Preparation method of polyester type catechin

Abstract

The invention relates to a preparation method of polyester type catechin, which comprises the following steps: S1, extracting tea enzyme from fresh castanopsis sclerophylla leaves, carrying out graded purification on the tea enzyme by using ammonium sulfate with the saturation degree of 30-80%, retaining an ammonium sulfate purification part with the saturation degree of 40-60%, and removing molecular weight lt; filtering to obtain an enzyme solution; the method comprises the following steps: S1, mixing catechin monomers according to a concentration ratio to prepare a catechin monomer solution with a concentration of 0.1-10 mg / mL, S2, mixing the catechin monomer solution and an enzyme solution, carrying out an enzymatic reaction, terminating the reaction, and carrying out centrifugation to obtain an enzymatic reaction product; and S3, loading the enzymatic reaction product on a macroporous resin chromatographic column, eluting with an ethanol solution, collecting the eluent, concentrating, and drying to obtain the polyester catechin. According to the method, the obtaining efficiency of the polyester type catechin can be remarkably improved, and compared with the prior art, the yield of the product is improved by 120%.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction and separation technology, specifically to a method for preparing polyester-type catechins using catechin monomers and tea enzymes. Background Technology

[0002] Polyester-type catechins (TSs) are a class of tea polyphenolic compounds with important biological activities. They are primarily catechin dimers formed during the processing of black tea through the oxidative polymerization of monomeric catechins catalyzed by tea enzymes. They significantly contribute to the color, flavor, and aroma of black tea and are one of the core products formed during black tea processing. Studies have shown that polyester-type catechins exhibit significant effects in antioxidation, anticancer, and antibacterial activities, and have broad application prospects in the food, pharmaceutical, and cosmetic fields.

[0003] There are multiple structures of TSs monomers. Eight polyester-type catechins have been isolated and identified from tea leaves, and their specific structures are as follows: .

[0004] Among them, polyester-type catechin A, CAS number: 89064-31-3, molecular formula: C 44 H 34 O 22 Molecular weight 914.73; Polyester-type catechin B, CAS No.: 89064-32-4, Molecular formula: C 37 H 30 O 18 Molecular weight 762.62; Polyester-type catechin C, CAS No.: 89013-69-4, Molecular formula: C 30 H 26 O 14 , molecular weight 610.52.

[0005] TSA is a light brown amorphous powder with the molecular formula C2. 44 H 34 O 22 The ¹H and ¹³C spectra indicate that it has a symmetrical molecular structure containing one biphenyl group; TSB is also a light brown amorphous powder with the molecular formula C2. 37 H 30 O 18 In addition, TSD, TSE, TSF, and TSG are all beige (approximately light brown). TSH is a brown amorphous powder with the molecular formula C2. 37 H 30 O8. Among them, TSA and TSD are mutually inhibited isomers, TSE is the S-type inhibited isomer of TSC, that is, S-TSE and R-TSC are mutually optical isomers, and TSH and TSB are also mutually inhibited isomers.

[0006] The extraction of polyester catechins currently faces numerous challenges. The most traditional method involves separation and purification using black tea as raw material. However, research by the Tea Research Institute of the Chinese Academy of Agricultural Sciences shows that the total amount of polyester catechins in many black tea samples is less than 2%, with most samples containing around 1%. The average TSA content of Jin Jun Mei (2020) was 0.38%, while the average of the control series of black teas was 0.21%, indicating a low content. Furthermore, tea samples contain a large number of compounds with similar structures and polarities to polyester catechins. Therefore, traditional methods for separating and purifying polyester catechins from black tea suffer from low extraction efficiency and high costs.

[0007] Existing technologies also include synthetic methods for preparing polyester-type catechins. For example, prior art CN109845852A discloses a highly efficient chemical oxidation synthesis method for polyester-type catechins, comprising the following steps: 1) preparing a tea polyphenol solution; 2) adding a copper salt chemical catalyst and reacting at a controlled temperature to promote the conversion of catechins to dehydropolyester-type catechins; 3) then adding vitamin C to continue the reaction, promoting the conversion of dehydropolyester-type catechins to polyester-type catechins; 4) after extraction, concentrating by vacuum rotary evaporation, and drying to obtain polyester-type catechins. However, this synthesis method requires the use of large amounts of organic solvents, posing environmental pollution problems, and the synthesis steps are complex, making it difficult to guarantee the purity of the product.

[0008] Existing technology CN118256569A discloses a low-temperature enzymatic method for preparing polyester-type catechin A. This method uses an enzyme extract or preparation of plant polyphenol oxidase as the enzyme source, epigallocatechin gallate (EGCG) as the substrate, and a buffer solution at a specific pH as the reaction system. Through low-temperature enzymatic reaction and subsequent steps such as column chromatography, distillation concentration, liquid phase preparation, and freeze-drying, polyester-type catechin A (TSA) is synthesized via targeted enzymatic catalysis. This method achieves the enzymatic oxidation of EGCG at low temperatures and the subsequent conversion reaction to obtain dimer TSA, laying a theoretical and practical foundation for the green, safe, efficient, and large-scale industrial production and utilization of TSA. However, this technology only involves the preparation of polyester-type catechin A and suffers from the problem of numerous impurities.

