KR102475100B1 - Preparing method of highly functional peptide derived from keratinocyte protein - Google Patents

Abstract

A novel peptide that exhibits skin wrinkle inhibitory activity as a highly functional peptide derived from natural fermentation is disclosed. The present invention provides a tetrapeptide exhibiting skin wrinkle inhibitory activity consisting of an amino acid sequence of glycine (Gly)-glutamin-valine (Valine, Val)-serine (Ser).

Description

Method for producing highly functional peptides derived from keratinocyte protein {PREPARING METHOD OF HIGHLY FUNCTIONAL PEPTIDE DERIVED FROM KERATINOCYTE PROTEIN}

The present invention relates to highly functional peptides, and more particularly to highly functional peptides derived from naturally fermented products.

The skin is the largest tissue in the human body and functions to protect the inside of the body from sunlight, physical and chemical stimuli, and is absolutely necessary for human life maintenance and is constantly regenerated to maintain homeostasis. The skin is composed of epidermis, dermis, and subcutaneous fat in order from the outside. have. Skin is a tissue that covers the entire body and has various functions. Since it has a barrier function between the inside and outside of the body, it has an immune system by the islets of Langerhans and has a moisture control function by the epidermis. In addition, it synthesizes vitamin D with appropriate sunlight and performs the function of lipid storage and excretion through sweat glands. The stratum corneum produced by the normal differentiation process in healthy skin has the function of maintaining moisture in the skin and protecting it from external environmental stimuli. This function is called the skin barrier function and can be said to be the most important role of the epidermis.

One of the areas of greatest interest in cosmetics is aging related to aging, pigmentation, loss of elasticity, skin dryness, hair loss, and lack of luster in hair. The skin undergoes various changes through aging. First, the thickness of the epidermis, dermis, and subcutaneous tissue, which are components of the skin, becomes thinner, and the extracellular matrix (ECM) component that gives skin elasticity changes. As collagen production rapidly decreases with age, it has a close relationship with aging generation, and collagen, elastin, proteoglycans, and glucosaminoglycans ( Glucosaminoglycan), laminin, fibronectin, etc. are oxidized and lose their functions, so the skin loses its elasticity and aging is excessively formed, resulting in aging skin.

The stratum corneum protects our body from the external environment and serves as the frontmost barrier to prevent loss of body fluids to the outside. During the differentiation of keratinocytes, their cell membrane is replaced with a unique structure called the 'cornified envelope'. In the membrane of keratinocytes, various insoluble proteins cross-link to form a structure, and in a state surrounded by a lipid membrane outside the cell, covalent bonds with some lipid components constituting the lipid membrane form a physical barrier to protect against external threats. to protect the human body. In particular, various structural proteins present in keratin tissue are known to play an important role in the formation of epidermal tissue and barrier function, and various skin diseases caused by the absence of structural proteins in keratinocytes exist.

Connective tissue fibers of the extracellular matrix include glial fibers (collagen fibers), reticular fibers, and elastic fibers (elastic fibers), of which collagen accounts for about 70% of skin connective tissue. is mostly formed in fibroblasts of the skin. With age, the collagen content in skin connective tissue is reduced, which is due to the decrease in collagen synthesis and the promotion of decomposition. Therefore, the decrease in collagen biosynthesis and the acceleration of collagen degradation can be said to be the biggest causes of skin aging.

The process of collagen biosynthesis is regulated by many factors involved in the transcription level and the post-translation level, resulting in changes, and collagen degradation is caused by collagenase, which degrades collagen by ultraviolet rays, etc. By promoting the expression of matrix metalloproteases (MMPs) such as collagenase, collagen degradation is promoted, and the collagen content is reduced. In addition, as the transformation of collagen is accelerated by the external environment, the aging of the skin increases and deepens. As a result, the skin aging phenomenon appears due to non-homogenization of cells, loss of elastin, destruction of collagen, decrease and delay of collagen synthesis, and the like. Therefore, although skin aging occurs in the epidermis, it can be seen as a phenomenon that occurs in the dermis rather than in the epidermis.

Various cosmetic compositions are being studied to solve the problem of skin aging, and some of the skin aging reduction effects are showing visible results. For example, retinoids widely used to improve skin aging, melasma, and pigmentation, and in particular, clinical results for removing skin aging for retinol have been reported in various ways, and cosmetics containing retinol can reduce skin aging caused by sunlight. Effectively improved weight, skin sagging, and loss of elasticity.

