JP2011102286A - Composition exhibiting activity such as fat-reducing activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition effective for preventing or eliminating obesity or lowering blood glucose level. <P>SOLUTION: There are provided (1) a composition containing a hot water extract from mushrooms belonging to the genus Grifola frondosa, Polyporaceae, Aphyllophorales, Basidiomycetes; (2) a composition containing a low molecular fraction obtained by separating a hot water extract from mushrooms belonging to the genus Grifola frondosa, Polyporaceae, Aphyllophorales, Basidiomycetes with a separation membrane having one in a fraction mol.wt. range of 30,000 to 10,000; and (3) a composition containing ergothioneine, a pharmacologically acceptable salt thereof, or a compound which is hydrolyzed to produce the ergothioneine, as a fat-reducing activity ingredient. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention exhibits at least one activity selected from the group consisting of visceral fat and blood triglyceride reducing activity, insulin secretion promoting activity and blood glucose lowering activity, ie, metabolic syndrome and diabetes The present invention relates to a composition derived from mushrooms belonging to the order Mushrooms, Sarnococcidae, and Maitake genus, and a composition containing ergothioneine, which is effective for prevention and treatment.

  In modern society, the number of patients with metabolic syndrome continues to increase. Obesity due to the accumulation of visceral fat has attracted attention as a factor that increases the risk of suffering from metabolic syndrome. Therefore, while taking a well-balanced meal and preventing obesity by exercising is important, it is often difficult to achieve.

  In modern society, type II (non-insulin dependent) diabetic patients and their reserves are steadily increasing due to changes in dietary habits, lack of exercise, and the addition of stress. Patients with type (insulin-dependent) diabetes are still not decreasing. Drugs prescribed for such diabetic patients include sulfonylurea (SU drug) and phenylalanine derivatives in addition to insulin used when type I diabetics and type II diabetics have significant hyperglycemia. Insulin secretion promoters such as α-glucosidase inhibitors (αGI drugs), glucose absorption inhibitors such as biguanides (BG drugs), thiazolidine derivatives (TZD drugs) and other insulin resistance improving drugs are known .

  Under such circumstances, many compositions effective for the prevention of obesity containing components derived from mushrooms have been proposed. Typical examples of active ingredients contained in such a composition are high molecular compounds such as β-D-glucan, chitosan, and glycoprotein. On the other hand, a composition effective for preventing obesity, which is a component derived from mushrooms and contains components other than the polymer compound, is also known.

  Patent Document 1 discloses a fat accumulation inhibitor containing a water-containing organic solvent extract or an organic solvent extract from a jellyfish that is a kind of mushrooms. Patent Document 1 does not describe what the active ingredient of this fat accumulation inhibitor is, but a description that it is a fat-soluble component and a case where a water-containing organic solvent is used for extraction because it is fat-soluble. Describes that the extraction efficiency decreases as the content of the aqueous component increases (paragraph [0021]).

  Patent Document 2 discloses a lipolysis promoter containing mushrooms such as lobster oyster mushrooms belonging to the octopus family, or extracts thereof, as active ingredients. Patent Document 2 exemplifies water, methanol, ethanol and the like as extraction solvents for the extract (paragraph number [0007]), but the solvent used in the examples is only 70% methanol (paragraph number). [0010]).

  Patent Document 3 describes that a cyclic peptide derived from a mushroom is effective as an anti-obesity agent. In the embodiment, the active ingredient is extracted from the bamboo shoot. According to FIG. 1, which describes the extraction process, extraction is first performed from dry oyster mushrooms with aqueous ethanol, and then the extract is subjected to partitioning with ethyl acetate / water. According to FIG. 1, the aqueous layer after this distribution showed no fat accumulation inhibitory activity. Therefore, it can be said that the cyclic peptide disclosed in Patent Document 3 is also a fat-soluble component.

  Patent Document 4 discloses that, with regard to eringi or hanabiratake, when a powder produced through drying, repair treatment using hot gas and pulverization process is given to a rat raised on a high fat diet together with water, It is described that the enhancement of the degradation action was recognized (Test Example 6). Further, it is also described that the hot water extraction fraction from the powder was expected to contain oligosaccharides, various glycosides, proteins and water-soluble polysaccharides (paragraph number [0033]). . However, Patent Document 4 does not describe what is a component that enhances the lipolytic action.

  Patent Document 5 discloses a composition having an action of suppressing the amount of neutral fat in blood, in which a seaweed extract and an extract of medicinal herbs are further added with an extract of salmon such as Mannentake and Agaricus spp. (Claim 2). In that example, hot water is used for extraction of active ingredients (paragraph number [0020]). However, Patent Document 5 does not describe that the hot water extract from sputum alone exhibits an effect of suppressing the amount of neutral fat in blood.

  Moreover, ergothioneine is mentioned as one of the physiologically active substances contained in many mushrooms. Claim 7 of Patent Document 6 states that “a therapeutically effective amount of an agent that increases the activity and / or protein level of a sirtuin in a cell is administered to a subject in need thereof to reduce the body weight of the subject. , Or a method for preventing weight gain of a subject ”, and ergothioneine is mentioned as an example of the agent. Further, claim 17 of Patent Document 7 states that “a method of treating obesity in a subject that consumes a high-fat diet and needs treatment, wherein a certain amount of a sirtuin-activating compound is administered to the subject. And ergothioneine as an example of a sirtuin activating compound. Further, Patent Document 8 discloses an invention relating to utilization of a sirtuin activating compound, and ergothioneine is mentioned as an example of a sirtuin activating compound. However, in Patent Document 8, the specification in the invention (claim 5) according to "use of a specific compound for dispensing a medicinal product that reduces a subject's body weight or prevents a patient's weight gain in a subject in need". This compound does not include ergothioneine. In any of Patent Documents 6 to 8, it is not demonstrated that ergothioneine is effective in preventing or treating obesity.

  On the other hand, mushrooms contain various components, and there are many documents describing that mushrooms themselves or extracts from mushrooms are effective in reducing blood sugar. Among such documents, Patent Documents 9 to 13 are mentioned as referring to changes in blood or pancreatic insulin levels due to mushroom intake.

In Patent Document 9, an extract from a mycelium or fruiting body of maitake or a composition obtained by mixing vitamins in a mycelium or fruiting body of maitake shows an improvement effect on type I and type II diabetes. The effect is described. In this example, when the above extract or composition was orally administered to a type II diabetes model mouse (KK-A ^y mouse), the amount of insulin in the blood and the blood were compared with those in the untreated group (non-administered group). It is described that the amount of triglyceride has decreased (paragraph numbers [0030] to [0036]).

Patent Document 10 describes that proteoglucan derived from maitake or choreimaitake is effective in improving insulin-independent (type II) diabetes. In the examples, it is described that in the experiment using spontaneously diabetic mice KK-A ^y , the blood sample insulin concentration was lower in the sample feed group fed with the proteoglucan-contaminated sample than in the control feed group. (Paragraph number [0043]).

  Patent Document 11 describes that a dried powder of Yamabushitake and a hot water extract from Yamabushitake are effective for the prevention and improvement of diabetes. In the examples, in the GOTO-KAKIZAKI rat (GK rat) which is a type II diabetes model animal, the pancreatic insulin level increased in the group ingested with the hot water extract from Yamabushitake compared with the non-administered group. The fact that it was recognized is described (paragraph number [0026]).

  Patent Document 12 describes that matsutake or an extract thereof has an effect of promoting insulin secretion, an effect of suppressing an increase in blood glucose level, and the like. In this example, in the control rats, the plasma insulin level reached a peak at 30 minutes after the meal in both the basic diet group and the experimental diet group (feed with added powder of matsutake mycelium), and then gently decreased. On the other hand, in the neonatal streptozotocin (STZ) -treated rat (nSTZ rat), which is a type II diabetes model animal, the plasma insulin level in the experimental diet group was almost the same as that in the control rat, but the basic diet group was 30 It is described that the insulin level was significantly low from minutes to 120 minutes (paragraph numbers [0176] to [0181]).

  Patent Document 13 describes that an active substance derived from Hanabiratake has insulin secretion promoting activity. In Example 2, in an oral glucose tolerance test using ICR mice, glucose load was performed after oral administration of dried mushroom powder, and then blood insulin concentration was measured. As a result, Hanabiratake dry powder or Maitake dry powder was administered. It is stated that the insulin concentration was increased in the group compared to the control group (no mushroom dry powder administration) (paragraph numbers [0029] to [0030]). In addition, in the glucose tolerance test using KK-Ay diabetic mice and Wistar rats, the insulin concentration after 30 minutes of glucose load was more particularly observed in the group administered with dry powder of Hanabiratake compared to the control group (no administration of dry mushroom powder). (Examples 4 and 5).

