KR102497294B1 - Skin External Composition For Preventing and Improving Skin Disease Containing Novel Peptide as Active Ingredient - Google Patents

Abstract

The present invention relates to an external skin preparation composition containing a novel peptide as an active ingredient, and more specifically, to an external skin preparation composition containing mini hBD-2, which is a novel peptide derived from beta-defensin-2, to have effects of preventing and alleviating skin diseases by reducing skin harmful bacteria and enhancing skin immunity.

Description

Skin External Composition For Preventing and Improving Skin Disease Containing Novel Peptide as Active Ingredient

The present invention relates to a composition for external application for skin containing a novel peptide as an active ingredient, and specifically, includes a novel peptide mini hBD-2 derived from beta-defensin-2 to reduce skin harmful bacteria and improve skin disease by improving skin immunity. It relates to a composition for external application for skin having an effect of preventing and improving.

The skin is the basis of the innate immunity that protects our body from external infections. The skin is a representative organ that exhibits an immune response as well as a primary protective film against the outside, and plays a role in protecting against invasion by various harmful factors by regulating immune responses and producing endogenous antimicrobial peptides as various cells. Antimicrobial peptides produced by the skin include beta-defensin and LL-37.

In addition, the skin is a complex ecosystem in which various microorganisms reside, and the correlation between the microbial population residing on the skin, the skin immune system, and the skin health condition is attracting attention.

Beneficial bacteria and harmful bacteria coexist in the microorganisms residing on the skin, and the distribution of beneficial bacteria is greater in healthy skin, and the distribution of harmful bacteria is greater in the case of atopic or acne-prone skin. In general, skin harmful bacteria include Pseudomonas aeruginosa , Staphylococcus aureus ( Staphylococcus aureus ), Candida albicans and the like, and these strains are known as causative bacteria that cause various skin troubles and inflammations. Therefore, the reduction of skin harmful bacteria can exhibit the effect of preventing skin diseases such as skin trouble and inflammation.

Korean Patent Registration No. 10-1811050 discloses a fusion polypeptide in which an anti-inflammatory polypeptide and a ferritin monomer fragment are conjugated, and a pharmaceutical composition for preventing and treating inflammatory diseases containing the same as an active ingredient.

In addition, Korean Patent Registration No. 10-1249702 discloses an antibacterial and anti-inflammatory composition comprising a peptide consisting of an amino acid sequence derived from human beta-defensin-2.

Therefore, the present inventors confirmed that the novel peptide derived from the nucleotide sequence of beta-defensin is more economical than beta-defensin, an existing antibacterial peptide, and has excellent effects in preventing and improving skin diseases by reducing skin harmful bacteria and enhancing skin immunity. invention was completed.

An object of the present invention is to provide a composition for external application for skin containing, as an active ingredient, a novel peptide that is more economical than beta-defensin, which is an existing antibacterial peptide, and has excellent effects in preventing and improving skin diseases by reducing skin harmful bacteria and enhancing skin immunity.

In order to achieve the above object, the present invention provides a composition for external application for skin for preventing and improving skin diseases, comprising, as an active ingredient, a peptide derived from beta-defensin-2 and having the amino acid sequence of SEQ ID NO: 1.

Preferably, the peptide may be included in an amount of 0.005 to 5.0% by weight based on the total weight of the composition for external application for skin.

Also preferably, the peptide has an effect of preventing and improving skin diseases by reducing skin harmful bacteria.

Also preferably, the peptide has an effect of preventing and improving skin diseases by promoting skin immunity through cell migration and immune cell regulation.

Also preferably, the composition for external application for skin is one selected from the group consisting of lotion, essence, lotion, cream, pack, gel, ointment, patch, foundation, makeup base, sunscreen, bath agent, soap, liquid cleanser and spray It can be characterized by the above formulation.

The composition for external application for skin containing the novel peptide according to the present invention as an active ingredient exhibits effects of preventing and improving skin diseases by reducing skin harmful bacteria and enhancing skin immunity.

Figure 1 shows the mechanism of inducing cell migration (chemotaxis) and regulating immune cells of human-derived beta-defensins.
Figure 2 shows the results of evaluating the cytotoxicity of HaCaT and HDFn cells of Example 1 of the present invention.
Figure 3 shows the results of evaluating the chemokine expression of Example 1 of the present invention.
Figure 4 shows the results of evaluating the MAPK expression of Example 1 of the present invention.
Figure 5 shows the results of evaluating the cell proliferation rate of Example 1 of the present invention.

