Anti-aging injection composition, Anti-aging injection, and use thereof

Abstract

An anti-aging injection composition, an anti-aging injection, and use thereof. The anti-aging injection composition contains hyaluronic acid or a salt thereof, glycine, proline, lysine hydrochloride, and leucine, which can synergistically promote the generation of collagen and elastin in the skin, thereby helping to correct skin wrinkles, improve skin elasticity, and enhance skin radiance. The anti-aging injection composition can be used to prepare an anti-aging injection, which has the effect of resisting skin aging.

Description

Anti-aging injection composition, anti-aging injection and use thereof Technical Field

[0001] The present application belongs to the field of medical cosmetology technology, and specifically relates to an anti-aging injection composition, an anti-aging injection solution and uses thereof. Background Art

[0002] As people's pursuit of a more youthful appearance increases, facial and neck aging is becoming a significant concern for those seeking beauty. Skin aging has numerous causes, including sun exposure, environmental pollution, and unhealthy lifestyle habits such as prolonged late nights, smoking, and excessive drinking, all of which can negatively impact skin health, leading to sagging, fine lines, coarse lines, dullness, and other aging-related issues. Skin aging can be largely attributed to fibroblast dysfunction and decreased biosynthetic activity in the skin. Aging fibroblasts produce less extracellular matrix, making it insufficient for maintaining the skin's youthful appearance.

[0003] Hyaluronic acid, also known as hyaluronic acid, is a macromolecular acidic mucopolysaccharide composed of D-glucuronic acid and N-acetylglucosamine disaccharide units. It is widely distributed throughout the human body, including tissues such as articular cartilage, the lens, and the dermis. The skin accounts for over half of the total hyaluronic acid in the body. As people age, the amount of hyaluronic acid in the skin gradually decreases, causing the skin to lose elasticity and become rough and dull. Because it is non-species-specific, endogenous hyaluronic acid deficiency can be supplemented with exogenous hyaluronic acid. Due to its natural water-retention, lubricity, and viscoelastic properties, hyaluronic acid is widely used in beauty, cosmetics, and pharmaceuticals.

[0004] Amino acids are a general term for a class of organic compounds containing amino and carboxyl groups. They are the most basic substances that make up biological proteins and are related to life activities. They are the basic units that make up protein molecules in organisms and are closely related to the life activities of organisms. They have special physiological functions in the body and are one of the indispensable nutrients in organisms. More than 300 natural amino acids have been discovered, of which approximately 22 are required by the human body. They are divided into essential amino acids (which the human body cannot synthesize on its own) and non-essential amino acids. In particular, glycine, proline, lysine, and leucine are the main amino acids that make up collagen. As nutrients, adding small amounts of amino acids to skin injection products has benefits for skin health and anti-aging.

[0005] Therefore, hyaluronic acid and amino acids can be combined to fight skin aging. For example, they can be added to injectable products in the medical aesthetics field as an effective anti-aging ingredient.

[0006] In the prior art, the document "ENHANCEMENT OF FIBROBLAST PROLIFERATION, COLLAGEN BIOSYNTHESIS AND PRODUCTION OF GROWTH FACTORS AS A RESULT OF COMBINING SODIUM HYALURONATE AND AMINOACIDS" (MAMARIGGIO, etc.) discloses a gel product containing hyaluronic acid and amino acids. This product is a surgical product and not an anti-aging injection in the medical beauty field. It is mainly used to accelerate wound healing and can be used to treat chronic skin wounds. In order to achieve the purpose of promoting wound healing, this product needs to add a higher concentration of amino acids to stimulate the proliferation of fibroblasts in the wound and promote the expression of inflammatory factors, thereby promoting wound healing. In the wound healing process, inflammatory factors have the function of clearing pathogens from the damaged area and promoting tissue repair, but inflammatory factors (TNF-α, IFN-β, IFN-γ, etc.) can induce epithelial cell senescence by producing ROS and activating the ATM / P53 / P21 (WAF1 / CIP1) signaling pathway. Therefore, the product disclosed in this document is not suitable for anti-aging. In addition, high-concentration amino acid formulas are not suitable for skin injection as water-light products. High concentrations of amino acids may increase the safety risks of the product, for example, causing redness, swelling, pain, etc.

[0007] Patent CN 117357699 A discloses a sodium hyaluronate composition with freckle removal efficacy. The patent verifies the improvement effect of sodium hyaluronate, peptides, freckle removal components, vitamins, buffers, and certain types of amino acids (glycine, alanine, proline) on fine lines, texture, and spots on facial skin. However, the formula is complex in composition and requires the addition of a large number of peptides and vitamins with anti-aging effects. This leads to high production costs, poor stability, and difficulty in storage and transportation. Summary of the Invention

[0008] The present application creatively discovered that combining hyaluronic acid or its salts with relatively low amounts of glycine, proline, lysine hydrochloride, and leucine not only does not produce an inflammatory response, but can actually reduce the expression of inflammatory factors, leading to a better anti-aging effect. Injecting an anti-aging injection containing this composition into the dermis of the skin not only promotes skin hydration, but also promotes the metabolic activity and maintenance of normal skin cell structure, stimulates the proliferation of skin fibroblasts, increases collagen fiber content, and repairs elastic fiber damage.

[0009] The anti-aging effect of the composition is better than that of a combination of hyaluronic acid or its salt and a higher amount of glycine, proline, lysine hydrochloride, leucine or other types of amino acids. Even without the addition of additional active ingredients such as peptides and vitamins, it can still exert a significant anti-aging effect.

[0010] The purpose of the present application is to provide an anti-aging injection composition, an anti-aging injection solution and uses thereof.

[0011] Specifically, this application involves the following aspects:

[0012] 1. An anti-aging injection composition comprising hyaluronic acid or a salt thereof and amino acids, wherein the amino acids comprise glycine, proline, leucine, and lysine hydrochloride.

