CN105055527B - A kind of preparation method of Olive leaf P.E - Google Patents
A kind of preparation method of Olive leaf P.E Download PDFInfo
- Publication number
- CN105055527B CN105055527B CN201510620159.8A CN201510620159A CN105055527B CN 105055527 B CN105055527 B CN 105055527B CN 201510620159 A CN201510620159 A CN 201510620159A CN 105055527 B CN105055527 B CN 105055527B
- Authority
- CN
- China
- Prior art keywords
- olive leaf
- oleuropein
- olive
- ethyl alcohol
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 240000007817 Olea europaea Species 0.000 title claims abstract description 52
- 235000002725 Olea europaea Nutrition 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 235000011576 oleuropein Nutrition 0.000 claims abstract description 33
- 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 32
- 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 32
- 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 32
- 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 32
- 238000000034 method Methods 0.000 claims abstract description 29
- 238000001035 drying Methods 0.000 claims abstract description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 61
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 37
- 235000019441 ethanol Nutrition 0.000 claims description 29
- 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
- 229960000583 acetic acid Drugs 0.000 claims description 18
- 229940088598 enzyme Drugs 0.000 claims description 18
- 239000012362 glacial acetic acid Substances 0.000 claims description 17
- 239000003513 alkali Substances 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 16
- 239000002994 raw material Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 239000003480 eluent Substances 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 108091005804 Peptidases Proteins 0.000 claims description 11
- 239000004365 Protease Substances 0.000 claims description 11
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000000843 powder Substances 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
- 150000001875 compounds Chemical class 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 7
- 230000007935 neutral effect Effects 0.000 claims description 7
- 229920005989 resin Polymers 0.000 claims description 7
- 239000011347 resin Substances 0.000 claims description 7
- 239000012535 impurity Substances 0.000 claims description 6
- 101710180319 Protease 1 Proteins 0.000 claims description 5
- 101710137710 Thioesterase 1/protease 1/lysophospholipase L1 Proteins 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 4
- 239000012267 brine Substances 0.000 claims description 3
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 claims description 3
- 239000012266 salt solution Substances 0.000 claims description 2
- 229920006395 saturated elastomer Polymers 0.000 claims description 2
- 238000000605 extraction Methods 0.000 abstract description 9
- 238000001764 infiltration Methods 0.000 abstract description 9
- 230000008595 infiltration Effects 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 238000000926 separation method Methods 0.000 abstract description 5
- 235000013305 food Nutrition 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 3
- 230000036541 health Effects 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 231100000252 nontoxic Toxicity 0.000 abstract description 2
- 230000003000 nontoxic effect Effects 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 13
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound 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
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 4
- 229930182470 glycoside Natural products 0.000 description 4
- 150000002338 glycosides Chemical class 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 235000002639 sodium chloride Nutrition 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 239000004382 Amylase Substances 0.000 description 3
- 102000013142 Amylases Human genes 0.000 description 3
- 108010065511 Amylases Proteins 0.000 description 3
- 102000004882 Lipase Human genes 0.000 description 3
- 239000004367 Lipase Substances 0.000 description 3
- 108090001060 Lipase Proteins 0.000 description 3
- 235000019418 amylase Nutrition 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 235000019421 lipase Nutrition 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 238000011160 research Methods 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
- 239000002904 solvent Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 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
- 230000009471 action Effects 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- UFJORDZSBNSRQT-UHFFFAOYSA-N 3-(4-oxo-2-phenylchromen-3-yl)-2-phenylchromen-4-one Chemical compound O1C2=CC=CC=C2C(=O)C(C=2C(C3=CC=CC=C3OC=2C=2C=CC=CC=2)=O)=C1C1=CC=CC=C1 UFJORDZSBNSRQT-UHFFFAOYSA-N 0.000 description 1
- 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
- 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
- 239000004480 active ingredient Substances 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000006286 aqueous extract Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000009084 cardiovascular function Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 208000013507 chronic prostatitis Diseases 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 150000002213 flavones Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000000874 microwave-assisted extraction Methods 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 201000007094 prostatitis Diseases 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- 239000000057 synthetic resin Substances 0.000 description 1
- 235000018553 tannin Nutrition 0.000 description 1
- 229920001864 tannin Polymers 0.000 description 1
- 239000001648 tannin Substances 0.000 description 1
- 238000009966 trimming Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Abstract
The invention discloses a kind of preparation method of Olive leaf P.E and its Olive leaf P.Es of preparation.By to the infiltration of olive leaf, enzymolysis and extraction, separation, drying technique, obtained oleuropein content be more than 90% Olive leaf P.E product.Entire method is only made of 4 steps, and it is common non-toxic components to use reagent, high income and at low cost, and acquired product can be directly used for preparing drug and health products, food, be highly suited for industrial method.
