KR20180019959A - Peptide for adsorbing and removing fine dust, and uses thereof - Google Patents

Abstract

The present invention relates to a peptide with excellent heavy metal and fine dust adsorption performance, and more particularly, to a new peptide consisting of a specific sequence and the use of the peptide. The peptide consists of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a peptide for adsorbing and removing fine dust,

The present invention relates to novel peptides having excellent heavy metal and fine dust adsorption effects.

Skin is the primary barrier of the human body. It protects the internal organs of the body from external stimuli such as changes in temperature and humidity, ultraviolet rays, and pollutants, and plays an important role in the maintenance of body homeostasis such as body temperature control.

Rapid industrialization, automobile exhaust gas, heavy metal powder from China and foreign particles such as fine dust pollute the skin and are considered to be the main cause of skin aging and trouble.

Removing heavy metals and fine dust from the skin through cleansing is an effective method for preventing skin troubles, but heavy metals and fine dusts are adsorbed in the cells and are not easily desorbed.

Accordingly, various materials have been studied to remove foreign substances in the skin and to improve the skin. However, recently developed cosmetic raw materials have various problems such as insufficient efficacy and skin side effects.

Even if the efficacy is excellent, current cosmetic raw materials have a limited range of action on the skin, and most of them have problems similar to the effect on the skin, such as promoting collagen synthesis, inhibiting collagen decomposition, and removing active oxygen.

Therefore, based on the latest theories of skin science, researches on raw materials and techniques that can identify and delay or delay new skin aging mechanisms are actively under way.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a novel peptide having excellent biostability and excellent in heavy metal and fine dust adsorbing ability.

According to one aspect of the present invention, there is provided a skin condition improving peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In one embodiment, the peptide is capable of releasing and adsorbing heavy metals or fine dust.

In one embodiment, the peptide may have anti-inflammatory and antioxidant activity.

According to another aspect of the present invention, there is provided a skin condition improving composition comprising, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In one embodiment, the composition may be for fine dust and heavy metal adsorption, antioxidant, anti-inflammatory.

According to another aspect of the present invention, there is provided an external preparation composition comprising the peptide as an active ingredient.

According to the present invention, since the peptide is small in size, the skin permeation rate is excellent and heavy metals and fine dust adsorbed on the skin can be effectively released and removed.

The peptide can be used not only for medical purposes for heavy metal removal and detoxification but also for cosmetic products such as quasi-drug compositions, health functional food compositions, and cosmetic compositions.

It should be understood that the effects of the present invention are not limited to the effects described above, but include all effects that can be deduced from the description of the invention or the composition of the invention set forth in the claims.

As used herein, the terminology used herein is intended to encompass all commonly used generic terms that may be considered while considering the functionality of the present invention, but this may vary depending upon the intent or circumstance of the skilled artisan, the emergence of new technology, and the like. Also, in certain cases, there may be a term selected arbitrarily by the applicant, in which case the meaning thereof will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term, not on the name of a simple term, but on the entire contents of the present invention.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

The numerical range includes numerical values defined in the above range. All numerical limitations of all the maximum numerical values given throughout this specification include all lower numerical limitations as the lower numerical limitations are explicitly stated. All the minimum numerical limitations given throughout this specification include all higher numerical limitations as the higher numerical limitations are explicitly stated. All numerical limitations given throughout this specification will include any better numerical range within a broader numerical range, as narrower numerical limitations are explicitly stated.

Hereinafter, embodiments of the present invention will be described in detail, but it should be apparent that the present invention is not limited by the following examples.

According to one aspect of the present invention, there is provided a skin condition improving peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The peptide refers to a polymer composed of one or more amino acids linked by amide bonds (or peptide bonds). For the purpose of the present invention, the peptide may refer to peptides or fragments thereof exhibiting fine dust and heavy metal adsorption and antioxidative effects.

The general rules for naming the peptides may be based on three letter or single letter amino acid codes, unless specifically indicated otherwise. For example, the center of the amino acid structure is represented by a three-letter code (e.g., Ala, Lys), and a D-stereotype (e.g., D-Ala, D-Lys) It is possible to assume an L-steric form. The amino acid residue constituting the peptide may be a natural or non-natural amino acid residue.

