A method for extracting epicatechin using ionic liquid and its application

Abstract

The present invention belongs to the technical field of separation of natural active ingredients, and specifically relates to a method and application of extracting epicatechin using ionic liquid. The preparation method comprises the following steps: (1) mixing tea residues and ionic liquids evenly, performing extraction reaction, and then filtering to remove impurities to obtain tea residue extract; (2) applying the tea residue extract to a column, separating and eluting by preparative column chromatography, collecting the fraction containing epicatechin according to the position of the chromatographic peak, and then freeze-drying to obtain epicatechin; the ionic liquid comprises 1-butyl-3-methylimidazole chloride and / or 1-ethyl-3-methylimidazole chloride. The present invention extracts tea residues under specific conditions by using specific ionic liquids, and can extract epicatechin from tea residues with extremely high efficiency, and the extraction performance is significantly improved compared with traditional reagents, and the operation is convenient, time-saving, and cost-effective, and it is easy to industrialize production, suitable for a wide range of application fields and has huge market value.

Description

Technical Field

[0001] The invention belongs to the technical field of natural active ingredient separation, and specifically relates to a method for extracting epicatechin by using ionic liquid and application thereof. Background Art

[0002] In China alone, 5 million tons of tea waste are produced each year, and most of these wastes are landfilled or burned. However, these waste tea wastes actually contain a large number of important bioactive substances that can be used for commercialization, such as tea polyphenols. As an antioxidant, there are many industries in the market that need tea polyphenols, such as health products, food, and even the pharmaceutical industry. If tea polyphenols can be extracted from tea wastes as much as possible, it will provide strong support for the innovation and development of related industries.

[0003] As a natural compound in tea polyphenols, epicatechin has multiple biological activities such as strong antioxidant, anti-inflammatory, antibacterial and anti-cancer effects. It has attracted much attention in the field of health and wellness. Its significant health benefits include protecting cardiovascular, preventing chronic diseases, enhancing the immune system and regulating metabolism. It has been widely used in functional foods, health products and cosmetics. With the continuous improvement of consumers' health awareness, the market demand for epicatechin products has grown rapidly, creating huge business opportunities for companies in the health industry.

[0004] Currently, the traditional reagents used to extract epicatechin components from tea residues are mostly organic reagents, but there are many problems. First, the extraction efficiency of traditional reagents is low, which not only makes the extraction cost high but also fails to effectively extract some substances; secondly, traditional organic reagents are mostly volatile reagents, which are easy to pollute the environment and cause harm to the operator's body during the extraction process.

[0005] Therefore, there is an urgent need to provide a method with simple process, green environmental protection and high extraction efficiency to effectively extract the active ingredient of epicatechin in tea residues, so as to promote the in-depth development of tea resources, which is of great strategic significance for the development of related industries. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method and application of extracting epicatechin using ionic liquid to achieve efficient extraction of epicatechin with simple process, environmental protection and mildness.

[0007] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0008] In a first aspect, the present invention provides a method for preparing epicatechin, comprising the following steps:

[0009] (1) Evenly mixing tea residues and ionic liquid, performing extraction reaction, and then filtering to remove impurities to obtain tea residue extract;

[0010] (2) applying the tea residue extract to a column, separating and eluting by preparative column chromatography, collecting the epicatechin-containing fractions according to the chromatographic peak positions, and freeze-drying the fractions to obtain epicatechin;

[0011] The ionic liquid includes 1-butyl-3-methylimidazolium chloride and / or 1-ethyl-3-methylimidazolium chloride.

[0012] As a new type of "green reagent", ionic liquids have unique advantages, such as low volatility, low pollution, strong solubility, low dissolved oxygen content, and the extracted substances are not easy to be oxidized. However, the current application of ionic liquids in extraction, especially the relevant research on the extraction of epicatechin in tea residues, is still blank. The inventors of this application have found through a large number of experimental studies that when 1-butyl-3-methylimidazolium chloride or 1-ethyl-3-methylimidazolium chloride ionic liquids are used to extract waste tea residues, the extraction efficiency of tea polyphenols in tea residues can be greatly improved compared with traditional reagents, especially epicatechin, which has a very high extraction value.

[0013] Preferably, the ionic liquid is 1-butyl-3-methylimidazolium chloride.

[0014] Through experimental research, it was found that 1-butyl-3-methylimidazolium chloride has the best extraction efficiency for epicatechin in waste tea residues, thus maximizing the utilization of the residual value of tea residues.

