Blood glucose level suppressants and glucose absorption inhibitors

Abstract

To provide novel blood glucose level elevation inhibitors and glucose absorption inhibitors. [Solution] A blood glucose level elevation inhibitor or glucose absorption inhibitor containing sapindoside B as an active ingredient.

Description

[Technical Field]

[0001] The present invention relates to a blood glucose level elevation inhibitor and a glucose absorption inhibitor. [Background technology]

[0002] Diabetes is one of the lifestyle-related diseases that is increasing worldwide. Diabetes is divided into two main categories: type 1, also known as insulin-dependent diabetes mellitus, and type 2, also known as non-insulin-dependent diabetes mellitus. In particular, the early stages of type 2 diabetes are characterized by insulin resistance, mainly in insulin-acting tissues such as the liver, skeletal muscle, and adipocytes. Insulin resistance in these tissues is related to excessive glucose production by the liver and impaired glucose utilization by peripheral tissues.

[0003] Type 2 diabetes, in particular, is a metabolic disorder related to lifestyle and can be prevented through daily dietary management. Controlling blood glucose levels is especially important for preventing hyperglycemia and subsequent diabetes-related complications, and therefore substances to control blood glucose levels have been developed. For example, Patent Document 1 describes that a substance obtained by extracting horse chestnut is effective in suppressing the rise in blood glucose levels. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Patent No. 4741899 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] As a result of diligent research, the inventors have discovered that a component derived from Akebia has the effect of suppressing the rise in blood glucose levels, and furthermore, inhibiting glucose absorption in the intestinal tract. Based on this novel finding, the present invention aims to provide a novel blood glucose level suppressant and glucose absorption inhibitor. [Means for solving the problem]

[0006] The present invention provides, for example, the following [1] to [6]. [1] A blood glucose-lowering agent containing sapindoside B as the active ingredient. [2] A glucose absorption inhibitor containing sapindoside B as the active ingredient. [3] The agent according to [1] or [2], comprising sapindoside B as an akebia extract. [4] The agent according to [3], wherein the akebi extract is an akebi leaf extract. [5] The agent according to [3] or [4], wherein the akebi extract is extracted using an extraction solvent containing alcohol. [6] The agent according to [5], wherein the alcohol comprises at least one selected from the group consisting of methanol and ethanol. [Effects of the Invention]

[0007] According to the present invention, novel blood glucose level elevation inhibitors and glucose absorption inhibitors are provided. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1(a) shows the results of carbohydrate loading tests using Akebia leaf extract, Figure 1(b) shows the results when soluble starch was used as the carbohydrate, Figure 1(b) shows the results when maltose was used, and Figure 1(c) shows the results when glucose was used. [Figure 2] These are the 1H NMR (Figure 2(a)) and 13C NMR (Figure 2(b)) spectra of the active ingredient compound in the Akebia leaf extract. [Figure 3] These are the results of a glucose tolerance test for sapindoside B. [Figure 4] Figure 4(a) is a graph showing the α-amylase inhibitory activity of sapindoside B, and Figure 4(b) is a graph showing the α-glucosidase inhibitory activity of sapindoside B. [Modes for carrying out the invention]

[0009] Hereinafter, embodiments for implementing the present invention will be described in detail. Note that the present invention is not limited to the following embodiments.

[0010] In the numerical ranges described step by step in this specification, the upper limit value or lower limit value of a numerical range at a certain step can be arbitrarily combined with the upper limit value or lower limit value of a numerical range at other steps. In the numerical ranges described in this specification, the upper limit value or lower limit value of the numerical range may be replaced with the value shown in the examples. The components or materials exemplified in this specification can be used alone or in combination of two or more, unless otherwise specified.

[0011] The blood glucose level elevation inhibitor according to this embodiment contains sapindoside B as an active ingredient.

[0012] Sapindoside B is a triterpenoid saponin compound contained in various plants, for example, plants of the family Sapindaceae. More specifically, it is a compound represented by the following formula (1).