  As described above, there are mushrooms that lower and increase the blood insulin level. Regarding maitake, Patent Documents 9 and 10 describe that the insulin concentration is decreased, while Patent Document 13 describes that the insulin concentration is increased.

JP2007-269939 JP 2000-26308 A JP2005-220074 JP2007-269641 JP 2006-36681 A Special table 2007-527418 Special table 2007-500377 Special table 2009-500331 JP-A-6-312936 JP-A-10-182702 JP-A-2005-179302 JP 2005-225801 A JP2008-230991

  The present inventors have considered that a safe and effective intake of ingredients effective in preventing obesity is a reliable way to implement measures for preventing obesity without difficulty. Therefore, the present inventors decided to search for foods containing ingredients effective in preventing obesity. In such search, attention was paid to edible mushrooms known to contain various useful ingredients. And among such edible mushrooms, the focus is on mushrooms belonging to the genus Basidiomycetes, Hymenoptera, Sarnosidaceae, Maitake genus, and whether such mushrooms contain an effective ingredient for obesity prevention I decided to study.

  As the extraction solvent, it is preferable that the animal is harmless even if it is ingested as it is. Therefore, it was first tried to use water (more specifically, hot water). And it was decided to verify whether the hot water extract from the said mushroom showed fat reduction activity. Furthermore, it was decided to verify whether or not ergothioneine was contained in the hot water extract from the mushroom and whether or not ergothioneine was effective in preventing obesity.

  In the course of the above study, the present inventors thought that the hot water extract from the mushrooms may also exhibit useful activities other than fat reducing activity. As with obesity, we focused on the increasing number of patients in modern society, and we examined whether the hot water extract from mushrooms is effective in preventing or treating diabetes. And since it became clear that the hot water extract from the said mushroom is effective in the treatment of diabetes, what component contained in the said hot water extract is used for the treatment of diabetes by what kind of action mechanism. The usefulness was also studied.

  Accordingly, an object of the present invention is to provide at least one activity selected from the group consisting of a fat-reducing activity, an insulin secretion-promoting activity, and a blood glucose-lowering activity derived from a mushroom belonging to the family Basidiomycetes, Hymenoptera, Sarnococcidae, and Maitake. It is in providing the composition which shows this. Another object of the present invention is to provide a group consisting of fat reducing activity, insulin secretion promoting activity, and blood glucose lowering activity, containing ergothioneine, a pharmacologically acceptable salt thereof, or a compound that becomes ergothioneine upon hydrolysis as an active ingredient. It is providing the composition which shows at least 1 activity selected from these.

  Furthermore, the objective of this invention is providing the preparation method of the said composition. An object of the present invention is to provide a method for reducing fat, enhancing insulin action, or lowering blood glucose level, using the above composition.

  The present inventors have studied whether or not a component exhibiting fat-reducing activity is contained in Anninkou (scientific name: Grifola galgal), which is a kind of mushrooms belonging to the genus Basidiomycetes, Hymenoptera, Sarnosidaceae, and Maitake. Thereafter, the inventors also studied whether or not a component derived from anninko also exhibited insulin secretion promotion and blood glucose lowering activity, and as a result, the present invention was completed.

  That is, the present invention comprises a group consisting of a fat-reducing activity, an insulin secretion-promoting activity, and a blood glucose-lowering activity, characterized by comprising a hot water extract from a mushroom belonging to the genus Basidiomycetes, Hymenoptera, Sarnosidaceae, Maitake Relates to a composition exhibiting at least one activity selected from (hereinafter, also referred to as “first composition”). In addition, the present invention is characterized by containing a low molecular fraction obtained by separating the hot water extract with a separation membrane having a fractional molecular weight in the range of 30,000 to 10,000. And a composition exhibiting at least one activity selected from the group consisting of fat-reducing activity, insulin secretion-promoting activity and blood glucose-lowering activity (hereinafter sometimes referred to as “second composition”).

  The mushrooms belonging to the basidiomycetes, Hydrangea, Sarnococcaceae, and Maitake are preferably the scientific name Grifola galgal whose Japanese name is Anninkou.

  The present invention also includes ergothioneine, a pharmacologically acceptable salt thereof, or a compound that becomes ergothioneine by hydrolysis as an active ingredient, and has a fat-reducing activity, an insulin secretion-promoting activity, and a blood glucose-lowering activity. It relates to a composition exhibiting at least one activity selected from the group consisting of (hereinafter sometimes referred to as “third composition”).

  The present invention separates the supernatant from the dried powder of mushrooms belonging to the genus Basidiomycetes, Hymenoptera, Sarnococcaceae, Maitake, with hot water from the mixture obtained in step (1) and step (1) A method for preparing a composition exhibiting at least one activity selected from the group consisting of a fat-reducing activity, an insulin secretion-promoting activity, and a blood glucose-lowering activity, comprising the step (2) and the step (3) of concentrating the supernatant. (Hereinafter sometimes referred to as “first preparation method”).

  The present invention separates the supernatant from the dried powder of mushrooms belonging to the genus Basidiomycetes, Hymenoptera, Sarnococcaceae, and Maitake, from the mixture obtained in step (I) and step (I). Step (II) and the step (III) of obtaining a low molecular fraction by fractionating the components in the supernatant with a separation membrane having a fractional molecular weight in the range of 30,000 to 10,000. And a method for preparing a composition exhibiting at least one activity selected from the group consisting of fat-reducing activity, insulin secretion-promoting activity and blood glucose-lowering activity (hereinafter also referred to as “second preparation method”).

  The present invention also relates to a method for reducing fat, enhancing insulin action, or lowering blood glucose level, wherein a hot water extract from a fungus belonging to the genus Basidiomycetes, Hymenoptera, Sarnochaeaceae, Maitake genus, The present invention relates to a method comprising administering in a pharmacologically effective amount (hereinafter sometimes referred to as “first administration method”).

  Furthermore, the present invention relates to a method for reducing fat, enhancing insulin action, or lowering blood glucose level, comprising extracting a hot water extract from a mushroom belonging to the genus Basidiomycetes, Hymenoptera, Sarnostocidae, and Maitake. A method comprising administering a pharmacologically effective amount of a low molecular fraction obtained by separation with a separation membrane having a molecular weight cut off within a range of 30,000 to 10,000 (hereinafter referred to as “No. The second administration method ”).

  The present invention provides a composition effective for reducing visceral fat and blood neutral fat and a method for preparing the same. The present invention also provides a composition having insulin secretion promoting activity or blood glucose lowering activity and a method for preparing the same. Furthermore, the present invention provides a method for reducing fat, a method for enhancing insulin action, and a method for lowering blood glucose level. The composition of the present invention is useful as a prophylactic / therapeutic agent for metabolic syndrome and as a prophylactic / therapeutic agent for diabetes. It is considered that at least a part of the blood glucose lowering activity is attributed to the activity of increasing the uptake of sugar (glucose) into cells exhibited by the composition. Therefore, the composition of the present invention is also useful as a promoter for the ability of cells to take up glucose.

  The composition of the present invention is useful for prevention and treatment of metabolic syndrome and for prevention and treatment of diabetes. Since the raw material or origin of the composition of the present invention is food, the composition is very safe and it is expected that no side effects will occur.

