Mitochondria function-improving agent, cosmetic, and mitochondria function-improving method

Abstract

To provide a mitochondria function-improving agent or the like which improves a mitochondria function in a skin cell.SOLUTION: A mitochondria function-improving agent that improves a mitochondria function in a cell contains components included in a swallow's nest as an active ingredient. According to each point of view of the present invention, a mitochondria function-improving agent or the like which improves mitochondria function in a skin cell can be provided.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to a mitochondrial function enhancer, a cosmetic, and a method for enhancing mitochondrial function.

Background Art

[0002] The skin covers the entire surface of the human body and is constantly exposed to the surrounding environment. The structure of the skin is divided into three layers: the epidermis, the dermis, and the subcutaneous fat layer. The epidermis further consists of the stratum corneum, the granular layer, the spinous layer, and the basal layer.

[0003] When the skin is exposed to external stimuli such as ultraviolet rays and chemical substances, reactive oxygen species are generated. Reactive oxygen species promote the decomposition of collagen in the epidermis and the production of melanin, causing skin aging such as wrinkles, spots, and sagging.

[0004] In addition, when the skin barrier function is reduced due to external stimuli, skin problems such as itching, eczema, and atopic dermatitis occur.

[0005] In order to improve these conditions, in recent years, many studies related to skin anti-aging have been conducted, and the inventors of the present application have been particularly searching for foods that are effective in improving the skin.

[0006] It has been clarified that SIRT1 and SIRT3 exhibit anti-aging effects in various tissues when activated.

[0007] When SIRT1 is activated, the activities of mitochondria, the external barrier, and the internal barrier downstream of the bioactive flow increase, leading to wrinkle suppression and improvement of the skin barrier function and water retention function.

[0008] In addition, when SIRT3 is activated, reactive oxygen species are eliminated, leading to promotion of whitening.

[0009] In recent years, it has been becoming clear that mitochondrial function is closely related to aging. In particular, mitochondrial dysfunction in skin cells is thought to lead to skin aging such as wrinkle formation and epidermal hyperplasia, as well as an increase in gray hair and hair loss. Suppression and improvement of wrinkles, suppression of epidermal hyperplasia, and suppression of aging such as gray hair and hair loss by improving mitochondrial function are expected (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0011] Therefore, an object of the present invention is to provide a mitochondrial function improving agent or the like that improves the function of mitochondria in skin cells or myoblasts.

Means for Solving the Problems

[0012] A first aspect of the present invention is a mitochondrial function improving agent that improves the function of mitochondria in cells, and contains a component contained in edible bird's nest as an active ingredient.

[0013] A second aspect of the present invention is the mitochondrial function improving agent according to the first aspect, wherein the active ingredient activates PGC1-α.

[0014] A third aspect of the present invention is the mitochondrial function improving agent according to the second aspect, wherein the active ingredient increases the number of mitochondria in cells.

[0015] A fourth aspect of the present invention is a mitochondrial function improver according to the third aspect, wherein the active ingredient increases the area of mitochondria.

[0016] A fifth aspect of the present invention is a cosmetic for improving the function of mitochondria in cells, which contains the active ingredient according to any one of the first to fourth aspects.

[0017] A sixth aspect of the present invention is a wrinkle improver for improving the function of mitochondria in cells, which contains the active ingredient according to any one of the first to fourth aspects.

[0018] A seventh aspect of the present invention is an anti-aging agent for improving the function of mitochondria in cells, which contains the active ingredient according to any one of the first to fourth aspects.

[0019] An eighth aspect of the present invention is a method for producing a mitochondrial function improver for improving the function of mitochondria in cells, which includes a step of adding a component contained in bird's nest as an active ingredient.

[0020] A ninth aspect of the present invention is a method for improving mitochondrial function for improving the function of mitochondria in cells, which includes a step of applying an external preparation containing a component contained in bird's nest as an active ingredient to a subject (excluding medical acts on humans).

[0021] A tenth aspect of the present invention is an exosome controller for controlling exosomes secreted from intestinal cells, which contains a component contained in bird's nest as an active ingredient.

[0022] An eleventh aspect of the present invention is a food composition for controlling exosomes secreted from intestinal cells, which contains a component contained in bird's nest as an active ingredient.

[0023] The twelfth aspect of the present invention is a method for producing an intestinal-derived exosome-containing composition containing intestinal-derived exosomes secreted from intestinal cells, the method for producing an intestinal-derived exosome-containing composition including a step of adding a solution containing components contained in edible bird's nest as an active ingredient to a medium containing the intestinal cells.

[0024] The thirteenth aspect of the present invention is a method for controlling exosomes that control exosomes secreted from intestinal cells, the method for controlling exosomes including a step of adding a solution containing components contained in edible bird's nest as an active ingredient to a medium containing the intestinal cells.

[0025] The fourteenth aspect of the present invention is an intestinal-derived exosome-containing composition containing intestinal-derived exosomes secreted from intestinal cells to which a solution containing components contained in edible bird's nest as an active ingredient has been added.

[0026] The fifteenth aspect of the present invention is a mitochondrial function enhancer for improving the function of mitochondria in cells, the mitochondrial function enhancer containing the intestinal-derived exosomes of the fourteenth aspect as an active ingredient.

[0027] The sixteenth aspect of the present invention is the mitochondrial function enhancer of the fifteenth aspect, which improves the function of mitochondria in epidermal keratinocytes, dermal fibroblasts, or myoblasts.

[0028] The seventeenth aspect of the present invention is a reactive oxygen species scavenger for scavenging reactive oxygen species, the reactive oxygen species scavenger containing the intestinal-derived exosomes of the fourteenth aspect as an active ingredient.

[0029] The eighteenth aspect of the present invention is a wrinkle-improving agent for improving skin wrinkles, the wrinkle-improving agent containing the intestinal-derived exosomes of the fourteenth aspect as an active ingredient.

[0030] A 19th aspect of the present invention is a skin moisturizing function improver for improving the moisturizing function of the skin, which contains the intestinal-derived exosome of the 14th aspect as an active ingredient.

[0031] A 20th aspect of the present invention is a muscle function activator that contains the intestinal-derived exosome of the 14th aspect as an active ingredient.

[0032] A 21st aspect of the present invention is a food composition for preventing aging-related diseases that contains the intestinal-derived exosome of the 14th aspect as an active ingredient.

Advantages of the Invention

[0033] According to each aspect of the present invention, it becomes possible to provide a mitochondrial function improver or the like that improves the function of mitochondria in skin cells or myoblasts.

[0034] In searching for foods that suppress skin aging, the inventors of the present invention focused on the longevity genes SIRT1 and SIRT3.

[0035] According to the 2nd aspect of the present invention, by activating SIRT1 and activating PGC1-α, it becomes possible to improve the function of mitochondria.

[0036] In particular, according to the 3rd and 4th aspects of the present invention, by increasing the number and area of mitochondria in skin cells, it becomes possible to improve the function and activity of mitochondria. In particular, there have been few reports of substances that increase both the number and area of mitochondria, which can be said to be an excellent effect of the present invention.

[0037] According to any one of the 5th to 7th aspects of the present invention, it becomes possible to provide a cosmetic, wrinkle improver, or aging inhibitor that improves mitochondrial function when applied to the skin.

