SKIN SOOTHING COMPOSITION INCLUDING EXOSOMES DERIVED FROM NATURAL EXTRACT

Abstract

Disclosed is a cosmetic composition for skin soothing. The cosmetic composition includes, as active ingredients, exosomes derived from naturally occurring deer antler velvet. The cosmetic composition can effectively soothe the skin whose condition has been abnormally altered (such as atopic dermatitis, inflammation, erythema, oxidation, cytotoxic agents in the skin, loss of moisture, melasma, itching, roughness or wrinkles) by various causes.

Claims

1. A method for soothing skin in a subject in need thereof, comprising administering to the subject a composition comprising deer antler velvet-derived exosomes as active ingredients.

2. The method according to claim 1, wherein the skin soothing is anti-inflammation, amelioration of atopic dermatitis, antioxidation or wrinkle improvement.

3. The method according to claim 1, wherein the exosomes have a diameter of 120 to 600 nm.

4. The method according to claim 1, wherein the composition is prepared into a powder, gel, cream, essence, lotion, sol-gel, emulsion, oil, wax, spray or mist.

5. The method according to claim 1, wherein the composition is prepared into a mask pack.

6. A method for ameliorating or treating dermatitis, atopic dermatitis or wrinkle in a subject in need thereof, comprising administering to the subject a composition comprising deer antler velvet-derived exosomes as active ingredients.

7. A method for preparing a skin soothing composition comprising deer antler velvet-derived exosomes as active ingredients, the method comprising a) washing and powdering deer antler velvet, b) dissolving the powder in an exosome extraction buffer, and c) isolating deer antler velvet-derived exosomes from the solution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0064] These and/or other aspects and advantages of the invention will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

[0065] FIGS. 1A, 1B and 1C show the contents of CD9 (FIG. 1A), CD63 (FIG. 1B), and CD81 proteins (FIG. 1C) in deer antler velvet-derived exosomes;

[0066] FIGS. 2A and 2B show the diameters of exosomes derived from deer antler velvet (FIG. 2A) and tortoise shell (FIG. 2B) determined by transmission electron microscopy and DLS.

[0067] FIG. 3 shows the effect of deer antler velvet-derived exosomes on cytotoxicity.

[0068] FIG. 4 shows the anti-atopic efficacy of deer antler velvet-derived exosomes (inhibitory efficacy on the production of overexpressed TARC induced by TNF-α and IFN-γ);

[0069] FIG. 5 shows the anti-atopic efficacy of deer antler velvet-derived exosomes (inhibitory efficacy on the production of overexpressed MDC induced by TNF-α and IFN-γ);

[0070] FIG. 6 shows the inhibitory efficacy of deer antler velvet-derived exosomes on nitric oxide production;

[0071] FIG. 7 shows the anti-inflammatory efficacy of deer antler velvet-derived exosomes; and

[0072] FIG. 8 shows the anti-wrinkle efficacy of deer antler velvet-derived exosomes.

DETAILED DESCRIPTION OF THE INVENTION

[0073] The present invention will be more specifically explained with reference to the following examples. It will be evident to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

EXAMPLES

1. Preparation of Deer Antler Velvet-Derived Exosomes (Experimental Example 1)

[0074] 10 g of deer antler velvet (Hans Medicine Co., Ltd., Korea) was washed with purified water, mixed, and dried to prepare a mixed sample. The mixed sample was pulverized into a finely divided powder, followed by drying. A 10-fold volume of a buffer (EXO-BB, System Biosciences, Palo Alto, Calif., USA) was added to the dry powder. The dry powder was dissolved by vortexing for 5 min. The solution was placed in a 2 mL collection tube, loaded onto a filter cartridge, and centrifuged at 18,000 G for 2 min. The down layer was transferred to a 1.5 mL microfuge tube. At that time, care was taken not to permit pellets to fall apart from the tube during centrifugation. A 2-fold volume of an exosome isolation buffer was added to the microfuge tube, followed by incubation at 4° C. for 48 h to increase the yield of exosomes. Thereafter, centrifugation was performed at 10,000 G for 60 min. The supernatant was removed from the microfuge tube to isolate deer antler velvet-derived exosomes in the form of pellets. The exosome pellets were redissolved in EXO-BB buffer using a sonicator and stored at 4° C. until use for subsequent experiments.

2. Preparation of Deer Antler Velvet Extract (Experimental Example 2)

[0075] A deer antler velvet extract was prepared, and its activity was compared with that of the deer antler velvet-derived exosomes. 100 g of deer antler velvet (Hans Medicine Co., Ltd., Korea) was washed with purified water, mixed, and dried to prepare a mixed sample. The mixed sample was pulverized into a finely divided powder, followed by drying. The dry powder was extracted by heating under reflux in 1 L of purified water as a solvent for 12 h, macerated, and filtered through a filter paper having a pore size of 1.2 μm. The filtrate was concentrated and dried under reduced pressure to prepare a deer antler velvet extract in the form of a dry powder.

