Composition for repairing immune barrier and soothing skin and its application in cosmetics

Abstract

The present invention provides a composition for repairing immune barrier and soothing skin and its application in cosmetics, belonging to the field of skin immunity technology. The composition provided by the present invention includes 0.5-0.8 parts of glycosaminoglycan and 0.8-2.3 parts of schizophyllan by weight, which can enhance skin immune response, remove harmful bacteria from the skin, transform from pro-inflammatory macrophages to inflammatory suppressive cells at the end of inflammation, and secrete inflammatory factors to inhibit inflammatory factors, while inhibiting the NF-kB inflammatory signal of cells to prevent skin damage caused by excessive immune response. The cosmetic composition provided by the present invention combines the five ingredients of ceramide, squalane, TRPV1 inhibitor, glycosaminoglycan and schizophyllan, and prepares a composition with repairing epidermal barrier and enhancing skin immune barrier through multi-pathway synergistic action, anti-inflammatory and soothing effects, which can be widely used in cosmetics for treating sensitive skin populations.

Description

Technical Field

[0001] The invention belongs to the technical field of skin immunity, and in particular relates to a composition for repairing immune barriers and soothing skin and an application thereof in cosmetics. Background Art

[0002] Sensitive skin is a type of skin on various parts of the body, especially the facial skin, which is affected by factors such as genetics, environment, cosmetics, psychological stress, hormones, etc., and may undergo irritation reactions or overactivation of the immune system, resulting in problems such as itching, redness, burning, stinging, tightness, and rash.

[0003] Current research suggests that the mechanism of sensitive skin is related to the skin barrier-neurovascular-immune inflammation. The stratum corneum, as a permeable barrier, can prevent irritants, allergens and microorganisms from entering the skin. The destruction of the epidermal barrier causes sensitive skin to react more strongly to irritation and is more sensitive than normal skin. Under the influence of internal and external factors, the skin barrier is damaged, and the sensory neurons located in the skin are activated, and the incoming signals increase, which are transmitted to the cerebral cortex through the spinal cord to produce sensations such as itching, tingling and burning; after the TPPV1 receptors of keratinocytes and mast cells are activated, calcium ions flow in, the cells become polarized, and inflammatory factors and chemokines are released, causing local inflammatory reactions and vasodilation and redness of the skin. At the same time, the skin epidermal microbial flora of people with sensitive skin is imbalanced, pathogenic bacteria overgrow and release too many signal molecules, which can cause skin inflammation, destroy the skin's immune barrier, and increase the sensitivity of the skin.

[0004] At present, the scientific mechanism for treating sensitive skin is mainly to avoid adding irritating ingredients to cosmetics, add mild skin care ingredients that help repair the epidermal barrier, or soothe and anti-inflammatory, and inhibit neurogenic inflammation (such as CN117860625A, CN118045019A, CN117982387A). In addition, CN117462437A discloses a mussel protein composition that has good skin barrier repair, inflammatory response inhibition, and subcutaneous nerve excessive feedback through multi-pathway synergy, and is suitable for sensitive skin. The mechanism of these products for treating sensitive skin is mainly based on repairing the epidermal barrier or reducing the susceptibility of peripheral sensory neurons in the skin. Although it can alleviate the sensitivity mediated by irritant reactions, it ignores the destruction of the skin's flora microenvironment (the normal flora of the sensitive skin surface flora, Staphylococcus epidermidis, which is beneficial to the skin, is reduced, while the skin pathogen Staphylococcus aureus is increased), and the importance of immune response disorders and immune balance regulation caused by it.

[0005] More and more research evidence shows that β-glucan can activate trained immunity through epigenetic reprogramming and metabolomics reprogramming to enhance the recognition and binding of the surface receptors of the skin's innate immune cells to lipopolysaccharides on the membrane of pathogens. In addition, under the continuous stimulation of pathogenic microorganisms, on the one hand, β-glucan can enhance the immune function of the skin through activated trained immunity, shorten the immune response time, enhance the activity of immune cell proliferation and differentiation and be recruited to the inflammatory site, promote the secretion of inflammatory factors, and quickly eliminate harmful bacteria; on the other hand, at the end of inflammation, the metabolites of β-glucan activated trained immunity can regulate the transformation of macrophages from inflammatory type to repair type, upregulate the expression of IL-10, an inhibitor of inflammatory factor, terminate the immune response in time, and relieve skin sensitivity symptoms.

[0006] In addition, many scientific studies have confirmed that immune response imbalance is the main pathological change of sensitive skin, which is even more common than irritation caused by damaged epidermal barrier. In addition, imbalance of flora diversity and function is related to the development of inflammatory skin diseases. However, the cosmetics currently available on the market only target sensitive skin induced by irritation reactions, and cannot solve sensitive skin caused by excessive growth of harmful bacteria on the skin.

