Composition for promoting collagen regeneration and application and product thereof

Abstract

The invention belongs to the technical field of cosmetics and biological medicines, and particularly relates to a composition for promoting collagen regeneration as well as application and a product thereof. The composition provided by the invention is prepared from collagen peptide, sodium hyaluronate and hydroxytyrosol, the combination of the collagen peptide, the sodium hyaluronate and the hydroxytyrosol has a synergistic effect. The composition has remarkable advantages in the aspects of collagen regeneration and skin improvement, the content of I, III, IV, VII and XVII type collagen can be remarkably increased, and multi-effect collagen regeneration promotion is achieved by inhibiting collagen degrading enzyme MMP-1 and up-regulating TGF-beta1; the content of AQP3 and HA can be increased, and water locking and moisturizing of the skin are enhanced; by activating an Nrf2 pathway, the SOD2 level is improved, IL-6 and IL-1alpha are inhibited, and the antioxidant and anti-inflammatory effects are achieved; the thickness of an epidermal layer and the density of dermal fibroblasts can be remarkably improved, wrinkle resistance and repairing are effectively achieved, and the skin structure is improved.

Description

Technical Field

[0001] This invention belongs to the fields of cosmetics and biomedicine, specifically relating to a composition that promotes collagen regeneration and its applications and products. Background Technology

[0002] In the fields of cosmetics and biomedicine, promoting collagen regeneration is a key technological approach to addressing issues such as skin aging, wound repair, and tissue fibrosis. As a core component of the extracellular matrix, the content and structural integrity of collagen directly affect skin elasticity, wound healing speed, and tissue function. However, current related technologies and products still have significant limitations and urgently require breakthroughs.

[0003] From the perspective of ingredient systems, existing compositions for promoting collagen regeneration have significant drawbacks. On the one hand, traditional active ingredients such as vitamin C and retinol, while promoting collagen synthesis by activating prolyl hydroxylase, are chemically unstable and easily oxidized and inactivated in formulations. Furthermore, high concentrations can irritate the skin, causing adverse reactions such as erythema and desquamation. On the other hand, bioactive ingredients, such as recombinant human epidermal growth factor (rhEGF), can efficiently induce collagen synthesis, but their production costs are high, and long-term use may induce abnormal cell proliferation, posing a potential carcinogenic risk. Furthermore, existing technologies, such as patent CN119367238B, provide an active composition for promoting collagen regeneration, consisting of *Desmodium styracifolium* extract, *Vermicomyces cerevisiae* extract, deer bone collagen, tripeptide-1, and oligopeptide-2. This invention achieves excellent collagen regeneration by combining various raw materials, resulting in a synergistic effect. However, it focuses primarily on regulating the synthesis of type I collagen, while paying insufficient attention to the synergistic effects of other subtypes such as type III collagen (involved in early skin repair), type IV collagen (maintaining the integrity of the basement membrane), and type VII collagen (anchoring the epidermis and dermis). Consequently, it is difficult to achieve comprehensive effects in skin repair, moisturizing, and barrier function improvement.

[0004] At the mechanism of action level, current technologies exhibit a clear single-path dependency. Most products promote collagen synthesis solely by upregulating the transforming growth factor-β (TGF-β) / Smad signaling pathway, neglecting the degradation effect of matrix metalloproteinases (MMPs) on collagen. Simultaneously, oxidative stress and inflammatory responses, as important triggers for accelerated collagen loss, are often overlooked by existing technologies. Reactive oxygen species (ROS) can damage collagen fiber structure and inhibit fibroblast activity, while inflammatory factors such as IL-6 and TNF-α exacerbate collagen degradation by activating MMP expression. Products lacking synergistic antioxidant and anti-inflammatory mechanisms struggle to address the root cause of collagen loss. Furthermore, existing products are often limited to single application scenarios, lacking multifunctional solutions that can accommodate both daily cosmetic care and clinical biopharmaceutical treatments.

[0005] In summary, developing a collagen regeneration composition with safe and controllable components, multi-dimensional synergistic mechanism of action, and wide applicability has become a core direction for breaking through current technological bottlenecks. Summary of the Invention

[0006] To address the above shortcomings, the present invention provides a composition for promoting collagen regeneration, its application, and products.

[0007] The technical solution of this invention is as follows: On one hand, the present invention provides a composition for promoting collagen regeneration, the composition comprising collagen peptides, sodium hyaluronate and hydroxytyrosol.

[0008] Specifically, the mass ratio of collagen peptides, sodium hyaluronate, and hydroxytyrosol in the composition is 78-82:4-10:0.396-0.528.

[0009] Preferably, the mass ratio of collagen peptides, sodium hyaluronate, and hydroxytyrosol in the composition is 78.0-78.5, 78.5-80.0, 80.0-80.5, 80.5-81.0, 81.0-81.5, or 81.5-82.0: 4.0-4.5, 4.5-5.0, 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, 8.5-9.0, 9.0-9.5, or 9.5-10.0: 0.396-0.400, 0.400-0.450, 0.450-0.500, or 0.500-0.528.

[0010] More preferably, the mass ratio of collagen peptides, sodium hyaluronate, and hydroxytyrosol in the composition is 80.5-81.0: 4.5-5.0, 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5, 8.5-9.0, 9.0-9.5, or 9.5-10.0: 0.400-0.450.

[0011] More preferably, the mass ratio of collagen peptides, sodium hyaluronate, and hydroxytyrosol in the composition is 80.647:4.839-9.679:0.403.

