Milk-derived exosome-like vesicles-containing topical skin preparation, collagen production promoter, and elastin production promoter

Abstract

To provide a novel topical preparation for skin containing milk-derived exosome-like vesicles, a collagen production promoter and an elastin production promoter.SOLUTION: A topical preparation for skin contains exosome-like vesicles derived from mammal milk.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an external skin preparation, a collagen production promoter, and an elastin production promoter, each containing milk-derived exosomes. [Background technology]

[0002] Exosomes are membrane vesicles secreted by various cells and are a type of extracellular vesicle. When first discovered, they were thought to be involved in the release of unwanted cellular contents, and their function remained largely unknown. However, in recent years, it has become clear that they encapsulate various proteins and genetic information and play a role in transmitting information to other cells, attracting attention. In particular, the development of biomarkers and therapies focusing on exosomes released by cancer cells is actively underway worldwide. Furthermore, research is being conducted on exosomes as drug delivery carriers, and their application in food and cosmetics is being considered.

[0003] Patent Document 1 (WO 2019 / 146612) proposes a composition for treating skin diseases, which contains adipose-derived stem cells encapsulating drug-encapsulated nanoparticles or contains exosomes secreted from adipose-derived stem cells. It discloses that when stem cells are made to contain drug-encapsulated nanoparticles in which a drug is encapsulated, the exosomes secreted from the stem cells are rich in components useful for treating various skin diseases, namely the drug encapsulated in the nanoparticles and various factors produced intracellularly in the stem cells, such as angiogenic factors, and that by using such exosomes as an active ingredient, it is possible to prepare a composition for treating skin diseases that can be used as an external preparation and has a significantly excellent therapeutic effect against skin diseases.

[0004] Patent Document 2 (International Publication No. 2020 / 158930) discloses that exosomes derived from yeast used in sake or miso enhance the expression of collagen and elastin in proliferated fibroblasts, thereby preventing a decrease in tissue elasticity and improving elasticity.

[0005] Patent Document 3 (JP 2018-531932 A) discloses that extracellular vesicles derived from plant juices have excellent skin permeability and are effective in wound treatment, skin moisturizing, skin whitening, wrinkle improvement, and anti-aging, as well as hair growth and hair restoration. However, it was not satisfactory in terms of the functions we were looking for and the odor unique to yeast and plant juices.

[0006] Exosomes are found in bodily fluids such as saliva, blood, urine, breast milk, and amniotic fluid, and as mentioned above, are also secreted from cultured cells. In the course of their research into exosomes, the present inventors discovered that milk-derived exosomes have an excellent effect of promoting collagen and elastin production, leading to the completion of the present invention. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2020 / 158930 [Patent Document 2] International Publication No. 2020 / 158930 [Patent Document 3] Special Publication No. 2018-531932 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention addresses the problem of providing novel topical skin preparations, collagen production promoters, and elastin production promoters that contain milk-derived exosome-like vesicles. [Means for solving the problem]

[0009] The main means for solving the problems of the present invention are as follows. 1. A topical skin preparation characterized by containing milk-derived exosome-like vesicles. 2. A collagen production promoter characterized by containing milk-derived exosome-like vesicles. 3. An elastin production promoter characterized by containing milk-derived exosome-like vesicles. 4. The topical skin preparation described in 1., characterized in that the milk-derived exosome-like vesicles contain sediment obtained by centrifugation at a rotational force of 10,000 x g or more. [Effects of the Invention]

