Thaflavin nano-composite with whitening and moisturizing effects as well as preparation method and application of theaflavin nano-composite

Abstract

The invention discloses a theaflavin nano-composite with whitening and moisturizing effects, which is mainly prepared from theaflavin as an active component, an encapsulation carrier and a synergistic component, and has a core-shell structure with theaflavin as a core and a three-dimensional network formed by the encapsulation carrier as a shell, the purity of the theaflavin is not lower than 80%, and the mass ratio of the theaflavin-3-gallate to the theaflavin-3 '-gallate is 1: (0.8-1.2); the encapsulation carrier comprises modified silk fibroin, activated tamarind seed polysaccharide and self-assembly oligopeptide with pH responsiveness, and a three-dimensional network is formed through reaction; the synergistic interaction components are 2-o-ethyl ascorbic acid and eugenol. The invention also discloses a preparation method and application of the compound. The theaflavin nano-composite prepared by the invention has the advantages of high stability, good bioavailability, capability of quickly releasing active matters on the surface of skin in a weakly acidic environment, and realization of whitening and moisturizing effects.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a theaflavins nanocomposite with whitening and moisturizing effects, its preparation method, and its application. Background Technology

[0002] Theaflavins (TFs) are benzophenone compounds formed by the oxidation and polymerization of catechins during the fermentation of black tea. Often referred to as "soft gold" in tea, they possess remarkable biological activity and pharmacological functions. Studies have shown that theaflavins not only exhibit significant antioxidant, anti-inflammatory, antibacterial, and anticancer activities, but also demonstrate various health benefits such as lowering blood lipids, lowering blood sugar, and protecting cardiovascular health. In the field of skin care, theaflavins demonstrate powerful whitening potential by inhibiting tyrosinase activity, reducing melanin production, and neutralizing free radicals; simultaneously, their antioxidant properties help protect the skin barrier function, indirectly enhancing skin's moisturizing ability.

[0003] However, theaflavins face two major challenges in practical applications: poor chemical stability (sensitive to temperature, pH, and light) and low bioavailability. At temperatures exceeding 70°C, theaflavins degrade by up to 56% in aqueous solutions, and are almost completely inactivated at 100°C; auto-oxidation accelerates under alkaline conditions (pH>7), and degradation is rapid at pH=9; the degradation rate exceeds 20% after 6 hours of light exposure. These characteristics severely limit the effectiveness of theaflavins in the cosmetics field. However, most existing technologies remain at the stage of simple composition, failing to effectively address the core issues of theaflavins' stability and low bioavailability. While a few patents employing liposome technology have made some improvements, they still exhibit significant shortcomings in encapsulation efficiency, loading capacity, and long-term stability.

[0004] Furthermore, theaflavins have poor skin permeability, mainly due to their large molecular weight and strong hydrophilicity, making it difficult to effectively penetrate the stratum corneum. This results in low bioavailability at the target site, making it difficult to achieve the desired whitening effect even at high concentrations. Current technologies attempt to encapsulate polyphenols using conventional nanocarriers (such as liposomes, ordinary protein particles, or polysaccharide microspheres), but these methods have significant shortcomings. For example, while unmodified silk fibroin has good biocompatibility, its binding to polyphenol molecules lacks specificity and strength, leading to unstable encapsulation rates and easy particle aggregation, hindering intelligent controlled release of the active ingredient. Ordinary physical mixing or simple compounding processes cannot solve the problems of poor compatibility and limited functionality between components; they often only involve a simple superposition of physical properties, failing to produce synergistic enhancement effects.

[0005] Furthermore, the cosmetics industry is increasingly demanding higher standards for the stability, safety, and efficacy of whitening ingredients. The market urgently needs an innovative technological solution that can simultaneously address the stability of active ingredients, skin penetration, targeted release, and synergistic effects of multiple benefits. Therefore, developing an advanced delivery system that can precisely encapsulate and protect theaflavins, intelligently release them in response to the skin's microenvironment, and simultaneously provide multiple skincare benefits has become a pressing technical challenge in this field. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a theaflavin nanocomplex with whitening and moisturizing effects, its preparation method and application, which improves the stability and skin permeability of the theaflavin nanocomplex, enabling it to quickly release active ingredients in the weakly acidic environment of the skin surface, thereby achieving whitening and moisturizing effects.

[0007] To address the above technical problems, this invention discloses a theaflavin nanocomposite with whitening and moisturizing effects. The theaflavin nanocomposite is characterized by being prepared primarily from theaflavins as active components, an encapsulating carrier, and synergistic ingredients, possessing a core-shell structure with theaflavins as the core and a three-dimensional network formed by the encapsulating carrier as the shell. The purity of the theaflavins is not less than 80%, preferably not less than 95%, wherein the mass ratio of theaflavins-3-gallate to theaflavins-3'-gallate is 1:0.8–1.2 (preferably 1:1). The encapsulating carrier comprises modified silk fibroin, activated soybean seed polysaccharide, and pH-responsive self-assembled short peptides, which react to form a three-dimensional network. The synergistic ingredients are 2-o-ethyl ascorbic acid and eugenol.

[0008] Furthermore, the modified silk fibroin has a β-sheet content ≥40%, calculated by fitting the amide I band peaks of the Fourier transform infrared spectrum; the free carboxyl content is increased by more than 15% compared with the unmodified silk fibroin, determined by potentiometric titration; the activated soybean seed polysaccharide has a weight-average molecular weight of 10-30 kDa and a degree of esterification ≤30%.