[0038] According to the 10th to 21st aspects of the present invention, it becomes possible to control the secretion of exosomes from intestinal cells that directly affects the enhancement of mitochondria in cells.

[0039] The 10th to 21st aspects can be conceived from the fact that the inventors found that exosomes secreted from intestinal cells, triggered by the absorption of components contained in edible bird's nest by intestinal cells, lead to the enhancement and activation of cell mitochondria.

Brief Description of the Drawings

[0040]

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Mode for Carrying Out the Invention

[0041] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Note that the examples of the present invention are not limited to the contents described below.

Examples

[0042] Chapter 1 Experimental Materials and Methods Section 1 Cell Culture Item 1 Human epidermal keratinocyte HaCaT cells In this study, human epidermal keratinocyte HaCaT cells were used as model cells for human epidermis. The HaCaT cells were subcultured in a cell culture dish (Corning, New York, USA) at 37°C in the presence of 5% CO2 using DMEM medium containing 10% fetal bovine serum (FBS) inactivated by heating in a constant temperature bath at 56°C for 30 minutes. The DMEM medium was prepared by dissolving 4.75 g of Dulbecco's modified Eagle's medium "Nissui" (2) (Nissui pharmaceutical, Tokyo, Japan) in 470 mL of Milli-Q water, sterilizing it by autoclaving, and then adding 1 mL of 100 U / mL penicillin (Meiji seika pharma, Tokyo, Japan), 1 mL of 0.1 mg / mL streptomycin (Meiji seika pharma, Tokyo, Japan), 6 mL of 10% NaHCO3 (FUJIFILM Wako), and L-glutamine filtered and sterilized with a 0.22 μm filter to a final concentration of 4 mM (10 mL of 0.2 M L(+)-glutamine (FUJIFILM Wako)). The medium was stored at 4°C.

[0043] Item 2. Human epidermal keratinocyte HaCaT (SIRT1 p-EGFP) cells HaCaT cells (SIRT1 p-EGFP) into which an EGFP expression vector (SIRT1 p-EGFP) whose expression is controlled by the SIRT1 promoter, established by previous research, was introduced into HaCaT cells were used. Similar to the HaCaT cells, they were subcultured at 37°C in the presence of 5% CO2 using DMEM medium supplemented with 10% FBS and a Petri dish.

[0044] Item 3. Human epidermal keratinocyte HaCaT (SIRT3 p-EGFP) cells HaCaT cells (SIRT3 p-EGFP) transfected with an EGFP expression vector (SIRT3 p-EGFP) whose expression is controlled by the SIRT3 promoter, established by previous research, were used. Similar to HaCaT cells, they were subcultured in DMEM medium supplemented with 10% FBS in a Petri dish at 37 °C in the presence of 5% CO2.

[0045] Section 2 Samples and Their Preparation Methods Item 1 Edible Bird's Nest Samples Edible bird's nest sample solutions A to C were prepared. As the preparation procedure for the edible bird's nest sample solution, first, dried and pulverized Malaysian swiftlet nests (with a water content of 10% or less) and a protease preparation solution were immersed at a ratio of 1:9, and then enzymatic hydrolysis was carried out for 48 hours while keeping the temperature in the range of 45 - 55 °C. After that, undissolved residues were removed by a 100-mesh sieve, and the sieved liquid was deactivated with enzymes under the conditions of 80 - 90 °C for 60 minutes to obtain an extract derived from edible bird's nest.

[0046] Here, the above protease preparation solution was prepared by completely dissolving protease derived from Aspergillus oryzae (titer: 30000 u / g (CFA method, pH 6.0)) in pure water treated with an RO membrane at a concentration in the range of 1.5 to 2.5% at room temperature.

[0047] Subsequently, the solution containing the extract derived from edible bird's nest (hereinafter referred to as the "edible bird's nest solution") was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was adjusted to concentrations of 150 μg / mL, 450 μg / mL, 1500 μg / mL, and 4500 μg / mL using 1×PBS as a solvent. It was stored at 4 °C and appropriately suspended for use.

[0048] Item 2 Positive Control Resveratrol Solution Resveratrol powder (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was prepared at 10 mM using dimethyl sulfoxide (DMSO) (FUJIFILM Wako), filtered and sterilized through a 0.20 μm × 13 mm filter (Merck Millipore), stored at -20°C, thawed as appropriate, suspended, and used.

[0049] AICAR solution AICAR powder (FUJIFILM Wako) was prepared at 100 mM using 1×PBS, filtered and sterilized through a 0.22 μm × 13 mm filter (Merck Millipore), stored at -20°C, thawed as appropriate, and used.

[0050] Section 3 Exploration of the functionality of bird's nest using IN Cell Analyzer 2200 Verification of SIRT1 and SIRT3 activation in Item 1 Reagent preparation (1) Cell fixation solution To the required amount of 1×PBS, 0.08 g / mL of paraformaldehyde (FUJIFILM Wako, Tokyo, Japan) and 2 μL / mL of 5 N NaOH were added, and an 8% paraformaldehyde solution was prepared by dissolving it in a 60°C water bath and stored at 4°C. (2) Nuclear staining solution Hoechst33342 was diluted to 2 μg / mL with 1×PBS and used in a light-shielded state.

[0051] The SIRT1 / 3 activity in HaCaT (SIRT1 / 3) cells supplemented with the bird's nest solution was evaluated. HaCaT (SIRT1 / 3p-EGFP) cells were seeded in a 96-well black plate (Greiner Bio-one, Tokyo, Japan) at a final concentration of 6.0×104 cells / mL and cultured in DMEM medium containing 10% FBS at 37 °C in the presence of 5% CO2. After 24 hours, resveratrol and the bird's nest samples were added as positive controls at a concentration of 0.1 μL / well each. Also, 1×PBS was added in the same amount as the sample addition as a control and cultured at 37 °C in the presence of 5% CO2. After 48 hours, 100 μL / well of cell fixative was added and left to stand at room temperature for 15 minutes in the dark. The cell culture solution and the cell fixative were combined and removed, washed twice with 1×PBS, and then 100 μL / well of nuclear staining solution was added. After leaving it to stand at room temperature for 20 minutes in the dark, the nuclear staining solution was removed and washed twice with 1×PBS. 1×PBS was added at 100 μL / well, and the EGFP fluorescence intensity was measured using an IN Cell Analyzer 2200.

[0052] Section 2 Verification of the effect on mitochondria (1) Mitochondrial staining solution Using DMEM medium containing 10% FBS, Mito TrackerRed CMXos was diluted to a final concentration of 0.25 μM and Mito TrackerGreen FM was diluted to a final concentration of 0.2 μM. (2) Nuclear staining solution The nuclear staining solution was prepared in the same procedure as in Section 3, Item 1.

