CN105055527A - Preparation method of olive leaf extract - Google Patents
Preparation method of olive leaf extract Download PDFInfo
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- CN105055527A CN105055527A CN201510620159.8A CN201510620159A CN105055527A CN 105055527 A CN105055527 A CN 105055527A CN 201510620159 A CN201510620159 A CN 201510620159A CN 105055527 A CN105055527 A CN 105055527A
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- oleuropein
- olive
- enzymolysis
- olive leaf
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- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 229940114496 olive leaf extract Drugs 0.000 title abstract description 6
- 240000007817 Olea europaea Species 0.000 claims abstract description 36
- RFWGABANNQMHMZ-HYYSZPHDSA-N Oleuropein Chemical compound O([C@@H]1OC=C([C@H](C1=CC)CC(=O)OCCC=1C=C(O)C(O)=CC=1)C(=O)OC)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O RFWGABANNQMHMZ-HYYSZPHDSA-N 0.000 claims abstract description 34
- RFWGABANNQMHMZ-UHFFFAOYSA-N 8-acetoxy-7-acetyl-6,7,7a,8-tetrahydro-5H-benzo[g][1,3]dioxolo[4',5':4,5]benzo[1,2,3-de]quinoline Natural products CC=C1C(CC(=O)OCCC=2C=C(O)C(O)=CC=2)C(C(=O)OC)=COC1OC1OC(CO)C(O)C(O)C1O RFWGABANNQMHMZ-UHFFFAOYSA-N 0.000 claims abstract description 33
- HKVGJQVJNQRJPO-UHFFFAOYSA-N Demethyloleuropein Natural products O1C=C(C(O)=O)C(CC(=O)OCCC=2C=C(O)C(O)=CC=2)C(=CC)C1OC1OC(CO)C(O)C(O)C1O HKVGJQVJNQRJPO-UHFFFAOYSA-N 0.000 claims abstract description 33
- RFWGABANNQMHMZ-CARRXEGNSA-N oleuropein Natural products COC(=O)C1=CO[C@@H](O[C@H]2O[C@@H](CO)[C@H](O)[C@@H](O)[C@@H]2O)C(=CC)[C@H]1CC(=O)OCCc3ccc(O)c(O)c3 RFWGABANNQMHMZ-CARRXEGNSA-N 0.000 claims abstract description 33
- 235000011576 oleuropein Nutrition 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 31
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 35
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 24
- 108010059892 Cellulase Proteins 0.000 claims description 19
- 229940106157 cellulase Drugs 0.000 claims description 19
- 102000004190 Enzymes Human genes 0.000 claims description 18
- 108090000790 Enzymes Proteins 0.000 claims description 18
- 229940088598 enzyme Drugs 0.000 claims description 18
- 239000002994 raw material Substances 0.000 claims description 17
- 235000002725 Olea europaea Nutrition 0.000 claims description 16
- 150000001875 compounds Chemical class 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 15
- 239000003480 eluent Substances 0.000 claims description 13
- 229960000583 acetic acid Drugs 0.000 claims description 12
- 239000012362 glacial acetic acid Substances 0.000 claims description 12
- 108091005658 Basic proteases Proteins 0.000 claims description 11
- 235000002639 sodium chloride Nutrition 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 239000000284 extract Substances 0.000 claims description 8
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- 238000001291 vacuum drying Methods 0.000 claims description 8
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 7
- 230000007935 neutral effect Effects 0.000 claims description 7
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- 238000009776 industrial production Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 13
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
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- 239000004382 Amylase Substances 0.000 description 3
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- 235000019421 lipase Nutrition 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- XHFLOLLMZOTPSM-UHFFFAOYSA-M sodium;hydrogen carbonate;hydrate Chemical compound [OH-].[Na+].OC(O)=O XHFLOLLMZOTPSM-UHFFFAOYSA-M 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- 241000795633 Olea <sea slug> Species 0.000 description 2
- 241000207834 Oleaceae Species 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
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- 230000002708 enhancing effect Effects 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
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- 239000002904 solvent Substances 0.000 description 2
- 244000147058 Derris elliptica Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- CDXSJGDDABYYJV-UHFFFAOYSA-N acetic acid;ethanol Chemical compound CCO.CC(O)=O CDXSJGDDABYYJV-UHFFFAOYSA-N 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
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Abstract
The invention discloses a preparation method of an olive leaf extract and the olive leaf extract prepared by the preparation method. An olive leaf extract product of which the oleuropein content is greater than 90% is obtained through the processes of infiltration, enzymatic extraction, separation and drying on olive leaves. The whole method only comprises four steps, the adopted reagents are common non-toxic ingredients, the yield is high, the cost is low, the obtained product can be directly used for preparing medicines, health products and foods, and the method is very suitable for industrial production.
