Cosmetic application of 7-ethyl-4, 5, 6, 7-tetrahydro-9-hydroxy-2, 7-methylene-2H-2-benzazon-1 (3H)-one

Abstract

The invention provides a cosmetic application of a benzazine derivative, the cosmetic application is selected from anti-aging, anti-inflammatory and / or anti-oxidation efficacy, and the benzazine derivative has a structure as shown in a formula I. The invention further provides a preparation method of the benzazine derivative. The invention also relates to application of the benzazoine derivative of formula I in preparation of skin external preparation with anti-aging, anti-inflammatory and / or anti-oxidation effects: # imgabs0 #

Description

Technical Field

[0001] The present invention relates to research on the cosmetic effects of benzodiazine derivatives, and specifically to the anti-aging, anti-inflammatory and / or antioxidant applications of benzodiazine derivatives, as well as the application of benzodiazine derivatives in the preparation of external skin preparations with anti-aging, anti-inflammatory and antioxidant effects. Background Art

[0002] The skin is the largest organ in the human body, and beauty and anti-aging are long-cherished goals. Wrinkles are one of the most common signs of aging. Many factors, including environment, aging, lifestyle, health, and psychological state, significantly influence the development of wrinkles. Collagen in the dermis is a major factor influencing skin aging. Type I collagen is a fibrous collagen found in large quantities in bones and skin, contributing to their elasticity. With aging, or due to external factors such as UV rays, collagen content in the skin gradually decreases, causing the skin to sag and wrinkles to appear. Therefore, promoting the expression of type I collagen is an effective method for improving skin wrinkles. Furthermore, for wrinkle reduction, particularly for dynamic expression lines, some ingredients work by inhibiting the release of acetylcholine at the neuromuscular junction, paralyzing muscles and reducing muscle contraction force.

[0003] At the same time, intrinsic and extrinsic aging of skin cells share a common molecular mechanism: oxidative stress caused by excessive accumulation of reactive oxygen species (ROS) within cells, which triggers a series of subsequent effects, causing skin cell damage and accelerating skin aging. Therefore, effectively inhibiting oxidative stress and delaying skin cell aging is an urgent need in dermatology and cosmetic medicine.

[0004] Furthermore, PGE2 (prostaglandin E2) is an important inflammatory mediator and a key contributor to skin inflammation and various skin diseases. For example, PGE2 dilates blood vessels, increases permeability, and promotes the infiltration of inflammatory cells, leading to typical inflammatory symptoms such as redness, swelling, and fever. Excessive PGE2 levels can damage the skin barrier, increasing water loss and the intrusion of external irritants, leading to dryness, sensitivity, and other problems. Therefore, inhibiting PGE2 is a strategy for improving adverse skin conditions.

[0005] Early studies such as Chinese patent application CN 202410825191.9 disclosed a compound (1) with a unique nitrogen-bridged ring structure, the molecular formula of which is C 15 H 19 NO2 can be named according to the systematic method: 7-ethyl-4,5,6,7-tetrahydro-9-hydroxy-2,7-methylene-2H-2-benzoazoline-1(3H)-one.

[0006]

[0007] Compound (1) is a chiral compound. Early studies involved the preparation of optically pure monomers (R configuration and S configuration) of compound (1) and its antioxidant effect (detected by the ORAC method).

[0008] In addition, the literature (Zheng Wei et al., "1-Ethyl-4-hydroxy-9-azatricyclo[7.4.1.0 2,7 ]tetradeca-2,4,6-trien-8-one", Acta Cryst. 2009, E65, o3008) reported the compound 1-ethyl-4-hydroxy-9-azatricyclo[7.4.1.0 2,7 ]Tetradeca-2,4,6-trien-8-one.

[0009] However, to date, no benzodiazepine derivatives have been found to possess anti-aging, anti-inflammatory and / or antioxidant cosmetic benefits. Summary of the Invention

[0010] The present invention provides a cosmetic application of a benzodiazine derivative, wherein the cosmetic application is selected from: anti-aging, anti-inflammatory and / or antioxidant effects, and the benzodiazine derivative has a structure of Formula I:

[0011]

[0012] In a preferred embodiment, the purity of the benzodiazine derivative is ≥98.5%.

[0013] In a preferred embodiment, the anti-aging effect is achieved by promoting type I collagen and / or inhibiting acetylcholine.

[0014] In a preferred embodiment, the anti-inflammatory effect is achieved by inhibiting the inflammatory factor PGE2.

[0015] In a preferred embodiment, the antioxidant effect is achieved by inhibiting ROS.

[0016] In a preferred embodiment, the benzodiazine derivative is used at a concentration of at least 3 ppm. In a preferred embodiment, the benzodiazine derivative is used at a concentration of less than 100 ppm. In a preferred embodiment, the benzodiazine derivative is used at a concentration of 3.125-50 ppm, preferably 12.5-50 ppm.

[0017] On the other hand, the present invention also relates to the use of a benzodiazine derivative in the preparation of a skin external preparation having anti-aging, anti-inflammatory and / or antioxidant effects, wherein the benzodiazine derivative has a structure of Formula I:

[0018]

[0019] In a preferred embodiment, the skin external preparation is selected from: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The fluorescence results of immunofluorescence detection of type I collagen in replicative senescent fibroblasts in Test Example 2 are shown. DETAILED DESCRIPTION

[0021] The present invention aims to provide an R-configured benzodiazepine derivative (e.g., a compound of Formula I) for use in skin care, which can be used in cosmetics as an anti-aging (e.g., promoting type I collagen, inhibiting acetylcholine to improve expression lines), anti-inflammatory (e.g., inhibiting the inflammatory factor PGE2), and / or antioxidant (inhibiting ROS) ingredient.

