JP7637708B2 - Composition for reducing serum TMAO - Google Patents

Description

The present invention relates to a composition for reducing serum levels of trimethylamine N-oxide (TMAO).

It is known that fish are rich in n-3 polyunsaturated fatty acids, which are unsaturated fatty acids. Many studies have been reported so far that have shown that eating fish frequently reduces the risk of developing cardiovascular disease. In particular, an epidemiological study in Greenland is famous as a pioneering epidemiological study that drew attention to the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) in the prevention and treatment of cardiovascular disease. In this epidemiological study, it was revealed that the indigenous Greenland Inuit people have a low incidence of acute myocardial infarction and a low mortality rate from ischemic heart disease. Furthermore, as a result of investigating their diet, it was found that they consume a lot of n-3 polyunsaturated fatty acids such as EPA and docosahexaenoic acid (DHA) due to their dietary habit of eating a lot of seals and fish, and conversely, they consume little n-6 polyunsaturated fatty acids, which are abundant in terrestrial animals and plants. It has been reported that such differences in the fatty acid composition ingested in the diet are the reason for the low incidence of acute myocardial infarction and the low mortality rate from ischemic heart disease in the Inuit (Non-Patent Documents 1 to 3). In addition, human clinical trials have demonstrated the triglyceride-lowering effect of EPA and DHA, but it is known that their cholesterol-lowering effect is either weak or not observed.

On the other hand, it has been reported that long-chain monounsaturated fatty acids (LC-MUFA) with carbon numbers of 20 and 22, which are known to be abundant in saury oil and Alaska pollock oil, have a cholesterol-lowering effect, unlike EPA and DHA (Patent Document 1). In addition, it has been found that the intake of saury oil or Alaska pollock oil reduces total cholesterol, LDL-cholesterol, triglycerides, and blood glucose levels in the blood, and improves insulin resistance, in diet-induced obese mice and type 2 diabetes model mice (Non-Patent Documents 4-6). Similar effects have been observed in LC-MUFA.
This has also been confirmed in oils and fats that are selectively concentrated (Non-Patent Document 5). In relation to this, a cohort study of 50,000 Japanese men and women has reported that men who consume more seafood have a lower risk of developing diabetes, and that the risk is particularly low in groups that consume large amounts of small and medium-sized fish such as saury and mackerel, and fatty fish such as salmon (Non-Patent Document 7).

Furthermore, a study using an arteriosclerosis model mouse reported that intake of LC-MUFA inhibited the formation of atherosclerotic lesions in the aortic sinus, and that this was likely the result of a combined protective effect of reducing blood inflammatory cytokine concentrations and promoting cholesterol removal from macrophages by stimulating the PPAR signaling pathway (Non-Patent Document 8).

The present inventors have also found that ingestion of oil rich in LC-MUFA improves flow-mediated vasodilation (Patent Document 2).

Trimethylamine N-oxide (TMAO) is a substance that is produced by metabolism of trimethylamine (TMA)-containing substances in foods, such as phosphatidylcholine, choline, and carnitine, through intestinal bacterial metabolism and metabolism in the liver. Associations between TMAO and the onset of various diseases, such as cardiovascular diseases such as atherosclerosis (Non-Patent Documents 9-11), heart failure (Non-Patent Document 12), thrombosis (Non-Patent Document 13), and chronic kidney disease (Non-Patent Document 14), have been reported.

Re-table No. 2012-121080 PCT/JP2018/019783

Dyerberg J et al. Am J Clin Nutr, 28(9):958-66, 1975. Dyerberg J et al. Lancet, 2(8081):117-9, 1978 Bang HO et al. Am J Clin Nutr, 33(12):2657-61, 1980. Yang ZH et al. Lipids, 46(5):425-34, 2011. Yang ZH et al. J Agric Food Chem, 59(13):7482-9, 2011. Yang ZH et al. Lipids Health Dis, 10:189-98, 2011. Nanri A et al. Am J Clin Nutr, 94(3):884-91, 2011. Yang ZH et al. Mol Nutr Food Res, 0:1-11, 2016. Wang Z et al. Nature, 472(7341):57-63, 2011. Tang WH et al. New Engl J Med, 368(17):1575-84, 2013. Koeth RA et al. Nat Med, 19(5):576-85, 2013. Tang WH et al. J Am Coll Cardiol, 64(18):1908-14, 2014. Zhu W et al. Cell, 165(1):111-124, 2016. Tang WH et al. Circ Res, 116(3):448-55, 2015.

