JPWO2008090999A1 - Glucosidase inhibitor - Google Patents

Abstract

The present invention provides an α-glucosidase inhibitor comprising a concentrated extract of shochu residue mash or a concentrated extract of the shochu residue mash filtrate or supernatant. The α-glucosidase inhibitor of the present invention can be used as a hypoglycemic agent alone or in combination with a fibrous substance.

Description

  The present invention relates to a glucosidase inhibitor.

Diabetes treatment is aimed at maintaining blood glucose levels in the normal range to prevent complications. Complications include nephropathy, retinopathy, neuropathy, and stroke and myocardial infarction due to the progression of arteriosclerosis. It has been found that arteriosclerosis has already progressed, especially from the postprandial hyperglycemic stage. Therefore, glycemic 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 α-glucosidase inhibitors can be used. The α-glucosidase inhibitor suppresses the decomposition of starch or carbohydrates contained in the meal and delays the absorption of glucose into the body, thereby suppressing the rapid increase in blood glucose level after the meal. As an α-glucosidase inhibitor, acarbose, voglibose, and miglitol are known.
Japanese Patent Application Laid-Open No. 2003-116486 describes a food containing a large amount of lactic acid as a food that suppresses an increase in blood glucose level after a meal. The above document describes that lactic acid exhibits an α-glucosidase inhibitory action and an α-amylase inhibitory action, and further suppresses an increase in blood glucose level after a meal. The above document exemplifies dairy products made using lactic acid bacteria, such as yogurt, lactic acid bacteria beverages, sour milk beverages, as foods containing lactic acid, and also in food production such as sake brewing, soft drinks, confectionery, etc. Although lactic acid is used, it is also described that there is no report that these foods have shown an action of suppressing postprandial blood glucose elevation.
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 problems have been discussed regarding the disposal of shochu residue moromi, and its effective use is required. Although it is known that shochu residue moromi is used as livestock feed, research on its usefulness is still ongoing.
Japanese Patent Application Laid-Open No. 2002-371003 describes that a barley fermented extract obtained by concentrating a filtrate of a distillation residue of barley shochu suppressed an increase in blood glucose level after a meal. However, it is described that the blood sugar level elevation inhibitory component described in the above-mentioned document is due to an insulin-like action, and no glucosidase and amylase inhibitory action was observed (paragraph number 0013).
Japanese Patent Application Laid-Open No. 2005-224205 describes a health food having a hypoglycemic effect using buckwheat shochu. The above document does not describe α-glucose inhibition.

An 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 α-glucosidase inhibitor comprising, as an active ingredient, a concentrated extract of shochu residue mash or a filtrate or supernatant extract of the shochu residue mash.
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 hypoglycemic agent comprising as an active ingredient a concentrated extract of shochu residue mash or a concentrated extract of the filtrate or supernatant of the shochu residue mash.
According to the present invention, a component having an α-glucosidase inhibitory activity is obtained from shochu residue mash, and an α-glucosidase inhibitor having this active component is provided.

FIG. 1 is a graph showing the inhibition rate for α-glucosidase activity of wheat, rice, rice bran and soba shochu residue mash supernatant fractions.
FIG. 2 is a graph showing the effect of administration of the wheat shochu residue mash supernatant fraction on the change in blood glucose level of normal rats after sucrose application.
FIG. 3 is a graph showing the inhibition rate against α-glucosidase activity of the wheat shochu residue mash supernatant fraction divided by molecular weight.
FIG. 4 is a graph showing the inhibition rate of α-glucosidase 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 administration of sucrose by administration of a shochu residue mash mash supernatant fraction combined with a fibrous substance.
FIG. 6 is a graph showing sucrose load in administration at various doses and changes in blood glucose level with time before administration of the test substance, after 30 minutes and 60 minutes after administration of the test substance.
FIG. 7 is a graph showing changes in blood glucose level with time when 0 g of a test substance is administered (control) and when 0.5 g is administered.
FIG. 8 is a graph showing blood insulin concentrations before administration and at 30 minutes after administration at the time of 0 g test administration (control) and 0.5 g administration.