[0009] Existing technology CN114015733B discloses an enzyme-salt coupled catalytic synthesis method for polyester-type catechins. Using green tea leaves as raw material, hot water extraction is performed. After cooling and centrifugation to remove impurities, the supernatant is the green tea extract. A composite oxidoreductase solution and salt solution are added to the green tea extract. The pH value, cation concentration and ratio of the salt solution, and the addition ratio and total amount of polyphenol oxidase solution and peroxidase solution are precisely controlled. Fermentation is carried out under controlled temperature conditions to obtain a fermentation broth. The fermentation broth is concentrated by membrane filtration and spray-dried to obtain the polyester-type catechin product. This technology achieves a polyester-type catechin content of over 30%, 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 enzymatic oxidation. These impurities and the oxides they form have similar polarity to the substrate catechins and the product polyester-type catechins, making the separation and purification of the product extremely difficult.

[0010] Existing technology CN118581173A discloses an efficient in vitro enzymatic synthesis method for polyester-type catechin A, comprising the following steps: 1) preparing an epigallocatechin gallate (EGCG) solution; 2) homogenizing potato tubers, filtering and centrifuging, precipitating with ammonium sulfate, dialyzing and freeze-drying to obtain potato polyphenol oxidase powder; 3) adding potato polyphenol oxidase powder to the EGCG solution, controlling the temperature and stirring to promote the conversion of EGCG to dehydropolyester-type catechin A; 4) after the reaction, adding vitamin C and heating the reaction solution to promote the conversion of dehydropolyester-type catechin A to polyester-type catechin A; 5) filtering the reaction solution through a membrane, concentrating by vacuum rotary evaporation, and drying to obtain the polyester-type catechin A product. However, this technology only involves the preparation of polyester-type catechin A and has the problem of many impurities.

[0011] Xue Jinjin et al. (DOI:10.13386 / j.issn1002-0306.2019.20.013.) studied the oxidation characteristics of catechins by enzymes in immature Satsuma mandarin oranges, Japanese pears, and loquats. They found that enzymes in immature Satsuma mandarin oranges selectively oxidize pyrogallol-type catechins to produce dehydropolyester-type catechins, while enzymes in Japanese pears and loquats can oxidize pyrogallol-type catechins to form dehydropolyester-type catechins, as well as oxidize pyrogallol-type catechins and catechol-type catechins to form TFs. In black tea and oolong tea, oolongtheanins and polyester-type catechins coexist in the tea leaves, which are the oxidation and reduction products of dehydropolyester-type catechins, respectively. These findings indicate that oxidases from different species exhibit significant differences in their preference for pyrogallol and catechol-type catechin substrates, as well as the types of products obtained (e.g., coupling products, benzo[a]pyrophenolone product formation types). Enzyme selection has a substantial impact on the formation of TFs and polyester-type catechins. Summary of the Invention

[0012] The purpose of this invention is to provide a method for preparing polyester-type catechins that is efficient, environmentally friendly, and low in cost.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing a polyester-type catechin 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.

[0014] The raw material of this invention is fresh leaves of *Zhuyeqi*. On the one hand, this invention can quickly and easily extract relatively pure enzymes from fresh *Zhuyeqi* leaves. Compared with the use of other raw materials, such as potato oxidase, the method of extracting enzymes from the raw materials of this invention is simpler. On the other hand, enzymes in different tea varieties have significant differences in molecular weight and oxidative activity. The tea enzyme extraction parameters of this invention are designed based on *Zhuyeqi* and are more suitable for *Zhuyeqi*.

[0015] This invention utilizes ammonium sulfate for fractional precipitation of tea enzymes. The tea enzymes are separated from each other according to their molecular weight, from largest to smallest, in an ammonium sulfate solution of increasing concentration, thus purifying the tea enzymes. After purification, the ammonium sulfate can be removed using a dialysis bag, making the operation simple and resulting in high purity of the tea enzymes. Furthermore, extensive experiments have shown that the ammonium sulfate purification fraction with a saturation level of 40%-60% exhibits the best effect in converting catechin monomers into enzymes.

[0016] Other methods for purifying enzymes, besides dialysis, also require steps such as ultrafiltration and column chromatography. These methods rely on equipment such as ultrafiltration membranes, packing materials, and chromatography columns. Too many purification steps can affect the purity and yield of the product.

[0017] This invention removes components with a molecular weight <3000 from enzymes to prevent low molecular weight substances from participating in oxidation, thereby increasing byproducts in the reaction system, reducing yield, and further increasing the cost of further purification.

[0018] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows:

[0019] In one preferred embodiment, the extraction of tea enzymes from fresh Castanopsis fargesii 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.

[0020] In one preferred embodiment, the mass ratio of powder to polyvinylpyrrolidone is 11.6:1.5-2.5.

