JP2023108900A - ORAL COMPOSITION FOR SUPPRESSING BLOOD SUGAR LEVEL ELEVATION, ORAL COMPOSITION FOR SUPPRESSING AGEs GENERATION, SUPPLEMENT, FUNCTIONAL FOOD AND PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

To provide an oral composition excellent in inhibitory effect on blood sugar level elevation, and in inhibitory effect on AGEs generation.SOLUTION: An oral composition contains Aomoji leaf extract and/or dry matter. The oral composition is excellent in inhibitory effect on blood sugar level elevation, and in inhibitory effect on AGEs generation, based on sugar absorption inhibitory action in small intestinal epithelium cells, sugar uptake acceleration action in liver cells, and DPP-4 enzyme activity inhibitory action, and can be used preferably for a supplement, a functional food, a pharmaceutical composition or the like.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to an oral composition for suppressing elevation of blood sugar level, an oral composition for suppressing AGE production, and applications thereof.

In recent years, due to changes in eating habits such as obesity, overeating, lack of exercise, stress, drinking, etc., the number of diabetic patients and potential diabetic patients is rapidly increasing, and this has become a serious problem worldwide. Diabetes is classified into type 1 and type 2 depending on the presence or absence of insulin dependence, and more than 90% of diabetic patients have type 2 diabetes. Diabetes is a disease in which hyperglycemia continues chronically due to insufficient action of insulin. Chronic continuation of such hyperglycemia increases the risk of complications such as myocardial infarction, arteriosclerosis, and stroke.

Thus, in order to prevent diabetes, it is necessary to prevent a hyperglycemic state and suppress an increase in blood sugar level, and for this purpose, the importance of improving diet and exercise is known. In particular, it is known that, in improving dietary habits, it becomes difficult for the blood sugar level to rise by considering the amount and selection of carbohydrates, the manner of eating, the combination of foods, and the like.

In addition, as a food material that exhibits the effect of suppressing the rise in blood sugar level, a composition containing Yukou, which is a citrus fruit of the Rutaceae family (Patent Document 1), an agent containing Kucho tea extract (Patent Document 2), cacao polyphenols and A composition containing lipid as an active ingredient (Patent Document 3), a composition containing a soybean extract as an active ingredient (Patent Document 4), and the like have already been disclosed.

In addition, many studies have been conducted on the causal relationship of diseases such as diabetes in Advanced Glycation End Products (AGEs). For example, Patent Literature 5 discloses that an extract of Betula annuus has an AGE production inhibitory effect.

JP 2019-137660 A JP 2021-138647 A JP 2019-180399 A JP 2018-172353 A JP 2019-43887 A

As described above, the plant-derived compositions disclosed in Patent Documents 1 to 4 and the like have been reported as compositions for suppressing elevation of blood sugar level, and Patent Document 5 discloses compositions having AGE production inhibitory action. However, the actual situation is that there is room for improvement in terms of its effects, production costs, and the like.

Under such circumstances, it is an object of the present invention to provide a novel oral composition and its applied product having a blood glucose level elevation suppressing action and an AGEs production suppressing action.

As a result of intensive research to solve the above problems, the present inventors have found that processed Aomoji leaves suppress blood sugar level elevation, suppress sugar absorption in small intestinal epithelial cells, promote sugar uptake in liver cells, and inhibit DPP-4 enzyme activity. , and found that it has an excellent effect in suppressing the production of AGEs, leading to the present invention.

That is, the present invention relates to the following inventions.
<1> An oral composition for suppressing elevation of blood sugar level, characterized by containing a leaf extract and/or dried product.
<2> The oral composition according to <1>, wherein the Aomoji leaf extract is a solvent extract of Aomoji leaf.
<3> The oral composition according to <2>, wherein the solvent extract of Aomoji leaves is a solvent extract of Aomoji leaves using a mixed solvent of water and ethanol.
<4> The oral composition according to <2>, wherein the solvent extract of Aomoji leaves is a hot water extract of Aomoji leaves.
<5> An oral composition for suppressing AGEs production containing a leaf extract and/or dried product.
<6> A supplement containing the oral composition according to any one of <1> to <5>.
<7> A functional food containing the oral composition according to any one of <1> to <5>.
<8> A pharmaceutical composition containing the oral composition according to any one of <1> to <5>.
<9> The pharmaceutical composition according to <8>, which is for preventing or improving diabetes.

