CN102249920A - Preparation method of high-purity salvianolic acid A - Google Patents
Preparation method of high-purity salvianolic acid A Download PDFInfo
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Abstract
The invention relates to a preparation method of high-purity salvianolic acid A. The preparation method is characterized in that: by taking lithospermum as a raw material, high-purity salvianolic acid A is obtained through the processes of water heating and extraction, acid regulation and transformation, resin absorption, elution and separation as well as purification and concentration. In the preparation method, the lithospermum is used as the raw material for preparing the salvianolic acid A, thereby expanding the sources of medicinal raw materials, saving the original salvia mitiorrhiza material and greatly reducing the use cost of the raw material; and simultaneously, the preparation method of the salvianolic acid A by taking the lithospermum as the raw material has a simple technological process and is suitable for large scale production, and the salvianolic acid A prepared by the method has high yield and purity and low cost.
Description
Technical field
The invention belongs to the medicinal plants field, particularly relate to the preparation method of a kind of high-purity danshinolic acid A.
Background technology
The red sage root is the Labiatae salvia, as the existing long clinical application history of China's conventional Chinese medicine material, has consequence in the treatment of cardiovascular disorder, and its significant curative effect has obtained extensive approval clinically.The active chemical of the red sage root mainly contains two big classes: fat-soluble tanshinone compound and water-soluble pressure differential self.Salvianolic acid has effect such as anti peroxidation of lipid and removing free radical widely, and the anti-oxidant activity of salvianolic acid compounds is the strongest in known phenolic compound, and is wherein the strongest with salviol acid A (abbreviation salvianolic acid A) anti-oxidant activity again.Salvianolic acid A has good liposoluble and saturating film ability, compares with most of phenolic acid in the red sage root, and its Premeabilisation of cells ability is stronger, and bioavailability is higher.Therefore, salvianolic acid A has very high medicinal exploitation value.
But, the natural content of the salvianolic acid A in the red sage root extremely low (be about red rooted salvia 0.01～0.06%), not only the crude drug cost is too high to extract salvianolic acid A from the red sage root, and the separation and purification difficulty is also big, seriously restrict the research and development of this product, becoming the bottleneck of its industrialization.
For solving the source problem that comes of salvianolic acid A, people attempt from the red sage root salvianolic acid A being transformed in recent years, find can obtain a certain amount of salvianolic acid A by the degraded of salvianolic acid B, and salvianolic acid B also are effective constituents important in the red sage root.Disclosing a kind of preparation method of danshen root salvianolic acid A as Chinese patent 200710001055.4, is by the salvianolic acid B degradation treatment is obtained salvianolic acid A.Owing to also contain important fat-soluble effective constituent such as Tanshinone II A, Cryptotanshinone etc. in the red sage root, obviously, the way that only obtains salvianolic acid A to sacrifice the numerous effective constituent of the red sage root does not meet scientific and reasonable, as to make full use of resource theory.In addition, this flavor medicinal material purposes of the red sage root is a lot, and a lot of Chinese patent medicines such as Radix Salviae Miltiorrhizae Injection, FUFANG DANSHEN PIAN, FUFANG DANSHEN DIWAN etc. all need to extract with the red sage root, need to consume a large amount of red rooted salvias.
In addition, point out in related discipline scholar's research: the cultivation of the red sage root is not easy to, and red sage root happiness is grown in and has a moderate climate, place sunny, with the humid air, and the growth optimum temperuture is 20 ~ 26 ℃, and the suitableeest relative air humidity is 80%.Its well developed root system, the fearness drought is avoided waterlogging again.The red sage root plant appropriate to the occasion selection soil layer deep, loose fertile, physical features is higher, the plantation of well-drained sand loam plot.The waterways will be done in the level land; The mountain region, should select on the sunny side low hillside to the mountain range, the gradient should not be too big, 10 ~ 30 degree better, the soil pH value is between 6.5 ~ 7.5.And the unsuitable continuous cropping of the red sage root, by condition survey discovery that 1 ~ 4 year different continuous cropping time limits plot red sage root is grown, the growth of the continuous cropping serious harm red sage root, show that mainly vegetative period in 6 ~ September, withered seedling rate rose significantly, on the ground, the underground part increment descends, root quantity, diameter and length reduce, output reduces, root system outward appearance deformity, active constituent content reduces, and this has also influenced the output of the red sage root to a certain extent.
