KR102475936B1 - A cosmetic composition comprising yeast expressing peptide LL-37 - Google Patents

Abstract

It relates to a cosmetic composition comprising yeast expressing the antimicrobial peptide LL-37. The cosmetic composition according to one aspect uses yeast expressing LL-37, which is produced only by skin wounds and infections, as an active ingredient, thereby suppressing the expression of inflammatory factors in the skin and promoting wound regeneration or skin regeneration to maintain healthy skin help to In addition, by expressing the natural antibacterial peptide LL-37 in yeast, it can be usefully used for improving skin-related conditions because it has low resistance and is harmless to the human body while maintaining anti-inflammatory and wound regeneration activity in vivo.

Description

A cosmetic composition comprising yeast expressing peptide LL-37

It relates to a cosmetic composition comprising yeast expressing the antimicrobial peptide LL-37.

The skin is a physical barrier that protects against the invasion of external harmful substances, and is directly exposed to chemical, mechanical, and biological stimuli, so that microbial infections, wounds, and inflammatory reactions related to immune responses frequently occur. Such skin infections and wounds are mainly treated with antibiotics containing ingredients such as mupirocin, fusidic acid, gentamicin, neomycin, and bacitracin, but antibiotics have the disadvantage of increasing resistance when used for a long time. .

As an alternative to this, research is being conducted to develop a new concept of antibiotics using antimicrobial peptides, which are natural materials and have little side effects. Anti-microbial peptides (AMPs) are one of the defense mechanisms used by organisms in nature, and are small-sized peptides composed of 10 to 50 amino acids, and act as a primary defense material when organisms are infected with pathogens. Human antimicrobial peptides typically include defensing and caselicidin, and human antimicrobial peptide LL-37 is the only casericidin-based protein found in humans.

On the other hand, the antimicrobial peptide LL-37 is only weakly expressed in chronic wounds, and its therapeutic usefulness in cells is limited due to lack of sufficient stability against proteases. Therefore, it is necessary to develop a new cosmetic composition that can be usefully used for skin-related conditions by maintaining anti-inflammation and cell regeneration ability similar to the existing antibiotics of antibacterial peptides even in the intracellular environment.

As a result of efforts to develop a cosmetic composition having excellent anti-inflammatory and wound regeneration effects while having stability in vivo, the inventors of the present invention completed the present invention by confirming the anti-inflammatory and skin cell regeneration effects of yeast expressing the antibacterial peptide LL-37 did

One aspect is to provide a cosmetic composition containing yeast expressing the human antimicrobial peptide LL-37.

Another aspect is to provide a quasi-drug composition for improving skin conditions containing yeast expressing the human antimicrobial peptide LL-37.

Another aspect is to provide a pharmaceutical composition for anti-inflammatory and skin regeneration comprising yeast expressing the human antimicrobial peptide LL-37.

Another aspect comprises preparing yeast expressing the human antimicrobial peptide LL-37; and

It is to provide a method for producing a cosmetic composition comprising the step of culturing the prepared yeast to obtain a culture medium or lysate.

One aspect provides a cosmetic composition comprising yeast expressing the human antimicrobial peptide LL-37.

The human antimicrobial peptide LL-37 is a human cathelicidin peptide, which is secreted from various epithelial cells or immune cells of the human body. Although LL-37 has various functions in the human body, such as neutralizing endotoxins and regulating inflammatory reactions, as well as antibacterial properties, it has potential cytotoxicity and can be hydrolyzed by various enzymes in the body. There are difficulties in use.

Information on the human antimicrobial peptide LL-37 protein sequence is, but is not limited to, available from the Uniprot database (Duplantier et al. 2013 Frontiers in Immunology 4(143): 1-14; UniProt ID: P49913), the LL-37 may be synthesized from the peptide sequence information.

In one embodiment, the human antimicrobial peptide LL-37 protein sequence used is as follows.

