WO2024071138A1 - Mitochondrial function improving composition - Google Patents

Abstract

The purpose of the present invention is to provide a novel mitochondrial function improving composition. The present invention provides a mitochondrial function improving composition including, as an active ingredient, a fat that contains a β position-palmitic acid. The present invention also provides a mitochondrial biosynthesis-related gene expression enhancing composition including, as an active ingredient, a fat that contains a β position-palmitic acid. The present invention brings about the effect of improving a mitochondrial function and has an advantage of being highly safe in that even continuous ingestion over a long period of time does not cause a concern for a side effect.

Description

Composition for improving mitochondrial function REFERENCE TO RELATED APPLICATIONS

This application benefits from the priority of an earlier Japanese application, Patent Application No. 2022-153569 (filing date: September 27, 2022), the entire disclosure of which is incorporated herein by reference.

The present invention relates to a composition for improving mitochondrial function. The present invention also relates to a composition for enhancing expression of mitochondrial biogenesis-related genes.

Mitochondria are intracellular organelles that not only produce ATP, which is essential for intracellular energy metabolism, through aerobic respiration, but also play a wide range of metabolic roles, such as regulating oxidative stress, regulating intracellular ^Ca2+ concentration, controlling cell death, synthesizing phospholipids, synthesizing heme, and synthesizing steroids.

Fetal babies are in a relatively hypoxic state and generate energy through anaerobic glycolysis, but immediately after birth, babies are exposed to oxygen in the air and switch to aerobic respiration through mitochondria. Aerobic respiration through mitochondria is more efficient at producing energy than anaerobic respiration through glycolysis. In glycolysis, two molecules of ATP are produced per glucose molecule, whereas in aerobic respiration through mitochondria, 36 molecules of ATP are produced per glucose molecule. Therefore, it is thought that it is important for newborns and infants to quickly adapt to aerobic mitochondrial respiration in order to provide the energy required for rapid growth.

On the other hand, mitochondrial dysfunction is more likely to occur with age. This leads to a decrease in ATP production by mitochondria and an increase in the production of reactive oxygen species (ROS). Furthermore, these mitochondrial dysfunctions cause intracellular metabolic disorders and the accumulation of mitochondrial DNA mutations, leading to a vicious cycle, and mitochondrial dysfunction is thought to be a factor in promoting aging. Mitochondrial dysfunction has also been reported to be associated with various diseases such as cancer, neurodegenerative diseases, arteriosclerosis, and diabetes (Non-Patent Documents 1, 2, and 3).

The present invention aims to provide a novel composition for improving mitochondrial function. The present invention also aims to provide a novel composition for enhancing the expression of mitochondrial biogenesis-related genes.

The present inventors have now found that the addition of β-palmitic acid to cultured human cells significantly increases ATP production compared to when β-palmitic acid is not added. The present inventors have also found that the addition of β-palmitic acid to cultured human cells significantly increases Sirt3 gene expression levels, Nfe2l2 gene expression levels, and Tfam gene expression levels compared to when β-palmitic acid is not added or when β-oleic acid is added, and that there is a tendency for Nrf1 gene expression levels and Pgc-1α gene expression levels to increase compared to when β-palmitic acid is not added. The present inventors further found that the addition of β-palmitic acid to cultured human cells significantly increases the expression level of the Sirt3 gene compared to the addition of β-myristic acid or β-stearic acid, that the expression levels of the Nfe2l2 gene and the Tfam gene significantly increase compared to the addition of β-myristic acid, palmitic acid, or β-stearic acid, and that the expression level of the Nrf1 gene significantly increases compared to the addition of β-myristic acid. The present invention is based on these findings.

According to the present invention, the following inventions are provided.
[1] A composition for improving mitochondrial function or an agent for improving mitochondrial function, comprising an oil or fat containing β-palmitic acid as an active ingredient.
[2] A composition for enhancing expression of a mitochondrial biogenesis-related gene or an agent for improving mitochondrial function, comprising an oil or fat containing β-palmitic acid as an active ingredient.
[3] The composition or agent described in [2] above, wherein the gene is one or more genes selected from the group consisting of Sirt3 gene, Nfe2l2 gene, and Tfam gene.
[4] The composition or agent according to any one of the above [1] to [3] for use in anti-aging.
[5] The composition or agent according to any one of [1] to [4] above, for use in promoting ATP production.
[6] The composition or agent according to any one of [1] to [5] above, which is a food composition.
[7] A method for improving mitochondrial function or enhancing expression of a mitochondrial biogenesis-related gene, comprising having a subject in need thereof ingest an oil or fat containing an effective amount of β-palmitic acid or a composition containing the same.
[8] A method for maintaining, improving, treating or preventing a disease or condition that can be maintained, improved, treated or prevented by improving mitochondrial function, comprising having a subject in need thereof ingest an effective amount of an oil or fat containing β-palmitic acid or a composition containing the same.
[9] Use of an oil or fat containing β-palmitic acid as an agent for improving mitochondrial function or an agent for enhancing mitochondrial biogenesis-related gene expression for the manufacture of an agent for improving mitochondrial function or an agent for enhancing mitochondrial biogenesis-related gene expression, or in a method for improving mitochondrial function or a method for enhancing mitochondrial biogenesis-related gene expression.
[10] Use of an oil or fat containing β-palmitic acid for the manufacture of an agent for maintaining, improving, treating, or preventing a disease or condition that can be maintained, improved, treated, or prevented by improving mitochondrial function, as an agent for maintaining, improving, treating, or preventing a disease or condition that can be maintained, improved, treated, or prevented by improving mitochondrial function, or in a method for maintaining, improving, treating, or preventing a disease or condition that can be maintained, improved, treated, or prevented by improving mitochondrial function.