[0053] The effect on mitochondria in HaCaT cells supplemented with the bird's nest solution was verified. HaCaT cells at a final concentration of 6.0×10 4They were seeded into a 96-well black plate at a density of [[cells / mL]], and cultured in DMEM medium containing 10% FBS at 37 °C in the presence of 5% CO2. After 24 hours, AICAR was added to a final concentration of 250 mM, and the bird's nest samples were added at a concentration of 0.1 μL / well for each concentration. As a control, 1×PBS was added in the same amount as the sample addition amount, and cultured at 37 °C in the presence of 5% CO2. After 24 hours, the cell culture medium was removed, and a mitochondrial staining solution was added at 100 μL / well and incubated at 37 °C for 15 minutes. After removing the mitochondrial staining solution, a nuclear staining solution was added at 100 μL / well and left to stand at room temperature in the dark for 30 minutes. After removing the nuclear staining solution, 1×PBS was added at 150 μL / well, and the number, area, and activity of mitochondria were measured using an IN Cell Analyzer 2200.

[0054] Section 6 Statistical Analysis Statistical analysis was performed by Student's t-test and Tukey-Krammer method (Statcel 4), and a significant difference was considered when p < 0.05.

[0055] Chapter 2 Results Section 1 Exploration of the functionality of bird's nest using IN Cell Analyzer 2200 Using HaCaT cells transfected with SIRT1 p-EGFP, the SIRT1 promoter activity when the bird's nest sample was added was evaluated by measuring the EGFP fluorescence intensity. Figure 1 shows a diagram indicating the EGFP fluorescence intensity by the SIRT1 promoter activity of the bird's nest sample. Resveratrol and the bird's nest sample were added as positive controls to HaCaT (SIRT1 p-EGFP) cells, and the fluorescence intensity of EGFP was measured using an IN Cell Analyzer 2200 after culturing for 48 hours.

[0056] As a result, as shown in Fig. 1, significantly higher fluorescence intensities than the control were obtained in all of the samples A to C of the swallow's nest. This suggests that the bird's nest sample quantitatively activates SIRT1. The quantitative activation of SIRT1 leads to the activation of PGC1-α.

[0057] Next, using HaCaT cells transfected with SIRT3 p-EGFP, the SIRT3 promoter activity when the bird's nest sample was added was evaluated by measuring the EGFP fluorescence intensity. Fig. 2 is a diagram showing the EGFP fluorescence intensity due to the SIRT3 promoter activity of the bird's nest sample. Similar to SIRT1, Resveratrol and the bird's nest sample were added to HaCaT (SIRT3 p-EGFP) cells, and after culturing for 48 hours, the EGFP fluorescence intensity was measured using an IN Cell Analyzer 2200.

[0058] As a result, as shown in Fig. 2, the sample of 4500 μg / mL of the swallow's nest A showed a significantly higher fluorescence intensity than the control.

[0059] The effect on mitochondria in HaCaT cells by the bird's nest sample was measured. Figs. 3, 4, and 5 are diagrams showing the number of mitochondria, mitochondrial area, and mitochondrial activity in HaCaT cells, respectively. AICAR was added to HaCaT cells as a positive control at a final concentration of 100 mM, and the bird's nest sample was added at 0.1 μL / well, respectively. After culturing for 48 hours, staining was performed with MitoTracker Green and MitoTracker Red, and the number, area, and activity of mitochondria were measured using an IN Cell Analyzer 2200.

[0060] As a result, as shown in Figs. 3 and 4, a significant increase in both the number and area was confirmed in all of the bird's nests A, B, and C compared to the control.

[0061] Also, as shown in Fig. 5, a tendency was obtained in which the fluorescence intensity of mitochondrial activity was also stronger compared to the control. In particular, high activity was observed in Sample A at 450 μg / mL, Sample B at 450 μg / mL and 4500 μg / mL, and Sample C at 4500 μg / mL.

[0062] Note that the mitochondrial activity shown in Fig. 5 reflects the number of mitochondria showing high activity and does not indicate the total amount of activity of all the mitochondria in the sample.

[0063] In this study, first, for the purpose of clarifying the molecular basis of skin improvement by bird's nest, research was conducted focusing on longevity genes. In the verification of SIRT1 and SIRT3 activation, it was confirmed that the bird's nest sample quantitatively activates SIRT1 and significantly activates SIRT3 compared to the control.

[0064] Also, in the verification of the effect on mitochondria downstream of SIRT1, multiple bird's nest samples were confirmed to significantly enhance the number and area of mitochondria and also enhance mitochondrial activity. From this, it is considered that bird's nest activates PGC1-α, which is downstream of SIRT1 and upstream of mitochondria.

[0065] HaCaT cells are a cell model of human epidermis without blood vessels. Therefore, the bird's nest sample according to this example is expected to have functions such as suppression and improvement of wrinkles, suppression of epidermal thickening, suppression of gray hair and hair loss, at least as an active ingredient of the applied cosmetics, wrinkle improver or anti-aging agent.

Example

[0066] Chapter 2 Experimental Materials and Methods Section 1 Cell Culture Item 1 Culture of Human Colorectal Cancer-Derived Cell Line Caco-2 Caco-2 cells were subcultured using Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% heat-inactivated Fetal Bovine Serum (FBS) (complement inactivated by heating in a 56°C water bath for 30 minutes) and cell culture dishes at 37°C in the presence of 5% CO₂. The DMEM medium was prepared by dissolving 4.75 g of DMEM medium "Nissui" (2) (Nissui Pharmaceutical, Tokyo, Japan) in 470 mL of Milli-Q water, sterilizing it by autoclaving, and then adding 1 mL of 100 U / mL penicillin (Meiji Seika Pharma, Tokyo, Japan), 1 mL of 0.1 mg / mL streptomycin (Meiji Seika Pharma), 6 mL of 10% NaHCO₃ (FUJIFILM Wako Pure Chemical), and L-glutamine filtered and sterilized with a 0.22 μm filter (Toyo Roshi) to a final concentration of 4 mM. The medium was stored at 4°C.

[0067] Item 2. Culture of human epidermal keratinocyte HaCaT cells Similar to Caco-2 cells, subculture was performed using DMEM medium supplemented with 10% FBS and cell culture dishes at 37°C in the presence of 5% CO₂.

[0068] Item 3. Culture of human epidermal keratinocyte HaCaT (SIRT1p-EGFP) cells HaCaT cells (SIRT1p-EGFP) into which the SIRT1-EGFP reporter vector had been introduced, established by previous research, were used. HaCaT cells (SIRT1p-EGFP) are a stable expression strain and are designed to express EGFP fluorescence in response to activation of the SIRT1 promoter. Similar to Caco-2 cells, subculture was performed using DMEM medium supplemented with 10% FBS and cell culture dishes at 37°C in the presence of 5% CO₂.

[0069] Item 4. Culture of immortalized human skin fibroblast - SV40, a human skin fibroblast cell line In this study, the immortalized human skin fibroblast - SV40 (hereinafter referred to as Skin Fibroblast cells) (Applied biological materials, Richmond, BC, Canada), a human skin fibroblast cell line, was used as a skin dermal fibroblast model. The Skin Fibroblast cells were subcultured at 37°C in the presence of 5% CO2 using DMEM medium containing 10% non-inactivated FBS and cell culture dishes.

[0070] Section 2 Samples and Their Preparation Methods - Swiftlet Nest Samples The swiftlet nest solution prepared in the same manner as in Example 1 was centrifuged at 10,000 rpm for 10 minutes, and its supernatant was adjusted to concentrations of 150 μg / mL, 450 μg / mL, 1500 μg / mL, and 4500 μg / mL using 1×PBS as a solvent. It was stored at -20°C and thawed as appropriate when used.

