Endoplasmic reticulum stress inhibitors, neurodegenerative disease prevention and improvement agents, dementia prevention, progression prevention and improvement agents, and foods.

Abstract

This invention provides an endoplasmic reticulum stress inhibitor and a neurodegenerative disease preventative and ameliorative agent that can be taken long-term and used as a food or medicine for prevention, symptom relief, or improvement. [Solution] Formula (I): TIFF2026091873000009.tif28170 (in the formula, R 1 , R 2 and R 3 Each of these is a saturated fatty acid residue, at least one of which is a pentadecanoic acid residue. ) A endoplasmic reticulum stress inhibitor containing a triglyceride represented as ( ) as an active ingredient.

Description

[Technical Field]

[0001] This invention relates to an endoplasmic reticulum (ER) stress inhibitor for improving the state of ER stress using a triglyceride composed mainly of a saturated fatty acid containing pentadecanoic acid. Furthermore, this invention relates to an agent for preventing, improving, and treating diseases whose onset is related to ER stress, and these agents can be used in foods, pharmaceutical compositions, etc. [Background technology]

[0002] Endoplasmic reticulum (ER) stress is a condition in which cells, exposed to various internal or external environmental changes, experience abnormal expression of the protein synthesis system within the ER lumen, leading to an increase in protein levels. This accumulation of proteins that cannot be properly eliminated results in the accumulation of defective proteins that do not fold correctly. Factors that cause ER stress include nutrient starvation, disruption of intracellular calcium concentration, hypoxia, expression of mutant proteins, and viral infection. When ER stress occurs, cells activate lipid synthesis and expand the ER to increase their protein folding capacity in order to maintain homeostasis. In stages where the accumulation of defective proteins is relatively minor, the ER stress response, a response mechanism to eliminate defective proteins, is performed. However, if the stress state is severe or persists for a long period, denatured proteins accumulate in the ER, leading to adverse effects on the cell. To avoid damage from ER stress and maintain homeostasis, cells induce apoptosis (programmed cell death) in tissues and organs throughout the body. When this reaction occurs in nerve tissue, it leads to the degeneration and loss of nerve fibers (neurons). Endoplasmic reticulum stress has been suggested to be involved in the development of neurodegenerative diseases (see Non-Patent Literature 1).

[0003] With Japan facing a super-aging society, the number of patients with neurodegenerative diseases is rapidly increasing, becoming a major social issue. Neurodegenerative diseases are illnesses in which specific groups of nerve cells in the central nervous system are damaged and lost, impairing nerve function. Representative neurodegenerative diseases include Alzheimer's disease and Creutzfeldt-Jakob disease, which cause cognitive impairment, and Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease, which present with motor dysfunction. Endoplasmic reticulum stress is known to be involved in the onset of these diseases. A common characteristic of these diseases is the accumulation of degenerated proteins, hence they are also called folding diseases. For example, in Alzheimer's disease, amyloid-beta is observed extracellularly, and tau protein is observed intracellularly. In Parkinson's disease, α-synuclein is accumulated, in ALS, mutant superoxide dismutase is accumulated, and in Huntington's disease, huntingtin protein is accumulated. Creutzfeldt-Jakob disease, classified as a prion disease, develops due to the accumulation of abnormal prions. Thus, although the proteins that accumulate differ depending on the disease, a commonality is that the accumulation of denatured proteins damages specific nerve cells and causes cell death.

[0004] To date, several food components have been reported that are expected to help prevent dementia, but their effectiveness remains unclear. Foods that suppress oxidative stress include polyphenols (Non-Patent Document 2), vitamins C and E (Non-Patent Document 3), and folic acid (Non-Patent Document 4). Antioxidants are thought to suppress neuronal cell death caused by oxidative stress by removing reactive oxygen species generated in the brain during the pre-dementia stage. Furthermore, it has been reported that fatty acids with 7 carbon atoms and the triglyceride trihepranoin increase circulating ketone bodies and reduce amyloid-beta deposition in Alzheimer's disease patients (Patent Document 1). However, the effectiveness of these is still unknown and is not yet sufficient. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Satoshi Kanemoto, Kazunori Imaizumi, Endoplasmic Reticulum Stress and Disease, Journal of Japanese Biochemical Society 90(1):51-59(2018) [Non-Patent Document 2] Costa C,et.al.,Current evidence on the ef fect of dietary polyphenols intake on chrnic diseases.Food Chem Toxicol 110:286-299(2017) [Non-Patent Document 3] Bhatti AB,et.al.,Vitamin supplementation as an adjuvant treatment for Alzheimer's disease.J Clin Diagn Res,10,7-11(2016) [Non-Patent Document 4] Cacciapuoti F.Lowering homocysteine ​​levels with folic acid and B-vitamins do not reduce early atherosclerosis, but could interfere with cognitive decline and Alzheimer's disease.J Thromb Thrombolysis,36,258-262(2013) [Patent Documents]

[0006] [Patent Document 1] Special table 2013-516416 publication [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] In conventional food and drink products, there is no known food and drink product that suppresses cell death by reducing endoplasmic reticulum stress and prevents and improves neurodegeneration. In addition, in dementia therapeutic drugs, there are problems such as insufficient effects and side effects, and there is a desire for food and drink products and drugs that can be taken long-term for prevention, symptom reduction, and improvement. Therefore, the problem to be solved by the present invention is to provide an endoplasmic reticulum stress inhibitor, a neurodegenerative disease prevention and improvement agent, and a dementia prevention, progression prevention, and improvement agent, as well as foods, that can be used as food and drink products and drugs suitable for such uses.

