JP2024051305A - Carbohydrate burning promoter - Google Patents

Abstract

The present invention provides a carbohydrate burning accelerator that exhibits an excellent carbohydrate burning promotion effect. The carbohydrate burning accelerator contains, as an active ingredient, an oil containing diacylglycerol in which 20% by mass or more of the constituent fatty acids are eicosapentaenoic acid.

Description

The present invention relates to a carbohydrate burning accelerator.

In recent years, "carbohydrate-restricted diets" that reduce carbohydrate intake have been attracting attention, and in order to prevent metabolic syndrome including obesity, the burning of not only lipids but also carbohydrates in the body is an important factor.
Exercise is an effective way to promote carbohydrate burning. Exercise increases the metabolism of the whole body and increases energy consumption. However, exercise is a heavy burden and not everyone can easily do it. Therefore, there is a demand for carbohydrate burning promoters that can be used easily and daily and have few side effects.

Eicosapentaenoic acid (C20:5, EPA) is a linear highly unsaturated fatty acid with 20 carbon atoms and five double bonds, and has been reported to have physiological effects such as anti-inflammatory effects and platelet aggregation inhibitory effects. It has also been reported that oral intake of ethyl esters of EPA and docosahexaenoic acid (C22:6, DHA) promotes lipid utilization (Non-Patent Document 1). On the other hand, it has been reported that their effect on carbohydrate utilization is conversely suppressed (Non-Patent Document 1).
Furthermore, it has been reported that oral ingestion of EPA esters and DHA esters between meals suppresses the rise in blood glucose level after sugar loading (Patent Document 1), but the effect is limited to after meals.

On the other hand, it has been reported that fats and oils containing 5% by weight or more of diglycerides and/or monoglycerides whose constituent acyl groups have an ω3 unsaturated acyl group content of 15% by weight or more have the effect of lowering fasting blood glucose levels and blood insulin concentrations (Patent Documents 2 and 3).
However, in the above-mentioned Patent Documents 2 and 3, the ω3 unsaturated acyl group is mainly a docosahexaenoyl group or an α-linoleyl group. There have been no reports so far on the effect of diacylglycerol-containing fats and oils containing a high concentration of eicosapentaenoic acid on carbohydrate burning.

International Publication No. 2012/063820 JP 2001-247457 A JP 2001-247473 A

Charlotte J Green et al., Hepatic de novo lipogenesis is suppressed and fat oxidation is increased by omega-3 fatty acids at the expense of glucose metabolism, BMJ Open Diabetes Res Care., 2020, Vol. 8, No. 1, e000871

The present invention relates to providing a carbohydrate burning promoter that exhibits excellent carbohydrate burning promotion effects.

In light of the above problems, the inventors conducted extensive research and discovered that carbohydrate burning significantly increased when mice were continuously fed fats and oils containing diacylglycerol with a high concentration of eicosapentaenoic acid.

That is, the present invention provides a carbohydrate burning promoter that contains, as an active ingredient, an oil or fat containing diacylglycerol in which 20% by mass or more of the constituent fatty acids are eicosapentaenoic acid.

The carbohydrate burning accelerator of the present invention consistently exerts an excellent carbohydrate burning promoting effect. Therefore, it is useful for preventing, improving, or treating obesity, diabetes, and metabolic syndrome.

This shows the amount of carbohydrates burned during the light period. This shows the amount of carbohydrates burned during the dark period. The total amount of carbohydrates burned (light and dark periods) is shown.

In the present invention, "oils and fats" refers to ester compounds of fatty acids and glycerin, and includes one or more of monoacylglycerol, diacylglycerol, and triacylglycerol. There is no particular restriction on the type of oil and fat, and any oil that can be used as an edible oil and fat may be used.

The fats and oils used in the present invention are fats and oils containing diacylglycerol in which 20% by mass or more of the constituent fatty acids are eicosapentaenoic acid. Hereinafter, in this specification, "fat and oil containing diacylglycerol in which 20% by mass or more of the constituent fatty acids are eicosapentaenoic acid" may be simply referred to as "fat and oil of the present invention."

