WO2008090999A1

WO2008090999A1 - Glucosidase inhibitor

Info

Publication number: WO2008090999A1
Application number: PCT/JP2008/051142
Authority: WO
Inventors: Hiroyoshi Inoue
Original assignee: Up Well Co. Ltd.
Priority date: 2007-01-22
Filing date: 2008-01-21
Publication date: 2008-07-31
Also published as: JP4947812B2; JPWO2008090999A1

Abstract

Disclosed is an α-glucosidase inhibitor comprising a concentrated extract of a shochu residue moromi mash or a concentrated extract of a filtrate or supernatant of the shochu residue moromi mash. The α-glucosidase inhibitor may be used singly or in combination with a fibrous material as a hypoglycemic agent.

Description

Dalcosidase inhibitor Technical Field

The present invention relates to a darcosidase inhibitor.

Light

Background art

The purpose of diabetes treatment is to maintain blood glucose levels within the normal range to prevent complications. Complications include nephropathy, retinopathy, neuropathy, and stroke and myocardial infarction due to progression of arteriosclerosis. Atherosclerosis has already been shown to progress, especially from the postprandial hyperglycemic stage. Therefore, a blood glucose control that improves postprandial hyperglycemia is also important to prevent the progression of arteriosclerosis.

In order to lower postprandial hyperglycemia, fast-acting insulin secretory drugs and glucosidase inhibitors can be used. α-Dalcosidase inhibitors suppress the breakdown of starch or carbohydrates in the diet and delay the absorption of budu sugar into the body, thereby suppressing a rapid increase in blood glucose level after eating. Known α-darcosidase inhibitors include carbose, poglibose, and miglitol. '

Japanese Patent Application Laid-Open No. 2000-0 1 1 6 4 8 6 describes a food containing a large amount of lactic acid as a food for suppressing an increase in blood glucose level after a meal. In the above document, it is described that lactic acid has an a-darcosidase inhibitory action and an o-amylase inhibitory action, and further suppresses an increase in blood glucose level after meals. The above documents exemplify dairy products made using lactic acid bacteria, such as yogurt, lactic acid bacteria drinks, and sour milk drinks, as foods containing lactic acid. Also, in food production such as sake brewing, soft drinks, confectionery, etc. Lactic acid is used, but these foods He also states that no reports have been found to show the effect of suppressing the increase. Shochu is produced by fermenting starchy raw materials (cereals, potatoes) or saccharide raw materials (brown sugar, natto palm) and then distilling them. In the production process of shochu, the residue mash after distillation is usually discarded. However, environmental issues regarding the disposal of shochu residue and mash are being discussed, and their effective use is required. The power of shochu residue moromi that is known to be used as animal feed is still being researched.

Japanese Patent Application Laid-Open No. 2002-371003 describes that a barley fermented extract obtained by concentrating a filtrate of a distilled residue of barley shochu suppressed an increase in blood glucose level after a meal. However, it is described that the blood glucose level elevation inhibitory component described in the above-mentioned document is due to an insulin-like action, and no darcosidase or amylase inhibitory action was observed (paragraph number 0013).

Japanese Patent Application Laid-Open No. 2005-224205 describes a health food having a blood glucose lowering effect using buckwheat shochu. The above document, _alpha _ Darukoshida - not described with respect peptidase inhibition. Disclosure of the invention

The object of the present invention is to find out the usefulness of shochu residue mash that is normally discarded in the production of shochu.

The present invention provides an a-darcosidase inhibitor containing, as an active ingredient, a concentrated extract of shochu residue moromi or a filtrate or supernatant extract of the shochu residue moromi.

In one embodiment, the concentrated extract is a fraction having a molecular weight of 6000 or less obtained from a shochu residue mash filtrate or supernatant.

The present invention further provides a concentrated extract of shochu residue moromi or a concentrated extract of the shochu residue moromi filtrate or supernatant and a fibrous substance as an active ingredient. A depressant is also provided.

