Method for jointly separating isochlorogenic acid A and isochlorogenic acid C based on extraction and chromatography

Abstract

The invention discloses a method for jointly separating isochlorogenic acid A and isochlorogenic acid C based on extraction and chromatography. The method comprises the following steps: preparing a stevia rebaudiana polyphenol solution, and adjusting the pH value of the stevia rebaudiana polyphenol solution to obtain a first treatment solution; extracting the first treating fluid by using an ethyl acetate saturated aqueous solution, collecting a raffinate phase and an extract phase, drying the raffinate phase, dissolving the raffinate phase in a methanol solution, and filtering to obtain a second treating fluid; separating the second treatment liquid in a chromatographic column; sequentially eluting by adopting a 92-94% v / v methanol solution, a 85-87% v / v methanol solution, a 78-80% v / v methanol solution and a 65-70% v / v methanol solution, and respectively collecting a first eluant, a second eluant, a third eluant and a fourth eluant; drying the second eluent, and sequentially crystallizing and recrystallizing to obtain high-purity isochlorogenic acid C; and drying the fourth eluent, and sequentially crystallizing and recrystallizing to obtain the high-purity isochlorogenic acid A. According to the method, high-content isochlorogenic acid A and isochlorogenic acid C are recovered from a stevia rebaudiana polyphenol product through extraction, chromatographic separation and crystallization, and the resource utilization rate is increased.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chlorogenic acid, and in particular to a method for separating iso-chlorogenic acid A and iso-chlorogenic acid C based on extraction and chromatographic separation. BACKGROUND

[0002] Stevia rebaudiana is rich in two types of high-value components, steviol glycosides and stevia polyphenols. However, the traditional process takes the extraction of steviol glycosides as the core target, and mostly uses water extraction or calcium / iron salt complexation to remove impurities, resulting in the polyphenol of stevia polyphenol. 2+ forming stable complexes. Because these complexes have poor water solubility, they precipitate out of solution, forming a precipitate. The polyphenol precipitate is discarded with the waste residue, resulting in a waste of resources.

[0003] Stevia polyphenols contain a certain amount of iso-chlorogenic acid A and iso-chlorogenic acid C, both of which are key intermediates for antiviral and anti-inflammatory drugs. The two are highly similar in structure, and the resolution of traditional chromatography is insufficient to effectively separate single polyphenols. Most of the obtained are mixed polyphenols, which cannot meet the needs of the medical and other fields. Therefore, how to extract high-purity iso-chlorogenic acid A and iso-chlorogenic acid C from stevia polyphenols is of great significance. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a method for separating iso-chlorogenic acid A and iso-chlorogenic acid C based on extraction and chromatography, which effectively adjusts the extraction, chromatographic separation, and crystallization conditions to recover high-content iso-chlorogenic acid A and iso-chlorogenic acid C from stevia rebaudiana polyphenol products, greatly improving resource utilization, and effectively utilizing waste liquid in the separation process, improving product recovery.

[0005] To solve the above technical problems, the technical solution of the present application is:

[0006] A method for separating iso-chlorogenic acid A and iso-chlorogenic acid C based on extraction and chromatography, comprising the following steps:

[0007] (1) preparing a stevia rebaudiana polyphenol solution and adjusting its pH to obtain a first treatment liquid;

[0008] (2) extracting the first treatment liquid with ethyl acetate saturated aqueous solution as the extractant for multiple times, collecting the raffinate phase, and combining the multiple extraction phases;

[0009] (3) the raffinate phase is dried, dissolved in a methanol solution, and filtered to obtain a second treatment liquid; the second treatment liquid is pumped into a chromatographic column for separation; after the feeding is completed, a 92-94% v / v methanol solution, a 85-87% v / v methanol solution, a 78-80% v / v methanol solution, and a 65-70% v / v methanol solution are used as a first eluent, a second eluent, a third eluent, and a fourth eluent, respectively, for elution, and a first eluate, a second eluate, a third eluate, and a fourth eluate are collected in sequence;

[0010] (4) the second eluate is dried, dissolved in purified water to obtain a third treatment liquid, the pH of the third treatment liquid is adjusted, and crystallization is performed under stirring, followed by filtration, collection of a first filter cake and a first filtrate, mixing of the first filter cake and purified water, heating and stirring until the solids are dissolved to obtain a fourth treatment liquid, crystallization of the fourth treatment liquid after cooling, and filtration to collect a second filter cake and a second filtrate; the second filter cake is dried to obtain high-purity iso-chlorogenic acid C;

[0011] (5) the fourth eluate is dried, dissolved in an ethyl acetate saturated aqueous solution to obtain a fifth treatment liquid, crystallization is performed under stirring to obtain a third filter cake and a third filtrate, the third filter cake is mixed with purified water to obtain a sixth treatment liquid, the sixth treatment liquid is subjected to beating treatment, and filtration is performed to obtain a fourth filter cake and a fourth filtrate; the fourth filter cake is dried to obtain high-purity iso-chlorogenic acid A.

[0012] Preferably, in step (1), when the stevia polyphenol solution is prepared, the mass ratio of the stevia polyphenol product to purified water is 1:3-8; and the pH is adjusted to 4.5-5.0.

[0013] Preferably, in step (2), when the extraction is performed, the volume ratio of the extractant to the first treatment liquid is (1-3):1, and the extraction is performed 2-4 times.

[0014] Preferably, in step (3), the concentration of the methanol solution is 95-98% v / v, and when the second treatment liquid is prepared, the mass ratio of the solids to the methanol solution is 1:(5-10).