[0021] In one preferred embodiment, the buffer is PBS or Tris buffer.

[0022] In one preferred embodiment, the washing solution is an aqueous acetone solution.

[0023] In one preferred embodiment, the volume concentration of the acetone aqueous solution used for grinding and washing is 70-90%.

[0024] In one preferred embodiment, the step of purifying tea enzymes using ammonium sulfate with a saturation of 30%-80% includes: First, add ammonium sulfate solution to the tea enzyme to make the ammonium sulfate saturation in the solution 30%. After mixing evenly, let the solution stand until precipitation is complete, then centrifuge to obtain the supernatant and precipitate. Take the precipitate and mark it as the 30% precipitate fraction. Continue to add ammonium sulfate solution to the supernatant until the ammonium sulfate saturation is 40%, 60%, and 80%, and repeat the standing and centrifugation operations to obtain the 40%, 60%, and 80% precipitate fractions respectively.

[0025] In a preferred embodiment of the present invention, the 40% precipitate portion and the 60% precipitate portion are combined to form the 40%-60% ammonium sulfate purification fraction.

[0026] Ammonium sulfate saturation refers to the percentage of the actual concentration of ammonium sulfate in a solution relative to its maximum solubility (i.e., saturation concentration) at that temperature. At 20°C (room temperature): the solubility of ammonium sulfate in water is approximately 70.6 g / 100 g water.

[0027] In one preferred embodiment, in step S2, the catechin monomer includes four of the following: epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC).

[0028] Extensive experimental evidence from this invention demonstrates that EGCG can yield TSA from catechin monomer raw materials, EGC can yield TSC, and a mixture of EGCG and EGC can yield TSA, TSB, and TSC. EGCG and EGC are essential substrates for the formation of TSA, TSB, and TSC. EC can reduce the reaction potential energy during the oxidation process of the raw materials, increasing reaction efficiency. Although EGC is not a direct substrate for the formation of polyester-type catechins, the presence of EGC in the oxidation system affects the oxidation balance of the entire catechin system, and its auto-oxidation products can also affect the yield of polyester-type catechins by influencing the redox disproportionation reaction. Simultaneously, EGC affects the formation of polyester-type catechins by influencing the microenvironment of catechin metabolism.

[0029] 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).

[0030] In step S2, the molar concentration ratio of epigallocatechin gallate, epigallocatechin, epicatechin gallate, and epicatechin is determined based on the ratio of catechin content in fresh Castanopsis cuspidatum leaves. Excessively high or low concentrations of any single raw material will lead to an increase in byproducts.

[0031] The following is an introduction to each raw material: Epigallocatechin gallate (EGCG), CAS No.: 989-51-5, Molecular formula: C 22 H 18 O 11 Molecular weight: 458.37.

[0032] Epigallocatechin gallate (EGC), CAS No.: 970-74-1, Molecular formula: C 15 H 14 O7, molecular weight: 306.27.

[0033] Epicatechin gallate (ECG), CAS No.: 1257-08-5, Molecular formula: C 22 H 18 O 10 Molecular weight: 442.37.

[0034] Epicatechin (EC), CAS No.: 14344-48-0, Molecular Formula: C 15 H 14 O6, molecular weight: 290.27.

[0035] In one preferred embodiment, in step S2, the mass ratio of catechin monomer solution to enzyme solution is 50-80:1.

[0036] In one preferred embodiment, in step S2, the enzymatic reaction temperature is 20-50°C, the pH of the reaction system is 3.5-7.5, and the reaction time is 0.5-6 hours.

[0037] In one preferred embodiment, step S2, the step of terminating the reaction is to inactivate the reaction system in a water bath at 90-100°C for 7-20 minutes.

[0038] In one preferred embodiment, in step S2, the centrifugation speed is 4000-8000 rpm and the centrifugation time is 10-20 minutes.

[0039] In one preferred embodiment, in step S3, the macroporous resin chromatography column is an HP-20 macroporous adsorption resin column.

[0040] In one preferred embodiment, in step S3, the concentration of the ethanol solution is 30-70%.

[0041] In one preferred embodiment, in step S3, the elution flow rate is 1-3 mL / min.

[0042] Compared with the prior art, the beneficial effects of the present invention are: 1. The method of this invention has low cost: Compared with using commercial polyphenol oxidase for enzymatic oxidation reactions, this invention utilizes catechin monomers and crude tea enzymes as raw materials, which are widely available. It also simplifies the steps of separating tea enzymes and regulating the activity of polyphenol oxidase and peroxidase, thus reducing production costs. The resulting product system consists entirely of polyester-type catechins with simple structures, which significantly reduces separation costs and improves separation efficiency compared to further purification.

