Extraction method of tea polyphenol of green tea

Abstract

The invention relates to the field of plant extraction, and particularly discloses a green tea polyphenol extraction method, which comprises: S1, washing a green tea raw material with acid water, separating with macroporous resin, and eluting with ethanol to prepare a tea polyphenol extraction liquid; s2, enabling the prepared tea polyphenol extracting solution to firstly pass through a cellulose column filled with a cellulose filler prepared by taking epicatechin as a template agent molecule, and then eluting with ethyl propionate to prepare an EC extract; s3, enabling the tea polyphenol extracting solution passing through the cellulose column to pass through a modified PCL-PHEMA copolymer resin column containing ether bonds, and then eluting by adopting an ethanol solution to prepare the EGCG extract. The method has the characteristics that tea polyphenol extraction of green tea is realized, and EGCG and EC separation and extraction are further realized.

Description

Technical Field

[0001] The present invention relates to the field of plant extraction, and more specifically, to a method for extracting green tea polyphenols. Background Art

[0002] Tea polyphenols, also known as antioxidants and vitamin polyphenols, are a type of polyhydroxy compounds contained in tea leaves, referred to as TP. The main chemical components are complexes of catechins (flavanols), flavonoids and flavonols, anthocyanins, phenolic acids and phenolic acids, polymerized phenols, etc. Tea polyphenols can account for 30% of the dry weight of tea leaves. Tea polyphenols have broad application prospects in oils, food, medicine, daily chemicals, light chemicals, cosmetics, health care and many other aspects. It has abundant raw material sources, high profits and high added value, so it has become a hot research issue at present.

[0003] At present, the extraction technology of tea polyphenols mainly includes solvent extraction, resin adsorption, precipitation separation, microwave method and membrane separation method, but they are currently limited to the extraction of tea polyphenols. Catechins are the main components of tea polyphenols, accounting for 65-80% of the total amount of tea polyphenols. Catechins mainly include epicatechin EC, epigallocatechin EGC, epigallocatechin gallate ECG and epigallocatechin gallate EGCG.

[0004] EGCG monomer is the main antioxidant component of tea polyphenols, and its reducing property can even reach 100 times that of L-isoascorbic acid. Studies on epicatechin EC have found that it has antioxidant effects and free radical scavenging effects. Increasing epicatechin levels can enhance the ability of plasma to scavenge free radicals and inhibit lipid peroxidation and damage. In addition, epicatechin EC extracted from green tea can also help improve the condition of diabetic patients because it can reduce acetylcholinesterase activity. In addition, it is also an ideal health product for athletes because it has anabolic and muscle strength-promoting effects. Studies have also found that epicatechin EC can promote heart health and has potential anti-cancer effects.

[0005] In addition, both EGCG and EC have a wide range of biological activities, such as anti-cancer, anti-viral, and antioxidant. Due to its richer content and stronger antioxidant capacity, EGCG has been widely concerned and applied in many studies. Therefore, it is of great significance to further separate and extract EGCG and EC during the extraction of green tea polyphenols. Summary of the invention

[0006] In order to achieve the extraction of green tea polyphenols and further achieve the separation and extraction of EGCG and EC, the present application provides a method for extracting green tea polyphenols.

[0007] In the first aspect, the present application provides a method for extracting green tea polyphenols, using the following technical solution: A method for extracting green tea polyphenols comprises the following steps: S1, washing the green tea raw material with acid water, separating it with a macroporous resin, and then eluting it with ethanol to obtain a tea polyphenol extract; S2, passing the obtained tea polyphenol extract through a cellulose column filled with cellulose filler prepared by using epicatechin as a template molecule, and then eluting with ethyl propionate to obtain an EC extract; S3. The tea polyphenol extract after passing through the cellulose column is passed through a modified PCL-PHEMA copolymer resin column containing an ether bond, and then eluted with an ethanol solution to obtain an EGCG extract.

[0008] By adopting the above technical scheme, in the present application, green tea is first rinsed in acid water to facilitate the dissolution of tea polyphenols, and then the tea polyphenols are selectively adsorbed by a macroporous resin, and then eluted with ethanol to obtain a tea polyphenol extract. The prepared tea polyphenol extract is then first passed through a cellulose column and then eluted with ethyl acetate. The cellulose column is prepared using epicatechin as a template molecule, so that the imprinted holes on the cellulose column are highly matched with epicatechin EC, achieving a high adsorption of epicatechin EC and the separation and extraction of EC monomers. Moreover, since the molecular weight of EGCG is larger, the adsorption of EGCG by the cellulose column is weak, but the adsorption of EC is strong.

