Soothing composition, application thereof and skin care product

Abstract

The invention belongs to the technical field of skin care, and discloses a soothing composition, application and a skincare product, the soothing composition comprises lactoferrin, oat extract and calcium gluconate, and the weight ratio of the lactoferrin to the oat extract to the calcium gluconate is (0.1-0.5): (40-60): (4-8); the lactoferrin, the oat extract and the calcium gluconate are compounded, so that the obvious effect of inhibiting the gene expression of the kallikrein-related enzyme 7 can be achieved, and the skin soothing effect of the composition is effectively improved by regulating the gene expression of the kallikrein-related enzyme 7.

Description

Technical Field

[0001] This application relates to the field of skin care, and more particularly to a soothing composition and its use, and skin care products. Background Technology

[0002] As the largest organ in the human body, the skin plays a vital protective role, making skin health and care increasingly important. In daily life, human skin comes into direct contact with the external environment. Pollutants such as dust, particles, harmful gases, and various bacteria in the air can damage the skin (especially facial skin), causing inflammation, aging, allergies, dryness, and other skin problems. Furthermore, unhealthy daily habits, increasing work and life stress, and changes in dietary structure further exacerbate and increase the frequency of skin problems and symptoms.

[0003] Skin soothing is an important topic in modern skincare. Its necessity lies in relieving skin inflammation caused by environmental stress (such as ultraviolet rays and pollution), chemical irritants (such as cosmetic ingredients), and internal factors (such as sensitive skin and damaged skin barrier), thereby maintaining a healthy skin condition.

[0004] Therefore, it is necessary to develop a skin care composition that can effectively soothe the skin, thereby maintaining the skin in a healthy state and preventing the aggravation and frequent occurrence of skin problems and symptoms. Summary of the Invention

[0005] Therefore, the primary objective of this application is to provide a soothing composition that can inhibit the increased expression of the kinin-releasing enzyme 7 gene, thereby effectively maintaining the skin in a healthy state.

[0006] Another objective of this application is to provide the use of the soothing composition in the preparation of skin care products that can effectively provide excellent skin soothing effects.

[0007] Another object of this application is to provide a skin care product containing the soothing composition of this application, which has excellent skin soothing effects.

[0008] To achieve the above objectives, this application provides a soothing composition comprising lactoferrin, oat extract, and calcium gluconate, wherein the weight ratio of lactoferrin, oat extract, and calcium gluconate is 0.1~0.5:40~60:4~8.

[0009] Lactoferrin can promote the expression of filaggrin (FLG) and aquaporin AQP-3, and inhibit the secretion of inflammatory factors such as TNF-α and IL-6. It also has a bidirectional regulatory effect on the skin microecology, inhibiting unfavorable bacteria and promoting the growth of beneficial bacteria. Oat extract can increase skin moisture content and reduce transepidermal water loss. Calcium gluconate can relieve symptoms such as redness and itching caused by allergies by increasing capillary density and reducing tissue fluid exudation. It can also reduce skin inflammation by inhibiting the release of inflammatory mediators such as histamine from mast cells.

[0010] This application utilizes a combination of lactoferrin, oat extract, and calcium gluconate to significantly inhibit the expression of the kallikrein-associated enzyme 7 gene. By regulating the expression of the kallikrein-associated enzyme 7 gene, the composition effectively enhances its skin-soothing efficacy.

[0011] The soothing composition of this application also includes Polygonatum sibiricum extract and phytosphingosine, wherein the weight ratio of lactoferrin, oat extract, calcium gluconate, Polygonatum sibiricum extract and phytosphingosine is 0.1~0.5:40~60:4~8:0.05~2.5:0.1~1.5.

[0012] This application also provides the use of the above-described soothing composition in the preparation of skin care products.

[0013] This application also provides a skin care product containing the above-mentioned soothing composition; Furthermore, the skin care product contains 0.01~99.99wt%, preferably 2~20%.

