Collagen peptide, composition and use thereof

Abstract

The present invention belongs to the field of biomedicine. Disclosed are a collagen peptide, a composition and the use thereof. Specifically disclosed are a type 21 recombinant humanized collagen peptide (ColpepA1 21) and a composition thereof, and the use of a collagen material in anti-aging treatments, skin care and wound repair. The recombinant collagen of the present invention can effectively promote the repair process after skin injury, enhance the hydration state and barrier function of skin, reduce fine lines and wrinkles, improve the firmness and elasticity of skin, thereby significantly promoting key biological reactions in the wound healing process. Further disclosed in the present invention is a composite collagen material, which can effectively promote wound healing and cell regeneration, and alleviate oxidative damage.

Description

A collagen peptide, a composition and its application

[0001] Cross-reference to related applications

[0002] This invention claims priority to Chinese Patent Applications Nos. 202411462453.6 and 202411462441.3, filed on October 18, 2024, the full text of which is incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of biomedicine, specifically relating to a collagen peptide, a composition, and its application. Background Technology

[0004] Collagen is not only one of the most abundant proteins in the human body, accounting for more than 30% of total protein, but it is also a cornerstone for maintaining the structure and function of various tissues. It exists in the skin, bones, tendons, blood vessels, and other connective tissues, forming a three-dimensional network structure that provides mechanical strength and stability. In the skin, collagen is responsible for maintaining firmness and elasticity, keeping the skin surface smooth and radiant. With age, the body's natural collagen production slows down, and existing collagen gradually degrades and is damaged by factors such as ultraviolet radiation, environmental pollution, and unhealthy lifestyle habits. This leads to the skin gradually losing its original elasticity and firmness, resulting in signs of aging such as sagging, fine lines, and wrinkles. Furthermore, the reduction of collagen in other tissues also affects bone density, tendon elasticity, and vascular stability, thus impacting overall health. Modern scientific research is actively seeking methods to slow down the degradation process of collagen and increase its production rate in the body, aiming to mitigate the effects of aging. Currently, various collagen supplements, skin care products, and medical aesthetic procedures on the market attempt to restore the youthful state of skin and other tissues by externally supplementing or stimulating the natural production of collagen. However, how to effectively improve the absorption and utilization of collagen in the body, and how to precisely target the specific types of collagen needed during the aging process, remain key research areas and challenges. In the field of wound repair, traditional treatment methods face challenges such as accelerating wound healing, reducing scar formation, and improving repair quality. Although various products exist on the market to promote wound healing, such as collagen-based dressings and growth factors, how to effectively promote wound healing, improve treatment outcomes, and alleviate patient suffering remains an important research direction.

[0005] Against this backdrop, in-depth research on specific types of collagen, such as collagen type 21 (COL21A1), is particularly important. Collagen type 21 is a non-fibrous collagen, relatively rare in the adult human body, accounting for less than 1% of total collagen. Compared to other more well-known collagen types, research on collagen type 21 is relatively limited. Belonging to the small molecule collagen family and as part of the FACIT (fibrous-associated collagen with a disrupted triple helix) family, collagen type 21 plays a crucial role in connecting extracellular matrix components. Collagen type 21 is primarily expressed in vascular smooth muscle cells and may play a role in extracellular matrix assembly during angiogenesis. Furthermore, the gene expression of collagen type 21 is regulated by developmental stages, with higher expression levels in the fetal stage, suggesting it may play an important role in the development of various tissues. Recent studies on collagen type 21 have revealed its potential functions in vascular health and blood pressure regulation. These properties give type 21 collagen unique application potential in the fields of skin health, anti-aging, and vascular health.

[0006] Currently, the market offers a variety of anti-aging and skin-whitening products, most of which focus on replenishing lost collagen in the skin using ordinary collagen or its derivatives. However, the effectiveness of these products is often limited by the size of collagen molecules and skin absorption rates, and it is difficult to optimize for the specific biological functions of collagen. Based on its unique biological characteristics and mechanisms of action, type 21 collagen may become one of the important directions for future anti-aging and skin-whitening research and applications. By understanding its specific biological functions and mechanisms of action, it is possible to develop targeted, highly bioavailable, and multi-functional anti-aging and skin-whitening therapies and products, which not only improve skin appearance but also promote overall health and slow down the aging process.

[0007] While research has explored the effects of type 21 collagen on vascular health and blood pressure regulation, its application in promoting wound healing remains in the exploratory stage. Developing novel composite materials based on type 21 collagen could not only provide more effective treatment options for wound repair but also potentially improve patients' recovery quality and quality of life by promoting high-quality tissue regeneration. Therefore, this invention aims to develop a novel composite collagen raw material and its preparation method. By deeply studying the biological characteristics of type 21 collagen and its mechanism of action in wound repair, this invention provides a more effective, safe, and biocompatible treatment option for wound healing. Furthermore, this invention explores the synergistic effects of type 21 collagen with other bioactive molecules to achieve precise regulation of the wound healing process, thereby accelerating wound healing, reducing scar formation, and improving the quality of repaired tissue. Summary of the Invention

[0008] This invention relates to a type 21 recombinant humanized collagen (ColpepA1 21) having a specific amino acid sequence, a composition thereof, and its application in the skin care field, particularly in facial anti-aging, whitening and brightening, and wound repair.

[0009] This invention provides a type 21 recombinant humanized collagen, wherein the amino acid sequence of the collagen is selected from SEQ ID No. 1:

[0010] In one aspect, the present invention also provides a type 21 recombinant humanized collagen, wherein the amino acid sequence of the collagen comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID No. 1 above.

[0011] This specific amino acid sequence of collagen is optimized to improve its bioavailability and efficacy in the skin.

[0012] In one embodiment, the collagen of the present invention can enhance skin moisture regulation and optimize barrier function.

[0013] In one embodiment, the collagen described in this invention can provide key support for anti-aging and skin rejuvenation.

[0014] In one implementation, the key support for anti-aging and skin rejuvenation includes reducing fine lines and wrinkles and / or increasing skin firmness.

[0015] In one embodiment, the collagen of the present invention can whiten skin and / or provide support for skin whitening and brightening.

[0016] In one embodiment, the support for skin whitening and brightening includes inhibiting tyrosinase activity.

[0017] In one embodiment, the support for skin whitening and brightening includes inhibiting melanin levels.

[0018] In one embodiment, the collagen of the present invention can promote the production of type I collagen.

[0019] In one embodiment, the collagen of the present invention can significantly increase the content of type I collagen.

[0020] In one embodiment, the promotion of type I collagen production and / or significant increase in type I collagen content can enhance skin structural strength and elasticity, particularly showing significant benefits in improving skin firmness and / or reducing signs of aging.

[0021] In one embodiment, the collagen of the present invention has the activity of promoting tissue repair and regeneration, especially accelerating the healing of damage and cell remodeling processes after skin injury.

[0022] In one embodiment, the collagen of the present invention has cell migration-promoting activity, thereby helping to shorten wound healing time and improve the skin's self-recovery ability.

[0023] In one embodiment, the collagen of the present invention can promote the expression of aquaporin AQP3, thereby improving the skin's water retention capacity and reducing water evaporation rate, maintaining a suitable hydration state of the wound and the surrounding skin, and promoting the wound repair process.

[0024] In one embodiment, the collagen of the present invention can enhance the stability and elasticity of skin structure, thereby playing a key role in the repair and regeneration of damaged skin.

[0025] In one embodiment, the collagen of the present invention can promote the formation and maintenance of the skin barrier, thereby playing a key role in the repair and regeneration of damaged skin.

[0026] In one embodiment, the collagen of the present invention can enhance the expression of FLG.

[0027] In one embodiment, the collagen of the present invention can enhance the expression of LOR.

[0028] In one embodiment, the collagen of the present invention can enhance the activity of TGM1.

[0029] In one embodiment, the collagen of the present invention can be used in combination with moisturizers such as hyaluronic acid and glycerin.

[0030] In one embodiment, the collagen of the present invention can be combined with antioxidants such as vitamin E and green tea extract to enhance the skin's ability to resist environmental oxidative damage.

[0031] In one embodiment, the collagen of the present invention may also be supplemented with stabilizers such as vitamin B3 to maintain the stability and activity of the collagen.

[0032] In one embodiment, the collagen of the present invention can also be used in combination with an emulsifier to stabilize the emulsion system of the product and ensure the uniform distribution of the active ingredients.

[0033] In one embodiment, the collagen of the present invention is used in conjunction with a penetration enhancer, such as liposomes or nanoparticles, to enhance the permeability of the collagen in the skin.

[0034] In one embodiment, the collagen of the present invention can be used in combination with excipients and fillers to improve the texture and appearance of the article.

[0035] In one embodiment, the moisturizing ingredients may include hyaluronic acid, glycerin, natural moisturizing factor (NMF), allantoin, polyols (such as butylene glycol, glycol, etc.), and plant extracts (such as aloe vera extract, green tea extract, etc.); the antioxidant ingredients may include vitamin C and its derivatives, vitamin E and its derivatives, conjugated linoleic acid, white tea extract, green tea extract, resveratrol, glutathione, etc.; the whitening ingredients may include arbutin, kojic acid, vitamin B3 (niacinamide), glycyrrhizic acid, fruit acids (such as citric acid, lactic acid, etc.), and other herbal extracts.

[0036] In another aspect, the invention also provides a nucleic acid molecule comprising a fragment encoding any of the aforementioned collagen proteins.

[0037] In another aspect, the present invention also provides a carrier containing the aforementioned nucleic acid molecules.

[0038] In another aspect, the present invention also provides a host cell containing the aforementioned nucleic acid molecule or vector.

[0039] In another aspect, the present invention also provides a method for preparing the aforementioned collagen, the method comprising the step of preparing the collagen using a Pichia pastoris expression system.

[0040] In one embodiment, the method includes one or more steps selected from: introducing the aforementioned vector into Pichia pastoris to form Pichia pastoris genetically engineered strains, fermenting and culturing the Pichia pastoris genetically engineered strains, and inducing and expressing the collagen.

[0041] In one embodiment, the carrier is pPIC9K or pGAPZα.

[0042] Preferably, the carrier is pPIC9K.

[0043] In one embodiment, the Pichia pastoris is Pichia pastoris GS115, X-33, KM71, etc.

[0044] Preferably, the Pichia pastoris is Pichia pastoris GS115.

[0045] In one embodiment, the introduction of the aforementioned vector into Pichia pastoris to form Pichia pastoris genetically engineered strains is achieved through electroporation, chemical transformation, or ultrasound-assisted transfection.

[0046] Preferably, the method used in the step is electroporation.

[0047] In one embodiment, the culture medium for fermentation is BMGY medium.

[0048] In one embodiment, the culture medium for induction and expression is BMMY medium.

[0049] The collagen synthesis process described in this invention covers the entire process from gene construction, cell culture to protein expression, ensuring efficient and stable protein production.

[0050] In one embodiment, the method for preparing collagen according to the present invention further includes a purification step. The purification step successfully extracts and purifies the collagen from the Pichia pastoris expression system.

[0051] In one embodiment, the purification step includes treating the fermentation broth supernatant with NaCl and adjusting the pH.

[0052] In one embodiment, the purification step includes a step of specifically purifying type 21 collagen using nickel affinity chromatography; preferably, the type 21 collagen is tagged with His.

[0053] In one embodiment, the purification step includes eluting the collagen with imidazole to obtain purified collagen.

[0054] The purification steps described above in this invention not only ensure the high purity of the target protein but also maintain its biological activity, providing high-quality protein samples for further application research and product development. The entire purification process is designed to balance efficiency and protein stability, forming an important foundation for applying recombinant proteins in the fields of anti-aging and skin care.

[0055] The present invention provides a method for preparing a collagen raw material for wound repair, wherein the method comprises the following steps: (1) mixing collagen with auxiliary components; (2) coagulating the collagen material; and (3) sterilizing the collagen material.

[0056] In one embodiment, the mixing described in this invention is carried out at room temperature or under heating conditions.

[0057] In one embodiment, the mixing described in this invention refers to being carried out at room temperature of 20-25°C; preferably, the mixing is carried out at any one or more temperatures selected from 20°C, 21°C, 22°C, 23°C, 24°C, and 25°C; preferably, the mixing is carried out at 25°C.

[0058] In one embodiment, the solidification molding of the present invention is carried out at room temperature or low temperature.

[0059] In one embodiment, the solidification molding of the present invention refers to being carried out at a low temperature of 2-15℃; preferably, the solidification molding is carried out at any one or more temperatures selected from 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, 11℃, 12℃, 13℃, 14℃, and 15℃.

[0060] In one embodiment, the solidification molding of the present invention refers to being carried out at room temperature of 20-25°C; preferably, the solidification molding is carried out at any one or more temperatures selected from 20°C, 21°C, 22°C, 23°C, 24°C, and 25°C; preferably, the solidification molding is carried out at 25°C.

[0061] In one embodiment, the sterilization method of the present invention includes any one of heat sterilization, chemical sterilization or radiation sterilization.

[0062] Preferably, the sterilization is gamma ray sterilization or plasma sterilization.

[0063] In one embodiment, the method steps of the present invention are as follows: cross-linking reaction of a mixture of collagen and auxiliary components in sodium acetate buffer at room temperature; transferring the mixture into a mold, allowing it to stand, and refrigerating it for 12-24 hours to form; low-temperature curing and plasma sterilization.

[0064] In one embodiment, the method steps of the present invention are as follows: pre-soaking collagen and auxiliary components in water and heating to dissolve; adding hyaluronic acid solution, stirring and coagulating at room temperature; low-temperature curing, and plasma sterilization.

[0065] In one embodiment, the method steps of the present invention are as follows: mixing collagen and auxiliary components, spin-coating onto a substrate; drying and peeling, and sterilizing with gamma rays.

[0066] In one embodiment, the method steps of the present invention are as follows: mixing collagen and auxiliary components, pouring into a mold, freezing and then freeze-drying; gas cross-linking, and gamma ray sterilization.