[0022]

[0023] The benzodiazine derivatives described herein may optionally be in finished packaging. In one embodiment, the packaging is a container, such as a plastic, metal, or glass tube or jar, containing the benzodiazine derivative. The product may also have packaging, such as a plastic or cardboard box, for storing the container. In one embodiment, the product contains the benzodiazine derivative and has instructions for the user to apply the benzodiazine derivative to the skin to treat signs of skin aging, as discussed below. Such instructions may be printed on the container, on a label insert, or on any other packaging.

[0024] As used herein, "topical application" means applying or spreading directly to the external skin, scalp, or hair, for example, using the hands or an applicator such as a wipe, roller, or sprayer.

[0025] As used herein, "cosmetic" refers to cosmetic substances or preparations that maintain, restore, impart, stimulate, or enhance the physical appearance or the appearance of increased beauty or youthfulness, especially as it relates to the appearance of tissue or skin.

[0026] As used herein, "cosmetically effective amount" means an amount of a physiologically active compound or composition sufficient to treat one or more skin conditions, but low enough to avoid serious side effects. The cosmetically effective amount of a compound or composition will vary depending on the specific condition being treated, the age and physical condition of the end user, the severity of the condition being treated / prevented, the duration of the treatment, the nature of other treatments, the specific compound or product / composition employed, the specific cosmetically acceptable carrier utilized, and the like.

[0027] In order to provide a more concise description, some quantitative expressions given herein are not modified by the term "about". It should be understood that, regardless of whether the term "about" is explicitly used, each quantity given herein is intended to refer to the actual given value and also to refer to the approximate value of these given values that can be reasonably estimated by a person of ordinary skill in the art, including the approximate value of these given values caused by experimental and / or measurement conditions.

[0028] To provide a more concise description, some quantitative expressions herein are described as a range of about X amount to about Y amount. It should be understood that when a range is described, the range is not limited to the upper and lower limits described, but should include the entire range of about X amount to about Y amount or any amount therebetween.

[0029] Benzodiazine derivatives

[0030] The present invention is based on the unexpected discovery that benzodiazine derivatives of Formula I exhibit anti-aging (e.g., promoting type I collagen production, inhibiting acetylcholine production, and improving expression lines), anti-inflammatory (e.g., inhibiting the inflammatory factor PGE2), and / or antioxidant (e.g., inhibiting ROS) effects. Therefore, benzodiazine derivatives have promising applications in cosmetics as topical skin preparations.

[0031] The present invention relates to the study of the cosmetic efficacy of benzodiazine derivatives. In a specific embodiment, the benzodiazine derivatives have the following structure:

[0032]

[0033] The compound of formula I is of R configuration and its chemical name is (7R)-ethyl-4,5,6,7-tetrahydro-9-hydroxy-2,7-methano-2H-2-benzoazoline-1(3H)-one. The molecular formula is C 15 H 19 NO2, molecular weight 245.31, white crystals, easily soluble in alcohol, acetone, etc.; optical rotation value (in MeOH) [α] D =+61.0° (c=0.35); HPLC purity assay>98.5%.

[0034] In some embodiments, the compound of formula I has anti-aging (promoting type I collagen, inhibiting acetylcholine to improve expression lines), anti-inflammatory (inhibiting inflammatory factor PGE2), and antioxidant (inhibiting ROS) effects, and can be added as an effective ingredient to skin topical preparations for use.

[0035] In some embodiments, the benzodiazine derivative is used at a concentration of at least about 3 ppm, preferably at least about 3.125 ppm. In preferred embodiments, the benzodiazine derivative is used at a concentration of less than about 100 ppm, preferably less than 50 ppm.

[0036] In some embodiments, the benzoazoline derivative is used at a concentration of about 3.125-50 ppm. In some embodiments, the benzoazoline derivative is used at a concentration of about 12.5-50 ppm.

[0037] In some embodiments, the benzoazoline derivative is used at a concentration of about 3.125-12.5 ppm. In some embodiments, the benzoazoline derivative is used at a concentration of about 3.125-6.25 ppm.

[0038] In one specific embodiment, the benzodiazepine derivative is used at a concentration of about 25 ppm.

[0039] Skin topical products

[0040] In some embodiments, the benzodiazine derivatives can be used to prepare a topical skin preparation. The topical skin preparation is preferably a cosmetic composition, including but not limited to products in the form of creams, lotions, gels, toners, essences, facial masks, eye creams, aerosols (cleansing foams), sprays, shower gels, and facial cleansers.

[0041] The term "external skin preparation" generally refers to any ingredient typically applied externally to the skin, and may include, for example, a cosmetic composition. Such cosmetic compositions may include base cosmetics, facial makeup, body makeup, hair care cosmetics, and the like. There are no particular limitations on the dosage form, and the dosage form may be selected based on the intended purpose. Depending on the dosage form and intended purpose, the cosmetic composition may also contain various cosmetically acceptable media or base excipients.

