PREPARING METHOD OF HIGHLY FUNCTIONAL PEPTIDE DERIVED FROM KERATINOCYTE PROTEIN

Abstract

Disclosed is a novel peptide, which is a highly functional peptide derived from a natural fermentation product exhibiting an anti-wrinkle activity. A tetrapeptide consists of an amino acid sequence of glycine (Gly)-glutamine (Gln)-valine (Val)-serine (Ser) (SEQ ID NO: 1). The peptide is synthesized by utilizing 2-(4-nitrophenyl)sulfonylethoxycarbonyl-amino acid (Nsc-amino acid) as an intermediate. The anti-wrinkle activity is exhibited through the inhibition of elastase. The anti-wrinkle activity is exhibited through collagen synthesis.

Claims

1. A tetrapeptide exhibiting an anti-wrinkle activity consisting of the amino acid sequence glycine (Gly)-glutamine (Gln)-valine (Val)-serine (Ser) (SEQ ID NO: 1), wherein the tetrapeptide is prepared by a method comprising: obtaining a ginseng fruit extract; fermenting the ginseng fruit extract to obtain a fermented ginseng fruit extract; constructing a peptide library comprising a 2-(4-nitrophenyl)sulfonylethoxycarbonyl-amino acid; and synthesizing the tetrapeptide of SEQ ID NO: 1 by combining the peptide library with the fermented ginseng fruit extract.

2. (canceled)

3. (canceled)

4. The tetrapeptide of claim 1, wherein the anti-wrinkle activity is exhibited through the inhibition of elastase.

5. The tetrapeptide of claim 1, wherein the anti-wrinkle activity is exhibited through increasing collagen synthesis.

6. An anti-wrinkle composition comprising the tetrapeptide of claim 1, wherein the composition is: a cosmetic in a form selected from one or more from the group consisting of a lotion, emulsion, cream essence, makeup ointment, spray, gel, pack, sun blocking agent, makeup base, foundation, powder, makeup remover, and cleansing agent; or a preparation in a form selected from the group consisting of an ointment, a patch, a gel, a cream, and a spray.

7. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. A method of manufacturing the tetrapeptide of claim 1 comprising: obtaining a ginseng fruit extract; fermenting the ginseng fruit extract to obtain a fermented ginseng fruit extract; constructing a peptide library comprising a 2-(4-nitrophenyl)sulfonylethoxycarbonyl-amino acid; synthesizing the tetrapeptide of SEQ ID NO: 1 by combining the peptide library with the fermented ginseng fruit extract.

12. The method of claim 11, wherein the step of obtaining a ginseng fruit extract comprises hot water extraction.

13. The method of claim 11, wherein the step of fermenting comprises adding a Lactobacillus paracasei strain to the ginseng fruit extract.

14. A method of inhibiting wrinkle formation comprising: a step of topically applying the anti-wrinkle composition of claim 6 to skin, thereby increasing collagen synthesis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a flowchart illustrating the overall peptide synthesis process in Example 1 of the present invention.

[0023] FIG. 2 is a graph illustrating the measurement results of collagen production in Example 3 of the present invention.

[0024] FIGS. 3 to 5 are graphs illustrating the experimental results of the cell proliferation ability in Example 4 of the present invention.

[0025] FIGS. 6 and 7 are graphs illustrating the HPLC analysis results of the tetrapeptides of Sample 10 and Sample 34 in Example 5 of the present invention, respectively.

[0026] FIGS. 8 and 9 are graphs illustrating the experimental results of thermal stability and photostability in Example 6 of the present invention.

DETAILED DESCRIPTION

[0027] Hereinafter, the present invention will be described in detail through preferable embodiments. First of all, it will be understood that words or terms used in the specification and claims shall not be interpreted as the meaning defined in commonly used dictionaries. It will be further understood that the words or terms should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the technical idea of the invention, based on the principle that an inventor may properly define the meaning of the words or terms to best explain the invention. Therefore, the features of the embodiments and drawings described herein are merely the most preferable exemplary embodiment for the purpose of illustrations only, not intended to represent all the technical concepts of the disclosure, and thus it should be understood that various modifications and equivalents could be made thereto at the time of present application. Furthermore, in the entire specification, when it is described that one part “includes” some components, it does not mean that other components are excluded but means that other elements may be further included if there is no specific contrary description.

