Method for simultaneously preparing high-content isochlorogenic acid A and high-content isochlorogenic acid C

Abstract

The invention discloses a method for simultaneously preparing high-content isochlorogenic acid A and high-content isochlorogenic acid C. The method comprises the following steps: dissolving a stevia rebaudiana polyphenol product in purified water, and adjusting the pH value to obtain a treating fluid 1; adsorbing the treating fluid 1 through macroporous adsorption resin, jacking the treating fluid 1 through purified water, and mixing effluent liquid during adsorption and purified water jacking to obtain effluent liquid 1; concentrating the effluent 1 by using a reverse osmosis membrane, adjusting the pH value to obtain a treating fluid 2, and extracting the treating fluid 2 by using an ethyl acetate saturated aqueous solution to obtain a raffinate phase I and an extract phase I; adjusting the pH value of the raffinate phase I, and extracting with an ethyl acetate-n-butyl alcohol saturated aqueous solution to obtain a raffinate phase II and an extract phase II; after drying the extract phase I, sequentially crystallizing and recrystallizing to obtain high-purity isochlorogenic acid C; and drying the extract phase II, and sequentially crystallizing, pulping and purifying to obtain the high-purity isochlorogenic acid A. The method comprises the following steps: removing impurities by adopting macroporous adsorption resin, extracting by adopting a solvent step by step, carrying out crude separation on the isochlorogenic acid A and the isochlorogenic acid C, and carrying out subsequent crystallization and refining to obtain the high-content isochlorogenic acid A and the high-content isochlorogenic acid C.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant extraction, in particular to a method for simultaneously preparing high content of isochlorogenic acid A and isochlorogenic acid C. BACKGROUND

[0002] Stevia rebaudiana is famous for its leaves containing natural sweeteners, steviol glycosides, which have high sweetness and low calories. The extraction and purification of steviol glycosides from the leaves of Stevia rebaudiana on an industrial scale is a complex process, during which a certain amount of by-product, stevia polyphenols, is separated. Stevia polyphenols mainly include a variety of phenolic acid compounds and flavonoids. Phenolic acid compounds include chlorogenic acid and a series of structurally related and more complex isomers (collectively referred to as chlorogenic acid compounds). Chlorogenic acid compounds include a certain amount of isochlorogenic acid A and isochlorogenic acid C, both of which have significant pharmacological activities such as antioxidant, anti-inflammatory, hypoglycemic, and anti-tumor activities. It is of great significance to recover high-purity isochlorogenic acid A and isochlorogenic acid C from stevia polyphenols.

[0003] Isochlorogenic acid A and isochlorogenic acid C are structural isomers of each other, have highly similar molecular structures, and close physicochemical properties, making them difficult to separate. Although techniques for simultaneously extracting steviol glycosides and chlorogenic acid compounds have been developed in recent years, most focus on the recovery of total phenols or mixed chlorogenic acids, and there are few studies on the specific separation of high-purity isochlorogenic acid A and isochlorogenic acid C monomers. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a method for simultaneously preparing high content of isochlorogenic acid A and isochlorogenic acid C, which addresses the deficiencies of the prior art. The method uses macroporous adsorption resin to remove impurities, specific solvents to extract step by step, and separately separates isochlorogenic acid A and isochlorogenic acid C, followed by subsequent crystallization to refine, obtaining high content of isochlorogenic acid A and isochlorogenic acid C. The method is simple to operate, the waste liquid is effectively recovered and reused, significantly improving resource utilization efficiency, and the purity and yield of the product are also significantly improved.

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

[0006] A method for simultaneously preparing high content of isochlorogenic acid A and isochlorogenic acid C, comprising the following steps:

[0007] (1) Dissolve the stevia polyphenol product in purified water, adjust the pH of the solution to obtain a treatment solution 1;

[0008] (2) Pump the treatment solution 1 into a resin column filled with a styrene-based macroporous adsorption resin for adsorption. After completion, use purified water to top up the column. Mix the effluent during the adsorption process and the effluent during the purified water topping-up process to obtain effluent 1. After the water topping-up is completed, use a methanol solution to elute to obtain effluent 2;

[0009] (3) Concentrating the effluent 1 by using reverse osmosis membrane, adjusting the pH of the concentrated solution to obtain a treatment liquid 2, and using ethyl acetate saturated aqueous solution to extract the treatment liquid 2 for multiple times to obtain a raffinate phase 1, and combining the extraction phases of multiple extractions to obtain an extraction phase 1;

[0010] (4) Adjusting the pH of the raffinate phase 1, and then using ethyl acetate-n-butanol saturated aqueous solution to extract for multiple times to obtain a raffinate phase 2, and combining the extraction phases of multiple extractions to obtain an extraction phase 2;

