CN101624607A - Method for preparing hydroxytyrosol - Google Patents
Method for preparing hydroxytyrosol Download PDFInfo
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- CN101624607A CN101624607A CN200910017367A CN200910017367A CN101624607A CN 101624607 A CN101624607 A CN 101624607A CN 200910017367 A CN200910017367 A CN 200910017367A CN 200910017367 A CN200910017367 A CN 200910017367A CN 101624607 A CN101624607 A CN 101624607A
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- hydroxytyrosol
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- hydroxytyrosols
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Abstract
The invention belongs to the technical field of bioengineering, and more particularly discloses a method for preparing hydroxytyrosol. The method takes p-hydroxy phenylethanol as raw material and polyphenol oxidase as catalyst; oxidation reaction is carried out under the condition of participation of oxygen; ascorbic acid is used for inhibiting the bisphenol activity of the polyphenol oxidase; the reaction process is controlled to obtain reaction liquid which is rich in the hydroxytyrosol; then, after membrane separation as well as adsorptive separation, extraction and column chromatography of macroporous absorption resin, the hydroxytyrosol product with high purity can be obtained. The method has the advantages of easily obtained raw material, continuous operation, environmental protection, easy purification of the product, high yield and the like, so as to be applicable to industrialized production.
Description
(1) technical field
The invention belongs to technical field of bioengineering, particularly a kind of preparation method of Hydroxytyrosol.
(2) background technology
The development of numerous disease all involves a large amount of dietary fats.Diseases such as atherosclerosis, coronary heart disease, mammary cancer, prostate cancer, ovarian cancer and colorectal carcinoma are all relevant with dietary fat separately, yet, evidence shows the quantity of being not only dietary fat important in the etiology of some cancer, also comprises the type of dietary fat.
Sweet oil is the main lubricant component of Mediterranean diet, relevant with the low sickness rate of coronary heart disease and some cancer, some test demonstration, the hydrolysis of the bitter glycosides of sweet oil phenols Fructus oleae europaeae and other material of the same clan can produce the little phenols component with strong antitumour activity, and Fructus oleae europaeae phenols Hydroxytyrosol can prevent low-density lipoprotein (LDL) oxidation, platelet aggregation and suppress 5-and the 12-lipoxygenase especially; In addition, we have found that Hydroxytyrosol produces restraining effect to the DNA base modification and the tyrosine nitration reaction of peroxynitrite decision, and it can remove the cytotoxicity that free active oxygen causes in various human cell system; At last, studies show that the absorption of Hydroxytyrosol digestion back dose-dependently in human body demonstrates its bioavailability.
Hydroxytyrosol has antiinflammation, and vasodilation and anti-microbial effect can prevent atherosclerosis, can reduce the sickness rate of some cancer (as mammary gland, prostate gland, uterine endometrium, digestive tube); Because Hydroxytyrosol has very strong anti-oxidant activity and reduces the effect of cancer morbidity, therefore has huge medical functions prospect.
(3) summary of the invention
The present invention is in order to remedy the deficiencies in the prior art, and providing a kind of is raw material with the p-hydroxyphenylethanol, the preparation method of biosynthesizing Hydroxytyrosol.
The present invention is achieved through the following technical solutions:
A kind of structure is characterized in that: comprise the steps: suc as formula the preparation method of the Hydroxytyrosol shown in (I)
(1) be that structure is a raw material suc as formula the p-hydroxyphenylethanol shown in (II) in 2～7.5 the damping fluid to be dissolved in the pH value, add structure suc as formula the xitix shown in (III), under the catalysis of polyphenoloxidase,, obtain containing the reaction solution of Hydroxytyrosol (I) with the oxygen reaction;
(2) be the nanofiltration membrane of 200Da～1000Da with reaction solution by molecular weight cut-off, macromolecular components in the isolate reactant;
(3) reaction solution is passed through styrene resin or polymeric amide chromatography post, use the purified water wash-out, collect the elutriant of hydroxyl tyrosol;
(4) elutriant is concentrated, use ethyl acetate extraction,, concentrate and reclaim ethyl acetate, obtain the Hydroxytyrosol crude product of faint yellow paste the ethyl acetate phase dehydration of extraction;
(5) make the Hydroxytyrosol crude product through column chromatography, adopt the eluent wash-out, collect hydroxyl tyrosol part, get high purity Hydroxytyrosol (I) product behind the recovery solvent.