Description
Technical field
The present invention relates to a kind of preparation methods of Olive leaf P.E, belong to natural plant extracts field.
Background technology
Olive (Olea europaea L.) belongs to Oleaceae (Oleaceae) Olea (Olea) aiphyllium, is
World-renowned woody oil tree species have the cultivation history of more than 4000 years.Olive is mainly distributed on 45 ° of north latitude and arrives 37 ° of south latitude
Between, central distribution is mediterranean country, and Greece, Italy, Tunisia, Spain include China now to concentrate the place of production
Countries in the world have introducing and planting.China is since 1964 introduce a fine variety olive, nearly ten years, in development of the West Regions policy support
Under, the plantation development of olive is very fast, and the construction of base in Gansu, Sichuan Er Sheng has begun to take shape, and sets up olive oil processing
Factory, olive fruit are fully used, but olive growing leaves discarded due to trimming every year are more than thousands of tons of, and are not obtained very
Good utilisation.
Secoiridoid and its glycosides, flavones and its glycosides, biflavone and its glycosides, low molecule tannin etc. are mainly contained in olive leaf
Ingredient, secoiridoid are main active.
Oleuropein (oleuropein) is the bitter principle in olive leaf, is a kind of bimolecular structure phenols and flavonoids object
Matter, molecular formula C25H32O13, molecular weight 540.52,87~89 DEG C of fusing point is easy to moisture absorption, is soluble in ethyl alcohol, acetone, pyridine etc., solvable
Yu Shui, butanol, ethyl acetate etc. are practically insoluble in ether, petroleum ether, chloroform etc..It is reported that oleuropein has natural resist
Oxidation, enhancing cardiovascular function, treatment chronic prostatitis, strengthen immunity, reconciles endocrine system at antitumor, anti-aging
The important healthcare function such as system, protect liver, anti-inflammatory, while also reducing blood lipid, hypoglycemic pharmacological action are widely used in food, change
The fields such as adornment and bio-pharmaceuticals.
Oleuropein is mainly extracted from olive leaf and is obtained.Oleuropein extraction point disclosed in document report or patent at present
More from technology, olive leaf extractive technique mainly includes solvent extractive technique, supercritical CO2Abstraction technique, ultrasonic wave extraction
Take technology and microwave-assisted extraction technique etc..But organic solvent extraction is with impurity content is high, solvent consumption is big, of high cost, pollution
The shortcomings of big.Supercritical CO2Extraction has many advantages, such as that extraction content is high, pollution-free, but puts into big, of high cost.Ultrasound involves micro-
Although wave extraction has many advantages, such as energy saving, efficient, large-scale application is difficult to realize in production.