[0042] The skin topical preparation comprising a benzodiazine derivative can be topically applied to human skin and / or hair. The skin topical preparation may also comprise a cosmetically acceptable topical carrier, which may be from about 50% to about 99.99% by weight of the skin topical preparation (e.g., from about 80% to about 99% by weight of the skin topical preparation). In a preferred embodiment of the present invention, the cosmetically acceptable topical carrier comprises water. The cosmetically acceptable topical carrier may comprise one or more selected from wetting agents, emollients, greases, moisturizers, and similar substances. In one embodiment, the cosmetically acceptable topical carrier comprises a substrate such as a nonwoven fabric or a membrane material.

[0043] Skin topical agents can be prepared into a variety of product types, including but not limited to lotions, creams, gels, sticks, sprays, ointments, cleansing liquid lotions and solid soaps, shampoos and hair conditioners, hair fixatives, pastes, foams, powders, mousses, shaving creams, wipes, patches, hydrogels, film-forming products, facial masks and skin films, films and cosmetics such as foundations and mascara. These product types can contain several types of cosmetically acceptable topical carriers, including but not limited to solutions, suspensions, emulsions (e.g., microemulsions and nanoemulsions), gels, solids and liposomes.

[0044] Skin topical preparations containing benzodiazine derivatives can be formulated as solutions. Solutions typically contain an aqueous solvent or an organic solvent (e.g., about 50% to about 99.99% or about 90% to about 99% of a cosmetically acceptable aqueous solvent or organic solvent). Examples of suitable organic solvents include propylene glycol, polyethylene glycol, polypropylene glycol, glycerol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol, ethanol, and mixtures thereof.

[0045] Skin topical preparations can be formulated as solutions containing emollients. Such skin topical preparations preferably contain from about 2% to about 50% of one or more emollients. As used herein, "emollient" refers to a substance used to prevent or alleviate dryness, for example, by preventing transdermal loss of skin moisture. Examples of emollients include, but are not limited to, vegetable oils, mineral oils, aliphatic esters, and the like.

[0046] Lotions can be prepared from such solutions.Lotions typically contain from about 1% to about 20% (e.g., from about 5% to about 10%) of one or more emollients and from about 50% to about 90% (e.g., from about 60% to about 80%) water.

[0047] Another type of product that can be formulated from a solution is a cream. Creams typically contain from about 5% to about 50% (e.g., from about 10% to about 20%) of one or more emollients and from about 45% to about 85% (e.g., from about 50% to about 75%) water.

[0048] Although it is preferred that the topical skin preparation containing the benzodiazine derivative contain water, the topical skin preparation may alternatively be anhydrous or an ointment containing no water but organic and / or silicone solvents, oils, lipids, and waxes. The ointment may contain a simple base of animal or vegetable oils or semisolid hydrocarbons. The ointment may contain from about 2% to about 10% of one or more emollients and from about 0.1% to about 2% of one or more thickeners.

[0049] The topical skin preparation can be formulated as an emulsion. If the topical carrier is an emulsion, about 1% to about 10% (e.g., about 2% to about 5%) of the topical carrier contains one or more emulsifiers. The emulsifier can be nonionic, anionic, or cationic. Examples of suitable emulsifiers include those commonly identified as suitable emulsifiers in the field of personal care and cosmetic formulations.

[0050] Lotions and creams can be formulated as emulsions. Typically, such lotions contain from 0.5% to about 5% of one or more emulsifiers. Such creams typically contain from about 1% to about 20% (e.g., from about 5% to about 10%) of one or more emollients; from about 20% to about 80% (e.g., from 30% to about 70%) of water; and from about 1% to about 10% (e.g., from about 2% to about 5%) of one or more emulsifiers.

[0051] Oil-in-water and water-in-oil single emulsion skin care preparations, such as lotions and creams, are well known in the cosmetics field and can be used in the present invention. Multiphase emulsion skin external preparations (such as water-in-oil-in-water type and oil-in-water-in-oil type) can also be used in the present invention. Generally, such single-phase emulsions or multiphase emulsions contain water, an emollient, and an emulsifier as their essential ingredients.

[0052] Skin topical preparations containing benzodiazine derivatives can also be formulated into gels (e.g., aqueous gels, alcoholic gels, alcohol / water gels, or oily gels using suitable gelling agents). Suitable gelling agents for aqueous and / or alcoholic gels include, but are not limited to, natural gums, acrylic acid and acrylate polymers and copolymers, and cellulose derivatives (e.g., hydroxymethyl cellulose and hydroxypropyl cellulose). Suitable gelling agents for oils (e.g., mineral oils) include, but are not limited to, hydrogenated butylene / ethylene / styrene copolymers and hydrogenated ethylene / propylene / styrene copolymers. Such gels typically contain between about 0.1% and 5% by weight of such gelling agents.

[0053] The external preparation for skin application comprising the benzoazoline derivative can also be formulated into a solid preparation (eg, a wax-based stick, a bar soap, a powder, or a wipe containing a powder).

[0054] In addition to the above-mentioned components, the external preparations for skin that can be used in the present invention may contain various other oil-soluble substances and / or water-soluble substances at levels conventionally established in the technical field thereof for use in external preparations for skin and hair.

[0055] The skin external preparation of the present invention may contain additional components commonly found in skin care compositions, such as moisturizers, skin conditioners, emulsifiers, preservatives, antioxidants, fragrances, chelating agents, etc., as long as they are physically and chemically compatible with other components in the skin external preparation and do not affect the effects of the benzodiazepine derivatives of the present invention.