The body weight at the time of dissection of the control group and the low-dose group of mice is shown. The body weight at the time of dissection of the control group and the high-dose group of mice is shown. The liver weight at the time of dissection of a control group and a low-dose group of mice is shown. The liver weight at the time of dissection of a control group and a high-dose group of mice is shown. The spleen weight at the time of dissection of the control group and the low-dose group of mice is shown. The spleen weight at the time of dissection of the control group and the high-dose group of mice is shown. Fig. 3 shows the weight of fat around the testis at the time of dissection of the control group and low-dose group of mice. Fig. 2 shows the weight of the testicular fat at the time of dissection of the control group and the high-dose group of mice. The amount of triglyceride in plasma at the time of dissection of a control group and a low-high dose group of mice is shown. The body weight at the time of dissection of a control group, a fruit body-added feed group, and a low-molecular-fraction-added feed group of mice is shown. The liver weight at the time of dissection of a control group, a fruit body-added feed group and a low molecular fraction-added feed group of mice is shown. The spleen weight at the time of dissection of a control group, a fruit body-added feed group, and a low-molecular-fraction-added feed group of mice is shown. Fig. 4 shows the weight of fat around the testis at the time of dissection of a control group, a fruit body-added feed group and a low-molecular-fraction-added feed group of mice. The amount of triglyceride in plasma at the time of dissection of a control group, a fruit-body-added feed group, and a low-molecular-fraction-added feed group of mice is shown. The feed intake of a control group of normal mice (described as “normal diet”, hereinafter the same) and a low molecular fraction added diet group (described as “sample-added diet”, hereinafter the same) are shown. The feed intake of the control group of obese mice and the low molecular fraction added feed group is shown. The amount of drinking water of the control group of normal mice and the low molecular fraction added feed group is shown. The amount of drinking water of the control group of obese mice and the low molecular fraction added feed group is shown. The blood glucose level of the control group of a normal mouse | mouth and an obese mouse | mouth, and a low molecular fraction addition feed group is shown. The blood glucose level before and after fasting of the control group of a normal mouse | mouth and an obese mouse | mouth, and a low molecular fraction addition feed group is shown. The body weight at the time of dissection of the control group of normal mice and obese mice and the low molecular fraction added diet group is shown. The liver weight at the time of dissection of the control group of normal mice and obese mice and the low molecular fraction added diet group is shown. The spleen weight at the time of dissection of the control group of normal mice and obese mice and the low molecular fraction added diet group is shown. The testicular fat weight at the time of dissection of a control group of normal mice and obese mice and a low molecular fraction added diet group is shown. The number of red blood cells (RBC) at the time of dissection of the control group of normal mice and obese mice and the low molecular fraction added diet group is shown. The hemoglobin (HGB) density | concentration at the time of dissection of the control group of a normal mouse | mouth and an obese mouse | mouth and a low molecular fraction addition feed group is shown. The hematocrit (HCT) value at the time of dissection of the control group of a normal mouse | mouth and an obese mouse | mouth and a low molecular fraction addition feed group is shown. The erythrocyte volume (MCV) at the time of dissection of a control group of normal mice and obese mice and a low molecular fraction added diet group is shown. The white blood cell (WBC) count at the time of dissection of the control group of normal mice and obese mice and the low molecular fraction-added feed group is shown. The number of platelets (PLT) at the time of dissection of the control group of normal mice and obese mice and the low molecular fraction added diet group is shown. The total protein (TP) amount in plasma at the time of dissection of a control group and a low molecular fraction added diet group of normal mice and obese mice is shown. The albumin / globulin (A / G) ratio in plasma at the time of dissection of the control group of normal mice and obese mice and the low molecular fraction added diet group is shown. The total cholesterol (T-Cho.) Value in plasma at the time of dissection of a control group and a low molecular fraction added diet group of normal mice and obese mice is shown. The amount of triglyceride (TG) in plasma at the time of dissection of the control group of normal mice and obese mice and the low molecular fraction added diet group is shown. The leptin concentration of plasma at the time of dissection of the control group of normal mice and obese mice and the low molecular fraction added diet group is shown. The adiponectin concentration of plasma at the time of dissection of the control group of normal mice and obese mice and the low molecular fraction added diet group is shown. The insulin concentration of the plasma at the time of dissection of the control group of normal mice and obese mice and the low molecular fraction added diet group is shown. The measurement result of sugar (glucose) uptake activity is shown.

  The first composition according to the present invention contains a hot water extract from mushrooms belonging to the genus Basidiomycetes, Hydrangea, Sarnococcidae, and Maitake, and comprises fat-reducing activity, insulin secretion-promoting activity, and blood glucose-lowering activity A composition exhibiting at least one activity selected from the group.

  Mushrooms belong to the family Basidiomycetes, Hymenoptera, Sarnococcidae, Maitake, and hot water extracts from the mushrooms have at least one activity selected from the group consisting of fat reduction activity, insulin secretion promoting activity and blood glucose lowering activity. As long as it contains the component shown, it is not particularly limited.

  Examples of mushrooms belonging to the basidiomycetes, Hydrangea, Sarnococcidae, and Maitake genus include Gryfora galgar (scientific name: Grifola gargal), Gryfora soldulenta (scientific name: Grifola sordulenta), : Grifola frontosa). The first two are mushrooms that grow in Argentina and Chile, especially in the Patagonia region of Chile. An artificial cultivation method of Anninkou is disclosed in, for example, Japanese Patent Application Laid-Open No. 2007-20560. Maitake mushrooms are widely artificially cultivated in Japan.

  Among mushrooms belonging to the family Basidiomycetes, Hydrangea, Sarnococcaceae, and Maitake genus, Anninkou is most preferred. Anninkou is particularly preferable when the composition of the present invention is expected to have insulin secretion promoting activity and / or blood glucose lowering activity. The mushroom may be a mycelium or a fruit body, but a fruit body is preferred.

  The “hot water extract” from the mushrooms is not limited to the hot water extract itself. For example, hot water extracts from the above mushrooms such as hot water extract concentrates and freeze-dried hot water extracts do not significantly impair fat-reducing activity, insulin secretion promoting activity or blood glucose-lowering activity. Such a treatment, that is, “derived from a hot water extract” from the above mushrooms is also included in the concept of “hot water extract”.

  The method for preparing the “hot water extract” from the mushrooms is not particularly limited except that the first stage is hot water extraction. A preferred method will be described later as the first preparation method.

  The second composition according to the present invention is a hot water extract from a mushroom belonging to the genus Basidiomycetes, Hymenoptera, Sarnococcidae, and Maitake, which has a fractional molecular weight in the range of 30,000 to 10,000. The composition contains a low molecular fraction obtained by separation with a separation membrane, and exhibits at least one activity selected from the group consisting of fat reduction activity, insulin secretion promoting activity and blood glucose lowering activity.

  The “low molecular fraction” refers to a fraction that permeates through the membrane when the hot water extract is fractionated at a certain molecular weight with an ultrafiltration membrane, for example. The “low molecular fraction” is not limited to the fraction permeated through the membrane itself. For example, the low molecular fraction, such as a concentrated solution of a low molecular fraction or a freeze-dried product of a low molecular fraction, is treated so that fat reducing activity, insulin secretion promoting activity or blood glucose lowering activity is not significantly impaired. In other words, the “low molecular fraction” is also included in the concept of “low molecular fraction”.

  In the “low molecular fraction” in the present invention, among the components contained in the hot water extract from the above mushrooms, the separation molecular weight is any one within the range of 30,000 to 10,000. The component which permeate | transmitted the film | membrane is contained. The molecular weight cut-off is preferably in the range of 20,000 to 10,000, more preferably in the range of 13,000 to 10,000, and 13,000. Is particularly preferred.

  The third composition according to the present invention contains ergothioneine, a pharmacologically acceptable salt thereof, or a compound that becomes ergothioneine by hydrolysis as an anti-inflammatory active ingredient. Here, ergothioneine is represented by the formula described in the following [Chemical Formula 1].

  The “pharmacologically acceptable salt” is, for example, an alkali or alkaline earth metal salt in which the hydrogen of the hydroxyl group is replaced with sodium, potassium, calcium or the like in the formula described in the above [Chemical Formula 1]. Or an ammonium salt substituted with an ammonium group, hydrogen of a hydroxyl group or hydrogen of an imino group, an inorganic acid such as hydrochloric acid or sulfuric acid, an organic acid such as p-toluenesulfonic acid, glycine An addition salt added with an amino acid such as hydrate or the like added with water. However, it is not limited to these, and any may be used as long as it is pharmacologically acceptable.

  The “compound that becomes ergothioneine by hydrolysis” means, for example, that the hydroxyl group in the formula described in the above [Chemical Formula 1] is protected by a protecting group, in other words, the hydrogen atom in the hydroxyl group is , A compound that is substituted by some group and becomes a hydroxyl group when hydrolyzed. Examples of such “hydroxyl groups protected by protecting groups” include methoxymethyl ether group, tetrahydropyranyl ether group, tertiary butyl ether group, allyl ether group, benzoate group, acetate group (acetyloxy group), Examples include a formate group, a crotonate group, a p-phenylbenzoic acid ester group, a trimethylacetyloxy group, a tertiary butyldimethylsilyloxy group, a tertiary butyldiphenylsilyloxy group, a trityloxy group, and a benzyloxy group.

  In the present invention, “containing ergothioneine, a pharmacologically acceptable salt thereof, or a compound that becomes ergothioneine by hydrolysis as an active ingredient” means that the third composition of the present invention shows fat-reducing activity, insulin With respect to at least one activity selected from the group consisting of secretory promoting activity and hypoglycemic activity, ergothioneine in an amount such that at least a portion of that activity is exhibited by ergothioneine, preferably primarily as indicated by ergothioneine, The pharmacologically acceptable salt or the compound which becomes ergothioneine by hydrolysis is contained in the composition.

  Ergothioneine can be synthesized, for example, by converting histidine into hercynin and reacting it with cysteine. Ergothionein and the like can also be extracted from natural products. The natural product used for extraction of ergothioneine and the like is not particularly limited as long as it contains ergothioneine and no toxic substances are extracted at the same time as the extraction of ergothioneine. Examples of natural products suitable for extraction of ergothioneine and the like include edible mushrooms. In addition, specific examples of edible mushrooms include basidiomycetes, cynomolgus mushrooms, sorghum mushrooms, and mushrooms described above in the description of the first composition of the present invention.

