JPWO2017159725A1 - Composition for promoting GLP-1 secretion and method for producing the same - Google Patents

Abstract

The present invention provides compositions useful for activation of GLP-1 secretion. This composition can be composed of a specific compound such as the following compound (1).

Such a specific compound can be efficiently obtained, for example, by heat-treating bitter gourd ingredients (for example, bitter gourd puree).

Description

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

GLP-1 [Glucagon-like peptide-1] is a gastrointestinal hormone secreted from the gastrointestinal mucosal epithelium.
As actions of such GLP-1, insulin synthesis and secretion stimulation, glucagon secretion inhibition, food intake inhibition, hyperglycemia reduction and the like are known. Therefore, the improvement of these actions can be expected by activating GLP-1.

  On the other hand, bitter gourd (bitter gourd) is a plant that is edible and cultivated, and studies on the physiological effects of bitter gourd are also being made.

  And there is also a report which suggests the relationship between such bitter gourd and GLP-1, for example, in nonpatent literature 1, the specific compound contained in bitter gourd has the activation action of GLP-1, and has a blood glucose regulation function. It is 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).
Another object of the present invention is to suppress an increase in blood glucose level, to suppress appetite, to suppress overeating, to improve glucose metabolism, to prevent or treat diabetes, to prevent or treat obesity, to reduce body weight, to body fat An object of the present invention is to provide a composition useful for reducing the rate.
Still another object of the present invention is to provide a method for efficiently producing a compound useful for activation of GLP-1 secretion or the like or 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 intensive studies to solve the above-mentioned problems, the present inventors focused on bitter gourd and examined in detail the components contained in bitter gourd. In addition to the compounds described in Non-Patent Document 1, GLP-1 activity There is a possibility that various compounds having an oxidative effect may exist, and surprisingly, such compounds are not contained in raw bitter gourd or very little, if any, are subjected to heat treatment. In addition, such various compounds include compounds that are particularly excellent in the action of promoting GLP-1 secretion or include new compounds that have not been known so far. As a result, the present invention has been completed.

  That is, the composition of the present invention includes at least one component (A) selected from the following formulas (1) to (11) (included as an active component). Since such a composition has an activation action for GLP-1 secretion, it can be used as a composition for promoting GLP-1 secretion (a 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 intended for actions / functions resulting from or related to the action. For example, the composition of the present invention is used for suppressing blood glucose level elevation, appetite suppression, overeating, glucose metabolism improvement, diabetes prevention or treatment, obesity prevention or treatment, weight reduction, and It may be a composition for use in at least one application selected from those for reducing body fat percentage.

  Further, the composition of the present invention is used for suppressing an increase 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 reducing body weight, and It may be a composition for use in at least one application selected from those for reducing body fat percentage. In such a composition, blood sugar level increase suppression (function), appetite suppression (function), overeating suppression (function), glucose metabolism improvement (function), diabetes prevention or treatment (function), obesity prevention or treatment At least one function (or action) selected from (function), weight reduction (function), and body fat percentage reduction (function) may not necessarily result from GLP-1 secretion promotion. -1 secretion may be promoted.

In the composition of the present invention, the component (A) may particularly include 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 component derived from bitter gourd (particularly, a component derived from heat-treated bitter gourd).
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) at a ratio of 0.005 ppm or more on a mass basis. In addition, the component (A) may particularly include at least one component (A1) selected from the above formulas (6) to (11), and typically, the above formulas (6) to ( At least one component (A1) selected from 11) may be contained in a proportion of 0.005 ppm or more based on mass.

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

  You may use the composition of this invention in order to add to food-drinks (or as an additive for food-drinks).

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

  The composition or food or drink of the present invention has a GLP-1 secretion promoting action, and also has a function resulting from or related to the action. Therefore, the composition or food / beverage product of the present invention may be a composition or food / beverage product with an indication of a function that promotes secretion of GLP-1 and / or a function caused by promotion of secretion of GLP-1. Examples of such a composition or food and drink include, for example, a blood glucose level increase suppressing function, an appetite suppressing function, an overeating suppression function, a glucose metabolism improving function, a diabetes prevention or treatment function, an obesity prevention or treatment function, The composition or the food / beverage products etc. which attached | subjected the display (functional display) of the at least 1 function selected from the reduction function and the reduction function of a body fat ratio (a function which can be equated with these) are contained.

The component (A) can be efficiently generated 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, the heating step for heat-treating the bitter gourd component Including methods.
The present invention also includes a method for producing the composition, which includes a heating step of heat-treating bitter gourd ingredients.

  In the method of the present invention, in the heating step, puree or beverage (especially puree) of bitter gourd ingredients may be heat-treated. Moreover, in a heating process, you may heat-process at 60 degreeC or more for 5 minutes or more. Furthermore, you may heat-process with a closed system in a heating process.

