CN118063319B - Method for preparing high-purity chlorogenic acid by using stevia rebaudiana extract - Google Patents
Method for preparing high-purity chlorogenic acid by using stevia rebaudiana extract Download PDFInfo
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- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 title claims abstract description 232
- 235000001368 chlorogenic acid Nutrition 0.000 title claims abstract description 218
- CWVRJTMFETXNAD-FWCWNIRPSA-N 3-O-Caffeoylquinic acid Natural products O[C@H]1[C@@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-FWCWNIRPSA-N 0.000 title claims abstract description 217
- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 title claims abstract description 217
- 229940074393 chlorogenic acid Drugs 0.000 title claims abstract description 217
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 title claims abstract description 217
- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 title claims abstract description 217
- CWVRJTMFETXNAD-JUHZACGLSA-N chlorogenic acid Chemical compound O[C@@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-JUHZACGLSA-N 0.000 title claims abstract description 215
- 238000000034 method Methods 0.000 title claims abstract description 17
- 244000228451 Stevia rebaudiana Species 0.000 title description 35
- 235000006092 Stevia rebaudiana Nutrition 0.000 title description 35
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 140
- 239000011347 resin Substances 0.000 claims abstract description 75
- 229920005989 resin Polymers 0.000 claims abstract description 75
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 47
- GYFFKZTYYAFCTR-JUHZACGLSA-N 4-O-trans-caffeoylquinic acid Chemical compound O[C@@H]1C[C@](O)(C(O)=O)C[C@@H](O)[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 GYFFKZTYYAFCTR-JUHZACGLSA-N 0.000 claims abstract description 32
- GYFFKZTYYAFCTR-UHFFFAOYSA-N 5-O-(6'-O-galloyl)-beta-D-glucopyranosylgentisic acid Natural products OC1CC(O)(C(O)=O)CC(O)C1OC(=O)C=CC1=CC=C(O)C(O)=C1 GYFFKZTYYAFCTR-UHFFFAOYSA-N 0.000 claims abstract description 32
- GYFFKZTYYAFCTR-LMRQPLJMSA-N cryptochlorogenic acid Natural products O[C@H]1C[C@@](O)(C[C@H](O)[C@H]1OC(=O)C=Cc2ccc(O)c(O)c2)C(=O)O GYFFKZTYYAFCTR-LMRQPLJMSA-N 0.000 claims abstract description 32
- 238000011084 recovery Methods 0.000 claims abstract description 21
- 238000001179 sorption measurement Methods 0.000 claims abstract description 21
- 241000544066 Stevia Species 0.000 claims abstract description 12
- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HELXLJCILKEWJH-NCGAPWICSA-N 0.000 claims abstract description 12
- 239000012535 impurity Substances 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 184
- 239000007787 solid Substances 0.000 claims description 63
- 238000006243 chemical reaction Methods 0.000 claims description 54
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical class CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 48
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- 239000002904 solvent Substances 0.000 claims description 21
- GWTUHAXUUFROTF-UHFFFAOYSA-N pseudochlorogenic acid Natural products C1C(O)C(O)C(O)CC1(C(O)=O)OC(=O)C=CC1=CC=C(O)C(O)=C1 GWTUHAXUUFROTF-UHFFFAOYSA-N 0.000 claims description 15
- CWVRJTMFETXNAD-NXLLHMKUSA-N trans-5-O-caffeoyl-D-quinic acid Chemical compound O[C@H]1[C@H](O)C[C@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-NXLLHMKUSA-N 0.000 claims description 15
- 238000011068 loading method Methods 0.000 claims description 11
- 238000000605 extraction Methods 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 8
- 238000002425 crystallisation Methods 0.000 claims description 7
- 230000008025 crystallization Effects 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 6
- 239000008346 aqueous phase Substances 0.000 claims 3
- 238000004458 analytical method Methods 0.000 abstract description 51
- 238000001035 drying Methods 0.000 abstract description 22
- 239000000047 product Substances 0.000 description 27
- 230000000052 comparative effect Effects 0.000 description 23
- 239000012043 crude product Substances 0.000 description 14
- 238000010992 reflux Methods 0.000 description 14
- 230000000694 effects Effects 0.000 description 11
- 239000007788 liquid Substances 0.000 description 10
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- 239000000126 substance Substances 0.000 description 7
- 238000000926 separation method Methods 0.000 description 6
- 238000001816 cooling Methods 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 239000002994 raw material Substances 0.000 description 5
- 239000006227 byproduct Substances 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 4
- -1 chlorogenic acid compound Chemical class 0.000 description 2
- 239000012527 feed solution Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 241000205585 Aquilegia canadensis Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000208689 Eucommia ulmoides Species 0.000 description 1
- 241000533293 Sesbania emerus Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/28—Preparation of carboxylic acid esters by modifying the hydroxylic moiety of the ester, such modification not being an introduction of an ester group
- C07C67/293—Preparation of carboxylic acid esters by modifying the hydroxylic moiety of the ester, such modification not being an introduction of an ester group by isomerisation; by change of size of the carbon skeleton
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/52—Separation; Purification; Stabilisation; Use of additives by change in the physical state, e.g. crystallisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/60—Separation; Purification; Stabilisation; Use of additives by treatment giving rise to chemical modification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/14—The ring being saturated