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 1)

In one embodiment, the yeast expressing the human antimicrobial peptide LL-37 may be transformed with an expression construct containing the LL-37 gene.

As used herein, the term "expression construct" refers to a nucleic acid molecule containing only minimal elements for protein expression in cells.

The expression construct may be a recombinant vector. As used herein, the term “vector” refers to a nucleic acid construct comprising essential regulatory elements that are operably linked to allow expression of a protein of interest in a suitable host, particularly recombinant yeast. The vectors may be plasmids, phage particles, or simply latent genomic inserts. Once transformed into a suitable host, the vector can replicate and function independently of the host genome or, in some cases, can integrate into the genome itself. In the present specification, “plasmid” and “vector” are sometimes used interchangeably. However, the present disclosure includes other forms of vectors having functions equivalent to those known or becoming known in the art.

The recombinant vector is not limited thereto, but preferably may be a vector in which a promoter, a nucleic acid or gene for transformation, a restriction enzyme recognition sequence, a transcription termination sequence, an antibiotic resistance gene, etc. are operably linked, and each of the above components is operably linked. As long as it is commonly used in the art, it can be used without limitation.

As used herein, the term "operably linked" means that an expression control sequence is linked to control transcription and translation of a polynucleotide sequence encoding a component such as a nucleic acid for transformation, and is controlled by an expression control sequence including a promoter. It may include maintaining the correct reading frame so that a component such as a nucleic acid for transformation to be encoded is generated. In addition, the term "antibiotic resistance gene" as used herein refers to a component that is linked to a nucleic acid for transformation to be expressed in order to determine and select whether a foreign nucleic acid is inserted during transformation, but is not limited thereto, but those skilled in the art Appropriate antibiotic resistance genes can be used depending on the purpose.

As a method of transforming the expression construct into yeast cells, a method of transforming a vector into a eukaryotic cell known in the art may be used, for example, a microinjection method, a calcium phosphate precipitation method, an electroporation method, or a liposome method. -mediated transfection method, DEAE-dextran treatment method, gene bombardment method, acetate-lithium DMSO method, etc., but are not limited thereto.

The transformed yeast may be cultured according to a conventional method in the art.

For example, the culture medium is a medium containing methanol, YPM (Bacto-peptone 2% (w/v), Bacto-Yeast Extract 1% (w/v), Methanol 3% (w/v)) medium, YPD (Bacto-peptone 2% (w/v), Bacto-Yeast Extract 1% (w/v), D-glucose 2% (w/v)), minimal medium YNBD (YNB without amino acids 0.67% (w/v) v), amino acid mixture, D-glucose 2% (w/v)), etc. may be used, and YPM medium may be preferably used, but is not limited thereto.

The culture carbon source is preferably methanol, and the concentration of methanol is 1% (w/v) to 10% (w/v), preferably 2% (w/v) to 5% (w/v), more preferably It may be 2% (w / v) to 4% (w / v), but is not limited thereto.

The culture temperature may be 25 to 45°C, preferably 30 to 40°C, more preferably 35 to 39°C, but is not limited thereto.

The culture pH may be 4.5 to 7.0, preferably 5.0 to 6.5, and more preferably 5.7 to 6.3, but is not limited thereto.

The culture stirring speed may be 100 to 300 rpm, preferably 150 to 250 rpm, and more preferably 180 to 220 rpm, but is not limited thereto.

In one embodiment, the yeast may be in the form of yeast culture, lysate, extract of culture, or a mixture thereof.

As used herein, the term "culture medium" may be used interchangeably with "culture supernatant", "condition culture medium" or "conditioned medium", and yeast expressing the human antimicrobial peptide LL-37 is capable of growing and surviving in vitro. It may mean the entire medium including the strain obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients, metabolites thereof, and extra nutrients. In addition, the culture solution may mean a culture solution obtained by removing the cells from the cell culture solution obtained by culturing the strain. On the other hand, the liquid from which the cells are removed from the culture medium is also called "supernatant", which is obtained by leaving the culture medium still for a certain period of time and taking only the liquid of the upper layer except for the part sunk in the lower layer, removing the cells through filtration, or centrifuging the culture medium It can be obtained by removing the lower sediment and taking only the upper liquid.

