Green preparation method and application of anti-glycation tea extract

Abstract

The invention relates to a green preparation method and application of an anti-saccharification tea extract, and belongs to the technical field of biology. The preparation method of the tea extract comprises the following steps: extracting tea by using a deep eutectic solvent to obtain the tea extract, and carrying out deproteinization and decoloration to obtain the tea extract. The hydrogen bond acceptor of the deep eutectic solvent comprises proline or betaine, and the hydrogen bond donor of the deep eutectic solvent comprises xylitol or glycerol. By utilizing the NaDES extraction technology, effective components can flow out by efficiently breaking cell walls, so that the extraction efficiency of tea active components represented by tea polyphenol is effectively improved; the selected NaDES solvent is natural, green and easy to obtain; the tea extract disclosed by the invention has an anti-saccharification effect, blocks glycosylation reaction, reduces and inhibits generation of AGEs, improves the problems of dark skin, skin laxity and aging caused by saccharification, reduces collagen loss, keeps skin elasticity and delays skin aging.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a green preparation method and application of an anti-glycation tea extract. Background Art

[0002] With the rapid economic development and the improvement of living standards, people pay more and more attention to body maintenance and facial skin care. According to investigations, due to external environments such as work pressure, eating habits and work and rest patterns, as well as endogenous factors such as oxidative stress, cell inflammation and aging, the skin care needs of the consumer group are becoming increasingly diverse. Anti-glycation, anti-oxidation, whitening and anti-aging are all key skin care needs of the current consumer group. Among them, anti-glycation skin care has received much attention in recent years, and the market share has gradually expanded.

[0003] As an essential nutrient for the human body, if sugar is ingested in excess or oxidized by the action of reactive oxygen species, it will combine with proteins in the body to undergo a glycation reaction, causing protein solidification, affecting melanin secretion, etc., and finally generating a series of advanced glycation end products - AGEs (glycation end products). Once AGEs are generated, they cannot be self-decomposed and excreted. After long-term accumulation, they will cause collagen and elastin to break and lose elasticity and support, form pigment adducts with proteins, make the skin become dull, yellow, loose, dry and rough, and accelerate the process of skin aging.

[0004] Under the background of "dual carbon", the ESG concept (environment, society and corporate governance) has begun to rise. Sustainable consumption and green products have gradually been recognized and concerned by the public. More and more consumers advocate "green skin care" and more recognize skin care products made from natural raw materials and free of harmful chemicals. Tea has a history of thousands of years in China and is an essential drink in people's daily life. It is rich in active ingredients such as tea polyphenols, amino acids, caffeine and tea polysaccharides, and has effects such as anti-oxidation, anti-inflammation, cholesterol reduction, weight loss, prevention of diabetes and heart disease. Investigations and studies have shown that tea extracts can inhibit the occurrence of glycation reactions by inhibiting the production of AGEs, thereby exerting an anti-glycation effect. At present, the extraction process of tea is mostly hot water extraction or organic solvent extraction, but hot water extraction will damage components such as tea polyphenols and flavonoids, and organic solvents are prone to residue and environmental pollution, which does not conform to the concept of green sustainable development. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a green preparation method and application of an anti-glycation tea extract.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for preparing a tea extract. Tea is extracted with a deep eutectic solvent to obtain a tea extract, and the tea extract is obtained by deproteinization and decolorization; the hydrogen bond acceptor of the deep eutectic solvent includes proline or betaine, and the hydrogen bond donor of the deep eutectic solvent includes xylitol or glycerol.

[0008] By selecting a deep eutectic solvent with a specific component ratio to extract tea, the active components in the obtained tea extract, including gallocatechin, epigallocatechin, epigallocatechin gallate, and epicatechin gallate, have high contents, have anti-glycation activity, and have no adverse reactions to the skin.

[0009] Furthermore, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is hydrogen bond acceptor: hydrogen bond donor = 1: (1 - 4).

[0010] In a specific embodiment of the present invention, in the deep eutectic solvent composed of proline and xylitol, the molar ratio of proline to xylitol is proline: xylitol = 1: 1; in the deep eutectic solvent composed of betaine and xylitol, the molar ratio of betaine to xylitol is betaine: xylitol = 1: 1; in the deep eutectic solvent composed of proline and glycerol, the molar ratio of proline to glycerol is proline: glycerol = 1: 4.

[0011] Furthermore, the mass ratio of the tea to the deep eutectic solvent is tea: deep eutectic solvent = 1: (10 - 60), more preferably 1: (19.8 - 20.2), and preferably 1: 20.

