CN102093345A - Method for separating out dehydrogenized turkey corn by applying silicagel column chromatography - Google Patents
Method for separating out dehydrogenized turkey corn by applying silicagel column chromatography Download PDFInfo
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- CN102093345A CN102093345A CN 201010598262 CN201010598262A CN102093345A CN 102093345 A CN102093345 A CN 102093345A CN 201010598262 CN201010598262 CN 201010598262 CN 201010598262 A CN201010598262 A CN 201010598262A CN 102093345 A CN102093345 A CN 102093345A
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- chloroform
- dehydrocorydaline
- methyl alcohol
- dehydrogenized
- turkey
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 30
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- 229940045641 monobasic sodium phosphate Drugs 0.000 claims description 5
- 239000000049 pigment Substances 0.000 claims description 5
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 5
- RHQQHZQUAMFINJ-GKWSUJDHSA-N 1-[(3s,5s,8s,9s,10s,11s,13s,14s,17s)-3,11-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-hydroxyethanone Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CC[C@H]21 RHQQHZQUAMFINJ-GKWSUJDHSA-N 0.000 claims description 4
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a method for separating out dehydrogenized turkey corn by applying silicagel column chromatography. The method comprises the following steps: (1) adding chloroform in taken 200-300 meshes silicagel, stirring and then pouring the mixture in a chromatography column, and mounting the chromatography column; (2) dissolving ethanol crude extract of rhizome corydalis with a mixed solvent of little chloroform and methyl alcohol at a volume ratio of 50:1, producing sample solution and loading the sample, successively carrying out gradient elution with eluant of chloroform and methyl alcohol at a volume ratio of 50:1, 40:1 and 30:1, wherein the flow speed of the eluant is 1.6-2.5 ml/min, collecting eluent in equi-volume, merging the eluent in which the relative front (Rf) value and retention time are the same as that of dehydrogenized turkey corn standard product and the purify of dehydrogenized turkey corn is larger than 90%, recovering solvent with a rotary evaporator so as to obtain crude dehydrogenized turkey corn; and (3) crystallizing the obtained crude dehydrogenized turkey corn by using methyl alcohol or chloroform after the crude dehydrogenized turkey corn is decolored, so that pure dehydrogenized turkey corn is obtained. The method has the advantages of simple process and high recovery, and the purity of the dehydrogenized turkey corn obtained by separation is high.
Description
(1) technical field
The present invention relates to the applying silicon plastic column chromatography and from the corydalis tuber crude extract, separate the method that obtains the high purity Dehydrocorydaline.
(2) background technology
Corydalis tuber (Corydalis yanhusuo W.T.Wang) is the dry tuber of papaveracease (Popaveraceae) plant Yanhusuo, nature and flavor suffering, hardship, temperature, return liver, the spleen channel, main product in Zhejiang, provinces such as Jiangsu, Anhui, Sichuan, corydalis tuber is one of famous " Zhejiang eight flavors ".Corydalis tuber have invigorate blood circulation, sharp gas, lenitive effect, can be used for the chest side of body, epigastric pain, through closing dysmenorrhoea, stasis of blood resistance in postpartum, tumbling and swelling.Modern study shows that corydalis tuber effective constituent mainly is alkaloid, it is reported, kind surplus the alkaloid nearly 40 in the corydalis tuber mainly comprises: tetrahydropalmatine, protopine, Dehydrocorydaline tens of kinds of alkaloids such as (Dehydrocorydaline).The modern pharmacology experiment has proved that tetrahydropalmatine is main analgesia, calm composition in the contained alkaloid of corydalis tuber; Dehydrocorydaline has the expansion coronary vasodilator; improve coronary flow; improve the myocardial nutrition volume of blood flow; strengthen effects such as myocardial hypoxia tolerance and protection myocardial ischemia, necrosis, be the effective constituent of treatment coronary heart disease, angina drug corydalis tuber preparation " Kedaling ".In addition, Dehydrocorydaline has also been obtained curative effect preferably in the treatment Peptic Ulcers.
At present, the separation of bibliographical information Dehydrocorydaline has two kinds: a kind of is the application standard high speed adverse current chromatogram, and this method applied sample amount is little, and the instrument that uses is expensive, is difficult to be used widely; Another kind is the various column chromatographies of integrated use (macroporous adsorbent resin, aluminum oxide column chromatography and gel column chromatographies), this method complex steps, and efficient is low, and through chromatography repeatedly, the dead absorption of sample is bigger.
(3) summary of the invention
The method that the purpose of this invention is to provide a kind of separating high-purity Dehydrocorydaline, this method technology is simple, and the separation efficiency height can be used for suitability for industrialized production.
