A recombinant humanized collagen composition, preparation method and application thereof

Abstract

The present application belongs to the technical field of cosmetics, and particularly relates to a recombinant humanized collagen composition, a preparation method and application. The present application first reports a recombinant humanized collagen composition, which is based on the independently developed recombinant humanized collagen, and through compounding chitosan, ectoine and other components, the application scene of the recombinant type IV collagen is effectively expanded; and the composition itself can reduce the ability of melanocytes to synthesize melanin, has the ability to scavenge free radicals, and can also reduce the amount of preservatives in the composition, effectively maintain the stability of the recombinant type IV collagen. The recombinant humanized collagen composition can be used for daily skin care, and can also help the recovery after medical and aesthetic surgery, and therefore has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a recombinant humanized collagen composition, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Currently, minimally invasive cosmetic procedures are popular, with treatments like hyaluronic acid injections and phototherapy addressing skin concerns. However, achieving perfect results depends not only on professional technique but also on the recovery period. Minimal cosmetic procedures typically involve superficial wounds, often densely packed. Damaged basement membranes cannot function properly, connecting the dermis and epidermis, transmitting signals, and delivering nutrients, directly leading to further damage to the wound and surrounding skin barrier. When the wound extends to the epidermis, it affects melanocyte activity. Diet, air pollution, ultraviolet radiation, and microbial contamination can all stimulate melanin production around the wound, resulting in pigmentation after healing. During the wound recovery period, the exposed skin is susceptible to free radicals from ultraviolet radiation and airborne impurities, slowing recovery and accelerating the aging of the surrounding skin.

[0004] Type IV collagen is involved in the formation of the basement membrane. As a major component of the basement membrane, recombinant type IV collagen is essential for direct supplementation during wound healing, which can accelerate wound recovery and improve prognosis.

[0005] Wound dressings are essential products for postoperative recovery. Products like face masks and lotions, which cannot be terminally sterilized, contain preservatives and antibacterial agents and can be reused multiple times after opening. However, this can irritate the wound, cause pain, and reduce patient compliance. Most preservatives affect the structure and aggregation behavior of collagen, limiting the widespread application of recombinant type IV collagen. Furthermore, the inventors have found that there is currently limited research on the application of recombinant humanized type IV collagen. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a recombinant humanized collagen composition, its preparation method, and its applications. Based on their independently developed recombinant humanized collagen, the inventors have expanded the application scenarios of recombinant type IV collagen by combining it with components such as chitosan and ectoine. Furthermore, the composition itself can reduce the ability of melanocytes to synthesize melanin, possesses free radical scavenging capabilities, and can reduce the amount of preservatives required. Based on these research findings, this invention was completed.

[0007] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a recombinant humanized collagen composition comprising at least recombinant humanized type IV collagen, chitosan, and ectoine; wherein the amino acid sequence of the recombinant humanized type IV collagen is shown in SEQ ID NO.1.

[0009] The recombinant humanized collagen composition may also contain other raw material ingredients permitted for addition in the cosmetic field, including emulsifiers, emollients, moisturizers, thickeners, lubricants, chelating agents, fillers, binders, antioxidants, preservatives, fragrances, dyes, buffers, pH adjusters, solvents, viscosity control agents, and opacifiers.

[0010] The preservatives include, but are not limited to, phenoxyethanol, ethylparaben, and propylparaben.

[0011] In another specific embodiment of the present invention, the recombinant humanized collagen composition comprises the following components in parts by weight:

[0012] Recombinant humanized type IV collagen 0.001-0.5 parts, chitosan 0.2-1.25 parts, ectoine 0.2-2 parts;

[0013] Furthermore, the recombinant humanized collagen composition also includes a preservative, wherein the amount of preservative added is 0.01-1 part.

[0014] Furthermore, the recombinant humanized collagen composition comprises the following components in mass fractions:

[0015] Recombinant humanized type IV collagen 0.001-0.5%, chitosan 0.2-1.25%, ectoine 0.2-2%, preservative 0.01-1%, balance water.

[0016] The amount of preservative added is 0.1-0.5%, and the preservative is phenoxyethanol.

