COMPOSITION FOR SKIN ELASTICITY ENHANCEMENT AND WRINKLE IMPROVEMENT COMPRISING MILK EXOSOMES

Abstract

The present invention relates to a composition for enhancing skin elasticity and/or ameliorating wrinkles, including milk exosomes, and a method for treating wrinkles by administering milk exosomes.

Claims

1. A composition for enhancing skin elasticity and ameliorating wrinkles, comprising milk exosomes.

2. The composition of claim 1, wherein the milk exosomes are derived from cows.

3. The composition of claim 1, further comprising an aging amelioration use.

4. The composition of claim 3, wherein the aging is photoaging.

5. A cosmetic composition for enhancing skin elasticity and ameliorating wrinkles, comprising the composition of claim 1.

6. The cosmetic composition of claim 5, wherein the milk exosomes are derived from cows.

7. The cosmetic composition of claim 5, further comprising an aging amelioration use.

8. The cosmetic composition of claim 7, wherein the aging is photoaging.

9. A food composition for enhancing skin elasticity and ameliorating wrinkles, comprising the composition of claim 1.

10. The food composition of claim 9, wherein the milk exosomes are derived from cows.

11. The food composition of claim 9, wherein the food composition is a health functional food.

12. A pharmaceutical composition for enhancing skin elasticity and preventing or treating wrinkles, comprising the composition of claim 1.

13. The pharmaceutical composition of claim 12, wherein the milk exosomes are derived from cows.

14. The pharmaceutical composition of claim 12, further comprising an aging prevention or treatment use.

15. A method for treating wrinkles, the method comprising administering the composition of claim 1 to a subject in need thereof.

16. The method of claim 15, wherein the milk exosomes are derived from cows.

17: The method of claim 15, further comprising aging treatment.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0074] FIG. 1 shows the results of confirming the size of milk exosomes and exosome markers.

[0075] FIG. 2 shows the results of confirming the morphology and size of milk exosomes using an electron microscope.

[0076] FIG. 3 shows the results of confirming the toxicity of milk exosomes through cell viability.

[0077] FIG. 4 shows the mRNA expression levels of genes involved in skin elasticity and wrinkle formation/suppression of milk exosomes.

[0078] FIG. 5 shows the results of measuring the production amount of collagen of milk exosomes.

[0079] FIG. 6 shows the results of measuring elastase inhibitory activity by treating human dermal fibroblasts with milk exosomes.

[0080] FIG. 7 shows the results of measuring collagenase inhibitory activity by treating human dermal fibroblasts with milk exosomes.

[0081] FIG. 8 shows the results of measuring the effect of milk exosomes on amelioration of the photoaging phenomenon.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0082] Hereinafter, the present invention will be described in detail through the Examples. However, the following Examples are only for exemplifying the present invention, and the present invention is not limited by the following Examples.

Example 1. Method for Preparing Milk Exosomes

[0083] Exosomes were extracted from cow-derived milk using a centrifuge. More specifically, milk was aliquoted into tubes, and the supernatant was collected by centrifugation at 2000 g and 10,000 g for 10 minutes, respectively. The collected supernatant was filtered through 0.45 μm and 0.2 μm filters, and then mixed with phosphate buffered saline (PBS). Thereafter, the Exoquick exosome precipitation solution (System Biosciences) was mixed with PBS, the resulting mixture was added thereto, and the resultant was allowed to stand for 30 minutes, and centrifuged again at 1,500 g for 30 minutes or 10,000 g for 10 minutes. The supernatant was removed, and an exosome pellet was dissolved in PBS and used for subsequent experiments.

Experimental Example 1: Confirmation of Characteristics of Milk Exosomes

[0084] 1-1. Measurement of Size of Milk Exosomes

[0085] In order to confirm the size of the milk exosomes, the size of the exosomes was measured through dynamic light scattering (DLS), and it was confirmed that the size of the milk exosomes was 80 to 190 nm (FIG. 1).

