Dihydroquercetin-collagen supermolecular system and preparation method and application thereof

Abstract

This invention discloses a dihydroquercetin-collagen supramolecular system, its preparation method, and its applications. The dihydroquercetin-collagen supramolecular system comprises dihydroquercetin and collagen, wherein dihydroquercetin and collagen are linked by non-covalent bonds, and the mass ratio of dihydroquercetin to collagen is (1-5):1. This invention uses a supramolecular preparation method to obtain the dihydroquercetin-collagen supramolecular system, which exhibits high water solubility, stability, bioactivity, and anti-hair loss and anti-aging effects.

Description

Technical Field

[0001] This invention relates to a collagen supramolecular system, particularly a dihydroquercetin-collagen supramolecular system, its preparation method, and its applications. Background Technology

[0002] Collagen, an important biomacromolecule, is widely distributed in connective tissues and is a major structural protein of the extracellular matrix. Its unique triple-helix structure endows it with excellent biocompatibility, biodegradability, and mechanical properties, showing broad application prospects in tissue engineering, drug delivery, wound dressings, and functional materials. In recent years, constructing supramolecular complexes or cocrystal materials with specific structures and functions by regulating the non-covalent interactions between collagen and functional molecules has become a research hotspot in the interdisciplinary field of biomaterials and chemical biology. This molecular-level recognition and assembly can not only expand the physicochemical properties of collagen but also promise to endow it with new biological functions, such as promoting cell adhesion, regulating release behavior, or enhancing mechanical properties.

[0003] However, collagen alone does not have typical antioxidant structures such as phenolic hydroxyl groups and conjugated double bonds, and therefore does not have the ability to directly scavenge free radicals and quench reactive oxygen species.

[0004] Dihydroquercetin, also known as piperidine, is a reduced derivative of quercetin. It is a naturally occurring flavonoid compound found primarily in larch, grape seeds, citrus fruits, and other plants, possessing various biological activities including antioxidant, anti-inflammatory, and liver-protective properties.

[0005] Dihydroquercetin has very low solubility at room temperature, is slightly soluble in cold water, and is almost insoluble in nonpolar solvents such as benzene and hexane. It cannot be used directly in aqueous formulations. After oral administration, it dissolves slowly, is poorly absorbed, and has low bioavailability, making it difficult to achieve effective blood drug concentrations. This severely limits its absorption and utilization in vivo. Furthermore, dihydroquercetin is easily oxidized and is sensitive to light. Formulations require complete protection from light, nitrogen purging, or the addition of antioxidants, increasing process and packaging costs. The finished product has a short shelf life and is prone to darkening, layering, and reduced activity.

[0006] Existing technologies for improving the water solubility and stability of dihydroquercetin mainly include nanodispersion, crystal engineering, chemical modification, and the formation of inclusion complexes with cyclodextrins, lecithin, etc. While cyclodextrin inclusion technology can improve solubility to some extent, it suffers from limited inclusion rates and poor biocompatibility. Chemical modification methods may disrupt the natural active structure of dihydroquercetin, reducing its biological efficacy. Nanodispersion technology involves complex preparation processes and high costs, hindering large-scale production.

[0007] Currently, existing technologies for combining dihydroquercetin and collagen disclose the physical mixing of the two, but these methods cannot effectively address the inherent problems of both dihydroquercetin and collagen themselves. Another method disclosed involves using a metal vapor synthesis technique to first synthesize a conjugate of silver nanoparticles and dihydroquercetin, which is then combined with collagen to construct a collagen-dihydroquercetin-silver nanoparticle ternary composite material. This ternary composite material possesses broad-spectrum antibacterial activity, selective antitumor activity, potent antioxidant capacity, and good biocompatibility.

[0008] However, there are no reports on the preparation of collagen-dihydroquercetin supramolecular collagen by directly combining dihydroquercetin with collagen using supramolecular methods. Summary of the Invention

[0009] The purpose of this invention is to provide a dihydroquercetin-collagen supramolecular system, its preparation method, and its applications. This invention utilizes a supramolecular preparation method to obtain a dihydroquercetin-collagen supramolecular system, which exhibits high water solubility, stability, and bioactivity, as well as anti-hair loss and anti-aging effects.

[0010] The technical solution of the present invention is a dihydroquercetin-collagen supramolecular system, comprising dihydroquercetin and collagen, wherein dihydroquercetin and collagen are linked by non-covalent bonds, and the mass ratio of dihydroquercetin to collagen is (1-5):1.

[0011] In the aforementioned dihydroquercetin-collagen supramolecular system, the collagen is animal collagen, plant collagen, hydrolyzed collagen, recombinant type I collagen, recombinant type III collagen, recombinant type VI collagen, recombinant type XVII collagen, or polypeptide.

[0012] In the aforementioned dihydroquercetin-collagen supramolecular system, the dihydroquercetin is (2R,3R)-dihydroquercetin.

[0013] In the aforementioned dihydroquercetin-collagen supramolecular system, the dihydroquercetin-collagen supramolecular system includes a solid system, a liquid system, a semi-solid system, or a gaseous system.

[0014] In the aforementioned dihydroquercetin-collagen supramolecular system, the preparation method of the solid system includes the following steps:

[0015] (1) Preparation of collagen solution;

[0016] (2) Activation of dihydroquercetin: Take dihydroquercetin powder, add anhydrous ethanol, and activate by ultrasonication to obtain dihydroquercetin activation solution;

[0017] (3) Mixing and dispersing: Disperse the collagen solution and dihydroquercetin activating solution evenly in proportion to obtain a mixed dispersion;

[0018] (4) Isothermal self-assembly reaction: The mixed dispersion is stirred in a gradient and protected by an inert gas throughout the process to obtain a supramolecular precursor;

[0019] (5) Gradient solvent evaporation: The supramolecular precursor is evaporated in stages to ensure that the residual ethanol content in the system is ≤5%;

[0020] (6) Stepwise drying and curing: The system after solvent evaporation is dried stepwise to obtain a dihydroquercetin-collagen supramolecular solid system.

[0021] In the aforementioned dihydroquercetin-collagen supramolecular system, step (1) specifically involves: taking collagen powder, adding water, and stirring in a water bath at 35~45℃ until completely dissolved to obtain a collagen solution.

[0022] In the aforementioned dihydroquercetin-collagen supramolecular system, in step (2), the ultrasonic conditions are: ultrasonic frequency of 40-60kHz, ultrasonic temperature of 30-40℃, and ultrasonic time of 15-20min.

[0023] In the aforementioned dihydroquercetin-collagen supramolecular system, in step (3), the collagen solution and the dihydroquercetin activation solution are evenly dispersed according to the mass ratio of dihydroquercetin to collagen of (1-5):1.

[0024] In the aforementioned dihydroquercetin-collagen supramolecular system, the dispersion conditions in step (3) are: rotation speed of 500-800 r / min, temperature of 35-45℃, and time of 20-30 min.

[0025] In the aforementioned dihydroquercetin-collagen supramolecular system, in step (4), the gradient stirring method is as follows: the stirring rate is 300 r / min for the first 30 min, 200 r / min for the middle 60 min, and 150 r / min for the last 30 min, with a total reaction time of 2 h and a stirring temperature of 40~50℃.

[0026] In the aforementioned dihydroquercetin-collagen supramolecular system, in step (5), the volatilization is divided into stages: the volatilization rate is 1-1.5 g / h for the first 2 hours; the volatilization rate is 0.8-1 g / h for the middle 2 hours; and the volatilization rate is 0.5-0.8 g / h for the last 2 hours, with a total volatilization time of 6 hours.

[0027] In the aforementioned dihydroquercetin-collagen supramolecular system, step (6) is a stepwise drying process: first, drying under ventilation conditions for 2-6 hours to remove residual free ethanol; then drying under vacuum conditions for 2-6 hours until the system is completely dry.

[0028] In the aforementioned dihydroquercetin-collagen supramolecular system, the liquid system further includes water and a hydrophilic medium, wherein the mass percentage of the hydrophilic medium in the liquid system is 5%-80%.