Therefore, the cultivation situation of the red sage root with and numerous purposes be doomed to make the red sage root supply falls short of demand, this point that the substantial appreciation of prices of the red sage root is verified in recent years.Make the preparation level of salvianolic acid A reach industrialization, must seek the source problem that comes that new resource solves salvianolic acid A.
Summary of the invention
Purpose of the present invention is intended to: provide a kind of salvianolic acid A that can obtain newly to originate, and the preparation method who therefrom obtains high-purity danshinolic acid A; Both solved the source problem that comes of salvianolic acid A, the while also provides a kind of yield height, cost is low, preparation process is simple, is beneficial to the processing method that high-purity danshinolic acid A is produced in industrialization.
The preparation method of this high-purity danshinolic acid A is characterized in that: it is raw material with the Asian puccoon, extracts and turning of acid, resin absorption, wash-out separation and purifying enrichment process acquisition high-purity danshinolic acid A by the water heating.
Concrete preparation method comprises the steps: that extract (1), (2) transform, and separate (3), (4) purifying;
(1) extract: is that secondary is extracted in heating at least in 70 ~ 100 ℃ the water with the section of medicinal material Asian puccoon root and rhizome in temperature, merges to filter the back and obtain extracting solution;
(2) transform: the extracting solution appropriateness is concentrated, and be that the inorganic acid for adjusting pH value of 0.1 ~ 2mol/L is to 1-6,100 ℃ of following back flow reaction with concentration; Or concentrated solution placed the autoclave reacting by heating, obtain to contain the conversion fluid of high density salvianolic acid A;
(3) separate: the adsorption column of conversion fluid is nonpolar with having filled, low-pole or Semi-polarity macroporous adsorbent resin adsorbs, after treating adsorption equilibrium, water carries out wash-out and removes impurity, separate with water/alcoholic solvent system wash-out then, collects the elutriant that i.e. acquisition behind 3～5 column volumes contains salvianolic acid A;
(4) purifying: this elutriant is gone up polymeric amide, dextrane gel Sephadex LH-20, MCI GEL CHP20P, C4, C8 or the preparation of C18 bonding phase silicagel column after concentrating, with water/alcoholic solvent system wash-out, or with the drop counter current chromatography purifying, collection contains stream part of high-purity danshinolic acid A, obtains high-purity danshinolic acid A product behind concentrate drying.
The mineral acid that adopts in the described step (2) is any acid in hydrochloric acid, sulfuric acid, nitric acid, the phosphoric acid; The following reaction times of reflux state is 2 ~ 24 hours; Reacting by heating condition in autoclave is: 101 ~ 152 ℃ of temperature, and pressure 0.1 ~ 0.5Mpa, the reaction times is 0.5 ~ 12h.
Nonpolar, low-pole that adopts in the described step (3) or Semi-polarity macroporous adsorbent resin, AB-8 for the production of sea light chemical plant, Tianjin, a kind of in D101, the DM301 polymeric adsorbent, or the anti-resin processing plant in Shandong, Shandong produce 330, a kind of in CAD45, DM130,860021, DM-18, the D312 type polymeric adsorbent.
Water is pressed by water in described step (3), (4)/alcoholic solvent system: alcohol=configuration in 95: 5 ~ 5: 95.
Drying means in the described step (4) comprises: lyophilize, spraying drying or vacuum-drying.
The preparation method of this kind high-purity danshinolic acid A that proposes according to above method, by selecting the starting material of medicinal material Asian puccoon for use as the preparation salvianolic acid A, not only expand medicinal raw-material source, saved original red sage root material, and can reduce raw-material use cost greatly; Simultaneously this Asian puccoon is the preparation method of the salvianolic acid A of raw material, and technological process is simple, and the salvianolic acid A yield of preparation and purity height, cost are low, are fit to scale operation.
