Composition containing palmitoyl tripeptide-5, skin care product and application

Abstract

The embodiment of the invention discloses a composition containing palmitoyl tripeptide-5, a skin care product and application, and the composition containing palmitoyl tripeptide-5 is prepared from acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 according to the mass ratio of (1.5-2): 2. According to the composition containing the palmitoyl tripeptide-5, through the synergistic effect of the acetyl tripeptide-30 citrulline and the palmitoyl tripeptide-5, the effect of protecting the skin can be achieved.

Description

Technical Field

[0001] The invention relates to the technical field of peptide-containing compositions, and in particular to a composition, a skin care product and uses thereof containing palmitoyl tripeptide-5. Background Art

[0002] When the skin is exposed to ultraviolet radiation, the expression of intercellular cell adhesion molecule-1 (ICAM-1 or ICAM1) increases, damaging skin cells. Specifically, ICAM-1 is a cell surface glycoprotein and adhesion receptor; after binding to its ligand (such as LFA-1), ICAM-1 can activate multiple signaling pathways, such as NF-kB and MAPK pathways; these signaling pathways can induce the production of inflammatory factors, such as tumor necrosis factor α (TNF-α), interleukin (IL-6), etc., thereby exacerbating the inflammatory response.

[0003] Palmitoyl Tripeptide-5 is a synthetic signal peptide that can promote the synthesis of collagen and elastin in the skin, improve skin elasticity and glossiness, and reduce wrinkles.

[0004] Currently, there is no application of palmitoyl tripeptide-5 in protecting the skin from the aspect of ICAM-1. Summary of the invention

[0005] 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.

[0006] Another purpose of the embodiments of the present application is to provide a composition, skin care product and use containing palmitoyl tripeptide-5, wherein the composition containing palmitoyl tripeptide-5 can protect the skin through the synergistic effect of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5.

[0007] The embodiments of the present application mainly achieve the above objectives through the following technical solutions.

[0008] In one aspect, the present invention provides a composition containing palmitoyl tripeptide-5, comprising:

[0009] Acetyl Tripeptide-30citrulline; and

[0010] Palmitoyl Tripeptide-5;

[0011] The mass ratio of acetyl tripeptide-30 citrulline to palmitoyl tripeptide-5 is (1.5-2):2.

[0012] In yet another aspect, the present application provides a skin care product, comprising:

[0013] The mass ratio of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 is (1.5-2):2.

[0014] In yet another aspect, the embodiments of the present application provide a use of the composition containing palmitoyl tripeptide-5 in the preparation of skin care products.

[0015] In yet another aspect, the present application provides an application of the palmitoyl tripeptide-5-containing composition in the preparation of a UV damage inhibitor.

[0016] In yet another aspect, the present application provides an embodiment of the use of the palmitoyl tripeptide-5-containing composition in the preparation of an ICAM-1 expression inhibitor of skin cells.

[0017] The beneficial effects of the embodiments of the present invention include:

[0018] In some embodiments, the palmitoyl tripeptide-5-containing composition provided in the embodiments of the present application contains acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 in a mass ratio of (1.5 to 2):2, which can play a synergistic role, thereby inhibiting UVB from stimulating the skin to cause the expression of ICAM-1 and protecting the skin.

[0019] In some embodiments, the mass ratio of acetyl tripeptide-30 citrulline to palmitoyl tripeptide-5 in the palmitoyl tripeptide-5-containing composition provided in the embodiments of the present application is (1.5-1.8):2, which can play a synergistic role, thereby inhibiting UVB from stimulating the skin to cause the expression of ICAM-1 and protecting the skin.

[0020] In some embodiments, the palmitoyl tripeptide-5-containing composition provided in the embodiments of the present application plays a role in reducing skin inflammation caused by UVB stimulation by inhibiting the expression of ICAM-1 caused by UVB stimulation to the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the relative expression level of an mRNA in the experimental example of this application;

[0022] Figure 2 This is another schematic diagram of relative mRNA expression levels in the experimental example of this application. DETAILED DESCRIPTION

