Application of composition in preparation of retinoic acid receptor gamma agonist and external skin care product containing composition

Abstract

The embodiment of the invention discloses application of a composition in preparation of a retinoic acid receptor gamma agonist and an external skin care product containing the composition. The composition comprises one or more of cyasterone, geraniin and corilagin. The embodiment of the invention further discloses application of the composition in preparation of a retinoic acid binding protein 2 agonist. The invention also discloses an application of the prepared retinoic acid receptor gamma agonist or retinoic acid binding protein 2 agonist in preparation of a photoaging skin cell protection composition.

Description

Technical Field

[0001] The embodiments of the present disclosure relate to the field of cosmetic technology, and more particularly to the use of a composition in preparing a retinoic acid receptor γ agonist, and a topical skin care product containing the composition. Background Art

[0002] Retinoic acid receptor γ (RARγ) belongs to the retinoic acid receptor (RAR) subfamily within the nuclear receptor superfamily. Together with the retinoid X receptor (RXR), it forms the core regulatory unit of the retinoic acid signaling pathway. When RXR function is normal, it regulates cellular functions crucial for skin health and aging. With aging, the expression and function of RARγ may change. For example, in aging skin, decreased RARγ activity may lead to weakened skin barrier function, accelerated collagen loss, increased inflammation, hyperpigmentation, and the risk of skin tumors.

[0003] It is known in the prior art that retinoic acid can act as an agonist of retinoic acid receptor (RARγ). The inventors attempted to provide a new agonist of retinoic acid receptor γ.

[0004] The above information disclosed in this Background section is only for enhancement of understanding of the background of the inventive concept and therefore it may contain information that does not form the prior art that is already known to a person of ordinary skill in the art. Summary of the Invention

[0005] The content of this disclosure is used to briefly introduce concepts that will be described in detail in the detailed description section below. The content of this disclosure is not intended to identify key features or essential features of the claimed technical solution, nor is it intended to limit the scope of the claimed technical solution.

[0006] Some embodiments of the present disclosure provide use of the composition in preparing a retinoic acid receptor γ agonist, and topical skin care products containing the composition, to solve one or more of the technical problems mentioned in the above background technology section.

[0007] In a first aspect, some embodiments of the present disclosure provide a use of a composition in preparing a retinoic acid receptor γ agonist, wherein the composition comprises one or more of cyasterone, geraniol, and corilagin.

[0008] In a second aspect, some embodiments of the present disclosure provide a use of a composition in preparing a retinoic acid binding protein 2 agonist, wherein the composition comprises one or more of cyasterone, geraniol, and corilagin.

[0009] In a third aspect, some embodiments of the present disclosure provide a use of an agonist in preparing a composition for protecting photoaging skin cells, wherein the agonist is a retinoic acid receptor γ agonist as described in the first aspect or a retinoic acid binding protein 2 agonist as described in the second aspect.

[0010] Optionally, the above-mentioned photoaging skin cell protection composition is specifically

[0011] A composition that inhibits UVB-induced increase in LDH content; or

[0012] A composition that inhibits UVB-induced increase in MMP-1 content; or

[0013] A composition that inhibits UVB-induced increase in MMP-9 content; or

[0014] A composition that inhibits UVB-induced decrease in GSH content; or

[0015] A composition for inhibiting the decrease in SOD enzyme content induced by UVB.

[0016] In a fourth aspect, some embodiments of the present disclosure provide a topical skin care product comprising the retinoic acid receptor γ agonist as described in the first aspect or the retinoic acid binding protein 2 agonist as described in the second aspect.

[0017] In a fifth aspect, some embodiments of the present disclosure provide a topical skin care product, wherein the topical skin care product includes one or more of: an aqueous solution, an emulsion, a cream, an ointment, a gel, a cleanser, and a facial mask.

[0018] The above embodiments of the present disclosure have the following technical effects:

[0019] The present disclosure verifies the interaction between cyasterone and CRABP2 protein and the interaction between geraniin and RARγ protein through molecular docking experiments, binding posture analysis and energy decomposition analysis.