[0014] The beneficial effects of this application are: This application utilizes a combination of lactoferrin, oat extract, and calcium gluconate to significantly inhibit the expression of the kallikrein-associated enzyme 7 gene. By regulating the expression of the kallikrein-associated enzyme 7 gene, the composition effectively enhances its skin-soothing efficacy. Attached Figure Description

[0015] Figure 1 The effect of different concentrations of the tested samples on the expression level of the KLK7 gene in RAWV264.7 cells; Where #P<0.05; #P<0.01; ###P<0.001; ###P<0.001; *P<0.05; **P<0.01; ***P<0.001; **^P<0.0001 Note: # indicates a significant difference between the model group and the blank group, and * indicates a significant difference between the tested sample and the model group at this concentration. Figure 2The effect of the tested samples on the expression of AOP3 in NHEK cells (green fluorescence indicates AOP3); Figure 3 This shows the trend of AOP3 protein content changes; Where: compared with the control, significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **. Detailed Implementation

[0016] In the description of this application, it should be noted that, where no specific conditions are specified in the embodiments, they shall be performed under conventional conditions or conditions recommended by the manufacturer.

[0017] In the following examples and comparative examples, the lactoferrin was purchased from Recomtein® LF provided by Guangzhou Jikemei Chuang Life Science Co., Ltd., containing 10% lactoferrin and 90% mannitol. The actual amount of lactoferrin used in the examples and comparative examples of this application was used as the basis for calculation. The oat extract was purchased from Zhejiang Chenhai Life Science Co., Ltd., with the product name Essenpure CBG. The calcium gluconate was purchased from Zhengzhou Ruipu Bioengineering Co., Ltd. The Polygonatum odoratum extract was purchased from Ruixi (Guangzhou) Trading Co., Ltd., with the product name PO-100. The phytosphingosine was purchased from Chongqing Zhihe Biomedical Co., Ltd.

[0018] Unless otherwise specified, the parts are by weight and the percentage is by weight percentage; it should be noted that in the following examples and comparative examples, water is used as a solvent and does not affect the effect of the composition.

[0019] Example 1

[0020] A soothing composition is prepared by mixing 0.1 parts lactoferrin, 60 parts oat extract, 4 parts calcium gluconate and 35.9 parts water.

[0021] Example 2

[0022] A soothing composition is prepared by mixing 0.5 parts lactoferrin, 40 parts oat extract, 8 parts calcium gluconate and 51.5 parts water.

[0023] Example 3

[0024] A soothing composition is prepared by mixing 0.2 parts lactoferrin, 50 parts oat extract, 5 parts calcium gluconate and 44.8 parts water.

[0025] Example 4

[0026] A soothing composition is prepared by mixing 0.2 parts lactoferrin, 50 parts oat extract, 5 parts calcium gluconate, 1 part Polygonatum odoratum extract, 0.5 parts phytosphingosine and 43.3 parts water.

[0027] Example 5

[0028] A soothing composition is prepared by mixing 0.2 parts lactoferrin, 50 parts oat extract, 5 parts calcium gluconate, 1.5 parts Polygonatum odoratum extract and 43.3 parts water.

[0029] Example 6

[0030] A soothing composition is prepared by mixing 0.2 parts lactoferrin, 50 parts oat extract, 5 parts calcium gluconate, 1.5 parts phytosphingosine and 43.3 parts water.

[0031] Comparative Example 1 A soothing composition is prepared by mixing 55.2 parts lactoferrin and 44.8 parts water.

[0032] Comparative Example 2 A soothing composition is prepared by mixing 55.2 parts of oat extract and 44.8 parts of water.

[0033] Comparative Example 3 A soothing composition is prepared by mixing 55.2 parts calcium gluconate and 44.8 parts water.

[0034] Comparative Example 4 A soothing composition is prepared by mixing 0.2 parts lactoferrin and 99.8 parts water.

[0035] Comparative Example 5 A soothing composition is prepared by mixing 5 parts calcium gluconate and 95 parts water.