The culture medium may include a culture medium itself obtained by culturing yeast, a concentrate thereof, a lyophilized product, or a culture supernatant obtained by removing yeast from the culture medium, a concentrate thereof, or a lyophilisate.

The culture medium may be obtained by culturing yeast in a medium at a temperature of higher than 10° C. or lower than 40° C. for a certain period of time, for example, 4 to 50 hours.

The culture supernatant may be obtained by centrifuging or filtering the yeast culture solution to remove yeast. The concentrate may be obtained by concentrating the yeast culture solution itself or the supernatant obtained after filtering the culture solution using a centrifugal separation or filter.

Culture medium and culture conditions for culturing the yeast can be appropriately selected or modified by those skilled in the art.

In this specification, the term "lysate" may refer to a product obtained by disrupting cell walls by chemical or physical forces in yeast itself. In the present specification, the "lysate" may be used interchangeably with "lysate", "lysate", or "lysate".

Any method for disrupting the yeast cultured in the present specification can be used without limitation as long as it can obtain the entire lysate of yeast cells. For example, crushing by sonication, crushing by glass beads, crushing using a surfactant, or a combination thereof may be used, but is not limited thereto.

As used herein, the term "extract of a culture solution" refers to an extract obtained from the culture solution or a concentrate thereof, and includes an extract solution, a diluted solution or concentrate of the extract solution, a dried product obtained by drying the extract solution, or a crude or purified product thereof, or a fraction obtained by fractionating the same. can do.

In this specification, the term "yeast" is a microorganism used to make beer, wine, makgeolli, etc., and is a collective term for unicellular organisms that are fungi or mushrooms, but have no hyphae and do not have photosynthetic performance or motility.

The yeast may be a microorganism of the Saccharomyces family (family), preferably Saccharomyces ( Saccharomyces ) genus (genus) or Pichia ( Pichia ) genus microorganisms, more preferably Saccharomyces It may be selected from the group consisting of Saccharomyces cerevisiae , Saccharomyces carlsbergensis , Saccharomyces servazzii , and Pichia pastoris , Most preferably, it may be Pichia pastoris , but is not limited thereto.

The yeast may be included in 1 to 30% by weight, preferably 5 to 25% by weight, based on the total weight of the composition in the form of yeast culture, lysate, culture extract or a mixture thereof, more preferably may be included in 10 to 20% by weight, more preferably 13 to 17% by weight, but is not limited thereto.

In one embodiment, the present inventors found that when human keratinocytes were treated with a yeast lysate expressing the human antibacterial peptide LL-37, the expression of IL-1β and IL-6, which are inflammatory inducing cytokines, was suppressed, and the lysate It was found that this skin inflammatory reaction can be suppressed, and furthermore, the migration of fibroblasts is rapidly increased to have excellent activity for wound healing and skin regeneration.

Therefore, the use of the cosmetic composition according to one aspect may include improving skin conditions, improving skin beauty, preventing, improving or treating skin diseases.

The skin condition may be one or more selected from the group consisting of skin wound, skin inflammation, skin aging, skin barrier, skin wrinkle, and skin elasticity.

As used herein, the term "skin aging" refers to both tangible and intangible changes that appear in the skin with age, such as thinning of the epidermis, number of cells or blood vessels in the dermis, DNA damage repair ability, cell replacement cycle, It refers to reduction in wound healing, skin barrier function, epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage defense, chemical substance elimination ability, immune response, sensory function, and thermoregulation.

In this specification, the term "skin wrinkles" refers to a state in which elasticity of the skin is lost and loosened, and for example, the skin may be folded. The "prevention or improvement of skin wrinkles" may mean any action of preventing or improving wrinkles by suppressing the expression of wrinkle-related factors or increasing the total amount of collagen.

