Method for continuously preparing neochlorogenic acid by two-phase system

Abstract

The invention discloses a method for continuously preparing neochlorogenic acid through a two-phase system, and belongs to the technical field of plant byproduct utilization. The method comprises the following steps: dissolving a raw material containing isochlorogenic acid in ethyl acetate, adding a proper amount of water, adding alkali to adjust the pH value to 7-11, and carrying out a first hydrolysis reaction in a water-ethyl acetate two-phase system reaction solution; after the first hydrolysis reaction is finished, adjusting the pH value of a reaction liquid water layer to 2-6, and separating the water layer from an ethyl acetate layer; adding water into the ethyl acetate layer, and continuing to repeat the hydrolysis reaction; collecting and combining water layers separated each time, performing macroporous adsorption resin purification and methanol desorption on the water layers, collecting desorption liquid, concentrating and spraying to obtain the neochlorogenic acid. The total content of chlorogenic acid in the prepared product can reach 75% and above, and the content of neochlorogenic acid 5-CQA can reach 50% and above. The method has the advantages of simple process, easiness in operation and the like, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of utilization of plant by-products, and particularly relates to a method for continuously preparing neochlorogenic acid in a two-phase system. Background Art

[0002] Chlorogenic acid (CA for short) is an ester compound formed by caffeic acid and quinic acid, belonging to phenylpropanoid compounds in plants. According to the binding position of caffeoyl on quinic acid, chlorogenic acid has multiple isomers. Among them, the three common mono-caffeoyl quinic acids are:

[0003] 3-CQA (3-caffeoyl quinic acid): The caffeoyl group is connected to the 3rd position of quinic acid.

[0004] 4-CQA (4-caffeoyl quinic acid): The caffeoyl group is connected to the 4th position of quinic acid.

[0005] 5-CQA (5-caffeoyl quinic acid, also called neochlorogenic acid): The caffeoyl group is connected to the 5th position of quinic acid.

[0006] These three isomers are usually collectively referred to as chlorogenic acid. In plants, 5-CQA is a common main component, but in some plants such as Houttuynia cordata Thunb. leaves, 3-CQA and 4-CQA are dominant.

[0007] Neochlorogenic acid, with the chemical formula C 16 H 18 O9 and a molecular weight of 354.31. In its structure, the caffeoyl group is connected to the 5th position of quinic acid, so it is also called 5-caffeoyl quinic acid (5-CQA). Neochlorogenic acid (5-CQA) exists in plants such as Eucommia ulmoides Oliv., Lonicera japonica Thunb., coffee, and Stevia rebaudiana Bertoni. Usually during the processing, a large amount of isochlorogenic acid by-products are generated while obtaining neochlorogenic acid (5-CQA).

[0008] Neochlorogenic acid is an important natural phenolic acid compound, and its unique structure endows it with excellent antioxidant properties. In the human body, neochlorogenic acid can scavenge free radicals, reduce oxidative stress reactions, thereby contributing to maintaining cell health and delaying the aging process. In addition, neochlorogenic acid also has various biological activities such as anti-inflammatory and antibacterial properties, and these characteristics make it have broad application prospects in the fields of medicine, health products, etc.

[0009] For example, in the pharmaceutical field, neochlorogenic acid is used to treat various inflammatory diseases due to its anti-inflammatory and antibacterial effects. Research shows that neochlorogenic acid can inhibit the production of inflammatory factors, reduce the inflammatory response, and has a certain therapeutic effect on inflammatory diseases such as arthritis and enteritis. At the same time, neochlorogenic acid also has anti-tumor effects, can inhibit the growth and spread of tumor cells, and provides new ideas for cancer treatment.

[0010] In the health product field, neochlorogenic acid has attracted much attention due to its antioxidant and anti-aging effects. By ingesting plant extracts rich in neochlorogenic acid, it can help the human body scavenge free radicals, improve antioxidant capacity, thereby delaying the aging process and maintaining youthful vitality.

[0011] The main components of isochlorogenic acid include three kinds: 3,5-dicaffeoylquinic acid (3,5-diCQA), 3,4-dicaffeoylquinic acid (3,4-diCQA), and 4,5-dicaffeoylquinic acid (4,5-diCQA).

[0012] The traditional method for preparing neochlorogenic acid is mainly to use Eucommia ulmoides or honeysuckle as raw materials, and obtain it through extraction and resin separation and purification. The content of neochlorogenic acid in the raw materials is less than 2%, and the neochlorogenic acid in the further refined product is only 5-10% (HPLC). High-content (content determined by HPLC above 90%) neochlorogenic acid products are mainly prepared by chemical synthesis methods, with complex reaction routes and requiring multiple functional group protections and deprotections.

[0013] The main product obtained in the processing of Stevia rebaudiana is stevioside. In addition to glycosides, an extract containing a relatively high content of isochlorogenic acid and a small amount of chlorogenic acid can also be obtained. The content of isochlorogenic acid in the extract containing isochlorogenic acid and chlorogenic acid can reach 65%-85%. This isochlorogenic acid mainly includes isochlorogenic acid A (3,5-diCQA) and isochlorogenic acid C (4,5-diCQA).