[0067] In one embodiment, the auxiliary ingredients of the present invention are selected from one or more of the following: antibacterial agents, anti-inflammatory agents, growth factors, moisturizers, antioxidants, emulsifiers, thickeners, etc.

[0068] In one embodiment, the concentration of collagen is 0.0001% to 50% based on the total weight of the collagen material.

[0069] Preferably, the concentration of the collagen is 1% to 20%.

[0070] In one embodiment, the weight ratio of collagen to auxiliary ingredients in this invention is selected from:

[0071] Collagen to antibacterial agent: 5:1 to 20:1; or

[0072] Collagen to anti-inflammatory agent: 20:1 to 100:1; or

[0073] Collagen to growth factor ratio: 50:1 to 200:1; or

[0074] Collagen to moisturizer ratio: 1:1 to 10:1; or

[0075] Collagen to antioxidant ratio: 10:1 to 50:1; or

[0076] Collagen and emulsifier or thickener: one or more combinations of 5:1 to 20:1.

[0077] In one embodiment, the method steps of the present invention are as follows: a mixture of type 21 collagen, hyaluronic acid and crosslinking agent is subjected to a crosslinking reaction in sodium acetate buffer at room temperature; the mixture is transferred to a mold, left to stand, and refrigerated for 12-24 hours to form; low-temperature curing and plasma sterilization are performed.

[0078] Preferably, the ratio is: 2-3% (w / v) type 21 collagen, 1% (w / v) hyaluronic acid, and 0.1% (w / v) crosslinking agent; more preferably, the proportion of type 21 collagen is 3% (w / v).

[0079] Preferably, the crosslinking agent is EDC.

[0080] In one embodiment, the method steps of the present invention are as follows: pre-soaking and heating a mixture of gelatin and collagen in water to dissolve it; adding hyaluronic acid solution, stirring and solidifying at room temperature; low-temperature curing, and plasma sterilization.

[0081] Preferably, the ratio is: 2% (w / v) type 21 collagen solution, 1% (w / v) hyaluronic acid, and 3% (w / v) gelatin.

[0082] In one embodiment, the method steps of the present invention are as follows: mixing type 21 collagen and PLGA in a certain ratio, spin-coating the mixture onto a substrate; drying and peeling, and sterilizing with gamma rays.

[0083] Preferably, the ratio is: 1.5% (w / v) type 21 collagen solution and 2% (w / v) PLGA.

[0084] In one embodiment, the method steps of the present invention are as follows: mixing type 21 collagen and chitosan in a certain ratio, pouring into a mold, freezing and then freeze-drying; gas cross-linking, and gamma ray sterilization.

[0085] Preferably, the ratio is: 2.5% (w / v) type 21 collagen solution and 1.5% (w / v) chitosan.

[0086] In another aspect, the present invention provides a novel composite collagen material prepared according to the above method, wherein the collagen material is used for wound repair after skin injury.

[0087] Another aspect of the present invention provides a novel composite collagen material, wherein the novel composite collagen material comprises type 21 recombinant humanized collagen, the amino acid sequence of which is selected from SEQ ID No. 1, and further comprises one or more auxiliary components selected from the following: antibacterial agents, anti-inflammatory agents, growth factors, moisturizers, antioxidants, emulsifiers, thickeners, etc.

[0088] In one embodiment, the novel composite adhesive raw material of the present invention has a collagen concentration of 0.0001% to 50% based on the total weight of the adhesive raw material.

[0089] Preferably, the concentration of the collagen is 1% to 20%.

[0090] In one embodiment, the novel composite adhesive raw material of the present invention is designed for wound repair, using collagen as an active ingredient to accelerate the wound healing process, reduce scar formation, and improve treatment efficacy.

[0091] In one embodiment, the collagen raw material of the present invention, wherein the weight ratio of the collagen to the auxiliary components is selected from one or more combinations thereof:

[0092] In one embodiment, the ratio of collagen to antibacterial agent is selected from 5:1 to 20:1; an appropriate concentration of antibacterial agent can effectively inhibit bacterial growth, but too high a concentration may cause skin irritation.

[0093] In one embodiment, the ratio of collagen to anti-inflammatory agent is selected from 20:1 to 100:1; the use of anti-inflammatory agent should be carefully controlled to avoid causing local or systemic side effects.

[0094] In one embodiment, the ratio of collagen to growth factor is selected from 50:1 to 200:1; the growth factor has high activity and can exert its effect with a small dose, while an excessive dose may accelerate tumor cell growth or other adverse reactions.

[0095] In one embodiment, the ratio of collagen to moisturizer is selected from 1:1 to 10:1; a higher amount of moisturizer can enhance the skin's moisturizing properties, but it should not be excessive to avoid affecting other functions of the formulation.

[0096] In one embodiment, the ratio of collagen to antioxidant is selected from 10:1 to 50:1; antioxidants can prevent the formulation from oxidizing, but their dosage should be controlled to avoid affecting the long-term stability of the product.

[0097] In one embodiment, the ratio of collagen to emulsifier or thickener is selected from 5:1 to 20:1; this helps improve the texture and stability of the product, but should be used in moderation to ensure the suitability and comfort of the final product.

[0098] In one embodiment, the novel composite collagen material of the present invention is suitable for wound repair after various types of skin injuries.

[0099] In one embodiment, the skin injury includes, but is not limited to, surgical incisions, burns, abrasions, or chronic wounds.

[0100] Another aspect of the present invention provides a method for preparing the above-mentioned collagen material.

[0101] The raw materials described in this invention can effectively accelerate the healing process and reduce scar formation.

[0102] In one embodiment, the novel composite collagen material of the present invention is in the form of, but is not limited to, solid, liquid, semi-solid, or gel formulations.

[0103] In one embodiment, the novel composite collagen material of the present invention includes, but is not limited to, topical gels, creams, dressings, sprays, oral / oral medications, or injections.

[0104] In one embodiment, the novel composite collagen material of the present invention is a collagen-hyaluronic acid hydrogel dressing, which is prepared by a chemical cross-linking method:

[0105] Material composition: Type 21 collagen: 2-3% (w / v), hyaluronic acid: 1% (w / v), EDC: 0.1% (w / v) (crosslinking agent);

[0106] Preparation process:

[0107] The mixture was cross-linked in sodium acetate buffer at room temperature.

[0108] Transfer the mixture into a mold, let it stand, and then refrigerate for 12-24 hours until it is completely gelled.

[0109] Low-temperature plasma sterilization and aseptic packaging.

[0110] In one embodiment, the novel composite collagen material of the present invention is a collagen-hyaluronic acid hydrogel dressing, which is prepared using gelatin as a crosslinking agent:

[0111] Material composition: Type 21 collagen solution: 2% (w / v), hyaluronic acid: 1% (w / v), gelatin: 3% (w / v);

[0112] Preparation process:

[0113] Gelatin and collagen are pre-soaked in water and then heated to dissolve;

[0114] Add hyaluronic acid solution, stir and solidify at room temperature;

[0115] Low-temperature curing and plasma sterilization.

[0116] In one embodiment, the novel composite collagen material of the present invention is a thin-film dressing, prepared by the following method:

[0117] Material composition: Type 21 collagen solution: 1.5% (w / v), PLGA: 2% (w / v);

[0118] Preparation process:

[0119] Mix collagen and PLGA, and spin-coat the substrate.

[0120] Drying and peeling, followed by gamma ray sterilization.

[0121] In one embodiment, the novel composite collagen material of the present invention is a sponge or fiber dressing, prepared by the following method:

[0122] Material composition: Type 21 collagen solution: 2.5% (w / v), chitosan: 1.5% (w / v);

[0123] Preparation process:

[0124] Mix collagen and chitosan, pour into a mold, freeze, and then freeze-dry.

[0125] Gas cross-linking, gamma ray sterilization.

[0126] In one embodiment, the novel composite collagen material of the present invention promotes rapid and high-quality wound repair, optimizes the healing process, reduces inflammatory response, accelerates skin regeneration, reduces scar formation, and improves treatment efficacy by providing ColpepA1 21.

[0127] In one aspect, the present invention provides a composition in which the active ingredient comprises the above-mentioned collagen; preferably, the collagen is type 21 recombinant humanized collagen.

[0128] In one embodiment, the composition comprises one or more auxiliary ingredients or excipients.

[0129] In a preferred embodiment, the auxiliary ingredient is selected from one or more of moisturizers, antioxidants, UV absorbers, penetration enhancers, and plant extracts.

[0130] In a preferred embodiment, the excipient is selected from one or more of stabilizers, emulsifiers, conditioning agents, diluents, fillers, binders, humectants, absorption promoters, surfactants, lubricants, flavorings, or seasonings.

[0131] In one embodiment, the compositions of the present invention include, but are not limited to, medical compositions, such as one or more of pharmaceutical compositions, food compositions, health care compositions, and dietary supplements. Specifically, the compositions of the present invention are in the form of one or more of tablets, capsules, powders, microparticles, solutions, lozenges, gels, creams, liniments, suspensions, tinctures, poultices, liniments, lotions, and aerosols, and can be prepared using commonly known preparation techniques. Appropriate pharmaceutical excipients, such as stabilizers, antioxidants, and preservatives, can be added to maintain the stability and efficacy of the product.

[0132] In one embodiment, the auxiliary ingredients or excipients are used to achieve skin moisturizing, anti-oxidation, or whitening effects.

[0133] In one embodiment, the auxiliary ingredient is a moisturizer.

[0134] The moisturizer described in this invention can increase the skin's moisture content, thereby improving the skin's softness and elasticity, and enhancing its effectiveness in reducing wrinkles and anti-aging.

[0135] In one embodiment, the moisturizer is selected from one or more of hyaluronic acid, glycerin, panthenol (vitamin B5), betaine, polysorbate, natural moisturizing factor (NMF), allantoin, and polyols (such as butylene glycol, glycol, etc.).

[0136] In one embodiment, the auxiliary ingredient is an antioxidant.

[0137] The antioxidants described in this invention can enhance the skin's ability to resist environmental oxidative damage, further improving their efficacy in anti-aging skincare.

[0138] In one embodiment, the antioxidant is selected from one or more of vitamin C and its derivatives, vitamin E and its derivatives, green tea extract, conjugated linoleic acid, coenzyme Q10, resveratrol, glutathione, etc.

[0139] In one embodiment, the auxiliary ingredient is a whitening component.

[0140] In one embodiment, the whitening component is selected from one or more of arbutin, nicotinamide kojic acid, tranexamic acid, glycyrrhizic acid, and fruit acids.

[0141] In one embodiment, the auxiliary component is an emulsifier.

[0142] The emulsifier described in this invention stabilizes the emulsion system of skincare products, ensuring the uniform distribution of active ingredients.

[0143] In one embodiment, the emulsifier is selected from one or more of polysorbate-60, glyceryl stearate, etc.

[0144] In one embodiment, the auxiliary ingredient is a plant extract.

[0145] The plant extracts described in this invention can provide skin soothing and additional skin care benefits.

[0146] In one embodiment, the plant extract is selected from one or more of aloe vera extract, white tea extract, green tea extract, cucumber extract, witch hazel extract, etc.

[0147] In one embodiment, the auxiliary component is an ultraviolet absorber.

[0148] The ultraviolet absorber described in this invention can provide sun protection and reduce the damage of ultraviolet rays to the skin.

[0149] In one embodiment, the ultraviolet absorber is selected from one or more of titanium dioxide, zinc oxide, ethylhexyl methoxycinnamate, etc.

[0150] In one embodiment, the auxiliary component is a penetration enhancer.

[0151] The penetration enhancer described in this invention can enhance the permeability of collagen in the skin.

[0152] In one embodiment, the penetration enhancer is selected from one or more of liposomes, nanoparticles, and transdermal enhancers such as dimethyl sulfoxide.

[0153] In one embodiment, the collagen composition of the present invention may also contain a stabilizer such as vitamin B3 to maintain the stability and activity of the collagen and ensure a long-lasting anti-aging effect.

[0154] In one embodiment, the composition of the present invention may be used in combination with excipients and / or fillers; preferably silica (dimethyl silicone oil), rice bran wax, etc. The use of such excipients and / or fillers can improve the texture and appearance of the article, providing a smooth feel and excellent spreadability.

[0155] In one embodiment, the collagen composition of the present invention may include flavorings and seasonings; preferably natural essential oils, synthetic flavorings, etc. The addition of flavorings and seasonings can enhance the sensory experience for the user.

[0156] The collagen composition of this invention, based on traditional efficacy, enhances its effects on skin moisturizing, elasticity improvement, fine line reduction, and anti-oxidation by combining with a variety of auxiliary ingredients, and can also provide a more comprehensive and personalized skin care solution.

[0157] In another aspect, the present invention provides a medical article comprising the aforementioned collagen, the aforementioned collagen material for wound repair, or the aforementioned composition.

[0158] In one embodiment, the medical product is a solid, liquid, semi-solid, or gel formulation.

[0159] In one embodiment, the formulation of the medical product is selected from one or more of the following: serum, mask, lotion, skin enhancer, topical gel, cream, dressing, spray, oral medication, or injection.

[0160] In one embodiment, the cream is a face cream.

[0161] In a preferred embodiment, the skin enhancer is an injectable skin enhancer.

[0162] In one embodiment, the formulation of the composition of the present invention is selected from one or more of topical gels, creams, dressings, sprays, oral medications, or injections.

[0163] In a preferred embodiment, the medical product is in the form of a dressing, including but not limited to hydrogel dressings, film dressings, sponge or fiber dressings, etc.

[0164] In a preferred embodiment, the medical product is in the form of a hydrogel dressing, more preferably a collagen-hyaluronic acid hydrogel dressing.

[0165] In one embodiment, the medical product may be selected from one or more forms such as face cream, serum, mask, gel, lotion, or skin booster, for improving skin elasticity, reducing fine lines, and increasing skin firmness and radiance.

[0166] In one embodiment, the injectable skin enhancer of the present invention comprises other active ingredients and auxiliary ingredients, including but not limited to: hyaluronic acid, vitamins, antioxidants, other peptides, amino acids or stabilizers, etc.