3. Identification of the Deer Antler Velvet-Derived Exosomes

[0076] 1) Identification Using Exosome Markers

[0077] The contents of CD9, CD63, and CD81 proteins in the deer antler velvet-derived exosomes prepared in Experimental Example 1 were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit (System Biosciences, Palo Alto, Calif., USA). CD9, CD63, and CD81 proteins are typical exosome markers. A tortoise shell extract (sample 1) and a musk extract (sample 2) were used as controls for comparative experiments. The tortoise shell extract and the musk extract were prepared in the same manner as in Experimental Example 1, except that tortoise shell (Daon Co., Ltd., Korea) and musk (Seosahyang Co., Ltd., Korea) were used as raw materials, respectively. Tortoise shell and musk have been used as animal-derived medicines in Korea. As a result, the deer antler velvet-derived exosomes prepared in Experimental Example 1 were found to contain significantly larger amounts of exosome markers compared to the tortoise shell and musk extracts (samples 1 and 2), indicating the presence of a much greater number of exosomes in the deer antler velvet than in the animal-derived medicines (FIG. 1A: CD9, FIG. 1B: CD63, and FIG. 1C: CD81).

[0078] 2) Analysis Using Transmission Electron Microscope

[0079] The shapes and sizes of the deer antler velvet-derived exosomes prepared in Experimental Example 1 and the tortoise shell-derived exosomes (sample 1) were analyzed by transmission electron microscopy and dynamic light scattering (DLS). As a result, the deer antler exosomes and the tortoise shell-derived exosomes were substantially spherical in shape and had diameters of ˜60-600 nm (FIG. 2A: deer antler velvet-derived exosomes; FIG. 2B: tortoise shell-derived exosomes).

4. Evaluation of Effect of the Deer Antler Velvet-Derived Exosomes on Cytotoxicity

[0080] The effects of the deer antler velvet-derived exosomes prepared in Experimental Example 1 and the deer antler velvet extract prepared in Experimental Example 2 on the growth of human immortalized keratinocytes (HaCaT, ATCC) were evaluated by MTT colorimetric assay. First, human immortalized keratinocytes were inoculated into Dulbecco's modified Eagle's medium (DMEM, GIBCO) as a dedicated medium supplemented with 10% fetal bovine serum (FBS, Cambrex) in a 24-well cell culture dish at a density of 1×10.sup.5 and cultured under humidified conditions at 37° C. and 5% CO.sub.2 for 24 h. After removal of the medium, the culture was treated with different concentrations (10 and 100 μg/ml) of the deer antler velvet-derived exosomes prepared in Example 1 diluted with serum-free DMEM, followed by further culture for 24 h. The resulting culture was treated with MTT reagent (1 mg/ml). 2 h later, formazan produced inside the cells as a result of the MTT treatment was dissolved using DMSO and absorbance was measured at 570 nm. For comparison, the culture was treated with tacrolimus (1 and 10 μg/ml) as a positive control. As a result, it was found that the deer antler velvet extract and the deer antler velvet-derived exosomes did not significantly affect the viability of human immortalized keratinocytes, indicating that the deer antler velvet extract and the deer antler velvet-derived exosomes did not show toxicity (FIG. 3).

