Black tea extract and application thereof in aspect of improving inflammation

Abstract

The invention discloses a black tea extract and application thereof in improving inflammation, and belongs to the field of skin care products. According to the method, a natural eutectic solvent system is adopted for the first time, and active ingredients of black tea leaves, tea stems and tea shells are synergistically extracted. Extraction process parameters are optimized through a response surface method, a green and efficient novel extraction method is established, the method can remarkably improve the yield of active ingredients such as total phenols, flavones and theanine in traditional waste-tea stems and tea shells, and the extraction efficiency of the method is remarkably superior to that of a traditional water extraction method. Further cell experiments prove that the obtained tea shell extract has excellent anti-inflammatory activity and can effectively inhibit expression of IL-6 in macrophages. Therefore, the invention not only provides an innovative technology for extraction of active ingredients of black tea, but also opens up a new way for high-value resource utilization of black tea processing byproducts, and particularly has a wide application prospect in the aspect of developing cosmetics or health care products with anti-oxidation and anti-inflammatory functions.

Description

TECHNICAL FIELD

[0001] The present application relates to black tea extract and its application in improving inflammation, belonging to the field of skin care products. BACKGROUND

[0002] In modern society, a variety of unhealthy living habits such as high-sugar and high-oil diet, irregular work and rest, persistent mental stress, and improper skin care methods, are increasingly becoming important inducements to damage the skin barrier function, cause microecological imbalance, and activate inflammatory pathways, directly leading to skin manifestations such as redness, sensitivity, acne recurrence, and premature aging. This modern pathological mechanism is in line with the discussion of inflammatory state in "Shengjing" and "hot skin" in "Huangdi Neijing".

[0003] In recent years, with the increasing demand for skin repair and the in-depth study of skin inflammation mechanism, the active ingredients in black tea have gradually attracted widespread attention. Black tea is rich in tea polyphenols (especially theaflavins and thearubigins), polysaccharides, theanine, and other functional ingredients, which show significant effects in anti-inflammatory, antioxidant, antibacterial, and immune regulation. Studies have shown that black tea extract can effectively inhibit the expression of inflammatory factors, relieve skin irritation, and enhance skin barrier function. Based on the systematic extraction and whole ingredient analysis of its functional ingredients, researchers have further verified the potential and mechanism of black tea active ingredients in soothing skin inflammation and improving skin sensitivity through molecular docking experiments and in vitro detection of inflammatory factors, providing a scientific basis for its development and application in repair-type skin care products.

[0004] The current utilization of black tea resources is severely wasted. During the refining process of black tea, about 5% to 10% of tea stems and tea shells and other by-products are produced. These parts are rich in active ingredients similar to high-grade tea leaves, but due to the low efficiency of traditional extraction technology, their value has not been effectively tapped, and most of them are discarded as agricultural waste or used in a low-value extensive way, which not only causes great waste of resources but also brings environmental pressure. Therefore, developing a technology that can efficiently extract high-value active ingredients from these by-products is a key problem to be solved for realizing the full utilization of black tea resources and promoting the sustainable development of the industry.

[0005] However, the bottleneck of extraction technology seriously restricts the maximization of the efficacy of black tea active ingredients (especially those in tea stems and tea shells) in the field of skin care. Currently, the mainstream extraction process has significant defects: Traditional tea leaf extraction process mainly relies on solid-liquid extraction (SLE) technology, which involves solvent use and solute leaching. Based on the principle of "like dissolves like", the target ingredients in tea leaves are extracted into solvents with polar or non-polar solvents to separate the target ingredients from tea leaves.

[0006] Conventional extraction of tea active substances is usually carried out by hot or cold water extraction. During the process, extraction temperature and time are the key factors affecting the concentration of the extract. High temperature is conducive to the wetting of tea samples and can improve the solubility of active substances, but long-term hot water extraction can easily cause thermal degradation of active substances. Cold water can reduce the risk of thermal degradation, but it takes a long time to extract and is more likely to be contaminated by microorganisms, which limits the application of active substances in the food and pharmaceutical industries to some extent.

[0007] In addition to traditional water extraction, organic solvent extraction, steam distillation, and reflux extraction are also widely used. Compared with water solvent, organic solvent has a higher extraction rate, but it can introduce impurities and the solvent has certain toxicity, which may pollute the environment. Steam distillation, as a traditional method for extracting essential oils and phenolic substances, involves hydrolysis, water diffusion, and heating decomposition steps. This method takes a long time, has a high temperature, and consumes a lot of energy, which is not suitable for compounds with poor thermal stability. Reflux extraction is a process of repeated evaporation and condensation of solvent at a constant temperature. The temperature selection depends on the solvent used, and it has the characteristics of less solvent requirement, short extraction time, and high efficiency.

[0008] In recent years, the use of green extraction technology to separate active ingredients from natural plants has become a trend. Compared with traditional methods, green extraction technology has many advantages, such as reducing solvent and energy consumption, and reducing time cost. Such innovative technologies include pressurized liquid extraction (PLE), supercritical fluid extraction (SFE), ultrasonic-assisted extraction (UAE), enzyme-assisted extraction (EAE), and deep eutectic solvent (DES) extraction (which can be used alone or in combination with the above technologies), among which DES opens up a more green and efficient path for tea extraction.

