NOVEL COMPOSITION COMPRISING GARDENIA JASMINOIDES-DERIVED EXOSOMES AS ACTIVE INGREDIENT

Abstract

A composition containing Gardenia jasminoides-derived exosomes as an active ingredient is provided for skin elasticity improvement, skin wrinkle reduction, skin regeneration, skin tone improvement, skin brightness improvement, skin whitening, wound healing and/or wound healing acceleration.

Claims

1. A method for skin elasticity improvement, skin wrinkle reduction, skin regeneration, skin tone improvement, skin brightness improvement or skin whitening, the method comprising topically administering a composition to a skin of a subject in need thereof, wherein the composition comprises exosomes derived from Gardenia jasminoides as an active ingredient.

2. The method of claim 1, wherein the composition is a shampoo, a soap, a rinse, a surfactant-containing cleanser, a cream, a lotion, an ointment, a tonic, a treatment, a conditioner, a suspension, an emulsion, a paste, a gel, an oil, a wax, a spray, an aerosol, a mist, or a powder.

3. The method of claim 1, wherein the composition is a cream or a lotion.

4. The method of claim 1, wherein the subject is at least one selected from the group consisting of a human, a dog, a cat, a rodent, a horse, a cattle, a monkey and a pig.

5. A method for skin elasticity improvement, skin wrinkle reduction, skin regeneration, skin tone improvement, skin brightness improvement or skin whitening, the method comprising: (a) (a1) applying a composition comprising exosomes derived from Gardenia jasminoides as an active ingredient to a skin of a subject in need thereof; or (a2) contacting or attaching a patch, a mask pack or a mask sheet, which has the composition applied thereto or soaked therein, to the skin; or (a3) sequentially performing (a1) and (a2).

6. The method of claim 5, wherein the composition is a lotion or a cream in step (a).

7. The method of claim 5, further comprising step (b) removing the patch, the mask pack or the mask sheet from the skin after step (a2) or (a3), and again applying the composition to the skin.

8. The method of claim 7, wherein the composition is a lotion or a cream in step (b).

9. The method of claim 5, wherein the subject is at least one selected from the group consisting of a human, a dog, a cat, a rodent, a horse, a cattle, a monkey and a pig.

10. A method for healing wound or accelerating wound healing of a subject in need thereof, the method comprising topically administering a composition to a skin or a wounded area of the subject, wherein the composition comprises exosomes derived from Gardenia jasminoides as an active ingredient.

11. The method of claim 10, wherein the composition is topically administered to a target area of the skin, by injection, microneedling, spread, spray, transdermal delivery using a patch or a sheet, iontophoresis, or a combination thereof.

12. The method of claim 10, wherein the composition is an injectable formulation, an infusion formulation, a spray formulation, a liquid formulation, or a patch formulation.

13. The method of claim 10, wherein the subject is at least one selected from the group consisting of a human, a dog, a cat, a rodent, a horse, a cattle, a monkey and a pig.

14. A method for healing wound or accelerating wound healing of a subject in need thereof, the method comprising administering an effective amount of a composition to a skin or a wounded area of the subject, wherein the composition comprises exosomes derived from Gardenia jasminoides as an active ingredient.

15. The method of claim 14 wherein the composition is administered to the subject by injection, microneedling, or a combination thereof.

16. The method of claim 14, wherein the composition is an injectable formulation, an infusion formulation, or a liquid formulation.

17. The method of claim 14, wherein the subject is at least one selected from the group consisting of a human, a dog, a cat, a rodent, a horse, a cattle, a monkey and a pig.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0019] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0020] FIG. 1 is a graph showing the particle size distribution and particle number obtained by performing nanoparticle tracking analysis (NTA) of exosomes derived from Gardenia jasminoides of the present invention.

[0021] FIG. 2 depicts fluorescence microscopic images of cells showing that fluorescence-stained exosomes derived from Gardenia jasminoides were delivered into human dermal fibroblasts (green: exosomes delivered into cells; and blue: cell nucleus).

[0022] FIG. 3 is a graph showing that the relative amount of collagen increased after human dermal fibroblasts were treated with exosomes derived from Gardenia jasminoides.

[0023] FIG. 4 is a graph showing that the migration of human dermal fibroblasts remarkably increased after scratch-wounds were treated with exosomes derived from Gardenia jasminoides.

