A method for preparing a polyester-type catechin

Abstract

The present application relates to a kind of preparation methods of polyester catechin, comprising the following steps: S1, from the leaf of Castanopsis fargesii fresh leaf extraction tea enzyme, using the ammonium sulfate with 30%-80% saturation fractionated purification tea enzyme, and keep the ammonium sulfate purification part with 40%-60% saturation, remove the impurity with molecular weight <3000Da, obtain enzyme liquid;Catechin monomer is mixed according to concentration ratio, and catechin monomer solution with concentration of 0.1-10mg / mL is prepared, S2, catechin monomer solution and enzyme liquid are mixed to carry out enzymatic reaction, and then terminate reaction, centrifugal, obtain enzymatic reaction product;S3, the enzymatic reaction product is on macroporous resin chromatographic column, eluted by ethanol solution, collect eluent, concentrate, dry and obtain the polyester catechin.The method of the present application can significantly improve the efficiency of polyester catechin, compared with prior art, the yield of product of the present application is increased by 120%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural product extraction and separation, and particularly relates to a method for preparing polyester catechin using catechin monomers and tea leaf enzymes. BACKGROUND

[0002] Polyester catechin (TSs) is a kind of tea polyphenol compound with important biological activity, which is mainly formed by oxidation and polymerization of catechin monomers under the catalysis of tea leaf enzymes during black tea processing. TSs has important contribution to the color, taste and aroma of black tea, and is one of the core products formed in the process of black tea processing. Studies have shown that TSs exhibits significant effects in antioxidant, anticancer, antibacterial and other aspects, and has broad application prospects in the fields of food, medicine, cosmetics and the like.

[0003] The structure of TSs monomer is multiple, and eight kinds of polyester catechin structures are separated and identified from tea leaves, and the specific structures are as follows:

[0004] .

[0005] Among them, the polyester catechin A has a CAS number of 89064-31-3, a molecular formula of C 44 H 34 O 22 , and a molecular weight of 914.73; the polyester catechin B has a CAS number of 89064-32-4, a molecular formula of C 37 H 30 O 18 , and a molecular weight of 762.62; the polyester catechin C has a CAS number of 89013-69-4, a molecular formula of C 30 H 26 O 14 , and a molecular weight of 610.52.

[0006] TSA is a light brown amorphous powder, and has a molecular formula of C 44 H 34 O 22 . The (1H) spectrum and (13C) spectrum show that it is a symmetrical molecular structure containing one biphenyl group; TSB is also a light brown amorphous powder, and has a molecular formula of C 37 H 30 O 18 . In addition, TSD, TSE, TSF and TSG are beige (close to light brown). TSH is a brown amorphous powder, and has a molecular formula of C 37 H 30 O8. Among them, TSA and TSD are each other's atropisomers, TSE is the S-type atropisomer of TSC, that is, S-TSE and R-TSC are each other's optical isomers, and TSH and TSB are also each other's atropisomers.

[0007] Currently, there are many challenges in obtaining polyphenols. The most traditional method is to separate and purify from black tea, but research by the Tea Research Institute of the Chinese Academy of Agricultural Sciences shows that the total amount of polyphenols in many black tea samples is less than 2%, and the total amount of polyphenols in most black tea samples is around 1%. The average TSA content of Jinjunmei (2020) is 0.38%, and the average of the control series of black tea is 0.21%, which is relatively low. There are also a large number of compounds in tea samples that are similar in structure and polarity to polyphenols. Therefore, the traditional method of separating and purifying polyphenols from black tea has the defects of low extraction efficiency and high cost.

[0008] There are also synthetic methods for preparing polyphenols in the prior art. For example, the prior art CN109845852A discloses a chemical oxidation high-efficiency synthesis method of polyphenols, which comprises the following steps: 1) preparing a tea polyphenol solution; 2) adding a chemical catalyst copper salt to control the temperature reaction and promote the conversion of catechin to dehydrogenated polyphenol; 3) then adding vitamin C to continue the reaction and promote the conversion of dehydrogenated polyphenol to polyphenol; 4) after extraction, vacuum rotary evaporation concentration, drying, and obtaining polyphenol. However, this synthesis method requires the use of a large amount of organic solvent, which causes environmental pollution, and the synthesis steps are complex, making it difficult to ensure the purity of the product.

[0009] The prior art CN118256569A discloses a method for preparing polyphenol A at low temperature by enzyme method. The method uses enzyme extract or polyphenol oxidase enzyme preparation as the enzyme source, uses epigallocatechin gallate (EGCG) as the substrate, and uses a certain pH buffer as the reaction system. After low-temperature enzymatic reaction and subsequent column chromatography, distillation concentration, liquid phase preparation, and freeze-drying steps, polyphenol A (TSA) is synthesized by directional enzyme catalysis. This method realizes the enzymatic oxidation of EGCG at low temperature and obtains the dimer TSA through conversion reaction, which lays a theoretical and practical basis for the green, safe, and efficient large-scale industrial production and utilization of TSA. However, this technology only involves the preparation of polyphenol A, and there are many impurities.

