Application of cyclic dipeptide to regulation of cytokines and preparation of anti-inflammatory soothing cosmetics

Abstract

The invention provides application of cyclic dipeptide to regulation of cytokines and preparation of anti-inflammatory soothing cosmetics, and belongs to the technical field of cosmetics. In a cell factor network participating in the inflammation process, key media such as TNF-alpha, IL-1beta, IL-6 and IL-10 play an important role in the skin level, and the cyclo (glycine-L-proline) dipeptide can regulate release of the cell factors or limit the effect of the cell factors, so that the cyclo (glycine-L-proline) dipeptide can have anti-inflammatory and soothing effects. According to the cosmetic disclosed by the invention, the cyclic dipeptide is added, so that the anti-inflammatory and soothing effects of the cosmetic can be greatly improved, at least one effect of reducing erythema, reducing percutaneous water loss, relieving stabbing pain and reducing an after-sun erythema index is realized, and the cosmetic disclosed by the invention can be suitable for sensitive skin.

Description

Technical Field

[0001] The present application relates to the field of cosmetic technology, and in particular to the use of a cyclic dipeptide in regulating cytokines and in preparing anti-inflammatory and soothing cosmetics. Background Art

[0002] Sensitive skin refers to skin that reacts to physical stimuli (such as temperature changes), chemicals, or emotional fluctuations, exhibiting transient or recurring redness, itching, burning, dryness, and scaling. This hyperreactive state may be accompanied by dilated capillaries or localized edema. This skin condition is particularly prevalent on exposed areas such as the face and neck.

[0003] Therefore, how to design a cosmetic suitable for sensitive skin is a problem that needs to be solved urgently. Summary of the Invention

[0004] The present application provides an application of a cyclic dipeptide in regulating cytokines and in preparing anti-inflammatory and soothing cosmetics. The cyclic dipeptide has anti-inflammatory and soothing effects and is suitable for sensitive skin.

[0005] The embodiment of the present application is implemented as follows:

[0006] In a first aspect, the present application provides an example of a cyclic dipeptide for use in regulating cytokines. The cytokines include at least one of TNF-α, IL-1β, IL-6, and IL-10, and the cyclic dipeptide is a cyclic (glycine-L-proline) dipeptide.

[0007] In the above technical solution, in the cytokine network involved in the inflammatory process, key mediators such as TNF-α, IL-1β, IL-6 and IL-10 play an important role at the skin level. Cyclic (glycine-L-proline) dipeptide can regulate the release of the above cytokines or limit their effects, thereby enabling it to have anti-inflammatory and soothing effects.

[0008] In some possible embodiments, the concentration of the cyclic dipeptide used to modulate TNF-α is 10 ppm to 1000 ppm.

[0009] In some possible embodiments, the cyclic dipeptide and asiaticoside are used together to regulate TNF-α, and the concentration of asiaticoside is 10 ppm to 1000 ppm.

[0010] In the above technical solution, the cyclic dipeptide can be used in combination with asiaticoside to regulate TNF-α and enhance the regulatory effect.

[0011] In some possible embodiments, the concentration of the cyclic dipeptide used to modulate IL-6 is 10 ppm to 1000 ppm.

[0012] In some possible embodiments, the cyclic dipeptide and the purslane extract are used together to regulate IL-6, and the concentration of the purslane extract is 10 ppm to 1000 ppm.

[0013] In the above technical solution, the cyclic dipeptide can be used in combination with the purslane extract to regulate IL-6 and enhance the regulatory effect.

[0014] In some possible embodiments, the concentration of the cyclic dipeptide used to modulate IL-1β is 10 ppm to 1000 ppm.

[0015] In some possible embodiments, cyclic dipeptide and acetyl dipeptide-1 cetyl ester are used together to regulate IL-1β, and the concentration of acetyl dipeptide-1 cetyl ester is 10 ppm to 1000 ppm.

[0016] In the above technical solution, cyclic dipeptide can be used in combination with acetyl dipeptide-1 cetyl ester to regulate IL-1β and enhance the regulatory effect.

[0017] In some possible embodiments, the concentration of the cyclic dipeptide used to modulate IL-10 is 10 ppm to 1000 ppm.

[0018] In some possible embodiments, the cyclic dipeptide and bisabolol are used together to regulate IL-10, and the concentration of bisabolol is 10 ppm to 1000 ppm.

[0019] In the above technical solution, the cyclic dipeptide can be used in combination with bisabolol to regulate IL-10 and enhance the regulatory effect.

[0020] In the second aspect, the present application provides an example of the use of a cyclic dipeptide in the preparation of anti-inflammatory and soothing cosmetics. The cyclic dipeptide is a cyclic (glycine-L-proline) dipeptide, and the cyclic dipeptide is used to regulate at least one of TNF-α, IL-1β, IL-6 and IL-10.

