Facial anti-aging collagen peptide, composition and application thereof

Abstract

The invention relates to the field of biological medicines, in particular to a facial anti-aging collagen peptide, a composition and an application of the facial anti-aging collagen peptide, and particularly relates to a 21-type recombinant humanized collagen peptide (ColpepA1 21) and an application of a composition of the 21-type recombinant humanized collagen peptide in anti-aging treatment and skin care. The recombinant collagen provided by the invention can effectively promote the repair process after skin injury, enhance the hydration state and barrier function of the skin, reduce fine lines and wrinkles, and improve the compactness and elasticity of the skin.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically 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 more than 30% of the total protein in the human body, but also the cornerstone of maintaining the structure and function of various tissues in the human body. It exists in the skin, bones, tendons, blood vessels and other connective tissues of the human body, forming a three-dimensional network structure that provides mechanical strength and stability of the tissue. In the skin, collagen is responsible for maintaining the firmness and elasticity of the skin and keeping the skin surface smooth and shiny. As we age, the natural production rate of collagen in the human body begins to slow down, and the existing collagen will gradually degrade and be damaged due to factors such as ultraviolet radiation, environmental pollution, and bad living habits. This causes the skin to gradually lose its original elasticity and firmness, and show signs of aging such as sagging, fine lines and wrinkles. In addition, the reduction of collagen in other tissues will also affect the density of bones, the elasticity of tendons, and the stability of blood vessels, thus affecting overall health. Modern scientific research is actively looking for ways to slow down the degradation process of collagen and increase its production rate in the body in order to slow down the effects of aging. There are currently a variety of collagen supplements, skin care products, and medical cosmetic procedures on the market that attempt to restore the youthfulness of the 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 accurately target the need for specific types of collagen during the aging process, remain research focuses and challenges.

[0003] In this context, it is particularly important to conduct in-depth research on specific types of collagen, such as type 21 collagen (COL21A1). 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. Compared with other more well-known types of collagen, type 21 collagen has been relatively less studied. Type 21 collagen 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 extracellular matrix components. According to the research of Chou MY and Li HC [Chou MY, Li HC (March 2002)], type 21 collagen is mainly expressed in vascular smooth muscle cells and may play a role in the assembly of the extracellular matrix during angiogenesis. In addition, the gene expression of type 21 collagen is regulated by the developmental stage, and the expression level is higher in the fetal stage, 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 function in vascular health and blood pressure regulation. Surendran et al. (2016) found through a large-scale genotype survey that the type 21 collagen gene is involved in the formation of collagen in various tissues such as the heart and aorta, and is associated with vascular remodeling and hypertension. This provides a new perspective for the study of COL21A1 in promoting skin health and anti-aging. These characteristics give type 21 collagen unique application potential in the fields of skin health, anti-aging and vascular health.

[0004] There are many anti-aging and whitening products on the market, most of which focus on using ordinary collagen or its derivatives to replenish the loss of collagen in the skin. However, the effectiveness of these products is usually limited by the size of collagen molecules and skin absorption rate, and it is difficult to optimize the biological functions of specific collagen. Based on its unique biological characteristics and mechanism of action, type 21 collagen may become one of the important directions for future anti-aging and whitening research and application. By understanding its specific biological functions and mechanisms of action, it is possible to develop anti-aging and whitening therapies and products with strong targeting, high bioavailability, and multiple functions, which are not limited to improving the appearance of the skin, but also include promoting overall health and delaying the aging process. Summary of the invention

[0005] The 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 facial anti-aging and whitening and brightening.

[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] On the one hand, 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 greater than 90%, 95%, 96%, 97%, 98%, 99% or higher with the amino acid sequence shown in the above SEQ ID No.1.

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

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

[0014] In one embodiment, the collagen described herein 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 described in the present invention can whiten the skin and / or provide support for skin whitening and lightening.

[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, the method comprising 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: introducing the aforementioned vector into Pichia pastoris to form a genetically engineered Pichia pastoris, fermenting and culturing the genetically engineered Pichia pastoris, 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 medium for the fermentation culture is BMGY medium.

