A method for extracting green tea polyphenols

Abstract

The application relates to the field of plant extraction, and particularly discloses a green tea tea polyphenol extraction method, which comprises the following steps: S1, macroporous resin separation is adopted after green tea raw materials are washed with acid water and then eluted with ethanol to prepare tea polyphenol extract; S2, the prepared tea polyphenol extract is first passed through a cellulose column filled with cellulose filler prepared by taking epicatechin as a template agent molecule, then eluted with ethyl propionate to prepare an EC extract; and 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, then eluted with an ethanol solution to prepare an EGCG extract. The application has the characteristics of realizing green tea tea polyphenol extraction and further realizing the separation and extraction of EGCG and EC.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant extraction, more particularly, it relates to a method for extracting green tea polyphenols. BACKGROUND

[0002] Tea polyphenols, also known as anti-oxidation and vitamin polyphenols, are a kind of polyhydroxy compounds contained in tea leaves, which are referred to as TP. The main chemical components are catechins (flavanols), flavones and flavonols, anthocyanins, phenolic acids and phenolic acid compounds, and polymeric phenols. Tea polyphenols can account for 30% of the dry weight of tea leaves. Tea polyphenols have broad application prospects in oil, food, medicine, daily chemical, light chemical, cosmetics, health care and many other fields. The raw material source is abundant, and the profit and added value are high, so it has become a research hotspot.

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

[0004] Among them, EGCG monomer is the main antioxidant component of tea polyphenols, and its reducing property can reach 100 times of L-isoascorbic acid. The research on epicatechin EC shows that it has antioxidant effect and free radical scavenging effect, and improving the level of epicatechin 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 activate acetylcholinesterase. In addition, it is an ideal health product for athletes, because it has anabolic and muscle strength promoting effect. In addition, it has been found that epicatechin EC can promote heart health and has potential anticancer effect.

[0005] In addition, EGCG and EC have wide biological activities such as anticancer, antiviral and antioxidant. Because EGCG has higher content and stronger antioxidant capacity, it has been widely concerned and applied in many studies. Therefore, it is of great significance to further separate and extract EGCG and EC from green tea tea polyphenols. SUMMARY

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

[0007] In a first aspect, the application provides a method for extracting green tea polyphenols, which adopts the following technical solution:

[0008] A method for extracting green tea polyphenols, comprising the following steps:

[0009] S1. After washing the green tea raw material with acid water, macroporous resin is used for separation, and then ethanol is used for elution to prepare a tea polyphenol extract;

[0010] S2. The prepared tea polyphenol extract is first passed through a cellulose column filled with cellulose packing prepared by using epicatechin as a template molecule, and then ethyl propionate is used for elution to prepare an EC extract;

[0011] 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 an ethanol solution is used for elution to prepare an EGCG extract.

[0012] By adopting the above technical solution, the green tea is first washed in acid water in the application, which is helpful for the dissolution of tea polyphenols, and then macroporous resin is used for selective adsorption of tea polyphenols, and then ethanol is used for elution to prepare a tea polyphenol extract. The prepared tea polyphenol extract is first passed through a cellulose column, and then ethyl acetate is used for elution. The cellulose column is prepared by using epicatechin as a template molecule, so that the imprint cavities on the cellulose column are highly matched with epicatechin EC, realizing high adsorption of epicatechin EC and separation and extraction of EC monomers. Moreover, due to the larger molecular weight of EGCG, the adsorption of the cellulose column to EGCG is weak, and the adsorption to EC is strong.

[0013] Based on the fact that the EGCG monomer structure contains more phenolic hydroxyl groups and galloyl groups, EGCG has strong polarity and hydrophilicity. The EC molecular structure is small, contains fewer phenolic hydroxyl groups, and does not contain ester groups. Based on the above two differences, 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, contains hydroxyl groups, and the ester functional groups make the copolymer have good adsorption performance to EGCG. EGCG is a monomer containing an ester bond in the catechin monomer of tea polyphenols. In combination with the introduction of ether bonds in the copolymer, it is easier to adsorb the ester-containing EGCG monomer molecules with similar polarity. Moreover, the ether bond can form a stable complex with the ester-containing substance through the matching of the spatial structure, enhancing the adsorption effect of EGCG. Finally, the separation of EGCG monomers is realized. Finally, the method provided in the application realizes the efficient separation and collection of EGCG monomers and EC monomers.