[0012] Furthermore, the preparation method of the deep eutectic solvent is: mixing the hydrogen bond acceptor and the hydrogen bond donor, reacting at 50 - 70 °C for 3 - 7 h, more preferably reacting at 59.8 - 60.2 °C for 5.8 - 6.2 h, and preferably reacting at 60 °C for 6 h.

[0013] Furthermore, the extraction temperature is 45 - 60 °C, and the time is 1.5 - 3 h. More preferably, the temperature is 59.8 - 60.2 °C, and the time is 2.3 - 2.7 h. Preferably, the temperature is 60 °C, and the time is 2.5 h.

[0014] Furthermore, after extraction, centrifuge at 7000 - 9000 rpm / min for 5 - 15 min, take the supernatant to obtain a tea extract; preferably centrifuge at 8000 rpm / min for 10 min.

[0015] Furthermore, the tea is dried to a constant weight at 50 - 60 °C, preferably dried to a constant weight at 55 °C.

[0016] In a specific embodiment of the present invention, after the tea is dried to a constant weight at 50 - 60 °C, it is pulverized to obtain tea powder.

[0017] Further, the mass concentration of the deep eutectic solvent is 5% to 60%, more preferably 4.8% to 5.2%, and preferably 5%.

[0018] Further, the type of the tea is at least one of white tea, green tea, dark tea, black tea, oolong tea, and yellow tea.

[0019] In a second aspect, the present invention also provides a tea extract prepared by the preparation method.

[0020] In a third aspect, the present invention also provides an application of the tea extract in the preparation of an anti-aging product.

[0021] Further, the anti-aging includes anti-glycation, which can significantly inhibit the generation of advanced glycation end products (AGEs), including carboxymethyllysine, thereby inhibiting matrix metalloproteinase-1, reducing the loss of collagen, maintaining skin elasticity, and delaying the skin aging process.

[0022] Further, the product includes cosmetics.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) Compared with the traditional extraction process, the NaDES extraction technology of the present invention can efficiently break the cell wall, so that the functional components flow out, effectively improving the extraction efficiency of tea active ingredients represented by tea polyphenols; and the NaDES solvent selected in the present invention is natural, green, easy to obtain, low in cost, and does not need to remove organic solvents, and can be widely used as a green solvent in food and cosmetics;

[0025] (2) The tea extract provided by the present invention has an anti-glycation effect, blocks the glycosylation reaction, reduces and inhibits the generation of AGEs, improves the problems of skin dullness, skin relaxation and aging caused by glycosylation, reduces collagen loss, maintains skin elasticity, and delays the skin aging process. Description of the Drawings

[0026] Figure 1 Shows the influence of different solvents on the extraction of tea extract;

[0027] Figure 2 Shows the anti-glycation activity of the tea extract obtained in Example 1; where A is the inhibition rate of AGEs production and B is the relative content of CML;

[0028] Figure 3 Shows the influence of the tea extract obtained in Example 1 on the content of MMP-1;

[0029] Figure 4 Shows the influence of the tea extract obtained in Example 1 on the collagen content. Detailed Embodiments

[0030] To better illustrate the objectives, technical solutions, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Other materials, reagents, etc. used in the embodiments can be obtained from commercial sources unless otherwise specified.

[0031] Example 1

[0032] (1) Dry white tea leaves in an environment of 55°C until constant weight, and crush to obtain tea powder;

[0033] (2) Prepare the NaDES solvent: Put proline and xylitol into the reactor according to a molar ratio of 1:1, heat at 60°C for 6 h to obtain a viscous liquid, and add a certain amount of deionized water to prepare a NaDES solvent with a total mass concentration of 5% of proline and xylitol;

[0034] (3) Put the tea powder in step (1) and the NaDES solvent into the extraction module of a high-efficiency continuous extraction device (the device disclosed in Chinese Patent CN216136732U, which is simple and efficient, directly connected to a deproteinization and decolorization module, and equipped with a centrifugal device, simplifies the process, reduces the loss of the liquid material, and reduces the material cost, suitable for large-scale production of products) according to a mass ratio of 1:20, adjust the extraction temperature to 60°C, the time to 2.5 h, centrifuge at 8000 rpm / min for 10 min, take the supernatant to obtain a tea extract; perform deproteinization and decolorization purification on the tea extract to obtain a tea extract.