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Crystallography & Structural Chemistry (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
本发明公开了一种利用甜叶菊提取液制备高纯绿原酸的方法，包括以下步骤：将甜叶菊提取液除杂后采用LSA‑900E树脂进行吸附，之后进行梯度解析，收集目标解析液；将目标解析液干燥后重新溶于水中得溶液A，将溶液A经HZ‑841树脂进行吸附，之后分别采用不同浓度的乙醇溶液进行梯度解析，再次收集目标解析液；将目标解析液干燥后于一定条件下进行转化反应，得隐绿原酸成品；将隐绿原酸成品再在一定条件下进行转化反应，制得高纯绿原酸。本发明制得的绿原酸回收率高，纯度高。
The present invention discloses a method for preparing high-purity chlorogenic acid using stevia extract, comprising the following steps: removing impurities from the stevia extract and then using LSA-900E resin for adsorption, then performing gradient analysis, and collecting the target analysis solution; drying the target analysis solution and re-dissolving it in water to obtain solution A, adsorbing solution A through HZ-841 resin, and then using ethanol solutions of different concentrations for gradient analysis, and collecting the target analysis solution again; drying the target analysis solution and performing a conversion reaction under certain conditions to obtain a cryptochlorogenic acid product; and performing a conversion reaction on the cryptochlorogenic acid product under certain conditions to obtain high-purity chlorogenic acid. The chlorogenic acid prepared by the present invention has high recovery rate and high purity.
Description
Technical Field
The invention relates to the technical field of chlorogenic acid preparation, in particular to a method for preparing high-purity chlorogenic acid by using stevia rebaudiana extract.
Background
Chlorogenic acid is an important bioactive substance, and has antibacterial, antiviral, leukocyte increasing, liver protecting, gallbladder function promoting, antitumor, blood pressure lowering, blood lipid reducing, free radical scavenging, and central nervous system exciting effects. Chlorogenic acid is mainly obtained from honeysuckle, eucommia ulmoides leaves, coffee beans and other plants, but the raw materials are limited and have high cost. Chlorogenic acid can be extracted from stevia rebaudiana leaves, and raw material cost can be reduced, but most of chlorogenic acid substances extracted at present are mixtures of chlorogenic acid and other byproducts such as hidden chlorogenic acid, neochlorogenic acid and the like, the purity of the extracted chlorogenic acid is not high, and the pressure of the byproducts in the extraction process on storage is increased. At present, little research is done on separating and purifying chlorogenic acid from stevia extract. In order to solve the above technical problems, it is necessary to provide a method for preparing high purity chlorogenic acid by using stevia extract.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: aiming at the defects existing in the prior art, the method for preparing the high-purity chlorogenic acid by using the stevia rebaudiana extractive solution is provided, the stevia rebaudiana extractive solution is used as a raw material, the preparation cost of the chlorogenic acid is reduced, chlorogenic acid, cryptochlorogenic acid and new chlorogenic acid in the stevia rebaudiana extractive solution can be effectively separated, and finally the cryptochlorogenic acid and the new chlorogenic acid are converted into the chlorogenic acid, so that the chlorogenic acid with high purity and high yield is obtained.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a method for preparing high purity chlorogenic acid by using stevia rebaudiana extract comprises the following steps:
(1) Dissolving stevia rebaudiana extract in water to obtain stevia rebaudiana extract solution, removing impurities, adsorbing with LSA-900E resin, performing gradient analysis with ethanol solutions of different concentrations, and collecting analysis solution of 55-65wt% ethanol solution;
(2) Drying the resolving solution of the 55-65wt% ethanol solution obtained in the step (1), then re-dissolving the resolving solution in water to obtain a solution A, adsorbing the solution A by HZ-841 resin, collecting effluent liquid, then respectively carrying out gradient resolving by adopting ethanol solutions with different concentrations, and respectively collecting resolving solution of the 15-25wt% ethanol solution and resolving solution of the 35-45wt% ethanol solution;
(3) Drying the resolving solution of the ethanol solution with the concentration of 15-25wt% collected in the step (2) to obtain a solid A, drying the resolving solution of the ethanol solution with the concentration of 35-45wt% to obtain a solid B, dissolving the solid B in a methanol solution to obtain a solution B, then adjusting the pH value of the solution B, carrying out reflux reaction, adjusting the pH value of the solution B after the reaction is finished, and finally drying to obtain a crude product of the chlorogenic acid; extracting and purifying the crude product of the cryptochlorogenic acid to obtain a finished product of the cryptochlorogenic acid;
(4) Dissolving the finished product of the chlorogenic acid in a methanol solution to obtain a solution C, then carrying out reaction under the negative pressure condition, and finally removing the solvent in vacuum to obtain a crude chlorogenic acid product;
(5) Mixing the solid A prepared in the step (3) with the chlorogenic acid crude product obtained in the step (4), and crystallizing by adopting a mixed solution of ethyl acetate and water to prepare the high-purity chlorogenic acid.