It has been reported that an increase in serum TMAO is associated with various diseases, and there is a need for a method for reducing serum TMAO levels that is highly safe and can be implemented over the long term.
In addition, among LC-MUFA, positional isomers such as C20:1n-11 and C22:1n-11 are also found in fish oils derived from fish species other than saury, such as Alaska pollock, mackerel, and salmon, but are hardly found in vegetable oils. Since saury and Alaska pollock are popular fish among Japanese people and have high industrial value, there is a strong demand for the development of new applications for fish oils rich in LC-MUFA prepared from these fish species.

The present invention aims to provide new uses of fish oil components containing LC-MUFA, particularly new uses relating to health promotion.

As a result of intensive research conducted by the present inventors to achieve the above object, it was found that the intake of saury oil rich in LC-MUFA reduces the serum level of TMAO. It was not known that oil rich in LC-MUFA affects the serum level of TMAO.
Based on these findings, the present inventors conducted further research and completed the present invention.
That is, the present invention is as follows.

[1] A composition for reducing trimethylamine N-oxide in serum, comprising:
The composition contains, as an active ingredient, a component selected from monounsaturated fatty acids having 20 or more carbon atoms, salts thereof, and esters thereof.
[2] The composition according to [1], wherein the active ingredient is a component selected from a monounsaturated fatty acid having 20 carbon atoms, a salt thereof and an ester thereof, a component selected from a monounsaturated fatty acid having 22 carbon atoms, a salt thereof and an ester thereof, or a combination thereof.
[3] The composition according to [1] or [2], wherein the active ingredient is a glyceride containing a monounsaturated fatty acid having 20 or more carbon atoms as a constituent fatty acid.
[4] The composition described in [3], wherein the glyceride is a triglyceride.
[5] A food composition comprising an n-3 polyunsaturated fatty acid and an n-6 polyunsaturated fatty acid, the weight ratio of which to the n-6 polyunsaturated fatty acid being 4.0 or more.
The composition according to any one of claims 1 to 4.
[6] The composition according to any one of [1] to [5], wherein the active ingredient is derived from fish oil.
[7] The composition described in [6], wherein the fish oil is saury oil.
[8] The composition according to any one of [1] to [7], characterized in that the composition is used so that 2 mg/kg body weight/day or more of the active ingredient calculated as a monounsaturated fatty acid having 20 or more carbon atoms is orally administered for 4 weeks or more.
[9] The composition according to any one of [1] to [8], which is used for reducing the risk of contracting a disease caused by an increase in serum level of trimethylamine N-oxide.
[10] The composition according to any one of [1] to [9], which is a food composition.
[11] The composition according to any one of [1] to [9], which is a pharmaceutical composition.

According to the present invention, it is possible to suppress serum TM containing components of fish oil rich in LC-MUFA, such as saury oil.
A composition for reducing AO is provided, which can reduce the risk of contracting a disease caused by elevated serum levels of TMAO.

1 is a graph showing the change in serum TMAO level before and after ingestion of subjects who took the saury oil capsule or the blended oil capsule of Example 1. Saury oil capsule group: p=4.3×10 ⁻⁹ ; blended oil capsule group: p=1.0×10 ⁻⁴ .

The present invention will now be described in more detail.
In the present specification, the following abbreviations may be used.
TMAO: Trimethylamine N-oxide
MUFA: Monounsaturated fatty acids
LC-MUFA: A general term for long-chain monounsaturated fatty acids, especially the C20:1 and C22:1 isomers.
PUFA: Polyunsaturated fatty acids

<Composition for reducing serum TMAO>
The present invention provides a composition for reducing serum TMAO, which contains, as an active ingredient, a component selected from monounsaturated fatty acids having 20 or more carbon atoms, their salts, and their esters (hereinafter, sometimes referred to as the composition of the present invention).