[0043] 2. The method of this invention is highly efficient: The tea enzyme prepared using the method of this invention, derived from fresh leaves of *Castanopsis fargesii*, exhibits high specificity for catalyzing the formation of polyester catechins from substrates with polyphenol oxidases from different species. By precisely controlling the activity of tea oxidase through optimized enzymatic reaction conditions, the efficiency of obtaining polyester catechins can be significantly improved. Compared with existing technologies, the yield of this invention is increased by 120%, reaching 1.54 mg / 100 ml, with TSA at 0.72 ± 0.04 mg / 100 ml, TSB at 0.42 ± 0.03 mg / 100 ml, and TSC at 0.40 mg / 100 ml. The purity of polyester catechins (polyester catechins A, B, and C) in the purified system can reach 43%, which is 20-40 times more efficient than extracting polyester catechins using black tea (the total amount of polyester catechins in black tea is 1-2%).

[0044] 3. This invention is environmentally friendly: This invention mainly uses relatively environmentally friendly reagents such as buffer solutions and ethanol, avoiding the use of large amounts of organic solvents and reducing environmental pollution. Attached Figure Description

[0045] Figure 1 This is an HPLC analysis chromatogram of four catechin monomers.

[0046] Figure 2 This is the HPLC analysis chromatogram of the product from Example 2.

[0047] Figure 3 This is the HPLC analysis chromatogram of the product from Example 3.

[0048] Figure 4 This is the HPLC analysis chromatogram of the product from Example 4.

[0049] Figure 5 This is a bar chart showing the product content obtained at different reaction pH values.

[0050] Figure 6 This is a bar chart showing the product content obtained at different reaction temperatures. Detailed Implementation

[0051] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0052] The reagents EGCG, EGC, ECG, and EC of this invention were all provided by the National Engineering Research Center for the Utilization of Functional Plant Components. Polyester-type catechins A, B, and C were isolated and prepared from black tea and verified by mass spectrometry and nuclear magnetic resonance.

[0053] The compound structure of polyester-type catechin A is analyzed as follows: Theasinensin A, chemical formula: C 44 H 34 O 22,

MH-

[0054] The compound structure of polyester-type catechin B is analyzed as follows: Theasinensin B, chemical formula: C 37 H 30 O 18 , [MH-]m / z 761.1425, 13C 166.43, 156.47, 156.34, 156.29, 156.14, 156.08, 155.98, 145.43, 14 5.24, 144.90, 144.25, 143.79, 138.45, 132.87, 132.61, 128.52, 128.4 1, 120.03, 111.65, 110.86, 108.93, 107.44, 106.52, 98.33, 97.71, 95. 21, 95.05, 94.65, 94.60, 76.16, 75.00, 67.72, 64.15, 27.80, 25.54; 1H 6.95, 6.93, 6.75, 5.96, 5.95, 5.92, 5.89, 5.88, 5.34, 5.33, 4.56, 3.97, 3.96, 3.38, 3.35, 3.35, 3.34, 3.34, 3.34, 2.83, 2.79, 2.69, 2.65, 2.59, 2.58, 2.41, 2.39, 2.36, 1.23, 1.21, 1.19, 0.04.

[0055] The compound structure of polyester-type catechin C is analyzed as follows: Theasinensin C, chemical formula: C 30 H 26 O 14 , [MH-] m / z 609.1309, 13C 155.51, 154.94, 145.21, 144.32, 132.38, 129.04, 112.77, 107.59, 99.54, 95.95, 95.39, 76.14, 64.23, 27.81; 1H 7.08, 6.96, 6.90, 6.70, 6.58, 6.02, 6.01, 5.80, 5.80, 4.00, 4.00, 3.63, 3.61, 3.33, 2.64, 2.60, 2.51, 2.50, 2.47, 2.46, 1.17, 1.15, 1.13.

[0056] Other reagents were purchased from Sinopharm Group.

[0057] Example 1

[0058] Tea enzymes were extracted from fresh Castanopsis chinensis leaves. The fresh leaves (one bud and one leaf) were harvested in summer from the Chang'an Base of the College of Horticulture, Hunan Agricultural University. The extraction process is as follows:

[0059] Accurately weigh 50.0g of fresh tea leaves for each test. Grind the fresh tea leaves in a pre-cooled acetone aqueous solution with a small amount of quartz sand in an ice bath. Filter the ground liquid separately in a pre-cooled acetone aqueous solution until the washing liquid is colorless, yielding four acetone powders. The concentrations of the acetone aqueous solution are 100 vt, 90 vt, 80 vt, and 70 vt, respectively. Add 1.76g of PVP (polyvinylpyrrolidone) and 0.2mL of PBS (pH=6.5), a small amount of quartz sand, and homogenize in an ice bath. Filter through gauze, and heat the filtrate at 1×10⁻⁶. 4 Centrifuge at rpm and 4℃ for 10 min, and collect the supernatant as crude enzyme solution.

[0060] Four samples of crude tea enzyme were purified using ammonium sulfate at different concentrations. 50 mL of the supernatant (tea enzyme) was added to ammonium sulfate solution to achieve an ammonium sulfate saturation of 30%. The mixture was thoroughly mixed, and after complete precipitation, it was centrifuged to obtain the precipitate and supernatant. The precipitate was labeled as the 30% fraction. Ammonium sulfate solution was added to the supernatant to achieve ammonium sulfate saturation of 40%, 60%, and 80%, respectively. This centrifugation process was repeated to obtain 40%, 60%, and 80% precipitates, respectively. The precipitate was dissolved in 10 mL of PBS (pH=6.5). The solution and crude enzyme solution were then dialyzed separately in Tris-HCl buffer (removing the molecular weight >3000) overnight until no SO4 was detected in the dialysate. 2- Different tea enzymes exist and are obtained.

