Application of fullerene material in preparation of product for improving collagen expression level

Abstract

The invention belongs to the field of chemical materials, and particularly relates to application of a fullerene material in preparation of a product for improving the expression level of collagen. The invention provides a novel application of a fullerene material, the fullerene material obviously improves the expression level of collagen, especially I-type, III-type and XVII-type collagen, and especially the XVII-type collagen can delay cell aging and repair damaged cells.

Description

Technical Field

[0001] The present invention belongs to the field of chemical materials, and particularly relates to the use of fullerene materials in the preparation of products for increasing the expression level of collagen. Background Art

[0002] Collagen XVII, also known as COL17 / BP180 / BPAG2, is a transmembrane protein rich in 1497 amino acids, encoded by the COL17a1 gene. Its N-terminus is located in the cytoplasm and its C-terminus is located in the extracellular matrix. Like other transmembrane collagens, it plays a crucial role in maintaining the connection between intracellular and extracellular structural elements involved in epidermal adhesion. Collagen XVII is expressed in a variety of tissues, including skin, colon, esophagus, placenta, small intestine, stomach, etc., and its expression level is the highest and most concentrated in the skin. Collagen XVII is a key factor in skin aging and wound repair, and plays an important role in keeping the skin "young", maintaining hair follicle stem cells (hair loss, gray hair, etc.), and promoting wound repair.

[0003] The role of COL17 in the skin. The basement membrane (BM) plays an important role in the attachment between basal keratinocytes of the epidermis and the underlying dermis. Cleavage of COL17 is crucial for the correct formation of the basement membrane. Deficiency of COL17 protein will reduce the adhesion of keratinocytes to the basement membrane, resulting in variable basement membrane adhesion, which is typically characterized by multiple erosive nevi and premature hair loss.

[0004] The role of COL17 in aging. The skin is a highly structured organ, and the coordination of stem cell self-renewal, cell proliferation and differentiation maintains a dynamic balance. Organism aging is defined as tissue damage caused by the accumulation of many internal and external factors that induce cell damage. Examples of human skin aging are changes in the dermis and skin appendages, such as thinning of the dermis, dryness, wrinkles, gray hair and hair loss. As a connector between the epidermis and the dermis, COL17 is expressed in basal cells. A decrease in the expression level of the COL17A1 gene results in the loss of skin homeostasis and skin aging. At the same time, when there is excessive or repeated stress (oxidation or ultraviolet irradiation), it will also trigger a DNA damage response, leading to the degradation of COL17 protein and further causing the degradation of hemidesmosomes, making it easier to detach from the basement membrane, thus forming a phenomenon of vertical splitting, resulting in skin aging.

[0005] The role of COL17 in hair loss. Hair loss is the result of the combined action of various internal and external factors, among which aging is an irresistible factor. The gradual aging of hair follicle stem cells (HFSCs) can lead to the gradual miniaturization of hair follicles and ultimately result in hair loss. HFSCs exist in the bulge area of the hair follicle and maintain hair cycle regeneration. In vivo analysis of HFSCs shows that their DNA damage response can activate ELANE (neutrophil elastase), which causes the degradation of COL17A1 / BP180, and COL17A1 is the key molecule for HFSCs to maintain their functions, ultimately leading to the aging of HFSCs.

[0006] As a transmembrane protein present between the epidermis and the dermis, COL17 plays an important role in basement membrane adhesion, skin aging mechanism and the maintenance of hair follicle stem cells, and is a potential focus for future research on anti-aging and anti-hair loss skin care products.

[0007] Fullerene (C 60 ), is the third allotrope of carbon. Inside, it forms a football-shaped hollow molecule composed of atoms. This molecule has 60 carbon atoms forming 60 vertices, 32 faces (including 12 regular pentagons and 20 regular hexagons, where the pentagons are not connected to each other and are only adjacent to hexagons), and 30 carbon-carbon double bonds. Each carbon atom is connected to three adjacent carbon atoms by sp2 hybrid orbitals, and the remaining P orbitals form π bonds on the periphery and inside cavity of the C60 molecule. Fullerene is known as the "free radical sponge", and existing research has shown that it has antioxidant activity and a broad-spectrum cell protection effect. Therefore, fullerene has great potential in anti-aging and skin care. Summary of the Invention

[0008] In order to achieve the above technical objectives, the present invention provides a new use of fullerene materials, and the technical solutions are as follows:

[0009] In the first aspect, the present invention provides the use of fullerene materials in increasing the expression level of collagen.

[0010] In the second aspect, the use of fullerene materials in the preparation of products for increasing the expression level of collagen.

[0011] In some embodiments, the fullerene materials are used for one or more of the uses selected from promoting skin cell renewal, enhancing skin elasticity, promoting skin repair, inhibiting ultraviolet damage, skin whitening, reducing wrinkles or firming the skin.

[0012] In some embodiments, the collagen is one or more selected from type I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI collagen or tropocollagen.

[0013] In some embodiments, the collagen is type I, III, XVII collagen, and further preferably type XVII collagen.

[0014] In some embodiments, the fullerene material includes, but is not limited to: fullerene, (encapsulated) metallofullerene, fullerene nanotube, hydrated fullerene, fullerene ring, fullerene megatube, fullerene cluster, fullerene derivative, fullerene modifier, and / or fullerene polymer.

[0015] In some preferred embodiments, the fullerene material is selected from one or more of the following:

[0016] Oil-soluble fullerene, oil-soluble metallofullerene, water-soluble fullerene, water-soluble metallofullerene, liposome (lipid-encapsulated) fullerene, or a physiologically acceptable salt or ester or acid or amine or alcohol thereof.

[0017] In some preferred embodiments, the metallofullerene is an endohedral metallofullerene in which a metal fullerene or a metal cluster is embedded in a hollow fullerene.