For example, Chinese patent CN1583052A discloses a kind of Olive leaf P.E extraction preparation method, by olive leaf through disappearing
After poison rinsing, drying, alcohol extracting is concentrated in vacuo, and addition tea polyphenols are anti-oxidant in concentrate, is extracted through the dry olive leaf that obtains
Object, but active constituent content is low in the Olive leaf P.E of its preparation.Chinese patent CN101003557A discloses one kind and is rich in
The preparation method of the extractive of olive growing leaves of high-purity oleuropein, extracted by olive leaf polar solvent, alkaline centrifugal sedimentation,
Centrifugal clear liquid, film integrated separation and etc. obtain the concentrate by NF membrane, then medium Adsorption and desorption is attached is eluted
Liquid, it is dry after vacuum concentration, it obtains olive growing leaves rich in high-purity oleuropein of the oleuropein content more than 50% and extracts
Object.But this method practical operation step is excessive, is unfavorable for industrialized production, and membrane separation plant first-phase investment is big, improves
Product cost.Japanese Unexamined Patent Publication 2002128678 is extracted with aqueous solvent, after synthetic resin processing, is dried to obtain containing oleuropein
More than 25% extract.It is carried more than secondary using alcohol solution heat in United States Patent (USP) US5714150, after extracting solution is concentrated in vacuo
The Olive leaf P.E of the 30-40% containing oleuropein is prepared in spray drying or vacuum drying.Olive in these extracts
The content of olive hardship glycosides is too low, cannot meet market demands.
Using more in the article " introducing a fine variety oleuropein extraction separation and Structural Identification in A Si olive growing leaves " of Wang Chengzhang etc.
Secondary organic solvent extraction, silica gel column chromatography and HPLC prepare chromatography, have isolated the Cleupin that content is 95%, but the method
It is limited to a small amount of sample preparation to the greatest extent.Olive leaf is worn into the fine powder of 0.05 to 1 millimeter of diameter, pH8.0 in European patent EP 1795201
Buck extracts；Aqueous extracts are extracted through organic solvent, and extract liquor vacuum dehydrating at lower temperature obtains the thick of the 40-45% containing oleuropein and carries
Object；This crude extract through fast silica gel chromatogram column purification, is collected the stripping liquid of the part containing oleuropein, is contained after recycling design again
The amorphous solid of 95-98% oleuropeins.But although higher oleuropein content is obtained in this method, cost is inclined
Height, more suitable for scientific research, it is difficult to accomplish scale production.
Therefore, present inventor is in order to propose that a kind of active constituent content is high, at low cost and method is easy, solvent
It is Ke Xunhuanliyong, environmental pollution is reduced, is easy to the method for industrialization, has made intensive studies, and then obtains the present invention.
Invention content
To achieve these goals, the present invention provides a kind of preparation method of Olive leaf P.E, include the following steps：
（1）After raw material olive leaf saline sook 1-2 hours, drying is ground into fine powder；
（2）3-8 times of water is added in into raw material, adds in cellulase：The complex enzyme of alkali protease, compound enzyme dosage are original
The 0.5-2 weight % of material, are maintained at 30-50 DEG C and digest 0.5-2h, and filtering obtains enzymolysis liquid；
（3）Enzymolysis liquid is directly by neutral alumina column, and resin column blade diameter length ratio is 1: 4~1: 15, respectively with 1-3 times of cylinder
Ponding, 1-2 times of 95% ethanol solution of column volume elution removal of impurities, with -50% ethyl alcohol of 0.2-0.5% glacial acetic acid of 6-10 times of column volume
（1:1）Solution elutes, and collects eluent；
（4）After flinging to the glacial acetic acid and ethyl alcohol in eluent, micro-wave vacuum obtains oleuropein content and is more than 90%
Olive leaf P.E product.
Step（1）In, brine can be saturated salt solution, and the granularity of crushing is sieved preferably through 40-60 mesh.
Step（2）In, water is preferably 4-6 times, more preferably 5 times；Complex enzyme is preferably cellulase：Alkali protease
1:3 complex enzyme.
Step（3）In, micro-wave vacuum temperature：40-60 DEG C, 2-4 DEG C, more than vacuum degree -0.07Mpa of return difference temperature,
Microwave power 10-80KW is 100-150 minutes dry.