[0014] Optionally, the cellulose packing in step S2 is prepared by the following method:

[0015] The epicatechin and methacrylic acid monomer, acrylamide monomer are dissolved in dimethyl sulfoxide, ultrasonic dispersion for 10-20 min, then microcrystalline cellulose, crosslinking agent and initiator are added, stirring reaction at 80-100 ℃ for 16-20 h, then stop the reaction and stand for 30-50 min, then filter, then elution by adding ethanol solution and drying to obtain the cellulose filler.

[0016] By adopting the technical scheme, the cellulose filler in the application uses epicatechin EC as a template agent molecule, microcrystalline cellulose as a cellulose matrix, and under the action of an initiator and a crosslinking agent, methacrylic acid monomers and acrylamide monomers can be self-polymerized and copolymerized, and the carboxyl and amino groups can also be crosslinked with the microcrystalline cellulose matrix containing a hydroxyl functional group to form a network structure, while embedding the template agent molecule. The difference in solubility between the prepared polymer and epicatechin EC in ethanol is used to elute the template agent with ethanol to form an imprint cavity matching the shape, size and functional group of epicatechin EC, and then a molecularly imprinted cellulose column is prepared, which is used for the separation of tea polyphenol extract and has excellent specific selective adsorption for epicatechin EC, realizing the separation and extraction of EC monomers.

[0017] Optionally, in the preparation process of the cellulose filler, the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is ammonium persulfate and azobisisobutyronitrile with a mass ratio of 1: (0.8-1.2).

[0018] Optionally, in the preparation process of the cellulose filler, the raw materials are added according to the following weight parts:

[0019] 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.

[0020] By adopting the above technical scheme, the molecularly imprinted cellulose filler prepared by using the above proportioned raw materials has better specific adsorption for EC.

[0021] Optionally, in step S2, the prepared cellulose filler is columned, then washed with dichloromethane, and then the tea polyphenol extract prepared in step S1 is passed through the cellulose column.

[0022] By adopting the above technical scheme, the prepared cellulose filler is washed with dichloromethane, and the weak polarity of dichloromethane further enhances the specific adsorption of the cellulose column for EC monomers due to the strong polarity of EGCG.

[0023] 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:

[0024] The PCL-PHEMA copolymer is mixed with dimethylformamide and acetone, and the mixture is stirred and heated to 70-80°C to obtain mixture A;

[0025] Sulfuric acid is added to the prepared mixture A under stirring, and then bromoethane is added, and the temperature is raised to 120-140°C. After 1-2h of reaction, toluene is added and the reaction is continued for 2-3h;

[0026] After cooling, neutralization, and evaporation of the solvent, the modified PCL-PHEMA copolymer resin is obtained by direct water washing in water at 1-5°C.

[0027] By using the above technical solution, in this application, PCL-PHEMA copolymer is used as the benchmark, which is first dissolved and heated, and the hydroxyl groups in the copolymer are activated under the condition of an acidic catalyst. The activated hydroxyl group attacks the carbon atom in the halogenated hydrocarbon as a nucleophile, causing the carbon-halogen bond to break, and at the same time, an ether bond is formed between the oxygen atom of the hydroxyl group and the broken carbon atom. Under the condition of an acid catalyst, proton transfer occurs, and finally a modified copolymer containing an ether bond is formed. Due to the difference in the position of the hydroxyl substituent between EGCG and ECG, the polarity of the two is different. Since EGCG has more phenolic hydroxyl groups and aromatic rings, it has stronger polarity and hydrogen bond formation ability. In this application, PCL-PHEMA copolymer is selected as the benchmark and an ether bond is introduced into the hydrophilic segment of the copolymer, which enhances the hydrophilicity and polarity of the modified copolymer, making the modified copolymer form stronger hydrogen bonds with EGCG and have stronger adsorption of EGCG.

[0028] In this application, PCL-PHEMA copolymer is selected as the resin filler, and during the modification process, toluene is added to form a pore structure in the copolymer. Then, after the subsequent evaporation of the solvent, rapid cooling is performed to adjust the pore structure, making the modified copolymer have stronger adsorption of EGCG with larger molecular weight, and more small molecular ECG can pass through the modified copolymer resin filler without being adsorbed. Then, the subsequent elution program is used to separate the two, and finally, high-efficiency collection of EGCG is realized.

