Composition containing teprenone, skin care product and application

Abstract

The embodiment of the invention discloses a teprenone-containing composition, a skin care product and application. The teprenone-containing composition and the skin care product can promote the expression of CD44. The composition containing the teprenone is prepared from the teprenone and acetyl tripeptide-30 citrulline, and the mass ratio of the teprenone to the acetyl tripeptide-30 citrulline is 50 to (15 to 25).

Description

Technical Field

[0001] The present invention relates to the technical field of compositions, and in particular to a composition containing teprenone, a skin care product and uses thereof. Background Art

[0002] Phagocytic glycoprotein-1 (Pgp-1), also known as CD44. As a cell surface glycoprotein, CD44 participates in biological processes such as cell proliferation, differentiation, migration and angiogenesis, and plays a key role in cell signal transduction and tissue homeostasis regulation. CD44 participates in cell adhesion and migration by binding to matrix components such as hyaluronic acid.

[0003] Teprenone, also known as pentaerythritol, is a chemical; its chemical name is 6,10,14,18-tetramethyl-5,9,13,17-nonadecatetraen-2-one, and its molecular formula is C 23 H 38 O, molecular weight is 330.547. The structural formula of teprenone is shown in the following formula (1):

[0004] Formula (1):

[0005]

[0006] Teprenone has previously been commonly used as a gastric mucosal protective drug, and is mainly used clinically to treat gastric ulcers and acute and chronic gastritis.

[0007] In recent years, researchers have also discovered that teprenone can upregulate telomere maintenance genes and promote the repair of cellular DNA, and can be used as an anti-aging agent in topical skin preparations.

[0008] Acetyl Tripeptide-30Citruline is a synthetic peptide that is often used as an anti-wrinkle agent and anti-aging agent in skin topical preparations.

[0009] However, no research has been found on the effect of the combination of teprenone and acetyl tripeptide-30 citrulline on CD44. Summary of the invention

[0010] One purpose of the embodiments of the present application is to solve at least one problem in the above background and provide corresponding beneficial effects.

[0011] Another object of the embodiments of the present application is to provide a composition, a skin care product and a use containing teprenone, which can be used to promote the expression of CD44.

[0012] The embodiments of the present application mainly achieve the above objectives through the following technical solutions.

[0013] In a first aspect, the present invention provides a composition containing teprenone, comprising:

[0014] Teprenone and acetyl tripeptide-30 citrulline,

[0015] The mass ratio of the teprenone to the acetyl tripeptide-30 citrulline is 50:15-25.

[0016] In some technical solutions, the composition containing teprenone further includes a solvent.

[0017] In some technical solutions, the sum of the mass concentrations of teprenone and acetyl tripeptide-30 citrulline in the composition may be 0.0001 to 10% of the total weight of the composition, and further, may be 0.001 to 5%.

[0018] In a second aspect, the present application provides a skin care product, comprising:

[0019] Teprenone and acetyl tripeptide-30 citrulline,

[0020] The mass ratio of the teprenone to the acetyl tripeptide-30 citrulline is 50:15-25.

[0021] In some technical solutions, the sum of the mass concentrations of teprenone and acetyl tripeptide-30 citrulline in the skin care product may be 0.0001 to 10% of the total weight of the skin care product, and further, may be 0.001 to 5%.

[0022] In some technical solutions, the skin care product further includes a solvent.

[0023] In another aspect, the embodiments of the present application provide a use of the composition containing teprenone according to the first aspect in preparing a CD44 expression promoter.

[0024] In another aspect, an embodiment of the present application provides a use of the composition containing teprenone according to the first aspect in the preparation of a hyaluronic acid promoter.

[0025] In another aspect, the embodiments of the present application provide a use of the composition containing teprenone according to the first aspect in preparing a skin care product.

[0026] In another aspect, an embodiment of the present application provides a use of teprenone and acetyl tripeptide-30 citrulline in the preparation of a CD44 expression promoter, a hyaluronic acid promoter or a skin care product; wherein the mass ratio of the teprenone to the acetyl tripeptide-30 citrulline is 50:15-25.

