Compositions, skin care and uses containing palmitoyl tripeptide-5

Abstract

This application discloses a composition containing palmitoyl tripeptide-5, a skin care product, and its uses. The composition containing palmitoyl tripeptide-5 comprises acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 in a mass ratio of (1.5-2):2. The composition containing palmitoyl tripeptide-5 can protect the skin through the synergistic effect of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5.

Description

Technical Field

[0001] This invention relates to the field of peptide composition technology, and more specifically to compositions containing palmitoyl tripeptide-5, skin care products, and uses. Background Technology

[0002] When skin is exposed to ultraviolet radiation, the expression of intercellular 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 the NF-κB and MAPK pathways. These signaling pathways can induce the production of inflammatory factors, such as tumor necrosis factor-α (TNF-α) and interleukin (IL-6), thereby exacerbating the inflammatory response.

[0003] Palmitoyl Tripeptide-5 is a synthetic signaling peptide that promotes the synthesis of collagen and elastin in the skin, improving skin elasticity and radiance, and reducing wrinkles.

[0004] There is currently no evidence of any application of palmitoyl tripeptide-5 in protecting the skin from ICAM-1. Summary of the Invention

[0005] One objective of this application is to solve at least one of the problems mentioned in the background above and to provide corresponding beneficial effects.

[0006] Another objective of this 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 this application mainly achieve the above objectives through the following technical solutions.

[0008] In one aspect, embodiments of this application provide 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 another aspect, embodiments of this application provide a skincare product, comprising:

[0013] Acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 in a mass ratio of (1.5–2):2.

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

[0015] In another aspect, embodiments of this application provide the use of the palmitoyl tripeptide-5-containing composition described above in the preparation of a UV damage inhibitor.

[0016] In another aspect, embodiments of this application provide the use of the palmitoyl tripeptide-5-containing composition described above in the preparation of an ICAM-1 expression inhibitor for skin cells.

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

[0018] In some embodiments, the composition containing palmitoyl tripeptide-5 provided in this 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 in inhibiting the expression of ICAM-1 caused by UVB stimulation of the skin and thus protecting the skin.

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

[0020] In some embodiments, the composition containing palmitoyl tripeptide-5 provided in this application can reduce skin inflammation caused by UVB stimulation by inhibiting the expression of ICAM-1 induced by UVB stimulation of the skin. Attached Figure Description

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

[0022] Figure 2 This is a schematic diagram of the relative expression level of another mRNA in the experimental examples of this application. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0024] The terms "first," "second," etc., used in the embodiments of this application are for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first," "second," etc., may explicitly or implicitly include at least one of that feature.

[0025] Furthermore, the terms “comprising” and “containing,” “including,” and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such process, method, product, or apparatus.

[0026] In addition to the above, it should be emphasized that the reference to "embodiment" herein means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0027] The epidermis plays a role in mitigating external stimuli and controlling the loss of internal components such as moisture. It consists of four consecutive layers: the basal layer (the lowest layer), the spinous layer, the granular layer, and the stratum corneum. Most cells in each layer are keratinocytes differentiated from those in the basal layer. These keratinocytes, dividing and proliferating in the basal layer, differentiate into keratinocytes as they pass through the spinous and granular layers, forming the stratum corneum, which is composed of keratin fibers with strong cross-linking bonds. This keratinocyte eventually sheds as dirt. The stratum corneum is the outermost layer of the skin, functioning as a physical barrier against external stimuli. To maintain this barrier function, the skin undergoes a cycle of keratinization (keratinization) every four weeks, a process that begins with the formation of keratinocytes in the basal layer and continues until they become dirt and shed. However, when skin is exposed to external factors such as ultraviolet radiation, intense dry air, excessive skin cleansing, and smoking, or as we age, the activity and proliferation of epidermal cells decrease, slowing down the epidermal turnover rate. This can lead to incomplete differentiation, such as thinning of the epidermis or thickening of the stratum corneum. Consequently, the skin's moisturizing function and elasticity decrease, abnormal exfoliation occurs, and the skin ages, resulting in wrinkles, dullness, loss of texture, and decreased elasticity.

[0028] When skin is exposed to ultraviolet radiation, the expression level of ICAM-1 increases, damaging skin cells. This application's embodiments found that acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5, at a specific mass ratio, can synergistically inhibit ICAM-1 expression.

[0029] Accordingly, the following improvements are proposed in the embodiments of this application.