[0015] Preferably, the chromatographic column is a forward silica gel filler amino column, the filler particle size is 30-50 μm, and the pore size type is: When the feeding is performed, 0.8-1.0 g of the raffinate phase solids is fed per 100 ml of the chromatographic filler, and the feeding flow rate is 0.5-1.0 BV / h based on the volume of the filler.

[0016] Preferably, in step (3), when the first eluent is used for elution, the amount of the first eluent is 1.5-2.0 BV based on the volume of the chromatographic filler, and the feeding flow rate is 1.0-1.5 BV / h based on the volume of the filler.

[0017] Preferably, in step (3), when eluted with the second eluent, the amount of the second eluent is 1.3-1.8 BV of the volume of the chromatographic packing, and the flow rate is 0.5-1 BV / h of the volume of the packing.

[0018] Preferably, in step (3), when eluted with the third eluent, the amount of the third eluent is 1.5-2.0 BV of the volume of the chromatographic packing, and the flow rate is 0.5-1 BV / h of the volume of the packing.

[0019] Preferably, in step (3), when eluted with the fourth eluent, the amount of the fourth eluent is 2.0-2.5 BV of the volume of the chromatographic packing, and the flow rate is 1.0-2.0 BV / h of the volume of the packing.

[0020] Preferably, the first eluent and the fourth eluent are combined into the extract phase of step (2) and dried to obtain steviol polyphenols.

[0021] Preferably, in step (4), when preparing the third treatment liquid, the mass ratio of the solid to purified water is 1:(2.0-3.5), the pH is adjusted to 3.0-3.5, and the stirring crystallization time is 6-10 h.

[0022] Preferably, in step (4), when preparing the fourth treatment liquid, the mass ratio of the first filter cake to purified water is 1:(3-5), the heating temperature is 70-80℃, the temperature for cooling crystallization is 20-30℃, and the time is 4-6 h.

[0023] Preferably, in step (5), when preparing the fifth treatment liquid, the mass ratio of the solid to ethyl acetate saturated aqueous solution is 1:(2.5-3.5), the crystallization temperature is room temperature, and the time is 4-8 h.

[0024] Preferably, in step (5), when preparing the sixth treatment liquid, the mass ratio of the third filter cake to purified water is 1:(1.5-2.0), the beating temperature is 40-45℃, and the time is 1-3 h.

[0025] Preferably, the first filtrate and the third filtrate are combined into the raffinate phase of step (2), the second filtrate is combined into the second eluent, and the fourth filtrate is combined into the third eluent.

[0026] With the technical scheme, the beneficial effects of the present application are as follows: the present application uses stevia polyphenol product as raw material, and adopts extraction and chromatography to separate and prepare iso-chlorogenic acid A and iso-chlorogenic acid C. Specifically, the present application first uses ethyl acetate saturated aqueous solution as an extractant to extract the first treatment liquid with a specific pH, and iso-chlorogenic acid A and iso-chlorogenic acid C are enriched in the raffinate phase due to their high solubility under the specific pH condition; then, chromatography is used to separate iso-chlorogenic acid A and iso-chlorogenic acid C in the raffinate phase, and during chromatography, normal phase silica gel bonded amino filler is used, and different concentrations of methanol solution are used for gradient elution, and the difference in polarity between iso-chlorogenic acid A and iso-chlorogenic acid C is used to realize efficient separation. Moreover, the present application performs extraction before chromatography, and removes part of the strong lipophilic impurities in stevia polyphenol in advance, reduces the burden of chromatography, improves the efficiency of chromatography, and also reduces the amount of solvent used during chromatography.

[0027] The filtrate generated in the partial crystallization process of the present application is recovered and combined with the raffinate phase, and the target product that fails to crystallize or soluble impurities are separated from the raffinate phase of the next batch, so that iso-chlorogenic acid A and iso-chlorogenic acid C are fully separated and the total yield is improved. The filtrate generated in the partial crystallization process of the present application is used for the crystallization process of the next batch, which realizes the recycling of the mother liquor, improves the material utilization efficiency and the total recovery rate of the target product. DETAILED DESCRIPTION

[0028] In order to more clearly understand the above objectives, features and advantages of the present application, the solutions of the present application will be further described below. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0029] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein; obviously, the embodiments in the description are only some embodiments of the present application, not all embodiments.

[0030] In order to further understand the present application, the preferred embodiments of the present application will be described below in combination with the embodiments, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present application, and are not limitations on the claims of the present application.

[0031] The stevia polyphenol product of the present application is a by-product in the process of extracting stevia glycosides from stevia, and the specific process is as follows:

[0032] Stevia is extracted with 20-60% v / v ethanol (the mass ratio of stevia to ethanol is 1:10-15) at 25-35℃ for four times, the multiple extraction liquids are combined and filtered, and then de-ethanolized with a 200-400 Da nanofiltration membrane, the cut-off liquid is adjusted to pH 3-4, and the treated liquid is obtained;

[0033] The liquid to be treated is saturated and adsorbed by a polyamide resin, the ratio of the volume of the resin to the weight of the stevia rebaudiana is 10 ml:(4-7) g, the flow rate of the liquid to be treated is 1-2 BV / h of the volume of the resin, after the feeding is completed, 1.5-3.5 BV of purified water of the volume of the resin is used for elution, the flow rate of the liquid to be treated is controlled to be 1-2 BV / h of the volume of the resin, after the elution of the water is completed, 20-30% v / v ethanol of 1.5-2.5 BV of the volume of the resin is used for impurity removal, the flow rate of the liquid to be treated is 1-2 BV / h of the volume of the resin, then 70-85% v / v ethanol of 2.0-4.0 BV of the volume of the resin is used for elution, the flow rate of the liquid to be treated is 1-3 BV / h of the volume of the resin, and the elution liquid is dried to obtain the stevia rebaudiana polyphenol product.