[0056] In some embodiments of the topical skin preparations of the present invention, one or more preservatives may be used. Suitable preservatives include p-hydroxyacetophenone, C1-C4 alkyl parahydroxybenzoate, and phenoxyethanol. The preservative is used in an amount of about 0.5 to about 2 weight percent, preferably about 0.5 to 1 weight percent, based on the total weight of the composition.

[0057] In one embodiment of the skin external preparation of the present invention, one or more antioxidants can be used. Suitable antioxidants include butylated hydroxytoluene (BHT), ascorbyl palmitate (BHA), butylated hydroxyanisole, phenyl-α-naphthylamine, hydroquinone, propyl gallate, nordihydroguaiaretic acid, vitamin E or a derivative of vitamin E, vitamin C and its derivatives, calcium pantothenate, green tea extract and mixed polyphenols, and mixtures of the above-mentioned substances. The antioxidant used is about 0.02 to 0.5 weight % of the total weight of the composition, and more preferably, an amount ranging from about 0.002 to 0.1 weight %.

[0058] In one embodiment of the topical skin preparation of the present invention, one or more emollients may be used. By virtue of their ability to remain on the skin surface or in the stratum corneum, they act as lubricants, thereby reducing flaking and improving the skin's appearance. Typical emollients include fatty esters, fatty alcohols, mineral oils, polyether siloxane copolymers, and the like. Suitable examples of emollients include, but are not limited to, polypropylene glycol (PPG)-15 stearyl ether, PPG-10 cetyl ether, Steareth-10, Oleth-8, PPG-4 lauryl ether, vitamin E acetate, lanolin, cetyl alcohol, cetearyl ethylhexanoate, cetostearyl alcohol, glyceryl stearate, octyl hydroxystearate, dimethicone, and combinations thereof. Cetyl alcohol, cetearyl ethylhexanoate, cetostearyl alcohol, glyceryl stearate, and combinations thereof are preferred. When used, the emollient is used in an amount ranging from about 0.1 to about 30% by weight, preferably about 1 to about 30% by weight, based on the total weight of the composition.

[0059] In one embodiment of the topical skin preparation of the present invention, one or more moisturizers may be used. Moisturizers, also known as humectants, help enhance the effectiveness of emollients, reduce flaking, stimulate the removal of scaly skin, and improve skin feel. Polyols may be used as moisturizers, including, but not limited to, glycerin, polyalkylene glycols, alkylene polyols and their derivatives, including butylene glycol, propylene glycol, dipropylene glycol, polyglycerol, polyethylene glycol and its derivatives, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1,3-dibutylene glycol, 1,2,6-hexanetriol, ethoxylated glycerin, propoxylated glycerin, and combinations thereof. When used, the moisturizer is used in an amount of about 0.1 to about 20% by weight, preferably about 1 to about 15% by weight, based on the total weight of the composition.

[0060] In an example of the skin external preparation of the present invention, one or more emulsifiers can be used. The emulsifier can be used within the scope of an effective stabilizing amount. Preferably, based on the total weight of the composition, the emulsifier is used in an amount of about 1.0 to about 10.0 wt %, more preferably about 3.0 to about 6.0 wt %. Any emulsifier compatible with the components in the composition can be used. Suitable emulsifiers include stearic acid, cetyl alcohol, glyceryl stearate, lecithin, stearyl alcohol, Steareth-2, Steareth-20, acrylic acid (esters) / C10-30 alkyl acrylate crosspolymer, and combinations thereof.

[0061] In one embodiment of the skin external preparation of the present invention, one or more pH adjusting agents may be used. A useful pH adjusting agent in the skin external preparation of the present invention includes tromethamine. When used, the pH adjusting agent is used in an amount of about 0.1 to about 2 weight %, preferably about 0.1 to about 1 weight %, based on the total weight of the composition.

[0062] In one embodiment of the present invention, the topical preparation for skin comprises acrylates / C10-30 alkyl acrylate crosspolymer, glycerin, p-hydroxyacetophenone, glyceryl stearate and lecithin, cetearyl alcohol, cetearyl ethylhexanoate, tromethamine, or a combination thereof.

[0063] Additional cosmetic active agents

[0064] In some embodiments, the topical skin preparation may further comprise an additional cosmetic active agent. As used herein, a "cosmetic active agent" is a compound (such as a synthetic compound or a compound isolated from a natural source or natural extract) that has a cosmetic or therapeutic effect on the skin or hair, including but not limited to anti-acne agents, oil control agents, antimicrobial agents, anti-inflammatory agents, antifungal agents, antiparasitic agents, topical analgesics, sunscreens, photoprotectants, antioxidants, keratolytic agents, surfactants, moisturizers, nutrients, vitamins, energy enhancers, antiperspirants, astringents, deodorants, firming agents, anti-keratin agents, and agents for hair and / or skin conditioning.

