Preparation method of high-purity isochlorogenic acid B

Abstract

The invention discloses a preparation method of high-purity isochlorogenic acid B. The preparation method specifically comprises the following steps: dissolving a stevia rebaudiana polyphenol product, then adjusting the pH value to obtain a to-be-treated liquid I, heating to carry out isomerization reaction, after the reaction is finished, adjusting the pH value, concentrating, and cooling to obtain a to-be-treated liquid II; extracting the to-be-treated liquid II for multiple times to obtain a raffinate phase I, and adjusting the pH value of the raffinate phase I to obtain to-be-treated liquid III; extracting the to-be-treated liquid III for multiple times by adopting a water-saturated ethyl acetate solution to obtain a raffinate phase II; adsorbing the raffinate phase II through non-polar macroporous adsorption resin, sequentially carrying out jacking and desorption treatment, adjusting the pH value of a desorption solution, and drying to obtain a crude product; and carrying out crystallization and recrystallization treatment on the crude product to obtain the high-purity isochlorogenic acid B. According to the method, a stevia rebaudiana polyphenol product is used as a raw material, high-purity isochlorogenic acid B is prepared through isomerization reaction, extraction, resin adsorption and fractional crystallization, waste liquid generated in the process is fully utilized, the resource utilization rate is increased, and the product yield is increased.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of preparation of chlorogenic acid isomers, and particularly relates to a preparation method of high-purity isochlorogenic acid B. BACKGROUND

[0002] Isochlorogenic acid B is an important phenolic acid compound in plants and is one of the isomers of chlorogenic acid. Modern pharmacological studies have shown that isochlorogenic acid B has significant antioxidant, anti-inflammatory, antiviral, liver protection, and alpha-glucosidase inhibition activities, and has broad application prospects in the fields of medicine, health products, and functional foods.

[0003] At present, the main source of isochlorogenic acid B is extracted and separated from natural plants. Sweetleaf is an important economic crop, and its leaves contain not only high-intensity sweetener steviol glycosides but also a variety of polyphenols, including chlorogenic acid compounds that can be used as raw materials for the preparation of isochlorogenic acid B. Therefore, obtaining high-value isochlorogenic acid B from sweetleaf processing by-products or polyphenol extracts is an effective way to realize the comprehensive utilization of sweetleaf resources.

[0004] However, there are still many challenges in preparing high-purity isochlorogenic acid B from sweetleaf processing by-products in the prior art. First, the content of isochlorogenic acid B in naturally occurring chlorogenic acid substances is usually low, and its structure is very similar to that of other isomers, and its physical and chemical properties are close, making separation and purification extremely difficult. Second, conventional preparation methods often involve multiple and complex column chromatography steps, requiring the use of a large amount of organic solvent, which not only has high production cost and long operation period, but also is difficult to realize large-scale production. In addition, the traditional crystallization process has limited improvement in the purity and yield of isochlorogenic acid B, and the product purity often cannot meet market demand. Moreover, how to effectively treat the mother liquor, recover valuable components and solvents during the separation process to reduce cost and environmental pollution is also a problem that has not been properly solved.

[0005] Therefore, it is necessary to provide a preparation method of high-purity isochlorogenic acid B to solve the above problems. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a preparation method of high-purity isochlorogenic acid B to solve the above problems.

[0007] To solve the above technical problems, the technical solution of the present application is:

[0008] A preparation method of high-purity isochlorogenic acid B, comprising the following steps:

[0009] (1) dissolving stevia polyphenol product and adjusting pH to obtain a first treated solution;

[0010] (2) heating the first treated solution for isomerization reaction, adjusting pH and concentrating after reaction, and cooling to obtain a second treated solution;

[0011] (3) extracting the second treated solution with water-saturated ethyl acetate for multiple times to obtain a first raffinate, adjusting pH of the first raffinate to obtain a third treated solution, and combining the extraction phases to obtain a first extraction phase, which is dried after desolventizing to obtain a low-content stevia polyphenol product;

[0012] (4) extracting the third treated solution with water-saturated ethyl acetate for multiple times to obtain a second raffinate, and combining the extraction phases to obtain a second extraction phase, which is used in step (1) after desolventizing;

[0013] (5) adsorbing the second raffinate of step (4) with non-polar macroporous adsorption resin, and then sequentially performing top material and elution treatment, and drying the eluate after adjusting pH to obtain a crude product;

[0014] (6) sequentially performing crystallization and recrystallization treatment on the crude product to obtain high-purity isochlorogenic acid B.

[0015] Preferably, in step (1), the stevia polyphenol product is dissolved in purified water, and the mass ratio of the stevia polyphenol product to purified water is 1: (10-12); and the pH of the first treated solution is 7.7-8.0.

[0016] Preferably, in step (2), the temperature of the heating isomerization reaction is 75-80°C, and the time is 5-6h; the pH of the second treated solution is 4.0-4.5, and the solid content is 35-40wt%.

[0017] Preferably, in step (3), the volume ratio of the second treated solution to water-saturated ethyl acetate solution is 1: (1-3), and the extraction times are 2-3 times; and the pH of the third treated solution is 2.5-2.8.

[0018] Preferably, in step (4), the volume ratio of the third treated solution to water-saturated ethyl acetate solution is 1: (1-3), and the extraction times are 4-6 times.

