Facial anti-aging collagen peptide, composition and application thereof

Abstract

The present invention relates to the field of biomedicine, particularly to anti-aging collagen peptides, compositions, and their applications for facial treatments. Specifically, it relates to a recombinant humanized collagen peptide type 21 (ColpepA121) and its compositions for use in anti-aging treatments and skin care. The recombinant collagen of this invention can effectively promote the repair process after skin damage, enhance skin hydration and barrier function, reduce fine lines and wrinkles, and improve skin firmness and elasticity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a facial anti-aging collagen peptide, a composition and an application thereof. Background Art

[0002] Collagen is not only one of the most abundant proteins in the human body, accounting for over 30% of the total protein content, but it is also the cornerstone for the structure and function of numerous tissues. It is present in the skin, bones, tendons, blood vessels, and other connective tissues, forming a three-dimensional network that provides mechanical strength and stability. In the skin, collagen maintains its firmness and elasticity, maintaining its smooth surface and radiance. With aging, the body's natural production of collagen slows, and existing collagen is gradually degraded and damaged by factors such as UV exposure, environmental pollution, and unhealthy lifestyle habits. This causes the skin to gradually lose its elasticity and firmness, resulting in signs of aging such as sagging, fine lines, and wrinkles. Furthermore, the reduction of collagen in other tissues can affect bone density, tendon elasticity, and blood vessel stability, thereby impacting overall health. Modern scientific research is actively seeking methods to slow collagen degradation and increase its production in the body in order to mitigate the effects of aging. Currently, a variety of collagen supplements, skin care products, and medical cosmetic procedures are available on the market, attempting to restore the youthfulness of skin and other tissues by externally supplementing or stimulating natural collagen production. However, how to effectively enhance collagen absorption and utilization in the body, and how to precisely target the specific collagen requirements during aging, remain key research areas and challenges.

[0003] In this context, in-depth research on specific collagen types, such as type 21 collagen (COL21A1), is particularly important. Type 21 collagen is a non-fibrillar collagen that is relatively rare in the adult human body, accounting for less than 1% of the total collagen composition. Compared to other, more well-known collagen types, type 21 collagen has been relatively understudied. Type 21 collagen belongs to the small molecule collagen family. As part of the FACIT (fibril-associated collagen with interrupted triple helices) family, type 21 collagen plays a crucial role in connecting extracellular matrix components. According to research by Chou MY and Li HC (March 2002), type 21 collagen is primarily expressed in vascular smooth muscle cells and may play a role in extracellular matrix assembly during angiogenesis. Furthermore, the gene expression of type 21 collagen is regulated by developmental stage, with higher expression levels during fetal development, suggesting that it may play an important role in the development of various tissues. In recent years, research on type 21 collagen has revealed its potential role in vascular health and blood pressure regulation. Surendran et al. (2016) conducted a large-scale genotyping survey and found that the type 21 collagen gene is involved in collagen formation in various tissues, including the heart and aorta, and is associated with vascular remodeling and hypertension. This provides new insights into the role of COL21A1 in promoting skin health and anti-aging. These properties give type 21 collagen unique potential for applications in skin health, anti-aging, and vascular health.

[0004] There are currently a variety of anti-aging and whitening products on the market, most of which focus on using regular collagen or its derivatives to replenish collagen loss in the skin. However, the effectiveness of these products is often limited by the size of the collagen molecules and their absorption rate through the skin, and it is difficult to optimize the biological functions of specific collagens. Due to its unique biological properties and mechanism of action, type 21 collagen may become a key area of ​​future anti-aging and whitening research and application. By understanding its specific biological functions and mechanisms of action, it is possible to develop highly targeted, bioavailable, and multi-functional anti-aging and whitening therapies and products, not only to improve skin appearance but also to promote overall health and slow the aging process. Summary of the Invention

[0005] The present invention relates to a type 21 recombinant humanized collagen (ColpepA121) with a specific amino acid sequence, and its application in the field of skin care, especially in anti-aging and whitening and brightening of the face.

[0006] In one aspect, the present invention provides a type 21 recombinant humanized collagen, wherein the amino acid sequence of the collagen is selected from SEQ ID No. 1:

[0007] GKPGLQGPKGDPGLPGNPGYPGQPGQDGKPGYQGIAGTPGVPGSPGIQGARGLPGYKGEPGRDGD

[0008] KGDRGLPGFPGLHGMPGSKGEMGAKGDKGSPGFYGKKGAKGEKGNAGFPGLPGPAGEPGRHGKD

[0009] GLMGSPGFKGEAGSPGAPGQDGTRGEPGIPGFPGNRGLMGQKGEIGPPGQQGKKGAPGMPGLMGS

[0010] NGSPGQPGTPGSKGSKGEPGIQGMPGASGLKGEPGATG

[0011] 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 a sequence identity of at least 90%, 95%, 96%, 97%, 98%, 99% or higher with the amino acid sequence shown in SEQ ID No. 1.

[0012] This specific amino acid sequence of collagen is optimized to increase its bioavailability and efficacy in the skin.

[0013] In one embodiment, the collagen of the present invention can enhance skin moisture regulation and optimize barrier function.

[0014] In one embodiment, the collagen of the present invention can provide key support for anti-aging and skin rejuvenation.

[0015] In one embodiment, providing key support for anti-aging and skin rejuvenation includes reducing skin lines and wrinkles and / or increasing skin firmness.

[0016] In one embodiment, the collagen of the present invention can whiten the skin and / or provide support for skin whitening and brightening.

[0017] In one embodiment, providing support for skin whitening and lightening comprises inhibiting tyrosinase activity.

[0018] In one embodiment, providing support for skin whitening and lightening includes suppressing the content of melanin.

[0019] In one embodiment, the collagen of the present invention can promote the production of type I collagen.

[0020] In one embodiment, the collagen of the present invention can significantly increase the content of type I collagen.

[0021] 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, and particularly show significant benefits in improving skin firmness and / or reducing signs of aging.

[0022] Another aspect of the present invention also provides a nucleic acid molecule comprising a fragment encoding any of the aforementioned collagen proteins.

[0023] Another aspect of the present invention also provides a vector containing the aforementioned nucleic acid molecule.

[0024] Another aspect of the present invention also provides a host cell containing the aforementioned nucleic acid molecule or vector.

[0025] Another aspect of the present invention further provides a method for preparing the aforementioned collagen, which comprises the step of preparing the collagen using a Pichia pastoris expression system.

[0026] In one embodiment, the method comprises one or more steps selected from the group consisting of: introducing the aforementioned vector into Pichia pastoris to form a genetically engineered Pichia pastoris bacterium, fermenting and culturing the genetically engineered Pichia pastoris bacterium, and inducing and expressing the collagen.

[0027] In one embodiment, the vector is pPIC9K or pGAPZα.

[0028] Preferably, the vector is pPIC9K

[0029] In one embodiment, the Pichia pastoris is Pichia pastoris GS115, X-33, KM71, etc.

[0030] Preferably, the Pichia pastoris is Pichia pastoris GS115.

