CN103772478B - A kind of single-column circulation separation system and method thereof preparing high-purity tanshinone compound - Google Patents
A kind of single-column circulation separation system and method thereof preparing high-purity tanshinone compound Download PDFInfo
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- 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 claims abstract description 124
- 229930183118 Tanshinone Natural products 0.000 title claims abstract description 55
- 238000000926 separation method Methods 0.000 title claims abstract description 55
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- -1 tanshinone compound Chemical class 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 23
- WDBJFWOOWQREKA-UHFFFAOYSA-N salvilenone Chemical compound CC1=CC=C2C(=O)C(C(C)C)=C3C4=C2C1=CC=C4C(C)(C)O3 WDBJFWOOWQREKA-UHFFFAOYSA-N 0.000 claims abstract description 138
- 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 claims abstract description 76
- 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 claims abstract description 76
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 claims abstract description 40
- 238000002360 preparation method Methods 0.000 claims abstract description 39
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- 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 claims abstract description 32
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- 238000000605 extraction Methods 0.000 claims abstract description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 57
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 32
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- 239000000284 extract Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000005304 joining Methods 0.000 claims description 15
- 150000001875 compounds Chemical class 0.000 claims description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000000741 silica gel Substances 0.000 claims description 10
- 229910002027 silica gel Inorganic materials 0.000 claims description 10
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 9
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- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000007791 liquid phase Substances 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
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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 description 2
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- 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 description 2
- 239000007787 solid Substances 0.000 description 2
- XDUXBBDRILEIEZ-LJQANCHMSA-N (6s)-6-(hydroxymethyl)-1,6-dimethyl-8,9-dihydro-7h-naphtho[1,2-g][1]benzofuran-10,11-dione Chemical compound O=C1C(=O)C2=C3CCC[C@](C)(CO)C3=CC=C2C2=C1C(C)=CO2 XDUXBBDRILEIEZ-LJQANCHMSA-N 0.000 description 1
- WTPPRJKFRFIQKT-UHFFFAOYSA-N 1,6-dimethyl-8,9-dihydronaphtho[1,2-g][1]benzofuran-10,11-dione;1-methyl-6-methylidene-8,9-dihydro-7h-naphtho[1,2-g][1]benzofuran-10,11-dione Chemical compound O=C1C(=O)C2=C3CCCC(=C)C3=CC=C2C2=C1C(C)=CO2.O=C1C(=O)C2=C3CCC=C(C)C3=CC=C2C2=C1C(C)=CO2 WTPPRJKFRFIQKT-UHFFFAOYSA-N 0.000 description 1
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- XDUXBBDRILEIEZ-UHFFFAOYSA-N Tanshinone IIB Natural products O=C1C(=O)C2=C3CCCC(C)(CO)C3=CC=C2C2=C1C(C)=CO2 XDUXBBDRILEIEZ-UHFFFAOYSA-N 0.000 description 1
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Abstract
The present invention relates to single-column circulation separation system and the method thereof of preparation high-purity tanshinone compound, single-column circulation separation system comprises three current chromatographic columns, three pumps, three detectors, three collectors and three six-way valves.Make initial separator column with the first current chromatographic column, the second current chromatographic column and the 3rd current chromatographic column do separate multi-cycle separation post, connect first and second current chromatographic column with the second six-way valve, the 3rd six-way valve connection first and the 3rd current chromatographic column; Tanshinone compound multidimensional multi-cycle separation can be realized by regulating the second six-way valve and the 3rd six-way valve.Tanshinone compound comprises dihydrotanshinone Ⅰ, three leaf salvilenone B, methyltanshinone ester, Cryptotanshinone, tanshinone Ⅰ and 1,2-dihydro red sage root quinone.Extraction and isolation of the present invention is simple, the time is short, and product purity is high, can continuous operation, is suitable for suitability for industrialized production.
Description
Technical field
The present invention relates to a kind of single-column circulation separation system and the method thereof of preparing high-purity tanshinone compound.
Background technology
Adverse current chromatogram (Counter-current chromatography CCC) is a kind of liquid liquid distribution chromatography not needing solid support, therefore can avoid the shortcoming that solid support brings, such as sample loss, activity change, hangover and pollution.Compared to high performance liquid chromatography (High performance liquid chromatographyHPLC), CCC has larger preparation loading capacity and better selectivity.Therefore, even industrial production from trace analysis to preparative separation, CCC is still a kind of important separating and purifying technology.But the resolving power of adverse current chromatogram is still far smaller than HPLC, gas-chromatography (Gas chromatography GC) and capillary electrophoresis (Capillary electrophoresis CE).But investigator takes a lot of method improves this shortcoming, up to the present, puts in order at pillar, improve centrifugal force, adopt the aspects such as different shapes coiling to improve.
Under normal circumstances, increase pillar length and can improve separation efficiency, but the simple physical length increasing pillar not only needs to take greater room, need to expend more costs increases instrument and solvent simultaneously.Multi-cycle separation is then a kind of effective solution, it does not increase the physical length of pillar, but tail end elutriant is entered head end carry out multi-cycle separation, so both space can be saved, reagent can be saved again, the maximum risk of multi-cycle separation be material separately because causing mixing after circulation, therefore how to avoid back-mixing to be the maximum technological difficulties utilizing multi-cycle separation.Mostly existing cyclical chromatography is to circulate with single-column, adopts packed columnchromatography simultaneously, and post tail end pressure causes more greatly material requirements high, also because dead absorption etc. makes sample easily lose; Detector is not put in the circulating cycle in some multi-cycle separation, therefore cannot detect separation case in real time.
The red sage root (Salvia miltiorrhiza Bunge) is dry root and the stem of the labiate red sage root, begins to be loaded in Shennong's Herbal, is listed in top grade.Its bitter, cold nature, the thoughts of returning home, liver two warp, have promoting blood circulation and removing obstruction in channels, and disappear carbuncle, the relieving restlessness of stasis-dispelling and pain-killing, cool blood such as to be calmed the nerves at the effect, and what the traditional Chinese medical science had " Danshen simply, the same SIWU TANG of merit " says.TANSHINONES (Tanshinones) is the fat-soluble diterpene-kind compound in the red sage root with orange-yellow and orange red feature, is divided into 10 Multiple components such as tanshinone Ⅰ, tanshinone IIA, tanshinone ⅡB, Cryptotanshinone, dihydrotanshinone Ⅰ, hydroxyl TANSHINONES, tanshinone methyl ester, isotanshinone, different Cryptotanshinone by its structure difference.Because quinones composition has been reduced into diphenols derivative, the latter is oxidized to quinone again, therefore plays the effect of electron transmission.They, as new product of new old generation, by promoting or disturbing biological multiple biochemical reaction, show multiple biological activity.Pharmacology activity research shows, TANSHINONES all has good therapeutic action in antitumor, cardiovascular disorder, antisepsis and anti-inflammation etc.
