KR20210081843A - Cosmetic composition for antibacterial comprising the smap-18 peptide as an active ingredient - Google Patents

Abstract

The present invention relates to an antibacterial cosmetic composition comprising the SMAP-18 peptide of SEQ ID NO: 1 as an active ingredient, and cosmetics comprising the same. The active ingredient of the present invention, the SMAP-18 peptide of SEQ ID NO: 1 exhibits high antibacterial activity against gram-positive and gram-negative bacteria as well as fungi, can be used as a cosmetic preservative because of having a much higher antibacterial effect than the cosmetic preservatives commonly used in the market today, and can manufacture cosmetics that do not irritate the skin because there is no harmfulness and toxicity to the skin.

Description

Antibacterial cosmetic composition comprising SMAP-18 peptide as an active ingredient {COSMETIC COMPOSITION FOR ANTIBACTERIAL COMPRISING THE SMAP-18 PEPTIDE AS AN ACTIVE INGREDIENT}

The present invention relates to an antibacterial cosmetic composition comprising the SMAP-18 peptide of SEQ ID NO: 1 as an active ingredient, and cosmetics comprising the same.

Cosmetics contain many organic compounds, such as glycerin or sorbitol, which are carbon sources for microorganisms, amino acid derivatives, and proteins, which are nitrogen sources, including water and oil, so there is a possibility that microorganisms may reproduce and change contents. If contaminated cosmetics are used on the skin, serious problems such as skin irritation, allergic reaction, phototoxicity, and photoallergy may occur. Therefore, preservatives such as parabens are essential for sterilization and preservation of cosmetics, but causes such as cytotoxicity and skin allergy are a problem.

Therefore, many studies are being conducted to develop natural preservatives with antibacterial substances to replace chemical preservatives, which are controversial due to their harmfulness to the human body.

Antimicrobial peptides (AMPs) represent a broad range of short, cationic or amphiphilic gene-encoding peptide antibiotics that can be found in virtually any organism. Different AMPs exhibit different properties, and many peptides of this class are intensively studied as templates for cell-penetrating peptides as well as as antibiotics.

Sheep myeloid antimicrobial peptide-29 (SMAP-29) is a 28 residue a-helical catericidin-derived AMP containing an amidated C-terminus. Another form of SMAP-29 exists that contains 29 residues and an unamidated carboxyl terminus.

SMAP-29 exhibits potent and broad-spectrum antibacterial activity against gram-negative and gram-positive bacteria and fungi. However, SMAP-29 is also highly toxic to both human red blood cells (hRBC) and human embryonic kidney (HEK) cells. The cytotoxicity of SMAP-29 to human normal cells has become a major barrier to developing it as a novel antimicrobial agent.

Republic of Korea Patent Registration No. 10-1985264

An object of the present invention is to provide a new cosmetic composition comprising SMAP-18 peptide exhibiting antibacterial activity as an active ingredient.

In order to solve the above problems, the present invention provides an antibacterial cosmetic composition comprising the SMAP-18 peptide of SEQ ID NO: 1 as an active ingredient;

[SEQ ID NO: 1]

RGLRRLGRKIAHGVKKYG-NH _2.

In a preferred embodiment of the present invention, the SMAP-18 peptide is Escherichia coli ( E. coli ), Salmonella typhimurium ( S. typhimurium ), Pseudomonas aeruginosa ( P. aeruginosa ), Staphylococcus aureus ( S) aureus ), Bacillus subtilis ( B. subilis ), Staphylococcus epidermidis ( S. epidermidis ) and Candida glabrata ( C. glabrata ) Antibacterial activity against one or more strains selected from the group consisting of characterized by indicating.

In a preferred embodiment of the present invention, the SMAP-18 peptide is compared to Staphylococcus aureus ( S. aureus ) and Candida glabrata ( C. glabrata ) E. coli , Salmonella typhimurium ( S typhimurium ), Pseudomonas aeruginosa ( P. aeruginosa ), Bacillus subtilis ( B. subilis ) and Staphylococcus epidermidis ( S. epidermidis ) It is characterized by showing a stronger antibacterial activity.

In a preferred embodiment of the present invention, the SMAP-18 peptide is 16 ~ compared to methyl praben, 1,2-hexanediol, golden extract or phenoxy ethanol. It is characterized by exhibiting 32 times stronger antibacterial activity.

