum drying (60 ℃ of baking temperatures, 1 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 10KW) dry 110 minutes, obtain salvianolic acid A.
Embodiment 4
Get red rooted salvia, be cut into decoction pieces, add 80 ℃ of water temperature lixiviates of 10 times of amounts at every turn and get 3 times, stir simultaneously with 15 revs/min of speed, each warm macerating extracts 3 hours, extracting solution is evaporated to relative density 1.15 (60 ℃), adds ethanol to make to contain alcohol amount 70%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 20mg, aqueous solution is adjusted pH to 5.4 with 10% sodium bicarbonate, adds 0.8%ZnCl 2as catalyst, 128 ℃ of temperature thermal conversions 4.0 hours, 20% sulphuric acid adjust pH to 2.6 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 40 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 7, uses respectively 4 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions, removes impurity, use again 5 times of column volume 40% ethanol elutions, HPLC detects, and collects the 40% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 6mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 10 with polyamide ratio, resin column blade diameter length ratio is 1: 15, use respectively 4 times of cylinder hydrops, 7 times of column volume 35% alcoholic solution eluting remove impurity, use 6 times of column volumes, 60% alcoholic solution eluting again, collect the 60% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 12mg aqueous solution, aqueous solution is adjusted pH to 2.8 with 20% sulphuric acid, and by the t-butyl methyl ether of 4 times of amounts, point 4 extractions, separate organic layer, and reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.5g, adds 2.5 times of amount silica gel, stirs, and volatilizes, be added on 9 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 8, take pentane-t-butyl methyl ether as eluant, gradient elution, use respectively 8 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 8 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 10 times of water gagings dissolvings, with microwave vacuum drying (80 ℃ of baking temperatures, 5 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 60KW) dry 100 minutes, obtain salvianolic acid A.
Embodiment 5
Get red rooted salvia, be ground into diameter 2mm granule, add 85 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir simultaneously with 20 revs/min of speed, each warm macerating extracts 2.5 hours, extracting solution is evaporated to relative density 1.12 (60 ℃), adds ethanol to make to contain alcohol amount 70%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 10mg, aqueous solution is adjusted pH to 5.5 with 20% sodium citrate, adds 0.4%ZnCl 2as catalyst, 132 ℃ of temperature thermal conversions 3.5 hours, 20% acetic acid adjust pH to 2.6 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 35 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 8, use respectively 3 times of cylinder hydrops, 4.5 times of column volume 25% ethanol elutions, remove impurity, use again 6 times of column volume 40% ethanol elutions, HPLC detects, the 40% ethanol elution part that collection contains salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 8mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 12 with polyamide ratio, resin column blade diameter length ratio is 1: 18, use respectively 4 times of cylinder hydrops, 8 times of column volume 30% alcoholic solution eluting remove impurity, use 5 times of column volumes, 65% alcoholic solution eluting again, collect the 65% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 15mg aqueous solution, aqueous solution is adjusted pH to 2.7 with 20% acetic acid, and by the t-butyl methyl ether of 5 times of amounts, point 5 extractions, separate organic layer, and reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.5g, adds 2 times of amount silica gel, stirs, and volatilizes, be added on 10 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 10, with pentane, t-butyl methyl ether is eluant, gradient elution, use respectively 8 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 8 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 12 times of water gagings dissolvings, with microwave vacuum drying (45 ℃ of baking temperatures, 3 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 60KW) dry 150 minutes, obtain salvianolic acid A.
Embodiment 6
Get red rooted salvia, be ground into diameter 2mm granule, add 88 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir simultaneously with 22 revs/min of speed, each warm macerating extracts 3.5 hours, extracting solution is evaporated to relative density 1.23 (60 ℃), adds ethanol to make to contain alcohol amount 75%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 13mg, aqueous solution is adjusted pH to 5.6 with 10% sodium hydroxide, adds 0.5%ZnCl 2as catalyst, 133 ℃ of temperature thermal conversions 4.5 hours, 10% hydrochloric acid adjust pH to 2.7 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 40 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 9, uses respectively 4 times of cylinder hydrops, 4 times of column volume 22% ethanol elutions, removes impurity, use again 6 times of column volume 43% ethanol elutions, HPLC detects, and collects the 43% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every ml containing 10mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 15 with polyamide ratio, resin column blade diameter length ratio is 1: 20, use respectively 4 times of cylinder hydrops, 8 times of column volume 30% alcoholic solution eluting remove impurity, use 5 times of column volumes, 60% alcoholic solution eluting again, collect the 60% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 12mg aqueous solution, aqueous solution is adjusted pH to 2.8 with 10% hydrochloric acid, and by the t-butyl methyl ether of 5 times of amounts, point 5 extractions, separate organic layer, and reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.5g, adds 3 times of amount silica gel, stirs, and volatilizes, be added on 12 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 10, with pentane, t-butyl methyl ether is eluant, gradient elution, use respectively 6 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 7 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 10 times of water gagings dissolvings, with microwave vacuum drying (70 ℃ of baking temperatures, 2 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 5KW) dry 120 minutes, , obtain salvianolic acid A.
Embodiment 7
Get red rooted salvia, be cut into decoction pieces, add 90 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir simultaneously with 25 revs/min of speed, each warm macerating extracts 3 hours, extracting solution is evaporated to relative density 1.20 (60 ℃), adds ethanol to make to contain alcohol amount 75%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 20mg, aqueous solution is adjusted pH to 5.0 with 10% sodium carbonate, adds 1.0%ZnCl 2as catalyst, 135 ℃ of temperature thermal conversions 4.5 hours, 15% sulphuric acid adjust pH to 3.0 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 36 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 9, uses respectively 3 times of cylinder hydrops, 4 times of column volume 25% ethanol elutions, removes impurity, use again 5 times of column volume 45% ethanol elutions, HPLC detects, and collects the 45% ethanol elution part that contains salvianolic acid A, and decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every 1ml containing 6mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 10 with polyamide ratio, resin column blade diameter length ratio is 1: 10, use respectively 4 times of cylinder hydrops, 8 times of column volume 45% alcoholic solution eluting remove impurity, use 8 times of column volumes, 65% alcoholic solution eluting again, collect the 65% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 10mg aqueous solution, aqueous solution is adjusted pH to 2.7 with 15% sulphuric acid, and by the t-butyl methyl ether of 4 times of amounts, point 4 extractions, separate organic layer, and reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.6g, adds 3 times of amount silica gel, stirs, and volatilizes, be added on 10 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 8, with pentane, t-butyl methyl ether is eluant, gradient elution, use respectively 8 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 8 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 8 times of water gagings dissolvings, with microwave vacuum drying (55 ℃ of baking temperatures, 2 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 70KW) dry 90 minutes, obtain salvianolic acid A.
Embodiment 8
Get red rooted salvia, be ground into diameter 2mm granule, add 85 ℃ of water temperature lixiviates of 9 times of amounts at every turn and get 3 times, stir simultaneously with 22 revs/min of speed, each warm macerating extracts 3 hours, extracting solution is evaporated to relative density 1.14 (60 ℃), adds ethanol to make to contain alcohol amount 70%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, be diluted with water to every 1ml containing salvianolic acid B 25mg, aqueous solution is adjusted pH to 4.2 with 10% sodium citrate, adds 0.4%ZnCl 2as catalyst, 133 ℃ of temperature thermal conversions 4.5 hours, 10% hydrochloric acid adjust pH to 2.8 for conversional solution, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 5mg, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 45 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 10, use respectively 5 times of cylinder hydrops, 6 times of column volume 20% ethanol elutions, remove impurity, use again 5 times of column volume 45% ethanol elutions, HPLC detects, the 45% ethanol elution part that collection contains salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste, aqueous solution is concentrated into the solution of every 1ml containing 8mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 12 with polyamide ratio, resin column blade diameter length ratio is 1: 8, use respectively 3 times of cylinder hydrops, 6 times of column volume 45% alcoholic solution eluting remove impurity, use 8 times of column volumes, 55% alcoholic solution eluting again, collect the 55% alcoholic solution part that contains salvianolic acid A, decompression recycling ethanol is also concentrated into every 1ml containing salvianolic acid A 10mg aqueous solution, aqueous solution is adjusted pH to 2.9 with 15% hydrochloric acid, and by the t-butyl methyl ether of 5 times of amounts, point 5 extractions, separate organic layer, and reclaim under reduced pressure t-butyl methyl ether is made the extract of every 1ml containing salvianolic acid A 0.6g, adds 2.5 times of amount silica gel, stirs, and volatilizes, be added on 12 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 8, with pentane, t-butyl methyl ether is eluant, gradient elution, use respectively 8 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 8 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 9 times of water gagings dissolvings, with microwave vacuum drying (40 ℃ of baking temperatures, 1 ℃ of return difference temperature, more than vacuum-0.07Mpa, microwave power 80KW) dry 130 minutes, obtain salvianolic acid A.
