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

Abstract

The application discloses a method for preparing high-purity theaflavins by using macroporous resin columns in combination, and belongs to the technical field of food engineering. A crude theaflavins solution with a purity of 15-30% is added to an LX-20B resin column, gradient elution is carried out according to a specific procedure, and after the eluate is concentrated by rotary evaporation and / or freeze-drying, a first-purification product is obtained; the first-purification product solution is added to an AB-8 resin column, gradient elution is carried out according to a specific procedure, and after the eluate is concentrated by rotary evaporation and freeze-drying, a high-purity theaflavins product with a purity of 95% or above is obtained, so that the demand for high-purity theaflavins in some high-end health product and medical research and development fields can be met, and the application range of theaflavins is widened.

Description

TECHNICAL FIELD

[0001] The 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 a 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.

[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. 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. SUMMARY

[0007] [TECHNICAL PROBLEM]

[0008] In the prior art, due to the similar structure of theaflavins and catechins and the large amount of other components in theaflavins crude product, 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.

[0009] [TECHNICAL SCHEME]

[0010] 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.

[0011] The technical scheme of the present application comprises the following:

[0012] The present application takes the crude theaflavins as raw material, dissolves in hot water, and then removes the filter residue after standing and cooling to obtain the first sample solution. The first sample solution is loaded into a resin column, and the theaflavins are adsorbed on the resin. Different gradient ethanol solutions are used for gradient elution 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 a resin column, and the theaflavins are adsorbed. Different gradient ethanol solutions are used for gradient elution to obtain the second purified product. After rotary evaporation and freeze-drying, high-purity theaflavins product is obtained.

[0013] 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:

[0014] (1) Dissolve the crude theaflavins in hot water, remove the filter residue after standing and cooling, and then take the filtrate to be loaded into a 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.

[0015] (2) Mix the first purified product with hot water, filter while hot, and then take the filtrate to be loaded into an 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.

[0016] In one embodiment, the purity of 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%.

[0017] In one embodiment, the crude theaflavins are dissolved in hot water in step (1) 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 (column bed volume / h).

[0018] 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. The loading flow rate in step (2) is 0.5-1.5 BV / h.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] In one embodiment, the temperature of the hot water in step (1) and / or step (2) is 60-90°C.

[0023] In one embodiment, after step (2), the method further comprises:

[0024] (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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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 above-mentioned macroporous resin columns.

[0030] [Advantages]

[0031] 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:

[0032] (1) The resin used is a domestic macroporous adsorption resin, which has relatively low cost;

[0033] (2) The eluent used is only ethanol aqueous solution, without other toxic and harmful organic reagents, which is safe and environmentally friendly;

[0034] (3) The operation is simple, the loading amount is large, and it is suitable for large-scale production;

[0035] (4) The macroporous resin regeneration treatment is simple, and can be repeatedly used for many times;

[0036] (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;

[0037] (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;

[0038] (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

[0039] Figure 1 is the structure of four main theaflavins;

[0040] Figure 2 is the static adsorption / desorption experiment result of different macroporous resin columns on theaflavins in Example 4. DETAILED DESCRIPTION

[0041] 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.

[0042] The determination method involved in the present application:

[0043] Determination of the contents of main catechins, caffeine and four main theaflavins

[0044] 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.

[0045] Table 1 High performance liquid chromatography gradient elution program

[0046]

[0047]

[0048] Table 2 Standard curve of 12 standard substances

[0049]

[0050] Example 1

[0051] (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 (LX-20B was packed inside) and Φ16 x 300 mm (AB-8 was packed inside), respectively, to obtain LX-20B resin columns and AB-8 resin columns;

[0052] (2) Take 10 g of crude theaflavins 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%) and dissolve it in 100 mL of 70°C hot water to prepare a solution with a concentration of 100 mg / mL. After standing and cooling for 6 h, remove the filter residue by suction filtration. Then, pass the filtrate through a peristaltic pump at a flow rate of 0.5 BV / h to load it into the LX-20B resin column;

[0053] (3) After the loading is completed, perform gradient elution 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 at an elution flow rate of 2.0 BV / h. Collect the eluate of 65% (v / v) ethanol aqueous solution, remove ethanol by rotary evaporation, and then 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 65.79 ± 0.91%, the yield is 51.41 ± 1.57%, and the total recovery rate is 84.28 ± 2.10%;

[0054] (4) Take 1.00 g of the first purified product and dissolve it in 100 mL of 70°C hot water to prepare a solution with a concentration of 10 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 load it into the AB-8 resin column.

