WO2020218382A1 - Glp-1 secretagogue - Google Patents

Abstract

It has been expected to develop a novel GLP-1 secretagogue which promotes the secretion of GLP-1. The present invention provides a GLP-1 secretagogue characterized by comprising 0.4-4,440 ppm of a steviol glycoside having a structure to which rhamnose is attached.

Description

GLP-1 secretagogue

The present invention relates to a GLP-1 secretagogue and the like. More specifically, the present invention relates to a GLP-1 secretagogue containing a steviol glycoside having a structure to which rhamnose is bound.

GLP-1 [Glucagon-like peptide-1] is a hormone secreted from the gastrointestinal mucosal epithelium and the like. Known actions of GLP-1 include stimulation of insulin synthesis and secretion, inhibition of glucagon secretion, inhibition of food intake, and reduction of hyperglycemia. By activating the secretion of GLP-1, these effects can be expected to be improved.

Patent Document 1 describes a GLP-1 secretion-promoting composition containing at least one component (A) selected from 11 types of curbitan-type triterpenes contained in bitter gourd. Further, Patent Document 2 describes that stevioside, rebaugioside A, rebaugioside B, and rebaugioside D have a property of promoting the secretion of GLP-1.

International Publication No. 2017/159725 International Publication No. 2017/018404

The development of a new GLP-1 secretion promoter that promotes GLP-1 secretion is expected.

One aspect of the invention provides:
[1] A GLP-1 secretagogue characterized by containing 0.4 to 4,440 ppm of a steviol glycoside having a structure to which rhamnose is bound.
[2] The GLP-1 according to the above [1], wherein the steviol glycoside contains at least one selected from the group consisting of rebaugioside C, rebaugioside K, zulucoside A, zulucoside C, zulucoside D and zulucoside F. Secretory promoter.
[3] The GLP-1 secretagogue according to the above [1], wherein the steviol glycoside contains rebaudioside C.
[4] The GLP-1 secretagogue according to any one of the above [1] to [3], which is used for improving glucose metabolism, suppressing appetite, or preventing or improving diabetes or obesity.
[5] A GLP-1 secretagogue beverage containing the GLP-1 secretagogue according to any one of the above [1] to [4].
[6] A GLP-1 secretion-promoting food containing the GLP-1 secretion-promoting agent according to any one of the above [1] to [4].
[7] A pharmaceutical composition comprising the GLP-1 secretagogue according to any one of the above [1] to [4].

The GLP-1 secretagogue of the present invention can be used in applications where promotion of GLP-1 secretion is desired.

It is a graph which shows the GLP-1 secretion amount of various sweeteners in Experimental Example 1. It is a graph which shows the relationship between the concentration of the sample containing rebaugioside C (hereinafter, rebaugioside may be abbreviated as Reb) obtained in Experimental Example 2 and the amount of GLP-1 secreted. It is a graph which shows the relationship between the concentration of the stevioside-containing sample obtained in Experimental Example 3 and the amount of GLP-1 secretion. It is a graph which shows the relationship between the concentration of the rebaugioside A-containing sample obtained in Experimental Example 4 and the amount of GLP-1 secretion. It is a graph which shows the relationship between the concentration of the RebA emulsion-containing sample obtained in Experimental Example 5 and the amount of GLP-1 secretion. It is a graph which shows the surface tension which various sweeteners have in Experimental Example 6. It is a graph which shows the flavor characteristic of RebA in Experimental Example 7. It is a graph which shows the relationship between the concentration of various samples obtained in Experimental Examples 8 to 13 and the amount of GLP-1 secretion. It is a graph which shows the relationship between the concentration of the RebA-containing sample obtained in Experimental Example 8 and the amount of GLP-1 secretion. It is a graph which shows the relationship between the concentration of the RebA emulsion-containing sample obtained in Experimental Example 9 and the amount of GLP-1 secretion. It is a graph which shows the relationship between the concentration of the emulsified particle-containing sample obtained in Experimental Example 10 and the amount of GLP-1 secretion. 6 is a graph showing the relationship between the concentration of the mixed solution of the RebA-containing sample and the emulsified particle-containing sample and the amount of GLP-1 secreted obtained in Experimental Example 11. It is a graph which shows the relationship between the concentration of the glycerin-containing sample obtained in Experimental Example 12 and the amount of GLP-1 secretion. It is a graph which shows the relationship between the concentration of the MCT oil-containing sample obtained in Experimental Example 13 and the amount of GLP-1 secretion. It is a graph which shows the cytotoxicity (indicator of dead cell) of various samples obtained in Experimental Examples 15-20. a is the result 2 hours after administration, and b is the result 24 hours after administration. It is a graph which shows the GLP-1 secretion amount of various samples obtained in Experimental Examples 21-31. a is the result 2 hours after administration, and b is the result 24 hours after administration. It is a photograph which shows the distribution ratio of each steviol glycoside obtained in Experimental Example 32, and the state during a test. It is a photograph which shows the state of the RebA containing solution after the test in Experimental Example 32. It is a figure which shows the influence of the storage temperature on the flavor characteristic in Experimental Example 33. It is a figure which shows the stability in the vibration environment in Experimental Example 33. It is a figure which shows the degree of decrease of the sweetness intensity by emulsification in Experimental Example 33. It is a figure which shows the GLP-1 secretion amount in Experimental Example 34.

According to the research of the present inventor, a steviol glycoside having a rhamnose-bound structure such as RebC acts on cells of humans or non-human animals having GLP-1 secretory ability by itself to secrete GLP-1. It was found that it has the effect of increasing GLP-1, that is, it has the ability to promote GLP-1 secretion. One aspect of the present invention relates to a GLP-1 secretagogue (GLP-1 secretagogue composition) containing a steviol glycoside having a structure to which rhamnose is bound.

Rhamnose is a hexose having the following structure and represented by the chemical formula C ₆ H ₁₂ O ₅ . Rhamnose is sometimes called 6-deoxymannose, mannomethyrose.

Preferably, the rhamnose-based steviol glycoside contains at least one selected from the group consisting of rebaugioside C, rebaugioside K, zulucoside A, zulucoside C, zulucoside D and zulucoside F. More preferably, the rhamnose-based steviol glycoside comprises rebaugioside C.

The structures of Zulcoside A, Zulcoside C, Zulcoside D and Zulcoside F are as follows.

The content ratio of the rhamnose-based steviol glycoside in the GLP-1 secretion promoter of the present invention is 0.4 to 4,440 ppm. Preferably, 0.4 to 3,440, 0.4 to 2,440, 0.4 to 1,440, 0.4 to 940, 0.4 to 440, 0.4 to 240, 0 on a mass basis. .4-40, 0.4-30, 0.4-20, 0.4-10, 0.4-5, 0.4-1, 1-4,440, 5-4,440, 10-4 , 440, 50-4,440, 100-4,440, 300-4,440, 500-4,440, 700-4,440, 900-4,440, 1,000-4,440, 2,000 To 4,440, 3,000 to 4,440, 1 to 3,440, 5 to 2,440, 10 to 1,440, 50 to 940 or 100 to 440 ppm. Alternatively, preferably, on a mass basis, 95 to 1,000, 95 to 900, 95 to 800, 95 to 700, 95 to 600, 95 to 500, 95 to 400, 95 to 200, 200 to 1,000, 300 to 1,000, 400 to 1,000, 500 to 1,000, 600 to 1,000, 700 to 1,000, 800 to 1,000, 900 to 1,000, 200 to 900, 300 to 800, It is 400 to 700 or 500 to 600 ppm.

Alternatively, preferably, the content ratio of the rhamnose-based steviol glycoside in the GLP-1 secretagogue of the present invention is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60. , 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% by mass or more, and a suitable upper limit value is 100% by mass or less.

Alternatively, preferably, the content ratio of the rhamnose-based steviol glycoside in the GLP-1 secretagogue of the present invention is 95 to 10,000 or 950 to 10,000 ppm on a mass basis.

In the present invention, a rhamnose-based steviol glycoside may be combined with another glycoside sweetener. Other glycoside sweeteners include, for example, steviol, rebaudioside A, rebaugioside B, rebaugioside D, rebaudioside F, rebaugioside M, rebaudioside N, rebaugioside O, rebaugioside E, rubusoside and other ramnorth-free steviol glycosides; Mogrosides such as mogroside IV, 11-oxo-MogV, siamenocid I, and mogroside V (hereinafter, mogroside may be abbreviated as Mog); glycyrrhizic acid glycosides; and the like.

Other glycoside sweeteners preferably contain those having GLP-1 secretagogue, and include steviol glycosides or mogrosides without ramnorose, provided that they have GLP-1 secretagogue. More preferably, it further preferably contains at least one selected from the group consisting of RebA, RebB, stevioside or mogroside V, and particularly preferably contains RebA.

When the ratio of other glycoside sweeteners to the rhamnose-based steviol glycoside is expressed by P (mass%), P is preferably in the following numerical range.
1-100,000, 5-100,000, 10-100,000, 30-100,000, 50-100,000, 70-100,000, 90-100,000, 100-100,000, 200-100,000, 300-100,000, 400-100,000, 500-100,000, 600- 100,000, 700-100,000, 800-100,000, 900-100,000, 1-9,000, 5-9,000, 10-9,000, 30-9,000, 50-9,000, 70-9,000, 90-9,000, 100-9,000, 200-9,000, 300-9,000, 400-9,000, 500-9,000, 600-9,000, 700-9,000, 800-9,000, 900-9,000, 1-8,000, 5-8,000, 10-8,000, 30-8,000, 50-8,000, 70- 8,000, 90-8,000, 100-8,000, 200-8,000, 300-8,000, 400-8,000, 500-8,000, 600-8,000, 700-8,000, 800-8,000, 900-8,000, 1-7,000, 5-7,000, 10-7,000, 30-7,000, 50-7,000, 70-7,000, 90-7,000, 100-7,000, 200-7,000, 300-7,000, 400-7,000, 500-7,000, 600-7,000, 700-7,000, 800- 7,000, 900-7,000, 1-6,000, 5-6,000, 10-6,000, 30-6,000, 50-6,000, 70-6,000, 90-6,000, 100-6,000, 200-6,000, 300-6,000, 400-6,000, 500-6,000, 600-6,000, 700-6,000, 800-6,000, 900-6,000, 1-5,000, 5-5,000, 10-5,000, 30-5,000, 50-5,000, 70-5,000, 90-5,000, 100- 5,000, 200-5,000, 300-5,000, 400-5,000, 500-5,000, 600-5,000, 700-5,000, 800-5,000, 900-5,000, 1-4,000, 5-4,000, 10-4,000, 30-4,000, 50-4,000, 70-4,000, 90-4,000, 1 00-4,000, 200-4,000, 300-4,000, 400-4,000, 500-4,000, 600-4,000, 700-4,000, 800-4,000, 900-4,000, 1-3,000, 5-3,000, 10-3,000, 30- 3,000, 50-3,000, 70-3,000, 90-3,000, 100-3,000, 200-3,000, 300-3,000, 400-3,000, 500-3,000, 600-3,000, 700-3,000, 800-3,000, 900-3,000, 1-2,000, 5-2,000, 10-2,000, 30-2,000, 50-2,000, 70-2,000, 90-2,000, 100-2,000, 200-2,000, 300-2,000, 400-2,000, 500-2,000, 600- 2,000, 700-2,000, 800-2,000 or 900-2,000.