<A1> An oral composition for suppressing absorption of sugar in small intestinal epithelial cells, containing a leaf extract and/or dried product of Aomoji.
<A2> An oral composition for promoting sugar uptake in liver cells, containing a leaf extract and/or dried product of Aomoji.
<A3> An oral composition for inhibiting DPP-4 enzyme activity, containing a leaf extract and/or dried product.

<B1> A method for producing a leaf extract of Aomoji, which comprises contacting Aomoji leaves with an extraction solvent that is a mixed solvent of water and ethanol to extract components contained in the Aomoji leaves.
<B2> A method for producing a leaf extract of Aomoji, which comprises contacting Aomoji leaves with hot water to extract components contained in the Aomoji leaves.

INDUSTRIAL APPLICABILITY According to the present invention, an oral composition having a useful effect in suppressing elevation of blood sugar level and an applied product thereof are provided.

2 is a graph showing the results of an evaluation test of the sugar permeability in small intestinal epithelial cells of the 50% ethanol extract of Aomoji leaves of Experimental Example 1, which was evaluated in Test 1-A. 2 is a graph showing the results of an evaluation test of the sugar permeability in small intestinal epithelial cells of a 50% ethanol extract from leaves of Aomoji in Experimental Example 1, which was evaluated in Test 1-B. 2 is a graph showing the results of an evaluation test of sugar uptake in liver cells of a 50% ethanol extract of Aomoji leaves of Experimental Example 2, which was evaluated in Test 2. FIG. 10 is a graph showing the results of an evaluation test of sugar uptake in liver cells of the hot water extract of Aomoji leaves of Experimental Example 3, which was evaluated in Test 2. FIG. 10 is a graph showing the results of an evaluation test on inhibition of DPP-4 enzymatic activity of the hot water extract from the leaf of Test Example 3 and the cold water extract from the leaf of Test Example 4, which were evaluated in Test 3. FIG. 4 is a graph showing the results of an evaluation test on suppression of formation of Advanced Glycation End Products (AGEs) of a 50% ethanol extract of Aomoji leaves of Experimental Example 2, which was evaluated in Test 4. FIG. 10 is a graph showing the results of an evaluation test on suppression of formation of Advanced Glycation End Products (AGEs) of the hot water extract from leaves of Aomoji leaves of Experimental Example 3, which was evaluated in Test 4. FIG. 10 is a graph showing the results of the evaluation test of the cytotoxicity of the 50% ethanol extract of Aomoji leaves of Experimental Example 1, which was evaluated in Test 5. FIG.

Hereinafter, the present invention will be described in detail with examples, etc., but the present invention is not limited to the following examples, etc., and can be arbitrarily modified without departing from the scope of the present invention. In this specification, "~" shall be used as an expression including numerical values or physical quantities before and after it. Also, in this specification, the expression "A and/or B" includes "only A," "only B," and "both A and B."

TECHNICAL FIELD The present invention relates to an oral composition for suppressing elevation of blood sugar level (hereinafter referred to as "oral composition for suppressing elevation of blood sugar level of the present invention") containing a leaf extract and/or dried product of Aomoji leaves.
In addition, in the present specification, the extract and/or the dried product of Aomoji leaves may be collectively referred to as a "processed product of Aomoji leaves".

In the present invention, the “oral composition for suppressing elevation of blood sugar level” is a composition capable of suppressing or alleviating elevation of blood sugar level by oral ingestion.
The processed Aomoji leaf according to the present invention has a sugar absorption inhibitory action in small intestinal epithelial cells, a sugar uptake promoting action in liver cells, and a DPP-4 enzymatic activity inhibitory action, as shown in Examples described later. As a result, the oral composition containing the processed product of Aomoji leaves has an effect of suppressing an increase in blood sugar level, and thus can be used as an oral composition for suppressing an increase in blood sugar level.

Here, in the present specification, the term "sugar absorption in small intestinal epithelial cells" means the action of absorbing sugar via a sugar transport protein (SGLT1) present in small intestinal epithelial cells. By suppressing the absorption of , the increase in blood sugar level is suppressed.
"Glucose uptake action in liver cells" means the action of taking up sugar in the liver by the action of insulin after a meal. be.
"DPP-4 enzymatic activity inhibitory action" means an inhibitory action on the enzymatic activity of degrading a hormone (GLP-1) that is synthesized in small intestinal cells and stimulates insulin secretion in the pancreas, and inhibits DPP-4 enzymatic activity. As a result, the blood concentration of GLP-1 is increased, insulin secretion is enhanced, and an increase in blood sugar level is suppressed.