[0031] In one embodiment, the aforementioned vector is introduced into Pichia pastoris to form genetically engineered Pichia pastoris by electroporation, chemical transformation, or ultrasound-assisted transfection.

[0032] Preferably, the method used in the step is electroporation.

[0033] In one embodiment, the fermentation culture medium is BMGY medium.

[0034] In one embodiment, the induction and expression medium is BMMY medium.

[0035] The collagen synthesis process of the present invention covers a full set of steps from gene construction, cell culture to protein expression, ensuring efficient and stable protein production.

[0036] In one embodiment, the method for preparing collagen of the present invention further comprises a purification step, wherein the purification step can successfully extract and purify the collagen from the Pichia pastoris expression system.

[0037] In one embodiment, the purification step comprises the steps of subjecting the fermentation broth supernatant to NaCl treatment and pH adjustment.

[0038] In one embodiment, the purification step comprises a step of specifically purifying type 21 collagen using nickel affinity chromatography technology; preferably, the type 21 collagen carries a His tag.

[0039] In one embodiment, the purification step comprises the step of obtaining the purified collagen by imidazole elution.

[0040] The purification steps described above not only ensure the high purity of the target protein but also maintain its biological activity, providing high-quality protein samples for further applied research and product development. The entire purification process, designed to balance efficiency and protein stability, is a crucial foundation for the application of recombinant proteins in anti-aging and skin care applications.

[0041] Another aspect of the present invention provides a composition, wherein the active ingredient of the composition comprises the collagen; preferably, the collagen is type 21 recombinant humanized collagen.

[0042] In one embodiment, the composition of the present invention further comprises auxiliary ingredients or excipients.

[0043] In one embodiment, the auxiliary ingredients or excipients are used to achieve skin moisturizing, anti-oxidation or whitening effects.

[0044] In one embodiment, the auxiliary ingredient is a moisturizer.

[0045] The moisturizing agent of the present invention can increase the moisture content of the skin, thereby improving the softness and elasticity of the skin and enhancing its effect in the fields of reducing wrinkles and anti-aging.

[0046] In one embodiment, the moisturizing agent is selected from one or more of hyaluronic acid, glycerin, panthenol (vitamin B5), betaine, polysorbate, natural moisturizing factor (NMF), allantoin, polyols (such as butylene glycol, glycol, etc.), etc.

[0047] In one embodiment, the auxiliary ingredient is an antioxidant.

[0048] The antioxidant of the present invention can enhance the skin's ability to resist environmental oxidative damage, further improving its efficacy in anti-aging skin care.

[0049] 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, and the like.

[0050] In one embodiment, the auxiliary ingredient is a whitening component.

[0051] In one embodiment, the whitening component is selected from one or more of arbutin, niacinamide, kojic acid, tranexamic acid, glycyrrhizic acid, fruit acid, and the like.

[0052] In one embodiment, the auxiliary ingredient is an emulsifier.

[0053] The emulsifier of the present invention stabilizes the emulsification system of the skin care product and ensures uniform distribution of the active ingredients.

[0054] In one embodiment, the emulsifier is selected from one or more of polysorbate 60, glyceryl stearate, and the like.

[0055] In one embodiment, the auxiliary ingredient is a plant extract.

[0056] The plant extracts of the present invention can provide skin soothing and additional skin care benefits.

[0057] 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, and the like.

[0058] In one embodiment, the auxiliary ingredient is a UV absorber.

[0059] The ultraviolet absorber of the present invention can provide sun protection and reduce the damage of ultraviolet rays to the skin.

[0060] In one embodiment, the ultraviolet absorber is selected from one or more of titanium dioxide, zinc oxide, ethylhexyl methoxycinnamate, and the like.

[0061] In one embodiment, the auxiliary ingredient is a penetration enhancer.

[0062] The penetration enhancer of the present invention can enhance the permeability of collagen in the skin.

[0063] In one embodiment, the penetration enhancer is selected from one or more of liposomes, nanoparticles, and transdermal enhancers such as dimethyl sulfoxide.

[0064] In one embodiment, the auxiliary materials include one or more of stabilizers, emulsifiers, conditioners, diluents, fillers, binders, wetting agents, absorption enhancers, surfactants, lubricants, fragrances or flavorings, etc.

[0065] In one embodiment, the collagen composition of the present invention may further be added with a stabilizer such as vitamin B3 to maintain the stability and activity of the collagen and ensure a long-lasting anti-aging effect.

[0066] In one embodiment, the composition of the present invention may be used in combination with excipients and / or fillers; preferably silicic acid (dimethicone), rice bran wax, etc. The use of such excipients and / or fillers can improve the texture and appearance of the product, providing a smooth feel and excellent spreadability.

[0067] In one embodiment, the collagen composition of the present invention may contain fragrances and flavorings, preferably natural essential oils, synthetic fragrances, etc. The addition of fragrances and flavorings can enhance the sensory experience of the user during use.

[0068] The collagen composition of the present invention not only enhances its traditional efficacy in terms of skin moisturizing, improving elasticity, reducing fine lines and anti-oxidation by combining it with a variety of auxiliary ingredients, but also provides a more comprehensive and personalized skin care solution.

[0069] 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, dietary supplements, and the like. Specifically, the compositions of the present invention may be in the form of one or more of tablets, capsules, powders, granules, solutions, lozenges, jellies, creams, spirits, suspensions, tinctures, poultices, liniments, lotions, and aerosols, and may be prepared using commonly known preparation techniques. Appropriate pharmaceutical excipients, such as stabilizers, antioxidants, and preservatives, may be added to maintain product stability and effectiveness.

[0070] Another aspect of the present invention provides a product comprising the aforementioned collagen or the aforementioned composition.

[0071] In one embodiment, the product can be selected from one or more forms of cream, essence, mask, smear gel, lotion or skin booster, etc., and is used to improve skin elasticity, reduce fine lines, increase skin firmness and glossiness.

[0072] In one embodiment, the skin enhancing agent of the present invention is an injectable skin enhancing agent.

[0073] In one embodiment, the injectable skin enhancing agent 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.

[0074] In one embodiment, the injectable skin enhancer described herein is administered via a variety of minimally invasive injection devices and methods, including acupuncture devices, microneedles, microneedle rollers, automated microneedle pens (e.g., Dermapen), needle-free injection systems, motorized microneedle systems (e.g., SkinPen), ultrasonic injection devices, laser-assisted delivery systems, and pneumatic injection devices (e.g., JetPeel). These devices can precisely deliver the skin enhancer directly to the mid- or deep layers of the skin to improve the overall quality and appearance of the skin. These diverse administration methods allow the most appropriate technique to be selected to achieve optimal therapeutic outcomes, depending on the treatment goals and specific patient needs.

[0075] In one embodiment, the injectable skin enhancing agent of the present invention is specially adjusted in proportions of ingredients for different administration modes to meet various treatment needs.

[0076] In one embodiment, the injectable skin enhancer described herein has a relatively high collagen content (5% to 10%) for applications such as microneedling and microneedle rollers. Microneedles physically create microchannels in the skin, and the higher collagen concentration helps directly target the newly formed microchannels, promoting rapid skin repair and collagen regeneration.