According to the present invention, a component having α-darcosidase inhibitory activity is obtained from the shochu residue mash, and an o-darcosidase inhibitor having this active component is provided. Brief Description of Drawings

FIG. 1 is a graph showing the inhibition rate of α-darcosidase activity in wheat, rice, rice bran, and buckwheat shochu residue mash supernatant fractions.

FIG. 2 is a graph showing the effect of administration of the wheat flour shochu residue mash supernatant fraction on changes in blood glucose levels of normal rats after sucrose application.

FIG. 3 is a graph showing the inhibition rate of α-darcosidase activity in the wheat bran residue residue mash supernatant fraction divided by molecular weight.

FIG. 4 is a graph showing the inhibition rate against α-darcosidase activity of the rice shochu residue mash supernatant fraction divided by molecular weight.

FIG. 5 is a graph showing changes over time in blood glucose levels of normal rats after sucrose administration by administration of a shochu residue mash mash supernatant fraction combined with a fibrous substance. FIG. 6 is a graph showing the sucrose load at various doses and the time course of blood glucose level before and after the test substance administration, 30 minutes after administration of the test substance, and 60 minutes after administration of the test substance.

FIG. 7 is a graph showing changes in blood glucose level with time when test substance O g was administered (control) and when 0.5 g was administered.

FIG. 8 is a graph showing blood insulin concentrations before and 30 minutes after administration of test substance O g (control) and 0.5 g. BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, “shochu residue moromi” means starchy raw materials (cereals, potatoes, etc.) Or moromi that remains after fermenting saccharide raw materials (brown sugar, natto palm, etc.) and then distilling to remove the alcohol. The production of shochu usually includes a starch saccharification process by koji mold, a sugar fermentation process by yeast, and an alcohol distillation process. For example, wheat or rice is treated in a conventional manner (washing, dipping, draining, steaming, allowing to cool, etc.), and this is inoculated with a seed pod of Aspergillus Kawachii that is usually used in shochu production. , And make the iron for an appropriate period at the appropriate temperature. To the koji obtained in this manner, the yeast (eg, Kagoshima yeast or Kumamoto yeast) used for normal baking production is added and mixed, and saccharified and fermented by a conventional method to obtain primary moromi. . In the primary moromi, baking yeast is propagated. Next, if necessary, add and mix the shochu main ingredients and water treated by ordinary methods (washing, dipping, draining, steaming, allowing to cool, etc.) to the primary mash as necessary. Continue saccharification and fermentation during the period to obtain secondary moromi. As the shochu main ingredient, any commonly used ingredient can be used, and examples include, but are not limited to, grains such as barley, wheat, rice, corn, and buckwheat, potatoes, brown sugar, and peanuts. Barley is preferable. In the above explanation, the yeast fermentation process (preparation) is in two stages, but it is not necessary to divide the preparation into two stages. The mash after fermentation is subjected to distillation, and the alcohol is recovered and fired. On the other hand, a residue mash from which the alcohol content has been removed is obtained.

The above shochu residue mash is concentrated as it is or separated into solid and liquid by filtration, centrifugation, etc., and then the filtrate or supernatant is heated and concentrated, freeze-dried or spray-dried to obtain a concentrated extract. obtain. Further, the concentrated extract can be used to concentrate the active ingredient using an appropriate column such as a silica gel column, an ODS column, an ion exchange resin, or an ultrafiltration membrane molecular sieve. Of these, the filtrate or supernatant fraction is preferred, and the fraction with a molecular weight of 600 or less is preferred.

In the concentrated extract, as shown in the examples described later, Inhibitory activity of lysase is observed. In the present invention, the inhibition rate of the enzyme reaction of α-darcosidase under the conditions described in Example 1 is 20 ° /. When it is above, it is defined as having a-vlucosidase inhibitory activity. The concentrated extract also suppresses an increase in postprandial blood glucose level, as shown in Examples described later. Therefore, the hypoglycemic effect of the concentrated extract is considered to be due to α-darcosidase inhibition, and the concentrated extract can be used as a ct-glucosidase inhibitor. In the concentrated extract, as shown in the Examples described later, in addition to the inhibitory activity of _darcosidase, the inhibitory activity of invertase and α_amylase is also observed. Thus, the concentrated extract or monodalcosidase inhibitor can inhibit a variety of glycolytic enzymes.