[0017] Pure chitosan is a white or grayish-white translucent flaky solid. It dissolves in dilute acid to form a viscous precipitate. In dilute acid, the β-1,4-glycosidic bonds of chitosan slowly hydrolyze, generating chitosan with a lower relative molecular mass. It dissolves in acidic solutions to form positively charged cationic groups. Chitosan exhibits strong adsorption properties in solution. The chitosan molecule contains amino groups, making it basic. The C3-OH, C2-NH2, and C6-OH functional groups of chitosan can form intramolecular and intermolecular hydrogen bonds. In 0.1 mmol / L dilute hydrochloric acid, the hydrolysis rate of chitosan acetyl groups is approximately equal to the depolymerization rate of chitosan.

[0018] In this invention, chitosan with a degree of deacetylation of 80% to 95%, acid solubility, and a molecular weight of ~100 kDa is preferably selected.

[0019] Ectocin has multiple functions such as moisturizing, repairing, and isolating, and can be added to cosmetics as a skin moisturizer.

[0020] In a second aspect, this invention provides a method for preparing the above-mentioned recombinant humanized collagen composition, the method being as follows:

[0021] S1. Dissolve chitosan in an acidic solution and heat and stir until completely dissolved;

[0022] S2. Add ectoine to the solution obtained in step S1, and continue heating and stirring until completely dissolved. After swelling, it becomes a homogeneous and transparent liquid.

[0023] S3. Add the recombinant humanized type IV collagen to water for pre-dispersion. After the solution in step S2 has cooled down, add the collagen dispersion to the solution and stir until it is evenly dispersed.

[0024] Furthermore, adjust the pH of the solution system obtained in step S3 to be weakly acidic to neutral, such as pH 6.0-7.0.

[0025] Furthermore, the preparation method also includes adding other raw material components permitted in the cosmetic field to the solution system obtained in step S3, including emulsifiers, emollients, moisturizers, thickeners, lubricants, chelating agents, fillers, binders, antioxidants, preservatives, fragrances, dyes, buffers, pH adjusters, solvents, viscosity control agents, and opacifiers.

[0026] In step S1, the pH of the acidic solution is 3.0-5.0 (preferably 4.0), and the specific temperature conditions for heating and stirring are 40-60℃ (preferably 50℃).

[0027] In step S2, the temperature for continued heating and stirring is 40-60℃ (preferably 50℃).

[0028] In step S3, the temperature is lowered to room temperature, such as 20-30°C.

[0029] A fourth aspect of the present invention provides the use of the above-described recombinant humanized collagen composition in the preparation of products.

[0030] The product may be a pharmaceutical or a cosmetic, with cosmetics being preferred. The cosmetic may be a skin care product, and the product has at least the effects of inhibiting melanin production by melanocytes and scavenging free radicals.

[0031] When the product is a pharmaceutical product, the pharmaceutical product may also include at least one inactive pharmaceutical ingredient.

[0032] The inactive ingredient of the drug can be a pharmaceutically acceptable carrier. Furthermore, it can be formulated into dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral administration, external use, suppositories, and sterile injectable solutions, according to conventional methods.

[0033] The pharmaceutically acceptable dose of the carrier should be harmless to the subject, and specifically includes, but is not limited to, buffers, antioxidants, preservatives, bactericides, chelating agents, tension modifiers, surfactants, etc., without specific limitations.

[0034] A fifth aspect of the present invention provides a cosmetic product comprising at least the above-described recombinant humanized collagen composition.

[0035] Furthermore, the cosmetic product can be a skin care product, which, in addition to being used for daily skin care, can also help with post-operative recovery from cosmetic surgery due to its excellent effects in inhibiting melanin production by melanocytes and scavenging free radicals.

[0036] Furthermore, the cosmetic dosage form can be any one or more of the following: aqueous solution, emulsion, cream, serum, gel, mask, spray, or base makeup, without any specific limitation.

[0037] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0038] The above technical solution is the first to report a recombinant humanized collagen composition. Based on the independently developed recombinant humanized collagen, it can effectively expand the application scenarios of recombinant type IV collagen by compounding chitosan, ectoine and other components. Moreover, the composition itself can reduce the ability of melanocytes to synthesize melanin, has the ability to scavenge free radicals, and can also reduce the amount of preservatives in the composition, effectively maintaining the stability of recombinant type IV collagen. Therefore, it has good practical application value. Attached Figure Description

[0039] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0040] Figure 1 This is a diagram showing the detection of recombinant collagen content using ultraviolet-visible spectrophotometry according to the present invention.