[0029] Optionally, during the preparation of the modified PCL-PHEMA copolymer resin, the raw materials are added according to the following weight parts:

[0030] 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 bromoethane, and 10-15 parts of toluene.

[0031] By adopting the technical scheme, the ether bond is formed in the copolymer, and a certain amount of hydroxyl is retained, so that the adsorption of EGCG monomers is enhanced, and then the control of the subsequent temperature change is realized by controlling the amount of toluene porogen, so that the adsorption of EGCG and ECG is realized, and then the control of the subsequent elution program is realized to collect EGCG.

[0032] Optionally, in step S3, after the tea polyphenol extract is passed through the column, 5-10% mass concentration of ethanol solution is used for elution to obtain eluate A, then 40-50% mass concentration of ethanol solution is used for elution to obtain eluate B, then 70-80% mass concentration of ethanol solution is used for elution to obtain eluate C, and then the eluate C is concentrated to remove ethanol to obtain the EGCG extract.

[0033] By adopting the technical scheme, first, the low-concentration ethanol solution is used for elution to remove impurities, then the medium-concentration ethanol is used for elution to remove a small amount of ECG, and then the high-concentration ethanol solution is used for elution to obtain the high-purity EGCG extract.

[0034] Optionally, the specific operation of step S1 is as follows:

[0035] In step S1, the green tea raw material is crushed and then washed with acid water with a pH of 3-4 for 4-6 hours, and then filtered, and the filtrate is concentrated and then passed through a macroporous resin, and then eluted with 60-70% ethanol aqueous solution to obtain a tea polyphenol extract.

[0036] By adopting the technical scheme, the green tea raw material is first crushed to increase the surface area, which is beneficial to the dissolution of the effective components in the subsequent process, and then the green tea raw material is washed in an acidic environment, the acidic environment destroys the cell structure of the tea leaves, promotes the dissolution of the tea polyphenol effective components, and inhibits the activity of the enzyme to prevent the tea polyphenols from being enzymatically degraded in the tea polyphenol extraction process, the dissolution rate of the tea polyphenols is improved by the above method, and then the filtrate containing the tea polyphenols is obtained, and then the macroporous resin is used to separate the tea polyphenols, and then the ethanol solution is used for elution to obtain the eluate rich in tea polyphenols.

[0037] Optionally, the macroporous resin in step S1 is selected from D101 type macroporous resin.

[0038] In summary, the present application has the following beneficial effects:

[0039] 1、The green tea is first rinsed in acid water to help dissolve tea polyphenols in the application, then the tea polyphenols are selectively adsorbed by macroporous resin, then the tea polyphenol extract is prepared by elution with ethanol, and then the prepared tea polyphenol extract is sequentially passed through a cellulose column and a modified PCL-PHEMA copolymer resin column to realize the separation and collection of EC monomers and EGCG monomers, respectively.

[0040] 2、In the application, the cellulose filler uses epicatechin EC as a template agent molecule, microcrystalline cellulose as a cellulose matrix, and under the action of an initiator and a crosslinking agent, the methyl methacrylate monomer and the acrylamide monomer can be self-polymerized and copolymerized, and the carboxyl and amino groups can also be crosslinked with the microcrystalline cellulose matrix containing hydroxyl functional groups to form a network structure, while embedding the template agent molecules. By taking advantage of the difference in solubility of the prepared polymer and epicatechin EC in ethanol, the template agent is eluted with ethanol to form an imprint cavity that matches the shape, size and functional groups of epicatechin EC, and then a molecularly imprinted cellulose column is prepared, which is used for the separation of tea polyphenol extract and has excellent specific selective adsorption for epicatechin EC, realizing the separation and extraction of EC monomers.

