CN106617060A - Fructus phyllanthi polyphenols enriching and purifying method - Google Patents
Fructus phyllanthi polyphenols enriching and purifying method Download PDFInfo
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- CN106617060A CN106617060A CN201611225093.3A CN201611225093A CN106617060A CN 106617060 A CN106617060 A CN 106617060A CN 201611225093 A CN201611225093 A CN 201611225093A CN 106617060 A CN106617060 A CN 106617060A
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- 150000008442 polyphenolic compounds Chemical class 0.000 title claims abstract description 82
- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 82
- 238000000034 method Methods 0.000 title claims abstract description 46
- 239000009609 fructus phyllanthi Substances 0.000 title abstract description 12
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 34
- 238000000605 extraction Methods 0.000 claims abstract description 33
- 239000002904 solvent Substances 0.000 claims abstract description 12
- 238000001179 sorption measurement Methods 0.000 claims abstract description 10
- 238000000746 purification Methods 0.000 claims abstract description 8
- 238000011049 filling Methods 0.000 claims abstract description 7
- 240000007817 Olea europaea Species 0.000 claims description 87
- 238000011068 loading method Methods 0.000 claims description 24
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 20
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 239000012071 phase Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 239000000284 extract Substances 0.000 claims description 17
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 14
- 238000001994 activation Methods 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 238000001291 vacuum drying Methods 0.000 claims description 12
- 244000119298 Emblica officinalis Species 0.000 claims description 9
- 235000015489 Emblica officinalis Nutrition 0.000 claims description 9
- 239000000047 product Substances 0.000 claims description 9
- 239000003960 organic solvent Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 239000004519 grease Substances 0.000 claims description 6
- 239000003208 petroleum Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 238000002203 pretreatment Methods 0.000 claims description 6
- 238000007873 sieving Methods 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- 239000003643 water by type Substances 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 125000001033 ether group Chemical group 0.000 claims description 2
- 239000007790 solid phase Substances 0.000 claims 1
- 239000011347 resin Substances 0.000 abstract description 10
- 229920005989 resin Polymers 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 9
- 239000000463 material Substances 0.000 abstract description 9
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 abstract description 6
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 abstract description 3
- 229940024606 amino acid Drugs 0.000 abstract description 3
- 150000001413 amino acids Chemical class 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 3
- 229960001948 caffeine Drugs 0.000 abstract description 3
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 abstract description 3
- 150000001720 carbohydrates Chemical class 0.000 abstract description 3
- 239000003205 fragrance Substances 0.000 abstract description 3
- 239000000049 pigment Substances 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 239000011782 vitamin Substances 0.000 abstract description 3
- 229940088594 vitamin Drugs 0.000 abstract description 3
- 229930003231 vitamin Natural products 0.000 abstract description 3
- 235000013343 vitamin Nutrition 0.000 abstract description 3
- 150000003722 vitamin derivatives Chemical class 0.000 abstract description 3
- 239000012141 concentrate Substances 0.000 abstract description 2
- 230000003213 activating effect Effects 0.000 abstract 1
- 239000004480 active ingredient Substances 0.000 abstract 1
- 239000004615 ingredient Substances 0.000 abstract 1
- 238000007781 pre-processing Methods 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 239000003814 drug Substances 0.000 description 10
- 244000269722 Thea sinensis Species 0.000 description 9
- 235000009569 green tea Nutrition 0.000 description 8
- 239000000796 flavoring agent Substances 0.000 description 5
- 238000007865 diluting Methods 0.000 description 4
- 235000019634 flavors Nutrition 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 244000061176 Nicotiana tabacum Species 0.000 description 3