Specifically, the stevia rebaudiana extract is prepared by taking stevia rebaudiana leaves as a raw material and ethanol solution as an extraction solvent. More preferably, the concentration of the ethanol solution is 50-70v/v%, and the mass ratio of the stevia leaves to the ethanol solution is 1: (8-12); and/or the extraction temperature is 20-30deg.C and the extraction time is 2-5h.
As an improved technical scheme, in the step (1), the solid content of the stevia rebaudiana extracting solution is 2-5%.
As an improved technical scheme, in the step (1), the LX-8 type resin is adopted for removing impurities when the stevia rebaudiana extract is subjected to impurity removal, the LX-8 type resin is adopted for removing impurities, and the LSA-900E resin is adopted for adsorption treatment, wherein the feeding speed of the stevia rebaudiana extract is controlled to be 1-2BV/h.
As an improved technical scheme, the feeding speed is 2-4BV/h when the ethanol solution is adopted for gradient analysis in the step (1), and the concentration of the ethanol solution adopted for gradient analysis is 15-25wt%, 55-65wt% and 75-85wt% respectively.
As an improved technical scheme, in the step (2), the solid content of the solution A is 5-10%, the feeding speed during adsorption is 1-2BV/h, and the loading amount is 8-12% of the volume of the resin.
As an improved technical scheme, in the step (2), the feeding speed is 2-4BV/h when the ethanol solution is adopted for gradient analysis, and the concentration of the ethanol solution adopted for gradient analysis is 15-25wt%, 35-45wt% and 55-65wt% respectively.
As an improved technical scheme, in the step (3), the concentration of the methanol solution is 40-60v/v%, and the solid content of the solution B is 25-30%; and/or the temperature of the reflux reaction is 50-70 ℃, and the time of the reflux reaction is 20-40min.
As an improved technical scheme, in the step (3), the pH value of the solution B is firstly adjusted to 7.5-8.5, the reflux reaction is carried out, and the pH value of the solution B is adjusted to 5-6 after the reaction is finished.
In the step (3), when the crude product of the chlorogenic acid is extracted and purified, the crude product of the chlorogenic acid is dissolved in water, then saturated ethyl acetate is adopted for multiple times of extraction, the water phases of the multiple times of extraction are combined, and then the solvent is removed in vacuum, so that the finished product of the chlorogenic acid is obtained.
As an improved technical scheme, in the step (4), the concentration of the methanol solution is 20-30v/v%, the solid content of the solution C is 30-40%, the pressure of the reaction is-0.05 MPa to-0.1 MPa, the temperature is 70-80 ℃, and the reaction time is 1-3h.
As an improved technical scheme, in the step (4), the pH of the solution is regulated to 7-8 during the reaction, and the pH of the reaction system is adjusted back to 3-4 after the reaction is finished, and then the solvent is removed.
As an improved technical scheme, in the step (5), in the mixed solution of ethyl acetate and water, the volume ratio of ethyl acetate to water is 1: (1-4); and/or the crystallization treatment is carried out by mixing the prepared solid A and the chlorogenic acid crude product obtained in the step (4) into a mixed solution of ethyl acetate and water, stirring, layering, heating the water phase, cooling to room temperature under normal pressure, and carrying out crystallization treatment for 20-30h.
Due to the adoption of the technical scheme, the invention has the beneficial effects that:
The method comprises the steps of taking stevia rebaudiana extract as a raw material, firstly adopting a certain type of resin to remove impurities, then adopting the resin to adsorb to enrich chlorogenic acid substances, adopting ethanol solutions with different concentrations to perform gradient analysis, collecting analysis liquid containing chlorogenic acid substances, then adopting a specific type of resin to further separate and purify the analysis liquid containing chlorogenic acid substances, and then adopting the ethanol solutions with different concentrations to perform gradient analysis to obtain analysis liquid containing chlorogenic acid and analysis liquid containing chlorogenic acid substances complex, thereby realizing primary separation of chlorogenic acid; the analytical solution containing chlorogenic acid compound is converted under specific conditions, so that chlorogenic acid and new chlorogenic acid in the chlorogenic acid compound are converted into chlorogenic acid, and finally chlorogenic acid is converted into chlorogenic acid under specific conditions. Through the process, the isomer of chlorogenic acid in the stevia rebaudiana extract is converted into chlorogenic acid, and the obtained chlorogenic acid is high in purity and the effective recovery rate is obviously improved.