[0020] Furthermore, in vitro cell experiments verified that cyasterone, geraniol, and corilagin could upregulate the expression of RABP2 and RARγ in photoaged keratinocytes; and also verified that cyasterone, geraniol, and corilagin could inhibit the levels of matrix metalloproteinase-1 / 9 (MMP-1 / 9) and lactate dehydrogenase (LDH) activity in photoaged keratinocytes, and increase the activities of glutathione (GSH) and superoxide dismutase (SOD). This indicates that the composition provided by the present disclosure, including cyasterone, geraniol, and / or corilagin, can promote skin repair and regeneration, maintain the barrier function of the skin, improve skin photoaging, relieve skin inflammation, and promote collagen synthesis.

[0021] The present disclosure also verifies through the CAMVA test that geraniol, corilagin and cyasterone are non-irritating and can be applied to the field of external skin preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a schematic diagram of the chemical structure of cyasterone;

[0023] Figure 2 is a schematic diagram of the chemical structure of geranin;

[0024] Figure 3 This is a schematic diagram of the chemical structure of corilagin;

[0025] Figure 4 Schematic diagram of the detection results of cellular viability absorbance values ​​of different concentrations of cyasterone;

[0026] Figure 5 Schematic diagram of the detection results of cell viability absorbance values ​​(Cellular viability) of different concentrations of geranyl;

[0027] Figure 6 Cellular viability is the absorbance value of different concentrations of corilagin;

[0028] Figure 7 Schematic diagram of the detection results of cellular viability absorbance values ​​at different concentrations of retinol;

[0029] Figure 8 Schematic diagram of the test results showing the immunofluorescence results;

[0030] Figure 9 Schematic diagram of the detection results of immunofluorescence intensity (Degree of Immunofluorescence);

[0031] Figure 10 Schematic diagram of the detection results of MMP-1 content;

[0032] Figure 11 Schematic diagram of the detection results of MMP-9 content;

[0033] Figure 12 Schematic diagram of the detection results of LDH content;

[0034] Figure 13 Schematic diagram of the detection results of GSH content;

[0035] Figure 14Schematic diagram of the detection results of SOD content;

[0036] Figure 15 Schematic diagram of the experimental results of the chicken embryo chorioallantoic membrane blood vessels. DETAILED DESCRIPTION

[0037] Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although certain embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be construed as being limited to the embodiments described herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present disclosure. It should be understood that the drawings and embodiments of the present disclosure are for illustrative purposes only and are not intended to limit the scope of protection of the present disclosure.

[0038] Unless otherwise noted, all technical and scientific terms used herein all have the implication that is commonly understood by those of ordinary skill in the art.In general, term used herein is well known and conventionally used in this area.When amount, concentration or other value or parameter are represented with the scope, preferred range or a series of upper limit preferred values ​​and lower limit preferred value limited range, it should be understood that specifically disclose all scopes formed by any pairing of any upper range limit or upper limit preferred value and any lower range limit or lower limit right suspension.And no matter whether this scope is disclosed separately, when numerical range is described in this article, unless otherwise noted, otherwise this scope should include its end value and all integers and fractions within the scope.

[0039] It should also be noted that, for ease of description, only the parts related to the invention are shown in the drawings. In the absence of conflict, the embodiments and features in the embodiments of the present disclosure may be combined with each other.

[0040] It should be noted that the modifications of "one" and "plurality" mentioned in the present disclosure are illustrative rather than restrictive, and those skilled in the art should understand that unless otherwise clearly indicated in the context, they should be understood as "one or more".

[0041] The main terms used in this disclosure are defined as follows:

[0042] The term "retinoic acid receptor γ (RARγ)" is a member of the nuclear receptor superfamily and belongs to the steroid / thyroid hormone receptor family. Under normal physiological conditions, RARγ and RXRγ form a heterodimer, bind to specific DNA sequences, regulate the expression of a series of genes, and play a key role in physiological and pathological processes such as skin development, metabolic balance, immune regulation and tumor suppression.

[0043] The term "CRABP2" stands for Cellular Retinoic Acid Binding Protein 2, also known as CRABP2 (e.g., in certain antibody products or literature). As a carrier protein for the RARγ agonist retinoic acid (RA), CRABP2 forms a complex with RA, protecting it from degradation and promoting its transport to the cell nucleus, where it regulates its binding to the retinoic acid receptor γ (RARγ), thereby protecting the skin.

[0044] The term "Cyasterone", molecular formula: C 27 H 44 O7, structure as Figure 1 As shown in , molecular weight: 492.64g / mol; the main plant source is Amaranthaceae, such as Cyathula officinalis, Cyathula capitata, etc. Figure 1 A schematic diagram of the chemical structure of cyasterone.