[0027] The beneficial effects of the embodiments of the present invention include:

[0028] 1. In some embodiments, the composition containing teprenone of the embodiments of the present application specifically combines acetyl tripeptide-30 citrulline with teprenone to produce a synergistic effect, thereby increasing the gene transcription level of CD44 protein and promoting the expression of CD44 protein.

[0029] 2. Hyaluronic acid is widely used in the fields of medicine and cosmetics. It can not only be applied topically to the surface of the skin to moisturize and keep it moisturized, but also can be taken orally to increase the content of hyaluronic acid in the body, which has a positive effect on the health of the skin and other tissues. In addition, hyaluronic acid is also used in medical fields such as ophthalmology and orthopedics. It is understandable that, in some embodiments, the composition containing teprenone in the embodiment of the present application can promote the generation of hyaluronic acid because it can promote the expression of CD44 protein.

[0030] 3. The epidermis is one of the largest organs in the human body. It not only covers and protects our body, but also performs a variety of important physiological functions such as protection, barrier function, temperature regulation, moisturizing, lubrication and immune function, and hyaluronic acid plays a role in protecting the skin. Therefore, it can also be understood that in some embodiments, the composition containing teprenone in the embodiments of the present application can protect the skin by promoting the production of hyaluronic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A comparative schematic diagram of the relative expression levels of CD44 mRNA in the blank control group, the drug administration experimental group 4, the drug administration experimental group 1, and the drug administration experimental group 5 in Experimental Example 1 of the present application;

[0032] Figure 2 A comparative schematic diagram of the relative expression levels of CD44 mRNA in the blank control group, the drug administration experimental group 4, the drug administration experimental group 2, and the drug administration experimental group 6 in Experimental Example 1 of the present application;

[0033] Figure 3 This is a comparative schematic diagram of the relative expression levels of CD44 mRNA in the blank control group, the drug-administered experimental group 4, the drug-administered experimental group 3, and the drug-administered experimental group 7 in Experimental Example 1 of the present application. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0035] The terms "first", "second", etc. in the embodiments of the present application are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first", "second", etc. may explicitly or implicitly include at least one of the features.

[0036] Furthermore, the terms "comprises", "comprising", "including" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to the process, method, product or apparatus.

[0037] In addition to the above, it is still necessary to emphasize that the reference to "embodiment" in this article means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0038] The examples of the present application found that teprenone and acetyl tripeptide-30 citrulline have a synergistic effect in promoting CD44 protein expression, and can be used to prepare CD44 expression promoters, hyaluronic acid promoters or skin care products. Based on this, the compositions and uses described in the following examples are further provided.

[0039] <Composition containing teprenone>

[0040] In a first aspect, the present application provides a composition containing teprenone, comprising: teprenone and acetyl tripeptide-30 citrulline,

[0041] The mass ratio of teprenone to acetyl tripeptide-30 citrulline is 50:15-25.

[0042] The composition containing teprenone in the embodiment of the present application specifically combines acetyl tripeptide-30 citrulline and teprenone to produce a synergistic effect, thereby increasing the gene transcription level of CD44 protein and promoting the expression of CD44 protein.

[0043] Hyaluronic acid is widely used in the fields of medicine and cosmetics. It can not only be applied topically to the surface of the skin to moisturize and retain moisture, but can also be taken orally to increase the content of hyaluronic acid in the body, which has a positive impact on the health of the skin and other tissues. In addition, hyaluronic acid is also used in medical fields such as ophthalmology and orthopedics. It is understandable that the composition containing teprenone in the embodiment of the present application can promote the generation of hyaluronic acid because it can promote the expression of CD44 protein.

[0044] The skin epidermis is one of the largest organs in the human body. It not only covers and protects our body, but also performs a variety of important physiological functions such as protection, barrier function, temperature regulation, moisturizing, lubrication and immune function, and hyaluronic acid plays a role in protecting the skin. Therefore, it can also be understood that the composition containing teprenone in the embodiment of the present application can protect the skin by promoting the production of hyaluronic acid.