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

The composition and food or drink of the present invention usually have a GLP-1 secretion promoting action (or 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 secretion of GLP-1). Since such a method can activate the secretion of GLP-1, it can be applied to symptoms caused by or related to the activation effect of GLP-1 secretion. For example, such a method is selected from suppression of an increase in blood glucose level, 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. Alternatively, at least one method may be used.
Further, in the present invention, the composition or food or drink is ingested, taken or administered, suppression of blood sugar level increase, suppression of appetite, suppression of overeating, improvement of glucose metabolism, prevention or treatment of diabetes, prevention or treatment of obesity Also included are methods of performing (or realizing) at least one selected from weight reduction and body fat percentage reduction. In such a method, it is not always necessary to involve activation of GLP-1 (secretion promotion), and it may be accompanied by activation of GLP-1 (secretion promotion).
In the above method, the subject of ingestion, administration or administration may be an animal, and may be a human or a non-human animal (dog, cat, etc.).

In the present invention, a composition useful for activation (or promotion) of GLP-1 (or GLP-1 secretion) can be provided. Since such a composition has a GLP-1 activating action, the action / function resulting from or related to the action, for example, a blood glucose level increase suppressing action, an appetite suppressing action, an overeating suppressing action, an action of improving sugar 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.
In the present invention, regardless of whether it is caused by the GLP-1 secretion-promoting function, it is used for suppressing blood glucose level increase, for suppressing appetite, for suppressing overeating, for improving glucose metabolism, for preventing or treating diabetes, obesity It is possible to provide a composition useful for the prevention or treatment of diseases, for weight reduction, for body fat percentage reduction, and the like.
Therefore, the composition of the present invention can also be used for obesity prevention / treatment, diet, diabetes prevention / treatment, and the like. In addition, the timing of ingestion (taking) of the composition of the present invention is not particularly limited, and can be used, for example, for before meals (for intake before meals), between meals, for after meals, and the like.

  In particular, since the composition of the present invention can promote the secretion of GLP-1 to improve glucose metabolism, it can be suitably used for diseases that require improved glucose metabolism. Such diseases can include diabetes and obesity and are effective in their prevention and treatment. Therefore, the composition of the present invention can be provided with an indication that the composition is used for the prevention and / or improvement of hyperglycemia and obesity, for example, and the subject has a high blood sugar level. It is extremely useful for a person who has a tendency to gain weight, a person who is overweight, a person who has a tendency to have metabolic syndrome, and the like.

  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 food and drink.

  Moreover, in this invention, the compound contained in the above compositions or the said composition and useful for activation of GLP-1 etc. can be manufactured efficiently.

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

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

[Component (A)]
The composition of this invention contains the component (A) selected from following formula (1)-(11).

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

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

In addition, 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-malonylcucurbita-5,24-dien-19-al
Compound 4: momordicoside W
Compound 5: momordicoside P

  Moreover, the composition of this invention may contain the at least 1 sort (s) of component (A1) selected from compound (6)-(11).

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

  Such a derivative may be derived, for example, from an extraction solvent for separating or separating the component (A) from bitter gourd or the like.

  Component (A) (or compounds (1) to (11) and derivatives thereof) may be synthesized by a conventional method, but efficiently from plants, especially bitter gourd (heat-treated bitter gourd), as will be described later. Can be obtained.

Therefore, the composition of the present invention may contain the component (A) as a bitter gourd-derived component (bitter gourd component), particularly as a heat-treated bitter gourd-derived component (bitter gourd component).
Such bitter gourd ingredients may usually be processed bitter gourd. Examples of processed bitter gourd products include purees, beverages, pulverized products, extracts, and dried products (dried products). The beverage may be juice. The bitter gourd component may be a mixture thereof.

[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 (such as powder) or liquid.

  In particular, the composition of the present invention may be liquid. As a liquid aspect, puree, a drink, etc. are mentioned, for example.

  As described above, the component (A) is easy to obtain as a bitter gourd-derived component, and even if the bitter gourd component (the bitter gourd-derived component) other than the component (A) is included, the GLP-1 activity may be impaired. Absent.

  Therefore, the composition of the present invention may in particular be bitter gourd (especially heat-treated bitter gourd) powder (for example, dried product), puree, beverage (particularly puree), and the like. In addition, you may concentrate or dilute a puree or a drink as needed.

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

  In the composition (for example, liquid composition) of the present invention, the proportion of the component (A) can be selected from a range of about 0.005 ppm or more (for example, 0.01 to 10,000 ppm), for example, on a mass basis. 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), or about 10 ppm or more (for example, about 20 to 800 ppm, preferably about 50 to 500 ppm). Also good.

  Moreover, in the composition (for example, liquid composition) of the present invention, the proportion of the component (A1) can be selected from a range of, for example, about 0.005 ppm or more (for example, 0.01 to 10000 ppm) based on mass. 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), or about 10 ppm or more (for example, 20 to 800 ppm, preferably about 50 to 500 ppm). May be.

[Use of composition]
The composition of the present invention has a GLP-1 activation action (or 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 activation effect).

  In addition, the composition of the present invention is caused by the GLP-1 activation action, and the action related to the action, for example, the blood glucose level increase suppressing action, the appetite suppressing action, the overeating suppressing action, the sugar metabolism improving action, diabetes It can be suitably used as a composition for the purpose of preventing or treating the above, for preventing or treating obesity, for reducing the body weight, for reducing the body fat percentage, and the like.