In the present invention, the content of eicosapentaenoic acid in the fatty acid constituting diacylglycerol is 20% by mass or more.The content of eicosapentaenoic acid in the fatty acid constituting diacylglycerol is 20% by mass or more, preferably 25% by mass or more, more preferably 27% by mass or more, and even more preferably 30% by mass or more, from the viewpoint of promoting carbohydrate combustion, and from the viewpoint of oxidation stability, it is preferably 99% by mass or less, more preferably 98% by mass or less, and even more preferably 95% by mass or less.In addition, the content of eicosapentaenoic acid in the fatty acid constituting diacylglycerol is preferably 20% by mass or more and 99% by mass or less, more preferably 25% by mass or more and 98% by mass or less, more preferably 27% by mass or more and 98% by mass or less, and even more preferably 30% by mass or more and 95% by mass or less.
In this specification, the amount of fatty acid is calculated as the amount of free fatty acid.

The constituent fatty acid other than eicosapentaenoic acid constituting the diacylglycerol is not particularly limited, and may be either a saturated fatty acid or an unsaturated fatty acid.
From the viewpoints of flavor and industrial productivity of the oil and fat, the content of unsaturated fatty acids in the fatty acids constituting the diacylglycerol is preferably 60 to 100% by mass, more preferably 70 to 99.8% by mass, and even more preferably 80 to 99.5% by mass. From the viewpoint of physiological effects, the number of carbon atoms of the unsaturated fatty acids is preferably 14 to 24, and more preferably 16 to 22.

In particular, the content of linoleic acid (C18:2) in the fatty acids constituting diacylglycerol is preferably 0.4% by mass or more, more preferably 0.5% by mass or more, even more preferably 1% by mass or more, even more preferably 1.2% by mass or more, from the viewpoint of industrial productivity of the oil and fat, and is preferably 30% by mass or less, more preferably 20% by mass or less, even more preferably 10% by mass or less.

The content of oleic acid (C18:1) in the fatty acids constituting the diacylglycerol is preferably 1% by mass or more, more preferably 5% by mass or more, and even more preferably 7% by mass or more, from the viewpoint of industrial productivity of the oil and fat, and is preferably 30% by mass or less, more preferably 25% by mass or less, and even more preferably 20% by mass or less.

In addition, the content of docosahexaenoic acid (C22:6) in the fatty acids constituting diacylglycerol is preferably 0.3% by mass or more, more preferably 3% by mass or more, even more preferably 4% by mass or more, even more preferably 5% by mass or more, from the viewpoint of industrial productivity of fats and oils, and is also preferably 30% by mass or less, more preferably 27% by mass or less, even more preferably 25% by mass or less, even more preferably 20% by mass or less. The content of docosahexaenoic acid in the fatty acids constituting diacylglycerol is preferably 30% by mass or less, more preferably 0.3 to 30% by mass, even more preferably 3 to 27% by mass, even more preferably 4 to 25% by mass, even more preferably 5 to 20% by mass.

The total content of saturated fatty acids in the fatty acids constituting the diacylglycerol is preferably 20% by mass or less, more preferably 15% by mass or less, even more preferably 13% by mass or less, and preferably 0.5% by mass or more, from the viewpoints of appearance, physiological effects, and industrial productivity of the oils and fats. The total content of saturated fatty acids in the fatty acids constituting the diacylglycerol is preferably 0 to 20% by mass, more preferably 0.5 to 15% by mass, and even more preferably 0.5 to 13% by mass.
In this specification, the saturated fatty acid preferably has 14 to 24 carbon atoms, more preferably 14 to 22 carbon atoms.

The content of diacylglycerol in the fats and oils of the present invention is not particularly limited, but from the viewpoint of effectively expressing the effects and from the viewpoint of physiological effects, it is preferably 40% by mass or more, more preferably 50% by mass or more, even more preferably 60% by mass or more, and even more preferably 65% by mass or more, and from the viewpoint of industrial productivity, it is preferably 99.5% by mass or less, more preferably 98% by mass or less, even more preferably 95% by mass or less, and even more preferably 90% by mass or less. The content of diacylglycerol in the fats and oils is preferably 40% by mass or more, more preferably 40 to 99.5% by mass, even more preferably 50 to 98% by mass, even more preferably 60 to 95% by mass, and even more preferably 65 to 90% by mass.

The fats and oils of the present invention may contain triacylglycerol, and the content thereof is preferably 1% by mass or more, more preferably 2% by mass or more, even more preferably 5% by mass or more, even more preferably 10% by mass or more, from the viewpoint of industrial productivity of the fats and oils, and is preferably 85% by mass or less, more preferably 80% by mass or less, even more preferably 75% by mass or less, even more preferably 50% by mass or less, even more preferably 40% by mass or less.

