CN100500689C - Method of separating and preparing wild jujube seed saponin - Google Patents
Method of separating and preparing wild jujube seed saponin Download PDFInfo
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- CN100500689C CN100500689C CNB2007100996811A CN200710099681A CN100500689C CN 100500689 C CN100500689 C CN 100500689C CN B2007100996811 A CNB2007100996811 A CN B2007100996811A CN 200710099681 A CN200710099681 A CN 200710099681A CN 100500689 C CN100500689 C CN 100500689C
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Abstract
The present invention relates to high speed countercurrent chromatography method for separating high purity monomer spine date seed saponin A and B from its extract which is spine date seed crude material extracted by butanol or the 70% ethanol:water ingredient through large pore resin ethanol gradient elution. It is characterized in that the two-phase system is aqueous system, and the solvent can be normal alkane, ether, aliphatic ester, aliphatic alcohol, aliphatic ketone, water etc. The aqueous solvent system is composed of three ingredients, in which A ingredient is selected from ligarine, normal hexane, normal pentane etc normal alkane or ethyl acetate, butyl acetate etc., B is selected from aliphatic alcohol, aliphatic ketone as acetonitrile, methanol, ethanol, acetone etc., C is water. The method is applicable in countercurrent chromatography of all kinds of type. Large amount of crude product or mixture can be separated well and the final product can achieve high purity.
Description
Technical field
The present invention relates to a kind of high-speed countercurrent chromatography that adopts and from the Spina Date Seed crude extract, separate the preparation separation method of preparing high-purity monomer jujuboside A and B.
Background technology
Spina Date Seed is the dry mature seed of Rhamnaceae plant wild jujube ZiziphusjujubaMill.Var.spinosa (Bunge) Hu exH.F.chou.Spina Date Seed is the first-selected tranquilizing by nourishing the heart Chinese medicine that Chinese Pharmacopoeia records, the effect of tonifying liver, the peaceful heart, arrest sweating are arranged, promoting the production of body fluid, and it is thirsty to be mainly used in treatment restlessness of asrhenia type and insomnia, palpitation with fear dreaminess, the empty hidrosis of body and Tianjin wound.Shennong's Herbal is classified as top grade, and Compendium of Material Medica is classified this category as, classifies the medicine for nourishing the heart and tranquilizing the mind class as in " Chinese materia medica " textbook.Clinical application and pharmacological evaluation prove that all Spina Date Seed has significant sedative-hypnotic effect.Along with the development of society, people's living pressure of working is increasing, and symptoms such as the anxiety of Chan Shenging, insomnia are also constantly perplexing people's orthobiosis thereupon, and is just urgent day by day for the research and development of medicine for nourishing the heart and tranquilizing the mind.Wherein jujuboside A and B are one of effective constituent of its tranquilizing soporific.
Existing bibliographical information adopts the method for column chromatography and high performance liquid chromatography to separate, through the reverse phase silica gel post, carbon 18 posts, and Sephadex LH-20 post crude product behind the n-butanol extraction separates and obtains jujuboside A, perhaps separate and obtain jujuboside A, B through the repeated multiple times silicagel column, technology is loaded down with trivial details, the cost height, and the rate of recovery is low.The adverse current chromatogram method for separating and preparing can reach high purity, cost is low, and rate of recovery height is better than traditional column chromatography and high performance liquid chromatography separation method both ways, there is not on the one hand solid state adhesion body not have the irreversible adsorption loss, on the other hand the direct upper prop of crude product.Because high speed adverse current chromatogram (High-speed Countercurrent Chromatography, HSCCC) be that a kind of successive that grew up in recent years need not the efficient of any solid support, liquid liquid distribution chromatography isolation technique fast, the variety of issue that it has avoided solid state adhesion body or carrier to bring---sample easily is adsorbed, loss and sex change, when using other liquid phase chromatography amount of being prepared to separate, its allocative efficiency can obviously reduce, solvent-oil ratio is big, HSCCC guarantees higher peak type resolution, fractional dose is big, the sample free of losses, rate of recovery height, isolating environment relaxes, and saves solvent.Counter current chromatograph can directly advance slightly get sample in a large number product or synthetic mixture, and separating resulting can reach quite high purity, even can directly connect instruments such as mass spectrograph, has been widely used in the preparation separation and the purifying of field chemical substances such as biology, medicine, environmental protection.