[0021] In the above technical solution, the cosmetics of the present application can greatly improve the anti-inflammatory and soothing effects of the cosmetics by adding cyclic dipeptides, and achieve at least one of the following effects: reduced erythema, reduced transepidermal water loss, stinging relief, and reduced post-sun erythema index. The cosmetics of the present application can be suitable for sensitive skin. DETAILED DESCRIPTION

[0022] The present application found that the pathogenesis of sensitive skin involves the interaction of multiple factors, in which the immune inflammatory response occupies a central position. TNF-α, as the initiator of the inflammatory cascade, can enhance vascular endothelial permeability, release elastase by activating neutrophils, and promote the secretion of chemokines such as IL-8 by lymphocytes. IL-6, through the gp130 / JAK / STAT3 pathway, not only stimulates B cells to differentiate into plasma cells, but also induces Th17 cell differentiation, thereby increasing epidermal IL-17A secretion. After IL-1β is activated by the NLRP3 inflammasome, it triggers NF-κB nuclear translocation through the MyD88 adaptor protein, prompting keratinocytes to release CXCL8 (IL-8) and attract neutrophil infiltration. IL-10 is interleukin-10, originally known as the "cytokine synthesis regulator". It can inhibit the synthesis of IFN-γ, IL-2 and other cytokines by T lymphocytes, and inhibit the production of TNF-α, IL-1α, IL-1β, IL-6, IL-8 and other factors by monocytes and macrophages. The IL-10 family of cytokines are essential effector and regulator factors in the immune system, limiting excessive inflammatory responses, upregulating innate immunity, and promoting tissue repair mechanisms. They are crucial for combating infection, regulating inflammation, maintaining tissue homeostasis, and influencing autoimmunity and cancer progression. Their signaling is primarily mediated through activation of the Jak kinase and STAT transcription factor pathways. Family members often rely on Jak1 and Tyk2 to activate the downstream key transcription factor STAT3, mediating target gene expression. Notably, IL-10 can also activate STAT1 and STAT5 in certain cell types. Based on their function, the IL-10 family is further subdivided into three subfamilies: the IL-10 subfamily (IL-10 only), the IL-20 subfamily (IL-19, IL-20, IL-22, IL-24, and IL-26), and the distantly related cytokines (i.e., type III interferons, IL-28A / IFN-λ2, IL-28B / IFN-λ3, and IL-29 / IFN-λ1).

[0023] Therefore, regulating the release of inflammatory factors or limiting their effects can soothe the skin and is important for people with sensitive skin or skin irritation symptoms.

[0024] Based on this, the present application provides an application of a cyclic dipeptide in regulating cytokines, wherein the cytokines include at least one of TNF-α, IL-1β, IL-6 and IL-10, and the cyclic dipeptide is a cyclic (glycine-L-proline) dipeptide.

[0025] As the smallest member of the cyclic peptide family, cyclic dipeptides exhibit unique stability advantages due to their ubiquitous rigid diketopiperazine cyclic configuration. This type of compound is widely distributed in animal, plant and microbial metabolites (for example, 17 natural cyclic dipeptides have been isolated from the marine fungus Aspergillus sp.). Their restricted molecular conformations can specifically bind to biological macromolecules (such as enzyme active pockets or receptor proteins), thereby exhibiting significant physiological activities in anti-tumor, antibacterial and neuromodulatory fields. As important functional molecules in nature, cyclic peptides are not only an important source of drug lead compounds, but also play a key role in cell signal transduction and pathogen defense mechanisms. Given the breakthrough potential of cyclic dipeptides in interdisciplinary applications such as medicinal chemistry, synthetic biology and nanomaterials, this field has become a key direction of global biomedical research.

[0026] The information of cyclo(glycine-L-proline) dipeptide is shown in Table 1 below:

[0027] Table 1 Information on cyclo(glycine-L-proline) dipeptide

[0028]

[0029] The cyclic (proline-L-glycine) dipeptide used in the examples of this application can be provided by Redlin. This application provides a method for preparing a cyclic (proline-L-glycine) dipeptide:

[0030] S1 resin pretreatment

[0031] 1. Weigh 1.0g of resin onto a peptide synthesis column;

[0032] 2. DCM swelling: 30 ml × 3 times, 10 minutes each time;

[0033] 3. DIEA activation: 20 ml of 5% DIEA / DCM solution, bubbling with nitrogen for 30 min.

[0034] S2 first amino acid loading

[0035] 1. Dissolve Fmoc-Gly-OH (3 eq) in 10 ml DCM;

[0036] 2. Add DIEA (6 eq) and stir under nitrogen for 2 h;

[0037] 3. End-capping: quench with MeOH / DCM (1:9) solution for 15 min;

[0038] 4. Washing: DCM → DMF 3 times each.

[0039] S3 second amino acid coupling

[0040] 1. Fmoc removal: 20% piperidine / DMF (containing 0.1 M HOBt) treatment for 2 × 10 min; 2. Activation of Fmoc-L-Pro-OH:

[0041] Dissolve 2.5 eq of amino acid in 8 ml of DMF;

[0042] Add 2.4eq HATU + 5eq Oxyma Pure;

[0043] After pre-activation for 5 minutes, inject into the reaction column;

[0044] 3. Coupling: Nitrogen bubbling reaction for 1 h (real-time monitoring of ninhydrin color development).

