Collagen production promoter

Abstract

To provide collagen production promoters that are highly safe and long-term applicable.SOLUTION: Provided are a collagen production promoter, a collagen production promoting food, a skin wrinkle or sagging preventive or ameliorating agent, and a skin wrinkle or sagging preventive or ameliorating food, containing a kale-derived exosome as an active ingredient.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a collagen production promoter that promotes collagen production. [Background technology]

[0002] There are various causes of skin aging such as wrinkles and sagging, one of which is a decrease in the amount of dermal matrix components. Collagen is the main component of the dermal matrix, which gives skin firmness and prevents sagging, but it is known that its amount decreases with age and exposure to ultraviolet rays (Non-Patent Document 1). The decrease in collagen amount is due to a decrease in the collagen production ability of dermal fibroblasts, which results in wrinkles and sagging of the skin.

[0003] Therefore, attempts have been made to search for natural products that promote collagen production, and it has been reported that various substances, including plant extracts (Patent Document 1, Patent Document 2) and their components (Patent Document 3), have the effect of promoting collagen production.

[0004] Exosomes are membrane vesicles surrounded by a lipid bilayer membrane, and were previously thought to function to release unnecessary intracellular components to the outside. However, in recent years, it has become clear that many cells, including immune cells and tumor cells, release exosomes, which act as important messengers for the exchange of proteins and lipids between secretory cells (exosome-releasing cells) and their target cells (exosome-receiving cells).

[0005] Exosomes contain messenger RNA (mRNA), microRNA (miRNA), double-stranded DNA, proteins, lipids, etc., and it is known that the composition of these RNAs and proteins varies depending on the type of cell that secretes the exosomes. Exosomes or exosome-like vesicles also exist in plants, but the activity and function of exosomes are thought to differ depending on their origin. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2010-235482 A [Patent Document 2] JP 2012-148991 A [Patent Document 3] JP 2010-030911 A [Non-patent literature]

[0007] [Non-Patent Document 1] American Journal of Pathology. 2006;168:1861-1868. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention relates to providing a collagen production promoter which is highly safe and can be used for a long period of time. [Means for solving the problem]

[0009] The inventors conducted research to find a material that promotes collagen production from among the many natural products, and discovered that exosomes derived from kale have the effect of promoting collagen production in fibroblasts, thereby completing the present invention.

[0010] That is, the present invention relates to the following 1) to 4). 1) A collagen production promoter whose active ingredient is exosomes derived from kale. 2) A food for promoting collagen production that contains kale-derived exosomes as an active ingredient. 3) An agent for preventing or improving wrinkles or sagging skin, containing kale-derived exosomes as an active ingredient. 4) A food for preventing or improving wrinkles or sagging skin, containing kale-derived exosomes as an active ingredient. Effect of the Invention

[0011] Kale-derived exosomes can be easily obtained from kale and can be administered or ingested over a long period of time. Therefore, according to the present invention, a highly safe collagen production promoter is provided, which is effective in preventing or improving wrinkles and sagging skin caused by or associated with a decrease in collagen, and in improving skin elasticity. [Brief description of the drawings]

[0012] [Figure 1] Transmission electron microscope (TEM) image of kale-derived exosomes, exosome particle number and particle size. [Diagram 2] Uptake of kale-derived exosomes into skin fibroblasts. [Diagram 3] Kale-derived exosomes promote type I collagen gene expression in skin fibroblasts. [Figure 4] The effect of kale-derived exosomes on collagen production in skin fibroblasts. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In the present invention, exosomes derived from kale are exosomes contained in kale and are synonymous with exosomes obtained from kale. In the present invention, kale refers to kale (Brassica oleracea var. acephala) of the Brassicaceae family, but the variety of kale is not particularly limited. For example, in addition to "Hi-Pearl" (Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 20555), which is a kale variety (Brassica oleracea L.convar. acephala (DC.)Alef. var. sabellica L.), other varieties such as High Crop, Esthe, Extra Curled Scotch, etc. can be used, but it is preferable to use "Hi-Pearl" (Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 20555) because of its strong collagen production promoting effect. In addition, new varieties belonging to kale with increased glucoraphanin or sulforaphane content can also be used. In addition, those that have been subjected to a blanching treatment may also be used.