[0019] Preferably, in step (5), the non-polar macroporous adsorption resin has a pore volume of 0.6-0.7cm 3 / g, a pore size of 3.0-3.5nm, and a particle size of 0.3-0.4mm; the feeding amount is 13-15g of the dry phase of the second raffinate of step (4) per 100ml of the resin, and the feeding flow rate is 0.5-1.0BV / h of the volume of the resin.

[0020] The non-polar macroporous adsorption resin of the present application can be selected from one of XDA-1G resin of Xi'an Blue Sky Technology Co., Ltd., LK-CGA1 resin and LK-CGA6 resin of Amicor Health (China) Biomedicine Co., Ltd.

[0021] Preferably, in step (5), purified water is used for the top material, the volume of the purified water is 2-3 BV of the volume of the resin, and the flow rate is 1.0-3.0 BV / h of the volume of the resin.

[0022] Preferably, in step (5), 60-80% ethanol solution is used for elution, the volume of the eluent is 3-5 BV of the volume of the resin, and the flow rate is 1.0-3.0 BV / h of the volume of the resin; the pH of the eluent is adjusted to 2.0-2.5.

[0023] Preferably, in step (6), 90-93% v / v isopropyl alcohol is used as the crystallization solvent, the mass ratio of isopropyl alcohol to the crude product is (2.5-3.5):1, the crystallization temperature is room temperature, and the crystallization time is 18-24 h.

[0024] Preferably, in step (6), after crystallization, filtration is performed, and the filtrate is combined with the to-be-processed liquid II after desolventization.

[0025] Preferably, in step (6), in the recrystallization, purified water is used as the recrystallization solvent, the mass ratio of the purified water to the crystallization filter cake is (3.0-5.0):1; in the recrystallization, the solid is dissolved by heating to above 70℃, and then the temperature is decreased to 25-30℃ at a rate of 3-5℃ / h, and then filtration is performed; the recrystallization filtrate is combined with the eluent, and the recrystallization filter cake is dried to obtain high-purity iso-chlorogenic acid B.

[0026] In the stevia polyphenol product of the present application, the total content of chlorogenic acid and its isomers is 60-65 wt%, the content of iso-chlorogenic acid A is 35-45 wt%, the content of iso-chlorogenic acid B is 3-6 wt%, and the content of iso-chlorogenic acid C is 5-10 wt%.

[0027] Due to the adoption of the above technical solutions, the present application has at least the following beneficial effects:

[0028] The present application provides a preparation method of high-purity iso-chlorogenic acid B, which uses low-cost stevia polyphenol products as raw materials, and through isomerization reaction, liquid-liquid extraction, resin adsorption and step-by-step crystallization, and optimization of the conditions of each step, high-purity iso-chlorogenic acid B is prepared, the product yield is high, the waste liquid is fully utilized, and the resource utilization rate is improved.

[0029] In the isomerization reaction, the present application controls the pH of the to-be-treated liquid one and the temperature and time of the isomerization reaction, so that the ester bond of the chlorogenic acid in the stevia polyphenol product is hydrolyzed and rearranged under weak alkaline and heating conditions, thereby converting to more stable isochlorogenic acid B, and the isochlorogenic acid B is enriched by preliminary chemical reaction.

[0030] In the extraction process, the present application uses water-saturated ethyl acetate as an extractant for multi-stage extraction to achieve efficient enrichment of isochlorogenic acid B. Specifically, water-saturated ethyl acetate can effectively reduce the loss of target products during extraction, making the extraction process more stable. In the first extraction, the present application controls the pH of the to-be-treated liquid two, so that part of other low-polarity impurities and a small part of low-polarity isochlorogenic acid A and isochlorogenic acid C in the to-be-treated liquid two are protonated and can be preliminarily extracted by the organic phase. In the second extraction, the pH of the raffinate phase one is adjusted to 2.5-2.8, at which most of the low-polarity isochlorogenic acid A and isochlorogenic acid C that have not been isomerized are completely protonated, greatly increasing their solubility in the organic phase. After the isomerized isochlorogenic acid A and isochlorogenic acid C are desorbed, they are combined with the raw material and subjected to isomerization again, greatly improving the yield of the target product, and the raffinate phase two in the extraction process is further enriched by resin.

[0031] In the resin adsorption, the present application uses a specific non-polar macroporous adsorption resin to further enrich isochlorogenic acid B. The non-polar macroporous adsorption resin selectively adsorbs isochlorogenic acid B through van der Waals force, and then elutes high-purity isochlorogenic acid B by a certain concentration of ethanol. Ethanol, as a polar solvent, can destroy the van der Waals force and hydrogen bond interactions between isochlorogenic acid B and the resin. DETAILED DESCRIPTION

[0032] In order to more clearly understand the above-mentioned purposes, features and advantages of the present application, the schemes 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.

