Method for preparing high-purity theaflavin by coupling and combining macroporous resin columns

Abstract

Provided is a method for preparing a high-purity theaflavin by coupling and combining macroporous resin columns, belonging to the technical field of food engineering. A crude theaflavin solution with a purity of 15%-30% is loaded onto an LX-20B resin column. Gradient elution is performed according to a specific procedure. The eluate is subjected to rotary evaporation concentration and / or freeze-drying to obtain a primary purified product. The primary purified product solution is loaded onto an AB-8 resin column. Gradient elution is performed according to a specific procedure. The eluate is subjected to rotary evaporation concentration and freeze-drying to obtain a high-purity theaflavin product with a purity of 95% or higher, which can meet the requirements of some high-end health products and fields such as medical research and development for high-purity theaflavins, and broaden the application range of theaflavins.

Description

Method for preparing high-purity theaflavins by coupling macroporous resin column TECHNICAL FIELD

[0001] The present application belongs to the technical field of food engineering, and relates to a deep processing technology of natural products, in particular to a method for preparing high-purity theaflavins by coupling macroporous resin column. BACKGROUND

[0002] Theaflavins (TFs) in tea leaves have multiple functions such as antioxidation, antitumor, antibacterial and viral, anti-inflammatory, blood lipid regulation, and neuroprotection, and are superior to catechins in some functional activities. Therefore, theaflavins have great commercial value. Theaflavins have a very broad application prospect in the food, pharmaceutical, daily chemical and other industries. The content of theaflavins in natural tea leaves is relatively low, accounting for about 0.3-1.5% of the dry weight of black tea. At present, the purity of commercial theaflavins is generally 20-40%, which cannot meet the demand of some high-end products and medical research for higher purity theaflavins.

[0003] There are more than 20 kinds of theaflavins in the existing reports. Among them, the contents of theaflavin (TF1) in non-ester theaflavins, theaflavin-3-monogallate (TF2A), theaflavin-3'-monogallate (TF2B) and theaflavin-3,3'-digallate (TF3) in ester theaflavins are the highest, accounting for about 96% of the total TFs. The structures of the four main theaflavins are shown in Figure 1. They are formed by oxidative coupling of epicatechin (EC) and epigallocatechin (EGC), EC and epigallocatechin gallate (EGCG), EGC and epicatechin gallate (ECG), and EGC and EGCG, respectively.

[0004] Due to the presence of a large amount of other components and catechins with similar structures in tea extract and crude theaflavins, it is a great technical challenge in the industry to prepare high-purity theaflavins.

[0005] At present, the purification methods of theaflavins mainly include high-speed counter-current chromatography, medium-pressure preparative liquid chromatography, gel chromatography, silica gel chromatography, cellulose chromatography and the like. Such methods have the disadvantages of high cost, complicated operation, great toxicity of organic reagents and the like, and cannot realize the large-scale and commercial production of theaflavins. The macroporous resin has the advantages of large adsorption capacity, fast adsorption speed, good selectivity, simple regeneration treatment and low cost of domestic resin. The macroporous resin method has also achieved certain results in the separation and purification of theaflavins. According to the reports, Yu Jun et al. use NKA-9 resin to increase the purity of theaflavins from 16.6% to 44.1% with a yield of 56.67%; Yang Mingqi uses HZ-818 resin to increase the purity of theaflavins from 20% to 53.6% with a yield of 33.2%; Yuan Bin uses HP-20 resin to increase the purity of theaflavins from 30.49% to 59.64% with a yield of 20%; and Liu Hongtao uses HZ-816 resin to increase the purity of theaflavins from 52% to 90.8% with a yield of 56%.

[0006] So far, there is no report on the use of domestic macroporous resin to purify theaflavins from a purity of 15-30% to more than 95%. The present application uses domestic macroporous resin column coupling to prepare high-purity theaflavins, with a purity of more than 95%, which lays a foundation for the research and development of high-end products using theaflavins as raw materials.

[0007] SUMMARY

[0008] [TECHNICAL PROBLEM]

[0009] In the prior art, due to the similar structure of theaflavins and catechins and the large amount of other components in the crude theaflavins, the separation is difficult, the theaflavins product obtained by using one kind of macroporous resin has low purity and small increase; in addition, the prior art has the disadvantages of complicated operation, high cost and great toxicity, and cannot adapt to the development of large-scale separation and purification of theaflavins.

