CN105085266A - Method for preparing salvianolic acid A from a plurality of salvia plants - Google Patents
Method for preparing salvianolic acid A from a plurality of salvia plants Download PDFInfo
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Abstract
The present invention relates to a method for preparing salvianolic acid A from a plurality of salvia plants, major steps include: (1) plant roots and rhizomes are sliced, water is poured into a closed container and the decoction pieces are heated for extraction under oxygen free conditions; and (2) the extract is concentrated under reduced pressure to an appropriate volume, refluxed for reaction at 100 DEG C under oxygen free conditions at atmospheric pressure, or placed in an autoclave or a microwave reactor for heating for reaction to obtain a salvianolic acid A conversion solution. The salvianolic acid A conversion solution is further purified by conventional separation techniques such as macroporous resin, reversed phase chromatography on silica gel, Sephadex LH-20, droplet countercurrent chromatography, and the like to obtain a salvianolic acid A product with the content of more than 90%, and the salvianolic acid A product is used for clinical treatment and research. The method is simple in process, higher in salvianolic acid A conversion rate, and suitable for industrial production.
Description
Technical field
The invention belongs to medicinal plants field, particularly relate to a kind of preparation method of salvianolic acid A.
Background technology
The red sage root (SalviamiltiorrhizaBunge) has long clinical application history as China's conventional Chinese medicine, in the treatment of cardiovascular and cerebrovascular diseases, occupy critical role, and its significant curative effect has obtained extensive accreditation clinically.The current red sage root has become the Chinese medicine that China's consumption is maximum, sales volume is the highest, preparation manufacturer is maximum, and the consumption of red rooted salvia is more than 1.5 ten thousand tons/year.The red sage formulation of Clinical practice mainly comprises: FUFANG DANSHEN DIWAN, compound injection of red sage root, FUFANG DANSHEN PIAN, poly phenolic acid of Radix Salviae Miltiorrhizae salt injection etc.These preparations particularly in injection main effective constituent be pressure differential self, have resist myocardial ischemia, the effect such as anti peroxidation of lipid and scavenging free radicals.In the red sage root, topmost salvianolic acid composition is salvianolic acid B, in addition containing alkannic acid, Prolithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid C, salvianolic acid E etc., wherein with the activity of salviol acid A (SalvianolicacidA) the strongest (Jiang, RW; Lau, KM; Hon, PM, etalChemistryandbiologicalactivitiesofcaffeicacidderivat ivesfromSalviamltiorrhiza.CurrentMedicinalChemistry, 2005,12 (2), 237-246.).The molecular structure of salvianolic acid A determines it and had both had good water-soluble, and have again good liposoluble and permeable membrane ability, therefore compared with phenolic acid most of in the red sage root, Premeabilisation of cells ability is stronger, and bioavailability is higher.The significant biological activity of salvianolic acid A has caused the extensive concern of people.Research display: salvianolic acid A was to the continuous gastric infusion of experimental diabetic rats 8 weeks, can significantly reduce its fasting plasma glucose, triacylglycerol, total cholesterol, low density lipoprotein cholesterol and free fatty acid content, significantly increase serum superoxide dismutase activities, reduce model group rats mda content.Show that salvianolic acid A not only has good regulating effect to experimental diabetic rats blood fat, the metabolism of blood glucose of experimental diabetic rats can also be improved, significantly reduce fasting plasma glucose (square lotus flower, Wang Yuehua, He Guorong, Du Guanhua.Salvianolic acid A is to the blood sugar reducing function of experimental diabetic rats and Initial Study of Mechanism thereof.2011，20(21)，2063-2068.)。Du Guanhua etc. utilize Isolated Perfused Rat Lungs heart (Langendorffheart) ischemia-reperfusion injury model to find containing salvianolic acid A 0.1, the K-HShi damping fluid perfused hearts of 1.0mmol/L, Reperfu-sion after ligation coronary artery 15min, except ventricular fibrillation and slight irregular pulse appear in indivedual heart, most electrocardio-activity is in standard state substantially, ventricular fibrillation incidence is reduced to 25% respectively, 20%, result prompting salvianolic acid A may have better protecting effect (Du Guanhua to myocardial ischemia, Qiu Yue, Zhang Juntian. the provide protection that salvianolic acid A damages rat myocardial ischemia and reperfusion. Acta Pharmaceutica Sinica, 1995, 30 (10), 731.).On myocardial infarction model or myocardial ischemia-reperfusion animal model, research finds further, the intravenous injection of 0.3 ~ 10mg/kg salvianolic acid A significantly can reduce ECG ST section and raise degree and Left ventricular end diastolic pressure, raise left ventricular contraction power and minimax left ventricular pressure, reduce myocardial infarction area, and find that the salvianolic acid B effect of the effect of 2.5mg/kg salvianolic acid A and 10mg/kg is suitable, 5, the effect that 10mg/kg salvianolic acid A improves myocardial ischemia in rats is obviously better than salvianolic acid B (WangSB, TianS, YangF, etal.CardioprotectiveeffectofsalvianolicacidAonisoproter enol-inducedmyocardialinfarctioninrats.EurJPharm, 2009, 615 (1/3): 125. Song Yangjing, Kong Lingshan, Wu Jing, Deng.Salvianolic acid A and salvianolic acid B improve myocardial ischemia in rats effect and compare. Chinese Chinese materia medica information magazine; 2007; 14 (9): 36. kingdoms shake, Yan Yuping, Zhu Changfu. the provide protection that salvianolic acid A/salvianolic acid B different ratio damages rat myocardial ischemia and reperfusion.Hebei Chinese materia medica journal, 2006,21 (2): 4.).Be that isolated experiment or integral experiment result all prove that salvianolic acid A has better protecting effect to myocardial ischemia, and effect is better than salvianolic acid B.