[0034] The total content of chlorogenic acid and its isomers in the stevia rebaudiana polyphenol product of the present application is 40-65 wt%, the content of iso-chlorogenic acid A is 25-35 wt%, and the content of iso-chlorogenic acid C is 10-15 wt%.

[0035] The preparation method of the stevia rebaudiana polyphenol product in the following examples and comparative examples is as follows:

[0036] 5 kg of stevia rebaudiana is extracted four times with 60 L of 40% v / v ethanol, the combined extract is filtered to obtain a filtered clear liquid, the filtered clear liquid is de-ethanized by a 400 Da nanofiltration membrane, the cut-off liquid is adjusted to a pH of 3.5, and is pumped into 10 L of polyamide resin for adsorption, the flow rate of the liquid to be treated is 1.5 BV / h of the volume of the resin; 2 BV of purified water of the volume of the resin is used for elution, the flow rate of the liquid to be treated is 1.5 BV / h of the volume of the resin; then 2 BV of 25% v / v ethanol of the volume of the resin is used for impurity removal, the flow rate of the liquid to be treated is 1.5 BV / h of the volume of the resin; 3 BV of 80% v / v ethanol of the volume of the resin is used for elution, the flow rate of the liquid to be treated is 3 BV / h of the volume of the resin, and the 80% ethanol elution liquid is dried to obtain the stevia rebaudiana polyphenol product.

[0037] The chromatographic column in the following examples and comparative examples is a forward silica gel filler amino column of Micro Pure Biotechnology (Guangzhou) Co., Ltd. Series, the particle size of the chromatographic filler is 30-50 μm, and the pore size type is:

[0038] Example 1

[0039] A method for separating iso-chlorogenic acid A and iso-chlorogenic acid C based on extraction and chromatography, comprising the following steps:

[0040] (1) 0.1 kg of stevia rebaudiana polyphenol product (the total content of chlorogenic acid and its isomers is 45.8 wt%, the content of iso-chlorogenic acid A is 25.8 wt%, and the content of iso-chlorogenic acid C is 10.8 wt%) and 0.3 kg of purified water are mixed, stirred until the solid is dissolved, and the pH of the solution is adjusted to 4.5 to obtain a first treatment liquid;

[0041] (2) The first treated solution of step (1) is extracted with ethyl acetate saturated aqueous solution, the volume ratio of ethyl acetate saturated aqueous solution to the first treated solution is controlled to be 1:1, extraction is carried out twice, the raffinate is collected, and the extraction phases in multiple extractions are combined;

[0042] (3) The raffinate of step (2) is dried and dissolved in a 95% v / v methanol solution (the mass ratio of solid to methanol solution is 1:5) to obtain a second treated solution; the second treated solution is pumped into a chromatographic column (the particle size of the chromatographic packing is 30 μm, and the pore size is: ) for separation; 0.8 g of solid in the raffinate is fed per 100 ml of chromatographic packing, and the feed flow rate is 0.5 BV / h of the packing volume; after the feeding is completed, first, a 92% v / v methanol solution is used as a first eluent (the amount is 1.5 BV of the packing volume, and the flow rate is 1.0 BV / h of the packing volume); the first eluent is collected, a 85% v / v methanol solution is used as a second eluent (the amount is 1.3 BV of the packing volume, and the flow rate is 1.0 BV / h of the packing volume); the second eluent is collected, a 78% v / v methanol solution is used as a third eluent (the amount is 1.5 BV of the packing volume, and the flow rate is 1.0 BV / h of the packing volume); the third eluent is collected, and finally, a 65% v / v methanol solution is used as a fourth eluent (the amount is 2.0 BV of the packing volume, and the flow rate is 1.0 BV / h of the packing volume); the fourth eluent is collected;

[0043] (4) The first eluent and the fourth eluent of step (3) are combined into the extraction phase of step (2) and dried to obtain stevia polyphenols;

[0044] (5) The second eluent of step (3) is dried and dissolved in purified water, the mass ratio of solid to purified water is controlled to be 1:2.0 to obtain a third treated solution, the pH of the third treated solution is adjusted to 3.0, and the solution is stirred to crystallize for 6 h; after crystallization, the first filter cake and the first filtrate are collected, the first filtrate is combined into the raffinate of step (2), the first filter cake is mixed with purified water (the mass ratio of the first filter cake to purified water is 1:3), and the mixture is heated to 70°C and stirred until the solid is dissolved to obtain a fourth treated solution; the fourth treated solution is cooled to 20°C, and the solution is stirred to crystallize for 4 h; after crystallization, the second filter cake and the second filtrate are collected, the second filtrate is combined into the second eluent of the next batch, and the second filter cake is dried to obtain high-purity iso-chlorogenic acid C;

[0045] (6) The fourth eluent of step (3) was dissolved in ethyl acetate saturated aqueous solution (the mass ratio of solid to ethyl acetate saturated aqueous solution was 1:2.5) after drying, to obtain a fifth treatment liquid. After stirring at room temperature for 4 h, the fifth treatment liquid was filtered to obtain a third filter cake and a third filtrate. The third filtrate was combined with the raffinate of step (2) to obtain a fourth treatment liquid. The fourth treatment liquid was treated by beating at 40°C for 1 h, and then filtered to obtain a fourth filter cake and a fourth filtrate. The fourth filtrate was combined with the third eluent of the next batch, and the fourth filter cake was dried to obtain high-purity iso-chlorogenic acid A.