[0033] In order to solve the problems in the background art, the present application discloses the following technical scheme:

[0034] A preparation method of high-purity isochlorogenic acid B, comprising the following steps:

[0035] (1) dissolving the stevia polyphenol product and adjusting the pH to obtain a to-be-treated liquid one;

[0036] (2) heating the to-be-treated liquid one for isomerization reaction, adjusting the pH after the reaction, and concentrating and cooling to obtain a to-be-treated liquid two;

[0037] (3) the treated liquid two is extracted for multiple times with water-saturated ethyl acetate as an extraction solvent to obtain a raffinate phase one, the raffinate phase one is adjusted in pH to obtain a treated liquid three; and the extraction phases are combined to obtain an extraction phase one, which is dried after desolventization to obtain a low-content stevia polyphenol product;

[0038] (4) the treated liquid three is extracted for multiple times again with water-saturated ethyl acetate solution to obtain a raffinate phase two; and the extraction phases are combined to obtain an extraction phase two, which is desolventized and then used in step (1);

[0039] (5) the raffinate phase two of step (4) is adsorbed by a nonpolar macroporous adsorption resin, and then subjected to top material, elution treatment in sequence; the elution liquid is dried after pH adjustment to obtain a crude product;

[0040] (6) the above crude product is subjected to crystallization and recrystallization in sequence to obtain high-purity isochlorogenic acid B.

[0041] With regard to step (1):

[0042] In some embodiments of the present application, the stevia polyphenol product is dissolved in purified water, and the mass ratio of the stevia polyphenol product to purified water is 1: (10-12). The purified water is used as a solvent in the present application, which is safe, environmentally friendly and low in cost. By reasonably adjusting the mass ratio of the stevia polyphenol product to purified water, the solution has a suitable viscosity, which is beneficial to subsequent mass transfer and reaction.

[0043] In some embodiments of the present application, the pH of the treated liquid one is 7.7-8.0. The chlorogenic acid contains an ester bond and an ortho-phenolic hydroxyl group. In an alkaline condition, OH⁻ ions attack the ester bond, causing hydrolysis and rearrangement reaction. If the pH is too low, the reaction rate is too slow; if the pH is too high, side reactions will occur, and the yield and purity of the product will decrease.

[0044] With regard to step (2):

[0045] In some embodiments of the present application, the temperature of the heating isomerization reaction is 75-80℃, and the time is 5-6h. Heating can provide the necessary activation energy for the isomerization reaction, thereby ensuring the smooth progress of the isomerization reaction. Reasonable control of the isomerization reaction time can ensure that the reaction can proceed sufficiently, so that other chlorogenic acid isomers in the raw material are converted into isochlorogenic acid B.

[0046] In some embodiments of the present application, the pH of the liquid to be treated II is 4.0-4.5, and the solid content is 35-40 wt%. In order to facilitate the smooth progress of the subsequent extraction process, the pH and solid content of the liquid to be treated II are reasonably adjusted. Within a certain pH range, part of other low-polarity impurities and a small part of low-polarity isochlorogenic acid A and isochlorogenic acid C that are not isomerized are protonated and can be preliminarily extracted by the organic phase. The selection of a reasonable solid content can improve the efficiency of the subsequent liquid-liquid extraction.

[0047] With regard to step (3):

[0048] In some embodiments of the present application, the volume ratio of the liquid to be treated II to the water-saturated ethyl acetate solution is 1: (1-3). The use of a water-saturated ethyl acetate solution as an extractant can prevent the organic phase from absorbing water from the aqueous phase during the extraction process, thereby ensuring the stable progress of the extraction process.

[0049] In some embodiments of the present application, the pH of the liquid to be treated III is 2.5-2.8. Under this pH condition, most of the low-polarity isochlorogenic acid A and isochlorogenic acid C that are not isomerized are completely protonated, greatly enhancing their solubility in the organic phase and being extracted and enriched.

[0050] With regard to step (4):

[0051] In some embodiments of the present application, in step (4), the volume ratio of the liquid to be treated III to the water-saturated ethyl acetate solution is 1: (1-3). After isochlorogenic acid A and isochlorogenic acid C in the extraction phase II are desorbed and combined into the raw material, secondary isomerization is performed, which greatly improves the yield of the target product. Isochlorogenic acid B is preliminarily enriched in the raffinate phase II during the extraction process.

[0052] With regard to step (5):

[0053] In some embodiments of the present application, the non-polar macroporous adsorption resin has a pore volume of 0.6-0.7 cm 3 / g, a pore size of 3.0-3.5 nm, and a particle size of 0.3-0.4 mm; the feed amount is 13-15 g of the raffinate phase I per 100 ml of the resin in step (4), and the feed flow rate is 0.5-1.0 BV / h of the volume of the resin. The non-polar macroporous adsorption resin can adsorb hydrophobic substances through van der Waals force, and although isochlorogenic acid B contains phenolic hydroxyl groups, its large conjugated system makes it have a certain degree of hydrophobicity and can be effectively adsorbed by the non-polar resin. The selection of the pore volume, pore size, and particle size of the resin can allow isochlorogenic acid B molecules to smoothly enter the pore channels inside the resin, while impurities with larger molecular weights or mismatched spatial structures are excluded, realizing selective adsorption.

[0054] In some embodiments of the present application, the top material is carried out with purified water, the volume of which is 2-3BV of the resin volume, and the flow rate is 1.0-3.0BV / h of the resin volume. The resin column is washed with purified water, so as to wash off the very large polar impurities that are not firmly adsorbed by the resin, and improve the purity of the product.

[0055] In some embodiments of the present application, the elution is carried out with 60-80% ethanol solution, the volume of which is 3-5BV of the resin volume, and the flow rate is 1.0-3.0BV / h of the resin volume; ethanol is a polar solvent, which can destroy the van der Waals force and hydrogen bond interaction between the product and the resin, so as to elute iso- chlorogenic acid B from the resin. If the concentration of ethanol is too high, it will bring safety risks and make solvent recovery difficult; if the concentration is too low, the interaction will be weakened, more ethanol is needed for elution, and the solution consumption is large and the elution is not complete.