In recent years, as human lifespan has increased and the standard of living has improved, people's interest in becoming healthy and beautiful has increased. It can be well known that

Domestic skin aging technology has a large technology gap with developed countries, and most of the aging research conducted by the Korean Society of Gerontology focuses on suppression of antioxidant activity as basic research on aging mechanisms, and the overall level of competitiveness in the field of anti-aging science is It is 75% of the world's best technology possessor (USA), and the technology gap is 4.5 years. is very diverse, and areas such as cranial nerves, blood vessels, joints, cognition and perception, cancer, diabetes, and obesity are established as active research areas, but skin-related aging research has insufficient interest, investment, and research compared to other research areas. It is a situation in which systematic and integrated research from the perspective of aging is absent, so improvement is needed.

Recently, as the use of anti-aging products has expanded to young women in their late 20s, they are actively developing and launching anti-aging products. In particular, in order to satisfy the various needs of consumers in the cosmetics industry, it is not limited to the cosmetic field, but is expanding its scope to nutricosmetics, nutritional supplements, aesthetics, high-functional cosmetics, and cosmetic procedures. We are conducting a wide range of research on the topic of aging, from research on the aging mechanism to policy research such as support for policy development related to society, medicine and welfare of the elderly. is investing in

In order to effectively prevent or improve skin aging and aging, it is necessary to develop a new anti-aging composition using raw materials whose efficacy has been accurately verified.

The present invention is to provide a novel peptide that exhibits skin wrinkle inhibitory activity as a highly functional peptide derived from naturally fermented product, and a cosmetic composition for suppressing skin wrinkles containing the same.

In order to solve the above problems, the present invention provides a tetra peptide showing skin wrinkle inhibitory activity consisting of an amino acid sequence of glycine (Gly) -glutamin -valine (Val) -serine (Serine, Ser) do.

In addition, a tetrapeptide showing skin wrinkle inhibitory activity consisting of an amino acid sequence of glycine (Gly)-glutamine-aspartic acid (Asp)-proline (Proline, Pro) is provided.

In addition, the peptide provides a tetra-peptide exhibiting skin wrinkle inhibitory activity, characterized in that it is synthesized using 2- (4-nitrophenyl) sulfonylethoxycarbonyl-amino acid (Nsc-amino acid) as an intermediate.

In addition, the skin wrinkle inhibitory activity provides a tetra-peptide exhibiting skin wrinkle inhibitory activity, characterized in that it appears through elastase inhibition.

In addition, the skin wrinkle inhibitory activity provides a tetra-peptide exhibiting the skin wrinkle inhibitory activity, characterized in that it appears through collagen production.

In order to solve the above another problem, the present invention provides a cosmetic composition for suppressing skin wrinkles containing the peptide.

The tetra-peptide according to the present invention exhibits excellent skin wrinkle inhibitory activity through elastase inhibition or collagen production, and thus can have various applications as a cosmetic composition.

1 is a process diagram showing the entire peptide synthesis process in Example 1 of the present invention.
Figure 2 is a graph showing the results of measuring collagen production in Example 3 of the present invention.
3 to 5 are graphs showing the results of cell proliferation ability test in Example 4 of the present invention.
6 and 7 are graphs showing the HPLC analysis results of Sample 10 and 34 tetra-peptides, respectively, in Example 5 of the present invention.
8 and 9 are graphs showing the thermal and light safety test results in Example 6 of the present invention.

Hereinafter, the present invention will be described in detail through preferred embodiments. Prior to this, the terms or words used in this specification and claims should not be construed as being limited to the usual or dictionary meaning, and the inventor appropriately uses the concept of the term in order to explain his/her invention in the best way. Based on the principle that it can be defined, it should be interpreted as meaning and concept consistent with the technical spirit of the present invention. Therefore, since the configurations of the embodiments described in this specification are merely the most preferred embodiments of the present invention and do not represent all of the technical spirit of the present invention, various equivalents and modifications that can replace them at the time of the present application It should be understood that there may be Also, throughout the specification, when a part "includes" a certain component, it means that it may further include other components, not excluding other components, unless otherwise stated.