Means for Solving the Problem

[0008] As a result of intensive studies to solve the above problems, the inventors of the present invention found that a triglyceride composed mainly of saturated fatty acids containing pentadecanoic acid (C15) (pentadecanoic acid triglyceride: hereinafter sometimes referred to as "PdATG") acts on hippocampal-derived neurons to suppress the accumulation of denatured proteins in the endoplasmic reticulum caused by stress damage, and as a result, reduces the resulting cell death (apoptosis), thereby preventing or improving neurodegenerative diseases, and completed the present invention.

[0009] The endoplasmic reticulum stress inhibitor according to the first aspect of the present invention for solving the above problems is represented by the following formula (I):

Chemical formula

[0010] In one embodiment of this endoplasmic reticulum stress inhibitor, in the triglyceride of formula (I), it is preferable that R 1 and R 2 or R 1 and R 3 are pentadecanoic acid residues. In another embodiment, R 1 , R 2and R 3 Any one of these may be a tridecyl acid (C13), a myristic acid residue (C14), a palmitic acid residue (C16), or a margaric acid residue (C17).

[0011] In another preferred embodiment, R 1 , R 2 and R 3 The triglyceride of formula (I) above, in which all are pentadecanoic acid residues, and R 1 , R 2 and R 3 A triglyceride of formula (I) in which any two of the residues are pentadecanoic acid residues and the other one is a myristic acid or palmitic acid residue may also be included.

[0012] In yet another preferred embodiment of the endoplasmic reticulum stress inhibitor of the present invention, the triglyceride of formula (I) is derived from algae of the genus Aurantiochytrium or Schizochytrium, where R 1 , R 2 and R 3 Each of these may be a triglyceride, wherein each is a saturated fatty acid residue, at least one of which is a pentadecanoic acid residue. Furthermore, the mixture may contain unsaturated fatty acids derived from algae of the genus Aurantiochytrium or Schizochytrium.

[0013] In a second aspect of the present invention, an agent for the prevention and improvement of neurodegenerative diseases is provided, comprising a triglyceride represented by the above formula (I) as an active ingredient.

[0014] In a third aspect of the present invention, an agent for preventing, slowing the progression of, and improving dementia is provided, comprising a triglyceride represented by the above formula (I) as an active ingredient.

[0015] Furthermore, in a fourth aspect of the present invention, a food product containing a triglyceride represented by formula (I) as an active ingredient is provided. This food product is preferably used, for example, as a health food, a functional food, or a food for specified health uses to improve the daily lives of people who are prone to forgetfulness. [Effects of the Invention]

[0016] The endoplasmic reticulum stress inhibitor of the present invention can suppress endoplasmic reticulum stress in mammalian cells, particularly nerve cells, and can provide long-term use of food, beverages, or pharmaceuticals for prevention, symptom reduction, and improvement. [Brief explanation of the drawing]

[0017] [Figure 1] Figure 1 shows the inhibitory effect of PdATG on cell death induced by tunicamycin, an endoplasmic reticulum stress inducer. The vertical axis represents cell viability (%), and the horizontal axis represents the tunicamycin treatment concentration. ** indicates a statistically significant difference compared to the control at a 1% significance level, and * indicates a statistically significant difference at a 5% significance level. + indicates a significant trend. [Modes for carrying out the invention]

[0018] Next, each embodiment of the present invention will be described with reference to the drawings. It should be noted that the embodiments described below are not intended to limit the invention as defined in the claims, and not all of the elements and combinations thereof described in each embodiment are necessarily essential to the solution of the present invention.

[0019] (Active ingredients) In this specification, PdATG means an ester of at least one pentadecanoic acid and glycerol, represented by the following formula (I) R 1 , R 2 and R 3 At least one of, preferably any two of, for example, R 1 and R 2 or R 1 and R 3 However, R is more preferably 1 , R 2 and R 3 The triglyceride contains three pentadecanoic acid residues. The pentadecanoic acid may be attached to any of the 1st, 2nd, or 3rd positions on the glyceride.

[0020] [ka]

[0021] (In the formula, R 1 , R 2 and R 3 Each of these is a saturated fatty acid residue, and at least one of them is a pentadecanoic acid residue. In the formula, R 1 , R 2 and R 3 Any one of the residues represented by may be a saturated fatty acid residue other than a pentadecanoic acid residue. "Saturated fatty acid" is a general term for fatty acids that do not have double or triple bonds in their molecule, C n H 2n+1 It is represented by the chemical formula COOH. These saturated fatty acids are linear or branched saturated fatty acids, including linear saturated fatty acids such as capric acid (C10), lauric acid (C12), tridecyl acid (C13), myristic acid (C14), pentadecanoic acid (C15), palmitic acid (C16), margaric acid (C17), stearic acid (C18), arachidic acid (C20), behenic acid (C22), lignoceric acid (C24), and cerotic acid (C26), as well as branched saturated fatty acids such as 2-hexyldecanoic acid (C16), 13-methylpentadecanoic acid (C16), and 16-methylheptadecanoic acid (C18).