Further, P is preferably in the following numerical range.
1-100, 5-100, 10-100, 30-100, 50-100, 70-100, 90-100, 1-90, 5-90, 10-90, 30-90, 50-90, 70- 90, 1-80, 5-80, 10-80, 30-80, 50-80, 70-80, 1-70, 5-70, 10-70, 30-70, 50-70, 1-60, 5-60, 10-60, 30-60, 50-60, 1-50, 5-50, 10-50, 30-50, 1-40, 5-40, 10-40, 30-40, 1- 30, 5-30, 10-30, 1-20, 5-20, 10-20 or 1-10.

Furthermore, P is preferably in the following numerical range.
100-10,000, 200-10,000, 300-10,000, 400-10,000, 500-10,000, 600-10,000, 700-10,000, 800-10,000, 900-10,000, 1,000-10,000, 2,000-10,000, 3,000-10,000, 4,000- 10,000, 5,000-10,000, 6,000-10,000, 7,000-10,000, 8,000-10,000, 9,000-10,000, 100-9,000, 200-9,000, 300-9,000, 400-9,000, 500-9,000, 600-9,000, 700-9,000, 800-9,000, 900-9,000, 1,000-9,000, 2,000-9,000, 3,000-9,000, 4,000-9,000, 5,000-9,000, 6,000-9,000, 7,000-9,000, 8,000-9,000, 100-8,000, 200-8,000, 300- 8,000, 400-8,000, 500-8,000, 600-8,000, 700-8,000, 800-8,000, 900-8,000, 1,000-8,000, 2,000-8,000, 3,000-8,000, 4,000-8,000, 5,000-8,000, 6,000-8,000, 7,000-8,000, 100-7,000, 200-7,000, 300-7,000, 400-7,000, 500-7,000, 600-7,000, 700-7,000, 800-7,000, 900-7,000, 1,000-7,000, 2,000-7,000, 3,000- 7,000, 4,000-7,000, 5,000-7,000, 6,000-7,000, 100-6,000, 200-6,000, 300-6,000, 400-6,000, 500-6,000, 600-6,000, 700-6,000, 800-6,000, 900-6,000, 1,000-6,000, 2,000-6,000, 3,000-6,000, 4,000-6,000, 5,000-6,000, 100-5,000, 200-5,000, 300-5,000, 400-5,000, 500-5,000, 600-5,000, 700-5,000, 800- 5,000, 900-5,000, 1,000-5,000, 2,000-5,000 0, 3,000-5,000, 4,000-5,000, 100-4,000, 200-4,000, 300-4,000, 400-4,000, 500-4,000, 600-4,000, 700-4,000, 800-4,000, 900-4,000, 1,000-4,000, 2,000-4,000, 3,000-4,000, 100-3,000, 200-3,000, 300-3,000, 400-3,000, 500-3,000, 600-3,000, 700-3,000, 800-3,000, 900-3,000, 1,000-3,000, 2,000- 3,000, 100-2,000, 200-2,000, 300-2,000, 400-2,000, 500-2,000, 600-2,000, 700-2,000, 800-2,000, 900-2,000, 1,000-2,000, 1500-10000, 1500-9000, 1500-8000, 1500-7000, 1500-6000, 1500-5000, 1500-4000, 1500-3000 or 1500-2000.

The GLP-1 secretagogue of the present invention may contain components other than the above-mentioned rhamnose-based steviol glycoside and other glycoside sweeteners appropriately contained, as long as the effects of the present invention are not impaired. .. Other ingredients include rhamnose-based steviol glycosides and other sweeteners other than glycoside sweeteners, emulsifiers, proteins, glycoproteins, excipients, binders, disintegrants, coatings, lubricants. , Colorants, flavoring agents, stabilizers, absorption promoters, pH adjusters, preservatives, antioxidants, fragrances, vitamins, trace metal components and the like.

Other sweeteners may be natural sweeteners, sugar alcohols, artificial sweeteners and the like. For example, glucose; fructose; maltose; sucrose; lactose; rare sugars; peptide-based sweeteners such as aspartame, neotheme, and ariteme; sucrose derivatives such as sucralose; synthetic sweeteners such as assesulfam K, saccharin, advantheme, ticlo, and zultin. Vegetable protein-based sweeteners such as monerin, curculin, brazein, and somatin; taumarin; neohesperidin dihydrocalcone; sugar alcohols such as erythritol, xylitol, sorbitol, martitol, and mannitol; high fructose syrup; High fructose corn syrup; oligo sugar; honey; sugar cane juice (brown sugar honey); sugar (sucrose, warm sugar, brown sugar, Japanese bonsai, etc.); maple syrup; moraces (sugar honey); water candy and the like.

The GLP-1 secretion-promoting agent of the present invention may have any shape, and may be, for example, solid (for example, powder), gel or liquid.

The GLP-1 secretagogue of the present invention can be used for the purpose of actions / functions caused by or related to the GLP-1 secretagogue action.

For example, the GLP-1 secretagogue of the present invention is used for suppressing blood glucose elevation, suppressing appetite, suppressing overeating, improving glucose metabolism, preventing or treating diabetes, preventing or treating obesity, and body weight. It can be used for at least one application selected from reduction and reduction of body fat percentage. Preferably, it can be used for at least one application selected from for improving glucose metabolism, for suppressing appetite, and for preventing or ameliorating diabetes or obesity.

In addition, for the purpose of promoting gastric emptying, suppressing gastric acid secretion, suppressing hepatic glucose release, cardioplegia, improving learning memory, preventing or treating postprandial hyperglycemia (hidden diabetes), preventing or treating ulcerative colitis, etc. It can also be used.

It can also be used to prevent or treat vascular diseases such as atherosclerosis, neurodegenerative diseases, non-alcoholic hepatitis, ulcers, and asthma (see: Young-Sun Lee et al., "Anti-Inflammatory". Effects of GLP-1-Based Therapies beyond Glucose Control ", Mediators of Inflammation Volume 2016, Article ID 3094642, 11 pages).

The GLP-1 secretagogue of the present invention can be used (ingested, taken or administered) directly on humans or non-human animals having GLP-1 secretagogue ability.

When used directly on humans or non-human animals, the GLP-1 secretion-promoting agent of the present invention includes for suppressing an increase in blood glucose level, for suppressing appetite, for suppressing overeating, for improving glucose metabolism, and for preventing or treating diabetes. , For prevention or treatment of obesity, for weight reduction, for reducing body fat ratio, for promoting gastric content excretion, for suppressing gastric acid secretion, for suppressing hepatic glucose release, for myocardial protection, for improving learning memory, vascular disease Foods for the prevention or treatment of, prevention or treatment of neurodegenerative diseases, prevention or treatment of non-alcoholic hepatitis, prevention or treatment of kanpan, or prevention or treatment of asthma, such as noodles (soba, udon, Chinese noodles) Noodles, instant noodles, etc.), tofu, confectionery (candy, gum, chocolate, snacks, biscuits, cookies, gummy, etc.), breads, marine or processed livestock foods (kamaboko, ham, sausage, etc.), dairy products (fermented milk) ), Fats and oils processed foods (salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressings, fat spreads, etc.), seasonings (sauce, sauce, etc.), cooked or semi-cooked foods (champles, etc.), Examples include retort foods (curry, stew, bowls, porridge, miscellaneous dishes, etc.), cold confectionery (ice cream, sherbet, shaved ice, etc.), powdered foods (powdered beverages, powdered soups, etc.), and enteric liquid foods. In addition to so-called health foods, the foods for the above-mentioned uses include foods for specified health use, foods with health claims such as foods with nutritional claims, supplements (dietary supplements), and feeds.
In addition, for suppressing the rise in blood glucose level, for suppressing appetite, for suppressing overeating, for improving glucose metabolism, for preventing or treating diabetes, for preventing or treating obesity, for weight reduction, for reducing body fat ratio, and excreting stomach contents. For promotion, suppression of gastric acid secretion, suppression of hepatic glucose release, myocardial protection, improvement of learning memory, prevention or treatment of vascular diseases, prevention or treatment of neurodegenerative diseases, prevention or treatment of non-alcoholic hepatitis Beverages for the prevention or treatment of kanpan, or for the prevention or treatment of asthma, such as tea drinks, soft drinks, carbonated drinks (including non-alcoholic beer), nutritional drinks, fruit drinks, lactic acid drinks, juices, drinks, Alcoholic beverages, processed milk, prepared soy milk; etc.

Alternatively, the GLP-1 secretion-promoting agent of the present invention can be used in a state of being contained in a beverage, food, pharmaceutical composition or the like by an oral, nasal, intestinal or other route or by a tube route. ..

