Collagen composite freeze-dried powder for fading lines as well as preparation and application thereof

Abstract

The invention relates to the field of freeze-dried powder, and discloses collagen composite freeze-dried powder for fading lines as well as preparation and application of the collagen composite freeze-dried powder. The collagen composite freeze-dried powder comprises recombinant XVII type human collagen, a chitosan derivative, superoxide dismutase, mannitol, a plant extract, palmitoyl pentapeptide-4 and water, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are used as cross-linking agents, and carboxymethyl cellulose quaternary ammonium salt and aminated chitosan are subjected to an amidation reaction, so that the chitosan derivative is prepared; the carboxymethyl cellulose quaternary ammonium salt is synthesized by reacting carboxymethyl cellulose and 3-chloro-2-hydroxypropyl trimethyl ammonium chloride in an aqueous solution; the aminated chitosan is prepared by using acryloyl chitosan prepared by reaction of acryloyl chloride and chitosan and further performing thiol-ene click reaction on the acryloyl chitosan and beta-mercaptoethylamine. The collagen composite freeze-dried powder prepared by the preparation method disclosed by the invention has good anti-wrinkle and firming effects and antibacterial performance.

Description

Technical Field

[0001] This invention belongs to the field of freeze-dried powder technology, specifically relating to a collagen composite freeze-dried powder for fading wrinkles and its preparation and application. Background Technology

[0002] The skin protects the body from harmful and exogenous substances, serving as the body's primary anatomical barrier. Skin aging is a biological process in which the skin degenerates under the influence of internal aging factors and the external environment, losing its physiological functions and structural integrity, thus exhibiting signs of aging. External environmental factors such as ultraviolet radiation, tobacco, alcohol, and diet can accelerate skin aging, with ultraviolet radiation being the main cause of skin damage, manifesting as loose skin, increased wrinkles, and decreased elasticity. Freeze-dried powder, as a novel form of skincare product, is highly favored due to its high activity and stability. However, existing freeze-dried powder products still have shortcomings in anti-wrinkle and firming effects, and the role of skin microecological balance in the anti-aging process is increasingly recognized. The colonization and reproduction of harmful bacteria can trigger a state of micro-inflammation in the skin, activating inflammatory pathways, accelerating the degradation of collagen and elastin, and forming a vicious cycle of "inflammation-aging." Existing freeze-dried powders do not possess good antibacterial properties, limiting the wider application of these products. Summary of the Invention

[0003] To address the shortcomings mentioned in the background art, the present invention aims to provide a collagen composite freeze-dried powder for fading wrinkles, its preparation and application, wherein the prepared collagen composite freeze-dried powder has good anti-wrinkle, firming and antibacterial effects.

[0004] The objective of this invention can be achieved through the following technical solutions: A collagen complex freeze-dried powder for fading wrinkles comprises the following components by weight: 20-30 parts recombinant type XVII human collagen, 10-20 parts chitosan derivative, 3-9 parts superoxide dismutase, 2-5 parts mannitol, 1-5 parts plant extract, 0.5-2 parts palmitoyl pentapeptide-4, and 60-85 parts water. The chitosan derivative is prepared by amidation reaction of carboxymethyl cellulose quaternary ammonium salt and aminated chitosan using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide as crosslinking agents; the carboxymethyl cellulose quaternary ammonium salt is synthesized by reacting carboxymethyl cellulose as raw material and 3-chloro-2-hydroxypropyltrimethylammonium chloride as etherifying agent in aqueous solution; the aminated chitosan is prepared by substitution reaction of acryloyl chloride with chitosan to prepare acryloyl chitosan, and then by thiol-ene click reaction of acryloyl chitosan with β-mercaptoethylamine.

[0005] Preferably, the plant extract is a mixture of black tea extract, red clover extract and pomegranate fruit extract in a mass ratio of 1~2:1~1.5:1~1.5.