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] Epidermis has the effect of mitigating external stimulation, controlling the loss of components in the body such as moisture, starting from the basal layer as the bottom layer, and is composed of the continuous 4-layer structure of basal layer, stratum spinosum, stratum granulosum, and stratum corneum. Most of the cells present in each layer are keratinocytes differentiated by the basal layer. The keratinocytes that divide and proliferate in the basal layer are differentiated by the stratum spinosum and stratum granulosum and become keratinocytes, forming the stratum corneum that is composed of the keratin fibers with firm cross-linking bonds, and finally fall off from the stratum corneum as dirt. The stratum corneum is present in the outermost layer of the skin, and plays a role as a physical barrier for the stimulation from the outside. In order to make the skin have this barrier function, usually with the cycle of 4 weeks, repeating from producing keratinocytes in the basal layer to the circulation (keratinization) until it becomes dirt and peels off, carries out the metabolism of the epidermis. However, when the skin is affected by external factors such as ultraviolet rays, strong air dryness, excessive skin washing, smoking, etc., or as we age, the activity and proliferation capacity of epidermal cells will decrease, and the turnover speed of the epidermis will be delayed, which will cause incomplete differentiation such as thinning of the epidermis or thickening of the stratum corneum. This will lead to reduced moisturizing function or elasticity of the skin, abnormal peeling of keratin, and aging of the skin with changes such as wrinkles, dullness, loss of texture, and reduced elasticity.

[0028] When the skin is exposed to ultraviolet radiation, the expression of ICAM-1 will increase, damaging skin cells. The present application example found that acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 can produce a synergistic effect at a specific mass ratio to inhibit the expression of ICAM-1.

[0029] Based on this, the embodiments of the present application propose the following improvements.

[0030] <Composition containing palmitoyl tripeptide-5>

[0031] The present application embodiment provides a composition containing palmitoyl tripeptide-5, comprising:

[0032] The mass ratio of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 is (1.5-2):2.

[0033] UVB is a type of ultraviolet light. When it irradiates the skin, it stimulates skin cells and induces inflammation in skin cells.

[0034] In the palmitoyl tripeptide-5-containing composition provided in the embodiment of the present application, when acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 are used in combination at a mass ratio of (1.5-2):2, a synergistic effect can be produced to reduce the inflammation of skin cells caused by UVB stimulation and play a role in protecting the skin.

[0035] In addition, acetyl tripeptide-30 citrulline is a bioactive peptide widely used in anti-wrinkle formulas. Therefore, the composition containing palmitoyl tripeptide-5 can also be applied to anti-wrinkle products.

[0036] In some embodiments, the skin cells are human keratinocyte line (HaCaT).

[0037] In some embodiments, the mass ratio of acetyl tripeptide-30 citrulline to palmitoyl tripeptide-5 is (1.8-2):2, optionally 1.8:2.

[0038] In some embodiments, the composition containing palmitoyl tripeptide-5 further includes: a solvent.

[0039] Furthermore, the solvent is a cosmetically acceptable solvent.

[0040] 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. In certain embodiments, the composition containing palmitoyl tripeptide-5 may contain at least one cosmetically acceptable adjuvant. The adjuvant is selected from one or more of surfactants and / or emulsifiers, preservatives, buffers, chelating agents, denaturants, sunscreens, pH regulators, reducing agents and stabilizers, thickeners, gelling agents, film-forming polymers, fillers, matting agents, glossing agents, pigments, dyes, fragrances, and mixtures thereof.

[0041] In certain embodiments, the composition containing palmitoyl tripeptide-5 may include at least one cosmetically acceptable active ingredient, the active ingredient being 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, tetrahydromethylpyrimidine carboxylic acid, glabridin, bisabolol, ceramides, phenylethylresorcinol, soluble collagen, adenosine, 4-butylresorcinol, hydrolyzed collagen, inositol, carbohydrate isomers, fibronectin, astaxanthin, madecassoside, rhamnose, resveratrol, amino acids, hydroxypropyl tetrahydropyrantriol, fermentation products, sodium hyaluronate, niacinamide, panthenol, and plant extracts.

[0042] It is understood that the composition containing palmitoyl tripeptide-5 can be used as a skin care product or further used to prepare a skin care product. Optionally, the skin care product is an external skin preparation.

[0043] In certain embodiments, the skin care product can be manufactured in the form of cream, liquid, powder, block, mud-like solid, wax-based, freeze-dried, spray, aerosol, base material, etc.

[0044] Optionally, the cream form is further in the form of milk, ointment, cream, honey, fat, milk, emulsion, milk, milk solution and the like.

[0045] Optionally, the liquid form is further in the form of dew, liquid, water, oil, oil-water separation, etc.