[0010] The milk-derived exosome-like vesicles used in the present invention have the effect of promoting collagen and elastin production, and novel topical skin preparations, collagen production promoters, and elastin production promoters (hereinafter collectively referred to as "agents") utilizing these vesicles can be provided as cosmetics, pharmaceuticals, quasi-drugs, and other products. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the relative expression levels of a gene (COL1A1) involved in collagen production by milk (goat)-derived exosome-like vesicles in Examples 1 to 3 and Comparative Example 1. [Figure 2] FIG. 1 shows the relative expression levels of a gene (COL7A1) involved in collagen production by milk (goat)-derived exosome-like vesicles in Examples 4 to 6 and Comparative Example 2. [Figure 3] FIG. 1 shows the relative expression levels of genes (ELN) involved in elastin production by milk (goat)-derived exosome-like vesicles in Examples 7 to 9 and Comparative Example 3. [Figure 4] FIG. 1 shows the relative expression levels of a gene (COL1A1) involved in collagen production by milk (cow)-derived exosome-like vesicles in Examples 10 and 11 and Comparative Example 4. [Figure 5] FIG. 1 shows the relative expression levels of a gene (COL7A1) involved in collagen production by milk (cow)-derived exosome-like vesicles in Examples 12 and 13 and Comparative Example 5. [Figure 6] FIG. 1 shows the relative expression levels of genes (ELN) involved in elastin production by milk (cow)-derived exosome-like vesicles in Examples 14 and 15 and Comparative Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0012] The agent of the present invention will be described in detail below. (Milk-derived exosome-like vesicles) The milk used in the present invention is milk obtained from mammals, including milk obtained from humans, cows, goats, sheep, pigs, monkeys, dogs, cats, rats, mice, hamsters, guinea pigs, etc. Milk obtained from goats, cows, horses, sheep, and pigs is preferred. Milk may be colostrum or mature milk, with mature milk being preferred. Milk may be processed by sterilization, powderization, defatting, deproteinization, etc., as long as the membrane structure of the exosome-like vesicles contained therein is not completely decomposed. However, unsterilized raw milk is preferred, and if sterilization is performed, low-temperature treatment is preferred. The low-temperature treatment of the present invention refers to a treatment in which heat is applied at a temperature of 50°C to 90°C.

[0013] Milk-derived exosome-like vesicles are exosome-like vesicles contained in milk and are synonymous with exosome-like vesicles obtained from milk. Specifically, they include not only exosomes, a type of extracellular endoplasmic reticulum, but also endoplasmic reticulum other than exosomes, lipid bilayer-enclosed microparticles, lipids, proteins, and the like. Their diameters are approximately 20 to 200 nm. The milk-derived exosome-like vesicles of the present invention may be intact or have a disrupted membrane structure. Furthermore, the milk-derived exosome-like vesicles contained in topical skin preparations, collagen production promoters, and elastin production promoters may be microparticles encased in lipid bilayers, or may have such structures disrupted, or may be a mixture of these.

[0014] The method for preparing exosome-like vesicles from milk is not particularly limited, but preferably includes the steps of removing a lipid fraction, a casein fraction, and a cellular fraction in order to efficiently recover exosome-like vesicles. Removing these from milk yields a whey fraction, and exosome-like vesicles are preferably recovered from this whey fraction. The method for recovering an exosome-like vesicle fraction from the resulting whey fraction is not particularly limited, and known methods such as centrifugation, ultracentrifugation, chromatography, ultrafiltration, filtration, density gradient centrifugation, sucrose cushion method, and electrophoresis can be used. For example, when an exosome-like vesicle fraction is obtained as a precipitate from a whey fraction by centrifugation, the torque is preferably 10,000 × g or more, more preferably 50,000 × g or more, and even more preferably 100,000 × g or more. The upper limit is preferably about 1,000,000 × g. The centrifugation time is preferably 20 minutes or more, more preferably 40 minutes or more, and even more preferably 60 minutes or more. Furthermore, it is preferable to perform a purification procedure such as filtration before or after centrifugation.

[0015] The topical skin preparation of the present invention includes cosmetics, quasi-drugs, and pharmaceuticals, and may be in the form of a solution, emulsion, or polymer gel preparation such as a lotion, milky lotion, cream, or gel. It may also be a foam preparation, a multi-layer preparation, a spray preparation, a sheet in which nonwoven fabric or the like is impregnated, or a gel pack preparation.

[0016] The topical skin preparation of the present invention preferably contains milk-derived exosome-like vesicles at a solids concentration of 0.0001% to 5% by weight, more preferably 0.001% to 3% by weight, and particularly preferably 0.01% to 1% by weight. The topical skin preparation of the present invention can optionally contain medicinal ingredients such as moisturizers, surfactants, thickeners, anti-inflammatory agents, vitamins, antioxidants, blood circulation promoters, wound healing agents, antibacterial substances, skin activators, resident flora control agents, active oxygen scavengers, and skin whitening agents, depending on the purpose.