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000000638 solvent extraction Methods 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000000622 liquid--liquid extraction Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 208000007848 Alcoholism Diseases 0.000 description 1
- 241000050051 Chelone glabra Species 0.000 description 1
- 206010013911 Dysgeusia Diseases 0.000 description 1
- 241000221017 Euphorbiaceae Species 0.000 description 1
- 244000078118 Ilex latifolia Species 0.000 description 1
- 235000008706 Ilex latifolia Nutrition 0.000 description 1
- 241000207836 Olea <angiosperm> Species 0.000 description 1
- 235000011517 Terminalia chebula Nutrition 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 201000007930 alcohol dependence Diseases 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
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- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000686 essence Substances 0.000 description 1
- 239000004744 fabric Substances 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
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
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- 238000011069 regeneration method Methods 0.000 description 1
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- 238000002470 solid-phase micro-extraction Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000000092 stir-bar solid-phase extraction Methods 0.000 description 1
- 235000013616 tea Nutrition 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention provides a fructus phyllanthi polyphenols enriching and purifying method and belongs to the technical field of natural active ingredient purification and separation. According to the method, dry fructus phyllanthi is utilized as a raw material, and a fructus phyllanthi polyphenols product can be obtained through the steps of preprocessing the fructus phyllanthi, preparing fructus phyllanthi crude polyphenols, filling a solid-phase extraction column, performing activating treatment on a LiChrolut EN solid-phase extraction column, feeding a sample and performing stage treatment. The method utilizes LiChrolut EN resin as a preferential adsorption material of solid-phase extraction, the LiChrolut EN resin has a better adsorption effect on fructus phyllanthi polyphenols and can only adsorb a little saccharides, pigment, amino acid, caffeine and vitamin which are richer in content, the measured yield of the fructus phyllanthi polyphenols can reach 247.9mg GAE/100g to the maximum, and taste capacity can reach 1.52 to the maximum and is improved by 2.2 to 2.8 times compared with the taste capacity of crude polyphenols. The method is more suitable for extracting a polyphenols ingredient which is little in fructus phyllanthi and large in concentrate and content span, and the finished fructus phyllanthi polyphenols has the advantages of zero solvent residue and special peculiar smell, fresh and strong fragrance and higher extraction rate.
Description
Technical field
The present invention relates to a kind of enrichment and purification method of Yunnan olive polyphenol, belongs to active skull cap components purifies and separates technology neck
Domain.
Background technology
Yunnan olive（Fructusphyllanthi）, also known as emblic, it is Euphorbiaceae phyllanthus plant Yunnan olive
（Phyllanthusemblica L）Fruit, it really eats sour-sweet crisp and micro-puckery raw, and aftertaste is sweet；The wild Yunnan olive point of China
Cloth is on Yunnan, Guangxi, Fujian, Hainan and other places；Used as a kind of conventional Tibetan medicine, with myrobalan, terminaliae billericae,fructus three is often claimed in Tibetan medicine
It is very high for " three big fruits " usage frequency,《Tibetan medicine standard》In 290 kinds of contained Tibetan medicine patent medicine, there are 72 kinds containing Yunnan olive,
The 25% of sum is accounted for, in the contained 200 kinds of patent medicine of the Sanitation Ministry medicine standard nineteen ninety-five version Tibetan medicine standard, there are 59 kinds of olives containing Yunnan, accounted for
29%, Yunnan olive is written into《Chinese Pharmacopoeia》Version one in 1977；Substantial amounts of research confirms that Yunnan olive has throat-clearing throat-moistening, appetizing
The effects such as helping digestion, relieve the effect of alcohol, adjusting blood fat, beauty, anti-aging.