[0009] Deep eutectic solvent extraction (DES) is a new type of ionic liquid analogue, which is a low-melting mixture formed by hydrogen bond acceptors and hydrogen bond donors. Compared with traditional solvents, DES has the characteristics of environmental protection, stability, low volatility, easy synthesis, wide polarity range, and strong designability. DES can effectively control the solvent properties, solute hydrogen bond formation ability, and interaction strength by adjusting the molar ratio and type of hydrogen bond acceptors and hydrogen bond donors. In recent years, DES has been used to extract various natural products such as phenols and terpenes. Studies have shown that compared with traditional methods, the total phenol content, catechin concentration, and antioxidant activity of the extract are significantly improved when UAE-DES is applied to tea extraction. However, existing DES research mostly focuses on high-quality tea, and its extraction potential for waste by-products such as tea stems and tea shells is severely overlooked. At the same time, the process of screening and synthesizing efficient DES formulations is complex, and the cost of raw materials is higher than that of traditional solvents. Moreover, many DES components are not clearly included in the cosmetics regulations, and there is a lack of long-term skin safety data, which directly hinders their application in terminal skin care products.

[0010] Therefore, there is an urgent need in the art to develop a new extraction process that can overcome the above-mentioned defects. This process is green, efficient, and low-cost, and can selectively enrich the core anti-inflammatory active ingredients in black tea. More importantly, it is also suitable for low-value by-products such as tea stems and tea shells, which can be converted into safe, efficient, and suitable raw materials for high-end skincare products, thereby truly realizing the full-value utilization of black tea resources. SUMMARY

[0011] To solve the above technical problems, the present application intends to provide a method for extracting black tea extract based on natural deep eutectic solvent combined with ultrasonic extraction. The DES components used are substances commonly used in cosmetic formulations, which are environmentally friendly and can efficiently extract active substances such as alkaloids, flavonoids, total phenols, and theanine from different parts of black tea, including tea leaves, tea stems, and tea shells.

[0012] The first object of the present application is to provide a method for preparing black tea extract using deep eutectic solvent, the method steps are as follows: S1: Preparation of natural deep eutectic solvent aqueous solution: mix deep eutectic solvent with water uniformly to prepare natural deep eutectic solvent aqueous solution; S2: Ultrasonic extraction: add black tea powder to the natural deep eutectic solvent aqueous solution prepared in S1, then ultrasonic extraction, centrifugal collection of supernatant to obtain black tea extract; Among them, the natural deep eutectic solvent includes one of glucose-lactic acid, betaine-citric acid, proline-malic acid, and betaine-glycerol.

[0013] In an embodiment of the present application, in S1, the water content of the natural deep eutectic solvent aqueous solution is 15% to 25%.

[0014] In an embodiment of the present application, in S1, the preparation method of the natural deep eutectic solvent is: Mix glucose and lactic acid according to a molar ratio of 1:(4-6), stir at 75-85℃ for 2.5-3.5 h to obtain a glucose-lactic acid natural deep eutectic solvent; or, Mix betaine and citric acid according to a molar ratio of 1:(1-3), stir at 75-85℃ for 2.5-3.5 h to obtain a betaine-citric acid natural deep eutectic solvent; or, Mix proline and malic acid according to a molar ratio of 1:(1-3), stir at 75-85℃ for 2.5-3.5 h to obtain a proline-malic acid natural deep eutectic solvent; or, Mix betaine and glycerol according to a molar ratio of 1:(1-3), stir at 75-85℃ for 2.5-3.5 h to obtain a betaine-glycerol natural deep eutectic solvent.

[0015] In one embodiment of the present application, in S1, the black tea powder is prepared by freeze-drying or drying the black tea raw material, and then crushing and sieving the black tea raw material; the black tea raw material comprises at least one of tea leaves, tea stems and tea shells.

[0016] In one embodiment of the present application, the black tea is sieved through a 40-mesh sieve.

[0017] In one embodiment of the present application, the black tea is selected from Qimen black tea, Yunnan black tea, Yingde black tea and Zhengshan Xiaozhong.

[0018] In one embodiment of the present application, in S2, the ratio of the black tea powder to the eutectic solvent aqueous solution is 1 g: (15-30) mL.

[0019] In one embodiment of the present application, in S2, the ultrasonic extraction is performed at 50-70℃ and 20-80 Hz for 40-100 min.

[0020] In one embodiment of the present application, in S2, the centrifugation is performed at 8000-12000 rpm for 5-20 min.

[0021] In one embodiment of the present application, the black tea extract contains total phenols, flavonoids, alkaloids and theanine.

[0022] A second object of the present application is to provide a black tea extract prepared by the above method.

[0023] In one embodiment of the present application, the black tea extract contains total phenols, flavonoids, alkaloids and theanine.

[0024] A third object of the present application is to provide an application of the method or the black tea extract in preparing a cosmetic product having the functions of anti-inflammation, anti-oxidation and glycosylation.

[0025] A fourth object of the present application is to provide a product containing the black tea extract, which is a cosmetic product or a pharmaceutical product.

[0026] In one embodiment of the present application, the black tea extract is added in an amount of 1%-10% of the total mass of the product.

[0027] In one embodiment of the present application, the cosmetic product is in any one of the forms of a dressing, an ointment, a cream, a lotion, a spray, a cream, a water, a gel, an oil, a patch, a film, a mud, a powder, a solution and a film.

[0028] [Advantages] The application utilizes different parts of black tea (including tea leaves, tea stems and tea shells) to extract active ingredients in black tea under a natural deep eutectic solvent system. The application significantly improves the extraction yield of total phenols, flavonoids, alkaloids and theanine, thereby converting these agricultural processing by-products into high-value raw materials, greatly improving the comprehensive utilization efficiency and economic value of black tea resources. 1. High-value utilization of black tea by-products: The application utilizes the high extraction capacity of natural deep eutectic solvent system (DES) for black tea resources, especially the traditionally neglected or discarded tea stems and tea shells. Compared with traditional extraction methods, the application can significantly improve the yield of total phenols, flavonoids, alkaloids and theanine in tea stems and tea shells, and the extraction efficiency is significantly improved compared with traditional water extraction and alcohol extraction (Table 1). After process optimization, the total phenols, flavonoids, alkaloids, theanine and active substances in the black tea leaf extract are 106 mg / g, 67 mg / g, 56 mg / g, 17 mg / g and 248 mg / g, respectively. The low-value agricultural by-products are successfully converted into high-value raw materials rich in active ingredients, significantly improving the comprehensive utilization efficiency of black tea resources.