Description
Technical field
The present invention relates to a kind of preparation method of Olive leaf P.E, belong to natural plant extracts field.
Background technology
Fructus oleae europaeae (OleaeuropaeaL.), belongs to Oleaceae (Oleaceae) Olea (Olea) aiphyllium, is world-renowned woody oil tree species, has the cultivation history of more than 4000 year.Fructus oleae europaeae is mainly distributed between north latitude 45 ° to 37 °, south latitude, and central distribution is mediterranean country, and Greece, Italy, Tunisia, Spain are for concentrating the place of production, and all there is introducing and planting the countries in the world comprising now China.Since China introduced a fine variety Fructus oleae europaeae in 1964, nearly ten years, under development of the West Regions policy support, the plantation development of Fructus oleae europaeae is very fast, the construction of base of Gansu, Sichuan Er Sheng begins to take shape, and sets up olive oil processing factory, and Fructus oleae europaeae fruit is fully used, but the annual leaf of Fructus oleae europaeae discarded because of pruning is more than thousands of tons of, and is utilized very well.
Main containing secoiridoid and glycosides, flavone and glycosides thereof, bisflavone and the composition such as glycosides, low molecule tannin thereof in Folium olive, secoiridoid is main active.
Oleuropein (oleuropein) is the bitter principle in Folium olive, is a kind of bimolecular structure phenols and Flavonoid substances, molecular formula C
25h
32o
13, molecular weight 540.52, fusing point 87 ~ 89 DEG C, easy moisture absorption, is soluble in ethanol, acetone, pyridine etc., and water soluble, butanols, ethyl acetate etc. are dissolved in ether, petroleum ether, chloroform etc. hardly.It is reported, oleuropein has natural antioxidation, antitumor, slow down aging, enhancing cardiovascular function, treats the important health care such as chronic prostatitis, enhancing immunity, conciliation hormonal system, hepatoprotective, antiinflammatory, also have blood fat reducing, hypoglycemic pharmacological action simultaneously, be widely used in the fields such as food, cosmetic and bio-pharmaceuticals.
Oleuropein mainly extracts and obtains from Folium olive.Disclosed in current bibliographical information or patent, oleuropein extraction and separation technology is more, and Folium olive extractive technique mainly includes machine solvent extraction techniques, supercritical CO
2abstraction technique, ultrasonic extraction technology and microwave-assisted extraction technique etc.But organic solvent extraction has that impurity content is high, solvent consumption is large, cost is high, pollute the shortcomings such as large.Supercritical CO
2extraction has extracts the advantages such as content is high, pollution-free, but have high input, cost is high.Involve microwave extracting ultrasonic and although there is the advantages such as energy-conservation, efficient, production is difficult to realize large-scale application.