  The method of using the composition of the present invention (application method) can be appropriately selected according to the form of the composition. For example, the composition of the present invention may be used as it is (taken, taken, administered, etc.), and the composition of the present invention is formulated with other components (carrier, excipient, etc.) and used (taken). , Taking, administering, etc.).

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

  In the case of formulation, 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 used (ingested, taken, administered, etc.) by adding (or containing) to a food or drink. The food and drink includes not only general foods including so-called health foods but also health foods such as foods for specified health use and functional foods for nutrition. The food and drink also includes supplements (dietary supplements), feeds and the like.

  As long as the food / beverage products contain the composition of this invention, they may contain other components, such as a food additive (food additive). The food additive is not particularly limited. Calcium silicate, etc.), binder (eg, pregelatinized starch, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc.), disintegrant (eg, cellulose, hydroxypropylcellulose, corn starch, etc.), fluidizing agent (eg, light anhydrous silicic acid) Sucrose fatty acid esters, etc.), oils (eg, vegetable oils such as soybean oil, sesame oil, olive oil, linseed oil, sesame oil, rapeseed oil, coconut oil, corn oil, etc.), nutrients (eg, various Minera , Various vitamins, amino acids), flavorings, sweeteners, flavoring agents, coloring agents, solvents (ethanol), salts, surfactants, pH regulators, buffers, antioxidants, stabilizers, gelling agents, thickening Agents, lubricants, encapsulating agents, suspending agents, coating agents, preservatives and the like. The food additives may be used alone or in combination of two or more.

  In addition, a non-bitter gourd origin component may be sufficient as another component normally.

  When the composition of the present invention is used in addition to (or contained in) food or drink, the food or drink is not particularly limited. For example, food [e.g., noodles (soba, udon, Chinese noodles, instant noodles, etc.) ), Tofu, confectionery (rice cakes, candy, gum, chocolate, snacks, biscuits, cookies, gummi, etc.), breads, fishery or livestock processed foods (kamaboko, ham, sausage, etc.), dairy products (processed milk, fermented milk) Etc.), fats and oils and processed foods (salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing, etc.), seasonings (sauce, sauce, etc.), cooked or semi-cooked products (chample, etc.), retort food ( Curry, stew, rice cake, porridge, miscellaneous cooking, etc.), frozen dessert (ice cream, sorbet, shaved ice, etc.)], beverage (tea Fee, soft drinks, carbonated drinks, energy drinks, fruit drinks, lactic acid beverages, juices, etc. drinks) and the like.

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

  In the food and drink, the ratio of the composition (or component (A)) of the present invention can be selected from a range of, for example, about 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), or about 10 ppm or more (for example, 20 to 800 ppm, preferably about 50 to 500 ppm). It may be.

  Moreover, in the food and drink, the ratio of the composition (or component (A)) of the present invention is, for example, in the range of about 0.005 ppm or more (for example, 0.01 to 10000 ppm) on the mass basis of the component (A1). 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), or about 10 ppm or more (for example, 20 to 800 ppm, preferably 50 to 500 ppm). ) Degree.

  In addition, when using the composition or food / beverage products of this invention, intake (dosage, dosage) is not specifically limited, Although it can select suitably according to object age, weight, health condition, etc., for example, 1 day Per capita intake 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), 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 composition and food and drink of the present invention can be used or applied for either therapeutic use (medical use) or non-therapeutic use. Specifically, regardless of whether it is classified as a pharmaceutical, a quasi-drug, or a cosmetic, the function that promotes secretion of GLP-1 and the function that results from promotion of secretion of GLP-1 are explicitly or It can be used or applied as any implicitly appealing composition or food or drink.
Moreover, you may display the function resulting from the secretion promotion of GLP-1 and the function which accelerates | stimulates the secretion of GLP-1 in the composition and food-drinks of this invention. Such a display is not particularly limited, but includes a GLP-1 secretion promoting function and a function resulting from the promotion of GLP-1 secretion, such as a blood glucose level increase suppressing function, an appetite suppressing function, an overeating suppressing function, and a glucose metabolism. Improvement function, diabetes prevention or treatment function, obesity prevention or treatment function, weight reduction function, body fat percentage reduction function, or display that can be equated with these.
In addition, according to the aspect of a composition or food / beverage products, such a display may be attached | subjected to a composition or food / beverage products itself, and may be attached to the container or packaging of a composition or food / beverage products.

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

  That is, many components (precursors) that generate the component (A) are contained in the bitter gourd component, and the component (A) is generated by heating such a precursor. Therefore, the present invention includes a method for producing the composition (or component (A)) through at least a heating step of heat-treating the bitter gourd component.

  Moreover, it can also be said that a component (A) expresses or increases in a bitter gourd ingredient through such a heating process. Therefore, the present invention also includes a method of expressing or increasing the component (A) in the bitter gourd component through at least the heating step.

  In the heating step, the bitter melon component is not particularly limited as long as it contains the component that generates the component (A), such as bitter gourd (bitter melon body), fruit (or pulp), skin, cotton, seed, seed coat, etc. Or a mixture thereof. In general, bitter gourd often uses at least a fruit-containing portion (eg bitter gourd, pulp). The bitter gourd component may be processed bitter gourd.

