Sodium-glucose cotransporter 2 inhibitor and promoter of glucose excretion through urine

Abstract

To identify a component with sodium-glucose cotransporter 2 inhibitory activity from among food-ingestible components and to provide a novel sodium-glucose cotransporter 2 inhibitor.SOLUTION: A sodium-glucose cotransporter 2 inhibitor comprises delphinidin and / or a glycoside thereof, or a maqui berry extract.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to sodium-glucose cotransporter 2 inhibitors and agents that promote glucose excretion via urine. [Background technology]

[0002] Sodium glucose cotransporter 2 (SGLT), which actively transports glucose in a sodium-dependent manner, has been identified as a glucose transporter that takes up glucose into the body. SGLTs have three subtypes: SGLT1, SGLT2, and SGLT3. SGLT2 is expressed in the proximal tubules of the kidney and is responsible for reabsorption of glucose from urine and taking up glucose into the body.

[0003] In recent years, it has been shown that inhibiting SGLT2 can inhibit or delay glucose reabsorption in the kidney, thereby suppressing the rise in blood glucose levels, and SGLT2 inhibitors are being developed as treatments for diabetes. Most of the SGLT2 inhibitors reported so far are synthetic compounds that do not occur in nature. Furthermore, although flavonoids and other food ingredients have been reported to have SGLT-2 inhibitory activity, there are few reported examples of food ingredients with SGLT2 inhibitory activity.

[0004] Meanwhile, an experimental system capable of evaluating SGLT2 activity using CHO cells that highly express human SGLT2 has been established, and by utilizing this experimental system, catechin gallate and nobiletin have been identified as food ingredients with SGLT2 inhibitory activity (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] KAKEN Chemical Research Grant Database, 2016 Research Report "Food Functionality Research on the Regulation of the Renal Glucose Reabsorption Transporter SGLT2," website address<https: / / kaken.nii.ac.jp / ja / grant / KAKENHI-PROJECT-15K14737 / > Summary of the Invention [Problem to be solved by the invention]

[0006] As the incidence of diseases involving hyperglycemia, such as diabetes, continues to increase, it is becoming increasingly important to prevent or improve hyperglycemia through the simple method of ingesting food. Therefore, an objective of the present disclosure is to find a component that has SGLT2 inhibitory activity from among components that can be ingested as food, and to provide a new SGLT2 inhibitor. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above-mentioned problems and have found that delphinidin and its glycosides have excellent SGLT2 inhibitory activity. Furthermore, the present inventors have found that maqui berry extract has excellent SGLT2 inhibitory activity and can promote urinary glucose excretion. Furthermore, the present inventors have found that SGLT2 inhibition inhibits or delays glucose reabsorption in the kidney, thereby promoting urinary glucose excretion. Therefore, the present inventors have found that delphinidin and its glycosides, as well as maqui berry extract, can be used as agents for promoting urinary glucose excretion. The present disclosure was completed based on these findings and further research.

[0008] The present disclosure relates to uses of delphinidin and its glycosides and provides the following aspects of the invention. Item 1-1. SGLT2 inhibitors containing delphinidin and / or its glycosides. Item 1-2. The SGLT2 inhibitor according to Item 1-1, wherein the delphinidin and / or glycoside thereof is a delphinidin glycoside having a sambubiose residue. Item 1-3. The SGLT2 inhibitor according to Item 1-1 or 1-2, which is a food or drink. Item 1-4. Use of delphinidin and / or its glycoside for the production of an SGLT2 inhibitor. Items 1-5. Delphinidin and / or its glycosides used in treatment for inhibiting SGLT2. Item 1-6. A method for inhibiting SGLT2, comprising ingesting or administering an amount of delphinidin and / or its glycoside effective for inhibiting SGLT2 to a person in need of SGLT2 inhibition. Item 1-7. A urinary glucose excretion promoter comprising delphinidin and / or its glycoside. Item 1-8. The agent for promoting urinary glucose excretion according to Item 1-7, wherein the delphinidin and / or glycoside thereof is a delphinidin glycoside having a sambubiose residue. Item 1-9. The agent for promoting urinary glucose excretion according to Item 1-7 or 1-8, which is a food or drink. Items 1-10. Use of delphinidin and / or its glycoside for producing an agent for promoting urinary glucose excretion. Items 1-11. Delphinidin and / or its glycosides for use in a treatment for promoting excretion of glucose via urine. Item 1-12. A method for promoting urinary glucose excretion, comprising ingesting or administering to a person in need of promoting urinary glucose excretion an amount of delphinidin and / or its glycoside effective for promoting urinary glucose excretion.