[0071] Section 3 Verification of Activation of Skin Longevity Genes via the Intestine Effect of SIRT1 Promoter Activity in Item 1 Preparation of Reagents (1) Cell Fixative It was diluted with 1×PBS to 8% paraformaldehyde, 5 μg / mL of 2 N sodium hydroxide solution was added, and it was dissolved in a 60°C water bath and stored at 4°C. (2) Nuclear Staining Solution Hoechst33342 was diluted with 1×PBS to 2 μg / mL and used in a light-shielded state. Also, it was prepared at the time of use.

[0072] Caco-2 Supernatant Addition Experiment Caco-2 cells were adjusted to a final concentration of 1.0×10 5They were seeded in a 24-well plate (FALCON) to a density of 4 6.0×10 cells / mL. After 24 hours, 1 μL / well of the swallow nest samples at each concentration was added. 1×PBS was used as a control. HaCaT (SIRT1p-EGFP) cells were seeded in a 96-well black plate to a density of 6.0×10

[0073] Item 2 Effect on endogenous longevity genes using RT-qPCR Next, using the samples with enhanced SIRT1 activity selected from the screening results and newly prepared samples, the analysis of the longevity genes of HaCaT cells was performed by RT-qPCR.

[0074] (1) Preparation of total RNA using the High Pure RNA Isolation Kit Total RNA was prepared using the High Pure RNA Isolation Kit (Roche, Basel, Switzerland) according to the product protocol. Caco-2 cells were seeded in a 24-well plate at a final concentration of 1.0×105 cells / mL. After 24 hours, the swallow's nest samples were added at 1 μL / well for each concentration. HaCaT cells were at a final concentration of 6.0×10 4They were seeded into a 5 mL dish to reach a density of cells / mL. After culturing for 24 hours, 2.5 mL / well of the medium was removed, and then the samples were added. 2.5 mL / well of Caco-2 cell culture medium was added, and the cells were cultured at 37 °C in the presence of 5% CO2 for 48 hours. After 48 hours, the medium was completely removed, and 2 mL of 1×PBS was added for washing. Then, 200 μL of 1×PBS and 400 μL of the Lysis-binding Buffer of the High Pure RNA Isolation Kit were added, and the plate was tilted for 4 minutes while swirling to spread the mixture evenly over the entire dish until it lost its viscosity. The bottom of the dish was rubbed on the back of the chip, and the cell lysate was collected into a 1.5 mL tube. The collected cell lysate was thoroughly suspended with a vortex mixer for 50 seconds. The High Pure filter tube and collection tube of the Kit were assembled, and the entire volume of the cell lysate was added to the filter tube. Centrifugation was performed at 10,000 rpm for 15 seconds, the solution discharged into the collection tube was discarded, and the filter tube and collection tube were assembled again. To a 1.5 mL tube, 90 μL of DNase Incubation Buffer per sample and 10 μL of DNase I were added in an amount one more than the number of samples and mixed. 100 μL of this mixture was added to the previous filter tube and left standing at room temperature for 15 minutes. After 15 minutes, 500 μL of Wash Buffer I of the Kit was added to the filter tube, and centrifugation was performed at 10,000 rpm for 15 seconds. The solution in the collection tube was discarded, the filter tube and collection tube were assembled again, 500 μL of Wash Buffer II was added to the filter tube, and centrifugation was performed at 10,000 rpm for 15 seconds. The solution in the collection tube was discarded, the filter tube and collection tube were assembled, 200 μL of Wash Buffer II was added to the filter tube, and centrifugation was performed at 14,000 rpm for 2 minutes.After centrifugation, the filter tube was set in a new sterilized 1.5 mL tube, 100 μL of Elusion Buffer was added to the center of the filter tube, and it was allowed to stand at room temperature for 3 minutes. After standing, it was centrifuged at 10,000 rpm for 1 minute to elute total RNA. The total RNA concentration in the solution was measured using a Nano Drop 2000 (Thermo Fisher SCIENTIFIC, Waltham, USA).

[0075] (2) RT-qPCR Each RNA was diluted and prepared to be 6.25 ng / μL using Nuclease-Free Water in a 0.2 mL tube, and further 10-fold diluted samples were prepared respectively. Also, primers for SIRT1 and SIRT3 were diluted to 2 μM in a 0.2 mL tube. Furthermore, considering the number of samples and genes, Go Taq qPCR Master Mix, Go Script RT Mix, and Nuclease-Free Water were suspended in a 1.5 mL tube at a ratio of 25:1:4 to make a premix. The 96-well plate for PCR was set on an ice plate, 12 μL of the premix, 2 μL each of the forward (F) and reverse (R) primers, and 4 μL each of the diluted RNA were added to all wells, and then sealed. It was centrifuged for 10 seconds using a plate centrifuge and set for PCR measurement. The sequences of each primer are shown in Table 1.

[0076]

Table 1

[0077] Section 4 Effects on Epidermal Keratinocytes via Intestinal Exosomes Using RT-qPCR Item 1 Exosome Purification (1) Cell Culture and Swallow's Nest Sample Processing in Exosome Purification Caco-2 cells were seeded in a 10 mL dish at a density of 1.4×106 cells / dish. The culture after seeding was carried out in DMEM medium containing 10% Exosome-depleted FBS Media Supplement Heat Inactivated (System Biosciences, Mountain View, CA, USA). The prepared swallow's nest samples were added at a concentration of 10 μL / well 24 hours later, and the culture supernatant was collected 24 hours later. Exosomes secreted by Caco-2 cells were purified by the following method.