When the GLP-1 secretagogue of the present invention is added (or contained) to a food or drink, the type of the food or drink is not particularly limited. Foods include, for example, noodles (soba, udon, Chinese noodles, instant noodles, etc.), tofu, confectionery (candy, gum, chocolate, snacks, biscuits, cookies, gummy, etc.), breads, marine or processed livestock foods (soba, margarine or processed livestock foods). Kamaboko, ham, sausage, etc.), dairy products (fermented milk, etc.), fats and oils and processed fats and oils (salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing, fat spread, etc.), seasonings (sauce, sauce, etc.) ), Cooked or semi-cooked foods (champles, etc.), retort foods (curry, stew, bowls, porridge, miscellaneous dishes, etc.), cold confectionery (ice cream, sherbet, shaved ice, etc.), powdered foods (powdered beverages, powdered soup, etc.) , Enteral liquid food and the like. In addition to so-called health foods, foods for specified health use, foods with health claims such as foods with nutritional function, supplements (dietary supplements), feeds, etc. are also included.
Beverages include, for example, tea beverages, soft drinks, carbonated beverages (including non-alcoholic beers), nutritional beverages, fruit beverages, lactic acid beverages, juices, drinks, alcoholic beverages, processed milk, prepared soy milk and the like.

The food and drink may contain other components such as a food additive (food additive) as long as the GLP-1 secretagogue of the present invention is contained. The food additive is not particularly limited, but for example, excipients (for example, wheat starch, corn starch, cellulose, lactose, sucrose, mannitol, sorbitol, xylitol, pregelatinized starch, casein, magnesium silicate aluminate, etc. Calcium silicate, etc.), excipients (eg, pregelatinized starch, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc.), disintegrants (eg, cellulose, hydroxypropylcellulose, corn starch, etc.), fluidizers (eg, light anhydrous silicic acid) Oils (eg, soybean oil, sesame oil, olive oil, flaxseed oil, sesame oil, rapeseed oil, coconut oil, corn oil and other vegetable oils or animal / fish-derived oils), nutrients (eg, various minerals, various vitamins, etc. Amino acids), fragrances, sweeteners other than ramnorse starch glycosyl, flavoring agents, coloring agents, solvents (eg ethanol), salts, pH adjusters, buffers, antioxidants, stabilizers, gelling agents, Examples include thickeners, starches, encapsulants, suspending agents, coating agents, preservatives, emulsifiers and the like. The food additive may be contained alone or in combination of two or more.

When the GLP-1 secretagogue of the present invention is contained in foods and drinks, it is preferable to use an emulsifier.
In particular, rhamnose-based steviol glycosides and other glycoside sweeteners used as needed are incorporated into the emulsified particles formed by the emulsifier, or rhamnose-based steviol glycosides are incorporated into the core of the emulsified particles. And it is preferable to include other glycoside sweeteners used as needed. In the present invention, it is not always necessary that a specific glycoside is always incorporated or continuously contained in a specific emulsified particle. Glycosides are incorporated or included in the majority of the emulsified particles throughout the system, while the glycosides are incorporated into, contained, and migrated to regions outside the emulsified particles. Is preferable.

The use of an emulsifier can be expected to have a further effect of promoting GLP-1 secretion or an effect of reducing the bitterness of rhamnose-based steviol glycosides.

It is expected that the above effects due to the use of emulsifiers will be maintained even after storage, especially after storage in harsh environments such as high temperature conditions, acidic conditions, and vibration environments.

As the emulsifier, a known emulsifier can be used. Known emulsifiers include glycerin fatty acid esters such as monoglyceride, organic acid monoglyceride, and polyglycerin fatty acid ester; sorbitan fatty acid ester; sucrose fatty acid ester; propylene glycol fatty acid ester; stearoyl lactate; polysolvate; plant lecithin, egg yolk lecithin, fractionated lecithin. , Lecithins such as enzyme-treated lecithin; Kiraya extract, Yukkafoam extract; vegetable sterol; sphingolipid; bile powder; animal sterol and the like.

The emulsifier preferably contains an oil-in-water emulsifier, more preferably contains an oil-in-water emulsifier having an HLB value of 7 to 18, and more preferably has an HLB value of 7 to 18. Underneath, it comprises sucrose fatty acid esters, lecithins, glycerin fatty acid esters or polyglycerins, more preferably lecithins under the condition that the HLB value is 7-18, and particularly preferably the HLB value. Includes enzyme-treated lecithin, provided that is 7-18.

When using an emulsifier, it is preferable to combine a component that can be present in the core of emulsified particles such as fat and oil composed of fatty acids having 6 to 12 carbon atoms and an aqueous base such as glycerin.

The proportion (ppm) of rhamnose-based steviol glycosides in foods and drinks is preferably based on mass, for example, 2-550, 25-550, 30-550, 35-550, 40-550, 45-550, 50-550, 55-550, 20-540, 25-540, 30-540, 35-540, 40-540, 45-540, 50-540, 55-540, 20-530, 25-530, 30- 530, 35-530, 40-530, 45-530, 50-530, 55-530, 20-520, 25-520, 30-520, 35-520, 40-520, 45-520, 50-520, 55-520, 20-510, 25-510, 30-510, 35-510, 40-510, 45-510, 50-510, 55-510, 20-505, 25-505, 30-505, 35- 505, 40-505, 45-505, 50-505, 55-505, 20-500, 25-500, 30-500, 35-500, 40-500, 45-500, 50-500, 55-500, 20-495, 25-495, 30-495, 35-495, 40-495, 45-495, 50-495, 55-495, 20-490, 25-490, 30-490, 35-490, 40- It can take values of 490, 45-490, 50-490 or 55-490 ppm. Also, preferably, on a mass basis, 2 to 1500, 2 to 1200, 5 to 1200, 2 to 1000, 5 to 1000, 10 to 1000, 2 to 900, 5 to 900, 10 to 900, 15 to 900, 20-900, 25-900, 30-900, 35-900, 40-900, 45-900, 50-900, 55-900, 2-800, 5-800, 10-800, 15-800, 20- 800, 25-800, 30-800, 35-800, 40-800, 45-800, 50-800, 55-800, 2-700, 5-700, 10-700, 15-700, 20-700, 25-700, 30-700, 35-700, 40-700, 45-700, 50-700, 55-700, 2-600, 5-600, 10-600, 15-600, 20-600, 25- 600, 30-600, 35-600, 40-600, 45-600, 50-600, 55-600, 2-540, 2-530, 2-520, 2-510, 2-505, 2-500, 2-495, 2-490, 5-550, 5-540, 5-530, 5-520, 5-510, 5-505, 5-500, 5-495, 5-490, 10-550, 10- 540, 10-530, 10-520, 10-510, 10-505, 10-500, 10-495, 10-490, 15-550, 15-540, 15-530, 15-520, 15-510, It can take values of 15-505, 15-500, 15-495 or 15-490 ppm. Also, preferably, on a mass basis, 0.4 to 4,440, 0.4 to 3,440, 0.4 to 2,440, 0.4 to 1,440, 0.4 to 940, 0.4 to 440, 0.4 to 240, 0.4 to 40, 0.4 to 30, 0.4 to 20, 0.4-10, 0.4-5, 0.4-1, 1-4,440, 5-4,440, 10-4,440, 50-4,440, 100-4,440, 300-4,440, 500-4,440, 700-4,440, 900-4,440, 1,000- It can take values of 4,440, 2,000-4,440, 3,000-4,440, 1-3,440, 5-2,440, 10-1,440, 50-940 or 100-440 ppm.

In the present invention, the intake amount (dose, dose) of the rhamnose-based steviol glycoside is not particularly limited, and can be appropriately selected according to the target age, body weight, health condition, and the like.

When the GLP-1 secretagogue of the present invention is formulated and used, the dosage forms include, for example, tablets, powders, fine granules, granules, dry syrups, coated tablets, orally disintegrating tablets, chewable tablets, and capsules. Agents, soft capsules, syrups, enteral nutritional supplements and the like.

The present invention can be used or applied to either therapeutic (medical) or non-therapeutic applications. Specifically, regardless of whether or not it is classified into pharmaceuticals, quasi-drugs, cosmetics, etc., the function of promoting the secretion of GLP-1 or the function caused by the promotion of the secretion of GLP-1 is explicitly or It can be used or applied as any composition or food or drink that is implicitly appealing. In addition, the product using the present invention may be labeled with a function of promoting the secretion of GLP-1 or a function caused by promoting the secretion of GLP-1. Such a display is not particularly limited, but is limited to a GLP-1 secretion promoting function and a function caused by GLP-1 secretion promoting, for example, a blood glucose level elevation suppressing function, an appetite suppressing function, an overeating suppressing function, and sugar metabolism. Improvement function, diabetes prevention or treatment function, obesity prevention or treatment function, weight reduction function, body fat ratio reduction function, stomach content excretion promotion function, gastric acid secretion suppression function, hepatic glucose release suppression function, myocardial protection Function, learning memory enhancement function, prevention or treatment function of vascular disease, prevention or treatment function of neurodegenerative disease, prevention or treatment function of non-alcoholic hepatitis, prevention or treatment function of pancreatic disease, prevention or treatment function of asthma, Alternatively, a display that can be equated with these can be mentioned.

The timing of use of the GLP-1 secretagogue of the present invention is not particularly limited, and may be, for example, before meals, during meals, between meals, after meals, and before bedtime.

One aspect of the invention relates to rhamnose-based steviol glycosides used at 0.4-4,440 ppm for the treatment of diseases that can be ameliorated by promoting GLP-1 secretion. In addition, one aspect of the present invention promotes the secretion of GLP-1 in a subject, which comprises administering a rhamnose-based steviol glycoside at a concentration of 0.4-4,440 ppm to a subject in need thereof. Or a method of treating a disease that can be ameliorated by promoting GLP-1 secretion in a subject. Furthermore, one aspect of the present invention relates to the use of 0.4-4,440 ppm of rhamnose-based steviol glycosides in the manufacture of a medicament for treating a disease that can be ameliorated by promoting GLP-1 secretion. In addition, each aspect described above may have various features described in the present specification.
Diseases include hyperglycemia, bulimia nervosa, hyperacidity, diabetes, obesity, vascular disease, neurodegenerative diseases, non-alcoholic hepatitis, pancreatic disease, asthma and the like.

