CN103936816A - Method for purifying tanshinone IIA in salvia miltiorrhiza crude extract through simulated moving bed chromatography - Google Patents
Method for purifying tanshinone IIA in salvia miltiorrhiza crude extract through simulated moving bed chromatography Download PDFInfo
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- tanshinone
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- ethanol
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- HYXITZLLTYIPOF-UHFFFAOYSA-N Tanshinone II Natural products O=C1C(=O)C2=C3CCCC(C)(C)C3=CC=C2C2=C1C(C)=CO2 HYXITZLLTYIPOF-UHFFFAOYSA-N 0.000 title claims abstract description 150
- 238000000034 method Methods 0.000 title claims abstract description 42
- 235000011135 Salvia miltiorrhiza Nutrition 0.000 title claims abstract description 16
- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 11
- 239000000287 crude extract Substances 0.000 title claims abstract description 7
- AIGAZQPHXLWMOJ-UHFFFAOYSA-N tanshinone IIA Natural products C1=CC2=C(C)C=CC=C2C(C(=O)C2=O)=C1C1=C2C(C)=CO1 AIGAZQPHXLWMOJ-UHFFFAOYSA-N 0.000 title abstract description 18
- 241000304195 Salvia miltiorrhiza Species 0.000 title abstract description 14
- AZEZEAABTDXEHR-UHFFFAOYSA-M sodium;1,6,6-trimethyl-10,11-dioxo-8,9-dihydro-7h-naphtho[1,2-g][1]benzofuran-2-sulfonate Chemical compound [Na+].C12=CC=C(C(CCC3)(C)C)C3=C2C(=O)C(=O)C2=C1OC(S([O-])(=O)=O)=C2C AZEZEAABTDXEHR-UHFFFAOYSA-M 0.000 title abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 77
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 59
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 28
- 238000010828 elution Methods 0.000 claims abstract description 16
- 238000001179 sorption measurement Methods 0.000 claims abstract description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 10
- 238000000926 separation method Methods 0.000 claims abstract description 8
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000012535 impurity Substances 0.000 claims abstract description 4
- GMWTXQKKRDUVQG-WOPPDYDQSA-N 4-amino-5-bromo-1-[(2r,3s,4s,5r)-4-hydroxy-5-(hydroxymethyl)-3-methyloxolan-2-yl]pyrimidin-2-one Chemical compound C[C@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(N)C(Br)=C1 GMWTXQKKRDUVQG-WOPPDYDQSA-N 0.000 claims description 44
- 238000000605 extraction Methods 0.000 claims description 36
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- 238000000746 purification Methods 0.000 claims description 20
- 238000007670 refining Methods 0.000 claims description 18
- 239000000706 filtrate Substances 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 15
- 240000007164 Salvia officinalis Species 0.000 claims description 13
- 239000002994 raw material Substances 0.000 claims description 13
- 235000005412 red sage Nutrition 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 238000011097 chromatography purification Methods 0.000 claims description 12
- 230000011218 segmentation Effects 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 6
- 238000001953 recrystallisation Methods 0.000 claims description 5
- 239000000377 silicon dioxide Substances 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 238000007598 dipping method Methods 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 238000000151 deposition Methods 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 238000004088 simulation Methods 0.000 claims description 3
- 238000002425 crystallisation Methods 0.000 claims description 2
- 230000008025 crystallization Effects 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 244000132619 red sage Species 0.000 claims 2
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 12
- 239000000284 extract Substances 0.000 abstract description 9
- 238000011084 recovery Methods 0.000 abstract description 2
- 239000000741 silica gel Substances 0.000 abstract description 2
- 229910002027 silica gel Inorganic materials 0.000 abstract description 2
- 239000011259 mixed solution Substances 0.000 abstract 1
- 238000004062 sedimentation Methods 0.000 abstract 1
- 229930183118 Tanshinone Natural products 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 238000010262 high-speed countercurrent chromatography Methods 0.000 description 5
- GVKKJJOMQCNPGB-UHFFFAOYSA-N Cryptotanshinone Natural products O=C1C(=O)C2=C3CCCC(C)(C)C3=CC=C2C2=C1C(C)CO2 GVKKJJOMQCNPGB-UHFFFAOYSA-N 0.000 description 4