[0009] Furthermore, the self-assembled short peptides are in a dispersed state at pH ≥ 6.5 and undergo conformational changes in a weakly acidic environment of pH 5.0–5.8. This property enables the obtained theaflavin nanocomplex to achieve a cumulative theaflavin release rate of ≥ 80% in PBS buffer at pH 5.5 after 24 hours, while the cumulative release rate is ≤ 40% in PBS buffer at pH 7.4 after 24 hours. It exhibits excellent pH response characteristics to improve the release and absorption rate of theaflavins in the weakly acidic environment of the skin.

[0010] Furthermore, the theaflavin nanocomposite has an average particle size of 70–150 nm, a polydispersity index ≤0.18, and an encapsulation efficiency ≥96%.

[0011] This invention also discloses a method for preparing the aforementioned theaflavin nanocomposite with whitening and moisturizing effects, comprising the following steps:

[0012] (1) Carrier pretreatment:

[0013] Silk fibroin protease modification: Purified silk fibroin (silk fibroin raw material with most non-fibrous components removed and a relatively concentrated molecular weight distribution) is dissolved in pH buffer (generally PBS buffer with pH 7.0-7.5 can be used) to obtain a purified silk fibroin solution with a mass-volume concentration of 0.5%-3.0%. The pH of the system is adjusted to 6.8-7.5, and glutamine transaminase is added at a mass ratio of 1%-3% (preferably 1.5%-3%) of the purified silk fibroin. The mixture is stirred at 35-45°C for 2-4 hours. After the reaction, the enzyme is inactivated, dialyzed, and freeze-dried to obtain modified silk fibroin powder. Through enzymatic modification, the conformation and surface chemical properties of silk fibroin are reshaped at the molecular level, enabling the modified silk fibroin to bind with theaflavins molecules in a high-strength and specific manner.

[0014] Physical activation of soybean seed polysaccharide: Soybean seed polysaccharide was prepared into an aqueous solution with a mass-volume concentration of 2%–5%. It was first ultrasonically treated at a power of 500–800 W and a frequency of 15–35 kHz for 30–60 minutes, followed by treatment under ultra-high pressure of 250–300 MPa for 5–10 minutes. The treated solution was collected to obtain an activated soybean seed polysaccharide solution. This synergistic physical action of ultrasonic treatment followed by ultra-high pressure treatment effectively breaks down the long polysaccharide chains, reduces the molecular weight, and exposes more active sites such as hydroxyl and carboxyl groups. This makes it easier for the polysaccharide to interact with modified silk fibroin in subsequent steps through electrostatic interactions, hydrogen bonds, and other intermolecular forces to jointly construct a stable three-dimensional network structure.

[0015] (2) Three-step sequential self-assembly:

[0016] Step 1: Weigh the modified silk fibroin powder obtained in step (1) above, dissolve it in pH buffer (PBS buffer with pH 6.8 to 7.2) to prepare a modified silk fibroin solution with a mass concentration of 1% to 5%; dissolve theaflavins in edible ethanol aqueous solution (the volume concentration of ethanol aqueous solution is generally 70%) to obtain a theaflavins solution with a mass concentration of 0.001 to 0.15 g / mL; slowly add the theaflavins solution to the modified silk fibroin solution under magnetic stirring, and at the same time slowly add sodium dimethylsilanol hyaluronic acid ester (as a primary stabilizer) accounting for 0.5% to 1% of the total mass of the system (theaflavins solution and modified silk fibroin solution), and vortex-incubate at 30 to 35°C for 30 to 60 minutes; the modified silk fibroin and theaflavins attract each other through van der Waals forces, hydrogen bonds and other interactions to form a uniformly dispersed solution system;

[0017] Step 2: Slowly add an equal volume of the activated soybean seed polysaccharide solution obtained in step (1) to the system after the first step of vortex-incubation to form a mixed system. After adjusting the pH to 6.0-6.5, add C at a mass of 0.2%-0.5% of the total mass of the mixed system. 8-16 Sodium isoalkyl succinyl lactoglobulin sulfonate (as an emulsification promoter) was then sheared and dispersed at 15,000–20,000 rpm for 5–10 minutes to form a uniform pre-emulsion; a three-dimensional network complex was formed between the modified silk fibroin and the activated soybean seed polysaccharide, and the theaflavins were encapsulated within it;

[0018] Step 3: Dissolve the self-assembled short peptide, 2-o-ethyl ascorbic acid, and eugenol together in an edible ethanol solution to obtain a mixed solution. The ethanol concentration used should be 70%–80%, and the amount used should be sufficient to dissolve the assembled short peptide, 2-o-ethyl ascorbic acid, and eugenol. Use hydrochloric acid solution (generally a molar concentration of 0.1%). M) or citric acid (generally a mass-volume concentration of 10%) solution is slowly added dropwise to the resulting mixed solution. The pH value is adjusted to 5.4-5.6 (preferably 5.5), and then slowly added to the pre-emulsion obtained in the second step. Then, 0.1%-0.3% of 4-butylresorcinol (as a crosslinking agent) is added to the total mass of the system. The entire system is then circulated through a microfluidic homogenizer or cavitation treatment to obtain a uniformly dispersed nano-dispersion. In this step, the self-assembled short peptide undergoes a conformational change, which initiates the self-assembly process. The conformationally changed self-assembled short peptide can be embedded in the outer cavity (the inner cavity is occupied by theaflavins) of the three-dimensional network structure composed of modified silk fibroin and activated soybean seed polysaccharide. Finally, the three synergistically construct a stable three-dimensional network structure, encapsulating theaflavins, while the synergistic synergistic components are dispersed in the gaps of the three-dimensional network structure, and play a regulatory role in the interaction between the self-assembled short peptide, modified silk fibroin and activated soybean seed polysaccharide.

[0019] (3) Product purification: The nano-dispersion obtained in step (2) is purified by dialysis bag with a molecular weight cutoff of 8000-10000 Da to remove unencapsulated components, and then spray-dried or freeze-dried to obtain theaflavin nanocomplex.