[0077] In one embodiment, the injectable skin enhancer described herein, specifically for a hydrating injection system, contains high concentrations of antioxidants, such as vitamins C and E, which help protect the skin from free radical damage and enhance its overall resistance. Furthermore, the use of small-molecule hyaluronic acid allows for easier penetration into the deeper layers of the skin, providing improved hydration and intercellular signaling, thereby effectively enhancing skin hydration and firmness.

[0078] In one embodiment, the product of the present invention may further contain a pharmaceutically, food-, health-care-, or dietary-acceptable carrier. For example, various compatible solid or liquid fillers, gels, or solvents may be used. These carriers should be suitable for human use, possess sufficient purity, and be low in toxicity. Common carriers include cellulose and its derivatives, polyols such as glycerol, and other pharmaceutical excipients such as talc and magnesium stearate.

[0079] Another aspect of the present invention provides use of the aforementioned type 21 recombinant humanized collagen, the aforementioned nucleic acid molecule, the aforementioned vector or the aforementioned host cell in preparing a medical composition.

[0080] Another aspect of the present invention provides use of any one of the aforementioned medical compositions in the preparation of medical products.

[0081] In one embodiment, the aforementioned medical composition and / or medical product is used for anti-aging.

[0082] In one embodiment, the aforementioned medical composition and / or medical product is used for whitening and brightening the skin.

[0083] The recombinant humanized collagen type 21 (ColpepA1 21) described in this invention demonstrates significant advantages in anti-aging and skin whitening and brightening. While providing essential collagen supplementation, ColpepA1 21's unique efficacy and optimized molecular properties make it stand out among other collagen products.

[0084] 1. ColpepA1 21 has demonstrated its importance in enhancing the structural integrity of the skin, particularly by promoting the production of type I and type III collagen. This not only improves the skin's resistance to stretching and compression, but also effectively combats UV-induced photoaging, maintaining the skin's youthful appearance. Type III collagen, in particular, is a key source of skin elasticity, and ColpepA1 21's role in promoting its production supports increased skin elasticity and reduced signs of aging.

[0085] 2. The molecular size and structure of ColpepA1 21 are optimized to improve its penetration and absorption rate in the skin, which means that its active ingredients can act more deeply into the skin, providing cells with necessary nutrients, thereby supporting skin health and anti-aging at the molecular level.

[0086] 3. Cell experiments have demonstrated the effectiveness of ColpepA1 21 in skin whitening and brightening. It can effectively inhibit tyrosinase and melanin. This mechanism will help reduce skin pigmentation and achieve a whitening effect, providing a basis for its application in skin whitening and brightening products.

[0087] 4. Clinical trial results further demonstrate the multiple anti-aging effects of ColpepA1 21, demonstrating significant improvements in reducing wrinkles and dark spots, increasing skin elasticity and firmness, and improving skin radiance and color. These cosmetic and physiological improvements in the volunteers' skin conditions validate the unique efficacy of ColpepA1 21 and provide a strong scientific basis for its use in anti-aging skincare products.

[0088] In summary, ColpepA1 21 has multiple anti-aging functions as well as whitening and optimization properties, which are at the forefront of skin care science and provide new solutions for skin health and beauty. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 The quantitative test results of the type I collagen content in each experimental group of cells are shown, and the ability of different concentrations of ColpepA1 21 to promote the production of type I collagen is demonstrated by comparison.

[0090] Figure 2 The quantitative test results of the type I collagen content in each experimental group of cells are shown, and the ability of ColpepA121 of different lengths to promote type I collagen production is demonstrated by comparison.

[0091] Figure 3 Shown are the results of tyrosinase activity test after α-MSH induction in B16F10 cells.

[0092] Figure 4 Shown are the results of melanin content testing in B16F10 cells after α-MSH induction.

[0093] Figure 5 and Figure 6 Shows the mean statistics of the area and volume of crow's feet on the volunteers' faces before and after using the product.

[0094] Figure 7 and Figure 8 Shows the mean statistics of the area and volume of the volunteers' facial nasolabial folds before and after using the product.

[0095] Figure 9 Shows the mean statistics of facial skin elasticity (R2 value) of volunteers before and after product use.

[0096] Figure 10 Shows the mean statistics of the volunteers' facial skin firmness (F4 value) before and after using the product.

[0097] Figure 11 Shows the mean statistics of volunteers' facial skin glossiness before and after product use.

[0098] Figure 12 Shows the mean statistics of the volunteers' facial skin brightness (cheek L value) before and after product use.

[0099] Figure 13 Shows the mean statistics of the optical density of facial skin spots of volunteers before and after using the product.

[0100] Figure 14 Shows the mean statistics of facial erythema area of ​​volunteers before and after using the product.

[0101] Figure 15 Shown are the mean statistics of the volunteers' facial lactic acid stinging scores before and after product use.

[0102] Figure 16Shows the mean statistics of the moisture content of the volunteers' scalp before and after product use.

[0103] Figure 17 Shows the mean statistics of transepidermal water loss (TEWL value) of volunteers' scalp before and after using the product.

[0104] Figure 18 Shows the mean statistics of the volunteers' scalp erythema values ​​(EI values) before and after using the product.

[0105] Related definitions

[0106] Unless otherwise specified, the following terms used in the specification and claims have the following meanings:

[0107] As used herein, the term "collagen (COL)" refers to a vital structural protein in the human body, found in a variety of tissues and organs, such as skin, bones, tendons, ligaments, and cornea. The primary function of these proteins is to provide strength, stability, and elasticity to body tissues, ensuring the normal functioning of various physiological functions. Currently, there are at least 28 known types of collagen, each with a unique role and function in the body, forming a complex and finely regulated biological system.

[0108] As used herein, the term "type 21 collagen (COL21A1)" refers to a type of collagen found in the human body that belongs to the small molecule collagen family. As part of the FACIT (fibril-associated collagen with interrupted triple helix) family, type 21 collagen plays a vital role in connecting components of the extracellular matrix. Through its unique molecular structure, this collagen 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 it is not as well-known as type I and type III collagen, it plays a role in specific tissues and physiological processes, involving the composition of the extracellular matrix and skin repair mechanisms. Although type 21 collagen is relatively rare in the adult human body, accounting for less than 1% of the total collagen, its possible role in cellular processes such as migration, proliferation, and differentiation, as well as its potential role in maintaining extracellular matrix structure and promoting tissue regeneration, are important directions for future research.

[0109] As used herein, the term "recombinant humanized collagen type 21 (ColpepA1 21)" refers to a protein expressed in Pichia pastoris via genetic recombination technology that mimics the type 21 collagen naturally found in the human body. This protein possesses similar biological functions and structural properties to natural type 21 collagen, and its amino acid sequence has a specific encoding, designed to improve skin health and anti-aging capabilities.