The blood glucose level elevation suppressing action of the processed Aomoji leaf in the present invention can be evaluated by the sugar absorption suppressing action in small intestinal epithelial cells, the sugar uptake promoting action in liver cells, the DPP-4 enzyme activity inhibitory action, and the like. will be described later.

Hereinafter, the oral composition for suppressing elevation of blood sugar level of the present invention will be described in more detail.

The "Aomoji" used as a raw material for the oral composition for suppressing elevation of blood sugar level of the present invention belongs to the family Lauraceae and the genus Tribulus terrestris, and the part to be used of the present invention is the leaves. The leaves used as a raw material can be used as a raw material at any time during the cultivation period as long as the effects of the present invention are not impaired. Aomoji leaves may be natural products or may be artificially cultivated.

As described above, Aomoji leaf processed product is a general term for Aomoji leaf extracts and/or dried products.
In the present invention, the term "extract" refers to a form in which the content of the active ingredient is increased by compressing or solvent-extracting the target raw material plant or, if necessary, drying and shredding it. It is a concept that summarizes the things of Specifically, it includes an extract obtained from the leaves of Aomoji as an extraction raw material, a diluted or concentrated solution of the extract, or a crude or purified product thereof. A dried product obtained by drying the extract is also considered as an extract.

Moreover, in the present invention, the term "dried matter" refers to the dried raw material plant. Specifically, Aomoji leaves are dried by a known drying method such as sun-drying, heat-drying, or freeze-drying. Drying conditions are not particularly limited as long as they do not impair the effects of the present invention.
From the viewpoint of usability, the dried product is usually pulverized and used as a dry pulverized product. The grinding method is not particularly limited, and a conventionally known grinder may be used. The particle size of the powder is appropriately determined depending on the mode of use.

The Aomoji leaf extract and the dried Aomoji leaf may be used alone, or may be used by mixing them in an arbitrary ratio.

Solvent extracts are preferred among the processed products of Aomoji leaves according to the present invention.
The solvent extract of Aomoji leaves can be obtained by extracting the leaves of Aomoji in accordance with a conventional method. The extraction method is not particularly limited as long as it does not impair the effects of the present invention, and an appropriate method can be arbitrarily selected and used according to the purpose.
For example, it is preferable to extract the components contained in the leaves of Aomoji by contacting the leaves of Aomoji as they are or the crushed, dried or pulverized products thereof with an extraction solvent.

The extraction solvent may contain additives within a range that does not impair the effects of the present invention. Examples include pH adjusters, viscosity adjusters, and the like.

A suitable example of the solvent extract of Aomoji leaves is a solvent extraction of Aomoji leaves with a mixed solvent of water and ethanol.
The solvent extract can be safely orally ingested and has an excellent effect of suppressing elevation of blood sugar level. The ratio of water and ethanol in the mixed solvent is arbitrary as long as the effect of the present invention is achieved. % or less.

A preferred example of a solvent extract of Aomoji leaves is a water extract of Aomoji leaves. Water used for extraction includes distilled water, pure water, and sterilized water. For example, tap water or water containing impurities may be used. Cold water, room temperature water, hot water, hot water, etc. can be used as the extraction solvent as long as the effects of the present invention are not impaired. , hot water is preferred. The hot water extract of Aomoji leaves can be safely taken orally and has an excellent effect of suppressing elevation of blood sugar level.

The extraction time is appropriately selected in consideration of the type of extraction solvent and the ratio of Aomoji leaves to the extraction solvent, but it is particularly limited as long as it is a time that does not impair the effects of the present invention and allows the active ingredient to be extracted by the extraction solvent. not to be The obtained extract may be used as it is, or may be subjected to dilution, concentration, drying, purification, etc. according to the usual methods. Moreover, you may remove a solvent by vacuum concentration and freeze-drying as needed.

The oral composition for suppressing elevation of blood sugar level of the present invention may use the processed Aomoji leaf of the present invention as it is, but usually, the processed Aomoji leaf is combined with other ingredients that do not impair the effects of the present invention. It is used as an oral composition containing (hereinafter sometimes referred to as "oral composition of the present invention").
The other ingredients may be any ingredients that are safe for humans to ingest, and are appropriately determined in consideration of the form of use of the oral composition of the present invention (for example, supplements, functional foods, etc., described later).
The mixing ratio of other components can be appropriately selected and determined according to the purpose, as long as the effects of the present invention are not impaired.

