CN102464586A - Preparation method of danshinolic acid A - Google Patents
Preparation method of danshinolic acid A Download PDFInfo
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- CN102464586A CN102464586A CN2010105416513A CN201010541651A CN102464586A CN 102464586 A CN102464586 A CN 102464586A CN 2010105416513 A CN2010105416513 A CN 2010105416513A CN 201010541651 A CN201010541651 A CN 201010541651A CN 102464586 A CN102464586 A CN 102464586A
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Abstract
The invention relates to a preparation method of danshinolic acid A. The method comprises the following steps of: I, extracting: adding ethanol into red-rooted salvia root for extracting; II, performing crude separation: performing crude separation on danshinolic acid A with a chromatographic column filled with AB-8 macroporous adsorption resin; III, decoloring: decoloring with a polyamide column; IV, purifying: further purifying with an MCI-GEL reversed-phase fine separation column and a dextrangel LH-20 column; and V, drying and performing freeze drying.
Description
Technical field
The present invention relates to medical technical field, be specifically related to a kind of preparation method of salvianolic acid A.
Background technology
The red sage root is one of common drug in China's traditional medicine, has long clinical application historical.The chemical ingredients of the red sage root mainly can be divided into water soluble component and fat-soluble component two parts.It is the fat-soluble component aspect of representative that the early stage research of 20th century mainly concentrates on the TANSHINONES, through effort decades, obtains great achievement.From early 1980s; China pharmaceuticals researcher studies red sage root water soluble ingredient; At first reported the structure of red sage root water soluble ingredient Salvianic acidA; After this found tens of kinds of water soluble components successively, and clear and definite its chemical structure, prove that salvia-soluble effective constituent mainly is phenolic acid compound.From salvianolic acid compounds pharmacological research; Salvianolic acid has many-sided effect; For example, the myocardial cell injury that salvianolic acid A causes ischemia-reperfusion has the significant protection effect, and total salvianolic acid shows stronger ischemia resisting and pours into the arrhythmia effect again; Salvianolic acid A, salvianolic acid B and total salvianolic acid have provide protection to the brain injury that the mouse brain ischemia-reperfusion causes, can reduce MDA content in the cerebral tissue; The salvianolic acid anti thrombotic action; Salvianolic acid is to the provide protection of liver, kidney; Salvianolic acid has very strong antioxygenation, can remove superoxide anion and release basic radical, suppresses peroxidatic reaction of lipid, or the like.(Du Guanhua etc., preclinical medicine and clinical, 2000,20 (5): 10～14)
Salvianolic acid A is as the main active ingredient of the red sage root, studies morely recently, and relevant salvianolic acid A preparing method's Chinese patent has:
The extracting and purifying method of 200,710,070,553 1 kinds of salvianolic acid As
The preparation method of 200,710,001,055 1 kinds of danshen root salvianolic acid As
The process for purification of 200,810,120,784 1 kinds of salvianolic acid As
The preparation method of 200610165779 danshen root salvianolic acid As
The preparation method of 200,910,169,900 1 kinds of danshen root salvianolic acid As
The preparation method of 200610052442 danshen root salvianolic acid As
The process for extracting of 200,610,012,615 1 kinds of salvianolic acid As
The preparation method of 200,610,114,138 1 kinds of salviol acid As
The method of the Radix Salviae Miltiorrhizae extract of salvianolic acid A is rich in 200810000904 preparations
The control method of 200610048130 preparation salvianolic acid As
The preparation method of 200,710,099,618 1 kinds of danshen root salvianolic acid As
Summary of the invention
The present invention on the basis of existing technology, the preparation method improves to salvianolic acid A, through method of the present invention; Can prepare the high salvianolic acid A of purity with the industriallization means, reduce the purifying cost, avoid the use of poisonous organic reagent; Can adapt in the production; Simple to operate, good reproducibility, the monomer transfer rate is high.
Salvianolic acid A preparation method of the present invention may further comprise the steps:
Step 1, extraction
The red sage root adds extraction using alcohol;
Step 2, roughing out
Chromatographic column with being filled with the AB-8 macroporous adsorbent resin is carried out roughing out to salvianolic acid A;
Step 3, decolouring
Decolour with polyamide column;
Step 4, purifying
Be further purified with MCI-GEL anti-phase fine separation post and sephadex lh-20 post;
Lyophilize.
