TW201808305A - Composition for promoting glp-1 secretion, and method for manufacturing same - Google Patents

Abstract

The present invention provides a composition useful for activating GLP-1 secretion. This composition can be configured from a specific compound such as compound (1). The specific compound of such description can be obtained with good efficiency by heat treating a bitter gourd component (e.g., bitter gourd puree or the like), for example.

Description

GLP-1 secretion-promoting composition and manufacturing method thereof

The present invention relates to a composition useful for activating (or promoting) 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 GLP-1 secretion) and the like.

GLP-1 [Glucagon-like peptide-1] is a digestive tube hormone secreted by the epithelium of the digestive tract.

As the effects of such GLP-1, it is known to stimulate insulin synthesis or secretion, inhibit glycocrine secretion, inhibit food intake, and reduce hyperglycemia. Therefore, by activating GLP-1, the effect of these effects can be expected to be enhanced.

On the other hand, bitter gourd (cool gourd) is a edible plant, and many people continue to study the physiological effects of bitter gourd.

In addition, there have been reports indicating the relationship between this bitter gourd and GLP-1. For example, non-patent document 1 reports that a specific compound contained in bitter gourd has GLP-1 activation and may have a blood glucose regulating function.

[Prior technical literature] [Non-patent literature]

[Non-Patent Document 1] 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 to provide a method for suppressing an increase in blood glucose level, an appetite suppressant, an overeating suppressant, an improvement in glucose metabolism, a prevention or treatment of diabetes, a prevention or treatment of obesity, a decrease in weight, and a decrease in body. Compositions such as fat ratio.

Another object of the present invention is to provide a compound useful for activation of GLP-1 secretion or the like, or a method for efficiently producing a composition containing the 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 research conducted by the inventors of the present case to solve the above-mentioned problems, focusing on bitter gourd, a detailed study of the ingredients contained in bitter gourd found that: In addition to the compounds described in Patent Document 1, there may be various compounds having GLP-1 activation; and, unexpectedly, such compounds are not contained in raw bitter gourd, or even contained a very small amount, and A large amount is produced by heat treatment. Furthermore, the present invention has been accomplished by including various compounds which are particularly excellent in promoting the secretion of GLP-1, or novel compounds which have not been known before.

That is, the composition of the present invention contains at least one component (A) selected from the following formulae (1) to (11) (including as an active ingredient). Such a composition has the effect of activating GLP-1 secretion, and therefore can be used as a composition for activating GLP-1 secretion (composition for activating GLP-1).

Since the composition of the present invention has the effect of promoting the secretion of GLP-1, it can also be used for the purpose of causing or related functions and functions related to the effect. For example, the composition of the present invention may also be used for the purpose selected from the group consisting of: inhibition of increase in blood glucose level, inhibition of appetite, inhibition of excessive diet, improvement of glucose metabolism, prevention or treatment of diabetes, prevention or treatment of obesity, reduction At least one use for weight and body fat reduction Of the composition.

In addition, the composition of the present invention can also be used for the purpose selected from the group consisting of: inhibition of increase in blood glucose level, inhibition of appetite, inhibition of excessive diet, improvement of glucose metabolism, prevention or treatment of diabetes, prevention or treatment of obesity, and weight reduction. And a composition for at least one use for reducing body fat rate. In this composition, it is selected from the group consisting of suppression of blood glucose rise (function), appetite suppression (function), suppression of excessive diet (function), improvement of glucose metabolism (function), prevention or treatment of diabetes (function), prevention or treatment of obesity At least one of the functions (functions), weight reduction (functions), and reduction of body fat rate (functions) may not necessarily be caused by the promotion of GLP-1 secretion, but may also be caused by the promotion of GLP-1 secretion.

In the composition of the present invention, the component (A) may further include at least one component (A1) selected from the formulae (6) to (11).

The composition of the present invention may also contain the component (A) as a component derived from bitter gourd (particularly a component derived from bitter gourd after heat treatment).

The form of the composition of the present invention may be, for example, a solid state (for example, a powder state) or a liquid state.

The present invention includes a liquid composition including at least one component (A) selected from the aforementioned formulae (1) to (11). Such a liquid composition can contain a component (A) in the ratio of 0.005 ppm or more on a mass basis. In addition, the component (A) may include at least one component (A1), particularly selected from the formulae (6) to (11). Typically, the component (A) may be selected from the formula (6) at a ratio of 0.005 ppm or more based on the mass basis. 6) At least one component (A1) of (11).

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

The composition of the present invention can also be used to add to food and drink (or as an additive for food and drink).

The present invention also includes a food and beverage product including the aforementioned composition. Such food and beverage can contain, for example, at least one component (A) selected from the above-mentioned formulas (1) to (11) in a proportion of 0.005 ppm or more on a mass basis. In addition, in general, foods and beverages may also contain ingredients not derived from bitter gourd. In particular, food and beverage can also be beverages.

The composition or food or drink of the present invention has a function of promoting the secretion of GLP-1, and also has a function of causing or related to the effect. Therefore, the composition or food or drink of the present invention may also be a composition or food or drink with a label added with a function of promoting the secretion of GLP-1 and / or a label resulting from the function of promoting the secretion of GLP-1. Such a composition or food or drink includes a function selected from, for example, a function of suppressing an increase in blood glucose level, a function of suppressing appetite, a function of suppressing excessive diet, a function of improving glucose metabolism, a function of preventing or treating diabetes, a preventive or A composition or a food or drink that has at least one of the functions (or even the equivalent functions) of the function of treating obesity, the function of reducing body weight, and the function of reducing body fat rate.