[0110] As used herein, "identity" refers to the percentage of identical (i.e., identical) amino acids between two or more polypeptides. The sequence identity between two or more polypeptides can be determined by the following method: the amino acid sequence of the polypeptide is aligned and the number of positions containing the same amino acid residue in the aligned polypeptide is scored, and compared with the number of positions containing different amino acid residues in the aligned polypeptide. Polypeptides can differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same amino acid residue by the total number of amino acid residues in the polypeptide. For example, the percent identity can be calculated by dividing the number of positions containing the same amino acid residue by the total number of amino acid residues in the polypeptide and multiplying by 100.

[0111] As used herein, the term "genetically engineered Pichia pastoris" refers to a Pichia pastoris strain that has been genetically manipulated to insert specific exogenous genes. This modification enables the yeast to express proteins that are not in its own genome, allowing for the production of various recombinant proteins.

[0112] As used herein, the term "expression system" refers to a biochemical environment and process for synthesizing 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 and a specific operating process to express recombinant proteins.

[0113] As used herein, the term "HaCaT (human keratinocytes)" refers to an immortalized human keratinocyte cell line that is widely used to study skin biology, skin pathology, and drug toxicity testing. It is considered a valuable research tool because it can mimic the behavior of normal epidermal cells.

[0114] As used herein, the term "HFF-1 (human fibroblasts)" refers to fibroblasts isolated from the forearm of human fetuses. They are an important component of skin structure, responsible for synthesizing collagen and other extracellular matrix proteins, and are often used to study skin aging, wound healing, and collagen synthesis.

[0115] As used herein, the term "composition" refers to a preparation containing recombinant humanized collagen type 21 and possibly other auxiliary ingredients such as moisturizers, antioxidants or whitening ingredients, which work together to provide a more comprehensive skin care effect.

[0116] As used herein, 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.

[0117] As used herein, the term "aquaporin AQP3" refers to an aquaporin protein that is primarily expressed in skin cells and is responsible for regulating cellular water transport, playing a key role in maintaining skin moisture balance and barrier function.

[0118] As used herein, 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 water within the skin layers, and is essential for maintaining skin health, elasticity and resistance to external stress.

[0119] As used herein, the term "barrier optimization" refers to the process of enhancing the skin barrier function, including strengthening the tight junction of the epidermal layer to block external stimuli and reduce water loss, thereby protecting the skin from environmental damage and maintaining its healthy state.

[0120] As used herein, the term "type I collagen" refers to one of the main types of proteins that make up the human body's skin, bones, tendons, and other structures. This type of collagen is composed of two α1 chains and one α2 chain, forming a triple helical structure that gives the tissue its high tensile strength. Type I collagen plays a vital role in maintaining the skin's elasticity, stability, and overall health. With aging and the influence of environmental factors, type I collagen gradually degrades, leading to a loss of skin elasticity and the appearance of signs of aging, such as fine lines and wrinkles.

[0121] As used herein, the term "type III collagen" refers to the type of collagen that coexists with type I collagen in the skin's structure and is particularly abundant in young skin. It plays a crucial role in maintaining skin elasticity and assisting in the healing of damaged skin. Following skin injury, a temporary increase in type III collagen helps repair and rebuild the skin's structure. A decrease in type III collagen with age may affect the skin's elasticity and repair capacity.

[0122] As used herein, the term "photoaging" refers to the aging of the skin caused by long-term exposure to ultraviolet (UV) radiation. UV radiation can penetrate the surface of the skin and directly damage the collagen and elastic fibers in the skin, accelerating their degradation, resulting in signs of aging such as sagging, fine lines, wrinkles, and hyperpigmentation. Photoaging is another major cause of skin aging, in addition to natural aging.

[0123] As used herein, the term "UVA radiation" refers to a portion of the ultraviolet spectrum with wavelengths between 320 and 400 nanometers. UVA radiation penetrates deeply into the dermis, damaging collagen and elastin fibers, and is one of the primary causes of photoaging. UVA radiation damages the skin cumulatively, and long-term exposure can accelerate skin aging and increase the risk of skin cancer.

[0124] As used herein, 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 lines can be measured using specific instruments or software to assess the effectiveness of anti-aging products in reducing fine lines around the eyes. A reduction in area and volume generally indicates a positive effect of the product on improving skin elasticity and reducing wrinkles.

[0125] As used herein, the term "nasolabial fold area and volume" refers to the wrinkles located on either side of the mouth, which typically become more pronounced with age. Measuring nasolabial fold area and volume can be used to assess the effectiveness of anti-aging products in smoothing deep facial wrinkles. A reduction in area and volume indicates that the product is effectively improving skin structure and reducing the signs of facial aging.

[0126] As used herein, the term "facial skin elasticity (R2 value)" refers to a value obtained using a skin elasticity measuring instrument, which reflects the skin's elasticity. The R2 value is determined by measuring the skin's ability to recover after mechanical stretching. A higher R2 value indicates greater skin elasticity and resistance to deformation during aging.

[0127] As used herein, the term "facial skin firmness (F4 value)" refers to a quantitative parameter used to assess skin firmness. The F4 value is measured using specialized skin testing instruments and reflects the skin's firmness and elasticity. A higher F4 value indicates greater skin firmness, which helps reduce skin sagging and wrinkle formation.

[0128] As used herein, the term "facial skin radiance" refers to the skin's surface's ability to reflect light, a property often associated with skin health and youthfulness. Skin with a higher radiance reflects light more effectively, creating a brighter, fresher appearance that is often viewed as a sign of health and vitality. In anti-aging product evaluations, increased radiance is considered a positive indicator of product effectiveness, indicating that the product helps improve the skin's surface properties and enhances the skin's natural radiance.

[0129] As used herein, the term "facial skin brightness (cheek L value)" refers to a parameter used to measure the brightness of skin color. In color science, the L value represents a brightness scale from black (0) to white (100), with higher L values ​​indicating brighter skin. In skin science research and cosmetic effect evaluation, the cheek L value is often used as a quantitative indicator to assess the effects of improving skin brightness and color. Improving the cheek L value usually means that the skin becomes brighter and more even-toned, which is one of the desired effects of whitening or anti-aging products.

[0130] As used herein, the term "facial skin spot optical density" refers to the color depth and distribution density of discolored areas on the skin. This metric, measured using specific instruments, is used to evaluate the effectiveness of whitening or spot-reducing products. A higher spot optical density indicates darker, more densely distributed spots, while a lower spot optical density indicates lighter, less densely distributed spots. Whitening or spot-reducing products are designed to reduce the spot optical density, promoting a more even skin tone.

[0131] As used herein, the term "facial erythema area" refers to the size of a red spot on the skin surface caused by inflammation or other causes. Erythema is often associated with inflammatory conditions, allergic reactions, or other skin conditions. 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.

[0132] As used herein, the term "Facial Lactic Acid Stinging Score" refers to a subjective rating of the skin irritation (e.g., stinging) caused by a product. During skin irritation testing, participants rated the degree of irritation they experienced after using the product (e.g., none, mild, moderate, strong). Lower scores indicate less irritation and greater suitability for sensitive skin.