TANSHINONES is soluble in the organic solvent such as ethanol, methyl alcohol.Traditional preparation method uses column chromatography (thin-layer chromatography, silica gel column chromatography, preparative liquid chromatography) enriching and purifying repeatedly after pulverizing soak extraction, but these preparation methods often consuming time and meeting cause sample loss because of the absorption of column packing, the method that patent CN200610017897.4 provides the sherwood oil of a kind of normal phase silicagel column, ethyl acetate, benzene, chloroform, 60-90 specification repeatedly to extract is separated TANSHINONES, it is too much that it is separated number of times, complex operation, and employ the larger solvent of the environmental pollution such as benzene, chloroform; Just the steps such as alcohol steep, refluxing extraction, concentrating under reduced pressure, pure water are used in the tanshinone extract provided in patent CN201210549910.6 and the preparation method of the water solubles thereof, in the product obtained, tanshinone IIA content only has 10.0% ~ 15.0%, and Cryptotanshinone substances content is 8.0% ~ 11.0%.
Summary of the invention
The object of the invention is to be separated for above-mentioned existing TANSHINONES that separation process in preparation process is long, solvent for use environmental hazard is large, the not high defect of the rate of recovery, and provide a kind of extraction and isolation simple, can the single-column circulation separation system of preparation high-purity tanshinone compound of continuous operation and method thereof.
The object of the present invention is achieved like this:
The single-column circulation separation system of preparation high-purity tanshinone compound of the present invention, comprises three current chromatographic columns, three pumps, three detectors, three collectors and three six-way valves; Wherein the first six-way valve is sampling valve, head end and first pump of this sampling valve and the first current chromatographic column are connected, the tail end of the first current chromatographic column is connected with the input terminus of the first detector, the output terminal of the first detector is connected with the IIIth node of the second six-way valve, IVth node of the second six-way valve is connected with the VIth node of the 3rd six-way valve, and the Vth node of the 3rd six-way valve is connected with the first collector; IIth node of the second six-way valve is connected with the input terminus of the second pump, second delivery side of pump is connected with the head end of the second current chromatographic column, the tail end of the second current chromatographic column is connected with the input terminus of the second detector, the output terminal of the second detector is connected with the Ith node of the second six-way valve, and the VIth node of the second six-way valve is connected with the second collector; Ith node of the 3rd six-way valve is connected with the input terminus of the 3rd pump, 3rd delivery side of pump is connected with the head end of the 3rd current chromatographic column, the tail end of the 3rd current chromatographic column is connected with the input terminus of the 3rd detector, the output terminal of the 3rd detector is connected with the IIth node of the 3rd six-way valve, and the IIIth node of the 3rd six-way valve is connected with the 3rd collector.
Single-column circulation separation system of the present invention is utilized to prepare the method for high-purity tanshinone compound, this tanshinone compound comprises structural formula (1) dihydrotanshinone Ⅰ, structural formula (2) three leaf salvilenone B and structural formula (3) methyltanshinone ester
Its preparation comprises the following steps:
(1) after red sage root rhizome being pulverized, with the abundant soak extraction of the ethanol of volumetric concentration 95%;
(2) concentrating under reduced pressure ethanol extract, the enriched material purification on normal-phase silica gel obtained mixes sample, carry out wash-out with sherwood oil, ethyl acetate, first alcohol and water successively again, obtain the tanshinone extract being rich in dihydrotanshinone Ⅰ, three leaf salvilenone B and methyltanshinone ester;
(3) quaternary solvent system is prepared: by normal hexane, ethyl acetate, first alcohol and water by volume per-cent is respectively 68.70%, 24.91%, the quaternary solvent system of 3.64% and 2.75% preparation is as upper phase, by volume per-cent is respectively 2.53%, 24.77%, the quaternary solvent system of 49.04% and 23.66% preparation is as lower phase, join in three current chromatographic columns up and down mutually respectively by what prepare, the amount joining upper phase in each current chromatographic column is 60% of its column volume, the amount of lower phase is 40% of its column volume, wherein go up the phase that fixes mutually, do moving phase mutually down,
(4) by obtained for step (2) being rich in dihydrotanshinone Ⅰ, first three leaf salvilenone B are separated through the first current chromatographic column with the tanshinone extract of methyltanshinone ester, obtain the elutriant containing dihydrotanshinone Ⅰ and contain the elutriant of three leaf salvilenone B and methyltanshinone ester;
(5) the first detector is observed, after the elutriant containing dihydrotanshinone Ⅰ washes out from the first current chromatographic column, the second six-way valve is regulated to be connected with the second current chromatographic column by the first current chromatographic column, directly online proceed to the second current chromatographic column by containing the elutriant of dihydrotanshinone Ⅰ, observe the first detector, after dihydrotanshinone Ⅰ elutriant enters the second current chromatographic column completely, regulate the second six-way valve the first current chromatographic column and the second current chromatographic column to be disconnected, make dihydrotanshinone Ⅰ at the second current chromatographic column internal recycle, observe the first detector, after the elutriant containing three leaf salvilenone B and methyltanshinone ester washes out from the first current chromatographic column, the 3rd six-way valve is regulated to be connected with the 3rd current chromatographic column by the first current chromatographic column, direct online the elutriant containing three leaf salvilenone B and methyltanshinone ester is proceeded to the 3rd current chromatographic column, observe the first detector, after three leaf salvilenone B and methyltanshinone ester elutriant enter the 3rd current chromatographic column completely, the 3rd six-way valve is regulated the first current chromatographic column and the 3rd current chromatographic column to be disconnected, make three leaf salvilenone B and methyltanshinone ester at the 3rd current chromatographic column internal recycle, second current chromatographic column and the 3rd current chromatographic column are when multi-cycle separation, and the first current chromatographic column residue elutriant collected by the first collector,
The chromatographic peak of (6) second detector on-line checkingi representation compounds is after completing a circulation realization separation, again regulate the second six-way valve to be connected with the second current chromatographic column by the first current chromatographic column, the dihydrotanshinone Ⅰ in the second current chromatographic column collected by the second collector;
The chromatographic peak of (7) the 3rd detector on-line checkingi representation compounds is after completing a circulation realization separation, again regulate the 3rd six-way valve to be connected with the 3rd current chromatographic column by the first current chromatographic column, the 3rd collector collects three leaf salvilenone B and the methyltanshinone ester of the 3rd current chromatographic column.