[0041] Figure 2 This is a standard curve for detecting the recombinant collagen content of this invention.

[0042] Figure 3 This is an SDS-polyacrylamide gel electrophoresis image of the recombinant collagen of this invention. Detailed Implementation

[0043] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0044] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0045] The present invention will now be further illustrated with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Unless otherwise specified, experimental conditions not explicitly stated in the examples are generally performed under conventional conditions or as recommended by the reagent company. Unless otherwise specified, all reagents and consumables used in the following examples are commercially available.

[0046] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and not for limiting the scope of the invention. The following examples illustrate test methods with specific conditions, which are generally performed under conventional conditions. Unless otherwise specified, all percentages mentioned in the examples are mass percentages.

[0047] The amino acid sequence of the recombinant humanized type IV collagen in this application embodiment is shown in SEQ ID NO.1. It can be prepared according to the method described in Chinese Patent CN202410255649.1, specifically by optimizing the coding gene based on the codon preference of *E. coli*. The nucleotide sequence of the codon-optimized coding gene is shown in SEQ ID NO.2. The coding gene is inserted into the expression vector pET28a to construct a recombinant plasmid. The recombinant plasmid is transformed into *E. coli* BL21(DE3) to construct a recombinant strain. The recombinant strain is cultured to the logarithmic growth phase, and expression is induced by IPTG. After cell disruption and purification, the recombinant humanized type IV collagen is obtained. Experiments have shown that this recombinant humanized type IV collagen has a stronger ability to promote COL-1 secretion and FLG mRNA expression than commercially available recombinant collagen, indicating that it has anti-wrinkle and skin barrier repair functions.

[0048] Examples 1-3: Preparation of compositions containing type IV collagen. The components are shown in Table 1. The preparation method is as follows: Step 1: First, dissolve chitosan in a solution with pH=4.0 and stir at 50-60°C for 2 hours until completely dissolved.

[0049] Step 2: Add ectoine and stir at 50-60°C until completely dissolved. After swelling, it will become a homogeneous and transparent liquid.

[0050] Step 3: Pre-disperse recombinant humanized type IV collagen in water. Cool the solution from Step 2 to 20-30°C, add the collagen dispersion to the solution, and stir slowly for 2-3 hours until uniformly dispersed. Then refrigerate at 0-8°C for 24 hours to allow the reaction to proceed. After cooling to room temperature, it can be added to the product. The pH value should be 4.0-5.0.

[0051] Step 4: Adjust the pH of the solution to 6.0.

[0052] Step 5: Add water to 100%.

[0053] Step 6: Add the preservative phenoxyethanol to the composition and stir for 30 minutes until homogeneous.

[0054] Preferably, the chitosan has a degree of deacetylation of 80% to 95%, is acid-soluble, and has a molecular weight of 100 kDa.

[0055] Specifically, the compositions of the embodiments and comparative examples are shown in Table 1, expressed as mass fractions.

[0056] Example 4: The preparation method is the same as in Examples 1-3, but without the addition of phenoxyethanol.

[0057] Table 1. Composition of Examples 1-4 and Comparative Examples 1-11

[0058]

[0059] Example 5: The composition and preparation method of a spray dressing containing the composition of the present invention are as follows:

[0060] Raw material name Content / Percentage Chitosan 0.5 Ikdoin 0.5 Recombinant type IV collagen 0.05 Phenoxyethanol 1.0 glycerin 2.1 Oligomeric sodium hyaluronate 0.01 water To100

[0061] Preparation method:

[0062] Step 1: Dissolve chitosan in a solution with pH=4.0 and stir at 50-60℃ for 2 hours until completely dissolved.

[0063] Step 2: Add ectoine and stir at 50-60°C until completely dissolved. After swelling, it will become a homogeneous and transparent liquid.