[0028] The present inventors have found that a tetrapeptide with a specific amino acid sequence derived from a fermented product of a ginseng fruit exhibits an excellent anti-wrinkle activity through inhibition of elastase or collagen production, thereby leading to the present invention.

[0029] Accordingly, the present invention discloses a tetrapeptide exhibiting an anti-wrinkle activity consisting of an amino acid sequence of glycine (Gly)-glutamine (Gln)-valine (Val)-serine (Ser) (SEQ ID NO: 1) and a tetrapeptide exhibiting an anti-wrinkle activity consisting of an amino acid sequence of glycine (Gly)-glutamine (Gln)-aspartic acid (Asp)-proline (Pro) (SEQ ID NO: 2).

[0030] In another aspect, the present invention provides an anti-wrinkle cosmetic composition containing the tetrapeptide above.

[0031] The tetrapeptide may be contained in an amount of 0.001 wt % to 10 wt %, preferably 0.01 wt % to 5 wt %, and more preferably 0.05 wt % to 1 wt %, based on the total cosmetic composition.

[0032] The cosmetic composition according to the present invention may be applied to a gel type, skin type, cream type, ointment type, etc., but is not limited thereto. The composition may be appropriately prepared by a known method by adding an appropriate conventional softening agent, emulsifying agent, thickening agent, or other materials known in the art according to their type.

[0033] The gel-type composition may be prepared by adding an emollient (e.g., trimethylolpropane, polyethylene glycol, glycerin, etc.), a solvent (e.g., propylene glycol, ethanol, and isostatic alcohol, etc.), purified water, etc.

[0034] The skin-type composition may be prepared by adding fatty alcohols (e.g., stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, isocetyl alcohol, etc.), butylene glycol, glycerin, allantoin, methylparaben, EDTA-2-sodium, xanthan gum, dimethicone, polyethylene glycol-60 hydrogenated castor oil, polysorbate 60 and, purified water, etc.

[0035] The cream-type composition may be prepared by adding fatty alcohols (e.g., stearyl alcohol, myristyl alcohol, behenyl alcohol, arachidyl alcohol, isostearyl alcohol, isocetyl alcohol, etc.), lipids (e.g., lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and derivatives thereof; emulsifying agents (e.g., glyceryl stearate, sorbitan palmitate, sorbitan stearate, etc.); natural fats or oils (e.g., avocado oil, apricot oil, babassu oil, borage oil, camellia oil, etc.); solvents (e.g., propylene glycol, etc.); purified water, etc.

[0036] The ointment-type composition may be prepared by adding an emollient, an emulsifying agent, and a wax (e.g., microcrystalline lead, paraffin, ceresin, beeswax, spermaceti, vaseline, etc.).

[0037] The cosmetic composition of the present invention may further contain one or more wrinkle-improving ingredients exhibiting the same or similar function in addition to the active ingredients. The wrinkle-improving ingredient may be any one or more selected from the group consisting of vitamin C, retinoic acid, TGF, animal placenta-derived proteins, betulinic acid, and a chlorella extract, but is not limited thereto. In addition, the cosmetic composition of the present invention may further contain excipients including a fluorescent material, a fungicide, a hydrotrope, a humectant, a fragrance, a fragrance carrier, a protein, a solubilizer, a sugar derivative, a sun blocking agent, vitamins, plant extracts, etc.

[0038] In the present invention, the cosmetic composition may be formulated into an emollient lotion, an astringent lotion, a nourishing lotion, an eye cream, a serum, a nourishing cream, a massage cream, a cleansing cream, a cleansing lotion, a cleansing foam, a cleansing water, a powder, an essence, a pack, a hair tonic, a hair treatment, a shampoo, or a conditioner.

[0039] The cosmetic composition may be formulated by a conventional method. With regard to the formulation of an external skin preparation, reference may be made to the contents disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton Pa.; and with regard to the formulation of a cosmetic composition, reference may be made to the contents disclosed in the International cosmetic ingredient dictionary, 6th ed. (The cosmetic, Toiletry and Fragrance Association, Inc., Washington, 1995).