[0014] Exosomes are a type of membrane vesicle, a microparticle covered with a lipid bilayer, that contains lipids, proteins, RNA (mRNA, miRNA), and DNA. The particle size of exosomes derived from kale was 3.0 × 10 10 ~4.0×10 12 (Per 5 g of powder), the average particle size is 115 to 260 nm.

[0015] Exosomes can be prepared from kale by appropriately combining known methods such as ultracentrifugation, density gradient centrifugation, sucrose cushion method, and filter method. For example, 1) add phosphate-buffered saline (PBS) to the powdered juice of kale and mix, then use a high-speed refrigerated centrifuge to gradually increase the speed of rotation, for example, 1,200 x g, 3,000 x g, and 10,000 x g, and collect the supernatant. 2) The collected supernatant is ultracentrifuged at 30,000 to 50,000 rpm for 2 to 24 hours using an ultracentrifuge, and the supernatant is removed to obtain a crude exosome fraction. Next, the obtained crude exosome fraction can be subjected to iodixanol density gradient centrifugation to isolate and purify exosomes. For example, an iodixanol gradient (concentrations 40%, 20%, 10%, 5%) is prepared in a tube, the crude exosome fraction is added on top of it, and ultracentrifugation is performed at 30,000 to 50,000 rpm to collect the layer corresponding to the density of exosomes (1.15 to 1.19 g / mL), which is used as the exosome fraction. Furthermore, ultracentrifugation is performed at 50,000 to 100,000 rpm to obtain the precipitate as purified exosomes.

[0016] The exosomes used in the present invention may be either purified exosomes or crude exosome fractions. The exosome fraction may be substantially exosomes or a solution or suspension thereof. The exosomes used in the present invention may be intact or may be disrupted in which the membrane vesicle structure is partially or completely destroyed.

[0017] As shown in the Examples below, the exosomes of the present invention are taken up by normal human dermal fibroblasts and promote collagen production. Therefore, the exosomes derived from kale can be used as a collagen production promoter to promote collagen production. In the present invention, promoting collagen production means promoting or improving the collagen production ability, preferably type I collagen production ability, of human skin dermis fibroblasts, thereby increasing the amount of collagen in human skin dermis. Increasing the amount of collagen in the dermis can improve or prevent wrinkles, and also improve skin elasticity. Therefore, kale-derived exosomes can also be used as an agent for preventing or improving wrinkles and sagging skin caused by or associated with a decrease in collagen.

[0018] The collagen production promoter can be a medicine, quasi-drug, cosmetic, supplement, or food (collagen production promoting food, skin wrinkle or sagging prevention or improvement food) for promoting collagen production and preventing or improving skin wrinkles and sagging, or can be a material or preparation to be added to these. In addition, since the kale-derived exosomes of the present invention are absorbed into the body when orally ingested, they are considered to have the same effect as the results of the cell evaluation test. In addition to general foods and beverages, the above foods also include foods that are based on the concept of collagen production and preventing or improving wrinkles and sagging skin, and are labeled as such as functional foods and foods for specified health uses, as well as other foods.

[0019] The above-mentioned pharmaceuticals (including quasi-drugs) containing the exosomes derived from kale of the present invention can be administered in any dosage form. When administering, the active ingredient can be mixed with a solid or liquid pharmaceutical non-toxic carrier suitable for administration methods such as oral, rectal, injection, and external use, and administered in the form of a conventional pharmaceutical formulation.

[0020] For example, oral preparations include solid preparations such as tablets, granules, powders, and capsules, liquid preparations such as solutions, suspensions, and emulsions, and freeze-dried preparations. These preparations can be prepared by conventional means in the field of formulation. Examples of the non-toxic pharmaceutical carriers include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid esters, amino acids, gelatin, albumin, water, and physiological saline. In addition, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonicity agents, and excipients can be added as necessary.

[0021] Furthermore, examples of the form of topical preparations (including cosmetics, pharmaceuticals, and quasi-drugs) include ointments, creams, and topical liquid preparations, as well as various cosmetic forms (for example, basic cosmetics such as lotions, milky lotions, various creams, packs, and serums; hair products such as shampoos, rinses, treatments, hair tonics, hair liquids, hair creams, and hair milks; bath cosmetics such as bath additives; makeup cosmetics such as foundations, lipsticks, mascaras, and eye shadows; sunscreens, etc.).