[0009] Based on the fact that EGCG monomer structure contains more phenolic hydroxyl groups and gallic acid groups, EGCG has strong polarity and hydrophilicity, while EC has a small molecular structure, fewer phenolic hydroxyl groups, and does not contain ester groups. Based on the difference between the above two, the tea polyphenol extract after passing through the cellulose column is passed through a modified PCL-PHEMA copolymer resin column. The modified PCL-PHEMA copolymer resin column is composed of polycaprolactone and polyhydroxyethyl methacrylate, and contains hydroxyl groups. The ester functional group makes the copolymer have good adsorption performance for EGCG. EGCG is a monomer containing an ether bond among the catechin monomers in tea polyphenols. The introduction of ether bonds in the copolymer makes it easier to adsorb ether-bonded EGCG monomer molecules with similar polarity. Moreover, the ether bonds can also form stable complexes with ether-bonded substances through matching of spatial structures, thereby enhancing the adsorption effect on EGCG, and finally achieving the separation of EGCG monomers. Finally, the method provided by the present application realizes the efficient separation and collection of EGCG monomers and EC monomers.

[0010] Optionally, the cellulose filler in step S2 is prepared by the following method: Epicatechin, methacrylic acid monomer and acrylamide monomer are dissolved in dimethyl sulfoxide, ultrasonically dispersed for 10-20 minutes, then microcrystalline cellulose, crosslinking agent and initiator are added, stirred and reacted at 80-100° C. for 16-20 hours, then the reaction is stopped, allowed to stand for 30-50 minutes and filtered, then eluted by adding ethanol solution and dried to obtain cellulose filler.

[0011] By adopting the above technical scheme, the cellulose filler in the present application uses epicatechin EC as a template molecule and microcrystalline cellulose as a cellulose matrix. Under the action of an initiator and a cross-linking agent, methacrylic acid monomers and acrylamide monomers can self-polymerize and copolymerize, and can also cross-link with a microcrystalline cellulose matrix containing a hydroxyl functional group by using carboxyl groups and amino groups to form a network structure. At the same time, the template molecules are embedded, and the solubility difference between the prepared polymer and epicatechin EC in ethanol is utilized. After the template is eluted with ethanol, an imprinted cavity matching the shape, size and functional group of epicatechin EC is formed, thereby preparing a molecularly imprinted cellulose column, which is used for the separation of tea polyphenol extracts, has excellent specific selective adsorption for epicatechin EC, and realizes the separation and extraction of EC monomers.

[0012] Optionally, during the preparation of the cellulose filler, ethylene glycol dimethacrylate is used as the crosslinking agent, and ammonium persulfate and azobisisobutyronitrile are used as the initiator in a mass ratio of 1:(0.8-1.2).

[0013] Optionally, during the preparation of the cellulose filler, the raw materials are added according to the following weight parts: 10-20 parts of epicatechin, 15-25 parts of methacrylic acid monomer, 10-20 parts of acrylamide monomer, 20-30 parts of dimethyl sulfoxide, 15-25 parts of microcrystalline cellulose, 1-3 parts of crosslinking agent, and 2-4 parts of initiator.

[0014] By adopting the above technical solution and selecting the above-mentioned raw materials in the above-mentioned proportion, the molecularly imprinted cellulose filler has better specific adsorption for EC.

[0015] Optionally, in step S2, the prepared cellulose filler is loaded onto a column and then rinsed with dichloromethane, and then the tea polyphenols extract prepared in step S1 is passed through the cellulose column.

[0016] By adopting the above technical solution, the prepared cellulose filler is washed with dichloromethane. Dichloromethane has a weak polarity. Since EGCG has a strong polarity, the cellulose column is washed in a weakly polar solvent to further enhance the specific adsorption of EC monomers.

[0017] Optionally, the filler of the modified PCL-PHEMA copolymer resin column is a modified PCL-PHEMA copolymer resin, and the modified PCL-PHEMA copolymer resin is prepared by the following method: Mix the PCL-PHEMA copolymer with dimethylformamide and acetone, heat to 70-80°C, and stir to obtain a mixture A; add sulfuric acid to the obtained mixture A under stirring, then add ethyl bromide, heat to 120-140°C, react for 1-2h, then add toluene, and continue to react for 2-3h; After cooling, the mixture is neutralized, and then the solvent is evaporated and directly put into water at 1-5°C for washing to obtain a modified PCL-PHEMA copolymer resin.

[0018] By adopting the above technical scheme, the PCL-PHEMA copolymer is used as a benchmark in the present application, which is first dissolved and then heated, and the hydroxyl groups and ethyl bromide in the copolymer are activated under acidic catalyst conditions, and the activated hydroxyl groups act as nucleophiles to attack the carbon atoms in the halogenated hydrocarbons, resulting in the breakage of the carbon-halogen bond. At the same time, an ether bond is formed between the oxygen atom of the hydroxyl group and the broken carbon atom, and then a proton transfer occurs under acidic catalytic conditions to finally form a modified copolymer containing an ether bond. The difference in polarity between EGCG and ECG due to the different positions of the hydroxyl substituents is utilized. Since EGCG has more phenolic hydroxyl groups and aromatic rings, it has stronger polarity and hydrogen bond formation ability. In the present application, the PCL-PHEMA copolymer is selected as a benchmark and after introducing ether bonds on the hydrophilic segments of the copolymer, the hydrophilicity and polarity of the modified copolymer are enhanced, so that the modified copolymer forms a stronger hydrogen bond with EGCG, and the adsorption of EGCG is stronger.