[0035] Example 2

[0036] Replace proline in Example 1 with betaine, and keep other conditions unchanged to prepare a tea extract.

[0037] Example 3

[0038] Replace xylitol in Example 1 with glycerol, and set the molar ratio of proline to glycerol to 1:4, and keep other conditions unchanged to prepare a tea extract.

[0039] Example 4

[0040] Set the mass ratio of the tea powder to the NaDES solvent in Example 1 to 1:60, and set the extraction time to 3 h, and keep other conditions unchanged to prepare a tea extract.

[0041] Example 5

[0042] Set the mass concentration of the NaDES solvent in Example 1 to 60%, and keep other conditions unchanged to prepare a tea extract.

[0043] Example 6

[0044] Set the extraction temperature in Example 1 to 45 °C and the extraction time to 3 h, and keep other conditions unchanged to prepare tea extract.

[0045] Comparative Example 1

[0046] Replace the NaDES solvent in Example 1 with deionized water, and keep other conditions unchanged to prepare tea extract.

[0047] Comparative Example 2

[0048] Replace the NaDES solvent in Example 1 with absolute ethanol, perform protein deproteinization and decolorization on the tea extract, centrifuge, take the supernatant, rotary evaporate to remove ethanol, and add deionized water with the same volume as the initially added absolute ethanol to obtain the prepared tea extract, with other conditions unchanged.

[0049] Comparative Example 3

[0050] Replace xylitol in Example 1 with matrine, and keep other conditions unchanged to prepare tea extract.

[0051] Comparative Example 4

[0052] Replace proline and xylitol in Example 1 with betaine and citric acid, and keep other conditions unchanged to prepare tea extract.

[0053] Comparative Example 5

[0054] Replace proline in Example 1 with matrine, and keep other conditions unchanged to prepare tea extract.

[0055] Comparative Example 6

[0056] Replace proline and xylitol in Example 1 with betaine and sorbitol, set the molar ratio of betaine to sorbitol to 1:3, and keep other conditions unchanged to prepare tea extract.

[0057] Effect of Different Solvents on the Extraction of Tea Extract in Example 4

[0058] I. Experimental Method

[0059] 1. Draw a standard curve using gallic acid as the standard, and determine the content of tea polyphenols in the tea extracts obtained by extracting with different NaDES solvents in Examples 1-3 and Comparative Examples 1-6 through an ultraviolet spectrophotometer.

[0060] 2. Perform HPLC liquid phase detection and analysis on the tea extracts obtained by treating with different NaDES solvents, and use gallocatechin, epigallocatechin, epigallocatechin gallate, and epicatechin gallate as standard reference substances to compare the differences in the main components of the tea extracts.

[0061] II. Experimental Results

[0062] As Figure 1 shown, in the traditional water extraction process (Comparative Example 1), the content of tea polyphenols is 76.70 mg / g, and in the anhydrous ethanol extraction process (Comparative Example 2), the content of tea polyphenols is 21.97 mg / g. In the tea extract obtained by treating with the NaDES solvent, when the NaDES solvent is proline-xylitol (Example 1), the content of tea polyphenols can be as high as 87.48 mg / g, which indicates that the NaDES solvent selected in the present invention can efficiently extract the active components in tea and obtain a tea extract with high content of substances. However, the tea polyphenol extraction rates of the NaDES solvents composed of proline and matrine (Comparative Example 3), the NaDES solvent composed of betaine and citric acid (Comparative Example 4), and the NaDES solvent composed of matrine and xylitol (Comparative Example 5) are much lower than those of the traditional water extraction process. The tea polyphenol extraction rate (77.26 mg / g) in the NaDES solvent composed of betaine-sorbitol (Comparative Example 6) is similar to that of the traditional water extraction process (Comparative Example 1), but there is a large gap compared with Examples 1-6. This shows that the extraction of target components by the NaDES solvent is specific, and the composition ligands, molar ratios, dosage amounts, etc. of the NaDES solvent need to be adjusted in the early stage to obtain the best NaDES solvent to achieve the best extraction efficiency.