Salvianolic acid A preparation method of the present invention preferably may further comprise the steps:
Step 1, extraction
The red sage root adds extraction using alcohol, filters, and filtrating is concentrated into does not have the alcohol flavor, promptly gets the extracting solution that is rich in salvianolic acid A;
Step 2, roughing out
The extracting solution adding is filled with on the chromatographic column of AB-8 macroporous adsorbent resin, uses ethanol elution, detect to there not being blue spot with the thin layer chromatography board that is sprayed with iron trichloride simultaneously, collect elutriant, be concentrated into medicinal extract;
Step 3, decolouring
Concentrated extract is used dissolve with ethanol, adds in the polyamide column, and ethanol elution has a tangible yellow colour band on the visible post, with this colour band of thin layer chromatography board monitoring that is sprayed with iron trichloride, begins to receive from blue spot is arranged, and to there not being blue spot, is concentrated into medicinal extract;
Step 4, purifying
Medicinal extract is used dissolve with ethanol, and last MCI-GEL anti-phase fine separation post is used ethanol elution, detects with the thin layer chromatography board that is sprayed with iron trichloride; From there being blue spot to begin to receive, till not having a blue spot, collect elutriant, use dissolve with ethanol again after being concentrated into medicinal extract; Last sephadex lh-20 post is used ethanol elution, detects with the thin layer chromatography board that is sprayed with iron trichloride, begins to receive from blue spot is arranged; Till not having a blue spot, collect elutriant, being concentrated into does not have alcohol;
Lyophilize promptly gets.
Salvianolic acid A preparation method of the present invention preferably may further comprise the steps:
Step 1, extraction
Salvia piece added extraction using alcohol 2 hours, filtered, and added ethanol in the dregs of a decoction and continued to extract 1 hour, and filtration merges extracted twice liquid, and being concentrated into does not have the alcohol flavor, adds water and processes the solution that contains 0.5g crude drug/mL, and 100 ℃ are incubated 16 hours.Promptly get the extracting solution that is rich in salvianolic acid A.
Step 2, roughing out
Radix Salviae Miltiorrhizae extract adding after transforming is filled with on the chromatographic column of AB-8 macroporous adsorbent resin, the water flushing, flow rate control meets 8BV down at 1.5-2.0BV/h, discards.Using ethanol elution instead, detect to there not being blue spot with the thin layer chromatography board that is sprayed with iron trichloride simultaneously, meet about 0.8BV down, be concentrated into medicinal extract, detect through HPLC, mainly is salvianolic acid A and a spot of salvianolic acid B.
Step 3, decolouring
Load crude drug-filler ratio and be 10: 1 100-200 purpose polyamide column, with Ethanol Treatment good after with moving phase, the concentrated extract in the step 2 use dissolve with ethanol, add in the post, first to use ethanol elution, flow velocity be 1.0-1.5BV/h, descends to meet 10BV, discards.Use ethanol elution again, on the visible post a tangible colour band is arranged, detect this colour band with the thin layer chromatography board that is sprayed with iron trichloride, begin to receive from blue spot is arranged, to there not being blue spot, about 5BV is concentrated into medicinal extract.
Step 4, purifying
Medicinal extract is with the dissolve with ethanol of 10 times of amounts, and last MCI-GEL anti-phase fine separation post (crude drug-filler was than 20: 1) is used ethanol elution; Flow velocity is 1BV/h, detects with the thin layer chromatography board that is sprayed with iron trichloride, begins to receive from blue spot is arranged; Till not having a blue spot, about 3BV.Use dissolve with ethanol again after being concentrated into medicinal extract, last sephadex lh-20 post (crude drug-filler was than 20: 1) is used ethanol elution, detects with the thin layer chromatography board that is sprayed with iron trichloride, from being arranged, blue spot begins to receive, and till not having a blue spot, about 5BV.Being concentrated into does not have alcohol, promptly gets.
Step 4 liquid concentrator lyophilize 24 hours promptly gets salvianolic acid A.
Salvianolic acid A preparation method of the present invention, most preferred may further comprise the steps:
Step 1, extraction
Salvia piece adds 6 times of 50% extraction using alcohol 2 hours, filters, and adds 5 times of 50% ethanol in the dregs of a decoction and continues to extract 1 hour, and filtration merges extracted twice liquid, and being concentrated into does not have the alcohol flavor, adds water and processes the solution that contains 0.5g crude drug/mL, and 100 ℃ are incubated 16 hours.Promptly get the extracting solution that is rich in salvianolic acid A.