[0167] In one embodiment, the injectable skin enhancer of the present invention is administered via various minimally invasive injection devices and methods, including hyaluronic acid injection devices, microneedles, microneedle rollers, automated microneedle pens (such as Dermapen), needle-free injection systems, electric microneedle systems (such as SkinPen), ultrasonic injection devices, laser-assisted delivery systems, and pneumatic injection devices (such as JetPeel). These devices can precisely deliver the skin enhancer directly to the middle or deep layers of the skin to improve overall skin quality and appearance. Through these different administration methods, the most suitable technique can be selected to achieve the best therapeutic effect based on the treatment goals and the specific needs of the patient.

[0168] In one embodiment, the injectable skin enhancer of the present invention has been specially formulated with different component ratios for different application methods to meet various treatment needs.

[0169] In one embodiment, for applications such as microneedling and microneedle rollers, the injectable skin enhancer of this invention contains a relatively high concentration of collagen (5%–10%). Microneedles physically create tiny channels in the skin, and the high concentration of collagen helps to directly act on the newly formed microchannel areas, promoting rapid skin repair and collagen regeneration.

[0170] In one embodiment, for a hyaluronic acid injection system, the injectable skin enhancer of the present invention contains a high concentration of antioxidants, such as vitamins C and E, which help protect the skin from free radical damage and enhance the skin's overall resistance. Simultaneously, the use of small-molecule hyaluronic acid allows for easier penetration into the deeper layers of the skin, providing better hydration and intercellular signaling, thereby effectively improving skin hydration and firmness.

[0171] In one embodiment, the medical product of the present invention may also contain pharmaceutically, food-grade, health supplement-grade, or dietaryally acceptable carriers. For example, various compatible solid or liquid fillers, gelling agents, or solvents may be used. These carriers should be suitable for human use, possessing sufficient purity and low toxicity. Commonly used carriers include cellulose and its derivatives, polyols such as glycerol, and other pharmaceutical excipients such as talc and magnesium stearate.

[0172] Another aspect of the present invention provides the use of the aforementioned type 21 recombinant humanized collagen, the aforementioned nucleic acid molecule, the aforementioned carrier, or the aforementioned host cell in the preparation of medical compositions.

[0173] Another aspect of the present invention provides the use of any of the foregoing compositions in the preparation of medical articles.

[0174] In one embodiment, the aforementioned medical composition and / or medical product is used for anti-aging.

[0175] In one embodiment, the aforementioned medical composition and / or medical product is used for whitening and brightening.

[0176] In one embodiment, the aforementioned medical composition and / or medical product is used for wound repair.

[0177] Another aspect of the present invention provides the application of the aforementioned collagen, the aforementioned nucleic acid molecule, the aforementioned carrier, the aforementioned host cell, or any of the aforementioned collagen materials in the preparation of a medical composition for wound repair; preferably, the medical composition comprises the aforementioned collagen material or prepared by the aforementioned methods.

[0178] The collagen and its composition of the present invention, or the novel composite adhesive raw material prepared thereof, have the following characteristics:

[0179] 1. Rarity and Unique Functions: Type 21 collagen is relatively rare in the adult human body, and research on it is relatively limited. Furthermore, research indicates its potential applications in skin and vascular health are unique. Developing wound repair materials using this rare collagen type demonstrates the innovation of exploring novel biomaterials.

[0180] 2. Promotes natural skin healing and HaCaT cell migration: Type 21 collagen accelerates the skin's natural healing process by promoting HaCaT cell migration, which is crucial for wound repair. Accelerating new skin formation and healing based on HaCaT cell migration is a key aspect of this innovative application.

[0181] 3. Enhanced AQP3 Expression: AQP3 is an important aquaporin, crucial for skin hydration and cell migration. Type 21 collagen can enhance AQP3 expression, which not only helps retain wound moisture but may also promote more efficient cell migration and wound healing.

[0182] Advantages of this invention

[0183] (I) Anti-aging and whitening / brightening aspects

[0184] The recombinant humanized collagen type 21 (ColpepA1 21) described in this invention exhibits significant advantages in skin anti-aging and brightening. While providing basic collagen supplementation, ColpepA1 21's unique efficacy and optimized molecular properties set it apart from numerous collagen products.

[0185] 1. ColpepA1 21 has demonstrated its importance in enhancing skin structural integrity, particularly in promoting the production of type I and type III collagen. This not only benefits the skin's tensile and compressive strength but also effectively combats photoaging caused by UV radiation, maintaining a youthful appearance. In particular, ColpepA1 21's role in promoting the production of type III collagen, a crucial source of skin elasticity, supports increased skin elasticity and reduces signs of aging.

[0186] 2. The molecular size and structure of ColpepA1 21 have been optimized to improve its penetration and absorption in the skin, which means that its active ingredients can work more deeply into the skin to provide cells with the necessary nutrients, thereby supporting skin health and anti-aging at the molecular level.

[0187] 3. Cell experiments have demonstrated the effects of ColpepA1 21 on skin whitening and brightening. It can effectively inhibit tyrosinase and melanin. This mechanism will help reduce skin pigmentation and thus achieve a whitening effect, providing a basis for its application in skin whitening and brightening products.

[0188] 4. Clinical trial results further demonstrate the multiple anti-aging effects of ColpepA1 21, showing significant improvements in reducing wrinkles and age spots, enhancing skin elasticity and firmness, and improving skin radiance and tone. These improvements in the volunteers' skin condition, both in appearance and physiologically, validate the unique efficacy of ColpepA1 21, providing a strong scientific basis for its application in anti-aging skincare products.

[0189] In conclusion, ColpepA1 21, with its multiple anti-aging functions, whitening and optimizing properties, is at the forefront of skincare science, providing a new solution for skin health and beauty.

[0190] (II) Wound Repair

[0191] 1. Promotes rapid healing: By promoting HaCaT cell migration and enhancing AQP3 expression, type 21 collagen can accelerate the wound healing process and shorten the patient's recovery time.

[0192] 2. Improve repair quality: Promotes the production of type I and type III collagen, which helps to build new skin tissue with stable structure and normal function, thus improving the quality of repair.

[0193] 3. Biocompatibility and low immunogenicity: As a protein that exists naturally in the human body, type 21 collagen has good biocompatibility and low immunogenicity, reducing the risk of triggering an immune response.

[0194] 4. Multifunctionality: In addition to directly promoting wound healing, type 21 collagen may also improve the skin's moisture retention capacity by influencing AQP3 expression, thus having potential added value for improving skin health. Attached Figure Description

[0195] Figure 1 shows the quantitative test results of type I collagen content in cells of each experimental group, demonstrating the ability of different concentrations of ColpepA1 21 to promote type I collagen production.

[0196] Figure 2 shows the quantitative test results of type I collagen content in cells of each experimental group, and demonstrates the ability of different lengths of ColpepA1 21 to promote type I collagen production by comparison.

[0197] Figure 3 shows the results of tyrosinase activity assay in B16F10 cells after α-MSH induction.

[0198] Figure 4 shows the results of melanin content testing in B16F10 cells after α-MSH induction.

[0199] Figures 5 and 6 show the average area and volume of crow's feet wrinkles on the faces of volunteers before and after product use.

[0200] Figures 7 and 8 show the average area and volume of nasolabial folds on the faces of volunteers before and after product use.

[0201] Figure 9 shows the mean values ​​of facial skin elasticity (R2 value) before and after product use for volunteers.

[0202] Figure 10 shows the mean statistics of facial skin firmness (F4 value) of volunteers before and after product use.

[0203] Figure 11 shows the average facial skin radiance of volunteers before and after product use.

[0204] Figure 12 shows the mean values ​​of facial skin brightness (cheek L value) of volunteers before and after product use.

[0205] Figure 13 shows the mean optical density of facial skin spots before and after product use in volunteers.

[0206] Figure 14 shows the average facial erythema area of ​​volunteers before and after product use.

[0207] Figure 15 shows the mean scores of facial lactic acid stinging on volunteers before and after product use.

[0208] Figure 16 shows the average moisture content of volunteers' scalps before and after product use.

[0209] Figure 17 shows the mean TEWL values ​​of scalp transcutaneous water loss before and after product use in volunteers.

[0210] Figure 18 shows the mean scalp erythema index (EI) values ​​of volunteers before and after product use.

[0211] Figure 19 shows the healing process of keratinocytes in a scratch test, including scratch closure at different time points.

[0212] Figures 20 and 21 show a comparison of keratinocyte scratch healing rates, demonstrating the quantitative analysis results of healing progress in different experimental tissues.

[0213] Figure 22 shows the content of aquaporin 3 in cells after different treatments, and demonstrates the effect of ColpepA1 21 on AQP3 expression by comparison.

[0214] Figure 23 shows the relative integrated optical density (IOD) values ​​of filaggrin (FLG). A t-test was used to examine the significance of differences between the experimental and negative control groups, where significance is indicated by * (p < 0.05) and ** (p < 0.01).

[0215] Figure 24 shows the relative integrated optical density (IOD) value of lobe protein (LOR).

[0216] Figure 25 shows the relative integrated optical density (IOD) value of glutamine transferase 1 (TGM1).

[0217] Figure 26 shows the effect of ColpepA1 21 hydrogel dressing on cell viability under oxidative stress.

[0218] Related definitions

[0219] Unless otherwise specified, the following terms used in the specification and claims shall have the following meanings:

[0220] As used in this article, the term "collagen (COL)" refers to a crucial structural protein in the human body, widely found in various tissues and organs such as skin, bones, tendons, ligaments, and the cornea. The main function of these proteins is to provide strength, stability, and elasticity to body tissues, ensuring the normal functioning of various physiological processes. At least 28 types of collagen are currently known, each playing a unique role and function in the body, forming a complex and finely regulated biological system.

[0221] As used in this article, the term "type 21 collagen (COL21A1)" refers to a type of collagen present in the human body, belonging to the small molecule collagen family. As part of the FACIT (fibrous-associated collagen with a disrupted triple helix) family, type 21 collagen plays a crucial role in connecting extracellular matrix components. This collagen, through its unique molecular structure, promotes interactions and linkages between different collagen types, thereby supporting the integrity and order of the extracellular matrix and its function in wound healing and tissue repair. Although less well-known than type I and type III collagen, it plays a role in specific tissue and physiological processes, involving extracellular matrix composition and skin repair mechanisms. While type 21 collagen is relatively scarce in the adult human body, accounting for less than 1% of total collagen, its potential roles in cellular processes such as migration, proliferation, and differentiation, as well as its potential role in maintaining extracellular matrix structure and promoting tissue regeneration, represent important directions for future research.

[0222] As used herein, the term "recombinant humanized collagen type 21 (ColpepA1 21)" refers to a protein expressed in Pichia pastoris using gene recombination technology that mimics naturally occurring collagen type 21 in the human body. This protein possesses similar biological functions and structural characteristics to natural collagen type 21, and its amino acid sequence is specifically encoded to enhance skin health and anti-aging capabilities.

[0223] As used herein, “identity” refers to the percentage of identical (i.e., common) amino acids between two or more polypeptides. Sequence identity between two or more polypeptides can be determined by aligning the amino acid sequences of the polypeptides and scoring the number of positions in the aligned polypeptide containing the same amino acid residues, comparing this to the number of positions in the aligned polypeptide containing different amino acid residues. Polypeptides can differ at a position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., insertions or deletions of one or two amino acids in the polypeptide). Sequence identity can be calculated by dividing the number of positions containing the same amino acid residues by the total number of amino acid residues in the polypeptide. For example, the percentage of identity can be calculated by dividing the number of positions containing the same amino acid residues by the total number of amino acid residues in the polypeptide and multiplying by 100.

[0224] As used in this article, the term "Pichia pastoris genetically engineered strain" refers to a Pichia pastoris strain that has undergone genetic manipulation to insert a specific foreign gene. This modification enables the yeast to express proteins not found in its own genome, allowing for the production of various recombinant proteins.

[0225] As used herein, the term "expression system" refers to a set of biochemical environments and processes used to synthesize a specific protein, including host cells, vectors, inducers, etc. In this article, the Pichia pastoris expression system refers to a system that uses Pichia pastoris as a host cell to express recombinant proteins through specific procedures.

[0226] As used in this article, the term "HaCaT (human keratinocytes)" refers to an immortalized human keratinocyte line that is widely used in research on skin biology, skin pathology, and drug toxicity testing, and is considered a valuable research tool because it can mimic the behavior of normal epidermal cells.

[0227] As used in this article, the term "HFF-1 (human fibroblasts)" refers to fibroblasts isolated from the forearm of a human fetus. They are an important part of the skin structure, responsible for synthesizing collagen and other extracellular matrix proteins, and are often used to study skin aging, wound healing, and collagen synthesis.

[0228] As used herein, the term "composition" refers to a formulation containing recombinant humanized collagen type 21 and possibly other auxiliary ingredients such as moisturizers, antioxidants, or skin brightening agents, which work together to provide a more comprehensive skin care effect.

[0229] As used in this article, the term “cell migration” refers to the process by which cells move from one location to another in an in vivo or in vitro environment, which is an important biological process in skin wound healing and tissue regeneration.

[0230] As used in this article, the term "aquaporin AQP3" refers to an aquaporin that is mainly expressed in skin cells and is responsible for regulating water transport in cells, playing a key role in maintaining skin moisture balance and barrier function.

[0231] As used in this article, the term "moisture regulation" refers to a series of physiological processes that maintain the skin's moisture balance, involving the absorption, distribution, and retention of moisture within the skin layers. These processes are crucial for maintaining healthy, elastic skin and resisting external stresses.

[0232] As used in this article, the term "barrier optimization" refers to the process of enhancing the skin barrier function, including strengthening the tightness of the epidermal layer to block external stimuli and reduce moisture loss, thereby protecting the skin from environmental damage and maintaining its healthy state.