In this specification, the compositions [1] to [6] above may be referred to as the "compositions of the present invention." Furthermore, in this specification, the agents [1] to [6] above may be referred to as the "agents of the present invention."

The active ingredient of the composition and agent of the present invention is fat or oil that has been used as a food ingredient for many years. Therefore, the composition and agent of the present invention not only has the effect of improving mitochondrial function, but also has the advantage of being highly safe, with no risk of side effects even when taken continuously over a long period of time.

Figure 1 shows the amount of ATP produced in the control group (no test substance added) and each test group with the addition of palmitic acid, 2-monopalmitin, and 2-monoolein in a test using HepG2 cells (n=3 per group). The measured values are shown as the mean ± standard deviation. ^* indicates a significant difference compared to the control group (p<0.05) (Tukey-Kramer test). Figure 2 shows the expression levels of Sirt3 gene (Figure 2A), Nfe2l2 gene (Figure 2B), Tfam gene (Figure 2C), Nrf1 gene (Figure 2D) and Pgc-1α gene (Figure 2E) in the control group (no test substance added) and each test group with the addition of 2-monopalmitin and 2-monoolein in a test using HepG2 cells (n=3 per group). Measurement values are shown as mean ± standard deviation. Lowercase letters indicate significant differences between different signs (p<0.05) (Tukey-Kramer test). Figure 3 shows the expression levels of Sirt3 gene (Figure 3A), Nfe2l2 gene (Figure 3B), Tfam gene (Figure 3C), Nrf1 gene (Figure 3D) and Pgc-1α gene (Figure 3E) in the control group (no test substance added) and each test group with the addition of 2-monomyristin, palmitic acid, 2-monopalmitin and 2-monostearin in a test using HepG2 cells (n=3 per group). Measurement values are shown as mean ± standard deviation. Lowercase letters indicate significant differences between different signs (p<0.05) (Tukey-Kramer test).

Description of the Invention

The composition and agent of the present invention contain an oil containing β-palmitic acid as an active ingredient. In the present invention, "β-palmitic acid" refers to a substance in which palmitic acid is bound to the β-position of a glyceride, and refers to all compounds that are identical in chemical formula. In other words, β-palmitic acid is acylglycerol with palmitic acid bound to at least the β-position, and includes acylglycerol (monoacylglycerol) with palmitic acid bound to the β-position and an optional fatty acid bound to either of the two α-positions, and acylglycerol (triacylglycerol) with palmitic acid bound to the β-position and an optional fatty acid bound to each of the two α-positions. In addition, β-palmitic acid may be a pure product or a mixture with other substances. For example, a raw material in which β-palmitic acid has been measured by a known measurement method and its presence has been confirmed may be used as is. A typical example of a raw material (mixture) rich in β-palmitic acid is lard, which can also be used in the composition and agent of the present invention. On the other hand, lard has a unique "animal odor," and in order to eliminate this odor, it is known that vegetable oils rich in palmitic acid are chemically or enzymatically modified to obtain raw materials rich in β-palmitic acid (JP-T-8-509620A, JP-T-8-509621A, JP-A-6-70786A). Alternatively, a commercially available product of β-palmitic acid (e.g., Betapol (Bunge, Roders, Krokran)) can be purchased and mixed with other raw materials as appropriate for use in the composition and agent of the present invention.

In fats and oils containing β-palmitic acid, which is the active ingredient of the present invention, the lower limit of the ratio (mass ratio) of palmitic acid bound to the β-position (sn-2 position) of the glyceride relative to the total amount of fatty acids bound to the β-position can be set to 10%, preferably 50%, and more preferably 74%, from the viewpoint of better exerting the effects of the present invention. The upper limit of the above ratio can be set to a range not exceeding 90%, preferably 85%, and more preferably 78%, from the viewpoint of stable production. These lower and upper limits can be combined in any desired manner, and the above ratio range can be, for example, 50% or more but less than 90%, 70-85%, or 74-78%.

In fats and oils containing β-palmitic acid, which is the active ingredient of the present invention, the lower limit of the ratio of palmitic acid at the β-position of glycerides to the total palmitic acid (bonding ratio of palmitic acid to the β-position, mass ratio) can be 50%, preferably 54%, more preferably 65%, even more preferably 67%, and particularly preferably 68%, from the viewpoint of better exerting the effects of the present invention. The upper limit of the above ratio can be in a range not exceeding 98%, preferably 75%, more preferably 74%, even more preferably 73%, and particularly preferably 72%, from the viewpoint of stable production. These lower and upper limits can be combined in any desired manner, and the above ratio range can be, for example, 50% or more but less than 98%, 54-75%, 65-74%, 67-73%, or 68-72%.

