The invention relates to 3, 3apos; extraction method and application of theaflavin digallate

Abstract

The invention relates to the field of plant extraction, and particularly discloses an extraction method and application of 3, 3 '-digallate theaflavin. According to the extraction method, by optimizing early-stage fermentation treatment of tea leaves, the structure of a target component in the tea leaves is promoted to be converted beneficial to extraction of TFDG, and a foundation is laid for subsequent efficient extraction; then, according to the physicochemical property of the TFDG, a special extracting agent is adopted to extract effective components from the fermented black tea at a low temperature, and the extracting efficiency is enhanced by utilizing the binding force formed by the extracting agent and galloyl of the TFDG and stronger than that of a conventional ethanol solvent; and finally, purifying to obtain a target product.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant extraction, and particularly relates to an extraction method of 3,3'-dicatechin gallate theaflavins and application thereof. BACKGROUND

[0002] 3,3'-dicatechin gallate theaflavins (TFDG) is a kind of polyphenol compound, and its natural source mainly exists in black tea. It is a kind of theaflavins, and is formed by the oxidation of catechins in the fermentation process of tea leaves. It accounts for 2-6% of the dry weight of polyphenols in black tea. In the artificial synthesis method, EGCG and epicatechin gallate are usually used as substrates to obtain TFDG through the catalysis of polyphenol oxidase.

[0003] At present, the main application fields of 3,3'-dicatechin gallate theaflavins include: (1) medicine field: TFDG can inhibit the proliferation of prostate cancer cells and induce their apoptosis; it is also found to inhibit the angiogenesis of ovarian cancer cells by down-regulating the expression of HIF-1α and VEGF. (2) Cardiovascular disease: TFDG can inhibit the restenosis after vascular injury, and protect the vascular structure by regulating the smooth muscle phenotype conversion. (3) Bone health: TFDG can promote the differentiation of osteoblasts and inhibit the formation of osteoclasts, thereby preventing osteoporosis and periprosthetic osteolysis. (4) Anti-inflammatory and antioxidant: TFDG has significant anti-inflammatory and antioxidant effects, can inhibit the activation of NF-κB, reduce the production of pro-inflammatory cytokines, and at the same time, can scavenge free radicals and protect cells from oxidative damage.

[0004] Considering the two sources of 3,3'-dicatechin gallate theaflavins, the artificial synthesis preparation method can provide products in large quantities, but there are still problems of complexity of synthesis route and cost to be solved, and the black tea extraction method also faces the challenge of low extraction amount. Therefore, the application provides a method for improving the extraction amount of 3,3'-dicatechin gallate theaflavins in black tea. SUMMARY

[0005] In order to overcome the deficiencies of the prior art, the application promotes the conversion of the structure of the target component in the tea leaves to be conducive to the extraction of TFDG by carrying out the pre-fermentation treatment of the tea leaves, thereby laying a foundation for the subsequent efficient extraction; then the effective component is extracted from the fermented black tea at a lower temperature according to the physicochemical properties of TFDG by using a special extractant; and finally the target product is obtained through purification.

[0006] To achieve the above purpose, the technical scheme adopted by the application is: The application provides a method for extracting 3,3'-dicatechin gallate theaflavins, which specifically comprises the following steps: (1) Pretreatment: the black tea and loquat leaves are crushed to pass through a 40-100 mesh sieve to obtain a black tea mixed powder; (2) Fermentation: the black tea mixed powder, a strain and purified water are mixed, oxygen is introduced at a flow rate of 0.05-0.3 L / min, static fermentation is performed for 15-25 h, and intermittent stirring is performed at 80-150 rpm for 4-10 min every 2-3 h to obtain a fermentation mixture; (3) Extraction: the fermentation mixture is mixed with an extracting agent, stirring is performed at 20-60℃ for 20-60 min, ultrasonic oscillation is performed for 2-8 min every 10-20 min during the stirring process, an ultrasonic frequency is 10-30 kHz, and filtration is performed to obtain an extract; (4) Purification: an equal volume of ethyl acetate is added to the extract, the mixture is shaken to separate into layers, the upper liquid is taken, rotary evaporation is performed until the weight is constant, then purified water at 5-15℃ is added, stirring is performed for 5-10 min, and filtration is performed to obtain a precipitate, the precipitate is washed with purified water at 5-15℃, and finally the product is vacuum freeze-dried to obtain the 3,3'-dicatechin gallate theaflavins.