Salvianolic acid A also has better protecting effect to cerebral ischemia; business great talents etc. observe salvianolic acid A 2.5 ~ 10mg/kg intravenously administrable can significantly improve middle cerebral artery occlusion model in rats (middlecerebralarteryocclusion; MCAO) rat functional defect state; reduce infarct size; alleviate cerebral edema; reduce related brain areas neure damage degree; comparatively salvianolic acid B has more obvious effect (Shang Hongcai; Cao Hongbo; vast sea; Deng. salviol acid A, B is to focal cerebral ischemia in rats injury protection Benefit Transfer.Pharmacology and Clinics of Chinese Materia Medica, 2007,23 (3), 15-18).The current research of salvianolic acid A has entered into a relatively comprehensively deep stage; its excellent physico-chemical property and various active as the protection heart, cerebrovascular trauma, anti-liver injury, anti-oxidant, prevent and treat diabetes and complication etc.; for the further exploitation of salvianolic acid A and clinical application provide abundant research data (Zhang Li; Zhang Weiku, Zhao Ying etc.The research and advances of salvianolic acid A. CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2011,36 (19), 2603-2609.) but, the natural content of salvianolic acid A is extremely low (being about 0.01 ~ 0.06% of red rooted salvia), thus makes the high cost extracting salvianolic acid A, and technique is loaded down with trivial details, separation and purification difficulty is excessive, seriously governs the research and development of this product.
Salvianolic acid A structure
Carry out source problem for what solve salvianolic acid A, many scholars attempts obtaining salvianolic acid A by transforming salvianolic acid B from the red sage root.Prior art discloses, and during heating, salvianolic acid B ruptures an ester bond, and a part Salvianic acidA is sloughed in hydrolysis, then can generate salvianolic acid A by the open loop of dihydrofuran ring, dehydration, decarboxylation.The Chinese patent of application number 201210488782.9 discloses a kind of preparation method of danshen root salvianolic acid A, by the method for water extraction, High Temperature High Pressure conversion, reverse-phase chromatography, normal-phase chromatography acquisition salvianolic acid A, but to salvianolic acid A destroy very large, preparation cost is higher, and finally on normal phase column, adopt the organic solvent purifyings such as alkane, t-butyl methyl ether, methyl acetate, cause that product loss is comparatively large, organic solvent residual serious, be difficult to the salvianolic acid A obtaining " patent medicine level ".The Chinese patent of application number 201310192210.0 discloses the preparation method of another kind of danshen root salvianolic acid A, high boiling organic solvent such as ethylene glycol, methyl-sulphoxide, dimethyl formamide etc. are adopted to transform Radix Salviae Miltiorrhizae extract, although can salvianolic acid A be transformed at ambient pressure, but due to an organic solvent as transforming solution, preparation cost is high, toxicity is large, technique is time-consuming, and due to use organic solvent boiling point many between 150 ~ 200 DEG C, still cannot overcome the serious problem of organic solvent residual.
In addition, using red rooted salvia to transform in the research of salvianolic acid A, the problem of existing technique ubiquity low conversion rate, but there is no a research at present and can in depth disclose the basic reason causing low conversion rate.Although some patent or document are attempted to overcome this defect, how from the viewpoint of change pH value, add catalyzer etc., think add catalyzer can fast reaction speed, shorten the time that salvianolic acid B is destroyed.As the Chinese patent " a kind of catalyzed conversion salvianolic acid B prepares the method for salvianolic acid A " of application number 201010143678.7, the method adding urea seeding is adopted to improve the productive rate of salvianolic acid A; And the Chinese patent of application number 201210487598.2 " a kind of catalyzed conversion salvianolic acid B prepares the method for salvianolic acid A " by name equally, think and add the productive rate that urea seeding can not improve salvianolic acid A, therefore adopt iron(ic) chloride, ruthenium trichloride, zinc chloride, Palladous chloride etc. as catalytic conversion salvianolic acid A, and think higher than the former transformation efficiency.In fact, the effect of catalyzer reduces reaction activity, can improve speed of reaction after adding, but do not help raising transformation efficiency.But the Chinese patent of application number 201210487598.2 is after adding catalyzer, reaction under high pressure temperature cited is in an embodiment still up to 120-135 DEG C, reaction times also reaches 2.5-4.5hr, have no catalyzer and play positive effect, and transformation efficiency also only reaches about 60%.
The Chinese patent of application number 201210334295.7 " a kind of medicinal material of salvianolic acid A high-content, preparation method and purposes ", different areas are used as the medicinal material containing salvianolic acid B of the red sage root, by high temperature, high pressure, high humidity treatment, the salvianolic acid B contained by medicinal material is made to change into salvianolic acid A in plant tissue cell, need to regulate pH at 1.0-12.0 with acid, alkali in treating processes, need medicinal material again dry after process.This process is not only loaded down with trivial details, and is not operate under homogeneous solution state owing to reacting, and controllability is not strong.The object of this invention is the medicinal material of the salvianolic acid A/salvianolic acid B in order to obtain different content ratio, and the method that its content indicates is the peak area ratio of salvianolic acid A/salvianolic acid B in HPLC collection of illustrative plates, is 0.6: 1 to 18: 1.But the transformation efficiency of salvianolic acid A can not be calculated according to this ratio.First, salvianolic acid A is widely different to the UV response value of salvianolic acid B in HPLC figure, the peak area response of salvianolic acid A is far away higher than salvianolic acid B, and this is determined by its constitutional features, therefore cannot calculate the content of salvianolic acid A from the radiometer of the two peak area; Secondly, not represent greatly the transformation efficiency of salvianolic acid A high for the peak area ratio of salvianolic acid A/salvianolic acid B.Experiment of the present invention discloses (contrast see Fig. 1, Fig. 2 and Fig. 5, Fig. 6), at conversion certain hour, the peak area of salvianolic acid B sharply declines close to the peak area of salvianolic acid A when disappearing, but the ratio of the two is very high, show on certain time point, the salvianolic acid A of salvianolic acid B and conversion is subject to more serious destruction simultaneously, and this invention does not provide the optimal conditions that salvianolic acid A transforms.