Single-column circulation separation system of the present invention is utilized to prepare the method for high-purity tanshinone compound, this tanshinone compound comprises structural formula (4) Cryptotanshinone, structural formula (5) tanshinone Ⅰ and structural formula (6) 1,2-dihydro red sage root quinone
Its preparation comprises the following steps:
(1) after red sage root rhizome being pulverized, with the abundant soak extraction of the ethanol of volumetric concentration 95%;
(2) concentrating under reduced pressure ethanol extract, the enriched material purification on normal-phase silica gel obtained mixes sample, then carries out wash-out with sherwood oil, ethyl acetate, first alcohol and water successively, obtains the tanshinone extract being rich in Cryptotanshinone, tanshinone Ⅰ and 1,2-dihydro red sage root quinone;
(3) quaternary solvent system is prepared: by normal hexane, ethyl acetate, first alcohol and water by volume per-cent is respectively 74.51%, 20.29%, the quaternary solvent system of 5.18% and 0.02% preparation is as upper phase, by volume per-cent is respectively 6.31%, 25.70%, the quaternary solvent system of 50.50% and 17.49% preparation is as lower phase, join in three current chromatographic columns up and down mutually respectively by what prepare, the amount joining upper phase in each current chromatographic column is 60% of its column volume, the amount of lower phase is 40% of its column volume, wherein go up the phase that fixes mutually, do moving phase mutually down.
(4) obtained for step (2) is rich in Cryptotanshinone, tanshinone Ⅰ and 1, first the tanshinone extract of 2-dihydro red sage root quinone is separated through the first current chromatographic column, obtain containing Cryptotanshinone, tanshinone Ⅰ and the elutriant containing 1,2-dihydro red sage root quinone;
(5) the first detector is observed, when containing Cryptotanshinone, after the elutriant of tanshinone Ⅰ washes out from the first current chromatographic column, the second six-way valve is regulated to be connected with the second current chromatographic column by the first current chromatographic column, online directly will containing Cryptotanshinone, the elutriant of tanshinone Ⅰ proceeds to the second current chromatographic column, observe the first detector, work as Cryptotanshinone, after tanshinone Ⅰ elutriant enters the second current chromatographic column completely, the second six-way valve is regulated the first current chromatographic column and the second current chromatographic column to be disconnected, make Cryptotanshinone, tanshinone Ⅰ is at the second current chromatographic column internal recycle,
(6) observe the first detector, after the elutriant containing 1,2-dihydro red sage root quinone washes out from the first current chromatographic column, be directly collected in the first collector;
The chromatographic peak of (7) second detector on-line checkingi representation compounds is after completing two circulation realization separation, again regulate the second six-way valve to be connected with the second current chromatographic column by the first current chromatographic column, the second collector collects Cryptotanshinone, tanshinone Ⅰ in the second current chromatographic column.
Utilize single-column circulation separation system of the present invention to prepare the method for high-purity tanshinone compound, this tanshinone compound comprises structural formula (1) dihydrotanshinone Ⅰ, structural formula (2) three leaf salvilenone B,
Its preparation comprises the following steps:
(1) after red sage root rhizome being pulverized, with the abundant soak extraction of the ethanol of volumetric concentration 95%;
(2) concentrating under reduced pressure ethanol extract, the enriched material purification on normal-phase silica gel obtained mixes sample, then carries out wash-out with sherwood oil, ethyl acetate, first alcohol and water successively, obtains the tanshinone extract being rich in dihydrotanshinone Ⅰ and three leaf salvilenone B;
(3) quaternary solvent system is prepared: by normal hexane, ethyl acetate, first alcohol and water by volume per-cent is respectively 74.51%, 20.29%, the quaternary solvent system of 5.18% and 0.02% preparation is as upper phase, by volume per-cent is respectively 6.31%, 25.70%, the quaternary solvent system of 50.50% and 17.49% preparation is as lower phase, join in three current chromatographic columns up and down mutually respectively by what prepare, the amount joining upper phase in each current chromatographic column is 60% of its column volume, the amount of lower phase is 40% of its column volume, wherein go up the phase that fixes mutually, do moving phase mutually down,
(4) first the dihydrotanshinone Ⅰ that is rich in that step (2) is obtained is separated through the first current chromatographic column with the tanshinone extract of three leaf salvilenone B, obtains the elutriant containing dihydrotanshinone Ⅰ and three leaf salvilenone B;
(5) the first detector is observed, after the elutriant containing dihydrotanshinone Ⅰ washes out from the first current chromatographic column, the second six-way valve is regulated to be connected with the second current chromatographic column by the first current chromatographic column, direct online the elutriant containing dihydrotanshinone Ⅰ and three leaf salvilenone B is proceeded to the second current chromatographic column, observe the first detector, after the elutriant containing dihydrotanshinone Ⅰ and three leaf salvilenone B enters the second current chromatographic column completely, the second six-way valve is regulated the first current chromatographic column and the second current chromatographic column to be disconnected, make dihydrotanshinone Ⅰ and three leaf salvilenone B at the second current chromatographic column internal recycle,
(6) the first detector is observed, after the first current chromatographic column wash-out is clean, second time sample introduction, be separated through the first current chromatographic column, obtain the elutriant containing dihydrotanshinone Ⅰ and three leaf salvilenone B, after dihydrotanshinone Ⅰ washes out from the first current chromatographic column, the 3rd six-way valve is regulated to be connected with the 3rd current chromatographic column by the first current chromatographic column, direct online the elutriant containing dihydrotanshinone Ⅰ and three leaf salvilenone B is proceeded to the 3rd current chromatographic column, observe the first detector, after the elutriant containing dihydrotanshinone Ⅰ and three leaf salvilenone B enters the 3rd current chromatographic column completely, the 3rd six-way valve is regulated the first current chromatographic column and the 3rd current chromatographic column to be disconnected, make dihydrotanshinone Ⅰ and three leaf salvilenone B at the 3rd current chromatographic column internal recycle,
The chromatographic peak of (7) second detector on-line checkingi representation compounds is after completing a circulation realization separation, again regulate the second six-way valve to be connected with the second current chromatographic column by the first current chromatographic column, the second collector collects dihydrotanshinone Ⅰ in the second current chromatographic column and three leaf salvilenone B;
The chromatographic peak of (8) the 3rd detector on-line checkingi representation compounds is after completing a circulation realization separation, the 3rd six-way valve is again regulated to be connected with the 3rd current chromatographic column by the first current chromatographic column, the dihydrotanshinone Ⅰ in the 3rd collector collection the 3rd current chromatographic column and three leaf salvilenone B.
