COMPOSITION, CONTAINING RGD MOTIF-CONTAINING PEPTIDE OR FRAGMENT THEREOF, FOR TREATING BURNS AND GLAUCOMA, ALLEVIATING SKIN WRINKLES, AND PROMOTING HAIR GROWTH

Abstract

The present invention relates to an RGD motif-containing peptide or a fragment thereof, which is used to effectively treat burns and glaucoma, obtain an excellent effect of alleviating skin wrinkles, and is effective in the promotion of hair restoration and hair growth as well as the prevention of hair loss. Therefore, the motif-containing peptide or the fragment thereof can be utilized for a cosmetic composition and a pharmaceutical composition.

Claims

1-8. (canceled)

9. A method for burn treatment; glaucoma treatment; skin wrinkle reduction; or hair loss prevention, scalp health improvement, or hair growth and hair restoration promotion, the method comprising: administering to a subject in need thereof an effective amount of a peptide consisting of an amino acid sequence of SEQ ID NO: 1 comprising an RGD motif (Arg-Gly-Asp motif) or its fragment.

10. The method according to claim 9, wherein the fragment is at least one selected from the group consisting of the following fragments: 1) a fragment consisting of 5-45 amino acids comprising the RGD motif of SEQ ID NO: 1, optionally with no disulfide bond or 3 or less disulfide bonds between cysteines in the fragment; 2) a fragment comprising 30th-38th amino acids in the amino acid sequence of SEQ ID NO: 1, optionally with no disulfide bond or 1 or more disulfide bonds between cysteines in the fragment; 3) a fragment comprising 32nd-43rd amino acids in the amino acid sequence of SEQ ID NO: 1, optionally with no disulfide bond or 1 or more disulfide bonds between cysteines in the fragment; 4) a fragment comprising 30th-44th amino acids in the amino acid sequence of SEQ ID NO: 1, optionally with no disulfide bond or 1 or more disulfide bonds between cysteines in the fragment; 5) a fragment comprising 18th-54th amino acids in the amino acid sequence of SEQ ID NO: 1, optionally with no disulfide bond or 1 or more disulfide bonds between cysteines in the fragment; and 6) a fragment comprising 10th-54th amino acids in the amino acid sequence of SEQ ID NO: 1, optionally with no disulfide bond or 1 or more disulfide bonds between cysteines in the fragment.

11. The method according to claim 10, wherein the fragment is at least one selected from the group consisting of the following fragments: 1) a fragment consisting of 9, 12 or 15 amino acids comprising the RGD motif of SEQ ID NO: 1, optionally with no disulfide bond or 3 or less disulfide bonds between cysteines in the fragment; 2) a fragment consisting of 30th-38th amino acids in the amino acid sequence of SEQ ID NO: 1; 3) a fragment consisting of 32nd-43rd amino acids in the amino acid sequence of SEQ ID NO: 1, with a disulfide bond between 36th cysteine and 42nd cysteine; 4) a fragment consisting of 30th-44th amino acids in the amino acid sequence of SEQ ID NO: 1, with no disulfide bond between cysteines in the fragment; or 5) a fragment consisting of 30th-44th amino acids in the amino acid sequence of SEQ ID NO: 1, with a disulfide bond between cysteines in the fragment.

12. The method according to claim 9, wherein the peptide or its fragment has a collagen synthesis promotion effect.

13. The method according to claim 9, wherein the peptide or its fragment is topically administered.

14. A cosmetic composition comprising a peptide consisting of an amino acid sequence of SEQ ID NO: 1 comprising an RGD motif (Arg-Gly-Asp motif) or its fragment as an active ingredient, and having at least one of the following effects: burn treatment; glaucoma treatment; skin wrinkle reduction; or hair loss prevention, scalp health improvement, or hair growth and hair restoration promotion.

15. The cosmetic composition according to claim 14, wherein the fragment is at least one selected from the group consisting of the following fragments: 1) a fragment consisting of 5-45 amino acids comprising the RGD motif of SEQ ID NO: 1, optionally with no disulfide bond or 3 or less disulfide bonds between cysteines in the fragment; 2) a fragment comprising 30th-38th amino acids in the amino acid sequence of SEQ ID NO: 1, optionally with no disulfide bond or 1 or more disulfide bonds between cysteines in the fragment; 3) a fragment comprising 32nd-43rd amino acids in the amino acid sequence of SEQ ID NO: 1, optionally with no disulfide bond or 1 or more disulfide bonds between cysteines in the fragment; 4) a fragment comprising 30th-44th amino acids in the amino acid sequence of SEQ ID NO: 1, optionally with no disulfide bond or 1 or more disulfide bonds between cysteines in the fragment; 5) a fragment comprising 18th-54th amino acids in the amino acid sequence of SEQ ID NO: 1, optionally with no disulfide bond or 1 or more disulfide bonds between cysteines in the fragment; and 6) a fragment comprising 10th-54th amino acids in the amino acid sequence of SEQ ID NO: 1, optionally with no disulfide bond or 1 or more disulfide bonds between cysteines in the fragment.

16. The cosmetic composition according to claim 15, wherein the fragment is at least one selected from the group consisting of the following fragments: 1) a fragment consisting of 9, 12 or 15 amino acids comprising the RGD motif of SEQ ID NO: 1, optionally with no disulfide bond or 3 or less disulfide bonds between cysteines in the fragment; 2) a fragment consisting of 30th-38th amino acids in the amino acid sequence of SEQ ID NO: 1; 3) a fragment consisting of 32nd-43rd amino acids in the amino acid sequence of SEQ ID NO: 1, with a disulfide bond between 36th cysteine and 42nd cysteine; 4) a fragment consisting of 30th-44th amino acids in the amino acid sequence of SEQ ID NO: 1, with no disulfide bond between cysteines in the fragment; or 5) a fragment consisting of 30th-44th amino acids in the amino acid sequence of SEQ ID NO: 1, with a disulfide bond between cysteines in the fragment.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0147] FIG. 1 shows the results of HU024 measured by the MALDI-TOF mass analysis method.

[0148] FIG. 2 shows the results of HU025 measured by the MALDI-TOF mass analysis method.

[0149] FIG. 3 shows the results of HU026 measured by the MALDI-TOF mass analysis method.

[0150] FIG. 4 shows the results of HU027 measured by the MALDI-TOF mass analysis method.

[0151] FIG. 5 shows the measurement results of weight changes of G1-G6.

[0152] (G1: Normal control, n=5

[0153] G2: Vehicle control, n=9-10

[0154] G3: Test article (3.3 μg/head/day), n=10

[0155] G4: Test article (10 μg/head/day), n=10

[0156] G5: Test article (30 μg/head/day), n=10

[0157] G6: Positive control (10 μg/head/day), n=9-10)

[0158] FIG. 6 shows the measurement results of burn wound area changes of G1-G6.

[0159] (G2: Vehicle control, n=9-10

[0160] G3: Test article (3.3 μg/head/day), n=10

[0161] G4: Test article (10 μg/head/day), n=10

[0162] G5: Test article (30 μg/head/day), n=10

[0163] G6: Positive control (10 μg/head/day), n=9-10)

[0164] FIG. 7 shows burn wound area reduction (%) of G2-G6.

[0165] (G1: Normal control, n=5

[0166] G2: Vehicle control, n=9-10

[0167] G3: Test article (3.3 μg/head/day), n=10

[0168] G4: Test article (10 μg/head/day), n=10

[0169] G5: Test article (30 μg/head/day), n=10

[0170] G6: Positive control (10 μg/head/day), n=9-10)

[0171] FIG. 8 shows changes in eschar formation and inflammatory cell infiltration of G1-G6.

[0172] (G1: Normal control, n=5

[0173] G2: Vehicle control, n=9-10

[0174] G3: Test article (3.3 μg/head/day), n=10

[0175] G4: Test article (10 μg/head/day), n=10

[0176] G5: Test article (30 μg/head/day), n=10

[0177] G6: Positive control (10 μg/head/day), n=9-10)

[0178] FIG. 9 shows changes in skin appendage regeneration, connective tissue growth, epidermal cell regeneration of G1-G6.

[0179] (G1: Normal control, n=5

[0180] G2: Vehicle control, n=9-10

[0181] G3: Test article (3.3 μg/head/day), n=10

[0182] G4: Test article (10 μg/head/day), n=10

[0183] G5: Test article (30 μg/head/day), n=10

[0184] G6: Positive control (10 μg/head/day), n=9-10)

[0185] FIG. 10 shows the extent of eschar formation and inflammatory cell infiltration of G1-G6.

[0186] (e: epidermis, d: dermis, hd: hypodermis)

[0187] (A: Normal control,

[0188] B: Vehicle control,

[0189] C: Test article (3.3 μg/head/day),

[0190] D: Test article (10 μg/head/day),

[0191] E: Test article (30 μg/head/day),

[0192] F: Positive control (10 μg/head/day))

[0193] FIG. 11 shows R1 numerical value changes after use of test product (HU022) and control product.

