Anti-wrinkle and anti-aging cosmetic composition comprising green fluorescent protein-human epidermal growth factor fusion protein as effective component

Abstract

A method for treating wrinkle or aging on a skin of a subject includes applying to the skin a composition that includes a green fluorescent protein-human epidermal growth factor fusion protein as an effective component. Since the fusion protein has an excellent anti-oxidation activity and a high skin cell proliferation effect, it can be advantageously used as a raw material of functional cosmetics having excellent skin regeneration effect like improvement of skin wrinkles and skin whitening.

Claims

1. A method for treating wrinkle or aging on a skin of a subject, the method comprising applying to the skin of the subject a composition comprising a green fluorescent protein-human epidermal growth factor fusion protein, wherein the green fluorescent protein-human epidermal growth factor fusion protein consists of the amino acid sequence of SEQ ID NO: 2.

2. The method of claim 1, wherein the green fluorescent protein-human epidermal growth factor fusion protein is included at 0.000002 to 0.002% by weight relative to the total weight of the composition.

3. The method of claim 1, wherein the composition has any one formulation selected from a group consisting of a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage crme, a nutrition crme, an eye crme, a moisturizing crme, a hand crme, an essence, a nutrition essence, a pack, a cleansing foam, a cleansing water, a cleansing lotion, a cleansing crme, a body lotion, a body cleanser, a soap, and a powder.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the result that expression of the green fluorescent protein-human epidermal growth factor fusion protein is confirmed by SDS-polyacrylamide gel in which GFP-EGF indicates the green fluorescent protein-human epidermal growth factor fusion protein.

(2) FIG. 2 shows the result of DPPH test to examine the anti-oxidation effect of the green fluorescent protein-human epidermal growth factor fusion protein.

(3) FIG. 3 is a photographic image from which the cell proliferation effect of the green fluorescent protein-human epidermal growth factor fusion protein in dermal fibroblasts is confirmed based on crystal violet staining.

DETAILED DESCRIPTION

(4) To achieve the object of the present invention, the present invention provides an anti-wrinkle and anti-aging cosmetic composition comprising a green fluorescent protein-human epidermal growth factor fusion protein as an effective component.

(5) The cosmetic composition of the present invention comprises, as an effective component, a fusion protein of the human epidermal growth factor protein having a skin regeneration effect and the green fluorescent protein having an excellent anti-oxidation activity, and thus it is a cosmetic composition having a function of improving wrinkles based on skin cell regeneration and also an anti-aging function based on anti-oxidation activity.

(6) With regard to the cosmetic composition according to one embodiment of the present invention, the green fluorescent protein may originate from a jellyfish, and preferably from a crystal jellyfish (Aequorea victoria), but it is not limited thereto.

(7) The scope of the green fluorescent protein originating from jellyfish according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 1, and also functional equivalents of the protein. The term functional equivalent indicates a protein having, as a result of addition, substitution, or deletion of an amino acid, at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% sequence homology with the amino acid sequence represented by SEQ ID NO: 1, and it indicates a protein exhibiting substantially the same activity as the protein represented by SEQ ID NO: 1. As described herein, the expression substantially the same activity means an anti-oxidation activity.

(8) The scope of the green fluorescent protein-human epidermal growth factor fusion protein according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 2, and also functional equivalents of the protein. The term functional equivalent indicates a protein having, as a result of addition, substitution, or deletion of an amino acid, at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% sequence homology with the amino acid sequence represented by SEQ ID NO: 2, and it indicates a protein exhibiting substantially the same activity as the protein represented by SEQ ID NO: 2. As described herein, the expression substantially the same activity means a wrinkle improving effect based on skin regeneration while maintaining the anti-oxidation activity.

(9) In the cosmetic composition according to one embodiment of the present invention, content of the fusion protein is preferably 0.000002 to 0.002% by weight relative to the total weight of the cosmetic composition.