In a preferred embodiment of the present invention, the SMAP-18 peptide is used in a lotion containing 3% (v/v) glycereth-26 and 0.05% (v/v) xanthan gum. E. coli ( Escherichia coli , E. coli ) and Staphylococcus aureus ( Staphylococcus aureus , S. aureus ) It is characterized in that it exhibits antibacterial activity, in which case the preferred concentration of SMAP-18 peptide is 100 ~ 200 μg / mL to be.

In another embodiment of the present invention, there is provided a cosmetic comprising the antibacterial cosmetic composition.

The SMAP-18 peptide of SEQ ID NO: 1, which is the active ingredient of the present invention, exhibits high antibacterial activity against not only gram-positive and gram-negative bacteria but also fungi, and exhibits a much higher antibacterial effect compared to cosmetic preservatives commonly used in the market. It can be used as a preservative, and since there is no harm or toxicity to the skin, it is possible to manufacture cosmetics that do not irritate the skin.

1 is a graph showing the results of an antimicrobial effect SMAP-18 peptides on Escherichia coli (Escherichia coli, E.coli) under a lotion formulation.
2 is a graph showing the results of the antibacterial effect of the SMAP-18 peptide on Staphylococcus aureus , S. aureus under the lotion formulation.
Figure 3 is a photograph showing the results of an antimicrobial effect SMAP-18 peptides on Escherichia coli (Escherichia coli, E.coli) under a lotion formulation.
4 is a photograph showing the results of the antibacterial effect of the SMAP-18 peptide against Staphylococcus aureus , S. aureus under the lotion formulation.

Hereinafter, the present invention will be described in detail in detail. Unless otherwise defined, terms used in this specification should be interpreted as content commonly understood by those of ordinary skill in the art. The drawings and embodiments of the present specification are for those skilled in the art to easily understand and practice the present invention, and content that may obscure the gist of the present invention may be omitted from the drawings and embodiments, and the present invention is limited to the drawings and embodiments it is not going to be

The antibacterial cosmetic composition of the present invention contains the SMAP-18 peptide of SEQ ID NO: 1 as an active ingredient;

[SEQ ID NO: 1]

RGLRRLGRKIAHGVKKYG-NH _2.

The SMAP-18 peptide of SEQ ID NO: 1, which is an active ingredient of the present invention, is a peptide consisting of 18 residues at the N-terminus of SMAP-29 and has reduced toxicity to mammalian cells compared to the SMAP-29 peptide. The SMAP-18 peptide of SEQ ID NO: 1 shows no cytotoxicity against mammalian cells such as RAW 264.7, NIH-3T3 and HeLa cells even at 100 mM.

The present inventors found that the SMAP-18 peptide is E. coli ( E. coli ), Salmonella typhimurium ( S. typhimurium ), Pseudomonas aeruginosa ( P. aeruginosa ), Staphylococcus aureus ( S. aureus ), Bacillus Subtilis ( B. subilis ), Staphylococcus epidermidis ( S. epidermidis ) and Candida glabrata ( C. glabrata ) It was confirmed that it exhibits antibacterial activity against one or more strains selected from the group consisting of, As a result of the experiment, the SMAP-18 peptide of SEQ ID NO: 1, which is the active ingredient of the present invention, is methyl praben, 1,2-hexanediol (1,2-Hexanediol), gold extract, which are currently commercially mainly used preservative compositions. Alternatively, it could be confirmed that 16 to 32 times stronger antibacterial activity than that of phenoxy ethanol.

MIC (minimal inhibitory concentration) test results (Table 1), the SMAP-18 peptide compared to Staphylococcus aureus ( S. aureus ) and Candida glabrata ( C. glabrata ) Escherichia coli ( E. coli ), Salmonella Typhi murium ( S. typhimurium ), Pseudomonas aeruginosa ( P. aeruginosa ), Bacillus subtilis ( B. subilis ) and Staphylococcus epidermidis showing stronger antibacterial activity against ( S. epidermidis ) It could be confirmed, and the experimental results indicate that the SMAP-18 peptide has a higher antibacterial effect on Gram-negative bacteria than on Gram-positive bacteria or fungi.