The method has the advantages of relatively simple process operation and low cost, greatly improves the conversion rate of the chlorogenic acid from the hidden chlorogenic acid to the chlorogenic acid by optimizing conditions, ensures stable conversion process, can realize recycling of chlorogenic acid byproducts in industrial production, and improves the yield of the chlorogenic acid while reducing the storage pressure caused by byproduct accumulation.
Drawings
FIG. 1 is an HPLC chart of chlorogenic acid product obtained in example 1.
Detailed Description
The invention is further illustrated below with reference to examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The effective recovery rate of chlorogenic acid in the following examples and comparative examples and the conversion rate of cryptochlorogenic acid in the conversion reaction of cryptochlorogenic acid to chlorogenic acid in step (5) were calculated as follows:
The conversion rate of chlorogenic acid (%) = { [ (crude chlorogenic acid mass×chlorogenic acid content in crude chlorogenic acid) - (mass of finished product of chlorogenic acid×chlorogenic acid content in finished product of chlorogenic acid) ]/(mass of finished product of chlorogenic acid×content of chlorogenic acid in finished product of chlorogenic acid) } ×100%;
effective chlorogenic acid recovery (%) = (high purity chlorogenic acid mass×chlorogenic acid content in high purity chlorogenic acid/stevia extract mass×chlorogenic acid content in stevia extract) ×100%.
Example 1
A method for preparing high purity chlorogenic acid by using stevia rebaudiana extract comprises the following steps:
(1) 700g of stevia rebaudiana extract (9.8% of chlorogenic acid, 2.0% of cryptochlorogenic acid and 0.7% of neochlorogenic acid) is taken and dissolved in water to obtain a stevia rebaudiana extract with 2% of solid content;
(2) Adding stevia rebaudiana extract into LX-8 resin at a speed of 1BV/h for impurity removal, adding effluent into LSA-900E resin at a speed of 1BV/h for adsorption treatment, then sequentially carrying out gradient analysis on LSA-900E resin by adopting 20wt%, 60wt% and 80wt% ethanol solution (the volume of the ethanol solution is 2BV of the resin volume), and collecting analysis solution of 60wt% ethanol solution;
(3) Drying the analysis solution of the 60wt% ethanol solution to obtain 110g of solid (the main components are 52.3% of chlorogenic acid, 11.2% of cryptochlorogenic acid and 1.6% of neochlorogenic acid), redissolving the solid into water to obtain a solution A with 5% of solid content, feeding the solution A into the model HZ-841 resin at a feeding speed of 1BV/h for adsorption, loading the solution A with 8% of the volume of the resin, and then sequentially carrying out gradient analysis on the resin by adopting 20wt%, 40wt% and 60wt% ethanol solutions (the volume of the ethanol solution is 2BV of the volume of the resin), and respectively collecting the analysis solution of the 20wt% ethanol solution and the analysis solution of the 40wt% ethanol solution;
(4) Drying the analysis solution of the 20wt% ethanol solution collected in the step (3) to obtain 59g of solid A (the main component is chlorogenic acid, the content of which is 78%), drying the analysis solution of the 40wt% ethanol solution collected in the step (3) to obtain 42g of solid B (the main component is 26.1% of chlorogenic acid, 23.5% of cryptochlorogenic acid and 5.4% of neochlorogenic acid), dissolving the solid B in 200ml of 50v/v% methanol solution to obtain solution B, then adjusting the pH of the solution B to 8.3, carrying out reflux reaction at 60 ℃ for 30min, adjusting the pH of the solution B to 5.5 after the reaction is finished, and finally drying to obtain 37g of crude cryptochlorogenic acid (the main component is 2.1% of chlorogenic acid and 48.1%); dissolving the crude product of the chlorogenic acid in 150ml of water, extracting with saturated ethyl acetate with the same volume, extracting repeatedly for three times, combining water phases extracted for three times, and removing the solvent in vacuum to obtain 18g of finished product of the chlorogenic acid, wherein the content of the chlorogenic acid is 89.7%, and the content of the chlorogenic acid is 3.1%;
(5) Dissolving a finished product of the chlorogenic acid in 80ml of methanol solution with the concentration of 20v/v% in a high-pressure reaction kettle to obtain a solution C, regulating the pH value of the solution C to 7, carrying out reflux reaction for 2 hours at the temperature of 80 ℃ below zero under the pressure of-0.05 MPa, after the reaction is finished, regulating the pH value of the solution to 4, and removing the solvent to obtain 16g of crude chlorogenic acid, wherein the content of the chlorogenic acid is 82.3%, and the content of the chlorogenic acid is 7.8%; the conversion rate of the chlorogenic acid is 78.1 percent;
(6) Adding the solid A obtained in the step (4) and the chlorogenic acid crude product obtained in the step (5) into a mixed solution of ethyl acetate and water (the volume ratio of the ethyl acetate to the water is 1:1), controlling the mass ratio of the solid to the solvent to be 1:2, stirring and mixing, layering, collecting a water phase, heating the water phase to 70 ℃ under normal pressure, cooling to room temperature at a speed of 20 ℃/h for crystallization treatment for 24 hours, and obtaining 49g of high-purity chlorogenic acid, wherein the chlorogenic acid content is 95.1%, and the effective recovery rate of the chlorogenic acid is 67.9%.