[0009] The present disclosure also provides the following aspects of the invention relating to uses of maqui berry extract. Item 2-1. SGLT2 inhibitors containing maqui berry extract. Item 2-2. The SGLT2 inhibitor according to Item 2-1, wherein the maqui berry extract is an extract of maqui berry fruit. Item 2-3. The SGLT2 inhibitor according to Item 2-1 or 2-2, which is a food or drink. Item 2-4. Use of maqui berry extract for the manufacture of an SGLT2 inhibitor. Item 2-5. Maqui berry extract used in a treatment for inhibiting SGLT2. Item 2-6. A method for inhibiting SGLT2, comprising ingesting or administering to a person in need of SGLT2 inhibition an amount of maqui berry extract effective for inhibiting SGLT2. Item 2-7. A urinary glucose excretion promoter containing maqui berry extract. Item 2-8. The urinary glucose excretion promoter according to Item 2-1, wherein the maqui berry extract is an extract of maqui berry fruit. Item 2-9. The urinary glucose excretion promoter according to Item 2-7 or 2-8, which is a food or drink. Item 2-10. Use of a maqui berry extract for the manufacture of an agent for promoting urinary glucose excretion. Item 2-11. A maqui berry extract used in a treatment to promote excretion of glucose via urine. Item 2-12. A method for promoting urinary glucose excretion, comprising ingesting or administering to a person in need of promoting urinary glucose excretion an amount of maqui berry extract effective for promoting urinary glucose excretion. [Effects of the Invention]

[0010] Furthermore, according to one embodiment of the present disclosure, SGLT2 can be inhibited by ingestion or administration of delphinidin and / or its glycoside, thereby enabling the prevention or treatment of diabetes, diabetic complications, etc. Delphinidin and / or its glycoside are naturally occurring components and highly safe, providing an SGLT2 inhibitor that can be ingested daily as a food, etc. Furthermore, while SGLT1 inhibition can cause diarrhea due to malabsorption of glucose and galactose, and simultaneous inhibition of SGLT1 and SGLT2 has the disadvantage of making hypoglycemia more likely to occur, delphinidin and / or its glycoside have low inhibitory activity against SGLT1 and can selectively inhibit SGLT2, thereby eliminating the problems associated with SGLT1 inhibition.

[0011] According to another embodiment of the present disclosure, ingesting or administering maqui berry extract can inhibit SGLT2, thereby enabling the prevention or treatment of diabetes, diabetic complications, etc. Furthermore, the maqui berry extract used in the present disclosure can be used as a food ingredient, providing a highly safe SGLT2 inhibitor that can be ingested daily as a food, etc. Furthermore, since maqui berry extract has low inhibitory activity against SGLT1 and can selectively inhibit SGLT2, it can eliminate the problems associated with SGLT1 inhibition. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of measuring the SGLT2 inhibitory activity of phlorizin and empagliflozin using CHO cells highly expressing human SGLT2. [Figure 2] FIG. 1 shows the results of measuring the SGLT2 inhibitory activity of delphinidin glycosides using CHO cells highly expressing human SGLT2. [Figure 3] FIG. 1 shows the results of culturing CHO cells with high expression of human SGLT2 in the presence of delphinidin glycosides and measuring the cell lethality. [Figure 4] FIG. 1 shows the results of measuring the SGLT2 inhibitory activity of various extracts using CHO cells highly expressing human SGLT2. [Figure 5] FIG. 1 shows the results of culturing CHO cells with high expression of human SGLT2 in the presence of maqui berry extract and measuring the cell viability. [Figure 6] FIG. 1 shows the results of administering maqui berry extract to diabetic model mice and measuring the amount of glucose in urine. [Figure 7] FIG. 1 shows the results of measuring the SGLT1 inhibitory activity of various extracts using CHO cells highly expressing human SGLT2. DETAILED DESCRIPTION OF THE INVENTION

[0013] 1.Definition In this disclosure, the expression "X to Y" regarding a numerical range refers to a range from X to Y.

[0014] In this disclosure, "SGLT2 inhibitor" refers to an ingredient or composition used to inhibit SGLT2.

[0015] In this disclosure, "agents that enhance the excretion of glucose via urine" refer to ingredients or compositions used to enhance the excretion of glucose from the body via urine.

[0016] 2.SGLT2 inhibitors (1) One embodiment of the SGLT2 inhibitor of the present disclosure is characterized in that it contains delphinidin and / or its glycoside as an active ingredient. Hereinafter, an SGLT2 inhibitor containing delphinidin and / or its glycoside may be referred to as "SGLT2 inhibitor (1)." The SGLT2 inhibitor (1) of the present disclosure will be described in detail below.