[0056] In some embodiments of the present application, the pH of the elution liquid is adjusted to 2.0-2.5. Before crystallization, the pH of the elution liquid needs to be adjusted. Under acidic conditions, iso-chlorogenic acid B exists in the form of molecules, and its solubility in the solvent is reduced, which is beneficial to subsequent crystallization treatment.

[0057] With regard to step (6):

[0058] In some embodiments of the present application, when crystallizing, 90-93% isopropyl alcohol is used as the crystallization solvent, the mass ratio of isopropyl alcohol to the crude product is (2.5-3.5):1, the crystallization temperature is room temperature, and the time is 18-24h.

[0059] In some embodiments of the present application, after crystallization, filtration is carried out, and the filtrate is combined with the to-be-treated liquid II after isopropyl alcohol is removed.

[0060] In some embodiments of the present application, when recrystallizing, purified water is used as the recrystallization solvent, and the mass ratio of purified water to the crystallization filter cake is (3.0-5.0):1; when recrystallizing, the temperature is heated to above 70℃ for solid dissolution, and the temperature is lowered for crystallization, the cooling rate is 3-5℃ / h, and after the temperature is lowered to 25-30℃, filtration is carried out, and the recrystallization filtrate is combined with the elution liquid, and the recrystallization filter cake is dried to obtain high-purity iso-chlorogenic acid B.

[0061] In the above 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 examples in the specification are only some of the embodiments of the present application, not all the embodiments.

[0062] In order to further understand the present application, the preferred embodiments of the present application are described below in conjunction with the examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present application, and are not a limitation on the claims of the present application.

[0063] In the following examples and comparative examples, the preparation method of the stevia polyphenol product is as follows:

[0064] S1: Stevia was extracted with 30% v / v ethanol (mass ratio of stevia to ethanol 1:15) at 30°C for 4 times, the extract was combined and filtered, the filtrate was de-alcoholized with a 200 Da nanofiltration membrane, and the retentate was adjusted to pH 3.3 to obtain a to-be-treated liquid;

[0065] S2: The to-be-treated liquid was saturatedly adsorbed by a polyamide resin, the feed amount was 10 ml of resin volume to 7 g of stevia, the feed flow rate was 1 BV / h of the resin volume, after the feeding was completed, 1.5 BV of the resin volume of purified water was used for elution, the feed flow rate was controlled to be 1 BV / h of the resin volume, after the water elution was completed, 2.5 BV of the resin volume of 20% v / v ethanol was used for impurity removal, the feed flow rate was 1 BV / h of the resin volume, then 4.0 BV of the resin volume of 80% v / v ethanol was used for elution, the feed flow rate was 1 BV / h of the resin volume, and the eluate was dried to obtain a stevia polyphenol product.

[0066] Example 1

[0067] A preparation method of high-purity iso-chlorogenic acid B, comprising the following steps:

[0068] (1) 2 kg of stevia polyphenol product (total content of chlorogenic acid and its isomers 63.6 wt%, content of iso-chlorogenic acid A 40.2 wt%, content of iso-chlorogenic acid B 4.5 wt%, and content of iso-chlorogenic acid C 9.3 wt%) was dissolved in 20 L of purified water, and the solution was adjusted to pH 7.7 to obtain a to-be-treated liquid one;

[0069] (2) The to-be-treated liquid one was heated to 75°C, and isomerization reaction was performed for 5.0 h, after the reaction was completed, the system pH was adjusted to 4.0, then the solution was concentrated to a solid content of 35 wt%, and then the temperature was lowered to room temperature to obtain a to-be-treated liquid two;

[0070] (3) The to-be-treated liquid two was extracted twice with water-saturated ethyl acetate as the extraction solvent to obtain a raffinate one, and in each extraction, the volume ratio of the to-be-treated liquid two to the water-saturated ethyl acetate solution was controlled to be 1:1; the extraction phases obtained by multiple extractions were combined to obtain extraction phase one, which was desolventized and dried to obtain a low-content stevia polyphenol product;

[0071] (4) The pH of the raffinate one of step (3) was adjusted to 2.5 to obtain a to-be-treated liquid three, and the to-be-treated liquid three was extracted again with water-saturated ethyl acetate solution for 4 times to obtain a raffinate two; and the extraction phases obtained by multiple extractions were combined to obtain extraction phase two, which was desolventized and used in step (1); in each extraction, the volume ratio of the to-be-treated liquid three to the water-saturated ethyl acetate solution was controlled to be 1:1;

[0072] (5) The raffinate phase II of step (4) is adsorbed by non-polar macroporous adsorption resin (the non-polar macroporous adsorption resin is XDA-1G resin of Xi'an Lanxiao Science and Technology Co., Ltd., the pore volume of the non-polar macroporous adsorption resin is 0.60 cm 3 / g, the pore size is 3.0 nm, and the particle size is 0.3-0.4 mm; the feeding amount is 13 g of dry raffinate phase II per 100 ml of resin, and the feeding flow rate is 0.5 BV / h of the volume of the resin), and then top charging (purified water with a volume of 2.0 BV of the resin is used for top charging, and the feeding flow rate is 1.0 BV / h of the volume of the resin), desorption treatment (60% ethanol solution with a volume of 3 BV of the resin is used for desorption, and the feeding flow rate is 1.0 BV / h), and drying after adjusting the pH of the desorption solution to 2.0 to obtain a crude product;