[0194] FIG. 12 shows R2 numerical value changes after use of test product (HU022) and control product.

[0195] FIG. 13 shows R3 numerical value changes after use of test product (HU022) and control product.

[0196] FIG. 14 shows R4 numerical value changes after use of test product (HU022) and control product.

[0197] FIG. 15 shows R5 numerical value changes after use of test product (HU022) and control product.

[0198] FIG. 16 shows Ra numerical value changes after use of test product (HU022) and control product.

[0199] FIG. 17 shows Rmax numerical value changes after use of test product (HU022) and control product.

[0200] FIG. 18 shows Rt numerical value changes after use of test product (HU022) and control product.

[0201] FIG. 19 shows Rp numerical value changes after use of test product (HU022) and control product.

[0202] FIG. 20 shows Rv numerical value changes after use of test product (HU022) and control product.

[0203] FIG. 21 shows changes in expert visual assessment scores after use of test product (HU022) and control product.

[0204] FIG. 22 shows R1 numerical value changes after use of test product (HU024) and control product.

[0205] FIG. 23 shows R2 numerical value changes after use of test product (HU024) and control product.

[0206] FIG. 24 shows R3 numerical value changes after use of test product (HU024) and control product.

[0207] FIG. 25 shows R4 numerical value changes after use of test product (HU024) and control product.

[0208] FIG. 26 shows R5 numerical value changes after use of test product (HU024) and control product.

[0209] FIG. 27 shows Ra numerical value changes after use of test product (HU024) and control product.

[0210] FIG. 28 shows Rmax numerical value changes after use of test product (HU024) and control product.

[0211] FIG. 29 shows Rt numerical value changes after use of test product (HU024) and control product.

[0212] FIG. 30 shows Rp numerical value changes after use of test product (HU024) and control product.

[0213] FIG. 31 shows Rv numerical value changes after use of test product (HU024) and control product.

[0214] FIG. 32 shows changes in expert visual assessment scores after use of test product (HU024) and control product.

[0215] FIG. 33 shows the measurement results of weight changes of G1-G10.

[0216] (G1: Vehicle control (Sterile distilled water for Injection), n=10;

[0217] G2: Test article I (HU024 20 ug/head/day), n=10;

[0218] G3: Test article I (HU024 60 ug/head/day), n=10;

[0219] G4: Test article II (HU025 20 ug/head/day), n=10;

[0220] G5: Test article II (HU025 60 ug/head/day), n=10;

[0221] G6: Test article III (HU026 20 ug/head/day), n=10;

[0222] G7: Test article III (HU026 60 ug/head/day), n=10;

[0223] G8: Test article IV (HU027 20 ug/head/day), n=10;

[0224] G9: Test article IV (HU027 60 ug/head/day), n=10;

[0225] G10: Reference control (Minoxyl 3%), n=10)

[0226] FIG. 34 shows the measurement results of hair area changes of G1-G10.

[0227] (G1: Vehicle control (Sterile distilled water for Injection), n=10;

[0228] G2: Test article I (HU024 20 ug/head/day), n=10;

[0229] G3: Test article I (HU024 60 ug/head/day), n=10;

[0230] G4: Test article II (HU025 20 ug/head/day), n=10;

[0231] G5: Test article II (HU025 60 ug/head/day), n=10;

[0232] G6: Test article III (HU026 20 ug/head/day), n=10;

[0233] G7: Test article III (HU026 60 ug/head/day), n=10;

[0234] G8: Test article IV (HU027 20 ug/head/day), n=10;

[0235] G9: Test article IV (HU027 60 ug/head/day), n=10;

[0236] G10: Reference control (Minoxyl 3%), n=10)

[0237] FIG. 35 shows the measurement results of hair weight changes.

[0238] (G1: Vehicle control (Sterile distilled water for Injection), n=10;

[0239] G2: Test article I (HU024 20 ug/head/day), n=10;

[0240] G3: Test article I (HU024 60 ug/head/day), n=10;

[0241] G4: Test article II (HU025 20 ug/head/day), n=10;

[0242] G5: Test article II (HU025 60 ug/head/day), n=10;

[0243] G6: Test article III (HU026 20 ug/head/day), n=10;

[0244] G7: Test article III (HU026 60 ug/head/day), n=10;

[0245] G8: Test article IV (HU027 20 ug/head/day), n=10;

[0246] G9: Test article IV (HU027 60 ug/head/day), n=10;

[0247] G10: Reference control (Minoxyl 3%), n=10)

[0248] FIG. 36 shows the measurement results of intraocular pressure changes in increased intraocular pressure animal model administered with samples 1-5.

[0249] Nor: normal mouse

[0250] Microbead: increased intraocular pressure animal model

[0251] Sample 1: Tafluprost+HU024 (20 ug/ml)

[0252] Sample 2: HU024 (20 ug/ml)

[0253] Sample 3: HU025 (20 ug/ml)

[0254] Sample 4: HU026 (20 ug/ml)

[0255] Sample 5: HU027 (20 ug/ml)

[0256] Pos Cont: Tafluprost

[0257] FIG. 37 shows the measurement results of changes of optic nerve cells in increased intraocular pressure animal model administered with samples 1-5.

[0258] Control: normal mouse

[0259] Microbead: increased intraocular pressure animal model

[0260] Sample 1: Tafluprost+HU024 (20 ug/ml)

[0261] Sample 2: HU024 (20 ug/ml)

[0262] Sample 3: HU025 (20 ug/ml)

[0263] Sample 4: HU026 (20 ug/ml)

[0264] Sample 5: HU027 (20 ug/ml)

[0265] (+)Control: Tafluprost

BEST MODE FOR CARRYING OUT THE INVENTION

[0266] Hereinafter, the present disclosure will be described in further detail through examples. These examples are intended to describe the present disclosure in more detail, and it is obvious to persons having ordinary skill in the art that the scope of the present disclosure is not limited to the scope of disclosure of the examples.

Example 1. Synthesis of Peptide

[0267] A polypeptide consisting of 452nd-509th amino acids of ADAM metallopeptidase domain 15 (ADAM 15) (GenBank accession no. AAH14566.1) is designated as HU022 (SEQ ID NO: 1), and comprises a portion of disintegrin.

[0268] Furthermore, a peptide consisting of a total of 15 amino acids that is designed to surround an RGD motif in the disintegrin region of ADAM15 protein (NCBI Reference Sequence: NM_001261464), comprise cysteines playing a key role in the 3-dimensional (3D) structure with the RGD motif centered. The peptide is designed to have a disulfide bond formed between cysteines, and is designated as HU024 (SEQ ID NO: 2).

[0269] Furthermore, a peptide designed to have the same amino acid sequence as HU024, but no disulfide bond, is designated as HU025 (SEQ ID NO: 3).

[0270] Furthermore, additionally, a peptide in a smaller size that is designed to surround an RGD motif in the disintegrin region of ADAM15 protein, consisting of 12 amino acids, is designated as HU026 (SEQ ID NO: 4), and consisting of 9 amino acids, is designated as HU027 (SEQ ID NO: 5).

[0271] Hereinafter, a method for producing the peptides is described in more detail.

[0272] For synthesis of a new peptide comprising an RGD motif according to the present disclosure, a solid phase peptide synthesis method (solid-phase method) was used (Merrifield, R. B., J. Am. Chem. Soc. 85:2149, 1963). Target peptides HU024, HU025, HU026, HU027 were synthesized on Rink amide MBHA resin using Fmoc chemistry, and the amino acid used was those having N-terminal and side chains protected using 9-fluorenylmethoxycarbonyl group. An amount of amino acids used in synthesis was 0.3 mmol that is 3 equivalents of 0.1 mmol resin. In coupling at each step, reaction was performed with an addition of DIC (N,N′-Diisoprpyl Cabodiimide) as a reagent for the activation of C-terminal and CI-HOBt (6-Chloro-1-hydroxybenzotriazole) as a reagent for suppressing the racemization in a sequential order according to each sequence, and the Fmoc group of the peptides was removed by treatment with 50% Piperidine/DMF (50/50,v/v) solution at room temperature for 10˜20 minutes. After peptide synthesis was completed, for release from solid phase and deprotection of side chain, treatment with TFA (Trifluoroacetic acid)/water mixed solution (9.5/0.5,v/v) was performed at room temperature for 2 hours. For disulfide bond formation in the peptide, a high dilution air oxidation method was used to form S—S bonds in molecules. To accelerate the reaction, a catalytic amount of DMSO was used.

[0273] The synthesized peptide was separated by HPLC using reverse phase C18 column, and 95% or more of purity was confirmed by HPLC for analysis. Also, the molecular weight of each peptide was confirmed by MALDI-TOF (matrix-associated laser desorption ionization) mass analysis method (FIG. 1, FIG. 2, FIG. 3 and FIG. 4).

[0274] The amino acid sequences and characteristics of the synthesized polypeptide are listed in Table 1.