Reaction principle of the present invention is as follows:
Preparation method of the present invention, in the step (1), described polyphenoloxidase is on-fixed enzyme or immobilized enzyme.The on-fixed enzyme is meant the adding of carrying out catalyzer in reaction raw materials by the mode of adding; Immobilized enzyme is meant enzyme to be combined with the water-insoluble macromolecular carrier or enzyme is embedded in method physics or chemistry and makes in the microcapsule of water-insoluble gel or semi-permeable membranes, as the surface support, crosslinked, embedding etc.; General stability increases behind the enzyme immobilization, easily separates from reactive system, and is easy to control, can repeated multiple times use.Preferably use immobilized enzyme in the present invention.
Polyphenoloxidase is a kind of proteoplast, participates in a series of chemical transformation that caused by the enzymatic activity in the course of processing of material, so the biological catalyst that is otherwise known as; Polyphenoloxidase is mainly derived from soil microorganisms, secretions from plant roots and plant and animal residues and decomposes the enzyme that discharges, and it is a kind of plyability enzyme; Polyphenoloxidase is mainly derived from potato, apple, banana, bacterium mushroom etc.
In the step (1), the concentration of p-hydroxyphenylethanol is 10mg/L～1000mg/L, preferred 50～200mg/L; The concentration of xitix is 10mg/L～1000mg/L, the pH preferred 4～7 of preferred 100mg/L～500mg/L damping fluid; In the step (2), the preferred 200Da～500Da of the molecular weight cut-off of nanofiltration membrane.
The elutriant that chromatography column adopts in the step (3) is a purified water, generally is the water that adopts distillation method, ion exchange method, reverse osmosis method or other suitable methods to make, and does not contain any additives.
In the step (4), elutriant is concentrated 10 times, use equal volume of ethyl acetate 3～5 times, concentrate the recovery ethyl acetate and obtain the Hydroxytyrosol crude product.
In the step (5), the used sorbent material of column chromatography is silica gel or neutral alumina; Eluent is one or more the mixed solution in water, methyl alcohol, ethanol, propyl alcohol, ethyl acetate, N-BUTYL ACETATE, chloroform, methylene dichloride, sherwood oil, the gasoline; Final Hydroxytyrosol product is the thick material of water white transparency.
The column chromatography method that adopts among the present invention is an adsorpting column chromatography, and it is stationary phase with the solid adsorbent, is that moving phase constitutes post with organic solvent or damping fluid, is used for the impurity of absorption reaction liquid, and reaction solution is carried out wash-out; Also relate to a kind of polymeric amide chromatography post among the present invention, polymeric amide is to form hydrogen bond between its energy and the separated object to the adsorption of polar material, and the power of this hydrogen bond has just determined the size of adsorptive power between separated object and the polyamide layer.
The present invention is raw material with the p-hydroxyphenylethanol, with the polyphenoloxidase is catalyzer, in the presence of oxygen, oxidizing reaction takes place, and, control reaction process by xitix inhibition polyphenoloxidase bis-phenol activity, obtain being rich in the reaction solution of Hydroxytyrosol, and then, obtain highly purified Hydroxytyrosol product through membrane sepn, absorption with macroporous adsorbent resin separation, extraction, column chromatography.
The present invention has raw material and is easy to get, operates serialization, environmental friendliness, the easy purifying of product, yield advantages of higher, is applicable to suitability for industrialized production.