[0045] S4 intramolecular cyclization

[0046] 1. Resin cleavage: TFE / DCM (1:4) solution treatment 3 × 5 min;

[0047] 2. Dissolve the linear peptide in 5 ml of anhydrous DMF (containing 0.1% TFA);

[0048] 3. Condensation system:

[0049] HATU (3eq) + HOAt (3eq);

[0050] DIEA (6 eq) was added dropwise (rate 0.5 ml / min).

[0051] 4. Cyclization reaction:

[0052] Temperature: -15°C (acetonitrile / dry ice bath);

[0053] Reaction time: 16 h (nitrogen protection);

[0054] Concentration control: 0.005M (high dilution prevents dimerization).

[0055] S5 cleavage and purification

[0056] 1. Resin cleavage: TFA / H2O / TIPS (95:2.5:2.5) treatment for 2 h;

[0057] 2. Precipitation: cold ether precipitation (-20℃ for 12h);

[0058] 3. Purification:

[0059] Semi-preparative HPLC conditions:

[0060] Chromatographic column: XBridge BEH C18 (5 μm, 10 × 250 mm);

[0061] Mobile phase: A: 0.1% TFA / H2O; B: 0.1% TFA / CAN;

[0062] Gradient: 15% B → 35% B / 30 min;

[0063] Freeze-drying: pre-freeze at -80℃ and then vacuum dry for 48h;

[0064] A cyclic (glycine-L-proline) dipeptide was obtained.

[0065] Optionally, the concentration of the cyclic dipeptide used to regulate TNF-α is 10 ppm to 1000 ppm.

[0066] As an example, the concentration of the cyclic dipeptide used to modulate TNF-α can be 10 ppm, 20 ppm, 50 ppm, 80 ppm, 100 ppm, 200 ppm, 500 ppm, or 1000 ppm.

[0067] Optionally, the cyclic dipeptide and asiaticoside are used together to regulate TNF-α, and the concentration of asiaticoside is 10 ppm to 1000 ppm.

[0068] As an example, the concentration of asiaticoside may be 10 ppm, 20 ppm, 50 ppm, 80 ppm, 100 ppm, 200 ppm, 500 ppm or 1000 ppm.

[0069] Cyclic dipeptide can be used in conjunction with Centella asiatica to regulate TNF-α and enhance the regulatory effect.

[0070] Optionally, the concentration of the cyclic dipeptide used to modulate IL-6 is 10 ppm to 1000 ppm.

[0071] As an example, the concentration of the cyclic dipeptide used to modulate IL-6 can be 10 ppm, 20 ppm, 50 ppm, 80 ppm, 100 ppm, 200 ppm, 500 ppm, or 1000 ppm.

[0072] Optionally, the cyclic dipeptide and purslane extract are used together to regulate IL-6, and the concentration of the purslane extract is 10 ppm to 1000 ppm.

[0073] As an example, the concentration of the Portulaca oleracea extract may be 10 ppm, 20 ppm, 50 ppm, 80 ppm, 100 ppm, 200 ppm, 500 ppm, or 1000 ppm.

[0074] Cyclic dipeptide can be used in conjunction with purslane extract to regulate IL-6 and enhance the regulatory effect.

[0075] Optionally, the concentration of the cyclic dipeptide used to modulate IL-1β is 10 ppm to 1000 ppm.

[0076] As an example, the concentration of the cyclic dipeptide used to modulate IL-1β can be 10 ppm, 20 ppm, 50 ppm, 80 ppm, 100 ppm, 200 ppm, 500 ppm, or 1000 ppm.

[0077] Optionally, the cyclic dipeptide and acetyl dipeptide-1 cetyl ester are used in combination to regulate IL-1β, and the concentration of acetyl dipeptide-1 cetyl ester is 10 ppm to 1000 ppm.

[0078] As an example, the concentration of acetyl dipeptide-1 cetyl ester can be 10 ppm, 20 ppm, 50 ppm, 80 ppm, 100 ppm, 200 ppm, 500 ppm, or 1000 ppm.

[0079] Cyclic dipeptide can be used in conjunction with acetyl dipeptide-1 cetyl ester to regulate IL-1β and enhance the regulatory effect.

[0080] Optionally, the concentration of the cyclic dipeptide used to modulate IL-10 is 10 ppm to 1000 ppm.

[0081] As an example, the concentration of the cyclic dipeptide used to modulate IL-10 can be 10 ppm, 20 ppm, 50 ppm, 80 ppm, 100 ppm, 200 ppm, 500 ppm, or 1000 ppm.

[0082] Optionally, the cyclic dipeptide and bisabolol are used together to regulate IL-10, and the concentration of bisabolol is 10 ppm to 1000 ppm.