The above concentrated extract (especially the molecular weight of the shochu residue mash filtrate or supernatant 60 °

The fraction of 0 or less contains succinic acid, pyroglutamic acid, and pyruvic acid. These organic acids, namely succinic acid, pyroglutamic acid, and pyruvic acid, have _darcosidase inhibitory activity.

The concentrated extract or a-darcosidase inhibitor can inhibit the action of α-darcosidase, which degrades carbohydrates, and delays the absorption of glucose from the small intestine. It can be administered orally for the treatment or prevention of various complications such as disorders, cataracts, kidney disorders, retinopathy, joint sclerosis, atherosclerosis, diabetic lobe. The dose depends on the method of administration and the degree of symptoms, patient age, body weight, etc., but it is usually per adult dose per concentrated dose (especially the molecular weight of the filtrate or supernatant of shochu residue mash) (Fraction of 6 00 or less) may be from 0.05 g to 10 g, preferably from 0.075 g to 5 g, more preferably from 0.1 _§ to 1 _§ . This dose can be appropriately determined by a known glucose tolerance test by humans.

The concentrated extract or ο; -darcosidase inhibitor can be in the form of a tablet, powder, or liquid. It can also be added to food or beverages. Thus, a food for health maintenance having an a-darcosidase inhibitory action or a glycolytic enzyme inhibitory action as described above can also be obtained. Examples of such foods include home-use diabetic foods, liquid foods, foods for the sick (combination foods for adjusting diabetic foods, etc.), foods for specified health use, diet foods, and foods based on carbohydrates. For example, but not limited to. Specific food forms include, but are not limited to, coffee, soft drinks, soups, fruit juices, jams, biscuits, breads, and pasta. Addition or processing to foods can be performed by methods commonly used by those skilled in the art. Addition to animals other than humans, such as livestock or pet food, is also possible. The concentrated extract or α; -darcosidase inhibitor may itself be contained as a hypoglycemic agent alone or in combination with a fibrous substance. It is particularly useful for suppressing postprandial blood glucose levels. By containing a fibrous substance, the blood glucose lowering effect is further enhanced. Examples of the fibrous material include cellulose and indigestible dextrin, and preferably indigestible dextrin is used. Such a hypoglycemic agent can also be made into a food as described above.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these exemplifications. Example

In this example, shochu residue mash from sweet potato, barley, rice or buckwheat was produced as follows.

(Production Example 1: Production of wheat shochu residue moromi)

Barley is shaved with a refining machine, water is added to this and steamed, then about 35-4

It was allowed to cool to 0 ° C. In addition to this, Aspergillus Kawachii; Nonosuke Shoten) was mixed and the bacilli were propagated after 5 days. Next, appropriate amounts of water and shochu yeast were added and mixed, and fermented at about 25 to 30 ° C. for 10 days to obtain moromi. The mash that had been fermented was distilled with a pot still to remove alcohol in the mash. The mash from which the alcohol is removed is the residue mash. The residue moromi was centrifuged at 100 g at room temperature for 30 minutes, and the supernatant was collected. This supernatant was subjected to an ultrafiltration membrane device (Asahi Kasei; pen-type UF membrane), and a solution having a molecular weight of 600 or less was collected and then freeze-dried. Further, the remaining solution was separated into a fraction having a molecular weight of 500000 or less and a fraction having a molecular weight exceeding 500000, and each solution was freeze-dried. The lyophilizate was used in the following examples.