As a result of intensive efforts to discover peptides having a biologically active activity, the inventors of the present invention have confirmed the excellent skin improving effect of the peptide, particularly the adsorption effect of fine dust including heavy metals in the skin.

The peptide may be composed of the amino acid sequence of Formula 1, or may include the amino acid sequence as a part.

The peptides can effectively release and adsorb heavy metals or fine dust in the skin.

Generally, heavy metals refer to metals having a specific gravity of 4 or more, such as mercury, cadmium, lead, or copper. Since they form biodegradable organic complexes with biomolecules, they are not released into the body for a long time, Can be accumulated.

Symptoms such as Fuji paralysis, abnormal movements, ataxia, pigmentation and keratinization of skin, atrophy of claw or hoof and hair, reproductive dysfunction, malformations, growth retardation, and impaired immune function when exposed to a certain amount of heavy metals .

The harmful heavy metal fine particles adsorbed on the skin cause a chemical reaction in the air to generate additional harmful substances such as nitrogen oxides (NO) and sulfur oxides (SO), so that inflammatory substances such as cytokines and the like are increased, . ≪ / RTI > In addition, the seed metal may damage hyaluronic acid which maintains skin moisturization, thereby adversely affecting the skin.

Fine dust penetrates into the alveoli of humans and accumulates in the bronchi and the lungs and can directly cause various respiratory diseases. The fine dust may lower the immune function of the body, cause asthma and dyspnea, and may increase the concentration of heavy metals in the rain or snow due to long-distance movement. In particular, fine dust can penetrate deep into skin pores due to its small diameter, and fine dust that is not removed can cause inflammation or trouble in the skin.

The peptide can effectively remove and remove heavy metals and fine dust adsorbed in skin pores or cells. In particular, the peptide has a high molecular weight and a small particle size, and thus has high skin permeability, and thus has excellent heavy metal and fine dust adsorption effect. The peptide can increase the activity of the cell, promote the discharge of foreign substances, bind with heavy metals and fine dust, and can be released to the outside of the cell.

In addition, the peptides can exhibit anti-inflammatory activity through the expression of inflammatory cytokines and inhibition of inflammatory cell proliferation, and can effectively remove active oxygen species in the cells.

On the other hand, the peptide can be produced biologically by extracting a protein in vivo and treating it with a protease to give a low molecular weight or by using a gene recombination and protein expression system, or by a chemical synthesis method using a peptide synthesizer or the like .

For example, a gene encoding a fusion partner and a fusion protein composed of the peptide may be prepared through gene manipulation, and the host microorganism may be transformed with the gene and expressed in the form of a fusion protein in the host microorganism.

The fusion protein can be cleaved and separated using proteolytic enzymes or compounds, and the peptide can be obtained. Specifically, a DNA sequence encoding an amino acid residue that can be cleaved by a proteolytic enzyme, such as Factor Xa or an enterokinase, a compound such as CNBr or hydroxylamine, can be inserted between the fusion partner and the gene of the peptide .

The N or C-terminal of the peptide may be bonded with an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group or polyethylene glycol (PEG). The stability of the peptide can be remarkably improved by the modification. The stability is a concept including not only in vivo stability but also storage stability. The protecting group can protect the peptide of the present invention from attack of a protein cleaving enzyme in vivo.

In addition, the peptide may comprise a functional equivalent of a peptide comprising the amino acid sequence. Said "functional equivalent" means that at least 40%, preferably at least 80%, more preferably at least 90%, more preferably at least 95% sequence identity with said amino acid sequence as a result of addition, substitution or deletion of amino acids Means a protein having homology and having a physiological activity substantially equivalent to the above amino acid sequence.

The "substantially homogenous physiological activity" means a skin condition improving activity such as anti-inflammatory activity due to the structural and functional homology of the peptide. The sequence homology is determined by comparing the two optimally arranged sequences with the comparison region, and some nucleic acid sequences in the comparison region may be added or deleted.

According to another aspect of the present invention, there is provided a skin condition improving composition comprising, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In one embodiment, the peptide may be for fine dust and heavy metal adsorption, antioxidant, anti-inflammation, skin wrinkle improvement, or skin regeneration. That is, since the peptide can effectively release and adsorb heavy metals or fine dusts, it can be used for removing skin troubles such as fine dust adsorbed in skin pore cells.