[0064] Step 3: Pre-disperse recombinant humanized type IV collagen in water. Cool the solution from Step 2 to 20-30°C, add the collagen dispersion to the solution, and stir slowly for 2-3 hours until uniformly dispersed. Then refrigerate at 0-8°C for 24 hours to allow the reaction to proceed, and then remove to room temperature.

[0065] Step 4: Adjust the pH of the solution to 6.0.

[0066] Step 5: Disperse oligomeric sodium hyaluronate in glycerin, add a small amount of water, heat and stir until dissolved and homogeneous.

[0067] Step 6: Add the liquid from step 5 to the liquid from step 4 and stir well.

[0068] Step 7: Add the preservative phenoxyethanol to the composition, add water to make up the volume, and stir for 30 minutes until homogeneous.

[0069] Comparative Examples 1-4:

[0070] The preparation process is the same as in Example 1.

[0071] Comparative Example 5: Only recombinant type IV collagen was added.

[0072] Steps 1 and 2 are deleted; the rest of the preparation process is the same as in Example 1.

[0073] Comparative Example 6: Recombinant type IV collagen was above the range of the composition.

[0074] The preparation process is the same as in Example 1.

[0075] Comparative Example 7: The spray dressing of Example 5 did not contain this composition.

[0076] Raw material name Content / Percentage Phenoxyethanol 1.0 glycerin 2.1 Sodium hyaluronate 0.01 Recombinant type IV collagen 0.01 water To100

[0077] Comparative Example 8: The spray dressing of Example 5 did not contain recombinant type IV collagen.

[0078] Raw material name Content / Percentage Phenoxyethanol 1.0 glycerin 2.1 Sodium hyaluronate 0.01 water To100

[0079] Comparative Examples 9–11: Contains no chitosan, ectoine, or collagen.

[0080] The preparation method is the same as in Example 1.

[0081] Conclusions: Examples 1-5 demonstrate that the composition exhibits good stability within the specified range even with high preservative concentrations. Example 5 shows that the composition is stable in formulations. Comparative Examples 1 and 2, with chitosan content exceeding the composition's range, are unstable; Comparative Examples 3 and 4, with ectoine content exceeding the composition's range, are unstable; Comparative Example 5 shows that recombinant type IV collagen cannot exist stably in the phenoxyethanol system; Comparative Example 6 shows that recombinant type IV collagen exceeding the composition's range cannot exist stably; Comparative Example 7, in a spray system containing only recombinant type IV collagen and lacking the other two components of the composition, is unstable, indicating that this composition can stabilize collagen in more difficult-to-stabilize aqueous systems; Comparative Example 8 shows that the spray system without collagen is stable. Comparative Examples 9-11 show that the absence of chitosan and ectoine in the composition both cause collagen instability, while the system without recombinant collagen is stable.

[0082] In summary, within the scope of the composition, recombinant type IV collagen can be stabilized in preservative systems.

[0083]

[0084] The content and purity of Examples 1-5 were tested.

[0085] Content detection method: The content of recombinant collagen was determined according to the 2,2'-biquinoline-4,4'-dicarboxylic acid method (BCA method). Protein molecules react with Cu in an alkaline solution. 2+ Reduced to Cu + 2,2'-Biquinoline-4,4'-dicarboxylic acid (BCA) and Cu +The proteins combine to form a purple complex. Within a certain range, the intensity of the color is directly proportional to the protein concentration. A standard curve is constructed using a protein reference solution, and the protein content in the test sample is determined by colorimetry. Preparation of the reference solution: A recombinant collagen raw material standard is selected as the reference, dissolved in water, and prepared into a solution containing 1.0 mg / ml. Preparation of the test sample solution: 1 ml of samples from Examples 1 and 4 is added to 9 ml of water; the original solutions from Examples 3 and 5 are used to prepare a test sample solution with a recombinant collagen content of approximately 0.5 mg / ml. Accurately measure 0.0 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, and 0.5 ml of the reference solution (the volume of the reference solution can be adjusted appropriately within the range of this method), and place them into stoppered test tubes. Add water to each tube to a final volume of 0.5 ml, then add 10.0 ml of copper-BCA reagent to each tube. Mix immediately, incubate at 37°C for 30 minutes, cool, and measure the absorbance at 565 nm using UV-Vis spectrophotometry (General Rule 0401). Simultaneously, use tube 0 as a blank. Calculate the linear regression equation based on the concentration of the reference solution and its corresponding absorbance. Separately, accurately measure an appropriate amount of the test solution and measure it using the same method. Calculate the protein concentration in the test solution from the linear regression equation and multiply by the dilution factor to obtain the final concentration.