[0065] In one embodiment, these cosmetic active agents are selected from (but are not limited to): hydroxy acids, benzoyl peroxide, D-panthenol, octyl methoxycinnamate, titanium dioxide, octyl salicylate, homosalate, avobenzone, carotenoids, free radical scavengers, spin traps, amines, retinoids such as retinol and retinyl palmitate, ceramides, polyunsaturated fatty acids, essential fatty acids, enzymes, enzyme inhibitors, minerals, hormones such as estrogen, steroids such as hydrocortisone, 2-dimethylaminoethanol, copper salts such as copper chloride, copper-containing peptides such as Cu:Gly-His-Lys, coenzyme Q10, peptides, amino acids such as proline, vitamins, lactobionic acid, acetyl-CoA, niacin, riboflavin, thiamine, ribose, electron transporters such as NADH and FADH2, and other plant extracts such as aloe vera, feverfew, oatmeal, and their derivatives and mixtures. The cosmetic active agent is typically present in an amount of from about 0.001% to about 20%, for example from about 0.005% to about 10%, such as from about 0.01% to about 5%, by weight of the topical skin preparation of the present invention.

[0066] Examples of vitamins include, but are not limited to, vitamin A, vitamin B (e.g., vitamin B3, vitamin B5, and vitamin B12), vitamin C, vitamin K, and different forms of vitamin E (e.g., α, β, γ, or Δ tocopherol), or mixtures thereof, and derivatives thereof.

[0067] Examples of hydroxy acids include, but are not limited to, glycolic acid, lactic acid, malic acid, salicylic acid, citric acid, and tartaric acid.

[0068] Examples of antioxidants include, but are not limited to, water-soluble antioxidants such as sulfhydryl compounds and their derivatives (e.g., sodium metabisulfite and N-acetylcysteine), lipoic acid and dihydrolipoic acid, resveratrol, lactoferrin, and ascorbic acid and ascorbic acid derivatives (e.g., ascorbyl palmitate and ascorbic acid polypeptide). Oil-soluble antioxidants suitable for use in the topical skin preparations of the present invention include, but are not limited to, butylated hydroxytoluene, retinoids (e.g., retinol and retinyl palmitate), tocopherols (e.g., tocopherol ethyl ester), tocotrienols, and ubiquinone. Natural extracts containing antioxidants suitable for use in the topical skin preparations of the present invention include, but are not limited to, extracts containing flavonoids and isoflavones and their derivatives (e.g., genistein and di-zein), extracts containing resveratrol, and the like. Examples of such natural extracts include grape seeds, green tea, pine bark, and propolis.

[0069] Example

[0070] The present invention will be further described below with reference to specific examples. It should be noted that the examples are intended only to further illustrate the present invention and are not to be construed as limiting the scope of the present invention. Those skilled in the art may make non-essential improvements and adjustments based on the above-described disclosure of the present invention. The test methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise indicated, all percentages and parts are by weight.

[0071] The compound of formula I (HPLC purity>98.5%) was provided by Shanghai Institute of Biomedical Technology.

[0072] Example 1:

[0073] The compound of formula I was accurately weighed and prepared with DMSO solution to a concentration of 5000 ppm as the test stock solution.

[0074] Example 2:

[0075] The solution of Example 1 was diluted with culture medium to a concentration of 50 ppm as a test solution.

[0076] Example 3:

[0077] The solution of Example 1 was diluted with culture medium to a concentration of 25 ppm as a test solution.

[0078] Example 4:

[0079] The solution of Example 1 was diluted with culture medium to a concentration of 12.5 ppm as a test solution.

[0080] Example 5:

[0081] The solution of Example 1 was diluted with culture medium to a concentration of 6.25 ppm as a test solution.

[0082] Example 6:

[0083] The solution of Example 1 was diluted with culture medium to a concentration of 3.125 ppm as a test solution.

[0084] Test Example 1: Fibroblast-based Collagen I Content Detection Method

[0085] 1. Test system

[0086] 1.1 Cells

[0087] The cells used in this test were fibroblasts, batch number: Fb19052002, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0088] 1.2 Main Reagents

[0089] DMEM culture medium (Gibco), PBS (Solarbio), TGF-β1 (Peprotech), DMSO (Sigma), CollagenI ELISA kit (CUSABIO).

[0090] 1.3 Main Equipment

[0091] CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), microplate reader (BioTek, Epoch), inverted microscope (Olympus, CKX53), UVA irradiator (Philips).

[0092] 2. Test Method

[0093] 1) Cell seeding: After thawing the cells, when the plating rate reaches about 60%, seed the cells into a 6-well plate and incubate in a CO2 incubator (37°C, 5% CO2) overnight.

[0094] 2) Liquid preparation: Test groups according to Table 1.

[0095] Table 1

[0096]

[0097] 3) Dosing: According to the test grouping, when the cell plating rate in the 6-well plate reaches 30% to 50%, group dosing is carried out. 2 mL of culture medium is added to each well of the blank control group and the negative control group; 2 mL of culture medium containing 100 ng / mL TGF-β1 is added to each well of the positive control group; 2 mL of culture medium containing the corresponding concentration of sample is added to each well of the sample group. Each group has 3 replicates. After dosing, place the 6-well plate in an incubator (37°C, 5% CO2) and incubate for 24 hours.

[0098] 4) UVA irradiation: According to the test group, the UVA irradiation group was irradiated at a dose of 30J / cm 2 After irradiation, the cells were placed in an incubator (37°C, 5% CO2) and cultured for 24 h.

[0099] 5) Sample collection: After irradiation, the cell culture fluid was collected for ELISA testing.

[0100] 6) ELISA test: Perform detection and analysis according to the operating instructions of the ELISA kit.