As used herein, the phrase " comprising as an active ingredient " may mean an effective amount of a substance capable of exhibiting a skin improving effect, for example, a degree capable of exhibiting fine dust and heavy metal adsorption, antioxidation or skin regeneration activity.

In addition, since the peptides have various skin condition improving effects as well as the fine dust and heavy metal adsorption effect, the compositions can be used for skin improvement and cosmetic use.

The peptides can be used for various applications such as pharmaceutical compositions, cosmetic compositions, and external skin preparations due to the skin improving activity.

The pharmaceutical composition may be of heavy metal or fine dust adsorption. Since the composition contains a heavy metal and a peptide having an excellent effect of adsorbing fine dust, the skin contaminated by various environmental factors such as fine dust can be effectively improved.

The pharmaceutical composition may form pharmaceutically acceptable acid addition salts according to methods conventional in the art. The free acid may be an organic acid or an inorganic acid. For example, the inorganic acid may be hydrochloric acid, bromic acid, sulfuric acid, or phosphoric acid. The organic acid may be citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid But are not limited to, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4- toluenesulfonic acid, benzenesulfonic acid, Nicotinic acid, isonicotinic acid, picolinic acid, glutamic acid, or aspartic acid.

The pharmaceutical composition may further comprise a commonly used carrier, excipient and diluent.

The compositions can be administered in the form of oral delivery , parenteral delivery. The peptide may be administered systemically or topically, and the administration may include oral administration and parenteral administration. When the compound is administered as a composition, it may be formulated with a suitable amount of a pharmaceutically acceptable vehicle or carrier to provide a suitable dosage form.

In addition, the composition comprising the peptide may further comprise a carrier, an excipient and a diluent which are used in the production of the pharmaceutical composition.

Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, But are not limited to, cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

In addition, the composition may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, external preparation, suppository and sterilized injection solution.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and the solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Or gelatin. In addition to the above excipients, lubricants such as magnesium stearate and talc may also be used.

Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, syrups, and the like. In addition to water and liquid paraffin which are simple diluents, various excipients such as wetting agents, sweeteners, have.

The preparation for parenteral administration may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository. Examples of the non-aqueous solvent and suspensions may include injectable esters such as propylene glycol, polyethylene glycol, vegetable oil such as olive oil and ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, and glycerogelatin.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is desirable to administer an amount that allows for all of the above factors to be maximally effected in a minimal amount without side effects, which can be readily determined by one skilled in the art.

On the other hand, the cosmetic composition or external preparation for skin may be formulated by a conventional method. In the formulation of the external preparation for skin, Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can refer to, and in the formulation of the cosmetic composition, International cosmetic ingredient dictionary, 6th ed., The cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1995, which is incorporated herein by reference.

Specifically, the cosmetic composition may be prepared in the form of a general emulsified formulation and a solubilized formulation. For example, lotion such as soft lotion or nutrition lotion; Lotion such as facial lotion, body lotion; Nourishing cream, moisture cream, cream such as eye cream; essence; Makeup ointment; spray; Gel; pack; Sunscreen; Makeup base; A foundation such as a liquid type, a solid type or a spray type; powder; Make-up removers such as cleansing creams, cleansing lotions and cleansing oils; Or a cleansing agent such as a cleansing foam, a soap, a body wash, and the like, but is not limited thereto. The external preparation for skin may be formulated into ointments, patches, gels, creams or sprays, but is not limited thereto.

The cosmetic composition or the external preparation may suitably contain other ingredients in addition to the essential ingredients in the respective formulations within the range not hindering the object of the present invention depending on the type of the formulation or the purpose of use.

The cosmetic composition or the external preparation may contain an acceptable carrier and may be suitably mixed with oil, water, a surfactant, a moisturizer, a lower alcohol, a thickener, a chelating agent, a pigment, a preservative, It is not.

The acceptable carrier may vary according to the formulation. For example, when formulated into ointments, pastes, creams or gels, there may be used an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc, zinc oxide, Mixture can be used

When the cosmetic composition or the external preparation is formulated with a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof may be used as a carrier component, Such as propane, butane, or dimethyl ether.

