Skin antioxidant repair composition and method for synergistically regulating NRF2 by hexapeptide modifier

Abstract

The invention discloses a skin anti-oxidation repairing composition for synergistically regulating NRF2 by a hexapeptide modifier and a method of the skin anti-oxidation repairing composition. The skin anti-oxidation repairing composition is prepared from the following active ingredients in percentage by mass: 0.1 to 5 percent of ergothioneine, 0.1 to 30 percent of hydroxypropyl tetrahydropyrantriol and 0.1 to 10 percent of ectoine, the composition also comprises a synergistic component, and the synergistic component is selected from 0.01-10% of a hexapeptide modifier PCB11. The invention relates to the technical field of cosmetics. According to the skin antioxidant repair composition for synergistically regulating and controlling the NRF2 through the hexapeptide modifier and the method thereof, an NRF2 channel is activated in a synergic targeting manner through hexapeptide PCB11 and ergothioneine (the expression of NQO1 is improved by 6.8 times), efficient entrapment and intelligent release of active ingredients are achieved through a vitamin E-lecithin composite carrier, the anti-wrinkle, repair and light protection effects of the composition are clinically verified, and the composition has good application prospects. The product is obviously superior to a traditional single-target product.

Description

Technical Field

[0001] The present invention relates to the field of cosmetic technology, and specifically to a skin antioxidant repair composition and method for synergistically regulating NRF2 with a hexapeptide modification. Background Art

[0002] As the largest exposed organ in the human body, the skin is exposed to long-term environmental stressors such as ultraviolet (UV) rays and pollutants, leading to oxidative stress, inflammatory cascades, and extracellular matrix (ECM) degradation. Existing technologies have bottlenecks such as insufficient synergy and low transdermal efficiency in single-target strategies (such as NRF2 activators). For example: 1. Patent CN112456789A uses sulforaphane to activate NRF2, but its inhibition of SASP is insufficient (no significant difference in IL-6 secretion, p>0.05); 2. US20210052321A1 combines resveratrol and vitamin C, but the two compete for the transdermal transporter (SVCT2), resulting in a 42% decrease in bioavailability; 3. Traditional liposomes have problems such as low encapsulation efficiency (<70%), poor photostability, and non-responsive release (for example, the soybean lecithin carrier in CN103340759A loses more than 40% of its activity after UV irradiation). Summary of the Invention

[0003] In response to the shortcomings of the existing technology, the present invention provides a skin antioxidant repair composition and method for synergistically regulating NRF2 with a hexapeptide modifier, which solves the bottleneck problems of single-target strategies (such as NRF2 activators) such as insufficient synergy and low transdermal efficiency.

[0004] To achieve the above objectives, the present invention is implemented through the following technical solutions: a skin antioxidant repair composition that synergistically regulates NRF2 with a hexapeptide modification, comprising the following active ingredients in the following mass percentages: Ergothioneine 0.1-5%, Hydroxypropyl Tetrahydropyrantriol 0.1-30%, Ectoine 0.1-10%; The invention also comprises a synergistic component, wherein the synergistic component is selected from the hexapeptide modification PCB110.01-10%.

[0005] Preferably, the PCB11 and ergothioneine are co-loaded in a vitamin E-lecithin composite nanocarrier, the particle size of the nanocarrier is 50-150 nm, and the encapsulation efficiency is ≥85%.

[0006] The hexapeptide modification PCB11 is a palmitoylated hexapeptide (structural formula: Palmitoyl-GHKSPH), and its preparation method refers to patent number CN117466973A. It is coupled with a C16 fatty acid chain by solid-phase synthesis and purified by HPLC (purity ≥95%).

[0007] The molecular structure of PCB11 contains a C16 fatty acid chain, and its binding energy with the Kelch domain of KEAP1 protein (PDB: 8IXS) is ≤-8.5 kcal / mol, which was verified by AutoDock4.2 molecular docking.

[0008] Preferably, the mass ratio of the PCB11 to ergothioneine satisfies; ; And the environmental response release kinetics under UVB irradiation is consistent with; ; Among them, the cumulative release rate triggered by UV for 24 hours is ≥90%, and the release rate under UV-free conditions is ≤20%.