[0012] More preferably, the mass ratio of collagen peptides, sodium hyaluronate, and hydroxytyrosol in the composition is 80.647:4.839:0.403 or 80.647:9.678:0.403.

[0013] Specifically, the collagen peptides contain at least 20% collagen tripeptides.

[0014] More preferably, the collagen peptides contain 20%-100% collagen tripeptides.

[0015] More preferably, the collagen peptides comprise 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100% collagen tripeptides.

[0016] On the other hand, the present invention provides a method for preparing the composition described in any of the above claims, the method comprising mixing collagen peptides, sodium hyaluronate and hydroxytyrosol in a certain proportion to obtain the composition.

[0017] In another aspect, the present invention provides a composition formulation comprising any of the compositions described above.

[0018] Specifically, the composition formulation further includes any one or more of the following: plant extracts, vitamins, bioactive peptides, enzymes, acids, polysaccharides, or mineral elements.

[0019] Preferably, the plant extracts include, but are not limited to, any one or more of the following: ginseng extract, grape seed extract, licorice extract, tea extract, aloe vera extract, witch hazel extract, rosemary extract, centella asiatica extract, peony extract, angelica extract, coix seed extract, poria cocos extract, polygonum cuspidatum extract, mulberry bark extract, honeysuckle extract, orange peel extract, lemon extract, peach blossom extract, ginkgo leaf extract, wolfberry extract, rose extract, lavender extract, chamomile extract, atractylodes macrocephala extract, peony root bark extract, scutellaria baicalensis extract, astragalus membranaceus extract, angelica dahurica extract, bletilla striata extract, oat extract, eucommia ulmoides extract, kudzu root extract, polygonatum odoratum extract, rehmannia glutinosa extract, eleutherococcus senticosus extract, cornus officinalis extract, salvia miltiorrhiza extract, chrysanthemum extract, cinnamon extract, magnolia officinalis extract, schisandra chinensis extract, amomum villosum extract, and cistanche deserticola extract.

[0020] Specifically, the vitamins mentioned include fat-soluble vitamins and / or water-soluble vitamins.

[0021] Preferably, the fat-soluble vitamins include, but are not limited to, any one or more of vitamin A, vitamin D, vitamin E, or vitamin K; Preferably, the water-soluble microorganisms include, but are not limited to, any one or more of the following: vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B12, vitamin C, choline, or inositol.

[0022] Preferably, the bioactive peptides include, but are not limited to, any one or more of the following: collagen peptides, elastin peptides, glutathione, casein phosphopeptides, hyaluronic acid hydrolysate peptides, ceramide peptides, soybean peptides, corn peptides, sea cucumber peptides, oyster peptides, bird's nest peptides, carnosine, or lactoferrin peptides.

[0023] Preferably, the enzymes include, but are not limited to, any one or more of the following: superoxide dismutase, catalase, glutathione peroxidase, bromelain, papain, and lysozyme.

[0024] Preferably, the acidic substances include, but are not limited to, any one or more of the following: vitamin A and its derivatives, vitamin C and its derivatives, fruit acids, salicylic acid, arbutin, tranexamic acid, kojic acid, ferulic acid, or azelaic acid.

[0025] Specifically, the polysaccharides include, but are not limited to, any one or more of the following: fungal polysaccharides, animal polysaccharides, plant polysaccharides, and algal polysaccharides.

[0026] Preferably, the fungal polysaccharides include, but are not limited to, any one or more of the following: Poria cocos polysaccharide, Tremella fuciformis polysaccharide, Enoki mushroom polysaccharide, Auricularia auricula-judae polysaccharide, Cordyceps sinensis polysaccharide, Phellinus linteus polysaccharide, and Trametes versicolor polysaccharide.

[0027] Preferably, the animal polysaccharide includes any one or more of the following: glycogen, chondroitin sulfate, hyaluronic acid, heparin, chitosan, chitin, or glycosaminoglycans; Preferably, the plant polysaccharides include any one or more of the following: cellulose, hemicellulose, pectin, inulin, konjac polysaccharide, astragalus polysaccharide, wolfberry polysaccharide, tea polysaccharide, kelp polysaccharide, or jujube polysaccharide; Preferably, the algal polysaccharide includes any one or more of green algal polysaccharides, red algal polysaccharides, or brown algal polysaccharides.

[0028] Preferably, the mineral elements include, but are not limited to, any one or more of the following: zinc, copper, iron, magnesium, calcium, or silicon.

[0029] More preferably, the composition further includes epigallocatechin gallate.

[0030] More preferably, the composition formulation comprises the composition described in any one of the above claims and gallic catechin gallate.

[0031] In another aspect, the present invention provides the use of the compositions described in any one of the foregoing claims and the formulations of the compositions described in any one of the foregoing claims in the preparation of products, wherein the products are selected from any one of the following: (1) Cosmetics that are anti-aging, hydrating, moisturizing, anti-inflammatory, or antioxidant; Or (2) anti-inflammatory, antioxidant, or skin hydration supplements; Or (3) Anti-aging, skin moisture improvement, anti-inflammatory or antioxidant drugs.

[0032] In another aspect, the present invention provides a cosmetic comprising the composition described in any one of the preceding claims and the formulation of the composition described in any one of the preceding claims.

[0033] Specifically, the cosmetics include, but are not limited to, any one or more of the following: face cream, face mask, eye cream, neck cream, hand cream, body lotion, moisturizer, serum, toner, shampoo, conditioner, shower gel, or facial cleanser.

[0034] Specifically, the cosmetics also include excipients acceptable in the cosmetics industry.