[0133] As used herein, the term "scalp hydration" refers to the ability of the scalp's surface layer to retain moisture. This metric is crucial for assessing the health of the scalp and overall skin. A properly hydrated scalp is more healthy and vibrant, with less dryness, itchiness, and other issues. Improving scalp hydration is a goal sought by many hair and skincare products.

[0134] As used herein, the term "transepidermal water loss (TEWL)" refers to the rate of water evaporation from the scalp surface and reflects the integrity of the skin's barrier function. A low TEWL value indicates good skin barrier function, effectively preventing excessive water 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 moisturizing capacity.

[0135] As used herein, the term "scalp erythema index (EI)" refers to an instrumentally measured indicator of scalp erythema, used to quantitatively assess scalp inflammation or sensitivity. A higher EI value indicates more severe scalp inflammation or sensitivity. It is an important indicator for evaluating the soothing effects of skincare and haircare products.

[0136] As used herein, the term "TGFβ1" (transforming growth factor β1) refers to a multifunctional cytokine that regulates cell proliferation, differentiation, and migration. In skin physiology, TGFβ1 has an important influence on collagen synthesis, wound healing processes, and anti-inflammatory effects.

[0137] As used herein, the term "HPR (phthalein oxalate)" is a synthetic retinol derivative commonly used in skin care products for its ability to promote cell renewal, enhance skin repair, and improve the condition of aging skin.

[0138] As used herein, the term "composition" or "preparation" refers to a mixture containing one or more collagen proteins described herein and other components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating the absorption of the active ingredient and thereby exerting its biological activity.

[0139] As used herein, the term "antioxidant" refers to a class of substances that can neutralize free radicals and slow down or prevent cell damage, which plays an important role in reducing oxidative stress caused by environmental factors and preventing skin aging.

[0140] As used herein, the term "emulsifier" refers to a substance that can help oil and water mix to form a stable emulsion, and is used in skin care products to improve the texture and stability of the product.

[0141] As used herein, the term "humectant" refers to a substance that attracts or locks in water, helping the skin retain moisture and plays a vital role in maintaining the skin's hydration state and preventing dryness.

[0142] As used herein, the term "ultraviolet absorber" or "sunscreen" refers to a compound that can absorb or reflect ultraviolet rays, protecting the skin from harmful UV radiation, which is very important for preventing skin aging and skin cancer.

[0143] 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 manufacture, storage, and use. 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, gamma-aminobutyric acid (GABA), opines, alanine, octopine, 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, sucrose, etc., can also play the role of controlling osmotic pressure. The stabilizer specifically used in the present invention is 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 salt (such as arginine hydrochloride), glycine, and proline. Preferred stabilizers are 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.

[0144] As used herein, the term "skin booster" refers to a specific class of skin care formulations designed to improve the skin's moisture retention, elasticity, and overall health by direct injection into the mid-layers of the skin. Skin boosters include, but are not limited to, the following ingredients:

[0145] 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;

[0146] 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, and thus achieve significant anti-aging effects.

[0147] Vitamins (such as vitamin E, vitamin C, vitamin B3, etc.): They have antioxidant properties that can help reduce free radical damage and promote skin brightening and pigmentation uniformity.

[0148] Other antioxidants (such as coenzyme Q10, etc.): protect the skin from environmental damage and enhance the skin's ability to repair.

[0149] Other peptides: stimulate collagen and elastin production to help reduce the appearance of fine lines and wrinkles.

[0150] Amino Acids: Act as the basic building blocks of skin cells, promoting skin repair and regeneration.

[0151] Stabilizers (such as sodium chloride, mannitol, sorbitol, sucrose and arginine hydrochloride, etc.): ensure that the active ingredients remain stable during manufacturing, storage and application, thereby optimizing the therapeutic effect and ensuring safe use.

[0152] As used herein, the term "melanin" is an important biological pigment that determines the color of skin, hair, and eyes in humans and other organisms, is involved in pigmentation, and has the function of protecting the skin from ultraviolet damage.

[0153] As used herein, the term "tyrosinase" is a copper-containing oxidase and is the rate-limiting enzyme that regulates melanin production. It is widely found in plants, animals, and microorganisms. In the human body, tyrosinase is mainly present in melanocytes, which can catalyze tyrosine to produce dopa and its further oxidation to produce dopaquinone, ultimately forming melanin. The expression and activity of tyrosinase determine the amount of melanin produced, thereby affecting the individual's skin color and sensitivity to ultraviolet rays. It is also related to the occurrence of diseases such as excessive melanin deposition such as freckles and brown spots in the human body. The excessive activity of tyrosinase is associated with certain types of skin cancer such as melanoma. Therefore, the formation of melanin can be reduced by inhibiting the activity of tyrosinase, reducing skin pigmentation, thereby achieving the effect of skin whitening and brightening. DETAILED DESCRIPTION

[0154] Example 1: Preparation and purification of recombinant humanized collagen type 21 (ColpepA1 21)

[0155] 1. Construction of genetically engineered Pichia pastoris:

[0156] Activate yeast GS115 by streaking on a YPD plate, select a single colony of activated Pichia pastoris GS115 on the YPD plate and inoculate it in YPD liquid, culture it at constant temperature and shaking; pipette 100uL of the bacterial solution and inoculate it into 100mL YPD liquid, culture it at 30℃, 220rpm, for 12-13h, and culture it until the OD 600 =1.3-1.5; observe under a microscope for bacterial contamination, divide the bacteria into sterile 50ml centrifuge tubes, and place on ice for 10-15 minutes.

[0157] Centrifuge twice, discard the supernatant, and resuspend the bacteria in sterile water; centrifuge twice more, discard the supernatant, resuspend the bacteria in sorbitol, and then aliquot to obtain competent cells; use electroporation to transfer the vector into the competent cells, and then culture on plates until clones are produced.

[0158] 2. Fermentation culture of genetically engineered Pichia pastoris:

[0159] Pick a single colony on the YPD plate and place it in a conical flask containing 50 mL of BMGY. Cultivate at 30°C and 220 rpm for 24 h. Confirm the end of cultivation by measuring the OD600 value. Centrifuge for 10 min, collect the bacteria, wash once with BMMY, centrifuge again, and dilute the bacteria to an OD value of 0. 600 =1.0.

[0160] 3. Induction and expression of recombinant human collagen:

[0161] Transfer the diluted bacterial solution to a 500-mL conical flask and induce yeast protein expression at 29°C and 220 rpm. Then, add methanol every 24 hours to a final methanol concentration of 1% for 84 hours of continuous induction. Examine the culture medium under a microscope to see if there is any bacterial contamination. Measure the pH of the fermentation liquid, which should be less than 6. Transfer the fermentation liquid to a 500-mL round-bottom centrifuge tube and centrifuge at 15,000 g at 4°C for 20 minutes to remove the supernatant. Remove 1 mL of the supernatant and add the corresponding loading buffer. Mix well, incubate at 95°C for 10 minutes, and store at -20°C.

