JP7329768B2 - Diabetes prophylactic and therapeutic agents, blood glucose level elevation inhibitors, blood glucose level spike inhibitors and glucose uptake inhibitors - Google Patents

Description

TECHNICAL FIELD The present invention relates to a natto-derived preventive/therapeutic agent for diabetes, an agent for suppressing an increase in blood sugar level, an agent for suppressing blood sugar level spike, and an agent for inhibiting glucose uptake.

At present, obesity due to excessive calorie intake and the subsequent onset of lifestyle-related diseases such as diabetes are problems. It is said that taking care not to increase the blood sugar level on a daily basis is important in suppressing the onset of lifestyle-related diseases. For the prevention of lifestyle-related diseases, diet therapy and exercise therapy are considered effective, and in diet therapy, calorie intake is restricted mainly so as not to raise blood sugar levels.
However, it may be psychologically difficult to continue such diet therapy, and substances (ingredients, agents, etc.) that suppress the rise in blood sugar levels and foods and drinks containing such substances (including health foods) The development is believed to be effective in implementing continuous therapy.

Natto is a traditional Japanese food made by fermenting soybeans with Buccilus subtilis var natto. When Bacillus natto proliferates on the surface of soybeans, various polysaccharides are produced and the soybeans become viscous.

As a technology related to Bacillus subtilis natto, nattokinase is known to be useful as a therapeutic drug for diabetes (Patent Document 1). However, nattokinase is a type of enzyme protein contained in natto, and is not a water-soluble sugar (chain component) in the first place. Ta.

An antidiabetic composition containing a composition obtained by fermenting soybean protein and rice bran with Bacillus natto and further treating the fermented product with protease is known (Patent Document 2).
Also, an anti-diabetic composition containing a fermented soybean composition obtained by fermenting a solid fraction of ground soybean is known (Patent Document 3).
However, these were not all water soluble or were antidiabetic drugs associated with increased insulin levels.

Regarding sugar contained in natto, the current situation is as follows.
That is, natto contains polysaccharides called polyglutamic acid and levan, and polyglutamic acid (poly-γ-glutamic acid) is used as a base material for drug delivery systems such as insulin. . However, there is no report that polyglutamic acid has an activity to suppress the elevation of blood sugar level after ingestion of sucrose.

In addition, levan has various functions such as moisturizing properties, and is used in the development of food additives, cosmetics, and pharmaceuticals. However, even if natto-derived levan is administered, no effect of lowering blood glucose levels is observed in normal rats or streptozotocin-treated diabetic rats, and it has been reported that there is no improvement in diabetic symptoms (Non-Patent Document 1 ).

That is, there is no report showing that the water-soluble viscous component (fraction) derived from natto has the effect of suppressing the increase in blood sugar level after ingestion of sucrose.

On the other hand, conventionally, mammals such as mice and rats have been used to suppress postprandial hyperglycemia and to evaluate preventive/therapeutic agents for diabetes. However, many problems have been pointed out in terms of not only cost but also animal welfare for subjecting a large number of mammals to experiments.
The present inventors established a method (screening method) for searching for an agent for suppressing an increase in human blood sugar level and an active substance for suppressing postprandial hyperglycemia in humans by an evaluation system using silkworms, and found that an inhibitor for postprandial hyperglycemia and new prophylactic/therapeutic agents for diabetes (Patent Documents 4, 5, Non-Patent Documents 2, 3, 4, etc.).

A transient increase in blood sugar level due to sucrose intake is called a blood sugar level spike, and is one of the factors that cause diabetes. Suppression of this blood sugar level spike is thought to be useful in preventing the onset of diabetes, and the development of substances (ingredients), drugs, health foods, food and drink, etc. having such effects is desired.

JP 2004-115434 A JP 2013-032334 A JP 2013-075883 A JP 2009-058500 A International Publication No. WO/2017/061353

de Melo FC, Zaia CT, Celligoi MA. Levan from Bacillus subtilis Natto: its effects in normal and in streptozotocin-diabetic rats. Braz J Microbiol. 2012; 43:1613-1619 Matsumoto Y, Ishii M, Sekimizu K. An in vivo invertebrate evaluation system for identifying substances that suppress sucrose-induced postprandial hyperglycemia. Scientific reports. 2016; 6:26354. Ishii M, Matsumoto Y, Sekimizu K. Bacterial polysaccharides inhibit sucrose-induced hyperglycemia in silkworms. Drug discoveries & therapeutics. 2018; 12:185-188. Ishii M, Matsumoto Y, Sekimizu K. Inhibitory effects of alpha-cyclodextrin and its derivative against sucrose-induced hyperglycemia in an in vivo evaluation system. Drug discoveries & therapeutics. 2018; 12:122-125

An object of the present invention is to provide an agent that contains a safe and secure naturally-derived ingredient and prevents or treats diabetes by suppressing elevation of blood sugar level through a mechanism of action different from that of promoting insulin secretion.

