Multifunctional yak collagen hydrotherapy needle and application thereof in skin photoaging

Abstract

The invention belongs to the technical field of skin repair, and particularly relates to a multifunctional yak collagen hydrotherapy needle and application thereof in skin photoaging. The invention provides a multifunctional yak collagen water-light injection. The water-light injection is prepared from the following components: yak collagen, vitamin C, alpha-arbutin, sodium hyaluronate, NMN, PDRN and copper blue peptide. The water-light needle has the remarkable effects of resisting oxidation, whitening, preserving moisture, promoting tissue repair and relieving; in addition, the yak collagen has excellent biological safety, no cytotoxicity, no whole-body toxicity, no pyrogenicity and no hemolysis risk, and the yak collagen keeps a complete triple-helix structure; the water-light needle can effectively improve skin texture, improve skin density and hydration, significantly reduce transdermal moisture loss, promote skin color to recover to a healthy state, significantly promote collagen regeneration and accelerate a photoaging skin recovery process, and has important significance in the field of skin photoaging repair.

Description

Technical Field

[0001] The present invention belongs to the technical field of skin repair, and in particular relates to a multifunctional yak collagen water light injection and its application in skin photoaging. Background Art

[0002] With the accelerated pace of modern life and the increase in environmental pressure, skin aging, especially facial aging, has become an increasingly prominent problem that needs to be overcome in the field of cosmetics research and development and dermatology. Anti-wrinkle and anti-aging are the current key directions of scientific research and industry. Due to the complexity of the aging mechanism, which involves multiple factors such as oxidative stress, cell function decline, protein damage and changes in the skin microenvironment, a single ingredient is often difficult to exert significant efficacy, and the screening of efficient combinations faces great challenges. In particular, in achieving antioxidant and anti-wrinkle functions, not only are the ingredients required to have excellent biological activity, but they also need to take into account stability, synergy and safety, which has long been the core problem that restricts technological progress in this field.

[0003] Common antioxidant ingredients include vitamin C, vitamin E, coenzyme Q10, glutathione, resveratrol, green tea extract, ferulic acid, astaxanthin, proanthocyanidins, arbutin, perilla extract, curcumin, cysteine, and alpha-lipoic acid. Whitening ingredients include tranexamic acid, kojic acid, glabridin, allantoin, hydroquinone, niacinamide, ellagic acid, 3-O-ethyl ascorbic acid, pomegranate extract, dipotassium glycyrrhizate, vitamin E, alpha-arbutin, chamomile extract, and nonapeptide. Hydrating and moisturizing ingredients include sodium hyaluronate, glycerin, butylene glycol, 1,2-hexanediol, trehalose, ceramide, sodium lactate, urea, beta-glucan, squalane, honey extract, propylene glycol, mannitol, betaine, and aloe vera extract. The anti-wrinkle ingredients include retinol, L-carnosine, beta-glucan, NMN (nicotinamide mononucleotide), PDRN (polydeoxyribonucleic acid), copper peptide, adenosine, bifid yeast extract, peptide-1, resveratrol, panthenol, lipoic acid, and coenzyme Q10. Soothing ingredients include dipotassium glycyrrhizate, NMN, PDRN, chamomile extract, ceramides, Centella asiatica extract, beta-glucan, bisabolol, D-panthenol, aloe vera extract, scutellaria baicalensis root extract, vitex agnus-castus extract, allantoin, xanthan gum, and avocado butter. Tissue repair ingredients include ceramides, epidermal growth factor, bifid yeast extract, peptide-1, copper peptide, PDRN, madecassoside, hydroxyproline, arbutin, licorice extract, allantoin, Centella asiatica extract, NMN, placenta extract, and D-panthenol.

[0004] While each of these ingredients possesses specific benefits, achieving the desired anti-wrinkle and antioxidant effects through simple compounding is often difficult. The complex interactions between ingredients, coupled with the limitations of the number of screening experiments and conditions, make the screening process full of uncertainty and serendipity. Summary of the Invention

[0005] Based on the above technical problems, the present invention combines the unique plasmid characteristics of yak collagen, and after multiple rounds of screening and optimization, finally unexpectedly screened out a multifunctional yak collagen complex solution, which shows excellent synergistic effects and excellent functions in anti-oxidation, anti-wrinkle and other aspects. It not only significantly improves the skin's antioxidant protection and repair capabilities, but also effectively relieves skin inflammation and sensitive reactions, achieving a balance between safety and efficacy that traditional skin care products find difficult to achieve, and provides an ideal solution to resolve the contradiction between safety and efficacy of traditional skin care products.

[0006] Specifically include the following:

[0007] In a first aspect, the present invention provides a yak collagen hydrating injection stock solution, which is composed of yak collagen, vitamin C, α-arbutin, sodium hyaluronate, NMN, PDRN and blue copper peptide.

[0008] Preferably, each milliliter of yak collagen hydrating injection solution contains 100-500μg yak collagen, 10-100μg vitamin C, 5-50μg α-arbutin, 500-5000μg sodium hyaluronate, 10-100μg NMN, 10-100μg PDRN and 10-50μg blue copper peptide.

[0009] Preferably, each milliliter of yak collagen water light injection solution contains 100μg yak collagen, 20μg vitamin C, 10μg α-arbutin, 1000μg sodium hyaluronate, 10μg NMN, 10μg PDRN and 10μg blue copper peptide.

[0010] Preferably, the yak collagen is yak tendon collagen.

[0011] In a second aspect, the present invention provides a use of the yak collagen hydrating injection concentrate described in the first aspect in the preparation of yak collagen hydrating injection.

[0012] In a third aspect, the present invention provides a yak collagen hydrating injection, which is prepared from the yak collagen hydrating injection stock solution described in the first aspect.

[0013] Preferably, the preparation method of the yak collagen water light injection is:

[0014] (1) purifying yak collagen to obtain a purified collagen solution;

[0015] (2) adding sodium hyaluronate to the purified collagen solution of step (1) and stirring at 25° C. until a transparent viscous solution is formed;

[0016] (3) adding vitamin C to the transparent viscous solution in step (2) under light-shielding conditions until it is evenly dispersed;

[0017] (4) Dissolve α-arbutin in sterile water, dissolve blue copper peptide in deionized water, dissolve PDRN freeze-dried powder in sterile water, and dissolve NMN in the dark, and then add them to the system formed in step (3);

[0018] (5) Use a 0.22 μm sterile filter membrane for sterile filtration to obtain a clear, sterile functional hydrating injection concentrate, which is the yak collagen hydrating injection.

[0019] Preferably, the step (1) is: adding yak tendon collagen to sterile deionized water containing 0.5% glacial acetic acid, and dissolving it at 4°C to obtain a uniform collagen solution; then using a dialysis bag with a molecular weight cutoff range of 8000 to 14000Da, dialyzing for 48 hours at 4°C, during which the deionized water is replaced 3 to 5 times to obtain a purified collagen solution.