[0020] Furthermore, the mass ratio of the theaflavins to the modified silk fibroin, activated soybean seed polysaccharide, and self-assembled short peptides is 1:5 to 10; the mass ratio of the modified silk fibroin to the activated soybean seed polysaccharide is 1:1 to 2.5; the mass of the self-assembled short peptides is 0.5% to 2.0% of the mass of theaflavins; the mass ratio of eugenol to 2-o-ethyl ascorbic acid is 1:2.5 to 10; and the mass of eugenol is 10% to 20% of the mass of theaflavins.

[0021] Furthermore, in step (1), the ultrasonic treatment for the physical activation of soybean seed polysaccharide is performed using a probe-type ultrasonic instrument. Continuous ultrasonic treatment or intermittent ultrasonic treatment is selected. When using continuous ultrasonic treatment, the frequency is 15-25 kHz; when using intermittent ultrasonic treatment, the frequency is 25-35 kHz, and the working cycle is 2-5 seconds of ultrasonic treatment followed by 3-4 seconds of intermittent treatment.

[0022] Furthermore, in step (1), the stirring speed of the stirring reaction during the silk fibroin protease modification is 200-400 rpm; in step (2), the rotation speed of the vortex apparatus used during the vortex-incubation in the first step is 300-500 rpm; in step (2), the micro-jet homogenizer is used with a pressure of 1500-1800 bar, a cycle of 5-8 times, and the heat exchanger temperature is always maintained at 25±2°C; in step (2), the cavitation treatment uses hydraulic cavitation with a cavitation number of 0.1-0.15, an inlet pressure maintained at 0.5-0.7 MPa, and a time of 10-20 minutes, or ultrasonic cavitation with a frequency of 16 kHz - 100 kHz and a time of 25-35 minutes.

[0023] The present invention further discloses the aforementioned theaflavin nanocomplex and the application of the theaflavin nanocomplex prepared by the aforementioned method in the preparation of topical cosmetic compositions for skin whitening, anti-oxidation and moisturizing.

[0024] Furthermore, the theaflavin nanocomplex is added at a mass of 0.5% to 3.0% of the total mass of the topical cosmetic composition.

[0025] In all mass-volume concentrations of this invention, when the mass unit is g, the corresponding volume unit is mL.

[0026] The beneficial effects of this invention are:

[0027] (1) Good stability: The dense composite shell constructed by enzymatically modified silk fibroin and physically activated soybean seed polysaccharide provides comprehensive physical and chemical protection for theaflavins. After 90 days of storage at 25°C in the dark, the theaflavins retention rate is as high as 90% or more, and the particle size increase is no more than 10%, which is far superior to conventional preparations.

[0028] (2) Significant effect of targeted release: 2-o-ethyl ascorbic acid and eugenol, as synergistic ingredients of this invention, are dispersed in the gaps of a three-dimensional network structure constructed from self-assembled short peptides, modified silk fibroin, and activated soybean seed polysaccharide. This not only effectively protects theaflavins (preventing them from being exposed and oxidized), but also allows theaflavins to exert their antioxidant, antibacterial, and anti-inflammatory effects before theaflavins themselves. At this time, the gaps in the three-dimensional network are vacated. In the weakly acidic environment (pH 4.5-5.5) of the skin surface, the self-assembled short peptides undergo pH-induced structural rearrangement, allowing theaflavins loaded in the core of the three-dimensional network structure to be released in a directional, controllable, and rapid manner through the vacated gaps, thus exerting their effects. After the theaflavins are released, water molecules re-enter the interior of the three-dimensional network structure, prolonging their residence time on the skin surface and reducing water evaporation, thereby achieving the antioxidant, whitening, and moisturizing effects of theaflavins "at the right place and at the right time".

[0029] The theaflavin nanocomplex of the present invention has a half-maximal inhibitory concentration (IC50) against tyrosinase. 50 The concentration was as low as 8.2 μg / mL, which is more than 40% lower than that of free theaflavins and their physical mixtures, and the inhibition rate of tyrosinase was as high as 85.2% at a concentration of 50 μg / mL.

[0030] (3) Highly efficient transdermal absorption: Theaflavins are encapsulated in a three-dimensional network structure composed of self-assembled short peptides, modified silk fibroin, and activated soybean seed polysaccharides. This structure has lipophilic properties and is compatible with the lipid structure of the stratum corneum, thus significantly enhancing the ability of theaflavins to penetrate the skin's stratum corneum barrier. Simultaneously, nanotechnology uniformly disperses theaflavin molecules within the lipophilic carrier, preventing the formation of large aggregates due to intermolecular forces, further improving penetration efficiency and bioavailability. In vitro transdermal experiments show that its transdermal absorption rate is at least 60% higher than that of free theaflavins.

[0031] (4) Green and controllable process: Water is used as the main solvent in the entire preparation process. Biological enzyme method and physical treatment are used in the key carrier modification stage to avoid the use of harmful chemical crosslinking agents. The process is environmentally friendly and safe as a whole, and the parameters are precise and controllable, making it easy to scale up production. Attached Figure Description

[0032] Figure 1 Scanning electron microscope (SEM) images of different theaflavin nanocomposites (A represents the nanocomposite without theaflavin, BE represents the theaflavin nanocomposites prepared from theaflavin solutions of different concentrations, namely 3.75, 5, 6.25, and 7.5 mg / mL, respectively, and F represents single theaflavin).

[0033] Figure 2The graph shows the changes in particle size and zeta potential of the theaflavins nanocomposite prepared in Example 1 after being redissolved in deionized water at different ion concentrations.

[0034] Figure 3 The graph shows the changes in particle size and zeta potential of the theaflavins nanocomposite prepared in Example 1 after being redissolved in deionized water under different pH conditions.

