Deep eutectic solvent-based polyphenol-rich plant extract, and preparation method and application thereof

Abstract

This invention relates to a polyphenol-rich plant extract based on a deep eutectic solvent, its preparation method, and its application. The preparation method includes: S1, pretreating plant raw materials rich in pigments or polyphenols to obtain plant powder; S2, mixing the plant powder with a deep eutectic solvent to obtain an extract; S3, separating and purifying the extract to obtain a plant polyphenol extract. Compared with existing technologies, this invention uses a deep eutectic solvent system to extract the active components of plant polyphenols, which is beneficial for the stability of pH-sensitive components such as anthocyanins. The resulting extract has a bright color and also exhibits good antioxidant properties and color fastness.

Description

Technical Field

[0001] This invention belongs to the field of natural product extract technology, and relates to a polyphenol-rich plant extract based on a deep eutectic solvent, its preparation method and application. Background Technology

[0002] Anthocyanins are water-soluble flavonoid polyphenols responsible for giving plant fruits, peels, or tubers their vibrant red, purple, or blue colors, and are widely found in berries. These pigments not only act as secondary metabolites in plants, helping them resist ultraviolet radiation, pests, and environmental stress, but also endow them with strong antioxidant properties.

[0003] Traditional methods for extracting polyphenols from plants (such as ethanol or water extraction) have significant limitations: such as poor pH stability, easy discoloration of the extract, and low extraction recovery rate, which limit the stability in application.

[0004] Deep eutectic (DES) solvent extraction is a rapidly developing green and efficient sample pretreatment technique in recent years. CN120241547A discloses a method for preparing Huangshan pine needle polyphenols and an acne-reducing and anti-inflammatory essence, using a mixture of choline chloride, glycerol, lactic acid, and water as the DES solvent to extract Huangshan pine needle polyphenols from Huangshan pine needle fermentation products. CN117126302A discloses a temperature-responsive deep eutectic aqueous two-phase system for simultaneously extracting polysaccharides and polyphenols from Schisandra chinensis, using betaine and urea as the DES solvent, combined with n-propanol aqueous two-phase extraction to extract polysaccharides and polyphenols from Schisandra chinensis fruits.

[0005] However, there is currently no suitable solvent for extracting anthocyanin-containing polyphenol extracts from berry plants that can improve the extraction rate and stability of the extract while also enhancing its properties, such as antioxidant properties and hair dyeing effects. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of traditional methods for extracting polyphenols from plants, such as poor pH stability, easy discoloration of the extract, low extraction recovery rate, and low safety. Therefore, this invention provides a polyphenol-rich plant extract based on a deep eutectic solvent, its preparation method, and its application.

[0007] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide a method for preparing a plant extract rich in polyphenols, comprising the following steps: S1. Pre-treat plant materials rich in polyphenols and / or pigments to obtain plant powder; S2. Mix the plant powder obtained in step S1 with a deep eutectic solvent and extract to obtain an extract; S3. Separate and purify the extract obtained in step S2 to obtain the plant extract.

[0008] In some specific embodiments, in step S1, the plant material rich in polyphenols and / or pigments is selected from any one or more combinations of berry plants, vegetable plants, flower plants, and herbaceous plants.

[0009] As a more preferred step, in step S1, the plant material rich in polyphenols and / or pigments is selected from any one or more combinations of mulberry, blueberry, grape, butterfly pea, black goji berry, blackcurrant, purple cabbage, purple sweet potato, rose, and calendula.

[0010] In some specific embodiments, in step S1, the pretreatment method is selected from any one or more combinations of drying, pulverizing, grinding, shearing, pressing, freeze drying, rotary evaporation, low-temperature vacuum concentration, and spray drying.

[0011] As a more preferred step, step S1, the pretreatment step is as follows: the plant raw material is frozen at -30℃ to 0℃, then freeze-dried under vacuum at -60℃ to -40℃ for 12 to 72 hours, pulverized and sieved to obtain plant powder with a particle size ≤ 80 mesh.

[0012] As a more preferred method, the vacuum freeze-drying temperature is -50°C and the time is 48 hours.

[0013] In some specific embodiments, in step S2, the deep eutectic solvent is composed of hydrogen bond acceptors and hydrogen bond donors in a molar ratio of 1:(0.5~5), and the water content of the deep eutectic solvent is 0~80%. The hydrogen bond acceptor is selected from any one or more combinations of betaine, choline chloride, acetylcholine, and carnitine. The hydrogen bond donor is selected from any one or more combinations of organic acids, polyols, urea, and sugars.

[0014] In some specific embodiments, in step S2, the organic acid is selected from any one or more of formic acid, acetic acid, propionic acid, butyric acid, valeric acid, lactic acid, glycolic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, malic acid, tartaric acid, citric acid, benzoic acid, phenylacetic acid, 3-phenylpropionic acid, salicylic acid, gallic acid, caffeic acid, ferulic acid, and gluconic acid.

