Agent for maintaining or improving cognitive function, agent for maintaining or improving memory, agent for improving depression, or agent for improving fatigue, or agent for activating gut-brain correlation

Abstract

To provide a new agent capable of activating intestinal cephalic phase.SOLUTION: An agent for maintaining or improving cognitive function, an agent for maintaining or improving memory, an agent for improving depression, or an agent for improving fatigue, comprising a koi extract as an active ingredient, wherein the koi extract is derived from a heating - and protease-treated product of koi, and the maintenance or improvement of cognitive function, the maintenance or improvement of memory, the improvement of depression, or the improvement of fatigue is caused by gut-brain interaction activation.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for maintaining or improving cognitive function, an agent for maintaining or improving memory, an agent for improving depression or fatigue, or an agent for activating gut-brain interaction. [Background technology]

[0002] The gut-brain axis is a concept that refers to the mutual influence between the intestine and the brain via the autonomic nervous system, humoral factors, etc. Non-Patent Document 1 discloses that exosomes secreted from intestinal cells treated with carnosine promote neurite outgrowth in nerve cells, i.e., carnosine can activate the gut-brain axis. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Katakura, Yoshinori. "Carnosine's memory function-improving effect and its molecular basis: Why does carnosine improve brain function?" Chemistry and Biology 57.10(2019): 596-600. Summary of the Invention [Problem to be solved by the invention]

[0004] Substances that can activate gut-brain interaction, such as carnosine, are thought to be effective in improving brain function. However, knowledge about substances that can activate gut-brain interaction is limited. Therefore, the present invention aims to provide a novel agent that can activate gut-brain interaction. [Means for solving the problem]

[0005] The present inventors discovered that a carp extract derived from heated and protease-treated carp can activate the gut-brain axis, leading to the completion of the present invention.

[0006] The present disclosure provides, for example, the inventions described in the following [1] to [6]. [1] A cognitive function maintenance or improvement agent, a memory maintenance or improvement agent, a depression improvement agent, or a fatigue improvement agent, comprising carp extract as an active ingredient, The carp extract is derived from a product of heating and protease treatment of carp; The agent, wherein the maintenance or improvement of cognitive function, the maintenance or improvement of memory, the improvement of depression, or the improvement of fatigue is due to activation of gut-brain correlation. [2] The agent according to [1], wherein the protease comprises an endopeptidase derived from Bacillus subtilis and an endopeptidase derived from Aspergillus oryzae. [3] The agent according to [1] or [2], wherein the activation of the gut-brain axis is accompanied by the extension of neurites of neurons and an increase in the membrane potential of mitochondria of neurons. [4] A gut-brain correlation activator containing carp extract as an active ingredient, The carp extract is derived from carp that has been heated and treated with protease. [5] The agent according to [4], wherein the protease comprises an endopeptidase derived from Bacillus subtilis and an endopeptidase derived from Aspergillus oryzae. [6] The agent according to [4] or [5], wherein the activation of the gut-brain axis is accompanied by the extension of neurites of neurons and an increase in the membrane potential of mitochondria of neurons. [Effects of the Invention]

[0007] According to the present invention, a novel agent for activating gut-brain interaction can be provided. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the results of evaluating neurite outgrowth when culture supernatant of human intestinal epithelial cells treated with carp extract was added to human neurons. [Figure 2](A) Figure showing the results of evaluating the number of mitochondria when culture supernatant of human intestinal epithelial cells treated with carp extract was added to human neurons. (B) Figure showing the results of evaluating the area of ​​mitochondria when culture supernatant of human intestinal epithelial cells treated with carp extract was added to human neurons. (C) Figure showing the results of evaluating the membrane potential of mitochondria when culture supernatant of human intestinal epithelial cells treated with carp extract was added to human neurons. [Figure 3] (A) and (B) show the results of evaluating the transcription level of the SIRT1 gene when the culture supernatant of human intestinal epithelial cells treated with carp extract was added to human neurons. [Figure 4] (A) Figure 1 shows the results of evaluating the transcription level of the BDNF gene when the culture supernatant of human intestinal epithelial cells treated with carp extract was added to human neurons. (B) Figure 1 shows the results of evaluating the transcription level of the NAMPT gene when the culture supernatant of human intestinal epithelial cells treated with carp extract was added to human neurons. [Figure 5] (A) Figure showing the results of evaluating neurite outgrowth when exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to the culture medium of human neurons so that the exosome content in the culture medium was 450 ng / mL in terms of exosomal protein. (B) Figure showing the results of evaluating neurite outgrowth when exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to the culture medium of human neurons so that the exosome content in the culture medium was 900 ng / mL in terms of exosomal protein. [Figure 6](A) Figure showing the results of assessing the number of mitochondria when exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to the culture medium of human neurons so that the exosome content in the culture medium was 450 ng / mL in terms of exosomal protein. (B) Figure showing the results of assessing the number of mitochondria when exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to the culture medium of human neurons so that the exosome content in the culture medium was 900 ng / mL in terms of exosomal protein. (C) Figure showing the results of assessing the area of ​​mitochondria when exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to the culture medium of human neurons so that the exosome content in the culture medium was 450 ng / mL in terms of exosomal protein. (D) Exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to the culture medium of human neurons at an exosome content of 900 ng / mL in terms of exosomal protein. (E) Exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to the culture medium of human neurons at an exosome content of 450 ng / mL in terms of exosomal protein. (F) Exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to the culture medium of human neurons at an exosome content of 900 ng / mL in terms of exosomal protein. [Figure 7](A) This figure shows the results of evaluating the transcription level of the SIRT1 gene when exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to human neurons. (B) This figure shows the results of evaluating the transcription level of the NAMPT gene when exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to human neurons. (C) This figure shows the results of evaluating the transcription level of the SIRT3 gene when exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to human neurons. (D) This figure shows the results of evaluating the transcription level of the BDNF gene when exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to human neurons. [Figure 8] (A) Figure showing the results of evaluating the transcription level of the TFAM gene when exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to human neurons. (B) Figure showing the results of evaluating the transcription level of the MITOL gene when exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to human neurons. (C) Figure showing the results of evaluating the transcription level of the Drp1 gene when exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract were added to human neurons. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, embodiments of the present disclosure will be described in detail.

[0010] The gut-brain correlation activator according to the present disclosure contains carp extract as an active ingredient, and the carp extract is derived from carp that has been heated and treated with protease.

[0011] Furthermore, the agent for maintaining or improving cognitive function, memory, depression, or fatigue according to the present disclosure contains the carp extract as an active ingredient, and the maintenance or improvement of cognitive function, memory, depression, or fatigue is achieved by activating the gut-brain correlation. Hereinafter, the agent for activating the gut-brain correlation according to the present disclosure and the agent for maintaining or improving cognitive function, memory, depression, or fatigue according to the present disclosure are collectively referred to as the "agent according to the present disclosure."

[0012] In this disclosure, carp refers to fish of the genus Cyprinus. Carp is preferably Cyprinus carpio, Cyprinus Rubrofuscus, or a hybrid of these, with the above hybrids being more preferred. Examples of such hybrids include Yamato carp. Carp may be wild carp, farmed carp, or fish-caught carp.

[0013] The carp extract may be an extract of carp from which the gallbladder (bitter balls) has been removed, or an extract of the whole carp, and is preferably an extract of carp from which the gallbladder (bitter balls) has been removed.

[0014] The carp extract is derived from carp that has been heated and treated with protease. The protease preferably contains an endopeptidase, more preferably an endopeptidase derived from Bacillus subtilis and an endopeptidase derived from Aspergillus oryzae.

[0015] The carp extract is derived from a product of carp that has been heated and treated with protease, but the extract may also be an extract from the product of carp that has been heated and treated with protease. The extract may also be obtained by subjecting the treated product to processing such as filtration, sterilization, concentration, solid-liquid separation, removal of metal foreign matter, drying, etc. For example, the extract may be selected from the liquid component obtained by removing solids from the treated product and / or a concentrate obtained by concentrating the treated product, or a dried product obtained by further drying these.

[0016] The carp extract may contain water, protein, lipids, carbohydrates, ash, minerals such as sodium, free amino acids such as hydroxyproline, free asparagine, and free glutamine, taurine, etc. In the present disclosure, protein is a concept that includes peptides.

[0017] The protein content of the carp extract may be 60 to 95% by mass, or 70 to 90% by mass, based on the total solid content of the carp extract. In the present disclosure, the protein content refers to the value determined by the nitrogen quantitative conversion method using a nitrogen conversion coefficient of 6.25. In this specification, the solid content refers to the components excluding solvents such as water.

[0018] The lipid content in the carp extract may be 0.05 to 3% by mass, or 0.2 to 1% by mass, based on the total solid content of the carp extract. In the present disclosure, the lipid content refers to the value determined by acid hydrolysis.