Such as, Chinese patent CN1583052A discloses a kind of Olive leaf P.E and extracts preparation method, by Folium olive after sterilization rinsing, drying, alcohol extraction, vacuum concentration, in concentrated solution, add tea polyphenols antioxidation, drying obtains Olive leaf P.E, but in its Olive leaf P.E prepared, active constituent content is low.Chinese patent CN101003557A discloses a kind of preparation method being rich in the olive leaf extract of high-purity oleuropein, the concentrated solution by NF membrane is obtained by steps such as the extraction of Folium olive polar solvent, alkaline centrifugal sedimentation, centrifugal clear liquid, film integrated separations, then media adsorbs, desorption obtain eluent, dry after vacuum concentration, obtain the olive leaf extract being rich in high-purity oleuropein that oleuropein content is greater than 50%.But the method practical operation step is too much, is unfavorable for industrialized great production, and membrane separation plant first-phase investment is large, improves product cost.Japanese Unexamined Patent Publication 2002128678 aqueous solvent extracts, and after synthetic resin process, drying obtains the extract containing oleuropein more than 25%.Adopt alcohol-water solution heat to carry more than secondary in US Patent No. 5714150, spraying dry or vacuum drying after extracting solution vacuum concentration, it prepares the Olive leaf P.E containing oleuropein 30-40%.In these extracts, the content of oleuropein is too low, all can not meet market demands.
Organic solvent extraction, silica gel column chromatography and HPLC preparative hplc is adopted repeatedly during the article of Wang Chengzhang etc. " introduces a fine variety oleuropein extraction and isolation and Structural Identification in A Si leaf of Fructus oleae europaeae ", isolated the cleupin that content is 95%, but this method is limited to the greatest extent a small amount of sample preparation.In European patent EP 1795201, Folium olive is worn into the fine powder of diameter 0.05 to 1 millimeter, pH8.0 potass extraction; Aqueous extracts is through organic solvent extraction, and extract vacuum dehydrating at lower temperature obtains the crude extract containing oleuropein 40-45%; This crude extract, again through fast silica gel chromatogram column purification, collects the stripping liquid containing oleuropein part, obtains the amorphous solid containing 95-98% oleuropein after recycling design.Although but in the method, obtaining higher oleuropein content, high expensive, is more suitable for scientific research, is difficult to accomplish scale production.
Therefore, present inventor, in order to propose that a kind of active constituent content is high, cost is low and method is easy, solvent reusable edible, reduces environmental pollution, is easy to the method for industrialization, conducts in-depth research, and then obtain the present invention.
Summary of the invention
To achieve these goals, the invention provides a kind of preparation method of Olive leaf P.E, comprise the steps:
(1) raw material Folium olive saline soak, after 1-2 hour, is dried, is ground into fine powder;
(2) in raw material, add 3-8 times of water, add cellulase: the compound enzyme of alkaline protease, compound enzyme consumption is the 0.5-2 % by weight of raw material, enzymolysis 0.5-2h at remaining on 30-50 DEG C, filters, obtains enzymolysis solution;
(3) enzymolysis solution directly passes through neutral alumina column, resin column blade diameter length ratio is 1: 4 ~ 1: 15, respectively with 1-3 times of cylinder hydrops, the remove impurity of 1-2 times column volume 95% alcoholic solution eluting, with 0.2-0.5% glacial acetic acid-50% ethanol (1:1) eluant solution of 6-10 times of column volume, collect eluent;
(4), after flinging to glacial acetic acid in eluent and ethanol, microwave vacuum drying, obtains the Olive leaf P.E product that oleuropein content is greater than 90%.
In step (1), saline can be saturated aqueous common salt, and the granularity of pulverizing is preferably through 40-60 mesh sieve.
In step (2), the water yield is preferably 4-6 doubly, is more preferably 5 times; Compound enzyme is preferably cellulase: the compound enzyme of alkaline protease 1:3.
In step (3), microwave vacuum drying temperature: 40-60 DEG C, return difference temperature 2-4 DEG C, more than vacuum-0.07Mpa, microwave power 10-80KW, dry 100-150 minute.
The present invention is by the infiltration to Folium olive, enzymolysis and extraction, be separated, dry technique, obtain the product being rich in oleuropein: through screening and the optimization of system, first raw material pre-treatment step is determined, infiltrate with saline before preparation, and by appropriate pulverizing, stripping effective ingredient more up hill and dale, further, adopt neutral alumina column, enzymolysis solution gets final product direct upper prop without the need to concentrated, significantly reduce energy consumption, after glacial acetic acid-ethanol elution eluting and drying, obtain the Olive leaf P.E product that oleuropein content is greater than 90%.Whole method is only made up of 4 steps, adopt reagent to be common non-toxic components, yield is high and cost is low, and the product that obtains can be directly used in prepares medicine and health product, food, is be very suitable for industrial method.
