a-GLUCOSIDASE INHIBITOR, INVERTASE INHIBITOR, AND SUGAR ABSORPTION INHIBITOR

Abstract

[Object] To provide a composition having an excellent α-glucosidase inhibitory effect or invertase inhibitory effect.

[Solution] A compound (I) contains a compound represented by a structural formula described below as an active ingredient,

##STR00001##

Claims

1. An α-glucosidase inhibitor comprising: a compound (I) represented by a structural formula described below as an active ingredient, ##STR00006##

2. The α-glucosidase inhibitor according to claim 1, which is obtained from sap of a tree belonging to a genus of Acer in a family of Aceraceae.

3. The α-glucosidase inhibitor according to claim 2, wherein the tree belonging to the genus of Acer in the family of Aceraceae is at least one species selected from the group consisting of a sugar maple, a painted maple, a black maple, a red maple, a silver maple, a striped maple, a mountain maple, and a Norway maple.

4. The α-glucosidase inhibitor according to claim 1, which inhibits an enzyme activity of maltase.

5. The α-glucosidase inhibitor according to claim 1, which inhibits an enzyme activity of isomaltase.

6. The α-glucosidase inhibitor according to claim 1, which inhibits an enzyme activity of sucrase.

7. A food comprising: the α-glucosidase inhibitor according to claim 1.

8. An invertase inhibitor comprising: a compound (I) represented by a structural formula described below as an active ingredient, ##STR00007##

9. The invertase inhibitor according to claim 8, which is obtained from sap of a tree belonging to a genus of Acer in a family of Aceraceae.

10. The invertase inhibitor according to claim 9, wherein the tree belonging to the genus of Acer in the family of Aceraceae is at least one species selected from the group consisting of a sugar maple, a painted maple, a black maple, a red maple, a silver maple, a striped maple, a mountain maple, and a Norway maple.

11. A food comprising: the invertase inhibitor according to claim 8.

12. A saccharide absorption inhibitor comprising: a compound (I) represented by a structural formula described below, ##STR00008##

13. A saccharide composition comprising: the saccharide absorption inhibitor according to claim 12; and sucrose.

14. A food comprising: the saccharide absorption inhibitor according to claim 12.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0029] FIG. 1 illustrates results obtained by performing PMP derivation with respect to maple syrup and performing capillary electrophoresis.

[0030] FIG. 2 illustrates results obtained by performing the PMP derivation with respect to invertase-digested maple syrup and performing the capillary electrophoresis.

[0031] FIG. 3 illustrates results obtained by performing the PMP derivation with respect to the invertase-digested maple syrup, adding invertase further, and performing the capillary electrophoresis.

[0032] FIG. 4 illustrates results obtained by ultra-filtrating maple syrup and performing HPLC.

[0033] FIG. 5 illustrates results obtained by performing the HPLC after performing acid-hydrolysis with respect to a fraction indicated by a * mark in FIG. 4 in the HPLC.

[0034] FIG. 6 illustrates screening results of inhibitory enzymes by a compound (I).

[0035] FIG. 7 illustrates changes with time of plasma glucose and insulin when normal rats were orally administered only with sucrose and when the normal rats were orally co-administered with sucrose and the compound (I).

[0036] FIG. 8 illustrates changes with time of plasma glucose and insulin when OLETF rats were orally administered only with sucrose and when the OLETF rats were orally co-administered with sucrose and the compound (I).

DESCRIPTION OF EMBODIMENTS

Examples

[0037] Hereinafter, the present invention is described in detail with reference to examples. It is a matter of course that the present invention is not limited to the examples described below.

[0038] [α-Glucosidase Inhibition]

[0039] Hereinafter, an α-glucosidase inhibitory effect was evaluated with respect to a compound (I).