[0029] 2. Green, efficient and synergistically optimized extraction process: The molar ratio of DES, water content of DES, extraction temperature, time and solid-liquid ratio were optimized and modeled by response surface method (see Figure 1 、 2 ), and the optimal extraction conditions were established. This process not only avoids the use of organic solvents, but also meets the requirements of green chemistry. Moreover, there is a significant synergistic effect between the factors, achieving efficient and stable extraction of active ingredients under mild conditions.

[0030] 3. Outstanding antioxidant, anti-sugar and anti-inflammatory potential: In vitro antioxidant experiments confirmed ( Figure 3 ), the extracts of different parts of black tea obtained by the application all showed strong free radical scavenging ability, among which the DPPH and ABTS free radical scavenging rates of tea shell extract were particularly outstanding, with IC50 values of 0.92 mg / mL and 1.61 mg / mL, respectively. It also has a significant anti-sugar effect, and the inhibition rate of black tea leaf extract at a concentration of 1 mg / mL is 48.5%. Cell experiment results show that ( Figure 4 、 5 ), within the safe concentration range, the black tea extract of the application, especially the extract from tea shells and tea stems, can significantly inhibit the release of key inflammatory factor IL-6 in LPS-induced RAW264.7 macrophages (the inhibition rate can be as high as 68.6%). This fully proves that these extracts from "waste" have great development and application value in skin inflammation relief and skin barrier repair in the skin care field. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 : Effects of different factors on the total extraction rate of active substances in black tea, (A) the effect of molar ratio, (B) the effect of water content, (C) the effect of extraction time, (D) the effect of extraction temperature, (E) the effect of solid-liquid ratio; Figure 2 : 3D / 2D response surface plots of the effects of the interaction of each factor of DES extraction method on the total extraction rate of active substances in black tea, (A-B) extraction temperature / solid-liquid ratio, (C-D) extraction time / extraction temperature, (E-F) extraction time / solid-liquid ratio; Figure 3 : Antioxidant performance of extracts from different parts of black tea: (A) DPPH free radical scavenging rate, (B) ABTS free radical scavenging rate Figure 4 : Effects of extracts from different parts of black tea on the activity of RAW264.7 macrophages, (A) black tea leaf extract, (B) tea shell extract, (C) tea stem extract; Figure 5 : Effects of extracts from different parts of black tea on the levels of inflammatory factors in RAW264.7 macrophages, (A) IL-6 inflammatory factor content, (B) TNF-α inflammatory factor content. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are for better explaining the present application and are not used to limit the present application.

[0033] The detection methods involved in the following examples are as follows: 1. Determination of flavonoid, total phenol, and total alkaloid content: Rutin was used as the standard, and the sodium nitrite-aluminum nitrate colorimetric method was used to determine the flavonoid content in the black tea extract. Gallic acid was used as the standard, and the Folin phenol colorimetric method was used to determine the total phenol content in the black tea extract. Hydrochloric berberine was used as the standard, and the sulfuric acid colorimetric method was used to determine the total alkaloid content in the black tea extract.

[0034] 2. Determination of theanine content: Theanine was used as the standard, and the high-performance liquid chromatography method was used to determine the theanine content in the black tea extract.

[0035] The chromatographic conditions are as follows: An Agilent 1100 series HPLC system (Agilent, San Jose, CA, USA) equipped with a diode array detector (DAD) was used for high-performance liquid chromatography-ultraviolet detection (HPLC-UV). The chromatographic column was a Diamonsil C18 reversed-phase column (250 mm × 4.6 mm, 5 μm), the flow rate was 1.0 mL·min -1The column temperature was set at 35℃. The mobile phase was 0.1% (v / v) phosphoric acid aqueous solution (A) and acetonitrile (B); Gradient elution: 0~12 min, 15%~25% B; 12~15 min, 25%~45% B; 15~20 min, 45%~30% B; 20~30 min, 30%~20% B; 30~32 min, 20%~15% B. The detection wavelength was 507 nm. The injection volume was 20 μL, and filtered through a 0.45 μm membrane filter before use.

[0036] The raw materials involved in the following examples are as follows: Glucose was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd., with CAS number 50-99-7; Proline was purchased from Shanghai Hao Hong Biomedicine Technology Co., Ltd., with CAS number 147-85-3; Glycerol was purchased from Shanghai Hao Hong Biomedicine Technology Co., Ltd., with CAS number 56-81-5; Betaine was purchased from Shanghai Hao Hong Biomedicine Technology Co., Ltd., with CAS number 107-43-7; Lactic acid was purchased from Shanghai Macklin Biochemical Technology Co., Ltd., with CAS number 50-21-5; Citric acid was purchased from Shanghai Hao Hong Biomedicine Technology Co., Ltd., with CAS number 77-92-9; Malic acid was purchased from Shanghai Macklin Biochemical Technology Co., Ltd., with CAS number 6915-15-7; Black tea powder: The black tea leaves purchased from Chongqing Shihuo Agricultural Development Co., Ltd. were dried, crushed and sieved through 40 mesh to obtain black tea powder; Black tea shell powder: The tea shell purchased from Chongqing Shihuo Agricultural Development Co., Ltd. was dried, crushed and sieved through 40 mesh to obtain tea shell powder; Black tea stem powder: The tea stem purchased from Chongqing Shihuo Agricultural Development Co., Ltd. was dried, crushed and sieved through 40 mesh to obtain tea stem powder.