[0017] [Active ingredient] The SGLT2 inhibitor (1) of the present disclosure uses delphinidin and / or its glycoside as an active ingredient for inhibiting SGLT2.

[0018] Delphinidin is a type of anthocyanidin, a natural component found in fruits and vegetables.

[0019] The type of glycocone in delphinidin glycosides is not particularly limited, and examples thereof include monosaccharides such as glucose, galactose, arabinose, and xylose; and disaccharides such as sambubiose, rutinose, and sophorose. Among these glycocones, glucose, sambubiose, and galactose are preferred, glucose and sambubiose are more preferred, and sambubiose is even more preferred. The number of glycocones bonded in delphinidin is, for example, 1 to 3, preferably 1 or 2. Furthermore, glycocone binding sites in delphinidin include one or more of the 3-position of the C ring, the 5-position of the A ring, and the 7-position of the A ring. Among these, the 3-position of the C ring and / or the 5-position of the A ring are preferred. Specific preferred examples of delphinidin glycosides include delphinidin 3-glucoside, delphinidin 3-sambubioside, delphinidin 3,5-diglucoside, delphinidin 3-sambubioside-5-glucoside, and delphinidin-3-galactoside.

[0020] Delphinidin and its glycosides are cationic and form salts with halide ions (such as chloride ions) as counter ions.

[0021] Delphinidin and its glycosides are known compounds present in natural products such as maqui berry, cranberry, pomegranate, concord, black currant, etc. In the SGLT2 inhibitor (2) of the present disclosure, delphinidin and its glycosides are preferably extracted or purified from natural products, but may also be synthesized by chemical synthesis.

[0022] In addition, in the SGLT2 inhibitor (1) of the present disclosure, delphinidin and / or its glycoside may be used in the form of an extract containing delphinidin and / or its glycoside. The extract containing delphinidin and / or its glycoside can be obtained by extracting a natural product containing delphinidin and / or its glycoside.

[0023] The SGLT2 inhibitor (1) of the present disclosure may be one selected from delphinidin and its glycosides, or two or more of these may be used in combination. Among delphinidin and its glycosides, from the viewpoint of more effectively exerting the SGLT2 inhibitory effect, preferred are delphinidin glycosides, more preferred are delphinidin glycosides having a sambubiose residue, even more preferred are delphinidin 3-sambubioside and delphinidin 3-sambubioside-5-glucoside, and particularly preferred is delphinidin 3-sambubioside.

[0024] [Dosage form / product form, etc.] The dosage form of the SGLT2 inhibitor (1) of the present disclosure is not particularly limited and may be any of solid, semi-solid, or liquid, and may be appropriately determined depending on the formulation form, administration method, etc. of the SGLT2 inhibitor (1).

[0025] The application method of the SGLT2 inhibitor (1) of the present disclosure may be, for example, any of oral ingestion, oral administration, intravascular (intra-arterial or intravenous) administration, transdermal administration, enteral administration, pulmonary administration, intranasal administration, etc. Among these application methods, oral ingestion or oral administration is preferable.

[0026] The SGLT inhibitors of the present disclosure can be provided in the form of foods, beverages, and pharmaceuticals.

[0027] When the SGLT2 inhibitor (1) of the present disclosure is provided in the form of a food or beverage, delphinidin and / or its glycoside may be combined with other food ingredients or additives to prepare the desired form. Examples of foods and beverages to which the SGLT2 inhibitor (1) of the present disclosure can be applied include general foods and beverages, as well as foods for specified health uses, foods with nutrient function claims, and foods with functional claims. The form of these foods and beverages is not particularly limited, but specific examples include supplements such as capsules (soft capsules, hard capsules), tablets, granules, powders, jellies, and liposome preparations; beverages such as energy drinks, fruit juice drinks, carbonated drinks, lactic acid drinks, jelly drinks, and energy drinks; and luxury items such as dumplings, ice cream, sherbet, gummies, and candies. Among these foods and beverages, supplements and beverages are preferred.

[0028] When the SGLT2 inhibitor (1) of the present disclosure is provided in the form of a food or drink, the content of delphinidin and / or glycosides thereof in the food or drink may be appropriately set depending on the form of the food or drink, the daily intake amount, etc., and the content of delphinidin and / or glycosides thereof contained in the food or drink may be, for example, 0.01 to 40% by weight, preferably 0.05 to 30% by weight, more preferably 0.1 to 20% by weight.