[0045] In some embodiments, the skin cells are human immortalized keratinocytes (HaCaT), that is, the composition containing teprenone can increase the expression of CD44 protein in HaCaT cells.

[0046] In some embodiments, the mass ratio of teprenone to acetyl tripeptide-30 citrulline is 50:15.

[0047] In some embodiments, the mass ratio of teprenone to acetyl tripeptide-30 citrulline is 50:20.

[0048] In some embodiments, the mass ratio of teprenone to acetyl tripeptide-30 citrulline is 50:25.

[0049] In some embodiments, the sum of the mass concentrations of teprenone and acetyl tripeptide-30 citrulline in the teprenone-containing composition may be 0.0001 to 10% of the total weight of the composition, and further, may be 0.001 to 5%.

[0050] In some embodiments, the composition containing teprenone further comprises: a solvent. Furthermore, the solvent is a solvent acceptable to cosmetics.

[0051] In some embodiments, the solvent can be selected from one or more of ethanol, isopropanol, glycerol, octanediol, 1,2-pentanediol, propylene glycol, butylene glycol, glycol, glycol ether, ethylene glycol, propylene glycol, butylene glycol, hexylene glycol, dipropylene glycol, diethylene glycol, 2-ethoxyethanol, diethylene glycol monomethyl ether, triethylene glycol monomethyl ether, and sorbitol.

[0052] In certain embodiments, the geranylgeranylacetone-containing composition may comprise at least one cosmetically acceptable adjuvant. The adjuvant is selected from one or more of surfactants and / or emulsifiers, preservatives, buffers, chelating agents, denaturing agents, sunscreen agents, pH regulators, reducing agents and stabilizers, thickening agents, gelling agents, film-forming polymers, fillers, matting agents, brightening agents, pigments, dyes, fragrances, and mixtures thereof.

[0053] In certain embodiments, the geranylgeranylacetone-containing composition may comprise at least one cosmetically acceptable active ingredient, which is selected from one or more of caffeine, caffeic acid, cyclic peptides, vitamin A and its derivatives, retinol, vitamin C and its derivatives, vitamin E and its derivatives, arbutin, cinnamic acid and its derivatives, ferulic acid, ergothioneine, ectoine, glabridin, bisabolol, ceramides, phenylethyl resorcinol, soluble collagen, adenosine, 4-butylresorcinol, hydrolyzed collagen, inositol, carbohydrate isomers, fibronectin, astaxanthin, asiaticoside, rhamnose, resveratrol, amino acids, hydroxypropyltetrahydropyrantriol, fermentates, sodium hyaluronate, niacinamide, panthenol, plant extracts.

[0054] It is understood that the geranylgeranylacetone-containing composition can be used as a skin care product or further used for preparing a skin care product.

[0055] Optionally, the skin care product is a topical skin preparation.

[0056] In certain embodiments, the skin care product can be manufactured in the form of creams, liquids, powders, blocks, mud-like solids, wax-based, freeze-dried, sprays, aerosols, substrates, etc.

[0057] Optionally, the cream form is further in the form of milk, paste, cream, honey, fat, milk, lotion, milk, milk lotion, etc.

[0058] Optionally, the liquid form is further in the form of lotion, liquid, water, oil, oil-water separation, etc.

[0059] Optionally, the cream form is further in the form of milk, paste, cream, honey, fat, milk, lotion, milk, milk lotion, etc.

[0060] Optionally, the powder form can be further in the form of gels such as gels, gums, loose powders, granules, etc.

[0061] Optionally, the block form can be further in the form of block powder, large solid blocks, etc.

[0062] Optionally, the wax-based is mainly based on wax.

[0063] Optionally, the freeze-dried form is further in the form of freeze-dried powder, freeze-dried tablets, etc.

[0064] Optionally, the spray contains no propellant.

[0065] Optionally, the aerosol contains a propellant.

[0066] Optionally, the substrate is a substrate such as a patch or a film for use with cosmetics.