The GLP-1 secretagogue of the present invention is produced by a known method according to its use, composition and the like. When the GLP-1 secretion promoter of the present invention contains an emulsifier, a step of mixing an oil-in-water emulsifier, a ramnorth-based steviol glycoside, and an aqueous medium to prepare a mixture, and the result. When the mixture is produced by adding a component that can be contained in the core of the emulsified particles such as fats and oils to the mixture and stirring the mixture, at least a part of the ramnorse-based steviol glycoside is incorporated into the emulsified particles, resulting in the result. It is preferable because the GLP-1 secretion promoting effect can be further improved. At this time, it is preferable to use a homomixer or the like and perform high-speed stirring under conditions more intense than mixing so that the fats and oils and the like are included in the core of the emulsified particles.

For high-speed stirring under conditions more intense than mixing, the stirring speed is preferably 5,000 to 10,000, 5,000 to 8,000, or 8,000 to 10,000 rpm.

In another embodiment, when the GLP-1 secretagogue of the present invention contains an emulsifier, a step of mixing an oil-in-water emulsifier and an aqueous medium to prepare a mixture, and the resulting mixture. It can also be produced through a step of adding a ramnorth-based steviol glycoside and a component that can be contained in the core of emulsified particles such as oil and fat and stirring. For stirring, it is preferable to use a homomixer or the like and stir at high speed under conditions more intense than mixing so that fats and oils are included in the core of the emulsified particles. For high speed stirring under conditions more intense than mixing, the stirring speed is preferably 5,000 to 10,000, 5,000 to 8,000 or 8,000 to 10,000 rpm.

As used herein, the term "subject" means any living organism capable of secreting GLP-1, preferably an animal, more preferably a mammal, and even more preferably a human. The subject may be healthy (for example, not having a specific or arbitrary disease) or may be suffering from any disease, but treatment of a disease that can be improved by promoting GLP-1 secretion, etc. When intended, it typically means a subject who has or is at risk of developing the disease.
As used herein, the term "treatment" shall include all types of medically acceptable prophylactic and / or therapeutic interventions aimed at the cure, temporary remission or prevention of disease. For example, "treatment" includes medically acceptable interventions for a variety of purposes, including delaying or stopping the progression of a disease, regressing or eliminating lesions, preventing the onset or recurrence of the disease, and the like. Therefore, the compositions of the present invention can be used for the treatment and / or prevention of diseases.

Hereinafter, the present invention will be described in more detail with reference to Examples. Experimental Example 14 is a missing number.
<Experimental example 1>
It was examined whether RebA, RebB, RebM, RebN, RebD, RebC, MogV and stevioside have a GLP-1 secretagogue effect. Specifically, the amount of GLP-1 secreted was measured by the following procedure, and the one having a higher amount of secretion than the control no sample was determined to have a GLP-1 secretion promoting effect.

(Adjustment of each test substance)
Each test substance (B, C, D, E, F, G, I, J) was dissolved with PBS (Thermo Fisher Scientific) (Cat. No. 14190250) to obtain a stock solution. The concentration of the stock solution is shown below.
B (RebA); 5 mM, C (RebB); 1M, D (RebM); 2.5 mM,
E (RebN); 1M, F (RebD); 2.5 mM, G (RebC); 2.5 mM,
I (MogV); 1M, J (stevioside); 1M

(Quantitative GLP-1)
H716 (ATCC) (Cat. No. ATCC® CCL-251) at RPMI1640 (10% FBS (Thermo Fisher Scientific) (Cat. No. 10439024), 1 mM Sodium Pyruvate) at 37 ° C., 5% CO ₂ Incubated in the incubator of.
Preculture was performed for 2 weeks to allow the cells to fully wake up, and then a stock of cells was prepared.
Cells were seeded on 96-well plates at 2 × 10 ⁵ cells / 90 μL (high) per well (N = 4). At this time, the FBS contained in the medium was reduced to 0.5% to suppress cell proliferation.
After 24 hours, 10 μL of the highest concentration sample (ie, stock solution) was added.
After an additional 24 hours, GLP-1 was quantified. The specific procedure followed the Human GLP-1 (7-36 amide) Immunoassay kit (Cat. No. AL359) (Lot number 2361115) user guide provided by PerkinElmer. PBS was used as a negative control (no sample).

The results are shown in Fig. 1. According to FIG. 1, it was RebA, RebB, RebC, MogV, and stevioside that secreted significantly more GLP-1 than no sample.

<Experimental example 2>
After adding 21 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) to 20 mg of RevC, the mixture was stirred by vortex and allowed to stand for several hours to obtain a stock solution having a RevC concentration of 1,000 mM. Using the obtained stock solution and PBS, a 10 mM solution was prepared.
The 10 mM solution was diluted with PBS containing 1% DMSO as needed to obtain Sample G-1 mM to Sample G-10 mM. Table 1 shows the concentration of RebC.

<Experimental example 3>
After adding 12.4 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) to 15 mg of stevioside, the mixture was stirred with vortex to obtain a stock solution having a stevioside concentration of 150 mM. A 15 mM solution was prepared using the obtained stock solution and PBS.
The 15 mM solution was diluted as needed with PBS containing 1% DMSO to give sample J-0.1 mM to sample J-15 mM. Table 1 shows the concentration of stevioside.

<Experimental Example 4>
To 20 mg of RebA, 8.27 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) was added, then 74.5 μL of PBS was added and stirred by vortex to obtain a stock solution having a RebA concentration of 250 mM. It was. A 25 mM solution was prepared using the obtained stock solution and PBS.
The 25 mM solution was diluted with PBS containing 1% DMSO as needed to obtain Sample B-1 mM to Sample B-25 mM. Table 1 shows the concentration of RebA.

<Experimental Example 5>
490 g of glycerin, 100 g of RebA, and 10 g of polyglycerin fatty acid ester were mixed and dissolved at a temperature of 70 ° C. to prepare an aqueous mixed solution. 300 g of MCT oil was added as fat and oil to this aqueous mixed solution, and emulsified with a homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) at a rotation speed of 8000 rpm. After the stirring was completed, the mixture was cooled to 40 ° C. and 100 g of water was added to obtain a RebA processing solution.
To 241.8 μL of the obtained RebA processing solution, 10 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 034-24053) and 748.2 μL of PBS were added and stirred by vortexing, and a stock solution having a RebA concentration of 25 mM was added. Got
The stock solution was diluted with PBS containing 1% DMSO as needed to obtain Sample B processing-0.25 mM to Sample B processing-25 mM. Table 1 shows the concentration of RebA.

<Measurement of GLP-1 secretion>
Under the conditions shown in Table 1, the samples obtained in Experimental Examples 2 to 5 were added to the medium, the cells were cultured in the obtained medium, and the amount of GLP-1 secreted after 2 hours was measured. The specific procedure is as follows.
H716 (human colon-derived cells) at RPMI1640 (Thermo Fisher Scientific, model number: 61870-136) (10% FBS (Hycrone, model number: SH30396.03), 1 mM Sodium Pyruvate) at 37 ° C., 5% CO ₂ . Cultured in an incubator.
Preculture was performed for 2 weeks to allow the cells to fully wake up, and then a stock of cells was prepared.
Cells were seeded on 96-well plates at 2 × 10 ⁵ cells / 90 μL per well. At this time, the FBS contained in the medium was reduced to 0.5% to suppress cell proliferation.
After 24 hours, 10 μL of each sample was added (sample B: n = 3-4, sample B processed, sample G, sample J: n = 4). The final concentration in the medium is shown in the "Sample FC (mM)" column of Table 1.
After a predetermined time (2 hours) had elapsed from the addition, a 96-well plate was collected.
The collected 96-well plate was centrifuged at 300 g × 5 minutes, and the supernatant was collected.
GLP-1 in the supernatant collected using the Human GLP-1 (7-36 amide) Immunoassay kit (manufactured by PerkinElmer, model number: AL359C) was quantified, and the results are shown in FIGS. 2 to 5. Figures 2 to 5 show the amount of GLP-1 secreted from a 1% DMSO-containing solution as a non-irritating control (negative control) and the water system of PMA (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 162-23591) as a positive control. The amount of GLP-1 secreted from the solution was also listed. For the aqueous solution of PMA, 16.21 μL of DMSO was added to 1 mg of PMA to prepare a stock solution, PBS was added to the stock solution to prepare a 1 mM solution, and this was diluted with PBS containing 1% DMSO. Obtained.

Table 1 is shown below. The positive control in Table 1 is also referred to as the positive control. Negative controls are also called negative controls.

<Experimental example 6: Measurement of surface tension>
In order to investigate whether there is a correlation between the GLP-1 secretion promoting effect and the surface tension of the emulsifier, an experiment was conducted according to the following procedure.
An aqueous solution containing each of RebA, RebD, and RevM (commercially available) was prepared. The blending amounts of RebA, RebD, and RevM were adjusted to Brix 10 in terms of sucrose. That is, RebA was 333 ppm, RebD was 351 ppm, and RebM was 351 ppm. The surface tension of the obtained aqueous solution was measured by a plate method using an automatic surface tension meter (CBVP-Z type, manufactured by Kyowa Interface Science Co., Ltd.). The same test was performed using water as a control. The results are shown in FIG.

<Experimental Example 7: Evaluation of flavor characteristics>
The flavor characteristics of the emulsified composition of Liver were evaluated by the following procedure.
RebA was dissolved in pure water to prepare 100 ppm and 300 ppm aqueous solutions, which were used as controls. Next, the RebA processing solution prepared in Experimental Example 5 was added to pure water to prepare 100 ppm and 300 ppm aqueous solutions in the same manner. Seven well-trained sensory panelists evaluated the flavor of the aqueous solution containing the RebA processing solution in 0.5-point increments with a control of 3 points, and the average value is shown in the figure. The flavor was evaluated based on sweetness intensity, sweetness aftertaste, and bitterness intensity.
The results are shown in FIG.

