JP6802256B2 - GLP-1 secretion promoting composition and method for producing the same - Google Patents

Description

The present invention relates to a composition useful for activating (or promoting) GLP-1 (or GLP-1 secretion), and a method for producing the same. The present invention also relates to a novel compound useful for activating GLP-1 secretion (or promoting GLP-1 secretion) and the like.

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

On the other hand, bitter melon (bitter melon) is a plant that is cultivated for food, and research on the physiological action of bitter melon is being conducted.

There is also a report suggesting such a relationship between bitter gourd and GLP-1. For example, in Non-Patent Document 1, a specific compound contained in bitter gourd has a GLP-1 activating effect and has a blood glucose regulating function. It has been reported that it may have.

Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 625892 13pages

An object of the present invention is to provide a composition useful for promoting or activating GLP-1 secretion (GLP-1 response).
Other objects of the present invention are for suppressing increase in blood glucose level, suppressing appetite, suppressing overeating, improving glucose metabolism, preventing or treating diabetes, preventing or treating obesity, reducing body weight, and body fat. It is an object of the present invention to provide a composition useful for reducing the rate.
Still another object of the present invention is to provide a compound useful for activating GLP-1 secretion and the like, or a method for efficiently producing a composition containing this compound.
Another object of the present invention is to provide a novel compound useful for promoting or activating GLP-1 secretion.

As a result of diligent studies to solve the above problems, the present inventors have focused on bitter gourd and examined the components contained in bitter gourd in detail. As a result, in addition to the compounds described in Non-Patent Document 1, GLP-1 activity There may be a variety of compounds that have a chemical effect, and, surprisingly, such compounds are not or, if any, in raw bitter gourd and are heat treated. In addition to being produced in large quantities over time, such various compounds include compounds that are exceptionally excellent in promoting GLP-1 secretion, and include novel compounds that have not been known in the past. We found that and completed the present invention.

That is, the composition of the present invention contains (includes as an active ingredient) at least one component (A) selected from the following formulas (1) to (11). Since such a composition has an action of activating GLP-1 secretion, it can be used as a composition for promoting GLP-1 secretion (composition for activating GLP-1).

Since the composition of the present invention has an action of promoting GLP-1 secretion, it may be suitably used for applications for the purpose of an action / function caused by or related to the action. For example, the composition of the present invention is used for suppressing an increase in blood glucose level, suppressing appetite, suppressing overeating, improving glucose metabolism, preventing or treating diabetes, preventing or treating obesity, reducing body weight, and the like. It may be a composition for use in at least one application selected from those for reducing body fat percentage.

In addition, the composition of the present invention is used for suppressing an increase in blood glucose level, suppressing appetite, suppressing overeating, improving glucose metabolism, preventing or treating diabetes, preventing or treating obesity, reducing body weight, and the like. It may be a composition for use in at least one application selected from those for reducing body fat percentage. In such compositions, blood glucose elevation suppression (function), appetite suppression (function), hypereating suppression (function), improvement of glucose metabolism (function), prevention or treatment of diabetes (function), prevention or treatment of obesity At least one function (or action) selected from (function), weight loss (function), and body fat percentage reduction (function) does not necessarily have to be due to the promotion of GLP-1 secretion, and GLP. It may be due to the promotion of secretion of -1.

In the composition of the present invention, the component (A) may particularly contain at least one component (A1) selected from the formulas (6) to (11).
In addition, the composition of the present invention may contain the component (A) as a bitter gourd-derived component (particularly, a heat-treated bitter gourd-derived component).
The form of the composition of the present invention may be, for example, solid (for example, powder) or liquid.

The present invention includes a liquid composition containing at least one component (A) selected from the formulas (1) to (11). Such a liquid composition may contain the component (A) in a proportion of 0.005 ppm or more on a mass basis. Further, the component (A) may particularly contain at least one component (A1) selected from the formulas (6) to (11), and typically the above formulas (6) to (11). At least one component (A1) selected from 11) may be contained in a proportion of 0.005 ppm or more on a mass basis.

The composition (or liquid composition) of the present invention may be, for example, a puree or a beverage, and in particular, a heat-treated bitter gourd puree or a beverage.

The composition of the present invention may be used for addition to food or drink (or as an additive for food or drink).

The present invention also includes foods and drinks containing the composition. Such foods and drinks may contain, for example, at least one component (A) selected from the above formulas (1) to (11) in a proportion of 0.005 ppm or more on a mass basis. In addition, food and drink may usually contain non-bitter gourd-derived components. The food and drink may be, in particular, a beverage.

The composition or food or drink of the present invention has an action of promoting GLP-1 secretion, and also has a function caused by or related to the action. Therefore, the composition or food or drink of the present invention may be a composition or food or drink with an indication of a function of promoting the secretion of GLP-1 and / or a function of promoting the secretion of GLP-1. Such compositions or foods and drinks include, for example, blood glucose elevation suppressing function, appetite suppressing function, overeating suppressing function, glucose metabolism improving function, diabetes prevention or treatment function, obesity prevention or treatment function, and body weight. A composition or a food or drink with a display (functional display) of at least one function (furthermore, a function that can be equated with these) selected from the reduction function and the body fat percentage reduction function is included.

The component (A) can be efficiently produced by heat-treating the bitter gourd component.
Therefore, the present invention is a method for expressing or increasing at least one component (A) selected from the above formulas (1) to (11) in the bitter gourd component, and is a heating step for heat-treating the bitter gourd component. Including methods including.
The present invention also includes a method for producing the composition, which comprises a heating step of heat-treating the bitter gourd component.

In the method of the present invention, the puree or beverage (particularly puree) of the bitter gourd component may be heat-treated in the heating step. Further, in the heating step, the heat treatment may be performed at 60 ° C. or higher for 5 minutes or longer. Further, in the heating step, heat treatment may be performed in a closed system.

Among the components (A), the compounds selected from the formulas (6) to (11) are novel compounds. Therefore, the present invention also includes compounds selected from the above formulas (6) to (11).

The composition and food and drink of the present invention usually have a GLP-1 secretion promoting action (or a GLP-1 activating action).
Therefore, the present invention also includes a method of ingesting, taking or administering the composition or food or drink to activate GLP-1 (or promote the secretion of GLP-1). Since GLP-1 secretion can be activated by such a method, it can be applied to symptoms caused or related to the activation action of GLP-1 secretion. For example, such a method is selected from suppression of blood glucose elevation, suppression of appetite, suppression of overeating, improvement of glucose metabolism, prevention or treatment of diabetes, prevention or treatment of obesity, weight reduction, and reduction of body fat percentage. It may be at least one method.
Further, in the present invention, the composition or food or drink is ingested, taken or administered to suppress an increase in blood glucose level, suppress appetite, suppress overeating, improve glucose metabolism, prevent or treat diabetes, prevent or treat obesity. Also included are methods of performing (or achieving) at least one selected from weight loss, and body fat percentage reduction. Such a method does not necessarily have to be accompanied by activation of GLP-1 (promotion of secretion), and may be accompanied by activation of GLP-1 (promotion of secretion).
In the above method, the target of ingestion, administration or administration may be any animal, and may be any human or non-human animal (dog, cat, etc.).