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

[0035] The synthesis process of collagen described in 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 using nickel affinity chromatography to specifically purify type 21 collagen; 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 aforementioned purification steps of the present 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 design of the entire purification process takes into account both efficiency and protein stability, which is an important basis for the application of recombinant proteins in the fields of anti-aging and skin care.

[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 adjuvants.

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

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

[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 field of reducing wrinkles and anti-aging.

[0046] 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, 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 ability of the skin to resist environmental oxidative damage, and further improve 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 described herein 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 flavoring agents, etc.

[0065] In one embodiment, the collagen composition of the present invention may also 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 can 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, provide a smooth touch and excellent spreadability.

[0067] In one embodiment, the collagen composition of the present invention may contain spices and flavoring agents, preferably natural essential oils, synthetic spices, etc. The addition of spices and flavoring agents can enhance the sensory experience of users when using the composition.

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

[0069] In one embodiment, the composition of the present invention includes but is not limited to medical compositions, such as one or more of pharmaceutical compositions, food compositions, health care compositions, dietary supplements, etc. Specifically, the composition of the present invention includes one or more of tablets, capsules, powders, microgranules, solutions, lozenges, jellies, cream preparations, spirits, suspensions, tinctures, poultices, liniments, lotions, and aerosols, etc., and can be prepared using commonly known preparation techniques, and appropriate pharmaceutical excipients, such as stabilizers, antioxidants, and preservatives, can be added to maintain the stability and effectiveness of the product.

[0070] Another aspect of the present invention provides a product, wherein the product comprises 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., for improving skin elasticity, reducing fine lines, and increasing 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 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.

[0074] In one embodiment, the injectable skin enhancer of the present invention is administered through a variety of minimally invasive injection devices and methods, including water light needle injectors, microneedles, microneedle rollers, automatic microneedle pens (such as Dermapen), needle-free injection systems, electric microneedle systems (such as SkinPen), ultrasonic injection devices, laser-assisted delivery systems, and air pressure injection devices (such as JetPeel). The devices can accurately deliver the skin enhancer directly to the middle or deep layers of the skin to improve the overall quality and appearance of the skin. Through these different administration methods, the most appropriate technology can be selected to achieve the best treatment effect according to the treatment goals and the specific needs of the patient.

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

[0076] In one embodiment, for the application of microneedles, microneedle rollers, etc., the collagen content in the injectable skin enhancer of the present invention is relatively high (5% to 10%). Microneedles form tiny channels on the skin by physical means, and the higher concentration of collagen helps to directly act on the newly formed microchannel area, promoting rapid skin repair and collagen regeneration.

[0077] In one embodiment, for the water light needle injection system, the injectable skin enhancer of the present invention contains a high concentration of antioxidants, such as vitamins C and E, which helps protect the skin from free radical damage and enhance the overall resistance of the skin. At the same time, the use of small molecule hyaluronic acid can more easily penetrate into the deep layer of the skin, provide better hydration and intercellular signal transmission, thereby effectively improving the moisture and firmness of the skin.

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

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

[0080] Another aspect of the present invention also 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.

[0083] The recombinant humanized collagen type 21 (ColpepA1 21) of the present invention has shown significant advantages in anti-aging and whitening and brightening the skin. While providing basic collagen supplementation, ColpepA1 21's unique efficacy and optimized molecular properties make it stand out among many collagen products.

[0084] 1. ColpepA1 21 has demonstrated its importance in enhancing the structural integrity of the skin, especially in promoting the production of type I and type III collagen. This is not only beneficial for improving the skin's resistance to stretching and compression, but also effectively combats UV-induced photoaging and maintains the skin's youthful state. In particular, type III collagen, as an important source of skin elasticity, ColpepA1 21's role in promoting its production provides support for increasing skin elasticity and reducing 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 deeper into the skin, providing cells with necessary nutrients, thereby supporting skin health and anti-aging at the molecular level.