In the present invention, “shochu residue moromi” refers to moromi that remains after fermenting starch raw materials (cereals, potatoes, etc.) or saccharide raw materials (brown sugar, natto palms, etc.) and then removing alcohol by distillation. Say. The production of shochu usually includes a starch saccharification step by koji mold, a sugar fermentation step by yeast, and an alcohol distillation step. For example, wheat or rice is treated by a conventional method (washing, soaking, draining, steaming, allowing to cool, etc.), and this is inoculated with a seed cake of white birch fungi (for example, Aspergillus Kawachii) commonly used in shochu production, Make iron for an appropriate period at a suitable temperature. To the koji obtained in this manner, water and yeast (for example, Kagoshima yeast or Kumamoto yeast) used for normal shochu production are added and mixed, and saccharified and fermented by a conventional method to obtain primary mash. In primary moromi, shochu yeast is grown. Next, if necessary, add and mix the shochu main ingredients and water treated by ordinary methods (water washing, dipping, draining, steaming, allowing to cool, etc.) to this primary mash as necessary. Further saccharification and fermentation is performed to obtain secondary moromi. As the shochu liquor main raw material, any commonly used raw material can be used, and examples include, but are not limited to, grains such as barley, wheat, rice, corn, and buckwheat, potatoes, brown sugar, and soy sauce. Preferably, it is barley. In the above explanation, the yeast fermentation process (preparation) is in two stages, but it is not always necessary to divide the preparation into two stages. The mash after fermentation is subjected to distillation, and the alcohol is recovered to make shochu, while the residue mash from which the alcohol has been removed is obtained.
The above shochu residue mash is concentrated as it is, or solid-liquid separated by filtration, centrifugation, etc., and then the filtrate or supernatant liquid is concentrated by heating, freeze-drying or spray-drying to obtain a concentrated extract. Furthermore, the concentrated extract can also concentrate the active ingredient using suitable columns, such as a silica gel column, ODS column, an ion exchange resin, and an ultrafiltration membrane molecular sieve. Of these, the filtrate or supernatant fraction is preferred, and the fraction having a molecular weight of 6000 or less is preferred.
The concentrated extract exhibits α-glucosidase inhibitory activity as shown in the Examples described later. In the present invention, it is defined as having an α-glucosidase inhibitory activity when the inhibition rate of the enzyme reaction of α-glucosidase under the conditions described in Example 1 is 20% or more. 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-glucosidase inhibition, and the concentrated extract can be used as an α-glucosidase inhibitor.
In the concentrated extract, in addition to α-glucosidase inhibitory activity, invertase and α-amylase inhibitory activity is also seen, as shown in the Examples described later. Thus, the concentrated extract or α-glucosidase inhibitor can inhibit various glycolytic enzymes.
The concentrated extract (particularly, a fraction having a molecular weight of 6000 or less of shochu residue residue mash or supernatant) contains succinic acid, pyroglutamic acid, and pyruvic acid. These organic acids, i.e., succinic acid, pyroglutamic acid, and pyruvic acid have α-glucosidase inhibitory activity.
The concentrated extract or the α-glucosidase inhibitor can inhibit the action of α-glucosidase that degrades carbohydrates and delay the absorption of glucose from the small intestine, so that neuropathy caused by diabetes and hyperglycemia caused therefrom, It can be administered orally for the treatment or prevention of various complications such as cataract, nephropathy, retinopathy, arteriosclerosis, atherosclerosis, diabetic gangrene. The dose depends on the method of administration and the degree of symptoms, the age and weight of the patient, etc., but usually a concentrated extract (especially the molecular weight of the filtrate or supernatant of shochu residue residue mash is 6000 or less per one adult dose). The fraction) may be 0.05 g to 10 g, preferably 0.075 g to 5 g, more preferably 0.1 g to 1 g. This dosage can be appropriately determined by a known glucose tolerance test by humans.
The concentrated extract or α-glucosidase inhibitor can be in the form of a tablet, powder, or liquid. Moreover, it can also be set as the food for health maintenance which has an alpha-glucosidase inhibitory action or a glycolytic enzyme inhibitory action as mentioned above by adding this to a foodstuff or a drink. Examples of such foods include home-use diabetic foods, liquid foods, foods for sick people (combination foods for adjusting diabetic foods, etc.), foods for specified health use, diet foods, and foods based on carbohydrates. However, it is not limited to these. Specific food forms include, but are not limited to, coffee, soft drinks, soup, fruit juice, jam, biscuits, bread, and pasta. Addition or processing to foods can be performed by methods commonly used by those skilled in the art. Addition to non-human animals such as livestock or pet feed is also possible.
The concentrated extract or the α-glucosidase inhibitor may be contained alone as a hypoglycemic agent, or may be contained in combination with a fibrous substance. It is particularly useful for suppressing postprandial blood glucose levels. By containing a fibrous substance, the hypoglycemic effect is further enhanced. Examples of the fibrous substance include cellulose and indigestible dextrin, but preferably indigestible dextrin is used. Such a hypoglycemic agent can also be made into a food as described above.
EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited to these illustrations.