[0233] As used in this article, the term "Type I collagen" refers to one of the main protein types that make up the structures of human skin, bones, tendons, etc., and is the most abundant type of collagen in the human body. This type of collagen consists of two α1 chains and one α2 chain, forming a triple helix structure that gives tissues high tensile strength. The main function of Type I collagen is to provide structural support and physical strength to these tissues, playing a crucial role in maintaining the elasticity, stability, and overall health of the skin. With age and the influence of environmental factors, Type I collagen gradually degrades, leading to loss of skin elasticity and the appearance of signs of aging, such as fine lines and wrinkles. In facial filler products, an increase in Type I collagen is considered an important indicator of product effectiveness, especially in anti-aging and improving skin structure.

[0234] As used herein, the term "Type III collagen" refers to a type of collagen that coexists with Type I collagen in the skin structure, particularly abundant in younger skin. Primarily found in the intercellular matrix of the skin, it plays a crucial role in maintaining skin elasticity and assisting in the healing of damaged skin. A temporary increase in Type III collagen after skin injury helps repair and rebuild skin structure. Its decreasing levels with age may affect skin elasticity and repair capacity. Increased Type III collagen content in facial filler products demonstrates the potential of these products to promote natural skin repair and enhance skin elasticity.

[0235] As used in this article, the term "photoaging" refers to skin aging caused by prolonged exposure to ultraviolet (UV) radiation. UV radiation can penetrate the skin's surface, directly damaging collagen and elastin fibers, accelerating their degradation, and leading to signs of aging such as sagging skin, fine lines, wrinkles, and pigmentation. Photoaging is another major cause of skin aging besides natural aging.

[0236] As used in this article, the term "UVA radiation" refers to the ultraviolet spectrum with wavelengths between 320 and 400 nanometers. UVA can penetrate deep into the dermis, causing damage to collagen and elastin fibers, and is one of the main causes of photoaging. The damage of UVA radiation to the skin is cumulative; long-term exposure can lead to accelerated skin aging and increase the risk of skin cancer.

[0237] As used in this article, the term "facial crow's feet area and volume" refers to fine lines located around the corners of the eyes, caused by facial expressions such as smiling or blinking. The area and volume of these fine lines can be measured using specific instruments or software to assess the impact of anti-aging products on reducing fine lines around the eyes. A reduction in area and volume generally indicates a positive effect of the product in improving skin elasticity and reducing wrinkles.

[0238] As used in this article, the term "facial nasolabial fold area and volume" refers to the wrinkles located on either side of the corners of the mouth, which typically become more pronounced with age. Measuring the area and volume of nasolabial folds allows for the assessment of the effectiveness of anti-aging products in smoothing deep facial wrinkles. Reduced area and volume indicate that the product effectively improves skin structure and reduces signs of facial aging.

[0239] As used in this article, the term "facial skin elasticity (R2 value)" refers to a value obtained using a skin elasticity measuring instrument, which reflects the degree of skin elasticity. The R2 value is determined by measuring the skin's ability to return to its original shape after being mechanically stretched. The higher the R2 value, the better the skin elasticity and the better it can resist deformation during the aging process.

[0240] As used in this article, the term "facial skin firmness (F4 score)" refers to a quantitative parameter for assessing the firmness of the skin. The F4 score is measured using specialized skin testing equipment and reflects the firmness and elasticity of the skin. A higher F4 score indicates better skin firmness, which helps reduce skin sagging and wrinkle formation.

[0241] As used in this article, the term "facial skin radiance" refers to the skin's ability to reflect light, a characteristic often associated with skin health and youthfulness. Skin with higher radiance reflects light more effectively, giving a bright, fresh visual effect and is generally considered a sign of health and vitality. In anti-aging product evaluations, increased radiance is considered a positive indicator of product effectiveness, suggesting that the product helps improve skin's surface properties and enhance its natural glow.

[0242] As used in this article, the term "facial skin brightness (cheek L-value)" refers to a parameter used to measure the lightness or darkness of skin color. In color science, the L-value represents a brightness level from black (0) to white (100), with a higher L-value indicating brighter skin. In dermatological research and cosmetic efficacy evaluation, the cheek L-value is often used as a quantitative indicator to assess the improvement in skin brightness and tone. Increasing the cheek L-value generally means that the skin becomes brighter and more even-toned, which is one of the desired effects of whitening or anti-aging products.

[0243] As used in this article, the term "facial skin pigmentation density" refers to the color depth and density of pigmented areas on the skin. This indicator is measured using specific instruments and is used to evaluate the effectiveness of skin whitening or pigmentation-reducing products. Higher pigmentation density indicates darker, more densely distributed pigmented areas, while lower density indicates lighter, more sparsely distributed pigmented areas. Skin whitening or pigmentation-reducing products aim to lower pigmentation density, resulting in a more even skin tone.

[0244] As used in this article, the term "facial erythema area" refers to the size of an area of ​​redness on the skin surface caused by inflammation or other reasons. The appearance of erythema is usually associated with skin inflammation, allergic reactions, or other skin problems. When evaluating the effectiveness of anti-inflammatory, soothing, or anti-allergic products, a reduction in erythema area is considered a positive result, indicating that the product effectively reduces skin inflammation and improves skin health.

[0245] As used in this article, the term "facial lactic acid stinging score" refers to a subjective rating of the skin irritation (such as stinging) caused by a product. During skin irritation testing, participants rate the level of irritation they experience after using the product (e.g., no, mild, moderate, strong). A lower score indicates less irritation and greater suitability for sensitive skin.

[0246] As used in this article, the term "scalp moisture content" refers to the scalp's ability to retain moisture. This metric is crucial for assessing the health of the scalp and overall skin; a scalp with sufficient moisture content is more likely to remain healthy and vibrant, reducing issues such as dryness and itching. Increasing scalp moisture content is a goal that many hair and skin care products attempt to achieve.

[0247] As used in this article, the term "transepidermal water loss (TEWL)" refers to the rate at which moisture evaporates from the scalp surface, reflecting the integrity of the skin barrier function. A lower TEWL value indicates good skin barrier function, effectively preventing excessive moisture evaporation and maintaining the skin's moisture balance. When evaluating the effectiveness of skincare and haircare products, a lower TEWL value is generally considered an indicator of improved skin barrier function and enhanced skin hydration.

[0248] As used in this article, the term "erythema index (EI value)" refers to an indicator of the degree of scalp erythema measured by an instrument, used to quantitatively assess the inflammation or sensitivity of the scalp. A higher EI value indicates a more severe degree of scalp inflammation or sensitivity. It is an important indicator in evaluating the calming and soothing effects of skincare or haircare products.

[0249] As used in this article, the term "TGFβ1 (transforming growth factor β1)" refers to a multifunctional cytokine that regulates cell proliferation, differentiation, and migration. In skin physiology, TGFβ1 plays a crucial role in collagen synthesis, wound healing, and anti-inflammatory effects.

[0250] As used in this article, the term "HPR (oxaloolphthalein oxalate)" is a synthetic retinol derivative commonly used in skin care products and wound repair preparations. It has the ability to promote cell renewal, enhance skin repair, and improve the appearance of aging skin. In wound repair preparations, it is used to promote cell renewal, enhance the repair capacity of wounds, and improve healing efficiency, while also improving the appearance of aging skin that may occur during the wound healing process.

[0251] As used herein, the terms "composition" or "formulation" refer to a mixture containing one or more of the collagen described in this application along with other components, such as physiologically pharmaceutically acceptable carriers and excipients or adjuvants such as humectants, antioxidants, etc. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, enhance the absorption of the active ingredient, and thereby exert its biological activity. In some embodiments, these components work together to provide a more comprehensive skin care effect. In some embodiments, during wound repair, topical application to the wound can be promoted to enhance the bioavailability of the active ingredient, thereby accelerating wound healing and tissue regeneration. The terms "medical composition" and "medical product" are used interchangeably herein.

[0252] As used in this article, the term "antioxidant" refers to a class of substances that can neutralize free radicals and slow down or prevent cell damage. This plays an important role in reducing oxidative stress caused by environmental factors and preventing skin aging. In the process of wound healing, it helps reduce cell damage caused by oxidative stress, supports a healthy cellular environment, and promotes rapid wound recovery.

[0253] As used in this article, the term "emulsifier" refers to a substance that helps oil and water mix to form a stable emulsion. In skincare products, it is used to improve the texture and stability of the product; in wound healing formulations, it is used to improve the texture and stability of the product and ensure that the active ingredients are evenly distributed for easy local application.

[0254] As used in this article, the term "humectant" refers to a substance that can attract or lock in moisture, helping the skin retain moisture. It plays a vital role in maintaining the skin's hydration and preventing dryness, maintaining a suitable moist environment in wound treatment areas, and accelerating cell repair and wound healing.

[0255] As used in this article, the terms "ultraviolet absorber" or "sunscreen" refer to compounds that can absorb or reflect ultraviolet rays, protecting the skin from UV radiation damage. These compounds are important for preventing skin aging and skin cancer.

[0256] As used herein, the term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacturing, storage, and application. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols, and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or its salts (e.g., arginine hydrochloride), glycine, alanine (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opins, alanine, strombine, and trimethylamine N-oxide (TMAO), human serum albumin (hsa), bovine serum albumin (bsa), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, and RNAase A. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, and sucrose, can also control osmotic pressure. The stabilizers specifically used in this invention are selected from one or more of polyols, amino acids, salts, and sugars. Preferred salts are sodium chloride, preferred sugars are sucrose and trehalose, and preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine or its salts (such as arginine hydrochloride), glycine, and proline. Preferred stabilizers include sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, sodium chloride-trehalose, arginine hydrochloride-mannitol, and arginine hydrochloride-sucrose.

[0257] As used herein, the term "skin booster" refers to a specific class of skin care preparations designed to improve the skin's moisture retention, elasticity, and overall health by direct injection into the middle layer of the skin. Skin boosters include, but are not limited to, the following ingredients:

[0258] Hyaluronic acid: As one of the main ingredients, hyaluronic acid can effectively improve the skin's water retention capacity, increase the skin's plumpness and radiance;

[0259] Collagen: As one of the active ingredients, the main function of collagen is to activate the basal layer of cells, stimulate the regeneration and repair of cells under the skin, improve the elasticity and firmness of the skin, thereby achieving a significant anti-aging effect.

[0260] Vitamins (such as vitamin E, vitamin C, B3, etc.): have antioxidant properties, which can help reduce free radical damage and promote brighter skin and even pigmentation.

[0261] Other antioxidants (such as coenzyme Q10) protect the skin from environmental damage and enhance its repair capabilities.

[0262] Other peptides: stimulate the production of collagen and elastin, helping to reduce fine lines and wrinkles.

[0263] Amino acids: As the basic building blocks of skin cells, they promote skin repair and regeneration.

[0264] Stabilizers (such as sodium chloride, mannitol, sorbitol, sucrose, and arginine hydrochloride) ensure the stability of the active ingredient during manufacturing, storage, and application, thereby optimizing therapeutic effects and ensuring safe use.

[0265] As used in this article, the term "melanin" is an important biological pigment that determines the color of the skin, hair, and eyes of humans and other organisms, participates in pigmentation, and protects the skin from ultraviolet radiation damage.

[0266] As used in this article, the term "tyrosinase" refers to a copper-containing oxidase, the rate-limiting enzyme regulating melanin production. It is widely found in plants, animals, and microorganisms. In humans, tyrosinase is primarily found in melanocytes, where it catalyzes the production of dopa from tyrosine and its further oxidation to dopaquinone, ultimately forming melanin. The expression and activity of tyrosinase determine the amount of melanin produced, thus affecting an individual's skin color and sensitivity to ultraviolet radiation. It is also associated with diseases such as freckles and age spots caused by excessive melanin deposition. Excessive tyrosinase activity is associated with certain types of skin cancer, such as melanoma. Therefore, inhibiting tyrosinase activity can reduce melanin formation, alleviate skin pigmentation, and achieve a skin whitening and brightening effect.

[0267] As used in this article, the term "barrier function" refers to a series of physiological functions of the skin that together form a defense line protecting the body from harmful external substances. This includes, but is not limited to, preventing pathogen invasion, reducing moisture evaporation, blocking ultraviolet radiation, and resisting chemical damage. The barrier function is accomplished through the coordinated efforts of multiple layers of the skin, including the stratum corneum of the epidermis, the sebum film, and the harmonious balance of the microbial community.

[0268] As used in this article, the term "skin barrier formation" refers to the process by which various cellular and molecular components in the skin, particularly the epidermis, interact to form a protective barrier through complex biochemical processes. This process includes the production, differentiation, and death of keratinocytes, as well as their complex interactions with lipids, thereby establishing an effective physical and chemical barrier.

[0269] As used in this article, the term "hydration status" refers to the level of moisture required by the skin to maintain its normal structure and physiological functions. Hydration status affects the skin's appearance, softness, elasticity, and resistance to irritation. The regulation of skin hydration status involves multiple factors, including water exchange within and between stratum corneum cells, the production of natural moisturizing factors, and the absorption and loss of external moisture.

[0270] As used in this article, the term "filaggrin (FLG)" refers to a protein belonging to the keratin family, primarily found in the stratum corneum of the epidermis. FLG plays a central role in the construction of the skin barrier, responsible for maintaining the moisture of the stratum corneum and, through its hydrolysis products, maintaining pH balance and an antimicrobial environment. FLG deficiency or dysfunction is often associated with various skin conditions, such as eczema and keratosis.

[0271] As used in this article, the term "LOR" refers to another structural protein in the stratum corneum of the epidermis, a major component of the skin barrier responsible for reinforcing the skin's structural integrity. LOR provides additional mechanical strength to the skin by cross-linking with other proteins within the stratum corneum and plays a crucial role in the skin's defense mechanisms.

[0272] As used herein, the term "glutamine transferase 1 (TGM1)" refers to an enzyme primarily expressed in the granular layer and stratum corneum of the epidermis, whose function is to promote the formation of stable cross-linked structures between skin proteins. The enzymatic reactions catalyzed by TGM1 are crucial for the integrity and protective capacity of the skin barrier, and its deficiency is associated with the development of certain hereditary skin diseases.