  When the composition of the present invention is a composition exhibiting a fat reducing activity, specific indicators of the presence or absence of fat reducing activity include, for example, the presence or absence of a decrease in visceral fat and the decrease in blood neutral fat (triglyceride). Or not. There is no particular limitation on the principle or mechanism of action that causes such fat loss.

  When the composition of the present invention is a composition showing insulin secretion promoting activity, the presence of insulin secretion promoting activity can be detected, for example, by measuring the insulin concentration in plasma. There are no particular limitations on the principle or mechanism of action that exerts insulin secretion promoting activity. In addition, when the composition of the present invention is a composition exhibiting a blood glucose lowering activity, the blood glucose lowering activity can be detected by measuring fasting blood glucose or a blood glucose level increase and decrease pattern after glucose load. I can do it. There is no particular limitation on the principle or mechanism of action in which the blood glucose lowering activity is expressed. However, it is considered that at least a part of the blood glucose lowering activity is expressed by the activity of increasing the sugar (glucose) uptake ability of cells exhibited by the composition of the present invention.

  In the case of the first composition, the composition of the present invention comprises the hot water extract described above, the low molecular fraction described above in the case of the second composition, and the low molecular fraction in the case of the third composition. Ergothioneine, a pharmacologically acceptable salt thereof, or a compound that becomes ergothioneine by hydrolysis is contained as an essential component. Other components may or may not be contained. Other components are not particularly limited as long as they do not inhibit the target activity.

  The composition according to the present invention is for oral administration, for example, in the form of tablets, capsules, granules, fine granules, powders, etc., and for parenteral administration, for example, injections, inhalants, It can be provided in a dosage form such as a suppository or a transdermal absorption agent. Therefore, additives, excipients, binders, disintegrants, solvents and the like that are generally used to obtain the dosage forms in such fields can be used as other components. In addition, other compounds exhibiting a synergistic or additive effect with the above essential components may also be used as other components.

  The composition of the present invention preferably contains the essential component as a main component. Here, “main component” means that 30% or more, preferably 50% or more, more preferably 70% or more of the target activity (for example, fat reduction activity) exhibited by the composition depends on the essential components. It is expressed.

  The intake amount of the first composition of the present invention is not particularly limited, but is about 0.3 to 2 g / kg body weight in terms of the amount of lyophilized powder of hot water extract per day for an adult. It is more preferable that it is about 0.5 to 1.2 g / kg body weight. The intake amount of the second composition of the present invention is not particularly limited, but is about 0.25 to 1.80 g / kg body weight in terms of the amount of lyophilized powder of the low molecular weight fraction per day for an adult. It is preferably about 0.40 to 1.10 g / kg body weight. The intake of the third composition of the present invention is not particularly limited, but is preferably 2 to 16 mg / kg body weight in terms of the amount of ergothioneine per day for an adult, and is preferably 2 to 8 mg / kg- More preferably it is body weight.

  The present invention also relates to a method for preparing a composition exhibiting at least one activity selected from the group consisting of fat reducing activity, insulin secretagogue activity and blood glucose lowering activity. The first method is a step (1) of extracting from a dry powder of mushrooms with hot water, a step (2) of separating the supernatant from the mixture obtained in step (1), and the supernatant. Step (3) of concentrating. The raw material used in this method is a dried powder of mushroom fruiting bodies or mycelium belonging to the order of the mushrooms, the moss family, and the genus Maitake. The method for drying mushrooms is, for example, applying warm air of 40 to 50 ° C. (preferably around 45 ° C.) to the mushroom for one day and then warm air of 60 to 80 ° C. (preferably around 70 ° C.) for about 1 hour, for example. It's what you hit. Powderization is performed, for example, by pulverizing the dried mushrooms with a mixer.

  In the method of the present invention, hot water is used as the extraction solvent. Hot water is water at about 80-100 ° C, preferably about 90-100 ° C. The hot water extraction is performed, for example, by putting a dry powder of mushrooms in hot water and stirring the mixture of mushrooms and hot water while maintaining the temperature (step (1)).

  Upon completion of extraction, that is, step (1), the supernatant is separated from the mixture obtained in step (1) (step (2)). This step is preferably performed after the dry powder of mushrooms has been precipitated by centrifugation. Further, this step may be performed by separating and removing the dried powder of mushrooms by passing the mixture obtained in step (1) through a sieve.

  Steps (1) and (2) may be performed once. Usually, fresh hot water is added to the mushroom remaining after step (2), and further, stirring extraction (step (1)) and supernatant liquid are added. Separation (step (2)) is performed. Steps (1) and (2) are preferably performed 2 to 5 times, particularly preferably 3 or 4 times, for example, if the stirring time in step (1) is about 30 minutes.

  Since the active ingredient concentration in the supernatant obtained in step (2) is low, the supernatant is concentrated in step (3). The concentration is not limited as long as the active ingredient, for example, ergothionein, is not decomposed.

  After the concentration step (3), a freezing or spray drying step (step (4)) is also preferably performed. Note that the freezing or spray drying may be performed under the conditions and methods normally performed in this field.

  It is also preferable to perform the step (5) of fractionating the concentrate obtained in the step (3) by, for example, reverse phase high performance liquid chromatography (reverse phase HPLC). Thereby, the active ingredient concentration can be increased. Furthermore, the fraction showing the target activity obtained in the step (5) is frozen or spray-dried in the same manner as in the step (6) and / or in the same manner as in the step (4). It is also preferable to carry out step (7).

  A method for preparing a composition exhibiting at least one activity selected from the group consisting of fat reducing activity, insulin secretagogue activity and blood glucose lowering activity of the present invention, the second method comprising: Extraction with hot water (I), step (II) of separating the supernatant from the mixture obtained in step (I), and components in the supernatant have a molecular weight cutoff of 30,000 to 10, And fractionating with a separation membrane in any of the range of 000 to obtain a low molecular fraction (III).

  Steps (I) and (II) of the second method are the same as steps (1) and (2) of the first method, respectively. In step (III), the components contained in the supernatant obtained in step (II) are fractionated by a separation membrane having a fractional molecular weight in the range of 30,000 to 10,000. Obtain the molecular fraction. The molecular weight cut-off is preferably in the range of 20,000 to 10,000, more preferably in the range of 13,000 to 10,000, and 13,000. Is particularly preferred. When fractionating the components contained in the supernatant with a separation membrane, the supernatant may be used as it is, after being concentrated to some extent, or once the supernatant is freeze-dried, An aqueous solution obtained by pulverizing by a method such as spray drying and dissolving the powder in water may be used.

  It is also preferable to carry out the concentration step (IV) and / or the freeze or spray drying step (V) after the fractionation step (III). Concentration, freezing or spray drying may be carried out under conditions and methods usually performed in this field.

  The invention also relates to a method of reducing fat, enhancing insulin action or lowering blood glucose levels. The first administration method is a method comprising administering a hot water extract from a mushroom belonging to the genus Basidiomycetes, Hymenoptera, Sarnococcidae, and Maitake in a pharmacologically effective amount. In addition, the second administration method is one in which the molecular weight cut off of a hot water extract from a mushroom belonging to the genus Basidiomycetes, Hymenoptera, Sarnococcidae, and Maitake is in the range of 30,000 to 10,000. This is a method comprising administering a low molecular fraction obtained by separation with a separation membrane in a pharmacologically effective amount. As the mushroom, anninninko is particularly preferable.

  In carrying out the administration method of the present invention, the hot water extract (in the case of the first administration method) or the low molecular fraction (in the case of the second administration method) described above is used as it is or in such a field. Along with commonly used additives, excipients, binders, disintegrants, solvents, etc., effective doses for subjects (humans and mammals) who intend to reduce fat, enhance insulin action or lower blood glucose levels To administer. An “effective amount” is an amount that achieves the purpose of administration, specifically, an amount that reduces fat, an amount that enhances insulin action, or an amount that lowers blood glucose level .

  The administration method is not particularly limited. Oral administration or parenteral administration may be used. Moreover, the dosage form at the time of administration is not particularly limited. The appropriate dosage form is selected according to the administration method

  Hereinafter, the present invention will be described specifically by way of examples.

(Example 1) Hot water extraction of active ingredients from Anninkou (Part 1)
(1) Preparation of dry powder of mushrooms The body of Anninkou (manufactured by Iwate Mycology Laboratory Co., Ltd.) was exposed to 45 ° C warm air all day and then 70 ° C warm air for 1 hour. . The obtained dried dried carrot body was put into a mixer and pulverized for about 1 minute to obtain a powder.

(2) Hot water extraction and fractionation by ultrafiltration membrane 10 g of dried powder of anninko fruiting body was mixed with 200 ml of hot water (about 90 ° C.), and the resulting mushroom / hot water mixture was gently stirred for 30 minutes. did. The mushroom / hot water mixture was centrifuged (10,000 rpm, 10 minutes) and divided into supernatant and precipitate.