[0046] Example 2

[0047] A method for separating iso-chlorogenic acid A and iso-chlorogenic acid C based on extraction and chromatography, comprising the following steps:

[0048] (1) 0.1 kg of stevia polyphenol product (total content of chlorogenic acid and its isomers was 45.8 wt%, content of iso-chlorogenic acid A was 25.8 wt%, and content of iso-chlorogenic acid C was 10.8 wt%) and 0.8 kg of purified water were mixed and stirred until the solid was dissolved. The pH of the solution was adjusted to 5.0 to obtain a first treatment liquid;

[0049] (2) The first treatment liquid of step (1) was extracted with ethyl acetate saturated aqueous solution as an extractant. The volume ratio of ethyl acetate saturated aqueous solution to the first treatment liquid was controlled to be 3:1, and the extraction was performed for 4 times. The raffinate was collected, and the extraction phases of multiple extractions were combined;

[0050] (3) The raffinate of step (2) was dried and then dissolved in a 98% v / v methanol solution (the mass ratio of solid to methanol solution was 1:10) to obtain a second treatment liquid. The second treatment liquid was pumped into a chromatography column (the particle size of the chromatography filler was 50 μm, and the pore size was: ) for separation. During feeding, 1.0 g of solid raffinate was fed per 100 ml of chromatography filler, and the feeding flow rate was 1.0 BV / h of the volume of the filler. After feeding was completed, 94% v / v methanol solution was used as the first eluent (the amount was 2.0 BV of the volume of the chromatography filler, and the flow rate was 1.5 BV / h of the volume of the filler). The first eluent was collected. Then, 87% v / v methanol solution was used as the second eluent (the amount was 1.8 BV of the volume of the chromatography filler, and the flow rate was 0.5 BV / h of the volume of the filler). The second eluent was collected. Then, 80% v / v methanol solution was used as the third eluent (the amount was 2.0 BV of the volume of the chromatography filler, and the flow rate was 0.5 BV / h of the volume of the filler). The third eluent was collected. Finally, 70% v / v methanol solution was used as the fourth eluent (the amount was 2.5 BV of the volume of the chromatography filler, and the flow rate was 2.0 BV / h of the volume of the filler). The fourth eluent was collected.

[0051] (4) The first eluent and the fourth eluent of step (3) are combined into the extraction phase of step (2) and dried to obtain steviol polyphenols;

[0052] (5) The second eluent of step (3) is dissolved in purified water after drying, and the mass ratio of solid to purified water is controlled to be 1:3.5 to obtain a third treatment liquid. The pH of the third treatment liquid is adjusted to 3.5, and the crystallization is stirred for 10 hours. After the crystallization is completed, filtration is performed, and the first filter cake and the first filtrate are collected. The first filtrate is combined into the raffinate phase of step (2). The first filter cake and purified water are mixed (the mass ratio of the first filter cake to purified water is 1:5), and then heated to 80°C and stirred until the solid is dissolved to obtain a fourth treatment liquid. The fourth treatment liquid is cooled to 30°C, and after crystallization for 6 hours, filtration is performed to collect the second filter cake and the second filtrate. The second filtrate is combined into the second eluent of the next batch, and the second filter cake is dried to obtain high-purity iso-chlorogenic acid C;

[0053] (6) The fourth eluent of step (3) is dissolved in ethyl acetate saturated aqueous solution (the mass ratio of solid to ethyl acetate saturated aqueous solution is 1:3.5) after drying to obtain a fifth treatment liquid. After stirring at room temperature for 8 hours, filtration is performed to obtain a third filter cake and a third filtrate. The third filtrate is combined into the raffinate phase of step (2). The third filter cake and purified water (the mass ratio of the third filter cake to purified water is 1:2.0) are mixed to obtain a sixth treatment liquid. The sixth treatment liquid is subjected to beating treatment at 45°C for 3 hours, and filtration is performed to obtain a fourth filter cake and a fourth filtrate. The fourth filtrate is combined into the third eluent of the next batch, and the fourth filter cake is dried to obtain high-purity iso-chlorogenic acid A.

[0054] Example 3

[0055] A method for separating iso-chlorogenic acid A and iso-chlorogenic acid C based on the combination of extraction and chromatography, comprising the following steps:

[0056] (1) 0.1 kg of stevia polyphenol product (total content of chlorogenic acid and its isomers is 45.8 wt%, content of iso-chlorogenic acid A is 25.8 wt%, and content of iso-chlorogenic acid C is 10.8 wt%) and 0.6 kg of purified water are mixed, and the solid is stirred to dissolve. The pH of the solution is adjusted to 4.8 to obtain a first treatment liquid;

[0057] (2) The first treatment liquid of step (1) is extracted with ethyl acetate saturated aqueous solution as an extractant. The volume ratio of ethyl acetate saturated aqueous solution to the first treatment liquid is controlled to be 2:1, and extraction is performed for 3 times. The raffinate phase is collected, and the extraction phases in multiple extractions are combined;