The present invention can provide a composition useful for activating (or promoting) GLP-1 (or GLP-1 secretion). Since such a composition has a GLP-1 activating action, an action / function caused by or related to the action, for example, an action of suppressing an increase in blood glucose level, an action of suppressing appetite, an action of suppressing overeating, an action of improving glucose metabolism, It can be suitably used as a composition for the purpose of preventing or treating diabetes, preventing or treating obesity, reducing body weight, reducing body fat percentage, and the like.
Further, in the present invention, regardless of whether or not it is caused by the secretion promoting function of GLP-1, for suppressing increase in blood glucose level, for suppressing appetite, for suppressing overeating, for improving glucose metabolism, for preventing or treating diabetes, and for obesity. It is possible to provide a composition useful for prevention or treatment of diabetes, for weight reduction, for reduction of body fat percentage, and the like.
Therefore, the composition of the present invention can also be used for the prevention / treatment of obesity, for dieting, for the prevention / treatment of diabetes, and the like. The timing of ingestion (ingestion) of the composition of the present invention is not particularly limited, and can be used, for example, for pre-meal (pre-meal ingestion), inter-meal, post-meal, and the like.

In particular, since the composition of the present invention can promote the secretion of GLP-1 and improve glucose metabolism, it can be suitably used for diseases requiring improvement of glucose metabolism. Such diseases include diabetes and obesity, which are effective in their prevention and treatment. Therefore, the composition of the present invention can be provided, for example, with an indication that it is used for prevention and / or improvement of hyperglycemia and obesity, and is an object having a high blood glucose level. It is extremely useful for people, those who are overweight, and those who have a tendency toward metabolic syndrome.

Moreover, the composition (or compound) of the present invention is stable against heat and long-term storage, and can be stably present in vivo (for example, under gastric acid exposure). Therefore, it can be suitably used as an additive for foods and drinks.

Further, in the present invention, the above composition or a compound contained in the composition and useful for activation of GLP-1 can be efficiently produced.

Furthermore, the present invention can provide a novel compound useful for activation of GLP-1 and the like.

FIG. 1 is a graph showing the amount of GLP-1 secreted by each component in Example 3 when the control is set to 100 in Example 3. FIG. 2 is a chromatogram of LC-MS analysis of the acetone extract and fraction 2 obtained in Example 3. FIG. 3 is a graph showing the change with time (100 ° C.) of each component amount in Example 6. FIG. 4 is a graph showing the change with time (120 ° C.) of each component amount in Example 6. FIG. 5 is a graph showing the results of the stability test of each component in Example 7. FIG. 6 is a graph showing the measurement results of the blood glucose level in Example 9.

[Component (A)]
The composition of the present invention contains a component (A) selected from the following formulas (1) to (11).

In the composition of the present invention, the component (A) may contain at least one compound of the compounds (1) to (11), and may contain a combination of two or more compounds.

Among the components (A), the compounds (6) to (11) are novel compounds, and the present invention also includes such novel compounds. In the present specification, the names (trivial names) of these novel compounds are as shown in the table below.

The names of the compounds (1) to (5) are as follows.
Compound 1: momordicoside I
Compound 2: 5β, 19-epoxycucurbita-6,23 (E) -diene-3β, 19,25-triol (5β, 19-epoxycucurbita-6,23 (E) -diene-3,19,25-triol )
Compound 3: 7,23-dihydroxy-3-O-malonyl cucurbita-5,24-dien-19-al (7,23-dihydroxy-3-O-malonylcucurbita-5,24-dien-19-al)
Compound 4: momordicoside W
Compound 5: momordicoside P

In addition, the composition of the present invention may contain at least one component (A1) selected from the compounds (6) to (11).

Further, the composition of the present invention may contain the component (A) (or the compounds (1) to (11)) in the form of a derivative as long as it has an action of activating GLP-1 secretion. Such derivatives include, for example, salts, solvates (hydrates, etc.), ethers [eg, in compounds (1) to (11), some or all of the hydroxyl groups are alkoxy groups (eg, methoxy groups). , C _1-10 alkoxy group such as ethoxy group, preferably C _1-4 alkoxy group)], ester [for example, in compounds (1) to (11), part or all of the hydroxyl groups are acyloxy. A group substituted with a group (for example, a C _1-10 acyloxy group such as an acetoxy group, preferably a C _1-4 acyloxy group)] and the like.

In addition, such a derivative may be derived from the extraction solvent at the time of separating or separating the component (A) from bitter gourd or the like, for example.

The component (A) (or the compounds (1) to (11) and their derivatives) may be synthesized by a conventional method, but efficiently from a plant, particularly a bitter gourd (heat-treated bitter gourd) as described later. Obtainable.

Therefore, the composition of the present invention may contain the component (A) as a bitter gourd-derived component (bitter gourd component), particularly a heat-treated bitter gourd-derived component (bitter gourd component).
Such a bitter gourd component may usually be a bitter gourd processed product. Examples of the bitter gourd processed product include puree, beverage, crushed product, extract, dried product (dried product) and the like. The beverage may be squeezed juice. The bitter gourd component may be a mixture of these.

[Aspect of composition]
The form of the composition of the present invention is not particularly limited as long as it contains the component (A), and may be solid (powder or the like), liquid or the like.

In particular, the form of the composition of the present invention may be liquid. Examples of the liquid mode include puree, beverage and the like.

As described above, the component (A) is easily obtained as a bitter gourd-derived component, and even if a bitter gourd component (bitter gourd-derived component) other than the component (A) is contained, the GLP-1 active action may be impaired. Absent.

Therefore, the composition of the present invention may be, in particular, a powder (for example, a dried product) of bitter gourd (particularly heat-treated bitter gourd), a puree, a beverage (particularly puree), or the like. The puree or beverage may be concentrated or diluted, if necessary.

The composition of the present invention may be composed of only the component (A) as long as it has a GLP-1 activating effect, or may contain other components (such as bitter gourd-derived components other than the component (A)). ..

In the composition of the present invention (for example, a liquid composition), the ratio of the component (A) can be selected from the range of, for example, about 0.005 ppm or more (for example, 0.01 to 10,000 ppm) on a mass basis, and 0. It may be about 5 ppm or more (for example, 0.6 to 6000 ppm), more preferably about 1 ppm or more (for example, 5 to 1000 ppm), and more preferably about 10 ppm or more (for example, 20 to 800 ppm, preferably 50 to 500 ppm). May be good.

Further, in the composition of the present invention (for example, a liquid composition), the ratio of the component (A1) can be selected from the range of, for example, about 0.005 ppm or more (for example, 0.01 to 10,000 ppm) on a mass basis, and is 0. It may be about 5.5 ppm or more (for example, 0.6 to 6000 ppm), more preferably about 1 ppm or more (for example, 5 to 1000 ppm), and more preferably about 10 ppm or more (for example, 20 to 800 ppm, preferably 50 to 500 ppm). You may.

[Use of composition]
The composition of the present invention has a GLP-1 activating action (or a GLP-1 secretion promoting action) and the like. Therefore, the composition of the present invention can be used for GLP-1 activation or the like (or as a composition having a GLP-1 activating action).

In addition, the composition of the present invention has an action related to the action related to the GLP-1 activating action, for example, an action of suppressing an increase in blood glucose level, an action of suppressing appetite, an action of suppressing overeating, an action of improving glucose metabolism, and diabetes. It can be suitably used as a composition for the purpose of prevention or treatment of obesity, prevention or treatment of obesity, weight reduction action, body fat percentage reduction action and the like.

The method of use (application method) of the composition of the present invention can be appropriately selected depending on the form of the composition and the like. For example, the composition of the present invention may be used as it is (ingestion, ingestion, administration, etc.), or the composition of the present invention may be formulated together with other components (carrier, excipient, etc.) and used (ingestion). , Ingestion, administration, etc.).