[0086] 3. Cell experiments have proven the effect of ColpepA1 21 on 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. The clinical trial results further demonstrated the multiple anti-aging effects of ColpepA1 21, which significantly improved the effects of reducing wrinkles and spots, improving skin elasticity and firmness, and improving skin radiance and color. These improvements in the appearance and physiology of the volunteers' skin conditions verified the unique efficacy of ColpepA1 21 and provided a strong scientific basis for its use in anti-aging skin care 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 group of cells in the experiment 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 group of cells in the experiment are shown, and the ability of ColpepA121 of different lengths to promote the production of type I collagen is demonstrated by comparison.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0104] Fig.18 The mean statistics of the volunteers' scalp erythema values ​​(EI values) before and after using the product are shown.

[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, which is widely present in various tissues and organs, such as skin, bones, tendons, ligaments and cornea. The main function of these proteins is to provide strength, stability and elasticity to body tissues, ensuring the normal functioning of various physiological functions. There are at least 28 known types of collagen, each of which plays a unique role and function in the body, forming a complex and finely regulated biological system.

[0108] As used herein, the term "collagen type 21 (COL21A1)" refers to a type of collagen present in the human body, which belongs to the family of small molecule collagens. As part of the FACIT (fibril-associated collagen with interrupted triple helix) family, collagen type 21 plays a vital role in connecting extracellular matrix components. This collagen, through its unique molecular structure, promotes interactions and links 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 collagen types I and III, it plays a role in specific tissues and physiological processes, involving the composition of the extracellular matrix and the mechanism of skin repair. Although collagen type 21 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 the structure of the extracellular matrix 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 by genetic recombination technology that simulates the type 21 collagen naturally present in the human body. The protein has similar biological functions and structural properties to natural type 21 collagen, and its amino acid sequence has a specific coding, which is intended to improve the health and anti-aging ability of the skin.

[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 sequences of the polypeptides are aligned and the number of positions containing the same amino acid residues in the aligned polypeptides is scored, and compared with the number of positions containing different amino acid residues in the aligned polypeptides. The polypeptides may 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 residues by the total number of amino acid residues in the polypeptide. For example, the identity percentage 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.

[0111] As used herein, the term "genetically engineered Pichia pastoris" refers to a Pichia pastoris strain that has been genetically manipulated and has had specific foreign genes inserted into it. This modification enables yeast to express proteins that are not in its own genome and is used to produce various recombinant proteins.

[0112] As used herein, the term "expression system" refers to a set of biochemical environments and processes for synthesizing specific proteins, 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 a specific operating process.

[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, etc., and is considered a valuable research tool because of its ability to 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 a human fetus, which are an important component of the skin structure, responsible for the synthesis of 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 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.

[0118] As used herein, the term "water regulation" refers to a series of physiological processes that maintain the moisture balance of the skin, involving the absorption, distribution and retention of water within the skin layers, which 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 connection of the epidermis to block external stimulation 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 collagen is composed of two α1 chains and one α2 chain, forming a triple helical structure that gives the tissue a high degree of tensile strength. Type Ⅰ collagen plays a vital role in maintaining the elasticity and stability of the skin and its overall health. As we age and due to environmental factors, type Ⅰ collagen gradually degrades, causing the skin to lose elasticity and show 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 structure, especially in young skin. It plays a vital role in maintaining skin elasticity and assisting in the healing process of damaged skin. After skin injury, a temporary increase in type III collagen helps repair and rebuild the skin's structure. Its decrease with age may affect the skin's elasticity and repair ability.

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

[0123] As used herein, the term "UVA radiation" refers to a section of the ultraviolet spectrum with a wavelength between 320-400 nanometers. UVA can penetrate deep into the dermis of the skin, causing damage to collagen and elastic fibers, and is one of the main causes of photoaging. The damage of UVA radiation to the skin is cumulative, and long-term exposure will lead to accelerated 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 fine lines can be measured by specific instruments or software to evaluate the effect of anti-aging products on reducing fine lines around the eyes. A reduction in area and volume generally indicates that the product has a positive effect on improving skin elasticity and reducing wrinkles.