The bitter gourd component may be heat-treated as it is, but for example, it is preferably heat-treated as puree, such as puree or beverage, particularly as puree. By heat-treating such bitter gourd puree or the like, the component (A) can be efficiently generated or expressed.
Puree and the like may be concentrated or diluted as necessary.
Further, the bitter gourd component may be subjected to heat treatment (or heat treatment of the bitter gourd component containing the acid component) 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 generated efficiently.

  In the heating step, the heating temperature can be appropriately selected according to 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), and more preferably. 80 degreeC or more (for example, about 80-120 degreeC) may be sufficient.

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

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

  Heating (heat treatment) may be performed under an active atmosphere (such as an air atmosphere) or may be performed under an inert atmosphere. Moreover, you may heat by a closed system. In particular, the component (A) can be efficiently generated by heating in a closed system.

  The bitter gourd component after the heating step (heated bitter gourd component) may be used as it is or may be purified according to 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. Further, when the composition is composed mainly of the component (A), a composition containing the component (A) in a high proportion from the bitter gourd component after the heat treatment may be obtained using a conventional technique such as extraction. .

  Hereinafter, the present invention will be described more specifically 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 by a person.

(Example 1) Separation and identification of compounds (1) to (11) (extraction and crude fractionation)
Bitter gourd chips (20 kg) were extracted with methanol (MeOH) to obtain a MeOH extract. The obtained MeOH extract was partitioned with 50% aqueous MeOH-hexane (1: 1, 60 L). The soluble part of 50% MeOH aqueous solution was adsorbed on an ion exchange resin [Made by Mitsubishi Chemical, Diaion HP-20 (resin amount: 30 L)], and eluted with 50% MeOH aqueous solution (100 L) and MeOH (100 L). The solvent was distilled off to obtain 50% MeOH fraction and MeOH fraction (114 g).
In addition, since the bitter gourd dry chip | tip is a heating air dried product, it is heat-processed.

(Purification of MeOH fraction)
The MeOH fraction (5.0 g) was separated into 19 fractions by 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) (manufactured by YMC, Trial C18, water-CH ₃ CN) to obtain compound (3) (25.0 mg). Fraction 9 was purified by HPLC (manufactured by YMC, Triart C18, water-CH ₃ CN) to isolate compound (7) (18.3 mg). Fractions 12 and 16 were similarly purified by HPLC (manufactured by YMC, Triart C18, water-CH ₃ CN), and from fraction 12, compound (11) (0.8 mg) was purified from fraction 16 to compound ( 6) (1.0 mg) was obtained.

(Heat treatment of MeOH fraction and purification of heat-treated product)
To the MeOH fraction (10.2 g), 2% acetic acid / 70% CH ₃ CN aqueous solution was added, and the reaction mixture heated under reflux for 5 hours was treated with MPLC (ODS, 20 mM CH ₃ COONH ₄ aqueous solution-CH ₃ CN, acetone). Fractionated into 17 fractions.
Fraction 4 was purified by HPLC (manufactured by YMC, Trial C18, water-CH ₃ CN) to obtain compound (9) (10.0 mg). Fraction 8 was purified by HPLC (manufactured by YMC, Trial C18, water-CH ₃ CN) to isolate compound (4) (19.7 mg). Fractions 9 and 10 were similarly purified by HPLC (manufactured by YMC, Trial C18, H ₂ O—CH ₃ CN). From fraction 9, compound (5) (17.1 mg) was purified from fraction 10 to compound ( 1) (43.5 mg) was obtained. Fractions 16 and 17 were 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, Cosmosil PBr, purified water _-CH 3 CN), compound (2) (10.7mg), to give the compound (10) (3.8mg), and the compound (8) (4.6mg).

  In addition, the structures of the compounds (1) to (11) were identified by mass spectrometry and NMR, and confirmed to be the structures as described above. An example of structural analysis data is shown below.

  [Compound (1)]

¹ H-NMR (Pyridine-d5): δ 0.77 (3H, s), 0.85 (3H, s), 0.95 (3H, s), 0.97 (3H, d, J = 5.6 Hz) ), 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 = 10.0 Hz), 3.62 (1 H, d, J = 7.9 Hz), 3.72 (1 H, br s), 3.75 (1 H, d, J = 7) .9 Hz), 4.01 (1 H, m), 4.04 (1 H, m), 4.24 (2 H, m), 4.44 (1 H, dd, J = 5.2, 11.5 HHz), 4.61 (1H, dd, J = 2.0, 11.5 Hz), 4.94 (1H, d, J = 7.7 Hz), 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): δ 15.5, 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 H-NMR (Pyridine-d5): δ 0.85 (3H, s), 0.92 (3H, d, J = 5.4 Hz), 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, 12.1 Hz), 3.64 (1 H, br s), 5.37 (1 H, m), 5.59 (1 H, dd, J = 3.4, 9.7 Hz), 5.96 ( 2H, m), 6.28 (1H, br d, J = 9.7 Hz)

¹³ C-NMR (Pyridine-d5): δ 15.6, 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 ₄ 435.3445, found 495.3427.