From existing document analysis, it can be found that in the effective component of relatively clearer and more definite olive and Yunnan olive relieving alcoholism and protecting liver extremely
Flavonoid substances containing part, alcohols material, polyphenols, but the discriminating of its specific composition and the system of proportioning less grind
Study carefully and also do not carry out.The domestic exploitation to Yunnan olive are extremely paid attention to, and numerous studies have mainly been done to Yunnan Fructus Canrii Albi extract by China
Work, and has been developed over numerous functional products, in health care of food, essence and flavoring agent and medicine clinically obtaining extensively should
With（《A kind of oral care formulations and preparation method thereof》, application number：201110291992.4；《With Yunnan olive, plum, Ilex Latifolia Thunb
The tobacco aromaticss prepared with tobacco and its application》, application number：201410520369.5；《Fructus phyllanthi compound tea and its preparation side
Method》, application number：201310593498.2）.Contain more polyphenol in the olive of Yunnan, at present separation is extracted to Yunnan olive polyphenol pure
Change less and polyphenol the yield of research not high.And, in newest research tendency, for polyphenol is in terms of flavour ability is improved
The input of effect is increasing.Thus, carry out targetedly Yunnan olive polyphenol and extract the research of new technology and improving flavour
Application effect research in terms of ability effect, can improve the using value of Yunnan olive.
At present, common Yunnan olive polyphenol collecting material method includes that the precipitation method, solvent extraction, ultrafiltration, macropore are inhaled
Attached resin method.There is the shortcomings of solvent consumption is big, cumbersome, material is difficult regeneration, Comparatively speaking, ultrafiltration in the above two methods
There is unique advantage with macroporous absorbent resin.In the comparative studies of these methods, it can be seen that it respectively has pluses and minuses.Liquid liquid
Extraction is most popular abstraction technique, and it has the advantages that, and treating capacity is big, solvent selects various, cycles of concentration higher, many
For qualitative analysis；Due to liquid-liquid extraction workload greatly, many organic solvent usage amounts and the shortcomings of poor dosing accuracy,
It is badly in need of more convenient, safe and efficient extraction collection technique at present.Head-space sampling, SPME and Stir Bar Sorptive Extraction tool
Have simple to operate, it is quick, solvent-free, the characteristics of be easily operated automatically, be suitable for the quantitative analysis of a large amount of samples, but due to inhaling
The selective preference difference of enclosure material, easily causes the loss of some type activity materials, and these methods are difficult to realize to Yunnan olive
Comprehensive extraction of olive polyphenol substance, enrichment.SPE is current the most widely used Sample Pretreatment Technique, is widely used in
The analysis of the field such as food, medicine, tobacco, environment sample.SPE has flexible operation, and sorbing material wide variety is molten
Agent usage amount is few, and micro Yunnan olive class and separation of the important polyphenol of trace, enrichment are particularly suitable for the features such as cycles of concentration is high.By
In Yunnan olive constituent species, the complexity of property, the levels span of polyphenol is big, there is presently no one kind and meets all demands
Pre-treating method, and disposably complete to concentrate the enrichment of all polyphenols.
The present invention relates to a kind of be based on special resin sorbing material（LiChrolut EN resins）Enrichment Yunnan olive polyphenol
Method, plays the purpose to Yunnan olive comprehensive utilization of resources, and technical process can as far as possible reduce the loss of target component, finally obtain
Natural Yunnan olive polyphenol extract.The selective absorption of LiChrolut EN resins, for polyphenol has preferable suction-operated,
And the adsorption effect such as carbohydrate, pigment, amino acid, caffeine, vitamin more rich to content in sample is poor, it is suitable for extraction
Yunnan olive polyphenol composition is taken, the method is characterized by finished product Yunnan olive polyphenol no solvent residue, without specific peculiar smell, fragrance is fresh,
Strong, yield is higher.
The content of the invention
Present invention aim at providing a kind of based on special resin sorbing material（LiChrolut EN resins）Enrichment Yunnan olive
The method of olive polyphenol, plays the purpose to Yunnan olive comprehensive utilization of resources, and technical process can as far as possible reduce target aroma component
Loss, finally obtains natural Yunnan olive polyphenol extract.The Yunnan olive polyphenol collection method, more suitable in extraction Yunnan olive
Composition is few and the big Polyphenols component of levels span, it is characterized in that finished product Yunnan olive polyphenol no solvent residue, without special
Peculiar smell, fragrance is fresh, strong, and recovery rate is higher.So as to the Yunnan olive polyphenol enrichment for providing a kind of environmental protection, having a extensive future
Purification technique.