[0273] As used in this article, the term "3D epidermal skin model" refers to a laboratory-constructed three-dimensional skin model that simulates the structure and function of human skin. These models typically consist of multiple cell types, including epidermal and dermal cells, and can be used to study skin biology, drug screening, and disease models.

[0274] As used herein, the term "immunofluorescence assay" refers to an experimental technique that uses specific antibodies bound to fluorescent labels to locate and quantify target antigens in tissue sections or cell products. This technique is extremely useful for visualizing the location and expression levels of specific proteins within cells, particularly in pathological and cell biology research.

[0275] As used herein, the term "Type 21 collagen hydrogel dressing" refers to a polymeric material formulated based on the Type 21 collagen of this patent and auxiliary ingredients such as hyaluronic acid, forming a three-dimensional network structure through the use of a chemical cross-linking agent. This hydrogel dressing plays a key role in wound repair and cell regeneration, improving the survival and activity of damaged cells, and exhibiting excellent cell protection effects, especially under oxidative stress. Detailed Implementation

[0276] In specific embodiments, the materials and methods used are as follows:

[0277] Cell lines: HaCaT (human keratinocytes) and HFF-1 (human fibroblasts), purchased from Fenghui Biotechnology.

[0278] Reagents: CCK-8 kit (Japan Dojin CK04) was used for cell viability assessment, and AQP3 (Shanghai ELISA) and type I collagen kit (Shanghai ELISA CMM2023H1) were used for related protein expression analysis.

[0279] Example 1: Preparation and purification of recombinant humanized collagen type 21 (ColpepA1 21)

[0280] 1. Construction of Pichia pastoris genetically engineered strain:

[0281] Activated yeast GS115 was streaked onto YPD plates. Single clones of activated Pichia pastoris GS115 from the YPD plates were selected and incubated in YPD liquid with constant temperature and shaking. 100 μL of the bacterial culture was pipetted into 100 mL of YPD liquid and incubated at 30°C, 220 rpm for 12-13 h until OD reached the target value. 600 =1.3-1.5; examine under a microscope for contamination, aliquot the bacterial cells into sterile 50ml centrifuge tubes, and incubate on ice for 10-15 minutes.

[0282] Centrifuge twice, discard the supernatant, and resuspend the bacterial cells in sterile water; centrifuge twice more, discard the supernatant, and resuspend the bacterial cells in sorbitol before aliquoting to obtain competent cells; transfer the vector into the competent cells using electroporation, and then culture on plates until clones are produced.

[0283] 2. Fermentation culture of Pichia pastoris genetically engineered strain:

[0284] Single colonies from the YPD plate were picked and transferred to a conical flask containing 50 mL of BMGY. The flask was incubated at 30°C and 220 rpm for 24 hours. The incubation was confirmed by measuring the OD600 value. After centrifugation for 10 minutes, the bacterial cells were collected, washed once with BMMY, centrifuged again, and diluted to the appropriate OD600 concentration. 600 =1.0.

[0285] 3. Induction and expression of recombinant human collagen:

[0286] Transfer the diluted bacterial culture to a 500 mL Erlenmeyer flask and induce yeast protein expression at 29 °C and 220 rpm. Add methanol every 24 h to maintain a final methanol concentration of 1% in the culture medium and continue induction culture for 84 h. Examine the culture under a microscope for contamination and check the pH of the fermentation broth; it should be less than 6. Transfer the fermentation broth to a 500 mL round-bottom centrifuge tube and centrifuge at 15000 g and 4 °C for 20 min. Collect the supernatant. Take 1 mL of the supernatant and add the appropriate loading buffer, mix well, incubate at 95 °C for 10 min, and store at -20 °C.

[0287] 4. Purification of recombinant human collagen:

[0288] The supernatant of the obtained fermentation broth was transferred to an Erlenmeyer flask, and NaCl was added to bring the final NaCl concentration to 150 mM. The pH of the fermentation broth supernatant was adjusted to 8.0 with NaOH or HCl. The flask was centrifuged at 15000 g for 30 min to ensure that the supernatant was clear and transparent. The column was equilibrated with 10 column bed volumes of binding buffer. Samples were loaded at a rate of 1 mL / min, and the percolation fluid was collected during loading. Unbound proteins and other proteins were eluted with 5 column bed volumes of binding buffer. The target protein was eluted with elution buffer (500 mM imidazole).

[0289] Preparation of protein detection samples: Add the purified protein, pre-column supernatant, and post-column liquid collected from each tube to the corresponding loading buffer, mix well, incubate at 95℃ for 10 min, and store at -20℃.

[0290] Through this series of steps, recombinant humanized collagen type 21 (ColpepA1 21) can be successfully prepared and purified, and high-purity, high-activity target protein can be obtained using the Pichia pastoris expression system.

[0291] For specific synthesis methods and purification techniques, please refer to patent CN114195884A.

[0292] Example 2: Formation of Type I Collagen

[0293] This study aims to investigate the ability of recombinant human collagen type 21 (ColpepA1 21) to promote the production of type I collagen. In the field of anti-aging, especially anti-photoaging research, the ability to produce type I collagen is widely considered an important indicator for evaluating the effectiveness of skincare ingredients. Type I collagen, as a major component of skin structure, is crucial for maintaining skin elasticity, stability, and overall health. With age and environmental factors, especially the effects of ultraviolet radiation (UVA / UVB), type I collagen in the skin gradually degrades, leading to skin laxity, fine lines, and wrinkles—the so-called photoaging phenomenon. This experiment aims to investigate the ability of recombinant human collagen type 21 (ColpepA1 21) to promote the production of type I collagen through specific experimental research. By simulating an environment where UVA radiation damages the skin, this study investigates whether ColpepA1 21 can effectively promote the synthesis of type I collagen in damaged skin, thereby supporting its role as an effective anti-photoaging ingredient.

[0294] Experimental Groups

[0295] Control group: No reagents were added and no UVA radiation treatment was performed.

[0296] Negative control group (5J / cm) 2 UVA): Cells were exposed to 5J / cm 2 UVA damage was treated without the addition of any therapeutic agents.

[0297] Positive control group (TGFβ1): 100 ng / mL TGFβ1 was added after UVA damage, which is known to promote the production of type I collagen.

[0298] ColpepA1 21 experimental group: After UVA damage, 10 ppm or 50 ppm of type 21 recombinant humanized collagen was added respectively.

[0299] ColpepA1 21 (long chain) experimental group: 50 ppm of type 21 recombinant humanized collagen ColpepA1 21 (long chain) was added after UVA damage.

[0300] The amino acid sequence of ColpepA1 21 (long chain) is selected from SEQ ID No. 2:

[0301] This specific amino acid sequence is derived from patent CN114195884A. This experimental group aims to further verify the potential efficacy of ColpepA1 21 in promoting type I collagen production. By comparing the effects of known long-chain collagen with the short-chain collagen of this patent, the difference in the effectiveness of short-chain versus long-chain collagen in promoting type I collagen synthesis is evaluated.

[0302] HFF-1 fibroblasts were cultured separately, plated and cultured for 24 h. When the cell confluence was 70-80%, UVA was induced and sample was added. After culturing for 24 h, the supernatant was collected and the type I collagen content was determined using an ELISA kit.

[0303] Data collection and analysis:

[0304] The content of type I collagen in cell supernatant was quantitatively analyzed using an ELISA kit.

[0305] Figure 1 shows the type I collagen content in the blank control group, negative control group, positive control group, and experimental groups with different concentrations of ColpepA1 21. A t-test was used to test for significant differences between the corresponding experimental groups and the negative control group, where * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. The results show that the blank control group exhibited normal baseline levels of type I collagen. After 5 J / cm 2 The negative control group treated with UVA showed a significant decrease in type I collagen content, indicating that UVA radiation damages the extracellular matrix. The TGFβ1 positive control group showed some recovery in type I collagen content compared to the negative group, but it remained lower than the untreated control group, suggesting that TGFβ1 has a certain repair effect. In the experimental groups supplemented with 10 ppm and 50 ppm ColpepA1 21, the type I collagen content was higher than that of the UVA-treated negative control group. Particularly in the 50 ppm ColpepA1 21 group, the type I collagen content was significantly higher than that of the negative control group, with a promotion rate of 23.21%, indicating that ColpepA1 21 has a significant promoting effect on type I collagen synthesis.

[0306] Figure 2 shows the content of type I collagen in the blank control group, negative control group, positive control group, and the ColpepA1 21 experimental group and the ColpepA1 21 (long chain) experimental group at a concentration of 50 ppm. The results showed that the blank control group exhibited normal baseline levels of type I collagen, while the negative control group showed a significant decrease in type I collagen content. After treatment with ColpepA1 21 (long chain), the content of type I collagen increased, by 34.0% compared to the negative control group. Although this increase was less than that in the positive control group, it indicates that ColpepA1 21 (long chain) has the ability to promote type I collagen synthesis in damaged skin. Of particular note is that compared to the ColpepA1 21 (long chain) group, the ColpepA1 21 group showed a more significant effect, with an average type I collagen content significantly higher than that of the ColpepA1 21 (long chain) group, increasing by 63.6% compared to the negative control group. This significant difference indicates that, thanks to its more optimized molecular structure, the short ColpepA1 21 provided by this patent has made a significant improvement over the long chain in promoting the synthesis of type I collagen, providing strong scientific evidence for its application in anti-photoaging and skin repair, and indicating its great potential in the development of future skin care products.

[0307] ColpepA1 21, a recombinant humanized collagen, significantly promotes the synthesis of type I collagen after UVA damage, which is crucial for maintaining and restoring the integrity of skin structure. This discovery provides strong scientific evidence for the application of ColpepA1 21 in the field of anti-aging, particularly its potential benefits in promoting damaged skin repair and improving skin elasticity. Regarding anti-aging collagen patents, ColpepA1 21 demonstrates its commercial value as an effective skin care ingredient by enhancing the production of key skin structural components, contributing to skin health and delaying the aging process.

[0308] Example 3: Effect of recombinant humanized collagen type 21 on tyrosinase activity

[0309] This embodiment aims to investigate the effect of recombinant human collagen type 21 (ColpepA1 21) on tyrosinase activity in B16F10 cells, in order to evaluate its subsequent effect on skin whitening and brightening.

[0310] The cell line used in this experiment was mouse skin melanoma cell line B16F10 (Qisai Biotechnology), and the cell passage number was 7-8.

[0311] Main reagents and consumables:

[0312] B16 cell culture medium (Qisai Biotechnology), high-quality fetal bovine serum (Gibco), phosphate-buffered saline (Basal Media, PBS), trypsin (Basal Media, 0.25%), dimethyl sulfoxide (Solarbio), cell culture flasks (Thermo, T75), cell culture plates (Corning, 12-well plates), pipettes (Thermo, 5mL, 15mL), centrifuge tubes (Corning, 15mL), disposable cell counting chamber (Countstar), melanocyte-stimulating hormone α-MSH (Yuanye Biotechnology), and mouse tyrosinase (TyR) ELISA kit (Yuanju Biotechnology).

[0313] Instruments and equipment:

[0314] Adjustable pipettes (Eppendorf), analytical balances (Sartorius), inverted microscopes (Motic), low-speed centrifuges (Heal Force), incubators (Heal Force), electric pipettes (Eppendorf), cell counters (Countstar), clean benches (Thermo), digital display constant temperature water baths (Lanbao Haibo Biotechnology), liquid nitrogen tanks, refrigerators (Haier), and multi-functional microplate readers (PerkinElmer).

[0315] 1. Cell seeding: Dilute B16F10 cells to 5×10⁻⁶. 4 At a seeding density of 1 cell / mL, cells were seeded onto 24-well plate slides at a dilution of 500 μL / well and incubated in a cell culture incubator (37℃, 5% CO2, 95% RH) for 24±2 h.

[0316] 2. Experimental grouping: The experiment included a blank control group, a negative control group, a positive control group, and a sample group.

[0317] 3. Solution preparation:

[0318] 1) Sample group: Prepare sample ColpepA1 21 with cell culture medium to 20 ppm;

[0319] 2) Positive control group: 377 (phenylethyl resorcinol) solution.

[0320] 4. Induction: Except for the blank control group, each well was incubated with 0.1 μM α-MSH for 48 h ± 2 h.

[0321] 5. Sample addition: After induction incubation, wash the cell plate with PBS. Add 500 μL of culture medium to each well of the blank control group; add 500 μL of culture medium to each well of the negative control group; add 500 μL of culture medium containing the corresponding concentration of 377 to each well of the positive control group; add 500 μL of culture medium containing the corresponding concentration of the sample to each well of the sample group. After sample addition, place the 24-well plate in an incubator (37℃, 5% CO2, 95% RH) and continue incubation for 24 ± 2 h.

[0322] 6. Detection of cellular tyrosinase activity: After incubation, cells were repeatedly frozen and thawed three times. The supernatant was centrifuged at 3000 rpm for 5 min to remove the precipitate. The changes in tyrosinase activity were detected using an enzyme-linked immunosorbent assay (ELISA) reader and a kit.

[0323] 7. Tyrosinase activity assay in B16F10 cells after α-MSH induction

[0324] As shown in Figure 3, the diagrams represent:

[0325] Blank control group: Added with an equal volume of cell culture medium; Negative control group: Added with an equal volume of 0.1 μM α-MSH-induced cell culture medium; Positive control group: Added with an equal volume of 0.1 μM α-MSH and cell culture medium containing 4 μM (approximately 0.86 ppm) 377; Sample group: Added with an equal volume of 0.1 μM α-MSH and cell culture medium containing the corresponding concentration of the sample; ### indicates P < 0.001 relative to the blank control group; ** indicates P < 0.01 relative to the negative control group; Values ​​are mean ± variance, n = 3B16F10.