[0017] The collagen production promoters and elastin production promoters of the present invention include cosmetics, quasi-drugs, and pharmaceuticals, and can be manufactured according to commonly used formulation methods. The collagen production promoters and elastin production promoters of the present invention preferably contain milk-derived exosome-like vesicles at a solids concentration of 0.0001% to 5% by weight, more preferably 0.001% to 3% by weight, and particularly preferably 0.01% to 1% by weight. Depending on the purpose, the collagen production promoters and elastin production promoters of the present invention can contain optional ingredients such as oils and fats such as vegetable oils, higher fatty acids, higher alcohols, silicones, anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, preservatives, sugars, sequestering agents, polymers such as water-soluble polymers, thickeners, powder components, UV absorbers, UV blockers, moisturizers such as hyaluronic acid, fragrances, pH adjusters, etc. In addition, other medicinal and physiologically active ingredients such as vitamins, skin activators, blood circulation promoters, resident bacteria control agents, active oxygen scavengers, anti-inflammatory agents, whitening agents, and disinfectants may also be contained.

[0018] The active ingredient may be any ingredient that has been conventionally used in pharmaceuticals, quasi-drugs, cosmetics, sanitary materials, etc., and is soluble or dispersible in water, and can be used without any particular limitation. Specific examples include angelica extract, avocado extract, hydrangea extract, althea extract, arnica extract, apricot extract, apricot kernel extract, fennel extract, turmeric extract, oolong tea extract, echinacea leaf extract, Scutellaria root extract, Phellodendron bark extract, barley extract, watercress extract, orange extract, seawater dried product, hydrolyzed elastin, hydrolyzed wheat powder, hydrolyzed silk, chamomile extract, carrot extract, artemisia capillaris extract, licorice extract, kalkade extract, kiwi extract, and cinchona extract. Cucumber extract, guanosine, kumazasa extract, walnut extract, grapefruit extract, clematis extract, yeast extract, burdock extract, comfrey extract, collagen, bilberry extract, budding extract, umbilical cord extract, salvia extract, soapwort extract, bamboo extract, hawthorn extract, shiitake mushroom extract, rehmannia root extract, lithospermum root extract, linden extract, meadowsweet extract, calamus root extract, white birch extract, horsetail extract, honeysuckle extract, ivy extract, se Hawthorn extract, elderberry extract, yarrow extract, peppermint extract, mallow extract, Swertia japonica extract, Chinese laurel extract, thyme extract, clove extract, Imperata cylindrica extract, tangerine extract, spruce extract, Houttuynia cordata extract, tomato extract, natto extract, carrot extract, wild rose extract, hibiscus extract, burdock root extract, parsley extract, honey, parietaria extract, burdock extract, bisabolol, coltsfoot extract, butterbur extract, bupleurum extract Examples of such extracts include jasmine extract, butcher's broom extract, grape extract, propolis, loofah extract, peppermint extract, linden extract, hop extract, pine extract, horse chestnut extract, skunk cabbage extract, soapberry extract, peach extract, cornflower extract, eucalyptus extract, yuzu extract, mugwort extract, lavender extract, apple extract, lettuce extract, lemon extract, astragalus extract, rose extract, rosemary extract, Roman chamomile extract, and royal jelly extract.

[0019] In addition, moisturizing agents such as amino acids, urea, sodium pyrrolidonecarboxylate, betaine, whey, and trimethylglycine; oily ingredients such as sphingolipids, ceramide, cholesterol, cholesterol derivatives, and phospholipids; anti-inflammatory agents such as ε-aminocaproic acid, glycyrrhizinic acid, β-glycyrrhetinic acid, lysozyme chloride, guaiazulene, and hydrocortisone; vitamins such as vitamins A, B2, B6, D, and E, calcium pantothenate, biotin, and nicotinamide; antioxidants such as tocopherol, carotenoids, flavonoids, tannins, lignans, and saponins; blood circulation promoters such as γ-oryzanol and vitamin E derivatives; wound healing agents such as retinol and retinol derivatives; biopolymers such as deoxyribonucleic acid, sodium chondroitin sulfate, collagen, elastin, chitin, chitosan, and hydrolyzed eggshell membrane; allantoin, diisopropylamine dichloroacetate, 4-aminomethylcyclohexyl methyl ... Active ingredients such as xancarboxylic acid; cepharanthine, capsicum tincture, hinokitiol, iodized garlic extract, pyridoxine hydrochloride, dl-α-tocopherol, dl-α-tocopherol acetate, nicotinic acid, nicotinic acid derivatives, D-pantothenyl alcohol, acetylpantothenyl ethyl ether, biotin, allantoin, isopropylmethylphenol, estradiol, ethinylestradiol, carpronium chloride, benzalkonium chloride, diphenhydramine hydrochloride, tacanal, camphor, salicylic acid, nonylic acid vanillylamide, nonanoic acid vanillylamide, piroctone olamine, glyceryl pentadecanoate, mononitroguaiacol, resorcinol, γ-aminobutyric acid, benzethonium chloride, mexiletine hydrochloride, auxin, female hormones, cantharides tincture, cyclosporine, hydrocortisone, polyoxyethylene sorbitan monostearate, etc. [Example]