[0029] This liquid system introduces a hydrophilic medium into the aqueous phase that can form hydrogen bonds with dihydroquercetin and / or collagen, enabling dihydroquercetin and collagen to form stable intermolecular association or self-assembled structures through non-covalent bonding. This allows a stable liquid composition to be obtained without completely removing the organic solvent and performing a curing step.

[0030] In the aforementioned dihydroquercetin-collagen supramolecular system, the hydrophilic medium is a low-molecular-weight compound capable of providing hydrogen bond donors or acceptors, selected from at least one of polyols, sugar alcohols, and amide moisturizers.

[0031] In this system, the hydrophilic medium can participate in the hydrogen bonding between the phenolic hydroxyl groups of dihydroquercetin and the collagen peptide chains, thereby promoting the formation of intermolecular associated structures.

[0032] In the aforementioned dihydroquercetin-collagen supramolecular system, the preparation method of the liquid system includes the following steps:

[0033] (1) Preparation of collagen solution;

[0034] (2) Activation of dihydroquercetin: Take dihydroquercetin powder, add a hydrophilic medium, and disperse or dissolve it under stirring or ultrasonic conditions to obtain an activated dihydroquercetin solution.

[0035] (3) Mixing and dispersing: Disperse the collagen solution and dihydroquercetin activating solution evenly in proportion to obtain a mixed dispersion;

[0036] (4) Isothermal self-assembly reaction: The mixed dispersion was stirred in a gradient and protected by an inert gas throughout the process to obtain a dihydroquercetin-collagen supramolecular liquid system.

[0037] This dihydroquercetin-collagen supramolecular liquid system has an intermolecular association structure or a self-assembled structure, and its appearance is uniform.

[0038] In the aforementioned dihydroquercetin-collagen supramolecular system, step (1) specifically involves: taking collagen powder, adding water, and stirring in a water bath at 35~45℃ until completely dissolved to obtain a collagen solution.

[0039] In the aforementioned dihydroquercetin-collagen supramolecular system, in step (2), the ultrasonic conditions are: frequency of 40-60kHz, temperature of 30-40℃, and time of 15-20min.

[0040] In the aforementioned dihydroquercetin-collagen supramolecular system, in step (3), the collagen solution and the dihydroquercetin activation solution are evenly dispersed according to the mass ratio of dihydroquercetin to collagen of (1-5):1.

[0041] In the aforementioned dihydroquercetin-collagen supramolecular system, the dispersion conditions in step (3) are: rotation speed of 500-800 r / min, temperature of 35-45℃, and time of 20-30 min.

[0042] In the aforementioned dihydroquercetin-collagen supramolecular system, in step (4), the gradient stirring method is as follows: the stirring rate is 300 r / min for the first 30 min, 200 r / min for the middle 60 min, and 150 r / min for the last 30 min, with a total reaction time of 2 h.

[0043] The present invention also provides the application of the above-mentioned dihydroquercetin-collagen supramolecular system in the preparation of anti-hair loss and / or anti-aging products.

[0044] In the aforementioned applications, the anti-aging includes anti-oxidation and / or anti-wrinkle and firming.

[0045] In the aforementioned applications, the products include cosmetics, pharmaceuticals, or health foods.

[0046] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0047] This invention employs a supramolecular preparation method to prepare a dihydroquercetin-collagen supramolecular system. In this system, dihydroquercetin and collagen are linked by non-covalent bonds in a supramolecular form, effectively enhancing the water solubility, stability, and bioactivity of dihydroquercetin. Furthermore, it increases the expression level of β-catenin in dermal papilla cells, exhibits a high DPPH free radical scavenging rate, and enhances the expression level of Collage I in fibroblasts. This results in superior anti-hair loss, antioxidant, and anti-wrinkle firming abilities, surpassing the efficacy of pure dihydroquercetin and physical mixtures of dihydroquercetin and collagen. This system can be applied in cosmetics, pharmaceuticals, and health foods with anti-hair loss and anti-aging effects. Moreover, this supramolecular system is available in various dosage forms, including solid, liquid, semi-solid, and gaseous systems. Specific preparation methods for both solid and liquid dosage forms are provided to meet diverse production and formulation needs.

[0048] The raw materials used in this invention (collagen and dihydroquercetin) are all readily available for industrial production. The process is based on a basic framework and does not require additional high equipment investment. Costs are controllable through parameter optimization, making it suitable for large-scale production. Attached Figure Description

[0049] Figure 1This is a comparison XRD pattern of Example 1 in Experiment 1.

[0050] Figure 2 This is a comparison XRD pattern of Example 2 in Experiment 1.

[0051] Figure 3 This is a comparison XRD pattern of Example 5 in Experiment 1.

[0052] Figure 4 This is a comparison of the infrared spectra of the components in Experiment Example 2.

[0053] Figure 5 This is a comparison chart of thermogravimetric analyses of the components in Experiment Example 3.

[0054] Figure 6 It refers to the solubility of each component sample in aqueous solution under the condition of equal dihydroquercetin content in Experiment Example 4.

[0055] Figure 7 It refers to the stability of dihydroquercetin monomer in aqueous solution at a dihydroquercetin content of 0.1%.

[0056] Figure 8 It is the stability of the dihydroquercetin-collagen physical mixture in aqueous solution at a dihydroquercetin content of 0.1%.

[0057] Figure 9 It measures the stability of dihydroquercetin-collagen supramolecular molecules in aqueous solution at a dihydroquercetin content of 0.1%.

[0058] Figure 10 This is a bar chart showing the effects of different concentrations of DHT on the viability of dermal papilla cells.

[0059] Figure 11 This is a graph showing the results of detecting the viability of dermal papilla cells at different concentrations of supramolecular groups, physical mixture groups, and collagen groups.

[0060] Figure 12 This is an immunofluorescence staining image of β-catenin on dermal papilla cells at 10 ppm for supramolecular groups, physical mixture groups, and collagen groups.

[0061] Figure 13 This is a statistical graph showing the fluorescence intensity of β-catenin in dermal papilla cells at 10 ppm for the supramolecular group, physical mixture group, and collagen group. In the graph, # indicates a significant difference compared to the BC group, and "####" indicates a P-value < 0.0001; * indicates a significant difference compared to the NC group, and "*" indicates a P-value < 0.05.

[0062] Figure 14This is a graph showing the DPPH free radical scavenging rate of the supramolecular group, the physical mixture group, and the collagen group. In the graph, △ indicates the significance of the supramolecular group compared with the physical mixture group, and "△" means p<0.05; # indicates the significance of the supramolecular group compared with the collagen group, and "###" means p<0.001.

[0063] Figure 15 This is a graph showing the results of fibroblast viability testing at different concentrations of supramolecular groups, physical mixture groups, and collagen groups.

[0064] Figure 16 These are immunofluorescence staining images of fibroblasts with Collage I at different concentrations of supramolecular groups, physical mixture groups, and collagen groups.

[0065] Figure 17 This is a statistical graph showing the fluorescence intensity of Collage I in dermal papilla cells at 10 ppm for the supramolecular group, the physical mixture group, and the collagen group. In the graph, # indicates a significant difference compared to the BC group, "####" indicates a P-value < 0.0001; * indicates a significant difference compared to the NC group, "**" indicates a P-value < 0.01, "***" indicates a P-value < 0.001, and "****" indicates a P-value < 0.0001.

[0066] Figure 18 This is the XRD pattern of the dihydroquercetin-collagen supramolecular solid system of Comparative Example 1.

[0067] Figure 19 These are HPLC comparison chromatograms of dihydroquercetin after treatment using the methods described in Example 1 and Comparative Example 2, respectively.

[0068] Figure 20 This is a comparison of XRD patterns of the dihydroquercetin-collagen supramolecular solid system in Comparative Example 5. Detailed Implementation

[0069] The dihydroquercetin-collagen supramolecular system comprises dihydroquercetin and collagen, wherein dihydroquercetin and collagen are linked by non-covalent bonds in a mass ratio of (1-5):1.

[0070] The collagen used is animal collagen, plant collagen, recombinant type I collagen, recombinant type III collagen, recombinant type VI collagen, recombinant type XVII collagen, or polypeptide. The molecular weight of the collagen is 10-50 kDa, and the purity is ≥95%. Recombinant type I collagen, recombinant type III collagen, recombinant type VI collagen, and recombinant type XVII collagen can be prepared using a yeast eukaryotic expression system.