[0326] The results of tyrosinase activity in cells after α-MSH induction are as follows:

[0327] (1) Compared with the blank control group, the tyrosinase activity of the negative control group was significantly increased, proving that the model was successfully constructed;

[0328] (2) Compared with the negative control group, 2 μM (about 0.43 ppm) 377 can significantly inhibit tyrosinase activity, further proving that the model was successfully constructed, with an inhibition rate of 54.60%;

[0329] (3) Compared with the negative control group, 20 ppm ColpepA1 21 significantly inhibited tyrosinase activity, with an inhibition rate of 56.49%.

[0330] Based on the above in vitro cell experiments, it is demonstrated that 20 ppm ColpepA1 21 can significantly inhibit tyrosinase activity.

[0331] Example 4: Effects of recombinant humanized collagen type 21 on melanin

[0332] This study aimed to investigate the effect of recombinant human collagen type 21 (ColpepA1 21) on melanin in B16F10 cells. Tyrosinase is a key enzyme in the melanin biosynthesis pathway, catalyzing the conversion of tyrosine to dopa, which in turn generates melanin. Controlling tyrosinase activity can reduce melanin production, thereby reducing skin pigmentation.

[0333] The cell line used in this experiment was mouse skin melanoma cell line B16F10 (Qisai Biotechnology), and the cell passage number was 7-8.

[0334] Main reagents and consumables:

[0335] B16 cell culture medium (Qisai Biotechnology), high-quality fetal bovine serum (Gibco), phosphate-buffered saline (Basal Media, PBS), trypsin (Basal Media, Trypsin, 0.25%), dimethyl sulfoxide (Solarbio), cell culture flasks (Thermo, T75), cell culture plates (Corning, 12-well plates), pipettes (Thermo, 5mL, 15mL), centrifuge tubes (Corning, 15mL), disposable cell counting chamber (Countstar), melanocyte-stimulating hormone α-MSH (Yuanye Biotechnology), and mouse melanin ELISA kit (Yuanju Biotechnology).

[0336] Instruments and equipment:

[0337] Adjustable pipettes (Eppendorf), analytical balances (Sartorius), inverted microscopes (Motic), low-speed centrifuges (Heal Force), incubators (Heal Force), electric pipettes (Eppendorf), cell counters (Countstar), clean benches (Thermo), digital display constant temperature water baths (Lanbao Haibo Biotechnology), liquid nitrogen tanks, refrigerators (Haier), and multi-functional microplate readers (PerkinElmer).

[0338] 1. Cell seeding: Dilute B16F10 cells to 5×10⁻⁶. 4 At a seeding density of 1 cell / mL, cells were seeded onto 24-well plate slides at a dilution of 500 μL / well and incubated in a cell culture incubator (37℃, 5% CO2, 95% RH) for 24±2 h.

[0339] 2. Experimental grouping: The experiment was set up with a blank control group, a negative control group, and a sample group.

[0340] 3. Solution preparation:

[0341] Sample group: Prepare sample ColpepA1 21 with cell culture medium to 20 ppm.

[0342] 4. Induction: Except for the blank control group, each well was incubated with 0.1 μM α-MSH for 48 h ± 2 h.

[0343] 5. Sample addition: After induction incubation, wash the cell plate with PBS. Add 500 μL of culture medium to each well of the blank control group; add 500 μL of culture medium to each well of the negative control group; add 500 μL of culture medium containing the corresponding concentration of sample to each well of the sample group. After sample addition, place the 24-well plate in an incubator (37℃, 5% CO2, 95% RH) and continue incubation for 24 ± 2 h.

[0344] 6. Detection of melanin content in cells: After incubation, cells were repeatedly frozen and thawed three times. The supernatant was centrifuged at 3000 rpm for 5 min to remove the precipitate. The changes in melanin content were detected using an ELISA reader and a kit.

[0345] 7. Melanin content assay in B16F10 cells after α-MSH induction:

[0346] As shown in Figure 4, the diagrams represent:

[0347] Blank control group: Added with an equal volume of cell culture medium; Negative control group: Added with an equal volume of 0.1 μM α-MSH-induced cell culture medium; Sample group: Added with an equal volume of 0.1 μM α-MSH and cell culture medium containing the corresponding concentration of the sample; ### indicates P < 0.001 relative to the blank control group; ** indicates P < 0.01 relative to the negative control group; Values ​​are mean ± variance, n = 3.

[0348] The melanin content of B16F10 cells after α-MSH induction is as follows:

[0349] (1) Compared with the blank control group, the melanin content of the negative control group was significantly increased, proving that the model was successfully constructed;

[0350] (2) Compared with the negative control group, 20 ppm of type 21 collagen can significantly inhibit melanin content, with an inhibition rate of 18.82%.

[0351] Based on the above in vitro cell experiments, it is demonstrated that 20 ppm ColpepA1 21 can significantly inhibit melanin production. These effects indicate that ColpepA1 21 can be used as an effective active ingredient in skin whitening and brightening products.

[0352] Example 5: Clinical Trial

[0353] This embodiment presents clinical trial results validating the effectiveness of recombinant human collagen type 21 (ColpepA1 21) in improving signs of skin aging in practical applications. Key signs of aging focused on include dull skin tone, age spots, sagging skin, and wrinkles. This clinical trial aimed to evaluate the efficacy of ColpepA1 21 in improving skin quality and appearance, supporting its commercial potential as an anti-aging product ingredient.

[0354] The experiment recruited 30 healthy volunteers with an average age of 45.3 ± 6.7 years. The volunteers presented with dull skin, age spots, poor skin firmness, and noticeable crow's feet and nasolabial folds. The sample group received an appropriate amount of lotion containing 500 ppm of type 21 recombinant human collagen; the control group received a lotion without type 21 recombinant human collagen. Volunteers were instructed to apply the corresponding product to the left and right sides of their face / scalp after cleansing, gently patting until fully absorbed. The product was used twice daily for 28 days. Follow-up visits were conducted on day 0 (D0), day 14 (D14), and day 28 (D28).

[0355] Data collection and analysis:

[0356] Facial measurements included crow's feet, nasolabial folds, skin elasticity, skin firmness, skin luster, skin pigmentation density, erythema area, and lactic acid stinging score; scalp measurements included moisture content, transepidermal water loss, and erythema value.

[0357] The T-test was used to test the significance of the difference between the sample group and the control group. The asterisk (*) in the figure indicates that p < 0.05.

[0358] Figures 5 and 6 show the mean area and volume of crow's feet wrinkles on the volunteers' faces before and after product use. The results demonstrate a significant improvement in crow's feet wrinkles after using the lotion containing ColpepA1 21. This improvement directly proves the effectiveness of ColpepA1 21 in reducing fine lines around the eyes, reflecting its positive impact on the deep structure of the skin. The reduced area and volume indicate a significant improvement in skin firmness and smoothness, which is related to ColpepA1 21 promoting collagen synthesis and strengthening the skin's supporting structure.

[0359] Figures 7 and 8 show the mean area and volume of nasolabial folds on the faces of volunteers before and after product use, demonstrating a reduction in nasolabial folds. This reduction not only improves the overall appearance of the face but also illustrates the efficacy of ColpepA1 21 in rebuilding skin elastic fibers. This may be because ColpepA1 21 activates the skin's natural repair mechanisms, promoting the production of collagen and elastin.

[0360] Figure 9 shows the mean R² values ​​of facial skin elasticity before and after product use in volunteers, demonstrating an increase in facial elasticity. Increased skin elasticity is a key indicator in anti-aging research, indicating that the skin can better resist mechanical deformation and return to its original state. ColpepA1 21 directly reflects its contribution to improving skin firmness and reducing wrinkle formation by enhancing skin elasticity.

[0361] Figure 10 shows the mean facial skin firmness (F4 value) of volunteers before and after product use, demonstrating an improvement in facial firmness. Decreased skin firmness is a common problem with age. The improved facial firmness shown in Figure 11 illustrates that ColpepA1 21 effectively combats skin sagging, restoring firmness and a youthful appearance.

[0362] Figures 11 and 12 show the mean statistical values ​​of facial skin glossiness and cheek brightness (L-value) before and after product use, respectively. The results demonstrate improvements in skin glossiness and brightness. These improvements are directly correlated with the reflectivity and smoothness of the skin surface. This indicates that ColpepA1 21 not only improves the internal structure of the skin but also optimizes the smoothness and gloss of the skin surface, enhancing the overall visual appearance of the skin.

[0363] Figure 13 shows the mean optical density of facial skin pigmentation before and after product use in volunteers, demonstrating a decrease in skin pigmentation optical density. Pigmentation is a common marker of UV exposure and photoaging of the skin. The efficacy of ColpepA1 21 in reducing pigmentation may be related to its influence on melanocyte activity and regulation of pigment production, thus helping to even out skin tone and reduce pigmentation.

[0364] Figure 14 shows the mean facial erythema area of ​​volunteers before and after product use, with the results indicating a reduction in facial erythema area. This reduction in erythema area reflects a decrease in skin inflammation, which may suggest that ColpepA1 21 has anti-inflammatory effects, reducing skin sensitivity and improving skin comfort.

[0365] Figure 15 shows the mean facial lactic acid stinging scores of volunteers before and after product use. The results show that a decrease in facial lactic acid stinging scores indicates improved skin comfort. The decrease in lactic acid stinging scores indicates that skin tolerance has improved after product use, reducing discomfort caused by external stimuli and enhancing skin barrier function.

[0366] Figures 16 and 17 show the mean scalp moisture content and mean transepidermal water loss (TEWL) values ​​of volunteers before and after product use, respectively. The results show that both scalp moisture content and TWEL values ​​improved, indicating enhanced skin barrier function. These improvements demonstrate that ColpepA1 21 can enhance the moisturizing ability of the scalp and the entire skin, reducing moisture loss, which is crucial for maintaining healthy skin and preventing dryness and aging.

[0367] Figure 18 shows the mean scalp erythema index (EI) values ​​of volunteers before and after product use. The results show a reduction in scalp erythema index, indicating its efficacy in reducing inflammation and alleviating skin sensitivity. The reduction in scalp erythema index further confirms the ability of ColpepA1 21 to reduce skin inflammation, which is particularly important for people with sensitive skin or poor skin conditions.

[0368] In summary, the results of this clinical trial provide strong data support for the anti-aging patent of ColpepA1 21 recombinant humanized collagen, demonstrating that ColpepA1 21 can effectively improve multiple signs of skin aging, including reducing wrinkles and age spots, increasing skin elasticity and firmness, and improving overall skin radiance and tone. These changes not only improved the appearance of the volunteers' skin but also physiologically enhanced skin health, reducing moisture loss and inflammation. These efficacy indicate that ColpepA1 21 can be used as an effective active ingredient in anti-aging products.

[0369] Example 6.1: Cell Scratch Assay

[0370] This study aimed to evaluate the role of recombinant human collagen type 21 (ColpepA1 21) in promoting wound healing, particularly its impact on skin cell migration. Cell migration is a crucial process for wound repair and regeneration, essential for rapid wound closure and high-quality healing. Using HaCaT keratinocytes and HFF-1 fibroblasts as models, the experiment explored how different concentrations of recombinant human collagen type 21 promote the migration and healing of these cells, in order to further investigate its application potential in wound repair.

[0371] Experimental Groups:

[0372] Control group: No therapeutic agents were added. Cell migration was directly observed after the scratch test to observe the natural cell migration or wound closure speed.

[0373] Positive control group (TGFβ1): 100 ng / mL TGFβ1 was added and used in scratch assays as a known cell migration and healing promoter to evaluate its effect on cell behavior.

[0374] Collagen control group (Col III): 50 ppm of type III collagen was added and used as a reference for the effect of the control collagen after scratch test.

[0375] ColpepA1 21 experimental group: 10 ppm and 50 ppm of type 21 recombinant humanized collagen (ColpepA1 21) were added respectively, and the effects of different concentrations were investigated after scratch testing.

[0376] ColpepA1 21 (long chain) experimental group: After adding 50 ppm of type 21 recombinant humanized collagen ColpepA1 21 (long chain) and conducting a scratch test, the difference in effect between it and the ColpepA1 21 (short chain) of this patent was examined.

[0377] The ColpepA1 21 (long chain) amino acid sequence is selected from SEQ ID No. 2.

[0378] This experimental group aims to further verify the efficacy of ColpepA1 21 in promoting cell migration. By comparing the effects of known long-chain collagen with the short-chain collagen of this patent, the differences in the effects of short chains on promoting cell migration and wound closure rate compared to long chains will be evaluated.

[0379] Keratinocytes (HaCaT) were cultured separately and plated for 24 hours. When the cell confluence reached 50%–60%, sample treatment was added. When the cell confluence reached 100%, scratching was performed. The difference between the five groups lay in the different conditions during the culture process. All groups were scratched and cultured for 36 hours, and photos were taken at 0, 12, 24, and 36 hours to compare the scratch healing progress.

[0380] Data collection and analysis:

[0381] 1. Visual assessment

[0382] As shown in Figure 19, the blank control group exhibited very limited cell migration in the scratch area throughout the time point, indicating baseline-level cell migration or wound closure rate. The 50 ppm type III collagen group served as a control to investigate the effect of standard type III collagen on cell migration. The TGFβ1 group showed enhanced cell migration compared to the blank control group, suggesting that TGFβ1 acts as a stimulator of cell migration, consistent with its known biological role in wound healing. The 10 ppm ColpepA1 21 group showed increased migration compared to the blank control group, but less than the 50 ppm ColpepA1 21 group, suggesting a dose-dependent response to ColpepA1 21. The 50 ppm ColpepA1 21 group showed a significant increase in cell migration in the scratch area, with a visually observable reduction in scratch width over time. Cell migration is a key step in wound healing; therefore, this finding highlights the potential role of type 21 collagen in accelerating wound closure, which has significant implications for the development of wound repair materials.

[0383] 2. Quantitative analysis

[0384] Figure 20 shows the percentage of scratch closure at different time points for the blank control group, positive control group, collagen control group, and experimental groups with different concentrations of ColpepA1 21. The bars represent the average closure percentage, and the error bars represent within-group variation.

[0385] 12 hours: All groups showed some degree of wound closure, with varying efficacy. The closure percentage in the 50ppm ColpepA1 21 group was higher than that in the blank control group and the lower concentration of ColpepA1 21.