- GVKKJJOMQCNPGB-JTQLQIEISA-N Cryptotanshinone Chemical compound O=C1C(=O)C2=C3CCCC(C)(C)C3=CC=C2C2=C1[C@@H](C)CO2 GVKKJJOMQCNPGB-JTQLQIEISA-N 0.000 description 4
- 230000002411 adverse Effects 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- ONUSCCKLNWURMA-UHFFFAOYSA-N 3,8-dimethylnaphtho[2,1-g][1]benzofuran-4,5-dione Chemical compound O=C1C(=O)C2=CC=C3C(C)=CC=CC3=C2C2=C1C(C)=CO2 ONUSCCKLNWURMA-UHFFFAOYSA-N 0.000 description 2
- KXNYCALHDXGJSF-UHFFFAOYSA-N 4,8-dimethyl-8,9-dihydronaphtho[2,1-f][1]benzofuran-7,11-dione Chemical compound CC1=CC=CC2=C(C(C=3OCC(C=3C3=O)C)=O)C3=CC=C21 KXNYCALHDXGJSF-UHFFFAOYSA-N 0.000 description 2
- XYKZSUXWBGUGQV-UHFFFAOYSA-N 4,8-dimethylnaphtho[2,1-f][1]benzofuran-7,11-dione Chemical compound CC1=CC=CC2=C(C(C=3OC=C(C=3C3=O)C)=O)C3=CC=C21 XYKZSUXWBGUGQV-UHFFFAOYSA-N 0.000 description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 239000012846 chemical reference substance Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 238000004237 preparative chromatography Methods 0.000 description 2
- 238000004262 preparative liquid chromatography Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- HARGZZNYNSYSGJ-UHFFFAOYSA-N 1,2 dihydrotanshinquinone Natural products C1=CC2=C(C)C=CC=C2C(C(=O)C2=O)=C1C1=C2C(C)CO1 HARGZZNYNSYSGJ-UHFFFAOYSA-N 0.000 description 1
- XDUXBBDRILEIEZ-UHFFFAOYSA-N 6-(hydroxymethyl)-1,6-dimethyl-8,9-dihydro-7h-naphtho[1,2-g][1]benzofuran-10,11-dione Chemical compound O=C1C(=O)C2=C3CCCC(C)(CO)C3=CC=C2C2=C1C(C)=CO2 XDUXBBDRILEIEZ-UHFFFAOYSA-N 0.000 description 1
- HARGZZNYNSYSGJ-JTQLQIEISA-N Dihydrotanshinone I Chemical compound C1=CC2=C(C)C=CC=C2C(C(=O)C2=O)=C1C1=C2[C@@H](C)CO1 HARGZZNYNSYSGJ-JTQLQIEISA-N 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 230000000274 adsorptive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000004185 countercurrent chromatography Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- LQRVVHITSCAECY-UHFFFAOYSA-N dichloromethane hexane methanol hydrate Chemical compound O.OC.ClCCl.CCCCCC LQRVVHITSCAECY-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- 239000003527 fibrinolytic agent Substances 0.000 description 1
- 230000003480 fibrinolytic effect Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- -1 hydroxyl methyl Chemical class 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- QHGPIJMPUOVBOL-UHFFFAOYSA-N isotanshinone IIA Natural products O=C1C2=CC=C(C(CCC3)(C)C)C3=C2C(=O)C2=C1C(C)=CO2 QHGPIJMPUOVBOL-UHFFFAOYSA-N 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000009438 liyan Substances 0.000 description 1
- 230000005906 menstruation Effects 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical group CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000003119 painkilling effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003815 supercritical carbon dioxide extraction Methods 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 230000002936 tranquilizing effect Effects 0.000 description 1
Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a method for purifying tanshinone IIA in a salvia miltiorrhiza crude extract through simulated moving bed chromatography (SMBC). The method is characterized in that basic zones of the SMBC are an elution zone, a refinement zone and an adsorption zone, the elution zone is independent, octadecylsilane (ODS) bonded silica gel is used as a stationary phase, a mixed solution of alcohol and water is used as a mobile phase, the SMBC separation is carried out, and previous impurities are removed at a raffinate outlet; tanshinone IIA extract liquid is obtained at an extract liquid outlet, and the tanshinone IIA product with the purity of 98.5% is obtained after the tanshinone IIA extract liquid is concentrated and subjected to water sedimentation and alcohol dissolving treatment. The method can be used for stably, continuously and efficiently purifying the tanshinone IIA in salvia miltiorrhiza alcohol extract liquid on a large scale through SMBC; the highest recovery rate of the salvia miltiorrhiza IIV in the extract liquid reaches 99.2%; the HPLC purity reaches 95%; the stationary phase and the mobile phase can be repeatedly used; the cost is reduced; the method belongs to the environment-friendly separation process.