[0086] Wavelength scanning revealed an absorption peak at 565 nm, so a standard curve was established at 565 nm.

[0087] Standard curve formula: Conc = -0.1676 + 2.3114 * Abs

[0088] Example 1 Example 3 Example 4 Example 5 0 days 0.4962％ 0.0503％ 0.5061％ 0.0502％ 121℃ 0.4974％ 0.0504％ 0.5011％ 0.0498％ 5 days 0.4981％ 0.0502％ 0.5062％ 0.0500％ 10 days 0.4930％ 0.0502％ 0.5048％ 0.0499％ 5 days 0.4955％ 0.0499％ 0.5062％ 0.0502％ 10 days 0.4967％ 0.0503％ 0.5027％ 0.0501％ change No significant changes No significant changes No significant changes No significant changes

[0089] Purity testing of recombinant collagen raw materials in the product was performed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A stacking gel and separating gel (8%) were prepared. SDS-PAGE is a denaturing polyacrylamide gel electrophoresis method. The principle of this method for protein separation is based on the fact that most proteins can bind to the anionic surfactant sodium dodecyl sulfate (SDS) in a specific weight ratio to form a complex. This causes the negative charge on the protein molecules to far exceed the net charge of the native protein molecules, eliminating the charge effect between different protein molecules and allowing proteins to be separated according to molecular size.

[0090] ① Prepare 10% gel polymerization catalyst (0.5g)

[0091] ② Gel preparation

[0092] Clean the glass plates with distilled water, then degrease them with 75% alcohol. After drying, fix them on the gel casting holder. Select the appropriate concentration of the lower gel according to the sample standard (generally 5 mL is sufficient). Add each component sequentially according to the formula, and mix thoroughly with a pipette. Quickly pour the mixture into the gaps between the installed glass plates (the liquid level should be below the comb slots), leaving approximately 1 cm of space for the stacking gel. Carefully add a small amount of double-distilled water to the gel surface for a water seal, avoiding agitation of the gel surface. At room temperature, the gel should polymerize within 30-50 minutes.

[0093] Prepare the concentrated gel according to the formula (approximately 2 mL per block), blow it evenly with a blower, pour off the water on the bottom layer of gel after it solidifies, and quickly pour in the concentrated gel. Immediately insert a clean comb, being careful not to introduce air bubbles. Inserting the comb into the accumulated gel at a certain angle can reduce the formation of air bubbles.

[0094] ④ Sample preparation

[0095] Prepare the sample solution according to the pharmacopoeia to the corresponding concentration (50 μL is sufficient). Add protein loading buffer (5x) to the sample solution and dilute the sample according to the sample volume: protein loading buffer volume (4:1). Mix well and boil in a metal bath at 100℃ for 5-10 min.

[0096] ⑤ Prepare the protein marker; 10uL is sufficient for each use.

[0097] ⑥ Preparation of SDS-PAGE electrophoresis buffer (Tris-Gly, 10x) (the remaining buffer can be stored at room temperature for 2-3 days, or at 4°C for 1-2 weeks)

[0098] ⑦ Sample loading and gel running

[0099] Turn on the electrophoresis apparatus and set the constant voltage (80V, 30min). Place the glass slide on the electrophoresis tank. Add electrophoresis buffer to the wells, ensuring it is completely submerged, and check for leaks. Remove the comb and add 5μL of protein marker and sample (10μL or adjust according to concentration). Place the electrophoresis tank into the plastic container (note the positions of the black and red connectors). Fill the wells with electrophoresis buffer to the maximum capacity, then fill the container to 2 Gels. Start the electrophoresis apparatus. Set the initial voltage to 80V. Once the sample migrates from the stacking gel to the separating gel (two bands appear on the marker, and the sample is compressed into a single line), change the voltage to 120V. Continue until the sample reaches the bottom of the gel. Turn off the power and disconnect the electrodes. Remove the glass slide from the apparatus. Use a small spatula to peel off the gel and cut off a corner to mark the gel orientation.