As a result of intensive studies to solve the above problems, the present inventors have found that the water-soluble viscous fraction of natto exhibits an inhibitory effect on the increase in blood sugar level caused by ingestion of sucrose or glucose in silkworms. Found it.
In addition, the present inventors have found that a polysaccharide fraction obtained by enzymatic treatment of the water-soluble viscous fraction of natto, that is, a sugar chain component also has an activity of suppressing an increase in blood sugar level after ingestion of sucrose, leading to the present invention.
In addition, as described above, it has been confirmed that suppression of blood sugar level elevation in humans and suppression of postprandial hyperglycemia in humans can be evaluated using an evaluation system using silkworms. Search methods (screening methods) for substances having the above effects on humans have also been established (Patent Documents 4, 5, Non-Patent Documents 2, 3, 4, etc.).
In the present invention, the above evaluation system for saccharides using silkworms is simply abbreviated as "silkworm evaluation system".

Furthermore, the present inventors demonstrated that the enzyme-treated polysaccharide fraction (sugar chain component) of natto inhibits glucose uptake in human intestinal cells using Caco-2 cells derived from the human intestinal tract. led to the present invention.

That is, the present invention provides a preventive and therapeutic agent for diabetes characterized by containing a water-soluble viscous fraction of natto or a sugar chain component contained in a water-soluble viscous fraction of natto.

In addition, the present invention provides a blood sugar elevation inhibitor characterized by containing a water-soluble viscous fraction of natto or a sugar chain component contained in a water-soluble viscous fraction of natto.

The present invention also provides an intestinal glucose uptake inhibitor comprising a water-soluble viscous fraction of natto or a sugar chain component contained in the water-soluble viscous fraction of natto. .

The present invention also provides a blood sugar level spike inhibitor comprising a water-soluble viscous fraction of natto or a sugar chain component contained in the water-soluble viscous fraction of natto.

In addition, the present invention is characterized by having a step of screening the type of natto using a silkworm evaluation system. The object is to provide a method for producing or screening an agent.

ADVANTAGE OF THE INVENTION According to this invention, the natto which is familiar as foodstuffs is used as a raw material, and the agent which can suppress a blood glucose level rise can be obtained from there. That is, according to the present invention, it is possible to provide a naturally derived (general food-derived) blood sugar level elevation inhibitor, glucose uptake (transport) inhibitor, etc. that are safe and give a sense of security.

In addition, since the agent of the present invention can inhibit glucose uptake from the intestinal tract, the blood glucose concentration can be suppressed.

All of the above-mentioned effects of the present invention relate to suppression of blood glucose concentration by ingestion of sugar such as sucrose. It is extremely effective in suppressing a blood sugar level spike, in which the blood sugar level rises sharply immediately after taking a meal.

Agents for treating diabetes include those that promote insulin secretion (improve hyposecretion), those that eliminate (improve) insulin resistance, and those that suppress glucose absorption. It is something that does.
The absorption and transport of sugar includes taking sugar into cells of the intestinal tract and releasing sugar from the cells of the intestinal tract into the blood. According to the present invention, as shown in Example 5, In particular, the former can be inhibited, that is, the uptake of glucose into intestinal cells can be inhibited.

The blood sugar level spike described above occurs even in healthy people. INDUSTRIAL APPLICABILITY The present invention can be applied not only to diabetic patients, but also to people who want to prevent diabetes, people who want to reduce diabetes, healthy people, and the like.

