Composition for inhibiting protein carbonylation as well as preparation method and application thereof

Abstract

The invention provides a composition for inhibiting protein carbonylation and application of the composition. The composition of the present application comprises a first component and a second component, the first component comprising an umbelliferae carrots extract, a cruciferae brassica extract, an azaleaceae vaccinium extract, and an amaranaceae spinach extract as active ingredients; the second component comprises tocopheryl acetate as an active ingredient, and the mass ratio of the active ingredient of the first component to the active ingredient of the second component is 1: 20000 to 3.2: 1. By utilizing the composition provided by the invention, cell protein carbonylation caused by oxidative stress can be delayed, so that the aging state of a human body is improved. The main components of the composition are plant extracts, so that the composition is low in skin irritation, safe, effective and environment-friendly, and a new direction is developed for the development of pure natural whitening and anti-aging cosmetics. The invention further provides a preparation method of the composition and application of the composition in preparation of products for inhibiting protein carbonylation.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and more specifically, to compositions capable of inhibiting protein carbonylation, methods of preparation, and applications thereof. Background Technology

[0002] Carbonylated proteins are a class of oxidatively modified proteins induced by oxidative stress, in which amino acid residues undergo carbonylation. Carbonylation refers to the oxidation of specific amino acids (such as lysine and histidine) in proteins into carbonyl compounds. Protein carbonylation is a non-enzymatic, irreversible carbonyl modification of proteins, classified as irreversible post-translational modification (PTM). With the aid of a metal ion (iron or copper) catalytic oxidation system in vivo, reactive oxygen species (ROS) can directly oxidize lysine, arginine, proline, and threonine residues on protein side chains to form carbonyl groups. Protein carbonylation can occur through direct ROS attack, metal-catalyzed oxidation, incorporation of lipid peroxidation products, or addition of advanced aging processes (AGEs). Air pollutants such as cigarette smoke and PM2.5, on the one hand, contain ROS and reactive oxygen species (RCS), which can directly lead to lipid oxidation and protein carbonylation; on the other hand, they can also stimulate the production of ROS, further inducing various oxidation processes and protein carbonylation. Reactive carbonyl species (RCS) play a crucial role in biological systems. They are direct pathogenic factors in the cytotoxic processes caused by oxidants and free radicals attacking biological systems. RCS are involved in the initiation and development of many diseases and stresses, and even participate in and initiate the aging process. Therefore, protein carbonylation is one of the most harmful irreversible oxidative protein modifications and is considered a major marker of oxidative stress-related diseases and aging.

[0003] Under normal physiological conditions, the production and clearance of reductase inhibitors (RCS) in an organism are in dynamic equilibrium. As the body ages, the RCS defense system gradually weakens. Under stress and disease, RCS production can significantly exceed the body's clearance capacity, disrupting the balance and leading to "carbonyl stress." Carbonyl stress refers to the production of RCS in a biological system exceeding the body's clearance capacity, resulting in carbonylation modification of biomolecules such as proteins. This causes structural changes and functional loss in biomolecules, leading to cellular and tissue dysfunction and accelerating the aging process.

[0004] Therefore, effectively inhibiting protein carbonylation may be a new approach to addressing disease and aging. However, there is currently a lack of extracts from purely natural substances as the main ingredient to prepare related products for inhibiting protein carbonylation and thus slowing down the aging process of the body (e.g., skin). Summary of the Invention

[0005] There is a need for cosmetic and / or skincare compositions derived from natural substances to inhibit the carbonylation of proteins in the skin. The object of this invention is to overcome at least one aspect of the problems and / or defects present in the prior art.

[0006] The primary objective of this application is to provide a composition for inhibiting protein carbonylation, comprising: a first component and a second component. The first component comprises extracts of *Carotenoides* (Apiaceae), *Brassica* (Brassica oleracea), *Vaccinium* (Ericaceae), and *Spinacia* (Amaranthaceae) as active ingredients; the second component comprises tocopheryl acetate as an active ingredient.

[0007] Specifically, the mass ratio of the active ingredient in the first component to the active ingredient in the second component is 1:20000 to 3.2:1.

[0008] Further, the first component comprises, by volume, ≥0.00025 w / v% and ≤5 w / v% of the following extracts: 0.00025 w / v% and ≤5 w / v% of the *Caragana* genus extract, ≥0.00025 w / v% and ≤5 w / v% of the *Brassica* genus extract, ≥0.00025 w / v% and ≤5 w / v% of the *Vaccinium* genus extract, and ≥0.00025 w / v% and ≤5 w / v% of the *Spinach* genus extract, all belonging to the Amaranthaceae family.

[0009] Furthermore, the second component contains greater than or equal to 0.0005 w / v% and less than or equal to 5 w / v% of tocopherol acetate, based on the volume of the composition.

[0010] Furthermore, the first component comprises, by volume, ≥0.0005 w / v% and ≤5 w / v% of the following extracts: 0.0005 w / v% and ≤5 w / v% of the *Caragana* genus extract, ≥0.0005 w / v% and ≤5 w / v% of the *Brassica* genus extract, ≥0.0005 w / v% and ≤5 w / v% of the *Vaccinium* genus extract, and ≥0.0005 w / v% and ≤5 w / v% of the *Spinach* genus extract, all belonging to the Amaranthaceae family.