[0006] Preferably, the method for preparing the chitosan derivative includes the following steps: A. Chitosan and methanesulfonic acid were added to the reactor under ice-water bath conditions and stirred to dissolve. Acryloyl chloride was then added dropwise and reacted for 8 hours. Subsequently, the reaction solution was precipitated with a mixture of diethyl ether and acetone to obtain acrylated chitosan. B. Acrylamide-modified chitosan, β-mercaptoethylamine and deionized water were added to the reactor at room temperature and stirred to dissolve. Then, the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone was added and reacted under 365nm ultraviolet light for 1 hour. The mixture was then dialyzed through a dialysis bag to obtain amino-modified chitosan. C. Disperse carboxymethyl cellulose in a medium of isopropanol and water, add sodium hydroxide aqueous solution to alkalize the reaction for 30 min, then add 3-chloro-2-hydroxypropyltrimethylammonium chloride, stir the reaction at 60℃ for 2 h, then cool the reaction solution, dialyze the reaction solution with distilled water for three days, concentrate by rotary evaporation and vacuum dry to obtain carboxymethyl cellulose quaternary ammonium salt; D. Add 2-(N-morpholino)ethanesulfonic acid buffer and carboxymethyl cellulose quaternary ammonium salt at pH 6.5 to the reactor and stir to mix. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide and continue stirring for 1 hour. Then add aminated chitosan and stir at 35-55℃ for 20-24 hours. After that, cool the reaction solution, dialyze the reaction solution with distilled water for three days, concentrate by rotary evaporation and vacuum dry to obtain chitosan derivative.

[0007] Preferably, the addition ratio of chitosan, methanesulfonic acid and acryloyl chloride in step A is 4g:30mL:15mL.

[0008] Preferably, in step B, the mass ratio of acryloylated chitosan, β-mercaptoethylamine, and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone is 2:3:0.5.

[0009] Preferably, the concentration of the sodium hydroxide aqueous solution in step C is 0.35~0.4 g / mL; the addition ratio of carboxymethyl cellulose, isopropanol, water, sodium hydroxide aqueous solution and 3-chloro-2-hydroxypropyltrimethylammonium chloride is 6 g: 75 mL: 25 mL: 10 mL: 5~8 g.

[0010] Preferably, in step D, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 2:1.

[0011] Preferably, in step D, the mass ratio of carboxymethyl cellulose quaternary ammonium salt to aminated chitosan is 1:2~3.

[0012] The preparation method of the collagen composite freeze-dried powder for fading wrinkles as described above includes the following steps: weigh each component according to the weight parts, stir and mix recombinant type XVII human collagen, chitosan derivative, superoxide dismutase, mannitol, plant extract and palmitoyl pentapeptide-4 evenly, freeze at -25~-20℃ for 6~10h, then freeze at -10~-5℃ for 8~12h, then freeze at -22℃ for 2~6h, and finally keep at 25~33℃ for 4~6h; the vacuum degree during the freeze-drying process is 0.6~0.8mbar, and the collagen composite freeze-dried powder for fading wrinkles is obtained.

[0013] The application of the collagen complex freeze-dried powder for reducing wrinkles as described above is that the collagen complex freeze-dried powder for reducing wrinkles is applied to skin care products.

