Application of schizophyllan as human beta-defensin-2 accelerant

Abstract

The invention provides an application of schizophyllan as a human beta-defensin-2 accelerant. According to the application, the effect of inducing human beta-defensin-2 (hBD-2) expression of the schizophyllan is found for the first time, and the schizophyllan can effectively stimulate an antimicrobial defense system on the surface of a human body to restore the stability of a natural internal environment, so that the schizophyllan is used for improving natural defense on the surface of the human body or improving microbiome balance on the surface of the human body; the active effects on prevention and / or reduction of skin infection, skin sensitivity, skin barrier damage and skin microecological disorder, prevention and improvement of pox muscles and the like are achieved, and the application field of the substance is widened.

Description

Technical Field

[0001] The present application belongs to the technical field of skin care, and in particular to the use of schizophyllan as a human β-defensin-2 promoter. Background Art

[0002] The epidermis establishes a barrier against microbial infection by producing antimicrobial peptides (AMPs). Antimicrobial peptides are a group of small cationic polypeptides. Antimicrobial peptides have a broad spectrum of antibacterial, fungal and viral effects and are effector molecules of innate immunity. In addition, these peptides regulate local inflammatory responses and activate cellular immunity and adaptive immunity. Defensins and cathelicidins are the main members of skin antimicrobial peptides. In addition, other skin peptides, such as protease inhibitors, chemokines, granulin family and neuropeptides, also show antimicrobial activity. They play an important role in skin immune defense.

[0003] Defensins contain six cysteine ​​residues, forming characteristic disulfide bridges. The order of arrangement and molecular structure of disulfide bridges divide defensins into three categories: α-defensins, β-defensins, and γ-defensins. Four β-defensins have been found in various human cells, including peripheral blood monocytes and epithelial cells, namely human β-defensins-1, -2, -3, and -4 (hBD-1 to hBD-4). hBD-2 is usually upregulated in infected, damaged, inflamed skin or under stressful conditions, and is absent in normal skin. However, for consumers, they do not want hBD-2 to be expressed only when the skin is infected, damaged, inflamed, or under emotional stress to play a protective role in the skin barrier.

[0004] Additionally, these peptides, when produced normally, are sufficient to limit the proliferation of microorganisms on the skin surface, thereby preventing injury or infection. However, when insufficient to limit bacterial proliferation occurs, infection or sepsis of the skin and / or epithelial mucosa is observed.

[0005] Therefore, there is a need for healthy skin, dry skin, sensitive skin, allergic skin, burned / traumatized skin, etc., which can stimulate the above-mentioned natural defense system to restore the natural internal environment stability, especially in the case of insufficient barrier function, making it possible to avoid any risk of undesirable bacterial proliferation.

[0006] Although when the skin is invaded by bacteria, fungi, or viruses, people usually think of using antibacterial substances to eliminate the adverse effects of harmful bacteria, fungi, or viruses, antibacterial substances are usually not selective and will kill or inhibit the growth of bacteria including beneficial bacteria, which will also disrupt the balance of the skin's own microbiome and weaken the skin's natural defense. Therefore, simple antibacterial agents cannot meet people's needs for skin care.

[0007] Although immunomodulation can also help resist bacterial, fungal, and viral infections, such immunomodulation is often accompanied by symptoms such as inflammation, redness, and sensitivity, and is therefore undesirable.

[0008] Substances that do not have immunomodulatory effects can also promote the expression of defensins. For example, Rhodiola rosea (RHODIOLAROSEA) extracts have immunomodulatory effects, but the applicant found in the study that Rhodiola rosea (RHODIOLAROSEA) extracts cannot promote the expression of human β-defensin-2. Therefore, they are ineffective in inducing antimicrobial skin defense systems.

[0009] Therefore, the search for active substances that are effective in inducing antimicrobial skin defense systems is difficult but crucial. Summary of the invention

[0010] Surprisingly and unexpectedly, schizophyllan has the effect of promoting the production of human β-defensin-2 (hBD-2), thereby effectively stimulating the antimicrobial defense system on the human surface to restore the natural homeostasis.

[0011] Specifically, this application involves the following aspects:

[0012] 1. Use of schizophyllan as a human β-defensin-2 promoter.

[0013] 2. Use of schizophyllan or a topical composition comprising schizophyllan for application to a human body surface to produce human beta-defensin-2 on said surface.

[0014] 3. The use according to item 1 or 2, wherein the schizophyllan is used to improve the natural defense of the human body surface.

[0015] 4. The use according to item 1 or 2, wherein the schizophyllan is used to improve the balance of the microbiome on the surface of the human body.