[0010] [TECHNICAL SCHEME]

[0011] In order to overcome the deficiencies of the prior art, the purpose of the present application is to provide a method for preparing high-purity theaflavins by macroporous resin column coupling, to solve the problems of high cost, complicated operation and great toxicity of the existing separation and purification technology, to use domestic macroporous resin and only use ethanol and water as eluent to obtain high-purity theaflavins product and to improve the yield of theaflavins. At the same time, the macroporous resin column can be used repeatedly and is suitable for large-scale production.

[0012] The technical scheme of the present application comprises the following:

[0013] The present application takes the theaflavins crude product as raw material, dissolves in hot water, and then removes the filter residue after standing and cooling to obtain the first sample solution. The theaflavins are adsorbed on the resin column, and then gradient elution is carried out using different gradient ethanol solutions to obtain the first purified product. The first purified product is concentrated by rotary evaporation and freeze-dried, then dissolved in hot water, and the filter residue is removed by suction filtration to obtain the second sample solution. The second sample solution is loaded into the resin column, and after the theaflavins are adsorbed, gradient elution is carried out using different gradient ethanol solutions to obtain the second purified product. After rotary evaporation and freeze-drying, the high-purity theaflavins product is obtained.

[0014] The first object of the present application is to provide a method for separating and purifying high-purity (more than 95%) theaflavins product, which specifically comprises the following steps:

[0015] (1) Dissolve the theaflavins crude product in hot water, remove the filter residue after standing and cooling, and then take the filtrate to the LX-20B resin column for chromatography. Elute with water, 10-30% ethanol aqueous solution by volume, and 40-70% ethanol aqueous solution by volume in sequence, collect the eluate of 40-70% ethanol aqueous solution by volume, and concentrate by rotary evaporation to obtain the first purified product;

[0016] (2) Mix the first purified product with hot water, filter while hot, and then take the filtrate to the AB-8 resin column for chromatography. Elute with water, 10-30% ethanol aqueous solution by volume, 30-50% ethanol aqueous solution by volume, and 50-70% ethanol aqueous solution by volume in sequence, collect the eluate of 50-70% ethanol aqueous solution by volume, and concentrate by rotary evaporation and freeze-drying to obtain high-purity theaflavins.

[0017] In one embodiment, the purity of the theaflavins in the theaflavins crude product in step (1) is 15-30%, and the purity of the high-purity theaflavins in step (2) is not less than 95%.

[0018] In one embodiment, the theaflavins crude product is dissolved in hot water in step (1) to obtain a solution with a concentration of 50-200 mg / mL; and the loading flow rate in step (1) is 0.5-1.5 BV / h (column bed volume / h).

[0019] In one embodiment, the first purified product is mixed with hot water in step (2) to obtain a solution with a concentration of 5-15 mg / mL; and the loading flow rate in step (2) is 0.5-1.5 BV / h.

[0020] In one embodiment, the height-diameter ratio of the LX-20B resin column in step (1) and / or the AB-8 resin column in step (2) is (10-20):1.

[0021] In one embodiment, in step (1), each gradient elution volume is 2-4 BV of water, 4-8 BV of 10-30% ethanol aqueous solution, and 3-6 BV of 40-70% ethanol aqueous solution, and each gradient elution flow rate is 1.0-2.5 BV / h.

[0022] In one embodiment, in step (2), each gradient elution volume is 2-4 BV of water, 3-6 BV of 10-30% ethanol aqueous solution, 3-6 BV of 30-50% ethanol aqueous solution, and 3-6 BV of 50-70% ethanol aqueous solution, and each gradient elution flow rate is 1.0-2.5 BV / h.

[0023] In one embodiment, the temperature of the hot water in step (1) and / or step (2) is 60-90°C.

[0024] In one embodiment, after step (2), the method further comprises:

[0025] (3) Resin regeneration and recycling: the LX-20B resin column and the AB-8 resin column are cleaned with 95% ethanol aqueous solution, and then rinsed with deionized water until the effluent has no alcohol smell; the LX-20B resin is further subjected to alkaline washing, water washing to neutral, acid washing, and water washing to neutral, and steps (1) and (2) are repeated for recycling.

[0026] In one embodiment, the parameters of the rotary evaporation concentration in step (1) and / or step (2) are: temperature 40-50°C, and rotation speed 70-100 rpm.