[0014] The currently market-recognized product is mainly neochlorogenic acid (5-CQA), so it is of great significance to extract high-purity neochlorogenic acid (5-CQA) products from plants. Converting isochlorogenic acid into neochlorogenic acid (5-CQA) has great economic value.

[0015] However, it is found in actual production that if the extracts of isochlorogenic acid and chlorogenic acid obtained from stevia are further hydrolyzed, 3,5-dicaffeoylquinic acid (3,5-diCQA) and 4,5-dicaffeoylquinic acid (4,5-diCQA) will be hydrolyzed to form neochlorogenic acid (5-CQA) and caffeic acid. At the same time, the generated 5-CQA will continue to be hydrolyzed to produce caffeic acid and quinic acid, resulting in the loss of neochlorogenic acid products. Therefore, high-purity neochlorogenic acid products cannot be obtained by simply hydrolyzing the extracts of isochlorogenic acid and chlorogenic acid from stevia, and other complex methods are not conducive to industrial promotion. Summary of the Invention

[0016] In order to overcome at least one deficiency of the prior art, one of the objectives of the present invention is to provide a method for continuously preparing neochlorogenic acid in a two-phase system.

[0017] Another objective of the present invention is to provide the neochlorogenic acid prepared by the above method.

[0018] The technical solution adopted by this application is as follows:

[0019] In the first aspect, this application provides a method for continuously preparing neochlorogenic acid in a two-phase system, and the method includes:

[0020] (1) Dissolve the raw material containing isochlorogenic acid in ethyl acetate, add an appropriate amount of water, and then add a base to adjust the pH to 7-11, and carry out the first hydrolysis reaction in the reaction solution of the water and ethyl acetate two-phase system; after the first hydrolysis reaction is completed, adjust the pH of the water layer of the reaction solution to 2-6, and carry out liquid-liquid extraction to separate the water layer and the ethyl acetate layer;

[0021] (2) Add water to the ethyl acetate layer, and then add a base to adjust the pH to 7-11, and carry out the second hydrolysis reaction in the reaction solution of the water and ethyl acetate two-phase system; after the second hydrolysis reaction is completed, adjust the pH of the water layer of the reaction solution to 2-6, and carry out liquid-liquid extraction to separate the water layer and the ethyl acetate layer;

[0022] (3) Collect and combine the water layers separated each time. After removing ethyl acetate, purify the water layer with macroporous adsorption resin, elute with methanol, collect the eluate, concentrate and spray to obtain neochlorogenic acid.

[0023] In some embodiments, the mass-volume ratio of the raw material containing isochlorogenic acid, ethyl acetate and water in step (1) is 1 kg: (1-3) L: (1-3) L.

[0024] In some preferred embodiments, the mass (kg)-volume (L) ratio of the raw material containing isochlorogenic acid, water and ethyl acetate in step (1) is 1 kg: (1-2) L: (1-2) L.

[0025] In some embodiments, in step (2), the volume ratio of ethyl acetate to water is (1-3):(1-3). Preferably, the volume ratio of ethyl acetate to water is (1-2):(1-2).

[0026] In some embodiments, the base is added in steps (1) and (2) to adjust the pH to 9-11.

[0027] In some embodiments, the base in steps (1) and (2) is one or more of sodium hydroxide, potassium hydroxide, and calcium hydroxide.

[0028] In some embodiments, the temperature of the hydrolysis reaction in steps (1) and (2) is 40°C-70°C, and the hydrolysis reaction time is 5-12 hours. Preferably, the temperature of the hydrolysis reaction is 40°C-60°C, and the hydrolysis reaction time is 5-10 hours.

[0029] In some preferred embodiments, in steps (1) and (2), the pH of the aqueous layer of the reaction solution is adjusted to 2-5.

[0030] In some embodiments, step (2) is repeated 1-2 times, preferably once.

[0031] In some embodiments, in the methanol decomposition in step (3), the methanol volume percentage is 50%-80% methanol, and the decomposition volume is 1-3 BV.

[0032] In some preferred embodiments, the methanol decomposition in step (3) is performed twice:

[0033] The first methanol decomposition is performed with methanol in a volume percentage of 10% to 30%, preferably 10% to 20% (V / V); and / or the decomposition volume is 1 to 3 BV, preferably 1 to 2 BV; the first decomposition solution is obtained and used to recover methanol for later use;

[0034] The second methanol analysis, methanol is 50%-80% methanol by volume, preferably 60%-80% (V / V) methanol, more preferably 70%-80% (V / V) methanol; and / or; the analysis volume is 1-3BV, preferably 2-3BV; the second analysis solution is obtained, which is used for concentration and spraying to obtain new chlorogenic acid.