[0015] In some specific embodiments, step S2 involves extraction using any one of ultrasound-assisted extraction, microwave-assisted extraction, or heating and stirring extraction.

[0016] In some specific embodiments, the extraction parameters for step S2 are: a material-to-liquid ratio of 1 g to (20–100) mL, an extraction time of (10–60) min, and an extraction temperature of (30–70) °C. The extraction power of ultrasound-assisted extraction and microwave-assisted extraction is (200~500) W.

[0017] As a more preferred method, the extraction parameters are: a solid-liquid ratio of 1 g to (30-70) mL, an extraction time of (10-50) min, and an extraction temperature of (30-70) °C. The extraction power of ultrasound-assisted extraction is 300 W.

[0018] In some specific embodiments, step S3, the separation and purification method is as follows: centrifuge the extract, collect the supernatant and then filter it; The centrifugation parameters are: rotation speed (5000~12000) r / min, time (5~20) min; The filtration parameters are: filtration through a 0.1–0.5 μm microporous membrane.

[0019] More preferably, the pore size of the filter membrane is 0.22~0.25 μm.

[0020] The second technical solution of the present invention is to provide a plant polyphenol extract, which is obtained by the preparation method described in one of the above technical solutions.

[0021] The third technical solution of the present invention is to provide an application of the plant polyphenol extract as described in the second technical solution above in antioxidant products, hair dye products, and skin care products.

[0022] In this invention, antioxidant products may include edible products such as health supplements and food additives.

[0023] The fourth technical solution of the present invention is to provide a hair dyeing product, comprising the following raw materials in parts by volume: 5-20 parts of plant polyphenol extract; 3-8 parts of development agent; Mordant 3-8 parts; The development agent is selected from any one or a combination of hydrogen peroxide solution and ascorbic acid aqueous solution; The mordant is a metal ion salt solution, and the metal ions are selected from Fe. 2+ Fe 3+ Cu 2+ Al 3+ Any one or combination thereof.

[0024] More preferably, the developer is selected from a 2% hydrogen peroxide solution or a 5% ascorbic acid aqueous solution.

[0025] More preferably, the hair dye product comprises raw materials in the following volumes: 10 parts plant polyphenol extract; Five parts of 5% ascorbic acid were used as a development agent; Five parts of 10% ferric chloride solution were used as mordants.

[0026] The hair dye product provided by this invention, composed of plant polyphenol extracts, can form a metal polyphenol network complex with metal ions through the plant polyphenol extracts, providing long-lasting coloring and hair care functions.

[0027] In some specific embodiments, the hair dye product further includes any one or more combinations of stabilizers, humectants, pH adjusters, or thickeners to enhance stability and dyeing effect.

[0028] The fifth technical solution of the present invention is to provide a method of using the hair dyeing product as described in the fourth technical solution above, which is to apply the hair dyeing product to the hair to be dyed, keep it warm for 10 to 60 minutes, wash it with water and let it dry.

[0029] In some specific embodiments, the hair to be dyed may be bleached hair, white hair, black hair, or other colored hair.

[0030] Compared with the prior art, the present invention has the following advantages: (1) The present invention uses a deep eutectic solvent system to extract active ingredients from plants, which can achieve a higher extraction rate and enhance the stability of pH-sensitive components such as anthocyanins, thus avoiding oxidative degradation.

[0031] (2) The deep eutectic solvent system used in this invention can provide a stable acidic environment, which is conducive to the stability of pH-sensitive components and makes the target plant components stable in color in the extract.

[0032] (3) The deep eutectic solvent system used in this invention has low toxicity and biodegradability, especially the system with betaine as hydrogen bond acceptor. As a naturally sourced hydrogen bond acceptor, betaine has no halogen ions compared to the traditional choline chloride system. It has GRAS food-grade safety status, higher biodegradability and lower cytotoxicity. After combining with organic acids, it forms a low-volatility solvent, reducing environmental pollution. It is expected to be applied to the food and cosmetic fields.

[0033] (4) The plant polyphenol extract obtained by this invention has natural coloring properties and can complex with metal ions to form metal polyphenol networks (MPNs) for color development, which can maintain good color fastness. At the same time, it can also reduce irritation to hair and scalp, which is in line with the development trend of green and natural hair dyeing. Attached Figure Description

[0034] Figure 1 The different betaine-organic acid deep eutectic solvents used in Example 1.

[0035] Figure 2 The extraction rate of mulberry polyphenols from different betaine-organic acid deep eutectic solutions in Example 1 is shown.

[0036] Figure 3 The effect of ultrasound time on the extraction rate of mulberry polyphenols in Example 2.

[0037] Figure 4 The effect of the material-to-liquid ratio on the extraction rate of mulberry polyphenols in Example 2.

[0038] Figure 5 The effect of water content in the betaine-anhydrous citric acid deep eutectic solvent on the extraction rate of mulberry polyphenols in Example 2.