Examples of other components include excipients, binders, disintegrants, coating agents, lubricants, colorants, flavoring agents, stabilizers, emulsifiers, absorption promoters, surfactants, and pH adjustments. Formulations, preservatives, antioxidants and the like. The formulation method is not particularly limited, and a conventional method can be applied.

When formulated, examples of the dosage form include tablets, powders, fine granules, granules, dry syrups, coated tablets, orally disintegrating tablets, chewable tablets, capsules, soft capsules, syrups and the like.

In addition, the composition of the present invention may be added (or contained) to foods and drinks for use (ingestion, ingestion, administration, etc.). Foods and drinks include general foods including so-called health foods, foods for specified health use, foods with nutritional function and other foods with health function. Foods and drinks also include supplements (dietary supplements) and feeds.

The food and drink may contain other ingredients such as food additives (food additives) as long as the composition 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) , Sucrose fatty acid esters, etc.), 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, amino acids), fragrances, sweeteners, flavoring agents, coloring agents, solvents (ethanol), salts, surfactants, pH adjusters, buffers, antioxidants, stabilizers, gelling agents, increase Examples include adhesives, starches, encapsulants, suspending agents, coating agents, preservatives and the like. The food additive may be used alone or in combination of two or more.

In addition, other components may be usually non-bitter gourd-derived components.

When the composition of the present invention is added (or contained) to a food or drink, the food or drink is not particularly limited, but for example, food [for example, noodles (soba, udon, Chinese noodles, instant noodles, etc.) ), Tofu, confectionery (candy, candy, gum, chocolate, snack confectionery, biscuits, cookies, gummy, etc.), breads, marine or livestock processed foods (kamaboko, ham, sausage, etc.), dairy products (processed milk, fermented milk, etc.) ), Fats and oils processed foods (salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressings, etc.), seasonings (sauce, sauce, etc.), cooked or semi-cooked foods (champles, etc.), retort foods (chample, etc.) Curry, stew, bowl, porridge, miscellaneous cooking, etc.), cold confectionery (ice cream, sherbet, shaved ice, etc.)], beverages (tea beverages, soft beverages, carbonated beverages, nutritional beverages, fruit beverages, lactic acid beverages, juices, drinks Etc.) and so on.

In particular, the food or drink may be a beverage (particularly, a beverage containing bitter gourd).

In foods and drinks, the proportion of the composition (or component (A)) of the present invention can be selected from the range of, for example, 0.005 ppm or more (for example, 0.01 to 10,000 ppm) based on the mass of the component (A). , 0.5 ppm or more (for example, 0.6 to 6000 ppm), more preferably about 1 ppm or more (for example, 5 to 1000 ppm), and more preferably about 10 ppm or more (for example, 20 to 800 ppm, preferably 50 to 500 ppm). It may be.

Further, in foods and drinks, the proportion of the composition (or component (A)) of the present invention is, for example, from the range of about 0.005 ppm or more (for example, 0.01 to 10,000 ppm) based on the mass of the component (A1). It can be selected and may be about 0.5 ppm or more (for example, 0.6 to 6000 ppm), more preferably 1 ppm or more (for example, 5 to 1000 ppm), and 10 ppm or more (for example, 20 to 800 ppm, preferably 50 to 500 ppm). ) May be the case.

When the composition or food or drink of the present invention is used, the intake amount (dose, dose) is not particularly limited and can be appropriately selected according to the target age, body weight, health condition, etc., for example, one day. The amount of intake per component (A) is 0.001 mg or more (for example, 0.001 to 10000 mg), preferably 0.01 mg or more (for example, 0.05 to 5000 mg), and more preferably 0.1 mg or more in terms of component (A). (For example, 0.5 to 1000 mg), particularly about 1 mg or more (for example, 5 to 500 mg) may be used.

The compositions and foods and drinks of 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 composition and food and drink of 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 indications are not particularly limited, but are limited to GLP-1 secretion promoting function and GLP-1 secretion promoting function, for example, blood glucose level elevation suppressing function, appetite suppressing function, overeating suppressing function, sugar metabolism. The function of improving, the function of preventing or treating diabetes, the function of preventing or treating obesity, the function of reducing weight, the function of reducing body fat percentage, or a display that can be equated with these.
In addition, such a label may be attached to the composition or the food or drink itself, or may be attached to the container or the packaging of the composition or the food or drink, depending on the mode of the composition or the food or drink.

[Method for producing composition]
The composition (or component (A)) of the present invention may be produced by a synthetic method, but in particular, it can be efficiently produced by using a bitter gourd component.

That is, a large amount of a component (precursor) that produces the component (A) is contained in the bitter gourd component, and the component (A) is produced by heating such a precursor. Therefore, the present invention includes a method for producing the composition (or the component (A)) through at least a heating step of heat-treating the bitter gourd component.

It can also be said that the component (A) is expressed or increased in the bitter gourd component through such a heating step. Therefore, the present invention also includes a method of expressing or increasing the component (A) in the bitter gourd component through at least a heating step.

In the heating step, the bitter gourd component is not particularly limited as long as it contains a component that produces the component (A), such as bitter gourd (bitter gourd body), fruit (or flesh), skin, cotton, seeds, and seed coat. It may be a mixture thereof. Usually, bitter gourd often uses at least a portion containing fruits (bitter gourd body, pulp, etc.). Moreover, the bitter gourd component may be a bitter gourd processed product.

The bitter gourd component may be heat-treated as it is, but for example, it is preferably heat-treated as a puree or a processed bitter gourd product such as a puree or a beverage. By heat-treating such a bitter gourd component puree or the like, the component (A) can be efficiently produced or expressed.
The puree and the like may be concentrated or diluted as needed.
Further, the bitter gourd component may be heat-treated (or the bitter gourd component containing the acid component is heat-treated) in the presence of an acid component (for example, an organic acid such as acetic acid or citric acid). By heat-treating in the presence of an acid component, the component (A) can be easily produced efficiently.

In the heating step, the heating temperature can be appropriately selected depending on the heating time and the like, but is, for example, 40 ° C. or higher (for example, 40 to 160 ° C.), preferably 60 ° C. or higher (for example, 60 to 140 ° C.), more preferably. May be about 80 ° C. or higher (for example, 80 to 120 ° C.).

The heating time is, for example, about 1 minute or more (for example, 1 to 60 minutes), preferably 5 minutes or more (for example, 5 to 40 minutes), and more preferably 10 minutes or more (for example, 10 to 30 minutes). It may be.

In the heating step, the heating method can be appropriately selected depending on the form of the bitter gourd component and the like, and is not particularly limited, and may be any of resistance heating, induction heating, dielectric heating (microwave heating, etc.) and the like. .. Further, the heating means can be appropriately selected according to the heating method.
Depending on the form of the bitter gourd component, it may be heated by boiling, cooking, baking, steaming, or boiling water.

The heating (heat treatment) may be performed in an active atmosphere (air atmosphere, etc.) or in an inert atmosphere. Alternatively, it may be heated in a closed system. In particular, the component (A) can be efficiently produced by heating in a closed system.

The bitter gourd component (heat-treated bitter gourd component) after the heating step may be used as it is or may be purified depending on the form of the composition. For example, when the composition is puree or the like, the bitter gourd component after the heat treatment may be concentrated or diluted as it is and used. Further, when the composition is composed mainly of the component (A), a composition containing a high proportion of the component (A) may be obtained from the bitter gourd component after the heat treatment by using a conventional method such as extraction. ..

Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited thereto, and many modifications have ordinary knowledge in the art within the technical idea of the present invention. It is possible depending on the person.

(Example 1) Separation and identification of compounds (1) to (11) (extraction and crude fractionation)
Dried bitter gourd chips (20 kg) were extracted with MeOH (MeOH) to obtain a MeOH extract. The obtained MeOH extract was partitioned with 50% aqueous MeOH solution-hexane (1: 1,60 L). The soluble part of the 50% MeOH aqueous solution is adsorbed on an ion exchange resin [Diaion HP-20 (resin amount: 30 L) manufactured by Mitsubishi Chemical Corporation], eluted with 50% MeOH aqueous solution (100 L) and MeOH (100 L), and under reduced pressure. The solvent was distilled off to obtain a 50% MeOH fraction and a MeOH fraction (114 g).
Since the bitter gourd-dried chip is a heated air-dried product, it has been heat-treated.

(Purification of MeOH fraction)
Divide the MeOH fraction (5.0 g) into 19 fractions with a medium pressure preparative liquid chromatograph (MPLC) (ODS, water-MeOH) and MPLC (ODS, 20 mM CH ₃ COONH ₄ aqueous solution-CH ₃ CN, acetone). I drew it.
Fraction 7 was purified by high performance liquid chromatography (HPLC) (YMC, Triart C18, water-CH ₃ CN) to obtain compound (3) (25.0 mg). In addition, fraction 9 was purified by HPLC (Triart C18 manufactured by YMC, water-CH ₃ CN), and compound (7) (18.3 mg) was isolated. Fractions 12 and 16 are also similarly purified by HPLC (YMC, Triart C18, water-CH ₃ CN) to obtain compound (11) (0.8 mg) from fraction 12 and compound (0.8 mg) from fraction 16. 6) (1.0 mg) was obtained.

(Heat treatment of MeOH fraction and purification of heat-treated product)
A 2% acetic acid / 70% CH ₃ CN aqueous solution was added to a MeOH fraction (10.2 g), and the reaction product was heated under reflux for 5 hours with MPLC (ODS, 20 mM CH ₃ COONH ₄ aqueous solution-CH ₃ CN, acetone). It was divided into 17 fractions.
Fraction 4 was purified by HPLC (YMC, Triart C18, water-CH ₃ CN) to obtain compound (9) (10.0 mg). Fraction 8 was purified by HPLC (YMC, Triart C18, water-CH ₃ CN) to isolate compound (4) (19.7 mg). Fractions 9 and 10 are also purified by HPLC (YMC, Triart C18, H ₂ O-CH ₃ CN) to obtain compound (5) (17.1 mg) from fraction 9 and compound (17.1 mg) from fraction 10. 1) (43.5 mg) was obtained. The fractions 16 and 17 are combined, MPLC (ODS, 20 mM CH ₃ COONH ₄ aqueous solution-CH ₃ CN, acetone), HPLC (YMC, Triart C18, water-CH ₃ CN) and HPLC (Nacalai Tesque, Inc., Purification with Cosmosil PBr, water-CH ₃ CN) gave compound (2) (10.7 mg), compound (10) (3.8 mg), and compound (8) (4.6 mg).

The structures of the compounds (1) to (11) were identified by mass spectrometry and NMR, and it was confirmed that they had the same structures as described above. An example of structural analysis data is shown below.

[Compound (1)]

^{1 1} H-NMR (Pyridine-d5): δ0.77 (3H, s), 0.85 (3H, s), 0.95 (3H, s), 0.97 (3H, d, J = 5.6Hz) ), 1.56 (3H, s), 1.569 (3H, s), 1.573 (3H, s), 2.28 (2H, m), 2.32 (1H, br s), 2. 43 (1H, br d, J = 10.0Hz), 3.62 (1H, d, J = 7.9Hz), 3.72 (1H, br s), 3.75 (1H, d, J = 7) .9Hz), 4.01 (1H, m), 4.04 (1H, m), 4.24 (2H, m), 4.44 (1H, dd, J = 5.2, 11.5Hz), 4.61 (1H, dd, J = 2.0, 11.5Hz), 4.94 (1H, d, J = 7.7Hz), 5.59 (1H, dd, J = 3.6, 9. 7Hz), 5.95 (3H, m), 6.22 (1H, d, J = 9.7Hz)

¹³ C-NMR (Pyridine-d5): δ15.5, 19.35, 19.41, 20.7, 21.6, 24.3, 26, 1, 28, 1, 28, 7, 31.4, 31.5, 33.9, 37.0, 39.6, 40.0, 40.6, 45.8, 45.9, 49.3, 50.7, 52.7, 63.5, 70. 2,72.3,76,3,78.8,78.9,80.6,86.0,86.3,107.4,124.7,130.5,134.6,142.2

HRMS (ESI, [M + Na] ⁺ ) calcd for C ₃₆ H ₅₈ NaO ₈ 641.4024, found 641.4025.

[Compound (2)]

^{1 1} H-NMR (Pyridine-d5): δ0.85 (3H, s), 0.92 (3H, d, J = 5.4Hz), 0.97 (3H, s), 1.00 (3H, s) ), 1.51 (3H, s), 1.58 (6H, s), 2.30 (1H, m), 2.34 (1H, br s), 2.46 (1H, dd, J = 6) .0, 12.1Hz), 3.64 (1H, br s), 5.37 (1H, m), 5.59 (1H, dd, J = 3.4,9.7Hz), 5.96 ( 2H, m), 6.28 (1H, br d, J = 9.7Hz)

¹³ C-NMR (Pyridine-d5): δ15.6, 18.4, 19.4, 21.8, 23.1, 24.9, 28.4, 28.6, 28.7, 31.4, 34.4, 37.1, 37.9, 39.1, 40.0, 45.8, 49.4, 50.5, 50.8, 70.2, 76.8, 86.7, 108. 3,124.7, 131.0, 134.4, 142.2

HRMS (ESI, M + Na ⁺ ) calcd for C ₃₀ H ₄₈ NaO ₄ 495.3445, found 495/3427.

[Compound (3)]

^{1 1} H-NMR (Pyridine-d5): δ0.84 (3H, s), 0.94 (3H, s), 1.04 (3H, m), 1.17 (3H, s), 1.34 ( 3H, s), 1.72 (3H, s), 1.73 (3H, s), 2.07 (1H, m), 2.11 (1H, m), 2.37 (1H, m), 2.70 (1H, m), 3.69 (1H, m), 3.70 (1H, m), 4.34 (1H, m), 4.83 (1H, m), 5.09 (1H) , M), 5.62 (1H, m), 6.23 (1H, m), 10.59 (1H, s)

¹³ C-NMR (Pyridine-d5): δ15.5, 18.59, 18.61, 19.6, 22.3, 23, 1, 25.6, 26.3, 26.8, 27.5 28.5, 30.0, 33.4, 35.3, 36.8, 40.6, 43.4, 45.9, 46.4, 48.8, 51.0, 51.2, 51. 7, 65.6, 65.9, 80.1, 125.2, 131.4, 132.4, 144.4, 168.0, 170.3, 208.1

HRMS (ESI, M + Na ⁺ ) calcd for C ₃₃ H ₅₀ NaO ₇ 581.3449, found 581.3444.