[0010] The prior art CN114015733B discloses a method for catalytic synthesis of enzyme salt coupling of polyestercatechin, which uses green tea leaf as raw material, hot water extraction, and the supernatant of the extraction liquid is green tea extract after cooling, centrifugal impurity removal treatment; adding complex oxidoreductase liquid and salt liquid to the green tea extract, precisely controlling the pH value of the reaction system, the cation concentration and the ratio of the salt liquid, and the addition ratio and total amount of polyphenol oxidase liquid and peroxidase liquid, and controlling the fermentation time under the optimum temperature condition to obtain the fermentation liquid; the fermentation liquid is concentrated by membrane filtration and spray dried to obtain the polyestercatechin product. The content of polyestercatechin obtained by this technology is more than 30%, which is significantly higher than the content obtained by the traditional single polyphenol oxidase method. However, in this method, the raw material contains not only catechin compounds, but also other small molecule phenolic impurities. These impurities form a variety of oxides under the oxidation of enzymes. The impurities and the oxides formed by the impurities have similar polarity to the substrate catechin and the product polyestercatechin, and the separation and purification of the product is extremely difficult.

[0011] The prior art CN118581173A discloses a method for efficient in vitro synthesis of polyestercatechin A, which comprises the following steps: 1) preparing epigallocatechin gallate (EGCG) solution; 2) filtering and centrifuging the homogenized potato tuber, and then dialyzing and freeze-drying after ammonium sulfate salting-out to obtain potato polyphenol oxidase powder; 3) adding potato polyphenol oxidase powder to the EGCG solution, and stirring and reacting at a controlled temperature to promote the conversion of EGCG to dehydro-polyestercatechin A; 4) adding vitamin C and heating the reaction liquid after the reaction is completed to promote the conversion of dehydro-polyestercatechin A to polyestercatechin A; 5) filtering the reaction liquid, concentrating by vacuum rotary evaporation, and drying to obtain the polyestercatechin A product. However, this technology only relates to the preparation of polyestercatechin A, and has the problem of many impurities.

[0012] Xue Jinjin et al. (DOI:10.13386 / j.issn1002-0306.2019.20.013.) studied the oxidation characteristics of enzymes in immature Wenzhou Citrus unshiu, Japanese pear and loquat fruits, and found that the enzymes in immature Wenzhou Citrus unshiu fruits selectively oxidize pyrogallol-type catechins to produce dehydro-polyestercatechin, while the enzymes in Japanese pear and loquat can oxidize pyrogallol-type catechins to form dehydro-polyestercatechin, and can also oxidize pyrogallol-type catechins and catechin to form TFs. In black tea and oolong tea, oolong theanins and polyestercatechin exist simultaneously in tea leaves, which are the oxidation and reduction products of dehydro-polyestercatechin, respectively. These indicate that the preference of oxidases from different species for pyrogallol and catechin substrates and the types of products obtained (such as the formation of coupling products, benzotropolone products) are very different. The selection of enzymes has a great influence on the formation of TFs and polyestercatechin. SUMMARY

[0013] The application aims to provide a preparation method of polyester catechin with high efficiency, environmental protection and low cost.

[0014] To achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows:

[0015] A preparation method of polyester catechin comprises the following steps:

[0016] S1, extracting tea leaf enzyme from fresh leaves of Castanopsis fargesii, purifying the tea leaf enzyme by using ammonium sulfate with a saturation degree of 30%-80%, and reserving the ammonium sulfate purification part with a saturation degree of 40%-60%, removing impurities with a molecular weight of less than 3000 Da, and obtaining enzyme liquid;

[0017] Mixing catechin monomers according to a concentration ratio to prepare catechin monomer solution with a concentration of 0.1-10 mg / mL;

[0018] S2, mixing the catechin monomer solution and the enzyme liquid to perform enzymatic reaction, then stopping the reaction, and centrifuging to obtain an enzymatic reaction product;

[0019] S3, loading the enzymatic reaction product into a macroporous resin chromatographic column, eluting with an ethanol solution, collecting the eluate, and concentrating and drying to obtain the polyester catechin.

[0020] The raw material of the application is fresh leaves of Castanopsis fargesii, on the one hand, the application can quickly and simply extract the required enzyme from the fresh leaves of Castanopsis fargesii, and the method of extracting the enzyme from the raw material is simpler than using other raw materials such as potato oxidase; on the other hand, the enzyme in different tea varieties has obvious differences in molecular weight and oxidation activity, and the tea enzyme extraction parameters of the application are designed according to Castanopsis fargesii, which is more suitable for Castanopsis fargesii.

[0021] The application uses ammonium sulfate to fractionally precipitate tea leaf enzyme, and the tea leaf enzyme is separated from low to high concentration of ammonium salt solution according to the difference in molecular weight, which plays a role in purifying the tea leaf enzyme. After purification, the ammonium salt can be removed by using a dialysis bag, which is simple to operate and has high purity of tea leaf enzyme. And the application finds through a large number of experiments that the enzyme in the ammonium sulfate purification part with a saturation degree of 40%-60% has the best effect on converting catechin monomers.

[0022] Other methods of purifying enzymes need steps such as ultrafiltration and column chromatography in addition to dialysis, which need to use ultrafiltration membranes, fillers and chromatographic columns, and have too many purification steps, which affect the purity and yield of the product.

[0023] The present application removes components with a molecular weight of less than 3000 in the enzyme, prevents low-molecular-weight substances from participating in oxidation, thereby increasing the byproducts of the reaction system, reducing the yield, and further increasing the cost of further purification.

[0024] According to the embodiments of the present application, the present application can be further optimized, and the following is the technical solution formed after optimization:

[0025] In one preferred embodiment, the tea enzyme extracted from the fresh leaves of Castanopsis fargesii includes the following steps:

[0026] In the fresh leaves of Castanopsis fargesii, add an aqueous acetone solution for grinding, then wash, filter, and obtain a powder; add polyvinylpyrrolidone and a buffer solution to the powder in an ice bath; then filter, centrifuge, and collect the supernatant to obtain the tea enzyme.

