CN101973854A - Method for efficiently enriching high-quality hydroxyl tyrosol from olive leaves - Google Patents
Method for efficiently enriching high-quality hydroxyl tyrosol from olive leaves Download PDFInfo
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Abstract
The invention discloses a method for efficiently enriching high-quality hydroxyl tyrosol from olive leaves. The method comprises the following steps of: sifting a raw production material with high content of hydroxyl tyrosol from 10 breeds of olive leaves; extracting by using ultrasonic wave or microwave; and carrying out macroporous resin adsorption and medium pressure column chromatogram separation to prepare an extract containing more than 90% of hydroxyl tyrosol. Compared with the traditional purifying method, the method has the characteristics of simplicity, convenience, quickness, economy, high yield, and the like and is suitable for industrial production.
Description
Technical field
The present invention relates to the plant milk extract technical field, particularly a kind of from leaf of Fructus oleae europaeae the method for the high-quality Hydroxytyrosol of efficiently concentrating.
Background technology
Fructus oleae europaeae (Olea europaea L.) is extensively planted in Mediterranean Zone as world-renowned woody oil tree species, and nowadays this area has become the main growth area of olive, accounts for 98% of whole world olive tree cultivated area.
Fructus oleae europaeae antioxidation biology activeconstituents mainly is Oleuropein (oleuropein), Hydroxytyrosol (hydroxytyrosol), flavonoid (flavonoids), lignan class (Lignan), caffeoyl phenylethyl alcohol glycoside (Caffeoyl phenylethanoid glycosides) etc., and the small molecules diphenols compound with Hydroxytyrosol precursor structure has the anti-oxidant activity effect most, and wherein Hydroxytyrosol and Oleuropein are the focuses of studying both at home and abroad.
Natural Hydroxytyrosol mainly is present in the processing waste water of Fructus oleae europaeae fruit, leaf and sweet oil, and especially Oleuropein and the Hydroxytyrosol content in leaf is apparently higher than fruit and bark.Hydroxytyrosol mainly is each position that has Fructus oleae europaeae with the form of carboxylate Oleuropein, accounts for 6-9% (mass ratio) in exsiccant olive leaf.The Oleuropein instability, very easily be subjected to the influence of factors such as air, sunlight, acid, alkali, enzyme, microorganism, oxygenant, high temperature, reaction such as oxidation, condensation, chelating takes place and wrecking, especially easily is degraded to Hydroxytyrosol and elemic acid (elenolic acid) under the effect of sour, alkali and enzyme.In the sweet oil course of processing, for removing sour and astringent taste in the sweet oil, add the mineral acid hydrolysis Oleuropein and can obtain polyphenolic compound, Hydroxytyrosol is the main component of these phenol mixtures.
Chemically the synthesis of hydroxy tyrosol is existing reports that people such as Baraldi adopt 3, and 4-dihydroxyphenyl acetic acid ester is that raw material passes through LiAlH
1Or NaBH
1Catalytic reduction, but scale, especially catalytic reagent costliness that preparation amount only restrains for the laboratory youngster, the preparation of industrialization difficulty.Juan Carlos adopts biological enzyme, is reductive agent with Vc, and tyrosol is converted into Hydroxytyrosol under tyrosinase catalysis, but by product is many, and Hydroxytyrosol separates because of difficulty.The difficult batch preparations Hydroxytyrosol of realizing of present synthetic method.
Discover that Hydroxytyrosol has a lot of aspects healthy bio-pharmacology activity that is beneficial to man, and is subjected to the attention on biomedical boundary deeply.Pharmacological research shows: a kind of very strong free radical scavenger during Hydroxytyrosol, can eliminate endogenous and exogenous butylation free radical and oxide compound effectively.The oxidation-resistance of Hydroxytyrosol and to the elimination of free radical can the some other synthetic of force rate and natural antioxidant all high, resistance of oxidation is Hydroxytyrosol＞Oleuropein＞coffic acid＞VE＞Vc＞tyrosol＞BHT.Because Hydroxytyrosol contains two phenolic hydroxyl groups; easier permeates cell membranes; can improve heart coronary blood flow, suppress collagen-induced platelet aggregation, more effectively protect the oxidative damage and the mitochondrial function imbalance of the retinal pigment epithelium that cigarette toxic ingredient propenal causes.