[0035] Preferably, the cosmetic excipients acceptable in the cosmetic field include, but are not limited to, one or more of the following: moisturizers, thickeners, emollients, skin conditioning agents, emulsifiers, preservatives, softeners, lubricants, and stabilizers.

[0036] In another aspect, the present invention provides a health product comprising any of the compositions or composition preparations described above.

[0037] Specifically, the dosage forms of the health products include solid dosage forms, semi-solid dosage forms, or liquid dosage forms.

[0038] Specifically, the health products also include nutritionally acceptable nutritional additives.

[0039] Preferably, the nutritional additives include, but are not limited to, any one or more of the following: dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0040] In another aspect, the present invention provides a medicine comprising any of the compositions or formulations described above.

[0041] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0042] Preferably, the pharmaceutically acceptable excipients include, but are not limited to: solvents, diluents, disintegrants, precipitation inhibitors, surfactants, flow aids, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavorings, sweeteners, ion exchangers, release agents, coating agents, flavoring agents, or antioxidants.

[0043] Specifically, the dosage form of the drug, depending on the route of administration, includes, but is not limited to, gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0044] Preferably, the gastrointestinal dosage forms include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0045] Preferably, the gastrointestinal dosage forms include, but are not limited to, injectable dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0046] A method for anti-aging, hydrating, moisturizing, improving skin moisture status, anti-inflammatory, or antioxidant purposes, the method comprising using any of the compositions, compositional formulations, cosmetics, health products, or pharmaceuticals described above.

[0047] The beneficial effects of this invention are as follows: (1) The composition of the present invention can significantly increase the content of type I, III, IV, VII and XVII collagen; at the same time, it inhibits the collagen degradation enzyme MMP-1 and upregulates the expression level of the pro-synthesis factor TGF-β1, indicating that the composition of the present invention can promote collagen regeneration in multiple ways.

[0048] (2) The composition of the present invention can significantly increase the content of AQP3 and HA, strengthen the skin's water-locking function, and has a highly effective moisturizing effect.

[0049] (3) The composition of the present invention can activate the Nrf2 pathway, increase the expression level of SOD2, and inhibit IL-6 and IL-1α inflammatory factors, and has strong antioxidant and anti-inflammatory effects. (4) The composition of the present invention increases the thickness of the epidermis by 47.81%-62.58% and the density of dermal fibroblasts by 217.65%-250.00%, effectively resists wrinkles and repairs skin structure.

[0050] (5) The composition of the present invention combines collagen peptides, sodium hyaluronate and hydroxytyrosol to prepare a composition that can play a synergistic role. Attached Figure Description

[0051] Figure 1 The results show the thickness of the epidermal viable cell layer; ## in the figure represents P < 0.01 compared with group BC; ** represents P < 0.01 compared with group NC.

[0052] Figure 2 The results show the density of dermal fibroblasts; ## in the figure represents P < 0.01 compared with group BC; ** represents P < 0.01 compared with group NC.

[0053] Figure 3The results show the Collagen I expression levels; ## in the figure represents P < 0.01 compared to group BC; ** represents P < 0.01 compared to group NC.

[0054] Figure 4 The results show the Collagen III expression levels; ## in the figure represents P < 0.01 compared to the BC group; ** represents P < 0.01 compared to the NC group.

[0055] Figure 5 The results show the Collagen IV expression levels; ## in the figure represents P < 0.01 compared to the BC group; ** represents P < 0.01 compared to the NC group; and *** represents P < 0.001 compared to the NC group.

[0056] Figure 6 The results show the expression levels of Collagen VII; ## in the figure represents P < 0.01 compared with the BC group; ** represents P < 0.01 compared with the NC group; and *** represents P < 0.001 compared with the NC group.

[0057] Figure 7 The results show the expression levels of Collagen XVII; ## in the figure represents P < 0.01 compared to group BC; ** represents P < 0.01 compared to group NC.

[0058] Figure 8 The results show the collagen fiber content determination; ## in the figure represents P < 0.01 compared with group BC; ** represents P < 0.01 compared with group NC.

[0059] Figure 9 The results are for the determination of TGF-β1 content; ## in the figure represents P < 0.01 compared with group BC; ** represents P < 0.01 compared with group NC.

[0060] Figure 10 The results are for the determination of MMP-1 content; ## in the figure represents P < 0.01 compared with group BC; ** represents P < 0.01 compared with group NC.

[0061] Figure 11 The results are for HA content determination; ## in the figure represents P < 0.01 compared with group BC; ** represents P < 0.01 compared with group NC; *** represents P < 0.001 compared with group NC.

[0062] Figure 12 The results are for Nrf2 content determination; ## in the figure represents P < 0.01 compared with group BC; ** represents P < 0.01 compared with group NC.

[0063] Figure 13The results are for SOD2 content determination; ## in the figure represents P < 0.01 compared with group BC; ** represents P < 0.01 compared with group NC.

[0064] Figure 14 The results are for AQP3 content determination; ## in the figure represents P < 0.01 compared with group BC; ** represents P < 0.01 compared with group NC.

[0065] Figure 15 The results are for IL-6 content determination; ## in the figure represents P < 0.01 compared with group BC; ** represents P < 0.01 compared with group NC.

[0066] Figure 16 The results are for IL-1α content determination; ## in the figure represents P < 0.01 compared with group BC; ** represents P < 0.01 compared with group NC. Detailed Implementation

[0067] The present invention will be further clearly and completely illustrated below through examples. These examples are only a portion of the embodiments of the present invention and are not intended to limit the invention, but are merely illustrative. Unless otherwise specified, the experimental methods used in the following examples are conventional experiments. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. The collagen peptides of the present invention were purchased from Haitongyuan Food Technology Co., Ltd., batch number TC240621A2.

