Green preparation method and application of anti-glycation tea extract

Abstract

The present application relates to a kind of green preparation method and application of anti-glycation tea extract, belong to the field of biotechnology.The preparation method of tea extract of the present application extracts tea with deep eutectic solvent, obtains tea extract, and obtains tea extract after deproteinization and decolorization;Hydrogen bond acceptor of the deep eutectic solvent includes proline or betaine, and hydrogen bond donor of the deep eutectic solvent includes xylitol or glycerol.The NaDES extraction technology of the present application can effectively break cell wall, make efficacy component flow out, effectively improve the extraction efficiency of tea active ingredient represented by tea polyphenol;NaDES solvent selected by the present application is natural, green, easy to obtain;The tea extract of the present application has anti-glycation efficacy, blocks glycosylation reaction, reduces and inhibits the generation of AGEs, improves the skin dullness, skin relaxation and aging problem caused by glycation, reduces collagen loss, maintains skin elasticity, delays skin aging.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a green preparation method and application of an anti-glycation tea extract. Background Technology

[0002] With rapid economic development and improved living standards, people are paying increasing attention to body maintenance and facial skincare. Surveys indicate that due to external environmental factors such as work pressure, dietary habits, and sleep patterns, as well as internal factors like oxidative stress, cellular inflammation, and aging, consumer skincare needs are becoming increasingly diversified. Anti-glycation, anti-oxidation, whitening, and anti-aging are among the key skincare demands of today's consumers. Among these, anti-glycation skincare has received significant attention in recent years, and its market share is gradually expanding.

[0003] Sugar, an essential nutrient for the human body, can lead to glycation if consumed in excess or oxidized by reactive oxygen species. This glycation process binds to proteins in the body, causing protein solidification, affecting melanin secretion, and ultimately generating a series of advanced glycation end products (AGEs). Once formed, AGEs cannot be broken down and eliminated naturally. Over time, they accumulate, causing collagen and elastin to break down, lose elasticity and support, and form pigment adducts with proteins, resulting in dull, loose, dry, and rough skin, accelerating the skin aging process.

[0004] Under the backdrop of "dual carbon" (carbon and energy), ESG (Environmental, Social, and Governance) principles are gaining popularity. Sustainable consumption and green products are gradually being recognized and valued by the public. More and more consumers are advocating "green skincare" and are increasingly accepting skincare products made from natural ingredients and free of harmful chemicals. Tea has a history of thousands of years in my country and is an essential beverage in people's daily lives. It is rich in active ingredients such as tea polyphenols, amino acids, caffeine, and tea polysaccharides, and has antioxidant, anti-inflammatory, cholesterol-lowering, weight-loss, and diabetes and heart disease prevention effects. Research shows that tea extracts can inhibit the production of AGEs, thereby inhibiting glycation reactions and exerting anti-glycation effects. Currently, tea extraction processes are mostly hot water extraction or organic solvent extraction. However, hot water extraction can destroy components such as tea polyphenols and flavonoids, while organic solvents are prone to residues and environmental pollution, which is inconsistent with the concept of green and sustainable development. Summary of the Invention

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

[0006] To achieve the above objectives, 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, wherein tea is extracted with a deep eutectic solvent to obtain a tea extract, and the extract is obtained by deproteinization and decolorization; wherein 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] This invention extracts tea using a deep eutectic solvent with a specific ratio of components. The resulting tea extract contains high levels of active components, including gallocatechin, epigallocatechin, epigallocatechin gallate, and epicatechin gallate, which exhibit anti-glycation activity and have no adverse effects on 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, the molar ratio of proline to xylitol in the eutectic solvent composed of proline and xylitol is proline:xylitol = 1:1; the molar ratio of betaine to xylitol in the eutectic solvent composed of betaine and xylitol is betaine:xylitol = 1:1; and the molar ratio of proline to glycerol in the eutectic solvent composed of proline and glycerol is proline:glycerol = 1:4.

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

[0012] Furthermore, the method for preparing the deep eutectic solvent is as follows: the hydrogen bond acceptor and the hydrogen bond donor are mixed and reacted at 50-70°C for 3-7 hours, more preferably at 59.8-60.2°C for 5.8-6.2 hours, and preferably at 60°C for 6 hours.

[0013] Further, the extraction temperature is 45-60℃ and the time is 1.5-3h, more preferably the temperature is 59.8-60.2℃ and the time is 2.3-2.7h, and preferably the temperature is 60℃ and the time is 2.5h.

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

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

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

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

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

[0019] Secondly, the present invention also provides a tea extract prepared by the aforementioned preparation method.

[0020] Thirdly, the present invention also provides the application of the tea extract in the preparation of anti-aging products.