The SMAP-18 peptide is Escherichia coli , E. coli in a lotion containing 3% (v/v) glycereth-26, 0.05% (v/v) xanthan gum. ) and Staphylococcus aureus ( Staphylococcus aureus , S. aureus ) It was confirmed that it exhibits antibacterial activity ( FIGS. 1 to 4 ). However, it can be confirmed that the SMAP-18 peptide exhibits a stronger antibacterial effect against Escherichia coli , E.coli than the Staphylococcus aureus , S.aureus , which is the MIC (minimal inhibitory concentration ) shows a similar pattern to the measurement result.

1 and 3, it was confirmed that E. coli (Escherichia coli , E. coli ) did not proliferate at all at a concentration of 100 μg/mL or more of the SMAP-18 peptide, and therefore 3% (v/v) glycerose-26 (glycereth-26), 0.05% (v / v) xanthan gum (xanthan gum) to at least 100μg / mL concentration in order to indicate the antibacterial effect on Escherichia coli (Escherichia coli, E.coli) in the lotion containing the SMAP- It is preferred to add 18 peptides.

Staphylococcus aureus of SMAP-18 peptide in lotion containing 3% (v/v) glycereth-26 and 0.05% (v/v) xanthan gum , S. aureus ) was confirmed to increase sharply when the concentration of 200 μg / mL was added, therefore, when using the SMAP-18 peptide of the present invention in the lotion of the composition 100 ~ 200 μg / It would be desirable to add a concentration of mL.

The antibacterial cosmetic composition comprising the SMAP-18 peptide of SEQ ID NO: 1 of the present invention as an active ingredient may be prepared by being included in any cosmetic formulation conventionally prepared in the art, for example, a solution, suspension, emulsion. , paste, gel, cream, lotion, powder, oil, soap, cleansing, shampoo, rinse, oil, powder foundation, emulsion foundation, wax foundation and spray, etc., but is not limited thereto.

In particular, the SMAP-18 peptide, which is the active ingredient of the present invention, is preferably added as a cosmetic preservative in consideration of the broad antibacterial spectrum shown for bacteria and fungi, and the antibacterial cosmetic composition of the present invention is currently commercially available. 16 to 32 times stronger antibacterial activity than methyl praben, 1,2-hexanediol, golden extract or phenoxy ethanol, which are preservative compositions mainly used as Therefore, it will be applicable as a cosmetic preservative to replace existing chemical preservatives in various cosmetic formulations.

Hereinafter, the present invention will be described in more detail through Examples, and the present invention is not limited by the Examples.

<Example>

(1) SMAP-18 peptide synthesis

SMAP-18 peptide including SEQ ID NO: 1 of Table 1 was synthesized by standard Fmoc solid phase method using Rink Amide 4-methylbenzhydrylamine Resin (Novagiochem, 0.54 mmol/g). DCC and HOBt were used as coupling reagents, and a 10-fold excess of Fmoc-amino acid was added during all coupling cycles. TFA/water/thioanisole/phenol/ethanedithiol/triisopropylsilane (81.5:5:5:5:5:5:1:1:v/v/v/v/v/ After cleavage and deprotection with a mixture of v), the crude peptide was repeatedly extracted with diethyl ether and prepared using a 0-90% water/acetonitrile gradient in the presence of 0.05% TFA (trifluoroacetic acid) Vydac C18 Purification by RP-HPLC on a column (20 mm x 250 mm, 300 Å, 15 mm particle size). The final purity (>95%) of the peptides was assessed by RP-HPLC on an analytical Vydac C18 column (4.6 x 250 mm, 300 Å, 5 mm particle size). The molecular weight of the synthetic peptide was determined by MALDI-TOF MS.

peptide amino acid sequence molecular weight MS SMAP-18 (SEQ ID NO: 1) RGLRRLGRKIAHGVKKYG-NH ₂ 2064.5

(2) strains and culture conditions of the strains used

E. coli ( Escherichia coli ) (KCTC 1682), S. typhimurium ( Salmonella typhimurium ) (KCTC 1926), P. aeruginosa ( Pseudomonas aeruginosa ) (KCTC 1637) and Representative Gram-positive bacteria S. aureus ( Staphylococcus aureus ) (KCTC 1621), B. subtilis ( Bacillus subtilis ) (KCTC 3068), S. epidermidis ( Staphylococcus epidermidis ) (KCTC 1917), and the fungus C. glabrata ( Candida glabrata ) (KCTC 7219) was purchased from the Korea Research Institute of Bioscience and Biotechnology and used. For solid culture, a plate of Mueller Hinton agar (Difco, USA) containing ^{the final 10 6 CFU/mL bacteria was prepared.} For liquid culture, the fungus C. glabrata was cultured in GPY medium (glucose 4%, peptone 0.5%, yeast extract 0.05%), and other strains were cultured in LB medium.