More deep abroad to the Fructus oleae europaeae The Study on Resources, particularly to the research of wherein aldehydes matter.A lot of research and utilization high resolution techniques are identified and quantitative analysis aldehydes matter in sweet oil, fruit and the leaf, Bouazizda etc. are at Fructus oleae europaeae in the ripening stage, phenols such as Oleuropein, Hydroxytyrosol in the research leaf of Fructus oleae europaeae, the Changing Pattern of flavonoids such as rutin, Quercetin; Ryan etc. studied in two vegetative period, different varieties, different sites, Hydroxytyrosol in the different year olive, several aldehydes matters such as Oleuropein.
Domestic to the also not further investigation of leaf of Fructus oleae europaeae resource, to the research of Hydroxytyrosol still less.The Chinese patent of the patent No. 03815986 is from the compoistion and method of use that is rich in Hydroxytyrosol of olive vegetation water, Oleuropein obtains Hydroxytyrosol in the utilization acid hydrolysis olive leaf, carry out chromatographic purifying again, some technology exists uses a large amount of organic solvents, there is solvent loss, be easy to generate problems such as environmental pollution, the Chinese patent of the patent No. 200710057290 extracts the method for Hydroxytyrosol from olive extract, wherein directly utilize the Fructus oleae europaeae plant extraction liquid of buying to carry out resin isolation, used the method for advanced molecular distillation to promote a technology of leaf of Fructus oleae europaeae resources development and utilization simultaneously, but its technology is too general, wherein raw material is not well selected, though in the separation and purification process, also used youngster to plant macroporous resin, but do not provide comprehensive data parameters, to compare, thereby in production application, there is a not minor issue, how to design the cover production technique that the economy of production application uses of fitting, can guarantee again simultaneously leaf of Fructus oleae europaeae effective constituent fully utilized becomes key.
Summary of the invention
The object of the present invention is to provide a kind of from leaf of Fructus oleae europaeae the method for the high-quality Hydroxytyrosol of efficiently concentrating, this method is not only simple and convenient, and economic environmental protection is effective, is fit to large batch of preparation.
Technical solution of the present invention is
A kind of from leaf of Fructus oleae europaeae the method for the high-quality Hydroxytyrosol of efficiently concentrating, form by following steps:
(1) raw materials for production of screening high-content Hydroxytyrosol from 10 Fructus oleae europaeae kind leaves;
(2) with the green oil olive leaf low-temperature dark drying of screening, drying temperature is lower than 20 ℃, is broken into the following powder of 0.5cm;
(3) with alcohol or the aqueous acetone solution extract oil olive leaf powder of C1-C3, alcohol or acetone mass percentage concentration are 30-100%, extract solution and are 1-30 with leaf of Fructus oleae europaeae powder quality ratio: 1, extracting temperature is 25-65 ℃, and the time is 10-60min, lixiviate number of times 1-5 time, filter united extraction liquid;
(4) vacuum concentration extracting solution under less than 65 ℃ of temperature reclaims organic solvent, and organic solvent content is lower than 1% in the control concentrated solution, thin up filters again, separates through macroporous resin, collect Hydroxytyrosol wash-out position, with the organic molten extraction of middle polarity 3-5 time, merge extraction phase, cryogenic vacuum concentrates, reclaim organic solvent, the molten liquid vacuum-drying of contracting, preparation leaf of Fructus oleae europaeae Hydroxytyrosol highly finished product, the content of Hydroxytyrosol is 10%-60%;
(5) adopt column chromatography to separate olive leaf Hydroxytyrosol highly finished product, enrichment 80%-98% Hydroxytyrosol.
The purity of the open HPLC external standard method Hydroxytyrosol of this patent.With the HPLC peak area is ordinate zou, and Hydroxytyrosol quality (μ g) is an X-coordinate, draws the HPLC typical curve, and get the HPLC equation of linear regression and be: Y=179663.8X-35982.8, its relation conefficient is r=0.9998.HPLC chromatographic condition: C
18ODS chromatographic column (Φ 4.6mm * 200mm, 5 μ m), moving phase: methanol-water (0.2% acetic acid) volume ratio 6: 94, wavelength 230nm, flow velocity: 1mL/min.
The accurate Hydroxytyrosol standard solution 10 μ L that draw repeat sample introduction 5 times, measure its peak area, RSD=0.93%.Accurately take by weighing leaf of Fructus oleae europaeae powder 1g, preparation method by Hydroxytyrosol solution in the experimental technique prepares its extracting solution, extracting solution is divided into 6 parts then, a copy of it is as blank sample, and all the other 5 parts of difference quantitatively add the Hydroxytyrosol standard solution of 0.67mg/mL, and sample introduction is measured then, each sample advances 3 times, get its peak area mean value, getting its average recovery is 100.5%, RSD=0.98%.The result shows that this method precision and circulation ratio are good.