[0058] (3) The raffinate phase of step (2) is dried and dissolved in a 97% v / v methanol solution (the mass ratio of solid to methanol solution is 1:8) and filtered to obtain a second treatment liquid. The second treatment liquid is pumped into a chromatographic column (the particle size of the chromatographic packing is 40 μm, and the pore size is: ) separation, the feed of 0.9g of the solid in the raffinate phase per 100ml of the chromatographic packing, the feed flow rate of 0.8BV / h of the volume of the packing; after the end of the feed, first elution is carried out using 93% v / v methanol solution as the first eluent (the amount of 1.8BV of the volume of the chromatographic packing, the flow rate of 1.2BV / h of the volume of the packing), the first eluate is collected, second elution is carried out using 86% v / v methanol solution as the second eluent (the amount of 1.6BV of the volume of the chromatographic packing, the flow rate of 0.6BV / h of the volume of the packing), the second eluate is collected, third elution is carried out using 79% v / v methanol solution as the third eluent (the amount of 1.8BV of the volume of the chromatographic packing, the flow rate of 0.6BV / h of the volume of the packing), the third eluate is collected, and finally fourth elution is carried out using 68% v / v methanol solution as the fourth eluent (the amount of 2.4BV of the volume of the chromatographic packing, the flow rate of 1.5BV / h of the volume of the packing), the fourth eluate is collected;

[0059] (4) the first eluate and the fourth eluate of step (3) are combined into the extract phase of step (2) and dried to obtain steviol polyphenols;

[0060] (5) the second eluate of step (3) is dried and dissolved in purified water, the mass ratio of the solid to the purified water is controlled to be 1:3.0 to obtain a third treatment liquid, the pH of the third treatment liquid is adjusted to 3.2, and the third treatment liquid is stirred to crystallize for 8h, after the crystallization is completed, filtration is carried out, the first filter cake and the first filtrate are collected, the first filtrate is combined into the raffinate phase of step (2), the first filter cake is mixed with purified water (the mass ratio of the first filter cake to the purified water is 1:4), and the mixture is heated to 75℃ and stirred until the solid is dissolved to obtain a fourth treatment liquid, the fourth treatment liquid is cooled to 25℃, and after crystallization for 5h, filtration is carried out, the second filter cake and the second filtrate are collected, the second filtrate is combined into the second eluate of the next batch, and the second filter cake is dried to obtain high-purity iso- chlorogenic acid C;

[0061] (6) the fourth eluate of step (3) is dried and dissolved in ethyl acetate saturated aqueous solution (the mass ratio of the solid to the ethyl acetate saturated aqueous solution is 1:3.3) to obtain a fifth treatment liquid, the fifth treatment liquid is stirred to crystallize at room temperature for 6h, and then filtration is carried out to obtain a third filter cake and a third filtrate, the third filtrate is combined into the raffinate phase of step (2), the third filter cake is mixed with purified water (the mass ratio of the third filter cake to the purified water is 1:1.8) to obtain a sixth treatment liquid, the sixth treatment liquid is subjected to beating treatment at 42℃ for 2h, and then filtration is carried out to obtain a fourth filter cake and a fourth filtrate, the fourth filtrate is combined into the third eluate of the next batch, and the fourth filter cake is dried to obtain high-purity iso- chlorogenic acid A.

[0062] Example 4

[0063] A method for separating iso-chlorogenic acid A and iso-chlorogenic acid C based on the combination of extraction and chromatography, comprising the following steps:

[0064] (1) 0.1 kg of stevia polyphenol product (total content of chlorogenic acid and its isomers is 45.8 wt%, content of isochlorogenic acid A is 25.8 wt%, and content of isochlorogenic acid C is 10.8 wt%) and 0.5 kg of purified water are mixed, stirred until the solids are dissolved, and the pH of the solution is adjusted to 4.6 to obtain a first treatment solution;

[0065] (2) The first treatment solution of step (1) is extracted with ethyl acetate saturated aqueous solution as the extractant, the volume ratio of ethyl acetate saturated aqueous solution to the first treatment solution is controlled to be 2:1, extraction is performed for 3 times, the raffinate phase is collected, and the extraction phases in multiple extractions are combined;

[0066] (3) The raffinate phase of step (2) is dried and dissolved in a 96% v / v methanol solution (mass ratio of solid to methanol solution is 1:7) and filtered to obtain a second treatment solution; the second treatment solution is pumped into a chromatographic column (chromatographic packing particle size is 40 μm, and pore size is: ) for separation, 0.9 g of solid in the raffinate phase per 100 ml of chromatographic packing is fed, and the feeding flow rate is 0.7 BV / h of the volume of the packing; after feeding is completed, 93% v / v methanol solution is first used as the first eluent (the amount is 1.6 BV of the volume of the chromatographic packing, and the feeding flow rate is 1.5 BV / h of the volume of the packing), elution is performed, the first eluent is collected, 86% v / v methanol solution is used as the second eluent (the amount is 1.5 BV of the volume of the chromatographic packing, and the feeding flow rate is 0.8 BV / h of the volume of the packing), elution is performed, the second eluent is collected, 79% v / v methanol solution is used as the third eluent (the amount is 1.7 BV of the volume of the chromatographic packing, and the feeding flow rate is 0.8 BV / h of the volume of the packing), elution is performed, the third eluent is collected, and finally 66% v / v methanol solution is used as the fourth eluent (the amount is 2.2 BV of the volume of the chromatographic packing, and the feeding flow rate is 1.5 BV / h of the volume of the packing), elution is performed, and the fourth eluent is collected;

[0067] (4) The first eluent and the fourth eluent of step (3) are combined into the extraction phase of step (2) and dried to obtain stevia polyphenol;