The oral composition according to the present invention is excellent in suppressing sugar absorption in small intestinal epithelial cells, promoting sugar uptake in liver cells, and inhibiting DPP-4 enzyme activity due to the processed Aomoji leaves contained therein. In order to exhibit the effect, as described above, it can be used as an oral composition for suppressing elevation of blood sugar level, as well as "oral composition for suppressing sugar absorption in small intestinal epithelial cells" and "oral composition for promoting sugar uptake in liver cells". , can be used individually as an "oral composition for inhibiting DPP-4 enzyme activity".
In addition, the oral composition of the present invention has an effect of suppressing sugar absorption in small intestinal epithelial cells or promoting sugar uptake in liver cells, and thus has an effect of suppressing obesity. Therefore, the oral composition according to the present invention can be used as an obesity-suppressing oral composition.

Furthermore, since the oral composition containing the processed product of Aomoji leaves according to the present invention exhibits an excellent effect of suppressing AGEs production, it can be used as an "oral composition for suppressing AGEs production".

As used herein, the term "AGE production inhibitory action" means an action to inhibit the production of Advanced Glycation End Products, and AGEs accumulate in the body upon reaction between sugars and biosubstances. is known to cause various diseases, and has been reported to be a causative agent of diabetic complications, inflammation, kidney disease, cancer, osteoporosis, Alzheimer's disease, cataracts, age spots, wrinkles, etc. ing. When blood sugar levels rise and AGEs increase in the blood and tissues, the functions of biological substances such as proteins are impaired, causing various diseases. can prevent AGEs from accumulating in the body.

As shown in the below-described Examples, the oral composition containing the processed Aomoji leaves has an excellent inhibitory effect on AGEs production, and therefore can be used as an oral composition for inhibiting AGEs production. Therefore, it can be used for supplements, functional foods, pharmaceutical compositions, and the like.

The oral composition of the present invention has excellent blood glucose level elevation inhibitory action (sugar absorption inhibitory action in small intestinal epithelial cells, sugar uptake promoting action in liver cells and DPP-4 enzyme activity inhibitory action) and AGEs production inhibitory action. Therefore, it can be used in supplements, functional foods, pharmaceutical compositions, and the like.

In addition, regarding the action or efficacy of the oral composition of the present invention, the following indications may be attached at the time of commercialization. For example, when labeling an action or effect on blood sugar levels, "for those who have begun to worry about their blood sugar levels", "for those who are concerned about their blood sugar levels", and "for those who are concerned about their postprandial blood sugar levels". It is possible to display "moderate increase in blood sugar level", "moderate sugar absorption", "suppress sugar absorption", and the like.

The intake amount of the oral composition of the present invention is not particularly limited as long as it is an amount that can exhibit the effects of the present invention, and the form of the oral composition of the present invention, the subject's sex, weight, health condition, etc. determined accordingly. The intake amount may be taken once or divided into several times a day. Moreover, in order to obtain the effects or efficacy of the oral composition of the present invention, it is preferable to take the oral composition of the present invention continuously.

Supplements, functional foods, food additives, and pharmaceutical compositions, which are preferred forms of the oral composition of the present invention, will be described below, but the use of the oral composition of the present invention is not limited to these.

The supplement of the present invention contains the oral composition (processed product of Aomoji leaves) of the present invention. The form of the supplement of the present invention is not particularly limited, and may be any form such as tablets, powders, granules, capsules, dragees, films, troches, chewables, solutions, emulsions and suspensions. .
The supplement of the present invention may contain any ingredient commonly used as a supplement, in addition to the oral composition of the present invention. Examples of such ingredients include amino acids, peptides; vitamins such as vitamin E, vitamin C, vitamin A, vitamin B, and folic acid; minerals; sugars; inorganic salts; citric acid or salts thereof; Nutrient tonic ingredients such as propolis, royal jelly and taurine; crude drug extracts such as ginger extract and ginseng extract; herbs: collagen and the like.

The ratio of the oral composition of the present invention to be blended into the supplement is arbitrary, but the blending ratio is selected within a range in which the action of suppressing the elevation of blood sugar level and the action of suppressing the production of AGEs are exhibited.