[0013] 2. The anti-aging injection composition according to item 1, wherein the molecular weight of the hyaluronic acid or its salt is 200,000-2.5 million Da.

[0014] 3. The anti-aging injection composition according to item 1 or 2, wherein the mass ratio of the amino acid to the hyaluronic acid or a salt thereof is 1:(2-20).

[0015] 4. The anti-aging injection composition according to any one of items 1 to 3, wherein the mass ratio of glycine, proline, leucine and lysine hydrochloride is (2-20):(1-15):(1-8):(1-8).

[0016] 5. The anti-aging injection composition according to any one of items 1 to 4, wherein the composition further comprises excipients and / or active ingredients acceptable in the field of medical aesthetics.

[0017] 6. An anti-aging injection composition comprising hyaluronic acid or a salt thereof, glycine, proline, leucine, and lysine hydrochloride.

[0018] 7. The anti-aging injection composition according to item 6, wherein the molecular weight of the hyaluronic acid or its salt is 200,000-2.5 million Da.

[0019] 8. The anti-aging injection composition according to item 6 or 7, wherein the mass ratio of the sum of glycine, proline, leucine, and lysine hydrochloride to the hyaluronic acid or a salt thereof is 1:(2-20).

[0020] 9. The anti-aging injection composition according to any one of items 6 to 8, wherein the mass ratio of glycine, proline, leucine and lysine hydrochloride is (2-20):(1-15):(1-8):(1-8).

[0021] 10. The anti-aging injection composition according to any one of items 6 to 9, wherein the composition further comprises excipients and / or active ingredients acceptable in the field of medical aesthetics.

[0022] 11. An anti-aging injection comprising the anti-aging injection composition according to any one of items 1 to 10.

[0023] 12. The anti-aging injection according to item 11, wherein the content of hyaluronic acid or its salt is 8-20 mg / mL, the content of glycine is 0.2-2 mg / mL, the content of proline is 0.1-1.5 mg / mL, the content of leucine is 0.1-0.8 mg / mL, and the content of lysine hydrochloride is 0.1-0.8 mg / mL.

[0024] 13. Use of the anti-aging injection composition according to any one of Items 1 to 10 or the anti-aging injection solution according to any one of Items 11 to 12 in anti-skin aging.

[0025] 14. The use according to item 13, wherein the anti-skin aging includes promoting collagen production in the skin, promoting elastin production in the skin, reducing skin wrinkles, improving skin elasticity, enhancing skin brightness, reducing collagen fiber breakage, damage, loss and degradation, reducing elastic fiber aging damage, promoting skin tissue regeneration, reducing oxidative stress caused by skin aging, reducing inflammatory response caused by skin aging, or increasing the expression of AQP-3 in the skin.

[0026] 15. Use of the anti-aging injection composition according to any one of items 1 to 10 or the anti-aging injection solution according to any one of items 11 to 12 in the preparation of an anti-skin aging product.

[0027] 16. The use according to item 15, wherein the anti-skin aging includes promoting collagen production in the skin, promoting elastin production in the skin, reducing skin wrinkles, improving skin elasticity, enhancing skin brightness, reducing collagen fiber breakage, damage, loss and degradation, reducing elastic fiber aging damage, promoting skin tissue regeneration, reducing oxidative stress caused by skin aging, reducing inflammatory response caused by skin aging, or increasing the expression of AQP-3 in the skin.

[0028] 17. A method for combating skin aging, comprising administering to a subject an effective amount of the anti-aging injection composition according to any one of items 1 to 10 or the anti-aging injection solution according to any one of items 11 to 12.

[0029] 18. The method according to item 17, wherein the anti-skin aging includes promoting collagen production in the skin, promoting elastin production in the skin, reducing skin wrinkles, improving skin elasticity, enhancing skin brightness, reducing collagen fiber breakage, damage, loss and degradation, reducing elastic fiber aging damage, promoting skin tissue regeneration, reducing oxidative stress response caused by skin aging, reducing inflammatory response caused by skin aging, or increasing the expression of AQP-3 in the skin.

[0030] The anti-aging injectable composition of the present application contains hyaluronic acid or its salts, glycine, proline, lysine hydrochloride, and leucine. The hyaluronic acid or its salts, glycine, proline, lysine hydrochloride, and leucine synergistically promote the production of collagen and elastin in the skin, thereby correcting wrinkles, improving skin elasticity, and enhancing skin brightness. This anti-aging injectable composition can be used to prepare anti-aging injections, such as fillers and anti-aging injections, to combat skin aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 shows the observation results of skin changes in mice in each test group;

[0032] FIG2 shows the HE staining results of mouse skin in each experimental group;

[0033] Figure 3 shows the results of Masson staining of mouse skin in each experimental group;

[0034] Figure 4 shows the electron microscopy results of the skin of mice in each experimental group;

[0035] FIG5 shows the results of resorcinol fuchsin staining of mouse skin in each experimental group;

[0036] FIG6 shows fluorescence images of MMP3-positive skin basal stem cells (red) in the skin of mice in each experimental group;

[0037] FIG7 shows the relative statistical results of the number of MMP3-positive skin basal cells in the skin of mice in each experimental group;

[0038] FIG8 shows fluorescence images of CK19-positive skin basal stem cells (red) in the skin of mice in each experimental group;

[0039] FIG9 shows the relative statistical results of the number of CK19-positive skin basal stem cells in the skin of mice in each experimental group;

[0040] FIG10 shows fluorescence images of CK14-positive skin basal stem cells (red) in the skin of mice in each experimental group;

[0041] FIG11 shows the relative statistical results of the number of CK14-positive skin basal cells in the skin of mice in each experimental group;

[0042] Figure 12 shows the expression results of SOD, CAT, HO-1, and NQO1 in the skin of mice in each experimental group;

[0043] FIG13 shows the expression results of TNF-α and IL-1β in the skin of mice in each experimental group;

[0044] FIG14 shows the water content of the skin of mice in each test group;

[0045] FIG15 shows the AQP-3 expression levels of mice in each experimental group. DETAILED DESCRIPTION

[0046] The present application is further described below with reference to examples. It should be understood that the examples are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0047] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as those commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific examples, which are not intended to limit the scope of this application.