[0020] In a fourth aspect, the present invention provides the use of the yak collagen hydrating injection described in the third aspect in the preparation of skin beauty products.

[0021] Preferably, the yak collagen water light injection is used for repairing photoaged skin.

[0022] The beneficial effects of the present invention are:

[0023] The present invention combines the unique plasmid characteristics of yak collagen, and after multiple rounds of screening and optimization, finally unexpectedly screened out a multifunctional yak collagen water light injection composed of yak collagen, vitamin C, α-arbutin, sodium hyaluronate, NMN, PDRN, and blue copper peptide. The water light injection has significant antioxidant, whitening, moisturizing, tissue repair promotion and soothing effects, and also has excellent biosafety, no cytotoxicity, no systemic toxicity, no pyrogenicity and no hemolytic risk, and the yak collagen maintains a complete triple helix structure; the water light injection can effectively improve skin texture, increase skin density and hydration, and significantly reduce transepidermal water loss, promote skin color to return to a healthy state, significantly promote collagen regeneration, and accelerate the recovery process of photoaged skin, which is of great significance in the field of skin photoaging repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The antioxidant capacity and tyrosinase inhibition ability of yak collagen combined with whitening ingredients at different concentrations.

[0025] Figure 2Screening of hydrating and moisturizing efficacy was conducted through zebrafish experiments; phenotypic diagram of the moisturizing effect of Col combined with hydrating and moisturizing ingredients on zebrafish (a), histogram of tail length (b), and histogram of tail fin area (c).

[0026] Figure 3 Anti-wrinkle efficacy screening was performed through zebrafish experiments; phenotypic diagram (a) and bar graph (b) of the anti-wrinkle effect of Col combined with anti-wrinkle ingredients on the zebrafish tail fin.

[0027] Figure 4 Soothing efficacy screening was performed through zebrafish experiments; the effects of Col combined with different soothing ingredients on the stimulation intensity (a) and the number of embryonic spins (b) in the zebrafish stimulation model.

[0028] Figure 5 The efficacy of promoting tissue repair was screened through zebrafish experiments; the phenotype (a) and the tail fin area bar graph (b) of the repair effect of Col combined with tissue repair promoting ingredients on the zebrafish tail fin area.

[0029] Figure 6 Circular dichroism characteristics of Col-M; CD spectrum of Col-M (a), CD thermal curve of Col-M (b), first derivative of the thermal curve of Col-M. (c).

[0030] Figure 7 Changes in body weight of cytotoxicity and systemic toxicity of Col-M at different concentrations (0%, 20%, 50%, 50%, 100%, 200%).

[0031] Figure 8 Hemolysis experiments of 0.9% Saline and Col-M: visual image (a) and bar graph (b).

[0032] Figure 9 DPPH free radical scavenging ability and tyrosinase inhibition ability of Col-M at different ratios.

[0033] Figure 10 The successful establishment of the mouse photoaging model was evaluated using dermatoscopy (a) and ultrasound (b).

[0034] Figure 11 The repair performance of Col-M was evaluated using ultrasound and dermatoscopy; dermatoscopy (a) and ultrasound skin imaging (b) of mice injected with different groups, including Normal group, Model group, Col group, Control group and Col-M group.

[0035] Figure 12Combo evaluation of Col-M performance; dermal density (a), melanin (b), hydration (c), and TEWL (d) of mouse skin in the Normal, Model, Col, Control, and Col-M groups.

[0036] Figure 13 The skin of mice in the Normal, Model, Col, Control, and Col-M groups was stained with H&E (a), and the epidermal thickness of the mouse skin was quantitatively analyzed using Image J (b).

[0037] Figure 14 Masson staining was used to evaluate the ability of Col-M to promote collagen regeneration; Masson staining images (a) and collagen fraction volume (b) of the Normal, Model, Col, Control, and Col-M groups at 2, 4, and 8 weeks.

[0038] Figure 15 Quantitative analysis of the hydroxyproline effect of Col-M on the repair of photoaged skin; Hyp content in the Normal group, Model group, Col group, Control group and Col-M group at week 8.

[0039] Figure 16 Quantitative analysis of antioxidant indicators of Col-M's effect on the repair of photoaged skin; MDA (a), SOD (b) and GSH (c) contents in the Normal group, Model group, Col group, Control group and Col-M group at week 8. DETAILED DESCRIPTION

[0040] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings. Obviously, the described embodiments are only part of the present invention, not the entire invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort shall fall within the scope of protection of the present invention.

[0041] It should be noted that, in the following examples, unless otherwise specified, the methods used are all conventional methods; the reagents used are all conventional reagents and can be purchased from the market.

[0042] In the following examples, yak collagen (Col) is used, and the extraction method is described in invention patent CN113520900B. However, the collagen described in the present invention is not limited to the above collagen.

[0043] The vitamin C described in the following examples is medical grade and purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.; blue copper peptide, medical grade, is purchased from Guangzhou Baiyu Biotechnology Co., Ltd.; glutathione, cysteine, nicotinamide, nonapeptide, α-arbutin, tranexamic acid, trehalose, glycerol, ceramide, β-glucan (PJT), bifida yeast extract, L-carnosine, NMN, allantoin, Centella asiatica extract, medical grade, are purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.; PDRN, medical grade, is purchased from Shandong Freda Biotechnology Co., Ltd.; sodium hyaluronate, medical grade, is purchased from Huaxi Biotechnology Co., Ltd.; 0.9% sodium chloride is purchased from Anhui Jiuqiang Pharmaceutical Co., Ltd.; Kunming mice, clean grade, are purchased from the Experimental Animal Center of Lanzhou University.

[0044] Example 1 Preparation of multifunctional yak collagen water light needle

[0045] 1. Antioxidant Activity Assay

[0046] (1) Preparation of composite solution:

[0047] VC+Col composite solution (each component is 0.1 mg / mL): Dissolve vitamin C (VC) in sterile water at the required concentration, slowly add collagen solution under light-proof conditions, and gently mix to obtain the solution.

[0048] GSH+Col composite solution (each component is 0.1 mg / mL): Prepare glutathione (GSH) into a solution and slowly add it to the collagen solution and mix well. Store in a dark place and at low temperature.

[0049] Cys+Col complex solution (each component is 0.1 mg / mL): dissolve cysteine (Cys) in sterile water and add collagen solution in proportion.

[0050] (2) DPPH free radical scavenging rate

[0051] A DPPH free radical solution was prepared in anhydrous ethanol to a final concentration of 0.10 mg / mL. 100 μL of each sample solution and an equal volume of DPPH working solution were added to a 96-well plate. After thorough mixing, the mixture was incubated in the dark for 30 minutes to complete the free radical scavenging reaction. The absorbance of the mixture was then measured at a wavelength of 517 nm. To enhance data reliability and reproducibility, three independent replicates were performed for each treatment condition.