  [Compound (3)]

¹ H-NMR (Pyridine-d5): δ 0.84 (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): δ 15.5, 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 H-NMR (CD ₃ OD): δ 0.90 (3H, s), 0.94 (3H, s), 0.94 (3H, d, J = 5.6 Hz), 0.95 (3H, s) ), 1.17 (3H, s), 1.28 (6H, s), 2.18 (1H, m), 2.48 (1H, dd, J = 5.4, 12.4 Hz), 2. 94 (1H, brs), 3.25 (1H, m), 3.26 (1H, m), 3.31 (1H, m), 3.39 (1H, m), 3.41 (1H, br s), 3.68 (1H, dd, J = 5.4, 11.7 Hz), 3.86 (1H, br d, J = 11.7 Hz), 4.27 (1H, d, J = 7) .8 Hz), 5.10 (2 H, s), 5.61 (3 H, m), 6.11 (1 H, br d, J = 9.7 Hz)

¹³ C-NMR (CD ₃ OD): δ 13.8, 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 6577.3973, found 657.3985.

  [Compound (5)]

¹ H-NMR (CD ₃ OD): δ 0.78 (3H, s), 0.81 (3H, s), 0.81 (3H, d, J = 5.6 Hz), 0.82 (3H, s) ), 1.06 (3H, s), 1.16 (6H, s), 2.06 (1H, m), 2.36 (1H, dd, J = 5.4, 12.4 Hz), 2. 82 (1H, br s), 3.24 (1 H, dd, J = 2.8, 7.9 Hz), 3.29 (1H, br s), 3.37 (1H, dd, J = 2.8) , 9.2 Hz), 3.53 (1 H, m), 3.56 (1 H, m), 3.72 (1 H, m), 3.97 (1 H, dd, J = 2.8, 2.8 Hz) ), 4.56 (1H, d, J = 7.9 Hz), 4.98 (2H, s), 5.48 (3H, m), 5.99 (1H, dd, J = 1.7, 9) .7Hz)

¹³ C-NMR (CD ₃ OD): δ 13.8, 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 6577.3973, found 6577.3975.

  [Compound (6)]

¹ H-NMR (CDCl ₃ ): δ 0.88 (3H, s), 0.91 (3H, s), 0.91 (3H, d, J = 5.8 Hz), 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, brs), 4.49 (1H, m), 5.16 (1H, m), 5.23 (1H, d, J = 8.4 Hz), 5.69 (1H, dd, J = 3) .8, 9.8 Hz), 6.11 (1H, dd, J = 2.2, 9.7 Hz)

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

  [Compound (7)]

¹ H-NMR (CD ₃ OD): δ 0.72 (3H, s), 0.84 (3H, s), 0.90 (3H, d, J = 5.6 Hz), 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, 8.7 Hz), 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, 11.7 Hz), 3.72 (1H, d, J = 11.7 Hz), 3.91 (1H, d, J = 5.0 Hz), 4.21 (1H, d, J = 7.7 Hz), 4.31 (1H, d t, J = 3.2, 8.5 Hz), 5.06 (1H, d, J = 8.5 Hz), 5.77 (1H, d, J = 5.0 Hz), 9.75 (1H, s) )

¹³ C-NMR (CD ₃ OD): δ 13.9, 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 H-NMR (Pyridine-d5): 0.87 (3H, s), 0.93 (6H, s), 0.98 (3H, d, J = 5.8 Hz), 1.53 (3H, s ), 1.89 (3H, s), 2.30 (1H, m), 2.31 (1H, m), 2.49 (1H, dd, J = 5.4, 12.4 Hz), 3. 64 (1H, brs), 3.89 (1H, m), 4.18 (1H, m), 4.39 (1H, m), 4.45 (1H, m), 4.51 (1H, m), 4.68 (1H, m), 5.39 (1H, d, J = 7.7 Hz), 5.01 (2H, br s), 5.64 (1 H, m), 5.75 ( 1H, ddd, J = 6.3, 8.2, 15.5 Hz), 6.23 (1H, dd, J = 2.2, 9.8 Hz), 6.29 (1H, d, J = 15. 5Hz)

¹³ C-NMR (Pyridine-d5) δ 14.7, 18.65, 18.70, 19.7, 21.0, 23.1, 24.8, 27.3, 28.2, 30.9, 33 .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 H-NMR (Pyridine-d5): δ 0.76 (3H, s), 0.84 (3H, s), 0.95 (3H, d, J = 6.0 Hz), 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 = 15.4 Hz), 3.69 (1H, d, J = 15.4 Hz), 4.31 (1H , D, J = 4.8 Hz), 5.07 (1 H, m), 5.91 (1 H, m), 5.93 (1 H, m), 6.19 (1 H, d, J = 4.8 Hz) ), 10.57 (1H, s)