Present invention also offers the Olive leaf P.E that above-mentioned preparation method is obtained.
Detailed description of the invention
R&D process of the present invention will be introduced in detail below.
One, the research of enzymolysis process
Experiment material: Folium olive powder, pectase, cellulase, alkaline protease, amylase, lipase, lysozyme
Experimental technique: get Folium olive powder, adds raw material weight 1.5-2 water doubly, and adds the enzyme of 1%, supersound process.Measure the content of oleuropein in the supernatant after enzymolysis.
Experimental result sees the following form.
| Enzyme class | Oleuropein (mg/100ml) |
| Cellulase | 24.32 |
| Alkaline protease | 19.06 |
| Pectase | 19.59 |
| Amylase | 20.28 |
| Lipase | 14.17 |
| Lysozyme | 15.89 |
| Cellulase: alkaline protease (1:1) | 28.45 |
| Cellulase: alkaline protease (1:2) | 30.25 |
| Cellulase: alkaline protease (1:3) | 39.54 |
| Cellulase: amylase (1:1) | 25.11 |
| Cellulase: lipase (1:1:1) | 20.19 |
As can be seen here, the stripping of compound enzyme to oleuropein of cellulase and alkaline protease is highly beneficial, particularly preferably cellulase: alkaline protease (1:3).
Two, the research of technique before enzymolysis
Experiment material: Folium olive, water, saline solution, lime water, sodium bicarbonate water, cellulase, alkaline protease
Experimental technique: get Folium olive, adds 5-10 times of water, saline solution, lime water, sodium bicarbonate water infiltration after 1 hour respectively, dries, carries out enzymolysis (cellulase: alkaline protease 1:3) after pulverizing, measures the content of oleuropein in enzymolysis supernatant.
Experimental result sees the following form.
| Technique | Oleuropein (mg/100ml) |
| Enzymolysis after water infiltration | 40.61 |
| Enzymolysis after saturated common salt water infiltration | 62.17 |
| Enzymolysis after lime water infiltrates | 51.59 |
| Enzymolysis after sodium bicarbonate water infiltration | 42.38 |
Inventor very surprisingly finds, the Folium olive after saturated common salt water infiltration, again through enzymolysis, greatly can improve the stripping of oleuropein.This reason is not yet clear and definite, seem different from adopting the method for lime water lixiviate in prior art, but inventor's conjecture is the infiltration because of saline solution in the Folium olive of comparatively plumpness, after cellulase and basic protein enzyme interacting, has played abnormal compound action and has caused.
Three, the screening of column chromatography
Experimental technique: get the supernatant after enzymolysis, after concentrated, put in the good neutral alumina column resin 100g of pretreatment, shaking table dynamic adsorption is after 8 hours, dress post, respectively with 1-3 times of cylinder hydrops, 30% ethanol, 50% ethanol, 80% ethanol, 0.5% glacial acetic acid, n-butyl alcohol, ethyl acetate solution or its mixed liquor eluting, collect eluent and carry out oleuropein assay, elution fractions evaporate to dryness calculates dry cream amount.
Experimental result sees the following form.
| Eluent kind | Dry cream amount (g) | Oleuropein content |
| Water | 3.82 | 55.21 |
| 30% ethanol | 4.24 | 79.09 |
| 50% ethanol | 4.65 | 82.35 |
| 80% ethanol | 4.11 | 77.26 |
| 0.5% glacial acetic acid | 4.97 | 80.42 |
| N-butyl alcohol | 3.89 | 67.09 |
| 30% ethanol-0.5% glacial acetic acid (1:1) | 5.49 | 85.21 |
| 50% ethanol-0.5% glacial acetic acid (1:1) | 6.25 | 92.89 |
| 80% ethanol-0.5% glacial acetic acid (1:1) | 5.02 | 78.54 |
| N-butyl alcohol-0.5% glacial acetic acid (1:1) | 4.85 | 75.67 |
Result shows, the effect of 50% ethanol-0.5% glacial acetic acid (1:1) is best.