[0046] Optionally, the cream form is further in the form of milk, ointment, cream, honey, fat, milk, emulsion, milk, milk solution and the like.

[0047] Optionally, the powder form can be further in the form of gel, glue, etc., loose powder, granules, etc.

[0048] Alternatively, the block form may be further in the form of block powder, bulk solid, and the like.

[0049] Optionally, the wax base uses wax as a main base material.

[0050] Optionally, the lyophilized form is further in the form of lyophilized powder, lyophilized tablets, etc.

[0051] Optionally, the spray contains no propellant.

[0052] Optionally, the aerosol contains a propellant.

[0053] Optionally, the substrate is a substrate such as a patch or a film for use with cosmetics.

[0054] In certain embodiments, the skin care products can be topically applied to the hair, body hair, trunk, head, face, eyes, lips, hands, feet, skin all over the body, fingernails, nails, etc.

[0055] In certain embodiments, the sum of the mass fractions of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 in the composition is 0.0001 to 10%, and further can be 0.001 to 5%.

[0056] In certain embodiments, the sum of the mass fractions of acetyl tripeptide-30 citrulline, palmitoyl tripeptide-5 and the solvent in the composition or skin care product is 0.0001 to 10%, and further, can be 0.001 to 5%.

[0057] <Purpose>

[0058] According to the aforementioned composition containing palmitoyl tripeptide-5, the present application embodiment can also provide a use of the composition containing palmitoyl tripeptide-5 in the preparation of an ICAM-1 expression inhibitor; wherein the mass ratio of acetyl tripeptide-30 citrulline to palmitoyl tripeptide-5 is (1.5-2):2. Optionally, it is (1.8-2):2. By inhibiting the expression of ICAM-1, it can play a role in reducing skin inflammation caused by UVB stimulation and achieve the effect of protecting the skin.

[0059] More specifically, the ICAM-1 expression inhibitor is an ICAM-1 expression inhibitor of skin cells.

[0060] Furthermore, the skin cells are human immortalized keratinocytes (human keratinocyte line, HaCaT).

[0061] It can be understood that the composition containing palmitoyl tripeptide-5 can reduce UVB damage by inhibiting the expression of ICAM-1, specifically, it can reduce or alleviate skin inflammation caused by UVB stimulation; therefore, the embodiment of the present application can also provide a use of the composition containing palmitoyl tripeptide-5 in the preparation of a UV damage inhibitor.

[0062] As can be seen from the foregoing, acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 can play a synergistic role in skin care, so in some embodiments, the present application embodiment can also provide a use of a composition containing palmitoyl tripeptide-5 in the preparation of a skin care product. Optionally, the skin care product is an external skin preparation.

[0063] <Experiment>

[0064] The following experiments will verify the synergistic effects of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5.

[0065] 1. Experimental reagents and instruments

[0066] 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.

[0067] Palmitoyl Tripeptide-5 stock solution: Mix palmitoyl tripeptide-5 powder into DMSO to form palmitoyl tripeptide-5 stock solution. The concentration can be adjusted as needed. Palmitoyl tripeptide-5 is sourced from GenScript Biotech Co., Ltd.

[0068] The first composition is a 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.

[0069] The second composition is a DMEM culture medium solution containing acetyl tripeptide-30 citrulline at a concentration of 180 μg / mL; specifically, the acetyl tripeptide-30 citrulline stock solution can be diluted with the first composition (DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin) to a concentration of acetyl tripeptide-30 citrulline of 180 μg / mL, thereby obtaining the second mixture.

[0070] The third composition is a DMEM culture medium solution containing acetyl tripeptide-30 citrulline at a concentration of 200 μg / mL; specifically, the acetyl tripeptide-30 citrulline stock solution can be diluted with the first composition (DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin) to a concentration of acetyl tripeptide-30 citrulline of 200 μg / mL, thereby obtaining the third mixture.

[0071] The fourth composition is a DMEM culture medium solution containing palmitoyl tripeptide-5 at a concentration of 200 μg / mL; specifically, the palmitoyl tripeptide-5 mother solution can be diluted with the first composition (DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin) to a palmitoyl tripeptide-5 concentration of 200 μg / mL, thereby obtaining the fourth mixture.

