Endoplasmic reticulum stress inhibitor, and preventive and improvement agent for chronic kidney disease, and food

Abstract

To provide endoplasmic reticulum stress inhibitors that are safe for long-term consumption and applicable as a food, beverage, or medication for prevention, symptom mitigation, or improvement, and also to provide preventive and improvement agents for diseases manifested through podocyte death.SOLUTION: A composition contains a triglyceride represented by formula (I) (where R1, R2 and R3 each represent a saturated fatty acid residue, where at least one thereof is a pentadecanoic acid residue) as an active ingredient.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an endoplasmic reticulum stress inhibitor for improving endoplasmic reticulum stress conditions.Furthermore, the present invention relates to an agent for preventing, improving, or treating a disease whose cause is associated with endoplasmic reticulum stress, in particular, an agent for preventing and improving chronic kidney disease, and a food containing such an agent. [Background technology]

[0002] ER stress is a state in which cells are exposed to various internal or external environmental changes, and the amount of protein increases in the ER lumen due to abnormal expression of the protein synthesis system, causing proteins that cannot be eliminated normally to accumulate, and proteins that cannot be folded normally and accumulate as defective proteins. Factors that cause ER stress include nutrient starvation, disturbance 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 the protein folding capacity to maintain homeostasis. When the accumulation of defective proteins is relatively minor, the ER stress response, which is a response mechanism for eliminating defective proteins, is performed. However, when the stress state is severe or continues for a long period of time, denatured proteins accumulate in the ER. This has a negative effect on the cells. In order to avoid damage caused by ER stress and maintain homeostasis, cell death (apoptosis) is induced in tissues and organs throughout the body.

[0003] Chronic kidney disease (hereinafter sometimes abbreviated as "CKD") is a chronic progressive disease with a relatively high prevalence, and refers to a condition in which either or both of the initial signs suggesting decreased renal function or renal dysfunction persist for more than three months. The number of Japanese adults with CKD is said to be as high as approximately 13.3 million (Non-Patent Document 1). Furthermore, CKD increases with age, and the prevalence rate of CKD among the elderly exceeds approximately 30%, so it is attracting attention as a new national disease (Non-Patent Document 1). As the stage of CKD progresses, treatment such as dialysis may become necessary, and it is also important to take measures to prevent the disease from worsening before the introduction of dialysis. It is known that endoplasmic reticulum stress is involved in the development of CKD. To date, dietary therapy such as salt restriction, protein restriction, and potassium restriction has been implemented as a treatment adjunct to prevent the progression of CKD through food. It has been reported that lactoferrin, a type of milk protein, and its hydrolysate have a renal function protecting effect (see Patent Document 1), and that D-serine, a type of amino acid (see Patent Document 2), D-alanine (see Patent Document 3), and lipids derived from herbs (see Patent Document 4) can improve CKD, but their effects are unknown and cannot be considered sufficient.

[0004] In addition, drugs to treat the cause include corticosteroids used when there is inflammation in the kidneys due to chronic glomerulonephritis, immunosuppressants used when abnormal deduction is thought to be the cause, and antiplatelet drugs and anticoagulants that improve blood flow and reduce the burden on the kidneys (see Non-Patent Document 2). However, although these drugs provide a temporary therapeutic effect, it is difficult to use them over the long term due to side effects, and the current situation is that they cannot be said to be sufficient.

[0005] Conventionally, compositions containing hesperidin as an active ingredient have been proposed as compositions for suppressing optic nerve cell death in glaucoma and for preventing and treating glaucoma (Patent Documents 1 and 2). In addition, a composition containing lactic acid bacteria, such as Lactobacillus paracasei KW3110 strain, has been proposed as a composition for preventing visual impairment caused by exposure to light such as blue light (Patent Document 3). It has been claimed that the ingestion of Lactobacillus paracasei KW3110 strain acts antagonistically against retinal cell death caused by blue light irradiation, thereby maintaining a significant retinal thickness and reducing eye fatigue, but the effect is not sufficient, and the mechanism of action of suppressing cell death has not been particularly clarified. In addition, it has been previously disclosed that various substances such as tauroursodeoxycholic acid (TUDCA), blueberry extract, and anthocyanin are effective against ocular tissue disorders, but the effect is not fully satisfactory. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Japanese Society of Nephrology (ed.): CKD Treatment Guide 2012, Tokyo Medical Press (2012) [Non-Patent Document 2] Foley RN, Parfrey PS, Sarnak MJ. Epidemiology of cardiovascular disease in chronic renal disease. J Am Soc Nephrol 1998 Dec;9(12 Suppl):S16-23.) [Patent documents]