[0024] FIG. 5 depicts fluorescence microscopic images of cells showing that fluorescence-stained exosomes derived from Gardenia jasminoides were delivered into melanoma cells (green: exosomes delivered into cells; and blue: cell nucleus).

[0025] FIG. 6 is a graph showing that the amount of melanin production decreased when melanoma cells were treated with exosomes derived from Gardenia jasminoides.

DETAILED DESCRIPTION OF INVENTION

[0026] The present invention provides a composition for skin elasticity improvement, skin wrinkle reduction, skin regeneration, skin tone improvement, skin brightness improvement, skin whitening, wound healing and/or wound healing acceleration, containing exosomes derived from Gardenia jasminoides as an active ingredient.

[0027] As used herein, the term topical administration for example means to apply or spread a composition that is suitable for topical application, onto the surface of skin including the scalp, not limited thereto. Thus, the topical administration includes administering a composition to the skin and/or scalp by topical routes such as injection, microneeding, spread, spray, transdermal delivery using a patch or a sheet, iontophoresis, and the like.

[0028] As used herein, the term exosomes refers to nano-sized vesicles secreted or released from plant cells into extracellular spaces and having a membrane structure, and is also referred to as exosome-like vesicles or exosome-like particles.

[0029] As used herein, the term skin elasticity refers to a feature in which skin deformed by an external force easily returns to its original shape when the external force is removed. The term skin wrinkles refers to fine lines caused by skin aging. Skin wrinkles may be caused by genetic factors, reduction in collagen and elastin present in the skin dermis, external environmental factors, or the like. Accordingly, the term skin wrinkle reduction or improvement as used herein refers to suppressing or inhibiting the formation of wrinkles on the skin, or reducing already formed wrinkles.

[0030] Meanwhile, the term skin tone, as used herein, refers to the darkness or lightness of skin color. As used herein, the term skin whitening includes increasing the brightness of the skin whose brightness has decreased due to an excess of pigments such as melanin, or maintaining the brightness of the skin at a certain level.

[0031] As used herein, the term wound means a condition in which a part or all of the body is injured, and is intended to encompass pathological conditions in which a tissue constituting an inside or an external surface of the body, for example, skin, muscle, nerve tissue, bone, soft tissue, internal organ, or blood vessel tissue, is damaged or destroyed. As an example, not limiting the present invention, examples of the wound include abrasion, laceration, stab wound, incised wound, avulsion, bedsore, tissue destruction caused by irradiation, penetrated wound, gunshot wound, burn, frostbite, surgical wound, sutures after plastic surgery, wound caused by chemical substance and so on, and may include any damage to any part of an individual.

[0032] As used herein, the term iontophoresis refers to a method of flowing a microcurrent through a skin to which an active ingredient has been applied, generating a potential difference thereby and changing the electrical environment of the skin, and thus allowing an ionized active ingredient to penetrate the skin by electrical repulsion. Examples of iontophoresis that is used in one embodiment of the present invention include: a method of introducing a microcurrent into a skin by allowing the microcurrent to flow from an external power source into an electrode patch on the skin, the microcurrent being generated by the external power source; a method of introducing a microcurrent into a skin, the microcurrent being generated by a battery provided in an electrode patch on the skin; and a method of introducing a microcurrent into a skin through a patch on the skin provided with a reverse electrodialysis device, the microcurrent being generated by the concentration difference between high concentration electrolyte solution and low concentration electrolyte solution in the reverse electrodialysis device. However, the present invention is not limited thereto, and various types of iontophoresis may, of course, be used.

[0033] As used herein, the term Chija refers to the fruit of Gardenia jasminoides.

[0034] As used herein, the term exosomes derived from Gardenia jasminoides is meant to include all exosomes isolated from, for example, a culture medium of Gardenia jasminoides plant cells, a culture medium of Gardenia jasminoides callus, a culture medium of Gardenia jasminoides plant stem cells, Chija or Gardenia jasminoides juice, or a biological solution of Gardenia jasminoides equivalent thereto, or derived (e.g., secreted or released) from plant cells or plant stem cells of Gardenia jasminoides.

[0035] In the composition according to one embodiment of the present invention, the exosomes derived from Gardenia jasminoides may be obtained by isolation and purification from a culture medium of Gardenia jasminoides flower-derived callus.

