A method for the preparation of theaflavins by Sephadex G molecular sieve tandem column chromatography

Abstract

The application discloses a method for preparing theabromine by using Sephadex G molecular sieve series connection column chromatography, and steps are as follows: 1) using low-grade black tea as raw material, hot water extraction, filtering the extraction liquid and cooling, obtaining the black tea crude extract; 2) vacuum concentration of the black tea crude extract, and putting into Sephadex G gel filler series connection chromatography column; 3) gradient elution, and collecting the eluent containing theabromine in sequence; 4) concentrating the eluent respectively, and vacuum drying, obtaining theabromine products with different molecular weights with high yield. The application can obtain theabromine products with different molecular weight intervals by using the series connection separation of different Sephadex G molecular sieve chromatography columns, and the yield of theabromine products with different molecular weight intervals reaches more than 93%, which is higher than that of the existing theabromine separation and preparation method.

Description

Technical Field

[0001] This invention belongs to the field of deep processing of tea, specifically a method for preparing theaflavins by tandem separation using Sephadex G molecular sieve column chromatography. Background Technology

[0002] Theabrownins are important chemical components in tea, significantly impacting its quality and function. Dark brown in color, theabrownins constitute a large proportion of the dry weight of fermented tea leaves and possess strong antioxidant properties, effectively scavenging free radicals and protecting cells from oxidative damage. Furthermore, theabrownins exhibit antibacterial, antiviral, and anti-inflammatory biological activities. However, the isolation and preparation of theabrownins are challenging due to their large molecular weight variations, unclear formation mechanisms, and complex chromatographic behavior, hindering research and development in areas such as flavor evaluation, biological activity, structure-activity relationships, and functional applications.

[0003] There are various methods for preparing theabrownins, including traditional extraction, liquid fermentation, and chromatographic separation. Traditional extraction methods mainly include hot water extraction and organic solvent extraction, supplemented by microwave, ultrasonic, or enzymatic techniques, and are currently the primary methods for obtaining theabrownins. These methods are suitable for separating target substances with less demanding operational requirements; however, the yield and content of the target substance prepared by these methods are not high, and even with optimization of process parameters such as extraction solvent, extraction temperature, and extraction time, the yield and content of theabrownins are still difficult to significantly increase. Liquid fermentation, which uses oxidoreductase catalysis to form theabrownins, also suffers from the drawback of low yield and content. Chromatographic separation is a method that utilizes the different partition coefficients of different substances between the stationary and mobile phases to separate different components in theabrownins. Yang Xinhe et al. used a dextran gel LH-20 column to classify the pigments (i.e., theabrownins) in the aqueous layer of Pu'er tea water extract after reagent purification. Six distinct bands were observed in the chromatographic column, with differences in color difference, reducing power, pH value, UV-Vis spectrum, and infrared spectrum. (Yang Xinhe, Wang Lili, Huang Jianan, et al. Preliminary study on the classification and related properties of theabrownins in Pu'er tea [J]. Tea Science, 2011, 31(3):187~194.). Yang Dapeng selected silica gel powder as the packing material and methanol solution of different concentrations as the eluent to classify theabrownin extract. The theabrownin content and absorbance of the 60% methanol extract were higher than those of other treatments, accounting for about 50% of the total (Yang Dapeng. Separation and structural identification of the main chemical components of theabrownins in Yunnan Pu'er tea [D]. Kunming: Yunnan Agricultural University, 2009.). Wang Tianlu selected AB-8 macroporous resin to separate theabrownins from Pu'er tea, obtaining two components, TBsP1 and TBsP2. He further used a DEAE-52 cellulose column to separate the TBsP1 component, obtaining six components (Wang Tianlu, Du Liping, Liu Yan, et al. Study on the separation of theabrownins from Pu'er tea [J]. Science of Food Industry, 2016, 37(16):136-145). Yi Lian et al. compared the effects of nine macroporous resins—D101, S-8, AB-8, HP-20, NKA-9, LSA-7, XAD-7HP, D201, and polyamide—on the separation and purification of tea polyphenols and theabrownins, concluding that D101 was more suitable for purifying theabrownins (Yi Lian. Study on the separation and function of Pu'er tea polyphenols and theabrownins [D]. Changsha: Hunan Agricultural University, 2010).