[0007] [Patent Document 1] Patent Publication No. 2022-18420 [Patent Document 2] JP 2019-214557 A [Patent Document 3] JP 2019-214558 A [Patent Document 3] Special Publication No. 2019-518034 Summary of the Invention [Problem to be solved by the invention]

[0008] Conventional foods and beverages are not known that suppress cell death, prevent or improve protein denaturation, or suppress defective proteins by suppressing endoplasmic reticulum stress reduction, and there are problems with side effects in kidney disease treatment drugs. For this reason, there is a demand for foods and beverages and medicines that can be taken for the long term to prevent, alleviate, and improve symptoms. The problem that the present invention aims to solve is to provide an endoplasmic reticulum stress inhibitor, an agent for preventing, preventing the progression of, and improving chronic kidney disease, and a food that can be used as foods and beverages and medicines suitable for such uses. [Means for solving the problem]

[0009] As a result of intensive research conducted by the inventors to solve the above-mentioned problems, they discovered that a triglyceride composed of saturated fatty acids mainly containing pentadecanoic acid (C15) (pentadecanoic acid triglyceride: hereinafter sometimes referred to as "PdATG") acts on renal glomerular cells to inhibit the accumulation of denatured proteins in the endoplasmic reticulum caused by damage due to stress, thereby reducing the resulting cell death (apoptosis), thereby effectively preventing or ameliorating chronic kidney disease, and thus completed the present invention.

[0010] In a first aspect of the present invention for solving the above problems, an endoplasmic reticulum stress inhibitor is a compound represented by the following formula (I):

[0011] [ka]

[0012] (In the formula, R 1 , R 2 and R 3 are saturated fatty acid residues, at least one of which is a pentadecanoic acid residue.) as an active ingredient.

[0013] In one embodiment of the endoplasmic reticulum stress inhibitor, the triglyceride of formula (I) is 1 and R 2 or R 1 and R 3 is preferably a pentadecanoic acid residue. 1 , R 2 and R 3 Any one of the above may be a tridecylic acid residue (C13), a myristic acid residue (C14), a palmitic acid residue (C16) or a margaric acid residue (C17).

[0014] In another preferred embodiment, R 1 , R 2 and R 3 wherein all of R are pentadecanoic acid residues; and 1 , R 2 and R 3 and a triglyceride of formula (I) in which any two of the above are pentadecanoic acid residues and the other is a myristic acid or palmitic acid residue.

[0015] In yet another preferred embodiment of the endoplasmic reticulum stress suppressant of the present invention, the triglyceride of formula (I) is derived from algae of the genus Aurantiochytrium or Schizochytrium, wherein R 1 , R 2 and R 3 and , each of which is a saturated fatty acid residue, at least one of which is a pentadecanoic acid residue. In addition, the mixture may contain unsaturated fatty acids derived from algae of the genus Aurantiochytrium or Schizochytrium.

[0016] In a second aspect of the present invention, there is provided an agent for preventing and improving glomerular dysfunction due to chronic kidney disease, which comprises the triglyceride represented by the above formula (I) as an active ingredient.

[0017] In a third aspect of the present invention, there is provided a food product containing the triglyceride represented by the above formula (I) as an active ingredient. This food product is preferably used for improving the daily life of people who are prone to swelling and fatigue, for example, as a health food, a functional food, or a food for specified health uses. Effect of the Invention

[0018] The endoplasmic reticulum stress inhibitor of the present invention can suppress endoplasmic reticulum stress in mammalian cells, particularly renal globules, and can provide foods, beverages, and medicines that can be taken over the long term for prevention, symptom relief, and improvement. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the details of an experiment conducted in one embodiment of the present invention to examine changes in glomerular area due to added drugs in a CKD animal model and a control group, in which (A) is a micrograph of a glomerular section after a test period has elapsed, and (B) is a graph showing the area of ​​the glomerulus. [Diagram 2] FIG. 1 shows the details of an experiment conducted to examine changes in renal function due to added drugs in a CKD animal model and a control group in one embodiment of the present invention, where (A) is a graph showing BUN values, and (B) is a graph showing Cre values. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Next, the present invention will be described in more detail based on the embodiments. Note that the embodiments described below do not limit the invention according to the claims, and all of the elements and combinations thereof described in each embodiment are not necessarily essential to the solution of the present invention.

[0021] (Active ingredient) As used herein, PdATG refers to at least one ester of pentadecanoic acid and glycerol, and is represented by the formula (I) R 1 , R 2 and R 3At least one, preferably any two, for example, R 1 and R 2 or R 1 and R 3 More preferably, R 1 , R 2 and R 3 The binding position of pentadecanoic acid to the glyceride may be any of positions 1 to 3.

[0022] [ka]

[0023] (In the formula, R 1 , R 2 and R 3 are each a saturated fatty acid residue, at least one of which is a pentadecanoic acid residue.