<Experimental Example 1> Minimal inhibitory concentration (MIC) measurement

In order to measure the antibacterial effect of the SMAP-18 peptide of SEQ ID NO: 1 synthesized in Example, the MIC (minimal inhibitory concentration) was measured for the 7 strains.

Each of the above strains were liquid-cultured, and the cultured strains were prepared by diluting the ^{cultured strains in 1% peptone to 4Х10 6 CFU/mL.} The SMAP-18 peptide of Example 1 was dissolved in a 1% peptone solution to a concentration of 409.6 μg/mL, and then 100 μL was added to a 96-well plate and serially diluted with 100 μL of a 1% peptone solution. After dispensing 100 μL of the 7 strains, the MIC value was confirmed by culturing for 16 hours in a 37° C. incubator. Through UV absorbance (600 nm) measurement, the lowest concentration of the extract without bacteria was expressed as the MIC value, and the results are shown in [Table 2].

As a comparative control, Bacillus amyloliquefaciens extract, KR-12-a3 peptide, melittin peptide, and methyl praben, 1,2-hexanediol, which are commonly used cosmetic preservatives (1,2-Hexanediol), gold extract or phenoxy ethanol (Phenoxy ethanol) was used, and Bacillus amyloliquefaciens MIC was measured under the same conditions as in the above experiment except for the extract. Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) extract was tested by diluting it so that the final concentration is 1250, 1000, 750, 500, 250, 125, 62.5, 31.3, 15.6 μg / mL, respectively.

strain MIC (μg/mL) BA
extract KR-12-a3 SMAP-18 melittin methyl
parabens 1,2-Hexanediol Gold
extract phenoxy
ethanol E. coli 750 1.6 6.4 5.7 > 204.8 > 204.8 > 204.8 > 204.8 S. typhimurium 750 1.6 6.4 5.7 > 204.8 > 204.8 > 204.8 > 204.8 P. aeruginosa 750 3.2 6.4 5.7 > 204.8 > 204.8 > 204.8 > 204.8 S. aureus 31.3 3.2 12.8 2.8 > 204.8 > 204.8 > 204.8 > 204.8 B. subtilis 750 3.2 6.4 5.7 > 204.8 > 204.8 > 204.8 > 204.8 S. epidermidis 750 3.2 6.4 5.7 > 204.8 > 204.8 > 204.8 > 204.8 C. glabrata 750 12.8 12.8 22.4 > 204.8 > 204.8 > 204.8 > 204.8

As a result of the experiment, SMAP-18 peptide was compared to methyl praben, 1,2-hexanediol, golden extract or phenoxy ethanol, which are commonly used cosmetic preservatives. It was confirmed that the antibacterial activity was 16 to 32 times stronger. In particular, general cosmetics for gram-negative bacteria E. coli ( Escherichia coli ), S. typhimurium ( Salmonella typhimurium ), P. aeruginosa ( Pseudomonas aeruginosa ) and gram-positive bacteria B. subtilis ( Bacillus subtilis ) and S. epidermidis ( Staphylococcus epidermidis ) It exhibited 32 times stronger antibacterial activity than preservatives.