[0011] Furthermore, the first component comprises, by volume, ≥0.0005 w / v% and ≤3 w / v% of the following extracts: ...

[0012] The second component contains ≥0.001 w / v% and ≤5 w / v% of tocopherol acetate, based on the volume of the composition.

[0013] Furthermore, the mass ratio of the active ingredient in the first component to the active ingredient in the second component is from 1:15000 to 0.8:1.

[0014] Specifically, carrot extract is preferred from the Apiaceae family, Brassica oleracea extract is preferred from the Brassica oleracea family, blueberry extract is preferred from the Ericaceae family, and spinach extract is preferred from the Amaranthaceae family.

[0015] In one embodiment, the first component and tocopherol acetate in the above-described protein carbonylation inhibition composition may be provided separately in individual packages or may be provided mixed together.

[0016] Furthermore, in addition to the extracts described above, the first and second components also contain physiologically acceptable mediators.

[0017] Preferably, the first component consists of extracts from the genus *Carotenoides* (Apiaceae family), *Brassica* (Brassica oleracea family), *Vaccinium* (Ericaceae family), *Spinach* (Amaranthaceae family), and the physiologically acceptable medium.

[0018] Preferably, the second component consists of the tocopherol acetate and the physiologically acceptable medium.

[0019] The second objective of this application is to provide a method for preparing the protein carbonylation inhibitory composition provided in the first objective, comprising the following steps: diluting each active ingredient of the first component with a physiologically acceptable medium to a first solution greater than or equal to 0.00025 w / v% and less than or equal to 2 w / v%, and adding the second component to the first solution to achieve a final concentration greater than or equal to 0.0005 w / v% and less than or equal to 5 w / v%, thereby obtaining the composition.

[0020] It should be noted that, in this application, the first component of the protein carbonylation inhibition composition of the present invention can also be prepared by separately preparing multiple solutions of the active ingredients in a solvent, and then mixing the solutions together to form the first component. The first component is also commercially available, for example, EL superfood blend (I6868337) from BIO COMPENT RESEARCH (which contains 1% carotene extract, 1% cauliflower extract, 1% blueberry extract and 1% spinach extract).

[0021] A third objective of this application is to provide the use of the composition in the preparation of products that inhibit protein carbonylation. Products include pharmaceuticals, medical devices, or cosmetics. Pharmaceuticals include, but are not limited to, dermal dosage forms: specifically, topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc. Ointments are specifically categorized as creams, lotions, ointments, and gels. Further, medical devices include medical dressings, specifically including: transparent film dressings, hydrocolloid dressings, alginate dressings, foam (sponge) dressings, soft silicone silver ion dressings, lipid hydrocolloid dressings, carbon-containing dressings, and hydrogels. Even further, cosmetic dosage forms include solid cosmetics, semi-solid cosmetics, and liquid cosmetics; specifically, but not limited to, facial cleansers, toners, serums, masks, lotions, face and neck creams, eye creams, sunscreens, foundations, and makeup removers.

[0022] The drug, medical device, or cosmetic that inhibits protein carbonylation also includes a composition for inhibiting protein carbonylation provided according to the first purpose of this application and pharmaceutically or cosmetically acceptable excipients.

[0023] Pharmaceutical or cosmetic excipients include, but are not limited to, crosslinking agents, flow aids, thickeners, lubricants, wetting agents, fillers, excipients, solubilizers, disintegrants, etc.

[0024] Tackifiers may include, but are not limited to, copolymers of methacrylate and n-butyl acrylate, copolymers of methyl acrylate and 2-ethylhexyl acrylate, polybutene, ester resins, terpene resins, alicyclic saturated hydrocarbon resins, etc.

[0025] The solubilizer may include, but is not limited to, pyrrolidone derivatives such as Crotamiton and N-methyl-2-pyrrolidone, peppermint oil, 1,3-butanediol, etc., and may contain one or more of them individually or in combination.

[0026] Excipients may include, but are not limited to, starch, glucose, lactose, sucrose, mannitol, trehalose, gelatin, malt, rice, cellulose, magnesium carbonate, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, ethylene glycol, water, ethanol, etc.

[0027] Diluents include, but are not limited to, aqueous diluents (such as water, glycerin, propylene glycol and other polyols), alcohol diluents (such as ethanol, isopropanol, n-propanol, etc.), ether diluents (such as diethyl ether, tetrahydrofuran, etc.), vegetable oils (such as olive oil, grapeseed oil, etc.), mineral oils (such as white petrolatum, etc.), and organic solvents (such as acetone, ethyl acetate, etc.).

[0028] Adhesives may include, but are not limited to, starch paste, methylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, ethylcellulose, povidone, syrup, glue, polyethylene glycol, etc.

[0029] Lubricants may include, but are not limited to, dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, and croscarmellose. Disintegrants may include starch, clay, cellulose, alginate, gum, and cross-linked polymers, such as croscarmellose or croscarmellose, croscarmellose calcium, soybean polysaccharide, and guar gum.

[0030] Fillers may include, but are not limited to, confectioner's sugar, compressible sugar, dextrates, dextrin, dextrose, lactose, mannitol, microcrystalline cellulose, powdered cellulose, sorbitol, sucrose, dicalcium phosphate, starch, pregelatinized starch, and talc.