[0071] The dihydroquercetin is (2R,3R)-dihydroquercetin, which is an off-white or light yellow powder with an optical purity of ≥99%.

[0072] The dihydroquercetin-collagen supramolecular system includes solid, liquid, semi-solid, or gaseous systems.

[0073] The preparation method of the dihydroquercetin-collagen supramolecular solid system includes the following steps:

[0074] (1) Collagen dissolution: Take collagen powder, add 20-40 times its weight of deionized water, place it in a constant temperature water bath at 35-45℃, stir for 30-40 minutes until completely dissolved, and obtain a collagen solution for later use;

[0075] Stirring temperature controlled at 35-45℃ can ensure that collagen is fully dissolved, while avoiding the loss of its biological activity due to high temperature, and preserving its hemidesmosome-related active sites and cell adhesion activity.

[0076] (2) Activation of dihydroquercetin: Take dihydroquercetin powder, add 30-50 times its mass of anhydrous ethanol, and activate by ultrasonication at a frequency of 40-60 kHz, an ultrasonic temperature of 30-40 ℃, and an ultrasonic time of 15-20 min to obtain an activated dihydroquercetin solution for later use.

[0077] The ultrasonic activation temperature is controlled at 30-40℃ to avoid changes in its chiral configuration caused by high temperature, while breaking the hydrophobic interaction between its molecules and enhancing its reactivity with collagen.

[0078] (3) Mixing and dispersing: Accurately weigh the pretreated dihydroquercetin activation solution and collagen solution, and place them in a high-speed disperser at a mass ratio of dihydroquercetin to collagen (1-5):1. Disperse for 20-30 minutes at a speed of 500-800 r / min and a temperature of 35-45℃ to obtain a mixed dispersion. Ensure that the components are uniformly mixed to lay the foundation for self-assembly.

[0079] High-speed dispersion is carried out at a temperature of 35-45℃ to avoid the destruction of the α-dihydroquercetin conformation and the loss of recombinant collagen activity.

[0080] (4) Isothermal self-assembly reaction: The mixed dispersion was transferred to an isothermal water bath reactor and stirred at 40~50℃ using a gradient stirring method: the stirring rate was 300r / min for the first 30min, 200r / min for the middle 60min, and 150r / min for the last 30min, with a total reaction time of 2h; an inert gas was introduced for protection during the stirring process, with a flow rate of 50ml / min, to form a supramolecular precursor;

[0081] Gradient stirring can avoid local concentration unevenness and ensure that the self-assembly reaction is uniform and sufficient. Inert gas is introduced during stirring to prevent the oxidative degradation of dihydroquercetin and at the same time promote the full self-assembly of the two through non-covalent bonds.

[0082] (5) Gradient solvent evaporation: After the reaction is completed, the beaker is transferred to a high-precision heating stage and the temperature is set to 50℃ (temperature fluctuation ≤ ±1℃). A segmented evaporation mode is adopted: the first 2 hours are kept in a ventilated state and the evaporation rate is controlled at 1-1.5 g / h; the middle 2 hours are kept in a semi-closed state and the evaporation rate is controlled at 0.8-1 g / h; the last 2 hours are kept in a slightly closed state and the evaporation rate is controlled at 0.5-0.8 g / h. The total evaporation time is 6 hours, so that the residual ethanol in the system is ≤5%.

[0083] Segmented evaporation can prevent the collapse of the supramolecular structure caused by rapid ethanol evaporation, while also reducing raw material loss.

[0084] (6) Stepwise drying and curing: The system after ethanol evaporation is placed in a 50℃ precision oven and a stepwise drying mode is adopted: first, it is dried under ventilation for 4 hours to remove residual free ethanol; then it is dried under vacuum (vacuum degree -0.07MPa) for 4 hours to ensure that the system is completely dry; finally, it is naturally cooled to room temperature to obtain supramolecular solid crude product.

[0085] Step-by-step drying can avoid damage to the supramolecular structure caused by high temperatures, thus ensuring product stability.

[0086] (7) Post-processing and quality control: The crude supramolecular solid was ground using a planetary ball mill at a speed of 250 r / min for 30 min and then passed through a 200-mesh sieve to obtain supramolecular powder.

[0087] The dihydroquercetin-collagen supramolecular liquid system includes dihydroquercetin, collagen, water, and a hydrophilic medium. Dihydroquercetin and collagen are connected by non-covalent bonds, and the mass ratio of dihydroquercetin to collagen is (1-5):1. The entire system is an intermolecular association structure or a self-assembled structure.

[0088] The hydrophilic medium is selected from one or more of the following:

[0089] Polyols: glycerol, propylene glycol, 1,3-propanediol, methylpropanediol, butanediol, pentanediol, hexanediol, 1,2-pentanediol, etc.

[0090] Sugar alcohols: sorbitol, xylitol, erythritol, etc.;

[0091] Amides: urea, hydroxyethyl urea, etc.;

[0092] Other hydrophilic small molecules capable of hydrogen bonding.

[0093] The hydrophilic medium accounts for 5%-80% of the mass percentage in the system.

[0094] The preparation method of the dihydroquercetin-collagen supramolecular liquid system includes the following steps:

[0095] (1) Preparation of collagen solution: Take collagen powder, add 20-40 times its weight of deionized water, place it in a constant temperature water bath at 35-45℃, stir for 30-40 minutes until completely dissolved, and obtain collagen solution;

[0096] (2) Activation of dihydroquercetin: Dihydroquercetin is added to a mixed solvent containing a hydrophilic medium and dispersed or dissolved under stirring or ultrasonic conditions. The ultrasonic conditions are: frequency of 40-60kHz, temperature of 30-40℃, and time of 15-20min to obtain an activated dihydroquercetin solution.

[0097] (3) Mixing and dispersing: Mix the collagen solution and the dihydroquercetin activation solution at a mass ratio of dihydroquercetin to collagen of (1-5):1, and stir for 20-30 minutes at a speed of 500-800 r / min and 35-45℃ to form a uniform mixed dispersion.

[0098] (4) Isothermal self-assembly reaction: The temperature was maintained in the same way as the mixing and dispersion steps, and the mixture was stirred at an isothermal gradient. The stirring rate was 300 r / min for the first 30 min, 200 r / min for the middle 60 min, and 150 r / min for the last 30 min. The total reaction time was 2 h. The entire process was carried out under inert gas protection to obtain the dihydroquercetin-collagen supramolecular liquid system.

[0099] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention. In the following embodiments, all raw materials used are commercially available conventional products.

[0100] Example 1:

[0101] The preparation method of the dihydroquercetin-collagen supramolecular solid system includes the following steps:

[0102] (1) Collagen dissolution: Take 0.2g of recombinant type XVII collagen powder, add 4g of deionized water, place in a 35℃ constant temperature water bath, stir for 30min until completely dissolved, and obtain collagen solution for later use.

[0103] (2) Activation of dihydroquercetin: Take 1g of (2R,3R)-dihydroquercetin powder, add 30 times its mass of anhydrous ethanol (30g), and activate by ultrasonication at a frequency of 40kHz, an ultrasonic temperature of 30℃, and an ultrasonic time of 15min to obtain an activated dihydroquercetin solution for later use.

[0104] (3) Mixing and dispersing: Accurately weigh the above dihydroquercetin activation solution and collagen solution, and place them in a high-speed disperser at a mass ratio of 5:1 for dihydroquercetin to collagen. Disperse for 20 minutes at a speed of 500 r / min and a temperature of 35℃ to obtain a mixed dispersion.

[0105] (4) Isothermal self-assembly reaction: The mixed dispersion was transferred to an isothermal water bath reactor and stirred at 45°C using a programmed stirring mode: the stirring rate was 300 r / min for the first 30 min, 200 r / min for the middle 60 min, and 150 r / min for the last 30 min, with a total reaction time of 2 h; nitrogen gas was introduced for protection during the stirring process at a flow rate of 50 ml / min to obtain the supramolecular precursor.