[0035] In some embodiments, the mass percentage of isochlorogenic acid in the raw material is not less than 50%.

[0036] In a second aspect, the present application provides a new chlorogenic acid prepared by the above method.

[0037] The beneficial effects of this application are:

[0038] The preparation method of neochlorogenic acid provided by this application uses isochlorogenic acid, a by-product of stevia, as the raw material. The raw material is easily obtainable and has a low cost, making it suitable for industrial production. Processing and utilizing plant by-products and converting them into high-value-added products improve the overall economic efficiency.

[0039] The method of this application has a low requirement for the content of isochlorogenic acid in the raw material, and the content of chlorogenic acid (3-CQA + 4-CQA + 5-CQA) in the prepared product can reach 75% and above, and the content of neochlorogenic acid 5-CQA can reach 50% and above (the results are determined by HPLC).

[0040] The method of this application has the advantages of simple process and easy operation, and is suitable for large-scale industrial production. Description of the Drawings

[0041] Figure 1 It is a picture of the product obtained in the embodiment of this application.

[0042] Figure 2 It is the HPLC chromatogram of the product obtained in Example 1. Detailed Embodiments

[0043] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the protection scope of the present invention.

[0044] To solve the problem of the low utilization rate of isochlorogenic acid by-products in the processing of stevia, this application has developed a method for preparing neochlorogenic acid from isochlorogenic acid, with the expectation of high-value utilization of the low-value by-product isochlorogenic acid.

[0045] This application provides a method for continuously preparing neochlorogenic acid in a two-phase system, and the method includes:

[0046] S1. Dissolve the raw material containing isochlorogenic acid in ethyl acetate, add an appropriate amount of water, and then add a base to adjust the pH to 7-11, and carry out the first hydrolysis reaction in the reaction solution of the water and ethyl acetate two-phase system; after the first hydrolysis reaction is completed, adjust the pH of the water layer of the reaction solution to 2-6, and separate the water layer and the ethyl acetate layer;

[0047] S2. Add water to the ethyl acetate layer, and then add a base to adjust the pH to 7-11, and carry out the second hydrolysis reaction in the reaction solution of the water and ethyl acetate two-phase system. After the second hydrolysis reaction is completed, adjust the pH of the water layer of the reaction solution to 2-6, and carry out liquid-liquid extraction to separate the water layer and the ethyl acetate layer;

[0048] S3. Add water to the ethyl acetate layer, then add base to adjust the pH to 7 - 11, and conduct the third hydrolysis reaction in the two - phase reaction system of water and ethyl acetate; after the third hydrolysis reaction is completed, adjust the pH of the aqueous layer of the reaction solution to 2 - 6, and conduct liquid - liquid extraction to separate the aqueous layer and the ethyl acetate layer;

[0049] S4. Collect and combine the aqueous layers separated three times. After removing ethyl acetate, purify the aqueous layer with macroporous adsorption resin, elute with methanol, collect the eluate, concentrate and spray to obtain neochlorogenic acid.

[0050] This application's research found that the solubility differences between neochlorogenic acid and isochlorogenic acid in water and ethyl acetate are large. Ethyl acetate is more suitable as an extraction solvent, which is beneficial for removing impurities and reducing the loss of the target product, thereby significantly improving the purity and yield of the target product neochlorogenic acid. Other common solvents such as alcohols and acetone are miscible with water. If solvents with low polarity such as alkanes are used, the solubility of isochlorogenic acid is too small. Therefore, ethyl acetate is finally determined to be used.

[0051] Among them, neochlorogenic acid is a water - soluble substance with good solubility in water, so neochlorogenic acid is obtained in water. Isochlorogenic acid is mainly obtained in the ethyl acetate layer.

[0052] In the said method, before adjusting the base, the raw material containing isochlorogenic acid is in an acidic form and soluble in ethyl acetate. After adding the base, it is in a salt form and soluble in water; after subsequent acid adjustment, it becomes an acid and is soluble in ethyl acetate.

[0053] This application's research found that through multiple hydrolysis reactions, isochlorogenic acid in the plant extract raw material can be efficiently hydrolyzed to produce neochlorogenic acid, and at the same time, the generated neochlorogenic acid can be prevented from further hydrolysis. Under alkaline conditions, the plant extract raw material of neochlorogenic acid reacts in the two - phase system of water and ethyl acetate, which can remove impurities such as flavonoids and phospholipids; by adjusting the pH for liquid - liquid extraction, chlorogenic acid (including 5 - CQA, 4 - CQA, and 3 - CQA) can be effectively separated from isochlorogenic acid, and then continuous reaction can be carried out, significantly improving the yield of the product neochlorogenic acid. Through resin purification, water - soluble impurities such as salts are removed, and finally neochlorogenic acid with a high content is obtained.

