JP2005220110A - Buckwheat chaff extract having starch degrading enzyme inhibitory activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a starch degradation inhibitor that can simply be produced from a readily available raw material in large amounts. <P>SOLUTION: The subject starch degrading enzyme inhibitor contains an extract from buckwheat chaff with water or an organic solvent or their mixture, as an active ingredient. Therapeutic and/or prophylactic agents for diabetes, hyperglycemia and/or therapeutic and/or prophylactic agents for obesity can contain the extract from buckwheat chaff. A functional food containing the chaff extract has functions to cure or prevent diabetes, hyperglycemia and/or obesity. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an extract of buckwheat husk having amylolytic enzyme inhibitory activity, an amylolytic enzyme inhibitor containing the extract as an active ingredient, and a treatment for diabetes, hyperglycemia and / or obesity containing the extract as an active ingredient, and / or Alternatively, it relates to a preventive drug, and further to a functional food containing the extract.

  Carbohydrates such as starch are decomposed by digestive enzymes (α-amylase, glucosidase, etc.) into glucose, which becomes a source of energy. However, if you eat too many meals, the diabetes reserve army or patient will have an increased blood sugar level. In addition, recent dietary habits tend to consume too much carbohydrate, and obesity has been highlighted as well as diabetes and hyperglycemia as described above. Under such circumstances, there is an increasing need for health foods that have a function of suppressing or preventing these symptoms in consumers.

  Conventionally, in order to treat and / or prevent such symptoms, attempts have been made to use extracts derived from synthetic products and natural products as components of pharmaceuticals and foods as anti-diabetic components, blood glucose level-suppressing components or anti-obesity components. ing. For example, guava leaf extract (refer to Patent Document 1), banaba extract (refer to Patent Document 2), cashew extract (refer to Patent Document 3) and the like have been reported as α-amylase inhibiting components, and sweet tea extract (as a glucosidase inhibiting component). Patent document 4), modern rice extract (see patent document 5), perilla extract (see patent document 6), and the like have been reported.

  However, conventional starch degradation inhibitors have various problems, such as those with insufficient effects, those with a small amount of production due to limited raw materials, and those that require time and cost for production. .

As for buckwheat husk extract and fractions, their use as lipid peroxide inhibitors, cholesterol-lowering agents, neutral fat-lowering agents or hyperlipidemia-improving agents are mentioned, but as starch-degrading enzyme inhibitors Is not mentioned at all (see Patent Document 7). Also, the activity of this buckwheat husk fraction is high in the fraction with a molecular weight of 10,000 or less, so it is clear that it is distinguished from that of the present invention.
International Patent Application Publication No. WO01 / 66714 JP 2003-095941 A JP 2002-051881 A JP 2003-252784 A JP 2002-316939 A JP 2000-102383 A JP-A-10-218786

  In view of such circumstances, there has been an urgent need to develop a starch degradation inhibitor that can be obtained in large quantities, is easy to manufacture, and that has an excellent effect.

  Accordingly, the present inventors have conducted extensive research to solve the above-mentioned problems, and found that an extract having high amylolytic enzyme inhibitory activity can be obtained by extracting buckwheat husk with water, an organic solvent or a mixture thereof. It came to complete.

That is, the present invention
(1) An extract having amylolytic enzyme inhibitory activity, obtained by extracting buckwheat husk with water, an organic solvent or a mixture thereof;
(2) The extract according to (1), which is a fraction having a molecular weight of 10,000 or more;
(3) The extract according to (1) or (2), which inhibits α-amylase;
(4) The extract according to any one of (1) to (3), wherein the organic solvent is ethanol;
(5) An amylolytic enzyme inhibitor comprising the extract according to any one of (1) to (4) as an active ingredient;
(6) A therapeutic and / or prophylactic agent for diabetes, hyperglycemia and / or obesity comprising the extract according to any one of (1) to (4) as an active ingredient;
(7) A functional food comprising the extract according to any one of (1) to (4) as an active ingredient; and (8) the functional food according to (7), which is buckwheat.

  According to the present invention, an extract having high starch-degrading enzyme activity can be obtained from buckwheat husk, so that a therapeutic and / or prophylactic agent for diabetes, hyperglycemia and / or obesity using this extract as an active ingredient can be produced. Moreover, the functional food which has the function of treatment and / or prevention of diabetes, hyperglycemia and / or obesity can also be obtained by using the extract of the present invention for foods and food materials.

  The extract having the amylolytic enzyme inhibitory activity of the present invention can be obtained by extracting buckwheat husk with water, an organic solvent or a mixture thereof. As can be seen from the following description, no special equipment or the like is required for the production of the extract of the present invention. Since buckwheat husks have not been used much as waste in the past, and the amount thereof is abundant, the present invention is also excellent from the viewpoint of effective utilization of unused biomass and is environmentally friendly It can be said that it is an invention.