[0068] Example 1 Composition The composition of Example 1 consists of collagen peptides, sodium hyaluronate, and hydroxytyrosol; the mass ratio of collagen peptides, sodium hyaluronate, and hydroxytyrosol in the composition is 80.647:4.839:0.403.

[0069] The composition of Example 1 was prepared by weighing collagen peptides, sodium hyaluronate and hydroxytyrosol respectively, mixing them well to obtain the composition.

[0070] Example 2 Composition The composition of Example 2 consists of collagen peptides, sodium hyaluronate, and hydroxytyrosol; the mass ratio of collagen peptides, sodium hyaluronate, and hydroxytyrosol in the composition is 80.647:9.678:0.403.

[0071] The composition of Example 2 was prepared by weighing collagen peptides, sodium hyaluronate and hydroxytyrosol respectively, mixing them well to obtain the composition.

[0072] Example 3 Composition The composition of Example 3 consists of collagen peptides, sodium hyaluronate, and hydroxytyrosol; the mass ratio of collagen peptides, sodium hyaluronate, and hydroxytyrosol in the composition is 78:10:0.396.

[0073] The composition of Example 3 was prepared by weighing collagen peptides, sodium hyaluronate and hydroxytyrosol respectively, mixing them well to obtain the composition.

[0074] Example 4 Composition The composition of Example 4 consists of collagen peptides, sodium hyaluronate, and hydroxytyrosol; the mass ratio of collagen peptides, sodium hyaluronate, and hydroxytyrosol in the composition is 80:4:0.528.

[0075] The composition of Example 4 was prepared by weighing collagen peptides, sodium hyaluronate and hydroxytyrosol respectively, mixing them well to obtain the composition.

[0076] Example 5 Composition Formulation The composition formulation of Example 5 comprises the composition of Example 1 and epigallocatechin gallate. The mass ratio of collagen peptides, sodium hyaluronate, hydroxytyrosol, and epigallocatechin gallate in the composition formulation is 80.647:4.839:0.403:1.199.

[0077] The preparation method of the composition formulation in Example 5 is as follows: epigallocatechin gallate is added to the composition of Example 1, mixed well, and the composition formulation is obtained.

[0078] Comparative Example 1 Composition The composition of Comparative Example 1 consists of collagen peptides and sodium hyaluronate; the mass ratio of collagen peptides to sodium hyaluronate in the composition is 80.647:4.839.

[0079] The composition of Comparative Example 1 was prepared by weighing collagen peptides and sodium hyaluronate separately, mixing them well to obtain the composition.

[0080] Comparative Example 2 Composition The composition of Comparative Example 2 consists of collagen peptides and hydroxytyrosol; the mass ratio of collagen peptides to hydroxytyrosol in the composition is 80.647:0.403.

[0081] The composition of Comparative Example 2 was prepared by weighing collagen peptides and hydroxytyrosol separately, mixing them well to obtain the composition.

[0082] Experimental Example 1 Add 3.7 mL of ex vivo skin model culture medium (Guangdong Boxi Biotechnology Shaanxi Branch) to each well and incubate at 37℃ in a 5% CO2 incubator, changing the medium daily. After 2 days of ex vivo skin model culture, the models were randomly divided into blank group (BC), negative control group (NC), positive control group (PC), THA group, J group, EG group, Q group, Example 1-Example 5 groups, and Comparative Example 1-Comparative Example 2 groups. Each group underwent the following drug treatments: Blank control (BC) group: Replace with fresh culture medium; Negative control (NC) group: Replace with fresh culture medium; Positive control (PC) group: Replace with fresh culture medium containing 0.01% m / v VC and 0.0007% m / v VE; THA group: Replace with fresh culture medium containing 0.004839% m / v sodium hyaluronate solution; Group J: Replace with fresh culture medium containing 0.080647% m / v collagen peptides; EG group: Replace with fresh culture medium containing 0.001199% m / v epigallocatechin gallate; Group Q: Replace with fresh medium containing 0.000403% m / v hydroxytyrosol, i.e., fresh medium containing 0.000305% v / v hydroxytyrosol; Example 1 group: Replace the fresh culture medium containing the composition of Example 1, that is, give fresh culture medium containing 0.080647% m / v collagen peptide, 0.004839% m / v sodium hyaluronate and 0.000403% m / v hydroxytyrosol; Example 2 group: Replace the fresh culture medium containing the composition of Example 2, that is, give fresh culture medium containing 0.080647% m / v collagen peptide, 0.009678% m / v sodium hyaluronate and 0.000403% m / v hydroxytyrosol; Example 3 group: Replace the fresh culture medium containing the composition of Example 3, that is, give fresh culture medium containing 0.078% m / v collagen peptide, 0.010% m / v sodium hyaluronate and 0.000396% m / v hydroxytyrosol; Example 4 group: Replace the fresh culture medium containing the composition of Example 4, that is, give fresh culture medium containing 0.080% m / v collagen peptide, 0.004% m / v sodium hyaluronate and 0.000528% m / v hydroxytyrosol; Example 5 group: Replace the fresh culture medium containing the composition of Example 5, that is, give fresh culture medium containing 0.080647% m / v collagen peptide, 0.004839% m / v sodium hyaluronate, 0.000403% m / v hydroxytyrosol and 0.001199% m / v epigallocatechin gallate; Comparative Example 1: Replace with fresh culture medium containing the composition of Comparative Example 1, i.e., give fresh culture medium containing 0.080647% m / v collagen peptides and 0.004839% m / v sodium hyaluronate; Comparative Example 2: Replace the culture medium with fresh medium containing the composition of Comparative Example 2, i.e., give fresh medium containing 0.080647% m / v collagen peptides and 0.000403% m / v hydroxytyrosol.