[0045] The term "geranin" is a natural polyphenol compound with the chemical formula: C 41 H 28 O 27 , the structure is as follows Figure 2 As shown in , the molecular weight is 952.64g / mol, and it mainly exists in medicinal plants such as Phyllanthus urinaria, Geranium, and Water Lily. Figure 2 It can be a schematic diagram of the chemical structural formula of geranyl.

[0046] The term "corilagin" has a molecular formula of C34H28O19, a molecular weight of 756.5 g / mol, and a structure such as Figure 3 As shown in . Since the structure of geranin consists of D-glucose pyranose and dehydrogenated hexacyclic biphenyl groups connected to the O-2 and O-4 positions of corilagin, corilagin is formed by the decomposition of geranin when it encounters water. Figure 3 A schematic diagram of the chemical structure of corilagin can be shown.

[0047] The term "reactive oxygen species" (ROS) refers to a class of highly reactive chemical species derived from oxygen molecules, including free radicals (such as superoxide anion O2- and hydroxyl radical ·OH) and non-free radicals (such as hydrogen peroxide H2O2). They have strong oxidizing properties and can attack biomolecules (such as DNA, proteins, and lipids), causing oxidative damage.

[0048] The term "Matrix Metalloproteinase-1 (MMP-1)" is also known as interstitial collagenase or collagenase-1. Overactive MMP-1 breaks down collagen fibers, leading to thinning and decreased skin elasticity. This breakdown of the collagen network causes the skin's supporting structure to collapse, resulting in fine lines and deep wrinkles.

[0049] The term "Matrix Metalloproteinase-9 (MMP-9)", excessive activity of MMP-9 degrades type IV collagen and laminin, resulting in loose epidermal-dermal connection and damaged skin barrier; it also cuts elastin, exacerbating skin sagging and wrinkles.

[0050] Lactate dehydrogenase (LDH) is an oxidoreductase and a key enzyme in the glycolysis pathway, catalyzing the reversible conversion of pyruvate to lactate. Active LDH can inhibit the expression of epidermal differentiation markers (such as loricrin and filaggrin), weakening the skin barrier function.

[0051] The term "glutathione (GSH)" is chemically named γ-glutamylcysteinylglycine. Glutathione can neutralize free radicals, such as reactive oxygen species (ROS), hydrogen peroxide (H2O2), and lipid peroxides. It reduces the activation of collagenases (such as MMP-1), protecting collagen and elastin fibers. It inhibits lipid peroxidation and prevents damage to the skin barrier.

[0052] The term "superoxide dismutase (SOD)" refers to a class of metalloenzymes that are widely present in organisms and can efficiently catalyze superoxide free radicals (O2 - ) into hydrogen peroxide (H2O2) and oxygen (O2), and is the core enzyme of the antioxidant defense system of organisms. It can remove superoxide free radicals (O2 - ), blocking free radical chain reactions and protecting cell membranes, proteins and DNA from oxidative damage.

[0053] In a first aspect, the present invention provides a use of a composition in preparing a retinoic acid receptor γ agonist, wherein the composition comprises one or more of cyasterone, geraniol, and corilagin.

[0054] In a second aspect, the present invention provides a use of a composition in preparing a retinoic acid binding protein 2 agonist, wherein the composition comprises one or more of cyasterone, geraniol, and corilagin.

[0055] In a third aspect, the present invention provides a use of an agonist in preparing a composition for protecting photoaged skin cells, wherein the agonist is the retinoic acid receptor γ agonist as described in the first aspect or the retinoic acid binding protein 2 agonist as described in the second aspect.

[0056] In some embodiments, the above-mentioned photoaging skin cell protection composition is specifically

[0057] A composition that inhibits UVB-induced increase in LDH content; or

[0058] A composition that inhibits UVB-induced increase in MMP-1 content; or

[0059] A composition that inhibits UVB-induced increase in MMP-9 content; or

[0060] A composition that inhibits UVB-induced decrease in GSH content; or

[0061] A composition for inhibiting the decrease in SOD enzyme content induced by UVB.

[0062] Therefore, it can be seen that the prepared photoaging skin cell protection composition has the effects of alleviating oxidative stress, protecting cell structure, protecting collagen, resisting photoaging, and maintaining the barrier function of the skin.