[0011] (5) After the extraction phase 1 is dried, dissolving it in an ethanol solution, adjusting the pH to obtain a treatment liquid 3, and stirring and crystallizing the treatment liquid 3 to obtain a filter cake 1 and a filtrate 1;

[0012] (6) Mixing the filter cake 1 and purified water, and stirring and heating until the solid is dissolved to obtain a treatment liquid 4, and crystallizing the treatment liquid 4 after cooling to obtain a filter cake 2 and a filtrate 2, and drying the filter cake 2 to obtain high-purity isochlorogenic acid C;

[0013] (7) After the extraction phase 2 of step (4) is dried, dissolving it in saturated ethyl acetate, adding isochlorogenic acid A seeds, stirring and crystallizing at room temperature, filtering to obtain a filter cake 3 and a filtrate 3; drying the filter cake 3, mixing it with purified water, and beating and treating to obtain a filter cake 4 and a filtrate 4; and drying the filter cake 4 to obtain high-purity isochlorogenic acid A.

[0014] Preferably, in step (1), the mass ratio of the stevia polyphenol product to purified water is 1:(30-50); and the pH of the treatment liquid 1 is 3.5-4.0.

[0015] Preferably, in step (2), the styrene-based macroporous adsorption resin has a resin pore volume of 0.6-0.7 cm 3 / g, a pore size of 4.1-4.5 nm, a particle size of 0.3-0.4 mm, and a specific surface area of 1000-1200 m2 / g; and the feeding amount during adsorption is 35-40 g of the stevia polyphenol product per 100 ml of the resin.

[0016] The styrene-based macroporous adsorption resin in the present application can be selected from one of LX-62 resin of Xi'an Lanxiao Science and Technology New Material Co., Ltd., LX-B14 resin of Xi'an Lanxiao Science and Technology New Material Co., Ltd., and DA201-CB1# resin of Jiangsu Suqing Water Treatment Engineering Group Co., Ltd.

[0017] Preferably, in step (2), the amount of purified water used when the purified water is topped is 2-3 BV of the volume of the resin.

[0018] Preferably, in step (2), the concentration of the methanol solution is 60-80% v / v, and the amount used is 2-4 BV of the volume of the resin.

[0019] Preferably, in step (2), the flow rate of the feed, water top-up, and feed liquid during elution is 1-2 BV / h of the volume of the resin.

[0020] Preferably, in step (3), the concentration of the reverse osmosis membrane concentrated effluent 1 is 25-35 wt%, and the pH of the treatment liquid 2 is 3.0-3.5.

[0021] Preferably, in step (3), during extraction, the volume ratio of ethyl acetate saturated aqueous solution to treatment liquid 2 is (1-3):1.

[0022] Preferably, in step (4), the pH of the raffinate phase 1 is adjusted to 2.0-2.5.

[0023] Preferably, in step (4), the mass ratio of ethyl acetate to n-butanol in the ethyl acetate-n-butanol saturated aqueous solution is (9.0-9.5):1; and during extraction, the volume ratio of the ethyl acetate-n-butanol saturated aqueous solution to the feed liquid is (2-3):1.

[0024] Preferably, in step (5), the concentration of the ethanol solution is 15-25% v / v, the addition amount of the ethanol solution is 2.0-2.5 BV of the mass of the dried extraction phase 1, the pH of the treatment liquid 3 is 2.5-3.0, and the stirring crystallization time is 6-10 h.

[0025] Preferably, in step (6), the mass ratio of the filter cake 1 to purified water is 1:(2-3), the heating and stirring temperature at which the solid is dissolved is 70-80℃, the cooling and crystallization temperature is 25-35℃, and the time is 4-6 h.

[0026] Preferably, in step (7), the amount of saturated ethyl acetate is 2-3 times the mass of the dried extraction phase 2; the addition amount of isocyanogenic acid A seed crystal is 3-5% of the mass of the dried extraction phase 2; and the stirring crystallization temperature at room temperature is 6-12 h.

[0027] Preferably, in step (7), the addition amount of purified water is 2.0-2.5 times the mass of the dried filter cake 3, the beating treatment temperature is 35-40℃, and the time is 2-3 h.

[0028] Preferably, the raffinate phase 2 is combined into the effluent 2, dried to obtain a stevia polyphenol product, the filtrate 1 and the filtrate 3 are combined into the effluent 1, the filtrate 2 is combined into the extraction phase 1, and the filtrate 4 is combined into the extraction phase 2.

[0029] The isocyanogenic acid A seed crystal in the present application can be selected from the last batch of un-dried filter cake 3 in the production process or commercially available isocyanogenic acid A with a purity of ≥98%, such as isocyanogenic acid A with a purity of ≥98% from Shanghai Yuan Ye Biotechnology Co., Ltd., batch number P11D11L134209.