(4) embodiment
Embodiment 1:
Take by weighing the 2.5kg p-hydroxyphenylethanol, be dissolved in the 100kg 0.1M pH=6.5 phosphate buffered saline buffer, add the 5kg xitix, add enzyme liquid 1L (than 500U/mg alive), in 50 ℃ of stirring reactions 60 minutes, reaction solution passes through nanofiltration membrane, dialyzate is by styrene resin post (about 100kg resin), wash with water to effluent liquid and detect no Hydroxytyrosol spot with TLC, this water lotion is concentrated into 50kg, uses 100kg ethyl acetate extraction 5 times, and acetic acid ethyl fluid concentrates and reclaims solvent, get the faint yellow dope of 2.5kg, Hydroxytyrosol content 88.3%.
Embodiment 2:
The polyphenoloxidase liquid of getting purifying is embedded in the chitosan and is shaped with glutaraldehyde cross-linking, measures its vigor 50U/mg; Get the 5kg p-hydroxyphenylethanol and be dissolved in 2000L 0.05mol/L phosphate buffered saline buffer (pH value 6.5), add the 10kg anticyclic acid, stirring and dissolving, add the 1kg immobilized enzyme, in 50 ℃ of stirring reactions 60 minutes, the reclaiming by centrifuge immobile polyphenol oxidase is also used in the reaction that obtains, reaction solution is passed through nanofiltration membrane, dialyzate is by styrene resin post (about 200kg resin), wash with water to effluent liquid with the no Hydroxytyrosol spot of TLC detection, this water lotion is concentrated into 100kg, with 100kg ethyl acetate extraction 5 times, acetic acid ethyl fluid concentrates and reclaims solvent, gets the faint yellow dope of 4kg, Hydroxytyrosol content 91.2%.
Embodiment 3:
The polyphenoloxidase liquid of getting purifying is embedded in the alginates with the crosslinked shaping of the rare diacid-ethene of anhydrous fourth, measures its vigor 35U/mg; Get the 5kg p-hydroxyphenylethanol and be dissolved in 2000L0.05mol/L phosphate buffered saline buffer (pH value 6.5), add the 10kg anticyclic acid, stirring and dissolving, add the 1.5kg immobilized enzyme, in 50 ℃ of stirring reactions 60 minutes, the reclaiming by centrifuge immobile polyphenol oxidase is also used in the reaction that obtains, reaction solution is passed through nanofiltration membrane, dialyzate is by styrene resin post (about 200kg resin), wash with water to effluent liquid with the no Hydroxytyrosol spot of TLC detection, this water lotion is concentrated into 100kg, with 100kg ethyl acetate extraction 5 times, acetic acid ethyl fluid concentrates and reclaims solvent, gets the faint yellow dope of 4.2kg, Hydroxytyrosol content 87.6%.
Embodiment 4:
The polyphenoloxidase liquid of getting purifying is embedded in the chitosan and is shaped with glutaraldehyde cross-linking, measures its vigor 50U/mg; Get 2kg immobilized enzyme pearl (Φ 20*2000cm) in the jacketed glass post of packing into, chuck is with 50 ℃ of hot water circulation; Get the 10kg p-hydroxyphenylethanol and be dissolved in 3000L0.05mol/L phosphate buffered saline buffer (pH value 6.5), add the 20kg anticyclic acid, this reaction solution passes through the enzyme post with the flow velocity of 10L/min, and continuously by styrene resin post (about 400kg resin), reaction solution has flowed the back and has added 100L 0.05mol/L phosphate buffered saline buffer (pH value 6.5) flushing enzyme post, after finishing enzyme post interface is separated with the resin column interface, resin column continues to wash to effluent liquid with purified water and detects no Hydroxytyrosol spot with TLC, this water lotion is concentrated into 200kg, with 200kg ethyl acetate extraction 5 times, acetic acid ethyl fluid concentrates and reclaims solvent, gets the faint yellow dope of 8.5kg, Hydroxytyrosol content 92.4%.
Embodiment 5:
Get Hydroxytyrosol crude product 1kg among the embodiment 4, after the silica gel adsorption, use ethyl acetate: sherwood oil (1: 1) wash-out, collect hydroxyl tyrosol part, reclaim solvent, get the transparent dope of 950g pink colour, Hydroxytyrosol 95.7%.