[0052]

[0053] Among them, A is the absorbance after the sample reacts with DPPH, B is the absorbance after the sample is mixed with ethanol, and C is the absorbance after the sample is mixed with water and DPPH.

[0054] The results are as follows Figure 1 As shown, DPPH radical scavenging activity increased significantly when the concentrations of VC, GSH, and Cys ranged from 0 mg / mL to 0.04 mg / mL. At a concentration of 0.04 mg / mL, the scavenging rate of the VC+Col solution was approximately 90%, while at the same concentration, the scavenging rates of the GSH+Col and Cys+Col solutions were less than 60%. These results demonstrate that VC+Col, GSH+Col, and Cys+Col solutions all effectively scavenged free radicals, with scavenging activity positively correlated with increasing concentration. The VC+Col composite solution exhibited the strongest DPPH radical scavenging and antioxidant capacity.

[0055] (2) ABTS free radical scavenging rate

[0056] Mix equal volumes of 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution, place in a brown volumetric flask, and let stand at room temperature for 12 to 16 hours in the dark to prepare ABTS·+ working solution. Before use, it needs to be diluted with ultrapure water until the absorbance of the dilution at a wavelength of 734 nm is stable within the range of 0.70±0.02. Subsequently, 180 μL of the diluted ABTS·+ solution is added to a 96-well plate, followed by 20 μL of the sample to be tested. After mixing, let the reaction stand at room temperature for 5 minutes, and then use a microplate reader to measure the absorbance of the final system in each well at 734 nm. Set up 3 parallel wells for each sample to ensure the accuracy of the data. The ABTS free radical scavenging rate is calculated according to the following formula:

[0057]

[0058] Where: M represents the absorbance of the sample solution in the experimental group after reacting with the ABTS·+ free radical solution; N represents the blank group, in which the absorbance of the sample solution is mixed with water; P represents the control group, in which the absorbance of water is mixed with the ABTS·+ free radical solution.

[0059] The results are as follows Figure 1 As shown, when the antioxidant concentration reached 0.06 mg / mL, the ABTS free radical scavenging rate of the VC + Col solution approached 100%. In contrast, the GSH + Col and Cys + Col solutions did not exceed 80%. These results indicate that the VC + Col, GSH + Col, and Cys + Col complex solutions all possessed strong ABTS free radical scavenging abilities, with the VC + Col complex exhibiting the strongest ABTS free radical scavenging and antioxidant capacity.

[0060] 2. Whitening activity assay

[0061] (1) Preparation of composite solution

[0062] An+Col composite solution (each component is 0.1 mg / mL): Dissolve α-arbutin (An) in an appropriate amount of sterile water, add to the collagen solution, mix well, and store in the dark.

[0063] TC+Col composite solution (each component is 0.1 mg / mL): Dissolve tranexamic acid (TC) directly in the collagen solution, mix well, and store in a dark and low temperature environment.

[0064] VB3+Col composite solution (each component is 0.1 mg / mL): dissolve nicotinamide (VB3) in the collagen solution and mix thoroughly.

[0065] H-9+Col composite solution (each component is 0.1 mg / mL): Dissolve nonapeptide (H-9) in sterile water, slowly add to collagen solution, mix thoroughly, and store at low temperature away from light.

[0066] (2) Tyrosinase inhibition rate

[0067] To prepare PBS buffer: Weigh 71.63 g of Na₂HPO₄·12H₂O and dissolve it in an appropriate amount of ultrapure water to make a solution. Dose to 1000 mL. After mixing, prepare 0.2 mol / L PBS buffer at a pH of 6.8. Store in a refrigerator at 4°C until use.

[0068] Preparation of L-tyrosine solution (concentration of 0.15 mmol / L): Accurately weigh 0.0272 g of L-tyrosine, add it to the prepared PBS buffer and pre-disperse it, sonicate for 30 minutes, and then dilute to 100 mL with PBS buffer.

[0069] To prepare the tyrosinase solution: Weigh 0.0010 g of tyrosinase powder and dissolve it in PBS buffer (pH 6.8). The volume is adjusted to 20 mL to obtain a 0.05 mg / mL tyrosinase solution. This solution should be stored at 4°C to maintain its activity.

[0070] Experimental Procedure: Add the appropriate amount of PBS buffer (pH = 6.8, 0.2 mol / L), sample solution, and L-tyrosine solution to each test tube in sequence and place in a 37°C water bath for 10 minutes. Subsequently, add the required amount of tyrosinase solution (0.05 mg / mL) and continue heating the reaction in a 37°C water bath for 10 minutes. Use a spectrophotometer to measure the absorbance of each solution at 475 nm and record the experimental results.

[0071] According to the above steps, the absorbance of each experimental solution at 475nm was measured and the inhibition rate of the sample on tyrosinase was calculated. The specific calculation formula is as follows:

[0072]

[0073] Where C1 represents the absorbance without adding sample and enzyme solution, C2 represents the absorbance without adding sample but after adding enzyme solution, T1 represents the absorbance with adding sample but not adding enzyme solution, and T2 represents the absorbance with adding sample and enzyme solution at the same time.

[0074] The results are as follows Figure 1 As shown, at a concentration of 0.1 mg / mL, the inhibition rate of An+Col was approximately 50%. In contrast, at the same concentrations, the inhibition rates of TC+Col, VB3+Col, and H-9+Col were all below 50%. These results indicate that An, TC, VB3, and H-9 all inhibit tyrosinase activity, and the tyrosinase inhibition rate increases with increasing concentrations of An, TC, VB3, and H-9. Among them, the An+Col complex solution had the most significant tyrosinase inhibitory effect, demonstrating excellent whitening results.

[0075] 3. Zebrafish Efficacy Assay

[0076] (1) Preparation of composite solution

[0077] HA+Col (each component is 0.1 mg / mL): Dissolve sodium hyaluronate (HA) in sterile water, fully swell and stir to a uniform solution, then slowly add it to the collagen solution and mix thoroughly to obtain a composite solution. Store at 4°C.

[0078] Ceramide + Col (each component is 0.1 mg / mL): Dissolve ceramide in sterile water evenly, then slowly add it to the collagen solution to form a composite solution.

[0079] DTd+Col (each component is 0.1 mg / mL): Slowly add glycerol (DTd) to the collagen solution in proportion and mix thoroughly to obtain a transparent composite solution. This system has good stability and can be stored at room temperature.

[0080] DG+Col (each component is 0.1 mg / mL): Dissolve trehalose (DG) in an appropriate amount of sterile water, heat and dissolve until completely transparent, cool to room temperature, slowly add to the collagen solution, mix well, and store at low temperature.