[0007] Therefore, there is an urgent need in the art to develop a cosmetic composition that can effectively eliminate the discomfort caused by sensitive skin and relieve adverse symptoms of sensitive skin such as facial redness, tightness, and scaling, so as to meet the needs of the increasing number of people with sensitive skin. Summary of the invention

[0008] In view of the above-mentioned deficiencies, the present invention provides a composition for repairing the immune barrier and soothing the skin and its application in cosmetics. The composition provided by the present invention includes 0.5-0.8 parts of glycosaminoglycan and 0.8-2.3 parts of schizophyllan by weight, which can enhance the skin immune response, remove harmful bacteria from the skin, transform from pro-inflammatory macrophages to inflammatory suppressive cells at the end of inflammation, and secrete inflammatory factors that inhibit inflammatory factors, while inhibiting the NF-kB inflammatory signal of the cell to prevent skin damage caused by excessive immune response. The cosmetic composition provided by the present invention combines the five ingredients of ceramide, squalane, TRPV1 inhibitor, glycosaminoglycan and schizophyllan, and prepares a composition with the function of repairing the epidermal barrier and enhancing the skin immune barrier through multi-pathway synergistic action. It has the effects of anti-inflammatory and soothing, and can be widely used in cosmetics for treating sensitive skin people.

[0009] The technical solution of the present invention includes:

[0010] In a first aspect, the present invention provides a use of β-glucan in preparing a composition for repairing immune barriers and soothing skin.

[0011] Specifically, the β-glucan is polymerized from D-glucose monomers connected by β-1,3 glycosidic bonds as the main chain.

[0012] More specifically, the β-1,3-D-glucan is secreted and produced by microorganisms.

[0013] Preferably, the microorganisms include but are not limited to Schizophora β-1,3-D-glucan, Candida albicans β-1,3-D-glucan, yeast β-1,3-D-glucan, oat β-1,3-D-glucan, seaweed β-1,3-D-glucan, barley β-1,3-D-glucan or medicinal mushroom β-1,3-D-glucan.

[0014] Further preferably, the yeast β-1,3-D-glucan includes but is not limited to baker's yeast β-1,3-D-glucan or brewer's yeast β-1,3-D-glucan.

[0015] Further preferably, the medicinal mushroom β-1,3-D-glucan includes but is not limited to Ganoderma lucidum β-1,3-D-glucan, turkey tail β-1,3-D-glucan, shiitake β-1,3-D-glucan or maitake β-1,3-D-glucan.

[0016] Further preferably, the β-1,3-D-glucan is secreted and produced by schizophyllan.

[0017] More specifically, the β-1,3-D-glucan is recognized by receptors on the surface of innate immune cells and plays an important role in human skin immune defense.

[0018] Preferably, the innate immune cells include but are not limited to neutrophils, macrophages or dendritic cells.

[0019] Preferably, the receptors are Dectin1 receptor and CR3 complement receptor.

[0020] More specifically, the glycosidic bonds of the β-1,3-D-glucan are β-1,3 glycosidic bonds or main chain β-1,3 glycosidic bonds and side chain β-1,6 glycosidic bonds or β-1,3 glycosidic bonds.

[0021] Preferably, the connection mode of the branched β-1,6 glycosidic bonds includes but is not limited to the following structure: one of every three β-1,3 glycosidic bond-linked sugar groups is linked via a branched β-1,6 glycosidic bond; or

[0022] One of every seven β-1,3-glycosidic-linked sugar moieties is linked via a branched β-1,6-glycosidic bond; or

[0023] One of every three β-1,3 glycosidic-linked sugar groups is connected by a branched β-1,6 glycosidic bond and two β-1,3 glycosidic bonds.

[0024] Further preferably, the connection mode of the branched β-1,6 glycosidic bonds is that one of the three β-1,3 glycosidic bond-linked sugar groups is connected by a branched β-1,6 glycosidic bond, forming a unique superhelical three-dimensional structure, which is easily recognized and bound by immune receptors, thereby enhancing the skin's immune barrier.

[0025] In certain embodiments, the structural formula of the 1,3-1,6 branched β-glucan is as follows:

[0026]

[0027] Wherein, n is an integer ≥ 1, preferably 500-3000, and more preferably 1000-2000.

[0028] Preferably, the degree of branching (DB) of the 1,3-1,6 branched β-glucan is 0.01-1; more preferably, the degree of branching (DB) of the 1,3-1,6 branched β-glucan is 0.3-0.6; more preferably, the degree of branching (DB) of the 1,3-1,6 branched β-glucan is 0.8-1.

[0029] Preferably, the molecular weight of the 1,3-1,6-branched β-glucan is ≥1KD; more preferably, the molecular weight of the 1,3-1,6-branched β-glucan is 500KD-4000KD; more preferably, the molecular weight of the 1,3-1,6-branched β-glucan is 1000KD-2000KD.

[0030] In a second aspect, the present invention provides a composition comprising: glycosaminoglycan and schizophyllan.