The present invention adopts the content rule of HPLC methods analyst Hydroxytyrosol, from the Chenggu No. 32, skin melon that, ground draws in section, fruit reaches peaceful, No. 6, nine peaks, how cleverly join, A Si, little Lay star, the Lay star, the raw materials for production of screening high-content Hydroxytyrosol in the leaf such as 10 Fructus oleae europaeae kinds such as No. 8 grades are planted in Hubei Province, the result shows, the leaf of Fructus oleae europaeae of same place of production contemporaneity different varieties, the Hydroxytyrosol massfraction is variant, the Hydroxytyrosol content range is between 0.3%～0.8% in 10 kind leaf of Fructus oleae europaeae, and Hydroxytyrosol content size order is followed successively by: Xiao Laixing＞section draws ground＞A Si＞Hubei Province to plant how spirit of No. 6＞Guo Daning＞Chenggu, No. 8＞nine peaks No. 32＞Lay star＞Pi Guaer＞join.
The content of Hydroxytyrosol in the table 1 different varieties leaf of Fructus oleae europaeae
The present invention chooses branded oil olive kind and carries out seasonal enrichment discipline research, screens high-load month, the age of tree, kind.
The selection result shows, Hydroxytyrosol content is annual in the leaf of Fructus oleae europaeae has 1 peak period and a low ebb phase in long-term, and the content in 5-6 month is the highest; The end in 11-12 month; Content difference is bigger between the different age of trees and different varieties, and following age of tree leaf of Fructus oleae europaeae Hydroxytyrosol content was apparently higher than age of tree leaf more than 18 years in 6 years; Spire is planted in 6 years living Hubei Province, and to reach 0.768%, 6 year life nine peak May minimum No. 6 December, is 0.058%; Therefore, the raw materials for production of high-content Hydroxytyrosol should gathered the bright leaf of Fructus oleae europaeae below 6 years the 5-6 month in every year, must guard against 11, gather December.Collection green oil olive leaf is lower than 20 ℃ of lucifuges ventilations and dries in the shade, or vacuum-drying below 50 ℃, preparation leaf of Fructus oleae europaeae cured leaf, and wherein water content is at 3%-8%, and Hydroxytyrosol content is greater than 0.5%.Content is greater than the veteran leaf in most of kind spire, No. 8＞Chenggu No. 32＞Lay star＞Fo Ao＞nine peaks is planted No. 6 in Hubei Province, in whole 1 year, the Hydroxytyrosol content from 0.058% to 0.768% in the leaf of Fructus oleae europaeae, and content is than Hydroxytyrosol content height in the leaf of Fructus oleae europaeae in the foreign literature.As table 2
Hydroxytyrosol content (%) in the table 2 different varieties difference month leaf of Fructus oleae europaeae
It is ultrasonic wave and microwave extraction that the present invention adopts leaf of Fructus oleae europaeae Hydroxytyrosol extracting method, extraction agent alcohol or the aqueous acetone solution of C1-C3, particular methanol and acetone, methyl alcohol or acetone quality branch concentration in vain are 30-100%, preferred 6080%, extract solution and be 1-30 with leaf of Fructus oleae europaeae powder quality ratio: 1, preferred 10-15: 1, extracting temperature is 25-65 ℃, preferred 30-45 ℃, time is 10-60min, preferred 20-30min. lixiviate number of times 2-3 time, and leaf of Fructus oleae europaeae Hydroxytyrosol extraction yield people is in 90%.
This patent research and design 4 factors, 4 level [L
16(4
4)] orthogonal test, be index examination ultrasonic extracting process with the content of Hydroxytyrosol, test-results sees Table 3.
Table 3 orthogonal experiments
By in the table as can be seen, the influence that Hydroxytyrosol is extracted is followed successively by: solid-liquid ratio＞power＞time＞concentration.Optimum extraction process is power 120W, and 90% methyl alcohol is extraction agent, time 20min, and solid-liquid ratio 1: 20 extracts 3 times.
The present invention adopts macroporous resin separation and purification Hydroxytyrosol, selects AB 8 types or DM130 type or D101 type or X-5 type or NKA-II.Select ethanol-water solution degree of passing wash-out, determining alcohol is 0-80%, preferred 0-40% alcohol-water solution wash-out part, enrichment Hydroxytyrosol wash-out part. preferred following steps: a wet method dress post, diameter=2cm, height=15cm, 30g AB-8 type or DM130 type or D101 type or X-5 type or NKA-II, the b sample dissolution adds a chromatography column.C adds elutriant, and with the washing of 2-3 times of volume, the 5%-40% ethanol with 2 times of column volumes carries out wash-out more earlier, and flow velocity 1.25mL/min collects elutriant.