[0081] β-glucan + Col (each component at 0.1 mg / mL);

[0082] Nicotinamide mononucleotide (NMN) + Col (each component at 0.1 mg / mL);

[0083] L-Carnosine + Col (each component is 0.1 mg / mL);

[0084] Polydeoxyribonucleotide (PDRN) + Col (each component 0.1 mg / mL);

[0085] Bifida yeast extract (BFE) + Col (each component was 0.1 mg / mL);

[0086] NMN+Col (each component was 0.1 mg / mL);

[0087] PDRN+Col (each component 0.1 mg / mL);

[0088] Allantoin + Col (each component is 0.1 mg / mL);

[0089] Centella asiatica extract (Cae) + Col (each component 0.1 mg / mL);

[0090] Ceramide+Col (each component 0.1 mg / mL);

[0091] BFE+Col (each component 0.1 mg / mL);

[0092] Allantoin + Col (each component 0.1 mg / mL);

[0093] Blue copper peptide (GHK) + Col (each component is 0.1 mg / mL);

[0094] The preparation method is the same as that of the above-mentioned composite solution.

[0095] (2) Zebrafish hydration and moisturizing experiment

[0096] The experiment was conducted using wild-type AB zebrafish (3 days post-fertilization) (dpf). Seventy 3dpf zebrafish were randomly divided into seven groups: normal group, model group, collagen group (Col), sodium hyaluronate-collagen group (HA+Col), trehalose-collagen group (DG+Col), glycerol-collagen group (DTd+Col), and ceramide-collagen group (Ceramide+Col). The normal group was incubated in fish embryo culture medium, the model group incubated in 1.5% sodium chloride solution, the Col group incubated in a mixture of 1.5% sodium chloride and 0.1mg / mL collagen, and the four moisturizing ingredient groups were incubated in solutions containing 1.5% sodium chloride, 0.1mg / mL collagen, and 0.1mg / mL moisturizing ingredient.

[0097] During the experiment, zebrafish embryos were placed in a 96-well cell culture plate, with one embryo per well, and 200 μL of the corresponding treatment solution was added. The embryos were incubated in a temperature-controlled incubator at 28 ± 1°C for 15 minutes. After incubation, the zebrafish were placed flat on a glass slide and images were captured using a microscope. Subsequently, the area and length of the zebrafish tails in each group were analyzed using Image J software, and the average tail area and length for each group were calculated according to the following formula:

[0098]

[0099] A: Length after dehydration. B: Length before dehydration. C: Caudal fin area after dehydration. D: Caudal fin area before dehydration.

[0100] The results are as follows Figure 2 As shown, compared with the normal group, the model group showed significant reductions in tail area and length, confirming the successful establishment of the zebrafish hydration assessment model. The Ceramide+Col, DTd+Col, DG+Col, and Col groups showed lower relative tail fin area and length compared to the HA+Col group. The HA+Col group showed the smallest changes in tail length and tail fin area, with relative tail fin area at 87.17% and relative tail length at 95.80%. These results demonstrate that HA+Col possesses the most effective hydration and moisturizing properties.

[0101] (3) Zebrafish anti-wrinkle experiment

[0102] The experiment was conducted using wild-type AB zebrafish at 2 days post-fertilization (dpf). Eighty zebrafish were randomly divided into eight groups and plated on six-well plates, with 10 zebrafish per well in a final volume of 3 mL. These groups included normal, model, Col (0.1 mg / mL), β-glucan (β-glucan) + Col (0.1 mg / mL + 0.1 mg / mL), nicotinamide mononucleotide (NMN) + Col (0.1 mg / mL + 0.1 mg / mL), L-carnosine (L-carnosine) + Col (0.1 mg / mL + 0.1 mg / mL), polydeoxyribonucleotide (PDRN) + Col (0.1 mg / mL + 0.1 mg / mL), and bifid yeast extract (BFE) + Col (0.1 mg / mL + 0.1 mg / mL). All groups, except the normal group, were exposed to UV light. The specific irradiation scheme is as follows: three irradiation phases, each phase lasting 15 minutes, with an intensity of 2.0-2.5 mW / cm 2 Each stage was followed by a 30-minute interval. All groups were incubated in the dark at 28°C for 22 hours. Ten zebrafish per group were observed under a microscope. Tail fin area was measured using Image J software and statistical analysis was performed.

[0103]

[0104] S represents the tail fin area of the irradiated sample group. M represents the tail fin area of the irradiated model group. N represents the tail fin area of the normal group.

[0105] The results are as follows Figure 3 As shown: In the normal group, zebrafish were not exposed to UV radiation and showed smooth and intact tail fins. In contrast, in the model group, zebrafish showed rough and wrinkled tail fins after UV radiation, thus confirming the successful establishment of the model. This experiment measured the tail fin area of zebrafish embryos after UV-induced wrinkling. The anti-wrinkle rate of the Col group was 63.4%, the anti-wrinkle rate of the β-glucan+Col group was 77.6%, the anti-wrinkle rate of the NMN+Col group was 95.00%, the anti-wrinkle rate of the L-Carnosine+Col group was 71.0%, the anti-wrinkle rate of the PDRN+Col group was 85.5%, and the anti-wrinkle rate of the BFE+Col group was 88.9%. The anti-wrinkle rates of the remaining groups were lower than the NMN+Col group. These results indicate that NMN+Col exhibits the strongest anti-wrinkle ability.

[0106] (4) Zebrafish soothing experiment

[0107] Healthy zebrafish embryos (70 in total) at 3 dpf were placed in a 96-well plate with two embryos per well. These zebrafish were divided into seven groups: normal group (treated with normal culture medium), model group (0.5 mg / mL SDS), Col group (0.5 mg / mL SDS and 0.1 mg / mL Col), NMN+Col (treated with 0.5 mg / mL SDS, 0.1 mg / mL NMN, 0.1 mg / mL), PDRN+Col (0.5 mg / mL SDS, 0.1 mg / mL PDRN, 0.1 mg / mL Col), Allantoin+Col (0.5 mg / mL SDS, 0.1 mg / mL Allantoin, 0.1 mg / mL Col), and Centella asiatica extract (Cae)+Col (0.5 mg / mL SDS, 0.1 mg / mL Cae, and 0.1 mg / mL Col). After 15 minutes of treatment, the embryos were observed under a stereomicroscope for 1 minute, and the number of rotational movements of the embryos in each group was recorded.

[0108] The results are as follows Figure 4As shown, compared with the normal group, the stimulation intensity and number of embryonic spins in the model group increased significantly, indicating that the zebrafish stimulation model was successfully established. The stimulation intensity of the blank group was 100%, the stimulation intensity of the model group was 636.4%, the stimulation intensity of the Col group was 424.2%, the stimulation intensity of the NMN+Col group was 233.6%, the stimulation intensity of the PDRN+Col group was 190.9%, the stimulation intensity of the Allantoin+Col group was 260.0%, and the stimulation intensity of the Cae+Col group was 236.3%. The stimulation intensity results showed that NMN+Col, PDRN+Col, Cae+Col, Allantoin+Col, and Col all effectively reduced the stimulation intensity in zebrafish embryos. The embryonic spin count in the PDRN+Col group was the lowest. In contrast, the NMN+Col, Cae+Col, Allantoin+Col, and Col groups all showed higher embryonic spin counts than the PDRN+Col group. These results indicate that PDRN+Col has the most effective soothing effect.