The present inventor has also carried out other many-sided screenings, comprise the screening etc. of the granularity of pulverizing, resin column model, finally obtain technical scheme of the present invention, obtain a kind of method easy, be easy to implement and obtain comprising the Olive leaf P.E more than more than 90% oleuropein, can be directly used in and prepare food, health product or medicine.
Embodiment 1: a kind of Olive leaf P.E, it obtains by the following method, comprises the steps:
(1) raw material Folium olive saline soak, after 1 hour, is dried, is ground into 40-60 order fine powder;
(2) in raw material, add 5 times of water, add cellulase: the compound enzyme of alkaline protease 1:3, compound enzyme consumption is 1 % by weight of raw material, enzymolysis 0.5h at remaining on 30-50 DEG C, filters, obtains enzymolysis solution;
(3) enzymolysis solution directly passes through neutral alumina column, resin column blade diameter length ratio is 1: 4 ~ 1: 15, respectively with 1-3 times of cylinder hydrops, the remove impurity of 1-2 times column volume 95% alcoholic solution eluting, with 0.2-0.5% glacial acetic acid-50% ethanol (1:1) eluant solution of 6 times of column volumes, collect eluent;
(4), after flinging to glacial acetic acid in eluent and ethanol, microwave vacuum drying, temperature: 40-60 DEG C, return difference temperature 2-4 DEG C, more than vacuum-0.07Mpa, microwave power 10-80KW, dry 100-150 minute, obtains product.After measured, in this product, oleuropein content is 96.03%.
Embodiment 2: a kind of Olive leaf P.E, it obtains by the following method, comprises the steps:
(1) raw material Folium olive saline soak, after 2 hours, is dried, is ground into fine powder;
(2) in raw material, add 3 times of water, add cellulase: the compound enzyme of alkaline protease, compound enzyme consumption is 2 % by weight of raw material, enzymolysis 0.5h at remaining on 30-50 DEG C, filters, obtains enzymolysis solution;
(3) enzymolysis solution directly passes through neutral alumina column, resin column blade diameter length ratio is 1: 4 ~ 1: 15, respectively with 1-3 times of cylinder hydrops, the remove impurity of 1-2 times column volume 95% alcoholic solution eluting, with 0.2-0.5% glacial acetic acid-50% ethanol (1:1) eluant solution of 10 times of column volumes, collect eluent;
(4), after flinging to glacial acetic acid in eluent and ethanol, microwave vacuum drying, temperature: 40-60 DEG C, return difference temperature 2-4 DEG C, more than vacuum-0.07Mpa, microwave power 10-80KW, dry 100-150 minute, obtains product.After measured, in this product, oleuropein content is 93.92%.
Embodiment 3: a kind of Olive leaf P.E, it obtains by the following method, comprises the steps:
(1) raw material Folium olive saline soak, after 1 hour, is dried, is ground into 50-60 order fine powder;
(2) in raw material, add 7 times of water, add cellulase: the compound enzyme of alkaline protease 1:3, compound enzyme consumption is 1 % by weight of raw material, enzymolysis 0.5h at remaining on 30-50 DEG C, filters, obtains enzymolysis solution;
(3) enzymolysis solution directly passes through neutral alumina column, resin column blade diameter length ratio is 1: 4 ~ 1: 15, respectively with 1-3 times of cylinder hydrops, the remove impurity of 1-2 times column volume 95% alcoholic solution eluting, with 0.2-0.5% glacial acetic acid-50% ethanol (1:1) eluant solution of 8 times of column volumes, collect eluent;
(4), after flinging to glacial acetic acid in eluent and ethanol, microwave vacuum drying, temperature: 40-60 DEG C, return difference temperature 2-4 DEG C, more than vacuum-0.07Mpa, microwave power 10-80KW, dry 100-150 minute, obtains product.After measured, in this product, oleuropein content is 95.40%.