¹³ C-NMR (Pyridine-d5): δ 14.8, 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 H-NMR (Pyridine-d5): 0.88 (3H, s), 0.93 (6H, s), 0.98 (3H, d, J = 5.8 Hz), 1.53 (3H, s ), 1.90 (3H, s), 2.30 (1H, m), 2.31 (1H, m), 2.49 (1H, dd, J = 5.4, 12.4 Hz), 3. 69 (1H, br s), 4.00 (2H, m), 4.20 (1H, dd, J = 8.7, 8.7 Hz), 4.24 (1H, dd, J = 8.7, 8.8 Hz), 4.40 (1 H, dd, J = 2.0, 11.4 Hz), 4.57 (1 H, dd, J = 5.0, 11.4 Hz), 4.93 (1 H, d) , J = 7.7 Hz), 4.96 (2H, br s), 5.65 (1 H, m), 5.75 (1 H, ddd, J = 6.3, 8.2, 15.5 Hz), 6.24 (1H dd, J = 2.2,9.8Hz), 6.30 (1H, d, J = 15.5Hz)

¹³ C-NMR (Pyridine-d5): δ 14.6, 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, 134.4, 142.3

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

  [Compound (11)]

¹ H-NMR (Pyridine-d5) (Pyridine-d5): 0.76 (3H, s), 0.83 (3H, s), 0.88 (3H, s), 0.95 (3H, d, J = 5.7 Hz), 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 = 7.7 Hz), 5.60 (1H, d, J = 3.3, 9.7 Hz), 5.91 (1H, m), 5.93 (1H, m), 6.30 (1H, d, J = 2.0, 9.7 Hz)

¹³ C-NMR (Pyridine-d5): δ 14.4, 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 6555.3817, found 6555.3823.

Example 2 Confirmation of GLP-1 Secretion Promoting Action 150 ml of water was added to dried bitter gourd chips (10.5 g) and heated to reflux for 1 hour. The reaction product was extracted by adding 150 ml of acetone and then filtered. The residue was re-extracted with 100 ml of acetone. 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 had GLP-1 secretion activity.

As an example, the following table shows the results of comparing the compounds (1) to (5) and (7) with the control. The table also shows the results of TPA (3 μM), which is known to have a GLP-1 secretion activation effect.
The GLP-1 secretion activation action was confirmed as follows.
Poly-L-Lysine 96-well plates are BD, PBS (+), antibiotics, Dulbecco's Modified Eagle's Medium (DMEM), glucose, carboxymethylcellulose Sodium salt (CMC-Na) is manufactured by Nacalai Tesque, TPA [12-O-tetradecanoylphorbol-13-acetate] is manufactured by Cell Signaling, and active GLP-1 ELISA kit is Merck Millipore, Fetal bovine serum (FBS) manufactured by Sigma, Sitagliptin phosphate (Santagliptin phosphate) a Cruz Biotechnology, NCI-H716 cells assigned from ATCC were used.
NCI-H716 cells suspended in DMEM medium (10% FBS, 2 mM glutamine, 1% antibiotics added) are seeded on a Poly-L-Lysine 96-well plate at 0.5 × 10 5 cells / well at 100 μL each. The cells were cultured in a 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 melon acetone extract = 1 mg / ml, 100 μL of PBS (+) solution added 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 secretion promoting active ingredient 150 ml of water was added to dried bitter gourd chips (10.5 g), and the mixture was heated to reflux for 1 hour. The reaction product was extracted by adding 150 ml of acetone and then filtered. The residue was re-extracted with 100 ml of acetone. 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 a water-eluted fraction (fraction 1: 3.1 g), an acetonitrile-eluted fraction (fraction 2: 210 mg), and an acetone-eluted fraction (fraction 3). : 140 mg).
Each fraction and acetone extract were confirmed to have GLP-1 secretion activity in the same manner as in Example 2.

  The results compared with the control are shown in FIG. In the figure, TPA (3 μM), which is known to have a GLP-1 activating action, is also shown. Fr means “fraction” and “bitter gourd” means an 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 (fraction 1-3) was confirmed, it showed the strongest activity in fraction 2, It was found that the active ingredient was contained in a large amount in fraction 2.

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

  As is apparent from the results in FIG. 2, the two MS chromatograms of the acetone extract and fraction 2 are almost the same, so that they are present in the acetone extract after the heat treatment and are effective in fraction 2. It was found to be concentrated.

  And it was also confirmed that fraction 2 contained compounds (1) to (11).

(Example 4) Quantification of compounds (1) to (5) In each sample shown below, compounds (1) to (5) were picked up from compounds (1) to (11) and analyzed by LC-MS. Was quantified.

(Heat-treated bitter gourd)
Bitter gourd freeze-dried powder (50 mg) was weighed into a test tube with a lid, and 3 ml of water was added and sealed. The mixture was heated at 120 ° C. for 20 minutes using a microwave synthesizer (CEM Discover). After heating, 7 ml of acetonitrile was added to the reaction product and extracted with ultrasound for 2 minutes. After standing, the supernatant was filtered with a 0.45 μm filter to obtain an analysis sample.