Description of drawings
Fig. 1 is the molecular structural formula of alkannic acid in the Asian puccoon of the present invention;
Fig. 2 is the molecular structural formula of salvianolic acid A of the present invention;
Fig. 3 is converted into the transformation mechanism figure of salvianolic acid A molecule for the alkannic acid molecule;
Fig. 4 is the liquid chromatogram that Asian puccoon obtains through extraction with aqueous solution;
Fig. 5 is that shikonin extract is through transforming the liquid chromatogram that obtains.
Embodiment
The Chinese medicinal materials Asian puccoon that utilizes that the present invention proposes is extracted the method for high-purity danshinolic acid A, solved at present and can only extract shortage of resources and the high predicament of resource valency that salvianolic acid A exists, proposed the resource evolutionary path for heavy industrialization obtains high-purity danshinolic acid A from the red sage root.This technical scheme is utilized the abundant total phenolic acid of Asian puccoon (mainly being alkannic acid) that belongs to the coffic acid polymkeric substance of content in the Asian puccoon, changes into salvianolic acid A efficiently under certain processing condition.This conversion utilizes mineral acid to realize that the mechanism of its conversion is represented by following molecular structural formula under certain processing condition:
In above-mentioned expression formula, the levoform on top is the alkannic acid molecular structural formula, and the bottom levoform is the molecular structural formula of salvianolic acid A, and the right formula on top is the alkannic acid molecular structure produces structural changes in the mineral acid treatment process a synoptic diagram.The process of its variation is: the alkannic acid molecule heats under acidic conditions, it is unstable that furan nucleus is wherein met acid, ehter bond in the ring is easy to hydrolysis, addition reaction takes place, generate a fat hydroxyl and a phenolic hydroxyl group respectively after making the Sauerstoffatom addition on the furan nucleus, fat hydroxyl and adjacent hydrogen take place to eliminate reaction under acid hot conditions then, slough a part water, slough a part carbonic acid gas after carboxyl on the former furan nucleus dissociates simultaneously, generate the salvianolic acid A molecule.
In the contriver studies for many years, the discovery Asian puccoon (
Lithospermum erythrorhizon) in contain abundant phenolic acid composition, wherein the content with alkannic acid is the highest, can reach 4 ~ 7%, and the total phenolic acid of Asian puccoon belongs to the coffic acid polymkeric substance,, the total phenolic acid of Asian puccoon (mainly being alkannic acid) can be changed into salvianolic acid A efficiently under certain condition.Because Asian puccoon is stronger to the natural condition adaptive faculty, general soil all can be grown, and is cold-resistant, be afraid of waterlogging, be born under mountain region thick grass and exsiccant stone matter hillside, mountain valley and the scrub growth more, soil is required very not strict, the germination optimum temperuture is 13～17 ℃, and percentage of germination can reach more than 80%, more easily cultivation.Extract from Asian puccoon and transform salvianolic acid A, yield can reach about 2%, has solved the source problem that comes of salvianolic acid A preferably.
This method of utilizing the Chinese medicinal materials Asian puccoon to obtain high-purity danshinolic acid A, its concrete steps are: extract (1), (2) transform, and separate (3), four steps of (4) purifying.
(1) extract: is that secondary is extracted in heating at least in 70 ~ 100 ℃ the water with the section of medicinal material Asian puccoon root and rhizome in temperature, merges to filter the back and obtain extracting solution;
(2) transform: the extracting solution appropriateness is concentrated, and be that the inorganic acid for adjusting pH value of 0.1 ~ 2mol/L is to 1-6,100 ℃ of following back flow reaction with concentration; Or concentrated solution placed the autoclave reacting by heating, obtain to contain the conversion fluid of high density salvianolic acid A;
(3) separate: the adsorption column of conversion fluid is nonpolar with having filled, low-pole or Semi-polarity macroporous adsorbent resin adsorbs, after treating adsorption equilibrium, water carries out wash-out and removes impurity, separate with water/alcoholic solvent system wash-out then, collects the elutriant that i.e. acquisition behind 3～5 column volumes contains salvianolic acid A;
(4) purifying: this elutriant is gone up polymeric amide, dextrane gel Sephadex LH-20, MCI GEL CHP20P, C4, C8 or the preparation of C18 bonding phase silicagel column after concentrating, with water/alcoholic solvent system wash-out, or with the drop counter current chromatography purifying, collection contains stream part of high-purity danshinolic acid A, obtains high-purity danshinolic acid A product behind concentrate drying.