[0072] The fifth composition is a DMEM culture medium solution containing 180 μg / mL of acetyl tripeptide-30 citrulline and 200 μg / mL of palmitoyl tripeptide-5; specifically, the palmitoyl tripeptide-5 mother solution and acetyl tripeptide-30 citrulline can be added to the first composition (DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin), and the concentration of palmitoyl tripeptide-5 is diluted to 200 μg / mL and the concentration of acetyl tripeptide-30 citrulline is diluted to 180 μg / mL, thereby obtaining the fifth mixture.

[0073] The sixth composition is a DMEM culture medium solution containing 200 μg / mL of acetyl tripeptide-30 citrulline and 200 μg / mL of palmitoyl tripeptide-5; specifically, palmitoyl tripeptide-5 mother solution and acetyl tripeptide-30 citrulline can be added to the first composition (DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin), and the concentration of palmitoyl tripeptide-5 is diluted to 200 μg / mL and the concentration of acetyl tripeptide-30 citrulline is diluted to 200 μg / mL, and the sixth mixture can be obtained.

[0074] 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.

[0075] Fetal bovine serum (FBS) was purchased from Gibco brand fetal bovine serum of Thermo Fisher Scientific (China) Co., Ltd., product number 10091148.

[0076] 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.

[0077] CCK8 mother solution is taken from Beyotime / Biyuntian C0040 Cell Counting Kit-8 (CCK-8 kit). The main chemical in CCK8 mother solution is WST-8, whose chemical name is 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt. In the presence of an electron coupling agent (that is, living cells undergoing aerobic respiration and energy metabolism), it can be oxidized and reduced by NAD+ to become a water-soluble yellow formazan product (Formazan). The more living cells there are, the more Formazan is produced, and the darker the color. Therefore, the depth of the color of the reaction product is proportional to the number of living cells. The faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color.

[0078] The CCK8 working solution can be prepared as follows: take 1 ml of CCK8 mother solution (Beyotime / Biyuntian C0040 Cell Counting Kit-8 (CCK-8 Kit)) and add it to 9 ml of DMEM culture medium to make 10 ml of CCK8 working solution.

[0079] PBS (phosphate buffered saline), pH 7.4, purchased from Thermo Fisher Scientific (China) Co., Ltd. Gibco brand PBS is a balanced salt solution suitable for various cell culture applications, such as washing cells before dissociation, transporting cells or tissues, diluting cells for counting and preparing reagents.

[0080] RNA extraction kit, specifically TransZol Up Plus RNA Kit (Transgen Biotech), purchased from Beijing Quanshijin Biotechnology Co., Ltd., is suitable for extracting total RNA from cells and tissues. The sample is lysed with TransZolUp, 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: TransZolUp 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.

[0081] 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.

[0082] 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 11202ES.

[0083] CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), microplate reader (BioTek, Epoch), micro-spectrophotometer (Thermo Scientific NanoDrop), ProFlex TM PCR instrument (Applied Biosystems SimpliAmp PCR thermal cycler), QPCR instrument (Applied Biosystems QuantStudio 1), UV crosslinker (UVB crosslinker, Luyor UCL-3500), high-speed refrigerated centrifuge (Eppendorf Centrifuge5418R).

[0084] 2. Experimental Methods

[0085] <Experimental Example 1, Cell Viability Detection Test>

[0086] The CCK8 (Cell Counting Kit CCK 8) kit was used to test the survival rate of HaCaT cells to detect the effects of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 on cell survival rate.

[0087] The test method includes the following steps S11 to S14:

[0088] Step S11, cell inoculation.

[0089] Specifically, immortalized human keratinocytes (HaCaT) were taken and cultured at 1×10 4 The cells were inoculated into a 96-well plate at a density of 100 μL / well, and 100 μL of complete culture medium (complete culture medium is DMEM culture medium containing 10% FBS and 1% penicillin / streptomycin) was added to each well, and then incubated in an incubator (the incubator contains 5% CO2 and the temperature is 37°C) for 24 h.

[0090] Step S12: medication administration.

[0091] Specifically, when the cell plating rate in the 96-well plate reached 60% to 70%, the cells in the 96-well plate were divided into a blank control group and 5 experimental groups, the 5 experimental groups were: drug administration experimental group 1, drug administration experimental group 2, drug administration experimental group 3, drug administration experimental group 4, and drug administration experimental group 5, each group had 5 replicate wells; 100 μL of the corresponding treatment substance was added to each well, and after the drug administration was completed, the cells were placed in an incubator (37° C., 5% CO2) and cultured for 24 h.