(Raw bitter gourd, bitter gourd puree, bitter gourd juice)
50 mg each of lyophilized products of raw bitter gourd, bitter gourd puree or bitter gourd juice were weighed into a test tube with a lid, added with 3 ml of water and 7 ml of acetonitrile, and extracted with ultrasound for 2 minutes. After standing, the supernatant was filtered with a 0.45 μm filter to obtain an analysis sample.
Samples of raw bitter gourd, bitter gourd puree and bitter gourd juice were obtained as follows.
Raw bitter gourd: Raw bitter gourd was obtained by cutting bitter gourd into about 1/2 to 1/4 and removing cotton and seeds.
Puree: bitter gourd was cut to about 1/2 to 1/4 to remove cotton and seeds. 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 sealed heat-resistant container and immersed in a thermostatic bath containing water. The set temperature of the thermostatic layer was set to 95 ° C., and it was gradually heated from the water state. It was maintained for 15 minutes from the time when the temperature of the thermostatic bath reached 95 ° C. to obtain a puree.
Juice: Water was added to the puree obtained above so that bitter gourd puree: water = 2: 3. The mixture was sterilized at 108 to 111 ° C. for about 65 seconds through a strainer of 80 mesh or more. Filled and cooled above 88 ° C.

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 conditions)
Column: Triart C18 (YMC, 150 × 2.1 mm ID, 3 μm)
Column temperature: 40 ° C
Flow rate: 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 following table.

  As is clear from the results in the above table, the compounds (1) to (5) are hardly contained in raw bitter gourd that has not been heat-treated, and are notably contained in bitter gourd or processed bitter gourd that has been heat-treated. I found out.

(Example 5) Comparison of contents of compounds (1) to (5) depending on the heating temperature The bitter gourd freeze-dried powder (50 mg) was weighed into a test tube with a lid, and 3 ml of water was added and sealed. Using a microwave synthesizer (CEM Discover), it was heated at 100, 120, 140 or 160 ° C. for 10 minutes. After heating, 7 ml of acetonitrile was added to the reaction product and extracted with ultrasound for 2 minutes. After standing, the supernatant was filtered with a 0.45 μm filter to obtain an analysis sample.
In each sample obtained, the 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 following table.

  As is clear from the results in the above table, the amount of each component increases with a rise in heating temperature and then tends to decrease. Therefore, in order to obtain compounds (1) to (5) at a high ratio, It turns out that adjustment is also necessary.

(Example 6) Time-dependent change in content of compounds (1) to (5) under heat treatment conditions Bitter gourd freeze-dried powder (50 mg) was weighed in a tester with a lid, and 3 ml of water was added and sealed. Heating was performed at 100 ° C. and 120 ° C. for 2, 5, 10, 20, 40 or 60 minutes using a microwave synthesizer (CEM Discover). After heating, 7 ml of acetonitrile was added to the reaction product and extracted with ultrasound for 2 minutes. After standing, the supernatant was filtered with a 0.45 μm filter to obtain an analysis sample.
In each sample obtained, the 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 FIG. 3 (100 ° C.) and FIG. 4 (120 ° C.).

  As is apparent from the results of FIGS. 3 and 4, since the amount of each component tends to increase with the heating time and then decreases, in order to obtain the compounds (1) to (5) at a high ratio, It turns out that adjustment is also necessary.

(Example 7) Stability test of compounds (1) to (5) 0.5 mL of 70% acetonitrile mixed 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 amount of the compounds (1) to (5) when allowed to stand at 40 ° C. was measured over time. The measurement was performed by LC-MS analysis in the same manner as in Example 4.

  Then, the amount of the compounds (1) to (5) in the sample is plotted as the amount immediately after preparation is taken as 100%. The results are shown in FIG.

  As is apparent from the results of FIG. 5, the half-lives of the compounds (1) to (5) were 2 hours or more under the conditions corresponding to those in the stomach, indicating that they were stable.

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

1. Animals used Wistar rats Male 8 weeks old Group composition Water, bitter gourd 3. 3. Substance to be administered and number of cases Control (water) 20 mL / kg 9 cases Heated puree 20 g (dosage volume 20 mL) / kg 9 cases Blood collection site Under isoflurane anesthesia, the right jugular vein of the rat was cannulated, and the cannula was subcutaneously removed from the back so that blood could be collected over time. The experiment was conducted at least one day after the cannulation surgery. The cannula was filled with heparin to prevent blood from clotting.
5. Blood collection and administration Blood collection was performed under light anesthesia with isoflurane, and 1 hour before administration and 1 hour after administration. Administration was orally administered immediately after blood collection before administration.
6). Measurement Items GLP-1 was measured using a GLP-1 ELISA kit (Wako High Sensitive).
7). Evaluation Evaluation was made 100% before administration, and the control group and the heated puree group were compared by the rate of increase after administration.

  As is apparent from the results in the above table, the heated puree group was found to have an action of activating GLP-1 secretion in vivo.

(Example 9) Confirmation of blood glucose level increase inhibitory action by heating puree Using the heated puree obtained in Example 4, the in vivo GLP-1 activation action was confirmed under the following conditions.

1. Animals used C57BL / 6 mice Male 6 weeks old Dosage and administration liquid volume The dosage was 10 and 20 g / kg, and the administration liquid volume was 20 mL / kg. For a dose of 20 g / kg, a heated puree stock solution was used as an administration solution, and for a 10 g / kg, the stock solution was diluted twice with purified water to prepare an administration solution.
3. Group composition The following table.