[0029] When the SGLT2 inhibitor (1) of the present disclosure is provided in the form of a pharmaceutical product, delphinidin and / or its glycoside may be formulated into a desired form, either as is or in combination with other additives, etc. Pharmaceutical products to which the SGLT2 inhibitor (1) of the present disclosure can be applied include, for example, oral pharmaceutical products such as drinks, capsules (soft capsules, hard capsules), tablets, granules, powders, jellies, and syrups; liposome preparations, injections, enteral preparations, drip infusions, nasal drops, injections, infusions, and suppositories. Among these pharmaceutical products, oral pharmaceutical products are preferred.

[0030] When the SGLT2 inhibitor (1) of the present disclosure is provided in the form of a pharmaceutical product, the content of delphinidin and / or its glycoside in the pharmaceutical product may be appropriately set depending on the form of the pharmaceutical product, the administration route, the daily dose, etc., and the content of delphinidin and / or its glycoside in the pharmaceutical product may be, for example, 0.1 to 90% by weight, preferably 0.5 to 80% by weight, and more preferably 1 to 70% by weight.

[0031] [Application] The SGLT2 inhibitor (1) of the present disclosure is used to inhibit SGLT2 in vivo. Because the SGLT2 inhibitor (1) of the present disclosure has low inhibitory activity against SGLT1, it is also useful as a selective inhibitor of SGLT2.

[0032] SGLT2 inhibition inhibits or delays glucose reabsorption in the kidney, thereby suppressing an increase in blood glucose level. Therefore, in one embodiment, the SGLT2 inhibitor (1) of the present disclosure can be used for suppressing an increase in blood glucose level and for preventing or ameliorating hyperglycemia.

[0033] Furthermore, SGLT2 inhibition inhibits or delays glucose reabsorption in the kidney, thereby making it possible to prevent or treat diseases associated with hyperglycemia. Therefore, in another aspect, the SGLT2 inhibitor (1) of the present disclosure can also be used as a preventive or therapeutic agent for diseases associated with hyperglycemia. Examples of diseases associated with hyperglycemia include diabetes (type 1 and type 2), diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, myocardial infarction, stroke, cirrhosis, arteriosclerosis obliterans, and chronic kidney disease. Among these, preferred examples of diseases for which the SGLT2 inhibitor (1) of the present disclosure is applicable include diabetes, diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy.

[0034] Furthermore, SGLT2 inhibition can inhibit or delay glucose reabsorption in the kidney, thereby promoting glucose excretion via urine. Therefore, in yet another embodiment, the SGLT2 inhibitor (1) of the present disclosure can be used as an agent for promoting glucose excretion via urine. In yet another embodiment, the SGLT2 inhibitor (1) of the present disclosure can also be used as an agent for increasing urinary glucose concentration (an agent used to increase urinary glucose concentration). When the SGLT2 inhibitor (1) of the present disclosure is used as an agent for promoting glucose excretion via urine or an agent for increasing urinary glucose concentration, its target populations include, for example, those seeking inhibition of blood glucose level increases, those seeking prevention or amelioration of hyperglycemia, and those seeking prevention or treatment of diseases associated with hyperglycemia.

[0035] [Intake or dosage] The intake or dosage of the SGLT2 inhibitor (1) of the present disclosure may be any amount effective for inhibiting SGLT2, and may be appropriately determined depending on the dosage form, administration method, age and symptoms of the target patient, etc. For example, the intake or dosage of delphinidin and / or its glycoside per day for an adult is about 0.1 to 500 mg, preferably about 1 to 100 mg.

[0036] 3.SGLT2 inhibitors (2) Another embodiment of the SGLT2 inhibitor of the present disclosure is characterized in that it contains maqui berry extract as an active ingredient. Hereinafter, an SGLT2 inhibitor containing maqui berry extract may be referred to as "SGLT2 inhibitor (2)." The SGLT2 inhibitor (2) of the present disclosure will be described in detail below.

[0037] [Active ingredient] The SGLT2 inhibitor (2) of the present disclosure uses maqui berry extract as an active ingredient for inhibiting SGLT2.

[0038] Maqui berry (Aristotelia chilensis) is a plant of the family Aristoteliaceae, order Malvaceae. Maqui berry extract can be obtained by subjecting maqui berry to an extraction process. Examples of raw maqui berry parts to be subjected to the extraction process include the fruit, seeds, flowers, leaves, roots, and stems. Among these parts, the fruit is preferred from the viewpoint of extracting components exhibiting SGLT2 inhibitory activity (e.g., delphinidin glycosides) at high concentrations. The raw maqui berry parts used as the extraction material may be raw, or may be subjected to pretreatment such as crushing, cutting, steaming, rolling, drying, or roasting, as necessary.

