CN118697662A - Composition for repairing immune barrier and relieving skin and application of composition in cosmetics - Google Patents

Abstract

The invention provides a composition for repairing immune barrier and relieving skin and application thereof in cosmetics, and belongs to the technical field of skin immunity. The composition provided by the invention comprises 0.5-0.8 part of glucosaminodextran and 0.8-2.3 parts of schizophyllan by weight, can enhance skin immune response, remove harmful bacteria of skin, convert and secrete inhibition inflammatory factors from pro-inflammatory macrophages to inflammation inhibition cells at the end of inflammation, and inhibit NF-kB inflammatory signals of the cells so as to prevent skin injury caused by excessive immune response. The cosmetic composition provided by the invention combines five components of ceramide, squalane, TRPV1 inhibitor, glucosaminodextran and schizophyllan, and prepares the composition with the effects of repairing epidermis barrier and enhancing skin immunity barrier through multi-channel synergistic effect, has anti-inflammatory and relieving effects, and can be widely applied to cosmetics for treating sensitive muscle groups.

Description

Composition for repairing immune barrier and relieving skin and application of composition in cosmetics

Technical Field

The invention belongs to the technical field of skin immunity, and particularly relates to a composition for repairing immune barrier and relieving skin and application of the composition in cosmetics.

Background

Sensitive skin is the skin of various parts of the body, especially the facial skin, is influenced by genetic, environmental, cosmetic, psychological pressure, hormone and other factors, and has the problems of itching, redness, burning, stinging, tightness, rash and the like caused by the stimulation reaction or the excessive activation of an immune system.

Current research suggests that the mechanism of development of sensitive muscle is associated with skin barrier-neurovascular-immune inflammation. The stratum corneum acts as a permeability barrier that prevents irritants, allergens and microorganisms from entering the skin. Whereas disruption of the epidermal barrier results in sensitive skin being more stimulated and more sensitive than normal skin. Under the action of internal and external factors, the skin barrier is damaged, the afferent signals are increased after the sensory neurons positioned on the skin are activated, and the afferent signals are transmitted into the cerebral cortex through the spinal cord to generate the sensations of itching, stinging, burning and the like; after the TPPV receptor of keratinocytes and mast cells is activated, calcium ions flow in, the cells are polarized, and inflammatory factors and chemokines are released, causing local inflammatory reactions and vasodilation redness of the skin. Meanwhile, the skin epidermis of the sensitive muscle group is dysbacteriosis, pathogenic bacteria excessively reproduce and release excessive signal molecules, so that skin inflammation can be caused, and the immune barrier of the skin is destroyed, thereby increasing the sensitivity of the skin.

Currently, the scientific mechanism for treating sensitive muscles is mainly to avoid adding irritant components, adding mild skin care components which help repair the epidermal barrier, or relieving inflammation, inhibiting neurogenic inflammation (such as CN117860625A, CN118045019a, CN117982387 a) in cosmetics. In addition, CN117462437a discloses a mussel protein composition which has good skin barrier repair, inflammatory reaction inhibition, and alleviation of subcutaneous nerve overfeedback through multi-channel synergy, and is suitable for sensitive skin. The mechanism of these products for treating sensitive muscle groups is mainly based on repairing the epidermal barrier or weakening the susceptibility of skin peripheral sensory neurons, but ignores the importance of disrupted skin flora microenvironment (reduced normal flora staphylococcus epidermidis beneficial to the skin and increased skin pathogenic bacteria staphylococcus aureus) and its induced immune response disorder and regulation of immune balance in sensitive skin surface flora.

There is growing evidence that beta-glucan can activate training immunity through epigenetic reprogramming and metabonomic reprogramming to enhance recognition and binding of lipopolysaccharide on pathogenic membranes by surface receptors of innate immune cells of the skin. In addition, under the continuous stimulation of pathogenic microorganisms, on one hand, beta-glucan enhances skin immunity through activated training immunity, shortens immune response time, enhances the proliferation and differentiation activities of immune cells and is recruited to inflammation sites, promotes the secretion of inflammatory factors and rapidly eliminates harmful bacteria; on the other hand, at the end of inflammation, beta-glucan activates immune-training metabolites to regulate the transformation of macrophages from inflammatory type to reparative type, up-regulates the expression of the inhibiting inflammatory factor IL-10, and timely terminates immune response to relieve skin sensitivity symptoms.

Furthermore, several scientific studies have demonstrated that immune imbalance is a major pathological change in sensitive skin and is even more prevalent than the stimulatory response initiated by an impaired epidermal barrier. Furthermore, an imbalance in flora diversity and function is associated with the development of inflammatory skin diseases. However, the cosmetics currently existing on the market are directed only to sensitive skin induced based on the irritant reaction, and cannot solve the sensitive skin caused by excessive multiplication of skin harmful bacteria.