[0055] (5) After the loading is completed, perform gradient elution 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 at an elution flow rate of 2.0 BV / h. Collect the eluate of 50% (v / v) ethanol aqueous solution, remove ethanol by rotary evaporation, and then 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 95.41 ± 0.77%, the yield is 55.34 ± 1.95%, and the total recovery rate is 95.83 ± 0.79%;

[0056] (6) Resin regeneration: use 2 BV of 95% (v / v) ethanol aqueous solution to clean the LX-20B resin column and the AB-8 resin column, respectively, and then use 4 BV of deionized water for elution until there is no obvious alcohol smell in the effluent. For the LX-20B resin column, first use 2 BV of 4% NaOH solution for elution, then wash with water until neutral, then use 2 BV of 2% HCl solution for elution, and finally wash with water until neutral. The elution flow rate is 1.0 BV / h. After the resin regeneration treatment is completed, steps (2) to (5) can be repeated for the next round of purification.

[0057] (7) Two resins were regenerated and recycled for 5 times, and the purity and yield of the theaflavins product obtained were not significantly decreased: the purity of the theaflavins product obtained was 95.10±0.69%, the yield was 30.28±1.51%, and the total recovery rate was 80.46±1.01% after the resins were regenerated and recycled for 5 times.

[0058] Example 2

[0059] (1) The pretreated LX-20B and AB-8 resins were wet-packed into glass chromatographic columns according to a height-diameter ratio of 15:1, and the glass chromatographic column specifications were Φ25×500 mm (LX-20B) and Φ16×300 mm (AB-8), respectively, to obtain LX-20B resin columns and AB-8 resin columns;

[0060] (2) 15 g of theaflavins crude product with a purity of 20% (main impurities and contents: catechin: 35.10±0.05%, caffeine: 1.55±0.03%, protein: 6.36±0.35%, and polysaccharide: 6.43±0.50%) were weighed and dissolved in 100 mL of hot water at 80°C to prepare a solution with a concentration of 150 mg / mL, which was allowed to stand and cool for 12 h, and then filtered to remove the filter residue. The filtrate was loaded into the LX-20B resin column through a peristaltic pump at a flow rate of 1.0 BV / h;

[0061] (3) After the loading was completed, gradient elution was performed in the order of 4 BV of water, 6 BV of 30% (v / v) ethanol aqueous solution, and 3 BV of 70% (v / v) ethanol aqueous solution, and the elution flow rate was 1.0 BV / h. The eluate of the 70% (v / v) ethanol aqueous solution was collected, concentrated by rotary evaporation to remove ethanol, and then freeze-dried to obtain a primary purified product. High-performance liquid chromatography analysis showed that the purity of the theaflavins product was 62.48±0.37%, the yield was 52.98±2.01%, and the total recovery rate was 86.19±3.54%;

[0062] (4) 1.50 g of the primary purified product was weighed and dissolved in 100 mL of hot water at 80°C to prepare a solution with a concentration of 15 mg / mL, which was filtered while hot to remove the filter residue. The filtrate was loaded into the AB-8 resin column through a peristaltic pump at a flow rate of 1.0 BV / h;

[0063] (5) After the end of the sample loading, gradient elution was performed in the order of 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, with an elution flow rate of 2.0 BV / h. The eluate of 60% (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 95.02 ± 0.39%, the yield was 58.06 ± 0.23%, and the total recovery rate was 95.98 ± 0.11%;

[0064] (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 5 BV of deionized water was used for elution, and the elution was stopped when there was no obvious alcohol smell in the effluent. For the LX-20B resin, 2 BV of 4% NaOH solution was first used for elution, then water was used for washing to neutral, 2 BV of 2% HCl solution was used for elution, and finally water was used for washing to neutral. The elution flow rate was 1.5 BV / h. After the resin regeneration treatment was completed, steps (2) to (5) could be repeated to perform the next round of purification.