[0078] (2) Purification of exosomes by phosphatidylserine affinity method 10 mL of the collected culture supernatant was centrifuged at 300×g for 5 minutes to remove cells in the culture supernatant. The supernatant was transferred to another tube and then centrifuged at 1,200×g for 20 minutes to remove cell fragments. The supernatant was transferred to another tube and centrifuged at 10,000×g for 30 minutes to remove extracellular vesicles larger than exosomes. The culture supernatant from which cells and large-sized extracellular vesicles were removed was concentrated approximately 40-fold using a centrifugal ultrafiltration unit with a filter having a molecular weight cut-off of 100,000 (AmiconUltra15 100K, Merck Millipore). Purification of exosomes from the concentrated culture supernatant was performed using the MagCapture Exosome Isolation PS Kit Ver.2 (FUJIFILM Wako). First, buffers were prepared. 0.55 mL of Exosome Immobilizing / Washing Buffer (10×) and 4.95 mL of purified water were added to a 15 mL tube, and further 11 μL of Exosome Binding Enhancer (500×) was added to prepare Exosome Immobilizing / Washing buffer (1×). 15 μL of Exosome Elution Buffer (10×) and 135 μL of purified water were added to a 1.5 mL tube to prepare Exosome Elution Buffer (1×). Next, Exosome Capture immobilized beads were prepared. 60 μL of Biotin Capture Magnetic Beads well-stirred with a vortex mixer was transferred to a 1.5 mL Reaction Tube, 500 μL of Exosome Immobilizing / Washing buffer (1×) was added, and the mixture was suspended with a vortex mixer. The tube was spun down and set on a magnetic stand and allowed to stand for 1 minute.Next, add 500 μL of Exosome Immobilizing / Washing buffer (1×) and 10 μL of Biotin-labeled Exosome Capture to the tube, remove it from the magnetic stand, suspend it with a vortex mixer, and react for 10 minutes at room temperature while inverting and mixing with a rotary shaker. Spin down the tube, set it on the magnetic stand again, and let it stand for about 1 minute. Once the magnetic beads have completely adhered to the tube wall, remove the supernatant with a pipette. (A) Add 500 μL of Exosome Immobilizing / Washing buffer (1×) to this tube, remove it from the magnetic stand, suspend it with a vortex mixer, spin down the tube, then set it on the magnetic stand again and let it stand for 1 minute. Once the magnetic beads have completely adhered to the tube wall, remove the supernatant with a pipette. Repeat the operation in (A) one more time. Through the above operations, Exosome Capture-immobilized beads were completed. Next, a process of reacting the Exosome Capture-immobilized beads with the culture supernatant concentrated as described above was performed. Transfer approximately 500 μL of the culture supernatant concentrated 40-fold to a sterilized 1.5 mL tube, add 1 / 500 volume of Exosome Binding Enhancer (500×) to the cell supernatant, and mix with a vortex mixer. Spin down this tube, transfer the sample to the tube containing the Exosome Capture-immobilized beads (Reaction Tube), and mix with a vortex mixer. React for 1 hour or more at room temperature while inverting and mixing with a rotary shaker. After spinning down the 1.5 mL Reaction Tube, set it on the magnetic stand and let it stand for about 1 minute. Once the magnetic beads have completely adhered to the tube wall, remove the supernatant with a pipette to obtain beads bound with exosomes. Next, the exosome-bound beads were washed.(a) Add 1 mL of Washing Buffer containing Exosome Binding Enhancer to a 1.5 mL Reaction Tube containing exosome-binding beads, suspend by vortex mixer, spin down the 1.5 mL Reaction Tube and set it on a magnetic stand. After standing for about 1 minute until the magnetic beads adhere completely to the tube wall, remove the supernatant. Repeat the operation in (a) two more times. By this operation, washed exosome-binding beads were obtained. The elution of exosomes was performed as follows. Add 50 μL of Exosome Elution Buffer (1×) to a 1.5 mL Reaction Tube containing the washed exosome-binding beads, remove from the magnetic stand and suspend by vortex mixer. Spin down the tube, set it on the magnetic stand, and after standing for 1 minute, when the magnetic beads adhere completely to the tube wall, collect the supernatant into a new sterile 1.5 mL tube. Add an additional 50 μL of Exosome Elution Buffer (1×) to the magnetic beads remaining in the 1.5 mL Reaction Tube, remove from the magnetic stand and suspend by vortex mixer. Spin down, set on the magnetic stand, and stand for 1 minute. When the magnetic beads adhere completely to the tube wall, collect the supernatant into a new sterile 1.5 mL tube (the above-mentioned one) to obtain a total of 100 μL of exosome solution.

[0079] (3) Quantification of exosome amount (BCA method) The amount of exosomes was quantified by measuring the protein concentration using the BCA method. For the measurement of protein concentration by the BCA method, the Micro BCA Protein Assay Kit (Thermo Fisher Scientific) was used. For the measurement of the exosome solution, a 10-fold diluted solution using 30 μL out of 100 μL was used. As the standard for the standard curve, a solution obtained by serially diluting BSA with PBS to a concentration of 0 to 200 μg / mL was used. With 2 wells of a 96-well plate (Thermo Fisher Scientific) as one assay, 150 μL each was added to each well in the order of standard and sample. Next, a mixture of Micro Reagent A, Micro Reagent B, and Micro Reagent C included in the Micro BCA Protein Assay Kit at a ratio of 25:24:1 was added to all wells at 150 μL each. The reaction was carried out in the dark at 37°C for 2 hours, and the measurement was performed based on the absorbance at 595 nm using an absorptiometer (Sunrise, TECAN). Quantification was performed based on the standard curve obtained by the measurement to determine the protein concentration.

[0080] Item 2. Effects on longevity genes (1) Preparation of total RNA using the High Pure RNA Isolation Kit HaCaT cells were adjusted to a final concentration of 3.0×10 4Cells were seeded in a 5 mL dish to a density of cells / mL. After culturing for 24 hours, 1 μg / well of the exosomes purified in Section 4 was added, and the cells were cultured for 48 hours at 37°C in the presence of 5% CO2. After 48 hours, the culture medium was completely removed, and 2 mL of 1×PBS was added for washing. 200 μL of 1×PBS and 400 μL of the Lysisbinding Buffer from the High Pure RNA Isolation Kit were added, and the solution was spread over the entire dish while tilting the plate for 4 minutes until it lost its viscosity. The bottom of the dish was rubbed on the back of the chip, and the cell lysate was collected into a 1.5 mL tube. The collected cell lysate was thoroughly suspended with a vortex mixer for 50 seconds. The High Pure filter tube and collection tube of the Kit were assembled, and the entire volume of the cell lysate was added to the filter tube. Centrifugation was performed at 10,000 rpm for 15 seconds, the solution discharged into the collection tube was discarded, and the filter tube and collection tube were reassembled. To a 1.5 mL tube, 90 μL of DNase Incubation Buffer per sample and 10 μL of DNase I were added in an amount one more than the number of samples and mixed. 100 μL of this mixture was added to the previous filter tube and left standing at room temperature for 15 minutes. After 15 minutes, 500 μL of Wash Buffer I of the Kit was added to the filter tube, and centrifugation was performed at 10,000 rpm for 15 seconds. The solution in the collection tube was discarded, the filter tube and collection tube were reassembled again, 500 μL of Wash Buffer II was added to the filter tube, and centrifugation was performed at 10,000 rpm for 15 seconds. The solution in the collection tube was discarded, the filter tube and collection tube were reassembled, 200 μL of Wash Buffer II was added to the filter tube, and centrifugation was performed at 14,000 rpm for 2 minutes. After centrifugation, the filter tube was set in a new sterilized 1.5 mL tube, 100 μL of Elusion Buffer was added to the center of the filter tube, and it was left standing at room temperature for 3 minutes.After standing, it was centrifuged at 10,000 rpm for 1 minute to elute total RNA. The total RNA concentration in the solution was measured using a Nano Drop 2000 (Thermo Fisher SCIENTIFIC).

[0081] (2) RT-qPCR It was carried out in the same procedure as in Section 3, Item 1(2). The sequences of each primer are shown in Table 1.

[0082] Item 3 Effect on genes related to the outer barrier (1) Preparation of total RNA using the High Pure RNA Isolation Kit It was carried out in the same procedure as in Section 4, Item 2(1). (2) RT-qPCR It was carried out in the same procedure as in Section 3, Item 1(2). The sequences of each primer are shown in Table 1.

[0083] Item 5 Effect on mitochondria (1) Mitochondrial staining solution Using DMEM medium containing 10% FBS, Mito TrackerRed CMXos was prepared at a final concentration of 0.25 μM and Mito TrackerGreen FM was prepared at a final concentration of 0.2 μM. (2) Nuclear staining solution The nuclear staining solution was prepared in the same procedure as in Section 3, Item 1(2).