<Experimental Example 8>
To 20 mg of RebA, 8.27 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) was added, then 74.43 μL of PBS was added and stirred by vortex to obtain a stock solution having a RebA concentration of 250 mM. It was. A 50 mM solution was prepared using the obtained stock solution and PBS.
The 50 mM solution was diluted with PBS containing 1% DMSO to obtain Sample B-1 mM to Sample B-25 mM. Table 2 shows the concentration of RebA.

<Experimental Example 9>
490 g of glycerin, 100 g of RebA, and 10 g of polyglycerin fatty acid ester were mixed and dissolved at a temperature of 70 ° C. to prepare an aqueous mixed solution. 300 g of MCT oil was added as fat and oil to this aqueous mixed solution, and emulsified with a homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) at a rotation speed of 8000 rpm. After the stirring was completed, the mixture was cooled to 40 ° C. and 100 g of water was added to obtain a RebA processing solution.
To 241.8 μL of the obtained RebA processing solution, 10 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 034-24053) and 748.2 μL of PBS were added and stirred by vortexing, and a stock solution having a RebA concentration of 25 mM was added. Got
The stock solution was diluted with PBS containing 1% DMSO as needed to obtain Sample B processing-1 mM to Sample B processing -25 mM. Table 2 shows the concentration of RebA.

<Experimental Example 10>
A solution C was obtained in the same manner as in the preparation of the RebA processing solution of Experimental Example 9, except that 490 g of glycerin, 10 g of polyglycerin fatty acid ester, 300 g of MCT oil and 200 g of water were used without using 100 g of RebA.
To 483.5 μL of the obtained C solution, 10 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) and 506.5 μL of PBS were added and stirred by vortexing to obtain a stock solution having a concentration equivalent to RebA of 50 mM. It was.
The obtained stock solution was diluted with PBS containing 1% DMSO to obtain Sample C-1 mM to Sample C-25 mM. Table 2 shows the concentration equivalent to RebA. The RebA equivalent concentration means that the concentration of glycerin, polyglycerin fatty acid ester and MCT oil in Sample C is the same as the concentration of glycerin, polyglycerin fatty acid ester and MCT oil in Sample B processing in Experimental Example 9. It means the concentration of RebA during the processing of the sample B.

<Experimental Example 11>
In the same manner as in Experimental Example 8, Sample B containing RebA having a concentration twice that of Sample B + C was obtained. That is, sample B-2 mM, sample B-5 mM, sample B-10 mM, sample B-20 mM, sample B-30 mM, sample B-40 mM, and sample B-50 mM were obtained. The concentration behind the hyphen indicates the concentration of RebA.
In the same manner as in Experimental Example 10, Sample C containing polyglycerin fatty acid ester and the like having a concentration twice that of Sample B + C was obtained. That is, Sample C-2 mM, Sample C-5 mM, Sample C-10 mM, Sample C-20 mM, Sample C-30 mM, Sample C-40 mM, and Sample C-50 mM were obtained. The concentration behind the hyphen indicates the concentration equivalent to RebA.
Sample B and sample C (for example, sample B-2 mM and sample C-2 mM) having the same concentration were mixed in equal amounts to obtain sample B + C-1 mM to sample B + C-25 mM. Table 2 shows the concentration of RebA.

<Experimental Example 12>
To 118.5 μL of glycerin, 10 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) and 871.5 μL of PBS were added and stirred by vortexing to obtain a stock solution having a concentration equivalent to RebA of 25 mM.
The obtained stock solution was diluted with PBS containing 1% DMSO as needed to obtain Sample D-1 mM to Sample D-25 mM. Table 2 shows the concentration equivalent to RebA. The RebA equivalent concentration means the concentration of RebA during the processing of the sample B when the glycerin concentration in the sample D and the glycerin concentration during the processing of the sample B of the experimental example 9 are the same.

<Experimental Example 13>
To 72.5 μL of MCT oil, 10 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) and 917.5 μL of PBS were added and stirred by vortexing to obtain a stock solution having a concentration equivalent to RebA of 25 mM. ..
The obtained stock solution was diluted with PBS containing 1% DMSO as needed to obtain Sample E-1 mM to Sample E-25 mM. Table 2 shows the concentration equivalent to RebA. The RebA equivalent concentration means the concentration of RebA during the processing of the sample B when the concentration of the MCT oil in the sample E and the concentration of the MCT oil during the processing of the sample B in Experimental Example 9 are the same.

<Measurement of GLP-1 secretion>
Under the conditions shown in Table 2, the samples obtained in Experimental Examples 8 to 13 were added to a medium, cells were cultured in the obtained medium, and the amount of GLP-1 secreted after 2 hours was measured. The specific procedure is as follows.
H716 (human colon-derived cells) at RPMI1640 (Thermo Fisher Scientific, model number: 61870-136) (10% FBS (Hycrone, model number: SH30396.03), 1 mM Sodium Pyruvate) at 37 ° C., 5% CO _2. Incubated in the incubator of.
Preculture was performed for 2 weeks to allow the cells to fully wake up, and then a stock of cells was prepared.
Cells were seeded on 96-well plates at 2 × 10 ⁵ cells / 90 μL per well. At this time, the FBS (manufactured by Hycrone, model number: SH30396.03) contained in the medium was reduced to 0.5% to suppress cell proliferation.
After 24 hours, 10 μL of each sample was added (n = 3-4). The final concentration in the medium is shown in the "Sample FC" column of Table 2.
After a predetermined time (2 hours) had elapsed from the addition, a 96-well plate was collected.
The collected 96-well plate was centrifuged at 300 g × 5 minutes, and the supernatant was collected.
GLP-1 in the collected supernatant was quantified using a Human GLP-1 (7-36 amide) Immunoassay kit manufactured by PerkinElmer. At this time, the standard range of the calibration curve is 30 to 100,000 pg / mL, and some values larger than 100,000 pg / mL cause an error. Therefore, a value considered to be 100,000 pg / mL or more is set to 100, Calculated as 000 pg / mL.
Outliers (p <0.01) were excluded by the Smirnov-Grabs test.
The results are shown in FIG. In FIG. 8, the amount of GLP-1 secreted from a 1% DMSO-containing solution as a non-irritating control and the GLP-1 secretion from an aqueous solution of PMA (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 162-23591) as a positive control. I also posted the amount. For the aqueous solution of PMA, 16.21 μL of DMSO was added to 1 mg of PMA to prepare a stock solution, PBS was added to the stock solution to prepare a 1 mM solution, and this was diluted with PBS containing 1% DMSO. Obtained.

Next, for confirmation, the amount of GLP-1 secretion was also measured on another plate. Specifically, using the same culture supernatant as in FIG. 8, the amount of GLP-1 was measured using samples B to D on one assay plate and sample E on another assay plate. The results of measuring the culture supernatant after 2 hours on the same assay plate are shown in FIGS. 9 to 14. The concentration of each sample used is shown in FIGS. 9-14. The concentrations of C, D, and E shown in FIGS. 11, 13, and 14 are the concentrations equivalent to RebA.
The range of the calibration curve was 30 to 100,000 pg / mL, but this time there was no error value with a value larger than 100,000 pg / mL, so values larger than 100,000 pg / mL are calculated as theoretical values. did.

Table 2 is shown below. The positive control in Table 2 is also referred to as the positive control. Negative controls are also called negative controls.

<Experimental Example 15>
After adding 9.4 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 034-24053) to 36.3 mg of RebA, 929.0 μL of PBS was added and stirred by vortex, and a stock solution having a RebA concentration of 40 mM was added. Got
The obtained stock solution and PBS were diluted with PBS containing 1% DMSO to obtain Sample B-0.31 mM to Sample B-20 mM. Table 3 shows the concentration of RebA.

<Experimental Example 16>
490 g of glycerin, 100 g of RebA, and 10 g of polyglycerin fatty acid ester were mixed and dissolved at a temperature of 70 ° C. to prepare an aqueous mixed solution. 300 g of MCT oil was added as fat and oil to this aqueous mixed solution, and emulsified with a homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) at a rotation speed of 8000 rpm. After the stirring was completed, the mixture was cooled to 40 ° C. and 100 g of water was added to obtain a RebA processing solution.
To 193.4 μL of the obtained RebA processing solution, 10 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 034-24053) and 796.6 μL of PBS were added and stirred by vortexing, and the stock solution having a RebA concentration of 20 mM was added. Got
The stock solution was diluted with PBS containing 1% DMSO as needed to obtain Sample B processing −0.31 mM to Sample B processing −20 mM. Table 3 shows the concentration of RebA.

<Experimental example 17>
A solution C was obtained in the same manner as in the preparation of the RebA processing solution of Experimental Example 16 except that 490 g of glycerin, 10 g of polyglycerin fatty acid ester, 300 g of MCT oil and 200 g of water were used without using 100 g of RebA.
To 386.8 μL of C solution, 10 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) and 603.2 μL of PBS were added and stirred by vortexing to obtain a stock solution having a concentration equivalent to RebA of 40 mM. ..
The stock solution was diluted with PBS containing 1% DMSO to obtain Sample C-0.31 mM to Sample C-20 mM. Table 3 shows the concentration equivalent to RebA. The RebA equivalent concentration is the concentration of RebA during the processing of Sample B when the concentration of the polyglycerin fatty acid ester in the sample C and the concentration of the polyglycerin fatty acid ester during the processing of the sample B in Experimental Example 16 are the same. means.

<Experimental Example 18>
In the same manner as in Experimental Example 15, Sample B containing RebA having a concentration twice that of Sample B + C was obtained. That is, sample B-0.62 mM, sample B-2.5 mM, sample B-10 mM, and sample B-40 mM were obtained. The concentration behind the hyphen indicates the concentration of RebA.
In the same manner as in Experimental Example 17, Sample C containing a polyglycerin fatty acid ester having a concentration twice that of Sample B + C was obtained. That is, sample C-0.62 mM, sample C-2.5 mM, sample C-10 mM, and sample C-40 mM were obtained. The concentration behind the hyphen indicates the concentration equivalent to RebA.
Sample B and sample C (for example, sample B-0.62 mM and sample C-0.62 mM) having the same concentration were mixed in equal amounts to obtain sample B + C-0.31 mM to sample B + C-20 mM. Table 3 shows the concentration of RebA.