Therefore, there is an urgent need in the art to develop a cosmetic composition that can effectively eliminate the uncomfortable feeling of the sensitive skin and alleviate the bad symptoms of redness, tightness, scales, etc. of the sensitive skin, so as to meet the demands of the increasingly sensitive muscle groups.

Disclosure of Invention

In order to overcome the defects, the invention provides a composition for repairing immune barrier and relieving skin and application thereof in cosmetics. The composition provided by the invention comprises 0.5-0.8 part of glucosaminodextran and 0.8-2.3 parts of schizophyllan by weight, can enhance skin immune response, remove harmful bacteria of skin, convert and secrete inhibition inflammatory factors from pro-inflammatory macrophages to inflammation inhibition cells at the end of inflammation, and inhibit NF-kB inflammatory signals of the cells so as to prevent skin injury caused by excessive immune response. The cosmetic composition provided by the invention combines five components of ceramide, squalane, TRPV1 inhibitor, glucosaminodextran and schizophyllan, and prepares the composition with the effects of repairing epidermis barrier and enhancing skin immunity barrier through multi-channel synergistic effect, has anti-inflammatory and relieving effects, and can be widely applied to cosmetics for treating sensitive muscle groups.

The technical scheme of the invention comprises the following steps:

In a first aspect, the invention provides the use of a beta-glucan in the preparation of a composition for repairing an immune barrier and soothing the skin.

Specifically, the beta-glucan is formed by polymerizing D-type glucose monomers with beta-1, 3 glycosidic bonds as a main chain.

Further specifically, the beta-1, 3-D-glucan is secreted by microorganisms.

Preferably, the microorganism includes, but is not limited to, schizophyllan beta-1, 3-D-glucan, candida albicans beta-1, 3-D-glucan, yeast beta-1, 3-D-glucan, oat beta-1, 3-D-glucan, seaweed beta-1, 3-D-glucan, barley beta-1, 3-D-glucan, or medicinal mushroom beta-1, 3-D-glucan.

Further preferred, the yeast beta-1, 3-D-glucan includes, but is not limited to, baker's yeast beta-1, 3-D-glucan or Saccharomyces cerevisiae beta-1, 3-D-glucan.

Further preferably, the medicinal mushrooms beta-1, 3-D-glucan comprises, but is not limited to, ganoderma lucidum beta-1, 3-D-glucan, turkey tail beta-1, 3-D-glucan, lentinus edodes beta-1, 3-D-glucan or Maitake mushroom beta-1, 3-D-glucan.

Further preferably, the beta-1, 3-D-glucan is secreted by schizophyllan.

Further specifically, the beta-1, 3-D-glucan is recognized by receptors on the surface of innate immune cells and plays an important role in the immune defenses of human skin.

Preferably, the innate immune cell surface includes but is not limited to neutrophils, macrophages or dendritic cells.

Preferably, the receptor is a Dectin1 receptor and a CR3 complement receptor.

More specifically, the glycosidic bond of the beta-1, 3-D-glucan is formed by connecting beta-1, 3 glycosidic bond or main chain beta-1, 3 glycosidic bond and branched beta-1, 6 glycosidic bond or beta-1, 3 glycosidic bond.

Preferably, the branched β -1,6 glycosidic linkages are linked in a manner including, but not limited to, the following structures: one of every 3 beta-1, 3 glycosidic bond linked glycosyl is formed by linking a branched beta-1, 6 glycosidic bond; or (b)

One of every 7 beta-1, 3 glycosidic bond linked glycosyl is formed by linking a branched beta-1, 6 glycosidic bond; or (b)

One of every 3 beta-1, 3 glycosidic bond linked glycosyl is formed by linking a branched beta-1, 6 glycosidic bond and 2 beta-1, 3 glycosidic bonds.

Further preferably, the branched beta-1, 6 glycosidic bond is formed by connecting one of every 3 beta-1, 3 glycosidic bond linked glycosyl groups through the branched beta-1, 6 glycosidic bond, so that a unique supercoiled three-dimensional structure is formed, and the structure is easy to recognize and combine by an immune receptor, and the immune barrier of skin is enhanced.

In certain embodiments, the 1,3-1,6 branched beta-glucan has the following structural formula:

wherein n is an integer of 1 or more, more preferably 500 to 3000, still more preferably 1000 to 2000.

Preferably, the branching Degree (DB) of the 1,3-1,6 branched-chain beta-glucan is 0.01-1; more preferably, the branching Degree (DB) of the 1,3-1,6 branched-chain beta-glucan is 0.3-0.6; more preferably, the branching Degree (DB) of the 1,3-1,6 branched beta-glucan is 0.8-1.

Preferably, the molecular weight of the 1,3-1,6 branched beta-glucan is more than or equal to 1KD; more preferably, the molecular weight of the 1,3-1,6 branched beta-glucan is 500KD-4000KD; more preferably, the molecular weight of the 1,3-1,6 branched beta-glucan is 1000KD-2000KD.