When the cosmetic composition or the external preparation is formulated into a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as the carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, Glycol, and 1,3-butylglycol oil may be used, and fatty acid esters of cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or sorbitan may be used have.

When the cosmetic composition or the external preparation is formulated as a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the cosmetic composition is formulated into soap, it is preferable to use, as a carrier component, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolizate, isethionate, a lanolin derivative, an aliphatic alcohol, a vegetable oil, Can be used.

The cosmetic composition or the external preparation for skin according to the present invention may be formulated into a lipid, an organic solvent, a solubilizer, a thickening agent, a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, a foaming agent ), Perfumes, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants, chelating agents, preservatives, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, Such as any other ingredients used in cosmetics or dermatology.

However, the adjuvant and the mixing ratio thereof can be appropriately selected so as not to affect the preferable properties of the cosmetic composition according to the present invention.

The present invention will be further described with reference to the following examples, but it should be apparent that the present invention is not limited by the following examples.

Preparation Example: Preparation of peptide

Peptides were synthesized using conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group of amino acid. Amino acid residues were extended using N-hydroxybenzotriazole (HOBt) and N, N'-diisopropylcarbodiimide (DIC) as an activator (Reference: Wang C. Chan, Perter D. White, 'Fmoc-solid phase peptide synthesis', Oxford].

The synthesized peptides were purified by C18 reverse phase high performance liquid chromatography (HPLC, Waters Associates, USA). The columns were ACQUITY UPLC BEH300 C18 (2.1 x 100 mm, 1.7 μm, Waters Co, USA).

The peptides synthesized according to the above method are shown in Table 1 below

division order Remarks Example 1 LYLCTRGSF SEQ ID NO: 1 Example 2 CTRGSGYQFIH SEQ ID NO: 2

Experimental Example 1: Fine dust decomposition test

The effect of decomposing fine particles by the peptides of Example 1 and Example 2 was confirmed. Peptide concentration of 50 mg / L was added to the solution mixed with 1% (w / w) of fine dust (PM10) collected using a HEPA filter, and the permeability (550 nm) was measured using a UV / VIS spectrophotometer Respectively. As a control, a solution containing MCT was used.

The fine dust permeability is calculated by dividing the permeability of the sample mixed with the fine dust by the permeability of the control sample. The higher the permeability, the more effectively the fine dust is dissolved and decomposed. The fine dust decomposition rate was calculated as (transmittance - 1) x 100, and the evaluation results are shown in Table 2 below.

division 0 minutes 5 minutes 10 minutes 15 minutes Example 1 4.8 13.7 26.3 37.5 Example 2 6.2 15.3 30.2 39.1 Control group 0.1 0.1 0.2 0.3

[Decomposition rate of fine dust (%)]

Referring to Table 2, the fine dust mixed with the sample was decomposed in a time-dependent manner according to the input of the peptide, and the results suggest that the peptide can effectively decompose and remove fine dust adsorbed on the skin.

Experimental Example 2: Heavy metal removal test

The effect of removing the heavy metals by the peptides of Example 1 and Example 2 was confirmed.

Lead (60ppm) and cadmium (30ppm) solutions and 50mg / L of peptides were mixed in equal volumes in separate wells and reacted three times. After three reactions, only the lead and cadmium solutions were separated and the residual heavy metal content of the fraction was measured by ICP-MS. As a control, a solution containing MCT was used.

The adsorption rate was calculated by dividing the concentration of the heavy metal and the adsorbed sample by the concentration of the control sample. The higher the adsorption rate, the more effective adsorption and removal of the heavy metal. The heavy metal removal rate was calculated as (1 - adsorption rate) × 100, and the evaluation results are shown in Table 3 below.

division Lead Removal Rate (%) Cadmium removal rate (%) Example 1 1 mg / L 6.8 8.3 10 mg / L 24.6 29.5 50 mg / L 39.2 43.5 Example 2 1 mg / L 7.8 6.3 10 mg / L 21.3 23.5 50 mg / L 35.6 41.7 Control group 0.1 0.2

As a result of the analysis, the heavy metals were removed in a concentration-dependent manner according to the input of the peptide, which suggests that the peptide can effectively adsorb and remove heavy metals adsorbed on the skin.