Used mineral acid is any acid in hydrochloric acid, sulfuric acid, nitric acid, the phosphoric acid in the described step (2); The following reaction times of reflux state is 2 ~ 24 hours; Reacting by heating condition in autoclave is: 101 ~ 152 ℃ of temperature, and pressure 0.1 ~ 0.5Mpa, the reaction times is 0.5 ~ 12h.
Nonpolar, the low-pole of described step (3) or Semi-polarity macroporous adsorbent resin, AB-8 for the production of sea light chemical plant, Tianjin, a kind of in D101, the DM301 polymeric adsorbent, or the anti-resin processing plant in Shandong, Shandong produce 330, a kind of in CAD45, DM130,860021, DM-18, the D312 type polymeric adsorbent.
Water is pressed by water in described step (3), (4)/alcoholic solvent system: alcohol=configuration in 95: 5 ~ 5: 95.
Detect (accompanying drawing 4,5) through HPLC, Asian puccoon transforms according to this technology after extracting, and alkannic acid wherein transforms into salvianolic acid A substantially.As seen from Figure 4, Asian puccoon is in the liquid chromatogram that extraction with aqueous solution obtains, and alkannic acid (retention time is 8.67 minutes peak) is a main component wherein; Fig. 5 is that shikonin extract is through transforming the liquid chromatogram that obtains.As seen, after transforming, the peak of former 8.67 minutes alkannic acids disappears substantially among the figure, and the main peak that salvianolic acid A occurs at 15.69 minutes shows that the alkannic acid major part changes into salvianolic acid A.The high-purity danshinolic acid A product that obtains adopts high-performance liquid chromatography method content, and the result shows that content is that yield is not less than 1.6% more than 90%.
The following pharmacodynamic experiment that the salvianolic acid A that obtains from Asian puccoon is carried out shows that salvianolic acid A has the better protecting effect to cardiovascular and cerebrovascular diseases.
Experimental technique:
Get 100 of Kunming mouses, male and female half and half, be divided into 5 groups at random, i.e. control group (giving the isometric(al) physiological saline of (containing N.F,USP MANNITOL 64.0mg/kg)), red sage root group (Radix Salviae Miltiorrhizae Injection 10000mg/kg), salvianolic acid A basic, normal, high (5,10,20mg/kg).Each group is all by the tail intravenously administrable, and the administration volume is 20ml/kg.Behind the intravenously administrable 10min, mouse is placed the airtight wide-necked bottle of 125ml (built-in sodica calx 20g).Stopping with the mouse mouth breathing is the dead mouse sign, the survival time of record mouse, surpass 60min in 60min.Experimental result sees Table 1.
Table 1 salvianolic acid A is to the provide protection of mouse normal pressure hypoxia tolerance
Annotate: compare with control group:
*P＜0.05,
*P＜0.01.
The animal model preparation:
(1), and lies on the back and be fixed on the operating table with 1.2% vetanarcol (i.p.) anesthetized rat;
(2), expose glandula submandibularis in neck median incision.Passivity is separated left and right sides glandula submandibularis, and the right side body of gland is pulled to side, exposes carotid triangle;
(3) passivity is isolated arteria carotis communis, notes not damaging vagus nerve;
(4) with 4#0 surgical thread ligation arteria carotis communis proximal part, clamp carotid artery distal end, temporary interruption blood flow with bulldog clamp;
(5) cut off a little otch at the arteria carotis communis distal end, with the preparation the bolt line by otch through arteria carotis communis, enter arteria carotis communis, unclamp bulldog clamp, the line bolt is sent into cranium gently encircle inaccessible arteria cerebri media initial part to Willians,, cause the arteria cerebri media ischemic.Line bolt head end is apart from the about 1cm of arteria carotis communis crotch, the ligation otch, and skin suture, its end of a thread size that is fit to of the animal of different weight has certain difference;
(6) sham operated rats is not except that inserting the line bolt, and all the other processing are the same;
(7) the clear-headed laggard capable study of behaviour scoring of surgeon's knot band animal, 0 ~ 0.5 is divided into model failure animal, gets rid of and the final data statistics.