[0083] After completing the above drug administration treatment, all groups except BC were irradiated (30 J / cm²). 2 UVA +50mJ / cm 2 (UVB). After irradiation, the ex vivo skin model was incubated at 37°C in 5% CO2 medium for 24 hours. This procedure was repeated four times. After the repeated procedures, each group was treated with the drug daily for three days.

[0084] 1. Anti-wrinkle index: Epidermal cell layer thickness measurement Skin models used for testing were fixed with 4% paraformaldehyde for 24 hours. After fixation, H&E staining was performed, and images were taken and observed under a microscope. The thickness of the epidermal cell layer was collected and analyzed.

[0085] The measurement results are as follows Figure 1 As shown, compared with the BC group, the epidermal viable cell layer thickness in the NC group was significantly decreased, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the epidermal viable cell layer thickness in the PC group was significantly increased, indicating that the positive control detection was effective. Compared with the NC group, the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly increased the epidermal viable cell layer thickness. Among them, the average epidermal layer thickness of Example 1-Example 5 groups were 72.48 μm, 73.56 μm, 72.09 μm, 70.35 μm, and 74.71 μm, respectively, with an increase rate of 57.81%-67.59% compared with the NC group.

[0086] 2. Anti-wrinkle index: Determination of dermal cell thickness Skin models used for testing were fixed with 4% paraformaldehyde for 24 hours. After fixation, H&E staining was performed, and images were taken and observed under a microscope. The thickness of the dermal live cell layer was collected and analyzed.

[0087] The measurement results are as follows Figure 2As shown, compared with the BC group, the dermal fibroblast density in the NC group was significantly decreased, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the dermal fibroblast density in the PC group was significantly increased, indicating that the positive control detection was effective. Compared with the NC group, the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly increased the dermal fibroblast density. Among them, the dermal fibroblast density of Example 1-Example 5 groups increased by 217.65%-250.00% compared with the NC group.

[0088] 3. Anti-wrinkle index: Collagen I expression level determination Skin models used for testing were fixed in 4% paraformaldehyde for 24 hours. Immunofluorescence detection (Collagen I antibody, CST) was then performed, and images were taken and analyzed under a microscope. The green fluorescent region represents Collagen I; the stronger the green fluorescence, the higher the Collagen I content.

[0089] The measurement results are as follows Figure 3 As shown, compared with the BC group, the Collagen I content in the NC group was significantly decreased, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the Collagen I content in the PC group was significantly increased, indicating that the positive control test was effective. Compared with the NC group, the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly increased the Collagen I content. Among them, the Collagen I content increase rate in Example 1-Example 5 groups was more significant than that in the other groups, with an increase rate of 155.88%-188.24% compared with the NC group.

[0090] 4. Anti-wrinkle index: Collagen III expression level determination Skin models used for testing were fixed with 4% paraformaldehyde for 24 hours, followed by immunofluorescence detection (Collagen III, Wuhan Sanying). Images were taken and analyzed under a microscope. The green fluorescent region represents Collagen III; stronger green fluorescence indicates a higher Collagen III content.

[0091] The measurement results are as follows Figure 4 As shown, compared with the NC group, the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly increased the Collagen III content. Among them, the Collagen III content of Example 1-Example 5 groups increased by 264.52%-303.23% compared with the NC group.

[0092] 5. Anti-wrinkle index: Collagen IV expression level determination Skin models used for testing were fixed in 4% paraformaldehyde for 24 hours. Immunofluorescence detection (Collagen IV antibody, Abcam) was then performed, and images were taken and analyzed under a microscope. The green fluorescent region represents Collagen IV; the stronger the green fluorescence, the higher the Collagen IV content.

[0093] The measurement results are as follows Figure 5 As shown, compared with the NC group, the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly increased the Collagen IV content. Among them, the Collagen IV content increase rate of Example 1-Example 5 groups was more significant than that of the other groups, with an increase rate of 117.78%-151.11% compared with the NC group.

[0094] 6. Anti-wrinkle index: Collagen VII expression level determination Skin models used for testing were fixed in 4% paraformaldehyde for 24 hours, followed by immunofluorescence detection (Collagen VII, Abcam). Images were taken and analyzed under a microscope. The green fluorescent region represents Collagen VII; stronger green fluorescence indicates a higher Collagen VII content.

[0095] The measurement results are as follows Figure 6 As shown, compared with the NC group, the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly increased the Collagen VII content, with improvement rates of 56.10%, 31.71%, 57.78%, 58.54%, 148.78%, 155.56%, 120.00%, 113.33%, 184.44%, 66.67%, and 106.67%, respectively. The combined improvement rate of the THA group, J group, and Q group compared to the NC group was 146.35%, while the improvement rate of the composition in Example 1 of this invention was 148.78%. This demonstrates the synergistic effect of the compositions of this invention.

[0096] 7. Anti-wrinkle index: Collagen XVII expression level determination Skin models used for testing were fixed in 4% paraformaldehyde for 24 hours. Immunofluorescence detection (using Collagen XVII antibody, Abcam) was then performed, and images were taken and analyzed under a microscope. The green fluorescent region represents Collagen XVII; stronger green fluorescence indicates a higher Collagen XVII content.