[0061] The activities of different tea enzymes in catalyzing the formation of polyester catechin A from EGCG were determined, and the optimal ammonium sulfate fractionation precipitation gradient was identified. The steps are as follows:

[0062] The catechin monomer EGCG was dissolved in a phosphate buffer solution with a pH of 6.5 to achieve an EGCG concentration of 0.1 mmol / L in the reaction solution. Different tea enzyme solutions were added to the pre-oxygenated EGCG solution at a mass ratio of 1:60. The enzymatic reaction was carried out at 35℃ and pH 6.5 for 4 hours. After the reaction, the reaction was terminated by a 100℃ water bath, followed by centrifugation at 6000 rpm for 15 minutes. The supernatant was collected to obtain a polyester-type catechin solution. The polyester-type catechin solution was purified using a macroporous adsorption resin HP-20 with a 50% (v / v) ethanol solution as the eluent at a flow rate of 2 mL / min. The eluent was collected, concentrated, and freeze-dried to obtain high-purity polyester-type catechins.

[0063] HPLC analysis was performed on the polyester-type catechin solution, with 272 nm as the detection wavelength. 5.0 mg of a mixed standard was weighed, containing the following mass fractions: TSA 10.0%, TSB 6.0%, TSC 4.0%, EGCG 37.5%, EGC 8.8%, EC 3.3%, ECG 9.9%. A 10 mL reference solution was prepared with water. 10.0 mg of the freeze-dried polyester-type catechin was weighed and prepared as a 10 mg / 100 mL working solution with pure water. The mobile phase consisted of A: 30% methanol / acetonitrile (v:v) and B: 0.3% phosphoric acid aqueous solution. 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; and the column was a Welchrom C18. The column (4.6×200mm, 5μm) was used for detection with a Shimadzu Prominence LC-20 instrument, repeated three times. The results are shown in the table below.

[0064] The results showed that, at the same volume fraction, the 40-60% ammonium sulfate-crude enzyme solution after washing with 70-90% acetone aqueous solution had a relatively high efficiency in catalyzing the formation of polyester-type catechins, especially the 35-50% ammonium sulfate-crude enzyme solution after washing with 80% acetone aqueous solution had the highest efficiency.

[0065] Meanwhile, the present invention also compared the catalytic effect of enzyme solutions before and after dialysis. After dialysis, the dialysis membrane removed the part with a molecular weight <3000Da, reducing impurities and oxidation byproducts, and reducing the workload of further purification. The average value of the catalytic TSA activity of tea enzyme after dialysis was the highest at 67.4mg / L. In terms of TSA production, dialysis increased the catalytic activity by about 39%.

[0066] Example 2

[0067] Four catechin monomers were dissolved in a phosphate buffer solution with a pH of 6.5 to make the final catechin concentration ratio in the reaction solution EGCG:EGC:ECG:EC=5:4:3:2, and the EGCG concentration was 0.1 mmol / L.

[0068] Tea enzymes were extracted from fresh Castanopsis chinensis leaves. The 40%-60% ammonium sulfate precipitate was collected and dissolved in 10 mL of PBS (pH=6.5). The 40%-60% ammonium sulfate precipitate was purified using a dialysis membrane to remove impurities with a molecular weight <3000 Da, yielding a tea enzyme solution. The specific steps were the same as in Example 1. The extracted tea enzyme solution was placed at -80 to -20°C. The obtained tea enzyme solution was added to a pre-oxygenated catechin monomer mixture at a mass ratio of 1:60. The enzymatic reaction was carried out at 35°C and pH 6.5 for 4 hours. After the reaction, the reaction was terminated by a 100°C water bath, followed by centrifugation at 6000 rpm for 15 minutes. The supernatant was collected to obtain a preliminarily separated polyester-type catechin solution.

[0069] The initially separated polyester catechin solution was purified by passing it through a macroporous adsorption resin HP-20, using a 50% (v / v) ethanol solution as the eluent at a flow rate of 2 mL / min. The eluent was collected, concentrated, and dried to obtain high-purity polyester catechins.