[0027] In one preferred embodiment, the mass ratio of the powder to polyvinylpyrrolidone is 11.6:1.5-2.5.

[0028] In one preferred embodiment, the buffer solution is PBS or Tris buffer solution.

[0029] In one preferred embodiment, the washing solution is an aqueous acetone solution.

[0030] In one preferred embodiment, the volume concentration of the aqueous acetone solution for grinding and washing is 70-90%.

[0031] In one preferred embodiment, the step of using ammonium sulfate with a saturation of 30%-80% to fractionally purify the tea enzyme includes:

[0032] First, add ammonium sulfate solution to the tea enzyme to make the saturation of ammonium sulfate in the solution 30%, mix uniformly, then let the solution stand until the precipitation is complete, centrifuge, obtain the supernatant and the precipitate, and take the precipitate as the 30% precipitate part; continue to add ammonium sulfate solution to the supernatant to make the saturation of ammonium sulfate 40%, 60%, and 80%, and repeat the standing and centrifugation operation to obtain the 40% precipitate part, the 60% precipitate part, and the 80% precipitate part in sequence.

[0033] In the preferred embodiment of the present application, the 40% precipitate part and the 60% precipitate part are combined to form the 40%-60% ammonium sulfate purification part.

[0034] The saturation of ammonium sulfate refers to the actual concentration of ammonium sulfate in the current solution as a percentage of the maximum solubility of ammonium sulfate at that temperature (i.e., the saturation concentration). At 20°C (room temperature), the solubility of ammonium sulfate in water is about 70.6 grams per 100 grams of water.

[0035] In one preferred embodiment, in step S2, the catechin monomers include four of epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC).

[0036] A large number of experiments prove that, in the catechin monomer raw material, EGCG can obtain TSA, EGC can obtain TSC, and mixed use of EGCG and EGC can obtain TSA, TSB, and TSC. EGCG and EGC are necessary substrates for generating TSA, TSB, and TSC, and EC can reduce the reaction potential in the oxidation process of the raw material and increase the reaction efficiency. Although EGC is not a direct substrate for forming polyester catechin, the presence of EGC in the oxidation system will affect the oxidation balance of the entire catechin system, and its autoxidation product can also affect the yield of polyester catechin by affecting the oxidation-reduction disproportionation reaction. At the same time, EGC affects the microenvironment of catechin metabolism and further affects the formation of polyester catechin.

[0037] In one preferred embodiment, in step S2, the molar concentration ratio of epigallocatechin gallate, epigallocatechin, epicatechin gallate, and epicatechin is (8-4):(6-3):(5-3):(3-1).

[0038] In step S2, the molar concentration ratio of epigallocatechin gallate, epigallocatechin, epicatechin gallate, and epicatechin is determined according to the ratio of catechin content in the fresh leaves of Quercus. Too high or too low concentration of a single raw material will increase the by-products.

[0039] The following is an introduction to each raw material:

[0040] Epigallocatechin gallate (EGCG), CAS No.: 989-51-5, molecular formula: C 22 H 18 O 11 , molecular weight: 458.37.

[0041] Epigallocatechin (EGC), CAS No.: 970-74-1, molecular formula: C 15 H 14 O7, molecular weight: 306.27.

[0042] Epicatechin gallate (ECG), CAS No.: 1257-08-5, molecular formula: C 22 H 18 O 10 , molecular weight: 442.37.

[0043] Epicatechin (EC), CAS No. 14344-48-0, molecular formula: C 15 H 14 O6, molecular weight: 290.27.

[0044] In one preferred embodiment, in step S2, the mass ratio of the epicatechin monomer solution and the enzyme solution is 50-80:1.

[0045] In one preferred embodiment, in step S2, the enzymatic reaction temperature is 20-50℃, the pH of the reaction system is 3.5-7.5, and the reaction time is 0.5-6 hours.

[0046] In one preferred embodiment, in step S2, the step of terminating the reaction is to inactivate the reaction system in a water bath at 90-100℃ for 7-20 minutes.

[0047] In one preferred embodiment, in step S2, the centrifugal speed is 4000-8000 rpm, and the centrifugal time is 10-20 minutes.

[0048] In one preferred embodiment, in step S3, the macroporous resin chromatography column is an HP-20 macroporous adsorption resin column.

[0049] In one preferred embodiment, in step S3, the concentration of the ethanol solution is 30-70%.

[0050] In one preferred embodiment, in step S3, the elution flow rate is 1-3 mL / min.

[0051] Compared with the prior art, the present application has the following beneficial effects:

[0052] 1. The method of the present application has low cost: compared with using polyphenol oxidase products for enzymatic oxidation reaction, the present application uses epicatechin monomers and tea crude enzymes as raw materials, which are widely available, and simplifies the steps of tea enzyme separation and regulation of polyphenol oxidase and oxidase activity, thereby reducing production cost. The product system obtained is a polymeric epicatechin, which has a simple structure and can greatly reduce separation cost and improve separation efficiency compared with further purification.

[0053] 2、The method has high efficiency: the tea leaf enzyme prepared by the method of the application has high specificity for catalyzing the formation of polyestercatechin from catechin, for polyphenol oxidases from different species; the activity of the tea leaf oxidase is precisely regulated by optimizing the enzyme reaction conditions, which can significantly improve the efficiency of obtaining polyestercatechin, compared with the prior art, the product yield of the application is increased by 120%, reaching 1.54 mg / 100 ml, TSA is 0.72±0.04 mg / 100 ml; TSB is 0.42±0.03 mg / 100 ml; TSC is 0.40 mg / 100 ml; the purity of polyestercatechin (polyestercatechin A, B and C) in the obtained system after purification can reach 43%, which is 20-40 times higher than the efficiency of using black tea to extract polyestercatechin (the total amount of polyestercatechin in black tea is 1-2%).