In a second aspect, the present invention provides a composition comprising: glucoaminoglucan and schizophyllan.

Specifically, the composition comprises 0.5-0.8 part of glucosaminodextran and 0.8-2.3 parts of schizophyllan by weight.

Preferably, the composition comprises 0.6 to 0.8 parts by weight of glucosaminodextran and 1.5 to 2.3 parts by weight of schizophyllan.

Preferably, the composition comprises 0.8 parts by weight of glucosaminodextran and 2.3 parts by weight of schizophyllan.

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

Specifically, the cosmetic composition consists of glucosaminodextran, schizophyllan, TRPV1 inhibitor, ceramide and squalane.

Preferably, the cosmetic composition comprises 0.5 to 0.8 part of glucosaminodextran, 0.8 to 2.3 parts of schizophyllan, 1 to 2 parts of TRPV1 inhibitor, 0.5 to 1 part of ceramide and 0.1 to 0.5 part of squalane in parts by weight.

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

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

In particular, the cosmetic is used for improving sensitive muscles.

Preferably, the cosmetic improves sensitive muscles by protecting against skin irritants, soothing skin discomfort, eliminating skin pathogenic microorganisms, balancing the micro-ecology of the skin surface or controlling skin immune inflammation.

Preferably, the cosmetic product includes, but is not limited to, a face cream, a mask, an eye cream, a neck cream, a hand cream, a body lotion, a moisturizing liquid, an essence, a toner, a shampoo, a conditioner, a body wash, or a face wash.

In a fifth aspect, the present invention provides a cosmetic comprising the above composition or a cosmetic composition.

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

Preferably, the weight percentage of the composition or the cosmetic composition in the cosmetic is 2%.

Specifically, the cosmetic also comprises auxiliary materials acceptable in the field of cosmetics.

Preferably, the adjuvants include, but are not limited to, one or more of moisturizers, thickeners, emollients, skin conditioners, emulsifiers, preservatives, emollients, lubricants, stabilizers.

The invention has the technical effects that:

(1) The molecular docking and qPCR experiments show that the schizophyllan provided by the invention is more stable in combination with an immune receptor, activates training immunity, regulates and controls inflammatory reaction, rapidly eliminates pathogens, and is applied to cosmetics as a component for enhancing skin immunity and regulating immune balance.

(2) The composition provided by the invention can enhance skin immune response, remove harmful bacteria of the skin, convert from pro-inflammatory macrophages to inflammatory suppressor cells at the end stage of inflammation and secrete suppressor inflammatory factors, and inhibit NF-kB inflammatory signals of the cells so as to prevent skin injury caused by excessive immune response.

(3) The invention provides a cosmetic composition for treating sensitive muscles, which is based on possible occurrence mechanisms of the sensitive muscles respectively: the unbalance of skin barrier-neurovascular-immune inflammation-flora targets a plurality of action paths, plays corresponding effects respectively, and is coordinated, so that the problems of barrier damage, neurogenic inflammation induced by nervous system activation and skin microecological unbalance-mediated immune regulation unbalance of sensitive skin which is found in the current research are solved in a multi-layer and multi-dimensional manner.

Drawings

FIG. 1 is a molecular docking image of schizophyllan and beta-glucan binding to Dectin-1, respectively; in the figure, A is a butting image of the binding of schizophyllan to Dectin-1; b is a molecular docking image of the binding of beta-glucan and Dectin-1.

FIG. 2 is a molecular docking image of schizophyllan and beta-glucan binding to CR3, respectively; in the figure, A is a molecular docking image of the binding of schizophyllan and CR 3; b is a molecular docking image of the binding of beta-glucan to CR 3.

FIG. 3 is a molecular docking image of chitin, alginic acid and pectin disaccharides bound to Dectin-1 and CR3, respectively; in the figure, A is a molecular docking image of the combination of chitin and Dectin-1; b is a 3D molecular docking image of the combination of chitin and CR 3; c is a molecular docking image of alginic acid combined with Dectin-1; d is a molecular docking image of alginic acid combined with CR 3; e is a molecular docking image of the binding of pectbiose to Dectin-1; f is a molecular docking image of the binding of pectbiose to CR 3.

FIG. 4 shows the combined treatment of THP-1 cells with 20nM LPS and 100mM schizophyllan for 12h.

FIG. 5 shows the combined treatment of THP-1 cells with 20nM LPS and 100mM schizophyllan for 24h.

FIG. 6 is a graph showing the effect of schizophyllan-glycosaminoglycan composition on the proliferation rate of Staphylococcus aureus.

FIG. 7 is a graph showing the effect of schizophyllan-glycosaminoglycan composition on the proliferation rate of Staphylococcus epidermidis.