[0101] 7) Calculation of improvement rate: Improvement rate (%) = [(sample group - negative control group) / negative control group] × 100%.

[0102] 8) Statistical Analysis: Graphs were generated using GraphPad Prism, and results are expressed as mean ± SD. Comparisons between groups were analyzed using the t-test. P < 0.05 was considered a significant difference, and P < 0.01 was considered a highly significant difference.

[0103] 3. Test results

[0104] Table 2

[0105]

[0106] Note: When the t-test method was used for statistical analysis, compared with the BC group, the significance was indicated by #, P value < 0.05 was indicated by #, and P value < 0.01 was indicated by ##; compared with the NC group, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by **.

[0107] From the experimental results, it can be seen that Example 3 exhibits a very strong effect in promoting type I collagen, with the enhancement rate being approximately 10 times greater than that of the positive control (growth factor).

[0108] Test Example 2: Immunofluorescence detection of type I collagen in replicative senescent fibroblasts

[0109] In order to further evaluate the effect of the examples in promoting collagen, tests on senescent fibroblasts were also performed.

[0110] (1) Cell information: Primary human dermal fibroblasts (FB, Boxi Fb19052002).

[0111] (2) Reagent information:

[0112] “Primary antibody”: Collagen I antibody (Abcam, cat. no. ab138492), 1:1000 dilution;

[0113] Secondary antibody: Labeled goat anti-rabbit IgG (Servicebio, catalog number: GB25303), diluted 1:800;

[0114] Buffer A: 85% PBS (Source Culture, Catalog No. B210KJ) + 5% goat serum (Biyuntian, Catalog No. C0265) + 10% Triton stock solution;

[0115] “buffer B”: 95% PBS (Source Culture, Catalog No. B210KJ) + 5% goat serum (Biyuntian, Catalog No. C0265);

[0116] Note: Triton solution (SIGMA, Cat. No.: T8787-50mL); Triton stock solution: 10% Triton solution, obtained by diluting Triton 10-fold with PBS. The stock solution can be stored at 4°C for 6 months.

[0117] (3) Fluorescence inverted microscope: LEICADMi8, magnification 10 times.

[0118] 1. Experimental grouping and drug addition:

[0119] The test set up a replicative senescent cell group and a test sample group. Among them, the replicative senescent cell group (cells with more than 50 generations) and the sample group (test samples, incubated for 72 hours).

[0120] 2. Immunofluorescence detection:

[0121] 2.1 Fixing cells

[0122] The cultured cells were fixed with 4% paraformaldehyde, and then the paraformaldehyde was aspirated and allowed to evaporate completely.

[0123] 2.2 Rehydration, sealing, and drilling

[0124] 1) Add PBS to the culture dish to rehydrate the cells that have been dehydrated and fixed with paraformaldehyde.

[0125] 2) After rehydration, add buffer A and let it stand at room temperature for 1 hour to complete the sealing and drilling work.

[0126] 2.3 Primary Antibody Treatment

[0127] Aspirate buffer A from the culture dish and immediately add buffer B containing the primary antibody. Place in a 4°C refrigerator overnight. The next day, remove the primary antibody, add PBS, let stand for 5 minutes, and then wash repeatedly with PBS several times.

[0128] 2.4 Secondary Antibody Treatment

[0129] Add Buffer B containing the corresponding secondary antibody and incubate at room temperature for 2 hours in the dark. Discard the solution and wash with PBS. Add Buffer C, let stand for 5 minutes, and then wash again with PBS.

[0130] 2.5 Sealing

[0131] 1) Aspirate the remaining liquid in the culture dish, dry it in the dark, add a drop of sealing agent, and cover with a coverslip.

[0132] 2) Place the mounting medium in a dark, dry place for 24 hours to dry in the shade, and observe the staining results under a fluorescent inverted microscope.

[0133] 3. Calculation of results:

[0134] Green fluorescence under the microscope indicates areas of Collagen I expression. Higher fluorescence intensity indicates higher Collagen I content and greater anti-aging efficacy. The IOD values of the green fluorescent areas were calculated using Image J software. The average IOD value was then calculated, and the improvement rate was calculated using the formula: Improvement rate (%) = [(average IOD value of sample group - average IOD value of replicative senescent cell group) / average IOD value of replicative senescent cell group] × 100%.

[0135] 4. Experimental results:

[0136] Table 3

[0137]

[0138] The experimental results show that Examples 2, 3, and 4 all exhibited a strong promoting effect on type I collagen, with enhanced green fluorescence. The effect became even stronger with increasing concentration, with a collagen enhancement rate of 254% achieved at 50 ppm. This indicates that the compound of Formula I, at varying concentrations, also exhibited a strong collagen-promoting effect on senescent fibroblasts.

[0139] Test Example 3: Acetylcholine content detection based on neuronal cells

[0140] 1. Test system

[0141] 1.1 Cells

[0142] The cells used in this test are neuronal cells, batch number: 210126, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0143] 1.2 Main Reagents

[0144] DMEM culture medium (Gibco), fetal bovine serum (Lanzhou Rongye), PBS (Solebol), DMSO (Sigma), and acetylcholine kit (Nanjing Jiancheng).

[0145] 1.3 Main Equipment

[0146] CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), and microplate reader (BioTek, Epoch).

[0147] 2. Test Method

[0148] 1) Cell seeding: After thawing cells, when the plating rate reaches about 60%, seed the cells into a 6-well plate and incubate in a CO2 incubator (37°C, 5% CO2) overnight.