  The precipitate was mixed with 200 ml of hot water (about 90 ° C.) and the resulting mushroom / hot water mixture was gently stirred for 30 minutes. The mushroom / hot water mixture was centrifuged (10,000 rpm, 10 minutes) and divided into supernatant and precipitate. This process was performed twice more.

  All supernatants were combined, a portion was taken, concentrated under reduced pressure, and then lyophilized. The remaining supernatant was filtered through an ultrafiltration membrane (Asahi Kasei UF Module Microza RACP; molecular weight cut off: 13,000) manufactured by Asahi Kasei Chemicals Corporation to obtain a low molecular fraction. This was concentrated to about 100 ml or less under reduced pressure, and then lyophilized.

  The ratio of the weight of the hot water extract after lyophilization to the weight of the dry powder of carrot fruit used as a raw material was about 45%, and the ratio of the weight of the low molecular weight fraction was about 40%.

(Example 2) Confirmation of the presence of ergothioneine in hot water extract from Anninkou and its quantification (1) Preparation of sample Anninkou (Iwate Mycology Research Institute, Inc., artificial cultivation in the same manner as in Example 1) The supernatant was obtained from the dried powder of the fruiting body by hot water extraction. All supernatants were combined and concentrated under reduced pressure, then made up to 100 ml.

(2) Examination of the presence or absence of ergothioneine A hot water extract (mesh-up to 100 ml) and an ergothioneine sample (purchased from Sigma) were added to Sep Pak (C18) (manufactured by Waters). ) And then subjected to HPLC. The HPLC conditions were as follows.

(HPLC conditions)
Column: ODS-HG-5 C18 (4.6 mmφ × 250 mm) (manufactured by Nomura Chemical Co., Ltd.), two were connected in tandem.
Eluent: H ₂ O / 0.1% triethylamine (TEA)
Flow rate: 0.8 ml / min Indicator for identification of fraction containing ergothioneine: Absorbance at 260 nm

  Both the hot water extract and the ergothioneine preparation were absorbed at a retention time of about 9 minutes. Therefore, it was revealed that the hot water extract contains ergothioneine.

(3) Determination of ergothioneine Aqueous solutions (plural) of ergothioneine preparations were prepared, their absorbance at 260 nm was measured, and a calibration curve was prepared. All the ergothioneine-containing fractions fractionated in (2) (derived from the hot water extract not containing the ergothioneine preparation) were combined, the absorbance at 260 nm was measured, and the ergothioneine concentration was determined from the calibration curve. The concentration of the ergothioneine-containing fraction was multiplied by the concentration to calculate the total amount of ergothioneine, and the result was divided by the weight of the dry carrot fruit body powder used to calculate the weight of ergothioneine per gram of the carrot fruit body dry powder. The ergothioneine content per gram of dried carrot fruit body was about 2.0 mg.

(Example 3) Measurement of visceral fat reducing activity and neutral fat reducing activity of hot water extract and low molecular fraction (Part 1)
Normal mice (ICR males) were directly orally administered (free administration of each component by gastric sonde) of the lyophilized product of the hot water extract prepared in Example 1 and the lyophilized product of the low molecular fraction. Similarly, ergothioneine was directly orally administered. Two weeks after the start of administration, the mice were dissected, and body weight, fat around the testis, and neutral fat (triglyceride) in blood were measured.

(1) Animals used ICR mice (slc: 6 weeks old, male, purchased from Japan SLC) were used. After 1 week of preliminary breeding, it was used for experiments at 7 weeks of age. There were 5 animals in each group.

(2) Samples and doses by gavage administration The samples and doses subjected to gavage administration are as follows.
(2-1) Anninkou hot water extract The freeze-dried product was administered at 1,000 mg / kg-body weight (A) or 4,000 mg / kg-body weight (B).
(2-2) Anninkou low molecular fraction The lyophilized product was administered at 1,000 mg / kg-body weight (A) or 4,000 mg / kg-body weight (B).
(2-3) Ergothioneine (purchased from Sigma)
5 mg / kg-body weight (A) or 20 mg / kg-body weight (B) was administered.

(3) Breeding method and administration method
Nosan experimental animal feed lab MR stock (LABO MR STOCK; 259.2 kcal / 100 g; Nippon Agricultural Industrial Co., Ltd.) was fed as a basic (normal) feed. The basic feed was fed to the control group and each gavage group.

  Forcible oral administration was performed from 10 am to 11 am 5 times a week (Tuesday and Saturday and Sunday) using a gastric sonde for mice. In the control group, tap water was similarly administered by oral gavage.

(4) Blood collection and dissection of mice (administration period: 2 weeks)
Two weeks after the start of the experiment, the mouse was killed by anesthesia with ethyl ether, the abdomen was immediately opened, and heparinized blood was collected from the abdominal vena cava with a 2.5 ml syringe. Prior to anesthesia, fasting and other measures were not taken, and the mice were under normal free feeding and drinking. The centrifuged plasma was stored in a freezer at −80 ° C., and triglyceride (neutral fat) was measured by an inspection company.

(5) Results (5-1) Anatomical findings No abnormalities such as bleeding and dirt around the anus were observed on the appearance of the mouse at the time of dissection, and there were no abnormalities such as bleeding or adhesions in the abdominal cavity and chest cavity. In addition, in any individual, a large amount of contents were observed in the stomach and cecum.

(5-2) Body weight FIG. 1 shows body weights at the time of dissection of the control group (Cont-2W) and the low-administration group (that is, the group (A) having a small dose for each administration sample). Moreover, the body weight at the time of dissection of a control group (Cont-2W) and a high-administration group (namely, group (B) with a large dose for each administration sample) is shown in FIG.

  Compared to the control group, there was a tendency for the body weight to decrease in the sample administration group, especially in the ergothioneine administration group, 6.1% (low administration group; A) or 8.0% (high administration group; B). It was a decline. However, these changes in body weight during dissection were not significant changes.

(5-3) Liver weight The liver weight was expressed as an absolute weight obtained by measuring the weight of the liver extracted at the time of dissection and a relative weight per 10 g of body weight obtained by dividing the weight of the liver by the individual body weight. The data for the control group (Cont-2W) and the low-dose group (A) are shown in FIG. 3, and the data for the control group (Cont-2W) and the high-dose group (B) are shown in FIG. Although a weight loss of 13.9% was observed in the ergothioneine-administered group B (high-administration group) relative to the control group, it was not a change with a significant difference.

(5-4) Spleen Weight The spleen weight was expressed as an absolute weight obtained by measuring the weight of the spleen removed at the time of dissection and a relative weight per 10 g of body weight obtained by dividing the spleen weight by the individual body weight. The data of the control group (Cont-2W) and the low administration group (A) are shown in FIG. 5, and the data of the control group (Cont-2W) and the high administration group (B) are shown in FIG. In any group, there was no significant change compared to the control group.

(5-5) Weight of fat (peritoneal fat) Since the fat around the testis was easily collected and the weight was constant, it was measured as a representative of intraperitoneal fat. The weight of fat extracted at the time of dissection was expressed as an absolute weight and a relative weight per 10 g of body weight obtained by dividing the weight of fat by individual body weight. The data of the control group (Cont-2W) and the low administration group (A) are shown in FIG. 7, and the data of the control group (Cont-2W) and the high administration group (B) are shown in FIG.

  The fat absolute weight of the control group is 1012 ± 55 mg (n = 5), the low molecular fraction administration group (A: 8.6%; B: 24.8%), the hot water extract administration group (A: 18) .6%; B: 20.3%) and ergothioneine-administered group (A: 29.7%; B: 35.1%) showed a weight reduction with a clear significant difference from the control group. It was. 7 and 8, * is P <0.05, and ** is P <0.01 (the same applies to the following drawings).

(5-6) Triglyceride (neutral fat)
The amount of triglyceride in plasma (unit: mg / ml) is shown in FIG. A decrease in the amount of triglyceride was observed in the low molecular fraction administration group, the hot water extract administration group, and the ergothioneine administration group compared to the average value (150 ± 12 mg / dl) of the control group (Cont-2W). It was. In particular, in the low molecular fraction administration group (A), the hot water extract administration groups (A) and (B), and the ergothioneine administration group (B), a decrease with an obvious significant difference was observed with respect to the control group. It was.

(5-7) Summary As described above, the hot water extract from the fruit body of Annin-ko fruit, the low molecular fraction with a molecular weight of 13,000 or less obtained from the hot water extract, and ergothioneine are visceral fat and blood triglyceride. It has been clarified that the effect of reducing (neutral fat) is exhibited, that is, the fat reducing activity is exhibited. Moreover, at least a part of the above-mentioned effects of the hot water extract from the fruit body of anninkou and low molecular fraction may be exhibited by ergothioneine contained therein.