[0019] The carbohydrate content of the carp extract may be 2 to 25% by mass, or 5 to 15% by mass, based on the total solid content of the carp extract. In the present disclosure, the carbohydrate content refers to the value obtained by subtracting the protein, lipid, and ash contents (% by mass) based on the total solid content of the carp extract from 100.

[0020] The ash content of the carp extract may be 1 to 20% by mass, or 3 to 12% by mass, based on the total solid content of the carp extract. In the present disclosure, the ash content refers to the value determined by the direct ashing method.

[0021] The sodium content of the carp extract may be 0.02 to 6% by mass, or 0.1 to 3% by mass, based on the total solid content of the carp extract. In the present disclosure, the sodium content refers to the value determined by the analytical method for nutritional components, etc., in the appendix to the Food Labeling Standards (revised November 30, 2020, Food and Nutrition Table No. 454).

[0022] The hydroxyproline content in the carp extract may be 0.1 to 15% by mass, or 0.5 to 8% by mass, based on the total solid content of the carp extract. In the present disclosure, the hydroxyproline content refers to the value determined by post-column derivatization using an LA8080 high-speed amino acid analyzer, AminoSAAYA (manufactured by Hitachi High-Tech Science Corporation).

[0023] The free asparagine content in the carp extract may be 0.002 to 0.6% by mass, or 0.01 to 0.3% by mass, based on the total solid content of the carp extract. In the present disclosure, the free asparagine content refers to the value determined by a post-column derivatization method using an LA8080 high-speed amino acid analyzer, AminoSAAYA (manufactured by Hitachi High-Tech Science Corporation).

[0024] The free glutamine content in the carp extract may be less than 0.01% by mass based on the total solid content of the carp extract. In this disclosure, the free glutamine content refers to the value determined by post-column derivatization using an LA8080 high-speed amino acid analyzer, AminoSAAYA (manufactured by Hitachi High-Tech Science Corporation).

[0025] The taurine content in the carp extract may be 0.02 to 6% by mass, or 0.1 to 3% by mass, based on the total solid content of the carp extract. In the present disclosure, the taurine content refers to the value determined by post-column derivatization using an LA8080 high-speed amino acid analyzer, AminoSAAYA (manufactured by Hitachi High-Tech Science Corporation).

[0026] The carp extract may contain, as its amino acid composition, isoleucine, leucine, lysine, methionine, cystine, phenylalanine, tyrosine, threonine, tryptophan, valine, histidine, arginine, alanine, aspartic acid, glutamic acid, glycine, proline, serine, etc. These amino acids may be contained as amino acid residues in the proteins contained in the carp extract.

[0027] The content of each amino acid in the carp extract as an amino acid composition may be, based on the total solid content of the carp extract, 1.5 to 5.5 mass% or 2 to 4 mass% for isoleucine, 3 to 11.5 mass% or 4 to 9 mass% for leucine, 3.5 to 13.5 mass% or 4.5 to 10 mass% for lysine, 1 to 4.5 mass% or 1.5 to 3 mass% for methionine, 0.2 to 0.9 mass% or 0.3 to 0.7 mass% for cystine, 1 to 5.5 mass% or 2 to 4 mass% for phenylalanine, 1 to 4 mass% or 1.5 to 3 mass% for tyrosine, 2 to 7.5 mass% or 2.5 to 5.5 mass% for threonine, and 2 to 7.5 mass% or 2.5 to 5.5 mass% for tryptophan. The amount may be 0.2 to 1% by mass or 0.3 to 0.8% by mass, valine may be 2 to 7.5% by mass or 2.5 to 5.5% by mass, histidine may be 1 to 4% by mass or 1.5 to 3% by mass, arginine may be 2.5 to 11.5% by mass or 3.5 to 8.5% by mass, alanine may be 3 to 13% by mass or 4.5 to 10% by mass, aspartic acid may be 3.5 to 15% by mass or 5 to 11% by mass, glutamic acid may be 6 to 25% by mass or 8 to 18.5% by mass, glycine may be 4.5 to 18.5% by mass or 6 to 14% by mass, proline may be 2.5 to 10% by mass or 3.5 to 7.5% by mass, and serine may be 2 to 8% by mass or 2.5 to 6% by mass. In the present disclosure, the content of the amino acids other than tryptophan in the amino acid composition among the above-mentioned amino acids means a value determined by post-column derivatization using an LA8080 high-speed amino acid analyzer AminoSAAYA (manufactured by Hitachi High-Tech Science Corporation). In the present disclosure, the content of tryptophan in the amino acid composition means a value determined by high-performance liquid chromatography.

[0028] The area percentage of each molecular weight fraction obtained by subjecting the above carp extract to size exclusion chromatography using the method described below may be 0.5 to 8% or 1 to 4% for the fraction with a molecular weight of 6000 or more, 2 to 20% or 4 to 12% for the fraction with a molecular weight of 3000 or more but less than 6000, 10 to 50% or 20 to 40% for the fraction with a molecular weight of 1000 or more but less than 3000, 5 to 40% or 10 to 30% for the fraction with a molecular weight of 500 or more but less than 1000, and 20 to 60% or 30 to 50% for the fraction with a molecular weight less than 500. The size exclusion chromatography was performed using a high-performance liquid chromatograph (LC-20AD, Shimadzu Corporation) with a UV-visible detector SPD-20A (Shimadzu Corporation) as the detector, a TSKgel G2500PWXL (Tosoh Corporation) column, a mixture of water, acetonitrile, and trifluoroacetic acid (volume ratio: water:acetonitrile:trifluoroacetic acid = 55:45:0.1) as the mobile phase, a column temperature of 40 °C, a flow rate of 0.5 mL / min, an injection volume of 20 μL, and a measurement wavelength (detection wavelength) of 220 nm. The molecular weight standards used were cytochrome C (molecular weight 12327), aprotinin (molecular weight 6512), bacitracin (molecular weight 1450), angiotensin II (molecular weight 1046), Gly-Gly-Tyr-Arg (molecular weight 451), and Gly-Gly-Gly (molecular weight 189).

[0029] The Brix value of the carp extract may be 60 to 80%, or 65 to 75%. The Brix value refers to the value obtained by handheld refractive index measurement (manufactured by ATAGO). The pH of the carp extract may be 5.5 to 7.5, or 6 to 6.6.

[0030] The solid content of the carp extract in the agent according to the present disclosure may be 1 to 99% by mass, or 10 to 90% by mass, relative to the total mass of the agent.

[0031] The dosage and administration of the agent according to the present disclosure can be determined appropriately depending on factors such as the health condition of the person taking it, the method of administration, and the combination with other agents. For example, an adult weighing 60 kg may take 1 to 20 g of the carp extract per day, or 0.2 to 1.6 g of the carp extract per day, calculated as solid content. The administration may be once a day or divided into multiple doses, such as twice or three times a day.

[0032] The agent according to the present disclosure is not particularly limited as long as it has the effect of activating the gut-brain correlation, but is preferably taken orally. That is, the agent according to the present disclosure is preferably an oral agent.

[0033] In addition to the carp extract, the agent of the present disclosure may contain other ingredients acceptable for food use, pharmaceutically acceptable ingredients, etc. Examples of acceptable ingredients include amino acids such as tryptophan, proteins such as yeast extract and fish collagen, plant extracts such as ginger extract, fermented black garlic extract, maca extract, kabosu juice, plum extract, grapefruit seed extract, and hythion extract, sweeteners such as maltose, sucralose, thaumatin, acesulfame potassium, aspartame-sucralose, starch syrup, granulated sugar, and galactooligosaccharide syrup, minerals such as calcium stearate, potassium chloride, and sodium chloride, citric acid, pH adjusters, agar, thickening polysaccharides, flavorings, microcrystalline cellulose, microcrystalline silicon dioxide, hydroxypropyl cellulose, silicone resins, and cyclic oligosaccharides. Pharmaceutically acceptable ingredients include excipients and emulsifiers.

[0034] The agent according to the present disclosure may be, for example, in powder form or liquid form, and the dosage form of the agent according to the present disclosure may be, for example, a tablet, a powder, or a drinkable preparation.

[0035] The agent according to the present disclosure may be used as a food composition, a pharmaceutical product, or a quasi-drug product itself, or as an ingredient in these products. Examples of food compositions include health foods, functional foods, nutritional compositions, dietary supplements, supplements, health foods, foods for specified health uses, foods with nutrient functions, and foods with functional claims. Furthermore, the food composition may be a beverage such as a soft drink or energy drink, a powdered beverage, a concentrated beverage, a seasoning, a candy, a jelly, or the like.