All technical terms used in the present invention, unless otherwise defined, have the following definitions and correspond to the meanings commonly understood by those of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention.

The term "about" means a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length of 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, means an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight or length that varies by 4, 3, 2 or 1%.

Throughout this specification, unless the context requires otherwise, the terms "comprise" and "comprising" include a given step or component, or group of steps or components, but any other step or component, or It is to be understood that steps or groups of components are not excluded.

The present invention relates to a composition for external application for skin containing mini hBD-2, a novel peptide composed of the amino acid sequence represented by SEQ ID NO: 1.

[SEQ ID NO: 1]: PVFCPRRYKQIGTCGLPGTKCCKKP

The novel peptide mini hBD-2 of the present invention is a similar peptide to beta-defensin-2, an existing antibacterial peptide. The novel peptide mini hBD-2 of the present invention contains a part of the sequence of beta-defensin-2, which exhibits superior antibacterial activity compared to the existing beta-defensin-2 and has economic characteristics.

On the other hand, beta-defensin is one of the representative antibacterial peptides, and as a mechanism of action, beta-defensin is known to directly act on viruses, germs and bacteria by destroying cell membranes, or to remove pathogens and regulate inflammation by regulating immune cells. The immune control mechanism of beta-defensins is to activate MAPKinase (ERK, p38, JNK) and NF-kB through Toll-like receptor (TLR), C-C chemokine receptor type 6 (CCR6), and G protein-coupled receptor (GPCR) to Induce expression of defensins. Produced beta-defensins bind to CCRs and GPCRs, activate MAPKinase, induce chemotaxis by expressing cytokines and chemokines such as CCL20, CCL22, and CXCL9, and regulate immune cell activity. It causes an adptive immune response. The mechanism of inducing cell migration (chemotaxis) and regulating immune cells of the beta-defensin described above is shown in FIG. 1 below.

In the present invention, the novel peptide mini hBD-2 may contain 0.005% to 5.0% by weight based on the total weight of the composition. When the content of the peptide is less than 0.005% by weight, the effect is insignificant, and when it exceeds 5.0% by weight, efficiency is low compared to the amount of material input, which is not economical.

In the present invention, the novel peptide mini hBD-2 can be characterized in that it has an effect of preventing and improving skin diseases by reducing skin harmful bacteria.

Preferably, the skin harmful bacteria may be Pseudomonas aeruginosa , Staphylococcus aureus , and Candida albicans, but are not limited thereto.

In the present invention, the novel peptide mini hBD-2 can be characterized in that it has an effect of preventing and improving skin diseases by enhancing skin immunity by increasing the expression of beta-defensin.

In the present invention, the composition for external application for skin may be prepared in various forms. Preferably, lotion, essence, gel, emulsion, lotion, cream (oil-in-water (O/W) type, water-in-oil (W/O) type, multi-phase), solution, suspension, anhydrous product, gel, mask, pack , Powder, capsules with coatings such as gelatin, ointments, patches, foundations, makeup bases, sunscreens, bath additives, soaps, liquid cleansers, spray formulations, and the like.

In the present invention, the skin is a concept that includes not only the face, but also the scalp and the whole body. As a skin external preparation composition that can be applied to the scalp, there are shampoo, rinse, treatment, hair growth agent, etc., body cleanser that can be applied to the whole body, etc. It can be manufactured in various forms for use.

In the present invention, the composition for external application for skin may be a cosmetic composition or a pharmaceutical composition.

Specifically, the cosmetic composition may contain adjuvants commonly used in the cosmetic field, such as hydrophilic or lipophilic activators, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, odor absorbers, dyes, conditioning agents, and the like. . The amount of these various adjuvants is an amount commonly used in the art, but in any case, the adjuvant and its ratio will be selected so as not to adversely affect the desired properties of the cosmetic composition according to the present invention.

Specifically, the pharmaceutical composition is a composition for treatment of skin diseases, wound healing and skin regeneration, and may be a transdermal dosage form such as lotion, ointment, gel, cream, patch or spray, but is not limited thereto.