[0037] The solvent of the solutions involved in the examples and comparative examples is water if not specified.

[0038] Example 1: Preparation of black tea extract A method for extracting active ingredients from black tea using a natural deep eutectic solvent, the steps are as follows: (1) Preparation of natural deep eutectic solvent Glucose and lactic acid were mixed in a round-bottom flask at a molar ratio of 1:5, stirred at 80℃ for 3 h, and a clear and transparent liquid was obtained, which was glucose-lactic acid deep eutectic solvent; (2) Extraction of active ingredients The glucose-lactic acid deep eutectic solvent in step (1) was mixed with water according to a mass ratio of 4:1 to prepare a glucose-lactic acid deep eutectic solvent aqueous solution (water content 20%), and the glucose-lactic acid deep eutectic solvent aqueous solution was placed in a conical flask and mixed with black tea powder, a solid-liquid ratio of 1 g:20 mL, 40 Hz, 60°C ultrasonic extraction for 60 min, 10,000 rpm centrifugation for 10 min, filtration, to obtain a black tea active ingredient extract. Example 2: Preparation of black tea extract A method for extracting active ingredients from black tea using a natural deep eutectic solvent, the steps are as follows: (1) Preparation of natural deep eutectic solvent Betaine-citric acid was mixed in a round-bottom flask according to a molar ratio of 1:2, stirred at 80°C for 3 h to obtain a clear and transparent liquid, and a betaine-citric acid deep eutectic solvent was obtained. (2) Extraction of active ingredients The betaine-citric acid deep eutectic solvent in step (1) was mixed with water according to a mass ratio of 9:1 to prepare a betaine-citric acid deep eutectic solvent aqueous solution (water content 10%), and the betaine-citric acid deep eutectic solvent aqueous solution was placed in a conical flask and mixed with black tea powder, a solid-liquid ratio of 1 g:10 mL, 20 Hz, 20°C ultrasonic extraction for 20 min, 8,000 rpm centrifugation for 5 min, filtration, to obtain a black tea active ingredient extract.

[0039] Example 3: Preparation of black tea extract A method for extracting active ingredients from black tea using a natural deep eutectic solvent, the steps are as follows: (1) Preparation of natural deep eutectic solvent Proline and malic acid were mixed in a round-bottom flask according to a molar ratio of 1:2, stirred at 80°C for 3 h to obtain a clear and transparent liquid, and a proline-malic acid deep eutectic solvent was obtained. (2) Extraction of active ingredients The proline-malic acid deep eutectic solvent was mixed with water according to a mass ratio of 7:3 to prepare a proline-malic acid deep eutectic solvent aqueous solution (water content 30%), and the proline-malic acid deep eutectic solvent aqueous solution was placed in a conical flask and mixed with black tea powder, a solid-liquid ratio of 1 g:30 mL, 80 Hz, 80°C ultrasonic extraction for 120 min, 12,000 rpm centrifugation for 20 min, filtration, to obtain a black tea active ingredient extract.

[0040] Example 4: Preparation of black tea extract On the basis of Example 1, the natural deep eutectic solvent in step (1) is changed to betaine and glycerol (molar ratio 1:2), and the remaining steps are consistent with Example 1, to prepare the active ingredient extract of black tea.

[0041] Comparative Example 1: Preparation of black tea extract On the basis of Example 1, the deep eutectic solvent in step (1) is changed to L-proline and lactic acid (molar ratio 1:5), and the remaining steps are consistent with Example 1.

[0042] Comparative Example 2: Preparation of black tea extract On the basis of Example 1, the deep eutectic solvent in step (1) is changed to L-proline and glycerol (molar ratio 1:5), and the remaining steps are consistent with Example 1.

[0043] Comparative Example 3: Preparation of black tea extract On the basis of Example 1, the deep eutectic solvent in step (1) is changed to glucose and citric acid (molar ratio 1:5), and the remaining steps are consistent with Example 1.

[0044] Comparative Example 4: Preparation of black tea extract On the basis of Example 1, the deep eutectic solvent in step (1) is changed to betaine and lactic acid (molar ratio 1:5), and the remaining steps are consistent with Example 1.

[0045] Comparative Example 5: Preparation of black tea extract Black tea powder is extracted with deionized water (water extraction) or 75% ethanol (alcohol extraction) at a solid-liquid ratio of 1:20 g / mL at 60°C for 2 h, and the extraction is repeated three times to obtain the extract.

[0046] Test Example 1: Deep eutectic solvent screening The active ingredient extracts of black tea prepared in Examples 1-4, Comparative Examples 1-5, water extraction, and alcohol extraction are tested for the extraction amounts of alkaloids, flavonoids, total phenols, and theanine, and the results are shown in Table 1.

[0047] Table 1: Active ingredient content in the extract

[0048] The deep eutectic solvent extraction experiment is performed using black tea samples to compare the total flavonoid content (TFC), total phenol content (TPC), alkaloid content, and theanine content under different process conditions, and to determine the optimal extraction process parameters for the black tea samples. The experimental results show that in Example 1 where glucose and lactic acid are used to make the deep eutectic solvent, the main active ingredients in the black tea extract are alkaloids, polyphenols, flavonoids, and theanine compounds, among which the polyphenol content is the highest, followed by the flavonoid content.