[0097] The measurement results are as follows Figure 7 As shown, compared with the NC group, the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly increased the content of Collagen XVII. Among them, the increase rate of Collagen XVII content in Example 1-Example 5 groups was more significant than that in the other groups, with an increase rate of 133.33%-153.33% compared with the NC group.

[0098] 8. Anti-wrinkle index: Collagen fiber content determination Skin models used for testing were fixed with 4% paraformaldehyde, stained with Masson's stain, and photographed under a microscope. The blue area represents collagen fibers; the larger the blue area, the higher the collagen fiber content.

[0099] The measurement results are as follows Figure 8 As shown, compared with the BC group, the collagen fiber content in the NC group was significantly decreased, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the Collagen XVII content in the PC group was significantly increased, indicating that the positive control test was effective. Compared with the NC group, the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly increased collagen fiber content. Among them, the collagen fiber increase rate of Example 1-Example 5 groups was more significant than that of the other groups, with an increase rate of 110.42%-120.83% compared with the NC group.

[0100] 9. Anti-wrinkle index: TGF-β1 content determination Skin models used for testing were fixed in 4% paraformaldehyde for 24 hours. Immunofluorescence detection (TGF-β1 antibody, Abcam) was then performed, and images were taken and analyzed under a microscope. The green fluorescent region represents TGF-β1; the stronger the green fluorescence, the higher the TGF-β1 content.

[0101] The measurement results are as follows Figure 9As shown, compared with the BC group, the TGF-β1 content in the NC group was significantly decreased, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the TGF-β1 content in the PC group was significantly increased, indicating that the positive control detection was effective. Compared with the NC group, the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly increased TGF-β1 expression. The improvement rates of the THA group, J group, Q group, and Example 1 group compared with the NC group were 85.37%, 53.66%, 117.07%, and 270.73%, respectively. Among them, the total improvement rate of the THA group + J group + Q group compared with the NC group was 256.10%, while the improvement rate of the composition of Example 1 of this invention was 270.73%. This demonstrates the synergistic effect of the composition of this invention.

[0102] 10. Anti-wrinkle index: MMP-1 content determination After the administration of the drug, the skin model culture medium was collected in centrifuge tubes. After collection, the samples used for ELISA detection were frozen at -80°C and analyzed according to the instructions of the MMP-1 ELISA kit (Abcam).

[0103] The measurement results are as follows Figure 10 As shown, compared with the BC group, the MMP-1 content in the NC group was significantly increased, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the MMP-1 content in the PC group was significantly decreased, indicating that the positive control detection was effective. Compared with the NC group, the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly reduced MMP-1 expression. Among them, Example 1-Example 5 groups showed the most significant inhibition of MMP-1 expression, with inhibition rates of 42.31%-46.36% compared with the NC group.

[0104] 11. Moisturizing index: HA content determination Biotin coloration assay: A skin model for testing was taken and fixed with 4% paraformaldehyde for 24 hours. The images were then photographed and analyzed (Hyaluronan Binding Protein, Biotin Conj., Amsbio). Green fluorescence represents HA; the stronger the green fluorescence, the higher the HA content.

[0105] The measurement results are as follows Figure 11As shown, compared with the BC group, the HA content in the NC group decreased significantly, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the HA content in the PC group increased significantly, indicating that the positive control detection was effective. Compared with the NC group, the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly increased the HA content. Among them, the improvement effect of Example 1-Example 5 groups was more obvious, with an improvement rate of 183.87%-261.29% compared with the NC group.

[0106] 12. Antioxidant index: Nrf2 content determination Skin models used for testing were fixed in 4% paraformaldehyde for 24 hours. Immunofluorescence detection (Nrf2 antibody, Abcam) was then performed, and images were taken and analyzed under a microscope. The green fluorescent region represents Nrf2; the stronger the green fluorescence, the higher the Nrf2 content.

[0107] The measurement results are as follows Figure 12 As shown, compared with the BC group, the Nrf2 content in the NC group decreased significantly, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the Nrf2 content in the PC group increased significantly, indicating that the positive control detection was effective. Compared with the NC group, the Nrf2 content in the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly increased the Nrf2 content. Among them, the improvement effect of Example 1-Example 5 groups was more obvious, with an improvement rate of 240.00%-263.33% compared with the NC group.

[0108] 13. Antioxidant index: SOD2 content determination Skin models used for testing were fixed in 4% paraformaldehyde for 24 hours. Immunofluorescence detection (SOD2 antibody, Abcam) was then performed, and images were taken and analyzed under a microscope. The green fluorescent region represents SOD2; the stronger the green fluorescence, the higher the SOD2 content.

[0109] The measurement results are as follows Figure 13 As shown, compared with the BC group, the SOD2 content in the NC group decreased significantly, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the SOD2 content in the PC group increased significantly, indicating that the positive control detection was effective. Compared with the NC group, the SOD2 content in the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly increased the SOD2 content. Among them, the improvement effect of Example 1-Example 5 groups was more obvious, with an improvement rate of 266.67%-326.67% compared with the NC group.