[0035] Figure 4 The graph shows the variation of the polydispersity index (PDI) of the theaflavins nanocomposite prepared in Example 1 under different pH conditions after being redissolved in deionized water.

[0036] Figure 5 The graph shows the change in particle size of the theaflavins nanocomposite prepared in Example 1 after being redissolved in deionized water at different storage times.

[0037] Figure 6 The graph shows the change in zeta potential at different storage times after the theaflavins nanocomposite prepared in Example 1 was redissolved in deionized water.

[0038] Figure 7 Photographs of the theaflavin nanocomplex prepared in Example 1 after being redissolved in deionized water and left at room temperature for 7 days (from left to right: aqueous solutions of the theaflavin nanocomplex at concentrations of 3, 4.5, 6, 7.5, and 9 mg / mL). Detailed Implementation

[0039] The present invention will be further explained below with reference to the embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0040] Example 1

[0041] (1) Carrier pretreatment:

[0042] Silk fibroin protein modification by protease method: Weigh 10g of purified silk fibroin protein, dissolve it in PBS buffer with pH 7.2, adjust the pH of the system to 7.0, and prepare a silk fibroin protein solution with a mass-volume concentration of 1%. Then add 0.15g of transglutaminase (accounting for 1.5% of the purified silk fibroin protein mass) to it. Stir at 200 rpm for 3 hours at 45℃. After the reaction is completed, heat in an 85℃ water bath for 10 minutes to inactivate the enzyme. Then dialyze at 4℃ using a dialysis bag with a molecular weight cutoff of 8000 Da for 48 hours. Freeze dry to obtain modified silk fibroin protein powder.

[0043] Physical activation of soybean seed polysaccharide: Weigh 20g of soybean seed polysaccharide, dissolve it in deionized water to prepare a 2% (w / v) soybean seed polysaccharide solution (about 1000mL). First, treat it with ultrasound at 800W power and 30kHz frequency with a working cycle of 2 seconds / 3 seconds interval for 30 minutes. Then, immediately treat it under 300 MPa ultra-high pressure for 10 minutes. Collect the treated solution to obtain the activated soybean seed polysaccharide solution.

[0044] (2) Three-step sequential self-assembly:

[0045] Step 1: Weigh 8g of the modified silk fibroin powder obtained in step (1) above, dissolve it in PBS buffer at pH 7.0, and prepare a modified silk fibroin solution with a mass concentration of 1% (about 800mL); weigh 5g of theaflavins with a purity of 95% (where the mass ratio of theaflavins-3-gallate to theaflavins-3'-gallate is 1:1), dissolve it in edible ethanol aqueous solution with a volume concentration of 70%, and obtain a theaflavins solution of 0.025g / mL (about 200mL); slowly add it dropwise to the modified silk fibroin solution under magnetic stirring, and at the same time add sodium dimethylsilanol hyaluronic acid ester at a mass of 0.8% of the total mass of the theaflavins solution and the modified silk fibroin solution, and vortex-incubate at 32°C and 450 rpm for 50 minutes;

[0046] Step 2: Slowly add the activated soybean seed polysaccharide solution obtained in step (1) to the system after vortex-incubation in step 1. After forming a mixed system, adjust the pH to 6.0-6.5, and add C at a mass of 0.3% of the total mass of the mixed system. 8-16 Sodium isoalkyl succinyl lactoglobulin sulfonate was immediately dispersed at high speed of 20,000 rpm for 8 minutes to form a uniform pre-emulsion.

[0047] Step 3: Weigh 0.075g of pH-responsive self-assembled short peptides (1.5% of the theaflavin mass), 0.75g of eugenol, and 3.75g of 2-o-ethyl ascorbic acid (eugenol accounts for 15% of the theaflavin mass, and the mass ratio of eugenol to 2-o-ethyl ascorbic acid is approximately 1:5). Dissolve the eugenol in 70% edible ethanol solution to obtain a mixed solution. Titrate the obtained mixed solution slowly with 0.1M hydrochloric acid to adjust its pH to 5.5. Then slowly add the pre-emulsion obtained in Step 2, followed by 0.2% of the total mass of the system at this point. The entire system is then circulated 8 times using a microfluidic homogenizer at 1600 bar, with the heat exchanger temperature maintained at 25 ± 2°C, to obtain a nano-dispersion.

[0048] (3) Product purification: The nano-dispersion obtained in step (2) is purified by dialysis bag with a molecular weight cutoff of 8000 Da to remove unencapsulated components, and then spray-dried or freeze-dried to obtain the theaflavin nanocomplex.

[0049] Example 2

[0050] The amount of transglutaminase added in step (1) silk fibroin protease modification was adjusted to 0.5% of the amount of purified silk fibroin protein, and the other conditions were the same as in Example 1.

[0051] Example 3

[0052] The amount of transglutaminase added in step (1) silk fibroin protease modification was adjusted to 0, and the other conditions were the same as in Example 1.

[0053] Table 1. Results of the effect of modified silk fibroin on the properties of theaflavin nanocomposite.

[0054]

[0055] Example 4

[0056] The ultra-high pressure treatment of the aqueous solution of soybean seed polysaccharide in step (1) physical activation of soybean seed polysaccharide is omitted. The other conditions are the same as in Example 1.

[0057] Example 5

[0058] The ultrasonic treatment of the aqueous solution of soybean seed polysaccharide in step (1) physical activation of soybean seed polysaccharide is omitted. The other conditions are the same as in Example 1.

[0059] Example 6

[0060] The ultrasonic treatment and ultra-high pressure treatment of the aqueous solution of soybean seed polysaccharide in step (1) physical activation of soybean seed polysaccharide are omitted. The other conditions are the same as in Example 1.

