CN106967136A - A kind of method of separating high-purity oleuropein - Google Patents
A kind of method of separating high-purity oleuropein Download PDFInfo
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- CN106967136A CN106967136A CN201710231654.9A CN201710231654A CN106967136A CN 106967136 A CN106967136 A CN 106967136A CN 201710231654 A CN201710231654 A CN 201710231654A CN 106967136 A CN106967136 A CN 106967136A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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Abstract
The present invention relates to the method that a kind of separation of utilization counter current chromatography prepares high-purity oleuropein.The present invention extracts olive growing leaves using ethanol solution first, and then extractive of olive growing leaves is separated by separation means of countercurrent chromatography, and method includes preparing the dicyandiamide solution for being constituted mobile phase and stationary phase with n-butanol ethyl acetate methanol water；Stationary phase is filled to the splitter of full counter-current chromatograph, and is pumped into mobile phase to two-phase and reaches balance；Mobile phase is eluted after sample introduction valve injection；According to detector collection of illustrative plates and combination HPLC detection collection target components；Oleuropein monomeric compound is obtained after flow point containing oleuropein is merged into recycling design.The oleuropein compound that purity is more than 90% can be obtained by this method.This method is simple and efficient, good separating effect, and preparation amount is big, and sample loss is small, and separation costs are low, and product purity is high, and whole separation method is suitable for industrialized production.
Description
Technical field
The invention belongs to Separation of Natural Products technical field, and in particular to a kind of method of separating high-purity oleuropein.
Background technology
Oleuropein (Oleuropein) is a kind of iridoid glycosides compound, and modern pharmacology research shows：Olive
Bitter glycosides has anti-inflammatory, antimycotic, antiviral, anti-oxidant, anticancer and the various active such as hypoglycemic.
In recent years, domestic and foreign scholars are benefited our pursuits to the method for extraction and purification of oleuropein.It is such as entitled
The Chinese invention patent of " a kind of method of purifying oleuropein " (Application No. 201010549802.X) discloses a kind of from oil
The method that oleuropein is extracted in olive leaf：Olive growing leaves are extracted with ethanol, activated carbon decolorizing is then added, reclaimed after ethanol
With extracting n-butyl alcohol, separated after extracting n-butyl alcohol phase recycling design with countercurrent chromatography instrument, purity can be obtained
More than 90% oleuropein sterling.But the patent, using activated carbon decolorizing and extracting n-butyl alcohol, solvent expends more, reclaims
N-butanol energy consumption is big, and the last process rate of recovery is relatively low, and whole technique oleuropein yield only reaches 1% or so.Invention name
Claim a kind of " preparation method of oleuropein " (application number：201510270831.5) Chinese invention patent disclose one kind and prepare
The method of oleuropein, is, using ethanol as entrainer, to be passed through CO by olive growing leaves2Extracted, after gained extract is concentrated
With macroporous resin adsorption, eluate is separated with high speed adverse current chromatogram, with chloroform-methanol-water (10:5:7, V:V) it is two-phase solvent,
Prepare oleuropein.The patent uses supercritical CO2Extracted, equipment and process costs are higher, constrain entering for it
One step develops.
The content of the invention
For defect present in above-mentioned prior art and deficiency, it is an object of the invention to provide a kind of separating high-purity
The method of oleuropein, on the basis of the high-purity oleuropein of acquisition, also obtains bigger yield, and by reducing technique
Step reduces production cost, so as to solve problems of the prior art.
The method of the separating high-purity oleuropein of the present invention, including the steps：
1) extractive of olive growing leaves is prepared：
Extracted using 60%-90% (volume ratio) ethanol from olive growing leaves and obtain extractive of olive growing leaves,
Described extracting method is soak extraction method or heating and refluxing extraction
2) selection of solvent system is separated：
N-butanol, ethyl acetate, first alcohol and water are mixed as separation solvent system, stratification after shaking will be upper and lower
Mutually separate respectively as stationary phase and mobile phase；
As the preferred of embodiment, wherein n-butanol, ethyl acetate, the volume ratio of first alcohol and water are 1:19:1:19；
3) stationary phase is full of to the splitter of counter-current chromatograph first, counter-current chromatograph main frame is then started, reaches setting
After rotating speed, then mobile phase is pumped into thereto, after two-phase reaches dynamic equilibrium in splitter, by sampling valve by olive growing leaves
Extract injects, and automatic fraction collector receives cut；
4) by the cut of collection by high performance liquid chromatography detection, merge after the cut containing oleuropein, recycling design
Obtain oleuropein.
Described step 2) in, the pH value of solvent for use system is 2-14.
Described step 2) in, can be using upper as stationary phase in solvent system, lower phase is mobile phase；Can also be with molten
Lower in agent system is mutually stationary phase, and upper phase is mobile phase.