[0019] In the present application, PCL-PHEMA copolymer is selected as the resin filler, and the addition of toluene during the modification process forms a pore structure in the copolymer, and the subsequent evaporation of the solvent is combined with rapid cooling to adjust the pore structure, so that the modified copolymer has a stronger adsorption effect on EGCG with a larger molecular weight, while more small molecule ECG will pass through the modified copolymer resin filler without being adsorbed, and then the subsequent elution procedure is used to separate the two, and finally achieve efficient collection of EGCG.

[0020] Optionally, when preparing the modified PCL-PHEMA copolymer resin, the raw materials are added according to the following weight parts: 30-40 parts of PCL-PHEMA copolymer, 10-20 parts of dimethylformamide, 8-15 parts of acetone, 2-5 parts of sulfuric acid, 20-30 parts of Ethyl bromide, 10-15 parts toluene.

[0021] By adopting the above technical scheme and controlling the ratio of the above raw materials, the final copolymer is formed with ether bonds while retaining certain hydroxyl groups to enhance the adsorption of EGCG monomer, and the subsequent temperature change is regulated after the amount of toluene porogen added to achieve different adsorption of EGCG and ECG, and the collection of EGCG is achieved by controlling the subsequent elution procedure.

[0022] Optionally, in step S3, after the tea polyphenol extract is passed through the column, it is first eluted with a 5-10% mass concentration ethanol solution to obtain eluent A, then eluted with a 40-50% mass concentration ethanol solution to obtain eluent B, and then eluted with a 70-80% mass concentration ethanol solution to obtain eluent C, and then the eluent C is concentrated to remove ethanol to obtain the EGCG extract.

[0023] By adopting the above technical scheme, a low-concentration ethanol solution is first used to elute and remove impurities, and then a small portion of ECG is eluted with a medium-concentration ethanol solution, and then a high-concentration ethanol solution is used to elute to obtain a high-purity EGCG extract.

[0024] Optionally, the specific operations of step S1 are: In step S1, the green tea raw material is crushed and washed with acid water with a pH of 3-4 for 4-6 hours, then filtered, the filtrate is concentrated and passed through a macroporous resin, and then eluted with a 60-70% ethanol aqueous solution to obtain a tea polyphenol extract.

[0025] By adopting the above technical scheme, in the present application, the green tea raw material is first crushed to increase the surface area, which is beneficial to the dissolution of effective ingredients in the subsequent processing process, and then the green tea raw material is rinsed in an acidic environment. The acidic environment destroys the cell structure of the tea leaves, promotes the dissolution of the effective ingredients of tea polyphenols, and inhibits the activity of enzymes to prevent the enzymatic hydrolysis of tea polyphenols during the re-extraction process. The dissolution rate of tea polyphenols is increased by the above method, and then the filtrate containing tea polyphenols is obtained by filtration. The tea polyphenols are then separated by a macroporous resin and eluted with an ethanol solution to obtain an eluate rich in tea polyphenols.

[0026] Optionally, the macroporous resin in step S1 is D101 macroporous resin.

[0027] In summary, this application has the following beneficial effects: 1. In the present application, green tea is firstly rinsed in acid water to facilitate the dissolution of tea polyphenols, and then the tea polyphenols are selectively adsorbed by a macroporous resin, and then eluted with ethanol to obtain a tea polyphenol extract, and then the obtained tea polyphenol extract is sequentially passed through a cellulose column and a modified PCL-PHEMA copolymer resin column to separate and collect EC monomers and EGCG monomers respectively; 2. In the present application, the cellulose filler uses epicatechin EC as the template molecule and microcrystalline cellulose as the cellulose matrix. Under the action of an initiator and a cross-linking agent, the methacrylic acid monomer and the acrylamide monomer can self-polymerize and copolymerize, and can also cross-link with the microcrystalline cellulose matrix containing hydroxyl functional groups by using carboxyl groups and amino groups to form a network structure. At the same time, the template molecule is embedded, and the difference in solubility between the prepared polymer and epicatechin EC in ethanol is used. After the template is eluted with ethanol, an imprinted cavity matching the shape, size and functional group of epicatechin EC is formed, thereby preparing a molecularly imprinted cellulose column, which is used for the separation of tea polyphenol extracts. It has excellent specific selective adsorption for epicatechin EC, and realizes the separation and extraction of EC monomers. 3. The modified PCL-PHEMA copolymer resin column in the present application is composed of polycaprolactone and polyhydroxyethyl methacrylate, and contains hydroxyl groups. The ester functional group makes the copolymer have good adsorption performance for EGCG. The introduction of ether bonds in the copolymer makes it easier to adsorb ether-bonded EGCG monomer molecules with similar polarity. Moreover, the ether bonds can also form stable complexes with ether-bonded substances through matching of spatial structures, thereby enhancing the adsorption effect of EGCG and finally achieving the separation of EGCG monomers. Finally, the method provided in the present application realizes the efficient separation and collection of EGCG monomers and EC monomers. DETAILED DESCRIPTION