[0065] (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 95.32 ± 0.44%, the yield was 30.49 ± 1.25%, and the total recovery rate was 81.72 ± 1.61%.

[0066] Example 3

[0067] (1) The pretreated AB-8 and LX-20B resins were wet-packed into glass chromatography columns (Φ16 x 400 mm) according to a height-to-diameter ratio of 20:1, to obtain an LX-20B resin column and an AB-8 resin column;

[0068] (2) 5 g 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%, and polysaccharide: 6.96 ± 0.66%) was 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 stand and cool for 8 h, and then the filtrate was obtained by suction filtration to remove the filter residue. The filtrate was loaded into the LX-20B resin column at a flow rate of 0.5 BV / h by using a peristaltic pump;

[0069] (3) After the end of loading, gradient elution was performed 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, 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 the product of the first purification. High performance liquid chromatography analysis showed that the purity of the product of the first purification was 63.14 ± 0.26%, the yield was 50.95 ± 1.21%, and the total recovery rate was 85.33 ± 2.61%;

[0070] (4) 0.25 g of the product of the first purification was 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 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.

[0071] (5) After the end of loading, 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, 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 the product of high-purity theaflavins. High performance liquid chromatography analysis showed that the purity of the product of high-purity theaflavins was 95.90 ± 0.59%, the yield was 57.44 ± 0.81%, and the total recovery rate was 94.94 ± 0.85%;

[0072] (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. The elution was stopped when there was no obvious alcohol smell in the effluent. For the LX-20B resin column, 2 BV of 4% NaOH solution was used for elution first, followed by water washing to neutral, then 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 in the above processes. After the end of the resin regeneration treatment, steps (2) to (5) could be repeated to perform the next round of purification.

[0073] (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 the resins were recycled for 5 times, the purity of the product of high-purity theaflavins was 95.11 ± 0.23%, the yield was 30.26 ± 0.97%, and the total recovery rate was 81.09 ± 1.27%.

[0074] Example 4 Screening of Macroporous Resin Column

[0075] Through static adsorption / desorption experiments, a macroporous resin LX-20B with high adsorption / desorption of teaflavins was screened from six different macroporous resins, and the specific experiment was as follows:

[0076] 1.00 g (dry weight) of the pretreated macroporous resin was placed in a 50 mL conical flask, 20 mL of a solution of teaflavins with a content of 100 mg / mL was added, and after adsorption for 12 h on a constant temperature shaker at 25 °C and 130 r / min, the saturated resin was filtered to obtain a filtrate; the saturated resin was washed with deionized water for 2-3 times, the surface water 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 on a constant temperature shaker at 25 °C and 130 r / min to obtain a desorption solution; the concentrations of teaflavins in the initial solution, the filtrate and the desorption solution were determined by HPLC method. The static equilibrium adsorption amount (Q e ), desorption amount (Q d ) and desorption rate (D) of the resin for teaflavins were calculated, and the appropriate resin was selected based on the above parameters. The calculation formulae were as follows:

[0077]

[0078] Q d = C2 x V2 / W

[0079]

[0080] In the formulae, Q e and Q d are the static adsorption amount and desorption amount of teaflavins (mg / g dry resin) respectively, D is the desorption rate (%), C0 is the initial concentration of teaflavins (mg / mL), C1 is the equilibrium concentration of teaflavins (mg / mL), C2 is the total content of teaflavins in the desorption solution (mg / mL), V1 is the adsorption solution volume of teaflavins (mL), V2 is the desorption solution volume of teaflavins (mL), and W is the dry weight of the resin (g).