[0048] In view of the problems existing in the prior art, the present application provides an anti-aging injection composition comprising hyaluronic acid or a salt thereof and amino acids, wherein the amino acids include glycine, proline, leucine, and lysine hydrochloride.

[0049] Those skilled in the art will appreciate that, in addition to the four amino acids glycine, proline, leucine, and lysine hydrochloride, the anti-aging injection composition of the present application may further comprise other amino acids.

[0050] In a specific embodiment, the amino acids consist of glycine, proline, leucine, and lysine hydrochloride.

[0051] In a specific embodiment, the molecular weight of the hyaluronic acid or its salt is 200,000 Da to 2.5 million Da, for example, 200,000 Da, 300,000 Da, 400,000 Da, 500,000 Da, 600,000 Da, 700,000 Da, 800,000 Da, 900,000 Da, 1,000,000 Da, 1,100,000 Da, 1,200,000 Da, 1,300,000 Da, 1,400,000 Da, 1,500,000 Da, 1,600,000 Da, 1,700,000 Da, 1,800,000 Da, 1,900,000 Da, 2,000,000 Da, 2,100,000 Da, 2,200,000 Da, 2,300,000 Da, 2,400,000 Da, 2,500,000 Da, and any value therebetween.

[0052] In a specific embodiment, the hyaluronic acid or its salt can be a combination of one or more, for example, it can be a combination of one hyaluronic acid or its salt, a combination of two hyaluronic acids or their salts, a combination of three hyaluronic acids or their salts, etc.

[0053] In a specific embodiment, the mass ratio of the amino acid to the hyaluronic acid or its salt is 1:(2-20), for example, it can be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, and any value therebetween.

[0054] In a specific embodiment, the mass ratio of glycine, proline, leucine, and lysine hydrochloride is (2-20): (1-15): (1-8): (1-8). 2-20 can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and any values ​​therebetween. 1-15 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and any values ​​therebetween. 1-8 can be 1, 2, 3, 4, 5, 6, 7, 8, and any values ​​therebetween.

[0055] Those skilled in the art will appreciate that, according to actual application requirements, the anti-aging injection composition may further include excipients and / or active ingredients acceptable in the field of medical aesthetics.

[0056] In one specific embodiment, the anti-aging injection composition comprises hyaluronic acid or a salt thereof and amino acids, wherein the amino acids include glycine, proline, leucine, and lysine hydrochloride. The molecular weight of the hyaluronic acid or a salt thereof is 2.0-2.5 million Da, and the mass ratio of the amino acids to the hyaluronic acid or a salt thereof is 1:(2-20).

[0057] In a specific embodiment, the anti-aging injection composition comprises hyaluronic acid or a salt thereof and amino acids, wherein the amino acids include glycine, proline, leucine, and lysine hydrochloride. The molecular weight of the hyaluronic acid or its salt is 2.0-2.5 million Da, and the mass ratio of the amino acids to the hyaluronic acid or its salt is 1:(2-20). The mass ratio of glycine, proline, leucine, and lysine hydrochloride is (2-20):(1-15):(1-8):(1-8).

[0058] In one specific embodiment, the anti-aging injection composition comprises hyaluronic acid or a salt thereof and amino acids, wherein the amino acids are composed of glycine, proline, leucine, and lysine hydrochloride. The molecular weight of the hyaluronic acid or a salt thereof is 2.0-2.5 million Da, and the mass ratio of the amino acids to the hyaluronic acid or a salt thereof is 1:(2-20).

[0059] In a specific embodiment, the anti-aging injection composition comprises hyaluronic acid or a salt thereof and amino acids, wherein the amino acids are composed of glycine, proline, leucine, and lysine hydrochloride. The molecular weight of the hyaluronic acid or its salt is 2.0-2.5 million Da, and the mass ratio of the amino acids to the hyaluronic acid or its salt is 1:(2-20). The mass ratio of the glycine, proline, leucine, and lysine hydrochloride is (2-20):(1-15):(1-8):(1-8).

[0060] In one specific embodiment, the anti-aging injection composition comprises hyaluronic acid or a salt thereof and amino acids, wherein the amino acids include glycine, proline, leucine, and lysine hydrochloride. The molecular weight of the hyaluronic acid or a salt thereof is 2.0-2.5 million Da, and the mass ratio of the amino acids to the hyaluronic acid or a salt thereof is 1:(2-20).

[0061] In a specific embodiment, the anti-aging injection composition is composed of hyaluronic acid or a salt thereof and amino acids, wherein the amino acids include glycine, proline, leucine, and lysine hydrochloride. The molecular weight of the hyaluronic acid or its salt is 2.0-2.5 million Da, and the mass ratio of the amino acids to the hyaluronic acid or its salt is 1:(2-20). The mass ratio of glycine, proline, leucine, and lysine hydrochloride is (2-20):(1-15):(1-8):(1-8).

[0062] In one specific embodiment, the anti-aging injection composition comprises hyaluronic acid or a salt thereof and amino acids, wherein the amino acids are glycine, proline, leucine, and lysine hydrochloride. The molecular weight of the hyaluronic acid or a salt thereof is 2.0-2.5 million Da, and the mass ratio of the amino acids to the hyaluronic acid or a salt thereof is 1:(2-20).