[0007] The present application promotes the conversion of the target component structure in the tea to facilitate the extraction of TFDG by performing a preliminary fermentation treatment on the tea, lays a foundation for subsequent efficient extraction, extracts the target component from the fermented black tea at a lower temperature according to the physicochemical properties of TFDG with the prepared extracting agent, and finally obtains the target product through purification.

[0008] In some embodiments, in step (1), the mass ratio of the black tea and loquat leaves is 1:(0.1-0.4).

[0009] The present application co-ferments the black tea and loquat leaves, accelerates the oxidation of catechins through the coffee quinic acid in the loquat leaves and polyphenol oxidase, and improves the yield of TFDG.

[0010] In some embodiments, in step (2), the mass ratio of the black tea mixed powder and the strain is 1:(0.001-0.1).

[0011] In some embodiments, in step (2), the strain comprises Lactobacillus bulgaricus and Streptococcus thermophilus.

[0012] The present application obtains a metabolite through the synergistic effect of Lactobacillus bulgaricus and Streptococcus thermophilus, promotes the isomerization of catechins, and accelerates the generation of TFDG.

[0013] In some embodiments, in step (3), the mass ratio of the fermentation mixture and the extracting agent is 1:(5-10).

[0014] In some embodiments, in step (2), the static fermentation is first carried out at 25-30 DEG C for 1-2 hours, and then the temperature is lowered to 20-25 DEG C for 15-24 hours.

[0015] Compared with constant temperature fermentation, the present application inhibits side reactions during TFDG conversion by controlling temperature in sections, thereby improving the yield.

[0016] In some embodiments, in step (3), the preparation of the extracting agent is as follows: the betaine and lactic acid are stirred with purified water until transparent and uniform, thereby obtaining the extracting agent.

[0017] The extracting agent configured in the present application forms a stable hydrogen bond network, has high extraction efficiency and safety, has high matching degree with TFDG in terms of polarity parameter, has high mass transfer efficiency, and the hydrogen bond formed by the carboxyl group of lactic acid and the galloyl group of TFDG is stronger than the van der Waals force of conventional ethanol solvents, thereby further improving the extraction efficiency.

[0018] In some embodiments, the molar ratio of the betaine and lactic acid is (0.8-1.5):1.

[0019] In some embodiments, the content of purified water in the extracting agent is 25-35 wt%.

[0020] By controlling the water content of the extracting agent, the present application avoids the high viscosity hindering molecular diffusion on the one hand, and avoids the hydrogen bond network from being destroyed, so that the polarity mismatch leads to a decrease in the extraction rate.

[0021] In some embodiments, in step (4), the resin adsorption uses a polyamide resin with pH=4-6.

[0022] By optimizing the pre-fermentation and the extracting agent, the present application improves the content of TFDG in the extracting solution, so that a TFDG with high purity can be obtained by simple extraction and resin adsorption in the purification step, and the purification efficiency of this method is higher than that of the preparation grade thin layer chromatography and / or high performance liquid chromatography purification, thereby reducing the process cost.

[0023] In some embodiments, the preparation method obtains 3,3'-dicatechin gallate with a purity of ≥92%.

[0024] Finally in the purification step, the application adds ethyl acetate, shakes and separates the upper ethyl acetate phase containing lactic acid and TFDG and the lower water phase containing betaine and impurities. After removing the upper ethyl acetate by rotary evaporation, low-temperature purified water is added to the residue to dissolve lactic acid, and then filtration is performed to leave a water-soluble low theaflavin mixture. Finally, a high-purity TFDG is obtained by resin adsorption, and the remaining impurities are isomers of TFDG, such as theaflavin (Theaflavin-TF1), theaflavin-3-gallate (Theaflavin-3-gallate-TF2a), theaflavin-3'-gallate (Theaflavin-3'-gallate-TF2b), and the like.