(Example 4) Measurement of visceral fat reducing activity and neutral fat reducing activity of annin carrot fruit body and low molecular fraction (part 2)
Normal mice (ICR males) were fed with the dried dried carrot fruit body (powder) prepared in Example 1 and the lyophilized product of the low molecular weight fraction. Four weeks after the start of administration, the mice were dissected and the body weight, peri-testicular fat weight, and blood neutral fat (triglyceride) were measured.

(1) Animals used ICR mice (slc: 6 weeks old, male, purchased from Japan SLC) were used. After 1 week of preliminary breeding, it was used for experiments at 7 weeks of age. There were 6 animals in each group.

(2) Bait The following food was given. In addition, tap water was used for drinking water and was freely consumed.
(2-1) Control group Nosan experimental animal feed lab MR stock (LABO MR STOCK; 259.2 kcal / 100 g; Nippon Nosan Kogyo Co., Ltd.) was given as a basic (normal) feed.
(2-2) Anninkou fruit body added feed group Feed obtained by adding 5.01% by weight of dried anninko fruit body prepared in Example 1 (powder) to Nosan experimental animal feed laboratory MR stock Gave.
(2-3) Anninkou low molecular fraction added feed group The lyophilized product of the low molecular fraction prepared in Example 1 was added at a ratio of 2.175% by weight to the feed laboratory MR stock for No-san experimental animals. Feeded.

(3) Breeding method
In each group, the feed and water were freely consumed.

(4) Blood collection and dissection of mice (administration period: 4 weeks)
Four weeks after the start of the experiment, the mice were killed by anesthesia with ethyl ether, the abdomen was immediately opened, and heparinized blood was collected from the abdominal vena cava with a 2.5 ml syringe. Prior to anesthesia, fasting and other measures were not taken, and the mice were under normal free feeding and drinking. The centrifuged plasma was stored in a freezer at −80 ° C., and triglyceride (neutral fat) was measured by an inspection company.

(5) Results (5-1) Anatomical findings No abnormalities such as bleeding and dirt around the anus were observed on the appearance of the mouse at the time of dissection, and there were no abnormalities such as bleeding or adhesions in the abdominal cavity and chest cavity. In addition, in any individual, a large amount of contents were observed in the stomach and cecum.

(5-2) Body weight The body weights at the time of dissection of the control group (Cont-4W), the fruit body-added feed group and the low molecular fraction-added feed group are shown in FIG. These three groups had approximately the same weight at the time of dissection.

(5-3) Liver weight The liver weight was expressed as an absolute weight obtained by measuring the weight of the liver extracted at the time of dissection and a relative weight per 10 g of body weight obtained by dividing the weight of the liver by the individual body weight. The results are shown in FIG. There was a weight increase of 9.4% in the fruit body-added feed group and 6.9% in the low-molecular-weight-added feed group compared to the control group (Cont-4W), but this was not a significant change. It was.

(5-4) Spleen Weight The spleen weight was expressed as an absolute weight obtained by measuring the weight of the spleen removed at the time of dissection and a relative weight per 10 g of body weight obtained by dividing the spleen weight by the individual body weight. The results are shown in FIG. Although a slight increase was observed in the low molecular fraction added feed group compared to the control group (Cont-4W), it was not a change with a significant difference.

(5-5) Weight of fat (peritoneal fat) Since the fat around the testis was easily collected and the weight was constant, it was measured as a representative of intraperitoneal fat. The weight of fat extracted at the time of dissection was expressed as an absolute weight and a relative weight per 10 g of body weight obtained by dividing the weight of fat by individual body weight. The results are shown in FIG.

  The absolute fat weight of the control group (Cont-4W) is 851 ± 92 mg (n = 6), which is a 16.5% weight reduction compared to the control group (breeding period: 2 weeks) in Example 3. there were. In addition, in the fruit-body-added feed group and the low-molecular-weight fraction-added feed group, the fat weight decreased with a significant difference compared to the control group (fruit-body-added feed group: 30.3%; low-molecular-weight fraction-added feed). Group: 37.7%).

(5-6) Triglyceride (neutral fat)
The amount of triglyceride in plasma (unit: mg / ml) is shown in FIG. The average value of the control group (Cont-4W) was 116 ± 16 mg / dl (n = 6), which was a 22% decrease compared to the control group in Example 2 (breeding period: 2 weeks). . In addition, the fruit body-added feed group and the low-molecular-weight fraction-added feed group showed a decrease in the amount of triglyceride with a clear significant difference compared to the control group (fruit-body-added feed group: 37.1%; low-molecular-weight fraction-added feed). Group: 21.6%).

(5-7) Summary From the above, the dry body powder of anninko and the low molecular fraction of the hot water extract from the dried fruit body of anninkou are contrasted with visceral fat and blood triglyceride (neutral fat). It became clear that the effect of reducing with a significant difference was demonstrated with respect to a group, ie, a fat reduction activity.

(Example 5) Examination of the effect of administration of a low molecular fraction on obese mice Obese mice (C57BL / 6JamSlc-ob / ob) and syngeneic normal mice (C57BL / 6CrS) are bred and measurement of feed intake, etc. Blood glucose level measurement, anatomical examination, blood biochemical value measurement, and hormone measurement were performed. Seven normal mice were fed a normal diet, eight normal mice were fed a low molecular fraction lyophilized product (prepared in the same manner as in Example 1), obese Ten mice were fed the normal feed, and ten obese mice were fed the low molecular fraction lyophilized product (prepared in the same manner as in Example 1). .

(1) Materials (1-1) Animals used and breeding period Obese mice (C57BL / 6JhamSlc-ob / ob; male; 6 weeks old; Japan SLC) and syngeneic normal mice (C57BL / 6CrS; male; 6 weeks old) Japan SLC) was purchased and subjected to the experiment at 7 weeks of age after 1 week of preliminary breeding. The experimental period was 6 weeks.

(1-2) Feed For control mice, as a basic (ordinary) feed, a feed laboratory MR stock for Nosan laboratory animals (LABO MR STOCK; 259.2 kcal / 100 g; Nippon Agricultural Industry Co., Ltd .; hereinafter referred to as “normal diet”) ) Was given. In the test mouse, a solid feed (Lot. No. S-090310; Nippon Agricultural Industry Co., Ltd.) in which 1.5% by mass of a lyophilized product of a low molecular fraction prepared in the same manner as in Example 1 was added to the basic feed. Production; hereinafter referred to as “sample-added food”).

(1-3) Breeding method Each group was allowed to ingest the feed and tap water freely.

(2) Measurement of feed intake and drinking water (2-1) Measurement method For each feed box attached to the breeding gauge, the weight of the ingested feed is measured three times a week and divided by the number of breeding animals. The daily feed intake per animal was determined. Tap water was used as the drinking water, and the weight of the drinking water taken from the water bottle was measured three times a week and divided by the number of breeding animals to obtain the amount of drinking water per animal per day. Eating and drinking spills were not considered and included in the intake. The measurement was performed from 10 am to 11 am.

(2-2) Results The feed intake of normal mice is shown in FIG. 15, the feed intake of obese mice is shown in FIG. 16, the amount of water consumed by normal mice is shown in FIG. 17, and the amount of water consumed by obese mice is shown in FIG.

(Feed intake) There was no significant difference between normal and obese mice between the normal diet group and the sample-added diet group. Throughout the experimental period, the sample-added food intake of normal mice was 3.59 g / day / mouse, and the intake of the low molecular fraction was 53.8 mg / day / mouse. The sample-fed food intake of obese mice was 6.09 g / day / mouse, and the intake of the low molecular fraction was 91.3 mg / day / mouse. When the intake of the low molecular fraction is converted per kg body weight, it is 2.02 g / kg / day for normal mice (average body weight: 26.6 g) and 1.75 g / kg for obese mice (average body weight: 52.1 g). / Day, and in terms of adult (weight: 50 kg), the intake is 88 to 101 g / day.
(Water consumption) Only normal mice showed a significant increase in water consumption in the sample-added food group compared to the normal food group.

(3) Measurement of blood glucose level (3-1) Measurement method At 10 to 11 am, blood was collected by incising the tail vein of a mouse. The blood glucose level was measured using a blood glucose measurement kit (PRECISION XCEED G3 ABBOTT JAPAN CO. LTD.). Blood was collected before the start of the experiment (week 0) and thereafter once a week. Only after 5 weeks of breeding, fasting for 16 hours (drinking water only) was performed, and blood was collected before and after fasting.

(3-2) Results FIG. 19 shows a graph showing changes in blood glucose level. Moreover, the bar graph which shows the blood glucose level before and after fasting five weeks after the experiment start is shown in FIG.
As is clear from FIG. 19, in the obese mice, the sample-added diet administration period was 2 weeks or more, and a decrease in blood glucose level was observed compared to the normal diet group. There was no such difference in normal mice.
Usually, the diet rhythm of mice is concentrated at night. However, in obese mice, there are individuals who ingest food in the morning and noon, and there may be individual differences in blood glucose levels at the time of blood collection (10 to 11 am). As shown in FIG. 20, individual differences in diet rhythm were eliminated by fasting, and both the normal and obese mice showed significantly lower blood glucose levels in the sample-added diet group than in the normal diet group.