According to the technical scheme that the present invention is provided：A kind of enrichment and purification method of Yunnan olive polyphenol, step is as follows：
（1）Yunnan olive pre-treatment：Weigh it is a certain amount of be dried Yunnan olive, be placed in 45 DEG C of vacuum drying chambers and be dried to close over dry,
Then vacuum drying chamber temperature is adjusted into 105 DEG C to dry to constant weight, and after sieving by 40 ~ 80 mesh granularity selection requirements, it is standby；
（2）The preparation of the thick polyphenol of Yunnan olive：Accurately weigh（1）The phyllanthus emblica powder end 5.00g for obtaining, is positioned over 500mL round bottoms burning
In bottle, the ethanol solution 300mL of mass fraction 60% is added by the solid-liquid ratio of the g/mL of 1 ︰, 20 ~ 1 ︰ 60, flow back in 40 DEG C of water-baths and carry
Take and filtered after 6h, the thick polyphenol of Yunnan olive before the as upper solid-phase extraction column of gained filtrate；
（3）The filling of solid-phase extraction column：Voluntarily load the solid-phase extraction column that there is good adsorption function to Yunnan olive polyphenol；
（4）The activation process of LiChrolut EN solid-phase extraction columns：First it is respectively adopted 20 mL dichloromethane before loading successively, 20
ML methyl alcohol, 30mL ultra-pure waters carry out activation process to extraction column；
（5）Loading：Take（2）The thick polyphenol of Yunnan olive for obtaining, after extension rate dilution of the deionized water according to 2 ~ 6 times of volumes
Loading LiChrolut EN solid-phase extraction columns, the loading flow velocity of 1.0 ~ 3.0 mL/min, first uses after completion of the sample in control process
Petroleum ether grease removal, is then extracted with organic solvent, obtains organic solvent phase extract and water phase residue, is extracted 3 times, every time extraction
Time 30min is taken, solvent load is 3 × 200mL；
（6）Classification is processed：Will（5）The organic solvent phase extract being obtained by extraction is concentrated in vacuo to 10mL at 35 DEG C, is subsequently placed in
In culture dish, and the liquid residued on extraction post jamb is removed with a small amount of pure water, freeze-drying after merging is final Yunnan olive
Olive polyphenol finished product.
Step（4）The solid-phase extraction column used selects to be Merck KGaA Millipore Corp. LiChrolut EN/1000
Mg, LiChrolut EN/500 mg or LiChrolut EN/200 mg.
Step（5）The extraction organic solvent is ether, ethyl acetate or n-butanol.
Beneficial effects of the present invention：The present invention using LiChrolut EN resins selective absorption, for polyphenol have compared with
Good suction-operated, and the adsorption effect such as carbohydrate, pigment, amino acid, caffeine, vitamin more rich to content in sample
Poor, the yield for determining gained Yunnan olive polyphenol can reach 247.9 mg GAE/100 g；Flavour ability can reach
1.52, improve 2.2～2.8 times than thick polyphenol flavour ability.
Specific embodiment
Embodiment given below, the invention will be further described.