[Compound (4)]

^{1 1} H-NMR (CD ₃ OD): δ0.90 (3H, s), 0.94 (3H, s), 0.94 (3H, d, J = 5.6Hz), 0.95 (3H, s) ), 1.17 (3H, s), 1.28 (6H, s), 2.18 (1H, m), 2.48 (1H, dd, J = 5.4, 12.4Hz), 2. 94 (1H, br s), 3.25 (1H, m), 3.26 (1H, m), 3.31 (1H, m), 3.39 (1H, m), 3.41 (1H, 1H, m) br s), 3.68 (1H, dd, J = 5.4, 11.7Hz), 3.86 (1H, br d, J = 11.7Hz), 4.27 (1H, d, J = 7) 8.8Hz), 5.10 (2H, s), 5.61 (3H, m), 6.11 (1H, br d, J = 9.7Hz)

¹³ C-NMR (CD ₃ OD): δ13.8, 17.8, 17.9, 18.8, 19.1, 22.6, 24.3, 26.5, 27.6, 28.6 28.8, 30.4, 33.3, 36.2, 38.3, 38.9, 41.2, 41.3, 44.8, 47.3, 49.9, 61.4, 69. 8,70.4,73.9,76.2,76.4,84.9,85.6,104.6,106.1,124.5,131.2,132.7,139.4

HRMS (ESI, M + Na ⁺ ) calcd for C ₃₆ H ₅₈ NaO ₉ 657.3973, found 657.3985.

[Compound (5)]

^{1 1} H-NMR (CD ₃ OD): δ0.78 (3H, s), 0.81 (3H, s), 0.81 (3H, d, J = 5.6Hz), 0.82 (3H, s) ), 1.06 (3H, s), 1.16 (6H, s), 2.06 (1H, m), 2.36 (1H, dd, J = 5.4, 12.4Hz), 2. 82 (1H, br s), 3.24 (1H, dd, J = 2.8, 7.9 Hz), 3.29 (1H, br s), 3.37 (1H, dd, J = 2.8) , 9.2Hz), 3.53 (1H, m), 3.56 (1H, m), 3.72 (1H, m), 3.97 (1H, dd, J = 2.8, 2.8Hz) ), 4.56 (1H, d, J = 7.9Hz), 4.98 (2H, s), 5.48 (3H, m), 5.99 (1H, dd, J = 1.7,9) .7Hz)

¹³ C-NMR (CD ₃ OD): δ13.8, 17.8, 17.9, 18.8, 19.3, 22.6, 24.3, 26.5, 27.6, 28.6 28.7, 30.4, 33.3, 36.2, 38.3, 38.9, 41.2, 41.3, 44.8, 47.3, 49.9, 61.8, 67. 6, 69.8, 71.2, 71.3, 73.9, 84.9, 85.6, 103.4, 104.6, 124.5, 131.2, 132.7, 139.4

HRMS (ESI, M + Na ⁺ ) calcd for C ₃₆ H ₅₈ NaO ₉ 657.3973, found 657.3975.

[Compound (6)]

^{1 1} H-NMR (CDCl ₃ ): δ0.88 (3H, s), 0.91 (3H, s), 0.91 (3H, d, J = 5.8Hz), 0.94 (3H, s) , 1.24 (3H, s), 1.72 (3H, s), 1.74 (3H, s), 2.50 (1H, m), 2.86 (1H, m), 3.43 ( 1H, br s), 4.49 (1H, m), 5.16 (1H, m), 5.23 (1H, d, J = 8.4Hz), 5.69 (1H, dd, J = 3) 8.8, 9.8Hz), 6.11 (1H, dd, J = 2.2, 9.7Hz)

HRMS (ESI, M + Na ⁺ ) calcd for C ₃₀ H ₄₈ NaO ₄ 495.3445, found 495.3439.

[Compound (7)]

^{1 1} H-NMR (CD ₃ OD): δ0.72 (3H, s), 0.84 (3H, s), 0.90 (3H, d, J = 5.6Hz), 0.98 (3H, s) ), 1.22 (3H, s), 1.57 (3H, s), 1.60 (3H, s), 1.83 (2H, m), 1.84 (1H, br s), 1. 98 (1H, m), 2.30 (1H, m), 2.49 (1H, dd, J = 3.5, 8.7Hz), 3.05 (1H, dd, J = 7.7, 8) .0Hz), 3.13 (1H, m), 3.16 (1H, m), 3.23 (1H, m), 3.45 (1H, br s), 3.53 (1H, dd, J) = 4.5, 11.7Hz), 3.72 (1H, d, J = 11.7Hz), 3.91 (1H, d, J = 5.0Hz), 4.21 (1H, d, J = 7.7Hz), 4.31 (1H, dt, J = 3.2,8.5Hz), 5.06 (1H, d, J = 8.5Hz), 5.77 (1H, d, J = 5) .0Hz), 9.75 (1H, s)

¹³ C-NMR (CD ₃ OD): δ13.9, 16.7, 17.3, 17.9, 21.6, 21.9, 24.5, 24.6, 26, 1, 27,3 28.9, 32.3, 34.2, 35.8, 41.1, 44.2, 45.4, 47.6, 49.9, 50.1, 50.6, 61.4, 65. 1,65.3.70.3,74.2,76.3,76.6,85.8,105.0,122.0,129.0,132.0,146.3,208.6

HRMS (ESI, M + Na ⁺ ) calcd for C ₃₆ H ₅₈ NaO ₉ 657.3973, found 657.3978.

[Compound (8)]

^{1 1} H-NMR (Pyridine-d5): 0.87 (3H, s), 0.93 (6H, s), 0.98 (3H, d, J = 5.8Hz), 1.53 (3H, s) ), 1.89 (3H, s), 2.30 (1H, m), 2.31 (1H, m), 2.49 (1H, dd, J = 5.4, 12.4Hz), 3. 64 (1H, br s), 3.89 (1H, m), 4.18 (1H, m), 4.39 (1H, m), 4.45 (1H, m), 4.51 (1H, 1H, m) m), 4.68 (1H, m), 5.39 (1H, d, J = 7.7Hz), 5.01 (2H, br s), 5.64 (1H, m), 5.75 ( 1H, ddd, J = 6.3,8.2,15.5Hz), 6.23 (1H, dd, J = 2.2,9.8Hz), 6.29 (1H, d, J = 15. 5Hz)

¹³ C-NMR (Pyridine-d5) δ14.7, 18.65, 18.70, 19.7, 21.0, 23.1, 24.8, 27.3, 28.2, 30.9, 33 8.8, 36.7, 38.8, 39.9, 41.6, 45.2, 47.7, 48.2, 50.6, 63.0, 69.0, 72.1, 73.1 , 75.9, 84.2, 103.5, 104.8, 114.4, 129.8, 131.1, 133.9, 134.4, 142.3

HRMS (ESI, M + Na ⁺ ) calcd for C ₃₆ H ₅₆ NaO ₈ 639.3867, found 639.3868.