Our research discloses, and cause the low major cause of salvianolic acid A yield to be to transform the oxidized destruction of salvianolic acid B in front and conversion reaction, thus the quantity making salvianolic acid B change into salvianolic acid A greatly reduces.The easily oxidized reason of salvianolic acid B is containing adjacent two phenolic hydroxyl groups of many groups in its molecule, even if also very easily oxidized under small amounts agent existence condition, therefore in atmosphere long-time expose can and the oxygen molecule generation oxidation-reduction reaction in air.In addition, in conversion process under aerobic conditions, the salvianolic acid A of salvianolic acid B and generation also can be partially oxidized.Therefore, the essential measure improving salvianolic acid A yield is whole extraction, conversion production process all must completely cut off air.
We are through studying discovery for many years, and the aqueous solution of salvianolic acid B, at a certain temperature can be oxidized very soon as exposed in atmosphere; And when isolated air, salvianolic acid B can more stably be present in the aqueous solution of more than 50 DEG C.This is found to be us and takes heating extractive technique from medicinal material, to propose salvianolic acid B fully provide experimental basis.Experiment shows, the method for isolated air can adopt nitrogen, argon shield, and the protected effect of argon gas is better than nitrogen.In addition, research shows, many salvias contain more rich salvianolic acid B, as originate in Yunnan Yunnan Salvia japonica Thunb. (SalviayunnanensisC.H.Wright), originate in Gansu Salvia przewalskii (SalviaprzewalskiiMaxim), originate in the Anhui, the pale reddish brown Zhejiang red sage root (SalviasinicaMigof.purpureaH.W.Li) in Zhejiang, originate in the Salvia miltiorrhiza f. (SalviamiltiorrhizaBungevar.albaC.Y.WuetH.W.Li) etc. in Shandong, the content of its salvianolic acid B can reach 2 ~ 5%.The content of part kind salvianolic acid B is higher, and as originated in the Salvia cavaleriei (SalviacavalerieiL é vl.) in Sichuan, the content of its salvianolic acid B reaches about 8%.Therefore, adopt this method, not only from the red sage root and other salvias, salvianolic acid B can be obtained efficiently, also obtain salvianolic acid A provide new available sources for transforming from these plants further.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of preparation method obtaining salvianolic acid A from multiple salvia, the method not only efficiently solves current salvianolic acid A and to originate single problem, and overcome the deficiencies in the prior art, preparation process is simple, yield is high, cost is low, is applicable to industrialization and produces.
Several plant of the present invention comprises: the red sage root (SalviamiltiorrhizaBunge); Radix Salviae Miltiorrhizae (SalviamiltiorrhizaBungefalbaC.Y.WuetH.W.Li); Salvia bowleyana Dunn (SalviabowleyanaDunn); Intend the red sage root (SalviasinicaMigo); Anhui, the pale reddish brown Zhejiang red sage root (SalviasinicaMigof.purpureaH.W.Li); Salvia japonica Thunb. (SalviajaponicaThunb); Yunnan Salvia japonica Thunb. (SalviayunnanensisC.H.Wright); Salvia przewalskii (SalviaprzewalskiiMaxim); Huangpu Salvia japonica Thunb. (SalviaprionitisHance); Field bund Salvia japonica Thunb. (SalviascapiformisHance); Brown hair Salvia przewalskii (SalivaprzewalskiiMaxim.var.mandarinorum (Diels) Stib); Threeleaf sage root (SalviatrijugaDiels.); Salvia chinensis (SalviachinensisBenth); Salvia cavaleriei (SalviacavalerieiL é vl.); Root of Unileaf sage. (SalviacavalerieiL é vl.Var.simplicifoliaStib); Arisaema balansae Engl. grass (SalviakwangsiensisH.W.Li); Salvia miltiorrhiza f. (SalviamiltiorrhizaBungevar.albaC.Y.WuetH.W.Li).These plants are natural existence throughout the country, at various places well-grown, is good natural medicine resource.
A kind of preparation method obtaining salvianolic acid A from several salvia of the present invention, comprises the steps:
(1) extract: the section of plant root and rhizome is placed in encloses container, add water dipped medicine materical crude slice surface, and under oxygen free condition, 50-100 DEG C of heating is extracted, and can obtain the extracting solution being rich in salvianolic acid B;
(2) transform: after extracting solution is evaporated to proper volume, under oxygen free condition, normal pressure is in 100 DEG C of back flow reaction, or is placed in autoclave or microwave reactor reacting by heating, can obtain the conversion fluid being rich in salvianolic acid A;
(3) salvianolic acid A conversion fluid the present invention obtained uses conventional isolation techniques further, as technology purifying such as macroporous resin, reversed-phase silica gel chromatography, dextrane gel SephadexLH-20, drop counter current chromatographies, the salvianolic acid A product that content reaches more than 90% can be obtained.The method technological process is simple, the salvianolic acid A yield of preparation and purity high, be applicable to scale operation.
The preparation method of a kind of salvianolic acid A provided by the invention, wherein: step (1) preferably adds thermal extraction method and is: get plant medicine materical crude slice, adds 6 ~ 15 times of water gagings at every turn, adopts steam heating or heating in water bath, extracts 3 times, extract 1 ~ 4 hour at every turn; Preferred, extraction time is 2 times; The water added is to the ratio of medicine materical crude slice for first time adds by 10 times amount, and second time adds by 8 times amount.
The method of step (1), (2) isolated air is as described below: in the encloses container selected, by the external rare gas element of T-valve, vacuumize 3 displaced air after passing into rare gas element, wherein rare gas element selects nitrogen and argon gas, preferred argon gas; Normal pressure lower 100 DEG C of times transformed are 8 ~ 24 hours, preferably 10 ~ 16 hours; Under high pressure, the transformation time of temperature 101 ~ 152 DEG C is 0.1-12 hour, wherein temperature preferably 115 ~ 145 DEG C, more preferably 130 ~ 140 DEG C; Transformation time preferably 0.3 ~ 3 hour, more preferably 0.5 ~ 2 hour.Microwave reaction temperature is 110 ~ 160 DEG C, and transformation time is 0.1-5 hour, wherein temperature preferably 115 ~ 140 DEG C, more preferably 120 ~ 130 DEG C; Transformation time preferably 0.2 ~ 3 hour, more preferably 0.5 ~ 1.5 hour.