[0024] In the formula, R 1 , R 2 and R 3 Any one of the residues represented by the formula (I) 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 a double bond or a triple bond in the molecule, and C n H 2n+1 The saturated fatty acids are represented by the chemical formula COOH. They are linear or branched saturated fatty acids, such as capric acid (C10), lauric acid (C12), tridecylic 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), and branched saturated fatty acids such as 2-hexyldecanoic acid (C16), 13-methylpentadecanoic acid (C16), and 16-methylheptadecanoic acid (C18).

[0025] In a preferred embodiment, PdATG is R 1 , R 2 and R 3wherein all of R are pentadecanoic acid residues; and 1 , R 2 and R 3 and a triglyceride in which any two of the above are pentadecanoic acid residues and the remaining one is a myristic acid or palmitic acid residue. The content 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. Each of these is contained in an amount of 10% by mass or more, preferably 20% by mass or more, based on the total amount of triglycerides. Furthermore, it is more preferable that the mixture of triglycerides containing two or more pentadecanoic acid residues is contained in an amount of 50% by mass or more in the oil or fat.

[0026] In a further preferred embodiment, PdATG is represented by the following formula (II) or (III):

[0027] [ka]

[0028] (However, in the above formulas (II) and (III), R is a C14 to C16 saturated fatty acid.) It is more preferable that the mixture of triglycerides containing two or more pentadecanoic acid residues is contained in the fat or oil at 50% by mass or more, but even if the content of triglycerides containing two or more pentadecanoic acid residues is 50% by mass or less, the purpose can be achieved by increasing the intake amount. Therefore, the active ingredient of the present invention may be present in the form of a mixture of triglycerides containing two or more pentadecanoic acid residues, and the mixture itself can function as an active ingredient so long as it is contained at a purity of at least 1% by mass, preferably 50% by mass or more, more preferably 90% by mass or more, based on the total amount of triglycerides.

[0029] The active ingredient of the present invention may be present in a state of a mixture with a triglyceride other than the compound of formula (I), and as long as it is contained at a purity of at least 1% by mass, preferably 50% by mass or more, and more preferably 90% by mass or more, based on the total amount of triglycerides, the mixture itself can exhibit the function of an active ingredient.

[0030] The active ingredient of the present invention has at least one, and preferably two or more, odd-chain fatty acids, particularly pentadecanoic acid, in its molecule, and it is believed that ingesting this ingredient will suppress abnormal proteins that accumulate in the endoplasmic reticulum due to endoplasmic reticulum stress, as described below, reduce cell death, and restore normality.

[0031] (Action and effect) The active ingredient of the present invention has the effect of alleviating endoplasmic reticulum stress in various cells, thereby improving the condition of diseases caused by endoplasmic reticulum stress and poor physical condition before the onset of the disease. The kidney is said to be a "silent organ," and in many cases, no obvious subjective symptoms appear unless the condition worsens. Even if the glomerular filtration function is reduced, there may be almost no subjective symptoms. By the time the abnormality is obvious and symptoms are noticed, the glomerular filtration function may have decreased to nearly half of what it was in health. CKD is known to be a disease that causes various complications, and it has been shown that patients whose disease has progressed to a stage where they require dialysis have a significantly high risk of developing cardiovascular diseases such as heart failure and cerebrovascular disease, and that the relative risk is 10-30 compared to elderly people living in the community (Non-Patent Document 2). It has also been reported that patients on dialysis are at higher risk of physical and cognitive decline, and the impact on life prognosis and ADL / QOL decline has been pointed out. Early detection and prevention of progression of CKD in the elderly are important, and it is extremely important to establish a preventive method from a time well before the onset of this condition. In addition, it is important to prevent the progression of CKD by taking preventative measures before the onset of the disease, and at the same time, to start treatment at an early stage when the onset of the disease is suspected.

[0032] CKD can be detected by assessing the glomerular filtration function, which examines the amount of protein in urine and creatinine in blood. Creatinine is a waste product in the blood. Normally, it is filtered by the kidneys and most of it is excreted in urine, but when glomerular filtration function declines, it accumulates in the blood without being excreted in urine. This creatinine in the blood is called serum creatinine, and is an indicator used in clinical settings. A decline in renal function can also be detected by a test that examines the amount of nitrogen in the blood, which is blood urea nitrogen (BUN). Urea nitrogen is a waste product that is produced after protein is used. Normally, it is filtered by the glomeruli of the kidneys and excreted in urine, but when renal function declines, it cannot be completely filtered and accumulates in the blood, causing the urea nitrogen level in the blood to increase. These are the main ways to assess glomerular function and renal function.