Detailed Description

In order to make the technical means, the creation features, the achievement of the purpose and the effect of the present invention easy to understand, the present invention will be further elucidated with reference to the specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all of them. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention. In the following examples, unless otherwise specified, the methods of operation used were conventional, the equipment used was conventional, and the materials used in the examples were the same.

The experimental materials used in the invention are shown in table 1:

TABLE 1 Experimental materials of the invention

EXAMPLE 1 molecular docking

1. Experimental materials

Receptor: PDB structure of Dectin-1 and CR3 proteins.

Ligand: schizophyllan, dextran, pectin disaccharide, citrus pectin and chitin numbering (CID, chemical Identifier).

Operation and analysis software: pyMOL; autodock4 Vina; autodock tools1.5.6; chemdraw.

2. Experimental methods and procedures

Downloading PDB structure of receptor protein to be tested, and storing file in PDB format. PDB structure of receptor: dectin-1 protein was 2Cl8 and CR3 protein was 7usl. Amino acid residues 843-1076 were truncated using software PyMOL.

Ligand preparation on pubchem website: schizophyllan is CID24777, beta-glucan is CID439262, alginic acid is CID5102882, chitin is CID6857375, and pectobiose is CID16738707.

Molecular docking calculations were performed using Autodock4 Vina software: matching the ligand with the receptor, defining a search area in the residue range of the receptor amino acid to be detected, respectively calculating lattice point energy by scanning different atoms, continuously adjusting the conformation, position and direction of the ligand, and scoring and sequencing to obtain the conformation with the lowest energy as an output result.

Molecular docking operations were performed with autodock tools1.5.6, and PyMOL was started to analyze the results.

Molecular docking 2D pictures were drawn with Chemdraw software.

3. Experimental results and discussion

3.1 Schizophyllan activates training immunity by binding to Dectin-1 protein C domain

Whether the conformation of Dectin1 receptor after binding to beta-glucan is stable or not is critical for activating training immunity. Molecular docking images of schizophyllan 1,3-1,6 branched beta-glucan and 1,3 linear beta-glucan binding to the C domain of Dectin1 receptor are shown in fig. 1, and binding free energies are shown in table 2.

TABLE 2 binding energy of schizophyllan and 1,3 Linear beta-glucan to Dectin-1 receptor

The binding free energy of schizophyllan was-8.8 Kcal/mol (A in FIG. 1), beta-glucan was-8.2 Kcal/mol (B in FIG. 1), and both were less than-5 Kcal/mol, indicating that the molecular docking results were authentic. The results show that compared with 1,3 straight-chain beta-glucan, 1,3-1,6 branched-chain beta-glucan of schizophyllan has lower binding energy and more stable structure, shows that the 1,3-1,6 branched-chain beta-glucan of the schizophyllan has stronger biological activity, induces conformational change after being combined with a receptor, leads the Dectin-1 molecule to form a dimer/oligomer, activates intracellular molecules to generate signal transduction, triggers training immunity, enhances the identification and binding capacity of immune cells to pathogenic microorganisms, and activates the immunoregulation function of skin cells.

3.2 Schizophyllan activates training immunity by binding to the Linctin-1 end domain of the CR3 complement receptor

The stability of the conformation of the CR3 complement receptor after binding to β -glucan is critical for activation of the training immunity. Molecular docking images of schizophyllan 1,3-1,6 branched beta-glucan and 1,3 linear beta-glucan binding to the CR3 receptor Linctin-type 1 domain are shown in FIG. 2, and binding free energies are shown in Table 3.

TABLE 3 binding energy of schizophyllan and 1,3 Linear beta-glucan to CR3 receptor

The binding energy of schizophyllan was-7.6 Kcal/mol (A in FIG. 2), and that of 1,3 linear beta-glucan was-6.8 Kcal/mol (B in FIG. 2), both less than-5 Kcal/mol, indicating that the molecular docking results were authentic. The results of this study are explained from the molecular energy and structural point of view: schizophyllan 1,3-1,6 branched beta-glucan has lower structural binding energy than beta-glucan. Therefore, the structure is more stable, the biological activity is stronger, the time of immune response is shortened, the proliferation and activation of skin immune cells are promoted, the inflammatory response is regulated and controlled, and the pathogen is rapidly cleared.

3.3 Schizophyllan 1,3-1,6 branched beta-glucan structure is more bioactive than other glucans

In order to verify that the beta-glucan with the 1,3-1,6 branched chains of schizophyllan has a molecular basis with stronger biological activity, three beta-glucans with moisturizing effects and different sources are selected for the study, and are divided into three groups for molecular docking tests. The molecular docking image is shown in fig. 3 and the binding free energy is shown in table 4.