[0061] Table 2. Results of the effect of soybean seed polysaccharide on the properties of theaflavins nanocomplex.

[0062]

[0063] Example 7

[0064] The microjet homogenization treatment in step (2) is adjusted to hydraulic cavitation treatment, with a cavitation number of 0.15, an inlet pressure of 0.6 MPa, a treatment time of 10 minutes, and other conditions being the same as in Example 1.

[0065] Example 8

[0066] The microfluidic homogenization process in step (2) was adjusted to traditional homogenization process, high-pressure shearing, with a rotation speed of 18000 rpm and a processing time of 10 minutes. The other conditions were the same as in Example 1.

[0067] Example 9

[0068] The microfluidic homogenization process in step (2) was changed to ultrasonic cavitation process. A probe-type ultrasonic cell disruptor was used to process the cells at a power of 800W with an ultrasonic cycle of 2 seconds / intermittent 3 seconds. The processing time was 15 minutes, and the other conditions were the same as in Example 1.

[0069] Table 3. Effects of nano-sizing on the properties of theaflavin nanocomposites

[0070]

[0071] Example 10

[0072] The amount of pH-responsive self-assembled short peptide added in step (2) 3 was adjusted to 0, and the other conditions were the same as in Example 1.

[0073] Example 11

[0074] Replace the pH-responsive self-assembled short peptide in step (2) 3 with a non-pH-responsive short peptide, and keep the other conditions the same as in Example 1.

[0075] Table 4. Effects of self-assembled short peptides on the theaflavins release performance of theaflavins nanocomposites

[0076]

[0077] Table 5. Effects of self-assembled short peptides on the skin permeability of theaflavins in theaflavins nanocomplexes.

[0078]

[0079] Example 12

[0080] The purity of theaflavins in step (2) was adjusted to 80%, and the remaining conditions were the same as in Example 1.

[0081] Example 13

[0082] The purity of theaflavins in step (2) was adjusted to 70%, and the remaining conditions were the same as in Example 1.

[0083] Example 14

[0084] The mass ratio of theaflavin-3-gallate to theaflavin-3'-gallate in step (2) was adjusted to 1:0.5, and the remaining conditions were the same as in Example 1.

[0085] Table 6. Effect of the purity of theaflavins extract on the properties of theaflavins nanocomposites

[0086]

[0087] Comparing Examples 1, 12, and 13, the purity of theaflavins was affected in the following ways:

[0088] (1) Effects on nanostructure and stability: Decreased purity directly led to a significant decrease in encapsulation efficiency (96.8% → 75.6%), a sharp increase in average particle size (85.2 nm → 165.3 nm), and an increase in polydispersity index (0.15 → 0.35). This indicates that impurities in low-purity raw materials interfered with the effective binding and orderly assembly between modified silk fibroin and theaflavins, resulting in non-uniform, loosely structured nanoparticles with poor physical stability.

[0089] (2) Impact on chemical stability: Zeta potential and storage stability: Decreased purity leads to a decrease in the absolute value of the zeta potential on the surface of nanoparticles (-32.5mV → -22.3mV), which means that the electrostatic repulsion between particles is weakened, making them more prone to aggregation and precipitation. This is directly reflected in the significant decrease in the 30-day retention rate (91.5% → 65.2%), proving that the chemical stability of low-purity samples cannot meet the shelf-life requirements of products.

[0090] (3) Impact on core efficacy: Whitening and antioxidant effects: Reduced purity leads to decreased tyrosinase inhibition capacity (IC50). 50 ) and DPPH free radical scavenging ability (EC 50 All were significantly reduced. IC 50 An increase from 8.2 μg / mL to 25.3 μg / mL means the whitening effect drops to about one-third; EC 50 The increase from 12.5 μg / mL to 38.9 μg / mL indicates a significant decrease in antioxidant capacity. This proves that the presence of impurities is not only unhelpful but also dilutes and interferes with the efficacy of theaflavins. Moisturizing performance: Skin hydration improvement rate decreased from 42.5% to 22.1%. This is not due to changes in moisturizing ingredients, but rather to the deterioration of the nanostructure (increased particle size and wider distribution), leading to decreased skin permeability and bioavailability, preventing the active ingredients and moisturizing components from effectively reaching and acting on the stratum corneum.

[0091] Example 15: Efficacy comparison with commercially available whitening ingredients

[0092] Step 1: Sample Preparation

[0093] Sample A: The theaflavin nanocomposite obtained in Example 1 was dissolved in the emulsion blank matrix to prepare a theaflavin solution with a mass concentration of 0.1%.

[0094] Sample B: 2% nicotinamide solution

[0095] Sample C: Arbutin solution with a mass concentration of 0.5%

[0096] Sample D: A 10% (w / w) solution of a vitamin C derivative

[0097] Step 2: In vitro efficacy testing

[0098] Apart from the 24-hour moisturizing rate, all other indicators were evaluated using in vitro activity: tyrosinase and antioxidant activity were determined using in vitro reagent methods; cytotoxicity was evaluated using absorbance methods; and transdermal absorption rate was evaluated using skin cell model experiments.

[0099] Table 7. Performance comparison of theaflavin nanocomplex with commercially available skin-whitening ingredients.

[0100]

[0101] Example 16: Regulation of release behavior by process parameters

[0102] Step 1: Multi-factor process optimization experiment.

[0103] Using L9(3) 4 Orthogonal array design experiment, considering the following factors:

[0104] Microjet pressure (A): 1000, 1500, 2000 bar

[0105] The pH values ​​(B) of the reaction system in step (2) are: 5.4, 5.5, and 5.6.

[0106] Self-assembled short peptide addition (C): 0.5%, 1.0%, 2.0% (based on theaflavin mass)

[0107] Step 2: Correlation analysis between release behavior and efficacy.