(Production Example 2: Production of rice shochu residue moromi)

Rice was shaved with a refiner, then water was added and steamed. The cooked rice was cooled to about 35 to 40 ° C. and mixed with Aspergillus Kawachii (Matsuyuki Higuchi Co., Ltd.) and allowed to propagate for 5 days. Next, an appropriate amount of mizuopi shochu yeast was added thereto and fermented at about 25 to 30 ° C. for 10 days to obtain moromi. Subsequently, a residue mash was produced in the same manner as in Production Example 1 above, fractionated according to molecular weight, and lyophilized. (Production Example 3: Production of mash shochu residue moromi)

The surface of the sweet potato was washed, then steamed with water, and then powdered. According to the description in Production Example 2 above, the seed gonococcus was planted in the rice for koji making. An appropriate amount of steamed sweet potato, water and shochu yeast were mixed with the smelted rice and fermented at about 25 to 30 ° C for 3 days to obtain moromi. Subsequently, the residue mash was produced in the same manner as in Production Example 1 above, fractionated according to molecular weight, and freeze-dried. (Production Example 4: Manufacture of soba shochu residue moromi)

The buckwheat rice was shaved with a refiner, then water was added and steamed. The steamed buckwheat rice was cooled to about 35-40 ° C and mixed with Aspergillus Kawachii (Matsunosuke Hamaguchi Co., Ltd.). To the buckwheat soba noodles, the buckwheat oat and rice of the above production examples 1 and 2 are added and mixed so that the ratio of rice: wheat: buckwheat is 1: 1: 1.2, and water is added. It was mixed with an appropriate amount of shochu yeast and fermented at about 25-30 ° C for 10 days to obtain moromi. Subsequently, the residue mash was produced in the same manner as in Production Example 1 above, fractionated according to molecular weight, and freeze-dried.

(Example 1: Effect of shochu residue moromi on a-darcosidase activity) In each of production examples 1 to 4, lyophilized products of fractions having a molecular weight of 6◦00 or less of wheat, rice, rice bran, and soba shochu residue mash As a control, 10 mg of carboxyl (trade name Darkopai (registered trademark); Bayer) was taken as l Omg and dissolved in 1 mL of 0.02 M phosphate buffer to obtain a test substance solution. In order to investigate the effect of the test substance on α-darcosidase activity, a reaction solution having the following composition was prepared: 0.4% ρ-Nitrophenyl a-D-darcoviranoside (Wako Pure Chemical Industries) 0.2 mL; Substance solution 0.2 mL; and 0.5 U / mL α-Dalcosidase (Toyobo) 0.1 mL. Here, one unit (U) of ct-dalcosidase is the amount of enzyme that liberates 1 μπιοΐ of glucose per minute from the non-reducing terminal side of the substrate under the following standard reaction conditions. The above reaction solution was incubated at 37 ° C for 15 minutes to allow the enzyme reaction to proceed. The reaction was stopped by adding 0.5 mL of 2M Tris solution (pH 7.0). 0.02 mL of the solution after stopping the reaction is taken, and the coloring reagent (glucose CII test kit; Wako Pure Chemical Industries, Ltd.) 3. Add (mL) and mix for 5 minutes at 37 ° C. And the absorbance was measured with a spectrophotometer (Beckman) at 500 nm. All measurements were performed twice.

Fig. 1 is a graph showing the inhibition rate of these shochu residue moromi to α-darcosidase activity. The results of carbose, which is known to inhibit ct-dalcosidase, are also shown. The vertical axis represents the inhibition rate (%) of α-darcosidase activity. All shochu residue and moromi showed inhibition of α-darcosidase activity, and in particular, wheat shochu residue and mash showed inhibition of more than 60%.

(Example 2: Effect of shochu residue moromi on postprandial blood glucose level)

Seven-week-old normal male rats (Kudo Co., Ltd.) were fasted for 12 hours. After fasting, the control group was orally ingested with 2 g / kg of sucrose, and the cellulose-administered group was orally ingested with 2 g / kg of sucrose and 2 Omg / kg of cenorelose was intragastrically administered. In addition, 2 Omg / kg of carbose was administered intragastrically with 2 gZkg of sucrose, and the residue of the burning residue was mixed with the supernatant fraction. 20 mgZkg of a lyophilized fraction having a molecular weight of 6 000 or less was administered intragastrically. There were 5 animals in each group. Blood glucose levels were measured before treatment, 30 minutes, 60 minutes, and 120 minutes after treatment. The blood glucose level here is the serum glucose concentration.