The oral composition (processed product of Aomoji leaves) of the present invention can be easily taken for a long time by mixing it with various foods and beverages to make it a functional food so that it can be easily taken orally on a daily basis. .
The term “functional food” as used herein refers to foods and/or beverages ingested for the purpose of maintaining health in addition to general foods. This concept includes functional foods, health foods, dietary supplements, nutritional insurance foods, and so on. Among these, foods with function claims, foods with health claims, and foods with nutrient function claims are preferred functional foods. In addition, when commercializing as a functional food, various additives used in food, specifically, coloring agents, preservatives, thickening stabilizers, antioxidant bleaching agents, antibacterial antifungal agents, Acidulants, seasonings, emulsifiers, enhancers, manufacturing agents, fragrances, etc. may be added.

Foods and beverages that are the target of the functional food of the present invention are not particularly limited. Examples of foods include sausages, hams, processed seafood products, jellies, candies, chewing gums and the like. Beverages include various teas, green juices, soft drinks, alcoholic beverages, nutritional drinks, and the like.

The ratio of adding the oral composition of the present invention to the functional food is arbitrary, but the mixing ratio is selected within a range in which the functional food has the effect of suppressing the increase in blood sugar level and the effect of suppressing the production of AGEs.

When the oral composition of the present invention is used as a food with functional claims, the oral composition of the present invention may be labeled with the action or efficacy brought about by the processed Aomoji leaf according to the present invention in the product. For example, it may be displayed on the main body of the product according to the present invention, a package, a container, a stored item such as a package, or an advertisement thereof.

In addition, the oral composition (processed product of Aomoji leaves) of the present invention can be used as a food additive by itself or by adding other ingredients thereto. Other ingredients are not particularly limited as long as they can be used as food and drink additives. Beverages and foods to which food additives are added are optional and not particularly limited.

The ratio of the oral composition of the present invention to be added to the food additive is arbitrary, but the ratio is selected so long as the inhibitory effect on blood sugar level rise and the production of AGEs is exhibited.

The oral composition of the present invention can also be applied to animals other than humans. Therefore, it can be added to supplements for animals such as pet food and functional foods.

Since the oral composition of the present invention has a blood glucose level elevation suppressing action and an AGE production suppressing action, the oral composition of the present invention is formulated into a pharmaceutical composition ( Hereinafter, it may be described as "the pharmaceutical composition of the present invention").

As used herein, the term "pharmaceutical composition" means an agent useful for at least one of prevention, treatment, and amelioration of symptoms of a target disease. As used herein, "prevention" includes suppression and delay of onset of the disease or symptom. "Treatment" also includes amelioration and remission of the condition of the disease or symptom, as well as suppression of progress of the disease or symptom. Moreover, in the present specification, the term "pharmaceutical composition" includes not only pharmaceuticals but also quasi-drugs.

Specific examples of diseases or symptoms targeted by the pharmaceutical composition of the present invention include, for example, diabetes, arteriosclerotic diseases, renal diseases, Alzheimer's disease, osteoporosis, skin diseases such as skin aging, cataracts, age-related macular degeneration, and the like. mentioned. Among these, diabetes is a preferred example of the disease or condition targeted by the pharmaceutical composition of the present invention.

The pharmaceutical composition of the present invention may contain, in addition to the oral composition of the present invention, other drugs and pharmacologically acceptable optional ingredients within a range that does not impair its efficacy. Also, the composition of the present invention itself may be used as the pharmaceutical composition of the present invention.

Forms of the pharmaceutical composition of the present invention include tablets, pills, granules, powders, liquids, suspensions, syrups, capsules, and the like. The form of the pharmaceutical composition of the present invention is not particularly limited, and may be solid or liquid, or solid and liquid. A preferred embodiment of the pharmaceutical composition of the present invention is an oral formulation.

The intake amount of the pharmaceutical composition of the present invention is not particularly limited as long as it is an amount capable of exhibiting the effects of the present invention, and is appropriately determined according to the subject's sex, body weight, health condition, and the like. The intake amount may be taken once or divided into several times a day. Moreover, from the viewpoint of prevention, treatment, and amelioration of symptoms of target diseases, it is preferable to continuously ingest the pharmaceutical composition of the present invention.

EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these.
In addition, "%" shown in mixed solvents in Examples indicates "% by volume" unless otherwise specified. In addition, when the extraction solvent is a mixed solvent, water, which is the balance, is omitted. For example, "50% ethanol" means a mixed solvent of 50% by volume ethanol and 50% by volume water.
<1. Preparation of Dried Aomoji Leaves>
After washing the collected Aomoji leaves with tap water, blanching was performed for 5 minutes using a steam convection oven (humidity 100%, temperature 100°C). Then, it was dried for 20 hours using a dryer (dry bulb 65°C, wet bulb 35°C). The dried Aomoji leaves were pulverized so as to pass through a 0.4 mm mesh to obtain dried and pulverized Aomoji leaves.

<2. Preparation of Aomoji Leaf Extract>
The above <1. Preparation of Dried Matter of Japanese Artemisia Leaves> was extracted using an extraction solvent to obtain extracts of Japanese artichoke leaves of Experimental Examples 1 to 4.

(Experimental example 1) Extraction 1 with 50% ethanol from dried matter
400 mL of a 50% ethanol solution was added to 4 g of dried pulverized Aomoji leaves, and extraction was performed with stirring at room temperature for 3 hours. Furthermore, 400 mL of 50% ethanol solution was added again to the extraction residue, and the mixture was extracted with stirring at room temperature for 3 hours, and then centrifuged (10,000×g, 30 minutes, 4° C.) to obtain an extract. The obtained extracts were mixed and then dried using a rotary evaporator, a centrifugal concentrator and a freeze dryer to obtain a dry powdery Aomoji leaf extract of Experimental Example 1.

(Experimental example 2) Extraction 2 with 50% ethanol from dried matter
The same dried pulverized Aomoji leaf as in Experimental Example 1 was used. 0.5 g of dry pulverized Aomoji leaves was immersed in 10 mL of a 50% ethanol solution and extracted with shaking (1,500 rpm) for 1 hour. A leaf extract of Aomoji leaves of Experimental Example 2 was obtained.

(Experimental Example 3) Extraction with Hot Water from Dried Matter The same dry pulverized matter of Aomoji leaves as in Experimental Example 1 was used. 0.5 g of dry pulverized Aomoji leaves was immersed in 35 mL of pure water warmed to 85° C. and allowed to stand for 5 minutes for immersion extraction (85° C.). 2 to obtain the extract of Aomoji leaves of Experimental Example 3.

(Experimental Example 4) Extraction with Cold Water from Dried Matter The same dry pulverized matter of Aomoji leaves as in Experimental Example 1 was used. 0.5 g of dried pulverized Aomoji leaves was immersed in 35 mL of pure water cooled to 4° C. and allowed to stand for 16 hours for immersion extraction (4° C.). 2 to obtain the extract of Aomoji leaves of Experimental Example 4.

<3. Evaluation>
(Test 1: Evaluation of sugar permeability in small intestinal epithelial cells)
The sugar permeability of the small intestine epithelial-like cell membrane was measured for the leaf extract (50% ethanol extract) of Aomoji leaves of Experimental Example 1. It should be noted that <Test 1-A> in the evaluation method described later assumes that the Aomoji leaf extract is ingested before meals, and <Test 1-B> assumes that the Aomoji leaf extract is ingested during meals. .

(Evaluation method)
<Test 1-A>
Caco-2 cells, a human colon cancer-derived cell line used as a small intestinal epithelial cell model, were seeded in a 24-well transwell at a seeding density of 2.5×10 ⁵ cells and cultured in a CO ₂ incubator for about 3 weeks. bottom.
The TEER value (transepithelial electrical resistance value) of the cells was measured, and after reaching 300Ω/cm ² , 500 μL of the 50% ethanol extract of Aomoji leaves of Experimental Example 1 was added to the lumen side. The concentrations of the Aomoji leaf extract added were 100 μg/mL and 200 μg/mL.
After addition, it was cultured for 24 hours in a CO ₂ incubator (5% CO ₂ , 37° C.). After 24 hours, cells were washed twice and pre-incubated for 30 minutes in a _CO2 incubator. A 25 mM glucose solution was then added to the luminal side and cultured in a CO ₂ incubator for 1 hour. After 1 hour, the solution on the basement membrane side was collected, and the glucose concentration was measured.
The glucose concentration was measured using Glucose Assay Kit-WST (a product of Dojindo Laboratories).
The obtained measurement results were calculated with the glucose concentration without addition of the leaf extract of Aomoji as a comparative example being 100.