[0041] 3、The modified PCL-PHEMA copolymer resin column in the application is composed of polycaprolactone and polyhydroxyethyl methacrylate, containing hydroxyl groups, and the ester functional group of the copolymer has good adsorption performance for EGCG. In addition, the introduction of ether bonds in the copolymer makes it easier to adsorb EGCG monomer molecules with similar polarity containing ester bonds, and the ether bonds can also form stable complexes with ester-containing substances through spatial structure matching, enhancing the adsorption effect of EGCG. Finally, the separation of EGCG monomers is realized, and the efficient separation and collection of EGCG monomers and EC monomers are realized through the method provided in the application. DETAILED DESCRIPTION

[0042] The application is further described in detail below in conjunction with the examples. It is particularly pointed out that: in the following examples, the specific conditions are not specified, and the conventional conditions or the conditions recommended by the manufacturer are used. In the following examples, the raw materials used can be obtained from ordinary commercial sources unless otherwise specified.

[0043] In the following preparation examples, PCL-PHEMA, i.e., polycaprolactone-polyhydroxyethyl methacrylate, is selected from polycaprolactone-polyhydroxyethyl methacrylate block copolymer purchased from Hangzhou Xinqiao Biological Technology Co., Ltd., with a Mw of 5000.

[0044] The following preparation example is a preparation example of a cellulose filler

[0045] Preparation Example 1

[0046] A preparation method of a cellulose filler, comprising the following steps:

[0047] 1.5 kg epicatechin and 2 kg methacrylic acid monomer, 1.5 kg acrylamide monomer were dissolved in 2.5 kg dimethyl sulfoxide, ultrasonic dispersion for 15 min, then 2 kg microcrystalline cellulose, 0.2 kg crosslinking agent and 0.3 kg initiator were added, stirring reaction at 90℃ for 18 h, then stop reaction and stand for 40 min, then filter, then elution by adding ethanol solution and drying, to prepare cellulose filler.

[0048] The crosslinking agent is selected from ethylene glycol dimethacrylate, and the initiator is selected from ammonium persulfate and azobisisobutyronitrile with a mass ratio of 1:1.

[0049] Preparation Example 2

[0050] A method for preparing a cellulose filler, comprising the following steps:

[0051] 1 kg epicatechin and 1.5 kg methacrylic acid monomer, 1 kg acrylamide monomer were dissolved in 2 kg dimethyl sulfoxide, ultrasonic dispersion for 10 min, then 1.5 kg microcrystalline cellulose, 0.1 kg crosslinking agent and 0.2 kg initiator were added, stirring reaction at 80℃ for 20 h, then stop reaction and stand for 30 min, then filter, then elution by adding ethanol solution and drying, to prepare cellulose filler.

[0052] The crosslinking agent is selected from ethylene glycol dimethacrylate, and the initiator is selected from ammonium persulfate and azobisisobutyronitrile with a mass ratio of 1:0.8.

[0053] Preparation Example 3

[0054] A method for preparing a cellulose filler, comprising the following steps:

[0055] 2 kg epicatechin and 2.5 kg methacrylic acid monomer, 2 kg acrylamide monomer were dissolved in 3 kg dimethyl sulfoxide, ultrasonic dispersion for 20 min, then 2.5 kg microcrystalline cellulose, 0.3 kg crosslinking agent and 0.4 kg initiator were added, stirring reaction at 100℃ for 16 h, then stop reaction and stand for 50 min, then filter, then elution by adding ethanol solution and drying, to prepare cellulose filler.

[0056] The crosslinking agent is selected from ethylene glycol dimethacrylate, and the initiator is selected from ammonium persulfate and azobisisobutyronitrile with a mass ratio of 1:1.2.

[0057] Preparation Example 4

[0058] A method for preparing a cellulose filler, according to the method in Preparation Example 1, except that no crosslinking agent is added in the raw materials.

[0059] The following preparation example is a preparation example of a modified PCL-PHEMA copolymer resin filler

[0060] Preparation Example 5

[0061] A preparation method of a modified PCL-PHEMA copolymer resin filler, comprising the following steps:

[0062] 3.5 kg of PCL-PHEMA copolymer was mixed with 1.5 kg of dimethylformamide and 1.2 kg of acetone, and the mixture was stirred and heated to 75°C to obtain mixture A;

[0063] 0.3 kg of sulfuric acid was added to the prepared mixture A under stirring, and then 2.5 kg of bromoethane was added, and the temperature was raised to 130°C, and after 1.5 h of reaction, 1.2 kg of toluene was added, and the reaction was continued for 2.5 h;

[0064] After cooling, neutralization, and evaporation of the solvent, the modified PCL-PHEMA copolymer resin was directly placed in water at 3°C for water washing, and the modified PCL-PHEMA copolymer resin filler was obtained after cutting and granulation.