[0067] In certain embodiments, the skin treatments can be topically applied to the hair, body hair, trunk, head, face, eyes, lips, hands, feet, general skin, fingernails, nails, and the like.

[0068] <Application>

[0069] According to the aforementioned composition containing teprenone, the present application embodiment may also provide a use of the composition containing teprenone in preparing a CD44 expression promoter;

[0070] The mass ratio of teprenone to acetyl tripeptide-30 citrulline is 50:15-25.

[0071] It is understandable that the composition containing teprenone can promote the expression of CD44 and the production of hyaluronic acid, thereby achieving the effect of protecting the skin.

[0072] More specifically, the CD44 expression promoter is a CD44 expression promoter of skin cells.

[0073] Furthermore, the skin cells are human immortalized keratinocytes (human keratinocyte line, HaCaT).

[0074] It can be understood that the composition containing teprenone can promote the production of hyaluronic acid by promoting the expression of CD44; therefore, the embodiment of the present application can also provide a use of the composition containing teprenone in the preparation of a hyaluronic acid promoter.

[0075] As can be seen from the foregoing, acetyl tripeptide-30 citrulline and teprenone can play a synergistic role in skin care. Therefore, in some embodiments, the present application can also provide a use of a composition containing teprenone in the preparation of a skin care product.

[0076] Optionally, the skin care product is a topical skin preparation.

[0077] Optionally, when teprenone and acetyl tripeptide-30 citrulline are used to prepare a skin care product, the sum of the mass concentrations of teprenone and acetyl tripeptide-30 citrulline in the skin care product may be 0.0001 to 10% of the total weight of the skin care product, and further, may be 0.001 to 5%.

[0078] <Experiment>

[0079] Experimental cells

[0080] Keratinocytes HaCaT.

[0081] Experimental reagents

[0082] Acetyl Tripeptide-30 Citrulline (GenScript Biotech Ltd.).

[0083] The molecular weight of acetyl tripeptide-30 citrulline is 647.7 g / mol, 1 μM = 1 umol / L.

[0084] Teprenone (Pure Biopharmaceutical Technology Co., Ltd.).

[0085] The molecular weight of teprenone is 330.547 g / mol.

[0086] DMEM (dulbecco's modified eagle medium) culture medium is a basic culture medium purchased from Gibco Biological Culture Medium of Thermo Fisher Scientific (China) Co., Ltd., and the product number is C11995500BT.

[0087] Fetal bovine serum (FBS) was purchased from Gibco brand fetal bovine serum of Thermo Fisher Scientific (China) Co., Ltd., product number 10091148.

[0088] Penicillin / streptomycin, Gibco brand penicillin / streptomycin purchased from Thermo Fisher Scientific (China) Co., Ltd., product number 25200114, penicillin / streptomycin solution (10,000 U / mL), which includes penicillin and streptomycin, with a concentration of 10,000 U / mL respectively.

[0089] PBS (Phosphate Buffered Saline), pH 7.4, is a balanced salt solution useful in a variety of cell culture applications such as washing cells prior to dissociation, transporting cells or tissues, diluting cells for counting, and preparing reagents.

[0090] RNA extraction kit, specifically ER501-01-V2 TransZol Up Plus RNA Kit (full gold, item number RE501-01-V2), is suitable for extracting total RNA from cells and tissues. The sample is lysed with TransZol Up, and after adding RNA Extraction Agent, the solution is divided into a colorless aqueous phase and a pink organic phase, and RNA is in the aqueous phase; the RNA in the aqueous phase is specifically adsorbed by a silica gel membrane centrifugal column. The RNA extraction kit specifically includes the following components: TransZol Up 100mL, RNA Extraction Agent 20mL, Clean Buffer 9 (CB9) 110mL, Wash Buffer 9 (WB9) 24mL, RNase-free Water 40mL, RNase-free Tube (1.5mL) 100each, RNA Spin Columns with Collection Tubes 100each.