[0020] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0021] (Preparation of exosome-like vesicles derived from milk (goat)) Pasteurized goat's milk (-30°C) from Hokkaido with a non-fat milk solids content of 7.5% or more and milk fat content of 2.5% or more was thawed at 4°C for one day and then allowed to return to room temperature one hour before processing. The goat's milk was added to a 50 mL centrifuge tube and centrifuged at room temperature for 10 minutes at 3,000 x g. The supernatant was collected and de-fat-treated. After de-fatting, acetic acid (1 / 100th the volume of the supernatant) was added, mixed by inversion, and allowed to stand at room temperature for 5 minutes. The tube was centrifuged at 10,000 x g for 10 minutes to precipitate protein components, and the supernatant was collected and de-proteinized. The supernatant after protein removal was passed through a 0.22 μm filter (Millipore) to completely remove precipitates. The supernatant was then ultracentrifuged at 4°C and a rotation speed of 100,000 × g (35,000 rpm) (Beckman, SW41Ti rotor) for 70 minutes. The supernatant was removed, and the pelleted exosome-like vesicles were suspended in an equal volume of PBS(-) and ultracentrifuged again under the same conditions. After removing the supernatant, the pelleted exosome-like vesicles were resuspended in PBS(-) and recovered to obtain milk-derived exosome-like vesicles 1 of the present invention.

[0022] Milk-derived exosome-like vesicles 2 of the present invention were obtained in the same manner as above, except that defatting was performed and protein removal treatment was not performed. Milk-derived exosome-like vesicles 3 of the present invention were obtained in the same manner as above, except that defatting was not performed and protein removal treatment was performed. A 0.45 μm filter (Millipore) was used for filtration. Milk-derived exosome-like vesicles 4 of the present invention were obtained in the same manner as above, except that neither the defatting process nor the protein removal process was performed, and filtration was not performed.

[0023] (Nanosight, NTA analysis) Milk-derived exosome-like vesicles 1–4 were collected by ultracentrifugation and their particle concentration and distribution were measured using a Nanosight LM10HS (Nanosight). Analysis was performed using Nanoparticle Tracking Analysis (NTA) software version 2.1 (Nanosight). The camera level, threshold, and focus were the same for all test samples. Protein concentration of the exosome-like vesicle solution was measured using the Qubit Protein Assay Kit (Thermo Fisher) according to the kit's protocol.

[0024] (result) The results are shown in Table 1. [Table 1] Both methods yielded milk-derived exosome-like vesicles with a particle diameter of approximately 170 nm. Furthermore, the particle mass per 1 ng of protein was higher for milk-derived exosome-like vesicles 1 and 3. This confirms that at least some protein removal treatment is necessary to obtain highly pure exosome-like vesicles.

[0025] (Cellular testing) Normal human dermal fibroblasts (NHDFs (LONZA)) were cultured in a 12-well plate at 0.5 × 10 5 24 hours after seeding, 0.3, 1.0, and 3.4 × 10 cells / well were seeded. 9 Cell culture medium containing goat milk-derived exosome-like vesicles 1 at a concentration of 1 / well (Examples 1 to 9) or cell culture medium containing the same volume of PBS(-) (Comparative Examples 1 to 3) was added to the cells and cultured for 24 hours. After that, the cells were washed with PBS(-) and mRNA was recovered from the cells.