[0022] In addition to the essential component, kale-derived exosomes, the topical preparation can also contain optional components that are usually included in topical preparations. Examples of such optional components include surfactants, oils, alcohols, moisturizers, thickeners, preservatives, antioxidants, chelating agents, pH adjusters, fragrances, dyes, UV absorbing / scattering agents, powders, vitamins, amino acids, water-soluble polymers, foaming agents, pigments, plant extracts, animal-derived components, seaweed extracts, various drugs, additives, water, etc.

[0023] In addition, the form of the above-mentioned food containing kale-derived exosomes may be various food compositions, as well as forms similar to the above-mentioned oral administration formulations (tablets, capsules, syrup, etc.). To prepare various types of food, the kale-derived exosomes of the present invention can be used alone or in appropriate combination with other food ingredients, solvents, softeners, oils, emulsifiers, preservatives, flavorings, stabilizers, colorants, antioxidants, moisturizers, thickeners, etc.

[0024] The content of kale-derived exosomes in the above-mentioned pharmaceuticals (including quasi-drugs), cosmetics, and foods varies depending on the form of use, but is usually 0.01 to 10 mass% and preferably 0.1 to 1 mass% in terms of exosomes of the total mass of the formulation.

[0025] When the kale-derived exosomes of the present invention are used as medicines, cosmetics or foods, or when incorporated into medicines or foods, the dosage may vary depending on the patient's condition, weight, sex, age, or other factors. In the case of oral administration, the daily dosage per adult is usually preferably 0.1 mg to 100 g, and more preferably 1 mg to 10 g, of exosomes. The above-mentioned preparation may be administered according to any administration regimen, but is preferably administered once or several times a day for several weeks to several months. EXAMPLES

[0026] Manufacturing example: Preparation of exosomes derived from kale (1) Isolation of exosomes by density gradient ultracentrifugation 5.0 g of juice powder from kale (blanched, variety: Hi-Crop) and glucoraphanin-rich kale (blanched, variety: Hi-Pal, hereafter referred to as "GEK") was weighed into a centrifuge tube, and 50 mL of phosphate-buffered saline (PBS) was added. After thoroughly mixing the juice powder and PBS, the mixture was centrifuged using a high-speed refrigerated centrifuge (himac CR21N, himac) at 1,200 x g for 20 minutes at 4°C, 3,000 x g for 20 minutes at 4°C, and 10,000 x g for 1 hour at 4°C, gradually increasing the speed of rotation, to collect the supernatant. The collected supernatant was filtered through a 0.45 μm filter and then dispensed into 10 mL portions in ultracentrifuge tubes (13PET TUBE, himac). The centrifuge tube was attached to a swing rotor (P40ST, Himac) and ultracentrifuged at 30,000 rpm, 4°C for 2 hours using an ultracentrifuge (himac CP75β, Himac). The supernatant was discarded, and 500 μL of PBS was added to the precipitate to obtain a crude exosome fraction. Next, exosomes were isolated by iodixanol density gradient centrifugation using Optiprep (60% iodixanol, Abbot Diagnostics Technologies AS). From the bottom of the centrifuge tube, layers of Optiprep diluted solutions with iodixanol concentrations of 40%, 20%, 10%, and 5% were layered to create an iodixanol gradient in the tube. 500 μL of the crude exosome fraction was added on top of the layers, and ultracentrifugation was performed at 30,000 rpm, 4°C for 18 hours. 1 mL was collected from the top, and the layer corresponding to the exosome density of 1.15 to 1.19 g / mL was collected as an index and used as the exosome fraction. Ultracentrifugation was then performed at 55,000 rpm and 4°C for 2 hours, and 500 μL of PBS was added to the resulting precipitate to obtain purified exosomes. The exosomes were aliquoted and stored at -80°C until use in experiments.

[0027] (2) Measurement of exosome particle concentration and particle size by nanosite analysis The particle concentration and size of exosomes were measured by nanosite analysis, which was outsourced to Fujifilm Wako Pure Chemical Corporation. As shown in Figure 1, the particle number of kale-derived exosomes was 3.9 × 10 12 (Per 5 g of powder), the average particle size was 200 ± 1.1 nm, and the number of GEK-derived exosome particles was 1.9 × 10 11 The average particle size (per 5 g of powder) was 176±2.1 nm.