[0033] (Kidney disease and endoplasmic reticulum stress) Endoplasmic reticulum stress progresses due to environmental factors such as aging, genetic factors, and lifestyle habits, and insufficient repair gradually leads to loss of homeostasis and cell death. When internal and external factors are combined and abnormally progress, it can be said to be an age-related disease, but each organ develops its own characteristic disease. It has become clear that glomerular epithelial cell damage plays an important role in the progression of glomerular damage in the kidney, regardless of the current disease, and it is being proposed that endoplasmic reticulum stress is involved (Yang Guochang, Special feature: Nephrotic syndrome: Elucidation of the mechanism of glomerular epithelial cell damage - from the perspective of endoplasmic reticulum stress - Journal of the Japanese Society of Nephrology 49(2), 72-76). It has become clear that medicines and food ingredients that can actually improve endoplasmic reticulum stress are essential for maintaining people's health.

[0034] (Method for producing triglyceride mixture) The triglyceride mixture, which is an active ingredient of the present invention, may be chemically synthesized or naturally occurring. If it is natural, its source is not particularly limited. Examples include lipids produced in the body of an organism, such as fats of livestock or poultry, oils and fats from seafood, 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 kingdom Stramenopile, etc.), green algae, diatoms, dinoflagellates, yeast, and fungi of the genera Mucor and Mortierella. Members of the microbial group Stramenopile include microalgae. Microalgae refer to organisms that perform oxygen-generating photosynthesis, excluding mosses, ferns, and seed plants, and have a cell size of 1 μm to 100 μm in diameter. Labyrinthula, a protist closely related to microalgae, is also included. Labyrinthulae are non-photosynthetic, heterotrophic marine eukaryotic microorganisms that are widely distributed, mainly in the subtropics and tropics. In general, Labyrinthulae are broadly divided into the Labyrinthulidae and Thraustochytriidae families, and include the genera Labyrinthula, Aurantiochytrium, Schizochytrium, Thraustochytrium, Aplanochytrium, oblongichytrium, Botryochytrium, and Japonochytrium.

[0035] As the Labyrinthulea to be cultured, the genus Aurantiochytrium, Schizochytrium, or Thraustochytrium is more preferable. These species have a relatively high ability to produce lipids and the like, and can produce hydrocarbons such as squalene, and are therefore suitable for use as food or as a raw material for biofuel.

[0036] The Labyrinthulea may be cultured by any of the culture methods such as batch culture, continuous culture, and fed-batch culture. The Labyrinthulea may be cultured by any suitable culture method such as shaking culture, aeration culture, aeration stirring culture, airlift culture, and stationary culture. Among these culture methods, aeration stirring culture or airlift culture is more preferable. As the culture device used for culturing Labyrinthulea, for example, a mechanical stirring type reactor, an airlift type reactor, a packed bed type reactor, a fluidized bed type reactor, etc. can be used. As the culture vessel, various vessels such as tanks, jar fermenters, flasks, dishes, culture bags, tubes, and test tubes can be used depending on the purpose of the culture, the culture volume, etc. The culture vessel may be made of suitable materials such as inorganic materials such as stainless steel and glass, and organic materials such as polystyrene, polyethylene terephthalate copolymer, and polypropylene.

[0037] Labyrinthulea can be cultured under appropriate temperature, pH, aeration, etc. The culture temperature is preferably from 5° C. to 40° C., more preferably from 10° C. to 35° C., and even more preferably from 10° C. to 30° C. The pH is preferably from 2 to 11, more preferably from 4 to 9, and even more preferably from 6 to 8.

[0038] The culture of Labyrinthules can be subcultured at appropriate intervals depending on the genus and species of Labyrinthules, medium composition, culture conditions, etc. For example, after the start of culture, Labyrinthules complete the logarithmic growth phase about 2 days later and enter the death phase about 7 days later. Therefore, the subculture of Labyrinthules is preferably performed at intervals of 1 day or more and 10 days or less, more preferably at intervals of 2 days or more and 7 days or less, and even more preferably at intervals of 2 days or more and 5 days or less. The culture time of Labyrinthules can be set as appropriate depending on the genus and species of Labyrinthules, medium composition, culture conditions, purpose of culture, etc. In particular, Aurantiochytrium algae of Labyrinthules algae are preferred because they are heterotrophic algae that live in brackish waters and have the characteristic of assimilating nutrients in water to produce lipids and accumulating them in their cells.

[0039] It is preferable to use a strain of Aurantiochytrium algae that has an excellent ability to produce the desired triglyceride. Such an algae strain may be one that has been collected and isolated from the wild, one that has been cloned through mutagenesis and screening, or one that has been established using recombinant gene technology. For example, Aurantiochytrium Sp.SA-96, NIES-3737, Aurantiochytrium NB6-3, or Aurantiochytrium mh1959 has 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) in the cells, and is therefore particularly preferable as a microorganism to be used for producing the pentadecanoic acid triglyceride of the present invention.