[0030] The stevia polyphenol product of the present application is a by-product in the process of extracting steviol glycosides from stevia, and the specific process is as follows:

[0031] Stevia is extracted with 20-60% v / v ethanol (the mass ratio of stevia to ethanol is 1:10-15) for 3-5 times at 25-35 DEG C, the multiple extraction solutions are combined, filtered, and then dealcoholized with a 200-400 Da nanofiltration membrane, the cut liquid is adjusted to pH 3-4 to obtain a to-be-treated liquid;

[0032] The to-be-treated liquid is adsorbed by a polyamide resin, the feed amount is 10 ml:(4-7) g of the ratio of the volume of the resin to the weight of stevia, the feed flow rate is 1-2 BV / h of the volume of the resin, after the feed is completed, the resin is topped with 1.5-3.5 BV of purified water, the feed flow rate is controlled to be 1-2 BV / h of the volume of the resin, after the water topping is completed, the resin is topped with 1.5-2.5 BV of 20-30% v / v ethanol to remove impurities, the feed flow rate is 1-2 BV / h of the volume of the resin, then the resin is eluted with 2.0-4.0 BV of 70-85% v / v ethanol, the feed flow rate is 1-3 BV / h of the volume of the resin, and the eluted liquid is dried to obtain the stevia polyphenol product.

[0033] The total content of chlorogenic acid and its isomers in the stevia polyphenol product of the present application is 45-65 wt%, the content of iso-chlorogenic acid A is 25-35 wt%, and the content of iso-chlorogenic acid C is 10-15 wt%.

[0034] Due to the adoption of the above technical solutions, the present application has the following beneficial effects:

[0035] The present application provides a method for simultaneously preparing high-content iso-chlorogenic acid A and iso-chlorogenic acid C, which takes stevia polyphenol as raw material, a by-product of steviol glycoside preparation, first separates chlorogenic acid substances by using a specific-pore-structure styrene macroporous adsorption resin to achieve preliminary enrichment and impurity removal, then extracts the separated chlorogenic acid substances under a certain pH condition with ethyl acetate as an extractant, uses ethyl acetate to preferentially extract components with relatively low polarity to enrich iso-chlorogenic acid C in the chlorogenic acid substances, and obtains iso-chlorogenic acid C crude product, and collects the raffinate phase in the extraction process, uses a specific ratio of ethyl acetate-n-butanol saturated aqueous solution to extract under a specific pH condition to enrich iso-chlorogenic acid A with relatively high polarity, and obtains iso-chlorogenic acid A crude product.

[0036] When refining iso-chlorogenic acid C, the present application dissolves it in a specific concentration of ethanol solution, and preliminarily crystallizes under a certain pH condition, and then recrystallizes with purified water as a crystallization solvent to effectively remove residual impurities, and obtain high-purity iso-chlorogenic acid C product with a purity of not less than 98%.

[0037] The present application selects a specific solvent during the refining of isochlorogenic acid A, adds an appropriate amount of crystal seed to induce crystallization, and then combines purified water for pulping and purification, thereby effectively removing impurities and obtaining high-purity isochlorogenic acid A products with a purity of not less than 98%.

[0038] The combined raffinate phase of the present application is recovered into the effluent 2, thereby preparing a by-product stevia polyphenol product and improving resource utilization. The filtrate 1 during crystallization and the filtrate 3 are combined into the effluent 1, the filtrate 2 during recrystallization is combined back into the extraction phase 1, and the filtrate 4 is combined back into the extraction phase 2, thereby further improving the total yield of the product. DETAILED DESCRIPTION

[0039] In order to enable a more clear understanding of the above-mentioned objects, 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.

[0040] In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application, but the present application can also be implemented in other manners different from those described herein; obviously, the embodiments described in the specification are only a part of the embodiments of the present application, and not all the embodiments.

[0041] 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 a limitation on the claims of the present application.

[0042] The preparation method of the stevia polyphenol product in the following examples and comparative examples is as follows:

[0043] 5 kg of stevia is extracted with 70 L of 30% v / v ethanol for 5 times, and the combined extract is filtered to obtain a filtered clear liquid. The filtered clear liquid is de-alcoholized by using a 400 Da nanofiltration membrane, the cut-off liquid is adjusted to pH 3.4, and then pumped into 9 L of polyamide resin adsorption at a flow rate of 1.5 BV / h of the resin volume. The resin volume is topped with 2 BV of purified water at a flow rate of 1.5 BV / h of the resin volume. Then, 2 BV of 22% v / v ethanol is used to remove impurities in sequence at a flow rate of 1.5 BV / h of the resin volume. 3 BV of 75% v / v ethanol is used for elution at a flow rate of 3 BV / h of the resin volume. The elution liquid of 80% ethanol is dried to obtain a stevia polyphenol product.

