KR102203424B1 - A zdhhc13-derived peptide, and uses thereof - Google Patents

Abstract

The present invention relates to a physiologically active peptide derived from a growth factor, and provides a physiologically active peptide having an excellent skin improvement effect composed of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4.

Description

Bioactive peptide derived from ZDHHC13, and uses thereof {A ZDHHC13-DERIVED PEPTIDE, AND USES THEREOF}

The present invention relates to a novel use of a physiologically active peptide derived from a growth factor.

Melanin refers to a black or brown pigment present in skin and hair. Melanin exists in the skin, protects the skin by blocking ultraviolet rays, and maintains the skin's body temperature.

There are two kinds of melanin: eumelanin and pheomelanin. The color of human skin is determined by the ratio of the amount and type of these melanin.

When melanin secretes various elements from keratinocytes exposed to ultraviolet light, melanocytes can recognize them and form melanin.

In particular, α-MSH (α-melanocyte-stimulating hormone) is activated by binding to MC1R (Melanocortin 1 receptor) present in melanocytes, and melanin is formed by lower factors.

In addition to the binding of α-MSH, a ligand, MC1R is known to be activated only when palmitoylation is performed on the 315th amino acid cysteine (Chen S et al., Nature. 2017, 21;549(7672), p399-403). . MC1R palmitoylation is achieved by the protein-acyl transferase ZDHHC13.

Meanwhile, ZDHHC13 is activated by phosphorylation by ATR after ultraviolet (UV) irradiation.

Until now, the technology development trend of whitening functional cosmetics is mainly focused on the development of raw materials centered on natural products.

In a situation where interest and demand for whitening functional cosmetics are increasing, technology to secure safety and stability by high concentration and density of effective drugs is applied through additional process variables such as fermentation to use natural products as raw materials, but skin absorption rate This is low and the whitening effect is not well implemented.

In addition, natural product-based cosmetic materials are difficult to control the quality of physiologically active unit substances and may cause side effects due to the specificity of an unclear mechanism.

Recently, the Nagoya Protocol, an international agreement that includes guidelines for sharing the benefits of using biological resources, has come into effect, and in the mid- to long-term, royalties for the use of overseas biological resources increase, raising concerns about the development of materials derived from natural products.

The Nagoya Protocol is an international agreement to share the benefits of using biological genetic resources fairly between the resource providing country and the user country (ABS: Access to genetic resources and benefit-sharing). It is putting more weight on securing domestic biomaterials and developing biomaterials.

Most of the cosmetic ingredients currently on the market are natural products, and arbutin, which has a mechanism to inhibit tyrosinase, is a representative whitening ingredient.

Since alpha arbutin is a glycosylated hydroquinone, excessive use may cause skin hypersensitivity reactions and acne, and in severe cases, it may cause ochronosis of skin deposits.

Therefore, there is a need to develop a technology capable of inducing a more efficient skin whitening effect, overcoming the limitations of existing whitening functional cosmetics that use natural substances-based materials as functional raw materials.

Not only can it overcome the shortcomings of natural products-based raw materials, such as low transdermal absorption rate, unclear mechanism of action in vivo, and limitations of use of biological resources, but also because it is synthesized based on the protein sequence in the human body, it is safe and easy to synthesize, so it can be efficiently developed Efficacy can be verified.

In addition, as mentioned above, the Nagoya Protocol was enacted and the development of bio-materials is receiving more attention due to restrictions on the use of biological genetic resources, so the cosmetic market using peptides is expected to grow further in the future.

The present invention has been conceived to solve the problems of the prior art, and an object of the present invention is to provide a bioactive peptide having excellent biological stability and excellent melanin formation inhibitory effect.

According to an aspect of the present invention, there is provided a physiologically active peptide consisting of one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 4.

In one embodiment, the peptide may inhibit melanogenesis in melanocytes.

In one embodiment, the peptide may inhibit pigmentation due to ultraviolet (UV) light.

In one embodiment, the peptide may inhibit pigmentation due to oxidative stress.

According to another aspect of the present invention, there is provided a physiologically active peptide mixture comprising two or more peptides consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4.

In one embodiment, the peptide mixture may inhibit melanogenesis in melanocytes.