The content of monoacylglycerol in the fats and oils of the present invention is preferably 5% by mass or less, more preferably 3% by mass or less, even more preferably 2% by mass or less, even more preferably 1.5% by mass or less, and also preferably more than 0% by mass, from the viewpoints of flavor, industrial productivity of the fats and oils, and oxidation stability. The content of monoacylglycerol in the fats and oils of the present invention may be 0% by mass.

The fats and oils of the present invention may contain free fatty acids or salts thereof as impurities. From the viewpoints of flavor and oxidation stability, the content of free fatty acids or salts thereof in the fats and oils is preferably 3% by mass or less, more preferably 2% by mass or less, even more preferably 1% by mass or less, and also preferably more than 0% by mass. The content of free fatty acids or salts thereof in the fats and oils may be 0% by mass.

In the present invention, the fatty acids constituting the oil or fat may be the same as or different from the fatty acids constituting the diacylglycerol described above, but from the viewpoint of effectively exerting the effect, it is preferable that they are the same.
The content of eicosapentaenoic acid in the fatty acids constituting the fats and oils is preferably 20 to 99 mass%, more preferably 20 to 98 mass%, even more preferably 25 to 97 mass%, and even more preferably 30 to 95 mass%.
The content of unsaturated fatty acids in the fatty acids constituting the fat or oil is preferably 60 to 100% by mass, more preferably 70 to 99.8% by mass, and even more preferably 80 to 99.5% by mass.
The content of docosahexaenoic acid in the fatty acids constituting the oil or fat is preferably 30% by mass or less, more preferably 0.3 to 30% by mass, even more preferably 3 to 27% by mass, even more preferably 4 to 25% by mass, and even more preferably 5 to 20% by mass.

The total content of saturated fatty acids in the fatty acids constituting the oil or fat is preferably 0 to 20% by mass, more preferably 0.5 to 17% by mass, and even more preferably 0.5 to 15% by mass.

The fat or oil of the present invention can be obtained by, for example, an esterification reaction between a fatty acid derived from a fat or oil and glycerin, or a transesterification reaction (glycerolysis) between a fat or oil and glycerin.
The esterification reaction and the glycerolysis reaction are roughly classified into a chemical method using a chemical catalyst such as an alkali metal or an alloy thereof, an oxide or hydroxide of an alkali metal or an alkaline earth metal, or an alkoxide of an alkali metal or an alkaline earth metal, and an enzymatic method using an enzyme such as lipase.
Among these, from the viewpoint of controlling the fatty acid composition, an esterification reaction between fractionated fatty acids derived from fats and oils, which will be described later, and glycerin is preferred.

In the present invention, the oils and fats (edible oils and fats) may be either vegetable oils and fats or animal oils and fats. Examples of such oils and fats include vegetable oils and fats such as soybean oil, rapeseed oil, safflower oil, rice oil, corn oil, sunflower oil, cottonseed oil, olive oil, sesame oil, peanut oil, pearl barley oil, wheat germ oil, perilla oil, linseed oil, perilla oil, chia seed oil, sacha inchi oil, walnut oil, kiwi seed oil, salvia seed oil, grape seed oil, macadamia nut oil, hazelnut oil, pumpkin seed oil, camellia oil, tea seed oil, borage oil, palm oil, palm olein, palm stearin, coconut oil, palm kernel oil, cacao butter, monkey fat, shea butter, and algae oil; animal oils and fats such as fish oil, seal oil, whale oil, lard, beef tallow, and butter fat; and oils and fats such as interesterified oils, hydrogenated oils, and fractionated oils thereof. These may be used alone or in combination of two or more.

Fatty acids derived from fats and oils can be obtained by hydrolyzing fats and oils. Methods for hydrolyzing fats and oils include high-temperature, high-pressure hydrolysis and enzymatic hydrolysis. The high-temperature, high-pressure hydrolysis method is a method in which water is added to fats and oils and reacted under high temperature and high pressure conditions to obtain fatty acids and glycerin. The enzymatic hydrolysis method is a method in which water is added to fats and oils and reacted under low temperature conditions using an oil hydrolase as a catalyst to obtain fatty acids and glycerin.
The hydrolysis reaction can be carried out according to a conventional method.

