CN103420838B - Method for separating and purifying chlorogenic acid by utilizing temperature to induce aqueous two-phase system - Google Patents

Method for separating and purifying chlorogenic acid by utilizing temperature to induce aqueous two-phase system Download PDF

Info

Publication number
CN103420838B
CN103420838B CN201310339674.XA CN201310339674A CN103420838B CN 103420838 B CN103420838 B CN 103420838B CN 201310339674 A CN201310339674 A CN 201310339674A CN 103420838 B CN103420838 B CN 103420838B
Authority
CN
China
Prior art keywords
water
chlorogenic acid
phase
organic phase
temperature
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.)
Expired - Fee Related
Application number
CN201310339674.XA
Other languages
Chinese (zh)
Other versions
CN103420838A (en
Inventor
霍清
薛明德
杨晓芳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
College of Biochemical Engineering of Beijing Union University
Original Assignee
College of Biochemical Engineering of Beijing Union University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by College of Biochemical Engineering of Beijing Union University filed Critical College of Biochemical Engineering of Beijing Union University
Priority to CN201310339674.XA priority Critical patent/CN103420838B/en
Publication of CN103420838A publication Critical patent/CN103420838A/en
Application granted granted Critical
Publication of CN103420838B publication Critical patent/CN103420838B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Abstract

The invention relates to a method for separating and purifying chlorogenic acid by utilizing temperature to induce an aqueous two-phase system, which comprises the following steps: (1) smashing folium cortex eucommiae to be placed in an extracting tank, adding 5 to 7 times of water in weight, heating and back flowing for 1.5 to 2 h, and concentrating until paste is soaked, that is chlorogenic acid crude product; (2) mixing EOPO and water according to the volume ratio of (1-3):(1-4), then adding salt in the mixture, uniformly mixing, adding the chlorogenic acid crude product in the solution, uniformly mixing, centrifugally separating water phase and organic phase rich in EOPO, enriching chlorogenic acid in organic phase, and enriching impurity in water phase; (3) water bathing the organic phase gained in the step (2) at the temperature of 50 to 70 DEG C for 10 to 30 min, grouping the solution into two phases automatically, centrifugally separating the organic phase and the water phase, enriching the chlorogenic acid in water phase, and enriching EOPO in organic phase to be recycled; (4) cooling the obtained water phase to the room temperature, placing overnight, dissolving out crystal, and filtering to obtain chlorogenic acid pure product. The method is simple in technology and short in time consumption, reduces the material consumption, facilitates industrial pilot plant test enlargement, and is higher in yield coefficient.

