Skin composition containing extracellular vesicles derived from koji

Abstract

We have developed exosomes useful as skin agents and provide skin compositions. [Solution] A skin composition containing extracellular vesicles derived from koji is provided. The composition is characterized in that the extracellular vesicles are exosomes and the koji is rice koji, barley koji, or soybean koji. A method for producing koji-derived extracellular vesicles is also provided, characterized by culturing koji mold and collecting extracellular vesicles from the resulting culture.

Description

Technical Field

[0001] The present invention relates to a skin composition containing koji-derived extracellular vesicles, particularly koji-derived exosomes.

Background Art

[0002] Exosomes are membrane vesicles secreted from cells, and their surface is composed of a lipid bilayer. Exosomes are responsible for intercellular information transmission locally or systemically by transporting intracellular proteins and genetic information (mRNA, microRNA, etc.) outside the cell. Their functions are diverse. For example, it has been reported that extracellular vesicles derived from Gram-negative bacteria have functions such as anti-cancer effects (Non-Patent Document 1: Kim OY et al., Nat Commun, 626, 2017). It has also been suggested that the functions of exosomes vary depending on the cells from which they are derived. For example, exosomes derived from mesenchymal stem cells such as adipose-derived stem cells are known to have functions such as suppressing inflammation and treating dementia (Non-Patent Document 2: Katsuda et al., Methods Mol Biol., 1212, 2015).

[0003] Therefore, it is considered that by using exosomes secreted from various cells, various physiological functions can be brought about and applied to the treatment of various diseases. However, the effects of exosomes on the skin, for example, the effects on skin melanocytes, are not clear.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

[0005] Under the circumstances described above, there was a need for the development of exosomes useful as skin agents. [Means for solving the problem]

[0006] The inventors of this invention conducted diligent research to solve the above problems and, as a result, discovered that exosomes derived from koji suppress melanocytes, thus completing the present invention.

[0007] In other words, the present invention is as follows: (1) A skin composition containing extracellular vesicles derived from koji. (2) The composition according to (1), wherein the extracellular vesicle is an exosome. (3) The composition according to (1) or (2), wherein the koji is rice koji, barley koji, or soybean koji. (4) A composition described in any one of items (1) to (3) used as a pharmaceutical, quasi-drug, or cosmetic. (5) A composition according to any one of items (1) to (4) for the purpose of skin whitening, anti-wrinkle, skin regeneration, skin wound healing, or dermatitis. (6) A method for producing extracellular vesicles derived from koji mold, characterized by culturing koji mold and collecting extracellular vesicles from the resulting culture. (7) The method according to (6), wherein the koji mold is Aspergillus oryzae, Aspergillus oryzae, Aspergillus oryzae, or Aspergillus purpurea. (8) The method according to (6) or (7), wherein the extracellular vesicle is an exosome. [Effects of the Invention]

[0008] The present invention provides extracellular vesicles derived from koji. The extracellular vesicles of the present invention (particularly koji-derived exosomes) are useful as topical skin preparations and cosmetics because they suppress genes involved in the activity, proliferation, and secretion of melanocytes. [Brief explanation of the drawing]

[0009] [Figure 1] This figure shows the melanocyte-inhibiting effect of extracellular vesicles derived from Dassai Amazake (rice koji). [Figure 2] This figure shows the results of a melanin synthesis inhibition test using B16 cells (Mouse B16 melanoma 4A5). [Modes for carrying out the invention]

[0010] 1. Extracellular vesicles derived from koji The present invention relates to a skin composition containing extracellular vesicles derived from koji. The composition of the present invention can be used as a pharmaceutical, quasi-drug, or cosmetic composition. The extracellular vesicles used in this invention may be exosomes, microvesicles, or apoptotic bodies, but exosomes are preferred. Exosomes are vesicles derived from endosomes, microvesicles are vesicles secreted directly from cells, and apoptotic bodies are cell fragments produced by cell death. The method for producing extracellular vesicles derived from koji according to the present invention will be explained in particular with exosomes as an example.

[0011] (1) Koji Koji is made by cultivating microorganisms effective for food fermentation, such as Aspergillus oryzae, on grains such as rice, wheat, and soybeans. Therefore, rice, wheat, and soybeans are among the raw materials for koji. The above-mentioned koji ingredients are fermented using koji mold. The koji mold used in this invention is a microorganism that has been used for a long time in the brewing of sake, miso, soy sauce, and other products. Because koji mold has the ability to secrete and produce high amounts of protein, several grams per liter of culture medium, it is also used for the production of useful proteins such as enzymes.