Beneficial effect of the present invention is:
1) adopt multi-cycle separation, not only can save solvent, also can simplify the process finding suitable solvent system, thus solvent that sample solubility is large can be used to strengthen applied sample amount, improve the output of unit time;
2) adopt two parallel multi-cycle separation of post, the purifying realizing complex sample when not increasing separator column physical length can be met;
3) with three adverse current chromatogram column combinations, two dimensional separation and multi-cycle separation are combined, by the switching regulating the second six-way valve, the 3rd six-way valve can realize two dimensional separation and multi-cycle separation, simple to operate, save disengaging time.
4) separator column and available adverse current chromatogram, also can with common high performance liquid chromatography, applied range.
Accompanying drawing explanation
Fig. 1 is single-column circulation separation system schematic diagram, in figure: A1 is the first current chromatographic column, and B1 is the second current chromatographic column, C1 is the 3rd current chromatographic column, and 1 is the first pump, and 2 is the second pump, 3 is the 3rd pump, and 4 is the first detector, and 5 is the second detector, 6 is the 3rd detector, and 7 is the first collector, and 8 is the second collector, 9 is the 3rd collector, 10 is the first six-way valve, and 11 is the second six-way valve, and 12 is the 3rd six-way valve.
Fig. 2 is that six-way valve is in position 1 view;
Fig. 3 is that six-way valve is in position 2 view;
Fig. 4 is embodiment 1 sample HPLC analysis chart; In figure: 1 is dihydrotanshinone Ⅰ, 2 is three leaf salvilenone B, and 3 is methyltanshinone ester;
Fig. 5 is embodiment 2 sample HPLC analysis chart; In figure: 4 is Cryptotanshinone, 5 is tanshinone Ⅰ, and 6 is 1,2-dihydro red sage root quinone;
Fig. 6 is embodiment 3 sample HPLC analysis chart; In figure: 1 is dihydrotanshinone Ⅰ, 2 is three leaf salvilenone B.
Fig. 7 is embodiment 1 sepn process on-line checkingi schematic diagram, wherein: figure (a) A1 system is separated; Figure (b) B1 system is separated; Figure (c) C1 system is separated.
Fig. 8 is embodiment 2 sepn process on-line checkingi schematic diagram, wherein: figure (a) A1 system is separated; Figure (b) B1 system is separated.
Fig. 9 is embodiment 3 sepn process on-line checkingi schematic diagram, wherein: figure (a) A1 system is separated; Figure (b) B1 system is separated; Figure (c) C1 system is separated.
Figure 10 is the dihydrotanshinone Ⅰ liquid-phase chromatographic analysis of purifying.
Figure 11 is three leaf salvilenone B liquid-phase chromatographic analysis of purifying.
Figure 12 is the methyltanshinone ester liquid-phase chromatographic analysis of purifying.
Figure 13 is the Cryptotanshinone liquid-phase chromatographic analysis of purifying.
Figure 14 is the tanshinone Ⅰ liquid-phase chromatographic analysis of purifying.
Figure 15 is 1,2-dihydro red sage root quinone liquid-phase chromatographic analysis of purifying.
Embodiment
Below by specific embodiment, technical scheme of the present invention is further described.
With reference to Fig. 1, the single-column circulation separation system of preparation high-purity tanshinone compound of the present invention, comprise three current chromatographic column A1, B1, C1, three pumps 1, 2, 3, three detectors 4, 5, 6, three collectors 7, 8, 9 and three six-way valves 10, 11, 12, wherein the first six-way valve 10 is sampling valve, head end and first pump 1 of this sampling valve and the first current chromatographic column A1 are connected, the tail end of the first current chromatographic column A1 is connected with the input terminus of the first detector 4, the output terminal of the first detector 4 is connected with the IIIth node of the second six-way valve 11, IVth node of the second six-way valve 11 is connected with the VIth node of the 3rd six-way valve 12, Vth node of the 3rd six-way valve 12 is connected with the first collector 7, IIth node of the second six-way valve 11 is connected with the input terminus of the second pump 2, the output terminal of the second pump 2 is connected with the head end of the second current chromatographic column B1, the tail end of the second current chromatographic column B1 is connected with the input terminus of the second detector 5, the output terminal of the second detector 5 is connected with the Ith node of the second six-way valve 11, and the VIth node of the second six-way valve 11 is connected with the second collector 8, Ith node of the 3rd six-way valve 12 is connected with the input terminus of the 3rd pump 3, the output terminal of the 3rd pump 3 is connected with the head end of the 3rd current chromatographic column C1, the tail end of the 3rd current chromatographic column C1 is connected with the input terminus of the 3rd detector 6, the output terminal of the 3rd detector 6 is connected with the IIth node of the 3rd six-way valve 12, and the IIIth node of the 3rd six-way valve 12 is connected with the 3rd collector 9.
Embodiment 1
Single-column circulation separation system is utilized to prepare the method for high-purity tanshinone compound, this tanshinone compound comprises structural formula (1) dihydrotanshinone Ⅰ, structural formula (2) three leaf salvilenone B and structural formula (3) methyltanshinone ester
Step is as follows:
1. extract and enrichment:
Get 9.5 kilograms, dry red sage root rhizome, be ground into 60 object meal, 30L volumetric concentration 95% alcohol steep is placed in 3 times under room temperature, each 24 hours, periodic agitation, merge vat liquor concentrating under reduced pressure, obtain red sage root crude extract, take out 30g purification on normal-phase silica gel and mix sample, then use sherwood oil, eluent ethyl acetate, collect the elutriant being rich in tanshinone compound, concentrating under reduced pressure freeze-drying, obtain the crude product (HPLC analysis chart is shown in Fig. 4) being rich in dihydrotanshinone Ⅰ, three leaf salvilenone B and methyltanshinone ester, for the preparation of separation.