[0004] Currently, column chromatography methods for separating theabrownins often employ dextran gel LH20 (separation range 0.1–4 kDa), silica gel (separation range <0.5 kDa), and macroporous adsorption resins. However, these methods only yield a few mixtures with relatively small molecular weights or similar polarities, and lack systematic separation targeting different molecular weight ranges. Therefore, they cannot effectively separate theabrownins with a wide molecular weight range. DEAE-52 cellulose column chromatography, based on the principle of ion exchange, also struggles to achieve high yields of theabrownins. Summary of the Invention

[0005] To address the problems existing in the above-mentioned background technology, the purpose of this invention is to provide a method for preparing theaflavins by tandem separation using Sephadex G molecular sieve column chromatography, specifically achieved through the following technical solution:

[0006] A method for preparing theaflavins by Sephadex G molecular sieve tandem column chromatography includes the following steps:

[0007] 1) Using medium- to low-grade black tea as raw material, hot water is used for extraction. The extract is filtered and cooled to obtain crude black tea extract.

[0008] 2) The crude extract of black tea was concentrated under vacuum and placed into a tandem chromatographic column packed with Sephadex G gel packing material of different molecular sieve pore sizes.

[0009] 3) The chromatography column is subjected to gradient elution with an elution solvent, wherein the elution solvent is a mixed solvent composed of acetone, acid and pure water, and the eluent containing theaflavins is collected sequentially.

[0010] 4) The eluents were concentrated and vacuum dried to obtain high-yield theabrownin products with different molecular weights.

[0011] Furthermore, in step 1), the material-to-liquid ratio for hot water extraction is 1g:10-12mL, the extraction time is 40-60min, and the extraction is performed twice.

[0012] Further, in step 2), the Sephadex G gel packing material is selected from Sephadex G10 (separation range <0.7 kDa) to Sephadex G200 (separation range 5 to 60 kDa), preferably Sephadex G10 (separation range <0.7 kDa) to Sephadex G200 (separation range 5 to 60 kDa), and more preferably Sephadex G75 (separation range 3 to 80 kDa) to Sephadex G200 mesh (separation range 5 to 300 kDa). The packing material is loaded into different chromatographic columns and the chromatographic columns are connected in series.

[0013] Further, in step 3), the concentration of the acetone solution is 25-85%; the flow rate of the chromatography process is 0.6-2.6 column bed volumes / hour, preferably 0.8-1.8 column bed volumes / hour.

[0014] Further, in step 3), the gradient elution specifically involves sequentially eluting with 25-35%, 45-58%, and 60-85% acetone solutions at room temperature, collecting the eluents at 1 / 8 to 1 / 16 of the column bed volume, and combining the eluted components.

[0015] Further, in step 3), the acid in the mixed solvent is propionic acid or butyric acid, and the amount added accounts for 0.02 to 0.09% of the volume of the mixed solvent.

[0016] This invention utilizes Sephadex G series gel packing materials with different molecular sieve effects for the systematic separation of theabrownins, thereby obtaining high-yield theabrownin products of different molecular weights to meet the needs of developing various products in the pharmaceutical, food, and chemical industries. The molecular sieve effect occurs during column chromatography separation, where large molecules cannot enter the gel interior and flow out of the column first along the gaps between gel particles, while small molecules can enter the porous network structure inside the gel particles, flowing slowly and ultimately flowing out of the column, thus separating substances of different molecular sizes in the sample.

[0017] Sephadex (dextran gel) is composed of dextran glycosides and 3-chloro-1,2-epoxypropane linked by ether bonds. Depending on its water absorption and separation range, gel chromatography materials with different specifications and molecular sieve effects exist. They are distinguished by the degree of crosslinking, using Sephadex G followed by a number; the smaller the number, the greater the degree of crosslinking, resulting in smaller pore sizes and a narrower separation range. The Sephadex G series packing materials offer a variety of separation ranges and particle sizes to choose from, including Sephadex G15 (separation range 0.7–1.5 kDa), Sephadex G25 (separation range 1–5 kDa), Sephadex G50 (separation range 1.5–30 kDa), Sephadex G75 (separation range 3–80 kDa), Sephadex G100 (separation range 4–150 kDa), Sephadex G150 (separation range 5–300 kDa), and Sephadex G200 (separation range 5–600 kDa).

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) By tandem separation of different Sephadex G molecular sieve chromatographic columns, theabrownin products with different molecular weight ranges can be obtained, including theabrownins with molecular weight ranges of 1-1.5 kDa, 3-5 kDa, 6-8 kDa, 12-16 kDa, 30-40 kDa, and 90-100 kDa.