[0022] In a preferred embodiment, PdATG is R 1 , R 2 and R 3 The triglyceride of formula (I) above, in which all are pentadecanoic acid residues, and R 1 , R 2 and R 3The mixture contains both triglycerides in which any two of the residues are pentadecanoic acid residues and the other one is a myristic acid or palmitic acid residue. The ratio of the two in this mixture is not particularly limited, but is preferably 1:2 to 2:1 by mass, and more preferably approximately 1:1. Furthermore, each of these is present in an amount of 10% by mass or more, preferably 20% by mass or more, relative to the total amount of triglycerides. Moreover, it is more preferable that the mixture of triglycerides containing two or more pentadecanoic acid residues is present in an amount of 50% by mass or more of the oil and fat.

[0023] In a more preferred embodiment, PdATG is represented by the following formula (II) or (III). [ka]

[0024] (However, in formulas (II) and (III) above, R is a saturated fatty acid of C14 to C16.) It is more preferable that a mixture of triglycerides containing two or more residues of pentadecanoic acid is present in an amount of 50% by mass or more in the oil and fat, but even if the content of triglycerides containing two or more residues of pentadecanoic acid is 50% by mass or less, the objective can be achieved by increasing the intake amount. Therefore, the active ingredient of the present invention may exist in the form of a mixture of triglycerides containing two or more residues of pentadecanoic acid, and the mixture itself can function as an active ingredient if it is present with a purity of at least 1% by mass, preferably 50% by mass or more, and more preferably 90% by mass or more, relative to the total amount of triglycerides.

[0025] The active ingredient of the present invention may exist in a mixture with triglycerides other than the compound of formula (I), and the mixture itself can function as an active ingredient if it is present with a purity of at least 1% by mass, preferably 50% by mass or more, and more preferably 90% by mass or more, relative to the total amount of triglycerides.

[0026] The active ingredient of the present invention has at least one, preferably two or more, odd-chain fatty acids, particularly pentadecanoic acid, in its molecule. Therefore, it is believed that ingesting it suppresses the accumulation of abnormal proteins in the endoplasmic reticulum due to endoplasmic reticulum stress, as described later, thereby reducing cell death and restoring normal function.

[0027] (Effects and Benefits) The active ingredient of this invention has the effect of improving diseases caused by endoplasmic reticulum stress and pre-disease physical conditions by alleviating endoplasmic reticulum stress in various cells. In the brains of patients with Alzheimer's disease, abnormal protein deposition such as senile plaques and neurofibrillary tangles is observed. Senile plaques are composed of amyloid-beta, a peptide aggregate of about 40 amino acids, and this aggregate induces neuronal cell death. Amyloid-beta deposition induces endoplasmic reticulum stress in nerve cells, which is the main cause of neuronal cell death (see Ogen-Shtern N, et. al., Protein aggregation and ER stress. Brain Res, 1648, 658-666 (2016)). At the same time, amyloid-beta deposition also causes the production of reactive oxygen species in the brain by glial cells, and this oxidative stress also causes neuronal cell death (see Angelova PR, & Abramov AY. Interaction of neurons and astrocytes underlies the mechanism of Aβ-induced neurotoxicity. Biochem Soc Trans, 42, 1286-1290 (2014)). Generally, the latency period from amyloid-beta deposition to the onset of dementia is said to be 20 to 30 years, and by the time dementia develops, a considerable number of nerve cells have already been lost, and a significant portion of the neural circuits have broken down. Therefore, it is considered almost impossible to regenerate the neural circuits in the brain and to completely cure the disease after its onset.

[0028] This means that establishing preventive measures well before the onset of symptoms is extremely important. One preventive measure is to prevent neurodegeneration by taking preventive and mitigating components before the onset of symptoms. At the same time, early treatment from the initial stages when the onset of symptoms is suspected is also important.

[0029] Furthermore, in kidney cells and other tissues, when insulin-secreting β-cells experience endoplasmic reticulum stress, insulin production ceases, leading to diabetes.

[0030] (Aging-related diseases and endoplasmic reticulum stress) As described above, endoplasmic reticulum (ER) stress progresses due to environmental factors such as aging, genetic factors, and lifestyle, and insufficient repair gradually leads to loss of homeostasis and cell death. Age-related diseases are characterized by abnormal progression due to the combination of internal and external factors, with specific diseases occurring in each organ. Among these, metabolic syndrome is cited as a risk factor for cerebrovascular and cardiovascular diseases and diabetes, increasing arteriosclerosis, decreased insulin sensitivity, and cancer incidence. While the oxidative stress hypothesis has been proposed as a risk factor for age-related progression, and the relationship between many diseases and reactive oxygen species has been debated, the reality is that ER stress is thought to play a significant role. The progression of ER stress leads to the manifestation of age-related diseases and age-related neurodegenerative diseases. The progression of ER stress in various tissues is considered a major cause of adult diseases, including cancer. In other words, it has become clear that pharmaceuticals and food components that can actually improve ER stress are essential for maintaining people's health.