By heating the bitter gourd component, the component (A) can be efficiently produced.

Therefore, the present invention includes a method for making bitter gourd ingredients A method for expressing or increasing at least one component (A) selected from the aforementioned formulae (1) to (11), which includes a heating step of heating a bitter gourd component.

The present invention also includes a method for producing the composition, which includes a heating step of heating a bitter gourd component.

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

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

The composition and food or drink of the present invention generally have a GLP-1 secretion promoting effect (or GLP-1 activating effect).

Therefore, the present invention also includes a method of ingesting, taking, or administering the aforementioned composition or food or drink to activate GLP-1 (or promote GLP-1 secretion). In this method, since the secretion of GLP-1 can be activated, it can be applied to symptoms that are caused by or related to the activation of GLP-1 secretion. For example, this method may be at least one method selected from the group consisting of suppressing an increase in blood glucose level, suppressing appetite, suppressing excessive diet, improving glucose metabolism, preventing or treating diabetes, preventing or treating obesity, reducing weight, and reducing body fat rate.

Furthermore, the present invention also includes a method for ingesting, taking, or administering the aforementioned composition or food or drink to effect (or achieve) an increase in blood glucose level At least one of suppression, appetite suppression, excessive diet suppression, improvement of glucose metabolism, prevention or treatment of diabetes, prevention or treatment of obesity, weight loss, and reduction in body fat rate. In this method, it is not necessary to accompany GLP-1 activation (secretion promotion), but it may also be accompanied by GLP-1 activation (secretion promotion).

In addition, in the method described above, the subject of ingestion, administration, or administration may be any animal as long as it is an animal, and may be any of a human and a non-human animal (dog, cat, etc.).

In the present invention, a composition useful for activating (or promoting) GLP-1 (or GLP-1 secretion) can be provided. This composition, due to the activation of GLP-1, is suitable for use as a cause or a function related to the function ‧ function, such as suppressing the increase in blood glucose level, suppressing appetite, suppressing excessive diet, improving glucose metabolism, preventing A composition for the purpose of treating diabetes, preventing or treating obesity, reducing weight, reducing body fat rate, and the like.

Moreover, in the present invention, regardless of whether or not it is caused by the secretion-promoting function of GLP-1, it can be provided for use in suppressing an increase in blood glucose level, suppressing appetite, suppressing excessive diet, improving glucose metabolism, preventing or treating diabetes. Compositions for use, prevention or treatment of obesity, weight reduction, body fat reduction and the like.

Therefore, the composition of the present invention can also be used for the prevention and treatment of obesity or diet, the prevention and treatment of diabetes and the like. In addition, The timing of ingestion (taking) of such a composition of the present invention is not particularly limited, and it can be used, for example, before meals (for ingestion), between meals, after meals, and the like.

In particular, since the composition of the present invention can promote the secretion of GLP-1 and improve the glucose metabolism, it can be applied to diseases that need to improve the glucose metabolism. Examples of such diseases include diabetes and obesity, and these preventive and therapeutic systems are effective. Therefore, the composition of the present invention may be provided with an indication for the purpose of prevention and / or improvement of hyperglycemia or obesity, for a target group with a high blood glucose level, a target group with a slight obesity, and a tendency toward metabolic syndrome. Target groups are extremely useful.

In addition, the composition (or compound) of the present invention is stable to heat or long-term storage, and can also stably exist in a living body (for example, when exposed to gastric acid). Therefore, it can also be used as an additive for food and drink.

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

Furthermore, in the present invention, novel compounds for the activation of GLP-1 and the like can be provided.

FIG. 1 is a graph showing the GLP-1 secretion amount of each component in Example 3 when the control group is set to 100 in Example 3. FIG.

Figure 2 is a chromatogram of the acetone extract obtained in Example 3 and LC-MS analysis of Division 2.

FIG. 3 is a graph showing a diachronic change (100 ° C.) of each component amount in Example 6. FIG.

FIG. 4 is a graph showing a diachronic change (120 ° C.) of each component amount in Example 6. FIG.

FIG. 5 is a graph showing the results of the stability test of each component in Example 7. FIG.

FIG. 6 is a graph showing measurement results of blood glucose levels in Example 9. FIG.

[Form of Implementing Invention] [Ingredient (A)]

The composition of the present invention contains a component (A) selected from the following formulae (1) to (11).

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

Among ingredients (A), compounds (6) to (11) are new This invention also includes such novel compounds. In this specification, the names (conventional names) of these novel compounds are shown in the following tables, respectively.

In addition, the names of compounds (1) to (5) are as follows:

Compound 1: Momordicoside I

Compound 2: 5β, 19-epoxycucurbitin-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-malonylcucurbitin-5,24-diene-19-aldehyde (7,23-dihydroxy-3-O-malonylcucurbita-5,24-dien-19 -al)

Compound 4: Momordicoside W

Compound 5: momordicoside P

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

In addition, the composition of the present invention can also contain the component (A) (or compounds (1) to (11)) in the form of a derivative as long as it has an activation effect of GLP-1 secretion. Examples of such derivatives include salts, solvates (such as hydrates), and ethers [for example, in compounds (1) to (11), a part or all of the hydroxyl group is substituted with an alkoxy group (for example, a methoxy group) Compounds such as C _1-10 alkoxy, ethoxy, and preferably C _1-4 alkoxy)], esters [for example, in compounds (1) to (11), a part or all of the hydroxyl groups are substituted with An ethoxy group (for example, a C _1-10 ethoxy group such as ethoxy group, preferably a C _1-4 ethoxy group) and the like.