[0018] In some more preferred embodiments, the metallofullerene includes M@C 2n , M2@C 2n , MA@C 2n , M3N@C 2n , M2C2@C 2n , M2S@C 2n , M2O@C 2n and M x A 3-x N@C 2n one or more of them, where M and A both represent metal elements and M and A are both selected from any one of Sc, Y, and lanthanide metal elements, where 30 ≤ n ≤ 60 and 0 ≤ x ≤ 3.

[0019] In some preferred embodiments, the oil-soluble fullerene includes a fullerene with an oil solution coated on the outer surface of the carbon cage.

[0020] In some preferred embodiments, the oil-soluble metallofullerene includes a metallofullerene with an oil solution coated on the outer surface of the carbon cage.

[0021] In some preferred embodiments, the oil solution includes, but is not limited to: saturated fats and / or unsaturated fats.

[0022] In some more preferred embodiments, the oil solution is selected from olive oil, linseed oil, sunflower oil, corn germ oil, soybean oil, fish oil and / or squalane, caprylic / capric triglyceride, hydrogenated polyisobutene, neopentyl glycol diheptanoate, isododecane, isocetane, etc.

[0023] In some embodiments, the oil-soluble fullerene or oil-soluble metal fullerene is obtained by modifying fullerene or metal fullerene with oil solubility.

[0024] In some embodiments, the oil solubility modification is to disperse fullerene or metal fullerene in the oil solution to obtain oil-soluble fullerene or oil-soluble metal fullerene.

[0025] In some embodiments, the water-soluble fullerene or water-soluble metal fullerene is selected from at least one of the following:

[0026] Fullerene or metal fullerene with hydrophilic groups modified on the outer surface of the carbon cage, fullerene or metal fullerene wrapped by hydrophilic biomolecules on the outer surface of the carbon cage, fullerene or metal fullerene loaded by a biocompatible carrier material, or water-soluble supramolecular system fullerene or metal fullerene formed by self-assembly.

[0027] In some preferred embodiments, the hydrophilic group includes at least one of hydroxyl, carboxyl, mercapto, amino or water-soluble amino acid residues.

[0028] In some embodiments, the water-soluble amino acid residue refers to the incomplete amino acid remaining after losing a part of the amino acid molecule when modifying fullerene with a water-soluble amino acid. Losing any part of the amino acid molecule is considered an amino acid residue, such as losing the hydrogen on the amino group of the amino acid, losing the hydrogen or hydroxyl group on the carboxyl group of the amino acid, etc. Optionally, the water-soluble amino acid residue is at least one of alanine residue, glycine residue, serine residue, arginine residue, lysine residue and aspartic acid residue.

[0029] In some more preferred embodiments, the hydrophilic group is hydroxyl.

[0030] In some preferred embodiments, the hydrophilic biomolecule includes at least one of water-soluble amino acid, water-soluble peptide chain or water-soluble polysaccharide.

[0031] In some preferred embodiments, the water-soluble amino acid is selected from at least one of alanine, glycine, serine, arginine, lysine or aspartic acid.

[0032] In some embodiments, the biocompatible carrier material is selected from at least one of liposomes, proteins, polymer micelles, or cell membrane carriers.

[0033] In some preferred embodiments, the protein includes albumin, ferritin, or transferrin.

[0034] In some preferred embodiments, the polymer micelles include at least one of poly (lactic-co-glycolic acid)-polyethylene glycol, polylysine, chitosan, sodium hyaluronate (hyaluronic acid), polysorbate, stearate.

[0035] In some preferred embodiments, the liposomes include at least one of lecithin liposomes, hydrogenated phospholipid liposomes, iron oxide liposomes, liposomes conjugated with targeting groups.

[0036] In some preferred embodiments, the cell membrane carriers include at least one of erythrocyte membrane carriers, neutrophil membrane carriers, macrophage membrane carriers, platelet carriers.

[0037] In some preferred embodiments, the fullerenes include, but are not limited to: C 20 、C 60 、C 70 、C 76 、C 80 、C 96 or C 240 .

[0038] In some more preferred embodiments, the fullerenes are C 60 or C 70 .

[0039] In some embodiments, the fullerene material is used to regulate the expression level of collagen.

[0040] In some preferred embodiments, the fullerene material is used to regulate the expression level of type XVII collagen (COL17).

[0041] In some embodiments, the product includes a pharmaceutical composition or a personal care composition.

[0042] In some embodiments, the dosage form of the product includes, but is not limited to: solid preparations, liquid preparations, gas preparations, or semi-solid preparations.

[0043] In some embodiments, the product includes, but is not limited to: facial cleanser, cleansing milk, cleansing soap, cleansing powder, cleansing foam, cleansing gel, cleansing cream, lotion, emulsion, cream, essence, serum, sunscreen, essence lotion, spray, permeating milk, essential oil, stock solution, gel, facial mask, dressing, nanoemulsion, micellar agent, etc.

[0044] In some embodiments, the product includes, but is not limited to: anti - hair - loss ampoules, hair - growth essences, scalp nutrient solutions, anti - hair - loss shampoos, anti - hair - loss hair conditioners, anti - hair - loss sprays, anti - hair - loss hair oils, or follicle nourishing stock solutions, etc.

[0045] In some embodiments, the product is used for preventing hair loss, strengthening hair, and promoting hair growth.

[0046] In some embodiments, the pharmaceutical composition or personal care composition comprises the following components by weight percentage: fullerene material 5% - 30%, thickener 0.1% - 5%, humectant 1% - 20%, preservative 0.1% - 30%, and the balance is water.

[0047] In some embodiments, the fullerene material is a fullerene complex.

[0048] In some embodiments, the weight percentage of the fullerene material is 5% - 30%, for example, it can be 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, or 30%, preferably 5% - 25%, more preferably 5% - 10% or 20% - 25%.