[0027] In one embodiment, the conditions of the freeze-drying are: vacuum degree 10-100 Pa, time 12 h-24 h, and temperature -40°C to -10°C.

[0028] In one embodiment, in step (3), the elution flow rate of the 95% ethanol aqueous solution, alkaline washing, acid washing, and water washing is 1.0-2.0 BV / h, the elution volume of the 95% ethanol aqueous solution, alkaline washing, and acid washing is 2-3 BV, and the elution volume of the water washing is 4-6 BV.

[0029] In one embodiment, in step (3), the alkaline washing solution is 2-4% NaOH solution, and the acid washing solution is 2-4% HCl solution.

[0030] The second object of the present application is to provide a method for separating and purifying a high-purity (more than 95%) theaflavins product by coupling the macroporous resin column described above.

[0031] [Beneficial effects]

[0032] The method of the application adopts two specific domestic macroporous resin columns in combination, and only uses water and ethanol aqueous solution as eluent, to prepare high-purity theaflavins (more than 95%), which can effectively realize the separation of theaflavins and main catechins and caffeine (CAF), and meet the demand of high-purity theaflavins in some high-end product and medical research and development fields. The method has the following advantages:

[0033] (1) The resin used is a domestic macroporous adsorption resin, which has relatively low cost;

[0034] (2) The eluent used is only ethanol aqueous solution, without other toxic and harmful organic reagents, which is safe and environmentally friendly;

[0035] (3) The operation is simple, the loading amount is large, and it is suitable for large-scale production;

[0036] (4) The macroporous resin regeneration treatment is simple, and can be repeatedly used for many times;

[0037] (5) The purity of the obtained theaflavins is more than 95%, the total recovery rate of theaflavins is greater than 80%, and the yield of 95% high-purity theaflavins is greater than 30%. Therefore, it can be well applied to theaflavins separation and purification process, and has wide application prospect. At the same time, it lays a foundation for widening the application range of theaflavins in high-end product field;

[0038] (6) The purification method of the application solves the problems of existing separation and purification technology, such as complicated operation, small processing capacity, high cost and high solvent toxicity, and greatly improves the purity of theaflavins;

[0039] (7) The application first adopts LX-20B resin column for primary purification, which is more conducive to improving the loading amount and purification efficiency compared with other macroporous resins, and is more suitable for the enrichment of low-concentration theaflavins; then adopts AB-8 resin column for secondary purification, which can remove caffeine and other impurities that cannot be removed by LX-20B resin column, and is more suitable for the preparation of higher-purity theaflavins in combination with specific elution gradient (water, 10-30% ethanol aqueous solution, 30-50% ethanol aqueous solution, 50-70% ethanol aqueous solution are used in turn for elution). BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is the structure of four main theaflavins;

[0041] Figure 2 is the static adsorption / desorption experiment result of different macroporous resin columns on theaflavins in Example 4. DETAILED DESCRIPTION

[0042] The specific embodiments of the present application are described in detail below with reference to the accompanying drawings. The described embodiments are merely part of the embodiments of the present application, and are intended to explain the present application, and cannot be understood as limiting the present application.

[0043] The determination method involved in the present application:

[0044] Determination of the contents of main catechins, caffeine and four main theaflavins

[0045] Quantitative analysis of the components was performed by high performance liquid chromatography. Quantitative analysis was performed using a high performance liquid chromatograph equipped with a Waters e2695 pump and a Waters PDA detector. The sensitivity of the response value of this method was 0.0001 Au, and the determination conditions of high performance liquid chromatography (HPLC) were as follows: liquid chromatography column: C 18 (5 μm in particle size, 150 mm x 4.6 mm); sample injection amount: 10 μL; column temperature: 30 °C; ultraviolet detector: λ = 280 nm; mobile phase A: 0.1% (v / v) formic acid / water; mobile phase B: acetonitrile solution; elution gradient as shown in the following table. The standard curve of the 12 standard substances involved is shown in Table 2.