[0068] (5) The second eluent of step (3) is dried and dissolved in purified water, the mass ratio of solid to purified water is controlled to be 1:2.5, to obtain a third treatment liquid, the pH of the third treatment liquid is adjusted to 3.3, and the third treatment liquid is stirred to crystallize for 8 hours. After crystallization, filtration is performed, a first filter cake and a first filtrate are collected, the first filtrate is combined with the raffinate of step (2), and the first filter cake is mixed with purified water (the mass ratio of the first filter cake to purified water is 1:4) and heated to 76°C and stirred until the solid is dissolved to obtain a fourth treatment liquid. The fourth treatment liquid is cooled to 27°C, and after crystallization for 5 hours, filtration is performed to collect a second filter cake and a second filtrate. The second filtrate is combined with the second eluent of the next batch, and the second filter cake is dried to obtain high-purity isochlorogenic acid C;

[0069] (6) The fourth eluent of step (3) is dried and dissolved in ethyl acetate saturated aqueous solution (the mass ratio of solid to ethyl acetate saturated aqueous solution is 1:2.8) to obtain a fifth treatment liquid. After stirring at room temperature for 5 hours, filtration is performed to obtain a third filter cake and a third filtrate. The third filtrate is combined with the raffinate of step (2), and the third filter cake is mixed with purified water (the mass ratio of the third filter cake to purified water is 1:1.7) to obtain a sixth treatment liquid. The sixth treatment liquid is subjected to beating treatment at 43°C for 2 hours, and filtration is performed to obtain a fourth filter cake and a fourth filtrate. The fourth filtrate is combined with the third eluent of the next batch, and the fourth filter cake is dried to obtain high-purity isochlorogenic acid A.

[0070] Comparative Example 1

[0071] The difference between this comparative example and Example 4 is that in step (1), the amount of purified water is 0.2 kg, and the other operations are the same as those in Example 4.

[0072] Comparative Example 2

[0073] The difference between this comparative example and Example 4 is that in step (1), the pH of the solution is 4.0, and the other operations are the same as those in Example 4.

[0074] Comparative Example 3

[0075] The difference between this comparative example and Example 4 is that in step (1), the pH of the solution is 5.5, and the other operations are the same as those in Example 4.

[0076] Comparative Example 4

[0077] The difference between this comparative example and Example 4 is that in step (3), when preparing the second treatment liquid, the concentration of the methanol solution is 90% v / v, and the other operations are the same as those in Example 4.

[0078] Comparative Example 5

[0079] The difference between this comparative example and Example 4 is that in step (3), a normal phase silica gel bonded amino filler with a pore size of 20-40 μm is used, and the other operations are the same as those in Example 4. The other operations are the same as those in Example 4.

[0080] Comparative Example 6

[0081] This comparative example differs from Example 4 in that in step (3), normal phase silica bonded cyano packing material is used instead of normal phase silica bonded amino packing material, and otherwise the procedure is the same as Example 4.

[0082] Comparative Example 7

[0083] This comparative example differs from Example 4 in that in step (3), the first eluent is a methanol solution at a concentration of 98% v / v, and otherwise the procedure is the same as Example 4.

[0084] Comparative Example 8

[0085] This comparative example differs from Example 4 in that in step (3), the first eluent is a methanol solution at a concentration of 90% v / v, and otherwise the procedure is the same as Example 4.

[0086] Comparative Example 9

[0087] This comparative example differs from Example 4 in that in step (3), the second eluent is a methanol solution at a concentration of 83% v / v, and otherwise the procedure is the same as Example 4.

[0088] Comparative Example 10

[0089] This comparative example differs from Example 4 in that in step (3), the second eluent is a methanol solution at a concentration of 88.5% v / v, and otherwise the procedure is the same as Example 4.

[0090] Comparative Example 11

[0091] This comparative example differs from Example 4 in that in step (3), the third eluent is a methanol solution at a concentration of 75% v / v, and otherwise the procedure is the same as Example 4.

[0092] Comparative Example 12

[0093] This comparative example differs from Example 4 in that in step (3), the third eluent is a methanol solution at a concentration of 82% v / v, and otherwise the procedure is the same as Example 4.

[0094] Comparative Example 13

[0095] This comparative example differs from Example 4 in that in step (5), the third treatment solution has a pH of 2.5, and otherwise the procedure is the same as Example 4.

[0096] Comparative Example 14

[0097] The comparative example differs from Example 4 in that in step (5), the pH of the third treatment solution is 4.0, and the other operations are the same as in Example 4.

[0098] Comparative Example 15

[0099] The comparative example differs from Example 4 in that in step (6), the temperature of the beating treatment is 30°C, and the other operations are the same as in Example 4.

[0100] Comparative Example 16

[0101] The comparative example differs from Example 4 in that in step (6), the temperature of the beating treatment is 50°C, and the other operations are the same as in Example 4.

[0102] The yields, the purities, and the purities of the iso-chlorogenic acid A and the iso-chlorogenic acid C produced in the above examples and comparative examples are shown in Table 1.

[0103] The yield of iso-chlorogenic acid A (%) = [(iso-chlorogenic acid A yield x iso-chlorogenic acid A purity) / (sweet stevia polyphenol product mass x iso-chlorogenic acid A content in the sweet stevia polyphenol product)] x 100%.

[0104] The yield of iso-chlorogenic acid C (%) = [(iso-chlorogenic acid C yield x iso-chlorogenic acid C purity) / (sweet stevia polyphenol product mass x iso-chlorogenic acid C content in the sweet stevia polyphenol product)] x 100%.

[0105] Table 1

[0106]

[0107]

[0108] As can be seen from the above test results, the iso-chlorogenic acid A and the iso-chlorogenic acid C are effectively separated from the sweet stevia polyphenol based on the extraction-chromatographic separation-crystallization method according to the present application, and by reasonably optimizing the conditions, the purity of the iso-chlorogenic acid A and the iso-chlorogenic acid C reaches more than 98%, and the yield is high.