[0073] (6) The above crude product is dissolved in 90% isopropyl alcohol, the mass ratio of isopropyl alcohol to the crude product is controlled to be 2.5:1, and stirring crystallization is performed at room temperature for 18 h; after the crystallization is completed, filtration is performed, the filtrate is desolventized, and then combined into the to-be-treated liquid II; and the filter cake is reserved;

[0074] (7) The filter cake collected in step (6) and purified water are mixed, the mass ratio of the purified water to the filter cake in step (6) is controlled to be 3:1, heating is performed to 70°C, stirring is performed until the solid is dissolved, then temperature reduction is performed to 25°C at a rate of 3°C / h, and then filtration is performed; the filtrate is combined into the desorption solution in step (5), and the filter cake is dried to obtain high-purity iso-chlorogenic acid B.

[0075] Example 2

[0076] A preparation method of high-purity iso-chlorogenic acid B, comprising the following steps:

[0077] (1) 2 kg of stevia polyphenol product (the total content of chlorogenic acid and its isomers is 63.6 wt%, the content of iso-chlorogenic acid A is 40.2 wt%, the content of iso-chlorogenic acid B is 4.5 wt%, and the content of iso-chlorogenic acid C is 9.3 wt%) is dissolved in 24 L of purified water, and the pH of the solution is adjusted to 8.0 to obtain a to-be-treated liquid I;

[0078] (2) The to-be-treated liquid I is heated to 80°C, and isomerization is performed for 6 h; after the reaction is completed, the pH of the system is adjusted to 4.5, then concentration is performed to a solid content of 40 wt%, and then temperature reduction is performed to room temperature to obtain a to-be-treated liquid II;

[0079] (3) The to-be-treated liquid II is extracted 3 times with water-saturated ethyl acetate as an extraction solvent to obtain raffinate phase I; in each extraction, the volume ratio of the to-be-treated liquid II to the water-saturated ethyl acetate solution is controlled to be 1:3; the extraction phases obtained by multiple extractions are combined to obtain extraction phase I, which is desolventized and dried to obtain a low-content stevia polyphenol product;

[0080] (4) adjust the pH of the raffinate phase one in step (3) to 2.8 to obtain a treated liquid three, and then extract the treated liquid three with water-saturated ethyl acetate solution for 6 times to obtain a raffinate phase two; combine the extraction phases obtained in the multiple extractions to obtain a extraction phase two, which is used in step (1) after desolventization; in each extraction, the volume ratio of the treated liquid three to the water-saturated ethyl acetate solution is controlled to be 1:3;

[0081] (5) adsorb the raffinate phase two in step (4) on a non-polar macroporous adsorption resin (the non-polar macroporous adsorption resin is LK-CGA6 resin of Aikangjian (China) Biomedicine Co., Ltd., the pore volume of the non-polar macroporous adsorption resin is 0.70 cm 3 / g, the pore size is 3.5 nm, and the particle size is 0.3-0.4 mm; the feeding amount is 15 g of dry raffinate phase two per 100 ml of resin, and the feeding flow rate is 1.0 BV / h of the volume of the resin), and then sequentially perform elution (using 3.0 BV of purified water in terms of the volume of the resin for elution, and the feeding flow rate is 3.0 BV / h of the volume of the resin), desorption treatment (using 5 BV of 80% ethanol solution in terms of the volume of the resin for desorption, and the feeding flow rate is 3.0 / h), and drying of the desorption liquid after adjusting the pH to 2.5 to obtain a crude product;

[0082] (6) dissolve the crude product in 93% isopropyl alcohol, control the mass ratio of isopropyl alcohol to the crude product to be 3.5:1, stir and crystallize at room temperature for 24 h, filter after crystallization, and combine the desolventized filtrate to the treated liquid two, and dry the filter cake;

[0083] (7) mix the filter cake collected in step (6) with purified water, control the mass ratio of the purified water to the filter cake in step (6) to be 5:1, heat to 85℃, stir until the solid is dissolved, then cool to 30℃ at a rate of 5℃ / h, filter, combine the filtrate to the desorption liquid in step (5), and dry the filter cake to obtain high-purity iso-chlorogenic acid B.

[0084] Example 3

[0085] A method for preparing high-purity iso-chlorogenic acid B, comprising the following steps:

[0086] (1) dissolve 2 kg of stevia polyphenol product (the total content of chlorogenic acid and its isomers is 63.6 wt%, the content of iso-chlorogenic acid A is 40.2 wt%, the content of iso-chlorogenic acid B is 4.5 wt%, and the content of iso-chlorogenic acid C is 9.3 wt%) in 23 L of purified water, and adjust the pH of the solution to 7.9 to obtain a treated liquid one;

[0087] (2) heat the treated liquid one to 76℃, and isomerize for 5.5 h, then adjust the pH of the system to 4.2 after the reaction is completed, concentrate to a solid content of 36 wt%, and cool to room temperature to obtain a treated liquid two;