[0046] Table 1 High performance liquid chromatography gradient elution program

[0047] Table 2 Standard curve of 12 standard substances

[0048] Example 1

[0049] (1) The pretreated LX-20B resin and AB-8 resin were wet-packed into glass chromatography columns according to a height-diameter ratio of 12:1, and the glass chromatography columns had specifications of Φ25 x 400 mm (with LX-20B packed inside) and Φ16 x 300 mm (with AB-8 packed inside), respectively, to obtain LX-20B resin columns and AB-8 resin columns;

[0050] (2) 10 g of a theaflavin crude product with a purity of 25% (main impurities and contents: catechin: 28.58 ± 0.05%, caffeine: 1.03 ± 0.06%, protein: 5.27 ± 0.26%, polysaccharide: 5.31 ± 0.58%) was dissolved in 100 mL of hot water at 70 °C to prepare a solution with a concentration of 100 mg / mL, and the solution was allowed to stand and cool for 6 h. The filtrate was removed by suction filtration, and the filtrate was loaded into the LX-20B resin column at a flow rate of 0.5 BV / h by using a peristaltic pump;

[0051] (3) After the end of loading, gradient elution was performed in the order of 3 BV of water, 6 BV of 25% (v / v) ethanol aqueous solution, and 3 BV of 65% (v / v) ethanol aqueous solution, with an elution flow rate of 2.0 BV / h, and the eluate of 65% (v / v) ethanol aqueous solution was collected. After removal of ethanol by rotary evaporation, the concentrated solution was freeze-dried to obtain the product of the first purification. High-performance liquid chromatography analysis showed that the purity of the product of the first purification was 65.79 ± 0.91%, the yield was 51.41 ± 1.57%, and the total recovery rate was 84.28 ± 2.10%;

[0052] (4) 1.00 g of the product of the first purification was dissolved in 100 mL of hot water at 70°C to prepare a solution with a concentration of 10 mg / mL. The solution was filtered while hot to remove the filter residue, and the filtrate was loaded into the AB-8 resin column at a flow rate of 1.0 BV / h by using a peristaltic pump;

[0053] (5) After the end of loading, gradient elution was performed in the order of 3 BV of water, 6 BV of 25% (v / v) ethanol aqueous solution, 6 BV of 35% (v / v) ethanol aqueous solution, and 4 BV of 50% (v / v) ethanol aqueous solution, with an elution flow rate of 2.0 BV / h, and the eluate of 50% (v / v) ethanol aqueous solution was collected. After removal of ethanol by rotary evaporation, the concentrated solution was freeze-dried to obtain the product of high-purity theaflavins. High-performance liquid chromatography analysis showed that the purity of the product of high-purity theaflavins was 95.41 ± 0.77%, the yield was 55.34 ± 1.95%, and the total recovery rate was 95.83 ± 0.79%;

[0054] (6) Resin regeneration: 2 BV of 95% (v / v) ethanol aqueous solution was used to clean the LX-20B resin column and the AB-8 resin column, respectively, and then 4 BV of deionized water was used for elution until there was no obvious alcohol smell in the effluent. For the LX-20B resin column, 2 BV of 4% NaOH solution was first used for elution, and then water was used for washing until neutral. Then, 2 BV of 2% HCl solution was used for elution, and finally water was used for washing until neutral. The elution flow rate was 1.0 BV / h. After the end of the resin regeneration treatment, steps (2) to (5) could be repeated to perform the next round of purification.

[0055] (7) After the two types of resins were recycled for 5 times, the purity and yield of the product of theaflavins did not decrease significantly. After 5 times of recycling, the purity of the product of high-purity theaflavins was 95.10 ± 0.69%, the yield was 30.28 ± 1.51%, and the total recovery rate was 80.46 ± 1.01%.

[0056] Example 2

[0057] (1) The pretreated LX-20B and AB-8 resins were packed into a glass chromatography column by wet method with a height-to-diameter ratio of 15:1. The glass chromatography column specifications were Φ25×500mm (LX-20B) and Φ16×300mm (AB-8), respectively, to obtain LX-20B resin column and AB-8 resin column.

[0058] (2) Weigh 15g of crude theaflavins with a purity of 20% (main impurities and contents: catechins: 35.10±0.05%, caffeine: 1.55±0.03%, protein: 6.36±0.35%, polysaccharides: 6.43±0.50%) and dissolve it in 100mL of hot water at 80℃ to prepare a solution of 150mg / mL. Let it stand and cool for 12h, filter to remove the filter residue, and load the filtrate into an LX-20B resin column at a flow rate of 1.0BV / h using a peristaltic pump.