The present inventors have found that tetrapeptides having a specific amino acid sequence derived from fermented ginseng fruit exhibit excellent skin wrinkle inhibitory activity through elastase inhibition or collagen production, and have led to the present invention.

Therefore, the present invention is a tetra peptide showing skin wrinkle inhibitory activity consisting of an amino acid sequence (SEQ ID NO: 1) of glycine (Gly)-glutamin-valine (Valine, Val)-serine (Serine, Ser), and glycine Disclosed is a tetrapeptide showing skin wrinkle inhibitory activity consisting of the amino acid sequence (SEQ ID NO: 2) of (Glycine, Gly)-Glutamine-Aspartic acid (Asp)-Proline (Proline, Pro).

In another aspect, the present invention provides a cosmetic composition for suppressing skin wrinkles containing the tetra peptide.

The tetra peptide may be contained in an amount of 0.001 to 10% by weight, preferably 0.01 to 5% by weight, more preferably 0.05 to 1% by weight, based on the total cosmetic composition.

The cosmetic composition according to the present invention may be applied in a gel type, skin type, cream type, ointment type, etc., but is not limited thereto. The above composition can be appropriately prepared by a known method by adding suitable conventional softeners, emulsifiers, thickeners or other materials known in the art according to its type.

The gel-type composition may be prepared by adding a softening agent such as trimethylolpropane, polyethylene glycol or glycerin, a solvent such as propylene glycol, ethanol, or isocetic alcohol, purified alcohol, or the like.

The skin type composition contains fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol and isocetyl alcohol, butylene glycol, glycerin, allantoin, methyl paraben, EDTA- It can be prepared by adding 2-sodium, xanthan gum, dimethicone, polyethylene glycol-60 hydrogenate, castol oil, polysorbate 60, and purified water.

The cream-type composition includes fatty alcohols such as stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, and isocetyl alcohol, lecithin, phosphatidylcholine, phosphatidylethanolamine, sofphatidylserine, sofphatidyl Lipids such as inositol, derivatives thereof, emulsifiers such as glyceryl stearate, sorbitan palmitate, and soribitan stearate, natural fats or oils such as avocado oil, apricot oil, babassu oil, vitreous borage oil, camellia oil, It can be prepared by adding a solvent such as propylene glycol and purified water.

The ointment-type composition may be prepared by adding a softener, an emulsifier, and a wax such as microcrystalline wax, paraffin, ceresin, beeswax, spermaceti, and vaseline.

The cosmetic composition of the present invention may further contain at least one skin wrinkle improvement component exhibiting the same or similar function in addition to the active ingredient. The skin wrinkle improvement component may be at least one selected from the group consisting of vitamin C, retinoic acid, TGF, animal placenta-derived protein, betulinic acid, and chlorella extract, but is not limited thereto. In addition, the cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrotropes inducing substances, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, and the like. can

In the present invention, the cosmetic composition is softening lotion, astringent lotion, nutrient lotion, eye cream, serum, nutrient cream, massage cream, cleansing cream, cleansing lotion, cleansing foam, cleansing water, powder, essence, pack, hair tonic, hair treatment It can be formulated as a ment, shampoo or rinse.

The cosmetic composition may be formulated by conventional methods. In the formulation of external skin preparations, reference may be made to the contents disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA, and in the formulation of cosmetic compositions, International Cosmetic Ingredient Dictionary, 6th ed (The cosmetic, Toiletry and Fragrance Association , Inc, Washington, 1995) may be referred to.

Specifically, the cosmetic composition may be prepared in a general emulsified formulation and solubilized formulation. For example, cosmetic lotion, such as softening lotion or nourishing lotion; emulsions such as facial lotion and body lotion; creams such as nourishing creams, moisture creams, and eye creams; 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 creams, cleansing lotions, and cleansing oils; Alternatively, it may be formulated as a cleanser such as a cleansing foam, soap, body wash, etc., but is not limited thereto. In addition, the skin external preparation may be formulated as an ointment, patch, gel, cream or spray, but is not limited thereto.

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

The cosmetic composition may include a conventionally acceptable carrier, and for example, oil, water, a surfactant, a moisturizer, a lower alcohol, a thickener, a chelating agent, a colorant, a preservative, a flavoring agent, and the like may be appropriately blended, but are not limited thereto. not.