[0028] (3) Observation using a transmission electron microscope (TEM) 25% glutaraldehyde was added to 500 μL of the exosome solution to a final concentration of 0.1% (w / v) for fixation, and the solution was left to stand overnight at 4°C. The solution was transferred to a 1.5 mL microtube for ultracentrifugation, and ultracentrifuged at 55,000 rpm and 4°C for 2 hours using an ultracentrifuge (himac CP75β, himac), and the supernatant was removed. 50 μL of absolute ethanol was added to the precipitate to wash the exosomes, and then the ethanol was removed using a centrifugal evaporator (CVE-3000, EYELA). 20 μL of PBS was added and mixed well to prepare a sample for TEM observation. 5 μL of this sample was dropped onto Carbon / Formvar film coated TEM grids (ALLIANCE Biosystems) and left to stand for 10 minutes. The added sample was removed, and 5 μL of 1% (w / v) phosphotungstic acid solution was dropped. After immediate removal, the solution was thoroughly dried to prepare a grid for TEM observation. A transmission electron microscope (JEM-1400, JEOL Ltd., Tokyo, Japan) was used, and the observations were performed according to the attached instruction manual. The observed images are shown in Figure 1.

[0029] Example 1 Evaluation of exosome uptake into human skin fibroblasts Human skin fibroblasts NB1RGB were obtained from the Cellular Materials Division of the RIKEN BioResource Research Center. NB1RGB was added at a final concentration of 5.0 × 10 5 The cells were seeded in a 24-well plate at 0.4 mL per well so that the cells were 10 cells / mL, and cultured in MEM-α containing 10% FBS for 24 hours. After washing with PBS, the medium was replaced with MEM-α containing 10% FBS. In addition, a sample containing DAPI (Cosmo Bio), a DNA-binding reagent, and a sample containing Vybrant were added.TM GEK-derived exosomes (final concentration 5 × 10) labeled with DiI Cell-Labeling Solution (Thermo Fisher) 7 Samples containing GEK-derived exosomes (particles / mL) and DAPI (Cosmo Bio), Vybrant TM Kale-derived exosomes (final concentration 5 × 10) labeled with DiI Cell-Labeling Solution (Thermo Fisher) 7 A sample (kale-derived exosomes) was prepared by adding DiI (DiI particles / mL) and DAPI (Cosmo Bio). After culturing for 3 hours, the sample was observed with a fluorescence microscope EVOS (Thermo Fisher) (DiI: excitation wavelength 550 nm, fluorescence wavelength 565 nm) (DAPI: excitation wavelength 360 nm, fluorescence wavelength 460 nm). As shown in Figure 2, it was confirmed that GEK-derived exosomes and kale-derived exosomes were taken up by human dermal fibroblasts.

[0030] Example 2: Expression analysis of type I collagen gene using real-time PCR method NB1RGB final concentration 2.0 x 10 5 The cells were seeded at 1 mL / well in a 12-well plate at 100 cells / mL and cultured in MEM-α containing 10% FBS for 24 hours. After washing with PBS, the cells were replaced with MEM-α without FBS and cultured in a serum-starved state for 24 hours. After that, exosomes were added to a final concentration of 1 × 10 8 The medium was added so that the final concentration of exosomes was 1 × 10 7 , 2.5×10 7 Culture medium adjusted to give 1000 particles / mL was added, and the cells were cultured for 12 hours in a CO2 incubator. All cell cultures were performed in a CO2 incubator at 37°C with a CO2 concentration of 5%. After removing the culture medium, the cells were washed with PBS, and 1 mL of TRIzol (Thermo Fisher) was added. RNA was extracted according to the instructions attached to TRIzol.

[0031] The concentration and purity of the extracted RNA was determined using NanoDrop TM The measurement was performed using a PCR Lite (Thermo Fisher). The ratio of A260 to A280 (A260 / A280), which indicates the purity of the nucleic acid, was confirmed to be 1.8 to 2.1, and cDNA synthesis was performed. The extracted total RNA was incubated at 65°C for 5 minutes and then rapidly cooled on ice. ReverTra Ace qPCR RT Master Mix gDNA Remover (TOYOBO) was used for cDNA synthesis. 6 μL of 4×DN Master Mix containing gDNA Remover and Nuclease-free Water were added to 1.5 μg of RNA template to make a total volume of 24 μL. The mixture was stirred well to make it homogenous, and incubated at 37°C for 5 minutes to perform the DNase reaction. The solution after the reaction was transferred to ice again, and 6 μL of 5×RT Mater Mix II was added and stirred well to make it homogenous. After that, the reverse transcription reaction was performed by incubating at 37°C for 15 minutes, 50°C for 5 minutes, and 98°C for 5 minutes, and cDNA was synthesized.