Infarct size is measured
(1) postoperative is 24 hours, behind the mensuration behavior scoring animal broken end is got brain;
(2) remove olfactory bulb, cerebellum and low brain stem, crown 5 cuttves of all cutting, totally 6;
(3) 6 cerebral tissues are placed in the 2%TTC solution, and 37 degree lucifuges are hatched 30min dyeing, and TTC can react with the Intramitochondrial succinodehydrogenase of active cells, generates red praising by the first moon, therefore is used to represent the vigor of cell;
(4) 10% formalin fixed 4h;
(5) take pictures, dyed back healthy tissues takes on a red color, and the infarct position is white in color;
(6) utilize the morphological analysis system-computed to go out infarct size, compare.
Above-mentioned experiment shows: salvianolic acid A has stronger cardiovascular and cerebrovascular activity, has very high medicinal exploitation and is worth.
Below in conjunction with specific embodiment, further set forth the present invention.
1, extract: take by weighing 2000g Asian puccoon medicinal material (root, stem section), heat under 80 ℃ of temperature condition with 10 times of water gagings (20000ml) solution and extracted 2 hours, extract 2 times, merging obtains extracting solution after filtering.
2, transform: extracting solution is concentrated into 4000ml, and regulating pH with 2mol/L hydrochloric acid is 3, backflow 7hr, and stopped reaction obtains to contain the conversion fluid of high density salvianolic acid A.
3, separate: take by weighing 2000g DM301 type macroporous resin, adorn post after the pre-treatment.Conversion fluid is added the chromatography column top, make it to enter fully the post bed.5 column volumes of water wash-out are removed impurity, with containing 3 column volumes of 70% alcoholic acid solvent elution, merge this part elutriant then, obtain to contain stream part of salvianolic acid A.
4, purifying: should flow part and be evaporated to proper volume, and be prepared purifying, and, collect stream part of containing salvianolic acid A, and remove the methyl alcohol postlyophilization, and obtain high-purity danshinolic acid A product through concentrating with 40% methanol-eluted fractions with C18 bonded silica gel post; It is 98.6% that product pick-up rate 1.6%, HPLC record purity.
Embodiment 2. (is example with 2000g Asian puccoon medicinal material)
1, extracts: take by weighing 2000g Asian puccoon medicinal material (root, stem are cut into slices), heat down at 100 ℃ with 10 times of water gaging solution and extracted 2 hours, extract 2 times, merge filtration and obtain extracting solution.
2, transform: extracting solution is concentrated into 4000ml, and regulating pH with 0.5mol/L sulfuric acid is 5, backflow 5hr, stopped reaction.
3, separate: take by weighing 2000g 330 type macroporous resins, adorn post after the pre-treatment.Conversion fluid is added the chromatography column top, make it to enter fully the post bed.5 column volumes of water wash-out are removed impurity, with containing 4 column volumes of 50% alcoholic acid solvent elution, merge this part elutriant then, obtain to contain stream part of salvianolic acid A.
4, purifying: should flow part through after being concentrated into proper volume, and be prepared purifying, and, collect stream part of containing salvianolic acid A, and remove the ethanol postlyophilization, and obtain high-purity danshinolic acid A product through concentrating with 45% ethanol elution with polyamide column; Yield 2.5%, it is 92.4% that HPLC records purity.
1, extracts: take by weighing 1000g Asian puccoon medicinal material (root, stem section), heat under 85 ℃ of temperature with 10 times of water gaging solution and extracted 2 hours, extract 2 times, merge filtration and obtain extracting solution.
2, transform: extracting solution is concentrated into 2000ml, and regulating pH with 0.2mol/L nitric acid is 6, backflow 24hr, stopped reaction.
3, separate: take by weighing 1000g CAD45 type macroporous resin, adorn post after the pre-treatment.Conversion fluid is added the chromatography column top, make it to enter fully the post bed.5 column volumes of water wash-out are removed impurity, use 3 column volumes of 60% methanol-eluted fractions then, merge this part elutriant, obtain to contain stream part of salvianolic acid A.