[0013]

Chemical formula

[0014] Sapindoside B has a hederagenin-type triterpene and three pyranosides as substructures, the three pyranosides being α-L-arabinose, α-L-lamonose, and β-D-xylose. Sapindoside B is caropanax saponin I, 23-hydroxy-3β-[[2-O-(3-O-β-D-xylopyranosyl-α-L-ramnopyranosyl)-α-L-arabinopyranosyl]oxy]oleana-12-eno-28-acid, 3β-[[2-O-(3-O-β-D-xylopyranosyl-α-L-ramnopyranosyl)-α-L-arabinopyranosyl]oxy]-23-hydroxyoleana-12-eno-28-acid, 3β-[2-O-(3-O-β-D-xylopyranosyl-α-L-ramnopyranosyl) It is also called -α-L-arabinopyranosyloxy]-23-hydroxyoleana-12-eno-28-acid, 3β-[[2-O-[6-deoxy-3-O-(β-D-xylopyranosyl)-α-L-mannopyranosyl]-α-L-arabinopyranosyl]oxy]-23-hydroxy-5α-oleana-12-eno-28-acid, and 3β-[2-O-[3-O-(β-D-xylopyranosyl)-α-L-ramnopyranosyl]-α-L-arabinopyranosyloxy]-23-hydroxyoleana-12-eno-28-acid.

[0015] Sapindoside B according to this embodiment is preferably derived from Akebia quinata, more preferably from the leaves, vines, or stems of Akebia quinata, and even more preferably from the leaves of Akebia quinata. Akebia quinata is a plant belonging to the Akebia family and is widely distributed in Southeast Asian countries, the People's Republic of China, the Republic of Korea, and Japan. Akebia leaves are palmate compound leaves, consisting of five oval leaflets with short petioles. Sapindoside B according to this embodiment may be derived from parts other than the pericarp of Akebia quinata.

[0016] The blood glucose-lowering agent according to this embodiment may contain commercially available sapindoside B, or it may contain sapindoside B purified or isolated from plants such as Akebia as an active ingredient. The blood glucose-lowering agent may also contain sapindoside B as Akebia extract. In other words, the blood glucose-lowering agent according to this embodiment can also be described as an agent containing Akebia extract as an active ingredient.

[0017] When a blood glucose-lowering agent contains akebi extract, the part of the akebi plant used to obtain the extract is not limited to the leaves, vines, stems, or any other part containing sapindoside B. The akebi extract according to this embodiment may be akebi leaf extract, akebi vine extract, or akebi stem extract, and is preferably akebi leaf extract. The akebi extract according to this embodiment does not need to contain akebi fruit peel extract.

[0018] If a blood glucose-lowering agent contains akebi extract, the method for producing the akebi extract is any method that extracts sapindoside B so that it is included in the extract. The extraction method is not particularly limited, but for example, as described in the examples below, it can be obtained by immersing the target material, such as akebi leaves, in an extraction solvent containing alcohol, such as methanol, ethanol, or isopropanol, for a predetermined time. In other words, the akebi extract may be an extract obtained using an extraction solvent containing alcohol. The alcohol preferably includes at least one selected from the group consisting of methanol and ethanol. Alternatively, the akebi extract can also be obtained by immersing the target material in hot water for a predetermined time, using hot water as the extraction solvent. The akebi extract obtained by these methods may be obtained after extraction with an extraction solvent, followed by operations such as concentration and / or drying.

[0019] The blood glucose-lowering agent according to this embodiment may consist solely of the active ingredient sapindoside B, or it may contain other ingredients that are acceptable as pharmaceuticals, quasi-drugs, or foods in addition to the active ingredient. If the blood glucose-lowering agent contains sapindoside B as an akebi extract, the blood glucose-lowering agent may consist solely of the akebi extract, or it may contain the above-mentioned other ingredients in addition to the akebi extract.

[0020] Other ingredients permitted as pharmaceuticals and quasi-drugs include, for example, bases, excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, suspending agents, antioxidants, and solubilizers. Other ingredients permitted as food include, for example, as excipients, carbohydrates such as starch, cellulose, CMC, lactose, sugar, and glucose; and for processing, preservation, taste adjustment, and solubility / disintegration adjustment, minerals, vitamins, flavonoids, quinones, polyphenols, saponins (excluding sapindoside B), amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, flavorings, stabilizers, preservatives, and surfactants.

[0021] The amount of the active ingredient in the blood glucose elevation inhibitor according to this embodiment is not particularly limited. For example, the amount of sapindoside B may be in the range of 0.01% by mass or more and 100% by mass or less based on the total amount of the blood glucose elevation inhibitor.