TABLE-US-00001 TABLE 1 Name (Sequence Character- number) Sequences istics HU024 (Sequence number 2) [00001] 15 aa,  1662.8Da, pI 4.56 Disulfide bond: 1 HU025 NH.sub.2—RPTRGDCDLPEFCPG—COOH 15 aa,  (Sequence 1662.8Da, number 3) pI 4.56 Disulfide  bond: 0 HU026 (Sequence number 4) [00002] 12 aa, 1352.5Da, pI 4.03 Disulfide bond: 1 HU027 NH.sub.2—RPTRGDCDL—COOH 9 aa,  (Sequence 1032.1Da, number 5) pI 5.95 Disulfide   bond: None Free Cystein

[0275] Furthermore, for target peptide HU022, the commercial product (EYEGENE Inc.) was used.

Example 2. Burn Treatment Effect

[0276] The experiment was carried out with 55 male SD (Sprague-Dawley) rats that were divided into five groups: 5 rats for normal group (1 group, G1), 9-10 rats for negative control (1 group, G2), 30 rats for experimental group (3 groups, 10 rats in each group, G3, G4, G5), and 9-10 rats for positive control (1 group, G6). In the remaining groups except normal group, burned male SD rats was used for experiment. After SD rats were anesthesized, 2-degree burns, 2 cm in diameter, were induced on the SD rats using an aluminum electric soldering iron.

[0277] Test substance HU022 (G3, G4, G5), normal saline solution (G2) and Fiblast (G6) were applied to the skin of the burned male SD rats repeatedly once a day seven times a week for 7 days. Fiblast was applied to control group (G6) in 10 μg/dose/head, and the experimental group was divided into 3 groups and test substance was applied to each group in 3.3 μg/dose/head (G3), 10 μg/dose/head (G4), and 30 μg/dose/head (G5).

[0278] After burn was induced, imaging was performed before test substance was applied, and imaging was performed on 1st, 4th, 8th, 11th, and 15th days, and after imaging, test substance was applied and the applied site was covered with gauze and wrapped with tegaderm. Furthermore, images taken for measuring the burn wound area were analyzed using Image analyzer program.

[0279] On the 14th day after burn, the burn site on the back was scraped from the dermis and fixed in 10% neutrally buffered formalin solution, and histopathology test was conducted through H&E staining. The skin tissue was observed with optical microscope (Olympus BX41, Olympus, Japan), and histological changes related to burn treatment in epidermis, dermis, hypodermis, and subcutaneous muscles were analyzed for each of the following six items: 1) eschar, (0, none; 1+, mild; 2+, moderate; 3+ severe; 4+, very severe), 2) infiltration of inflammatory cells (0, none; 1+, epidermis infiltration; 2+, dermis infiltration; 3+, hypodermis infiltration, 4+, muscle infiltration), 3) angiogenesis (randomly selecting 3 zones at a magnification of ×400, and counting and averaging the number of all blood vessels observed), 4) skin appendage regeneration (pilosebaceous), (0, none; 1+, the extent of regeneration is low; 2+, regeneration is observed at several zones; 3+, regeneration of appendages is normal in shape, but the number is less than normal; 4+; the same level as normal group), 5) connective tissue growth (fibroplasia) (0, normal group level; 1+, only observed in some areas of the dermis or hypodermis; 2+, observed in the dermis and hypodermis; 3+, observed over a wide area of the dermis and hypodermis; 4+, observed over the entire burn wound area), 6) epidermal regeneration (0, regeneration is not observed; 1+, basal cells are observed as a layer in some areas; 2+, basal cells are observed in row, 3+, basal cells are observed as a layer or multiple layers in most regions of damaged skin and the spinous layer or granular layer is identified, 4+, normal group epidermis structure), and after assessment, averages were calculated for each group.

[0280] With the measurement results, parametric comparison determined significance via Student's t-test and One-Way ANOVA, and significance testing relative to negative control was conducted. Furthermore, with the histopathology test results of burns, Kruskal-Wallis' H-test was conducted, and when significance between groups was identified, comparison between groups was performed through Mann-Whitney U-test. The computation program for statistics was SPSS 10.1 K. P<0.05 was determined to be statistically significant.

[0281] As a result, any abnormal syndrome was not observed after test substance was applied, and on the 8th day (Day 8), a statistically significant weight reduction (P<0.01) was observed in burn group when compared to normal group, but the weight reduction was the result of burn (Table 2, FIG. 5).

TABLE-US-00002 TABLE 2 MALE BODY WEIGHTS (g) Day 1 Day 8 Day 15 G1 255.31 ± 8.84 288.69 ± 5.72  302.88 ± 4.53  G2  260.02 ± 13.82 .sup. 266.12 ± 16.86.sup.## 299.07 ± 16.75 G3  261.24 ± 10.05 269.41 ± 17.57 300.31 ± 20.36 G4 260.59 ± 9.27 267.86 ± 5.20  296.55 ± 8.86  G5 257.94 ± 9.23 259.49 ± 15.34 284.87 ± 17.06 G6 255.56 ± 7.60 260.49 ± 12.77 293.06 ± 13.16 Data are expressed as Mean ± S.D. The results were statistically analyzed student't-test. .sup.##significantly different from G1 versus G2, P < 0.01

[0282] As a result of measuring the burn wound area, in comparing absolute values of areas, a statistically significant change was not observed in all experimental groups as compared to negative control (Table 3, FIG. 6). As a result of measuring the reduction (%) in burn wound area, a statistically significant area reduction was observed in test substance 30 μg/head/day group (G5) on Day 4 when compared to negative control (P<0.05), and any difference was not observed in the remaining test substance-administered groups (Table 4, FIG. 7).

TABLE-US-00003 TABLE 3 MALE BURN WOUNDED AREA (cm.sup.2) Day 1 Day 4 Day 8 Day 11 Day 15 G1 — — — — — G2 4.5 ± 1.2 3.8 ± 0.9 2.6 ± 0.4 2.0 ± 0.6 1.7 ± 0.6 G3 4.8 ± 0.8 3.8 ± 0.6 2.9 ± 0.6 2.5 ± 0.7 1.9 ± 0.6 G4 4.3 ± 0.9 3.6 ± 0.8 2.9 ± 0.5 2.4 ± 0.3 2.2 ± 0.4 G5 5.2 ± 0.4 3.5 ± 0.5 2.9 ± 0.3 2.3 ± 0.1 1.8 ± 0.4 G6 4.6 ± 0.9 3.1 ± 0.8 2.5 ± 0.7 1.9 ± 0.6 1.8 ± 0.5 Data are expressed as Mean ± S.D. The results were statistically analyzed student't-test.

TABLE-US-00004 TABLE 4 MALE BURN WOUNDED AREA (%) Day 1 Day 4 Day 8 Day 11 Day 15 G1 — — — — — G2 100.0 ± 26.9 84.3 ± 20.7 58.3 ± 9.2 44.8 ± 13.8 37.3 ± 13.5 G3 100.0 ± 16.3  79.2 ± 13.571  60.8 ± 11.9 51.6 ± 15.7 40.1 ± 12.8 G4 100.0 ± 23.0 82.7 ± 18.0  68.2 ± 12.3 56.8 ± 7.7  51.9 ± 8.9  G5 100.0 ± 7.0  67.6 ± 9.3* 55.9 ± 5.1 43.9 ± 2.6  35.6 ± 8.4  G6 100.0 ± 19.7 68.5 ± 18.5 53.922 ± 15.7  43.5 ± 12.6 39.4 ± 11.3 Data are expressed as Mean ± S.D. The results were statistically analyzed student't-test *significantly different from G2, P < 0.05

[0283] The histopathology test results are as follows (Table 5, FIG. 8, FIG. 9 and FIG. 10):

1) Eschar

[0284] In burn wound skin epidermis, fibrin, white blood cell, coagulated exudate and dead tissue are coagulated together to form eschar. The extent of eschar formation was assessed using 5 levels (0-4 points). Normal group G1 was normal skin tissue where eschar is not observed. In negative control (G2) and test substance 3.3 μg/head/day administered group (G3), eschar as thick as mean 4.0±0.0 and 4.0±0.0 points was observed over the entire burned area and assessed as being very severe. In test substance 10 μg/head/day administered group (G4), eschar scored 3.2±0.6 points corresponding to the level of about 80% of the entire burned area, and in test substance 30 μg/head/day administered group (G5) and positive control (G6), eschar scored 2.7±0.7 and 2.6±0.5 points, indicating reduced eschar formation. Furthermore, in test substance 10 μg/head/day administered group (G4), 30 μg/head/day administered group (G5) and positive control (G6), reduced eschar formation and reduced eschar thickness were observed, and the statistical analysis results revealed that there was significance compared to negative control (G2) (P<0.01).