Technical scheme of the present invention is as follows:
A kind of applying silicon plastic column chromatography separates the method for Dehydrocorydaline, said method comprising the steps of:
(1) gets 200～300 purpose silica gel, add chloroform and stir, place the ultrasonator vibration not adorn post, obtain silicagel column to having to pour chromatography column into behind the bubble;
(2) with the ethanol crude extract minimum of chloroform of corydalis tuber, the methyl alcohol volume ratio is 50: 1 blended dissolution with solvents, make need testing solution, get need testing solution as sample on the sample solution, make sample solution be adsorbed on the silica gel fully, in the described sample solution in the ethanol crude extract of corydalis tuber and the silicagel column mass ratio of silica gel be 1: 30～105, use chloroform successively, the methyl alcohol volume ratio is eluent wash-out 150～250min of 50: 1, chloroform, eluent wash-out 150～250min of 40: 1 of methyl alcohol volume ratio, chloroform, 30: 1 eluent of methyl alcohol volume ratio is eluted to thin-layer chromatography and follows the tracks of and to detect no Dehydrocorydaline component to the elutriant, the eluent flow velocity is 1.6～2.5ml/min, equal-volume is collected elutriant, the elutriant that collection is obtained detects with thin-layer chromatography and high performance liquid chromatography, merges and Dehydrocorydaline standard substance R
fValue and retention time is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain the Dehydrocorydaline crude product;
(3) behind the Dehydrocorydaline crude product depigmentation that obtains of step (2) with methyl alcohol or chloroform crystallization, obtain the pure product of Dehydrocorydaline.
The ethanol crude extract of described corydalis tuber makes as follows: get the corydalis tuber pulverizing medicinal materials, with 60～98% alcohol reflux 1～5 time, the extracting solution that merges each time, the suction filtration rear filtrate is evaporated to the medicinal extract shape, with the acid solution adjust pH is 2～3, left standstill 10～30 hours, and got supernatant liquid filtering and remove precipitation; Filtrate is used chloroform extraction 1～5 time, collects the combined chloroform layer, and rotary evaporation reclaims chloroform, gets the ethanol crude extract of corydalis tuber.
Described acid solution is that mass concentration is 1～2% aqueous hydrochloric acid or 1～2% aqueous sulfuric acid.
In the described step (3), the method for Dehydrocorydaline crude product depigmentation is one of following:
(A) the Dehydrocorydaline crude product is used dropper sucking-off pigment with a small amount of anhydrous alcohol solution pigment, and residuum with methyl alcohol or chloroform crystallization, obtains the pure product of Dehydrocorydaline again;
(B) the Dehydrocorydaline crude product is dissolved in methyl alcohol, add small amount of activated, the quality consumption of gac is counted 0.01～0.02g/ml with the gained liquor capacity usually, stir 45～60min, remove by filter gac, get the filtrate rotary evaporation except that desolvating, residuum with methyl alcohol or chloroform crystallization, obtains the pure product of Dehydrocorydaline again.
The dress post is to add chloroform to stir in silica gel in the described step (1), places the ultrasonator vibration not adorn post to having to pour chromatography column into behind the bubble, and is forced into the silica gel face with the duplex ball and no longer descends.
Going up sample in the described step (2) is after treating chloroform and the silica gel face flushing, and drips sample solution to sample with dropper and is adsorbed on the silica gel fully.
Ethanol crude extract with corydalis tuber in the described step (2) is 50: 1 blended dissolution with solvents with minimum of chloroform, methyl alcohol volume ratio, and the described consoluet amount of ethanol crude extract that just can make corydalis tuber that is meant on a small quantity gets final product, and this is that those skilled in the art can understand.
The testing conditions of high performance liquid chromatography is in the described step (2): ODS C
18Column (4.6 * 250mm, 5 μ m); Column temperature: 30 ℃; Moving phase: acetonitrile-0.03mol/L SODIUM PHOSPHATE, MONOBASIC (32: 68, v/v), isocratic elution; Flow velocity: 0.6ml/min; Detect wavelength: 345nm; Sample size: 20 μ l.
The pure product of the Dehydrocorydaline that obtains can further detect with above-mentioned high performance liquid chromatography testing conditions, further determine that it is Dehydrocorydaline, and purity is greater than 98%.
Beneficial effect of the present invention is: the present invention separates the Dehydrocorydaline purity height that obtains, and technology is simple, and rate of recovery height plays an important role to making full use of of corydalis tuber medicinal material.