[0106] (5) Gradient solvent evaporation: After the reaction was completed, the beaker was transferred to a high-precision heating stage and the temperature was set to 50℃ (temperature fluctuation ±0.8℃). A segmented evaporation mode was adopted: the first 2 hours were kept in a ventilated state with an evaporation rate of 1.0 g / h; the middle 2 hours were kept in a semi-closed state with an evaporation rate of 0.8 g / h; and the last 2 hours were kept in a slightly closed state with an evaporation rate of 0.5 g / h. The total evaporation time was 6 hours, and the residual ethanol content of the system was measured to be 4.8%.

[0107] (6) Step-by-step drying and curing: After the ethanol evaporates, the system is placed in a 50°C precision oven and a step-by-step drying mode is adopted: first, it is dried for 4 hours under ventilation conditions to remove residual free ethanol; then it is dried for 4 hours under vacuum conditions (vacuum degree -0.07MPa) to ensure that the system is completely dry; finally, it is naturally cooled to room temperature to obtain the supramolecular solid crude product.

[0108] (7) Post-processing and quality control: The supramolecular solid crude product is ground by a planetary ball mill at a speed of 250 r / min for 30 min and then passed through a 200 mesh sieve to obtain the supramolecular powder product.

[0109] Tests showed that the water solubility of the supramolecular powder product was 30.5 mg / ml, and the supramolecular structure was stable, with no clumping or degradation.

[0110] Example 2:

[0111] The preparation method of the dihydroquercetin-collagen supramolecular solid system includes the following steps:

[0112] (1) Collagen dissolution: Take 0.3g of recombinant type XVII collagen powder, add 40 times its weight of deionized water (12g), place it in a constant temperature water bath at 45℃, stir for 40min until completely dissolved, and obtain collagen solution for later use.

[0113] (2) Activation of dihydroquercetin: Take 0.3g of (2R,3R)-dihydroquercetin powder, add 50 times its mass of anhydrous ethanol (15g), and activate by ultrasonication at a frequency of 60kHz, an ultrasonic temperature of 40℃, and an ultrasonic time of 20min to obtain an activated dihydroquercetin solution for later use.

[0114] (3) Mixing and dispersing: Accurately weigh the above dihydroquercetin activation solution and collagen solution, and place them in a high-speed disperser at a mass ratio of (2R,3R)-dihydroquercetin to collagen of 5:1. Disperse for 30 minutes at a speed of 800 r / min and a temperature of 45℃ to obtain a mixed dispersion.

[0115] (4) Isothermal self-assembly reaction: The mixed dispersion was transferred to an isothermal water bath reactor and stirred at 45°C using a programmed stirring mode: the stirring rate was 300 r / min for the first 30 min, 200 r / min for the middle 60 min, and 150 r / min for the last 30 min, with a total reaction time of 2 h; nitrogen gas was introduced for protection during the stirring process at a flow rate of 50 ml / min to obtain the supramolecular precursor.

[0116] (5) Gradient solvent evaporation: After the reaction was completed, the beaker was transferred to a high-precision heating stage and the temperature was set to 50℃ (temperature fluctuation ±0.7℃). A segmented evaporation mode was adopted: the first 2 hours were kept in a ventilated state with an evaporation rate of 1.5 g / h; the middle 2 hours were kept in a semi-closed state with an evaporation rate of 1.0 g / h; and the last 2 hours were kept in a slightly closed state with an evaporation rate of 0.8 g / h. The total evaporation time was 6 hours, and the residual ethanol content of the system was measured to be 2.9%.

[0117] (6) Step-by-step drying and curing: After the ethanol evaporates, the system is placed in a 50°C precision oven and a step-by-step drying mode is adopted: first, it is dried for 4 hours under ventilation conditions to remove residual free ethanol; then it is dried for 4 hours under vacuum conditions (vacuum degree -0.07MPa) to ensure that the system is completely dry; finally, it is naturally cooled to room temperature to obtain the supramolecular solid crude product.

[0118] (7) Post-processing and quality control: The supramolecular solid crude product is ground by a planetary ball mill at a speed of 250 r / min for 30 min and then passed through a 200 mesh sieve to obtain the supramolecular powder product.

[0119] Tests showed that the water solubility of the supramolecular powder product was 36.7 mg / ml, and the supramolecular structure was stable with no clumping or degradation.

[0120] Example 3:

[0121] The preparation method of the dihydroquercetin-collagen supramolecular solid system includes the following steps:

[0122] (1) Collagen dissolution: Take 0.12g of recombinant type XVII collagen powder, add 35 times its weight of deionized water (4.2g), place in a constant temperature water bath at 42℃, stir for 38min until completely dissolved, and obtain collagen solution for later use.

[0123] (2) Activation of dihydroquercetin: Take 0.6g of (2R,3R)-dihydroquercetin powder, add 45 times its mass of anhydrous ethanol (27g), and activate by ultrasonication at a frequency of 55kHz, an ultrasonic temperature of 38℃, and an ultrasonic time of 19min to obtain an activated dihydroquercetin solution for later use.

[0124] (3) Mixing and dispersing: Accurately weigh the above dihydroquercetin activation solution and collagen solution, and place them in a high-speed disperser at a mass ratio of (2R,3R)-dihydroquercetin to collagen of 5:1. Disperse for 28 minutes at a speed of 700 r / min and a temperature of 42℃ to obtain a mixed dispersion.

[0125] (4) Isothermal self-assembly reaction: The mixed dispersion was transferred to an isothermal water bath reactor and stirred at 45°C using a programmed stirring mode: the stirring rate was 300 r / min for the first 30 min, 200 r / min for the middle 60 min, and 150 r / min for the last 30 min, with a total reaction time of 2 h; nitrogen gas was introduced for protection during the stirring process at a flow rate of 50 ml / min to obtain the supramolecular precursor.

[0126] (5) Gradient solvent evaporation: After the reaction was completed, the beaker was transferred to a high-precision heating stage and the temperature was set to 50℃ (temperature fluctuation ±0.8℃). A segmented evaporation mode was adopted: the first 2 hours were kept in a ventilated state with an evaporation rate of 1.3 g / h; the middle 2 hours were kept in a semi-closed state with an evaporation rate of 0.95 g / h; and the last 2 hours were kept in a slightly closed state with an evaporation rate of 0.7 g / h. The total evaporation time was 6 hours, and the residual ethanol content of the system was measured to be 3.2%.

[0127] (6) Step-by-step drying and curing: After the ethanol evaporates, the system is placed in a 50°C precision oven and a step-by-step drying mode is adopted: first, it is dried for 4 hours under ventilation conditions to remove residual free ethanol; then it is dried for 4 hours under vacuum conditions (vacuum degree -0.07MPa) to ensure that the system is completely dry; finally, it is naturally cooled to room temperature to obtain the supramolecular solid crude product.

[0128] (7) Post-processing and quality control: The supramolecular solid crude product is ground by a planetary ball mill at a speed of 250 r / min for 30 min and then passed through a 200 mesh sieve to obtain the supramolecular powder product.

[0129] Tests showed that the water solubility of the supramolecular powder product was 35.8 mg / ml, and the supramolecular structure was stable with no clumping or degradation.

[0130] Example 4:

[0131] The preparation method of the dihydroquercetin-collagen supramolecular solid system includes the following steps:

[0132] (1) Collagen dissolution: Take 0.15g of recombinant type XVII collagen powder, add 4.5g of deionized water, place in a constant temperature water bath at 40℃, stir for 35min until completely dissolved, and obtain collagen solution for later use.

[0133] (2) Activation of dihydroquercetin: Take 0.75g of (2R,3R)-dihydroquercetin powder, add 40 times its weight of anhydrous ethanol (30g), and activate by ultrasonication at a frequency of 50kHz, an ultrasonic temperature of 35℃, and an ultrasonic time of 18min to obtain an activated dihydroquercetin solution for later use.

[0134] (3) Mixing and dispersing: Accurately weigh the above dihydroquercetin activation solution and collagen solution, and place them in a high-speed disperser at a mass ratio of (2R,3R)-dihydroquercetin to collagen of 5:1. Disperse for 25 minutes at a speed of 650 r / min and a temperature of 40℃ to obtain a mixed dispersion.