[0083] As an example, the concentration of bisabolol can be 10 ppm, 20 ppm, 50 ppm, 80 ppm, 100 ppm, 200 ppm, 500 ppm, or 1000 ppm.

[0084] Cyclic dipeptide can be used in conjunction with bisabolol to regulate IL-10 and enhance the regulatory effect.

[0085] In the cytokine network involved in the inflammatory process, key mediators such as TNF-α, IL-1β, IL-6 and IL-10 play an important role at the skin level. Cyclic (glycine-L-proline) dipeptide can regulate the release of the above cytokines or limit their effects, thereby enabling it to have anti-inflammatory and soothing effects.

[0086] The present application also provides an application of a cyclic dipeptide in the preparation of anti-inflammatory and soothing cosmetics. The cyclic dipeptide is a cyclic (glycine-L-proline) dipeptide, and the cyclic dipeptide is used to regulate at least one of TNF-α, IL-1β, IL-6 and IL-10.

[0087] Cosmetics can be selected from the following forms: cream, lotion, gel, toner, serum, mask, eye cream, aerosol cleansing foam, spray, shower gel or facial cleanser.

[0088] The embodiments of the present application will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, the conditions are carried out according to conventional conditions or manufacturer recommendations. The reagents or instruments used are not specified by the manufacturer and are conventional products that can be purchased commercially.

[0089] Example 1

[0090] Cytotoxicity experiment: Cell experiments have shown that the cyclic dipeptide has no cytotoxicity. The method and results are as follows: RAW264.7 cells in the logarithmic growth phase grown in DMEM medium were cultured at a rate of (5-6)×10 4 Cells were seeded into a 96-well plate at a volume of 100 μL / well. An equal amount of PBS was added to the wells around the 96-well plate to prevent evaporation of the culture medium and affect the experimental results. The plates were incubated in a 37°C, 5% CO2 incubator. After 24 hours, the original culture medium was aspirated with a pipette, the plates were washed once with complete culture medium, and 100 μL of new culture medium was added. 10 μL of sample was added to each well and the plates were cultured in an incubator. After 24 hours, the culture medium was discarded, the plates were washed once with PBS, and 100 μL of culture medium containing 10% CCK8 solution was added to each well. The plates were incubated in a 37°C, 5% CO2 incubator for 1-4 hours. The color was observed at any time. When it darkened, the absorbance was measured using a microplate reader. Dual wavelengths (upper 450, lower 650) and zones were set for cytotoxicity detection.

[0091] Example 2

[0092] Anti-inflammatory and soothing efficacy of RAW264.7 cells TNF-α protein content test (ELISA method):

[0093] Group 1 is 10ppm of cyclic dipeptide, group 2 is 100ppm of cyclic dipeptide, group 3 is 10ppm of asiaticoside, group 4 is 100ppm of asiaticoside, group 5 is 10ppm of cyclic dipeptide + 10ppm of asiaticoside, group 6 is 100ppm of cyclic dipeptide + 100ppm of cyclic dipeptide, and group 7 is 100ppm of cyclic dipeptide + 100ppm of asiaticoside.

[0094] The test method is as follows:

[0095] RAW264.7 cells in the logarithmic growth phase grown in DMEM medium were cultured at a rate of (10-15)×10 4 Cells were seeded at 100 μL / well in a 100 mm cell culture dish. 10 mL of culture medium was added to each well and incubated in a 37°C, 5% CO2 incubator. After 24 hours, 100 μL of sample was added to each well. Three replicates were set for each concentration and incubation continued. After 24 hours of incubation, cells were harvested by pipetting, the supernatant discarded, and the tubes were washed twice with PBS, and the supernatant discarded. Whole blood was collected into a tube without anticoagulant and allowed to stand at room temperature for 1 hour. After the whole blood naturally coagulated and serum was separated, the tube was centrifuged at approximately 1500 g for 10 minutes at 4°C. The yellow supernatant was obtained as serum. Whole blood was collected into a tube with anticoagulant, mixed, and placed on ice. The tube was centrifuged at approximately 1500 g for 10 minutes at 4°C. The yellow or light yellow supernatant was obtained as plasma. 100 μL of capture antibody solution was added to each well, covered with a plate sealer, and incubated at 4°C overnight. After overnight, the plate solution was discarded. Wash each well five times with 300-400 μL of wash buffer, patting dry on thick absorbent paper for the final rinse. Dilute the sample with sample diluent at 1:5, 1:10, 1:50, 1:100, 1:1000, and 1:2000 to determine the optimal TNF-α concentration for detection. Prepare a gradient of TNF-α protein standards with working concentrations of 1200 pg / mL, 600 pg / mL, 300 pg / mL, 150 pg / mL, 75 pg / mL, 37.5 pg / mL, 18.75 pg / mL, and 9.38 pg / mL for plotting a standard curve. Add 100 μL / well of sample, standard, and sample diluent to the corresponding wells as the standard, test, and blank groups. Add 50 μL / well of biotin-conjugated anti-human TNF-α antibody to all wells, cover with sealing film, and incubate at room temperature in the dark for 2 hours. Wash the plate five times with 300-400 μL of wash buffer per well, patting dry on thick absorbent paper for the final time. Add 100 μL of diluted streptavidin-HRP to all wells, cover with film, and incubate at room temperature in the dark for 1 hour. Add 300-400 μL of wash buffer to each well, patting dry on thick absorbent paper for the final time. Add 100 μL of TMB colorimetric solution to all wells. Cover with film and incubate at room temperature in the dark for 30 minutes. Add 50 μL of stop solution to all wells, mix thoroughly, and immediately measure the A450 value. Calculate the average absorbance of each replicate standard and sample. The number of replicates should be within 20% of the average. Plot a standard curve for the TNF-α standard. Plot the standard concentration on the horizontal axis and the A450 value on the vertical axis, connecting the coordinate points of each standard with a smooth line. Calculate the corresponding sample concentration using the sample absorbance and the standard curve.