[0040] Specifically, the cosmetic composition may be prepared into general emulsified formulations and solubilized formulations. For example, the cosmetic composition may be prepared into a lotion (e.g., an emollient lotion or a nourishing lotion); an emulsion (e.g., a facial lotion, a body lotions, etc.); a cream (e.g., a nourishing cream, a moisturizing cream, an eye cream, etc.); an essence; a makeup ointment; a spray; a gel; a pack; a sun blocking agent; a makeup base; a foundation (e.g., a liquid type, a solid type, a spray type, etc.); a powder; a makeup remover (e.g., a cleansing cream, a cleansing lotion, a cleansing oil, etc.); or a cleansing agent (e.g., a cleansing foam, a soap, a body wash, etc.), but is not limited thereto. In addition, the external preparation for skin may be formulated into an ointment, a patch, a gel, a cream or spray, but is not limited thereto.

[0041] In the cosmetic composition, in addition to the essential ingredients in each formulation, other ingredients may appropriately be mixed within the range that does not impair the purpose of the present invention according to the type of formulation, purpose of its use, etc.

[0042] The cosmetic composition may include a conventionally acceptable carrier, for example, an oil fraction, water, a surfactant, a humectant, a lower alcohol, a thickener, a chelating agent, a colorant, a preservative, a fragrance, etc. may appropriately be mixed, but is not limited thereto no.

[0043] The acceptable carrier may vary depending on the formulation. For example, when the composition is formulated into an ointment, paste, cream, or gel, an animal oil, a vegetable oil, a wax, paraffin, starch, tragacanth, a cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, or a mixture thereof may be used.

[0044] When the cosmetic composition is formulated into a powder or spray, as a carrier component, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used. In the case of a spray, a propellant (e.g., chlorofluorohydrocarbon, propane, butane, and dimethyl ether) may be further included.

[0045] When the cosmetic composition is formulated into a solution or emulsion, as a carrier component, a solvent, a solubilizing agent, or an emulsifying agent may be used. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, and 1,3-butylglycol oil may be used; and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fatty esters, or fatty acid esters of polyethylene glycol or sorbitan may be used.

[0046] When the cosmetic composition is formulated into a suspension, as a carrier component, a liquid diluents (e.g., water, ethanol, and propylene glycol); a suspending agent (e.g., ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters, and polyoxyethylene sorbitan esters); microcrystalline cellulose; aluminum metahydroxide; bentonite; agar or tragacanth, etc. may be used.

[0047] The cosmetic composition, depending on the quality or function of the final product, may further include a fatty material, an organic solvent, a solubilizing agent, a thickening agent, a gelling agents, a softening agent, an antioxidant, a suspending agent, a stabilizing agent, a foaming agent, a fragrance, a surfactant, water, an ionic or non-ionic type emulsifying agent, a filler, a sequestering agent, a chelating agent, a preservative, a blocking agent, a humectant, an essential oil, a dye, a pigment, a hydrophilic or lipophilic activating agent commonly used in the industry; adjuvants commonly used in the field of cosmetology or dermatology, such as any other ingredients commonly used in cosmetics. However, the adjuvant and a mixing ratio thereof may appropriately be selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

[0048] Hereinafter, the present invention will be described by way of specific embodiments according to the present invention.

Preparation Example: Preparation of Fermented Product of Ginseng Fruit

[0049] After adding purified water (2 kg) to dried ginseng fruit (200 g), hot water extraction was performed at 120° C. for 6 hours. After extraction, a ginseng fruit extract having a sugar content of 6 to 8 birx was used. After the extraction, a Lactobacillus paracasei strain (purchased from the Korean Cell Line Bank (KCLB)) was cultured and the liquid culture was inoculated into the ginseng fruit extract in an amount of 1 wt % at a concentration of 1.0×10.sup.9 cfu/mL (measured by a spectrophotometer), and allowed to ferment at 37° C. for 3 days. The medium used for the cultivation was a medium consisting of glucose (0.6 wt %), an enzyme extract (0.3 wt %), soytone (0.1 wt %), and distilled water. After the fermentation, the fermentation liquid was centrifuged at 4,000 rpm for 10 minutes to separate lactic acid bacteria and a culture thereof, and then the culture was filtered through a 0.2 μm filter to prepare a fermentation product of ginseng fruit.