According to another aspect of the present invention, there is provided a cosmetic composition comprising the peptide or the peptide mixture as an active ingredient.

In one embodiment, the cosmetic composition may be for skin whitening or inhibiting pigmentation by ultraviolet (UV) light.

According to another aspect of the present invention, there is provided a composition for external application for skin comprising the peptide or the peptide mixture as an active ingredient.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating pigmentary diseases comprising the peptide or the peptide mixture as an active ingredient.

In one embodiment, the pigmented disease may be selected from the group consisting of melasma, freckles, black spots, birthmarks, spots, and hyperpigmentation during pregnancy.

According to an aspect of the present invention, as a small sized peptide, it has excellent skin penetration rate, thereby improving skin whitening and pigmentation inhibition effects.

Due to its physiological activity, the peptide can be used in products for various purposes, such as pharmaceuticals, quasi-drugs, health functional foods, and cosmetics.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a graph showing the intracellular melanin inhibitory effect of a peptide (Example 1) according to an embodiment of the present invention.
2 is a graph showing the intracellular melanin inhibitory effect in melanocytes treated with a peptide (Example 1) according to an embodiment of the present invention.
3 is a graph showing the intracellular melanin inhibitory effect in melanocytes treated by mixing a peptide according to an embodiment of the present invention.

Terms used in the present specification have selected general terms that are currently widely used as possible while considering functions in the present invention, but this may vary depending on the intention or precedent of a technician working in the field, the emergence of new technologies, and the like. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning of the terms will be described in detail in the description of the corresponding invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall contents of the present invention, not a simple name of the term.

Unless otherwise defined, 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 the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in this application. Does not.

The numerical range includes the numerical values defined in the above range. All maximum numerical limits given throughout this specification include all lower numerical limits as if the lower numerical limits were expressly written. All minimum numerical limits given throughout this specification are inclusive of all higher numerical limits as if the higher numerical limits were expressly written. All numerical limits given throughout this specification will include all better numerical ranges within the wider numerical range, as if the narrower numerical limits were expressly written.

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

One aspect of the present invention provides a physiologically active peptide consisting of one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 4.

As a result of diligently trying to discover a peptide having a biologically effective activity, the present inventors have identified the physiological activity of the peptide in vivo, and in particular, the activity of the peptide to inhibit skin whitening and pigmentation.

The peptide is derived from ZDHHC13 in the human body and has high skin permeability due to its small molecular weight and size, and has excellent skin whitening and pigmentation inhibitory effects.

In addition, the physiologically active peptide is a generic term for a peptide having a predetermined physiological action in a living body. By adjusting genetic expression and physiological function, abnormal conditions due to lack or excessive secretion of substances involved in function regulation in the living body It can play a role of correcting when it is shown.

The peptide may consist of one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 4, or may include the amino acid sequence as a part.

The peptide (peptide) refers to a polymer composed of one or more amino acids linked by an amide bond (or a peptide bond). For the purposes of the present invention, the peptide may mean a peptide or a fragment thereof exhibiting an effect of improving wrinkles and improving skin elasticity.

The general rules for naming the peptides may be based on a three-letter or one-letter amino acid code in the absence of specifically indicated exceptions. For example, the central part of the amino acid structure is indicated by a 3-letter code (eg, Ala, Lys), and the D-stereotype (eg, D-Ala, D-Lys) is specifically specified by writing “D-” in front of the 3-letter code. If not indicated, it can be assumed to be in L-stereotype. The amino acid residues constituting the peptide may be natural or non-natural amino acid residues.

The peptide may inhibit the formation of melanin in melanocytes, and may inhibit the activity of tyrosinase.

The melanin is a dark brown pigment that is present in the eyes, skin, and hair, and blocks the penetration of ultraviolet rays in a manner that absorbs more than a certain amount of ultraviolet rays from the body, thereby protecting the body or maintaining body temperature.

However, excess melanin may be secreted by exposure to excessive ultraviolet rays, and skin color may be discolored. Melanin can be produced by a combination of hormones and melanogenic enzymes, including tyrosinase.

The peptide can effectively inhibit the activity of the tyrosinase and inhibit the production of melanin, thereby improving pigmentation and whitening the skin.

The peptide can effectively inhibit pigmentation due to ultraviolet (UV) light and pigmentation due to oxidative stress.