[0088] (3) The treated liquid two is twice extracted with water-saturated ethyl acetate to obtain raffinate phase one, and the volume ratio of the treated liquid two to the water-saturated ethyl acetate solution is controlled to be 1:3 in each extraction; the extraction phases obtained in multiple extractions are combined to obtain extraction phase one, which is dried after desolventizing to obtain a low-content stevia polyphenol product;

[0089] (4) The pH of the raffinate phase one obtained in step (3) is adjusted to 2.7 to obtain a treated liquid three, and the treated liquid three is extracted with water-saturated ethyl acetate solution for 5 times to obtain raffinate phase two; and the extraction phases obtained in multiple extractions are combined to obtain extraction phase two, which is desolventized and then used in step (1); the volume ratio of the treated liquid three to the water-saturated ethyl acetate solution is controlled to be 1:2 in each extraction;

[0090] (5) The raffinate phase two in step (4) is adsorbed by a non-polar macroporous adsorption resin (the non-polar macroporous adsorption resin is LK-CGA1 resin of Amicor (China) Biomedicals Co., Ltd., the pore volume of the non-polar macroporous adsorption resin is 0.63 cm 3 / g, the pore size is 3.3 nm, and the particle size is 0.3-0.4 mm; the feeding amount is 14 g of dry raffinate phase two per 100 ml of resin, and the feeding flow rate is 0.8 BV / h of the volume of the resin), and then top charging (pure water with a volume of 2.2 BV of the resin is used for top charging, and the feeding flow rate is 2 BV / h of the volume of the resin), desorption treatment (65% ethanol solution with a volume of 4 BV of the resin is used for desorption, and the feeding flow rate is 2.0 BV / h), and drying after adjusting the pH of the desorption liquid to 2.3 to obtain a crude product;

[0091] (6) The above crude product is dissolved in 92% isopropyl alcohol, the mass ratio of isopropyl alcohol to the crude product is controlled to be 2.8:1, and the product is stirred at room temperature for 20 h to crystallize, then filtered, and the filtrate is desolventized and combined with the treated liquid two, and the filter cake is reserved;

[0092] (7) The filter cake collected in step (6) and purified water are mixed, the mass ratio of the purified water to the filter cake in step (6) is controlled to be 4:1, heated to 80°C, stirred until the solid is dissolved, then cooled to 27°C at a rate of 4°C / h, filtered, and the filtrate is combined with the desorption liquid in step (5), and the filter cake is dried to obtain high-purity iso-chlorogenic acid B.

[0093] Example 4

[0094] A method for preparing high-purity iso-chlorogenic acid B, comprising the following steps:

[0095] (1) 2 kg of stevia polyphenol product (the total content of chlorogenic acid and its isomers is 63.6 wt%, the content of iso-chlorogenic acid A is 40.2 wt%, the content of iso-chlorogenic acid B is 4.5 wt%, and the content of iso-chlorogenic acid C is 9.3 wt%) is dissolved in 22 L of purified water, and the pH of the solution is adjusted to 7.8 to obtain a treated liquid one;

[0096] (2) heating the to-be-processed liquid one to 78°C, isomerization for 5.5 h, adjusting the pH of the system to 4.3 after the reaction is completed, then concentrating to a solid content of 38 wt%, and cooling to room temperature to obtain to-be-processed liquid two;

[0097] (3) extracting to-be-processed liquid two twice with water-saturated ethyl acetate as the extraction solvent to obtain raffinate one, and the volume ratio of to-be-processed liquid two to water-saturated ethyl acetate solution is controlled to be 1:2 during each extraction; combining the extraction phases obtained by multiple extractions to obtain extraction phase one, and drying after desolventizing to obtain a low-content stevia polyphenol product;

[0098] (4) adjusting the pH of raffinate one in step (3) to 2.6 to obtain to-be-processed liquid three, and water-saturated ethyl acetate solution is used to extract to-be-processed liquid three again for 5 times to obtain raffinate two; and combining the extraction phases obtained by multiple extractions to obtain extraction phase two, which is desolventized and then used in step (1); and the volume ratio of to-be-processed liquid three to water-saturated ethyl acetate solution is controlled to be 1:2 during each extraction;

[0099] (5) adsorbing raffinate two in step (4) on a non-polar macroporous adsorption resin (the non-polar macroporous adsorption resin is LK-CGA1 resin from Amicor (China) Biomedicals Co., Ltd., the pore volume of the non-polar macroporous adsorption resin is 0.63 cm 3 / g, the pore size is 3.3 nm, and the particle size is 0.3-0.4 mm; the feeding amount is 14 g of dry raffinate two per 100 ml of resin, and the feeding flow rate is 0.6 BV / h of the volume of the resin), and then sequentially performing elution (using 2.7 BV of purified water of the volume of the resin for elution, and the liquid feeding flow rate is 2 BV / h of the volume of the resin), and desorption treatment (using 4 BV of 70% ethanol solution of the volume of the resin for desorption, and the liquid feeding flow rate is 2.0 BV / h); drying after adjusting the pH of the desorption liquid to 2.2 to obtain a crude product;

[0100] (6) dissolving the above crude product in 91% isopropyl alcohol, controlling the mass ratio of isopropyl alcohol to crude product to be 3.2:1, stirring at room temperature for 22 h, filtering after crystallization is completed, and combining the desolventized filtrate to to-be-processed liquid two, and the filter cake is reserved;

[0101] (7) mixing the filter cake collected in step (6) and purified water, controlling the mass ratio of purified water to the filter cake in step (6) to be 4:1, heating to 80°C, stirring until the solid is dissolved, then cooling to 28°C at a rate of 4°C / h, and filtering, and combining the filtrate to the desorption liquid in step (5), and drying the filter cake to obtain high-purity iso-green acid B.