[0109] (5) Zebrafish tissue repair experiment

[0110] The experiment was conducted using healthy zebrafish embryos at 3 days post-birth weight (dpf). After anesthetizing the zebrafish larvae with 0.016% tricaine, their caudal fins were removed distally from the spine using a scalpel under a stereomicroscope. Eighty caudal fin-removed embryos were randomly divided into eight groups: normal, model, Col (0.1 mg / mL), Cae + Col (0.1 mg / mL + 0.1 mg / mL), Ceramide + Col (0.1 mg / mL + 0.1 mg / mL), BFE + Col (0.1 mg / mL + 0.1 mg / mL), Allantoin + Col (0.1 mg / mL + 0.1 mg / mL), and GHK + Col (0.1 mg / mL + 0.1 mg / mL). These embryos were transferred to a 96-well plate, with one embryo per well. The normal and model groups were each given 0.2 mL of embryo culture medium, while the sample groups were given 0.2 mL of their respective sample solutions. Embryos were placed in a constant temperature incubator at 28 ± 1°C for 72 hours and then photographed under a microscope. The tail fin area was measured using Image J software and statistical analysis was performed.

[0111] The results are as follows Figure 5 As shown in the figure, the caudal fin area of the GHK+Col group was the largest, at 318,226 pixels, while the caudal fin areas of ceramide+Col, BFL+Col, allantoin+Col, Cae+Col, and Col were all smaller. These results indicate that the GHK+Col complex solution has the strongest tissue repair ability.

[0112] 4. Preparation of Multifunctional Yak Collagen Hydrating Injection (Col-M)

[0113] Composition of the multifunctional yak collagen water light injection (Col-M): Each milliliter of yak collagen water light injection stock solution contains yak collagen 100μg, vitamin C 20μg, α-arbutin 10μg, sodium hyaluronate 1000μg, NMN 10μg, PDRN10μg and blue copper peptide 10μg.

[0114] Preparation method: Yak tendon collagen was added to sterile deionized water containing 0.5% glacial acetic acid and dissolved under magnetic stirring at 4°C for 48 hours to obtain a homogeneous collagen solution. The solution was then dialyzed using a dialysis bag with a molecular weight cutoff range of 8,000 to 14,000 Da at 4°C for 48 hours, with the deionized water changed 3 to 5 times to effectively remove residual acetic acid and obtain a purified collagen solution. Sodium hyaluronate was slowly added to the collagen solution in a proportional amount and stirred at 25°C for 2 to 4 hours until completely dissolved, forming a transparent, viscous solution. Pre-weighed vitamin C was added in the dark, stirring gently to ensure thorough dispersion and prevent oxidative inactivation. α-Arbutin was pre-dissolved in a small amount of sterile water and slowly added to the system with stirring. Blue copper peptide was pre-dissolved in deionized water and slowly added dropwise to the main system with gentle mixing. PDRN lyophilized powder was dissolved in a small amount of sterile water and added to the system with stirring for 10 minutes to ensure uniform distribution. Dissolve NMN quickly in the dark and add it to the main system. Continue stirring for 10 minutes. Finally, filter the solution through a 0.22μm sterile filter membrane to obtain a clear, sterile functional hydrating solution (Col-M). Aseptically dispense the solution into pre-sterilized glass bottles in a clean area. Seal the bottles and store in the dark at a low temperature (2-8°C).

[0115] Example 2 Characterization and in vitro activity detection of multifunctional yak collagen water light needle (Col-M)

[0116] 1. Centrifugal stability

[0117] A certain amount of Col-M was weighed and placed in a centrifuge tube. The centrifuge was set at 2500 rpm, 5000 rpm, 7500 rpm, and 10000 rpm for 30 minutes at 4°C. After centrifugation, the pH value and extrusion force were measured. The appearance of the sample was also observed, including any significant changes in color and odor, and the presence of precipitation.

[0118] After centrifugation, the color and odor did not change significantly, and no precipitation was present. The pH value and extrusion force were unchanged, as shown in Table 1.

[0119] Table 1 Centrifugal stability test

[0120]

[0121] 2. Circular dichroism

[0122] The structural characteristics of yak skin and tendon collagen were analyzed using circular dichroism spectroscopy. Prior to measurement, the sample solution had a concentration of 0.5 mg / mL and was allowed to stand at 4°C for at least 24 hours. The solution was then placed in a 1 mm thick cuvette and scanned over a wavelength range of 190 to 260 nm, with a wavelength step size of 1 nm.

[0123] The results are as follows Figure 6 As shown, the CD spectrum of Col-M exhibits a maximum absorption peak at 225 nm and a minimum absorption peak at 198 nm, indicating that Col-M maintains a triple helical structure. Furthermore, analysis of the thermal transition curve further confirms that Col-M possesses excellent stability, with a thermal transition temperature of 43.93°C.

[0124] 3. Biosafety of Col-M

[0125] (1) Cytotoxicity

[0126] The CCK-8 method was used to evaluate the cytotoxicity of Col-M on human dermal fibroblasts (HDF). HDF cells were cultured in a constant temperature incubator at 37°C and 5% CO2. The culture medium was MEM containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin. In the experiment, high concentration Col-M was first diluted with MEM to different concentrations (0%, 20%, 50%, 100%, 200%). Then, the cells were digested with 0.25% (w / w) trypsin. The cell digestion process could be observed under a microscope during the digestion process. After the digestion was completed, they were seeded into 96-well plates at a cell density of 7×10 3 and then cultured at 37°C for 24 hours.

[0127] Then, different concentrations of Col-M samples were added to the 96-well plate and incubated for 24 hours. After the incubation, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 1 to 3 hours. The culture medium was used as a blank control and the optical density (OD) was measured at a wavelength of 450 nm using a microplate reader. 450 ). The calculation formula of cell survival rate is as follows:

[0128]

[0129] A represents the OD value of the sample. B represents the OD value of the blank group. C represents the OD value of the culture medium.

[0130] The results are as follows Figure 7As shown in the figure: Under the conditions of concentration range of 20% to 200%, the survival rate of HDF cells by Col-M exceeded 100%, which was significantly higher than that of the blank control group. This result shows that Col-M has no obvious toxic effect on HDF cells within this concentration range.

[0131] (2) Systemic toxicity

[0132] Twenty Kunming mice (half male and half female, 6-8 weeks old, weighing 17-23 g) were selected and provided by the Animal Experimentation Center of Lanzhou University. Mice were randomly divided into experimental and control groups, with each group consisting of 5 female and 5 male mice. All mice were injected via the tail vein with a dose of 50 mL / kg. The experimental group received a saline dilution of Col-M, while the control group received an equal volume of saline. All procedures were identical.