(10) In the cosmetic composition of the present invention, components that are typically used for a cosmetic composition can be included in addition to the effective components that are described above. Examples thereof include a lipid material, an organic solvent, a dissolution agent, a condensation agent, a gelling agent, a softening agent, an anti-oxidant, a suspension agent, a stabilizer, a foaming agent, an aroma, a surface active agent, water, an ionic or non-ionic emulsifier, a filler, a metal ion sequestering agent, a chelating agent, a preservative, vitamin, a blocking agent, a moisturizing agent, essential oil, a dye, a pigment, a hydrophilic or lipophilic activating agent, a common auxiliary agent such as lipid vesicle, and a carrier.

(11) The composition of the present invention can be prepared in any formulation which is generally prepared in the pertinent art. For example, the composition may be formulated into a solution, a suspension, an emulsion, a paste, a gel, a crme, a lotion, a powder, an oil, a powder foundation, an emulsion foundation, a wax foundation, a spray, or the like, but not limited thereto.

(12) More specifically, it can be prepared as a cosmetic formulation like a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage crme, a nutrition crme, an eye crme, a moisturizing crme, a hand crme, an essence, a nutrition essence, a pack, a cleansing foam, a cleansing water, a cleansing lotion, a cleansing crme, a body lotion, a body cleanser, a soap, and a powder.

(13) In a case in which the cosmetic composition of the present invention has a formulation type of paste, crme, or gel, it is possible to use animal oil, plant oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide as a carrier component.

(14) In a case in which the cosmetic composition of the present invention has a formulation type of powder or spray, it is possible to use lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder as a carrier component. In particular, in a case in which the cosmetic composition is a spray, a propellant such as chlorofluorohydrocarbon, propane/butane, or dimethyl ether may be additionally included.

(15) In a case in which the cosmetic composition of the present invention has a formulation type of solution or emulsion, a solvent, a dissolution agent, or an emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, and fatty acid ester of sorbitan.

(16) In a case in which the cosmetic composition of the present invention has a formulation type of suspension, it is possible to use, as a carrier component, a liquid phase diluent such as water, ethanol, or propylene glycol, a suspension agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth.

(17) Hereinbelow, the present invention is explained in greater detail in view of the Examples. However, it is evident that the following Examples are given only for exemplification of the present invention and by no means the present invention is limited to the following Examples.

Example 1. Induced Expression and Isolation of Green Fluorescent Protein-Human Epidermal Growth Factor Fusion Protein

(18) As a method for preparing a gene construct to produce the green fluorescent protein-human epidermal growth factor fusion protein and a method for producing the green fluorescent protein-human epidermal growth factor fusion protein by using the gene construct, E. coli ROSETTA2 (DE3) pLysS, which is transformed E. coli provided in Korean Patent Registration No. 1482187 (NEXGEN Biotechnologies, Inc.), was cultured, in case of batch culture, in 1 l LB or BSB medium till to have OD.sub.600 value of 0.6 to 0.8, or cultured using a 20 l, fermenter till to have OD.sub.600 value of 15 to 20 in case of continuous culture. Thereafter, by adding 1 to 5 mM IPTG or 2% lactose, expression of the cloned gene was induced. Culture was further continued for 4 to 5 hours after inducing the gene expression, and then the cells were collected by centrifuge. The obtained cells were thoroughly suspended in a buffer solution (phosphate buffered saline, NaCl 8 g, KCl 0.2 g, Na.sub.2HPO.sub.4 1.44 g, KH.sub.2PO.sub.4 0.24 g/t, pH 7.4). By disrupting the cells using an ultrasonic homogenizer, a solution containing the intracellular proteins was obtained. Thereafter, the protein-containing cell solution was subjected to a further procedure using Sepabeads Resin Brominated Styrenic Adsorbent (Sorbent Technologies, Inc., USA) to finally isolate and purify GFP-EGF.

(19) In order to determine the presence or absence of a fusion protein in the solution, 15% SDS-polyacrylamide gel electrophoresis was carried out. As a result, it was possible to confirm that the green fluorescent protein-human epidermal growth factor fusion protein is present in the sample (FIG. 1).

Example 2. Measurement of Anti-Oxidation Activity of Green Fluorescent Protein-Human Epidermal Growth Factor Fusion Protein

(20) In order to measure the anti-oxidation activity of green fluorescent protein-human epidermal growth factor fusion protein, DPPH (1,1-diphenyl-2-picryl hydrazyl) analysis, which is one of the methods to measure the free radical scavenging effect, was carried out.