Hydroxytyrosol is an oily matter, be soluble in ethyl acetate and propyl carbinol, the present invention adopts the mixed solution of a kind of or two kinds of any ratios in ethyl acetate and the propyl carbinol repeatedly to extract macroporous resin enrichment Hydroxytyrosol wash-out part, ethyl acetate or n-butanol extraction with equivalent, extract 2-4 time, merge organic layer, concentrate, reclaim organic solvent, obtain the Hydroxytyrosol highly finished product.
For the 90% above Hydroxytyrosol of enrichment from leaf of Fructus oleae europaeae, positive pressure phase column purification during this patent adopts, leaf of Fructus oleae europaeae hydroxytyrosol extract and middle compression leg filler are pressed mass ratio 1: 15-50 absorption, in the compression leg filler can select a kind of in 200-500 purpose silica gel and the aluminum oxide or two kinds arbitrarily than, eluent is that eluent is a sherwood oil: ethyl acetate=10: 0.1-5 (v/v), preferred 10: 2-3 (v/v).Middle compression leg column length 20-300cm, column diameter 2-30cm, post press and are to detect wavelength 220-280nm by 3-20MPa, flow velocity 2-200ml/min, and enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 80% above Hydroxytyrosol; The present invention simultaneously adopts high pressure liquid chromatography column purification Hydroxytyrosol, and preparation condition is: permaphase ODS C18 (Φ 10mm * 250mm, 5 μ m); Moving phase: methanol-water (0.2% acetic acid) volume ratio 6-20: 94-80, wavelength 220-250nm, ultraviolet 220-270nm, flow velocity: 1-3mL/min.Enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 90% above Hydroxytyrosol.
Identify its chemical structure through IR, MS, NMR etc.
The spectroscopic identification result is as follows:
MS:153.2 is [M-H], determines that this compound molecular weight is 154, conforms to the molecular weight of Hydroxytyrosol.
1H-NMR (ppm): δ 3.69 is 1a-H, and δ 3.66 is 1b-H, and δ 2.68 is 2a-H, and δ 2.65 is 2b-H, and δ 6.67 is 4H, and δ 6.69 is 7-H, and δ 6.66 is 8-H, and δ 3.3 places are the methanol solvate peak.During unsubstituted, δ is 7.27 on the phenyl ring, and phenyl ring H quilt-OH replaces, and the δ of H on its ortho position is reduced.Be connected with alcoholic extract hydroxyl group on 1 C, its δ increases.General phenolic hydroxyl group δ is 4～7.5, and alcoholic extract hydroxyl group is between 1～5.Consistent with the Hydroxytyrosol H-MNR of bibliographical information.
13C-NMR: δ 64.55 is C-1, and δ 39.62 is C-2, and δ 121.20 is C-3, and δ 117.05 is C-4, and δ 146.08 is C-5, and δ 144.55 is C-6, and δ 116.30 is C-7, and δ 131.79 is C-8.Annotate: δ 49.1 places are the methanol solvate peak.During the phenyl ring unsubstituted, δ is 128.5, occurs 6 peaks between δ 110～165ppm, and they be the chemical shift of the C on the phenyl ring as can be known, because 5,6 for being connected with phenolic hydroxyl group, the cloud density of carbon reduced, the chemical shift δ increase of corresponding carbon.δ 64.55, are the chemical shift of carbon for being connected with 1 of alcoholic extract hydroxyl group as can be known according to chemical shift.Consistent with the Hydroxytyrosol C-MNR of bibliographical information.
Through the comparison of MS and NMR and Hydroxytyrosol standard substance spectral data, determine that Compound I is a Hydroxytyrosol.
The present invention obtains following technique effect:
1. set up the high-throughout screening method of leaf of Fructus oleae europaeae Hydroxytyrosol first, especially pass through the HPLC method to the seasonal enrichment discipline analysis of the different age of trees of different varieties, best industrial raw materials for production acquisition time and quality requirements have been determined, reduce production costs, for suitability for industrialized production provides optimum feed stock.
2. adopt ultrasonic wave and the microwave extraction technology green oil olive cured leaf extract at low temperature to screening first, by organic solvent extraction, resin gradient elution, integrated technologies such as middle compression leg separation prepare 90% hydroxytyrosol extract.Compare with purification process in the past, this kind method has easy, quick, economical, and characteristics such as yield height are fit to suitability for industrialized production.