As shown in figure 1, the chlorogenic acid content in the high-purity chlorogenic acid is up to 95.1% by High Performance Liquid Chromatography (HPLC).
Example 2
A method for preparing high purity chlorogenic acid by using stevia rebaudiana extract comprises the following steps:
(1) 700g of stevia rebaudiana extract (9.8% of chlorogenic acid, 2.0% of cryptochlorogenic acid and 0.7% of neochlorogenic acid) is taken and dissolved in water to obtain a stevia rebaudiana extract with 2% of solid content;
(2) Adding stevia rebaudiana extract into LX-8 resin at a speed of 1BV/h for impurity removal, adding effluent into LSA-900E resin at a speed of 1BV/h for adsorption treatment, then sequentially carrying out gradient analysis on LSA-900E resin by adopting 20wt%, 60wt% and 80wt% ethanol solution (the volume of the ethanol solution is 2BV of the resin volume), and collecting analysis solution of 60wt% ethanol solution;
(3) Drying the analysis solution of the 60wt% ethanol solution to obtain 108g of solid (the main components are 53.1% of chlorogenic acid, 12.5% of cryptochlorogenic acid and 1.2% of neochlorogenic acid), redissolving the solid into water to obtain a solution A with 6% of solid content, feeding the solution A into the model HZ-841 resin at a feeding speed of 1BV/h for adsorption, collecting effluent with a loading amount of 9% of the volume of the resin, and then sequentially carrying out gradient analysis on the resin by adopting 20wt%, 40wt% and 60wt% ethanol solutions (the volume of the ethanol solution is 2BV of the volume of the resin), and respectively collecting the analysis solution of the 20wt% ethanol solution and the analysis solution of the 40wt% ethanol solution;
(4) Drying the analysis solution of the 20wt% ethanol solution collected in the step (3) to obtain 56g of solid A (the content of chlorogenic acid is 75 percent), drying the analysis solution of the 40wt% ethanol collected in the step (3) to obtain 44g of solid B (the main component is 25.6 percent of chlorogenic acid, 24.3 percent of cryptochlorogenic acid and 5.1 percent of neochlorogenic acid), dissolving the solid B in 200ml of 50v/v percent methanol solution to obtain solution B, then adjusting the pH of the solution B to 7.8, carrying out reflux reaction at 60 ℃ for 30min, adjusting the pH of the solution B to 5.5 after the reaction is finished, and finally drying to obtain 36g of crude cryptochlorogenic acid (the main component is 3.6 percent of chlorogenic acid and 51.2 percent of cryptochlorogenic acid); dissolving the crude product of the chlorogenic acid in 150ml of water, extracting with saturated ethyl acetate with the same volume, extracting repeatedly for three times, combining water phases extracted for three times, and removing the solvent in vacuum to obtain 18.6g of finished product of the chlorogenic acid, wherein the content of the chlorogenic acid is 88.2% and the content of the chlorogenic acid is 0.5%;
(5) Dissolving a finished product of the chlorogenic acid in 80ml of methanol solution with the concentration of 20v/v% in a high-pressure reaction kettle to obtain a solution C, regulating the pH value of the solution C to 7, carrying out reflux reaction for 2 hours at the temperature of 70 ℃ below zero under the pressure of-0.05 MPa, after the reaction is finished, regulating the pH value of the solution to 3, and removing the solvent to obtain 15.1g of a crude chlorogenic acid product, wherein the chlorogenic acid content is 83.4% and the chlorogenic acid content is 6.1%; the conversion rate of the chlorogenic acid is 76.2%;
(6) Adding the solid A obtained in the step (4) and the chlorogenic acid crude product obtained in the step (5) into a mixed solution of ethyl acetate and water (the volume ratio of the ethyl acetate to the water is 1:1.2), controlling the mass ratio of the solid to the solvent to be 1:2, stirring and mixing, layering, collecting a water phase, heating the water phase to 70 ℃ under normal pressure, cooling to room temperature at a speed of 20 ℃/h for crystallization treatment for 24 hours, and obtaining 52g of high-purity chlorogenic acid, wherein the chlorogenic acid content is 93.3%, and the effective chlorogenic acid recovery rate is 70.7%.