Embodiment 1
（1）Yunnan olive pre-treatment：Weigh it is a certain amount of be dried Yunnan olive, be placed in 45 DEG C of vacuum drying chambers and be dried to close over dry,
Then vacuum drying chamber temperature is adjusted into 105 DEG C to dry to constant weight, and after sieving by the mesh granularity selection requirement of Yunnan olive 60, it is standby；
（2）The preparation of the thick polyphenol of Yunnan olive：Accurately weigh（1）The phyllanthus emblica powder end 5.00g of 60 mesh for obtaining, is positioned over 500mL
It is the ethanol solution 300mL that the g/mL of 1 ︰ 40 add mass fraction 60% by solid-liquid ratio in round-bottomed flask, flows back in 40 DEG C of water-baths and carry
Take and filtered after 6h, the thick polyphenol of Yunnan olive before the as upper solid-phase extraction column of gained filtrate；It is polyphenols using Dot value methods
Concentration/respective concentration threshold determine and calculate the thick polyphenol green tea value of Yunnan olive for 0.62；
（3）The filling of solid-phase extraction column：Voluntarily load the LiChrolut EN that there is good adsorption function to Yunnan olive polyphenol
（500 mg）Solid-phase extraction column；
（4）The activation process of LiChrolut EN solid-phase extraction columns：First it is respectively adopted successively before loading with 20 mL dichloromethane,
20 mL methyl alcohol, 30mL ultra-pure waters carry out activation process to extraction column；
（5）Loading：Take a certain amount of（2）The thick polyphenol of Yunnan olive for obtaining, loading LiChrolut EN after diluting 4 times with deionized water
Solid-phase extraction column, loading flow velocity is 2.0 mL/min in control process, and petroleum ether grease removal is first used after completion of the sample, then uses positive fourth
Alcohol is extracted, and obtains n-butyl alcohol phase extract and water phase residue, is extracted 3 times, each extraction time 30min, and solvent load is 3 ×
200mL；
（6）Classification is processed：Will（5）The n-butyl alcohol phase extract being obtained by extraction is concentrated in vacuo to 10mL or so at 35 DEG C, then puts
In culture dish, and the liquid residued on extraction post jamb is removed with a small amount of pure water, freeze-drying after merging is final Yunnan
Olive polyphenol finished product.
Yield is determined and calculated with Folin-Ciocalteu methods as 247.9 mg GAE/100 g；It is using Dot value methods
The concentration of polyphenols/respective concentration threshold is determined and calculates green tea value for 1.52.
Embodiment 2
（1）Yunnan olive pre-treatment：Weigh it is a certain amount of be dried Yunnan olive, be placed in 45 DEG C of vacuum drying chambers and be dried to close over dry,
Then vacuum drying chamber temperature is adjusted into 105 DEG C to dry to constant weight, and after sieving by the mesh granularity selection requirement of Yunnan olive 80, it is standby；
（2）The preparation of the thick polyphenol of Yunnan olive：Accurately weigh（1）The phyllanthus emblica powder end 5.00g of 80 mesh for obtaining, is positioned over 500mL
It is the ethanol solution 300mL that the g/mL of 1 ︰ 40 add mass fraction 60% by solid-liquid ratio in round-bottomed flask, flows back in 40 DEG C of water-baths and carry
Take and filtered after 6h, the thick polyphenol of Yunnan olive before the as upper solid-phase extraction column of gained filtrate；It is polyphenols using Dot value methods
Concentration/respective concentration threshold determine and calculate the thick polyphenol green tea value of Yunnan olive for 0.59；
（3）The filling of solid-phase extraction column：Voluntarily load the LiChrolut EN that there is good adsorption function to Yunnan olive polyphenol
（500 mg）Solid-phase extraction column；
（4）The activation process of LiChrolut EN solid-phase extraction columns：First it is respectively adopted before loading with 20 mL dichloromethane, 20 mL
Methyl alcohol, 30mL ultra-pure waters carry out activation process to extraction column；
（5）Loading：Take a certain amount of（2）The thick polyphenol of Yunnan olive for obtaining, loading LiChrolut EN after diluting 6 times with deionized water
Solid-phase extraction column, loading flow velocity is 2.0 mL/min in control process, and petroleum ether grease removal is first used after completion of the sample, then uses acetic acid
Ethyl ester is extracted, and obtains ethyl acetate phase extract and water phase residue, is extracted 3 times, each extraction time 30min, solvent load
For 3 × 200mL；
（6）Classification is processed：Will（5）The ethyl acetate phase extract being obtained by extraction is concentrated in vacuo to 10mL or so at 35 DEG C, then
In being placed in culture dish, and the liquid residued on extraction post jamb is removed with a small amount of pure water, freeze-drying after merging is final
Yunnan olive polyphenol finished product.