[0125] As used herein, the term "facial nasolabial fold area and volume" refers to the wrinkles located on both sides of the mouth corners that generally become more pronounced with age. By measuring the area and volume of nasolabial folds, the effectiveness of anti-aging products in smoothing deep facial wrinkles can be evaluated. Reduced area and volume indicate that the product is effective in improving skin structure and reducing signs of facial aging.

[0126] As used herein, the term "facial skin elasticity (R2 value)" refers to a value obtained by a skin elasticity measuring instrument, which is used to reflect the elasticity of the skin. The R2 value is determined by measuring the ability of the skin to recover to its original shape after mechanical stretching. The higher the R2 value, the better the skin elasticity and the better it can resist deformation during the aging process.

[0127] As used herein, the term "facial skin firmness (F4 value)" refers to a quantitative parameter for evaluating skin firmness. The F4 value is measured by a special skin testing instrument and reflects the firmness and elasticity of the skin. The higher the F4 value, the better the skin firmness, which helps to reduce skin sagging and wrinkle formation.

[0128] As used herein, the term "facial skin radiance" refers to the ability of the skin surface to reflect light, a property that is often associated with skin health and youthfulness. Skin surfaces with higher radiance reflect light more effectively, giving a bright, fresher visual appearance, and are 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 level from black (0) to white (100), and the higher the L value, the brighter the skin. In skin science research and cosmetic effect evaluation, the cheek L value is often used as a quantitative indicator to evaluate the effect of improving skin brightness and color. Improving the cheek L value usually means that the skin becomes brighter and more even, which is one of the effects that whitening products or anti-aging products hope to achieve.

[0130] As used herein, the term "optical density of facial skin spots" refers to the color depth and distribution density of the spots on the skin. This indicator is measured by a specific instrument and is used to evaluate the effectiveness of whitening or spot reduction products. A higher optical density of spots indicates that the spots are darker and more densely distributed, while a lower optical density indicates that the spots are lighter and more sparsely distributed. Whitening or spot reduction products are designed to reduce the optical density of spots and make the skin tone more even.

[0131] As used herein, the term "facial erythema area" refers to the size of the erythema area on the skin surface due to inflammation or other reasons. The appearance of erythema is usually related to skin inflammation, allergic reactions or other skin problems. When evaluating the effect of anti-inflammatory, sedative or anti-allergic products, a reduction in the area of ​​erythema is considered a positive result, indicating that the product can effectively reduce the skin inflammation state and improve skin health.

[0132] As used herein, the term "facial lactic acid stinging score" refers to a subjective score that evaluates the skin irritation (e.g., stinging) caused by a product. When conducting a skin irritation test, participants give a score based on the degree of irritation they feel after using the product (e.g., no, mild, moderate, strong). The lower the score, the less irritating the product is to the skin and the more suitable it is for sensitive skin.

[0133] As used herein, the term "scalp moisture content" refers to the ability of the scalp's surface layer to retain moisture. This indicator is extremely important for assessing the health of the scalp and the skin as a whole. A scalp with adequate moisture content can remain healthier and more vibrant, with less dryness, itchiness and other problems. Improving scalp moisture content is a goal that many hair care and skin care products attempt to achieve.

[0134] As used herein, the term "scalp transepidermal water loss (TEWL value)" refers to the rate of evaporation of water from the scalp surface, which reflects the integrity of the skin barrier function. A lower TEWL value indicates that the skin barrier function is good, effectively preventing excessive evaporation of water and maintaining the moisture balance of the skin. When evaluating the effects of skin care and hair care products, reducing the TEWL value is generally regarded as an indicator of improving the skin barrier function and enhancing the skin's moisturizing ability.

[0135] As used herein, the term "scalp erythema index (EI value)" refers to an index of the degree of scalp erythema measured by an instrument, which is used to quantitatively evaluate the inflammation or sensitivity of the scalp. The higher the EI value, the more severe the scalp inflammation or sensitivity. It is an important index in evaluating the calming and soothing effects of skin care or hair care products.

[0136] As used herein, the term "TGFβ1" (transforming growth factor β1) refers to a multifunctional cytokine that is able to regulate 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, which has the ability to promote cell renewal, enhance skin repair and improve the aging state of the skin.