TABLE 4 binding energy of beta-glucans of different Structure to Dectin-1 receptor and CR3 receptor

The first group examined chitin, which had a binding energy of-6.1 Kcal/mol (A in FIG. 3) to Dectin-1 receptor, and a binding energy of-5.1 Kcal/mol (B in FIG. 3) to CR3 receptor. The second group examined alginic acid, which was analyzed for its binding energy to Dectin-1 receptor of-6 Kcal/mol (C in FIG. 3) and CR3 receptor of-5.1. 5.1 Kcal/mol (D in FIG. 3). The third group examined pectbiose, which was analyzed for its binding energy to Dectin-1 receptor of-7.9 Kcal/mol (E in FIG. 3), and CR3 receptor of-6.3 Kcal/mol (F in FIG. 3).

Comparison of the binding energy of the molecular docking with-5 Kcal/mol shows that the results of the first three groups of samples are authentic. The above results demonstrate that the binding energy between schizophyllan 1,3-1,6 branched-chain structure beta-glucan and Dectin-1 and CR3 receptors is the lowest compared with four beta-glucans with conventional moisturizing effects, so that the stability of the molecular structure is the highest, the higher biological activity is achieved, and the schizophyllan has the potential effect of activating training immunity to enhance body immunity.

Example 2 qPCR experiment

1. Cell culture

Collecting and centrifuging THP-1 cells of a dish of human monocytic leukemia cells with moderate p60 density and good growth vigor, re-suspending, diluting to 24-pore plates, treating the THP-1 cells with the concentration of 1.5x10ζ5/pore with 50nM phorbol ester PMA for 24 hours until the cells are completely adhered to the wall, and establishing a cell inflammation model. The medium was discarded and cells were harvested after 12h and 24h treatment with 20nM lipopolysaccharide LPS and 100nM schizophyllan (LPS+schizophyllan) or 20nM lipopolysaccharide LPS and phosphate buffer (LPS+phosphate buffer).

2. RNA extraction

RNA was extracted from the above cells using an RNA extraction kit (commercially available from Biyun Tian Biotechnology, cat. No. R0017M), and the concentration and purity of the RNA were measured using a NANO-400A ultra-micro nucleic acid analyzer.

3. Primer synthesis

Primers GAPDH (internal control), IL-1. Beta., TNF. Alpha., IL-6, IL-10, ARG1, CD80, CD86 (Table 5) were designed using NCBI and Primer Blast, respectively, and sequences were biosynthesized from the family of Practidae.

Primer sequences of Table 5

Note that: in the table, F represents the forward primer and R represents the reverse primer.

4. RT-PCR reaction

RT-PCR reaction: the reverse transcription reaction system was prepared on ice according to the instructions of the reverse transcription kit (purchased from the company of the following holy biotechnology (Shanghai) Co., ltd., product No. 14601ES 10). After reverse transcription of RNA into cDNA on a PCR instrument according to the instructions on the kit, a real-time fluorescent quantitative PCR (RT-PCR) reaction system was formulated on ice: a96-well plate dedicated to fluorescent quantitative PCR was prepared by taking 5. Mu.L of SYBR Green dye (purchased from the company of Santa Clara Biotechnology, inc. (Shanghai) and 0.2. Mu.L of each of the upstream and downstream primers, 1. Mu.L of cDNA of the sample, 3.6. Mu.L of double distilled water (ddH ₂ O), and RT-PCR reaction conditions of Bio-rad: pre-denaturation at 95℃for 2 min, denaturation at 95℃for 10 s, annealing/extension at 60℃for 30 s,40 cycles, ct values were obtained after the end of the experiment, and after repeating the experiment three times, statistical analysis was performed on the results using 2 ^-△△Ct.

5. Experimental results and discussion

The relative amounts of mRNA expressed by GAPDH (reference), IL-1β, TNFα, IL-6, IL-10, ARG1, CD80, CD86 after treatment of THP-1 cells with 100nM schizophyllan and 20nM lipopolysaccharide LPS for 12h and 24h are shown in FIGS. 4 and 5, respectively. When the schizophyllan is used for treating THP-1 cells for 12 hours, the expression quantity of the pro-inflammatory factors IL-1 beta, TNF alpha and IL-8 is obviously increased. CD80/CD86 is a marker on the surface of pro-inflammatory macrophages, indicating that schizophyllan can enhance the innate immune function of the skin and rapidly clear pathogens. IL-10 is an anti-inflammatory cytokine with immunomodulating effects, which restores the immune system to a steady state at the end of inflammation with a slight increase. ARG1 is a marker on the surface of repair type macrophages, and slightly increases, which indicates that macrophages are transformed from a pro-inflammatory type to a repair type, and gradually regulate immune response; when the schizophyllan is used for treating THP-1 cells for 24 hours, the expression quantity of pro-inflammatory factors IL-1 beta, TNF alpha, IL-6 and CD80/CD86 is obviously reduced compared with 12 hours, and the expression quantity of IL-10 and ARG1 is obviously up-regulated compared with 24 hours, which shows that at the end of inflammation, the schizophyllan can regulate the immune balance, down-regulate skin inflammatory reaction and prevent skin from generating immune overexcitation, and shows that macrophages are mainly reparative macrophages, the inflammatory reaction of the skin is controlled, and the immune barrier is repaired.