The present invention by the infiltration of olive leaf, enzymolysis and extraction, separation, drying technique, obtained rich in oleuropein
Product：By the screening and optimization of system, it is first determined raw material pre-treatment step is infiltrated before preparation with brine,
And by appropriate crushing, active ingredient is more thoroughly dissolved out, further, using neutral alumina column, enzymolysis liquid need not
Concentration can direct upper prop, significantly reduce energy consumption, most afterwards through the elution of glacial acetic acid-ethanol eluate and it is dry after, obtain
Oleuropein content is more than 90% Olive leaf P.E product.Entire method is only made of 4 steps, and institute is using reagent
Common non-toxic components, high income and at low cost, acquired product can be directly used for preparing drug and health products, food, right and wrong
It is adapted to industrial method.
The present invention also provides Olive leaf P.Es made from above-mentioned preparation method.
Specific embodiment
It will be described in detail the R＆D process of the present invention below.
First, the research of enzymolysis process
Experiment material：Olive leaf powder, pectase, cellulase, alkali protease, amylase, lipase, lysozyme
Experimental method：Olive leaf powder is taken, the water of 1.5-2 times of raw material weight is added in, and add in 1% enzyme, is ultrasonically treated.
Measure the content of oleuropein in the supernatant after enzymolysis.
Experimental result see the table below.
| Enzyme class | Oleuropein（mg/100ml） |
| Cellulase | 24.32 |
| Alkali protease | 19.06 |
| Pectase | 19.59 |
| Amylase | 20.28 |
| Lipase | 14.17 |
| Lysozyme | 15.89 |
| Cellulase：Alkali protease（1:1） | 28.45 |
| Cellulase：Alkali protease（1:2） | 30.25 |
| Cellulase：Alkali protease（1:3） | 39.54 |
| Cellulase：Amylase（1:1） | 25.11 |
| Cellulase：Lipase（1:1:1） | 20.19 |
It can be seen that the complex enzyme of cellulase and alkali protease is highly beneficial to the dissolution of oleuropein, it is especially excellent
Select cellulase：Alkali protease（1:3）.
2nd, the research of technique before digesting
Experiment material：Olive leaf, water, saline solution, lime water, sodium bicarbonate water, cellulase, alkali protease
Experimental method：Olive leaf is taken, is separately added into 5-10 times of water, saline solution, lime water, sodium bicarbonate water infiltration 1 hour
Afterwards, it dries, is digested after crushing（Cellulase：Alkali protease 1:3）, measure oleuropein in enzymolysis supernatant and contain
Amount.
Experimental result see the table below.
| Technique | Oleuropein（mg/100ml） |
| It is digested after water infiltration | 40.61 |
| It is digested after saturated common salt water infiltration | 62.17 |
| It is digested after lime water infiltration | 51.59 |
| It is digested after sodium bicarbonate water infiltration | 42.38 |
Inventor extremely surprisingly has found that the olive leaf after saturated common salt water infiltration through enzymolysis, can be greatly improved again
The dissolution of oleuropein.The reason is not yet clear and definite, seems different, but send out from the method for using lime water extraction in the prior art
Person of good sense's conjecture be in more plump olive leaf because of the infiltration of saline solution, with cellulase and basic protein enzyme interacting after,
Having played abnormal compound action causes.
3rd, the screening of column chromatography
Experimental method：The supernatant after enzymolysis is taken, after concentration, is put in the neutral alumina column resin 100g pre-processed,
After shaking table Dynamic Adsorption 8 hours, column is filled, respectively with 1-3 times of cylinder ponding, 30% ethyl alcohol, 50% ethyl alcohol, 80% ethyl alcohol, 0.5% ice
Acetic acid, n-butanol, ethyl acetate solution or the elution of its mixed liquor, collect eluent and carry out oleuropein assay, partly wash
De- liquid, which is evaporated, calculates dry cream amount.