[0149] 2) Liquid preparation: Test groups according to the table below.

[0150] Table 4

[0151]

[0152] 3) Dosing: The cells were divided into groups according to Table 4. When the cell plating rate in the 6-well plate reached 40% to 60%, the cells were dosed in groups. The dosage per well was 2 mL. Each group had 3 replicate wells. The cells were incubated in an incubator (37°C, 5% CO2) for 24 h.

[0153] 4) Detection: Collect the cell culture supernatant and perform the test according to the instructions of the detection kit.

[0154] 5) Calculation of inhibition rate: inhibition rate (%) = [(blank control group - sample group) / blank control group] × 100%.

[0155] 6) Statistical Analysis: Graphs were generated using GraphPad Prism, and results are expressed as mean ± SD. Comparisons between groups were analyzed using the t-test. All statistical analyses were two-tailed. P < 0.05 was considered a significant difference, and P < 0.01 was considered a highly significant difference.

[0156] 3. Test results

[0157] Table 5

[0158]

[0159] Note: When the t-test method was used for statistical analysis, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by ** compared with the BC group.

[0160] The experimental results showed that, based on neuronal cells, compared with the blank group BC, Example 5 at a concentration of 6.25 ppm was able to significantly inhibit acetylcholine content, with a statistically significant inhibition rate of 10.59%, suggesting its role in improving dynamic expression lines.

[0161] Test Example 4: Detection of inflammatory factors based on UVB irradiation of keratinocytes

[0162] 1. Test system

[0163] 1.1 Cells

[0164] The cells used in this test were keratinocytes, batch number: EP23081602, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0165] 1.2 Main Reagents

[0166] KcGrowth (Guangdong Boxi Biotechnology), PBS (Solaibao), dexamethasone (China Food and Drug Administration), PGE2 ELISA kit (ENZO), DMSO (Sigma).

[0167] 1.3 Main Equipment

[0168] CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), microplate reader (BioTek, Epoch), inverted microscope (Olympus, CKX53), UVB irradiator (Philips).

[0169] 2. Test Method

[0170] 2.1 Grouping

[0171] Table 6

[0172]

[0173] 2.2 Experimental steps

[0174] 1) Cell seeding: After thawing cells, when the plating rate reaches about 60%, seed the cells into a 6-well plate and incubate in a CO2 incubator (37°C, 5% CO2) overnight.

[0175] 2) Dosing: Assemble the cells according to the test grouping table above. When the cell plating rate in the 6-well plate reaches 40% to 60%, perform group dosing, with three replicates per group. Add 2 mL of culture medium to each well of the blank control group and the negative control group, 2 mL of culture medium containing dexamethasone to each well of the positive control group, and 2 mL of culture medium containing the corresponding concentration of the test sample to each well of the sample group. After dosing, place the 6-well plate in a CO2 incubator (37°C, 5% CO2) and continue incubation for 24 hours.

[0176] 3) UVB irradiation: The test groups were divided into groups according to the table. Except for the blank control group, the other groups were irradiated with UVB at a dose of 300 mJ / cm2. After irradiation, the cells were placed in a CO2 incubator (37°C, 5% CO2) and cultured for 24 hours.

[0177] 4) ELISA assay: After the incubation, the cell culture supernatant was collected and ELISA assay was performed according to the ELISA kit instructions.

[0178] 5) Calculation of inhibition rate: Inhibition rate (%) = [(negative control group - sample group) / negative control group] × 100%

[0179] 6) Statistical Analysis: Graphs were generated using GraphPad Prism, and results are expressed as mean ± SD. Comparisons between groups were analyzed using the t-test. All statistical analyses were two-tailed.

[0180] 3. Test results

[0181] Table 7

[0182]

[0183] Note: When the t-test method was used for statistical analysis, compared with the BC group, the significance was indicated by #, P value < 0.05 was indicated by #, and P value < 0.01 was indicated by ##; compared with the NC group, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by **.

[0184] The results showed that compared with the BC group, the PGE2 content in the NC group increased significantly, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the PGE2 content in the PC group decreased significantly, indicating that the positive control in this test was effective. Compared with the NC group, the PGE2 content in the Example 6 group decreased significantly, indicating that the Example has a soothing effect on the inflammatory factor PGE2, with an inhibition rate of up to 49.43%.

[0185] Test Example 5: Flow Cytometry Detection of Reactive Oxygen Species (ROS) Content

[0186] 1. Test system

[0187] 1.1 Cells

[0188] The cells used in this test were keratinocytes, batch number: 21072702, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0189] 1.2 Main Reagents

[0190] KC2500 culture medium (Boxi Biotechnology), PBS (Boster), fetal bovine serum (FBS, Lanzhou Rongye), DMSO (Sigma), vitamin E (VE, Sigma), ROS kit (Biyuntian), DCFH-DA probe (Biyuntian).

[0191] 1.3 Main Equipment

[0192] CO2 incubator (Thermo, HF151UV), clean bench (Sujing Antai, SW-CJ-1F), flow cytometer (Beckman, CytoFLEX), UVB irradiator (Boxi Biotechnology), and microplate reader (BioTek, Epoch).

[0193] 2. Test Method

[0194] 1) Cell seeding: 2.2×10 5 Keratinocytes were seeded into 6-well plates at a seeding density of 100 cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0195] 2) Liquid preparation: Test groups according to Table 8.