Example 3 verification of the Effect of schizophyllan-glycosaminoglycan composition

1. Experimental grouping

(1) Composition 1:0.6 part of glucosaminodextran and 1.5 parts of schizophyllan.

(2) Composition 2:0.8 part of glucosaminodextran and 2.3 parts of schizophyllan.

(3) Composition 3:1 part of glucosaminodextran and 2.3 parts of schizophyllan.

(4) Composition 4:0.8 part of glucosaminodextran and 2.5 parts of schizophyllan

(5) Schizophyllan group: 2.3 parts of schizophyllan.

(6) Glucosaminodextran group: 0.8 parts of glucosaminodextran.

2. Action on skin immune Barrier repair

Aiming at the increase of staphylococcus aureus on the skin surface of sensitive muscle groups and the decrease of staphylococcus epidermidis beneficial to the skin health of human bodies, the invention provides a schizophyllan-glucosaminodextran composition capable of improving the sensitive muscle groups. The following experiments will verify the proliferation of compositions 1-4, schizophyllan group and glucosaminodextran group against staphylococcus aureus and staphylococcus epidermidis by in vitro studies of the respective infections of staphylococcus aureus and staphylococcus epidermidis with mouse macrophage RAW264.7, i.e. whether the above-mentioned test substances modulate the skin micro-ecological balance by activating the activity of skin immune cells. The specific experimental method is as follows:

(1) Preparation of bacterial suspension

Staphylococcus aureus (Staphylococcus aureus, ATCC 6538) and Staphylococcus epidermidis (Staphylococcus epidermidis, ATCC 700296) from the Guangdong province institute of microorganisms were inoculated in TSA (Tryptic Soy Agar) slant culture-based (37.+ -. 1 ℃) incubator for activation for 24 hours. After the completion of the activation, the test slant medium was diluted with 10mL of sterile physiological saline to give a bacterial content of 10 ⁶ CFU/mL.

(2) Culture of RAW264.7 mouse macrophages

One dish of RAW264.7 mice with moderate p60 density and good growth vigor are digested, collected and centrifuged, and diluted to 24-well plates after being resuspended, and the concentration of RAW264.7 mice macrophages is 1.5X10 ⁵/well. Cells from 21 wells were divided into 7 groups and each group was repeated three times. The medium was discarded (medium without antibiotics) and compositions 1-4, schizophyllan group and glucosaminodextran group were added, respectively, and PBS was co-cultured with mouse macrophage RAW264.7 as a control group while staphylococcus aureus and staphylococcus epidermidis were inoculated to infect RAW264.7 cells.

(3) RAW264.7 mice macrophage inner bacterial survival experiment

The same number of RAW264.7 mouse macrophages were infected with Staphylococcus aureus and Staphylococcus epidermidis, respectively, at a ratio of multiplicity of infection (MOI) of 1. High concentrations (50 mg/mL) of gentamicin were used to kill bacteria outside the mouse macrophages. The lipids were then incubated with 10mg/mL gentamicin with cells. At 24 hours post infection (hpi), cells were washed thoroughly and lysed in ice-cold 0.025% Triton X-100 (ultrapure water) to release intracellular bacteria, pellet was obtained by centrifugation at 8000 rpm, and resuspended in Phosphate Buffer (PBS). Aliquots were plated on TSA broth agar plates and counted for Colony Forming Units (CFU) after overnight incubation at 37℃and the average was taken by repeating 3 experiments. Calculation of proliferation rates of staphylococcus aureus and staphylococcus epidermidis: proliferation rate (%) = (CFU _{Control group} -CFU _{test set} ）/CFU _{Control group} ×100), data results were statistically analyzed using software graphpaprism 9.0 and SPSS STATISTICS 26.0.0.

The results of the effect of the inventive compositions 1, 2, 3 and 4, schizophyllan group and glucosaminodextran group on the proliferation of staphylococcus aureus and staphylococcus epidermidis are shown in figures 6 and 7, respectively.

The above experimental results show that composition 1 and composition 2 significantly promote the proliferation of staphylococcus epidermidis and effectively inhibit the proliferation of staphylococcus aureus, compared with composition 3, composition 4, schizophyllan group and glucosaminodextran group. Further, the effects of the 0.6 part of glucosaminodextran and 1.5 part of schizophyllan, 0.8 part of schizophyllan and 2.3 parts of glucosaminodextran composition on the inhibition of the proliferation of staphylococcus aureus and the promotion of the proliferation of staphylococcus epidermidis were more remarkable than those of the schizophyllan group from the compositions 1 and 2. From the results of in vitro experiments of composition 1 and composition 2, it can be seen that the effect of stimulating the macrophages of mice to inhibit the proliferation of staphylococcus aureus and promoting staphylococcus epidermidis under the condition of composition 2 (0.8 part of glucosaminodextran and 2.3 parts of schizophyllan) is better.