[0031] Specifically, the composition comprises 0.5-0.8 parts of glycosaminoglycan and 0.8-2.3 parts of schizophyllan by weight.

[0032] Preferably, the composition comprises 0.6-0.8 parts of glycosaminoglycan and 1.5-2.3 parts of schizophyllan by weight.

[0033] Preferably, the composition comprises 0.8 parts of glycosaminoglycan and 2.3 parts of schizophyllan by weight.

[0034] In a third aspect, the present invention provides a cosmetic composition comprising the above composition.

[0035] Specifically, the cosmetic composition consists of glycosaminoglycan, schizophyllan, a TRPV1 inhibitor, ceramide and squalane.

[0036] Preferably, the cosmetic composition comprises, by weight, 0.5-0.8 parts of glycosaminoglycan, 0.8-2.3 parts of schizophyllan, 1-2 parts of TRPV1 inhibitor, 0.5-1 parts of ceramide and 0.1-0.5 parts of squalane.

[0037] Further preferably, the cosmetic composition comprises, by weight, 0.8 parts of glycosaminoglycan, 2.3 parts of schizophyllan, 2 parts of TRPV1 inhibitor, 1 part of ceramide and 0.5 parts of squalane.

[0038] In a fourth aspect, the present invention provides use of the above composition or cosmetic composition in the preparation of cosmetics.

[0039] Specifically, the cosmetics are used to improve sensitive skin.

[0040] Preferably, the cosmetic improves sensitive skin by defending against skin irritants, soothing skin discomfort, removing skin pathogenic microorganisms, balancing the microecology on the skin surface, or controlling skin immune inflammation.

[0041] Preferably, the cosmetics include but are not limited to facial cream, facial mask, eye cream, neck cream, hand cream, body lotion, moisturizing lotion, essence, toner, shampoo, conditioner, shower gel or cleanser.

[0042] In a fifth aspect, the present invention provides a cosmetic comprising the above-mentioned composition or cosmetic composition.

[0043] Specifically, the weight percentage content of the composition in the cosmetic or the cosmetic composition is 2%-10%.

[0044] Preferably, the weight percentage content of the composition in the cosmetic or the cosmetic composition is 2%.

[0045] Specifically, the cosmetics also include auxiliary materials acceptable in the cosmetics field.

[0046] Preferably, the auxiliary materials include, but are not limited to, one or more of moisturizers, thickeners, emollients, skin conditioners, emulsifiers, preservatives, emollients, lubricants, and stabilizers.

[0047] The technical effects achieved by the present invention are:

[0048] (1) Through molecular docking and qPCR experiments, the present invention found that the mycophenolic acid provided by the present invention binds more stably to immune receptors, activates and trains immunity, regulates inflammatory responses, and quickly eliminates pathogens. It can be used in cosmetics as a component for enhancing skin immune function and regulating immune balance.

[0049] (2) The composition provided by the present invention can enhance the skin immune response, remove harmful bacteria from the skin, transform pro-inflammatory macrophages into anti-inflammatory cells at the end of inflammation and secrete anti-inflammatory factors, while inhibiting the NF-kB inflammatory signal of the cells to prevent skin damage caused by excessive immune response.

[0050] (3) The present invention provides a cosmetic composition for treating sensitive skin, which targets multiple action pathways based on the possible mechanisms of sensitive skin: skin barrier-neurovascular-immune inflammation-microbial imbalance, and plays a corresponding role respectively, and coordinates with each other to achieve a multi-level and multi-dimensional solution to the barrier damage, neurogenic inflammation induced by nervous system activation, and immune regulation imbalance mediated by skin microecological imbalance found in sensitive skin in current research. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 These are the molecular docking images of schizophyllan and β-glucan binding to Dectin-1 respectively; Figure A is the docking image of schizophyllan binding to Dectin-1; Figure B is the molecular docking image of β-glucan binding to Dectin-1.

[0052] Figure 2 The molecular docking images of schizophyllan and β-glucan binding to CR3 respectively; Figure A is the molecular docking image of schizophyllan binding to CR3; Figure B is the molecular docking image of β-glucan binding to CR3.

[0053] Figure 3 Molecular docking images of chitin, alginate and pectin disaccharide binding to Dectin-1 and CR3 respectively; A is the molecular docking image of chitin binding to Dectin-1; B is the 3D molecular docking image of chitin binding to CR3; C is the molecular docking image of alginate binding to Dectin-1; D is the molecular docking image of alginate binding to CR3; E is the molecular docking image of pectin disaccharide binding to Dectin-1; F is the molecular docking image of pectin disaccharide binding to CR3.

[0054] Figure 4 THP-1 cells were treated with 20 nM LPS and 100 mM mycoplasmin for 12 h.

[0055] Figure 5THP-1 cells were treated with 20 nM LPS and 100 mM mycoplasmin for 24 h.