[0044] Example 1

[0045] A method for simultaneously preparing high-content isochlorogenic acid A and isochlorogenic acid C, comprising the following steps:

[0046] (1) 300 g of stevia polyphenol product (total content of chlorogenic acid and its isomers 59.8 wt%, content of isochlorogenic acid A 30.3 wt%, content of isochlorogenic acid C 10.7 wt%) was dissolved in 9.0 kg of purified water, and the pH of the solution was adjusted to 3.5 to obtain treatment solution 1;

[0047] (2) The treatment solution 1 of step (1) was pumped into a resin column containing a polystyrene macroporous adsorption resin (resin type LX-B14 resin from Xi'an Lanxiao Science and Technology New Material Co., Ltd., resin pore volume 0.7 cm 3 / g, pore size 4.1 nm, particle size 0.3-0.4 mm, specific surface area 1200 m2 / g) to perform adsorption, and the feeding amount was 35 g of stevia polyphenol product per 100 ml of resin during adsorption. After completion, purified water was used for top charging, and the amount of purified water was 2 BV of the volume of the resin. The effluent during the adsorption process and the effluent during the purified water top charging process were mixed to obtain effluent 1. After the water top charging was completed, a methanol solution with a concentration of 60% v / v (amount of 2 BV of the volume of the resin) was used for elution to obtain effluent 2, and the stevia polyphenol was obtained by drying the effluent 2;

[0048] (3) The effluent 1 was concentrated to a solid content of 25 wt% using a reverse osmosis membrane, the pH of the concentrated solution was adjusted to 3.0 to obtain treatment solution 2, and the treatment solution 2 was extracted 3 times with ethyl acetate saturated aqueous solution, and the volume ratio of ethyl acetate saturated aqueous solution to treatment solution 2 was 1:1 during extraction. The raffinate phase 1 was collected, and the extraction phases were combined to obtain extraction phase 1;

[0049] (4) The pH of the raffinate phase 1 of step (3) was adjusted to 2.0, and then it was extracted 3 times with ethyl acetate-n-butanol saturated aqueous solution (mass ratio of ethyl acetate to n-butanol 9.0:1), and the raffinate phase 2 was collected and combined into effluent 2. The extraction phases were combined to obtain extraction phase 2;

[0050] (5) The extraction phase 1 of step (3) was dried and dissolved in an ethanol solution with a concentration of 15% v / v (added amount 2.0 times the mass of the dried extraction phase 1), and the pH was adjusted to 3.0 to obtain treatment solution 3. After stirring and crystallization for 6 h, the treatment solution 3 was filtered to obtain filter cake 1 and filtrate 1. The filtrate 1 was combined into effluent 1;

[0051] (6) The filter cake 1 of step (5) and purified water (mass ratio of filter cake 1 to purified water 1:2) were mixed, heated to 80°C, and stirred to dissolve the solids to obtain treatment solution 4. The treatment solution 4 was cooled to 25°C, and after crystallization for 4 h, it was filtered to obtain filter cake 2 and filtrate 2. The filter cake 2 was dried to obtain high-purity isochlorogenic acid C. The filtrate 2 was combined into extraction phase 1;

[0052] (7) The extraction phase two of step (4) was dissolved in saturated ethyl acetate (the amount of addition was 2.0 times the mass of the dried extraction phase two), and isocyanogenic acid A seeds (the amount of addition was 3wt% of the mass of the dried extraction phase two, and the seeds were the filter cake 3 of the previous batch) were added. The mixture was stirred at room temperature for 12h, and then filtered to obtain the filter cake 3 and the filtrate 3. The filtrate 3 was combined with the effluent 1. The filter cake 3 was dried and mixed with purified water (the amount of addition was 2.0 times the mass of the dried filter cake 3), and then treated by beating at 35℃ for 2h. The mixture was filtered to obtain the filter cake 4 and the filtrate 4. The filter cake 4 was dried to obtain high-purity isocyanogenic acid A. The filtrate 4 was combined with the extraction phase two.

[0053] Example 2

[0054] A method for simultaneously preparing high-content isocyanogenic acid A and isocyanogenic acid C, comprising the following steps:

[0055] (1) 300g of stevia polyphenol product (total content of chlorogenic acid and its isomers was 59.8wt%, content of isocyanogenic acid A was 30.3wt%, and content of isocyanogenic acid C was 10.7wt%) was dissolved in 15.0kg of purified water, and the pH of the solution was adjusted to 4.0 to obtain a treatment liquid 1.

[0056] (2) The treatment liquid 1 of step (1) was pumped into a resin column filled with a polystyrene macroporous adsorption resin (resin type was LX-62 resin from Xi'an Lanxiao Science and Technology New Material Co., Ltd., resin pore volume was 0.6cm 3 / g, pore size was 4.5nm, particle size was 0.3-0.4mm, and specific surface area was 1000㎡ / g) to perform adsorption. The feeding amount was 40g of stevia polyphenol product per 100ml of resin during adsorption. After completion, purified water was used for top charging, and the amount of purified water was 3BV of the resin. The effluent during adsorption and the effluent during top charging with purified water were mixed to obtain an effluent 1. After the end of top charging with water, an 80% v / v methanol solution (the amount of use was 4BV of the resin) was used for elution to obtain an effluent 2. The effluent 2 was dried to obtain stevia polyphenol.