Example 4 verification of the Effect of a cosmetic composition prepared from a schizophyllan-Glucoaminoglucan composition

1. Experimental grouping

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

(2) Cosmetic composition 2:0.8 part of glucosaminodextran, 2.3 parts of schizophyllan, 2 parts of TRPV1 inhibitor and 1 part of ceramide.

(3) Cosmetic composition 3:0.8 part of glucosaminodextran, 2.3 parts of schizophyllan, 2 parts of TRPV1 inhibitor.

(4) Cosmetic composition 4:0.8 part of glucosaminodextran, 2.3 parts of schizophyllan, 1 part of ceramide and 0.5 part of squalane.

2. Preparation of essence

Mixing materials 1, 2, 3, 4, 5, and 6, heating to 85deg.C, homogenizing, cooling, adding materials 7, 8, 9, and 10, stirring, adding 2% cosmetic composition 1-4 or water, and stirring to obtain essence 1-5. The formulations of the essences 1 to 5 are shown in Table 6.

TABLE 6 essence 1-5 mass ratio of each component

3. Soothing efficacy verification of essence 1-5

The experimental method comprises the following steps: according to the method provided by the cosmetic soothing efficacy-zebra fish embryo neutrophil test method, 24 wild type strain zebra fish embryos are randomly selected in the research, 5mL of zebra fish embryo culture solution (blank control group) and 5mL of fish embryo culture solution (model control group) containing 10 mu M anhydrous copper sulfate are respectively added into a 3cm culture dish to induce the damage of nerve hillock cells in the lateral line region of the zebra fish embryo, and the neutrophils are aggregated due to immune response, so that a zebra fish skin soothing model is established. Randomly selecting 24 zebra fish embryos to a 3cm culture dish, and adding 5mL of zebra fish embryo culture solution containing 10 mu M anhydrous copper sulfate and 10 mu M indomethacin to serve as a positive control. 1.0mg/mL essence is respectively added into 5mL of fish embryo culture solution containing 10 mu M anhydrous copper sulfate for 1-5, and the fish embryo culture solution is cultured in a constant temperature box at 28+/-1 ℃ for 40-45 min. The number of neutrophils in zebra fish embryos was counted under a split microscope to evaluate the soothing efficacy of the essences 1-5.

Calculating a formula of the inhibition rate of neutrophil aggregation: inhibition = (M-S)/M100%; wherein, the ratio S represents the average value of the number of the neutrophils in the zebra fish embryo of the sample treatment group; m represents the average value of the number of the neutrophils in the zebra fish embryo of the model control group. The test values of the serum 1-5 groups and the model control group were analyzed for statistical differences (p < 0.05).

The measurement results are shown in Table 7.

TABLE 7 number of granulocytes in the center of zebra fish embryos

Compared with a blank control group, the number of the zebra fish embryo neutrophils in the model control group is obviously increased, which indicates that the experiment successfully builds a zebra fish soothing model. The essence 1 has the most obvious inhibition rate of the neutrophil aggregation of the zebra fish embryo of 48 percent, the second is the essence 2-4 respectively, and the worst inhibition effect of the neutrophil aggregation of the zebra fish embryo is the essence 5. And compared with the model group, p is less than 0.01, which shows that the essence 1-4 has a soothing effect on zebra fish embryos and has statistically significant differences.

4. Verification of the effect of essence

The essences 1-4 are respectively used for carrying out consumer tests of sensitive muscle application and sensitive muscle treatment, and the specific implementation and the result analysis are as follows:

Principle of: the subjects required to be tested according to the bulletin of the "cosmetic efficacy claim evaluation Specification" (No. 50 of 2021) and its annex must be volunteers who respond positively to the lactic acid stinging screening or who self-identify as sensitive muscles in a questionnaire, and support the sensitive muscles application claim whenever the product is approved to be suitable. 132 healthy men or women (lactic acid stinging reaction positive and dermatologists identified as skin sensitive) who were 18-55 years old and met the conditions of the group were selected during the test. All subjects were divided into 4 groups, 33 persons/group, and the applicable acceptance ratio for sensitive muscles was 100% and the acceptance ratio for improving symptoms such as dry skin, scales, burning, stinging, redness, tightness, dry itching and the like was more than 50% in the 28-day product use process, so that the cosmetic composition of the invention was considered to be applicable to sensitive muscles and capable of effectively treating sensitive muscle groups.

The verification method comprises the following steps: the tester can use the product according to the requirement in normal face-cleaning thickness, and other skin-refreshing and skin-moisturizing products cannot be used during the test. The serum was used once each after the early and late face washes, and the serum was massaged to promote absorption, and all subjects remained consistent in use habits during the 28 day test period. Subjects filled in the questionnaire dispensed on days 0, 14, and 28, respectively, to verify whether the cosmetics used by the subjects improved their skin condition.

