Composition containing acetyl dipeptide-1 cetyl ester, skin care product and application

Abstract

The embodiment of the invention discloses a composition containing acetyl dipeptide-1 cetyl ester, a skin care substance and application. The composition is prepared from acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4, wherein the mass ratio of the acetyl dipeptide-1 cetyl ester to the palmitoyl pentapeptide-4 is (2.5 to 5) to (25 to 50); the effect of protecting skin cells can be achieved.

Description

Technical Field

[0001] The invention relates to the technical field of applying acetyl dipeptide-1 cetyl ester, and in particular to a composition containing acetyl dipeptide-1 cetyl ester, a skin care product and a use thereof. Background Art

[0002] 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.

[0003] 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.

[0004] Intercellular cell adhesion molecule-1 (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.

[0005] When the skin is exposed to ultraviolet rays, the expression of ICAM-1 increases, damaging skin cells.

[0006] Acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4 are both raw materials that can be used in the cosmetics field. At present, the application of acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4 in protecting skin cells from the aspect of ICAM-1 has not been found. Summary of the invention

[0007] 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.

[0008] The inventors of the present application unexpectedly discovered that the combination of acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4 can synergistically inhibit the expression of ICAM-1 in HaCaT cells, that is, produce a synergistic effect in inhibiting the inflammation of HaCaT cells.

[0009] Therefore, another object of the embodiments of the present application is to provide a use of acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4 for the preparation of an inflammatory inhibitor.

[0010] Another object of the embodiments of the present application is to provide a composition comprising acetyl dipeptide-1 cetyl ester, which can be used for anti-inflammatory purposes.

[0011] The embodiments of the present application mainly achieve the above objectives through the following technical solutions.

[0012] In one aspect, the embodiments of the present application provide a composition comprising: acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4.

[0013] In another aspect, the present invention provides a skin care product, comprising: acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4.

[0014] In another aspect, the present application provides a use of acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4 in synergistically preparing an anti-inflammatory preparation.

[0015] The beneficial effects of the embodiments of the present invention include:

[0016] In some embodiments, the present application provides a use of acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4 in synergistically preparing an anti-inflammatory preparation.

[0017] In some embodiments, the present application provides an application of acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4 to synergistically reduce the expression of ICAM1 in skin cells (keratinocytes).

[0018] In some embodiments, the present application provides a composition, including: acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4. When acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4 act together, they can synergistically inhibit and synergistically reduce the inflammatory response caused by UVB irradiation in skin cells (keratinocytes), so that the composition has the effect of inhibiting inflammation.

[0019] In some embodiments, the mass ratio of acetyl dipeptide-1 cetyl ester to palmitoyl pentapeptide-4 is (2.5-5): (25-50), which can have a good anti-inflammatory effect.

[0020] In some embodiments, the present application also provides a skin care product, including acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4, which can have anti-inflammatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A comparative diagram of the relative expression levels of ICAM-1 gene mRNA in the blank control group, UVB control experimental group, and drug-administered experimental group samples 3, 1, and 5 in the experimental examples of this application;

[0022] Figure 2 It is a comparative schematic diagram of the relative expression levels of ICAM-1 gene mRNA in the blank control group, UVB control experimental group, and drug administration experimental group samples 4, 2, and 6 in the experimental examples 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", "third", 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 "include", "comprise", "contain", "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device 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 these processes, methods, products or devices.

[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] <Synergistic Use of Acetyl Dipeptide-1 Cetyl Ester and Palmitoyl Pentapeptide-4>

[0028] In the first aspect, the embodiments of the present application provide a use of acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4 in synergistically preparing an anti-inflammatory preparation.