[0096] The sample information and test contents of the TNF-α protein content test for the anti-inflammatory and soothing effects of RAW264.7 cells are shown in Table 2:

[0097] Table 2

[0098]

[0099] Example 3

[0100] Anti-inflammatory and soothing efficacy of RAW264.7 cells IL-6 protein content test (ELISA method):

[0101] Group 1 is 10ppm of cyclic dipeptide, group 2 is 100ppm of cyclic dipeptide, group 3 is 10ppm of purslane extract, group 4 is 100ppm of purslane extract, group 5 is 10ppm of cyclic dipeptide + 10ppm of purslane extract, group 6 is 100ppm of cyclic dipeptide + 100ppm of cyclic dipeptide, and group 7 is 100ppm of cyclic dipeptide + 100ppm of purslane extract.

[0102] The test method is as follows:

[0103] RAW264.7 cells in the logarithmic growth phase grown in DMEM medium were cultured at a rate of (10-15)×10 4Cells were seeded at 100 μL / well in a 100 mm cell culture dish. 10 mL of culture medium was added to each well and incubated in a 37°C, 5% CO2 incubator. After 24 hours, 100 μL of sample was added to each well. Three replicates were set for each concentration and incubation continued. After 24 hours of incubation, cells were harvested by pipetting, the supernatant discarded, and the tubes were washed twice with PBS, and the supernatant discarded. Whole blood was collected into a tube without anticoagulant and allowed to stand at room temperature for 1 hour. After the whole blood naturally coagulated and serum was separated, the tube was centrifuged at approximately 1500 g for 10 minutes at 4°C. The yellow supernatant was obtained as serum. Whole blood was collected into a tube with anticoagulant, mixed, and placed on ice. The tube was centrifuged at approximately 1500 g for 10 minutes at 4°C. The yellow or light yellow supernatant was obtained as plasma. 100 μL of capture antibody solution was added to each well, covered with a plate sealer, and incubated at 4°C overnight. After overnight, the plate solution was discarded. Wash each well five times with 300-400 μL of wash buffer, patting dry on thick absorbent paper for the final wash. Dilute the sample with sample diluent at 1:5, 1:10, 1:50, 1:100, 1:1000, and 1:2000 to determine the optimal IL-6 concentration for detection. Prepare a gradient of IL-6 protein standards with working concentrations of 1200 pg / mL, 600 pg / mL, 300 pg / mL, 150 pg / mL, 75 pg / mL, 37.5 pg / mL, 18.75 pg / mL, and 9.38 pg / mL for plotting a standard curve. Add 100 μL / well of sample, standard, and sample diluent to the corresponding wells as the standard, test, and blank groups. Add 50 μL / well of biotin-conjugated anti-human IL-6 antibody to all wells, cover with sealing film, and incubate at room temperature in the dark for 2 hours. Wash the plate five times with 300-400 μL of wash buffer per well, patting dry on thick absorbent paper for the final time. Add 100 μL of diluted streptavidin-HRP to all wells, cover with film, and incubate at room temperature in the dark for 1 hour. Wash the plate five times with 300-400 μL of wash buffer per well, patting dry on thick absorbent paper for the final time. Add 100 μL of TMB colorimetric solution to all wells. Cover with film and incubate at room temperature in the dark for 30 minutes. Add 50 μL of stop solution to all wells, mix thoroughly, and immediately measure the A450 value. Calculate the average absorbance of each replicate standard and sample. The number of replicates should be within 20% of the average. Plot a standard curve for the IL-6 standard. Plot the standard concentration on the horizontal axis and the A450 value on the vertical axis, connecting the coordinate points of each standard with a smooth line. Calculate the corresponding sample concentration based on the sample absorbance and the standard curve.