[0031] The endoplasmic reticulum (ER) stress response is one of the mechanisms of cellular stress adaptation and plays an important role in the survival of cancer cells in the tumor microenvironment, such as hypoxia and low glucose levels. Cancer is the leading cause of death in Japan. The characteristics of cancer are 1) autonomous proliferative capacity, 2) invasive and metastatic capacity, and 3) cachexia (a state of malnutrition where nutrients are depleted by cancer tissue despite adequate nutrient intake). Cancer cells can survive and proliferate even in hypoxic and nutrient-poor environments. This hypoxic and nutrient-poor environment is precisely the environment that induces ER stress, and increased expression of ER molecular chaperones is observed in cancer cells, which is involved in the proliferation and metastasis of cancer cells. In breast cancer cells and hepatocellular carcinoma, increased expression of XBP-1 is thought to contribute to the survival of cancer cells. Suppressing XBP-1, which is abnormally expressed due to increased ER stress within cells, may also have the potential to suppress cancer development. Furthermore, it is known that XBP-1 (X-box binding protein 1) undergoes splicing of its mRNA during endoplasmic reticulum stress response, and that the spliced ​​form of XBP-1 acts as a nuclear transcription factor, efficiently signaling the accumulation of abnormal proteins (Yanagitani, K., et al. Molelular Cell vol.34, 191-200 (2009)).

[0032] (Method for producing triglyceride mixtures) The triglyceride mixture, which is the active ingredient of the present invention, may be chemically synthesized or naturally occurring. If it is naturally occurring, its source is not particularly limited. Examples include lipids produced by organisms in their bodies, such as the fats of livestock and poultry, the oils and fats of fish and shellfish, vegetable oils, or lipid-producing microorganisms. From the viewpoint of industrial productivity, microorganisms such as algae, bacteria, fungi (including yeast), and / or protists are preferred. Preferred microorganisms include those selected from the group consisting of golden algae (microorganisms of the stramenopil kingdom, etc.), green algae, diatoms, dinoflagellates, yeasts, and fungi of the genera Mucor and Mortierella. The members of the microbial group stramenopil include microalgae. Microalgae are defined as organisms with a cell size of 1 μm to 100 μm in diameter, which are the remaining organisms from among photosynthetic organisms that produce oxygen, excluding mosses, ferns, and seed plants. Labyrinthulae, which are protists closely related to microalgae, are also included. Labyrinthuli are heterotrophic marine eukaryotic microorganisms that do not perform photosynthesis and are widely distributed mainly in subtropical and tropical regions. Generally, Labyrinthuli are broadly classified into the families Labyrinthulidae and Thraustochytriidae, and include genera such as Labyrinthula, Aurantiochytrium, Schizochytrium, Thraustochytrium, Aplanochytrium, oblongichytrium, Botryochytrium, and Japonochytrium.

[0033] Among the Labyrinthula species to be cultured, Aurantiochytrium, Schizochytrium, or Slaustchytrium genera are more preferable. These species have relatively high lipid production capabilities and can produce hydrocarbons such as squalene, making them suitable for use in food products and as raw materials for biofuels.

[0034] Labyrinthura can be cultured using any of the following methods: batch culture, continuous culture, or fed-batch culture. Furthermore, Labyrinthura can be cultured using any appropriate culture method, such as shaking culture, aeration culture, aeration-stirred culture, air-lift culture, or static culture. Among these culture methods, aeration-stirred culture or air-lift culture is more preferred. Culture apparatus used for culturing Labyrinthura can include, for example, mechanically agitated reactors, air-lift reactors, packed-bed reactors, and fluidized-bed reactors. Various containers such as tanks, jar fermenters, flasks, dishes, culture bags, tubes, and test tubes can be used as culture vessels, depending on the purpose of cultivation and the required culture volume. Culture vessels may be made of appropriate materials, such as inorganic materials like stainless steel and glass, or organic materials like polystyrene, polyethylene terephthalate copolymer, and polypropylene.

[0035] Labyrinthula can be cultured under appropriate temperature, pH, and aeration conditions. The culture temperature is preferably between 5°C and 40°C, more preferably between 10°C and 35°C, and even more preferably between 10°C and 30°C. The pH is preferably between 2 and 11, more preferably between 4 and 9, and even more preferably between 6 and 8.

[0036] Labyrinthuria can be cultured by subculturing at appropriate intervals depending on the genus and species of Labyrinthuria, the culture medium composition, and the culture conditions. For example, Labyrinthuria complete their logarithmic growth phase in about 2 days after the start of culture and enter the death phase in about 7 days. Therefore, it is preferable to subculture Labyrinthuria at intervals of 1 to 10 days, more preferably at intervals of 2 to 7 days, and even more preferably at intervals of 2 to 5 days. The culture time for Labyrinthuria can be set to an appropriate time depending on the genus and species of Labyrinthuria, the culture medium composition, the culture conditions, and the purpose of the culture. In particular, Aurantiochytrium algae of the Labyrinthuria genus are preferred because they are heterotrophic algae that inhabit brackish water areas and have the characteristic of assimilating nutrients in the water to produce lipids and accumulating them in the cells.

[0037] It is preferable to use strains of Aurantiochytrium algae that have a superior ability to produce the desired triglycerides. Such algal strains may be naturally collected and isolated, cloned after mutagenesis and screening, or established using genetic engineering techniques. For example, Aurantiochytrium Sp.SA-96, NIES-3737, Aurantiochytrium NB6-3, or Aurantiochytrium mh1959 strains are particularly preferred as microorganisms for the production of pentadecanoic acid triglyceride in the present invention because they have the property of accumulating large amounts of triglycerides containing the odd-chain fatty acid pentadecanoic acid (PDA) and triglycerides containing the highly unsaturated fatty acids docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) within their cells.