[0036] The composition containing exosomes derived from Gardenia jasminoides as an active ingredient according to one embodiment of the present invention may exhibit at least one effect of skin elasticity improvement, skin wrinkle reduction, skin regeneration, skin tone improvement, skin brightness improvement, skin whitening, wound healing and/or wound healing acceleration.

[0037] The composition containing exosomes derived from Gardenia jasminoides as an active ingredient according to the present invention may be a cosmetic composition or a pharmaceutical composition.

[0038] The present invention provides a cosmetic composition for skin elasticity improvement, skin wrinkle reduction, skin regeneration, skin tone improvement, skin brightness improvement and/or skin whitening, containing exosomes derived from Gardenia jasminoides as an active ingredient. For example, the cosmetic composition may be a shampoo, a soap, a rinse, a surfactant-containing cleanser, a cream, a lotion, an ointment, a tonic, a treatment, a conditioner, a suspension, an emulsion, a paste, a gel, an oil, a wax, a spray, an aerosol, a mist, or a powder, and is preferably a lotion or a cream.

[0039] The present invention also provides a pharmaceutical composition for wound healing and/or wound healing acceleration, containing exosomes derived from Gardenia jasminoides as an active ingredient.

[0040] As an example not limiting the present invention, the pharmaceutical composition according to one embodiment of the present invention may be administered or treated by injection, microneedling, iontophoresis, application, or a combination thereof. For example, the pharmaceutical composition may be an injectable formulation, an infusion formulation, a spray formulation, a liquid formulation, or a patch formulation.

[0041] In one embodiment of the present invention, when the composition is used as a pharmaceutical composition, it may include pharmaceutically acceptable carriers, excipients or diluents. The carriers, excipients and dilutes include, but are not limited to, lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, the effective amount of the pharmaceutical composition according to one embodiment of the present invention means the amount required for administration in order to achieve the effects of wound healing and/or wound healing acceleration.

[0042] The content of the pharmaceutical composition according to one embodiment in a formulation may be suitably selected depending on the kind, amount, form and the like of additional components as described above. For example, the pharmaceutical composition of the present invention may be contained in an amount of about 0.1 to 99 wt %, preferably about 10 to 90 wt %, based on the total weight of an injectable formulation. Furthermore, the suitable dose of the pharmaceutical composition according to one embodiment of the present invention may be adjusted depending on the severity of disease, the type of formulation, formulating method, patient's age, sex, body weight, health condition, diet, excretion rate, the period of administration, and the regime of administration. For example, when the pharmaceutical composition according to one embodiment of the present invention is administered to an adult, it may be administered once to several times at a dose of 0.001 mg/kg to 100 mg/kg per day.

[0043] Meanwhile, when the composition according to one embodiment of the present invention is prepared as a cosmetic composition, it may suitably contain components which are generally used in cosmetic products, for example, moisturizers, antioxidants, oily components, UV absorbers, emulsifiers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, and various skin nutrients, etc., as needed, within the range that does not impair the effect of the present invention.

[0044] Furthermore, the cosmetic composition according to one embodiment of the present invention may include, in addition to the exosomes derived from Gardenia jasminoides, an agent for improving skin condition, an antioxidant, an anti-aging agent, a whitening agent and/or a moisturizer, which have been used in the prior art, within the range that does not impair the effects (e.g., skin elasticity improvement, skin wrinkle reduction, skin regeneration, skin tone improvement, skin brightness improvement, skin whitening, skin beauty, etc.).

[0045] The cosmetic composition according to one embodiment of the present invention may be used in various forms, for example, a patch, a mask pack, a mask sheet, a cream, a tonic, an ointment, a suspension, an emulsion, a paste, a lotion, a gel, an oil, a pack, a spray, an aerosol, a mist, a foundation, a powder, and an oilpaper.

[0046] The cosmetic composition according to one embodiment of the present invention may be used for the purpose of skin elasticity improvement, skin wrinkle reduction, skin regeneration, skin tone improvement, skin brightness improvement and/or skin whitening, and may be prepared as any formulation which is generally prepared in the art. For example, it may be formulated as a patch, a mask pack, a mask sheet, a skin softener, a nutrition, an astringent lotion, a nourishing cream, a massage cream, an eye cream, a cleansing cream, an essence, an eye essence, a cleansing lotion, a cleansing foam, a cleansing water, a sunscreen, a lipstick, a soap, a shampoo, a surfactant-containing cleanser, a bath preparation, a body lotion, a body cream, a body oil, a body essence, a body cleanser, a hairdye, a hair tonic, etc., without being limited thereto.