[0054] In addition, considering production and benefits, multiple (three - time) hydrolysis is easier to achieve continuous production, and the resulting neochlorogenic acid product has better benefits. If the number of hydrolysis reactions exceeds three, the economic benefits will decrease, and the yield of neochlorogenic acid will not increase further. Therefore, to optimize cost - effectiveness, the optimal number of hydrolysis reactions is three.

[0055] In some of the embodiments, the raw material containing isochlorogenic acid is a plant - derived isochlorogenic acid extract, and the mass percentage content of isochlorogenic acid in the isochlorogenic acid extract is not less than 50% (HPLC).

[0056] More preferably, the extract contains 50%-80% by mass of isochlorogenic acid, the balance being inevitable impurities and possibly also containing a small amount of chlorogenic acid. The plant includes at least one of Eucommia ulmoides, Lonicera japonica, coffee, and Stevia rebaudiana. More preferably, the plant is Stevia rebaudiana.

[0057] In some embodiments, the temperature of the hydrolysis reaction in steps S1-S3 is 40°C-70°C, and the hydrolysis reaction time is 5-12 h; preferably, the temperature of the hydrolysis reaction is 40°C-60°C, the pH of the hydrolysis reaction is 9-11, and the hydrolysis reaction time is 5-10 h.

[0058] When the system pH is 7-9, the system pH is relatively low, the reaction rate is slightly slower, and the hydrolysis reaction time needs to be relatively extended, which may cause loss of the product. Therefore, more preferably, the pH is 9-11, and the hydrolysis reaction time is 5-10 h.

[0059] If the system pH is higher than 11, excessive use of the base will cause further reaction of neochlorogenic acid and result in loss of the product.

[0060] In some embodiments, the base used in the base hydrolysis reaction is one or more of sodium hydroxide, potassium hydroxide, and calcium hydroxide.

[0061] The amount of the base added can be calculated according to the pH value. When adding the base, stirring is required. After the system is homogeneous, the pH value is measured again.

[0062] In some embodiments, in step S1, the mass (kg) to volume (L) ratio of the raw material containing isochlorogenic acid, water, and ethyl acetate is 1 kg:(1-3) L:(1-3) L; preferably 1 kg:(1-2) L:(1-2) L.

[0063] In some embodiments, in steps S2 and S3, the volume ratio of ethyl acetate to water is (1-3):(1-3). Preferably, the volume ratio of ethyl acetate to water is (1-2):(1-2).

[0064] In some embodiments, in steps S1, S2, and S3, before separating the aqueous layer and the ethyl acetate layer by liquid-liquid extraction, the pH of the aqueous layer of the reaction solution is adjusted to 2-6 with an acid, preferably adjusted to pH 3-5, to facilitate better separation of the generated 5-CQA and isochlorogenic acid. When adjusting the pH value, stir appropriately, let it stand for stratification, and measure whether the pH of the aqueous layer reaches the requirement after stratification.

[0065] There is no special limitation on the acid used for pH adjustment in this application. Exemplarily, sulfuric acid can be used for pH adjustment; the concentration of sulfuric acid is preferably 50%-80%.

[0066] Macroporous adsorption resins can selectively adsorb target compounds (neochlorogenic acid 5-CQA, 3-CQA, and 4-CQA) through their porous structures and chemical properties. The macroporous adsorption resins are not limited in this application, and conventional finished macroporous adsorption resins can be selected. For example, AB-8, D101, HPD722, LX-8, DA-201, XDA-8, and NKA-9. D101 and DA201 are preferred.

[0067] Methanol, as an organic solvent, has strong dissolving ability and can effectively desorb the target components adsorbed by the resin. The concentrations of methanol mentioned in this application are all volume percentage concentrations.

[0068] In some embodiments, in the methanol desorption in step S4, the methanol is methanol with a volume percentage of 50%-80%, and the desorption volume is 1-3BV.

[0069] In some preferred embodiments, the methanol desorption in step S4 is two-stage methanol desorption:

[0070] For the first methanol desorption, the methanol is methanol with a volume percentage of 10%-30%, preferably methanol with a volume percentage of 10%-20%; the desorption volume is 1-3BV (1-3 times the resin bed volume), preferably 1-2BV (1-2 times the resin bed volume), and it is desorbed through the resin column at an appropriate flow rate (such as 1BV / h) to obtain the first desorbed solution for recovering methanol for standby;

[0071] For the second methanol desorption, the methanol is methanol with a volume percentage of 50%-80%, preferably methanol with a volume percentage of 70%-80%, the desorption volume is 1-3BV, preferably 2-3BV, and it is desorbed through the resin column at an appropriate flow rate (such as 1-1.5BV / h), and the second desorbed solution is collected, concentrated, and spray-dried to obtain neochlorogenic acid.

[0072] This application finds that macroporous adsorption resins have good adsorption effects on chlorogenic acid. When using low-concentration methanol for desorption, water-soluble and small-molecule impurities in the system are preferentially desorbed. When using high-concentration methanol, macromolecular chlorogenic acid in the system can be desorbed.