[0049] In some embodiments, the weight percentage of the thickener is 0.1% - 5%, for example, it can be 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4%, preferably 0.1% - 4%, more preferably 0.1% - 0.2% or 0.4% - 4%.

[0050] In some embodiments, the weight percentage of the humectant is 1% - 20%, for example, it can be 1%, 2%, 3%, 4%, 5%, 5.4%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, or 20%, preferably 5% - 20%, more preferably 5% - 10% or 10% - 20%.

[0051] In some embodiments, the weight percentage of the preservative is 0.1% - 30%, for example, it can be 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, preferably 0.1% - 25%, more preferably 0.4% - 25% or 0.4% - 5%.

[0052] In some alternative embodiments, the pharmaceutical composition or personal care composition comprises a chelating agent, and the weight percentage of the chelating agent is 0-1%, for example, it can be 0, 0.05%, 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, preferably 0-0.5%, more preferably 0-0.1%.

[0053] In some alternative embodiments, the pharmaceutical composition or personal care composition comprises an emulsifier, and the weight percentage of the emulsifier is 0-10%, for example, it can be 0, 1%, 2%, 3%, 3.8%, 4%, 5%, 6%, 6.8%, 7%, 8%, 9%, 10%, preferably 0-7%, more preferably 3%-7%.

[0054] In some alternative embodiments, the pharmaceutical composition or personal care composition comprises a emollient, and the weight percentage of the emollient is 0-10%, for example, it can be 0, 1%, 2%, 3%, 4%, 5%, 5.9%, 6%, 7%, 8%, 9%, 10%, preferably 0-7%, more preferably 5%-6%.

[0055] In some alternative embodiments, the pharmaceutical composition or personal care composition comprises a flavoring agent, and the weight percentage of the flavoring agent is 0-1%, for example, it can be 0, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, preferably 0-0.5%, more preferably 0-0.1%.

[0056] In some alternative embodiments, the pharmaceutical composition or personal care composition comprises a skin conditioner, for example, it can be 0, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, and the weight percentage of the skin conditioner is 0-1%, preferably 0-0.5%, more preferably 0-0.1%.

[0057] In some alternative embodiments, the pharmaceutical composition or personal care composition comprises an antioxidant, and the weight percentage of the antioxidant is 0-1%, for example, it can be 0, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, preferably 0-0.5%, more preferably 0-0.1%.

[0058] In some embodiments, the fullerene material is C 60 or C 70 , more preferably C 60 .

[0059] In some embodiments, the thickener comprises one or more selected from carbomer, xanthan gum, cetearyl alcohol, acryloyldimethyltaurate / VP copolymer crosspolymer.

[0060] In some embodiments, the humectant comprises one or more selected from glycerin, ethylhexylglycerin, 1,2 - hexanediol, butanediol, arginine, glucose.

[0061] In some embodiments, the chelating agent comprises one or more selected from disodium edetate, sodium phytate, octanohydroxamic acid, preferably disodium edetate.

[0062] In some embodiments, the emulsifier comprises one or more selected from glyceryl stearate, PEG - 100 stearate, cetearyl alcohol, arachidyl alcohol, behenyl alcohol, arachidyl glucoside.

[0063] In some embodiments, the emollient comprises one or more selected from C10 - 18 triglycerides, polydimethylsiloxane, ethylhexylglycerin.

[0064] In some embodiments, the preservative comprises one or more selected from ethylhexylglycerin, 1,2 - hexanediol, p - hydroxyacetophenone, ethanol, sodium bisulfite.

[0065] In some embodiments, the flavoring agent comprises essence.

[0066] In some embodiments, the skin conditioner comprises one or more selected from allantoin, sodium hyaluronate, ceramide, preferably allantoin.

[0067] In some embodiments, the antioxidant comprises one or more selected from sodium bisulfite, vitamin C, vitamin E, preferably sodium bisulfite.

[0068] In some preferred embodiments, the pharmaceutical composition or personal care composition comprises a thickener, a humectant, a chelating agent, an emulsifier, an emollient, a preservative, a flavoring agent, and the balance is water.

[0069] In some preferred embodiments, the pharmaceutical composition or personal care composition comprises the following components in weight percentages: thickener 0.4% - 4%, humectant 5% - 10%, preservative 0.4% - 5%, chelating agent 0 - 0.1%, emulsifier 3% - 7%, emollient 5% - 6%, flavoring agent 0 - 0.1%.

[0070] In some preferred embodiments, the pharmaceutical composition or personal care composition comprises a thickener, a humectant, a skin conditioner, a preservative, an antioxidant, and the balance is water.

[0071] In some preferred embodiments, the pharmaceutical composition or personal care composition comprises the following ingredients in weight percentages: thickener 0.1%-0.2%, humectant 10%-20%, preservative 0.4%-25%, skin conditioner 0-0.1%, antioxidant 0-0.1%.

[0072] The present invention provides a new use of fullerene materials in increasing the expression level of collagen, wherein the fullerene materials significantly increase the expression level of collagen, especially type XVII collagen, and can delay cell aging and repair damaged cells. Description of the Drawings

[0073] Figure 1 Shows C 60 Effect on the relative expression level of type XVII collagen in HACAT cells. Where * indicates P<0.05; ** indicates P<0.01; ***P<0.001; ****P<0.0001; ns indicates P>0.05.

[0074] Figure 2 Shows C 70 Effect on the relative expression level of type XVII collagen in HEK-a cells. Where * indicates P<0.05; ** indicates P<0.01; ***P<0.001; ****P<0.0001; ns indicates P>0.05.

[0075] Figure 3 Shows Case 1 of the improvement of the skin by the fullerene cream in Example 3.

[0076] Figure 4 Shows Case 2 of the improvement of the skin by the fullerene cream in Example 3.