[0022] The blood glucose-lowering agent according to this embodiment may be in any form, such as solid (powder, granules, etc.), liquid (solution, suspension, etc.), or paste. The dosage form of the blood glucose-lowering agent according to this embodiment may be any dosage form, such as tablets (plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, etc.), capsules, powders, granules, or liquid preparations (syrups, jellies, etc.). These preparations can be obtained, for example, by mixing the active ingredient (sapindoside B or akebia extract) with other ingredients as needed and then molding them.

[0023] The blood glucose level-lowering agent according to this embodiment can be prepared as a food composition (beverage and food), a quasi-drug, or a pharmaceutical product. That is, as one embodiment of the present invention, a food composition (beverage and food), a quasi-drug, or a pharmaceutical product for suppressing blood glucose level-lowering, containing sapindoside B as an active ingredient, is provided. The food composition may be, for example, a food for specified health uses, a food for special dietary uses, a nutritional supplement, or a functional food.

[0024] The target recipients of the blood glucose level-increasing inhibitor according to this embodiment may be humans, or animals other than humans (for example, non-human mammals).

[0025] The method of administering the blood glucose-lowering agent according to this embodiment is not particularly limited and can be appropriately set according to the type, condition, and age of the target. The method of administration may be oral or parenteral, but oral or enteral administration is preferred, and administration via the small intestine after administration is also acceptable.

[0026] An example of the dosage of the blood glucose-lowering agent according to this embodiment is that, when the target of administration is a human, the dosage of sapindoside B may be in the range of 1 mg or more, 10 mg or more, or 50 mg or more per day, or the dosage of sapindoside B may be in the range of 500 mg or less, 300 mg or less, or 100 mg or less per day.

[0027] When the blood glucose elevation inhibitor according to this embodiment contains akebi extract as the active ingredient, an example of the dosage of the blood glucose elevation inhibitor may be in the range of 1 g or more, 10 g or more, or 30 g or more per day as the dosage of akebi extract, or in the range of 100 g or less, 80 g or less, or 50 g or less per day as the dosage of akebi extract.

[0028] The blood glucose level-lowering agent according to this embodiment may be administered once a day, or divided into two, three, or more doses per day, so that the daily amount of active ingredient is within the above-mentioned range.

[0029] The blood glucose-raising inhibitor according to this embodiment can also be used to suppress glucose absorption in the intestinal tract, since its active ingredient, sapindoside B, inhibits glucose absorption in the intestinal tract. In other words, one embodiment of the present invention can be considered as a glucose absorption inhibitor containing sapindoside B as an active ingredient. The specific embodiments of the glucose absorption inhibitor according to this embodiment can be the embodiments of the blood glucose-raising inhibitor described above.

[0030] The glucose absorption inhibitor according to this embodiment suppresses glucose absorption in the intestinal tract and can therefore be used for purposes such as suppressing fat accumulation, suppressing vascular endothelial inflammation (maintaining vascular flexibility), suppressing atherosclerosis and non-atherosclerosis, protecting against peripheral vascular damage, suppressing cognitive deterioration, improving skin quality, improving bowel movements, promoting urination, improving edema, stopping bleeding, and improving the oral environment. [Examples]

[0031] The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to the following examples.

[0032] [Example 1: Preparation of Akebia Extract (Akebia Leaf Extract)] Akebia leaves collected in Okayama Prefecture were used as the basis for this study. 50g of fresh akebia leaves were mixed with 10 times the volume (500mL) of methanol, and the mixture was allowed to stand at room temperature for one week to extract the akebia leaves. The resulting methanol extract was concentrated under reduced pressure to obtain a crude extract as a green powder. The recovery rate of the crude extract from the methanol extract was 5.0 w / w%. The obtained powder was stored at 20°C and used as the akebia leaf extract in the following tests.

[0033] [Test Example 1: Carbohydrate Load Test of Akebia Leaf Extract] The experiment used 6-week-old male ddY mice (SLC Japan Co., Ltd.) that had undergone a one-week acclimatization period. The mice weighed 25-30g and were housed in an isolator cage system maintained at a temperature of 24±1℃ and humidity of 50±10% with a 12-hour light-dark cycle. During rearing, the mice were given ad libitum access to tap water and standard laboratory feed.