[0056] Figure 6 The effect of the schizophyllan-glucosaminoglycan composition on the proliferation rate of Staphylococcus aureus.

[0057] Figure 7 The effect of the schizophyllan-glycosaminoglycan composition on the proliferation rate of Staphylococcus epidermidis. DETAILED DESCRIPTION

[0058] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0059] The experimental materials used in the present invention are shown in Table 1:

[0060] Table 1 Experimental materials of the present invention

[0061]

[0062] Example 1 Molecular docking

[0063] 1. Experimental Materials

[0064] Receptors: PDB structures of Dectin-1 and CR3 proteins.

[0065] Ligand: Chemical Identifier (CID) of schizophyllan, dextran, pectobiose, citrus pectin and chitin.

[0066] Operation and analysis software: PyMOL; Autodock4 Vina; AutodockTools1.5.6; Chemdraw.

[0067] 2. Experimental methods and steps

[0068] Download the PDB structure of the receptor protein to be tested, and save the files in .pdb format. The PDB structure of the receptor: Dectin-1 protein is 2CL8, and CR3 protein is 7usl. Use the software PyMOL to intercept the amino acid residues 843-1076.

[0069] Ligand preparation was performed on the pubchem website: schizophyllan was CID24777, β-glucan was CID439262, alginate was CID5102882, chitosan was CID6857375, and pectin disaccharide was CID16738707.

[0070] The molecular docking calculations were performed using Autodock4 Vina software: the ligands were matched with the receptors, a search region was defined within the residue range of the receptor amino acids to be tested, the grid energy was calculated by scanning different atoms, the conformation, position and orientation of the ligands were continuously adjusted, and scoring and sorting were performed to obtain the conformation with the lowest energy as the output result.

[0071] Molecular docking was performed using Autodock Tools 1.5.6, and PyMOL was launched to analyze the results.

[0072] The 2D images of molecular docking were drawn using Chemdraw software.

[0073] 3. Experimental results and discussion

[0074] 3.1 Schizophyllan activates trained immunity by binding to the C-type domain of Dectin-1 protein

[0075] Whether the conformation of the Dectin1 receptor is stable after binding to β-glucan is the key to activating trained immunity. Molecular docking images of 1,3-1,6 branched β-glucan and 1,3 straight β-glucan binding to the C-type domain of the Dectin1 receptor are shown in Figure 1. Figure 1 The binding free energy is shown in Table 2.

[0076] Table 2 Binding energy of schizophyllan and 1,3 linear β-glucan to Dectin-1 receptor

[0077]

[0078] The binding free energy of schizophylline is -8.8 Kcal / mol ( Figure 1 A in), β-glucan is -8.2 Kcal / mol ( Figure 1 B in the figure) are all less than -5 Kcal / mol, indicating that the molecular docking results are credible. The results show that compared with 1,3 linear β-glucan, the 1,3-1,6 branched β-glucan of schizophyllan has lower binding energy to Dectin-1 protein and a more stable structure, showing stronger biological activity. After binding to the receptor, it induces conformational changes, causing Dectin-1 molecules to form dimers / oligomers, activating intracellular molecules to undergo signal transduction, triggering trained immunity, enhancing the recognition and binding ability of immune cells to pathogenic microorganisms, and activating the immune regulation function of skin cells.

[0079] 3.2 Schizophyllan activates trained immunity by binding to the Linctin-1 terminal domain of the CR3 complement receptor

[0080] The stability of the conformation of the CR3 complement receptor after binding to β-glucan is the key to activating trained immunity. Molecular docking images of 1,3-1,6 branched β-glucan and 1,3 linear β-glucan binding to the CR3 receptor Linctin-1 domain are shown in Figure 1. Figure 2 The binding free energy is shown in Table 3.

[0081] Table 3 Binding capacity of schizophyllan and 1,3 linear β-glucan to CR3 receptor

[0082]

[0083] The binding energy of schizophylline is -7.6 Kcal / mol ( Figure 2 A in), 1,3 linear β-glucan is -6.8Kcal / mol ( Figure 2 B), are all less than -5 Kcal / mol, indicating that the molecular docking results are credible. The results of this study are explained from the perspective of molecular energy and structure: compared with β-glucan, the 1,3-1,6 branched β-glucan structure of schizophyllan is less binding energy. Therefore, its structure is more stable and its biological activity is stronger, which shortens the time of immune response, promotes the proliferation and activation of skin immune cells, regulates inflammatory response, and quickly eliminates pathogens.

[0084] 3.3 The 1,3-1,6 branched β-glucan structure of schizophyllan is more biologically active than other glucans

[0085] In order to verify the molecular basis of the stronger biological activity of 1,3-1,6-branched β-glucan from schizophyllan, this study selected three β-glucans with moisturizing effects and different sources and divided them into three groups for molecular docking tests. Figure 3 The binding free energy is shown in Table 4.