Description
Technical field
The present invention relates to the method for purification of natural phant active principle, especially a kind of three-section simulated moving bed chromatography purification Tanshinone II A method.
Background technology
Tanshinone II A is one of effective constituent of the red sage root, there is boundless potential applicability in clinical practice, except effect of traditional promoting blood flow to regulate menstruation, stasis-dispelling and pain-killing, tranquilizing by nourishing the heart, in recent years pharmacological research show Tanshinone II A also have anti-inflammatory, anti-oxidant, antitumor, antibacterial, protect the liver and reduce blood viscosity, anticoagulant, promotion fibrinolytic, anticoagulant, prolongation thrombosis and promote thrombolysis, regulate body's immunity, improve the pharmacologically actives such as anti-diabetic microvascular complication (time precious traditional Chinese medical science traditional Chinese medicines .2010,21:7,1770-1772).
Tanshinone II A is the fat-soluble component of the red sage root.Except Tanshinone II A, in the fat-soluble component of the red sage root, also has the diterpene-kind compound of other TANSHINONES type, as Tanshinone I, Tanshinone II B, Cryptotanshinone, hydroxyl TANSHINONES, red sage root hydroxyl methyl esters, dihydrotanshinone I, and isotanshinone I, isotanshinone II, different Cryptotanshinone, dihydroisotanshinone I etc.
At present, the major technique of acquisition high purity (higher than 90%) Tanshinone II A monomer has: supercritical carbon dioxide extraction method, high-speed countercurrent chromatography, preparative liquid chromatography.
Open (bulletin) number: the CN1369485 of Chinese patent, has provided " a kind of method of extracting and refining tanshinone IIA by supercritical CO 2 ", adopts supercritical CO
2from the red sage root, extract Tanshinone II A, the disposable extraction and fractionation that carries out is refining, have nontoxic, quick, inexpensive, cold operation, meet the advantages such as environmental requirement, but while extracting high purity (higher than 90%) Tanshinone II A monomer, yield is low.
High-speed countercurrent chromatography does not need to use solid packing, the immiscible solvent systems high speed planetary motion in supporting tube of application two-phase carrys out separating natural product, open (bulletin) number: the CN1337397 of Chinese patent, in " method of application high speed adverse current chromatogram separation and purification TANSHINONES ", solvent system is normal hexane, ethyl acetate, methyl alcohol, water, after separating, secondary HSCCC chromatographic instrument obtains Tanshinone II A, each disengaging time needs 5～6 hours, and disengaging time is long, open (bulletin) number: the CN1394870 of Chinese patent, the solvent system of " a kind of method of separation and purification TANSHINONES " is sherwood oil, ethyl acetate, methyl alcohol, water, adopt HSCCC chromatographic instrument, flash liberation can obtain Cryptotanshinone, Tanshinone I and Tanshinone II A, but obtain 8～9 hours time of Tanshinone II A, still very long, Min Zhang etc., at " Development of a strategy and process parameters for a green process in counter-current chromatography:Purification of tanshinone IIA and cryptotanshinone from Salvia miltiorrhiza Bunge as a case study " (Journal of Chromatography A, 2011, 1218:6031 – 6037) in a literary composition with normal hexane-methylene chloride-methanol-water, and the second adverse current chromatography (CCC) of normal hexane-alcohol-water is extracted Tanshinone II A, disengaging time shortens, product purity is high, but yield is very low.Although high-speed countercurrent chromatography is without irreversible adsorption, or disengaging time is long, or the rate of recovery is low, efficiency is still needed and is further improved.
In preparative liquid chromatography, open (bulletin) number: the CN101210041 of Chinese patent, provide " a kind of method for separating and preparing of tanshinone IIA chemical reference substance ", taking weight content 50～97% Tanshinone II A extracts as raw material, taking methanol-water solution as elutriant, taking C8 or C18 bonding filler as stationary phase purification tanshinone IIA chemical reference substance; Open (bulletin) number: the CN1670019 of Chinese patent, " a kind of extracting method of TANSHINONES " of proposition adds to the alcohol extract of the red sage root on macroporous adsorptive resins, adopts respectively certain density alcohol gradient elution, obtains Tanshinone II A in extract; Open (bulletin) number: the CN101200490 of Chinese patent, " preparation method of high-purity tanshinone Long LiYan " mainly adopts normal-phase chromatography (taking silica gel or aluminum oxide as stationary phase Tanshinone raw material, sherwood oil/methylene dichloride is moving phase), separate by Multifunctional layered analysis system, elutriant is concentrated, recrystallization, obtains high purity Tanshinone II A.These column chromatographies are all batch process, and eluent consumption is large, can not automatically produce continuously.