[0009] Preferably, the composition satisfies the inhibition rate of skin aging-associated secretory phenotype (SASP) factors; ; The data were determined by ELISA in a 3D skin model.

[0010] The present invention also discloses a skin antioxidant repair composition and method for synergistically regulating NRF2 with a hexapeptide modification, comprising the following steps: a. Vitamin E-lecithin composite nanocarriers were prepared by thin film hydration method. Lecithin and vitamin E (mass ratio 1:0.5) were dissolved in chloroform and rotary evaporated to form a lipid film. The hydration medium was phosphate buffer (pH 6.5) containing ergothioneine and PCB11. b. high-pressure homogenization (pressure 10,000-15,000 psi, 3-5 cycles) to form nanocarriers with a particle size of 50-150 nm; c. Ectoin is embedded into the nanocarrier bilayer membrane through hydrophobic interaction, with an adsorption density of 2.8-4.1 molecules / nm²; d. The pH of the final product is adjusted to 5.5-6.0, and pharmaceutically acceptable excipients are added.

[0011] Preferably, the high-pressure homogenization process parameters in step b include: Lipid concentration 2-5% w / v; Homogenizing pressure 10,000 psi; The particle size distribution of the final product is PDI≤0.25, and the Zeta potential is -15±5mV.

[0012] Preferably, the composition achieves the following effects in human clinical trials: The SOD activity of the stratum corneum increased by ≥200% within 28 days (measured by colorimetry); Transepidermal water loss (TEWL) reduced by ≥30% (tested by Tewameter TM300); Forehead wrinkle volume reduction ≥15% (PRIMOS 3D imaging analysis).

[0013] The present invention provides a skin antioxidant repair composition and method for synergistically regulating NRF2 with a hexapeptide modification. Compared with the prior art, it has the following advantages: This skin antioxidant repair composition and method for synergistically regulating NRF2 with a hexapeptide modifier, targets and activates the NRF2 pathway through synergistic targeting of hexapeptide PCB11 and ergothioneine (NQO1 expression increased by 6.8 times), combined with a vitamin E-lecithin composite carrier (encapsulation efficiency ≥85%, UV-triggered release rate ≥89%). Clinically, TEWL decreased by ≥28% and wrinkle volume decreased by ≥15% after 28 days, achieving multi-dimensional anti-aging effects of antioxidant (SOD activity +213%), anti-inflammatory (IL-6 inhibition rate 69.2%), and collagen regeneration (synthesis amount +42%). Clinically, wrinkles (-18.3%) and skin barrier (TEWL↓31.7%) were significantly improved after 28 days, and the production process is stable (activity retention rate 95%), breaking through the limitations of traditional single targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a molecular docking diagram of PCB11 and KEAP1 of the present invention; Figure 2 Schematic diagram of NQO1 mRNA expression level (qPCR) of the present invention; Figure 3 This is a schematic diagram of the IL-6 and MMP-1 inhibition rates of the present invention; Figure 4 Schematic diagram of molecular docking of the present invention; Figure 5 Schematic diagram of the UV-triggered release curve of the present invention. DETAILED DESCRIPTION

[0015] The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0016] See also Figure 1-Figure 5 , the embodiment of the present invention provides a technical solution: a skin antioxidant repair composition that synergistically regulates NRF2 with a hexapeptide modification, comprising the following active ingredients in the following mass percentages: Ergothioneine 0.1-5%, Hydroxypropyl Tetrahydropyrantriol 0.1-30%, Ectoine 0.1-10%; The invention also comprises a synergistic component, wherein the synergistic component is selected from the hexapeptide modification PCB110.01-10%.

[0017] Furthermore, the PCB11 and ergothioneine are co-loaded in a vitamin E-lecithin composite nanocarrier, the particle size of the nanocarrier is 50-150 nm, and the encapsulation efficiency is ≥85%.

[0018] The molecular structure of PCB11 contains a C16 fatty acid chain, and its binding energy with the Kelch domain of KEAP1 protein (PDB: 8IXS) is ≤-8.5 kcal / mol, which was verified by AutoDock4.2 molecular docking.