Embodiment 9
Get the salvianolic acid A 80g that embodiment 3 makes, inject water 2800ml and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.5, add mannitol 60g to make to dissolve, add again vitamin C 0.5g, be stirred to dissolve, mix, add active carbon 2g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-40 ℃ with 20 ℃/h speed, be incubated freezing 16 hours; Be evacuated to below 0.3mbar, in 10 hours, the salvianolic acid A formulation temperature freezing risen to-25 ℃, maintain-25 ℃ of vacuum dryings 8 hours; Continue to heat up, be evenly warming up to 41 ℃ with 0.5 ℃/min, maintain 41 ℃ and be dried after 3 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 10
Get the salvianolic acid A 20g that embodiment 4 makes, inject water 1900ml and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.8, add mannitol 20g to make to dissolve, add again vitamin C 0.8g, be stirred to dissolve, mix, add active carbon 0.5g stirring and adsorbing, filter, remove active carbon; Inject water to 2000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-45 ℃ with 25 ℃/h speed, be incubated freezing 15 hours; Be evacuated to below 0.3mbar, in 12 hours, the salvianolic acid A formulation temperature freezing risen to-25 ℃, maintain-25 ℃ of vacuum dryings 8 hours; Continue to heat up, be evenly warming up to 40 ℃ with 0.8 ℃/min, maintain 40 ℃ and be dried after 3 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 11
Get the salvianolic acid A 10g that embodiment 5 makes, inject water 1500ml and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.6, add mannitol 20g to make to dissolve, add again vitamin C 0.8g, be stirred to dissolve, mix, add active carbon 1.2g stirring and adsorbing, filter, remove active carbon; Inject water to 2000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-42 ℃ with 25 ℃/h speed, be incubated freezing 12 hours; Be evacuated to below 0.3mbar, in 13 hours, the salvianolic acid A formulation temperature freezing risen to-23 ℃, maintain-23 ℃ of vacuum dryings 7 hours; Continue to heat up, be evenly warming up to 42 ℃ with 0.8 ℃/min, maintain 42 ℃ and be dried after 4 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 12
Get the salvianolic acid A 30g that embodiment 6 makes, inject water 2000ml and be stirred to dissolve, with 20% sodium carbonate adjust pH 4.2, add mannitol 20g, lactose 10g to make to dissolve, add again thiourea 0.5g, be stirred to dissolve, mix, add active carbon 1.0g stirring and adsorbing, filter, remove active carbon; Inject water to 2000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-44 ℃ with 29 ℃/h speed, be incubated freezing 14 hours; Be evacuated to below 0.3mbar, in 11 hours, the salvianolic acid A formulation temperature freezing risen to-26 ℃, maintain-26 ℃ of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 43 ℃ with 0.6 ℃/min, maintain 43 ℃ and be dried after 3.5 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 13
Get the salvianolic acid A 35g that embodiment 7 makes, inject water 2500ml and be stirred to dissolve, with 10% potassium hydroxide adjust pH 4.5, add glucose 30g to make to dissolve, add again sodium sulfite 3g, be stirred to dissolve, mix, add active carbon 1.5g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-43 ℃ with 27 ℃/h speed, be incubated freezing 11 hours; Be evacuated to below 0.3mbar, in 12.5 hours, the salvianolic acid A formulation temperature freezing risen to-24 ℃, maintain-24 ℃ of vacuum dryings 7.5 hours; Continue to heat up, be evenly warming up to 40 ℃ with 0.7 ℃/min, maintain 40 ℃ and be dried after 5 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 14
Get the salvianolic acid A 40g that embodiment 8 makes, injecting water 2700ml is stirred to dissolve, with 10% sodium hydroxide adjust pH 4.3, add glucose 20g, lactose 20g to make to dissolve, then add sodium pyrosulfite 4g, be stirred to dissolve, mix, add active carbon 1.5g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-41 ℃ with 28 ℃/h speed, be incubated freezing 13 hours; Be evacuated to below 0.3mbar, in 13.5 hours, the salvianolic acid A formulation temperature freezing risen to-25.5 ℃, maintain-25.5 ℃ of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 44 ℃ with 0.9 ℃/min, maintain 44 ℃ and be dried after 3.5 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 15
Get the salvianolic acid A 60g that embodiment 1 makes, inject water 2700ml and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.0, add mannitol 40g to make to dissolve, add again Catergen g, be stirred to dissolve, mix, add active carbon 2g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-42.5 ℃ with 24.5 ℃/h speed, be incubated freezing 11.5 hours; Be evacuated to below 0.3mbar, in 13.5 hours, the salvianolic acid A formulation temperature freezing risen to-24.5 ℃, maintain-24.5 ℃ of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 42.5 ℃ with 0.55 ℃/min, maintain 42.5 ℃ and be dried after 2.5 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Embodiment 16
Get the salvianolic acid A 70g that embodiment 2 makes, inject water 2800ml and be stirred to dissolve, with 10% sodium hydroxide adjust pH 5.0, add mannitol 40g to make to dissolve, add again Catergen g, be stirred to dissolve, mix, add active carbon 2g stirring and adsorbing, filter, remove active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-44.5 ℃ with 28.5 ℃/h speed, be incubated freezing 11.5 hours; Be evacuated to below 0.3mbar, in 13.5 hours, the salvianolic acid A formulation temperature freezing risen to-24.5 ℃, maintain-24.5 ℃ of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 43.5 ℃ with 0.75 ℃/min, maintain 43.5 ℃ and be dried after 3 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
Experimental example 1: salvianolic acid A, the research of salvianolic acid B analytical method
1. instrument and reagent
Instrument: Waters e2695 high performance liquid chromatograph, Empower2 chromatographic work station, 2998 diode array detector; Sartorius cp225D 100,000/electronic balance.
Chromatographic column: YMC C 18chromatographic column (250 × 4.6mm, 5 μ are m);
Reagent: methanol is chromatographically pure, water is ultra-pure water prepared by Millipore, other reagent are analytical pure.
Salvianolic acid B reference substance (lot number 111562-201009) is all purchased from Nat'l Pharmaceutical & Biological Products Control Institute, for assay; Salvianolic acid A reference substance, for self-control, is 99.52% through purity mark content.
2. the preparation of reference substance solution and need testing solution
The preparation of 2.1 reference substance solution: precision takes salvianolic acid B, the about 10mg of salvianolic acid A reference substance respectively, puts in 100ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, in contrast product stock solution; Precision is drawn above-mentioned each 1ml respectively again, puts in same 10ml measuring bottle, adds methanol and is diluted to scale, shakes up, as mixing reference substance solution.
The preparation precision of 2.3 need testing solutions takes embodiment 1 sample (being approximately equivalent to salvianolic acid A 10mg) and following " 1.3 salvianolic acid B raw material preparations in experimental example 2 " obtain sample (being approximately equivalent to salvianolic acid B 10mg), put in 100ml measuring bottle, add dissolve with methanol and be diluted to scale, shake up, to obtain final product.
3. chromatographic condition and system suitability
Take octadecylsilane chemically bonded silica as filler; Flow velocity 1.0ml/min; Detect wavelength: get mixing reference substance solution, carry out UV scanning, result has absorption maximum at 286nm wavelength place, therefore determine that detecting wavelength is 286nm; 30 ℃ of column temperatures; Number of theoretical plate calculates and should be not less than 10000 by salvianolic acid A peak.
0-10 minute time, the ratio of methanol is down to 60% by 30% ratio that rises to 40%, 0.1-0.5% phosphate aqueous solution by 70%; 10-30 minute time, the ratio of methanol is down to 45% by 40% ratio that rises to 55%, 0.1-0.5% phosphate aqueous solution by 50%; 30-60 minute time, the ratio of methanol is down to 20% by 55% ratio that rises to 80%, 0.1-0.5% phosphate aqueous solution by 45%.
Under these conditions, the chromatographic peak retention time of salvianolic acid A, salvianolic acid B reference substance and Radix Salviae Miltiorrhizae extract, salvianolic acid catalyzed conversion liquid is in table 1, and HPLC collection of illustrative plates is shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4.
The chromatographic peak retention time result of the each reference substance of table 1.
Figure BSA00000811649800251
4, the investigation of linear relationship
Accurate above-mentioned mixing reference substance solution 0.1ml, 0.2ml, 0.5ml, 1ml, 2ml, the 5ml of drawing, put respectively in 10ml measuring bottle, add methanol and be diluted to following concentration and be about: the series standard solution of 0.001mg/ml, 0.002mg/ml, 0.005mg/ml, 0.01mg/ml, 0.02mg/ml, 0.05mg/ml.The accurate each 10 μ l injection liquid chromatographies of above-mentioned standard solution of drawing, calculate peak area by " 3. chromatographic condition and system suitability " lower chromatographic condition, take peak area integrated value as vertical coordinate, each concentration reference substance sample size is abscissa, drawing standard curve respectively.Result shows to mix reference substance solution and become good linear relationship, table 2 in following scope.
Table 2. mixes reference substance solution linear relationship result
Figure BSA00000811649800252
5. precision test
Get mixing reference substance solution, measure according to " 3. chromatographic condition and system suitability " lower chromatographic condition, repeat sample introduction 6 times.Result shows that the precision of the method is good, in table 3.
Table 3. Precision test result
Figure BSA00000811649800261
6. stability test
Get need testing solution, measure according to " 3. chromatographic condition and system suitability " lower chromatographic condition, at regular intervals sample introduction once, in result need testing solution each composition in 24 hours peak area without significant change, show having good stability of need testing solution, in table 4.
Table 4. stability test result
Figure BSA00000811649800262
7. replica test
Get this salvianolic acid A raw material, add methanol and make 6 parts of need testing solutions, measure according to " 3. chromatographic condition and system suitability " lower chromatographic condition.Result shows that the method repeatability is good, in table 5.
Table 5. replica test result
Figure BSA00000811649800263
8. recovery test
Adopt application of sample recovery test, get the salvianolic acid A raw material 10mg of known content, accurately weighed, parallel 6 parts, add a certain amount of reference substance solution in the ratio of each constituent content-reference substance (1: 1) respectively, by 2.3 below legal system available test sample solutions, measure and calculate average recovery and the RSD of each composition.Result shows that the accuracy of the method is good, in table 6.
Table 6 recovery test result
Figure BSA00000811649800271
Experimental example 2: the extraction of salvianolic acid B
1.1 extract the confirmation of solvent and extracting method
Take red rooted salvia 400g, measure content of danshinolic acid B by method under Chinese Pharmacopoeia Radix Salviae Miltiorrhizae item, be divided into 4 parts, test by following testing program:
Experiment 1: get red rooted salvia 100g, add 8 times of water gagings at every turn and extract 1.5 hours at 80 ℃ of warm macerating, warm macerating extracts three times altogether, and merge extractive liquid, is measured salvianolic acid B and calculates extraction ratio, and result is as table 7.
Experiment 2: get red rooted salvia 100g, add 8 times of water gagings at every turn and extract 1.5 hours at 80 ℃ of warm macerating, stir simultaneously with 10～50 revs/min of speed, warm macerating stirs and extracts three times altogether, and merge extractive liquid, is measured salvianolic acid B and calculates extraction ratio, and result is as table 7.
Experiment 3: get red rooted salvia 100g, add 8 times of water gagings at every turn and decoct 1.5 hours, decoct altogether three times, merge extractive liquid,, measures salvianolic acid B and calculate extraction ratio, and result is as table 7.
Experiment 4: get red rooted salvia 100g, add 8 times of amount 50% alcohol reflux 1.5 hours at every turn, extract altogether three times, merge extractive liquid,, measures salvianolic acid B and calculate extraction ratio, and result is as table 7.
Table 7. extracts solvent and extracting method experimental result
Figure BSA00000811649800272
Figure BSA00000811649800281
Above-mentioned experimental result shows, adopt 50% ethanol to make extraction solvent salvianolic acid B extraction ratio is had to impact, 50% ethanol extraction is better than water extraction, but with water temperature soak add stir extract difference little, two kinds of extracting modes that stir in water extraction process and do not stir have impact to the extraction ratio of salvianolic acid B, decocting extraction may be because temperature is higher, and salvianolic acid B is extracted and has impact.
The preferred extraction process of 1.2 orthogonal experiment
1.2.1 the optimization of water extraction process research: according to above result of the test, water extraction process using extraction time (A), extraction time (B), quantity of solvent (C), extract four of temperature (D) as investigation factor, each factor is established three levels, carry out Orthogonal Experiment and Design (table 8, table 9) by L9 (34) orthogonal table, the extraction ratio that investigation index is salvianolic acid B.
Table 8. extraction factor water-glass
Figure BSA00000811649800282
Table 9. extraction process orthogonal experiments table
Figure BSA00000811649800291
Table 10. the results of analysis of variance
Figure BSA00000811649800292
F 0.05(2，2)＝19.00 F 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 ℃, warm macerating extracts 1～3 time, stir with 10～50 revs/min of speed simultaneously, extract 1～4 hour at every turn or add 3～15 times of amounts at every turn and decoct extraction, each extraction 1～4 hour, extracts 1～3 time altogether.
1.2.2 the optimization of alcohol extraction technique research: according to above result of the test, alcohol extraction technique is using four of extraction times (A), extraction time (B), quantity of solvent (C), concentration of alcohol (D) as investigation factor, each factor is established three levels, by L9 (3 4) orthogonal table carries out Orthogonal Experiment and Design (table 11, table 12), the extraction ratio that to investigate index be salvianolic acid B.
Table 11. extraction factor water-glass
Figure BSA00000811649800293
Table 12. extraction process orthogonal experiments
Table 13. the results of analysis of variance
Figure BSA00000811649800302
F 0.05(2，2)＝19.00 F 0.01(2，2)＝99.00
Alcohol reflux extracts the results of analysis of variance and shows, each experimental factor, to the extraction rate of transform of salvianolic acid B there are no significant difference, therefore the extraction conditions of this salvianolic acid B is measured 30%～60% alcohol reflux 1～3 time for adding 3～15 times, extracts 1～4 hour at every turn.