[0073] If only one desorption is carried out with methanol, the desorption effect is poor. The gradient desorption method explored in this application is convenient for increasing the content of neochlorogenic acid in the product.

[0074] In terms of methanol concentration, when it is lower than 10%, the desorption effect is poor and a larger column volume is used. When it is higher than 80%, it is not conducive to solvent recovery and utilization in production.

[0075] The present application provides a finished product of neochlorogenic acid prepared by the above method. The finished product of neochlorogenic acid contains neochlorogenic acid (5-CQA) with a mass percentage of 50% or more, and also contains a small amount of 3-CQA and 4-CQA. The total chlorogenic acid (3-CQA + 4-CQA + 5-CQA) content is not less than 75%. The product has a loose texture and is brown in color. The picture of the product is shown in Figure 1 。

[0076] The isochlorogenic acid raw material used in the following examples is the by-product isochlorogenic acid obtained from the extraction of stevia. The content of isochlorogenic acid in the raw material is 50%-80% (HPLC).

[0077] The stevia processing method is to use a pure solvent or water for extraction. After concentration, liquid-liquid separation is carried out using ethyl acetate to separate stevioside from isochlorogenic acid, and stevioside is further refined. Isochlorogenic acid is refined to obtain a raw material containing 50%-80% isochlorogenic acid. For details, please refer to the method mentioned in Chinese Patent CN 109265346 A (publication date: January 25, 2019).

[0078] The macroporous adsorption resin used in the examples is a commercially available product without special restrictions.

[0079] In the research and development process of the present application, various experiments of small-scale and medium-scale specifications were carried out. The labels of the examples do not represent the order of experiments in the research and development process.

[0080] For those not specifying the specific technology or conditions in the examples, they shall be in accordance with the technology or conditions described in the literature in this field or in accordance with the product instructions. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be purchased through regular channels.

[0081] The present application will be described below in conjunction with examples.

[0082] Example 1

[0083] This example provides a method for continuously preparing neochlorogenic acid in a two-phase system, and the steps are as follows:

[0084] Add 200 kg of isochlorogenic acid raw material with a content of 50.2% into the reaction tank, dissolve it with 200 L of ethyl acetate, then add 200 L of water, add sodium hydroxide to adjust the pH to 10, and react at 55 °C for 8 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 3, separate the aqueous layer, and the remaining in the reaction tank is the ethyl acetate layer. Continue to add 200 L of water to the reaction tank, add sodium hydroxide to adjust the pH to 10, and react at 55 °C for 8 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 3, separate the aqueous layer, and the remaining in the reaction tank is the ethyl acetate layer. Continue to add 200 L of water to the reaction tank, add sodium hydroxide to adjust the pH to 10, and react at 55 °C for 8 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 3, and separate the aqueous layer.

[0085] Combine the aqueous layers separated three times, remove ethyl acetate, then carry out macroporous adsorption resin purification, elute with 10% methanol for 2 BV, then elute with 70% methanol for 3 BV, concentrate the eluate of the second methanol elution, and spray dry to obtain the finished product.

[0086] Figure 2 It is the HPLC chromatogram of the product obtained in Example 1. The content of neochlorogenic acid (5-CQA) in the finished product is 55.1%, and the content of total chlorogenic acid (3-CQA + 4-CQA + 5-CQA) is 85.2%.

[0087] Example 2

[0088] This example provides a method for continuously preparing neochlorogenic acid in a two-phase system, and the steps are as follows:

[0089] Add 200 kg of isochlorogenic acid raw material with a content of 50.2% into the reaction tank, dissolve it with 200 L of ethyl acetate, then add 400 L of water, add sodium hydroxide to adjust the pH to 11, and react at 40 °C for 6 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 4, separate the aqueous layer, and the remaining in the reaction tank is the ethyl acetate layer. Continue to add 400 L of water to the reaction tank, add sodium hydroxide to adjust the pH to 11, and react at 40 °C for 6 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 4, and separate the aqueous layer.

[0090] Combine the aqueous layers separated twice, remove ethyl acetate, then carry out macroporous adsorption resin purification, elute with 15% methanol for 2 BV, then elute with 70% methanol for 3 BV, concentrate the eluate of the second methanol elution, and spray dry to obtain the finished product.

[0091] The content of neochlorogenic acid (5-CQA) in the finished product is 50.3%, and the content of total chlorogenic acid (3-CQA + 4-CQA + 5-CQA) is 75.4%.

[0092] Because only two hydrolysis reactions are carried out in this embodiment, the contents of neochlorogenic acid and chlorogenic acid in the finished product are relatively low.