5. Evaluation of Anti-Atopic Efficacy of the Deer Antler Velvet-Derived Exosomes

[0081] Human immortalized keratinocyte cell line (HaCaT, 5×10.sup.5/well) was cultured in FBS DMEM for 24 h. Cells were co-treated with different concentrations (1% and 10% (v/v)) of the deer antler velvet-derived exosomes prepared in Experimental Example 1 and TNF-α and IFN-γ (10 ng/ml) diluted with FBS-free DMEM, followed by further culture for 24 h. The above procedure was repeated except that the deer antler velvet extract prepared in Experimental Example 2 was used instead of the deer antler velvet-derived exosomes prepared in Experimental Example 1. The supernatant of each well was quantified using a human TARC ELISA kit (R&D system) and a human MDC ELISA kit (R&D system). A group treated with the same amount of distilled water was used as a negative control and a group treated with tacrolimus was used as a positive control. Tacrolimus is a calcineurin inhibitor and is known to inhibit the phosphatase activity of calcineurin (Sieber, M., Karanik, M., Brandt, C., Blex, C., et al., 2007, Inhibition of calcineurin NFAT signaling by the pyrazolopyrimidine compound NCI3., European Journal of Immunology, 37, 2617-2626). A recent research has shown that tacrolimus administration is effective in patients with atopic dermatitis resistant to corticosteroids (Russell, J. J., 2002, Topical tacrolimus: a new therapy for atopic dermatitis. American Family Physician, 66, 1899-1902). Referring to FIG. 4, the treatment with the antler velvet-derived exosomes significantly suppressed the production of overexpressed TARC induced by TNF-α and IFN-γ. This effect was significantly improved in the test group treated with the antler velvet-derived exosomes at a concentration of 10% (v/v) compared to that in the positive control treated with tacrolimus. These results indicate that the deer antler velvet-derived exosomes can inhibit the expression of thymus and activation-regulated chemokine (TARC), a specific factor for atopic dermatitis, to effectively prevent and improve atopic dermatitis. Referring to FIG. 5, the treatment with the antler velvet-derived exosomes significantly suppressed the production of overexpressed MDC induced by TNF-α and IFN-γ. This effect was significantly improved in the test group treated with the antler velvet-derived exosomes at a concentration of 10% (v/v) compared to that in the positive control treated with tacrolimus. These results indicate that the deer antler velvet-derived exosomes are effective over tacrolimus as an immunosuppressant that has been used to treat atopic dermatitis and they can be used as natural medicinal ingredients to replace tacrolimus.

6. Evaluation of Inhibitory Efficacy of the Deer Antler Velvet-Derived Exosomes on Nitric Oxide Production

[0082] To evaluate the anti-inflammatory effect of the deer antler velvet-derived exosomes, the inhibition of production of nitric oxide (NO), a representative cytotoxic agent involved in inflammation induction, was measured. First, RAW 264.7 cells of a macrophage cell line were obtained from the Korean Cell Line Bank (KTCC, Seoul, Korea). Specifically, the cells were inoculated into Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 100 μg/ml penicillin, and 100 μg/ml streptomycin and cultured at 37° C. and 5% CO.sub.2. More specifically, the RAW 264.7 cells were seeded in a 96-well plate at a density of 1×10.sup.6 cells/mL (in DMEM) and cultured for 24 h. Cells were co-treated with different concentrations (1% and 10% (v/v)) of the deer antler velvet-derived exosomes prepared in Experimental Example 1 and a fresh medium containing LPS (1 μg/ml), which is known as an endotoxin, followed by further culture for 24 h. The above procedure was repeated except that the deer antler velvet extract prepared in Experimental Example 2 was used instead of the deer antler velvet-derived exosomes prepared in Experimental Example 1. Then, 100 μl of the cell culture supernatant was mixed with 100 μl of Griess reagent [1% (w/v) sulfanilamide and 0.1% (w/v) naphthylethylenediamine in 2.5% (v/v) phosphoric acid] and incubated in a 96-well plate for 10 min. Absorbance was measured at 540 nm using an ELISA reader to determine the amount of nitric oxide produced. The concentration of nitrite produced was calculated from a standard curve of sodium nitrite in DMEM. The inhibitory activity of each sample on nitric oxide production was evaluated based on a difference in the amount of nitrite produced between the LPS-treated control (Cont) and the untreated control (Nor). As a result, the deer antler velvet-derived exosomes were observed to suppress the production of nitric oxide in all treated groups compared to in the LPS-treated control (Cont), unlike the deer antler velvet extract, indicating their excellent anti-inflammatory effect (FIG. 6).

7. Evaluation of Anti-Inflammatory Efficacy of the Deer Antler Velvet-Derived Exosomes

[0083] RAW 264.7 cells of a macrophage cell line were obtained from the Korean Cell Line Bank (KTCC, Seoul, Korea). Specifically, the cells were inoculated into Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 100 μg/ml penicillin, and 100 μg/ml streptomycin and cultured at 37° C. and 5% CO.sub.2. More specifically, the RAW 264.7 cells were seeded in a 6-well plate at a density of 1×10.sup.6 cells/mL (in DMEM) and cultured for 24 h. Cells were co-treated with different concentrations (1% and 10% (v/v)) of the deer antler velvet-derived exosomes prepared in Experimental Example 1 and a fresh medium containing LPS (1 μg/ml), which is known as an endotoxin, followed by further culture for 24 h. The above procedure was repeated except that the deer antler velvet extract prepared in Experimental Example 2 was used instead of the deer antler velvet-derived exosomes prepared in Experimental Example 1. The mRNA expression level of the COX-2 gene was determined by RT-PCR. The primers used were as follows:

TABLE-US-00001 COX-2: Forward (SEQ ID No. 1) 5′-AAG ACT TGC CAG GCT GAA CT-3′, Reverse (SEQ ID No. 2) 5′-CTT CTG CAG TCC AGG TTC AA-3′. GAPDH: Forward (SEQ ID No. 3) 5′-TGA AGG TCG GTG TGA ACG GAT TTT GGC-3′, Reverse (SEQ ID No. 4) 5′-TGG TTC ACA CCC ATC ACA AAC ATG G-3′.