[0014] The beneficial effects of this invention are: This invention utilizes acryloyl chloride to introduce carbon-carbon double bonds into chitosan molecules to prepare acryloylated chitosan. Then, it uses acryloylated chitosan to undergo a thiol-ene click reaction with β-mercaptoethylamine to prepare aminated chitosan. Simultaneously, this invention uses carboxymethyl cellulose as a raw material and 3-chloro-2-hydroxypropyltrimethylammonium chloride as an etherifying agent to synthesize a quaternary ammonium salt of carboxymethyl cellulose in aqueous solution. Then, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide as crosslinking agents, it prepares a product by amidation reaction of the carboxymethyl cellulose quaternary ammonium salt and aminated chitosan. Chitosan derivatives, in which chitosan, as a natural polymer material, possesses properties such as non-toxicity, harmlessness, antibacterial properties, safety, and biodegradability, are obtained by amylating the hydroxyl groups on the chitosan molecule to obtain more amino groups. The abundant amino and quaternary ammonium salt groups in the chitosan derivatives significantly improve the defect that the original chitosan can only dissolve under acidic conditions. Furthermore, it not only provides an excellent moisturizing environment but also constructs a biomimetic network similar to the extracellular matrix through amide bonds, providing a good scaffold for the adhesion and expression of recombinant human type XVII collagen. Simultaneously, this chitosan derivative and recombinant human type XVII collagen... Collagen exhibits excellent biocompatibility, preventing protein aggregation and precipitation caused by charge differences or hydrophobic interactions, thus significantly extending the product's shelf life. Furthermore, this chitosan derivative, through quaternized cellulose segments, effectively promotes the orderly arrangement of epidermal cells and inhibits the growth of harmful microorganisms on the skin surface, providing a healthy microenvironment for fading wrinkles. Additionally, during freeze-drying, this chitosan derivative forms a hydrogen-bonded complex with palmitoyl pentapeptide-4, significantly enhancing the chemical stability of palmitoyl pentapeptide-4 in formulations and its bioactivity after reconstitution, achieving continuous and stable release of palmitoyl pentapeptide-4 from the skin surface. It provides long-lasting stimulation of collagen regeneration. The added plant extracts (black tea extract, red clover extract, and pomegranate fruit extract) are rich in flavonoids, which not only have antioxidant and anti-inflammatory effects, but also protect the skin from UV damage, thereby delaying aging. Furthermore, under the attraction of the positive charge in the chitosan derivative, the plant extracts can be more evenly dispersed in the freeze-dried framework, avoiding the problem of uneven distribution of effective ingredients in traditional freeze-dried powders. Superoxide dismutase is not only an independent antioxidant ingredient, but also a key regulator that protects the collagen scaffold, enhances the efficacy of plant extracts, and maintains the redox balance of the entire system. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a transmission electron microscope image of the dermal-epidermal continuity of the collagen composite freeze-dried powder for fading wrinkles according to the present invention. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1: A method for preparing a chitosan derivative includes the following steps: A. Under ice-water bath conditions, add 4g of chitosan and 30mL of methanesulfonic acid to the reactor and stir to dissolve. Then add 15mL of acryloyl chloride dropwise and react for 8h. Subsequently, precipitate the reaction solution with a mixture of diethyl ether and acetone (volume ratio of 1:1) to obtain acryloylated chitosan. B. At room temperature, add 2g of acrylamide chitosan, 3g of β-mercaptoethylamine and 50mL of deionized water to the reactor and stir to dissolve. Then add 0.5g of photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone and react under 365nm ultraviolet light for 1h. Then dialyze the mixture with a dialysis bag (MWCO=8000) to obtain aminoated chitosan. C. Disperse 6g of carboxymethyl cellulose in a medium of 75mL isopropanol and 25mL water, add 10mL of sodium hydroxide aqueous solution with a concentration of 0.37g / mL for alkalization reaction for 30min, then add 6.3g of 3-chloro-2-hydroxypropyltrimethylammonium chloride, stir and react at 60℃ for 2h, then cool the reaction solution, dialyze the reaction solution with distilled water for three days, concentrate by rotary evaporation and vacuum dry to obtain carboxymethyl cellulose quaternary ammonium salt; D. Add 50 mL of 0.2 mol / L 2-(N-morpholino)ethanesulfonic acid buffer (pH=6.5) and 5 g of carboxymethyl cellulose quaternary ammonium salt to the reactor and stir to mix. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a molar ratio of 2:1 and continue stirring for 1 h. Then add 12 g of aminated chitosan and stir at 45 °C for 24 h. After that, cool the reaction solution, dialyze the reaction solution with distilled water for three days, concentrate by rotary evaporation and vacuum dry to obtain chitosan derivative.

[0019] Example 2 A collagen complex freeze-dried powder for fading wrinkles, comprising the following components by weight: 21 parts recombinant type XVII human collagen, 12 parts chitosan derivative prepared in Example 1, 3.5 parts superoxide dismutase (Cu / Zn-SOD), 2.5 parts mannitol, 1.4 parts plant extract (a mixture of black tea extract, red clover extract and pomegranate fruit extract in a mass ratio of 1:1:1), 0.6 parts palmitoyl pentapeptide-4, and 66 parts water.