Water-soluble viscous fraction of natto, voglibose, which is an α-glycosidase inhibitor in humans, was mixed with a feed containing sucrose and administered to silkworms, and then the glucose concentration in body fluids of silkworms was measured Graph showing the results is. 1 is a graph showing the dose dependence of the glucose concentration in body fluids of silkworms after an evaluation sample was mixed with a feed containing sucrose and administered to the silkworms. A. Graph showing dose dependence of water-soluble viscous fraction of natto B. Graph showing dose dependence of indigestible dextrin 2 is a graph showing the results of measuring the glucose concentration in body fluids of silkworms after sugar chain components (enzyme-treated polysaccharide fraction) in natto were mixed with sucrose-containing feed and administered to silkworms. Fig. 2 is a graph showing the results of measuring the glucose concentration in body fluids of silkworms after a water-soluble viscous fraction of natto was mixed with a sucrose-containing feed or a glucose-containing feed and administered to silkworms. 2 is a graph showing that addition of a sugar chain component (enzyme-treated polysaccharide fraction) in natto inhibits glucose uptake of Caco-2 cells, which are human intestinal cells (control is indigestible dextrin).

The present invention will be described below, but the present invention is not limited to the following specific embodiments, and can be arbitrarily modified within the scope of the technical idea.

The present invention is a preventive and therapeutic agent for diabetes characterized by containing a water-soluble viscous fraction of natto or a sugar chain component contained in a water-soluble viscous fraction of natto.
Further, the present invention is also a blood sugar elevation inhibitor characterized by containing a water-soluble viscous fraction of natto or a sugar chain component contained in a water-soluble viscous fraction of natto.

Although the present invention is not limited, it is also a preventive/therapeutic agent for diabetes that is not for promoting insulin secretion or a blood glucose elevation suppressing agent that is not for promoting insulin secretion. That is, the present invention is not excluded from the present invention until some insulin secretion is concomitantly involved in the mechanism of action, but the present invention is a non-insulin secretion-promoting diabetes preventive and therapeutic agent or a non-insulin secretion promoting agent. It is also an insulin secretion-promoting agent for suppressing elevation of blood sugar level.
In other words, the present invention is a non-insulin-dependent preventive/therapeutic agent for diabetes mellitus or a non-insulin-dependent inhibitor of blood glucose level elevation, which is not for promoting insulin secretion but for improving insulin resistance (or reducing resistance). It is also a preventive/therapeutic agent for diabetes mellitus or an agent for suppressing elevation of blood sugar levels.

According to the present invention, it was found that the water-soluble viscous fraction of natto or the sugar chain component contained in the water-soluble viscous fraction of natto suppresses blood sugar spikes and inhibits glucose uptake from the intestinal tract. Ta.
None of the above-described effects are related to insulin secretion, so the present invention is a preventive/therapeutic agent for diabetes mellitus or an agent for suppressing elevation of blood sugar level, which is not for promoting insulin secretion.

Since the promotion of insulin secretion by β-cell stimulation may lead to β-cell exhaustion and side effects on the pancreas, the present invention has the above-mentioned effects and is used for the above-mentioned purposes. Diabetes prophylactic/therapeutic agents or blood glucose elevation inhibitors are extremely useful.

As shown in the examples of the present application, inhibition of glucose uptake from the intestinal tract is useful for prevention of diabetes, suppression of blood sugar level rise, suppression of blood sugar level spikes, etc. Therefore, the preventive and therapeutic agent for diabetes of the present invention or blood sugar level The increase suppressing agent can be suitably applied not only to type 2 diabetes patients but also to potential type 2 diabetes patients, type 1 diabetes patients, healthy individuals who want to prevent them, and the like.

The type of natto used as a raw material for the "water-soluble viscous fraction of natto" and "the sugar chain component contained in the water-soluble viscous fraction of natto" is not particularly limited as long as it exhibits the above-described effects of the present invention. do not have. As shown in Example 1, some of the evaluated natto #1, #2, #3 and #4 greatly suppressed the increase in blood glucose concentration after ingestion of sucrose (Fig. 1 ).

However, among those evaluated in Example 1, natto #4 was the most effective (there was a difference between the types of natto), so multiple natto were evaluated to adopt highly effective natto. is preferred. "Natto" contains beans and Bacillus natto as raw materials. Therefore, in the present invention, "types of natto" refer to at least types such as soybeans and types of Bacillus subtilis natto.
Each type of natto had different inhibitory effects on the increase in blood glucose level after sucrose ingestion in silkworms. Therefore, it is considered that the type of natto with higher activity can be searched for using the silkworm evaluation system.
Therefore, the present invention uses a silkworm evaluation system and has a step of screening natto that exhibits the above effects or the effects of the following agents. It is also a production method or a screening method for inhibitors or blood sugar level spike inhibitors.