In addition, such a derivative may be derived from an extraction solvent when the component (A) is fractionated or separated from, for example, bitter gourd.

Ingredient (A) (or compounds (1) to (11) and derivatives thereof) can be synthesized by conventional methods; it can be obtained from plants, especially, as described later, from bitter gourd (heat-treated bitter gourd) with high efficiency. .

Therefore, the composition of the present invention may also contain the component (A) as a component derived from bitter gourd (bitter gourd component), and particularly a component subjected to heat treatment (bitter gourd component).

Such a bitter gourd ingredient is generally a bitter gourd processed product. Examples of processed bitter gourd include mud, beverages, crushed matter, extracts, dried matter (dried product), and the like. In addition, the beverage can also be juiced. Bitter melon ingredients can also be a mixture of these.

[Form of composition]

The form of the composition of the present invention is not particularly limited as long as it contains the component (A). They are not particularly limited, and may be in a solid state (powder state, etc.), a liquid state, or the like.

In particular, the form of the composition of the present invention may be liquid. Examples of the liquid form include mud, drinks, and the like.

The component (A) is as described above, and is easily obtained as a component derived from bitter gourd, and even if it contains a bitter gourd component (component from bitter gourd) other than the component (A), it does not impair GLP-1 activity.

Therefore, the composition of the present invention can be powder (for example, dried product), fruit puree, beverage (especially puree), etc. of bitter gourd (especially heat-treated bitter gourd). In addition, purees or beverages can be concentrated or diluted as required.

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

In the composition (for example, a liquid composition) of the present invention, the proportion of the component (A) may be selected, for example, from a range of about 0.005 ppm or more (for example, 0.01 to 10000 ppm) on a mass basis, or may be 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).

In addition, the proportion of the component (A1) in the composition (for example, a liquid composition) of the present invention may be selected from a range of about 0.005 ppm or more (for example, 0.01 to 10,000 ppm) on a mass basis, or may be 0.5. ppm or more (e.g. 0.6 to 6000 ppm), more preferably about 1 ppm or more (e.g. 5 to 1000 ppm), or 10 ppm or more (e.g. (E.g., 20 to 800 ppm, preferably 50 to 500 ppm).

[Use of composition]

The composition of the present invention has GLP-1 activation effect (or GLP-1 secretion promotion effect) and the like. Therefore, the composition of the present invention can be used for GLP-1 activation and the like (or as a composition having GLP-1 activation).

In addition, the composition of the present invention can be suitably used as an effect caused by and related to the activation effect of GLP-1, such as an effect of suppressing an increase in blood glucose level, an effect of suppressing appetite, an effect of suppressing excessive diet, an effect of improving glucose metabolism, prevention or A composition for treating diabetes, preventing or treating obesity, reducing body weight, reducing body fat rate, and the like.

The use method (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 directly (ingestion, administration, administration, etc.), or the composition of the present invention may be co-formulated with other ingredients (carriers, excipients, etc.) and used (ingestion, administration, administration, etc.) I waited).

Examples of the other components include excipients, binders, disintegrating agents, coating agents, lubricants, colorants, flavoring and deodorizing agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH modifier, preservative, antioxidant, etc. The formulation method is not particularly limited, and a conventional method can be applied.

In the formulation, examples of the dosage form include lozenges, powders, fine granules, granules, dry suspensions, coated lozenges, and oral cavity. Disintegrating tablets, chewing tablets, capsules, soft capsules, syrups, etc.

In addition, the composition of the present invention may be added to (or contained in) food and drink and used (ingested, taken, administered, etc.). Food and beverages include health foods such as specific health foods, nutritional foods, and the like in addition to general foods including so-called health foods. The food and beverage also includes supplements (nutritional supplement foods), feeds, and the like.

The food and beverage may include other ingredients such as food additives (food additives) as long as the composition of the present invention is included. The food additive is not particularly limited, and examples thereof include an excipient (for example, wheat starch, corn starch, cellulose, lactose, sucrose, mannitol, sorbitol, xylitol, alpha starch, casein, silicon Magnesium aluminum, calcium silicate, etc.), binders (e.g. alpha starch, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, etc.), disintegrants (e.g. cellulose, hydroxypropyl cellulose, corn starch) Etc.), plasticizers (such as light anhydrous silicic acid, sucrose fatty acid esters, etc.), oils (such as soybean oil, sesame oil, olive oil, linseed oil, perilla oil, rapeseed oil, coconut oil, corn oil, etc. Vegetable oil or animal / fish-derived oil), nutrients (such as various minerals, various vitamins, amino acids), spices, sweeteners, flavoring agents, colorants, solvents (ethanol), salts, surfactants, pH adjusting agent, buffering agent, antioxidant, stabilizer, gelling agent, viscosity increasing agent, lubricant, encapsulating agent, suspending agent, coating agent, preservative, etc. Food additives can be used alone or in combination of two or more.