[0102] Comparative Example 1

[0103] The difference between this comparative example and Example 4 is that in step (1), the pH of to-be-processed liquid one is 6.5, and the other operations are the same as those in Example 4.

[0104] Comparative Example 2

[0105] This comparative example differs from Example 4 in that in Step (1), the pH of the to-be-treated liquid one is 9.0, and other operations are the same as those in Example 4.

[0106] Comparative Example 3

[0107] This comparative example differs from Example 4 in that in Step (2), the pH of the to-be-treated liquid two is 3.0, and other operations are the same as those in Example 4.

[0108] Comparative Example 4

[0109] This comparative example differs from Example 4 in that in Step (2), the pH of the to-be-treated liquid two is 5.5, and other operations are the same as those in Example 4.

[0110] Comparative Example 5

[0111] This comparative example differs from Example 4 in that in Step (2), the solid content of the to-be-treated liquid two is 50 wt%, and other operations are the same as those in Example 4.

[0112] Comparative Example 6

[0113] This comparative example differs from Example 4 in that in Step (4), the pH of the to-be-treated liquid three is 1.5, and other operations are the same as those in Example 4.

[0114] Comparative Example 7

[0115] This comparative example differs from Example 4 in that in Step (4), the pH of the to-be-treated liquid three is 3.5, and other operations are the same as those in Example 4.

[0116] Comparative Example 8

[0117] This comparative example differs from Example 4 in that in Step (5), the pH of the elution liquid is adjusted to 1.0, and other operations are the same as those in Example 4.

[0118] Comparative Example 9

[0119] This comparative example differs from Example 4 in that in Step (5), the pH of the elution liquid is adjusted to 3.5, and other operations are the same as those in Example 4.

[0120] Comparative Example 10

[0121] This comparative example differs from Example 4 in that in Step (7), the cooling rate is 7°C / min, and other operations are the same as those in Example 4.

[0122] The performance parameters of the products in the above examples and comparative examples are shown in Table 1.

[0123] The yield of isochlorogenic acid B (%) = yield of isochlorogenic acid B x purity / [weight of stevia polyphenol product x (purity of isochlorogenic acid A in stevia polyphenol + purity of isochlorogenic acid B in stevia polyphenol + purity of isochlorogenic acid C in stevia polyphenol)] x 100%.

[0124] Table 1

[0125]

[0126] As can be seen from the test results in Table 1, the present application uses low-cost stevia polyphenol product as raw material, and through isomerization reaction, liquid-liquid extraction, resin adsorption and step-by-step crystallization, and optimization of the conditions of each step, high-purity isochlorogenic acid B is prepared, the product yield is high, the waste liquid is fully utilized, and the resource utilization rate is improved.

[0127] Compared with the examples, the pH of the first treated liquid in Comparative Example 1 is too low, which causes the isochlorogenic acid A and isochlorogenic acid C in the raw material not to be directionally isomerized to isochlorogenic acid B, resulting in too low concentration of isochlorogenic acid B in the subsequent crystallization system, which cannot be crystallized. The pH of the first treated liquid in Comparative Example 2 is too high, which can cause side reactions, resulting in loss of target product and significant decrease in product purity.

[0128] After the isomerization reaction, the pH and solid content of the system need to be reasonably adjusted before extraction, so that part of the low-polarity impurities are protonated, which is convenient for subsequent extraction removal, and the appropriate solid content ensures the efficiency of liquid-liquid extraction. In Comparative Example 3, the pH of the second treated liquid is too low, which causes a large amount of isochlorogenic acid A and isochlorogenic acid C to be protonated and partially removed in extraction, thereby reducing the amount of isochlorogenic acid A and isochlorogenic acid C that are not isomerized and the product yield. In Comparative Example 4, the pH of the second treated liquid is too high, which causes insufficient protonation of impurities, resulting in an increase in residual impurities and a decrease in product purity. In Comparative Example 5, the solid content of the second treated liquid is too high, which increases the solution viscosity, making subsequent extraction difficult, and the yield and purity of the product are both decreased to a certain extent.

[0129] When performing secondary extraction, the pH of the third treated liquid needs to be reasonably adjusted. Under specific solvents and specific pH ranges, isochlorogenic acid A and C are fully protonated and can be extracted by the organic phase, while isochlorogenic acid B is retained in the aqueous phase for preliminary enrichment. In Comparative Example 6, the pH of the third treated liquid is too low, which causes part of the isochlorogenic acid B to be protonated and extracted by the organic phase, resulting in loss of target product and a decrease in purity. In Comparative Example 7, the pH of the third treated liquid is too high, which causes insufficient protonation of isochlorogenic acid A and C, resulting in ineffective removal of impurities, more residual impurities, and decreases in product purity and yield.