[0038] The cultivation of the above-mentioned Aurantiochytrium algae is carried out by methods established in the art. That is, normal maintenance cultivation is carried out by seeding algae in a culture medium with appropriately prepared components and following standard procedures. The culture medium for cultivating Aurantiochytrium algae essentially contains salinity, a carbon source, and a nitrogen source. Generally, so-called GTY medium (artificial seawater 10-40 g / L, D(+) glucose 20-100 g / L, tryptone 10-60 g / L, yeast extract 5-40 g / L) is used for cultivating microalgae.

[0039] Carbon sources include sugars such as glucose, fructose, and sucrose. These carbon sources are added, for example, at a concentration of 20 to 120 g per liter of culture medium.

[0040] Aurantiochytrium algae are marine algae, and an appropriate amount of artificial seawater is added to the culture medium. Preferably, the artificial seawater is added so that the final salinity of the culture medium is about 10% (v / v) to about 100% (v / v) of seawater (salinity 3.4% (w / v)), for example, a salinity of about 1.0 to 3.0% (w / v).

[0041] Generally, various nitrogen sources can be added to the culture medium for microalgae, such as organic nitrogen (monosodium glutamate, urea), inorganic nitrogen (ammonium acetate, ammonium sulfate, ammonium chloride, sodium nitrate, ammonium nitrate), or biodigests (yeast extract, corn steep liquor, polypeptone, peptone, tryptone, etc.). In particular, cell extracts obtained by extracting liquid components from various animal cells are preferred as nitrogen sources to be added to the culture medium for Aurantiochytrium algae. When cells must be cultured on an industrial scale to obtain cultured cell products, the use of cell extracts, which are rich in nutrients such as amino acids, nucleic acids, vitamins, and minerals derived from cells and are available at low cost, is extremely advantageous.

[0042] However, as described above, using a culture medium prepared based on cell extracts significantly reduces the proportion of odd-chain fatty acids in the triglycerides produced by cultured algae. Therefore, cell extracts could not be used as a nitrogen source for the culture medium to efficiently produce the target product of the present invention. Accordingly, the inventors have already reported a method for producing triglycerides containing odd-chain fatty acids as the main component by culturing Aurantiochytrium algae in an algal culture medium prepared by adding strongly acid-treated cell extracts, and by dramatically increasing the production of odd-chain fatty acids compared to when untreated cell extracts were added (Japanese Patent Publication No. 2017-063633).

[0043] Furthermore, in a preferred embodiment of the present invention, the basic culture medium for cultivating Aurantiochytrium algae is prepared by adding 10-50 mM valine and 10-50 mM sodium propionate to a medium containing 2% or more glucose, 0.5-4% monosodium glutamate, 0.1-2% yeast extract, 1-3.3% sea salt, and 2-20% whey (animal or plant-based). Tofu whey (soybean whey) is preferred as the animal or plant-based whey. To this basic culture medium, 2% or more of a culture solution of Aurantiochytrium that has been pre-cultured at 20-30°C for 72 hours with 2% or more glucose, 0.5-4% monosodium glutamate, 0.1-2% yeast extract, 1-3.3% sea salt, and 2-20% whey (animal or plant-based) is added. Air is then passed through this Aurantiochytrium-added culture solution and it is gently stirred. The culture should be carried out at 20-30°C and maintained at a pH of 5.0-8.5 (using a 1.0 M NaOH solution for pH adjustment) for 48-200 hours. After culturing, Aurantiochytrium cells that have produced pentadecanoic acid triglyceride can be recovered by centrifugation (see WO2020 / 054804 pamphlet).

[0044] The pellets recovered from the culture medium obtained by the above method by centrifugation or filtration are dried by freeze-drying or drying by heating. Alternatively, the culture medium in which the algal cells are suspended after cultivation may be used directly in the triglyceride extraction step. Extraction may be carried out multiple times using different organic solvents. As organic solvents, a mixture of a polar solvent and a weakly polar solvent such as an n-hexane-ethanol mixed solvent, a chloroform-methanol mixed solvent, or an ethanol-diethyl ether mixed solvent can be used. The obtained extract is purified by methods known to those skilled in the art.

[0045] Methods for separating triglycerides employ fractionation techniques known to those skilled in the art. Separation and purification may be performed by utilizing various physicochemical properties of the triglyceride molecules to be fractionated, such as polarity, solubility in the solvent, melting point, specific gravity, and molecular weight, and column chromatography is preferably used. The conditions for the triglyceride separation means can be set by ordinary condition studies by those skilled in the art, depending on the composition of the triglyceride mixture and the type of triglyceride to be fractionated.

[0046] Algae of the genera Schizochytrium and Aurantiochytrium can synthesize and accumulate both odd-chain fatty acid triglycerides and highly unsaturated fatty acid triglycerides within their cells. Therefore, ethanol, hexane, or ethyl acetate is added to the obtained algal cells to extract the lipids, and the solvent is removed by distillation to obtain algal lipids. By allowing these lipids to stand at 5°C, pentadecanoic acid triglyceride can be precipitated. The composition of the purified pentadecanoic acid triglyceride "PdATG" can be analyzed by HPLC-MS, HPLC, gas chromatography, etc.