[0029] Acetyl Dipeptide-1cetyl ester, CAS No. 196604-48-5, chemical formula C 33 H 57 N5O5, the structure is shown in formula 1 below;

[0030] Formula 1:

[0031]

[0032] Palmitoyl pentapeptide-4, also known as pentapeptide (Palmitoyl pentapeptide), CAS number is 214047-00-4, chemical formula is C 39 H 75 N7O 10 , the structure is shown in Formula 2 below;

[0033] Formula 2:

[0034]

[0035] HaCaT cells (Human Immortalized Keratinocytes) are immortalized keratinocytes, a type of skin cell.

[0036] Specifically, when acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4 act together, they can synergistically inhibit the expression of ICAM1 in HaCaT cells, that is, they can produce an anti-inflammatory effect on HaCaT cells.

[0037] In some embodiments, the mass ratio of acetyl dipeptide-1 cetyl ester to palmitoyl pentapeptide-4 is (2.5-5): (25-50), which can have a good anti-inflammatory effect.

[0038] In addition, acetyl dipeptide-1 cetyl ester has a lubricating effect. When applied to products, it can make the products easier to spread on the skin and increase the comfort of use.

[0039] <Composition>

[0040] In a second aspect, the present application provides a composition comprising:

[0041] Acetyl dipeptide-1 cetyl ester; and

[0042] Palmitoyl Pentapeptide-4.

[0043] In some embodiments, the mass ratio of acetyl dipeptide-1 cetyl ester to palmitoyl pentapeptide-4 is (2.5-5): (25-50), which can have a good anti-inflammatory effect.

[0044] The combination of acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4 in a specific mass ratio can synergistically inhibit the expression of ICAM1 in HaCaT cells, that is, it can produce an anti-inflammatory effect on HaCaT cells.

[0045] It is easy to understand that due to the anti-inflammatory effect on HaCaT cells, it can further inhibit or avoid inflammation of the epidermis and play a role in protecting the skin.

[0046] In addition, acetyl dipeptide-1 cetyl ester has a lubricating effect. When applied to the composition, it can make the composition easier to spread on the skin and increase the comfort of use.

[0047] UVB is a type of ultraviolet light with a wavelength of 320nm-400nm, also known as medium-wave erythema effect ultraviolet light. UVB ultraviolet light has a stimulating effect on the human body, and excessive exposure can cause inflammatory reactions in the skin. It is understood that in some embodiments, the composition can be used to prepare a UV damage inhibitor to inhibit inflammation caused by UVB.

[0048] In some embodiments, the composition further comprises a solvent.

[0049] In some embodiments, the solvent is a cosmetically acceptable solvent.

[0050] In some embodiments, 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.

[0051] <Skin Care Products>

[0052] The present application also provides a skin care product, comprising the composition described in the embodiment of the second aspect.

[0053] In some embodiments, the skin care product can be in the form of a cream such as milk, ointment, cream, honey, fat, milk, lotion, milk, and milk solution.

[0054] In some embodiments, the skin care product can be in the form of lotion, liquid, water, oil, oil-water separation and other liquid forms.

[0055] In some embodiments, the skin care product may also be in the form of a gel such as gel or glue, or a powder such as loose powder or granules.

[0056] In some embodiments, the skin care product can be in the form of a block of powder, a large solid block, or the like.

[0057] In some embodiments, the skin care product may be in the form of a puree-like solid.

[0058] In some embodiments, the skin care product may be in the form of a wax-based product with wax as the main base material.

[0059] In some embodiments, the skin care product can be in the form of freeze-dried powder, freeze-dried tablets, and other freeze-dried forms.

[0060] In some embodiments, the skin care product may be in the form of a spray with or without a propellant.

[0061] In some embodiments, the skin care product can be in the form of a patch, a film, or the like containing a base material for use with cosmetics.

[0062] In some embodiments, the skin care products can be topically applied to the hair, body hair, trunk, head, face, eyes, lips, hands, feet, whole body skin, fingernails, and nails.

[0063] In some embodiments, the skin care product may contain at least one cosmetically acceptable auxiliary agent, which 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.