Step 2, roughing out
Radix Salviae Miltiorrhizae extract adding after transforming is filled with on the chromatographic column of AB-8 macroporous adsorbent resin, the water flushing, flow rate control meets 8BV (8 times of column volumes) down at 1.5-2.0BV/h, discards.Using 95% ethanol elution instead, detect to there not being blue spot with the thin layer chromatography board that is sprayed with iron trichloride simultaneously, meet about 0.8BV down, be concentrated into medicinal extract, detect through HPLC, mainly is salvianolic acid A and a spot of salvianolic acid B.
Step 3, decolouring
Load crude drug-filler ratio and be 10: 1 100-200 purpose polyamide column, moving phase is adjusted into 40% ethanol after good, the concentrated extract in the step 2 is used 40% dissolve with ethanol with Ethanol Treatment; Add in the post, using 40% ethanol elution, flow velocity earlier is 1.0-1.5BV/h; Under meet 10BV, discard.Use 60% ethanol elution again, on the visible post a tangible colour band is arranged, detect this colour band with the thin layer chromatography board that is sprayed with iron trichloride, begin to receive from blue spot is arranged, to there not being blue spot, about 5BV is concentrated into medicinal extract.
Step 4, purifying
Medicinal extract is with 20% dissolve with ethanol of 10 times of amounts, and last MCI-GEL anti-phase fine separation post (crude drug-filler was than 20: 1) is used 20% ethanol elution; Flow velocity is 1BV/h, detects with the thin layer chromatography board that is sprayed with iron trichloride, begins to receive from blue spot is arranged; Till not having a blue spot, about 3BV.Use 20% dissolve with ethanol again after being concentrated into medicinal extract, last sephadex lh-20 post (crude drug-filler was than 20: 1) is used 20% ethanol elution, detects with the thin layer chromatography board that is sprayed with iron trichloride, from being arranged, blue spot begins to receive, and till not having a blue spot, about 5BV.Being concentrated into does not have alcohol, promptly gets.
The step 4 liquid concentrator is through pre-freeze more than 4 hours, and-50 ℃ of dryings 24 hours promptly get salvianolic acid A.
Salvianolic acid A preparation method of the present invention, wherein most preferred method obtains through screening, and screening process is following:
(1) extraction process
Adopt water, 0.45%NaHCO
3The aqueous solution, 50% ethanol, 80% ethanol and five kinds of solvents of 95% ethanol extract simultaneous test, find to have only 0.45%NaHCO
3The aqueous solution can extract and obtain salvianolic acid A, but content is lower, and is main with salvianolic acid B all in all the other four kinds of solvent extraction liquid.
Through investigating optimum solvent, best pH and the Best Times that transforms, obtain following extraction process:
Salvia piece adds 6 times of 50% extraction using alcohol 2 hours, filters, and adds 5 times of 50% ethanol in the dregs of a decoction and continues to extract 1 hour, and filtration merges extracted twice liquid, and being concentrated into does not have the alcohol flavor, and adding water becomes the solution that contains 0.5g crude drug/mL, and 100 ℃ are incubated 16 hours.Promptly get the extracting solution that is rich in salvianolic acid A.
(2) purifying process
Step 1, roughing out
When early-stage Study salvianolic acid L and M, find that the AB-8 macroporous adsorbent resin is better than all the other water soluble components to the adsorptive power of salvianolic acid A, and desorb is fast, desorption efficiency is high, thus this step to have selected AB-8. crude drug-resin ratio be 3: 1.Concrete technology is following:
Radix Salviae Miltiorrhizae extract adding after transforming is filled with on the chromatographic column of AB-8 macroporous adsorbent resin, and with 30% flushing, flow rate control meets 8BV (8 times of column volumes) at 1.5-2.0BV/h, discards.Use 95% ethanol elution instead, flow rate control detects to there not being blue spot with the thin layer chromatography board that is sprayed with iron trichloride at 1.5-2.0BV/h simultaneously, meets about 0.8BV down, is concentrated into medicinal extract, detect through HPLC, and mainly be salvianolic acid A minute quantity salvianolic acid B, see Fig. 1.