[0086] 1-2. Confirmation of CD9 Protein Expression

[0087] The expression of CD9, which is an exosome marker, was confirmed using western blot, and as illustrated in FIG. 1, CD9 protein, which is an exosome marker, was clearly observed in exosomes, and it was confirmed that CD9 was not expressed in the supernatant other than the exosomes.

[0088] Proteins were isolated from milk exosomes using a 10% SDS polyacrylamide gel. After the isolated protein was transferred to a nitrocellulose membrane (GE Healthcare), it was reacted with a 5% skim milk powder for 1 hour to prevent the non-specific binding of an antibody. A CD9 (Abcam) primary antibody was diluted at a ratio of 1:1000 and bound to the membrane at 4° C. for 12 to 18 hours. Thereafter, it was combined with an HRP-tagged anti-rabbit antibody and reacted at room temperature for 30 minutes. Protein bands were observed using an ECL kit (Santa Cruz Biotechnology).

[0089] 1-3. Confirmation of Morphology and Size of Exosomes

[0090] The morphology and size of the milk exosomes extracted in Example 1 were observed using an electron microscope. As a result, as illustrated in FIG. 2, it was confirmed that the exosomes were present in a spherical shape within a size of 200 nm in diameter.

Experimental Example 2. Confirmation of Cytotoxicity of Milk Exosomes

[0091] 2-1. Human Dermal Fibroblast Culture

[0092] Normal human dermal fibroblasts (NHDF), which are human dermal fibroblasts, were purchased from Lonza (New Jersey, USA) and used, and cultured in an incubator at 37° C. and 5% CO2 using an FGM-2 medium (Lonza, N.J., USA) supplemented with 2% fetal bovine serum (FBS), 0.1% insulin, 0.1% recombinant human fibroblast growth factor-0, and 0.1% GA-1000.

[0093] 2-2. Confirmation of Cytotoxicity

[0094] In order to determine whether the milk exosomes of the present invention are toxic, cell viability was measured by treating NHDF cells with 20 μg/ml and 50 μg/ml of milk exosomes for 24 hours, respectively. A group not treated with milk exosomes was used as a control (Mock).

[0095] NHDF cells were aliquoted at a volume of 5×10.sup.4 cells/ml in a 96-well plate and cultured in an incubator under conditions of 37° C. and 5% CO2 for 24 hours. The cultured cells were treated with milk exosomes and cultured for 24 hours, and then the effect on cell viability was measured using an EZ-Cytox cell viability assay kit (Daeil Lab Service) according to the manufacturer's method.

[0096] As a result, as illustrated in FIG. 3, there was no change in viability in both the control and the group treated with milk exosomes, confirming that the milk exosome of the present invention was not toxic.

Experimental Example 3. Confirmation of Effect of Milk Exosomes on Skin Elasticity Improvement and Wrinkle Amelioration

[0097] 3-1. Confirmation of Expression Patterns of Skin Elasticity and Wrinkle-Related Genes

[0098] In order to investigate the expression patterns of skin elasticity and wrinkle-related genes according to treatment with milk exosomes, NHDF cells were treated with milk exosomes, and expression patterns of Timp1, procollagen, CD34, elastin and MMP-1 were confirmed by qRT-PCR.

[0099] NHDF cells were treated with milk exosomes and the cells were collected after 24 hours. Total RNA was isolated by reacting the collected cells with Tri reagent (Bioline). In order to perform a reverse transcription reaction, dNTP, M-MLV reverse-transcriptase (Promega), and the like were added to 1 μg of RNA, and then reacted at 37° C. for 1 hour to synthesize cDNA. A real-time polymerase chain reaction was performed using SYBR Green PCR Master mix (Bioline) to measure Timp1, procollagen, CD34, elastin and MMP-1 mRNA expression. PCR conditions for amplifying a specific gene were performed as follows. After reaction at 95° C. for 10 minutes, the gene was amplified for 40 cycles with a cycle of 15 seconds at 95° C., 15 seconds at 60° C., and 15 seconds at 72° C. A target gene mRNA expression level was corrected with the expression level relative to the actin expression level. The primers used are as shown in the following Table 1.