[0030] Compositions containing palmitoyl tripeptide-5

[0031] This application provides a composition containing palmitoyl tripeptide-5, comprising:

[0032] Acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 in a mass ratio of (1.5–2):2.

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

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

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

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

[0037] In some embodiments, the mass ratio of acetyl tripeptide-30 citrulline to palmitoyl tripeptide-5 is (1.8 to 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 may be selected from one or more of ethanol, isopropanol, glycerol, octanediol, 1,2-pentanediol, propylene glycol, butylene glycol, glycol, glycol, glycol ether, ethylene glycol, propylene glycol, butylene glycol, hexanediol, dipropylene glycol, diethylene glycol, 2-ethoxyethanol, diethylene glycol monomethyl ether, triethylene glycol monomethyl ether, and sorbitol. In some 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, opacifiers, pH adjusters, reducing agents and stabilizers, thickeners, gelling agents, film-forming polymers, fillers, matting agents, gloss agents, pigments, dyes, fragrances, and mixtures thereof.

[0041] In some embodiments, the composition containing palmitoyl tripeptide-5 may contain at least one cosmetically acceptable active ingredient selected from 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, glycyrrhizin, bisabolol, ceramides, phenylethyl resorcinol, soluble collagen, adenosine, 4-butylresorcinol, hydrolyzed collagen, inositol, saccharide isomers, fibronectin, astaxanthin, asiaticoside, rhamnose, resveratrol, amino acids, hydroxypropyltetrahydropyrantriol, fermentation products, sodium hyaluronate, nicotinamide, panthenol, and plant extracts, one or more of these.

[0042] It is understood that the composition containing palmitoyl tripeptide-5 can be used as a skin care product or further used in the preparation of skin care products. Optionally, the skin care product is a topical skin preparation.

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

[0044] Optionally, the cream or lotion may be further in the form of emulsion, paste, cream, honey, fat, lotion, milk, milk, milk liquid, etc.

[0045] Optionally, the liquid form may be further divided into dew, liquid, water, oil, oil-water separation, etc.

[0046] Optionally, the cream or lotion may be further in the form of emulsion, paste, cream, honey, fat, lotion, milk, milk, milk liquid, etc.

[0047] Alternatively, the powder form can be further divided into gels such as jelly and glue, loose powder, granules, etc.

[0048] Alternatively, the block form can be further divided into block powder, large solid blocks, etc.

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

[0050] Optionally, the freeze-drying form can be further divided into freeze-dried powder, freeze-dried tablets, etc.

[0051] Optionally, the spray does not contain propellant.

[0052] Optionally, the aerosol contains a propellant.

[0053] Optionally, the substrate may be a film, a paste, or other substrate used in conjunction with cosmetics.

[0054] In some implementations, the skin care product can be applied topically to hair, body hair, torso, head, face, eyes, lips, hands, feet, whole body skin, fingers (toes), fingernails (toenails), etc.

[0055] In some 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 more preferably 0.001 to 5%.

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

[0057] <Application>

[0058] According to the aforementioned composition containing palmitoyl tripeptide-5, this application embodiment can also provide the use of the palmitoyl tripeptide-5 composition 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 ICAM-1 expression, it can reduce skin inflammation caused by UVB stimulation, thereby achieving a skin protection effect.

[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 keratinocyte lines (HaCaT).

[0061] It is understood that the composition containing palmitoyl tripeptide-5 can reduce UVB damage by inhibiting the expression of ICAM-1, specifically reducing or alleviating skin inflammation caused by UVB stimulation; therefore, this application embodiment can also provide the application of the composition containing palmitoyl tripeptide-5 in the preparation of UV damage inhibitors.

[0062] As mentioned above, acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 can have a synergistic skin-care effect. Therefore, in some embodiments, this application may also provide the use of a composition containing palmitoyl tripeptide-5 in the preparation of skin care products. Optionally, the skin care product is a topical skin preparation.

[0063] <Experiment>

[0064] The synergistic effect of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 will be verified through experiments in the following section.

[0065] I. Experimental Reagents and Instruments

[0066] Acetyl tripeptide-30 citrulline stock solution: Acetyl tripeptide-30 citrulline is mixed into PBS to form acetyl tripeptide-30 citrulline stock solution; the concentration can be adjusted as needed. (Acetyl tripeptide-30 citrulline is sourced from Genscript Biotech Inc.)