In addition, other ingredients may generally be ingredients not derived from bitter gourd.

When the composition of the present invention is added to (or contained in) food or drink, the food or drink is not particularly limited, and examples thereof include food [for example, noodles (buckwheat noodles, udon noodles, Chinese noodles, instant noodles, etc.) ), Tofu, sweets (sugar, candy, chewing gum, chocolate, snacks, biscuit, cookies, jelly fudge, etc.), bread, aquatic or animal processed food (fish plate, ham , Sausages, etc.), dairy products (processed milk, fermented milk, etc.), fats and oils processed foods (salad oil, fried oil, margarine, mayonnaise, butter, foaming cream, salad dressing, etc.), seasonings (sauces) , Sauces, etc.), conditioned or semi-conditioned products (Okinawa miscellaneous stir-fry, etc.), conditioned package foods (curry, stew, rice bowl, porridge, chowder, etc.), ice products (ice cream, juice slush, tartar, etc. ), Etc.], beverages (tea beverages, refreshing beverages, carbonated beverages, nutritional beverages, fruit beverages, lactic acid beverages, fruit juices, drinks, etc.) and the like.

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

In the food and drink, the proportion of the composition (or component (A)) of the present invention may be selected from a range of about 0.005 ppm or more (for example, 0.01 to 10,000 ppm) on the basis of the quality of the component (A), or may be 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).

In addition, the ratio of the composition (or component (A)) in the food and beverage of the present invention may be selected from a range of about 0.005 ppm or more (for example, 0.01 to 10,000 ppm) based on the mass standard of the component (A1), or It may be 0.5 ppm or more (for example, 0.6 to 6000 ppm), more preferably 1 ppm or more (for example, 5 to 1,000 ppm), or 10 ppm or more (for example, 20 to 800 ppm, preferably 50 to 500 ppm).

In addition, when using the composition or food or drink of the present invention, the intake amount (dosage amount, administration amount) is not particularly limited, and can be appropriately selected according to the age, weight, health status, etc. of the subject. A) Conversion can be 0.001 mg or more (for example, 0.001 to 10,000 mg), preferably 0.01 mg or more (for example, 0.05 to 5000 mg), more preferably 0.1 mg or more (for example, 0.5 to 1000 mg), and especially 1 mg or more (for example, 5 to 500 mg) about.

The composition and food or drink of the present invention can be used or applied to any of therapeutic use (medical use) and non-therapeutic use. Specifically, whether or not it is classified as a pharmaceutical, a quasi-drug, a cosmetic, etc., can be used or applied as a function that expressly or implicitly seeks to promote the secretion of GLP-1, or is caused by a function that promotes the secretion of GLP-1 Functional composition or food and drink.

In addition, the composition and food or drink of the present invention may be marked with a function of promoting the secretion of GLP-1 or a function resulting from the function of promoting the secretion of GLP-1. Examples of such indications include, but are not particularly limited to, the function of promoting the secretion of GLP-1, or the function of promoting the secretion of GLP-1, such as the function of suppressing the increase in blood glucose level, the function of suppressing appetite, and the function of suppressing excessive diet Function, function to improve glucose metabolism, function to prevent or treat diabetes, function to prevent or treat obesity, function to reduce body weight, function to reduce body fat rate, or other equivalent labels.

In addition, such labels may be added to the composition or food or drink itself, or to the container or packaging of the composition or food or drink, depending on the form of the composition or food or drink.

[Manufacturing method of composition]

The composition (or component (A)) of the present invention can be produced by a synthetic method, and in particular, it can be produced efficiently using a bitter gourd ingredient.

That is, the bitter gourd component contains a large amount of a component (precursor) that can generate the component (A), and by heating such a precursor, the component (A) can be generated. Therefore, the present invention includes a method for producing the aforementioned composition (or component (A)) by at least a heating step of heat-treating a bitter gourd component.

In addition, after such a heating step, the component (A) in the balsam pear component can be expressed or increased. Therefore, the present invention also includes a method for expressing or increasing the component (A) in the balsam pear 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 can generate the component (A), and may be bitter gourd (bitter gourd main body), fruit (or pulp), skin, melon, seeds, seed coat, etc. Mixtures of this type are also possible. Generally, a portion containing at least fruits (bitter gourd main body, pulp, etc.) is mostly used. The bitter gourd component may be a processed bitter gourd.

Bitter melon ingredients can be directly heated. However, for example, for processed fruits such as fruit puree and beverages, it is especially used as a puree for heating. Handling is better. By heating the puree or the like of this bitter gourd component, the component (A) can be efficiently produced or expressed.

In addition, fruit puree can be concentrated or diluted as required.

The bitter gourd component may be heat-treated (or the bitter gourd component containing an acid component may be heat-treated) in the presence of an acid component (for example, an organic acid such as acetic acid and citric acid). By performing a heat treatment in the presence of an acid component, the component (A) is easily produced with high efficiency.

In the heating step, the heating temperature may be appropriately selected according to the heating time, for example, it may be 40 ° C or higher (for example, 40 to 160 ° C), preferably 60 ° C or higher (for example, 60 to 140 ° C), and more preferably Above 80 ° C (for example, 80 ~ 120 ° C).

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

In addition, in the heating step, the heating method may be appropriately selected depending on the morphology of the bitter gourd component and the like, and is not particularly limited, and may be any of resistance heating, induction heating, and dielectric heating (such as microwave heating). The heating means may be appropriately selected depending on the heating method.