[0040] The cultivation of the Aurantiochytrium algae is carried out by a method established in the art. That is, normal maintenance cultivation is carried out by seeding the algae in a medium with appropriately prepared ingredients, and following a standard method. The medium for culturing the Aurantiochytrium algae essentially contains salt, a carbon source, and a nitrogen source. Generally, the 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 culturing microalgae.

[0041] Carbon sources include sugars such as glucose, fructose, sucrose, etc. These carbon sources are added at a concentration of, for example, 20 to 120 g per 1 liter of medium.

[0042] Aurantiochytrium algae are marine algae, and an appropriate amount of artificial seawater is added to the medium. Preferably, the artificial seawater is added so that the final salinity of the medium is about 10% (v / v) to about 100% (v / v) of seawater (salinity 3.4% (w / v)), for example, about 1.0 to 3.0% (w / v).

[0043] In general, various nitrogen sources may be added to the culture medium for microalgae, such as organic nitrogen such as sodium glutamate and urea, inorganic nitrogen such as ammonium acetate, ammonium sulfate, ammonium chloride, sodium nitrate, and ammonium nitrate, and biological digests such as yeast extract, corn steep liquor, polypeptone, peptone, and tryptone. In particular, cell extracts obtained by extracting liquid components from various animal cells are preferably used as nitrogen sources to be added to the medium used for culturing Aurantiochytrium algae. When cells must be mass-cultured on an industrial scale to obtain cultured cell products, the use of cell extracts, which are rich in nutrients such as cell-derived amino acids, nucleic acids, vitamins, and minerals and are available at low cost, is extremely advantageous.

[0044] However, as described above, when a medium prepared based on a cell extract is used, the proportion of odd-numbered fatty acids in the triglycerides produced by the cultured algae is significantly reduced, and therefore, when the target product of the present invention is efficiently produced, the cell extract cannot be used as a nitrogen source for the medium. Therefore, the present inventors have cultured Aurantiochytrium algae in an algal culture medium prepared by adding a cell extract treated with a strong acid, and have found that the production amount of odd-numbered fatty acids is dramatically increased compared to the case where a cell extract not treated with the strong acid is added, and have already reported a method for producing triglycerides containing odd-numbered fatty acids as a main component (JP Patent Publication No. 2017-063633).

[0045] Furthermore, in a preferred embodiment of the present invention, the basal medium for culturing Aurantiochytrium algae is a medium containing 2% or more glucose, 0.5-4% sodium glutamate, 0.1-2% yeast extract, 1-3.3% sea salt, and 2-20% whey (animal or vegetable), to which 10-50 mM valine and 10-50 mM sodium propionate are added. The animal or vegetable whey is preferably tofu whey (soybean whey). To this basal medium, 2% or more of a culture solution of Aurantiochytrium pre-cultured at 20-30°C for 72 hours is added. Air is circulated through this Aurantiochytrium-added culture solution and the mixture is gently stirred. The culture is carried out for 48 to 200 hours at 20 to 30° C. and pH maintained at 5.0 to 8.5 (pH is adjusted using 1.0 M NaOH solution). After the culture, the Aurantiochytrium cells that produced pentadecanoic acid triglyceride can be collected by centrifugation (see WO2020 / 054804 pamphlet).

[0046] The pellets collected by centrifugation or filtration from the culture solution obtained by the above method are dried by freeze-drying or drying by heating. Alternatively, the culture medium in which the cultured algal cells are suspended may be used as is for the triglyceride extraction step. Extraction may be performed multiple times using different organic solvents. As the organic solvent, a mixture of a polar solvent and a weakly polar solvent, such as a mixture of n-hexane and ethanol, a mixture of chloroform and methanol, or a mixture of ethanol and diethyl ether, may be used. The obtained extract is purified by a method known to those skilled in the art.

[0047] A method for separating triglycerides may employ a fractionation method 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 a solvent, melting point, specific gravity, and molecular weight, and preferably, column chromatography technology is used. The conditions for the triglyceride separation means can be set by those skilled in the art through ordinary condition studies depending on the composition of the triglyceride mixture and the type of triglyceride to be fractionated.

[0048] The algae of the genus Schizochytrium and Aurantiochytrium can synthesize and accumulate both odd-chain fatty acid triglycerides and highly unsaturated fatty acid triglycerides within the cells. Therefore, ethanol, hexane, or ethyl acetate is added to the obtained algal cells to extract lipids, and the solvent is then distilled off to obtain algal lipids. Pentadecanoic acid triglyceride can be precipitated by leaving this lipid at rest at 5°C. The composition of the purified pentadecanoic acid triglyceride "PdATG" can be analyzed by HPLC-MS, HPLC, gas chromatography, etc.