[0084] RT-PCR was performed using the above primers. The experimental results are summarized in FIG. 7. A significant inhibitory effect on COX-2 gene expression was observed in the group treated with the deer antler velvet-derived exosomes compared to in the negative control treated with LPS only and the group treated with the deer antler velvet extract, indicating that the deer antler velvet-derived exosomes can be used to treat and prevent inflammatory diseases or are effective in skin soothing.

8. Evaluation of Anti-Wrinkle Effect of the Deer Antler Velvet-Derived Exosomes

[0085] Normal human dermal fibroblasts (NHDFs) were obtained by skin biopsy from healthy young male volunteers. Cells were plated in a 100 mm tissue culture plate and cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin in a humid environment of 5% CO.sub.2 at 37° C. All experiments were conducted using only cells between passages 6 and 10. The normal human dermal fibroblasts were seeded in 40-mm tissue culture plates (1.2×10.sup.5 cells), cultured to a confluence of 80%, and washed twice with phosphate buffered saline (PBS), after which 1980 μl of fresh serum-free medium and 20 μl of sample were added to each well. After culture for 3 days, the culture was harvested and centrifuged at 7,500 rpm and 4° C. for 5 min. A change in the expression level of MMP-1 protein (Human Total MMP-1 kit (R&D Systems, Inc., Minneapolis, Minn., USA)) was monitored by ELISA. The treatment with the deer antler velvet-derived exosomes prepared in Experimental Example 1 led to a marked reduction in the expression of MMP-1 protein. Specifically, the inhibitory effects of the groups treated with the deer antler velvet-derived exosomes at concentration of 0.01%, 0.1%, and 1% were improved by 41%, 45%, and 47%, respectively, compared to those of the untreated groups (FIG. 8). These results indicate that the deer antler velvet-derived exosomes can effectively suppress the production of MMP-1 protein, which is the cause of wrinkles, to prevent and ameliorate skin wrinkles.

PREPARATIVE EXAMPLES

Preparative Example 1: Preparation of Cosmetic Product

[0086] According to a suitable process known in the art, a cosmetic product was produced to have the following composition: 2 ml of the deer antler velvet-derived exosomes, 7.0 mg of 1,3-butylene glycol, 1.0 mg of glycerin, 1.5 mg of polysorbate 60, 2.0 mg of lipophilic glyceryl stearate, 4.0 mg of mineral oil, 3.0 mg of cetearyl alcohol, a small amount of a preservative, an appropriate amount of a combined fragrance, and purified water (up to a total of 100 mg).

Preparative Example 2: Preparation of Pharmaceutical Products

[0087] A 0.9% isotonic solution (pH 7.0) was prepared using 2 ml of the deer antler velvet-derived exosomes, additives such as NaCl and NaOH, and 900 ml of water for injection. The isotonic solution was filtered three times through a PES 0.1 filter. After glass vial bottles, rubber stoppers, and aluminum caps were sterilized, the isotonic solution was divided into small portions (each 20 ml) and placed in the vial bottles. The vial bottles were capped with the rubber stoppers and the aluminum caps and sterilized in an autoclave under high pressure. After foreign matter was inspected using a foreign matter inspection machine, microbiological and endotoxin tests were conducted. Thereafter, the vials were labeled and kept refrigerated.

Preparative Example 3: Preparation of Granules

[0088] According to a suitable process known in the art, 2 ml of the deer antler velvet-derived exosomes, an appropriate amount of a vitamin mixture, 10 μg of biotin, 1.7 mg of nicotinamide, 50 μg of folic acid, 0.5 mg of calcium pantothenate, an appropriate amount of a mineral mixture, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate monobasic, 55 mg of dibasic calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed to prepare a health functional food.

Preparative Example 4: Preparation of Mask Packs

[0089] According to a suitable process known in the art, 0.05 wt % of the deer antler velvet-derived exosomes, 5.0 wt % of glycerin, 4.0 wt % of propylene glycol, 15.0 wt % of polyvinyl alcohol, 8.0 wt % of ethanol, 1.0 wt % of polyoxyethylene ether, 0.2 wt % of methyl para-oxybenzoate, an appropriate amount of a dye, and an appropriate amount of a fragrance were mixed to prepare mask packs.

[0090] Although the particulars of the present invention have been described in detail, it will be obvious to those skilled in the art that such particulars are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.