The compositions and agents of the present invention may contain fats and oils containing β-palmitic acid alone, or may contain fats and oils containing β-palmitic acid in a mixture with other ingredients. The content of fats and oils containing β-palmitic acid in the compositions and agents of the present invention may be, for example, 0.1 to 20% by mass, and is preferably 1 to 9% by mass.

The composition and agent of the present invention may contain other fats and oils, such as fats and oils containing medium-chain fatty acids (e.g., medium-chain fatty acid glycerides) and general edible fats and oils (e.g., fats and oils mainly composed of long-chain fatty acids, such as olive oil and soybean oil), in addition to fats and oils containing β-palmitic acid. The content of fats and oils other than fats and oils containing β-palmitic acid in the composition and agent of the present invention can be, for example, 0 to 99% by mass, and is preferably 10 to 40% by mass.

The composition and agent of the present invention may contain other fats and oils, such as fats and oils containing medium-chain fatty acids (e.g., medium-chain fatty acid glycerides) and general edible fats and oils (e.g., fats and oils mainly composed of long-chain fatty acids, such as olive oil and soybean oil), in addition to fats and oils containing β-palmitic acid. The content of fats and oils other than fats and oils containing β-palmitic acid in the composition and agent of the present invention can be, for example, 0 to 99% by mass, and is preferably 10 to 40% by mass.

According to the Examples below, it was confirmed that β-palmitic acid increases mitochondrial ATP production.According to the Examples below, it was also confirmed that β-palmitic acid increases the expression levels of the Sirt3 gene, Nfe2l2 gene, Tfam gene, Nrf1 gene, and Pgc-1α gene.In this specification, "one or more genes selected from the group consisting of the Sirt3 gene, Nfe2l2 gene, Tfam gene, Nrf1 gene, and Pgc-1α gene" are referred to as "mitochondrial biogenesis-related genes," and the specific functions of each gene are as follows:

The SIRT3 protein encoded by the "Sirt3 gene" is activated by NAD ⁺ , activates Foxo3 and ROS decomposition enzymes that decompose reactive oxygen species (ROS) by deacetylation, and eliminates ROS to suppress damage to mitochondrial DNA (mtDNA) and cells. It has also been reported that transcription of the Sirt3 gene is promoted by the NFE2L2 protein (Yue Qian, et al., Oxid Med Cell Longev., 26; 9423593 (2020)., Judith Hagenbuchner, et al., Front Physiol., 20; 4: 147 (2013)., F Kyle Satterstrom, et al., Aging Cell., 14(5): 818-25 (2015).).

It has been reported that the NFE2L2 protein encoded by the "Nfe2l2 gene" promotes mitochondrial biogenesis by contributing to the maintenance of the expression levels of the Nrf1 gene and PGC-1α gene, contributes to the maintenance of mitochondrial structure and functionality, that transcription of the Nfe2l2 gene is promoted by activated PGC-1α, and that the Nfe2l2 gene is also activated by reactive oxygen species, inflammatory cytokines, and endoplasmic reticulum stress (Di nkova-Kostova AT, et al., Free Radic Biol Med. 88 (Pt B): 179-188 (2015); Makiko Ohtsuji, et al., J Biol Chem., 283(48): 33554-62 (2008); Richard C Scarpulla, Biochim Biophys Acta. 1813(7): 1269-78 (2011).

TFAM protein, encoded by the "Tfam gene," is a transcription factor encoded by nuclear DNA that is responsible for controlling mitochondrial function. It has been reported that upon transfer to mitochondria, it stabilizes mtDNA, promotes the synthesis of electron transport chain complexes encoded by mtDNA, and promotes mtDNA transcription and replication, and that the transcription of the Tfam gene is promoted by NRF1 and NFE2L2 proteins (Yue Qian, et al., Oxid Med Cell Longev., 26; 9423593 (2020)., Melania Collu-Marchese, et al., Biosci Rep. 35(3):e00221(2015).).

It has been reported that the NRF1 protein encoded by the "Nrf1 gene" promotes the transcription of mitochondrial transcription factor A (mtTFA), an mtDNA transcription and amplification factor, and the transcription of mitochondrial proteins encoded in the nucleus, and that the transcription of Nrf1 is promoted by activated PGC-1α (Katsutaro Morino, et al., Diabetes 49(11): 837-840 (2006); Richard C. Scarpulla, Biochim Biophys Acta. 1813(7): 1269-78 (2011)).

It has been reported that the PGC-1α protein encoded by the "Pgc-1α gene" is activated through phosphorylation by AMPK, deacetylation by SIRT1, and phosphorylation by p38MAPK, and that activated PGC-1α contributes to mitochondrial biogenesis by promoting the biosynthesis of NRF1, NFE2L2, and TFAM (Katsutaro Morino et al., Diabetes, 49(11): 837-840(2006); Melania Collu-Marchese, et al., Biosci Rep. 35(3):e00221(2015); Richard C Scarpulla, Biochim Biophys Acta. 1813(7): 1269-78(2011)).