Description of drawings
Hydroxytyrosol HPLC in the accompanying drawing 1 different varieties sample.
Hydroxytyrosol HPLC in the accompanying drawing 2 different month duplicate samples.
The pure product HPLC of Hydroxytyrosol of accompanying drawing 3 preparations.
Accompanying drawing 4 is the monomeric MS spectrogram of Hydroxytyrosol.
Accompanying drawing 5 is that Hydroxytyrosol is monomeric
1The H-NMR spectrogram.
Accompanying drawing 6 is that Hydroxytyrosol is monomeric
13The C-NMR spectrogram.
Embodiment
Following examples are more of the present invention giving an example, and should not regarded as limitation of the invention.
(1), pulverizes with leaf of Fructus oleae europaeae low-temperature dark drying;
(2) get leaf of Fructus oleae europaeae supersound extraction after the pulverizing, the ethanol consumption be 1-30 doubly, determining alcohol is 30-100%, extraction temperature is 25-65 ℃, extraction time is 10-30min, the lixiviate number of times is three times;
(3) leach liquor is filtered, vacuum concentration, the vacuum concentration temperature is no more than 65 ℃, gets the olive leaf crude extract.With crude extract thin up after-filtration, remove impurity;
(4) step (3), in the leaf of Fructus oleae europaeae leaf-alcohol extract crude product that obtains further carry out ion-exchange and separate, method may further comprise the steps:
A wet method dress post, diameter=2cm, height=15cm, 30g AB-8;
The b sample dissolution adds a chromatography column;
C adds elutriant, and with the washing of 2-3 times of volume, 10% ethanol with 2 times of column volumes carries out wash-out more earlier, and flow velocity 1.25mL/min collects elutriant, and uses ethyl acetate extraction.
Described olive leaf extract and preparation method thereof is characterized in that, among the step c, containing of Hydroxytyrosol can reach more than the 25%-60%.
(5) adopt column chromatography to separate olive leaf Hydroxytyrosol highly finished product,
The method of middle compression leg purification of hydroxy tyrosol is pressed mass ratio absorption in 1: 1550 with leaf of Fructus oleae europaeae Hydroxytyrosol highly finished product and middle compression leg filler, and middle compression leg filler can be selected 300 purpose silica gel, and eluent is that eluent is a sherwood oil: ethyl acetate.Middle compression leg column length 40cm, column diameter 3cm, post press and are to detect wavelength 230nm by 3MPa, flow velocity 10ml/min, and enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 80% above Hydroxytyrosol.
The method of high-pressure chromatographic column purification of hydroxy tyrosol is carried out high pressure liquid chromatography with 80% above leaf of Fructus oleae europaeae hydroxytyrosol extract and is separated preparation, and preparation condition is: permaphase ODS C18 (Φ 10mm * 250mm, 5 μ m); Moving phase: methanol-water (0.2% acetic acid) volume ratio 10: 90, wavelength 230nm, flow velocity: 3mL/min.Enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 95% above Hydroxytyrosol.
(1), pulverizes with leaf of Fructus oleae europaeae low-temperature dark drying;
(2) get leaf of Fructus oleae europaeae supersound extraction after the pulverizing, the ethanol consumption be 1-30 doubly, determining alcohol is 30-100%, extraction temperature is 25-65 ℃, extraction time is 10-30min, the lixiviate number of times is three times;
(3) leach liquor is filtered, vacuum concentration, the vacuum concentration temperature is no more than 65 ℃, gets the olive leaf crude extract.With crude extract thin up after-filtration, remove impurity;
(4) step (3), in the leaf of Fructus oleae europaeae leaf-alcohol extract crude product that obtains further carry out ion-exchange and separate, method may further comprise the steps:
A wet method dress post, diameter=2cm, height=15cm, 30g DM130;
The b sample dissolution adds a chromatography column;
C adds elutriant, and with the washing of 2-3 times of volume, 10% ethanol with 2 times of column volumes carries out wash-out more earlier, and flow velocity 1.25mL/min collects elutriant, and uses ethyl acetate extraction.
Described olive leaf extract and preparation method thereof is characterized in that, among the step c, the content of Hydroxytyrosol can reach more than the 20%-60%.
(5) adopt column chromatography to separate olive leaf Hydroxytyrosol highly finished product,
The method of middle compression leg purification of hydroxy tyrosol is pressed mass ratio 1: 15-50 absorption with leaf of Fructus oleae europaeae Hydroxytyrosol highly finished product and middle compression leg filler, and middle compression leg filler can be selected 300 purpose silica gel, and eluent is that eluent is a sherwood oil: ethyl acetate.Middle compression leg column length 40cm, column diameter 3cm, post press and are to detect wavelength 230nm by 3MPa, flow velocity 10ml/min, and enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 80% above Hydroxytyrosol.