[0020] The preparation method of the above-mentioned collagen composite freeze-dried powder for fading wrinkles includes the following steps: weigh each component according to the weight parts, stir and mix recombinant type XVII human collagen, chitosan derivative, superoxide dismutase, mannitol, plant extract and palmitoyl pentapeptide-4 evenly, freeze at -22℃ for 8h, then freeze at -6℃ for 10h, then freeze at -22℃ for 4h, and finally keep at 30℃ for 5h; the vacuum degree during the freeze-drying process is 0.7mbar, and the collagen composite freeze-dried powder for fading wrinkles is obtained.

[0021] Example 3 A collagen complex freeze-dried powder for fading wrinkles, comprising the following components by weight: 25 parts recombinant type XVII human collagen, 16 parts chitosan derivative prepared in Example 1, 6.6 parts superoxide dismutase (Cu / Zn-SOD), 3.8 parts mannitol, 2.7 parts plant extract (a mixture of black tea extract, red clover extract and pomegranate fruit extract in a mass ratio of 1:1:1), 1.1 parts palmitoyl pentapeptide-4, and 70 parts water.

[0022] The preparation method of the collagen composite freeze-dried powder for fading wrinkles is the same as in Example 2.

[0023] Example 4 A collagen complex freeze-dried powder for fading wrinkles, comprising the following components by weight: 28 parts of recombinant type XVII human collagen, 18 parts of chitosan derivative prepared in Example 1, 8.4 parts of superoxide dismutase (Cu / Zn-SOD), 4.7 parts of mannitol, 4.4 parts of plant extracts (a mixture of black tea extract, red clover extract and pomegranate fruit extract in a mass ratio of 1:1:1), 1.7 parts of palmitoyl pentapeptide-4, and 82 parts of water.

[0024] The preparation method of the collagen composite freeze-dried powder for fading wrinkles is the same as in Example 2.

[0025] Comparative Example 1: A collagen complex freeze-dried powder for fading wrinkles, comprising the following components by weight: 28 parts of recombinant type XVII human collagen, 18 parts of amino-modified chitosan prepared in Example 1, 8.4 parts of superoxide dismutase (Cu / Zn-SOD), 4.7 parts of mannitol, 4.4 parts of plant extracts (a mixture of black tea extract, red clover extract and pomegranate fruit extract in a mass ratio of 1:1:1), 1.7 parts of palmitoyl pentapeptide-4, and 82 parts of water.

[0026] The preparation method of the collagen composite freeze-dried powder for fading wrinkles is the same as in Example 2.

[0027] Comparative Example 2: A collagen complex freeze-dried powder for fading wrinkles, comprising the following components by weight: 28 parts recombinant type XVII human collagen, 8.4 parts superoxide dismutase (Cu / Zn-SOD), 4.7 parts mannitol, 4.4 parts plant extract (a mixture of black tea extract, red clover extract and pomegranate fruit extract in a mass ratio of 1:1:1), 1.7 parts palmitoyl pentapeptide-4, and 82 parts water.

[0028] The preparation method of the collagen composite freeze-dried powder for fading wrinkles is the same as in Example 2.

[0029] Performance testing A. In vitro efficacy testing: (1) Immerse the freshly obtained skin tissue in 75% alcohol for 30 seconds, then wash three times with sterile PBS buffer, and cut the skin into (24±2) mm pieces. 2 The tissue blocks, with the epidermis facing up and the dermis facing down, were placed in a culture mold. The culture mold was then transferred into a 6-well plate, and 3.7 mL of culture medium was added to each well. The plates were incubated at 37°C in a 5% CO2 incubator, with the medium changed daily. (2) Two days after ex vivo skin tissue culture, irradiation and drug administration were initiated according to the test groups and corresponding treatment conditions in Table 1; the irradiation dose was UVA (30 J / cm²). 2 ) and UVB (50mJ / cm 2 The samples were irradiated continuously for 4 days, with fresh culture medium replaced after each irradiation and drug administration performed. The positive control (VC+VE) was administered via subsoil, while the test samples were administered via surface. After 4 days of continuous irradiation, the ex vivo skin tissue was cultured for another 3 days without irradiation, and only the samples were drugged.