[0064] In some embodiments, the skin care product may include at least one cosmetically acceptable active ingredient. Exemplarily, the active ingredient is 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, 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 one or more of plant extracts.

[0065] In some embodiments, the mass concentration of the composition in the skin care product can be 0.0001 to 10% of the total weight of the skin care product, and further, can be 0.001 to 5% of the total weight of the skin care product.

[0066] <Use of the composition>

[0067] It is understandable that, in some embodiments, the composition can be used to prepare skin care products. Therefore, the present application embodiment provides a use of the composition described in the second aspect embodiment in preparing skin care products.

[0068] In some embodiments, the composition can be used as an anti-inflammatory preparation. Therefore, the embodiments of the present application can also provide a use of the composition containing acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4 as an anti-inflammatory preparation.

[0069] In some embodiments, the composition can be used to inhibit the expression of ICAM1 in skin cells. Therefore, the present application embodiment can also provide a use of the composition containing acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4 as an ICAM1 expression inhibitor.

[0070] <Experimental example>

[0071] The following samples were prepared for subsequent Experimental Examples 1 and 2.

[0072] 1. Blank control sample:

[0073] The DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin is prepared by mixing DMEM culture medium, FBS and penicillin / streptomycin; wherein the volume fraction of FBS in the DMEM culture medium solution is 10%, and the volume fraction of penicillin / streptomycin in the DMEM culture medium solution is 1%.

[0074] 2. UVB control sample:

[0075] The DMEM culture medium solution containing 10% FBS and 1% penicillin / streptomycin is prepared by mixing DMEM culture medium, FBS and penicillin / streptomycin; wherein the volume fraction of FBS in the DMEM culture medium solution is 10%, and the volume fraction of penicillin / streptomycin in the DMEM culture medium solution is 1%.

[0076] 3. Sample 1 of the drug administration experimental group:

[0077] The DMEM culture medium solution containing acetyl dipeptide-1 cetyl ester at a concentration of 2.5 μg / mL, 10% FBS and 1% penicillin / streptomycin is prepared by mixing DMEM culture medium, acetyl dipeptide-1 cetyl ester, FBS and penicillin / streptomycin; wherein the volume fraction of FBS in the DMEM culture medium solution is 10%, and the volume fraction of penicillin / streptomycin in the DMEM culture medium solution is 1%.

[0078] 4. Sample 2 of the drug administration experimental group:

[0079] The DMEM culture medium solution containing acetyl dipeptide-1 cetyl ester at a concentration of 5 μg / mL, 10% FBS and 1% penicillin / streptomycin is prepared by mixing DMEM culture medium, acetyl dipeptide-1 cetyl ester, FBS and penicillin / streptomycin; wherein the volume fraction of FBS in the DMEM culture medium solution is 10%, and the volume fraction of penicillin / streptomycin in the DMEM culture medium solution is 1%.

[0080] 5. Sample 3 of the drug administration experimental group:

[0081] The DMEM culture medium solution containing palmitoyl pentapeptide-4 at a concentration of 50 μg / mL, 10% FBS and 1% penicillin / streptomycin is prepared by mixing DMEM culture medium, palmitoyl pentapeptide-4, FBS and penicillin / streptomycin; wherein the volume fraction of FBS in the DMEM culture medium solution is 10%, and the volume fraction of penicillin / streptomycin in the DMEM culture medium solution is 1%.

[0082] 6. Sample 4 of the drug administration experimental group:

[0083] The DMEM culture medium solution containing palmitoyl pentapeptide-4 at a concentration of 25 μg / mL, 10% FBS and 1% penicillin / streptomycin is prepared by mixing DMEM culture medium, palmitoyl pentapeptide-4, FBS and penicillin / streptomycin; wherein the volume fraction of FBS in the DMEM culture medium solution is 10%, and the volume fraction of penicillin / streptomycin in the DMEM culture medium solution is 1%.