[0028] The present application is further described in detail below in conjunction with the examples. It is particularly noted that if no specific conditions are specified in the following examples, the experiments are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

[0029] In the following preparation examples, PCL-PHEMA, i.e., polycaprolactone-polyhydroxyethyl methyl acrylate, is a polycaprolactone-polyhydroxyethyl methyl acrylate block copolymer purchased from Hangzhou Xinqiao Biotechnology Co., Ltd., and its Mw is 5000.

[0030] The following preparation example is a preparation example of cellulose filler Preparation Example 1 A method for preparing a cellulose filler comprises the following steps: 1.5 kg epicatechin, 2 kg methacrylic acid monomer and 1.5 kg acrylamide monomer were dissolved in 2.5 kg dimethyl sulfoxide, and ultrasonically dispersed for 15 minutes. Then, 2 kg microcrystalline cellulose, 0.2 kg cross-linking agent and 0.3 kg initiator were added, and the mixture was stirred and reacted at 90° C. for 18 hours. Then, the reaction was stopped, allowed to stand for 40 minutes, and then filtered. Then, ethanol solution was added for elution and then dried to obtain a cellulose filler.

[0031] Among them, ethylene glycol dimethacrylate is selected as the cross-linking agent, and ammonium persulfate and azobisisobutyronitrile with a mass ratio of 1:1 are selected as the initiator.

[0032] Preparation Example 2 A method for preparing a cellulose filler comprises the following steps: 1 kg of epicatechin, 1.5 kg of methacrylic acid monomer and 1 kg of acrylamide monomer were dissolved in 2 kg of dimethyl sulfoxide, and ultrasonically dispersed for 10 minutes. Then, 1.5 kg of microcrystalline cellulose, 0.1 kg of cross-linking agent and 0.2 kg of initiator were added, and the mixture was stirred and reacted at 80° C. for 20 hours. Then, the reaction was stopped, allowed to stand for 30 minutes, and then filtered. Then, an ethanol solution was added for elution and then dried to obtain a cellulose filler.

[0033] Among them, ethylene glycol dimethacrylate is selected as the cross-linking agent, and ammonium persulfate and azobisisobutyronitrile are selected as the initiator in a mass ratio of 1:0.8.

[0034] Preparation Example 3 A method for preparing a cellulose filler comprises the following steps: 2kg of epicatechin, 2.5kg of methacrylic acid monomer and 2kg of acrylamide monomer were dissolved in 3kg of dimethyl sulfoxide, and ultrasonically dispersed for 20 minutes. Then, 2.5kg of microcrystalline cellulose, 0.3kg of cross-linking agent and 0.4kg of initiator were added, and the mixture was stirred and reacted at 100°C for 16 hours. Then, the reaction was stopped, allowed to stand for 50 minutes, and then filtered. Then, ethanol solution was added for elution and then dried to obtain a cellulose filler.

[0035] Among them, ethylene glycol dimethacrylate is selected as the cross-linking agent, and ammonium persulfate and azobisisobutyronitrile with a mass ratio of 1:1.2 are selected as the initiator.

[0036] Preparation Example 4 A method for preparing a cellulose filler is carried out according to the method in Preparation Example 1, except that no crosslinking agent is added to the raw material.

[0037] The following preparation example is a preparation example of modified PCL-PHEMA copolymer resin filler Preparation Example 5 A method for preparing a modified PCL-PHEMA copolymer resin filler comprises the following steps: 3.5 kg of PCL-PHEMA copolymer was mixed with 1.5 kg of dimethylformamide and 1.2 kg of acetone, heated to 75° C., and stirred to obtain a mixture A; Under stirring conditions, 0.3 kg of sulfuric acid was added to the obtained mixture A, and then 2.5 kg of ethyl bromide was added, and the temperature was raised to 130°C. After reacting for 1.5 hours, 1.2 kg of toluene was added, and the reaction was continued for 2.5 hours; After cooling, the mixture was neutralized, and then the solvent was evaporated and directly put into 3° C. water for washing to obtain a modified PCL-PHEMA copolymer resin, which was then cut and granulated to obtain a modified PCL-PHEMA copolymer resin filler.