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

When the cosmetic composition 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 in the case of a spray, chlorofluoro It may further contain a propellant such as hydrocarbon, propane, butane or dimethyl ether.

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

When the cosmetic composition is formulated as a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like can be used.

The cosmetic composition includes fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, and fragrances commonly used in the industry depending on the quality or function of the final product. , surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives commonly used in cosmetics It may additionally contain adjuvants commonly used in cosmetology or dermatology, such as any other ingredient. However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect desirable properties of the cosmetic composition according to the present invention.

Hereinafter, specific embodiments according to the present invention will be described.

Preparation Example: Preparation of fermented ginseng fruit

2 kg of purified water was added to 200 g of dried ginseng berries, and hot water extraction was performed at 120° C. for 6 hours. After extraction, ginseng fruit extract having a sugar content of 6 to 8 birx was used. After extraction, a strain of Lactobacillus paracasei (purchased from the Korea Cell Line Bank) was cultured, and the ginseng fruit extract was inoculated with 1% by weight of the culture medium having a concentration of 1.0×10 ⁹ cfu/ml as measured by a spectrophotometer, and 37° C. It was fermented for 3 days, and the medium used for the culture was a medium composed of glucose 0.6% by weight, enzyme extract 0.3% by weight, soytone 0.1% by weight and distilled water. After fermentation, the fermentation broth was centrifuged at 4,000 rpm for 10 minutes to separate the lactic acid bacteria and the culture medium, and then the culture medium was filtered through a 0.2 μm filter to prepare a fermented ginseng fruit product.

Example 1: Synthesis of Peptide Library

A total of 42 peptide sequences were derived by purification and separation from the prepared fermented ginseng fruit, and the sequences of the peptides are shown in Table 1 below.

To construct a peptide library for synthesizing the peptides in Table 1, 19 nsc-amino acids (nsc-Ala, nsc-Arg (pbf), nsc-Asp (OtBu), nsc-Asn (trt), nsc-Gly, nsc -Glu(OtBu), nsc-Gln(trt), nsc-His(trt), nsc-Ser(tBu), nsc-Thr(tBu), nsc-Tyr(tBu), nsc-Trp(Boc), nsc- Chloro trityl chloride resin (CTL resin, Novabiochem Cat No. 01-64-0021) into a 96-well Teflon reactor in each of 19 lines by series, 50 mg was added, 1 ml of methylene chloride (MC) was added, and the mixture was stirred for 3 minutes. The solution was removed, 1 ml of dimethylformamide (DMF) was added, stirred for 3 minutes, and then the solvent was removed again. The prepared tranexamic acid-peptidyl resin was washed three times each with DMF, MC, and methanol, dried by slowly flowing nitrogen air, and then completely dried under reduced pressure in vacuum under P2O5. To the prepared resin, a fugitive solution [trifluroacetic acid (TFA) 81.5%, distilled water 5%, thioanisole 5%, phenol 5%, EDT (1,2-Ethanedithiol) 2.5% and TIS (Triisopropylsilane) 1% included] 30 ml was added, and the reaction was maintained for 1 hour in an ice bath with occasional shaking at room temperature. The resin was filtered, washed with a small amount of TFA solution and combined with the mother liquor. After this, a tetra peptide was obtained. An overall schematic diagram of peptide synthesis is shown in FIG. 1 .

As a result of the above synthesis and purification, 42 kinds of tetrapeptides having different sequences were obtained (Table 1). The yield of the synthesized tetra-peptide differed depending on the difference in physical properties according to the sequence of the peptide, but showed an average yield of about 20%. The elution time during column separation was also different, but a peak was identified in the 6 to 8 minute range.