Description

A kind of method of utilizing temperature-induced double-aqueous phase system separation and purification chlorogenic acid
Technical field
The present invention relates to a kind of method of utilizing temperature-induced double-aqueous phase system separation and purification chlorogenic acid, belong to technical field of biochemical industry.
Background technology
Chlorogenic acid, is the depside being generated by coffic acid and quinic acid, is phenylpropanoids.In plant Japanese Honeysuckle, the bark of eucommia, content is higher, soluble in water, ethanol, acetone and other organic solvent, and traditional extraction process primary isoamyl alcohol method, utilizes its acidic extraction.Because chlorogenic acid is the fat that coffic acid and quininic acid form, facile hydrolysis while extracting with soda acid, productive rate is not high.
Patent documentation CN101781211A discloses a kind of method for extracting chlorogenic acid from sunflower meal, the method is pulverized sunflower meal, adding concentration is the proteolytic enzyme that 1% cellulase and concentration are 5%, cellulase and proteolytic enzyme enzyme liquor ratio are 4: 1, sunflower meal and two kinds of enzyme liquor ratios are 1: 8, adjust pH is 4, and enzymolysis time is 1.2h, and hydrolysis temperature is 49 ℃; 45%～55% ethanolic soln lixiviate that is 3～6 by pH value again, is fully dissolved in vat liquor the chlorogenic acid in sunflower meal, and is cooled to room temperature; Then, then use Amberlyst process conventional processing, through absorption, wash-out, the technique such as dry, make chlorogenic acid, chlorogenic acid yield can reach 1.91%.
Patent documentation CN101781211A discloses a kind of total flavones, chlorogenic acid extraction process prepared from Chinese medicine Folium Eucommiae, adopts the two enzyme enzymolysis of Sumizyme MP-composite flavor enzyme, membrane sepn and spray drying technology to obtain chlorogenic acid product.Patent documentation CN102476996A discloses a kind of extraction process of Chlorogenic Acid of Flos Lonicerae, utilize ethanol refluxing process to extract the former green acids in Japanese Honeysuckle, its processing step is: microwave treatment packs Japanese Honeysuckle in the 250mL microwave special container of prolong is housed, the pre-soaking time is 8～24h, setting microwave power is 600～800W, and radiated time is 1～3min, starts microwave oven, after extraction finishes, united extraction liquid, concentrate drying, obtains crude extract; Enzymolysis and extraction, to the enzymolysis solution that adds 0～3.0% in crude extract, hydrolysis temperature is 25～60 ℃, enzymolysis time is 1～4h; Adding solid-liquid ratio is the ethanol of 1: 8～1: 12, and the concentration of ethanol is 65～75%, backflow lixiviate 0.5～1.5h; In chlorogenic acid crude extract, adding solid-liquid ratio is the water of 1: 2～1: 5, and precipitation, filtration, filtrate concentrate drying, obtain finished product.
Aqueous two phase extraction technique is a kind of novel isolation technique, and the hydrophilic polymer aqueous solution can form double-aqueous phase system under certain condition, distributes difference by separated object in two-phase, just can realize separation.This technology, since research and development, has been widely used in product separation and the extraction in the fields such as biological chemistry, cytobiology and biochemical industry.Aqueous two phase extraction technique facility investment is few, simple to operate, and does not have the remaining problem of organic solvent, therefore in natural drug separation and acquisition, also has good application prospect.But, due to polymer recovery difficulty, so hindered to a certain extent the application of Two-phase system.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing temperature-induced double-aqueous phase system separation and purification chlorogenic acid, the method technique is simple, does not use poisonous organic solvent, and prepared chlorogenic acid purity is high.
For achieving the above object, the present invention is by the following technical solutions:
A method of utilizing temperature-induced double-aqueous phase system separation and purification chlorogenic acid, the method comprises the following steps:
(1) chlorogenic acid extracting crude product: put into extractor after Folium Eucommiae is pulverized, add wherein the water of 5～7 times of weight, reflux 1.5～2h, filters, and filtrate is concentrated into medicinal extract, is chlorogenic acid crude product;
(2) double water-phase is separated: by PEP-101 and water 1～3: 1～4 mixing by volume, then add wherein salt, mix, in gained solution, the concentration of salt is 0.05～0.15g/mL, in solution, by the amount of 2.0～5.0mg/mL, add chlorogenic acid crude product, after mixing, take rotating speed as 1000～2000r/min centrifugation, 3～5min, water phase separated and the organic phase that is rich in PEP-101, chlorogenic acid is in organic phase enrichment, and impurity is in water enrichment;
(3) temperature-induced double water-phase is separated: the organic phase that step (2) is obtained is at 50～70 ℃ of water-bath 10～30min, solution is divided into two-phase automatically, take rotating speed as 1000～2000r/min centrifugation, 3～5min, separated organic phase and water, chlorogenic acid is in water enrichment, and PEP-101 is in organic phase enrichment and reclaimed;
(4) concentrate drying: the water that step (3) is obtained is cooled to room temperature, and placement is spent the night, crystallization, filters and obtains chlorogenic acid sterling.
In the method for the invention, PEP-101 under normal temperature (EOPO) forms double-aqueous phase system with salt brine solution, and chlorogenic acid is enriched to EOPO phase.Because EOPO has lower cloud point, rising EOPO phase temperature, when temperature rises to the cloud point temperature of EOPO when above, solution is divided into water and organic phase automatically, and chlorogenic acid is assigned to water again, and the organic phase that is rich in EOPO is reclaimed.
Wherein, the molecular-weight average of the PEP-101 in described step (2) is preferably 2000～2870.Described salt is dipotassium hydrogen phosphate, potassium primary phosphate or anhydrous sodium sulphate.In described step (2) volume ratio of PEP-101 and water be preferably 1: 1,1: 2,1: 3,1: 4,2: 3,3: 2 or 3: 4.
In temperature-induced double water-phase sepn process, operating environment remains at the temperature more than cloud point of EOPO, and the system temperature in centrifugal separation processes remains on 50～70 ℃, and preferably controlling is 60 ℃, and centrifugal separation processes can operate at twice.
The invention has the advantages that:
The present invention adopts EOPO/ salt double water-phase to distribute in conjunction with temperature induced phase-separable from natural phant extracting chlorogenic acid from Eucommia leaves, and technique is simple, consuming time short, reduces material consumption, be beneficial to industry test and amplify, and yield is higher.Chlorogenic acid purity prepared by the present invention is high, is rich in one of EOPO simultaneously and can reclaims and recycle mutually, technique environmental protection.
Embodiment
Below by specific embodiment, the present invention will be further described.The reagent using in following examples is SILVER REAGENT.
Embodiment 1
1) chlorogenic acid extracting crude product: 1.5kg Folium Eucommiae is pulverized, put into extractor, add wherein 10L water, reflux 2h, is concentrated into medicinal extract, is chlorogenic acid crude product, and content is 13.37%.
2) double water-phase is separated: preparation EOPO/ salt double-aqueous phase system, and EOPO (molecular weight 2000) 10mL wherein, water 15mL, the concentration of dipotassium hydrogen phosphate is 0.1g/mL.In double-aqueous phase system, add chlorogenic acid crude product 100mg, after mixing, take rotating speed as 2000r/min centrifugation 3min, separated organic phase (being rich in EOPO) and water, chlorogenic acid is in organic phase enrichment (upper phase), and impurity is in water enrichment.
3) temperature-induced double water-phase is separated: organic phase (being rich in EOPO) is carried out to temperature induced phase-separable.After 60 ℃ of water-bath 20min, solution is divided into two-phase automatically, wherein goes up and is rich in mutually EOPO, the lower water that is rich in mutually, in whizzer with 2000r/min centrifugation 5min, separated organic phase (being rich in EOPO) and water, chlorogenic acid is in water enrichment, and organic phase EOPO is reclaimed.
4) water is cooled to room temperature, and placement is spent the night, and crystallization filters and obtains chlorogenic acid sterling.Adopt aforesaid operations, only change EOPO model SDP28, SDP30, SDP35, molecular weight is respectively 2000,2500,2750, and experimental data is in Table 1.
The impact of table 1EOPO molecular weight on partition ratio and yield
The volume ratio of the upper and lower phase of the temperature-induced phase system of R-;
The temperature-induced phase system partition ratio of K-;
C 2bthe mass concentration of phase Content of Chlorogenic Acid under-temperature-induced phase system, mg/mL;
Y 1-double-aqueous phase system chlorogenic acid is at upper yield in mutually, %;
Y-is temperature-induced balances each other chlorogenic acid at lower yield in mutually, %.
M-chlorogenic acid sterling quality, mg;
The purity of F-chlorogenic acid sterling, %;
From table 1, all certain in the quality of dipotassium hydrogen phosphate, water, when the molecular weight of EOPO is 2750 (SDP35), yield is maximum, reaches 59.7%.Separating effect is best.
Embodiment 2
1) chlorogenic acid extracting crude product: 1.5kg Folium Eucommiae is pulverized, put into extractor, add wherein 10L water, reflux 2h, is concentrated into medicinal extract, is chlorogenic acid crude product, and content is 13.37%.
2) double water-phase is separated: preparation EOPO/ salt double-aqueous phase system, and EOPO (SDP35, molecular weight 2750) 10mL wherein, water 15mL, salt is selected respectively KH 2pO 4, K 2hPO 4, Na 2sO 4, NH 4cl, concentration is respectively 0.1g/mL.In double-aqueous phase system, add chlorogenic acid crude product 100mg, after mixing, take rotating speed as 2000 turn/min centrifugation 3min, separated organic phase (being rich in EOPO) and water, chlorogenic acid is in organic phase enrichment (upper phase), and impurity is in water enrichment.
3) temperature-induced double water-phase is separated: organic phase (being rich in EOPO) is carried out to temperature induced phase-separable.After 60 ℃ of water-bath 20min, solution is divided into two-phase automatically, wherein goes up and is rich in mutually EOPO, the lower water that is rich in mutually, in whizzer with 2000r/min centrifugation 5min, separated organic phase (being rich in EOPO) and water, chlorogenic acid is in water enrichment, and organic phase EOPO is reclaimed.
4) water is cooled to room temperature, and placement is spent the night, and crystallization filters and obtains chlorogenic acid sterling.
In double water-phase separation and temperature-induced double water-phase sepn process, measure respectively the impact of different salt pair partition ratio and chlorogenic acid yield, experimental data is in Table 2.In double water-phase sepn process, when adopting NH 4during Cl salt, not stratified.
The impact of the kind of table 2 salt on partition ratio and yield
Embodiment 3
1) chlorogenic acid extracting crude product: 1.5kg Folium Eucommiae is pulverized, put into extractor, add wherein 10L water, reflux 2h, is concentrated into medicinal extract, is chlorogenic acid crude product, and content is 13.37%.
2) double water-phase is separated: preparation EOPO/ salt double-aqueous phase system, EOPO (SDP35 wherein, molecular weight 2750) 5,10,15mL, water 10,15,20mL, dipotassium hydrogen phosphate 1.0,1.5,2.0g, in double-aqueous phase system, add chlorogenic acid crude product 100mg, after mixing, take rotating speed as 2000r/min centrifugation 3min, separated organic phase (being rich in EOPO) and water, chlorogenic acid is in organic phase enrichment (upper phase), and impurity is in water enrichment.
3) temperature-induced double water-phase is separated: organic phase (being rich in EOPO) is carried out to temperature induced phase-separable.After 60 ℃ of water-bath 20min, solution is divided into two-phase automatically, wherein goes up and is rich in mutually EOPO, the lower water that is rich in mutually, in whizzer with 2000r/min centrifugation 5min, separated organic phase (being rich in EOPO) and water, chlorogenic acid is in water enrichment, and organic phase EOPO is reclaimed.
4) water is cooled to room temperature, and placement is spent the night, and crystallization filters and obtains chlorogenic acid sterling 11.2mg, and purity is 81.4%, and yield reaches 68.2%.
Utilize Three factors-levels orthogonal experiment, investigate temperature-induced SDP35/K 2hPO 4double-aqueous phase system, determines the top condition of separating chlorogenic acid.Quadrature factor level is in Table 3, and experimental result is in Table 4, and quadrature analysis is in Table 5.
Table 3 Three factors-levels orthogonal design table
Table 4 orthogonal test
The analysis of table 5 orthogonal experiment
Extreme difference maximum in table 5 is 0.837, and the separated maximum effect factor of temperature-induced double water-phase is EOPO amount.Work as K 2hPO 42.0g, water yield 20mL, SDP3515mL can reach optimal separation effect.