The general approved viewpoint of current one is: salvianolic acid B is easily by high temperature in leaching process, and therefore Extracting temperature often arranges very low.As the Chinese patent " a kind of extracting method of effective component in red sage " of application number 02110817.X, the isolated at low temperatures air of ultrasonic technology is adopted to extract pressure differential self, adopt in an embodiment " supersound process 1-2 hour; nitrogen is filled in local; constantly add appropriate ice block cooling during ultrasonication ", this mode does not obviously reach the requirement of normalizing operation, is not suitable for suitability for industrialized production.In addition, though the temperature that this invention sets in the claims is less than 60 DEG C, actual extracting temperature controls at 4-10 DEG C, and obviously, the too low meeting of temperature makes extraction efficiency greatly reduce.The present invention breaches the intrinsic notion that salvianolic acid B can not extract at relatively high temperatures on extracting method, namely can 50-100 DEG C of heating extract under anaerobic, salvianolic acid B extracts completely, ultrasound assisted extraction technique under the isolated air low temperature that extraction yield adopts far above the Chinese patent of application number 02110817.X.The present invention is with the extraction yield of salvianolic acid B for evaluation index, and under nitrogen protection, compare ultrasonic cryogenic and extract and heat the effect extracted, experimental data sees the following form 1.
Take salvia piece six parts, every part of 50g, add 500ml water respectively; under nitrogen protection, a employing supersound extraction, temperature controls at 40 DEG C; another five parts adopt heating in water bath to extract; temperature controls at 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C and 100 DEG C respectively, each extraction 2 times, each 1hr; filter; merging filtrate, is settled to 1000ml, measures content of danshinolic acid B.The results are shown in Table 1.
The result that table 1 Different Extraction Method obtains
As can be seen here, in leaching process, as long as isolated air, avoid the oxidized destruction of salvianolic acid B, high temperature is conducive to the abundant proposition of salvianolic acid B, and the mindset that salvianolic acid B can not at high temperature extract has been broken in this discovery, has unforeseeable technique effect.
Equally, in salvianolic acid A conversion process, isolated air not only can ensure that salvianolic acid B is destroyed to greatest extent less, thus is converted into salvianolic acid A as much as possible, and can avoid the oxidized destruction of salvianolic acid A that generated.The impact that the isolated air of contrast experiment's sufficient proof that the present invention does at ambient pressure transforms salvianolic acid A: by Salvia miltiorrhiza Bge water extract 100 DEG C of reflux under nitrogen protection, respectively in 9h, 11h, 12h, 13.5h, 15h, 16.5h, 18h, 19h, 21.5h, 23h sampling and measuring, record salvianolic acid B and be slowly converted into salvianolic acid A in 24 hours, wherein salvianolic acid A content generates the highest time point is 12hr (Fig. 1, the Rt=25.06min of salvianolic acid A; The Rt=16.77min of salvianolic acid B), and salvianolic acid B transforms maximum time points at 19hr (Fig. 2, the Rt=24.94min of salvianolic acid A, the Rt=16.64min of salvianolic acid B).Salvianolic acid A is about 50% at the transformation efficiency of 12hr.Under argon shield, the transformation efficiency of salvianolic acid A is higher, and it transforms the highest time point is 13.5hr, and transformation efficiency can reach about 60% (Fig. 3, the Rt=24.80min of salvianolic acid A, the Rt=16.62min of salvianolic acid B).This is because ordinary nitrogen is due to the restriction by preparation method; oxygen still containing minute quantity; argon gas then can ensure oxygen-free gas, thus avoids salvianolic acid A a small amount of in conversion process and B to be destroyed, and therefore under argon shield, the transformation efficiency of salvianolic acid A is higher.Experiment proves simultaneously, if Salvia miltiorrhiza Bge water extract to be directly exposed to 100 DEG C of reflux in air, then, when 12hr, salvianolic acid B and salvianolic acid A are all destroyed totally.
On this basis, for improving the transformation efficiency of salvianolic acid A further, the present invention has carried out again High Temperature High Pressure under isolated air and has transformed the experiment of salvianolic acid A.By Salvia miltiorrhiza Bge water extract reacting by heating at 0.2MPa/120 DEG C in autoclave under nitrogen protection, respectively at 0min, 30min, 60min, 120min, 180min, 240min, (Fig. 4 is the HPLC collection of illustrative plates of 0min to 300min sampling and measuring, the Rt=27.013min of salvianolic acid B), record salvianolic acid B and be substantially converted into salvianolic acid A in 300min, wherein salvianolic acid A content generates the highest time point is 180min (Fig. 5, the Rt=31.513min of salvianolic acid A), and salvianolic acid B transforms maximum time points at 300min (Fig. 6, the Rt=31.532min of salvianolic acid A).The transformation efficiency of salvianolic acid A when 180min is about 62%.Under argon shield, the transformation efficiency of salvianolic acid A is higher, and the transformation efficiency when 180min can reach about 70% (Fig. 7, the Rt=31.635min of salvianolic acid A).Simultaneously, this experiment is contrasted with the Chinese patent of application number 201210487598.2 and finds, when isolated air, the time of the two reaction has no significant difference, and the transformation efficiency of this experiment is higher, illustrate that the key affecting this reaction is isolated air, and adding of catalyzer is optional.
The present invention is compared with the prior art, do not need to carry out concentration to extracting solution, the adjustment of pH value etc., do not need to add catalyzer yet, whole process does not use high boiling organic solvent, avoid the organic solvent residual of product, not only technological process is easier compared with prior art, save cost, shorten the production cycle, and by optimizing High Temperature High Pressure anaerobic reaction conditions, the transformation efficiency making salvianolic acid B be converted into salvianolic acid A reaches about 70%, have also exceeded any prior art, therefore, the present invention achieves unexpected technique effect, there is significant creativeness.