  The raw buckwheat husk may be any one. Noodle varieties and localities do not significantly affect the amylolytic enzyme inhibitory activity. In addition, in this specification, the buckwheat husk extract obtained by this invention may be named generically "the extract of this invention." Therefore, in the present specification, the term “the extract of the present invention” refers to a product obtained by simply extracting buckwheat husk with water or an organic solvent or a mixture thereof, and a product obtained by further purifying the extract (a product obtained by molecular weight fractionation). Including those processed and processed into various forms as described later.

  The buckwheat husk may be subjected to extraction as it is, or may be subjected to a treatment such as pulverization or immersion in an extraction solvent before extraction in order to increase extraction efficiency. A known grinder or mill can be used as the pulverizer.

  A preferred form of extraction of buckwheat husk is solid-liquid extraction. As the solvent, water, an organic solvent, or a mixture thereof is used. Those blending ratios can be determined by those skilled in the art through simple preliminary experiments. The organic solvent is preferably a hydrophilic one. Of these, alcohols such as methanol, ethanol, propanol, i-propanol, and n-butanol are preferable. In consideration of using the buckwheat husk extract of the present invention for pharmaceuticals and foods, that is, safety and extraction efficiency, Most preferred. The extraction temperature is not particularly limited and is preferably 80 ° C. to 100 ° C., but may be room temperature. The solid-liquid extraction method and apparatus may be widely known in the art, and can be appropriately selected and used by those skilled in the art. For example, a Soxhlet extractor may be used in the laboratory, and industrially, a type of apparatus in which the liquid phase is osmotically refluxed in a fixed solid phase, or a type in which a solid phase is dispersed in the liquid phase. Various devices such as a device can be used.

  The buckwheat husk extract obtained by such an extraction operation may be used as it is as the extract of the present invention, or may be used after further purification. Various means and methods can be used to purify the extract of the present invention, and those skilled in the art can easily select or use them in combination. For example, various chromatographic methods such as ion exchange chromatography and gel filtration chromatography, ultrafiltration methods using cellulose membranes and synthetic membranes, reverse osmosis methods, adsorption methods, organic solvent fractionation and other separation methods can be used. . Moreover, you may centrifuge the produced precipitate as needed. As described later, since most of the amylolytic enzyme inhibitory activity in the extract of the present invention is present in a fraction having a molecular weight of about 10,000 or more, it is preferable to include a molecular weight fractionation step in the purification process. The molecular weight fractionation method includes a gel filtration method and a method using an ultrafiltration membrane, and those skilled in the art can appropriately select and use them.

  The form of the extract of the present invention can be various depending on its use. For example, the extract of the present invention may be concentrated and used by evaporation with a rotary evaporator, or may be diluted with water or the like. The liquid extract of the present invention may be used as a solid or powder by a method such as evaporation to dryness or freeze drying. Such techniques are well known to those skilled in the art. Below, the use of the extract of this invention is illustrated and demonstrated.

  One use of the extract of the present invention is as an active ingredient in amylolytic enzyme inhibitors. In that case, the extract of the present invention may be used after being concentrated or diluted as appropriate, and may be used after being processed into a liquid or solid (powder, granules, etc.). The dosage form of the amylolytic enzyme inhibitor of the present invention may be any, and may be a liquid, powder, tablet, capsule, syrup and the like. Examples of carriers or excipients used for such drugs include, but are not limited to, aqueous carriers such as water, ethanol, glycerol, and molasses, and solid carriers such as sucrose, talc, and microcrystalline cellulose. In addition to the extract of the present invention, a substance that has been confirmed to have amylolytic enzyme inhibitory activity may be used in the amylolytic enzyme inhibitor. Since the dose of the extract of the present invention is a natural product and is not considered to be toxic, there is no particular upper limit, but it is usually up to about 20 g per day for an adult with a body weight of 70 kg, converted to the raw buckwheat husk. I will.

  A further use of the extract of the invention is as an active ingredient in a remedy and / or preventive for conditions that can be treated and / or prevented by inhibiting amylolytic enzymes, for example diabetes, hyperglycemia and / or obesity. Is to use. Such drugs are preferably administered orally. The dosage form may be any form, and may be a liquid, powder, tablet, capsule, syrup and the like. Carriers or excipients used for such drugs include, but are not limited to, aqueous carriers such as water, ethanol, glycerol, molasses, and solid carriers such as sucrose, talc, and microcrystalline cellulose. Moreover, you may use together with the other substance by which the effect is confirmed regarding the treatment and / or prevention of diabetes, hyperglycemia, and / or obesity. Since the dose of the extract of the present invention is a natural product and is not considered to be toxic, there is no particular upper limit, but it is usually up to about 20 g per day for an adult with a body weight of 70 kg, converted to the raw buckwheat husk. I will.