[0040] [PMP Derivatization Processing of Sample]

[0041] The compound (I) was obtained from sap of trees belonging to a genus of Acer in a family of Aceraceae and maple syrup (BASCOM MAPLE FARMS INC.: hereinafter, also simply referred to as “sap and the like”). Specifically, 50 μL of 0.3 mol/L sodium hydroxide solution and 50 μL of 0.5 mol/L 1-phenyl-3-methyl-5-pyrazolone (hereinafter also referred to as “PMP”: manufactured by Kishida Chemical Co., Ltd.) methanolic solution were added to a dried sample of sap equivalent to 200 μL (10 μL of maple syrup or 10 mg of maple sugar), and the mixed liquid was heated at 70° C. for 30 minutes. The heated mixed liquid was neutralized by adding 50 μL of 0.3 mol/L hydrochloric acid, diluted with 100 μL of distilled water, and extraction was performed three times with 200 μL of chloroform to remove an excessive PMP reagent. By this process, PMP-derivatives for capillary electrophoresis were obtained.

Purification of Compound (I)

[0042] The sap and the like were ultra-filtrated with a 10 kDa filter to remove proteins, and the obtained filtrate was gel-filtrated for performing further molecular weight fractionation. Gel filtration was performed using water as a mobile phase using Sephadex G-15 having a length of 1000 mm and an internal diameter of 28 mm, and fractions were collected by a fraction collector (Model 2110 manufactured by Bio-Rad Laboratories, Inc.). The obtained fractions were purified by high performance liquid chromatography (hereinafter, also referred to as “HPLC”). The peak corresponding to the compound (I) was observed between 32 and 33 minutes, and a fraction containing the compound (I) in high purity was collected with the peak as a criterion. The obtained solution was liophylized and used as a standard substance. 100 μg of the compound (I) was dissolved in 100 μL of water, and then the HPLC was performed.

[0043] [Capillary Electrophoresis]

[0044] Agilent 3D capillary electrophoresis system (Model G1600A manufactured by Waldbronn) equipped with a diode array UV detector was used. Samples were injected under a pressure of 50 mbar for 4 seconds. Separation was performed in a fused silica capillary column (manufactured by GL Science Inc., total length: 58.5 cm, effective length: 50 cm, internal diameter: 50 μm) having a non-treated inner surface. 200 mmol/L of a borate buffer solution for a background electrolyte (BGE) was obtained by adding pellets and 0.1 mol/L of a sodium hydroxide aqueous solution to a borate aqueous solution having the concentration slightly higher than 200 mmol/L, adjusting the pH to 10.5 using a pH meter, and adjusting the volume to 200 mmol/L using a volumetric flask. A voltage of 15 kV was applied to both ends of the capillary. Before injecting each sample, the capillary was conditioned by continuous rinsing with 0.5 mol/L sodium hydroxide for one minute and with the BGE for five minutes using a flush mode of the system. Detection was performed by monitoring UV absorption at 245 nm. Measurement was performed at 25±1° C.

[0045] [HPLC]

[0046] An HPLC system is constituted by a pump (Model LC-10AD manufactured by Shimadzu Corporation), a degasser (Model DGU-12A manufactured by Shimadzu Corporation), and a corona Veo detector (manufactured by Thermo Fisher Scientific, Inc.). Asahipak NH2P-50 4E column (5 μm, 4.6 mm internal diameter×250 mm, manufactured by Showa Denko K.K.) was used, and acetonitrile/water (3:1; v/v) was used as a mobile phase. Elution was performed at a flow rate of 1 ml/min at room temperature (about 23° C.). 20 μL of the sample was injected. In purification and fractionation, Asahipak NH2P-50 column (5 μm, 10.0 mm internal diameter×250 mm, manufactured by Showa Denko K.K.) was used and the flow rate was set to 2 mL/min. Using an adjustable splitter (manufactured by Thermo Fisher Scientific, Inc.) and setting the split ratio to 1:20, detection was performed at a low flow rate, and fractionation was performed at a high flow rate.

[0047] [Structural Analysis of Compound (I)]

[0048] LC-ESI-MS/MS analysis was performed using Finnigan LTQ linear ion trap mass spectrometer (manufactured by Thermo Fisher Scientific, Inc.) equipped with an ES ion source, Paradigm MS4 pump (manufactured by Michrom Bioresources Inc.), and an autosampler (HTCPAL, CTC Analytics). The conditions for ionization were as follows.