[0039] Figure 6 The effect of ultrasonic time (A) and material-liquid ratio (B) on the extraction rate of mulberry polyphenols in Example 3.

[0040] Figure 7 The effect of ultrasonic time (A) and water content (C) of betaine-anhydrous citric acid deep eutectic solvent on the extraction rate of mulberry polyphenols in Example 3.

[0041] Figure 8 The effect of the material-liquid ratio (B) and the water content (C) of the betaine-anhydrous citric acid deep eutectic solvent on the extraction rate of mulberry polyphenols in Example 3.

[0042] Figure 9 The DPPH scavenging rate of the deep co-crystallized extract of mulberry polyphenols.

[0043] Figure 10 The ABTS scavenging rate of the deep co-crystallized extract of mulberry polyphenols.

[0044] Figure 11 The image shows the color fastness test results of hair dyeing with mulberry polyphenol deep eutectic extract.

[0045] Figure 12 This is a color diagram illustrating the extraction effect of mulberry polyphenol deep eutectic extract. Detailed Implementation

[0046] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0048] Unless otherwise specified, the materials and processes described in the following embodiments or examples are conventional materials and processes used in the art to achieve the corresponding functions.

[0049] Example 1: This embodiment provides a method for preparing polyphenol extract from mulberry fruit, comprising the following steps: (1) The mulberry fruit was frozen at -20℃ and dried at -50℃ for 48 h in a vacuum freeze dryer. The powder was then ground by machine and passed through an 80-mesh sieve to obtain freeze-dried mulberry fruit powder.

[0050] (2) Take freeze-dried mulberry fruit powder and betaine-organic acid deep eutectic solvent, mix them evenly and dissolve them according to a certain material-liquid ratio, and extract them in an ultrasonic extraction device by water bath method; (3) After sonication, centrifuge at 8000 r / min for 10 minutes, filter the liquid in the upper layer through a 0.22~0.25 μm filter membrane, and use the filtrate as the deep co-crystal extract of mulberry polyphenols.

[0051] In step (2), the parameters of the betaine-organic acid deep eutectic solvent system are set as follows: Using betaine as a hydrogen bond acceptor and organic acid as a hydrogen bond donor, the two are preheated and mixed evenly in a specific ratio until they become transparent. Water is then added to dilute the mixture, resulting in a deep eutectic solvent of betaine and organic acid. The organic acid can be selected from citric acid, lactic acid, glacial acetic acid, or tartaric acid.

[0052] In the betaine-organic acid deep eutectic solvent, the molar ratio of betaine to organic acid is 1:1 to 1:3. The preheating temperature of the betaine-organic acid deep eutectic solvent is 60~80℃, and the water content in the betaine-organic acid deep eutectic solvent is 30%.

[0053] Pretreated freeze-dried and sieved mulberry powder was mixed with a betaine-organic acid deep eutectic solution at a ratio of 1 g:30 mL. Ultrasonic extraction was then performed with an ultrasonic power of 360 W for 30 min at 50℃. Compared with water extraction and 70% ethanol volume solution, the resulting extract was centrifuged at 8000 r / min for 10 minutes. The supernatant was filtered through a 0.22–0.25 μm filter membrane. A standard curve was established using gallic acid, and the polyphenol content of the extract was determined using the Folin-Ciocalteu method, in triplicate.

[0054] Table 1 Example of a deep eutectic system of betaine and organic acid Figure 1 The figures show the various betaine-organic acid deep eutectic systems in Table 1. As can be seen from the figures, the betaine-organic acid deep eutectic solvents are clear and transparent, the systems are homogeneous, and the hydrogen bond acceptors and donors are well-bonded. Specifically, the betaine-citric acid group is a transparent, colorless, viscous liquid; the betaine-lactic acid group is a transparent, pale yellow liquid; the betaine-glacial acetic acid system is a transparent, colorless liquid; and the betaine-tartaric acid group is a transparent, slightly yellow, viscous liquid.

[0055] Figure 2 The table shows the polyphenol extraction rates of mulberry by various systems. As can be seen from the figure, the betaine-organic acid system has a higher extraction rate than water extraction, indicating that the betaine-organic acid deep eutectic system is more efficient in extracting plant polyphenols. This is because organic acids provide an acidic environment, which gently disrupts plant cell walls, facilitating polyphenol dissolution; the strong hydrogen bonding also contributes to good solubility of mulberry polyphenols. The polyphenol extraction rates of the DES-BC, DES-BL, and DES-BA groups also surpassed those of traditional ethanol extraction. Specifically, the polyphenol extraction rate of the DES-BC-1 group reached 41.72 mg / g, and the DES-BA-1 group reached 38.41 mg / g, both higher than the 32.86 mg / g polyphenol extraction rate of the 70% ethanol group.

[0056] Example 2: This embodiment provides a method for preparing polyphenol extracts from mulberry fruit using a betaine-anhydrous citric acid deep eutectic solvent, comprising the following steps: (1) The mulberry fruit was frozen at -20℃ and dried at -50℃ for 48 h in a vacuum freeze dryer. The powder was then ground by machine and passed through an 80-mesh sieve to obtain freeze-dried mulberry fruit powder.