The above functions of mitochondrial biogenesis-related genes suggest that mitochondrial biogenesis is promoted by the <"Nfe2l2 gene"-"Sirt3 gene"> pathway and the <"Pgc-1α gene"-"Nrf1 gene/Nfe2l2 gene"-"Tfam gene"> pathway, respectively. And, without being bound by the following theory, in light of the results of the examples described below, it is presumed that β-palmitic acid acts on the expression of the Nfe2l2 gene and/or the Pgc-1α gene (particularly the expression of the Nfe2l2 gene), resulting in upregulation of the expression of the Sirt3 gene and the Tfam gene.

In this way, the intake of β-palmitic acid can increase the amount of mitochondrial ATP production and enhance the expression of mitochondrial biogenesis-related genes. In other words, β-palmitic acid can be used to promote mitochondrial ATP production and enhance the expression of mitochondrial biogenesis-related genes (preferably one or more genes selected from the group consisting of the Sirt3 gene, the Nfe2l2 gene, and the Tfam gene), and can also be used to improve mitochondrial function.

In the present invention, "mitochondrial function" refers to mitochondrial functions such as ATP production, mitochondrial biogenesis (e.g., synthesis of electron transport complexes), activation of electron transport complexes, oxidative stress regulation (e.g., suppression of damage to mitochondrial DNA and cells caused by reactive oxygen species (ROS)), and control of cell death. In addition, in the present invention, "improvement of mitochondrial function" refers to enhancing the mitochondrial function of a subject. Subjects include humans and non-human animals, and non-human animals are preferably non-human mammals (e.g., mice, rats, guinea pigs, horses, cows, monkeys, rabbits, pigs, dogs, and cats).

The compositions and agents of the present invention can be provided in the form of medicines and quasi-drugs (e.g., pharmaceutical compositions), foods (e.g., food compositions), feeds (e.g., feed compositions), etc., and can be implemented as described below.

In the present invention, the oils and fats containing β-palmitic acid can be orally administered to humans and non-human animals. Oral preparations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. These preparations can be formulated using pharma- ceutical acceptable carriers by methods commonly used in the field. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, flavorings, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc.

In the present invention, the oils and fats containing β-palmitic acid can be administered to humans and non-human animals by methods other than oral administration, such as feeding tube administration and nasal feeding tube administration, depending on the form of the composition and agent of the present invention. For example, by making the composition and agent of the present invention into a viscous liquid composition containing oils and fats containing β-long-chain saturated fatty acids, or a semi-solid composition containing oils and fats containing β-long-chain saturated fatty acids, it can be ingested or administered to humans and non-human animals who have reduced chewing and swallowing functions and are unable to take orally or administer the composition or agent. By having the composition and agent of the present invention ingested or administered by methods other than oral intake, it is expected that the mitochondrial function of the subject to be ingested or administered can be improved, even if the chewing and swallowing functions of the subject to be ingested or administered have decreased due to aging, etc.

In the present invention, fats and oils containing β-palmitic acid can be orally ingested by humans and non-human animals. When fats and oils containing β-palmitic acid are orally ingested, they may be in an isolated, purified or crude form, or in the form of a food or food ingredient containing fats and oils containing β-palmitic acid.

In the present invention, when fats and oils containing β-palmitic acid are provided as food, the fats and oils can be contained in the food, and such foods contain an effective amount of fats and oils containing β-palmitic acid. In the present invention, when fats and oils containing β-palmitic acid are provided as food, foods or ingredients that already contain the fats and oils can be provided as the food of the present invention, and such foods contain an effective amount of fats and oils containing β-palmitic acid. Here, "containing an effective amount" of fats and oils containing β-palmitic acid refers to a content such that fats and oils containing β-palmitic acid are ingested in the range described below when the amount normally consumed in each food is ingested. In addition, "food" is used to mean health foods, functional foods, health functional foods (e.g., foods for specified health uses, foods with nutritional functions, foods with functional claims), foods for special dietary uses (e.g., foods for people with difficulty swallowing, infant formula, powdered milk for pregnant and lactating women, foods for sick people), nutritional supplements, and foods for infants.

The form of the "food" is not particularly limited, and may be, for example, a liquid form such as a beverage or liquid food, a paste, semi-liquid, gel, or a solid, bar, or powder form. When the composition and agent of the present invention are used in the form of a powder, they can be manufactured by using means such as spray drying or freeze drying.

When the composition and agent of the present invention are provided as a food containing fats and oils containing β-palmitic acid, they can be manufactured according to a normal food manufacturing method, except for blending fats and oils containing β-palmitic acid. That is, the food of the present invention can be prepared by adding fats and oils containing β-palmitic acid, regardless of their form (liquid, solid, powder, etc.), to various foods (e.g., milk, soft drinks, fermented milk, yogurt, chocolate, gummies, cheese, bread, biscuits, cookies, crackers, pizza crust, jelly, ice cream, high-energy supplements, high-energy pastes, modified milk powder, liquid diets, special dietary foods, foods for sick people, complete nutritional foods, dietary supplements, frozen foods, processed foods, and other commercially available foods) or their ingredients. In addition, subjects can ingest fats and oils containing β-palmitic acid in various forms (liquid, solid, powder, paste, etc.) by adding them to water, food, drink, or meals. In particular, in the present invention, by blending an effective amount of oils and fats containing β-palmitic acid into a liquid diet, it is possible to produce a food that also has the effect of improving mitochondrial function, and such a food is advantageous because it can be ingested or administered to sick or elderly people with reduced chewing or swallowing functions.