Depending on the shape of the bitter gourd ingredient, it can be heated by boiling, cooking, grilling, steaming, water heating, and the like.

Heating (heat treatment) may be performed in an active environment (air environment, etc.), and may also be performed in an inert environment. It can also be heated in a closed system. In particular, by heating in a closed system, the component (A) can be efficiently produced.

The bitter gourd component (heated bitter gourd component) after the heating step may be used directly or purified according to the form of the composition. For example, when the composition is a puree or the like, the bitter gourd component after the heat treatment may be directly concentrated or diluted and used. When the composition is composed of the component (A) as the main component, a composition containing the component (A) in a high proportion can also be obtained from the balsam pear component after heat treatment using a conventional method such as extraction.

[Example]

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to these, and many variations can be implemented by those skilled in the art within the technical idea of the present invention.

(Example 1) Isolation and identification of compounds (1) to (11) (Extraction and coarse division)

A dried bitter gourd (20 kg) was extracted with methanol (MeOH) to obtain a MeOH extract. The resulting MeOH extract was separated in 50% MeOH in water-hexane (1: 1, 60 L). A 50% aqueous solution of MeOH was adsorbed on an ion-exchange resin [Diaion HP-20 (resin amount: 30L)] produced by Mitsubishi Chemical Corporation, and then dissolved in a 50% aqueous solution of MeOH (100L) and MeOH (100L). The solvent was distilled off under reduced pressure to obtain 50% MeOH fraction and MeOH fraction (114 g).

In addition, since the dried bitter gourd is a heated and air-dried product, it is not heat-treated.

(MeOH-differentiated purification)

The MeOH fraction (5.0 g) was separated into 19 fractions by medium pressure liquid chromatography (MPLC) (ODS, water-MeOH) and MPLC (ODS, 20 mM CH ₃ COONH ₄ aqueous solution-CH ₃ CN, acetone).

7 was purified by high performance liquid chromatography (HPLC) (manufactured by YMC, Triart C18, water-CH ₃ CN) to obtain compound (3) (25.0 mg). Further, in order to HPLC (YMC Corporation, Triart C18, water -CH ₃ CN) 9 distinguish purified, isolated compound (7) (18.3mg). Divisions 12 and 16 were also purified by HPLC (Triart C18, water-CH ₃ CN, manufactured by YMC) in the same manner. Compound (11) (0.8 mg) was obtained from division 12, and compound (6) was obtained from division 16 ( 1.0 mg).

(Heat treatment by MeOH and purification of heat treated products)

The reactant obtained by adding 2% acetic acid and 70% CH ₃ CN aqueous solution to MeOH (10.2 g) and heating and refluxing for 5 hours was separated by MPLC (ODS, 20 mM CH ₃ COONH ₄ aqueous solution-CH ₃ CN, acetone). 17 distinctions.

4 was purified by HPLC (manufactured by YMC, Triart C18, water-CH ₃ CN) to obtain compound (9) (10.0 mg). Purification was performed by HPLC (Triart C18, water-CH ₃ CN, manufactured by YMC), and compound (4) (19.7 mg) was isolated. Division 9 and 10 were also purified by HPLC (manufactured by YMC, Triart C18, H ₂ O-CH ₃ CN), and compound (5) (17.1 mg) was obtained from division 9 and compound (1) (10) was obtained from division 10. 43.5 mg). MPLC (ODS, 20 mM CH ₃ COONH ₄ aqueous solution-CH ₃ CN, acetone), HPLC (manufactured by YMC, Triart C18, water-CH ₃ CN), and HPLC (manufactured by Nacalai Tesque, Cosmosil PBr, water-CH) ₃ CN) Purification and differentiation of 16 and 17 gave compound (2) (10.7 mg), compound (10) (3.8 mg), and compound (8) (4.6 mg).

In addition, the structures of the compounds (1) to (11) were identified by mass spectrometry and NMR, and the structures were confirmed as described above. An example of the 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 = 5.6Hz), 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 = 5.2,11.5HHz), 4.61 (1H, dd, J = 2.0,11.5HHz), 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 H-NMR (Pyridine-d5): δ 0.85 (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): 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 ₄ 495.3445, 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 = 5.6Hz), 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, br s), 3.68 (1H, dd, J = 5.4,11.7Hz), 3.86 (1H, br d, J = 11.7Hz) , 4.27 (1H, d, J = 7.8Hz), 5.10 (2H, s), 5.61 (3H, m), 6.11 (1H, br d, J = 9.7Hz)

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 ₉ 657.3973, 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 = 5.6Hz), 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 = 2.8,7.9Hz ), 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): 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 ₉ 657.3973, found 657.3975.

[Compound (6)]

¹ H-NMR (CDCl ₃ ): δ 0.88 (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 = 3.8,9.8Hz), 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 H-NzMR (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, d, 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): 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 = 5.8Hz), 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, 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, 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 = 6.0Hz), 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): 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 = 5.8Hz), 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): 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): δ0.76 (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): 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 ₉ 655.3817, found 655.3823.

(Example 2) Confirmation of effect of promoting GLP-1 secretion

150 ml of water was added to the dried bitter gourd slice (10.5 g), and the mixture was heated under reflux for 1 hour. The reaction mixture was extracted with 150 ml of acetone and then filtered. 100 ml of acetone was added to the residue, and re-extraction was performed. After filtration, the filtrate was 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 in the manner described above had GLP-1 secretion activity.