[0084] 7. Sample 5 of the drug administration experimental group:

[0085] The DMEM culture medium solution containing 2.5 μg / mL acetyl dipeptide-1 cetyl ester, 50 μg / mL palmitoyl pentapeptide-4, 10% FBS and 1% penicillin / streptomycin is prepared by mixing DMEM culture medium, acetyl dipeptide-1 cetyl ester, palmitoyl pentapeptide-4, FBS and penicillin / streptomycin; wherein the volume fraction of FBS in the DMEM culture medium solution is 10%, and the volume fraction of penicillin / streptomycin in the DMEM culture medium solution is 1%.

[0086] 8. Sample 6 of the drug administration experimental group:

[0087] The DMEM culture medium solution containing 5 μg / mL acetyl dipeptide-1 cetyl ester, 25 μg / mL palmitoyl pentapeptide-4, 10% FBS and 1% penicillin / streptomycin is prepared by mixing DMEM culture medium, acetyl dipeptide-1 cetyl ester, palmitoyl pentapeptide-4, FBS and penicillin / streptomycin; wherein the volume fraction of FBS in the DMEM culture medium solution is 10%, and the volume fraction of penicillin / streptomycin in the DMEM culture medium solution is 1%.

[0088] <Experimental Example 1, Cell Viability Detection Test>

[0089] The CCK8 (Cell Counting Kit CCK 8) experiment was used to test the survival rate of HaCaT cells to detect the synergistic effect of acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4.

[0090] The test method includes the following steps S101-104:

[0091] Step S101, cell inoculation.

[0092] Specifically, human immortalized keratinocytes (HaCaT) were taken and cultured at 2×10 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.

[0093] Step S102: medication administration.

[0094] Specifically, when the cell plating rate in the 96-well plate reached 40% to 50%, the cells in the 96-well plate were divided into a blank control group and 6 experimental groups, the 6 experimental groups 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, and drug administration experimental group 6, each with 6 replicate wells; except for the blank control group, 100 μL of the corresponding drug administration treatment was added to each well of the other experimental groups, and after the drug administration was completed, the cells were placed in an incubator (37° C., 5% CO2) and cultured for 24 h.

[0095] Among them, the medication treatments corresponding to the blank control group, medication experimental group 1, medication experimental group 2, medication experimental group 3, medication experimental group 4, medication experimental group 5, and medication experimental group 6 are: blank control sample, medication experimental group sample 1, medication experimental group sample 2, medication experimental group sample 3, medication experimental group sample 4, medication experimental group sample 5, and medication experimental group sample 6.

[0096] Step S103: CCK8 detection.

[0097] After drug treatment, after the cells were incubated for 24 hours, the supernatant was discarded, 100 μL of CCK8 working solution (prepared in DMEM medium) was added, and incubated at 37°C in the dark for 2 hours. After the incubation, the OD values ​​were read at 450nm and 650nm, and the OD values ​​were used as the relative activity of each group of cells.

[0098] Step S104, calculating the cell survival rate of each experimental group.

[0099] 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. The calculation formula for the cell survival rate of each experimental group is as follows:

[0100] Cell viability = (relative viability of the experimental group - relative viability of the blank control group) / relative viability of the blank 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, the drug administration experimental group 5, and the drug administration experimental group 6 were all greater than 80%, 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 S201 to S204.

[0108] S201, cell seeding:

[0109] Specifically, human immortalized keratinocytes (HaCaT) were taken and cultured at 80×10 4 The cells were inoculated into a 6-well plate at a density of 100 μL / well, and 100 μL of complete medium was added to each well (the complete medium was DMEM medium containing 10% FBS and 1% penicillin / streptomycin);

[0110] Then, the cells were incubated in an incubator (containing 5% CO 2 and at a temperature of 37° C.) for 24 h.

