ANTI-AGING AND ANTIOXIDANT COSMETIC COMPOSITION CONTAINING BROCCOLI EXOSOMES AS ACTIVE INGREDIENT AND FUNCTIONAL COSMETICS COMPRISING SAME

Abstract

The present disclosure relates to a functional cosmetic composition using broccoli exosomes as an active ingredient and a functional cosmetic product containing the same. The broccoli exosomes have a lipid bilayer structure and are well absorbed into skin cells, and the absorbed broccoli exosomes remove reactive oxygen within skin cells and stimulate anti-oxidant signaling mechanisms, so they show anti-oxidant efficacy, and promote hyaluronic acid synthesis, and thus, they can be useful for preparation of functional cosmetics for skin aging prevention, skin whitening, wrinkle improvement and skin cell regeneration.

Claims

1. A method of exhibiting a cosmetic efficacy on a subject in need thereof, comprising administering isolated broccoli exosomes to the subject.

2. The method according to claim 1, wherein the broccoli exosomes are isolated from at least one selected from the group consisting of broccoli flowers, broccoli stems and broccoli sprouts.

3. The method according to claim 1, wherein the broccoli exosomes are comprised in a composition in an amount of 10.sup.6 to 10.sup.13 particles per 1 mL of the composition, and the administration of the broccoli exosomes is carried out by administering the composition to the subject.

4. The method according to claim 3, wherein the composition is formulated as one selected from the group consisting of aqueous solution, suspension, emulsion, cream, freezing solution, spray-dried powder and freeze-dried powder.

5. The method according to claim 1, wherein the broccoli exosomes comprise polyphenol.

6. The method according to claim 1, wherein the broccoli exosomes have an anti-aging or anti-oxidant use.

7. The method according to claim 3, wherein the composition is a cosmetic composition.

8. The method according to claim 7, wherein the cosmetic composition is a formulation selected from the group consisting of mist, serum, nourishing cosmetic water, soft cosmetic water, soft water, emulsion, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, powder, makeup base, essence, nourishing essence, packs, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, face wash, treatment, cosmetic liquid, cosmetic packs, ointment, gel, liniment, liquid, patches, spray, bath preparations, sunscreens, sun oil and hair products.

9. The method according to claim 1, wherein the cosmetic efficacy is at least one efficacy selected from the group consisting of skin aging prevention, skin whitening, wrinkle improvement, skin cell regeneration, skin moisturization, skin barrier improvement and anti-inflammation.

10. The method according to claim 7, wherein the cosmetic composition is prepared by a method, comprising: crushing broccoli; performing centrifugation; and performing tangential-flow filtration (TFF).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 is a schematic diagram of the process of the method for isolating plant exosomes.

[0043] FIG. 2 is the result of morphology analysis (TEM) of broccoli sprout exosomes.

[0044] FIG. 3 is the result of characteristic analysis (NTA and protein assay) of broccoli sprout exosomes.

[0045] FIG. 4 is the result of analysis of the polyphenol content and copper reducing ability of broccoli sprout exosomes.

[0046] FIG. 5 is the result of evaluation of melanin formation inhibitory ability of broccoli sprout exosomes.

[0047] FIG. 6 is the result of evaluation of melanin formation inhibitory ability in a cell model in which melanin formation is promoted by a-MSH of broccoli sprout exosomes.

[0048] FIG. 7 is the result of evaluation of collagen producing ability of broccoli sprout exosomes.

[0049] FIG. 8 is the result of evaluation of hyaluronic acid producing ability of broccoli sprout exosomes.

[0050] FIG. 9 is the result of evaluation of NO production inhibitory ability of broccoli sprout exosomes.

[0051] FIG. 10 is the result of evaluation of cytotoxicity of broccoli sprout exosomes on human fibroblasts (HDF), B16F10 Melanoma, and mouse macrophages (RAW 264.7).