[0034] Mice were divided into two groups: a control group and an akebia leaf extract administration group. All mice were fasted for 24 hours. The control group was given 1000 mg / kg of soluble starch as a carbohydrate. The akebia leaf extract administration group was given 1000 mg / kg of soluble starch and 1000 mg / kg of akebia leaf extract. Blood was collected from the tail veins of the mice at 0, 30, 60, 90, 120, and 240 minutes after ingestion of the above samples. The collected blood was centrifuged at 5000 × g to prepare serum. Blood glucose levels (serum glucose levels) were measured using the prepared serum with a commercially available clinical reagent (Glucose Test Wako, Fujifilm Wako Pure Chemical Industries, Ltd.). The same tests were also performed by replacing soluble starch as a carbohydrate with maltose or glucose.

[0035] The test results are shown in Figure 1. Figure 1(a) shows the results when soluble starch was used, Figure 1(b) shows the results when maltose was used, and Figure 1(c) shows the results when glucose was used, with the mean ± standard deviation (n=6) shown. Thirty minutes after carbohydrate loading, akebia leaf extract significantly suppressed the rise in blood glucose levels compared to the control group for all three types of carbohydrates (p<0.001). If akebia leaf extract only inhibited carbohydrate-degrading enzymes and did not inhibit glucose absorption, it should not have shown a suppression effect on blood glucose levels in glucose-loaded mice. However, according to the results of this test, akebia leaf extract also showed a suppression effect on blood glucose levels in glucose-loaded mice, suggesting that akebia leaf extract has at least an effect of inhibiting glucose absorption in the intestinal tract.

[0036] [Example 2: Isolation of Sapindoside B] One of the active ingredients contained in Akebia leaves was isolated by bioassay-induced fractionation using glucose-loaded mice. 2.5 g of the crude extract prepared in Example 1 was dissolved in water and partitioned with ethyl acetate. The aqueous layer was then dried and further partitioned with water and butanol. As a result, activity was confirmed in the butanol layer. A portion of the recovered butanol layer was separated using an ODS gel column. The fraction showing specific activity was further purified by repeated separation using reverse-phase HPLC (column: Develosil HG-5, Nomura Chemical Co., Ltd.). As a result, 10.6 mg of compound (referred to as "compound 1") was purified. The purity of the finally purified compound 1 was confirmed by thin-layer chromatography (stationary phase: ODS plate, mobile phase: 70% methanol, detection conditions: immersion in 10% sulfuric acid and heating at 110°C). Under these conditions, compound 1 was detected as a single spot with a retention factor of 0.10. From this result, it was confirmed that pure compound 1 was obtained.

[0037] [Example 3: NMR data analysis of the active ingredient] The molecular formula of "Compound 1" was determined by HR-FABMS (High-Resolution Fast Atom Bombardment Mass Spectrometry) using C 46 H 74 O 16 That was the decision. Next, 1 1H NMR, 13 Based on detailed analysis of NMR data such as 13C NMR, DEPT (Distortionless Enhancement by Polarization Transfer), and 2D NMR (COSY (COrrelation SpectroscopY), HMQC (Heteronuclear Multiple Quantum Correlation), HMBC (Heteronuclear Multiple Bond Correlation), ROESY (Rotating-frame Overhauser Effect SpectroscopY)), the planar structure of compound 1, including its relative stereochemistry, was determined. 1 The 1H NMR spectrum is shown in Figure 2(a). 13The 13C NMR spectrum is shown in Fig. 2(b). For "Compound 1", 1 the 1H NMR and 13 13C NMR spectral data are in good agreement with the structure of sapindoside B. Finally, Compound 1, one of the active ingredients in the extract of Akebia leaves, was identified as sapindoside B represented by the following formula (1).

[0038] [Chemical formula]

[0039] [Test Example 2: Glucose tolerance test of sapindoside B] Mice were divided into three groups: a control group, a low-dose group, and a high-dose group. After fasting all mice for 24 hours, the control group was given 1000 mg / kg of glucose. On the other hand, the low-dose group was given 1000 mg / kg of glucose and 1 mg / kg of purified sapindoside B. Also, the high-dose group was given 1000 mg / kg of glucose and 10 mg / kg of purified sapindoside B. Blood was collected from the tail veins of the mice 0, 30, 60, 90, and 120 minutes after ingestion of the above specimens. The blood glucose levels of the mice were measured in the same manner as in Test Example 1 for the collected blood.