[0110] Experimental Example 2 Add 3.7 mL of ex vivo skin model culture medium (Guangdong Boxi Biotechnology Shaanxi Branch) to each well and incubate at 37℃ in a 5% CO2 incubator, changing the medium daily. After 2 days of ex vivo skin model culture, the models were randomly divided into blank group (BC), negative control group (NC), positive control group (PC), THA group, J group, EG group, Q group, Example 1-Example 5 groups, and Comparative Example 1-Comparative Example 2 groups. Each group underwent the following drug treatments: Blank control (BC) group: Replace with fresh culture medium; Negative control (NC) group: Replace with fresh culture medium; Positive control (PC) group: Replace with fresh culture medium containing 0.0033% m / v CaCl2; THA group: Replace with fresh culture medium containing 0.004839% m / v sodium hyaluronate solution; Group J: Replace with fresh culture medium containing 0.080647% m / v collagen peptides; EG group: Replace with fresh culture medium containing 0.001199% m / v epigallocatechin gallate; Group Q: Replace with fresh medium containing 0.000403% m / v hydroxytyrosol, i.e., fresh medium containing 0.000305% v / v hydroxytyrosol; Example 1 group: Replace the fresh culture medium containing the composition of Example 1, that is, give fresh culture medium containing 0.080647% m / v collagen peptide, 0.004839% m / v sodium hyaluronate and 0.000403% m / v hydroxytyrosol; Example 2 group: Replace the fresh culture medium containing the composition of Example 2, that is, give fresh culture medium containing 0.080647% m / v collagen peptide, 0.009678% m / v sodium hyaluronate and 0.000403% m / v hydroxytyrosol; Example 3 group: Replace the fresh culture medium containing the composition of Example 3, that is, give fresh culture medium containing 0.078% m / v collagen peptide, 0.010% m / v sodium hyaluronate and 0.000396% m / v hydroxytyrosol; Example 4 group: Replace the fresh culture medium containing the composition of Example 4, that is, give fresh culture medium containing 0.080% m / v collagen peptide, 0.004% m / v sodium hyaluronate and 0.000528% m / v hydroxytyrosol; Example 5 group: Replace the fresh culture medium containing the composition of Example 5, that is, give fresh culture medium containing 0.080647% m / v collagen peptide, 0.004839% m / v sodium hyaluronate, 0.000403% m / v hydroxytyrosol and 0.001199% m / v epigallocatechin gallate; Comparative Example 1: Replace with fresh culture medium containing the composition of Comparative Example 1, i.e., give fresh culture medium containing 0.080647% m / v collagen peptides and 0.004839% m / v sodium hyaluronate; Comparative Example 2: Replace the culture medium with fresh medium containing the composition of Comparative Example 2, i.e., give fresh medium containing 0.080647% m / v collagen peptides and 0.000403% m / v hydroxytyrosol.

[0111] After completing the above drug administration treatment, all groups except BC were irradiated (30 J / cm²). 2 UVA +50mJ / cm 2 (UVB). After irradiation, the ex vivo skin model was incubated at 37°C in 5% CO2 medium for 24 hours. This procedure was repeated four times. After the repeated procedures, each group was treated with the drug daily for three days.

[0112] Moisturizing index: AQP3 content determination Skin models used for testing were fixed in 4% paraformaldehyde for 24 hours. Immunofluorescence detection (AQP3 antibody, Abcam) was then performed, and images were taken and analyzed under a microscope. The green fluorescent region represents AQP3; the stronger the green fluorescence, the higher the AQP3 content.

[0113] The measurement results are as follows Figure 14 As shown, compared with the BC group, the AQP3 content in the NC group was significantly lower, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the AQP3 content in the PC group was significantly higher, indicating that the positive control detection was effective. Compared with the NC group, the AQP3 content in the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly increased the AQP3 content. Among them, the improvement effect of Example 1-Example 5 groups was more obvious, with an improvement rate of 142.22%-177.78% compared with the NC group.

[0114] Experimental Example 3 Add 3.7 mL of ex vivo skin model culture medium (Guangdong Boxi Biotechnology Shaanxi Branch) to each well and incubate at 37℃ in a 5% CO2 incubator, changing the medium daily. After 2 days of ex vivo skin model culture, the models were randomly divided into blank group (BC), negative control group (NC), positive control group (PC), THA group, J group, EG group, Q group, Example 1-Example 5 groups, and Comparative Example 1-Comparative Example 2 groups. Each group underwent the following drug treatments: Blank control (BC) group: Replace with fresh culture medium; Negative control (NC) group: Replace with fresh culture medium; Positive control (PC) group: Replace with fresh culture medium containing 0.01% m / v dexamethasone; THA group: Replace with fresh culture medium containing 0.004839% m / v sodium hyaluronate solution; Group J: Replace with fresh culture medium containing 0.080647% m / v collagen peptides; EG group: Replace with fresh culture medium containing 0.001199% m / v epigallocatechin gallate; Group Q: Replace with fresh medium containing 0.000403% m / v hydroxytyrosol, i.e., fresh medium containing 0.000305% v / v hydroxytyrosol; Example 1 group: Replace the fresh culture medium containing the composition of Example 1, that is, give fresh culture medium containing 0.080647% m / v collagen peptide, 0.004839% m / v sodium hyaluronate and 0.000403% m / v hydroxytyrosol; Example 2 group: Replace the fresh culture medium containing the composition of Example 2, that is, give fresh culture medium containing 0.080647% m / v collagen peptide, 0.009678% m / v sodium hyaluronate and 0.000403% m / v hydroxytyrosol; Example 3 group: Replace the fresh culture medium containing the composition of Example 3, that is, give fresh culture medium containing 0.078% m / v collagen peptide, 0.010% m / v sodium hyaluronate and 0.000396% m / v hydroxytyrosol; Example 4 group: Replace the fresh culture medium containing the composition of Example 4, that is, give fresh culture medium containing 0.080% m / v collagen peptide, 0.004% m / v sodium hyaluronate and 0.000528% m / v hydroxytyrosol; Example 5 group: Replace the fresh culture medium containing the composition of Example 5, that is, give fresh culture medium containing 0.080647% m / v collagen peptide, 0.004839% m / v sodium hyaluronate, 0.000403% m / v hydroxytyrosol and 0.001199% m / v epigallocatechin gallate; Comparative Example 1: Replace with fresh culture medium containing the composition of Comparative Example 1, i.e., give fresh culture medium containing 0.080647% m / v collagen peptides and 0.004839% m / v sodium hyaluronate; Comparative Example 2: Replace the culture medium with fresh medium containing the composition of Comparative Example 2, i.e., give fresh medium containing 0.080647% m / v collagen peptides and 0.000403% m / v hydroxytyrosol.