Experimental Example 3: Polycyclic Aromatic Hydrocarbon Removal Test

The adsorption effect of polycyclic aromatic hydrocarbons (PAH), which is a harmful atmospheric substance contained in a large amount of fine dust, was tested.

Porcine sheath (1 cm x 1 cm) with hair removed was prepared and washed with PBS for 10 minutes. After washing, 600ppm of Naphtalene, Acenaphthylene, Acenaphthene, Fluorene, Fluoranthene, Pyrene, Chrysene, Benzopyrene (PH 11) for 2 hours and then dried at room temperature. The experimental group was applied with different concentration of peptide and reacted for 3 hours. The control group was applied with PBS and reacted for 3 hours.

After washing with PBS, the specimen was cut to a predetermined weight and then crushed with a homogenizer. The pulverized tissue was dried with anhydrous sodium sulfate (Na ₂ SO ₄ ), and the polycyclic aromatic hydrocarbons were extracted from the tissue by Sohxlet extraction and liquid-liquid extraction (Husain et al. 1997; Takatsuki et al., 1985) and quantitated by HPLC. The quantification results are shown in Table 4 below, and the results are expressed as a ratio of the control group to the experimental group.

division Example 1 Example 2 1 mg / L 10 mg / L 50 mg / L 1 mg / L 10 mg / L 50 mg / L naphthalene 29.7 15.6 2.2 30.3 14.4 2.5 Acenaphthylene 32.4 15.7 1.7 33.5 16.2 19 Fluorene 36.2 17.4 3.2 39.6 18.8 4.5 Fluoranthene 34.6 14.7 2.5 37.3 15.3 2.7 Pyrene 30.4 13.2 0.9 36.4 13.9 1.2 Crissen 28.5 13.8 1.1 29.2 14.2 1.1 Benzopyran 18.4 6.5 3.2 21.5 7.1 3.5

[% of control]

As a result of the analysis, the polycyclic aromatic hydrocarbons were removed in a concentration-dependent manner according to the peptide treatment, and the results suggest that the peptides can effectively remove harmful substances that contain large amounts of fine dust and air and cause skin troubles .

Experimental Example 4: Test for decomposition of active oxygen

Free radical scavenging activity using DPPH (1, 1-diphenyl-2-picryl hydrazyl).

4 mL of methanol was added to the test tube, and the peptide was added by concentration. 1 mL of 0.15 mM DPPH solution was added, and the reaction was allowed to proceed at room temperature for 30 minutes. Then, the absorbance at 520 nm was measured.

Initial (Ai) and blank (Ab) without substrate and DPPH were measured, and α-tocopherol and BHT were used as positive control. The results are shown in Table 5 below.

division 0 mg / L 1 mg / L 10 mg / L 50 mg / L Example 1 0 41.7 65.7 75.3 Example 2 0 43.8 67.5 87.7 alpha-tocopherol 0 45.2 64.4 79.6 BHT 0 38.1 52.6 67.2

 [DPPH erase rate (%)]

The peptides of Examples 1 and 2 were evaluated to have higher or equal free radical scavenging activity than α-tocopherol and BHT, which are well known in the art. These results suggest that the peptide can effectively promote the decomposition of active oxygen, and that it is excellent in preventing skin aging and improving skin condition due to antioxidant activity.

Experimental Example 5: Measurement of active oxygen species in cells (DCF-DA assay)

The removal of reactive oxygen species (ROS) was assessed by comparing the amount of reactive oxygen species produced in the cells stimulated with silica after the peptide treatment with the negative control (Cho et al., 1999; Kim et al., 2002).

Raw 264.7 cells were inoculated into 96 well plates at a concentration of 1 × 10 ⁴ cells / well. After replacing the medium with fresh medium, cells were cultured for 24 hours by treatment with a certain concentration of the peptide.

10.0 μL of WST-1 solution (EZ-CYTOX, DOGEN, Korea) was added to each well and incubated for 1 hour. Absorbance was measured with a measurement wavelength of 450 nm and a reference wavelength of 655 nm using an ELISA reader Respectively.