[0030]

[0031] ① Results of dermal-epidermal junction (DEJ) continuity test like Figure 1 As shown, Figure 1Continuity images of the DEJ were captured using a transmission electron microscope (Hitachi). Arrows indicate the DEJ structure, and the NC group marks the damage area.

[0032] Compared with the BC group, the DEJ structure in the NC group was damaged (fractured), indicating that the stimulus conditions in this test were effective.

[0033] Compared with the NC group, the DEJ structure of the PC group showed better continuity, indicating that the positive control in this test was effective.

[0034] Compared with the NC group, the DEJ structure of samples 2-4 showed better continuity, indicating that the samples had an ameliorative effect on DEJ damage.

[0035] ② Immunofluorescence analysis results of integrin α6 content

[0036] Note: Integrated optical density (IOD) reflects the content of integrin α6. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by #); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0037] As shown in the table above, the integrin α6 content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective.

[0038] Compared with the NC group, the PC group showed a significant increase in integrin α6 content, indicating that the positive control in this test was effective.

[0039] Compared with the NC group, the integrin α6 content of samples 2-4 increased significantly, with increases of 193.75%, 150.00%, and 171.88%, respectively.

[0040] ③ Immunofluorescence analysis results of laminin 332 content

[0041] Note: Integrated optical density (IOD) / Area reflects the content of Laminin 332. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by #); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0042] As shown in the table above, the content of Laminin332 in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective.

[0043] Compared with the NC group, the content of Laminin 332 in the PC group was significantly increased, indicating that the positive control in this test was effective.

[0044] Compared with the NC group, the content of Laminin 332 in samples 2-4 increased significantly, with increases of 45.90%, 54.10%, and 95.08%, respectively.

[0045] ④ Immunofluorescence analysis results of type IV collagen content

[0046] Note: Integrated optical density (IOD) / Area reflects the content of Collagen IV. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by #); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0047] As shown in the table above, the Collagen IV content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective.

[0048] Compared with the NC group, the Collagen IV content in the PC group was significantly increased, indicating that the positive control in this test was effective.

[0049] Compared with the NC group, the Collagen IV content in samples 2-4 increased significantly, with increases of 44.07%, 64.41%, and 91.53%, respectively.

[0050] Immunofluorescence analysis results of type 5 and 7 collagen content

[0051] Note: Integrated optical density (IOD) / Area, the value reflects Collagen VII is indicated by #, P-value < 0.01 is indicated by #; compared with the NC group, significance is indicated by *, P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **.

[0052] As shown in the table above, the Collagen VII content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective.

[0053] Compared with the NC group, the Collagen VII content in the PC group was significantly increased, indicating that the positive control in this test was effective.

[0054] Compared with the NC group, the Collagen VII content of samples 2-4 increased significantly, with increases of 27.27%, 42.42%, and 45.45%, respectively.

[0055] ⑥ Results of immunofluorescence analysis of nestin content

[0056] Note: Integrated optical density (IOD) reflects the content of Nidogen. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by #); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0057] As shown in the table above, the Nidogen content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective.

[0058] Compared with the NC group, the Nidogen content in the PC group was significantly increased, indicating that the positive control in this test was effective.

[0059] Compared with the NC group, the Nidogen content of samples 2-4 increased significantly, with increases of 200.00%, 384.21%, and 384.21%, respectively.

[0060] ⑦ Results of immunofluorescence analysis of reticulin content

[0061] Note: Integrated optical density (IOD) reflects the content of Plectin. When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by # (P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by #); significance compared to the NC group is indicated by * (P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **).

[0062] As shown in the table above, the Plectin content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective.

[0063] Compared with the NC group, the PC group showed a significant increase in Plectin levels, indicating that the positive control in this test was effective.

[0064] Compared with the NC group, the Plectin content of samples 2-4 increased significantly, with increases of 172.22%, 261.11%, and 311.11%, respectively.