[0047] Algae of the genus Aurantiochytrium can synthesize and accumulate both odd-chain fatty acid triglycerides and highly unsaturated fatty acid triglycerides within their cells. Therefore, after extracting lipids by adding hexane or ethyl acetate to the obtained Aurantiochytrium cells, the unsaturated fatty acids are oxidatively decomposed by adding hydrogen peroxide to this lipid solution or by passing ozone through it. After the reaction is complete, oxides are removed using sodium bicarbonate and sodium carbonate or ion exchange resin to obtain pentadecanoic acid triglyceride "PdATG". The composition of the purified pentadecanoic acid triglyceride "PdATG" can be analyzed by HPLC-MS, HPLC, gas chromatography, etc.

[0048] (Endoplasmic reticulum stress inhibitor) The first aspect of the present invention relates to an endoplasmic reticulum (ER) stress inhibitor, which includes a formulation that suppresses neuronal cell death caused by the accumulation of abnormal proteins in the ER. Here, neuronal cell death encompasses necrosis and apoptosis of nerve cells. The term "suppression of neuronal cell death" includes mitigating, reducing, or eliminating neuronal cell death, inhibiting the progression of neuronal cell death, and preventing or avoiding it. It can be used to prevent, slow the progression of, and / or improve disorders (diseases and age-related changes) that manifest as neuronal cell death due to ER stress, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, Creutzfeldt-Jakob disease, and amnesia.

[0049] (Pharmaceuticals) The neurodegenerative disease prevention and improvement agent according to the second aspect of the present invention and the dementia prevention, progression prevention, and improvement agent according to the third aspect contain pentadecanoic acid triglyceride represented by the above formula (I) as an active ingredient and can be used to improve the symptoms of neurodegenerative diseases such as Alzheimer's disease, and is useful as a pharmaceutical for that purpose. Here, "pharmaceutical" means a therapeutic drug for preventing, preventing the progression, and / or improving the symptoms of dementia, etc., in patients with Alzheimer's disease, etc., by suppressing endoplasmic reticulum stress.

[0050] When manufacturing pharmaceuticals using pentadecanoic acid triglyceride represented by the above formula (I), any auxiliary agents such as sugars like dextrin and starch; proteins like gelatin, soy protein, and corn protein; amino acids like alanine, glutamine, and isoleucine; polysaccharides like cellulose and acacia gum; and oils and fats like soybean oil and medium-chain triglyceride can be added to formulate the product into any dosage form.

[0051] The amount of pentadecanoic acid triglyceride represented by the above formula (I) in the pharmaceutical product according to the present invention is not particularly limited, but it is preferable to adjust it so that the daily intake of pentadecanoic acid triglyceride for adults, which is the concentration that shows efficacy, is about 10 to 1000 mg.

[0052] The pharmaceutical product of this embodiment may contain only the compound of formula (I) as an active ingredient, or it may contain other components as long as they do not inhibit the neuronal cell death inhibitory effect. The other components may be, for example, conventionally used therapeutic or prophylactic drugs for neurodegenerative diseases. Therefore, in a further embodiment, the endoplasmic reticulum stress inhibitor of this embodiment provides a pharmaceutical composition for preventing and improving neurodegenerative diseases.

[0053] (food) The food according to the fourth aspect of the present invention contains pentadecanoic acid triglyceride represented by the above formula (I) as an active ingredient and can be consumed over a long period of time as a preventive food or beverage even before the onset of neurodegenerative diseases, making it useful as a health food for that purpose. Pentadecanoic acid, which constitutes PdATG, has been reported to be present in small amounts in the meat of cattle, pigs, chickens, and sheep, as well as in fish living in rivers and the sea, and in edible parts of mushrooms, and it is also presumed that PdATG is present in trace amounts and is highly safe based on many years of experience in consumption.

[0054] Therefore, the food of this embodiment is useful as a health food to be consumed for health promotion. Here, "health food" refers to food and beverages intended for use in promoting health in daily life, or for preventing, slowing the progression of, alleviating or improving age-related forgetfulness, decline in comprehension and judgment, memory impairment, disorientation, executive function impairment, aphasia, apraxia, agnosia, and dementia. It refers to a broad definition of "health food" that includes functional foods, nutrient function foods, or foods for specified health uses under the "Health Functional Food System" that meet the safety and effectiveness standards set by the government.

[0055] When manufacturing food products using pentadecanoic acid triglyceride represented by the above formula (I), any auxiliary agents such as sugars like dextrin and starch; proteins like gelatin, soy protein, and corn protein; amino acids like alanine, glutamine, and isoleucine; polysaccharides like cellulose and acacia gum; and oils and fats like soybean oil and medium-chain triglyceride can be added to formulate the product into any dosage form.

[0056] Furthermore, while the amount of pentadecanoic acid triglyceride represented by formula (I) in the food of the present invention is not particularly limited, it is preferable to adjust it so that the daily intake of pentadecanoic acid triglyceride for adults is approximately 1 to 100 mg, taking into consideration the general intake of the food to which it is added.

[0057] Specific examples of the above-mentioned foods include, for example, beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks (including concentrated liquids and powders for adjusting these beverages); frozen desserts such as ice cream, ice sherbet, and shaved ice; noodles such as soba, udon, vermicelli, gyoza wrappers, shumai wrappers, Chinese noodles, and instant noodles; confectionery such as candy, candy, gum, chocolate, snacks, biscuits, jelly, jam, cream, and baked goods; processed seafood and livestock products such as kamaboko, ham, and sausage; dairy products such as processed milk and fermented milk; oils and processed oils such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings; seasonings such as sauces and dips; health and nutritional supplements in various forms such as tablets and granules; and other items such as soups, stews, salads, side dishes, and pickles.