[0063] In a fourth aspect, the present invention provides a topical skin care product comprising the retinoic acid receptor γ agonist described in the first aspect or the retinoic acid binding protein 2 agonist described in the second aspect.

[0064] In a fifth aspect, some embodiments of the present disclosure provide a topical skin care product, wherein the topical skin care product includes one or more of: an aqueous solution, an emulsion, a cream, an ointment, a gel, a cleanser, and a facial mask.

[0065] Among them, external skin care products may refer to care products that act on the skin surface by applying, covering, etc.

[0066] Optionally, the topical skin care product includes one or more of: lotions, emulsions, creams, ointments, gels, facial cleansers, and facial masks. In practice, lotions may include one or more of: skin care lotions, toners, and serums. Emulsions may include lotions. Creams may include facial creams. Facial creams may be transparent or opaque. Ointments may include skin care creams. Facial cleansers may include one or more of: facial cleansing foams, facial cleansing waters, and facial cleansing liquids. When the topical skin care product is a facial mask, a base material commonly used in the art and the skin care composition of the present disclosure applied or impregnated onto the base material may be used. Base materials include, but are not limited to, silk facial mask paper, cotton facial mask paper, polyester facial mask paper, or blended facial mask paper. The choice of base material can be determined by those skilled in the art based on actual needs. The aforementioned facial masks may take the form of, but are not limited to, jelly masks, cream masks, and essential oil masks.

[0067] The present disclosure will be described in detail below with reference to embodiments.

[0068] [Experiment 1: In vitro cell experiment]

[0069] Further in vitro cell experiments can verify the regulatory effects of amaranthone and geraniol on CRABP2 and RARγ proteins, respectively.

[0070] Since the structure of geraniol is composed of D-glucose pyranose and dehydrogenated hexacyclic biphenyl groups connected to the O-2 and O-4 positions of corilagin, and corilagin is formed by the decomposition of geraniol in water, and its stability is similar to that of geraniol in molecular docking and surface plasmon resonance experiments, we also explored the regulatory effect of corilagin on RARγ protein.

[0071] 1.1 Drug concentration detection in cell validation experiments

[0072] Cell viability can be detected using methods well known in the art, such as the MTT assay (also known as the MTT colorimetric assay, a method for detecting cell survival and growth). The MTT assay can be performed according to the experimental methods described in "Molecular Biology Experimental Techniques," "Cell Experiment Guide," "Cell Biology Experimental Course," or "Biochemistry and Molecular Biology Experiments."

[0073] Experimental group information:

[0074] Blank control group: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin.

[0075] Sample 1-1: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including 0.1 μM cyasterone.

[0076] Sample 1-2: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including 1 μM cyasterone.

[0077] Sample 1-3: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including 10 μM cyasterone.

[0078] Samples 1-4: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including 100 μM cyasterone.

[0079] Sample 2-1: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including geranylgeraniol at a concentration of 0.12 μM.

[0080] Sample 2-2: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including geranylgeraniol at a concentration of 1.2 μM.

[0081] Sample 2-3: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including geranin at a concentration of 12 μM.

[0082] Sample 2-4: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including geranin at a concentration of 120 μM.

[0083] Sample 3-1: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including corilagin at a concentration of 0.12 μM.

[0084] Sample 3-2: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including corilagin at a concentration of 1.2 μM.

[0085] Sample 3-3: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including corilagin at a concentration of 12 μM.

[0086] Sample 3-4: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including corilagin at a concentration of 120 μM.

[0087] Sample 4-1: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including retinol at a concentration of 0.1 μM.

[0088] Sample 4-2: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including retinol at a concentration of 1 μM.

[0089] Sample 4-3: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including retinol at a concentration of 10 μM.

[0090] Sample 4-4: DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin, including retinol at a concentration of 100 μM.

[0091] like Figure 4 As shown, compared with the blank control group, increasing the concentration of cyasterone at 1 μM had neither negative nor positive effects on cell viability, so 1 μM was selected to further verify the ability of cyasterone to regulate CRBP2.

[0092] like Figure 5 As shown, compared with the blank control group, it can be seen that when the concentration of geranyl is 12 μM, it has neither negative effect nor promoting effect on cell viability. 12 μM geranyl was selected to verify the regulatory effect on RARγ.

[0093] like Figure 6 As shown, compared with the blank control group, it can be seen that when the concentration of geranyl is 12 μM, it has neither negative effect nor promoting effect on cell viability. 12 μM of corilagin was selected to verify the regulatory effect on RARγ.