[0133] We continuously monitored the physiological status, body weight changes, toxic reactions, and mortality of mice 24, 48, and 72 hours after injection. The acute toxicity of Col-M was assessed based on body weight changes, toxic symptoms, and mortality.

[0134] The results are shown in Tables 2-3 and Figure 7 As shown, the average weight of mice before saline injection was 25.37 g; after saline injection, the average weight was 27.05 g, 28.47 g, and 29.36 g at 24, 48, and 72 hours, respectively. For the Col-M group, the average weight of mice before administration was 25.42 g; after Col-M injection, the average weight was 26.96 g, 28.03 g, and 29.12 g at 24, 48, and 72 hours, respectively. These results indicate that the Col-M solution increased the weight of mice after injection without exhibiting systemic toxicity.

[0135] Table 2 Observation of systemic reactions in mice after injection

[0136]

[0137] Table 3 Toxicological evaluation of Col-M: Mouse body weight

[0138]

[0139] (3) Pyrogen test

[0140] Six female New Zealand white rabbits weighing approximately 2.0 kg were purchased from the Animal Experimental Center of Lanzhou University. Before the experiment, the rabbits' body temperatures were recorded every 30 minutes, and a normal temperature range was established, requiring a variation of no more than 0.2°C. Next, 2 mL of 0.9% saline and Col-M were injected into the rabbits' ear veins, respectively, and six temperature readings were recorded. Temperature change was calculated by subtracting the normal temperature from the highest recorded temperature.

[0141] If the temperature variation of each rabbit remained within 0.6°C and the total temperature difference of all rabbits in the group was less than 1.3°C, the biomaterial was considered non-pyrogenic.

[0142] The results, shown in Table 4, show that the body temperatures of the three rabbits in the 0.9% saline group increased by 0°C, 0.1°C, and 0°C, respectively; in the Col-M group, the rabbits' temperatures increased by 0.15°C, 0.35°C, and 0.25°C, respectively. The temperature changes in all experimental animals did not exceed 0.6°C. The cumulative temperature increases in the 0.9% saline and Col-M groups were 0.1°C and 0.75°C, respectively, both significantly below 1.3°C. These data indicate that yak collagen hydration injections are not pyrogenic.

[0143] Table 4 Heat source experiments of 0.9% Saline and Col-M

[0144]

[0145] (4) Hemolysis test

[0146] Take 1mL of fresh rabbit blood and mix it with 5mL of anticoagulant, which is 0.1mg / mL heparin sodium solution, prepared with normal saline and recorded as solution A. Centrifuge solution A with a low-temperature centrifuge at a speed of 2500rpm for 10 minutes. After the centrifugation, collect the red blood cells. Then, use 2mL of normal saline to wash the red blood cells 3 to 5 times until the red color no longer appears in the supernatant. Add 2mL of 0.9% normal saline to dilute the washed red blood cell suspension. Mix the dilution with 0.9% normal saline, deionized water and Col-M solution respectively, incubate at 37°C for 1 hour, and continue incubation at room temperature for 3 hours. Centrifuge the supernatant and measure the absorbance (OD value) of hemoglobin at a wavelength of 545nm. Finally, the hemolysis rate (HR, %) formula is as follows:

[0147]

[0148] OD a : OD value of the test sample. OD b : OD value of 0.9% saline. OD c : OD value of ultrapure water.

[0149] The results are as follows Figure 8 As shown: During the experiment, the supernatant in the saline group remained clear and colorless, while a significant red precipitate accumulated at the bottom. After shaking, a sand-like suspension formed, indicating that the red blood cells had not been hemolyzed. Similarly, the Col-M group showed a similar phenomenon. The upper layer of the liquid remained clear and colorless, and a clear red blood cell precipitate was also visible at the bottom. As a control group, the hemolysis rate of 0.9% saline was 0%, while the hemolysis rate of yak collagen hydration injection was only 0.07%, far below the safety threshold of 5%. This result shows that yak collagen hydration injection has excellent blood compatibility and does not have the potential risk of causing hemolysis.

[0150] 4. In vitro efficacy experiments of Col-M

[0151] (1) DPPH free radical scavenging rate experiment

[0152] The yak collagen water light needle was prepared into Col-M solutions of different concentrations, and the DPPH free radical scavenging activity of the test solutions was studied in vitro according to the method in Example 1. The results are as follows: Figure 9 As shown, within the Col-M concentration range of 20% to 100%, the DPPH radical scavenging ability significantly increased with increasing sample concentration, exhibiting an overall rapid growth trend. At concentrations between 100% and 500% Col-M, the data plateaued, reaching a peak scavenging rate of 96.42%, approaching 100% DPPH radical scavenging. These results demonstrate that Col-M possesses significant DPPH radical scavenging ability.

[0153] (2) Tyrosinase inhibition rate experiment

[0154] According to the method in Example 1, the in vitro tyrosinase inhibition rate of the test solution was studied. Figure 9 As shown, within the 20%-100% Col-M ratio range, the inhibitory effect of Col-M on tyrosinase activity increases with increasing concentration. At 100% Col-M, the tyrosinase inhibition rate reaches a maximum of 95.00%, after which it levels off. These results demonstrate that yak collagen hydration injections have excellent tyrosinase-scavenging capabilities and are highly effective for whitening.

[0155] Example 3 Study on the in vivo activity and photoaging skin repair performance of Col-M

[0156] 1. Photoaging Mouse Model

[0157] Ninety female Kunming mice aged 6 to 8 weeks were purchased (Lanzhou Animal Experimental Center). Before the experiment, the mice were shaved on their backs and placed in special cages equipped with eye protection. The mice were randomly divided into five groups: the normal group consisted of healthy mice that did not receive any treatment; the model group consisted of mice that received UV irradiation but no treatment; the Col group received UV irradiation and was injected with 200 μL of a 0.1 mg / mL collagen solution; the Control group received UV irradiation and was injected with 200 μL of Filorga water light needle solution; and the Col-M group received UV irradiation and was injected with 200 μL of the Col-M solution described in this application.

[0158] During the experiment, mice were exposed to ultraviolet radiation three times a week for 8 consecutive weeks, with each exposure lasting 20 minutes. The cumulative exposure doses of UVA and UVB were 20.4 J / cm 2 and 1.44 J / cm 2 At weeks 2, 4, and 8 of the experiment, the mice were shaved and anesthetized with 10% sodium pentobarbital solution for skin examination. Subsequently, the mice were euthanized, and skin tissue samples from the injection sites were collected for subsequent analysis.

[0159] The results are as follows Figure 10 As shown, the back skin texture of mice in the Normal group was smooth and delicate, while that of mice in the Model group showed significant dryness, roughness, and increased wrinkling. Ultrasound analysis of the Normal group revealed more yellowish-white areas, while the Model group showed pronounced wrinkles, with fewer yellowish-white areas and larger green areas. This indicates that the photoaging model in mice was successfully established.