When the composition and agent of the present invention are provided in the form of a food or raw material (particularly a processed raw material) that already contains fats and oils containing β-palmitic acid, examples of such foods and raw materials include edible fats and oils such as lard and vegetable oils and fats.

The composition and agent of the present invention can be administered to subjects of any age, but are suitable for administration to subjects in which improved mitochondrial function is desired. Preferred subjects include, for example, infants, who are expected to quickly adapt to aerobic mitochondrial respiration, and middle-aged and elderly people (e.g., 65 years or older), who have a high need to suppress the decline in mitochondrial function with age. It has also been reported that the mRNA expression level of the Sirt3 gene, a mitochondrial biogenesis-related gene, decreases with age (Hongguang Lu, et al., Ann Vasc Surg.; 64: 303-317 (2020)), and that the expression and activity of the Nfe2l2 gene decrease with age, suggesting that this promotes aging (Silva-Palacios A, et al., Ageing Res Rev.; 47: 31-40 (2018)). Since the transcription of the Tfam gene is promoted by the Nfe2l2 gene, it is believed that the expression of the Tfam gene also decreases with age. For this reason, middle-aged and elderly people, who are likely to have reduced expression of these genes, are suitable candidates for taking the composition and agent of the present invention.

　Subjects who take or administer the compositions and agents of the present invention can be subjects in a state of reduced mitochondrial function. In the present invention, subjects with reduced mitochondrial function can be identified, for example, using as an indicator the mRNA expression level and/or protein expression level of mitochondrial biogenesis-related genes in a biological sample (e.g., blood, saliva). For example, if a biological sample is collected from a subject before the start date of intake or administration, and the mRNA expression level and/or protein expression level of mitochondrial biogenesis-related genes in the biological sample is lower than a reference value, the subject can be identified as a subject in a state of reduced mitochondrial function.

In the present invention, the "reference value" can be determined, for example, by calculation from the mRNA expression level and/or protein expression level of the mitochondrial biogenesis-related gene in a biological sample of a subject with normal mitochondrial function (normal subject). The normal subject is typically a healthy person. In the above-mentioned method for determining the reference value, the average value, median value, percentile value, maximum value, or minimum value of the normal subject can be used. The percentile value can be any value, for example, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 85, 90, or 95. The number of normal subjects when calculating the reference value is preferably multiple, for example, 2 or more, 5 or more, 10 or more, 20 or more, 50 or more, or 100 or more. The reference value is a value for identifying a subject with reduced mitochondrial function, and in this sense can be called a cutoff value or a boundary value.

In the present invention, the "reference value" can also be calculated based on the mRNA expression level and/or protein expression level of the mitochondrial biogenesis-related gene in a biological sample from a subject with normal mitochondrial function (normal subject) and the mRNA expression level and/or protein expression level of the mitochondrial biogenesis-related gene in a biological sample from a subject with reduced mitochondrial function (subject with reduced function). For example, the reference values can be set for the subjects with reduced mitochondrial function and normal subjects by performing statistical analysis such as ROC (Receiver Operating Characteristic) analysis using the mRNA expression level and/or protein expression level of the mitochondrial biogenesis-related gene in the biological sample. The creation of ROC curves and the setting of reference values based on ROC curves are well known, and can be set appropriately by a person skilled in the art from the standpoint of sensitivity and specificity.

In the present invention, for example, when the mRNA expression level and/or protein expression level of a mitochondrial biogenesis-related gene in a biological sample from a subject is lower than the average expression level of the gene in normal subjects, or is about 0.9 times or less, about 0.85 times or less, about 0.8 times or less, about 0.75 times or less, about 0.7 times or less, about 0.65 times or less, about 0.6 times or less, about 0.55 times or less, about 0.5 times or less, about 0.45 times or less, about 0.4 times or less, or about 0.35 times or less compared to the average expression level, the subject can be identified as having reduced mitochondrial function. Here, in comparison with the average expression level of the mRNA expression level and/or protein expression level of a mitochondrial biogenesis-related gene in normal subjects, the number of mitochondrial biogenesis-related genes may be one or more, preferably three or more, more preferably four or more, and even more preferably five.

Furthermore, mitochondrial function declines with age, but mitochondrial function of a subject can be improved by ingesting the composition and agent of the present invention, and therefore the composition and agent of the present invention can be used for anti-aging or suppression of cellular aging. When the composition and agent of the present invention are used for suppressing cellular aging, the cells to be targeted for aging suppression include, but are not limited to, cells that constitute metabolically active organs (e.g., liver, kidney, muscle, brain, heart), and specifically include liver cells, kidney cells, muscle cells, brain cells, cardiac cells, etc.