In this example, the residue mash of sweet potatoes made from sweet potato, barley, rice or buckwheat was produced as follows.
(Production Example 1: Production of wheat shochu residue moromi)
Barley was shaved with a refining machine, water was added to this and steamed, and then allowed to cool to about 35-40 ° C. This was mixed with Aspergillus Kawachii (Matsunosuke Hamaguchi Co., Ltd.), and the Aspergillus was propagated after 5 days. Next, water and shochu yeast were added in an appropriate amount to this, mixed, and fermented at about 25-30 ° C. for 10 days to obtain moromi. The mash that had been fermented was distilled with 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 10,000 g for 30 minutes at room temperature, and the supernatant was collected. This supernatant was subjected to an ultrafiltration membrane device (Asahi Kasei; pen-type UF membrane), and a solution of a fraction having a molecular weight of 6000 or less was collected and then freeze-dried. Further, the remaining solution was separated into a fraction having a molecular weight of 50000 or less and a fraction having a molecular weight exceeding 50000, 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 (Matsunosuke Hamaguchi Co., Ltd.), and the bacilli were propagated after 5 days. Next, appropriate amounts of water and shochu yeast were 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 potato shochu residue moromi)
The sweet potatoes were washed on the surface, then steamed with water and then ground. According to the description in Production Example 2 above, the seed gonococcus was planted in the rice and kneaded. Steamed sweet potatoes, water and shochu yeast were mixed in appropriate amounts with the koji rice and fermented at about 25-30 ° C. for 3 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 4: Production 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 to 40 ° C. and mixed with Aspergillus Kawachii (Matsunosuke Hamaguchi Co., Ltd.), and the koji mold was allowed to propagate for 5 days. To the smelted buckwheat, the stalked wheat and rice of the above production examples 1 and 2 are added and mixed so that the ratio of rice: wheat: soba is 1: 1: 1.2, and water and shochu are further mixed. It was mixed with an appropriate amount of yeast and fermented at about 25-30 ° 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.
(Example 1: Effect of shochu residue moromi on α-glucosidase activity)
100 mg each of freeze-dried fractions having a molecular weight of 6000 or less of the wheat, rice, rice bran, and buckwheat shochu residue moromi of Production Examples 1 to 4 and 10 mg of acarbose (trade name Glucoby (registered trademark); Bayer) as a control, The test substance solution was obtained by dissolving in 1 mL of 0.02 M phosphate buffer. In order to investigate the action of the test substance on the α-glucosidase activity, a reaction solution having the following composition was prepared: 0.4% p-nitrophenyl α-D-glucopyranoside (Wako Pure Chemical) 0.2 mL; Solution 0.2 mL; and 0.5 U / mL α-glucosidase (Toyobo) 0.1 mL. Here, 1 unit (U) of α-glucosidase is the amount of enzyme that liberates 1 μmol of glucose per minute from the non-reducing terminal side of the substrate under the following standard reaction conditions. The 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). Take 0.02 mL of the solution after stopping the reaction, add 3.0 mL of a coloring reagent (glucose CII test Wako; Wako Pure Chemicals), mix and incubate at 37 ° C. for 5 minutes, and then 505 nm with a spectrophotometer (Beckman). Absorbance was measured at. All measurements were performed twice.
FIG. 1 is a graph showing the inhibition rate of these shochu residue moromi to α-glucosidase activity. The results for acarbose, which is known to inhibit α-glucosidase, are also shown. The vertical axis represents the inhibition rate (%) of α-glucosidase activity. All shochu residue and moromi showed inhibition of α-glucosidase activity, and in particular, wheat shochu residue and mash showed inhibition exceeding 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, the cellulose-administered group was orally ingested with 2 g / kg of sucrose and 20 mg / kg of cellulose intragastrically, and the acarbose-administered group was 20 mg / kg acarbose was administered intragastrically with 2 g / kg of sucrose orally, and the fraction freezing fraction of the wheat shochu residue mash of Production Example 1 having a molecular weight of 6000 or less was added to the shochu residue mash mash supernatant fraction administration group. A dry product of 20 mg / kg was intragastrically administered. Each group had 5 animals. The blood glucose level was measured before treatment, after 30 minutes, after 60 minutes, and after 120 minutes. The blood glucose level here is the serum glucose concentration.