<Experimental Example 19>
To 94.8 μL of glycerin, 10 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) and 895.2 μL of PBS were added and stirred by vortexing to obtain a stock solution having a concentration equivalent to RebA of 20 mM.
The obtained stock solution was diluted with PBS containing 1% DMSO as needed to obtain Sample D-0.31 mM to Sample D-20 mM. Table 3 shows the concentration equivalent to RebA. The RebA equivalent concentration means the concentration of RebA during the processing of the sample B when the concentration of the glycerin in the sample D and the concentration of the glycerin during the processing of the sample B of the experimental example 16 are the same.

<Experimental Example 20>
To 58.0 μL of MCT oil, 10 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) and 932.0 μL of PBS were added and stirred by vortexing to obtain a stock solution having a concentration equivalent to RebA of 20 mM. ..
The obtained stock solution was diluted with PBS containing 1% DMSO as needed to obtain Sample E-0.31 mM to Sample E-20 mM. Table 3 shows the concentration equivalent to RebA. The RebA equivalent concentration means the concentration of RebA during the processing of the sample B when the concentration of the MCT oil in the sample E and the concentration of the MCT oil during the processing of the sample B of the experimental example 16 are the same.

<Cytotoxicity>
Under the conditions shown in Table 3, the samples obtained in Experimental Examples 15 to 20 were added to the medium, the cells were cultured in the obtained medium, and the cytotoxicity was measured after 2 hours and 24 hours. The specific procedure is as follows.
H716 cells were cultured in RPMI1640 (Thermo Fisher Scientific, model number: 61870-136) (10% FBS (Gibco, model number: SH102770), 1 mM Sodium Pyruvate) in an incubator at 37 ° C. and 5% CO ₂ . .. H716 cells were seeded on 96-well plates at 2 × 10 ⁵ cells / 90 μL / well (for evaluation of dead cells) and on 48-well plates at 4 × 10 ⁵ cells / 180 μL / well (for morphological observation). The FBS in the medium when seeded on the plate was 0.5%. Twenty-four hours after cell seeding, samples of each concentration were added at 10 μL / well (96 well plate) or 20 μL / well (48 well plate). The final concentration in the medium is shown in the "Sample FC" column of Table 3. Two hours after sample addition, the highest concentration of each sample (# 1, # 6, # 10, # 14, # 18, # 22, # 26, # 30 shown in Table 3) was evaluated.

(Evaluation using dead cells as an index)
After 2 hours and 24 hours after sample addition, the cells were centrifuged at 100 × g for 5 minutes to remove the supernatant. Propidium iodide solution (0.66 μg / mL, diluted 1: 1500 with PBS) was added in 50 μL / well portions, and the cells were allowed to stand at room temperature for 15 minutes to stain dead cells. After suspending the cells by pipetting, phase difference images and fluorescence images were taken under a microscope. The total number of cells was measured by ImageJ based on the phase difference image, and the number of dead cells was measured based on the fluorescence image. The dead cell rate was calculated using the measured number of cells.
Dead cell rate (%) = number of dead cells / total number of cells x 100
The dead cell rate at the time of adding each sample is 0% when not added 24 hours after cell seeding, and 100% when 70% ethanol is added and allowed to stand on ice for 30 minutes or more. Corrected. The results are shown in FIG. FIG. 15a shows the result after 2 hours, and FIG. 15b shows the result after 24 hours.

From FIG. 15a showing the results after 2 hours, it can be seen that cell death was not induced in all the samples.

In addition, as can be seen from FIG. 15b showing the results after 24 hours, the dead cell rate was the highest when sample B + C (RebA concentration 2 mM) was added, but the dead cell rate was equivalent to the lowest concentration of PMA. there were. Except for sample B + C, the dead cell rate was equal to or lower than that of the negative control (-) (also referred to as negative control). Therefore, it is unlikely that cell death was induced in sample B + C, and cell death was not induced in other samples.

(Evaluation using cell morphology as an index)
Two hours and 24 hours after the sample was added, the cells were photographed under a microscope to confirm the presence or absence of morphological changes. Since sample B processing, C and B + C are cloudy, it is difficult to judge under a microscope when 0.5 mM and 2 mM are added, but for other samples, even if they are added to H716 cells, cell contraction and rupture occur. No morphological change was observed.

Table 3 is shown below. The positive control in Table 3 is also referred to as the positive control. Negative controls are also called negative controls.

<Experimental Example 21>
After adding 34.7 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) to 134.3 mg of RebA, 3438 μL of PBS was added and stirred by vortex to obtain a stock solution having a RebA concentration of 40 mM. It was.
The obtained stock solution was diluted with PBS containing 1% DMSO to obtain Sample B-1 mM to Sample B-20 mM. Table 4 shows the concentration of RebA.

<Experimental Example 22>
The C-1 solution was prepared in the same manner as in Experimental Example 16 except that 100 g of RebA was replaced with 100 g of water and 10 g of sucrose fatty acid ester was used instead of 10 g of polyglycerin fatty acid ester as an emulsifier. Got Specifically, 490 g of glycerin and 10 g of sucrose fatty acid ester (HLB = 16 manufactured by Mitsubishi Chemical Foods) were mixed and dissolved at a temperature of 70 ° C. to prepare an aqueous mixed solution. 300 g of MCT oil was added as fat and oil to this aqueous mixed solution, and emulsified with a homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) at a rotation speed of 8000 rpm. After the stirring was completed, the mixture was cooled to 40 ° C. and 200 g of water was added to obtain a C-1 solution.
To 580.2 μL of the obtained C-1 solution, 15 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) and 904.8 μL of PBS were added and stirred by vortex, and the concentration equivalent to RebA was 40 mM. A stock solution was obtained.
The obtained stock solution was diluted with PBS containing 1% DMSO to obtain Sample C-1-1 mM to Sample C-1-20 mM. Table 4 shows the concentration equivalent to RebA. The RebA equivalent concentration is the concentration of RebA in the sample B + C-1 when the concentration of the glycerin and the emulsifier in the sample C-1 is the same as the concentration of the glycerin and the emulsifier in the sample B + C-1 of Experimental Example 25. Means concentration.

<Experimental Example 23>
Sample C-2-in the same manner as in Experimental Example 22 except that 10 g of enzymatically decomposed lecithin (HLB = 12.0 manufactured by Taiyo Kagaku Co., Ltd.) was used instead of 10 g of sucrose fatty acid ester (HLB = 16 manufactured by Mitsubishi Chemical Foods). From 1 mM to sample C-2-20 mM was obtained. Table 4 shows the concentration equivalent to RebA. The RebA equivalent concentration is the concentration of RebA in Sample B + C-2 when the concentration of glycerin and emulsifier in Sample C-2 is the same as the concentration of glycerin and emulsifier in Sample B + C-2 of Experimental Example 26. Means concentration.

<Experimental Example 24>
Sample C-3-similar to Experimental Example 22 except that 10 g of organic acid monoglyceride (HLB = 9.0 manufactured by Taiyo Kagaku Co., Ltd.) was used instead of 10 g of sucrose fatty acid ester (HLB = 16 manufactured by Mitsubishi Chemical Foods). From 1 mM to sample C-3-20 mM was obtained. Table 4 shows the concentration equivalent to RebA. The RebA equivalent concentration is the concentration of RebA in Sample B + C-3 when the concentration of glycerin and emulsifier in Sample C-3 is the same as the concentration of glycerin and emulsifier in Sample B + C-3 of Experimental Example 27. Means concentration.

<Experimental Example 25>
In the same manner as in Experimental Example 21, Sample B containing RebA having a concentration twice that of Sample B + C-1 was obtained. That is, sample B-2 mM, sample B-5 mM, sample B-10 mM, sample B-20 mM, sample B-30 mM, and sample B-40 mM were obtained. The concentration behind the hyphen indicates the concentration of RebA.
In the same manner as in Experimental Example 22, a C-1 sample containing a sucrose fatty acid ester having a concentration twice that of sample B + C-1 was obtained. That is, sample C-1-2 mM, sample C-1-5 mM, sample C-1-10 mM, sample C-1-20 mM, sample C-1-30 mM, and sample C-1-40 mM were obtained. The concentration behind the hyphen indicates the concentration equivalent to RebA.
Sample B and sample C (for example, sample B-1-2 mM and sample C-1-2 mM) having the same concentration were mixed in equal amounts to obtain sample B + C-1-1 mM to sample B + C-1-20 mM. Table 4 shows the concentration of RebA.

<Experimental Example 26>
Sample B + C-2-in the same manner as in Experimental Example 25, except that 10 g of enzymatically decomposed lecithin (HLB = 12.0 manufactured by Taiyo Kagaku Co., Ltd.) was used instead of 10 g of sucrose fatty acid ester (HLB = 16 manufactured by Mitsubishi Chemical Foods). From 1 mM to sample B + C-2-20 mM was obtained. Table 4 shows the concentration of RebA.

<Experimental example 27>
Sample B + C-3-similar to Experimental Example 25, except that 10 g of organic acid monoglyceride (HLB = 9.0 manufactured by Taiyo Kagaku Co., Ltd.) was used instead of 10 g of sucrose fatty acid ester (HLB = 16 manufactured by Mitsubishi Chemical Foods). From 1 mM to sample B + C-3-20 mM was obtained. Table 4 shows the concentration of RebA.

<Experimental Example 28>
Add 339 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) to 13.1 mg of sucrose fatty acid ester, vortex, and further dilute with DMSO 20 times to stock a RebA equivalent concentration of 2,000 mM. Obtained liquid. PBS was added to the obtained stock solution to obtain a 20 mM solution.
The obtained 20 mM solution was diluted with PBS containing 1% DMSO as needed to obtain Sample G-1-1 mM to Sample G-1-20 mM. Table 4 shows the concentration equivalent to RebA. The RebA equivalent concentration is the concentration of the sucrose fatty acid ester in the sample G-1 and the concentration of the sucrose fatty acid ester in the sample B + C-1 of Experimental Example 25 when the concentration is the same in the sample B + C-1. It means the concentration of RebA.

