CN1136210C - High-speed countercurrent chromatographic process of separating and purifying tanshinol - Google Patents
High-speed countercurrent chromatographic process of separating and purifying tanshinol Download PDFInfo
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- CN1136210C CN1136210C CNB001209868A CN00120986A CN1136210C CN 1136210 C CN1136210 C CN 1136210C CN B001209868 A CNB001209868 A CN B001209868A CN 00120986 A CN00120986 A CN 00120986A CN 1136210 C CN1136210 C CN 1136210C
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Abstract
The present invention relates to a high-speed countercurrent chromatographic method for separating and purifying tanshinol. The method comprises the steps that (1), a solvent system forming fixed phases and mobile phases is prepared so that the fixed phases are filled in a countercurrent chromatography column; the mobile phases are pumped into the column and sampled by a sampling valve; a target ingredient is received; the solvent system is prepared from n-hexane, ethanol and water; (2), part of separated objects obtained by (1) is separated and purified by a high-speed countercurrent chromatography; the solvent system comprises the n-hexane, ethyl acetate, methanol and water. An HSCCC chromatography is used, and the proper solvent system is matched. The method has the advantages of large preparation amount, high separation efficiency and short separation time, and the purities of tanshinol IIA, cryptotanshinone and tanshinol I which are obtained by separation are all more than 95%.
Description
Technical field
The present invention relates to a kind of method of using high speed adverse current chromatogram separation and purification TANSHINONES.Particularly a kind of application high speed adverse current chromatogram separation and purification Tanshinone II A, Cryptotanshinone, the method for Tanshinone I.
Technical background
The red sage root (Salivia miltiorrhiza Bunge) is a kind of conventional Chinese medicine of China, has many-sided curative effect clinical, as analgesia, antipruritic, liver cirrhosis and splenomegaly that treatment neurasthenia aypnia, advanced schistosomiasis are caused, the liver aspect that contracts of edema, gynaecopathia and icteric infectious hepatitis has certain curative effect, pharmacological research in recent years proves that the red sage root also has the effect of improving coronary circulation, suppressing the anti-inflammation detumescence of thrombotic diseases, existing patent medicine listing.Tanshinone compound is the effective ingredient in the fat soluble ingredient of red sage root, is the various index compositions that contain red sage root medicine.
But how fast, efficiently separating also from the crude extract of red sage root crude drug, the purifying tanshinone compounds is the problem that everybody relatively is concerned about.
Summary of the invention
The purpose of this invention is to provide a kind of from the red sage root crude extract method of separation and purification Tanshinone II A, Cryptotanshinone, Tanshinone I, this method technology is easy, the efficient height, product purity is greater than 95%.
For achieving the above object, the present invention takes following design: a kind of method of using high speed adverse current chromatogram separation and purification TANSHINONES, it comprises: (1), preparation constitutes stationary phase, the solvent system of moving phase, make in the counter current chromatograph pillar and be full of stationary phase, its main frame is rotated, again moving phase is pumped in the post, by the sampling valve sample introduction, according to detector spectrogram receiving target composition, described solvent system is by normal hexane, ethanol, water is formed, (2), do separation and purification by the part isolate that (1) obtains with high-speed counter-current chromatograph, described solvent system is a normal hexane, ethyl acetate, methyl alcohol, water, solvent system normal hexane described in the step (1), ethanol, the consumption volume ratio of water is 4: 2.5: 2, in the described step (2), the isolate that separates " I " that obtain from step (1) remakes separation and purification, the retention time scope of this isolate I is 2.7-3.3 hour, normal hexane in the used solvent system: ethyl acetate: methyl alcohol: the consumption volume ratio of water is 3: 2: 2.5: 1.5.
In the described step (2), the isolate that separates " III " that obtain from step (1) remakes separation and purification, the retention time scope of this isolate III is 5.3-6.4 hour, normal hexane in the solvent system of used solvent: ethyl acetate: methyl alcohol: the consumption volume ratio of water is 3: 2: 2.5: 2.
The invention will be further described below in conjunction with embodiment and accompanying drawing.
Description of drawings
Fig. 1 is (HSCCC) color atlas of half countercurrent chromatography of red sage root crude extract
Fig. 2 is (HSCCC) color atlas of the high speed adverse current chromatogram of I part in the separation graph 1
Fig. 3 is (HSCCC) color atlas of the high speed adverse current chromatogram of III part in the separation graph 1
Embodiment
1, with the red sage root crude drug of 1000g in 65% aqueous ethanolic solution of 4000ml after ultrasonic 1 hour, soaked 24 hours, suction filtration goes out the aqueous solution, adds 65% aqueous ethanolic solution of 4000ml in filter residue again, repeats above operation, carry out altogether three times, the extracting solution merging is become medicinal extract with the vacuum rotary evaporator concentrating under reduced pressure, and thickening temperature is 40 ℃, drying under reduced pressure, drying temperature is 40 ℃, gets extract 205g.
2,65% extract is dissolved in the 500ml water, extracts with the 500ml sherwood oil.
The aqueous solution that is about to extract mixes with sherwood oil, shakes up, and extract dissolves in sherwood oil, removes sherwood oil, and remainder is used petroleum ether extraction again, repetitive operation 10 times, evaporate to dryness.Get ligroin extraction 101g.