Summary of the invention
The objective of the invention is Spina Date Seed crude product with n-butanol extraction and be raw material or adopt macroporous resin from content only be 0.3% jujuboside A and B to bring up to about 50% be raw material, the method for high speed adverse current chromatogram obtains jujuboside A and the B of purity more than 95% then.The solution of the present invention is: the Spina Date Seed crude product raw material of n-butanol extraction or this crude product adopt macroporous resin to obtain 70% ethanol by ethanol gradient elution: the water elution component is separated preparation high purity acid jujube kernel saponin A and B by counter current chromatography: selective solvent two combined, available two-phase system is an aqueous solvent system.Aqueous solvent system is made of three components, and the A component can be selected from normal paraffin or ester classes such as ethyl acetate, butylacetate such as sherwood oil, normal hexane, Skellysolve A.Fatty Alcohol(C12-C14 and C12-C18) and aliphatic ketones such as the optional acetonitrile of B component, methyl alcohol, ethanol, acetone.The C component is a water.
Adopt aqueous solvent system, below be stationary phase mutually, be moving phase mutually down, under guaranteeing that phase volume ratio is less than 1 prerequisite up and down, according to solubility constant, do not destroying under the system equilibrated situation, regulate the volume ratio of A: B: C three components: 10-5: 1: 10-5 obtains jujuboside A and B through a counter-current separation.
Experiment condition is fit to room temperature 15-30 ℃, and room temperature is little to the separation efficiency influence.In the said temperature scope, when room temperature was higher, appearance time slightly shifted to an earlier date, and separation efficiency changes little, and jujuboside A and B peak shape are not had influence.
At first by volume above-mentioned solvent system is disposed in the separating funnel, shakes up the back standing demix.Ready to balance after for some time separates upper and lower phase.Adopt analysis mode or half countercurrent chromatography instrument, be furnished with the NS-1007 pump, 2mL or 20mL sampling valve, tetrafluoroethylene post, column volume are 40 or 240mL, the 8823A-UV UV-detector, Yokogawa 3057 potable recording instrument, the FC-95 automatic fraction collector.(70% ethanol: water) elution fraction is dissolved in the moving phase stand-by with the jujuboside crude extract or by the acquisition of macroporous resin ethanol gradient elution.Before the sample introduction, earlier be filled with whole pillar with stationary phase, the adjustment engine speed is 500-1000rpm (half preparation type instrument rotating speed) 1500-2000rpm (analysis mode instrument rotating speed), flow velocity with 0.5-3.0mL/min pumps into moving phase in the post, after treating that whole system is set up running balance, by the sampling valve sample introduction; According to the detector uv atlas, the receiving target composition obtains solid then.
Jujuboside A and its purity of B of extracting with present method can reach more than 95%.Be applicable to that the counter current chromatograph with various models separates preparation jujuboside A and B monomer, can directly advance slightly get sample in a large number product or synthetic mixture, separating resulting can reach quite high purity.
Embodiment
Embodiment 1:
Choose normal hexane-alcohol-water on half preparation type counter current chromatograph separation and purification jujuboside extract (the Spina Date Seed crude product raw material of n-butanol extraction or this crude product adopt macroporous resin to obtain 70% ethanol by ethanol gradient elution: the water elution component), at first above-mentioned solvent composition is disposed in the separating funnel, shakes up the back standing demix by 8: 1: 8 volume ratios.Ready to balance after for some time separates upper and lower phase, takes off as stationary phase, and is last as moving phase.Adopt half countercurrent chromatography instrument, be furnished with the NS-1007 pump, the 20mL sampling valve, tetrafluoroethylene post, column volume are 240mL, 8823A-UV UV-detector, Yokogawa 3057 potable recording instrument, FC-95 automatic fraction collector.The Spina Date Seed crude product material dissolution that takes by weighing the 300mg n-butanol extraction is stand-by in 20mL moving phase.Before the sample introduction, earlier be filled with whole pillar with stationary phase, the adjustment engine speed is 800rpm, with the flow velocity of 2.0mL/min moving phase is pumped in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction; Then according to the detector uv atlas, the receiving target composition, its HPLC purity reaches about 96%.
Embodiment 2:
Choose butylacetate-methanol-water and on half preparation type counter current chromatograph, separate the jujuboside extract.By 5: 1: 5 volume ratios above-mentioned solvent composition is disposed in the separating funnel earlier, shakes up the back standing demix.Ready to balance after for some time separates upper and lower phase, takes off as stationary phase, and is last as moving phase.Adopt half countercurrent chromatography instrument, be furnished with the NS-1007 pump, the 20mL sampling valve, tetrafluoroethylene post, column volume are 240mL, 8823A-UV UV-detector, Yokogawa 3057 potable recording instrument, FC-95 automatic fraction collector.The Spina Date Seed crude product that takes by weighing the 100mg n-butanol extraction adopts macroporous resin to obtain 70% ethanol by ethanol gradient elution: the water elution components dissolved is stand-by in 20mL moving phase, before the sample introduction, earlier be filled with whole pillar with stationary phase, the adjustment engine speed is 800rpm, with the flow velocity of 2.0mL/min moving phase is pumped in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction; Then according to the detector uv atlas, the receiving target composition, its HPLC purity reaches about 97%.