Simulated moving bed chromatography, on the basis of preparative chromatography, has been introduced continuously, the operating mechanism of adverse current, rectifying, makes chromatographic separation become continuous operation from discontinuous operation, can carry out automatization, mass-producing, high efficiency separation, is the effective means of purification compound.Not yet find at present the report of simulated moving bed chromatography technology separating-purifying Tanshinone II A.
Summary of the invention
The invention provides a kind of method of three-section simulated moving bed chromatography purification Tanshinone II A, the Tanshinone II A of can purifying continuously, automatically, efficiently.
The method of a kind of three-section simulated moving bed chromatography purification Tanshinone II A provided by the invention, utilizes simulated moving bed chromatography to be called for short the SMBC Tanshinone II A of purifying from red sage root crude extract, the theing contents are as follows of the method:
(1) SMBC equipment
Comprise 3～10 root chromatogram columns, elution band moving phase transferpump, refining band moving phase transferpump, feeding liquid transferpump, valve, many logical, connection lines and container for storing liquid;
The stationary phase of chromatographic column uses octadecylsilane chemically bonded silica ODS, 10～60 μ m;
(2) SMBC working conditions
Operational mode: three band SMBC are set, and three bands comprise elution band, refining band and adsorption zone, a is elution band chromatographic column number, 1≤a≤2; B is refining band chromatographic column number, 1≤b≤4; C is adsorption zone chromatographic column number, 2≤c≤4, and operational mode represents with a-b-c;
Mobile phase composition: the moving phase P of elution band is the mixing solutions of ethanol and water, the percentage composition C of ethanol
pbe 50～100%; The moving phase D of refining band and adsorption zone is the mixing solutions of ethanol and water, the percentage composition C of ethanol
dbe 50～80%; Sample introduction liquid: obtain filtrate after the alcohol dipping of 5～20 times of quality of the broken rear use of salvia miltiorrhiza raw material medicinal powder, be the solution of red sage root crude extract, filtrate or directly as sample introduction liquid, or be sample introduction liquid containing the aqueous solution of ethanol 0～50%, the required aqueous solution volume containing ethanol 0～50% is less than 2 times of filtrate volumes;
Flow rate of mobile phase: in refining band and adsorption zone, elutriant D flow velocity Q
dfor 4～30 times of column volumes per hour, i.e. 4～30BV/h, as elutriant D flow velocity Q
dunit is mL/min, and chromatographic column radius r unit is cm, and when column's length L unit is cm, 1 BV/h is equivalent to 60Q
d/ (π r
2l); Sample introduction liquid F flow velocity Q
ffor 0.1Q
d～1.5Q
d, raffinate R flow velocity Q
r=Q
d+ Q
f; In elution band, elutriant P flow velocity Q
pfor 0.5Q
d～2QD, extraction liquid E flow velocity Q
e=Q
p;
Switching time T
s: 9～40min;
Simulation moving-bed service temperature: room temperature;
Sample introduction pattern: continuous sample introduction, or segmentation sample introduction;
(3) SMBC purification step
Use SMBC equipment as described in (1) under the SMBC working conditions as described in (2), to carry out SMBC separation, from raffinate R outlet, remove various front impurity; From extraction liquid E outlet, obtain Tanshinone II A extraction liquid;
(4) extraction liquid aftertreatment
Will be concentrated containing Tanshinone II A extraction liquid, through the molten processing of depositing in water alcohol, add water by concentrated solution, be precipitated, then add dissolve with ethanol precipitation, crystallization, obtains Tanshinone II A product.
(5) detection method
High performance liquid chromatograph: the L-2000 of Hitachi; Analyze chromatographic column: Agilent Extend-C18,5 μ m, 150mm × 4.6mm I.D.; Moving phase: V(methyl alcohol): V(water)=85:15; Detect wavelength: 270nm flow velocity: 1.0mL/min; Column temperature: 30 DEG C.Demarcated the content of liquid Tanshinone II A to be measured by Tanshinone II A standard substance.
The method of described SMBC purification Tanshinone II A, at each switching cycle 0～T
sin, continuous sample introduction.
The method of described SMBC purification Tanshinone II A, at each switching cycle 0～T
sin, segmentation sample introduction, 0 < t < T
s, sample introduction in 0～t (min), 0.25min < T
s-t < 2min.