The present invention is compared by experiment repeatedly, investigates, finally determine the technique of high temperature extraction under oxygen free condition, high pressure converted, optimize processing parameter from each technological process extracted, transform on the possible factor affecting salvianolic acid A transformation efficiency.According to the preparation method of this kind of high-purity danshinolic acid A that above method proposes, by selecting multiple salvia as the starting material preparing salvianolic acid A, not only expand medicinal raw-material source, changed and only rely on the red sage root to be the present situation that raw material obtains salvianolic acid A; Simultaneously this salvia is the preparation method of the salvianolic acid A of raw material, and technological process is simple, the salvianolic acid A yield of preparation and purity is high, cost is low, is applicable to scale operation.
On the other hand, the present invention also provides a kind of method measuring salvianolic acid A and content of danshinolic acid B, and adopt high performance liquid chromatography (HPLC) to measure salvianolic acid A, salvianolic acid B, the method effectively can be separated each salvianolic acid constituents, and condition determination is as follows:
With octadecylsilane key and silica gel for stationary phase, determined wavelength: 286nm; Detector: PAD; Flow velocity: 1ml/min; Column temperature 30 DEG C; Theoretical plate number should be not less than 3000 by salvianolic acid A;
The preparation of reference substance solution: precision takes salvianolic acid A and salvianolic acid B 20mg, accurately weighed, is placed in 10mL measuring bottle, is diluted to scale with dissolve with methanol, shakes up; Precision measures 1ml solution, be placed in 10mL measuring bottle, is diluted to scale, shakes up, obtain reference substance solution with dissolve with methanol.
Prepared by need testing solution: precision measures and is equivalent to 20mg salvianolic acid A and salvianolic acid B sample in 100ml volumetric flask, adds methanol dilution, constant volume, crosses 0.22 μm of millipore filtration, as need testing solution.
Wash-out take acetonitrile as mobile phase A, with 0.1 ~ 0.67% formic acid for Mobile phase B, carries out gradient elution by following condition, runs 40min;
0 ~ 10min, maintenance acetonitrile ratio is the ratio of 15%, 0.1 ~ 0.67% aqueous formic acid is 85%;
10 ~ 25min, acetonitrile ratio rises to 25% by 15%, and the ratio of 0.1 ~ 0.67% aqueous formic acid drops to 75% by 85%;
25 ~ 40min, the ratio keeping acetonitrile ratio 25%, 0.1 ~ 0.67% aqueous formic acid is 75%.
Assay method: accurate absorption reference substance solution and each 20 μ l of need testing solution respectively, injection liquid chromatography, measures content.
Extract according to this technique the multiple salvia containing salvianolic acid B and transform, result salvianolic acid A all obtains higher transformation efficiency.Carry out separation and purification to the extracting solution after transforming, obtain salvianolic acid A product, adopt the method for high performance liquid chromatography to measure content, result display content is more than 90%, specifically sees embodiment.
Accompanying drawing explanation
Fig. 1 is atmospheric pressure reflux 12 hours under Salvia miltiorrhiza Bge water extract nitrogen protection;
Fig. 2 is atmospheric pressure reflux 19 hours under Salvia miltiorrhiza Bge water extract nitrogen protection;
Fig. 3 is atmospheric pressure reflux 13.5 hours under Salvia miltiorrhiza Bge water extract argon shield;
Fig. 4 is the HPLC collection of illustrative plates before Salvia miltiorrhiza Bge water extract high-temperature high-voltage reaction;
Fig. 5 is the HPLC collection of illustrative plates of reacting by heating 180min under 0.2MPa in autoclave under Salvia miltiorrhiza Bge water extract nitrogen protection;
Fig. 6 is the HPLC collection of illustrative plates of reacting by heating 300min under 0.2MPa in autoclave under Salvia miltiorrhiza Bge water extract nitrogen protection;
Fig. 7 is the HPLC collection of illustrative plates of reacting by heating 180min under 0.2MPa in autoclave under Salvia miltiorrhiza Bge water extract argon shield.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1:
1. extract: take 100g salvia bowleyana Dunn pharmaceutical decocting piece, add the 70 DEG C of heating extractions 2 hours under nitrogen protection of 1000ml water, extracts 2 times, second time adds water 800ml, and merging filtration obtains extracting solution.
2. transform: extracting solution is concentrated into 200ml, atmospheric pressure reflux 15hr under argon shield, stopped reaction.The content measuring salvianolic acid B in extracting solution is 1.36%, and in conversion fluid, the content of salvianolic acid A is 0.53%, and transformation efficiency is 56.6%.
3. column chromatography for separation: take 200gDM301 type macroporous resin, fill post after pre-treatment.Conversion fluid is added above chromatography column, after entering post bed completely, wash 3 column volumes with water, discard this elution fractions; Use 70% ethanol elution, 3 column volumes again, merge this elution fractions.
4. column chromatography purification: elutriant is evaporated to proper volume, is prepared purifying with C18 bonded silica gel post, with 40% methanol-eluted fractions, collects the elutriant containing salvianolic acid A, through concentrated removing methyl alcohol postlyophilization, obtains salvianolic acid A product.It is 94.6% that HPLC records purity.
Embodiment 2:
1. extract: take 100g and intend red rooted salvia medicine materical crude slice, add the 60 DEG C of heating extractions 2 hours under argon shield of 1000ml water, extracts 2 times, second time adds water 800ml, and merging filtration obtains extracting solution.
2. transform: extracting solution is concentrated into 200ml, and reflux under nitrogen protection 8hr, stopped reaction.The content measuring salvianolic acid B in extracting solution is 1.12%, and in conversion fluid, the content of salvianolic acid A is 0.41%, and transformation efficiency is 53.2%.