After the hydrolysis of the oil or fat, it is preferable to remove solids by fractionating the hydrolysis reaction product. Examples of fractionation methods include solvent fractionation, natural fractionation (dry fractionation), and wetting agent fractionation.
The precipitated solid may be removed by means of static separation, filtration, centrifugation, mixing the fatty acid with an aqueous solution of a wetting agent, or the like.

The esterification reaction between fatty acids derived from fats and oils and glycerin is preferably carried out under mild conditions by an enzymatic method, which is excellent in terms of flavor and the like.
The amount of the enzyme used can be appropriately determined taking into consideration the activity of the enzyme. When an immobilized enzyme is used, the amount of the enzyme used is preferably 1 to 30% by mass, more preferably 2 to 20% by mass, based on the total mass of the raw materials for the esterification reaction, from the viewpoint of improving the reaction rate.
The reaction temperature of the esterification reaction is preferably 0 to 100° C., more preferably 20 to 80° C., and even more preferably 30 to 60° C., from the viewpoint of improving the reaction rate and suppressing the deactivation of the enzyme. Moreover, the reaction time is preferably within 15 hours, more preferably 1 to 12 hours, and even more preferably 2 to 10 hours, from the viewpoint of industrial productivity of fats and oils.
The fatty acid and glycerin can be brought into contact with each other by immersion, stirring, or passing the mixture through a column packed with immobilized lipase using a pump or the like.

The transesterification reaction (glycerolysis) between fats and oils and glycerin is preferably carried out by a chemical method from the viewpoint of reactivity.
From the viewpoint of reactivity, the amount of the catalyst used is preferably 0.001 to 3 mass %, more preferably 0.005 to 2 mass %, and even more preferably 0.01 to 1 mass %, based on the mass of the reaction raw materials.

The reaction temperature is preferably 120 to 200° C., more preferably 150 to 180° C., from the viewpoint of reactivity and suppression of formation of by-products. The reaction time is preferably 6 hours or less, more preferably 4 hours or less, from the viewpoint of reactivity.
In the present invention, the pressure in the reaction system during the ester exchange reaction is not particularly limited, and the reaction can be carried out under normal pressure or reduced pressure. In the case of normal pressure, it is preferable to carry out the reaction under a nitrogen gas flow from the viewpoint of reactivity.

After the esterification reaction or transesterification reaction (glycerolysis), a refining process that is usually used for oils and fats may be carried out. Specifically, such processes include distillation, acid treatment, water washing, bleaching, and deodorization.

As shown in the examples below, when the fats and oils of the present invention were orally administered to obese/type 2 diabetes model mice (db/db mice), a significant increase in carbohydrate burning was observed in both the light period, the dark period, and the overall period (light period and dark period). That is, the fats and oils of the present invention have a carbohydrate burning promotion effect, and the effect is constant. Here, constant carbohydrate burning promotion means that the effect of carbohydrate burning promotion continues for a longer period, unlike temporary carbohydrate burning promotion that disappears within a few hours at most after a meal. If carbohydrate burning is promoted by the fats and oils of the present invention, energy consumption will increase, and it will be possible to prevent, improve, or treat the onset or progression of diseases or symptoms such as obesity, metabolic syndrome (visceral fat syndrome), and type 2 diabetes.
Therefore, the fat or oil of the present invention can be a carbohydrate burning accelerator, can be used for the acceleration of carbohydrate burning, and can be used to produce a carbohydrate burning accelerator.

In the present invention, "promotion of carbohydrate burning" is a concept including increasing the degree of carbohydrate burning compared to when the oil or fat of the present invention is not administered or ingested. "Carbohydrate burning" refers to metabolizing carbohydrates ingested from food in each organ and using them as an energy source, and the amount of carbohydrate burned can be calculated, for example, by measuring breath using the formula shown in the Examples below. Note that carbohydrates are synonymous with carbohydrates and are classified into monosaccharides, disaccharides, oligosaccharides, and polysaccharides.
"Use" may be for humans or non-human animals, and may be therapeutic or non-therapeutic. "Non-therapeutic" is a concept that does not include medical procedures, i.e., methods of surgery, therapy, or diagnosis of humans, and more specifically, does not include methods of surgery, therapy, or diagnosis performed by a physician, or a medical professional, or a person under the direction of a physician, on humans.
"Prophylaxis" refers to preventing or delaying the onset of a disease or condition in an individual, or reducing an individual's risk of developing a disease or condition.
"Amelioration" refers to improving a disease, symptom or condition, preventing or slowing the worsening of a disease, symptom or condition, or reversing, preventing or slowing the progression of a disease or symptom.
Furthermore, "treatment" includes not only complete cure of a disease but also amelioration of symptoms.