[0036] The agent according to the present disclosure can activate the gut-brain axis. In the present disclosure, whether a test substance can activate the gut-brain axis can be determined by the following method. First, an appropriate amount of the test substance is added to intestinal epithelial cells and cultured for 24 hours, and then the culture supernatant is collected. An equal amount of the culture supernatant (test substance group) or medium (control group n) is added to neurons and cultured for 24 hours. If the neurite length of the neurons in the test substance group is longer than that of the control group (e.g., if the average neurite length is longer than that of the control group, or if the p-value relative to the control group obtained by Student's t-test is less than 0.05), and if the mitochondrial membrane potential is higher (e.g., if the average mitochondrial membrane potential is higher than that of the control group, or if the p-value relative to the control group obtained by Student's t-test is less than 0.05), it can be determined that the test substance can activate the gut-brain axis. For example, a human colon cancer-derived cell line can be used as the intestinal epithelial cells. The nerve cells may be, for example, a cell line derived from human neuroblastoma. Specifically, whether or not a test substance can activate the gut-brain axis can be confirmed by the method described in the Examples below.

[0037] Activation of the gut-brain axis by the agent according to the present disclosure is preferably accompanied by neurite outgrowth in neurons and an increase in the membrane potential of neurons' mitochondria, which can more effectively maintain or improve cognitive function, memory, depression, or fatigue.

[0038] Activation of the gut-brain axis by the agent according to the present disclosure is preferably accompanied by an increase in the transcription levels of the SIRT1 gene and / or the TFAM gene in neurons, which can more effectively maintain or improve cognitive function, memory, depression, or fatigue.

[0039] SIRT1 has been reported to activate PGC-1α by deacetylating it, enhance mitochondrial function, promote efficient ATP production from smaller energy sources, promote differentiation of neural stem cells into neurons, promote autophagy, promote mitophagy, reduce reactive oxygen species, and improve cellular stress resistance (Canto, Carles, et al. "AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity." Nature 458.7241(2009):1056-1060. Yoshiyuki Horio. "Function of the protein deacetylase SIRT1 and its involvement in pathogenesis." Sapporo Medical Journal 87.1-6 (2018):1-8). It has also been reported that SIRT1 can regulate the differentiation of neural progenitor cells (Hisahara, Shin, et al. "Histone deacetylase SIRT1 modulates neuronal differentiation by its nuclear translocation." Proceedings of the National Academy of Sciences 105.40 (2008): 15599-15604.). Based on the above, increasing the transcription level of the SIRT1 gene in neurons promotes the maintenance or improvement of neuronal function and mitochondrial function in neurons, and may result in the maintenance or improvement of cognitive function, memory, depression, or fatigue.

[0040] Additionally, it has been reported that resveratrol, an agonist of SIRT1, can exert a strong neuroprotective effect in microglia by reducing amyloid-β-induced NF-kB signaling (Chen, Jennifer, et al. "SIRT1 protects against microglia-dependent amyloid-β toxicity through inhibiting NF-kB signaling." Journal of Biological Chemistry 280.48 (2005):40364-40374.). Amyloid-β is known to be involved in the development of Alzheimer's disease. It has also been reported that SIRT1 deacetylase can promote neuronal survival in cell models of Alzheimer's disease or amyotrophic lateral sclerosis, and can protect against neurodegeneration in transgenic mice carrying the p25 gene (Kim, Dohoon, et al., "SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis," The EMBO journal, 26.13 (2007): 3169-3179).Additionally, it has been reported that SIRT1 can suppress amyloid-β production in neurons through various mechanisms (Qin, Weiping, et al. "Neuronal SIRT1 activation as a novel mechanism underlying the prevention of Alzheimer's disease amyloid neuropathology by calorie restriction." Journal of Biological Chemistry 281.31 (2006):21745-21754. Qin, Weiping, et al. "Regulation of forkhead transcription factor FoxO3a contributes to calorie restriction-induced prevention of Alzheimer's disease-type amyloid neuropathology and spatial memory deterioration." Annals of the New York Academy of Sciences 1147.1 (2008):335-347.). Based on these findings, increasing the transcription level of the SIRT1 gene in neurons may be able to maintain or improve cognitive function, in particular.

[0041] TFAM is known to protect mitochondria from oxidative stress by, for example, maintaining the function of mitochondrial DNA (mtDNA) (Oka, Sugako, et al. "Human mitochondrial transcription factor A breaks the mitochondria-mediated vicious cycle in Alzheimer's disease." Scientific Reports 6.1 (2016): 37889.). Consequently, increased transcription of the TFAM gene in neurons promotes the maintenance or improvement of mitochondrial function in neurons, thereby promoting the maintenance or improvement of neuronal function, and may result in the maintenance or improvement of cognitive function, memory, depression, or fatigue, particularly cognitive function.

[0042] It has also been reported that in a mouse model of Alzheimer's disease, expression of TFAM reduces 8-oxoguanine, an oxidized form of guanine, in mtDNA, reduces intracellular amyloid-β accumulation, and increases transthyretin expression, significantly improving cognitive function (ibid.). Transthyretin is known to inhibit intracellular amyloid-β accumulation. It has also been reported that addition of TFAM to Alzheimer's disease model neurons harboring the PSEN1 (P117L) gene suppresses the impaired neurite outgrowth exhibited by these cells (ibid.). Based on these findings, increasing the transcriptional level of the TFAM gene in neurons may maintain or improve cognitive function, in particular.

[0043] The agent according to the present disclosure inhibits the BDNF (brain-derived neurotrophic factor) gene and NAMPT (NAD + The agent according to the present disclosure can activate the gut-brain axis without increasing the transcription level of the BDNF gene or NAMPT gene.

[0044] Although the mechanism by which the agent according to the present disclosure activates the gut-brain axis is not entirely clear, the present inventors believe that the agent according to the present disclosure acts on intestinal epithelial cells to produce extracellular vesicles such as exosomes, cytokines, soluble factors such as growth factors, and the like, and that these then act on neurons to at least partially contribute to the activation of the gut-brain axis. For example, the exosomes are thought to contribute to the increase in mitochondrial membrane potential of neurons caused by the agent according to the present disclosure. Furthermore, exosomes are thought not to contribute to the neurite outgrowth of neurons caused by the agent according to the present disclosure (i.e., the neurite outgrowth of neurons caused by the agent according to the present disclosure is thought to be exosome-independent), and factors other than exosomes (e.g., soluble factors such as the cytokines and growth factors mentioned above) are thought to contribute. Furthermore, if the agent according to the present disclosure increases the transcription level of the SIRT1 gene in neurons, the exosomes are thought to contribute to this increase in the transcription level of the SIRT1 gene.

[0045] The agent according to the present disclosure can be prepared by mixing the carp extract with other ingredients as needed, and then molding it into the dosage form as needed.

[0046] The carp extract can be produced by heating and treating carp with protease, preferably by heating the carp before the protease treatment.

[0047] The carp may be pulverized before heating and treating with protease to obtain a pulverized carp product. By preparing the pulverized carp product, the protease treatment can be carried out efficiently. Pulverization of the carp may be carried out, for example, by subjecting the carp to a pulverizer, mixer, blender, food processor, or the like. The pulverized carp product may have a maximum particle size of 40 mm or less, or may have a maximum particle size of 30 mm or less. The pulverized carp product may also be in liquid form.

[0048] The heat treatment may be carried out in the presence of water. In this case, the heat treatment may be carried out in the presence of 10 to 90 parts by mass or 15 to 60 parts by mass of water per 33.4 parts by mass of ground carp. The heat treatment may be carried out by raising the temperature to 90 to 93°C. For example, the heat treatment may be carried out by continuing heating from 60°C for 20 minutes until the temperature reaches 90 to 93°C. By carrying out the heat treatment, autolysis during protease treatment can be suppressed.

[0049] The protease treatment may be carried out under conditions that allow for appropriate hydrolysis of carp-derived proteins. For example, the protease treatment may be carried out in a system containing ground carp, a solvent such as water, and a protease. The amount of protease in the system may be appropriately determined depending on the properties of the protease, such as its titer. The temperature of the protease treatment may be the optimal temperature for the protease used, and the time of the protease treatment may be appropriately determined depending on the properties of the protease, such as its titer, and the amount of protease. For example, when the protease contains a Bacillus subtilis-derived endopeptidase and an Aspergillus oryzae-derived endopeptidase, the content of the Bacillus subtilis-derived endopeptidase in the system where the protease treatment is performed may be 0.001 to 0.05% by mass or 0.004 to 0.014% by mass, and the content of the Aspergillus oryzae-derived endopeptidase may be 0.005 to 0.25% by mass or 0.02 to 0.06% by mass. In this case, the content of ground carp in the system may be 13 to 53% by mass or 23 to 43% by mass, and the content of water may be 46 to 86% by mass or 56 to 76% by mass. In this case, the temperature for the protease treatment may be 55 to 65°C or 57 to 63°C, the time for the protease treatment may be 3 to 5 hours, and the pH for the protease treatment may be 6 to 7.5 or 6.5 to 6.8. The protease may be added to the system by adding cells that express the protease to the system. Examples of such cells include cells of prokaryotes such as bacteria and eukaryotes such as yeast.