Sample order Sample order One MSYQ 22 FSSG 2 QKKQ 23 GQLE 3 PTPQ 24 ELPE 4 PPVD 25 EQQE 5 DCVK 26 LELP 6 VKTS 27 GQLK 7 KTSG 28 KHLE 8 SGGS 29 EHQE 9 GSGY 30 EGQL 10 GQVS 31 LEVP 11 GYVS 32 PEEQ 12 SQQV 33 QMGQ 13 TQTS 34 GQDP 14 CAPQ 35 QLKY 15 QPSY 36 YLEQ 16 YGGG 37 EQQE 17 SSGG 38 TKGE 18 GGSG 39 VLLP 19 GSSG 40 VEHQ 20 GCFS 41 QQKQ 21 CFSS 42 EVQW

Example 2: Screening of tetrapeptides

In order to confirm the skin wrinkle inhibitory effect of the tetra peptide, an elastase inhibition test was conducted. Using N-succinyl-(L-Aal)3-p-nitroanilide as a substrate, the amount of p-nitroanilide produced was measured at 37°C for 30 minutes. 100 μg/ml of each test peptide and negative and positive controls were taken in a test tube, 50 μg/ml of pancreatic solution (Type I: derived from porcine pancreas, 0.6 unit/ml, Sigma Aldrich, USA) was added, and 50 mM as a substrate 100 μg/ml of N-succinyl-(LAla)3-p-nitroanilide (1 mg/ml) dissolved in Tris-HCl buffer (pH 8.6) was added, reacted for 30 minutes, and absorbance was measured at 410 nm using a microplate reader. measured. Elastase inhibitory activity was expressed as the absorbance decrease rate of the sample solution added and unadded as shown in Equation 1 below.

[Equation 1]

After treating 42 kinds of synthetic peptides, the elastase activity inhibitory effect was measured, and the results are shown in Table 2 below.

Sample Elastase inhibition rate
(%) Sample Elastase inhibition rate (%) One 79.4 22 69.1 2 88.7 23 73.2 3 69.3 24 90.5 4 72.5 25 53.6 5 118.4 26 78.2 6 66.3 27 56.0 7 44.5 28 79.3 8 49.6 29 66.3 9 56.3 30 69.7 10 33.9 31 70.6 11 78.5 32 64.0 12 49.3 33 55.6 13 59.0 34 34.5 14 77.2 35 60.5 15 63.5 36 72.3 16 67.1 37 64.2 17 77.0 38 59.3 18 103.5 39 60.7 19 58.3 40 55.7 20 55.2 41 72.3 21 47.4 42 49.5 untreated group 100 positive control
(Ursonic acid) 28.8

Referring to Table 2, synthetic tetrapeptides with excellent elastase inhibitory ability (%) were found to be superior to Ursonic acid, an existing elastase inhibitory functional material. From these results, it is confirmed that when a factor inducing wrinkle formation is applied to the skin, the synthesized tetrapeptide inhibits wrinkle formation through an elastase inhibitory effect and is effective in preventing skin aging.

Candidate peptides showing good titer by screening were divided into separate groups, and experiments were performed after mass synthesis.

Example 3: Intracellular collgen production ability test of tetrapeptide

The intracellular collagen production ability test was conducted on 2 types of samples, 10 samples and 34 samples selected in the elastase inhibitory test. Human dermal fibroblast (HDF) cells to be used for the test were prepared by adding 1X LSGS (Low Serum Growth Supplement, cascade biologics S-003-10) to Medium 106 (cascade biologics, M-106-500). ℃, 5% CO ₂ It was cultured under conditions. After culturing the cells in a 24-well plate at a concentration of 1X 10 ⁵ cells/well and confirming cell adhesion, the control group was treated without any treatment and only the solvent was added. In the dish, 4 kinds of experimental groups and 10 μg/ml of TGF were added to the control group. The treatment was performed to a concentration of ml. After adding the test substance to each dish, it was cultured for 2 days. After 2 days, the medium was collected and collagen production was measured using the Procollagen Type I C-peptide (PIP) EIA kit (takara MK101), and the results are shown in FIG. 2 .

Referring to FIG. 2, as a result of measuring collagen production after treating the two experimental groups by concentration, collagen production was insufficient in most experimental groups compared to TGF, but higher collagen production was confirmed than that of the negative control group. From these results, it is confirmed that collagen production in the skin can be suppressed by increasing collagen production according to the sequence of the tetrapeptide compared to the case of using a single raw material.

Example 4: Test for confirming cell proliferation promoting ability of tetra peptide

In order to confirm the cell proliferation ability of the two tetrapeptides selected in Examples 2 and 3, experiments were conducted in the following manner, and the results are shown in FIGS. 3 to 5. Cell proliferation ability test was measured using MTT assay.