The UV rays can be divided into UV-A, B, and C depending on the length of the wavelength. Of these, UV-A is less irritating to the skin and causes less damage to the skin than UV-B. It causes pigmentation such as age spots and intensifies skin aging, and in severe cases, it can also lead to skin cancer.

In addition, reactive oxygen species caused by oxidative stress is a cause of damage and pigmentation of skin cells, and may cause skin aging by direct loss of functions such as lipids, proteins, nucleic acids, and enzymes constituting skin cells.

The present inventors have found that the peptide can effectively inhibit pigmentation that may be caused by various factors, and through this, various uses have been derived.

The peptides can be prepared according to chemical synthesis methods known in the art, in particular solid-phase synthesis techniques (see Merrifield, J. Amer. Chem. 1963, Soc. 85:2149-54; Stewart, et al. Solid Phase Peptide Synthesis, 2nd.ed., Pierce Chem. 1984, Co.: Rockford, 111).

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

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

The N or C-terminus of the peptide may be an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or a polyethylene glycol (PEG) group. Stability of the peptide may be significantly 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 by protein cleavage enzymes in vivo.

The peptide may contain a functional equivalent of a peptide comprising the amino acid sequence. The "functional equivalent" is a sequence of at least 40% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more of the amino acid sequence as a result of addition, substitution or deletion of amino acids. By having homology, it means a protein having substantially the same physiological activity as the amino acid sequence.

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

The peptide can be produced by extracting a protein in a living body and then treating it with a proteolytic enzyme to reduce molecular weight, or biologically using a gene recombination and protein expression system, or by a chemical synthesis method using a peptide synthesizer. .

According to another aspect of the present invention, there is provided a physiologically active peptide mixture comprising two or more peptides consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4.

The peptide mixture may exhibit more excellent physiological activity in vivo through a synergistic effect between the peptides compared to the case where the peptide is treated alone.

According to another aspect of the present invention, there is provided a cosmetic composition comprising the peptide or the peptide mixture as an active ingredient.

The cosmetic composition may be for skin whitening or for inhibiting pigmentation by ultraviolet (UV) light.

The "skin whitening" suppresses the production of various biomolecules, such as melanin, oxidation-reduction hemoglobin, carotene, and melanoids, which affect skin color, thereby alleviating skin pigmentation phenomena such as blemishes, freckles, and spots. It means the effect of improving skin brightness and uniformity by relieving redness.

The “included as an active ingredient” may mean containing an effective amount capable of exhibiting an effect of improving skin, for example, improving skin whitening and pigmentation by reducing melanin synthesis in the skin.

According to another aspect of the present invention, there is provided a composition for external application for skin comprising the peptide or the peptide mixture as an active ingredient.

The skin external preparation or cosmetic composition may be formulated by a conventional method. Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can refer to the contents disclosed in the formulation of external preparations for skin, and the International cosmetic ingredient dictionary, 6th ed., The cosmetic, Toiletry and Fragrance in the formulation of cosmetic compositions. Association, Inc., Washington, 1995.

Specifically, the cosmetic composition can be prepared in the form of a general emulsion formulation and solubilization formulation. For example, a lotion such as a flexible lotion or a nutritional lotion; Emulsions such as facial lotion and body lotion; Creams such as nourishing cream, moisture cream, and eye cream; essence; Cosmetic ointment; spray; Gel; pack; Sunscreen; Makeup base; Foundations such as liquid type, solid type or spray type; powder; Makeup removers such as cleansing cream, cleansing lotion, and cleansing oil; Alternatively, it may be formulated with a detergent such as a cleansing foam, soap, or body wash, but is not limited thereto. In addition, the external preparation for skin may be formulated as an ointment, patch, gel, cream, or spray, but is not limited thereto.

In each formulation, the cosmetic composition or the external preparation may be appropriately blended with other components within a range that does not impair the object according to the present invention depending on the type of formulation or purpose of use, in addition to the essential components.

The cosmetic composition or external preparation may include a carrier that is generally acceptable, and for example, oil, water, surfactant, moisturizer, lower alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. may be appropriately blended, but limited thereto. It does not become.