1.3 salvianolic acid B raw material 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 ℃ of warm macerating, stir with 10～50 revs/min of speed simultaneously, warm macerating stirs and extracts 3 times altogether, filters merging filtrate, extracting solution is evaporated to relative density 1.10～1.25 (60 ℃), add ethanol to make to contain alcohol amount 60%, filter, decompression filtrate recycling ethanol is also concentrated into without alcohol taste, vacuum drying, salvianolic acid extract 4.15kg, recording content of danshinolic acid B is 10.25%.
Experimental example 3: salvianolic acid B transforms the comparison of salvianolic acid A technique
Experiment 1: get the about 30g of salvianolic acid B raw material of preparation under 1.3, be mixed with 200ml solution, add 10% sodium hydroxide solution adjust pH to 4.5;
Experiment 2: get the about 30g of salvianolic acid B raw material of preparation under 1.3, be mixed with 200ml solution, add 10% sodium hydroxide solution adjust pH to 4.5, add 1.0%ZnCl 2;
Experiment 3: get containing the about 6g of salvianolic acid B (content 51.54%) raw material, adding purified water, to be diluted to salvianolic acid B concentration be 15mg/ml solution, adds carbamide, and making it with the mol ratio of salvianolic acid B is 0.5;
Above-mentioned each experimental group is placed in same autoclave, reacts 4.0 hours at 120 ℃, cooling, calculates salvianolic acid A productive rate.
Table 14. salvianolic acid B transforms salvianolic acid A experimental result
Figure BSA00000811649800311
The demonstration of table 14 result, salvianolic acid B high-purity affects not transforming, does not need to be purified to more than 50% to transform, and salvianolic acid B is converted in salvianolic acid A process, regulates pH value, adds 1.0%ZnCl 2, can greatly improve the productive rate of salvianolic acid A.
Experimental example 4: salvianolic acid B transforms salvianolic acid A process optimization
Above-mentioned experimentation proves, salvianolic acid B purity is not to affect salvianolic acid B to transform the factor of salvianolic acid A, but adds certain catalysts influence salvianolic acid B to transform salvianolic acid A, and therefore, we all add 1%ZnCl to each experimental group 2as catalyst, be converted into other factors of salvianolic acid A to affecting salvianolic acid B: salvianolic acid B concentration (A), pH (B), temperature (C), time (D) are carried out orthogonal test, each factor is established three levels, carry out EXPERIMENTAL DESIGN (table 15, table 16) by L9 (34) orthogonal table, the productive rate that investigation index is salvianolic acid A.
Table 15. transforming agent water-glass
Figure BSA00000811649800321
Table 16. conversion process orthogonal experiments
Figure BSA00000811649800322
Table 17. the results of analysis of variance table
Figure BSA00000811649800331
*F 0.05(2，2)＝19.00 △F 0.01(2，2)＝99.00
The results of analysis of variance demonstration, the optimum condition that this orthogonal test is optimized according to intuitive analysis is A 3b 2c 2d 2, factor A (salvianolic acid B concentration) has certain influence to salvianolic acid A productive rate, analyzes from K value, concentration is converted into salvianolic acid A on improving salvianolic acid B effect higher than 30mg/ml does not affect, and the impurity forming is more, therefore, before transforming, salvianolic acid B concentration should be selected 1～30mg/ml; Factor B (pH), factor C (temperature) have utmost point significant difference to salvianolic acid A productive rate, in prompting salvianolic acid B conversion process, should strictly control pH and temperature, can successfully realize transforming to salvianolic acid A of salvianolic acid B higher yields.
Experimental example 5: catalyst Z nCl 2consumption is on the impact transforming
Transform salvianolic acid A process optimization condition by above-mentioned salvianolic acid B, get salvianolic acid B raw material, add purified water 200ml, make the solution that salvianolic acid B concentration is 15mg/ml, regulating pH is 4.5, and conversion temperature is 120 ℃, and transformation time is 4 hours, by with salvianolic acid B molar percentage, add respectively not commensurability catalyst Z nCl 2, the results are shown in Table 18.
Table 18. catalyst Z nCl 2consumption affects experimental result to transforming
Figure BSA00000811649800332
Figure BSA00000811649800341
Catalyst Z nCl 2consumption affects experimental result to conversion and shows, catalyst amount>=0.02% can produce 55.65% conversion ratio, preferred catalyst consumption 0.1%-3.0%, more preferably, after 0.5%～2.0%. catalyst amount > 3%, conversion ratio is no longer significantly improved.
Experimental example 6: catalyst transforms the catalytic action of salvianolic acid A to salvianolic acid B
Transform salvianolic acid A process optimization condition by above-mentioned salvianolic acid B, get salvianolic acid B raw material, add purified water 200ml, make the solution that salvianolic acid B concentration is 15mg/ml, regulating pH is 4.5, and conversion temperature is 120 ℃, and transformation time is 4 hours, add respectively the catalyst of different cultivars and various dose to transform, the results are shown in Table 19.
Table 19. different catalysts transforms the impact of red A on red B
Figure BSA00000811649800342
Table 19 result shows, above 4 kinds of catalyst all can be used as the catalyst in salvianolic acid B conversion reaction, and each catalyst amount and salvianolic acid B molar percentage be 0.5%～3.0%, productive rate >=40% of salvianolic acid A.Conversion ratio exceedes 60%, but Comprehensive Assessment adopts ZnCl 2optimum.
Experimental example 7: the purification with macroreticular resin of salvianolic acid A
Get salvianolic acid A and transform 13 parts of solution, put in each model macroporous resin 100g that pretreatment is good, after shaking table dynamic adsorption 8 hours, dress post, wash successively 3 column volumes of eluting, 5 column volumes of 20% ethanol elution, 5 column volumes of 70% ethanol elution with water, collect 70% ethanol containing salvianolic acid A eluent part, carry out salvianolic acid A assay, eluent evaporate to dryness calculates dry cream amount, the results are shown in Table 20.
The confirmation of table 20. macroporous resin model
Result shows: above 13 kinds of macroporous resins are good to the adsorption effect of salvianolic acid A, and can well remove impurity with the ethanol gradient elution of variable concentrations, obtain the salvianolic acid A eluent that content is greater than 75%, with the HPD-100 amount of getting dry extract maximum, content is high, and resin absorption amount is large.
Experimental example 8: the polyamide column chromatography purification of salvianolic acid A
Get 3 parts of salvianolic acid A solution after purification with macroreticular resin, put in each model resin 100g that pretreatment is good, after shaking table dynamic adsorption 8 hours, dress post, wash successively 3 column volumes of eluting, 5 column volumes of 20% ethanol elution, 5 column volumes of 70% ethanol elution with water, collect 70% ethanol elution and carry out salvianolic acid A assay, part eluent evaporate to dryness calculates dry cream amount, the results are shown in Table 21.
The confirmation of table 21. resin model
Figure BSA00000811649800361
Result shows: above 3 kinds of resins are good to the adsorption effect of salvianolic acid A, and can well remove impurity with the ethanol gradient elution of variable concentrations, obtains content and is about 90% salvianolic acid A eluent; Maximum with the polyamide amount of getting dry extract, content high adsorption capacity is large.
Experimental example 9: the abstraction purification of salvianolic acid A
By 70% ethanol elution decompression recycling ethanol after column chromatography purification for the second time, adjust pH 2.0～4.0, use again n-butyl alcohol, t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate, Ethyl formate, ethanol extraction 5 times, Separation of Organic phase, reclaim solvent, dry, obtain salvianolic acid A, measure salvianolic acid A content, the results are shown in Table 22.
The confirmation of organic solvent for table 22. extraction
Figure BSA00000811649800362
Table 22 result shows: n-butyl alcohol is because polarity is large, salvianolic acid A amount is maximum, but its salvianolic acid A content is not improved significantly, ether is because polarity is less than normal, water-solubility impurity is few, and salvianolic acid A content is high, but the salvianolic acid A amount obtaining is few, the salvianolic acid A amount that other extraction solvent t-butyl methyl ether, methyl acetate, ethyl acetate, butyl acetate, Ethyl formate obtain is larger, and salvianolic acid A content is all improved; Obtain with t-butyl methyl ether that extract is many, salvianolic acid A content is high.
Experimental example 10: the purification by silica gel column chromatography of salvianolic acid A
Get 12 parts of salvianolic acid A extracts after abstraction purification, every part containing salvianolic acid A 10g, adds the silica gel of 20g, stirs, and volatilizes; Be added on the dry silicagel column of the 100g having installed stirring sample silica gel, the two-phase solvent forming take petroleum ether, pentane, normal heptane, ethyl acetate, methyl acetate, Ethyl formate, t-butyl methyl ether is respectively as eluant, HPLC or thin layer chromatography detect, collect salvianolic acid A eluent, eluent evaporate to dryness, get dry extract, measure salvianolic acid A content, the results are shown in Table 23.
The confirmation of table 23. eluant
Figure BSA00000811649800371
Result shows: when salvianolic acid A is used normal phase silica gel column chromatography, adopting the two-phase solvent of pentane-t-butyl methyl ether composition is eluant, and gradient elution, can well remove impurity, obtains highly purified salvianolic acid A eluent.
Experimental example 11: salvianolic acid A drying means research
Get 4 parts of eluents after silica gel purification eluting, every part containing salvianolic acid A 100g, is concentrated into without thick paste shape, adds after 10 times of water gagings dissolve, to adopt respectively that vacuum drying, lyophilisation, spraying are dry, microwave vacuum drying obtains salvianolic acid A, this salvianolic acid A is detected, the results are shown in Table 24.
Table 24. salvianolic acid A drying means testing result
Figure BSA00000811649800381
Result shows: adopt lyophilisation overlong time, and high cost, and organic solvent residual is serious; The vacuum drying time is slightly long, and Drying Time of Vertical Spray Dryer is short but instantaneous temperature is higher; Microwave vacuum drying baking temperature is low, and the time is short, and gained salvianolic acid A indices is good.
Through further experiment is definite, microwave vacuum drying optimum range is chosen as temperature: 20-100 ℃, and return difference temperature 1-5 ℃, more than vacuum-0.07Mpa, microwave power 1-100KW, dry 10-200 minute.
Experimental example 12: the Preliminary screening of salvianolic acid A freeze-dried powder adjuvant
Freeze-dried powder generally need add appropriate amount of auxiliary materials, adjuvant mainly comprises filler and antioxidant, be mainly used in following object: the dissolubility that increases composition in injection, when the molding of powder pin, need add filler as supporter, improve mouldability, regulate powder pin admittedly containing thing weight, regulate osmotic pressure, add antioxidant to keep principal agent stable simultaneously.
Get salvianolic acid A raw material by table 25 and make freeze-dried powder from different filleies and vitamin C.
The table 25. filler screening table of writing out a prescription
Figure BSA00000811649800382
Get salvianolic acid A by table 25 formula, add the water for injection of making total amount 90% and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.5, add adjuvant to make to dissolve, mix, add active carbon 2g stirring and adsorbing, filtration, removes active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-42 ℃ with 28 ℃/h speed, be incubated freezing 12 hours; Be evacuated to below 0.3mbar, in 11 hours, the salvianolic acid A formulation temperature freezing risen to-25 ℃, maintain-25 ℃ of vacuum dryings 6.5 hours; Continue to heat up, be evenly warming up to 40 ℃ with 0.8 ℃/min, maintain 40 ℃ and be dried after 5 hours, sample temperature is down to room temperature, add a cover, observe mouldability, in table 26.