[0100] ⑧ Staining, decolorization

[0101] After removing the electrophoresis gel slides, place them in a glass dish with excess Coomassie Brilliant Blue staining solution and shake gently for 2-4 hours. Then, place the stained gel slides in a plastic container, add an appropriate amount of boiling water, and microwave for 15 minutes (medium-high heat). Discard the liquid, add another appropriate amount of boiling water, and microwave for another 10 minutes. Remove and discard the liquid. Next, add destaining solution and allow to destain for 1.5 hours before photographing. The results are as follows. Figure 3 As shown in the table below, lanes 1-6 each represent the following:

[0102]

[0103] In summary: Recombinant type IV collagen alone is difficult to maintain stability in high-concentration preservative systems, but it becomes stable after the addition of chitosan and ectoine. Furthermore, the detection of recombinant collagen in the composition allows for quality control of the product. Content results showed no significant change in the recombinant collagen content during the observation period, indicating that high concentrations of preservatives do not affect the collagen content in the system. Purity results before and after sterilization showed that although there was a slight degradation of recombinant collagen, the main band was still clear, and the purity change was minimal. Conventional methods can be used for product quality control, making the application more convenient.

[0104] Irritation test: The test was conducted in accordance with the cosmetic standard "Eye irritation test: Chicken embryo chorioallantoic membrane test".

[0105] Finish line score Irritant Classification ES≤12 Non-irritating / mildly irritating 12<ES<16 moderate irritation ES≥16 Severe irritation

[0106]

[0107] Results: Eye irritation results showed that the composition itself had no / mild eye irritation; adding the composition to systems with high preservative content, at high, medium, and low proportions, all reduced irritation. Example 4 showed that the composition itself was non-irritating; Comparative Example 6 showed that the system without added collagen showed no / mild irritation, but was close to the limit of moderate irritation; Comparative Example 8 showed that the spray dressing without this composition was moderately irritating, indicating that this composition has a significant ability to reduce the irritation of preservatives and antibacterial agents. Wounded skin is prone to irritation, and further irritation should be avoided as much as possible.

[0108]

[0109]

[0110] In addition to stabilizing recombinant humanized type IV collagen and reducing preservative irritation, the composition of this invention also has the ability to reduce melanin production by melanocytes. The following is a melanin content detection experiment: Experimental method: Cells were seeded in 6-well plates and cultured for 8 hours. Then, α-MSH at a final concentration of 100 μg / mL and samples of different concentrations were added. 100 μg / mL α-arbutin was used as the control group, and no substances were added to the control group. After 24 hours of culture, cells were collected and lysed, with shaking during the process. After centrifugation, the precipitate and supernatant were separated and stored at -20℃. 100 μL of 1M NaOH (containing 10% DMSO) was added to the precipitate, and the mixture was stirred and boiled in boiling water for 10 minutes. After cooling, 90 μL of each solution was taken and the absorbance was measured at 475 nm. The melanin content was calculated using the following formula (Asample represents the absorbance of the sample solution, and Ablank represents the absorbance of the blank control):

[0111] Melanin content = (Sample A / Blank A) x 100%.

[0112] Results: Examples 4-9 show that as the amount of the composition added increases, the melanin content decreases significantly; Comparative Example 9, compared with Example 4, shows that when the chitosan content is below the composition range, the melanin content increases, and Example 5 shows that melanin production can also be inhibited in the product; Comparative Example 10, compared with Example 4, shows that when the chitosan content is above the composition range, the melanin content does not decrease significantly, and Comparative Example 2 shows that the product is unstable and therefore not used; Comparative Examples 11 and 12, compared with Example 4, show that when the ectoine content is below the composition range, the melanin content increases significantly, and when it is above the composition range, ectoine precipitates, so the experiment cannot be conducted; Comparative Example 13, compared with Example 4, shows that recombinant type IV collagen alone has virtually no ability to inhibit melanocytes; Comparative Examples 14-16 show that the absence of any component in the composition has no significant ability to inhibit melanin synthesis.