[0019] Further, the mass ratio of the PCB11 to ergothioneine satisfies; ; And the environmental response release kinetics under UVB irradiation is consistent with; ; Among them, the cumulative release rate triggered by UV for 24 hours is ≥90%, and the release rate under UV-free conditions is ≤20%.

[0020] Furthermore, the composition satisfies the inhibition rate of skin aging-associated secretory phenotype (SASP) factors; ; The data were determined by ELISA in a 3D skin model.

[0021] The present invention also discloses a method for preparing a skin antioxidant repair composition for synergistically regulating NRF2 with a hexapeptide modification, comprising the following steps: a. Vitamin E-lecithin composite nanocarriers were prepared by thin film hydration method. Lecithin and vitamin E (mass ratio 1:0.5) were dissolved in chloroform and rotary evaporated to form a lipid film. The hydration medium was phosphate buffer (pH 6.5) containing ergothioneine and PCB11. b. high-pressure homogenization (pressure 10,000-15,000 psi, 3-5 cycles) to form nanocarriers with a particle size of 50-150 nm; c. Ectoin is embedded into the nanocarrier bilayer membrane through hydrophobic interaction, with an adsorption density of 2.8-4.1 molecules / nm²; d. The pH of the final product is adjusted to 5.5-6.0, and pharmaceutically acceptable excipients are added.

[0022] Furthermore, the high pressure homogenization process parameters in step b include: Lipid concentration 2-5% w / v; Homogenizing pressure 10,000 psi; The particle size distribution of the final product is PDI≤0.25, and the Zeta potential is -15±5mV.

[0023] Furthermore, the composition achieved the following effects in human clinical trials: The SOD activity of the stratum corneum increased by ≥200% within 28 days (measured by colorimetry); Transepidermal water loss (TEWL) reduced by ≥30% (tested by Tewameter TM300); Forehead wrinkle volume reduction ≥15% (PRIMOS 3D imaging analysis).

[0024] Example 1: Preparation of core anti-aging composition Recipe composition: Ergothioneine (EGT): 0.5%; Hydroxypropyl tetrahydropyrantriol (HPT): 5%; Ectoin: 1%; Hexapeptide modification PCB11: 0.1%; Carrier system: lecithin 1.2% + vitamin E 0.6%; Deionized water: balance.

[0025] Preparation method: Liposome encapsulation Preparation of lipid film: Lecithin (1.2 g) and vitamin E (0.6 g) were dissolved in 20 mL of chloroform and rotary evaporated (40 °C, 100 rpm) to form a uniform film.

[0026] Hydration: Add PBS buffer (pH 6.5, 50 mL) containing ergothioneine (0.5 g) and PCB11 (0.1 g), vortex and then sonicate (50°C, 200 W, 15 min).

[0027] High pressure homogenization A high-pressure homogenizer (Nano DeBEE, 10,000 psi) was used for four cycles to obtain nanocarriers with uniform particle size distribution.

[0028] Ectoin modification Ectoin (1 g) was mixed with the nanocarrier suspension, incubated at 50 °C for 2 h, and centrifuged to remove unbound components. Characterization data: Particle size: D50=105±12nm (dynamic light scattering method); Encapsulation efficiency: ergothioneine 89.2%, PCB11 86.5% (ultrafiltration centrifugation-HPLC combination); Photostability: Activity retention rate after ultraviolet irradiation (UVB 30mJ / cm²) ≥92%.

[0029] Example 2: Verification of PCB11-KEAP1 targeted binding Experimental design: Molecular docking Protein processing: The crystal structure of the KEAP1 Kelch domain was downloaded from the PDB database (PDB ID: 8IXS), water molecules and original ligands were removed, and hydrogen atoms and charges were added using AutoDock Tools.

[0030] Ligand processing: The PCB11 structure was optimized by Gaussian 16 (M062X / LANL2DZ basis set) and a pdbqt file was generated.

[0031] Docking parameters were as follows: the center coordinates of the docking space (x = 5.52, y = 13.58, z = 5.00), a grid size of 25 × 25 × 25 Å, and a depletion value of 10.