[0036] Comparative Example 6 A soothing composition is prepared by mixing 50.2 parts of oat extract, 5 parts of calcium gluconate and 44.8 parts of water.

[0037] Comparative Example 7 A soothing composition is prepared by mixing 2.1 parts lactoferrin, 53.1 parts calcium gluconate and 44.8 parts water.

[0038] Comparative Example 8 A soothing composition is prepared by mixing 0.3 parts lactoferrin, 54.9 parts oat extract and 44.8 parts water.

[0039] Efficacy test I. Expression of the inflammatory cytokine KLK7 1. Experimental Background Kallikrein-associated peptidase 7 (KLK7) is a member of the kallikrein (KLK) gene family, belonging to the serine protease family, and plays an important role in skin barrier function and inflammation regulation. KLK7 promotes normal stratum corneum shedding by hydrolyzing intercellular junction proteins, participating in the physiological desquamation process of the skin. If the skin is damaged, KLK7 expression is upregulated, exacerbating inflammatory responses and barrier dysfunction. In eczema patients, due to impaired skin barrier function, abnormal expression or activity of KLK7 may worsen the condition, leading to excessive dryness and inflammation.

[0040] 2. Equipment and Materials 2.1 Equipment: Multifunctional microplate reader (TECAN, SPARK), CO2 incubator (Thermo), Real-time PCR instrument (Bio-Rayet, CFX ConnectOotics odule), PCR amplification instrument (Longi, A300). 2.2 Cell line: RAW264.7 (mouse mononuclear macrophage leukemia cells) 2.3 Reagents: Dexamethasone (Merck), LPS (lipopolysaccharide, Maclean's) RNA Rapid Extraction Kit (MegBio), qPCR Mix Kit (SYBR Green) 3 Sample processing The test sample was diluted to 0.5% and 1% concentration.

[0041] 4 Experimental Methods KLK7 genetic testing Cells were resuspended and seeded in 6-well plates.

[0042] Drug administration and induction: Discard the old culture medium in the 6-well plates. Except for the control group, all other groups were stimulated with culture medium containing LPS. After stimulation, the test sample groups were added with basal culture medium containing 1% of the test substance, the positive control group was added with basal culture medium containing dexamethasone, and the control group was added with only basal culture medium. After drug administration, the 6-well plates were placed in a CO2 incubator for further incubation.

[0043] After culture, cells were collected, total mRNA was extracted, quantified, and cDNA was obtained by reverse transcription for Real-Time PCR. A 2... -△△CT The relative expression levels of SOD and CAT genes were analyzed using a method. The average Ct value of three replicates for each gene per cDNA sample was used as the amplification result. The amplification level of GAPDH gene was used as an internal reference gene. The cycle threshold ΔCt was calculated as Ctgene - CtGAPDH, and the relative gene expression level ΔΔCt = ΔCttest - ΔCtblank control. -△△CTThe ratio of expression levels in the test group to those in the control group was analyzed and calculated.

[0044] The samples of Example 3 (0.5%, 1%) were tested according to the above detection method, and the results are shown in Table 1 and... Figure 1 As shown, Figure 1 The three-mu cover described herein is Embodiment 3 of this application; Table 1. Effects of different concentrations of the sample from Example 3 on the expression level of the KLK7 gene in RAW264.7 cells. Gene expression ratio Blank group 1 Positive control group 1.84 0.5% Sample from Example 3 3.63 1% Example 3 Sample 3.19 The samples of Examples 1-6 and Comparative Examples 1-8 were tested according to the above testing methods, and the results are shown in Table 2. Table 2 shows the effects of samples from Examples 1-6 and Comparative Examples 1-8 on KLK7 gene expression levels in RAW264.7 cells. Gene expression ratio Blank group 1 Positive control group 1.84 Example 1 3.19 Example 2 3.14 Example 3 3.08 Example 4 2.64 Example 5 2.96 Example 6 3.10 Comparative Example 1 3.23 Comparative Example 2 5.06 Comparative Example 3 3.11 Comparative Example 4 4.82 Comparative Example 5 4.49 Comparative Example 6 4.45 Comparative Example 7 3.60 Comparative Example 8 4.57 According to the results in Table 2: According to the data from Comparative Examples 1-5, both high concentrations of lactoferrin and calcium gluconate showed significant inhibitory effects on KLK7 gene expression. While high concentrations of oat extract also exhibited some inhibitory effect on KLK7 gene expression, it was significantly weaker than that of lactoferrin and calcium gluconate. Furthermore, low concentrations of lactoferrin and calcium gluconate showed slightly higher inhibitory effects on KLK7 gene expression than high concentrations of oat extract. This indicates that lactoferrin and calcium gluconate play a major role in inhibiting KLK7 gene expression.