[0015] Preferably, the extraction reaction conditions are: extraction at 50-70° C. for 4-6 hours.

[0016] Through experimental exploration, it was found that when the specific ionic liquid is used to extract epicatechin from tea dregs to obtain epicatechin, the extraction temperature and time will have a significant impact on the extraction effect. If the extraction time is less than 4h, the ionic liquid cannot fully contact the epicatechin active ingredient in the tea dregs, resulting in a decrease in the dissolution rate of epicatechin. Once the extraction time exceeds 6h, the dissolved epicatechin will react with other components in the tea dregs unnecessarily, thereby affecting the purity and quality of epicatechin. As for the choice of temperature, when the extraction temperature is lower than 50°C, the diffusivity of the ionic liquid will be affected, which is insufficient to effectively dissolve the epicatechin component in the tea dregs, resulting in low extraction efficiency. When the temperature is higher than 70°C, the excessively high temperature may reduce the thermal stability of the ionic liquid and even cause thermal degradation of epicatechin, thereby affecting the selectivity and yield of the extraction.

[0017] Preferably, the extraction reaction conditions are: extraction at 70° C. for 4 hours.

[0018] When the tea residues are extracted under the specific extraction conditions of 70°C for 4 hours, the optimal extraction efficiency of epicatechin can be effectively guaranteed, while the degradation of the extracted epicatechin can be effectively avoided.

[0019] Preferably, the mass ratio of the tea residue to the ionic liquid is tea residue: ionic liquid = 1: (5-15).

[0020] Preferably, the mass ratio of the tea residue to the ionic liquid is tea residue:ionic liquid=1:10.

[0021] Through experimental exploration, it was found that when the mass ratio of tea residue to the ionic liquid adopts the above preferred ratio range of 1: (5-15), the extraction efficiency of epicatechin can be significantly improved. When the amount of ionic liquid is too high, it will cause waste of resources and increase production costs. At the same time, it may cause the dissolution or reaction of other non-target compounds, thereby reducing the selective extraction efficiency of epicatechin; and when the amount of ionic liquid is too low, the epicatechin in the tea residue cannot be fully dissolved, resulting in a low extraction efficiency of epicatechin. When the mass ratio of tea residue to ionic liquid is 1:10, it can ensure excellent epicatechin extraction efficiency while minimizing costs and improving the quality of epicatechin components.

[0022] Preferably, the water content of the tea residue is controlled below 5%.

[0023] During the ionic liquid extraction, the water content of the tea residue is kept as low as possible to not exceed 5%. Otherwise, too high a water content may limit the solubility of the ionic liquid, and the water competitively interacts with the ionic liquid, making it difficult for the ionic liquid to effectively react or dissolve the epicatechin in the tea residue, thereby reducing the extraction rate of epicatechin.

[0024] Preferably, the concentration of the ionic liquid is 40-60% (w / v).

[0025] In a second aspect, the present invention provides use of an ionic liquid in the preparation of epicatechin, wherein the ionic liquid comprises 1-butyl-3-methylimidazolium chloride and / or 1-ethyl-3-methylimidazolium chloride.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention uses a specific ionic liquid to extract tea residues, which can not only "turn waste into treasure" and maximize the residual value of tea residues, but also play a good role in protecting the environment. Under specific extraction conditions, the extraction performance of the ionic liquid is much better than that of traditional reagents, especially for epicatechin, an antioxidant widely used in various fields.

[0028] 2. It is difficult to extract epicatechin with traditional reagents, but the specific ionic liquid of the present invention can extract 17 mg of epicatechin from each gram of tea residue with extremely high efficiency, so it has extremely high extraction value, demonstrating the great prospect of commercial application of the specific ionic liquid in tea residue extraction.

[0029] 3. The extraction technology of the present invention is not only low in cost and convenient to operate, but also short in time (extraction can be completed within a few hours), high in extraction efficiency, and easy for enterprises to operate, thereby reducing the extraction cost. The extracted antioxidant, epicatechin, has a wide range of applications and huge market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a qualitative detection diagram of epicatechin in Example 1 in high pressure liquid chromatography. DETAILED DESCRIPTION

[0031] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] Unless otherwise specified, all raw materials involved in the following examples and comparative examples are commercially available general materials. Experimental operations not specifically described in the examples are all routine operations in the art or can be understood or known by those skilled in the art based on the prior art or common knowledge they master.