[0063] In a specific embodiment, the anti-aging injection composition is composed of hyaluronic acid or a salt thereof and amino acids, wherein the amino acids are composed of glycine, proline, leucine, and lysine hydrochloride. The molecular weight of the hyaluronic acid or its salt is 2.0-2.5 million Da, and the mass ratio of the amino acids to the hyaluronic acid or its salt is 1:(2-20). The mass ratio of glycine, proline, leucine, and lysine hydrochloride is (2-20):(1-15):(1-8):(1-8).

[0064] In a specific embodiment, the anti-aging injection composition comprises hyaluronic acid or a salt thereof and glycine, proline, leucine, and lysine hydrochloride. The molecular weight of the hyaluronic acid or its salt is 2-2.5 million Da, and the mass ratio of the sum of the mass of the glycine, proline, leucine, and lysine hydrochloride to the mass of the hyaluronic acid or its salt is 1:(2-20). Furthermore, the mass ratio of the glycine, proline, leucine, and lysine hydrochloride is (2-20):(1-15):(1-8):(1-8).

[0065] In a specific embodiment, the anti-aging injection composition is composed of hyaluronic acid or a salt thereof and glycine, proline, leucine, and lysine hydrochloride. The molecular weight of the hyaluronic acid or its salt is 2.0-2.5 million Da, and the mass ratio of the sum of the mass of the glycine, proline, leucine, and lysine hydrochloride to the mass of the hyaluronic acid or its salt is 1:(2-20). Furthermore, the mass ratio of the glycine, proline, leucine, and lysine hydrochloride is (2-20):(1-15):(1-8):(1-8).

[0066] In a specific embodiment, in the anti-aging injection composition, the content of hyaluronic acid or its salt is 8-20 mg / mL, for example, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, and any value therebetween; the content of glycine is 0.2-2 mg / mL, for example, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL. / mL, 1.9 mg / mL, 2 mg / mL, and any value therebetween; the content of proline is 0.1-1.5 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL. g / mL, 1.4 mg / mL, 1.5 mg / mL, and any values ​​therebetween; the leucine content is 0.1-0.8 mg / mL, and the lysine hydrochloride content is 0.1-0.8 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, and any values ​​therebetween.

[0067] The present application also provides an anti-aging injection composition comprising hyaluronic acid or a salt thereof, glycine, proline, leucine, and lysine hydrochloride.

[0068] The type, molecular weight, and content of hyaluronic acid or its salt, and the mass ratio and content of glycine, proline, leucine, and lysine hydrochloride are as described above.

[0069] In a specific embodiment, the mass ratio of the sum of glycine, proline, leucine, and lysine hydrochloride to the hyaluronic acid or its salt is 1:(2-20), for example, it can be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, and any value in between these values.

[0070] The present application also provides an anti-aging injection composition, the active ingredients of which include: hyaluronic acid or its salt, glycine, proline, leucine, and lysine hydrochloride.

[0071] The type, molecular weight, and content of hyaluronic acid or its salt, and the mass ratio and content of glycine, proline, leucine, and lysine hydrochloride are as described above.

[0072] The active ingredients refer to ingredients that can resist skin aging, and the anti-skin aging effects include promoting collagen production in the skin, promoting elastin production in the skin, reducing skin wrinkles, improving skin elasticity, enhancing skin brightness, reducing collagen fiber breakage, damage, loss and degradation, reducing elastic fiber aging damage, promoting skin tissue regeneration, reducing oxidative stress caused by skin aging, reducing inflammatory response caused by skin aging, or increasing the expression of AQP-3 in the skin.

[0073] The present application also provides an anti-aging injection, comprising any one of the above-mentioned anti-aging injection compositions.

[0074] In a specific embodiment, in the anti-aging injection, the content of hyaluronic acid or its salt is 8-20 mg / mL, for example, it can be 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, and any value therebetween; the content of glycine is 0.2-2 mg / mL, for example, it can be 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL. mL, 1.9 mg / mL, 2 mg / mL, and any value therebetween; the content of proline is 0.1-1.5 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, and any values ​​therebetween; the leucine content is 0.1-0.8 mg / mL, and the lysine hydrochloride content is 0.1-0.8 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, and any values ​​therebetween.

[0075] Those skilled in the art will understand that the injection solution also needs to include water for injection, a buffer solution, an osmotic pressure regulator and / or an anesthetic, and also needs to be sterilized accordingly.

[0076] The present application also provides use of any one of the above-mentioned anti-aging injection compositions or anti-aging injection solutions in anti-skin aging.

[0077] The present application also provides use of any of the above-mentioned anti-aging injection compositions or anti-aging injection solutions in the preparation of anti-skin aging products.

[0078] The present application also provides a method for anti-skin aging, comprising administering an effective amount of any one of the above-mentioned anti-aging injection compositions or anti-aging injection solutions to a subject.

[0079] In the above-mentioned uses or methods, the anti-skin aging can cover various types of skin aging known in the art, such as promoting collagen production in the skin, promoting elastin production in the skin, reducing skin wrinkles, improving skin elasticity, enhancing skin brightness, reducing collagen fiber breakage, damage, loss and degradation, reducing elastic fiber aging damage, promoting skin tissue regeneration, reducing oxidative stress response caused by skin aging, reducing inflammatory response caused by skin aging, or increasing the expression of AQP-3 in the skin, etc.

[0080] The anti-aging injection composition of the present application contains hyaluronic acid or its salt, glycine, proline, lysine hydrochloride, and leucine. The hyaluronic acid or its salt, glycine, proline, lysine hydrochloride, and leucine synergistically promote the production of collagen and elastin in the skin. Therefore, the anti-aging injection composition of the present application is beneficial for correcting skin wrinkles, improving skin elasticity, enhancing skin brightness, and other effects that resist skin aging. It can be used to prepare anti-aging injection solutions, such as fillers and anti-aging injections.