[0057] (3) The effluent 1 was concentrated to a solid content of 35wt% by using a reverse osmosis membrane. The pH of the concentrated liquid was adjusted to 3.5 to obtain a treatment liquid 2. The treatment liquid 2 was extracted 4 times with ethyl acetate saturated aqueous solution. The volume ratio of ethyl acetate saturated aqueous solution to treatment liquid 2 was 3:1 during extraction. The raffinate phase one was collected, and the extraction phases were combined to obtain an extraction phase one.

[0058] (4) The pH of the raffinate phase one of step (3) was adjusted to 2.5, and then the extraction was performed 5 times with ethyl acetate-n-butanol saturated aqueous solution (the mass ratio of ethyl acetate to n-butanol was 9.5:1). The raffinate phase two was collected and combined with the effluent 2. The extraction phases were combined to obtain an extraction phase two.

[0059] (5) The dried extraction phase 1 of step (3) is dissolved in an ethanol solution with a concentration of 25% v / v (the amount of ethanol added is 2.5 times the mass of the dried extraction phase 1), and the pH is adjusted to 2.5 to obtain a treatment liquid 3. The treatment liquid 3 is stirred and crystallized for 10 h, and then filtered to obtain a filter cake 1 and a filtrate 1; the filtrate 1 is combined with the effluent 1;

[0060] (6) The filter cake 1 of step (5) is mixed with purified water (the mass ratio of the filter cake 1 to the purified water is 1:3), heated to 70°C, and stirred to dissolve the solid to obtain a treatment liquid 4. The treatment liquid 4 is cooled to 35°C, crystallized for 6 h, and then filtered to obtain a filter cake 2 and a filtrate 2. The filter cake 2 is dried to obtain high-purity iso-chlorogenic acid C; the filtrate 2 is combined with the extraction phase 1;

[0061] (7) The dried extraction phase 2 of step (4) is dissolved in saturated ethyl acetate (the amount of ethyl acetate added is 3.0 times the mass of the dried extraction phase 2), and iso-chlorogenic acid A seeds (the amount of seeds added is 5 wt% of the mass of the dried extraction phase 2, and the seeds are the filter cake 3 of the previous batch) are added. The mixture is stirred at room temperature for 6 h, and then filtered to obtain a filter cake 3 and a filtrate 3. The filtrate 3 is combined with the effluent 1. The filter cake 3 is dried and mixed with purified water (the amount of purified water added is 2.5 times the mass of the dried filter cake 3), and then treated by beating at 40°C for 3 h. The mixture is filtered to obtain a filter cake 4 and a filtrate 4. The filter cake 4 is dried to obtain high-purity iso-chlorogenic acid A; and the filtrate 4 is combined with the extraction phase 2.

[0062] Example 3

[0063] A method for simultaneously preparing high-content iso-chlorogenic acid A and iso-chlorogenic acid C, comprising the following steps:

[0064] (1) 300 g of a stevia polyphenol product (the total content of chlorogenic acid and its isomers is 59.8 wt%, the content of iso-chlorogenic acid A is 30.3 wt%, and the content of iso-chlorogenic acid C is 10.7 wt%) is dissolved in 12.0 kg of purified water, and the pH of the solution is adjusted to 3.8 to obtain a treatment liquid 1;

[0065] (2) The treatment liquid 1 of step (1) is pumped into a resin column filled with a polystyrene macroporous adsorption resin (DA201-CB1# from Jiangsu Suqing Water Treatment Engineering Group Co., Ltd., the resin pore volume is 0.65 cm 3 / g, the pore size is 4.2 nm, the particle size is 0.3-0.4 mm, and the specific surface area is 1100 m2 / g) to perform adsorption. During the adsorption, the amount of stevia polyphenol product fed per 100 ml of resin is 37 g, and after the adsorption is completed, purified water is used for top charging, and the amount of purified water used is 2.5 BV of the volume of the resin. The effluent during the adsorption and the effluent during the top charging of purified water are mixed to obtain an effluent 1. After the top charging of water is completed, a methanol solution with a concentration of 70% v / v (the amount used is 3 BV of the volume of the resin) is used for elution to obtain an effluent 2. The effluent 2 is dried to obtain a stevia polyphenol;

[0066] (3) Concentrate the effluent 1 to a solid content of 30 wt% by using reverse osmosis membrane, adjust the pH of the concentrated solution to 3.2 to obtain a treatment liquid 2, extract the treatment liquid 2 with saturated ethyl acetate aqueous solution for 4 times, the volume ratio of saturated ethyl acetate aqueous solution to treatment liquid 2 is 2:1 during extraction, collect the raffinate phase one, combine the extraction phases of multiple times to obtain the extraction phase one;