The carbohydrate combustion promoter of the present invention can itself be a medicine, quasi-drug, cosmetic, food, or feed for promoting carbohydrate combustion, or can be a material or preparation that is mixed with the medicine, quasi-drug, cosmetic, food, or feed to be used.

The pharmaceutical (including quasi-drugs, the same applies below) contains the fat or oil of the present invention as an active ingredient for promoting carbohydrate burning. Furthermore, the pharmaceutical may contain a pharma- ceutically acceptable carrier, or other active ingredients, pharmacological ingredients, etc., as necessary, so long as the function of the active ingredient is not lost.
The pharmaceutical agent may be administered in any dosage form. Examples of dosage forms include oral solid preparations such as tablets (including chewable tablets), capsules, granules, powders, and lozenges, oral liquid preparations such as oral liquids and syrups, and parenteral preparations such as injections, suppositories, inhalants, transdermal agents, and external preparations. The preferred dosage form is oral administration. The size of the dosage form can be adjusted as desired depending on the purpose of use.
Preparations of such various dosage forms can be prepared according to standard methods by appropriately combining, as necessary, pharma- ceutically acceptable carriers such as excipients, binders, bulking agents, disintegrants, surfactants, lubricants, dispersants, buffers, osmotic pressure regulators, pH adjusters, emulsifiers, preservatives, stabilizers, thickeners, flow improvers, flavoring agents, foaming agents, fragrances, coating agents, diluents, and other medicinal ingredients.

The cosmetic contains the oil of the present invention as an active ingredient for promoting carbohydrate burning, and may further contain a carrier acceptable for cosmetics, or other active ingredients, medicinal ingredients, cosmetic ingredients, etc., as necessary, so long as the function of the active ingredient is not lost.
Preferred examples of cosmetics containing the oil or fat of the present invention include face and body cosmetics (for example, lotions, gels, creams, packs, etc.), make-up cosmetics, face or body cleansers, etc.
Each of the preparations of such cosmetics can be manufactured according to a conventional method. Examples of carriers acceptable for cosmetics include various oils, surfactants, gelling agents, buffers, preservatives, antioxidants, solvents, dispersants, chelating agents, thickeners, UV absorbers, emulsion stabilizers, pH adjusters, colorants, fragrances, etc.
Other active ingredients, medicinal ingredients, and cosmetic ingredients include, for example, plant extracts, bactericides, moisturizers, anti-inflammatory agents, antibacterial agents, keratolytic agents, cooling agents, antiseborrheic agents, cleansing agents, and makeup ingredients.

The food contains the oil or fat of the present invention as an active ingredient for promoting carbohydrate burning.
Foods include foods that claim to promote carbohydrate burning and have been approved or notified to be labeled as such, as necessary (foods for specified health uses, foods with functional claims, foods for special dietary uses, etc.). Examples of such claims include "promotes carbohydrate burning,""increases the ability to metabolize carbohydrates," and "makes carbohydrates easier to consume." Foods that have been approved or notified to be labeled as such can be distinguished from general foods.
The food may be in the form of a solid, semi-solid, or liquid (e.g., beverage). Examples include various food compositions (breads, cakes, noodles, confectioneries, frozen foods, ice creams, candies, toppings, soups, dairy products, shakes, beverages, seasonings, etc.), and nutritional supplement compositions in the same form as the oral preparations described above (solid preparations such as granules, powders, tablets, capsules, microcapsules, and lozenges).
Foods of various forms can be prepared according to standard methods by appropriately combining the oil or fat of the present invention with any food material, or other active ingredients, or additives acceptable for foods (e.g., solvents, softeners, oils, emulsifiers, preservatives, acidulants, sweeteners, bittering agents, pH adjusters, stabilizers, colorants, UV absorbers, antioxidants, moisturizers, thickeners, adhesives, dispersants, flow improvers, humectants, aromatics, seasonings, flavor adjusters), etc.