[0049] Ion source voltage: 4.5 kV

[0050] Capillary temperature: 275° C.

[0051] Capillary voltage: 25 V

[0052] Sheath gas (N2 gas): Flow rate of 50

[0053] Auxiliary gas (N2 gas): Flow rate of 5

[0054] Tube lens offset voltage: 90 V

[0055] Helium gas was used as a collision gas for Collision Induced Dissolution (CID) analysis. The normalized collision energy and the activation Q value were set to 35% and 0.18, respectively. As an LC column, TSK gel ODS-100S (manufactured by Tosoh Corporation, 5 μm, 150 mm×2.0 mm internal diameter) was used. .sup.1H and .sup.13C-NMR were obtained using JNM-ECA800 instrument operating at 800 MHz and 200 MHz, respectively. NMR measurement samples were dissolved in heavy water.

[0056] [Invertase Digestion of Maple Syrup]

[0057] Enzyme reaction was performed by adding 40 μL of 5 mmol/L acetate buffer solution with the pH of 4.5 and 5 μL of 100 U/mL invertase to 10 mg of maple syrup, and incubating the mixture at 37° C. for 30 minutes. The reaction mixture was heated in a water bath for one minute to inactivate the enzyme. After evaporated and dried at room temperature, an invertase-digested substance was PMP-derivatized under the same conditions as those of an undigested sample. 50 μL of the PMP-derivatives were evaporated and dried, and then redissolved in 45 μL of 5 mmol/L acetate buffer solution with the pH of 4.5, and pre-incubation was performed for five minutes. Then, 5 μL of 100 U/mL invertase solution was added, and incubation was performed for 15 minutes. The reaction liquid was heated in a water bath for one minute to inactivate the enzyme, and analyzed by the capillary electrophoresis.

[0058] [Analysis of Invertase Inhibition by Compound (I)]

[0059] For invertase inhibition analysis, 100 μg of sucrose as a substrate and 1 μg, 10 μg, or 100 μg of the compound (I) being the invertase inhibitor were added to 50 μL of 100 mmol/L acetate buffer solution, and pre-incubation was performed at 37° C. for five minutes. Thereafter, 50 μL of 0.2 U/mL invertase solution was added, and incubation was performed for 15 minutes. Thereafter, 10 μL of the reaction mixture was heated in a water bath to inactivate the enzyme and evaporated and dried, and the PMP derivatization was performed. A substance subjected to the enzyme reaction under the same conditions without adding the inhibitor was used as a blank. In each sample, the concentration required for the compound (I) to inhibit 50% of the enzyme was defined as IC.sub.50. An inhibition rate by the compound (I) was calculated using a following equation.

Inhibition rate (%)=[D1−(D2−D3)/1]×100  (Equation 1)

[0060] D1: Peak area of glucose in blank sample

[0061] D2: Peak area of glucose in each sample after enzyme reaction

[0062] D3: Peak area of glucose contained in inhibitor as an impurity

[0063] The IC.sub.50 value was calculated from a dose-response curve of the compound (I) used as the inhibitor.

[0064] [Screening of Inhibition Analysis by Compound (I)]

[0065] As a crude enzyme mixture, 50 mg of rat intestinal acetone powder was added to 450 μL of 50 mmol/L phosphate buffer solution (pH 6.0), the mixture was stirred for 30 seconds, and then homogenized. Thereafter, the mixture was centrifuged (10000 rpm, 4° C., 20 minutes), and the supernatant was used as a purified enzyme (50 mg/450 μL). Sucrose and two types of glucose disaccharides (maltose and isomaltose) having different bonds were used as substrates. 3.4 mg of each of the two types of substrates was dissolved in 100 μL of phosphate buffer solution, and each solution was used as a substrate solution. A solution obtained by dissolving 3.4 mg of the compound (I) as a competitive inhibitor in 1 mL of phosphate buffer solution and diluting 100 times, was used as an inhibitor solution. 100 μL of the substrate solution and 10 μL of the inhibitor solution were mixed, and pre-incubation was performed for five minutes. Then, 90 μL of an enzyme solution was added to start incubation. Five hours later, 10 μL of the reaction solution was heated in a water bath for 10 minutes to stop the reaction, followed by the PMP derivatization, and then the capillary electrophoresis. An enzyme reaction solution under the same conditions without the inhibitor was used as a blank. The inhibition rate was calculated for each substrate using the same equation as that in the invertase inhibition analysis.