(21) For DPPH analysis, L-ascorbic acid was used as a control. As for the test method, each of L-ascorbic acid, the green fluorescent protein-human epidermal growth factor fusion protein, and human epidermal growth factor protein was prepared at 10 M concentration while DPPH was prepared to have final concentration of 200 M. After mixing each of the above proteins with DPPH at a ratio of 1:1, they were allowed to stand for 30 minutes at 37 C. Thereafter, absorbance at 520 nm was measured by using an ELISA reader. The free radical scavenging activity (%) was calculated based on the following formula 1, and the results are shown in FIG. 2.
Free radical scavenging activity (%)=100((B/A)100)(Formula 1)

(22) [A: Absorbance of control group not treated with sample

(23) B: Absorbance of control group or test group treated with sample]

(24) As a result, it was found that the green fluorescent protein-human epidermal growth factor fusion protein exhibited the free radical scavenging activity which is about 2.5 times higher than the ascorbic acid as a control group. Furthermore, even when compared to the human epidermal growth factor protein which has not been fused with the green fluorescent protein, it exhibited the free radical scavenging activity which is at least about 2.5 times higher (FIG. 2). Accordingly, it was possible to confirm that the fusion protein of the present invention can have a high anti-oxidation effect due to the green fluorescent protein, and this means that an anti-skin aging effect can be exhibited based on the excellent anti-oxidation effect of the fusion protein.

Example 3. Cell Proliferation Effect of Green Fluorescent Protein-Human Epidermal Growth Factor Fusion Protein

(25) A fraction from which the presence of the green fluorescent protein-human epidermal growth factor fusion protein finally isolated and purified in Example 1 has been confirmed was selected as a sample, and used for determination of the cell proliferation effect in HDFa cell (Human Dermal Fibroblasts adult).

(26) To measure the cell proliferation activity of the green fluorescent protein-human epidermal growth factor fusion protein, cultured HDFa cells were further cultured for 3 days at 37 C. after treating them with the fusion protein or human epidermal growth factor protein not fused with green fluorescent protein at a concentration of 0, 0.02 ppm, or 0.2 ppm. Thereafter, presence or absence of cell proliferation was determined based on crystal violet staining method. As a result, it was possible to confirm the cell proliferation effect in HDFa cells in accordance with an increase in the treatment concentration of fusion protein. It was also confirmed that the cell proliferation effect of the fusion protein is more excellent than the cell proliferation obtained by a treatment with human epidermal growth factor protein alone (FIG. 3).

(27) Based on the above results, it was confirmed that the green fluorescent protein-human epidermal growth factor fusion protein has not only an excellent anti-oxidation effect but also an anti-wrinkle effect.

Test Example 1. Sensory Skin Irritation Test Regarding Skin Regeneration Activity Like Wrinkle Improvement and Skin Whitening and Anti-Oxidation Effect

(28) Cosmetic compositions of Preparation examples 1, 2, 3, and 4, in which the green fluorescent protein-human epidermal growth factor fusion protein isolated and purified in Example 1 is contained as an effective component, and cosmetic compositions of Comparative example 1, 2, 3, and 4 were prepared and used for a sensory test.

(29) Specifically, in order to determine an anti-wrinkle effect among the items relating to skin regeneration activity, total 30 people including men and women between the age of 30 and 59 (i.e., 10 people in 30 s, 10 people in 40 s, and 10 people in 50 s) were selected as a subject, and they were asked to apply the control group (Comparative example) to the area around the left eye or the test group (Preparation example) to the area around the right eye, once a day for 2 weeks continuously. The evaluation was carried out in terms of wrinkle smoothing around eye. Furthermore, with regard to the skin whitening as one of the activity-related items described above, the degree of a change in skin tone was also evaluated in the same manner as above. Furthermore, as for the anti-oxidation-related matter, the anti-aging effect was analyzed as an aging retardation effect, and relative comparison was made based on naked eye observation. As for the skin irritation, the sensory test was also carried out in the same manner as above regarding itchiness, stinging feeling, erythema of skin or the like. The evaluation was carried out based on the following evaluation criteria, i.e., 5-point scale including quite excellent (5 points), excellent (4 points), favorable (3 points), poor (2 points), and very poor (1 point). The results are shown in the following Tables 2, 4, 6 and 8.