3T3 cells, HACAT cells, and DP cells were seeded in a 24-well plate at a cell number of 4 × 10 ⁴ cells/24well per well, and then incubated for 24 hours at 37° C. under 5% CO ₂ conditions. After washing twice with PBS (phosphate buffer saline), each sample was treated with 10 ug/ml and 100 ng/ml of TGF-β (Transforming Growth Factor-β) as a control, followed by incubation for 24 hours. After cultivation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 0.5% in DPBS (Dulbecco's Phosphate-Buffered Saline) was mixed with the culture medium at a ratio of 1:9 (v/v). After addition, incubate in a CO ₂ incubator for 2 hours. Then, the produced formazan was dissolved in dimethyl sulfoxide (DMSO) and absorbance was measured at 570 nm using ELISA.

Referring to Figures 3 to 5, for HACAT cells, 3T3 cells and DP cells, the two peptides treated from low to high concentrations were confirmed to have cell proliferation ability in all skin cells in a concentration-dependent manner, and microscopic observation of the cells No significant change was observed in either.

Through this, it can be seen that the tetra peptide according to the present invention has no toxicity to skin-related cells, and appears to have a skin cell proliferation effect. Therefore, it can be predicted that the cosmetic composition containing the tetra peptide according to the present invention will not have a significant effect even if the skin is treated.

Example 5: HPLC analysis of tetra peptides

HPLC analysis conditions for accurately analyzing and quantifying the two selected tetrapeptides were established.

Each peptide was analyzed using waters HPLCwaters 2695) and waters C18 (Waters Xterra MS C18 column (L: 250 mm, LD: 4.6 mm, 5 μm)) column. At this time, the wavelength of the HPLC detector was 216 nm. Acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) and water containing 0.1% TFA were used as solvents for the mobile phase of HPLC, and by changing the concentration of ACN over time, peptide The purity analysis was observed, and the results are shown in FIGS. 6 and 7. Conditions required for peptide material analysis are shown in Table 3 below.

Condition of HPLC analysis Column waters Xterra MS column (L : 250 mm, LD : 4.6 mm, 5 μm) Detector waters 2487 dual absorbance detector Detection wavelength 216 nm Flow rate 1.0 mL/min Injection volume 50 µL Mobile phase
conditions for HPLC gradient-elution Program order Time (min) 0.1% TFAs
in DW (%) 0.1% TFAs
in ACN (%) One 0 100 0 2 10 75 25 3 20 50 50 4 30 25 75 5 40 0 100 6 45 100 0

Referring to FIGS. 6 and 7, as a result of HPLC analysis, it was confirmed that two types of tetra peptides could be accurately identified under the above HPLC analysis conditions and the correct concentrations thereof were known.

Example 6: Confirmation of thermal and light stability of tetra peptide

In order to confirm the heat and light stability of the two tetrapeptides, experiments were conducted in the following manner, and the results are shown in FIGS. 8 and 9.

Dissolve each sample in 50 mM Tris-HCl (pH 8.0) buffer so that each sample is 10 mg / ml, dispense into glass vials, store at 50 ° C for 4 weeks, and measure the loss of peptides in the composition due to heat and the stability was measured under daylight conditions in the same way.

9 and 10, as a result of measuring the loss of peptides due to heat and measuring the stability under daylight conditions, only less than 10% of the peptides were lost for up to 4 weeks in two tetrapeptides, indicating that the heat and sunlight storage period It was confirmed that it has high stability.

Example 7: Preparation of cosmetic composition containing tetra peptide

A cream-type composition containing tetra peptide was prepared by the following method. As a cream-type composition, a formulation was configured as shown in Table 4 below, including 1 part by weight of the tetra peptide of Sample 10.

1) [Prime B] Manufacturing

While warming water to 60 to 70 ° C. while stirring, powdery raw materials are gradually added sequentially, stirred with a disper mixer until thickened, evenly dissolved, then cooled, and prepared in advance for ease of processing.

2) [Phase A] Manufacturing

After sufficiently impregnating the powdery raw material in glycerin, water is added and heated to 50 to 60° C. to dissolve evenly with a disper mixer.

3) [Phase A] + [Phase B] mixing and stirring preparation

Water phase B prepared in advance is added to water phase A and warmed while mixing evenly. Prepare so that 65 to 75 ° C can be maintained.