Example 3
A method for preparing high purity chlorogenic acid by using stevia rebaudiana extract comprises the following steps:
(1) 700g of stevia rebaudiana extract (9.8% of chlorogenic acid, 2.0% of cryptochlorogenic acid and 0.7% of neochlorogenic acid) is taken and dissolved in water to obtain a stevia rebaudiana extract with 2% of solid content;
(2) Adding stevia rebaudiana extract into LX-8 resin at a speed of 1.5BV/h for impurity removal, adding effluent into LSA-900E resin at a speed of 1.5BV/h for adsorption treatment, and then sequentially carrying out gradient analysis on LSA-900E resin by adopting 20wt%, 60wt% and 80wt% ethanol solution (the volume of the ethanol solution is 2BV of the resin volume), and respectively collecting analysis liquid of 40wt% ethanol solution and analysis liquid of 60wt% ethanol solution;
(3) Drying the analysis solution of the 60wt% ethanol solution to obtain 113g of solid (the main components are 51.2% of chlorogenic acid content, 10.3% of cryptochlorogenic acid and 1.5% of neochlorogenic acid), redissolving the solid into water to obtain a solution A with 7% of solid content, feeding the solution A into the model HZ-841 resin at a feeding speed of 1.5BV/h for adsorption, collecting effluent with a loading amount of 9% of the resin volume, and then sequentially carrying out gradient analysis on the resin by adopting 20wt%, 40wt% and 60wt% ethanol solutions (the volume of the ethanol solution is 2BV of the resin volume), and respectively collecting analysis solutions of 20wt% ethanol solution and 40wt% ethanol solution;
(4) Drying the analysis solution of the 20wt% ethanol solution collected in the step (3) to obtain 57.3g of solid A (the main component chlorogenic acid is 77.3%), drying the analysis solution of the 40wt% ethanol solution collected in the step (3) to obtain 44g of solid B (the main component chlorogenic acid is 25.3%, the chlorogenic acid is 24.6%, and the chlorogenic acid is 5%), dissolving the solid B in 200ml of 50v/v% methanol solution to obtain solution B, then adjusting the pH of the solution B to 8.0, carrying out reflux reaction at 60 ℃ for 20min, adjusting the pH of the solution B to 6 after the reaction is finished, and finally drying to obtain 35.1g of crude chlorogenic acid (the main component chlorogenic acid is 3.3%, and the chlorogenic acid is 50.2%); dissolving the crude product of the chlorogenic acid in 150ml of water, extracting with saturated ethyl acetate with the same volume, extracting repeatedly for three times, combining water phases extracted for three times, and removing the solvent in vacuum to obtain 19.6g of finished product of the chlorogenic acid, wherein the content of the chlorogenic acid is 86.3% and the content of the chlorogenic acid is 0.065%;
(5) Dissolving a finished product of the chlorogenic acid in 80ml of methanol solution with the concentration of 20v/v% in a high-pressure reaction kettle to obtain a solution C, regulating the pH value of the solution C to 7, carrying out reflux reaction for 2 hours at the temperature of 70 ℃ below zero under the pressure of-0.1 MPa, after the reaction is finished, regulating the pH value of the solution to 4, and removing the solvent to obtain 15g of a crude chlorogenic acid product, wherein the chlorogenic acid content is 80.6% and the chlorogenic acid content is 11.3%; the conversion rate of the chlorogenic acid is 71.4%;
(6) Adding the solid A obtained in the step (4) and the chlorogenic acid crude product obtained in the step (5) into a mixed solution of ethyl acetate and water (the volume ratio of the ethyl acetate to the water is 1:1.3), controlling the mass ratio of the solid to the solvent to be 1:2, stirring and mixing, layering, collecting a water phase, heating the water phase to 70 ℃ under normal pressure, cooling to room temperature at a speed of 20 ℃/h for crystallization treatment for 24 hours, and obtaining 54.5g of high-purity chlorogenic acid, wherein the chlorogenic acid content is 91.5%, and the effective chlorogenic acid recovery rate is 72.7%.