[0057] (2) The deep eutectic solvent of betaine-anhydrous citric acid is a mixture of betaine and anhydrous citric acid in a molar ratio of 1:1, with a water content of 20%~60%.

[0058] Take freeze-dried mulberry fruit powder and betaine-anhydrous citric acid deep eutectic solvent, mix them evenly and dissolve them at a material-liquid ratio of 1:30~1:70 (g:mL), and extract them in an ultrasonic extraction device by water bath method. The ultrasonic power is 360 W, the ultrasonic time is 10~50 min, and the ultrasonic temperature is 50℃. (3) After sonication, centrifuge at 8000 r / min for 10 minutes, filter the liquid in the upper layer through a 0.22~0.25 μm filter membrane, and use the filtrate as the deep co-crystal extract of mulberry polyphenols.

[0059] The polyphenol content of the obtained extract was determined by the Folin-Ciocalteu method, in triplicate.

[0060] A one-way variable analysis was performed on the above preparation methods: (1) Ultrasound time The material-to-liquid ratio was set at 1 g:30 mL, the ultrasonic power at 360 W, the ultrasonic temperature at 50 °C, and the water content of the betaine-anhydrous citric acid deep eutectic solvent was 30%. The effect of ultrasonic time of 10–50 min on the extraction rate of mulberry polyphenols was determined in triplicate.

[0061] like Figure 3 As shown in the figure, when the ultrasonic extraction time is controlled within the range of 10 to 50 minutes, the extraction rate of mulberry polyphenols initially increases after further increasing the ultrasonic time. When the ultrasonic time reaches 20 minutes, the extraction rate of mulberry polyphenols reaches its highest level. After exceeding 20 minutes, the extraction rate of mulberry polyphenols decreases accordingly.

[0062] (2) Material-liquid ratio The ultrasonic time was set to 30 min, the ultrasonic power to 360 W, the ultrasonic temperature to 50℃, and the water content of the betaine-anhydrous citric acid deep eutectic solvent to be 30%. The effect of a material-liquid ratio of 1:30 to 1:70 (g / mL) on the extraction rate of mulberry polyphenols was determined in triplicate.

[0063] like Figure 4 As shown in the figure, when the material-liquid ratio is controlled within the range of 1:30 to 1:70 (g / mL), the extraction rate of mulberry polyphenols initially increases with the increase of the material-liquid ratio. When the material-liquid ratio reaches 1g:50mL, the extraction rate of mulberry polyphenols reaches its highest level. After further increasing the ratio of betaine-anhydrous citric acid deep eutectic solvent, the extraction rate of mulberry polyphenols decreases accordingly.

[0064] (3) Water content of betaine-anhydrous citric acid deep eutectic solvent The material-to-liquid ratio was set at 1 g: 30 mL, the ultrasonic power at 360 W, the ultrasonic temperature at 50 ℃, and the ultrasonic time at 30 min. The effect of 20-60% water content in the betaine-anhydrous citric acid deep eutectic solvent on the extraction rate of mulberry polyphenols was determined in triplicate.

[0065] like Figure 5 As shown in the figure, when the water content of the betaine-anhydrous citric acid deep eutectic solvent is controlled within the range of 20% to 60%, the extraction rate of mulberry polyphenols initially increases with the increase of water content in the betaine-anhydrous citric acid deep eutectic solvent. When the water content reaches 40%, the extraction rate of mulberry polyphenols reaches its highest level. After further increasing the water content, the extraction rate of mulberry polyphenols decreases accordingly.

[0066] Example 3: This embodiment provides a method for preparing polyphenol extracts from mulberry fruit using a betaine-anhydrous citric acid deep eutectic solvent, comprising the following steps: (1) The mulberry fruit was frozen at -20℃ and dried at -50℃ for 48 h in a vacuum freeze dryer. The powder was then ground by machine and passed through an 80-mesh sieve to obtain freeze-dried mulberry fruit powder.

[0067] (2) The betaine-anhydrous citric acid deep eutectic solvent is a mixture of betaine and anhydrous citric acid in a molar ratio of 1:1, with a water content of 30%~50%.

[0068] Take freeze-dried mulberry fruit powder and betaine-anhydrous citric acid deep eutectic solvent, mix them evenly and dissolve them at a material-liquid ratio of 1:40~1:60 (g:mL), and extract them in an ultrasonic extraction device by water bath method. The ultrasonic power is 360 W, the ultrasonic time is 10~30 min, and the ultrasonic temperature is 50℃.

[0069] (3) After sonication, centrifuge at 8000 r / min for 10 minutes, filter the liquid in the upper layer through a 0.22~0.25 μm filter membrane, and use the filtrate as the deep co-crystal extract of mulberry polyphenols.

[0070] The polyphenol content of the obtained extract was determined by the Folin-Ciocalteu method, in triplicate.