The method of high-pressure chromatographic column purification of hydroxy tyrosol is carried out high pressure liquid chromatography with 80% above leaf of Fructus oleae europaeae hydroxytyrosol extract and is separated preparation, and preparation condition is: permaphase ODS C18 (Φ 10mm * 250mm, 5 μ m); Moving phase: methanol-water (0.2% acetic acid) volume ratio 10: 90, wavelength 230nm, flow velocity: 3mL/min.Enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 95% above Hydroxytyrosol.
(1), pulverizes with leaf of Fructus oleae europaeae low-temperature dark drying;
(2) get leaf of Fructus oleae europaeae supersound extraction after the pulverizing, the ethanol consumption be 1-30 doubly, determining alcohol is 30-100%, extraction temperature is 25-65 ℃, extraction time is 10-30min, the lixiviate number of times is three times;
(3) leach liquor is filtered, vacuum concentration, the vacuum concentration temperature is no more than 65 ℃, gets the olive leaf crude extract.With crude extract thin up after-filtration, remove impurity;
(4) step (3), in the leaf of Fructus oleae europaeae leaf-alcohol extract crude product that obtains further carry out ion-exchange and separate, method may further comprise the steps:
A wet method dress post, diameter=2cm, height=15cm, 30g D101;
The b sample dissolution adds a chromatography column;
C adds elutriant, and with the washing of 2-3 times of volume, 10% ethanol with 2 times of column volumes carries out wash-out more earlier, and flow velocity 1.25mL/min collects elutriant, and uses ethyl acetate extraction;
Described olive leaf extract and preparation method thereof is characterized in that, among the step c, the content of Hydroxytyrosol can reach more than the 15%-50%.
(5) adopt column chromatography to separate olive leaf Hydroxytyrosol highly finished product,
The method of middle compression leg purification of hydroxy tyrosol is pressed mass ratio 1: 15-50 absorption with leaf of Fructus oleae europaeae Hydroxytyrosol highly finished product and middle compression leg filler, and middle compression leg filler can be selected 300 purpose silica gel, and eluent is that eluent is a sherwood oil: ethyl acetate.Middle compression leg column length 40cm, column diameter 3cm, post press and are to detect wavelength 230nm by 3MPa, flow velocity 10ml/min, and enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 80% above Hydroxytyrosol.
The method of high-pressure chromatographic column purification of hydroxy tyrosol is carried out high pressure liquid chromatography with 80% above leaf of Fructus oleae europaeae hydroxytyrosol extract and is separated preparation, and preparation condition is: permaphase ODS C18 (Φ 10mm * 250mm, 5 μ m); Moving phase: methanol-water (0.2% acetic acid) volume ratio 10: 90, wavelength 230nm, flow velocity: 3mL/min.Enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 95% above Hydroxytyrosol.
Embodiment 4
(1), pulverizes with leaf of Fructus oleae europaeae low-temperature dark drying;
(2) get leaf of Fructus oleae europaeae supersound extraction after the pulverizing, the ethanol consumption be 1-30 doubly, determining alcohol is 30100%, extraction temperature is 25-65 ℃, extraction time is 10-30min, the lixiviate number of times is three times;
(3) leach liquor is filtered, vacuum concentration, the vacuum concentration temperature is no more than 65 ℃, gets the olive leaf crude extract.With crude extract thin up after-filtration, remove impurity;
(4) step (3), in the leaf of Fructus oleae europaeae leaf-alcohol extract crude product that obtains further carry out ion-exchange and separate, method may further comprise the steps:
A wet method dress post, diameter=2cm, height=15cm, 30g X-5;
The b sample dissolution adds a chromatography column;
C adds elutriant, and with the washing of 2-3 times of volume, 10% ethanol with 2 times of column volumes carries out wash-out more earlier, and flow velocity 1.25mL/min collects elutriant, and uses ethyl acetate extraction.
Described olive leaf extract and preparation method thereof is characterized in that, among the step c, the content of Hydroxytyrosol can reach more than the 15%-55%.