Step 2, decolouring
The salvianolic acid solution that from a last step, obtains has darker color; Therefore need to solve the decolouring problem; This step selected D101, LS-18, LS-300, ADS-7, D301, D201,201 * 4 with eight kinds of fillers of polymeric amide; The result shows that D101, LS-18, LS-300, ADS-7 do not have decolorization basically.And D301, D201,201 * 4 pairs of salvianolic acid As have very strong dead adsorption, all can't be by wash-out after being adsorbed.Polymeric amide had both had good decolorization, but also had good separating effect.
In sum, it is best decolouring filler that this step is selected polymeric amide, and concrete technology is following:
Load crude drug-filler ratio and be 10: 1 100-200 purpose polymeric amide, moving phase is adjusted into 40% ethanol after good, the concentrated extract in the step 2 is used 40% dissolve with ethanol with Ethanol Treatment; Add in the post, using 40% ethanol elution, flow velocity earlier is 1.0-1.5BV/h; Under meet 10BV, discard.Use 60% ethanol elution again, on the visible post a tangible yellow colour band is arranged, with this colour band of thin layer chromatography board monitoring that is sprayed with iron trichloride, begin to receive from blue spot is arranged, to there not being blue spot, about 5BV is concentrated into medicinal extract.
Step 3, purifying
Under with an organic solvent prerequisite not, alternative filler is more limited in the purge process, after having compared sephadex lh-20 and MCI; Through TE; Finally selected first process MCI, and then the method for process polydextran gel, purifying process is specific as follows:
Medicinal extract is with 20% dissolve with ethanol of 10 times of amounts, and last MCI-GEL anti-phase fine separation post (crude drug-filler was than 20: 1) is used 20% ethanol elution; Flow velocity is 1BV/h, detects with the thin layer chromatography board that is sprayed with iron trichloride, begins to receive from blue spot is arranged; Till not having a blue spot, about 3BV.Use 20% dissolve with ethanol again after being concentrated into medicinal extract, last sephadex lh-20 post (crude drug-filler was than 20: 1) is used 20% ethanol elution, detects with the thin layer chromatography board that is sprayed with iron trichloride, from being arranged, blue spot begins to receive, and be last to there not being blue spot, about 5BV.Being concentrated into does not have alcohol, promptly gets.
Step 4 is dry
Because therefore the easy oxidized variable color of salvianolic acid A in direct drying under reduced pressure process has selected lyophilize at last.Technology is: pre-freeze is more than 4 hours, and-50 ℃ of dryings 24 hours promptly get salvianolic acid A.Detection figure sees Fig. 2.
The present invention also comprises a kind of purification process of salvianolic acid A, may further comprise the steps:
Get the salvianolic acid A that aforesaid method obtains and use dissolve with ethanol, add and be filled with in the post of sephadex lh-20, use ethanol elution; Thin layer chromatography board to be sprayed with iron trichloride detects, and begins to receive from blue spot is arranged, till not having a blue spot; Being concentrated into does not have alcohol, and lyophilize promptly gets.
Preferred purification process may further comprise the steps:
Get the salvianolic acid A that aforesaid method obtains, use 20% dissolve with ethanol, adding is filled with in the post of sephadex lh-20; Use 20% ethanol elution, detect, begin to receive from blue spot is arranged with the thin layer chromatography board that is sprayed with iron trichloride; Till not having a blue spot; Being concentrated into does not have alcohol, and lyophilize promptly gets.
The preparation technology that the present invention obtains is simple to operate, adopts ethanol-water system, and nontoxic no organic contamination can prepare in enormous quantities.Can realize preparing content 90% above bulk drug.Therefore the technology of the present invention production physical condition that is closely connected, can realize industrialization.
Description of drawings
Sample HPLC after Fig. 1 roughing out detects figure
Fig. 2 salvianolic acid A monomer HPLC detects figure
Embodiment
Below in conjunction with embodiment medicine of the present invention is further specified, following each embodiment only is used to the present invention is described and is not limitation of the present invention.
Embodiment 1
1, content is greater than the preparation of 90% sample
The 2kg salvia piece, twice (2h 1h), filters, and united extraction liquid is concentrated into RD1.20 (80 ℃), adds water to 4L, and 100 ℃ are incubated 16 hours with 6 times, 5 times amount 50% extraction using alcohols.Promptly get the extracting solution that is rich in salvianolic acid A.