Example 1: Synthesis of Peptide Library

[0050] The fermentation product of ginseng fruit was purified and isolated, and a total of 42 peptide sequences were derived therefrom, and these peptide sequences are shown in Table 1 below.

[0051] In order to construct a peptide library for synthesizing the peptides of Table 1, a chlorotrityl chloride resin (CTL resin, Novabiochem Cat. No. 01-64-0021), to which 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-Leu, nsc-Ile, nsc-Val, nsc-Phe, nsc-Met, nsc-Lys(Boc), and nsc-Pro) are attached, was in series into a 96-well Teflon reactor in an amount of 50 mg for each of 19 lines, and methylene chloride (MC, 1 mL) was added thereto, stirred for 3 minutes. After removing the solution, 1 mL of dimethylformamide (DMF) was added thereto, and the mixture was stirred for 3 minutes, and the solvent was removed again. The prepared tranexamic acid-peptidyl resin was washed 3 times with DMF, MC, and methanol, respectively, dried by slowly flowing nitrogen air thereto, and then dried completely by reducing under vacuum in the presence of P.sub.2O.sub.5. After adding 30 mL of an eluting solution (containing trifluroacetic acid (TFA) (81.5%), distilled water (5%), thioanisole (5%), phenol (5%), 1,2-ethanedithiol (EDT) (2.5%), and triisopropylsilane (TIS, 1%) to the prepared resin, and the reaction was maintained for one 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. Thereafter, a tetrapeptide was obtained. The overall schematic diagram for the peptide synthesis is shown in FIG. 1.

[0052] As a result of the synthesis and purification above, 42 types of tetrapeptides having different sequences were obtained (Table 1). Although the yield of the synthesized tetrapeptides varied depending on the difference in physical properties due to the difference in the sequences of the peptides, the average yield was about 20%. From the column separation, it was confirmed that the elution time also varied, but the peaks were shown in the region of 6-8 minutes.

TABLE-US-00001 TABLE 1 Sample Sequence SEQ ID NO 1 MSYQ 3 2 QKKQ 4 3 PTPQ 5 4 PPVD 6 5 DCVK 7 6 VKTS 8 7 KTSG 9 8 SGGS 10 9 GSGY 11 10 GQVS 1 11 GYVS 12 12 SQQV 13 13 TQTS 14 14 CAPQ 15 15 QPSY 16 16 YGGG 17 17 SSGG 18 18 GGSG 19 19 GSSG 20 20 GCFS 21 21 CFSS 22 22 FSSG 23 23 GQLE 24 24 ELPE 25 25 EQQE 26 26 LELP 27 27 GQLK 28 28 KHLE 29 29 EHQE 30 30 EGQL 31 31 LEVP 32 32 PEEQ 33 33 QMGQ 34 34 GQDP 2 35 QLKY 35 36 YLEQ 36 37 EQQE 37 38 TKGE 38 39 VLLP 39 40 VEHQ 40 41 QQKQ 41 42 EVQW 42

Example 2: Screening of Tetrapeptides

[0053] In order to confirm the anti-wrinkle effect of tetrapeptides, a test of inhibiting elastase was performed. The amount of p-nitroanilide produced was measured using N-succinyl-(L-Aal)3-p-nitroanilide as a substrate at 37° C. for 30 minutes. After adding each of the peptides to be tested and negative and positive controls in an amount of 100 μg/mL into test tube, respectively, a pancreatic solution (type I: derived from porcine pancreas, 0.6 unit/mL, Sigma Aldrich, USA) in an amount of 50 μg/mL was added thereto. Thereafter, a substrate, in which N-succinyl-(L-Ala)3-p-nitroanilide (1 mg/mL) was dissolved in 50 mM Tris-HCl buffer (pH 8.6), was added in an amount of 100 μg/mL thereto and allowed to react for 30 minutes and the absorbance was measured at 410 nm using a microplate reader. The elastase inhibitory activity was expressed as a decrease rate in absorbance of the groups with and without the addition of the sample solution as shown in Equation 1 below.