[0066] [Oral Glucose Tolerance Testing (OGT Testing) Using Sucrose]

[0067] Glucose tolerance testing of normal Wistar type rats and OLETF rats with diabetes was performed using the compound (I). After fasted for 14 hours, glucose was loaded, and blood taken from tail veins of the rats was subjected to various tests.

[0068] An aqueous solution containing 0.5 mg/ml of sucrose (hereinafter, also referred to as “A Liquid”) and an aqueous solution containing 0.5 mg/ml of sucrose and 0.085 mg/ml of the compound (I) (hereinafter, also referred to as “B Liquid”) were prepared. Six normal 7-week-old rats (male) were divided into two groups. In one group, the A liquid was orally administered such that an amount of sucrose was 1.5 g/kg based on the rat body weight. In the other group, the B liquid was orally administered such that the amount of sucrose was 1.5 g/kg based on the rat body weight. Blood was taken from the tail veins of the rats before the administration and in 30 minutes, 60 minutes, 90 minutes, and 120 minutes after the administration, and then plasma was obtained by centrifugation. For the obtained plasma, glucose and insulin were quantified.

[0069] [Evaluation]

[0070] FIG. 1 illustrates results obtained by performing the PMP derivation with respect to the maple syrup and analyzing by the capillary electrophoresis. As a result, a plurality of peaks was detected, and each peak was identified as glucose, xylose, arabinose, mannose, and ribose. Further, the compound (I) indicated by a * mark was identified.

[0071] FIG. 2 illustrates results of the capillary electrophoresis with respect to the invertase-digested maple syrup. As a result of invertase-digesting the maple syrup, peak areas of glucose, xylose, and the compound (I) (* mark) increased as compared with those in the results obtained in the invertase-undigested maple syrup (FIG. 1).

[0072] FIG. 3 illustrates results obtained by further adding invertase to the invertase-digested maple syrup and similarly analyzing by the capillary electrophoresis. When invertase was added to the invertase-digested maple syrup, the peak area of the compound (I) decreased, but significant changes were not observed in the peak areas of other saccharides. Based on this result, the compound (I) was presumed to interact with invertase.

[0073] FIG. 4 illustrates results obtained by ultra-filtrating the maple syrup and analyzing by the HPLC. With respect to the results in FIG. 4, peaks of sucrose, fructose, and glucose were identified using standard substances. A fraction indicated by the peak (* mark) between 16 to 17 minutes was fractionated, PMP-derivatized, and then analyzed by the capillary electrophoresis. As a result, the substance contained in the peak between 16 to 17 minutes in the HPLC coincided with the peak of oligosaccharide interacting with invertase.

[0074] FIG. 5 illustrates results obtained by analyzing by the HPLC after performing acid-hydrolysis with respect to the fraction indicated by the * mark in the HPLC. Two major peaks illustrated in FIG. 5 were confirmed to correspond to fructose and glucose. Since the two peak areas were almost equal, the compound (I) was presumed to be disaccharide containing fructose and glucose.

[0075] As a result of analyzing the PMP-derivatives of the compound (I) by the LC-ESI-MS/MS and measuring the molecular weight, the mass was m/z 673.26 as [M+H].sup.+, which coincided with the mass of PMP-derivatized disaccharide. The product ion was m/z 511.33, which coincided with the mass of PMP-derivatized monosaccharide.