[0092] Among them, the corresponding treatment substances of the blank control group, dosing experimental group 1, dosing experimental group 2, dosing experimental group 3, dosing experimental group 4, and dosing experimental group 5 are: the first composition, the second composition, the third composition, the fourth composition, the fifth composition, and the sixth composition, respectively.

[0093] Step S13: CCK8 detection.

[0094] After drug treatment, the cells were incubated for 24 h, the supernatant was discarded, 100 μL of CCK8 working solution (prepared in DMEM medium) was added, and the cells were incubated at 37° C. in the dark for 2 h. After the incubation, the absorbance value (OD value) of each group was read at 450 nm.

[0095] Step S14, calculating the cell survival rate of each experimental group.

[0096] The cell survival rate of the blank control group was set as 100% as a benchmark to further determine the cell survival rate of each experimental group.

[0097] The calculation formula of cell survival rate in each experimental group is as follows:

[0098] Cell survival rate = [(OD experimental well - OD zeroing well) / (OD control well - OD zeroing well)] × 100%;

[0099] Among them, the OD experimental well is the absorbance value of the experimental group, the OD control well is the absorbance value of the blank control group, and the OD zero adjustment well is the absorbance value of the calculation control group.

[0100] The absorbance value of the control group can be determined by the following method: a zero well without cells is set in a 96-well plate, only 1X CCK-8 working solution is added, and the OD value is read at 450 nm as the absorbance value of the control group.

[0101] Experimental results and analysis :

[0102] The cell survival rate of each group is shown in Table 1.

[0103] Table 1. Cell survival rate of each group

[0104]

[0105] According to Table 1, compared with the blank control group, the cell survival rates of the drug administration experimental group 1, the drug administration experimental group 2, the drug administration experimental group 3, the drug administration experimental group 4, and the drug administration experimental group 5 were not significantly reduced, indicating that acetyl dipeptide-1 cetyl ester, palmitoyl pentapeptide-4 and their combination will not cause adverse effects on cells.

[0106] <Experimental Example 2, mRNA expression level detection>

[0107] The experimental method includes the following steps S101 to S104.

[0108] S101, cell seeding:

[0109] Specifically, immortalized human keratinocytes (HaCaT) were taken and cultured at 80×10 4 The cells were inoculated into a 6-well plate at a density of 100 cells / well, and 2 ml of complete culture medium (complete culture medium is DMEM culture medium containing 10% FBS and 1% penicillin / streptomycin) was added to each well; then incubated in an incubator (the incubator contains 5% CO2 and the temperature is 37°C) for 24 hours.

[0110] S102, drug administration:

[0111] When the cell plating rate in the 6-well plate reaches 70% to 80%, a blank control group and 6 experimental groups are set up on the 6-well plate. The 6 experimental groups are: UVB control experimental group, drug administration experimental group 1, drug administration experimental group 2, drug administration experimental group 3, drug administration experimental group 4, and drug administration experimental group 5;

[0112] Except for the blank control group, the six experimental groups were treated with 50 mJ / cm 2 The cells were irradiated with a UVB dose of 100 μg / mL; the culture medium of the blank control group and the six experimental groups was then discarded, and the corresponding treatment substances were added to each group for drug treatment. Each group had three replicate wells, and after the drug treatment, the cells were placed in an incubator (37°C, 5% CO2) and cultured for 24 hours.

[0113] Among them, the treatment substances corresponding to the blank control group, UVB control experimental group, dosing experimental group 1, dosing experimental group 2, dosing experimental group 3, dosing experimental group 4, and dosing experimental group 5 are: the first composition, the first composition, the second composition, the third composition, the fourth composition, the fifth composition, and the sixth composition, respectively.

[0114] S103, RNA extraction:

[0115] After culturing for 24 hours, RNA was extracted from the cells in each group, which specifically included the following steps (1) to (10).

[0116] (1) After 24 hours of culture, pour out the culture medium and rinse once with PBS. 2 Add 1 ml of TansZolUp to the growing cultured cells, place horizontally for a while to evenly distribute the lysate on the cell surface and lyse the cells, then use a pipette to blow the cells to make them fall off. Transfer the cell lysate to a centrifuge tube, add 0.2 ml of RNAExtractionAgent, and repeatedly blow and suck with a pipette until there is no obvious precipitation in the lysate, and vortex at room temperature for 5 minutes.