[0086] Table 4 Binding energy of β-glucans with different structures to Dectin-1 receptor and CR3 receptor

[0087]

[0088] The first group detected chitin, whose binding energy to the Dectin-1 receptor was -6.1 Kcal / mol ( Figure 3 The binding energy of the CR3 receptor is -5.1 Kcal / mol ( Figure 3 The second group tested alginate and analyzed its binding energy with Dectin-1 receptor to be -6 Kcal / mol ( Figure 3 C in the figure), and the binding energy to the CR3 receptor is -5.1 Kcal / mol ( Figure 3 The third group tested pectin disaccharide and analyzed its binding energy with Dectin-1 receptor as -7.9 Kcal / mol ( Figure 3 The binding energy of the CR3 receptor is -6.3 Kcal / mol ( Figure 3 F in.

[0089] The binding energy of the above molecular docking was compared with -5 Kcal / mol, indicating that the results of the first three groups of samples are credible. The above results confirm that compared with the four conventional moisturizing β-glucans, the binding energy between schizophyllan and Dectin-1 and CR3 receptors is the lowest. Therefore, its molecular structure is the most stable and has stronger biological activity, indicating that schizophyllan has the potential to activate training immunity to enhance the body's immunity.

[0090] Example 2 qPCR experiment

[0091] 1. Cell culture

[0092] A dish of p60 human monocytic leukemia THP-1 cells with moderate density and good growth was collected, centrifuged, resuspended and diluted to a 24-well plate at a concentration of 1.5*10^5 THP-1 cells / well, treated with 50nM phorbol ester PMA for 24h until completely attached to the wall to establish a cell inflammation model. The culture medium was discarded, and the cells were collected after being treated with 20nM lipopolysaccharide LPS and 100nM schizophyllan (LPS+schizophyllan) or 20nM lipopolysaccharide LPS and phosphate buffer (LPS+phosphate buffer) for 12h and 24h.

[0093] 2. RNA Extraction

[0094] The RNA in the above cells was extracted using an RNA extraction kit (purchased from Biotime Biotechnology, catalog number R0017M), and the RNA concentration and purity were detected using a NANO-400A ultra-micro nucleic acid analyzer.

[0095] 3. Primer synthesis

[0096] Primers for GAPDH (internal reference), IL-1β, TNFα, IL-6, IL-10, ARG1, CD80, and CD86 ( Table 5 ) were designed using NCBI and Primer Blast, and the sequences were synthesized by Qingke Biotechnology.

[0097] Table 5 Primer sequences

[0098]

[0099] Note: In the table, F represents forward primer and R represents reverse primer.

[0100] 4. RT-PCR reaction

[0101] RT-PCR reaction: According to the instructions of the reverse transcription kit (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 14601ES10), the reverse transcription reaction system was prepared on ice. After reverse transcribing RNA into cDNA on the PCR instrument according to the instructions on the kit, the real-time fluorescence quantitative PCR (RT-PCR) reaction system was prepared on ice: 5 μL of (RT-PCR) SYBR Green dye (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd. (company, catalog number 11203ES08) was taken on a 96-well plate dedicated to fluorescence quantitative PCR, 0.2 μL of each upstream and downstream primers, 1 μL of sample cDNA, and 3.6 μL of double distilled water (ddH2O); Bio-rad RT-PCR reaction conditions: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 10 s, 60℃ annealing / extension for 30 s, and 40 cycles. After the experiment, the Ct value was obtained and used 2 -△△Ct Calculation and Analysis,After repeating the experiment three times, the results were subjected to statistical analysis.

[0102] 5. Experimental results and discussion

[0103] THP-1 cells were treated with 100 nM schizophyllan and 20 nM LPS for 12 h and 24 h. GAPDH (Internal reference) 、 IL-1β, TNFα, IL-6, IL-10, ARG1, CD80, CD86 The relative mRNA expression levels of Figure 4 and Figure 5 When schizophyllan was treated with THP-1 cells for 12 h, pro-inflammatory factors IL-1β, TNFα, IL-8 The expression levels increased significantly. CD80 / CD86 It is a marker on the surface of pro-inflammatory macrophages, indicating that schizophyllan can enhance the innate immune function of the skin and quickly eliminate pathogens. IL-10 is an anti-inflammatory cytokine with immunomodulatory effects. It can restore the immune system to homeostasis at the end of inflammation and increase slightly. ARG1 It is a marker on the surface of repair macrophages. The slight increase indicates that macrophages change from pro-inflammatory to repair and gradually regulate the immune response. When THP-1 cells are treated with schizophyllan for 24 hours, the pro-inflammatory factor IL-1β, TNFα, IL-6 and CD80 / CD86 The expression level of IL-10 and ARG1The expression of was significantly upregulated compared with that at 24h, indicating that at the end of inflammation, mycobacterium schizophyllum can regulate the immune balance, downregulate the inflammatory response of the skin, and prevent the skin from immune overexcitation. This shows that macrophages gradually become dominated by repair macrophages, the inflammatory response of the skin is controlled, and the immune barrier is repaired.