[0108] The performance of theaflavin nanocomposites prepared under different process conditions was tested, and the results are shown in the table below.

[0109] Table 8. Effects of microfluidic pressure, system pH, and amount of self-assembled short peptides added on the properties of theaflavins nanocomposites.

[0110]

[0111] Step 3: Single-factor effect analysis

[0112] To clarify the independent influence of each parameter, range analysis was performed on the orthogonal experimental results to extract the average performance of each factor at different levels.

[0113] Single-factor effect of microjet pressure (A)

[0114] Fixed conditions: system pH (B) = 5.5, self-assembled short peptide addition amount (C) = 1.0%.

[0115] Table 9. Effect of microfluid pressure on the properties of theaflavin nanocomposites

[0116]

[0117] At a microjets pressure of 1500 bar, the theaflavins nanocomposite exhibits the smallest average particle size and most uniform distribution, resulting in the highest targeted release rate and whitening activity. Too low a pressure leads to insufficient nano-sizing; too high a pressure may cause excessive local aggregation of particles, degrading performance.

[0118] Single-factor effect of system pH (B)

[0119] Fixed conditions: microjet pressure (A) = 1500 bar, self-assembled short peptide addition amount (C) = 1.0%.

[0120] Table 10 Effect of pH on the properties of theaflavin nanocomposites

[0121]

[0122] When the system pH is too low (5.0), the self-assembled short peptides assemble too quickly, forming a porous structure that leads to severe leakage; when the pH is too high (6.0), the self-assembled short peptides are inhibited, and both the smart release properties and efficacy are significantly reduced. This strictly limits the optimal pH window.

[0123] Single-factor effect of the amount of self-assembled short peptide added (C)

[0124] Fixed conditions: microjet pressure (A) = 1500 bar, system pH (B) = 5.5.

[0125] Table 11 Effect of self-assembled short peptide addition amount on the properties of theaflavins nanocomposites

[0126]

[0127] The highest targeted release efficiency and system stability (highest absolute value of Zeta potential) can be achieved when the self-assembled short peptide is added at an amount of 1.0%. Insufficient addition results in weak driving force; excessive addition may lead to an overly dense internal structure, which is not conducive to the efficient release of theaflavins.

[0128] Example 17: Application in whitening and moisturizing serum

[0129] Step 1: Serum Formulation and Processing

[0130] Basic formula composition:

[0131] Oil phase: 5 wt% squalane + 3 wt% jojoba oil + 2 wt% vitamin E acetate;

[0132] Emulsion system: 2 wt% glyceryl stearate + 1 wt% PEG-100 stearate;

[0133] Active ingredient: 1.5 wt% the theaflavins nanocomplex of Example 1;

[0134] Preservative system: 0.8 wt% phenoxyethanol + 0.2 wt% ethylhexylglycerin;

[0135] The remainder is deionized water, which is the aqueous phase.

[0136] Preparation process:

[0137] Squalane, jojoba oil, vitamin E acetate, and glyceryl stearate and PEG-100 stearate as emulsifiers were mixed to form an oil phase. Deionized water, which served as the aqueous phase, was heated to 75°C. The oil phase was then slowly added to the aqueous phase and homogenized at 2000 rpm for 3 minutes. After the system cooled to 45°C, theaflavin nanocomplex, phenoxyethanol, and ethylhexylglycerin were added. The mixture was stirred at 100 rpm at room temperature for 30 minutes and then cooled to 35°C to obtain a whitening and moisturizing essence.

[0138] Step 2: Human efficacy testing (30 volunteers, 28 days)

[0139] Table 12 Comparison of the effects of serums containing theaflavins nanocomplexes with commercially available similar serums.

[0140]

[0141] Example 18: Application in moisturizing and repairing face masks

[0142] Step 1: Mask formulation and manufacturing process.

[0143] Facial mask liquid formula:

[0144] Moisturizer: 5wt% glycerin + 3wt% butylene glycol + 2wt% sodium hyaluronate;

[0145] Thickener: 0.8wt% Carbomer 940;

[0146] Active ingredient: 2wt% theaflavins nanocomposite of Example 1 of this invention;

[0147] Preservative: 0.5wt% phenoxyethanol;

[0148] The remainder is deionized water.

[0149] The above uses a neutralizing agent: triethanolamine aqueous solution to adjust the pH to 5.5.

[0150] Preparation process:

[0151] Carbomer 940 was uniformly dispersed in deionized water and allowed to swell for 24 hours to form an acidic dispersion. Glycerin, butylene glycol, and sodium hyaluronate were then added and stirred until homogeneous. While continuously stirring, a suitable amount of 10% triethanolamine aqueous solution was slowly added dropwise to the above system to precisely adjust the pH of the entire mixture to 5.5 ± 0.1, at which point the system transformed into a transparent gel matrix. The system was heated to 45 ± 2°C, and then the theaflavin nanocomposite obtained in Example 1 and phenoxyethanol were added. The mixture was stirred at 300–500 rpm for 20–30 minutes at 45°C until completely homogeneous, yielding the final moisturizing and repairing mask liquid. A facial mask sheet was then soaked in the mask liquid before use.

[0152] Step 2: Efficacy Verification (35 volunteers)

[0153] Table 13 Comparison of the effects of face masks containing theaflavins nanocomplexes and commercially available face masks (1 hour after use)

[0154]

[0155] Table 14 Comparison of the effects of face masks containing theaflavins nanocomplexes and commercially available face masks (after 4 weeks of continuous use).

[0156]

[0157] The above embodiments, through detailed process parameters and system performance data, fully demonstrate the significant advantages and innovations of the theaflavins nanocomposite of the present invention in terms of raw material selection, process optimization, and efficacy.