[Compound (9)]

^{1 1} H-NMR (Pyridine-d5): δ0.76 (3H, s), 0.84 (3H, s), 0.95 (3H, d, J = 6.0Hz), 1.13 (3H, s) ), 1.31 (3H, s), 1.526 (3H, s), 1.531 (3H, s), 2.07 (1H, m), 2.22 (1H, m), 2.33 (1H, s), 2.66 (1H, m), 3.64 (1H, d, J = 15.4Hz), 3.69 (1H, d, J = 15.4Hz), 4.31 (1H) , D, J = 4.8Hz), 5.07 (1H, m), 5.91 (1H, m), 5.93 (1H, m), 6.19 (1H, d, J = 4.8Hz) ), 10.57 (1H, s)

¹³ C-NMR (Pyridine-d5): δ14.8, 17.8, 18.7, 21.6, 22.3, 24.9, 26.0, 26.8, 27.4, 29.1, 30.6, 34.6, 36.1, 36.3, 39.3, 39.8, 42.7, 45.5, 47.9, 49.9, 50.2, 50.5, 65. 2,69.5,79.4,123.9, 124.5, 141.5, 143.7, 167.3, 169.5, 207.3

HRMS (ESI, M + Na ⁺ ) calcd for C ₃₃ H ₅₀ NaO ₇ 581.3449, found 581.3433.

[Compound (10)]

^{1 1} H-NMR (Pyridine-d5): 0.88 (3H, s), 0.93 (6H, s), 0.98 (3H, d, J = 5.8Hz), 1.53 (3H, s) ), 1.90 (3H, s), 2.30 (1H, m), 2.31 (1H, m), 2.49 (1H, dd, J = 5.4, 12.4Hz), 3. 69 (1H, br s), 4.00 (2H, m), 4.20 (1H, dd, J = 8.7, 8.7Hz), 4.24 (1H, dd, J = 8.7, 8.8Hz), 4.40 (1H, dd, J = 2.0, 11.4Hz), 4.57 (1H, dd, J = 5.0, 11.4Hz), 4.93 (1H, d) , J = 7.7Hz), 4.96 (2H, br s), 5.65 (1H, m), 5.75 (1H, ddd, J = 6.3,8.2,15.5Hz), 6.24 (1H, dd, J = 2.2, 9.8Hz), 6.30 (1H, d, J = 15.5Hz)

¹³ C-NMR (Pyridine-d5): δ14.6, 18.65, 18.70, 19.7, 21.0, 23.1, 24.8, 27.2, 28.1, 30.8, 33.7, 36.7, 38.9, 39.9, 41.6, 45.2, 47.7, 48.2, 50.6, 62.8, 71.6, 75.8, 78. 0,78.3,84,2,85.0,104.9,106.2,114.4,129.8,131.1,133.9,134.4142.3

HRMS (ESI, M + Na ⁺ ) calcd for C ₃₆ H ₅₆ NaO ₈ 639.3867, found 639.3847.

[Compound (11)]

^{1 1} H-NMR (Pyridine-d5) (Pyridine-d5): 0.76 (3H, s), 0.83 (3H, s), 0.88 (3H, s), 0.95 (3H, d, J = 5.7Hz), 1.52 (3H, s), 1.54 (6H, s), 1.88 (1H, m), 2.23 (1H, m), 3.65 (1H, br) s), 3.88 (1H, m), 4.10 (1H, m), 4.36 (1H, m), 4.45 (1H, m), 4.54 (1H, m), 4. 62 (1H, m), 5.29 (1H, d, J = 7.7Hz), 5.60 (1H, d, J = 3.3, 9.7Hz), 5.91 (1H, m), 5.93 (1H, m), 6.30 (1H, d, J = 2.0, 9.7Hz)

¹³ C-NMR (Pyridine-d5): δ14.4, 18.5, 18.7, 19.1, 20.8, 23.6, 28.0, 30.6, 30.8, 33.7, 36.3, 38.1, 39.2, 40.5, 44.6, 47.7, 48.2, 50.1, 50.5, 63.1, 68.9, 72.1, 72. 9,75.7,84.9,86.9,104.7,123.9,132.3,132.7,141.4,181.9

HRMS (ESI, M + Na ⁺ ) calcd for C ₃₆ H ₅₆ NaO ₉ 655.38317, found 655.3823.

(Example 2) Confirmation of GLP-1 secretion promoting action 150 ml of water was added to a bitter gourd dried chip (10.5 g), and the mixture was heated under reflux for 1 hour. 150 ml of acetone was added to the reaction product for extraction, and the mixture was filtered. 100 ml of acetone was added to the residue and re-extracted. After filtration, the filtrates were combined and concentrated under reduced pressure to obtain 3.46 g of an acetone extract.
It was confirmed that the compounds (1) to (11) obtained in Example 1 and the acetone extract obtained as described above have a GLP-1 secretory activity.

As an example, the results of comparison with the controls for compounds (1) to (5) and (7) are shown in the table below. The table also shows the results of TPA (3 μM), which is known to have a GLP-1 secretion activating effect.
The GLP-1 secretion activating action was confirmed as follows.
Poly-L-Lycine 96-well plate is BD, PBS (+), antibiotics, Dulvecco's Modified Eagle's Medium (DMEM), glucose, carboxymethyl cellulose. The sodium salt (CMC-Na) is manufactured by Nakaraitesk Co., Ltd., the TPA [12-O-tetradecanoylformol-13-Acatete] is manufactured by Cell Signaling Co., Ltd., and the active GLP-1 ELISA kit is Merck Millipore, Fetal glucose serum (FBS) was manufactured by Sigma, sitagliptin phosphate was manufactured by Santa Cruz Biotechnology, and NCI-H716 cells were transferred from ATCC.
NCI-H716 cells suspended in DMEM medium (10% FBS, 2 mM glutamine, 1% antibiotics added) were seeded on Poly-L-Lycine 96-well plates at 0.5 × 105 cells / well in 100 μL increments. , CO2 incubator (manufactured by espec) for 48 hours.
After washing with PBS (+), the final concentrations are compound (1) = 0.71 μg / mL, compound (2) = 1.03 μg / mL, compound (3) = 0.52 μg / mL, compound (4) = 1.92 μg / mL, compound (5) = 1.43 μg / mL, compound (7) = 6.35 μg / mL bitter gourd acetone extract = 1 mg / ml, 100 μL of PBS (+) solution supplemented with 10 μM Sitagliptin phosphate. Added to cells. After 1 hour, the added solution was recovered and the amount of active GLP-1 in the solution was measured using an ELISA kit.

(Example 3) Preparation method of fraction containing a large amount of GLP-1 secretagogue active ingredient 150 ml of water was added to bitter gourd dried chips (10.5 g), and the mixture was heated under reflux for 1 hour. 150 ml of acetone was added to the reaction product for extraction, and the mixture was filtered. 100 ml of acetone was added to the residue and re-extracted. After filtration, the filtrates were combined and concentrated under reduced pressure to obtain 3.46 g of an acetone extract.
The obtained acetone extract was fractionated by MPLC (ODS), and the water-eluted fraction (fraction 1: 3.1 g), the acetonitrile-eluted fraction (fraction 2: 210 mg) and the acetone-eluted fraction (fraction 3) were fractionated. : 140 mg) was obtained.
It was confirmed that each fraction and the acetone extract had a GLP-1 secretory activity in the same manner as in Example 2.

The result of comparison with the control is shown in FIG. In the figure, TPA (3 μM) known to have a GLP-1 activating effect is also shown, Fr means “fraction”, and “bitter gourd” means acetone extract.

As is clear from the results of FIG. 1, when the GLP-1 secretion promoting activity of the acetone extract (bitter gourd) and each fraction (fractions 1-3) was confirmed, the strongest activity was shown in the fraction 2. It was found that the active ingredient was contained in a large amount in fraction 2.

In addition, LC-MS analysis of the acetone extract and fraction 2 was performed. The results are shown in FIG.