Hereinafter, the contents of the present invention will be described in more detail in the following examples or experimental examples, but the scope of the present invention is not limited only to the following examples or experimental examples, and includes modifications of equivalent technical ideas.

Example 1: Synthesis of novel peptide mini hBD-2

A novel peptide mini hBD-2 consisting of the amino acid sequence represented by SEQ ID NO: 1 was synthesized by requesting Peptron Co., Ltd.

[SEQ ID NO: 1]: PVFCPRRYKQIGTCGLPGTKCCKKP

Comparative Example 1: Human-derived beta-defensin-2

Beta-defensin-2 standard, which is the parent of the novel peptide mini hBD-2, was purchased from Peprotech.

Experimental Example 1: Measurement of antibacterial activity

In this experimental example, the antibacterial activity of Example 1 and Comparative Example 1 was measured through the ultra-sensitive radial diffusion assay (URDA) method. Antibacterial activity was tested against skin harmful bacteria Pseudomonas aeruginosa , Staphylococcus aureus and fungus Candida albicans.

After culturing the strain in tryptic soy broth (TSB) (bacterial medium) or potato dextrose broth (PDB) (fungal medium) at an appropriate temperature (25 ° C or 37 ° C) for 16 hours, the bacterial concentration was 1 × 10 ⁸ It was adjusted to be CFU/ml (bacterial medium) or 1×10 ⁶ CFU/ml (fungal medium). After that, 0.5 ml of the bacterial solution diluted in each concentration was added to 9.5 ml of an underlay gel containing 0.03% TSB or 0.03% PDB, 1% Type I agarose and 10 mM phosphate buffer (pH 6.5), mixed, and then spread flat on a plate. It was poured and hardened. After punching wells in the hardened plate using a punch, 5 μl of sample was injected. After incubating the plate at an appropriate temperature for 3 hours, pour an overlay gel containing 10 ml of 6% TSB or 6% PDB, 1% Type I agarose, and 10 mM phosphate buffer (pH 6.5) on the underlay gel for 16 hours. Incubated at appropriate temperature. Antibacterial activity was confirmed by measuring the size of the clear zone. Each sample was tested at a concentration of 0.01%, and the results are shown in Table 1 below.

strain Clear zone (mm) Example 1 Comparative Example 1 P. aeruginosa 12 7 S. aureus 11 6 C. albicans 6 -

As shown in Table 1, the novel peptide mini hBD-2 of Example 1 exhibited antibacterial activity against both bacteria and fungi among harmful skin bacteria, and exhibited excellent antibacterial activity compared to Comparative Example 1.

Experimental Example 2: Evaluation of cell migration (chemotaxis) induction and immune cell regulation

In this experimental example, the mechanism of cell migration (chemotaxis) induction and immune cell regulation of Example 1 was evaluated. The mechanism was confirmed through cytotoxicity evaluation, chemokine expression evaluation, mitogen-activated protein kinase (MAPK) expression evaluation, and cell proliferation evaluation.

<Evaluation of cytotoxicity>

Human keratinocytes (HaCaT) and human fibroblasts (HDFn) were dispensed into a 96 well plate at a concentration of 2 × 10 ⁴ cells/well, and then cultured in a 37° C. incubator for one day. Samples were treated by concentration and reacted in a 37° C. incubator for 24 hours. Each well was treated with 20 ul of MTT reagent at a concentration of 5 mg/ml and reacted for 2 hours. After the reaction was complete, the supernatant was removed, and cells were completely dissolved by treatment with 100 ul of DMSO, and cytotoxicity was confirmed by measuring absorbance at 550 nm. In addition, the significance of the test substance was confirmed according to the biological statistical standard (significance level (α): 1%) through a two sample t-test.

The results are shown in FIG. 2 below, and it was confirmed that the novel peptide mini hBD-2 of Example 1 did not show cytotoxicity to both HaCaT and HDFn.