[0067] Palmitoyl Tripeptide-5 Mother Liquor: Palmitoyl Tripeptide-5 powder is mixed into DMSO to form a palmitoyl Tripeptide-5 mother liquor. The concentration can be adjusted as needed. Palmitoyl Tripeptide-5 is sourced from Genscript Biotech Inc.

[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 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 (a DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin) to a concentration of 180 μg / mL to obtain the second mixture.

[0070] The third composition is a DMEM 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 (a DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin) to a concentration of 200 μg / mL to obtain the third mixture.

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

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

[0073] The sixth composition is a DMEM medium solution containing 200 μg / mL acetyl tripeptide-30 citrulline and 200 μg / mL palmitoyl tripeptide-5. Specifically, palmitoyl tripeptide-5 stock solution and acetyl tripeptide-30 citrulline can be added to the first composition (DMEM medium solution containing 10% FBS and 1% penicillin / streptomycin) to dilute the concentration of palmitoyl tripeptide-5 to 200 μg / mL and the concentration of acetyl tripeptide-30 citrulline to 200 μg / mL, thus obtaining the sixth mixture.

[0074] DMEM (dulbecco's modified eagle medium) is a basic culture medium purchased from Gibco biological medium of Thermo Fisher Scientific (China) Co., Ltd., product code C11995500BT.

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

[0076] The penicillin / streptomycin was purchased from Thermo Fisher Scientific (China) Co., Ltd. as Gibco brand penicillin / streptomycin, catalog number 25200114, penicillin / streptomycin solution (10,000 U / mL), which includes penicillin and streptomycin at concentrations of 10,000 U / mL.

[0077] The CCK8 stock solution was obtained from Beyotime C0040 Cell Counting Kit-8 (CCK-8 reagent kit). The most important chemical in the CCK8 stock solution is WST-8, chemically named 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt. In the presence of electron coupling agents (i.e., aerobic respiration and energy metabolism in living cells), it can be oxidized and reduced by NAD+ to a water-soluble yellow formazan product. The more living cells there are, the more formazan is produced, and the darker the color will be. Therefore, the intensity of the reaction product color is directly 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 stock solution (Beyotime C0040Cell Counting Kit-8) and add it to 9 ml of DMEM medium to prepare 10 ml of CCK8 working solution.

[0079] PBS (phosphate buffer), pH 7.4, purchased from Thermo Fisher Scientific (China) Co., Ltd., 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] An RNA extraction kit, specifically the TransZol Up Plus RNA Kit (Transgen Biotech), purchased from Beijing TransGen Biotech Co., Ltd., is suitable for extracting total RNA from cells and tissues. The sample is lysed using TransZolUp, and after adding the RNA Extraction Agent, the solution separates into a colorless aqueous phase and a pink organic phase, with RNA in the aqueous phase. A silica gel membrane centrifuge column is used to specifically adsorb the RNA from the aqueous phase. 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) 100 each, and RNA Spin Columns with Collection Tubes 100 each.

[0081] Using an M-MLV reverse transcriptase kit, the Evo M-MLV Reverse Transcription Premix Kit (with gDNA removal reagent for qPCR) Ver.2 (Agbio, AG11728) was selected. This kit 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 (with gDNA removal reagent for qPCR) Ver.2 contains all the components required for the reverse transcription reaction.

[0082] The premixed solution for 2× real-time quantitative PCR amplification (English name: 2×qPCR SYBR Green MasterMix, abbreviated as SYBR Mix) contains hot-start DNA polymerase. DNA Polymerase, SYBR Green I, deoxyribonucleoside triphosphates (dNTPs), magnesium ions (Mg) 2+ ) qPCR SYBR Green Master Mix can be selected in specific ways. qPCR SYBR Green Master Mix (No Rox) is available from Yeasen under product number 11202ES.

[0083] CO2 incubator (Thermo, 150I), laminar flow hood (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 (Luyor UCL-3500), and high-speed refrigerated centrifuge (Eppendorf Centrifuge 5418R).

[0084] II. Experimental Methods

[0085] <Experiment Example 1: Cell Viability Detection Assay>

[0086] HaCaT cell viability was tested using the CCK8 (Cell Counting Kit) to detect the effects of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 on cell viability.

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

[0088] Step S11: Cell seeding.