[00001]$\begin{array}{cc}\mathrm{Elastase}\mathrm{inhibition}\mathrm{rate}\left(\%\right)=\frac{\mathrm{absorbance}\mathrm{of}\mathrm{control}-\mathrm{absorbance}\mathrm{of}\mathrm{sample}}{\mathrm{absorbance}\mathrm{of}\mathrm{control}}\times 100& \left[\mathrm{Equation}1\right]\end{array}$

[0054] After treating the 42 kinds of synthesized peptides, their effects on inhibition of elastase were measured. The results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Elastase Inhibition Sample Rate (%) 1 79.4 2 88.7 3 69.3 4 72.5 5 118.4 6 66.3 7 44.5 8 49.6 9 56.3 10 33.9 11 78.5 12 49.3 13 59.0 14 77.2 15 63.5 16 67.1 17 77.0 18 103.5 19 58.3 20 55.2 21 47.4 Untreated Group 100 22 69.1 23 73.2 24 90.5 25 53.6 26 78.2 27 56.0 28 79.3 29 66.3 30 69.7 31 70.6 32 64.0 33 55.6 34 34.5 35 60.5 36 72.3 37 64.2 38 59.3 39 60.7 40 55.7 41 72.3 42 49.5 Positive Group 28.8 (Ursonic acid)

[0055] Referring to Table 2, it was found that the synthesized tetrapeptides having excellent elastase inhibitory ability (%) are even superior to that of ursonic acid, which is an existing functional material for inhibiting elastase. From this result, it can be seen that the synthesized tetrapeptides are effective in preventing skin aging by inhibiting wrinkle formation through their effects of inhibiting elastase when factors that induce wrinkle formation are applied to the skin.

[0056] Candidate peptides showing good titer by screening were classified into separate groups and synthesized on a large scale and were subjected to experiments.

Example 3: Test of Intracellular Collagen Production Ability of Tetrapeptides

[0057] For the two types of tetrapeptides selected from the test of elastase inhibition ability (i.e., Sample 10 and Sample 34), their intracellular collagen production abilities were tested. The human dermal fibroblast (HDF) cells to be used in the test were cultured in a medium, in which 1× Low Serum Growth Supplement (Cascade Biologics™, S-003-10) was added to Medium 106 (Cascade Biologics™, M-106-500), under the conditions of 37° C. and 5% CO.sub.2. The cells were cultured in a 24-well plate at a concentration of 1×10.sup.5 cells/well and the cell adhesion was examined. For the control group, only a solvent was added without any treatment. The dishes were added 4 types of experimental groups and a control group, which were each treated with TGF to a concentration of 10 μg/mL. After adding the test material to each dish, it was cultured for 2 days. After 2 days, each medium was collected and collagen production therein was measured using a Procollagen Type I C-peptide (hereinafter “PIP”) EIA kit (Takara MK101). The results are shown in FIG. 2.

[0058] Referring to Table 2, it was found that as a result of measuring collagen production after treating the two experimental groups according to concentration, the amount of collagen production was insufficient in most of the experimental groups compared to the control group (i.e., TGF); however, the amount of collagen production was higher than that of the negative control group. From these results, it can be seen that the wrinkles on the skin can be inhibited by increasing collagen production according to the sequences of these tetrapeptides, as compared to the case where a single raw material is used.

Example 4: Test of Cell Proliferation Promoting Ability of Tetrapeptides

[0059] In order to confirm the cell proliferation ability of the two tetrapeptides selected in Examples 2 and 3, an experiment was performed as follows, and the results are shown in FIGS. 3 to 5. In this experiment, the cell proliferation ability was measured using the MTT assay.