[0104] The sample information and test contents of the IL-6 protein content test for the anti-inflammatory and soothing effects of RAW264.7 cells are shown in Table 3:

[0105] Table 3

[0106]

[0107] Example 4

[0108] Anti-inflammatory and soothing efficacy of RAW264.7 cells IL-1β protein content test (qPCR method):

[0109] Group 1 is 10ppm of cyclic dipeptide, group 2 is 100ppm of cyclic dipeptide, group 3 is 10ppm of acetyl dipeptide-1 cetyl ester, group 4 is 100ppm of acetyl dipeptide-1 cetyl ester, group 5 is 10ppm of cyclic dipeptide + 10ppm of acetyl dipeptide-1 cetyl ester, and group 6 is 10ppm of cyclic dipeptide + 10ppm of cyclic dipeptide.

[0110] The test method is as follows:

[0111] RAW264.7 cells in the logarithmic growth phase grown in DMEM medium were cultured at a rate of (10-15)×10 4Cells were seeded at 100 μL / well in a 100 mm cell culture dish. 10 mL of culture medium was added to each well and incubated in a 37°C, 5% CO2 incubator. After 24 hours, 100 μL of sample was added to each well. Three replicates were set for each concentration and incubation continued. After 24 hours of incubation, cells were harvested by pipetting, the supernatant discarded, and the tubes were washed twice with PBS, and the supernatant discarded. Whole blood was collected into a tube without anticoagulant and allowed to stand at room temperature for 1 hour. After the whole blood naturally coagulated and serum was separated, the tube was centrifuged at approximately 1500 g for 10 minutes at 4°C. The yellow supernatant was obtained as serum. Whole blood was collected into a tube with anticoagulant, mixed, and placed on ice. The tube was centrifuged at approximately 1500 g for 10 minutes at 4°C. The yellow or light yellow supernatant was obtained as plasma. 100 μL of capture antibody solution was added to each well, covered with a plate sealer, and incubated at 4°C overnight. After overnight, the plate solution was discarded. Wash each well five times with 300-400 μL of wash buffer, patting dry on thick absorbent paper for the final wash. Dilute the sample with sample diluent at 1:5, 1:10, 1:50, 1:100, 1:1000, and 1:2000 to determine the optimal IL-1β concentration for detection. Prepare a gradient of IL-1β protein standards with working concentrations of 1200 pg / mL, 600 pg / mL, 300 pg / mL, 150 pg / mL, 75 pg / mL, 37.5 pg / mL, 18.75 pg / mL, and 9.38 pg / mL for plotting the standard curve. Add 100 μL / well of sample, standard, and sample diluent to the corresponding wells as the standard, test, and blank groups, respectively. Add 50 μL / well of biotin-conjugated anti-human IL-1β antibody to all wells, cover with a sealing film, and incubate at room temperature in the dark for 2 hours. Wash the plate five times with 300-400 μL of wash buffer per well, patting dry on thick absorbent paper for the final time. Add 100 μL of diluted streptavidin-HRP to all wells, cover with a sealing film, and incubate at room temperature in the dark for 1 hour. Wash the plate five times with 300-400 μL of wash buffer per well, patting dry on thick absorbent paper for the final time. Add 100 μL of TMB colorimetric solution to all wells. Cover with a sealing film and incubate at room temperature in the dark for 30 minutes. Add 50 μL of stop solution to all wells, mix thoroughly, and immediately measure the A450 value. Calculate the average absorbance of each replicate standard and sample. The number of replicates should be within 20% of the average. Construct a standard curve for the IL-1β standard. Use the standard concentration as the horizontal axis and the A450 value as the vertical axis, and connect the coordinate points of each standard with a smooth line. Calculate the corresponding concentration of the sample based on the sample's absorbance value and the standard curve.

[0112] The sample information and test contents of the IL-1β protein content test for the anti-inflammatory and soothing effects of RAW264.7 cells are shown in Table 4:

[0113] Table 4

[0114]

[0115]

[0116] Example 5

[0117] Anti-inflammatory and soothing efficacy of RAW264.7 cells IL-10 protein content test (ELISA method):

[0118] Group 1 is 10 ppm of cyclic dipeptide, group 2 is 100 ppm of cyclic dipeptide, group 3 is 10 ppm of bisabolol, group 4 is 100 ppm of bisabolol, group 5 is 10 ppm of cyclic dipeptide + 10 ppm of bisabolol, group 6 is 100 ppm of cyclic dipeptide + 100 ppm of cyclic dipeptide, and group 7 is 100 ppm of cyclic dipeptide + 100 ppm of bisabolol.

[0119] The test method is as follows:

[0120] RAW264.7 cells in the logarithmic growth phase grown in DMEM medium were cultured at a rate of (10-15)×10 4Cells were seeded at 100 μL / well in a 100 mm cell culture dish. 10 mL of culture medium was added to each well and incubated in a 37°C, 5% CO2 incubator. After 24 hours, 100 μL of sample was added to each well. Three replicates were set for each concentration and incubation continued. After 24 hours of incubation, cells were harvested by pipetting, the supernatant discarded, and the tubes were washed twice with PBS, and the supernatant discarded. Whole blood was collected into a tube without anticoagulant and allowed to stand at room temperature for 1 hour. After the whole blood naturally coagulated and serum was separated, the tube was centrifuged at approximately 1500 g for 10 minutes at 4°C. The yellow supernatant was obtained as serum. Whole blood was collected into a tube with anticoagulant, mixed, and placed on ice. The tube was centrifuged at approximately 1500 g for 10 minutes at 4°C. The yellow or light yellow supernatant was obtained as plasma. 100 μL of capture antibody solution was added to each well, covered with a plate sealer, and incubated at 4°C overnight. After overnight, the plate solution was discarded. Wash each well five times with 300-400 μL of wash buffer, patting dry on thick absorbent paper for the final wash. Dilute the sample with sample diluent at 1:5, 1:10, 1:50, 1:100, 1:1000, and 1:2000 to determine the optimal IL-10 concentration for detection. Prepare a gradient of IL-10 protein standards with working concentrations of 1200 pg / mL, 600 pg / mL, 300 pg / mL, 150 pg / mL, 75 pg / mL, 37.5 pg / mL, 18.75 pg / mL, and 9.38 pg / mL for plotting a standard curve. Add 100 μL / well of sample, standard, and sample diluent to the corresponding wells as the standard, test, and blank groups, respectively. Add 50 μL / well of biotin-conjugated anti-human IL-10 antibody to all wells, cover with film, and incubate at room temperature in the dark for 2 hours. Wash the plate five times with 300-400 μL of wash buffer per well, patting dry on thick absorbent paper for the final time. Add 100 μL of diluted streptavidin-HRP to all wells, cover with film, and incubate at room temperature in the dark for 1 hour. Wash the plate five times with 300-400 μL of wash buffer per well, patting dry on thick absorbent paper for the final time. Add 100 μL of TMB colorimetric solution to all wells. Cover with film and incubate at room temperature in the dark for 30 minutes. Add 50 μL of stop solution to all wells, mix thoroughly, and immediately measure the A450 value. Calculate the average absorbance of each replicate of the standard and sample. The number of replicates should be within 20% of the average. Construct a standard curve for the IL-10 standard. Use the standard concentration as the horizontal axis and the A450 value as the vertical axis, and connect the coordinate points of each standard with a smooth line. Calculate the corresponding concentration of the sample based on the sample's absorbance value and the standard curve.

[0121] The sample information and test contents of the IL-10 protein content test for the anti-inflammatory and soothing effects of RAW264.7 cells are shown in Table 5:

[0122] Table 5

[0123]

[0124] Application Example 1

[0125] The present invention provides a soothing cream and a preparation method thereof, the formula of which is shown in Table 6:

[0126] Table 6

[0127]

[0128] To prepare the soothing cream:

[0129] S1. Mix and stir phase A and heat to 80 degrees Celsius to dissolve evenly;

[0130] S2. Mix phase B, heat to 80 degrees Celsius and stir evenly (B1 is dissolved in advance);

[0131] S3, mix phase A and phase B, homogenize at 8000 rad for 2 min;

[0132] S4. Mix phase C with a small amount of water and heat to 70 degrees Celsius to dissolve, then add to the material.

[0133] Application Example 2

[0134] The present invention provides an essence and a preparation method thereof, the formula of which is shown in Table 7:

[0135] Table 7

[0136]

[0137] The preparation method of the essence is as follows:

[0138] S1. Heat phase A to 80 degrees Celsius and stir evenly;

[0139] S2. Heat the mixture of phase B to 80 degrees Celsius and stir evenly;

[0140] S3, mix phase A and phase B, homogenize at 8000 rad for 2 min;

[0141] S4. Mix phase C with a small amount of water and heat to 70 degrees Celsius to dissolve, then add to the material.

[0142] Application Example 3

[0143] The present invention provides an emulsion and a preparation method thereof, the formula of which is shown in Table 8:

[0144] Table 8

[0145]

[0146] The preparation method of the emulsion is as follows:

[0147] S1. Mix and stir phase A and heat to 80 degrees Celsius to dissolve evenly; wait until the raw materials are evenly dissolved before adding and stirring to dissolve to prevent bubbles;

[0148] S2, heating the mixed materials of phase B to 80 degrees Celsius;

[0149] S3. Add phase B to phase A and homogenize at 7000 rad for 2 min.

[0150] S4. Dissolve phase C in water and add it to the material and stir evenly;

[0151] S5. Mix phase D with a small amount of water and heat to 70 degrees Celsius to dissolve, then add to the material.

[0152] Experimental Example 1

[0153] The results of the cytotoxicity experiment are shown in Table 9.

[0154] Table 9 Cytotoxicity test results

[0155] Cyclic dipeptide concentration (ppm) Cell viability (Mean±SD) Blank control 100.0±2.5％ 1ppm 107±8.4％ 10ppm 107±2.8％ 100ppm 107±2.1％ 1000ppm 108±5.4％

[0156] As shown in Table 9, the cyclic dipeptide has no cytotoxicity and does not cause a decrease in cell viability.

[0157] Experimental Example 2

[0158] The results of TNF-α protein content test (ELISA method) are shown in Table 10.