The different filleies of table 26 affect result to mouldability
Figure BSA00000811649800392
Figure BSA00000811649800401
From table 26 experimental result, add not commensurability filler, larger on the impact of mouldability, filler mannitol, glucose, lactose are better to lyophilized formulations effect, lyophilized powder sedimentation is good, porous nickel, therefore as selecting adjuvant, but general lower than 20mg mouldability and solubility.
Experimental example 13: different filleies and the antioxidant impact on lyophilized formulations type
According to above-mentioned experimental result, select respectively mannitol, glucose, lactose to carry out confirmatory study, and antioxidant is compared to research, select respectively vitamin C, thiourea, sodium sulfite, sodium pyrosulfite as antioxidant, experimental program is in table 27, and compare with not adding antioxidant prescription, experimental result is in table 28.
The different filleies of table 27 and antioxidant Preliminary screening
Figure BSA00000811649800402
Get salvianolic acid A by table 27 formula, add the water for injection of making total amount 90% and be stirred to dissolve, with 10% sodium hydroxide adjust pH 4.5, add filler and antioxidant to make dissolving, mix, add active carbon 2g stirring and adsorbing, filtration, removes active carbon; Inject water to 3000ml, detect intermediate content, pH value, detect qualified rear use 0.22 μ m microporous filter membrane and filter, filtrate fill in aseptic cillin bottle, part butyl rubber bung beyond the Great Wall, make altogether 1000 bottles, sabot, after fill completes, sends into freeze dryer, close chamber door, open freeze dryer, be cooled to-42 ℃ with 25 ℃/h speed, be incubated freezing 14 hours; Be evacuated to below 0.3mbar, in 10～14 hours, the salvianolic acid A formulation temperature freezing risen to-25 ℃, maintain-23 ℃ of vacuum dryings 7 hours; Continue to heat up, be evenly warming up to 44 ℃ with 0.6 ℃/min, maintain 44 ℃ and be dried after 2 hours, sample temperature is down to room temperature, add a cover, observe mouldability, in table 28.
The different filleies of table 28 and the antioxidant result that affects on mouldability and the quality of the pharmaceutical preparations
Figure BSA00000811649800411
Table 28 experimental result shows, adds not commensurability filler mannitol, glucose, lactose better to preparations shaping, and lyophilized powder sedimentation is good, porous nickel, consumption select 20mg～40mg/2ml～3ml mouldability and solubility all better.Add content on main constituent of water-soluble vitamin c, thiourea, sodium sulfite, sodium pyrosulfite and other compositions all without impact, add salvianolic acid A stable content after antioxidant, other compositions are unchanged, and preparation effect is more excellent.
Experimental example 14: lyophilisation condition research
3.1 lyophilisation conditions are preferred: in freezing conditions, mainly contain two factors: the impact on mouldability: the temperature of freezing body before freezing speed, vacuum drying; Impact on freeze drying rate: heating curve when vacuum freeze-drying.
Preferably following on freeze dryer:
A, medicinal liquid are cooled to-40 ℃～-45 ℃ with the chilling rate of 20-30 ℃/h in freeze dryer, be incubated freezing 10～16 hours, be evacuated to below 0.3mbar, in 10～14 hours, the salvianolic acid A formulation temperature freezing is risen to-23 ℃～-27 ℃, maintain-23 ℃～-27 ℃ vacuum dryings 6～8 hours;
B, medicinal liquid are first cooled to-25 ℃ with the chilling rate of 20-30 ℃/h in freeze dryer, be incubated freezing 5～8 hours, be refrigerated to again-40 ℃～-45 ℃, be incubated freezing 5～8 hours, be evacuated to below 0.3mbar, in 10～14 hours, the salvianolic acid A formulation temperature freezing is risen to-23 ℃～-27 ℃, maintain-23 ℃～-27 ℃ vacuum dryings 6～8 hours;
C, medicinal liquid are cooled to-15 ℃ with the chilling rate of 20-30 ℃/h in freeze dryer, be incubated freezing 5～8 hours, be refrigerated to again-40 ℃～-45 ℃, be incubated freezing 5～8 hours, be evacuated to below 0.3mbar, in 10～14 hours, the salvianolic acid A formulation temperature freezing is risen to-23 ℃～-27 ℃, maintain-23 ℃～-27 ℃ vacuum dryings 6～8 hours;
Above three kinds of result of the tests: 1, medicinal liquid is directly refrigerated to-40 ℃～-45 ℃ with 20-30 ℃/h, and powder body is more even, and without crack, drying time is short.2, powder body is inhomogeneous, has crack, and 3, freeze-drying time is long, powder body is inhomogeneous, has crack.Therefore, while selecting medicinal liquid freezing, be directly down to-40 ℃～-45 ℃ with the chilling rate of 20-30 ℃/h, powder body is more even, without crack, can save freeze-drying time.
3.2 pilot scale freeze-drying curves
Freeze-drying curve refers to the relation curve of product temperature in freeze-drying process or shelf temperature time to time change.The number of the shape of freeze-drying curve and the performance of product, loading amount, the many factors such as kind and appointed condition of separation container are relevant.The freeze dryer that we select in the time of middle trial production is 4000 bottles of scales, substantially meets large production requirement.Cooled the temperature to-40 ℃～-45 ℃ at 1～4 hour, at this temperature, keep 10～16 hours, again 10～14 hours by temperature rise to-23 ℃～-27 ℃ and keep 6～8 hours, then at 6～9 hours by temperature rise to 40 ℃～45 ℃ and keep 2～5 hours, freeze-drying curve is as shown in Figure 5 and Figure 6.
Experimental example 15: preparation stabilization Journal of Sex Research
Get embodiment 10,11,12 samples, by the requirement of stability of drug products experimentation relevant technologies, by above three lot number samples at ambient temperature, by 0 month, 1 month, 2 months, 3 months, 6 months intervals, the situation of change of making regular check on sample property, solubility and clarity, pH value, salvianolic acid A content, other compositions, the results are shown in Table 29.
Table 29 stability experiment result
The demonstration of table 29 stability experiment observed result, lyophilized injectable powder at ambient temperature salvianolic acid A is stable, and other compositions are also without significant change, and preparation appearance character is good, and preparation solubility is good, and pH value is stable, solution clarification.
Below experiment all gets with salvianolic acid A freeze-dried powder the sample that embodiment 14 obtains.
Experimental example 16: the protective effect of salvianolic acid A freeze-dried powder to the reperfusion injury of SD isolated rat heart
Male SD rat, body weight (280 ± 20) g; Quality certification SCXK (capital) 2007-0006 is provided by Beijing Vital River Experimental Animals Technology Co., Ltd..After intraperitoneal anesthesia, open rapidly breast, mention gently heart, cut off superior and inferior vena cava, pulmonary artery, aorta and heart tissue around, rapidly heart is taken out together with one section of aorta, aortic root retains the approximately length of 0.5～1cm, and standby heart cathetrization is used.Move to immediately containing in oxygen K-H liquid (4 ℃ of left and right) of getting ready in advance, push gently heart intraventricular residual blood is discharged, clean heart remained blood, hang 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) ℃; 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 to left ventricle by atrioventricular orifice.Latex water pocket is full of ultra-pure water with in the conduit being connected, and the other end of conduit, by T joint pressure transducer, is thoroughly removed inner size bubble.Regulate the water yield of water pocket in ventricle to make heart ventricle end diastolic pressure maintain 4-6mmHg scope by fine setting injector.Left indoor pressure is tested after stablizing 30min again.Record the coronary flow of each minute heart with graduated cylinder; Pressure transducer signal is inputted multitrack recording system by bridge amplifier, carries out live signal acquisition and processing.
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 freeze-dried powder (1.0,0.5,0.25mg/L), positive drug group (diltiazem hydrochloride 0.3mg/L): use the pastille infusion liquid perfusion of variable concentrations in the time recovering perfusion, and continue whole refilling process.
Before each group record stops filling with and after filling with again since 10min, every maximum the climbing speed (+dp/dt of coronary flow (CBF), left ventricular diastolic pressure (LVEDP), left indoor pressure of 10min max) and the maximum fall off rate (dp/dt of left indoor pressure mma), n=8.
Table 30. salvianolic acid A freeze-dried powder pours into the hemodynamic impact of 60min again on isolated rat heart
(
Figure BSA00000811649800441
n＝8)
Note: with Normal group comparison: #P < 0.01, ##P < 0.001; With model group comparison: △ P < 0.05, * P < 0.01, * * P < 0.001
With Normal group comparison, model group ischemia-reperfusion 60min, CBF ,+dp/dt max,-dp/dt maxobviously decline (P < 0.01 or P < 0.001), LVEDP significantly increases (P < 0.001).With model group comparison, positive drug group, the high, medium and low dosage group of salvianolic acid A freeze-dried powder These parameters all have clear improvement, and can suppress the maximum climbing speed decline of coronary flow minimizing, the rising of left chamber diastolic pressure, left indoor pressure, the maximum fall off rate decline of left indoor pressure that ischemia-reperfusion causes.And salvianolic acid A freeze-dried powder group high dose group is suitable with positive drug group effect.Between salvianolic acid A freeze-dried powder various dose group, there is dose dependent.Experimental result shows that salvianolic acid A freeze-dried powder can alleviate isolated rat heart ischemical reperfusion injury degree, and prompting salvianolic acid A freeze-dried powder can obviously improve coronary flow, improve systolic and diastolic function, the cardioprotection ischemical reperfusion injury of isolated heart aorta diastolic function and damaged myocardium.
Experimental example 17: the impact of salvianolic acid A freeze-dried powder on 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 freeze-dried powder (2.5,5.0,10.0mg/kg) at random; Establish sham operated rats simultaneously.
20% urethane intraperitoneal anesthesia rat, fixing.After tracheal intubation, connect respirator, exhale by the frequency of 10～12ml tidal volume, 80 times/min, continuous positive pressure breathing, inhales: exhale than being 1: 1.Now connect electrocardiograph and measure rat normal ECG.Along between left border of sternum the 3rd, 4 ribs, open breast, extrude heart, between left auricle and pulmonary conus, find out left anterior descending coronary artery, under ramus descendens anterior arteriae coronariae sinistrae initial part, 2mm sentences circular noinvasive sewing needle 6-0 silk thread threading, rapidly ligation ramus descendens anterior arteriae coronariae sinistrae (a sham operated rats not ligation of threading), and with one the little plastics pipe pad with groove at ligation position, the ligation thereon of two rear line heads, sew up thoracic wall, recover autonomous respiration.The complete Rat Ecg of at once observing of performing the operation, take left chamber antetheca be that cyanosis and II lead electrocardiogram show that the S-T section back of a bow is upwards obviously raised and lasting 30min above for modeling successfully.
Each treated animal is tail vein injection relative medicine (sham operated rats and model group are injected isopyknic normal saline) after modeling success, injects twice every day, continuously 7d.30min after last administration, animal intraperitoneal anesthesia, through common carotid artery intubate to left ventricle, connect multiple tracks intelligence physiological signal acquisition analysis system by pressure converter, postoperative stable 30min, recorded heart rate (HR), arterial pressure, left ventricular systolic pressure (LVSP), maximum the climbing speed (+dp/dt of left indoor pressure max) and the maximum fall off rate (dp/dt of left indoor pressure max); Insert limb electrode monitoring standard II lead electrocardiogram simultaneously.