Radix Salviae Miltiorrhizae extract adding after transforming is filled with on the chromatographic column of 700gAB-8 macroporous adsorbent resin, uses 30% alcohol flushing, flow rate control meets 9L down at 1.5-2.0BV/h, discards.Use 95% ethanol elution instead, detect to there not being blue spot with the thin layer chromatography board that is sprayed with iron trichloride simultaneously, meet 1L down, be concentrated into medicinal extract.
Get 100g100-200 purpose polymeric amide, moving phase is adjusted into 40% ethanol after good, concentrated extract is used 40% dissolve with ethanol, add in the post, use earlier 40% ethanol elution, time meet 5L, discard with Ethanol Treatment.Use 60% ethanol elution again, on the visible post a tangible colour band is arranged, detect this colour band with the thin layer chromatography board that is sprayed with iron trichloride, begin to receive from blue spot is arranged, to there not being blue spot, about 2.5L is concentrated into medicinal extract.
Medicinal extract is with 20% dissolve with ethanol of 10 times of amounts, on the post of 100mL MCI-GEL anti-phase fine separation is housed, use 20% ethanol elution, detect with the thin layer chromatography board that is sprayed with iron trichloride, begin to receive from blue spot is arranged, till not having a blue spot, meet 500ml down.Being concentrated into does not have alcohol, and lyophilize gets 6.5g salvianolic acid A monomer, lot number: 20080920.
2, content is greater than the preparation of 98% sample
Get the salvianolic acid A 3g more than 90%, use 20% dissolve with ethanol, adding is filled with in the post of 100g sephadex lh-20; Use 20% ethanol elution, detect, begin to receive from blue spot is arranged with the thin layer chromatography board that is sprayed with iron trichloride; Till not having a blue spot, about 1L.Being concentrated into does not have alcohol, and lyophilize promptly gets, and is total to 1.68g, lot number: 20090927.
3, assay
1, instrument and chromatographic condition
Instrument: Agilent 1100 high performance liquid chromatographs, quaternary pump, UV-detector.
Chromatographic column: Agilent C18 analytical column (4.6mm * 250mm, 5 μ m); Moving phase: adopt gradient elution, see table 1; Flow velocity: 1mLmin
-1Detect wavelength 280nm; Column temperature: 30 ℃.
Table 1 chromatographic condition gradient
2, the preparation of reference substance solution agent testing sample solution
Get salvianolic acid A reference substance (purchasing the one side science and technology in Tianjin) 10mg, the accurate title, decide, and puts in the 100ml volumetric flask, uses dissolve with methanol, and fixed dissolving to scale, promptly gets the reference substance solution that concentration is 0.11mg/ml.
Get salvianolic acid A bullion and high-purity danshinolic acid A10mg respectively, each sample is parallel gets two parts, and accurate the title decides, and puts in the 100ml volumetric flask, uses dissolve with methanol, and fixed dissolving to scale, shakes up, to be measured.
3, measure
Get each 10 μ L of reference substance solution and sample solution, measure, each kind be parallel to advance two pins.
4, result sees table 2.
Table 2 salvianolic acid A samples contg is measured the result
Embodiment 2
Step 1, extraction
Salvia piece adds 95% extraction using alcohol 2 hours, filters, and adds 95% ethanol in the dregs of a decoction and continues to extract 1 hour, and filtration merges extracted twice liquid, and being concentrated into does not have the alcohol flavor, adds water and processes the solution that contains 0.5g crude drug/mL, and 100 ℃ are incubated 16 hours.Promptly get the extracting solution that is rich in salvianolic acid A.
Step 2, roughing out
Radix Salviae Miltiorrhizae extract adding after transforming is filled with on the chromatographic column of AB-8 macroporous adsorbent resin, the water flushing, flow rate control meets 8BV down at 1.5-2.0BV/h, discards.Using 50% ethanol elution instead, detect to there not being blue spot with the thin layer chromatography board that is sprayed with iron trichloride simultaneously, meet about 0.8BV down, be concentrated into medicinal extract, detect through HPLC, mainly is salvianolic acid A and a spot of salvianolic acid B.
Step 3, decolouring
Load crude drug-filler ratio and be 10: 1 100-200 purpose polyamide column, with 50% Ethanol Treatment good after with moving phase, the concentrated extract in the step 2 is used 50% dissolve with ethanol; Add in the post, using 50% ethanol elution, flow velocity earlier is 1.0-1.5BV/h; Under meet 10BV, discard.Use 50% ethanol elution again, on the visible post a tangible colour band is arranged, detect this colour band with the thin layer chromatography board that is sprayed with iron trichloride, begin to receive from blue spot is arranged, to there not being blue spot, about 5BV is concentrated into medicinal extract.