[0065] Preparation Example 6

[0066] A preparation method of a modified PCL-PHEMA copolymer resin filler, comprising the following steps:

[0067] 3 kg of PCL-PHEMA copolymer was mixed with 1 kg of dimethylformamide and 0.8 kg of acetone, and the mixture was stirred and heated to 70°C to obtain mixture A;

[0068] 0.2 kg of sulfuric acid was added to the prepared mixture A under stirring, and then 2 kg of bromoethane was added, and the temperature was raised to 120°C, and after 2 h of reaction, 1 kg of toluene was added, and the reaction was continued for 2 h;

[0069] After cooling, neutralization, and evaporation of the solvent, the modified PCL-PHEMA copolymer resin was directly placed in water at 1°C for water washing, and the modified PCL-PHEMA copolymer resin filler was obtained after cutting and granulation.

[0070] Preparation Example 7

[0071] A preparation method of a modified PCL-PHEMA copolymer resin filler, comprising the following steps:

[0072] 4 kg of PCL-PHEMA copolymer was mixed with 2 kg of dimethylformamide and 1.5 kg of acetone, and the mixture was stirred and heated to 80°C to obtain mixture A;

[0073] After cooling, neutralization, and evaporation of the solvent, the modified PCL-PHEMA copolymer resin was obtained by water washing at room temperature.

[0074] After cooling, neutralization, and evaporation of the solvent, the modified PCL-PHEMA copolymer resin was obtained by water washing at room temperature.

[0075] Preparation Example 8

[0076] A method for preparing a modified PCL-PHEMA copolymer resin filler, which was prepared according to the method in Preparation Example 5, except that the modified PCL-PHEMA copolymer resin was directly obtained by water washing at room temperature after neutralization and evaporation of the solvent.

[0077] Comparative Preparation Example 1

[0078] A method for preparing a PCL-PHEMA copolymer resin filler, comprising the following steps:

[0079] 3.5 kg of PCL-PHEMA copolymer was mixed with 1.5 kg of dimethylformamide and 1.2 kg of acetone, and mixture A was prepared by heating to 75°C and stirring.

[0080] Under stirring, 1.2 kg of toluene was added, and the temperature was raised to 130°C and stirred for 2.5 h.

[0081] After cooling, neutralization, and evaporation of the solvent, the modified PCL-PHEMA copolymer resin was obtained by water washing at room temperature.

[0082] Example 1

[0083] A method for extracting green tea polyphenols, comprising the following steps:

[0084] S1, the green tea raw material was crushed and passed through a 60 mesh sieve to obtain green tea raw material powder, 10 kg of green tea raw material powder was washed with hydrochloric acid aqueous solution at pH 3.5 at room temperature for 5 h, the green tea raw material powder was completely immersed in the hydrochloric acid aqueous solution, then filtered, the filtrate was concentrated to 1 / 5 of the original volume, then passed through a D101 macroporous resin at a flow rate of 3 BV / h, then eluted with 65% mass concentration of ethanol aqueous solution at a flow rate of 2 BV / h for 3 BV, and the eluate was collected to obtain a tea polyphenol extract;

[0085] 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, then eluted with 6 BV of ethyl propionate as eluent at a flow rate of 2 BV / h, to prepare an EC extract, then the prepared EC extract is poured into a rotary evaporator, and the ethyl propionate is evaporated at 75°C and 250 mmHg, then methanol is added to obtain an EC treatment solution with methanol as the solvent, then the EC treatment solution is crystallized and dried to obtain an EC extract;

[0086] S3, the tea polyphenol extract after passing through the cellulose column is passed through a column filled with 1 kg of modified PCL-PHEMA copolymer resin prepared in Preparation Example 5, then eluted with 8% ethanol solution to obtain eluent A, then eluted with 75% ethanol solution to collect eluent B, and eluent B is concentrated to remove alcohol and dried to obtain an EGCG extract.

[0087] In the above steps S2 and S3, the cellulose column and the modified PCL-PHEMA copolymer resin column are first washed with water to balance the cellulose column and the modified PCL-PHEMA copolymer resin column before passing through the tea polyphenol extract, and the volume of water added is 3 times the bed volume of the cellulose column.