[0138] As used herein, the term "composition" or "preparation" refers to a mixture containing one or more collagens 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, facilitate the absorption of active ingredients, and thus exert 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 is important for 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 light absorber" or "sunscreen agent" refers to a compound that can absorb or reflect ultraviolet light and protect 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 (such as arginine hydrochloride), glycine, alanine (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ-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 RNAaseA. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., can also play a role in 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 ability to retain water, increase the skin's plumpness and radiance;

[0146] Collagen: As one of the active ingredients, the main function of collagen is to activate the cell basal layer, stimulate the regeneration and repair of cells under the skin, and improve the elasticity and firmness of the skin, thereby achieving a significant anti-aging effect.

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

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

[0149] Other Peptides: Stimulate the production of collagen and elastin 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, eyes, etc. 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 for regulating melanin production, widely present in plants, animals and microorganisms, and in the human body, tyrosinase is mainly present in melanocytes, which can catalyze tyrosine to generate dopa and further oxidize it to generate dopaquinone, and finally form melanin. The expression and activity of tyrosinase determine the amount of melanin produced, thereby affecting the skin color and sensitivity to ultraviolet rays of the individual, and it is also related to the occurrence of diseases such as excessive melanin deposition such as human freckles and brown spots, and the excessive activity of tyrosinase is related to certain types of skin cancer such as melanoma. Therefore, the formation of melanin can be reduced by inhibiting the activity of tyrosinase, and skin pigmentation can be reduced, 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 and streak culture on YPD plate. Select single clone of Pichia GS115 activated on YPD plate and culture in YPD liquid. Culture at constant temperature and shaking. Pipette 100uL bacterial solution and inoculate it in 100mL YPD liquid. Culture at 30℃, 220rpm, 12-13h until 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 package them 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 OD 600 =1.0.

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

[0161] Transfer the diluted bacterial solution to a 500mL conical flask and induce yeast to express protein at 29℃ and 220rpm. Then add methanol every 24h to make the final concentration of methanol in the culture medium 1% and continue inducing culture for 84h. Check under a microscope to see if there is any bacterial contamination and measure the pH of the fermentation liquid, which should be less than 6. Transfer the fermentation liquid to a 500mL round-bottom centrifuge tube and centrifuge at 15000g and 4℃ for 20min to remove the supernatant. Remove 1mL of supernatant and add corresponding loading buffer. Mix well and incubate at 95℃ for 10min and store at -20℃.

[0162] 4. Purification of recombinant human collagen:

[0163] Transfer the obtained fermentation broth supernatant to a conical flask, add NaCl to make the final concentration of NaCl 150mM, adjust the pH of the fermentation broth supernatant to 8.0 with NaOH or HCl; centrifuge at 15000g for 30min to ensure that the supernatant is clear and transparent, balance the column with 10 nickel column bed volumes of binding buffer; load the sample at a speed of 1mL / min, and collect the column liquid while loading the sample. Elute the unbound protein and impurity protein with 5 nickel column bed volumes of binding buffer; elute the target protein with elution buffer (500mM imidazole).

[0164] Preparation of protein detection samples: Add the collected purified proteins, supernatant before column penetration, and liquid after column penetration into the corresponding loading buffer, mix well, incubate at 95℃ for 10min, 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 a high-purity, high-activity target protein.

[0166] For specific synthesis methods 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 activity assessment, AQP3 (Shanghai ELISA) and type I collagen kit (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), type I collagen in the skin will gradually degrade, causing skin sagging, 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 Grouping

[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 agents.

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

[0179] Fibroblasts HFF-1 were cultured separately and plated for 24 hours. When the cell confluence reached 70-80%, samples were treated with UVA induction and cultured for 24 hours. The supernatants were 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 is 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 show that the blank control group showed a normal baseline level of type I collagen. After 5J / cm 2The type I collagen content in the negative control group treated with UVA was significantly reduced, indicating that UVA radiation has a damaging effect on the extracellular matrix. The type I collagen content in the TGFβ1 positive control group recovered compared with the negative group, but it was still lower than the control group without radiation, indicating that TGFβ1 has a certain repair effect. In the experimental groups with 10ppm and 50ppm ColpepA1 21 added, the type I collagen content was higher than that in the negative control group treated with UVA. In particular, in the 50ppm ColpepA1 21 group, the type I collagen content was significantly higher than that in the negative control group, and the promotion rate was 23.21%. This result shows that ColpepA1 21 has a significant promoting effect on the synthesis of type I collagen.