4) [Oil Phase] Mixing and Stirring Manufacturing

Weigh each raw material in an individual beaker and dissolve it by heating and stirring (at least 80 ° C). The melted phase is prepared so that the temperature can be maintained above 75 ℃.

5) Whole Cream Preparation

The oil phase is gradually added to the prepared water phase mixture, and the emulsification process is performed by vigorously stirring (6,000 rpm) for about 10 minutes. At this time, the temperature is maintained at 65 ~ 75 ℃.

After completion, additive A is prepared to proceed with pH adjustment and neutralization process. At this time, since the viscosity of the contents rises, stir vigorously (6,000 rpm) evenly for 5 minutes or more.

When the contents cool down below 50℃, add additive B and mix slowly for 2 minutes (3,000~4,000 rpm). After stirring, the contents are collected through a degassing/filtration process.

After completion, the pH adjustment and neutralization process proceeds. At this time, since the viscosity of the contents rises, stir vigorously (6,000 rpm) evenly for more than 5 minutes.

When the contents cool down below 50℃, add a preservative and slowly mix for 2 minutes (3,000~4,000rpm). When the temperature is below 40℃, add tetra peptide and flavoring, stir slowly for 2 minutes (3,000~4,000rpm), and degas /Collect the contents through the filtration process.

The prepared cream was stored at low temperature (4℃), high temperature (50℃), room temperature (25℃), and sunlight conditions to check the stability of the cream. Discoloration, odor change, phase change, and pH change of the cream for 4 weeks under all conditions. The same phenomenon was not found, and it was confirmed that the stability of the cream was maintained through this. In particular, the stability of the tetra peptide was maintained, and as a result of HPLC analysis, it was confirmed that the content was maintained at 90% or more for 4 weeks under all conditions.

division Raw material name / ingredient name Ingredients / INCI Name parts by weight Award A DI WATER Water 48.04 GLYCERIN Glycerin 10.00 Keltrol-F Xanthan Gum 0.10 Adenosine Adenosine 0.04 Award B DI Water Water 30.00 beta-glucan beta-glucan 0.20 Carbopol 940 Carbomer 0.02 Paid Kalcol 6870 Cetearyl Alcohol 2.50 GMS-105 Glyceryl Stearates 1.50 Arlacel 165V Glyceryl Stearate, Peg-100 Stearate 1.20 Tween 80 Polysorbate 80 1.00 Arlacel 83 Sorbitan Sesquioleate 0.70 Phytosqualane Squalane 5.00 DUB MCTs Caprylic/Capric Triglycerides 3.00 Additive A DI Water Water 3.00 L-Arginine (pH 4.50 ~ 5.50) Arginine 0.50 Additive B 1,2-Hexanediol 1,2-Hexanediol 2.00 Additive C Lutein Solution Lutein 1.00 Additive D (Fragrance) Perfume Fragrance 0.10 post-appointed peptide GQVS peptide 0.1

Above, preferred embodiments of the present invention have been described in detail. The description of the present invention is for illustrative purposes, and those skilled in the art will understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention.

Therefore, the scope of the present invention is indicated by the following claims rather than the above detailed description, and all changes or modifications derived from the meaning, scope and equivalent concept of the claims are included in the scope of the present invention. should be interpreted

<110> A & PEP INC. <120> PREPARING METHOD OF HIGHLY FUNCTIONAL PEPTIDE DERIVED FROM KERATINOCYTE PROTEIN <130> NP20-11155 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 4 <212> PRT <213> artificial sequence <220> <223> Tetra Peptide <400> 1 Gly Gln Val Ser One <210> 2 <211> 4 <212> PRT <213> artificial sequence <220> <223> Tetra Peptide <400> 2 Gly Gln Asp Pro One

Claims (6)

A cosmetic composition for suppressing skin wrinkles containing as an active ingredient a tetrapeptide showing skin wrinkle inhibitory activity consisting of an amino acid sequence of Glycine (Gly)-Glutamine-Valine (Val)-Serine (Ser) . For suppressing skin wrinkles containing as an active ingredient a tetra peptide showing skin wrinkle inhibitory activity consisting of the amino acid sequence of Glycine (Gly) - Glutamine - Aspartic acid (Asp) - Proline (Proline) cosmetic composition.
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