[0089] Specifically, immortalized keratinocytes (HaCaT) are taken and processed at a concentration of 1×10⁻⁶. 4 Inoculate the plates at a density of 100 μL / well into 96-well plates, add 100 μL of complete culture medium (complete culture medium is DMEM medium containing 10% FBS and 1% penicillin / streptomycin) to each well, and then incubate in an incubator (containing 5% CO2 at a temperature of 37°C) for 24 h.

[0090] Step S12, drug administration.

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

[0092] Among them, the treatments corresponding to the blank control 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 are respectively: the first composition, the second composition, the third composition, the fourth composition, the fifth composition, and the sixth composition.

[0093] Step S13, CCK8 detection.

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

[0095] Step S14: Calculate the cell survival rate of each experimental group.

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

[0097] The formulas for calculating cell viability in each experimental group are as follows:

[0098] Cell viability = [(OD experimental wells - OD zeroing wells) / (OD control wells - OD zeroing wells)] × 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 zeroing well is used to calculate the absorbance value of the control group.

[0100] The absorbance value of the control group can be determined as follows: a cell-free well is set in a 96-well plate, and only CCK-8 working solution containing 1X is added. The OD value is read at 450 nm and used as the absorbance value of the control group.

[0101] Experimental Results and Analysis :

[0102] The cell survival rates for each group are shown in Table 1.

[0103] Table 1. Cell viability of each group

[0104]

[0105] As shown in Table 1, compared with the blank control group, the cell survival rate of the drug-treated experimental groups 1, 2, 3, 4, and 5 was not significantly reduced, indicating that acetyl dipeptide-1 cetyl ester, palmitoyl pentapeptide-4 and their combinations do not have adverse effects on cells.

[0106] <Experimental Example 2: Detection of mRNA Expression Levels>

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

[0108] S101, Cell Seeding:

[0109] Specifically, immortalized keratinocytes (HaCaT) were taken and processed at a concentration of 80 × 10⁻⁶. 4 Inoculate the plates at a density of 10 cells / well into 6-well plates, add 2 ml of complete culture medium (complete culture medium is DMEM medium containing 10% FBS and 1% penicillin / streptomycin) to each well; then incubate in an incubator (containing 5% CO2 at 37℃) for 24 h.

[0110] S102, Drug administration:

[0111] When the cell deposition rate in the 6-well plate reaches 70%–80%, a blank control group and 6 experimental groups are set up in 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 Cells were irradiated with UVB dose; then the culture media of the blank control group and 6 experimental groups were discarded, and the corresponding treatments were added to each group for drug administration. Each group was set up with 3 replicates. After drug administration, the cells were placed in an incubator (37℃, 5% CO2) for 24h.

[0113] Among them, the treatments corresponding to the blank control group, 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 are respectively: first composition, first composition, second composition, third composition, fourth composition, fifth composition, and sixth composition.

[0114] S103, RNA extraction:

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

[0116] (1) After culturing for 24 hours, pour out the culture medium and rinse once with PBS. Every 10cm 2 Add 1 ml of TansZolUp to the growing cultured cells, place them horizontally for a moment to allow the lysis buffer to distribute evenly on the cell surface and lyse the cells, then use a pipette to detach the cells. Transfer the cell lysis buffer to a centrifuge tube, add 0.2 ml of RNAExtractionAgent, and repeatedly pipette until there is no obvious precipitate in the lysis buffer. Vortex at room temperature for 5 minutes.

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

[0118] At this point, the sample is divided into three layers: a colorless aqueous phase (upper layer), a middle layer, and a pink organic phase (lower layer); the RNA is in the aqueous phase, and the volume of the aqueous phase is approximately 50% to 60% of the volume 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 obtained solution and precipitate together into a centrifuge column, centrifuge at 12,000g for 30 seconds at room temperature, and discard the effluent.

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

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

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

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

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

[0126] (10) The concentration of RNA was determined using a micro spectrophotometer (Thermo Scientific NanoDrop) to obtain RNA samples with measured concentrations for each group, and stored at -80℃.

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

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

[0129] (a) The RNA samples with measured concentrations in each group were reverse transcribed to obtain reverse transcribed samples.

[0130] Specifically, reverse transcription is performed using RT-PCR: the RNA sample with the measured concentration is used to calculate the corresponding RNA volume in 2 μg, and then the corresponding volume of RNase-free water (RNase-free H2O) and reverse transcriptase mix is ​​added. The total volume of the RT-PCR reaction system is 20 μL.