The acceptable carrier may vary depending on the formulation. For example, when formulated as an ointment, paste, cream or gel, as a carrier component, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, or their Mixtures can be used

When the cosmetic composition or external preparation is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component, and chlorofluoro Propellants such as rohydrocarbon, propane, butane or dimethyl ether may further be included.

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

When the cosmetic composition or external preparation is formulated as a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester are suspended. First, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.

When the cosmetic composition is formulated with soap, as a carrier component, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolyzate, isethionate, a lanolin derivative, an aliphatic alcohol, vegetable oil, glycerol, sugar, etc. Can be used.

The cosmetic composition or skin external preparation is a fatty substance, organic solvent, solubilizer, thickener, gelling agent, emollient, antioxidant, suspending agent, stabilizer, foaming agent, which are commonly used in the industry, depending on the quality or function of the final product. ), fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic activators, common in cosmetics It may additionally contain adjuvants commonly used in the field of cosmetology or dermatology, such as any other ingredients used as.

However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating pigmentary diseases comprising the peptide or the peptide mixture as an active ingredient.

The "pigmented disease" refers to pigmentation such as freckles, hereditary contralateral pigmentation, reticulosis pigmentation, birthmarks such as congenital pigmentation such as flat birthmarks, pigmented birthmarks, and acquired symptoms such as melasma, senile pigmentation, etc. Pigmentation, and hereditary skin diseases such as vitiligo, Badenburg syndrome, acquired skin diseases such as post-inflammatory white nasal convulsions, unexplained droplets hypomelanin, melasma, skin diseases due to drug treatment such as minocycline, bleo May include mycin, busulfan, zidovudine, and skin diseases that have been metastasized through infection, such as, but is preferably one from the group consisting of melasma, freckles, black spots, birthmarks, spots and gestational hyperpigmentation. More than one can be selected.

The "prevention" means any action that reduces the frequency or severity of pathological symptoms. Prevention can be complete or partial. In this case, it may mean a phenomenon in which the symptoms of pigmented diseases in the individual decrease compared to the case where the composition is not used.

The “treatment” refers to any action that clinically intervenes in order to change the natural process of a target or cell to be treated, and may be performed while a clinical pathology is in progress or to prevent it.

The desired therapeutic effect is to prevent the occurrence or recurrence of the disease, alleviate symptoms, reduce all direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, or It may include alleviating or temporarily alleviating the condition, or improving the prognosis.

The composition may be administered in the form of oral delivery or parenteral delivery. The composition may be administered systemically or locally, and the administration may include oral administration and parenteral administration. The composition may be formulated with an appropriate amount of a pharmaceutically acceptable vehicle or carrier to provide an appropriate dosage form.

The composition may further include a carrier, an excipient, and a diluent used in the preparation of a pharmaceutical composition. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil may be mentioned, but the present invention is not limited thereto.

In addition, the composition may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injectable solutions.

Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient, such as starch, calcium carbonate, sucrose, and lactose, in the compound and its fractions. Or it can be prepared by mixing gelatin or the like. In addition, lubricants such as magnesium stearate and talc may be used in addition to the excipients.

Liquid preparations for oral administration may include suspensions, liquid solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., may be used in addition to simple diluents such as water and liquid paraffin.

Formulations for parenteral administration may be sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories.

The non-aqueous solvent and suspension may be propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or injectable ester such as ethyl oleate. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, and glycerogelatin may be used.

The pharmaceutical composition may be administered to a subject in a pharmaceutically effective amount. The “pharmaceutically effective amount” refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, drug activity, drug Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-used drugs and other factors well known in the medical field.

The pharmaceutical composition may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is preferable to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.

It is apparent that the present invention is further elaborated through the following examples, but the present invention is not limited by the following examples.

Preparation Example: Peptide

Link amide methylbenzhydryl amine resin (147mg, 0.1mmol) was added to 3mL of dimethylformamide (DMF) anhydrous organic solvent and swelled for about 30 minutes.

In 1.5 mL of DMF anhydrous organic solvent, 0.3 mmol of Fmoc-Lys(Boc)-OH (141 mg), carboxyl activator N, N-Diisopropyl carbodiimde (DIC, 48 µl), and addition adjuvant N-Hydrobenzotriazole (HOBt, 40 mg) ) Was added and mixed to activate for about 20 minutes.