[0050] After the protease treatment, the protease may be inactivated, for example, by heating the mixture to 90 to 93°C. For example, the mixture may be heated from 60°C for 20 minutes until the temperature reaches 90 to 93°C.

[0051] After heating and protease treatment, the extract may be subjected to filtration, sterilization, concentration, solid-liquid separation using a strainer or the like, removal of metal contaminants using a magnet or the like, drying, or other suitable procedures depending on the dosage form and intended use of the agent of the present disclosure. Liquid carp extract can be obtained, for example, by concentrating the extract after heating and protease treatment, preferably by performing filtration, sterilization, concentration, solid-liquid separation using a strainer or the like, and removal of metal contaminants using a magnet or the like, in this order. Powdered carp extract can be obtained, for example, by concentrating the extract after heating and protease treatment, followed by drying, preferably by performing filtration, sterilization, concentration, solid-liquid separation using a strainer or the like, removal of metal contaminants using a magnet or the like, and drying, in this order, after heating and protease treatment.

[0052] The filtration is not particularly limited, and may be performed sequentially using a plurality of filtration membranes with different pore sizes. Filtration may also be performed by squeeze filtration using a filter aid such as diatomaceous earth or ore.

[0053] Sterilization may be carried out, for example, at 120 to 130°C for 2 to 4 seconds.

[0054] Concentration is preferably carried out so that the Brix value of the carp extract reaches the desired Brix value. The Brix value is the value obtained using a handheld refractive index analyzer (manufactured by ATAGO). Concentration may be carried out by evaporation, membrane concentration, freeze concentration, or a combination of multiple methods.

[0055] The solid-liquid separation using a strainer is preferably carried out using a strainer with a mesh size of 35 to 45.

[0056] The removal of metallic foreign matter by the magnet is preferably carried out with a magnetic force of 8000 gauss or more, and the magnetic force of the magnet may be 14000 gauss or less.

[0057] Drying may be carried out by spray drying, freeze drying, vacuum drying, etc., and is preferably carried out by spray drying. By drying, the carp extract can be made into a powder.

[0058] The carp extract produced by the above procedure may be stored frozen, preferably by quick freezing. [Example]

[0059] The present invention will be described in more detail with reference to the following examples, but is not limited to these examples. In the following examples, Caco-2 (a human colon cancer-derived cell line) was used as a human intestinal epithelial cell model, and SH-SY5Y (a human neuroblastoma-derived cell line) was used as a human neuronal cell model. In Figures 1 to 4, "*," "**," and "***" indicate that the p-values ​​relative to the control in the Student's t-test were p<0.05, p<0.01, and p<0.001, respectively. In Figures 1 to 4, the absence of "*," "**," or "***" indicates that the p-value relative to the control in the Student's t-test was p≧0.05. In Figures 5 to 8, "*," "**," and "***" indicate that the p-values ​​relative to the control in the Dunnett's test were p<0.05, p<0.01, and p<0.001, respectively. In Figures 1 to 4, when there is no "*", "**", or "***", it means that the p-value relative to the control in Dunnett's test is p≧0.05.

[0060] [Caco-2 and SH-SY5Y] Caco-2 and SH-SY5Y were subcultured in DMEM medium (FBS-containing DMEM medium) containing 10% by volume of inactivated fetal bovine serum (Capricorn Scientific, hereafter referred to as "FBS") in a cell culture dish (Corning) at 37°C in the presence of 5% CO2. The FBS used was inactivated by heating in a 56°C water bath for 30 minutes. The DMEM medium was prepared by dissolving 4.75 g of Dulbecco's Modified Eagle's Medium "Nissui" (2) (Nissui Pharmaceutical Co., Ltd.) in 470 mL of Milli-Q water, sterilizing the solution by autoclaving, and then adding 10 mL of 0.2 mol / L L(+)-glutamine (Fujifilm Wako Pure Chemical Industries, Ltd.), 10 mL of 10 w / v% NaHCO3 aqueous solution (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 mL of 50,000 U / mL penicillin (Meiji Seika Pharma Co., Ltd.), and 1 mL of 0.05 mg / mL streptomycin (Meiji Seika Pharma Co., Ltd.). The 0.2 mol / L L(+)-glutamine was filter-sterilized, and the 10 w / v% NaHCO3 solution was autoclaved.

[0061] [Preparation of carp extract] Yamato carp, from which the gallbladder (bitter balls) had been removed, was prepared as the carp raw material. The carp raw material was crushed using a pulper grinder to a maximum particle size of approximately 20 mm, yielding crushed carp material. 33.4 parts of crushed carp material was added to 66.5 parts of water and heated to 90-93°C. The mixture was then immediately cooled to 60°C. 0.004-0.014 parts of Bacillus subtilis endopeptidase and 0.02-0.06 parts of Aspergillus oryzae endopeptidase were then added to the 33.4 parts of crushed carp material and incubated at approximately 58-62°C and pH 6.5-6.8 for 4 hours. The mixture was then heated from 60°C to 90°C for approximately 20 minutes to inactivate the two endopeptidases. The mixture was then coarsely filtered using a 1 mm mesh screen. Then, diatomaceous earth and ore were added as filter aids, and the mixture was squeezed and filtered to a filtration permeability of 42.0 ml / cm 2After that, diatomaceous earth and ore were added as filter aids, and the mixture was squeezed and filtered at a filtration permeability of 10.0 ml / cm. 2 The filtrate was sterilized at 125°C for 3 seconds. The sterilized filtrate was concentrated until its Brix value reached 68-71%. The Brix value was determined using a handheld refractive index analyzer (manufactured by ATAGO). To remove foreign matter from the concentrated liquid, the liquid was passed through a 40-mesh strainer for solid-liquid separation. Metallic foreign matter was then removed using a magnet with a magnetic force of 8000 gauss or more. The carp extract obtained was then flash-frozen to below -30°C and stored at below -18°C.

[0062] The composition of the carp extract was determined. The water content of the carp extract was determined to be 40.2 g / 100 g by heating at 105°C until constant mass was reached. The nitrogen conversion method, using a nitrogen conversion factor of 6.25, determined the protein content of the carp extract to be 49.9 g / 100 g. The acid hydrolysis method determined the lipid content of the carp extract to be 0.3 g / 100 g. The direct incineration method determined the ash content of the carp extract to be 4.0 g / 100 g. The subtraction method, i.e., subtracting the sum of the water, protein, lipid, and ash contents (g / 100 g) from 100 g, determined the carbohydrate content of the carp extract to be 5.6 g / 100 g. Sodium content was determined based on the analytical method for nutritional components in the appendix to the Food Labeling Standards (No. 454, revised November 30, 2020). The sodium content of the carp extract was 650 mg / 100 g. Free amino acids were quantified using the post-column derivatization method with the LA8080 AminoSAAYA high-speed amino acid analyzer (Hitachi High-Tech Science). The hydroxyproline content was 1700 mg / 100 g, the free asparagine content was 82 mg / 100 g, the free glutamine content was less than 5 g / 100 g, and the taurine content was 780 mg / 100 g.

[0063] The amino acid composition of the carp extract was determined. Quantitative analysis of the amino acid composition was performed using a LA8080 high-speed amino acid analyzer, AminoSAAYA (Hitachi High-Tech Science Corporation), by post-column derivatization. The amino acid content of the carp extract was found to be as follows: isoleucine 1700 mg / 100 g, leucine 3500 mg / 100 g, lysine 4100 mg / 100 g, methionine 1300 mg / 100 g, cystine 260 mg / 100 g, phenylalanine 1600 mg / 100 g, and tyrosine 1200 mg / 100 g. The carp extract contained 00mg / 100g of tryptophan, 2200mg / 100g of threonine, 2200mg / 100g of valine, 1200mg / 100g of histidine, 3400mg / 100g of arginine, 3900mg / 100g of alanine, 4400mg / 100g of aspartic acid, 7400mg / 100g of glutamic acid, 5500mg / 100g of glycine, 3000mg / 100g of proline, and 2300mg / 100g of serine. Furthermore, the tryptophan content of the carp extract was quantified by high-performance liquid chromatography to be 300mg / 100g.