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

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

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

[0137] (c) Take cDNA samples for qPCR reaction and analyze the relative mRNA expression level of related genes (ICAM-1 gene) in each group.

[0138] Specifically, cDNA samples were used as cDNA templates for qPCR reactions. Three auxiliary wells were prepared for each gene in each cDNA sample for detection. After sample loading, the qPCR program was run. After the qPCR reaction, the reaction data were processed, and the 2-ΔΔCt method was used to analyze the relative mRNA expression levels of the relevant gene (ICAM-1 gene) in each group. Data between groups were analyzed using T-TEST statistical analysis. P < 0.05 indicated significant differences.

[0139] The volume of each well in the qPCR reaction system is 10 μL, as shown in Table 4.

[0140] The qPCR reaction procedure 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 10µM primers (forward + reverse) 0.6μL

[0143] Table 5. qPCR reaction procedure

[0144]

[0145]

[0146] Experimental Results and Analysis :

[0147] For detailed experimental results, please see Figure 1 and Figure 2 . Figure 1 A schematic diagram showing the relative expression levels of ICAM-1 mRNA in the blank control group, UVB control group, drug-treated group 1, drug-treated group 3, and drug-treated group 4. Figure 2 This is a schematic diagram showing the relative expression levels of ICAM-1 mRNA in the blank control group, UVB control group, drug-treated group 2, drug-treated group 3, and drug-treated group 5.

[0148] 1. The experimental results of the blank control group and the UVB control group show that ICAM-1 was strongly induced to be expressed on HaCaT cells after UV irradiation, indicating that UVB irradiation of skin cells will increase the risk of inflammation.

[0149] 2. In experimental groups 1 and 2, after UVB treatment, the second and third compositions, respectively, were added. However, because the second and third compositions only contained acetyl tripeptide-30-citrulline and lacked palmitoyl tripeptide-5, ICAM-1 was strongly induced to express on HaCaT cells, with a higher expression level than the UVB control group. This indicates that acetyl tripeptide-30-citrulline, when used alone, is insufficient to inhibit the increase in ICAM-1 expression induced by UVB irradiation.

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

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

[0152] In summary, the combination of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 cetyl ester can effectively inhibit the increase in ICAM-1 expression in HaCaT cells induced by UVB, thus demonstrating that the combination of acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 cetyl ester can suppress the aggravation of inflammation.

[0153] Furthermore, persistent inflammation can damage the skin's collagen and elastin fibers, leading to signs of aging such as wrinkles, sagging, and pigmentation. Therefore, compositions containing acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 cetyl ester can inhibit inflammation-induced aging, producing anti-aging effects.

[0154] <Application Example>

[0155] An 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 emulsion includes the following steps:

[0157] S201. Heat phase A to 85°C and allow it to swell uniformly, and heat phase B to 85°C and allow it to dissolve uniformly.

[0158] S202, then add the heated phase A into the heated phase B and homogenize for 2-3 minutes to obtain the first mixture;

[0159] S203. Add C phase homogenization to the first mixture for 1-2 min, stir and cool to below 40℃ to obtain the second mixture;

[0160] S204. Add phase D and the dissolved phase E to the second mixture and stir until homogeneous to obtain an emulsion.

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

[0162] Table 6. Emulsion Formulation

[0163]

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

[0165] The above detailed embodiments have described the present invention in detail, but these are not intended to limit the invention. The scope of protection of the present invention is not limited to the above embodiments; any equivalent modifications or variations made by those skilled in the art based on the disclosure of the present invention should be included within the scope of protection set forth in the claims.

Claims

1. A composition containing palmitoyl tripeptide-5, characterized in that, include: Acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 in a mass ratio of (1.5~2):

2.

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

2.

4. The composition containing palmitoyl tripeptide-5 according to claim 1, characterized in that, It also includes 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, butanediol, ethylene glycol, hexanediol, dipropylene glycol, diethylene glycol, 2-ethoxyethanol, diethylene glycol monomethyl ether, triethylene glycol monomethyl ether, and sorbitol.

6. The composition containing palmitoyl tripeptide-5 according to claim 4, characterized in that, The solvent is selected from one or more of glycols and glycol ethers.

7. A skincare product, characterized in that, include: Acetyl tripeptide-30 citrulline and palmitoyl tripeptide-5 in a mass ratio of (1.5~2):

2.

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

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