After the activated solution and the resin were stirred for 2 hours to react, the reaction solution was filtered, and the resin was washed several times with DMF and methanol, and a kaiser test was performed.

If the kaiser test was positive, the above activation solution was prepared and participated.

If the test result is negative, it is mixed with 3 mL of piperidine solution dissolved in a DMF solvent at a concentration of 20% by weight, reacted for about 15 minutes to remove the N-terminal Fmoc group, and then activated the following amino acids in the same way to activate Reacted with the amino acid bound to.

After the synthesis of the amino acid portion of the last sequence was completed, the N-terminal Fmoc group was removed using 20% by weight piperidine/DMF, and the resin was washed several times with DMF and methanol, and then dried.

The peptide bound to the resin was reacted with cleavage solution (trifluoroacetic acid (80%), triisopropylsilane (5%), Thioanisole (5%), 1,2-Ethanedithiol (5%) Water (5%)) at room temperature (4 hours). ) The side chain protection group of the amino acid was removed and the peptide was isolated from the resin.

Trifluoroacetic acid in the peptide solution was removed by passing nitrogen gas, and diethyl ether cooled to -20°C was added, and then the precipitated peptide was centrifuged (3,000 rpm, 20 minutes). ).

The obtained peptide was purified using reverse phase HPLC (C-18 column C18-120A, 50mm*250mm) (Eluent: water/acetonitrile in 0.1% TFA (gradient): Flow Rate 50mL/min). A material having a molecular weight of 1308 was identified using an ESI-Mass mass spectrometer.

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

division order Remark Example 1 RRRRRRGSQCRNHSHG SEQ ID NO: 1 Example 2 TLKEVLTCSW SEQ ID NO: 2 Example 3 RYHSIVTLPR SEQ ID NO: 3 Example 4 SIFYALRYHSIVTLPR SEQ ID NO: 4

Experimental Example 1: Evaluation of cytotoxicity The toxicity of the peptide synthesized in Preparation Example on cells was measured. The peptide obtained in Preparation Example was suspended in purified water, prepared by concentration, and then cell viability was measured.

B16F10 cells were dispensed into each well of a 96-well plate to become 2 x 10 ³ cells, and then cultured for 18 hours in a cell incubator under conditions of 5% CO ₂ and 37°C.

The obtained samples were treated by concentration and cultured for 48 hours. When conducting a toxicity test for a mixture of peptides, the highest concentration that did not show toxicity in a single peptide was treated.

After removing the medium, WST-1 solution was added, reacted for 30 minutes, and absorbance was measured at 450 nm.

Survival rate (%) Concentration (nM) 0 50 100 200 300 400 500 Example 1 100 102.3 107.4 123.5 135.79 129.2 132.1 Example 2 100 104.1 107.4 123.6 135.8 129.2 132.4 Example 3 100 103.3 107.3 113.1 119.8 114.0 108.8 Example 4 100 106.2 107.7 118.8 123.6 112.7 103.2

division Survival rate (%) Control 100 Positive control 98 Example 1 99 Example 2 97 Example 3 98 Example 4 94 Example 1 + Example 2 93 Example 1 + Example 3 95 Example 1 + Example 4 94 Example 2 + Example 3 93 Example 2 + Example 4 95 Example 3 + Example 4 93 Example 2 + Example 3 + Example 4 94 Example 1 + Example 2 + Example 3 + Example 4 93

Referring to Tables 2 and 3, little cytotoxicity was observed at all concentrations of the sample. The above results suggest that the peptide of the present application is not only harmless to the human body, but also has excellent human safety.

Experimental Example 2: Evaluation of melanin production inhibitory activity

In order to evaluate the melanogenesis inhibitory effect of the peptide synthesized in Preparation Example, melanocyte B16F10 was cultured.

B16F10, a melanocyte, was dispensed into each well of a 6-well plate so as to be 8 x 10 ⁴ cells, and then cultured for 18 hours in a cell incubator under conditions of 5% CO ₂ and 37°C.

After 18 hours, the medium of the B16F10 cells was removed, and the synthesized peptide was treated at the same concentration and cultured for 2 days in a cell incubator under conditions of 5% CO ₂ and 37°C.