[0012] In the present invention, for example, the following koji molds can be used. Yellow koji mold: Aspergillus oryzae, Aspergillus sojae, etc. Black koji mold: Aspergillus nigar (Aspergillus niger), etc. White koji mold: Aspergillus kawachii (Aspergillus kawachii), etc. Red koji mold: Fungus belonging to the genus Monascus (Monascus genus)

[0013] (2) Cultivation First, wash the rice until there is no white turbidity. After discarding the water used for washing, add fresh water and soak the rice until it is completely immersed. The standard time for soaking in water is not particularly limited, but it can be adjusted according to the season. For example, in summer it is 3 - 5 hours, in spring and autumn it is 6 - 12 hours, and in winter it is 15 - 20 hours. After soaking, lift the rice in a colander and drain for about 2 - 4 hours, and further mix it about 1 - 2 times per hour so that the rice is turned up and down. Then, transfer the rice to a steamer and steam it. The steamed rice is quickly spread on a tray sterilized with alcohol (or shochu), and use a rice paddle or the like to cut the rice to quickly remove the surface moisture and residual heat of the rice. Sprinkle the seed koji when the temperature of the steamed rice reaches about 36 degrees. The amount of seed koji used is, for example, 2 g of seed koji for 1 kg of rice. Quickly gather the rice mixed with koji and wrap it in a clean cloth. Keep the rice wrapped in the cloth at a temperature of about 30 - 32 degrees. Koji will be ready 45 - 48 hours after inoculation. The koji has a strong fragrance like chestnuts, the mycelium grows, and the rice grains stick together to form a plate-like shape. The standard for completion is that it can be easily loosened. For this rice koji (raw material), add water (1 L of water for 100 g of rice koji), and leave it at 4°C for 8 - 16 hours to obtain koji water containing extracellular vesicles. Alternatively, leaving it overnight at 30 degrees to 37 degrees will also result in the completion of rice koji containing extracellular vesicles.

[0014] (3) Recovery Methods for recovering exosomes from the culture (fermented products such as rice koji) include ultracentrifugation, immunoprecipitation, affinity method using magnetic beads, density gradient centrifugation, etc. Commercially available reagents for exosome isolation can also be used to isolate and recover exosomes.

[0015] In the case of ultracentrifugation, for example, centrifugation can be performed in several steps in advance at a centrifugal force of about 100 g to 10,000 g, and then ultracentrifugation can be performed at a centrifugal force of about 120,000 g for 70 minutes to obtain high-purity exosomes. However, those skilled in the art can appropriately set the centrifugation method and conditions.

[0016] Confirmation that the recovered exosomes are the target ones can be carried out by morphological observation using a transmission electron microscope, measurement of the number and particle size of particles using a nanoparticle tracking system, gene analysis using PCR, immunological methods (ELISA, FACS, etc.), Western blot, and the like. Also, confirmation is made that the exosomes are the target ones using as an index the proteins recognized as exosome markers. For example, CD9, CD63, etc. can be used as markers for detection.

[0017] 2. Skin composition The present invention provides a skin composition containing extracellular vesicles derived from koji, particularly exosomes derived from koji. The skin composition of the present invention is used as a cosmetic, a pharmaceutical product or a quasi-drug. The disorders or diseases targeted for use of the skin composition of the present invention are not particularly limited.

[0018] The skin composition of the present invention can include various pharmaceutically (medicinally) acceptable carriers according to the usage form as long as it contains an effective amount of exosomes as an active ingredient without losing its activity. For example, generally used carriers can be applied as diluents, excipients, etc. [[ID=2G]]

[0019] WID=23]]For example, water, physiological saline (such as PBS), and various organic solvents can be mentioned. Examples of the organic solvent include aqueous solutions of alcohol (such as ethanol), glycerol, olive oil, etc. Furthermore, various fillers, extenders, binders, surfactants, dyes, fragrances, etc. can be added.

[0020] When the extracellular vesicles derived from koji according to the present invention are used as a pharmaceutical composition, they can be in the form of parenteral preparations such as liquids, suspensions, emulsions, ointments, aqueous gels, sheets, and microneedles, or in the form of oral preparations such as granules, powders, tablets, and capsules.

[0021] Furthermore, for use in injections, etc., it can be prepared as a freeze-dried or granulated product that is dissolved in physiological saline or a suitable buffer solution (e.g., PBS) immediately before use to prepare a drug solution.

[0022] The compositions of the present invention can be used in forms, methods, and dosages appropriate for therapeutic purposes and patients. The amount and frequency of addition are expected to vary depending on the type of skin disease or disorder, as well as conditions such as the culture conditions and culture medium used to obtain exosomes. Therefore, those skilled in the art can appropriately determine the dosage and administration. For example, the effective amount of exosomes is a concentration in the culture medium of 1 × 10⁻⁶ 9 The concentration should be 5 μg / mL to 20 μg / mL or higher.

[0023] The skin compositions of the present invention can be manufactured as various compositions according to classifications such as cosmetics, or quasi-drugs (medicinal cosmetics) which are positioned between cosmetics and pharmaceuticals. The efficacy of the skin composition of the present invention includes, for example, whitening, anti-wrinkle, rough skin, chapped skin, heat rash, chilblains, cracks, chapped skin, acne, oily skin, razor burn, sunspots and freckles, redness after sunburn or snowburn, skin tightening, skin cleansing, skin conditioning, keeping skin healthy, skin moisturizing, skin protection, skin dryness prevention, and reducing the appearance of fine lines caused by dryness. Furthermore, the skin composition of the present invention can be used for skin cancer, skin trauma, burns, skin regenerative medicine, skin wound healing, atopic dermatitis, and the like.