[0064] When the above carp extract was subjected to size exclusion chromatography under the following conditions, the area percentages of each molecular weight fraction were 2% for molecular weights of 6000 or more, 8% for molecular weights of 3000 or more but less than 6000, 31% for molecular weights of 1000 or more but less than 3000, 19% for molecular weights of 500 or more but less than 1000, and 40% for molecular weights less than 500. <Size exclusion chromatography conditions> Model: LC-20AD (Shimadzu Corporation) Detector: UV-visible detector SPD-20A (Shimadzu Corporation) Column: TSKgel G2500PWXL, φ7.8 mm x 300 mm (Tosoh Corporation) Column temperature: 40℃ Mobile phase: A mixture of water, acetonitrile, and trifluoroacetic acid (volume ratio of water:acetonitrile:trifluoroacetic acid = 55:45:0.1) Flow rate: 0.5mL / min Measurement wavelength: 220nm Injection volume: 20μL Molecular weight standards: cytochrome C (molecular weight 12327), aprotinin (molecular weight 6512), bacitracin (molecular weight 1450), angiotensin II (molecular weight 1046), Gly-Gly-Tyr-Arg (molecular weight 451), and Gly-Gly-Gly (molecular weight 189).

[0065] The Brix value of the carp extract was measured using a handheld refractive index analyzer (manufactured by ATAGO) and was found to be 69.4%. The pH of the carp extract diluted 100 times with water was measured and found to be 6.26.

[0066] [Obtaining culture supernatant of human intestinal epithelial cells treated with carp extract] The Caco-2 cells subcultured in the above [Caco-2 and SH-SY5Y] were placed in a 24-well plate (Corning, 353504) at 1 × 10 5 Cells were seeded at 1000 cells / well and cultured in FBS-containing DMEM medium. 24 hours after seeding, the carp extract prepared in the above section "Preparation of Carp Extract" was added to the culture medium at concentrations of 1 vol%, 0.3 vol%, 0.1 vol%, or 0.03 vol%. After 24 hours, 1 mL of culture supernatant was collected and used as the culture supernatant of human intestinal epithelial cells treated with carp extract. Hereinafter, the culture supernatants obtained by adding carp extract to the culture medium at 1 vol%, 0.3 vol%, 0.1 vol%, and 0.03 vol% are referred to as "CP1%, CP0.3%, CP0.1%, and CP0.03%, respectively.

[0067] [Exosome preparation from culture supernatant of human intestinal epithelial cells treated with carp extract] (Preparation of Carp Peptide Solution) 24 μL of the carp extract prepared in the above section [Preparation of carp extract] was dissolved in 11.976 mL of DMEM medium containing 10% by volume of Exosome-depleted FBS Media Supplement Heat Inactivated (System Biosciences), suspended using a vortex mixer, and then sterilized by filtration using a 0.22 μm × 33 mm filter to prepare a 0.2% by volume carp peptide solution.

[0068] (Human intestinal epithelial cells treated with carp extract) Caco-2 cells subcultured in the above [Caco-2 and SH-SY5Y] were placed in a 10 mL dish at 1.4 × 10 5 Cells were seeded at 1000 cells / mL. They were cultured in DMEM medium containing 10% (volume) of Exosome-depleted FBS Media Supplement Heat Inactivated (System Biosciences). After 24 hours, 5 mL of the medium was removed from each dish, and 5 mL of the 0.2% (volume) Carp peptide solution prepared as described above (Preparation of Carp Peptide Solution) was added to each dish to a final concentration of 0.3% or 0.1% (volume). Culture supernatants were collected 24 hours after addition (hereinafter referred to as the "CP0.1% group" and "CP0.3% group," respectively). Control groups were also cultured using the same procedure as for the CP0.1% and CP0.3% groups, except that instead of removing 5 mL of the medium from each dish and adding the Carp peptide solution, 72 μL of the medium was removed from each dish and 72 μL of sterile water was added. In the above procedure, the number of dishes in each group was n=4.

[0069] (Exosome purification) Exosomes were purified from the culture supernatants collected from the 0.1% CP group, 0.3% CP group, and control group using the phosphatidylserine affinity method. Specifically, 40 mL of each culture supernatant was centrifuged at 300 × g for 5 minutes to remove cells. The resulting supernatant was transferred to another tube and centrifuged at 1,200 × g for 20 minutes to remove cell debris. The resulting supernatant was transferred to another tube and centrifuged at 10,000 × g for 30 minutes to remove extracellular vesicles larger than exosomes. The resulting culture supernatant, from which cells and large extracellular vesicles had been removed, was concentrated approximately 20-fold using a centrifugal ultrafiltration unit with a molecular weight cutoff of 100,000 (AmiconUltra-15 100K, Merck Millipore), to obtain concentrated supernatants for the 0.1% CP group, 0.3% CP group, and control group.

[0070] Exosome purification from the concentrated supernatant was performed using the MagCapture Exosome Isolation PS Kit Version 2 (Fujifilm Wako). Specifically, Exosome Capture-immobilized beads were prepared as follows: 60 μL of Streptavidin Magnetic Beads were transferred to the attached Reaction Tube, and 500 μL of Exosome Capture Immobilizing Buffer was added to the Reaction Tube and suspended using a vortex mixer. The Reaction Tube was then spun down and placed on a magnetic stand for 1 minute. Once the magnetic beads were completely attached to the wall of the Reaction Tube, the supernatant was removed with a pipette. Next, 500 μL of Exosome Capture Immobilizing Buffer and 10 μL of Biotin-labeled Exosome Capture were added to the Reaction Tube, which was then removed from the magnetic stand and suspended using a vortex mixer. The mixture was then incubated at 2°C–10°C for 10 minutes with end-over-end mixing. The Reaction Tube was then spun down and placed back on the magnetic stand and left to stand for 1 minute. Once the magnetic beads had completely adhered to the wall of the Reaction Tube, the supernatant was removed with a pipette. 500 μL of Exosome Capture Immobilizing Buffer (1×) was then added to the Reaction Tube, which was then removed from the magnetic stand and suspended using a vortex mixer. The Reaction Tube was then spun down, placed back on the magnetic stand and left to stand for 1 minute. Once the magnetic beads had completely adhered to the wall of the Reaction Tube, the supernatant was removed with a pipette (hereinafter, this procedure will also be referred to as procedure (A)). Procedure (A) above was then repeated once more. Exosome Capture-immobilized beads were obtained through these procedures.

[0071] Next, the resulting Exosome Capture-immobilized beads were reacted with the concentrated supernatant as follows: First, approximately 500 μL of the 40-fold concentrated culture supernatant was transferred to a sterilized 1.5 mL tube. Exosome Binding Enhancer (500x) was added at a volume of 1 / 500 of the cell supernatant volume and mixed using a vortex mixer. The tube was then spun down, and the sample was transferred to the Reaction Tube containing the Exosome Capture-immobilized beads and mixed using a vortex mixer. The reaction was then allowed to proceed at room temperature for at least 1 hour while mixing by end-over-end mixing using a rotary mixer. The Reaction Tube was then spun down, placed on a magnetic stand, and left to stand for approximately 1 minute. Once the magnetic beads were completely attached to the wall of the Reaction Tube, the supernatant was removed with a pipette to obtain the Exosome Capture-immobilized beads (hereinafter referred to as "exosome-bound beads").

[0072] Next, the exosome-bound beads were washed using the following procedure. First, 1 mL of Washing Buffer containing Exosome Binding Enhancer was added to the Reaction Tube containing the exosome-bound beads and suspended using a vortex mixer. The Reaction Tube was then spun down and placed on a magnetic stand. After leaving the tube for approximately 1 minute until the magnetic beads had completely adhered to the tube wall, the supernatant was removed (hereinafter, this procedure will also be referred to as procedure (i)). Then, procedure (i) above was repeated two more times. This yielded washed exosome-bound beads.

[0073] Next, exosome elution was performed as follows: 50 μL of Exosome Elution Buffer (1x) was added to the reaction tube containing the washed exosome-bound beads, and the tube was removed from the magnetic stand and suspended using a vortex mixer. The reaction tube was spun down, placed on the magnetic stand, and left to stand for 1 minute. Once the magnetic beads had completely adhered to the tube wall, the supernatant was collected into a new, sterile 1.5 mL tube. An additional 50 μL of Exosome Elution Buffer (1x) was added to the magnetic beads remaining in the reaction tube, removed from the magnetic stand, and suspended using a vortex mixer. The tube was then spun down, placed on the magnetic stand, and left to stand for 1 minute. Once the magnetic beads had completely adhered to the tube wall, the supernatant was collected into the 1.5 mL tube, yielding a total of 100 μL of exosome-containing solution.