[0049] Aurantiochytrium algae can synthesize and accumulate both odd-chain fatty acid triglycerides and highly unsaturated fatty acid triglycerides within the cells. Therefore, hexane or ethyl acetate is added to the obtained Aurantiochytrium cells to extract lipids, and then hydrogen peroxide is added to the lipid solution or ozone is passed through to oxidize and decompose the unsaturated fatty acids. After the reaction is completed, the oxidized products 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.

[0050] (Endoplasmic reticulum stress inhibitor) The endoplasmic reticulum stress inhibitor according to the first aspect of the present invention includes a preparation that inhibits glomerular epithelial cell death caused by abnormal protein accumulation in the endoplasmic reticulum. Here, glomerular epithelial cell death includes necrosis and apoptosis of glomerular epithelial cells. It includes alleviating, reducing, or eliminating cell death, inhibiting the progression of cell death, and preventing or preventing it. It can be used to prevent, prevent the progression of, and / or improve disorders (diseases and age-related changes) that are manifested by glomerular epithelial cell death caused by endoplasmic reticulum stress, such as kidney diseases including CKD, tubulointerstitial nephritis, hyperuricemia, gout, nephrolithiasis, foot gangrene, and other diseases related to blood vessels throughout the body (cardiovascular diseases), and is useful as a pharmaceutical for this purpose.

[0051] (Drugs) In addition, the term "pharmaceutical product" means a therapeutic drug for preventing, preventing the progression of, and / or ameliorating symptoms in patients with disorders manifested by glomerular epithelial cell death as described above and related disorders by suppressing endoplasmic reticulum stress.

[0052] The composition according to the present invention and the medicine made from the same are effective not only for humans but also for mammals including livestock such as cows, horses, pigs and goats, and pets such as dogs and cats.

[0053] The composition according to the present invention or the pharmaceutical product thereof may contain only the compound of formula (I) as an active ingredient, or may contain other ingredients as long as they do not inhibit the glomerular epithelial cell injury inhibitory effect. The other ingredients may be, for example, therapeutic or preventive drugs that have been used in the past. Therefore, in a further aspect, the endoplasmic reticulum stress inhibitor of this embodiment provides a pharmaceutical composition for preventing and improving glomerular epithelial cell injury diseases.

[0054] The composition according to the present invention or the pharmaceutical product thereof can be administered orally, and can be prepared in the form of granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, suspensions, etc., as dosage forms suitable for oral administration. These preparations can be formulated using pharma- ceutical acceptable carriers by methods commonly used in the art. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, flavors, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc.

[0055] Although not particularly limited, more specifically, for example, when a pharmaceutical product is manufactured by blending pentadecanoic acid triglyceride represented by the above formula (I), any auxiliary agent can be added, for example, sugars such as dextrin, starch, etc.; proteins such as gelatin, soy protein, corn protein, etc.; amino acids such as alanine, glutamine, isoleucine, etc.; polysaccharides such as cellulose, gum arabic, etc.; fats and oils such as soybean oil, medium-chain fatty acid triglyceride, etc., to formulate the pharmaceutical product into any dosage form.

[0056] The amount of pentadecanoic acid triglyceride represented by the above formula (I) in the pharmaceutical of the present invention is not particularly limited, but it is preferable to adjust the amount of pentadecanoic acid triglyceride consumed per day by an adult, which is a concentration that shows efficacy, to about 5 to 1000 mg per day.

[0057] Furthermore, the composition according to the present invention or the pharmaceutical product thereof is not limited to an oral administration form, and may also be a parenteral administration form, for example, an injection, an infusion, etc. In this case, too, it can be formulated using pharma- ceutical agents, carriers, etc., according to a method commonly used in this field.

[0058] (food) The food according to the third aspect of the present invention contains the pentadecanoic acid triglyceride represented by the above formula (I) as an active ingredient, and can be taken as a preventive food and drink for a long period of time before the onset of CKD, and is useful as a health food for that purpose. Pentadecanoic acid, which constitutes PdATG, is reported to be contained in small amounts in edible parts of meat of cows, pigs, chickens, sheep, etc., fish living in rivers and seas, mushrooms, etc., and PdATG is also contained in extremely small amounts, and it is presumed to be highly safe based on long-term dietary experience.

[0059] Therefore, the food of this embodiment is useful as a health food to be taken for health promotion. Here, "health food" means food and drink intended for use in daily life to promote health and to prevent, prevent the progression of, alleviate or improve symptoms such as swelling due to aging, swelling of fingers, face, shins and insteps, changes in urine, difficulty in urinating, frequent urination, abnormal foaming of urine, urine that is brownish in color due to blood, heavy body, lethargy, fatigue, cystitis, etc., and refers to "health food" in a broad sense including functional labeling foods, nutrient functional foods, or foods for specified health uses under the "health food system" that meet the standards for safety and efficacy set by the government.