[0183] Figure 2 The contents of type I collagen in the blank control group, negative control group, positive control group, ColpepA1 21 experimental group and ColpepA1 21 (long chain) experimental group at a concentration of 50 ppm are shown. The results showed that the blank control group showed a normal baseline level of type I collagen, and the type I collagen content in the negative control group was significantly reduced. After treatment with ColpepA1 21 (long chain), the content of type I collagen increased by 34.0% compared with the negative control group. Although it was not as high as the positive control group, this showed that ColpepA1 21 (long chain) had the ability to promote the synthesis of type I collagen in damaged skin. It is worth noting that compared with the ColpepA1 21 (long chain) group, the ColpepA1 21 group showed a more significant effect, and the average type I collagen content was significantly higher than that of the ColpepA1 21 (long chain) group, which was 63.6% higher than the negative control group. This significant difference shows 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 application in anti-photoaging and skin repair, and heralding its great potential in the development of future skin care products.

[0184] Recombinant humanized collagen type 21 can significantly promote the synthesis of type I collagen after UVA damage, which is of great significance for maintaining and restoring the structural integrity of the skin. This discovery provides a strong scientific basis for the application of ColpepA1 21 in the field of anti-aging, especially its potential benefits in promoting the repair of damaged skin and improving skin elasticity. In terms of anti-aging collagen patents, ColpepA1 21 demonstrates its commercial value as an effective skin care ingredient by enhancing the production of the main structural components of the skin, which helps promote skin health and delay 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, so as to evaluate its subsequent effect on skin whitening and brightening.

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

[0188] Main reagents and consumables:

[0189] B16 cell-specific culture medium (Qisai Biotechnology), high-quality fetal bovine serum (Gibco), phosphate buffered saline (BasalMedia, 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 plates (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 inoculated at a density of 100 μL / mL and 500 μL / well of cell dilution 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: The sample ColpepA1 21 was prepared into 20 ppm using cell culture medium;

[0196] 2) Positive control group: 377 (phenylethylresorcinol) 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 the induction incubation is completed, wash the cell plate with PBS, add 500 μL of culture solution to each well of the blank control group; add 500 μL of culture solution to each well of the negative control group; add 500 μL of culture solution containing the corresponding concentration of 377 to each well of the positive control group; add 500 μL of culture solution containing the corresponding concentration of sample to each well of the sample group. After the addition of samples, place the 24-well plate in an incubator (37°C, 5% CO2, 95% RH) and continue to culture for 24±2h.

[0199] 6. Cell tyrosinase activity detection: After the incubation, freeze and thaw the cells three times repeatedly, take the supernatant and centrifuge at 3000rpm for 5min to remove the precipitate, and detect the changes in tyrosinase activity using an ELISA instrument 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: adding an equal amount of cell culture medium; negative control group: adding an equal amount of 0.1 μM α-MSH to induce cell culture medium; positive control group: adding an equal amount of 0.1 μM α-MSH and cell culture medium containing 4 μM (about 0.86 ppm) 377; sample group: adding 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) 377 can significantly inhibit 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 is proved that 20ppm ColpepA1 21 can significantly inhibit the activity content of tyrosinase.

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

[0209] This example aims to explore the effect of recombinant humanized collagen type 21 (ColpepA1 21) on melanin in B16F10 cells. Since tyrosinase is a key enzyme in the melanin biosynthesis pathway, it catalyzes the conversion of tyrosine to DOPA, thereby generating melanin. Controlling the activity of tyrosinase can reduce the production of melanin, thereby reducing skin pigmentation.