As an example, the results of comparison of the compounds (1) to (5) and (7) with the control group are shown in the following table. The table also shows the results of TPA (3 μM) known to have GLP-1 secretion activation.

The GLP-1 secretion activation was confirmed as follows.

Poly-L-Lysine 96-well well plate system uses BD Corporation; PBS (+), antibiotics, Dulbecco's Modified Eagle's Medium (DMEM) , Glucose, sodium carboxymethyl cellulose (CMC-Na) are produced by Nacalai Tesque; TPA [12-O-tetradecanoylphorbol-13-Acetate] Produced by Cell Signaling; active GLP-1 ELISA kit It was produced by Merck Millipore; Fetal bovine serum (FBS) was produced by Sigma; Sitagliptin phosphate was produced by Santa Cruz Biotechnology; NCI-H716 cells were produced by ATCC Transferor.

NCI-H716 cells suspended in DMEM medium (added with 10% FBS, 2 mM glutamic acid, and 1% antibiotics) were seeded in Poly-L-Lysine 96-well wells at 0.5 × 105 cells / well at 100 μL each. The cells were cultured in a CO ₂ incubator (manufactured by espec) for 48 hours.

After washing with PBS (+), as the final concentration, compound (1) = 0.71 μg / mL, compound (2) = 1.03 μg / mL, compound (3) = 0.52 μg / mL, and 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 a 10 μM solution of Sitagliptin phosphate in PBS (+) was added to the cells . The added solution was recovered after 1 hour, and the amount of active GLP-1 in the solution was measured using an ELISA kit.

(Example 3) A modulation method including differentiation of a large amount of GLP-1 secretion-promoting active ingredients

150 ml of water was added to the dried bitter gourd slice (10.5 g), and the mixture was heated under reflux for 1 hour. The reaction mixture was extracted with 150 ml of acetone and then filtered. 100 ml of acetone was added to the residue, and re-extraction was performed. After filtration, the filtrate was concentrated under reduced pressure to obtain 3.46 g of an acetone extract.

The obtained acetone extract was separated by MPLC (ODS) to obtain a water-soluble fraction (Division 1: 3.1 g), an acetonitrile-dilute fraction (Division 2: 210 mg), and an acetone-dilute fraction (Division 3: 140 mg).

For each division and acetone extract, in the same manner as in Example 2, it was confirmed that it has a GLP-1 secretion activity.

The results of comparison with the control group are shown in FIG. 1. In addition, TPA (3 μM), which is known to have GLP-1 activation, is also shown in the figure. Fr means "differentiation" and "bitter gourd" means acetone extract.

The results in Figure 1 are clear, confirming the acetone extract (Bitter gourd) and the GLP-1 secretion-promoting activity of each of the divisions (Divisions 1-3) showed the strongest activity in Division 2, and it was found that the active ingredient was contained in Division 2 in a large amount.

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

It is clear from the results in FIG. 2 that the MS chromatograms of the acetone extract and the division 2 are almost the same, and it can be seen that the acetone extract existing in the heat-treated acetone extract can be efficiently concentrated in the division 2.

It was also confirmed that the compounds (1) to (11) were contained in the division 2.

(Example 4) Quantification of compounds (1) to (5)

In each sample shown below, compounds (1) to (5) were selected from the compounds (1) to (11) and quantified by LC-MS analysis.

(Heat treated bitter gourd)

A freeze-dried powder (50 mg) of bitter gourd was measured in a test tube with a lid, and 3 ml of water was added to seal it. Using a microwave synthesizer (Discover manufactured by CEM), heating was performed at 120 ° C for 20 minutes. After heating, 7 ml of acetonitrile was added to the reaction mixture, and the mixture was subjected to ultrasonic extraction for 2 minutes. After standing, the supernatant was filtered through a 0.45 μm filter to prepare an analysis sample.

(Raw bitter gourd, bitter gourd puree, bitter gourd juice)

In a test tube with a lid, 50 mg each of the freeze-dried product of raw bitter gourd, bitter gourd puree or bitter gourd juice was measured, and 3 ml of water and 7 ml of acetonitrile were added for 2 minutes for ultrasonic extraction. After standing, the supernatant was filtered through a 0.45 μm filter to prepare an analysis sample.

In addition, samples of raw bitter gourd, bitter gourd puree, and bitter gourd juice were obtained as follows.

Raw bitter gourd: Cut bitter gourd into about 1/2 to 1/4, remove the bitter gourd and seeds as raw bitter gourd.

Puree: Cut bitter gourd into about 1/2 to 1/4, remove melon and seeds. Add water to make bitter gourd: water = 4: 1, and crush it with a juice machine to obtain a puree puree. In addition, the puree is cooled after sterilization and stored frozen until use.

The puree puree is placed in a sealed heat-resistant container and immersed in a thermostatic bath filled with water. The set temperature of the thermostatic layer was set to 95 ° C, and the temperature was gradually heated from the state of water. It was maintained for 15 minutes from the time when the temperature of the thermostatic bath reached 95 ° C, and a puree was obtained.

Juice: add water to the puree obtained above, and add water to make bitter gourd puree: water = 2: 3. Pass through a 80-mesh strainer and sterilize at 108 ° C to 111 ° C for about 65 seconds. Fill and cool above 88 ° C.