[0386] 24 hours: The differences in wound closure rates between different treatment groups became more pronounced, with the 50ppm ColpepA1 21 group showing better healing compared to the blank control group and the type III collagen group.

[0387] At 36 hours: the 50ppm ColpepA1 21 group showed the highest wound closure rate, approximately 63.85%, which was significantly higher than the blank control group and the 10ppm ColpepA1 21 group.

[0388] Quantitative data strongly demonstrate that recombinant humanized collagen peptide type 21 (ColpepA1 21) significantly enhances HaCaT cell migration and wound closure, particularly at a high concentration of 50 ppm. The blank control group served as a baseline for natural wound closure, while the TGFβ1 group confirmed the known positive effects of this growth factor on cell migration. The increased migration observed in the 50 ppm ColpepA1 21 group not only demonstrates the peptide's effectiveness in promoting wound healing but also suggests a possible dose-dependent mechanism, where higher concentrations lead to greater cell migration.

[0389] Figure 21 shows the percentage of scratch closure at 12 hours in the blank control group, positive control group, and the ColpepA1 21 (long chain) experimental group at a concentration of 50 ppm. At the 12-hour time point, each experimental group exhibited varying degrees of cell migration and wound closure. The blank control group (Control), serving as baseline, showed the lowest scratch closure rate, averaging 3.89%. This result is expected, as the control group received no collagen or growth factor treatment and only reflected the natural migration capacity of cells. The positive control group (TGFβ1) showed an average scratch closure rate of 33.93%, a significant improvement consistent with the known promoting effects of TGF-β1 on cell migration and healing. The average scratch closure rate in the ColpepA1 21 (long chain) experimental group was 28.19%, lower than the TGFβ1-treated positive control group but significantly higher than the naturally healing blank control group. This indicates that ColpepA1 21 (long chain) can promote cell migration and scratch healing, although its effect is slightly less than that of known growth factors. It is worth noting that the ColpepA1 21 experimental group of this patent showed an average scratch closure rate of 41.44%, which was significantly higher than that of the blank control group. It was not only higher than that of the ColpepA1 21 (long chain) experimental group, but also showed a stronger promoting effect compared with the positive control group.

[0390] The results of this experiment demonstrate that the recombinant humanized collagen type 21 (ColpepA1 21) of this patent can significantly promote skin cell migration and accelerate wound closure, especially at higher concentrations, where its effect is more pronounced and superior to some known promoting factors. This highlights the potential application value of ColpepA1 21 in the development of wound repair materials and provides important scientific basis for the design and development of novel composite materials based on collagen type 21. Furthermore, its ability to promote cell migration and accelerate wound healing further confirms the significant advantages of ColpepA1 21 in improving the quality and speed of wound healing, especially in situations requiring rapid wound healing.

[0391] Example 6.2: Generation of aquaporin 3

[0392] This study aimed to explore the effect of recombinant human collagen type 21 (ColpepA1 21) on the expression of aquaporin 3 (AQP3), a key factor in promoting wound healing. AQP3 is essential for maintaining skin moisture balance, promoting cell proliferation, and accelerating wound healing. By evaluating the promoting effect of ColpepA1 21 on AQP3 expression, the study aimed to reveal its potential application in wound repair and promoting skin regeneration.

[0393] Experimental Groups:

[0394] Blank control group (Control): No reagents were added; used for comparison of baseline levels.

[0395] Positive control group (TGFβ1): 100 ng / mL TGFβ1 was added as a positive control known to promote AQP3 expression.

[0396] Collagen control group (Col III): 50 ppm of type III collagen was added to compare the effects of different types of collagen.

[0397] ColpepA1 21 experimental group: 10 ppm of type 21 recombinant humanized collagen was added to evaluate its effect on AQP3 expression.

[0398] All groups were cultured with HaCaT keratinocytes and plated for 24 h. When the cell confluence reached 90%, samples were added and cultured for another 24 h. Cells were then harvested and lysed on ice. After high-speed centrifugation (10,000 rpm), the supernatant was collected and used for AQP3 expression level detection with an ELISA kit.

[0399] Data collection and analysis:

[0400] Samples are added to microplates pre-coated with specific antibodies, followed by the addition of a secondary antibody that binds to the target protein. A color reaction is then generated by adding substrate, the intensity of which is proportional to the protein content in the sample. The intensity of the color reaction in each well is read using a microplate reader, and the specific AQP3 content is calculated using a standard curve.

[0401] Figure 22 shows the aquaporin 3 (AQP3) content in each experimental group. The results indicate that the control group showed the lowest AQP3 expression level, providing a baseline AQP3 content in untreated cells. The TGFβ1 group significantly increased AQP3 content, a result consistent with the role of TGFβ1 in promoting cell proliferation and migration in cell biology, suggesting that TGFβ1 can effectively enhance AQP3 expression. The 50 ppm type III collagen group (Col III) also increased AQP3 content, but its effect appeared to be less than that of the TGFβ1 group. The 10 ppm recombinant humanized collagen type 21 group (ColpepA1 21) increased AQP3 content by 16.93% compared with the control group, indicating that this low concentration of ColpepA1 21 can promote AQP3 expression.

[0402] In the field of wound repair, maintaining adequate wound moisture is considered a key factor in promoting healing. Since AQP3 plays a central role in regulating skin water transport and maintaining cellular water balance, increased AQP3 expression is crucial for promoting rapid wound healing and maintaining the hydration state of damaged skin. This is significant for maintaining adequate wound moisture, accelerating cell migration and proliferation during wound healing. The results of this embodiment demonstrate that even at low concentrations, ColpepA1 21 can significantly promote AQP3 expression, proving its highly efficient promoting effect. This is particularly important for developing dose-efficiency and cost-effective wound repair materials. This embodiment showcases the potential value of ColpepA1 21 in promoting wound healing and enhancing skin barrier function.

[0403] Therefore, this embodiment not only demonstrates the application value of recombinant humanized collagen type 21 in wound repair and skin regeneration, but also provides a solid scientific foundation for further research on the potential mechanisms of ColpepA1 21 in wound repair. This discovery is expected to promote the development of novel composite materials based on collagen type 21, providing more effective treatment options for wound repair.

[0404] Example 6.3: 3D Epidermal Skin Model Testing

[0405] This embodiment aims to evaluate the effect of collagen type 21 (ColpepA1 21) on wound repair in a 3D epidermal skin model, specifically by measuring changes in the levels of filaggrin (FLG), lobelin (LOR), and glutaminase 1 (TGM1). These three proteins play a crucial role in the formation and maintenance of the skin barrier, and changes in their levels directly reflect improvements in skin repair and barrier function.

[0406] The experimental groups are as follows:

[0407] Blank control group (BC): No treatment or drug administration was performed, and it was used to provide baseline data for the experiment.

[0408] Negative control group (NC): Received 600 mJ / cm² 2 UVB irradiation without any drug treatment was used to demonstrate the damaging effects of UVB irradiation.

[0409] Positive control group (PC): Received 600 mJ / cm² 2 UVB irradiation and treatment with WY14643 (50 μM), a known repair promoter, were used to demonstrate the effectiveness of the experimental system's response.

[0410] Sample group (collagen type 21, ColpepA1 21): received 600 mJ / cm 2 UVB irradiation was followed by treatment with 0.0125% (v / v) type 21 collagen to evaluate its repair effect on UVB-induced damage.

[0411] Testing System: 3D Epidermal Skin Model The batch number is ES231107, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0412] Main reagents: EpiGrowth culture medium (Guangdong Boxi Biotechnology), PBS (Solepro), WY14643 (Sigma), FLG antibody (Abeam), LOR antibody (Abeam), TGM1 antibody (Abeam), paraformaldehyde (Biosharp).

[0413] Main equipment: CO2 incubator (Thermo, 1501), clean bench (Suzhou Antai, SW-CJ-1F), fluorescence microscope (Leica, DM2500).

[0414] Experimental procedure:

[0415] 1. Model preparation: Transfer the 3D epidermal skin model to a 6-well plate, add 0.9 mL of EpiGrowth culture medium to each well beforehand, and label each test group number on the 6-well plate.

[0416] 2. Sample Preparation: According to the grouping, evenly apply the sample stock solution to the model surface. For all groups requiring UVB irradiation, apply 600 mJ / cm². 2 After UVB irradiation treatment, the model was placed in a CO2 incubator and incubated at 37°C and 5% CO2 for 24 hours.

[0417] 3. Model cleaning: After incubation, clean the model surface with sterile PBS to remove residual test material, and then gently wipe away any residual liquid inside and outside the model with sterile cotton swabs.

[0418] 4. Immunofluorescence assay: After model treatment and incubation, the model used for detection was circumcised and fixed with 4% paraformaldehyde for 24 hours. After fixation, immunofluorescence detection of FLG, LOR, and TGM1 was performed. Images were taken using a fluorescence microscope, and relevant image data were collected for analysis.

[0419] 5. Improvement Rate Calculation: Based on the data obtained from immunofluorescence detection, the improvement rate of each experimental group was calculated using the following formula to evaluate the repair effect of ColpepA1 21:

[0420] Improvement rate (%) = (Experimental group - Blank control group) / Blank control group × 100%

[0421] Figure 23 shows the relative integrated optical density (IOD) values ​​of filaggrin (FLG), reflecting the expression levels of FLG under different experimental conditions. A t-test was used to examine the significant differences between the experimental groups and the negative control group, where significance is indicated by * (p < 0.05) and ** (p < 0.01) (Figures 24 and 25 are similar). FLG is an indispensable component of the stratum corneum structure, promoting skin hydration and enhancing barrier function. FLG hydrolysis products are natural moisturizing factors, effectively maintaining skin moisture, skin barrier function, and overall skin health. The basal control group (BC) had the highest FLG expression level, indicating normal skin barrier function. In contrast, the IOD value of FLG in the negative control group (NC) decreased significantly after UVB irradiation, revealing the effects of UVB irradiation on keratinocytes and its induced barrier function damage. In the positive control group (PC), after treatment with WY14643, FLG expression increased significantly, thus confirming the repair effect of the positive drug on stratum corneum damage. Meanwhile, the application of type 21 collagen (ColpepA121 0.0125%) in sample group significantly increased FLG expression, suggesting its potential role in promoting stratum corneum recovery and barrier function.

[0422] Figure 24 shows the relative integrated optical density (IOD) value of lobelin (LOR). LOR is one of the main proteins in epidermal keratinocytes, constituting the outer barrier of the skin. It enhances the structural strength and barrier function of the skin by cross-linking with other proteins, generating resistance to external physical, chemical, and biological stimuli. Under UVB irradiation, the negative control group (NC) showed a significant reduction in LOR expression, indicating the damage to skin structural strength and barrier function caused by external stimuli. After treatment with WY14643, the positive control group (PC) showed a significant increase in LOR expression, indicating the role of the positive drug in the recovery of stratum corneum barrier function. The LOR level in the sample group containing type 21 collagen (ColpepA1 21 0.0125%) was significantly higher than that in the NC group, further emphasizing the importance of type 21 collagen in improving skin barrier and defense capabilities, which is particularly crucial for wound healing.

[0423] Figure 25 shows the relative integrated optical density (IOD) values ​​of glutaminase 1 (TGM1). TGM1 is a key enzyme involved in the cross-linking process of stratum corneum proteins, promoting terminal differentiation of keratinocytes and forming a robust skin barrier. Its activity is crucial for maintaining the integrity and function of the skin barrier. As shown, the TGM1 level in the negative control group (NC) was significantly reduced by UVB irradiation, highlighting the negative impact of irradiation on the cross-linking process of stratum corneum proteins. The positive control group (PC) showed a significantly increased TGM1 level compared to the negative control group, suggesting that WY14643 may promote TGM1 expression. The significant promoting effect of collagen type 21 (ColpepA1 21 0.0125%) on TGM1 (an increase of 315.63%) revealed its positive impact on accelerating skin barrier formation and terminal differentiation of keratinocytes, which has significant benefits for rapid wound healing and reduced infection risk.

[0424] In summary, our findings clearly demonstrate that collagen type 21 (ColpepA1 21) has a significant positive regulatory effect on the expression of skin barrier proteins FLG, LOR, and TGM1. These proteins are key components of skin hydration, structural integrity, and barrier function, and their enhanced expression is crucial for skin repair and wound healing after UVB irradiation. The application of collagen type 21 can promote the recovery of these key factors, provide necessary barrier protection for wounds, and potentially accelerate the healing process. Therefore, collagen type 21 not only demonstrates its potential clinical applications in skin biology and wound treatment but also provides valuable ingredient information for the development of skin care products. These findings support further clinical research and product innovation to translate the benefits of collagen type 21 into effective wound treatment strategies.

[0425] In summary, the above experimental results demonstrate that recombinant humanized collagen type 21 (ColpepA1 21) exhibits significant promoting effects on key biological processes in wound repair, providing comprehensive support for wound healing and skin regeneration.

[0426] Promoting Cell Migration: ColpepA1 21, especially at higher concentrations (50 ppm), demonstrated a significant ability to accelerate healing through HaCaT cell scratch assays, which is crucial for rapid wound closure and shortening the healing cycle. At the dermal callus level, ColpepA1 21 effectively promotes the key cell migration process in wound repair.

[0427] Enhanced hydration: ColpepA1 21 significantly increased AQP3 expression, highlighting its importance in maintaining the hydration environment of the wound and surrounding tissues, and in preserving normal cell function during wound healing, thus enhancing the hydration capacity of skin cells. In wound repair, enhanced AQP3 expression helps maintain adequate moisture in the wound area, thereby promoting normal cell function and effective wound healing.

[0428] Enhancing the rapid formation of the epidermal barrier: ColpepA121 revealed its potential role in promoting keratinocyte migration and shortening the healing cycle by significantly increasing the expression of the key protein FLG. Furthermore, enhanced expression of LOR indicates the effectiveness of ColpepA121 in strengthening epidermal barrier structure and improving the skin's defense against external stimuli. Further, the increased TGM1 activity underscores the crucial role of ColpepA121 in the terminal differentiation of keratinocytes and epidermal barrier formation.