[0021] Furthermore, the anti-aging effect includes anti-glycation. It can significantly inhibit the formation of advanced glycation end products (AGEs), including carboxymethyl lysine, thereby inhibiting matrix metalloproteinase-1, reducing collagen loss, maintaining skin elasticity, and delaying the skin aging process.

[0022] Furthermore, the products include cosmetics.

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

[0024] (1) Compared with traditional extraction processes, the present invention utilizes NaDES extraction technology to efficiently break down cell walls, allowing the active components to flow out, thus effectively improving the extraction efficiency of tea active ingredients, represented by tea polyphenols; and the NaDES solvent used in the present invention is natural, green, readily available, and inexpensive, and does not require the removal of 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 anti-glycation effect, blocks glycation reaction, reduces and inhibits the production of AGEs, improves skin dullness, skin laxity and aging caused by glycation, reduces collagen loss, maintains skin elasticity and delays the skin aging process. Attached Figure Description

[0026] Figure 1 The effect of different solvents on the extraction of tea extracts;

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

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

[0029] Figure 4 The effect of the tea extract obtained in Example 1 on collagen content. Detailed Implementation

[0030] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, other materials and reagents used in the embodiments are commercially available.

[0031] Example 1

[0032] (1) Dry the white tea leaves at 55℃ to constant weight and then pulverize them to obtain tea powder;

[0033] (2) Preparation of NaDES solvent: Proline and xylitol were added to the reactor in a molar ratio of 1:1 and heated at 60°C for 6 hours to obtain a viscous liquid. A certain amount of deionized water was added to prepare NaDES solvent with a total mass concentration of 5% for proline and xylitol.

[0034] (3) The tea powder and NaDES solvent from step (1) are added to the extraction module of a high-efficiency continuous extraction device (disclosed in Chinese patent CN216136732U, which is simple and efficient, directly connected to the deproteinization and decolorization module, and equipped with a centrifuge device, which simplifies the process, reduces material loss, and lowers material costs, and is suitable for large-scale production of products) at a mass ratio of 1:20. The extraction temperature is controlled at 60℃, the time is 2.5h, and the mixture is centrifuged at 8000rpm / min for 10min. The supernatant is taken to obtain the tea extract. The tea extract is then deproteinized and decolorized to obtain the tea extract.

[0035] Example 2

[0036] In Example 1, proline was replaced with betaine, and other conditions remained unchanged to prepare a tea extract.

[0037] Example 3

[0038] In Example 1, xylitol was replaced with glycerol, and the molar ratio of proline to glycerol was set to 1:4, while other conditions remained unchanged, to prepare a tea extract.

[0039] Example 4

[0040] The tea extract was prepared by setting the mass ratio of tea powder to NaDES solvent in Example 1 to 1:60, the extraction time to 3 hours, and keeping other conditions unchanged.

[0041] Example 5

[0042] The NaDES solvent concentration in Example 1 was set to 60%, and other conditions remained unchanged to prepare tea extract.

[0043] Example 6

[0044] The extraction temperature in Example 1 was set to 45°C and the extraction time was set to 3 hours, while other conditions remained unchanged, to prepare tea extract.

[0045] Comparative Example 1

[0046] The tea extract was prepared by replacing the NaDES solvent in Example 1 with deionized water while keeping other conditions unchanged.

[0047] Comparative Example 2

[0048] The NaDES solvent in Example 1 was replaced with anhydrous ethanol to deproteinize and decolorize the tea extract. After centrifugation, the supernatant was collected, and the ethanol was removed by rotary evaporation. An equal volume of deionized water as initially added anhydrous ethanol was added to obtain the prepared tea extract, with other conditions remaining unchanged.

[0049] Comparative Example 3

[0050] By replacing xylitol with matrine in Example 1, and keeping other conditions unchanged, a tea extract was prepared.

[0051] Comparative Example 4

[0052] Tea extract was prepared by replacing proline and xylitol in Example 1 with betaine and citric acid, while keeping other conditions unchanged.

[0053] Comparative Example 5

[0054] By replacing proline with matrine in Example 1, and keeping other conditions unchanged, a tea extract was prepared.

[0055] Comparative Example 6

[0056] In Example 1, proline and xylitol were replaced with betaine and sorbitol, and the molar ratio of betaine to sorbitol was set to 1:3. Other conditions remained unchanged, and tea extract was prepared.

[0057] Example 4: Effect of different solvents on the extraction of tea extract

[0058] I. Experimental Methods

[0059] 1. A standard curve was plotted using gallic acid as a standard, and the content of tea polyphenols in the tea extracts extracted with different NaDES solvents in Examples 1-3 and Comparative Examples 1-6 was determined by ultraviolet spectrophotometer.