As is clear from the results of FIG. 2, since the two MS chromatograms of the acetone extract and the fraction 2 are almost the same, they are present in the acetone extract after the heat treatment and are efficient for the fraction 2. It was found that it was concentrated in.

It was also confirmed that the fraction 2 contained the compounds (1) to (11).

(Example 4) Quantification of compounds (1) to (5) In each of the samples shown below, compounds (1) to (5) were picked up from compounds (1) to (11) and subjected to LC-MS analysis. Quantified by.

(Heat-treated bitter gourd)
The lyophilized powder of bitter gourd (50 mg) was weighed in a test tube with a lid, 3 ml of water was added, and the mixture was sealed. It was heated at 120 ° C. for 20 minutes using a microwave synthesizer (Discover manufactured by CEM). After heating, 7 ml of acetonitrile was added to the reaction product, and the mixture was extracted with ultrasonic waves for 2 minutes. After standing, the supernatant was filtered through a 0.45 μm filter to prepare an analytical sample.

(Raw bitter gourd, bitter gourd puree, bitter gourd juice)
50 mg each of a frozen dried product of raw bitter gourd, bitter gourd puree or bitter gourd juice was weighed in a test tube with a lid, 3 ml of water and 7 ml of acetonitrile were added, and the mixture was extracted by ultrasonic waves for 2 minutes. After standing, the supernatant was filtered through a 0.45 μm filter to prepare an analytical sample.
Samples of raw bitter gourd, bitter gourd puree, and bitter gourd juice were obtained as follows.
Raw bitter gourd: The bitter gourd was cut into about 1/2 to 1/4, and cotton and seeds were removed to obtain raw bitter gourd.
Puree: Bitter gourd was cut to about 1/2 to 1/4 and cotton and seeds were removed. Water was added so that bitter gourd: water = 4: 1 and crushed with a mixer to obtain a puree stock solution. The puree was sterilized, cooled, and stored frozen until use.
The puree stock solution was placed in a closed heat-resistant container and immersed in a constant temperature bath containing water. The set temperature of the constant temperature layer was set to 95 ° C., and the temperature was gradually heated from the state of water. The temperature of the constant temperature bath was maintained for 15 minutes from the time when the temperature reached 95 ° C. to obtain puree.
Juice: The puree obtained above was hydrated so that bitter gourd puree: water = 2: 3. It was sterilized at 108 ° C. to 111 ° C. for about 65 seconds through a strainer of 80 mesh or more. It was filled and cooled at 88 ° C. or higher.

The analysis conditions were as follows.
(LCMS measurement)
Each sample was analyzed by LCMS-IT-TOF and quantified. The quantitative value was obtained from a calibration curve prepared in advance using a standard.
(LC condition)
Column: Triart C18 (YMC, 150 x 2.1 mm ID, 3 μm)
Column temperature: 40 ° C
Flow velocity: 0.3 ml / min
Gradient: 30% → 100% (30-39 minutes) B in A
A: 0.1% formic acid aqueous solution B: 0.1% formic acid acetonitrile injection amount: 2 μl

The results of quantifying compounds (1) to (5) in each sample are shown in the table below.

As is clear from the results in the above table, the compounds (1) to (5) are hardly contained in the raw bitter melon that has not been heat-treated, and are significantly contained in the bitter gourd or the processed bitter gourd product that has undergone the heat treatment. It turned out that

(Example 5) Comparison of content of compounds (1) to (5) depending on difference in heating temperature A freeze-dried powder (50 mg) of bitter gourd was weighed in a test tube with a lid, and 3 ml of water was added to seal the mixture . Using a microwave synthesizer (Discover manufactured by CEM), heating was performed at 100, 120, 140 or 160 ° C. for 10 minutes. After heating, 7 ml of acetonitrile was added to the reaction product, and the mixture was extracted with ultrasonic waves for 2 minutes. After standing, the supernatant was filtered through a 0.45 μm filter to prepare an analytical sample.
In each of the obtained samples, compounds (1) to (5) were quantified by LC-MS analysis in the same manner as in Example 4.

The results of quantifying compounds (1) to (5) in each sample are shown in the table below.

As is clear from the results in the above table, the amount of each component tends to increase as the heating temperature rises and then decrease. Therefore, in order to obtain the compounds (1) to (5) at a high ratio, the heating temperature must be adjusted. It turns out that adjustments are also needed.

(Example 6) Changes in content of compounds (1) to (5) over time under heat treatment conditions A freeze-dried powder (50 mg) of bitter gourd was weighed by a lidded examiner, and 3 ml of water was added and sealed. Using a microwave synthesizer (Discover manufactured by CEM), heating was performed at 100 ° C. and 120 ° C. for 2, 5, 10, 20, 40 or 60 minutes. After heating, 7 ml of acetonitrile was added to the reaction product, and the mixture was extracted with ultrasonic waves for 2 minutes. After standing, the supernatant was filtered through a 0.45 μm filter to prepare an analytical sample.
In each of the obtained samples, compounds (1) to (5) were quantified by LC-MS analysis in the same manner as in Example 4.

The results of quantifying the compounds (1) to (5) in each sample are shown in FIGS. 3 (100 ° C) and 4 (120 ° C).

As is clear from the results of FIGS. 3 and 4, the amount of each component tends to increase with the heating time and then decrease. Therefore, in order to obtain the compounds (1) to (5) at a high ratio, the heating time is required. It turns out that adjustments are also needed.

(Example 7) Stability test of compounds (1) to (5) 0.5 mL of 70% acetonitrile mixed aqueous solution and 0.2N hydrochloric acid aqueous solution containing compounds (1) to (5) at a concentration of 4 μg / mL, respectively. A 5 mL mixed solution was prepared, and the amounts of the compounds (1) to (5) when left at 40 ° C. were measured over time. The measurement was carried out by LC-MS analysis in the same manner as in Example 4.

Then, with the amount immediately after preparation as 100%, how the amounts of the compounds (1) to (5) in the sample change was plotted. The results are shown in FIG.

As is clear from the results of FIG. 5, the half-life of the compounds (1) to (5) was 2 hours or more under the conditions corresponding to the stomach, and it was found to be stable.

(Example 8) Confirmation of GLP-1 activating effect by heated puree Using the bitter gourd puree (heated puree) obtained in Example 4, the GLP-1 activating effect in vivo was confirmed under the following conditions.

1. 1. Animals used Wistar rat male 8 weeks old 2. Group composition Water, bitter gourd puree 3. Substances to be administered and number of cases Control (water) 20 mL / kg 9 cases Heated puree 20 g (administration liquid volume 20 mL) / kg 9 cases 4. Blood collection site Under isoflurane anesthesia, a cannula was inserted into the right jugular vein of the rat, and the cannula was pulled out from the back through the subcutaneous vein so that blood could be collected over time. The experiment was conducted one day or more after the cannulation surgery. The cannula was also filled with heparin to prevent blood from coagulating.
5. Blood collection and administration Blood collection was performed under light anesthesia of isoflurane, and was performed before and 1 hour after administration. The administration was oral immediately after blood sampling before administration.
6. Measurement item For the measurement of GLP-1, the total GLP-1 was measured using the GLP-1 ELISA kit (Wako High Sensitive).
7. Evaluation Evaluation was set to 100% before administration, and the control group and the heated puree group were compared according to the rate of increase after administration.

As is clear from the results in the above table, it was found that the heated puree group has an action of activating the secretion of GLP-1 in vivo.