[0162] 4. Purification of recombinant human collagen:

[0163] Transfer the resulting fermentation broth supernatant to a conical flask, add NaCl to a final concentration of 150 mM, and adjust the pH of the fermentation broth supernatant to 8.0 with NaOH or HCl. Centrifuge at 15,000 g for 30 minutes to ensure clarity. Equilibrate the column with 10 bed volumes of binding buffer per nickel column. Load the sample at 1 mL / min, collecting the flow-through as the sample is loaded. Elute unbound proteins and contaminants with 5 bed volumes of binding buffer per nickel column. Elute the target protein with elution buffer (500 mM imidazole).

[0164] Prepare protein test samples: add the collected purified protein, supernatant before column loading, and liquid after column loading to the corresponding loading buffer, mix well, incubate at 95℃ for 10 minutes, and store at -20℃.

[0165] Through this series of steps, recombinant humanized collagen type 21 (ColpepA1 21) can be successfully prepared and purified, and the Pichia pastoris expression system can be used to obtain high-purity and high-activity target proteins.

[0166] For specific synthesis and purification methods, please refer to patent CN114195884A.

[0167] In a specific embodiment, the materials and methods used are as follows:

[0168] Cell lines: HaCaT (human keratinocytes) and HFF-1 (human fibroblasts) were purchased from Fenghui Biotechnology.

[0169] Reagents: CCK-8 kit (Japan Tongren CK04) was used for cell viability assessment, and AQP3 (Shanghai ELISA) and type I collagen kits (Shanghai ELISA CMM2023H1) were used for related protein expression analysis.

[0170] Example 2: Production of Type I Collagen

[0171] This example aims to explore the ability of recombinant humanized 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 to be one of the important indicators for evaluating the effectiveness of skin care ingredients. Type I collagen, as the main component of skin structure, is essential for maintaining the elasticity, stability and overall health of the skin. With age and the influence of environmental factors, especially ultraviolet rays (UVA / UVB), the type I collagen in the skin will gradually degrade, leading to sagging skin, fine lines and wrinkles, the so-called photoaging phenomenon. This experiment aims to explore the ability of recombinant humanized collagen type 21 (ColpepA1 21) to promote the production of type I collagen through specific experimental studies. By simulating the environment of UVA radiation damaging the skin, it is studied whether ColpepA1 21 can effectively promote the synthesis of type I collagen in damaged skin, thereby supporting its use as an effective anti-photoaging ingredient.

[0172] Experimental groups

[0173] Blank control group (Control): No reagent was added and no UVA radiation treatment was performed.

[0174] Negative control group (5J / cm 2 UVA): cells are exposed to 5J / cm 2 UVA damage treatment without any added therapeutic agent.

[0175] Positive control group (TGFβ1): 100 ng / mL TGFβ1 was added after UVA damage, which is known to promote type I collagen production.

[0176] ColpepA1 21 experimental group: 10ppm or 50ppm of type 21 recombinant humanized collagen was added after UVA damage.

[0177] ColpepA1 21 (long chain) experimental group: 50 ppm of type 21 recombinant humanized collagen ColpepA121 (long chain) was added after UVA damage.

[0178] The amino acid sequence of ColpepA1 21 (long chain) is selected from SEQ ID No. 2, a specific amino acid sequence derived from patent CN114195884A. This experimental group aims to further verify the potential effectiveness of ColpepA1 21 in promoting type I collagen production. By comparing the effects of known long-chain collagens with the short-chain collagen of this patent, the difference in the effectiveness of the short-chain collagen compared to the long-chain collagen in promoting type I collagen synthesis was evaluated.

[0179] Fibroblasts HFF-1 were cultured separately, plated and cultured for 24 hours. When the cell confluence reached 70-80%, samples were added for UVA induction and cultured for 24 hours. The supernatant was collected and the type I collagen content was determined using an ELISA kit.

[0180] Data Collection and Analysis:

[0181] The content of type I collagen in the cell supernatant was quantitatively analyzed by using an ELISA kit.

[0182] Figure 1 The content of type I collagen in the blank control group, negative control group, positive control group and experimental groups with different concentrations of ColpepA1 21 is shown. T-test was used to test the significant difference between the corresponding experimental group and the negative control group, where * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. The results showed that the blank control group showed normal baseline levels of type I collagen. After 5J / cm 2The type I collagen content in the UVA-treated negative control group was significantly reduced, indicating that UVA radiation damages the extracellular matrix. The type I collagen content in the TGFβ1-positive control group recovered somewhat compared to the negative group, but was still lower than that in the unirradiated control group, suggesting that TGFβ1 has a certain repair effect. In the experimental groups supplemented with 10ppm and 50ppm ColpepA1 21, the type I collagen content was higher than that in the UVA-treated negative control group. In particular, in the 50ppm ColpepA1 21 group, the type I collagen content was significantly higher than that in the negative control group, with a promotion rate of 23.21%. This result indicates that ColpepA1 21 significantly promotes the synthesis of type I collagen.

[0183] Figure 2 The results show the type I collagen content in the blank control group, negative control group, positive control group, and the ColpepA1 21 experimental group and ColpepA1 21 (long chain) experimental group at a concentration of 50 ppm. The results show that the blank control group showed normal baseline levels of type I collagen, while the negative control group had a significantly decreased type I collagen content. After treatment with ColpepA1 21 (long chain), type I collagen content increased by 34.0% compared to the negative control group. Although not as high as the positive control group, this suggests that ColpepA1 21 (long chain) has the ability to promote type I collagen synthesis in damaged skin. Of particular note, the ColpepA1 21 group showed a more significant effect than the ColpepA1 21 (long chain) group, with average type I collagen content significantly higher than 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 significant progress in promoting the synthesis of type I collagen compared to the long chain, providing strong scientific evidence for its applications in anti-photoaging and skin repair, and indicating its great potential in the development of future skin care products.

[0184] Recombinant humanized collagen type 21 significantly promotes the synthesis of type I collagen after UVA damage, which is important for maintaining and restoring the structural integrity of the skin. This discovery provides strong scientific evidence for the application of ColpepA1 21 in the anti-aging field, particularly its potential benefits in promoting the repair of damaged skin 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 the skin's key structural components, helping to promote skin health and slow the aging process.

[0185] Example 3: Effect of recombinant humanized collagen type 21 on tyrosinase activity

[0186] This example aims to explore the effect of recombinant humanized collagen type 21 (ColpepA1 21) on tyrosinase activity in B16F10 cells, in order to evaluate its subsequent effect on skin whitening and brightening.

[0187] The cell line used in this experiment was mouse skin melanoma cell B16F10 (Qisai Biotechnology), and the cell generation number was 7-8.

[0188] Main reagents and consumables:

[0189] B16 cell-specific 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, 5 mL, 15 mL), centrifuge tubes (Corning, 15 mL), disposable cell counting chambers (Countstar), melanocyte-stimulating hormone α-MSH (Yuanye Biotechnology), mouse tyrosinase (TyR) ELISA detection kit (Yuanju Biotechnology).

[0190] Instruments and equipment:

[0191] Adjustable pipette (Eppendorf), analytical balance (Sartorius), inverted microscope (Motic), low-speed centrifuge (Heal force), constant temperature incubator (Heal force), electric pipette (Eppendorf), cell counter (Countstar), clean bench (Thermo), digital constant temperature water bath (Lanburg Haibo Biotechnology), liquid nitrogen tank, refrigerator (Haier), multifunctional microplate reader (PerkinElmer).