The impact of table 31. salvianolic acid A freeze-dried powder on rats with myocardial ischemia electrocardio and cardiac function ( n=10)
Figure BSA00000811649800461
Note: with sham operated rats comparison: #P < 0.01; With model group comparison: * P < 0.05, * * P < 0.01
Experimental result demonstration, with sham operated rats comparison, following coronary artery occlusion causes the long-term myocardial infarction and ischemia model Rat Ecg of rat 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 rat heart muscle contractile function obviously reduces.With model group comparison, 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, in each medicine group with the middle and high dosage of salvianolic acid A freeze-dried powder the electrocardio to long-term rats with myocardial ischemia and parameters of left ventricular function to improve effect the most obvious, between the each dosage group of salvianolic acid A freeze-dried powder, be a certain amount of effect relationship, prompting salvianolic acid A freeze-dried powder can reduce rats with myocardial ischemia ST section rising, improve heart rate, increase its arterial pressure, improve the cardiac muscle contracting power of relaxing, electrocardio and cardiac function to rats with myocardial ischemia improve significantly.
Experimental example 18: the impact of salvianolic acid A freeze-dried powder on enzyme work and Radical Metabolism in rats with myocardial ischemia serum
The each group of experimental example 17 rat is after electrocardio and parameters of left ventricular function are measured and finished, abdominal aortic blood, get serum, by the operation of detection kit description, measure superoxide dismutase (SOD), malonaldehyde (MDA) content and aspartate amino transferase (AST), creatine kinase (CK), lactic acid dehydrogenase (LDH) activity in serum.
Table 32. salvianolic acid A freeze-dried powder is lived and metabolism of free radical to enzyme in rats with myocardial ischemia serum
(
Figure BSA00000811649800462
n＝10)
Figure BSA00000811649800471
Note: with sham operated rats comparison: #P < 0.01; With model group comparison: * P < 0.05, * * P < 0.01
When myocardial ischemia, produce a large amount of oxygen-derived free radicals, cardiac muscle is caused to damage, SOD, MDA are the objective indicators of reflection oxygen free radical injury; Extensively be present in myocardium enzyme AST, the LDH in myocardial cell, the activity of CK can reflect the degree of injury of myocardial ischemia.With sham operated rats comparison, in myocardial infarction and ischemia model group rat blood serum, SOD obviously reduces, MDA, AST, LDH, CK significantly raise (P < 0.01); With model group comparison, 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 particularly remarkable with high, the middle dosage group of salvianolic acid A freeze-dried powder and positive drug group drug effect.Experimental result prompting; salvianolic acid A freeze-dried powder 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 freeze-dried powder can suppress oxygen-derived free radicals and produce; improve the oxidation resistance of cardiac muscular tissue; protecting myocardial cell film, reduces overflowing of enzyme, improves myocardium energy supply; reduce ischemia to myocardium degree of injury, thereby play ischemic myocardial protection effect.
Experimental example 19: the impact of salvianolic acid A freeze-dried powder on rats with myocardial ischemia myocardial infarct size
Get the rat after experimental example 18 blood sampling finishes, core immediately dirty, normal saline cleans, and cuts off right atrium, isolates left ventricle, claims left ventricle weight in wet base after sucking excessive moisture.Below ligature, parallel coronary sulcus direction is cut into 5 of equal thickness, put into 1%TTC dye liquor, 37 ℃ of constant temperature dyeing 10min, infarct is not dyed to redness, and infarct is not colored and is canescence, and infarct is weighed, calculate infarct and heavily account for the heavy percentage ratio of left ventricle, i.e. myocardial infarct size.
The impact of table 33. salvianolic acid A freeze-dried powder on rats with myocardial ischemia myocardial infarct size (
Figure BSA00000811649800472
n=10)
Figure BSA00000811649800473
Figure BSA00000811649800481
Note: with sham operated rats comparison: #P < 0.01; With model group comparison: * * P < 0.01
There is obvious myocardial ischemia infarction in myocardial infarction and ischemia model group rat, infarction size reaches 53% left and right; Compared with model group, after various dose salvianolic acid A freeze-dried powder drug administration by injection 7d, the myocardial infarct size of rat significantly reduces (P < 0.01), and presents certain dose-effect relationship.Prompting salvianolic acid A freeze-dried powder can dwindle myocardial infarct size, alleviates treating myocardial ischemia damage and heart infarction degree, and myocardial ischemia is had to obvious therapeutic action.
Experimental example 20: salvianolic acid A freeze-dried powder is on rats with myocardial ischemia cardiac muscle histopathology and Ultrastructural impact
Animal model and grouping administration are with experimental example 17.Administration finishes latter second day, and sacrifice of animal is cored dirty, and normal saline flushing, cuts left ventricle, gets at random the left ventricle of Some Animals for each group, and in 10% formaldehyde, after fixing, flushing, dehydration, dimethylbenzene soaks into, paraffin embedding, section, HE dyeing, mounting.The microstructure of light Microscopic observation cardiac muscle.Get the left ventricle of random residual animal, fixing in 3% glutaraldehyde solution, through dehydrations at different levels, soak into epoxy resin Epon812 embedding, 65 ℃ of polymerizations, section, electron microscopic observation Myocardial ultrastructure with expoxy propane.
Result shows, light Microscopic observation sham operated rats animal cardiac muscle marshalling, and heart band is clear, only has extremely indivedual slight hypertrophy of flesh core, and myocardium interstitial has no cell infiltration, without hemorrhagic necrosis.Model group and sham operated rats comparison, cardiac muscle fiber obvious tumefaction, degeneration, arrangement disorder, cardiac muscle cross striation is most of to disappear, the obvious hypertrophy of flesh core, spatium intermusculare fibroplasia is more, a large amount of cell infiltration, the necrosis of part specimen cardiac muscular tissue, the dissolved sample of muscle glycogen, has obvious kitchen range shape myocardial infarction.Various dose salvianolic acid A freeze-dried powder group, positive drug (hydrochloride for injection diltiazem) group and model group comparison; pathological changes all obviously alleviates; cardiac muscle fiber mild swelling; arrange still orderly; band is more clear; hypertrophy that flesh core is slight, myocardium interstitial, without hemorrhage, has a small amount of inflammatory cell infiltration not waiting; Paired observation between each treatment group, high, the middle dosage of salvianolic acid A freeze-dried powder and the cardiac muscle swelling of positive drug group and inflammatory infiltration are the lightest, and ischemic myocardial tissue is had to more obvious protective effect.
Under transmission electron microscope, observe visible sham operated rats animal cardiac muscle cell Z line rule, each muscle segment is clear, and myofilament is evenly distributed, arranges closely neat, and triplet is obvious, and structure of mitochondria is complete, and ridge is many, clear and arrange orderly.Model group and sham operated rats comparison, sarcostyle arrangement disorder, and visible downright bad muscle fiber, mitochondrion obviously increases, vacuolar degeneration, ridge rareness or fall into disarray, and unintelligible, 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 freeze-dried powder low dose group structure of mitochondria is substantially complete, part mitochondrial vacuolar degeneration, and myofilament is arranged still neat, and part ridge is smudgy, and Ultrastructural change is obviously lighter than model group; High, the middle dosage group of salvianolic acid A freeze-dried powder, positive drug group myocardial cell Z line fundamental rule, substantially complete, the few part mitochondrial swelling of structure of mitochondria, ridge is substantially clear, and myofilament is arranged substantially neat, nucleus structural integrity, ultrastructure shows pathological changes is obviously improved.
Light microscopic and Electronic Speculum results suggest, salvianolic acid A freeze-dried powder can obviously improve the myocardium pathological change of rats with myocardial ischemia, alleviates myocardium pathology damage, can be used for the treatment of ischemic heart desease.
Experimental example 21: the impact of salvianolic acid A freeze-dried powder on 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 freeze-dried powder (10.0,5.0,2.5mg/kg).
20% urethane intraperitoneal anesthesia rat, fixing.After tracheal intubation, connect respirator, exhale by the frequency of 10～12ml tidal volume, 80 times/min, continuous positive pressure breathing, inhales: exhale than being 1: 1.Now connect electrocardiograph and measure rat normal ECG.Along between left border of sternum the 3rd, 4 ribs, open breast, extrude heart, between left auricle and pulmonary conus, find out left anterior descending coronary artery, under ramus descendens anterior arteriae coronariae sinistrae initial part, 2mm sentences circular noinvasive sewing needle 6-0 silk thread threading, ligation ramus descendens anterior arteriae coronariae sinistrae (a sham operated rats not ligation of threading) rapidly, and with one the little plastics pipe pad with groove at ligation position, the ligation thereon of two rear line heads, recording ecg.Take left chamber antetheca be that cyanosis and II lead electrocardiogram show that the S-T section back of a bow is upwards obviously raised and lasting 20min above for modeling successfully; After myocardial ischemia 90 minutes, unclamp line, take out pipe pad, realize and pouring into again.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 again 6h, 24h, 48h anesthetized animal (fill with again 24,48h treated animal begins still twice of drug administration by injection every day for second day after operation 4 administrations on the same day finish, until draw materials), fixing, cut open breast and core dirty, separate left ventricle, claim left ventricle weight in wet base after sucking excessive moisture.Below ligature, crosscut becomes 5 of equal thickness, puts into 1%TTC dye liquor, 37 ℃ of constant temperature dyeing 10min, infarct is not dyed to redness, and infarct is not colored and is canescence, infarct is weighed, and calculates infarct and heavily accounts for the heavy percentage ratio of left ventricle, i.e. myocardial infarct size.
Table 34. salvianolic acid A freeze-dried powder is on myocardial ischemia in rats-fill with the again impact of different time myocardial infarct size
(
Figure BSA00000811649800491
n＝6)
Figure BSA00000811649800501
Note: with sham operated rats comparison: #P < 0.01; With model group comparison: * * P < 0.01
Model group myocardial ischemia in rats 1.5h, then after pouring into 6h, myocardial infarction is serious, and in recovery perfusion 48h, infarction size progressively increases, myocardial damage aggravation.With model group comparison, various dose salvianolic acid A freeze-dried powder can obviously dwindle myocardial infarct size (P < 0.01), alleviate myocardial ischemia-reperfusion damage, and myocardial infarct size does not expand with Ischemia Reperfusion time lengthening; Between each dosage group, there is certain agent effect relationship.Prompting salvianolic acid A freeze-dried powder can dwindle expeirmental myocardial ischemia and pour into the myocardial infarct size of rat again, alleviates Myocardial injury degree, and Myocardial Ischemia Reperfusion Injury is had to good preventive and therapeutic effect.
Experimental example 22: the impact of salvianolic acid A freeze-dried powder on Myocardial Ischemia Reperfusion Injury SD metabolism of free radicals in rats
Modeling and medication are with experimental example 21.Pour into again after 24h in recovery, 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, superoxide dismutase (SOD), xanthine oxidase (XOD), glutathion peroxidase (GSH-Px), catalase (CAT) activity in tissue.
Table 35. salvianolic acid A freeze-dried powder in rat tissue Radical Metabolism relevant enzyme live impact ( n=10)
Figure BSA00000811649800503
Figure BSA00000811649800511
Note: with sham operated rats comparison: #P < 0.01; With model group comparison: * P < 0.05, * * P < 0.01
Experimental result shows, with sham operated rats comparison, model group rat is at ischemia-reperfusion 24h, the SOD of cardiac muscular tissue, GSH-Px, CAT enzyme are lived and are significantly declined, MDA, XOD enzyme are lived and are significantly raise (P < 0.01), and prompting myocardial ischemia in rats-after pouring into, Peroxidation Product is piled up serious again, myocardial clearance Peroxidation Product ability significantly declines, and Ischemia Reperfusion causes that a large amount of oxygen-derived free radicals generations cause myocardial damage to increase the weight of.Each medicine group and model group comparison, the SOD of cardiac muscular tissue, GSH-Px, CAT enzyme are lived and are raise in various degree, MDA, XOD enzyme are lived and are reduced in various degree (P < 0.05 or P < 0.01), and high, the middle dosage group of salvianolic acid A freeze-dried powder effect is the most obvious.Prompting salvianolic acid A can strengthen myocardial clearance free radical ability, suppresses oxygen-derived free radicals and produces, and strengthens rat heart muscle oxidation resistance, suppresses Myocardial Ischemia Reperfusion Injury.