[0026] (mRNA recovery and gene amplification by RT-PCR) QIAzol Lysis Buffer (QIAGEN) was added to the cells at 350 μl / well and thoroughly disrupted by pipetting. The resulting cell homogenate was collected in a 1.5 ml tube. After allowing the mixture to stand at room temperature for 5 minutes to promote protein dissociation, chloroform (1 / 5 the volume of the lysis reagent) was added and vortexed vigorously for 15 seconds. After allowing the mixture to stand at room temperature for 3 minutes, the mixture was centrifuged at 12,000 × g for 15 minutes at 4°C, and the top layer was collected in a new 2 ml tube. 1.5 volumes of 100% ethanol (1.5 times the volume of the collected solution) were added to the tube and mixed by pipetting. The mixture was then applied to a column and centrifuged at 8,000 × g for 15 seconds at room temperature. 700 μL of RWT solution was then added to the column and centrifuged at 8,000 × g for 15 seconds at room temperature. 500 μL of RPE solution was then added and centrifuged at 8,000 × g for 15 seconds at room temperature. After washing, 500 μL of RPE solution was added and centrifuged at 8,000 × g for 2 minutes at room temperature. The column was then transferred to a new 2 mL tube and centrifuged at full speed for 1 minute to air dry. 30 μL of RNeasy Water was added, and the column was left to stand for 1 minute. Then, total RNA was recovered by centrifugation at 8,000 × g for 1 minute (miRNeasy Mini Kit (QIAGEN)).

[0027] RNA was quantified using a NanoDrop 2000C (Thermo Fisher Scientific), the concentration was adjusted, and PrimeScript was used. tm cDNA synthesis was performed using an RT reagent kit (TAKARA). Subsequently, after preparation using Power SYBR Green Master Mix (Thermo Fisher), RT-qPCR was performed using a QuantStudio 5 PCR (Thermo Fisher). Expression of each gene was calculated using the comparative CT method (ΔΔCT) with ribosomal protein S18 as an endogenous control. Table 2 shows the primer information for each gene obtained from TAKARA. [Table 2]

[0028] (result) The results are shown in Tables 3 to 5 and Figures 1 to 3. [Table 3] [Table 4]

[0029] [Table 5] It was confirmed that milk-derived exosome-like vesicles obtained from the goat's milk of the present invention promote the expression of collagen and elastin genes.

[0030] (Preparation of milk (cow)-derived exosome-like vesicles) Milk-derived exosome-like vesicles 5 were obtained in the same manner as in the preparation of milk-derived exosome-like vesicles 1, except that milk (raw milk pasteurized at 75°C for 15 minutes) was used.

[0031] (Cellular testing) Normal human dermal fibroblasts (NHDFs (LONZA)) were cultured in a 12-well plate at 0.5 × 10 5 24 hours after seeding, 1.0, 3.4 × 10 cells / well were seeded. 9 Cell culture medium containing milk-derived exosome-like vesicles 5 per well (Examples 10 to 15) or cell culture medium containing the same volume of PBS(-) (Comparative Examples 4 to 6) was added to the cells and cultured for 24 hours. After that, the cells were washed with PBS(-) and mRNA was recovered from the cells. (mRNA recovery and gene amplification by RT-PCR) RT-qPCR was performed in the same manner as described above using milk-derived exosome-like vesicles 1.

[0032] The results are shown in Tables 6 to 8 and Figures 4 to 6. [Table 6] [Table 7]

[0033] [Table 8] It was confirmed that milk-derived exosome-like vesicles obtained from the milk of the present invention promote the expression of collagen and elastin genes.

Claims

1. A topical skin preparation characterized by containing goat milk-derived exosome-like vesicles at a solids concentration of 0.01% by weight or more.

2. A collagen production promoter characterized by containing goat milk-derived exosome-like vesicles at a solids concentration of 0.01% by weight or more.

3. An elastin production promoter characterized by containing goat milk-derived exosome-like vesicles at a solids concentration of 0.01% by weight or more.

4. A topical skin preparation containing goat's milk-derived exosome-like vesicles, wherein the milk-derived exosome-like vesicles contain sediment obtained by centrifugation at a rotational force of 10,000 x g or more.

5. A collagen production promoter characterized by containing goat milk-derived exosome-like vesicles, wherein the milk-derived exosome-like vesicles contain sediment obtained by centrifugation at a rotational force of 10,000 x g or more.

6. An elastin production promoter characterized by containing goat's milk-derived exosome-like vesicles, wherein the milk-derived exosome-like vesicles contain sediment obtained by centrifugation at a rotational force of 10,000 x g or more.

7. A collagen production promoter consisting of exosome-like vesicles derived from goat's milk.

8. An elastin production promoter consisting of exosome-like vesicles derived from goat's milk.

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