[0067] (4) Adjust the pH of the raffinate phase one of step (3) to 2.2, then extract with ethyl acetate-n-butanol saturated aqueous solution (the mass ratio of ethyl acetate to n-butanol is 9.3:1) for 4 times, collect the raffinate phase two and combine to the effluent 2, combine the extraction phases of multiple times to obtain the extraction phase two;

[0068] (5) After drying the extraction phase one of step (3), dissolve it in an ethanol solution with a concentration of 20% v / v (the added amount is 2.2 times the mass of the dried extraction phase one), adjust the pH to 2.8 to obtain a treatment liquid 3, stir the treatment liquid 3 to crystallize for 8 h, then filter to obtain a filter cake 1 and a filtrate 1; combine the filtrate 1 to the effluent 1;

[0069] (6) Mix the filter cake 1 of step (5) and purified water (the mass ratio of filter cake 1 to purified water is 1:3), heat to 75℃, stir to dissolve the solid, obtain a treatment liquid 4, cool the treatment liquid 4 to 30℃, crystallize for 5 h, then filter to obtain a filter cake 2 and a filtrate 2, dry the filter cake 2 to obtain high-purity isochlorogenic acid C; combine the filtrate 2 to the extraction phase one;

[0070] (7) After drying the extraction phase two of step (4), dissolve it in saturated ethyl acetate (the added amount is 2.3 times the mass of the dried extraction phase two), add isochlorogenic acid A seed crystal (the added amount is 4 wt% of the mass of the dried extraction phase two, the seed crystal is isochlorogenic acid A with a purity of ≥98% from Shanghai Yuanye Biotechnology Co., Ltd., batch number P11D11L134209), stir to crystallize at room temperature for 8 h, filter to obtain a filter cake 3 and a filtrate 3; combine the filtrate 3 to the effluent 1; mix the filter cake 3 after drying with purified water (the added amount is 2.2 times the mass of the filter cake 3 after drying), treat with beating at 38℃ for 3 h, filter to obtain a filter cake 4 and a filtrate 4; dry the filter cake 4 to obtain high-purity isochlorogenic acid A; combine the filtrate 4 to the extraction phase two.

[0071] Comparative Example 1

[0072] The difference between this comparative example and Example 3 is that in step (1), the pH of the treatment liquid is 3.0, and the other operations are the same as those of Example 3.

[0073] Comparative Example 2

[0074] The difference between this comparative example and Example 3 is that in step (1), the pH of the treatment liquid is 4.5, and the other operations are the same as those of Example 3.

[0075] Comparative Example 3

[0076] The difference between this comparative example and Example 3 is that in step (3), the effluent 1 is concentrated using a reverse osmosis membrane to a solid content of 40 wt%, while the other operations are the same as in Example 3.

[0077] Comparative Example 4

[0078] The difference between this comparative example and Example 3 is that in step (3), the pH of the treatment solution 2 is 2.5, and the other operations are the same as in Example 3.

[0079] Comparative Example 5

[0080] The difference between this comparative example and Example 3 is that in step (3), the pH of the treatment solution 2 is 4.0, and the other operations are the same as in Example 3.

[0081] Comparative Example 6

[0082] The difference between this comparative example and Example 3 is that in step (4), the pH of the raffinate phase 1 in step (3) is adjusted to 1.5, and the other operations are the same as in Example 3.

[0083] Comparative Example 7

[0084] The difference between this comparative example and Example 3 is that in step (4), the pH of the raffinate phase 1 in step (3) is adjusted to 3.0, and the other operations are the same as in Example 3.

[0085] Comparative Example 8

[0086] The difference between this comparative example and Example 3 is that in step (4), the mass ratio of ethyl acetate to n-butanol in the saturated aqueous solution of ethyl acetate-n-butanol is 10:1, and the other operations are the same as in Example 3.

[0087] Comparative Example 9

[0088] The difference between this comparative example and Example 3 is that in step (4), the mass ratio of ethyl acetate to n-butanol in the saturated aqueous solution of ethyl acetate-n-butanol is 8:1, and the other operations are the same as in Example 3.

[0089] Comparative Example 10

[0090] The difference between this comparative example and Example 3 is that in step (5), the concentration of the ethanol solution is 10% v / v, and the other operations are the same as in Example 3.

[0091] Comparative Example 11

[0092] The difference between this comparative example and Example 3 is that in step (5), the concentration of the ethanol solution is 30% v / v, and the other operations are the same as in Example 3.

[0093] Comparative Example 12

[0094] The difference between the present comparative example and Example 3 is that in step (5), the pH of the treatment liquid 3 is 2.0, and other operations are the same as those in Example 3.

[0095] Comparative Example 13

[0096] The difference between the present comparative example and Example 3 is that in step (5), the pH of the treatment liquid 3 is 3.5, and other operations are the same as those in Example 3.