[0135] (4) Isothermal self-assembly reaction: The mixed dispersion was transferred to an isothermal water bath reactor and stirred at 45°C using a programmed stirring mode: the stirring rate was 300 r / min for the first 30 min, 200 r / min for the middle 60 min, and 150 r / min for the last 30 min, with a total reaction time of 2 h; nitrogen gas was introduced for protection during the stirring process at a flow rate of 50 ml / min to obtain the supramolecular precursor.

[0136] (5) Gradient solvent evaporation: After the reaction was completed, the beaker was transferred to a high-precision heating stage and the temperature was set to 50℃ (temperature fluctuation ±0.9℃). A segmented evaporation mode was adopted: the first 2 hours were kept in a ventilated state with an evaporation rate of 1.2 g / h; the middle 2 hours were kept in a semi-closed state with an evaporation rate of 0.9 g / h; and the last 2 hours were kept in a slightly closed state with an evaporation rate of 0.6 g / h. The total evaporation time was 6 hours, and the residual ethanol content in the system was measured to be 3.6%.

[0137] (6) Step-by-step drying and curing: In strict accordance with the process requirements, the system after ethanol evaporation is placed in a 50℃ precision oven and a step-by-step drying mode is adopted: first, it is dried under ventilation for 4 hours to completely remove the residual free ethanol; then it is dried under vacuum (vacuum degree -0.07MPa) for 4 hours to ensure that the system is completely dry; finally, it is naturally cooled to room temperature to obtain the supramolecular solid crude product.

[0138] (7) Post-processing and quality control: The supramolecular solid crude product is ground by a planetary ball mill at a speed of 250 r / min for 30 min and then passed through a 200 mesh sieve to obtain the supramolecular powder product.

[0139] The water solubility of the supramolecular powder product was found to be 35.3 mg / ml, and the supramolecular structure was stable with no clumping or degradation.

[0140] This embodiment strictly follows a step-by-step drying process, first using ventilation to remove ethanol, and then vacuum drying. This effectively avoids the collapse of the supramolecular structure and the oxidation of raw materials. Compared with direct vacuum drying or direct ventilation drying, it significantly improves product stability and yield, highlighting the necessity of the step-by-step drying process.

[0141] Example 5:

[0142] The preparation method of the dihydroquercetin-collagen supramolecular solid system in this embodiment differs from that in Example 1 in that the recombinant type XVII collagen is replaced with hydrolyzed collagen.

[0143] The water solubility of the supramolecular powder product was found to be 41.4 mg / ml, and the supramolecular structure was stable with no clumping or degradation.

[0144] Example 6:

[0145] The preparation method of the dihydroquercetin-collagen supramolecular liquid system includes the following steps:

[0146] (1) Collagen dissolution: Take 0.2g of recombinant type XVII collagen powder, add 4g of deionized water, place in a 35℃ constant temperature water bath, stir for 30min until completely dissolved, and obtain collagen solution for later use.

[0147] (2) Activation of dihydroquercetin polyol: Take 1g of (2R,3R)-dihydroquercetin powder, add 30g of propylene glycol / glycerol mixed polyol, and activate by ultrasonication at a frequency of 40kHz, an ultrasonic temperature of 30℃, and an ultrasonic time of 15min to obtain a uniform and transparent dihydroquercetin polyol activation solution.

[0148] (3) Mixing and dispersing: The dihydroquercetin polyol activating solution and collagen solution were mixed at a mass ratio of dihydroquercetin: collagen = 5:1 and dispersed at high speed. The dispersion conditions were: rotation speed 500 r / min, temperature 35℃, time 20 min, to obtain a mixed dispersion.

[0149] (4) Isothermal self-assembly: The mixed dispersion was stirred at 45℃ for 2 hours. The stirring rate was 300 r / min for the first 30 minutes, 200 r / min for the middle 60 minutes, and 150 r / min for the last 30 minutes. Nitrogen protection was maintained throughout the process. The mixture was filtered to remove impurities, and a clear and transparent dihydroquercetin-collagen supramolecular liquid system with uniform texture was obtained.

[0150] Experimental Example 1: X-ray powder diffraction (XRD) characterization.

[0151] Instruments and parameters: X-ray diffractometer model D8 ADVANCE Da Vinci; phase analysis was performed using X-ray diffractometer, with the following test conditions: scanning range 5-60°, scanning rate 10° / min, Cu-Kα radiation source (λ = 1.54178 Å). The crystal structure was analyzed based on the XRD data and graphs, and the presence of new peaks was determined.

[0152] Test method: Powder X-ray diffraction tests were performed on (2R,3R)-dihydroquercetin, recombinant type XVII collagen, hydrolyzed collagen, and the dihydroquercetin-collagen supramolecular proteins prepared in Examples 1, 2 and 5, respectively. The data results of each sample were then compared and analyzed.

[0153] XRD data of each sample are as follows Figure 1 , Figure 2 and Figure 3 As shown, Figure 1 The supramolecular protein of Example 1 Figure 2 The supramolecular strain of Example 2 Figure 3The XRD data for the supramolecular collagen in Example 5 are shown below. XRD pattern analysis clearly demonstrates that the methods in Examples 1, 2, and 5 can successfully form supramolecular collagen from (2R,3R)-dihydroquercetin (DHQ) and recombinant type XVII collagen (Col). Specifically, the XRD patterns of the dihydroquercetin-collagen supramolecular collagen in Examples 1, 2, and 5 show pure DHQ (typical characteristic peaks at 2θ angles of approximately 10.5°, 15.3°, 20.1°, and 25.6°) and pure Col ... respectively. The novel characteristic diffraction peaks (2θ angles of approximately 19.8° and 22.5°) not found in the original diffuse peaks of pure DHQ (2θ angles of approximately 17.6° and 23.3°) show a significant decrease in intensity, a broadening of the full width at half maximum (FWHM), and a tendency towards diffusion. The diffuse peaks of pure Col show a slight shift in position and a slight increase in intensity. All of these characteristics indicate that DHQ and Col are not simply physically mixed, but rather undergo self-assembly through hydrogen bonding, hydrophobic interactions, and π-π stacking interactions to form a supramolecular structure with a new crystal structure.

[0154] Experimental Example 2: FTIR characterization.

[0155] Test methods: Infrared spectroscopy was used to characterize (2R,3R)-dihydroquercetin, recombinant type XVII collagen, and the dihydroquercetin-collagen supramolecular protein prepared in Example 1. The test parameters were transmittance and wavenumbers of 500 cm⁻¹. -1 -4000cm -1 The resolution is 0.2cm. -1 The test mode was tablet compression.

[0156] FTIR spectrum analysis results are as follows Figure 4 As shown, the method in Example 1 successfully formed a supramolecular structure between (2R,3R)-dihydroquercetin (DHQ) and recombinant type XVII collagen (Col). The core evidence is that the FTIR spectrum of the dihydroquercetin-collagen supramolecular structure in Example 1 showed a new characteristic absorption peak not seen in pure DHQ or pure Col, while the characteristic absorption peak of the hydroxyl group (-OH) in pure DHQ (approximately 3200-3600 cm⁻¹) was also observed. - ¹) Characteristic absorption peak of benzene ring (C=C) (approximately 1600-1650 cm⁻¹) - ¹) The intensity is significantly reduced and the peak position shifts; the characteristic absorption peak of the amide bond (-CONH-) in pure Col (approximately 1650 cm⁻¹) is lost. - ¹、1540cm - ¹) Significant shifts and broadening also occurred. These phenomena confirm that the two were not simply physically mixed, but rather self-assembled through hydrogen bonds, hydrophobic interactions, and π-π stacking interactions to form a supramolecular system with novel infrared absorption characteristics, further corroborating the successful formation of supramolecular structures.

[0157] Experimental Example 3: Differential Scanning Calorimetry (DSC) Characterization.

[0158] Test Method: DSC was performed using a Netzsch DSC 200F3 instrument. (2R,3R)-dihydroquercetin, collagen, and the supramolecular dihydroquercetin-collagen supramolecular prepared in Example 1 were placed in alumina crucibles, covered with perforated lids, and heated at a rate of 10°C / min to the set temperature. The test temperature range was 30°C to 300°C. Nitrogen was used as the purge and protective gas. The melting point changes were analyzed in detail based on the results.