[0063] It can be concluded from Table 1 that the types of catechins extracted in Examples 1-6 and Comparative Examples 1-6 are relatively consistent. Compared with Comparative Examples 1-6, the contents of catechin components in Examples 1-6 after extraction with the NaDES solvent have all increased to a certain extent. Taking Comparative Example 1 as a comparison, the epigallocatechin gallate and epicatechin gallate in Example 1 are the most significant, rising from 0.8981 mg / mL and 0.1858 mg / mL to 1.3335 mg / mL and 0.2801 mg / mL respectively, with growth rates of 48.48% and 50.75% respectively. At the same time, by comparing the components in the tea extracts extracted with different NaDES solvents, it can be further shown that there are significant differences in the active components in the tea extracts treated with the NaDES solvents provided in Examples 1-6 of the present invention. Compared with the NaDES solvents treated in the comparative examples, the NaDES solvents provided in Examples 1-6 are more applicable. The differences in active ingredients also have a certain impact on the efficacy and activity of the tea extract.

[0064] Table 1 Component analysis of tea extracts extracted with different solvents

[0065]

[0066] Antiglycation activity of the tea extract of Example 5

[0067] I. Experimental method

[0068] A glycosylation reaction was constructed using fructose and BSA protein to verify the inhibitory effect of the tea extract obtained in Example 1 (with mass concentrations of 0.05%, 0.1%, 0.5%, 1% and 5%, prepared with PBS buffer) on the generation level of AGEs.

[0069] In sample group A1, 1 mL of sample solution and 1 mL of fructose solution were added. After mixing, the mixture was incubated in a 37°C constant temperature water bath for 2 h, and then 1 mL of BSA solution (4 mg / mL) was added. It was placed in a 37°C constant temperature incubator and incubated for 3 days. Then, the fluorescence intensity of the sample was measured under the conditions of an excitation wavelength of 370 nm and an emission wavelength of 440 nm. In sample background group A2, PBS buffer was used instead of fructose solution. In simulation system group A3, PBS buffer was used instead of sample solution. In BSA single incubation group A4, PBS buffer was used instead of sample solution and fructose solution. The inhibition rate of advanced glycation end products (in the following formula, A1, A2, A3, and A4 represent the fluorescence values of the corresponding groups):

[0070]

[0071] Human skin fibroblasts were treated with methylglyoxal (MGO) to establish a glycation model. The anti-glycation effect of the tea extracts with different concentrations obtained in Example 1 (with mass concentrations of 0.0625%, 0.125% and 0.25%, prepared with PBS buffer) was evaluated by detecting the concentration of carboxymethyllysine (CML) using a CML-ELISA kit.

[0072] Human skin fibroblasts were cultured in DMEM medium containing 10% (v / v) fetal bovine serum in an incubator (37°C, 5% CO2) to prepare a cell suspension with a density of 4×10 4 cells / mL. The cell suspension was inoculated into a 24-well plate and incubated for 18 - 24 h. Then, the original medium was discarded, 0.5 mmol / L MGO was added to construct a glycation model, and the negative control was not treated. Subsequently, different concentrations of sample solutions were added. The positive control group was added with 0.1 mmol / L aminoguanidine sulfate, and the negative control and model groups were not treated. After incubation for 48 h, the medium was discarded, the cells were washed with PBS, lysed at 4°C, and detected using a CML-ELISA kit.

[0073] II. Experimental Results

[0074] As Figure 2 shown in A, there is a concentration-dependent relationship between the inhibition rate of AGEs generation and the tea extract. After calculation, the IC 50 of the tea extract obtained in Example 1 for the ability to inhibit AGEs generation is 0.17%.

[0075] As Figure 2As shown in Figure B, tea extracts with mass concentrations of 0.0625%, 0.125%, and 0.25% can effectively inhibit the expression of CML, which is one of the structural forms of AGEs.

[0076] Based on the comprehensive experimental results of anti-glycation activity detection, the tea extract of Example 1 effectively inhibits the generation of AGEs, thereby exerting anti-glycation activity.

[0077] Effect of the tea extract of Example 6 on the content of MMP-1

[0078] I. Experimental method

[0079] Excessive AGEs are produced in the glycation reaction. AGEs will affect the activity of matrix metalloproteinases, activate MMP-1 (matrix metalloproteinase 1), and the overexpression of MMP-1 will exacerbate skin aging and collagen degradation. Tea extracts can inhibit the expression of MMP-1 by slowing down or inhibiting the production of AGEs.

[0080] Human skin fibroblasts were cultured in DMEM medium containing 10% (v / v) fetal bovine serum in an incubator (37°C, 5% CO2) for 24 h. Then, different concentrations of tea extracts (mass concentrations were 0.025% and 0.1%, prepared with PBS buffer) were added according to the experimental groups and incubated for 4 h. After the incubation, except for the negative control group, the others were irradiated with UVA (4.8 J / cm 2 ) irradiation. After the irradiation, the culture medium containing different concentrations of tea extracts was replaced with fresh medium. The negative control group and the model group were replaced with fresh medium, and the positive control group was replaced with fresh medium containing 10 ng / mL TGF-β1. After incubating for 24 h, the cell culture supernatant was collected for ELISA detection to calculate the content of MMP-1.