Claims (7)
1. a preparation method for Olive leaf P.E, comprises the steps:
(1) raw material Folium olive saline soak, after 1-2 hour, is dried, is ground into fine powder;
(2) in raw material, add 3-8 times of water, add cellulase: the compound enzyme of alkaline protease, compound enzyme consumption is the 0.5-2 % by weight of raw material, enzymolysis 0.5-2h at remaining on 30-50 DEG C, filters, obtains enzymolysis solution;
(3) enzymolysis solution directly passes through neutral alumina column, resin column blade diameter length ratio is 1: 4 ~ 1: 15, respectively with 1-3 times of cylinder hydrops, the remove impurity of 1-2 times column volume 95% alcoholic solution eluting, with 0.2-0.5% glacial acetic acid-50% ethanol (1:1) eluant solution of 6-10 times of column volume, collect eluent;
(4), after flinging to glacial acetic acid in eluent and ethanol, microwave vacuum drying, obtains the Olive leaf P.E product that oleuropein content is greater than 90%.
2. method as claimed in claim 1, in step (1), saline can be saturated aqueous common salt.
3. method as claimed in claim 1, in step (1), the granularity of pulverizing is preferably through 40-60 mesh sieve.
4. method as claimed in claim 1, in step (2), the water yield is preferably 4-6 doubly, is more preferably 5 times.
5. method as claimed in claim 1, in step (2), compound enzyme is preferably cellulase: the compound enzyme of alkaline protease 1:3.
6. method as claimed in claim 1, in step (3), microwave vacuum drying temperature: 40-60 DEG C, return difference temperature 2-4 DEG C, more than vacuum-0.07Mpa, microwave power 10-80KW, dry 100-150 minute.
7. the extract being rich in oleuropein that said method obtains.
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Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106046078A (en) * | 2016-06-28 | 2016-10-26 | 郭舒洋 | Method for extracting oleuropein from olea europaea leaves |
| CN106421741A (en) * | 2016-09-26 | 2017-02-22 | 南京中生生物科技有限公司 | Water-soluble olive leaf extract health-care product and preparation method thereof |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003557A (en) * | 2006-12-12 | 2007-07-25 | 中国林业科学研究院林产化学工业研究所 | Method for preparing extractive of olive leaves rich in oleuropein in high purity |
| KR20100033087A (en) * | 2008-09-19 | 2010-03-29 | (주)아모레퍼시픽 | Hair coloring composition using iridoid glucosides |
| CN103333213A (en) * | 2013-07-24 | 2013-10-02 | 桂林茗兴生物科技有限公司 | Method for extracting oleuropein from olive leaves |
| US20130272974A1 (en) * | 2012-04-12 | 2013-10-17 | Ahmad Alkayali | Hydroxytyrosol Product, Method of Making, and Uses Thereof |
| CN103571890A (en) * | 2013-10-28 | 2014-02-12 | 桂林茗兴生物科技有限公司 | Method of preparing hydroxytyrosol by using oleuropein |
-
2015
- 2015-09-25 CN CN201510620159.8A patent/CN105055527B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003557A (en) * | 2006-12-12 | 2007-07-25 | 中国林业科学研究院林产化学工业研究所 | Method for preparing extractive of olive leaves rich in oleuropein in high purity |
| KR20100033087A (en) * | 2008-09-19 | 2010-03-29 | (주)아모레퍼시픽 | Hair coloring composition using iridoid glucosides |
| US20130272974A1 (en) * | 2012-04-12 | 2013-10-17 | Ahmad Alkayali | Hydroxytyrosol Product, Method of Making, and Uses Thereof |
| CN103333213A (en) * | 2013-07-24 | 2013-10-02 | 桂林茗兴生物科技有限公司 | Method for extracting oleuropein from olive leaves |
| CN103571890A (en) * | 2013-10-28 | 2014-02-12 | 桂林茗兴生物科技有限公司 | Method of preparing hydroxytyrosol by using oleuropein |
Non-Patent Citations (2)
| Title |
|---|
| JIAO-JIAO YUAN等: "Enzymatic Hydrolysis of Oleuropein from Olea europea (Olive) Leaf Extract and Antioxidant Activities", 《MOLECULES》 * |
| 谢普军等: "油橄榄叶提取物中橄榄苦苷稳定性及", 《食品工业科技》 * |
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