[0049] Example 5: Preparation of black tea extract On the basis of Example 1, the molar ratio of natural deep eutectic solvent in step (1) was changed to 1:2, 1:3, 1:4, 1:6, and the remaining steps were consistent with Example 1.

[0050] The active ingredient extract solution of black tea obtained in Example 1 and Example 5 was taken to detect the extraction amount of alkaloids, flavonoids, total phenols and theanine, and the results are shown in Table 2 and Figure 1 (A). With the increase of molar ratio, the total amount of active substances first increased and then stabilized.

[0051] Table 2 Active ingredient content in the extract solution

[0052] Example 6: Preparation of black tea extract On the basis of Example 1, the water content in step (1) was changed to 10%, 15%, 25%, 30%, and the remaining steps were consistent with Example 1.

[0053] The active ingredient extract solution of black tea obtained in Example 1 and Example 6 was taken to detect the extraction amount of alkaloids, flavonoids, total phenols and theanine, and the results are shown in Table 3 and Figure 1 (B). When the water content exceeds 20%, the total amount of active substances gradually decreases, which may be due to the fact that the interaction between DES and target components is reduced due to the excessive water content, resulting in a decrease in the total amount of active substances.

[0054] Table 3 Active ingredient content in the extract solution

[0055] Example 7: Preparation of black tea extract On the basis of Example 1, the extraction time in step (2) was changed to 20 min, 40 min, 80 min, 100 min, and the remaining steps were consistent with Example 1.

[0056] The active ingredient extract solution of black tea obtained in Example 1 and Example 7 was taken to detect the extraction amount of alkaloids, flavonoids, total phenols and theanine, and the results are shown in Table 4 and Figure 1 (C). With the increase of ultrasonic time, the extraction content of active ingredients of black tea first increased and then tended to be stable. In order to ensure the extraction efficiency, the extraction time of 40, 60, 80 min was selected for subsequent response surface experiment.

[0057] Table 4 Active ingredient content in the extract solution

[0058] Example 8: Preparation of black tea extract On the basis of Example 1, the extraction temperature in step (2) was changed to 40°C, 50°C, 60°C, 70°C, 80°C, and the other steps were the same as in Example 1.

[0059] The active ingredient extracts of black tea obtained in Example 1 and Example 8 were taken to detect the extraction amounts of alkaloids, flavonoids, total phenols, and theanine, and the results are shown in Table 5 and Figure 1 (D). It was found that the total extraction amount increased when the extraction temperature was in the range of 40°C to 60°C. The extraction rate decreased as the temperature rose to 70°C. High temperature would cause the decomposition of polyphenols and flavonoids. Therefore, the extraction time of 50, 60, and 70°C was selected for the subsequent response surface experiment.

[0060] Table 5 Content of active ingredients in the extract

[0061] Example 9: Preparation of black tea extract On the basis of Example 1, the solid-liquid ratio (g:mL) in step (2) was changed to 1:10, 1:15, 1:25, 1:30, and the other steps were the same as in Example 1.

[0062] The active ingredient extracts of black tea obtained in Example 1 and Example 9 were taken to detect the extraction amounts of alkaloids, flavonoids, total phenols, and theanine, and the results are shown in Table 6 and Figure 1 (E). The solid-liquid ratio affects the mass transfer driving force. When the ratio is 1:20, the amount of solvent is sufficient to ensure efficient mass transfer. When the ratio is too low, the solvent is insufficient, and the extraction is not complete; when the ratio continues to increase, although the driving force increases, the yield improvement is no longer significant, and the economic angle is no longer optimal.

[0063] Table 6 Content of active ingredients in the extract

[0064] Example 10: Preparation of black tea extract This example provides a response surface method optimization experiment for ultrasonic-assisted extraction of active substances of black tea Figure 2 ).

[0065] On the basis of Example 1, according to the results of the single-factor experiment, the effects of extraction time, solid-liquid ratio, and ultrasonic temperature on the total extraction amount of black tea were further optimized. The Design-Expert 13 software was used to perform response surface analysis on the extraction factors of black tea, and the factor and level table for response surface analysis is shown in Table 7.

[0066] Table 7 Factors and levels for response surface analysis

[0067] The results are shown in Table 8, and a quadratic polynomial regression equation of total active substances of black tea leaves is obtained from the independent variables X1 extraction temperature, X2 solid-liquid ratio, and X3 extraction time: Y = 2245.92 + 2.4625 X1- 5.3875 X2+ 1.1 X3- 0.675 X1X2- 1.65 X1X3- 0.4 X2X3- 12.5725 X1 2 - 12.0225 X2 2 - 3.5475 X3 2 Table 8 Response surface design and its response values

[0068] The results of variance analysis and fitting analysis of the above regression model are shown in Table 9. F test shows that the regression model has a high F value (F = 46.16) and a low P value (P < 0.0001), indicating that the model is significant. The equation misfit term is not significant (P = 0.09597), R 2 = 0.9834, indicating that the established quadratic regression model can be used to analyze and predict the process conditions of ultrasonic-assisted DES extraction of active substances of black tea leaves. The results of coefficient evaluation and significance test of the regression model show that all the first-order terms and second-order terms have a significant effect on the active substances of black tea leaves.

[0069] The best simulation results of the established target black tea total extraction model are: extraction temperature (60.94°C), extraction time (64.36 min), and solid-liquid ratio (1:18.85 g / mL). Under this parameter, the predicted extraction rate is 246.7 mg / g. In order to facilitate the experiment, the extraction temperature, extraction time, and solid-liquid ratio are set to 61°C, 64 min, and 1 g:19 mL, respectively.