[0031] The flow aid may include, but is not limited to, colloidal silica (e.g., colloidal silica), magnesium trisilicate, starch, talc, calcium phosphate, magnesium stearate, aluminum stearate, calcium stearate, magnesium carbonate, magnesium oxide, polyethylene glycol, powdered cellulose, and microcrystalline cellulose.

[0032] Cosmetic excipients include, but are not limited to, surfactants, moisturizers, plasticizers, emulsifiers, fragrances, pigments, preservatives, and antioxidants. Surfactants include, but are not limited to: anionic surfactants such as sodium dioctyl sulfosuccinate, alkyl sulfates, sodium 2-ethylhexyl alkyl sulfate, and sodium dodecylbenzene sulfonate; cationic surfactants such as hexadecyltrimethylammonium chloride, octadecyldimethylbenzylammonium chloride, and polyoxyethylene dodecyl monomethylammonium chloride; and also nonionic or amphoteric surfactants. Moisturizers include, but are not limited to: concentrated glycerin, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol, polypropylene glycol, liquid paraffin, 1,3-propanediol, 1,3-butanediol, maltitol, xylitol, and other polyols, which may contain one or a combination of two or more.

[0033] Moisturizers include, but are not limited to: sucrose, sorbitol, glycerin, 1,3-butanediol, propylene glycol, dipropylene glycol, and other sucrose or polyols; polyether compounds formed by the addition polymerization of a polyol core with three or more hydroxyl groups in its molecule with 2-100 moles of propylene oxide and less than 50 moles of ethylene oxide; ethyl glucosyl ether-10; chondroitin sulfate; hyaluronic acid; sulfated mucopolysaccharides; carboxylic acid; telopeptide-free collagen; cholesterol-12-hydroxystearate; bile acid monosalts; dl-pyrrolidone carboxylic acid monosalts; short-chain soluble collagen; Rosa roxburghii extract; and Achillea millefolium. Extracts of *Millettia speciosa*, propylene glycol, dipropylene glycol, 1,3-butanediol, 1,4-butanediol, diethylene glycol, triethylene glycol, and polyethylene glycols with molecular weights above these limits; glycerol, diglycerol, and polyglycerols with molecular weights above these limits; sugar alcohols such as sorbitol, mannitol, maltitol, xylitol, and erythritol; sugars such as fructose, glucose, galactose, maltose, lactose, and trehalose; and polypropylene glycol / polyethylene glycol copolymers or their dimethyl ethers.

[0034] Antioxidants include, but are not limited to, tocopheryl acetate, ascorbic acid and / or its derivatives, sodium sulfite, butylated hydroxytoluene, etc., which may contain one or more individually or in combination. Plasticizers include, but are not limited to, lipophilic oily substances, such as fatty acid esters with 6 to 18 carbon atoms, diesters with 6 to 10 carbon atoms, higher alcohols with 10 to 18 carbon atoms, or castor oil, etc. Fatty acid esters with 6 to 18 carbon atoms include, for example, higher fatty acid esters such as hexyl laurate, isopropyl myristate, isopropyl palmitate, or glycerol fatty acid esters such as medium-chain triglycerides. Diesters with 6 to 10 carbon atoms include, for example, diisopropyl adipate, dioctyl adipate, diethyl adipate, diisopropyl sebacate, diethyl sebacate, etc. Higher alcohols with 10 to 18 carbon atoms include, for example: hexyldecyl alcohol, myristol, lauryl alcohol, oleyl alcohol, octyldodecyl alcohol, etc.

[0035] Plasticizers include, but are not limited to: alkyl phthalates and allyl phthalates; naphthalene esters; lactates (e.g., sodium, ammonium, and potassium salts); sorbitol polyoxyethylene ether-30; urea; lactic acid; sodium pyrrolidone carboxylate (PCA); sodium hyaluronate or hyaluronic acid; soluble collagen; modified proteins; monosodium glutamate; α- and β-hydroxy acids such as glycolic acid, lactic acid, citric acid, maleic acid, and salicylic acid; polyglycerol methacrylate; polymer plasticizers such as polyquaternium salts; proteins and amino acids such as glutamic acid, aspartic acid, and lysine; hydrolyzed starch products; other low molecular weight esters (e.g., esters of C2-C10 alcohols and acids); and mixtures thereof.

[0036] Preservatives include, but are not limited to, methylparaben, butylparaben, propylparaben, thymol, etc., which may contain one or more of these substances individually or in combination.

[0037] As a pigment, there are no particular restrictions on its types. Pigments listed in the legal pigment manual can be added, and they can be used alone or in combination of two or more.

[0038] The excipients used in medical dressings include water-soluble synthetic polymers and water-soluble natural polymers. The water-soluble synthetic polymers include polyacrylamide, polyacrylic acid, polyvinylpyrrolidone, polymaleic anhydride, polyquaternium salts, polyethylene glycol, modified cellulose, and modified starch. The water-soluble natural polymers include collagen, gelatin, chitosan, and hyaluronic acid.