[0093] Example 3

[0094] This embodiment provides a method for continuously preparing neochlorogenic acid in a two-phase system, and the steps are as follows:

[0095] Add 200 g of isochlorogenic acid raw material with a content of 50.2% into a reaction vessel, add 400 mL of ethyl acetate to dissolve it, then add 200 mL of water, add sodium hydroxide to adjust the pH to 9, and react at 60 °C for 6 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 2, separate the water layer, and the remaining in the reaction vessel is the ethyl acetate layer. Continue to add 200 mL of water to the reaction vessel, add sodium hydroxide to adjust the pH to 9, and react at 60 °C for 6 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 2, separate the water layer, and the remaining in the reaction vessel is the ethyl acetate layer. Continue to add 400 mL of water to the reaction vessel, add sodium hydroxide to adjust the pH to 9, and react at 60 °C for 6 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 2, and separate the water layer.

[0096] Combine the water layers separated three times, remove ethyl acetate, then carry out macroporous adsorption resin purification, elute with 10% methanol for 2 BV, then elute with 70% methanol for 2 BV, concentrate the eluate of the second methanol elution, and spray dry to obtain the finished product. The content of neochlorogenic acid (5-CQA) in the finished product is 65.1%, and the content of total chlorogenic acid (3-CQA + 4-CQA + 5-CQA) is 90.2%.

[0097] Example 4

[0098] This embodiment provides a method for continuously preparing neochlorogenic acid in a two-phase system, and the steps are as follows:

[0099] Add 200 kg of isochlorogenic acid raw material with a content of 79.8% into a reaction tank, add 400 L of ethyl acetate to dissolve it, then add 400 L of water, add potassium hydroxide to adjust the pH to 11, and react at 50 °C for 5 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 3, separate the water layer, and the remaining in the reaction tank is the ethyl acetate layer. Continue to add 400 L of water to the reaction tank, add potassium hydroxide to adjust the pH to 11, and react at 50 °C for 5 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 3, separate the water layer, and the remaining in the reaction tank is the ethyl acetate layer. Continue to add 400 L of water to the reaction tank, add potassium hydroxide to adjust the pH to 11, and react at 50 °C for 5 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 3, and separate the water layer.

[0100] Combine the aqueous layers after three separations. After removing ethyl acetate, perform purification on macroporous adsorption resin. Elute with 10% methanol for 1 BV, then elute with 70% methanol for 3 BV. Concentrate the eluate from the second methanol elution and perform spray drying to obtain the finished product. The content of neochlorogenic acid (5-CQA) in the said finished product is 68.2%, and the content of total chlorogenic acid (3-CQA + 4-CQA + 5-CQA) is 93.1%.

[0101] Example 5

[0102] This example provides a method for continuously preparing neochlorogenic acid in a two-phase system, and the steps are as follows:

[0103] Add 200 kg of isochlorogenic acid raw material with a content of 79.8% into a reaction tank, add 400 L of ethyl acetate to dissolve it, then add 200 L of water, add sodium hydroxide to adjust the pH to 10, and react at 70 °C for 5 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 3, and separate the aqueous layer. What remains in the reaction tank is the ethyl acetate layer. Continue to add 200 L of water to the reaction tank, add sodium hydroxide to adjust the pH to 10, and react at 70 °C for 5 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 3, and separate the aqueous layer. What remains in the reaction tank is the ethyl acetate layer. Continue to add 200 L of water to the reaction tank, add sodium hydroxide to adjust the pH to 10, and react at 70 °C for 5 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 3, and separate the aqueous layer.

[0104] Combine the aqueous layers after three separations. After removing ethyl acetate, perform purification on macroporous adsorption resin. Elute with 10% methanol for 2 BV, then elute with 75% methanol for 2 BV. Concentrate the eluate from the second methanol elution and perform spray drying to obtain the finished product. The content of neochlorogenic acid (5-CQA) in the said finished product is 66.3%, and the content of total chlorogenic acid (3-CQA + 4-CQA + 5-CQA) is 89.6%.

[0105] Example 6

[0106] This example provides a method for continuously preparing neochlorogenic acid in a two-phase system, and the steps are as follows:

[0107] Add 200 g of isochlorogenic acid raw material with a content of 79.8% into a reaction vessel, dissolve it with 400 mL of ethyl acetate, then add 200 mL of water, add sodium hydroxide to adjust the pH to 8, and react at 70 °C for 12 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 3, separate the aqueous layer, and the remaining in the reaction vessel is the ethyl acetate layer. Continue to add 200 mL of water to the reaction vessel, add sodium hydroxide to adjust the pH to 8, and react at 70 °C for 12 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 3, separate the aqueous layer, and the remaining in the reaction vessel is the ethyl acetate layer. Continue to add 200 mL of water to the reaction vessel, add sodium hydroxide to adjust the pH to 8, and react at 70 °C for 12 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 3, and separate the aqueous layer.

[0108] Combine the aqueous layers separated three times. After removing ethyl acetate, carry out macroporous adsorption resin purification. Use 10% methanol to elute 2 BV, then use 70% methanol to elute 2 BV. Concentrate the eluate of the second methanol elution, and spray dry to obtain the finished product. The content of neochlorogenic acid (5-CQA) in the obtained finished product is 65.2%, and the content of total chlorogenic acid (3-CQA + 4-CQA + 5-CQA) is 88.7%.