[0070] Table 9 Results of response surface variance analysis

[0071] Example 11: Preparation of black tea leaf extract under the optimal extraction process conditions and component analysis 1. Preparation of black tea leaf extract On the basis of Example 1, the ultrasonic extraction temperature, extraction time, and solid-liquid ratio in step (2) are changed to 61°C, 64 min, and 1 g:19 mL, respectively, and the other steps are consistent with Example 1.

[0072] 2. Total component analysis of black tea leaf extract (1) Experimental procedure To determine the contents of catechins (EGCG, GCG, EGC, ECG, EC, GC, C, gallic acid, purine alkaloids (CAF, THB) and theaflavins (TF1, TF2a, TF2b, TF3) in black tea leaf extract, a UHPLC (Thermo Scientific Dionex UltiMate 3000, USA) system was used, which included a degasser, an autosampler (WPS3000RS), a binary pump (HPG-3400RS), a column compartment (TCC-100) and a diode array detector (DAD, DAD-3000). The separation of these compounds was performed on an Acquity UPLC® BEH Shield RP18 column (2.1 x 50 mm, 1.7 um, Waters, USA) at a temperature of 40 °C and a flow rate of 0.25 mL / min. The injection volume was set to 1 uL and the detection wavelength was set to 278 nm for the elution gradient of mobile phase A (0.1% formic acid-water, by volume) and mobile phase B (acetonitrile) as follows: 0-2.5 min, 2% B; 2.5-3 min, 2%~5% B; 3-8 min, 5%~15% B; 8-14 min, 15%~30% B; 14-16 min, 30%~50% B; 16-18 min, 50%~2% B; 18-22 min, 2% B.

[0073] From the composition of black tea leaf extract, the content of caffeine was as high as 28.29 mg / g, which was significantly higher than other components, indicating that the DES used had a very strong extraction capacity for alkaloid components. Among the catechins, the content of ECG (8.75 mg / g) was significantly higher than that of EGCG (1.76 mg / g), and the complete detection of theaflavins components showed that DES could effectively retain the characteristic oxidation products of black tea. Among them, the contents of TF2a (1.08 mg / g) and TF3 (1.59 mg / g) were higher, and these two components were closely related to the briskness and strength of black tea soup, suggesting that the DES extract had an advantage in maintaining the quality characteristics of black tea. The simultaneous detection of gallic acid (2.18 mg / g) and theabrownine (2.04 mg / g) reflected the integrity of the oxidation gradient of phenolic components in the extract.

[0074] Table 10 Main active ingredients in black tea leaf extract

[0075] Example 12: Preparation of black tea shell extract under the optimal extraction process conditions On the basis of Example 11, the raw material (black tea leaf powder) of step (1) was changed to tea stem powder, and the remaining steps were consistent with Example 11.

[0076] Example 13: Preparation of black tea stem extract under optimal extraction process conditions On the basis of Example 11, the raw material (black tea leaf powder) of step (1) was changed to tea stem powder, and the remaining steps were consistent with Example 11.

[0077] Test Example 2: Detection of active ingredients in different black tea parts 1. Active ingredient analysis The active ingredient extract solutions obtained from different black tea parts in Examples 11-13 were detected for the extraction amounts of alkaloids, flavonoids, total phenols, and theanine, and the results are shown in Table 11.

[0078] Table 11: Active ingredient content in extract solutions of different black tea parts

[0079] To verify the prediction results of the above response surface method and facilitate actual operation, the optimized extraction process of Examples 11-13 was as follows: molar ratio 1:5; water content 20%; solid-liquid ratio 1 g:19 mL; ultrasonic extraction temperature 61℃; and ultrasonic extraction time 64 min. The active substances in different parts of black tea were extracted under the above conditions, and the total amount of active substances extracted from black tea leaves was 248 mg / g, which was basically consistent with the predicted value. In addition, the optimal process was applied to tea shells and tea stems, and it was found that tea shells were the preferred part for specific enrichment of flavonoid components.

[0080] 2. Antioxidant activity test The extract solutions of different parts of black tea (tea leaves, tea shells, and tea stems) in Examples 11-13 were diluted with water to 0.5, 1, 3, 5, 8, and 10 mg / mL for antioxidant activity tests.

[0081] (1) DPPH free radical scavenging experiment 8.0 mg of 1,1-diphenyl-2-picrylhydrazine was weighed using a micro-balance and weighing paper, directly poured into a 100 mL volumetric flask, and dissolved and made up to volume with anhydrous ethanol to prepare a 0.2 mmol / L DPPH solution (which needs to be prepared and used immediately, and used up within 3.5 h). 0.5 mL of extract solution of different concentrations was taken, 0.5 mL of 0.2 mmol / L DPPH solution was added, and it was vortexed uniformly and placed in the dark for 30 min. The OD517 nm absorbance value was measured using a 96-well plate and a microplate reader, Ai, the absorbance value of the mixture of DPPH solution and an equal volume of anhydrous ethanol was measured, A0, and the absorbance value of the mixture of black tea extract solution and an equal volume of anhydrous ethanol was measured, Aj.

[0082] (2) ABTS radical scavenging experiment Take 0.2 mL of different concentrations of extraction solution, add 0.8 mL of ABTS working solution, vortex uniformly, and then place it in the dark for 6 min. Use 96-well plates and a microplate reader to measure OD734 nm, denoted as Ai, measure the absorbance value of 0.8 mL ABTS working solution mixed with 0.2 mL deionized water as A0, and the absorbance value of 0.2 mL extraction solution mixed with 0.8 mL anhydrous ethanol as Aj.