[0060] 2. The tea extracts obtained by treatment with different NaDES solvents were analyzed by HPLC. Gallocatechin, epigallocatechin, epigallocatechin gallate and epicatechin gallate were used as standard references to compare the differences in the main components of the tea extracts.

[0061] II. Experimental Results

[0062] like Figure 1 As shown, the tea polyphenol content in the traditional water extraction process (Comparative Example 1) is 76.70 mg / g, and the tea polyphenol content in the anhydrous ethanol extraction process (Comparative Example 2) is 21.97 mg / g. However, the tea extract obtained by treatment with NaDES solvent (proline-xylitol, Example 1) has a tea polyphenol content as high as 87.48 mg / g. This indicates that the NaDES solvent selected in this invention can efficiently extract the active components in tea and obtain a tea extract with high content. However, the extraction rates of tea polyphenols using NaDES solvents composed of proline and matrine (Comparative Example 3), betaine and citric acid (Comparative Example 4), and matrine and xylitol (Comparative Example 5) were significantly lower than those using traditional water extraction. The extraction rate of tea polyphenols using NaDES solvent composed of betaine and sorbitol (Comparative Example 6) (77.26 mg / g) was similar to that of traditional water extraction (Comparative Example 1), but significantly lower than that of Examples 1-6. This indicates that NaDES solvent extraction of the target component is specific, and the optimal NaDES solvent composition, ligand composition, molar ratio, and dosage must be adjusted in the early stages to obtain the best extraction efficiency.

[0063] Table 1 shows that the types of catechins extracted in Examples 1-6 and Comparative Examples 1-6 are quite consistent. Compared with Comparative Examples 1-6, the content of catechin components after extraction with NaDES solvent in Examples 1-6 all showed a certain increase. Among them, taking Comparative Example 1 as a comparison, the epigallocatechin gallate and epicatechin gallate in Example 1 showed the most significant increases, 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%. Furthermore, comparing the components in tea extracts extracted with different NaDES solvents further demonstrates that the active components in the tea extracts treated with NaDES solvent provided in Examples 1-6 of this invention have significant differences. Compared with the NaDES solvent treatment in the comparative examples, the NaDES solvent provided in Examples 1-6 is more suitable. The differences in active components also have a certain impact on the efficacy and activity of the tea extracts.

[0064] Table 1. Component analysis of tea extracts after extraction with different solvents.

[0065]

[0066] Example 5: Anti-glycation activity of tea extract

[0067] I. Experimental Methods

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

[0069] For sample group A1, 1 mL of sample solution and 1 mL of fructose solution were added, mixed, and incubated in a 37°C water bath for 2 hours. Then, 1 mL of BSA solution (4 mg / mL) was added, and the mixture was incubated in a 37°C incubator for 3 days. The fluorescence intensity of the samples was then measured at an excitation wavelength of 370 nm and an emission wavelength of 440 nm. For sample background group A2, PBS buffer was used instead of fructose solution; for simulated system group A3, PBS buffer was used instead of sample solution; and for BSA-only incubation group A4, PBS buffer was used instead of both sample solution and fructose solution. The inhibition rate of advanced glycation end products (A1, A2, A3, and A4 in the following formulas represent the fluorescence values ​​of the corresponding groups) is calculated as follows:

[0070]

[0071] Human skin fibroblasts were treated with methylglyoxal (MGO) to induce glycation, and the anti-glycation effect of different concentrations of tea extract obtained in Example 1 (mass concentrations of 0.0625%, 0.125%, and 0.25%, prepared with PBS buffer) was evaluated by detecting the concentration of carboxymethyl lysine (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 cells at a density of 4 × 10⁶ cells / year. 4 Cell suspensions of 100 cells / mL were seeded into 24-well plates and incubated for 18–24 h. The plates were then removed, the original culture medium was discarded, and 0.5 mmol / L MgO was added to construct a glycosylation model. The negative control group received no treatment. Subsequently, different concentrations of sample solutions were added. The positive control group received 0.1 mmol / L aminoguanidine sulfate, while the negative control and model groups received no treatment. Cells were incubated for 48 h. After 48 h, the culture medium was discarded, cells were washed with PBS, lysed at 4°C, and detected using a CML-ELISA kit.

[0073] II. Experimental Results

[0074] like Figure 2 As shown in Figure A, the inhibition rate of AGEs formation is concentration-dependent with respect to the tea extract. The IC50 of the tea extract obtained in Example 1 for inhibiting AGEs formation was calculated. 50 At 0.17%.

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

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

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

[0078] I. Experimental Methods

[0079] Glycation produces excessive AGEs, which affect the activity of matrix metalloproteinases (MMP-1). MMP-1 overexpression exacerbates skin aging and collagen degradation. Tea extract can inhibit MMP-1 expression by slowing down or suppressing the production of AGEs.