[0158] By changing the amount of theaflavins added in step (2) of Example 1, while keeping other conditions unchanged, theaflavins solutions with concentrations of 3.75, 5, 6.25, and 7.5 mg / mL were obtained, and finally, the following results were obtained: Figure 1 The theaflavin nanocomposite shown in BE. Compared with the theaflavin-free nanocomposite A and the single theaflavin F, A and F are both blocky layered structures, while BE has an ellipsoidal or spherical structure, indicating that the theaflavin is encapsulated in a shell.

[0159] The theaflavin nanocomposite prepared in Example 1 was redissolved in deionized water, and different ions were added to obtain aqueous solutions of the theaflavin nanocomposite with ion concentrations of 0, 20, 50, 100, 200, and 300 mmol / L. The aqueous solutions of the theaflavin nanocomposite obtained above were measured to obtain the following results: Figure 2 The change diagram shown:

[0160] With increasing ion concentration, the particle size of the theaflavins nanocomposite showed an increasing trend, increasing from 275.1 nm to 908 nm, while the zeta potential showed a trend of first decreasing and then stabilizing, eventually maintaining at around -8.359 mV. The absolute value of the zeta potential of this composite remained at a certain level within the range of ion concentrations of 50–300 mmol / L to maintain charge repulsion, and there was no significant increase in particle size aggregation. This indicates that the theaflavins nanocomposite prepared in this invention has a uniform dispersion state under different ion concentrations, strong resistance to ion interference, and good stability.

[0161] The theaflavin nanocomposite prepared in Example 1 was redissolved in deionized water, and the pH was adjusted to obtain aqueous solutions of the theaflavin nanocomposite with pH values ​​of 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, and 8.0. The aqueous solutions of the theaflavin nanocomposite obtained above were measured to obtain the following results: Figure 3 and Figure 4 The change diagram shown:

[0162] As the pH value increased from 3.0 to 5.0, the particle size of the theaflavins nanocomposite showed an increasing trend, increasing from 457.8 nm to 773.8 nm. The zeta potential first decreased and then stabilized, eventually remaining at 10.063 mV. The polydispersity index (PDI) showed significant changes at pH 3 and 8, reaching values ​​of 0.0627 and 0.0247, respectively. The zeta potential of the composite remained at a certain level to maintain charge repulsion, and the generally low PDI indicated a uniform particle size distribution and no significant increase in particle size aggregation. This demonstrates that the theaflavins nanocomposite prepared in this invention exhibits stable dispersion under different pH conditions, strong resistance to pH fluctuations, and good stability.

[0163] The theaflavin nanocomposite prepared in Example 1 was redissolved in deionized water to obtain aqueous solutions of the theaflavin nanocomposite with concentrations of 0.5 and 4.5 mg / mL; single theaflavin was dissolved in deionized water to obtain an aqueous solution of single theaflavin. The aqueous solutions obtained above were measured to obtain the following results: Figure 5 and Figure 6 The change diagram shown:

[0164] like Figure 5As shown, during the 10-day storage period, the particle size of single theaflavins fluctuated significantly and generally showed an upward trend, while the particle size of the theaflavins nanocomposite prepared in Example 1 remained relatively stable, maintaining between 212 and 340 nm and 848.7 and 932 nm. These results indicate that the dispersion state of free single theaflavins is relatively unstable during storage (481–1145 nm), while the theaflavins nanocomposite prepared in this invention showed no obvious aggregation, demonstrating better stability.

[0165] like Figure 6 As shown, during the 10-day storage period, the zeta potential of single theaflavins fluctuated significantly, remaining between -6.965 and 2.148 mV. In contrast, the zeta potential of the theaflavins nanocomposite prepared in Example 1 was relatively stable, remaining around 12.139-16.626 mV and 20.002-24.714 mV. These results indicate that the zeta potential of single theaflavins is relatively unstable and fluctuates greatly during storage. However, the zeta potential of the theaflavins nanocomposite prepared in this invention did not show significant decay during storage, the charge repulsion effect was maintained, and there was no risk of aggregation due to potential decrease, demonstrating good stability.

[0166] like Figure 7 As shown, the theaflavins nanocomplex prepared in Example 1 was redissolved in deionized water to obtain aqueous solutions of theaflavins nanocomplex with concentrations of 3, 4.5, 6, 7.5, and 9 mg / mL from left to right. After standing at room temperature for 7 days, all five tubes of theaflavins nanocomplex aqueous solutions remained clear, exhibiting a uniform brownish-red color, and no precipitation or stratification occurred, indicating that the theaflavins nanocomplex prepared in this invention has good stability.

[0167] Of course, the present invention may have other various embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and modifications according to the present invention, but these corresponding changes and modifications should all fall within the protection scope of the claims of the present invention.

Claims

1. A theaflavin nanocomplex with whitening and moisturizing effects, characterized in that, The theaflavin nanocomposite is mainly prepared from theaflavins as active components, an encapsulating carrier, and synergistic ingredients, and has a core-shell structure with theaflavins as the core and a three-dimensional network formed by the encapsulating carrier as the shell; the purity of the theaflavins is not less than 80%, wherein the mass ratio of theaflavins-3-gallate to theaflavins-3'-gallate is 1:0.8-1.2; the encapsulating carrier includes modified silk fibroin, activated soybean seed polysaccharide, and pH-responsive self-assembled short peptides, which form a three-dimensional network through reaction; the synergistic ingredients are 2-o-ethyl ascorbic acid and eugenol.

2. The theaflavin nanocomposite according to claim 1, characterized in that, The modified silk fibroin has a β-sheet content ≥40% and a free carboxyl content that is more than 15% higher than that of the unmodified silk fibroin; the activated soybean seed polysaccharide has a weight-average molecular weight of 10-30 kDa and a degree of esterification ≤30%.