[0016] 5. Use of schizophyllan in the preparation of a product for improving the natural defenses of the human body surface or improving the balance of the microbiome on the human body surface.

[0017] 6. The use according to item 5, wherein the schizophyllan promotes the production of human β-defensin-2 on the surface of the human body.

[0018] 7. The use according to any one of items 4 to 6, wherein the product is a topical product.

[0019] 8. The use according to item 7, wherein the product is a cosmetic or a medicine.

[0020] Effects of the Invention

[0021] The present application discovers for the first time that schizophyllan has the effect of inducing the expression of β-defensin-2 (hBD-2). Therefore, it can effectively stimulate the antimicrobial defense system on the human body surface to restore the stability of the natural internal environment, thereby being used to improve the natural defense on the human body surface or improve the balance of the microbiome on the human body surface. This has a positive effect on preventing and / or reducing skin infections, skin sensitivity, damaged skin barrier, skin microecological disorders, preventing / improving acne skin, etc., and broadens the application scope of this substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the relative fluorescence quantitative result of HBD-2 expression in keratinocytes. DETAILED DESCRIPTION

[0023] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0024] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those generally understood by those skilled in the art. Although similar or identical methods and materials as described herein may be used in experiments or practical applications, materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, materials, methods and examples are provided for illustration only and are not restrictive. The present application is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present application.

[0025] In the present application, Schizophyllan (SPG) has a main chain of β-1,3-pyranose, and one of every three glycosyl groups is connected to a β-1,6-pyranose. SPG dissolves in water and becomes a trimer composed of three sugar chains of similar length forming a rod-shaped rigid triple helical structure.

[0026] Schizophyllan can be purchased commercially or prepared by methods known in the art.

[0027] In some specific embodiments, the weight average molecular weight (Mw) of schizophyllan can be greater than 10 kDa, preferably greater than 110 kDa, preferably 110 kDa-2000 kDa.

[0028] The present application found that schizophyllan can promote the expression of human β-defensin-2. Therefore, in the present application, schizophyllan is used as a human β-defensin-2 promoter.

[0029] In the present application, the invention relates to the use of schizophyllan in the preparation of a human β-defensin-2 promoter.

[0030] In the present application, the use of schizophyllan or a topical composition comprising schizophyllan is applied to the surface of the human body to produce human β-defensin-2 on the surface.

[0031] According to the above uses, schizophyllan is used to improve the natural defense of the human body surface.

[0032] According to the above uses, schizophyllan is used to improve the balance of the microbiome on the surface of the human body.

[0033] Preferably, the human body surface includes at least one of the skin, the scalp including the hair, the armpits and the oral cavity.

[0034] The use of the above aspects may be therapeutic or non-therapeutic. Preferably, the use is non-therapeutic, including cosmetic aspects.

[0035] The human body surface is a barrier to prevent the invasion and reproduction of pathogens. For example, the skin's antimicrobial defense mechanism includes the integrity of the stratum corneum, the production of dry sebum and lysozyme, acidity, and defensins. Improving the natural defense of the human body surface or the balance of the microbiome on the human body surface has a positive effect on preventing and / or reducing skin infections, skin sensitivity, damaged skin barriers, skin microecological disorders, preventing / improving acne, etc.

[0036] Skin infection is an immune process caused by pathogenic microorganisms invading the human body and interacting with the host immune system. Depending on the pathogenic microorganisms, skin infections are divided into viral infections, bacterial infections, and fungal infections.

[0037] Skin diseases may be infectious complications of dermatology and may include viral skin diseases, bacterial skin diseases, and fungal skin diseases.

[0038] Fungal infection of the skin includes superficial fungal diseases and deep fungal diseases. Superficial fungal diseases include fungal diseases of the surface stratum corneum such as tinea versicolor, tinea capitis, tinea corporis, tinea cruris, tinea pedis and onychomycosis caused by dermatophytes, and fungal diseases of the subcutaneous tissue such as sporotrichosis and pigmented fungal diseases. Deep fungal diseases include candidiasis, histoplasmosis, Penicillium marnefelis, cryptococcosis, etc.

[0039] Bacterial infectious skin diseases include coccal infections, bacillary infections, spirochete infections, mycoplasma infections and chlamydia infections.

[0040] Staphylococcus aureus and Streptococcus pyogenes are the main pathogens of the skin, and can cause coccal infectious skin diseases. Infectious skin diseases caused by Staphylococcus aureus and Streptococcus pyogenes are divided into epidermal infections, dermal infections and hair follicle infections. Epidermal infections include impetigo, deep impetigo, and staphylococcal scalded skin syndrome (SSSS). Dermal infections include erysipelas, cellulitis, and necrotizing fasciitis. Hair follicle infections include folliculitis, furunculosis, and carbuncle. Other infections include staphylococcal toxic shock syndrome, staphylococcal scalded skin syndrome (SSSS), suppurative hidradenitis, acute paronychia, and vesicular distal dactylitis.