3. column chromatography for separation: take 200g330 type macroporous resin, fill post after pre-treatment.Conversion fluid is added above chromatography column, after entering post bed completely, wash 3 column volumes with water, discard this elution fractions; Use 60% methanol-eluted fractions, 3 column volumes again, merge this elution fractions.
4. column chromatography purification: by elutriant after being concentrated into proper volume, is prepared purifying with polyamide column, with 40% ethanol elution, collects the elutriant containing salvianolic acid A, through concentrated removing ethanol postlyophilization, obtains salvianolic acid A product.It is 92.1% that HPLC records purity.
Embodiment 3:
1. extract: take Anhui, 100g pale reddish brown Zhejiang red rooted salvia medicine materical crude slice, add 1000ml water and heat extraction 2 hours at 50 DEG C under nitrogen protection, extracts 2 times, second time adds water 800ml, and merging filtration obtains extracting solution.
2. transform: extracting solution is concentrated into 200ml, and reflux under argon shield 24hr, stopped reaction.The content measuring salvianolic acid B in extracting solution is 1.41%, and in conversion fluid, the content of salvianolic acid A is 0.53%, and transformation efficiency is 54.6%.
3. column chromatography for separation: take 200gCAD45 type macroporous resin, fill post after pre-treatment.Conversion fluid is added above chromatography column, after entering post bed completely, wash 3 column volumes with water, discard this elution fractions; Use 50% ethanol elution, 3 column volumes again, merge this elution fractions.
4. column chromatography purification: by elutriant after being concentrated into proper volume, be prepared purifying with dextrane gel SephadexLH-20 post, with 85% methanol-eluted fractions, collect the elutriant containing salvianolic acid A, dry through concentrated removing methyl alcohol final vacuum, obtain salvianolic acid A product.It is 92.8% that HPLC records purity.
Embodiment 4:
1. extract: take 100g red rooted salvia medicine materical crude slice, add the 75 DEG C of heating extractions 2 hours under nitrogen protection of 1000ml water, extracts 2 times, second time adds water 800ml, and merging filtration obtains extracting solution.
2. transform: extracting solution is concentrated into 200ml, is placed in microwave reactor, under nitrogen protection 160 DEG C of reaction 0.1hr, stopped reaction.The content measuring salvianolic acid B in extracting solution is 2.46%, and in conversion fluid, the content of salvianolic acid A is 1.23%, and transformation efficiency is 72.7%.
3. column chromatography for separation: take 200gAB-8 type macroporous resin, fill post after pre-treatment.Conversion fluid is added above chromatography column, after entering post bed completely, wash 3 column volumes with water, discard this elution fractions; Use 70% ethanol elution, 3 column volumes again, merge this elution fractions.
4. column chromatography purification: elutriant is evaporated to proper volume, is prepared purifying with C18 bonded silica gel post, with 45% methanol-eluted fractions, collects the elutriant containing salvianolic acid A, through concentrated removing methyl alcohol postlyophilization, obtains salvianolic acid A product.It is 92.4% that HPLC records purity.
Embodiment 5:
1. extract: take 300g Salvia japonica Thunb. medicinal material, with the 90 DEG C of heating extractions 2 hours under argon shield of 3000ml water, extracts 2 times, second time adds water 2400ml, and merging filtration obtains extracting solution.
2. transform: extracting solution is concentrated into 600ml, is placed in microwave reactor, 110 DEG C of reaction 5hr under argon shield, stopped reaction.The content measuring salvianolic acid B in extracting solution is 1.06%, and in conversion fluid, the content of salvianolic acid A is 0.45%, and transformation efficiency is 61.7%.
3. column chromatography for separation: take 600gD101 type macroporous resin, fill post after pre-treatment.Conversion fluid is added above chromatography column, after entering post bed completely, wash 3 column volumes with water, discard this elution fractions; Use 40% ethanol elution, 5 column volumes again, merge this elution fractions.
4. column chromatography purification: be prepared purifying with MCIGELCHP20P post, with 35% ethanol elution, collects the elutriant containing salvianolic acid A, and spraying dry after concentrated removing ethanol, obtains salvianolic acid A product.It is 93.3% that HPLC records purity.
Embodiment 6:
1. extract: take 200g Yunnan Salvia japonica Thunb. medicinal material, with the 70 DEG C of heating extractions 2 hours under argon shield of 2000ml water, extracts 2 times, second time adds water 1600ml, and merging filtration obtains extracting solution.
2. transform: extracting solution is concentrated into 400ml, is placed in autoclave, under argon shield 112 DEG C, react 6hr, stopped reaction under 0.15Mpa.The content measuring salvianolic acid B in extracting solution is 1.27%, and in conversion fluid, the content of salvianolic acid A is 0.64%, and transformation efficiency is 73.2%.
3. column chromatography for separation: take 400gDM130 type macroporous resin, fill post after pre-treatment.Conversion fluid is added above chromatography column, after entering post bed completely, wash three column volumes with water, discard this elution fractions; Use 70% ethanol elution, 5 column volumes again, merge this elution fractions.
4. column chromatography purification: elutriant is evaporated to proper volume, is prepared purifying with C4 bonded-phase silica post, with 65% methanol-eluted fractions, collects the elutriant containing salvianolic acid A, through concentrated removing methyl alcohol postlyophilization, obtains salvianolic acid A product.It is 90.7% that HPLC records purity.
Embodiment 7:
1. extract: take 500g Salvia przewalskii medicinal material, with the 80 DEG C of heating extractions 2 hours under nitrogen protection of 5000ml water, extracts 2 times, second time adds water 4000ml, and merging filtration obtains extracting solution.
2. transform: extracting solution is concentrated into 1000ml, is placed in autoclave, under argon shield 122 DEG C, 0.21Mpa reacts 3hr, stopped reaction.The content measuring salvianolic acid B in extracting solution is 1.55%, and in conversion fluid, the content of salvianolic acid A is 0.72%, and transformation efficiency is 67.5%.