[0104] Example 3 Verification of the effect of the schizophyllan-glycosaminoglycan composition

[0105] 1. Experimental Grouping

[0106] (1) Composition 1: 0.6 parts of glycosaminoglycan and 1.5 parts of schizophyllan.

[0107] (2) Composition 2: 0.8 parts of glycosaminoglycan and 2.3 parts of schizophyllan.

[0108] (3) Composition 3: 1 part of glycosaminoglycan and 2.3 parts of schizophyllan.

[0109] (4) Composition 4: 0.8 parts of glycosaminoglycan and 2.5 parts of schizophyllan

[0110] (5) Schizophylline group: 2.3 parts of schizophylline.

[0111] (6) Glycosaminoglycan group: 0.8 parts of glycosaminoglycan.

[0112] 2. Effect on repairing the skin immune barrier

[0113] In view of the increase of Staphylococcus aureus on the skin surface of people with sensitive skin, and the decrease of Staphylococcus epidermidis, which is beneficial to human skin health, the present invention provides a composition of schizophyllin-glycosaminoglycan that can improve the sensitive skin of people. The following experiment will verify the proliferation of Staphylococcus aureus and Staphylococcus epidermidis by in vitro studies of Staphylococcus aureus and Staphylococcus epidermidis respectively to verify whether the above-mentioned test substances regulate the balance of skin microecology by activating the activity of skin immune cells. The specific experimental method is as follows:

[0114] (1) Preparation of bacterial suspension

[0115] Staphylococcus aureus (ATCC6538) and Staphylococcus epidermidis (ATCC700296) from Guangdong Institute of Microbiology were inoculated in TSA (Tryptic Soy Agar) slant culture medium and cultured in an incubator (37±1℃) for 24 hours. After activation, the slant culture medium was diluted with 10mL sterile saline to make a bacterial count of 10 6 CFU / mL.

[0116] (2) Cultivation of RAW264.7 mouse macrophages

[0117] A dish of RAW264.7 mouse macrophages with moderate p60 density and good growth was digested, collected and centrifuged, resuspended and diluted to a 24-well plate. The concentration of RAW264.7 mouse macrophages was 1.5×10 5 Cells from 21 wells were taken and divided into 7 groups, each group was repeated three times. The culture medium (the culture medium does not contain antibiotics) was discarded and the compositions 1-4, the mycoplasma group and the glycosaminoglycan group were added, and PBS was used as a control group to co-culture with mouse macrophages RAW264.7, and Staphylococcus aureus and Staphylococcus epidermidis were inoculated to infect RAW264.7 cells.

[0118] (3) Bacterial survival experiment in RAW264.7 mouse macrophages

[0119] Equal numbers of RAW264.7 mouse macrophages were infected with Staphylococcus aureus and Staphylococcus epidermidis at a multiplicity of infection (MOI) of 1, respectively. High concentration (50 mg / mL) of gentamicin was used to kill bacteria outside mouse macrophages. Then, lipids and 10 mg / mL gentamicin were incubated with cells. At 24 hours post infection (hpi), cells were washed extensively and lysed in ice-cold 0.025% Triton X-100 (ultrapure water) to release intracellular bacteria, and the pellet was obtained by centrifugation at 8000 rpm and resuspended in phosphate buffered saline (PBS). Aliquots were inoculated on TSA broth agar plates and colony forming units (CFU) were counted after overnight incubation at 37°C. The experiment was repeated 3 times and the average value was taken. Calculation of proliferation rate of S. aureus and S. epidermidis: Proliferation rate (%) = (CFU 对照组 -CFU 测试组 ) / CFU 对照组 ×100%. Graphpadprism9.0 and SPSS Statistics 26.0 were used to perform statistical analysis on the data.

[0120] The effects of the compositions 1, 2, 3 and 4 of the present invention, the schizophyllan group and the glycosaminoglycan group on the proliferation of Staphylococcus aureus and Staphylococcus epidermidis are shown in the following table: Figure 6 and Figure 7 shown.

[0121] The above experimental results show that compared with composition 3, composition 4, schizophyllin group and glucosaminoglucan group, composition 1 and composition 2 can significantly promote the proliferation of Staphylococcus epidermidis and effectively inhibit the proliferation of Staphylococcus aureus. In addition, compared with the effects of composition 1 and composition 2 and the schizophyllin group, the compositions of 0.6 parts of glucosaminoglucan and 1.5 parts of schizophyllin, 0.8 parts of schizophyllin and 2.3 parts of glucosaminoglucan are more effective in inhibiting the proliferation of Staphylococcus aureus and promoting the proliferation of Staphylococcus epidermidis. From the in vitro experimental results of composition 1 and composition 2, it can be seen that under the conditions of composition 2 (0.8 parts of glucosaminoglucan and 2.3 parts of schizophyllin), the effect of stimulating mouse macrophages to inhibit the proliferation of Staphylococcus aureus and promote Staphylococcus epidermidis is better.