2) Infiltration of Inflammatory Cells

[0285] After burn was induced, an acute inflammatory reaction happens, and lymphocyte and neutrocyte infiltration is primarily observed. The extent of inflammatory cell infiltration was assessed using 5 levels (0-4 points). The normal group (G1) was normal tissue where inflammatory cell infiltration was not observed. In negative control (G2), all test substance administered groups (G3, G4, G5) and positive control (G6), infiltration of inflammatory cells such as lymphocyte, neutrocyte, and monocyte was observed in epidermis, dermis, and hypodermis layer, and any difference between each group was not observed.

3) Skin Appendage Regeneration (Pilosebaceous)

[0286] The extent to which skin appendages located in the dermis layer, such as follicles, sweat glands, and glandulae sebaceae are regenerated after burn is an important standard for determining the extent of skin regeneration with epidermal regeneration. The skin appendages are composed of epidermal cells, and undifferentiated cells located in the appendages are differentiated and move to epidermis, and participate in the regeneration process of epidermis. The extent of regeneration of skin appendages was assessed using 5 levels (0-4 points). In normal group (G1), skin appendages such as follicles and glandulae sebaceae were observed as a normal structure and assessed as 4 points. In negative control (G2), regeneration of skin appendages was not observed at all, or the extent of regeneration was observed as being very low. In test substance 10 ug/head/day administered group (G4) and test substance 30 μg/head/day administered group (G5), test substance concentration-dependent regeneration of skin appendages was found as getting promoted, and the skin appendage regeneration level was found as being statistically significant compared to negative control (G2) (P<0.05 or P<0.01). In positive control (G6), regeneration promotion of skin appendages was also observed, and was statistically significant (P<0.01).

4) Connective Tissue Growth (Fibroplasia)

[0287] After burn was induced, fibroblast growth and an increase in collagen produced from its cell in the therapy process is observed. The extent of connective tissue growth was assessed using 5 levels (0-4 points). Following fiber cell growth and collagen production in dermis at high density together with skin regeneration, when regeneration is completed, grown fiber cell and increased collagen slowly reduces and reaches a normal level. The normal group (G1) was normal tissue where fibroblast was not observed. In negative control (G2), all test substance administered groups (G3, G4, G5) and positive control (G6), very active fiber cell growth and collagen increase in dermis and hypodermis was observed, and it was assessed that there was no difference between each group.

5) Epidermal Regeneration

[0288] The epidermal cell regeneration is the process of regeneration of the damaged or lost epidermis with basal cell growth and differentiation into skin epidermal cells. For the epidermal regeneration capacity, the extent of epidermis formation and basal cell growth was observed and assessed using 5 levels (0-4 points). In normal group (G1), normal epidermal tissue of the basal layer, spinous layer, granular layer, and cornified layer was observed. In negative control (G2), epidermal regeneration was not observed in most of the animals. In test substance 3.3 μg/head/day administered group (G3), a very low level of basal layer cell regeneration was observed in some regions, and in test substance 10 μg/head/day administered group (G4) and 30 μg/head/day administered group (G5) and positive control (G6), regeneration of a basal layer was observed over the wider area, and it was assessed as having a statistically significant promotion effect of the regeneration of epidermal cells compared to negative control (P<0.05).

TABLE-US-00005 TABLE 5 Infiltration of Inflammatory Epidermal Group Eschar cells Pilosebaceous Fibroplasia regeneration G1 0 0 4.0 ± 0.0  0 4.0 ± 0.0  G2 4.0 ± 0.0.sup.##  .sup. 2.9 ± 0.4.sup.##  0 ± 0.0.sup.## .sup. 3.0 ± 0.0.sup.##  0.0 ± 0.0.sup.## G3 4.0 ± 0.0  3.0 ± 0.0 0.1 ± 0.3  3.1 ± 0.3 0.1 ± 0.0  G4 3.2 ± 0.6** 3.0 ± 0.0 0.9 ± 1.1*  3.0 ± 0.5 0.6 ± 0.5* G5 2.7 ± 0.7** 3.0 ± 0.0 1.1 ± 1.0** 3.3 ± 0.5 0.6 ± 0.7* G6 2.6 ± 0.5** 2.7 ± 0.5 0.4 ± 0.5** 3.2 ± 0.4 0.6 ± 0.5* Data are expressed as Mean ± S.D. The results were statistically analyzed by box-plot and Kruscal-wallis methods. *significantly different from G1 value, P < 0.05 **significantly different from G2 value, P < 0.01 .sup.##significantly different from G1 versus G2, P < 0.01

Example 3. Wrinkle Reduction Treatment

[0289] Women aged 30˜65 having wrinkles around eyes were tested. For 4 weeks, test product (HU022 or HU024) and control product (D.i-water) were randomly given to use the products on each of left and right sides of the face twice daily, and at the first visit, the subjects received test product and control product to use for 4 weeks, and at the same time, assessment and investigation before use was completed. They had second and third visits at the interval of 2 weeks after the start of use, and at each visit, were required to respond to various types of assessment and investigation defined in the experiment.

[0290] The assessment items, test method and its results are as follows:

(1) Assessment of Wrinkle Reduction Effect of Test Product HU022

[0291] The extent of wrinkles around eyes was measured using equipment. A target site to measure the wrinkle extent was regions around the eyes of the subjects, and the left side and the right side of the face were set as a measurement site. To measure the wrinkle extent, the subjects were allowed to wash the measurement site with water, and take a rest for 30 minutes in the waiting room under constant temperature and constant humidity conditions of indoor temperature 20˜25° C. and humidity 40˜60% to acclimate the skin surface temperature and humidity to the environment of the measurement space, and were not permitted to drink water during the rest. For objective measurement, measurements were performed by one researcher, and the same site was measured at each time of the first visit before use and the second and third visits in 2 and 4 weeks after use. Transparency profilometry analysis was performed using Visiometer SV600 (Courage-Khazaka electronic GmbH, Germany) on skin replica of the site to which test product and control product were applied before use and 2 and 4 weeks after use, and to identify if there is any difference in each of control group and experimental group after use, a change (%) in R1, R2, R4, R5 measurement values in 2 and 4 weeks after use relative to before use was analyzed.

[0292] The wrinkle extent parameters are as follows:

TABLE-US-00006 TABLE 6 R1 Skin roughness The difference between highest peak and lowest valley of wrinkle profile R2 Maximum roughness The largest of five R1 values obtained for each of five equal subdivisions in wrinkle profile R3 Average roughness Arithmetic average of a difference between maximum and minimum measured in each subdivision after the wrinkle profile is divided into five equal subdivisions along X-axis Free of artifacts as opposed to R1 and R2 R4 Smoothness depth The integral of the area between highest peak of wrinkle profile and the profile, divided by the length of mean line of the profile Average depth of skin wrinkle R5 Arithmetic average roughness The integral of the area between mean line of profile and the profile, divided by the length of mean line of the profile Average roughness of skin

[0293] To identify changes in R3 numerical value after the use of test product (HU022), R3 numerical values were measured before use, 2 weeks after use, and 4 weeks after use. The R3 numerical value of test product was 34.24±3.51 before use, 24.41±4.16 in 2 weeks after use, and 18.62±2.79 in 4 weeks after use, and the R3 numerical value of control product was 22.48±2.74 before use, 20.70±1.76 in 2 weeks after use, and 18.13±0.91 in 4 weeks after use, and to clearly identify changes in R3 numerical value, a change (%) in R3 was calculated each time zone (Table 7, FIG. 13).

TABLE-US-00007 TABLE 7 (Mean ± SD) Visit Test product Control product Before use 34.24 ± 3.51 22.48 ± 2.74 2 weeks after use 24.41 ± 4.16 20.70 ± 1.76 4 weeks after use 18.62 ± 2.79 18.13 ± 0.91 Change(%) Before use-2 weeks −23.71  −7.91 after use Before use-4 weeks −45.62 −19.33 after use *Change(%) = {(After − Before)/Before}*100

[0294] To identify changes in R1 numerical value after the use of test product (HU022), R1 numerical values were measured before use, 2 weeks after use, and 4 weeks after use. The R1 numerical value of test product was 76.23±12.15 before use, 54.59±10.99 in 2 weeks after use, and 51.06±19.28 in 4 weeks after use, and the R1 numerical value of control product was 55.74±12.48 before use, 56.17±11.11 in 2 weeks after use, and 50.86±4.67 in 4 weeks after use (Table 8, FIG. 11).

TABLE-US-00008 TABLE 8 (Mean ± SD) Visit Test product Control product Before use 76.23 ± 12.15 55.74 ± 12.48 2 weeks after use 54.59 ± 10.99 56.17 ± 11.11 4 weeks after use 51.06 ± 19.28 50.86 ± 4.67  Change(%) Before use-2 weeks −28.38  0.77 after use Before use-4 weeks −33.02 −8.76 after use *Change(%) = {(After − Before)/Before}*100

[0295] To identify changes in R2 numerical value after use of test product (HU022), R2 numerical values were measured before use, 2 weeks after use, and 4 weeks after use. The R2 numerical value of test product was 54.26±5.70 before use, 36.87±6.02 in 2 weeks after use, and 29.16±2.74 in 4 weeks after use, and the R2 numerical value of control product was 35.24±5.12 before use, 31.64±4.37 in 2 weeks after use, and 27.57±2.89 in 4 weeks after use (Table 9, FIG. 12).