[0159] The results of DSC spectrum analysis are as follows: Figure 5 As shown, the method in Example 1 successfully formed a supramolecular system from (2R,3R)-dihydroquercetin (DHQ) and recombinant type XVII collagen (Col). The core evidence is as follows: the DSC spectrum of the dihydroquercetin-collagen supramolecular system in Example 1 showed a new endothermic peak not present in pure DHQ and pure Col. At the same time, the intensity of the endothermic melting peak (corresponding to its crystalline melting) unique to pure DHQ was significantly reduced and the peak position shifted. The endothermic peak of thermal denaturation of pure Col also showed a significant shift and broadening. These phenomena confirm that the two are not simply physically mixed, but self-assembled through hydrogen bonding, hydrophobic interactions, and π-π stacking interactions to form a supramolecular system with novel thermal behavior characteristics. Its thermal stability is significantly different from that of the single raw material, further confirming the successful formation of the supramolecular system.

[0160] Experiment Example 4: Solubility Test.

[0161] Test method: Three concentration gradients of dihydroquercetin were set up (0.05%, 0.1%, and 0.2%, mass-volume ratio), with three parallel samples in each group, corresponding to the three sample groups: Group 1 was pure dihydroquercetin, Group 2 was a physical mixture of dihydroquercetin and collagen, and Group 3 was the dihydroquercetin-collagen supramolecular mixture prepared in Example 1. The dihydroquercetin used was (2R,3R)-dihydroquercetin, and the collagen used was recombinant type XVII collagen.

[0162] The results are as follows Figure 6As shown: At a dihydroquercetin concentration of 0.05%, all three solutions exhibited good transparency. At a dihydroquercetin concentration of 0.1%, the turbidity of the pure dihydroquercetin solution increased significantly, with some undissolved precipitate appearing; the physically mixed group solution was slightly turbid, with a significant decrease in transparency; the supramolecular group solution remained clear and transparent, with no precipitate formation and stable transparency. At a dihydroquercetin concentration of 0.2%, the pure dihydroquercetin solution was completely turbid, with a large amount of precipitate aggregated, making it impossible to observe the solution's transmittance; the physically mixed group solution was severely turbid, with obvious precipitate and extremely low transparency; the supramolecular group solution remained clear and transparent, with good transmittance. In summary, at the same dihydroquercetin concentration, the supramolecular solution exhibited significantly better transparency than the pure dihydroquercetin group and the physical mixture group. Furthermore, this difference in transparency became more pronounced as the dihydroquercetin concentration increased. This phenomenon is highly consistent with the solubility measurement results, further confirming that the dihydroquercetin-collagen supramolecular structure can significantly enhance the solubility of dihydroquercetin, allowing it to dissolve more fully in water and form a clear and stable solution.

[0163] Experiment Example 5: Stability Experiment.

[0164] Experimental Groups: Three groups of samples were set up, each prepared with an aqueous solution containing 0.1% dihydroquercetin. Group 1 was the pure dihydroquercetin group, Group 2 was the dihydroquercetin-collagen physical mixture group, and Group 3 was the dihydroquercetin-collagen supramolecular group. The dihydroquercetin used was (2R,3R)-dihydroquercetin, and the collagen used was recombinant type XVII collagen.

[0165] Stability testing conditions: Accelerated stability testing was conducted, and five storage conditions were set: low temperature (4℃), high temperature (45℃), strong light (light intensity 4000lx), room temperature and light protection (25℃), and cycling (-20~45℃). Samples were taken at 0d, 7d, and 14d of storage.

[0166] Test results (from left to right: 4℃, 25℃, 45℃, light intensity, and cyclic conditions) are as follows: Figure 7-9As shown in the figure, the pure dihydroquercetin solutions all became increasingly turbid after 14 days of storage, with a large amount of precipitate agglomerating at the bottom of the tubes and exhibiting no light transmittance. This indicates that severe oxidative degradation occurred during storage, and a large amount of undissolved components precipitated out, resulting in extremely poor stability. The dihydroquercetin-collagen physical mixture solutions were all clear and transparent at 0D, but after 2W, except for those transparent at 25℃, all others became turbid, with a large amount of precipitate at the bottom of the tubes, poor light transmittance, and obvious discoloration at high temperatures. This indicates that the simple physical mixing of the two can only slightly alleviate the degradation and precipitation of dihydroquercetin and cannot play an effective protective role. The dihydroquercetin-collagen supramolecular solutions, on the other hand, remained clear and transparent after 14 days of storage under different conditions, with no visible precipitate or suspended impurities, good light transmittance, and no precipitate agglomeration at the bottom of the tubes. This indicates that the supramolecular structure can effectively encapsulate and protect dihydroquercetin, significantly reducing its oxidative degradation and precipitation. The differences in appearance of the three groups of samples in the picture directly demonstrate that the stability of dihydroquercetin is significantly improved after it self-assembles with collagen to form supramolecular structures, and the improvement effect is far better than that of pure dihydroquercetin and simple physical mixing of the two.

[0167] Experiment 6: Anti-hair loss ability test.

[0168] Test method: β-catenin target regulation in dermal papilla cells (DP)

[0169] 1.1) After cell resuscitation, observe cell growth. Count the cells when cell coverage reaches 80% or higher, and seed them into 96-well and 24-well plates. The 96-well plates are used to detect cell viability, and the 24-well plates are used to detect β-catenin expression levels. Incubate the plates overnight in a CO2 incubator (37°C, 5% CO2).

[0170] 1.2) Solution preparation: Prepare the working solution for the sample.

[0171] 1.3) When the DP cell plating rate reaches about 60%, administer the drug to groups. Each group has 3 replicates.

[0172] 1.3.1) The concentration of dihydrotestosterone (DHT) for modeling experiments was explored according to the grouping in Table 1 below, and the DHT concentration of 1~1000μM was selected for the experiment;

[0173] Table 1. Experimental Protocol for Dihydrotestosterone (DP) Cell Concentration

[0174]

[0175] 1.3.2) Subsequently, according to the groups shown in Table 2 below, the DP cell experimental concentrations of the dihydroquercetin-collagen supramolecular group, the dihydroquercetin-collagen physical mixture group, and the collagen group in Example 1 were explored, and concentrations of 0.0005~0.1% by mass / volume percentage were selected for the experiments; among them, the dihydroquercetin used was (2R,3R)-dihydroquercetin, and the collagen used was recombinant type XVII collagen.

[0176] Table 2. Experimental protocols for DP cell concentrations of supramolecular collagen, physical mixtures, and collagen.

[0177]

[0178] 1.4) Toxicity test results:

[0179] 1.4.1) Results of DHT concentration on dermal papilla cell viability detection: Figure 10 As shown, DHT concentrations in the range of 1–100 μM indicate that dermal papilla cell viability is above 70% and toxicity is low, suggesting that further experiments can be conducted.

[0180] 1.4.2) Detection of dermal papilla cell viability at different concentrations of supramolecular groups, physical mixtures, and collagen, such as... Figure 11 As shown, supramolecular collagen, physical mixtures, and macromolecular collagen, within a mass-volume percentage concentration range of 0.0005–0.001%, all exhibited hair papilla cell viability exceeding 80% and showed no cytotoxicity.

[0181] 1.5) The optimal DHT concentration for modeling was determined to be 100 μM. Immunofluorescence detection of β-catenin was performed on supramolecular, physically mixed, and collagen groups at a safe concentration of 10 μg / mL, as shown in Table 3. Supramolecular collagen and 100 µM DHT were co-cultured for 24 h. After drug administration, the 24-well plates were incubated in an incubator (37°C, 5% CO2) for 24 h.

[0182] Table 3. Immunofluorescence detection protocol

[0183]

[0184] 1.6) Immunofluorescence staining: Fixation, blocking, addition of primary antibody, addition of secondary antibody, counterstaining with DAPI, mounting, and then photographing using a fluorescence microscope. Quantitative analysis of fluorescence intensity was performed using Image Pro Plus software.

[0185] 1.7) Immunofluorescence detection results:

[0186] 1.7.1) 100 μM DHT significantly reduced the expression level of β-catenin compared with the blank control group, with an inhibition rate of 76.48%;

[0187] 1.7.2) 10 μM minoxidil significantly increased the expression level of β-catenin compared with the DHT group, with an increase of 44.12%.