FIG. 2 is a graph showing the change in blood glucose level over time in each treatment group after sucrose application. The horizontal axis represents the postprandial time (minutes), and the vertical axis represents the blood glucose level (serum darose concentration: mgZmL). In the figure, the black circle is the control group, the white circle is the cellulose administration group, the white triangle is the carbose administration group, and the white square is the shochu residue mash supernatant fraction administration group. In the control group in which sucrose was orally administered, the blood glucose level rapidly increased by 30 minutes after the treatment, and gradually decreased thereafter. The cellulose administration group showed the same tendency as the control group. In contrast, in the carbohydrate administration group and in the shochu residue mash supernatant fraction administration group, blood was 30 minutes after treatment. The sugar level increased, but the increase was significantly lower than that of chondrol (in Fig. 2, **; r <0. 05).

(Example 3: Inhibition of barley shochu residue moromi a-darcosidase activity)

The molecular weight of the wheat shochu residue moromi obtained in Production Example 1 is not more than 600.000; the freezing of the fraction having a molecular weight of more than 60.000 and less than 5.000; and a molecular weight of more than 500.000 Using the dried product, the effect on the single dalcosidase activity was examined according to the procedure described in Example 1 above.

FIG. 3 is a graph showing the inhibition rate for the a-darcosidase activity of the mash supernatant fraction of the wheat shochu residue. The vertical axis represents the inhibition rate (%) of a-darcosidase activity. In the figure, the fraction with a molecular weight of 6 0 0 0 or less is MW <6 0 0 0; the fraction with a molecular weight of more than 6 0 0 0 but less than 5 0 0 0 0 is 6 0 0 0 MW <5 0 0 0 0; and a fraction having a molecular weight exceeding 5 0 0 0 0 is represented by 5 0 0 0 minus MW. In any fraction, inhibition of a-darcosidase activity was shown, but the inhibition rate was particularly high in fractions having a molecular weight of 600 or less.

Example 4: Rice shochu residue moromi-darcosidase activity inhibition

The molecular weight of the rice shochu residue moromi obtained in Production Example 2 is not more than 600.000; the freezing of the fraction having a molecular weight of more than 60.000 and less than 5.000; and a molecular weight of more than 500.000 Using the dried product, the effect on a-darcosidase activity was examined according to the procedure described in Example 1 above.

FIG. 4 is a graph showing the inhibition rate for the one dalcosidase activity of the mash residue obtained from the rice baking residue. The vertical axis represents the inhibition rate (%) of a-darcosidase activity. In the figure, the fraction with a molecular weight of 6 0 0 0 or less is MW <6 0 0 0; the fraction with a molecular weight of more than 6 0 0 0 but less than 5 0 0 0 0 is 6 0 0 0 MW + 5 0 0 0 0; and fractions with molecular weights greater than 5 0 0 0 0 at 5 0 0 0 0 <MW To express. In all fractions, including those with a molecular weight of 6 000 or less, 50 ° /. Inhibition of a-darcosidase activity was observed.