The monounsaturated fatty acid having 20 or more carbon atoms, its salt, or its ester used in the present invention is not particularly limited, and may be any that is acceptable for pharmaceutical or food use. Glycerides containing monounsaturated fatty acids having 20 or more carbon atoms as constituent fatty acids can be produced by known methods, and can also be produced as oils and fats derived from natural products, for example, by the boiling method described in WO1996/26647. Examples of oils and fats derived from natural products include marine oils (for example, fish oils such as saury oil, oils and fats from mammals such as seals and whales), microbial oils, etc. As described later, fish oils have a high content of monounsaturated fatty acids having 20 or more carbon atoms. Therefore, it is preferable that the active ingredient in the composition of the present invention is derived from fish oil (for example, saury oil). In addition, free monounsaturated fatty acids having 20 or more carbon atoms, their salts, and esters other than glycerides can be prepared by known methods, for example, using the above-mentioned glycerides as raw materials.

For example, pacific saury crude oil is usually extracted in the same way as other fish oils: Whole pacific saury or processing residues such as fish heads, skin, backbone, and innards generated during seafood processing are crushed and steamed, then pressed to separate the broth (stick water, SW) and pressed meal. The fats and oils obtained with the broth are separated from the broth by centrifugation.

The 2015 edition (7th revision) of the Standard Tables of Food Composition in Japan states that the fatty acids in Pacific saury (skinned, raw) contain 26.0% docosenoic acid (C22:1) by weight, 17.6% eicosenoic acid (C20:1) by weight, and 54.2% total monounsaturated fatty acids. Pacific saury oil is characterized by its high content of monounsaturated fatty acids among fish oils.

Crude fish oil is refined into fish oil through refining processes such as degumming, deacidification, bleaching, and deodorization. This refined fish oil can also be used as a source of components selected from monounsaturated fatty acids having 20 or more carbon atoms, their salts, and their esters.

In one embodiment, an oil having an increased concentration of monounsaturated fatty acids may be used as a source of a component selected from monounsaturated fatty acids having 20 or more carbon atoms, their salts, and their esters. In this case, a triglyceride enriched in MUFA can be obtained by a method of concentrating the monounsaturated fatty acids using a lipase reaction, or a method of concentrating the monounsaturated fatty acid ethyl esters after ethyl esterification, and then transesterifying them with glycerin to reconstitute them into triglycerides.

In one embodiment, the composition of the present invention may be a fatty acid composition mainly composed of free fatty acids, a fatty acid salt composition mainly composed of fatty acid salts, or a fatty acid ester composition mainly composed of fatty acid esters. Examples of fatty acid salts include potassium salts and sodium salts. Examples of fatty acid esters include esters of lower alcohols having 5 or less carbon atoms (i.e., alkyl esters; for example, methyl esters, ethyl esters, n-propyl esters, i-propyl esters, n-butyl esters, s-butyl esters, t-butyl esters, n-pentyl esters, etc.), esters of glycerin such as monoglycerides, diglycerides, and triglycerides (i.e., glycerides), and phospholipids. In a preferred embodiment, the composition of the present invention is a fatty acid alkyl ester composition or a fatty acid glyceride composition, and more preferably a fatty acid ethyl ester composition or a fatty acid triglyceride composition.

Monounsaturated fatty acids with 20 or more carbon atoms include monounsaturated fatty acids with 20 carbon atoms (C20:1) (
For example, C20:1 n-11, C20:1 n-9, C20:1 n-7), monounsaturated fatty acids with 22 carbon atoms (C22:1)
Examples include C22:1 n-11, C22:1 n-9, C22:1 n-7, C22:1 n-13), monounsaturated fatty acids having 24 carbon atoms (C24:1) (e.g., C24:1 n-9), etc. In a preferred embodiment, the active ingredient of the composition of the present invention is a component selected from C20:1, a salt thereof and an ester thereof, a component selected from C22:1, a salt thereof and an ester thereof, or a combination thereof.

The content of C22:1 and C20:1 varies depending on the fish species, but examples of fish species with high content of these include Pacific cod, Alaska pollock, Atlantic cod, and sablefish, salmonids such as chum salmon, coho salmon, sockeye salmon, pink salmon, Pacific salmon, and rainbow trout, smelts such as capelin and shishamo, and herring. In addition, fish such as sand lance, tuna, mackerel, and alfonsino contain relatively large amounts of these compounds. They are also found in large amounts in the liver oil of sharks such as the spiny dogfish, basking shark, and chimaera shark. In the present invention, fish oils prepared from these fish can be used as is, purified, or concentrated.

