CN101712669B - Method for separating and purifying luteolin - Google Patents
Method for separating and purifying luteolin Download PDFInfo
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- CN101712669B CN101712669B CN2009100042291A CN200910004229A CN101712669B CN 101712669 B CN101712669 B CN 101712669B CN 2009100042291 A CN2009100042291 A CN 2009100042291A CN 200910004229 A CN200910004229 A CN 200910004229A CN 101712669 B CN101712669 B CN 101712669B
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Abstract
The invention aims to provide a material for preparing a molecular imprinted polymer by taking luteolin as a template and adopting a molecular imprinting technique method, and a method for separating and purifying the luteolin in a natural product, and belongs to the field of separation and purification of effective components from the natural product. The method comprises the following steps: (1) crude extraction, namely sequentially washing, drying, crushing and sieving peanut shells, extracting the peanut shell powder with 70 percent ethanol time by time under the assistance of ultrasonic waves, and concentrating and drying the extract; (2) preparation of the polymer, namely weighing the pure luteolin, a functional monomer, a cross-linking agent and an initiating agent in proportion, dissolving the materials in a solvent, performing ultrasonic processing, degassing and polymerization on the solution to obtain the block polymer, and sequentially grinding, sieving and eluting the block polymer to obtain the molecular imprinted polymer of the luteolin; and (3) purification, namely loading the prepared molecular imprinted polymer into a solid-phase extraction column, loading the crude extract, washing off the luteolin left on the column after removing impurities by eluting, concentrating and drying the luteolin to obtain the pure product of the luteolin.
Description
Affiliated technical field:
What patent of the present invention related to is the separation and the purifying of luteolin in the natural phant, belongs to active ingredient of natural product separation and purification field.
Luteolin is a kind of very representative natural flavone, belongs to slightly acidic kaempferol compounds, and it is wider to distribute in vegitabilia, has very high biological activity.
Luteolin has very strong anti-oxidant activity, has effect antibiotic, antiviral and reduce fat and SUV in vivo; Luteolin also is antineoplastic startup factor, and research confirms that it has remarkable restraining effect to multiple solid tumor, ascites carcinoma and leukemia cell; Luteolin can also the multiple antiapoptotic factors that causes of enhanced sensitivity, has the antineoplastic potentiality of many target spots.
Inflammation plays an important role in many neurodegenerative disorders; Research shows that luteolin (can hinder the inflammatory reaction of several kinds of cells beyond the neural system, thereby reduce the interior inflammatory reaction of the brain relevant with aging to improve the forgetful and fragmentation that causes because of aging.This discovery possibly exert an influence to the disease like senile dementia and multiple sclerosis and so on.
Luteolin extracts from plant, and the purity of the luteolin that conventional separation method obtains is not high, and medicinal luteolin requires purity very high, and therefore, it is just extremely important to set up separation purification method efficient and luteolin cheaply.
Background technology:
Luteolin belongs to flavonoid; Flavonoid substances has the natural product that the C6-C3-C6 structure is basic parent nucleus; They have similar physicochemical property; They are present in the natural phant simultaneously, as except that containing luteolin, also containing Flavonoid substances (see figure 1)s such as eriodictyol, dihydroxyl chromone in the Pericarppium arachidis hypogaeae.
The method of from natural phant, extracting luteolin has lixiviate, ooze cross, refluxing extraction, Suo Shi extraction, supercritical fluid extraction, enzymolysis, ultrasonic extraction, the microwave extracting etc. of helping.But the extraction product that adopts above-mentioned each method to obtain is a total flavonoid, can't obtain the higher luteolin of purity.Method to the further separation and purification of luteolin has HPLC and chromatography at present; Though these two kinds of methods have higher separation efficiency, the liquid chromatography instrument that liquid phase chromatography needs is expensive, and extracted amount is limited; Chromatographically pure moving phase consumption is big, and working cost is very high.The silica gel of chromatography can't be reused, and needs a large amount of solvents, so separation costs is also very high.