[0054] 3、The application has good environmental protection: the application mainly uses relatively environmentally friendly reagents such as buffer solution and ethanol, avoiding the use of a large amount of organic solvents and reducing environmental pollution. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 is the HPLC analysis diagram of four catechin monomers.

[0056] Figure 2 is the HPLC analysis diagram of the product of example 2.

[0057] Figure 3 is the HPLC analysis diagram of the product of example 3.

[0058] Figure 4 is the HPLC analysis diagram of the product of example 4.

[0059] Figure 5 is the column diagram of the content of the product obtained at different reaction pHs.

[0060] Figure 6 is the column diagram of the content of the product obtained at different reaction temperatures. DETAILED DESCRIPTION

[0061] The application is not limited to the following specific embodiments, and those skilled in the art can use other various specific embodiments to implement the application according to the disclosed content of the application, or any simple changes or modifications made by using the design structure and ideas of the application, all fall within the protection scope of the application. It should be noted that the embodiments in the application and the features in the embodiments can be combined with each other without conflict.

[0062] The reagent EGCG, EGC, ECG and EC of the present application are provided by National Plant Functional Component Utilization Engineering Technology Research Center, and the polymeric catechins A, B and C are prepared by separation from black tea and verified by mass spectrometry and nuclear magnetic resonance.

[0063] The compound structure of polymeric catechin A is as follows:

[0064] Theasinensin A, chemical formula: C 44 H 34 O 22 ,

M-H-

[0065] The compound structure of polymeric catechin B is as follows:

[0066] Theasinensin B, chemical formula: C 37 H 30 O 18,

M-H-

[0067] The compound structure of the polyestercatechin C is as follows:

[0068] Theasinensin C, chemical formula: C 30 H 26 O 14 ,

M-H-

[0069] Other reagents were purchased from National Pharmaceutical Group.

[0070] Example 1

[0071] Tea leaf enzyme was extracted from fresh leaves of Castanopsis fargesii, and the fresh leaves (one bud and one leaf) of Castanopsis fargesii were picked from the Chang'an base of the Horticulture College of Hunan Agricultural University in summer. The extraction process is as follows:

[0072] Accurately weigh 4 powder of fresh tea leaves to be tested, 50.0 g each. Grind the fresh tea leaves in an ice bath using a small amount of quartz sand in a pre-cooled acetone solution. Filter the grinding liquid using a pre-cooled acetone solution until the washing liquid is colorless. Obtain 4 acetone powders. The concentration of the acetone solution is 100 vol%, 90 vol%, 80 vol%, and 70 vol% respectively. Add 1.76 g of PVP (polyvinylpyrrolidone) and 0.2 mL of PBS (pH = 6.5) to the acetone powder, a small amount of quartz sand, and homogenize in an ice bath. Filter through gauze. Centrifuge the filtrate at 1 x 10 4 rpm and 4°C for 10 min. Collect the supernatant as the crude enzyme solution.

[0073] Purify the 4 tea leaf crude enzymes obtained using different concentrations of ammonium sulfate. Add ammonium sulfate solution to 50 mL of the supernatant (tea leaf enzyme) to make the saturation of ammonium sulfate in the solution 30%, mix well, and centrifuge after the solution is completely precipitated. Obtain the precipitate and supernatant. Label the precipitate as the 30% precipitate fraction. Continue to add ammonium sulfate solution to the supernatant to make the saturation of ammonium sulfate in the solution 40%, 60%, and 80%, and repeat the centrifugation operation to obtain the 40%, 60%, and 80% precipitate fractions in sequence. Dissolve the precipitate using 10 mL of PBS (pH = 6.5) solution, and then dialyze the solution and the crude enzyme solution in Tris-HCl buffer (cut off molecular weight > 3000) overnight until no SO4 2- exists in the dialysis buffer. Obtain different tea leaf enzymes.

[0074] Determine the optimal ammonium sulfate fractionation gradient by measuring the activity of different tea leaf enzymes in catalyzing the formation of polyestercatechin A from EGCG.

[0075] Dissolve catechin monomer EGCG in a phosphate buffer solution with a pH value of 6.5 to make the concentration of EGCG in the reaction solution 0.1 mmol / L. Add the different tea leaf enzyme solutions obtained to the pre-oxygenated EGCG solution, and the mass ratio of the tea leaf enzyme solution to the EGCG solution is 1:60. Perform the enzymatic reaction at 35°C and a pH value of 6.5, and the reaction time is 4 hours. After the reaction is completed, terminate the reaction in a 100°C water bath, and then centrifuge at a speed of 6000 rpm for 15 min. Take the supernatant to obtain a polyestercatechin solution. Purify the polyestercatechin solution through a macroporous adsorption resin HP-20 using an ethanol solution with a volume fraction of 50% as the eluent, and the elution flow rate is 2 mL / min. Collect the eluate, concentrate, and freeze-dry to obtain high-purity polyestercatechin.