The ratio of monounsaturated fatty acids having 20 or more carbon atoms to the total fatty acids in the composition of the present invention can be 10% by weight or more, for example, 11% by weight or more, 12% by weight or more, 13% by weight or more, 14% by weight or more, 15% by weight or more, 16% by weight or more, 17% by weight or more, 18% by weight or more, 19% by weight or more, 20% by weight or more, 21% by weight or more, 22% by weight or more, 23% by weight or more, 24% by weight or more, 25% by weight or more, 26% by weight or more, 27% by weight or more, 28% by weight or more, 29% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more.
In the present invention, the ratio of a specific fatty acid to the total fatty acids in the composition (or the content of a specific fatty acid to the total weight of fatty acids) is determined based on the fatty acid composition. The fatty acid composition can be determined according to a conventional method. Specifically, the oil to be measured is esterified using a lower alcohol and a catalyst to obtain a fatty acid lower alkyl ester. The obtained fatty acid lower alkyl ester is then analyzed using a gas chromatograph equipped with a flame ionization detector (FID). Peaks corresponding to each fatty acid are identified in the obtained gas chromatography chart, and the peak area of each fatty acid is determined using the Agilent ChemStation integration algorithm (revision C.01.03 [37], Agilent Technologies). The fatty acid composition is determined by the percentage of each peak area relative to the sum of the peak areas of the fatty acids. The area percentage obtained by the above-mentioned measurement method is the same as the weight percentage of each fatty acid in the sample. Please refer to the Japan Oil Chemists' Society (JOCS) Standard Testing Methods for Analysis of Fats, Oils and Related Materials, 2013 Edition, 2.4.2.1-2013 Fatty Acid Composition (FID Constant Temperature Gas Chromatography) and 2.4.2.2-2013 Fatty Acid Composition (FID Programmed Temperature Gas Chromatography).
The analytical conditions for gas chromatography are as follows.
Equipment: Agilent 6890N GC system (Agilent)
Column: DB-WAX (Agilent Technologies, 30m x 0.25mm ID, 0.25μm film thickness)
Carrier gas: Helium (1.0 mL/min, constant flow)
Injection port temperature: 250℃
Injection volume: 1 μL
Injection method: Split Split ratio: 20:1
Column oven: 180℃ - 3℃/min - 230℃
Detector: FID
Detector temperature: 250℃

In this specification, the fatty acid content refers to the fatty acid content (wt%) determined based on the fatty acid composition described above.

In the present invention, examples of the salts of monounsaturated fatty acids having 20 or more carbon atoms include potassium salts and sodium salts. Examples of the esters of monounsaturated fatty acids having 20 or more carbon atoms include esters of lower alcohols having 5 or less carbon atoms (e.g., methyl esters, ethyl esters,
Examples of the glycerin ester include glycerin esters (e.g., n-propyl ester, i-propyl ester, n-butyl ester, s-butyl ester, t-butyl ester, n-pentyl ester, etc.), esters with glycerin such as monoglycerides, diglycerides, and triglycerides (i.e., glycerides), and phospholipids. In a preferred embodiment, the active ingredient of the composition of the present invention is a glyceride containing a monounsaturated fatty acid having 20 or more carbon atoms as a constituent fatty acid, and more preferably a triglyceride.

As the monounsaturated fatty acid having 20 or more carbon atoms in the present invention, fats and oils derived from natural products containing free monounsaturated fatty acids having 20 or more carbon atoms may be used as they are, or after purification or concentration. As the ester of monounsaturated fatty acids having 20 or more carbon atoms, fats and oils derived from natural products containing glycerides having monounsaturated fatty acids having 20 or more carbon atoms as constituent fatty acids may be used as they are, or after purification or concentration.

In addition, as the ester of monounsaturated fatty acids having 20 or more carbon atoms, fractionated oils obtained by esterifying fats and oils derived from natural products and then fractionating them by high performance liquid chromatography or the like can also be used. Fractionated oils include fractionated oils enriched in C20:1, fractionated oils enriched in C22:1, and fractionated oils enriched in C20:2.
Examples include 4:1 concentrated fractionated oils.