In the present invention, "aging" generally refers to changes (decline) in physiological functions that occur after maturity, and more specifically includes, for example, changes in the cardiovascular system, such as coronary artery sclerosis and a decrease in maximum cardiac output during exercise; changes in the respiratory system, such as a decrease in the number of alveoli and a decrease in lung elasticity; changes in the digestive system, such as aspiration pneumonia due to a decrease in chewing or swallowing ability, constipation and abnormal bowel movements due to a decrease in gastrointestinal motility, and reflux esophagitis due to the reflux of gastric contents into the esophagus; changes in the renal and urinary systems, such as loss of glomeruli, decreased renal blood flow, and decreased filtration rate; changes in the skeletal system, such as osteoporosis and fractures due to a decrease in bone mass and bone density, and arthritis due to a decrease in joint fluid and decreased elasticity of the synovial membrane; and changes in audiovisual functions, such as decreased eyesight and hearing loss. In the present invention, "anti-aging" includes maintaining the state before and after aging changes (i.e., at least maintaining the state before and after aging changes as they are, and preferably inhibiting or preventing changes (e.g., deterioration) in the state before and after aging changes), as well as improving the state before and after aging changes (i.e., improving the state before and after aging changes).

In the present invention, "cellular senescence" refers to a state in which mitochondrial function of cells is reduced with age and a state in which mitochondrial biogenesis in cells is reduced. In the present invention, "inhibition of cellular senescence" includes maintaining the state before and after changes due to cellular senescence (i.e., maintaining at least the state before and after changes due to cellular senescence as they are, and preferably inhibiting or preventing changes (e.g., deterioration) in the state before and after changes due to cellular senescence), as well as improving the state before and after changes due to cellular senescence (i.e., improving the state before and after changes due to cellular senescence).

The compositions and agents of the present invention can be taken not only by healthy individuals, but also by subjects who have developed a disease associated with decreased mitochondrial function or subjects who have decreased mitochondrial function. In other words, β-palmitic acid can be used to maintain, improve, treat or prevent diseases and conditions that can be maintained, improved, treated or prevented by improving mitochondrial function.

In the present invention, "improvement" means making a certain condition better. In the present invention, "maintenance" means keeping a certain condition at least as it is, and includes suppressing or preventing a certain condition from changing. "A certain condition" includes a changed condition and a normal condition, and "normal condition" includes the condition before the change. Furthermore, "change" is, for example, deterioration. In the present invention, one aspect of "improvement" is the recovery of a deteriorated condition. In the present invention, one aspect of "maintenance" is the suppression or prevention of deterioration of a deteriorated condition or a normal condition.

In the present invention, for example, oils and fats containing β-palmitic acid can be combined with other ingredients and provided as compositions or supplements aimed at improving mitochondrial function.

The daily intake or administration amount of the composition and agent of the present invention as a medicine or food is not particularly limited, since it varies depending on the pathology, age, symptoms, weight, and use of the subject. In the case of intake and administration for the purpose of improving mitochondrial function, the daily intake or administration amount (converted to solid content) of fats and oils containing β-palmitic acid for an adult is not particularly limited, but the lower limit is, for example, 0.01 g, preferably 0.1 g. The upper limit is, for example, 65 g or 60 g, preferably 5 g or 1 g. These lower and upper limits can be combined arbitrarily. The number and frequency of intake or administration can be appropriately determined depending on the degree of improvement of mitochondrial function required. The intake amount and administration amount, as well as the intake interval and administration interval, can be determined by the subject's doctor, pharmacist, registered dietitian, nutritionist, care worker, care manager, helper, staff of a care facility, caregivers such as the subject's family, or the subject himself/herself.

The composition and agent of the present invention may be used in combination with other compositions and agents that can be taken orally without any restrictions. For example, the effect of improving mitochondrial function can be further enhanced by using the composition and agent in combination with materials or compositions that are expected to have anti-aging effects, such as antioxidants.

The composition and agent of the present invention can be provided as a composition and agent with a daily intake amount effective for improving mitochondrial function. In this case, the composition and agent of the present invention may be packaged so that an effective daily intake amount of oils and fats containing β-palmitic acid can be ingested, and the packaging form may be a single package or multiple packages as long as an effective daily intake amount can be ingested. When provided in a packaged form, it is desirable that the package has a description regarding the intake amount so that an effective daily intake amount can be ingested, or that a document with such description is provided together with the composition and agent. Furthermore, when providing an effective daily intake amount in multiple packages, multiple packages of the effective daily intake amount can be provided as a set for convenience of ingestion.

The packaging form for providing the compositions and agents of the present invention is not particularly limited as long as it specifies a certain amount, and examples include wrapping paper, bags, soft bags, paper containers, cans, bottles, capsules, and other containers that can accommodate the composition and agents.

In order to maximize the effects of the compositions and agents of the present invention, it is desirable to administer or ingest them continuously for at least one week, and the administration and ingestion period is preferably two weeks or more (e.g., 2 to 14 weeks), and more preferably three weeks or more (e.g., 3 to 21 weeks). Here, "continuously" means that the compositions and agents of the present invention are administered or ingested continuously at least once per week (e.g., 1 to 7 times). When the compositions and agents of the present invention are provided in a packaged form, they may be provided as a set containing an effective intake amount for a certain period (e.g., one week) for continuous ingestion.