[0113] In summary: Systems containing only recombinant type IV collagen and those without chitosan and ectoine have limited ability to inhibit melanin production; systems containing all three ingredients can achieve the effect of inhibiting melanin production.

[0114] After cosmetic procedures, superficial wounds appear on the skin. External stimuli, diet, and emotions can all lead to increased melanocyte activity. The common approach is physical sun protection, but it is difficult to track the individual behavior of the patient. Using this composition during the wound healing period can effectively reduce melanin production and result in a better postoperative outcome for cosmetic procedures.

[0115]

[0116]

[0117] The composition has the ability to scavenge free radicals:

[0118] Hydroxyl radicals are generated using the Fenton reaction: H₂O₂ + Fe 2+ =·OH + H₂O + Fe 3+ Salicylic acid was added to the reaction system. The hydroxyl radicals generated in the Fenton reaction reacted with the salicylic acid to form 2,3-dihydroxybenzoic acid, which has a specific absorption at 510 nm. Adding an analyte with hydroxyl radical scavenging capabilities to the reaction system reduced the generation of hydroxyl radicals, thus correspondingly reducing the amount of colored compound formed. The absorbance of the reaction solution containing the analyte was measured at 510 nm using a fixed reaction time method and compared with a blank solution to determine the analyte's hydroxyl radical scavenging effect. Vitamin C was used as a positive control.

[0119] The formula for calculating its scavenging rate is: Hydroxyl radical scavenging rate (%) = A0 - (A x -A x0 ) / A 0* 100

[0120] Conclusion: Examples 4-9 show that the free radical scavenging rate gradually increases with increasing composition concentration. Comparative Examples 9-12 show that a certain free radical scavenging rate is achieved at concentrations below or above the composition's range. However, Comparative Examples 1-4 show that the system is unstable after adding preservatives and are therefore not used. Comparative Examples 13-16 show that no significant free radical scavenging rate is achieved without any component in the composition. Therefore, the composition has the ability to enhance the scavenging of free radicals.

[0121] Free radicals exacerbate inflammatory responses and damage collagen and elastin in the skin. The wound healing period is a time of collagen growth, and free radicals caused by external stimuli such as sun exposure and environmental pollution can prolong wound healing time. Inflammatory stimulation can also cause discomfort such as itching and stinging. Therefore, controlling free radicals during the wound healing period is essential. The composition of this invention has a significant ability to scavenge free radicals, making it suitable for the wound repair period, accelerating wound healing, improving healing quality, and reducing discomfort during the healing process.

[0122]

[0123] Preservative challenge tests were conducted on formulations incorporating the composition, revealing that the composition of the present invention can significantly reduce the amount of preservative required.

[0124] The compositions of Examples 6 and 7 are shown in the table below, and the preparation process is the same as that of Example 1.

[0125] percentage% Chitosan Ikdoin Recombinant type IV collagen Phenoxyethanol water Example 10 0.6 1 0.5 0.5 To100 Example 11 0.6 1 0.5 0.35 To100

[0126] The composition of Comparative Example 9 is shown in the table below, and the preparation process is the same as that of Example 5:

[0127]

[0128]

[0129] Meanwhile, 0.35%, 0.5%, and 1% aqueous solutions of phenoxyethanol were prepared as controls.

[0130] Referring to the Chinese Pharmacopoeia (2020) Part IV, 1121 Method for Testing Antibacterial Efficacy, and ISO 11930 Test Methods and Evaluation Standards,

[0131] Experimental bacterial strains: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, all of which were fourth-generation cultures;

[0132] Fungi: Aspergillus niger and Candida albicans, both of which are third-generation cultures.

[0133] Experimental steps:

[0134] ① Prepare bacterial suspensions by inoculating the cryopreserved strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa into tryptic soy peptone liquid medium and diluting to 10⁻⁶. 8 Prepare a large-scale bacterial suspension; inoculate the above-mentioned cryopreserved Candida albicans strain into Sabouraud dextrose liquid medium and dilute to 10⁻⁶. 6 Prepare a large-scale bacterial suspension; inoculate the above-mentioned cryopreserved Aspergillus niger strain onto Sabouraud dextrose agar slants and dilute to 10⁻⁶. 6 Prepare orders of magnitude bacterial suspensions for later use.