[0060] When producing a food by blending the pentadecanoic acid triglyceride represented by the above formula (I), any auxiliary agent can be added, for example, sugars such as dextrin, starch, etc.; proteins such as gelatin, soy protein, corn protein, etc.; amino acids such as alanine, glutamine, isoleucine, etc.; polysaccharides such as cellulose, gum arabic, etc.; fats and oils such as soybean oil, medium-chain fatty acid triglyceride, etc., and the like, to formulate the food into any dosage form.

[0061] In addition, the amount of pentadecanoic acid triglyceride represented by the above formula (I) in the food of the present invention is not particularly limited, but it is preferable to adjust the daily intake of pentadecanoic acid triglyceride per adult to about 1 to 100 mg per day, taking into consideration the general intake of the food to which it is added.

[0062] Specific examples of the above foods include 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 skins, shumai skins, Chinese noodles, and instant noodles; sweets such as candy, candy, gum, chocolate, snacks, biscuits, jellies, jams, creams, and baked goods; processed seafood and livestock foods such as kamaboko, ham, and sausages; dairy products such as processed milk and fermented milk; oils and fats and oil-processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings; seasonings such as sauces and sauces; health and nutritional supplements in various forms such as tablets and granules; and other examples include soups, stews, salads, side dishes, and pickles.

[0063] The food of 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.

[0064] The content concentration of pentadecanoic acid triglyceride in the food according to the present invention is about 0.00001 to 100% by mass (hereinafter expressed in %), preferably about 0.0005 to 50%, in terms of solid content, to ensure ease of use and good effects.

[0065] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the following examples, the unit % of the numerical values ​​showing the amount of each component added means % by mass. EXAMPLES

[0066] (Production Example 1) Production of pentadecanoic acid triglyceride using Aurantiochytrium Aurantichytrium mh1959 strain (purchased from Professor Masahiro Hayashi, Faculty of Agriculture, University of Miyazaki, National University Corporation) was precultured at 25°C for 72 hours using a medium containing 3.6% glucose, 0.5% sodium glutamate, 0.2% yeast extract, 1% sea salt, and 10% whey. This was added to the following basic medium at 2%, aerated, and gently stirred. 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% sodium glutamate, 0.2% yeast extract, 1% sea salt, and 10% whey. The culture was maintained at 25°C and pH was kept at 7.40 to 7.75 (pH was adjusted using 1.0 M NaOH solution) for 72 to 96 hours.

[0067] After cultivation, the mixture was centrifuged at 3000 rpm for 15 minutes to recover approximately 20 g of algae. Hexane or ethyl acetate was added to the 20 g of the resulting Aurantichytrium algae to extract lipids. Hydrogen peroxide was added to the extracted lipid solution (water was added as necessary), and ozone was aerated at room temperature. After the reaction was completed, oxides were removed using sodium bicarbonate and sodium carbonate or ion exchange resin, and 2 g of a mixture of pentadecanoic acid triglycerides was obtained, which precipitated as the temperature decreased.

[0068] (Composition analysis of pentadecanoic acid triglyceride) 0.50 mL of 14% BF3-methanol and 0.25 mL of methyl acetate were added to the lipid containing pentadecanoic acid triglyceride obtained in Production Example 1, and the mixture was heated at 70°C for 30 minutes to obtain fatty acid methyl esters (FAMEs). Exactly 1.0 mL of n-hexane and 5 mL of physiological saline were added to the reaction solution and mixed vigorously. The mixture was centrifuged at 2800 rpm for 10 minutes, and the n-hexane layer was used as a sample for gas chromatography.

[0069] The above samples were analyzed using a Shimadzu gas chromatograph GC-2025. The analysis conditions were as follows: Agilent J&W GC column DB-23 (30m x 0.25mm), 1μL of sample was injected, and detection was performed with a FID (flame ionization detector) using carrier gas (He, 14psi). The molecular species of FAME was identified based on the retention time of a fatty acid methyl ester standard (GL Science). The fatty acid composition was calculated from the area ratio. The calculated composition is a mass ratio. The proportion of odd-chain fatty acids was calculated by multiplying the total amount of fatty acids by the proportion (%) of odd-chain fatty acids (C13, C15, C17). The results are shown in Table 1 below.

[0070] [Table 1]

[0071] From the results shown in Table 1, the content of odd-chain fatty acids in the triglyceride obtained in Production Example 1 was 68.3% by mass. In addition, it was found that the triglyceride was mainly composed of pentadecanoic acid residues (C15) and palmitic acid residues (C16) as fatty acids.