[0083] To quantify the antioxidant activity of black tea extract, the sample concentration required to scavenge 50% of free radicals (IC50) was measured. The smaller the IC50 value, the greater the free radical scavenging ability. The experimental results show (Table 12, Figure 3 A), all parts of the extract showed significant DPPH free radical scavenging ability, and the scavenging effect was concentration-dependent. Among them, the tea shell extract showed the strongest scavenging ability, with the lowest IC50 value of 0.92 mg / mL; the scavenging ability of tea leaf extract was second (IC50 = 1.50 mg / mL), and the activity of tea stem extract was relatively weak (IC50 = 3.13 mg / mL). The results show that the tea shell extract prepared by the method of the present application has the most excellent DPPH free radical scavenging activity.

[0084] Table 12 DPPH detection results

[0085] ABTS detection results (Table 13, Figure 3 B) further confirmed that the extract of the present application has strong antioxidant potential. Consistent with the trend of DPPH experiment results, the scavenging ability of all samples increased with increasing concentration. The tea shell extract also performed best in this model, with an IC50 value of 1.61 mg / mL; the activity of tea leaf extract was second (IC50 = 2.48 mg / mL), and the activity of tea stem extract was relatively lowest (IC50 = 5.30 mg / mL).

[0086] Table 13 ABTS detection results

[0087] 3. Anti-glycation effect detection Bovine serum albumin (BSA, 10 mg / mL) and glucose (1.0 M) were mixed in a phosphate buffer system at pH 7.4. Blank control (BSA only), model control (BSA + glucose), sample groups (containing different concentrations of the test extract), and positive control group (containing aminoguanidine, 1 mg / mL) were set up. The reaction system was incubated at 55℃ in the dark for 7–14 days. The characteristic fluorescence intensity of advanced glycation end products (AGEs) was detected using a fluorescence spectrophotometer at an excitation wavelength of 370 nm and an emission wavelength of 440 nm. By comparing the fluorescence values ​​of each group, the inhibition rate of AGEs formation was calculated, and the half-maximal inhibitory concentration (IC50) was further obtained through dose-response curves to objectively evaluate the strength of its anti-glycation activity. This model can be effectively applied to evaluate the anti-glycation efficacy of DES extracts such as black tea leaves and tea husks.

[0088] AGEs inhibition rate (%) = [1-(FI) 样品组 -FI 空白组） ÷ (FI 模型组 -FI 空白组） ]×100%.

[0089] As shown in Table 14, all DES extracts exhibited varying degrees of anti-glycation activity. Among them, the black tea leaf extract showed the most outstanding anti-glycation ability, with an inhibition rate of 48.5% at a concentration of 1 mg / mL, which was significantly better than other raw material extracts. Furthermore, the total phenol content was positively correlated with the anti-glycation activity. Therefore, the raw materials with higher total phenol content among different parts of black tea extracts showed stronger AGEs inhibition ability.

[0090] Table 14 Anti-glycation test results

[0091] 4. Anti-inflammatory bioactivity assay (1) Cell viability experiment According to 1×10 4Macrophages (RAW264.7) were cultured in 96-well cell culture plates containing DMEM medium for 24 h. After culturing, the supernatant was discarded. Tea leaf, tea husk, and tea stem extracts were diluted to 1.25–20, 2.5–50, and 0.3125–5 μg / mL, respectively, using DMEM medium as sample wells. A blank control group (no extract) and a zero-adjustment group (no cells or samples) were used. Multiple replicates were set up for each well. After 24 h of culture, the supernatant was discarded, and the cells were washed with PBS. 100 μL of freshly prepared 0.5 mg / mL MTT solution (dissolved in DMEM medium) was added to each well. After 4 h of incubation, the supernatant was discarded, and 100 μL of DMSO was added to each well to dissolve the crystals. The plates were shaken at 37°C for 5 min, and the absorbance was read at 490 nm using a microplate reader. Cell proliferation was calculated using the following formula: Cell proliferation rate (%) = [(A1-A0) / (A2-A0)] × 100; In the formula: A0 represents the absorbance of the solvent group; A1 represents the absorbance of the sample group; A2 represents the absorbance of the blank control group.

[0092] Experimental results show that... Figure 4 As shown, RAW264.7 cells were treated with extracts from different parts of black tea for 24 h, and cell viability was detected using the MTT assay. The results showed that the cell viability was greater than 100% at concentrations less than 2.5, 5, and 0.625 μg / mL, respectively. These concentrations can be used as reference thresholds for subsequent drug concentration determinations.

[0093] (2) Detection of cellular inflammatory factor content According to 1×10 6 After seeding RAW264.7 cells into 12-well plates and culturing for 24 h, the black tea extracts from Examples 11-13 were diluted to 0.625 μg / mL with 1 μg / mL lipopolysaccharide (LPS) DMEM solution and incubated for 24 h. The cell supernatant was collected, and the secretion of inflammatory factors by RAW264.7 cells was detected according to the ELISA kit instructions.

[0094] Table 15. Test results of IL-6 inflammatory factor content in extracts from different parts of black tea.

[0095] Table 16. Test results of IL-6 inflammatory factor content in extracts from different parts of black tea.

[0096] Experimental results show that... Figure 5As shown, there were obvious differences in the inhibitory effects of different parts of black tea extracts on inflammatory factors. In terms of IL-6, tea leaf, tea shell and tea stem extracts all showed significant inhibitory effects (P<0.05), among which the inhibitory effect of tea shell extract was the most prominent, with an inhibition rate of 68.6%, and its IL-6 content (212 pg / mL) was significantly lower than that of other treatment groups. For TNF-a, the inhibition rate of tea stem extract was only 4.5%, and the inhibition rate of black tea leaf extract was only 9.6%, indicating that the inhibitory effect of these two extracts on TNF-a was weak. Tea shell extract showed excellent effect on the inhibition of two kinds of inflammatory factors. The inhibition rate of TNF-a (63.9%) was also significantly higher than that of other treatment groups. This excellent broad-spectrum anti-inflammatory effect is closely related to its unique chemical composition - the flavonoid content in tea shell extract is as high as 89.5 mg / g, which is significantly higher than that of other raw materials.