[0077] Figure 5 Shows the improvement results of the fullerene product on the overall hair density in two cases of Example 4.

[0078] Figure 6 Shows the improvement results of the fullerene product on the local hair density in two cases of Example 4.

[0079] Figure 7 Shows the effect of fullerene on type I collagen in fibroblasts in Example 5. Where compared with the model group, * indicates a statistically significant difference, P<0.05, ** indicates P<0.01, *** indicates P<0.001; compared with the fullerene group, # indicates a statistically significant difference, P<0.05, ## indicates P<0.01.

[0080] Figure 8It shows the effect of fullerene on type III collagen in fibroblasts in Example 5. Compared with the model group, * indicates that the difference is statistically significant, P < 0.05. Detailed implementation manners

[0081] To make the objectives, technical solutions, and advantages of the embodiments of the present disclosure clearer, the technical solutions of the embodiments of the present disclosure will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present disclosure. Apparently, the described embodiments are some, but not all, of the embodiments of the present disclosure. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present disclosure without creative efforts shall fall within the scope of protection of the present invention.

[0082] The present invention can be implemented in other specific forms without departing from its basic attributes. It should be understood that, on the premise of no conflict, any and all implementation manners of the present invention can be combined with the technical features in any other implementation manner or multiple other implementation manners to obtain additional implementation manners. The present invention includes such additional implementation manners obtained by such combination.

[0083] All publications and patents mentioned in the present disclosure are hereby incorporated by reference in their entirety into the present disclosure. If the uses or terms used in any incorporated publications and patents conflict with the uses or terms used in the present disclosure, then the uses and terms of the present disclosure shall prevail.

[0084] The section titles used herein are only for the purpose of organizing the article and should not be construed as limiting the subject matter described.

[0085] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0086] Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural referents. For example, referring to "a cell" includes multiple such cells and equivalents known to those skilled in the art, and so on.

[0087] As shown in the present disclosure, the term "about" represents a range of ±20% of the value following it. In some embodiments, the term "about" represents a range of ±10% of the value following it. In some embodiments, the term "about" represents a range of ±5% of the value following it.

[0088] The numerical ranges used in the present disclosure should be understood to have enumerated all the numbers within that range. For example, the range from 1 to 20 should be understood to include any number, combination of numbers, or sub-range from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0089] As shown in the present disclosure, the term "comprises" or "comprising" means "including but not limited to". This term is intended to be open-ended, to specify the presence of any of the stated features, elements, integers, steps, or components, but not to exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the application, particularly in the claims, may be replaced by the term "consisting of".

[0090] As shown in the present disclosure, the terms "optionally", "either", "any", or "any one" mean that the subsequently described event or circumstance may but need not occur, and this description includes the instances where the event or circumstance occurs or does not occur. As used in the present invention, "a" and "an" are used in the present invention to refer to one or more than one grammatical object.

[0091] As shown in the present disclosure, "and / or" should be understood to mean any one of the alternatives or any combination of any two or more of the alternatives.

[0092] As used in the present disclosure, the term "collagen (COL)" refers to a crucial structural protein in the human body, which is widely present in various tissues and organs, such as skin, bone, tendon, ligament, and cornea, etc. The main functions of these proteins are to provide strength, stability, and elasticity to body tissues, ensuring the normal progress of various physiological functions. Currently, at least 28 types of collagen are known, and each type plays a unique role and function in the body, forming a complex and finely regulated biological system. In some embodiments, the collagen is one or more selected from type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX, type XXI collagen or small collagen; in some preferred embodiments, the collagen is type XVII collagen.

[0093] As used in the present disclosure, the term "preservative", also known as "bacteriostatic agent", refers to a chemical substance used to inhibit the growth of microorganisms and is a common additive in non-solid preparations. Generally, it can be classified into acids, alcohols, quaternary ammonium salts, parabens, organic mercury compounds, etc. In some embodiments, the preservative comprises one or more selected from ethylhexylglycerin, 1,2 - hexanediol, p - hydroxyacetophenone, ethanol, sodium bisulfite.

[0094] As used in the present disclosure, the term "antioxidant" refers to a class of substances that can neutralize free radicals and slow down or prevent cell damage, which plays an important role in reducing oxidative stress caused by environmental factors and preventing skin aging. In some embodiments, the antioxidant comprises one or more selected from sodium bisulfite, vitamin C, vitamin E, preferably sodium bisulfite.

[0095] As used in the present disclosure, the term "emulsifier" refers to a substance that can help mix oil and water to form a stable emulsion and is used in beauty or hair care products to enhance the texture and stability of the products. In some embodiments, the emulsifier comprises one or more selected from glyceryl stearate, PEG - 100 stearate, cetearyl alcohol, arachidyl alcohol, behenyl alcohol, arachidyl glucoside.

[0096] As used in the present disclosure, the term "humectant" refers to a substance that can attract or lock in moisture and help the skin retain moisture, which is crucial for maintaining the hydrated state of the skin and preventing dryness. In some embodiments, the humectant comprises one or more selected from glycerol, ethylhexylglycerin, 1,2 - hexanediol, butanediol, arginine, glucose.

[0097] As used in the present disclosure, the term "thickener" refers to a substance that can increase the viscosity or consistency of a product. By changing the fluidity and texture of the product, it can improve the user experience and effect of the product. In some embodiments, the thickener comprises one or more selected from carbomer, xanthan gum, cetearyl alcohol, acryloyldimethyltaurate / VP - copolymer.

[0098] As used in the present disclosure, the term "chelating agent" refers to a class of substances that can bind to metal ions to prevent these ions from undergoing oxidation, decomposition and other reactions in the product, thereby maintaining the stability of the product. In some embodiments, the chelating agent comprises one or more selected from disodium ethylenediaminetetraacetate, sodium phytate, octanoyl hydroxamic acid, preferably disodium ethylenediaminetetraacetate.