Peptides were diluted with dimethylsulfoxide (DMSO, Sigma-Aldrich, USA) to a final concentration of 20 μM and DMSO was used as a blank test (negative control). The experiment was repeated 3 times to ensure reliability.

The experiment was carried out under the same cell line and the same cell culture conditions as the WST-1 assay, a cell activity assay. The cultured Raw 264.7 cells were washed once with phosphate buffered saline (PBS; sodium chloride 137 mM, phosphate buffer 10 mM, potassium chloride 2.7 mM, all from Biopure, Canada), and Raw 264.7 cells were harvested. The cells were suspended in 15 mL of PBS, and 15 μL of 20 mM 2 ', 7'-Dichlorofluorescein diacetate was added and incubated for 1 hour.

After centrifugation at 1200 rpm for 2 minutes, the cells were washed once with PBS solution, diluted to 1 × 10 ⁵ cells / mL, treated with the control substance and the peptide, and cultured for 10 minutes. 20 mg / mL of silica was treated with 50 μL and incubated for 1 hour. The cell pellet obtained by centrifuging at 6000 rpm for 5 minutes was dispersed in 200 μL PBS solution and inoculated into a 96-well plate.

Fluorescence was measured at 485 nm excitation / 535 nm emission using a fluorescence microplate reader to evaluate the amount of reactive oxygen species.

The peptides treated in the measurement of reactive oxygen species to the concentration of the test solution were used as the concentration of the test solution set through the cell activity measurement. As a negative control, DMSO was used instead of the sample. As a positive control, ascorbic acid (Sigma-Aldrich) was diluted to 100 mg / mL in DMSO and used at a concentration of 100 μg / mL.

Based on the above results, a test for the measurement of reactive oxygen species at the same concentration was carried out, and the treated peptide was used at a concentration of 50 mg / L. The experiment was repeated 4 times to ensure reliability. The measurement results are shown in Table 6 below.

division Measures Example 1 74.7 Example 2 72.8 Ascorbic acid 75.4

 [ROS generation inhibition rate (% of control)]

The free radical scavenging activity of the peptides of Examples 1 and 2 above was evaluated at an equivalent level compared to ascorbic acid, which is well known in the art.

That is, the above results suggest that the peptide is excellent in the ability to remove reactive oxygen species and has excellent antioxidative effect due to free radical scavenging activity.

Experimental Example 6: NF-kB transcription-inhibitory effect measurement test

The NF-kB reporter assay was used to measure the inhibitory effect of NF-kB transcription. INOS, COX-2, IFN-? Or TNF-? Induced by LPS are expressed by the transcription factor NF-kB. The NF-kB is inactivated in complex with IkB in the cytoplasm, and when I-kB-α is phosphorylated by I-kB-α kinase, the bound complex is degraded and activated as NF-kB, )do. Induces the expression of the target gene of NF-kB and causes inflammation.

Also, TRIF, which acts on inflammatory diseases such as multiple sclerosis, is a receptor that responds to the activity of TLRs (toll-like-receptors) in the TIP-domain with receptors that induce interferon-beta, And activates the immune response to the antigen.

The receptors recognize a well-preserved pathogenic pattern and activate the signal to stimulate the release of inflammatory cytokines. IRF-3 (interferon-stimulating factor 3) is a transcription factor induced by interferon alpha / beta stimulation, which binds to the interferon-stimulated response sequence (ISRE) in the transcriptional regulatory region of most interferon inducible genes and activates the transcription.

According to the manufacturer's protocol (Jin et al., 2009), 12-well plates were coated with NF-kB according to the presence of receptor molecules such as? -Galactosidase (MyD88 and TRIF) -Luc or TRIF was transfected into HEK293 (1 x ¹⁰⁶ cells / ml) cells and cultured for 24 hours.