[0059] (3) After the sample loading was completed, gradient elution was performed in the order of 4 BV water, 6 BV 30% (v / v) ethanol aqueous solution, and 3 BV 70% (v / v) ethanol aqueous solution. The elution flow rate was 1.0 BV / h. The eluent of 70% (v / v) ethanol aqueous solution was collected, and the ethanol was removed by rotary evaporation. The concentrate was then freeze-dried to obtain the first purified product. The high performance liquid chromatography analysis showed that the purity of theaflavins in the first purified product was 62.48±0.37%, the yield was 52.98±2.01%, and the total recovery rate was 86.19±3.54%.

[0060] (4) Weigh 1.50g of the first-purified product and dissolve it in 100mL of 80℃ hot water to prepare a 15mg / mL solution. Filter while hot to remove the residue. Load the filtrate into the AB-8 resin column at a flow rate of 1.0BV / h using a peristaltic pump.

[0061] (5) After the sample loading was completed, gradient elution was performed in the following order: 3 BV water, 6 BV 20% (v / v) ethanol aqueous solution, 4 BV 40% (v / v) ethanol aqueous solution, and 3 BV 60% (v / v) ethanol aqueous solution. The elution flow rate was 2.0 BV / h. The eluent of 60% (v / v) ethanol aqueous solution was collected, and the ethanol was removed by rotary evaporation. The concentrate was then freeze-dried to obtain a high-purity theaflavins product. The high-purity theaflavins product had a purity of 95.02±0.39%, a yield of 58.06±0.23%, and a total recovery rate of 95.98±0.11%.

[0062] (6) Resin regeneration: 2BV 95% (v / v) ethanol aqueous solution was used to clean the LX-20B resin column and the AB-8 resin column, respectively, and then 5BV deionized water was used for elution, and the flow-out liquid was stopped when there was no obvious alcohol smell; for the LX-20B resin, 2BV 4% NaOH solution was used for elution first, then water was used for washing to neutral, then 2BV 2% HCl solution was used for elution, and finally water was used for washing to neutral, and the elution flow rate was 1.5BV / h; after the resin regeneration treatment was completed, steps (2)-(5) could be repeated for the next round of purification;

[0063] (7) After the two resins were regenerated and recycled for 5 times, the purity and yield of the theaflavins product obtained did not decrease significantly: after the two resins were regenerated and recycled for 5 times, the purity of the high-purity theaflavins product obtained was 95.32±0.44%, the yield was 30.49±1.25%, and the total recovery rate was 81.72±1.61%.

[0064] Example 3

[0065] (1) The pretreated AB-8 and LX-20B resins were wet-packed into glass chromatography columns (Φ16×400mm) according to the height-diameter ratio of 20:1, to obtain the LX-20B resin column and the AB-8 resin column;

[0066] (2) 5g of theaflavins crude product with a purity of 15% (main impurities and contents: catechin: 42.63±0.04%, caffeine: 1.38±0.03%, protein: 6.95±0.40%, polysaccharide: 6.96±0.66%) was dissolved in 25mL of 90℃ hot water to prepare a solution with a concentration of 200mg / mL, and then the solution was cooled for 8h, and the filtrate was obtained by suction filtration to remove the filter residue, and then the filtrate was loaded into the LX-20B resin column at a flow rate of 0.5BV / h by using a peristaltic pump;

[0067] (3) After the loading was completed, gradient elution was performed in the order of 3BV water, 8BV 30% (v / v) ethanol aqueous solution, and 6BV 50% (v / v) ethanol aqueous solution, and the elution flow rate was 2.0BV / h, the eluate of 50% (v / v) ethanol aqueous solution was collected, and then the ethanol was removed by rotary evaporation, and then the concentrated solution was freeze-dried to obtain the first purification product; high performance liquid chromatography analysis showed that the purity of the theaflavins in the first purification product was 63.14±0.26%, the yield was 50.95±1.21%, and the total recovery rate was 85.33±2.61%;

[0068] (4) 0.25g of the first purification product was dissolved in 50mL of 90℃ hot water to prepare a solution with a concentration of 5mg / mL, and then the solution was filtered while hot to remove the filter residue, and then the filtrate was loaded into the AB-8 resin column at a flow rate of 1.0BV / h by using a peristaltic pump;