The analysis conditions are as follows:

(LCMS measurement)

Each sample was analyzed by LCMS-IT-TOF and quantified. The quantitative value is obtained from a calibration curve prepared using a standard in advance.

(LC conditions)

Column: Triart C18 (manufactured by YMC, 150 × 2.1mmI.D., 3μm)

Column temperature: 40 ℃

Flow rate: 0.3ml / min

Gradient: 30% → 100% (30-39 points) B in A

A: 0.1% formic acid aqueous solution

B: 0.1% formic acid acetonitrile

Injection volume: 2μl

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

From the results in the table above, it is clear that the compounds (1) to (5) are hardly contained in the raw bitter gourd which is not heat-treated, but are significantly contained in the heat-treated bitter gourd or bitter gourd processed product.

(Example 5) Comparison of the contents of compounds (1) to (5) due to the difference in heating temperature

A freeze-dried powder (50 mg) of bitter gourd was measured in a test tube with a lid, and 3 ml of water was added to seal it. Using a microwave synthesizer (Discover 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 mixture, and the mixture was subjected to ultrasonic extraction for 2 minutes. After standing, the supernatant was filtered through a 0.45 μm filter to prepare an analysis 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 the following table.

From the results in the table above, it is clear that the components tend to increase in quantity and then decrease with the rise in heating temperature. Therefore, it is known that in order to obtain compounds (1) to (5) in a high ratio, it is necessary to adjust the heating temperature.

(Example 6) Changes in the content of compounds (1) to (5) over time under heat treatment conditions

Measure the freeze-dried powder (50 mg) of bitter gourd in a covered test tube. Add 3ml of water to seal. 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 mixture, and the mixture was subjected to ultrasonic extraction for 2 minutes. After standing, the supernatant was filtered through a 0.45 μm filter to prepare an analysis 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 Figure 3 (100 ° C) and Figure 4 (120 ° C).

It can be seen from the results of Figures 3 and 4 that the amount of each component increases with heating time and then decreases. Therefore, it is known that to obtain compounds (1) to (5) at a high ratio, adjustment is also required. heating time.

(Example 7) Stability test of compounds (1) to (5)

A mixed solution containing 0.5 mL of a 70% acetonitrile mixed aqueous solution containing compounds (1) to (5) and 0.5 mL of a 0.2 N aqueous hydrochloric acid solution was prepared at a concentration of 4 μg / mL, and the compound (1) was measured over time at 40 ° C. ~ (5) amount. The measurement was performed by LC-MS analysis in the same manner as in Example 4.

Then, set the amount immediately after the preparation to 100%, and plot how the amounts of the compounds (1) to (5) in the sample change. The results are shown in FIG. 5.

As can be seen from the results in Figure 5, Under these conditions, the half-lives of compounds (1) to (5) showed a stability of more than 2 hours.

(Example 8) Confirmation of GLP-1 activation by heating fruit puree

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

1. Use of animals

Wistar rat male 8 weeks old

Group composition

Water, bitter gourd puree

3.Substances and cases

Control (water) 20mL / kg 9 cases

Heated fruit puree 20g (dosage volume 20mL) / kg 9 cases

4. Blood drawing site

Under isoflurane anesthesia, a cannula was inserted into the rat's right jugular vein, allowing the cannula to pass under the skin and out through the back, and blood can be drawn over time. The experiment was performed after the cannula was inserted more than 1 day after the operation. The cannula is filled with heparin to prevent blood clotting.

5. Blood drawing and administration

The blood was drawn under mild anesthesia of isoflurane, before and 1 hour after administration. Administration was performed orally following blood draw before administration.

6. Measurement items

The measurement of GLP-1 was performed using a GLP-1 ELISA kit (Wako High Sensitive) to measure total GLP-1.

7. Evaluation

The evaluation was set to 100% before the administration, and the control group and the heated puree group were compared according to the increase rate after the administration.

It is clear from the results in the above table that when the fruit puree group is heated, it is known that it has the function of activating GLP-1 secretion in vivo.

(Example 9) Confirmation of the effect of suppressing an increase in blood glucose level by heating fruit puree

Using the heated fruit puree obtained in Example 4, the activation of GLP-1 in vivo was confirmed under the following conditions.

1. Use of animals

C57BL line / 6 mice 6 weeks old

2. Dosing amount, dosing amount

The dosage was 10 and 20 g / kg. The dosage was 20 mL / kg. The dosing amount of 20 g / kg is a heated puree puree as the dosing liquid; 10 g / kg is diluted twice with pure water to prepare a dosing liquid.

3. Group composition

As shown in the table below.

4. Oral glucose tolerance test (OGTT)

The blood glucose values of the mice that were fasted for about 5 hours were measured, and the blood glucose values were divided into a control group, a low-dose group, and a high-dose group by stratified sampling method, and then uniformized. Orally administer the substance to each group, orally after 0.5 hours 2 g / kg of glucose (20% solution, 10 mL / kg) was given. Blood glucose levels are drawn from the tail vein under non-anaesthesia before administration of the substance (-0.5 hours), immediately before administration of glucose (0 hours), and 0.5, 1, 2 and 3 hours after administration of glucose. Blood glucose level measurement device (Glutest Neo Super, Sanwa Chemical Research Institute (stock)).

The results are shown in FIG. 6.