4, purifying: should flow part through after being concentrated into proper volume, be prepared purifying with dextrane gel Sephadex LH-20 post,, collect stream part of containing salvianolic acid A with 85% methanol-eluted fractions, remove methyl alcohol final vacuum drying through concentrating, obtain high-purity danshinolic acid A product; Obtain yield 2.8%, it is 90.8% that HPLC records purity.
Embodiment 4 (is example with 3000g Asian puccoon medicinal material)
1, extracts: take by weighing 3000g Asian puccoon medicinal material (root, stem are cut into slices), heat down at 75 ℃ with 10 times of water gaging solution and extracted 2 hours, extract 2 times, merge filtration and obtain extracting solution.
2, transform: extracting solution is concentrated into 6000ml, and regulating pH with 1mol/L phosphoric acid is 1.0, backflow 2hr, stopped reaction.
3, separate: take by weighing 3000g D101 type macroporous resin, adorn post after the pre-treatment.Conversion fluid is added the chromatography column top, make it to enter fully the post bed.5 column volumes of water wash-out are removed impurity, use 5 column volumes of 40% ethanol elution then, merge this part elutriant, obtain to contain stream part of salvianolic acid A.
4, purifying: should flow part through after being concentrated into proper volume, be prepared purifying,, collect stream part of containing salvianolic acid A with 35% ethanol elution with MCI GEL CHP20P post, through concentrated remove ethanol after spraying drying, obtain high-purity danshinolic acid A product.Yield 2.2%, it is 93.1% that HPLC records purity.
Embodiment 5 (is example with 200g Asian puccoon medicinal material)
1, extracts: take by weighing 200g Asian puccoon medicinal material (root, stem are cut into slices), heat down at 90 ℃ with 10 times of water gaging solution and extracted 2 hours, extract 2 times, merge filtration and obtain extracting solution.
2, transform: extracting solution is concentrated into 400ml, and regulating pH with 1mol/L hydrochloric acid is 5.5, reacts 3hr, stopped reaction under 112 ℃, 0.15Mpa.
3, separate: take by weighing 200g DM130 type macroporous resin, adorn post after the pre-treatment.Conversion fluid is added the chromatography column top, make it to enter fully the post bed.5 column volumes of water wash-out are removed impurity, use 5 column volumes of 70% ethanol elution then, merge this part elutriant, obtain to contain stream part of salvianolic acid A.
4, purifying: should flow part and be evaporated to proper volume, and be prepared purifying, and separate, and collect stream part of containing salvianolic acid A, and remove the methyl alcohol postlyophilization, and obtain high-purity danshinolic acid A product through concentrating with 65% methanol-eluted fractions with C4 bonding phase silicagel column.Yield 2.6%, it is 90.3% that HPLC records purity.
Embodiment 6 (is example with 500g Asian puccoon medicinal material)
1, extracts: take by weighing 500g Asian puccoon medicinal material (root, stem are cut into slices), heat down at 70 ℃ with 10 times of water gaging solution and extracted 2 hours, extract 2 times, merge filtration and obtain extracting solution.
2, transform: extracting solution is concentrated into 1000ml, and regulating pH with 0.5mol/L sulfuric acid is 6, reacts 8hr, stopped reaction under 121 ℃, 0.2Mpa.
3, separate: take by weighing 500g AB-8 type macroporous resin, adorn post after the pre-treatment.Conversion fluid is added the chromatography column top, make it to enter fully the post bed.5 column volumes of water wash-out are removed impurity, separate 3 column volumes with 70% methanol-eluted fractions then, merge this part elutriant, obtain to contain stream part of salvianolic acid A.
4, purifying: should flow part and be evaporated to proper volume, and be prepared purifying, and, collect stream part of containing salvianolic acid A, and remove the methyl alcohol postlyophilization, and obtain high-purity danshinolic acid A product through concentrating with 40% methanol-eluted fractions with polyamide column; Obtain yield 1.9%, it is 95.6% that HPLC records purity.