[0025] The second aspect of the application provides the use of 3,3'-digallate theaflavin obtained by the above preparation method in health products.

[0026] In the design of health product formulations, the application scientifically compounds TFDG with other synergistic functional ingredients, so that the ingredients have synergistic effects, thereby effectively improving the health care effect of the health product.

[0027] Compared with the prior art, the application has the following beneficial effects: 1. The extraction method of 3,3'-digallate theaflavin provided by the application includes pretreatment, fermentation, extraction, and purification steps. In the overall process, the tea leaves are first subjected to preliminary fermentation treatment, the coffee acyl quinic acid is provided by loquat leaves to accelerate the oxidation of catechins by polyphenol oxidase, and the yield of TFDG is improved. Then, the synergistic effect of Lactobacillus bulgaricus and Streptococcus thermophilus is promoted to promote the isomerization of catechins and accelerate the generation of TFDG, thereby improving the yield. The combination of the two promotes the structural transformation of the target components in the tea leaves, which is conducive to the extraction of TFDG, and lays a foundation for subsequent efficient extraction.

[0028] 2. According to the physicochemical properties of TFDG, the target components are extracted from the fermented black tea at a lower temperature using an extraction agent that has high extraction efficiency and safety. The hydrogen bond formed between the lactic acid carboxyl group in the extraction agent and the gallate acyl group in TFDG is stronger than the van der Waals force of the conventional ethanol solvent, thereby enhancing the extraction efficiency.

[0029] 3. In the final purification step, the application improves the content of TFDG in the extraction liquid through preliminary fermentation and optimization of the extraction agent, so that high-purity TFDG can be obtained by simple extraction and resin adsorption. The purification efficiency of this method is higher than that of preparative thin-layer chromatography and / or high-performance liquid chromatography purification, thereby reducing the process cost. DETAILED DESCRIPTION

[0030] The present application will be described in detail below with reference to specific embodiments. It should be noted that the following examples are illustrative of specific embodiments of the present application and are not intended to limit the scope of the present application. Other combinations and sub-combinations of the disclosed features and elements, and other modifications, can occur to those skilled in the art without departing from the spirit or scope of the present application. It should be noted that the raw materials used in the following preparation examples and examples can be obtained from any manufacturer on the market unless otherwise specified.

[0031] Preparation Example 1 The preparation steps of the extraction agent A are as follows: 1 mol of betaine, 1 mol of lactic acid and purified water are stirred to be transparent and uniform, and the content of purified water is ensured to be 30 wt%, i.e. the extraction agent A is obtained.

[0032] Preparation Example 2 The preparation steps of the extraction agent B are as follows: The difference between this preparation example and Preparation Example 1 is that the amount of lactic acid is 0.7 mol.

[0033] Preparation Example 3 The preparation steps of the extraction agent C are as follows: The difference between this preparation example and Preparation Example 1 is that the amount of lactic acid is 1.6 mol.

[0034] Example 1 An extraction method of 3,3'-dicatechin gallate, specifically comprising the following steps: (1) Pretreatment: 8 kg of black tea and 2 kg of loquat leaves are mixed and crushed through an 80-mesh sieve to obtain a black tea mixed powder; (2) Fermentation: 10 kg of black tea mixed powder, 74 g of Lactobacillus bulgaricus and 26 g of Streptococcus thermophilus, and 50 kg of purified water are mixed, the oxygen flow rate is 0.1 L / min, and the fermentation is carried out at 27℃ for 1.5 h, and then the temperature is lowered to 22℃ for 19 h of fermentation, and every 2 h, the mixture is intermittently stirred at 120 rpm for 8 min, to obtain a fermentation mixture; (3) Extraction: the fermentation mixture is mixed with the extraction agent A at a mass ratio of 1:7, stirred at 40℃ for 40 min, and in the stirring process, ultrasonic oscillation is performed every 20 min for 5 min, the ultrasonic frequency is 25 kHz, and then filtration is performed, to obtain an extraction liquid; (4) Purification: an equal volume of ethyl acetate is added to the extraction liquid, the mixture is shaken to separate the layers, the upper liquid is taken, rotary evaporation is performed to a constant weight, then 10℃ of purified water is added, stirring is performed for 8 min, and then filtration is performed, to obtain a precipitate, the precipitate is washed with 10℃ of purified water, and finally, the product is vacuum freeze-dried through pH=5 polyamide resin adsorption, to obtain 3,3'-dicatechin gallate.