[0020] 2) The yield of theabrownin products in different molecular weight ranges reached over 93%, which is higher than the existing methods for separating and preparing theabrownin. Detailed Implementation

[0021] Example 1

[0022] 1) Weigh 1000g of low-grade black tea raw material, extract it with hot water at a material-to-liquid ratio of 1g:10mL for 40min, extract twice, filter and cool to obtain black tea extract;

[0023] 2) Pack Sephadex G25 and Sephadex G75 gel packing materials into the chromatographic columns respectively, connect the two columns in series, each column has a diameter of 46 mm and a length of 800-1000 mm, and each column has a packed bed volume of 650 ml. Elute the columns sequentially with 50% ethanol, 100% ethanol and water to equilibrate the column bed.

[0024] 3) The crude extract of black tea was concentrated under vacuum. The concentrate was added to a tandem chromatographic column packed with Sephadex G25 and Sephadex G75 gel packing materials. Gradient elution was performed with acetone:propionic acid:water = 26.8%:0.02%:73%, acetone:propionic acid:water = 50.7%:0.03%:49%, and acetone:propionic acid:water = 71.6%:0.04%:28%, respectively. The elution rate was 1.3 BV / h (BV: column volume, h: hour).

[0025] 4) Collect the eluent at 1 / 9 of the column bed volume, combine the eluent components, concentrate by vacuum rotary evaporation and freeze-dry to obtain theabrownin products with molecular weight ranges of 1-1.2 kDa and 3-3.5 kDa, respectively. The product yields are shown in Table 1.

[0026] Example 2

[0027] 1) Weigh 1000g of low-grade black tea raw material, extract it with hot water at a material-to-liquid ratio of 1:10 for 40 minutes, extract twice, filter and cool to obtain black tea extract;

[0028] 2) Pack Sephadex G50 and Sephadex G100 gel packing materials into the chromatographic columns respectively, connect the two columns in series, each column has a diameter of 46 mm and a length of 800-1000 mm, and each column has a packed bed volume of 650 ml. Elute the columns sequentially with 50% ethanol, 100% ethanol and water to equilibrate the column bed.

[0029] 3) The crude extract of black tea was concentrated under vacuum and added to a tandem chromatographic column packed with Sephadex G25 and Sephadex G75 gel packing materials. Gradient elution was performed with acetone:propionic acid:water = 28.7%:0.03%:71%, acetone:propionic acid:water = 53.7%:0.03%:46%, and acetone:propionic acid:water = 75.6%:0.04%:24%, respectively, at a rate of 1.1 BV / h (BV: column volume, h: hour).

[0030] 4) Collect the eluent at 1 / 10 of the column bed volume, combine the eluent components, concentrate by vacuum rotary evaporation and freeze-dry to obtain theabrownin products with molecular weight ranges of 4-5 kDa and 13-14 kDa, respectively. The product yields are shown in Table 1.

[0031] Example 3

[0032] 1) Weigh 1200g of low-grade black tea raw material, extract it with hot water at a material-to-liquid ratio of 1:12 for 50 minutes, extract twice, filter and cool to obtain black tea extract;

[0033] 2) Pack Sephadex G100 and Sephadex G150 gel packing materials into the chromatographic columns respectively, connect the two columns in series, each column has a diameter of 46 mm and a length of 800-1000 mm, and each column has a packed bed volume of 650 ml. Elute the columns sequentially with 50% ethanol, 100% ethanol and water to equilibrate the column bed.

[0034] 3) The crude extract of black tea was concentrated under vacuum and added to a tandem chromatographic column packed with Sephadex G100 and Sephadex G150 gel packing materials. Gradient elution was performed with acetone:propionic acid:water = 32.7%:0.03%:71%, acetone:propionic acid:water = 56.5%:0.05%:43%, and acetone:propionic acid:water = 79.5%:0.05%:20%, respectively, at a rate of 1.0 BV / h (BV: column volume, h: hour).

[0035] 4) Collect the eluent at 1 / 12 of the column bed volume, combine the eluent components, concentrate by vacuum rotary evaporation and freeze-dry to obtain theabrownin products with molecular weight ranges of 15-16 kDa and 35-36 kDa, respectively. The product yields are shown in Table 1.

[0036] Comparative Example 1

[0037] 1) Weigh 1000g of low-grade black tea raw material, extract it with hot water at a material-to-liquid ratio of 1:10 for 40 minutes, extract twice, filter and cool to obtain black tea extract;

[0038] 2) Pack the LH20 dextran gel into the chromatographic column. The column specifications are 46 mm in diameter and 800-1000 mm in length, with a packed bed volume of 650 ml. Elute the column bed sequentially with 50% ethanol, 100% ethanol, and water to equilibrate it.