[0039] The fourth objective of this application is to provide the use of the protein carbonylation-inhibiting composition of the first objective and the protein carbonylation-inhibiting product of the third objective in the preparation of anti-aging and / or whitening products that inhibit protein carbonylation. The products include, but are not limited to, anti-skin aging and / or whitening products, including cosmetics, specifically including but not limited to toners, serums, masks, lotions, creams, eye creams, foundation products, or sunscreen products.

[0040] The fifth objective of this application is to provide a method for inhibiting protein carbonylation in mammalian somatic cells, comprising contacting a subject's somatic cells with an effective dose of the composition according to the first objective of this application or the product for inhibiting protein carbonylation according to the third objective. The effective dose is the amount that effectively inhibits protein carbonylation in somatic cells.

[0041] In one embodiment, the contact is performed in vivo, in vitro, or ex vivo.

[0042] In another embodiment, the contact can occur before or after the carbonylation of proteins in somatic cells.

[0043] In one embodiment, the somatic cells may be skin fibroblasts. When the target is skin fibroblasts, the above-described method for inhibiting somatic protein carbonylation may include contacting the product with the subject's skin. Furthermore, contact with the skin can achieve an anti-skin aging effect. Specifically, methods of application include, but are not limited to, applying, spraying, or patching to the subject's skin surface.

[0044] In one embodiment, at the contact, the composition is used in an amount of 0.00064 mg / 10,000 cells to 6.35 mg / 10,000 cells.

[0045] Beneficial effects of the invention

[0046] This application unexpectedly discovers that combining carrot extract, cauliflower extract, blueberry extract, spinach extract, and tocopheryl acetate at specific concentrations creates a series of organically linked components that exhibit strong synergistic effects. Ultimately, these components can inhibit the carbonylation of proteins in somatic cells (e.g., skin fibroblasts) in a low-concentration, highly synergistic manner. This inhibition significantly reduces cellular oxidation, thereby slowing down cellular aging. The main components of this composition are all natural, edible plant extracts, exhibiting low irritation to the body (especially the skin), safety, effectiveness, and environmental friendliness. Based on the compositions of this invention, new ideas and platforms can be opened up for the research and development of all-natural anti-aging and / or whitening products. Attached Figure Description

[0047] The above features and advantages of this application will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, wherein:

[0048] Figure 1 The results of immunofluorescence detection of carbonylation intensity in untreated cells under an inverted fluorescence microscope according to this application are shown.

[0049] Figure 2 The results of carbonylation intensity detection under an inverted fluorescence microscope after the addition of the methanal inducer (ACR) according to this application are shown.

[0050] Figure 3 The results of carbonylation intensity detection under an inverted fluorescence microscope after adding ACR and 0.5% of the first component according to this application are shown.

[0051] Figure 4 The results of carbonylation intensity detection under an inverted fluorescence microscope after adding ACR and 0.25% tocopherol acetate (VE Acetate) according to this application are shown.

[0052] Figure 5 The results of carbonylation intensity detection under an inverted fluorescence microscope after adding ACR and 0.5% first component + 0.25% tocopheryl acetate (first component: tocopheryl acetate = 2:1) are shown.

[0053] Figure 6 The diagram shows a comparison of cell fluorescence intensity and carbonylation intensity after treating cells with ACR, ACR + 0.5% first component, ACR + 0.25% tocopherol acetate, and ACR + 0.5% first component + 0.25% tocopherol acetate, respectively, according to this application. Detailed Implementation

[0054] Terminology Definition

[0055] Unless otherwise defined, all terms used herein have the same meaning as understood by one of ordinary skill in the art. For specific definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel).

[0056] The term “Carotenoides extract” as used in this article refers to concentrated components isolated from Carotenoid plants (such as the roots, leaves, and seeds of carrots) by physical or chemical methods. These components include volatile components (such as essential oils and terpenoids), non-volatile components (such as carotene, polyphenols, flavonoids, and polysaccharides), and other bioactive substances (such as vitamins and minerals).

[0057] As used in this article, "Brassica extract" refers to concentrated active ingredients isolated from the seeds, leaves, stems, flowers, or roots of plants in the genus Brassica (such as rapeseed, mustard greens, cabbage, cauliflower, and broccoli) of the Brassica family (Brassica genus) through physical or chemical methods. This genus of plants is rich in glucosinolates, which, upon hydrolysis, yield isothiocyanates (ITCs) (such as sulforaphane), exhibiting antioxidant, anti-inflammatory, and anticancer activities. Brassica extract mainly contains glucosinolates and their derivatives (e.g., sulforaphane), phenolic compounds (e.g., quercetin), terpenoids, vitamins, volatile oils (e.g., aromatic alcohols), and polysaccharides, among other active substances.

[0058] As used in this article, "Vaccinium extract" refers to a concentrated mixture of active ingredients isolated from the fruits, leaves, or roots of plants in the genus Vaccinium (Ericaceae) (such as blueberries, cranberries, and blueberries) through physical or chemical methods. This extract is rich in antioxidants and anti-inflammatory components, primarily including anthocyanins (such as delphinidin and cyanidin), proanthocyanidins (PACs) (especially type A PACs from cranberries), flavonoids (such as quercetin and myricetin), organic acids (such as benzoic acid and quinic acid), and vitamins and minerals (vitamins C, K, manganese, etc.).