[0035] Example 2 The extraction method of 3,3'-dicatechin gallate in this example is the same as that in Example 1, except that step (2) is replaced by: (2) Fermentation: 10 kg of black tea mixed powder, 74 g of Lactobacillus bulgaricus and 26 g of Streptococcus thermophilus, and 50 kg of purified water were mixed, the oxygen flow rate was 0.1 L / min, and static fermentation was carried out at 25°C for 20 h, with intermittent stirring at 120 rpm for 8 min every 2 h to obtain a fermentation mixture.

[0036] Example 3 The extraction method of 3,3'-dicatechin gallate in this example is the same as that in Example 1, except that step (2) is replaced by: (2) Fermentation: 10 kg of black tea mixed powder, 74 g of Lactobacillus bulgaricus and 26 g of Streptococcus thermophilus, and 50 kg of purified water were mixed, the oxygen flow rate was 0.1 L / min, and static fermentation was carried out at 25°C for 20 h, with intermittent stirring at 120 rpm for 8 min every 2 h to obtain a fermentation mixture.

[0037] Example 4 The extraction method of 3,3'-dicatechin gallate in this example is the same as that in Example 1, except that step (2) is replaced by: (2) Fermentation: 10 kg of black tea mixed powder, 74 g of Lactobacillus bulgaricus and 26 g of Streptococcus thermophilus, and 50 kg of purified water were mixed, the oxygen flow rate was 0.1 L / min, and static fermentation was carried out at 25°C for 20 h, with intermittent stirring at 120 rpm for 8 min every 2 h to obtain a fermentation mixture.

[0038] Example 5 The extraction method of 3,3'-dicatechin gallate in this example is the same as that in Example 1, except that in step (3), the same amount of extractant B is used instead of extractant A.

[0039] Example 6 The extraction method of 3,3'-dicatechin gallate in this example is the same as that in Example 1, except that in step (3), the same amount of extractant C is used instead of extractant A.

[0040] Comparative Example 1 The extraction method of 3,3'-dicatechin gallate in this example is the same as that in Example 1, except that step (1) is replaced by: (1) Pretreatment: 8 kg of black tea mixed powder was crushed to 80 mesh to obtain black tea powder.

[0041] Comparative Example 2 The extraction method of 3,3'-digalloyl theaflavins in the present comparative example is the same as that in Example 1, except that the extraction agent A in step (3) is replaced by the same amount of anhydrous ethanol.

[0042] Performance test Liquid phase detection purity: the purity of 3,3'-digalloyl theaflavins obtained in each example and comparative example was tested by HPLC analysis method, and lactic acid residue test was also conducted. In the lactic acid residue test, if the detection amount is ≤5000 ppm (food grade), the data is marked as "N.D.".

[0043] Table 1 Performance test results As can be seen from the data in Table 1, the lactic acid content in 3,3'-digalloyl theaflavins of Examples 1-6 and Comparative Examples 1-2 meets the food grade standard, and can be added in health products for compounding with other active ingredients having synergistic effect to improve efficacy. The purity of TFDG in Example 1 is 92.7%, which indicates that the extraction method provided by the present application can obtain TFDG with high purity through simple extraction and resin adsorption by optimizing the pre-fermentation and extraction agent.