The food of the present invention may be labeled with a label indicating that it has an effect of improving mitochondrial function. In this case, in order to make the labeling easy for consumers to understand, the food of the present invention may be labeled with, for example, some or all of the following labeling:
・For those who want to extend their healthy lifespan ・For those who are concerned about aging ・To slow down the changes that accompany cellular aging

In another aspect of the present invention, there is provided a method for improving mitochondrial function or a method for enhancing expression of mitochondrial biogenesis-related genes, comprising ingesting or administering to a subject in need thereof an effective amount of an oil or fat containing β-palmitic acid or a composition containing the same. The present invention also provides a method for maintaining, improving, treating or preventing a disease or condition that can be maintained, improved, treated or prevented by improving mitochondrial function, comprising ingesting or administering to a subject in need thereof an effective amount of an oil or fat containing β-palmitic acid or a composition containing the same. The method of the present invention can be carried out according to the description of the composition and agent of the present invention.

According to another aspect of the present invention, there is provided the use of an oil containing β-palmitic acid or a composition comprising the same as an agent for improving mitochondrial function or an agent for enhancing expression of mitochondrial biogenesis-related genes, or in a method for improving mitochondrial function or a method for enhancing expression of mitochondrial biogenesis-related genes, for the manufacture of an agent for improving mitochondrial function or an agent for enhancing expression of mitochondrial biogenesis-related genes, or in a method for improving mitochondrial function or a method for enhancing expression of mitochondrial biogenesis-related genes. According to the present invention, there is also provided the use of an oil containing β-palmitic acid or a composition comprising the same as an agent for maintaining, improving, treating or preventing a disease or condition that can be maintained, improved, treated or prevented by improving mitochondrial function, for the manufacture of an agent for maintaining, improving, treating or preventing a disease or condition that can be maintained, improved, treated or prevented by improving mitochondrial function, or in a method for maintaining, improving, treating or preventing a disease or condition that can be maintained, improved, treated or prevented by improving mitochondrial function. The use of the present invention can be carried out according to the description of the composition and agent of the present invention and the method of the present invention.

According to yet another aspect of the present invention, there is provided an oil containing β-palmitic acid or a composition comprising the same for use in improving mitochondrial function or enhancing expression of mitochondrial biogenesis-related genes. The present invention also provides an oil containing β-palmitic acid or a composition comprising the same for use in maintaining, improving, treating or preventing a disease or condition that can be maintained, improved, treated or prevented by improving mitochondrial function. These inventions can be carried out in accordance with the descriptions of the compositions and agents of the present invention and the methods of the present invention.

The methods and uses of the present invention may be for use in mammals, including humans, and both therapeutic and non-therapeutic uses are intended. As used herein, "non-therapeutic" does not mean to include surgery, therapy, or diagnosis of humans (i.e., medical procedures on humans), and specifically does not include methods of surgery, therapy, or diagnosis performed on humans by a physician or a person under the direction of a physician.

The present invention will be explained in more detail based on the following examples, but the present invention is not limited to these examples.

Example 1: Effect of β-palmitic acid on the amount of ATP produced In Example 1, the effect of β-palmitic acid on the amount of ATP produced was examined.

(1) Method Human liver-derived cell line HepG2 cells (JCRB cell bank) were seeded in a 96-well plate at 2,000 cells/0.1 mL/well, and the test was performed after 24 hours of culture. Palmitic acid (CAS number: 57-10-3, Cayman Chemical Company), 2-monopalmitin (CAS number: 23470-00-0, Cayman Chemical Company), and 2-monoolein (CAS number: 3443-84-3, Olbracht Serdary Research Laboratories) were added at 50 μM to each test group as test substances, and the control group was a culture medium without the addition of the test substance (n = 3 for each group). In addition, in all test groups and control groups, the final concentration of BSA was 0.1%, and the final concentration of EtOH was 0.01%. Four hours after the addition of the test substance, the amount of ATP production was measured using the ATP Assay Kit-Luminescence (Dojindo Laboratories) according to the kit protocol. Statistical analysis was performed by one-way ANOVA followed by multiple comparison test by Tukey-Kramer method.

(2) Results The results are shown in Figure 1. It was confirmed that the amount of ATP production increased significantly in the test group to which 2-monopalmitin (β-palmitic acid) was added, compared to the control group. On the other hand, no effect was observed on the amount of ATP production in the test groups to which palmitic acid and 2-monoolein (β-oleic acid) were added. This result demonstrated that β-palmitic acid promotes ATP production.

Example 2: Effect of β-palmitic acid on the expression levels of mitochondrial biogenesis-related genes (1)
In Example 2, the effect of β-palmitic acid on the expression level of mitochondrial biogenesis-related genes was examined.