[0059] As used in this article, "Spinacia extract" refers to a mixture of active ingredients extracted from plants of the genus Spinacia in the family Amaranthaceae using physical or chemical methods. For example, concentrated extracts of active ingredients from the leaves, stems, or seeds of spinach (Spinacia oleracea) may contain the following main active ingredients: chlorophyll; carotenoids such as lutein and zeaxanthin; vitamins such as vitamin K, vitamin C, and folic acid (B9); minerals such as iron, calcium, and magnesium; and phenolic compounds such as spinacetin and quercetin.

[0060] As used herein, the term "physiologically acceptable medium" refers to a fluid, more or less, which may include, but is not limited to, any additives or co-solvents commonly used in the cosmetics field, as well as adjuvants, humectants, surfactants, emulsifiers, etc., necessary for their formulation, suitable for contact with skin or mucous membranes without causing toxic or intolerance reactions. The physiologically acceptable medium is physiologically inert to the active properties of the compositions of the present invention. For example, it may include: aqueous media, such as pure water, deionized water, distilled water; water-soluble solvents, such as glycerin (moisturizing), propylene glycol (penetration enhancer), butylene glycol (moisturizing), polyethylene glycol (viscosity adjuster); oily media, such as vegetable oils (jojoba oil, olive oil, coconut oil), silicone oil, mineral oil (such as petrolatum); emulsifiers, including nonionic emulsifiers, anionic emulsifiers, cationic emulsifiers, and amphoteric emulsifiers; thickeners, including carbomer, xanthan gum, sodium alginate, and natural plant gums; and other physiologically acceptable media, such as alcohols, esters, sugars, amino acids, and inorganic salts. In this application, physiologically acceptable media can also be cell culture media, such as DMEM, RMPI-1640, and other commonly used cell culture media. It should be understood that although culture media are used as physiologically acceptable media in the following examples, those skilled in the art will understand that when applied to mammalian (e.g., human) skin, aqueous media such as deionized water can be used to dilute the various active ingredients in the compositions of this invention. Alternatively, to improve the skin permeability of the compositions of this invention, oily media or emulsifiers can also be used to dilute the various active ingredients.

[0061] The term “protein carbonylation” used in this article refers to the non-enzymatic irreversible carbonyl modification of proteins. Based on the involvement of ROS (Reactive Oxygen Species), the pathways for the generation of carbonyl proteins are divided into two main categories: (1) ROS directly oxidizes the side chain amino acids of proteins to generate carbonyl proteins (i.e., free radical oxidation); (2) proteins generate carbonyl proteins after lipid oxidation and non-enzymatic glycosylation (i.e., glycosylation).

[0062] The term "deionized water" as used in this article refers to pure water after ion exchange resins have been used to remove ionic impurities such as anions and cations from the water.

[0063] The term "effective dose" as used in this article refers to the dose of a pharmacological agent that is pharmacologically effective in achieving a therapeutic effect.

[0064] The term “VE Acetate” as used in this article refers to a fat-soluble vitamin whose hydrolysis product is tocopherol, which has antioxidant properties and is soluble in organic solvents such as fats and ethanol.

[0065] To enable those skilled in the art to better understand the technical solutions of this application, the following embodiments and test examples will be used to further describe this application in detail. These embodiments and test examples are for illustrative purposes only and are not intended to limit the scope of this application. The test materials used in the embodiments of this application are all conventional test materials in the art, which can be purchased through commercial channels or prepared by existing technical methods. If no specific preparation method is provided in the embodiments, it is a conventional substance or material that can be purchased through commercial channels.

[0066] As an optional embodiment, the above-mentioned extract can be obtained by the following specific extraction method:

[0067] The preparation steps of carrot extract are as follows: Fresh carrots are sliced ​​and extracted with 70% ethanol or water (50-60℃) for 2-3 hours. After filtration, the extract is concentrated under reduced pressure and freeze-dried to obtain powder, from which the applicable components are obtained: polyphenols, vitamin C, and polysaccharides.

[0068] The preparation steps of cauliflower extract are as follows: First, fresh cauliflower inflorescences are quick-frozen and then pulverized to avoid enzyme inactivation. Then, 0.1% myrosinase (or mustard seed powder) is added, and the mixture is reacted at pH 6.0 and 37℃ for 1 hour for enzymatic activation (sulforaphane exists in its precursor form (sulforaphane glycosides), which requires myrosinase hydrolysis for activation). Next, extraction is performed with 70% ethanol (containing 0.1% ascorbic acid for oxidation prevention) with shaking for 2 hours. Finally, adsorption is performed using a macroporous resin (such as AB-8), followed by elution with 50% ethanol, and concentration and purification.

[0069] The preparation steps of blueberry extract are as follows: First, freeze-dry blueberry fruit powder (avoiding heat damage) and pass it through a 60-mesh sieve. Then, extract with 60% ethanol (v / v) containing 0.1% hydrochloric acid at a material-to-liquid ratio of 1:15 at 50°C with shaking for 1 hour (protected from light). Finally, purify by adsorption with AB-8 macroporous resin, elute impurities with 40% ethanol, desorb anthocyanins with 70% ethanol, concentrate under vacuum, and spray dry.