[0032] Results: The binding energy between PCB11 and KEAP1 is -8.5 kcal / mol, and the hydrogen bonding involves Arg483, Ser508 ( Figure 1 ).

[0033] SPR binding assay The KEAP1 Kelch domain was immobilized on a CM5 sensor chip with a PCB11 concentration gradient of 0.1-100 nM and a flow rate of 30 μL / min.

[0034] Data: Binding constant KD = 13.2 nM, dissociation rate koff = 1.2 × 10⁻³ s⁻¹ (Biacore T200 analysis).

[0035] Example 3: Verification of anti-inflammatory and antioxidant effects Experimental groups: blank control group (NC); UVB irradiation model group (UV, 60mJ / cm²); PCB11 group (0.1%); EGT group (0.5%); EGT+PCB11 group (0.5%+0.1%).

[0036] Detection method: 1. NQO1 expression (qPCR) Total RNA was extracted from HaCaT cells and reverse transcribed into cDNA. Primer sequences: NQO1-F:5'-CAGTCAGCGACCTCATCTCC-3'; NQO1-R:5'-TCCAGCCGTTTCTTCCATCT-3'.

[0037] Results: The NQO1 mRNA expression level in the EGT+PCB11 group was 6.8 times that in the NC group (p<0.01), and was significantly higher than that in the single group (3.1 times in the EGT group and 2.9 times in the PCB11 group).

[0038] The sequence listing is as follows; SEQUENCE_LISTING SEQUENCE_ID: 1 SEQUENCE_NAME: NQO1-F LENGTH: 20 TYPE: DNA SEQUENCE_DATA: cag tca gcg acc tca tct cc SEQUENCE_ID: 2 SEQUENCE_NAME: NQO1-R LENGTH: 20 TYPE: DNA SEQUENCE_DATA: tcc agc cgt ttc ttc cat ct.

[0039] Note: The primer sequences used in this experiment (SEQ ID NO: 1-2) were designed based on the human NQO1 gene (GenBank: NM_000903.2). They are only used to verify the technical effect and do not constitute the protection content of this invention. The standard sequence listing has been submitted in addition.

[0040] 2. IL-6 and MMP-1 inhibition rate (ELISA) The cell supernatant was collected and detected using Human IL-6 / MMP-1 ELISA Kit (R&D Systems).

[0041] Table 1: Results Example 4: Preparation of environmentally responsive facial mask Recipe composition: Vitamin E-lecithin composite nanocarrier (loaded with PCB11+EGT): 3%; Titanium dioxide-coated mesoporous silica nanoparticles (UV-triggered units): 1.5%; Sodium hyaluronate: 0.5%.

[0042] Preparation method: 1. Preparation of UV-responsive carrier: Mesoporous silica (pore size 5 nm) was immersed in a mixed solution of PCB11+EGT (mass ratio 1:5) and vacuum adsorbed for 12 h.

[0043] The surface is coated with a titanium dioxide layer (thickness 10 nm) and calcined and solidified (300 ° C, 2 hours).

[0044] When triggered by ultraviolet rays, titanium dioxide produces reactive oxygen species that increase the permeability of the lipid membrane, with a 24-hour release rate of 89%.

[0045] The lipid carrier itself has a photoprotective effect (vitamin E antioxidant), reducing UV degradation of the active ingredients.

[0046] 2. Mask preparation: The core composition was mixed with sodium hyaluronate, and the UV responsive carrier was added and magnetically stirred until homogeneous.

[0047] Soak the Tencel fiber mask base material for 30 minutes and then divide it into aluminum foil bags.

[0048] Effect verification: UV-triggered release: Under UVB irradiation (30mJ / cm²), the release rate is 89% in 24 hours (HPLC detection); Instant moisturizing: Skin moisture content increased to 158% after 15 minutes of use (measured by Corneometer CM825).

[0049] Example 5: Application of Repair Cream Formulation Recipe composition: Example 1 Core composition: 80%; Acetyl Tripeptide-1: 0.01% Palmitoyl Pentapeptide-4: 0.01% Blue copper peptide (GHK-Cu): 0.05%; Carnosine: 0.2% Shea butter: 5%; Palmitoylethanolamide: 0.05% Emulsifier (cetearyl alcohol): 3%.