[0088] Example 2

[0089] A method for extracting green tea polyphenols, comprising the following steps:

[0090] S1, the green tea raw material is crushed and passed through a 60 mesh sieve to obtain green tea powder, 10 kg of green tea powder is washed with pH 3 hydrochloric acid aqueous solution at room temperature for 4 h, the green tea powder is completely immersed in the hydrochloric acid aqueous solution, then filtered, the filtrate is concentrated to 1 / 5 of the original volume, then passed through a D101 type macroporous resin at a flow rate of 3 BV / h, then eluted with 3 BV of 60% mass concentration ethanol aqueous solution at a flow rate of 2 BV / h, and the tea polyphenol extract is collected;

[0091] 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 2, then eluted with 6 BV of ethyl propionate as eluent at a flow rate of 2 BV / h, to prepare an EC extract, then the prepared EC extract is poured into a rotary evaporator, and the ethyl propionate is evaporated at 70°C and 200 mmHg, then methanol is added to obtain an EC treatment solution with methanol as the solvent, then the EC treatment solution is crystallized and dried to obtain an EC extract;

[0092] S3, the tea polyphenol extract after passing through the cellulose column is passed through a modified PCL-PHEMA copolymer resin column prepared in Preparation Example 6, and then eluted with 5% ethanol solution to obtain eluate A, and then eluted with 70% ethanol solution to obtain eluate B. Eluate B is concentrated to remove alcohol and dried to obtain an EGCG extract.

[0093] In the above steps S2 and S3, the cellulose column and the modified PCL-PHEMA copolymer resin column are first washed with water to balance the cellulose column and the modified PCL-PHEMA copolymer resin column before passing the tea polyphenol extract, and the volume of water added is 3 times the bed volume of the cellulose column.

[0094] Example 3

[0095] A method for extracting tea polyphenols from green tea, comprising the following steps:

[0096] S1, green tea raw materials are crushed and passed through a 60-mesh sieve to obtain green tea powder. 10 kg of green tea powder is washed with a pH 4 hydrochloric acid aqueous solution at room temperature for 6 h, and the green tea powder is completely immersed in the hydrochloric acid aqueous solution. Then, the filtrate is concentrated to 1 / 5 of the original volume, passed through a D101 macroporous resin at a flow rate of 3 BV / h, and then eluted with 70% ethanol aqueous solution at a flow rate of 2 BV / h for 3 BV. The eluate is collected to obtain a tea polyphenol extract;

[0097] S2, the tea polyphenol extract collected in step S1 is first passed through a cellulose column packed with 1 kg of cellulose packing prepared in Preparation Example 3, and then eluted with ethyl propionate as an eluent at a flow rate of 2 BV / h for 6 BV to prepare an EC extract. The prepared EC extract is then evaporated in a rotary evaporator at 80°C and 350 mmHg to remove ethyl propionate, and then methanol is added to obtain an EC treatment solution with methanol as the solvent. The EC treatment solution is crystallized and dried to prepare an EC extract;

[0098] S3, the tea polyphenol extract after passing through the cellulose column is passed through a modified PCL-PHEMA copolymer resin column prepared in Preparation Example 7, and then eluted with 10% ethanol solution to obtain eluate A, and then eluted with 80% ethanol solution to obtain eluate B. Eluate B is concentrated to remove alcohol and dried to obtain an EGCG extract.

[0099] In the above steps S2 and S3, the cellulose column and the modified PCL-PHEMA copolymer resin column are first washed with water to balance the cellulose column and the modified PCL-PHEMA copolymer resin column before passing the tea polyphenol extract, and the volume of water added is 3 times the bed volume of the cellulose column.

[0100] Example 4

[0101] A method for extracting green tea polyphenols, which is carried out according to the method in Example 1, except that the cellulose column is filled with the cellulose packing prepared in Preparation Example 4 in step S2.

[0102] Example 5

[0103] A method for extracting green tea polyphenols, which is carried out according to the method in Example 1, except that the cellulose packing is first washed with water and then with dichloromethane after being packed in step S2, and the tea polyphenol extract prepared in step S1 is then passed through the cellulose column, and the amount of dichloromethane added is 2 times the volume of the cellulose column.