[0099] As used in the present disclosure, the term "emollient" refers to a substance that can keep the skin smooth, soft and elastic. By forming a continuous closed oil film on the skin surface, it can prevent the evaporation of moisture in the deeper layer of the skin to keep the skin moisturized. In some embodiments, the emollient comprises one or more selected from triglycerides of C10-18 fatty acids, polydimethylsiloxane, and ethylhexylglycerin.

[0100] As used in the present disclosure, the term "fragrance" refers to substances that impart a specific aroma to the product. In some embodiments, the fragrance comprises essence.

[0101] As used in the present disclosure, the term "skin conditioner" refers to a class of functional ingredients that can improve the skin condition and promote skin health, such as helping to improve the skin's moisture retention ability, antioxidant ability, soothe inflammation, promote regeneration, etc. In some embodiments, the skin conditioner comprises one or more selected from allantoin, sodium hyaluronate, and ceramide, preferably allantoin.

[0102] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For all reagents or instruments without indicating the manufacturer, they are all conventional products that can be purchased commercially. To better illustrate the present invention, numerous specific details are given in the following embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessarily confusing the concepts of the present invention. Such structures and technologies have also been described in the publication of many publications.

[0103] In the embodiments of the present invention, fullerene is administered to cells through a complexation and solubilization technology, wherein the excipient includes 2% sodium hyaluronate (HA). The corresponding amounts of fullerene and HA are respectively added to water. Each time a component is added, continuous stirring is required. After mixing evenly, another component is added. The homogenization speed is 15,000 rpm and the homogenization time is 10 min to obtain the product; the blank excipient only contains the excipient part.

[0104] Example 1: C 60 Effect on the expression level of COL17

[0105] HaCaT cells (from the Cell Bank of the Chinese Academy of Sciences) were selected for the experiment. They were divided into a normal group, an excipient group, a vitamin C group, and a C 60Group: The normal group was not irradiated with a UVB (mid-band ultraviolet) instrument; the excipient group was a blank excipient containing no active ingredients without fullerene, and the addition amount of each excipient was the same as that of the excipient in the fullerene group; the excipient in the vitamin C group contained vitamin C with a concentration of 40 μg / mL, which was the same as the fullerene concentration. The treatment method was to dissolve vitamin C in sterile water first and then dilute it to 40 μg / mL with complete medium; C 60 The C 60 group had fullerene C

[0106] 1. Inoculate a confocal culture dish: Inoculate the cells into the confocal dish, with 200000 cells per dish, incubate overnight, and wait until the cells cover 50% of the confocal dish before use.

[0107] 2. Add the test substance: Aspirate the old medium in the confocal dish, wash it once with physiological saline, add 1.5 mL of the test substance to each well, incubate for 1 h, irradiate with a UVB instrument for 3 min, and then incubate overnight.

[0108] 3. Fixation: Aspirate the old medium in the confocal dish, wash it 3 times with TBST, 3 min each time. Add 800 μL of 4% paraformaldehyde to each dish to fix the cells at room temperature for 20 min, then aspirate it, and wash it 3 times with TBST, 3 min each time;

[0109] 4. Permeabilization

[0110] ① Preparation of 0.1% Triton X-100

[0111] Take 10 μL from a 100% Triton X-100 solution and dissolve it in 10 mL of TBST to obtain 10.1 mL of a 0.1% Triton X-100 solution;

[0112] ② Add 0.1% Triton X-100 to the confocal dish, 1 mL per dish, permeabilize the cells at room temperature for 13 min, then aspirate it, and wash it 3 times with TBST, 3 min each time;

[0113] 5. Blocking: Add 5% BSA to the confocal dish to block the non-specific binding of antibodies, 1 mL per dish, block the cells at room temperature for 30 min, then aspirate it, and do not wash;

[0114] 6. Incubate with the primary antibody (CollagenⅩⅦ): Dilute the primary antibody with TBST at a ratio of 1:200, add 200 μL to each dish, wrap it with tin foil, incubate the cells at 4 °C overnight, recover the primary antibody, and wash it 3 times with TBST, 3 min each time;

[0115] 7. Incubate the secondary antibody (Alexa Fluor 488): Dilute the secondary antibody 1:1000 with TBST, add 500 μL to each dish, wrap it with tin foil, incubate the cells at room temperature for 1 h, recover the secondary antibody, wash 3 times with TBST, 3 min for each wash;

[0116] 8. Stain the nucleus (Hoechst): Dilute Hoechst 1:1000 with PBS, add 200 μL to each dish, wrap it with tin foil, incubate the cells at room temperature for 5 min, aspirate, wash 3 times with TBST, 3 min for each wash, add 1 mL PBS to each dish.

[0117] The results are as Figure 1 shown. After C 60 treatment of the cells, the expression level (fluorescence intensity) of COL17 was significantly increased compared with the excipient group and the vitamin group, and was close to that of the normal group, with no significant difference.

[0118] Example 2: C 70 Effect on the expression level of COL17

[0119] HEKa cells (from the ATCC cell bank) were used in the experiment. They were divided into a normal group, an excipient group, a vitamin C group, and a C 70 group: The normal group was not irradiated with a UVB instrument; the excipient group was a blank excipient without active ingredients containing no fullerene, and the addition amount of each excipient was the same as that of the excipient in the fullerene group; the vitamin C group contained vitamin C in the excipient, with a concentration of 40 μg / mL, which was the same as the fullerene concentration. The treatment method was to dissolve vitamin C with sterile water first and then dilute it to 40 μg / mL with complete medium; the C 70 group contained fullerene C 70 in the excipient, with a concentration of 40 μg / mL. The remaining treatment methods were the same, and the steps were as follows:

[0120] 1. Inoculate a confocal culture dish: Inoculate the cells into a confocal dish, with 200,000 cells per dish, incubate overnight, and wait until the cells grow to 50% of the confocal dish before use.