The feed contains the oil or fat of the present invention as an active ingredient for promoting carbohydrate burning.
The feed is preferably in the form of pellets, flakes, mash or liquid, and examples thereof include livestock feed for cows, pigs, chickens, sheep, horses, etc., small animal feed for rabbits, rats, mice, etc., and pet food for dogs, cats, small birds, etc.
Feed can be prepared according to standard methods by appropriately combining the oil or fat of the present invention with other feed ingredients such as meat, protein, grains, brans, lees, sugars, vegetables, vitamins, minerals, gelling agents, shape-retaining agents, pH adjusters, seasonings, preservatives, nutritional supplements, etc.

The content of the oil or fat of the present invention in the formulation cannot be generalized because it varies depending on the form of the formulation, but for example, based on the total amount of the formulation, it is preferably 0.5 mass% or more and preferably 99.8 mass% or less.

The amount of the oil or fat of the present invention to be administered or used may be an amount that can achieve the effects of the present invention. The amount of the oil or fat to be administered or used may vary depending on the species, weight, sex, age, condition, or other factors of the subject, but in the case of oral administration (ingestion), the amount of the oil or fat of the present invention to be administered once per adult (60 kg) is, for example, 0.1 to 15 g.
The carbohydrate burning accelerator of the present invention may be administered or used according to any administration schedule, and may be administered or used repeatedly and continuously, once or multiple times a day, for one day or more, preferably for seven days or more, more preferably for 14 days or more, and even more preferably for 28 days or more.

The carbohydrate burning accelerator of the present invention is preferably administered or used in the form of an oil or fat composition.
When the carbohydrate combustion promoter is in the form of an oil composition, the content of the oil of the present invention in the oil composition is, from the viewpoint of usability, preferably 90% by mass or more, more preferably 95% by mass or more, and is preferably 100% by mass or less, more preferably 99.9% by mass or less.

The oil and fat composition preferably contains an antioxidant from the viewpoints of flavor, oxidation stability, coloring inhibition, etc. The content of the antioxidant in the oil and fat composition is preferably 0.005 to 1.0 mass%, more preferably 0.04 to 0.75 mass%, and even more preferably 0.08 to 0.5 mass%, from the viewpoints of flavor, oxidation stability, coloring inhibition, etc.
The antioxidant is not particularly limited, but is preferably at least one selected from natural antioxidants, lecithin, tocopherol, ascorbyl palmitate, ascorbyl stearate, dibutylhydroxytoluene (BHT), butylhydroxyanisole (BHA), and the like.

The subject to which the carbohydrate burning promoter of the present invention is administered or used is not particularly limited, so long as it is a human or non-human animal that requires or desires it. Preferred examples of subjects include mongoloids, etc. Non-human animals include non-human mammals such as apes and other primates.

Preparation of test oils Oil 1: 328 parts by mass of fatty acid obtained by enzymatic hydrolysis of DD Oil Type 2 (manufactured by Nippon Suisan Co., Ltd.) was mixed with 50 parts by mass of glycerin, and esterification reaction was carried out using 1,3-position selective lipase (manufactured by Novozymes Co., Ltd.) immobilized on ion exchange resin as a catalyst, and the immobilized enzyme was filtered off. Then, molecular distillation, acid treatment and water washing were carried out to obtain a treated oil. The treated oil was deodorized to obtain oil 1.
Oil and fat 2: 80 parts by mass of algae oil (manufactured by DSM) and 25 parts by mass of glycerin were mixed and subjected to a glycerolysis reaction using calcium hydroxide as a catalyst. Then, molecular distillation, acid treatment and water washing were performed to obtain a treated oil. The treated oil was deodorized to obtain oil and fat 2.
The glyceride compositions of oils and fats 1 and 2, the major fatty acid compositions in the oils and fats, and the major fatty acid compositions in diacylglycerol are shown in Table 1.

The methods for analyzing the glyceride composition and fatty acid composition are as follows.
(i) Glyceride Composition of Fats and Oils Fats and oils were dissolved in a mixed solvent of chloroform/methanol (1/9) to a concentration of 1 mg/ml, diluted 10-fold with methanol, and then subjected to high performance liquid chromatography (HPLC) together with a standard sample for correction to analyze.
<Standard sample>
(1) For diacylglycerol: triolein/diolein/monoolein = 5/80/15
(Weight ratio)
(2) For triacylglycerol: triolein/diolein/monoolein = 90/5/5 (weight ratio)
<HPLC analysis conditions>
(conditions)
Apparatus: UHPLC (Thermo Fisher Scientific)
Column: L-column C8 (2.1 x 35 mm x 5 μm) (manufactured by Chemicals Evaluation and Research Institute, Japan)
Column temperature: 40°C
Sample injection volume: 10 μL
Flow rate: 0.5 mL / min
Mobile phase: (A) water/acetonitrile = 9/1 (B) isopropanol