[0045] According to the data from Examples 1-3, when lactoferrin, oat extract and calcium gluconate are combined, even if the concentrations of lactoferrin and calcium gluconate are low, the inhibitory effect on KLK7 gene expression is higher than that of Comparative Examples 1-5. This indicates that the technical solution of this application uses lactoferrin, oat extract and calcium gluconate to produce a synergistic effect in inhibiting KLK7 gene expression.

[0046] A comparison of the data from Examples 5, 6, and 3 shows that Polygonatum sibiricum extract has a certain inhibitory effect on KLK7 gene expression, but its effect is not significant. Phytosphingosine does not inhibit KLK7 gene expression. However, it can be seen that in Example 4, when Polygonatum sibiricum extract and phytosphingosine are added simultaneously, the KLK7 gene expression inhibition effect of the composition is significantly improved compared to Example 3. This indicates that adding Polygonatum sibiricum extract and phytosphingosine to the combination of lactoferrin, oat extract, and calcium gluconate can produce a further synergistic effect. The mechanism is speculated to be that Polygonatum sibiricum extract has the effect of inhibiting the formation of advanced glycation end products (AGEs), while phytosphingosine protects the collagen structure through anti-glycation, indirectly maintaining the integrity of the skin barrier, thereby indirectly improving the anti-inflammatory effect and affecting the KLK7 gene expression inhibition effect. In the presence of Polygonatum sibiricum extract, the anti-glycation effect of phytosphingosine is further enhanced, thereby improving the KLK7 gene expression inhibition effect.

[0047] Based on the data comparison of Example 3 and Comparative Examples 6-8, it can be seen that when any one of the components lactoferrin, oat extract, or calcium gluconate is missing in Examples 6-8, the KLK7 gene expression inhibition effect is significantly reduced. Combined with the data analysis of Comparative Examples 1-5, it can be seen that the technical solution of this application is based on the synergistic enhancement of KLK7 gene expression inhibition effect produced by the combination of lactoferrin, oat extract, and calcium gluconate.

[0048] II. Moisturizing Efficacy Test 1. Experimental Principle Aquaporin 3 (AQP3) is a member of the aquaporin (AQP) family, and it has diverse functions in cells, including water balance, metabolic regulation, and cell signaling. AQP3 can efficiently regulate the osmotic balance of water inside and outside the cell by forming transmembrane channels, maintaining cell volume stability. AQP3 also participates in moisture retention in the stratum corneum of the skin, contributing to epidermal moisturization, while also helping to maintain elasticity and repair the skin barrier.

[0049] The moisturizing efficacy of the test samples was evaluated by detecting changes in AQP3 in human epidermal keratinocytes after sample treatment using immunofluorescence (IF) technology.

[0050] Immunofluorescence (IF): A fluorescent dye that does not affect the activity of the antigen and antibody is labeled on the antibody (or antigen). After binding with the corresponding antigen (or antibody), it exhibits a specific fluorescent reaction under a fluorescence microscope.

[0051] 2. Equipment and Materials 2.1 Equipment: Multifunctional microplate reader (TECAN, SPARK), CO2 incubator (Thermo, 150I), Olympus microscope (CK53) 2.2 Cell line: NHEK (normal human epidermal keratinocytes, Shanghai Boson Biotechnology Co., Ltd.) 2.3 Reagents: BSA (BioFox), AQP3 antibody (Abbkine), fluorescent secondary antibody (Abbkine) 3. Sample processing The test sample was filtered through a 0.22 μm filter membrane, and the filtrate was collected as the test sample mother liquor.