2. prepare solvent systems:
According to the volume percent of normal hexane, ethyl acetate, methyl alcohol, water be respectively 68.70%, 24.91%, 3.64%, 2.75% proportions on phase 800ml, phase 2L under the proportions of 2.53%, 24.77%, 49.04%, 23.66% respectively according to four volume percent, wherein go up the phase that fixes mutually, do moving phase mutually down.
3. single-column circulation adverse current chromatogram preparation:
(1) after preparing solvent systems, upper and lower phase pushes three current chromatographic column (A1-220ml with the first pump 1 respectively simultaneously, B1-180ml, C1-210ml), in, the amount joining upper phase in the first current chromatographic column A1 is 132ml, and the amount of lower phase is 88ml, the amount joining upper phase in the second current chromatographic column B1 is 108ml, the amount of lower phase is 72ml, and the amount joining upper phase in the 3rd current chromatographic column C1 is 126ml, and the amount of lower phase is 84ml.
(2) sampling valve is injected by the tanshinone extract 61mg 5ml being rich in dihydrotanshinone Ⅰ, three leaf salvilenone B and methyltanshinone ester obtained for step (1) with after phased soln under 5ml, open three current chromatographic columns, do moving phase mutually and with 3ml/min flow velocity wash-out down.Second six-way valve 11 and the 3rd six-way valve 12 are all placed in position 1 (as Fig. 2), first sample enters the first current chromatographic column A1 and is separated, observe the first detector 4, during 36min, separated object starts to wash out, during 54min, dihydrotanshinone Ⅰ starts to wash out, the second six-way valve 12 is regulated to be in position 2 (as Fig. 3), 3rd six-way valve 11 is still in position 1, first current chromatographic column A1 is connected with the second current chromatographic column B1, makes to enter the second current chromatographic column B1 containing dihydrotanshinone Ⅰ elutriant; Observe the first detector 4, during 67min, three leaf salvilenone B start to wash out, regulate the second six-way valve 11 home position 1,3rd six-way valve 12 is in position 2, disconnected with the second current chromatographic column B1 by first current chromatographic column A1 and be connected with the 3rd current chromatographic column C1, make the elutriant containing three leaf salvilenone B and methyltanshinone ester enter the 3rd current chromatographic column C1, the second current chromatographic column B1 carries out multi-cycle separation simultaneously; After observing the first detector 4,86min, regulate the 3rd six-way valve 12 home position 1, the first current chromatographic column A1 and the 3rd current chromatographic column C1 is disconnected, and the 3rd current chromatographic column C1 carries out multi-cycle separation; When observing second detector 5,133min, regulate the second six-way valve 11 to be in position 2, first current chromatographic column A1 and be connected with the second current chromatographic column B1, and collect dihydrotanshinone Ⅰ at the second collector 8; Observe the 3rd detector 6, during 182min, regulate the second six-way valve 11 home position 1, the 3rd six-way valve 12 is regulated to be in position 2, first current chromatographic column A1 is connected with the 3rd current chromatographic column C1, and collects three leaf salvilenone B and methyltanshinone ester (sepn process on-line checkingi figure is shown in Fig. 7) at the 3rd collector 9.
Concentrated freeze-dried rear mensuration purity, the purity of dihydrotanshinone Ⅰ is 100% (see Figure 10); The purity of three leaf salvilenone B is 97.6% (see Figure 11); The purity of methyltanshinone ester is 95.6% (see Figure 12).
Embodiment 2
Single-column circulation separation system is utilized to prepare the method for high-purity tanshinone compound, the tanshinone compound of preparation comprises structural formula (4) Cryptotanshinone, structural formula (5) tanshinone Ⅰ and structural formula (6) 1,2-dihydro red sage root quinone
Step is as follows:
1. extract and enrichment:
Get 9.5 kilograms, dry red sage root rhizome, be ground into 60 object meal, 30L volumetric concentration 95% alcohol steep is placed in 3 times under room temperature, each 24 hours, periodic agitation, merge vat liquor concentrating under reduced pressure, obtain red sage root crude extract, take out 30g purification on normal-phase silica gel and mix sample, then use sherwood oil, eluent ethyl acetate, collect the elutriant being rich in tanshinone compound, concentrating under reduced pressure freeze-drying, obtain the crude product (HPLC analysis chart is shown in Fig. 5) being rich in Cryptotanshinone, tanshinone Ⅰ and 1,2-dihydro red sage root quinone, for the preparation of separation.
2. prepare solvent systems:
By normal hexane, ethyl acetate, first alcohol and water by volume per-cent be respectively the upper phase 800ml of 74.51%, 20.29%, 5.18% and 0.02% preparation, by volume per-cent is respectively the lower phase 2L of 6.31%, 25.70%, 50.50% and 17.49% preparation.
3. single-column circulation adverse current chromatogram preparation:
(1) after preparing solvent systems, upper and lower phase pushes three current chromatographic column (A1-220ml with the first pump 1 respectively simultaneously, B1-180ml, C1-210ml) in, the amount joining upper phase in the first current chromatographic column A1 is 132ml, the amount of lower phase is 88ml, the amount joining upper phase in the second current chromatographic column B1 is 108ml, the amount of lower phase is 72ml, the amount joining upper phase in the 3rd current chromatographic column C1 is 126ml, the amount of lower phase is 84ml, wherein goes up the phase that fixes mutually, does moving phase mutually down.
(2) obtained for step (1) is rich in Cryptotanshinone, tanshinone Ⅰ and 1, on the tanshinone extract 80mg 5ml of 2-dihydro red sage root quinone with phased soln under 5ml after, inject sampling valve, open three current chromatographic columns, do moving phase mutually and with 3ml/min flow velocity wash-out down.Second six-way valve 11 and the 3rd six-way valve 12 are all placed in position 1 (as Fig. 2), first sample enters the first current chromatographic column A1 and is separated, observe the first detector 4, during 30min, separated object starts to wash out, during 65min, Cryptotanshinone starts to wash out, the second six-way valve 11 is regulated to be in position 2 (as Fig. 3), 3rd six-way valve 12 is in position 1 (as Fig. 2) and is connected with the second current chromatographic column B1 by the first current chromatographic column A1, makes the elutriant containing Cryptotanshinone and tanshinone Ⅰ enter the second current chromatographic column B1; Observe the first detector 4, after 78min, 1,2-dihydro red sage root quinone starts to wash out, the second six-way valve 11 home position 1 is regulated to be disconnected by first current chromatographic column A1 and the second current chromatographic column B1, Cryptotanshinone is separated at the second current chromatographic column B1 internal recycle with tanshinone Ⅰ, directly collects 1,2-dihydro red sage root quinone by the first collector 7; Observe the second detector 5, during 230min, regulate the second six-way valve 11 to be in position 2, the first current chromatographic column A1 is connected with the second current chromatographic column B1, collect Cryptotanshinone and tanshinone Ⅰ (sepn process on-line checkingi figure is shown in Fig. 8) by the second collector 8.