[0091] Using the reverse transcription kit of M-MLV reverse transcriptase, the Evo M-MLV reverse transcription premix kit (including gDNA removal reagent, for qPCR) Ver.2 (Agbio, AG11728) was selected, which contains 5X Evo M-MLV RT Reaction Mix, and the cDNA product can be directly used for qPCR detection. The Evo M-MLV reverse transcription premix kit (including gDNA removal reagent, for qPCR) Ver.2 contains all the components required for the reverse transcription reaction.

[0092] 2× real-time quantitative PCR amplification premix solution (English name is 2×qPCR SYBR Green MasterMix, referred to as SYBR Mix), which contains hot start DNA polymerase ( DNA Polymerase), SYBRGreen I, deoxyribonucleoside triphosphates (dNTPs), magnesium ions (Mg 2+ ). qPCR SYBR Green Master Mix can be selected qPCR SYBR Green Master Mix (No Rox) can be purchased from Yeasen, catalog number 11202ES08.

[0093] Experimental equipment

[0094] CO 2Incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), micro-spectrophotometer (Thermo Scientific NanoDrop), real-time fluorescence quantitative PCR instrument (Thermo Fisher, Quantstudio 1Plus) and high-speed refrigerated centrifuge (Eppendorf Centrifuge 5418R).

[0095] Experimental samples

[0096] Sample 1: DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin; wherein the mass fraction of FBS is 10%, and the mass fraction of penicillin / streptomycin is 1%; specifically, the DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin can be obtained by mixing FBS, penicillin / streptomycin and DMEM culture medium.

[0097] Acetyl Tripeptide-30 Citrulline Stock Solution: Acetyl Tripeptide-30 Citrulline was mixed into PBS to form an Acetyl Tripeptide-30 Citrulline Stock Solution. The concentration can be adjusted as needed. Acetyl Tripeptide-30 Citrulline was obtained from GenScript Biotech Co., Ltd.

[0098] Teprenone stock solution: Teprenone is mixed into DMSO (dimethyl sulfoxide) to form a teprenone stock solution, and the concentration can be adjusted as needed. Teprenone is sourced from Purui Biopharmaceutical Technology Co., Ltd.

[0099] Sample 2: DMEM culture medium solution containing acetyl tripeptide-30 citrulline at a concentration of 15 μg / mL; specifically, the acetyl tripeptide-30 citrulline stock solution was diluted with sample 1 (DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin) until the concentration of acetyl tripeptide-30 citrulline was 15 μg / mL, thereby obtaining sample 2.

[0100] Sample 3: DMEM culture medium solution containing acetyl tripeptide-30 citrulline at a concentration of 20 μg / mL; specifically, the acetyl tripeptide-30 citrulline stock solution was diluted with sample 1 (DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin) until the concentration of acetyl tripeptide-30 citrulline was 20 μg / mL, thereby obtaining sample 3.

[0101] Sample 4: A DMEM culture medium solution containing acetyl tripeptide-30 citrulline at a concentration of 25 μg / mL, comprising: specifically, the acetyl tripeptide-30 citrulline stock solution is diluted with sample 1 (DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin) until the concentration of acetyl tripeptide-30 citrulline is 25 μg / mL, thereby obtaining sample 4.

[0102] Sample 5: DMEM medium solution containing 50 μg / mL of teprenone; specifically, sample 5 was obtained by diluting the teprenone stock solution with sample 1 (DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin) to a teprenone concentration of 50 μg / mL.

[0103] Sample 6: a DMEM medium solution containing 50 μg / mL teprenone and 15 μg / mL acetyl tripeptide-30 citrulline; specifically, sample 1 (a DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin) was used to dilute the teprenone stock solution to a teprenone concentration of 50 μg / mL, and the acetyl tripeptide-30 citrulline stock solution was simultaneously diluted to a acetyl tripeptide-30 citrulline concentration of 15 μg / mL, thereby obtaining sample 6;

[0104] Sample 7: a DMEM medium solution containing 50 μg / mL teprenone and 20 μg / mL acetyl tripeptide-30 citrulline; specifically, sample 1 (a DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin) was used to dilute the teprenone stock solution to a teprenone concentration of 50 μg / mL, and the acetyl tripeptide-30 citrulline stock solution was simultaneously diluted to a acetyl tripeptide-30 citrulline concentration of 20 μg / mL, thereby obtaining sample 7;