[0060] 3T3 cells, HACAT cells, and DP cells were seeded to a 24-well plate so that each well can have a cell number of 4×10.sup.4 cells/well, and then incubated at a constant temperature under the conditions of 37° C. and 5% CO.sub.2 for 24 hours. After washing twice with phosphate buffer saline (PBS), the cells were treated with each sample at a concentration of 10 μg/mL and the control group was treated with transforming growth factor-13 (TGF-β) at a concentration of 100 ng/mL and incubated at a constant temperature for 24 hours. After the cultivation, 0.5% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in Dulbecco's Phosphate-Buffered Saline (DPBS) was mixed with the culture medium at a 1:9 (v/v) ratio and added thereto, and the mixture was cultured in a CO.sub.2 incubator for 2 hours. Thereafter, the generated formazan was dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm using ELISA.

[0061] Referring to FIGS. 3 to 5, with regard to the HACAT cells, 3T3 cells, and DP cells, it was confirmed that the two types of peptides, which were treated from low to high concentrations, have a cell proliferation ability in all of the skin cells in a concentration-dependent manner, and no significant changes were observed in the microscopic observation of these cells.

[0062] From the foregoing, it can be seen that the tetrapeptides according to the present invention have no toxicity to skin-related cells, and the tetrapeptides appear to have an effect on skin cell proliferation. Therefore, it can be predicted that the cosmetic composition containing these tetrapeptides according to the present invention will not have any significant side-effect even when it is treated on the skin.

Example 5: HPLC Analysis of Tetrapeptides

[0063] HPLC analysis conditions for accurate analysis and quantification of the two selected tetrapeptides were established.

[0064] Each peptide was analyzed using the HPLC 2695 (the Waters™) and C18 column (Waters™, Xterra MS C18 column; L: 250 mm, LD: 4.6 mm, 5 μm). In particular, the wavelength of the detector of HPLC used was 216 nm. As solvents for the mobile phase of HPLC, acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) and water containing 0.1% TFA were used. The purity analysis of the peptides was observed by changing the concentration of ACN with time, and the results are shown in FIGS. 6 and 7. The conditions required for the analysis of peptide materials are shown in Table 3 below.

TABLE-US-00003 TABLE 3 Conditions 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 0.1% TFA 0.1% TFA Program Time in DW in ACN order (min) (%) (%) Mobile phase 1 0 100 0 conditions for HPLC 2 10 75 25 gradient-elution 3 20 50 50 4 30 25 75 5 40 0 100 6 45 100 0

[0065] Referring to FIGS. 6 and 7, as a result of HPLC analysis, it was confirmed that the two types of tetrapeptide could be accurately identified under the conditions of the HPLC analysis and their accurate concentrations could be known.

Example 6: Confirmation of Thermal and Photo Stabilities of Tetrapeptides

[0066] In order to confirm the thermal and photo stabilities of the two types of tetrapeptides, experiments were conducted in the following manner, and the results are shown in FIGS. 8 and 9.

[0067] Each sample was dissolved in 50 mM Tris-HCl (pH 8.0) buffer to a concentration of 10 mg/mL and then seeded into a glass vial. Each vial was stored at 50° C. for 4 weeks, and the loss of peptides in the composition due to heat was measured, and stability under sunlight conditions was measured in the same manner.

[0068] Referring to FIGS. 9 and 10, as a result of measuring the loss of peptides due to heat and measuring the stability under sunlight conditions, the two tetrapeptides merely showed a peptide loss of less than 10% up to 4 weeks, confirming that these tetrapeptides had high stabilities during the storage period under heat and sunlight.

Example 7: Preparation of Cosmetic Composition Containing Tetrapeptides

[0069] A cream-type composition containing a tetrapeptide was prepared by the following method. As a cream type composition, one part by weight of the tetrapeptide of Sample 10 was included to constitute formulations as shown in Table 4 below.

1) Preparation of [Aqueous Phase B]

[0070] Powdered raw materials are slowly added in sequence while heating water and stirring at 60° C. to 70° C., stirred with a disper mixer until the mixture becomes thickened and loosened evenly, allowed to cool, and prepared in advance for ease of processing.

2) Preparation of [Aqueous Phase A]

[0071] After the powdered raw materials are sufficiently impregnated with glycerin, water is added thereto and heated to 50° C. to 60° C. to dissolve the mixture evenly with a disper mixer.