Detector: CAD (charged particle detector)

(ii) Fatty acid composition of fats and oils Fatty acid methyl esters were prepared according to "Preparation method of fatty acid methyl esters (2.4.1.-1996)" in "Standard Methods for the Analysis of Fats, Oils and Related Materials" edited by the Japan Oil Chemists' Society, and the fatty acid composition of the obtained fat and oil samples was measured in accordance with American Oil Chemists. Society Official Method Ce 1f-96 (GLC method).
<GLC analysis conditions>
Apparatus: Agilent 7890B (Agilent Technologies)
Column: CP-SIL88 50 m x 0.25 mm x 0.2 μm (Agilent J&W)
Carrier gas: 1.0 mL He/min
Injector: Split (1:50), T = 300 °C
Detector: FID, T = 300 ° C.
Oven temperature: 150°C for 5 min, increase at 1°C/min, increase at 160°C for 5 min, increase at 2°C/min, increase at 200°C for 10 min, increase at 10°C/min, increase at 220°C for 5 min

Test method overview Six-week-old obese/type 2 diabetes model mice (db/db mice, male) were pre-bred for one week to acclimate them to the environment, and then divided into three groups of five mice each with similar body weight: a high-fat/high-sucrose diet group (group 1), a high EPA-containing DAG group (group 2), and a high DHA-containing DAG group (group 3), and bred on each of the diets listed in Table 3. The breeding environment was a room temperature of 23±2°C, a humidity of 55±10%, and lighting hours from 7:00 a.m. to 7:00 p.m.

After 4 weeks of feeding, the mice were placed in the chamber of an exhaled gas analyzer (Arco 2000 system, Arco Systems) and allowed to acclimate for 24 hours. After that, the amount of carbohydrate burned was measured by measuring the exhaled breath for another 24 hours.
The carbohydrate burning rate was calculated with reference to Jequier's formula (described in Parenter Enteral Nutr, Vol. 11, pp. 86-89, 1987). That is, the oxygen consumption (VO ₂ ) and carbon dioxide discharge (VCO ₂ ) measured using an exhaled gas analyzer were applied to the following formula.

Carbohydrate burning rate (mg/min)
= 1.689 × _VO2 (mL/min) - 1.689 × _VCO2 (mL/min) - 0.324 × P (mg/min)
_VO2 : Oxygen consumption rate _VCO2 : Carbon dioxide excretion rate P: Protein combustion rate (In this test, the protein combustion rate was calculated assuming that it was 12.5% of the energy consumption rate (EE). EE = 3.9 x _VO2 (mL/min) + 1.1 x _VCO2 (mL/min).)

Test Results The results of the measurements are shown in Figures 1 to 3. Figure 1 shows the amount of carbohydrate burned per body weight during the light period (rest period), Figure 2 shows the dark period (active period), and Figure 3 shows the total amount (light and dark periods, 24 hours). Compared to the high-fat, high-sucrose diet group (Group 1), the high-DHA-containing DAG group (Group 3) did not show an increase in the amount of carbohydrate burned during the light period, dark period, or total period, but the high-EPA-containing DAG group (Group 2) showed a significant increase in carbohydrate burned during the light period, dark period, and total period.
Significant differences were tested using an unpaired t-test (*P<0.05), and the analysis results were expressed as mean values ± standard error.

Claims (5)

A carbohydrate burning promoter whose active ingredient is an oil containing diacylglycerol, the constituent fatty acids of which are 20% by mass or more of eicosapentaenoic acid. The carbohydrate combustion promoter according to claim 1, wherein the content of diacylglycerol in the oil or fat is 40% by mass or more. The carbohydrate combustion promoter according to claim 1 or 2, wherein the total content of saturated fatty acids in the fatty acids constituting the diacylglycerol is 20% by mass or less. The carbohydrate burning promoter according to any one of claims 1 to 3, wherein the content of docosahexaenoic acid in the fatty acids constituting the diacylglycerol is 30% by mass or less. The carbohydrate burning enhancer according to any one of claims 1 to 4, which is used as a medicine or food.