[0071] This embodiment aims to further determine the optimal process parameters for extracting mulberry polyphenols. (1) Response surface optimization Based on the single-factor experimental results of Example 2, the optimal parameters for ultrasonic extraction are: ultrasonic temperature 50℃, ultrasonic power 360W, ultrasonic extraction time 10~30 min, material-liquid ratio 1:40~1:60 (g / mL), and deep eutectic solvent water content 30~50%.

[0072] The factors and levels of the response surface methodology are shown in Table 2.

[0073] Table 2 Factor and Level Design of Response Surface Experiments Based on Table 2, the Box-Benhnken method on Design Expert 8.0.6 software was used to perform data regression analysis to determine the optimal parameters for the extraction process of mulberry polyphenols. The experimental design and results are shown in Table 3.

[0074] Table 3 Response Surface Experimental Design and Results The multiple regression equations for the total phenol content in the deep eutectic extract of mulberry polyphenols on extraction time (A), solid-liquid ratio (B), and water content (C) were obtained using software fitting: Polyphenol extraction rate = 74.06 - 0.53*A + 0.73*B + 1.37*C + 1.36*A*B + 0.21*A*C + 0.95*B*C - 2.27*A^2 - 6.08*B^2 - 2.77*C^2.

[0075] The analysis of variance for the regression model is shown in Table 4.

[0076] Table 4. Analysis of Variance of Regression Model As shown in Table 4, the p-value of the model is greater than 0.0001, indicating that the model is significant and has a good fit. The p-value of the lack of fit term for total phenols is 0.8644 > 0.05, indicating that the lack of fit is not significant and the model fit is good. The R-squared determination coefficient of total phenols is 0.9444, and the corrected R-squared determination coefficient Adj is 0.8729.

[0077] The above results indicate that the response surface model data fits well with the actual deep eutectic extraction of mulberry polyphenols, and can accurately reflect the relationship between model factors and actual conditions.

[0078] (2) Characterization of response surface interaction results Three-dimensional response surface plots and two-dimensional contour plots provide intuitive graphical interpretations of regression equations. Three-dimensional response surface plots can show the mutual influence of independent variables on the response variable; two-dimensional response contour plots are used to clarify the interactions between independent variables and the significance of these interactions: if the contour plot is circular, it indicates that the interaction between corresponding variables is negligible; if it is elliptical, it indicates that there is a significant interaction between corresponding variables. Three-dimensional response surface plots can also visually demonstrate the strength of the interaction between experimental factors: the steeper the surface and the greater the curvature, the higher the level of interaction.

[0079] All three sets of interaction diagrams show that the extraction rate has a significant nonlinear response to various factors, and the material-liquid ratio and water content have the most significant impact on the extraction rate. The ultrasonic time is conducive to dissolution in a short range, and the polyphenol extraction rate first increases and then decreases with time, indicating that excessive ultrasonic time will lead to degradation or re-adsorption of the target components.

[0080] Figure 6 To illustrate the effect of ultrasonic time (A) and material-to-liquid ratio (B) on the extraction rate of mulberry polyphenols, the following methods were used: Figure 6 It can be seen that when the water content is constant, as the solid-liquid ratio increases from low to high, the extraction rate initially rises and then plateaus or declines. The ultrasonic time is sensitive to improving the extraction rate in a short period of about 10–20 minutes, but declines after exceeding a threshold of about 20–30 minutes. This phenomenon indicates that the solid-liquid ratio determines the contact efficiency between the solvent and the solid phase, and while ultrasonic time can promote cell rupture and dissolution, it may also reduce the effective yield due to thermal effects or degradation caused by free radicals.

[0081] Figure 7 The effects of ultrasound time (A) and BC-1 moisture content (C) on the extraction rate of mulberry polyphenols were investigated. Figure 7 It can be seen that, under a fixed material-to-liquid ratio, the water content exhibits a significant peak response to the extraction rate, with moderate water content being optimal. When the water content is too low, the viscosity of DES is high, and diffusion is limited; when the water content is too high, the solvent polarity is close to that of water, disrupting the hydrogen bonding between DES and polyphenols and reducing solubility. Therefore, there exists an optimal water content window, which in this study was approximately 40%, within which the positive effect of ultrasonic time is most pronounced.

[0082] Figure 8 To illustrate the effects of the material-to-liquid ratio (B) and the water content of BC-1 (C) on the extraction rate of mulberry polyphenols, the following methods were used: Figure 8 It can be seen that the interaction between the solid-liquid ratio and water content shows that increasing the water content has limited effect on improving the extraction rate at low solid-liquid ratios; while at medium to high solid-liquid ratios, a moderate water content can significantly amplify the extraction efficiency. This interaction suggests a synergistic effect—sufficient solvent volume adjusted by water content, combined with appropriate solvent polarity, can maximize polyphenol dissolution.