[0074] (Evaluation of exosome quantity) The amount of exosomes in the exosome-containing solution was assessed by measuring the protein concentration using the bicinconic acid assay (BCA). Protein concentration was measured using the Micro BCA Protein Assay Kit (Thermo Fisher Scientific). The exosome-containing solution was diluted 10-fold before the BCA assay. For the standard curve, bovine serum albumin (BSA) was serially diluted with PBS from 0 to 200 μg / mL. Two wells of a 96-well plate (Thermo Fisher Scientific) were used as one assay, and 50 μL of the standard and sample were added to each well. Next, 50 μL of a mixture of Micro Reagent A, Micro Reagent B, and Micro Reagent C included in the Micro BCA Protein Assay Kit (volume ratio: 25:24:1) was added to all wells. The mixture was then incubated at 37°C for 2 hours in the dark, and the absorbance at 595 nm was measured using a multimode plate reader (PerkinElmer). Quantitation was performed based on the standard curve obtained from the measurement, and the protein concentration was determined, and the amount of exosomes was evaluated based on this.

[0075] Example 1: Evaluation of the neuronal activation effect of culture supernatant of human intestinal epithelial cells treated with carp extract Example 1-1 Evaluation of the neurite outgrowth effect of culture supernatant of human intestinal epithelial cells treated with carp extract (Addition of culture supernatant of human intestinal epithelial cells treated with carp extract to SH-SY5Y) The SH-SY5Y subcultured in the above [Caco-2 and SH-SY5Y] was 4.0 × 10 5Cells were seeded into a 96-well black plate (Greiner Bio-one) at a concentration of 0.1 cells / mL and cultured in FBS-containing DMEM medium. 24 hours after seeding, the culture supernatant obtained in the above section "Preparation of culture supernatant from human intestinal epithelial cells treated with carp extract" was added to each well at 30% by volume (30 μL). As a control, medium was added to each well at 30% by volume instead of the culture supernatant. As a positive control, retinoic acid (also referred to as "RA," Fujifilm Wako) was added to each well at a final concentration of 10 μM instead of the culture supernatant. Experiments were performed with n=3 wells per group.

[0076] (Immunostaining 1) Twenty-four hours after the addition of the culture supernatant, sterile water, or retinoic acid, 100 μL of 8 w / v% paraformaldehyde solution was added to each well over the culture medium and allowed to stand at room temperature for 15 minutes to fix the cells. After washing three times with PBS, 100 μL of blocking buffer was added to each well and allowed to stand at room temperature for 1 hour. The blocking buffer was then removed, and 50 μL of a primary antibody (Milli-Mark Pan Neuronal Marker, Merck Millipore) diluted 250-fold in antibody dilution buffer was added to each well. The antibody was incubated overnight at 4°C in the dark. After overnight incubation, the wells were washed three times with 1x PBS. A secondary antibody (F(ab')2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555, Thermo Fisher Scientific) diluted 1:1000 in antibody dilution buffer was added to each well at 50 μL / well and incubated at room temperature for 1 hour in the dark. After incubation, the wells were washed three times with 1x PBS. Cellstain Hoechst 33342 (Dojindo Laboratories), a nuclear staining dye, was added at 100 μL / well in the dark at room temperature for 15 minutes. The wells were then washed once with 1x PBS, and 150 μL / well of 1x PBS was added to each well. The neurite length of SH-SY5Y cells was then quantified using an IN Cell Analyzer 2200 (GE Healthcare). The ratio of neurite length to the control is shown in Figure 1. In FIG. 1, "Ctrl" indicates the control, and "RA" indicates the positive control.

[0077] The 8 w / v% paraformaldehyde solution was prepared by adding 0.80 g of paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd.), 10 mL of PBS, and 20 μL of 5 mol / L NaOH to a 15 mL centrifuge tube, mixing thoroughly, and dissolving in a water bath at 60°C. The blocking buffer was prepared by adding 10 mL of PBS, 0.50 mL of normal goat serum (Fujifilm Wako Pure Chemical Industries, Ltd.), and 30 μL of Triton X-100 (Sigma-Aldrich) to a 15 mL centrifuge tube and mixing thoroughly. The antibody dilution buffer was prepared by adding 0.10 g of BSA (Fujifilm Wako Pure Chemical Industries, Ltd.), 10 mL of PBS, and 30 μL of Triton X-100 (Sigma-Aldrich) to a 15 mL centrifuge tube and mixing thoroughly.

[0078] As shown in Figure 1, when CP 1%, CP 0.3%, CP 0.1%, or CP 0.03% culture supernatant was added to SH-SY5Y, the average neurite length was longer than that of the control group, with P values ​​less than 0.01 for all results. Therefore, it was demonstrated that the carp extract acts on the intestinal tract and can extend neurites in neural cells.

[0079] Example 1-2: Evaluation of the effect of culture supernatant of human intestinal epithelial cells treated with carp extract on increasing mitochondrial membrane potential in nerve cells (Addition of culture supernatant of human intestinal epithelial cells treated with carp extract to SH-SY5Y 2) SH-SY5Y 4.0 x 10 5 Instead of seeding 6.0 × 10 SH-SY5Y cells / mL, 5 The culture supernatant, sterile water, or retinoic acid was added to SH-SY5Y in the same manner as described above (addition of culture supernatant of human intestinal epithelial cells treated with carp extract to SH-SY5Y), except that cells / mL were seeded.

[0080] (Mitochondrial co-staining 1) Twenty-four hours after the addition of the culture supernatant, sterile water, or retinoic acid, the medium was removed from each well, and 100 μL / well of MitoTracker Red CMXRos (Thermo Fisher Scientific) solution diluted 4000-fold with FBS-containing DMEM medium at 37°C was added to each well and incubated at 37°C for 30 minutes. The solution was then removed from each well, and 100 μL / well of MitoTracker Green FM solution (Thermo Fisher Scientific) diluted 5000-fold with FBS-containing DMEM medium at 37°C was added to each well and incubated at 37°C for 30 minutes. The solution was then removed from each well, and 100 μL / well of Cellstain Hoechst 33342 (Dojindo Laboratories) solution diluted 500-fold with the above-mentioned DMEM medium was added, and incubated at room temperature for 30 minutes in the dark. The solution was then removed from each well, and 150 μL / well of 1× PBS was added to each well. After photographing using the "Mito TEST" protocol on an IN Cell Analyzer 2200 (GE Healthcare), the number, area, and membrane potential of mitochondria in SH-SY5Y were evaluated. The ratios of mitochondrial number, area, and membrane potential to the control are shown in Figure 2(A) to (C), respectively. In Figure 2, "Ctrl" indicates the control, and "RA" indicates the positive control.

[0081] As shown in Figure 2(C), when CP 1%, CP 0.3%, CP 0.1%, or CP 0.03% culture supernatant was added to SH-SY5Y, the average mitochondrial membrane potential was higher than that of the control group. Therefore, it was demonstrated that the carp extract can increase the mitochondrial membrane potential of neurons by acting on the intestinal tract. Meanwhile, as shown in Figures 2(A) and (B), the number and area of ​​mitochondria were similar in the control group and the culture supernatants containing CP 1%, CP 0.3%, CP 0.1%, or CP 0.03%.

[0082] The results of Examples 1-1 and 1-2 showed that the carp extract can activate the gut-brain axis.

[0083] Example 2: Evaluation of the effect of culture supernatant of human intestinal epithelial cells treated with carp extract on promoting the expression of SIRT1 gene and TFAM gene in neurons (Addition of culture supernatant of human intestinal epithelial cells treated with carp extract to SH-SY5Y 3) The SH-SY5Y subcultured in the above [Caco-2 and SH-SY5Y] was added to 6.0 × 10 5 The cells were seeded in 5 mL dishes at 1000 cells / mL and cultured in FBS-containing DMEM medium. The next day, CP 0.3% or CP 0.1% culture supernatant was added to each well at 30% (volume) (1.5 mL). As a control, medium was added to each well at 30% (volume) instead of the culture supernatant. As a positive control, retinoic acid (Fujifilm Wako) was added to each well at a final concentration of 10 μM instead of the culture supernatant.