Described step 3) in, counter-current chromatograph can use co-rotating mode, it would however also be possible to employ reversing mode.
Advantage of the present invention is that separation costs are low, safe preparation process, and separation process can be carried out continuously, operation letter
Single, oleuropein product purity can reach more than 90%.Compared with the method such as column chromatography, it without using solid-state carrier, because
Sample Irreversible Adsorption and the loss that solid carrier is caused is not present in this, and separative efficiency is high, and whole separating technology is suitable for industry
Metaplasia is produced, and raw material can be utilized to greatest extent, reduction production cost is a kind of efficient quick from extractive of olive growing leaves
The method of separating high-purity oleuropein.
Embodiment
Separation equipment counter-current chromatograph used in the embodiment of the present invention, can according to the difference of required target fractional dose
With selection analysis type, semi-preparative and preparative counter-current chromatograph.
The method of counter current chromatography separating high-purity oleuropein, including with n-butanol-acetate-methanol-water according to
Certain volume constitutes adverse current chromatogram stationary phase, the dicyandiamide solution of mobile phase than stratification after mixing；By stationary phase with larger stream
Speed is pumped into countercurrent chromatography separation column；After stationary phase is full of in post, start makes counter-current chromatograph main frame turn with setting speed
It is dynamic, then mobile phase is pumped into splitter with certain flow rate；After two-phase reaches dynamic equilibrium in splitter, pass through sample introduction
Valve injection；Automatic fraction collector collects cut；With high performance liquid chromatography detection, the cut containing oleuropein composition is merged
Afterwards oleuropein is obtained after recycling design.
The present invention is described in detail with reference to embodiment.
Embodiment 1
1. sample：Take 1kg olive growing leaves, add the ethanol of 15 liter 90% (volume ratio), heating and refluxing extraction 2 times, every time
2h, is recovered under reduced pressure ethanol after extract solution is merged, obtains extractive of olive growing leaves.
2. instrument：DE HPCCC Spectrum counter-current chromatographs, DE companies of Britain.
3. preparation method：By n-butanol-acetate-methanol-water according to 1:19:1:19(v:V) ratio is sufficiently mixed
It is placed in separatory funnel and stands overnight after uniform.Ultrasound 20min deaerates after upper and lower phase is released respectively, and the above is mutually fixation
Phase, lower phase is mobile phase.Stationary phase is pumped into counter-current chromatograph helix tube first, flow velocity 6mL/min treats that helix tube fills completely
After full stationary phase, counter-current chromatograph main frame is opened, slow adjustment helix tube rotating speed is to 1600r/min, while with 6mL/min stream
Speed is pumped into mobile phase.When system reaches balance, the foregoing olive extracts of 120mg are dissolved in the mixed solution of the upper and lower phases of 6mL
In, injected by sampling valve in helix tube, it is 30 DEG C to set instrument temperature.Automatic fraction collector connects sample, and 3mL is portion, warp
HPLC is detected, merges the cut containing oleuropein, off-white powder is obtained after being concentrated under reduced pressure.Reflected by nuclear magnetic resonance, mass spectrum
Surely it is oleuropein, determines purity >=90%.The oleuropein yield of whole implementation process reaches 83.2%.
Embodiment 2
1. sample：Take 1kg olive growing leaves, add the ethanol of 12 liter 80% (volume ratio), heating and refluxing extraction 2 times, every time
2h, is recovered under reduced pressure ethanol after extract solution is merged, obtains extractive of olive growing leaves.
2. instrument：TBE-300B counter-current chromatographs, Chinese Shanghai is with field Bioisystech Co., Ltd.
3. preparation method：By n-butanol-acetate-methanol-water according to 1:19:1:19(v:V) ratio is sufficiently mixed
Stratification in separatory funnel is placed on after uniform.Ultrasound 20min deaerates after upper and lower phase is released respectively, and the above is mutually fixation
Phase, lower phase is mobile phase.Stationary phase is pumped into counter-current chromatograph helix tube first, flow velocity 20mL/min treats that helix tube is complete
After stationary phase, counter-current chromatograph main frame is opened, slow adjustment helix tube rotating speed is to 950r/min, while with 1.5mL/min
Flow pump enter mobile phase.When helix tube tail end flows out mobile phase, system reaches balance, the foregoing kansu crazyweed herbs of 400mg is always given birth to
Alkaloids sample is dissolved in the mixed solution of the upper and lower phases of 20mL, is injected by sampling valve in helix tube, and it is 25 to set water bath with thermostatic control
DEG C, UV-detector Detection wavelength is 254nm, and automatic fraction collector connects sample, and 5mL is portion, and HPLC detections, merging contains olive
The cut of olive hardship glycosides, off-white powder is obtained after being concentrated under reduced pressure.It is oleuropein by nuclear magnetic resonance, Mass Spectrometric Identification, determines
Purity >=90%.The oleuropein yield of whole implementation process reaches 82.6%.