Claims (4)

1. a method of utilizing temperature-induced double-aqueous phase system separation and purification chlorogenic acid, is characterized in that, the method comprises the following steps:
(1) chlorogenic acid extracting crude product: put into extractor after Folium Eucommiae is pulverized, add wherein the water of 5～7 times of weight, reflux 1.5～2h, filters, and filtrate is concentrated into medicinal extract, is chlorogenic acid crude product;
(2) double water-phase is separated: PEP-101 and water are mixed 1～3:1～4 by volume, then add wherein salt, described salt is dipotassium hydrogen phosphate, potassium primary phosphate or anhydrous sodium sulphate; Mix, in gained solution, the concentration of salt is 0.05～0.15g/mL, in solution, by the amount of 2.0～5.0mg/mL, add chlorogenic acid crude product, after mixing, take rotating speed as 1000～2000r/min centrifugation, 3～5min, water phase separated and the organic phase that is rich in PEP-101, chlorogenic acid is in organic phase enrichment, and impurity is in water enrichment;
(3) temperature-induced double water-phase is separated: the organic phase that step (2) is obtained is at 50～70 ℃ of water-bath 10～30min, solution is divided into two-phase automatically, take rotating speed as 1000～2000r/min centrifugation, 3～5min, separated organic phase and water, chlorogenic acid is in water enrichment, and PEP-101 is in organic phase enrichment and reclaimed;
(4) concentrate drying: the water that step (3) is obtained is cooled to room temperature, and placement is spent the night, crystallization, filters and obtains chlorogenic acid sterling.
2. the method for utilizing temperature-induced double-aqueous phase system separation and purification chlorogenic acid according to claim 1, is characterized in that, the molecular-weight average of the PEP-101 in described step (2) is 2000～2870.
3. the method for utilizing temperature-induced double-aqueous phase system separation and purification chlorogenic acid according to claim 1, it is characterized in that, in described step (2), the volume ratio of PEP-101 and water is 1:1,1:2,1:3,1:4,2:3,3:2 or 3:4.
4. the method for utilizing temperature-induced double-aqueous phase system separation and purification chlorogenic acid according to claim 1, is characterized in that, the bath temperature of described step (3) is 60 ℃.
CN201310339674.XA 2013-08-06 2013-08-06 Method for separating and purifying chlorogenic acid by utilizing temperature to induce aqueous two-phase system Expired - Fee Related CN103420838B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310339674.XA CN103420838B (en) 2013-08-06 2013-08-06 Method for separating and purifying chlorogenic acid by utilizing temperature to induce aqueous two-phase system