[0121] 2. Add the test substance: Aspirate the old medium in the confocal dish, wash once with normal saline, add 1.5 mL of the test substance to each well, incubate for 1 h, irradiate with a UVB instrument for 3 min, and then incubate overnight.

[0122] 3. Fixation: Aspirate the old medium in the confocal dish, wash 3 times with TBST, 3 min for each wash, add 800 μL of 4% paraformaldehyde to each dish, fix the cells at room temperature for 20 min, then aspirate, wash 3 times with TBST, 3 min for each wash;

[0123] 4. Permeabilization

[0124] ① Preparation of 0.1% Triton X-100

[0125] Take 10 μL from the 100% Triton X-100 solution and dissolve it in 10 mL of TBST to obtain 10.1 mL of 0.1% Triton X-100 solution;

[0126] ② Add 0.1% Triton X-100 to the confocal dish, 1 mL per dish. After permeabilizing the cells at room temperature for 13 min, aspirate and discard it, and wash 3 times with TBST, 3 min each time;

[0127] 5. Blocking: Add 5% BSA to the confocal dish to block non-specific binding of antibodies, 1 mL per dish. After blocking the cells at room temperature for 30 min, aspirate and discard it, without washing;

[0128] 6. Incubate with primary antibody (Collagen XVII): Dilute the primary antibody 1:200 with TBST, then add 200 μL per dish, wrap it with tin foil, incubate the cells overnight at 4 °C, recover the primary antibody, and wash 3 times with TBST, 3 min each time;

[0129] 7. Incubate with secondary antibody (Alexa Fluor 488): Dilute the secondary antibody 1:1000 with TBST, then add 500 μL per dish, wrap it with tin foil, incubate the cells at room temperature for 1 h, recover the secondary antibody, and wash 3 times with TBST, 3 min each time;

[0130] 8. Stain nuclei (Hoechst): Dilute Hoechst 1:1000 with PBS, then add 200 μL per dish, wrap it with tin foil, incubate the cells at room temperature for 5 min, aspirate and discard it, wash 3 times with TBST, 3 min each time, and add 1 mL of PBS per dish;

[0131] The results are as Figure 2 shown, C 70 After treating the cells, compared with the excipient group and the vitamin group, the expression level (fluorescence intensity) of COL17 was significantly increased and was close to that of the normal group, with no significant difference.

[0132] Example 3: Efficacy of fullerene products on the skin

[0133] Collagen XVII is a key factor for the health of skin cells. When skin cells are healthy, the skin shows a healthy state. Therefore, a cream containing only fullerene as the active ingredient was prepared, and people aged 35 - 55 with age spots were recruited to test the effects of the pure fullerene cream on reducing wrinkles, firming, pore shrinking, and whitening and removing age spots; the product usage period was 90 days. The test instrument was Antera3D

[0134] The fullerene cream formula is shown in Table 1. The form of fullerene can be fullerene hyaluronic acid, fullerene chitosan, oil-soluble fullerene, polysorbate (tween) fullerene, olive oil-based fullerene, and the fullerene content is 400 ppm.

[0135] Table 1. Fullerene Cream Formula

[0136]

[0137]

[0138] The test results are shown in Table 2. It can be seen that compared with before using the product, the skin whiteness has improved. At the same time, the anti-wrinkle, freckle-lightening, skin-tightening, and firming effects are obvious, and no allergic reactions occurred during the overall test process, with a sensitivity rate of 0. Figure 3 and Figure 4 are two specific cases. As Figure 3 shown, it can be seen that the skin firmness of the users has been significantly improved, local freckles have disappeared, wrinkles have significantly reduced, and the skin has become whiter and brighter; while Figure 4 for the users, the wrinkles have significantly become shallower and reduced, and the eye skin has become firmer. The above results have all proved the improvement effect of fullerene products on the skin condition.

[0139] Table 2. Test Results of Fullerene Cream

[0140]

[0141] Example 4: The Efficacy of Fullerene Products on Hair

[0142] Hair loss and graying are related to the health of the scalp, and collagen is also a factor in hair health. Therefore, in this example, the efficacy of fullerene products on hair was tested, and the MoleMax HD hair growth analysis and management system was used as the test instrument. The product formula is shown in Table 3 below. The form of fullerene can be fullerene hyaluronic acid, fullerene chitosan, oil-soluble fullerene, polysorbate (tween) fullerene, olive oil-based fullerene, and the fullerene content is 400 ppm.

[0143] Table 3. Formula of Fullerene Anti-Hair Loss and Volumizing Serum

[0144]

[0145] Product usage plan: After washing hair and drying the scalp, apply 5 ml evenly on the whole head close to the scalp and let it absorb naturally.

[0146] For the selected subjects, the basic hair values were evaluated before using the product, including hair loss count, hair density evaluation, and image taking, and recorded; the same evaluations and tests were carried out again 4 weeks, 8 weeks, and 12 weeks after using the product.

[0147] Hair loss count: At each visit of the subject, the staff used the 60 - combing method to comb the subject's hair, count the fallen hair, and record it.

[0148] Hair density assessment: Hair density assessment includes overall hair density assessment and local hair density assessment. The specific methods are as follows:

[0149] (1) Overall hair density: Through image assessment, at each visit, a photo of the subject's full - head hair was taken.

[0150] (2) Local hair density: A haircut area of at least 1 cm * 1 cm was fixed on the subject's head for positioning. At each visit, ensure that the haircut area is consistent, and the hair was cut to a residual length of no more than 1 mm. Place the skin imager in the center of the haircut area to take a local hair image. Use image - analysis software to count the local hair density and hair diameter and record them.