[0041] Bacillus infectious skin diseases. The pathogenic bacteria are mainly Mycobacterium tuberculosis, Mycobacterium leprae and atypical mycobacteria, which can infect the skin and cause skin tuberculosis, leprosy and swimming pool granuloma.

[0042] Spirochete infectious skin diseases are mainly chancre symptoms caused by syphilis infection in the human body.

[0043] Various microorganisms on the skin surface, such as bacteria, fungi, and viruses, together with tissues, cells, various secretions, and microenvironments on the skin surface, constitute the skin microecology. Microorganisms on the skin surface can usually be divided into resident bacteria and transient bacteria. Resident bacteria can directly or indirectly protect the body from the invasion of pathogenic bacteria, thereby regulating the balance of the skin microecology.

[0044] In the present application, it is not intended to apply schizophyllan in pathological conditions, but to make the skin reach a better state or assist the skin to reach a better state from the perspective of beauty and skin care.

[0045] On the other hand, the present application also provides the use of schizophyllan in preparing a product for improving the natural defense of the human body surface or improving the balance of the microbiome on the human body surface.

[0046] Wherein, the schizophyllan promotes the production of human β-defensin-2 on the surface of the human body.

[0047] In some embodiments, the product is a topical product, i.e., a product that can be applied to the surface of the human body. Preferably, the surface of the human body includes at least one of the skin, the scalp including the hair, the armpits, and the oral cavity. Preferably, the topical product can be, for example, a cosmetic, a medicine, etc., preferably a cosmetic.

[0048] Those skilled in the art will appreciate that, depending on the type of product, the product may also include various types of physiologically acceptable media. Physiologically acceptable media may, for example, be solvents, diluents, excipients, buffers, fillers, binders, disintegrants, preservatives, lubricants, odorants, flavoring agents, sweeteners, etc.

[0049] In some specific embodiments, the product is a cosmetic, for example, a face cream, a face mask, an eye cream, a neck cream, a hand cream, a body lotion, a moisturizer, an essence, a toner, a shampoo, a conditioner, a scalp care solution, a shower gel, a facial cleanser, a mouthwash or a toothpaste.

[0050] Example

[0051] The present application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage. The reagents or instruments used without indicating the manufacturer are all conventional reagent products that can be obtained commercially.

[0052] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0053] Among them, schizophyllan 1 was purchased from Bioyouth TM -SPG schizophyllan gel, LOT: 20240501, Mw: 1.647×10 6 (±10.678%)Da), schizophyllan 2 was purchased from Bioyouth TM -SPG schizophyllan gel, LOT: 20240710, Mw: 1.461×10 6 (±9.763%)Da); DMEM medium was purchased from Gibco; Staphylococcus aureus ATCC29213 was purchased from American Type Culture Collection (ATCC).

[0054] Example 1 Antimicrobial peptide hBD-2 expression experiment

[0055] Different experimental sample solutions (different experimental samples are shown in Table 1) were prepared with schizophyllan in DMEM culture medium to detect its effect on the expression of antimicrobial peptide hBD-2 in primary keratinocytes.

[0056] The specific method includes: primary keratinocytes (human epidermal keratinocytes (HEKn) were isolated from neonatal foreskin by the laboratory according to the conventional method and frozen at the end of primary culture) were co-cultured with different experimental sample solutions for 1 day, the experimental sample solution was removed, the cells were fixed with methanol, and then blocked with blocking solution for 1 hour. After washing with PBS, hBD-2 primary antibody was added, incubated at 4°C overnight, diluted secondary antibody was added for staining for 1.5 hours, and then anti-fluorescence quenching agent was used to seal the slides, and photos were taken under a fluorescence microscope. ImageJ fluorescence semi-quantitative analysis was used to calculate the relative fluorescence intensity. The results of the experimental groups are shown in the figure. Figure 1 As shown in Table 1. On the basis of meeting the requirements of significant difference, the stronger the relative fluorescence intensity, the better the cell's own defense effect.

[0057] Table 1 Expression results of antimicrobial peptide HBD-2

[0058] Group Processing Relative fluorescence intensity% P-value Significance Control group DMEM medium 100 - - Experimental Group 1 0.001%(w / v)Schizophyllanthrin 1 123 0.0047 ** Experimental Group 2 0.005% (w / v) schizophyllanthrin 1 129 <0.0001 *** Experimental Group 3 0.01%(w / v)Schizophyllanthrin 1 130 <0.0001 *** Experimental Group 4 0.05% (w / v) schizophyllanthus 1 149 <0.0001 *** Experimental Group 5 0.01%(w / v)Schizophyllanthrin 2 134 <0.0001 ***

[0059] From Table 1 and Figure 1 It can be seen that treatment of keratinocytes with schizophyllan significantly promoted the expression of antimicrobial peptide hBD-2.