[0039] The extraction process for obtaining the maqui berry extract may be a general extraction method used in the production of ordinary plant extracts, such as solvent extraction, supercritical extraction, steam distillation, etc. Among these, solvent extraction is preferred.

[0040] Examples of extraction solvents used in solvent extraction treatment include water; lower monohydric alcohols having 1 to 4 carbon atoms, such as methanol, ethanol, n-propanol, isopropanol, and n-butanol; polyhydric alcohols, such as propylene glycol and 1,3-butylene glycol; and mixed solvents thereof. Among these extraction solvents, water, lower monohydric alcohols, and mixed solvents thereof are preferred, more preferably water, ethanol, and mixed solvents thereof, and even more preferably water. When a mixed solvent of water and lower monohydric alcohol is used as the extraction solvent, the ratio of water to lower monohydric alcohol is not particularly limited, but the weight ratio of water to lower monohydric alcohol is 10:90 to 90:10, preferably 20:80 to 80:20.

[0041] The solvent extraction treatment may be carried out by immersing or refluxing the raw material part of the maqui berry in the extraction solvent. After the extraction treatment, the solid matter is removed by solid-liquid separation to obtain a maqui berry extract. The obtained maqui berry extract may be used as a maqui berry extract as is, or, if necessary, a concentrate or a dried product may be used after removing part or all of the solvent.

[0042] [Dosage form / product form, etc.] The dosage form of the SGLT2 inhibitor (2) of the present disclosure is not particularly limited and may be any of solid, semi-solid, or liquid, and may be appropriately determined depending on the formulation form, administration method, etc. of the SGLT2 inhibitor (2). The application method of the SGLT2 inhibitor (2) of the present disclosure is the same as that of the SGLT2 inhibitor (1). The SGLT inhibitor (2) of the present disclosure can be provided in the form of a food or beverage or a pharmaceutical product, as in the case of the SGLT2 inhibitor (1).

[0043] When the SGLT2 inhibitor (2) of the present disclosure is provided in the form of a food or drink, specific examples of the food or drink are the same as those of the SGLT2 inhibitor (1). When the SGLT2 inhibitor (2) of the present disclosure is provided in the form of a food or drink, the content of delphinidin and / or its glycoside in the food or drink may be appropriately set depending on the form of the food or drink, the daily intake amount, etc., and for example, the content of maqui berry extract contained in the food or drink may be 5 to 60 wt %, preferably 10 to 50 wt %, more preferably 20 to 30 wt % in terms of dry weight.

[0044] When the SGLT2 inhibitor (2) of the present disclosure is provided in the form of a pharmaceutical product, specific examples of the pharmaceutical product are the same as those of the SGLT2 inhibitor (1). When the SGLT2 inhibitor (2) of the present disclosure is provided in the form of a pharmaceutical product, the content of the maqui berry extract in the pharmaceutical product may be appropriately determined depending on the form of the pharmaceutical product, the administration route, the daily dose, etc., and, for example, the content of the maqui berry extract contained in the pharmaceutical product may be 10 to 60 wt %, preferably 15 to 50 wt %, and more preferably 25 to 30 wt % in terms of dry weight.

[0045] [Application] The SGLT2 inhibitor (2) of the present disclosure is used to inhibit SGLT2 in vivo. Because the SGLT2 inhibitor (2) of the present disclosure has low inhibitory activity against SGLT1, it is also useful as a selective inhibitor of SGLT2. Specific examples of uses of the SGLT2 inhibitor (2) of the present disclosure are the same as those of the SGLT2 inhibitor (1) described above.

[0046] [Intake or dosage] The intake or dosage of the SGLT2 inhibitor (2) of the present disclosure may be any amount effective for inhibiting SGLT2, and may be appropriately determined depending on the dosage form, administration method, age and symptoms of the target patient, etc. For example, the daily intake or dosage for an adult may be set to about 0.1 to 500 g, preferably about 1 to 100 g, calculated as the dry weight of maqui berry extract. [Example]

[0047] The present disclosure will be specifically described below using examples, but the present invention is not limited to these examples.