[0081] II. Experimental results

[0082] As Figure 3 shown, the relative content of MMP-1 decreased after treatment with the tea extracts obtained from Example 1 (mass concentrations of 0.025% and 0.1%, prepared with PBS buffer), showing a dose-dependent relationship, and the tea extract with a mass concentration of 0.1% inhibited the protease activity of MMP-1 similar to the positive control.

[0083] Effect of the tea extract of Example 7 on collagen

[0084] I. Experimental method

[0085] AGEs not only have a direct impact on matrix metalloproteinases such as MMP-1, but also have a negative impact on collagen through the MMP-1 pathway or directly, damaging collagen and elastic fibers. Therefore, in this invention, the anti-glycation activity of tea extract was further verified by detecting the content of type I collagen (collagen I).

[0086] Human skin fibroblasts were cultured in DMEM medium containing 10% (v / v) fetal bovine serum in an incubator (37 °C, 5% CO2) for 24 h. Then, according to the experimental groups, different concentrations of tea extract (mass concentrations were 0.025% and 0.1% respectively, prepared with PBS buffer) were added and incubated for 4 h. After the incubation, except for the negative control group, the rest were irradiated with UVA (30 / cm 2 ). After the irradiation, the medium was replaced with fresh medium containing different concentrations of tea extract. The negative control group and the model group were replaced with fresh medium, and the positive control group was replaced with fresh medium containing 10 ng / mL TGF-β1. After incubating for 24 h, the cell culture supernatant was collected for ELISA detection to calculate the content of type I collagen.

[0087] II. Experimental results

[0088] As Figure 4 shown, the type I collagen treated with the tea extract obtained in Example 1 could still maintain a collagen content similar to that of normal undamaged cells (negative control) after the model was constructed. It can be shown that during the glycation reaction, the tea extract effectively avoids the impact of collagen degradation indirectly caused by the glycation reaction, effectively maintains the skin collagen content, keeps the skin elastic, and delays the skin aging process.

[0089] Human patch test of the tea extract in Example 8

[0090] I. Experimental method

[0091] Using the tea extract obtained in Example 1 as the test substance, 30 volunteers were recruited and pasted on the flexor side of the arm. After 24 hours, the test substance was removed, and the skin reactions were observed at 0.5, 24, and 48 h, and the results were recorded according to the skin reaction grading standard in the "Technical Specifications for Cosmetics Safety" (2015 edition).

[0092] II. Experimental results

[0093] As shown in Table 2 and Table 3, 0 cases of skin adverse reactions occurred among 30 volunteers, and it can be regarded as a safe cosmetic raw material for use.

[0094] Table 2 Skin reaction grading standard for skin occlusive patch test

[0095]

[0096] Summary of the results of the human patch test for cosmetics

[0097]

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a tea extract, characterized in that: The tea is extracted with a deep eutectic solvent to obtain a tea extract, and the tea extract is obtained by deproteinization and decolorization; the hydrogen bond acceptor of the deep eutectic solvent comprises proline or betaine, and the hydrogen bond donor of the deep eutectic solvent comprises xylitol or glycerol.

2. The preparation method according to claim 1, characterized in that: The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is hydrogen bond acceptor: hydrogen bond donor = 1: (1-4).

3. The preparation method according to claim 1, characterized in that: The mass ratio of tea to deep eutectic solvent is tea:deep eutectic solvent=1:(10-60).

4. The preparation method according to claim 1, characterized in that: The preparation method of the deep eutectic solvent is: mixing the hydrogen bond acceptor and the hydrogen bond donor, and reacting at 50-70° C. for 3-7 hours.

5. The preparation method according to claim 1, characterized in that: The extraction temperature is 45-60° C. and the extraction time is 1.5-3 hours.

6. The preparation method according to claim 1, characterized in that: The mass concentration of the deep eutectic solvent is 5% to 60%.

7. A tea extract, characterized in that It is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the tea extract according to claim 7 in the preparation of anti-aging products.

9. The use according to claim 8, characterized in that: The anti-aging includes anti-glycation.

10. The use according to claim 8, characterized in that: The products include cosmetics.

Images