TABLE-US-00009 TABLE 9 (Mean ± SD) Visit Test product Control product Before use 54.26 ± 5.70 35.24 ± 5.12 2 weeks after use 36.87 ± 6.02 31.64 ± 4.37 4 weeks after use 29.16 ± 5.12 27.57 ± 2.89 Change(%) Before use-2 weeks −32.04 −10.23 after use Before use-4 weeks −46.25 −21.75 after use *Change(%) = {(After − Before)/Before}*100

[0296] To identify changes in R4 numerical value after use of test product (HU022), R4 numerical values were measured before use, 2 weeks after use, and 4 weeks after use. The R4 numerical value of test product was 30.41±7.01 before use, 23.64±5.11 in 2 weeks after use, and 22.94±10.03 in 4 weeks after use, and the R4 numerical value of control product was 24.50±5.43 before use, 25.06±5.49 in 2 weeks after use, and 24.42±4.57 in 4 weeks after use (Table 10, FIG. 14).

TABLE-US-00010 TABLE 10 (Mean ± SD) Visit Test product Control product Before use 30.41 ± 7.01 24.50 ± 5.43 2 weeks after use 23.64 ± 5.11 25.06 ± 5.49 4 weeks after use  22.94 ± 10.03 24.42 ± 4.57 Change(%) Before use-2 weeks −22.27  2.31 after use Before use-4 weeks −24.57 −0.33 after use *Change(%) = {(After − Before)/Before}*100

[0297] To identify changes in R5 numerical value after use of test product (HU022), R5 numerical values were measured before use, 2 weeks after use, and 4 weeks after use. The R5 numerical value of test product was 13.57±3.65 before use, 9.44±2.44 in 2 weeks after use, and 10.10±5.97 in 4 weeks after use, and the R5 numerical value of control product was 11.02±2.94 before use, 11.44±3.14 in 2 weeks after use, and 10.51±1.90 in 4 weeks after use (Table 11, FIG. 15).

TABLE-US-00011 TABLE 11 (Mean ± SD) Visit Test product Control product Before use 13.57 ± 3.65 11.02 ± 2.94 2 weeks after use  9.44 ± 2.44 11.44 ± 3.14 4 weeks after use 10.10 ± 5.97 10.51 ± 1.90 Change(%) Before use-2 weeks −30.43  3.79 after use Before use-4 weeks −26.52 −4.64 after use *Change(%) = {(After − Before)/Before}*100

[0298] Furthermore, the wrinkle extent was measured by 3D imaging. A target site to measure the wrinkle extent was regions around the eyes of the subjects, and the right side of the face was set as a measurement site. To measure the wrinkle extent, the subjects were allowed to wash the measurement site with water, and take a rest for 30 minutes in the waiting room under constant temperature constant humidity conditions of indoor temperature 20˜25° C. and humidity 40˜60% to acclimate the skin surface temperature and humidity to the environment of the measurement space, and were not permitted to drink water during the rest. For objective measurement, measurements were performed by one researcher, and the same site was measured at each time of the first visit before use and the second and third visits in 2 and 4 weeks after use. Before use and in 2 and 4 weeks after use, images of the site to which the test product (HU022) was applied were formed using 3D skin imaging machine (PRIMOS Premium), and to verify if there is any difference in each of control group and experimental group in 4 weeks after use, wrinkle parameters, Ra, Rmax, Rt, Rp, Rv values, in the stored images of 2 weeks and 4 weeks after use relative to before use were analyzed.

[0299] The wrinkle extent parameters are as follows:

TABLE-US-00012 TABLE 12 Ra Roughness average of wrinkle profile Rmax Maximum height of wrinkle profile Rt Distance between highest peak and lowest valley of wrinkle profile Rp Maximum profile peak height of wrinkle profile Rv Maximum profile valley depth of wrinkle profile

[0300] To identify changes in Ra numerical value after use of test product, Ra numerical values were measured before use, 2 weeks after use, and 4 weeks after use.

[0301] The Ra numerical value of test product was 19.19±4.27 before use, 18.20±3.94 in 2 weeks after use, and 17.00±3.88 in 4 weeks after use, and the Ra numerical value of control product was 14.86±4.48 before use, 14.94±4.73 in 2 weeks after use, and 14.60±4.58 in 4 weeks after use (Table 13, FIG. 16).

TABLE-US-00013 TABLE 13 (Mean ± SD) Visit Test product Control product Before use 19.19 ± 4.27 14.86 ± 4.48 2 weeks after use 18.20 ± 3.94 14.94 ± 4.73 4 weeks after use 17.00 ± 3.88 14.60 ± 4.58 Change(%) Before use-2 weeks  −5.15  0.54 after use Before use-4 weeks −11.42 −1.76 after use *Change(%) = {(After − Before)/Before}*100

[0302] To identify changes in Rmax numerical value after use of test product, Rmax numerical values were measured before use, 2 weeks after use, and 4 weeks after use. The Rmax numerical value of test product was 176.29±27.79 before use, 168.83±29.61 in 2 weeks after use, and 168.80±33.21 in 4 weeks after use, and the Rmax numerical value of control product was 162.81±64.15 before use, 159.52±61.85 in 2 weeks after use, and 158.36±62.80 in 4 weeks after use (Table 14, FIG. 17).

TABLE-US-00014 TABLE 14 (Mean ± SD) Visit Test product Control product Before use 176.29 ± 27.79 162.81 ± 64.15 2 weeks after use 168.83 ± 29.61 159.52 ± 61.85 4 weeks after use 168.80 ± 33.21 158.36 ± 62.80 Change(%) Before use-2 weeks −4.23 −2.02 after use Before use-4 weeks −4.25 −2.73 after use *Change(%) = {(After − Before)/Before}*100

[0303] To identify changes in Rt numerical value after use of test product, Rt numerical values were measured before use, 2 weeks after use, and 4 weeks after use. The Rt numerical value of test product was 195.05±31.31 before use, 187.85±34.10 in 2 weeks after use, and 184.63±36.83 in 4 weeks after use, and the Rt numerical value of control product is 174.12±68.96 before use, 171.13±66.76 in 2 weeks after use, and 169.86±65.80 in 4 weeks after use (Table 15, FIG. 18).

TABLE-US-00015 TABLE 15 (Mean ± SD) Visit Test product Control product Before use 195.05 ± 31.31 174.12 ± 68.96 2 weeks after use 187.85 ± 34.10 171.13 ± 66.76 4 weeks after use 184.63 ± 36.83 169.86 ± 65.80 Change(%) Before use-2 weeks −3.69 −1.72 after use Before use-4 weeks −5.35 −2.45 after use *Change(%) = {(After − Before)/Before}*100

[0304] To identify changes in Rp numerical value after use of test product, Rp numerical values were measured before use, 2 weeks after use, and 4 weeks after use. The Rp numerical value of test product was 122.73±31.55 before use, 116.47±35.75 in 2 weeks after use, and 121.88±38.06 in 4 weeks after use, and the Rp numerical value of control product was 123.29±57.51 before use, 120.32±53.57 in 2 weeks after use, and 119.57±56.77 in 4 weeks after use (Table 16, FIG. 19).

TABLE-US-00016 TABLE 16 (Mean ± SD) Visit Test product Control product Before use 122.73 ± 31.55 123.29 ± 57.51 2 weeks after use 116.47 ± 35.75 120.32 ± 53.57 4 weeks after use 121.88 ± 38.06 119.57 ± 56.77 Change(%) Before use-2 weeks −5.10 −2.41 after use Before use-4 weeks −0.70 −3.02 after use *Change(%) = {(After − Before)/Before}*100

[0305] To identify changes in Rv numerical value after use of test product, Rv numerical values were measured before use, 2 weeks after use, and 4 weeks after use. The Rv numerical value of test product was 72.32±17.16 before use, 71.38±16.44 in 2 weeks after use, and 62.75±11.92 in 4 weeks after use, and the Rv numerical value of control product was 50.83±16.13 before use, 50.81±16.29 in 2 weeks after use, and 50.29±11.75 in 4 weeks after use (Table 17, FIG. 20).