Example 4
A method for preparing high purity chlorogenic acid by using stevia rebaudiana extract comprises the following steps:
(1) Taking 700g of stevia rebaudiana extract (9.8% of chlorogenic acid, 2.0% of cryptochlorogenic acid and 0.7% of neochlorogenic acid), and dissolving the stevia rebaudiana extract in water to obtain a stevia rebaudiana extract solution with 3% of solid content;
(2) Adding stevia rebaudiana extract into LX-8 resin at a speed of 2BV/h for impurity removal, adding effluent into LSA-900E resin at a speed of 2BV/h for adsorption treatment, and then sequentially carrying out gradient analysis on LSA-900E resin by adopting 20wt%, 60wt% and 80wt% ethanol solutions (the volume of the ethanol solution is 2BV of the resin volume), and respectively collecting analysis solution of 40wt% ethanol solution and analysis solution of 60wt% ethanol solution;
(3) Drying the analysis solution of the 60wt% ethanol solution to obtain 105g of solid (the main components are 56.2% of chlorogenic acid, 13.1% of cryptochlorogenic acid and 1.9% of neochlorogenic acid), redissolving the solid into water to obtain a solution A with the solid content of 8%, feeding the solution A into the model HZ-841 resin at the feeding speed of 2BV/h for adsorption, collecting effluent with the sample loading amount of 8% of the volume of the resin, and then sequentially carrying out gradient analysis on the resin by adopting 20wt%, 40wt% and 60wt% ethanol solutions (the volume of the ethanol solution is 2BV of the volume of the resin), and respectively collecting the analysis solution of the 20wt% ethanol solution and the analysis solution of the 40wt% ethanol solution;
(4) Drying the analysis solution of the 20wt% ethanol solution collected in the step (3) to obtain 61g of solid A (the content of chlorogenic acid as a main component is 75.2%), drying the analysis solution of the 40wt% ethanol solution collected in the step (3) to obtain 39g of solid B (the main component is 28% of chlorogenic acid, 24.1% of cryptochlorogenic acid and 5% of neochlorogenic acid), dissolving the solid B in 200ml of 50v/v% methanol solution to obtain solution B, then adjusting the pH of the solution B to 8.5, carrying out reflux reaction at 60 ℃ for 30min, adjusting the pH of the solution B to 6 after the reaction is finished, and finally drying to obtain 32g of crude cryptochlorogenic acid (the main component is 4.1% of chlorogenic acid and 51.2%); dissolving the crude product of the chlorogenic acid in 150ml of water, extracting with saturated ethyl acetate with the same volume, extracting repeatedly for three times, combining water phases extracted for three times, and removing the solvent in vacuum to obtain 17.6g of finished product of the chlorogenic acid, wherein the content of the chlorogenic acid is 86 percent and the content of the chlorogenic acid is 6.5 percent;
(5) Dissolving a finished product of the chlorogenic acid in 80ml of methanol solution with the concentration of 20v/v% in a high-pressure reaction kettle to obtain a solution C, regulating the pH value of the solution C to 7, carrying out reflux reaction for 2 hours at 80 ℃ under-0.1 MPa, after the reaction is finished, regulating the pH value of the solution to 4, and removing the solvent to obtain 15.1g of a crude chlorogenic acid product, wherein the chlorogenic acid content is 84.1% and the chlorogenic acid content is 8.4%; the conversion rate of the chlorogenic acid is 76.3 percent;
(6) Adding the solid A obtained in the step (4) and the chlorogenic acid crude product obtained in the step (5) into a mixed solution of ethyl acetate and water (the volume ratio of the ethyl acetate to the water is 1:1), controlling the mass ratio of the solid to the solvent to be 1:2, stirring and mixing, layering, collecting a water phase, heating the water phase to 70 ℃ under normal pressure, cooling to room temperature at a speed of 20 ℃/h for crystallization treatment for 24 hours, and obtaining 56.3g of high-purity chlorogenic acid, wherein the chlorogenic acid content is 94.2%, and the effective recovery rate of the chlorogenic acid is 77.3%.
In order to further verify the effect of the present invention, the following is further explained according to a plurality of comparative examples.
Comparative example 1
In step (1) as compared with example 1, the solid content of stevia extract was 8%, and the yield of high purity chlorogenic acid was 43g, wherein the chlorogenic acid content was 85% and the effective chlorogenic acid recovery was 53.3% as calculated in the same conditions as in example 1.
From the results of comparative example 1 and example 1, it can be seen that the purity and recovery rate of chlorogenic acid are greatly reduced due to the excessively high solid content, mainly because chromatographic crossover occurs due to the excessively high solid content, thereby affecting the separation effect. In order to ensure good separation effect of each component, the invention controls the solid content of stevia rebaudiana extracting solution to be 2-5%.
Comparative example 2
In step (2) the effluent of LX-8 resin was fed to LSA-900E resin at a rate of 5BV/h for adsorption treatment as compared with example 1, and the yield of high purity chlorogenic acid was 24g, calculated to be 80% chlorogenic acid and 28.0% chlorogenic acid recovery, as in example 1.
Comparative example 3
In the step (2), the T81 resin was used instead of the LSA-900E resin for adsorption treatment, and the other conditions were the same as in example 1, and the yield of high purity chlorogenic acid was calculated to be 10g, wherein the chlorogenic acid content was 67%, and the effective recovery rate of chlorogenic acid was 9.8%.
Comparative example 4
In the step (2), the D331 resin was used instead of the LSA-900E resin for adsorption treatment, and the other conditions were the same as in example 1, and the yield of high purity chlorogenic acid was calculated to be 16g, wherein the chlorogenic acid content was 72%, and the effective recovery rate of chlorogenic acid was 16.8%.