[0083] (3) Determination and verification of optimal process conditions Response surface methodology optimization yielded the optimal extraction conditions for mulberry polyphenol deep eutectic extract as follows: extraction time 19.15 min, solid-liquid ratio 1 g: 50.70 mL, betaine-anhydrous citric acid deep eutectic solvent with a water content of 42.56%, and predicted total phenol content of 74.28 mg / g.

[0084] Under the above-described response surface methodology, this embodiment provides the following method for preparing polyphenol extracts from mulberry fruit for verification: Mulberry fruits were frozen at -20℃ and dried at -50℃ for 48 h in a vacuum freeze dryer. The resulting powder was ground and passed through an 80-mesh sieve to obtain mulberry fruit freeze-dried powder. The betaine-anhydrous citric acid deep eutectic solvent was a mixture of betaine and anhydrous citric acid at a molar ratio of 1:1, with a water content of 42.56%. The mulberry fruit freeze-dried powder and the betaine-anhydrous citric acid deep eutectic solvent were uniformly mixed and dissolved at a material-to-liquid ratio of 1:50.70 (g:mL). Extraction was performed using an ultrasonic extraction device via a water bath method. The ultrasonic power was 360 W, the ultrasonic time was 19.15 min, and the ultrasonic temperature was 50℃. After ultrasonication, the mixture was centrifuged at 8000 r / min for 10 minutes. The supernatant was filtered through a 0.22–0.25 μm filter membrane, and the resulting filtrate was used as the mulberry polyphenol deep eutectic extract.

[0085] The polyphenol content was 73.51 mg / g obtained by the Folin-Ciocalteu method. The result was well-fitted with the response surface methodology, indicating that the model can predict the polyphenol content well.

[0086] Extraction effect and packaging example of the obtained mulberry polyphenol deep eutectic extract Figure 12 As shown, by Figure 12 It can be seen that the extract from the ethanol extraction group has a darker color, while the mulberry polyphenol deep eutectic extract obtained by the method of this invention has a brighter color and exhibits its natural purplish-red hue.

[0087] The prepared mulberry polyphenol deep eutectic extract was subjected to the following performance tests: (1) Antioxidant activity determination Mulberry polyphenol deep eutectic extract was diluted with distilled water to a specified concentration to obtain diluted mulberry polyphenol deep eutectic extract solutions with concentrations of 2, 1, 0.5, 0.25, and 0.125 mg / mL, respectively, for antioxidant activity experiments.

[0088] (1-1) Determination of DPPH free radical scavenging rate Weigh 2.3 mg of DPPH reagent, dissolve it in ethanol, and bring the volume to 50 mL. Store in the dark at low temperature for later use. When using the DPPH solution, repeatedly calibrate the absorbance of the solution using a UV spectrophotometer to ensure that the OD value of the prepared DPPH solution is between 1.2 and 1.3 at the absorption peak of λ=517 nm.

[0089] For each concentration gradient sample, three centrifuge tubes were used to perform parallel experiments as shown in Table 5. The reaction was carried out in the dark for 30 min. Ionized water was used as a blank control and vitamin C (VC) was used as a positive control. The final OD value of the reaction was measured at λ=517 nm.

[0090] Calculate its clearance rate using the following formula: in: A1: OD value of the sample after it has fully reacted with DPPH; A2: OD value after the sample has fully reacted with distilled water: A3: OD value after distilled water and DPPH have fully reacted; A0: OD value of distilled water at λ=517 nm.

[0091] Table 5 Experimental parameters for DPPH removal rate The DPPH scavenging rate of mulberry polyphenol deep co-crystal extract is as follows: Figure 9 As shown, DPPH scavenging rate was tested using a gradient of sample concentrations from 0.125 to 2 mg / mL, with an equivalent concentration of VC used as a positive control.

[0092] Depend on Figure 9 It can be seen that the DPPH scavenging rate of the mulberry polyphenol deep cocrystal extract is positively correlated with its concentration; the DPPH scavenging rate increases with increasing sample concentration. At a polyphenol concentration of 2 mg / mL, the sample achieved a DPPH scavenging rate of 90.51%. The ethanol-extracted samples showed a similar concentration-dependent trend, with an overall scavenging rate lower than that of the DES-extracted group. At a polyphenol concentration of 2 mg / mL, the DPPH scavenging rate of the ethanol sample was only 71.23%, lower than the 90.51% of the DES sample group. Although the mulberry polyphenol deep cocrystal extract did not exhibit the strong DPPH scavenging rate of the VC positive control, the experimental results indicate that the mulberry polyphenol deep cocrystal extract prepared according to Example 3 possesses excellent antioxidant activity.

[0093] (1-2) Determination of ABTS free radical scavenging rate An ABTS cation radical working solution was prepared by mixing 7 mmol / L ABTS solution with 245 mmol / L potassium persulfate at a volume ratio of 1:1, and then stored under light-protected conditions for 12 hours before use.