[0076] The HPLC detection was performed on the polyester type catechin solution, 272 nm was selected as the detection wavelength, 5.0 mg of the mixed standard was weighed, and the mass fractions of the contained substances were as follows: TSA 10.0%, TSB 6.0%, TSC 4.0%, EGCG 37.5%, EGC 8.8%, EC 3.3%, and ECG 9.9%, and water was added to prepare a 10 mL control solution for standby; 10.0 mg of the polyester type catechin obtained by freeze-drying was weighed, and pure water was used to prepare a 10 mg / 100 ml working solution, and A: 30% methanol / acetonitrile (v:v) and B: 0.3% phosphoric acid aqueous solution were used as the mobile phase; the detection gradient was as follows: A 0-22 min, 13%-21%; 22-35 min, 21-25%; 35-55 min, 25-25%; 55-56 min, 25-13%; the column temperature was 35 ℃; the flow rate was 0.8 mL / min; the wavelength was 272 nm; the chromatographic column was welchrom C18 column (4.6*200 mm, 5 μm); and the instrument was Shimadzu Prominence LC-20 for detection, and the detection was repeated three times. The results are shown in the following table.

[0077] The results show that, under the same volume fraction, the 40-60% ammonium sulfate-enzyme solution part after washing with 70-90% acetone aqueous solution has relatively high catalytic formation efficiency of polyester type catechin, and especially the 35-50% ammonium sulfate-enzyme solution part after washing with 80% acetone aqueous solution has the highest efficiency.

[0078] Meanwhile, the catalytic effect of the enzyme solution before and after dialysis is compared, the part with a molecular weight <3000 Da is removed by using a dialysis membrane after dialysis, the impurities and oxidation byproducts are reduced, and the workload during further purification is reduced, and the average value of the catalytic TSA activity of the tea enzyme after dialysis is up to 67.4 mg / L, and from the TSA generation amount, the catalytic activity is increased by about 39% after dialysis.

[0079] Example 2

[0080] The four catechin monomers were dissolved in a phosphate buffer solution with a pH value of 6.5, so that the final concentration ratio of catechin in the reaction solution was EGCG:EGC:ECG:EC=5:4:3:2, and the EGCG concentration was 0.1 mmol / L.

[0081] Tea leaf enzyme was extracted from fresh leaves of Castanopsis fargesii, the 40%-60% ammonium sulfate precipitate was collected, the precipitate was dissolved with 10 mL PBS (pH=6.5) solution, the 40%-60% ammonium sulfate precipitate was purified by dialysis membrane to remove impurities with molecular weight <3000 Da, and a tea leaf enzyme solution was obtained. The specific steps were the same as those in Example 1. The obtained tea leaf enzyme solution was placed in an environment of-80 to-20℃. The obtained tea leaf enzyme solution was added to the previously oxygenated catechin monomer mixed solution, and the mass ratio of the tea leaf enzyme solution to the catechin monomer mixed solution was 1:60. The enzymatic reaction was carried out at 35℃ and pH 6.5 for 4 hours. After the reaction was completed, the reaction was terminated in a 100℃ water bath, and then centrifuged at a speed of 6000 rpm for 15 minutes. The supernatant was taken to obtain a preliminarily separated polyester catechin solution.

[0082] The preliminarily separated polyester catechin solution was purified by macroporous adsorption resin HP-20, and an ethanol solution with a volume fraction of 50% was used as an eluent at an elution flow rate of 2 mL / min. The eluate was collected, concentrated and dried to obtain high-purity polyester catechin.

[0083] HPLC detection was performed on the four catechin monomers and high-purity polyester catechin, and 272 nm was selected as the detection wavelength. 5.0 mg of mixed standard was weighed, and the mass fractions were TSA 10.0%, TSB 6.0%, TSC 4.0%, EGCG 37.46%, EGC 8.82%, EC 3.29%, and ECG 9.94%. Water was added to prepare a 10 mL control solution for standby. 10.0 mg of the obtained polyester catechin was weighed, and water was added to prepare a 10 mg / 100 ml working solution. A: 30% methanol / acetonitrile (v:v), and B: 0.3% phosphoric acid aqueous solution were used as the mobile phase. The detection gradient was A 0-22 min, 13%-21%; 22-35 min, 21-25%; 35-55 min, 25-25%; 55-56 min, 25-13%. The column temperature was 35℃. The flow rate was 0.8 mL / min. The wavelength was 272 nm. The chromatographic column was welchrom C18 column (4.6 x 200 mm, 5 μm). The instrument was Shimadzu Prominence LC-20. The detection was repeated three times. The HPLC spectrum of the substrate is shown in Figure 1 The HPLC spectrum of the product is shown in Figure 2

[0084] ​The total yield of the product was 43.2%, in which the yield of TSC was 10.2%, the yield of TSB was 14.9%, and the yield of TSA was 18.1%; the purity of TSs in the sample was 57.7%. The yield of the polyester catechin was defined as: yield of polyester catechin = content of polyester catechin in the obtained product (%) x mass of the obtained product x 100% / mass of the processing raw material x content of polyester catechin (%).

[0085] Example 3

[0086] The four catechin monomers were dissolved in a phosphate buffer solution with a pH value of 6.5, so that the final concentration ratio of catechins in the reaction solution was EGCG:EGC:ECG:EC=8:5:4:3, and the concentration of EGCG was 0.1 mmol / L.