<Evaluation of chemokine expression>

Human keratinocytes (HaCaT) were seeded in a 6 well plate at a concentration of 4 × 10 ⁵ cells/well and stabilized for one day. Samples were treated by concentration and reacted in a 37° C. incubator for 6 hours. Cells were collected using a scraper and washed once with 1 x PBS solution. RNA was isolated using the Trizol method. Cells were lysed by adding 800 ul of Trizol, reacted at room temperature for 5 minutes, voltexed by adding 200 ul of chloroform, and then reacted at room temperature for 5 minutes. Centrifuged at 12,000 rpm, 4 ^o C for 15 minutes, transferred only the supernatant to a new e-tube, added the same amount of isopropanol, mixed 2-3 times, and reacted at room temperature for 10 minutes. After centrifugation at 12,000 rpm, 4 ^o C for 15 minutes, supernatant was removed, 800 ul of cold 70% ethanol was added to wash, and centrifugation was performed again at 12,000 rpm, 4 ^o C for 15 minutes. After completely removing the supernatant and drying the pellet until it became transparent, DEPC DW was added according to the amount of RNA. RNA was quantified using Nanodrop, PCR was performed, and bands were detected by electrophoresis on a 2% agarose gel. The band was quantified using Image J 1.47 software, and the expression increase rate (%) was expressed as a percentage compared to the control. In addition, the significance of the test substance was confirmed according to the biological statistical standard (significance level (α): 1%) through a two sample t-test.

The results are shown in FIG. 3, and it was confirmed that the novel peptide mini hBD-2 of Example 1 increased the expression of CCL20, CCL22, and CXCL5 in a concentration-dependent manner.

<MAPK expression evaluation>

Human keratinocytes (HaCaT) were seeded in a 6 well plate at a concentration of 4 × 10 ⁵ cells/well and stabilized for one day. Samples were treated by concentration and reacted in a 37° C. incubator for 30 minutes. Cells were collected using a scraper, washed once with 1 x PBS solution, and then cells were lysed using 1 x cell lysis buffer. Separated proteins were obtained by centrifugation at 12,000 rpm, 15 min, 4 ^° C. After quantifying the protein using the BCA quantification method, SDS-PAGE was performed using 20 μ of the protein. After electrophoresis, the gel was transferred to a polyvinylidene fluoride (PVDF) membrane and blocked for 1 hour at room temperature using 5% skim milk. After washing 3-5 times with TTBS buffer, the primary antibody was reacted overnight at 4 ^° C. After the primary antibody reaction, the cells were washed 3-5 times with TTBS buffer and then reacted with the secondary antibody for 2 hours at room temperature using 5% skim milk. After washing 3-5 times with TTBS buffer, the membrane was reacted with an ECL (enhanced chemiluminescence) solution and a luminescent band was identified using a Chemi Doc machine. The band was quantified using Image J 1.47 software, and the expression increase rate (%) was expressed as a percentage compared to the control. In addition, the significance of the test substance was confirmed according to the biological statistical standard (significance level (α): 1%) through a two sample t-test.

The results are shown in FIG. 4, and it was confirmed that the novel peptide mini hBD-2 of Example 1 expresses MAPKs (p-ERK, p-JNK, p-p38).

<Evaluation of cell proliferation>

Human fibroblasts (HDFn) were dispensed into a 96 well plate at a concentration of 3 × 10 ⁴ cells/ml and then cultured in a 37°C incubator for one day. Samples were treated by concentration and reacted in a 37° C. incubator for 48 hours. Each well was treated with 20 ul of MTT reagent at a concentration of 5 mg/ml and reacted for 2 hours. After the reaction was complete, the supernatant was removed, and cells were completely dissolved by treatment with 100 ul of DMSO, and cell proliferation was confirmed by measuring absorbance at 550 nm. In addition, the significance of the test substance was confirmed according to the biological statistical standard (significance level (α): 1%) through a two sample t-test.

The results are shown in FIG. 5, and the novel peptide mini hBD-2 of Example 1 exhibited a concentration-dependent cell proliferation increasing activity.

Preparation Example 1: Preparation of ointment containing novel peptide mini hBD-2

A cosmetic composition in an ointment formulation containing 1.0% by weight of the novel peptide mini hBD-2 of Example 1 was prepared with the composition shown in Table 2 below, and was referred to as Preparation Example 1, and Comparative Preparation Example 1 was prepared as a control.