[0122] Example 4 Verification of the effect of a cosmetic composition prepared from a schizophyllan-glycosaminoglycan composition

[0123] 1. Experimental Grouping

[0124] (1) Cosmetic composition 1: 0.8 parts of glycosaminoglycan, 2.3 parts of schizophyllan, 2 parts of TRPV1 inhibitor, 1 part of ceramide, and 0.5 parts of squalane.

[0125] (2) Cosmetic composition 2: 0.8 parts of glycosaminoglycan, 2.3 parts of schizophyllan, 2 parts of TRPV1 inhibitor, and 1 part of ceramide.

[0126] (3) Cosmetic composition 3: 0.8 parts of glycosaminoglycan, 2.3 parts of schizophyllan, and 2 parts of TRPV1 inhibitor.

[0127] (4) Cosmetic composition 4: 0.8 parts of glycosaminoglycan, 2.3 parts of schizophyllan, 1 part of ceramide, and 0.5 parts of squalane.

[0128] 2. Preparation of Essence

[0129] Mix raw materials 1, 2, 3, 4, 5, and 6 and heat to 85°C for homogenization. After cooling, add raw materials 7, 8, 9, and 10 and stir evenly. Add 2% of cosmetic composition 1-4 or water respectively and stir evenly to prepare essence 1-5. The formula of essence 1-5 is shown in Table 6.

[0130] Table 6 Mass ratio of each component of essence 1-5

[0131]

[0132] 3. Verification of the soothing effect of essence 1-5

[0133] Experimental method: According to the method provided in "Cosmetic Soothing Effect-Zebrafish Embryo Neutrophil Test Method", this study randomly selected 24 wild-type AB strain zebrafish embryos and placed them in 3 cm culture dishes. 5 mL of zebrafish embryo culture medium (blank control group) and 5 mL of fish embryo culture medium containing 10 µM anhydrous copper sulfate (model control group) were added to induce neuromast cell damage in the lateral line region of zebrafish embryos, and neutrophils aggregated in response to immune responses, so as to establish a zebrafish skin soothing model. 24 zebrafish embryos were randomly selected and placed in 3 cm culture dishes. 5 mL of zebrafish embryo culture medium containing 10 µM anhydrous copper sulfate and 10 µM indomethacin were added as positive controls. 1.0 mg / mL essences 1-5 were given to the 5 mL fish embryo culture medium group containing 10 µM anhydrous copper sulfate, and cultured in a constant temperature box at 28°C ± 1°C for 40 to 45 minutes. The number of zebrafish embryo neutrophils was observed and counted under a stereo microscope to evaluate the soothing effect of essences 1-5.

[0134] The formula for calculating the inhibition rate of neutrophil aggregation is: inhibition rate = (MS) / M*100%; where S represents the average number of neutrophils in zebrafish embryos in the sample treatment group; M represents the average number of neutrophils in zebrafish embryos in the model control group. Analyze whether the detection values ​​of the essence 1-5 groups and the model control group are statistically different (p<0.05).

[0135] The measurement results are shown in Table 7.

[0136] Table 7 Number of centrosomes in zebrafish embryos

[0137]

[0138] Compared with the blank control group, the number of neutrophils in zebrafish embryos in the model control group increased significantly, indicating that this experiment successfully established a soothing model for zebrafish. Essence 1 had the most significant inhibition rate of neutrophil aggregation in zebrafish embryos at 48%, followed by essences 2-4, and the worst inhibitory effect on neutrophil aggregation in zebrafish embryos was essence 5. Compared with the model group, essences 1-4 had p<0.01, indicating that essences 1-4 had a soothing effect on zebrafish embryos, with statistically significant differences.

[0139] 4. Verification of the effect of the essence

[0140] Serums 1-4 were tested for sensitive skin and treatment of sensitive skin. The specific implementation plan and result analysis are as follows:

[0141] Principle: According to the announcement of the "Evaluation Standards for Cosmetic Efficacy Claims" (No. 50 of 2021) and its annexes, the subjects must be volunteers who have a positive lactic acid stinging screening reaction or have self-identified as sensitive skin in the questionnaire survey. As long as they recognize that the product is suitable, the claim for sensitive skin is supported. During this test, 132 healthy men or women aged 18-55 years who met the inclusion criteria were screened (positive lactic acid stinging reaction and identified as sensitive skin by dermatologists). All subjects were divided into 4 groups, 33 people / group. During the 28-day product use process, the recognition rate for sensitive skin was 100%, and the recognition rate for improving symptoms such as dry skin, scaling, burning, stinging, redness, tightness, dryness and itching was greater than 50%. It can be considered that the cosmetic composition of the present invention is suitable for sensitive skin and can effectively treat people with sensitive skin.