The "skin disease" includes all diseases occurring on the skin, including wounds, acne, dermatitis, atopic dermatitis, allergic dermatitis, contact dermatitis, acne dermatitis, heat rash, It can be skin aging, skin wrinkles, or skin scars. The term "prevention" includes inhibiting the development of a disease. The term "treatment" includes the inhibition, alleviation, or elimination of the development of a disease.

The skin barrier improvement means to improve all changes in the skin due to the decrease or damage of the skin barrier function, and the skin barrier function decrease means, for example, increased skin wrinkles, dryness, acne, dermatitis, atopic dermatitis, allergy sexual dermatitis and the like.

As used herein, the term "included as an active ingredient" means that the yeast of the present specification, its culture medium, lysate, or an extract of its culture medium is added to the extent that the above-mentioned effects can be exhibited, and drug delivery and stabilization, etc. It means that it is formulated in various forms by adding various components as subcomponents for the purpose.

The cosmetic composition may have, for example, softening lotion, nutrient lotion, massage cream, nutrient cream, essence, pack, gel, ampoule, or skin-adhesive cosmetic formulation.

Ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions other than the composition as active ingredients, for example, conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and flavors. can include

Another aspect provides a quasi-drug composition for improving skin condition containing yeast expressing human antimicrobial peptide LL-37.

As used herein, the term "quasi-drug" refers to items that are not instruments, machines, or devices among items used for the purpose of diagnosing, treating, mitigating, treating, or preventing human or animal diseases, and pharmacologically affecting the structure and function of humans or animals. It means items other than instruments, machines or devices among items used for the purpose of stringing.

The quasi-drug composition is not particularly limited thereto, but preferably may be a disinfectant cleaner, shower foam, gargreen, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler. In addition, the quasi-drug composition may include external skin preparations and personal care products. The external skin preparation is not particularly limited thereto, but is preferably a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof. The external skin preparation is a component usually used in external preparations for skin such as cosmetics or pharmaceuticals, for example, water-based components, oil-based components, powder components, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , colorants, various skin nutrients, or combinations thereof and may be suitably blended as needed. The external skin preparations include metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridin, and calin. Hot-water extracts of fruits, various herbal medicines, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts and other drugs, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can also be mix|blended suitably.

Another aspect provides a pharmaceutical composition for anti-inflammatory and skin regeneration comprising yeast expressing human antimicrobial peptide LL-37.

The use of the pharmaceutical composition according to one aspect may include one or more improvement or treatment selected from the group consisting of wounds, acne, atopic dermatitis, contact dermatitis, and heat rash.

The pharmaceutical composition may additionally include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and magnesium stearate, talc, or a combination thereof as a lubricant. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated for oral or parenteral administration. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or combinations thereof. Parenteral dosage forms may be injections.

A preferred dosage of the pharmaceutical composition varies depending on the condition and body weight of the patient, the severity of the disease, the type of drug, the route and duration of administration, but can be appropriately selected by those skilled in the art. However, for more effective prevention or treatment, the composition is preferably administered at 0.0001 to 100 mg/kg per day, preferably 0.001 to 100 mg/kg, and administration may be administered once a day or several times. It can also be administered in divided doses.

Another aspect provides a method for preventing, ameliorating, or treating a condition in a subject comprising treating or administering to a subject in need thereof an effective amount of a composition described above.

The condition of the subject may be a skin related condition.

As used herein, the terms "administering," "introducing," and "implanting" are used interchangeably and according to one embodiment into a subject by a method or route that results in at least partial localization of a composition to a desired site. It may mean the arrangement of the composition according to one embodiment of.

Administration may be administered by a method known in the art. Administration can be administered directly to a subject by any means, for example, by routes such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. can The administration may be administered systemically or locally.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be a subject requiring an effect of suppressing the expression of inflammatory factors in the skin, regenerating wounds, regenerating the skin, strengthening the skin barrier, or improving skin wrinkles.