Ingredients (Unit:% by weight) Preparation Example 1 Comparative Preparation Example 1 Example 1 1.0 - Cetostearyl Alcohol 2.0 2.0 Self-emulsifying monostearic acid 2.0 2.0 stearic acid 1.0 1.0 wax 4.0 4.0 squalane 7.0 7.0 Glycerin Monostearate 3.0 3.0 Sorbitan Monostearate 1.0 1.0 Polysorbate 80 3.0 3.0 glycerin 5.0 5.0 propylene glycol 4.0 4.0 Spices Appropriate amount Appropriate amount vaseline Appropriate amount Appropriate amount Purified water to 100 to 100

Experimental Example 3: Acne alleviation effect evaluation

In this experiment, the acne alleviating effect was measured with respect to Preparation Example 1.

Specifically, 20 men and women between the ages of 20 and 45 were randomly divided into 2 groups (A and B) of 10 people each, and then the experiment was conducted with 10 people in the experimental group (A) and 10 people in the control group (B). The skin conditions of the test subjects who participated in the experiment were tested only with oily skin subjects with acne and a lot of sebum. The composition of Preparation Example 1 was applied to group A and the composition of Comparative Preparation Example 1 to group B was applied to the face for 4 weeks after cleansing in the morning and evening. The degree of acne relief was evaluated by the subjects after the experiment was completed (-3: very worse, -2: worse, -1: slightly worse, 0: no change, 1: slightly improved, 2: improved, 3: very improved) According to the sensory evaluation, it is shown in Table 3 below.

subject Degree of Acne Relief Group A group B One 2 -One 2 3 One 3 3 0 4 2 0 5 One -One 6 2 -3 7 2 -One 8 3 -2 9 One 0 10 3 0 average 2.2 -0.7

As a result, the composition of Preparation Example 1 was found to have an excellent acne alleviating effect by including the novel peptide mini hBD-2 as an active ingredient.

Preparation Examples 2 to 4: Preparation of composition for external application for skin containing novel peptide mini hBD-2

A composition for external application for skin (cosmetic composition, pharmaceutical composition) containing 1.0% by weight of the novel peptide mini hBD-2 of Example 1 was prepared. However, the present invention can be applied to various formulations, and this preparation example is only intended to specifically describe the present invention, not to limit it.

<Production Example 2: Skin Toner>

ingredient content
(unit: % by weight) Example 1 1.0 glycerin 5.0 1,3-butylene glycol 8.0 squalane 10.0 Polyoxyethylene sorbitan monooleate 2.0 triethanolamine 1.5 Glyceryl Stearate 0.5 Stearyl Glycyrrhetinate 0.2 carboxyvinyl polymer 0.1 arginine 0.1 Spices Appropriate amount antiseptic Appropriate amount Purified water to 100

<Preparation Example 3: Lotion (emulsion)>

ingredient content
(unit: % by weight) Example 1 1.0 glycerin 5.0 1,3-butylene glycol 3.0 ethanol 5.0 Polyoxyethylene nonylphenyl ether 0.5 Spices Appropriate amount antiseptic Appropriate amount Purified water to 100

<Preparation Example 4: Gel>

ingredient content
(unit: % by weight) Example 1 1.0 glycerin 4.0 propylene glycol 4.0 ethanol 10 Polyoxyethylene Hydrogenated Castor Oil 0.1 carboxypolymer 0.3 triethanolamine 0.3 Spices Appropriate amount antiseptic Appropriate amount Purified water to 100

Claims (5)

A composition for external application for skin for preventing and improving skin diseases, comprising as an active ingredient a peptide derived from beta-defensin-2 and consisting of the amino acid sequence of SEQ ID NO: 1. According to claim 1,
The peptide is a composition for external application for skin, characterized in that contained in 0.005 to 5.0% by weight relative to the total weight of the composition for external application for skin. According to claim 1,
The peptide composition for external application for skin, characterized in that it has an effect of preventing and improving skin diseases by reducing skin harmful bacteria. According to claim 1,
The peptide composition for external application for skin, characterized in that it has an effect of preventing and improving skin diseases by enhancing skin immunity through cell migration and immune cell regulation. According to claim 1,
The composition for external application for skin is one or more formulations selected from the group consisting of lotion, essence, lotion, cream, pack, gel, ointment, patch, foundation, makeup base, sunscreen, bath agent, soap, liquid cleanser, and spray. A composition for external application for skin.

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