[0081] In this example, the exchange capacities of six macroporous resins for four kinds of teaflavins were tested, and whether the resins were suitable for the separation and purification of teaflavins was judged by investigating the static adsorption / desorption amount and desorption rate of the resins for teaflavins. The results are shown in Table 1. Figure 2 The adsorption amount and desorption amount of LX-20B resin for teaflavins were significantly higher than those of other resins. Considering that the desorption rates of different resins for teaflavins were not significantly different and were all greater than 80%, LX-20B resin was considered as the preferred resin for the purification of teaflavins.

[0082] Comparative Example 1

[0083] Through dynamic elution experiment, AB-8 resin which is more suitable for the experiment is screened from macroporous resins suitable for separating and removing caffeine, and the specific experiment is as follows:

[0084] Compared with example 3, the difference is only that the AB-8 resin column is replaced by the LX-8 resin column, and high-purity theaflavins product is obtained; it can be known through high performance liquid chromatography analysis that the purity of theaflavins in the high-purity theaflavins product is 73.16±1.58%, the yield is 18.48±0.69%, and the total recovery rate is 35.50±0.93%.

[0085] Compared with example 3, the difference of comparative example 1 is only that the resin column used in secondary purification is different, and it can be found from the comparison result that the purification effect (theaflavins purity, yield and total recovery rate) of theaflavins under the same conditions by using LX-20B resin combined with LX-8 resin is much worse than that by using LX-20B resin combined with AB-8 resin in the application.

[0086] Comparative example 2

[0087] (1) The pretreated AB-8 and LX-20B resins are respectively wet-packed into glass chromatographic columns (Φ16×400mm) according to the height-diameter ratio of 20:1, and LX-20B resin column and AB-8 resin column are obtained;

[0088] (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%) is dissolved in 25mL of 90℃ hot water to prepare a solution with a concentration of 200mg / mL, and the solution is cooled for 8h, then the filtrate is obtained by suction filtration after removing the filter residue, and the filtrate is loaded into the LX-20B resin column at a flow rate of 0.5BV / h by using a peristaltic pump;

[0089] (3) After the loading is completed, gradient elution is carried out in the order of 3BV of water, 8BV of 10%(v / v) ethanol aqueous solution and 6BV of 35%(v / v) ethanol aqueous solution, the elution flow rate is 2.0BV / h, the eluate of 35%(v / v) ethanol aqueous solution is collected, and the ethanol is removed by rotary evaporation and concentration, and then the concentrated solution is freeze-dried to obtain the first purification product; it can be known through high performance liquid chromatography analysis that the purity of theaflavins in the first purification product is 23.26±0.59%, the yield is 32.91±1.28%, and the total recovery rate is 37.31±2.09%;

[0090] (4) 0.25g of the first purification product is dissolved in 50mL of 90℃ hot water to prepare a solution with a concentration of 5mg / mL, and the filter residue is removed by hot filtration, and the filtrate is loaded into the AB-8 resin column at a flow rate of 1.0BV / h by using a peristaltic pump;

[0091] (5) After the end of the loading, gradient elution was carried out 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, with an elution flow rate of 2.0 BV / h, and the eluate of 50% (v / v) ethanol aqueous solution was collected. After the ethanol was removed by rotary evaporation and concentration, the concentrated solution was freeze-dried to obtain a high-purity theaflavins product. High-performance liquid chromatography analysis showed that the purity of theaflavins in 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%.

[0092] Comparative Example 3

[0093] (1) The pretreated AB-8 and LX-20B resins were respectively wet-packed into a glass chromatography column (Φ16 × 400 mm) according to a height-diameter ratio of 20:1 to obtain an LX-20B resin column and an AB-8 resin column;

[0094] (2) 5 g 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%, and polysaccharide: 6.96 ± 0.66%) was dissolved in 25 mL of hot water at 90°C to prepare a solution with a concentration of 200 mg / mL. After being cooled for 8 h, the filtrate was obtained by suction filtration to remove the filter residue, 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;

[0095] (3) After the end of the loading, gradient elution was carried out 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, with an elution flow rate of 2.0 BV / h, and the eluate of 50% (v / v) ethanol aqueous solution was collected. After the ethanol was removed by rotary evaporation and concentration, the concentrated solution was freeze-dried to obtain a first purification product. High-performance liquid chromatography analysis showed that the purity of 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%;