FIG. 2 is a graph showing changes over time in blood glucose levels in each treatment group after sucrose was given. The horizontal axis represents the postprandial time (minutes), and the vertical axis represents the blood glucose level (serum glucose concentration: mg / mL). In the figure, a black circle is a control group, a white circle is a cellulose administration group, a white triangle is an acarbose administration group, and a white square is a shochu residue 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 acarbose administration group and the shochu residue residue mash supernatant fraction administration group, the blood glucose level increased 30 minutes after the treatment, but the increase was significantly lower than the control (** in FIG. 2; r <0.05).
(Example 3: Inhibition of α-glucosidase activity of wheat shochu residue moromi)
Using the lyophilized product of the fraction with a molecular weight of 6000 or less; a molecular weight of more than 6000 and less than 50,000; and a molecular weight of more than 50000 Thus, the influence on α-glucosidase activity was examined.
FIG. 3 is a graph showing the inhibition rate for α-glucosidase activity of these wheat shochu residue mash supernatant fractions. The vertical axis represents the inhibition rate (%) of α-glucosidase activity. In the figure, a fraction having a molecular weight of 6000 or less is represented by M.P. W. <6000; a fraction having a molecular weight of more than 6000 and not more than 50,000 is expressed as 6000 <M. W. <50000; and fractions with molecular weights greater than 50000 W. Represented by In any fraction, inhibition of α-glucosidase activity was shown, but the inhibition rate was particularly high in the fraction having a molecular weight of 6000 or less.
(Example 4: Inhibition of α-glucosidase activity of rice shochu residue moromi)
The procedure described in Example 1 above, using the lyophilized product of the fraction of the rice shochu residue moromi obtained in Production Example 2 with a molecular weight of 6000 or less; a molecular weight of more than 6000 and less than 50,000; and a molecular weight of more than 50,000 Thus, the influence on α-glucosidase activity was examined.
FIG. 4 is a graph showing the inhibition rate for α-glucosidase activity of the rice shochu residue mash supernatant fraction. The vertical axis represents the inhibition rate (%) of α-glucosidase activity. In the figure, a fraction having a molecular weight of 6000 or less is represented by M.P. W. <6000; a fraction having a molecular weight of more than 6000 and not more than 50,000 is 6000 <M. W. <50000; and fractions with molecular weights greater than 50000 W. Represented by In all fractions including a fraction having a molecular weight of 6000 or less, α-glucosidase activity inhibition exceeding 50% 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 ingested with 2 g / kg sucrose, and nothing else was administered. To the shochu residue residue mash supernatant fraction administration group, 2 g / kg of sucrose was orally ingested, and 5 mg / kg of the fraction lyophilized fraction of molecular weight 6000 or less of the wheat shochu residue residue mash of Production Example 1 was administered intragastrically. In addition, 2 g / kg sucrose was orally ingested into the cellulose addition group, and 5 mg / kg of the lyophilized fraction of molecular weight 6000 or less of the barley shochu residue mash of Production Example 1 above and 5 times the amount of cellulose were mixed. 5 mg / kg of the lyophilized fraction having a molecular weight of 6000 or less of the wheat bran residue of the above-mentioned Production Example 1 and 5 times the amount of the indigestible dextrin Was administered intragastrically. Each group had 5 animals. 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 giving sucrose. The vertical axis represents blood glucose level (serum glucose concentration: mg / mL). In the figure, the white circle is the sucrose intake group, the × is the group to which the fraction lyophilized product having a molecular weight of 6000 or less of the barley shochu residue mash is administered (the barley shochu residue mash supernatant fraction administration group), and the white triangle is the above production example 1. A group in which cellulose was added together with a lyophilized fraction having a molecular weight of 6000 or less of the barley shochu residue mash, and a white square was a lyophilized fraction having a molecular weight of 6000 or less in the barley shochu residue mash of Production Example 1 above. This is a group to which indigestible dextrin is added (indigestible dextrin added group). The increase in blood glucose level after a meal was further suppressed in the cellulose-added group and the indigestible dextrin-added group compared to the wheat shochu residue mash residue fraction administration group. In particular, an excellent hypoglycemic effect was observed by combining an indigestible dextrin with the wheat flour shochu residue mash residue fraction.