[0048] The plant materials or extracts used in the following test examples are as follows: Maqui berry extract : Extract (dried product) obtained by extracting maqui berry (Aristotelia chilensis) fruit with water (delphinidin-3-sambubioside-5-glucoside: 22.0%, delphinidin-3-sambubioside: 6.3%, delphinidin-3,5-diglucoside: 34.8%, delphinidin-3-glucoside: 19.2%, cyanidin-3-sambubioside-5-glucoside: 7.6%, cyanidin-3-sambubioside: 2.3%, cyanidin-3,5-: 4.8%, cyanidin-3-glucoside: 3.0%) Yuzu polyphenol extract : Extract (dried material) obtained by extracting yuzu fruit and peel with ethanol ·Ginseng extract : Extract (dried material) obtained by extracting ginseng rhizomes with ethanol Maca extract: Extract (dried product) obtained by extracting maca with hydroalcohol Bird's nest extract : Extract (dried material) obtained by hydrolyzing swiftlet nests and extracting with water Olive extract : Extract (dried material) obtained by extracting olive leaves, fruits and seeds with water

[0049] Reference Test Example 1: Evaluation system for SGLT2 inhibitory activity 1. Test Method A method for evaluating SGLT2 inhibitory activity was confirmed using CHO cells with high human SGLT2 expression (recombinant cells in which a human SGLT2 expression vector was introduced into CHO cells). The CHO cells with high human SGLT2 expression used in this study are a cell line known to have high sodium-dependent 2-deoxyglucose (2-DG) uptake activity (4B7 line described in Non-Patent Document 1). Specifically, using phlorizin and empagliflozin, which are known to have SGLT2 inhibitory activity, the following procedures were performed to confirm the evaluation of SGLT2 inhibitory activity: (1) intracellular uptake of 2-DG, (2) preparation of cell lysates, and (3) measurement of the amount of 2-DG in the cell lysates.

[0050] (1) Uptake of 2-DG into cells 2.0 × 10 human SGLT2 highly expressing CHO cells were placed in each well of a 24-well plate using Ham's F-12 (FBS, G418, P / S mixture) medium. 5 Cells were seeded at 1000 cells / well and cultured overnight at 37°C. Ham's F-12 (FBS, G418, P / S mixture) medium was then removed from each well, and each well was washed with 0.5 ml of Krebs Ringer HEPES (KRH) buffer. A KRH buffer containing the prescribed amount of test substance shown in Table 1 and 2 mM 2-DG was then added to each well, and the wells were incubated at 37°C for 10 minutes to allow 2-DG to be taken up into the cells. [Table 1]

[0051] (2) Preparation of cell lysate The supernatant was removed from the wells, and the cells were washed twice with 0.5 mL of ice-cold KRH Buffer. 0.3 mL of 0.1 M NaOH was added per well, and the cells were solubilized by heating at 60°C for 1 hour. After solubilization, the cells were neutralized with an equal volume of 0.1 M HCl, and the cell lysate was collected in a 1.5 mL tube.

[0052] (3) Measurement of 2-DG levels in cell lysates To each well of a 96-well plate, 0.025 ml of 2DG detection solution (Glucose Uptake-Glo Assay kit, Promega) was added, followed by 0.025 ml of the cell lysate obtained above, and the wells were incubated at 37°C for 30 minutes. Chemiluminescence measurement was then performed using an enzymatic method to calculate the amount of 2-DG taken up into the cells. The amount of 2-DG taken up into the cells after each test substance was calculated as the 2-DG uptake rate (%), relative to the amount of 2-DG taken up into the cells in the control group (defined as 100).

[0053] 2. Test Results The results are shown in Figure 1. As a result, the 2-DG uptake rate decreased when phlorizin and empagliflozin, which are known to have SGLT2 inhibitory activity, were added, confirming that SGLT2 inhibitory activity can be appropriately evaluated by the above-mentioned evaluation method using CHO cells with high human SGLT2 expression.

[0054] Test Example 1: Screening of ingredients with SGLT2 activity (1) 1. Test Method The test substances shown in Table 2 were used to evaluate the SGLT2 inhibitory activity in the same manner as in Reference Test Example 1 above.

[0055] [Table 2]

[0056] 2. Test Results The results are shown in Figure 2. When delphinidin glycoside was added, the 2-DG uptake rate was significantly reduced, indicating that delphinidin and its glycoside have SGLT2 inhibitory activity. Furthermore, the SGLT2 inhibitory activity of Example 2 was higher than that of Example 1, and the SGLT2 inhibitory activity of Example 4 was higher than that of Example 3. In other words, it was found that delphinidin glycosides having sambubiose residues have higher SGLT2 inhibitory activity.