[0094] like Figure 7 As shown, compared with the blank control group, it can be seen that when the concentration of retinol is 10 μM, it has neither negative effect nor promoting effect on cell viability. The regulatory effect of 10 μM retinol on RARγ was selected as a positive control.

[0095] 1.2 Establishment of UVB radiation-induced photoaging model of HACAT cells

[0096] UVB radiation of cells will lead to a decrease in the expression of CRABP2 and RARγ proteins. Based on this, a UVB radiation-induced photoaging model of HACAT cells was established. Based on this photoaging model, the inhibition of drugs on the UVB radiation-induced decrease in CRABP2 and RARγ protein expression can be explored, and the role of drugs in promoting the expression of CRABP2 and RARγ proteins and inhibiting photoaging damage can be verified.

[0097] The UVB radiation-induced HACAT cell photoaging model specifically includes the following steps:

[0098] Step 1: Cell seeding:

[0099] Human immortalized keratinocytes (HaCaT) were obtained. 4 Inoculate the cells into a 96-well plate at a seeding density of 100 μL / well, and add 200 μL complete medium (complete medium is DMEM medium containing 10% FBS and 1% penicillin / streptomycin) to each well. Incubate in an incubator (the incubator contains 5% CO 2 , temperature is 37°C) and incubated for 24 h.

[0100] Step 2: Drug administration:

[0101] When the cell plating rate in the 96-well plate reaches 70% to 80%, the cells are divided into a blank control group, a UVB control group and at least one experimental group. 2 The cells were irradiated with a UVB dose of 100 μg / mL. The culture medium of the blank control group, UVB control group, and each experimental group was discarded, and the corresponding treatment substances were added to each group for drug treatment. After the drug treatment, the cells were placed in an incubator (37°C, 5% CO2) and cultured for 24 h.

[0102] 1.3 Verification of the inhibition of reduced CRABP2 and RARγ protein expression:

[0103] This experimental example uses immunofluorescence staining experiments to verify that cyasterone, geraniol, and corilagin upregulate the expression of CRABP2 and RARγ proteins in photoaged skin cells.

[0104] The specific steps include:

[0105] In the first step, the cell slides were placed in a 24-well plate, and the above steps were used to establish a UVB radiation-induced HACAT cell photoaging model, including a blank control group, a UVB control group, and 7 experimental groups.

[0106] In the second step, the culture medium was discarded and the cells were gently washed three times with PBS.

[0107] The third step was to fix the cells with 4% paraformaldehyde at room temperature for 10 minutes.

[0108] Step 4: Wash three times with PBS, 3 minutes each time.

[0109] In the fifth step, the cells were treated with 0.5% Triton X-100 for 20 minutes to permeabilize them, and then washed three times with PBS for 3 minutes each time.

[0110] Step 6: Block the cells with 2% BSA at room temperature for 30 minutes.

[0111] In step 7, after removing the blocking solution, add the primary antibody and incubate overnight at 4°C. The primary antibody can be a CRABP2 antagonist and / or a RARγ antagonist, the CRABP2 antagonist is CRABP2 Antibody (YA1652), and the RARγ antagonist is BMS453 (BMS-189453).

[0112] Step 8. After incubation, remove the primary antibody and wash the cells three times with PBS for 5 minutes each time.

[0113] In step 9, 200 μL of fluorescently labeled secondary antibody was added to each well, and the samples were incubated at room temperature in the dark for 1 hour. The secondary antibody can be SITS (4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonicacid disodium).

[0114] Step 10. After incubation with the secondary antibody, remove the mixture and wash the cells three times with PBS for 5 minutes each time.

[0115] Step 11: Add Hoechst 33342 staining solution to stain the cell nuclei for 10 minutes. After washing twice with PBS, observe and photograph under a fluorescence microscope.

[0116] The drug concentrations and corresponding primary antibodies in the blank control group, blank control group and 12 experimental groups during drug administration are as follows:

[0117] Blank control group [Control]: No drugs were added, and the primary antibodies were 200 μL CRABP2 and 200 μL RARγ antagonist.

[0118] UVB control group [UAmodel]: no drugs were added, and the primary antibodies were 200 μL CRABP2 and 200 μL RARγ antagonist.