[0038] Preparation Example 6 A method for preparing a modified PCL-PHEMA copolymer resin filler comprises the following steps: 3 kg of PCL-PHEMA copolymer was mixed with 1 kg of dimethylformamide and 0.8 kg of acetone, heated to 70° C., and stirred to obtain a mixture A; Under stirring conditions, 0.2 kg of sulfuric acid was added to the obtained mixture A, and then 2 kg of ethyl bromide was added, and the temperature was raised to 120°C. After reacting for 2 hours, 1 kg of toluene was added, and the reaction was continued for 2 hours; After cooling, the mixture was neutralized, and then the solvent was evaporated and directly put into 1°C water for washing to obtain a modified PCL-PHEMA copolymer resin, which was then cut and granulated to obtain a modified PCL-PHEMA copolymer resin filler.

[0039] Preparation Example 7 A method for preparing a modified PCL-PHEMA copolymer resin filler comprises the following steps: 4 kg of PCL-PHEMA copolymer was mixed with 2 kg of dimethylformamide and 1.5 kg of acetone, heated to 80° C., and stirred to obtain a mixture A; Under stirring conditions, 0.5 kg of sulfuric acid was added to the obtained mixture A, and then 3 kg of ethyl bromide was added, and the temperature was raised to 140°C. After reacting for 1 hour, 1.5 kg of toluene was added, and the reaction was continued for 3 hours; After cooling, the mixture is neutralized, and then the solvent is evaporated and directly put into 5° C. water for washing to obtain a modified PCL-PHEMA copolymer resin, which is then cut and granulated to obtain a modified PCL-PHEMA copolymer resin filler.

[0040] Preparation Example 8 A method for preparing a modified PCL-PHEMA copolymer resin filler is carried out according to the method in Preparation Example 5, except that the modified PCL-PHEMA copolymer resin is directly prepared by neutralizing after cooling, evaporating the solvent and washing with water at room temperature.

[0041] Comparative Preparation Example 1 A method for preparing a PCL-PHEMA copolymer resin filler comprises the following steps: 3.5 kg of PCL-PHEMA copolymer was mixed with 1.5 kg of dimethylformamide and 1.2 kg of acetone, heated to 75° C., and stirred to obtain a mixture A; Under stirring conditions, add 1.2 kg of toluene, raise the temperature to 130°C, and stir for 2.5 hours; After cooling, the mixture was neutralized and then directly put into water at 3°C ​​for washing to obtain a modified PCL-PHEMA copolymer resin and a modified PCL-PHEMA copolymer resin filler.

[0042] Example 1 A method for extracting green tea polyphenols comprises the following steps: S1. Grind the green tea raw material and pass it through a 60-mesh sieve to obtain green tea raw material powder. Take 10 kg of the green tea raw material powder and rinse it in a hydrochloric acid aqueous solution with a pH of 3.5 at room temperature for 5 hours. The green tea raw material powder is completely immersed in the hydrochloric acid aqueous solution, and then filter it. The filtrate is concentrated to 1 / 5 of the original volume and then passed through a D101 macroporous resin at a flow rate of 3BV / h. Then, 3BV is eluted with a 65% mass concentration of ethanol aqueous solution at a flow rate of 2BV / h, and the eluate is collected to obtain a tea polyphenol extract; S2, the tea polyphenol extract collected in step S1 is first passed through a cellulose column filled with 1 kg of cellulose filler prepared in Preparation Example 1, and then 6 BV is eluted at a flow rate of 2 BV / h using ethyl propionate as an eluent to obtain an EC extract, and then the obtained EC extract is poured into a rotary evaporator, and ethyl propionate is evaporated at 75° C. and 250 mmHg, and then methanol is added to obtain an EC treatment solution with methanol as a solvent, and then the EC treatment solution is crystallized and dried to obtain an EC extract; S3. The tea polyphenol extract after passing through the cellulose column is passed through a 1 kg modified PCL-PHEMA copolymer resin column prepared in Preparation Example 5, and then eluted with an 8% ethanol solution to obtain an eluent A, and then eluted with a 75% ethanol solution to collect the eluent B, and the eluent B is concentrated to be alcohol-free and dried to obtain the EGCG extract.

[0043] In the above steps S2 and S3, after loading the cellulose column and the modified PCL-PHEMA copolymer resin column, the cellulose column and the modified PCL-PHEMA copolymer resin column are first rinsed with water for equilibrium and then passed through the tea polyphenols extract, and the added volume of water is 3 times the volume of the cellulose column bed.