[0109] In Comparative Examples 1-3, the solid-liquid ratio of the first treatment solution is too high, the pH is too low or too high, and the purity and yield of iso-chlorogenic acid A and iso-chlorogenic acid C are all decreased. When the solid-liquid ratio of the first treatment solution is too high, the stevia polyphenols cannot be fully dispersed in the purified water, the selectivity is poor during the subsequent ethyl acetate extraction, and part of the isomers with similar polarity cannot be effectively separated, so part of the iso-chlorogenic acid A and iso-chlorogenic acid C enters the extraction phase, resulting in a decrease in the effective components in the raffinate phase and a change in the components, which interferes with the chromatographic separation. When the pH of the first treatment solution is too low, the phenolic hydroxyl groups in the iso-chlorogenic acid A and iso-chlorogenic acid C are inhibited from ionization, and show stronger liposolubility, so part of them are extracted into the extraction phase by the saturated ethyl acetate solution, which reduces the product yield. When the pH is too high, the polarity of the solute molecules in the first treatment solution is increased, the target product cannot be effectively enriched during the extraction stage, and the hydrogen bond adsorption force of the amino filler is weakened during the chromatographic separation stage, so that the impurities and the target product are eluted together.

[0110] The pore size of the chromatographic packing in Comparative Example 5 is too large, and part of the macromolecular impurities also enter the pore channel while the chromatographic packing adsorbs the small molecular target product, which weakens the selective adsorption of the chromatographic packing to the target product. In Comparative Example 6, the cyano packing is used instead of the amino packing, and the cyano packing has low polarity and insufficient adsorption force to the effective product, so that the yield of the target product is reduced. In the chromatographic separation, the separation of isochlorogenic acid A and isochlorogenic acid C depends on the polarity difference in the gradient elution. The first eluent is a high-concentration methanol solution used for eluting weakly polar impurities, which facilitates the subsequent elution of isochlorogenic acid A and isochlorogenic acid C. The second eluent is used for eluting isochlorogenic acid C, and the third eluent is used for eluting isochlorogenic acid A. In Comparative Example 7, the concentration of the first eluent is too high, the elution strength is insufficient, part of the weakly polar impurities such as polysaccharides and polyphenol polymers cannot be effectively removed, the impurities occupy the active sites of the chromatographic packing, and the hydrogen bond interaction between isochlorogenic acid A and isochlorogenic acid C and the packing is hindered, which leads to overlapping elution and makes it difficult to separate isochlorogenic acid A and isochlorogenic acid C. In Comparative Example 8, the concentration of the first eluent is too low, and the elution strength is too large, so that part of the isochlorogenic acid C is eluted too early. In Comparative Example 9, the concentration of the second eluent is too low, so that the polarity difference between isochlorogenic acid A and isochlorogenic acid C is small, and it is difficult to effectively separate them in the gradient elution. In Comparative Example 10, the concentration of the second eluent is too high, and isochlorogenic acid C cannot be effectively resolved in the chromatographic column, part of which remains in the chromatographic column and is eluted by the third eluent, which leads to a reduced yield of isochlorogenic acid C and a significant decrease in the purity of isochlorogenic acid A. In Comparative Example 11, the concentration of the third eluent is too low, and the strongly polar polyphenol impurities remaining in the chromatographic column are eluted, causing cross contamination and a decrease in the purity of isochlorogenic acid A. In Comparative Example 12, the concentration of the third eluent is too high, and isochlorogenic acid A cannot be effectively resolved, which leads to a significant decrease in the yield of isochlorogenic acid A. In order to purify isochlorogenic acid C, the present application performs preliminary crystallization on the effluent during the elution of the second eluent, and the crystallinity of isochlorogenic acid C in the solution is adjusted by effectively controlling the pH of the solution during crystallization. In Comparative Example 13, the pH of the third treatment liquid is too low, and the phenolic hydroxyl groups in the chlorogenic acid isomers and other polyphenols are inhibited from dissolving, which leads to a significant decrease in the solubility of the impurities in water, and part of the impurities are precipitated during crystallization, which leads to a decrease in the purity of isochlorogenic acid C. In Comparative Example 14, the pH of the third treatment liquid is too high, part of the isochlorogenic acid molecules are dissociated and have a stronger interaction with water molecules, which leads to a significant increase in the solubility of the molecules in water, and part of the effective product cannot be completely precipitated during crystallization, which leads to a decrease in the yield of isochlorogenic acid C.

[0111] In order to purify isochlorogenic acid A, the present application mixes the crude isochlorogenic acid A obtained by preliminary purification and crystallization with purified water, and performs beating treatment at a certain temperature. In Comparative Example 15, the beating temperature is too low, and part of the impurities cannot be completely dissolved in the purified water, which leads to a decrease in the purity of isochlorogenic acid A. In Comparative Example 16, the beating treatment temperature is too high, and the solubility of isochlorogenic acid A in the purified water increases, which leads to a loss of part of isochlorogenic acid A and a decrease in the yield.

[0112] The principles and implementations of the present application are illustrated and described herein with reference to specific examples. The examples described above are presented merely to facilitate understanding of the methods of the present application and its core ideas, including the best mode, and also to enable any person skilled in the art to practice the present application, including manufacturing and using any device or system, and implementing any combined method. It should be noted that, for those skilled in the art, certain improvements and modifications can be made to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application. The scope of patent protection of the present application is defined by the claims, and can include other embodiments that can be conceived by those skilled in the art. If these other embodiments have structural elements similar to the literal expressions of the claims, or if they include equivalent structural elements that are not substantially different from the literal expressions of the claims, then these other embodiments should also be included in the scope of the claims.