TABLE-US-00017 TABLE 17 (Mean ± SD) Visit Test product Control product Before use 72.32 ± 17.16 50.83 ± 16.13 2 weeks after use 71.38 ± 16.44 50.81 ± 16.29 4 weeks after use 62.75 ± 11.92 50.29 ± 11.75 Change(%) Before use-2 weeks  −1.30 −0.05 after use Before use-4 weeks −13.24 −1.07 after use *Change(%) = {(After − Before)/Before}*100

[0306] Furthermore, visual assessment was performed. At each visit before and after use, experts assessed the wrinkle extent by scoring using global photodamage score (Br J Dermatol. 2010; 162(3):497-502). To verify if there is any difference in each of control group and experimental group in 4 weeks after use, a change (%) in visual assessment scores of 2 weeks and 4 weeks after use relative to before use was analyzed. The visual assessment scores are as follows (Table 18):

TABLE-US-00018 TABLE 18 Severity Wrinkle grading 0 none 1 none/mild 2 mild 3 mild/moderate 4 moderate 5 moderate/severe 6 severe 7 very severe

[0307] As a result, the expert visual assessment score of test product was 2.17±0.75 before use, 2.17±0.75 in 2 weeks after use, and 1.67±1.03 in 4 weeks after use, and the expert visual assessment score of control product was 2.33±1.03 before use, 2.33±1.03 in 2 weeks after use, and 2.33±1.03 in 4 weeks after use (Table 19, FIG. 21).

TABLE-US-00019 TABLE 19 (Mean ± SD) Visit Test product Control product Before use 2.17 ± 0.75 2.33 ± 1.03 2 weeks after use 2.17 ± 0.75 2.33 ± 1.03 4 weeks after use 1.67 ± 1.03 2.33 ± 1.03 Change(%) Before use-2 weeks 0  0 after use Before use-4 weeks −23.08 0 after use *Change(%) = {(After − Before)/Before}*100

[0308] Furthermore, surveying the subjects about effectiveness was conducted. The subjects were asked to personally make responses about the extent to which the subjects felt that wrinkles reduced after use of test product using 5 levels: better (4), a little bit better (3), no change (2), a little bit worse (1), worse (0). The researcher determined if test product has efficacy by calculating the percentage of the number of subjects for each response.

[0309] As a result of survey assessment about wrinkle reduction, at the third visit in 4 weeks after use, the subjects responded whether both test product and control product reduced wrinkles, and particularly, test product was better at reducing wrinkles than control product. As can be seen from this, the subjects having participated in the test found that the wrinkle reduction efficacy of test product was superior (Table 20).

TABLE-US-00020 TABLE 20 Number of subjects (percentage, %) Standard 4* 3* 2* 1* 0* Mean deviation Test 0 5 1 0 0 2.83 0.41 product (83.3) (16.7) Control 3 2 1 0 0 3.33 0.82 product (50.0) (33.3) (16.7) *4: very good, 3: good, 2: fair 1: bad, 0: very bad

[0310] Furthermore, survey assessment about tactile sensation and preference was performed, and the subjects were asked to respond personally to survey materials about tactile sensation the subjects felt in 4 weeks after use of clinical test product. Investigation was carried out based on the assessment items including the degree of moisture sensation, soft sensation, absorption performance, general tactile sensation, and fragrance, and assessment was carried out on 5 levels of very good, good, fair, bad, and very bad.

[0311] As a result of the investigation, it was found that the subjects generally had positive preference for test product. Particularly, the subjects had higher preference for test product than control product in terms of sticky sensation (Table 21).

TABLE-US-00021 TABLE 21 Preference score (mean ± Standard deviation) Moisture Soft Spreading Absorption Sticky sensation sensation performance performance sensation Fragrance Test 3.17 ± 0.75 3.17 ± 0.75 3.33 ± 0.52 3.17 ± 0.75 3.00 ± 0.63 2.50 ± 0.55 product Control 3.33 ± 0.82 3.33 ± 0.82 3.00 ± 0.63 2.50 ± 0.75 2.83 ± 0.75 2.50 ± 0.55 product *4: very good, 3: good, 2: fair 1: bad, 0: very bad

[0312] Furthermore, compliance assessment was performed, and at the second and third visits, records of product for test were collected, the number of uses of test product was identified, and the compliance was calculated. At each visit, any subject having the compliance of less than 80% or not having used 6 consecutive times or more was ruled out of the test. As a result, at the end of the test, the total compliance was 100%, the highest compliance was 100.00%, and the lowest compliance was 100.00%. In this test, there was no subject having the compliance of 80% or less, and data of all the subjects was used to analyze the results.

[0313] Furthermore, safety assessment was performed, and for safety of clinical test product, in all subjects having used the test product at least once, abnormal responses found at the second week and fourth week after use and abnormal responses reported during the test period were all used as safety assessment data of the product. There was no report about abnormal response, and on the dermatologist's physical examination, any skin abnormal sign, for example, redness, rash, and itching sensation was not observed. Accordingly, it could be seen that test product was a safe product not causing irritation to the skin.

(2) Assessment of Wrinkle Reduction Effect of Test Product HU024

[0314] The assessment items and test method were the same as those used for assessment of test product HU022.

[0315] After the extent of wrinkle was measured using equipment, to identify changes in R3 numerical value after use of test product, R3 numerical values were measured before use, 2 weeks after use, and 4 weeks after use. The R3 numerical value of test product was 29.15±5.56 before use, 20.80±3.97 in 2 weeks after use, and 17.81±4.00 in 4 weeks after use, and the R3 numerical value of control product was 24.43±3.12 before use, 23.56±3.53 in 2 weeks after use, and 18.57±1.86 in 4 weeks after use (Table 22, FIG. 24).

TABLE-US-00022 TABLE 22 (Mean ± SD) Visit Test product Control product Before use 29.15 ± 5.56 24.43 ± 3.12 2 weeks after use 20.80 ± 3.97 23.56 ± 3.53 4 weeks after use 17.81 ± 4.00 18.57 ± 1.86 Change(%) Before use-2 weeks −28.66 −3.55 after use Before use-4 weeks −54.55 −23.99 after use *Change(%) = {(After − Before)/Before}*100

[0316] The R1 numerical value of test product was 71.33±15.8 before use, 57.29±3.21 in 2 weeks after use, and 45.23±13.77 in 4 weeks after use, and the R1 numerical value of control product was 58.01±21.09 before use, 56.17±15.89 in 2 weeks after use, and 45.03±14.74 in 4 weeks after use (Table 23, FIG. 22).

TABLE-US-00023 TABLE 23 (Mean ± SD) Visit Test product Control product Before use 71.33 ± 15.86 58.01 ± 21.09 2 weeks after use 57.29 ± 3.21  56.17 ± 15.89 4 weeks after use 45.23 ± 13.77 45.03 ± 14.74 Change(%) Before use-2 weeks −19.68 −3.17 after use Before use-4 weeks −36.59 −22.37 after use *Change(%) = {(After − Before)/Before}*100

[0317] The R2 numerical value of test product was 46.06±11.62 before use, 30.71±6.57 in 2 weeks after use, and 28.67±7.47 in 4 weeks after use, and the R2 numerical value of control product was 35.97±5.82 before use, 37.15±4.96 in 2 weeks after use, and 26.68±3.30 in 4 weeks after use (Table 24, FIG. 23).

TABLE-US-00024 TABLE 24 (Mean ± SD) Visit Test product Control product Before use  46.06 ± 11.62 35.97 ± 5.82 2 weeks after use 30.71 ± 6.57 37.15 ± 4.96 4 weeks after use 28.67 ± 7.47 26.68 ± 3.30 Change(%) Before use-2 weeks −33.33 3.27 after use Before use-4 weeks −37.74 −25.84 after use *Change(%) = {(After − Before)/Before}*100

[0318] The R4 numerical value test product was 31.44±11.04 before use, 26.24±2.90 in 2 weeks after use, and 20.64±6.07 in 4 weeks after use, and the R4 numerical value of control product was 26.22±11.38 before use, 24.11±8.82 in 2 weeks after use, and 20.67±8.43 in 4 weeks after use (Table 25, FIG. 25).

TABLE-US-00025 TABLE 25 (Mean ± SD) Visit Test product Control product Before use  31.44 ± 11.04  26.22 ± 11.38 2 weeks after use 26.24 ± 2.90 24.11 ± 8.82 4 weeks after use 20.64 ± 6.07 20.67 ± 8.43 Change(%) Before use-2 weeks −16.54 −8.04 after use Before use-4 weeks −34.36 −21.17 after use *Change(%) = {(After − Before)/Before}*100

[0319] The R5 numerical value of test product was 13.52±5.43 before use, 11.58±0.89 in 2 weeks after use, and 8.42±3.48 in 4 weeks after use, and the R5 numerical value of control product was 10.93±6.72 before use, 10.68±5.56 in 2 weeks after use, and 8.79±4.95 in 4 weeks after use (Table 26, FIG. 26).

TABLE-US-00026 TABLE 26 (Mean ± SD) Visit Test product Control product Before use 13.52 ± 5.43 10.93 ± 6.72 2 weeks after use 11.58 ± 0.89 10.68 ± 5.56 4 weeks after use  8.42 ± 3.48  8.79 ± 4.95 Change(%) Before use-2 weeks −14.36 −2.26 after use Before use-4 weeks −37.71 −19.59 after use *Change(%) = {(After − Before)/Before}*100

[0320] After the wrinkle extent was measured by 3D imaging, to identify changes in Ra numerical value after use of test product, Ra numerical values were measured before use, 2 weeks after use, and 4 weeks after use.