[0097] Example 14: Application of black tea extract Through systematic formulation design (Table 17), a black tea extract serum was designed to improve inflammatory skin. The results of multi-center human efficacy evaluation showed that the black tea extract serum had the effects of repair, soothing, moisturizing and brightening. The experimental group was the product group, which used 1.5-2.0 mL of black tea extract serum each time, twice a day. The negative control group did not use the black tea extract serum.

[0098] In the experiment, compared with the negative control (without using the serum), the product group significantly improved the skin transepidermal water loss rate (P<0.001), the skin stratum corneum water content (P<0.001), and the skin smoothness (P<0.001) after 15 min and 14 days of use, and it was considered that the black tea extract serum had the effects of repair and moisturizing under the experimental conditions; According to the skin color, it can be represented by the CIELAB color system. In this study, by comparing the skin color a value and b value of different parts before and after using the product, the decrease of test values indicated that the product had a certain yellow-removing and brightening effect. In the open use of the product test, after 15 min and 14 days of use, the skin a value of the product group was significantly reduced (P<0.05) and the b value was very significantly reduced (P<0.01), which proved that the product had a significant yellow-removing effect; The skin glossiness test determines that the product group significantly improves the skin lightening (P<0.001); through expert evaluation, compared with before using the product, the proportion of the red area of the skin of the subject after using the product is significantly improved (P<0.05), and it is considered that the black tea extract essence water has a soothing effect under the experimental conditions.

[0099] The self-evaluation of the subject further supports the improvement of the black tea extract essence water on the sleepless skin: 91.88% agree with the moisturizing effect; 100% agree with the skin soothing effect: 96.50% agree with the immediate hydration effect; 98.88% agree with the sensitive skin symptom repair effect; 93.75% agree with the uniform skin lightening effect; 87.50% agree with the darkening and yellowing removal effect.

[0100] Table 17 Component formula of the black tea extract essence water

[0101] Although the present application has been disclosed in the above with preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for preparing black tea extract using a deep eutectic solvent, characterized by, The method steps are as follows: S1: Preparation of natural deep eutectic solvent aqueous solution: mix the deep eutectic solvent with water uniformly to prepare the natural deep eutectic solvent aqueous solution; S2: Ultrasonic extraction: after adding the black tea powder into the natural deep eutectic solvent aqueous solution prepared in S1, ultrasonic extraction is performed, and the supernatant is collected by centrifugation to obtain the black tea extract; The natural deep eutectic solvent includes one of glucose-lactic acid, betaine-citric acid, proline-malic acid, and betaine-glycerol; In S1, the water content of the natural deep eutectic solvent aqueous solution is 15%-25%; In S2, the solid-liquid ratio of the black tea powder to the deep eutectic solvent aqueous solution is 1 g:(15-30) mL; the ultrasonic extraction conditions are 50-70℃, 20-80 Hz extraction for 40-100 min; In S2, the black tea powder is prepared from the black tea raw material, and the black tea raw material includes at least one of tea leaves, tea stems, and tea shells.

2. The method of claim 1, wherein, In S1, the preparation method of the natural deep eutectic solvent is as follows: mix glucose and lactic acid at a molar ratio of 1:(4-6), stir at 75-85℃ for 2.5-3.5 h to obtain a glucose-lactic acid natural deep eutectic solvent; or, mix betaine and citric acid at a molar ratio of 1:(1-3), stir at 75-85℃ for 2.5-3.5 h to obtain a betaine-citric acid natural deep eutectic solvent; or, mix proline and malic acid at a molar ratio of 1:(1-3), stir at 75-85℃ for 2.5-3.5 h to obtain a proline-malic acid natural deep eutectic solvent; or, mix betaine and glycerol at a molar ratio of 1:(1-3), stir at 75-85℃ for 2.5-3.5 h to obtain a betaine-glycerol natural deep eutectic solvent.

3. The method of claim 1, wherein, In S1, the water content of the natural deep eutectic solvent aqueous solution is 15%-20%; the solid-liquid ratio of the black tea powder to the deep eutectic solvent aqueous solution is 1 g:19 mL; and the ultrasonic extraction conditions are 61℃, 20-80 Hz extraction for 64 min.

4. The method of claim 1, wherein, In S2, the black tea powder is obtained by freeze-drying or drying, crushing, and sieving of the black tea raw material.

5. The method of claim 1, wherein, In S2, the centrifugation conditions are 8000-12000 rpm centrifugation for 5-20 min.

6. A black tea extract prepared by the method of any one of claims 1 to 5, wherein the black tea extract is characterized by, The black tea extract contains total phenols, flavonoids, alkaloids, and theanine.

7. Use of the method according to any one of claims 1 to 5 or the black tea extract according to claim 6 for the manufacture of a cosmetic product, characterized in that, The cosmetic has the functions of anti-inflammatory, antioxidant, and anti-glycosylation.

8. A product comprising the black tea extract of claim 6, wherein, The product is a cosmetic or a drug.

9. The product of claim 8, wherein, The addition amount of the black tea extract is 1%-10% of the total mass of the product.

10. The product of claim 8, wherein, The dosage form of the cosmetic is any one of dressing, ointment, cream, emulsion, spray, cream, water, gel, oil, patch, film, mud, powder, solution, and film coating.

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