Experimental result see the table below.
| Eluent type | Dry cream amount（g） | Oleuropein content |
| Water | 3.82 | 55.21 |
| 30% ethyl alcohol | 4.24 | 79.09 |
| 50% ethyl alcohol | 4.65 | 82.35 |
| 80% ethyl alcohol | 4.11 | 77.26 |
| 0.5% glacial acetic acid | 4.97 | 80.42 |
| N-butanol | 3.89 | 67.09 |
| - 0.5% glacial acetic acid of 30% ethyl alcohol（1:1） | 5.49 | 85.21 |
| - 0.5% glacial acetic acid of 50% ethyl alcohol（1:1） | 6.25 | 92.89 |
| - 0.5% glacial acetic acid of 80% ethyl alcohol（1:1） | 5.02 | 78.54 |
| - 0.5% glacial acetic acid of n-butanol（1:1） | 4.85 | 75.67 |
The result shows that -0.5% glacial acetic acid of 50% ethyl alcohol（1:1）Effect it is best.
The present inventor has also carried out other various screenings, the screening etc. of the granularity including crushing, resin column type number,
Technical scheme of the present invention is finally obtained, a kind of method simplicity has been obtained, has been easy to implement and obtains comprising more than more than 90%
The Olive leaf P.E of oleuropein can be directly used for preparing food, health products or drug.
Embodiment 1：A kind of Olive leaf P.E, is made by the following method, includes the following steps：
（1）After raw material olive leaf saline sook 1 hour, drying is ground into 40-60 mesh fine powders；
（2）5 times of water are added in into raw material, add in cellulase：Alkali protease 1:3 complex enzyme, compound enzyme dosage are
1 weight % of raw material, is maintained at 30-50 DEG C and digests 0.5h, and filtering obtains enzymolysis liquid；
（3）Enzymolysis liquid is directly by neutral alumina column, and resin column blade diameter length ratio is 1: 4~1: 15, respectively with 1-3 times of cylinder
Ponding, 1-2 times of 95% ethanol solution of column volume elution removal of impurities, with -50% ethyl alcohol of 0.2-0.5% glacial acetic acid of 6 times of column volumes（1:1）
Solution elutes, and collects eluent；
（4）After flinging to the glacial acetic acid and ethyl alcohol in eluent, micro-wave vacuum, temperature：40-60 DEG C, return difference temperature 2-4
DEG C, more than vacuum degree -0.07Mpa, microwave power 10-80KW are 100-150 minutes dry, obtain product.After measured, in the product
Oleuropein content is 96.03%.
Embodiment 2：A kind of Olive leaf P.E, is made by the following method, includes the following steps：
（1）After raw material olive leaf saline sook 2 hours, drying is ground into fine powder；
（2）3 times of water are added in into raw material, add in cellulase：The complex enzyme of alkali protease, compound enzyme dosage are raw material
2 weight %, be maintained at 30-50 DEG C and digest 0.5h, filter, obtain enzymolysis liquid；
（3）Enzymolysis liquid is directly by neutral alumina column, and resin column blade diameter length ratio is 1: 4~1: 15, respectively with 1-3 times of cylinder
Ponding, 1-2 times of 95% ethanol solution of column volume elution removal of impurities, with -50% ethyl alcohol of 0.2-0.5% glacial acetic acid of 10 times of column volumes（1:
1）Solution elutes, and collects eluent；
（4）After flinging to the glacial acetic acid and ethyl alcohol in eluent, micro-wave vacuum, temperature：40-60 DEG C, return difference temperature 2-4
DEG C, more than vacuum degree -0.07Mpa, microwave power 10-80KW are 100-150 minutes dry, obtain product.After measured, in the product
Oleuropein content is 93.92%.
Embodiment 3：A kind of Olive leaf P.E, is made by the following method, includes the following steps：
（1）After raw material olive leaf saline sook 1 hour, drying is ground into 50-60 mesh fine powders；
（2）7 times of water are added in into raw material, add in cellulase：Alkali protease 1:3 complex enzyme, compound enzyme dosage are
1 weight % of raw material, is maintained at 30-50 DEG C and digests 0.5h, and filtering obtains enzymolysis liquid；
（3）Enzymolysis liquid is directly by neutral alumina column, and resin column blade diameter length ratio is 1: 4~1: 15, respectively with 1-3 times of cylinder
Ponding, 1-2 times of 95% ethanol solution of column volume elution removal of impurities, with -50% ethyl alcohol of 0.2-0.5% glacial acetic acid of 8 times of column volumes（1:1）
Solution elutes, and collects eluent；
（4）After flinging to the glacial acetic acid and ethyl alcohol in eluent, micro-wave vacuum, temperature：40-60 DEG C, return difference temperature 2-4
DEG C, more than vacuum degree -0.07Mpa, microwave power 10-80KW are 100-150 minutes dry, obtain product.After measured, in the product
Oleuropein content is 95.40%.