(4) Anatomy, measurement of blood biochemistry and obesity-related hormones (4-1) Blood collection Six weeks after the start of the experiment, at 10 am to 11 am, mice were killed by ethyl ether overanesthesia, and the abdomen was immediately opened. Then, heparinized blood was collected from the abdominal vena cava with a 2.5 ml syringe. Prior to anesthesia, fasting and other measures were not taken, and the mice were under normal free feeding and drinking. A part of the blood was used for blood count measurement, and the rest was centrifuged to obtain plasma, which was stored in a freezer at -30 ° C.

(4-2) Anatomy At the time of dissection, abnormalities such as organ position, bleeding and adhesion were examined closely. In addition, body weight, liver weight, spleen weight and testicular fat weight were measured.

(4-3) Measurement of blood cell count The collected blood is immediately subjected to an automatic blood cell counter (PocH-100i; manufactured by Sysmex Corporation), and the red blood cell count, hemoglobin concentration, hematocrit value, red blood cell volume, white blood cell count and platelet count are measured. did.

(4-4) Measurement of blood biochemical values For plasma, total protein amount, albumin / globulin ratio, total cholesterol amount and triglyceride amount were measured by SRL Corporation.

(4-5) Measurement of obesity-related hormones For plasma, the leptin concentration was measured using Mouse Leptin ELISA KIT, the high molecular weight adiponectin concentration was measured using Mouse / Rat High Weight Adipectin ELISA KIT, and Mouse InSulinITK was also used. Insulin concentration was measured using -011T). All of these kits are manufactured by Shibayagi Co., Gunma Prefecture, Japan.

(4-6) Results
(4-6-1) Anatomical findings No abnormalities such as body hair and dirt around the anus were observed in the appearance of the mouse at the time of dissection, and there were no abnormalities such as bleeding or adhesions in the abdominal cavity and chest cavity. In addition, in any individual, a large amount of contents were observed in the stomach and cecum.

(4-6-2) Body weight, liver weight, spleen weight and peri-testicular fat weight The body weight is shown in FIG. 21, the liver weight is shown in FIG. 22, the spleen weight is shown in FIG. 23, and the peri-testicular fat weight is shown in FIG. The body weight, liver weight, and spleen weight were not affected by the sample-added diet. Regarding the testicular fat weight, the relative weight per 10 g body weight of both normal and obese mice was significantly lower in the sample-added diet group than in the normal diet group.

(4-6-3) Blood cell count The red blood cell (RBC) number is shown in FIG. 25, the hemoglobin (HGB) concentration is shown in FIG. 26, the hematocrit (HCT) value is shown in FIG. 27, the red blood cell volume (MCV) is shown in FIG. The (WBC) number is shown in FIG. 29, and the platelet (PLT) number is shown in FIG. Only in normal mice, the sample-added diet group showed significantly lower values for the red blood cell count, hemoglobin concentration, hematocrit value, and white blood cell count than the normal diet group.

(4-6-4) Blood Biochemical Value FIG. 31 shows the total protein (TP) amount in plasma, FIG. 32 shows the albumin / globulin (A / G) ratio, and FIG. 32 shows the total cholesterol (T-Cho.) Amount. 33 shows the amount of triglyceride (TG). Regarding the amount of triglyceride, the normal food group and the obese mouse showed significantly lower values in the sample-added food group than in the normal food group.

(4-6-5) Measurement of Hormone FIG. 35 shows the leptin concentration in plasma, FIG. 36 shows the high molecular weight adiponectin (Adiponectin) concentration, and FIG. 37 shows the insulin (Insulin) concentration.
Leptin is synthesized and secreted mainly by adipocytes and works in the hypothalamus and participates in feeding regulation. From FIG. 35, it is considered that obese mice are in a leptin-resistant state in which the appetite due to leptin is not suppressed, although the leptin concentration in plasma is higher than that in normal mice in both the normal diet and the sample-added diet. It was. In addition, the lower leptin concentration in the sample-added diet group compared to the normal diet group seemed to reflect the amount of fat accumulated in the abdominal cavity.

  Adiponectin is expressed specifically in adipose tissue. Adiponectin expression and secretion decreases with obesity and adipocyte hypertrophy. Since adiponectin exhibits an insulin sensitivity enhancing action, it becomes insulin resistant in the case of hypoadiponectinemia. As shown in FIG. 36, the adiponectin concentration in obese mice was about twice that in normal mice, but there was no significant difference between the normal diet group and the sample-added diet group. That is, it was considered that the low molecular fraction had no significant effect on insulin sensitivity.

  As shown in FIG. 37, in the normal diet group, the plasma insulin concentration of obese mice was 20 times or more that of normal mice. In the sample-added diet group of obese mice, the blood insulin concentration was 2.5 times that of the normal diet group. Therefore, it was clarified that the low molecular fraction derived from the sample, ie, the hot water extract of Anninkou, exhibits an insulin secretion enhancing action. And it was thought that the blood glucose level decreased in the sample-added diet group of obese mice as compared with the normal diet group of obese mice (see FIGS. 19 and 20).

(5) Summary From the above, it has been clarified that the low molecular fraction of the hot water extract from dried ginseng fruit body has insulin secretion-promoting activity, thereby exhibiting a hypoglycemic effect. In addition, it was revealed that the low molecular fraction also has an effect of reducing visceral fat and blood triglyceride (neutral fat) with a significant difference from the control group.

(Example 6) Hot water extraction of active ingredients from ginseng (part 2)
(1) Preparation of dry powder of mushrooms The body of Anninkou (manufactured by Iwate Mycology Laboratory Co., Ltd.) was exposed to 45 ° C warm air all day and then 70 ° C warm air for 1 hour. . The obtained dried dried carrot body was put into a mixer and pulverized for about 1 minute to obtain a powder.

(2) Hot water extraction and fractionation by ultrafiltration membrane 10 g of dried powder of anninko fruiting body was mixed with 200 ml of hot water (about 90 ° C.), and the resulting mushroom / hot water mixture was gently stirred for 30 minutes. did. The mushroom / hot water mixture was centrifuged (10,000 rpm, 10 minutes) and divided into supernatant and precipitate.

  The precipitate was mixed with 200 ml of hot water (about 90 ° C.) and the resulting mushroom / hot water mixture was gently stirred for 30 minutes. The mushroom / hot water mixture was centrifuged (10,000 rpm, 10 minutes) and divided into supernatant and precipitate. This process was performed twice more.

  Combine all the supernatants, take half of them, and filter with an ultrafiltration membrane (Asahi Kasei UF Module Microza RACP; molecular weight cut off: 13,000) manufactured by Asahi Kasei Chemicals Corporation. (1) was obtained. The remaining supernatant was filtered through an ultrafiltration membrane (Pellicon XL device; fractional molecular weight: 10,000) manufactured by Millipore to obtain a low molecular fraction (2). These were each concentrated to about 100 ml or less under reduced pressure conditions, and then lyophilized.

Example 7 Measurement of Cellular Sugar (Glucose) Uptake Activity
According to the method of Yamamoto et al. (Yamamoto N., et al., Anal. Biochem., 2006, April 1, 351 (1), pp 139-145), insulin and the low molecular fraction derived from anninko prepared in Example 6 ( The effects of 1) and (2) on the uptake of sugar (glucose) by cells were examined.

(1) Measurement (1-1) Production of L6 skeletal muscle cells MEM medium A (10% (v / v) fetal bovine serum-containing MEM medium with L6 myoblasts at a concentration of 2 × 10 ⁴ cells / ml ). The obtained cell dispersion medium was placed in a 96-well tissue culture plate at 200 μl per well and cultured at 37 ° C. for 2 days in a 5% carbon dioxide incubator. After becoming confluent, the medium was removed by aspiration and changed to 200 μl of MEM medium B (MEM medium containing 2% (v / v) fetal calf serum) and cultured at 37 ° C. for 5 days in a 5% carbon dioxide incubator. . In this way, L6 skeletal muscle cells were prepared. Again, the medium was removed by aspiration and changed to 200 μl of MEM medium C (MEM medium containing 0.2% (w / v) bovine serum albumin (BSA)) and cultured at 37 ° C. for 18 hours in a 5% carbon dioxide incubator. After that, it was used for measurement of sugar (glucose) uptake activity.