The administration is 0.1 mg to 1,000 mg per day of the composition according to one embodiment, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg , 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg may be administered. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art can Dosage can be appropriately adjusted in consideration of these factors. The number of administrations can be once a day or two or more times within the range of clinically acceptable side effects, and administration can be performed at one or two or more sites, daily or at intervals of 2 to 5 days. The number of administration days may be administered from 1 day to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period. For non-human animals, the same dosage per kg as for humans is used, or the above dosage is converted by the volume ratio (eg, average value) of the organ (heart, etc.) between the target animal and the human. A single dose can be administered.

Another aspect comprises preparing yeast expressing the human antimicrobial peptide LL-37; And it provides a method for producing a cosmetic composition comprising the step of culturing the prepared yeast to obtain a culture medium or lysate.

The cosmetic composition according to one aspect uses yeast expressing LL-37, which is produced only by skin wounds and infections, as an active ingredient, thereby suppressing the expression of inflammatory factors in the skin and promoting wound regeneration or skin regeneration to maintain healthy skin help to In addition, by expressing the natural antibacterial peptide LL-37 in yeast, it can be usefully used for improving skin-related conditions because it has low resistance and is harmless to the human body while maintaining anti-inflammatory and wound regeneration activity in vivo.

1 is a graph confirming the expression levels of IL-1β and IL-6, which are inflammatory response factors, in human keratinocytes.
Figure 2 is a graph showing the degree of wound regeneration and recovery of cells treated with the yeast lysate expressing the antimicrobial peptide LL-37 of Example 1 and the general yeast lysate of Comparative Example 1 after scraping human fibroblasts. .

It will be described in more detail through the following examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of Yeast Expressing Human Antimicrobial Peptide LL-37

1-1. Preparation of transformed yeast

A yeast strain expression vector was constructed and introduced into the yeast strain to prepare transformed yeast expressing the human antimicrobial peptide LL-37. Specifically, in the case of Example 1, an expression vector for a yeast strain was constructed to include an inducible promoter, a human antimicrobial peptide LL-37 sequence (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES, SEQ ID NO: 1), and a transcription termination sequence. After transforming ( Pichia pastoris ) purchased commercially with the prepared vector, a strain line having an expression vector was selected, and LL-37 peptide expression was confirmed by Western blotting and used in the experiment. .

In the case of Comparative Example 1, Pichia pastoris, in which the human antimicrobial peptide LL-37 expression vector was not introduced, was used.

1-2. Transgenic yeast lysate preparation

Transformed yeast was grown for 30 days in YPM medium (Bacto-peptone 2% (w/v), Bacto-Yeast Extract 1% (w/v), Methanol 3% (w/v)) supplemented with 1.5% agar. Cultivated for 24 hours under conditions. The YPM medium was used after sterilizing the components except for methanol, cooling to 45° C., and mixing with filter-sterilized methanol. The cultured colony was placed in a 250mL baffle containing 25mL YPD (Bacto-peptone 2% (w/v), Bacto-Yeast Extract 1% (w/v), D-glucose 2% (w/v)) medium. After inoculation in a flask (baffled flask), incubation was performed at 30° C. at 250 rpm. Cultivation was performed for 12 to 16 hours until the degree of suspension of the cells reached an OD of 2 to 6 at a wavelength of 600 nm. Then, the culture medium was centrifuged at room temperature at 3000 rpm for 5 minutes to recover only the cells. The recovered transformed yeast cells were washed once with YNBD (YNB without amino acids 0.67% (w/v), amino acid mixture, D-glucose 2% (w/v)), a sterile minimal medium, and centrifuged again. and the supernatant was removed. The concentration of the initial cultured cells was cultured in a 500mL baffle flask containing 100mL YNBD culture medium at 30 °C and 250rpm conditions so that the concentration of the cells could be OD 0.5 at a wavelength of 600nm. 100% methanol was added every 24 hours to a final concentration of 0.5% (v/v). After centrifuging the culture solution cultured for up to 48 hours at 3000 rpm for 20 minutes, only the yeast cells were recovered by removing the supernatant. The obtained yeast cells were lysed using a Q 700 ultrasonic disperser (QSONICA, USA). The temperature was maintained below 30 ° C., the output of ultrasonic waves was 100 to 500 watts, and the pulse on / off was set to 10 s / 10 s to obtain a lysate, and the lysate (transformed yeast lysate) was used in subsequent experiments.