[0057] Test Example 2: Examination of the effect of delphinidin glycosides on cell viability 1. Test Method In Test Example 1, it was confirmed that delphinidin glycosides reduced the 2-DG uptake rate. To confirm that this result was not affected by cell viability, the effect of delphinidin glycosides on the viability of CHO cells highly expressing human SGLT2 was examined. The viability of CHO cells highly expressing human SGLT2 was measured by the lactate dehydrogenase (LDH) method. LDH measurement was performed using the LDH-Cytotoxicity Test Wako (Wako Pure Chemical Industries, Ltd.). The specific test procedure is as follows.

[0058] 5.0 x 10 human SGLT2 highly expressing CHO cells in a 96-well plate 3 After seeding cells / well, the test substance shown in Table 3 was added and incubated at 37°C for 15 minutes. The plate was then stirred and centrifuged, and the supernatant was collected. A luminescence reagent was then added to the supernatant in each well, and the reaction was allowed to proceed for 45 minutes. The reaction was then stopped with 0.5 M HCl. The absorbance at a wavelength of 560 nm was then measured. The cell lethality for each example was calculated by setting the absorbance of the negative control as 0% cell lethality and the absorbance of the positive control as 100% cell lethality.

[0059] [Table 3]

[0060] 2. Test Results The results are shown in Figure 3. As a result, when delphinidin glycoside was added, a sufficiently low cell lethality was observed. That is, from these results, it was confirmed that the decrease in 2-DG uptake rate caused by delphinidin glycoside in Test Example 1 was not affected by cell viability but was due to the SGLT2 inhibitory activity of delphinidin glycoside.

[0061] Test Example 3: Screening of ingredients with SGLT2 activity (2) 1. Test Method The test substances shown in Table 4 were used to evaluate the SGLT2 inhibitory activity in the same manner as in Reference Test Example 1 above.

[0062] [Table 4]

[0063] 2. Test Results The results are shown in Figure 4. The addition of maqui berry extract significantly reduced the 2-DG uptake rate, demonstrating that maqui berry extract has SGLT2 inhibitory activity.

[0064] Test Example 4: Examination of the effect of maqui berry extract on cell viability 1. Test Method In Test Example 3, it was confirmed that maqui berry extract reduced the 2-DG uptake rate. To confirm that this result was not affected by cell viability, the effect of maqui berry extract on the viability of CHO cells highly expressing human SGLT2 was examined. The specific test procedure is as follows.

[0065] 2 x 10 human SGLT2 highly expressing CHO cells were cultured in each well of a 24-well plate using Ham's F-12 (FBS, G418, P / S mixture) medium. 5Cells were seeded at 1000 cells / well and cultured overnight at 37°C. After removing the medium from each well, each well was washed with 0.5 ml of KRH buffer. Then, KRH buffer containing the test substance (as shown in Table 5) and 2 mM 2-DG was added to each well and incubated at 37°C for 10 minutes. The buffer in the wells was then removed, and the cells were washed twice with PBS. After this, 0.5 ml of medium and 50 μl of MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium Bromide) labeling reagent were added to each well and incubated at 37°C for 3 hours at 5-6.5% CO2. Then, 200 μL of DMSO (C2H6OS) was added to each well. After confirming that the purple formazan crystals were completely solubilized, the absorbance of each well at 530 nm was measured using a microplate reader. The absorbance in the case of the control was set to 100, and the absorbance in the case of each test substance was calculated as the cell viability (%).

[0066] [Table 5]

[0067] 2. Test Results The results are shown in Figure 5. As a result, when maqui berry extract was added, the cell viability was equivalent to that of the control. In other words, from these results, it was confirmed that the decrease in 2-DG uptake rate caused by maqui berry extract in Test Example 3 was not affected by cell viability, but was due to the SGLT2 inhibitory activity of maqui berry extract.

[0068] Test Example 3: Examination of the effect of maqui berry extract on urinary glucose levels 1. Test Method Ten 8-week-old male diabetic model mice (KK-Ay type 2 diabetic mice) were divided into two groups: a control group (n=6) and an Example 11 group (maqui berry extract-administered group) (n=4). The control group received a single oral administration of saline, while the maqui berry extract-administered group received a single oral administration of saline containing maqui berry extract (400 mg / kg of maqui berry extract). After oral administration, mice from both groups were allowed to drink water and eat food ad libitum, and urine was collected 4, 8, and 12 hours after oral administration. The collected urine volume and urinary glucose concentration were measured using a glucose measurement kit (Fujifilm Wako Pure Chemical Corporation), and the amount of urinary glucose was calculated.

[0069] 2. Test Results The results of adding up the glucose levels in urine collected 4, 8, and 12 hours after oral administration are shown in Figure 6. As a result, the amount of glucose in urine was increased in the maqui berry extract group compared to the control group. This indicates that administration of maqui berry extract inhibits SGLT2, thereby promoting glucose excretion via urine.