(4) embodiment
The present invention will be further described with specific embodiment below, but protection scope of the present invention is not limited thereto.
Embodiment 1
Get corydalis tuber medicinal material 1500g and pulverize the back with 70% alcohol reflux of 22.5L 30 minutes, by extracting 3 times with quadrat method, merge 3 times extracting solution, the suction filtration rear filtrate is evaporated to the medicinal extract shape, is 3 with 2% hydrochloric acid soln adjust pH, leaves standstill 15h, suction filtration, filtrate is reclaimed chloroform with 4 * 400mL chloroform extraction, combined chloroform layer with Rotary Evaporators, gets corydalis tuber crude extract 10.4g.
Take by weighing 200-300 order silica gel 25g, add the 300mL chloroform, stir, place the ultrasonator vibration not adorn post, obtain silicagel column to having to pour chromatography column into behind the bubble.
Take by weighing corydalis tuber crude extract 239mg (sample and silica gel ratio are 1: 105),, get sample solution with 4mL eluent (chloroform-methanol=50: 1 (v/v)) dissolving.
With dropper whole sample solutions are added drop-wise to the silica gel surface and make it be adsorbed in silica gel fully.
Be 50: 1 40: 1 eluent wash-out 250min of eluent wash-out 250min, chloroform, methyl alcohol volume ratio successively with chloroform, methyl alcohol volume ratio, chloroform, 30: 1 eluent of methyl alcohol volume ratio are eluted to thin-layer chromatography and follow the tracks of and to detect no Dehydrocorydaline component to the elutriant, the eluent flow velocity is 2.5ml/min, collect elutriant with Erlenmeyer flask, every 50ml collects one bottle, the elutriant that collection is obtained detects with thin-layer chromatography and high performance liquid chromatography, merges and Dehydrocorydaline standard substance R
fValue and retention time is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain the Dehydrocorydaline crude product.
Dehydrocorydaline crude product 3mL anhydrous alcohol solution pigment, with dropper with its sucking-off, remaining composition 5mL methanol crystallization, obtain yellow crystals 26mg, the crystal that obtains is carried out high performance liquid chromatography to be detected, further determine that it is Dehydrocorydaline, purity is 99.6%, and the rate of recovery is 84.9% (in the corydalis tuber crude extract).
The testing conditions of high performance liquid chromatography is: ODS C
18Column (4.6 * 250mm, 5 μ m); Column temperature: 30 ℃; Moving phase: acetonitrile-0.03mol/L SODIUM PHOSPHATE, MONOBASIC (32: 68, v/v), isocratic elution; Flow velocity: 0.6ml/min; Detect wavelength: 345nm; Sample size: 20 μ l.
Embodiment 2
Take by weighing 200-300 order silica gel 20g, add the 250mL chloroform, stir, place the ultrasonator vibration not adorn post, obtain silicagel column to having to pour chromatography column into behind the bubble.
Take by weighing embodiment 1 and extract the corydalis tuber crude extract 368mg (sample and silica gel ratio are 1: 54) that obtains,, get sample solution with 5mL eluent (chloroform-methanol=50: 1 (v/v)) dissolving.
With dropper whole sample solutions are added drop-wise to the silica gel surface and make it be adsorbed in silica gel fully.
Be 50: 1 40: 1 eluent wash-out 200min of eluent wash-out 150min, chloroform, methyl alcohol volume ratio successively with chloroform, methyl alcohol volume ratio, chloroform, 30: 1 eluent of methyl alcohol volume ratio are eluted to thin-layer chromatography and follow the tracks of and to detect no Dehydrocorydaline component to the elutriant, the eluent flow velocity is 2.0ml/min, collect elutriant with Erlenmeyer flask, every 50ml collects one bottle, the elutriant that collection is obtained detects with thin-layer chromatography and high performance liquid chromatography, merges and Dehydrocorydaline standard substance R
fValue and retention time is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain the Dehydrocorydaline crude product.
Dehydrocorydaline crude product 40mL dissolve with methanol, add the 0.4g gac, stir 45min, remove by filter gac, get the filtrate rotary evaporation except that desolvating, residuum is used the 6mL methanol crystallization again, obtain yellow crystals 37mg, determine that it is Dehydrocorydaline, purity 99.6%, the rate of recovery 78.4% (in the corydalis tuber crude extract) through the high performance liquid phase detection.
The testing conditions of high performance liquid chromatography is: ODS C
I8Column (4.6 * 250mm, 5 μ m); Column temperature: 30 ℃; Moving phase: acetonitrile-0.03mol/L SODIUM PHOSPHATE, MONOBASIC (32: 68, v/v), isocratic elution; Flow velocity: 0.6ml/min; Detect wavelength: 345nm; Sample size: 20 μ l.