(Example 6: Composition analysis of shochu residue moromi)
Dissolve 2.0 g of the lyophilized fraction of the molecular weight of 6000 or less of the wheat shochu residue residue of Production Example 1 in ultrapure water (millQ water), silica gel (Silica Gel 60N (spherical, neutral), 63-210 μm; Kanto; Chemical Co., Ltd.) was adsorbed to 10 g and dried. A column of 2 cm × 60 cm with a filter (No. 2) (VIDTEC) was packed with about 50 g of the silica gel, and the wheat shochu residue adsorbed with the silica gel was placed on the mash. A single solvent (CHCl ₃ : methanol: H ₂ O = 5: 3: 0.4) was allowed to flow and fractionated. Each fraction was subjected to thin layer chromatography (TLC: Partisil (registered trademark) K5F Silica Gel 150Å, 20 × 20 cm, Whatman) and sprayed with a color former (p-anisaldehyde, ethanol solution, Tokyo Chemical Industry Co., Ltd.). And spotted. Spots with the same Rf value were collected, concentrated and further purified by high performance liquid chromatography (HPLC).
HPLC was performed under the following conditions:
Equipment used: High performance liquid chromatography SHIMADZU LC-10A
Pump: LC-10AD x 2 detectors: CDD-6A (conductivity detector)
Controller: SCL-10A
Column oven: CTO-10A
Auto injector: SIL-10A
Chromatopack:
Column: Shim-Pack SCR-102H 300 × 8 mm (inner diameter) (Shimadzu Corporation)
Mobile phase:
5 mM p-toluenesulfonic acid flow rate: 0.8 mL / min Temperature: 45 ° C.
Analysis time: 40 minutes Detection: conductivity
Detection is based on a post-column buffering method Buffer: 5 mM p-toluenesulfonic acid and 100 μM EDTA, 20 mM Bis-Tris solution Flow rate: 0.8 mL / min Polarity: +
Response: slow
The specimen was prepared by dissolving 2.0 mg of the sample in 2000 μL of millQ water and further filtering this with a 0.45 μm membrane filter. The injection amount was 10 μL.
As a result of HPLC, peaks of fractions fr1-2 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, 10 μL was injected using the following standard solution of organic acid: phosphoric acid (546.9 mg / L), citric acid (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 (218.4 mg / L) . As a result, 1 mg of fr1-2 to 1-3 was found to contain 0.693 mg of succinic acid, 0.068 mg of pyroglutamic acid, and 0.014 mg of pyruvic acid (the remaining amount of about 25% is unknown). Further, when the influence of succinic acid, pyroglutamic acid, and pyruvic acid on α-glucosidase activity was examined according to the procedure described in Example 1 above, it was confirmed that they had glucosidase inhibitory activity (succinic acid: IC50 4 .99 mg / mL, and pyroglutamic acid: IC50 7.38 mg / mL, pyruvate: IC50 4.84 mg / mL).
(Example 7: Effect of shochu residue mash on the activity of various glycolytic enzymes)
(7-1. Invertase (sucrase) inhibitory activity)
The sample solution was added with 0.1 M acetate buffer (pH 5.0) (also used as a solvent in the following solutions) to 100 mg / mL of the fraction of molecular weight 6000 or less of the wheat shochu residue residue of Production Example 1 to obtain various concentrations. Adjusted. A 5% sucrose solution was used as a substrate, and 0.5 U / mL invertase was used as an enzyme solution.
Substrate 0.2 mL, enzyme solution 0.1 mL, and sample solution 0.2 mL were mixed and incubated at 37 ° C. for 15 minutes. Then, it heated for 10 minutes with the water bath at 90 degreeC or more, and reaction was stopped. 0.05 mL was taken from the solution after the reaction, and 3.0 mL of a coloring reagent (glucose CII test Wako; Wako Pure Chemical Industries, Ltd.) was added, mixed, incubated at 37 ° C. for 5 minutes, and 505 nm with a spectrophotometer (Beckman). Absorbance was measured at. All measurements were performed twice. As a result, inhibition of enzyme activity was observed with increasing concentration of the sample solution. When the concentration of the sample solution was 40 mg / mL, the inhibition rate was about 70%.
(7-2. Α-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 solution (pH 5.0) (also used as a solvent in the following solutions) to 100 mg / mL of the fraction having a molecular weight of 6000 or less in the wheat shochu residue residue of Production Example 1. did. A solution of 2-chloro-4-nitrophenyl-6-azido-6-deoxy-β-maltopentaoside as a substrate, a solution of glucoamylase and β-glucosidase as a coloring enzyme solution, and 0.28 U / as a test enzyme solution mL of Aspergillus oryzae α-amylase solution was prepared.