<Experimental Example 29>
Add 540 μL of PBS to 20.9 mg of enzymatically decomposed lecithin, vortex it, and dilute it 20-fold with DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) to stock a RebA equivalent concentration of 2,000 mM. Obtained liquid. PBS and DMSO were mixed with the stock solution to prepare a 20 mM solution (1% DMSO).
The obtained 20 mM solution was diluted with PBS containing 1% DMSO as needed to obtain Sample G-2-1 mM to Sample G-2-20 mM. Table 4 shows the concentration equivalent to RebA. The RebA equivalent concentration is the concentration of RebA in the sample B + C-2 when the concentration of the enzymatically decomposed lecithin in the sample G-2 is the same as the concentration of the enzymatically decomposed lecithin in the sample B + C-2 of Experimental Example 26. Means concentration.

<Experimental Example 30>
The organic acid monoglyceride was dissolved at 60 ° C., 905 μL of DMSO was added to 35.0 mg for vortexing, and the mixture was further diluted 20-fold with DMSO to obtain a stock solution having a concentration equivalent to RebA of 2,000 mM. PBS was added to the stock solution to obtain a 20 mM solution.
The obtained 20 mM solution was diluted with PBS containing 1% DMSO as needed to obtain Sample G-3-1 mM to Sample G-3-20 mM. Table 4 shows the concentration equivalent to RebA. The RebA equivalent concentration is the concentration of RebA in Sample B + C-3 when the concentration of organic acid monoglyceride in Sample G-3 is the same as the concentration of organic acid monoglyceride in Sample B + C-3 of Experimental Example 27. Means concentration.

<Experimental Example 31>
Polyglycerin fatty acid ester is dissolved at 60 ° C., 259 μL of DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 037-24053) is added to 50 μL, and 984 μL of PBS is added for vortexing, and the concentration equivalent to RebA is 400 mM. Stock solution was obtained.
A 20 mM solution was prepared using the obtained stock solution and PBS. The obtained 20 mM solution was diluted with PBS containing 1% DMSO as needed to obtain Sample G-4-1 mM to Sample G-4-20 mM. Table 4 shows the concentration equivalent to RebA. The RebA equivalent concentration means the concentration of RebA in the sample B + C when the concentration of the polyglycerin fatty acid ester in the sample G-4 and the concentration of the emulsifier in the sample B + C of Experimental Examples 25 to 27 are the same. To do.

<GLP-1 secretion amount>
The samples obtained in Experimental Examples 21 to 31 were given to cells, and the amount of GLP-1 secreted was measured 2 hours and 24 hours later. The specific procedure is as follows.
H716 cells were cultured in RPMI1640 (Thermo Fisher Scientific, model number: 61870-136) (10% FBS (Gibco, model number: SH102770), 1 mM Sodium Pyruvate) in an incubator at 37 ° C. and 5% CO ₂ . .. H716 cells were seeded on 96-well plates in 2 × 10 ⁵ cells / 90 μL / well. The FBS in the medium when seeded on the plate was 0.5%.
Twenty-four hours after cell seeding, as shown in Table 4, the samples obtained in Experimental Examples 21 to 31 were added to the medium at 10 μL / well. The final concentration in the medium is shown in the "Sample FC" column of Table 4.
Two hours and 24 hours after the addition of the sample, the 96-well plate was centrifuged at 300 × g for 5 minutes, and the culture supernatant was collected.
GLP-1 in the culture supernatant recovered using a Human GLP-1 (7-36 amide) Immunoassay kit (manufactured by PerkinElmer, model number: AL359C) was quantified. For quantitative values, values that exceeded the range of the calibration curve and resulted in an error were excluded. In addition, a Box plot was created using statistical analysis software R, and cases where a value smaller than the first quartile or larger than the third quartile was 1.5 quartiles or more were excluded as outliers.
The results are shown in FIG. In FIG. 16, the amount of GLP-1 secreted in a 1% DMSO-containing solution as a non-irritating control and the amount of GLP-1 secreted in an aqueous solution of PMA (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 162-23591) as a positive control are also shown. I put it. For the aqueous solution of PMA, 16.21 μL of DMSO was added to 1 mg of PMA to prepare a stock solution, PBS was added to the stock solution to prepare a 1 mM solution, and this was diluted with PBS containing 1% DMSO. Obtained.

Table 4 is shown below. The positive control in Table 4 is also referred to as the positive control. Negative controls are also called negative controls.

<Experimental Example 32>
The distribution ratios of stevioside, RebA, RebD, and RebM were measured. For reference, the structural formulas of stevioside, RebA, RebD, and RebM are shown below.

Specifically, the following operations were performed.
About 100 mL of pure water and about 100 mL of toluene were placed in a separatory funnel, and then 30 mg of each steviol glycoside was added. The liquid was sealed so as not to leak, and the separating funnel was allowed to stand after stirring with a shaker for about 30 minutes. When the liquid layer was separated into two layers, the distribution ratio was measured by LCMS after sampling from each layer.
The measurement results and photographs during the test are shown in FIG. A photograph of the RebA-containing liquid after the test is shown in FIG.

<Experimental Example 33>
1. 1. Sample preparation Samples SA-70A, SA-70B, SA-200A, SA-200B, SA-206A, SA-206B, SA-207A, and SA-207B were prepared by the following procedure. The sample ending in A is a sample prepared by dissolving RebA in an aqueous system. The sample ending in B is a sample prepared by dispersing RebA in an oil system.

(SA-70A)
487 g of glycerin, 103 g of RebA and 10 g of polyglycerin fatty acid ester (manufactured by Taiyo Kagaku Co., Ltd., HLB = 16.7) were mixed and dissolved at a temperature of 70 ° C. to obtain an aqueous mixed solution. 300 g of MCT was added as fat and oil to this aqueous mixed solution, and emulsified with a homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) at a rotation speed of 8000 rpm. After the stirring was completed, the mixture was cooled to 40 ° C. and 100 g of water was added to obtain an emulsified composition SA-70A.

(SA-70B)
487 g of glycerin and 10 g of polyglycerin fatty acid ester (manufactured by Taiyo Kagaku Co., Ltd., HLB = 16.7) were mixed and dissolved at a temperature of 70 ° C. to obtain an aqueous mixed solution. Further, 300 g of MCT and 103 g of RebA were mixed at room temperature to obtain an oil-based mixed solution. An oil-based mixed solution was added to the aqueous mixed solution, and the solution was emulsified with a homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) at a rotation speed of 8000 rpm. After the stirring was completed, the mixture was cooled to 40 ° C. and 100 g of water was added to obtain an emulsified composition SA-70B.

(SA-200A)
567 g of glycerin, 103 g of RebA and 30 g of polyglycerin fatty acid ester (manufactured by Taiyo Kagaku Co., Ltd., HLB = 16.7) were mixed and dissolved at a temperature of 70 ° C. to prepare an aqueous mixed solution. 200 g of MCT was added as fat and oil to this aqueous mixed solution, and emulsified with a homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) at a rotation speed of 8000 rpm. After the stirring was completed, the mixture was cooled to 40 ° C. and 100 g of water was added to obtain an emulsified composition SA-200A.

(SA-200B)
567 g of glycerin and 30 g of polyglycerin fatty acid ester (manufactured by Taiyo Kagaku Co., Ltd., HLB = 16.7) were mixed and dissolved at a temperature of 70 ° C. to prepare an aqueous mixed solution. Further, 200 g of MCT and 103 g of RebA were mixed at room temperature to obtain an oil-based mixed solution. An oil-based mixed solution was added to the aqueous mixed solution, and the solution was emulsified with a homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) at a rotation speed of 8000 rpm. After the stirring was completed, the mixture was cooled to 40 ° C. and 100 g of water was added to obtain an emulsified composition SA-200B.

(SA-206A)
537 g of glycerin, 103 g of RebA and 60 g of polyglycerin fatty acid ester (manufactured by Taiyo Kagaku Co., Ltd., HLB = 16.7) were mixed and dissolved at a temperature of 70 ° C. to prepare an aqueous mixed solution. 200 g of MCT was added as fat and oil to this aqueous mixed solution, and emulsified with a homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) at a rotation speed of 8000 rpm. After the stirring was completed, the mixture was cooled to 40 ° C., 100 g of water was added to obtain a pre-emulsified composition, and then fine emulsification was further performed at a pressure of 150 MPa with a wet atomizing device to obtain an emulsified composition SA-206A.

(SA-206B)
578 g of glycerin and 55 g of polyglycerin fatty acid ester (manufactured by Taiyo Kagaku Co., Ltd., HLB = 16.7) were mixed and dissolved at a temperature of 70 ° C. to prepare an aqueous mixed solution. Further, 182 g of MCT and 94 g of RebA were mixed at room temperature to obtain an oil-based mixed solution. An oil-based mixed solution was added to the aqueous mixed solution, and the solution was emulsified with a homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) at a rotation speed of 8000 rpm. After the stirring was completed, the mixture was cooled to 40 ° C., 91 g of water was added to obtain a pre-emulsified composition, and then fine emulsification was further performed at a pressure of 150 MPa with a wet atomizing device to obtain an emulsified composition SA-206B.

(SA-207A)
706 g of glycerin, 34 g of RebA and 60 g of polyglycerin fatty acid ester (manufactured by Taiyo Kagaku Co., Ltd., HLB = 16.7) were mixed and dissolved at a temperature of 70 ° C. to prepare an aqueous mixed solution. 100 g of MCT was added as fat and oil to this aqueous mixed solution, and emulsified with a homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) at a rotation speed of 8000 rpm. After the stirring was completed, the mixture was cooled to 40 ° C., 100 g of water was added to obtain a pre-emulsified composition, and then fine emulsification was further performed at a pressure of 150 MPa with a wet atomizing device to obtain an emulsified composition SA-207A.