[0094] Take a sample of ABTS cationic free radical working solution and dilute it with deionized water to an absorbance of approximately 0.70 ± 0.02. Following Table 6 below, using vitamin C (VC) as a positive control, perform parallel experiments with three centrifuge tubes for each concentration sample. Allow the samples to react completely at room temperature in the dark for 6 minutes, and measure the OD value at λ = 734 nm.

[0095] Calculate the ABTS free radical scavenging rate (%) for each group of samples according to the following formula: in, A1: OD value of the sample after it has fully reacted with ABTS; A2: OD value after the sample has fully reacted with distilled water: A3: OD value after distilled water and ABTS have fully reacted; A0: OD value of distilled water at λ=734 nm.

[0096] Table 6 Experimental Procedure for ABTS Scavenging Rate The ABTS scavenging rate of the deep co-crystal extract of mulberry polyphenols is shown in Figure 10. The ABTS scavenging rate was tested using a gradient of samples with concentrations ranging from 0.125 to 2 mg / mL, with an equivalent concentration of vitamin C as a positive control.

[0097] Depend on Figure 10 It can be seen that the ABTS scavenging rate of the mulberry polyphenol deep co-crystal extract is positively correlated with concentration; the ABTS scavenging rate increases with increasing sample concentration. At a polyphenol concentration of 2 mg / mL, the sample achieves an ABTS scavenging rate of 97.89%. The ethanol-extracted samples show a similar concentration-dependent trend, with an overall scavenging rate lower than that of the DES-extracted group. At low concentrations, the ABTS scavenging rates of ethanol and DES samples are similar, but as the concentration increases, the ABTS scavenging rate of the DES sample group surpasses that of the ethanol sample group. At a polyphenol concentration of 2 mg / mL, the DPPH scavenging rate of the ethanol sample is only 83.88%, lower than the 97.89% of the DES sample group. Although the mulberry polyphenol deep co-crystal extract does not exhibit the strong ABTS scavenging rate of the VC positive control, the experimental results indicate that the mulberry polyphenol deep co-crystal extract prepared according to Example 3 possesses excellent antioxidant activity.

[0098] (2) Staining performance determination (2-1) Hair dyeing effect Hair sample preparation: Purchase black human hair, wash it with shampoo and air dry it.

[0099] Bleaching treatment: Bleach with 6% hydrogen peroxide + 10% potassium persulfate (hair to bleach ratio is hair: bleach = 1:25w / v) for 10 minutes until the hair turns yellow.

[0100] Standardized processing: Cut the bleached hair into 3±0.5 cm lengths, tie them with rubber bands, each bundle weighing approximately 0.5g, and store at 25℃±2℃.

[0101] Table 7. Preparation of hair dye Prepare 20 mL hair dye solutions (numbers 1-4) according to Table 7 for hair dyeing. Prepare samples of equal concentration of deep eutectic extract and ethanol extract for hair dyeing experiments. Apply the hair dye solution evenly to bleached hair, wrap it for 30 minutes, and gently massage the hair to promote dyeing. After completing the dyeing process, rinse off any excess dye with water, air dry the hair, and then use an XD-110 colorimeter to test the dyeing effect. Perform three parallel tests for evaluation.

[0102] The hair dyeing effect is shown in Table 8. The total color deviation of the three-dimensional differences in L (brightness), a (red-green axis), and b (yellow-blue axis) is used to evaluate the hair dyeing effect.

[0103] L (lightness / brightness parameter) is one of the core dimensions, directly reflecting the "brightness" of the sample. The larger the L value, the brighter and lighter the sample; the smaller the value, the darker and deeper the sample. That is, a decrease in the L value can reflect the effect of hair dye in covering gray hair and dyeing hair black.

[0104] Table 8 Evaluation of Hair Dyeing Effects The hair dyeing effects are shown in Table 8. Dye group #1 showed the best dyeing effect on bleached hair and the most significant improvement in hair brightness. The DES sample group reduced the L value of bleached hair to 33.12 ± 0.13, while the ethanol sample group reduced the L value of bleached hair to the lowest level of 37.81 ± 0.32. The dyeing effect of the ethanol sample group was not as good as that of the DES extract sample group. The mulberry polyphenol deep co-crystal extract significantly altered the L value, resulting in the lowest possible L value for the hair, and thus had the best effect on covering gray hair and dyeing hair black.

[0105] Therefore, 10 mL of mulberry polyphenol deep eutectic extract, 5 mL of 5% ascorbic acid as a developing agent, and 5 mL of 10% ferric chloride solution as a mordant were mixed to form 20 mL of hair dye as the optimal group for subsequent color fastness testing.

[0106] (2-2) Color fastness Mix 10 mL of mulberry polyphenol deep eutectic extract, 5 mL of 5% ascorbic acid as a developing agent, and 5 mL of 10% ferric chloride solution as a mordant to form 20 mL of hair dye. Apply the dye to bleached hair for 30 minutes. After the treatment, wash off the excess dye and let it air dry.