[0111] S202, drug administration:

[0112] When the cell plating rate in the 6-well plate reaches 70% to 80%, a blank control group and 7 experimental groups are set up on the 6-well plate. The 7 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, drug administration experimental group 5, and drug administration experimental group 6;

[0113] In addition to the blank control group, the seven experimental groups were treated with 50 mJ / cm 2The cells were irradiated with a UVB dose, and then the culture medium was discarded and the corresponding drug treatment was added. Three replicate wells were set up in each group. After the drug administration was completed, the cells were placed in an incubator (37°C, 5% CO2) and cultured for 24 hours.

[0114] Among them, the medication treatments corresponding to the blank control group, UVB control experimental group, medication experimental group 1, medication experimental group 2, medication experimental group 3, medication experimental group 4, medication experimental group 5, and medication experimental group 6 are: blank control sample, UVB control sample, medication experimental group sample 1, medication experimental group sample 2, medication experimental group sample 3, medication experimental group sample 4, medication experimental group sample 5, and medication experimental group sample 6.

[0115] S203, RNA extraction:

[0116] After culturing for 24 hours, RNA was extracted from the cells in each group, specifically including the following steps S2031-S20310.

[0117] S2031. 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.

[0118] S2032, centrifuge at 10,000 g and 2-8°C for 15 minutes.

[0119] 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.

[0120] S2033. Transfer the colorless aqueous phase to a new centrifuge tube, add an equal volume of anhydrous ethanol, and gently invert to mix.

[0121] S2034. Add the obtained solution and precipitate into a centrifugal column, centrifuge at 12,000 g for 30 seconds at room temperature, and discard the effluent.

[0122] S2035, then add 500 μl of CB9, centrifuge at 12,000 g for 30 seconds at room temperature, discard the flow-through, and then repeat this step once.

[0123] S2036. Then add 500 μl of WB9, centrifuge at 12,000 g for 30 seconds at room temperature, discard the flow-through, and repeat the step once.

[0124] S2037, centrifuge at 12,000g for 2 minutes at room temperature to completely remove residual ethanol.

[0125] S2038. Place the centrifuge column in the RNase-free tube, add 50-200 μl RNase-free water in the center of the centrifuge column, and let it stand at room temperature for 1 minute.

[0126] S2039, centrifuge at 12,000 g for 1 minute at room temperature to elute RNA.

[0127] S20310. Use a micro-spectrophotometer (Thermo Scientific NanoDrop) to measure the concentration of RNA, obtain RNA samples with measured concentrations in each group, and store them at -80°C.

[0128] S204. Perform qPCR on the RNA samples with measured concentrations in each group to determine the relative expression level of mRNA.

[0129] Step S204 specifically includes steps S2041 to S2043:

[0130] S2041, reverse transcribe the RNA samples with measured concentrations in each group to obtain reverse transcribed samples,

[0131] 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.

[0132] 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.

[0133] Table 2. RT-PCR reaction system

[0134] name volume RNA 2ug <![CDATA[RNase Free H2O]]> Up to 20μL 5X Evo M-MLV RT Master Mix 4μL

[0135] Table 3. RT-PCR reaction procedure

[0136] temperature time 37℃ 40min 85℃ 5s 12℃ ∞

[0137] S2042. Then dilute 20 μL of the reverse transcription sample to 400 μL to obtain a cDNA sample with a final concentration of 5 ng / μL.

[0138] S2043. Take cDNA samples for qPCR reaction and analyze the relative expression level of mRNA of relevant genes (ICAM-1 gene) in each group.

[0139] 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.

[0140] Among them, the volume of each well of the qPCR reaction system is 10 μL, as can be seen in Table 4;

[0141] The qPCR reaction program can be found in Table 5.

[0142] Table 4. qPCR reaction system

[0143] 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

[0144] Table 5. qPCR reaction procedure

[0145] temperature time 95℃ 5min 95℃ 10s 60℃ 20s 72℃(scan) 30s 40X Melt(scan) 15s

[0146] Experimental results and analysis:

[0147] The experimental results are shown in Figure 1-2 .