[0160] (2) Combo evaluation of mouse skin condition

[0161] The Combo comprehensive skin analysis system was used for multi-dimensional testing. First, the Combo dermatoscope module was used to image the skin's appearance on the mouse's back. Multiple representative examination points were selected within the pre-defined measurement area, and three replicate measurements were performed at each point to ensure data consistency and accuracy. Upon pressing the start button, the device automatically captured and saved images of the skin's surface for subsequent analysis. Subsequently, the Combo system's high-frequency ultrasound imaging function was used to assess the density of the skin's internal structure. Before ultrasound scanning, a small amount of deionized water was evenly applied to the probe tip to optimize sound wave transmission. The probe was then gently applied to the target skin area to obtain clear cross-sectional images, revealing changes in collagen tissue firmness and subcutaneous structures. Skin elasticity was assessed using the Combo device's elasticity measurement module. Before measurement, the target skin surface should be thoroughly cleaned and kept dry. A dedicated probe was then applied perpendicularly to the skin for testing. The system automatically recorded the degree of skin deformation under mechanical stimulation, reflecting its elasticity. Finally, to evaluate the effect of Col-M on skin barrier function, transepidermal water loss (TEWL) measurements were performed using the Combo device's transepidermal water loss (TEWL) probe. During the test, the probe is tightly attached to the skin surface, and the instrument will monitor and record the water loss rate in real time, thereby indirectly reflecting the integrity and repair status of the skin barrier.

[0162] The results are as follows Figure 11 As shown, the backs of the Normal group were smooth and wrinkle-free, while those of the Model group showed noticeable wrinkles and a rough texture. The skin of the Col and Control groups showed mild wrinkling, while the skin of the Col-M group was comparable to the Normal group, showing no signs of wrinkling. These results indicate that Col-M effectively reduced wrinkle formation. Ultrasound was used to measure skin density to assess the repair effect of Col-M on photoaged mouse skin. In ultrasound images, signal intensity appears as white, yellow, red, blue, green, and black, in descending order. Skin density decreased significantly in the Model group and slightly in the Col group, while the Control and Col-M groups showed improvement, approaching normal skin levels. At week 8, skin density in the Col group increased. However, that in the Col-M group exceeded normal skin levels. These results indicate that Col-M can accelerate the recovery of photoaged skin, returning it to normal thickness and density.

[0163] The effect of Col-M on repairing photoaged skin was evaluated by measuring density, color, moisture and TEWL values. Figure 12As shown, throughout the experiment, the skin density of the Normal group remained high, while the skin density of the Model group gradually decreased due to UV damage and collagen degradation. At week 2, the skin density of the Col, Control, and Col-M groups was close to that of the Normal group. At weeks 4 and 8, the skin density of the Col-M group gradually increased. Notably, starting from week 4, the skin density of the Col and Control groups began to decrease compared to the Normal group. These results indicate that Col-M improves skin density in photoaged skin.

[0164] At week 2, melanin levels in the Model, Col, Control, and Col-M groups were higher than in the Normal group. By weeks 4 and 8, the skin color of the Control and Col-M groups gradually approached that of the Normal group, with the Col-M group showing a lighter complexion than the Control group. These results suggest that Col-M may help restore the complexion of photoaged skin.

[0165] At week 2, the water content of the Model and Col groups was lower than that of the Normal group, while the water content of the Control group was comparable to that of the Normal group. The water content of the Col-M group was greater than that of the Normal group. At weeks 4 and 8, the water content of the Col and Control groups remained lower than that of the Normal group, while the water content of the Col-M group was comparable to that of the Normal group. These results suggest that Col-M helps restore the water level of photoaged skin.

[0166] By week 2, TEWL values in the Col and Control groups were slightly higher than those in the Normal group, while the TEWL value in the Col-M group remained consistent with that of the Normal group. By weeks 4 and 8, TEWL values in the Col and Control groups increased significantly, while those in the Col-M group remained at comparable levels. These results suggest that Col-M can effectively repair the skin barrier in photoaged skin and promote the restoration of normal TEWL levels.

[0167] (3) Histological evaluation

[0168] Mouse skin tissue was stained with hematoxylin and eosin (H&E) and histologically analyzed to evaluate the inflammatory response induced by yak collagen hyaluronic acid injection. At weeks 2, 4, and 8 of the experiment, mice were anesthetized by intraperitoneal injection of 10% sodium pentobarbital solution. After ensuring adequate anesthesia, the mice were shaved and euthanized. Subsequently, skin tissue samples were removed and immediately placed in 4% paraformaldehyde solution and fixed for 48 hours to maintain the integrity of the tissue structure. After fixation, 5 μm thick sections were prepared after dehydration with graded ethanol, clearing with xylene, and embedding in paraffin. After dewaxing and hydration, H&E staining was performed to observe histological changes.

[0169] To evaluate the effect of yak collagen hydration therapy on collagen regeneration, Masson's trichrome staining was performed. At weeks 2, 4, and 8 of the experiment, dorsal skin samples were obtained from mice after euthanasia and fixed in 4% paraformaldehyde for 48 hours before being embedded in paraffin. Five-micron-thick tissue sections were prepared, dewaxed, and hydrated, and then stained with Masson's trichrome to assess collagen deposition.

[0170] The results are as follows Figure 13 As shown: At 2, 4, and 8 weeks after injection, no inflammatory response was observed in the Normal group, while inflammatory cell infiltration was observed in the Model group. No significant inflammation or granulomas were observed in the Col, Control, and Col-M groups. These results indicate that Col-M injection does not induce a significant inflammatory response.

[0171] At week 2, no significant differences were observed between the groups except for the Model group. By week 4, epidermal thickness had increased significantly in the Model group, while that in the Control group had increased somewhat. However, epidermal thickness in the Col and Col-M groups remained similar to that in the Normal group. By week 8, epidermal thickness in the Model group remained elevated, while that in the Control group had improved. The increase in thickness in the Col and Col-M groups only slightly exceeded that in the Normal group. These results suggest that Col-M helps restore epidermal thickness in photoaged skin.

[0172] To analyze the effect of Col-M in promoting collagen fiber repair and regeneration in photodamaged skin, Masson staining was used for evaluation. Figure 14As shown: The dermal collagen structure of the Normal group showed a regular wavy distribution throughout the experiment. At week 2, varying degrees of collagen destruction and structural loosening were observed in the Model, Col, Control, and Col-M groups, with the Model group showing the most severe damage. By week 4, the damaged collagen fibers in the Col, Control, and Col-M groups showed progressive repair and more organized arrangement compared with the Model group. By week 8, the Model group still showed obvious collagen fiber disruption, while the dermal collagen fibers in the Col, Control, and Col-M groups were denser and more neatly arranged. Collagen volume fractions obtained from Masson staining analysis using Image J showed a similar trend of collagen regeneration. These results suggest that Col-M can improve collagen structure and accelerate the remodeling process of photoaged skin.