Molecular imprinting is in the presence of linking agent; To have the radical-initiated polymerisation of uv-radiation or heat effect; Function monomer carries out polymerization round template molecule or microsphere, after polyreaction is accomplished, or with hydrolysis or carry out gentleness with suitable solvent and extract and remove covalent linkage or non covalent bond bonded template molecule; The polymkeric substance of the tridimensional network that obtains, i.e. molecularly imprinted polymer (MIPs) with special identification hole.Hole or the trace stayed on the polymkeric substance are complementary with template molecule on shape, size and function, and permanent memory effect is arranged.Molecularly imprinted polymer has the special recognition site of affinity structure for the template molecule that is adopted, and can with template molecule with the mode of action of host-guest bonding again optionally.Therefore this polymkeric substance has good effect for template molecule is separated from the complex system that contains template molecule.Separating or analyze medium and compare with conventional and traditional, is that separated object or analyte are had selectivity highly based on the outstanding feature of the molecularly imprinted polymer of molecular recognition.Simultaneously molecularly imprinted polymer prepares easyly, has good physical and chemical stability, can withstand high temperatures, high pressure, soda acid organic solvent etc., and easy storing can be preserved several years under conventional envrionment temperature and not reduced post and imitate.Therefore, polymkeric substance can reuse up to a hundred times and preserve " memory " effect, has unique advantage.Be easy to realize suitability for industrialized production.
Summary of the invention:
The purpose of this invention is to provide a kind of molecular imprinting method that adopts is the material that template prepares molecularly imprinted polymer with the luteolin, and with this plant extraction liquid that contains luteolin is carried out the separation and purification luteolin.
Technical scheme of the present invention is:
This is used to separate, the moity and the weight proportion of the molecular engram material of purifying luteolin are following:
Template molecule is a luteolin: 2～3%;
Function monomer is acrylic amide (2.5～3.0%), also can adopt 4-vinylpridine (4.0～4.5%), 2-vinylpyridine (4.0～4.5%) or 2, and 6-diacrylamine pyridine (3.0～4.0%) etc. are as function monomer of the present invention.
Linking agent is an ethylene glycol dimethacrylate (EDMA) (30～35%); Also can adopt N, N '-methylene diacrylamine (23～27%), divinylbenzene (20～25%), pentaerythritol triacrylate (PETRA) (30～35%) and trimethoxy propane trimethyl acrylic ester (TRIM) materials such as (28～33%) also can be used as linking agent of the present invention.
Initiator can be selected Diisopropyl azodicarboxylate (AIBN), ABVN or Lucidol for use, and consumption all is about 0.1%.
Solvent is selected acetone or acetonitrile for use, and after the content of above-mentioned each composition was confirmed, adding solvent was 100%.
Specific embodiments:
1, the pre-treatment of raw material
(1) Pericarppium arachidis hypogaeae is cleaned up the back oven dry, pulverizes, cross 40 mesh sieves, be stored in the dry environment, subsequent use.
(2) take by weighing peanut hull meal, get 70% ethanol that 10-15 doubly measures, at normal temperatures, adopt the UW auxiliary law, divide three subgradients to extract, each 15min through 45min as extracting solvent.
(3) be evaporated near doing to three ethanol extracts collected with rotatory evaporator, with liquid concentrator nature or vacuum-drying, the Pericarppium arachidis hypogaeae ethanol extraction, put into moisture eliminator after the sealing and preserve.
2, the preparation of luteolin molecularly imprinted polymer
In container (test tube), take by weighing luteolin, function monomer, linking agent and initiator by the above-mentioned proportioning raw materials that provides, fully be dissolved in the solvent, it was carried out ultrasonic 10-30 minute; To fill the container sealing of above-mentioned solution then, logical argon gas or nitrogen carry out deoxidation, with the container of good seal 60 ℃ of following constant temperature polymerizations 24 hours; Polymerization is ground the bulk polymer in the container, sieve after accomplishing, and uses 90% (v/v) methanol acetic acid solvent elution to remove template molecule, do not have polymeric function monomer, linking agent and initiator then, obtains luteolin molecularly imprinted polymer (MIP).
The process of the preparation of non-template imprinted polymer (NMIP) as a comparison is not except that adding the luteolin template molecule, and other step is identical with the preparation of imprinted polymer (MIP).
3, separation and the purifying to containing the luteolin crude extract
With the molecularly imprinted polymer dress post that makes, and use wetted with methanol.The plant extraction liquid upper prop that will contain luteolin; And the impurity in the crude extract is eluted (checking with thin-layer chromatography) fully with 10% methanol aqueous solution; Use methyl alcohol then: the luteolin that the solution of acetate (9: 1) will be retained on the post elutes (checking with thin-layer chromatography), obtains the luteolin straight product through concentrated, drying.
The present invention has following advantage:
1, molecularly imprinted polymer of the present invention is the template preparation with the luteolin; The hole of polymkeric substance and luteolin bulk of molecule are consistent; Its binding site also is complementary with the luteolin molecule, has very high selectivity, can reach good separating effect.
2, the polymkeric substance of preparation has very high intensity and stability, can reuse.Avoided the mass consumption problem of the silica gel in the silica gel separation and purification.