[0196] Table 8

[0197]

[0198] 3) Dosing: According to the test protocol in the table, when the cell plating rate in the 6-well plate reaches 40%-60%, group dosing is performed, with 2 mL of sample added to each well, and three replicates are set up for each group. After dosing, the 6-well plate is placed in an incubator (37°C, 5% CO2) and incubated for 24 hours.

[0199] 4) UVB irradiation: According to the experimental grouping, the UVB irradiation group was subjected to 300mJ / cm2 of UVB irradiation.

[0200] 5) Flow Cytometry: After irradiation, wash each well of cells three times with PBS. Add 1 mL of 10 μM DCFH-DA probe to each well and incubate in an incubator (37°C, 5% CO2) for 30 min. Discard the culture medium containing DCFH-DA, wash the cells three times with PBS, digest the cells with 0.25% trypsin, wash the cells once with PBS, add a certain amount of fresh PBS, and analyze by flow cytometry.

[0201] 7) Statistical Analysis: Graphs were generated using GraphPad Prism, and results are expressed as mean ± SD. Comparisons between groups were analyzed using the t-test. All statistical analyses were two-tailed. P < 0.05 was considered a significant difference, and P < 0.01 was considered a highly significant difference.

[0202] 3. Test results

[0203] Table 9

[0204]

[0205] Note: When the t-test method was used for statistical analysis, compared with the BC group, the significance was indicated by #, P value < 0.05 was indicated by #, and P value < 0.01 was indicated by ##; compared with the NC group, the significance was indicated by *, P value < 0.05 was indicated by **, and P value < 0.01 was indicated by **.

[0206] The results showed that compared with the BC group, the ROS level in the NC group increased significantly, indicating that the stimulation conditions in this experiment were effective. Compared with the NC group, the ROS level in the PC group decreased significantly, indicating that the positive control test was effective. Compared with the NC group, the ROS level in the Example 6 group decreased significantly, with an inhibition rate of 33.75%, indicating the antioxidant effect of the example.

[0207] The benzodiazine derivatives of the present invention can be used as functional additives in the preparation of cosmetics. The topical skin preparations are preferably cosmetic compositions, including but not limited to creams, lotions, gels, toners, essences, facial masks, eye creams, aerosols (cleansing foams), sprays, shower gels, and facial cleansers. The weight percentage of the benzodiazine derivatives in the topical skin preparations is 0.00156%-10% (w / w). The preferred weight percentage is 0.0001%-20% (w / w). The preferred weight percentage is 0.001%-10% (w / w). The more preferred weight percentage is 0.001%-5% (w / w). The most preferred weight percentage is 0.01%-5% (w / w). In some embodiments, the weight percentage of the benzodiazine derivatives in the topical skin preparations is 0.03125%-5% (w / w). The more preferred weight percentage is 0.00625%-1% (w / w). The most preferred weight percentage is 0.00625%-0.05% (w / w).

[0208] The following are specific application examples of benzodiazepine derivatives in topical skin preparations, as well as the formulations and preparation methods of these dosage forms. Specific application examples are as follows:

[0209] Application Example 1: Preparation of facial cream

[0210]

[0211] Application Example 2: Preparation of Emulsion

[0212]

[0213] Application Example 3: Preparation of Gel

[0214]

[0215]

[0216] Application Example 4: Preparation of Toner

[0217]

[0218] Application Example 5: Preparation of Essence

[0219]

[0220]

[0221] Application Example 6: Preparation of facial mask

[0222]

[0223]

[0224] Application Example 7: Preparation of Eye Cream

[0225]

[0226] Application Example 8: Preparation of Aerosol (Cleaning Bubble)

[0227]

[0228]

[0229] Application Example 9: Spray Preparation

[0230]

[0231] Application Example 10: Preparation of shower gel

[0232]

[0233]

[0234] Application Example 11: Preparation of Facial Cleanser

[0235]

[0236]

Claims

1. A cosmetic application of a benzodiazine derivative, wherein the cosmetic application is selected from the group consisting of anti-aging, anti-inflammatory and / or antioxidant effects, wherein the benzodiazine derivative has a structure of Formula I:

2. The use according to claim 1, characterized in that The purity of the benzodiazepine derivative is ≥98.5%.

3. The use according to claim 1, characterized in that The anti-aging effects are achieved by promoting type I collagen and / or inhibiting acetylcholine.

4. The use according to claim 1, wherein The anti-inflammatory effect is achieved by inhibiting the inflammatory factor PGE2.

5. The use according to claim 1, characterized in that The antioxidant effect is achieved by inhibiting ROS.

6. The use according to claim 1, wherein The benzodiazepine derivative is used at a concentration of at least 3 ppm.

7. The use according to claim 1, wherein The use concentration of the benzodiazepine derivative is below 100 ppm.

8. The use according to claim 1, wherein The benzodiazine derivative is used at a concentration of 3.125-50 ppm.

9. The use according to claim 1, wherein The use concentration of the benzodiazine derivative is 12.5-50 ppm.

10. Use of a benzodiazine derivative in the preparation of a topical skin preparation having anti-aging, anti-inflammatory and / or antioxidant effects, wherein the benzodiazine derivative has a structure of Formula I:

11. The use according to claim 10, characterized in that The skin external preparation is selected from: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser.

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