[0130] The present application can better control the solubility of the product in the crystallization solvent by adjusting the pH of the elution liquid before crystallization, thereby ensuring the smooth progress of the crystallization process. The pH of the elution liquid in Comparative Example 8 is too low, which can cause the solubility of isochlorogenic acid B and part of the polar impurities to decrease at the same time, and the separation degree decreases in the subsequent crystallization process, degrades or forms insoluble impurities, resulting in a decrease in product purity and yield. In Comparative Example 9, the pH of the elution liquid is too high, which causes isochlorogenic acid B to exist in the form of ions, and the solubility increases, which leads to difficulties in subsequent crystallization, resulting in a decrease in product yield and purity.

[0131] In Comparative Example 10, the cooling rate is too fast during recrystallization, which causes the crystallized isochlorogenic acid B to wrap a certain amount of impurities, and the purity of the product decreases significantly.

[0132] The principles and implementation modes of the present application are described by applying specific examples in the present application. The above examples are only used to help understand the method of the present application and its core idea, including the best mode, and also 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, some 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 the patent protection of the present application is defined by the claims, and can include other embodiments that can be thought of by those skilled in the art. If these other embodiments have structural elements similar to the textual expression of the claims, or if they include equivalent structural elements that are not substantially different from the textual expression of the claims, then these other embodiments should also be included in the scope of the claims.

Claims

1. A method for preparing high purity isochlorogenic acid B, characterized in that, The method comprises the following steps: (1) dissolving the stevia polyphenol product and adjusting the pH to obtain a first treated solution; (2) isomerizing the first treated solution, adjusting the pH after the reaction, and concentrating and cooling to obtain a second treated solution; (3) extracting the second treated solution with water-saturated ethyl acetate for multiple times to obtain a first raffinate, adjusting the pH of the first raffinate to obtain a third treated solution, and combining the extraction phases to obtain a first extraction phase, which is dried after desolventizing to obtain a low-content stevia polyphenol product; (4) extracting the third treated solution with water-saturated ethyl acetate for multiple times to obtain a second raffinate, and combining the extraction phases to obtain a second extraction phase, which is dried after desolventizing and used in step (1); (5) adsorbing the second raffinate of step (4) on a nonpolar macroporous adsorption resin, and then performing top feeding and elution treatment, and drying the eluate after adjusting the pH to obtain a crude product; (6) performing crystallization and recrystallization on the crude product to obtain high-purity isochlorogenic acid B. 2.The method for preparing high-purity iso- chlorogenic acid B according to claim 1, characterized in that, In step (1), the stevia polyphenol product is dissolved in purified water, and the mass ratio of the stevia polyphenol product to the purified water is 1: (10-12); the pH of the first treated solution is 7.7-8.

0. 3.The method for preparing high-purity iso- chlorogenic acid B according to claim 1, characterized in that, In step (2), the temperature of the isomerization reaction is 75-80°C, and the time is 5-6 h; the pH of the second treated solution is 4.0-4.5, and the solid content is 35-40 wt%. 4.The method for preparing high-purity iso- chlorogenic acid B according to claim 1, characterized in that, In step (3), the volume ratio of the second treated solution to the water-saturated ethyl acetate solution is 1: (1-3), and the extraction is performed for 2-3 times; the pH of the third treated solution is 2.5-2.

8. 5.The method for preparing high-purity iso- chlorogenic acid B according to claim 1, characterized in that, In step (4), the volume ratio of the third treated solution to the water-saturated ethyl acetate solution is 1: (1-3), and the extraction is performed for 4-6 times. 6.The method for preparing high-purity iso- chlorogenic acid B according to claim 1, characterized in that, In step (5), the pore volume of the nonpolar macroporous adsorption resin is 0.6-0.7 cm3 / g, the pore size is 3.0-3.5 nm, and the particle size is 0.3-0.4 mm; the feeding amount is 13-15 g of the dry phase of the second raffinate per 100 ml of the resin, and the feeding flow rate is 0.5-1.0 BV / h of the resin volume.

7. The method for preparing high purity of iso- chlorogenic acid B according to claim 1, characterized in that, In step (5), purified water is used for top feeding, and the volume of the purified water is 2-3 BV of the resin volume, and the flow rate is 1.0-3.0 BV / h of the resin volume. 8.The method for preparing high-purity iso- chlorogenic acid B according to claim 1, characterized in that, In step (5), 60-80% ethanol solution is used for elution, the volume of the eluent is 3-5 BV of the resin volume, and the flow rate is 1.0-3.0 BV / h of the resin volume; the pH of the eluate is adjusted to 2.0-2.

5. 9.The method for preparing high-purity iso- chlorogenic acid B according to claim 1, characterized in that, In step (6), at least one of the following features is included during crystallization: 90-93% v / v isopropyl alcohol is used as the crystallization solvent; the mass ratio of isopropyl alcohol to the crude product is (2.5-3.5): 1; the crystallization temperature is room temperature, and the time is 18-24 h; after crystallization, the filtrate is desolventized and combined with the second treated solution. 10.The method for preparing high-purity iso- chlorogenic acid B according to claim 1, characterized in that, In step (6), the purified water is used as the recrystallization solvent, and the mass ratio of the purified water to the crystalline filter cake is (3.0-5.0):1; the temperature is heated to above 70°C to dissolve the solid, then the temperature is decreased to 25-30°C at a rate of 3-5°C / h, and then filtered; the recrystallization filtrate is combined to the elution liquid, and the recrystallization filter cake is dried to obtain high-purity isochlorogenic acid B.

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