  The extract of the present invention can be used in various foods and food materials to obtain functional foods having a function of treating and / or preventing diabetes, hyperglycemia and / or obesity. Any shape of the functional food may be used. The extract of the present invention may be contained in any food or food material, but it is preferably used in noodles, particularly in buckwheat. The extract of the present invention may be used as a food additive or as a seasoning. Their shape is not particularly limited. For example, it may be contained in the sauce or may be contained in the sprinkling.

  The present invention will be described in more detail and specifically with reference to the following examples, but the examples are not intended to limit the present invention.

1. Screening of starch-degrading enzyme inhibitors from food processing wastes Degreased soybeans, buckwheat husk, rice bran, soy sauce cake, wine lees, chanterelle, plum seeds, cheese whey 20g each (dry weight) using Soxhlet extractor Extraction was performed with 150 ml of water or 150 ml of ethanol for 10 hours, and the extract was evaporated to dryness on a rotary evaporator, and then water was added to obtain 2.1 g of water extract and ethanol extract as samples. The above raw materials were obtained from general food processing manufacturers except for wine lees. Wine bottles were obtained from Mercian.
Next, α-amylase inhibitory activity and α-glucosidase inhibitory activity of these samples were measured. The measurement method is as follows.
(I) Measurement of α-amylase inhibitory activity Measurement was performed using Amylase Test Wako (Wako Pure Chemical Industries). The final sample concentration was 1 mg / ml (w / v), and the final amylase concentration was 0.098 U / ml. A system to which no sample was added was used as a control.
(Ii) Measurement of α-glucosidase inhibitory activity The maltose aqueous solution (10%) was used as a substrate, and the mixture was reacted at 37 ° C. for an appropriate time, and the amount of released glycol was measured. The final sample concentration was 1 mg / ml (w / v), and the final α-glucosidase concentration was 0.25 U / ml. A system to which no sample was added was used as a control. The amount of glucose was measured using Glucose CII-Test Wako (Wako Pure Chemical Industries).
The α-amylase inhibitory activity of each extract is shown in FIG. The buckwheat husk extract inhibited α-amylase 100% in the above measurement irrespective of the extraction solvent (water or ethanol). Subsequently, the α-glucosidase inhibitory activity of the ethanol extract of wine lees and the ethanol extract of defatted soybean was strong. FIG. 1B shows the α-glucosidase inhibitory activity of each extract. Among the test samples, the buckwheat husk extract showed the strongest α-glucosidase inhibitory activity in the above measurement irrespective of the extraction solvent (water, ethanol). Next, the α-glucosidase inhibitory activity of the soy sauce cake extract and the defatted soybean extract was strong.
From these results, it was considered that the buckwheat husk extract and the ethanol extract showed high inhibitory activity against α-amylase and α-glucosidase, and effectively inhibited starch degradation. In particular, the buckwheat husk extract showed strong inhibitory activity against α-amylase.

2. Preparation of purified buckwheat husk extract The production scheme of the purified buckwheat husk extract of the present invention is shown in FIG. In the method described in 1 above, 150 g of water was used as an extraction solvent to obtain 2.1 g of oat husk water extract from 20 g of oat husk (also described below using 150 ml of ethanol as an extraction solvent) Similar results were obtained). This buckwheat husk water extract is put into an ultrafiltration membrane (cellophane tube with a molecular weight cut off of 10,000, Wako Pure Chemical Industries) and dialyzed against water to obtain a fraction with a molecular weight of 10,000 or more and a fraction with a molecular weight of 10,000 or less. divided. 0.71 g of a fraction having a molecular weight of 10,000 or more was obtained, and 1.39 g of a fraction having a molecular weight of 10,000 or less was obtained. A fraction rotary evaporator having a molecular weight of 10,000 or more was evaporated to dryness and then dissolved in 150 ml of ethanol. This ethanol solution was extracted with a Soxhlet extractor for 10 hours, the extract was evaporated to dryness using a rotary evaporator, dissolved in water, and 0.027 g of a purified fraction having a molecular weight of 10,000 or more (hereinafter referred to as “purification”). This is called “buckwheat shell extract”. In addition, you may perform the evaporation-drying operation by said rotary evaporator instead of freeze-drying. The water used in the final stage is preferably purified water (for example, Milli Q water).