[0115] After completing the above drug administration treatment, all groups except BC were irradiated (30 J / cm²). 2UVA +50mJ / cm 2 (UVB). After irradiation, the ex vivo skin model was incubated at 37°C in 5% CO2 medium for 24 hours. This procedure was repeated four times. After the repeated procedures, each group was treated with the drug daily for three days.

[0116] After drug administration, the skin model culture medium was collected in centrifuge tubes. After collection, the samples used for ELISA detection were frozen at -80°C and analyzed according to the instructions of the IL-1α ELISA kit (Abcam) and IL-6 ELISA kit (Abcam).

[0117] 1. Anti-inflammatory marker: IL-6 content measurement results The measurement results are as follows Figure 15 As shown, compared with the BC group, the IL-6 level in the NC group was significantly increased, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the IL-6 level in the PC group was significantly decreased, indicating that the positive control detection was effective. Compared with the NC group, the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly reduced IL-6 expression, with the improvement effect of Example 1-Example 5 groups being more significant than the other groups, with an inhibition rate of 70.38%-75.50% compared with the NC group.

[0118] 2. Anti-inflammatory marker: IL-1α content determination The measurement results are as follows Figure 16 As shown, compared with the BC group, the IL-1α level in the NC group was significantly increased, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the IL-1α level in the PC group was significantly decreased, indicating that the positive control detection was effective. Compared with the NC group, the THA group, J group, EG group, Q group, Comparative Example 1 group, Comparative Example 2 group, and Example 1-Example 5 groups all significantly reduced IL-1α expression, with the improvement effect of Example 1-Example 5 groups being more significant than the other groups, with an inhibition rate of 66.41%-69.42% compared with the NC group.

[0119] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A composition for promoting collagen regeneration, characterized in that, The composition consists of collagen peptides, sodium hyaluronate, and hydroxytyrosol.

2. The composition according to claim 1, characterized in that, The mass ratio of collagen peptides, sodium hyaluronate, and hydroxytyrosol in the composition is 78-82:4-10:0.396-0.

528.

3. The composition according to claim 2, characterized in that, The mass ratio of collagen peptides, sodium hyaluronate, and hydroxytyrosol in the composition is 80.647:4.839-9.678:0.

403.

4. A method for preparing the composition according to any one of claims 1-3, characterized in that, The preparation method includes mixing collagen peptides, sodium hyaluronate, and hydroxytyrosol in parts by weight to obtain a composition.

5. A composition formulation comprising the composition according to any one of claims 1-3.

6. The composition formulation according to claim 5, characterized in that, The composition formulation further includes any one or more of the following: plant extracts, vitamins, bioactive peptides, enzymes, acids, polysaccharides, or mineral elements.

7. The composition formulation according to claim 6, characterized in that, The composition formulation also includes epigallocatechin gallate.

8. The use of the composition according to any one of claims 1-3 or the formulation of the composition according to any one of claims 5-7 in the preparation of a product, characterized in that, The product mentioned is selected from any one of the following products: (1) Cosmetics that are anti-aging, hydrating, moisturizing, anti-inflammatory, or antioxidant; Or (2) anti-inflammatory, antioxidant, or skin hydration supplements; Or (3) Anti-aging, skin moisture improvement, anti-inflammatory or antioxidant drugs.

9. A cosmetic product, characterized in that, The cosmetic product comprises the composition according to any one of claims 1-3 or the composition formulation according to any one of claims 5-7.

10. The cosmetic product according to claim 9, characterized in that, The cosmetics mentioned include any one or more of the following: face cream, face mask, eye cream, neck cream, hand cream, body lotion, moisturizer, serum, toner, shampoo, conditioner, shower gel, or facial cleanser.

11. The cosmetic product according to claim 9, characterized in that, The cosmetics mentioned also include excipients acceptable in the cosmetics industry.

12. The cosmetic product according to claim 11, characterized in that, The cosmetic excipients acceptable in the field include one or more of the following: moisturizers, thickeners, emollients, skin conditioning agents, emulsifiers, preservatives, softeners, lubricants, and stabilizers.

13. A health product, characterized in that, The cosmetic product comprises the composition according to any one of claims 1-3 or the composition formulation according to any one of claims 5-7.

14. The health product according to claim 13, characterized in that, The dosage forms of the health products include solid dosage forms, semi-solid dosage forms, or liquid dosage forms.

15. The health product according to claim 13, characterized in that, The health products mentioned also include nutritionally acceptable nutritional additives.

16. The health product according to claim 13, characterized in that, The nutritional additives include any one or more of the following: dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

17. A medicine, characterized in that, The pharmaceutical product comprises the composition according to any one of claims 1-3 or the composition formulation according to any one of claims 5-7.

18. The pharmaceutical product according to claim 17, characterized in that, The medicine also includes pharmaceutically acceptable excipients.

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