TABLE-US-00001 TABLE 1 SEQ ID NO Primer Sequence 1 Timp1 Forward 5′-ACC ACC TTA TAC CAG CGT TAT GA-3′ 2 Timp1 Reverse 5′-GGT GTA GAC GAA CCG GAT GTC-3′ 3 Procollagen Forward 5′-GTG CGA TGA CGT GAT CTG TGA-3′ 4 Procollagen Reverse 5′-CGG TGG TTT CTT GGT CGG T-3′ 5 CD34 Forward 5′-CTA CAA CAC CTA GTA CCC TTG GA-3′ 6 CD34 Reverse 5′-GGT GAA CAC TGT GCT GAT TAC A-3′ 7 Elastin Forward 5′-GCA GGA GTT AAG CCC AAG G-3′ 8 Elastin Reverse 5′-TGT AGG GCA GTC CAT AGC CA-3′ 9 MMP1 Forward 5′-ATT GGA GCA GCA AGA GGC TGG GA-3′ 10 MMPI Reverse 5′-TTC CAG GTA TTT CTG GAC TAA GT-3′

[0100] As a result, as illustrated in FIG. 4, it was confirmed that the mRNA expression of the MMP-1 gene was suppressed in a concentration-dependent manner in NHDF cells treated with milk exosomes. Meanwhile, in the case of TIMP1, which is a gene involved in collagen and elastin protection, it was confirmed that the expression level was increased according to the treatment with milk exosomes, and it was also confirmed that the expression levels of procollagen, CD34, and elastin were also increased. The above results suggest that milk exosomes promote the expression of genes involved in wrinkle amelioration, while there is an excellent effect on skin wrinkle amelioration by regulating the expression level of collagen-degrading enzyme-related genes.

[0101] 3-2. Confirmation of Production Amount of Type I Collagen

[0102] In order to confirm the collagen synthesis promoting effect of milk exosomes, the amount of collagen synthesis was confirmed after NHDF cells were treated with the milk exosomes. The production amount of collagen was measured by treating NHDF cells with milk exosomes at 20 μg/ml and 50 μg/ml, respectively, and a group not treated with milk exosomes was used as a control (Mock).

[0103] Specifically, NHTDF cells were aliquoted at 1.5×10.sup.5 cells/well, pre-cultured for 24 hours, then treated with milk exosomes at 20 μg/ml and 50 μg/ml, and cultured for 48 hours. Thereafter, a culture medium was centrifuged at 3000 rpm for 10 minutes, and then a Sircol collagen assay kit (Bioclolor, UK) was used. The obtained supernatant was treated with the isolation & concentration reagent provided in the kit and maintained at 4° C. for 16 hours or more, and then centrifuged at 12,000 rpm for 10 minutes to concentrate collagen. After the supernatant was removed, 1 ml of the provided sircol dye reagent was added to the pellet and the pellet was maintained for 30 minutes. After centrifugation at 12,000 rpm for 10 minutes, the pellet was washed with an acid-salt wash reagent, and then centrifuged again under the above conditions. After centrifugation, a dye adsorbed to collagen was dissolved by treatment with an alkali reagent, and absorbance was measured at a wavelength of 555 nm.

[0104] As a result, as illustrated in FIG. 5, it was confirmed that the amount of collagen synthesis was increased in the case of treatment with milk exosomes, and in particular, it was confirmed that the collagen synthesis promoting effect of the milk exosomes was significantly increased in a concentration-dependent manner.

[0105] 3-3. Confirmation of Elastase Activity Inhibitory Effect

[0106] Elastase inhibitory activity was measured according to the Cannell method (R. J. Cannell, S. J. Kellam, A. M. Owsianka, & J. M. Walker, 1988). By using N-succinyl-(L-Ala)3-p-nitroanilide (Sigma Aldrich Colo., USA) as a substrate, the production amount of p-nitroanilide produced from the substrate was measured at 415 nm at 37° C. for 20 minutes.