[0047] The cosmetic composition according to one embodiment of the present invention may contain components which are commonly used in cosmetic products. For example, the cosmetic composition may contain conventional adjuvants and carriers, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances. In addition, other components in each formulation for the cosmetic composition may be suitably selected without difficulty by those skilled in the art depending on the type or intended use of the cosmetic composition.

[0048] Another embodiment of the present invention provides a cosmetic method for regulating mammalian skin conditions, except for treatment purposes, using the cosmetic composition. In the cosmetic method of the present invention, the expression regulating skin conditions means improving skin conditions and/or prophylactically regulating skin conditions, and the expression improving skin conditions means a visually and/or tactilely perceivable positive change in the appearance and feeling of skin tissue. For example, the expression improving skin conditions may include skin elasticity improvement, skin wrinkle reduction, skin regeneration, skin tone improvement, skin brightness improvement, and/or skin whitening.

[0049] The cosmetic method according to one embodiment of the present invention includes: (a) applying the cosmetic composition directly to a mammalian skin; or (b) contacting or attaching a patch, a mask pack or a mask sheet, which has the cosmetic composition applied thereto or soaked therein, to the mammalian skin; or sequentially performing (a) and (b). In step (a), the cosmetic composition may be a lotion or a cream.

[0050] Alternatively, the cosmetic method according to one embodiment of the present invention may further comprise (c) removing the patch, the mask pack or the mask sheet from the mammalian skin after step (b), and applying the cosmetic composition to the mammalian skin. In step (c), the cosmetic composition may be a lotion or a cream.

[0051] In the cosmetic method according to one embodiment of the present invention, the mammal may be a human, a dog, a cat, a rodent, a horse, a cattle, a monkey, or a pig.

[0052] In addition, the present invention provides a method for healing wound or accelerating wound healing, the method comprising administering a therapeutically effective amount of the pharmaceutical composition to a mammal, or applying the pharmaceutical composition to a skin or a wounded area. The mammal may be a human, a dog, a cat, a rodent, a horse, a cattle, a monkey, or a pig.

Advantageous Effects

[0053] The composition according to the present invention is less likely to contain impurities such as residual solvents or growth regulators compared to conventional hot-water extracts or solvent extracts of fruits or flowers of Gardenia jasminoides, filtrates of such extracts, and culture media of Gardenia jasminoides callus, and has excellent effects on skin elasticity improvement, skin wrinkle reduction, skin regeneration, skin tone improvement, skin brightness improvement, skin whitening, wound healing, or wound healing acceleration.

[0054] It should be understood that the scope of the present invention is not limited to the aforementioned effects.

EXAMPLES

[0055] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only to illustrate the present invention and are not intended to limit or restrict the scope of the present invention. Those that can be easily inferred by those skilled in the art from the detailed description and examples of the present invention are interpreted as falling within the scope of the present invention. References referred to in the present invention are incorporated herein by reference.

[0056] Throughout the present specification, it is to be understood that, when any part is referred to as comprising any component, it does not exclude other components, but may further include other components, unless otherwise specified.

Example 1: Preparation of Culture Media of Gardenia jasminoides Callus

[0057] According to preparation and culture methods for plant stem cells known in the art, calluses were induced from Gardenia jasminoides flowers, and cells of the induced Gardenia jasminoides callus were cultured. In addition, a callus having a good growth state was selected and cultured in large amounts, thereby preparing culture media (conditioned media) of Gardenia jasminoides callus.

Example 2: Preparation of Gardenia jasminoides-Derived Exosomes

[0058] The culture media of Gardenia jasminoides callus prepared as described in Example 1 were purchased from Biospectrum Corporation (located in Gyeonggi-do, Korea and supplying culture media of Gardenia jasminoides callus). The culture media of Gardenia jasminoides callus were filtered through a 0.22 ?m filter to remove impurities such as cell debris, waste products and large particles. Gardenia jasminoides-derived exosomes were isolated from the filtered culture media by tangential flow filtration (TFF) method.