[0052] 4 Experimental Methods 4.1 Cell viability test After culturing NHEK cells in good growth condition in 96-well plates for 24 hours, the medium was replaced with a medium containing different concentrations of the test samples. After 24 hours, the OD570 and OD630 values ​​were measured by MTT assay to analyze the effect of the test samples on the tolerance of NHEK cells.

[0053] 4.2 Cellular AQP3 Immunofluorescence Assay Healthy NHEK cells were seeded into 12-well plates. Once the cells reached 85% confluence, the old culture medium was discarded, and new culture medium containing different concentrations of the test samples was added, followed by 48 hours of incubation. The old culture medium was then discarded, and the cells were washed three times with PBS, with 4% paraformaldehyde added to each well for fixation. After washing with PBS, AQP3 antibody dilution buffer was added and the cells were incubated overnight. After washing with PBST, fluorescent secondary antibody dilution buffer was added and the cells were incubated again. Observation was performed using a fluorescence microscope, and images were collected and analyzed to obtain data.

[0054] The samples provided in Example 3 were diluted to 0.1% and 0.2% volume concentrations according to the above detection methods, and the results are shown in Table 3. Figure 2 , Figure 3 As shown, Figure 2 , Figure 3 The three Mugai and TS mentioned above are both embodiments 3 of this application.

[0055] Table 3. Detection results of AQP3 protein content in samples provided in Example 1 at different concentrations. Relative IOD / average cell count Improvement rate Blank control 1.00 / 0.1% Example 3 3.00 200% 0.2% Example 3 4.06 306% The above detection methods were used to test Examples 1-6, Comparative Examples 1-3 and Comparative Examples 6-8 after being diluted to a certain concentration. The results are shown in Table 4. Table 4 Results of AQP3 protein content detection Relative IOD / average cell count Improvement rate Blank control 1.00 / 0.2% Example 1 4.29 329% 0.2% Example 2 3.92 292% 0.1% Example 3 3.00 200% 0.2% Example 3 4.06 306% 0.2% Example 4 4.13 313% 0.2% Example 5 4.08 308% 0.2% Example 6 4.11 311% 0.2% Comparative Example 1 2.78 178% 0.2% Comparative Example 2 3.21 221% 0.2% Comparative Example 3 2.57 157% 0.2% Comparative Example 6 3.03 203% 0.2% vs. Example 7 2.69 169% 0.2% vs. Example 8 3.37 237% According to the results in Table 4: Based on the data from 0.1% Example 3 and 0.2% Examples 1-3, it can be seen that this application has a high AQP3 enhancement effect; among them, based on the data from 0.2% Examples 1-3, it can be inferred that oat extract plays a relatively important role in enhancing AQP3 protein content.

[0056] The 0.2% comparative examples 1-3 further validated the hypothesis. According to the data comparison of the 0.2% comparative examples 1-3, oat extract > lactoferrin > calcium gluconate in terms of increasing AQP3 protein content.

[0057] However, in Example 3 of this application, which reduced the content of oat extract, the combination of lactoferrin and calcium gluconate resulted in a higher and more significant increase in AQP3 protein content than in Comparative Example 2 of the 0.2% formulation. This indicates that the combination of oat extract, lactoferrin, and calcium gluconate in this application produced a synergistic effect in increasing AQP3 protein content.

[0058] Based on the data comparison of 0.2% Example 3 and 0.2% Examples 4-6 of this application, it can be seen that the addition of Polygonatum odoratum extract and phytosphingosine in the technical solution of this application does not significantly increase the content of AQP3 protein.