The composition of the present invention may further comprise an n-3 polyunsaturated fatty acid and an n-6 polyunsaturated fatty acid, in which case the weight ratio of the n-3 polyunsaturated fatty acid to the n-6 polyunsaturated fatty acid is 4.0 or more, for example 5.0 or more, 6.0 or more, 7.0 or more, 8.0 or more, 9.0 or more, or 10.0 or more. In this specification, n-3 polyunsaturated fatty acid refers to an unsaturated fatty acid having 18 or more carbon atoms and having two or more carbon-carbon double bonds, in which the first double bond is between the third and fourth carbon atoms counting from the carbon of the methyl group at the end of the fatty acid chain. Examples of such fatty acids include α-linolenic acid (C18:3n-3), octadecatetraenoic acid (C18:4n-3), eicosatetraenoic acid (C20:4n-3), eicosapentaenoic acid (C20:5n-3), docosapentaenoic acid (C22:5n-3), and docosahexaenoic acid (C22:6n-3). The n-6 polyunsaturated fatty acid is an unsaturated fatty acid having 18 or more carbon atoms and two or more carbon-carbon double bonds, and the first double bond is between the 6th and 7th carbon atoms counting from the carbon of the methyl group at the end of the fatty acid chain, for example, linoleic acid (C18:2n-6) and arachidonic acid (C20:4n-6). In this specification, the weight ratio of fatty acids refers to the weight ratio based on the fatty acid composition determined as described above. The analytical conditions for gas chromatography are as described above.

The composition of the present invention can reduce the serum level of TMAO in a subject.Therefore, in one aspect, a method for reducing the serum level of TMAO in a subject is provided, comprising administering the composition of the present invention to the subject.
In healthy individuals, the plasma level of TMAO is less than 2.5 μM in the fasting state. Prospective follow-up of individuals with high plasma TMAO levels has revealed a higher incidence of cardiovascular events, with TMAO levels above 6.2 μM associated with a significantly increased risk of major adverse cardiovascular events (analysis of approximately 4,000 subjects divided into quartiles (<2.43, 2.43-3.66, 3.67-6.18, and >6.18) based on plasma TMAO concentration; see Non-Patent Document 10). The composition of the present invention can prevent serum levels of TMAO from increasing to a level that increases the risk of developing cardiovascular disease (e.g., 2.5 μM or more, 3.7 μM or more, or 6.2 μM or more). The compositions of the present invention can also reduce serum levels of TMAO to less than 2.5 μM, less than 2.4 μM, less than 2.3 μM, less than 2.2 μM, less than 2.1 μM, less than 2.0 μM, less than 1.9 μM, less than 1.8 μM, less than 1.7 μM, less than 1.6 μM, or less than 1.5 μM. The compositions of the present invention can also reduce serum levels of TMAO after ingestion by 0.10 μM or more, 0.15 μM or more, 0.20 μM or more, 0.25 μM or more, 0.29 μM or more, 0.50 μM or more, 1.0 μM or more, 1.5 μM or more, 2.0 μM or more, 2.5 μM or more, 3.0 μM or more, 3.5 μM or more, or 4.0 μM or more, compared to serum levels of TMAO before ingestion.
Known methods for measuring the serum level of TMAO include, for example, stable isotope dilution mass spectrometry (see Patent Document 9) and ELISA. In a preferred embodiment of the present invention, the serum level of TMAO is measured by ELISA. Measurement by ELISA can be performed by a standard method using an antibody against TMAO. Alternatively, a commercially available kit such as HUMAN Trimethylamine-N-oxide (TMAO) ELISA KIT (Glory Science CO., LTD) may be used.

The test in the following examples is conducted on healthy subjects, and the reduction of TMAO serum level does not mean the treatment of diseases such as cardiovascular disease, but it can reduce the risk of suffering from these diseases.Therefore, in one aspect, the composition of the present invention is a composition for reducing the risk of suffering from diseases caused by an increase in TMAO serum level.In another aspect, a method for reducing the risk of suffering from diseases caused by an increase in TMAO serum level is provided, comprising administering the composition of the present invention to a subject.
Here, examples of diseases attributable to an increase in serum level of TMAO include cardiovascular diseases such as atherosclerosis, heart failure, and thrombosis, and chronic kidney disease.
Furthermore, the present invention provides a novel use of LC-MUFA that was not previously known, based on the discovery that LC-MUFA reduces serum levels of TMAO. Thus, in one aspect, the composition of the present invention can be a composition for reducing the risk of contracting a disease caused by an increase in serum levels of TMAO, excluding arteriosclerosis. In yet another aspect, a method for reducing the risk of contracting a disease caused by an increase in serum levels of TMAO, excluding arteriosclerosis, is provided, which comprises administering the composition of the present invention to a subject.