[0065] The test results above show that, based on ex vivo skin tissue, compared with the control group, the dermal-epidermal junction (DEJ) structure of samples 2-4 has better continuity, and the contents of type IV collagen, integrin α6, and laminin 332 are significantly increased. This indicates that the samples can improve dermal-epidermal junction (DEJ) damage, increase the contents of type IV collagen, integrin α6, and laminin 332, and have a firming effect.

[0066] Based on ex vivo skin tissue, compared with the control group, the contents of type VII collagen, Nidogen, and Plectin in samples 2-4 were significantly increased, indicating that the samples can increase the contents of type VII collagen, Nidogen, and Plectin and have anti-wrinkle effects.

[0067] B. Antibacterial Performance Test: *Escherichia coli* (E. coli) and *Staphylococcus aureus* (S. aureus) were pre-inoculated into Erlenmeyer flasks containing LB liquid medium and cultured (37℃, 24h) to obtain bacterial suspensions. 0.1 mL of each *E. coli* and *S. aureus* bacterial suspension was evenly spread onto LB solid medium. Filter paper (12 mm in diameter) soaked in a 0.05% (w / w) sample aqueous solution was then flatly placed in the center of the medium. The flasks were sealed with sealing film and incubated in a biochemical incubator (37℃, 24h). The inhibition zones of each experimental group were observed and recorded. The data results are shown in the table below:

[0068] As can be seen from the table above, the samples prepared in Examples 2-4 of this invention have superior antibacterial activity compared with Comparative Examples 1-2.

[0069] C. Human efficacy testing: (1) Subject Screening: Strictly following the Declaration of Helsinki, subjects were informed of the test details, voluntarily participated in the test, and signed the informed consent form. The informed consent form clearly explained the possible risks, benefits, rights, and obligations related to participation in the study. Subject Inclusion Criteria: Healthy Chinese women aged 18-60 years (N=30); self-identified as having dry, loose, inelastic, and sensitive facial skin; randomized unilateral crow's feet grade 3-6 (according to SGS standard atlas); able to cooperate well with the test according to the test protocol; and able to maintain good lifestyle habits during the study period. Exclusion Criteria: Those with skin diseases at the test site; those who are pregnant, breastfeeding, or planning to conceive; those deemed unsuitable for participation by the researchers; those who have used antihistamines within the past week or immunosuppressants within the past month; those who have participated in human clinical trials of cosmetics within the past month; (2) Product usage and testing requirements: Subjects should use the product once each morning and evening after cleansing. Apply an appropriate amount of the product evenly to the face and gently massage until absorbed. During the testing period, subjects should discontinue the use of other similar products. Use continuously for 28 days, with follow-up visits on day 0 and day 28. On the day of the test, subjects should cleanse their face with the cleansing product provided by the laboratory and sit quietly for 30 minutes. Subsequently, researchers will test the outer corner of the eye on one random side. The entire test will be conducted in a constant temperature environment with a temperature of (21±2.0)℃ and a relative humidity of 50%±10%. (3) Skin anti-wrinkle test: The number, volume, area, and length of crow's feet wrinkles were measured using Primos CR after 28 days. The data results are shown in the table below:

[0070] As shown in the table above, compared with before using the product, the number, volume, area and length of wrinkles at the crow's feet were significantly improved after 28 days of using the samples prepared in Examples 2-4, and the improvement effect of Examples 2-4 was more obvious than that of Comparative Examples 1-2.

[0071] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0072] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A collagen composite freeze-dried powder for fading wrinkles, characterized in that, The product comprises the following components by weight: 20-30 parts recombinant human type XVII collagen, 10-20 parts chitosan derivative, 3-9 parts superoxide dismutase, 2-5 parts mannitol, 1-5 parts plant extract, 0.5-2 parts palmitoyl pentapeptide-4, and 60-85 parts water. The chitosan derivative is prepared by amidation reaction of carboxymethyl cellulose quaternary ammonium salt and aminated chitosan using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide as crosslinking agents; the carboxymethyl cellulose quaternary ammonium salt is synthesized by reacting carboxymethyl cellulose as raw material and 3-chloro-2-hydroxypropyltrimethylammonium chloride as etherifying agent in aqueous solution; the aminated chitosan is prepared by substitution reaction of acryloyl chloride with chitosan to prepare acryloyl chitosan, and then by thiol-ene click reaction of acryloyl chitosan with β-mercaptoethylamine.