[0173] (3) Determination of hydroxyproline content

[0174] The hydroxyproline (Hyp) content was determined using the chloramine T method. See Table 5 for the solution preparation method for determining the hydroxyproline standard curve.

[0175] Table 5 Solution preparation method for determining hydroxyproline standard curve by chloramine T method

[0176]

[0177] First, 20 mg of defatted skin tissue was thoroughly minced and then hydrolyzed at 85°C for 12 hours in 2 mL of 6 M hydrochloric acid. After the hydrolysis process, the sample was cooled at room temperature and the pH was adjusted to 7.0 using 10 mol / L NaOH. Next, hydroxyproline standard solutions of varying concentrations (5.0, 7.5, 10, 15, and 20 μg / mL) were prepared, and a standard curve was constructed.

[0178] Then, chloramine-T was added to the sample solution and incubated at room temperature for 20 minutes to catalyze the oxidation reaction of hydroxyproline. Next, Ehrlich's reagent was added and further incubated at 85°C for 10 minutes to promote the staining reaction. After the reaction was completed, the sample was quickly cooled to room temperature and incubated for 30 minutes. Finally, the absorbance was measured by spectrophotometer at a wavelength of 560 nm, and the concentration of hydroxyproline in the sample was calculated in combination with the standard curve. Finally, the content of hydroxyproline was expressed as the concentration per mg of dry weight tissue. The calculation formula is as follows:

[0179]

[0180] Where W represents the hydroxyproline content (μg / mg), which is calculated from the standard curve concentration C, sample volume V and mass m.

[0181] The results are as follows Figure 15 As shown: After 8 weeks of treatment, the relative hydroxyproline content in the Normal group was 99.92%, while the hydroxyproline content in the Model group was significantly lower, at 44.17%. Hydroxyproline content reached 87.26% in the Col group, and 80.12% in the Control group. Notably, the relative hydroxyproline content in the Col-M group reached 101.10%, highlighting the significant potential of Col-M in promoting collagen regeneration.

[0182] (4) Determination of antioxidant indicators

[0183] Xanthine can generate superoxide anion (O2 - ), the free radical further reacts with WST-1 reagent to generate a soluble yellow formazan product, whose maximum absorption peak appears at a wavelength of 450nm. SOD can remove O2 in the system. - , thereby effectively inhibiting the formation of formazan. Therefore, the color development of the yellow product in the reaction system can be used as an indirect indicator of the SOD enzyme activity level. The lighter the color, the more O2 - The more effectively it is cleared, the higher the SOD activity. Conversely, a darker color indicates lower SOD activity. This method is sensitive and intuitive and is commonly used in SOD function evaluation and antioxidant capacity testing.

[0184] SOD activity and MDA content were measured in dorsal skin samples from mice using a colorimetric assay to assess oxidative stress. Testing was performed at 2, 4, and 8 weeks. Skin tissue samples were added to the extract at a concentration of 0.1 g / mL, homogenized in an ice bath, and centrifuged at 8,000 × g for 10 minutes at 4°C. The supernatant was used for subsequent experiments. The absorbance of each sample was measured using a microplate reader, and SOD activity and MDA concentration were calculated using a standard curve.

[0185] The results are as follows Figure 16Long-term exposure to UV radiation leads to elevated levels of reactive oxygen species (ROS) and decreased levels of superoxide dismutase (SOD) and glutathione (GSH). Furthermore, UV radiation promotes lipid peroxidation, leading to increased production of malondialdehyde (MDA). The SOD activities of the Normal, Model, Col, Control, and Col-M groups were 221.14, 60.28, 173.97, 199.65, and 210.81 U / mg, respectively. The corresponding MDA levels were 1.24, 2.97, 1.63, 1.45, and 1.25 nmol / mg, respectively, and the GSH contents were 135.33, 64.57, 117.57, 119.76, and 129.19 μg / mg, respectively. These results suggest that Col-M increases SOD activity and GSH levels while decreasing MDA levels, thereby effectively counteracting UV-induced skin aging.

Claims

1. A yak collagen water light needle stock solution, characterized in that: The yak collagen hydrating injection stock solution is composed of yak collagen, vitamin C, α-arbutin, sodium hyaluronate, NMN, PDRN and blue copper peptide.

2. The yak collagen water light needle stock solution according to claim 1, characterized in that Each milliliter of yak collagen hydrating injection solution contains 100-500μg yak collagen, 10-100μg vitamin C, 5-50μg α-arbutin, 500-5000μg sodium hyaluronate, 10-100μg NMN, 10-100μg PDRN and 10-50μg blue copper peptide.

3. The yak collagen water light needle stock solution according to claim 2, characterized in that Each milliliter of yak collagen hydrating injection solution contains 100μg yak collagen, 20μg vitamin C, 10μg α-arbutin, 1000μg sodium hyaluronate, 10μg NMN, 10μg PDRN and 10μg blue copper peptide.

4. The yak collagen water light injection solution according to claim 1, characterized in that The yak collagen is yak tendon collagen.

5. Use of the yak collagen hydrating injection concentrate as described in any one of claims 1 to 4 in the preparation of yak collagen hydrating injection.

6. A yak collagen water light injection, characterized in that: The yak collagen hydrating injection is prepared from the yak collagen hydrating injection stock solution described in any one of claims 1-4.

7. The yak collagen water light injection according to claim 6, characterized in that The preparation method of the yak collagen water light injection is as follows: (1) purifying yak collagen to obtain a purified collagen solution; (2) adding sodium hyaluronate to the purified collagen solution of step (1) and stirring at 25° C. until a transparent viscous solution is formed; (3) adding vitamin C to the transparent viscous solution in step (2) under light-shielding conditions until it is evenly dispersed; (4) Dissolve α-arbutin in sterile water, dissolve blue copper peptide in deionized water, dissolve PDRN freeze-dried powder in sterile water, and dissolve NMN in the dark, and then add them to the system formed in step (3); (5) Use a 0.22 μm sterile filter membrane for sterile filtration to obtain a clear, sterile functional hydrating injection concentrate, which is the yak collagen hydrating injection.

8. The yak collagen water light injection according to claim 7, characterized in that The step (1) comprises: adding yak tendon collagen to sterile deionized water containing 0.5% glacial acetic acid, and dissolving the collagen at 4°C to obtain a uniform collagen solution; then dialyzing the collagen at 4°C for 48 hours using a dialysis bag with a molecular weight cutoff range of 8000 to 14000 Da, during which the deionized water is replaced 3 to 5 times to obtain a purified collagen solution.

9. Use of the yak collagen water light injection according to any one of claims 6 to 8 in the preparation of skin beauty products.

10. The use according to claim 9, characterized in that The yak collagen water light injection is used for repairing photoaged skin.

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