[0107] The dyed hair sample was placed in a 50 mL centrifuge tube, and 30 mL of distilled water was added. The mixture was stirred thoroughly to wash the hair. After washing for 1 minute, the hair was removed and dried. The color difference of the hair was then tested using an XD110 colorimeter. This process was repeated multiple times to test the color fastness of the hair dye. The color fastness test results are shown in Table 9 below. Figure 11 As shown.

[0108] Table 9 Colorfastness Test Results From the data in Table 9 and Figure 11 The results show that the hair dye and corresponding hair treatment process prepared according to this embodiment have good hair dyeing effect and color fastness. After 10 water washing tests, it still has a black color and is significantly different from the bleached hair control group. It has a good effect of covering black hair and maintaining color fastness.

[0109] This invention applies the aforementioned plant extracts to hair dyeing. The polyphenols and / or pigments within them possess natural color-developing properties, capable of complexing with metal ions to form metal polyphenol networks (MPNs) for color development, thus achieving a long-lasting and stable dyeing effect. Simultaneously, the antioxidant properties of polyphenols help protect hair from free radical damage, enhancing the shine and health of the dyed hair. Compared to traditional chemical dyes, the deep eutectic polyphenol hair dyeing system is gentler, replacing traditional synthetic dyes containing harmful ingredients such as ammonia and phenylenediamine.

[0110] The key to metal-polyphenol network (MPN) hair dyeing lies in its ability to form a stable color layer by depositing it on the hair surface or in the cuticle, providing long-lasting color while also conditioning the hair. It is particularly suitable for dyeing natural black hair, maintaining good colorfastness even after 10 washes. It reduces irritation to the hair and scalp, aligning with the trend towards green and natural hair dyeing.

[0111] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing a plant extract rich in polyphenols, characterized in that, Includes the following steps: S1. Pre-treat plant materials rich in polyphenols and / or pigments to obtain plant powder; S2. Mix the plant powder obtained in step S1 with a deep eutectic solvent and extract to obtain an extract; S3. Separate and purify the extract obtained in step S2 to obtain the plant extract.

2. The method for preparing polyphenol-rich plant extracts according to claim 1, characterized in that, In step S1, the plant material rich in polyphenols and / or pigments is selected from any one or more combinations of berry plants, vegetable plants, flower plants, and herbaceous plants.

3. The method for preparing polyphenol-rich plant extracts according to claim 1, characterized in that, In step S1, the pretreatment method is selected from any one or more combinations of drying, pulverizing, grinding, shearing, pressing, freeze drying, rotary evaporation, low-temperature vacuum concentration, and spray drying.

4. The method for preparing polyphenol-rich plant extracts according to claim 1, characterized in that, In step S2, the deep eutectic solvent is composed of hydrogen bond acceptors and hydrogen bond donors in a molar ratio of 1:(0.5~5), and the water content of the deep eutectic solvent is 0~80%. The hydrogen bond acceptor is selected from any one or more combinations of betaine, choline chloride, acetylcholine, and carnitine; the hydrogen bond donor is selected from any one or more combinations of organic acids, polyols, urea, and sugars.

5. The method for preparing polyphenol-rich plant extracts according to claim 4, characterized in that, In step S2, the organic acid is selected from any one or more of formic acid, acetic acid, propionic acid, butyric acid, valeric acid, lactic acid, glycolic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, malic acid, tartaric acid, citric acid, benzoic acid, phenylacetic acid, 3-phenylpropionic acid, salicylic acid, gallic acid, caffeic acid, ferulic acid, and gluconic acid.

6. The method for preparing polyphenol-rich plant extracts according to claim 1, characterized in that, In step S2, the extraction method can be selected from any one of ultrasound-assisted extraction, microwave-assisted extraction, or heating and stirring extraction. The extraction parameters were: material-to-liquid ratio 1 g : (20–100) mL, extraction time (10–60) min, and extraction temperature (30–70) ℃. The extraction power of ultrasound-assisted extraction and microwave-assisted extraction is (200~500) W.

7. The method for preparing polyphenol-rich plant extracts according to claim 1, characterized in that, Step S3, the separation and purification method is as follows: centrifuge the extract, collect the supernatant and then filter it; The centrifugation parameters are: rotation speed (5000~12000) r / min, time (5~20) min; The filtration parameters are: filtration through a 0.1–0.5 μm microporous membrane.

8. A plant polyphenol extract, characterized in that, It is obtained by the preparation method described in any one of claims 1 to 7.

9. The application of the plant polyphenol extract as described in claim 8 in antioxidant products and hair dye products.

10. A hair dyeing product, characterized in that, Raw materials including the following volume parts: 5-20 parts of plant polyphenol extract; 3-8 parts of development agent; Mordant 3-8 parts; The development agent is selected from any one or a combination of hydrogen peroxide solution and ascorbic acid aqueous solution; The mordant is a metal ion salt solution, and the metal ions are selected from Fe. 2+ Fe 3+ Cu 2+ Al 3+ Any one or combination thereof.

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