[0070] Four catechin monomers and high-purity polyester catechins were analyzed by HPLC with 272 nm as the detection wavelength. 5.0 mg of a mixed standard was weighed, with the following mass fractions: TSA 10.0%, TSB 6.0%, TSC 4.0%, EGCG 37.46%, EGC 8.82%, EC 3.29%, and ECG... 9.94%, diluted with water to prepare a 10 mL reference solution for later use; weigh 10.0 mg of the freeze-dried polyester catechin, and prepare a 10 mg / 100 mL working solution with pure water. Use A: 30% methanol / acetonitrile (v:v) and B: 0.3% phosphoric acid aqueous solution as the mobile phase; detection gradient: A 0-22 min, 13%-21%; 22-35 min, 21-25%; 35-55 min, 25-25%; 55-56 min, 25-13%; column temperature 35℃; flow rate 0.8 mL / min; wavelength 272 nm; column: Welchrom C18 column (4.6 × 200 mm, 5 μm); instrument: Shimadzu Prominence LC-20, repeated three times. The HPLC chromatogram of the substrate is shown below. Figure 1 As shown, the HPLC chromatogram of the product is as follows. Figure 2 As shown.

[0071] The total yield of polyester catechins in the product was 43.2%, with TSC yield at 10.2%, TSB at 14.9%, and TSA at 18.1%; the purity of TSs in the sample was 57.7%. The yield of polyester catechins was defined as: Polyester catechin yield = Polyester catechin content in the obtained product (%) × Product mass × 100% / Raw material mass × Polyester catechin content (%).

[0072] Example 3

[0073] Four catechin monomers were dissolved in a phosphate buffer solution with a pH of 6.5 to make the final catechin concentration ratio in the reaction solution EGCG:EGC:ECG:EC=8:5:4:3, and the EGCG concentration was 0.1 mmol / L.

[0074] Tea enzymes were extracted from fresh Castanopsis chinensis leaves. The 40%-60% ammonium sulfate precipitate was collected and dissolved in 10 mL of PBS (pH=6.5). The 40%-60% ammonium sulfate precipitate was purified using a dialysis membrane to remove impurities with a molecular weight <3000 Da, yielding the tea enzymes. The specific steps were the same as in Example 1. The extracted tea enzymes were placed in an environment of -80 to -20°C. The obtained tea enzyme solution was added to a pre-oxygenated catechin mixture at a mass ratio of 1:60. The enzymatic reaction was carried out at 35°C and pH 6.5 for 4 hours. After the reaction, the reaction was terminated by a 100°C water bath, followed by centrifugation at 6000 rpm for 15 minutes. The supernatant was collected to obtain a preliminarily separated polyester-type catechin solution.

[0075] The initially separated polyester catechin solution was purified using HP-20 macroporous adsorption resin with 50% ethanol solution as the eluent at a flow rate of 2 mL / min. The eluent was collected, concentrated, and dried to obtain high-purity polyester catechins. HPLC analysis was performed on the four catechin monomers and the initially separated polyester catechin solution, following the same procedure as in Example 2. The HPLC results of the product are shown below. Figure 3 As shown.

[0076] The total yield of polyester catechins in the product was 36.7%, of which TSC yield was 8.6%, TSB yield was 11.8%, and TSA yield was 16.3%; the purity of TSs in the sample was 50.3%.

[0077] Example 4

[0078] Four catechin monomers were dissolved in a phosphate buffer solution with a pH of 6.5 to achieve a final catechin concentration ratio of EGCG:EGC:ECG:EC = 5:4:1:1, with EGCG concentration at 0.1 mmol / L. Tea enzymes were extracted from fresh Castanopsis fargesii leaves. The 40%-60% ammonium sulfate precipitate was collected and dissolved in 10 mL of PBS (pH 6.5). The 40%-60% ammonium sulfate precipitate was purified using a dialysis membrane to remove impurities with a molecular weight <3000 Da, yielding the tea enzyme. The specific steps were the same as in Example 1. The obtained tea enzyme solution was added to a pre-oxygenated catechin mixture solution at a mass ratio of tea enzyme to catechin monomers of 1:50. The enzymatic reaction was carried out at 35°C and pH 6.5 for 4 hours. After the reaction, the reaction was terminated by a high-temperature water bath, followed by centrifugation at 5000 rpm for 12 minutes. The supernatant was collected to obtain a preliminarily separated polyester-type catechin solution.

[0079] The initially separated polyester catechin solution was purified using HP-20 macroporous adsorption resin with 40% (v / v) ethanol as the eluent at a flow rate of 1.5 mL / min. The eluent was collected, concentrated, and dried to obtain high-purity polyester catechins. HPLC analysis was performed on the four catechin monomers and the initially separated polyester catechin solution, following the same procedure as in Example 2. The HPLC results of the products are shown below. Figure 4 As shown.

[0080] The test results showed that the total yield of polyester catechins in the product was 31.7%, of which TSC accounted for 7.3% of the total mass, TSB for 9.6%, and TSA for 14.8%; the purity of TSs in the sample was 58.2%.

[0081] Example 5

[0082] Four catechin monomers were dissolved in phosphate buffer solution to make the final catechin concentration ratio in the reaction solution EGCG:EGC:ECG:EC=4:3:3:1, where the concentration of EGCG was 0.1 mmol / L.