3) Preparation by Mixed Stirring of [Aqueous Phase A]+[Aqueous Phase B]

[0072] Aqueous phase B prepared in advance is added to aqueous phase A, and the mixture is heated while mixing them the mixture evenly. The mixture is prepared to be maintained at a temperature between 65° C. and 75° C.

4) Preparation by Mixed Stirring of [Emulsion Phase]

[0073] Each raw material is weighed in an individual beaker and melted by heating and stirring (80° C. or higher). The molten phase is prepared so that the temperature can be maintained at 75° C. or higher.

5) Preparation of Whole Cream

[0074] The emulsion phase is slowly added to the prepared aqueous phase mixture and stirred vigorously (6,000 rpm) for about 10 minutes to proceed with the emulsification process. In particular, the temperature should be maintained at a temperature between 65° C. and 75° C.

[0075] After the completion of the above process, additive A is prepared and the process of pH adjustment and neutralization is performed. In particular, since the viscosity of the contents increases, the resultant is stirred vigorously (6,000 rpm) evenly for 5 minutes or more.

[0076] When the contents become cooled down to 50° C. or below, additive B is added thereto and mixed slowly for about 2 minutes (3,000 rpm to 4,000 rpm). When the temperature reaches 40° C. or below, additive C and additive D are added sequentially, and the mixture is stirred slowly (3,000 rpm to 4,000 rpm) for about 2 minutes, and thereafter, the contents are collected through a degassing/filtration process.

[0077] After the completion of the above process, the process of pH adjustment and neutralization is performed. In particular, since the viscosity of the contents increases, the resultant is stirred vigorously (6,000 rpm) and evenly for 5 minutes or more.

[0078] When the contents become cooled down to 50° C. or below, a preservative is added thereto and mixed slowly for about 2 minutes (3,000 rpm to 4,000 rpm). When the temperature reaches 40° C. or below, a tetrapeptide and a fragrance are added thereto, and the mixture is stirred slowly (3,000 rpm to 4,000 rpm) for 2 minutes, thereafter, the contents are collected through a degassing/filtration process.

[0079] The prepared cream was stored under conditions of a low temperature (4° C.), a high temperature (50° C.), room temperature (25° C.), and sunlight to confirm the stability of the cream. No phenomena such as discoloration, an altered odor, a phase change, and a pH change of the cream were observed for 4 weeks under all of the conditions above, confirming that the stability of the cream was maintained. In particular, the stabilities of the tetrapeptides were maintained, and the results of HPLC analysis confirmed that the contents were maintained at 90% or more for 4 weeks under all of the conditions above.

TABLE-US-00004 TABLE 4 Name of Raw Parts Materials/Name Name of by Category of Ingredients Ingredients/INCI Name weight Aqueous DI water water 48.04 Phase A Glycerin glycerin 10.00 Keltrol F xanthan gum 0.10 Adenosine adenosine 0.04 Aqueous DI water water 30.00 Phase B Beta-Glucan beta-glucan 0.20 Carbopol 940 carbomer 0.02 Emulsion Kalcol 6870 cetearyl alcohol 2.50 Phase Gms-105 glyceryl stearate 1.50 Arlacel 165V glyceryl stearate, peg-100 1.20 stearate Tween 80 polysorbate 80 1.00 Arlacel 83 sorbitan sesquioleate 0.70 Phytosqualane squalane 5.00 Dub Mct caprylic/capric triglyceride 3.00 Additive A DI Water water 3.00 L-Arginine (pH arginine 0.50 4.50 to pH 5.50) Additive B 1,2-Hexanediol 1,2-hexanediol 2.00 Additive C Lutein Solution lutein 1.00 Additive D Perfume fragrance 0.10 (fragrance) Peptide to be GQVS peptide 0.1 added later (SEQ ID NO: 1)

[0080] Heretofore, preferred embodiments of the present invention have been described in detail with reference to the accompanying drawings. The description of the present invention is illustrative and those skilled in the art could understand that modification into other specific forms could easily be made without changing the spirit and essential features of the present invention.

[0081] Thus, the scope of the present invention is represented not by the description of the present invention but by claims to be set forth later, and it should be understood that all the modified or changed forms derived from the meaning, scope, and equivalents thereto are included in the scope of the present invention.