(5) adopt column chromatography to separate olive leaf Hydroxytyrosol highly finished product,
The method of middle compression leg purification of hydroxy tyrosol is pressed mass ratio 1: 15-50 absorption with leaf of Fructus oleae europaeae Hydroxytyrosol highly finished product and middle compression leg filler, and middle compression leg filler can be selected 300 purpose silica gel, and eluent is that eluent is a sherwood oil: ethyl acetate.Middle compression leg column length 40cm, column diameter 3cm, post press and are to detect wavelength 230nm by 3MPa, flow velocity 10ml/min, and enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 80% above Hydroxytyrosol.
The method of high-pressure chromatographic column purification of hydroxy tyrosol is carried out high pressure liquid chromatography with 80% above leaf of Fructus oleae europaeae hydroxytyrosol extract and is separated preparation, and preparation condition is: permaphase ODS C18 (Φ 10mm * 250mm, 5 μ m); Moving phase: methanol-water (0.2% acetic acid) volume ratio 10: 90, wavelength 230nm, flow velocity: 3mL/min.Enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 95% above Hydroxytyrosol.
Embodiment 5
(1), pulverizes with leaf of Fructus oleae europaeae low-temperature dark drying;
(2) get leaf of Fructus oleae europaeae supersound extraction after the pulverizing, the ethanol consumption be 1-30 doubly, determining alcohol is 30-100%, extraction temperature is 25-65 ℃, extraction time is 10-30min, the lixiviate number of times is three times;
(3) leach liquor is filtered, vacuum concentration, the vacuum concentration temperature is no more than 65 ℃, gets the olive leaf crude extract.With crude extract thin up after-filtration, remove impurity;
(4) step (3), in the leaf of Fructus oleae europaeae leaf-alcohol extract crude product that obtains further carry out ion-exchange and separate, method may further comprise the steps:
A wet method dress post, diameter=2cm, height=15cm, 30g NKA-II;
The b sample dissolution adds a chromatography column;
C adds elutriant, and with the washing of 2-3 times of volume, 10% ethanol with 2 times of column volumes carries out wash-out more earlier, and flow velocity 1.25mL/min collects elutriant, and uses ethyl acetate extraction.
Described olive leaf extract and preparation method thereof is characterized in that, among the step c, the content of Hydroxytyrosol can reach more than the 10%-50%.
(5) adopt column chromatography to separate olive leaf Hydroxytyrosol highly finished product,
The method of middle compression leg purification of hydroxy tyrosol is pressed mass ratio 1: 15-50 absorption with leaf of Fructus oleae europaeae Hydroxytyrosol highly finished product and middle compression leg filler, and middle compression leg filler can be selected 300 purpose silica gel, and eluent is that eluent is a sherwood oil: ethyl acetate.Middle compression leg column length 40cm, column diameter 3cm, post press and are to detect wavelength 230nm by 3MPa, flow velocity 10ml/min, and enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 80% above Hydroxytyrosol.
The method of high-pressure chromatographic column purification of hydroxy tyrosol is carried out high pressure liquid chromatography with 80% above leaf of Fructus oleae europaeae hydroxytyrosol extract and is separated preparation, and preparation condition is: permaphase ODS C18 (Φ 10mm * 250mm, 5 μ m); Moving phase: methanol-water (0.2% acetic acid) volume ratio 10: 90, wavelength 230nm, flow velocity: 3mL/min.Enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 95% above Hydroxytyrosol.
Claims (11)
1. the method for the high-quality Hydroxytyrosol of efficiently concentrating from leaf of Fructus oleae europaeae, form by following steps:
(1) raw materials for production of screening high-content Hydroxytyrosol from 10 Fructus oleae europaeae kind leaves;
(2) with the green oil olive leaf low-temperature dark drying of screening, drying temperature is lower than 20 ℃, is broken into the following powder of 0.5cm;
(3) with alcohol or the aqueous acetone solution extract oil olive leaf powder of C1-C3, alcohol or acetone mass percentage concentration are 30-100%, extract solution and are 1-30 with leaf of Fructus oleae europaeae powder quality ratio: 1, extracting temperature is 25-65 ℃, and the time is 10-60min, lixiviate number of times 1-5 time, filter united extraction liquid;
(4) vacuum concentration extracting solution under less than 65 ℃ of temperature reclaims organic solvent, and organic solvent content is lower than 1% in the control concentrated solution, thin up filters again, separates through macroporous resin, collect Hydroxytyrosol wash-out position, with the organic molten extraction of middle polarity 3-5 time, merge extraction phase, cryogenic vacuum concentrates, reclaim organic solvent, concentrated solution vacuum-drying, preparation leaf of Fructus oleae europaeae Hydroxytyrosol highly finished product, the content of Hydroxytyrosol is 10%-60%;
(5) adopt column chromatography to separate olive leaf Hydroxytyrosol highly finished product, enrichment 80%-98% Hydroxytyrosol.