(Example 5: Effect of shochu residue mash combined with fibrous substance on postprandial blood glucose level)

Seven-week-old normal male rats (Kudo Co., Ltd.) were fasted for 12 hours. After fasting, the sucrose intake group was orally given 2 g / kg sucrose and nothing else was administered. To the shochu residue mash supernatant fraction administration group, 2 gZk g of sucrose was orally ingested, and 5 mg Zkg of the wheat shochu residue mash fraction lyophilized fraction of the above production example 1 was intragastrically administered. In addition to the oral intake of 2 g / kg sucrose, the cellulose-added group includes 5 mg Zkg of the lyophilized fraction of the malt residue of the wheat shochu residue of Production Example 1 above, with a molecular weight of 600 ◦ or less. In the group to which indigestible dextrin was added, the fraction freeze-dried product having a molecular weight of 600 000 or less and 5 times the amount of difficult Digested dextrin was mixed and administered into the stomach. There were 5 animals in each group. The blood glucose level was measured before the treatment and 30 minutes after the treatment. The blood glucose level here is the serum glucose concentration. FIG. 5 is a graph showing the change in blood glucose level over time in each treatment group after sucrose application. The vertical axis represents the blood glucose level (serum glucose concentration · mgZmL). In the figure, the white circle is the sucrose intake group, X is the group administered with the freeze-dried fraction with a molecular weight of 6000 or less of the barley burning residue mash (the barley shochu residue mash supernatant fraction administration group), the white triangle is the above production example A group in which cellulose was added together with a fraction freeze-dried fraction with a molecular weight of 6000 or less of the wheat shochu residue mash (cellulose addition group). And the white square is a fraction with a molecular weight of 6000 or less of the mash of the wheat shochu residue in Production Example 1 above. This is a group to which indigestible dextrin is added together with the lyophilized product (indigestible dextrin added group). Cellulose added group and indigestible dextrin added group are: Compared with the clear fraction administration group, the increase in blood glucose level after meal was further suppressed. In particular, the combination of indigestible dextrin and the wheat flour shochu residue mash supernatant fraction showed an excellent hypoglycemic effect. (Example 6: Composition analysis of shochu residue moromi)

Dissolve 2. O g of the lyophilized fraction of the molecular weight of the wheat shochu residue moromi of Production Example 1 in a fraction of 600 or less in ultrapure water (millQ water), silica gel (Silica Gel 60N (sph erical, neutral), 63-210 μm; Kanto Chemical Co., Ltd.) l O g was adsorbed and dried. A column of 2 cm X 60 cm with a filter (No. 2) (VIDTEC) was packed with about 50 g of the above-mentioned siri-force gel, and the above-mentioned wheat burning residue adsorbed with silica gel was placed on the mash. Single solvent (CHC1 _3: methanol: 0 = 5: ^3: 0.4) flowed was fractionated. Each fraction was subjected to thin layer chromatography (TLC: Partisil (registered trademark) K5F Silica Gel 150 A, 20 X 20 cm, Whatman), and with a color former (p-anisaldehyde, ethanol solution, Tokyo Chemical Industry Co., Ltd.) Fog and heated to confirm spot. Spots with the same R f value were collected, concentrated and further purified by high performance liquid chromatography (HPLC).

HPLC was performed under the following conditions:

Equipment used: High-speed liquid mouthmatography SHIMADZU LC-10A

Pump: LC-10AD X 2 units

Detector: CDD-6A (conductivity detector)

Controller: SCL—10A

Column oven: CT0-10A

Autoinjector: SIL-10A

Chroma knock:

Column: Shim- Pack SCR-102H 300 X 8mm (inner diameter) (Shimadzu Corporation)

Mobile phase: 5 mM p-tonoreens norephonic acid

Flow rate: 0.8mLZ min

Temperature: 45 ° C

Analysis time: 40 minutes

Detection: conductivity

Detection is by column column loose seed purification method.

Buffer: 20 mM Bis-Tris solution with 5 mM p-toluenesulfonic acid and 100 μΜ EDTA

Flow rate: 0.8mL / min

Polarity: +

Response: slow

The specimen was prepared by dissolving Sample 2. Omg in 2000 μL of millQ water and filtering it through a 0.45 μπι membrane filter. The injection volume was 10 μL.