[0084] Exosome addition experiment The effect of exosomes derived from Caco-2 cells treated with swallow nest solution on mitochondria in HaCaT cells was verified. The HaCaT cells had a final concentration of 6.0×10 4Cells were seeded in a 96-well black plate to a density of cells / mL and cultured in DMEM medium containing 10% FBS at 37 °C in the presence of 5% CO2. After 24 hours, exosomes were added to a concentration of 90 ng / well. As a control, 0.1 μL / well of 1×PBS was added and the cells were cultured at 37 °C in the presence of 5% CO2. After 48 hours, the cell culture medium was removed, 100 μL / well of mitochondrial staining solution was added, and the cells were incubated at 37 °C for 15 minutes. After removing the mitochondrial staining solution, 100 μL / well of nuclear staining solution was added and the cells were left standing at room temperature in the dark for 30 minutes. After removing the nuclear staining solution, 150 μL / well of 1×PBS was added and the mitochondrial activity was measured using an IN Cell Analyzer 2200.

[0085] Preparation of the reagent for testing the effect of ROS scavenging ability in Item 6 (1) HBSS 9.7 g of Hanks’ Balanced Salts (SIGMA) and 0.35 g of NaHCO3 (FUJIFILM Wako Pure Chemical Corporation) were placed in 900 mL of ultrapure water and stirred with a stirrer. After the powder had completely dissolved, the pH was adjusted to 7.0 (±0.1 - 0.3), made up to 1 L with a volumetric flask. Then, after sterilization with a 0.22 μm filter (Merck KGaA, Darmstadt, Germany), it was shielded from light and stored at 4 °C. (2) BES-H2O2-Ac 1 mg of BES-H2O2-Ac was dissolved in 750 μL of ethanol, shielded from light, and stored at 4 °C. When in use, it was appropriately diluted 500-fold with 10 mM HEPES (pH 7.4). (3) Nuclear staining solution The nuclear staining solution was prepared in the same procedure as in Section 3, Item 1.

[0086] Exosome addition experiment Subconfluent HaCaT cells were cultured in a 10 mL dish and exposed to UV-B 10 mJ / cm in a UV crosslinker (CL-1000 Ultraviolet Crosslinker, UVP, Upland, CA, USA) with the dish lid removed. 2 After washing with 1× PBS, the cells were detached with trypsin and plated in a 96-well plate at 1.2×10 5 After confirming that the cells had started to adhere, exosomes derived from Caco-2 cells treated with bird's nest extract were added at 90 ng / well. 1×PBS was used as a control. The cells were cultured for 24 hours at 37°C in the presence of 5% CO2. After that, the intracellular active oxygen scavenging ability and cell number were measured by BES staining using an IN Cell Analyzer 2200. After removing all the supernatant after culture, the cells were washed once with HBSS, and 100 μL / well of BES-H2O2-Ac diluted 400-fold with 10 mM HEPES and Hoechst 33342 solution diluted 500-fold were added, and the cells were incubated at 37°C in the dark for 30 minutes. After 30 minutes, the staining solution was removed, the cells were washed once with HBSS, and 100 μL / well of HBSS was added and measured using an IN Cell Analyzer 2200. Images were analyzed using IN Cell Investigator High-content image analysis software (GE Healthcare). The protocol used was "HaCaT-eGFP 20190627," and the intracellular reactive oxygen scavenging ability was examined by measuring the amount of GFP fluorescence.

[0087] Section 5. Effects on dermal fibroblasts (exosome experiment) Item 1. Exosome purification (1) Cell culture and bird's nest sample processing for exosome purification The procedure was the same as in Section 4, Paragraph 1 (1). (2) Purification of exosomes by phosphatidylserine affinity method The procedure was the same as that in Section 4, Paragraph 1(2). (3) Quantification of exosome amount (BCA method) The procedure was the same as that in Section 4, Paragraph 1(3).

[0088] Paragraph 2 Effect on longevity genes (1) Preparation of total RNA using High Pure RNA Isolation Kit The procedure was the same as that in Section 4, Paragraph 2(1). (2) RT-qPCR The procedure was the same as that in Section 3, Paragraph 1(2). The sequences of each primer are shown in Table 1.

[0089] Paragraph 3 Effect on wrinkle-related genes (1) Preparation of total RNA using High Pure RNA Isolation Kit The procedure was the same as that in Section 4, Paragraph 2(1). (2) RT-qPCR The procedure was the same as that in Section 3, Paragraph 1(2). The sequences of each primer are shown in Table 1 (Figure 21).

[0090] Section 6 Microarray analysis The exosome solution purified by the phosphatidylserine affinity method in Section 10, Paragraph 1 was used for microarray analysis. The microarray experiment was entrusted to Kanagawa Science (Kanagawa, Japan). Based on the obtained data, miRNAs with a ratio of 2.0 or more when compared with the control and a signal value of either of the two samples exceeding 100 were extracted as miRNAs with variable transcript levels. For the miRNAs selected by the above method, TargetScan Human 8.0 (http: / / www.targetscan.org / vert_80 / ) was used to search for target genes, and the functions of each gene were inferred by the online analysis software DAVID (https: / / david.ncifcrf.gov / summary.jsp).

[0091] Section 7 Statistical Analysis Statistical analysis was performed using Student’s t-test and Tukey-Krammer method (Statcel 4), and a p-value < 0.05 was considered to indicate a significant difference.

[0092] Chapter 3 Results Section 1 Verification of Activation of Skin Longevity Genes via the Intestinal Tract Item 1 Effect on the SIRT1 Promoter Samples were added to Caco-2 cells, and the supernatant was added to HaCaT cells. After culturing for 48 hours, the change in EGFP fluorescence intensity in HaCaT (SIRT1p-EGFP) cells was measured using an IN Cell Analyzer 2200. As a result, an increasing trend in the fluorescence value of SIRT1 promoter EGFP was observed at G150, G450, and G1500, so these were selected as SIRT1 promoter activation samples (Figure 6).

[0093] Item 2 Effect on Endogenous Longevity Genes Using RT-qPCR Samples selected in Item 1 of Section 1 and newly prepared samples were added to Caco-2 cells, and the mRNA expression levels of SIRT1 and SIRT3 in HaCaT cells after culturing for 48 hours with the added supernatant were measured by RT-qPCR. As a result, significant enhancement of expression was observed for L1500 and L4500 in SIRT1, and L4500 in SIRT3. From these results, L1500 and L4500 were selected as longevity gene activation samples (Figure 7).

[0094] Section 2 Effect on Epidermal Keratinocytes via Intestinal Tract-Derived Exosomes Using RT-qPCR Item 1 Effect on Longevity Genes Caco-2 cell-derived exosomes treated with the honeybee nest extract selected in Section 1 were added to HaCaT cells, and the mRNA expression levels of SIRT1 and SIRT3 after 48 hours of culture were measured by RT-qPCR. As a result, a tendency for enhanced expression was confirmed for SIRT1 and SIRT3 (Figure 8).