Experimental Example 1. Confirmation of anti-inflammatory effect

When the skin is damaged by bacterial infections, wounds, physical stimuli, or chemicals, an inflammatory response occurs to defend against it. At this time, various immune cells and an inflammation-inducing cytokine, IL-1β 1β), IL-6 (Interleukin-6), etc. are involved.

In order to confirm the anti-inflammatory effect of the cosmetic composition containing yeast expressing the antimicrobial peptide LL-37, human keratinocytes were used to examine the cytokine IL-1β and IL-6 involved in the inflammatory response. Expression levels were measured at the mRNA level.

1-1. Human keratinocyte culture

HaCaT cells, which are human keratinocytes, were cultured in 100 mm dishes containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin. The samples prepared in Example 1 and Comparative Example 1 were prepared by filtering with a 0.2 μm syringe filter, and each sample was added to the cell culture medium to a final concentration of 10% (v/v). As a positive control group, dexamethasone, which has an anti-inflammatory effect, was used. The culture solution containing the sample was cultured for 24 hours in an incubator maintained at 37°C, 5% CO ₂ concentration and 95% humidity, and then treated with 10 μg/ml of Poly I:C, an immunostimulant, and cultured for 4 hours.

1-2. Expression analysis of inflammatory cytokines

The cells cultured in Example 1-1 were recovered, washed with chilled phosphate buffered saline (PBS), and total RNA was extracted using PureLink® RNA Mini Kit (Thermo Fisher Scientific). Specific RNA extraction was performed according to the manual provided with the product. Changes in gene expression were measured by quantitative real time PCR (qRT-PCR), which binds a fluorescent substance to a DNA product amplified in the polymerase chain reaction (PCR) and continuously detects the fluorescent substance. In order to quantify the expression of IL-1β, an inflammatory response factor, and IL-6 genes, fluorescence values emitted by the PCR products were measured using SYBR Green (Thermo Fisher Scientific). A reaction solution was prepared by mixing 0.2 μM primer, 50 mM KCl, 20 mM Tris/HCl pH 8.4, 0.8 mM dNTP, 0.5 U Extaq DNA polymerase, 3 mM MgCl ₂ , and 1ХSYBR green in a PCR tube. After primary denaturation at 94°C for 3 minutes using a PCR machine, denaturation, annealing, and polymerization were performed at 94°C for 30 seconds, 58°C for 30 seconds, and 72°C. A total of 40 cycles were performed for 30 seconds, and fluorescence intensity was measured after each cycle was completed. The fluorescence intensities of the IL-1β and IL-6 genes were standardized with actin values, converted to 100% of the control group, and differences were compared to measure the expression level of each gene. The results are shown in FIG. 1 . Primer sequences used in the experiment are shown in Table 1 below.

gene forward primer reverse primer IL-1β 5′-GTCATTCGCTCCCCACATTCT-3′ 5′-ACTTCTTGCCCCCTTTGAAT-3′ IL-6 5′-TACCCCCAGGAGAAGATTCC-3′ 5′-TTTTCTGCCAGTGCCTCTTT-3′ Actin 5′-GGCCATCTCTTGCTCGAAGT-3′ 5′-GACACCTTCAACACCCCAGC-3′

As shown in FIG. 1, during Poly I:C treatment, the expression levels of the inflammatory response factor IL-β1 and IL-6 increased by 3.5 and 11 times, respectively, compared to the untreated group, and the antibacterial activity according to Example 1 increased. When the yeast lysate expressing the peptide LL-37 was treated, it was confirmed that the expression of IL-1β and IL-6 were significantly reduced. In addition, it was confirmed that the expression of IL-1β and IL-6 were significantly reduced even when compared to Comparative Example 1 treated with normal yeast culture and the positive control treated with dexamethasone.