[0069] (5) After the end of the loading, gradient elution was performed in the order of 3 BV water, 3 BV 30% (v / v) ethanol aqueous solution, 3 BV 40% (v / v) ethanol aqueous solution, and 3 BV 50% (v / v) ethanol aqueous solution, with an elution flow rate of 2.0 BV / h. The eluate of 50% (v / v) ethanol aqueous solution was collected, concentrated by rotary evaporation to remove ethanol, and then the concentrated solution was freeze-dried to obtain a high-purity theaflavins product. High-performance liquid chromatography analysis showed that the purity of the high-purity theaflavins product was 95.90 ± 0.59%, the yield was 57.44 ± 0.81%, and the total recovery rate was 94.94 ± 0.85%;

[0070] (6) Resin regeneration: 2 BV of 95% (v / v) ethanol aqueous solution was used to clean the LX-20B resin column and the AB-8 resin column, respectively, and then 6 BV of deionized water was used for elution, and the elution was stopped when there was no obvious alcohol smell. For the LX-20B resin column, 2 BV of 4% NaOH solution was used for elution, followed by water washing to neutral, 2 BV of 2% HCl solution was used for elution, and finally water washing to neutral. The elution flow rate was 2.0 BV / h. After the resin regeneration treatment was completed, steps (2) to (5) could be repeated for the next round of purification;

[0071] (7) After the two types of resins were regenerated and recycled for 5 times, the purity and yield of the theaflavins product obtained did not decrease significantly. After 5 times of regeneration and recycling, the purity of the high-purity theaflavins product obtained was 95.11 ± 0.23%, the yield was 30.26 ± 0.97%, and the total recovery rate was 81.09 ± 1.27%.

[0072] Example 4: Screening of macroporous resin columns

[0073] Through static adsorption / desorption experiments, a macroporous resin LX-20B with high adsorption / high desorption for theaflavins was selected from six different macroporous resins. The specific experiments are as follows:

[0074] 1.00 g (dry weight) of pretreated macroporous resin was placed in a 50 mL conical flask, and 20 mL of a theaflavins crude product solution with a concentration of 100 mg / mL was added. After adsorption for 12 h at 25°C and 130 r / min on a constant temperature shaker, the saturated resin was filtered to obtain a filtrate. The saturated resin was washed with deionized water for 2-3 times, and then the surface moisture of the washed resin was absorbed with filter paper. 20 mL of 95% (v / v) ethanol solution was added, and desorption was continued for 12 h at 25°C and 130 r / min on a constant temperature shaker to obtain a desorption solution. The concentrations of theaflavins in the initial solution, the filtrate, and the desorption solution were determined by HPLC. The static equilibrium adsorption capacity (Q e ) and desorption capacity (Q d), desorption rate (D), and then select the appropriate resin. The calculation formula is as follows:

[0075] Q d = C2 x V2 / W

[0076] In the formula: Q e , Q d are the static adsorption capacity and desorption capacity of theaflavins (mg / g dry resin), respectively, D is the desorption rate (%), C0 is the initial concentration of theaflavins (mg / mL), C1 is the equilibrium concentration of theaflavins (mg / mL), C2 is the total content of theaflavins in the desorption solution (mg / mL), V1 is the adsorption solution volume of theaflavins (mL), V2 is the desorption solution volume of theaflavins (mL), and W is the dry weight of the resin (g).

[0077] This example tests the exchange capacity of six kinds of macroporous resins for four kinds of theaflavins, and whether they are suitable for the separation and purification of theaflavins is judged by investigating the static adsorption / desorption capacity and desorption rate of different macroporous resins for theaflavins. The results are shown in Figure 2. The adsorption capacity and desorption capacity of LX-20B resin for theaflavins are significantly higher than those of other resins. In consideration of the fact that the desorption rates of different resins for theaflavins are not much different and are all greater than 80%, LX-20B resin is considered as the preferred resin for purifying theaflavins.

[0078] Comparative Example 1

[0079] Through a dynamic elution experiment, AB-8 resin is selected from macroporous resins suitable for separating and removing caffeine and is more suitable for use in this experiment. The specific experiment is as follows:

[0080] Compared with Example 3, the only difference is that the AB-8 resin column is replaced by an LX-8 resin column, and a high-purity theaflavin product is obtained. High-performance liquid chromatography analysis shows that the purity of theaflavins in the high-purity theaflavin product is 73.16 ± 1.58%, the yield is 18.48 ± 0.69%, and the total recovery rate is 35.50 ± 0.93%.