Concentrated freeze-dried rear mensuration purity, the purity of Cryptotanshinone is 94.7% (see Figure 13); The purity of tanshinone Ⅰ is 93.3% (see Figure 14); The purity of 1,2-dihydro red sage root quinone is 94.7% (see Figure 15).
Embodiment 3
Utilize single-column circulation separation system to prepare the method for high-purity tanshinone compound, the tanshinone compound of preparation comprises structural formula (1) dihydrotanshinone Ⅰ, structural formula (2) three leaf salvilenone B,
Step is as follows:
1. extract and enrichment:
Get 9.5 kilograms, dry red sage root rhizome, be ground into 60 object meal, 30L volumetric concentration 95% alcohol steep is placed in 3 times under room temperature, each 24 hours, periodic agitation, merge vat liquor concentrating under reduced pressure, obtain red sage root crude extract, take out 30g purification on normal-phase silica gel and mix sample, then use sherwood oil, eluent ethyl acetate, collect the elutriant being rich in tanshinone compound, concentrating under reduced pressure freeze-drying, obtain the crude product (HPLC analysis chart is shown in Fig. 6) being rich in dihydrotanshinone Ⅰ and three leaf salvilenone B, for the preparation of separation.
2. prepare solvent systems:
By normal hexane, ethyl acetate, first alcohol and water by volume per-cent be respectively the upper phase 800ml of 74.51%, 20.29%, 5.18% and 0.02% preparation, by volume per-cent is respectively the lower phase 2L of 6.31%, 25.70%, 50.50% and 17.49% preparation.
3. single-column circulation adverse current chromatogram preparation:
(1) after preparing solvent systems, upper and lower phase pushes three current chromatographic column (A1-220ml with the first pump 1 respectively simultaneously, B1-180ml, C1-210ml) in, the amount joining upper phase in the first current chromatographic column A1 is 132ml, the amount of lower phase is 88ml, the amount joining upper phase in the second current chromatographic column B1 is 108ml, the amount of lower phase is 72ml, the amount joining upper phase in the 3rd current chromatographic column C1 is 126ml, the amount of lower phase is 84ml, wherein goes up the phase that fixes mutually, does moving phase mutually down.
(2) sampling valve is injected by the tanshinone extract 60mg 5ml being rich in dihydrotanshinone Ⅰ and three leaf salvilenone B obtained in step (1) with after phased soln under 5ml, open three current chromatographic columns, do moving phase mutually and with 3ml/min flow velocity wash-out down.Second six-way valve 11 and the 3rd six-way valve 12 are all placed in position 1 (as Fig. 2), first sample enters the first current chromatographic column A1 and is separated, observe the first detector 4, during 30min, separated object starts to wash out, during 48min, dihydrotanshinone Ⅰ starts to wash out, the second six-way valve 11 is regulated to be in position 2 (as Fig. 3), 3rd six-way valve 12 is in position 1 (as Fig. 2) and is connected with the second current chromatographic column B1 by the first current chromatographic column A1, makes the elutriant containing dihydrotanshinone Ⅰ and three leaf salvilenone B enter the second current chromatographic column B1, observe the first detector 4, during 58min, dihydrotanshinone Ⅰ and three leaf salvilenone B elutriants enter the second current chromatographic column B1 completely, the second six-way valve 11 home position 1 is regulated to be disconnected by first current chromatographic column A1 and the second current chromatographic column B1, dihydrotanshinone Ⅰ and three leaf salvilenone B multi-cycle separation in the second current chromatographic column B1, observe the first detector 4, during 65min, after first current chromatographic column A1 is clean by wash-out, second time repeats sample introduction 60mg, the second six-way valve 11 and the 3rd six-way valve 12 is regulated to be in position 1, first sample enters the first current chromatographic column A1 and is separated, observe the first detector 4, during 87min, separated object starts to wash out, during 107min, dihydrotanshinone Ⅰ starts to be washed out, the second six-way valve 11 is regulated to be in position 1, 3rd six-way valve 12 is in position 2 and is connected with the 3rd current chromatographic column C1 by first current chromatographic column A1, make containing dihydrotanshinone Ⅰ, the elutriant of three leaf salvilenone B enters the 3rd current chromatographic column C1, observe the first detector 4, during 121min, dihydrotanshinone Ⅰ and three leaf salvilenone B elutriants enter the 3rd current chromatographic column C1 completely, the 3rd six-way valve 12 home position 1 is regulated to be disconnected by first current chromatographic column A1 and the 3rd current chromatographic column C1, dihydrotanshinone Ⅰ and three leaf salvilenone B multi-cycle separation in the 3rd current chromatographic column C1, observe the second detector 5, during 180min, dihydrotanshinone Ⅰ in second current chromatographic column B1 and three leaf salvilenone B complete two circulate after realize being separated, the second six-way valve 11 is regulated to be in position 2,3rd six-way valve is in position 1, first current chromatographic column A1 is connected with the second current chromatographic column B1, collects dihydrotanshinone Ⅰ and three leaf salvilenone B by the second collector 8, observe the 3rd detector 6, during 242min, dihydrotanshinone Ⅰ in 3rd current chromatographic column C1 and three leaf salvilenone B complete two circulate after realize being separated, the second six-way valve 11 is regulated to be in position 1,3rd six-way valve is in position 2, first current chromatographic column A1 is connected with the 3rd current chromatographic column C1, collects dihydrotanshinone Ⅰ and three leaf salvilenone B (sepn process on-line checkingi figure is shown in Fig. 9) by the 3rd collector 9.
Concentrated freeze-dried rear mensuration purity, the purity of dihydrotanshinone Ⅰ is 100% (see Figure 10); The purity of three leaf salvilenone B is 97.6% (see Figure 11).