[0087] Tea leaf enzyme was extracted from fresh leaves of Castanopsis fargesii, and the 40%-60% ammonium sulfate precipitate fraction was collected. The precipitate was dissolved in 10 mL of PBS (pH=6.5) solution, and the 40%-60% ammonium sulfate precipitate fraction was purified using a dialysis membrane to remove impurities with a molecular weight of <3000 Da to obtain tea leaf enzyme. The specific steps were the same as in Example 1. The extracted tea leaf enzyme was placed in an environment of -80 to -20°C. The obtained tea leaf enzyme solution was added to the previously oxygenated catechin mixed solution, and the mass ratio of tea leaf enzyme to catechin was 1:60. The enzymatic reaction was carried out at 35°C and a pH value of 6.5, and the reaction time was 4 hours. After the reaction was completed, the reaction was terminated in a 100°C water bath, and then centrifuged at a speed of 6000 rpm for 15 minutes. The supernatant was taken to obtain a preliminarily separated polyester catechin solution.

[0088] The preliminarily separated polyester catechin solution was purified by macroporous adsorption resin HP-20, and a 50% volume fraction of ethanol solution was used as the eluent with an elution flow rate of 2 mL / min. The eluate was collected, concentrated and dried to obtain high-purity polyester catechin. HPLC detection was performed on the four catechin monomers and the preliminarily separated polyester catechin solution, and the detection process was the same as in Example 2. The HPLC detection results of the product are shown in Table 2. Figure 3

[0089] The total yield of the product was 43.2%, in which the yield of TSC was 10.2%, the yield of TSB was 14.9%, and the yield of TSA was 18.1%; the purity of TSs in the sample was 57.7%. The yield of the polyester catechin was defined as: yield of polyester catechin = content of polyester catechin in the obtained product (%) x mass of the obtained product x 100% / mass of the processing raw material x content of polyester catechin (%).

[0090] Example 4

[0091] ​The four catechin monomers were dissolved in a phosphate buffer solution with a pH value of 6.5, so that the final concentration ratio of catechin in the reaction solution was EGCG:EGC:ECG:EC=5:4:1:1, and the concentration of EGCG was 0.1 mmol / L. Tea leaf enzyme was extracted from fresh leaves of Castanopsis fargesii, and the 40%-60% ammonium sulfate precipitate fraction was collected. The precipitate was dissolved in 10 mL of PBS (pH=6.5) solution, and the 40%-60% ammonium sulfate precipitate fraction was purified using a dialysis membrane to remove impurities with a molecular weight of <3000 Da, thereby obtaining tea leaf enzyme. The specific steps were the same as in Example 1. The obtained tea leaf enzyme solution was added to the catechin mixed solution that had been subjected to oxygen treatment in advance, and the mass ratio of tea leaf enzyme to catechin monomers was 1:50. The enzymatic reaction was performed at 35°C and a pH value of 6.5, and the reaction time was 4 hours. After the reaction was completed, a high-temperature water bath was used to terminate the reaction, and then centrifugation was performed at a speed of 5000 rpm for 12 minutes. The supernatant was obtained, thereby obtaining a preliminarily separated polyester catechin solution.

[0092] The preliminarily separated polyester catechin solution was purified by macroporous adsorption resin HP-20, and an ethanol solution with a volume fraction of 40% was used as an eluent at an elution flow rate of 1.5 mL / min. The eluate was collected, and high-purity polyester catechin was obtained after concentration and drying. HPLC detection was performed on the four catechin monomers and the preliminarily separated polyester catechin solution, and the detection process was the same as in Example 2. The HPLC detection results of the product are shown in Table 2. Figure 4

[0093] It was detected that the total yield of polyester catechin in the product was 31.7%, and the total mass of TSC was 7.3%, the total mass of TSB was 9.6%, and the total mass of TSA was 14.8%. The purity of TSs in the sample was 58.2%.

[0094] Example 5

[0095] The four catechin monomers were dissolved in a phosphate buffer solution, so that the final concentration ratio of catechin in the reaction solution was EGCG:EGC:ECG:EC=4:3:3:1, and the concentration of EGCG was 0.1 mmol / L.

[0096] ​Phosphate buffer solution set 5 reaction pH (pH = 3.5, 4.5, 5.5, 6.5, 7.5), from the extraction of tea leaf enzyme, collect 40%-60% ammonium sulfate precipitation part, precipitate using 10 mL of PBS (pH = 6.5) solution for dissolution, using dialysis membrane for 40%-60% ammonium sulfate precipitation part purification, remove the molecular weight <3000 Da impurities, obtain tea enzyme. The specific steps are the same as example 1. The obtained tea enzyme solution is added to the previously oxygen treated catechin mixed solution, the mass ratio of tea enzyme to catechin monomer is 1:50, the enzymatic reaction is carried out at 35℃, pH is 3.5, 4.5, 5.5, 6.5, 7.5 respectively, and the reaction time is 5 hours. Take out the reaction system, inactivate and determine the content (n = 3). The same as example 4. The product is detected, and the results are shown in Figure 5 .

[0097] From Figure 5 it can be seen that the total amount of polyester catechin is the highest when the pH of the reaction system is 6.5, and TSB is relatively strict to the pH of the reaction system. TSB is generated in a lower or higher pH environment or is in a lower amount, or cannot exist stably; TSC and TSA can be generated and exist in a relatively wide pH range, and the stock of TSA and TSB is the highest when pH is 6.5, which is 0.19 mg / 100 mL and 0.23 mg / 100 mL respectively; the content of theaflavin is relatively high at this time, so the buffer solution with pH of 6.0-6.5 is selected as the reaction liquid for subsequent experiments.

[0098] Example 6

[0099] On the basis of example 4, the enzymatic reaction temperature is adjusted to 20℃, 30℃, 40℃, 50℃ respectively, the reaction system is placed in the corresponding temperature shaker for 4h, the reaction system is taken out, inactivated and determined, and the results are shown in Figure 6 .