Preparation Example 1 and Comparative Example 1

(30) With or without adding the green fluorescent protein-human epidermal growth factor fusion protein (GFP-EGF) as an effective component, a skin composition was prepared with the components and content that are described in the following Table 1.

(31) TABLE-US-00001 TABLE 1 Skin composition Preparation example 1 Comparative example 1 Component (% by weight) (% by weight) GFP-EGF 0.002 Amino acid stock 0.1 0.1 Mixture of minerals 0.0007 0.0007 Purified water Balance Balance

(32) Result of the sensory test using Preparation example 1 and Comparative example 1 of above Table 1 is described in the following Table 2.

(33) TABLE-US-00002 TABLE 2 Result of Sensory Test of Preparation example 1 and Comparative example 1 Skin regeneration Skin Wrinkle improvement Whitening irritation Preparation Comparative Preparation Preparation Preparation Number example example example example example 30s 1 4 3 4 3 4 2 4 3 4 3 5 3 4 3 3 3 4 4 4 3 3 3 4 5 3 2 3 2 3 6 4 2 4 3 4 7 4 3 4 3 4 8 3 2 4 3 4 9 4 2 4 3 5 10 3 3 4 3 4 40s 11 4 2 5 3 4 12 4 2 5 2 4 13 4 2 4 3 5 14 4 3 5 2 5 15 5 2 4 2 5 16 4 3 4 2 4 17 5 2 5 2 5 18 4 3 4 3 5 19 4 3 4 3 5 20 4 3 5 2 4 50s 21 5 2 4 3 5 22 5 2 5 2 5 23 5 2 5 2 5 24 4 3 4 2 5 25 4 2 4 2 4 26 5 3 5 2 4 27 5 2 5 3 5 28 5 2 4 2 5 29 4 2 5 3 5 30 4 2 5 2 4 Mean value 4.1 2.4 4.2 2.5 4.5

Preparation Example 2 and Comparative Example 2

(34) With or without adding the green fluorescent protein-human epidermal growth factor fusion protein (GFP-EGF) as an effective component, an essence was prepared with the components and content that are described in the following Table 3.

(35) TABLE-US-00003 TABLE 3 Essence composition Preparation example 2 Comparative example 2 Component (% by weight) (% by weight) GFP-EGF 0.002 Amino acid stock 0.05 0.05 Mixture of minerals 0.0007 0.0007 Glycerol 5 5 1,3-Butylene glycol 10 10 Carbopol 940 0.3 0.3 Purified water Balance Balance

(36) Result of the sensory test using Preparation example 2 and Comparative example 2 of above Table 3 is described in the following Table 4.

(37) TABLE-US-00004 TABLE 4 Result of Sensory Test of Preparation example 2 and Comparative example 2 Skin regeneration Skin Wrinkle improvement Whitening irritation Preparation Comparative Preparation Preparation Preparation Number example example example example example 30s 1 4 2 4 3 4 2 4 2 4 3 5 3 4 3 4 3 4 4 4 2 3 3 4 5 3 2 3 3 4 6 4 2 4 3 4 7 4 3 4 3 4 8 3 2 4 3 4 9 4 2 5 2 5 10 4 3 4 3 4 40s 11 5 2 5 2 5 12 5 3 5 2 5 13 4 2 5 2 5 14 5 3 5 2 5 15 5 2 4 2 5 16 5 2 4 2 4 17 5 2 5 2 5 18 4 3 4 3 5 19 5 2 4 2 5 20 4 3 5 2 5 50s 21 5 2 4 2 5 22 5 2 5 2 5 23 5 2 5 2 5 24 4 2 4 2 5 25 4 2 4 3 5 26 5 3 5 2 4 27 5 2 5 3 5 28 5 2 4 2 5 29 4 2 5 3 5 30 5 1 5 2 4 Mean value 4.4 2.2 4.3 2.4 4.6

Preparation Example 3 and Comparative Example 3

(38) With or without adding the green fluorescent protein-human epidermal growth factor fusion protein (GFP-EGF) as an effective component, a lotion was prepared with the components and content that are described in the following Table 5.