Claims (7)
1. a kind of method of separating high-purity oleuropein, it is characterised in that described method includes the steps：
1) extractive of olive growing leaves is prepared：
Extracted using ethanol water from olive growing leaves and obtain extractive of olive growing leaves,
2) selection of solvent system is separated：
N-butanol, ethyl acetate, first alcohol and water are mixed as separation solvent system, stratification after shaking, by upper and lower phase point
Open respectively as stationary phase and mobile phase；
3) stationary phase is full of to the splitter of counter-current chromatograph first, counter-current chromatograph main frame is then started, reaches setting speed
Afterwards, then thereto mobile phase is pumped into, after two-phase reaches dynamic equilibrium in splitter, is extracted olive growing leaves by sampling valve
Thing injects, and automatic fraction collector receives cut；
4) by the cut of collection by high performance liquid chromatography detection, obtained after merging the cut containing oleuropein, recycling design
Oleuropein.
2. the method as described in claim 1, it is characterised in that described step 1) in ethanol water be 60%-90%
Ethanol.
3. the method as described in claim 1, it is characterised in that described step 1) in extracting method be soak extraction method or add
Circumfluence distillation.
4. the method as described in claim 1, it is characterised in that described step 2) in the pH value of solvent for use system be 2-
14。
5. the method as described in claim 1, it is characterised in that described step 2) in, it is solid using upper in solvent system
Determine phase, lower phase is mobile phase；Or so that, down for stationary phase, upper phase is mobile phase in solvent system.
6. the method as described in claim 1, it is characterised in that described step 2) in n-butanol, ethyl acetate, first alcohol and water
Volume ratio be 1:19:1:19.
7. the method as described in claim 1, it is characterised in that described step 3) in, counter-current chromatograph can be using rotating forward
Pattern, it would however also be possible to employ reversing mode.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710231654.9A CN106967136A (en) | 2017-04-11 | 2017-04-11 | A kind of method of separating high-purity oleuropein |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109400658A (en) * | 2018-11-26 | 2019-03-01 | 中国科学院兰州化学物理研究所 | The method of separated in synchronization purifying oleuropein and hydroxytyrosol from olive growing leaves |
| EP3818837A1 (en) | 2019-11-07 | 2021-05-12 | Michel Rumiz | Method for reducing the concentration of oleuropein and for formation and preservation of the concentration of hydroxytyrosol and tyrosol in olives |
| CN113754712A (en) * | 2021-09-03 | 2021-12-07 | 中国科学院兰州化学物理研究所 | Method for separating and preparing high-purity oleuropein |
| CN114085258A (en) * | 2021-11-29 | 2022-02-25 | 中国科学院兰州化学物理研究所 | Method for separating oleuropein from olive leaves by continuous sample injection through countercurrent chromatography |
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| CN102464684A (en) * | 2010-11-19 | 2012-05-23 | 苏州宝泽堂医药科技有限公司 | Method for purifying oleuropein |
| CN104910223A (en) * | 2015-05-26 | 2015-09-16 | 南京泽朗医药科技有限公司 | Preparation method of oleuropein |
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Patent Citations (2)
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| CN102464684A (en) * | 2010-11-19 | 2012-05-23 | 苏州宝泽堂医药科技有限公司 | Method for purifying oleuropein |
| CN104910223A (en) * | 2015-05-26 | 2015-09-16 | 南京泽朗医药科技有限公司 | Preparation method of oleuropein |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109400658A (en) * | 2018-11-26 | 2019-03-01 | 中国科学院兰州化学物理研究所 | The method of separated in synchronization purifying oleuropein and hydroxytyrosol from olive growing leaves |
| CN109400658B (en) * | 2018-11-26 | 2022-01-11 | 中国科学院兰州化学物理研究所 | Method for synchronously separating and purifying oleuropein and hydroxytyrosol from olive leaves |
| EP3818837A1 (en) | 2019-11-07 | 2021-05-12 | Michel Rumiz | Method for reducing the concentration of oleuropein and for formation and preservation of the concentration of hydroxytyrosol and tyrosol in olives |
| CN113754712A (en) * | 2021-09-03 | 2021-12-07 | 中国科学院兰州化学物理研究所 | Method for separating and preparing high-purity oleuropein |
| CN114085258A (en) * | 2021-11-29 | 2022-02-25 | 中国科学院兰州化学物理研究所 | Method for separating oleuropein from olive leaves by continuous sample injection through countercurrent chromatography |
| CN114085258B (en) * | 2021-11-29 | 2023-10-24 | 中国科学院兰州化学物理研究所 | Method for separating oleuropein from olive leaves by countercurrent chromatography continuous sampling |
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