[0081] Example

[0082] Example 1

[0083] To prepare the anti-aging injection, weigh appropriate amounts of phosphate, sodium chloride, glycine, proline, leucine, lysine hydrochloride, and sodium hyaluronate, then add water for injection and stir to dissolve completely. Fill the container and sterilize with moist heat (121°C for 10 minutes) to obtain a sterile anti-aging injection. The concentration of sodium hyaluronate (molecular weight 1.45 million Da) is 8 mg / mL, the concentration of glycine is 0.2 mg / mL, the concentration of proline is 0.1 mg / mL, the concentration of lysine hydrochloride is 0.1 mg / mL, and the concentration of leucine is 0.1 mg / mL.

[0084] Example 2

[0085] To prepare the anti-aging injection, weigh appropriate amounts of phosphate, sodium chloride, glycine, proline, leucine, lysine hydrochloride, and sodium hyaluronate, then add water for injection and stir to dissolve completely. Fill the container and sterilize with moist heat (121°C for 10 minutes) to obtain a sterile anti-aging injection. The concentration of sodium hyaluronate (molecular weight 1.45 million Da) is 12 mg / mL, the concentration of glycine is 0.8 mg / mL, the concentration of proline is 0.6 mg / mL, the concentration of lysine hydrochloride is 0.2 mg / mL, and the concentration of leucine is 0.2 mg / mL.

[0086] Example 3

[0087] An anti-aging injection was prepared. The difference from Example 1 was the content of each component: the concentration of sodium hyaluronate (molecular weight 1.45 million Da) was 20 mg / mL, the concentration of glycine was 2 mg / mL, the concentration of proline was 1.5 mg / mL, the concentration of lysine hydrochloride was 0.75 mg / mL, and the concentration of leucine was 0.75 mg / mL.

[0088] Example 4

[0089] An anti-aging injection was prepared. The difference from Example 2 was that the molecular weight of sodium hyaluronate was 300,000 Da.

[0090] Example 5

[0091] An anti-aging injection was prepared. The difference from Example 2 was that the molecular weight of sodium hyaluronate was 2.2 million Da.

[0092] Example 6

[0093] To prepare the anti-aging injection, weigh appropriate amounts of phosphate, sodium chloride, glycine, proline, leucine, lysine hydrochloride, and sodium hyaluronate, then add water for injection and stir to dissolve completely. Fill the container and sterilize with moist heat (121°C for 10 minutes) to obtain a sterile anti-aging injection. The concentration of sodium hyaluronate (molecular weight 1.64 million Da) is 13.3 mg / mL, the concentration of glycine is 0.8 mg / mL, the concentration of proline is 0.6 mg / mL, the concentration of lysine hydrochloride is 0.2 mg / mL, and the concentration of leucine is 0.2 mg / mL.

[0094] Example 7

[0095] Preparation of anti-aging injection: Weigh appropriate amounts of phosphate, sodium chloride, glycine, proline, leucine, lysine hydrochloride, and sodium hyaluronate, then add water for injection, stir to dissolve completely, fill, and then sterilize with high-temperature moist heat (121°C, 10 minutes) to obtain a sterile anti-aging injection. The concentration of sodium hyaluronate (molecular weight 1.45 million Da) is 10 mg / mL, the concentration of sodium hyaluronate (molecular weight 300,000 Da) is 2 mg / mL, the concentration of glycine is 0.8 mg / mL, the concentration of proline is 0.6 mg / mL, the concentration of lysine hydrochloride is 0.2 mg / mL, and the concentration of leucine is 0.2 mg / mL.

[0096] Comparative Example 1

[0097] The anti-aging injection was prepared, which was different from Example 1 in that no amino acids were added.

[0098] Comparative Example 2

[0099] The anti-aging injection was prepared, which was different from Example 2 in that no amino acids were added.

[0100] Comparative Example 3

[0101] An anti-aging injection was prepared, but unlike Example 3, no amino acids were added.

[0102] Comparative Example 4

[0103] The anti-aging injection was prepared, which was different from Example 4 in that no amino acids were added.

[0104] Comparative Example 5

[0105] The anti-aging injection was prepared, which was different from Example 5 in that no amino acids were added.

[0106] Comparative Example 6

[0107] The anti-aging injection was prepared, which was different from Example 6 in that no amino acids were added.

[0108] Comparative Example 7

[0109] An anti-aging injection was prepared, which differed from Example 1 in that sodium hyaluronate was not added.

[0110] Comparative Example 8

[0111] An anti-aging injection was prepared, which differed from Examples 2 and 4-6 in that sodium hyaluronate was not added.

[0112] Comparative Example 9

[0113] An anti-aging injection was prepared, which differed from Example 3 in that sodium hyaluronate was not added.

[0114] Comparative Example 10

[0115] To prepare the anti-aging injection, weigh appropriate amounts of phosphate, sodium chloride, glycine, proline, leucine, lysine hydrochloride, and sodium hyaluronate, then add water for injection and stir to dissolve completely. Fill the container and sterilize with moist heat (121°C for 10 minutes) to obtain a sterile anti-aging injection. The concentration of sodium hyaluronate (molecular weight 1.45 million Da) is 12 mg / mL, the concentration of glycine is 0.1 mg / mL, the concentration of proline is 0.05 mg / mL, the concentration of leucine is 1.6 mg / mL, and the concentration of lysine hydrochloride is 0.05 mg / mL.

[0116] Comparative Example 11

[0117] To prepare the anti-aging injection, weigh appropriate amounts of phosphate, sodium chloride, glycine, proline, leucine, lysine hydrochloride, and sodium hyaluronate, then add water for injection and stir to dissolve completely. Fill the container and sterilize with moist heat (121°C for 10 minutes) to obtain a sterile anti-aging injection. The concentration of sodium hyaluronate (molecular weight 1.64 million Da) is 13.3 mg / mL, the concentration of glycine is 10 mg / mL, the concentration of proline is 7.5 mg / mL, the concentration of leucine is 1.5 mg / mL, and the concentration of lysine hydrochloride is 1 mg / mL.