3, the ligroin extraction that separates TANSHINONES with high-speed counter-current chromatograph (New Technique Application Inst., Beijing City product):
A, applied sample amount 200mg, solvent system and amount ratio are normal hexane: ethanol: water=4: 2.5: 2 (volume), column volume: 200ml, rotating speed 800rpm, on be stationary phase mutually, is moving phase mutually down, flow velocity 2ml/min, the stationary phase retention value: 52%, detect: 254nm, see Fig. 1
Operation steps is: press all kinds of SOLVENTS volume ratio preparation solvent system, standing demix in separating funnel is told phase up and down, on be stationary phase mutually, time is moving phase mutually.Make in the counter current chromatograph pillar to be full of stationary phase, its main frame is rotated, again moving phase is pumped in the post, by the sampling valve sample introduction, according to detector spectrogram receiving target composition.
B, a separated " I " portion of product obtain with HSCCC (high-speed counter-current chromatograph) separation and purification.
Chromatographic condition: column volume: 200ml; Rotating speed: 800rpm
Solvent system: normal hexane: ethyl acetate: methyl alcohol: water=3: 2: 2.5: 1.5 (volume ratios)
Applied sample amount: 100mg, on be stationary phase mutually, is moving phase mutually down, flow velocity 2ml/min, the same a of working method, the stationary phase retention value: 55%, see Fig. 2, A is a Cryptotanshinone among the figure, B is a TANSHINONES, C the unknown.
C, the III portion of product HSCCC separation and purification that separation is obtained
Chromatographic condition: column volume: 200ml, rotating speed 800rpm.
Solvent system: normal hexane: ethyl acetate: methyl alcohol: water=3: 2: 2.5: 2 (volume ratios)
Being stationary phase mutually on the applied sample amount 80mg, is moving phase down mutually, flow velocity 2ml/min, the same a of working method, stationary phase retention value: 58%.See that the D peak is a Tanshinone II A among Fig. 3, the figure.
By the Tanshinone II A that above-mentioned condition obtains, the purity of Cryptotanshinone and Tanshinone I reaches more than 99%.
Analyze with HPLC: chromatographic condition: pillar be Intersil ODS-3 (4.6 * 15mm), moving phase is:
| Time (min) | Methyl alcohol (%) | Water (%) |
| 0.01 | 75 | 25 |
| 10 | 75 | 25 |
| 22 | 100 | 0 |
| 25 | 100 | 0 |
With detect down purity with ultraviolet 254nm.
The characteristics such as advantage of the present invention: the present invention adopts the HSCCC chromatograph, and is equipped with suitable dicyandiamide solution, and it is large to have a preparation amount, and separative efficiency is high, and disengaging time is short are separated the purity of the tanshinone IIA, Cryptotanshinone and the Tanshinone I that obtain all more than 95%.
Claims (2)
1, a kind of method of using high speed adverse current chromatogram separation and purification TANSHINONES, it is characterized in that: it comprises: (1), preparation constitutes stationary phase, the solvent system of moving phase, make in the counter current chromatograph pillar and be full of stationary phase, its main frame is rotated, again moving phase is pumped in the post, by the sampling valve sample introduction, according to detector spectrogram receiving target composition, described solvent system is by normal hexane, ethanol, water is formed, and (2) do separation and purification by the part isolate that (1) obtains with high-speed counter-current chromatograph, described solvent system is a normal hexane, ethyl acetate, methyl alcohol, water, solvent system normal hexane described in the step (1), ethanol, the consumption volume ratio of water is 4: 2.5: 2, and in the described step (2), the isolate that separates " I " that obtain from step (1) remakes separation and purification, the retention time scope of this isolate I is 2.7-3.3 hour, normal hexane in the used solvent system: ethyl acetate: methyl alcohol: the consumption volume ratio of water is 3: 2: 2.5: 1.5.
2, the method for application high speed adverse current chromatogram separation and purification TANSHINONES according to claim 1, it is characterized in that: in the step (1), the consumption volume ratio of described solvent system normal hexane, ethanol, water is 4: 2.5: 2, in the described step (2), the isolate that separates " III " that obtain from step (1) remakes separation and purification, the retention time scope of this isolate III is 5.3-6.4 hour, normal hexane in the solvent system of used solvent: ethyl acetate: methyl alcohol: the consumption volume ratio of water is 3: 2: 2.5: 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001209868A CN1136210C (en) | 2000-08-07 | 2000-08-07 | High-speed countercurrent chromatographic process of separating and purifying tanshinol |
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| CNB001209868A CN1136210C (en) | 2000-08-07 | 2000-08-07 | High-speed countercurrent chromatographic process of separating and purifying tanshinol |
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| CN1337397A CN1337397A (en) | 2002-02-27 |
| CN1136210C true CN1136210C (en) | 2004-01-28 |
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| CNB001209868A Expired - Fee Related CN1136210C (en) | 2000-08-07 | 2000-08-07 | High-speed countercurrent chromatographic process of separating and purifying tanshinol |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100393709C (en) * | 2004-03-17 | 2008-06-11 | 天津天士力现代中药资源有限公司 | Process for extracting tanshinone |
| CN1303052C (en) * | 2004-03-18 | 2007-03-07 | 中国科学院大连化学物理研究所 | Process for preparing Danshensu |
| CN1297267C (en) * | 2005-03-07 | 2007-01-31 | 四川大学 | Medicinal composition containing tanshinon II A sodium sulfonate and its quality control method |
| CN100422188C (en) * | 2006-08-25 | 2008-10-01 | 浙江大学 | Method for separating preparing tripterygium wilfordii monomer from tripterygium wilfordii by countercurrent flow chromatography |
| CN102167721B (en) * | 2011-03-24 | 2012-10-10 | 聊城大学 | Method for extracting and purifying tanshinone monomeric compounds from red sage root |
| CN105399796A (en) * | 2014-09-11 | 2016-03-16 | 宁波润沃生物科技有限公司 | Method for preparing high-purity tanshinone IIA and tanshinone I |
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