[0070] The preparation steps of spinach extract are as follows: First, fresh spinach is homogenized and then extracted with hot water at 50℃ for 1 hour at a material-to-liquid ratio of 1:10. The supernatant is then collected by centrifugation. Next, 3 times the volume of 95% ethanol is added to the supernatant, and the mixture is allowed to stand at 4℃ for 12 hours for alcohol precipitation purification to obtain precipitated polysaccharides. The filtrate is passed through a cation exchange resin column, and betaine is eluted with ammonia.

[0071] The carrot, cauliflower, blueberry, and spinach extracts mentioned above are also available from BIOCOMPENT RESEARCH. For example, the product code for a mixture of carrot, cauliflower, blueberry, and spinach extracts is I6868337.

[0072] For the purpose of illustrating this invention, any plants of the same genus and species should be included in this reference. It is understood that this invention may use extracts from plants of the genera *Carrot* (Apiaceae), *Brassica* (Brassica oleracea), *Vaccinium* (Ericaceae), and *Spina* (Amaranthaceae), and any tocopherol derivatives with similar efficacy.

[0073] The technical solution described in this application consists of the following parts in chronological order:

[0074] (1) Preparation of skin fibroblast cells and compositions;

[0075] (2) The protective effect of the composition on human skin fibroblasts was detected by cell immunofluorescence reaction;

[0076] (3) Evaluation of the effects of the composition.

[0077] Preparation Example

[0078] 1. Cell preparation: Asian human fibroblasts (Biocells) cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Corning) containing 10% fetal bovine serum were digested into single cells using TrypLE-Express enzyme (Life Technologies), and seeded in 24-well plates at 2 x 10⁶ cells / wells. 4 / Each well contains human skin fibroblasts (HDF), which are cultured adherently for 24 hours.

[0079] 2. Dilute acrolein (S107362, Aladdin) to 60 μm with DMEM and store for later use.

[0080] 3. Radish extract, cauliflower extract, blueberry extract, and spinach extract are mixed with DMEM to form an edible plant mixture, namely the first component, for later use. The mixture comprises (by weight / volume %): 1% carrot extract + 1% cauliflower extract + 1% blueberry extract + 1% spinach extract. It is then diluted according to the target ratio to the following concentrations (by weight / volume %): 0.5%, 0.25%, 0.1%, 0.002%, 0.001%, 0.0005%, and 0.00025%, respectively.

[0081] 4. Using DMEM, dilute VITAMIN E, USP, FCC, CODE0420085 (VE Acetate - Tocopheryl Acetate) (i.e., the second component) purchased from Independent Chemical Corp to a higher storage concentration of 10 wt% (v / v), and then further dilute to the following concentrations (w / v%): 0.25%, 0.1%, 0.01%, 0.001%, 0.0025%, and 0.00025%.

[0082] 5. Dilute glycerol (purchased from Wilmar) with DMEM to the following concentrations (volume ratio): 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, and 0.00005%.

[0083] Setting synergy comparison criteria

[0084] The synergistic effects of experimental data were evaluated using the isobole method described in “Synergistic Effects of Plant Derivatives and Conventional Chemotherapeutic Agents: An Update on the Cancer Perspective,” Medicina 2019, 55(4), 110.” According to the isobole method, when OE refers to the observed effect, and da and db represent the doses of component a and component b respectively (therefore, OE(da, db) refers to the combined observed effect of component a at da and component b at db; OE(da) refers to the observed effect of component a at da, and OE(db) refers to the observed effect of component b at db), the following three mathematical formulas can be derived:

[0085] OE(da, db)=OE(da)+OE(db) (1)

[0086] OE(da, db)>OE(da)+OE(db) (2)

[0087] OE(da, db) <OE(da)+OE(db) (3)

[0088] If the observed effect satisfies equation (1), then the effect of the two components is a simple addition of the individual effects, the two components do not interact, and therefore there is no synergistic effect.

[0089] If the observed effect satisfies equation (2), the result represents a true synergistic effect (or synergistic effect), meaning that the effect of the two components is greater than the simple sum of the individual effects.

[0090] If the observed effect satisfies equation (3), the result indicates the opposite effect of synergy, that is, the effect of the two components is less than the simple sum of the individual effects.

[0091] Example 1: Inhibition of protein carbonylation in skin fibroblasts using different concentrations of glycerol

[0092] Different concentrations of glycerol solutions from the preparation examples were mixed with 60 μM acrolein (ACR) induction solution and added to DMEM. Then, 500 μL of this solution was added to 24-well plates containing adherent human fibroblasts (HDF) cultured for 24 hours. Cells were treated at 37°C and 5% CO2 for 48 hours. After removing the DMEM medium and washing with 1×PBS, cells were fixed with 4% paraformaldehyde (PFA) solution. Immunofluorescence staining was then performed according to the method described in emula, V., Z. Ni, and M. Fedorova, Fluorescence labeling of carbonylated lipids and proteins in cells using coumarin-hydrazide. Redox Biol, 2015.5: p.195-204. Finally, images were captured using the PerkinElmer Operetta CLS high-content imaging system. For each group, at least three fields of view were captured, and the fluorescence intensity was calculated as the average gray value from the images. Meanwhile, each sample was replicated and the average value was taken. The wells containing only DMEM medium without any other substances were used as blank control wells, the wells containing only acrolein were used as negative control wells (induction wells), and the wells containing acrolein solution mixed with the test substance (glycerol of different concentrations) were used as test sample wells.