3. column chromatography for separation: take 1kgAB-8 type macroporous resin, fill post after pre-treatment.Conversion fluid is added above chromatography column, after entering post bed completely, wash three column volumes with water, discard this elution fractions; Use 70% methanol-eluted fractions, 3 column volumes again, merge this elution fractions.
4. column chromatography purification: elutriant is evaporated to proper volume, is prepared purifying with polyamide column, with 40% methanol-eluted fractions, collects the elutriant containing salvianolic acid A, through concentrated removing methyl alcohol postlyophilization, obtains salvianolic acid A product.It is 94.7% that HPLC records purity.
Embodiment 8:
1. extract: take 150g Salvia cavaleriei medicinal material, with the 95 DEG C of heating extractions 2 hours under argon shield of 1500ml water, extracts 2 times, second time adds water 1200ml, and merging filtration obtains extracting solution.
2. transform: extracting solution is concentrated into 300ml, is placed in autoclave, under argon shield 102 DEG C, 0.11Mpa reacts 12hr, stopped reaction.The content measuring salvianolic acid B in extracting solution is 3.28%, and in conversion fluid, the content of salvianolic acid A is 1.46%, and transformation efficiency is 64.7%.
3. column chromatography for separation: take 300g860021 type macroporous resin, fill post after pre-treatment.Conversion fluid is added above chromatography column, after entering post bed completely, wash three column volumes with water, discard this elution fractions; Use 75% ethanol elution, 4 column volumes again, merge this elution fractions.
4. column chromatography purification: elutriant is evaporated to proper volume, is prepared purifying with C8 bonded-phase silica post, with 50% methanol-eluted fractions, collects the elutriant containing salvianolic acid A, through concentrated removing methyl alcohol postlyophilization, obtains salvianolic acid A product.It is 93.4% that HPLC records purity.
Embodiment 9:
1. extract: take 200g Salvia miltiorrhiza f. medicinal material, add the 70 DEG C of heating extractions 2 hours under argon shield of 2000ml water, extracts 2 times, second time adds water 1600ml, and merging filtration obtains extracting solution.
2. transform: extracting solution is concentrated into 400ml, is placed in autoclave, 135 DEG C under nitrogen protection, 0.31Mpa reacts 2hr, stopped reaction.The content measuring salvianolic acid B in extracting solution is 0.95%, and in conversion fluid, the content of salvianolic acid A is 0.47%, and transformation efficiency is 71.9%.
3. column chromatography for separation: take 400gDM-18 type macroporous resin, fill post after pre-treatment.Conversion fluid is added above chromatography column, after entering post bed completely, wash three column volumes with water, discard this elution fractions; Use 60% ethanol elution, 4 column volumes again, merge this elution fractions.
4. column chromatography purification: elutriant is evaporated to proper volume, is prepared purifying with C18 post, with 45% ethanol elution, collects the elutriant containing salvianolic acid A, and spraying dry after concentrated removing ethanol, obtains salvianolic acid A product.It is 96.2% that HPLC records purity.
Embodiment 10:
1. extract: take 100g threeleaf sage root medicinal material, with the 100 DEG C of heating extractions 2 hours under nitrogen protection of 1000ml water, extracts 2 times, second time adds water 800ml, and merging filtration obtains extracting solution.
2. transform: extracting solution is concentrated into 200ml, is placed in autoclave, argon shield, 152 DEG C, react 0.1hr, stopped reaction under 0.52Mpa.The content measuring salvianolic acid B in extracting solution is 0.86%, and in conversion fluid, the content of salvianolic acid A is 0.41%, and transformation efficiency is 69.3%.
3. column chromatography for separation: take 200gD312 type macroporous resin, fill post after pre-treatment.Conversion fluid is added above chromatography column, after entering post bed completely, wash three column volumes with water, discard this elution fractions; Use 75% methanol-eluted fractions, 4 column volumes again, merge this elution fractions.
4. column chromatography purification: elutriant is evaporated to proper volume, is prepared purifying with C8 bonded silica gel post, with 55% methanol-eluted fractions, collects the elutriant containing salvianolic acid A, through concentrated removing methyl alcohol postlyophilization, obtains salvianolic acid A product.It is 92.7% that HPLC records purity.
Embodiment 11:
1. extract: take 100g root of Unileaf sage. medicinal material, add the 100 DEG C of heating extractions 2 hours under argon shield of 1000ml water, extracts 2 times, second time adds water 800ml, and merging filtration obtains extracting solution.
2. transform: extracting solution is concentrated into 300ml, is placed in autoclave, argon shield, 145 DEG C, react 0.5hr, stopped reaction under 0.41Mpa.The content measuring salvianolic acid B in extracting solution is 2.26%, and in conversion fluid, the content of salvianolic acid A is 1.07%, and transformation efficiency is 68.8%.
3. column chromatography for separation: take 200gD312 type macroporous resin, fill post after pre-treatment.Conversion fluid is added above chromatography column, after entering post bed completely, wash three column volumes with water, discard this elution fractions; Use 75% methanol-eluted fractions, 3 column volumes again, merge this elution fractions.
Drop counter current chromatography purifying: elutriant is evaporated to proper volume, purifying is prepared with high-speed counter-current chromatograph (HSCCC), with hexane-ethylacetate-methanol-water 4: 6: 5: 5 for solvent systems, using upper strata wherein as moving phase, lower floor is as stationary phase, collect the elutriant that salvianolic acid A purity is higher, spraying dry after concentrated removing organic solvent, obtains salvianolic acid A product.It is 96.1% that HPLC records purity.