[0118] Comparative Example 12

[0119] An anti-aging injection was prepared, which differed from Comparative Example 11 in that sodium hyaluronate was not added.

[0120] Comparative Example 13

[0121] To prepare the anti-aging injection, weigh appropriate amounts of phosphate, sodium chloride, glycine, proline, alanine, and sodium hyaluronate, add water for injection, stir until completely dissolved, fill, and sterilize with moist heat (121°C for 10 minutes) to obtain a sterile anti-aging injection. The concentration of sodium hyaluronate (molecular weight 1.1 million Da) is 6 mg / mL, the concentration of glycine is 0.15 mg / mL, the concentration of proline is 0.2 mg / mL, and the concentration of alanine is 0.1 mg / mL.

[0122] The specific component contents of the above examples and comparative examples are shown in Table 1.

[0123] Table 1

[0124] Experimental Example 1: Effect of Samples on Proliferation of Human Skin Fibroblasts

[0125] Using human skin fibroblasts (FB cells) as a model, the proliferation effects of the samples of Examples 1-7 and Comparative Examples 1-13 on FB cells were detected.

[0126] Sample preparation: Taking Example 1 as an example, the sample solution was diluted with 2% FBSDMEM culture medium to a concentration of 5% of the test product. The test products of Examples 2-7 and Comparative Examples 1-13 with different concentrations were prepared in the same manner.

[0127] Experimental method: FB cells were cultured in a T75 culture flask with 10% FBSDMEM medium. When the cell density reached about 80%, they were digested with 0.05% trypsin and centrifuged at 1000 rpm for 5 minutes. After resuspension, the cells were plated at 3×10 4Seed 10 cells / well into a 96-well plate; 24 hours after seeding, aspirate the culture medium and add the corresponding test article concentrations. After 24 hours, aspirate the culture medium, rinse each well with 200 μl of PBS, add 200 μl of freshly prepared 0.5 mg / ml MTT solution, and incubate in a CO2 incubator protected from light. After 3 hours, carefully aspirate the MTT solution, add 100 μl of DMSO to each well to dissolve the MTT solution, and measure absorbance at 550 nm using a microplate reader.

[0128] Data analysis and test results:

[0129] The absorbance of MTT was analyzed using Excel software, and cell activity was calculated using the following formula:

[0130] Cell activity growth rate (%) = (cell activity of sample - cell activity of 2% FBS-DMEM medium) × 100%

[0131] 2% FBS-DMEM culture medium was used as the negative control group sample.

[0132] The results of the effects of the samples prepared in Examples 1-7 and Comparative Examples 1-13 on FB cell proliferation are shown in Table 2.

[0133] Table 2 Test results of the effects of the test products on FB cell proliferation

[0134] It can be seen that the growth rates of cell activity in Examples 1-7 are all higher than those in the comparative examples, indicating that a synergistic effect occurs between hyaluronic acid and amino acids; Comparative Example 10 uses the same total amount of amino acids as in Example 2, but after adjusting the ratio of glycine, proline, leucine, and lysine hydrochloride, the growth rate of cell activity is significantly reduced; Comparative Example 11 uses the same amount of sodium hyaluronate as in Example 6, but after the amount of amino acid added is reduced, the growth rate of cell activity is significantly reduced; Comparative Example 13 does not achieve a good cell proliferation effect after replacing amino acids with other types.

[0135] In order to further verify the synergistic effect of hyaluronic acid and amino acids, the following COLBY formula was used for verification: E = X + Y - XY / 100

[0136] Where X and Y are the cell activity growth rates after single component treatment, E is the theoretical cell activity growth rate after combination treatment, and E is the actual cell activity growth rate after combination treatment. E>E indicates synergism; E<E indicates antagonism; and E =E indicates addition.

[0137] For example, when the concentration of sodium hyaluronate is 12 mg / mL, the cell activity growth rate is 6%, as shown in Comparative Example 2; when the concentration of glycine is 0.8 mg / mL, the concentration of proline is 0.6 mg / mL, the concentration of lysine hydrochloride is 0.2 mg / mL, and the concentration of leucine is 0.2 mg / mL, the cell activity growth rate is 2%, as shown in Comparative Example 8; when the concentration of sodium hyaluronate is 12 mg / mL and is combined with the concentration of glycine at 0.8 mg / mL, the concentration of proline at 0.6 mg / mL, the concentration of lysine hydrochloride at 0.2 mg / mL, and the concentration of leucine at 0.2 mg / mL, the cell activity growth rate is 26%, as shown in Example 2;

[0138] but,

[0139] X = cell activity growth rate when the concentration of sodium hyaluronate is 12 mg / mL = 6%;

[0140] Y = glycine concentration of 0.8 mg / mL, proline concentration of 0.6 mg / mL, lysine hydrochloride concentration of 0.2 mg / mL, leucine concentration of 0.2 mg / mL = 2%;

[0141] Eactual = Cell activity growth rate when sodium hyaluronate is 12 mg / mL in combination with glycine, proline, lysine hydrochloride, and leucine at 0.8 mg / mL, 0.6 mg / mL, 0.2 mg / mL, and 0.2 mg / mL, = 26%.

[0142] Substituting into the formula, we can obtain E=X+Y-XY / 100=7%<Eactual, which shows that Example 2 has a synergistic effect compared with Comparative Examples 2 and 8.

[0143] Similarly, the synergistic effect of Example 1 with Comparative Example 1 and Comparative Example 7, Example 3 with Comparative Example 3 and Comparative Example 9, Example 4 with Comparative Example 4 and Comparative Example 8, Example 5 with Comparative Example 5 and Comparative Example 8, Example 6 with Comparative Example 6 and Comparative Example 8, and Comparative Example 11 with Comparative Example 6 and Comparative Example 12 was verified.