Experimental example 23: the impact of salvianolic acid A freeze-dried powder on 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 freeze-dried powder (6,3,1.5mg/kg).Pentobarbital sodium 30mg/kg intravenous injection anesthesia, fixing, tracheal intubation connects respirator, separates left carotid, the intubate recording blood pressure that is connected with polygraph through pressure transducer, separation right side external jugular vein, along left side, the 4th intercostal is opened breast, makees pericardium bed.Separate aortic arch, place electromagnetic flowmeter probe, measure cardiac output.After opening breast, intubate is to right ventricle, records center venous pressure.Separate M-LAD, the standby ligation use of lead-in wire, seam is put 16 epicardial leads of leading and is connected ecg amplifier, records visceral pericardium electrocardiogram.Apex of the heart intubate, Bonding pressure transducer, measures intraventricular pressure.Record indices stable after, 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 is injected administration, administration volume 1ml/kg, and 10min injection is complete.Model group and sham operated rats wait the normal saline of capacity.After ischemia 3h, the solution bolt that bursts at the seams, realizes and pouring into.Respectively before ligation, ligation 15min (before being administration), 30,60,120,180min, fills with 30min, the variation of filling with 60min and recording visceral pericardium ECG ST section again, blood oxygen again.The total mV number (∑-ST) raising with the ST section of each each mapping point of moment represents myocardial ischemia journey.
The impact of table 36. salvianolic acid A freeze-dried powder on dog Myocardial Ischemia Reperfusion Injury degree of myocardial ischemia (∑-ST)
(
Figure BSA00000811649800512
mV，n＝6)
Note: with sham operated rats comparison: #P < 0.001; With model group comparison: * P < 0.05, * * P < 0.01
Experimental result shows, after ligation dog coronary artery, 5min starts, and respectively organizes dog epicardial electrogram ST section and raises sum (∑-ST) and obviously raise; Model group dog has been elevated to a metastable level to 15min after ligation, and realizing after perfusion again, although ∑-ST compared with declining between ischemic stage, significantly increases (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 (before being administration), respectively organizing ∑-ST and model group does not relatively have diversity (P > 0.05); And after ligation 30min begin (being 15min after administration), each medicine group ∑-ST starts to decline, degree of ischemia alleviates, but except salvianolic acid A freeze-dried powder high dose group and model group relatively have (P < 0.05) notable difference, other medicines group declines does not have statistical significance; But after to 60min after ligation to ligation during 180min (be after administration 45min to during 165min after medicine), still continuous decrease of each medicine group ∑-ST, and more all have significant difference (P < 0.05 or P < 0.01) with model group; Recover after perfusion again, each medicine group ∑-ST and model group significantly reduce.Between the each dosage group of salvianolic acid A freeze-dried powder, have a certain amount of effect relationship, in salvianolic acid A freeze-dried powder, dosage group ∑-ST fall is suitable with 0.5mg/kg hydrochloride for injection diltiazem group, and has trend slightly.Prompting salvianolic acid A freeze-dried powder can alleviate the degree of myocardial ischemia of experiment ischemia-reperfusion dog.Point out it to damage by Ischemic myocardium, ischemic heart desease is had to good therapeutical effect.
Experimental example 24: the impact of salvianolic acid A freeze-dried powder on dog Myocardial Ischemia Reperfusion Injury myocardial ischemia scope
Modeling, grouping administration are with described in experimental example 23.Record the variation of the visceral pericardium ECG ST section of each each time period of treated animal, represent myocardial ischemia scope with the mapping point sum (N-ST) of each moment ST section rising >=2mV.The impact of table 37. salvianolic acid A freeze-dried powder on dog Myocardial Ischemia Reperfusion Injury myocardial ischemia scope (N-ST)
(
Figure BSA00000811649800522
n＝6)
Figure BSA00000811649800531
Note: with sham operated rats comparison: #P < 0.001; With model group comparison: * P < 0.05, * * P < 0.01
Experimental result demonstration, after ligation dog coronary artery, respectively organizing dog epicardial electrogram N-ST obviously increases; Model group dog is to 15min after ligation, N-ST has been increased to a metastable level, and ischemia scope is basicly stable, and is realizing after perfusion again, although N-ST reduces during compared with ligation, but still the utmost point is significantly higher than sham operated rats (P < 0.001).In the 15min of each medicine group before ligation and after ligation (before being administration), respectively organizing N-ST and model group does not relatively have diversity; And after ligation 30min begin (being 15min after administration), each medicine group N-ST starts to have the trend of minimizing, but except salvianolic acid A freeze-dried powder high dose group slip and model group relatively have (P < 0.05) notable difference, other medicines group no difference of science of statistics; But after to 60min after ligation to ligation during 180min (be after administration 45min to during 165min after medicine), 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; Recover after perfusion again, each medicine group N-ST and model group significantly reduce.Between the each dosage group of salvianolic acid A freeze-dried powder, have a certain amount of effect relationship, in salvianolic acid A freeze-dried powder, dosage group N-ST slip is suitable with 0.5mg/kg hydrochloride for injection diltiazem group.Prompting salvianolic acid A freeze-dried powder can reduce the myocardial ischemia scope of experiment ischemia-reperfusion dog.Point out it to damage by Ischemic myocardium, ischemic heart desease is had to good therapeutical effect.
Experimental example 25: the impact of salvianolic acid A freeze-dried powder on dog Myocardial Ischemia Reperfusion Injury myocardial infarct size
Modeling, grouping administration are with described in experimental example 23.Each group dog ischemia 180min, then pour into after 60min, to core dirty, normal saline flushing, weighs heavy whole-heartedly.Cut off trunk and atrium, satisfactory chamber is heavy, is on average cut into five from the apex of the heart to ligation point, in 0.5% nitro blue tetrazolium (N-TB) dye liquor, dye, and 15min, infarcted region is not painted, and skipper is dyed in non-infarcted region.Separate Ji Fei infarcted region, infarcted region and weigh, accounting for heavy, infarcted myocardium whole-heartedly with infarcted myocardium and account for the percentage calculation infarction size that ventricle is heavy.
The impact of table 38. salvianolic acid A freeze-dried powder on dog Myocardial Ischemia Reperfusion Injury myocardial infarct size
(
Figure BSA00000811649800541
n＝6)
Figure BSA00000811649800542
Note: with sham operated rats comparison: #P < 0.01; With model group comparison: * P < 0.05, * * P < 0.01
Experimental result demonstration, after model group dog ischemia-reperfusion, myocardial infarction is heavier.Salvianolic acid A freeze-dried powder, hydrochloride for injection diltiazem all can significantly reduce experimental dog myocardial infarct size, and with salvianolic acid A freeze-dried powder high dose group dog myocardial infarction ratio minimum.With model group comparison; high, the middle dosage group of salvianolic acid A freeze-dried powder can extremely significantly reduce the proportion (P < 0.01) that infarcted myocardium accounts for heart and ventricle; the proportion that salvianolic acid A freeze-dried powder low dosage dog myocardial infarction district accounts for heart and ventricle also obviously reduces (P < 0.05); prompting salvianolic acid A freeze-dried powder energy dose dependent ground dwindles experimental dog myocardial infarct size; energy Ischemic myocardium-reperfusion injury, is used for the treatment of ischemic heart desease.
Experimental example 26: the impact of salvianolic acid A freeze-dried powder on dogs with acute myocardial ischemia coronary flow
Modeling, grouping, administration time and dosage are with method described in experimental example 23.Animal opens after breast, separates LCA, places electromagnetic flowmeter probe, measures the interior different time sections heart coronary flow (CBF) of ischemic stage 240min before ligation, after ligation.Experiment finishes 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.
The impact of table 39. salvianolic acid A freeze-dried powder on Myocardial Ischemia Reperfusion Injury dog coronary flow
(
Figure BSA00000811649800543
n＝6)
Figure BSA00000811649800544
Figure BSA00000811649800551
Note: after administration, blood flow amplification is with model group amplification comparison: * P < 0.05, * * P < 0.01
Experimental result demonstration, the each time period coronary flow of sham operated rats dog does not have significant change; From before the each time period coronary flow of model group dog and ligation relatively and respectively organize comparative result before coronary flow before dog administration and ligation, ligation dog coronary artery forms after myocardial ischemia, coronary flow has short time compensatory to increase, and increasing degree is in 10% left and right; After the each dosage of salvianolic acid A freeze-dried powder, the each administration of hydrochloride for injection diltiazem dog, before each time period coronary flow and administration, relatively there is increase in various degree, each medicine group is 15min (being ligation 30min) after administration, coronary flow amplification is between 13%～24%, except salvianolic acid A high dose group amplification has notable difference, other each medicine groups increase and are not obvious; And 45min, to 225min (being ligation 60min to 240min) after administration, relatively, has increased between 25.3%～52.6% before each medication therapy groups coronary flow and administration after administration; The most remarkable with high, the middle dosage group of salvianolic acid A freeze-dried powder, hydrochloride for injection diltiazem group, amplification is respectively 52.6%, 40.2%, 36.7%.Prompting salvianolic acid A freeze-dried powder can promote collateral circulation open, the coronary flow while increasing myocardial ischemia, thus to resisting myocardial ischemia, Ischemic myocardium damage, points out it aspect control ischemic heart desease, having certain purposes.
Experimental example 27: salvianolic acid A freeze-dried powder is on myocardial ischemia dog cardiac function and hemodynamic impact
Method described in experimental example 23 is pressed in modeling, grouping administration; Aortic root is placed electromagnetic flowmeter probe, measures cardiac output (CO); Apex of the heart intubate is to left ventricle, and Bonding pressure transducer, measures left constant pressure, left indoor pressure maximum collapse and diastole rate of change (± dp/dt max); Recording ecg (ECG); After coronary ligation operation, stablize 15min administration; Polygraph record, the variation of measuring and calculating the each leading indicator of different time in dog ischemia 240min: CO, left ventricular end diastolic presssure (LVEDP), ± dp/dt max, the acting of left chamber (LVW), total peripheral resistance (TPR).