[0096] (4) 0.25 g of the first purification product was dissolved in 50 mL of hot water at 90°C to prepare a solution with a concentration of 5 mg / mL. After being filtered while hot to remove the filter residue, the filtrate was loaded into the AB-8 resin column at a flow rate of 1.0 BV / h by using a peristaltic pump;

[0097] (5) After the end of the sample, according to the order of 3BV water, 3BV 10% (v / v) ethanol aqueous solution, 3BV 30% (v / v) ethanol aqueous solution, 3BV 40% (v / v) ethanol aqueous solution, gradient elution was carried out, the elution flow rate was 2.0BV / h, the eluate of 30% (v / v) ethanol aqueous solution was collected, after removing ethanol by rotary evaporation and concentration, the concentrated liquid was freeze-dried to obtain high-purity theaflavins product; it can be known by high performance liquid chromatography analysis that the purity of theaflavins in the high-purity theaflavins product was 42.57±0.78%, the yield was 37.50±0.99%, and the total recovery rate was 40.17±0.72%.

[0098] The above provided examples are not used to limit the scope covered by the present application, and the described steps are not used to limit the execution order. The improvements of the present application made by those skilled in the art in combination with the existing common knowledge are also within the protection scope defined by the claims of the present application.

Claims

1. A method for preparing high purity theaflavins by coupling a macroporous resin column, characterized in that, Comprising the following steps: (1) Dissolve the crude theaflavins in hot water, remove the filter residue after standing and cooling, and take the filtrate to chromatograph in an LX-20B resin column; sequentially use water, 10-30% ethanol aqueous solution by volume, and 40-70% ethanol aqueous solution by volume for elution, collect the eluate of 40-70% ethanol aqueous solution by volume, and concentrate by rotary evaporation to obtain a first purified product; Dissolve the crude theaflavins in hot water to obtain a solution with a concentration of 50-200 mg / mL; The loading flow rate is 0.5-1.5 BV / h; The gradient elution volumes are 2-4 BV of water, 4-8 BV of 10-30% ethanol aqueous solution by volume, and 3-6 BV of 40-70% ethanol aqueous solution by volume, and the gradient elution flow rate is 1.0-2.5 BV / h; (2) Mix the first purified product with hot water, filter while hot, and take the filtrate to chromatograph in an AB-8 resin column; sequentially use water, 10-30% ethanol aqueous solution by volume, 30-50% ethanol aqueous solution by volume, and 50-70% ethanol aqueous solution by volume for elution, collect the eluate of 50-70% ethanol aqueous solution by volume, and concentrate by rotary evaporation and freeze-drying to obtain high-purity theaflavins; Mix the first purified product with hot water to obtain a solution with a concentration of 5-15 mg / mL; The loading flow rate is 0.5-1.5 BV / h; The gradient elution volumes are 2-4 BV of water, 3-6 BV of 10-30% ethanol aqueous solution by volume, 3-6 BV of 30-50% ethanol aqueous solution by volume, and 3-6 BV of 50-70% ethanol aqueous solution by volume, and the gradient elution flow rate is 1.0-2.5 BV / h.

2. The method of claim 1, wherein, The purity of 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 of claim 1, wherein, 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.

4. The method of claim 1, wherein, The temperature of the hot water in step (1) and / or step (2) is 60-90 ℃.

5. The method of claim 1, wherein, After step (2), further comprising: (3) Resin regeneration and recycling: use 95% ethanol aqueous solution by volume to clean the LX-20B resin column and the AB-8 resin column, and then use deionized water to rinse until the effluent has no alcohol smell; the LX-20B resin column also needs to be subjected to alkali washing → water washing to neutralization → acid washing → water washing to neutralization operation; repeat steps (1) and (2) for recycling.

6. The method of claim 1, wherein, The parameters for rotary evaporation concentration in step (1) and / or step (2) are: temperature 40-50 ℃, and rotation speed 70-100 rpm.

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