[0100] The results show that the contents of TSA and TSC reach the peak value at 50℃, which are 0.33 mg / 100 mL and 0.40 mg / 100 mL respectively, but the content of TSB is low. Combined with the optimum reaction temperature of the total amount of TSC, TSA and TSB, 30-40℃ is selected as the temperature for subsequent reaction, and the total amount of polyester catechin is 0.83 mg / 100 mL at 30℃.

[0101] Comparative example 1

[0102] On the basis of Example 2, the added tea enzyme solution is replaced by a tea enzyme solution without dialysis to remove small molecules, i.e. tea enzymes are extracted from fresh leaves of Cleyera japonica, the tea enzymes are precipitated with 40%-60% ammonium sulfate, the precipitate is dissolved with 10 mL of PBS (pH=6.5) solution to obtain an enzyme solution. The enzyme solution is added to the previously oxygenated catechin mixed solution for reaction, and the others are the same as in Example 2. Preliminarily separated polyester catechins are obtained. The preliminarily separated polyester catechin solution is purified by macroporous adsorption resin HP-20, with an ethanol solution with a volume fraction of 50% as an eluent, an elution flow rate of 2 mL / min, collection of eluate, and concentration and drying to obtain high-purity polyester catechins. HPLC detection of the oxidation product shows that the total yield of polyester catechins in the product is 30.4%, of which the yield of TSC is 7.6%, the yield of TSB is 10.3%, and the yield of TSA is 12.5%; the purity of TSs in the sample is 40.92%.

[0103] Comparative Example 2

[0104] On the basis of Example 2, the catechin monomers are adjusted to 3 catechin monomers (EGCG, ECG, EC), the 3 catechin monomers are dissolved in a phosphate buffer solution with a pH value of 6.5 which is treated with oxygen in advance, the final concentration ratio of catechins in the reaction solution is EGCG:EGC:ECG:EC=5:0:3:2, and the concentration of EGCG is 0.1 mmol / L. The others are the same as in Example 2, and high-purity polyester catechins are obtained. Detection shows that the yield of polyester catechins in the product is 16.5%, and all of them are TSA.

[0105] Comparative Example 3

[0106] On the basis of Example 2, the catechin monomers are adjusted to 2 catechin monomers (EGCG, EGC), the 2 catechin monomers are dissolved in a phosphate buffer solution with a pH value of 6.5, the final concentration ratio of catechins in the reaction solution is EGCG:EGC:ECG:EC=5:4:0:0, and the concentration of EGCG is 0.1 mmol / L. The others are the same as in Example 2, and high-purity polyester catechins are obtained. Detection shows that the total yield of polyester catechins in the product is 24.3%, of which the yield of TSC is 5.3%, the yield of TSB is 8.2%, and the yield of TSA is 10.8%; the purity of TSs in the sample is 46.5%.

[0107] Comparative Example 4

[0108] On the basis of Example 2, the catechin monomers were adjusted to three catechin monomers (EGC, ECG, EC), and the three catechin monomers were dissolved in the phosphate buffer solution with pH value of 6.5 which was treated by oxygen in advance, so that the final concentration ratio of catechin in the reaction solution was ECGC:ECG:EC = 0:4:3:2, wherein the concentration of EGC was 0.08 mmol / L. The other was the same as Example 2, and high-purity polyester catechin was obtained. High-purity polyester catechin was obtained. It was detected that the total yield of polyester catechin in the product was 11.2%, and all were TSC.

[0109] Comparative Example 5

[0110] On the basis of Example 2, the catechin monomers were adjusted to three catechin monomers (EGCG, EGC, ECG), and the three catechin monomers were dissolved in the phosphate buffer solution with pH value of 6.5 which was treated by oxygen in advance, so that the final concentration ratio of catechin in the reaction solution was ECGC:ECG:EC = 0:4:3:2, wherein the concentration of EGC was 0.08 mmol / L. The other was the same as Example 2, and high-purity polyester catechin was obtained. High-purity polyester catechin was obtained. It was detected that the total yield of polyester catechin in the product was 11.2%, and all were TSC.

[0111] Comparative Example 6

[0112] On the basis of Example 2, the catechin monomers were adjusted to three catechin monomers (EGCG, EGC, ECG), and the three catechin monomers were dissolved in the phosphate buffer solution with pH value of 6.5 which was treated by oxygen in advance, so that the final concentration ratio of catechin in the reaction solution was ECGC:ECG:EC = 0:4:3:2, wherein the concentration of EGC was 0.08 mmol / L. The other was the same as Example 2, and high-purity polyester catechin was obtained. High-purity polyester catechin was obtained. It was detected that the total yield of polyester catechin in the product was 11.2%, and all were TSC.

[0113] Comparative Example 7

[0114] Four catechin monomers were respectively dissolved in four portions of phosphate buffer solution with pH value of 6.5, and only one catechin monomer was added to each portion of the phosphate buffer solution, so that the concentration ratio of the four catechin monomers was ECGC:ECG:EC = 5:4:3:2, wherein the concentration of ECGC was 0.1 mmol / L.

[0115] The tea enzyme solution was extracted as in Example 2. The same tea enzyme solution as in Example 2 was added to four reaction vessels, respectively, and then one of EGCG, EGC, EC and GCG was added to the four reaction vessels, respectively, to conduct enzymatic reaction for 4 hours. The other conditions were the same as in Example 2. Four preliminary separated polyesters of catechins solutions were obtained.