[0188] 1.7.3) Dihydroquercetin-collagen supramolecular structure can regulate DHT-induced changes in marker levels: such as Figure 12 and Figure 13 As shown, the 10 μg / mL dihydroquercetin-collagen supramolecular group can significantly upregulate the decrease in β-catenin caused by 100 μM DHT (p<0.001), with an increase of 49.61%, and has an anti-hair loss effect, which is significantly better than physical mixture and collagen at the same concentration.

[0189] As shown in Experiment 6, the anti-hair loss ability of dihydroquercetin-collagen supramolecular group is better than that of physical mixing and macromolecular collagen. It can be used in the preparation of cosmetics with anti-hair loss effect, and the addition ratio can be 10μg / mL.

[0190] Experiment Example 7: Antioxidant Capacity Test.

[0191] Test substance preparation: Prepare samples with gradient concentrations according to Table 4. After sample preparation, prepare reaction system according to the amount of each reagent added in Table 5. Add water to water-soluble samples C and C0, and use anhydrous ethanol for oil-soluble test substances. Mix well. Set up 3 replicates for each concentration and 1 background control well.

[0192] Table 4. Sample Concentration Preparation Table

[0193]

[0194] Table 5. DPPH free radical scavenging test reaction system

[0195]

[0196] All sample volumes mentioned above are in μL.

[0197] The reaction system was placed at room temperature and reacted in the dark for 30 minutes. After the reaction was completed, the absorbance OD value was read at 517 nm.

[0198] The scavenging rate of DPPH free radicals of the sample at each test concentration is calculated according to the following formula. At the same time, the standard deviation (SD) between the scavenging rates of each group of parallel tubes is calculated. The parallelism of the test is considered to be valid if the SD value is ≤3%.

[0199] .

[0200] Where: T represents the absorbance of the sample tube, that is, the absorbance of the solution after the sample reacts with DPPH;

[0201] T0 represents the background absorbance of the sample;

[0202] C represents the average of three absorbance readings of the DPPH tube, i.e., the absorbance of the DPPH solution without any sample added;

[0203] C0 represents the solvent background absorbance.

[0204] Experimental results:

[0205] like Figure 14 As shown, at a concentration of 125 μg / mL, the DPPH radical scavenging rate of the dihydroquercetin-collagen supramolecular group was 65.53%, the DPPH radical scavenging rate of the dihydroquercetin-collagen physical mixture group was 60.12%, and the DPPH radical scavenging rate of the collagen group was 3.43%. That is, the DPPH radical scavenging rate of the dihydroquercetin-collagen supramolecular group was significantly higher than that of the dihydroquercetin-collagen physical mixture group and the collagen group.

[0206] As shown in Experiment 7, the antioxidant capacity of the dihydroquercetin-collagen supramolecular group is better than that of the dihydroquercetin-collagen physical mixture group and the collagen group. It can be used in the preparation of cosmetics with antioxidant capacity, and the addition ratio can be 20~125 μg / mL.

[0207] Experiment Example 8: Anti-wrinkle and firming function test.

[0208] Test method: Fibroblast Collage I target regulation.

[0209] 1.1) After resuscitating HFF-1 cells, observe cell growth. Count the cells when cell coverage reaches 80% or higher, and seed them into 96-well and 24-well plates. The 96-well plates are used to detect cell viability, and the 24-well plates are used to detect Collage I expression levels. Incubate the plates overnight in a CO2 incubator (37°C, 5% CO2).

[0210] 1.2) Solution preparation: Prepare the working solution for the sample.

[0211] 1.3) When the HFF-1 cell plating rate reaches about 60%, administer the drug to different groups. Each group has 3 replicates.

[0212] 1.3.1) According to the groups in Table 6 below, the experimental concentrations of HFF-1 cells for the dihydroquercetin-collagen supramolecular group, the dihydroquercetin-collagen physical mixture group, and the collagen group in Example 1 were explored, and concentrations of 0.01~200μg / mL were selected for the experiments; among them, the dihydroquercetin used was (2R,3R)-dihydroquercetin, and the collagen used was recombinant type XVII collagen.

[0213] Table 6. Experimental Protocol for HFF-1 Cell Concentration

[0214]

[0215] 1.4) Concentration Experiment Results:

[0216] The effects of dihydroquercetin-collagen supramolecular groups, dihydroquercetin-collagen physical mixture groups, and collagen groups at different concentrations on fibroblast viability assays were as follows: Figure 15 As shown, within the concentration range of 0.01~100μg / mL, the fibroblast viability was above 90% and there was no cytotoxicity.

[0217] 1.5) Immunofluorescence detection of Collage I was performed on dihydroquercetin-collagen supramolecular groups, dihydroquercetin-collagen physical mixture groups, and collagen groups within a safe concentration range of 100 μg / mL. The detection protocol is shown in Table 7, 8 J / cm 2 After UVA irradiation, the drugs were administered in groups. After drug administration, the 24-well plates were placed in an incubator (37°C, 5% CO2) and incubated for 24 hours.

[0218] Table 7. Immunofluorescence Detection Protocol

[0219]

[0220] 1.6) Immunofluorescence staining: Fixation, blocking, addition of primary antibody, addition of secondary antibody, counterstaining with DAPI, mounting, and then photographing using a fluorescence microscope. Quantitative analysis of fluorescence intensity was performed using Image Pro Plus software.

[0221] 1.7) Immunofluorescence detection results:

[0222] 1.7.1) 8J / cm 2 Compared with the blank control group, UVA significantly reduced the expression level of Collage I, with an inhibition rate of 50%;

[0223] 1.7.2) Dihydroquercetin-collagen supramolecular structure can regulate UVA-induced changes in marker levels: such as Figure 16 and Figure 17As shown, the 10 μg / mL and 100 μg / mL dihydroquercetin-collagen supramolecular groups significantly upregulated the decrease in Collage I caused by UVA (p<0.001), with growth rates of 47% and 67%, respectively, demonstrating anti-wrinkle and firming effects, and were significantly superior to the physical mixture of dihydroquercetin-collagen and the collagen group at the same concentration.

[0224] As shown in Experiment 8, the anti-wrinkle and firming ability of the dihydroquercetin-collagen supramolecular group is better than that of the dihydroquercetin-collagen physical mixture group and the collagen group. It can be used in the preparation of cosmetics with anti-wrinkle and firming effects, and the addition ratio can be 10 or 100 μg / mL.

[0225] Comparative Example 1:

[0226] The preparation method of the comparative dihydroquercetin-collagen supramolecular solid system differs from that of Example 1 in that: in step (3), the mass ratio of (2R,3R)-dihydroquercetin to collagen is 10:1.

[0227] Results: The water solubility of the prepared supramolecular powder was tested, and the result was 17.3 mg / ml. X-ray powder diffraction (XRD) analysis was performed, and the results are shown below. Figure 18 As shown, from Figure 18 It can be seen that no characteristic peaks were detected in the dihydroquercetin-collagen supramolecular of Comparative Example 1, indicating that deviation in the feed ratio will lead to insufficient supramolecular formation and a significant decrease in performance.

[0228] Comparative Example 2:

[0229] The preparation method of the comparative dihydroquercetin-collagen supramolecular solid system differs from that of Example 1 in that: in step (2), the ultrasonic temperature is 60°C when dihydroquercetin is ultrasonically activated.

[0230] Results: The water solubility of the prepared supramolecular powder was tested, and the result was 16.8 mg / ml. (2R,3R)-dihydroquercetin was subjected to high-temperature treatment according to the methods in Example 1 and Comparative Document 2, respectively, followed by high-performance liquid chromatography (HPLC) analysis. The results are shown below. Figure 19 As shown in the figure. The results indicate that the impurity content in dihydroquercetin after high-temperature treatment in Comparative Example 2 increased significantly, indicating that the molecular structure of dihydroquercetin underwent degradation under high-temperature ultrasound, resulting in extremely low self-assembly efficiency with collagen and a severe decline in product performance.