After 2 days, cells were recovered from the plate using a scraper and centrifuged for 1 minute to obtain pellet-shaped cells. 1N NaOH was added to the obtained cells and reacted at 95° C. for 10 minutes to dissolve melanin.

After 10 minutes, the amount of melanin was measured at a wavelength of 475 nm using a microplate reader, and the amount of protein was measured at a wavelength of 595 nm using a BCA assay.

Each experimental group was quantified as “absolute amount of melanin / absolute amount of protein”, and the experimental group and the control group were relative quantified.

Α-MSH was used as a melanogenesis inducer, and arbutin was used as a positive control.

division Melanin relative value (%) Control 100 Positive control 52 Example 1 51 Example 2 84 Example 3 77 Example 4 86 Example 1 + Example 2 46 Example 1 + Example 3 41 Example 1 + Example 4 43 Example 2 + Example 3 80 Example 2 + Example 4 82 Example 3 + Example 4 81 Example 2 + Example 3 + Example 4 68 Example 1 + Example 2 + Example 3 + Example 4 27

Referring to Table 4, the peptide of Example 1 had excellent melanin inhibitory activity compared to Examples 2 to 4, and in particular, synergistic activity was implemented with Examples 2 to 4.

In particular, when the peptides of Examples 1 to 4 were simultaneously applied, the melanin inhibitory activity was further increased.

Experimental Example 3: Evaluation of tyrosinase inhibitory activity

In order to evaluate the melanogenesis inhibitory effect of the peptide synthesized in Preparation Example, melanocyte B16F10 was cultured.

CO₂, 37℃ 조건의 세포배양기에서 18시간 동안 배양하였다.Dispense melanocyte B16F10 into each well of a 6-well plate so that it becomes 8 x 10 ⁴ cells, and then 5%

CO ₂ , cultured for 18 hours in a cell incubator at 37°C.

CO₂, 37℃ 조건의 세포배양기에서 2일 동안 배양하였다.After 18 hours, the medium of B16F10 cells was removed, and the synthesized peptide was treated at the same concentration to 5%

CO ₂ , cultured for 2 days in a cell incubator at 37°C.

After 2 days, cells were recovered from the plate using a scraper and centrifuged for 1 minute to obtain pellet-shaped cells. The obtained cells were lysed using RIPA lysis buffer.

L-DOPA and cell lysate were added to a 96-well plate and reacted at 37°C for 1 hour. Then, absorbance was measured at 450 nm.

division Tyrosinase activity relative value (%) Control 100 Positive control 31 Example 1 52 Example 2 77 Example 3 79 Example 4 73 Example 1 + Example 2 44 Example 1 + Example 3 39 Example 1 + Example 4 41 Example 2 + Example 3 70 Example 2 + Example 4 74 Example 3 + Example 4 69 Example 2 + Example 3 + Example 4 63 Example 1 + Example 2 + Example 3 + Example 4 29

Referring to Table 5, the peptide of Example 1 was superior in skin whitening activity compared to Examples 2 to 4, and when applied together with Examples 2 to 4, the skin whitening effect was further enhanced.

In particular, when the peptides of Examples 1 to 4 were simultaneously applied, it was evaluated that the skin whitening activity was the most excellent.

Experimental Example 4: Evaluation of activity to inhibit skin pigmentation by ultraviolet rays

In order to evaluate the effect of inhibiting skin pigmentation by ultraviolet (UV) radiation of the peptide synthesized in Preparation Example, melanocyte B16F10 was cultured.

B16F10, a melanocyte, was dispensed into each well of a 6-well plate so as to be 8 x 10 ⁴ cells, and then cultured for 18 hours in a cell incubator under conditions of 5% CO ₂ and 37°C.

After 18 hours, the medium of the B16F10 cells was removed, and the peptide synthesized in Preparation Example was treated at the same concentration and cultured for 2 days in a cell incubator under conditions of 5% CO ₂ and 37°C.

After 2 days, cells were recovered from the plate using a scraper and centrifuged for 1 minute to obtain pellet-shaped cells.

1N NaOH was added to the obtained cells and reacted at 95° C. for 10 minutes to dissolve melanin.

After 10 minutes, the amount of melanin was measured at a wavelength of 475 nm using a microplate reader, and the amount of protein was measured at a wavelength of 595 nm using a BCA assay.