Data analysis:

(1) Testing crowd satisfaction

Satisfaction (%) = (comparative satisfaction + very satisfaction)/(test total) 100%

(2) Differential analysis

Performing binomial distribution test analysis on the variability by using SPSS, setting the satisfaction expectation value as 50%, and performing analysis on statistical significance of the mild performance index result by using a 95% confidence interval; p > 0.05 indicates no significant difference; p < 0.05 indicates significant variability; p < 0.01 indicates that the variability is very significant; p < 0.001 indicates that the variability is extremely pronounced.

(3) The soothing effect is declared

The essence composition provided by the invention has a significant difference after a period of time compared with the expected satisfaction value of 50%, and the sample is judged to support efficacy, otherwise, the sample is judged not to have the efficacy.

(4) The sensitivity muscle proper efficacy is declared

When 100% of the subjects consider the essence composition provided by the invention to be suitable for sensitive muscles, the sample is judged to support the efficacy, otherwise, the sample is judged not to have the efficacy.

The measurement results are shown in Table 8.

Table 8 evaluation of the effects of 132 subjects after use of the essences 1 to 4

Evaluation of results: after 14 days and 28 days of using the essence, 132 subjects had improved facial skin dryness, scales, redness, itching and stinging to different degrees, and the statistical analysis had significant differences. Among them, the subject had the highest satisfaction with concentrate 1. After 28 days of using the essence composition provided by the essence 1, 100% of subjects consider that the essence composition can significantly improve the dry skin, scales, redness, dry itching and stinging symptoms, and has a soothing effect. 100% of the subjects believe that the concentrate composition supports the efficacy of sensitive muscle adaptation.

The above detailed description is directed to a specific description of one possible embodiment of the invention, which is not intended to limit the scope of the invention. It should be noted that all equivalent implementations or modifications that do not depart from the spirit and scope of the present invention are intended to be included within the scope of the present invention. The scope of the invention should therefore be determined by the appended claims.

Claims (15)

1. A composition comprising, by weight, 0.5-0.8 parts of glucosaminodextran and 0.8-2.3 parts of schizophyllan.

2. The composition of claim 1, wherein the composition comprises 0.6-0.8 parts by weight of glucosaminodextran and 1.5-2.3 parts by weight of schizophyllan.

3. The composition of claim 1, wherein the composition comprises 0.8 parts by weight of glucosaminodextran and 2.3 parts by weight of schizophyllan.

4. A cosmetic composition characterized in that, the cosmetic composition comprising the composition of any one of claims 1 to 3.

5. The cosmetic composition according to claim 4, wherein the cosmetic composition consists of glucosaminodextran, schizophyllan, TRPV1 inhibitors, ceramides and squalane.

6. The cosmetic composition according to claim 5, wherein the cosmetic composition comprises 0.5 to 0.8 parts by weight of glucosaminodextran, 0.8 to 2.3 parts by weight of schizophyllan, 1 to 2 parts by weight of TRPV1 inhibitor, 0.5 to 1 part by weight of ceramide and 0.1 to 0.5 part by weight of squalane.

7. The cosmetic composition according to claim 6, wherein the cosmetic composition comprises 0.8 parts by weight of glucosaminodextran, 2.3 parts by weight of schizophyllan, 2 parts by weight of TRPV1 inhibitor, 1 part by weight of ceramide and 0.5 part by weight of squalane.

8. Use of the composition of any one of claims 1 to 3 or the cosmetic composition of any one of claims 4 to 7 in the preparation of a cosmetic.

9. The use according to claim 8, wherein the cosmetic is for improving sensitive muscles.

10. The use according to claim 9, wherein the cosmetic improves sensitive muscles by defending skin irritants, soothing skin discomfort, eliminating skin pathogenic microorganisms, balancing skin surface micro-ecology or controlling skin immune inflammation.

11. The use according to claim 8, wherein the cosmetic comprises a face cream, a face mask, an eye cream, a neck cream, a hand cream, a body lotion, a moisturizing lotion, an essence, a toner, a shampoo, a conditioner, a body wash or a face wash.

12. A cosmetic product, characterized in that it comprises a composition according to any one of claims 1 to 3 or a cosmetic composition according to any one of claims 4 to 7.

13. The cosmetic product according to claim 12, wherein the composition or cosmetic composition is present in an amount of from 2% to 10% by weight.

14. The cosmetic product of claim 12, wherein the cosmetic product further comprises a cosmetically acceptable adjuvant.

15. The cosmetic product of claim 14 wherein the adjuvant comprises one or more of moisturizers, thickeners, emollients, skin conditioners, emulsifiers, preservatives, emollients, lubricants, stabilizers.