[0044] Example 2 A method for extracting green tea polyphenols comprises the following steps: S1. Grind the green tea raw material and pass it through a 60-mesh sieve to obtain green tea raw material powder. Take 10 kg of the green tea raw material powder and rinse it in a hydrochloric acid aqueous solution with a pH of 3 at room temperature for 4 hours. The green tea raw material powder is completely immersed in the hydrochloric acid aqueous solution, and then filter it. The filtrate is concentrated to 1 / 5 of the original volume and then passed through a D101 macroporous resin at a flow rate of 3BV / h. Then, 3BV is eluted with a 60% mass concentration of ethanol aqueous solution at a flow rate of 2BV / h, and the eluate is collected to obtain a tea polyphenol extract; S2, the tea polyphenol extract obtained in step S1 is first passed through a cellulose column filled with 1 kg of cellulose filler obtained in Preparation Example 2, and then 6 BV is eluted at a flow rate of 2 BV / h using ethyl propionate as an eluent to obtain an EC extract, and then the obtained EC extract is poured into a rotary evaporator, and ethyl propionate is evaporated at 70° C. and 200 mmHg, and then methanol is added to obtain an EC treatment solution with methanol as a solvent, and then the EC treatment solution is crystallized and dried to obtain an EC extract; S3. The tea polyphenol extract after passing through the cellulose column is passed through a 1 kg modified PCL-PHEMA copolymer resin column prepared in Preparation Example 6, and then eluted with a 5% ethanol solution to obtain an eluent A, and then eluted with a 70% ethanol solution to collect the eluent B, and the eluent B is concentrated to be alcohol-free and dried to obtain the EGCG extract.

[0045] In the above steps S2 and S3, after loading the cellulose column and the modified PCL-PHEMA copolymer resin column, the cellulose column and the modified PCL-PHEMA copolymer resin column are first rinsed with water for equilibrium and then passed through the tea polyphenols extract, and the added volume of water is 3 times the volume of the cellulose column bed.

[0046] Example 3 A method for extracting green tea polyphenols comprises the following steps: S1. Grind the green tea raw material and pass it through a 60-mesh sieve to obtain green tea raw material powder. Take 10 kg of the green tea raw material powder and rinse it in a hydrochloric acid aqueous solution with a pH of 4 at room temperature for 6 hours. The green tea raw material powder is completely immersed in the hydrochloric acid aqueous solution, and then filter it. The filtrate is concentrated to 1 / 5 of the original volume and then passed through a D101 macroporous resin at a flow rate of 3BV / h. Then, 70% mass concentration of ethanol aqueous solution is used to elute 3BV at a flow rate of 2BV / h, and the eluate is collected to obtain a tea polyphenol extract; S2, the tea polyphenol extract collected in step S1 is first passed through a cellulose column filled with 1 kg of cellulose filler prepared in Preparation Example 3, and then 6 BV is eluted at a flow rate of 2 BV / h using ethyl propionate as an eluent to obtain an EC extract, and then the obtained EC extract is poured into a rotary evaporator, and ethyl propionate is evaporated at 80° C. and 350 mmHg, and then methanol is added to obtain an EC treatment solution with methanol as a solvent, and then the EC treatment solution is crystallized and dried to obtain an EC extract; S3. The tea polyphenol extract after passing through the cellulose column is passed through a 1 kg modified PCL-PHEMA copolymer resin column prepared in Preparation Example 7, and then eluted with a 10% ethanol solution to obtain an eluent A, and then eluted with an 80% ethanol solution to collect the eluent B, and the eluent B is concentrated to be alcohol-free and dried to obtain the EGCG extract.

[0047] In the above steps S2 and S3, after loading the cellulose column and the modified PCL-PHEMA copolymer resin column, the cellulose column and the modified PCL-PHEMA copolymer resin column are first rinsed with water for equilibrium and then passed through the tea polyphenols extract, and the added volume of water is 3 times the volume of the cellulose column bed.

[0048] Example 4 A method for extracting tea polyphenols from green tea is carried out according to the method in Example 1, except that the filler in the cellulose column in step S2 is the cellulose filler prepared in Preparation Example 4.

[0049] Example 5 A method for extracting tea polyphenols from green tea is carried out according to the method in Example 1, except that in step S2, after the cellulose filler is loaded onto the column, it is first rinsed with water and then rinsed with dichloromethane, and then the tea polyphenol extract obtained in step S1 is passed through the cellulose column, and the amount of dichloromethane added is twice the volume of the cellulose column.

[0050] Example 6 A method for extracting tea polyphenols from green tea is carried out according to the method in Example 1, except that in step S2, the prepared EC extract is directly evaporated and crystallized to obtain the EC extract.

[0051] Example 7 A method for extracting green tea polyphenols is carried out according to the method in Example 1, except that the modified PCL-PHEMA copolymer resin filler in step S3 is the modified PCL-PHEMA copolymer resin filler prepared in Preparation Example 8.

[0052] Comparative Example 1 A method for extracting tea polyphenols from green tea is carried out according to the method in Example 1, except that in step S1, 10 kg of green tea raw material powder is added with 8 times the mass of ethanol with a mass concentration of 70%, heated and refluxed for extraction twice, each time for 3 hours, the filtrate is combined after filtration, the pressure is reduced to no alcohol, the supernatant is taken after centrifugation, and then passed through D101 macroporous resin.