The method of described SMBC purification Tanshinone II A, at each switching cycle 0～T
sin, segmentation sample introduction, 0 < t < T
s, at t～T
s(min) interior sample introduction, 0.5min < t < 5min.
The method of described SMBC purification Tanshinone II A, at each switching cycle 0～T
sin, segmentation sample introduction, 0 < t
1< t
2< T
s, at t
1～t
2(min) interior sample introduction, 0.5min < t
1< 5min, 0.25min < T
s-t
2< 2min.
The method of described SMBC purification Tanshinone II A, in extraction liquid last handling process, will add water after concentrating containing Tanshinone II A extraction liquid, be precipitated thing, after water washing and precipitating thing, add dissolve with ethanol throw out, recrystallization, obtains the Tanshinone II A product of purity >=98%.
The method of described SMBC purification Tanshinone II A, in SMBC working conditions, adopts the mixing solutions of methyl alcohol and water as moving phase; In SMBC working conditions, salvia miltiorrhiza raw material medicinal powder is broken rear with obtaining filtrate after methyl alcohol dipping; In extraction liquid last handling process, adopt dissolve with methanol crystalline deposit thing.。
The present invention is compared with existing preparative chromatography isolation technique, and its significant beneficial effect is embodied in:
The method of three-section simulated moving bed chromatography purification Tanshinone II A; can mass-producing, the Tanshinone II A of purifying from the red sage root steadily and surely, continuously and automatically and efficiently; product recovery rate reaches 99.2%; HPLC purity reaches 95%; stationary phase and mobile being on good terms are recycled; reduce cost for purification, belonged to environmental protection separation engineering.
Brief description of the drawings
Fig. 1 is the HPLC spectrogram (4-Tanshinone II A chromatographic peak) of the sample introduction liquid of salvia miltiorrhiza raw material medicine A.
Fig. 2 is the HPLC spectrogram (4-Tanshinone II A chromatographic peak) of the sample introduction liquid of salvia miltiorrhiza raw material medicine B.
Fig. 3 is three-section simulated moving bed chromatography system.
Fig. 4 is the HPLC spectrogram of SMBC raffinate.
Fig. 5 is the HPLC spectrogram of SMBC extraction liquid.
Fig. 6 is that SMBC extraction liquid is through HPLC spectrogram concentrated, Tanshinone II A that depositing in water alcohol is molten, recrystallization obtains after processing.
Embodiment
Describe the present invention below in conjunction with accompanying drawing in detail with embodiment.
Embodiment 1
A method for three-section simulated moving bed chromatography purification Tanshinone II A, utilizes the SMBC Tanshinone II A of purifying from the ethanol extraction of the red sage root, the theing contents are as follows of the method:
(1) SMBC equipment
SMBC equipment is made up of 6 root chromatogram columns, moving phase D transferpump, stock liquid F pump, moving phase P transferpump, 36 internally piloted valves, a set of single chip computer automatic control system, 5 container for storing liquids and Duo Tong and connection line, wherein the flow of stock liquid F pumping capacity and moving phase P transferpump is 0～10mL/min, pressure is 0～30Mpa, and moving phase D, stock liquid F, moving phase P, extraction liquid E and raffinate R are respectively with a container for storing liquid;
Chromatographic column specification: diameter 1cm, long 10cm;
Stationary phase uses octadecylsilane chemically bonded silica ODS, 20～30 μ m.
(2) SMBC working conditions
Operational mode is 2-2-2, i.e. elution band: 2 root chromatogram columns; Refining band: 2 root chromatogram columns; Adsorption zone: 2 root chromatogram columns;
Mobile phase composition: the moving phase P of elution band is 95% ethanol; The moving phase D of refining band and adsorption zone is the mixing solutions of ethanol and water, the percentage composition C of ethanol
dbe 75%; Sample introduction liquid: 30g salvia miltiorrhiza raw material medicine A is with the filtrate after 120mL extraction using alcohol, and wherein Tanshinone II A concentration is 0.42mg/mL, filtrate by the proportioning dilution of moving phase D after as sample introduction liquid, the HPLC spectrogram of sample introduction liquid is shown in Fig. 1;
Flow rate of mobile phase: in elution band, elutriant P flow velocity Q
pfor 1.5mL/min, extraction liquid E flow velocity Q
e=Q
p; In refining band and adsorption zone, elutriant D flow velocity Q
dfor 1.5mL/min, sample introduction liquid F flow velocity Q
ffor 0.2mL/min, raffinate R flow velocity Q
r=Q
d+ Q
f;
Switching time: 12.1min;
Simulation moving-bed service temperature: room temperature;
Sample introduction pattern: continuous sample introduction, as shown in Figure 3.