3. The theaflavin nanocomposite according to claim 1, characterized in that, The self-assembled short peptides are in a dispersed state at pH ≥ 6.5 and undergo conformational changes in a weakly acidic environment of pH 5.0–5.

8.

4. The theaflavin nanocomposite according to claim 1, characterized in that, The theaflavin nanocomposite has an average particle size of 70–150 nm, a polydispersity index ≤0.18, and an encapsulation efficiency ≥96%.

5. A method for preparing a theaflavin nanocomposite with whitening and moisturizing effects as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Carrier pretreatment: Silk fibroin protein modification by protease method: The purified silk fibroin protein is dissolved in pH buffer to obtain a purified silk fibroin protein solution with a mass-volume concentration of 0.5% to 3.0%. The pH of the system is adjusted to 6.8 to 7.5, and glutamine transaminase is added at a mass ratio of 1% to 3% of the purified silk fibroin protein. The mixture is stirred at 35 to 45°C for 2 to 4 hours. After the reaction is completed, the enzyme is inactivated, dialyzed, and freeze-dried to obtain modified silk fibroin protein powder. Physical activation of soybean seed polysaccharide: Soybean seed polysaccharide was prepared into an aqueous solution with a mass-volume concentration of 2% to 5%. It was first subjected to ultrasonic treatment at a power of 500 to 800 W and a frequency of 15 to 35 kHz for 30 to 60 minutes, followed by treatment under ultra-high pressure of 250 to 300 MPa for 5 to 10 minutes. The treated solution was collected to obtain an activated soybean seed polysaccharide solution. (2) Three-step sequential self-assembly: Step 1: Weigh the modified silk fibroin powder obtained in step (1) above, dissolve it in pH buffer solution to prepare a modified silk fibroin solution with a mass concentration of 1% to 5%; dissolve theaflavins in edible ethanol aqueous solution to obtain a theaflavins solution with a mass concentration of 0.001 to 0.15 g / mL; slowly add the theaflavins solution to the modified silk fibroin solution under magnetic stirring, and at the same time slowly add sodium dimethylsilanol hyaluronic acid ester with a mass of 0.5% to 1% of the total mass of the system, and vortex-incubate at 30 to 35°C for 30 to 60 minutes; Step 2: Slowly add an equal volume of the activated soybean seed polysaccharide solution obtained in step (1) to the system after the first step of vortex-incubation to form a mixed system. After adjusting the pH to 6.0-6.5, add C at a mass of 0.2%-0.5% of the total mass of the mixed system. 8-16 Sodium isoalkyl succinyl lactoglobulin sulfonate was then dispersed at high speed of 15,000–20,000 rpm for 5–10 minutes to form a uniform pre-emulsion. Step 3: Dissolve the self-assembled short peptide, 2-o-ethyl ascorbic acid, and eugenol together in an edible ethanol solution to obtain a mixed solution; slowly add hydrochloric acid or citric acid solution dropwise to the obtained mixed solution, adjust its pH value to 5.4-5.6, and then slowly add it to the pre-emulsion obtained in step 2, followed by adding 0.1%-0.3% of 4-butylresorcinol by the total mass of the system. Subsequently, the entire system is circulated through a microfluidic homogenizer or cavitation treatment to obtain a uniformly dispersed nano-dispersion. (3) Product purification: The nano-dispersion obtained in step (2) is purified by dialysis bag with a molecular weight cutoff of 8000-10000 Da to remove unencapsulated components, and then spray-dried or freeze-dried to obtain theaflavin nanocomplex.

6. The preparation method according to claim 5, characterized in that, The mass ratio of theaflavins to modified silk fibroin, activated soybean seed polysaccharide, and self-assembled short peptides is 1:5-10; the mass ratio of modified silk fibroin to activated soybean seed polysaccharide is 1:1-2.5; the mass of the self-assembled short peptides is 0.5%-2.0% of the mass of theaflavins; the mass ratio of eugenol to 2-o-ethyl ascorbic acid is 1:2.5-10; and the mass of eugenol is 10%-20% of the mass of theaflavins.

7. The preparation method according to claim 5, characterized in that, In step (1), the ultrasonic treatment for the physical activation of soybean seed polysaccharide is performed using a probe-type ultrasonic instrument. Continuous ultrasonic treatment or intermittent ultrasonic treatment is selected. When using continuous ultrasonic treatment, the frequency is 15-25 kHz; when using intermittent ultrasonic treatment, the frequency is 25-35 kHz, and the working cycle is 2-5 seconds of ultrasonic treatment followed by 3-4 seconds of intermittent treatment.

8. The preparation method according to claim 5, characterized in that, In step (1), the stirring speed of the stirring reaction during the silk fibroin protease modification is 200-400 rpm; in step (2), the rotation speed of the vortex apparatus used during the vortex-incubation in the first step is 300-500 rpm; in step (2), the pressure of the micro-jet homogenizer during the treatment is 1500-1800 bar, and the number of cycles is 5-8; in step (2), the cavitation treatment in the third step uses hydraulic cavitation with a cavitation number of 0.1-0.15 and a time of 10-20 minutes or ultrasonic cavitation with a frequency of 16 kHz-100 kHz and a time of 25-35 minutes.

9. The use of the theaflavin nanocomposite as described in any one of claims 1-4 or the theaflavin nanocomposite obtained by the preparation method according to any one of claims 5-8 in the preparation of topical cosmetic compositions for skin whitening, anti-oxidation and moisturizing.

10. The application according to claim 9, characterized in that, The theaflavin nanocomplex is added at a mass of 0.5% to 3.0% of the total mass of the topical cosmetic composition.

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