[0040] The test results are shown in Fig. 3. The measured values are shown as the mean ± standard deviation (n = 8). At 30 minutes after glucose loading, the blood glucose level in the low-dose group that ingested sapindoside B was significantly lower than that in the control group (p < 0.05). Similarly, in the high-dose group, the blood glucose level was also significantly lower than that in the control group (p < 0.001). This indicates that sapindoside B has a certain effect of suppressing the increase in blood glucose level, reducing the peak of blood glucose level after glucose loading, and also suppressing the rapid decrease in blood glucose level. That is, it was found that sapindoside B is one of the active ingredients in the extract of Akebia leaves.

[0041] [Test Example 3: Carbohydrate-degrading enzyme inhibitory action of sapindoside B] [α-Amylase activity inhibition test] 30 μL of potassium phosphate buffer (100 mM, pH=6.9) and 20 μL of sapindoside B were mixed, and 20 μL of α-amylase (0.5 U / mL) was added to this mixture (As 1 Similarly, samples in which sapindoside B was replaced with potassium phosphate buffer (100 mM, pH=6.9) were used as controls (Ac 1 ) was used as a sample blank (Bs 1 ) or control blank (Bc 1 The mixture was used as the inhibitor. After pre-incubating the mixture at 37°C for 5 minutes, 100 μL of starch solution (0.5 mg / mL) was added to initiate the reaction, and the mixture was incubated at 37°C for 10 minutes. Subsequently, 150 μL of iodine solution (containing 5 mM and 1 mM hydrogen chloride) was added to stop the reaction. Enzyme activity was determined by measuring the absorbance at 590 nm using a microplate reader (Thermomax, Molecular Devices). The above method was repeated using acarbose as a positive control. The inhibitory activity of α-amylase was calculated using the following formula (A). Inhibition rate (%)=100×[(Bc 1 -Ac 1 )-(Bs 1 -As 1 )] / (Bc 1 -Ac 1 )···(A)

[0042] <α-glucosidase activity inhibition test> 50 μL of potassium phosphate buffer (100 mM, pH=7.0) and 20 μL of sapindoside B were mixed, and 20 μL of yeast α-glucosidase (0.1 U / mL) was added to this mixture (As 2 Similarly, samples in which sapindoside B was replaced with potassium phosphate buffer (100 mM, pH=7.0) were used as controls (Ac 2 ) was used as a sample blank (Bs 2 ) or control blank (Bc 2The mixture was used as a positive control. After pre-incubating the mixture at 37°C for 5 minutes, 20 μL of p-NPG solution (1 mM) was added to initiate the reaction, and the mixture was incubated at 37°C for 10 minutes. Subsequently, 50 μL of sodium carbonate solution (200 mM) was added to stop the reaction. Enzyme activity was determined by measuring the absorbance at 405 nm using a microplate reader (Thermomax, Molecular Devices). The above method was repeated using acarbose as a positive control. The inhibitory activity of α-glucosidase was calculated using the following formula (B). Inhibition rate (%)=100×[(Ac 2 -Bc 2 )-( As 2 -Bs 2 )] / (Ac 2 -Bc 2 )···(B)

[0043] Figure 4 shows the change in enzyme activity inhibition rate with respect to sapindoside B concentration. Figure 4(a) shows the change in inhibition rate against α-amylase, and Figure 4(b) shows the change in inhibition rate against α-glucosidase. Each point represents the mean ± standard deviation (n=3). At a concentration of 500 μg / mL, the inhibition rate of acarbose, the positive target, was 87.4 ± 0.75% for α-amylase and 59.5 ± 1.25% for α-glucosidase. On the other hand, the inhibition rates of sapindoside B against both α-amylase and α-glucosidase were less than 10%, indicating that sapindoside B does not have an inhibitory effect on carbohydrate-degrading enzymes. From these results, it is considered that the effect of sapindoside B in suppressing blood glucose elevation is not achieved by inhibiting carbohydrate-degrading enzymes, but by suppressing glucose absorption in the intestinal tract.

Claims

1. A blood glucose level-lowering agent containing sapindoside B as its active ingredient.

2. A glucose absorption inhibitor containing sapindoside B as the active ingredient.

3. The agent according to claim 1 or 2, wherein the aforementioned sapindoside B is included as an akebia extract.

4. The agent according to claim 3, wherein the akebi extract is an akebi leaf extract.

5. The agent according to claim 3, wherein the akebi extract is an extract obtained using an extraction solvent containing alcohol.

6. The agent according to claim 5, wherein the alcohol comprises at least one selected from the group consisting of methanol and ethanol.

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