In one embodiment, the above-mentioned method of lowering serum levels of TMAO in a subject and the method of reducing the risk of contracting a disease caused by an elevated serum level of TMAO may be a non-therapeutic method. Here, "non-therapeutic" is a concept that does not include medical procedures, i.e., surgery, treatment, or diagnosis performed by a physician (including a person under the instruction of a physician) on a human being.

The subject of administration of the composition of the present invention is a mammal, preferably a human. The age of the subject is not particularly limited as long as the effects of the present invention can be obtained, and is, for example, 20 years or older. In one embodiment, the subject may be a healthy individual. In another embodiment, the subject may be a mammal that does not suffer from a disease caused by an increase in serum levels of TMAO. In yet another embodiment, the subject may be a mammal that does not suffer from a disease caused by an increase in serum levels of TMAO, excluding arteriosclerosis.

The composition of the present invention may be prepared in a form suitable for pharmaceutical compositions, food compositions (e.g., functional foods, health foods, supplements, etc.), for example, in the form of various solid preparations such as granules (including dry syrups), capsules (soft capsules, hard capsules), tablets (including chewable tablets, etc.), powders (powders), and pills, or in the form of liquid preparations such as oral liquids (including liquids, suspensions, and syrups). For example, the composition of the present invention can be formulated as a soft capsule in which purified fish oil is filled into a gelatin membrane.

Examples of additives used in formulation include excipients, lubricants, binders, disintegrants, flow agents, dispersants, wetting agents, preservatives, thickeners, pH adjusters, colorants, flavorings, surfactants, and dissolution aids. When preparing a liquid formulation, thickeners such as pectin, xanthan gum, and guar gum can be added. A coating agent can be used to make a coated tablet, or a paste-like glue can be used. Furthermore, when preparing other forms, conventional methods can be followed.

The composition of the present invention can take the form of a food composition. In the present invention, the food composition means food in general, including beverages, and includes general foods, including health foods such as supplements, as well as specific health foods and functional foods as specified by the Consumer Affairs Agency's Health Foods System. For example, functional foods are provided that are labeled as having the effect of lowering serum levels of TMAO. For example, foods containing fish oil can be provided as is. The food composition of the present invention also includes food materials that can be added to, mixed with, or applied to other foods to impart the effect of lowering serum levels of TMAO. In addition to foods, the food composition can also be provided as animal feed, etc.

When the composition of the present invention is in the form of a food, the food is not particularly limited and may be beverages, confectioneries, breads, soups, etc., and includes, for example, general retort foods, frozen foods, instant foods (such as noodles), canned foods, sausages, cookies, biscuits, cereal bars, crackers, snacks (such as potato chips), pastries, cakes, pies, candies, chewing gums (including pellets and sticks), jellies, soups, ice cream, dressings, yogurt, etc., supplements in the form of tablets, capsules, emulsions, etc., soft drinks, etc. The manufacturing method of these foods is not particularly limited as long as it does not impair the effects of the present invention, and may be in accordance with the method used by those skilled in the art for each food.

Selling the product of the present invention by displaying the effects of the composition of the present invention on packaging containers (boxes, bags, cans, bottles, etc.), product instructions, and pamphlets is also included in the practice of the present invention. In addition, advertising and selling the product of the present invention by displaying the effects of the present invention on television, Internet websites, pamphlets, newspapers, magazines, etc. is also included in the practice of the present invention.