3, because the molecularly imprinted polymer of preparation has good recognition performance to luteolin, the solvent load of drip washing and wash-out also greatly reduces, and has reduced separation costs.
1, the preparation of luteolin molecularly imprinted polymer
Taking by weighing 0.731g luteolin, 0.706g acrylic amide, 10g pentaerythritol triacrylate (PETRA) and 0.03g ABVN is dissolved in the 20ml acetone; Ultrasonic in beaker; Then solution is poured in the sealable container (test tube), sealed, logical Ar
260 ℃ water bath with thermostatic control polymerization is put into the container of good seal in deoxidation.The preparation of blank polymkeric substance (NMIPs) is not except that adding the template molecule luteolin, and other is all identical.Polymerization is being pounded into fritter with the bulk polymer in the container, and is being ground after accomplishing, and obtains particulate polymers.Use methyl alcohol: the solution of acetate (9: 1) carries out wash-out to be removed and does not participate in polymeric solvent, initiator and template molecule, obtains the luteolin molecularly imprinted polymer.
2, the separation of luteolin liquid concentrator and purifying
The good luteolin imprinted polymer that takes by weighing the 3g wash-out is packed in the solid-phase extraction column, adds a little silica sand on the post upper strata after having adorned post, uses the wetting pillar of amount of methanol then.Pipette 15ml Pericarppium arachidis hypogaeae ethanol extract (0.5g/ml) upper prop, and, add leacheate (10% methyl alcohol) when treating liquid level and carry out drip washing, until the impurity in the Pericarppium arachidis hypogaeae ethanol extraction is eluted (checking with thin-layer chromatography) fully a little more than solidus surface to the post pressurization.Use methyl alcohol then: acetate (9: 1) eluant solution is adsorbed on the luteolin (checking with thin-layer chromatography) on the column extractor, obtains the luteolin straight product through concentrated, drying.
1, the preparation of luteolin molecularly imprinted polymer
Take by weighing 0.713g luteolin, 0.883g 2,6-diacrylamine pyridine, 10g EDMA and 0.03g AIBN are dissolved in the 20ml acetone, and be ultrasonic in beaker, then solution poured in the sealable container (flask at the bottom of the garden), seals, logical Ar
260 ℃ water bath with thermostatic control polymerization is put into the container of good seal in deoxidation.The preparation of blank polymkeric substance (NMIPs) is not except that adding the template molecule luteolin, and other is all identical.Polymerization is being pounded into fritter with the bulk polymer in the container, and is being ground after accomplishing, and obtains particulate polymers.Use methyl alcohol: the solution of acetate (9: 1) carries out wash-out to be removed and does not participate in polymeric solvent, initiator and template molecule, obtains the luteolin molecularly imprinted polymer.
2, the separation of luteolin liquid concentrator and purifying
The good luteolin imprinted polymer that takes by weighing the 2g wash-out is packed in the solid-phase extraction column, adds a little silica sand on the post upper strata after having adorned post, uses the wetting pillar of amount of methanol then.Pipette 10ml Pericarppium arachidis hypogaeae ethanol extract (0.6g/ml) upper prop, and, when treating liquid level, carry out drip washing, until the impurity in the Pericarppium arachidis hypogaeae ethanol extraction is eluted (checking with thin-layer chromatography) fully with 10% methyl alcohol leacheate a little more than solidus surface to the post pressurization.Use methyl alcohol then: acetate (9: 1) eluant solution is adsorbed on the luteolin (checking with thin-layer chromatography) on the column extractor, obtains the luteolin straight product through concentrated, drying.