[0144] The verification results are shown in Table 3.

[0145] Table 3

[0146] It can be seen that the E values ​​of Examples 1-6 are all smaller than E actual. Therefore, a synergistic effect is produced between the sodium hyaluronate and glycine, proline, leucine, and lysine hydrochloride in the examples of the present application, which can significantly promote the proliferation of human skin fibroblasts. The E values ​​of Comparative Example 11 are not much different from E actual. That is, although the amount of amino acid added in Comparative Example 11 is increased on the basis of Example 6, no synergistic effect is produced between the components, and the proliferation of human skin fibroblasts cannot be significantly promoted.

[0147] Experimental Example 2 Effects of Samples on Collagen and Elastin RNA Expression in FB Cell Model

[0148] Using FB cells as a model, the effects of the samples of Example 2, Comparative Example 2, and Comparative Example 8 on the RNA expression of collagen and elastin in FB cells were detected.

[0149] Test samples: The sample of Example 2 was diluted with 10% FBSDMEM culture medium to prepare a 5% concentration of the sample of Example 2; 5% of the sample of Comparative Example 2 and 5% of the sample of Comparative Example 8 were prepared in the same manner.

[0150] Control sample: 10% FBSDMEM medium

[0151] Experimental method: FB cells were cultured in a T75 culture flask with 10% FBSDMEM medium. When the cell density reached about 80%, they were digested with 0.05% trypsin and centrifuged at 1000 rpm for 5 minutes. After resuspension, 5×10 5 Cells were seeded / dish in 6-cm dishes. After 24 hours of seeding, the culture medium was aspirated and the corresponding concentrations of test samples or control samples were added. After 24 hours of treatment, each dish was washed three times with 2 ml of PBS and then 1 ml of Trizol was added and perfused. Cell samples were then collected for real-time fluorescence quantitative PCR analysis.

[0152] Data processing and test results:

[0153] Image J software was used to quantitatively analyze the fluorescence data of the photos and obtain the average fluorescence intensity. The relative fluorescence intensity was calculated using the following formula:

[0154] Data were processed using Graphpad Prism software, and the results are expressed as mean ± SD. Student's t-test was used for comparisons between groups. *P < 0.05 indicates a statistically significant difference, **P < 0.01 indicates a statistically significant difference, and ***P < 0.001 indicates an extremely significant difference.

[0155] The test results are shown in Table 3. Compared with Comparative Examples 2 and 8, it can be seen that the sample of Example 2 has a very good promoting effect on the RNA expression of type I and type III collagen and elastin.

[0156] Table 3 Effects of samples on RNA expression of type I, type III collagen and elastin

[0157] Experimental Example 3: Study on the efficacy of the sample of Example 2 through animal experiments

[0158] Healthy experimental mice were divided into three groups: a blank control group (Control), a model group (D-Gal), and a test group. The mice were depilated and given 5% D-galactose (D-Gal) (500 mg / kg) subcutaneously in the neck and back of the mice except the blank group every day for 42 consecutive days to establish a subacute aging model in mice; the blank control group was subcutaneously injected with an equal volume of normal saline every day. Treatment began 14 days after modeling. The test group was given a 200 μL dose of the sample in Example 2, and the blank control group was injected with an equal volume of normal saline. The treatment was once every 7 days for 28 consecutive days. After the treatment, the appearance of the mice's skin was observed, and the experimental mice were subjected to tissue sampling, tissue sectioning, staining, and other processing for tissue morphology observation and quantitative analysis.

[0159] 1) Observe the changes in the anti-aging effects of the test product on the skin

[0160] After continuous injection of D-Gal for 42 days, compared with the control group, the model group had more wrinkles on the neck and back, a significant increase in the number of keratinized epidermal cells, and a dull skin color. The skin on the neck and back of the mice in the experimental group treated with the test product of Example 2 was smooth and rosy, with good elasticity and strong skin gloss (as shown in Figure 1).

[0161] 2) Effects of the test product on the morphology of aging skin tissue

[0162] After administering D-Gal to construct a skin aging model, the epidermis of the mice in the model group showed uneven thickness and incomplete structure, with significantly thinner collagen fibers, loose arrangement, many breaks, and uneven distribution. The epidermis of the mice in the control group was regular, with uniform thickness, tightly arranged epidermal cells, normal morphology, and a clear boundary between the epidermis and dermis. After treatment with the test article, the epidermis of the mice was uniform in thickness, with intact tissue structure and relatively neat fiber arrangement (as shown in Figure 2), indicating that the test article has an improving effect on the tissue morphology of aging skin.

[0163] 3) Effects of the test product on collagen fibers in aging skin

[0164] Transmission electron microscopy observations showed that the collagen fibers in the skin of the model group mice were significantly reduced, and the arrangement was sparse, disordered, and broken. After treatment with the test product, the collagen fiber content in the skin of the test group mice increased and was arranged more densely (as shown in Figures 3 and 4), indicating that the test product can reduce the breakage, damage, and loss of collagen fibers.

[0165] 4) Effects of the test product on elastic fibers in aging skin

[0166] Resorcinol fuchsin staining revealed that the dermal elastic fibers in the control group were bluish-purple, slender, and evenly distributed in a wavy pattern. In the model group, the elastic fibers were broken, twisted, and accumulated into a disordered, tangled structure. Compared with the model group, the elastic fibers in the experimental group were more evenly distributed, with fewer broken fibers (as shown in Figure 5). This suggests that the experimental product has a significant effect on alleviating the aging-related damage to elastic fibers in skin.