Claims (4)
1. prepare the single-column circulation separation system of high-purity tanshinone compound for one kind, it is characterized in that comprising three current chromatographic columns (A1, B1, C1), three pumps (1,2,3), three detectors (4,5,6), three collectors (7,8,9) and three six-way valves (10,11,12); Wherein the first six-way valve (10) is sampling valve, head end and first pump (1) of this sampling valve and the first current chromatographic column (A1) are connected, the tail end of the first current chromatographic column (A1) is connected with the input terminus of the first detector (4), the output terminal of the first detector (4) is connected with the IIIth node of the second six-way valve (11), IVth node of the second six-way valve (11) is connected with the VIth node of the 3rd six-way valve (12), and the Vth node of the 3rd six-way valve (12) is connected with the first collector (7); IIth node of the second six-way valve (11) is connected with the input terminus of the second pump (2), the output terminal of the second pump (2) is connected with the head end of the second current chromatographic column (B1), the tail end of the second current chromatographic column (B1) is connected with the input terminus of the second detector (5), the output terminal of the second detector (5) is connected with the Ith node of the second six-way valve (11), and the VIth node of the second six-way valve (11) is connected with the second collector (8); Ith node of the 3rd six-way valve (12) is connected with the input terminus of the 3rd pump (3), the output terminal of the 3rd pump (3) is connected with the head end of the 3rd current chromatographic column (C1), the tail end of the 3rd current chromatographic column (C1) is connected with the input terminus of the 3rd detector (6), the output terminal of the 3rd detector (6) is connected with the IIth node of the 3rd six-way valve (12), and the IIIth node of the 3rd six-way valve (12) is connected with the 3rd collector (9).
2. utilize the single-column circulation separation system described in claim 1 to prepare the method for high-purity tanshinone compound, this tanshinone compound comprises structural formula (1) dihydrotanshinone Ⅰ, structural formula (2) three leaf salvilenone B and structural formula (3) methyltanshinone ester
its preparation comprises the following steps:
(1) after red sage root rhizome being pulverized, with the abundant soak extraction of the ethanol of volumetric concentration 95%;
(2) concentrating under reduced pressure ethanol extract, the enriched material purification on normal-phase silica gel obtained mixes sample, carry out wash-out with sherwood oil, ethyl acetate, first alcohol and water successively again, obtain the tanshinone extract being rich in dihydrotanshinone Ⅰ, three leaf salvilenone B and methyltanshinone ester;
(3) quaternary solvent system is prepared: by normal hexane, ethyl acetate, first alcohol and water by volume per-cent is respectively 68.70%, 24.91%, the quaternary solvent system of 3.64% and 2.75% preparation is as upper phase, by volume per-cent is respectively 2.53%, 24.77%, the quaternary solvent system of 49.04% and 23.66% preparation is as lower phase, three current chromatographic column (A1 are joined mutually respectively up and down by what prepare, B1, C1) in, the amount joining upper phase in each current chromatographic column is 60% of its column volume, the amount of lower phase is 40% of its column volume, wherein go up the phase that fixes mutually, do moving phase mutually down,
(4) by obtained for step (2) being rich in dihydrotanshinone Ⅰ, first three leaf salvilenone B are separated through the first current chromatographic column (A1) with the tanshinone extract of methyltanshinone ester, obtain the elutriant containing dihydrotanshinone Ⅰ and contain the elutriant of three leaf salvilenone B and methyltanshinone ester;
(5) the first detector (4) is observed, after the elutriant containing dihydrotanshinone Ⅰ washes out from the first current chromatographic column (A1), the second six-way valve (11) is regulated to be connected with the second current chromatographic column (B1) by the first current chromatographic column (A1), directly online proceed to the second current chromatographic column (B1) by containing the elutriant of dihydrotanshinone Ⅰ, observe the first detector (4), after dihydrotanshinone Ⅰ elutriant enters the second current chromatographic column (B1) completely, the second six-way valve (11) is regulated the first current chromatographic column (A1) and the second current chromatographic column (B1) to be disconnected, make dihydrotanshinone Ⅰ at the second current chromatographic column (B1) internal recycle, observe the first detector (4), after the elutriant containing three leaf salvilenone B and methyltanshinone ester washes out from the first current chromatographic column (A1), the 3rd six-way valve (12) is regulated to be connected with the 3rd current chromatographic column (C1) by the first current chromatographic column (A1), direct online the elutriant containing three leaf salvilenone B and methyltanshinone ester is proceeded to the 3rd current chromatographic column (C1), observe the first detector (4), after three leaf salvilenone B and methyltanshinone ester elutriant enter the 3rd current chromatographic column (C1) completely, the 3rd six-way valve (12) is regulated the first current chromatographic column (A1) and the 3rd current chromatographic column (C1) to be disconnected, make three leaf salvilenone B and methyltanshinone ester at the 3rd current chromatographic column (C1) internal recycle, second current chromatographic column (B1) and the 3rd current chromatographic column (C1) are when multi-cycle separation, and the first collector (7) is collected the first current chromatographic column (A1) and remained elutriant,
The chromatographic peak of (6) second detector (5) on-line checkingi representation compounds is after completing a circulation realization separation, again regulate the second six-way valve (11) to be connected with the second current chromatographic column (B1) by the first current chromatographic column (A1), the dihydrotanshinone Ⅰ in the second current chromatographic column (B1) collected by the second collector (8);
The chromatographic peak of (7) the 3rd detector (6) on-line checkingi representation compounds is after completing a circulation realization separation, again regulate the 3rd six-way valve (12) to be connected with the 3rd current chromatographic column by the first current chromatographic column (A1), the 3rd collector (9) collects three leaf salvilenone B and the methyltanshinone ester of the 3rd current chromatographic column (C1).