[0076] Table 1 illustrates results of analyzing the compound (I) by the NMR. Based on this chemical shift, it was concluded that the compound (I) had a structure represented by a structural formula described below.

TABLE-US-00001 TABLE 1 Chemical shift Oligosaccharide Residu Position δH δC J H, H Type Residu Position H (proton) C (carbon13) J (Hz) Type Glc α 1.sup.  5.07 94.72 3.8 d Glc β 1.sup.  4.49 98.57 8 d 2.sup.  3.38 74.05 3.8, 9.8 dd 2.sup.  3.09 76.67 8.0, 9.3 dd 3.sup.  3.54 75.25 — m 3.sup.  3.33 78.23 9.3, 9.3 dd 4.sup.  3.29 72.32 9.0, 9.0 dd 4.sup.  3.29 72.25 9.0, 9.0 dd 5.sup.  3.77 73.27 1.9, 9.2, 9.7 ddd 5.sup.  3.40 77.58 2.1, 8.7, 9.7 ddd 6.sup.  3.82 63.38  2.0, 11.0 dd 6.sup.  3.87 63.38  2.1, 10.9 dd Fru β 1′ 3.60 62.75 — m Fru β 1′ 3.54 62.72 — m 2′ — 106.31 — — 2′ — 106.34 — — 3′ 4.03 79.42 8.5 d 3′ 4.02 79.52 8.5 d 4′ 3.97 77.06 8.1, 8.2 dd 4′ 3.96 77.21 8.0, 8.3 dd 5′ 3.72 83.75 7.4 t 5′ 3.72 83.81 7.4 t 6′ 3.54 64.98 — m 6′ 3.66 65.10 — m

##STR00005##

[0077] Table 2 illustrates results of invertase inhibition analysis by the compound (I). The inhibition rates when 1 μg, 10 μg, and 100 μg of the compound (I) were added were 40.3%, 43.6%, and 65.2%, respectively, and a linearity (R.sup.2=0.99984) was observed between the concentration of the compound (I) and the inhibition rate. The IC.sub.50 calculated using the straight line was 1.17 mmol/L.

TABLE-US-00002 TABLE 2 Table. 2. Inhibitory effects of maplehiose against glycolytic enzyme inhibitor (μg) Peak response (Glc) Peak response (b-Glc) Inhibition (%) IC50 (mmol/1) invertase — 30.5 — 1 18.2 N.D 40.3 1.17 10 17.2 N.D 43.6 100 23.1 N.D 65.2 Maltase — 44.3 — α-(1-4)glucosidase 1 27 N.D 39.1 1.72 10 22.4 N.D 49.4 100 20.3 N.D 54.2 N.D = Not detectable

[0078] FIG. 6 illustrates screening results of an enzyme inhibitory activity by the compound (I). The inhibition rates by the compound (I) when sucrose, maltose, and isomaltose were used as the substrate were 12.3%, 9.4%, and 3.3%, respectively. Table 2 illustrates results of analyzing maltase inhibition by the compound (I). The inhibition rates when 1 μg, 10 μg, and 100 μg of the compound (I) were added were 39.1%, 49.4%, and 54.2%, respectively. The IC.sub.50 calculated using a straight line calculated from the concentrations and the inhibition rates of the compound (I) was 1.72 mmol/L.

[0079] FIG. 7 illustrates changes in plasma glucose and insulin when normal rats were orally co-administered with sucrose and the compound (I). Changes with time of insulin were similar regardless of the presence or absence of the compound (I). However, the plasma glucose value was significantly lower in the rats administered with the compound (I) than in the rats not administered with the compound (I). In FIG. 7, the compound (I) is indicated as Maplebiose.

[0080] FIG. 8 illustrates changes in plasma glucose and insulin when OLETF rats with diabetes were orally co-administered with sucrose and the compound (I). Changes with time of insulin were similar regardless of the presence or absence of the compound (I). However, the plasma glucose value in the rats administered with the compound (I) decreased to about 50% of that in the rats not administered with the compound (I). In FIG. 8, the compound (I) is indicated as Maplebiose.