<Test 1-B>
The same method as <Test 1-A> was performed, except that the 50% ethanol extract of Aomoji leaves of Experimental Example 1 adjusted to 100 μg/mL was added at the same time as the 25 mM glucose solution was added.

(result)
The results are shown in FIGS. 1 and 2. FIG. As shown in FIG. 1, the sugar permeability in <Test 1-A>, in which glucose was added after adding 50% ethanol extract of Aomoji leaves in Experimental Example 1, was 5.1 when 100 μg/mL of Aomoji leaf extract was added. % inhibition, and 10.3% inhibition when 200 μg/mL was added.
Further, as shown in FIG. 2, the sugar permeability in <Test 1-B>, in which the 50% ethanol extract of Aomoji leaves in Experimental Example 1 was added at the same time as glucose, was as follows: was suppressed by 21.9%.

From the above, it was shown that the addition of the 50% ethanol extract of Aomoji leaves before or at the same time as the addition of glucose was effective in suppressing the absorption of sugar in the small intestine.

(Test 2: Evaluation of sugar uptake in liver cells)
Assuming glucose uptake in the liver, the amount of glucose uptake in liver-derived cells was evaluated for the extracts of Aomoji leaves of Experimental Example 2 (50% ethanol extract) and Experimental Example 3 (hot water extract).

(Evaluation method)
HepG2 cells, a human liver-derived cell line, were seeded in a 96-well plate at a seeding density of 9.0×10 ⁴ cells/cm ² , cultured in a CO ₂ incubator for 24 hours, then 1% BSA-DMEM was added, Further cultured in a _CO2 incubator for 24 hours. Thereafter, 1% BSA-DMEM was removed, and the 50% ethanol extract from experimental example 2 adjusted to 50 μg/mL or the hot water extract from experimental example 3 adjusted to 35 μg/mL was added. After addition, it was washed after culturing for 24 hours in a _CO2 incubator.
Sugar uptake was measured using Glucose Uptake Assay Kit-Green (a product of Dojindo Laboratories). A fluorescence-labeled glucose derivative was added to the washed cells, incubated for 15 minutes in a CO ₂ incubator, washed, and fluorescence intensity was measured at excitation wavelength/fluorescence wavelength = 488 nm/520 nm.

(result)
The results are shown in FIGS. 3 and 4. FIG. As shown in FIG. 3, when the 50% ethanol extract from Aomoji leaves was added, the amount of sugar uptake increased by 1.25 times compared to when no Aomoji leaf extract was added, and as shown in FIG. When the extract was added, sugar uptake increased 1.14-fold compared to when the Aomoji leaf extract was not added.

From the above, it was shown that the 50% ethanol extract of the leaves of Aomoji and the hot water extract of the leaves of Aomoji had excellent effects on glucose uptake in the liver.

(Test 3: Evaluation of DPP-4 enzymatic activity inhibition)
The DDP-4 enzymatic activity was measured for the Aomoji leaf extracts of Experimental Example 3 (hot water extract) and Experimental Example 4 (cold water extract).

(Evaluation method)
The concentrations of the hot water extract from the leaves of Experimental Example 3 and the cold water extract from the leaves of Experimental Example 4 were 25, 50, 100, 200 and 300 μg/mL, respectively. The enzymatic activity of DDP-4 was measured using DPP(IV) Inhibitor Screening (product of Cayman Chemical). Each concentration of hot water extract from leaves of Aomoji was added to a 96-well plate attached to the kit, and fluorescence intensity was measured at excitation wavelength/fluorescence wavelength = 355 nm/455 nm.

(result)
The results are shown in FIG. The enzymatic activity of DPP-4 was inhibited by 18.8% when 25 μg/mL of the hot water extract of the leaf of Experimental Example 3 was added, compared with the case where the leaf extract of the leaf was not added. Further inhibition was 24.0% at 50 μg/mL, 35.8% at 100 μg/mL, 50.3% at 200 μg/mL and 62.7% at 300 μg/mL.
Further, when 25 μg/mL of the cold water extract of the leaves of Aomoji of Experimental Example 4 was added, the enzymatic activity of DPP-4 was inhibited by 16.3%. Further inhibition was 22.5% at 50 μg/mL, 29.5% at 100 μg/mL, 42.9% at 200 μg/mL and 50.1% at 300 μg/mL.

From the above, it has been shown that the hot water extract of Aomoji leaves and the cold water extract of Aomoji leaves have an excellent effect on inhibiting the enzymatic activity of DPP-4, and inhibit the enzymatic activity of DPP-4 in a concentration-dependent manner. was done.