Yield is determined and calculated with Folin-Ciocalteu methods as 240.6 mg GAE/100 g；It is using Dot value methods
The concentration of polyphenols/respective concentration threshold is determined and calculates green tea value for 1.48.
Embodiment 3
（1）Yunnan olive pre-treatment：Weigh it is a certain amount of be dried Yunnan olive, be placed in 45 DEG C of vacuum drying chambers and be dried to close over dry,
Then vacuum drying chamber temperature is adjusted into 105 DEG C to dry to constant weight, and after sieving by the mesh granularity selection requirement of Yunnan olive 60, it is standby；
（2）The preparation of the thick polyphenol of Yunnan olive：Accurately weigh（1）The phyllanthus emblica powder end 5.00g of 60 mesh for obtaining, is positioned over 500mL
It is the ethanol solution 300mL that the g/mL of 1 ︰ 60 add mass fraction 60% by solid-liquid ratio in round-bottomed flask, flows back in 40 DEG C of water-baths and carry
Take and filtered after 6h, the thick polyphenol of Yunnan olive before the as upper solid-phase extraction column of gained filtrate；It is polyphenols using Dot value methods
Concentration/respective concentration threshold determine and calculate the thick polyphenol green tea value of Yunnan olive for 0.53；
（3）The filling of solid-phase extraction column：Voluntarily load the LiChrolut EN that there is good adsorption function to Yunnan olive polyphenol
（1000 mg）Solid-phase extraction column；
（4）The activation process of LiChrolut EN solid-phase extraction columns：First successively respectively with 20 mL dichloromethane, 20 mL before loading
Methyl alcohol, 30mL ultra-pure waters carry out activation process to extraction column；
（5）Loading：Take a certain amount of（2）The thick polyphenol of Yunnan olive for obtaining, loading LiChrolut EN after diluting 2 times with deionized water
Solid-phase extraction column, loading flow velocity is 3.0 mL/min in control process, and petroleum ether grease removal is first used after completion of the sample, then uses ether
Extraction, obtains ether phase extract and water phase residue, extracts 3 times, each extraction time 30min, and solvent load is 3 ×
200mL；
（6）Classification is processed：Will（5）The ether phase extract being obtained by extraction is concentrated in vacuo to 10mL or so at 35 DEG C, is subsequently placed in
In culture dish, and the liquid residued on extraction post jamb is removed with a small amount of pure water, freeze-drying after merging is final Yunnan olive
Olive polyphenol finished product.
Yield is determined and calculated with Folin-Ciocalteu methods as 238.2 mg GAE/100 g；It is using Dot value methods
The concentration of polyphenols/respective concentration threshold is determined and calculates green tea value for 1.41.
Embodiment 4
（1）Yunnan olive pre-treatment：Weigh it is a certain amount of be dried Yunnan olive, be placed in 45 DEG C of vacuum drying chambers and be dried to close over dry,
Then vacuum drying chamber temperature is adjusted into 105 DEG C to dry to constant weight, and after sieving by the mesh granularity selection requirement of Yunnan olive 40, it is standby；
（2）The preparation of the thick polyphenol of Yunnan olive：Accurately weigh（1）The phyllanthus emblica powder end 5.00g of 40 mesh for obtaining, is positioned over 500mL
It is the ethanol solution 300mL that the g/mL of 1 ︰ 20 add mass fraction 60% by solid-liquid ratio in round-bottomed flask, flows back in 40 DEG C of water-baths and carry
Take and filtered after 6h, the thick polyphenol of Yunnan olive before the as upper solid-phase extraction column of gained filtrate；It is polyphenols using Dot value methods
Concentration/respective concentration threshold determine and calculate the thick polyphenol green tea value of Yunnan olive for 0.49；
（3）The filling of solid-phase extraction column：Voluntarily load the LiChrolut EN that there is good adsorption function to Yunnan olive polyphenol
（2000 mg）Solid-phase extraction column；
（4）The activation process of LiChrolut EN solid-phase extraction columns：First successively respectively with 20 mL dichloromethane, 20 mL before loading
Methyl alcohol, 30mL ultra-pure waters carry out activation process to extraction column；
（5）Loading：Take a certain amount of（2）The thick polyphenol of Yunnan olive for obtaining, loading LiChrolut EN after diluting 4 times with deionized water
Solid-phase extraction column, loading flow velocity is 1.0 mL/min in control process, and petroleum ether grease removal is first used after completion of the sample, then uses positive fourth
Alcohol is extracted, and obtains n-butyl alcohol phase extract and water phase residue, is extracted 3 times, each extraction time 30min, and solvent load is 3 ×
200mL；
（6）Classification is processed：Will（5）The n-butyl alcohol phase extract being obtained by extraction is concentrated in vacuo to 10mL or so at 35 DEG C, then puts
In culture dish, and the liquid residued on extraction post jamb is removed with a small amount of pure water, freeze-drying after merging is final Yunnan
Olive polyphenol finished product.