[0151] The test results are shown in Table 4. It can be seen that after using the fullerene product, the conditions of hair loss and hair density have both improved, and with the increase in the use time, the effect is more significant. Figure 5 and Figure 6 Two specific cases are presented, where Figure 5 show the overall hair density of two users. It can be seen that with the increase in the use time, the hair loss on the top of the head has improved significantly, and the hair parting has become smaller. Figure 6 shows the local hair density of the two users, and it can also be seen that there is obvious hair growth. The above results all prove the efficacy of the fullerene product in preventing hair loss and promoting hair growth.

[0152] Table 4. The efficacy of the fullerene product on hair

[0153]

[0154] Note: P≥0.05 indicates no statistical difference (denoted as "n.s."); P<0.05 indicates a significant difference (denoted as "*"); P<0.01 indicates a highly significant difference (denoted as "**").

[0155] Example 5: The effect of fullerene on collagen

[0156] To explore the effect of fullerene on other collagens, type I collagen and type III collagen were also selected as targets for experiments.

[0157] Inoculate P3 - P7 generation NIH3T3 mouse embryonic fibroblasts into a 6 - well plate. First, add 1 mL of medium to each well, and then spread 1 mL of cell dilution solution. The cell quantity is 400,000 cells per dish, and incubate overnight. Group design: There are 3 replicate wells in each group, which are respectively set as the normal group, the model group (UVB), the C 60 group (UVB + fullerene C 60 ), the VC ethyl ether group (UVB + vitamin C ethyl ether), the C 60 + VC ethyl ether group (UVB + fullerene C 60 + vitamin C ethyl ether). When the cells grow to 80% - 90% confluence in the dish, aspirate the old medium in the 6 - well plate, wash it once with normal saline, add 1.5 mL of the test substance to each well, incubate for 1 h, irradiate with a UVB instrument for 3 min, and then incubate overnight. Then collect the cells.

[0158] 1. Protein extraction: Add 200 μL of RIPA lysis buffer to each well in the 6 - well plate, lyse at 4°C for 15 min, then transfer to a 1.5 - mL centrifuge tube and centrifuge at 12,000 rpm at 4°C for 15 min. Take 10 μL of the supernatant, add 90 μL of normal saline to dilute it 10 times, and measure the protein concentration by the BCA method. Take the remaining 80 μL of the supernatant and transfer it to a new centrifuge tube, freeze it at - 20°C for storage for later use, and adjust the samples to the same concentration. Add 1 / 4 volume of the Loading buffer to the total volume of the sample and mix well for subsequent observation. Boil the sample in a metal bath at 100°C for 5 min.

[0159] (1) Preparation of BCA protein standard

[0160] Take 1.2 ml of protein standard preparation solution and add it to a tube of protein standard (30 mg BSA). After fully dissolving, prepare a 25 mg / ml protein standard solution. Take 20 μL of the 25 mg / ml protein standard and add 980 μL of normal saline to dilute it to a protein standard with a final concentration of 0.5 mg / ml.

[0161] (2) Preparation of BCA working solution

[0162] According to the number of samples, prepare an appropriate amount of BCA working solution by mixing 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1). For example, add 5 ml of BCA reagent A and 100 μl of BCA reagent B, mix well, and prepare 5.1 ml of BCA working solution. The BCA working solution is stable at room temperature within 24 hours.

[0163] (3) Protein concentration measurement

[0164] a. Add the standard products in volumes of 0, 1, 2, 4, 8, 12, 16, and 20 μl to the standard product wells of a 96-well plate, and make up to 20 μl with normal saline, corresponding to standard product concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml respectively.

[0165] b. Add 20 μl of the protein sample diluted 10-fold to the sample wells of a 96-well plate.

[0166] c. Add 200 μl of BCA working solution to each well and incubate at 37°C for 30 minutes.

[0167] d. Measure the absorbance at other wavelengths between 562 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0168] e. Calculate the protein concentration of the sample based on the standard curve and the volume of the sample used.

[0169] 2. Gel Preparation

[0170] The molecular weight of type I collagen (Collagen I) is 139 kDa, and the molecular weight of type III collagen (Collagen III) is 150 kDa. Select a 6% polyacrylamide gel for preparation. First, prepare the lower layer of the gel and pour it into the gel mold. Press it flat with absolute ethanol and let it stand for 15 minutes. After the gel solidifies, prepare the upper layer of the gel, pour it into the gel mold, insert the comb, and let it stand until it solidifies.

[0171] 3. Sample Loading

[0172] Place the prepared gel in the electrophoresis tank, add the electrophoresis buffer, remove the comb, and pipette 10 μL of each lane with a 10-μL pipette gun. Add 10-20 μL of the sample (judged according to the protein concentration measured by BCA).

[0173] 4. Electrophoresis Separation

[0174] Constant voltage electrophoresis: Usually start with a lower voltage of 70 V until the sample enters the lower layer of the gel, and then adjust to a higher voltage of 120 V until the dye approaches the bottom of the gel. Monitor the electrophoresis process: Judge whether the electrophoresis is completed by observing the position of the dye front.

[0175] 5. Blotting

[0176] Membrane preparation: Select a PVDF (polyvinylidene fluoride) or nitrocellulose membrane and pre-soak it in absolute ethanol for activation. Assembly of the blotting device: Place the gel, membrane, and filter paper in sequence in the blotting tank and moisten with the electrotransfer buffer. Electrotransfer: Set the current to 300 V. Heat will be generated during the electrotransfer process, and it needs to be carried out on ice for 1.5 h.

[0177] 6. Blocking

[0178] Block the membrane with 5% BSA (non-specific protein) at room temperature for 1 hour to reduce non-specific binding.