The activity of NF-kB and IRF-3 activity were measured 24 hours after treatment of LPS and the peptide using Luciferase assays. The luciferase assay was performed using a luciferase assay system (Promega Co., USA) reported in the manufacturer's protocol (Jung et al., 2009).

division NF-kB activity IRF-3 activity Control group (Blank group) 1.00 1.00 LPS
100 ng / ml 1st
Peptides 0 mg / L 3.80 4.50 1 mg / L 2.34 2.95 10 mg / L 1.85 1.73 50 mg / L 1.32 1.21 Second
Peptides 0 mg / L 3.80 4.50 1 mg / L 2.56 3.05 10 mg / L 2.23 1.74 50 mg / L 1.51 1.36

 [Fold, Luciferase activity]

Experimental Example 7: Quantitative test for inflammation-related protein mRNA

TNF-a, iNOS, and COX-2 were analyzed to evaluate the anti-inflammatory activity of the peptides.

RNA was prepared from RAW264.7 cells treated with LPS. The method was prepared using Trizol Reagent (Gibco BRL) as described in Lee et al., 2008. All RNAs were stored at -70 ° C before use. Semi-quantitative RT reactions were performed using MuLV reverse transcriptase.

All RNA (1 μg) was incubated with oligo-dT15 at 70 ° C for 5 minutes and mixed with 5 × first strand buffer, 10 mM dNTPs and 0.1 M DTT. The reaction mixture was further incubated at 37 ° C for 5 minutes and then incubated for 60 minutes after addition of MuLV reverse transcriptase (2 U).

The reaction was terminated by incubating at 70 DEG C for 10 minutes. (Conditions: 2 μL cDNA, 4 μM 5 'and 3' primer, 10 × buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl and 0.1 % Triton X-100), 250 μM dNTPs, 25 mM MgCl ₂ and 1 unit of Taq DNA polymerase (Promega, USA).

The PCR reaction was denatured at 94 DEG C for 30 seconds, annealed at 55 DEG C to 60 DEG C for 30 seconds, polymerized at 72 DEG C for 42 seconds, and finally polymerized at 72 DEG C for 5 minutes.

The primers for TNF-a, iNOS, and COX-2 are shown in Table 8 below.

division Forward primer Reverse primer iNOS 5'-FGGAGCCTTTAGACCTCAACAGA-3 ' 5'-RTGAACGAGGAGGGTGGTG-3 ' TNF-a 5'-FTGCCTATGTCTCAGCCTCTTC-3 ' 5'-RGAGGCCATTTGGGAACTTCT-3 ' COX-2 5'-FCACTACATCCTGACCCACTT-3 ' 5'-RATGCTCCTGCTTGAGTATGT-3 '

The results of measurement of mRNA expression levels according to the peptide treatments of Examples 1 and 2 are shown in Table 9 below. Analysis of RAW264.7 cell mRNA revealed that the expression of TNF-α, iNOS, and COX-2 was increased by the addition of LPS. On the other hand, the expression of TNF-a, iNOS, and COX-2 induced by the LPS decreased in a concentration-dependent manner when the peptide was treated.

division iNOS
Expression level TNF-a
Expression level COX-2 expression level Control group (Blank group) 1.00 1.00 1.00 LPS
100 ng / ml 1st
Peptides 0 mg / L 3.45 2.95 3.30 1 mg / L 2.56 2.36 2.63 10 mg / L 2.03 1.94 2.23 50 mg / L 1.63 1.52 1.96 Second
Peptides 0 mg / L 3.45 2.84 3.23 1 mg / L 2.46 2.27 2.73 10 mg / L 2.27 1.96 2.62 50 mg / L 1.71 1.48 1.67

 [Fold, GAPDH normalized]

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

<110> AHN, INSOOK <120> PEPTIDE FOR ADSORBING AND REMOVING FINE DUST, AND USES THEREOF <130> 16PP11160 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> SEQ ID NO1 <400> 1 Leu Tyr Leu Cys Thr Arg Gly Ser Phe   1 5 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <220> SEQ ID NO 2 <400> 2 Cys Thr Arg Gly Ser Gly Tyr Gln Phe Ile His   1 5 10

Claims (6)

A peptide for improving skin condition comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The method according to claim 1,
Peptides that release and adsorb heavy metals or fine dusts. The method according to claim 1,
Peptides with anti-inflammatory and antioxidant activity. A composition for improving skin condition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient. 5. The method of claim 4,
Micro dust and heavy metal adsorption, antioxidant, anti-inflammation, skin wrinkle improvement, or skin regeneration. An external-use composition comprising the peptide of any one of claims 1 to 3 as an active ingredient.