[0192] 1. Cell inoculation: B16F10 cells were diluted to 5×10 4 The cells were seeded at a density of 100 cells / mL and 500 μL / well of cell dilution solution were inoculated onto a 24-well plate slide and incubated in a cell culture incubator (37°C, 5% CO2, 95% RH) for 24±2 h.

[0193] 2. Experimental grouping: The experiment set up blank control group, negative control group, positive control group and sample group.

[0194] 3. Liquid preparation:

[0195] 1) Sample group: ColpepA1 21 sample was prepared into 20 ppm using cell culture medium;

[0196] 2) Positive control group: 377 (phenylethyl resorcinol) solution.

[0197] 4. Induction: Except for the blank control group, 0.1 μM α-MSH was added to each well and incubated for 48 h ± 2 h.

[0198] 5. Sample Addition: After induction and 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; and 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°C, 5% CO2, 95% RH) and continue incubation for 24 ± 2 hours.

[0199] 6. Cell tyrosinase activity detection: After the incubation, freeze and thaw the cells three times. Centrifuge the supernatant at 3000 rpm for 5 minutes to remove the precipitate. Detect the changes in tyrosinase activity using a microplate reader and a kit.

[0200] 7. Tyrosinase activity test after α-MSH induction in B16F10 cells

[0201] like Figure 3 As shown, the diagrams represent:

[0202] Blank control group: added with an equal amount of cell culture medium; negative control group: added with an equal amount of 0.1 μM α-MSH to induce cell culture medium; positive control group: added with an equal amount of 0.1 μM α-MSH and cell culture medium containing 4 μM (about 0.86 ppm) 377; sample group: added with an equal amount of 0.1 μM α-MSH and cell culture medium containing samples of corresponding concentrations; ### 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.

[0203] The results of tyrosinase activity after cell α-MSH induction are as follows:

[0204] (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;

[0205] (2) Compared with the negative control group, 2 μM (about 0.43 ppm) of 377 significantly inhibited tyrosinase activity, further proving that the model was successfully constructed, with an inhibition rate of 54.60%;

[0206] (3) Compared with the negative control group, 20 ppm ColpepA1 21 could significantly inhibit tyrosinase activity with an inhibition rate of 56.49%.

[0207] Based on the above in vitro cell test results, it was proved that 20ppm ColpepA1 21 could significantly inhibit the activity of tyrosinase.

[0208] Example 4: Effect of recombinant humanized collagen type 21 on melanin

[0209] This example investigates the effects of recombinant humanized collagen type 21 (ColpepA1 21) on melanin production 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.

[0210] The cell line used in this experiment was mouse skin melanoma cell B16F10 (Qisai Biotechnology), and the cell generation number was 7-8.

[0211] Main reagents and consumables:

[0212] B16 cell-specific 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, 5 mL, 15 mL), centrifuge tubes (Corning, 15 mL), disposable cell counting chambers (Countstar), melanocyte-stimulating hormone α-MSH (Yuanye Biotechnology), mouse melanin ELISA detection kit (Yuanju Biotechnology).

[0213] Instruments and equipment:

[0214] Adjustable pipette (Eppendorf), analytical balance (Sartorius), inverted microscope (Motic), low-speed centrifuge (Heal force), constant temperature incubator (Heal force), electric pipette (Eppendorf), cell counter (Countstar), clean bench (Thermo), digital constant temperature water bath (Lanburg Haibo Biotechnology), liquid nitrogen tank, refrigerator (Haier), multifunctional microplate reader (PerkinElmer).

[0215] 1. Cell inoculation: B16F10 cells were diluted to 5×10 4The cells were seeded at a density of 100 cells / mL and 500 μL / well of cell dilution solution were inoculated onto a 24-well plate slide and incubated in a cell culture incubator (37°C, 5% CO2, 95% RH) for 24±2 h.

[0216] 2. Experimental grouping: The experiment set up a blank control group, a negative control group and a sample group.

[0217] 3. Liquid preparation:

[0218] Sample group: Sample ColpepA1 21 was prepared into 20 ppm using cell culture medium.

[0219] 4. Induction: Except for the blank control group, 0.1 μM α-MSH was added to each well and incubated for 48 h ± 2 h.

[0220] 5. Sample Addition: After incubation, wash the 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; and add 500 μL of culture medium containing the corresponding sample concentration to each well of the sample group. After sample addition, place the 24-well plate in an incubator (37°C, 5% CO2, 95% RH) and continue incubation for 24 ± 2 hours.

[0221] 6. Detection of cell melanin content: After incubation, freeze and thaw the cells three times repeatedly. Centrifuge the supernatant at 3000 rpm for 5 minutes to remove the precipitate. Detect changes in melanin content using a microplate reader and a kit.

[0222] 7. Melanin content test after α-MSH induction in B16F10 cells:

[0223] like Figure 4 As shown, the diagrams represent:

[0224] Blank control group: added with an equal amount of cell culture medium; negative control group: added with an equal amount of 0.1 μM α-MSH-induced cell culture medium; sample group: added with an equal amount of 0.1 μM α-MSH and cell culture medium containing samples of corresponding concentrations; ### 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.

[0225] The results of melanin content in B16F10 cells after α-MSH induction are as follows:

[0226] (1) Compared with the blank control group, the melanin content in the negative control group was significantly increased, proving that the model was successfully constructed;

[0227] (2) Compared with the negative control group, 20 ppm type 21 collagen can significantly inhibit the melanin content, with an inhibition rate of 18.82%.

[0228] Based on the above in vitro cell test results, it is proven that 20ppm of ColpepA1 21 can significantly inhibit melanin content. The above efficacy shows that ColpepA1 21 can be used as an effective active ingredient in skin whitening and brightening products.

[0229] Example 5: Clinical trial

[0230] This example presents clinical trial results demonstrating the effectiveness of recombinant humanized collagen type 21 (ColpepA1 21) in improving skin aging in real-world applications. Key aging symptoms of interest include dull skin tone, dark spots, sagging skin, and wrinkles. This clinical trial aims to evaluate the efficacy of ColpepA1 21 in improving skin quality and appearance, supporting its commercial potential as an ingredient in anti-aging products.

[0231] The experiment recruited 30 healthy volunteers with an average age of 45.3±6.7 years. They had dull skin, pigmentation, poor skin firmness, obvious crow's feet, and nasolabial folds. The sample group used an appropriate amount of lotion containing 500ppm of recombinant humanized collagen type 21; the control group used a lotion without recombinant humanized collagen type 21. After cleansing the face / scalp, the volunteers were asked to apply the corresponding product to the left and right areas of the face / scalp, gently patting until completely absorbed. The product was used twice a day for a total of 28 days. Return visits were made on day 0 (D0), day 14 (D14), and day 28 (D28).