[0107] Specifically, after 20 μl of milk exosomes were treated with 120 μl of 50 mM Tris-HCl buffer (pH 8.0), 10 μl of elastase (1 unit/ml) and 50 μl of N-succinyl-(L-Ala)3-p-nitroanilide as an elastase substrate were added thereto and the resulting mixture was reacted at 37° C. for 20 minutes. After the reaction, absorbance was measured at 415 nm.

Inhibition rate (%)=[1−(absorbance of sample added group/absorbance of no addition group)]×100  [Equation 1]

[0108] As a result, as illustrated in FIG. 6, since it was confirmed that in the case of treatment with milk exosomes, the elastase activity inhibitory effect was remarkably increased compared to the control not treated with milk exosomes, it was confirmed that the elastase activity inhibitory effect was excellent.

[0109] 3-4. Confirmation of Collagenase Activity Inhibitory Effect

[0110] Collagenase inhibitory activity was measured according to the method of Wunsch and Heindrich (E. Wunsch, & H. G Heidrich, 1963).

[0111] Specifically, 4 mM CaCl.sub.2 was added to a 0.1 M Tris-HCL buffer (pH 7.5), 0.15 ml of 0.2 mg/mL collagenase (Sigma Aldrich Colo., USA) was added to a mixed solution of 0.25 ml of a substrate solution in which 0.3 mg/mL of 4-phenyl azobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Sigma Aldrich Colo., USA) was dissolved and 0.1 ml of milk exosomes, the resulting mixture was allowed to stand at room temperature for 20 minutes, then the reaction was stopped by adding 500 μL of 6% citric acid thereto, and then 2 mL of ethyl acetate was added thereto to measure the absorbance at 320 nm using a Vmax microplate spectrophotometer (Molecular Devices, Sunnyvale, Calif., USA). The collagenase inhibitory activity was expressed as the absorbance inhibition rate of the groups with and without the sample solution.

Inhibition rate (%)=[1−(absorbance of sample added group/absorbance of no addition group)]×100  [Equation 2]

[0112] As a result, as illustrated in FIG. 7, since it was confirmed that the collagenase activity inhibitory effect was remarkably increased in the case of treatment with milk exosomes, it was confirmed there is an excellent effect on the inhibition of the collagenase enzyme activity by the milk exosomes.

Experimental Example 4. Confirmation of Aging Amelioration Effect of Milk Exosomes

[0113] To investigate the effect of milk exosomes on a photoaging phenomenon caused by ultraviolet UVB, the amount and morphology of collagen fibers were observed by the Masson's trichrome staining method.

[0114] First, Neoderm-ED (Tego Science), which is an artificial skin composed of the epidermis and the dermis, was purchased and irradiated with 0.05 J/cm.sup.2 of ultraviolet rays once a day for 8 days, and treated with 50 μg/ml of milk exosomes once every other day for 8 days.

[0115] As a result, as illustrated in FIG. 8, it was confirmed that in the case of the treatment with the milk exosomes, the density of collagen fibers was dense and the arrangement was regular, whereas in the control not treated with the milk exosomes, the collagen fibers were destroyed, the density was sparse, and the arrangement was irregular, and the amount of collagen fibers was reduced.

[0116] Through the above results, it was confirmed that the milk exosomes had an excellent effect on alleviating skin aging caused by ultraviolet rays.

[0117] The above-described description of the present invention is provided for illustrative purposes, and those skilled in the field to which the present invention pertains will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are only exemplary in all aspects and are not restrictive. For example, each constituent element which is described as a singular form may be implemented in a distributed form, and similarly, constituent elements which are described as being distributed may be implemented in a combined form.

[0118] The scope of the present invention is represented by the following claims, and it should be interpreted that the meaning and scope of the claims and all the changes or modified forms derived from the equivalent concepts thereof fall within the scope of the present invention.