[0044] Compared with Example 1, Example 2 uses constant temperature static fermentation instead of stage cooling fermentation in the fermentation step, which is not conducive to inhibiting side reactions, resulting in a decrease in TFDG yield. Examples 3 and 4 use single strain in fermentation, which has less effect on the purification process, but affects the conversion of catechin to TFDG, resulting in a decrease in yield. Examples 5 and 6 change the amount of lactic acid used in the extraction agent, which may affect the selectivity of extraction, resulting in the retention of certain impurities in the product after purification. Comparative Example 1 does not add loquat leaves to black tea powder, and although the purity changes little, it is not conducive to the conversion of TFDG, resulting in a decrease in yield; Comparative Example 2 uses conventional anhydrous ethanol as the extraction agent, which reduces the matching degree with TFDG, resulting in a decrease in yield.

[0045] The above examples and comparative examples do not limit the present application in any form, although the present application has been disclosed as above with preferred embodiments, however, it is not intended to limit the present application, any skilled person in the art can make some changes or modifications to the above disclosed technical content without departing from the scope of the technical solution of the present application, and equivalent embodiments with equivalent changes are obtained, as long as they do not deviate from the technical solution of the present application, and any simple modification, equivalent change and modification of the above examples according to the technical essence of the present application are still within the scope of the technical solution of the present application.

Claims

1. A method for extracting 3,3'-digalloylflavonol, characterized by, Specifically comprising the following steps: (1) Pretreatment: crushing black tea and loquat leaves through a 40-100 mesh sieve to obtain a black tea mixture; (2) Fermentation: mixing the black tea mixture, a strain and purified water, with an oxygen flow rate of 0.05-0.3 L / min, static fermentation for 15-25 h, with intermittent stirring at 80-150 rpm for 4-10 min every 2-3 h to obtain a fermentation mixture; (3) Extraction: mixing the fermentation mixture with an extraction agent, stirring at 20-60℃ for 20-60 min, with ultrasonic oscillation for 2-8 min every 10-20 min during the stirring process, filtering to obtain an extract; (4) Purification: adding an equal volume of ethyl acetate to the extract, oscillating to separate the layers, taking the upper layer, rotary evaporation to constant weight, then adding purified water at 5-15℃, stirring for 5-10 min, filtering to obtain a precipitate, washing the precipitate with purified water at 5-15℃, and finally vacuum freeze-drying the product to obtain the 3,3'-dicatechin gallate theaflavins.

2. The extraction method of 3,3'-digalloyltheaflavin according to claim 1, characterized by, In step (1), the mass ratio of the black tea to the loquat leaves is 1:(0.1-0.4).

3. The extraction method of 3,3'-digalloyltheaflavin according to claim 1, characterized by, In step (2), the mass ratio of the black tea mixture to the strain is 1:(0.001-0.1).

4. The extraction method of 3,3'-digalloyltheaflavin according to claim 1, characterized by, In step (2), the strain comprises Lactobacillus bulgaricus and Streptococcus thermophilus.

5. The extraction method of 3,3'-digalloyltheaflavins according to claim 1, characterized by, In step (3), the mass ratio of the fermentation mixture to the extraction agent is 1:(5-10).

6. The extraction method of 3,3'-digalloyltheaflavins according to claim 1, characterized by, In step (2), the static fermentation is first carried out at 25-30℃ for 1-2 h, and then carried out at 20-25℃ for 15-24 h.

7. The extraction method of 3,3'-digalloyltheaflavins according to claim 1, characterized by, In step (3), the extraction agent is prepared by stirring betaine, lactic acid and purified water until they are transparent and uniform.

8. The extraction method of 3,3'-digalloyltheaflavin according to claim 7, characterized by, The molar ratio of the betaine to the lactic acid is (0.8-1.5):

1.

9. The extraction method of 3,3'-digalloyltheaflavin according to claim 7, characterized by, The content of the purified water in the extraction agent is 25-35 wt%.

10. The extraction method of 3,3'-digalloyltheaflavins according to claim 1, characterized by, In step (4), the resin adsorption uses a polyamide resin with a pH of 4-6.

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