Claims (8)
1. a kind of preparation method of Olive leaf P.E, includes the following steps：
(1) by after raw material olive leaf saline sook 1-2 hours, drying is ground into fine powder；
(2) 3-8 times of water is added in into raw material, adds in cellulase：The complex enzyme of alkali protease, compound enzyme dosage are raw material
0.5-2 weight % are maintained at 30-50 DEG C and digest 0.5-2h, and filtering obtains enzymolysis liquid；
(3) enzymolysis liquid is directly by neutral alumina column, and resin column blade diameter length ratio is 1: 4~1: 15, respectively with 1-3 times of column volume
Water, 1-2 times of 95% ethanol solution of column volume elution removal of impurities, -50% ethyl alcohol of 0.2-0.5% glacial acetic acid with 6-10 times of column volume are molten
Liquid elutes, and collects eluent, and the wherein ratio of -50% ethyl alcohol of 0.2-0.5% glacial acetic acid is 1:1；
(4) after flinging to the glacial acetic acid and ethyl alcohol in eluent, micro-wave vacuum obtains the olive that oleuropein content is more than 90%
Leaf extract product.
2. the method as described in claim 1, in step (1), brine is saturated salt solution.
3. the method as described in claim 1, in step (1), the granularity of crushing is sieved preferably through 40-60 mesh.
4. the method as described in claim 1, in step (2), water is 4-6 times.
5. method as claimed in claim 4, in step (2), water is 5 times.
6. the method as described in claim 1, in step (2), complex enzyme is cellulase: alkali protease 1:3 it is compound
Enzyme.
7. the method as described in claim 1, in step (3), micro-wave vacuum temperature：40-60 DEG C, return difference temperature 2-4
DEG C, more than vacuum degree -0.07Mpa, microwave power 10-80KW are 100-150 minutes dry.
8. claim 1-7 any one of them method extract obtained for being rich in oleuropein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510620159.8A CN105055527B (en) | 2015-09-25 | 2015-09-25 | A kind of preparation method of Olive leaf P.E |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510620159.8A CN105055527B (en) | 2015-09-25 | 2015-09-25 | A kind of preparation method of Olive leaf P.E |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105055527A CN105055527A (en) | 2015-11-18 |
| CN105055527B true CN105055527B (en) | 2018-06-29 |
Family
ID=54485209
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510620159.8A Active CN105055527B (en) | 2015-09-25 | 2015-09-25 | A kind of preparation method of Olive leaf P.E |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105055527B (en) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106822214A (en) * | 2015-12-03 | 2017-06-13 | 李飞燕 | A kind of Olive leaf P.E preparation method |
| 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 |
| CN106819324B (en) * | 2016-12-26 | 2020-09-01 | 福建农林大学 | A method for inhibiting formation of olive precipitate |
| CN106798068B (en) * | 2016-12-26 | 2021-03-05 | 福建农林大学 | A method for inhibiting formation of olive precipitate |
| CN107149080A (en) * | 2017-05-11 | 2017-09-12 | 郑州科技学院 | A kind of microcapsule food preservative and preparation method thereof |
| CN107252093B (en) * | 2017-05-26 | 2020-10-27 | 华南理工大学 | Guava leaf rich in soluble polyphenol and flavonoid aglycone, preparation method and application |
| CN107652333A (en) * | 2017-11-02 | 2018-02-02 | 成都金川田农机制造有限公司 | A kind of oleuropein extracting method |
| CN107898685A (en) * | 2017-11-29 | 2018-04-13 | 马斌祥 | A kind of preparation method and application of Olive leaf P.E |
| CN109043261A (en) * | 2018-08-20 | 2018-12-21 | 德昌泓源农林科技发展有限公司 | A kind of drink and production process |