[0105] Sample 8: A DMEM culture medium solution containing 50 μg / mL of teprenone and 25 μg / mL of acetyl tripeptide-30 citrulline; specifically, sample 1 (a DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin) was used to dilute the teprenone stock solution to a teprenone concentration of 50 μg / mL, and the acetyl tripeptide-30 citrulline stock solution was simultaneously diluted to a acetyl tripeptide-30 citrulline concentration of 25 μg / mL, to obtain sample 8.

[0106] <Experimental Example 1, mRNA expression level detection>

[0107] The relative mRNA expression level of the CD44 gene of HaCaT cells was measured by real-time fluorescence quantitative PCR test method to further analyze the efficacy of the composition containing teprenone in the examples of the present application.

[0108] The real-time fluorescence quantitative PCR test method includes the following steps S101 to S105.

[0109] S101, cell seeding:

[0110] Specifically, HaCaT cells were taken and cultured at 1×10 6Inoculate the cells into a 6-well plate at a density of 100 cells / well, add 2 mL of complete medium (DMEM medium containing 10% FBS and 1% penicillin / streptomycin) to each well, and then place the cells in an incubator (the incubator contains 5% CO 2 , temperature is 37°C) for 24h.

[0111] S102, Cell treatment:

[0112] When the cell plating rate in the 6-well plate reached 70% to 80%, the cells were divided into a blank control group and 7 experimental groups, which were: drug administration experimental group 1, drug administration experimental group 2, drug administration experimental group 3, drug administration experimental group 4, drug administration experimental group 5, drug administration experimental group 6, and drug administration experimental group 7;

[0113] According to Table 1, 2 mL of the corresponding sample was added to each group for treatment. Three replicate wells were set up for each group. After treatment, the cells were placed in an incubator (37°C, 5% CO 2 ) for 24 h.

[0114] Table 1

[0115]

[0116]

[0117] S103, RNA extraction:

[0118] After 24 hours of culture, RNA was extracted from the cells of each group using an RNA extraction kit to obtain a sample solution containing total RNA (Total RNA); specifically, the following steps (1) to (10) were included. The RNA extraction kit is the ER501-01-V2 TransZol Up Plus RNA Kit Animal Tissue / Cell Total RNA Extraction Kit

[0119] (1) After 24 hours of culture, pour out the culture solution and rinse the culture plate once with PBS. Then add 1 mL of TansZolUp to each sample well (the sample well is the culture well with the sample added), place it horizontally for a while to allow the lysis solution to evenly distribute on the cell surface and lyse the cells, then use a pipette to blow the cells off to obtain the lysed cell solution.

[0120] (2) Transfer the lysed cell solution to a centrifuge tube, add 0.2 ml of RNA Extraction Agent to each sample well, and repeatedly pipette until there is no obvious precipitation in the lysed cell solution. Then vortex and shake at room temperature for 5 minutes to obtain the shaken cell solution.

[0121] (3) Then, the shaken cell solution was centrifuged in a high-speed centrifuge to obtain a clarified supernatant, wherein the centrifuge setting parameters are as follows: speed 12000 rpm, temperature 4°C and running time 15 minutes. The top transparent phase was transferred to a new centrifuge tube, and an equal volume of anhydrous ethanol was added (precipitation may occur at this time), and the mixture was gently inverted to obtain a mixed solution.

[0122] (4) Transfer the mixed solution to the centrifuge column provided by the RNA extraction kit and centrifuge to obtain a precipitate. The centrifugation parameters are set to: 12,000 rpm, centrifuge at room temperature for 30 seconds, and discard the effluent. Add 500uL CB9 to the precipitate corresponding to each group of samples, centrifuge at 12,000 rpm at room temperature for 30 seconds, discard the effluent, and obtain the first precipitate. Add 500uL CB9 to the first precipitate corresponding to each group of samples again, centrifuge at 12,000 rpm at room temperature for 30 seconds, and discard the effluent to obtain the second precipitate.