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

[0211] Main reagents and consumables:

[0212] B16 cell-specific culture medium (Qisai Biotechnology), high-quality fetal bovine serum (Gibco), phosphate buffered saline (BasalMedia, 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 plates (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 inoculated at a density of 100 μL / mL and 500 μL / well of cell dilution 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 at 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 the induction incubation is completed, wash the cell plate with PBS, add 500 μL of culture solution to each well of the blank control group; add 500 μL of culture solution to each well of the negative control group; add 500 μL of culture solution containing the corresponding concentration of sample to each well of the sample group. After the addition of samples, place the 24-well plate in an incubator (37°C, 5% CO2, 95% RH) and continue to culture for 24±2h.

[0221] 6. Detection of cell melanin content: After incubation, freeze and thaw the cells three times repeatedly, take the supernatant and centrifuge at 3000rpm for 5min to remove the precipitate, and use an ELISA instrument and a kit to detect changes in melanin content.

[0222] 7. Test of melanin content in B16F10 cells after α-MSH induction:

[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 to induce 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 proved that 20ppm ColpepA1 21 can significantly inhibit the melanin content. The above effects show 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 shows the results of a clinical trial to verify the effect of recombinant humanized collagen type 21 (ColpepA1 21) on improving skin aging in practical applications. The aging phenomena of focus include dull skin tone, spots, sagging skin, wrinkles and other problems. This clinical trial aims to evaluate the efficacy of ColpepA1 21 in improving skin quality and appearance to support 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 old, with dull skin, spots, poor skin firmness, obvious crow's feet, nasolabial folds, etc. The sample group used an appropriate amount of lotion containing 500ppm recombinant humanized collagen type 21; the control group used a lotion without recombinant humanized collagen type 21. Volunteers were asked to clean their face / scalp, apply the corresponding products on the left and right areas of the face / scalp, and gently pat until completely absorbed. Use it twice a day for a total of 28 days. Follow-up visits were conducted on day 0 (D0), day 14 ((D14)) and day 28 (D28).

[0232] Data Collection and Analysis:

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

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

[0235] Figure 5 and Figure 6 The mean statistics of the area and volume of crow's feet on the volunteers' faces before and after using the product are shown. The results show that the crow's feet on the volunteers' faces were significantly improved after using the lotion containing ColpepA1 21. The improvement of these two indicators 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 that the firmness and smoothness of the skin have been significantly improved, which is related to ColpepA1 21 promoting collagen synthesis and strengthening the skin's support structure.

[0236] Figure 7 and Figure 8 The mean statistics of the area and volume of the facial nasolabial folds of the volunteers before and after using the product are shown, and the results show a reduction in facial nasolabial folds. The reduction of nasolabial folds not only improves the overall appearance of the face, but also illustrates the effectiveness of ColpepA1 21 in rebuilding skin elastic fibers. This may be due to ColpepA1 21 activating the skin's natural repair mechanism and promoting the production of collagen and elastin.

[0237] Fig. 9 The mean statistics of the facial skin elasticity (R2 value) of volunteers before and after product use show 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. ColpepA1 21 directly reflects its contribution to improving skin firmness and reducing wrinkle formation by enhancing skin elasticity.

[0238] Fig.10 The mean statistics of the volunteers' facial skin firmness (F4 value) before and after product use are shown, and the results show an improvement in facial firmness. As we age, a decrease in skin firmness is a common problem. Fig.11 The improvement in facial firmness shown demonstrates that ColpepA1 21 is effective in combating the phenomenon of skin sagging and restoring the skin's firmness and youthfulness.

[0239] Fig.11 and Fig.12 The mean statistics of the glossiness of the volunteers' facial skin before and after using the product and the mean statistics of the brightness of the volunteers' facial skin (cheek L value) before and after using the product are shown respectively, and the results show the improvement of skin glossiness and brightness. The improvement of glossiness and brightness is directly related to the reflectivity and smoothness of the skin surface. This shows that ColpepA1 21 not only improves the internal structure of the skin, but also optimizes the smoothness and gloss of the skin surface, improving the overall visual effect of the skin.