[0072] (Mass spectrometry of pentadecanoic acid triglyceride) The lipids containing the pentadecanoic acid triglyceride obtained in Production Example 1 were analyzed by mass spectrometry using a Thermo Fischer Orbitrap mass spectrometer Exactive Plus (DART ion source manufactured by AMR). As a result, from the fragment composition of the main mass spectrum peak, it was found that the pentadecanoic acid triglyceride obtained in Production Example 1 was a triglyceride mixture mainly containing a triglyceride formed only of pentadecanoic acid residue (C15) and a triglyceride containing two units of pentadecanoic acid residue (C15) and one unit of palmitic acid residue (C16).

[0073] (Example 1) Examination of glomerular function in CKD animal models using PdATG To examine the effect of PdATG, the following experiment was conducted using a solution prepared by dissolving the triglyceride of pentadecanoic acid obtained in Production Example 1 in ethanol as the test drug.

[0074] · Test method For the CKD animal model, C57BL / 6JJmsSlc male mice were used, and 5 / 6 nephrectomized mice (Nx mice) were prepared by removing 2 / 3 of the left kidney at 8 weeks of age and the entire right kidney at 9 weeks of age. Sham-operated mice (Sham group) only underwent skin and muscle incision. Renal function was determined from serum urea nitrogen (BUN) and serum creatinine (Cre) values. Hematoxylin and eosin staining was used for histological verification of renal glomeruli.

[0075] · Results Triglyceride of pentadecanoic acid (0.2 g / L) was suspended in drinking water for animals and orally administered to Nx mice for 8 weeks. The changes in body weight gain, water intake, and food intake over time were observed, but no changes due to pentadecyl administration were found. Also, a log-rank test was performed on the survival rate, but no significant difference was found.

[0076] · Effect of chronic administration of PdATG on renal function decline <Effect on glomerular area> As shown in Figures 1(A) and (B), the PdATG-administered group showed a tendency to decrease the area per glomerulus, which increased due to nephrectomy, compared with the water-administered group.

[0077] <Effect on CRE and BUN> As shown in Figures 2(A) and (B), the BUN and Cre values indicating renal function in Nx mice were significantly increased compared with those of sham-operated mice, but triglyceride of pentadecanoic acid significantly suppressed this increase. On the other hand, the administration of triglyceride of pentadecanoic acid had no effect on the increase in glomerular area occurring in Nx mice.

[0078] These results suggest that pentadecanoic acid triglyceride has the effect of preventing the worsening of uremia by partially suppressing the progression of renal dysfunction in CKD model mice. Therefore, it was confirmed that pentadecanoic acid triglyceride is useful for the prevention and treatment of kidney diseases associated with decreased renal function. [Industrial Applicability]

[0079] The PdATG of the present invention can be used to prevent, inhibit the progression of, and / or ameliorate disorders (diseases and age-related changes) that are manifested by glomerular epithelial cell death due to endoplasmic reticulum stress, such as tubulointerstitial nephritis, hyperuricemia, gout, nephrolithiasis, foot gangrene, and other systemic vascular diseases (cardiovascular diseases), and provides an endoplasmic reticulum stress preventive and inhibitor that has no or minimal side effects.

Claims

1. The following formula (I): 【Chemistry 1】 (In the formula, R １ , R ２ and R ３ are saturated fatty acid residues, at least one of which is a pentadecanoic acid residue.

2. R in formula (1) １ and R ２ Or R １ and R ３ The endoplasmic reticulum stress inhibitor according to claim 1, which contains as an active ingredient a triglyceride in which is a pentadecanoic acid residue.

3. R in formula (1) １ , R ２ and R ３ 3. An endoplasmic reticulum stress inhibitor as described in claim 1 or 2, comprising as an active ingredient a triglyceride in which any one of the above is a tridecylic acid residue (C13), a myristic acid residue (C14), a palmitic acid residue (C16) or a margaric acid residue (C17).

4. R in formula (1) １ , R ２ and R ３ in which all of R in formula (1) are pentadecanoic acid residues, １ , R ２ and R ３ and a triglyceride in which any two of the above are pentadecanoic acid residues and the remaining one is a myristic acid residue or a palmitic acid residue.

5. The endoplasmic reticulum stress inhibitor according to claim 1 or 2, wherein the triglyceride of formula (I) is derived from algae of the genus Aurantiochytrium or Schizochytrium.

6. 2. A preventive and ameliorative agent for diseases manifested by glomerular epithelial cell death, comprising the triglyceride according to claim 1 as an active ingredient.

7. 2. An agent for preventing, inhibiting the progression of, or ameliorating tubulointerstitial nephritis, hyperuricemia, gout, nephrolithiasis, or foot gangrene, comprising the triglyceride according to claim 1 as an active ingredient.

8. A food product comprising the triglyceride according to claim 1 as an active ingredient.

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