[0117] (2) Centrifuge at 10,000 g and 2-8°C for 15 minutes.

[0118] At this time, the sample is divided into three layers, a colorless aqueous phase (upper layer), a middle layer and a pink organic phase (lower layer); wherein RNA is in the aqueous phase, and the volume of the aqueous phase is about 50% to 60% of the TransZol Up reagent used.

[0119] (3) Transfer the colorless aqueous phase to a new centrifuge tube, add an equal volume of anhydrous ethanol, and gently invert to mix.

[0120] (4) Add the resulting solution and precipitate to a centrifuge column and centrifuge at 12,000 g for 30 seconds at room temperature. Discard the flow-through.

[0121] (5) Then add 500 μl of CB9 (Clean Buffer 9), centrifuge at 12,000 g for 30 seconds at room temperature, discard the flow-through, and repeat this step once.

[0122] (6) Then add 500 μl of WB9 (Wash Buffer 9), centrifuge at 12,000 g for 30 seconds at room temperature, discard the flow-through, and repeat the step once.

[0123] (7) Centrifuge at 12,000 g for 2 min at room temperature to completely remove residual ethanol.

[0124] (8) Place the centrifuge column in the RNase-free tube, add 50-200 μl of RNase-free water to the center of the centrifuge column, and let it stand at room temperature for 1 minute.

[0125] (9) Centrifuge at 12,000 g for 1 minute at room temperature to elute the RNA.

[0126] (10) The RNA concentration was measured using a micro-spectrophotometer (Thermo Scientific NanoDrop), and RNA samples of each group were obtained and stored at -80°C.

[0127] S104. Perform qPCR on the RNA samples with measured concentrations in each group to determine the relative expression level of mRNA.

[0128] Step S104 specifically includes steps (a) to (c):

[0129] (a) Reverse transcribe the RNA samples with measured concentrations in each group to obtain reverse transcribed samples.

[0130] Specifically, RT-PCR reaction was used for reverse transcription: the RNA sample with measured concentration was used to calculate the corresponding RNA volume based on 2ug, and then the corresponding volume of RNase-free water (RNase Free H2O) and reverse transcriptase mix were added. The total volume of the RT-PCR reaction system was 20μL.

[0131] The specific system of RT-PCR reaction can be found in Table 2, and the specific procedure of RT-PCR reaction can be found in Table 3.

[0132] Table 2. RT-PCR reaction system

[0133] name volume RNA 2ug <![CDATA[RNase Free H2O]]> Up to 20μL 5X Evo M-MLV RT Master Mix 4μL

[0134] Table 3. RT-PCR reaction procedure

[0135] temperature time 37℃ 40min 85℃ 5s 12℃ ∞

[0136] (b) Then 20 μL of the reverse transcribed sample was diluted to 400 μL to obtain a cDNA sample with a final concentration of 5 ng / μL.

[0137] (c) cDNA samples were taken for qPCR reaction to analyze the relative expression level of mRNA of relevant genes (ICAM-1 gene) in each group.

[0138] Specifically, cDNA samples were used as cDNA templates for qPCR reaction, and 3 duplicate wells were made for each gene of each cDNA sample for detection. After adding the samples, the qPCR program was performed on the machine. After the qPCR reaction, the reaction data was processed, and the 2-△△Ct method was used to analyze the relative mRNA expression level of related genes (ICAM-1 gene) in each group. The data between the groups were analyzed using T-TEST statistics, and P < 0.05 indicated a significant difference, which was statistically significant.

[0139] Among them, the volume of each well of the qPCR reaction system is 10 μL, as can be seen in Table 4;

[0140] The qPCR reaction program can be found in Table 5.

[0141] Table 4. qPCR reaction system

[0142] name volume cDNA template 4μL 2X qPCR SYBR Green Master Mix 5μL <![CDATA[Double distilled water (ddH2O)]]> 0.4μL 10uM primer (forward + reverse) 0.6μL

[0143] Table 5. qPCR reaction procedure

[0144]

[0145]

[0146] Experimental results and analysis :

[0147] The experimental results are detailed in Figure 1 and Figure 2 . Figure 1 It is a schematic diagram of the relative expression levels of ICAM-1 mRNA in the blank control group, UVB control experimental group, drug administration experimental group 1, drug administration experimental group 3, and drug administration experimental group 4; Figure 2 It is a schematic diagram of the relative expression levels of ICAM-1 mRNA in the blank control group, UVB control experimental group, drug administration experimental group 2, drug administration experimental group 3, and drug administration experimental group 5.