[0084] (Total RNA extraction 1) Twenty-four hours after the addition of the culture supernatant, sterile water, or retinoic acid, cell lysates were collected and total RNA was extracted using the High Pure RNA Isolation Kit (Roche Diagnostics GmbH) according to the following procedure. All reagents and equipment used from total RNA extraction to RT-qPCR were RNase-free. First, the medium was aspirated and washed with 1 mL of 1x PBS. Then, 200 μL of 1x PBS and 400 μL of the kit's cell lysis solution (lysis-binding buffer) were added. The dish was tilted and rocked for 2 minutes until the lysate became viscous. Holding the tip of a sterilized 100-1000 μL blue tip, the base of the tip was rubbed against the dish for 30 seconds, and the resulting cell lysate was collected into a 1.5 mL sample tube. The collected sample was then mixed in a vortex mixer for 1 minute. The filter tubes and collection tubes from the kit were assembled, and the entire volume of each cell lysate after mixing was added to the assembled filter tubes and centrifuged at 10,000 × g for 15 seconds. The waste liquid from the collection tubes was discarded. Next, 90 μL of DNase Incubation Buffer and 10 μL of DNase I were mixed in a sterilized reaction tube, and the resulting mixture was added to each filter tube and incubated at room temperature for 15 minutes. Next, 500 μL of Wash Buffer I from the kit was added to each filter tube and centrifuged at 10,000 × g for 15 seconds. The waste liquid from the collection tubes was discarded. Next, 500 μL of Wash Buffer II from the kit was added to each filter tube and centrifuged at 10,000 × g for 15 seconds. The waste liquid from the collection tubes was discarded. Next, 200 μL of Wash Buffer II from the kit was added to each filter tube and centrifuged at 13,000 × g for 2 minutes. The filter tubes were then inserted into new sterilized reaction tubes. Then, 50 μL of the elution buffer from the kit was added to each filter tube, and the tubes were incubated at room temperature for 3 minutes, followed by centrifugation at 10,000 × g for 1 minute. The resulting eluate was used as an RNA solution.The RNA concentration in the RNA solution was calculated based on the absorbance value at 260 nm using a NanoDrop 2000 / 2000c spectrophotometer (Thermo Fisher Scientific).

[0085] (RT-qPCR1) RT-qPCR was performed using the GoTaq 1-Step RT-qPCR System (Promega). Specifically, each RNA solution was diluted with nuclease-free water to 6.25 ng / μL or 0.625 ng / μL of RNA. Primers consisting of the nucleotide sequences of SEQ ID NOS: 1 to 6 were diluted with nuclease-free water to 2 μM each to obtain primer solutions. SEQ ID NOS: 1 and 2 represent the forward and reverse primers for the β-actin gene, respectively; SEQ ID NOS: 3 and 4 represent the forward and reverse primers for the SIRT1 gene, respectively; and SEQ ID NOS: 5 and 6 represent the forward and reverse primers for the TFAM gene, respectively. The β-actin gene was used as an endogenous control because its mRNA level was not affected by the addition of culture supernatant of human intestinal epithelial cells treated with carp extract to SH-SY5Y mice (3). Additionally, 10 μL of Go Taq qPCR Master Mix, 0.4 μL of Go Script RT Mix, and 1.6 μL of Nuclease-Free Water (all from the GoTaq 1-Step RT-qPCR System) were mixed per well to prepare a premix for each well. The premix was added to each well of a 96-well PCR plate placed on an ice plate at 12 μL / well, and 2 μL / well of the primer solution (for the forward primer and its corresponding reverse primer) was applied to the wall of each well. Subsequently, 4 μL / well of each diluted RNA solution was applied to the wall of each well. A negative control was also prepared by adding only the premix, the forward primer, and its corresponding reverse primer to each well. A qPCR sticker was then attached to the top of the plate, and the plate was centrifuged for 20 seconds in a plate centrifuge. Real-time qPCR was then performed using the Thermal Cycler Dice Real Time System (Takara Bio).The reaction conditions were one cycle of 37°C for 15 minutes, followed by one cycle of 95°C for 10 minutes, followed by 45 cycles of 95°C for 10 seconds, followed by 60°C for 30 seconds, followed by 72°C for 30 seconds, followed by 95°C for 15 seconds, followed by 60°C for 30 seconds, followed by 95°C for 15 seconds. Detection was based on FAM (6-carboxyfluorescein) fluorescence. The mean ΔCt values ​​were calculated by subtracting the Ct values ​​of the β-actin gene from the Ct values ​​of the SIRT1 gene or TFAM gene. The mean ΔCt values ​​of the control (sterilized water was added instead of the culture supernatant of human intestinal epithelial cells treated with carp extract) were then subtracted from each ΔCt value to calculate the ΔΔCt value. The mean and standard deviation (SD) of the obtained ΔΔCt values ​​were then calculated. -ΔΔCt The transcription levels of the target genes were calculated by substituting the values ​​into the expression vectors. The ratios of the transcription levels of the SIRT1 gene and the TFAM gene relative to the control are shown in Figure 3(A) and (B), respectively. In Figure 3, "Ctrl" indicates the control, and "RA" indicates the positive control.

[0086] As shown in Figures 3(A) and (B), when CP 0.3% or CP 0.1% culture supernatant was added to SH-SY5Y mice, the transcription levels of SIRT1 and TFAM genes were higher than those of the control group. Furthermore, the P values ​​for SIRT1 and TFAM gene transcription levels in CP 0.3% and CP 0.1% cultures compared to the control group were less than 0.05. Therefore, it was demonstrated that the carp extract acts on the intestinal tract to increase the transcription levels of SIRT1 and TFAM genes in neurons. In Figure 3, the ratios of SIRT1 and TFAM gene transcription levels in CP 0.3% and CP 0.1% cultures were approximately 1.16, 1.03, 1.14, and 1.18, respectively.

[0087] Example 3: Evaluation of the effect of culture supernatant of human intestinal epithelial cells treated with carp extract on promoting expression of BDNF gene and NAMPT gene in neurons RNA solutions were obtained by the same procedures as those described above (Addition of culture supernatant of human intestinal epithelial cells treated with carp extract to SH-SY5Y cells 3) and (Total RNA extraction) except that CP0.1% was used instead of CP0.3% or CP0.1%. Next, the same procedure as above (RT-qPCR) was performed, except that forward and reverse primers for BDNF and NAMPT were used instead of those for SIRT1 and TFAM. The transcription levels of BDNF and NAMPT were determined. The ratios of the transcription levels of BDNF and NAMPT to the control are shown in Figures 4(A) and 4(B), respectively. In Figure 4, "Ctrl" indicates the control, and "RA" indicates the positive control.

[0088] As shown in Figures 4(A) and (B), the addition of 0.1% CP culture supernatant to SH-SY5Y mice did not result in an increase in the transcription levels of the BDNF and NAMPT genes compared to the control group. Therefore, it was demonstrated that the activation of the gut-brain axis by the carp extract was mediated by a pathway that did not increase the transcription levels of the BDNF or NAMPT genes.

[0089] Examples 4 and 5 described below were carried out with the aim of verifying whether exosomes contained in the culture supernatant of human intestinal epithelial cells treated with carp extract are involved in the activation of the gut-brain axis by carp extract shown in Examples 1 to 3.

[0090] Example 4: Evaluation of the neuronal activation effect of exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract Example 4-1 Evaluation of the neurite outgrowth effect of exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract (Exosome addition to SH-SY5Y 1) The SH-SY5Y subcultured in the above [Caco-2 and SH-SY5Y] was 4.0 × 10 5Cells were seeded into each well of a 96-well black plate (Greiner Bio-One) at a concentration of 100 cells / mL. Twenty-four hours after seeding, the exosome-containing solutions from the CP0.1% group, CP0.3% group, and control group, obtained in the above section "Preparation of exosomes from culture supernatant of human intestinal epithelial cells treated with carp extract," were added to the wells so that the exosome content in the culture supernatant was 900 ng / mL or 450 ng / mL, calculated as protein content as determined in the above section (Evaluation of exosome quantity). As a positive control, RA (Fujifilm Wako) was added to each well in place of the exosomes to a final concentration of 100 μM. Each group was tested with n=3 wells.

[0091] (Immunostaining 2) Twenty-four hours after the addition of exosomes and RA to each group, the same procedures as described above (Immunostaining 1) were used to fix the cells and quantify the neurite length of SH-SY5Y cells. The ratio of neurite length to the control group is shown in Figures 5(A) and 5(B). In Figures 5(A) and 5(B), "Ctrl" indicates the control group, and "RA" indicates the positive control group. Figure 5(A) shows the results of adding each exosome-containing solution to the wells in the CP 0.1% group, the CP 0.3% group, and the control group so that the exosome content in the culture supernatant was 450 ng / mL in terms of protein. Figure 5(B) shows the results of adding each exosome-containing solution to the wells in the CP 0.1% group, the CP 0.3% group, and the control group so that the exosome content in the culture supernatant was 900 ng / mL in terms of protein.

[0092] As shown in Figure 5, the neurite length ratios in the CP0.1% and CP0.3% groups were comparable to those in the control group, regardless of whether the exosome-containing solution was added at 900 ng / mL or 450 ng / mL. Therefore, it was confirmed that exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract do not elongate neuronal neurites. Based on this, it is believed that the neuronal neurite elongation induced by the culture supernatant of human intestinal epithelial cells treated with carp extract shown in Example 1-1 (particularly Figure 1) is due to factors other than exosomes. Such factors could be, for example, soluble factors such as cytokines and growth factors.