[0059] The size and concentration of the isolated Gardenia jasminoides-derived exosomes were analyzed by nanoparticle tracking analysis (NTA) using NS300 (purchased from Malvern Panalytical) (FIG. 1).

Example 3: Evaluation of Delivery Ability of Gardenia jasminoides-Derived Exosomes into Dermal Fibroblasts

[0060] In order to examine whether the Gardenia jasminoides-derived exosomes would be delivered into human dermal fibroblasts (purchased from ATCC), the following analysis was performed. To fluorescence-stain the membrane of the Gardenia jasminoides-derived exosomes prepared in Example 2, the exosomes were allowed to react with PKH67 fluorescence dye (purchased from Sigma-Aldrich). After the reaction, the reaction solution was fractionated with a MiniTrap-25 column (purchased from Cytiva) to remove free PHK67 that was not stained in the exosome membrane. A negative control was prepared by allowing PKH67 fluorescence dye to react with a buffered solution and fractionating the reaction product with a MiniTrap-25 column. The exosomes stained with PKH67 were incubated with pre-cultured human dermal fibroblasts, and then whether the exosomes would be delivered into the cells over time was observed using a fluorescence microscope. Hoechst fluorescence dye (purchased from Thermo Fisher) was used to stain the cell nucleus, and CellMask Orange fluorescence dye (purchased from Thermo Fisher) was used to stain the cytoplasm. As a result of examining whether the exosomes would be delivered into the cells, it was confirmed that the fluorescence-stained exosomes were delivered into the cells and green fluorescence accumulated in the cells over time (FIG. 2).

Example 4: Evaluation of Effect of Stimulating Collagen Production

[0061] Human dermal fibroblasts (purchased from ATCC) dispersed in DMEM medium containing fetal bovine serum were dispensed into a multiwell plate, and then cultured for 24 hours. Thereafter, the culture media of Gardenia jasminoides callus prepared in Example 1 or the Gardenia jasminoides-derived exosomes prepared in Example 2 were diluted in serum-free medium, and then the human dermal fibroblasts were treated with each of the dilutions and cultured for 24 hours. To evaluate the collagen production effect using human dermal fibroblasts, experimental groups were classified as follows:

[0062] (1) Negative control group (indicated as N.C. in FIG. 3): an experimental group treated with a serum-free medium alone;

[0063] (2) Group treated with culture media of Gardenia jasminoides callus (indicated as Gardenia Callus CM in FIG. 3): an experiment group treated with the culture media of Gardenia jasminoides callus (prepared in Example 1) diluted in serum-free medium (treatment concentrationslow concentration: 1?10.sup.8 particles/mL, and high concentration: 1?10.sup.9 particles/mL); and

[0064] (3) Group treated with Gardenia jasminoides-derived exosomes (indicated as Gardenia Exosome in FIG. 3): an experiment group treated with the Gardenia jasminoides-derived exosomes (prepared in Example 2) diluted in serum-free medium (treatment concentrationslow concentration: 1?10.sup.8 particles/mL, and high concentration: 1?10.sup.9 particles/mL).

[0065] According to each experimental group described above, the human dermal fibroblasts were treated and cultured for 24 hours. The culture media were collected and centrifuged, and then the centrifuged media were prepared. The amount of collagen, which was synthesized from the human dermal fibroblasts and accumulated in the culture media, was measured using an EIA kit (purchased from Takara) for procollagen type I C-peptide (PIP). The measured amount of collagen was normalized by dividing it by the total number of cells measured using an MTT assay kit (purchased from Sigma-Aldrich) to determine the relative amount of collagen.

[0066] As a result, it was confirmed that when the human dermal fibroblasts were treated with a high concentration of the Gardenia jasminoides-derived exosomes of the present invention, the collagen synthesis in the human dermal fibroblasts remarkably increased, compared to the negative control, and the Gardenia jasminoides-derived exosomes was superior to the culture media of Gardenia jasminoides callus in view of the effect of increasing collagen synthesis (FIG. 3).

[0067] From the above experimental results, it can be seen that the Gardenia jasminoides-derived exosomes of the present invention have better effects on increasing the collagen synthesis, that is, skin elasticity improvement, wrinkle reduction and/or skin regeneration, than the culture media of Gardenia jasminoides callus.