[0095] Item 2. Effects on externally related barrier genes Caco-2 cell-derived exosomes treated with the honeybee nest extract selected in Section 1 were added to HaCaT cells, and the mRNA expression levels of externally related barrier genes after 48 hours of culture were measured by RT-qPCR. As a result, the expression levels were significantly enhanced, at least for Filaggrin, Involucrin, and TGM1 at L4500 and for LOR at L1500. (Figure 9).

[0096] Item 4. Effects on mitochondria Caco-2 cell-derived exosomes treated with the honeybee nest extract selected in Section 1 were added to HaCaT cells, and after 48 hours of culture, they were stained with MitoTracker Green and MitoTracker Red, and the mitochondrial activity was measured using an IN Cell Analyzer 2200. As a result, it was confirmed that the fluorescence intensity was enhanced for both L1500 and L4500 (Figure 10).

[0097] Item 5. Effects of ROS scavenging ability Caco-2 cell-derived exosomes treated with the honeybee nest extract selected in Section 1 were added to HaCaT cells irradiated with 10 mJ / cm 2 of UVB. After 24 hours of culture, the change in BES fluorescence intensity in HaCaT cells was measured using an IN Cell Analyzer 2200. As a result, it was confirmed that L4500 significantly reduced the fluorescence intensity compared to the control irradiated with UVB (Figure 11).

[0098] Section 3: Effects on Dermal Fibroblasts via Intestinal Exosomes Using RT-qPCR Item 1: Effects on Longevity Genes Caco-2 cell-derived exosomes treated with the extracted solution from the swallow's nest selected in Section 1 were added to human skin fibroblasts (Skin Fibroblast), and the mRNA expression levels of SIRT1 and SIRT3 after 48 hours were measured by RT-qPCR. As a result, it was confirmed that the expression levels increased in SIRT1 and SIRT3 (Figure 12).

Example

[0099] For us who are facing various problems such as the current aging society and stress society, it is recommended to incorporate appropriate exercise into our lives. Exercise can bring not only physical effects such as maintaining and improving skeletal muscles and cardiopulmonary function, but also mental effects such as mood swings and stress relief. As many previous studies have shown, exercise is known to be effective against various disease risks such as aging, depression, stress, lifestyle diseases, and dementia.

[0100] These effects are closely related to the functions of skeletal muscles stimulated by exercise and the mitochondria present inside them. Skeletal muscle fibers can be broadly divided into two types: slow-twitch muscle fibers and fast-twitch muscle fibers. Slow-twitch muscle fibers are characterized by a small maximum output but rich endurance and contain a large number of mitochondria. On the other hand, fast-twitch muscle fibers have a large maximum output and are rich in explosive power, but have poor endurance and mitochondrial content. Although both types of fibers are important for normal physical activities, in order to maintain normal mitochondria, it is necessary to protect and enhance slow-twitch muscle fibers.

[0101] In addition, mitochondria have the effect of suppressing the generation of reactive oxygen species (ROS) generated in the living body, and since most aging-related diseases are caused by ROS, it can be said that maintaining the homeostasis of mitochondria plays an important role in realizing anti-aging. That is to say, continuous exercise can lead to the prevention of aging-related diseases by stimulating slow-twitch muscle fibers.

[0102] However, if one becomes bedridden due to the decline of the legs and waist caused by aging, the worsening of pre-existing diseases, or the increased disease risk due to stress, it becomes difficult to continue appropriate exercise due to physical limitations. In this state, the disease risk further increases, and one falls into a vicious cycle where exercise is restricted.

[0103] Therefore, the present inventors focused on and verified the changes in mitochondrial function due to the ingestion of swallow nest extract.

[0104] Experimental Materials and Methods Section 1: Cell Culture Item 1: Culture of Human Colorectal Cancer-Derived Cell Line Caco-2 In this study, the human colorectal cancer-derived cell line Caco-2 was used as a human intestinal epithelial cell model. Caco-2 cells were subcultured using Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% inactivated Fetal Bovine Serum (FBS) (complement inactivated by heating in a constant temperature bath at 56°C for 30 minutes) at 37°C in the presence of 5% CO2. The DMEM medium was prepared by dissolving 4.75 g of Dulbecco's modified Eagle medium "Nissui" (2) (Nissui pharmaceutical, Tokyo, Japan) in 470 mL of Milli-Q water, and adding 1 mL of 50,000 U / mL penicillin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 1 mL of 0.05 g / mL streptomycin (Meiji seika pharma, Tokyo, Japan), 15 mL of 50 g / L NaHCO3 (FUJIFILM Wako), 10 mL of 0.2 M L-glutamine (FUJIFILM Wako), and 3 mL of sterilized water.

[0105] Item 2: Culture of Mouse Skeletal Muscle-Derived Myoblast Cell Line C2C12 In this study, the mouse skeletal muscle-derived myoblast cell line C2C12 was used as a skeletal muscle cell model. C2C12 cells were cultured in the same manner as the culture of Caco-2 cells shown above.

[0106] Section 2 Samples and Their Preparation Methods Item 1 Preparation of Bee Nest Extract The bee nest extract used was prepared by the applicant of the present application in the same manner as in Example 1. The bee nest extract was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was adjusted to concentrations of 150 μg / mL, 450 μg / mL, 1500 μg / mL, and 4500 μg / mL using 1×PBS as a solvent. It was stored at -20°C and thawed as appropriate when used.

[0107] Item 2 Preparation of AICAR Solution AICAR powder (FUJIFILM Wako) was prepared to 100 mM using 1×PBS, filtered and sterilized with a 0.22 μm filter (Merck Millipore), stored at -20°C, and thawed as appropriate for use.

[0108] Section 3 Verification of Mitochondrial Activation Effect in Undifferentiated C2C12 Cells via the Intestinal Tract Caco-2 cells were seeded in a 24-well plate (FALCON) at 1×105 cells / well. After culturing for 24 hours, bee nest extract was added to a final concentration of 150 ng / mL, 450 ng / mL, 1500 ng / mL, and 4500 ng / mL. For the control, 1×PBS equal in volume to the extract addition amount was added, and for the positive control, AICAR was added to a final concentration of 200 μM. One milliliter of the culture supernatant was collected 24 hours after the addition. C2C12 cells were seeded in a μClear Fluorescence Black Plate (Greiner bio-one, Tokyo, Japan) at a concentration of 1.0×105 cells / mL. After culturing for 24 hours, the culture supernatant of the recovered Caco-2 cells was added to be 10% in the medium. Twenty-four hours after the addition of the supernatant, the culture supernatant was aspirated and removed, and 100 μL / well of MitoTracker Red CMXRos (Thermo Fisher Scientific., Waltham, MA, USA) diluted to 250 nM in DMEM medium containing 10% FBS was added. The cells were incubated for 15 minutes at 37°C in the presence of 5% CO2 under light protection. Then, 100 μL / well of MitoTracker Green FM (Thermo Fisher Scientific Inc) diluted to 200 nM in DMEM medium containing 10% FBS was added, and the cells were incubated for 15 minutes at 37°C in the presence of 5% CO2 under light protection. Subsequently, the staining solution was aspirated and removed, and 100 μL / well of Cellstain Hoechst 33342 solution (DOJINDO, Kumamoto, Japan) diluted 500-fold in DMEM medium was added. The cells were allowed to stand for 30 minutes under light protection for nuclear staining. The nuclear staining solution was aspirated and removed, 150 μL / well of 1×PBS was added, and fluorescence detection was performed using the IN Cell Analyzer 2200 (GE Healthcare Japan, Tokyo, Japan). The images were analyzed using the IN Cell Developer Toolbox 1.9.2 (GE Healthcare Bioscience), and the changes in the total number of mitochondria, mitochondrial area, and mitochondrial activity in the cells were examined by measuring the fluorescence intensities of MitoTracker Red, MitoTracker Green, and Hoechst 33342.