[0097] Comparative Example 14

[0098] The difference between the present comparative example and Example 3 is that in step (7), the amount of isochlorogenic acid A seed added is 2wt% of the mass of the dried extraction phase, and other operations are the same as those in Example 3.

[0099] Comparative Example 15

[0100] The difference between the present comparative example and Example 3 is that in step (7), the temperature of the beating treatment is 25℃, and other operations are the same as those in Example 3.

[0101] Comparative Example 16

[0102] The difference between the present comparative example and Example 3 is that in step (7), the temperature of the beating treatment is 50℃, and other operations are the same as those in Example 3.

[0103] The yield, purity and yield of isochlorogenic acid A and isochlorogenic acid C in the above examples and comparative examples are shown in Table 1.

[0104] Table 1

[0105]

[0106]

[0107] From the above test results, it can be seen that the examples of the present application realize the efficient separation of isochlorogenic acid A and isochlorogenic acid C by the multi-step cooperative separation of resin adsorption, multi-stage extraction and fractional crystallization, and the optimization of the separation conditions of each stage.

[0108] Before being adsorbed by the macroporous adsorption resin, the pH of the treatment liquid 1 needs to be reasonably adjusted. In Comparative Example 1, the pH of the treatment liquid 1 is too low, the acidity is too strong, the structure of the target molecule is destroyed, and the yield of isochlorogenic acid C decreases significantly. In Comparative Example 2, the pH is too high, and under weak acid conditions, impurities and target molecules are co-dissolved, making subsequent separation difficult and reducing the purity of isochlorogenic acid C.

[0109] The pH of the treatment liquid is effectively adjusted in the present application in the primary extraction and the secondary extraction. The pH of the treatment liquid 2 or the raffinate phase I in Comparative Example 4 and Comparative Example 6 is too low, which can reduce the solubility of the target molecule and the polyphenol impurities in the solution, increase the extraction yield, but reduce the content of the effective components in the extract, and bring difficulties to the crystallization and purification process, and reduce the yield of the target product. The pH of the treatment liquid 2 or the raffinate phase I in Comparative Example 5 and Comparative Example 7 is too high, which can affect the extraction selectivity of the target molecule, and reduce the purity of the target product.

[0110] In the secondary extraction, the present application effectively controls the ratio of ethyl acetate and n-butanol to control the selective separation of the product. In Comparative Example 8, the ratio of ethyl acetate is too high, the polarity of the extractant is weak, the solubility of the target product is low, the extraction yield is low, and the yield of the product is reduced. In Comparative Example 9, the ratio of ethyl acetate is too low, which affects the selective separation of the product, and the purity of the product is reduced.

[0111] In the crystallization and separation of iso-chlorogenic acid C, the present application first dissolves the iso-chlorogenic acid C crude product in an ethanol solution of a certain concentration to remove impurities. In Comparative Example 10, the concentration of the ethanol solution is too low, and the impurities cannot be removed sufficiently. In Comparative Example 11, the concentration of the ethanol solution is too high, which can inhibit the crystallization of iso-chlorogenic acid C, and reduce the yield of the product.

[0112] In the crystallization and separation of iso-chlorogenic acid A, the present application adds a certain amount of crystal seeds to promote crystallization. In Comparative Example 14, the insufficient addition of the crystal seeds leads to incomplete crystallization. Furthermore, in the further purification of the preliminary crystallized iso-chlorogenic acid A, the present application uses water as a purification reagent, and performs beating treatment under certain conditions to remove impurities. In Comparative Example 15, the beating treatment temperature is too low, which can lead to the wrapping of the impurities. In Comparative Example 16, the beating treatment temperature is too high, which can lead to the dissolution of the product.

[0113] The principles and implementation modes of the present application are described by using specific examples in the present article. The above description of the examples is only used to help understand the method of the present application and its core idea, including the best mode, and also enables 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 those skilled in the art can make some improvements and modifications 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 literal expressions of the claims, or if they include equivalent structural elements that are not substantially different from the literal expressions of the claims, these other embodiments should also be included in the scope of the claims.