<Experimental Example 2> Preparation of lotion with antibacterial ingredients and preservative power test

As the strains used, representative gram-negative bacteria E. coli and gram-positive bacteria S. aureus were used. Each strain was pre-cultured on LB agar medium, inoculated colonies into LB liquid medium, and subcultured at 37° C. twice, followed by LB The medium was ^{diluted to 4Х10 8} CFU/mL. Each of the two strains ^{was inoculated to a final concentration of 1Х10 7} CFU/mL, and the SMAP-18 peptide of Example 1 was added to the lotion formulation to have a final concentration of 0, 25, 50, 10 and 200 μg/mL. Then, incubate for 24 hours in a 37°C incubator, take 100 μL of each strain sample and dilute it in PBS (Phosphate-buffered saline) buffer to make 10-fold and 100-fold dilution series, and spread the step-by-step dilution series by 100 µL on LB agar medium. After culturing for 16 hours in a 37 ° C incubator, the number of bacteria was measured. The lotion formulation was prepared by adding 3% (v/v) glycereth-26 and 0.05% (v/v) xanthan gum to water.

Methyl praben, 1,2-Hexanediol and Phenoxy ethanol, which are commonly used cosmetic preservatives, were used as comparative controls, and the experiment was performed under the same conditions as above.

1 and 3 show the results of the antibacterial effect of the SMAP-18 peptide on Escherichia coli , E. coli under the lotion formulation, and FIGS. 2 and 4 are Staphylococcus aureus under the lotion formulation. , resulting in the antibacterial effect of SMAP-18 peptide against S. aureus ).

1 and 3, E. coli (Escherichia coli , E. coli ) did not proliferate at all at a concentration of 100 μg/mL or more of the SMAP-18 peptide, but Methyl praben, 1,2-Hexanediol and Phenoxy ethanol were at a concentration of 200 μg. Even when it was increased to /mL, there was no antibacterial effect on E. coli (Escherichia coli , E. coli).

2 and 4 , it will be confirmed that the SMAP-18 peptide exhibits a strong antibacterial effect against Staphylococcus aureus , S. aureus , similarly to Escherichia coli , E. coli ). could In addition, Methyl praben, 1,2-Hexanediol and Phenoxy ethanol as in the E. coli ( Escherichia coli , E. coli ) Staphylococcus aureus ( Staphylococcus aureus , S. aureus ) even when the concentration is increased to 200 μg/mL There was no antibacterial effect on

However, it can be confirmed that the SMAP-18 peptide exhibits a stronger antibacterial effect against Escherichia coli , E. coli than the Staphylococcus aureus , S. aureus, which is the Minimal inhibitory concentration (MIC). ) shows a similar pattern to the measurement result.

Claims (7)

Antibacterial cosmetic composition comprising the SMAP-18 peptide of SEQ ID NO: 1 as an active ingredient;
[SEQ ID NO: 1]
RGLRRLGRKIAHGVKKYG-NH _2.
According to claim 1,
The SMAP-18 peptide is E. coli ( E. coli ), Salmonella typhimurium ( S. typhimurium ), Pseudomonas aeruginosa ( P. aeruginosa ), Staphylococcus aureus ( S. aureus ), Bacillus subtilis ( B. subilis ), Staphylococcus epidermidis ( S. epidermidis ) and Candida glabrata ( C. glabrata ) Antibacterial cosmetic, characterized in that it exhibits antibacterial activity against one or more strains selected from the group consisting of composition.
According to claim 1,
The SMAP-18 peptide is compared to Staphylococcus aureus ( S. aureus ) and Candida glabrata ( C. glabrata ), E. coli ), Salmonella typhimurium ( S. typhimurium ), Pseudomonas aeruginosa Nosa ( P. aeruginosa ), Bacillus subtilis ( B. subilis ) and Staphylococcus epidermidis ( S. epidermidis ) Antibacterial cosmetic composition, characterized in that it exhibits stronger antibacterial activity.
According to claim 1,
The SMAP-18 peptide showed 16 to 32 times stronger antibacterial activity than methyl praben, 1,2-hexanediol, gold extract or phenoxy ethanol. Antibacterial cosmetic composition, characterized in that.
According to claim 1,
The SMAP-18 peptide is Escherichia coli , E. coli in a lotion containing 3% (v/v) glycereth-26, 0.05% (v/v) xanthan gum. ) and Staphylococcus aureus ( Staphylococcus aureus , S. aureus ) Antibacterial cosmetic composition, characterized in that it exhibits antibacterial activity.
6. The method of claim 5,
The SMAP-18 peptide is an antibacterial cosmetic composition, characterized in that it is added at a concentration of 100 ~ 200μg / mL.
A cosmetic comprising the antibacterial cosmetic composition of claims 1 to 6.

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