[0321] The Ra numerical value of test product was 18.64±3.19 before use, 18.33±2.12 in 2 weeks after use, and 17.71±2.43 in 4 weeks after use, and the Ra numerical value of control product was 15.49±2.77 before use, 15.23±2.51 in 2 weeks after use, and 15.53±2.46 in 4 weeks after use (Table 27, FIG. 27).

TABLE-US-00027 TABLE 27 (Mean ± SD) Visit Test product Control product Before use 18.64 ± 3.19 15.49 ± 2.77 2 weeks after use 18.33 ± 2.12 15.23 ± 2.51 4 weeks after use 17.71 ± 2.43 15.53 ± 2.46 Change(%) Before use-2 weeks −0.75 1.27 after use Before use-4 weeks −4.10 0.75 after use *Change(%) = {(After − Before)/Before}*100

[0322] The Rmax numerical value of test product was 206.55±40.54 before use, 198.77±39.74 in 2 weeks after use, and 196.85±39.02 in 4 weeks after use, and the Rmax numerical value of control product was 162.86±35.17 before use, 158.09±40.68 in 2 weeks after use, and 165.26±39.77 in 4 weeks after use (Table 28, FIG. 28).

TABLE-US-00028 TABLE 28 (Mean ± SD) Visit Test product Control product Before use 206.55 ± 40.54 162.86 ± 35.17 2 weeks after use 198.77 ± 39.74 158.09 ± 40.68 4 weeks after use 196.85 ± 39.02 165.26 ± 39.77 Change(%) Before use-2 weeks −3.77 −2.93 after use Before use-4 weeks −4.69 1.47 after use *Change(%) = {(After − Before)/Before}*100

[0323] The Rt numerical value of test product was 225.94±45.00 before use, 216.37±41.76 in 2 weeks after use, and 210.60±38.61 in 4 weeks after use, and the Rt numerical value of control product was 174.55±34.66 before use, 167.65±41.28 in 2 weeks after use, and 176.14±39.43 in 4 weeks after use (Table 29, FIG. 29).

TABLE-US-00029 TABLE 29 (Mean ± SD) Visit Test product Control product Before use 225.94 ± 45.00 174.55 ± 34.66 2 weeks after use 216.37 ± 41.76 167.65 ± 41.28 4 weeks after use 210.60 ± 38.61 176.14 ± 39.43 Change(%) Before use-2 weeks −4.24 −3.95 after use Before use-4 weeks −6.79 0.91 after use *Change(%) = {(After − Before)/Before}*100

[0324] The Rp numerical value of test product was 160.85±37.51 before use, 155.24±43.18 in 2 weeks after use, and 153.78±36.12 in 4 weeks after use, and the Rp numerical value of control product was 118.80±39.02 before use, 112.77±44.16 in 2 weeks after use, and 123.39±42.81 in 4 weeks after use (Table 30, FIG. 30).

TABLE-US-00030 TABLE 30 (Mean ± SD) Visit Test product Control product Before use 160.85 ± 37.51 118.80 ± 39.02 2 weeks after use 155.24 ± 43.18 112.77 ± 44.16 4 weeks after use 153.78 ± 36.12 123.39 ± 42.81 Change(%) Before use-2 weeks −3.48 −5.07 after use Before use-4 weeks −4.40 3.86 after use *Change(%) = {(After − Before)/Before}*100

[0325] The Rv numerical value of test product was 65.09±11.95 before use, 61.12±8.02 in 2 weeks after use, and 56.82±12.01 in 4 weeks after use, and the Rv numerical value of control product was 55.75±17.98 before use, 54.87±21.72 in 2 weeks after use, and 52.75±15.00 in 4 weeks after use (Table 31, FIG. 31).

TABLE-US-00031 TABLE 31 (Mean ± SD) Visit Test product Control product Before use 65.09 ± 11.95 55.75 ± 17.98 2 weeks after use 61.12 ± 8.02  54.87 ± 21.72 4 weeks after use 56.82 ± 12.01 52.75 ± 15.00 Change(%) Before use-2 weeks −6.10 −1.57 after use Before use-4 weeks −12.71 −5.37 after use *Change(%) = {(After − Before)/Before}*100

[0326] To identify changes in visual assessment score, expert visual assessment scores were assessed before use, 2 weeks after use, and 4 weeks after use.

[0327] The expert visual assessment score of test product was 2.00±0.63 before use, 2.00±0.63 in 2 weeks after use, and 1.67±0.82 in 4 weeks after use, and the expert visual assessment score of control product was 2.00±0.63 before use, 2.00±0.63 in 2 weeks after use, and 2.00±0.63 in 4 weeks after use (Table 32, FIG. 32).

TABLE-US-00032 TABLE 32 (Mean ± SD) Visit Test product Control product Before use 2.00 ± 0.63 2.00 ± 0.63 2 weeks after use 2.00 ± 0.63 2.00 ± 0.63 4 weeks after use 1.67 ± 0.82 2.00 ± 0.63 Change(%) Before use-2 weeks 0 0 after use Before use-4 weeks −16.67 0 after use *Change(%) = {(After − Before)/Before}*100

[0328] As a result of survey assessment about wrinkle reduction, at the third visit in 4 weeks after use, the subjects responded that both test product and control product reduced wrinkles, and particularly, test product was better at reducing wrinkles than control product. As can be seen from this, the subjects having participated in the test found that the wrinkle reduction efficacy of test product was superior.

TABLE-US-00033 TABLE 33 Number of subjects (percentage, %) Standard 4* 3* 2* 1* 0* Mean deviation Test 1 4 1 0 0 3.00 0.63 product (16.7) (66.7) (16.7) Control 1 3 2 0 0 2.83 0.75 product (16.7) (50.0) (33.3) *4: very good, 3: good, 2: fair 1: bad, 0: very bad

[0329] As a result of investigating the subjects' preference in terms of moisture sensation, soft sensation, spreading performance, absorption performance, sticky sensation and fragrance of test product, it was found that the subjects generally had positive preference for test product. Particularly, the preference for test product was higher than control product in terms of sticky sensation.

TABLE-US-00034 TABLE 34 Preference score (mean ± standard deviation) Moisture Soft Spreading Absorption Sticky sensation sensation performance performance sensation Fragrance Test 3.17 ± 0.75 3.17 ± 0.75 3.33 ± 0.52 3.17 ± 0.75 3.00 ± 0.63 2.50 ± 0.55 product Control 3.17 ± 0.75 3.17 ± 0.75 3.33 ± 0.82 3.17 ± 0.75 2.83 ± 0.75 2.50 ± 0.55 product *4: very good, 3: good, 2: fair 1: bad, 0: very bad

[0330] Furthermore, there was no report about abnormal response while the subjects were using the test product for 4 weeks, and on the dermatologist's physical examination, any skin abnormal sign, for example, redness, rash and itching sensation was not observed. Accordingly, it could be seen that test product was a safe product not causing irritation to the skin.

Example 4. Hair Growth Test Effect

[0331] This test was conducted to assess the influence on hair growth of C57BL/6 mouse when 4 types of test substances, HU024, HU025, HU026, and HU027, were applied to the skin for 14 days.

[0332] The group design was set to negative control (sterile water for injection G1), test substance HU024 20 ug/head/day (G2), test substance HU024 60 ug/head/day (G3), test substance HU025 20 ug/head/day (G4), test substance HU025 60 ug/head/day (G5), test substance HU026 20 ug/head/day (G6), test substance HU026 60 ug/head/day (G7), test substance HU027 20 ug/head/day (G8), test substance HU027 60 ug/head/day (G9) and positive control minoxyl 3% (G10) administered groups. Each group had 10 animals.

[0333] After the animal was put under anesthesia using zoletil (1 mL/kg, i.p), the back of the animal was primarily shaved using a depilator, and Veet depilatory was evenly applied on the back. After about 5 minutes, the applied site was cleaned up using gauze moistened with sterile water for injection. Only animal whose shaved back was scarlet in color was used for group separation. The test substance was applied to the same site daily once per day, 7 times per week for 14 days, and the test substance was evenly applied to the shaved site (on the back) by rubbing with a glass rod about 10 times.

[0334] The test item was measurements of dead animal, general syndrome, weight change, hair growing area and hair weight, and comparison with negative control was conducted.

[0335] The hair growing area (%) relative to the test substance-applied area was measured and determined, and Image analyzer program (Image Pro Plus ver. 6.3, Cybermetics, USA) was used to measure. It was measured on the 1st, 3rd, 7th, 10th and 14th days after test substance administration. Imaging was performed together with the measurements on the date when the hair area was measured. On the next day after the end of application, the animals were killed by CO2 gas, and hair on the hair growing area was removed by a depilator and hair weight was measured.

TABLE-US-00035 TABLE 35 Score Extent of hair growth 0 None 1 0~25% hair growth at shaved site 2 26~50% hair growth at shaved site 3 51~75% hair growth at shaved site 4 76~100% hair growth at shaved site

[0336] The measurement results were analyzed using SPSS (ver. 10.1K), Duncan t-test for weight changes was conducted through one-way analysis of variance, hair weight was compared between groups using Student's t-test, and hair growing area was compared between groups through nonparametric multiple comparison procedures Whitney-Mann methods. P<0.05 was determined to be statistically significant.