Embodiment 3:
Get ethyl acetate-methanol-water component and on the analysis mode counter current chromatograph, separate the jujuboside extract.At first above-mentioned solvent composition is disposed in the separating funnel, shakes up the back standing demix by 10: 1: 10 volume ratios.Ready to balance after for some time separates upper and lower phase, takes off as stationary phase, and is last as moving phase.Adopt the analysis mode high-speed counter-current chromatograph, be furnished with the NS-1007 pump, the 1mL sampling valve, tetrafluoroethylene post, column volume are 40mL, 8823A-UV UV-detector, Yokogawa 3057 potable recording instrument, FC-95 automatic fraction collector.The Spina Date Seed dissolving crude product that takes by weighing the 15mg n-butanol extraction is stand-by in 1mL moving phase.Before the sample introduction, earlier be filled with whole pillar with stationary phase, the adjustment engine speed is 1600rpm, with the flow velocity of 1.0mL/min moving phase is pumped in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction; Then according to the detector uv atlas, the receiving target composition, its HPLC purity reaches more than 96%.
Embodiment 4:
Choose ethyl acetate-acetonitrile-water and on half preparation type counter current chromatograph, separate the jujuboside extract, at first above-mentioned solvent composition is disposed in the separating funnel, shake up the back standing demix by 6: 1: 8 volume ratios.Ready to balance after for some time separates upper and lower phase, takes off as stationary phase, and is last as moving phase.Adopt half countercurrent chromatography instrument, be furnished with the NS-1007 pump, the 20mL sampling valve, tetrafluoroethylene post, column volume are 240mL, 8823A-UV UV-detector, Yokogawa 3057 potable recording instrument, FC-95 automatic fraction collector.The Spina Date Seed crude product that takes by weighing the 370mg n-butanol extraction adopts macroporous resin to obtain 70% ethanol by ethanol gradient elution: the water elution components dissolved is stand-by in 20mL moving phase.Before the sample introduction, earlier be filled with whole pillar with stationary phase, the adjustment engine speed is 800rpm, with the flow velocity of 2.0mL/min moving phase is pumped in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction; Then according to the detector uv atlas, the receiving target composition, its HPLC purity reaches about 97%.
Embodiment 5:
Choose ethyl acetate, alcohol and water and on half preparation type counter current chromatograph, separate the jujuboside extract.At first above-mentioned solvent composition is disposed in the separating funnel, shakes up the back standing demix by 5: 1: 8 volume ratios.Ready to balance after for some time separates upper and lower phase, takes off as stationary phase, and is last as moving phase.Adopt half countercurrent chromatography instrument, be furnished with the NS-1007 pump, the 20mL sampling valve, tetrafluoroethylene post, column volume are 240mL, 8823A-UV UV-detector, Yokogawa 3057 potable recording instrument, FC-95 automatic fraction collector.The Spina Date Seed dissolving crude product that takes by weighing the 360mg n-butanol extraction is stand-by in 20mL moving phase.Before the sample introduction, earlier be filled with whole pillar with stationary phase, the adjustment engine speed is 800rpm, with the flow velocity of 2.0mL/min moving phase is pumped in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction; Then according to the detector uv atlas, the receiving target composition, its HPLC purity reaches about 96%.
Embodiment 6:
Choose isopropyl acetate-acetonitrile-water and on half preparation type counter current chromatograph, separate the jujuboside extract, at first above-mentioned solvent composition is disposed in the separating funnel, shake up the back standing demix by 6: 1: 6 volume ratios.Ready to balance after for some time separates upper and lower phase, takes off as stationary phase, and is last as moving phase.Adopt half countercurrent chromatography instrument, be furnished with the NS-1007 pump, the 20mL sampling valve, tetrafluoroethylene post, column volume are 240mL, 8823A-UV UV-detector, Yokogawa 3057 potable recording instrument, FC-95 automatic fraction collector.The Spina Date Seed crude product that takes by weighing the 365mg n-butanol extraction adopts macroporous resin to obtain 70% ethanol by ethanol gradient elution: the water elution components dissolved is stand-by in 20mL moving phase.Before the sample introduction, earlier be filled with whole pillar with stationary phase, the adjustment engine speed is 800rpm, with the flow velocity of 2.0mL/min moving phase is pumped in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction; According to the detector uv atlas, obtain yellow solid then, its HPLC purity reaches about 97%.
