Application of Ectoine or derivative thereof as human beta-defensin-2 expression promoter

Abstract

The invention provides an application of Ectoine or a derivative thereof as a human beta-defensin-2 expression promoter. According to the application, it is found for the first time that Ectoine or the derivative thereof has the effect of inducing cell expression of beta-defensin 2 (hBD-2), so that Ectoine or the derivative thereof is used for improving the defensive mechanism of skin antimicrobial.

Description

Technical Field

[0001] The present application belongs to the technical field of skin care, and in particular to the use of ectoine or its derivatives as human β-defensin-2 expression promoters. 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] Therefore, it is crucial to find active substances that are effective in inducing antimicrobial skin defense systems. Summary of the invention

[0008] Through experiments, the present application unexpectedly discovered that ectoine or its derivatives, which have beneficial effects on the skin and do not have antibacterial effects themselves, can promote skin cells to express human β-defensin-2 without causing infection, damage, inflammation or stress in skin cells, thereby enhancing the natural defense capabilities of the human body surface and maintaining and / or improving the balance of the microbiome on the human body surface.

[0009] Specifically, this application involves the following aspects:

[0010] 1. Use of ectoine or its derivatives as human β-defensin-2 expression promoter.

[0011] 2. Use of ectoine or its derivatives for application on the surface of human body to produce human β-defensin-2 on said surface.

[0012] 3. According to the use described in item 1 or 2, the ectoine or its derivative is used to maintain and / or improve the natural defense of the human body surface.

[0013] 4. According to the use of item 1 or 2, the ectoine or its derivative is used to maintain and / or improve the balance of the microbiome on the surface of the human body.

[0014] 5. Use of ectoine or its derivatives in the preparation of products for maintaining and / or improving the natural defense of the human body surface or maintaining and / or improving the balance of the microbiome on the human body surface.

[0015] 6. According to the use described in item 5, the ectoine or its derivative promotes the production of human β-defensin-2 on the surface of the human body.

[0016] 7. The use according to any one of items 4 to 6, wherein the product is a topical product.

[0017] 8. The use according to item 7, wherein the product is a cosmetic or a medicine, preferably a cosmetic.

[0018] Effects of the Invention

[0019] The present application discovers for the first time that ectoine or its derivatives have the effect of inducing the expression of β-defensin-2 (hBD-2), and therefore 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 maintain and / or improve the natural defense on the human body surface or maintain and / or improve the balance of the microbiome on the human body surface, which has beneficial effects on alleviating skin sensitivity, preventing / improving acne, etc., and broadens the application scope of the substance. DETAILED DESCRIPTION

[0020] 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.

[0021] 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.

[0022] The present application found that ectoine or its derivatives can promote the expression of human β-defensin 2 (hBD-2). Therefore, the present application provides the use of ectoine or its derivatives as a promoter for the expression of human β-defensin-2.

[0023] The present application also provides the use of ectoine or its derivatives in preparing a human β-defensin-2 expression promoter.

[0024] In some specific embodiments, ectoine or a derivative thereof is used for administration to a human body surface to produce human β-defensin-2 on the surface.

[0025] According to the above uses, ectoine or its derivatives are used to maintain and / or improve the natural defense of the human body surface.

[0026] According to the above uses, ectoine or its derivatives are used to maintain and / or improve the balance of the microbiome on the surface of the human body.

[0027] Preferably, the human body surface includes at least one of the skin, the scalp including the hair, the armpits and the oral cavity.

[0028] The use of the above aspects may be therapeutic or non-therapeutic. Preferably, the use is non-therapeutic, including cosmetic aspects.

[0029] In some specific embodiments, the ectoine derivative includes hydroxyectoine, ectoine sodium salt or ectoine potassium salt.

[0030] The human body surface is a barrier that prevents 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 skin's antimicrobial defense mechanism can prevent and / or reduce skin infections, skin sensitivity, damaged skin barriers, and skin microecological disorders.