4). Oral glucose tolerance test (OGTT)
The blood glucose level of the mice fasted for about 5 hours was measured, and the control group, the low-dose group, and the high-dose group were distributed to the control group, the low-dose group, and the high-dose group by the stratified extraction method using the blood glucose level. The administered substance was orally administered to each group, and 0.5 g of glucose (20% solution, 10 mL / kg) was orally administered 0.5 hours later. Blood glucose levels were collected from the tail vein under non-anaesthesia before administration of the administered substance (-0.5 hours), immediately before glucose administration (0 hours), and 0.5, 1, 2, and 3 hours after glucose administration. The measurement was performed using a measuring device (Glutest Neo Super, Sanwa Chemical Laboratory Co., Ltd.).

  The results are shown in FIG.

  As is apparent from the results of FIG. 6, the blood glucose level of the control group showed an increase that peaked at 0.5 hours after glucose administration, and then decreased. Both the low-dose group and the high-dose group of heat 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 significant low blood glucose level. The tendency was observed up to 1 hour after glucose administration.

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

(Example 11) Manufacture of coffee beverage Coffee extract was prepared in a conventional manner using 65 g of boiling water to 5 g of ground commercial coffee powder, and 5 g of bitter gourd pure obtained in Example 4 was added. A beverage was produced.

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

(Example 13) Manufacture of biscuits Using conventional methods, 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 obtained by freeze-drying the bitter gourd pure obtained in Example 4 Biscuits were manufactured.

(Example 14) Production of tofu 600 ml of well-cooled soymilk (no adjustment), 15 ml of bittern, 30 ml of water, 5 g of bitter gourd puree obtained in Example 4 were poured into a heat-resistant tapper and mixed. Wrapped and steamed for about 15 minutes on low heat with a steamed steamer to produce tofu.

  In the present invention, a composition useful for activation of GLP-1 secretion can be provided. In the present invention, such a composition can be produced efficiently.

Claims (27)

A composition for promoting GLP-1 secretion, comprising at least one component (A) selected from the following formulas (1) to (11).
  It contains at least one component (A) selected from the formulas (1) to (11) according to claim 1 and is used for suppressing an increase in blood glucose level, suppressing appetite, suppressing overeating, improving glucose metabolism, A composition for use in at least one application selected from prevention or treatment, prevention or treatment of obesity, weight reduction, and body fat percentage reduction.   At least one function selected from suppression of an increase in blood glucose level, 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 is GLP The composition according to claim 2, which results from promotion of secretion of -1.   The composition in any one of Claims 1-3 in which a component (A) contains the at least 1 sort (s) of component (A1) selected from Formula (6)-(11).   The composition in any one of Claims 1-4 which contains a component (A) as a component derived from bitter gourd.   The composition in any one of Claims 1-5 which contain a component (A) as a component derived from the bitter gourd which heat-processed.   The composition according to any one of claims 1 to 6, which is powdery or liquid.   A liquid composition comprising at least one component (A) selected from the formulas (1) to (11) according to claim 1 in a proportion of 0.005 ppm or more on a mass basis.   The composition according to claim 8, wherein the component (A) comprises at least one component (A1) selected from the formulas (6) to (11).   The composition of Claim 8 or 9 which contains at least 1 sort (s) of components (A1) selected from Formula (6)-(11) in the ratio of 0.005 ppm or more on a mass basis.   It is a puree or a drink, The composition in any one of Claims 8-10.   The composition according to any one of claims 8 to 11, which is a heat-treated bitter gourd puree or beverage.   The composition in any one of Claims 1-12 for adding to food-drinks.   The composition according to any one of claims 1 to 13, which is labeled with a function of promoting secretion of GLP-1 and / or a function resulting from promotion of secretion of GLP-1.   Selected from the function to suppress blood sugar level increase, appetite suppression function, overeating suppression function, glucose metabolism improvement function, diabetes prevention or treatment function, obesity prevention or treatment function, weight reduction function, and body fat percentage reduction function The composition according to any one of claims 1 to 14, which is labeled with at least one function.   Food / beverage products containing the composition in any one of Claims 1-15.   A food or drink comprising 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.   The food / beverage products of Claim 16 or 17 containing a non-bitter gourd origin component.   It is a drink, The food-drinks in any one of Claims 16-18.   The food / beverage products in any one of Claims 16-19 which attached | subjected the display of the function which accelerates | stimulates the secretion of GLP-1, and / or the function resulting from the secretion promotion of GLP-1.   Selected from the function to suppress blood sugar level increase, appetite suppression function, overeating suppression function, glucose metabolism improvement function, diabetes prevention or treatment function, obesity prevention or treatment function, weight reduction function, and body fat percentage reduction function The food-drinks in any one of Claims 16-20 which attached | subjected the display of the at least 1 function.   A method of expressing or increasing at least one component (A) selected from the formulas (1) to (11) according to claim 1 in the bitter gourd component, the method comprising a heating step of heat-treating the bitter gourd component .   A method for producing the composition according to claim 1, comprising a heating step of heat-treating bitter gourd ingredients.   The method according to claim 22 or 23, wherein the puree of bitter gourd is heat-treated in the heating step.   The method according to any one of claims 22 to 24, wherein the heating step is performed at 60 ° C or more for 5 minutes or more.   The method according to any one of claims 22 to 25, wherein in the heating step, heat treatment is performed in a closed system.   A compound selected from formulas (6) to (11) according to claim 1.