MODE FOR INVENTION

[0052] Hereinafter, the present disclosure will be described in more detail by examples. These examples are intended to illustrate the present disclosure more specifically only, and it will be obvious to those skilled in the art to which the present disclosure pertains that the scope of the present disclosure is not limited by these examples.

EXAMPLE

<Example 1> Plant Exosome Separation

[0053] As raw materials, using broccoli and broccoli sprouts, aloe vera peels and spinach, plant exosomes were isolated therefrom using ultracentrifugation and tangential flow filtration (TFF) methods. Specifically, the cleanly washed plant materials were crushed and homogenized by using a blender in a weight ratio of 1:1(w/w) to 1:10(w/w) with phosphate-buffered saline (PBS). The supernatant was collected by centrifugation at 1,000g for 10 minutes, and the supernatant was centrifugated sequentially at 2000g for 20 minutes, at 3,000g for 30 minutes, and at 10,000g for 60 minutes. Then, the supernatant was ultracentrifuged at 4 C. at 100,000g for 70 minutes, and through this process, vacuoles were removed. Finally, the supernatant was discarded, and the remaining pellet was suspended, and the exosomes were purified through the TFF system. Specifically, the exosomes were purified by using a filter having a molecular cut off of 100 to 500 kDa in the TFF system, and other impurity particles smaller than pores of the filter were removed and the solution containing the exosomes was concentrated. The separated exosomes were stored frozen at 70 C. or less until use (FIG. 1).

<Example 2> Exosome Protein Content Evaluation

[0054] The protein content comprised in the broccoli sprout exosomes, aloe exosome and spinach exosomes was measured using Micro BCA Protein Assay Kit of Thermo Scientific company. Two groups of exosomes were used for comparative study, one is the exosome samples immediately after isolation and the other one is the exosome sample stored at least 70 C. for one month. Specifically, using a pipette, each standard and sample 1.0 mL were out in a test tube, and WR 1.0 mL was added to each tube, and then mixed. The tube was covered and incubated in a water bath at 37 C. for 30 minutes and then cooling was conducted at a room temperature. The instrument was zeroed in a cuvette filled with distilled water with a spectrophotometer set to 562 nm, and the absorbance of the samples was measured within 10 minutes. A standard curve was prepared by measuring total protein concentration versus protein standard and plotting average blank corrected 562 nm reading values for each standard. Using the standard curve, the protein content of the samples was measured and the result was shown in Table 1 below.

TABLE-US-00001 TABLE 1 Sample Protein content First measurement Broccoli sprout exosome 983.32 g/mL (immediately after Aloe exosome 299.19 g/mL exosome separation) Spinach exosome 542.74 g/mL Second measurement Broccoli sprout exosome 102.59 g/mL (after thawing) Aloe exosome 50.21 g/mL Spinach exosome 52.35 g/mL

[0055] As a result of the experiment, it was confirmed that the broccoli sprout exosomes contained protein of 100 g/mL or more per unit volume of 1 mL even after thawing. Further characteristic evaluation was performed using the broccoli sprout exosome which was confirmed as having a high protein content.

<Example 3> Characteristic Evaluation of Broccoli Sprout Exosome

[0056] For the broccoli sprout exosome isolated in Example 1, further exosome characteristic evaluation was conducted through the following method. As the shape of the exosome, a spherical microstructure of 200 nm or less was confirmed through a scanning electron microscope (TEM) (scale bar 50 nm) (FIG. 2). The microparticle size was analyzed through nanoparticle tracking analysis (NTA), and it was confirmed that it had a size of 50 to 300 nm (FIG. 3). Through nanoparticle tracking analysis (NTA), the exosome concentration of 110.sup.8 to 110.sup.13 per unit volume of 1 mL was confirmed (FIG. 3). The protein content in the exosome was further measured several times using Micro BCA Protein Assay Kit of Thermo Scientific company, and it was confirmed that it contained protein of 10 to 1,000 g/mL per unit volume of 1 mL (FIG. 3).