[0033] The tea residues described in the embodiments of the present invention and the comparative examples all come from milk tea shops, such as "Leyancha". These tea residues are unused tea waste left in the process of boiling tea leaves to extract tea water to make milk tea. The collected tea residues are air-dried overnight and then dried to constant weight in a vacuum oven at 105°C to ensure that the water content of the tea residues is controlled below 5%. The calculation formula of moisture content (%) is as follows: (1) MC (%) = [M] i - [M] f , where MC is the calculated moisture content, [M] i is the initial weight of tea waste, [M] f is the final weight after drying.

[0034] The 1-butyl-3-methylimidazolium chloride was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (CAS registration number 79917-90-1, purity>97%), and the 1-ethyl-3-methylimidazolium chloride was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (CAS registration number 65039-09-0, purity>98%).

[0035] Example 1

[0036] This embodiment provides a method for preparing epicatechin, comprising the following steps:

[0037] (1) mixing tea residue with 1-butyl-3-methylimidazolium chloride ionic liquid having a concentration of 50% (w / v), extracting at 70° C. for 4 hours, and then filtering to remove large particle impurities to obtain a tea residue extract, wherein the mass ratio of the tea residue to the 1-butyl-3-methylimidazolium chloride is 1:10;

[0038] (2) The tea residue extract is loaded onto a column and separated by preparative column chromatography, with gradient elution performed using an eluent. During the chromatographic separation process, the chromatographic peaks are monitored in real time, and the fractions containing high-purity epicatechin are accurately collected. The fractions are then qualitatively and quantitatively analyzed by high performance liquid chromatography (HPLC) to ensure the purity and quality of the epicatechin obtained. Finally, the solvent and other trace impurities are removed by freeze drying to obtain high-purity epicatechin.

[0039] The preparative column chromatography uses an Agilent 1290 liquid chromatography system, and the chromatographic column is an AgilentPrep 100AC18 chromatographic column (50 x 250 mm, 10µm). The eluent includes 0.1% formic acid (liquid A) and pure methanol (liquid B), and the two eluents are mixed and flowed in different proportions at different times. The specific proportion and elution time can refer to the following HPLC elution program (75% A within 0-2 minutes, 75%-66% A within 2-9 minutes, 66%-58% A within 9-9.1 minutes, 58%-55% A within 9.1-30 minutes, 55% A within 30-40 minutes, 55%-75% A within 40-40.1 minutes, 75% A within 40.1-60 minutes; the flow rate is 0.3ml / min, the sample injection volume is 20μl, and the detection is performed at 278nm.

[0040] Example 2

[0041] This embodiment provides a method for preparing epicatechin, which differs from Embodiment 1 in that:

[0042] In the step (1), 1-ethyl-3-methylimidazolium chloride ionic liquid is used to extract the tea residue, and the remaining operations are the same.

[0043] Example 3

[0044] This embodiment provides a method for preparing epicatechin, which differs from Embodiment 1 in that:

[0045] In the step (1), the extraction temperature is 50° C., the mass ratio of tea residue to 1-butyl-3-methylimidazole chloride is 1:5, and the remaining operations are the same.

[0046] Example 4

[0047] This embodiment provides a method for preparing epicatechin, which differs from Embodiment 1 in that:

[0048] In the step (1), the extraction temperature is 6 hours, the mass ratio of tea residue to 1-butyl-3-methylimidazole chloride is 1:15, and the remaining operations are the same.

[0049] Comparative Example 1

[0050] This comparative example provides a method for preparing epicatechin, which differs from Example 1 in that the mass ratio of the tea residue to 1-butyl-3-methylimidazolium chloride is 1:20, and the rest are the same.

[0051] Comparative Example 2

[0052] This comparative example provides a method for preparing epicatechin, which differs from Example 1 in that the mass ratio of the tea residue to 1-butyl-3-methylimidazolium chloride is 1:2, and the rest are the same.

[0053] Comparative Example 3

[0054] This comparative example provides a method for preparing epicatechin, which differs from Example 1 in that the extraction conditions are: extraction at 30° C. for 3 h, and the rest are the same.

[0055] Comparative Example 4

[0056] This comparative example provides a method for preparing epicatechin, which differs from Example 1 in that the extraction conditions are: extraction at 80° C. for 7 h, and the rest are the same.