(39) TABLE-US-00005 TABLE 5 Lotion composition Preparation example 3 Comparative example 3 Component (% by weight) (% by weight) GFP-EGF 0.002 Amino acid stock 0.05 0.05 Mixture of minerals 0.0007 0.0007 Glycerol 3 3 1,3-Butylene glycol 10 10 Mineral oil 5 5 Cetyl alcohol 2 2 Xanthan gum 0.5 0.5 Purified water Balance Balance

(40) Result of the sensory test using Preparation example 3 and Comparative example 3 of above Table 5 is described in the following Table 6.

(41) TABLE-US-00006 TABLE 6 Result of Sensory Test of Preparation example 3 and Comparative example 3 Skin regeneration Skin Wrinkle improvement Whitening irritation Preparation Comparative Preparation Preparation Preparation Number example example example example example 30s 1 4 2 4 3 5 2 4 2 4 2 5 3 4 3 4 3 4 4 3 2 3 3 4 5 3 2 4 3 4 6 3 2 4 2 4 7 4 3 4 3 5 8 3 2 4 3 4 9 4 2 5 2 5 10 4 2 4 2 4 40s 11 5 2 5 2 5 12 5 3 5 2 5 13 5 2 5 2 5 14 5 2 5 2 5 15 5 2 4 1 5 16 5 1 4 2 4 17 5 2 5 2 5 18 4 3 4 3 5 19 5 2 5 1 5 20 5 1 5 2 5 50s 21 5 2 4 2 5 22 5 2 5 2 5 23 5 2 5 2 5 24 4 2 4 2 5 25 4 2 4 1 5 26 5 3 5 2 4 27 5 2 5 3 5 28 5 2 5 2 5 29 5 2 5 3 5 30 5 1 5 1 5 Mean value 4.4 2.0 4.4 2.1 4.7

Preparation Example 4 and Comparative Example 4

(42) With or without adding the green fluorescent protein-human epidermal growth factor fusion protein (GFP-EGF) as an effective component, a crme was prepared with the components and content that are described in the following Table 7.

(43) TABLE-US-00007 TABLE 7 Creme composition Preparation example 4 Comparative example 4 Component (% by weight) (% by weight) GFP-EGF 0.002 Amino acid stock 0.05 0.05 Mixture of minerals 0.0007 0.0007 Glycerol 2 2 Mineral oil 10 10 Olive emulsifying wax 3 3 Cetyl alcohol 2 2 Purified water Balance Balance

(44) Result of the sensory test using Preparation example 4 and Comparative example 4 of above Table 7 is described in the following Table 8.

(45) TABLE-US-00008 TABLE 8 Result of Sensory Test of Preparation example 4 and Comparative example 4 Skin regeneration Skin Wrinkle improvement Whitening irritation Preparation Comparative Preparation Preparation Preparation Number example example example example example 30s 1 4 3 3 2 5 2 4 2 4 2 5 3 4 3 4 2 4 4 4 2 3 3 4 5 3 3 3 3 4 6 3 2 3 2 4 7 4 3 4 3 5 8 4 2 4 3 4 9 4 2 4 2 5 10 4 3 4 2 4 40s 11 5 3 5 2 5 12 5 3 5 2 5 13 5 2 4 2 5 14 5 2 5 2 5 15 5 2 5 3 5 16 5 2 4 2 4 17 5 2 4 2 5 18 4 3 4 3 5 19 5 2 5 2 5 20 5 2 4 2 5 50s 21 5 2 4 2 5 22 5 2 5 2 5 23 5 2 5 2 5 24 4 2 5 2 5 25 4 2 4 2 5 26 5 3 5 2 4 27 5 2 5 3 5 28 5 2 5 2 5 29 5 2 5 3 5 30 5 2 5 2 5 Mean value 4.5 2.2 4.3 2.2 4.7