[0231] Comparative Example 3:

[0232] The preparation method of the comparative dihydroquercetin-collagen supramolecular solid system differs from that of Example 1 in that: in step (3), the high-speed dispersion is carried out at a rotation speed of 400 r / min and a time of 15 min.

[0233] Results: The water solubility of the prepared supramolecular powder was tested and found to be 21.1 mg / ml. After the aqueous solution was left to stand for a period of time, precipitation and turbidity occurred, indicating that the supramolecular formation was uneven. This suggests that insufficient mixing and dispersion parameters can lead to uneven mixing of components, affecting the sufficiency of the self-assembly reaction and thus reducing the supramolecular properties.

[0234] Comparative Example 4:

[0235] The preparation method of the comparative dihydroquercetin-collagen supramolecular solid system differs from that of Example 1 in that steps (5) and (6) are changed to placing the supramolecular precursor directly into a 70°C precision oven and drying it rapidly with ventilation throughout the process until the system is completely dry.

[0236] Results: The water solubility of the prepared supramolecular powder was tested, and the result was 12.3 mg / ml, indicating severely impaired water solubility. This suggests that the rapid evaporation of ethanol at high temperatures caused the supramolecular network structure to be destroyed before it could solidify, resulting in the loss of not only the active ingredients but also the destruction of its sustained-release and water-soluble properties.

[0237] Comparative Example 5:

[0238] The preparation method of the comparative dihydroquercetin-collagen supramolecular solid system differs from that of Example 1 in that: in step (3), the mass ratio of (2R,3R)-dihydroquercetin to collagen is 1:2.

[0239] Results: The obtained supramolecular powder was analyzed by X-ray powder diffraction (XRD). The results are shown in the figure below. Figure 20 As shown, from Figure 20 It can be seen that no characteristic peaks were detected in the dihydroquercetin-collagen supramolecular composition of Comparative Example 5, indicating that deviations in the feed ratio will lead to insufficient supramolecular formation and a significant decrease in performance.

Claims

1. A dihydroquercetin-collagen supramolecular system, characterized in that: It includes dihydroquercetin and collagen, wherein dihydroquercetin and collagen are linked by non-covalent bonds, and the mass ratio of dihydroquercetin to collagen is (1-5):

1.

2. The dihydroquercetin-collagen supramolecular system according to claim 1, characterized in that: The collagen used is animal collagen, plant collagen, hydrolyzed collagen, recombinant type I collagen, recombinant type III collagen, recombinant type VI collagen, recombinant type XVII collagen, or polypeptide.

3. The dihydroquercetin-collagen supramolecular system according to claim 1, characterized in that: The dihydroquercetin is (2R,3R)-dihydroquercetin.

4. The dihydroquercetin-collagen supramolecular system according to any one of claims 1-3, characterized in that: The dihydroquercetin-collagen supramolecular system includes solid, liquid, semi-solid, or gaseous systems.

5. The dihydroquercetin-collagen supramolecular system according to claim 4, characterized in that: The preparation method of the solid system includes the following steps: (1) Preparation of collagen solution; (2) Activation of dihydroquercetin: Take dihydroquercetin powder, add anhydrous ethanol, and activate by ultrasonication to obtain dihydroquercetin activation solution; (3) Mixing and dispersing: Disperse the collagen solution and dihydroquercetin activating solution evenly in proportion to obtain a mixed dispersion; (4) Isothermal self-assembly reaction: The mixed dispersion is stirred in a gradient and protected by an inert gas throughout the process to obtain a supramolecular precursor; (5) Gradient solvent evaporation: The supramolecular precursor is evaporated in stages to ensure that the residual ethanol content in the system is ≤5%; (6) Stepwise drying and curing: The system after solvent evaporation is dried stepwise to obtain a dihydroquercetin-collagen supramolecular solid system.

6. The dihydroquercetin-collagen supramolecular system according to claim 5, characterized in that: Step (1) is as follows: Take collagen powder, add water, and stir in a water bath at 35~45℃ until completely dissolved to obtain collagen solution.

7. The dihydroquercetin-collagen supramolecular system according to claim 5, characterized in that: In step (2), the ultrasonic conditions are: ultrasonic frequency of 40-60kHz, ultrasonic temperature of 30-40℃, and ultrasonic time of 15-20min.

8. The method for preparing dihydroquercetin-collagen supramolecular according to claim 5, characterized in that: In step (3), the collagen solution and dihydroquercetin activation solution are dispersed evenly according to the mass ratio of dihydroquercetin to collagen of (1-5):

1.

9. The dihydroquercetin-collagen supramolecular system according to claim 5, characterized in that: In step (3), the dispersion conditions are: rotation speed of 500-800 r / min, temperature of 35-45℃, and time of 20-30 min.

10. The dihydroquercetin-collagen supramolecular system according to claim 5, characterized in that: In step (4), the gradient stirring method is as follows: the stirring rate is 300 r / min for the first 30 min, 200 r / min for the middle 60 min, and 150 r / min for the last 30 min, with a total reaction time of 2 h.

11. The dihydroquercetin-collagen supramolecular system according to claim 5, characterized in that: In step (5), the volatilization is divided into stages: the volatilization rate is 1-1.5 g / h for the first 2 hours; the volatilization rate is 0.8-1 g / h for the middle 2 hours; and the volatilization rate is 0.5-0.8 g / h for the last 2 hours, with a total volatilization time of 6 hours.

12. The dihydroquercetin-collagen supramolecular system according to claim 5, characterized in that: Step (6) is to dry in stages: first, dry under ventilation conditions for 2-6 hours to remove residual free ethanol; then dry under vacuum conditions for 2-6 hours until the system is completely dry.

13. The dihydroquercetin-collagen supramolecular system according to claim 4, characterized in that: The liquid system further includes water and a hydrophilic medium, wherein the hydrophilic medium accounts for 5%-80% of the mass percentage of the liquid system.

14. The dihydroquercetin-collagen supramolecular system according to claim 4, characterized in that: The hydrophilic medium is selected from at least one of polyols, sugar alcohols, and amides.

15. The dihydroquercetin-collagen supramolecular system according to claim 4, characterized in that: The method for preparing the liquid system includes the following steps: (1) Preparation of collagen solution; (2) Activation of dihydroquercetin: Take dihydroquercetin powder, add a hydrophilic medium, and disperse or dissolve it under stirring or ultrasonic conditions to obtain an activated dihydroquercetin solution. (3) Mixing and dispersing: Disperse the collagen solution and dihydroquercetin activating solution evenly in proportion to obtain a mixed dispersion; (4) Isothermal self-assembly reaction: The mixed dispersion was stirred in a gradient and protected by an inert gas throughout the process to obtain a dihydroquercetin-collagen supramolecular liquid system.

16. The dihydroquercetin-collagen supramolecular system according to claim 15, characterized in that: Step (1) is as follows: Take collagen powder, add water, and stir in a water bath at 35~45℃ until completely dissolved to obtain collagen solution.

17. The dihydroquercetin-collagen supramolecular system according to claim 15, characterized in that: In step (2), the ultrasonic conditions are: frequency of 40-60kHz, temperature of 30-40℃, and time of 15-20min.

18. The dihydroquercetin-collagen supramolecular system according to claim 15, characterized in that: In step (3), the collagen solution and dihydroquercetin activation solution are dispersed evenly according to the mass ratio of dihydroquercetin to collagen of (1-5):

1.

19. The dihydroquercetin-collagen supramolecular system according to claim 15, characterized in that: In step (3), the dispersion conditions are: rotation speed of 500-800 r / min, temperature of 35-45℃, and time of 20-30 min.

20. The dihydroquercetin-collagen supramolecular system according to claim 15, characterized in that: In step (4), the gradient stirring method is as follows: the stirring rate is 300 r / min for the first 30 min, 200 r / min for the middle 60 min, and 150 r / min for the last 30 min, with a total reaction time of 2 h.

21. The use of the dihydroquercetin-collagen supramolecular system according to any one of claims 1-3 in the preparation of anti-hair loss and / or anti-aging products.

22. The application according to claim 21, characterized in that: The anti-aging measures include anti-oxidation and / or anti-wrinkle and firming.

23. The application according to claim 21, characterized in that: The products mentioned include cosmetics, pharmaceuticals, or health foods.

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