[0337] The test results are as follows:

[0338] With regard to weight changes, any difference was not observed in test substance administered group compared to negative control. Rather, in comparison with negative control on the 7th and 11st days after administration, a statistically significant weight increase was observed in control substance minoxyl 3% administered group (P<0.05 or P<0.01). The increased weight in control substance minoxyl 3% administered group was thought to be weight increase that is an adverse effect of the active ingredient of minoxyl, minoxidil (Table 36, FIG. 33).

TABLE-US-00036 TABLE 36 MALE BODY WEIGHTS (g) Day 1 Day 7 Day 11 G1 21.41 ± 0.91 22.62 ± 1.19 23.64 ± 1.29 G2 21.34 ± 0.60 22.64 ± 0.76 24.15 ± 0.86 G3 21.54 ± 0.53 23.02 ± 0.74 24.30 ± 1.08 G4 21.20 ± 1.23 22.63 ± 1.01 23.77 ± 1.17 G5 21.53 ± 0.63 23.20 ± 0.83 24.23 ± 1.02 G6 21.20 ± 0.77 22.48 ± 1.12 23.75 ± 1.27 G7 21.58 ± 0.61 22.80 ± 1.03 23.83 ± 1.30 G8 21.71 ± 0.59 23.27 ± 0.71 23.98 ± 0.79 G9 21.77 ± 0.87 23.07 ± 1.13 23.97 ± 1.14 G10 20.70 ± 1.24  23.85 ± 0.75*  25.73 ± 1.13** Data are expressed as Mean ± S.D. The results were statistically analyzed by Student's t-test. *significantly different from G1, P < 0.05 **significantly different from G1, P < 0.01

[0339] As a result of measuring the hair growing area, in comparison of test substance administered group and negative control, any difference was not observed until the 7th day after administration. On the 10th and 14th days, a statistically significant increase in hair growing area was observed in test substance HU025 60 ug/head/day administered group (G5) compared to negative control (P<0.05). The trend toward increased hair growing area by about 20% or more was also observed in the other test substance administered groups except test substance HU025 20 ug/head/day administered group on the 10th and 14th day when compared to negative control. As compared to negative control, a statistically significant increase in hair growing area was observed in control substance minoxyl 3% administered group on the 4th, 10th and 14th days (P<0.01) (Table 37, FIG. 34).

TABLE-US-00037 TABLE 37 MALE HAIR AREA (%) Day 1 Day 4 Day 7 Day 10 Day 14 G1 0.05 ± 0.04 0.02 ± 0.02 0.92 ± 0.33 2.74 ± 1.02 43.30 ± 9.48  G2 0.02 ± 0.01 0.06 ± 0.04 0.44 ± 0.19 7.55 ± 2.56 67.34 ± 9.03  G3 0.04 ± 0.04 0.00 ± 0.00 0.85 ± 0.75 6.56 ± 3.04 55.69 ± 12.45 G4 0.04 ± 0.03 0.06 ± 0.05 0.47 ± 0.22 2.04 ± 0.66 39.88 ± 8.74  G5 0.04 ± 0.04 0.42 ± 0.33 0.72 ± 0.51 19.61 ± 9.01* 75.45 ± 6.65* G6 0.10 ± 0.05 0.13 ± 0.07 0.43 ± 0.30 7.67 ± 4.54 56.64 ± 12.26 G7 0.07 ± 0.04 0.22 ± 0.13 0.69 ± 0.37 13.54 ± 4.75  65.41 ± 11.62 G8 0.13 ± 0.13 0.32 ± 0.14 0.44 ± 0.17 8.81 ± 3.57 56.95 ± 9.83  G9 0.17 ± 0.11 0.33 ± 0.22 0.50 ± 0.26 8.20 ± 3.43 61.95 ± 11.30 G10 0.01 ± 0.01  0.77 ± 0.26** 1.78 ± 1.07  29.78 ± 8.09**  90.51 ± 4.50** Data are expressed as Mean ± S.E.M. The results were statistically analyzed by Whittney-Mann methods. *significantly different from G1, P < 0.05 **significantly different from G1, P < 0.01

[0340] As a result of measuring hair weight, in comparison with negative control, a statistically significant increase in hair weight was observed in test substance HU026 60 ug/head/day administered group (P<0.05). Among the other test substance administered groups, in comparison with negative control, a weight increase by about 100% or more was observed in the remaining test substance administered groups except HU025 20 ug/head/day and HU027 20 ug/head/day administered group. As compared to negative control, a statistically significant increase hair weight was observed in control substance minoxyl 3% administered group (P<0.01) (Table 38, FIG. 35)

TABLE-US-00038 TABLE 38 MALE HAIR WEIGHTS (g) G1 0.008 ± 0.004 G2 0.016 ± 0.003 G3 0.023 ± 0.008 G4 0.005 ± 0.002 G5 0.021 ± 0.007 G6 0.021 ± 0.008 G7  0.026 ± 0.007* G8 0.010 ± 0.003 G9 0.016 ± 0.005 G10  0.031 ± 0.006** Data are expressed as Mean ± S.E.M. The results were statistically analyzed by Student's t-test. *significantly different from G1, P < 0.05 **significantly different from G1, P < 0.01

Example 5. Glaucoma Test Effect

[0341] For control drug, Taflotan eye drops (Santen Pharmaceutical Co., Ltd.) were used, and for test drug, Taflotan eye drops+HU024 (sample 1), HU024 (sample 2), HU025 (sample 3), HU026 (sample 4), and HU027 (sample 5) were used, and testing was performed on glaucoma animal model having microbead-induced elevated intraocular pressure. In experimental animal model, Rompun solution for injection and zoletil were mixed and used to put the experimental animal under anesthesia, topical anesthetic Alcaine was applied to eye, 1×106 microbead/mL 1 μL was injected into anterior chamber using micro glass needle to induce an increase in intraocular pressure for 2 weeks (IOVS 2011 Vol. 52 36-).

[0342] The control drug and 5 samples were provided to the experimental animal having the elevated intraocular pressure for 2 weeks, 5 samples were applied to eye once daily, comparative verification of efficacy was performed, and intraocular pressure reduction and optic nerve cell in the retina was observed through administration for 4 weeks.

[0343] The intraocular pressure was measured using a contact-type intraocular pressure meter Tono lab's tonometer TV02 model. Furthermore, to analyze optic nerve cell in the retina of glaucoma animal model, the scalp of the anesthetized rats was enucleated and got exposed, then the coordinates of superior culiculus in the brain connected to optic nerve were determined using Bregma and Lamda as reference points and were marked, a hole was created, and 3% Fluorogold was injected. In 5-7 days after fluorescent probe injection, the eye was enucleated and fixed in 4% paraformaldehyde at room temperature for 4 hours. The retina was separated from choroid of the fixed eye and mounted on a slide. In each experimental group slide, optic nerve cell expressing fluorescence was detected using fluorescence microscope, images were taken at a magnification of ×40 and ×200, and data was collected.

[0344] As a result of conducting the intraocular pressure test, the intraocular pressure increased to 21.9±8.3 mmHg for 2 weeks. After the elevated intraocular pressure was identified, the results of administering samples 1˜5 were shown in FIG. 36. A reduction in intraocular pressure was observed in samples 2 and 3, and significance was not observed in the remaining samples. It is considered that a slight reduction in intraocular pressure on the whole is attributed to the microbead model properties and is not relevant to the intraocular pressure decrease effect of the sample. In positive control, the intraocular pressure reduced as reported, and the intraocular pressure numerical value (15.9±2.9, 20.89%) considerably reduced as compared to negative control (FIG. 36).

[0345] As a result of analyzing optic nerve cell, significant optic nerve cell death was observed in negative control as compared to control group, and in samples 3 and 4 administered group and positive control, or Tafluprost applied group, protection from optic nerve cell death was observed. In the case of sample 3, it is thought to be result of the reduced intraocular pressure similar to FIG. 36, and in the case of sample 4, the intraocular pressure did not reduce but the optic nerve cell protection effect was found (FIG. 37).

[0346] In conclusion, sample 3, or HU025 had an effect in reducing the intraocular pressure featuring glaucoma. In addition, in many cases of domestic glaucoma patients, vision loss occurs due to optic nerve damage though the intraocular pressure is not high, and it is expected that HU025 sample can be used as drug to treat glaucoma because of having a repair efficacy for damaged optic nerve.

INDUSTRIAL APPLICABILITY

[0347] Using the peptide or its fragment according to the present disclosure, it is possible to effectively treat burns and glaucoma, obtain an excellent effect in reducing skin wrinkles, and it is effective in the promotion of hair restoration and hair growth as well as the prevention of hair loss, so it can be used for a cosmetic composition and a pharmaceutical composition.