[0058] The food according to the present invention may contain various food additives, such as antioxidants, flavorings, various esters, organic acids, organic acid salts, inorganic acids, inorganic acid salts, inorganic salts, pigments, emulsifiers, preservatives, seasonings, sweeteners, acidulants, fruit juice extracts, vegetable extracts, nectar extracts, pH adjusters, and quality stabilizers, either alone or in combination.

[0059] In the food according to the present invention, the concentration of pentadecanoic acid triglyceride as solid content is approximately 0.00001 to 100% by mass (hereinafter expressed in %), preferably approximately 0.0005 to 50%, to obtain good usability and effects.

[0060] Specific examples of the above foods include: for diabetes, foods for people with elevated blood sugar levels; for eye diseases, foods to improve glaucoma and retinitis pigmentosa; for neurodegenerative diseases, foods for dementia, improving memory, and improving cerebral blood flow; for Alzheimer's disease, foods to maintain and support memory, which is a part of cognitive function; for Creutzfeldt-Jakob disease, foods for dementia, mental stability, forgetfulness, memory, and improving cerebral blood flow; for Parkinson's disease, foods for muscle stiffness, reflex impairment, lightheadedness, dizziness, and insomnia; and for Huntington's disease, foods for difficulty swallowing and the ability to perceive things (thinking, judgment, etc.). Foods for memory loss, difficulty controlling emotions (depression, emotional outbursts, irritability, etc.), prion diseases, multiple sclerosis, amyotrophic lateral sclerosis, muscle atrophy, speech disorders, muscle strengthening, and swallowing disorders, metabolic diseases, obesity, dyslipidemia, and hyperlipidemia, foods for those with high levels of lipids such as cholesterol and triglycerides in the blood, as excess lipids in the blood increase the risk of arteriosclerosis, myocardial infarction, and stroke, and are therefore considered effective in improving these conditions. Foods for hypertension, headaches, nausea, vomiting, impaired consciousness, seizures, and those with elevated blood pressure, foods for kidney damage and chronic kidney disease, when waste products cannot be excreted through urine and the body's fluid balance (water and salt levels) cannot be regulated, and foods for normalizing growth such as height in cases of skeletal malformations (cartilage formation), are also included, but are not limited to these.

[0061] The present invention will now be described in more detail with reference to examples, but the present invention is not limited in any way to these examples. In the following examples, the unit % in the numerical values ​​indicating the amount of each component added means mass %. [Examples]

[0062] (Manufacturing Example 1) Production of pentadecanoate triglyceride using aurantiochytrium Aurantichytrium mh1959 strain (purchased from Professor Masahiro Hayashi, Faculty of Agriculture, Miyazaki University) was pre-cultured for 72 hours at 25°C in a medium containing 3.6% glucose, 0.5% monosodium glutamate, 0.2% yeast extract, 1% sea salt, and 10% whey. This was added to the following basic medium to a concentration of 2%, aerated, and gently mixed. 1 kg of basic medium was prepared by adding 50 mM valine and 25 mM sodium propionate to a medium containing 3.6% glucose, 0.5% monosodium glutamate, 0.2% yeast extract, 1% sea salt, and 10% whey. The culture was maintained at 25°C and pH 7.40-7.75 (1.0 M NaOH solution was used for pH adjustment) for 72-96 hours.

[0063] After culturing, the algae were centrifuged at 3000 rpm for 15 minutes to collect approximately 20 g of algae. Hexane or ethyl acetate was added to the obtained 20 g of Aurantichytrium algae to extract the lipids. Hydrogen peroxide was added to the extracted lipid solution (water was added if necessary), and ozone was passed through at room temperature. After the reaction was complete, oxides were removed using sodium bicarbonate and sodium carbonate or ion exchange resin to obtain 2 g of a mixture of pentadecanoic acid triglycerides that precipitated as the temperature decreased.

[0064] (Compositional analysis of pentadecanoic acid triglyceride) To the lipid containing pentadecanoic acid triglyceride obtained in Production Example 1, 0.50 mL of 14% BF3-methanol and 0.25 mL of methyl acetate were added, and the mixture was heated at 70°C for 30 minutes to obtain the fatty acid methyl ester (FAME). Exactly 1.0 mL of n-hexane and 5 mL of physiological saline were added to the reaction mixture and mixed vigorously. The mixture was centrifuged at 2800 rpm for 10 minutes, and the n-hexane layer was used as the sample for gas chromatography.

[0065] The above samples were analyzed using a Shimadzu GC-2025 gas chromatograph. The analysis conditions involved using an Agilent J&W GC column DB-23 (30m × 0.25mm), injecting 1 μL of the sample, and detecting it with a carrier gas (He, 14 psi) using a flame ionization detector (FID). FAME molecular species were identified based on the retention time of fatty acid methyl ester standards (GL Sciences). The fatty acid composition was determined from the area ratio. The determined composition is expressed as a mass ratio. The proportion of odd-chain fatty acids was calculated by multiplying the total fatty acid amount by the proportion (%) of odd-chain fatty acids (C13, C15, C17). The results are shown in Table 1 below.