[0109] Section 4 Statistical Analysis Statistical analysis was performed using Student's t-test, and a p value < 0.05 was considered to indicate a significant difference.

[0110] Results Section 1 Verification of the Mitochondrial Activation Effect in Myoblasts via the Intestinal Tract by Swallow Nest Extract Item 1 Measurement of the Number and Area of Intracellular Mitochondria C2C12 cells treated with the Caco-2 cell culture supernatant for 1 day were stained for mitochondria using the fluorescent reagent MitoTracker Green FM, and the fluorescence intensity was analyzed using an IN Cell Analyzer 2200. Note that AICAR was used as a positive control and C2C12 cells were treated with 200 μM for 1 day. As a result, compared with the control (1×PBS), an increasing trend in the number of mitochondria was observed with AICAR, and a significant increase was also observed in the Caco-2 cell culture supernatant supplemented with the swallow's nest extract (Figure 13).

[0111] Regarding the area of mitochondria, a significant increase was observed in the Caco-2 cell culture supernatant supplemented with the swallow's nest extract (Figure 14).

[0112] Item 2 Measurement of intracellular mitochondrial activity In the process of mitochondria generating ATP through oxidative phosphorylation, a membrane potential is generated on the inner and outer sides of the inner mitochondrial membrane. The fluorescent reagent MitoTracker Red CMXRos is taken up into mitochondria by the proton gradient under this situation and emits fluorescence. That is, the mitochondria stained by MitoTracker Red CMXRos are the activated mitochondria that carry out ATP production by oxidative phosphorylation among the total mitochondria in the cell. Based on this principle, C2C12 cells treated with the Caco-2 cell culture supernatant for 1 day were stained using MitoTracker Red CMXRos, and the fluorescence intensity was analyzed using an IN Cell Analyzer 2200 to verify the mitochondrial activation effect mediated by the intestinal cells of the swallow's nest extract. Note that AICAR was also used as a positive control in this measurement, and C2C12 cells were treated with 200 μM for 1 day. As a result, compared with the control (1×PBS), a significant enhancement of mitochondrial membrane potential activity was observed with AICAR, and an enhancement was also observed in the Caco-2 cell culture supernatant supplemented with the swallow's nest extract (Figure 15).

[0113] Discussion In this study, the mitochondrial function was verified for the muscle activation effect of swallow nest extract through the intestinal tract. As an in vitro experimental system for verifying the functionality on muscle through the intestinal tract, an experiment was conducted in which the culture supernatant of Caco-2 cells was added to C2C12 cells. In skeletal muscle with high energy consumption, since the function of mitochondria responsible for energy production is very important, the functionality was verified using mitochondria as an indicator. As a result, the activation of mitochondria in C2C12 cells was observed with the culture supernatant of Caco-2 cells treated with swallow nest extract. From these results, it is considered that swallow nest extract can promote energy production in mitochondria through the intestinal tract and activate muscle function.

Claims

1. A mitochondrial function enhancer for enhancing the function of mitochondria in cells, comprising a component contained in edible bird's nest as an active ingredient. A mitochondrial function enhancer containing a component contained in edible bird's nest as an active ingredient.

2. The mitochondrial function enhancer according to Claim 1, wherein the active ingredient activates PGC1-α.

3. The mitochondrial function enhancer according to Claim 2, wherein the active ingredient increases the number of mitochondria in cells.

4. The mitochondrial function enhancer according to Claim 3, wherein the active ingredient increases the area of mitochondria.

5. A cosmetic for enhancing the function of mitochondria in cells, containing any one of the active ingredients according to Claims 1 to 4.

6. A wrinkle-improving agent for enhancing the function of mitochondria in cells, containing any one of the active ingredients according to Claims 1 to 4.

7. An anti-aging agent for enhancing the function of mitochondria in cells, containing any one of the active ingredients according to Claims 1 to 4.

8. A method for producing a mitochondrial function enhancer for enhancing the function of mitochondria in cells, comprising the step of adding a component contained in edible bird's nest as an active ingredient. A method for producing a mitochondrial function enhancer, comprising the step of adding a component contained in edible bird's nest as an active ingredient.

9. A method for enhancing the function of mitochondria in cells, comprising the step of applying an external preparation containing a component contained in edible bird's nest as an active ingredient to a subject (excluding medical acts on humans). A method for enhancing the function of mitochondria (excluding medical acts on humans), comprising the step of applying an external preparation containing a component contained in edible bird's nest as an active ingredient to a subject.

10. An exosome controller for controlling exosomes secreted from intestinal cells, containing a component contained in edible bird's nest as an active ingredient. An exosome controller containing a component contained in edible bird's nest as an active ingredient.

11. An exosome control food composition for controlling exosomes secreted from intestinal cells, containing a component contained in edible bird's nest as an active ingredient.

12. A method for producing an intestinal-derived exosome-containing composition containing intestinal-derived exosomes secreted from intestinal cells, comprising the step of adding a solution containing a component contained in edible bird's nest as an active ingredient to a medium containing the intestinal cells. A method for producing an intestinal-derived exosome-containing composition, comprising the step of adding a solution containing a component contained in edible bird's nest as an active ingredient to a medium containing the intestinal cells.

13. An exosome control method for controlling exosomes secreted from intestinal cells. An exosome control method comprising a step of adding a solution containing a component contained in edible bird's nest as an active ingredient to a medium containing the intestinal cells.

14. An intestinal-derived exosome-containing composition containing intestinal-derived exosomes secreted from intestinal cells to which a solution containing a component contained in edible bird's nest as an active ingredient has been added.

15. A mitochondrial function improver for improving the function of mitochondria in cells, A mitochondrial function improver containing the intestinal-derived exosomes according to Claim 14 as an active ingredient.

16. The mitochondrial function improver according to Claim 15, which improves the function of mitochondria in epidermal keratinocytes, dermal fibroblasts, or myoblasts.

17. A reactive oxygen species scavenger for scavenging reactive oxygen species, A reactive oxygen species scavenger containing the intestinal-derived exosomes according to Claim 14 as an active ingredient.

18. A wrinkle improver for improving skin wrinkles, A wrinkle improver containing the intestinal-derived exosomes according to Claim 14 as an active ingredient.

19. A skin moisturizing function improver for improving the skin moisturizing function, A skin moisturizing function improver containing the intestinal-derived exosomes according to Claim 14 as an active ingredient.

20. A muscle function activator containing the intestinal-derived exosomes according to Claim 14 as an active ingredient.

21. A food composition for preventing aging-related diseases containing the intestinal-derived exosomes according to Claim 14 as an active ingredient.

Images