[0070] Test Example 4: Examination of SGLT1 inhibitory activity of maqui berry extract 1. Test Method SGLT1 inhibitory activity was evaluated using CHO cells with high human SGLT1 expression (recombinant cells in which a human SGLT1 expression vector was introduced into CHO cells). The CHO cells with high human SGLT1 expression used in this study are a cell line known to have high sodium-dependent 2-DG uptake activity (4G8 cell line, described in the reference below). Specifically, using phlorizin and empagliflozin, which are known to have SGLT2 inhibitory activity, the following procedures were performed to confirm the evaluation of SGLT1 inhibitory activity: (1) intracellular 2-DG uptake, (2) preparation of cell lysates, and (3) measurement of the amount of 2-DG in the cell lysates. Reference: Hideo Satsu et al., Inhibitory Effect of Tangeretin and Cardamonin on Human Intestinal SGLT1 Activity In Vitro and Blood Glucose Levels in Mice In Vivo, Nutrients 2021, 13, 3382. https: / / doi.org / 10.3390 / nu13103382

[0071] (1) Uptake of 2-DG into cells Human SGLT1-highly expressing CHO cells were seeded into each well of a 24-well plate at 2.0 × 10 cells / well using Ham's F-12 (FBS, G418, P / S mixture) medium and cultured overnight at 37°C. Next, Ham's F-12 (FBS, G418, P / S mixture) medium was removed from each well, and each well was washed with 0.5 ml of KRH buffer. Further, KRH buffer containing the predetermined amount of test substance shown in Table 6 and 2 mM 2-DG was added to each well, and the wells were incubated at 37°C for 10 minutes to allow 2-DG to be taken up into the cells.

[0072] [Table 6]

[0073] (2) Preparation of cell lysate The supernatant was removed from the wells, and the cells were washed twice with 0.5 mL of ice-cold KRH Buffer. 0.3 mL of 0.1 M NaOH was added per well, and the cells were solubilized by heating at 60°C for 1 hour. After solubilization, the cells were neutralized with an equal volume of 0.1 M HCl, and the cell lysate was collected in a 1.5 mL tube.

[0074] (3) Measurement of 2-DG levels in cell lysates To each well of a 96-well plate, 0.025 ml of 2DG detection solution (Glucose Uptake-Glo Assay kit, Promega) was added, followed by 0.025 ml of the cell lysate obtained above, and the mixture was incubated at 37°C for 30 minutes. Chemiluminescence measurement was then performed using an enzymatic method to calculate the amount of 2-DG taken up into the cells. The amount of 2-DG taken up into the cells for each test substance was calculated as the 2-DG uptake rate (%), relative to the amount of 2-DG taken up into the cells for the control, which was set at 100.

[0075] 2. Test Results The results are shown in Figure 7. The addition of maqui berry extract maintained a high 2-DG uptake rate, indicating that maqui berry extract has weak SGLT1 inhibitory activity and can selectively inhibit SGLT2. The expression level of SGLT1 in CHO cells with high human SGLT1 expression differs from the expression level of SGLT2 in CHO cells with high human SGLT2 expression. Comparing CHO cells with high human SGLT1 expression reveals differences in the susceptibility of sugar absorption by other transporters. Therefore, the evaluation criteria for the presence or absence of SGLT1 inhibitory activity differ from the evaluation criteria for the presence or absence of SGLT2 inhibitory activity. This explains why, when comparing Example 11 of Test Example 4 with Example 1 of Test Example 1, the former was evaluated as having low SGLT1 inhibitory activity and the latter as having high SGLT2 inhibitory activity, despite the 2-DG uptake rates being similar. Furthermore, since maqui berry extract contains delphinidin glycosides, these results indicate that delphinidin and / or its glycosides can also selectively inhibit SGLT2.

Claims

1. A sodium-glucose cotransporter 2 inhibitor comprising delphinidin and / or its glycoside.

2. The sodium-glucose cotransporter 2 inhibitor according to claim 1, wherein the delphinidin and / or its glycoside is a delphinidin glycoside having a sambubiose residue.

3. A sodium-glucose cotransporter 2 inhibitor, including maqui berry extract.

4. A urinary glucose excretion promoter comprising delphinidin and / or its glycoside.

5. The agent for promoting glucose excretion via urine according to claim 4, wherein the delphinidin and / or its glycoside is a delphinidin glycoside having a sambubiose residue.

6. A urinary glucose excretion promoter containing maqui berry extract.

Images