<Example 4> Evaluation of Polyphenol Content of Broccoli Sprout Exosome

[0057] For the broccoli sprout exosome isolated in Example 1, the polyphenol content in the exosome was evaluated through the following method. The polyphenol content in the broccoli exosome was measured using Phenolic Compounds Assay Kit of BioVision company, and the measurement method was performed according to the Kit manufacturer's protocol. It was confirmed that polyphenol of 50 to 500 pmol was contained inside the broccoli exosomes of 10.sup.8 (FIG. 4).

<Example 5> Evaluation of Anti-Oxidant Activity of Broccoli Sprout Exosome

[0058] For the broccoli sprout exosome isolated in Example 1, the polyphenol content in the exosome was evaluated through the following method. The anti-oxidant ability of the broccoli exosomes was evaluated using OxiSelect Total Antioxidant Capacity (TAC) Assay Kit of CELL BIOLABS inc. company, and the measurement method was performed according to the kit manufacturer's protocol. As a result of the evaluation, it was confirmed that the broccoli sprout exosome had ability to reduce Cu(II) ions of 10 to 100 M per unit volume of 1 mL into Cu(I) ions (FIG. 4).

<Example 6> Evaluation of In Vitro Whitening Efficacy of Broccoli Sprout Exosome

[0059] In order to evaluate the anti-oxidant efficacy of the broccoli sprout exosome isolated in Example 1, evaluation of whitening efficacy of the exosome was representatively carried out. Skin melanoma (B16F10) cells of 510.sup.3 to 110.sup.4 were treated with the sample comprising the broccoli exosomes, and after culturing for 3 days, the intracellular and extracellular melanin contents were measured. To determine the intracellular melanin contents, the absorbance of the solution at 475 nm was measured using a UV-visible spectrophotometer after destroying and lysing the cells. For the extracellular melanin, after collecting the cell culture solution cultured for 3 days, the absorbance of the solution at 490 nm was measured using a UV-visible spectrophotometer. As a negative control group, a growth medium (GM) without the exosomes was used, and as a positive control group, 0.5 mM arbutin which is widely used for whitening efficacy evaluation was used.

[0060] It was confirmed that the broccoli sprout exosome inhibited extracellular and intracellular melanin formation at a capacity of 510.sup.7 or more, and in particular, when 510.sup.8 was treated, there was a significant effect on inhibition of melanin formation (p<0.05) (FIG. 5).

[0061] For the whitening efficacy of the broccoli sprout exosome separated in Example 1, evaluation was further conducted using a model in which skin melanoma (B16F10) cells were treated with 100 nM -MSH to promote melanin synthesis and secretion. After treating the cell culture solution comprising the -MSH and broccoli exosomes, it was cultured for 3 days, and the total contents of intracellular and extracellular melanin were measured using a UV-visible spectrophotometer. For protein content measurement, after quantifying it according to Bradford method, the melanin amount was converted into the melanin amount per protein. As a negative control group, a cell culture medium without treating the exosome was used, and as a positive control group, 0.36 mM arbutin which is widely used for whitening efficacy evaluation was used. The broccoli sprout exosome inhibited the total melanin formation at a capacity of 210.sup.7 or more (p<0.05) (FIG. 6).

<Example 7> Evaluation of In Vitro Collagen Formation of Broccoli Sprout Exosome

[0062] In order to evaluate the efficacy on collagen formation of the broccoli sprout exosome separated in Example 1, the exosome was treated to human dermal fibroblasts (HDFs) to confirm the amount of produced intracellular collagen. The cell culture solution comprising the broccoli exosomes was treated to the HDFs cells, and after culturing them for 2 days, the amount of procollagen was measured using the supernatant by centrifugation. For the amount of procollagen, the absorbance at 450 nm was measured using Procollagen Type I C-Peptide (PIP) ELISA Kit. As a negative control group, the cell culture solution without any treatment (GM) was used, and as a positive control group, TGF-1 which is known to induce collagen production was used. It was confirmed that the broccoli sprout exosome had a significant effect on increasing intracellular collagen production compared to the negative control group at a concentration of 1.310.sup.6 to 110.sup.7 (p<0.05) (FIG. 7). This has efficacy similar to the positive control group, TGF-1.