[0060] Example 2 Antibacterial efficacy experiment

[0061] 1. Sample preparation:

[0062] 1.1 Test group sample solution

[0063] The sample solution was obtained by diluting schizophyllan 1 with DMEM medium (Gibco#100569010) to a concentration of 0.05% (w / v), which was used as test group A.

[0064] Keratinocytes (epidermal keratinocytes (HEKn) were isolated from neonatal foreskin by conventional methods in the laboratory and cryopreserved at the end of primary culture) were co-cultured with DMEM culture medium or sample solution at 37°C, 5% CO2 for 1 day, and then the solution was removed, PBS was added for washing, and RPMI lysis solution (Solarbio) was added to lyse the cells, and the cell supernatant was collected by centrifugation as experimental group B and experimental group C.

[0065] 1.2 Control group sample solution

[0066] Sterile PBS was used as the control group.

[0067] 2. Preparation of bacterial suspension:

[0068] Staphylococcus aureus ATCC29213 was cultured in nutrient broth at 37°C with shaking for 48 h, and Pseudomonas aeruginosa ATCC9027 was cultured in nutrient broth at 37°C with shaking for 18 h, and diluted with sterile PBS to prepare 1×10 5 -9×10 5 CFU / mL of bacterial suspension.

[0069] 3. Detection of antibacterial effect on Staphylococcus aureus:

[0070] The control group, experimental group A, experimental group B, and experimental group C were mixed with Staphylococcus aureus suspension at a ratio of 1000μL:100μL (total volume 3mL) to obtain a mixed solution. The mixed solutions of each experimental group and the control group were placed in a 37℃ incubator for incubation for 3h. 500μL of the mixed solution was taken respectively and diluted with sterilized PBS to 1 / 10 and 1 / 100 sample solutions. 100μL of the sample solution was taken respectively, and the plate was cultured at 32.5℃ for 41h. TSA agar (trypticase soy agar) was used for plate counting. Three plates were prepared for each sample solution, and the average value of the three parallel plate counts was calculated to calculate the antibacterial rate. The specific antibacterial results are shown in Table 2.

[0071] Bacterial inhibition rate (%) = (average value of control group - average value of test group) ÷ average value of control group × 100%.

[0072] With reference to the "Antibacterial and Antibacterial Effect Evaluation Method" standard number: WS / T 650-2019 issued by the National Health Commission, if the inhibition rate is ≥50%-90%, it is judged to have antibacterial effect; if the inhibition rate is ≥90%, it is judged to have a strong antibacterial effect; if it is less than 50%, it is considered to have no antibacterial effect.

[0073] Table 2 Antibacterial results of keratinocyte lysate treatment group

[0074] Group Processing Staphylococcus aureus inhibition rate Control group Sterile PBS 10.05％ Experimental Group A 0.05% schizophylline 16.73％ Test Group B DMEM cell lysis buffer 30.61％ Test Group C Cell lysate after treatment with 0.05% schizophyllan 79.59％

[0075] As can be seen from Table 2, the antibacterial rate of the control group was 10.05%, the antibacterial rate of the experimental group B was 30.61%, and the antibacterial rate of the experimental group A was 16.73%, indicating that schizophyllin itself has no antibacterial effect. The antibacterial rate of the experimental group C was 79.59%, indicating that the cell lysate treated with schizophyllin can inhibit the growth of Staphylococcus aureus, promote endogenous antibacterial activity of cells, and thus enhance the natural defense ability of the cells themselves.

Claims

1. Use of schizophyllan as a human β-defensin-2 promoter.

2. Use of schizophyllan or a topical composition comprising schizophyllan for application to a human body surface to produce human beta-defensin-2 on said surface.

3. The use according to claim 1 or 2, wherein the schizophyllan is used to improve the natural defense of the human body surface.

4. The use according to claim 1 or 2, wherein the schizophyllan is used to improve the balance of the microbiome on the surface of the human body.

5. Use of schizophyllan in the preparation of a product for improving the natural defenses of the human body surface or improving the balance of the microbiome on the human body surface.

6. The use according to claim 5, wherein the schizophyllan promotes the production of human β-defensin-2 on the surface of the human body.

7. The use according to any one of claims 4 to 6, wherein the product is a topical product.

8. The use according to claim 7, wherein the product is a cosmetic or a medicine.

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