[0068] As can be seen from the above results, the Gardenia jasminoides-derived exosomes of the present invention has a useful functional activity as a functional cosmetic for skin elasticity improvement, wrinkle reduction and/or skin regeneration, that is, an activity of increasing the collagen synthesis. Thus, the Gardenia jasminoides-derived exosomes of the present invention are useful as an active ingredient of a cosmetic composition for skin elasticity improvement, wrinkle reduction and/or skin regeneration.

Example 5: Evaluation of Skin Regeneration Effect Using Dermal Fibroblasts

[0069] To evaluate whether the exosomes prepared as described in Example 2 promotes wound healing in human dermal fibroblasts (purchased from ATCC), scratch-wound assay was performed. Human dermal fibroblasts dispersed in a DMEM containing fetal bovine serum were seeded into a culture plate for wound induction (ImageLock Plate; purchased from EssenBio) at a density of 5,000 cells/well and cultured for 24 hours at 37? C. under 5% CO.sub.2. After the cells reached a confluency of 90% or more, scratches were made using a WoundMaker (purchased from EssenBio). To evaluate the skin regeneration effect using human dermal fibroblasts, experimental groups were classified as follows:

[0070] (1) Negative control group (indicated as N.C. in FIG. 4): an experimental group treated with a serum-free medium alone;

[0071] (2) Positive control group (indicated as P.C. in FIG. 4): an experimental group treated with a medium containing 10% fetal bovine serum;

[0072] (3) Group treated with culture media of Gardenia jasminoides callus (indicated as Gardenia Callus CM in FIG. 4): an experiment group treated with the culture media of Gardenia jasminoides callus (prepared in Example 1) diluted in serum-free medium (treatment concentrationslow concentration: 5?10.sup.8 particles/mL, and high concentration: 1?10.sup.9 particles/mL); and

[0073] (4) Group treated with Gardenia jasminoides-derived exosomes (indicated as Gardenia Exosome in FIG. 4): an experiment group treated with the Gardenia jasminoides-derived exosomes (prepared in Example 2) diluted in serum-free medium (treatment concentrationslow concentration: 5?10.sup.8 particles/mL, and high concentration: 1?10.sup.9 particles/mL).

[0074] Thereafter, each of the experimental groups was subjected to Scratch-Wound, and the human dermal fibroblasts were cultured at 37? C. under 5% CO.sub.2 for 12 hours. The wound healing efficacy in each experimental group was measured using Incucyte (purchased from Sartorius).

[0075] As a result of measuring the wound healing efficacy, it was confirmed that the Gardenia jasminoides-derived exosomes (low and high concentrations) of the present invention remarkably increased the migration of the human dermal fibroblasts, compared to the negative control, and was superior to the same concentration of the culture media of Gardenia jasminoides callus (low and high concentrations) in view of the effect of increasing the migration of the human skin fibroblasts (FIG. 4).

[0076] As can be seen from the above experimental results, the Gardenia jasminoides-derived exosomes of the present invention have an excellent effect of promoting the migration of human dermal fibroblasts, that is, an excellent wound healing effect or skin regeneration effect, as compared with the culture media of Gardenia jasminoides callus.

[0077] Therefore, the Gardenia jasminoides-derived exosomes of the present invention are useful as an active ingredient of a cosmetic composition for skin elasticity improvement, wrinkle reduction and/or skin regeneration, and as an active ingredient of a pharmaceutical composition for wound healing or wound healing acceleration.

Example 6: Evaluation of Delivery Ability of Gardenia jasminoides-Derived Exosomes into Melanoma Cells

[0078] In order to examine whether the Gardenia jasminoides-derived exosomes would be delivered into mouse melanoma cells (B16F10; purchased from ATCC), the following analysis was performed. To fluorescence-stain the membrane of the Gardenia jasminoides-derived exosomes prepared in Example 2, the exosomes were allowed to react with PKH67 fluorescence dye (purchased from Sigma-Aldrich). After the reaction, the reaction solution was fractionated with a MiniTrap-25 column (purchased from Cytiva) to remove free PHK67 fluorescence dye that was not stained in the exosome membrane. A negative control was prepared by allowing PKH67 fluorescence dye to react with a buffered solution and fractionating the reaction product with a MiniTrap-25 column. The exosomes stained with PKH67 were incubated with pre-cultured mouse melanoma cells, and then whether the exosomes would be delivered into the cells over time was observed using a fluorescence microscope. Hoechst fluorescence dye (purchased from Thermo Fisher) was used to stain the cell nucleus, and CellMask Orange fluorescence dye (purchased from Thermo Fisher) was used to stain the cytoplasm. As a result of examining whether the exosomes would be delivered into the cells, it was confirmed that the fluorescence-stained exosomes were delivered into the cells and green fluorescence accumulated in the cells over time (FIG. 5).