[0119] Experimental group 1 [Retinol (binded with CRABP2), CRABP2 positive control I]: the drug was 10 μM retinol, and the primary antibody was 200 μL CRABP2 antagonist.

[0120] Experimental group 2 [Retinol (binded with RARγ), RARγ positive control I]: the drug was 10 μM retinol, and the primary antibody was 200 μL RARγ antagonist.

[0121] Experimental group 3 [Retinol+CRABP2 inhibitor, CRABP2 positive control II]: The drugs were 10 μM retinol and 20 nM CRABP2 inhibitor (CRABP2 Human Pre-designed siRNA Set A), and the primary antibody was 200 μL CRABP2 antagonist.

[0122] Experimental group 4 [Retinol + RARγ inhibitor, RARγ positive control II]: 10 μM retinol and 1 μM RARγ inhibitor (RARγ1 (D3A4) Rabbit mAb), and the primary antibody was 200 μL of RARγ antagonist.

[0123] Experimental group 5 [Cyasterone]: the drug was 1 μM cyasterone, and the primary antibody was 200 μL CRABP2 antagonist.

[0124] Experimental group 6 [Cyasterone+CRABP2 inhibitor]: The drugs were 1 μM cyasterone and 20 nM CRABP2 inhibitor, and the primary antibody was 200 μL CRABP2 antagonist.

[0125] Experimental group 7 [Geraniine]: the drug was 12 μM geraniine, and the primary antibody was 200 μL RARγ antagonist.

[0126] Experimental group 8 [Geraniin+RARγinhibitor]: the drugs were 12 μM geraniin and 1 μM RARγ inhibitor, and the primary antibody was 200 μL RARγ antagonist.

[0127] Experimental group 9 [Corilagin]: the drug was 12 μM corilagin, and the primary antibody was 200 μL RARγ antagonist.

[0128] Experimental group 10 [Corilagin+RARγinhibitor]: the drugs were 12 μM corilagin and 1 μM RARγ inhibitor, and the primary antibody was 200 μM RARγ antagonist.

[0129] Experimental group 11 [Corilagin+CRABP2 inhibitor]: the drugs were 12 μM corilagin and 20 nM CRABP2 inhibitor, and the primary antibody was 200 μL CRABP2 antagonist.

[0130] Experimental group 12 [Geraniin+CRABP2 inhibitor]: the drugs were 12 μM geraniin and 20 nM CRABP2 inhibitor, and the primary antibody was 200 μL CRABP2 antagonist.

[0131] The results are as follows Figure 8-9 As shown, the immunofluorescence results (Degree of Immunofluorescence) showed that compared with the blank control group and the UVB control group, the green fluorescence intensity of the cyasterone, geranyl, corilagin and retinol groups increased, and the green fluorescence intensity of the cyasterone, geranyl and corilagin groups was downregulated by the inhibitors of CRABP2 and RARγ proteins, indicating that cyasterone, geranyl and corilagin can upregulate the expression of CRABP2 and RARγ proteins in photoaged skin cells, proving that cyasterone, geranyl and corilagin can serve as expression agonists of CRABP2 and RARγ proteins.

[0132] [Experiment 2: Verifying the protective effects of cyasterone, geraniol, and corilagin on photoaged skin cells]

[0133] 2.1 Detection of LDH and MMP-1 Content

[0134] The above steps were used to establish a UVB radiation-induced photoaging model of HACAT cells. 200 μL of culture supernatant was collected from each well and transferred to a 1.5 mL sterile centrifuge tube for measuring LDH and MMP-1 levels.

[0135] 2.2 Protein detection reagents

[0136] In the first step, the above steps were used to establish a UVB radiation-induced photoaging model of HACAT cells, and the cells were washed once with PBS.

[0137] In the second step, 150 μL of RIPA lysis buffer was added to each well and the cells were lysed at 4°C for 15 minutes.

[0138] In the third step, 500 μL PBS was then added and the cells were washed repeatedly for 2 minutes.

[0139] Step 4: Transfer the lysate to a 1.5 mL centrifuge tube and centrifuge at 1000 rpm for 3 minutes.

[0140] Step 5: Collect the supernatant and determine the protein concentration using a BCA protein assay kit.

[0141] Step 6: According to the instructions of the corresponding detection kit, the supernatant was used to measure the MMP-9 content, GSH content and SOD enzyme content.