As a result of HPLC, peaks of fractions 1-2 to 1-3 were obtained. The obtained peak was compared with the peak of the standard solution chart to determine the component and its content. To prepare the standard solution chart, the following organic acid standard solutions were used and 10 μL was injected: phosphoric acid (546. 9 mg / L), citrate (220. 6 mg / L), malic acid (220 4 mg / L), succinic acid (298. 8 mg / L), lactic acid (606.0 mg / L), acetic acid (575. 9 mg / L), pyroglutamic acid (288. 5 mg / L), pyruvic acid (218. 4mg / L). As a result, it was found that 1 mg of fr 1 1-2 1-2 1 contained succinic acid 0 · 693 nx g, pyroglutamic acid 0.068 mg, and pyruvic acid 0.004 mg (about The remaining 25% is unknown). Furthermore, according to the procedure described in Example 1 above, the effects of succinic acid, pyroglutamic acid, and pyruvic acid on the activity of one darcosidase were examined. It was confirmed to have an inhibitory activity (succinic acid: IC 50 4.99 mg / mL, and pyroglutamic acid: IC 50 7.38 mgZmL, pyruvate: IC 50 4.84 mgZmL).

(Example 7: Effect of shochu residue moromi on activity of various glycolytic enzymes) (7-1. Invertase (sucrase) inhibitory activity)

Add the sample solution to the various concentrations by adding the fraction 1O OmgZmU O. 1M acetic acid buffer (pH 5.0) (also used as a solvent in the following solutions) to the molecular weight of the wheat shochu residue mash in Production Example 1 below 6000 It was adjusted. A 5 ° / ₀ sucrose solution was used as a substrate, and 0.5 U / mL invertase was used as an enzyme solution. The substrate (0.2 mL), the enzyme solution (0.1 mL), and the sample solution (0.2 mL) were mixed and incubated at 37 ° C for 15 minutes. Thereafter, the reaction was stopped by heating in a water bath at 90 ° C or higher for 10 minutes. Take 0.05 mL of the solution after the reaction and add a coloring reagent (glucose CII test kit; Wako Pure Chemicals) 3. Add OmL, mix, incubate at 37 ° C for 5 minutes, and place in spectrophotometer (Beckman). The absorbance was measured at 5 05 nm. All measurements were performed twice. As a result, inhibition of enzyme activity was observed as the concentration of the sample solution increased. When the concentration of the sample solution was 40 mg / mL, the inhibition rate was about 70%.

(7-2. Amylase inhibitory activity)

Using an amylase measurement kit (Kikkoman), the protocol was modified as follows, and α-amylase inhibitory activity was measured. The sample solution was adjusted to various concentrations by adding 10 mM acetate buffer (pH 5.0) (also used as a solvent in the following solutions) to fraction 10 OmgZmL of molecular weight of 6000 or less of the wheat shochu residue mash in Production Example 1. did. 2-Chloro-4-nitroph as substrate; Nyl-6-azido-6-deoxy-maltopentaoside solution, Glue as coloring enzyme solution As a solution of core amylase and / 3-darcosidase and a test enzyme solution, a 0.28 U / mL Aspergillus oryzae a-amylase solution was prepared.

First, add 25 L of the substrate solution and 25 μL of the coloring enzyme solution to the plate, then add 25 μL of the sample solution, and then add 25 L of the α-amylase solution of the test enzyme solution. Incubated at ° C for 15 minutes. Thereafter, 100 sodium carbonate solution was added to stop the reaction. Absorbance was measured at 400 nm with a spectrophotometer (Beckman) using 405 nm as a plate reader. All measurements were performed twice.

As a result, inhibition of enzyme activity was observed as the concentration of the sample solution increased. As a control, carbose (trade name Dalcobay (registered trademark); Bayer) exhibited an α-amylase activity inhibition rate of about 50.5% at a concentration of 0.003 mgZmL. In the sample solution, the inhibition rate was about 43% at a concentration of 5 mg ZmL. Compared with carbose, _α -amylase inhibitory activity was remarkably low. (Example 8 _: Effect of shochu residue moromi on postprandial blood glucose level) '

Volunteers were recruited to examine the effects of shochu residue moromi on postprandial blood glucose levels, and 7 men and women aged 20 to under 50 years were registered (1 male; 6 females; average age of all 33.4 + 6) 6 years old). The registration was conducted under the condition that there was no history of diabetes and that the patient had no special illness at the time of the examination, and received a comprehensive diagnosis by the responsible physician. Special diseases include 1) severe liver / kidney-cardiac dysfunction, 2) drug sensitivity, 3) mental or mental disabilities that lack judgment ability, 4) pregnant women, lactating women and this study 5) Others who are determined to be inappropriate for this study by the attending physician.