[0081] Compared with Example 3, the only difference between Comparative Example 1 is the different resin column used in the secondary purification. The comparison results show that the purification effect (theaflavin purity, yield, and total recovery rate) of LX-20B resin combined with LX-8 resin is much worse than that of LX-20B resin combined with AB-8 resin used in this application under the same conditions.

[0082] Comparative Example 2

[0083] (1) The pretreated AB-8 and LX-20B resins were respectively wet-packed into a glass chromatographic column (Φ16×400mm) according to a height-diameter ratio of 20:1, to obtain an LX-20B resin column and an AB-8 resin column;

[0084] (2) 5 g of a crude theaflavins product with a purity of 15% (main impurities and contents: catechin: 42.63±0.04%, caffeine: 1.38±0.03%, protein: 6.95±0.40%, polysaccharide: 6.96±0.66%) was weighed and dissolved in 25 mL of hot water at 90°C to prepare a solution with a concentration of 200 mg / mL. The solution was allowed to cool for 8 h, and then filtered to remove the residue. The filtrate was loaded onto the LX-20B resin column at a flow rate of 0.5 BV / h by using a peristaltic pump;

[0085] (3) After the loading was completed, gradient elution was performed in the order of 3 BV of water, 8 BV of 10% (v / v) ethanol aqueous solution, and 6 BV of 35% (v / v) ethanol aqueous solution, at an elution flow rate of 2.0 BV / h. The eluate of the 35% (v / v) ethanol aqueous solution was collected, concentrated by rotary evaporation to remove ethanol, and then freeze-dried to obtain a first purified product. High-performance liquid chromatography analysis showed that the theaflavins purity of the first purified product was 23.26±0.59%, the yield was 32.91±1.28%, and the total recovery rate was 37.31±2.09%;

[0086] (4) 0.25 g of the first purified product was weighed and dissolved in 50 mL of hot water at 90°C to prepare a solution with a concentration of 5 mg / mL. The solution was filtered while hot to remove the residue. The filtrate was loaded onto the AB-8 resin column at a flow rate of 1.0 BV / h by using a peristaltic pump;

[0087] (5) After the loading was completed, gradient elution was performed in the order of 3 BV of water, 3 BV of 30% (v / v) ethanol aqueous solution, 3 BV of 40% (v / v) ethanol aqueous solution, and 3 BV of 50% (v / v) ethanol aqueous solution, at an elution flow rate of 2.0 BV / h. The eluate of the 50% (v / v) ethanol aqueous solution was collected, concentrated by rotary evaporation to remove ethanol, and then freeze-dried to obtain a high-purity theaflavins product. High-performance liquid chromatography analysis showed that the theaflavins purity of the high-purity theaflavins product was 68.63±0.81%, the yield was 56.22±0.93%, and the total recovery rate was 94.15±0.96%.

[0088] Comparative Example 3

[0089] (1) The pretreated AB-8 and LX-20B resins were respectively wet-packed into a glass chromatographic column (Φ16×400mm) according to a height-diameter ratio of 20:1, to obtain an LX-20B resin column and an AB-8 resin column;

[0090] (2) Take 5 g of the crude product of theaflavins with a purity of 15% (main impurities and contents: catechin: 42.63±0.04%, caffeine: 1.38±0.03%, protein: 6.95±0.40%, polysaccharide: 6.96±0.66%) and dissolve it in 25 mL of hot water at 90°C to prepare a solution with a concentration of 200 mg / mL, and then let it stand and cool for 8 h. Remove the filter residue by suction filtration, and then pass the filtrate through a peristaltic pump at a flow rate of 0.5 BV / h to be loaded into the LX-20B resin column;

[0091] (3) After the loading is completed, perform gradient elution in the order of 3 BV of water, 8 BV of 30% (v / v) ethanol aqueous solution, and 6 BV of 50% (v / v) ethanol aqueous solution at an elution flow rate of 2.0 BV / h, collect the eluate of 50% (v / v) ethanol aqueous solution, and then concentrate and remove ethanol by rotary evaporation. Freeze-dry the concentrated solution to obtain the first purified product. High-performance liquid chromatography analysis shows that the purity of theaflavins in the first purified product is 63.14±0.26%, the yield is 50.95±1.21%, and the total recovery rate is 85.33±2.61%;

[0092] (4) Take 0.25 g of the first purified product and dissolve it in 50 mL of hot water at 90°C to prepare a solution with a concentration of 5 mg / mL. Remove the filter residue by hot filtration, and then pass the filtrate through a peristaltic pump at a flow rate of 1.0 BV / h to be loaded into the AB-8 resin column.