Thereafter, each experimental group was numerically expressed as "absolute amount of melanin / absolute amount of protein", and the experimental group and the control group were relative quantified.

Cells were irradiated with UV as a melanogenesis inducer, and arbutin was used as a positive control.

division Melanin relative value (%) Control 100 Positive control 48 Example 1 58 Example 2 73 Example 3 79 Example 4 83 Example 1 + Example 2 44 Example 1 + Example 3 39 Example 1 + Example 4 43 Example 2 + Example 3 70 Example 2 + Example 4 72 Example 3 + Example 4 72 Example 2 + Example 3 + Example 4 65 Example 1 + Example 2 + Example 3 + Example 4 27

Referring to Table 6, the peptide of Example 1 was excellent in inhibiting skin pigmentation by ultraviolet rays compared to Examples 2 to 4, and a synergistic effect was confirmed when applied together with Examples 2 to 4.

In particular, when the peptides of Examples 1 to 4 were applied simultaneously, the activity of inhibiting skin pigmentation by ultraviolet rays was further increased.

Experimental Example 5: Evaluation of activity to inhibit pigmentation by oxidative stress

In order to evaluate the effect of inhibiting skin pigmentation by ultraviolet rays of the peptide synthesized in Preparation Example, melanocyte B16F10 was cultured.

B16F10, a melanocyte, was dispensed into each well of a 6-well plate so as to be 8 x 10 ⁴ cells, and then cultured for 18 hours in a cell incubator under conditions of 5% CO ₂ and 37°C.

After 18 hours, the medium of the B16F10 cells was removed, and the peptide synthesized in Preparation Example was treated at the same concentration and cultured for 2 days in a cell incubator under conditions of 5% CO ₂ and 37°C.

After 2 days, cells were recovered from the plate using a scraper and centrifuged for 1 minute to obtain pellet-shaped cells.

1N NaOH was added to the obtained cells and reacted at 95° C. for 10 minutes to dissolve melanin.

After 10 minutes, the amount of melanin was measured at a wavelength of 475 nm using a microplate reader, and the amount of protein was measured at a wavelength of 595 nm using a BCA assay.

Each experimental group was expressed numerically as “absolute melanin/absolute protein”, and the experimental group and the control group were quantitatively quantified.

Cells were treated with H ₂ O ₂ as a melanogenesis inducer, and arbutin was used as a positive control.

division Melanin relative value (%) Control 100 Positive control 59 Example 1 63 Example 2 88 Example 3 89 Example 4 85 Example 1 + Example 2 54 Example 1 + Example 3 48 Example 1 + Example 4 50 Example 2 + Example 3 79 Example 2 + Example 4 77 Example 3 + Example 4 75 Example 2 + Example 3 + Example 4 68 Example 1 + Example 2 + Example 3 + Example 4 39

Referring to Table 6, the peptide of Example 1 was excellent in inhibiting skin pigmentation due to oxidative stress, and synergistic effects were realized when applied together with Examples 2 to 4.

In particular, when the peptides of Examples 1 to 4 were applied simultaneously, the activity of inhibiting skin pigmentation due to oxidative stress was further enhanced.

Experimental Example 6: Evaluation of skin brightness improvement activity

After applying the peptide to the skin, the effect of improving skin brightness was confirmed.

A cosmetic composition of an essence formulation containing the peptide of the preparation example was prepared, and 50 subjects were divided into 10 groups and applied, respectively, and changes in skin brightness were confirmed.

Before and after washing, formulated cosmetics were applied and used daily for 3 weeks. Skin brightness was checked with a LAB measuring device (Konica Minolta CR-300 Chroma Meter).

Then, the difference (L*) between the skin color at the measurement point and the application start point after application of each test substance was calculated according to Equation 1 below, and the results are shown in Table 8 below.

The control group was formulated excluding the peptide, and the positive control group added arbutin instead of the peptide.

The whitening effect is determined by comparing the L* of the sample application site and the control site. When the L* value is about 2, the whitening effect of the deposited pigment is obvious, and when it is about 1.5 or more, it can be determined that there is a whitening effect.