[0123] (5) Then, add 500uL WB9 to the second precipitate corresponding to each sample group, centrifuge at 12,000rpm for 30 seconds at room temperature, and discard the effluent. Repeat step (5) once.

[0124] (6) Centrifuge at 12,000 rpm for 2 minutes at room temperature to completely remove residual ethanol.

[0125] (7) Place the centrifuge column in the RNase-free Tube (provided in the kit), add 30uL RNase-free Water in the center of the centrifuge column, and let it stand at room temperature for 1 minute.

[0126] (8) Centrifuge at 12,000 rpm for 1 minute at room temperature to obtain RNA samples of each group.

[0127] (9) Use Nanodrop 2000 to prepare the sample solution containing 500 ng Total RNA.

[0128] S104, reverse transcription: using Evo-M-MLV reverse transcription reagent premix, PCR reverse transcription was performed on the sample solution to be tested, and finally a cDNA sample with a cDNA concentration of 5 ng / μL was obtained.

[0129] The reaction system of PCR reverse transcription is shown in Table 2, and the reaction conditions of PCR reverse transcription are shown in Table 3.

[0130] Table 2. PCR reverse transcription reaction system

[0131] Component name Usage 5×Evo-M-MLV RT Master Mix 4μL <![CDATA[Total RNA ×1 > - RNase free water Up to 20μL

[0132] Table 3. Reaction conditions for PCR reverse transcription

[0133]

[0134]

[0135] S105, Real-time PCR:

[0136] In step S105, cDNA samples of each group were taken for qPCR reaction to analyze the relative mRNA expression level (Relative mRNA Level) of CD44 gene of each group. The cDNA samples of each group were taken as cDNA templates, and qPCR SYBRGreen Mix was used to perform 3 parallel wells for each sample and each gene for detection.

[0137] In the specific test, add the sample first, then put it on the machine (Applied Biosystems TM QuantStudio TM 1) Perform qPCR program; the qPCR reaction system is shown in Table 4, the primer gene sequence is shown in Table 5, and the qPCR reaction program is shown in Table 6.

[0138] Table 4. qPCR reaction system

[0139] Solution volume 2XqPCR SYBR Green Master Mix 5μL 10μmol / mL primer (forward + reverse) 0.6μL cDNA template 4μL <![CDATA[ddH 2 The]]> 0.4μL

[0140] Table 5. Primer gene sequences

[0141]

[0142]

[0143] Table 6. qPCR reaction procedure

[0144] stage Temperature settings Pre-denaturation 95℃,5min Cycle (40cycle) 95℃, 10s→60℃, 60s Melting curve 60℃→95℃, temperature rise 0.3℃ every 15s

[0145] Experimental results and analysis :

[0146] The experimental results are shown in Figures 1 to 3 .

[0147] Figure 1 It is a comparative schematic diagram of the relative expression levels of CD44 mRNA in the blank control group, the dosing experimental group 4, the dosing experimental group 1, and the dosing experimental group 5. In the figure, A represents the blank control group, E represents the dosing experimental group 4 (teprenone 50 μg / mL), B represents the dosing experimental group 1 (acetyl tripeptide-30 citrulline 15 μg / mL), and F represents the dosing experimental group 5 (teprenone 50 μg / mL + acetyl tripeptide-30 citrulline 15 μg / mL).

[0148] Figure 2It is a comparative schematic diagram of the relative expression levels of CD44 mRNA in the blank control group, the dosing experimental group 4, the dosing experimental group 2, and the dosing experimental group 6. In the figure, A represents the blank control group, E represents the dosing experimental group 4 (teprenone 50 μg / mL), C represents the dosing experimental group 2 (acetyl tripeptide-30 citrulline 20 μg / mL), and G represents the dosing experimental group 6 (teprenone 50 μg / mL + acetyl tripeptide-30 citrulline 20 μg / mL).