[0240] Fig.13 The mean statistics of the optical density of facial skin spots of volunteers before and after using the product are shown, and the results show a reduction in the optical density of skin spots. Spots are a common sign of UV exposure and photoaging of the skin. The efficacy of ColpepA1 21 in reducing spots may be related to its influence on melanocyte activity and regulation of pigment production, which helps to even out skin tone and reduce spots.

[0241] Fig.14 The mean statistics of facial erythema area of ​​volunteers before and after using the product are shown, and the results show a reduction in facial erythema area. The reduction in erythema area reflects a reduction in skin inflammatory response, which may indicate that ColpepA1 21 has an anti-inflammatory effect, can reduce skin sensitivity and improve skin comfort.

[0242] Fig.15 The average statistics of the facial lactic acid stinging scores of volunteers before and after using the product are shown. The results show that the facial lactic acid stinging score has decreased, which means that the skin comfort has improved. The decrease in the lactic acid stinging score shows that the skin tolerance has improved after using the product, reducing the discomfort caused by external stimuli and enhancing the skin barrier function.

[0243] Fig.16 and Fig.17 The mean statistics of the moisture content of the volunteers' scalp before and after using the product and the mean statistics of the transepidermal water loss (TEWL) of the volunteers' scalp before and after using the product are shown respectively. The results show that both the moisture content of the scalp and the transepidermal water loss value have improved, indicating the strengthening of the skin barrier function. The improvement of these two indicators shows that ColpepA121 can enhance the moisturizing capacity of the scalp and even the entire skin and reduce water loss, which is crucial to maintaining the healthy state of the skin and preventing dryness and aging.

[0244] Fig.18 The mean statistics of the scalp erythema value (EI value) of the volunteers before and after using the product are shown. The results show a reduction in the scalp erythema value, indicating the efficacy of reducing inflammation and reducing skin sensitivity. The reduction in the scalp erythema value further confirms the ability of ColpepA1 21 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 anti-aging patent of recombinant humanized collagen type 21, indicating that ColpepA1 21 can effectively improve multiple signs of skin aging, including reducing wrinkles and spots, increasing skin elasticity and firmness, and improving the overall radiance and color of the skin. These changes not only improved the skin condition of the volunteers in appearance, but also improved the health of the skin physiologically, reducing water loss and inflammatory response. These effects show that ColpepA1 21 can be used as an effective active ingredient in anti-aging products.

[0246] The specific embodiments of the present invention are described in detail above, but they are only 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 made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention are all within the scope of the present invention.

Claims

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

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

3. A carrier, wherein The vector contains the nucleic acid molecule according to claim 2.

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

5. Use of the collagen according to claim 1, the nucleic acid molecule according to claim 2, the vector according to claim 3 or the host cell according to claim 4 in the preparation of an anti-aging medical composition.

6. A method for preparing collagen according to claim 1, wherein: The preparation method comprises the steps of preparing the collagen by using a Pichia pastoris expression system; Preferably, the preparation method comprises one or more steps selected from the group consisting of introducing the vector of claim 3 into Pichia pastoris to form genetically engineered Pichia pastoris, fermenting and culturing the genetically engineered Pichia pastoris, and inducing and expressing the collagen.

7. A composition, wherein the active ingredient of the composition comprises the collagen according to claim 1; Preferably, the composition comprises one or more auxiliary ingredients or excipients; More preferably, the auxiliary ingredients are selected from one or more of moisturizers, antioxidants, ultraviolet absorbers, penetration enhancers, and plant extracts; More preferably, the auxiliary material is selected from one or more of stabilizers, emulsifiers, conditioners, diluents, fillers, adhesives, wetting agents, absorption enhancers, surfactants, lubricants, fragrances or flavoring agents.

8. A medical product, wherein: The medical product comprises the collagen according to claim 1 or the composition according to claim 7; Preferably, the medical product is in the form of one or more selected from a facial cream, a serum, a facial mask, a smear gel, a lotion or a skin booster; More preferably, the skin enhancing agent is an injectable skin enhancing agent.

9. Use of the composition according to claim 7 or the medical product according to claim 8 in the preparation of anti-aging medical products.

Images