[Equation 1]

ΔL = L value at the time of measurement after application-L value at the start of application

division Brightness change after 3 weeks (ΔL) Control - Positive control 2.8 Example 1 2.5 Example 2 1.8 Example 3 1.6 Example 4 2.0 Example 1 + Example 2 2.9 Example 1 + Example 3 2.8 Example 1 + Example 4 2.7 Example 2 + Example 3 2.2 Example 2 + Example 4 2.1 Example 3 + Example 4 2.0 Example 2 + Example 3 + Example 4 2.4 Example 1 + Example 2 + Example 3 + Example 4 3.3

Referring to Table 8, when the peptide of Preparation Example was applied directly to the skin, skin brightness was significantly improved after 3 weeks, and a synergistic effect was realized when the peptide of Example 1 was applied simultaneously with the peptides of Examples 2 to 4. .

Experimental Example 7: Evaluation of activity to improve spots and freckles

50 female subjects aged 30 years or older (average 40.6±3.5 years old) who have started to produce spots, freckles, or dark spots are selected, and the cosmetic composition of the essence formulation containing the peptide of the above preparation example is applied every morning and evening for 12 weeks. Was applied.

50 subjects were divided into 10 groups of 5 subjects and applied for each experimental group, and a questionnaire evaluation in steps 0 to 5 on whether or not the blackness of the skin was improved at 8 weeks after use (not improved at all: 0 to very improved: 5 ), and the results are shown in Table 9 below.

The control group was formulated excluding the peptide, and the positive control group added arbutin instead of the peptide.

division Blackness improvement (after 8 weeks) Control - Positive control 3.05 Example 1 2.93 Example 2 2.21 Example 3 1.97 Example 4 2.46 Example 1 + Example 2 3.52 Example 1 + Example 3 3.44 Example 1 + Example 4 3.32 Example 2 + Example 3 2.57 Example 2 + Example 4 2.58 Example 3 + Example 4 2.46 Example 2 + Example 3 + Example 4 2.85 Example 1 + Example 2 + Example 3 + Example 4 4.16

Referring to Table 9, the peptides of the present application exhibit an excellent skin whitening effect, and may be usefully used in the treatment of pigmented diseases such as spots and freckles.

In particular, the peptide of Example 1 not only has excellent skin whitening effect, but also has a synergistic effect when applied simultaneously with the peptides of Examples 2 to 4.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and the concept of equivalents thereof should be construed as being included in the scope of the present invention.

SEQUENCE LISTING <110> GENECELLPHARM <120> A ZDHHC13-DERIVED PEPTIDE, AND USES THEREOF <130> P19-40002 <160> 4 <170> PatentIn version 3.2 <210> 1 <211> 16 <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> ARTIFICIAL SEQUENCE <400> 1 Arg Arg Arg Arg Arg Arg Gly Ser Gln Cys Arg Asn His Ser His Gly 1 5 10 15 <210> 2 <211> 10 <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> ARTIFICIAL SEQUENCE <400> 2 Thr Leu Lys Glu Val Leu Thr Cys Ser Trp 1 5 10 <210> 3 <211> 10 <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> ARTIFICIAL SEQUENCE <400> 3 Arg Tyr His Ser Ile Val Thr Leu Pro Arg 1 5 10 <210> 4 <211> 16 <212> PRT <213> ARTIFICIAL SEQUENCE <220> <223> ARTIFICIAL SEQUENCE <400> 4 Ser Ile Phe Tyr Ala Leu Arg Tyr His Ser Ile Val Thr Leu Pro Arg 1 5 10 15

Claims (11)

Bioactive peptide consisting of the amino acid sequence of SEQ ID NO: 1. The method of claim 1,
A peptide that inhibits the formation of melanin in melanocytes. The method of claim 1,
Peptides that inhibit pigmentation caused by ultraviolet (UV) rays. The method of claim 1,
Peptides that inhibit pigmentation caused by oxidative stress. A cosmetic composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. The method of claim 5,
Cosmetic composition for skin whitening or inhibiting pigmentation by ultraviolet (UV) light. The method of claim 5,
A cosmetic composition further comprising a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to 4. Comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient
A pharmaceutical composition for the prevention or treatment of one or more pigmented diseases selected from the group consisting of spots, freckles, black spots, birthmarks, spots and hyperpigmentation during pregnancy. The method of claim 8,
A pharmaceutical composition further comprising a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to 4. delete delete

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