[0429] These experimental results collectively highlight the application potential of ColpepA1 21 in wound repair, especially as a key active ingredient in novel composite adhesives. ColpepA1 21 not only promotes rapid skin repair and restoration of structural integrity but also enhances the skin's natural barrier function and hydration, making it an ideal choice for developing highly effective wound repair treatment strategies and skin regeneration products. Therefore, ColpepA1 21 is expected to become a key factor driving advancements in skin health and repair science in the future development of wound repair materials and skin care products.

[0430] Example 7: Preparation of ColpepA1 21 Collagen Hydrogel Dressing

[0431] Material:

[0432] ColpepA1 21 Collagen Type 21 Solution: 3% (w / v)

[0433] Hyaluronic acid: 1% (w / v)

[0434] Crosslinking agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC): 0.1% (w / v)

[0435] Mixing steps:

[0436] Collagen and hyaluronic acid were dissolved separately in sodium acetate buffer (pH 5.5) at room temperature. Hyaluronic acid was slowly added to the collagen solution while continuously stirring to ensure thorough mixing. EDC was slowly added as a cross-linking agent to initiate the cross-linking reaction, and stirring was continued at room temperature for 1 hour to promote full cross-linking and form a preliminary cross-linked network.

[0437] Gel formation:

[0438] Transfer the mixture to a mold and let it stand at room temperature for 1 hour. Then, move the mold to 4°C and refrigerate for 24 hours to promote complete gelation. Remove the gel from the mold and soak it in sterile cold physiological saline for 12 hours to remove unreacted crosslinking agents and byproducts.

[0439] Sterilization and packaging:

[0440] Low-temperature sterilization methods, such as plasma sterilization, are used to reduce potential heat damage. Under aseptic conditions, the hydrogel is cut to the required size and sealed in aseptic packaging material, stored at 4°C, away from light and high temperatures, to maintain product stability and bioactivity, and to prevent microbial contamination and drying.

[0441] Comparative Example 1: Preparation of Type III Collagen Hydrogel Dressing

[0442] Material:

[0443] Type III collagen solution: 3% (w / v)

[0444] Hyaluronic acid: 1% (w / v)

[0445] Crosslinking agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC): 0.1% (w / v)

[0446] The preparation method is the same as in Example 4.

[0447] Example 8: Experiment on the promotion of wound healing and cell regeneration by type 21 collagen hydrogel dressing

[0448] This embodiment aims to evaluate the effect of type 21 collagen hydrogel dressing on the activity of barrier keratinocytes under cell culture conditions, particularly its protective effect under oxidative stress. By comparing it with type III collagen hydrogel dressing, the potential of type 21 collagen in promoting wound healing and cell regeneration is further explored.

[0449] Experimental methods:

[0450] Cell seeding: Culture keratinocytes (HaCaT) and dilute the cells to 3 × 10⁻⁶. 5 At a seeding density of cells / ml, the cell dilution was seeded into 96-well plates at a density of 100 μl / well and incubated in a cell culture incubator (37℃, 5% CO2, 95% RH) for 24±2 h.

[0451] Experimental Groups:

[0452] Control and zeroing groups: contain only culture medium;

[0453] H2O2 model group (NC): Cells were treated with H2O2 to simulate oxidative stress;

[0454] Based on the H2O2 modeling group, the conditions for each sample group are as follows:

[0455] Sample group A: Added 1000 ppm ColpepA1 21;

[0456] Sample group B: Type III collagen hydrogel prepared by the method of Comparative Example 1 with the addition of an equal amount of 1000 ppm of type III collagen;

[0457] Sample group C: Type 21 collagen mixed hydrogel prepared by the method in Example 4 with the addition of an equal amount of 1000 ppm ColpepA1 21;

[0458] The activity test experiment was set up with a zeroing group, a normal group, an H2O2 modeling group, and a sample group. Each group had 3 replicate wells.

[0459] Solution preparation:

[0460] Sample: Dissolve the sample in cell culture medium (add DMSO to dissolve samples that cannot be dissolved, but the final concentration of DMSO shall not exceed 0.5%), and then dilute with cell culture medium; H2O2 working solution: Prepare H2O2 working solution by using serum-free medium with H2O2 stock solution.

[0461] CCK-8 working solution: Add 1 ml of CCK-8 stock solution to 9 ml of serum-free culture medium and mix well to prepare 10 ml of CCK-8 working solution.

[0462] H2O2 induction: When the cell confluence in the 96-well plate reaches 50%–70%, remove the cell culture medium from each group, wash with PBS once and discard the solution, then add H2O2 working solution.

[0463] Sample loading: After induction, wash twice with PBS. Add 100 μl of culture medium to each well of the normal group; add 100 μl of culture medium containing the corresponding concentration of sample to each well of the sample group; no cell seeding is done in the zeroing group, only 100 μl of cell culture medium is added. After sample loading, place the 96-well plate in an incubator (37℃, 5% CO2, 95% RH) and incubate for 24±2 h.

[0464] Cell viability assay: After culturing cells for 24±2h, the cell state was first observed under a microscope, and then the supernatant was discarded. 100μl of CCK-8 working solution was added to each well of each group and incubated at 37℃ in the dark for 2±0.5h. After incubation, the OD value was read at 450nm, and the percentage of cell viability was calculated according to the following formula.

[0465] Cell viability (%) = (OD of sample group - OD of zero-adjustment group) / (OD of blank group - OD of zero-adjustment group) × 100%.

[0466] Figure 26 shows the cell viability data of the control group, the H2O2 model group, and the three sample groups. The results show that the control group serves as a baseline, representing the cell survival status under natural conditions without any external intervention. Cell viability in the H2O2 model group significantly decreased to 71.16%, indicating significant oxidative damage under oxidative stress, which reduced cell viability. Cell viability in sample group A, with the addition of 1000 ppm ColpepA121, increased to 119.26%, indicating that type 21 collagen significantly improved the viability of damaged cells, exceeding levels under normal physiological conditions. Cell viability in sample group B, with the addition of 1000 ppm type III collagen mixed hydrogel, was 111.22%, also showing some mitigation effect on oxidative damage, but slightly less than that of pure type 21 collagen. Cell viability in sample group C, with the addition of 1000 ppm type 21 collagen mixed hydrogel, was the highest, reaching 145.89%, indicating that the composite material of type 21 collagen and other components has a synergistic effect, further improving cell recovery and survival.

[0467] The high cellular activity of type 21 collagen hydrogel indicates that it not only protects cells from oxidative stress damage caused by H2O2 but also promotes the recovery of damaged cells. In particular, the highest cellular activity shown in sample group C suggests that ColpepA1 21 may trigger cell signaling pathways in specific formulations, thereby enhancing the antioxidant and repair capabilities of cells. These properties of type 21 collagen hydrogel dressings, especially their superior effects in higher concentrations and composite materials, provide strong scientific support for their clinical application in treating inflammation and promoting wound healing. Due to its excellent cell-activating effects, type 21 collagen hydrogel can serve as a potential therapeutic material for promoting wound healing, reducing inflammatory responses, and accelerating skin regeneration.

[0468] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention are covered within the scope of the present invention.

Claims

1. A 21-type recombinant humanized collagen, wherein, The amino acid sequence of the collagen includes the amino acid sequence shown in SEQ ID No. 1 or contains an amino acid sequence that has at least 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID No.

1.

2. A nucleic acid molecule, wherein, The nucleic acid molecule contains a fragment encoding the collagen according to claim 1.

3. A vector, wherein, The carrier contains the nucleic acid molecule as described in claim 2.

4. A host cell, wherein, The host cell contains the nucleic acid molecule of claim 2 or the vector of claim 3.

5. The use of the collagen of claim 1, the nucleic acid molecule of claim 2, the carrier of claim 3, or the host cell of claim 4 in the preparation of anti-aging and / or whitening and brightening medical compositions.

6. A method of preparing the collagen of claim 1, wherein, The preparation method includes the step of preparing the collagen using the expression system of Pichia pastoris; Preferably, the preparation method includes one or more steps selected from introducing the vector of claim 3 into Pichia pastoris to form Pichia pastoris genetically engineered bacteria, fermenting and culturing the Pichia pastoris genetically engineered bacteria, and inducing and expressing the collagen.

7. A collagen material for wound repair, wherein, The collagen material comprises the collagen as described in claim 1.

8. The collagen material for wound repair according to claim 7, wherein, The collagen material is prepared by the following steps: (1) Mix collagen with auxiliary ingredients; (2) Coagulation and molding of collagen materials; (3) Sterilize the collagen material.

9. The collagen material according to claim 8, wherein: The mixing is carried out at room temperature or under heating conditions; and / or The solidification process is carried out at room temperature or low temperature; and / or The sterilization includes any one of the following methods: heat sterilization, chemical sterilization, or radiation sterilization; Preferably, the sterilization is gamma ray sterilization or plasma sterilization.

10. The collagen material of claim 8 or 9, wherein, The method steps are selected from any one of the following methods (1)-(4): Method (1): The mixture of collagen and auxiliary ingredients was cross-linked in sodium acetate buffer at room temperature; the mixture was transferred to a mold, left to stand, and refrigerated for 12-24 hours to form. Low-temperature curing, plasma sterilization; or Method (2): Pre-soak collagen and auxiliary ingredients in water and heat to dissolve; add hyaluronic acid solution, stir and solidify at room temperature; Low-temperature curing, plasma sterilization; or Method (3): Mix collagen and auxiliary ingredients, spin-coat onto a substrate; dry and peel off, then sterilize with gamma rays; or Method (4): Mix collagen and auxiliary ingredients, pour into a mold, freeze, and then freeze-dry; Gas cross-linking, gamma ray sterilization.

11. The collagen material of any one of claims 8-10, wherein, The auxiliary ingredients are selected from one or more of the following: antibacterial agents, anti-inflammatory agents, growth factors, moisturizers, antioxidants, emulsifiers, thickeners, etc.

12. The collagen material of any one of claims 8-11, wherein, The concentration of collagen is 0.0001% to 50% based on the total weight of the collagen material. Preferably, the concentration of the collagen is 1% to 20%.

13. The collagen material of any one of claims 8-12, wherein, The weight ratio of collagen to auxiliary ingredients is selected from: Collagen to antibacterial agent: 5:1 to 20:1; or Collagen to anti-inflammatory agent: 20:1 to 100:1; or Collagen to growth factor ratio: 50:1 to 200:1; or Collagen to moisturizer ratio: 1:1 to 10:1; or Collagen to antioxidant ratio: 10:1 to 50:1; or Collagen and emulsifier or thickener: one or more combinations of 5:1 to 20:

1.

14. The collagen material of any one of claims 8-13, wherein, The method steps are selected from any one of the following methods (1)-(4): Method (1): A mixture of type 21 collagen, hyaluronic acid, and a cross-linking agent was subjected to a cross-linking reaction in sodium acetate buffer at room temperature; the mixture was transferred to a mold, allowed to stand, and refrigerated for 12-24 hours to form; it was then cured at low temperature and sterilized by plasma; or Method (2): The mixture of gelatin and collagen was pre-soaked in water and heated to dissolve; hyaluronic acid solution was added, stirred and solidified at room temperature; low-temperature curing and plasma sterilization were performed; or Method (3): Mix type 21 collagen and PLGA in a specific ratio, spin-coat the mixture onto a substrate; dry and peel off, then sterilize with gamma rays; or Method (4): Mix type 21 collagen and chitosan in a specific ratio, pour into a mold, freeze, and then freeze-dry. Gas cross-linking, gamma ray sterilization; Preferably, the ratio in method (1) is: 2-3% (w / v) type 21 collagen, 1% (w / v) hyaluronic acid, and 0.1% (w / v) crosslinking agent; more preferably, the proportion of type 21 collagen is 3% (w / v); Preferably, the crosslinking agent in method (1) is EDC; Preferably, the ratio in method (2) is: 2% (w / v) type 21 collagen solution, 1% (w / v) hyaluronic acid, and 3% (w / v) gelatin; Preferably, the ratio in method (3) is: 1.5% (w / v) type 21 collagen solution and 2% (w / v) PLGA; Preferably, the ratio in method (4) is: 2.5% (w / v) type 21 collagen solution and 1.5% (w / v) chitosan.

15. A method for preparing the collagen material according to any one of claims 7-14.

16. A composition wherein the active ingredient of the composition comprises the collagen of claim 1; Preferably, the composition comprises one or more auxiliary ingredients or excipients; More preferably, the auxiliary ingredient is selected from one or more of moisturizers, antioxidants, ultraviolet absorbers, penetration enhancers, and plant extracts; More preferably, the excipients are selected from one or more of stabilizers, emulsifiers, conditioning agents, diluents, fillers, binders, humectants, absorption promoters, surfactants, lubricants, flavorings or seasonings.

17. A medical article, wherein, The medical product comprises the collagen of claim 1, the collagen material of any one of claims 7-14 or prepared by the method of claim 15, or the composition of claim 16; Preferably, the medical product is a solid, liquid, semi-solid, or gel preparation; Preferably, the formulation of the medical product is selected from one or more of the following: serum, face mask, lotion, skin enhancer, topical gel, cream, dressing, spray, oral medication, or injection. More preferably, the cream is a face cream; More preferably, the skin enhancer is an injectable skin enhancer; More preferably, the medical product is in the form of a dressing, including but not limited to hydrogel dressings, film dressings, sponge or fiber dressings, etc. More preferably, the medical product is in the form of a hydrogel dressing, and even more preferably, a collagen-hyaluronic acid hydrogel dressing.

18. The composition of claim 16 or the medical product of claim 17 is used for anti-aging, whitening and brightening and / or wound repair.

19. The use of the collagen of claim 1, the nucleic acid molecule of claim 2, the carrier of claim 3, or the host cell of claim 4 in the preparation of a medical composition for wound repair; Preferably, the wound repair medical composition comprises collagen material prepared by any one of claims 7-14 or by the method of claim 15.

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