[0059] Meanwhile, based on the data comparison of 0.2% Example 3 and 0.2% Comparative Examples 6-8, combined with the data analysis of 0.2% Comparative Examples 1-3, it can be concluded that oat extract and lactoferrin have a certain degree of synergistic enhancement effect in improving AQP3 protein content. However, the synergistic enhancement effect is significantly different from the technical solution of oat extract, lactoferrin and calcium gluconate used in this application. When oat extract and calcium gluconate are combined, or when lactoferrin and calcium gluconate are combined, no synergistic enhancement effect is produced.

[0060] III. Chicken Embryo Viral Allantoic Membrane (CAM) Experiment 1. Experimental Principle The chorioallantoic membrane (CAM) is a respiratory membrane surrounding the chicken embryo. Due to the rich blood supply on its surface, the CAM can be considered a complete organism. This experiment utilizes the intact, clear, and transparent vascular system of the mid-stage chorioallantoic membrane in hatched chicken embryos. A certain amount of the test substance was directly exposed to the chorioallantoic membrane, and after a period of time, changes in chorioallantoic membrane toxicity indicators (such as hemorrhage, coagulation, and vascularization) were observed and scored to evaluate the potential eye irritation of the test substance.

[0061] 2. Equipment and Materials Incubator, stereomicroscope, electronic balance, etc. BWEL-SPF Fertilized Chicken Embryos (Xinxing Dahuannong Poultry Egg Co., Ltd.) 3 Sample Preparation The test samples were serially diluted with normal saline.

[0062] 4 Experimental Methods 4.1 Test Grouping Negative control: Normal saline Positive control: 0.1M sodium hydroxide Test substance group: Take a certain amount of sample dilution 4.2 Preparation of Chicken Embryos Incubate the chicken embryos until the 8th day. Check with an egg candler and select chicken embryos with well-developed blood vessels. Mark the position of the air chamber on the eggshell surface.

[0063] 4.3 Test Procedure Start the test on the 9th day. Carefully peel off the eggshell part of the air chamber with a dental curved scissors. Drop a few drops of normal saline on the surface of the eggshell membrane to fully moisten it. After pouring out, carefully remove the eggshell membrane with forceps to ensure the integrity of the exposed chorioallantoic membrane. Take 0.3 ml of the diluted test substance and directly apply it to the chorioallantoic membrane. Observe the CAM reaction and record the time when each toxic effect appears within 5 minutes.

[0064] 4.4 Result Calculation and Standard Judgment 4.4.1 Calculation Formula

[0065] In the formula: SecH (bleeding time, s) - The average time when bleeding starts to occur observed on the CAM membrane; SecL (vascular lysis time, s) - The average time when vascular lysis starts to occur observed on the CAM membrane; SecC (coagulation time, s) - The average time when coagulation starts to occur observed on the CAM membrane.

[0066] 4.4.2 Judgment Standard IS < 1 indicates non-irritating, 1 ≤ IS < 5 indicates mild irritation, 5 < IS < 9 indicates moderate irritation, and IS ≥ 10 indicates strong irritation / corrosion.

[0067] Perform the test on Example 3 according to the above method, and the results are shown in Table 5; Table 5 Results of Irritation Test

[0068] The above embodiments are preferred embodiments of this application, but the implementation of this application is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of this application shall be considered equivalent substitutions and shall be included within the protection scope of this application.

Claims

1. A soothing composition, characterized in that, It includes lactoferrin, oat extract and calcium gluconate, wherein the weight ratio of lactoferrin, oat extract and calcium gluconate is 0.1~0.5:40~60:4~8.

2. The soothing composition according to claim 1, characterized in that, It also includes Polygonatum odoratum extract and phytosphingosine.

3. The soothing composition according to claim 2, characterized in that, The weight ratio of lactoferrin, oat extract, calcium gluconate, Polygonatum sibiricum extract and phytosphingosine is 0.1~0.5:40~60:4~8:0.05~2.5:0.1~1.

5.

4. Use of the soothing composition as described in claim 1 in the preparation of skin care products.

5. A skincare product, characterized in that, The skincare product contains the soothing composition as described in claim 1.

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