Embodiment 7 (is example with 150g Asian puccoon medicinal material)
1, extract: take by weighing 150g Asian puccoon medicinal material, heat down at 80 ℃ with 10 times of water gaging solution and extracted 2 hours, extract 2 times, merging is filtered and is obtained extracting solution.
2, transform: extracting solution is concentrated into 300ml, and regulating pH with 2mol/L phosphoric acid is 3.5, reacts 12hr, stopped reaction under 101 ℃, 0.1Mpa.
3, separate: take by weighing 200g 860021 type macroporous resins, adorn post after the pre-treatment.Conversion fluid is added the chromatography column top, make it to enter fully the post bed.5 column volumes of water wash-out are removed impurity, use 4 column volumes of 75% ethanol elution then, merge this part elutriant, obtain to contain stream part of salvianolic acid A.
4, purifying: should flow part and be evaporated to proper volume, and be prepared purifying, and, collect stream part of containing salvianolic acid A, and remove the methyl alcohol postlyophilization, and obtain high-purity danshinolic acid A product through concentrating with 50% methanol-eluted fractions with C8 bonding phase silicagel column; It is 92.6% that pick-up rate 2.0%, HPLC record purity.
Embodiment 8 (is example with 800g Asian puccoon medicinal material)
1, extracts: take by weighing 800g Asian puccoon medicinal material (root, stem are cut into slices), heat down at 80 ℃ with 10 times of water gaging solution and extracted 2 hours, extract 2 times, merge filtration and obtain extracting solution.
2, transform: extracting solution is concentrated into 1600ml, and regulating pH with 1mol/L phosphoric acid is 5.5, reacts 12hr, stopped reaction under 152 ℃, 0.5Mpa.
3, separate: take by weighing 800g DM-18 type macroporous resin, adorn post after the pre-treatment.Conversion fluid is added the chromatography column top, make it to enter fully the post bed.5 column volumes of water wash-out are removed impurity, use 4 column volumes of 60% ethanol elution then, merge this part elutriant, obtain to contain stream part of salvianolic acid A.
4, purifying: should flow and part be evaporated to proper volume, be prepared purifying,, collect stream part of containing salvianolic acid A with 45% ethanol elution with the C18 post, through concentrated remove ethanol after spraying drying, obtain high-purity danshinolic acid A product.Yield 1.8%, it is 98.5% that HPLC records purity.
Embodiment 9 (is example with 600g Asian puccoon medicinal material)
1, extracts: take by weighing 600g Asian puccoon medicinal material (root, stem are cut into slices), heat down at 100 ℃ with 10 times of water gaging solution and extracted 2 hours, extract 2 times, merge filtration and obtain extracting solution.
2, transform: extracting solution is concentrated into 1200ml, and regulating pH with 1mol/L sulfuric acid is 4.5, reacts 0.5hr, stopped reaction under 152 ℃, 0.5Mpa.
3, separate: take by weighing 600g D312 type macroporous resin, adorn post after the pre-treatment.Conversion fluid is added the chromatography column top, make it to enter fully the post bed.5 column volumes of water wash-out are removed impurity, use 3 column volumes of 75% methanol-eluted fractions then, merge this part elutriant, obtain to contain stream part of salvianolic acid A.
4, purifying: should flow part and be evaporated to proper volume, and be prepared purifying, and, collect stream part of containing salvianolic acid A, and remove the methyl alcohol postlyophilization, and obtain high-purity danshinolic acid A product through concentrating with 55% methanol-eluted fractions with C8 bonded silica gel post.Yield 2.1%, it is 95.7% that HPLC records purity.
Embodiment 10 (is example with 600g Asian puccoon medicinal material)
1, extracts: take by weighing 600g Asian puccoon medicinal material (root, stem are cut into slices), heat down at 100 ℃ with 10 times of water gaging solution and extracted 2 hours, extract 2 times, merge filtration and obtain extracting solution.
2, transform: extracting solution is concentrated into 1200ml, and regulating pH with 1mol/L hydrochloric acid is 5.5, reacts 1hr, stopped reaction under 152 ℃, 0.5Mpa.