[0135] ② Sample preparation: Weigh 3 portions of each sample, and weigh 120g of each portion into a sterile conical flask for later use.

[0136] ③ Sample inoculation: Take the three weighed samples above, and inoculate them with 1.2 ml of bacterial mixed suspension, 1.2 ml of Candida albicans suspension and 1.2 ml of Aspergillus niger spore suspension respectively. Mark them, mix them evenly, and store the samples at room temperature.

[0137] ④ Sample testing: The prepared samples were taken on days 0, 2, 7, 14 and 28, and the number of colonies in the samples was recorded to determine the antibacterial efficacy of the product.

[0138] Evaluation criteria:

[0139]

[0140] The results are shown in the table below:

[0141]

[0142]

[0143] The anti-corrosion challenge test showed that 1% of the preservative in the aqueous solution was required to pass the test; in Example 6 and Comparative Example 9, with the same amount of phenoxyethanol added, Example 6 passed the test. After adding the composition of the present invention, the anti-corrosion test (Examples 6 and 7) was passed with a low amount of preservative added.

[0144] In summary, the composition, containing 0.2-1.25% chitosan, 0.2-2% ectoine, and 0.001-0.1% recombinant type IV collagen, can reduce the irritation of the formulation itself and improve user experience in a phenoxyethanol addition system of 1.0%. The composition also ensures the stable existence of recombinant type IV collagen in preservative systems, expanding its application scenarios. Furthermore, the composition itself can reduce the ability of melanocytes to synthesize melanin, has free radical scavenging capabilities, and can reduce the amount of preservative required. These multiple effects work together to accelerate wound healing and improve post-treatment quality.

[0145] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A recombinant humanized collagen composition, characterized in that, The recombinant humanized collagen composition is made of components with the following mass fractions: 0.001-0.5% of recombinant humanized type IV collagen, 0.2-1.25% of chitosan, 0.2-2% of ectoine, 0.01-1% of preservative, and the balance of water, wherein the amino acid sequence of the recombinant humanized type IV collagen is shown in SEQ ID NO.

1.

2. The recombinant humanized collagen composition of claim 1, wherein, The preservative is phenoxyethanol.

3. The recombinant humanized collagen composition of claim 1, wherein, The recombinant humanized collagen composition further comprises other auxiliary materials allowed to be added in the cosmetic field.

4. The method of claim 1-3, wherein the recombinant humanized collagen composition is prepared by the steps of: a) obtaining a humanized collagen protein; b) obtaining a recombinant humanized collagen protein; c) mixing the humanized collagen protein and the recombinant humanized collagen protein; and d) purifying the collagen protein. The preparation method is as follows: S1, dissolving chitosan in an acidic solution and heating and stirring until completely dissolved; S2, adding ectoine to the solution prepared in step S1 and continuing to heat and stir until completely dissolved, and the solution is a uniform transparent liquid after swelling; S3, adding the recombinant humanized type IV collagen to water for pre-dispersion, and after the solution in step S2 is cooled, adding the collagen dispersion liquid to the solution and stirring until uniformly dispersed; Further, adjusting the pH of the solution system prepared in step S3 to weakly acidic to neutral, including pH 6.0-7.

0.

5. The production method according to claim 4, wherein In the step S1, the pH of the acidic solution is 3.0-5.0, and the heating and stirring temperature condition is 40-60℃; In the step S2, the temperature condition for continuing to heat and stir is 40-60℃; In the step S3, the temperature is cooled to room temperature.

6. Use of the above recombinant humanized collagen composition according to any one of claims 1-3 in the preparation of a pharmaceutical or cosmetic product, wherein the pharmaceutical or cosmetic product has the effects of inhibiting melanocytes from producing melanin and scavenging free radicals.

7. A cosmetic product, characterized by The cosmetic product is made of the recombinant humanized collagen composition according to any one of claims 1-3; The cosmetic product is a skin care product; The cosmetic product dosage form is any one or more of an aqueous preparation, a paste, a cream, an essence, a jelly, a mask, a spray, or a foundation.

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