[0039] 3) The crude extract of black tea was concentrated under vacuum. The concentrate was added to a chromatographic column packed with Sephadex LH20 packing material and eluted with 40% acetone solution at a rate of 1.1 BV / h (BV: column volume, h: hour).

[0040] 4) Collect the eluent at 1 / 10 of the column bed volume, combine the eluent components, concentrate by vacuum rotary evaporation and freeze-dry to obtain theabrownin products with molecular weight range of 0.4-0.6 kDa. The product yields are shown in Table 1.

[0041] Comparative Example 2

[0042] 1) Weigh 1000g of low-grade black tea raw material, extract it with hot water at a material-to-liquid ratio of 1:10 for 40 minutes, extract twice, filter and cool to obtain black tea extract;

[0043] 2) Pack the AB-8 packing material into the chromatographic column. The column specifications are 46 mm in diameter and 800-1000 mm in length, with a packed bed volume of 650 ml. Elute the column bed sequentially with 50% ethanol, 100% ethanol, and water to equilibrate it.

[0044] 3) The crude extract of black tea was concentrated under vacuum. The concentrate was added to a chromatographic column packed with AB-8 packing material and eluted with 60% methanol solution at a rate of 1.2 BV / h (BV: column volume, h: hour).

[0045] 4) Collect the eluent at 1 / 10 of the column bed volume, combine the eluent components, concentrate by vacuum rotary evaporation and freeze-dry to obtain theabrownin products with molecular weight range of 0.5-0.7 kDa. The product yields are shown in Table 1.

[0046] Comparative Example 3

[0047] 1) Weigh 1000g of low-grade black tea raw material, extract it with hot water at a material-to-liquid ratio of 1:10 for 40 minutes, extract twice, filter and cool to obtain black tea extract;

[0048] 2) Pack the silica gel into the chromatographic column. The column specifications are 46 mm in diameter and 800-1000 mm in length, with a packed bed volume of 650 ml. Elute the column bed sequentially with 50% ethanol, 100% ethanol, and water to equilibrate it.

[0049] 3) The crude extract of black tea was concentrated under vacuum. The concentrate was added to a chromatographic column packed with silica gel and eluted with 60% methanol solution at a rate of 1.0 BV / h (BV: column volume, h: hour).

[0050] 4) Collect the eluent at 1 / 12 of the column bed volume, combine the eluent components, concentrate by vacuum rotary evaporation and freeze-dry to obtain theabrownin products with molecular weight range of 0.8-0.9 kDa. The product yields are shown in Table 1.

[0051] Table 1. Theabrownin products and yields of the examples and comparative examples.

[0052]

Claims

1. A method for the preparation of the theabromine by Sephadex G molecular sieve column chromatography in series, characterized in that Includes the following steps: 1) Using medium- to low-grade black tea as raw material, hot water is used for extraction. The extract is filtered and cooled to obtain crude black tea extract. 2) The crude extract of black tea was concentrated under vacuum and placed into a tandem chromatographic column packed with Sephadex G gel packing material of different molecular sieve pore sizes. The Sephadex G gel packing material of different molecular sieve pore sizes was used to separate the different molecular weight components in theabrownin by size exclusion. The Sephadex G gel packing material used ranges from Sephadex G10 to Sephadex G200. Different types of Sephadex G gel packing material are packed into different chromatographic columns and the columns are connected in series. 3) The chromatography column is subjected to gradient elution with an elution solvent, wherein the elution solvent is a ternary mixed solvent composed of acetone, acid, and pure water, and the acid is propionic acid or butyric acid, and the amount added accounts for 0.02-0.09% of the volume of the mixed solvent; the gradient elution is specifically performed at room temperature by sequentially eluting with 25-35%, 45-58%, and 60-85% acetone solutions, and the eluent is collected at 1 / 8 to 1 / 16 of the column bed volume, and the eluent components are combined; 4) After concentrating and vacuum drying the eluents, theabrownin products with different molecular weights were obtained, and the yield of theabrownin products in different molecular weight ranges reached more than 93%.

2. The method for preparing theaflavins by Sephadex G molecular sieve tandem column chromatography as described in claim 1, characterized in that... In step 1), the material-to-liquid ratio for hot water extraction is 1g:10-12mL, the extraction time is 40-60 min, and the extraction is performed twice.

3. The method for preparing theaflavins by Sephadex G molecular sieve tandem column chromatography as described in claim 1, characterized in that... In step 3), the concentration of the acetone solution is 25-85%; the flow rate of the chromatography process is 0.6-2.6 column bed volumes / hour.

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