[0053] Comparative Example 2 A method for extracting green tea polyphenols is carried out according to the method in Example 1, except that the modified PCL-PHEMA copolymer resin filler in step S3 is replaced by an equal amount of the PCL-PHEMA copolymer resin filler prepared in Comparative Preparation Example 1.

[0054] Comparative Example 3 A method for extracting tea polyphenols from green tea is carried out according to the method in Example 1, except that in step S2, the tea polyphenol extract is passed through a cellulose column and then eluted with 25% mass concentration ethanol to obtain an EC extract.

[0055] Comparative Example 4 A method for extracting tea polyphenols from green tea is carried out according to the method in Example 1, except that in step S1, 10 kg of green tea raw material powder is added with 8 times the mass of ethanol with a mass concentration of 70%, and the mixture is heated under reflux and extracted twice, each time for 3 hours. The filtrate is combined after filtration, evaporated to be free of alcohol, and then diluted with water to a mass volume ratio of green tea raw material powder to water of 0.03 g:1 mL, and then the pH is adjusted to 3.5 with hydrochloric acid, and then passed through a D101 macroporous resin.

[0056] Performance Testing The above method was performed on the same batch of green tea raw materials (with a tea polyphenol content of 35%), and the tea polyphenol extract obtained in step S1 of Examples 1-3, Comparative Example 1 and Comparative Example 4 was concentrated and dried to obtain a tea polyphenol extract. The tea polyphenol content and yield in the tea polyphenol extract were tested. The test results are shown in Table 1 below.

[0057] Table 1: Test items Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 4 Tea polyphenols content / % 98.7 98.6 98.7 98.3 98.4 Tea polyphenols yield / % 82.3 81.6 81.9 72.9 77.6 Referring to the test results in Table 1 above, the extraction method in the present application has a good extraction effect on tea polyphenols, the tea polyphenols content in the tea polyphenols extract in step S1 is relatively high, and the method provided in the embodiment of the present application has a high yield of tea polyphenols. Referring to the detection methods of Example 1 and Comparative Example 1 and Comparative Example 4, the acid solution extraction in the present application is compared with the traditional alcohol extraction method in Comparative Example 1, and the yield of tea polyphenols is significantly improved. Combined with the detection method in Comparative Example 4, the pH value is adjusted after alcohol extraction in Comparative Example 4 for subsequent macroporous resin purification, and the yield of tea polyphenols is still lower than that in Example 1.

[0058] In addition, the yields of the EC extract and the EGCG extract obtained in steps S2 and S3 were statistically analyzed, and the statistical results are shown in Table 2 below: Table 2: Test items Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 EC yield / % 82.3 80.4 81.6 76.1 86.6 82.1 EGCG yield / % 92.3 90.7 91.6 86.8 92.1 92.0 Test items Example 7 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 — EC yield / % — 56.8 — 68.9 60.3 — EGCG yield / % 85.1 75.7 77.6 — 79.4 — Moreover, the contents of EC and EGCG in the EC extract and EGCG extract obtained in the above embodiments were determined by HPLC method. The contents of EC and EGCG in the extracts obtained in the above embodiments 1-5 were both above 98%. Referring to the test results in Table 2 above, it can be seen that the yields of EC monomer and EGCG monomer obtained in step S2 were also relatively high. The method provided in the present application not only obtains tea polyphenols with high content and high yield, but also obtains EGCG monomer and EC monomer with high purity and high yield through subsequent specific separation.

[0059] Referring to the test results of Example 1 and Example 4, it can be seen that when the cross-linking agent is not added during the preparation of the cellulose filler in Example 4, the subsequent adsorption effect on EC is reduced, and its yield is reduced. It may be because the polarity and pore structure distribution of the cellulose filler are also affected when the cross-linking agent is not added. It is found that the yield of EGCG is also reduced, which may be due to the partial loss of EGCG during the column in step S2. In addition, combined with the test results of Example 1 and Example 5, the cellulose filler is also rinsed with dichloromethane after the column is equilibrated, and the yield of EC monomer is further significantly improved without reducing the yield of EGCG, and its specific adsorption performance for EC monomer is better.

[0060] Referring to the test results of Example 1 and Example 6, the EC extract obtained in step S2 of Example 6 was directly evaporated and crystallized, and the EC content in the obtained EC extract was reduced to 92.3% without solvent exchange post-treatment. Combined with the test results of Example 7, when the modified PCL-PHEMA copolymer resin filler was prepared in step S3, toluene was added during the modification process to form pores, but after the subsequent evaporation of the solvent, it was not quickly and directly placed in low-temperature water for treatment, and the pore structure was not adjusted, which affected the adsorption performance of EGCG, resulting in a decrease in the final EGCG yield.