[0148] 1. Comparison of the experimental results of the blank control group and the UVB control experimental group showed that after ultraviolet irradiation, ICAM-1 was strongly induced to express on HaCaT cells, indicating that UVB irradiation of skin cells will increase the risk of inflammation.

[0149] 2. After UVB treatment, the second and third compositions were added to the drug administration experimental groups 1 and 2 respectively; however, since the second and third compositions only contained acetyl tripeptide-30 citrulline and no palmitoyl tripeptide-5, ICAM-1 was strongly induced to express on HaCaT cells, with a higher expression level than that of the UVB control experimental group. This indicates that acetyl tripeptide-30 citrulline, when used alone, is difficult to inhibit the increase in ICAM-1 expression caused by UVB irradiation.

[0150] 3. After UVB treatment, the fourth composition was added to the drug administration experimental group 3 for treatment; the fourth composition only added palmitoyl tripeptide-5, which had a certain promoting effect on inhibiting the expression of ICAM-1 on HaCaT cells.

[0151] 4. After UVB irradiation, the combined addition of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 cetyl ester to the administration experimental group 4 (the fifth composition was added) and the administration experimental group 5 (the sixth composition was added) showed a strong inhibitory effect on the expression of ICAM-1, which was stronger than that of the administration experimental group 3 to which palmitoyl tripeptide-5 was added alone.

[0152] In summary, the combination of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 cetyl ester can have a good inhibitory effect on the increase in the expression of ICAM-1 on HaCaT cells induced by UVB, which further proves that the combination of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 cetyl ester can inhibit the aggravation of inflammation.

[0153] In addition, continuous inflammatory response can damage the collagen and elastic fibers of the skin, leading to aging phenomena such as wrinkles, sagging, and pigmentation. Therefore, the composition containing acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 cetyl ester can inhibit the aging phenomena caused by inflammation and produce an anti-aging technical effect.

[0154] <Application Examples>

[0155] The application example provides a composition containing acetyl tripeptide-30 citrulline, wherein the composition containing acetyl tripeptide-30 citrulline is an emulsion.

[0156] The preparation method of the emulsion comprises the following steps:

[0157] S201, heat phase A to 85°C and swell evenly, and heat phase B to 85°C and dissolve evenly;

[0158] S202, then adding the heated phase A to the heated phase B and homogenizing for 2-3 minutes to obtain a first mixture;

[0159] 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;

[0160] S204, adding phase D and dissolved phase E to the second mixture and stirring evenly to obtain an emulsion.

[0161] The formula of the emulsion is shown in Table 6 below.

[0162] Table 6. Formula of emulsion

[0163]

[0164] It can be understood that the emulsion contains acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 cetyl ester, so it can inhibit the increase in the expression of ICAM-1 on HaCaT cells induced by UVB, thereby inhibiting the aging phenomenon caused by inflammation and producing an anti-aging technical effect.

[0165] 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 palmitoyl tripeptide-5, characterized in that: include: The mass ratio of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 is (1.5-2):

2.

2. The composition containing palmitoyl tripeptide-5 according to claim 1, characterized in that The mass ratio of the acetyl tripeptide-30 citrulline to the palmitoyl tripeptide-5 is (1.8-2):

2.

3. The composition containing palmitoyl tripeptide-5 according to claim 1, characterized in that The mass ratio of the acetyl tripeptide-30 citrulline to the palmitoyl tripeptide-5 is 1.8:

2.

4. The composition containing palmitoyl tripeptide-5 according to claim 1, characterized in that Also included are solvents.

5. The composition containing palmitoyl tripeptide-5 according to claim 4, characterized in that: The solvent is 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.

6. A skin care product, characterized in that: include: The mass ratio of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 is (1.5-2):

2.

7. Use of the composition containing palmitoyl tripeptide-5 according to any one of claims 1 to 3 in the preparation of skin care products.

8. Use of the composition containing palmitoyl tripeptide-5 according to any one of claims 1 to 3 in the preparation of a UV damage inhibitor.

9. Use of the palmitoyl tripeptide-5-containing composition according to any one of claims 1 to 3 in the preparation of an ICAM-1 expression inhibitor for skin cells.

10. The use according to claim 9, wherein the skin cells are human immortalized keratinocytes.

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