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310339674.XA CN103420838B (en) 2013-08-06 2013-08-06 Method for separating and purifying chlorogenic acid by utilizing temperature to induce aqueous two-phase system

Publications (2)

Publication Number Publication Date
CN103420838A CN103420838A (en) 2013-12-04
CN103420838B true CN103420838B (en) 2014-12-03

Family

ID=49646270

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310339674.XA Expired - Fee Related CN103420838B (en) 2013-08-06 2013-08-06 Method for separating and purifying chlorogenic acid by utilizing temperature to induce aqueous two-phase system

Country Status (1)

Country Link
CN (1) CN103420838B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103951562B (en) * 2014-05-09 2016-04-20 四川九章生物化工科技发展有限公司 A kind of chlorogenic acid crystal formation and preparation method thereof
CN111307969B (en) * 2020-03-06 2022-03-11 湖南省中医药研究院 Method for simultaneously extracting phenolic acid glycosides and polysaccharides in traditional Chinese medicine

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102001947A (en) * 2010-12-08 2011-04-06 广西金宏昕生物科技有限公司 Method for preparing honeysuckle chlorogenic acid

Also Published As

Publication number Publication date
CN103420838A (en) 2013-12-04

Similar Documents

Publication Publication Date Title
CN104530173B (en) A kind of extract the technique of tea saponin in cake of camellia oleifera seeds
CN103951718A (en) Method used for preparing high-purity gardenoside and crocin from gardenia jasminoides ellis
CN104326981B (en) A kind of high efficiency extraction separation method of bulleyaconitine A
CN104311620A (en) Method for purifying chenodeoxycholic acid
CN102730721A (en) Recovering method of by-product sodium chloride in polyphenylene sulfide production
CN103420838B (en) Method for separating and purifying chlorogenic acid by utilizing temperature to induce aqueous two-phase system
CN103012518B (en) Production process for simultaneously extracting asperuloside and chlorogenic acid from folium cortex eucommiae
CN102532332A (en) Method for extracting and preparing low molecular weight fucoidin from marine brown algae
CN101851221B (en) Method for preparing dihydroquercetin from larches
CN105985315A (en) Method for extracting nicotine from tobacco waste
CN106810564A (en) The method for separating eurycomanone is extracted in a kind of root from Tongkat Ali
CN103044442B (en) A kind of method of separation and purification GA, GB and bilobalide from Folium Ginkgo extract
CN104069191B (en) A kind of extraction process of water polygonum flaccidum general flavone
CN105272989A (en) Method for preparing chlorophyll sodium copper from mulberry leaves
CN105272887A (en) Method for extracting taurine and polysaccharides from abalone's viscera simultaneously
CN107759467A (en) A kind of preparation method for improving carnosic acid content in rosemary fat-soluble antioxidant
CN106832037A (en) A kind of notoginseng polysaccharide extracting method rapidly and efficiently
CN105399795B (en) Method for extracting astragaloside from radix astragali
CN107496508A (en) A kind of preparation method of Fourstamen Stephania Root alkaloid
CN104710492A (en) Method for extracting androstenedione in bi-liquid phases system
CN101792394A (en) Extraction separation method of L-synephrine
CN102146083B (en) Method for separating and extracting cepharanthine
CN103772186A (en) Refining method of fermented organic acid
CN110922413A (en) Extraction and separation method of glabridin
CN104292212A (en) Method for separation and purification of nicotine by electrodialysis method

Legal Events

Date Code Title Description
PB01 Publication
C06 Publication
SE01 Entry into force of request for substantive examination
C10 Entry into substantive examination
GR01 Patent grant
C14 Grant of patent or utility model
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20141203

Termination date: 20160806

CF01 Termination of patent right due to non-payment of annual fee