(3) SMBC purification step
Use SMBC equipment as described in (1) under the SMBC working conditions as described in (2), to carry out SMBC purification.From raffinate outlet, i.e. R mouth, removes various front impurity, and raffinate HPLC spectrogram as shown in Figure 4; From extraction liquid outlet, i.e. E mouth, obtains Tanshinone II A extraction liquid, and as shown in Figure 5, the chromatographic purity of Tanshinone II A is 94.5% to its HPLC spectrogram, yield 98.8%.
(4) extraction liquid aftertreatment
After will be concentrated containing Tanshinone II A raffinate, add water, be precipitated, wash with water after precipitation, with dissolve with ethanol precipitation, recrystallization 2 times, obtain purity and be 98.7% Tanshinone II A product, its HPLC spectrogram as shown in Figure 6.
(5) detection method
High performance liquid chromatograph: the L-2000 of Hitachi; Analyze chromatographic column: Agilent Extend-C18,5 μ m, 150mm × 4.6mm I.D.; Moving phase: V(methyl alcohol): V(water)=85:15; Detect wavelength: 270nm flow velocity: 1.0mL/min; Column temperature: 30 DEG C.Demarcated the content of liquid Tanshinone II A to be measured by Tanshinone II A standard substance.
Embodiment 2
Except following condition, other condition is all with embodiment 1;
Operational mode: 1-3-2;
Switching time Ts:11min;
SMBC purification result: the chromatographic purity of extraction liquid Tanshinone II A is 93%, yield is 92%.
Embodiment 3
Except following condition, other condition is all with embodiment 1;
5 root chromatogram columns, 30 internally piloted valves;
Chromatographic column specification: diameter 2cm, long 20cm;
Operational mode: 1-2-2;
Stationary phase: octadecylsilane chemically bonded silica ODS, 30～40 μ m;
Flow rate of mobile phase: elutriant D flow velocity Q
dfor 6mL/min, sample introduction liquid F flow velocity Q
ffor 0.8mL/min, elutriant P flow velocity Q
pfor 6mL/min;
Switching time Ts:19min;
SMBC purification result: the chromatographic purity of extraction liquid Tanshinone II A is 96%, yield is 92.7%.
Embodiment 4
Except following condition, other condition is all with embodiment 1;
5 root chromatogram columns, 30 internally piloted valves;
Chromatographic column specification: diameter 2cm, long 20cm;
Operational mode: 1-2-2;
Stationary phase: octadecylsilane chemically bonded silica ODS, 30～40 μ m;
Mobile phase composition: the percentage composition C of ethanol in the moving phase D of refining band and adsorption zone
dbe 77%;
Flow rate of mobile phase: elutriant D flow velocity Q
dfor 6mL/min, sample introduction liquid F flow velocity Q
ffor 0.8mL/min, elutriant P flow velocity Q
pfor 6mL/min;
Switching time Ts:16.5min;
SMBC separating resulting: the chromatographic purity of extraction liquid Tanshinone II A is 92.5%, yield is 100.0%.
Embodiment 5
Except following condition, other condition is all with embodiment 1;
Sample introduction liquid: 24g salvia miltiorrhiza raw material medicine B uses the filtrate after 120mL extraction using alcohol, and directly as sample introduction liquid, the HPLC spectrogram of sample introduction liquid is shown in Fig. 2, and wherein Tanshinone II A concentration is 0.21mg/mL;
Sample introduction liquid F flow velocity Q
f: 0.1mL/min
Switching time Ts:10.5min;
Sample introduction pattern: segmentation sample introduction, sample introduction during 1.5min to 10min;
SMBC separating resulting: the chromatographic purity of extraction liquid Tanshinone II A is 90.6%, yield is 94.6%.
Embodiment 6
Except following condition, other condition is all with embodiment 1;
Sample introduction liquid: 36g salvia miltiorrhiza raw material medicine B uses the filtrate after 120mL extraction using alcohol, and directly as sample introduction liquid, wherein Tanshinone II A concentration is 0.30mg/mL;
Sample introduction liquid F flow velocity Q
ffor 0.1mL/min;
Switching time Ts:10min;
Sample introduction pattern: segmentation sample introduction, sample introduction during 1min to 9.5min;
SMBC purification result: the chromatographic purity of extraction liquid Tanshinone II A is 90.4%, yield is 98.2%.