[0179] 7. Primary antibody incubation (Collagen I, Collagen III)

[0180] Cut out the target protein and the corresponding molecular weight of GAPDH on the membrane and mark them to distinguish the front and back sides. Then place them separately into the primary antibody and GAPDH diluted 1:1000 with 5% BSA and incubate with shaking overnight at 4°C (incubate for more than 12 hours).

[0181] 8. Secondary antibody incubation

[0182] Recover the primary antibody and GAPDH and store them frozen at -20°C. Wash the membrane 3 times with TBST, 10 minutes each time. Add the secondary antibody labeled with horseradish peroxidase (HRP) and incubate at room temperature for 1 - 2 hours. Then wash the membrane 3 times with TBST, 10 minutes each time.

[0183] 9. Color development and analysis

[0184] Use chemiluminescence or fluorescence substrate for color development, capture the signal through an imaging system, analyze the band intensity, and quantify the expression level of the target protein.

[0185] The results are as Figure 7 and Figure 8 shown. Compared with the model group, the experimental group containing fullerene can significantly increase the expression levels of type I collagen and type III collagen in fibroblasts, especially for the increase in type I collagen. Even compared with the VC ethyl ether group and the C 60 + VC ethyl ether group, there is also a significant increase. Therefore, the above experiments prove that fullerene can significantly increase the collagen expression level in fibroblasts, not limited to the type of collagen.

[0186] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement made by those of ordinary skill in the art to the technical solution of the present invention shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Use of fullerene materials in the preparation of products for enhancing the expression level of collagen.

2. The use according to claim 1, characterized in that, The fullerene materials are used for one or more of the following purposes: promoting skin cell renewal to enhance skin elasticity, promoting skin repair, inhibiting ultraviolet damage, skin whitening, reducing wrinkles, or firming the skin.

3. The use according to claim 1 or 2, characterized in that, The collagen is one or more selected from type I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XX, XXI collagen or small collagen; Preferably, the collagen is type I, III, or XVII collagen, and more preferably type XVII collagen.

4. The use according to any one of claims 1 to 3, characterized in that, The fullerene materials include fullerenes, metal fullerenes, fullerene nanotubes, hydrated fullerenes, fullerene rings, fullerene megatubes, fullerene clusters, fullerene derivatives, fullerene modifiers, and / or fullerene polymers; Preferably, the fullerene materials are selected from one or more of the following: Oil-soluble fullerenes, oil-soluble metal fullerenes, water-soluble fullerenes, water-soluble metal fullerenes, liposome fullerenes, or their physiologically acceptable salts or esters or acids or amines or alcohols.

5. The use according to claim 4, characterized in that, The metal fullerenes include M@C 2n , M2@C 2n , MA@C 2n , M3N@C 2n , M2C2@C 2n , M2S@C 2n , M2O@C 2n and M x A 3-x N@C 2n or more of them, wherein M and A both represent metal elements and M and A are each selected from any one of Sc, Y and lanthanide metal elements, wherein 30 ≤ n ≤ 60 and 0 ≤ x ≤ 3.

6. The use according to claim 4 or 5, characterized in that, The water-soluble fullerene or water-soluble metal fullerene is selected from at least one of the following: Fullerenes or metal fullerenes with hydrophilic groups modified on the outer surface of the carbon cage, fullerenes or metal fullerenes wrapped by hydrophilic biomolecules on the outer surface of the carbon cage, fullerenes or metal fullerenes loaded with biocompatible carrier materials, or water-soluble supramolecular system fullerenes or metal fullerenes formed by self-assembly.

7. The use according to claim 6, wherein The hydrophilic groups include at least one of hydroxyl, carboxyl, mercapto, amino, or water-soluble amino acid residues; and / or, the hydrophilic biomolecules include at least one of water-soluble amino acids, water-soluble peptide chains, or water-soluble polysaccharides; and / or, the biocompatible carrier materials are selected from at least one of liposomes, proteins, polymer micelles, or cell membrane carriers; 8. The use according to claim 7, wherein The water-soluble amino acid residues are at least one of alanine residue, glycine residue, serine residue, arginine residue, lysine residue, and aspartic acid residue; and / or, the water-soluble amino acids are at least one of alanine, glycine, serine, arginine, lysine, or aspartic acid; and / or, the proteins include albumin, ferritin, or transferrin; and / or, the polymer micelles include at least one of poly (lactic-co-glycolic acid)-polyethylene glycol, polylysine, chitosan, sodium hyaluronate, polysorbate, or stearate molecules; and / or, the liposomes include at least one of lecithin liposomes, hydrogenated phospholipid liposomes, iron oxide liposomes, or liposomes conjugated with targeting groups; and / or, the cell membrane carriers include at least one of erythrocyte membrane carriers, neutrophil membrane carriers, macrophage membrane carriers, or platelet carriers.

9. The use according to any one of claims 1-8, characterized in that, The fullerenes include C 20 , C 60 , C 70 , C 76 , C 80 , C 96 or C 240 , preferably C 60 or C 70 .

10. Use according to any one of claims 1-9, characterized in that, The product includes a pharmaceutical composition or a personal care composition; Preferably, the product includes facial cleanser, facial wash, facial soap, cleansing powder, facial foam, facial gel, facial cream, lotion, emulsion, cream, essence, serum, sunscreen, essence lotion, spray, penetration milk, essential oil, stock solution, gel, mask, dressing, nanoemulsion, micelle agent; Preferably, the product includes anti - hair - loss ampoule, hair - growth essence, scalp nutrient solution, anti - hair - loss shampoo, anti - hair - loss hair conditioner, anti - hair - loss spray, anti - hair - loss hair oil or hair follicle nourishing stock solution; Preferably, the product is used for preventing hair loss, strengthening hair and promoting hair growth; Preferably, the dosage form of the product includes solid preparation, liquid preparation, gas preparation or semi - solid preparation.

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