| CN109381385A (en) * | 2018-09-07 | 2019-02-26 | 南京医科大学附属口腔医院 | A kind of auxiliary ultrasonic, which cleans one's teeth, dispels tartar oral care composition, preparation method and application |
| CN109619118B (en) * | 2018-11-28 | 2021-07-09 | 韩纯伟 | Rooting agent for promoting rooting of magnolia denudata strips |
| CN111518618A (en) * | 2020-04-30 | 2020-08-11 | 江苏恒正合生命科学有限公司 | Preparation process of oleuropein-containing grease antioxidant |
| CN112409437A (en) * | 2020-12-12 | 2021-02-26 | 南京康齐生物科技有限公司 | Method for extracting and separating high-purity maslinic acid from olive leaves |
| CN113416354B (en) * | 2021-04-30 | 2023-06-23 | 百事基材料（青岛）股份有限公司 | Large biological butyronitrile gloves |
| CN114304465B (en) * | 2022-01-27 | 2023-01-10 | 陇南市祥宇油橄榄开发有限责任公司 | Olive leaf extract effervescent tablet and preparation method thereof |
Citations (3)
| 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 |
| 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 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101055075B1 (en) * | 2008-09-19 | 2011-08-05 | (주)아모레퍼시픽 | Hair Dye Composition Using Iridoid Glycosides |
| US20130272974A1 (en) * | 2012-04-12 | 2013-10-17 | Ahmad Alkayali | Hydroxytyrosol Product, Method of Making, and Uses Thereof |
-
2015
- 2015-09-25 CN CN201510620159.8A patent/CN105055527B/en active Active
Patent Citations (3)
| 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 |
| 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 |
|---|
| Enzymatic Hydrolysis of Oleuropein from Olea europea (Olive) Leaf Extract and Antioxidant Activities;Jiao-Jiao Yuan等;《molecules》;20150211(第20期);第2903-2921页 * |
| 油橄榄叶提取物中橄榄苦苷稳定性及;谢普军等;《食品工业科技》;20131231(第4期);第133-136页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105055527A (en) | 2015-11-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105055527B (en) | A kind of preparation method of Olive leaf P.E | |
| CN102451235B (en) | Preparation method of olive leaf extract | |
| CN103451022B (en) | Method for integrally extracting volatile oil, polysaccharide and flavone from elsholtzia haichowensis sun | |
| CN105754014B (en) | Extraction method of pectin and polyphenol in orange peel | |
| CN104906153A (en) | Technological method for efficiently extracting ginkgo flavone | |
| CN106008442A (en) | Method for extracting anthocyanin from nitraria tangutorum peel residues | |
| CN100590109C (en) | Technique for extracting cynamorlum herba cistanches medicinal materials effective component | |
| CN104173438A (en) | Preparation method of general flavone of purple perilla | |
| CN103142662A (en) | Method for extracting and purifying polyphenol from choerospondias axillaris peel | |
| CN104523767A (en) | Method for extracting flavones substances in cacumen biotae | |
| CN106822214A (en) | A kind of Olive leaf P.E preparation method | |
| CN102233013A (en) | Preparation method for total saponins of pulsatilla chinensis | |
| CN102731592A (en) | Method for extracting cleupin and amentoflavone from olive leaf | |
| CN104940280A (en) | Method for extracting total flavones from radix puerariae employing enzyme preparation | |
| CN106008647A (en) | Novel extraction method of momordica saponins | |
| CN103463160A (en) | Preparation method of high-content total flavones of chrysanthemum | |
| CN105175426B (en) | A kind of method of the extraction purification Bergenin from treebine stem | |
| CN107325139A (en) | A kind of rapidly and efficiently method of extraction purification anthocyanin from indigo fruit | |
| CN1962650A (en) | Process for preparing high-purity dehydrated andrographolide | |
| CN102311466A (en) | Method for extracting phenylethanoid glycoside active components from semenplantaginis | |
| CN102670935B (en) | Method for extracting total saponins from allium chinense | |