[0024] Preferably, the effects include skin whitening, anti-wrinkle, skin regeneration, skin wound healing, and atopic dermatitis.

[0025] Dosage forms of the skin composition of the present invention include, for example, liquids, sprays, ointments, creams, gels, and patches. When the skin composition is used as a topical skin preparation or cosmetic, examples include basic cosmetics (lotions, creams, emulsions, serums, facial cleansers, etc.), makeup cosmetics (foundations, etc.), hair care products (shampoos, conditioners, hair styling products, etc.), and other cosmetics (whitening agents, depilatory agents, etc.). Furthermore, as described above, it may be in the form of an oral preparation such as granules, powder, tablets, or capsules.

[0026] The skin composition of the present invention may contain components other than exosomes, depending on its classification (cosmetics, quasi-drugs, etc.) and dosage form. Examples of components other than exosomes include additives commonly used in the manufacture of cosmetics or quasi-drugs (especially topical skin preparations and transdermal compositions), such as stabilizers, preservatives, buffers, pH adjusters, and excipients. The content of such components other than exosomes can also be appropriately adjusted by those skilled in the art. If necessary, it is also possible to use drugs that are known to be incorporated into topical skin preparations, such as cosmetics and quasi-drugs, not as components to be encapsulated in exosomes, but as components not encapsulated in exosomes. Examples of such drugs include vitamins A, C, B, E and their derivatives, and estrogens.

[0027] Examples The present invention will be described in more detail below with reference to examples. However, the scope of the present invention is not limited to these examples. [Examples]

[0028] (1) Changes in melanocyte gene expression due to extracellular vesicles derived from rice koji Human melanocytes include normal human epidermal melanocytes (cryopreserved human melanocytes). <nhem-c>Funakoshi, C-12400, origin: foreskin of a minor was used. For culturing melanocytes, the Melanocyte Growth Medium Kit (Funakoshi, C-24110) was used, and the culture method followed the instructions in the attached manual.

[0029] T-25 (Nunc EasYFlask156367, pre-treated for cell culture) was used as the culture flask. After growing cells to 80% confluence, extracellular vesicles derived from rice koji were added. The concentration added was 100 to 1000 vesicles per cell. Extracellular vesicles derived from rice koji were added to the cell culture medium and cultured in a CO2 incubator for 72 hours. After culturing, the cells were harvested using a cell scraper, and mRNA was recovered using Qiagen's mRNA purification kit (RNAeasy mini kit).

[0030] Based on this mRNA, the gene expression levels of Tyrosinase, ROS, EPR, ETBR, and Mc1r were quantified by quantitative PCR (QuantiTect Probe PCR Kit (200), Qiagen). The relative expression levels after 72 hours were determined by setting the gene expression level in cells before the addition of extracellular vesicles as 100. As a result, it was found that the expression of all genes related to melanin synthesis was suppressed (Figure 1). The figure shows the average value for N=2.

[0031] (2) Melanin synthesis inhibition test using B16 cells (Mouse B16 melanoma 4A5) The cells used were mouse-derived B16 melanoma 4A5 cell line (Merck: 94042254). The culture medium used was Gibco's DMEM + 2 mM glutamine + 10% fetal bovine serum (FBS). B16 cells were cultured in a 6-well plate (Corning), and when they reached 85% confluence, extracellular vesicles derived from rice koji were added.

[0032] The added concentration ranged from 10 to 1000 cells, and comparisons were conducted within this range. After adding extracellular vesicles to the cell culture medium, the cells were cultured in a CO2 incubator for 72 hours. After culturing, melanin contained in the cells was extracted (dissolved in 1 M NaOH (Wako Pure Chemical Industries)) and absorbance was measured (OD420nm) using a spectrophotometer. The measurement results were expressed as relative values, with the melanin amount of untreated B16 cells set to 100. The average value for N=3 is shown. As a control group, PBS(-) and vitamin C (10 mg / ml) supplementation groups were used.

[0033] As a result, when extracellular vesicles derived from rice koji were added, the amount of melanin pigment was reduced in a dose-dependent manner (Figure 2).

Claims

1. A skin composition containing extracellular vesicles derived from koji.

2. The composition according to claim 1, wherein the extracellular vesicle is an exosome.

3. The composition according to claim 1 or 2, wherein the koji is rice koji, barley koji, or soybean koji.

4. A composition according to any one of claims 1 to 3, to be used as a pharmaceutical, quasi-drug, or cosmetic.

5. A composition according to any one of claims 1 to 4, for the purpose of skin whitening, anti-wrinkle, skin regeneration, skin wound healing, or dermatitis.

6. A method for producing extracellular vesicles derived from koji mold, characterized by culturing koji mold and collecting extracellular vesicles from the resulting culture.

7. The method according to claim 6, wherein the koji mold is Aspergillus oryzae, Aspergillus oryzae, Aspergillus oryzae, or Aspergillus purpurea.

8. The method according to claim 6 or 7, wherein the extracellular vesicle is an exosome.

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