3. utilize the single-column circulation separation system described in claim 1 to prepare the method for high-purity tanshinone compound, this tanshinone compound comprises structural formula (4) Cryptotanshinone, structural formula (5) tanshinone Ⅰ and structural formula (6) 1,2-dihydro red sage root quinone
its preparation comprises the following steps:
(1) after red sage root rhizome being pulverized, with the abundant soak extraction of the ethanol of volumetric concentration 95%;
(2) concentrating under reduced pressure ethanol extract, the enriched material purification on normal-phase silica gel obtained mixes sample, then carries out wash-out with sherwood oil, ethyl acetate, first alcohol and water successively, obtains the tanshinone extract being rich in Cryptotanshinone, tanshinone Ⅰ and 1,2-dihydro red sage root quinone;
(3) quaternary solvent system is prepared: by normal hexane, ethyl acetate, first alcohol and water by volume per-cent is respectively 74.51%, 20.29%, the quaternary solvent system of 5.18% and 0.02% preparation is as upper phase, by volume per-cent is respectively 6.31%, 25.70%, the quaternary solvent system of 50.50% and 17.49% preparation is as lower phase, three current chromatographic column (A1 are joined mutually respectively up and down by what prepare, B1, C1) in, the amount joining upper phase in each current chromatographic column is 60% of its column volume, the amount of lower phase is 40% of its column volume, wherein go up the phase that fixes mutually, do moving phase mutually down,
(4) obtained for step (2) is rich in Cryptotanshinone, tanshinone Ⅰ and 1, first the tanshinone extract of 2-dihydro red sage root quinone is separated through the first current chromatographic column (A1), obtain containing Cryptotanshinone, tanshinone Ⅰ and the elutriant containing 1,2-dihydro red sage root quinone;
(5) the first detector (4) is observed, when containing Cryptotanshinone, after the elutriant of tanshinone Ⅰ washes out from the first current chromatographic column (A1), the second six-way valve (11) is regulated to be connected with the second current chromatographic column (B1) by the first current chromatographic column (A1), online directly will containing Cryptotanshinone, the elutriant of tanshinone Ⅰ proceeds to the second current chromatographic column (B1), observe the first detector (4), work as Cryptotanshinone, after tanshinone Ⅰ elutriant enters the second current chromatographic column (B1) completely, the second six-way valve (11) is regulated the first current chromatographic column (A1) and the second current chromatographic column (B1) to be disconnected, make Cryptotanshinone, tanshinone Ⅰ is at the second current chromatographic column (B1) internal recycle,
(6) observe the first detector (4), after the elutriant containing 1,2-dihydro red sage root quinone washes out from the first current chromatographic column (A1), be directly collected in the first collector (7);
The chromatographic peak of (7) second detector (5) on-line checkingi representation compounds is after completing two circulation realization separation, again regulate the second six-way valve (11) to be connected with the second current chromatographic column (B1) by the first current chromatographic column (A1), the second collector (8) collects Cryptotanshinone, tanshinone Ⅰ in the second current chromatographic column (B1).
4. utilize the single-column circulation separation system described in claim 1 to prepare the method for high-purity tanshinone compound, this tanshinone compound comprises structural formula (1) dihydrotanshinone Ⅰ and structural formula (2) three leaf salvilenone B,
Its preparation comprises the following steps:
(1) after red sage root rhizome being pulverized, with the abundant soak extraction of the ethanol of volumetric concentration 95%;
(2) concentrating under reduced pressure ethanol extract, the enriched material purification on normal-phase silica gel obtained mixes sample, then carries out wash-out with sherwood oil, ethyl acetate, first alcohol and water successively, obtains the tanshinone extract being rich in dihydrotanshinone Ⅰ and three leaf salvilenone B;
(3) quaternary solvent system is prepared: by normal hexane, ethyl acetate, first alcohol and water by volume per-cent is respectively 74.51%, 20.29%, the quaternary solvent system of 5.18% and 0.02% preparation is as upper phase, by volume per-cent is respectively 6.31%, 25.70%, the quaternary solvent system of 50.50% and 17.49% preparation is as lower phase, three current chromatographic column (A1 are joined mutually respectively up and down by what prepare, B1, C1) in, the amount joining upper phase in each current chromatographic column is 60% of its column volume, the amount of lower phase is 40% of its column volume, wherein go up the phase that fixes mutually, do moving phase mutually down,
(4) first the dihydrotanshinone Ⅰ that is rich in that step (2) is obtained is separated through the first current chromatographic column (A1) with the tanshinone extract of three leaf salvilenone B, obtains the elutriant containing dihydrotanshinone Ⅰ and three leaf salvilenone B;
(5) the first detector (4) is observed, after the elutriant containing dihydrotanshinone Ⅰ washes out from the first current chromatographic column (A1), the second six-way valve (11) is regulated to be connected with the second current chromatographic column (B1) by the first current chromatographic column (A1), direct online the elutriant containing dihydrotanshinone Ⅰ and three leaf salvilenone B is proceeded to the second current chromatographic column (B1), observe the first detector (4), after the elutriant containing dihydrotanshinone Ⅰ and three leaf salvilenone B enters the second current chromatographic column (B1) completely, the second six-way valve (11) is regulated the first current chromatographic column (A1) and the second current chromatographic column (B1) to be disconnected, make dihydrotanshinone Ⅰ and three leaf salvilenone B at the second current chromatographic column (B1) internal recycle,
(6) the first detector (4) is observed, after the first current chromatographic column (A1) wash-out is clean, second time sample introduction, be separated through the first current chromatographic column (A1), obtain the elutriant containing dihydrotanshinone Ⅰ and three leaf salvilenone B, after dihydrotanshinone Ⅰ washes out from the first current chromatographic column (A1), the 3rd six-way valve (12) is regulated to be connected with the 3rd current chromatographic column (C1) by the first current chromatographic column (A1), direct online the elutriant containing dihydrotanshinone Ⅰ and three leaf salvilenone B is proceeded to the 3rd current chromatographic column (C1), observe the first detector (4), after the elutriant containing dihydrotanshinone Ⅰ and three leaf salvilenone B enters the 3rd current chromatographic column (C1) completely, the 3rd six-way valve (12) is regulated the first current chromatographic column (A1) and the 3rd current chromatographic column (C1) to be disconnected, make dihydrotanshinone Ⅰ and three leaf salvilenone B at the 3rd current chromatographic column (C1) internal recycle,
The chromatographic peak of (7) second detector (5) on-line checkingi representation compounds is after completing a circulation realization separation, again regulate the second six-way valve (11) to be connected with the second current chromatographic column (B1) by the first current chromatographic column (A1), the second collector (8) collects dihydrotanshinone Ⅰ in the second current chromatographic column (B1) and three leaf salvilenone B;
The chromatographic peak of (8) the 3rd detector (6) on-line checkingi representation compounds is after completing a circulation realization separation, the 3rd six-way valve (12) is again regulated to be connected with the 3rd current chromatographic column (C1) by the first current chromatographic column (A1), the dihydrotanshinone Ⅰ in the 3rd collector (9) collection the 3rd current chromatographic column (C1) and three leaf salvilenone B.
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