| CN104892696A (en) | Method for extracting salidroside from Tibetan natural rhodiola rosea | |
| CN102351928B (en) | Method for extracting iridoid active ingredients from plantain seeds | |
| CN102653537B (en) | Method for extracting and preparing high-purity silibinin from polygonum capitatum herb | |
| CN104910172A (en) | Preparation method and application of five stilbene tripolymers |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20160603 Address after: 011517 the Inner Mongolia Autonomous Region Helingeer County of Hohhot city Shengle Economic Zone Applicant after: INNER MONGOLIA CHANGHUI BIOLOGICAL TECHNOLOGY Co.,Ltd. Address before: 010010, building 3, unit 7, Wanda District, Wanda Plaza, Xinhua East Street, the Inner Mongolia Autonomous Region, Hohhot, 602, China Applicant before: Bai Xinliang |
|
| CB02 | Change of applicant information |
Address after: 011517 Hohhot city Helingeer County Inner Mongolia Shengle economic Park Applicant after: INNER MONGOLIA CHANGHUI BIOTECHNOLOGY CO.,LTD. Address before: 011517 Hohhot city Helingeer County Inner Mongolia Shengle economic Park Applicant before: INNER MONGOLIA CHANGHUI BIOLOGICAL TECHNOLOGY Co.,Ltd. |
|
| COR | Change of bibliographic data | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 028400 no.0427, Dongjiao Industrial Park, Kailu Town, Kailu County, Tongliao City, Inner Mongolia Autonomous Region Patentee after: INNER MONGOLIA CHANGHUI BIOTECHNOLOGY CO.,LTD. Address before: 011517 Hohhot city Helingeer County Inner Mongolia Shengle economic Park Patentee before: INNER MONGOLIA CHANGHUI BIOTECHNOLOGY CO.,LTD. |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A preparation method of olive leaf extract Effective date of registration: 20210222 Granted publication date: 20180629 Pledgee: Agricultural Bank of China Limited Helingeer County sub branch Pledgor: INNER MONGOLIA CHANGHUI BIOTECHNOLOGY Co.,Ltd. Registration number: Y2021150000016 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20220321 Granted publication date: 20180629 Pledgee: Agricultural Bank of China Limited Helingeer County sub branch Pledgor: INNER MONGOLIA CHANGHUI BIOTECHNOLOGY CO.,LTD. Registration number: Y2021150000016 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A preparation method of olive leaf extract Effective date of registration: 20220330 Granted publication date: 20180629 Pledgee: Agricultural Bank of China Limited Helingeer County sub branch Pledgor: INNER MONGOLIA CHANGHUI BIOTECHNOLOGY CO.,LTD. Registration number: Y2022150000027 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20230301 Granted publication date: 20180629 Pledgee: Agricultural Bank of China Limited Helingeer County sub branch Pledgor: INNER MONGOLIA CHANGHUI BIOTECHNOLOGY CO.,LTD. Registration number: Y2022150000027 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A preparation method of olive leaf extract Effective date of registration: 20230307 Granted publication date: 20180629 Pledgee: China Minsheng Banking Corp Hohhot branch Pledgor: INNER MONGOLIA CHANGHUI BIOTECHNOLOGY CO.,LTD. Registration number: Y2023150000031 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20231031 Granted publication date: 20180629 Pledgee: China Minsheng Banking Corp Hohhot branch Pledgor: INNER MONGOLIA CHANGHUI BIOTECHNOLOGY CO.,LTD. Registration number: Y2023150000031 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A preparation method for olive leaf extract Effective date of registration: 20231101 Granted publication date: 20180629 Pledgee: China Minsheng Banking Corp Hohhot branch Pledgor: INNER MONGOLIA CHANGHUI BIOTECHNOLOGY CO.,LTD. Registration number: Y2023150000154 |