It is clear from the results in FIG. 6 that the blood glucose level of the control group shows a change that peaks at 0.5 hours after glucose administration and then decreases. The low-dose and high-dose groups administered with heated puree showed the same changes as the control group, but the tendency to suppress the rise in blood glucose was seen in the low-dose group, and the significant hypoglycemia was seen in the high-dose group This tendency can be seen up to 1 hour after the administration of glucose.

(Example 10) Production of tea beverages

To 200 ml of a tea extract (those prepared by adding commercially available powdered green tea to 0.3% by mass), 20 g of the bitter gourd puree obtained in Example 4 was added to prepare a sencha drink.

(Example 11) Production of coffee beverage

About 5 g of ground commercial coffee powder, 65 g of boiling water was used to prepare a coffee extract according to a common method, and 5 g of bitter gourd puree obtained in Example 4 was added to prepare a coffee beverage.

(Example 12) Production of vegetable drink

One banana, one carrot, one lettuce, 30 g of bitter gourd puree obtained in Example 4, 200 ml of water, and 3 teaspoons of sugar were added to a food processor, and poured into a juice machine to obtain a vegetable drink.

(Example 13) Production of pasta

85g of wheat flour, 30g of sugar, 1g of baking powder, 1g of salt, 25g of sesame oil, 20g of cow's milk, and 1g of bitter gourd puree powder obtained in Example 4 were freeze-dried bitter gourd puree powder.

(Example 14) Production of tofu

600 ml of sufficiently cooled soybean milk (no adjustment), 15 ml of brine, 30 ml of water, and 5 g of bitter gourd puree obtained in Example 4 were poured into a heat-resistant freshness box and blended. Cover with cling film and cook in a steamer over a low heat for about 15 minutes to make tofu.

[Industrial availability]

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

Claims (27)

A composition for promoting GLP-1 secretion, comprising at least one component (A) selected from the following formulae (1) to (11): A composition comprising at least one component (A) selected from the formulae (1) to (11) as claimed in claim 1, for use in the group selected from the group consisting of a suppression of an increase in blood glucose level, an appetite suppression, an overeating suppression, At least one of uses for improving glucose metabolism, preventing or treating diabetes, preventing or treating obesity, reducing body weight, and reducing body fat rate. The composition according to claim 2, which is at least one selected from the group consisting of inhibition of an increase in blood glucose level, inhibition of appetite, inhibition of overeating, improvement of glucose metabolism, prevention or treatment of diabetes, prevention or treatment of obesity, weight loss, and reduction of body fat rate. The function is due to the promotion of GLP-1 secretion. The composition according to any one of claims 1 to 3, wherein the component (A) contains at least one component (A1) selected from the formulae (6) to (11). The composition according to any one of claims 1 to 4, which contains component (A) as a component derived from bitter gourd. The composition according to any one of claims 1 to 5, which contains the component (A) as a component derived from bitter gourd after heat treatment. 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 formulae (1) to (11) as claimed in 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) contains at least one component (A1) selected from the formulae (6) to (11). For example, the composition of claim 8 or 9 includes at least one component (A1) selected from the formulae (6) to (11) in a proportion of 0.005 ppm or more on a mass basis. If the composition of any one of claims 8 to 10, it is a puree or a beverage. The composition of any one of claims 8 to 11, which is heat treated Puree or drink of bitter gourd. The composition according to any one of claims 1 to 12, which is added to food and beverage. The composition according to any one of claims 1 to 13 is labeled with a function to promote the secretion of GLP-1 and / or a function resulting from the function to promote the secretion of GLP-1. The composition according to any one of claims 1 to 14, which is added with a function selected from the function of suppressing an increase in blood glucose level, the function of suppressing appetite, the function of suppressing excessive diet, the function of improving glucose metabolism, and the function of preventing or treating diabetes. Labeling of at least one of the functions of preventing or treating obesity, the function of reducing weight, and the function of reducing body fat rate. A food and drink product comprising the composition according to any one of claims 1 to 15. A food or drink product comprising at least one component (A) selected from the formulae (1) to (11) as claimed in claim 1 in a proportion of 0.005 ppm or more on a mass basis. The food or beverage of claim 16 or 17 contains ingredients not derived from bitter gourd. The food and beverage of any one of claims 16 to 18 is a beverage. The food or drink according to any one of claims 16 to 19 is added with a label that promotes the function of secreting GLP-1 and / or a function resulting from the function of promoting the secretion of GLP-1. The food or drink according to any one of claims 16 to 20, which is added with a function selected from the function of suppressing an increase in blood glucose level, the function of suppressing appetite, the function of suppressing excessive diet, the function of improving glucose metabolism, and the function of preventing or treating diabetes Labeling of at least one of the functions of preventing or treating obesity, the function of reducing weight, and the function of reducing body fat rate. A method for expressing or increasing at least one component (A) selected from the formulae (1) to (11) in the bitter gourd ingredient, including heating the bitter gourd ingredient by heat treatment step. A method for manufacturing a composition according to any one of claims 1 to 15, comprising a heating step of heating a bitter gourd ingredient. The method according to claim 22 or 23, wherein in the heating step, the puree of the balsam pear component is heat-treated. The method according to any one of claims 22 to 24, wherein in the heating step, heat treatment is performed at 60 ° C or higher 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 the group consisting of formulae (6) to (11) as in claim 1.