2. The collagen composite freeze-dried powder for fading wrinkles according to claim 1, characterized in that, The plant extract is composed of black tea extract, red axle extract and pomegranate fruit extract in a mass ratio of 1~2:1~1.5:1~1.

5.

3. The collagen composite freeze-dried powder for fading wrinkles according to claim 1, characterized in that, The preparation method of the chitosan derivative includes the following steps: A. Chitosan and methanesulfonic acid were added to the reactor under ice-water bath conditions and stirred to dissolve. Acryloyl chloride was then added dropwise and reacted for 8 hours. Subsequently, the reaction solution was precipitated with a mixture of diethyl ether and acetone to obtain acrylated chitosan. B. Acrylamide-modified chitosan, β-mercaptoethylamine and deionized water were added to the reactor at room temperature and stirred to dissolve. Then, the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone was added and reacted under 365nm ultraviolet light for 1 hour. The mixture was then dialyzed through a dialysis bag to obtain amino-modified chitosan. C. Disperse carboxymethyl cellulose in a medium of isopropanol and water, add sodium hydroxide aqueous solution to alkalize the reaction for 30 min, then add 3-chloro-2-hydroxypropyltrimethylammonium chloride, stir the reaction at 60℃ for 2 h, then cool the reaction solution, dialyze the reaction solution with distilled water for three days, concentrate by rotary evaporation and vacuum dry to obtain carboxymethyl cellulose quaternary ammonium salt; D. Add 2-(N-morpholino)ethanesulfonic acid buffer and carboxymethyl cellulose quaternary ammonium salt at pH 6.5 to the reactor and stir to mix. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide and continue stirring for 1 hour. Then add aminated chitosan and stir at 35-55℃ for 20-24 hours. After that, cool the reaction solution, dialyze the reaction solution with distilled water for three days, concentrate by rotary evaporation and vacuum dry to obtain chitosan derivative.

4. The collagen composite freeze-dried powder for fading wrinkles according to claim 3, characterized in that, In step A, the addition ratio of chitosan, methanesulfonic acid, and acryloyl chloride is 4g:30mL:15mL.

5. The collagen composite freeze-dried powder for fading wrinkles according to claim 3, characterized in that, In step B, the mass ratio of acryloylated chitosan, β-mercaptoethylamine, and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone is 2:3:0.

5.

6. The collagen composite freeze-dried powder for fading wrinkles according to claim 3, characterized in that, In step C, the concentration of the sodium hydroxide aqueous solution is 0.35~0.4 g / mL; the addition ratio of carboxymethyl cellulose, isopropanol, water, sodium hydroxide aqueous solution and 3-chloro-2-hydroxypropyltrimethylammonium chloride is 6 g: 75 mL: 25 mL: 10 mL: 5~8 g.

7. The collagen composite freeze-dried powder for fading wrinkles according to claim 3, characterized in that, In step D, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 2:

1.

8. The collagen composite freeze-dried powder for fading wrinkles according to claim 3, characterized in that, In step D, the mass ratio of carboxymethyl cellulose quaternary ammonium salt to aminated chitosan is 1:2~3.

9. A method for preparing a collagen composite freeze-dried powder for fading wrinkles according to any one of claims 1 to 8, characterized in that, The process includes the following steps: weighing each component according to weight, mixing recombinant type XVII human collagen, chitosan derivative, superoxide dismutase, mannitol, plant extract and palmitoyl pentapeptide-4 evenly, freezing at -25~-20℃ for 6~10h, then freezing at -10~-5℃ for 8~12h, then freezing at -22℃ for 2~6h, and finally holding at 25~33℃ for 4~6h; the vacuum degree during the freeze-drying process is 0.6~0.8mbar, and a collagen composite freeze-dried powder for fading wrinkles is prepared.

10. The application of the collagen composite freeze-dried powder for fading wrinkles according to any one of claims 1 to 8, characterized in that, The collagen complex freeze-dried powder for fading wrinkles is used in skincare products.

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