Embodiment 7
Except following condition, other condition is all with embodiment 1;
4 root chromatogram columns, 24 internally piloted valves;
Operational mode: 1-1-2;
Mobile phase composition: the moving phase D of refining band and adsorption zone is the mixing solutions of ethanol and water, the percentage composition C of ethanol
dbe 60%; Sample introduction liquid: 24g salvia miltiorrhiza raw material medicine A uses the filtrate after 120mL extraction using alcohol, and wherein Tanshinone II A concentration is 0.33mg/mL, adds water filtrate to configure sample introduction liquid, and adding volume of water is 1 times of filtrate volume;
Flow rate of mobile phase: elutriant D flow velocity Q
dfor 3mL/min, sample introduction liquid F flow velocity Q
ffor 3mL/min, elutriant P flow velocity Q
pfor 3mL/min;
Switching time Ts:17min;
Sample introduction pattern: segmentation sample introduction, sample introduction during 0～16.5min;
SMBC separating resulting: the chromatographic purity of extraction liquid Tanshinone II A is 93.9%, yield is 95.1%.
Embodiment 8
Except following condition, other condition is all with embodiment 1;
Chromatographic column specification: diameter 1cm, long 20cm;
Sample introduction liquid: 24g salvia miltiorrhiza raw material medicine B uses the filtrate after 120mL extraction using alcohol, and wherein Tanshinone II A concentration is 0.21mg/mL, directly as sample introduction liquid;
Mobile phase composition: the percentage composition C of ethanol in the moving phase D of refining band and adsorption zone
dbe 70%;
Flow rate of mobile phase: elutriant D flow velocity Q
dfor 3mL/min, sample introduction liquid F flow velocity Q
ffor 0.3mL/min, elutriant P flow velocity Q
pfor 3mL/min;
Switching time Ts:13.7min;
Sample introduction pattern: segmentation sample introduction, sample introduction during 4min to 13.2min;
SMBC separating resulting: the chromatographic purity of extraction liquid Tanshinone II A is 95.0%, yield is 99.2%.
Claims (7)
1. a method for three-section simulated moving bed chromatography purification Tanshinone II A, is characterized in that utilizing simulated moving bed chromatography to be called for short the SMBC Tanshinone II A of purifying from red sage root crude extract, the theing contents are as follows of the method:
(1) SMBC equipment
Comprise 3～10 root chromatogram columns, elution band moving phase transferpump, refining band moving phase transferpump, feeding liquid transferpump, valve, many logical, connection lines and container for storing liquid;
The stationary phase of chromatographic column uses octadecylsilane chemically bonded silica ODS, 10～60 μ m;
(2) SMBC working conditions
Operational mode: three band SMBC are set, and three bands comprise elution band, refining band and adsorption zone, a is elution band chromatographic column number, 1≤a≤2; B is refining band chromatographic column number, 1≤b≤4; C is adsorption zone chromatographic column number, 2≤c≤4, and operational mode represents with a-b-c;
Mobile phase composition: the moving phase P of elution band is the mixing solutions of ethanol and water, the percentage composition C of ethanol
pbe 50～100%; The moving phase D of refining band and adsorption zone is the mixing solutions of ethanol and water, the percentage composition C of ethanol
dbe 50～80%; Sample introduction liquid: obtain filtrate after the alcohol dipping of 5～20 times of quality of the broken rear use of salvia miltiorrhiza raw material medicinal powder, be the solution of red sage root crude extract, filtrate or directly as sample introduction liquid, or be sample introduction liquid containing the aqueous solution of ethanol 0～50%, the required aqueous solution volume containing ethanol 0～50% is less than 2 times of filtrate volumes;
Flow rate of mobile phase: in refining band and adsorption zone, elutriant D flow velocity Q
dfor 4～30 times of column volumes per hour, i.e. 4～30BV/h, as elutriant D flow velocity Q
dunit is mL/min, and chromatographic column radius r unit is cm, and when column's length L unit is cm, 1 BV/h is equivalent to 60Q
d/ (π r
2l); Sample introduction liquid F flow velocity Q
ffor 0.1Q
d～1.5Q
d, raffinate R flow velocity Q
r=Q
d+ Q
f; In elution band, elutriant P flow velocity Q
pfor 0.5QD～2Q
d, extraction liquid E flow velocity Q
e=Q
p;
Switching time T
s: 9～40min;
Simulation moving-bed service temperature: room temperature;
Sample introduction pattern: continuous sample introduction, or segmentation sample introduction;
(3) SMBC purification step
Use SMBC equipment as described in (1) under the SMBC working conditions as described in (2), to carry out SMBC separation, from raffinate R outlet, remove various front impurity; From extraction liquid E outlet, obtain Tanshinone II A extraction liquid;
(4) extraction liquid aftertreatment
Will be concentrated containing Tanshinone II A extraction liquid, through the molten processing of depositing in water alcohol, add water by concentrated solution, be precipitated, then add dissolve with ethanol, crystallization, obtains Tanshinone II A product.