Embodiment 6:
Get Hydroxytyrosol crude product 1kg among the embodiment 4, after the silica gel adsorption, use chloroform: methyl alcohol (8: 2) wash-out, collect hydroxyl tyrosol part, reclaim solvent, get 908g water white transparency dope, Hydroxytyrosol content 98.6%.
Embodiment 7:
Get Hydroxytyrosol crude product 1kg among the embodiment 4, after the alumina adsorption, methanol-eluted fractions is collected hydroxyl tyrosol part, reclaims solvent, gets 920g water white transparency dope, Hydroxytyrosol content 98.2%.
Claims (10)
1. a structure is characterized in that: comprise the steps: suc as formula the preparation method of the Hydroxytyrosol shown in (I)
(1) be that structure is a raw material suc as formula the p-hydroxyphenylethanol shown in (II) in 2～7.5 the damping fluid to be dissolved in the pH value, add structure suc as formula the xitix shown in (III), under the catalysis of polyphenoloxidase,, obtain containing the reaction solution of Hydroxytyrosol (I) with the oxygen reaction;
(2) be the nanofiltration membrane of 200Da～1000Da with reaction solution by molecular weight cut-off, macromolecular components in the isolate reactant;
(3) reaction solution is passed through styrene resin or polymeric amide chromatography post, use the purified water wash-out, collect the elutriant of hydroxyl tyrosol;
(4) elutriant is concentrated, use ethyl acetate extraction,, concentrate and reclaim ethyl acetate, obtain the Hydroxytyrosol crude product of faint yellow paste the ethyl acetate phase dehydration of extraction;
(5) make the Hydroxytyrosol crude product through column chromatography, adopt the eluent wash-out, collect hydroxyl tyrosol part, get high purity Hydroxytyrosol (I) product behind the recovery solvent.
2. the preparation method of Hydroxytyrosol according to claim 1, it is characterized in that: in the step (1), described polyphenoloxidase is on-fixed enzyme or immobilized enzyme.
3. the preparation method of Hydroxytyrosol according to claim 2, it is characterized in that: in the step (1), the concentration of described p-hydroxyphenylethanol is 10mg/L～1000mg/L, preferred 50～200mg/L.
4. according to the preparation method of claim 1,2 or 3 described Hydroxytyrosols, it is characterized in that: in the step (1), the concentration of described xitix is 10mg/L～1000mg/L, preferred 100mg/L～500mg/L.
5. according to the preparation method of claim 1,2 or 3 described Hydroxytyrosols, it is characterized in that: in the step (1), the pH of described damping fluid is 4～7.
6. according to the preparation method of claim 1,2 or 3 described Hydroxytyrosols, it is characterized in that: in the step (2), the molecular weight cut-off of described nanofiltration membrane is 200Da～500Da.
7. according to the preparation method of claim 1,2 or 3 described Hydroxytyrosols, it is characterized in that: in the step (4), elutriant is concentrated 10 times, use equal volume of ethyl acetate 3～5 times, concentrate the recovery ethyl acetate and obtain the Hydroxytyrosol crude product.
8. according to the preparation method of claim 1,2 or 3 described Hydroxytyrosols, it is characterized in that: in the step (5), the used sorbent material of described column chromatography is silica gel or neutral alumina.
9. according to the preparation method of claim 1,2 or 3 described Hydroxytyrosols, it is characterized in that: in the step (5), described eluent is one or more the mixed solution in water, methyl alcohol, ethanol, propyl alcohol, ethyl acetate, N-BUTYL ACETATE, chloroform, methylene dichloride, sherwood oil, the gasoline.
10. according to the preparation method of claim 1,2 or 3 described Hydroxytyrosols, it is characterized in that: described Hydroxytyrosol product is the thick material of water white transparency.