Step 4, purifying
Medicinal extract is with 50% dissolve with ethanol of 10 times of amounts, and last MCI-GEL anti-phase fine separation post (crude drug-filler was than 20: 1) is used 50% ethanol elution; Flow velocity is 1BV/h, detects with the thin layer chromatography board that is sprayed with iron trichloride, begins to receive from blue spot is arranged; Till not having a blue spot, about 3BV.Use 50% dissolve with ethanol again after being concentrated into medicinal extract, last sephadex lh-20 post (crude drug-filler was than 20: 1) is used 50% ethanol elution, detects with the thin layer chromatography board that is sprayed with iron trichloride, from being arranged, blue spot begins to receive, and till not having a blue spot, about 5BV.Being concentrated into does not have alcohol, promptly gets.
Step 4 liquid concentrator lyophilize 24 hours promptly gets salvianolic acid A.
Embodiment 3
Step 1, extraction
Salvia piece adds 50% extraction using alcohol 2 hours, filters, and adds 50% ethanol in the dregs of a decoction and continues to extract 1 hour, and filtration merges extracted twice liquid, and being concentrated into does not have the alcohol flavor, adds water and processes the solution that contains 0.5g crude drug/mL, and 100 ℃ are incubated 16 hours.Promptly get the extracting solution that is rich in salvianolic acid A.
Step 2, roughing out
Radix Salviae Miltiorrhizae extract adding after transforming is filled with on the chromatographic column of AB-8 macroporous adsorbent resin, the water flushing, flow rate control meets 8BV down at 1.5-2.0BV/h, discards.Using 50% ethanol elution instead, detect to there not being blue spot with the thin layer chromatography board that is sprayed with iron trichloride simultaneously, meet about 0.8BV down, be concentrated into medicinal extract, detect through HPLC, mainly is salvianolic acid A and a spot of salvianolic acid B.
Step 3, decolouring
Load crude drug-filler ratio and be 10: 1 100-200 purpose polyamide column, with 50% Ethanol Treatment good after with moving phase, the concentrated extract in the step 2 is used 50% dissolve with ethanol; Add in the post, using 50% ethanol elution, flow velocity earlier is 1.0-1.5BV/h; Under meet 10BV, discard.Use 50% ethanol elution again, on the visible post a tangible colour band is arranged, detect this colour band with the thin layer chromatography board that is sprayed with iron trichloride, begin to receive from blue spot is arranged, to there not being blue spot, about 5BV is concentrated into medicinal extract.
Step 4, purifying
Medicinal extract is with 20% dissolve with ethanol of 10 times of amounts, and last MCI-GEL anti-phase fine separation post (crude drug-filler was than 20: 1) is used 20% ethanol elution; Flow velocity is 1BV/h, detects with the thin layer chromatography board that is sprayed with iron trichloride, begins to receive from blue spot is arranged; Till not having a blue spot, about 3BV.Use 20% dissolve with ethanol again after being concentrated into medicinal extract, last sephadex lh-20 post (crude drug-filler was than 20: 1) is used 20% ethanol elution, detects with the thin layer chromatography board that is sprayed with iron trichloride, from being arranged, blue spot begins to receive, and till not having a blue spot, about 5BV.Being concentrated into does not have alcohol, promptly gets.
Step 4 liquid concentrator lyophilize 24 hours promptly gets salvianolic acid A.
Claims (6)
1. the preparation method of a salvianolic acid A is characterized in that, may further comprise the steps:
Step 1, extraction
The red sage root adds extraction using alcohol;
Step 2, roughing out
Chromatographic column with being filled with the AB-8 macroporous adsorbent resin is carried out roughing out to salvianolic acid A;
Step 3, decolouring
Decolour with polyamide column;
Step 4, purifying
Be further purified with MCI-GEL anti-phase fine separation post and sephadex lh-20 post;
Step 5, drying
Lyophilize.