As can be seen from the results of comparative example 2 and example 1, when LSA-900E resin was subjected to adsorption treatment, the feeding speed was increased, and the yield and purity of the product were greatly reduced, mainly because the feeding speed was too high, which was detrimental to adsorption of the effective components in the feed liquid by the resin. Therefore, in order to ensure good separation effect, the feeding speed is controlled to be 1-2BV/h when LSA-900E resin is adopted for adsorption treatment.
From the results of comparative example 3, comparative example 4 and example 1, it can be seen that the type of resin is extremely critical to the separation effect in separating chlorogenic acids, and the yield and content of the product are greatly reduced when the adsorption treatment is performed using T81 resin and using D331 resin instead of LSA-900E resin. Therefore, in order to ensure high purity and high yield of the product, LSA-900E resin is selected to separate chlorogenic acid substances.
Comparative example 5
In step (3), the solid content of the solution A was 12% as compared with example 1, and the yield of high purity chlorogenic acid was 46g as calculated in the same manner as in example 1, wherein the chlorogenic acid content was 80.2% and the effective chlorogenic acid recovery was 53.8%.
As can be seen from the results of comparative example 5 and example 1, when the HZ-841 resin is used for enriching the chlorogenic acid, the solid content of the feed liquid has a certain effect on the enriching effect, and the solid content of the feed liquid is increased in comparative example 5 compared with example 1, but the purity and yield of the high purity chlorogenic acid of the target product are reduced, mainly because chromatographic crossover occurs when the solid content is too high, thereby reducing the separating effect. In order to ensure high purity and high yield of chlorogenic acid, the solid content of the solution A is controlled to be 5-10%.
Comparative example 6
In step (3), the feed rate at the time of adsorption with HZ-841 resin was 5BV/h, and the yield of high purity chlorogenic acid was 36.5g as calculated in example 1, wherein the chlorogenic acid content was 83% and the effective chlorogenic acid recovery was 44.2%, as compared with example 1.
Comparative example 7
In step (3) as compared with example 1, LX-48 resin was used instead of HZ-841 resin, and the other conditions were the same as in example 1, and it was tested that the yield of high purity chlorogenic acid was 33g, wherein the chlorogenic acid content was 81%, and the effective recovery rate of chlorogenic acid was 39.0%.
Comparative example 8
In step (3), the loading was 15% by volume of the resin, and the yield of high purity chlorogenic acid was 39.6g, wherein the chlorogenic acid content was 76% and the chlorogenic acid yield was 43.9%, as compared with example 1, under the same conditions as in example 1.
As can be seen from the results of comparative examples 6, 7, 8 and 1, when enriching the chlorogenic acid, the selection of the resin type, the feeding speed and the loading amount all have influence on the separation effect, and further influence the purity and the yield of the high-purity chlorogenic acid, and when the selection of the resin type is improper, the feeding speed is too high and the loading amount is too high, the purity and the yield of the chlorogenic acid are greatly reduced. In order to ensure high purity and high yield of chlorogenic acid, the application selects the resin type enriched with the cryptochlorogenic acid to be HZ-841 resin, the loading speed is 1-2BV/h, and the loading amount is 8-12% of the volume of the resin.
Comparative example 9
In step (4), the solvent was directly removed without adjusting the pH of the solution after the completion of the reflux reaction, as compared with example 1, and the yield of high purity chlorogenic acid was calculated to be 35g, wherein the chlorogenic acid content was 82.6%, and the effective chlorogenic acid recovery was 42.1%, as in example 1.
Compared with comparative example 9 and example 1, in example 1, the pH of the reaction solution was adjusted after the reflux reaction was completed, and the yield and purity of chlorogenic acid were significantly improved. This is mainly because if the reaction feed solution is directly concentrated without adjusting the pH of the reaction feed solution, the pH of the solution increases during concentration, which may affect the destruction of the product, thereby reducing the purity and yield of chlorogenic acid.
Comparative example 10
In step (5), the reaction conditions were normal pressure conditions, and the other conditions were the same as in example 1, and the yield of high purity chlorogenic acid was calculated to be 21g, wherein the chlorogenic acid content was 79%, and the effective chlorogenic acid recovery was 24.2%, as compared with example 1.
Comparative example 11
In step (5), the solvent was directly removed without adjusting the pH of the solution after the completion of the reaction, as compared with example 1, and the yield of high purity chlorogenic acid was 38g, wherein the chlorogenic acid content was 84% and the effective recovery rate of chlorogenic acid was 46.5%, as calculated in the same manner as in example 1.
As can be seen from the results of comparative examples 10, 11 and 1, the reaction conditions are extremely critical in the process of converting chlorogenic acid into chlorogenic acid, the conversion effect is not obvious, the purity and yield of chlorogenic acid are greatly reduced in comparative example 10, and the pH of the solution needs to be properly adjusted after the conversion is finished, mainly because the pH of the solution is increased during the concentration of the solution without adjusting the pH of the solution, and the product is damaged if the pH of the solution is too large, thereby influencing the recovery of the product.
Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
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