Description of drawings:
The structure of letones in Fig. 1 Pericarppium arachidis hypogaeae
The basic parent nucleus b of a, Flavonoid substances, the structure of luteolin
The structure of the structure d of c, eriodictyol, dihydroxyl chromone
The synthetic synoptic diagram of the plain molecularly imprinted polymer of Fig. 2 rhinoceros grass
The mode of appearance sem photograph of Fig. 3 polymkeric substance
A and c, luteolin imprinted polymer b and d, non-imprinted polymer
The equilibrium adsorption figure of Fig. 4 polymkeric substance
A, imprinted polymer absorption b, the absorption of non-imprinted polymer
The ultraviolet-visible spectrogram of Fig. 5 luteolin in 0.01% methanol solution solution
The luteolin that a, luteolin standard substance b, separation obtain
Fig. 6 separates the infrared spectrogram of the luteolin that obtains
Claims (1)
1. the method that the extracting solution of the natural product that contains luteolin is carried out the separation and purification of luteolin is characterized in that, may further comprise the steps:
(1) slightly carries: Pericarppium arachidis hypogaeae is cleaned up the back oven dry, pulverizes, cross 40 mesh sieves; Take by weighing peanut hull meal, get 70% ethanol that 10-15 doubly measures, divide three extractions, extract ultrasonic 15min at every turn as extracting solvent; Extracting solution is collected, concentrated, obtain containing the crude extract of luteolin;
(2) constituent material of molecularly imprinted polymer: the template molecule of molecularly imprinted polymer adopts luteolin (2～3%); Function monomer adopts acrylic amide (2.5～3.0%), 4-vinylpridine (4.0～4.5%), 2-vinylpyridine (4.0～4.5%) or 2,6-diacrylamine pyridine (3.0～4.0%); Linking agent adopts ethylene glycol dimethacrylate (30～35%), uses N, N '-methylene diacrylamine (23～27%), divinylbenzene (20～25%), pentaerythritol triacrylate (30～35%) or trimethoxy propane trimethyl acrylic ester (28～33%); Initiator is selected Diisopropyl azodicarboxylate (0.1%), ABVN or Lucidol (0.1%) for use; Solvent is selected acetone or acetonitrile for use;
(3) preparation of molecularly imprinted polymer: the requirement proportioning by (2) in claims 1 takes by weighing luteolin, function monomer, linking agent and initiator, and adding solvent is 100%, through ultrasonic dissolution; Remove oxygen; 60 ℃ of following polymerizations 24 hours, obtain bulk polymer, it is ground, sieves; Employing methyl alcohol and acetate volume ratio are that 9: 1 solvent elution is removed template molecule, do not had polymeric function monomer, linking agent and initiator, obtain the luteolin molecularly imprinted polymer;
(4) purifying: with the molecularly imprinted polymer that the makes solid-phase extraction column of packing into; And with appearance on the crude extract; The methanol-eluted fractions of employing 10% is removed the impurity in the crude extract; Using methyl alcohol and acetate volume ratio again is that the luteolin that 9: 1 solution will be retained on the post washes, through concentrating, dry, obtain the luteolin straight product.
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Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102040579A (en) * | 2011-01-25 | 2011-05-04 | 彭国平 | Method for extracting luteolin from peanut roots, stems, leaves and shells |
| CN102671639A (en) * | 2012-05-24 | 2012-09-19 | 国际竹藤中心 | Bamboo-leaf-flavonoids molecular imprinted solid-phase extraction column, preparation and application thereof |
| CN102659982B (en) * | 2012-05-24 | 2014-09-10 | 国际竹藤中心 | Flavone magnetic molecularly imprinted polymer, preparation of flavone magnetic molecularly imprinted polymer, and application of flavone magnetic molecularly imprinted polymer to bamboo-leaf flavone separation |
| CN103933111A (en) * | 2013-05-02 | 2014-07-23 | 杭州耐奇睿生物医药科技有限公司 | Application of peanut extract |
| CN103524677A (en) * | 2013-09-30 | 2014-01-22 | 山西医科大学 | Galangin molecular imprinting polymer, and preparation method and application thereof |
| CN104546997A (en) * | 2013-10-16 | 2015-04-29 | 浙江中医药大学 | Method for extracting and purifying effective parts of peanut shells |
| CN104906171A (en) * | 2015-04-13 | 2015-09-16 | 沈阳北苑医药有限公司 | Process for extracting total flavonoids from peanut shells |
| CN105130939B (en) * | 2015-08-31 | 2017-09-22 | 桂林三宝生物科技有限公司 | A kind of method that cyanidenon is extracted from peanut shell |
| CN106588848B (en) * | 2016-11-15 | 2019-10-18 | 浙江工业大学 | The method of luteolin is extracted in a kind of peanut shell |
| CN108864021A (en) * | 2017-10-13 | 2018-11-23 | 昌邑市银江生物科技有限公司 | A method of high-purity luteolin is prepared using zinc salt |
| CN108864022A (en) * | 2017-10-13 | 2018-11-23 | 昌邑市银江生物科技有限公司 | A method of high-purity luteolin is prepared using ferrous salt |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1919843A (en) * | 2006-09-19 | 2007-02-28 | 天津大学 | Preparation method of siliceous inorganic flavonoid molecular engram microsphere |
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| CN1919843A (en) * | 2006-09-19 | 2007-02-28 | 天津大学 | Preparation method of siliceous inorganic flavonoid molecular engram microsphere |
Non-Patent Citations (1)
| Title |
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| 许志锋等.以桑色素为模板的分子烙印聚合物的制备、结合特性及其在薄层色谱固定相方面的应用.《高等学校化学学报》.2005,第26卷(第11期),2031-2036. * |
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