[0142] Depend on Figure 10-14As shown in the results, after UVB irradiation, the levels of matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-9 (MMP-9) and lactate dehydrogenase (L-LDH) activity in HACAT cells were significantly increased, while the level of glutathione (GSH) and superoxide dismutase (SOD) activity were significantly decreased after UVB irradiation.

[0143] Depend on Figure 10-14 As shown in the results, compared with the UVB group, the MMP-1 content, MMP-9 content and L-LDH activity of the experimental group added with cyasterone, geraniol and corilagin were significantly decreased, and the GSH content and SOD activity were significantly increased (P<0.05), which proved that the addition of cyasterone, geraniol and corilagin can inhibit UVB radiation-induced photoaging of HACAT cells.

[0144] This study also included experimental groups that simultaneously administered the drug and either an RARγ inhibitor or a CRABP2 inhibitor. A comparison of experimental groups 5 and 6 revealed that the anti-photoaging effect of experimental group 6, which received both cyasterone and a CRABP2 inhibitor, was significantly reduced compared to experimental group 5, which received only cyasterone. A comparison of experimental groups 7 and 8 revealed that the anti-photoaging effect of experimental group 8, which received both geraniol and a RARγ inhibitor, was significantly reduced compared to experimental group 7, which received only cyasterone. A comparison of experimental groups 9 and 10 revealed that the anti-photoaging effect of experimental group 8, which received both corilagin and a RARγ inhibitor, was significantly reduced compared to experimental group 9, which received only corilagin. This further demonstrates that cyasterone, geraniol, and corilagin can act as agonists for the expression of CRABP2 and RARγ proteins.

[0145] [Experiment 5: Chicken embryo chorioallantoic membrane vascular experiment]

[0146] The experiment was conducted according to the standard “SN / T 2329-2009 Chicken chorioallantoic membrane test for eye irritation / corrosion of cosmetics”.

[0147] Regarding the skin irritation of cyasterone, geraniol and corilagin, Figure 15 As shown in the chicken embryo chorioallantoic membrane vascular test (CAMVA), cyasterone at a concentration of 1 μM, geraniol and corilagin at a concentration of 12 μM, and retinol at a concentration of 10 μM did not irritate the chicken embryo chorioallantoic membrane, indicating that they are also safe for human skin. Figure 15 It can be a schematic diagram of the experimental results of the chicken embryo chorioallantoic membrane blood vessels.

[0148] The above-mentioned embodiments of the present disclosure have the following technical effects: the composition for preparing a retinoic acid receptor γ agonist disclosed in the present disclosure, wherein the composition of cyasterone, geraniol and corilagin has the effect of inhibiting photoaging of the skin and promoting collagen synthesis.

[0149] The above description is only an illustration of some preferred embodiments of the present disclosure and the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the embodiments of the present disclosure is not limited to the technical solutions formed by the specific combination of the above-mentioned technical features, but should also cover other technical solutions formed by any combination of the above-mentioned technical features or their equivalent features without departing from the above-mentioned inventive concept. For example, the above-mentioned features are replaced with (but not limited to) technical features with similar functions disclosed in the embodiments of the present disclosure.

Claims

1. Use of a composition in the preparation of a retinoic acid receptor γ agonist, wherein the composition comprises one or more of cyasterone, geraniol, and corilagin.

2. Use of a composition in the preparation of a retinoic acid binding protein 2 agonist, wherein the composition comprises one or more of cyasterone, geraniol, and corilagin.

3. Use of an agonist in preparing a composition for protecting photoaged skin cells, wherein the agonist is the retinoic acid receptor γ agonist as claimed in claim 1 or the retinoic acid binding protein 2 agonist as claimed in claim 2.

4. The use according to claim 3, wherein the photoaging skin cell protection composition is specifically A composition that inhibits UVB-induced increase in LDH content; or A composition that inhibits UVB-induced increase in MMP-1 content; or A composition that inhibits UVB-induced increase in MMP-9 content; or A composition that inhibits UVB-induced decrease in GSH content; or A composition for inhibiting the decrease in SOD enzyme content induced by UVB.

5. A skin care product for external use, comprising the retinoic acid receptor gamma agonist as claimed in claim 1 or the retinoic acid binding protein 2 agonist as claimed in claim 2.

6. The external skin care product according to claim 5, wherein The external skin care products include: one or more of: water solution, emulsion, cream, ointment, gel, facial cleanser, and facial mask.

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