First, 25 μL of a substrate solution and 25 μL of a coloring enzyme solution were added to a plate, then 25 μL of a sample solution was added, then 25 μL of an α-amylase solution of a test enzyme solution was added, and incubated at 37 ° C. for 15 minutes. Thereafter, 100 μL of sodium carbonate solution was added to stop the reaction. Absorbance was measured at 400 nm using a spectrophotometer (Beckman) with a plate reader filter at 405 nm. All measurements were performed twice.
As a result, inhibition of enzyme activity was observed with increasing concentration of the sample solution. Acarbose used as a control (trade name Glucobay (registered trademark); Bayer) exhibited an α-amylase activity inhibition rate of about 50.51% at a concentration of 0.003 mg / mL. In the sample solution, the inhibition rate was about 43% at a concentration of 5 mg / mL. Compared to acarbose, 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 were registered (1 male; 6 females; average age of all 33.4 ± 6. 6 years old). Upon registration, the patient underwent a comprehensive diagnosis by the responsible physician on the condition that there was no history of diabetes and that there was no special illness at the time of the test. Special diseases include 1) severe liver / kidney / cardiac dysfunction, 2) drug hypersensitivity, 3) mental disorders and impaired consciousness, 4) pregnant women, lactating women and this study. Those who plan to become pregnant, 5) Others who are judged unsuitable 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 received 0 g (control), 0.1 g, or 0.5 g of the test substance at the same time as 25 g of sucrose (Wako Pure Chemical Industries) was loaded at the start of the test. did. The blood sugar level was measured before sucrose loading and test substance administration, and 30 minutes and 60 minutes after test substance administration. At 0 g administration (control) and 0.5 g administration, blood collection for blood test was also performed before administration and 30 minutes after administration. Each test was performed at intervals of 24 hours or more, and excessive feeding suppression to the subjects was avoided.
Average values and standard deviations were obtained for all test items, and Student t test was performed for the difference between before and after administration. In all cases, the significance level was 5% or less.
The results for blood glucose levels related to dose effects are shown in FIG. In FIG. 6, the measured value is expressed as a relative value with the blood glucose level before sucrose loading as 100%. In the figure, a black circle represents a control, a triangle represents administration of 0.1 g of the test substance, and a white circle represents administration of 0.5 g of the test substance. With a load of 25 g sucrose, in the 0 g administration (control) of the test substance, the blood glucose level increased by about 30% after 30 minutes and remained elevated by 10% or more after 60 minutes. In contrast, the administration of 0.1 g of the test substance showed a significantly lower value 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 no difference was observed from the case of 0.1 g. This is thought to be because it is difficult to see the dose-effect relationship because the blood sugar elevation level is suppressed to about 10% increase with 0.1 g administration.
On the day different from the test of FIG. 6, the change in blood glucose level with time when 0 g of the test substance was administered (control) and 0.5 g was measured. The result is shown in FIG. 7 in terms of a relative value 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, the blood glucose level returned to the fasting level (before sucrose loading) at 120 minutes after administration.
Furthermore, blood samples were collected at the time of administration of 0 g of the test substance (control) and at the time of 0.5 g administration, before and 30 minutes after administration, and blood insulin concentration was measured. The result is shown in FIG. The blood insulin concentration was increased by the 25 g sucrose load, but the test substance did not affect the insulin concentration increase.

  The α-glucosidase 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 shochu residue, which is normally discarded in the production of shochu, for which environmental problems have been discussed.

Claims (3)

An α-glucosidase inhibitor comprising, as an active ingredient, a concentrated extract of shochu residue moromi, or a filtrate or supernatant concentrate of the shochu residue moromi. The α-glucosidase inhibitor according to claim 1, wherein the concentrated extract is a fraction having a molecular weight of 6000 or less obtained from a shochu residue mash filtrate or supernatant. A hypoglycemic agent comprising, as an active ingredient, a concentrated extract of shochu residue moromi or a filtrate or supernatant concentrate of the shochu residue moromi and a fibrous substance.

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