[0104] Example 6

[0105] A method for extracting green tea polyphenols, which is carried out according to the method in Example 1, except that the EC extract prepared in step S2 is directly evaporated and crystallized to prepare the EC extract.

[0106] Example 7

[0107] A method for extracting green tea polyphenols, which is carried out according to the method in Example 1, except that the modified PCL-PHEMA copolymer resin packing prepared in Preparation Example 8 is used in step S3.

[0108] Comparative Example 1

[0109] A method for extracting green tea polyphenols, which is carried out according to the method in Example 1, except that 10 kg of green tea raw material powder is added to 8 times its mass of 70% mass concentration ethanol and heated to reflux for 2 times, 3 hours each time, the filtrate is combined after filtration, and then distilled under reduced pressure to remove the alcohol, the supernatant is taken after centrifugation, and then passed through a D101 macroporous resin.

[0110] Comparative Example 2

[0111] A method for extracting green tea polyphenols, which is carried out according to the method in Example 1, except that the modified PCL-PHEMA copolymer resin packing is replaced with an equal amount of the PCL-PHEMA copolymer resin packing prepared in Comparative Preparation Example 1 in step S3.

[0112] Comparative Example 3

[0113] A method for extracting green tea polyphenols, which is carried out according to the method in Example 1, except that the tea polyphenol extract is eluted with 25% mass concentration ethanol after passing through the cellulose column in step S2 to prepare the EC extract.

[0114] Comparative Example 4

[0115] A method for extracting green tea tea polyphenols, according to the method in Example 1, the difference is that 10 kg of green tea raw material powder is added to 8 times the mass concentration of 70% ethanol heated to reflux extraction 2 times, 3h each time, after filtration, the filtrate is combined, evaporated to no alcohol, then diluted with water to the mass volume ratio of green tea raw material powder and water is 0.03g:1mL, then adjust the pH to 3.5 with hydrochloric acid, then pass through D101 type macroporous resin.

[0116] Performance test

[0117] The same batch of green tea raw material (tea polyphenol content of 35%) is operated according to the above method, and the tea polyphenol extract is prepared by concentrating and drying the tea polyphenol extract prepared in step S1 of Examples 1-3, Comparative Example 1 and Comparative Example 4. The tea polyphenol content and yield of the tea polyphenol extract are detected, and the detection results are shown in Table 1 below.

[0118] Table 1:

[0119]

[0120] Referring to the detection results in Table 1 above, the extraction method in the present application has good extraction effect on tea polyphenols, and the tea polyphenol content in the tea polyphenol extract in step S1 is high. Moreover, the method provided in the present application has high yield of tea polyphenols. Referring to the detection method of Examples 1 and Comparative Examples 1 and 4, the acid solution extraction method in the present application has significantly improved the yield of tea polyphenols compared to the traditional alcohol extraction method in Comparative Example 1. In addition, the detection method in Comparative Example 4, after alcohol extraction and pH adjustment for subsequent macroporous resin purification, the yield of tea polyphenols is still lower than that of Example 1.

[0121] In addition, the yield of EC extract and EGCG extract obtained in steps S2 and S3 is counted, and the counting results are shown in Table 2 below:

[0122] Table 2:

[0123]

[0124] Moreover, the contents of EC and EGCG in the EC extract and EGCG extract prepared in the above examples are determined by HPLC method. The contents of EC and EGCG in the extracts prepared in Examples 1-5 above are all above 98%. Referring to the detection results in Table 2 above, it can be seen that the yields of EC monomers and EGCG monomers prepared in step S2 are also high. Through the method provided in the present application, not only high content and high yield of tea polyphenols are prepared, but also high purity and high yield of EGCG monomers and EC monomers are prepared through subsequent specific separation.

[0125] From the detection results of Example 1 and Example 4, it can be seen that when the cellulose filler is prepared without adding a crosslinking agent in Example 4, the subsequent adsorption effect on EC is reduced, the yield is reduced, and it may be that the addition of the crosslinking agent affects the polarity and pore structure distribution of the cellulose filler, and it is found that the yield of EGCG is also reduced, which may be due to the loss of EGCG during column chromatography in step S2. In addition, combined with the detection results of Example 1 and Example 5, the cellulose filler is washed with dichloromethane after equilibration, which does not reduce the yield of EGCG, but significantly improves the yield of EC monomer, and the specific adsorption performance of the cellulose filler for EC monomer is better.