Embodiment 3
Take by weighing 200-300 order silica gel 9g, add the 200mL chloroform, stir, place the ultrasonator vibration not adorn post, obtain silicagel column to having to pour chromatography column into behind the bubble.
Take by weighing embodiment 1 and extract the corydalis tuber crude extract 296mg (sample and silica gel ratio are 1: 30) that obtains,, get sample solution with 4mL eluent (chloroform-methanol=50: 1 (v/v)) dissolving.
With dropper whole sample solutions are added drop-wise to the silica gel surface and make it be adsorbed in silica gel fully.
Use chloroform successively, the methyl alcohol volume ratio is 50: 1 eluent wash-out 150min, chloroform, 40: 1 eluent wash-out 150min of methyl alcohol volume ratio, chloroform, 30: 1 eluent of methyl alcohol volume ratio is eluted to thin-layer chromatography and follows the tracks of and to detect no Dehydrocorydaline component to the elutriant, the eluent flow velocity is 1.6ml/min, collect elutriant with Erlenmeyer flask, every 50ml collects one bottle, the elutriant that collection is obtained detects with thin-layer chromatography and high performance liquid chromatography, merge with Dehydrocorydaline standard substance Rf value and retention time is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain the Dehydrocorydaline crude product.
Dehydrocorydaline crude product 55mL dissolve with methanol, add the 1.1g gac, stir 60min, remove by filter gac, get the filtrate rotary evaporation except that desolvating, residuum is used the crystallization of 5mL chloroform again, obtain yellow crystals 30mg, be defined as Dehydrocorydaline through the high performance liquid phase detection, purity 99.4%, the rate of recovery are 78.9% (in the corydalis tuber crude extract).
The testing conditions of high performance liquid chromatography is: ODS C
18Column (4.6 * 250mm, 5 μ m); Column temperature: 30 ℃; Moving phase: acetonitrile-0.03mol/L SODIUM PHOSPHATE, MONOBASIC (32: 68, v/v), isocratic elution; Flow velocity: 0.6ml/min; Detect wavelength: 345nm; Sample size: 20 μ l.
Claims (5)
1. an applying silicon plastic column chromatography separates the method for Dehydrocorydaline, it is characterized in that said method comprising the steps of:
(1) gets 200～300 purpose silica gel, add chloroform and stir, place the ultrasonator vibration not adorn post, obtain silicagel column to having to pour chromatography column into behind the bubble;
(2) with the ethanol crude extract minimum of chloroform of corydalis tuber, the methyl alcohol volume ratio is 50: 1 blended dissolution with solvents, make need testing solution, get need testing solution as sample on the sample solution, make sample solution be adsorbed on the silica gel fully, in the described sample solution in the ethanol crude extract of corydalis tuber and the silicagel column mass ratio of silica gel be 1: 30～105, use chloroform successively, the methyl alcohol volume ratio is eluent wash-out 150～250min of 50: 1, chloroform, eluent wash-out 150～250min of 40: 1 of methyl alcohol volume ratio, chloroform, 30: 1 eluent of methyl alcohol volume ratio is eluted to thin-layer chromatography and follows the tracks of and to detect no Dehydrocorydaline component to the elutriant, the eluent flow velocity is 1.6～2.5ml/min, equal-volume is collected elutriant, the elutriant that collection is obtained detects with thin-layer chromatography and high performance liquid chromatography, merges and Dehydrocorydaline standard substance R
fValue and retention time is identical and Dehydrocorydaline purity greater than 90% elutriant, reclaim solvent with Rotary Evaporators, obtain the Dehydrocorydaline crude product;
(3) behind the Dehydrocorydaline crude product depigmentation that obtains of step (2) with methyl alcohol or chloroform crystallization, obtain the pure product of Dehydrocorydaline.
2. the method for claim 1, the ethanol crude extract that it is characterized in that described corydalis tuber makes as follows: get the corydalis tuber pulverizing medicinal materials, with 60～98% alcohol reflux 1～5 time, the extracting solution that merges each time, the suction filtration rear filtrate is evaporated to the medicinal extract shape, with the acid solution adjust pH is 2～3, leaves standstill 10～30 hours, gets supernatant liquid filtering and removes precipitation; Filtrate is used chloroform extraction 1～5 time, collects the combined chloroform layer, and rotary evaporation reclaims chloroform, gets the ethanol crude extract of corydalis tuber.