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| CN200910017367A CN101624607B (en) | 2009-08-03 | 2009-08-03 | Method for preparing hydroxytyrosol |
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| CN200910017367A CN101624607B (en) | 2009-08-03 | 2009-08-03 | Method for preparing hydroxytyrosol |
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| CN101624607B CN101624607B (en) | 2012-09-19 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104099379A (en) * | 2013-04-08 | 2014-10-15 | 中国科学院天津工业生物技术研究所 | Method for biosynthesis of tyrosol in Escherichia coli and application of tyrosol |
| CN104628528A (en) * | 2015-01-05 | 2015-05-20 | 康美（北京）药物研究院有限公司 | Preparation method of tyrosol |
| CN106631709A (en) * | 2016-09-23 | 2017-05-10 | 中国林业科学研究院林产化学工业研究所 | Hydroxytyrosol extraction and nitration method |
| CN114350717A (en) * | 2022-01-17 | 2022-04-15 | 南京合谷生命生物科技有限公司 | Method for preparing 3, 4-dihydroxy phenethyl alcohol by using biological enzyme catalysis |
| CN115010583A (en) * | 2022-06-15 | 2022-09-06 | 四川大学 | Method for synthesizing hydroxytyrosol by simulating enzyme catalytic reaction |
| WO2022184255A1 (en) | 2021-03-03 | 2022-09-09 | Wacker Chemie Ag | Method for producing a hydroxytyrosol |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101298411B (en) * | 2007-04-30 | 2010-05-19 | 天津市尖峰天然产物研究开发有限公司 | Method for extracting hydroxy tyrosol from olive extract |
-
2009
- 2009-08-03 CN CN200910017367A patent/CN101624607B/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104099379A (en) * | 2013-04-08 | 2014-10-15 | 中国科学院天津工业生物技术研究所 | Method for biosynthesis of tyrosol in Escherichia coli and application of tyrosol |
| CN104099379B (en) * | 2013-04-08 | 2019-04-30 | 中国科学院天津工业生物技术研究所 | A kind of method and application of the biosynthesis tyrosol in Escherichia coli |
| CN104628528A (en) * | 2015-01-05 | 2015-05-20 | 康美（北京）药物研究院有限公司 | Preparation method of tyrosol |
| CN106631709A (en) * | 2016-09-23 | 2017-05-10 | 中国林业科学研究院林产化学工业研究所 | Hydroxytyrosol extraction and nitration method |
| CN106631709B (en) * | 2016-09-23 | 2020-01-10 | 中国林业科学研究院林产化学工业研究所 | Method for extracting and nitrating hydroxytyrosol |
| WO2022184255A1 (en) | 2021-03-03 | 2022-09-09 | Wacker Chemie Ag | Method for producing a hydroxytyrosol |
| CN114350717A (en) * | 2022-01-17 | 2022-04-15 | 南京合谷生命生物科技有限公司 | Method for preparing 3, 4-dihydroxy phenethyl alcohol by using biological enzyme catalysis |
| CN115010583A (en) * | 2022-06-15 | 2022-09-06 | 四川大学 | Method for synthesizing hydroxytyrosol by simulating enzyme catalytic reaction |
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Owner name: SHANDONG CHENNONG HEALTH INDUSTRY CO., LTD. Free format text: FORMER NAME: SHANDONG JUYE CHENENG NATURAL PRODUCT CO., LTD. |
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Address after: Town Economic Development Zone 274933 Shandong city of Heze province Juye County Council Patentee after: SHANDONG CHENNONG HEALTH INDUSTRY CO., LTD. Patentee after: Liu Fujin Address before: Town Economic Development Zone 274933 Shandong city of Heze province Juye County Council Patentee before: Shandong Juye Cheneng Natural Product Co., Ltd. Patentee before: Liu Fujin |
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Effective date of registration: 20180528 Address after: 274900 Road East, north section of Gong Zhong Road, Juye County, Heze, Shandong Patentee after: Shandong morning Long Sheng Biotechnology Co., Ltd. Address before: 274933 Economic Development Zone, Dong Guan Tun Town, Juye County, Heze, Shandong Co-patentee before: Liu Fujin Patentee before: SHANDONG CHENNONG HEALTH INDUSTRY CO., LTD. |
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