Embodiment 7:
Choose sherwood oil-alcohol-water and on half preparation type counter current chromatograph, separate the jujuboside extract, at first above-mentioned solvent composition is disposed in the separating funnel, shake up the back standing demix by 7: 1: 7 volume ratios.Ready to balance after for some time separates upper and lower phase, takes off as stationary phase, and is last as moving phase.Adopt half countercurrent chromatography instrument, be furnished with the NS-1007 pump, the 20mL sampling valve, tetrafluoroethylene post, column volume are 240mL, 8823A-UV UV-detector, Yokogawa 3057 potable recording instrument, FC-95 automatic fraction collector.The Spina Date Seed crude product that takes by weighing the 300mg n-butanol extraction adopts macroporous resin to obtain 70% ethanol by ethanol gradient elution: the water elution components dissolved is stand-by in 20mL moving phase.Before the sample introduction, earlier be filled with whole pillar with stationary phase, the adjustment engine speed is 800rpm, with the flow velocity of 2.0mL/min moving phase is pumped in the post; After treating that whole system is set up running balance, by the sampling valve sample introduction; According to the detector uv atlas, obtain yellow solid then, its HPLC purity reaches about 97%.
Claims (6)
1. the method for separating and preparing of jujuboside A and B, it is characterized in that: it is to adopt counter current chromatography to separate from the jujuboside extract to prepare high purity acid jujube kernel saponin A and B, its solvent can be normal paraffin, Fatty Alcohol(C12-C14 and C12-C18), aliphatic ketone, fatty ester, nitrile, ethers, water, and the component of solvent can be made of three components wherein;
The Spina Date Seed crude product raw material that wherein said jujuboside extract is a n-butanol extraction or this crude product adopt macroporous resin to obtain 70% ethanol by ethanol gradient elution: the water elution component.
2. the method for separating and preparing of a kind of jujuboside A according to claim 1 and B, it is characterized in that: the aqueous solvent system that its three components constitute is to be made of normal paraffin or fatty ester, acetonitrile or Fatty Alcohol(C12-C14 and C12-C18) or aliphatic ketone, water, and volume ratio is followed successively by: 10-5:1:10-5.
3. the method for separating and preparing of a kind of jujuboside A according to claim 1 and B is characterized in that: normal paraffin is normal hexane, normal heptane, Skellysolve A.
4. the method for separating and preparing of a kind of jujuboside A according to claim 1 and B, it is characterized in that: Fatty Alcohol(C12-C14 and C12-C18), aliphatic ketone are: methyl alcohol, ethanol, acetone.
5. the method for separating and preparing of a kind of jujuboside A according to claim 1 and B is characterized in that: fatty ester is ethyl acetate, propyl acetate, isopropyl acetate, n-butyl acetate.
6. the method for separating and preparing of a kind of jujuboside A according to claim 1 and B is characterized in that: ethers is ether, sherwood oil, methyl tertiary butyl ether.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007100996811A CN100500689C (en) | 2007-05-28 | 2007-05-28 | Method of separating and preparing wild jujube seed saponin |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2007100996811A CN100500689C (en) | 2007-05-28 | 2007-05-28 | Method of separating and preparing wild jujube seed saponin |
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| CN101054404A CN101054404A (en) | 2007-10-17 |
| CN100500689C true CN100500689C (en) | 2009-06-17 |
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| CNB2007100996811A Expired - Fee Related CN100500689C (en) | 2007-05-28 | 2007-05-28 | Method of separating and preparing wild jujube seed saponin |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101926865B (en) * | 2010-09-11 | 2012-01-25 | 天津医科大学 | Spina date seed depression-resolving and nerve-soothing composition and preparation method thereof |
| CN109953995A (en) * | 2017-12-14 | 2019-07-02 | 复旦大学 | A kind of pharmaceutical composition and its preparing the purposes in hypnotic drug |
| CN113214349B (en) * | 2021-05-14 | 2022-07-26 | 华南理工大学 | Spina date seed saponin, preparation thereof and application thereof in preparing hypoglycemic drugs |
| CN114487181B (en) * | 2022-01-20 | 2024-03-19 | 广西壮族自治区食品药品检验所 | Method for measuring content of spine date seed saponin A and saponin B in Tianwang heart tonifying preparation |
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Granted publication date: 20090617 Termination date: 20120528 |