2. the method for a kind of three-section simulated moving bed chromatography purification Tanshinone II A according to claim 1, is characterized in that at each switching cycle 0～T
sin, continuous sample introduction.
3. the method for a kind of three-section simulated moving bed chromatography purification Tanshinone II A according to claim 1, is characterized in that at each switching cycle 0～T
sin, segmentation sample introduction, 0 < t < T
s, sample introduction within 0～t time period.
4. the method for a kind of three-section simulated moving bed chromatography purification Tanshinone II A according to claim 3, is characterized in that at each switching cycle 0～T
sin, segmentation sample introduction, 0 < t < T
s, at t～T
ssample introduction in time period.
5. the method for a kind of three-section simulated moving bed chromatography purification Tanshinone II A according to claim 3, is characterized in that at each switching cycle 0～T
sin, segmentation sample introduction, 0 < t
1< t
2< T
s, at t
1～t
2sample introduction in time period.
6. the method for a kind of three-section simulated moving bed chromatography purification Tanshinone II A according to claim 1, it is characterized in that in extraction liquid last handling process, after concentrating containing Tanshinone II A extraction liquid, add water, be precipitated thing, after water washing and precipitating thing, add dissolve with ethanol throw out, recrystallization, obtains the Tanshinone II A product of purity >=98%.
7. the method for a kind of three-section simulated moving bed chromatography purification Tanshinone II A according to claim 1, is characterized in that in SMBC working conditions, adopts the mixing solutions of methyl alcohol and water as moving phase; In SMBC working conditions, salvia miltiorrhiza raw material medicinal powder is broken rear with obtaining filtrate after methyl alcohol dipping; In extraction liquid last handling process, adopt dissolve with methanol crystalline deposit thing.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106518940A (en) * | 2016-11-07 | 2017-03-22 | 辽宁科技大学 | Method for purifying vincoside lactam |
| TWI661856B (en) * | 2017-02-18 | 2019-06-11 | I-Shou University | Separation method for crude extract |
| TWI697671B (en) * | 2019-07-18 | 2020-07-01 | 梁明在 | Method of purifying tanshinone-based compound |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1369485A (en) * | 2001-12-27 | 2002-09-18 | 广州美晨药业有限公司 | Process for extracting and refining tanshinone IIA by supercritical CO2 |
| CN1394870A (en) * | 2001-07-05 | 2003-02-05 | 北京天纯维通生物技术有限公司 | Method for separating and purifying tanshinone |
| CN101200490A (en) * | 2007-12-13 | 2008-06-18 | 上海朗萨医药科技有限公司 | Method for preparing high-purity tanshinoneIIA |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1394870A (en) * | 2001-07-05 | 2003-02-05 | 北京天纯维通生物技术有限公司 | Method for separating and purifying tanshinone |
| CN1369485A (en) * | 2001-12-27 | 2002-09-18 | 广州美晨药业有限公司 | Process for extracting and refining tanshinone IIA by supercritical CO2 |
| CN101200490A (en) * | 2007-12-13 | 2008-06-18 | 上海朗萨医药科技有限公司 | Method for preparing high-purity tanshinoneIIA |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106518940A (en) * | 2016-11-07 | 2017-03-22 | 辽宁科技大学 | Method for purifying vincoside lactam |
| CN106518940B (en) * | 2016-11-07 | 2020-09-01 | 辽宁科技大学 | Method for purifying vincoside-lactam |
| TWI661856B (en) * | 2017-02-18 | 2019-06-11 | I-Shou University | Separation method for crude extract |
| TWI697671B (en) * | 2019-07-18 | 2020-07-01 | 梁明在 | Method of purifying tanshinone-based compound |
| CN112239486A (en) * | 2019-07-18 | 2021-01-19 | 梁明在 | Method for purifying tanshinone compounds |
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