Claims

1. A method for simultaneously preparing high content of iso- chlorogenic acid A, iso-chlorogenic acid C, characterized in that, The method comprises the following steps: (1) dissolving the stevia polyphenol product in purified water, adjusting the pH of the solution to obtain a treatment liquid 1; (2) pumping the treatment liquid 1 into a resin column filled with styrene-based macroporous adsorption resin for adsorption, and then using purified water to top the material after the adsorption is completed; mixing the effluent during the adsorption and the effluent during the water topping to obtain effluent 1; and using a methanol solution to elute after the water topping is completed to obtain effluent 2; (3) concentrating the effluent 1 by using a reverse osmosis membrane, adjusting the pH of the concentrated solution to obtain a treatment liquid 2, and using ethyl acetate saturated aqueous solution to extract the treatment liquid 2 for multiple times to collect raffinate phase 1, and then combining the extraction phases of multiple extractions to obtain extraction phase 1; (4) adjusting the pH of the raffinate phase 1, and then using ethyl acetate-n-butanol saturated aqueous solution to extract for multiple times to collect raffinate phase 2, and then combining the extraction phases of multiple extractions to obtain extraction phase 2; (5) drying the extraction phase 1 and dissolving it in an ethanol solution, adjusting the pH to obtain a treatment liquid 3, stirring and crystallizing the treatment liquid 3, and then filtering to obtain filter cake 1 and filtrate 1; (6) mixing the filter cake 1 and purified water, heating and stirring until the solids are dissolved to obtain a treatment liquid 4, and then cooling and crystallizing the treatment liquid 4, filtering to obtain filter cake 2 and filtrate 2, and drying the filter cake 2 to obtain high-purity isochlorogenic acid C; (7) drying the extraction phase 2 of step (4), dissolving it in saturated ethyl acetate, adding isochlorogenic acid A crystal seeds, stirring and crystallizing at room temperature, filtering to obtain filter cake 3 and filtrate 3; drying the filter cake 3, mixing it with purified water, and beating and treating to obtain filter cake 4 and filtrate 4; and drying the filter cake 4 to obtain high-purity isochlorogenic acid A.

2. The method according to claim 1, wherein the method for simultaneously preparing high content of iso- chlorogenic acid A and iso-chlorogenic acid C is characterized in that, In step (1), the mass ratio of the stevia polyphenol product to purified water is 1:(30-50); and the pH of the treatment liquid 1 is 3.5-4.

0.

3. The method according to claim 1, wherein the method for simultaneously preparing high content of iso- chlorogenic acid A and iso-chlorogenic acid C is characterized in that, In step (2), the styrene-based macroporous adsorption resin has a pore volume of 0.6-0.7 cm 3 / g, a pore size of 4.1-4.5 nm, a particle size of 0.3-0.4 mm, and a specific surface area of 1000-1200 m2 / g; the feed amount during adsorption is 35-40 g of steviol glycoside product per 100 ml of resin; and the amount of purified water used during the purified water elution is 2-3 BV of the volume of the resin.

4. The method according to claim 1, wherein the method for simultaneously preparing high content of iso- chlorogenic acid A and iso-chlorogenic acid C is characterized in that, In step (2), the concentration of the methanol solution is 60-80% v / v, and the amount used is 2-4 BV of the resin volume; the feeding, water topping, and elution flow rates are all 1-2 BV / h of the resin volume.

5. The method according to claim 1, wherein the method is characterized in that, In step (3), the reverse osmosis membrane is used to concentrate the effluent 1 to a solid content of 25-35 wt%, the pH of the treatment liquid 2 is 3.0-3.5; and during the extraction, the volume ratio of ethyl acetate saturated aqueous solution to treatment liquid 2 is (1-3):

1.

6. The method according to claim 1, wherein the method is characterized in that, In step (4), the pH of the raffinate phase 1 is adjusted to 2.0-2.5; the mass ratio of ethyl acetate to n-butanol in the ethyl acetate-n-butanol saturated aqueous solution is (9.0-9.5):1; and during the extraction, the volume ratio of ethyl acetate-n-butanol saturated aqueous solution to the feed liquid is (2-3):

1.

7. The method according to claim 1, wherein the method is characterized in that, In step (5), the concentration of the ethanol solution is 15-25% v / v, the amount of the ethanol solution added is 2.0-2.5 BV of the mass of the dried extraction phase 1, the pH of the treatment liquid 3 is 2.5-3.0, and the stirring and crystallization time is 6-10 h.

8. The method for simultaneously preparing high-content isochlorogenic acid A and isochlorogenic acid C according to claim 1, characterized in that, In step (6), the mass ratio of the filter cake 1 to purified water is 1:(2-3), the temperature for heating and stirring until the solids are dissolved is 70-80°C, the temperature for cooling and crystallizing is 25-35°C, and the time is 4-6 h.

9. The method according to claim 1, wherein the method is for simultaneously preparing high content of iso- chlorogenic acid A and iso-chlorogenic acid C. In step (7), the amount of saturated ethyl acetate used is 2-3 times the mass of the dried extraction phase 2; the amount of isochlorogenic acid A crystal seeds added is 3-5% of the mass of the dried extraction phase 2; and the temperature for stirring and crystallizing at room temperature is 6-12 h.

10. The method according to claim 1, wherein the method for simultaneously preparing high content of iso- chlorogenic acid A and iso-chlorogenic acid C is characterized in that, In step (7), the amount of purified water added is 2.0-2.5 times the mass of the dried filter cake 3, and the temperature of the beating treatment is 35-40°C, and the time is 2-3 h.

Images