[0232] Data Collection and Analysis:

[0233] The face is measured for crow's feet, nasolabial folds, skin elasticity, skin firmness, skin gloss, skin spot optical density, erythema area, and lactic acid stinging score; the scalp is measured for water content, transepidermal water loss, and erythema value.

[0234] T-test was used to test the significant difference between the sample group and the control group, and * in the figure indicates p < 0.05.

[0235] Figure 5 and Figure 6 The data show the mean area and volume of crow's feet on volunteers' faces before and after product use, demonstrating a significant improvement in crow's feet after using the lotion containing ColpepA1 21. These improvements directly demonstrate the effectiveness of ColpepA1 21 in reducing fine lines around the eyes, reflecting its positive impact on the deeper layers of the skin. The reduction in area and volume indicates a significant improvement in skin firmness and smoothness, which is attributed to ColpepA1 21 promoting collagen synthesis and strengthening the skin's support structure.

[0236] Figure 7 and Figure 8 The mean statistical data for the area and volume of facial nasolabial folds of volunteers before and after product use show a reduction in facial nasolabial folds. This reduction not only improves the overall appearance of the face but also demonstrates the effectiveness of ColpepA1 21 in rebuilding skin elastic fibers. This is likely due to ColpepA1 21 activating the skin's natural repair mechanisms and promoting the production of collagen and elastin.

[0237] Figure 9 The mean statistical values ​​of facial skin elasticity (R² value) before and after product use are shown, demonstrating an increase in facial elasticity. Increased skin elasticity is a key indicator in anti-aging research, indicating that the skin is better able to resist mechanical deformation and return to its original shape. By enhancing skin elasticity, ColpepA1 21 directly contributes to improving skin firmness and reducing wrinkle formation.

[0238] Figure 10 The results show the average statistics of the volunteers' facial skin firmness (F4 value) before and after using the product, showing an improvement in facial firmness. As we age, the loss of skin firmness is a common problem. Figure 10 The improvement in facial firmness shown demonstrates that ColpepA1 21 is effective in combating the appearance of sagging skin and restoring its firmness and youthfulness.

[0239] Figure 11 and Figure 12 The mean statistical values ​​for facial skin gloss and brightness (cheek L value) of volunteers before and after product use are shown, respectively. The results show improvements in skin gloss and brightness. Improvements in gloss and brightness are directly related to the reflectivity and smoothness of the skin surface. This suggests that Colpep Al 21 not only improves the skin's internal structure but also optimizes the smoothness and radiance of the skin surface, enhancing the skin's overall visual appearance.

[0240] Figure 13 The results show the mean optical density of facial skin pigmentation on volunteers before and after product use, demonstrating a reduction in the optical density of skin pigmentation. Pigmentation is a common sign of UV exposure and photoaging. ColpepA1 21's effectiveness in reducing pigmentation may be related to its effects on melanocyte activity and regulation of pigment production, helping to even out skin tone and reduce pigmentation.

[0241] Figure 14 The mean values ​​of facial erythema area for volunteers before and after product use are shown, showing a reduction in facial erythema area. This reduction in erythema area reflects a decrease in skin inflammation, which may indicate that ColpepA1 21 has anti-inflammatory effects, reducing skin sensitivity and improving skin comfort.

[0242] Figure 15 The results show the mean lactic acid sting scores of volunteers before and after using the product. The results show a decrease in the lactic acid sting score, indicating improved skin comfort. This decrease in lactic acid sting score indicates improved skin tolerance after product use, reduced discomfort caused by external stimuli, and enhanced skin barrier function.

[0243] Figure 16 and Figure 17 The results show the mean scalp moisture content and transepidermal water loss (TEWL) values ​​of volunteers before and after product use, respectively. The results show that both scalp moisture content and TEWL values ​​improved, indicating a strengthening of the skin barrier function. The improvement in these two indicators shows that Colpep A121 can enhance the moisturizing capacity of the scalp and the entire skin, reducing water loss, which is crucial for maintaining skin health and preventing dryness and aging.

[0244] Figure 18 The results show the mean scalp erythema index (EI) of volunteers before and after product use, showing a reduction in scalp erythema, indicating the efficacy of reducing inflammation and reducing skin sensitivity. The reduction in scalp erythema further confirms ColpepA1 21's ability to reduce skin inflammation, which is particularly important for people with sensitive skin or poor skin conditions.

[0245] In summary, the results of this clinical trial provide strong data support for the patented anti-aging properties of recombinant humanized collagen type 21, demonstrating that ColpepA1 21 effectively improves multiple signs of skin aging, including reducing wrinkles and dark spots, increasing skin elasticity and firmness, and improving overall skin radiance and color. These changes not only improved the appearance of the volunteers' skin but also physiologically enhanced skin health, reducing water loss and inflammation. These benefits suggest that ColpepA1 21 can be used as an effective active ingredient in anti-aging products.

[0246] While the specific embodiments of the present invention have been described in detail above, these are merely exemplary and the present invention is not limited thereto. It will be apparent to those skilled in the art that any equivalent modifications and substitutions to the present invention fall within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are encompassed within the scope of the present invention.

Claims

1. A type 21 recombinant humanized collagen, wherein: The amino acid sequence of the collagen is the amino acid sequence shown in SEQ ID No.

1.

2. The collagen according to claim 1, wherein The collagen is used for anti-aging and / or skin whitening and brightening.

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

4. A carrier, wherein The vector contains the nucleic acid molecule according to claim 3.

5. A host cell, wherein The host cell contains the nucleic acid molecule according to claim 3 or the vector according to claim 4.

6. A method for preparing collagen according to claim 1 or 2, wherein: The preparation method comprises the step of preparing the collagen by utilizing the expression system of Pichia pastoris.

7. The preparation method according to claim 6, wherein The preparation method comprises one or more steps selected from the group consisting of introducing the vector of claim 4 into Pichia pastoris to form genetically engineered Pichia pastoris, fermenting and culturing the genetically engineered Pichia pastoris, and inducing and expressing the collagen. A composition, wherein the active ingredient of the composition comprises the collagen according to claim 1.

9. The composition according to claim 8, wherein The compositions may comprise one or more auxiliary ingredients or excipients.

10. The composition according to claim 9, wherein The auxiliary ingredients are selected from one or more of moisturizers, antioxidants, ultraviolet absorbers, penetration enhancers, and plant extracts.

11. The composition according to claim 9, wherein The auxiliary material is selected from one or more of stabilizers, emulsifiers, conditioners, diluents, fillers, adhesives, wetting agents, absorption promoters, surfactants, lubricants, spices or flavorings.

12. The composition according to any one of claims 8 to 11, wherein The composition is used for anti-aging and / or skin whitening and brightening.

13. A medical product, wherein: The medical product comprises the collagen according to claim 1 or 2 or the composition according to any one of claims 8 to 12.

14. The medical product according to claim 13, wherein The medical product is in the form of one or more selected from the group consisting of a cream, an essence, a mask, a smear gel, an emulsion, and a skin enhancer.

15. The medical product according to claim 14, wherein The skin enhancer is an injectable skin enhancer.

16. The medical product according to claim 13, wherein The medical product is used for anti-aging and / or skin whitening and brightening.

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