[0032] The expression level of the type I collagen gene col1a1 was quantified by real-time PCR. The reaction solution consisted of 2μL of cDNA diluted 10-fold with ultrapure water, 10μL of KAPA SYBR FAST Universal qPCR Kit (Nihon Genetics), 0.4μL each of forward and reverse primer solution (10μmol / μL), and 7.2μL of ultrapure water, making a total volume of 20μL. The reaction conditions were preincubation at 95℃ for 1 minute, followed by 40 cycles of 2step PCR (95℃, 5 seconds and 60℃, 30 seconds). The expression level of each gene was analyzed by the comparative Ct method (ΔΔCt method) using the housekeeping gene 18S rRNA as an internal standard.

[0033] [Table 1]

[0034] As shown in Figure 3, analysis of the expression of type I collagen gene col1a1 showed that the addition of exosomes significantly increased the expression level of col1a1 in both kale and GEK.

[0035] Example 3 Evaluation of collagen production ability using human dermal fibroblasts NB1RGB final concentration 2.0 x 10 5 1 mL of exosomes were seeded into each well of a 12-well plate at a concentration of 1 × 10 cells / mL and cultured in MEM-α containing 10% FBS for 24 hours. After washing with PBS, the medium was replaced with MEM-α without FBS and cultured in a serum-starved state for 24 hours. 7 ~2×10 8 After adding 1000g / mL of 1000g of 2-mercaptoethanol, the medium was removed from the cells cultured for 18 hours and washed twice with PBS. TGF-β (final concentration 50ng / mL), which has been reported to promote collagen production, was used as a positive control, and H2O2 (final concentration 1mM), which has been reported to induce collagen reduction, was used as a negative control. 80μL of RIPA Lysis Buffer (Santa Cruz) was added, and the collected cell liquid was transferred to a 1.5mL microtube and mixed with a vortex mixer for 30 seconds, then centrifuged at 12,000×g and 4℃ for 10 minutes to collect the supernatant. After adding and mixing with SDS sample buffer containing 10% 2-mercaptoethanol, the mixture was incubated at 100℃ for 5 minutes and used as a sample for SDS-PAGE. Collagen expression analysis was performed by Western blotting as shown below.

[0036] The SDS-PAGE samples were subjected to SDS-PAGE so that the protein concentration in each lane was 20 μg. For SDS-PAGE, a separation gel containing 10% acrylamide and a concentration gel containing 5% acrylamide were used. Electrophoresis was performed at 100 V and 100 mA for 120 minutes. After electrophoresis, the gel was immersed in a transfer solution for 10 minutes and transferred to a semi-dry transfer device (ATTO) at 30 V and 1.5 mA / cm. 2The proteins were transferred to a PVDF membrane (Merck) for 90 minutes. The PVDF membrane with the transferred proteins was washed three times for 5 minutes with PBST (PBS containing 0.05% (w / v) Tween 20) and blocked. The blocking agent was 3% (w / v) skim milk / PBST, and the membrane was permeated twice for 5 minutes at room temperature. The primary antibody was rabbit anti-COLI polyclonal antibody (2,000-fold dilution, Abcam) or mouse anti-β-actin monoclonal antibody HRP conjugate (10,000-fold dilution, Cell Signaling Technology), and the membrane was incubated overnight at 4°C with shaking. The secondary antibody for collagen detection was goat anti-rabbit IgG horseradish peroxidase antibody (4,000-fold dilution, Bio Rad), and the membrane was incubated for 1 hour with shaking. Finally, images were taken using a chemiluminescence imager EZ-Capture MG (ATTO), and the target protein was quantified using the software Image J.

[0037] As shown in Figure 4, an increase in collagen was observed with the positive control TGF-β, while a decrease in collagen was observed with the negative control H2O2. In contrast, it was shown that both kale and GEK significantly increased collagen production with the addition of exosomes, with GEK having a greater collagen production promoting effect than kale.

[0038] These results demonstrated that kale- and GEK-derived exosomes promote collagen production in human skin fibroblasts.

Claims

1. A type I collagen production promoter consisting of exosomes derived from kale.

2. A food product made from exosomes derived from kale that promotes the production of type I collagen.

Images