According to claim 1 described a kind of from leaf of Fructus oleae europaeae the method for the high-quality Hydroxytyrosol of efficiently concentrating, the raw materials for production that it is characterized in that the high-content Hydroxytyrosol are for gathering the bright leaf of Fructus oleae europaeae below 6 years the 5-6 month in every year, being lower than 20 ℃ of lucifuges ventilations dries in the shade, or vacuum-drying below 50 ℃, preparation leaf of Fructus oleae europaeae cured leaf, wherein water content is at 3%-8%, and Hydroxytyrosol content is greater than 0.5%.
According to claim 1 described a kind of from leaf of Fructus oleae europaeae the method for the high-quality Hydroxytyrosol of efficiently concentrating, it is characterized in that the Hydroxytyrosol content analysis adopts HPLC method, HPLC chromatographic condition: C
18ODS chromatographic column (Φ 4.6mm * 200mm, 5 μ m), moving phase: methanol-water (0.2% acetic acid) volume ratio 6: 94, wavelength 220-250nm, flow velocity: 1mL/min.
According to claim 1 described a kind of from leaf of Fructus oleae europaeae the method for the high-quality Hydroxytyrosol of efficiently concentrating, it is characterized in that 10 Fructus oleae europaeae kinds are that No. 32, Chenggu, Pi Guaer, section are drawn ground, Guo Daning, No. 6, nine peaks, joined many spirits, A Si, Xiao Laixing, Lay star, Hubei Province plants No. 8.
According to claim 1 described a kind of from leaf of Fructus oleae europaeae the method for the high-quality Hydroxytyrosol of efficiently concentrating, it is characterized in that leaf of Fructus oleae europaeae Hydroxytyrosol extracting method is ultrasonic wave and microwave extraction described in the step (3).
According to claim 1 described a kind of from leaf of Fructus oleae europaeae the method for the high-quality Hydroxytyrosol of efficiently concentrating, it is characterized in that the macroporous resin described in the step (4) is selected AB-8 type or DM130 type or D101 type or X-5 type or NKA-II.
According to claim 1 described a kind of from leaf of Fructus oleae europaeae the method for the high-quality Hydroxytyrosol of efficiently concentrating, it is characterized in that, macroporous resin described in the step (4) separates selects alcohol-water solution degree of passing wash-out, and determining alcohol is 0-80%, enrichment 0-40% alcohol-water solution wash-out part.
According to claim 1 described a kind of from leaf of Fructus oleae europaeae the method for the high-quality Hydroxytyrosol of efficiently concentrating, it is characterized in that the organic solvent described in the step (4) is the mixed solution of a kind of or two kinds of any ratios in ethyl acetate and the propyl carbinol.
According to claim 1 described a kind of from leaf of Fructus oleae europaeae the method for the high-quality Hydroxytyrosol of efficiently concentrating, it is characterized in that compression leg and high-pressure chromatographic column purifying during step 5 center pillar chromatographic separation can be selected.
According to claim 9 described in the method for compression leg purification of hydroxy tyrosol, the compression leg purifying is that 10%-60% leaf of Fructus oleae europaeae hydroxytyrosol extract and middle compression leg filler are adsorbed by mass ratio 1: 15-50 in it is characterized in that, in the compression leg filler can select a kind of in 200-500 purpose silica gel and the aluminum oxide or two kinds arbitrarily than, eluent is that eluent is a sherwood oil: ethyl acetate=10: 0.1-5 (v/v), preferred 10: 2-3 (v/v).Middle compression leg column length 20-300cm, column diameter 2-30cm, post press and are to detect wavelength 220280nm by 3-20MPa, flow velocity 2-200ml/min, and enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 80% above Hydroxytyrosol.
11. method according to the described high-pressure chromatographic column purification of hydroxy of claim 9 tyrosol, it is characterized in that 10%-60% leaf of Fructus oleae europaeae hydroxytyrosol extract is carried out high pressure liquid chromatography separates preparation, preparation condition is: permaphase ODS C18 (Φ 10mm * 250mm, 5 μ m); Moving phase: methanol-water (0.2% acetic acid) volume ratio 6-20: 94-80, wavelength 220-250nm, ultraviolet 220-270nm, flow velocity: 1-3mL/min.Enrichment Hydroxytyrosol position, the room temperature solvent recovered under vacuum is analyzed through HPLC, prepares 90% above Hydroxytyrosol.
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| 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 |
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