3. Various characteristics of the extract of the present invention The extract of buckwheat husk described in 2 above, the fraction having a molecular weight of 10,000 or more after ultrafiltration, the fraction having a molecular weight of 10,000 or less, and the α-amylase inhibitory activity of the extract of the purified buckwheat husk I investigated. Α-Amylase inhibitory activity was measured by the method described in 1 (i) above, and expressed as a sample concentration (referred to as “IC ₅₀ ”) necessary for inhibiting α-Amylase activity by 50%. IC ₅₀ values of the buckwheat husk extract, the fraction having a molecular weight of 10,000 or more, the fraction having a molecular weight of 10,000 or less, and the purified buckwheat husk extract are 65 μg / ml (w / v), 31 μg / ml (w / v), They were 2 mg / ml (w / v) and 7.2 μg / ml (w / v) (FIG. 2).
From the above results, since most of the α-amylase inhibitory activity of the extract of the present invention was in a fraction having a molecular weight of 1000 or more, it was suggested that the inhibitor may be a high molecular weight substance.
Therefore, the amount of polyphenol in the sample at each stage described in 2 above was measured. The Folin-Ciocalteau method was used to measure the amount of polyphenol. That is, 200 μl of a sample and 3.2 ml of water were placed in a test tube and stirred. 400 μl of saturated sodium carbonate solution was added thereto and left for 30 minutes. Thereafter, the prepared solution was measured for absorbance at a wavelength of 760 nm using a spectrophotometer. A calibration curve was prepared using catechin. The amount of polyphenols in the buckwheat husk extract, the fraction with a molecular weight of 10,000 or more after ultrafiltration, and the purified buckwheat husk extract is 22%, 32%, and 46% (w / v, catechin equivalent) of the sample, respectively. there were.
In this way, as the purification stage progressed, the amount of polyphenols contained in the sample increased, and when the sample was fractionated with organic solvents (benzene, toluene, chloroform), inhibitory activity against these organic solvents was observed. Since the substance is soluble (data not shown), the amylolytic enzyme inhibitory substance in the extract of the present invention was considered to be polyphenols.
The α-amylase inhibitory activity of the water extract of buckwheat husk and the ethanol extract of buckwheat husk obtained by the method of 1 above is the buckwheat extract marketed as an anti-diabetic functional extract and the buckwheat marketed as an extract for improving coldness and stiffness. Compared with leaf extract. All samples were measured as 1 mg / ml. The α-amylase inhibitory activity of the buckwheat husk water extract and the buckwheat husk ethanol extract was not inferior to these commercially available products, but exceeded their inhibitory activity (FIG. 3).
Furthermore, the α-amylase inhibitory activity of the buckwheat husk extract has recently been revealed to have an α-amylase inhibitory effect, and guava leaf water extract (100 g guava leaf (dry weight) added to commercial beverages) ) Was extracted with 2 liters of distilled water at 80 ° C. for 30 minutes). The IC ₅₀ value of the α-amylase inhibitory activity of the buckwheat husk extract of the present invention was 65 μg / ml (w / v), whereas that of the guava leaf water extract was 600 μg / ml (w / v). .

  The extract obtained by extracting the buckwheat husk of the present invention has a high starch degrading enzyme inhibitor activity and can be widely used in the fields of anti-diabetic, anti-hyperglycemia, anti-obesity pharmaceuticals and functional foods.

FIG. 1 is a diagram showing screening results of starch degrading enzyme inhibitors from various food processing wastes. The left panel (a) shows α-amylase inhibitory activity, and the right panel (b) shows α-glucosidase inhibitory activity. FIG. 2 is a diagram showing a production process of a buckwheat husk extract and a purified buckwheat husk extract of the present invention. 20 g of buckwheat husk was used as a raw material. The weight of the formulation obtained in each step and the IC ₅₀ value for α-amylase are also shown in the figure. FIG. 3 is a comparison of the α-amylase inhibitory activity of the buckwheat husk extract and the ethanol extract of the present invention with a commercially available product.

Claims (8)

  An extract having amylolytic enzyme inhibitory activity, obtained by extracting buckwheat husk with water, an organic solvent or a mixture thereof.   The extract according to claim 1, which is a fraction having a molecular weight of 10,000 or more.   The extract according to claim 1 or 2, which inhibits α-amylase.   The extract according to any one of claims 1 to 3, wherein the organic solvent is ethanol.   An amylolytic enzyme inhibitor comprising the extract according to any one of claims 1 to 4 as an active ingredient.   A therapeutic and / or prophylactic agent for diabetes, hyperglycemia and / or obesity comprising the extract according to any one of claims 1 to 4 as an active ingredient.   A functional food comprising the extract according to any one of claims 1 to 4 as an active ingredient. The functional food according to claim 7, which is soba.

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