(Example 9) Confirmation of blood glucose elevation inhibitory effect by heated puree Using the heated puree obtained in Example 4, the GLP-1 activating effect in vivo was confirmed under the following conditions.

1. 1. Animals used C57BL strain / 6 mice Male 6 weeks old 2. Dosage and solution amount The dose was 10 and 20 g / kg, and the dose was 20 mL / kg. For a dose of 20 g / kg, a heated puree stock solution was used as the administration solution, and for 10 g / kg, the stock solution was diluted 2-fold with purified water to prepare the administration solution.
3. 3. Group composition is as shown in the table below.

4. Oral glucose tolerance test (OGTT)
The blood glucose levels of the mice fasted for about 5 hours were measured, and the blood glucose levels were sorted into a control group, a low dose group, and a high dose group by a stratified sampling method to homogenize them. The administration substance was orally administered to each group, and 0.5 hours later, 2 g / kg of glucose (20% solution, 10 mL / kg) was orally administered. Blood glucose levels were collected from the tail vein before administration of the substance to be administered (-0.5 hours), immediately before glucose administration (0 hours), and 0.5, 1, 2, and 3 hours after glucose administration without anesthesia. The measurement was performed using a measuring device (Glucose Neo Super, Sanwa Kagaku Kenkyusho Co., Ltd.).

The results are shown in FIG.

As is clear from the results of FIG. 6, the blood glucose level in the control group showed an increase peaking at 0.5 hours after glucose administration and then decreasing. Both the low-dose group and the high-dose group of heated puree administration showed the same transition as the control group, but the low-dose group showed a tendency to suppress the increase in blood glucose level, and the high-dose group showed a significantly low blood glucose level. The tendency was observed up to 1 hour after glucose administration.

(Example 10) Production of tea beverage 20 g of bitter gourd puree obtained in Example 4 is added to 200 ml of a tea extract (prepared to 0.3% by mass by adding water to commercially available powdered green tea) to make a sencha beverage. Manufactured.

(Example 11) Production of coffee beverage A coffee extract is prepared by a conventional method using 65 g of boiling water to 5 g of crushed commercially available coffee powder, and 5 g of bitter gourd puree obtained in Example 4 is added to make coffee. Made a beverage.

(Example 12) Production of vegetable beverage Add 1 banana, 1 carrot, 1 bunch of salad vegetables, 30 g of bitter gourd puree obtained in Example 4, 200 ml of water, and 3 tablespoons of sugar to a food processor, and mix the vegetable beverage with a mixer. Obtained.

(Example 13) Production of biscuits Using 85 g of wheat flour, 30 g of sugar, 1 g of baking powder, 1 g of salt, 25 g of sesame oil, 20 g of milk, and 1 g of bitter gourd puree powder obtained by freeze-drying the bitter gourd puree obtained in Example 4, by a conventional method. Manufactured biscuits.

(Example 14) Production of tofu 600 ml of well-chilled soymilk (unadjusted), 15 ml of bittern, 30 ml of water, and 5 g of bitter gourd puree obtained in Example 4 were poured into a heat-resistant tapper and mixed. Wrapped and steamed in a steamer with steam for about 15 minutes on low heat to produce tofu.

The present invention can provide compositions useful for activating GLP-1 secretion. Further, in the present invention, such a composition can be efficiently produced.

Claims (31)

The following formulas (1) to at least one component selected from (11) (A) to including a GLP-1 secretagogue composition, at least selected from the following formulas (6) - (10) A composition containing one compound .
A composition containing at least one component (A) selected from the formulas (1) to (11) according to claim 1, and at least selected from the formulas (6) to (10) according to claim 1. Contains one compound, for blood sugar elevation suppression, appetite suppression, overeating suppression, glucose metabolism improvement, diabetes prevention or treatment, obesity prevention or treatment, weight loss, and body fat A composition for use in at least one application selected from for reducing rate. At least one function selected from suppression of blood glucose elevation, suppression of appetite, suppression of overeating, improvement of glucose metabolism, prevention or treatment of diabetes, prevention or treatment of obesity, weight loss, and reduction of body fat percentage is GLP. The composition according to claim 2, which is caused by the promotion of secretion of -1. The composition according to any one of claims 1 to 3, which comprises the compound of the formula ( 11 ) . The composition according to any one of claims 1 to 4, which comprises at least one compound selected from the formulas (1) to (2). The invention according to any one of claims 1 to 5, which comprises at least one compound selected from the formulas (1) to (2) and at least one compound selected from the formulas (3) to (5). Composition. The composition according to any one of claims 1 to 6, which comprises all the compounds of the formulas (1) to (5). It includes all compounds of formula (1) to (5), and the composition according to claim 1 comprising a compound of formula (11). The composition according to any one of claims 1 to 8, which contains the component (A) as a bitter gourd-derived component. The composition according to any one of claims 1 to 9, which comprises the component (A) as a heat-treated bitter gourd-derived component. The composition according to any one of claims 1 to 10, which is powdery or liquid. A liquid composition containing at least one component (A) selected from the formulas (1) to (11) according to claim 1 at a ratio of 0.005 ppm or more on a mass basis, according to claims 1 to 11. The composition according to any. The composition according to claim 12 , which comprises the compound of formula ( 11). The composition according to claim 12 or 13, which contains at least one component (A1) selected from the formulas (6) to (11) in a proportion of 0.005 ppm or more on a mass basis. The composition according to any one of claims 12 to 14, which is a puree or a beverage. The composition according to any one of claims 12 to 15, which is a heat-treated bitter gourd puree or beverage. The composition according to any one of claims 1 to 16, for addition to food and drink. A food or drink containing the composition according to any one of claims 1 to 17 . At least one component selected from the formulas of Claim 1, wherein (1) - (11) (A), a food or drink in a proportion of more than 0.005ppm by weight, of請Motomeko 1, wherein A food or drink containing at least one compound selected from the formulas ( 6 ) to ( 10 ). The component (A) is at least one compound selected from the formulas (1) to (2) according to claim 1 and at least one selected from the formulas (3) to (5) according to claim 1. The food or drink according to claim 18 or 19, which comprises the compound of the above. The food or drink according to any one of claims 18 to 20, wherein the component (A) contains all the compounds of the formulas (1) to (5) according to claim 1. The component (A) contains all the compounds of the formulas (1) to (5) according to claim 1, and at least one component (A1) selected from the formulas (6) to (11) according to claim 1. The food and drink according to any one of claims 18 to 21. The food or drink according to any one of claims 18 to 22, which contains a non-bitter gourd-derived component. The food or drink according to any one of claims 18 to 23, which is a beverage. The food or drink according to any one of claims 18 to 24 for promoting the secretion of GLP-1. Selected from blood glucose elevation suppression, appetite suppression, overeating suppression, glucose metabolism improvement, diabetes prevention or treatment, obesity prevention or treatment, body weight reduction, and body fat percentage reduction. The food or drink according to any one of claims 18 to 25, which is used for at least one purpose. A method for producing the composition according to any one of claims 1 to 17, which comprises a heating step of heat-treating the bitter gourd component. The method according to claim 27, wherein the puree of the bitter gourd component is heat-treated in the heating step. The method according to claim 27 or 28, wherein in the heating step, heat treatment is performed at 60 ° C. or higher for 5 minutes or longer. The method according to any one of claims 27 to 29, wherein the heat treatment is performed in a closed system in the heating step. A compound selected from the formulas (6) to (10) according to claim 1.