Yield is determined and calculated with Folin-Ciocalteu methods as 230.1 mg GAE/100 g；It is using Dot value methods
The concentration of polyphenols/respective concentration threshold is determined and calculates green tea value for 1.36.
Claims (3)
1. a kind of enrichment and purification method of Yunnan olive polyphenol, is characterized in that step is as follows：
（1）Yunnan olive pre-treatment：Weigh it is a certain amount of be dried Yunnan olive, be placed in 45 DEG C of vacuum drying chambers and be dried to close over dry,
Then vacuum drying chamber temperature is adjusted into 105 DEG C to dry to constant weight, and after sieving by 40 ~ 80 mesh granularity selection requirements, it is standby；
（2）The preparation of the thick polyphenol of Yunnan olive：Accurately weigh（1）The phyllanthus emblica powder end 5.00g for obtaining, is positioned over 500mL round bottoms burning
In bottle, the ethanol solution 300mL of mass fraction 60% is added by the solid-liquid ratio of the g/mL of 1 ︰, 20 ~ 1 ︰ 60, flow back in 40 DEG C of water-baths and carry
Take and filtered after 6h, the thick polyphenol of Yunnan olive before the as upper solid-phase extraction column of gained filtrate；
（3）The filling of solid-phase extraction column：Voluntarily load the solid-phase extraction column that there is good adsorption function to Yunnan olive polyphenol；
（4）The activation process of LiChrolut EN solid-phase extraction columns：First it is respectively adopted 20 mL dichloromethane before loading successively, 20
ML methyl alcohol, 30mL ultra-pure waters carry out activation process to extraction column；
（5）Loading：Take（2）The thick polyphenol of Yunnan olive for obtaining, after extension rate dilution of the deionized water according to 2 ~ 6 times of volumes
Loading LiChrolut EN solid-phase extraction columns, the loading flow velocity of 1.0 ~ 3.0 mL/min, first uses after completion of the sample in control process
Petroleum ether grease removal, is then extracted with organic solvent, obtains organic solvent phase extract and water phase residue, is extracted 3 times, every time extraction
Time 30min is taken, solvent load is 3 × 200mL；
（6）Classification is processed：Will（5）The organic solvent phase extract being obtained by extraction is concentrated in vacuo to 10mL at 35 DEG C, is subsequently placed in
In culture dish, and the liquid residued on extraction post jamb is removed with a small amount of pure water, freeze-drying after merging is final Yunnan olive
Olive polyphenol finished product.
2. the enrichment and purification method of Yunnan olive polyphenol as claimed in claim 1, it is characterised in that step（4）The solid phase used
Extraction column select as Merck KGaA Millipore Corp. LiChrolut EN/1000 mg, LiChrolut EN/500 mg or
LiChrolut EN/200 mg。
3. the enrichment and purification method of Yunnan olive polyphenol as claimed in claim 1, it is characterised in that step（5）The extraction is with having
Machine solvent is ether, ethyl acetate or n-butanol.
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