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 PDFInfo
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- 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
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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
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 ℃.
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