[0080] Human skin fibroblasts were cultured in DMEM medium containing 10% (v / v) fetal bovine serum for 24 h in an incubator (37°C, 5% CO2). Then, different concentrations of tea extract (0.025% and 0.1% by mass, prepared with PBS buffer) were added according to the experimental groups, and incubated for 4 h. After incubation, except for the negative control group, the remaining cells were subjected to UVA (4.8 J / cm²) incubation. 2 Irradiation was performed, and after irradiation, the culture medium was replaced with fresh medium containing different concentrations of tea extract. The negative control group and model group were replaced with fresh medium, while the positive control group was replaced with fresh medium containing 10 ng / mL TGF-β1. The cells were incubated for 24 hours. After incubation, the cell culture supernatant was collected and analyzed by ELISA to calculate the MMP-1 content.

[0081] II. Experimental Results

[0082] like Figure 3 As shown, the relative content of MMP-1 decreased after treatment with the tea extracts obtained in Example 1 (0.025% and 0.1% by mass, prepared with PBS buffer) in a dose-dependent manner, and the inhibition of MMP-1 protease activity by the 0.1% tea extract was similar to that of the positive control.

[0083] Example 7: Effects of Tea Extract on Collagen

[0084] I. Experimental Methods

[0085] AGEs not only directly affect matrix metalloproteinases such as MMP-1, but also negatively impact collagen through the MMP-1 pathway or a direct pathway, damaging collagen and elastin fibers. Therefore, this invention further verifies the anti-glycation activity of tea extract by detecting the content of type I collagen.

[0086] Human skin fibroblasts were cultured in DMEM medium containing 10% (v / v) fetal bovine serum for 24 h in an incubator (37°C, 5% CO2). Then, different concentrations of tea extract (0.025% and 0.1% by mass, prepared with PBS buffer) were added according to the experimental groups, and incubated for 4 h. After incubation, except for the negative control group, the remaining cells were subjected to UVA (30 / cm²) incubation. 2 Irradiation was performed, and after irradiation, the culture medium was replaced with fresh medium containing different concentrations of tea extract. The negative control group and model group were replaced with fresh culture medium, while the positive control group was replaced with fresh culture medium containing 10 ng / mL TGF-β1. The cells were incubated for 24 hours. After incubation, the cell culture supernatant was collected and analyzed by ELISA to calculate the type I collagen content.

[0087] II. Experimental Results

[0088] like Figure 4 As shown, the type I collagen treated with the tea extract obtained in Example 1 can still maintain a collagen content similar to that of normal undamaged cells (negative control) after model construction. This indicates that when glycation occurs, the tea extract effectively avoids the collagen degradation indirectly caused by glycation, effectively maintains skin collagen content, preserves skin elasticity, and delays the skin aging process.

[0089] Example 8: Human patch test of tea extract

[0090] I. Experimental Methods

[0091] Using the tea extract obtained in Example 1 as the test substance, 30 volunteers were recruited to apply the test substance to the flexor side of their arms. The test substance was removed after 24 hours, and the skin reaction was observed after 0.5, 24 and 48 hours. The results were recorded according to the skin reaction grading standard in the "Cosmetic Safety Technical Specifications" (2015 edition).

[0092] II. Experimental Results

[0093] As shown in Tables 2 and 3, 0 of the 30 volunteers experienced adverse skin reactions, indicating that the raw materials are safe for use in cosmetics.

[0094] Table 2. Grading Criteria for Skin Reactions in Closed Patch Tests

[0095]

[0096] Table 3 Summary of Cosmetic Human Patch Test Results

[0097]

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention 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, Tea was extracted using a deep eutectic solvent to obtain a tea extract, which was then subjected to deproteinization and decolorization to obtain a tea extract; the hydrogen bond acceptor of the deep eutectic solvent was proline, and the hydrogen bond donor of the deep eutectic solvent was xylitol. The active components in the obtained tea extract include gallocatechin, epigallocatechin, epigallocatechin gallate and epicatechin gallate; Adding water yields a deep eutectic solvent with a mass concentration of 5%. The molar ratio of proline to xylitol is: proline: xylitol = 1:

1.

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

3. The preparation method according to claim 1, characterized in that, The method for preparing the deep eutectic solvent is as follows: mix the hydrogen bond acceptor and the hydrogen bond donor, and react at 50-70°C for 3-7 hours.

4. 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.

5. A tea extract, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 4.

6. The use of the tea extract according to claim 5 in the preparation of anti-glycation products.

7. The application according to claim 6, characterized in that, The products include cosmetics.

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