[0050] Preparation method: The oil phase (shea butter, emulsifier) ​​and the water phase (core composition, GHK-Cu) were heated to 75°C separately.

[0051] The oil phase was slowly added to the water phase and homogenized (5,000 rpm, 5 min).

[0052] When cooled to 40℃, add EGF, stir until it reaches room temperature and then fill.

[0053] Wound repair effect; Skin defect model: After 7 days of using the repair cream, the healing area reached 85% (45% in the control group, p<0.001); Collagen density: Masson staining showed that the density of collagen fibers recovered to 96% of normal skin.

[0054] The present invention uses hexapeptide PCB11 and ergothioneine to synergistically target and activate the NRF2 pathway (NQO1 expression increased by 6.8 times), combined with a vitamin E-lecithin composite carrier intelligent delivery system (encapsulation rate > 90%, UV-triggered release rate ≥ 90%), to achieve multi-dimensional anti-aging effects of antioxidant (SOD activity +213%), anti-inflammatory (IL-6 inhibition rate 69.2%) and collagen regeneration (synthesis amount +42%). Clinically, wrinkles (-18.3%) and skin barrier (TEWL↓31.7%) were significantly improved in 28 days, and the production process is stable (activity retention rate 95%), breaking through the limitations of traditional single targets.

[0055] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0056] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A skin antioxidant repair composition that synergistically regulates NRF2 with a hexapeptide modification, characterized by: It is composed of the following active ingredients in the following percentages by mass: Ergothioneine 0.1-5%, Hydroxypropyl Tetrahydropyrantriol 0.1-30%, Ectoine 0.1-10%; The invention also comprises a synergistic component, wherein the synergistic component is selected from the hexapeptide modification PCB110.01-10%.

2. The skin antioxidant repairing composition according to claim 1, wherein the hexapeptide modification synergistically regulates NRF2, is characterized in that: The PCB11 and ergothioneine are co-loaded in a vitamin E-lecithin composite nanocarrier, the particle size of the nanocarrier is 50-150 nm, and the encapsulation efficiency is ≥85%.

3. The skin antioxidant repairing composition according to claim 1, wherein the hexapeptide modification synergistically regulates NRF2, is characterized in that: The mass ratio of the PCB11 to ergothioneine satisfies; ； And the environmental response release kinetics under UVB irradiation is consistent with; ； Among them, the cumulative release rate triggered by UV for 24 hours is ≥90%, and the release rate under UV-free conditions is ≤20%.

4. The skin antioxidant repairing composition according to claim 1, wherein the hexapeptide modification synergistically regulates NRF2, is characterized in that: The composition satisfies the inhibition rate of skin aging-associated secretory phenotype (SASP) factors; ； The data were determined by ELISA in a 3D skin model.

5. A method for preparing a skin antioxidant repairing composition for synergistically regulating NRF2 with a hexapeptide modification according to any one of claims 1 to 4, characterized in that: The following steps are involved: a. Vitamin E-lecithin composite nanocarriers were prepared by thin film hydration method. Lecithin and vitamin E were dissolved in chloroform at a mass ratio of 1:0.5 and rotary evaporated to form a lipid film. The hydration medium was a phosphate buffer solution (pH 6.5) containing ergothioneine and PCB11. b. high-pressure homogenization treatment, pressure 10,000-15,000 psi, 3-5 cycles, to form nanocarriers with a particle size of 50-150 nm; c. Ectoin is embedded into the nanocarrier bilayer membrane through hydrophobic interaction, with an adsorption density of 2.8-4.1 molecules / nm²; d. The pH of the final product is adjusted to 5.5-6.0, and pharmaceutically acceptable excipients are added.

6. The method for preparing a skin antioxidant repairing composition for synergistically regulating NRF2 with a hexapeptide modification according to claim 5, characterized in that: The high pressure homogenization process parameters in step b include: Lipid concentration 2-5% w / v; Homogenizing pressure 10,000 psi; The particle size distribution of the final product is PDI≤0.25, and the Zeta potential is -15±5mV.

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