Example 7: Melanogenesis Inhibitory Effect of Gardenia jasminoides-Derived Exosomes

[0079] The whitening effect of the Gardenia jasminoides-derived exosomes was evaluated through the degree of inhibition of melanogenesis in melanoma cells. The melanoma cells are cells (B16F10; purchased from ATCC) derived from mouse melanoma and are cells that secrete a black pigment called melanin. Melanoma cells were seeded into a 48-well plate at 8,000 cells per unit area and cultured for 24 hours at 37? C. under 5% CO.sub.2.

[0080] Thereafter, the culture media of Gardenia jasminoides callus prepared in Example 1 or the Gardenia jasminoides-derived exosomes prepared in Example 2 were diluted in a medium mixed with a-MSH, a melanin synthesis stimulant, and then melanoma cells were treated with the diluted culture media or exosomes and cultured for 48 hours. To evaluate the melanogenesis inhibitory efficacy using melanoma cells, the experimental groups were classified as follows:

[0081] (1) Negative control group (indicated as N.C. in FIG. 6): an experimental group treated with a medium mixed with the melanin synthesis stimulant a-MSH;

[0082] (2) Positive control group (indicated as P.C. in FIG. 6): an experimental group treated with a medium mixed with the melanin synthesis stimulant a-MSH and arbutin (final concentration 1 mM);

[0083] (3) Group treated with culture media of Gardenia jasminoides callus (indicated as Gardenia Callus CM in FIG. 6): an experimental group treated with a medium mixed with the culture media of Gardenia jasminoides callus (prepared in Example 1) and the melanin synthesis stimulant a-MSH (treatment concentrations of the culture media of Gardenia jasminoides calluslow concentration: 300 ?g/mL, and high concentration: 600 ?g/mL); and

[0084] (4) Group treated with Gardenia jasminoides-derived exosomes (indicated as Gardenia Exosome in FIG. 6): an experimental group treated with a medium mixed with the Gardenia jasminoides-derived exosomes (prepared in Example 2) and the melanin synthesis stimulant a-MSH (treatment concentrations of the Gardenia jasminoides-derived exosomeslow concentration: 300 ?g/mL, and high concentration: 600 ?g/mL).

[0085] The melanoma culture medium according to each experimental group was recovered. The recovered melanoma culture medium was mixed with CCK-8 assay reagent (purchased from Dojindo) and the mixture was incubated for 2 hours at 37? C. under 5% CO.sub.2. Then, the supernatant was transferred to a 96-well plate and the absorbance at 450 nm was measured to measure the total number of the cells. After washing the melanoma cells in the 48-well plate with a washing solution, the washed melanoma cells were treated with 1N NaOH (purchased from Merck-Millipore) mixed with 10% DMSO, and then the plate was sealed and heated for 20 minutes at 85? C. to extract melanin in the melanoma cells. The amount of extracted melanin was calculated by measuring the absorbance at 405 nm and normalized by the absorbance value measured through the CCK-8 assay.

[0086] As a result, it was confirmed that the culture media of Gardenia jasminoides callus had no effect of inhibiting melanin synthesis in the melanoma cells, whereas when the melanoma cells were treated with the high concentration of the Gardenia jasminoides-derived exosomes of the present invention, the melanin synthesis in the melanoma cells remarkably decreased, compared to the negative control (FIG. 6).

[0087] Therefore, a cosmetic composition containing the Gardenia jasminoides-derived exosomes as an active ingredient according to the present invention has a whitening effect, and the Gardenia jasminoides-derived exosomes of the present invention is useful as an active ingredient of a cosmetic composition for skin tone improvement, skin brightness improvement and/or skin whitening.

[0088] Although the present invention has been described with reference to the embodiments, the scope of the present invention is not limited to these embodiments. Any person skilled in the art will appreciate that various modifications and changes are possible without departing from the spirit and scope of the present invention and these modifications and changes also fall within the scope of the present invention.