[0083] Five reaction pH values ​​(pH=3.5, 4.5, 5.5, 6.5, 7.5) were set in phosphate buffer solution to extract tea enzymes from fresh Castanopsis fargesii leaves. The 40%-60% ammonium sulfate precipitate was collected and dissolved in 10 mL of PBS (pH=6.5). The 40%-60% ammonium sulfate precipitate was purified using a dialysis membrane to remove impurities with a molecular weight <3000 Da, yielding the tea enzyme. The specific steps were the same as in Example 1. The obtained tea enzyme solution was added to a pre-oxygenated catechin mixture solution. The mass ratio of tea enzyme to catechin monomers was 1:50. Enzymatic reactions were carried out at 35℃ and pH values ​​of 3.5, 4.5, 5.5, 6.5, and 7.5 for 5 hours. The reaction system was removed, inactivated, and then the content was determined (n=3). Other steps were the same as in Example 4. The product was analyzed, and the results are as follows: Figure 5 As shown.

[0084] Depend on Figure 5 It can be seen that the total amount of polyester catechins is the highest when the reaction system pH=6.5. TSB has strict requirements on the acidity and alkalinity of the reaction system. TSB is either generated in low or high pH environments, or cannot exist stably. TSC and TSA can be generated and exist in a relatively wide pH range. The highest content of TSA and TSB is at pH=6.5, which are 0.19mg / 100mL and 0.23mg / 100mL, respectively. The theaflavins content is relatively high at this time. Therefore, a buffer solution with pH 6.0-6.5 is selected as the reaction solution for subsequent experiments.

[0085] Example 6

[0086] Based on Example 4, the enzyme-catalyzed reaction temperatures were adjusted to 20℃, 30℃, 40℃, and 50℃, respectively. The reaction system was placed in a shaker at the corresponding temperature and reacted for 4 hours. The reaction system was then removed, inactivated, and the content was determined. The results are as follows: Figure 6 As shown.

[0087] The results showed that the contents of TSA and TSC reached their peak at 50℃, at 0.33 mg / 100 mL and 0.40 mg / 100 mL, respectively, but the contents of TSB were relatively low. Considering the optimal reaction temperature for the total amounts of TSC, TSA, and TSB, 30-40℃ was selected as the temperature for subsequent reactions. At 30℃, the total amount of polyester-type catechins was 0.83 mg / 100 mL.

[0088] Comparative Example 1

[0089] Based on Example 2, the added tea enzyme solution was replaced with a tea enzyme solution that does not require dialysis to remove small molecules. Specifically, tea enzymes were extracted from fresh Castanopsis chinensis leaves, precipitated with 40%-60% ammonium sulfate, and the precipitate was dissolved in 10 mL of PBS (pH=6.5) to obtain an enzyme solution. The enzyme solution was added to a pre-oxygenated catechin mixture for reaction, following the same procedure as in Example 2. Preliminary separation of polyester catechins was obtained. The preliminarily separated polyester catechin solution was purified using a macroporous adsorption resin HP-20, with 50% ethanol as the eluent at a flow rate of 2 mL / min. The eluent was collected, concentrated, and dried to obtain high-purity polyester catechins. HPLC analysis of the oxidation products showed that the total yield of polyester catechins was 30.4%, with TSC yielding 7.6%, TSB 10.3%, and TSA 12.5%; the purity of TSs in the sample was 40.92%.

[0090] Comparative Example 2

[0091] Based on Example 2, the catechin monomers were adjusted to three monomers (EGCG, ECG, and EC). These three monomers were dissolved in a phosphate buffer solution with a pH of 6.5 that had been pre-oxygenated, resulting in a final catechin concentration ratio of EGCG:EGC:ECG:EC = 5:0:3:2, with EGCG concentration at 0.1 mmol / L. Other steps were the same as in Example 2, yielding high-purity polyester-type catechins. Testing showed that the yield of polyester-type catechins in the product was 16.5%, and all were TSA.

[0092] Comparative Example 3

[0093] Based on Example 2, the catechin monomers were adjusted to two types (EGCG and EGC). These two monomers were dissolved in a phosphate buffer solution with a pH of 6.5, resulting in a final catechin concentration ratio of EGCG:EGC:ECG:EC = 5:4:0:0, with EGCG concentration at 0.1 mmol / L. Other steps were the same as in Example 2, yielding high-purity polyester-type catechins. Analysis showed that the total yield of polyester-type catechins in the product was 24.3%, with TSC yielding 5.3%, TSB 8.2%, and TSA 10.8%; the purity of TSCs in the sample was 46.5%.

[0094] Comparative Example 4

[0095] Based on Example 2, the catechin monomers were adjusted to three monomers (EGC, ECG, and EC). These three monomers were dissolved in a phosphate buffer solution with a pH of 6.5 that had been pre-oxygenated, resulting in a final catechin concentration ratio of EGCG:EGC:ECG:EC = 0:4:3:2, with EGC concentration at 0.08 mmol / L. Other steps were the same as in Example 2, yielding high-purity polyester-type catechins. The total yield of polyester-type catechins in the product was 11.2%, all of which were TSC.