[0159] Table 10 TNF-α protein content test (ELISA method) results

[0160]

[0161] According to the results in the above table, compared with the NC group, the TNF-α gene expression levels were significantly downregulated, with the downregulation rates of Group 1, Group 2, Group 5, Group 6 and Group 7 being 21.0%, 65.4%, 36.0%, 76.7% and 74.1%, respectively. Among them, the downregulation rate of Group 6 was higher than that of Group 7, indicating that cyclic dipeptide has a significant inhibitory effect on TNF-α expression in the range of 10 to 100 ppm and has a skin soothing effect.

[0162] Experimental Example 3

[0163] The results of IL-6 protein content test (ELISA method) are shown in Table 11.

[0164] Table 11 IL-6 protein content test (ELISA method) results

[0165]

[0166] According to the results in the above table, compared with the NC group, the IL-6 gene expression levels were significantly downregulated, with the downregulation rates of Group 1, Group 2, Group 5, Group 6 and Group 7 being 22.6%, 63.1%, 33.7%, 78.0% and 74.2%, respectively. Among them, the downregulation rate of Group 6 was higher than that of Group 7, indicating that cyclic dipeptide has a significant inhibitory effect on IL-6 expression in the range of 10 to 100 ppm and has a skin soothing effect.

[0167] Experimental Example 4

[0168] The results of IL-1β expression test are shown in Table 12.

[0169] Table 12 IL-1β expression test results

[0170]

[0171]

[0172] According to the results in the above table, compared with the NC group, the IL-1β gene expression levels were significantly downregulated, with the downregulation rates of Group 1, Group 2, Group 5 and Group 6 being 28%, 61%, 42% and 35%, respectively. Among them, the downregulation rate of Group 5 was higher than that of Group 6, indicating that cyclic dipeptide has a significant inhibitory effect on IL-1β expression in the range of 10 to 100 ppm and has a skin soothing effect.

[0173] Experimental Example 5

[0174] The results of IL-10 protein content test (ELISA method) are shown in Table 13.

[0175] Table 13L-10 protein content test (ELISA method) results

[0176]

[0177] According to the results in the above table, compared with the NC group, the IL-10 gene expression levels were significantly upregulated, with the upregulation rates of Group 1, Group 2, Group 5, Group 6 and Group 7 being 50.3%, 249.0%, 101.3%, 350.9% and 271.5%, respectively. Among them, the upregulation rate of Group 6 was higher than that of Group 7, indicating that cyclic dipeptide has a significant effect of promoting IL-10 expression in the range of 10 to 100 ppm and has a skin soothing effect.

[0178] The foregoing description is merely a specific embodiment of the present application and is not intended to limit the present application. Persons skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A use of a cyclic dipeptide in regulating cytokines, wherein the cytokines include at least one of TNF-α, IL-1β, IL-6 and IL-10, and the cyclic dipeptide is a cyclic (glycine-L-proline) dipeptide.

2. The use of the cyclic dipeptide according to claim 1 in regulating cytokines, characterized in that The concentration of the cyclic dipeptide used to regulate TNF-α is 10 ppm to 1000 ppm.

3. The use of the cyclic dipeptide according to claim 1 in regulating cytokines, characterized in that The cyclic dipeptide and asiaticoside are used together to regulate TNF-α, and the concentration of asiaticoside is 10ppm to 1000ppm.

4. The use of the cyclic dipeptide according to claim 1 in regulating cytokines, characterized in that The concentration of the cyclic dipeptide used to regulate IL-6 is 10 ppm to 1000 ppm.

5. The use of the cyclic dipeptide according to claim 1 in regulating cytokines, characterized in that The cyclic dipeptide and the purslane extract are used together to regulate IL-6, and the concentration of the purslane extract is 10ppm to 1000ppm.

6. The use of the cyclic dipeptide according to claim 1 in regulating cytokines, characterized in that The concentration of the cyclic dipeptide used to regulate IL-1β is 10 ppm to 1000 ppm.

7. The use of the cyclic dipeptide in regulating cytokines according to claim 1, characterized in that: The cyclic dipeptide and acetyl dipeptide-1 cetyl ester are used in combination to regulate IL-1β, and the concentration of the acetyl dipeptide-1 cetyl ester is 10 ppm to 1000 ppm.

8. The use of the cyclic dipeptide in regulating cytokines according to claim 1, characterized in that: The concentration of the cyclic dipeptide used to regulate IL-10 is 10 ppm to 1000 ppm.

9. The use of the cyclic dipeptide according to claim 1 in regulating cytokines, characterized in that The cyclic dipeptide and bisabolol are used together to regulate IL-10, and the concentration of the bisabolol is 10 ppm to 1000 ppm.

10. Use of a cyclic dipeptide in the preparation of anti-inflammatory and soothing cosmetics, wherein the cyclic dipeptide is a cyclic (glycine-L-proline) dipeptide, and the cyclic dipeptide is used to regulate at least one of TNF-α, IL-1β, IL-6 and IL-10.

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