[0066] [Table 1]

[0067] As shown in Table 1, the content of odd-chain fatty acids in the triglycerides obtained in Production Example 1 was 68.3% by mass. Furthermore, it was found that the fatty acids mainly consisted of triglycerides composed of pentadecanoic acid residues (C15) and palmitic acid residues (C16).

[0068] (Mass spectrometry of pentadecanoate triglyceride) The lipids containing pentadecanoic acid triglyceride obtained in Production Example 1 were analyzed by mass spectrometry using a Thermo Fischer Orbitrap Exactive Plus mass spectrometer (AMR DART ion source). As a result, from the fragment composition of the main mass spectral peaks, it was found that the pentadecanoic acid triglyceride obtained in Production Example 1 is a triglyceride mixture mainly consisting of triglycerides formed only from pentadecanoic acid residues (C15) and triglycerides containing two units of pentadecanoic acid residues (C15) and one unit of palmitic acid residue (C16).

[0069] (Example 1) Investigation of the effect of PdATG on suppressing cell death by reducing endoplasmic reticulum stress. To investigate the effects of PdATG, a solution of pentadecanoic acid triglyceride obtained in Production Example 1 dissolved in ethanol was used as the test drug, and the following experiment was conducted. • Test method Mouse hippocampal neurons (HT22: hippocampal-derived cell line) were cultured in 4 × 10 cm culture dishes. 5 Two plates were seeded at a density of cells / plate and incubated overnight in a 37°C, 5% CO2 incubator. Subsequently, the cells were divided into two groups: one treated with the test drug (50 μg / mL) and the other with a solvent (ethanol). The culture medium was treated with either the test drug or the solvent, and incubated for 72 hours (3 days) in a 37°C, 5% CO2 incubator. After confirming that both groups were conflected to over 90% (confirming that the drug treatment did not affect cell division), the cells were newly seeded into 96-well plates at a density of 4000 cells / well and incubated in a 37°C, 5% CO2 incubator.

[0070] After 24 hours of incubation, tunicamycin (Sigma), an endoplasmic reticulum stress inducer, was added to the culture medium at concentrations of 0.1, 1, 5, 10, 25, or 50 μg / mL, and the cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. Subsequently, cell viability was measured using the MTT method. The viability of cells in the solvent-treated group (those not treated with tunicamycin) was set as 100%, and the cell viability in the other wells was calculated accordingly. The results are shown in Table 2 and Figure 1. The MTT method is a colorimetric detection method that analyzes the number of viable cells by adding MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide) during cell culture and measuring the amount of MTT that is degraded. Specifically, when cells grown on a culture dish are incubated in an MTT solution, water-insoluble formazan dyes are produced, mainly depending on the enzymatic activity of mitochondria. After solubilizing the formazan dye, measurements are taken using a spectrophotometer. Since the obtained absorbance is proportional to the number of living cells, the degree of cell proliferation can be compared relatively.

[0071] [Table 2]

[0072] The results in Table 2 and Figure 1 show that cell death induced by endoplasmic reticulum stress by tunicamycin increased in a concentration-dependent manner in both cells prepared from culture dishes treated with the test drug and cells prepared from solvent-treated culture dishes. However, compared to cells prepared from solvent-treated culture dishes, the increase in cell death (decrease in survival rate) caused by low-concentration tunicamycin treatment was significantly suppressed in cells prepared from culture dishes treated with the test drug (a significant difference was observed with Student's t-test, p<0.01, when 0.1 μg / mL of tunicamycin was added), indicating that pretreatment with the test drug provides resistance to cell death induced by endoplasmic reticulum stress.

[0073] From these results, it has become clear that the present invention makes it possible to easily prevent and control cell death caused by endoplasmic reticulum stress.

[0074] (Example 2) Pancreatic cells in an endoplasmic reticulum stress state can be created by treating them with tunicamycin. The expression level of the transcription factor XBP1 expressed in these pancreatic cells was measured by real-time PCR after treatment with the pentadecanoic acid triglyceride (PdATG) mixture obtained in Production Example 1. Expression levels are expressed as relative levels, with the expression level in normal cells set to 100.

[0075] [Table 3]

[0076] The results in Table 2 confirm that the expression level of XBP-1, which was elevated by the endoplasmic reticulum stress response, approached normal levels upon the addition of PdATG. This phenomenon is thought to be effective in various cells, and PdATG is expected to improve diseases and cell damage caused by endoplasmic reticulum stress. [Industrial applicability]

[0077] The PdATG according to the present invention can be used to prevent, slow the progression of, and improve forgetfulness, decreased comprehension and judgment, memory impairment, disorientation, executive function disorders, aphasia, apraxia, agnosia, and dementia, and provides an endoplasmic reticulum stress preventive and inhibitory agent with few or no side effects. It is useful as a health food and pharmaceutical for preventing and reducing neuronal cell death, preventing the onset of dementia, slowing the progression of symptoms, and alleviating and improving symptoms.

Claims

[Claim 1] Formula (I) below: 【Chemistry 1】 (In the formula, R １ , R ２ and R ３ Each of these is a saturated fatty acid residue, at least one of which is a pentadecanoic acid residue. A preventive and ameliorative agent for neurodegenerative diseases containing a triglyceride represented by () as an active ingredient.

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