[0031] 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.

[0032] Skin diseases may be infectious complications of dermatology and may include viral skin diseases, bacterial skin diseases, and fungal skin diseases.

[0033] 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.

[0034] Bacterial infectious skin diseases include coccal infections, bacillary infections, spirochete infections, mycoplasma infections and chlamydia infections.

[0035] 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.

[0036] Bacillus infectious skin diseases. The pathogenic bacteria are mainly Propionibacterium of mycobacteria (such as Propionibacterium acnes), Mycobacterium tuberculosis, Mycobacterium leprae and atypical mycobacteria, which can infect the skin and cause skin acne, blackheads, pimples, blackheads, inflammatory papules, pustules, enlarged pores, dull skin color, nodules, abscesses, dandruff, tuberculosis, leprosy and swimming pool granuloma.

[0037] Spirochete infectious skin diseases are mainly chancre symptoms caused by syphilis infection in the human body.

[0038] 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.

[0039] In the present application, it is not intended to apply ectoine or its derivatives under 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.

[0040] On the other hand, the present application also provides the use of ectoine or its derivatives in the preparation of products for maintaining and / or improving the natural defense of the human body surface or maintaining and / or improving the balance of the microbiome on the human body surface.

[0041] Wherein, the ectoine or its derivatives promote the production of human β-defensin-2 on the surface of the human body.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] Example

[0046] 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.

[0047] Example 1

[0048] Experimental materials: Ecdoin (Huaxi Biotechnology), DMEM culture medium (Gibco#100569010).

[0049] Experimental sample solution: Ectoin was diluted with DMEM culture medium to prepare experimental sample solutions with different final concentrations, and the concentrations are shown in Table 1.

[0050] Methods: After keratinocytes (human epidermal keratinocytes (HEKn) were isolated from neonatal foreskin by conventional methods in the laboratory 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, MTT was added, and the supernatant was carefully aspirated after 3 hours, dimethyl sulfoxide (DMSO) was added, and the absorbance of each well at 550nm was detected by a microplate reader; the untreated well was used as the control, the absorbance of the sample well was defined as T, the absorbance of the untreated well was defined as NT, NT was taken as 100%, and the ratio of T / NT of the sample well was taken as the cell activity, and the results are shown in Table 1. On the basis of meeting the requirements of significant difference, the larger the cell activity %, the better the cell activity.

[0051] Table 1 Cell activity results

[0052]

[0053]

[0054] As can be seen from the above table, the cell activities after treatment with different concentrations of ectoine were 97% and 95%, which would not cause cell damage and can be used for subsequent experiments.

[0055] Example 2

[0056] Method: After keratinocytes (human 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 the experimental sample solution prepared in Example 1 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, hBD2 primary antibody was added, incubated overnight at 4 degrees, and diluted secondary antibody was added for staining for 1.5 hours. The slides were sealed with anti-fluorescence quencher, and photographed under a fluorescence microscope. ImageJ fluorescence semi-quantitative analysis was used to calculate the relative fluorescence intensity, and the results are shown in Table 2. On the basis of meeting the requirements of significant difference, the stronger the relative fluorescence intensity, the stronger the induction effect of hBD2 expression and the better the cell self-defense effect.

[0057] Table 2 Expression results of cell antimicrobial peptide hBD2

[0058] Group sample Fluorescence intensity % Significance Control group DMEM medium 84 - Experimental Group 1 Ecdoin - 0.1% 120 ** Experimental Group 2 Ecdoin - 0.5% 143 ***

[0059] As can be seen from the above table, the fluorescence intensity of experimental groups 1 and 2 was significantly different from that of the control group, indicating that Ecdoin has the effect of promoting the expression of antimicrobial peptide hBD2.

[0060] Example 3

[0061] Experimental materials: Ecdoin (Huaxi Biotechnology), DMEM culture medium (Gibco#100569010).