[0061] Combining the test results of Example 1 with those of Comparative Examples 1 and 4, it can be seen that when the tea polyphenols extraction method in step S1 results in a low tea polyphenols yield, the yields of EC monomer and EGCG monomer are also significantly reduced. Combining the test results of Comparative Examples 2 and 3, the PCL-PHEMA copolymer resin filler in Comparative Example 2 has not been reacted with halogenated hydrocarbons under strong sulfuric acid conditions to form ether bonds, and the adsorption of EGCG monomer is reduced, and its yield is significantly reduced. In Comparative Example 3, the tea polyphenols extract is eluted with ethanol after passing through a cellulose column, and the yield of EC monomer is significantly reduced, and the EC content is also significantly reduced to 78.5%.

[0062] The horizontal bars in Example 7, Comparative Example 2 and Comparative Example 3 indicate that only the post-elution treatment operation in step S2 has no effect on the EGCG yield in step S3, or only the filler in step S3 is changed, which will not affect the EC yield in step S2.

[0063] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for extracting green tea polyphenols, characterized in that: The following steps are involved: S1, washing the green tea raw material with acid water, separating it with a macroporous resin, and then eluting it with ethanol to obtain a tea polyphenol extract; S2, passing the obtained tea polyphenol extract through a cellulose column filled with cellulose filler prepared by using epicatechin as a template molecule, and then eluting with ethyl propionate to obtain an EC extract; S3. The tea polyphenol extract after passing through the cellulose column is passed through a modified PCL-PHEMA copolymer resin column containing an ether bond, and then eluted with an ethanol solution to obtain an EGCG extract.

2. The method for extracting green tea polyphenols according to claim 1, characterized in that: In step S2, the cellulose filler is prepared by the following method: Epicatechin, methacrylic acid monomer and acrylamide monomer are dissolved in dimethyl sulfoxide, ultrasonically dispersed for 10-20 minutes, then microcrystalline cellulose, crosslinking agent and initiator are added, stirred and reacted at 80-100° C. for 16-20 hours, then the reaction is stopped, allowed to stand for 30-50 minutes and filtered, then eluted by adding ethanol solution and dried to obtain cellulose filler.

3. The method for extracting green tea polyphenols according to claim 2, characterized in that: During the preparation of the cellulose filler, each raw material is added according to the following weight parts: 10-20 parts of epicatechin, 15-25 parts of methacrylic acid monomer, 10-20 parts of acrylamide monomer, 20-30 parts of dimethyl sulfoxide, 15-25 parts of microcrystalline cellulose, 1-3 parts of a cross-linking agent, and 2-4 parts of an initiator.

4. The method for extracting green tea polyphenols according to claim 1, characterized in that: In step S2, the prepared cellulose filler is loaded onto a column and then rinsed with dichloromethane, and then the tea polyphenol extract prepared in step S1 is passed through the cellulose column.

5. The method for extracting green tea polyphenols according to claim 2, characterized in that: In the process of preparing the cellulose filler, ethylene glycol dimethacrylate is selected as the crosslinking agent, and ammonium persulfate and azobisisobutyronitrile are selected as the initiator in a mass ratio of 1: (0.8-1.2).

6. The method for extracting green tea polyphenols according to claim 1, characterized in that: The filler of the modified PCL-PHEMA copolymer resin column is a modified PCL-PHEMA copolymer resin, and the modified PCL-PHEMA copolymer resin is prepared by the following method: Mixing PCL-PHEMA copolymer with dimethylformamide and acetone, heating to 70-80° C., and stirring to obtain a mixture A; Under stirring conditions, sulfuric acid is added to the obtained mixture A, and then ethyl bromide is added, and the temperature is raised to 120-140°C. After reacting for 1-2 hours, toluene is added, and the reaction is continued for 2-3 hours; After cooling, the mixture is neutralized, and then the solvent is evaporated and directly put into water at 1-5°C for washing to obtain a modified PCL-PHEMA copolymer resin.

7. The method for extracting green tea polyphenols according to claim 6, characterized in that: When preparing the modified PCL-PHEMA copolymer resin, each raw material is added according to the following weight parts: 30-40 parts of PCL-PHEMA copolymer, 10-20 parts of dimethylformamide, 8-15 parts of acetone, 2-5 parts of sulfuric acid, 20-30 parts of ethyl bromide, 10-15 parts of toluene.

8. The method for extracting green tea polyphenols according to claim 1, characterized in that: In step S3, after the tea polyphenol extract is passed through the column, it is first eluted with a 5-10% ethanol solution to obtain an eluent A, and then eluted with a 70-80% ethanol solution to obtain an eluent B, and then the eluent B is concentrated to remove ethanol to obtain an EGCG extract.

9. The method for extracting green tea polyphenols according to claim 1, characterized in that: In step S1, the green tea raw material is crushed and washed with acid water with a pH of 3-4 for 4-6 hours, and then filtered. The filtrate is concentrated and passed through a macroporous resin, and then eluted with a 60-70% ethanol aqueous solution to obtain a tea polyphenol extract.

10. The method for extracting green tea polyphenols according to claim 9, characterized in that: The macroporous resin in step S1 is D101 macroporous resin.