[0093] Example 4-2 Evaluation of the effects of exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract on the number, area, and membrane potential of mitochondria in neurons (Exosome addition to SH-SY5Y 2) The SH-SY5Y subcultured in the above [Caco-2 and SH-SY5Y] was added to 6.0 × 10 5 Cells were seeded into each well of a 96-well black plate (Greiner Bio-One) at a concentration of 100 cells / mL. Twenty-four hours after seeding, the exosome-containing solutions from the CP0.1% group, CP0.3% group, and control group, obtained in the above section "Preparation of exosomes from culture supernatant of human intestinal epithelial cells treated with carp extract," were added to the wells so that the exosome content in the culture supernatant was 900 ng / mL or 450 ng / mL, calculated as protein content, as determined in the above section (Evaluation of exosome quantity). As a positive control, AICAR (Fujifilm Wako) was added to each well to a final concentration of 100 μM instead of the culture supernatant. Each group was tested with n=3 wells.

[0094] (Mitochondrial Co-staining 2) Twenty-four hours after the addition of exosomes and AICAR, the SH-SY5Y cells in each well were co-stained to assess mitochondrial number, area, and membrane potential. Mitochondrial co-staining and assessment of mitochondrial number, area, and membrane potential were performed using the same procedures as described above (Mitochondrial Co-staining 1), except that the incubation time after the addition of diluted MitoTracker Red CMXRos and diluted MitoTracker Green FM solutions was changed from 30 minutes to 15 minutes. The ratios of mitochondrial number, area, and membrane potential to those of the control group are shown in Figures 6(A)–(F). In Figures 6(A)–(F), "Ctrl" indicates the control group, and "AICAR" indicates the positive control group. Figures 6(A), (C), and (E) show the results of adding each exosome-containing solution to the wells in the CP 0.1% group, the CP 0.3% group, and the control group so that the exosome content in the culture supernatant was 450 ng / mL in terms of protein. Figures 6(B), (D), and (F) show the results of adding each exosome-containing solution to the wells in the CP 0.1% group, the CP 0.3% group, and the control group so that the exosome content in the culture supernatant was 900 ng / mL in terms of protein.

[0095] As shown in Figures 6(A) and (B), the mitochondrial number ratio in the CP0.1% group, where the exosome-containing solution was added at 450 ng / mL and 900 ng / mL, was significantly higher than that in the control group. Therefore, it was confirmed that exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract can increase the number of mitochondria in neurons.

[0096] As shown in Figures 6(C) and 6(D), the mitochondrial area ratios in the CP0.1% group (450 ng / mL and 900 ng / mL exosome-containing solution) and the CP0.3% group (900 ng / mL exosome-containing solution) were significantly higher than those in the control group. Therefore, it was confirmed that exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract can increase the mitochondrial area of ​​neurons. Based on this, it is likely that exosomes contributed to the significantly higher mitochondrial area ratios in the "CP0.3%" and "CP1%" groups compared to the "Ctrl" group in Figure 2(B) of Example 1-2.

[0097] As shown in Figures 6(E) and (F), the mitochondrial membrane potential ratios in the CP0.1% group treated with 450 ng / mL and 900 ng / mL of the exosome-containing solution, as well as in the CP0.1% group treated with 450 ng / mL of the exosome-containing solution, were significantly higher than those in the control group. Therefore, it was confirmed that exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract can increase the mitochondrial membrane potential of neurons. Based on this, it is likely that exosomes contributed to the increase in the mitochondrial membrane potential of neurons induced by the culture supernatant of human intestinal epithelial cells treated with carp extract, as shown in Figure 2(C) of Example 1-2.

[0098] From the results of Example 4 above, it is thought that exosomes contribute in part to the activation of the gut-brain axis by carp extract, but that factors other than exosomes also contribute.

[0099] Example 5: Evaluation of the effect of exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract on the expression of various genes in neurons. (Addition of exosomes to SH-SY5Y 3) 7.5 × 10 SH-SY5Y subcultured in the above [Caco-2 and SH-SY5Y] 4Cells were seeded onto 2 mL dishes at a concentration of 100 cells / mL. The following day, the exosome-containing solutions from the CP0.1% group, CP0.3% group, and control group, obtained in the above section "Preparation of exosomes from culture supernatant of human intestinal epithelial cells treated with carp extract," were added to the dishes so that the exosome content in the culture supernatant was 450 ng / mL, calculated as protein content as determined in the above section (Evaluation of exosome quantity). As a positive control, AICAR (Fujifilm Wako Co., Ltd.) was added to the dishes instead of the culture supernatant to a final concentration of 100 μM.

[0100] (Total RNA extraction 2) Twenty-four hours after the addition of exosomes and AICAR, total RNA was extracted from each of the SH-SY5Y dishes to obtain the respective RNA solutions. Total RNA extraction was performed using the same procedure as described above (Total RNA Extraction 1), except that after adding the cell lysis solution to the SH-SY5Y, the dish was tilted and shaken for 2 to 4 minutes until the viscosity of the cell lysis solution disappeared.

[0101] (RT-qPCR2) Each RNA solution prepared in (Total RNA Extraction 2) above was subjected to RT-qPCR using the same procedure as (RT-qPCR 1) above to determine the transcription levels of the target genes. In (RT-qPCR 1), the transcription levels of the SIRT1 and TFAM genes were evaluated. In addition, the transcription levels of the TFAM gene (forward primer: SEQ ID NO: 5, reverse primer: SEQ ID NO: 6), BDNF gene (forward primer: SEQ ID NO: 7, reverse primer: SEQ ID NO: 8), SIRT3 gene (forward primer: SEQ ID NO: 9, reverse primer: SEQ ID NO: 10), NAMPT gene (forward primer: SEQ ID NO: 11, reverse primer: SEQ ID NO: 12), MITOL gene (forward primer: SEQ ID NO: 13, reverse primer: SEQ ID NO: 14), and Drp1 gene (forward primer: SEQ ID NO: 15, reverse primer: SEQ ID NO: 16) were also evaluated. The endogenous control was the same as (RT-qPCR 1) above. The ratios of the transcription levels of SIRT1, TFAM, SIRT3, BDNF, TFAM, MITOL, and Drp1 to those of the control group are shown in Figures 7(A) to 7(D) and 8(A) to 8(C), respectively. In Figures 7 and 8, "Ctrl" indicates the control group, and "AICAR" indicates the positive control group.

[0102] As shown in Figures 7(A), (B), and (D) and Figure 8(C), the transcription levels of SIRT1, NAMPT, BDNF, and Drp1 genes in the 450 ng / mL CP0.1% group were significantly higher than those in the control group. Therefore, it was confirmed that exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract can increase the transcription levels of SIRT1, NAMPT, BDNF, and Drp1 genes in neurons. Based on this, it is suggested that exosomes contributed to the increase in SIRT1 gene transcription levels in neurons induced by the culture supernatant of human intestinal epithelial cells treated with carp extract, as shown in Example 2. Furthermore, as shown by comparing Figures 3 and 4 with Figures 7 and 8, the effects of the culture supernatant of human intestinal epithelial cells treated with carp extract and the exosomes on the transcription levels of each gene did not necessarily match. This also indicates that the culture supernatant contains factors other than exosomes that affect neurons.

[0103] Furthermore, exosomes purified from the culture supernatant of human intestinal epithelial cells treated with carp extract as described above were able to increase the number of mitochondria, mitochondrial area, mitochondrial membrane potential, and transcription levels of the SIRT1, NAMPT, BDNF, and Drp1 genes in neurons, suggesting that these exosomes may be able to improve neurodegenerative diseases.

Claims

1. An agent for maintaining or improving cognitive function, an agent for maintaining or improving memory, an agent for improving depression, or an agent for improving fatigue, comprising carp extract as an active ingredient, The carp extract is derived from a carp that has been heated and treated with protease; The agent, wherein the maintenance or improvement of cognitive function, the maintenance or improvement of memory, the improvement of depression, or the improvement of fatigue is due to activation of gut-brain correlation.

2. The agent according to claim 1 , wherein the protease comprises an endopeptidase derived from Bacillus subtilis and an endopeptidase derived from Aspergillus oryzae.

3. The agent according to claim 1 or 2, wherein the activation of the gut-brain axis is accompanied by the extension of neurites of neurons and an increase in the membrane potential of mitochondria of neurons.

4. A gut-brain correlation activator comprising carp extract as an active ingredient, The carp extract is derived from carp that has been heated and treated with protease.

5. The agent according to claim 4, wherein the protease comprises an endopeptidase derived from Bacillus subtilis and an endopeptidase derived from Aspergillus oryzae.

6. The agent according to claim 4 or 5, wherein the activation of the gut-brain axis is accompanied by the extension of neurites of neurons and an increase in the membrane potential of mitochondria of neurons.

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