2. method as claimed in claim 1 is characterized in that, may further comprise the steps:
Step 1, extraction
The red sage root adds extraction using alcohol, filters, and filtrating is concentrated into does not have the alcohol flavor, promptly gets the extracting solution that is rich in salvianolic acid A;
Step 2, roughing out
The extracting solution adding is filled with on the chromatographic column of AB-8 macroporous adsorbent resin, uses ethanol elution, detect to there not being blue spot with the thin layer chromatography board that is sprayed with iron trichloride simultaneously, collect elutriant, be concentrated into medicinal extract;
Step 3, decolouring
Concentrated extract is used dissolve with ethanol, adds in the polyamide column, and ethanol elution has a tangible yellow colour band on the visible post, with this colour band of thin layer chromatography board monitoring that is sprayed with iron trichloride, begins to receive from blue spot is arranged, and to there not being blue spot, is concentrated into medicinal extract;
Step 4, purifying
Medicinal extract is used dissolve with ethanol, and last MCI-GEL anti-phase fine separation post is used ethanol elution, detects with the thin layer chromatography board that is sprayed with iron trichloride; From there being blue spot to begin to receive, till not having a blue spot, collect elutriant, use dissolve with ethanol again after being concentrated into medicinal extract; Last sephadex lh-20 post is used ethanol elution, detects with the thin layer chromatography board that is sprayed with iron trichloride, begins to receive from blue spot is arranged; Till not having a blue spot, collect elutriant, being concentrated into does not have alcohol;
Step 5, drying
Lyophilize promptly gets.
3. method as claimed in claim 2 is characterized in that, may further comprise the steps:
Step 1, extraction
Salvia piece added extraction using alcohol 2 hours, filtered, and added ethanol in the dregs of a decoction and continued to extract 1 hour, and filtration merges extracted twice liquid, and being concentrated into does not have the alcohol flavor, adds water and processes the solution that contains 0.5g crude drug/mL, and 100 ℃ are incubated 16 hours, promptly get the extracting solution that is rich in salvianolic acid A,
Step 2, roughing out
Radix Salviae Miltiorrhizae extract adding after transforming is filled with on the chromatographic column of AB-8 macroporous adsorbent resin, the water flushing, flow rate control is at 1.5-2.0BV/h; Under meet 8BV, discard, use ethanol elution instead; Detect to there not being blue spot with the thin layer chromatography board that is sprayed with iron trichloride simultaneously, meet about 0.8BV down, be concentrated into medicinal extract; Detecting through HPLC, mainly is salvianolic acid A and a spot of salvianolic acid B
Step 3, decolouring
Load crude drug-filler ratio and be 10: 1 100-200 purpose polyamide column, with Ethanol Treatment good after with moving phase, the concentrated extract in the step 2 is used dissolve with ethanol, in the adding post; Earlier using ethanol elution, flow velocity is 1.0-1.5BV/h, meets 10BV down, discards; Use ethanol elution again, on the visible post a tangible colour band is arranged, detect this colour band, begin to receive from blue spot is arranged with the thin layer chromatography board that is sprayed with iron trichloride; To there not being blue spot, about 5BV is concentrated into medicinal extract
Step 4, purifying
Medicinal extract is with the dissolve with ethanol of 10 times of amounts, last MCI-GEL anti-phase fine separation post, and crude drug-filler was used ethanol elution than 20: 1; Flow velocity is 1BV/h, detects with the thin layer chromatography board that is sprayed with iron trichloride, begins to receive from blue spot is arranged, till not having a blue spot; About 3BV uses dissolve with ethanol after being concentrated into medicinal extract again, last sephadex lh-20 post, and crude drug-filler was than 20: 1; Use ethanol elution, detect, begin to receive, till not having a blue spot from blue spot is arranged with the thin layer chromatography board that is sprayed with iron trichloride; About 5BV, being concentrated into does not have alcohol, promptly gets
Step 5, drying
Step 4 liquid concentrator lyophilize 24 hours promptly gets salvianolic acid A.