The impact of table 40. salvianolic acid A freeze-dried powder on myocardial ischemia dog CO ( l/min, n=6)
Figure BSA00000811649800553
Figure BSA00000811649800561
Note: with before self ischemia relatively, #P < 0.05, ##P < 0.01; With model group comparison, * P < 0.05, * * P < 0.01
The impact of table 41. salvianolic acid A freeze-dried powder on myocardial ischemia dog LVEDP (
Figure BSA00000811649800562
mmHg, n=6)
Figure BSA00000811649800563
Note: with before self ischemia relatively, #P < 0.05, ##P < 0.01; With model group comparison, * P < 0.05, * * P < 0.01
Table 42. salvianolic acid A freeze-dried powder is to myocardial ischemia dog+dp/dt maximpact (
Figure BSA00000811649800564
mmHg/s, n=6)
Figure BSA00000811649800565
Note: with before self ischemia relatively, #P < 0.05, ##P < 0.01; With model group comparison, * P < 0.05, * * P < 0.01
Table 43. salvianolic acid A freeze-dried powder is to myocardial ischemia dog-dp/dt maximpact (
Figure BSA00000811649800566
mmHg/s, n=6)
Figure BSA00000811649800571
Note: with before self ischemia relatively, #P < 0.05, ##P < 0.01; With model group comparison, * P < 0.05, * * P < 0.01
The impact of table 44. salvianolic acid A freeze-dried powder on the left chamber of myocardial ischemia dog acting (LVW) (
Figure BSA00000811649800572
n=6)
Figure BSA00000811649800573
Note: with before self ischemia relatively, #P < 0.05, ##P < 0.01; With model group comparison, * P < 0.05, * * P < 0.01
The impact of table 45. salvianolic acid A freeze-dried powder on myocardial ischemia dog total peripheral resistance (TPR) (
Figure BSA00000811649800574
n=6)
Figure BSA00000811649800581
Note: with before self ischemia relatively, #P < 0.05, ##P < 0.01; With model group comparison, * P < 0.05, * * P < 0.01
Experimental result shows, after ligation coronary ischemia 30min, before dog cardiac output (CO) and ischemia, significantly declines, LVEDP significantly raises, ± dp/dt maxsignificantly decline, left chamber acting (LVW) reduce, total peripheral resistance (TPR) enlarges markedly (P < 0.05 or P < 0.01), illustrate after dog coronary ligation, myocardial function is impaired obviously, myocardial contraction and diastolic function decline, hypokinemia, cardiac pumping function reduces, blood oxygen supply deficiency, and the impaired cardiac function that causes of myocardial ischemia-anoxemia declines.Can significantly suppress LVEDP rising, inhibition ± dp/dt with model group comparison: 15min after the administration of high dose salvianolic acid A freeze-dried powder (being ischemia 30min) maxdecline, suppress dog LVW and reduce, reduce TPR, 45min after administration (after being ischemia 60min) can significantly suppress CO and decline; 15min (after the ischemia 30min) dog ± dp/dt that can raise after the administration of middle dosage salvianolic acid A freeze-dried powder max, TPR is declined, 45min after administration (after being ischemia 60min) can significantly suppress dog LVW and reduce, reduces LVEDP, increases 105min (being ischemia 120min) dog CO after administration, its action effect is suitable with hydrochloride for injection diltiazem; After the administration of salvianolic acid A freeze-dried powder low dose group, 105min can obviously suppress dog CO decline, and after administration, 45min can reduce dog TPR, rising ± dp/dt maxand increase LVW, and after administration after 105min (being ischemia 120min) can obviously reduce dog LVEDP.After the administration of results suggest salvianolic acid A freeze-dried powder, can improve myocardial ischemia dog hemodynamic index, the myocardial contraction of Enhancement test dog, improve myocardium systolic and diastolic function, reduce Peripheral resistance, suppress the blood-pumping function that cardiac output reduces, improves heart, improve myocardial blood supply, improve myocardial ischemia and cardiac function, performance function of resisting myocardial ischemia.
Experimental example 28: the impact of salvianolic acid A freeze-dried powder on myocardial ischemia dog myocardium enzyme
Modeling, grouping administration are with method described in experimental example 23.Administration after the postoperative stable 15min of coronary ligation, after ligation, 240min (being 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.
The impact of table 46. salvianolic acid A freeze-dried powder on myocardial ischemia dog myocardium enzyme (
Figure BSA00000811649800582
u/L, n=6)
Figure BSA00000811649800583
Note: with model group comparison, * P < 0.01, * * P < 0.001
In serum, CK, LDH, AST, CK-MB are the coherent detection myocardial zymetology as Hypoxicichemic myocardial lesion of commonly using clinically, can reflecting myocardium degree of injury.Wherein CK-MB is Cardiac-specific enzyme, the most responsive in myocardial damage.Experimental result demonstration, with sham operated rats comparison, in myocardial infarction and ischemia model group dog serum, CK, LDH, AST, CK-MB significantly raise, illustrate after ligation coronary ischemia, dog myocardial mitochondria is destroyed, myocardial cell injury, and myocardium enzyme is released the rising alive of the clear center of hyperamization creatase.The each dosage group of salvianolic acid A freeze-dried powder and model group comparison, can reduce to some extent after dog myocardial ischemia 4h CK, LDH, AST, CK-MB activity (P < 0.01 or P < 0.001) in serum, and be dose dependent.Experimental result prompting salvianolic acid A freeze-dried powder can be stablized myocardial cell membrane, and the overflowing of myocardium enzyme when minimizing treating myocardial ischemia damage, has obvious protective effect to ischemic myocardium.
Experimental example 29: the impact of salvianolic acid A freeze-dried powder on myocardial ischemia dog fatty acid metabolism and radical metabolism
Modeling, grouping administration are with method described in experimental example 23.Administration after the postoperative stable 15min of coronary ligation, after ligation, 240min (being 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).
The impact of table 47. salvianolic acid A freeze-dried powder on myocardial ischemia dog fatty acid metabolism and radical metabolism
( n＝6)
Note: with model group comparison, * P < 0.05, * * P < 0.01
When myocardial ischemia, can cause that disorder in various degree appears in a series of myocardial metabolisms, thereby cause cardiac insufficiency.The main reason for the treatment of myocardial ischemia damage is owing to causing Power supply obstacle to make myocardial damage even downright bad 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 raise and can suppress the oxidation of glucose, increase the gathering of lactic acid in cell, therefore can make the Efficiency Decreasing of heart acting, cardiac muscle is produced to adverse influence.Experimental result shows, with sham operated rats comparison, model group dog is after ischemia 4h, the significantly rising (P < 0.01) of serum free fatty acid (FFA), lipid peroxide (LPO), after prompting myocardial ischemia there is obstacle in Myocardial Fatty Acids metabolism, will suppress the activity of glucose oxidase Major Enzymes, thereby increase myocardial oxygen consumption, expand myocardial infarct size.And the each dosage group of salvianolic acid A freeze-dried powder and model group comparison can significantly reduce FFA in myocardial ischemia dog serum, LPO content (P < 0.05 or P < 0.01), and be dose-dependence.Prompting salvianolic acid A freeze-dried powder can significantly suppress FFA level in serum and raise, and improves Myocardial Fatty Acids metabolism, reduces the accumulation of lactic acid; the production capacity that increases unit oxygen consumption, reduces myocardial oxygen consumption, thereby improves cardiac function; suppress myocardial infarct size, Ischemic myocardium damage.
With sham operated rats comparison, after model group dog myocardial ischemia 4h, in serum, MDA content significantly raises, active significantly decline (the P < 0.01) of SOD, GSH-Px, after prompting dog myocardial ischemia, oxygen-derived free radicals generates obviously to be increased, in body, remove the enzyme significantly reduction alive of oxygen-derived free radicals, and oxygen-derived free radicals can affect by a series of oxidation reactions normal configuration and the function of cell, aggravation treating myocardial ischemia damage degree.The each dosage group of salvianolic acid A freeze-dried powder and model group comparison, can significantly reduce the content of MDA in myocardial ischemia dog serum, make the active obviously enhancing (P < 0.05 or P < 0.01) of SOD in serum, GSH-Px simultaneously, and be certain agent effect relationship; Prompting salvianolic acid A freeze-dried powder can suppress lipid peroxidation process by certain mechanism, reducing oxygen-derived free radicals generates, simultaneously can improve endogenous activities of antioxidant enzymes again and alleviate oxygen-derived free radicals to myocardium damage, improving the oxidation resistance of ischemic myocardium and bring into play function of resisting myocardial ischemia.
Experimental example 30: the impact of salvianolic acid A freeze-dried powder on myocardial ischemia dog myocardial oxygen consumption
Prepare myocardial ischemia dog: modeling, grouping, dosage and time are with method described in experimental example 23.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%.
The impact (ml/min) of table 48. salvianolic acid A freeze-dried powder on myocardial ischemia dog myocardial oxygen consumption (
Figure BSA00000811649800611
n=6)
Figure BSA00000811649800612
Note: with model group comparison, * P < 0.05, * * P < 0.01
The impact (%) of table 49. salvianolic acid A freeze-dried powder on myocardial ischemia dog cardiac muscle coefficient of oxygen utilization (
Figure BSA00000811649800613
n=6)
Figure BSA00000811649800614
Note: with model group comparison, * P < 0.05, * * P < 0.01
Oxygen metabolism imbalance, oxygen supply and aerobic imbalance are the main causes of myocardial ischemia.Experimental result shows, model group dog causes after myocardial ischemia at ligation arteria coronaria, before myocardial oxygen consumption and coefficient of oxygen utilization and ischemia, relatively there is no significant change, but known in conjunction with the aforementioned experiment about myocardial ischemia left chamber acting in this explanation, after myocardial ischemia, the acting of left chamber 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.With model group comparison, the salvianolic acid A freeze-dried powder of various dose can reduce myocardial oxygen consumption and coefficient of oxygen utilization to some extent, and wherein high, the middle dosage group of salvianolic acid A freeze-dried powder ischemia 120min (being 105min after administration) coefficient of oxygen utilization has reduced respectively nearly 20%, 16% before compared with administration.In conjunction with in this explanation about the experimental result of salvianolic acid A freeze-dried powder to parameters of left ventricular function such as myocardial ischemia dog coronary flow and ventricle actings; prompting salvianolic acid A freeze-dried powder can reduce myocardial oxygen consumption and coefficient of oxygen utilization; improve the equilibrium of supply and demand of myocardium oxygen; improve ventricular function; the effect of performance protection ischemic myocardium, control myocardial ischemia.
Experimental example 31: salvianolic acid A freeze-dried powder 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 23.After ischemia 240min, draw blood 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.
The impact of table 50. salvianolic acid A freeze-dried powder on myocardial ischemia dog whole blood viscosity, plasma viscosity (
Figure BSA00000811649800621
n=6)
Figure BSA00000811649800622
Note: with model group comparison, * P < 0.05
With sham operated rats comparison, whole blood viscosity, plasma viscosity that ligation arteria coronaria causes myocardial ischemia dog obviously raise, and plasma fibrinogen content obviously rises, and packed cell volume and platelet adhesion rate obviously increase (P < 0.05); With model group comparison, in experiment, various dose salvianolic acid A freeze-dried powder 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; Between each dosage group, be certain agent effect trend; Prompting salvianolic acid A freeze-dried powder can reduce blood viscosity, suppresses platelet adhesion and assembles, and prevention Intravascular Thrombus forms, and improves myocardial ischemia dog hemorheology, improves microcirculation, can be used for preventing and treating ischemic heart desease.
Experimental example 32: the impact that salvianolic acid A freeze-dried powder forms rat thrombus in vivo
SD rat, (280 ± 20) g, divides 6 groups: normal saline group matched group, the high, medium and low dosage of salvianolic acid A freeze-dried powder (20,10,5mg/kg) group, aspirin group (10mg/kg) at random.Each group tail vein injection administration every day 1 time (matched group gives isometric(al) normal saline), 7d continuously, 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, with three sections of polyethylene tubes connections.Put into the long 5cm operation silk thread of having weighed in polyethylene tube stage casing.Be full of polyethylene tube with heparin-saline solution (5u/mL).Left external jugular vein is inserted in one end of pipe, and the other end is connected with right common carotid artery.Open bulldog clamp, blood returns to left jugular vein by the right carotid polyethylene tube of flowing through.Herba Clinopodii in after open blood flow 15min, takes out rapidly silk thread, and filter paper sucks supernatant blood, weighs, and gross weight subtracts line and heavily obtains wet weight of thrombus; Then put in 60 ℃ of baking ovens freeze-day with constant temperature to constant weight, weigh after cooling, be thrombosis dry weight.