As a test substance, a fraction having a molecular weight of 6000 or less of the wheat shochu residue mash of Production Example 1 was used. Subjects fasted for 10 hours before the start of the test (free drinking), and at the start of the test, 25 g of sucrose (Wako Pure Chemical Industries, Ltd.) was loaded at the same time as O g (control), 0. lg or 0.5 g test substance was taken and administered. Sucrose loading was measured before administration of the test substance, and 30 and 60 minutes after administration of the test substance. At the time of O _g administration (control) and 0.5 g administration, blood was collected for blood tests before and 30 minutes after administration. Each test was performed at intervals of 24 hours or longer, and excessive suppression of food intake was avoided.

For all test items, mean values and standard deviations were obtained, and differences before and after administration were subjected to Student t test. In all cases, the significance level was 5% or less.

Figure 6 shows the results of blood glucose levels related to dose effects. In Fig. 6, the measured values are expressed as relative values with the blood glucose level before sucrose loading as 100%. In the figure, black circles represent controls, triangles represent test substance 0.1 g administration, and white circles represent test substance 0.5 g administration. With 25 g sucrose loading, the test substance O g administration (control) increased the blood glucose level by approximately 30% after 30 minutes, and remained elevated by more than 10% after 60 minutes. In contrast, 0.1 g of the test substance showed significantly lower values than the control at both 30 minutes and 60 minutes after administration (P 0.05). The amount of the test substance was increased to 0.5 g, but there was no difference from the case of 0.1 lg. This is thought to be because it is difficult to see the dose-response relationship because the blood glucose elevation level was suppressed to an increase of about 10% after administration of 0.1 g. On the day different from the test of FIG. 6 above, changes in blood glucose level over time were measured at the time of test substance O g administration (control) and 0.5 g administration. The results are shown in FIG. 7 in terms of relative values with the blood glucose level before sucrose loading as 100%. In the figure, black circles represent controls, and white circles represent 0.5 g of the test substance. 30 minutes after administration, the increase in blood glucose of about 30% caused by loading with 25 g sucrose was reduced to about 16% by administration of 0.5 g of the test substance. However, no statistically significant difference was observed between the two groups. In both groups, blood glucose levels returned to fasting levels (before sucrose loading) 120 minutes after administration. Furthermore, blood was collected at the time of administration of the test substance O g (control) and at the time of 0.5 g, at 30 minutes before administration and at 30 minutes after administration, and the blood insulin concentration was measured. The results are shown in Fig. 8. The blood insulin concentration increased with the 25 g sucrose load, but the test substance did not affect this increase in insulin concentration. Industrial applicability

The _α -darcosidase inhibitor of the present invention is useful for the treatment or prevention of diabetes and its complications. In addition, according to the present invention, it is possible to effectively use the residue of fired residue that is normally discarded in the production of shochu, where environmental problems have been discussed.

Claims

The scope of the claims

1. Shochu residue _ Darukoshidaze inhibitors _α comprising as an active ingredient concentrated Ekisu or the shochu residue mash filtrate or supernatant in the concentrated extract of the mash.

2. The a-darcosidase inhibitor according to claim 1, wherein the concentrated extract is a fraction having a molecular weight of 600 or less obtained from a shochu residue mash filtrate or supernatant.

3. A hypoglycemic agent comprising, as an active ingredient, a concentrated extract of shochu residue mash or a filtrate or supernatant extract of the shochu residue mash and a fibrous substance.