In the present invention, the amount of the component selected from monounsaturated fatty acids having 20 or more carbon atoms, their salts and their esters that the subject ingests is not particularly limited, and for example, the amount is ingested in an amount equal to or greater than the effective amount for obtaining the desired effect. Here, the effective amount for obtaining the desired effect refers to the amount required to reduce the serum level of TMAO. For example, in the case of adults, depending on the age, weight, health condition, and other conditions of the subject, the daily intake may be 2 mg/kg body weight/day or more, e.g., 3 mg/kg body weight/day or more, 4 mg/kg body weight/day or more, 5 mg/kg body weight/day or more, 6 mg/kg body weight/day or more, 7 mg/kg body weight/day or more, 8 mg/kg body weight/day or more, 9 mg/kg body weight/day or more, 10 mg/kg body weight/day or more, 11 mg/kg body weight/day or more, 12 mg/kg body weight/day or more, 13 mg/kg body weight/day or more, 14 mg/kg body weight/day or more, 15 mg/kg body weight/day or more, 16 mg/kg body weight/day or more, 17 mg/kg body weight/day or more, 18 mg/kg body weight/day or more, 19 mg/kg body weight/day or more, 20 mg/kg body weight/day or more, 21 mg/kg body weight/day or more, 22 mg/kg body weight/day or more, 23 mg/kg body weight/day or more, 24 mg/kg body weight/day or more, calculated as monounsaturated fatty acids having 20 or more carbons. The animal may be administered at least 25 mg/kg body weight/day, at least 30 mg/kg body weight/day, at least 40 mg/kg body weight/day, at least 50 mg/kg body weight/day, at least 100 mg/kg body weight/day, or at least 200 mg/kg body weight/day of the active ingredient for 4 weeks or more, for example, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks.
Furthermore, the ingredient selected from monounsaturated fatty acids having 20 or more carbon atoms, their salts, and their esters does not have any severe side effects, so there is no limit to the daily intake amount.

The present invention will now be described in further detail with reference to examples, but the present invention is not limited to these examples.
In the examples, all percentages are by weight unless otherwise specified.

(Example 1) Intervention Study (1) Study Method a) Test Food: Sanma oil capsules contain 0.35 g of sanma-derived fish oil containing LC-MUFA at a ratio of about 29% to total fatty acids. Blended oil capsules contain 0.35 g of tuna oil and refined olive oil mixed at a weight ratio of 1:1 (containing LC-MUFA at a ratio of about 1.8% to total fatty acids). The total of EPA+DPA+DHA in the fatty acid composition of the sanma oil capsules and blended oil capsules was 19.4% and 19.7%, respectively, which was almost the same.
The fatty acid composition in the saury oil capsules and blended oil capsules is shown in Table 1. The fatty acid composition was calculated by the following method. The oil to be measured was esterified using a lower alcohol and a catalyst to obtain fatty acid lower alkyl esters. The obtained fatty acid lower alkyl esters were then analyzed using a gas chromatograph equipped with a flame ionization detector (FID). Peaks corresponding to each fatty acid were identified in the obtained gas chromatography chart, and the peak area of each fatty acid was calculated using the Agilent ChemStation integration algorithm (revision C.01.03 [37], Agilent Technologies). The fatty acid composition was determined by the percentage of each peak area relative to the sum of the peak areas of the fatty acids. The obtained area percentage was the same as the weight percentage of each fatty acid in the sample. Please refer to the Japan Oil Chemists' Society (JOCS) Standard Methods for Analysis of Fats, Oils and Related Materials, 2013 Edition, 2.4.2.1-2013 Fatty Acid Composition (FID Constant Temperature Gas Chromatography) and 2.4.2.2-2013 Fatty Acid Composition (FID Programmed Temperature Gas Chromatography). The analytical conditions for gas chromatography are as follows.
Equipment: Agilent 6890N GC system (Agilent)
Column: DB-WAX (Agilent Technologies, 30m x 0.25mm ID, 0.25μm film thickness)
Carrier gas: Helium (1.0 mL/min, constant flow)
Injection port temperature: 250℃
Injection volume: 1 μL
Injection method: Split Split ratio: 20:1
Column oven: 180℃ - 3℃/min - 230℃
Detector: FID
Detector temperature: 250℃

b) Subjects: 52 healthy adults, including students from Tokushima University, aged 20 years or older, were divided into two groups (a group that ingested saury oil capsules and a group that ingested blended oil capsules). Twenty-six participants, with an average age of 21 years, participated in the saury oil capsule group (average body weight: 54.2 kg (before ingestion) and 54.8 kg (after ingestion)), while 26 participants, with an average age of 21 years, participated in the blended oil capsule group (average body weight: 52.8 kg (before ingestion) and 53.0 kg (after ingestion)) (Table 2).