(1-2) Preparation of sample (a) Anninkou-derived low molecular fraction (1) Lyophilized product and (b) Anninkou-derived low molecular fraction (2) Lyophilized product was dissolved in ultrapure water, MEM medium C was diluted 100-fold, and MEM medium containing 250 μg / ml, 500 μg / ml, or 1,000 μg / ml of any of the lyophilized products was prepared. Further, as a negative control, MEM medium C not containing an anninkou-derived low molecular fraction was prepared. Furthermore, as a positive control, insulin was added to MEM medium C to prepare MEM medium with insulin concentrations of 1 nM, 10 nM, and 100 nM. A sample for the co-exposure test of anninkou and insulin was prepared as follows. Lyophilized product of low molecular fraction (1) or (2) derived from Anninkou was dissolved in ultrapure water and diluted 100-fold in MEM medium with insulin concentrations of 1 nM, 10 nM, and 100 nM prepared as a positive control. Then, an MEM medium containing 500 μg / ml of the lyophilized product together with insulin was prepared.

(1-3) Measurement of sugar uptake activity in L6 skeletal muscle cells (1-3-1) Preparation of measurement sample 96-well tissue culture plate of L6 skeletal muscle cells prepared as described in (1-1) Then, after removing the medium in each well by suction, 200 μl of any of the samples prepared in (1-2) was added per well, and cultured at 37 ° C. for 4 hours in a 5% carbon dioxide incubator.
Thereafter, Krebs-Ringer-Hepes (KRH) buffer (50 mM HEPES, pH 7.4; 137 mM NaCl, 4.8 mM KCl, 1.85 mM CaCl ₂ , 1.3 mM containing 0.1% (w / v) BSA MgSO ₄ containing) used in per well 150 [mu] l / dose and was washed 2 times. Next, 100 μl of KRH buffer containing 1 mM 2-deoxyglucose (2-DG) and 0.1% (w / v) BSA was added per well, and 37% in a 5% carbon dioxide incubator. Incubated for 20 minutes. Subsequently, washing was performed twice using KRH buffer containing 0.1% (w / v) BSA at 200 μl / well per well.
50 μl of 0.1N sodium hydroxide aqueous solution was added per well, heat-treated for 10 minutes with a 60 ° C. dry heat sterilizer, and stirred with a microplate mixer to lyse the cells. Next, the 96-well tissue culture plate was left in a dry heat sterilizer at 80 ° C. for 50 minutes to dry each hole. 50 μl of 0.1N hydrochloric acid aqueous solution was added per well, and then 50 μl of 200 mM triethanolamine (TEA) aqueous solution (pH 8.1) was added per well, followed by stirring with a microplate mixer. In this way, a measurement sample was prepared.

(1-3-2) Measurement of 2-DG 10 μl per well of a measurement sample was added to a 96-well plate, and then assay cocktail (50 mM TEA, pH 8.1; 50 mM KCl, 0.02% (w / V) BSA, 0.1 mM NADP, 2 μM resazurin, 2 units / ml diaphorase, 150 units / ml glucose-6-phosphate dehydrogenase) 100 μl was added. After stirring with a microplate mixer, it was incubated at 37 ° C. for 50 minutes in a 5% carbon dioxide incubator.
Immediately thereafter, fluorescence intensity was measured with a microplate reader (excitation wavelength: 530 nm; fluorescence wavelength: 570 nm). A calibration curve was prepared using 2-DG-6-phosphate as a standard substance, and 2-DG uptake per well was calculated for each sample.

(2) Results 2-DG uptake (ratio of 2-DG uptake) when cells were treated with low molecular fraction derived from Anninkou and insulin when the negative control 2-DG uptake was 1.0. Is shown in FIG. In FIG. 38, the negative control is “Control”, the Anninkou-derived low molecular fraction (1) is lyophilized product “Galgar (1)”, and the Anninkou-derived low molecular fraction (2) lyophilized product is “ "Galgal (2)".
As is clear from FIG. 38, both the Ginseng-derived low molecular fraction (1) lyophilized product and the Ginseng-derived low molecular fraction (2) lyophilized product promoted glucose uptake by L6 skeletal muscle cells as in the case of insulin. Moreover, under the co-exposure with insulin, both the low molecular fraction derived from Annin (1) and the low molecular fraction derived from Annin (2) and the freeze dried product showed additive glucose uptake. The components inside were found to have the ability to induce glucose uptake through an insulin-independent pathway.

  The present invention can be applied to pharmaceuticals and health foods for reducing visceral fat and blood neutral fat. The present invention can also be applied to pharmaceuticals and health foods for lowering blood glucose levels.

Claims (21)

At least selected from the group consisting of fat-reducing activity, insulin secretion-promoting activity, and blood glucose-lowering activity, characterized by containing a hot water extract from a basidiomycete, Hydrangea, Sarnococcidae, Mushrooms belonging to the genus Maitake A composition exhibiting one activity. The composition according to claim 1, which exhibits fat-reducing activity. The composition according to claim 1 or 2, wherein the mushrooms belonging to the family Basidiomycetes, Hydrangea, Sarnococcidae, and Maitake are Anninko (scientific name: Grifola galgal). The composition according to any one of claims 1 to 3, which exhibits an activity of increasing cellular sugar uptake. It is obtained by separating a hot water extract from a mushroom belonging to the genus Basidiomycetes, Hydrangea, Sarnococcidae, and Maitake, with a separation membrane having a fractional molecular weight in the range of 30,000 to 10,000. A composition exhibiting at least one activity selected from the group consisting of fat-reducing activity, insulin secretion-promoting activity and blood glucose-lowering activity, comprising a low molecular fraction. 6. A composition according to claim 5, which exhibits fat reducing activity. The composition according to claim 5 or 6, wherein the mushrooms belonging to the family Basidiomycetes, Hydrangea, Sarnococcidae, and Maitake are Anninko (scientific name: Grifola galgal). The composition according to any one of claims 5 to 7, which exhibits an activity of increasing cellular sugar uptake. Ergothioneine, a pharmacologically acceptable salt thereof, or a compound that becomes ergothioneine by hydrolysis as an active ingredient, and is selected from the group consisting of fat-reducing activity, insulin secretion-promoting activity, and blood glucose-lowering activity A composition exhibiting at least one activity. 10. A composition according to claim 9, which exhibits fat reducing activity. The composition according to claim 9 or 10, wherein the composition contains a hot water extract from a mushroom belonging to the genus Basidiomycetes, Hymenoptera, Sarnosidaceae, and Maitake. The composition is a separation membrane having a molecular weight cut-off within a range of 30,000 to 10,000 from a hot water extract from a mushroom belonging to the genus Basidiomycetes, Hymenoptera, Sarnosidaceae, and Maitake. The composition of Claim 9 or 10 containing the low molecular fraction obtained by isolate | separating. The composition according to any one of claims 9 to 12, which exhibits an activity of increasing cellular sugar uptake. Step (1) for extracting with hot water from dried powder of mushrooms belonging to the family Basidiomycetes, Hydrangea, Sarnococcidae, Maitake, and step (2) for separating the supernatant from the mixture obtained in step (1) And a method for preparing a composition exhibiting at least one activity selected from the group consisting of fat-reducing activity, insulin secretion-promoting activity, and blood glucose-lowering activity, comprising the step (3) of concentrating the supernatant. The method according to claim 14, wherein the mushroom belonging to the genus Basidiomycetes, Hyderna mushrooms, Sarnococcaceae, and Maitake is Anninkou (scientific name: Grifola galgal). Step (I) for extracting with hot water from dried powder of mushrooms belonging to the family Basidiomycetes, Hydrangea, Sarnococcidae, Maitake, and step (II) for separating the supernatant from the mixture obtained in step (I) And fractionation of the components in the supernatant with a separation membrane having a molecular weight cut off within the range of 30,000 to 10,000 to obtain a low molecular fraction (III) A method for preparing a composition exhibiting at least one activity selected from the group consisting of activity, insulin secretion-promoting activity and blood glucose lowering activity. The preparation method according to claim 16, wherein the mushrooms belonging to the genus Basidiomycetes, Hyderna mushrooms, Sarnococcidae, and Maitake are ninko (scientific name: Grifola galgal). A method for reducing fat, enhancing insulin action, or lowering blood glucose level, comprising a pharmacologically effective amount of a hot water extract from a basidiomycete, Physcomitrella, Sarnococcidae, Mushrooms belonging to the genus Maitake Administering. The method according to claim 18, wherein the mushroom belonging to the basidiomycetes, Hyderna mushrooms, Sarnococcidae, and Maitake mushrooms is Anninkou (scientific name: Grifola galgal). A method for reducing fat, enhancing insulin action, or lowering blood glucose level, comprising a hot water extract from a fungus belonging to the genus Basidiomycetes, Hymenoptera, Sarnococcidae, Mushrooms, having a molecular weight cut off of 30,000 A method comprising administering, in a pharmacologically effective amount, a low molecular fraction obtained by separation with a separation membrane in a range of 1 to 10,000. 21. The method according to claim 20, wherein the mushrooms belonging to the basidiomycetes, Hyderna mushrooms, Sarnococcidae, and Maitake genus are Anninko (scientific name: Grifola galgal).

Images