[0148] Figure 1 A comparative diagram of the relative expression levels of ICAM-1 gene mRNA in the blank control group, UVB control experimental group, and drug-treated experimental group samples 3, 1, and 5. Figure 1 In the figure, the five bars (A, B, E, C, G) from left to right correspond to the blank control group, the UVB control experimental group, the drug administration experimental group sample 3 (containing 50 μg / mL of palmitoyl pentapeptide-4), the drug administration experimental group sample 1 (containing 2.5 μg / mL of acetyl dipeptide-1 cetyl ester), and the drug administration experimental group sample 5 (containing 2.5 μg / mL of acetyl dipeptide-1 cetyl ester and 50 μg / mL of palmitoyl pentapeptide-4). Figure 2 A comparative diagram of the relative expression levels of ICAM-1 gene mRNA in the blank control group, UVB control experimental group, and drug-treated experimental group samples 4, 2, and 6. Figure 2In the figure, the five bars (A, B, F, D, H) from left to right correspond to the blank control group, the UVB control experimental group, the drug administration experimental group sample 4 (containing 25 μg / mL of palmitoyl pentapeptide-4), the drug administration experimental group sample 2 (containing 5 μg / mL of acetyl dipeptide-1 cetyl ester), and the drug administration experimental group sample 6 (containing 5 μg / mL of acetyl dipeptide-1 cetyl ester and 25 μg / mL of palmitoyl pentapeptide-4).

[0149] like Figure 1 As shown, after adding palmitoyl pentapeptide-4 or acetyl dipeptide-1 cetyl ester to samples 1 and 3 of the UVB-treated dosing experimental group, ICAM-1 was strongly induced to express on HaCaT cells, and the expression level was higher than that of the UVB control experimental group that was not treated with UVB. However, surprisingly, after adding palmitoyl pentapeptide-4 or acetyl dipeptide-1 cetyl ester in combination, sample 5 of the UVB-treated dosing experimental group showed inhibition of ICAM-1 expression. That is, the combination of palmitoyl pentapeptide-4 and acetyl dipeptide-1 cetyl ester can inhibit the increase in the expression of ICAM-1 on HaCaT cells induced by UVB, which further proves that the combination of palmitoyl pentapeptide-4 and acetyl dipeptide-1 cetyl ester can inhibit inflammation to a certain extent.

[0150] like Figure 2 As shown, after adding palmitoyl pentapeptide-4 or acetyl dipeptide-1 cetyl ester to samples 2 and 4 of the UVB-treated drug administration group, ICAM-1 was strongly induced to express on HaCaT cells, and the expression level was higher than that of the UVB group treated with UVB without drug addition. However, after adding palmitoyl pentapeptide-4 or acetyl dipeptide-1 cetyl ester in combination, sample 6 of the UVB-treated drug administration group showed an inhibition of ICAM-1 expression. In other words, the combination of palmitoyl pentapeptide-4 and acetyl dipeptide-1 cetyl ester can inhibit the increase in the expression of ICAM-1 on HaCaT cells induced by UVB to a certain extent, and further prove that the combination of palmitoyl pentapeptide-4 and acetyl dipeptide-1 cetyl ester can inhibit the aggravation of inflammation. In addition, it is easy to understand that a continuous inflammatory response can destroy the collagen and elastic fibers of the skin, causing wrinkles, sagging, pigmentation and other aging phenomena on the skin. Therefore, the combination of palmitoyl pentapeptide-4 and acetyl dipeptide-1 cetyl ester can inhibit the aging phenomenon caused by inflammation and achieve the technical effect of anti-aging.

[0151] In addition, when the above experiments involve the following reagents or equipment, they can be implemented with reference to the following standards:

[0152] 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.