3. method as claimed in claim 2 is characterized in that described acid solution is that mass concentration is 1～2% aqueous hydrochloric acid or 1～2% aqueous sulfuric acid.
4. the method for claim 1 is characterized in that in the described step (3), and the method for Dehydrocorydaline crude product depigmentation is one of following:
(A) the Dehydrocorydaline crude product is used dropper sucking-off pigment with a small amount of anhydrous alcohol solution pigment, and residuum with methyl alcohol or chloroform crystallization, obtains the pure product of Dehydrocorydaline again;
(B) the Dehydrocorydaline crude product is dissolved in methyl alcohol, adds small amount of activated, stirs 45～60min, removes by filter gac, gets the filtrate rotary evaporation except that desolvating, and residuum with methyl alcohol or chloroform crystallization, obtains the pure product of Dehydrocorydaline again.
5. the method for claim 1 is characterized in that the testing conditions of high performance liquid chromatography in the described step (2) is: ODS C
18Column (4.6 * 250mm, 5 μ m); Column temperature: 30 ℃; Moving phase: acetonitrile-0.03mol/L SODIUM PHOSPHATE, MONOBASIC (32: 68, v/v), isocratic elution; Flow velocity: 0.6ml/min; Detect wavelength: 345nm; Sample size: 20 μ l.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010598262 CN102093345B (en) | 2010-12-21 | 2010-12-21 | Method for separating out dehydrogenized turkey corn by applying silicagel column chromatography |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010598262 CN102093345B (en) | 2010-12-21 | 2010-12-21 | Method for separating out dehydrogenized turkey corn by applying silicagel column chromatography |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103616344A (en) * | 2013-12-12 | 2014-03-05 | 山东阿如拉药物研究开发有限公司 | Detection method of corydalis impatiens medicinal material |
| CN109503571A (en) * | 2018-12-06 | 2019-03-22 | 金华职业技术学院 | A kind of method of dehydrocorydaline in D101 macroporous resin purification rhizoma corydalis |
| CN114088829A (en) * | 2021-11-04 | 2022-02-25 | 天津中医药大学 | Detection method and application of dehydrocorydaline and metabolite thereof |
| CN116602966A (en) * | 2023-05-11 | 2023-08-18 | 华宝民康(广东)医药集团有限公司 | Application of 13-methyl-palmatine in preparation of medicines for resisting myocardial ischemia injury and heart fibrosis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1634295A (en) * | 2004-09-27 | 2005-07-06 | 方芳 | Preparation method for climbing nightshade extract |
| CN101480394A (en) * | 2008-01-09 | 2009-07-15 | 上海安普生物科技有限公司 | Anti-tumor pharmaceutical composition |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1634295A (en) * | 2004-09-27 | 2005-07-06 | 方芳 | Preparation method for climbing nightshade extract |
| CN101480394A (en) * | 2008-01-09 | 2009-07-15 | 上海安普生物科技有限公司 | Anti-tumor pharmaceutical composition |
Non-Patent Citations (3)
| Title |
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| 《中国中医药科技》 20090331 徐世芳 延胡索中盐酸脱氢紫堇碱对照品的制备与鉴定 129-130页 1-5 第16卷, 第2期 * |
| 《时珍国医国药》 20090228 白雪 延胡索中紫堇碱标准品的制备及含量测定 374-375页 1-5 第20卷, 第2期 * |
| 《时珍国医国药》 20090430 杨杰 延胡索中去氢紫堇碱对照品的制备及含量测定 1000-1002页 1-5 第20卷, 第4期 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103616344A (en) * | 2013-12-12 | 2014-03-05 | 山东阿如拉药物研究开发有限公司 | Detection method of corydalis impatiens medicinal material |
| CN103616344B (en) * | 2013-12-12 | 2016-03-16 | 山东金诃药物研究开发有限公司 | A kind of detection method of Tibetan medicine material Corydalis impatiens (Pall.) Fisch |
| CN109503571A (en) * | 2018-12-06 | 2019-03-22 | 金华职业技术学院 | A kind of method of dehydrocorydaline in D101 macroporous resin purification rhizoma corydalis |
| CN114088829A (en) * | 2021-11-04 | 2022-02-25 | 天津中医药大学 | Detection method and application of dehydrocorydaline and metabolite thereof |
| CN116602966A (en) * | 2023-05-11 | 2023-08-18 | 华宝民康(广东)医药集团有限公司 | Application of 13-methyl-palmatine in preparation of medicines for resisting myocardial ischemia injury and heart fibrosis |
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| CN102093345B (en) | 2013-03-27 |
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