[0109] In this example, when the pH value is adjusted down to 8, it is necessary to extend the hydrolysis time to achieve a better hydrolysis effect. However, the total hydrolysis reaction for three times takes 36 hours in total, which will increase the cost from the perspective of production.

[0110] Comparative Example 1

[0111] The difference between this comparative example and the method of Example 3 is only that: the reaction system is changed to n-hexane and water, that is, ethyl acetate is replaced by n-hexane, and other steps remain unchanged. The results show that the content of neochlorogenic acid (5-CQA) in the obtained finished product is 41.1%, and the content of total chlorogenic acid (3-CQA + 4-CQA + 5-CQA) is 65.2%.

[0112] Because the reaction system is changed to n-hexane and water, and n-hexane has poor solubility for isochlorogenic acid and impurities, it is difficult to separate isochlorogenic acid from neochlorogenic acid, resulting in a lower content of neochlorogenic acid.

[0113] Comparative Example 2

[0114] In this comparative example, the pH and hydrolysis time of the reaction system in the method of Example 6 are adjusted: the pH of the reaction system is adjusted to 12 and reacted for 10 h.

[0115] The specific steps are as follows:

[0116] Add 200 g of isochlorogenic acid raw material with a content of 79.8% into a reaction vessel, dissolve it with 400 mL of ethyl acetate, then add 200 mL of water, add sodium hydroxide to adjust the pH to 12, and react at 70 °C for 10 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 3, separate the aqueous layer, and the remaining in the reaction vessel is the ethyl acetate layer.

[0117] After removing ethyl acetate from the aqueous layer of the first hydrolysis reaction, carry out macroporous adsorption resin purification. Elute with 10% methanol for 2 BV, then elute with 70% methanol for 2 BV. Concentrate the eluate of the second methanol elution and spray dry. The results show that neochlorogenic acid is not obtained.

[0118] Because the pH of the system was adjusted to 12 and reacted for 10 h, the isochlorogenic acid in the system was completely hydrolyzed into caffeic acid and quinic acid, so neochlorogenic acid could not be obtained.

[0119] Comparative Example 3

[0120] Add 200 kg of isochlorogenic acid raw material with a content of 50.2% into a reaction tank, add 200 L of water, add sodium hydroxide to adjust the pH to 9, and react at 60 °C for 6 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 2, add 200 L of ethyl acetate, carry out liquid-liquid extraction, and separate the aqueous layer.

[0121] After removing ethyl acetate from the aqueous layer of the first hydrolysis reaction, carry out macroporous adsorption resin purification. Elute with 10% methanol for 2 BV, then elute with 70% methanol for 3 BV. Concentrate the eluate, and the content of neochlorogenic acid (5-CQA) in the obtained finished product is 22.1%, and the content of total chlorogenic acid (3-CQA + 4-CQA + 5-CQA) is 45.2%.

[0122] Hydrolysis is carried out under the condition of a single water system, and impurities in the raw materials will affect the reaction. For example, some impurities will react with the base, resulting in low reaction efficiency and low content of neochlorogenic acid in the product.

[0123] Comparative Example 4

[0124] Add 200 g of isochlorogenic acid raw material with a content of 50.2% to the reaction vessel, dissolve it with 400 mL of ethyl acetate, then add 200 mL of water, add sodium hydroxide to adjust the pH to 9, and react at 60 °C for 6 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 2, separate the aqueous layer, and the remaining in the reaction vessel is the ethyl acetate layer. Continue to add 200 mL of water to the reaction vessel, add sodium hydroxide to adjust the pH to 9, and react at 60 °C for 6 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 2, separate the aqueous layer, and the remaining in the reaction vessel is the ethyl acetate layer. Continue to add 400 mL of water to the reaction vessel, add sodium hydroxide to adjust the pH to 9, and react at 60 °C for 6 h to obtain a reaction solution. Add 60% sulfuric acid to the reaction solution to adjust the pH to 2, and separate the aqueous layer.

[0125] Combine the aqueous layers separated three times, remove ethyl acetate, then carry out macroporous adsorption resin purification, elute with 70% methanol for 3 BV, concentrate the eluate, and spray dry to obtain the finished product. The content of neochlorogenic acid in the finished product is 45.1%, and the content of chlorogenic acid (3-CQA + 4-CQA + 5-CQA) is 65.3%.

[0126] Using only 70% methanol for a single elution fails to separate impurities and the product in the system well, resulting in low contents of neochlorogenic acid and chlorogenic acid in the obtained product.

[0127] In summary, the preparation method of neochlorogenic acid provided in this application uses isochlorogenic acid, a by-product of stevia, as the raw material. The raw material is easily available and has a low cost, making it more suitable for industrial production. The method processes and utilizes plant by-products and converts them into neochlorogenic acid products with high added value, enhancing the overall economic benefits.