[0093] After obtaining the test values, calculate the protection rate using the following formula.

[0094] Protection rate (%) = 100% × (Induced value - Sample value) / (Induced value - Blank control value)

[0095] The detection results of inhibiting protein carbonylation using different concentrations of glycerol are shown in Table 1.

[0096] Table 1

[0097]

[0098] The results in Table 1 show that, compared with the negative control wells containing acrolein, glycerol at all concentrations did not show any protective effect against protein carbonylation. The p-values ​​were all greater than 0.05, failing to meet the significance standard, thus demonstrating that glycerol did not inhibit acrolein-induced protein carbonylation. Although the calculated protection rates at 0.001% and 0.01% concentrations of glycerol were relatively high, considering factors such as within-group variance, the statistical p-values ​​were all greater than 0.05, indicating no significant difference. Therefore, it is concluded that glycerol had no protective effect at these concentrations.

[0099] Example 2: Testing the effects of different concentrations of the first component, VE Acetate, and a combination of both on inhibiting protein carbonylation in skin fibroblasts.

[0100] Following the experimental method in Example 1, 0.1% and 0.001% mixtures (i.e., the first component), 0.1%, 0.01% and 0.001% VE Acetate were mixed with acrolein, or compositions of the first component at each concentration and VE Acetate in different proportions were combined with 60 μm acrolein and applied to HDF cells to verify the ability of different concentrations of the first component, VE Acetate and different compositions to inhibit protein carbonylation. The test results are shown in Table 2.

[0101] Table 2

[0102]

[0103]

[0104] The results in Table 2 show that different concentrations of VE Acetate and 0.1% of the first component all exhibited a certain inhibitory effect on protein carbonylation. When VE Acetate at three concentrations was combined with 0.1% or 0.001% of the first component, a strong synergistic effect was observed, resulting in a stronger inhibitory effect on protein carbonylation. When 0.001% of the first component and 0.001% of VE Acetate were used alone, the protection rates were -1.45% and 5.6%, respectively. When used in combination, the protection rate increased to 17.89%. In particular, when the ratio of 0.001% of the first component to 0.01% of VE Acetate was 1:10, the protection rate was above 28%, and when the ratio was 1:100, the protection rate reached above 35%.

[0105] Example 3: Inhibitory effect of compositions formed by combining 0.25% of the first component with different concentrations of VE Acetate.

[0106] To further explore the concentrations and ratios that can produce a synergistic effect, this embodiment increased the concentration of the first component to 0.25% and combined it with several lower concentrations of VE Acetate, while simultaneously increasing the ratio of the first component to VE Acetate. The protective effects of applying the two components separately or in combination were compared. The test results are shown in Table 3.

[0107] Table 3

[0108]

[0109]

[0110] As shown in Table 3, the protection rate of using 0.25% of the first component alone was 21.52%, and the protection rate of using 0.0025% VE Acetate was 3.11%. However, when the concentration of the first component was increased to 0.25% and the concentration of VE Acetate was reduced to 0.0025%, the protection rate was only 20.81%. This indicates that even when the concentration of the first component is significantly increased, the synergistic effect is not strong when the concentration of VE Acetate is very low.

[0111] Example 4: The inhibitory effect of reducing the concentration of the first component and increasing the concentration of VE Acetate on the formation of a composite.

[0112] According to the results of Example 2, the protection rate was 24.01% when the VE Acetate concentration was 0.1% and 17.72% when the VE Acetate concentration was 0.01%. The first component alone had no protective effect. The protection rate of 0.001% first component + 0.01% VE Acetate was 28.69%, and the synergistic protection rate of the composition of 0.001% first component + 0.1% VE Acetate reached 35.19%. Therefore, this example further compared the protective effects of VE Acetate at a concentration of 0.25% with different concentrations of the first component, and also compared the protective effects of VE Acetate at a concentration of 0.001% and then combined with the first component at a concentration of 0.002%. The results are shown in Table 4. Table 4

[0113]

[0114]

[0115] As shown in Table 4, the protection rate of 0.5% of the first component alone is 23.71%, and the protection rate of 0.25% of VE Acetate alone is 16.71%. The protection rate is highest at 46.26% when the combination of 0.5% of the first component and 0.25% of VE Acetate is formed. The protection rate of 0.00025% of the first component alone is 1.37%, but the protection rate of the combination of 0.00025% of the first component and 0.25% of VE Acetate is only 15.46%, which is lower than the protection rate of 0.25% of VE Acetate alone. The protection rate of 0.0005% of the first component alone is only 7.75%, but the protection rate is highest at 25.33% when the combination of 0.0005% of the first component and 0.25% of VE Acetate is formed, showing a slight improvement in protection rate. The protection rate is 14.36% when 0.002% of the first component is used alone, and 4.93% when 0.001% of VE Acetate is used alone. The protection rate is as high as 27.02% when the composition of 0.002% of the first component and 0.001% of VE Acetate is formed. Figure 6 The bar chart also shows that the untreated cell group had the lowest relative fluorescence intensity, approximately 1. The acrolein group (inducing protein carbonylation) had the highest relative fluorescence intensity, approximately 1.35, showing a highly significant difference compared to the untreated group. The relative fluorescence intensity of the 0.5% first-component treatment group was somewhat lower than that of the acrolein group. The relative fluorescence intensity of the 0.25% second-component treatment group was slightly higher than that of the 0.5% first-component treatment group, but still significantly different from that of the acrolein (ACR) group. However, the relative fluorescence intensity of the 1:2 treatment group (0.5% first-component + 0.25% second-component) was significantly lower than that of the 0.5% first-component and 0.25% second-component treatment groups, and highly significantly lower than that of the acrolein group, while its fluorescence intensity was close to that of the untreated group.