Through the above results, it was confirmed that the yeast lysate expressing the antimicrobial peptide LL-37 exhibited a high anti-inflammatory effect and could effectively improve skin inflammation.

Experimental Example 2. Confirmation of wound regeneration effect

The wound regeneration effect of the cosmetic composition containing yeast expressing the antimicrobial peptide LL-37 was confirmed. Specifically, the human dermal fibroblast cell line (Human dermal fibroblast, Hs68) was inoculated in a 6-well plate at a density of 4 x 10 ⁵ , and then cultured for 24 hours in an incubator at 37° C. and 5% CO ₂ conditions to reach 100% saturation. made it into a confluent state. Thereafter, the center of the cultured cell was scratched using a cell scrapper, and then the samples prepared in Example 1 and Comparative Example 1 (0.01% (w/w) lysate) were added for 12 hours. further cultured. As a control, cells not treated with yeast culture were used. After 12 hours, the degree of recovery of the wounds randomly made to the cells was photographed every 2 hours with a fluorescence microscope in the case of including and not including the sample, and the results are shown in FIG. 2 .

As shown in Figure 2, when the yeast lysate expressing the antimicrobial peptide LL-37 according to Example 1 is treated, cells treated with the sample according to Comparative Example 1, migration of fibroblasts to the damaged area compared to the untreated group It was confirmed that the relative wound density increased significantly as this increased.

Through the above results, it was confirmed that the yeast lysate expressing the antimicrobial peptide LL-37 rapidly increases the migration of skin fibroblasts, is effective in wound healing, and promotes skin regeneration.

<110> COSMAX, INC. <120> A cosmetic composition comprising yeast expressing anti-microbial Peptide LL-37 <130> PN136147KR <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 37 <212> PRT <213> artificial sequence <220> <223> antimicrobial peptide LL-37 <400> 1 Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5 10 15 Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val 20 25 30 Pro Arg Thr Glu Ser 35

Claims (12)

A cosmetic composition for improving skin condition containing yeast expressing human antimicrobial peptide LL-37,
The yeast is Pichia pastoris ,
The yeast is in the form of yeast culture, lysate, extract of culture, or a mixture thereof,
The skin condition is a skin wound or skin inflammation, the cosmetic composition. delete delete delete The cosmetic composition according to claim 1, wherein the yeast is contained in an amount of 0.01 to 30% by weight based on the total weight of the composition. delete delete A quasi-drug composition for improving skin conditions containing yeast expressing human antimicrobial peptide LL-37,
The yeast is Pichia pastoris ,
The yeast is in the form of yeast culture, lysate, extract of culture, or a mixture thereof,
The skin condition is a skin wound or skin inflammation, quasi-drug composition. delete An anti-inflammatory pharmaceutical composition comprising yeast expressing human antimicrobial peptide LL-37,
The yeast is Pichia pastoris ,
The yeast is in the form of yeast culture, lysate, extract of culture, or a mixture thereof, a pharmaceutical composition. The pharmaceutical composition according to claim 10, wherein the composition is used for one or more improvement or treatment selected from the group consisting of wounds, acne, atopic dermatitis, contact dermatitis, and heat rash. preparing yeast expressing human antimicrobial peptide LL-37; and
As a method for producing a cosmetic composition for improving skin conditions comprising the step of culturing the prepared yeast to obtain a culture medium or a lysate,
The yeast is Pichia pastoris ,
Wherein the skin condition is a skin wound or skin inflammation.

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