[0093] (5) After the loading is completed, perform gradient elution in the order of 3 BV of water, 3 BV of 10% (v / v) ethanol aqueous solution, 3 BV of 30% (v / v) ethanol aqueous solution, and 3 BV of 40% (v / v) ethanol aqueous solution at an elution flow rate of 2.0 BV / h, collect the eluate of 30% (v / v) ethanol aqueous solution, and then concentrate and remove ethanol by rotary evaporation. Freeze-dry the concentrated solution to obtain the high-purity theaflavins product. High-performance liquid chromatography analysis shows that the purity of theaflavins in the high-purity theaflavins product is 42.57±0.78%, the yield is 37.50±0.99%, and the total recovery rate is 40.17±0.72%.

[0094] The above embodiments are not intended to limit the scope of the present application, and the described steps are not intended to limit the execution order. Those skilled in the art can make obvious improvements to the present application based on the existing common knowledge, and such improvements also fall within the protection scope defined by the claims of the present application.

Claims

1. A method for preparing high-purity theaflavins using macroporous resin column coupling, characterized in that, Includes the following steps: (1) Dissolve the crude theaflavins in hot water, let it stand and cool, then filter to remove the residue. Take the filtrate and load it into an LX-20B resin column for chromatography. Elute with water, 10-30% ethanol aqueous solution and 40-70% ethanol aqueous solution in volume. Collect the eluent of 40-70% ethanol aqueous solution in volume, and concentrate it by rotary evaporation to obtain the first purified product. (2) The purified product was mixed with hot water and filtered while hot. The filtrate was then loaded into an AB-8 resin column for chromatography. The product was eluted sequentially with water, 10-30% ethanol aqueous solution, 30-50% ethanol aqueous solution, and 50-70% ethanol aqueous solution. The eluent of 50-70% ethanol aqueous solution was collected, concentrated by rotary evaporation, and freeze-dried to obtain high-purity theaflavins.

2. The method according to claim 1, characterized in that, The purity of the theaflavins in the crude theaflavins in step (1) is 15-30%, and the purity of the high-purity theaflavins in step (2) is not less than 95%.

3. The method according to claim 1, characterized in that, In step (1), the crude theaflavins are dissolved in hot water to obtain a solution with a concentration of 50-200 mg / mL; the loading flow rate in step (1) is 0.5-1.5 BV / h.

4. The method according to claim 1, characterized in that, In step (2), the purified product is mixed with hot water to obtain a solution with a concentration of 5-15 mg / mL; the loading flow rate in step (2) is 0.5-1.5 BV / h.

5. The method according to claim 1, characterized in that, The height-to-diameter ratio of the LX-20B resin column in step (1) and / or the AB-8 resin column in step (2) is (10-20):

1.

6. The method according to claim 1, characterized in that, In step (1), the elution volumes for each gradient are 2–4 BV of water, 4–8 BV of 10–30% ethanol aqueous solution, and 3–6 BV of 40–70% ethanol aqueous solution, and the elution flow rate for each gradient is 1.0–2.5 BV / h.

7. The method according to claim 1, characterized in that, In step (2), the elution volumes for each gradient are 2-4 BV of water, 3-6 BV of 10-30% ethanol aqueous solution, 3-6 BV of 30-50% ethanol aqueous solution, and 3-6 BV of 50-70% ethanol aqueous solution, and the elution flow rate for each gradient is 1.0-2.5 BV / h.

8. The method according to claim 1, characterized in that, The temperature of the hot water in step (1) and / or step (2) is 60 to 90°C.

9. The method according to claim 1, characterized in that, The process after step (2) also includes: (3) Resin regeneration and recycling: The LX-20B resin column and the AB-8 resin column are cleaned with a 95% ethanol aqueous solution and then rinsed with deionized water until the effluent has no alcohol odor; the regeneration of the LX-20B resin column also requires alkaline washing → water washing to neutral → acid washing → water washing to neutral; repeat steps (1) and (2) for recycling.

10. The method according to claim 1, characterized in that, The parameters for rotary evaporation concentration described in step (1) and / or step (2) are: temperature of 40-50°C and rotation speed of 70-100 rpm.

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