Table 51. salvianolic acid A freeze-dried powder on rat thrombus in vivo form impact (
Figure BSA00000811649800631
n=6)
Figure BSA00000811649800632
Note: with the comparison of normal saline group, * P < 0.05, * * P < 0.01
With the comparison of normal saline group, 60,30,15mg/kg salvianolic acid A freeze-dried powder can significantly alleviate that rat suppository is wet, dry weight (P < 0.05 or P < 0.01), and thrombosis is had to obvious inhibitory action; Between each dosage group, be certain dose-effect relationship.Prompting salvianolic acid A freeze-dried powder has and suppresses the effect that rat thrombus in vivo forms, and points out it to can be used for preventing and treating the ischemic heart desease due to thrombosis.

Claims (22)

1. the preparation method of salvianolic acid A freeze-dried powder, it is characterized in that the medicine of described salvianolic acid A freeze-dried powder for the preparation of prevention and treatment ischemic heart desease, described its weight proportion of salvianolic acid A freeze-dried powder is: salvianolic acid A 10g～80g, filler 10g～80g, antioxidant is to make 0.01%～0.2% of total amount; The preparation method of described salvianolic acid A freeze-dried powder is:
Get red rooted salvia, be cut into decoction pieces or be ground into diameter 1mm～5mm granule, add 3～15 times of amounts, 45～95 ℃ of water temperature lixiviates are got at every turn, stir with 10～50 revs/min of speed simultaneously, or add 3～15 times of amount water boiling and extraction, and extract altogether 1～3 time, extract 1～4 hour at every turn; Extracting solution is evaporated to relative density 1.0～1.25 (60 ℃), adds ethanol to make to contain alcohol amount 50%～85%, leaves standstill, and filters, and decompression filtrate recycling ethanol is also concentrated into without alcohol taste, obtains Radix Salviae Miltiorrhizae extract; Or
Get red rooted salvia, be cut into decoction pieces or be ground into diameter 1mm～5mm granule, add 3～15 times of amount 30%～60% alcohol reflux at every turn, extract 1～4 hour at every turn, extract altogether 1～3 time; Decompression recycling ethanol is also concentrated into without alcohol taste, obtains Radix Salviae Miltiorrhizae extract;
Above-mentioned Radix Salviae Miltiorrhizae extract was diluted with water to every 1ml containing salvianolic acid B 1～30mg, and adjusting PH with base to 3.5 for aqueous solution～6.5 add with salvianolic acid B molar percentage 0.1～3% zinc chloride as catalyst, 100～140 ℃ of temperature thermal conversions 1～6 hour;
Conversional solution adjust pH to 2.5～4.5, leave standstill, centrifugal, supernatant is evaporated to every 1ml containing salvianolic acid A 1～10mg, separate through HPD-100 macroporous resin column chromatography, salvianolic acid A applied sample amount is 1: 35～1: 70 with macroporous adsorbent resin ratio, resin column blade diameter length ratio is 1: 4～1: 30, use respectively 1～8 times of cylinder hydrops, 1～10 times of column volume 10%～40% ethanol elution, remove impurity, use again 2～10 times of column volume 20%～60% ethanol elutions, HPLC detects, 20%～60% ethanol elution part that collection contains salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste,
Aqueous solution is concentrated into the solution of every 1ml containing 1-10mg salvianolic acid A, separate by polyamide chromatography post, salvianolic acid A applied sample amount is 1: 5～1: 25 with polyamide ratio, resin column blade diameter length ratio is 1: 4～1: 25, use respectively 1～10 times of cylinder hydrops, 5～20 times of column volume 20%～60% alcoholic solution eluting remove impurity, use again 4～15 times of column volumes, 40%～90% alcoholic solution eluting, 40%～90% alcoholic solution part that collection contains salvianolic acid A, decompression recycling ethanol is also concentrated into without alcohol taste aqueous solution;
Aqueous solution is concentrated, acid adjustment pH to 2.0～4.0, the t-butyl methyl ether of doubly measuring with aqueous solution 1-8, point 2～6 extractions, separate organic layer, reclaim under reduced pressure t-butyl methyl ether, makes the extract of every 1ml containing salvianolic acid A 1g～10g, add 1～3 times of amount silica gel, stir, volatilize;
Be added on 5～20 times of dry silicagel columns of amount that installed stirring sample silica gel, silicagel column blade diameter length ratio is 1: 4～1: 25, take pentane-t-butyl methyl ether as eluant, gradient elution, use respectively 6～30 times of column volumes of pentane-t-butyl methyl ether (4: 6) eluting, 6～30 times of column volumes of pentane-t-butyl methyl ether (6: 4) eluting, reclaim under reduced pressure eluant, the salvianolic acid A reclaiming after organic solvent adds 5～20 times of water gagings dissolvings, microwave vacuum drying, obtains described salvianolic acid A;
Getting described salvianolic acid A 10g～80g injects water 1500～2800ml and is stirred to dissolve, by adjusting PH with base value 4.0～5.0, add described filler to make its dissolving, add again described antioxidant, be stirred to dissolve and mix, then add active carbon 0.5～2g stirring and adsorbing, filter and remove active carbon, inject fill after water and become bottle, send into and in freeze dryer, carry out lyophilization;
Described lyophilization comprises the steps:
A, freezing: be cooled to-40 ℃～-45 ℃ with 20 ℃～30 ℃/h speed, be incubated freezing 10～16 hours;
B, distillation: be evacuated to below 0.3mbar, in 10～14 hours, the salvianolic acid A formulation temperature freezing risen to-23 ℃～-27 ℃, maintain-23 ℃～-27 ℃ vacuum dryings 6～8 hours;
C, dry: continue to heat up, be evenly warming up to 40 ℃～45 ℃ with 0.5 ℃～1.0 ℃/min, maintain 40 ℃～45 ℃ and be dried after 2～5 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
2. preparation method according to claim 1, wherein said filler is selected from any one or a few in mannitol, glucose, lactose, and consumption is 20mg～40mg/2ml～3ml.
3. preparation method according to claim 1 and 2, wherein said antioxidant is selected from any one or a few in vitamin C, thiourea, sodium sulfite, sodium pyrosulfite.
4. preparation method according to claim 3, its weight proportion is: salvianolic acid A 20g, filler mannitol 20g, antioxidant vitamin C 0.8g; Its preparation method is:
Get described salvianolic acid A 20g, inject water 1900ml and be stirred to dissolve, with sodium hydroxide sodium adjust pH 4.0～5.0, add mannitol 20g to make to dissolve, then add 0.8g vitamin C, be stirred to dissolve and mix, add active carbon 0.5g stirring and adsorbing, filter and remove active carbon; Inject water to 2000ml, fill becomes 1000 bottles, lyophilization;
Described lyophilization comprises the steps:
A, freezing: be cooled to-45 ℃ with 25 ℃/h speed, be incubated freezing 15 hours;
B, distillation: be evacuated to below 0.3mbar, in 12 hours, the salvianolic acid A formulation temperature freezing risen to-25 ℃, maintain-25 ℃ of vacuum dryings 8 hours;
C, dry: continue to heat up, be evenly warming up to 40 ℃ with 0.8 ℃/min, maintain 40 ℃ and be dried after 3 hours, sample temperature is down to room temperature, add a cover, obtain salvianolic acid A freeze-dried powder.
5. a kind of preparation method according to claim 1, is characterized in that microwave vacuum drying temperature: 20-100 ℃, return difference temperature 1-5 ℃, and more than vacuum-0.07MPa, microwave power 1-100KW, dry 10-200 minute.
6. a kind of preparation method according to claim 5, is characterized in that microwave vacuum drying temperature: 50-85 ℃, return difference temperature 2-4 ℃, and more than vacuum-0.07MPa, microwave power 10-80KW, dry 100-150 minute.
7. a kind of preparation method according to claim 6, is characterized in that microwave vacuum drying temperature: 55-80 ℃, return difference temperature 2-3 ℃, and more than vacuum-0.07MPa, microwave power 25-60KW, dry 120-140 minute.
8. according to the preparation method of claim 1, wherein said can be used for treating one or more of following disease for the medicine preventing and treat ischemic heart desease: coronary heart diseases and angina pectoris, ischemic cardiomyopathy, myocardial infarction, myocardial ischemia apoplexy, acute coronary syndrome, coronary stricture, myocardial ischemia reperfusion injury, atherosclerosis.
9. according to the preparation method of claim 1 or 8, wherein said salvianolic acid A freeze-dried powder is used for improving the hemodynamic purposes of Ischemic Heart.
10. according to the preparation method of claim 9, wherein said salvianolic acid A freeze-dried powder is for improving the purposes of ischemic heart cardiac function.
11. according to the preparation method of claim 9, and wherein said salvianolic acid A freeze-dried powder is for increasing the purposes of the coronary flow of ischemic heart.
12. according to the preparation method of claim 1 or 8, and wherein said salvianolic acid A freeze-dried powder is for reducing the purposes of ischemic heart myocardial infarct size.
13. according to the preparation method of claim 12, and wherein said salvianolic acid A freeze-dried powder is for dwindling the purposes of myocardial ischemia scope.
14. according to the preparation method of claim 12, and wherein said salvianolic acid A freeze-dried powder is for alleviating the purposes of degree of myocardial ischemia.
15. according to the preparation method of claim 12, and wherein said salvianolic acid A freeze-dried powder regulates the purposes of Enzyme Activities for the protection of myocardial cell membrane structure.
16. according to the preparation method of claim 12, and wherein said salvianolic acid A freeze-dried powder is for suppressing the purposes of ischemic myocardial tissue inflammatory reaction.
17. according to the preparation method of claim 12, and wherein said salvianolic acid A freeze-dried powder is for the protection of the purposes of myocardial mitochondria damage.
18. according to claim 1 or 8 preparation method, wherein said salvianolic acid A freeze-dried powder is for improving the purposes of myocardial metabolism.
19. according to the preparation method of claim 18, and wherein said salvianolic acid A freeze-dried powder comprises for improving one or more of following myocardial metabolism for the purposes of improving myocardial metabolism: myocardium radical metabolism, fatty acid metabolism, energy metabolism.
20. according to the preparation method of claim 1 or 8, and wherein said salvianolic acid A freeze-dried powder is for reducing the purposes of myocardial oxygen consumption.
21. according to the preparation method of claim 1 or 8, and wherein said salvianolic acid A freeze-dried powder is used for improving hemorheological purposes.
22. according to the preparation method of claim 1 or 8, and wherein said salvianolic acid A freeze-dried powder is used for suppressing thrombotic purposes.
CN201210487270.0A 2012-11-20 2012-11-20 Salvianolic acid A lyophilized powder and application thereof in preparing medicaments Active CN102973544B (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102210666A (en) * 2010-04-06 2011-10-12 山东靶点药物研究有限公司 Medical use of salvianolic acid A

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102210666A (en) * 2010-04-06 2011-10-12 山东靶点药物研究有限公司 Medical use of salvianolic acid A

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* Cited by examiner, † Cited by third party
Title
丹酚酸A的研究与进展;张莉等;《中国中药杂志》;20111030;第36卷(第19期);第2603-2609页 *
张莉等.丹酚酸A的研究与进展.《中国中药杂志》.2011,第36卷(第19期),第2603-2609页.

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