[0096] Comparative Example 5

[0097] Based on Example 2, the catechin monomers were adjusted to three monomers (EGCG, EGC, and ECG). These three monomers were dissolved in a phosphate buffer solution with a pH of 6.5 that had been pre-oxygenated, resulting in a final catechin concentration ratio of EGCG:EGC:ECG:EC = 5:4:3:0, with EGCG concentration at 0.1 mmol / L. Other steps were the same as in Example 2, yielding high-purity polyester-type catechins. The total yield of polyester-type catechins in the product was 28.1%, with TSC yielding 6.1%, TSB yielding 9.3%, and TSA yielding 12.7%.

[0098] Comparative Example 6

[0099] Based on Example 2, the catechin monomers were adjusted to three monomers (EGCG, EGC, and EC). These three monomers were dissolved in a phosphate buffer solution with a pH of 6.5 that had been pre-oxygenated, resulting in a final catechin concentration ratio of EGCG:EGC:ECG:EC = 5:4:0:2, with EGCG concentration at 0.1 mmol / L. Other steps were the same as in Example 2, yielding high-purity polyester-type catechins. The yield of polyester-type catechins in the product was 28.6%, with TSA yielding 12.2%, TSB yielding 10.8%, and TSC yielding 5.6%.

[0100] Comparative Example 7

[0101] Four catechin monomers were dissolved in four phosphate buffer solutions with a pH of 6.5. Only one catechin monomer was added to each phosphate buffer solution, so that the ratio of EGCG:EGC:ECG:EC in the four catechin monomer solutions was 5:4:3:2, and the concentration of EGCG was 0.1 mmol / L.

[0102] The extraction of tea enzyme solution was the same as in Example 2. The same tea enzyme solution as in Example 2 was added to four reaction vessels, followed by the addition of one of EGCG, EGC, EC, or GCG to each vessel, and the enzymatic reaction was carried out for 4 hours. Other procedures were the same as in Example 2. Four preliminarily separated polyester-type catechin solutions were obtained.

[0103] The initially separated polyester catechin solution was purified by using HP-20 macroporous adsorption resin with 50% ethanol solution as eluent at a flow rate of 2 mL / min. The eluent was collected, concentrated, dried, and then the four products were analyzed.

[0104] TSA was obtained from the EGCG reaction cell, TSC from the EGC reaction cell, and no polyester catechins were obtained from the separate ECG and EC reaction cells. After mixing the four products together, the total yield of polyester catechins was 23.9%, with TSC at 9.8% and TSA at 14.1%, but TSB was not obtained.

[0105] Using the "increase in TSs yield (R)" combined with the "statistical test p-value (significance)" as the synergistic effect indicator, and through the correlation between "factor adjustment and effect change", the corresponding Examples 2-3 and Comparative Examples 2-7 were compared to quantify the synergistic effect of raw material composition on TSs formation. The following formula was used for calculation: ; Where i is the number, R is the TSs yield, CI < 0.9 indicates a synergistic effect, 0.90 ≤ CI < 0.95 indicates a weak synergistic effect, 0.95 ≤ CI < 1 indicates an additive effect, and CI > 1 indicates an antagonistic effect. D1 and D2 represent the actual amounts of EGCG and EGC substrates used in the actual mixture, respectively. X1 and D X2 These are the doses required to achieve equivalent effects when using EGCG and EGC substrates alone, respectively. EGC&EC K represents the coefficient of the promoting effect of EGC and EC on TSs, calculated from the yield of TSs in the single variable group with or without the addition of a fixed proportion of EGC and EC. EGC&EC= R 含EGC&EC / R 不含EGC&EC When the group does not contain EC, K EGC&EC =K EGC =0.937; when the group does not contain EGC, K EGC&EC =K EC =0.91; when the group contains EC and EGC, K EGC&EC =0.845. The results are shown in the table below.

[0106] The results showed that dialysis, adding EGC and EC to the EGCG and EGC substrates, and using conditions of 30-40℃ and pH 6.5 could effectively and directionally catalyze the formation of TSs from EGCG and EGC. Among them, the treatment of Group 1 (dialysis, EGCG / EGC / ECG / EC substrate ratio of 5:4:3:2, 35℃, pH 6.5) had the best effect, with the highest TSs yield and exhibited a strong synergistic effect.

[0107] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

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.

2. The preparation method according to claim 1, characterized in that, 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.

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

5.

4. The preparation method according to claim 2, characterized in that, The washing solution is an aqueous acetone solution; the volume concentration of the aqueous acetone solution used for grinding and washing is 70-90%.

5. The preparation method according to claim 2, 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.

6. The preparation method according to claim 1, characterized in that, In step S1, the catechin monomers include four of the following: epigallocatechin gallate, epigallocatechin, epicatechin gallate, and epicatechin.

7. The preparation method according to claim 6, characterized in that, In step S1, the molar concentration ratio of epigallocatechin gallate, epigallocatechin, epicatechin gallate, and epicatechin is (8-4):(6-3):(5-3):(3-1).

8. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of catechin monomer solution to enzyme solution is 50-80:1; the enzyme 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.

9. 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.

10. The preparation method according to claim 1, characterized in that, 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.

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