[0126] Referring to the detection results of Example 1 and Example 6, in Example 6, the prepared EC extract solution is directly evaporated and crystallized in step S2, and the content of EC in the prepared EC extract is reduced to 92.3% without solvent exchange post-treatment. Combined with the detection results of Example 7, in step S3, the modified PCL-PHEMA copolymer resin filler is prepared by adding toluene for pore formation during the modification process, but after evaporation of the solvent, it is not directly placed in low-temperature water for treatment, and the pore structure is not adjusted, which affects the adsorption performance of EGCG, resulting in a decrease in the yield of EGCG.

[0127] Combined with the detection results of Example 1 and Comparative Examples 1 and 4, it can be seen that when the tea polyphenol extraction method in step S1 results in a low yield of tea polyphenol, the yields of EC monomer and EGCG monomer are also significantly reduced. Combined with the detection results of Comparative Examples 2 and 3, in Comparative Example 2, the PCL-PHEMA copolymer resin filler is not reacted with a halogenated hydrocarbon to form an ether bond under strong acid conditions with sulfuric acid, which reduces the adsorption of EGCG monomer, and the yield is significantly reduced. In Comparative Example 3, after the tea polyphenol extract passes through the cellulose column, ethanol is used for elution, which significantly reduces the yield of EC monomer, and the content of EC is also significantly reduced to 78.5%.

[0128] Among them, the horizontal bar in Example 7, Comparative Example 2 and Comparative Example 3 indicates that only the elution post-treatment operation in step S2 has no effect on the yield of EGCG in step S3, or only the change of the filler in step S3 does not affect the yield of EC in step S2.

[0129] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for extracting green tea tea polyphenols, characterized by, The method comprises the following steps: S1, crushing green tea raw materials, then washing with acid water with pH of 3-4 for 4-6 hours, then filtering, concentrating the filtrate, then passing through a macroporous resin, then eluting with 60-70% ethanol aqueous solution to obtain a tea polyphenol extract; S2, the tea polyphenol extract obtained is first passed through a cellulose column filled with cellulose filler prepared by using epicatechin as a template molecule, then eluted 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, then eluted with 5-10% ethanol solution to obtain an eluate A, then eluted with 70-80% ethanol solution to obtain an eluate B, then removing ethanol from the eluate B by concentration to obtain an EGCG extract; The cellulose filler in step S2 is prepared by the following method: Dissolve epicatechin, methacrylic acid monomer and acrylamide monomer in dimethyl sulfoxide, ultrasonic dispersion for 10-20 min, then add microcrystalline cellulose, crosslinking agent and initiator, stir and react at 80-100℃ for 16-20 h, then stop the reaction, stand for 30-50 min, then filter, then elute with an ethanol solution, and then dry to obtain the cellulose filler; 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; The crosslinking agent is selected from ethylene glycol dimethacrylate, and the initiator is selected from ammonium persulfate and azobisisobutyronitrile with a mass ratio of 1: (0.8-1.2); The filler of the modified PCL-PHEMA copolymer resin column is a modified PCL-PHEMA copolymer resin, which is prepared by the following method: Mix PCL-PHEMA copolymer with dimethylformamide and acetone, heat to 70-80℃, and stir to obtain a mixture A; Under stirring, add sulfuric acid to the prepared mixture A, then add bromoethane, heat to 120-140℃, react for 1-2 h, then add toluene and continue to react for 2-3 h; After cooling, neutralize, then evaporate the solvent, and then directly put into water at 1-5℃ for water washing to obtain the modified PCL-PHEMA copolymer resin; During the preparation of 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 bromoethane, and 10-15 parts of toluene.

2. The method for extracting tea polyphenols from green tea according to claim 1, characterized in that: In step S2, the prepared cellulose filler is columned, then washed with dichloromethane, and then the tea polyphenol extract prepared in step S1 is passed through the cellulose column.

3. The method for extracting tea polyphenols from green tea according to claim 1, characterized in that: The macroporous resin in step S1 is selected from D101 type macroporous resin.

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