[0116] The above test results fully demonstrate that the first component and VE Acetate only produce excellent synergistic inhibition of protein carbonylation when they form a combination at a certain concentration and within a specific ratio range.

[0117] In summary, the composition provided in this application, consisting of extracts from various edible plants and vitamin E in a specific ratio, can produce a synergistic effect, effectively inhibiting protein carbonylation in skin cells and ultimately alleviating cellular oxidative stress, thus improving cellular aging. The main components of the composition are extracts from edible plants, which are widely available, safe, reliable, low-cost, and environmentally friendly.

[0118] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A composition for inhibiting protein carbonylation, comprising: a first component and a second component; in, The first component contains extracts of Carotenoides (Apiaceae), Brassica (Brassica oleracea), Vaccinium (Ericaceae), and Spinach (Amaranthaceae) as active ingredients; the second component contains tocopheryl acetate as an active ingredient. The mass ratio of the active ingredient in the first component to the active ingredient in the second component is from 1:20000 to 3.2:

1.

2. The composition according to claim 1, characterized in that, In the composition, the content of the active ingredient of the second component, based on the volume of the composition, is greater than or equal to 0.0005 w / v% and less than or equal to 5 w / v.

3. The composition according to claim 1, characterized in that, The first component comprises, by volume, greater than or equal to 0.00025 w / v% and less than or equal to 5 w / v% of the following extracts: Apiaceae (Carota genus), Brassica genus, Rhus chinensis genus, Ericaceae (Vaccinium genus), and Spinach genus, Amaranthaceae (Spina genus), with the balance being physiologically acceptable media.

4. The composition according to claim 3, characterized in that, The first component comprises, by volume, ≥0.0005 w / v% and ≤5 w / v% of the following extracts: <0.0005 w / v% and ≤5 w / v% of the *Carotenoides* genus extract, ≥0.0005 w / v% and ≤5 w / v% of the *Brassica* genus extract, ≥0.0005 w / v% and ≤5 w / v% of the *Vaccinium* genus extract, and ≥0.0005 w / v% and ≤5 w / v% of the *Spinach* genus extract, based on the volume of the first component:

5. The composition according to claim 4, characterized in that, The first component comprises, by volume, ≥0.0005 w / v% and ≤3 w / v% of the following extracts: 0.0005 w / v% and ≤3 w / v% of the following extracts: ...

6. The composition according to claim 5, characterized in that, In the composition, the content of the active ingredient of the second component, based on the volume of the composition, is greater than or equal to 0.001 w / v% and less than or equal to 5 w / v% of tocopherol acetate, wherein the mass ratio of the active ingredient of the first component to the active ingredient of the second component is from 1:15000 to 0.8:

1.

7. A method for preparing the composition according to any one of claims 1-6, characterized in that, The method includes the following steps: diluting each active ingredient of the first component with a physiologically acceptable medium to a first solution greater than or equal to 0.00025 w / v% and less than or equal to 2 w / v%, and adding the second component to the first solution to achieve a final concentration greater than or equal to 0.0005 w / v% and less than or equal to 5 w / v%, thereby obtaining the composition.

8. Use of the composition according to any one of claims 1-6 in the preparation of products that inhibit protein carbonylation.

9. The application according to claim 8, characterized in that, The product in question is a drug, medical device, or cosmetic.

10. The application according to claim 8 or 9, characterized in that, The product in question is an anti-aging and / or skin whitening product.

11. A method for inhibiting the carbonylation of proteins in mammalian somatic cells, comprising the step of contacting the composition according to any one of claims 1-6 with the somatic cells of the mammal.

12. The method according to claim 11, characterized in that, The contact occurs before or after the carbonylation of proteins in the somatic cells.

13. The method according to claim 11, characterized in that, The somatic cells are skin fibroblasts.

14. The method according to claim 11, characterized in that, During the contact, the composition is used in an amount of 0.00064 mg / 10,000 cells to 6.35 mg / 10,000 cells.

15. The method according to any one of claims 11-14, characterized in that, The contact can be performed in vivo, in vitro, or outside the body.

16. A product for preventing, inhibiting, preventing or antagonizing protein carbonylation in mammalian somatic cells, comprising the composition according to any one of claims 1-6 and pharmaceutically or cosmetically acceptable excipients.

17. The product according to claim 16, characterized in that, The product in question is a cosmetic, a pharmaceutical, or a medical device.

18. The product according to claim 17, characterized in that, The product in question is an anti-aging and / or skin whitening product.

Images