[0153] Fetal bovine serum (FBS) was purchased from Gibco brand fetal bovine serum of Thermo Fisher Scientific (China) Co., Ltd., product number 10091148.

[0154] 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.

[0155] CCK8 working solution is taken from Beyotime / Biyuntian C0040 Cell Counting Kit-8 (CCK-8 Kit). The main chemical in CCK8 working 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.

[0156] 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.

[0157] TransZol Up Plus RNA Kit (Transgen) is suitable for extracting total RNA from cells and tissues. The sample is lysed with TransZol Up. After adding RNA Extraction Agent, the solution is divided into a colorless aqueous phase and a pink organic phase. RNA is in the aqueous phase. A silica gel membrane centrifugal column is used to specifically adsorb RNA in the aqueous phase.

[0158] 5X Evo M-MLV RT Reaction Mix (Agbio, AG11728) is a reverse transcription kit using M-MLV reverse transcriptase. The cDNA product can be directly used for qPCR detection. 5X Evo M-MLV RT Reaction Mix Ver.2 contains all the components required for reverse transcription reaction.

[0159] qPCR SYBR Green Master Mix is ​​a premixed solution for 2× real-time quantitative PCR amplification. The Mix contains a hot start DNA Polymerase, SYBR Green I, dNTPs, Mg2+. qPCR SYBR Green MasterMix can be selected qPCR SYBR Green Master Mix (No Rox) can be purchased from Yeasen, catalog number 11202ES.

[0160] 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).

[0161] <Application Examples>

[0162] Application Example 1

[0163] Prepare the emulsion as follows:

[0164] Preparation method of emulsion:

[0165] Heat phase A to 85°C and swell evenly, heat phase B to 85°C and dissolve evenly. After heating, add phase A to phase B and homogenize for 2-3 minutes, then add phase C and homogenize for 1-2 minutes. Stir and cool to below 40°C, add phase D and dissolved phase E and stir evenly to obtain an emulsion.

[0166] The formula of the emulsion is shown in Table 6 below.

[0167] Table 6. Emulsion formula

[0168]

[0169] Since the emulsion of Application Example 1 contains palmitoyl pentapeptide-4 and acetyl dipeptide-1 cetyl ester, it has anti-inflammatory effect and can protect the skin.

[0170] Application Example 2

[0171] A composition was prepared as follows:

[0172] Preparation method of the composition: heat phase A to swell evenly, then stir and cool to below 40°C, add phase B and stir evenly, then add dissolved phase C and stir evenly, then add phase D and stir evenly to obtain the composition.

[0173] Table 7. Composition formula

[0174]

[0175] The composition of Application Example 2 has anti-inflammatory effects and can protect the skin because it contains palmitoyl pentapeptide-4 and acetyl dipeptide-1 cetyl ester.

[0176] 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 acetyl dipeptide-1 cetyl ester, characterized in that: include: Acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4; The mass ratio of the acetyl dipeptide-1 cetyl ester to the palmitoyl pentapeptide-4 is (2.5-5): (25-50).

2. The composition containing acetyl dipeptide-1 cetyl ester according to claim 1, characterized in that: Also included are solvents.

3. The composition containing acetyl dipeptide-1 cetyl ester according to claim 2, 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.

4. A skin care product, characterized in that include: Acetyl dipeptide-1 cetyl ester and palmitoyl pentapeptide-4; The mass ratio of the acetyl dipeptide-1 cetyl ester to the palmitoyl pentapeptide-4 is (2.5-5): (25-50).

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

6. The composition containing acetyl dipeptide-1 cetyl ester according to claim 5, 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.

7. Use of the composition containing acetyl dipeptide-1 cetyl ester according to claim 1 as an anti-inflammatory preparation.

8. Use of the composition containing acetyl dipeptide-1 cetyl ester according to claim 1 as an ICAM1 expression inhibitor.

9. Use of the composition containing acetyl dipeptide-1 cetyl ester according to claim 1 for preparing skin care products.

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