(Test 4: Evaluation of suppression of Advanced Glycation End Products (AGEs) production)
The AGEs production inhibition rate was measured for the leaf extracts of Aomoji leaves of Experimental Example 2 (50% ethanol extract) and Experimental Example 3 (hot water extract).

(Evaluation method)
500 μL of 400 mM glucose solution was placed in a 1.5 ml Eppendorf tube, and 100 μL of 80 mg/mL BSA solution was added. Various concentrations (Example 2 (50% ethanol extract): 0.4, 2 and 10 mg/mL, Example 3 (hot water extract): 0.14, 0.7 and 3.5 mg) were added to the Eppendorf tube. 20 μL of Aomoji leaf extract and 380 μL of PBS were added, and the Eppendorf tube was heated at 60° C. for 48 hours (stirred 3 times a day). A positive control was prepared by adding aminoguanidine, which is an anti-glycation agent, instead of the leaf extract of Aomoji. The reaction solution obtained after heating was dispensed into a 96-well plate, and fluorescence intensity was measured at excitation wavelength/fluorescence wavelength=370 nm/440 nm. The AGE production suppression rate was calculated from the measurement result of the obtained fluorescence intensity, and the 50% inhibitory activity concentration (IC ₅₀ : mg/mL) was calculated using the calculated value.

(result)
The results are shown in FIGS. 6 and 7. FIG. As shown in FIG. 6, the concentration of AGEs 50% production inhibitory activity in the 50% ethanol extract from Aomoji leaves was 1.3 mg/mL, and as shown in FIG. was 0.41 mg/mL. All extracts showed extremely low values compared to aminoguanidine, which is an anti-glycation agent.

From the above, it was shown that the 50% ethanol extract from the leaves of the genus Aomoji and the hot water extract from the leaves of the genus Aomoji have remarkably excellent effects on suppressing the production of AGEs.

(Test 5: Evaluation of cytotoxicity of Aomoji leaf extract)
Cell viability was measured for the Aomoji leaf extract (50% ethanol extract) of Experimental Example 1.

(Evaluation method)
Caco-2 cells, a human colon cancer-derived cell line used as a small intestinal epithelial cell model, were seeded in a 96-well plate at a seeding density of 5.0×10 ⁴ cells/well, and cultured for 24 hours in a CO ₂ incubator. bottom.
The medium was removed and 100 μL of 50% ethanolic extract of Aomoji leaves was added. The concentrations of the Aomoji leaf extract added were 1000, 800, 600, 400, 200, 100, 50 and 25 μg/mL. After addition, it was cultured in a _CO2 incubator for 24 hours. After 24 hours, the Aomoji leaf extract was removed, the cell viability was measured using Cell Counting Kit-8 (product of Dojindo Laboratories), and the cytotoxicity of the Aomoji leaf extract was evaluated.

(result)
The results are shown in FIG. A 50% ethanol extract of Aomoji leaves showed no cytotoxicity at concentrations up to 1000 μg/mL.

The oral composition containing the leaf extract and/or dried product of the present invention suppresses blood sugar level elevation, suppresses absorption of sugar in small intestinal epithelial cells, promotes sugar uptake in liver cells, inhibits DPP-4 enzyme activity, and produces AGEs. It has an excellent effect on inhibition, and can be used by blending in supplements, functional foods, pharmaceutical compositions, and the like.

Claims (9)

An oral composition for suppressing elevation of blood sugar level, characterized by containing a leaf extract and/or a dried product. 2. The oral composition according to claim 1, wherein the Aomoji leaf extract is a solvent extract of Aomoji leaves. 3. The oral composition according to claim 2, wherein the solvent extract of Aomoji leaves is a solvent extract of Aomoji leaves with an extraction solvent of a mixed solvent of water and ethanol. 3. The oral composition according to claim 2, wherein the solvent extract of Aomoji leaves is a hot water extract of Aomoji leaves. 1. An oral composition for suppressing AGEs production, comprising a leaf extract and/or a dried product. A supplement characterized by containing the oral composition according to any one of claims 1 to 5. A functional food comprising the oral composition according to any one of claims 1 to 5. A pharmaceutical composition comprising the oral composition according to any one of claims 1 to 5. The pharmaceutical composition according to claim 8, which is used for preventing or improving diabetes.