[0116] The preliminary separated polyesters of catechins solutions were purified by macroporous adsorption resin HP-20, and 50% ethanol solution was used as eluent at a flow rate of 2 mL / min. The eluate was collected, concentrated and dried, and the four products were detected.

[0117] TSA was obtained from the EGCG reaction pool, TSC was obtained from the EGC reaction pool, and no polyesters of catechins were obtained from the ECG and EC reaction pools. After the four products were mixed and detected, the total yield of the polyesters of catechins in the total product was 23.9%, of which the yield of TSC was 9.8%, and the yield of TSA was 14.1%, but no TSB was obtained.

[0118] The "TSs yield (R) improvement range" combined with the "statistical test P value (significance)" was used as the synergistic effect index, and the correlation between "element adjustment to effect change" was used to compare the corresponding Examples 2-3 and Comparative Examples 2-7, to quantify the synergistic effect of raw material composition on the formation of TSs. The following formula was used for calculation: ;

[0119] Wherein, i is the number, R is the yield of TSs, CI <0.9 indicates synergistic effect, 0.90≤CI<0.95 has weak synergistic effect, 0.95≤CI<1 has additive effect, CI>1 has antagonistic effect, D1 and D2 represent the actual amount of EGCG and EGC substrates in the actual mixture. X1 and D X2 are the dosages required to achieve the same effect when EGCG and EGC substrates are used alone, respectively. EGC&EC K EGC&EC= represents the promotion effect coefficient of EGC and EC on TSs, which is calculated from the yield of TSs in the single variable group with or without the addition of a fixed proportion of EGC and EC. 含EGC&EC R 不含EGC&EC / R EGC&EC When the group does not contain EC, K EGC =K EGC&EC =0.937; when the group does not contain EGC, K EC =K EGC&EC =0.91; when the group contains EC and EGC, K

[0120] The results show that by dialysis, increasing EGC and EC in the EGCG and EGC substrate, using 30-40℃, pH 6.5 conditions can effectively direct the catalysis of EGCG, EGC to form TSs, wherein the treatment of the first group (dialysis, EGCG / EGC / ECG / EC substrate ratio 5:4:3:2, 35℃, pH 6.5) with the best effect has the highest TSs yield, and shows strong synergistic effect.

[0121] It should be noted that the above examples are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can also be made by those of ordinary skill in the art. Here, it is impossible to exhaust all the embodiments. Any obvious changes or variations derived from the technical scheme of the present application are still within the scope of protection of the present application.

Claims

1. A method for preparing polyester-type catechins, characterized in that, Includes the following steps: S1. Extract tea enzymes from fresh Castanopsis fargesii leaves. Purify the tea enzymes using ammonium sulfate with a saturation of 30%-80% by grade, while retaining the ammonium sulfate purification fraction with a saturation of 40%-60%. Remove impurities with a molecular weight <3000Da to obtain the enzyme solution. Catechin monomers were mixed according to concentration ratios to prepare catechin monomer solutions with a concentration of 0.1-10 mg / mL. S2. Mix the catechin monomer solution and enzyme solution to carry out the enzymatic reaction, then terminate the reaction, centrifuge, and obtain the enzymatic reaction product. S3. The enzyme-catalyzed reaction product is loaded onto a macroporous resin chromatography column, eluted with ethanol solution, the eluent is collected, concentrated and dried to obtain the polyester-type catechin. Extracting tea enzymes from fresh Castanopsis chinensis leaves includes the following steps: Acetone aqueous solution was added to fresh Castanopsis chinensis leaves and the mixture was ground, washed, filtered, and powdered. Polyvinylpyrrolidone and buffer solution were added to the powder and mixed in an ice bath. The mixture was then filtered, centrifuged, and the supernatant was collected to obtain tea enzyme. The washing solution is an aqueous acetone solution; the volume concentration of the aqueous acetone solution used for grinding and washing is 70-90%. In step S1, the catechin monomers are four of the following: epigallocatechin gallate, epigallocatechin, epicatechin gallate, and epicatechin. The molar ratio of epigallocatechin gallate, epigallocatechin, epigallocatechin gallate, and epigallocatechin is (8-4):(6-3):(5-3):(3-1). In step S2, the mass ratio of catechin monomer solution to enzyme solution is 50-80:1; the enzyme-catalyzed reaction temperature is 20-50℃, the pH of the reaction system is 3.5-7.5, and the reaction time is 0.5-6 hours. In step S3, the concentration of the ethanol solution is 30-70%; the elution flow rate is 1-3 mL / min; and the macroporous resin chromatography column is an HP-20 macroporous adsorption resin column.

2. The preparation method according to claim 1, characterized in that, The mass ratio of powder to polyvinylpyrrolidone is 11.6:1.5-2.

5.

3. The preparation method according to claim 1, characterized in that, The steps for purifying tea enzymes using ammonium sulfate with a saturation of 30%-80% include: Add ammonium sulfate solution to tea enzymes to make the ammonium sulfate saturation in the solution 30%. After mixing evenly, let the solution stand until precipitation is complete, centrifuge to obtain supernatant and precipitate, and take the precipitate and mark it as 30% precipitate fraction. Ammonium sulfate solution was added to the supernatant until the ammonium sulfate saturation reached 40%, 60%, and 80%, respectively. The standing and centrifugation operations were repeated to obtain 40%, 60%, and 80% precipitates, respectively.

4. The preparation method according to claim 1, characterized in that, In step S2, the reaction is terminated by inactivating the reaction system in a water bath at 90-100°C for 7-20 minutes.

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