[0128] In the continuous preparation of neochlorogenic acid in a two-phase system of this application, isochlorogenic acid is converted into neochlorogenic acid with higher economic value. The setting of the two-phase system and the results of the corresponding hydrolysis reaction are the result of the joint control of the reaction pH value, reaction temperature, and reaction time. The higher the reaction pH value and the reaction temperature, the shorter the reaction time. In Example 3, the reaction pH is 9, but the reaction temperature is 5 °C higher than that in Example 1. Under the combined action of several parameters, the yield is relatively high. Similarly, in Examples 4, 5, and 6, within the range defined in this application, different temperature and pH conditions are adjusted, and according to liquid phase monitoring, neochlorogenic acid products with a relatively stable content reaching 50% and above are obtained.

[0129] The research of this application also finds that when the hydrolysis temperature is set at 75 °C, ethyl acetate in the system is extremely volatile, and the operation difficulty is relatively large.

[0130] When the pH is adjusted to 7 or below, the system is difficult to react. When the system pH is adjusted to 8, the hydrolysis time needs to be appropriately extended, otherwise the reaction yield will be low.

[0131] Except for n-hexane, other solvents such as methanol and acetone are soluble in water and it is difficult to form a two-phase system. Solvents with relatively low polarity such as toluene have low solubility for isochlorogenic acid and chlorogenic acid, and the liquid-liquid separation effect is poor.

[0132] In the two-step analytical process, if a solvent with a high degree is used in the first step, it is easy to cause loss of the product chlorogenic acid.

[0133] In summary, the method of this application has advantages such as simple process and easy operation, and is suitable for large-scale industrial production.

[0134] The above is a further detailed description of the present invention, and it should not be regarded as a limitation to the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention belongs, simple deductions or substitutions without departing from the concept of the present invention are within the protection scope of the present invention.

Claims

1. A method for continuously preparing neochlorogenic acid in a two-phase system, characterized in that: The method includes: (1) Dissolve the raw material containing isochlorogenic acid in ethyl acetate, add an appropriate amount of water, and then add a base to adjust the pH to 7 - 11, and conduct the first hydrolysis reaction in the reaction solution of the water and ethyl acetate two-phase system; after the first hydrolysis reaction ends, adjust the pH of the water layer of the reaction solution to 2 - 6, and conduct liquid-liquid extraction to separate the water layer and the ethyl acetate layer; (2) Add water to the ethyl acetate layer, and then add a base to adjust the pH to 7 - 11, and conduct the second hydrolysis reaction in the reaction solution of the water and ethyl acetate two-phase system; after the second hydrolysis reaction ends, adjust the pH of the water layer of the reaction solution to 2 - 6, and conduct liquid-liquid extraction to separate the water layer and the ethyl acetate layer; repeat this step 0 - 2 times; (3) Collect and combine the water layers separated each time, after removing ethyl acetate, purify the water layer with macroporous adsorption resin, elute with methanol, collect the eluate, concentrate and spray to obtain neochlorogenic acid.

2. The method according to claim 1, wherein In step (1), the mass-volume ratio of the raw material containing isochlorogenic acid, ethyl acetate and water is 1 kg:(1 - 3) L:(1 - 3) L.

3. The method according to claim 1, wherein In step (2), the volume ratio of ethyl acetate to water is (1 - 3):(1 - 3).

4. The method according to claim 1, wherein The base in steps (1) and (2) is one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, and / or, adding a base to adjust the pH to 7 - 11, preferably pH 9 - 11; and / or, after the hydrolysis reaction ends, adjust the pH of the water layer of the reaction solution to 2 - 6, preferably adjust to 2 - 5.

5. The method according to any one of claims 1-4, characterized in that, The temperature of the hydrolysis reaction in steps (1) and (2) is 40°C - 70°C, and the time of the hydrolysis reaction is 5 - 12 h; preferably the temperature of the hydrolysis reaction is 40°C - 60°C, and the time of the hydrolysis reaction is 5 - 10 h.

6. The method according to claim 5, characterized in that, Repeat step (2) 1 - 2 times, preferably 1 time.

7. The method according to claim 1, wherein In the methanol elution in step (3), the methanol is methanol with a volume percentage of 50% - 80%, and the elution volume is 1 - 3 BV.

8. The method according to claim 1 or 7, characterized in that The methanol elution in step (3) is two-stage methanol elution: For the first methanol elution, the methanol is methanol with a volume percentage of 10% - 30%, and the elution volume is 1 - 3 BV, preferably 1 - 2 BV, to obtain the first eluate for recycling methanol for standby; For the second methanol elution, the methanol is methanol with a volume percentage of 50% - 80%, and the elution volume is 1 - 3 BV, preferably 2 - 3 BV, to obtain the second eluate for concentration and spraying to obtain neochlorogenic acid.

9. The method according to claim 1, wherein The mass percentage content of isochlorogenic acid in the raw material is not less than 50%.

10. Neochlorogenic acid prepared by the method according to any one of claims 1 - 9.

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