3, separate: take by weighing 600g D312 type macroporous resin, adorn post after the pre-treatment.Conversion fluid is added the chromatography column top, make it to enter fully the post bed.5 column volumes of water wash-out are removed impurity, use 3 column volumes of 75% methanol-eluted fractions then, merge this part elutriant, obtain to contain stream part of salvianolic acid A.
4, purifying: should flow part and be evaporated to proper volume, (HSCCC) is prepared purifying with high-speed counter-current chromatograph, with hexane-ethyl acetate-methanol-water 4:6:5:5 is solvent systems, with wherein upper strata as moving phase, lower floor is as stationary phase, collection contains stream part of salvianolic acid A, through concentrate remove organic solvent after spraying drying, obtain high-purity danshinolic acid A product.Yield 2.4%, it is 98.7% that HPLC records purity.
Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.After the content of having read the present invention's instruction, 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 institute restricted portion equally.
Claims (6)
1. the preparation method of a high-purity danshinolic acid A, it is characterized in that: it is raw material with the Asian puccoon, extracts and turning of acid, resin absorption, wash-out separating treatment and purifying enrichment process obtain high-purity danshinolic acid A by the water heating.
2. the preparation method of a kind of high-purity danshinolic acid A as claimed in claim 1, concrete preparation method comprises the steps: that extract (1), (2) transform, separate (3), (4) purifying;
(1) extract: is that secondary is extracted in heating at least in 70 ~ 100 ℃ the water with the section of medicinal material Asian puccoon root and rhizome in temperature, merges to filter the back and obtain extracting solution;
(2) transform: the extracting solution appropriateness is concentrated, and be that the inorganic acid for adjusting pH value of 0.1 ~ 2mol/L is to 1-6,100 ℃ of following back flow reaction with concentration; Or concentrated solution placed the autoclave reacting by heating, obtain to contain the conversion fluid of high density salvianolic acid A;
(3) separate: the adsorption column of conversion fluid is nonpolar with having filled, low-pole or Semi-polarity macroporous adsorbent resin adsorbs, after treating adsorption equilibrium, water carries out wash-out and removes impurity, separate with water/alcoholic solvent system wash-out then, collects the elutriant that i.e. acquisition behind 3～5 column volumes contains salvianolic acid A;
(4) purifying: this elutriant is gone up polymeric amide, dextrane gel Sephadex LH-20, MCI GEL CHP20P, C4, C8 or the preparation of C18 bonding phase silicagel column after concentrating, with water/alcoholic solvent system wash-out, or with the drop counter current chromatography purifying, collection contains stream part of high-purity danshinolic acid A, obtains high-purity danshinolic acid A product behind concentrate drying.
3. the preparation method of a kind of high-purity danshinolic acid A as claimed in claim 2 is characterized in that: used mineral acid is any acid in hydrochloric acid, sulfuric acid, nitric acid, the phosphoric acid in the described step (2); The following reaction times of reflux state is 2 ~ 24 hours; Reacting by heating condition in autoclave is: 101 ~ 152 ℃ of temperature, and pressure 0.1 ~ 0.5Mpa, the reaction times is 0.5 ~ 12h.
4. the preparation method of a kind of high-purity danshinolic acid A as claimed in claim 2, it is characterized in that: nonpolar, the low-pole of described step (3) or Semi-polarity macroporous adsorbent resin, AB-8 for the production of sea light chemical plant, Tianjin, a kind of in D101, the DM301 polymeric adsorbent, or the anti-resin processing plant in Shandong, Shandong produce 330, a kind of in CAD45, DM130,860021, DM-18, the D312 type polymeric adsorbent.
5. the preparation method of a kind of high-purity danshinolic acid A as claimed in claim 2 is characterized in that: water is pressed by the water in described step (3), (4)/alcoholic solvent system: alcohol=configuration in 95: 5 ~ 5: 95.
6. the preparation method of a kind of high-purity danshinolic acid A as claimed in claim 1 is characterized in that: the drying means in the described step (4) comprises: lyophilize, spraying drying or vacuum-drying.
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| CN105085266A (en) * | 2014-05-14 | 2015-11-25 | 南京虹桥医药技术研究所 | Method for preparing salvianolic acid A from a plurality of salvia plants |
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