c) Study content: Subjects took 12 capsules per day for 4 weeks. Each of the 12 saury oil capsules contained 4.2g of saury oil, meaning that subjects took in approximately 1.1g of LC-MUFA and approximately 0.7g of n-3 polyunsaturated fatty acids (EPA + DPA + DHA) per day. Each of the 12 blended oil capsules contained 2.1g of tuna oil and 2.1g of refined olive oil, meaning that subjects took in approximately 0.07g of LC-MUFA and approximately 0.7g of n-3 polyunsaturated fatty acids (EPA + DPA + DHA) per day. Thus, although the intake of LC-MUFA differed in each capsule group, the intake of n-3 polyunsaturated fatty acids (EPA + DPA + DHA) was approximately the same.
Blood was collected at the start of the study and after 4 weeks of intake. Subjects were required to fast from the night before blood collection until collection. Blood was collected using a vacuum blood collection tube (Terumo) and left at room temperature, then centrifuged at 2000 rpm for 10 minutes and stored at -80°C until measurement. Serum TMAO was measured using an ELISA kit (HUMAN Trimethylamine-N-oxide (TMAO) ELISA KIT (Glory Science CO., LTD)).
Measurements were performed according to the manufacturer's instructions. Anti-TMAO antibody was added to purified human TMAO immobilized on a microplate and allowed to react. After reaction with enzyme-labeled antibody, the plate was washed and colored with TMB (3,3',5,5'-tetramethylbenzidine) to measure absorbance. TMAO concentration was calculated using a calibration curve obtained from a standard sample.
Statistical processing was performed using the statistical software Graphpad Prism (MDF Ltd.). For comparison between two groups, an unpaired t-test was used, and for comparison within a group, a paired t-test, which is a comparison of two corresponding groups, was used.
d) Implementation period: October 17th to December 7th, 2017

(2) Test results As shown in Table 2 and Figure 1, serum TMAO levels were significantly reduced in both the saury oil capsule intake group and the blended oil capsule intake group, but the saury oil capsule intake group showed a lower p value (saury oil capsule group: p=4.3× ^10-9 ; blended oil capsule group: p=1.0× ^10-4 ). Since the amount of n-3 polyunsaturated fatty acids in each capsule was the same, it was suggested that the action of LC-MUFA may reduce serum TMAO levels more significantly.
Non-Patent Document 10 describes that an increase in TMAO concentration is associated with an increased risk of new major adverse cardiovascular events. In addition, a paper published in 2017 (European Heart Journal 2017 38 2948-2956) shows the results of a systematic review and meta-analysis, and describes that there is a clear association between plasma TMAO levels and increased cardiovascular risk and mortality in all subjects analyzed. Therefore, it is believed that risk reduction or preventive effects can be obtained for cardiovascular disease by reducing serum TMAO levels through LC-MUFA intake in healthy individuals.

The present invention provides food and pharmaceutical compositions that reduce the risk of contracting diseases caused by elevated serum levels of TMAO.

Claims (8)

A food composition for reducing serum trimethylamine N-oxide, comprising:
The present invention comprises, as an active ingredient, a component selected from monounsaturated fatty acids having 20 or more carbon atoms, salts thereof, and esters thereof,
The food composition (excluding food compositions for reducing the risk of contracting cardiovascular disease) , wherein the active ingredient is derived from fish oil. The food composition according to claim 1, wherein the active ingredient is a component selected from monounsaturated fatty acids having 20 carbon atoms, salts thereof and esters thereof, a component selected from monounsaturated fatty acids having 22 carbon atoms, salts thereof and esters thereof, or a combination thereof. The food composition according to claim 1 or 2, wherein the active ingredient is a glyceride containing monounsaturated fatty acids having 20 or more carbon atoms as constituent fatty acids. The food composition according to claim 3, wherein the glyceride is a triglyceride. The food composition according to any one of claims 1 to 4, further comprising n-3 polyunsaturated fatty acids and n-6 polyunsaturated fatty acids, the weight ratio of n-3 polyunsaturated fatty acids to n-6 polyunsaturated fatty acids being 4.0 or more. The food composition according to any one of claims 1 to 5, wherein the fish oil is saury oil. The food composition according to any one of claims 1 to 6, characterized in that the active ingredient is orally administered at 2 mg/kg body weight/day or more, calculated as a monounsaturated fatty acid having 20 or more carbon atoms, for 4 weeks or more. The food composition according to any one of claims 1 to 7, characterized in that it is ingested by healthy individuals.

Images