[0062] Experimental sample preparation: Ectoin was dissolved and diluted with DMEM culture medium to prepare a sample solution A with a final concentration of 0.5%.

[0063] After the keratinocytes were co-cultured with sample solution A for 1 day, the sample solution was removed, PBS was added for washing, and then RPMI was added to lyse the cells. The supernatant was collected by centrifugation to obtain sample solution B.

[0064] Preparation of Propionibacterium acnes suspension: Propionibacterium acnes ATCC6919 was cultured in a reinforced Clostridium medium (Haibo Bio RCM medium, catalog number: HB0316) at 37°C for 144 h, and diluted with sterile PBS to prepare 1×10 5 ~9×10 5 CFU / mL of bacterial suspension.

[0065] Preparation of Staphylococcus epidermidis suspension: Staphylococcus epidermidis ATCC12228 was cultured in nutrient broth at 37°C with shaking for 48 h, and diluted with sterile PBS to prepare 1×10 5 ~9×10 5 CFU / mL of bacterial suspension.

[0066] Sample solution A and sample solution B were mixed evenly with the Propionibacterium acnes suspension at a ratio of 1000 μL:100 μL, and the mixtures were used as experimental group A and experimental group B, respectively.

[0067] Sample solution A and sample solution B were evenly mixed with the Staphylococcus epidermidis suspension at a ratio of 1000 μL:100 μL, and the samples were used as experimental group C and experimental group D, respectively.

[0068] Sterile PBS was used instead of the sample solution and mixed evenly with the bacterial suspension at a ratio of 1000 μL:100 μL as the control group.

[0069] Experimental group A, experimental group B, experimental group C, experimental group D and control group were placed in a 37℃ incubator for 3h at the same time. 500μL of the mixed solution was taken from each of the experimental group A, experimental group B, experimental group C, experimental group D and control group, and diluted to 1 / 10 and 1 / 100 sample solutions with sterile PBS. 100μL of the 1 / 10 and 1 / 100 sample solutions were taken respectively, and the plates were incubated at 32.5℃ for 41h. TSA agar (trypticase soy agar) was used for plate counting, and 3 plates were prepared for each sample solution.

[0070] Bacterial inhibition rate (%) = (average value of control group - average value of experimental group) ÷ average value of control group × 100%.

[0071] Table 3 Antibacterial results

[0072] Serial number sample Antibacterial rate Control group PBS 10％ Experimental Group A Sample solution A 16％ Experimental Group B Sample solution B 89％ Experimental Group C Sample solution A 18％ Experimental Group D Sample solution B 29％

[0073] 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.

[0074] As can be seen from Table 3 above, the antibacterial rate of experimental group A is 16%, and the antibacterial rate of experimental group B is 89%. This shows that the cell lysate treated with ectoin can inhibit the growth of Propionibacterium acnes, but ectoin itself has no antibacterial effect.

[0075] At the same time, it can be seen from Table 3 above that the inhibition rates of experimental groups C and D are both lower than 50%, which indicates that the cell lysate treated with ectoine and ectoine itself have no inhibitory effect on Staphylococcus epidermidis, and will not inhibit the growth of non-pathogenic bacteria such as Staphylococcus epidermidis.

Claims

1. Use of ectoine or its derivatives as human β-defensin-2 expression promoter.

2. Use of ectoine or its derivatives for application on the surface of human body to produce human β-defensin-2 on said surface.

3. The use according to claim 1 or 2, wherein the ectoine or its derivatives are used to maintain and / or improve the natural defense of the human body surface.

4. The use according to claim 1 or 2, wherein the ectoine or its derivatives are used to maintain and / or improve the balance of the microbiome on the surface of the human body.

5. Use of ectoine or its derivatives in the preparation of products for maintaining and / or improving the natural defense of the human body surface or maintaining and / or improving the balance of the microbiome on the human body surface.

6. The use according to claim 5, wherein the ectoine or its derivative 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, preferably a cosmetic.

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