[0057] Comparative Example 5

[0058] This comparative example provides a method for preparing epicatechin, which differs from Example 1 in that 1-butyl-3-methylimidazolium bromide is used as the ionic liquid to extract the tea residue, and the rest is the same.

[0059] Comparative Example 6

[0060] This comparative example provides a method for preparing epicatechin, which differs from Example 1 in that 70% ethanol is used to extract tea residues, and the rest are the same.

[0061] Comparative Example 7

[0062] This comparative example provides a method for preparing epicatechin, which differs from Example 1 in that pure water is used to extract tea residues, and the rest are the same.

[0063] Effect example

[0064] In this effect example, the tea residue extract obtained by the extraction method described in Examples 1-4 and Comparative Examples 1-7 was used as a sample, and the main components and contents thereof were determined by high performance liquid chromatography. The specific method is as follows.

[0065] Analyses were performed using an Agilent 1220 LC system equipped with an InfinityLab Poroshell 120 SB-C18 column (4.6 x 250 mm, 4µm). All detections were performed at 30°C. Prior to analysis, samples were diluted 10-fold with MilliQ water and then filtered through a 0.45µm PVDF syringe filter. The mobile phase consisted of 0.01% formic acid in water (A) and methanol (B). The gradient used was: 75% A in 0–2 min, 75%-66% A in 2–9 min, 66%–58% A in 9–9.1 min, 58%-55% A in 9.1–30 min, 55% A in 30–40 min, 55%-75% A in 40–40.1 min, and 75% A in 40.1–60 min. The flow rate was 0.3 ml / min. The sample injection volume was 20 μl and detection was performed at 278 nm.

[0066] The specific results are shown in Table 1.

[0067] Table 1

[0068]

[0069] Note: The table includes epicatechin (EC), epigallocatechin (EGC), epigallocatechin gallate (EGCG), and epicatechin epigallate (ECG).

[0070] It can be seen from the results in Table 1 that the technical solution of the present invention can effectively improve the extraction efficiency of tea polyphenols in tea residues, and the content of epicatechin (EC) in the extract is significantly improved compared with traditional extraction reagents. In particular, the epicatechin content obtained by the extraction method described in Example 1 reaches about 17 mg / g, and the extraction effect is extremely high. When the extraction conditions described in Comparative Examples 1-4 exceed the specified range of the present invention, the content of epicatechin in the extract is significantly reduced compared with that in the examples, and the extraction efficiency decreases, indicating that the extraction temperature and extraction time have a significant impact on the extraction effect of epicatechin. When the process conditions exceed the preferred range of the present invention, epicatechin cannot be fully dissolved or the dissolved epicatechin is degraded, thereby significantly reducing the yield of epicatechin. When other ionic liquids are used to extract tea residues in Comparative Example 5, the extraction efficiency of epicatechin is also reduced compared with that of Example 1-2 using the specific ionic liquids of 1-butyl-3-methylimidazolium chloride or 1-ethyl-3-methylimidazolium chloride. The above results all show that the present invention uses specific ionic liquids to extract tea residues under specific process conditions, which can effectively improve the extraction efficiency of epicatechin in tea residues and maximize the residual value of tea residues.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing epicatechin, characterized in that: The following steps are involved: (1) Evenly mixing tea residues and ionic liquid, performing extraction reaction, and then filtering to remove impurities to obtain tea residue extract; (2) applying the tea residue extract to a column, separating and eluting by preparative column chromatography, collecting the epicatechin-containing fractions according to the chromatographic peak positions, and freeze-drying the fractions to obtain epicatechin; The ionic liquid is selected from 1-butyl-3-methylimidazolium chloride and / or 1-ethyl-3-methylimidazolium chloride; The conditions of the extraction reaction are: extraction at 50-70°C for 4-6h; The mass ratio of the tea residue to the ionic liquid is tea residue: ionic liquid = 1: (5-15).

2. The preparation method according to claim 1, characterized in that The ionic liquid is 1-butyl-3-methylimidazolium chloride.

3. The preparation method according to claim 1, characterized in that: The extraction reaction conditions are: extraction at 70° C. for 4 h.

4. The preparation method according to claim 1, characterized in that: The mass ratio of the tea residue to the ionic liquid is tea residue:ionic liquid=1:

10.

5. The preparation method according to claim 1, characterized in that: The water content of the tea residue is controlled below 5%.

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