Claims (4)
1. from several salvia, obtain a preparation method for salvianolic acid A, it is characterized in that comprising the steps:
(1) extract: the section of selected plant root and rhizome is placed in encloses container, add water dipped medicine materical crude slice surface, and lower more than the 50 DEG C heating of oxygen free condition are extracted, and can obtain the extracting solution being rich in salvianolic acid B;
(2) transform: after extracting solution is evaporated to proper volume, under oxygen free condition, normal pressure is in 100 DEG C of back flow reaction, or is placed in autoclave or microwave reactor reacting by heating, can obtain the conversion fluid being rich in salvianolic acid A;
(3) salvianolic acid A conversion fluid the present invention obtained uses conventional isolation techniques further, as technology purifying such as macroporous resin, reversed-phase silica gel chromatography, dextrane gel SephadexLH-20, drop counter current chromatographies, the salvianolic acid A product that content reaches more than 90% can be obtained.The method technological process is simple, the salvianolic acid A yield of preparation and purity high, be applicable to suitability for industrialized production.
2. the preparation method of a kind of salvianolic acid A according to claim 1, is characterized in that described several plant comprises: the red sage root (SalviamiltiorrhizaBunge); Radix Salviae Miltiorrhizae (SalviamiltiorrhizaBungefalbaC.Y.WuetH.W.Li); Salvia bowleyana Dunn (SalviabowleyanaDunn); Intend the red sage root (SalviasinicaMigo); Anhui, the pale reddish brown Zhejiang red sage root (SalviasinicaMigof.purpureaH.W.Li); Salvia japonica Thunb. (SalviajaponicaThunb); Yunnan Salvia japonica Thunb. (SalviayunnanensisC.H.Wright); Salvia przewalskii (SalviaprzewalskiiMaxim); Huangpu Salvia japonica Thunb. (SalviaprionitisHance); Field bund Salvia japonica Thunb. (SalviascapiformisHance); Brown hair Salvia przewalskii (SalivaprzewalskiiMaxim.var.mandarinorum (Diels) Stib); Threeleaf sage root (SalviatrijugaDiels.); Salvia chinensis (SalviachinensisBenth); Salvia cavaleriei (SalviacavalerieiL é vl.); Root of Unileaf sage. (SalviacavalerieiL é vl.Var.simplicifoliaStib); Arisaema balansae Engl. grass (SalviakwangsiensisH.W.Li); Salvia miltiorrhiza f. (SalviamiltiorrhizaBungevar.albaC.Y.WuetH.W.Li).
3. the preparation method of a kind of salvianolic acid A according to claim 1, is characterized in that oxygen free condition adopts the method for nitrogen or argon shield to carry out, preferred argon shield.
4. the preparation method of a kind of salvianolic acid A according to claim 1, the method for wherein said mensuration salvianolic acid A and content of danshinolic acid B adopts high performance liquid chromatography, and condition determination is as follows:
(1) with octadecylsilane key and silica gel for stationary phase, determined wavelength: 286nm; Detector: PAD; Flow velocity: 1ml/min; Column temperature 30 DEG C; Theoretical plate number should be not less than 3000 by salvianolic acid A;
(2) preparation of reference substance solution: precision takes salvianolic acid A and salvianolic acid B 20mg, accurately weighed, is placed in 10mL measuring bottle, is diluted to scale with dissolve with methanol, shakes up; Precision measures 1ml solution, be placed in 10mL measuring bottle, is diluted to scale, shakes up, obtain reference substance solution with dissolve with methanol;
(3) need testing solution preparation: precision measures and is equivalent to 20mg salvianolic acid A and salvianolic acid B sample in 100ml volumetric flask, adds methanol dilution, constant volume, crosses 0.22 μm of millipore filtration, as need testing solution;
(4) wash-out take acetonitrile as mobile phase A, with 0.1 ~ 0.67% formic acid for Mobile phase B, carries out gradient elution by following condition, runs 40min;
0 ~ 10min, maintenance acetonitrile ratio is the ratio of 15%, 0.1 ~ 0.67% aqueous formic acid is 85%;
10 ~ 25min, acetonitrile ratio rises to 25% by 15%, and the ratio of 0.1 ~ 0.67% aqueous formic acid drops to 75% by 85%;
25 ~ 40min, the ratio keeping acetonitrile ratio 25%, 0.1 ~ 0.67% aqueous formic acid is 75%;
(5) assay method: accurate absorption reference substance solution and each 20 μ l of need testing solution respectively, injection liquid chromatography, measures content.
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| CN109528825A (en) * | 2018-12-18 | 2019-03-29 | 江西赣隆药业有限公司 | A kind of salvia bowleyana Dunn granule and preparation method thereof with activating microcirculation and removing stasis medicinal and menstruction regulating and pain relieving |
| CN109748793A (en) * | 2018-12-29 | 2019-05-14 | 正大青春宝药业有限公司 | A method of different salviandic acid A 1 and different salviandic acid A 2 in removal salviandic acid A sodium |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106431915A (en) * | 2016-09-07 | 2017-02-22 | 山东省分析测试中心 | Method for preparing salvianolic acid A |
| CN106431923A (en) * | 2016-09-07 | 2017-02-22 | 山东省分析测试中心 | Subcritical-water-conversion and zone-refining-counter-current-chromatography-separation combined preparing method for danshinolic acid A |
| CN106431923B (en) * | 2016-09-07 | 2019-04-19 | 山东省分析测试中心 | The salviandic acid A preparation method that subcritical water conversion combined area is separated with adverse current chromatogram |
| CN106431915B (en) * | 2016-09-07 | 2019-04-23 | 山东省分析测试中心 | A kind of preparation method of salviandic acid A |
| CN109528825A (en) * | 2018-12-18 | 2019-03-29 | 江西赣隆药业有限公司 | A kind of salvia bowleyana Dunn granule and preparation method thereof with activating microcirculation and removing stasis medicinal and menstruction regulating and pain relieving |
| CN109748793A (en) * | 2018-12-29 | 2019-05-14 | 正大青春宝药业有限公司 | A method of different salviandic acid A 1 and different salviandic acid A 2 in removal salviandic acid A sodium |
| CN109748793B (en) * | 2018-12-29 | 2021-07-09 | 正大青春宝药业有限公司 | Method for removing isosalvianolic acid A1 and isosalvianolic acid A2 from sodium salvianolic acid A |
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