4. method as claimed in claim 3 is characterized in that, may further comprise the steps:
Step 1, extraction
Salvia piece adds 6 times of 50% extraction using alcohol 2 hours, filters, and adds 5 times of 50% ethanol in the dregs of a decoction and continues to extract 1 hour, filters; Merge extracted twice liquid, being concentrated into does not have the alcohol flavor, adds water and processes the solution that contains 0.5g crude drug/mL; 100 ℃ are incubated 16 hours, promptly get the extracting solution that is rich in salvianolic acid A
Step 2, roughing out
Radix Salviae Miltiorrhizae extract adding after transforming is filled with on the chromatographic column of AB-8 macroporous adsorbent resin, the water flushing, flow rate control is at 1.5-2.0BV/h; Under connect 8 times of column volumes, discard, use 95% ethanol elution instead; Detect to there not being blue spot with the thin layer chromatography board that is sprayed with iron trichloride simultaneously, meet about 0.8BV down, be concentrated into medicinal extract; Detecting through HPLC, mainly is salvianolic acid A and a spot of salvianolic acid B
Step 3, decolouring
Load crude drug-filler ratio and be 10: 1 100-200 purpose polyamide column, moving phase is adjusted into 40% ethanol after good, the concentrated extract in the step 2 is used 40% dissolve with ethanol, in the adding post with Ethanol Treatment; Earlier using 40% ethanol elution, flow velocity is 1.0-1.5BV/h, meets 10BV down; Discard, use 60% ethanol elution again, on the visible post a tangible colour band is arranged; Detect this colour band with the thin layer chromatography board that is sprayed with iron trichloride, begin to receive from blue spot is arranged, to there not being blue spot; About 5BV is concentrated into medicinal extract
Step 4, purifying
Medicinal extract is with 20% dissolve with ethanol of 10 times of amounts, last NCI-GEL anti-phase fine separation post, and crude drug-filler was used 20% ethanol elution than 20: 1; Flow velocity is 1BV/h, detects with the thin layer chromatography board that is sprayed with iron trichloride, begins to receive from blue spot is arranged, till not having a blue spot; About 3BV uses 20% dissolve with ethanol after being concentrated into medicinal extract again, last sephadex lh-20 post, and crude drug-filler was than 20: 1; Use 20% ethanol elution, detect, begin to receive, till not having a blue spot from blue spot is arranged with the thin layer chromatography board that is sprayed with iron trichloride; About 5BV, being concentrated into does not have alcohol, promptly gets
Step 5, drying
The step 4 liquid concentrator is through pre-freeze more than 4 hours, and-50 ℃ of dryings 24 hours promptly get salvianolic acid A.
5. method as claimed in claim 4 is characterized in that, may further comprise the steps:
The salvianolic acid A that the weighting profit requires the 1-3 method to obtain is used dissolve with ethanol, adds to be filled with in the post of sephadex lh-20, uses ethanol elution; Thin layer chromatography board to be sprayed with iron trichloride detects, and begins to receive from blue spot is arranged, till not having a blue spot; Being concentrated into does not have alcohol, and lyophilize promptly gets.
6. method as claimed in claim 5 is characterized in that, may further comprise the steps:
The salvianolic acid A that the weighting profit requires the 1-3 method to obtain is used 20% dissolve with ethanol, adds to be filled with in the post of sephadex lh-20; Use 20% ethanol elution, detect, begin to receive from blue spot is arranged with the thin layer chromatography board that is sprayed with iron trichloride; Till not having a blue spot; Being concentrated into does not have alcohol, and lyophilize promptly gets.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105523926A (en) * | 2015-12-29 | 2016-04-27 | 山东大学 | An extracting, separating and purifying method of salvianolic acid A and a preparing method of salvianolic acid salts |
| CN107899085A (en) * | 2017-11-30 | 2018-04-13 | 太原理工大学 | A kind of preparation method of nanometer hydroxyapatite/PA6 composite materials |
| CN109678719A (en) * | 2019-02-22 | 2019-04-26 | 兰州和盛堂制药股份有限公司 | A kind of extraction preparation method and applications of high-purity monomer salviol acid A |
| 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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| CN1958555A (en) * | 2006-10-30 | 2007-05-09 | 王煜 | Method for preparing salviol acid A |
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| CN1958555A (en) * | 2006-10-30 | 2007-05-09 | 王煜 | Method for preparing salviol acid A |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105523926A (en) * | 2015-12-29 | 2016-04-27 | 山东大学 | An extracting, separating and purifying method of salvianolic acid A and a preparing method of salvianolic acid salts |
| CN107899085A (en) * | 2017-11-30 | 2018-04-13 | 太原理工大学 | A kind of preparation method of nanometer hydroxyapatite/PA6 composite materials |
| CN107899085B (en) * | 2017-11-30 | 2020-11-20 | 太原理工大学 | Preparation method of nano hydroxyapatite/PA 6 composite material |
| 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 |
| CN109678719A (en) * | 2019-02-22 | 2019-04-26 | 兰州和盛堂制药股份有限公司 | A kind of extraction preparation method and applications of high-purity monomer salviol acid A |
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