[0167] 5) The test product has the effect of reducing collagen fiber degradation

[0168] The expression of matrix metalloproteinase 3 (MMP3) is related to the degradation of collagen fibers. The results of immunofluorescence staining (Figure 6) showed that the expression of MMP3 in the skin tissue of the model group was significantly increased, and the expression of MMP3 was reduced after treatment with the test product. The results of Masson staining and resorcinol fuchsin staining showed that the test product group was able to promote the regeneration of collagen fibers and reduce the degradation of collagen fibers (as shown in Figure 7).

[0169] 6) The test product has the effect of promoting epidermal and dermal stem cells

[0170] The results of immunofluorescence staining showed that the number of CK19-positive cells in the skin tissue of the model group was significantly reduced, indicating that the number of epidermal stem cells in aging skin decreased. The number of CK19-positive cells increased after treatment with the test product. The results showed that the HA test group could promote the proliferation of epidermal stem cells in the skin, thereby promoting skin tissue regeneration (as shown in Figures 8 and 9).

[0171] The results of immunofluorescence staining showed that the number of CK14-positive cells in the skin tissue of the model group was significantly reduced, and the number of CK14-positive cells increased after treatment with the HA test product. The results showed that the test group was able to promote the proliferation of basal stem cells in the skin, thereby promoting skin tissue regeneration (as shown in Figures 10 and 11).

[0172] 7) Antioxidant effect of the test product

[0173] qPCR results showed that the test article treatment significantly increased the expression of antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT), as well as the expression of antioxidant-related genes heme oxygenase 1 (HO-1) and NAPDH quinone oxidoreductase 1 (NQO1), indicating that the test article treatment can reduce the oxidative stress response caused by skin aging, thereby delaying skin aging (as shown in Figure 12).

[0174] 8) Anti-inflammatory effect of the test article

[0175] The qPCR results showed that the HA test product treatment significantly reduced the expression of inflammatory factors TNF-α and IL-1β, indicating that the HA test product treatment can reduce the inflammatory response caused by skin aging (as shown in Figure 13).

[0176] 9) Effects of the test product on the skin moisture content of aging mice

[0177] The results of skin moisture content measurement showed that compared with the Control group, the skin moisture content of the model group was significantly decreased, and compared with the model group, the skin moisture content of the test product group was significantly increased (P<0.05), indicating that the test product was of great significance in improving skin moisture content (as shown in Figure 14).

[0178] By detecting the expression of water channel protein AQP-3 in the skin of each group of mice, it was found that compared with the control group, the expression of AQP-3 in the model group was significantly reduced. After treatment with the test product, the expression of AQP-3 was significantly increased (P<0.001), indicating that the test product can increase the expression of AQP-3 in the skin (as shown in Figure 15).

Claims

1. An anti-aging injection composition comprising hyaluronic acid or a salt thereof, glycine, proline, leucine, and lysine hydrochloride.

2. The anti-aging injection composition according to claim 1, wherein The molecular weight of the hyaluronic acid or its salt is 200,000-2.5 million Da.

3. The anti-aging injection composition according to claim 1 or 2, wherein: The mass ratio of the sum of glycine, proline, leucine, and lysine hydrochloride to the hyaluronic acid or its salt is 1:(2-20).

4. The anti-aging injection composition according to any one of claims 1 to 3, wherein The mass ratio of glycine, proline, leucine and lysine hydrochloride is (2-20):(1-15):(1-8):(1-8).

5. The anti-aging injection composition according to any one of claims 1 to 4, wherein The composition further comprises excipients and / or active ingredients acceptable in the field of medical aesthetics.

6. An anti-aging injection comprising the anti-aging injection composition according to any one of claims 1 to 5.

7. The anti-aging injection according to claim 6, wherein: In the anti-aging injection, the content of hyaluronic acid or its salt is 8-20 mg / mL, the content of glycine is 0.2-2 mg / mL, the content of proline is 0.1-1.5 mg / mL, the content of leucine is 0.1-0.8 mg / mL, and the content of lysine hydrochloride is 0.1-0.8 mg / mL.

8. Use of the anti-aging injection composition according to any one of claims 1 to 5 or the anti-aging injection solution according to any one of claims 6 to 7 in anti-skin aging.

9. The use according to claim 8, wherein the anti-skin aging effect comprises promoting collagen production in the skin, promoting elastin production in the skin, reducing skin wrinkles, improving skin elasticity, enhancing skin brightness, reducing collagen fiber breakage, damage, loss and degradation, reducing elastic fiber aging damage, promoting skin tissue regeneration, reducing oxidative stress caused by skin aging, reducing inflammatory response caused by skin aging, or increasing the expression of AQP-3 in the skin.

10. Use of the anti-aging injection composition according to any one of claims 1 to 5 or the anti-aging injection solution according to any one of claims 6 to 7 in the preparation of an anti-skin aging product.

11. The use according to claim 10, wherein the anti-skin aging effect comprises promoting collagen production in the skin, promoting elastin production in the skin, reducing skin wrinkles, improving skin elasticity, enhancing skin brightness, reducing collagen fiber breakage, damage, loss and degradation, reducing elastic fiber aging damage, promoting skin tissue regeneration, reducing oxidative stress caused by skin aging, reducing inflammatory response caused by skin aging, or increasing the expression of AQP-3 in the skin.

12. A method for anti-skin aging, comprising administering to a subject an effective amount of the anti-aging injection composition according to any one of claims 1 to 5 or the anti-aging injection solution according to any one of claims 6 to 7.

13. The method according to claim 12, wherein the anti-skin aging comprises promoting collagen production in the skin, promoting elastin production in the skin, reducing skin wrinkles, improving skin elasticity, enhancing skin brightness, reducing collagen fiber breakage, damage, loss and degradation, reducing elastic fiber aging damage, promoting skin tissue regeneration, reducing oxidative stress caused by skin aging, reducing inflammatory response caused by skin aging, or increasing the expression of AQP-3 in the skin.

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