<Example 8> Evaluation of In Vitro Hyaluronic Acid Production of Broccoli Sprout Exosome

[0063] In order to evaluate the efficacy on hyaluronic acid production of the broccoli sprout exosome isolated in Example 1, the exosome was treated to human dermal fibroblasts (HDFs) to confirm the amount of produced intracellular hyaluronic acid. The sample comprising the broccoli sprout exosomes was treated to the HDFs, and after culturing them for 2 days, the supernatants were collected by centrifugation and used as the test samples to measure the amount of produced hyaluronic acids. For determination of the amount of produced hyaluronic acid, the absorbance at 450 nm was measured using Human Hyaluronic Acid (HA) ELISA Kit. As a negative control group, the growth media without exosomes (GM) was used, and as a positive control group, retinoic acid which is used to promote hyaluronic acid production was used. It was confirmed that the broccoli sprout exosome had a significant effect on increasing intracellular hyaluronic acid production compared to the negative control group at a concentration of 2.510.sup.6 to 110.sup.7 (p<0.05) (FIG. 8). This has efficacy similar to the positive control group, retinoic acid.

<Example 9> Evaluation of In Vitro Nitric Oxide Production of Broccoli Sprout Exosome

[0064] In order to evaluate the efficacy of inhibiting Nitric Oxide (NO) production of the broccoli sprout exosome isolated in Example 1, the exosome was treated to mouse macrophages (RAW 264.7 cells) to confirm whether it inhibits NO production. After culturing the RAW 264.7 cells of 1.810.sup.5, it was exchanged with a new cell culture solution comprising lipopolysaccharide (LPS) 1 g/mL and the broccoli sprout exosomes, and then it was cultured for 24 hours. In 24 hours, the culture solution and an NO detection reagent were reacted for 10 minutes, and then the absorbance of the solution at 540 nm was measured. The amount of produced NO was evaluated by correcting the total protein amount, and as a positive control group, 7.8 g/mL of dexamethasone, which has an effect of inflammation inhibition, was used.

[0065] It was confirmed that the broccoli sprout exosome had efficacy to inhibit NO production at 210.sup.7 or more (p<0.05) (FIG. 9). Preferably, it effectively inhibits NO production of cells in the exosomes of 210.sup.7 to 410.sup.7, and this has efficacy similar to the positive control group, dexamethasone.

<Example 10> Evaluation of Cytotoxicity of Broccoli Sprout Exosome

[0066] In order to confirm the cytotoxicity of the broccoli sprout exosome isolated in Example 1, after treating the exosome, the cell viability of 2 kinds of human cells and mouse macrophages was evaluated using MTT assay.

[0067] After inoculating human fibroblasts at a concentration of 510.sup.4 cells/well and culturing them for 24 hours, the broccoli sprout exosomes were treated and then they were further cultured for 24 hours. After inoculating B16F10 melanoma cells at a concentration of 1.510.sup.4 cells/well and culturing them for 24 hours, the broccoli exosomes were treated and then they were cultured for 72 hours. After inoculating mouse macrophages (RAW 264.7) at a concentration of 1.810.sup.5 cells/well and culturing them for 20 hours, the broccoli exosomes were treated and then they were cultured for 24 hours. An MMT reagent was treated to each well at a concentration of 0.05% of the cell culture medium, and then they were incubated for 4 hours. After removing the culture solution, DMSO was inoculated by 1 mL/well, and then it was stirred for 10 minutes, and then the DMSO solution was recovered to measure the absorbance at 540 nm. The cell viability was written as a percentage compared to the exosome untreated group (FIG. 10). As a result of the experiment, it was confirmed that the broccoli sprout exosome did not exhibit the cytotoxicity at all concentrations.