[0149] Figure 3 It is a comparative diagram of the relative expression levels of CD44 mRNA in the blank control group, the dosing experimental group 4, the dosing experimental group 3, and the dosing experimental group 7. In the figure, A represents the blank control group, E represents the dosing experimental group 4 (teprenone 50 μg / mL), D represents the dosing experimental group 3 (acetyl tripeptide-30 citrulline 25 μg / mL), and H represents the dosing experimental group 7 (teprenone 50 μg / mL + acetyl tripeptide-30 citrulline 25 μg / mL). According to Figures 1 to 3 It can be seen that when teprenone acts on skin cells alone (see drug administration experimental group 4), it can promote the expression of CD44, but the expression level is limited, while when acetyl tripeptide-30 citrulline acts on skin cells alone (see drug administration experimental groups 1 to 3), it inhibits the expression of CD44.

[0150] From the results of the drug administration experimental groups 5 to 7, it can be seen that when teprenone and acetyl tripeptide-30 citrulline act together on skin cells, the mRNA expression level of CD44 can be greatly increased, and the effect is much greater than when teprenone acts alone. This shows that teprenone and acetyl tripeptide-30 citrulline can act synergistically to increase the mRNA expression level of CD44.

[0151] In more specific experimental results, it can also be seen that when the mass ratio of teprenone to acetyl tripeptide-30 citrulline is 50:15-25, it has a significant promoting effect on the increase of CD44 mRNA expression level.

[0152] Application examples:

[0153] The application example provides a composition containing teprenone, wherein the composition containing teprenone is an emulsion.

[0154] The preparation method of the emulsion comprises the following steps:

[0155] S201, heat phase A to 85°C and swell evenly, and heat phase B to 85°C and dissolve evenly;

[0156] S202, then adding the heated phase A to the heated phase B and homogenizing for 2-3 minutes to obtain a first mixture;

[0157] S203, adding phase C to the first mixture and homogenizing for 1-2 minutes, stirring and cooling to below 40° C. to obtain a second mixture;

[0158] S204, adding phase D and dissolved phase E to the second mixture and stirring evenly to obtain an emulsion.

[0159] The formula of the emulsion is shown in Table 7 below.

[0160] Table 7

[0161]

[0162] It is understandable that the emulsion contains acetyl tripeptide-30 citrulline and teprenone, and thus can inhibit and promote the expression of CD44, thereby promoting the production of hyaluronic acid and achieving the effect of protecting the skin.

[0163] The above specific embodiments have been described in detail for the present invention, but these do not constitute limitations on the present invention. The protection scope of the present invention is not limited to the above embodiments, and any equivalent modifications or changes made by ordinary technicians in the field based on the content disclosed by the present invention should be included in the protection scope recorded in the claims.

Claims

1. A composition containing teprenone, characterized in that include: Teprenone and acetyl tripeptide-30 citrulline, The mass ratio of the teprenone to the acetyl tripeptide-30 citrulline is 50:15-25.

2. The composition containing teprenone according to claim 1, characterized in that Also included are solvents.

3. A skin care product, characterized in that include: Teprenone and acetyl tripeptide-30 citrulline, The mass ratio of the teprenone to the acetyl tripeptide-30 citrulline is 50:15-25.

4. The skin care product according to claim 3, characterized in that Also included are solvents.

5. Use of the composition containing teprenone according to claim 1 in the preparation of a CD44 expression promoter. 6 . The use according to claim 5 , wherein the CD44 expression promoter is a CD44 expression promoter for skin cells, and the skin cells are human immortalized keratinocytes.

7. Use of the composition containing teprenone according to claim 1 in the preparation of a hyaluronic acid promoter.

8. The use according to claim 7, wherein the composition containing teprenone is used as a CD44 expression promoter.

9. Use of the composition containing teprenone according to claim 1 in preparing a skin care product.

10. Use of teprenone and acetyl tripeptide-30 citrulline in the preparation of a CD44 expression promoter, a hyaluronic acid promoter or a skin care product; wherein, The mass ratio of the teprenone to the acetyl tripeptide-30 citrulline is 50:15-25.

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