Claims

1. A method for separating isochlorogenic acid A and isochlorogenic acid C based on a combination of extraction and chromatography, characterized in that, Includes the following steps: (1) Prepare a stevia polyphenol solution and adjust its pH to obtain the first treatment solution; (2) The first treatment solution was extracted multiple times using saturated aqueous ethyl acetate as the extractant, the raffinate phase was collected, and the multiple extract phases were combined. (3) After drying the raffinate, dissolve it in methanol solution and filter to obtain the second treatment solution; pump the second treatment solution into the chromatographic column for separation; after the feed is completed, use 92-94% v / v methanol solution, 85-87% v / v methanol solution, 78-80% v / v methanol solution, and 65-70% v / v methanol solution as the first eluent, second eluent, third eluent and fourth eluent in sequence for elution, and collect the first eluent, second eluent, third eluent and fourth eluent in sequence; (4) After the second eluent is dried, it is dissolved in purified water to obtain the third treatment solution. The pH of the third treatment solution is adjusted and stirred to crystallize. Then it is filtered, and the first filter cake and the first filtrate are collected. The first filter cake and purified water are mixed and heated and stirred until the solid dissolves to obtain the fourth treatment solution. The fourth treatment solution is cooled and crystallized, and then filtered to collect the second filter cake and the second filtrate. The second filter cake is dried to obtain high-purity isochlorogenic acid C. (5) After the fourth eluent is dried, it is dissolved in a saturated aqueous solution of ethyl acetate to obtain the fifth treatment solution. After stirring and crystallizing, it is filtered to obtain the third filter cake and the third filtrate. The third filter cake is mixed with purified water to obtain the sixth treatment solution. The sixth treatment solution is pulped and filtered to obtain the fourth filter cake and the fourth filtrate. The fourth filter cake is dried to obtain high-purity isochlorogenic acid A.

2. The method for separating isochlorogenic acid A and isochlorogenic acid C based on extraction and chromatography according to claim 1, characterized in that, In step (1), when preparing the stevia polyphenol solution, the mass ratio of stevia polyphenol product to purified water is 1:3-8; and its pH is adjusted to 4.5-5.

0.

3. The method for separating isochlorogenic acid A and isochlorogenic acid C based on extraction and chromatography according to claim 1, characterized in that, In step (2), during extraction, the volume ratio of the extractant to the first treatment liquid is (1-3):1, and the number of extractions is 2-4.

4. The method for separating isochlorogenic acid A and isochlorogenic acid C based on extraction and chromatography according to claim 1, characterized in that, In step (3), the concentration of the methanol solution is 95-98% v / v, and the mass ratio of the solid to the methanol solution is 1:(5-10) when preparing the second treatment solution.

5. The method for separating isochlorogenic acid A and isochlorogenic acid C based on extraction and chromatography according to claim 1, characterized in that, In step (3), the chromatographic column is a normal silica-filled amino column with a particle size of 30-50 μm and a pore size type of: When feeding, 0.8-1.0 g of residual solid phase is added per 100 ml of chromatographic packing material, and the feed flow rate is 0.5-1.0 BV / h based on the packing material volume.

6. The method for separating isochlorogenic acid A and isochlorogenic acid C based on extraction and chromatography according to claim 1, characterized in that, In step (3), when eluting with the first eluent, the amount of the first eluent is 1.5-2.0 BV of the chromatographic packing volume, and the influent flow rate is 1.0-1.5 BV / h of the packing volume; When using a second eluent, the amount of the second eluent is 1.3-1.8 BV of the chromatographic packing volume, and the influent flow rate is 0.5-1 BV / h of the packing volume. When using a third eluent, the amount of the third eluent is 1.5-2.0 BV of the chromatographic packing volume, and the influent flow rate is 0.5-1 BV / h of the packing volume. When using a fourth eluent, the amount of the fourth eluent is 2.0-2.5 BV of the chromatographic packing volume, and the influent flow rate is 1.0-2.0 BV / h of the packing volume.

7. The method for separating isochlorogenic acid A and isochlorogenic acid C based on extraction and chromatography according to claim 1, characterized in that, The first and fourth eluents were combined into the extract phase of step (2) and dried to obtain stevia polyphenols.

8. The method for separating isochlorogenic acid A and isochlorogenic acid C based on extraction and chromatography according to claim 1, characterized in that, In step (4), when preparing the third treatment solution, the mass ratio of solid to purified water is 1:(2.0-3.5), the pH is adjusted to 3.0-3.5, and the stirring and crystallization time is 6-10h; When preparing the fourth treatment solution, the mass ratio of the first filter cake to purified water is 1:(3-5), the heating temperature is 70-80℃, the cooling crystallization temperature is 20-30℃, and the time is 4-6h. When preparing the fifth treatment solution, the mass ratio of solid to saturated aqueous solution of ethyl acetate is 1:(2.5-3.5), the crystallization temperature is room temperature, and the time is 4-8 hours.

9. The method for separating isochlorogenic acid A and isochlorogenic acid C based on extraction and chromatography according to claim 1, characterized in that, In step (5), when preparing the sixth treatment solution, the mass ratio of the third filter cake to purified water is 1:(1.5-2.0), the pulping temperature is 40-45℃, and the time is 1-3h.

10. The method for separating isochlorogenic acid A and isochlorogenic acid C based on extraction and chromatography according to claim 1, characterized in that, The first and third filtrates are combined into the raffinate phase of step (2), the second filtrate is combined into the second eluent, and the fourth filtrate is combined into the third eluent.

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