[0142] Verification method: The testers can use the product according to the required amount during normal facial cleansing, and other toners and moisturizers are not allowed to be used during the test. The essence is used once in the morning and evening after cleansing, and massage is performed to promote the absorption of the essence. All subjects maintain consistent usage habits during the 28-day test. The subjects fill out the questionnaires distributed on the 0th, 14th, and 28th days to verify whether the cosmetics used by the subjects can improve their skin condition.

[0143] Data Analysis:

[0144] (1) Satisfaction of test population

[0145] Satisfaction (%) = (number of relatively satisfied people + number of very satisfied people) / (total number of test takers) * 100%

[0146] (2) Difference analysis

[0147] SPSS was used to perform binomial distribution test to analyze the differences. The expected value of satisfaction was set at 50%, and the 95% confidence interval was used to analyze the statistical significance of the mild performance index results; p>0.05 indicated no significant difference; p<0.05 indicated significant difference; p<0.01 indicated very significant difference; p<0.001 indicated extremely significant difference.

[0148] (3) Soothing effect claims

[0149] Compared with the expected satisfaction value of 50%, there is a significant difference after using the essence composition provided by the present invention for a period of time, and the sample is judged to support the efficacy claim, otherwise it is judged that the sample does not have the efficacy.

[0150] (4) Efficacy claims for sensitive skin

[0151] When 100% of the subjects believe that the essence composition provided by the present invention is suitable for sensitive skin, the sample is judged to support the efficacy claim; otherwise, the sample is judged not to have the efficacy.

[0152] The measurement results are shown in Table 8.

[0153] Table 8 Effect evaluation of 132 subjects after using essence 1-4

[0154]

[0155] Results evaluation: After using Essence 1-4 for 14 days and 28 days, the 132 subjects' facial skin dryness, scaling, redness, itching, and tingling were improved to varying degrees, and statistical analysis showed significant differences. Among them, the subjects were most satisfied with Essence 1. After using the essence composition provided by Essence 1 for 28 days, 100% of the subjects believed that the essence composition could significantly improve the symptoms of dry skin, scaling, redness, itching, and tingling, and had a soothing effect. 100% of the subjects believed that the essence composition supported the efficacy claim of being suitable for sensitive skin.

[0156] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A composition, characterized in that The composition consists of 0.6-0.8 parts of glycosaminoglycan and 1.5-2.3 parts of schizophyllan by weight.

2. The composition according to claim 1, characterized in that The composition consists of 0.8 parts of glycosaminoglycan and 2.3 parts of schizophyllan by weight.

3. A cosmetic composition, characterized in that The cosmetic composition comprises the composition according to any one of claims 1 to 2.

4. The cosmetic composition according to claim 3, characterized in that The cosmetic composition consists of glycosaminoglycan, schizophyllan, TRPV1 inhibitor, ceramide and squalane.

5. The cosmetic composition according to claim 4, characterized in that The cosmetic composition comprises, by weight, 0.5-0.8 parts of glycosaminoglycan, 0.8-2.3 parts of schizophyllan, 1-2 parts of TRPV1 inhibitor, 0.5-1 parts of ceramide and 0.1-0.5 parts of squalane.

6. The cosmetic composition according to claim 5, characterized in that The cosmetic composition comprises, by weight, 0.8 parts of glycosaminoglycan, 2.3 parts of schizophyllan, 2 parts of TRPV1 inhibitor, 1 part of ceramide and 0.5 part of squalane.

7. Use of the composition according to any one of claims 1 to 2 or the cosmetic composition according to any one of claims 3 to 6 in the preparation of cosmetics.

8. The use according to claim 7, characterized in that: The cosmetics are used for improving sensitive skin.

9. The use according to claim 8, characterized in that: The cosmetics improve sensitive skin by defending against skin irritants, soothing skin discomfort, removing skin pathogenic microorganisms, balancing the microecology on the skin surface or controlling skin immune inflammation.

10. The use according to claim 9, characterized in that: The cosmetics include face cream, face mask, eye cream, neck cream, hand cream, body lotion, moisturizing lotion, essence, toner, shampoo, conditioner, shower gel or cleanser.

11. A cosmetic, characterized in that: The cosmetic comprises the composition according to any one of claims 1 to 2 or the cosmetic composition according to any one of claims 3 to 6.

12. The cosmetic according to claim 11, characterized in that The weight percentage content of the composition or cosmetic composition in the cosmetic is 2%-10%.

13. The cosmetic according to claim 11, characterized in that The cosmetics also include auxiliary materials acceptable in the cosmetics field.

14. The cosmetic according to claim 13, characterized in that The auxiliary materials include one or more of moisturizers, thickeners, emollients, skin conditioners, emulsifiers, preservatives, emollients, lubricants, and stabilizers.

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