(1) Method Human liver-derived cell line HepG2 cells (JCRB cell bank) were seeded in a 24-well plate at 100,000 cells/0.5 mL/well, and after 48 hours of culture, it was confirmed that the cells reached 80% confluence, and the test was performed. In each test group, 2-monopalmitin (CAS number: 23470-00-0, Cayman Chemical Company) and 2-monoolein (CAS number: 3443-84-3, Olbracht Serdary Research Laboratories) were added at 500 μM as test substances, and the control group was a culture medium without the addition of test substances (n = 3 for each group). In addition, in all test groups and control groups, the final concentration of BSA was 1% and the final concentration of EtOH was 0.1%. Four hours after the addition of the test substances, RNA was extracted and purified using the Maxwell RSC simplyRNA Cells Kit (Promega) according to the kit protocol. Then, using Takara PrimeScript RT Master Mix (Takara Bio), cDNA was synthesized by reverse transcription according to the kit's protocol. Next, using 7500 Fast Real-Time PCR System (Applied Biosystems), real-time quantitative PCR was performed to measure the expression levels of five genes, Sirt3 gene, Nfe2l2 gene, Tfam gene, Nrf1 gene, and Pgc-1α gene. Table 1 shows the sequence of the primers used, Table 2 shows the composition of the PCR reaction solution, and Table 3 shows the PCR conditions. After PCR was completed, melting curve analysis was performed, and the expression levels between samples were corrected by the expression level of the Actb gene. In addition, statistical analysis was performed by one-way analysis of variance (one-way ANOVA), followed by multiple comparison test by the Tukey-Kramer method.

(2) Results The results are shown in FIG. 2. In the test group to which 2-monopalmitin (β-palmitic acid) was added, the expression levels of three genes, Sirt3, Nfe2l2, and Tfam, were significantly increased compared to the control group (FIGS. 2A-C), and the expression levels of Nrf1 and Pgc-1α tended to increase (FIGS. 2D-E). On the other hand, in the test group to which 2-monoolein (β-oleic acid) was added, no genes were found to have a significantly increased expression level compared to the control group (FIGS. 2A-E). These results indicate that there are genes whose expression is enhanced by β-palmitic acid.

Example 3: Effect of β-palmitic acid on the expression levels of mitochondrial biogenesis-related genes (2)
In Example 3, the test substances were increased and the effect of β-palmitic acid on the expression level of mitochondrial biogenesis-related genes was further examined.

(1) Method In each test group, the test was carried out in the same manner as in Example 2(1) above, except that 2-monopalmitin (CAS number: 23470-00-0, Cayman Chemical Company), 2-monomyristin (CAS number: 3443-83-2, Larodan AB), palmitic acid (CAS number: 57-10-3, Cayman Chemical Company), and 2-monostearin (CAS number: 621-61-4, Larodan AB) were used as test substances.

(2) Results The results are shown in FIG. 3. In the test group to which 2-monopalmitin (β-palmitic acid) was added, the same results as in Example 2 were obtained. On the other hand, in the test groups to which 2-monomyristin (β-myristic acid), palmitic acid, or 2-monostearic acid (β-stearic acid) was added, no genes were found to have increased expression levels compared to the control group. The results of Examples 2 and 3 demonstrated the existence of genes whose expression is enhanced by β-palmitic acid.

Example 4: Preparation of infant food composition In Example 4, an infant food composition (formulated powdered milk) was prepared according to the composition (per 100 kcal) in Table 4. In addition to formulated powdered milk, beverages, jelly drinks, powdered drinks, solid preparations, powdered preparations, etc. can be produced by known methods.

Example 5: Preparation of food composition for the elderly In Example 5, a food composition for the elderly (liquid diet) was prepared according to the composition (per 100 ml) in Table 5. In addition to liquid diets, beverages, jelly drinks, powdered drinks, solid preparations, powdered preparations, etc. can be produced by known methods.

The present invention makes it possible to provide a method for producing a food for improving mitochondrial function that contains β-palmitic acid as an active ingredient.

Claims (10)

1. A composition for improving mitochondrial function, comprising an oil containing β-palmitic acid as an active ingredient.
2. A composition for enhancing expression of mitochondrial biogenesis-related genes, comprising an oil containing β-palmitic acid as an active ingredient.
3. The composition according to claim 2, wherein the gene is one or more genes selected from the group consisting of the Sirt3 gene, the Nfe2l2 gene, and the Tfam gene.
4. The composition according to claim 1 or 2 for use in anti-aging.
5. The composition according to claim 1 or 2 for use in promoting ATP production.
6. The composition according to claim 1 or 2, which is a food composition.
7. A method for improving mitochondrial function or enhancing expression of mitochondrial biogenesis-related genes, comprising administering to a subject in need thereof an effective amount of an oil or fat containing β-palmitic acid or a composition containing the same.
8. A method for maintaining, improving, treating or preventing a disease or condition that can be maintained, improved, treated or prevented by improving mitochondrial function, comprising having a subject in need ingest an effective amount of an oil or fat containing β-palmitic acid or a composition containing the same.
9. 　Use of an oil containing β-palmitic acid as an agent for improving mitochondrial function or an agent for enhancing expression of mitochondrial biogenesis-related genes for the manufacture of an agent for improving mitochondrial function or an agent for enhancing expression of mitochondrial biogenesis-related genes, or in a method for improving mitochondrial function or a method for enhancing expression of mitochondrial biogenesis-related genes.
10. 　Use of oils and fats containing β-palmitic acid for the manufacture of an agent for maintaining, improving, treating or preventing a disease or condition that can be maintained, improved, treated or prevented by improving mitochondrial function, as an agent for maintaining, improving, treating or preventing a disease or condition that can be maintained, improved, treated or prevented by improving mitochondrial function, or in a method for maintaining, improving, treating or preventing a disease or condition that can be maintained, improved, treated or prevented by improving mitochondrial function.

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