(SA-207B)
706 g of glycerin and 60 g of polyglycerin fatty acid ester (manufactured by Taiyo Kagaku Co., Ltd., HLB = 16.7) were mixed and dissolved at a temperature of 70 ° C. to prepare an aqueous mixed solution. Further, 100 g of MCT and 34 g of RebA were mixed at room temperature to obtain an oil-based mixed solution. An oil-based mixed solution was added to the aqueous mixed solution, and the solution was emulsified with a homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.) at a rotation speed of 8000 rpm. After the stirring was completed, the mixture was cooled to 40 ° C., 100 g of water was added to obtain a pre-emulsified composition, and then fine emulsification was further performed at a pressure of 150 MPa with a wet atomizing device to obtain an emulsified composition SA-207B.

2. Evaluation of emulsification stability 1. The stability was evaluated using the sample prepared in. The specific procedure is as follows.

2.1 Measurement of emulsified particle size immediately after adjustment The average particle size of the emulsified particles in each sample immediately after preparation was measured. Specifically, each sample immediately after preparation is appropriately diluted with ion-exchanged water so that the laser scattering intensity is about 1%, and then a laser diffraction scattering type particle size distribution measuring instrument (Spectris Co., Ltd. Malvern Panasonic Business). Part).

2.2 Measurement of emulsified particle size after refrigerated storage Each sample was allowed to stand in a refrigerator (4 ° C.) for 2 months. Then, the emulsified particle size was measured in the same manner as in 2.1.

The results are shown in the table below.

3. 3. Evaluation of flavor characteristics 1. Flavor characteristics were evaluated using the sample prepared in. Specifically, the evaluation was performed according to the following procedure.
RebA was dissolved in pure water to prepare an aqueous solution having a RebA concentration of 467 ppm, which was used as a control. Next, 1. Pure water was added to each sample prepared in (1) to prepare an aqueous solution having a RebA concentration of 467 ppm. Seven well-trained sensory panelists evaluated the flavor of each aqueous solution in 0.5-point increments with a control of 3 points, calculated the average value, and then ranked them. The flavor was evaluated based on sweetness intensity, sweetness aftertaste, and bitterness intensity. Those with the same score were given the same ranking. The ranking is shown in the column of each evaluation item in the table below. The sum of the rankings is shown in the total column of the table below.

4. Evaluation of various characteristics after storage 1. Emulsification stability, flavor characteristics, and vibration stability after storage were evaluated using the sample prepared in. Specifically, the evaluation was performed according to the following procedure.
3,483 ml of pure water was added to 16990.3 mg of sample SA-70A to obtain an evaluation liquid 70A. An evaluation liquid 206A was obtained in the same manner except that sample SA-206A was used instead of sample SA-70A. The composition of each evaluation liquid is as follows.

The obtained evaluation liquid 70A and evaluation liquid 206A were allowed to stand in a refrigerator having an internal temperature of 5 ° C. for 4 weeks. Further, another evaluation liquid 70A and an evaluation liquid 206A were allowed to stand in a constant temperature storage at 55 ° C. for 4 weeks.
Further, the pH of the evaluation liquid 70A and the evaluation liquid 206A was adjusted to 2.5 using citric acid anhydride, and the mixture was allowed to stand in a refrigerator having an internal temperature of 5 ° C. for 4 weeks. The pH of another evaluation solution 70A and evaluation solution 206A was adjusted to 2.5 using citric acid anhydride, and the mixture was allowed to stand in a constant temperature storage at 55 ° C. for 4 weeks.

4-1. Emulsification stability after storage The appearance of the liquid level of the evaluation liquid after standing for 4 weeks was observed. The results are shown in the table below.

4-2. Effect of storage temperature on flavor characteristics In addition, the effect of standing temperature on flavor was confirmed. Specifically, it was confirmed by the following procedure.
The evaluation solution 70A with no pH adjustment and a standing temperature of 5 ° C. was used as a control. When the control was evaluated at 5 points, the flavor of the evaluation solution 70A without pH adjustment and at a standing temperature of 55 ° C. was evaluated in 0.5 point increments. The panelists were four well-trained sensual panelists. The average value of the evaluation of each panelist was calculated.
Similarly, the evaluation liquid 206A without pH adjustment at a standing temperature of 5 ° C. was used as a control, and the flavor of the evaluation liquid 206A without pH adjustment at a standing temperature of 55 ° C. was evaluated in 0.5 point increments.
Similarly, the evaluation liquid 70A having a pH of 2.5 and a standing temperature of 5 ° C. was used as a control, and the flavor of the evaluation liquid 70A having a pH of 2.5 and a standing temperature of 55 ° C. was evaluated in 0.5 point increments.
Similarly, the evaluation liquid 206A having a pH of 2.5 and a standing temperature of 5 ° C. was used as a control, and the flavor of the evaluation liquid 206A having a pH of 2.5 and a standing temperature of 55 ° C. was evaluated in 0.5 point increments.

The results are shown in FIG. In addition, the comments of the panelists are described below.
70A, no pH adjustment;
The bitterness is removed and it is mellow, milky, and slightly clear. It became mellow and the bitterness decreased. Milky has increased. It's easier to drink. It feels a little fresh, but there is no critical deterioration.
206A, no pH adjustment;
The bitterness is removed and it is mellow, milky, and slightly clear. It became mellow and the bitterness decreased. Milky has increased. The sweetness does not change. No change. It feels a little fresh, but there is no critical deterioration.
70A, pH2.5;
Sourness is suppressed. The sourness increased. There are thorns. It became a mellow acidity. good. It becomes slightly mellow and has less sour thorns.
206A, pH2.5;
Sourness is suppressed. The sourness increased. But it's easier to drink. No change. The bitterness is slightly reduced. good. It becomes slightly mellow and has less sour thorns.

4-3. Vibration Accelerated Test The absorbance of the evaluation solution after standing at 5 ° C. for 4 weeks was measured at a wavelength of 680 nm using a spectrophotometer (UV1800 UV spectrophotometer manufactured by Shimadzu Corporation).
Next, the evaluation solution was centrifuged at 3000 rpm for 30 minutes. The absorbance of the evaluation solution after centrifugation was measured at a wavelength of 680 nm. The difference in absorbance Δabs before and after centrifugation was calculated, and those with Δabs of 0.02 or less were evaluated as “good” because they were excellent in emulsification stability during vibration.
The results are shown in FIG.

5. Degree of decrease in sweetness intensity 1. The degree of decrease in sweetness intensity due to emulsification was evaluated using the samples SA-70A and SA-206A prepared in. Specifically, the evaluation was performed according to the following procedure.
RebA was dissolved in pure water to prepare an aqueous solution of 467 ppm, which was used as a control. Next, 1. Each sample adjusted in 1 was added to pure water to prepare an aqueous solution having a RebA concentration of 467 ppm. Four well-trained sensory panelists were asked to choose which of the sucrose aqueous solutions Brix 2, 5, 8, 11, 14 each aqueous solution was close to. The average value of the evaluation results of each panelist was calculated. The results are shown in FIG.

<Experimental Example 34>
Evaluation liquid 70A and evaluation liquid 206A were obtained in the same manner as in “4. Evaluation of various characteristics after storage” of Experimental Example 33.
The obtained evaluation liquid 70A and evaluation liquid 206A were diluted with PBS containing DMSO to prepare a diluted liquid having a RebA concentration shown in the column of addition concentration in the table below.
Further, another evaluation liquid 70A and an evaluation liquid 206A were allowed to stand in a constant temperature storage at 55 ° C. for 4 weeks. The evaluation solution 70A and the evaluation solution 206A after standing were diluted with PBS containing DMSO to prepare a diluted solution having a RebA concentration shown in the column of addition concentration in the table below.
A 1% DMSO-containing solution was prepared as a non-irritating control (negative control). Further, as a positive control, PMA (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number: 162-23591) was adjusted by diluting with 1% DMSO so as to have the concentration shown in the addition concentration column in the table below.

Under the conditions shown in the above table, the diluent No. 1-27 was added to the medium, and the cells were cultured in the obtained medium. The specific procedure is as follows.
H716 cells were cultured in RPMI1640 (Thermo Fisher Scientific, model number: 61870-136) (10% FBS (Gibco, model number: SH102770), 1 mM Sodium Pyruvate) in an incubator at 37 ° C. and 5% CO ₂ . .. H716 cells were seeded on 96-well plates at 2 × 10 ⁵ cells / 90 μL / well. The FBS in the medium when seeded on the plate was 0.5%. After 24 hours from cell seeding, the diluent No. 1-27 was added to the wells in an amount of 10 μL / well. The final concentration of RebA in the medium after the addition of the diluted solution (PMA concentration in the case of Nos. 2 and 3) is shown in the column of "Final conc (mM)" in the above table.

The cell viability was confirmed 2 hours and 24 hours after the addition of the diluent. There was no problem with either sample.

Two hours and 24 hours after the addition of the diluent, the 96-well plate was centrifuged at 300 × g for 5 minutes, and the culture supernatant was collected.
GLP-1 in the collected culture supernatant was quantified using a Human GLP-1 (7-36 amide) Immunoassay kit (manufactured by PerkinElmer, model number: AL359C). For quantitative values, values that exceeded the range of the calibration curve and resulted in an error were excluded. In addition, a Box plot was created using statistical analysis software R, and cases where a value smaller than the first quartile or larger than the third quartile was 1.5 quartiles or more were excluded as outliers.
The results are shown in FIG.

Claims (7)

1. A GLP-1 secretagogue characterized by containing 0.4 to 4,440 ppm of steviol glycoside having a structure in which rhamnose is bound.
2. The GLP-1 secretion-promoting agent according to claim 1, wherein the steviol glycoside contains at least one selected from the group consisting of rebaugioside C, rebaugioside K, dulcoside A, dulcoside C, dulcoside D and dulcoside F.
3. The GLP-1 secretagogue according to claim 1, wherein the steviol glycoside contains rebaugioside C.
4. The GLP-1 secretagogue according to any one of claims 1 to 3, which is for improving glucose metabolism, suppressing appetite, or preventing or improving diabetes or obesity.
5. A GLP-1 secretion-promoting beverage containing the GLP-1 secretion-promoting agent according to any one of claims 1 to 4.
6. A GLP-1 secretagogue food containing the GLP-1 secretagogue according to any one of claims 1 to 4.
7. A pharmaceutical composition comprising the GLP-1 secretagogue according to any one of claims 1 to 4.

Images