"Water-soluble viscous fraction of natto" means adding water to natto in an amount 2 to 10 times the weight of the natto, stirring with a stirrer at 20 ° C., and then filtering out the remaining soybeans. , refers to a precipitate obtained when an aqueous solution, which is a filtrate, is obtained and ethanol is added to the aqueous solution in an amount of 2 volumes.
However, if it is obtained as described above, it is the "water-soluble viscous fraction of natto" in the present invention, and it is not limited to what is actually obtained by the above specific acquisition method.

"Sugar chain component contained in the water-soluble viscous fraction of natto" is synonymous with "enzyme-treated polysaccharide fraction", and the above-described water-soluble viscous fraction of natto is treated with an enzyme as shown in Example 3. It refers to the precipitate formed by adding ethanol after treatment with
However, if it is obtained as described above, it is the "sugar chain component contained in the water-soluble viscous fraction of natto" and the "enzyme-treated polysaccharide fraction" in the present invention, which is obtained by the above-described specific method. It is not limited to what is actually obtained.

The present invention is an intestinal glucose uptake inhibitor comprising a water-soluble viscous fraction of natto or a sugar chain component contained in the water-soluble viscous fraction of natto, and suppresses blood sugar spikes. Also a drug.
Example 5 demonstrated that the "sugar chain component (enzyme-treated polysaccharide fraction) contained in the water-soluble viscous fraction of natto" inhibits glucose uptake (or transport) from the intestinal tract. Therefore, the "water-soluble viscous fraction of natto" containing it can also be a glucose uptake inhibitor.

Furthermore, the fact that the "water-soluble viscous fraction of natto" can be a "glucose uptake inhibitor from the intestinal tract" was found in Example 4 to suppress the increase in blood sugar level even when not only sucrose but also glucose was ingested. It has also been proved by what

In addition, in all of Examples 1 to 4, the blood glucose concentration was measured 1 hour after the meal (feeding), and the effect of suppressing the increase in glucose concentration was confirmed, so the water-soluble viscous fraction of natto It was demonstrated that sugar chain components (enzyme-treated polysaccharide fractions) contained in the water-soluble viscous fraction of natto can also serve as blood sugar spike inhibitors. The water-soluble viscous fraction of natto suppresses postprandial hyperglycemia by inhibiting glucose uptake by intestinal cells, and is therefore useful as a postprandial hyperglycemia inhibitor.

Moreover, it is clear that if at least glucose uptake is inhibited, it can be used as an agent for suppressing elevation of blood sugar levels and a preventive and therapeutic agent for diabetes.

The agent of the present invention can be used by being contained in general food and drink, health food and drink, drugs, and the like. The agent of the present invention may be in a wet state by containing water or the like, or may be in a powder state after being dried.

When the agent of the present invention is used by being contained in general food and drink, the agent itself may be eaten or eaten, and it is also preferable to dilute with a liquid such as water or an edible solid before use. In addition, when it is contained in general food and drink, it may be blended during cooking, and it is also preferable to use it as sprinkles, seasonings, and the like.

The types of the general food and drink are not particularly limited, but for example, confectionery such as jelly, candy, chocolate, biscuits, gummy; Dairy products such as cream and lacto ice; Processed vegetables and fruits such as vegetable drinks, fruit drinks, and jams; Liquid foods such as soup; Processed grain products such as rice, bread, and noodles; foods; side dishes such as meat, fish, and vegetables; and the like.

There is no particular limitation on the dosage form when used by containing it in health foods, drinks, drugs, etc. Specifically, for example, oral solid preparations (tablets, coated tablets, granules, powders, hard capsules, soft capsules agents, etc.), oral liquids (oral solutions, syrups, elixirs, etc.), injections (solvents, suspensions, etc.), and the like.
Among them, oral solid preparations and oral liquid preparations are preferable because they are simple and easily exhibit the above effects.

As the oral solid preparation, the "water-soluble viscous fraction of natto or the sugar chain component contained in the water-soluble viscous fraction of natto" of the present invention includes, for example, excipients, binders, disintegrants, and lubricants. , coloring agents, flavoring agents, storage stabilizers, enteric agents, disintegration retardants, etc., and can be produced by a conventional method.

Examples of excipients include lactose, white sugar, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid and the like.
Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like. be done.
Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, lactose and the like.
Examples of the lubricant include refined talc, stearate, borax, polyethylene glycol and the like.
Examples of the coloring agent include titanium oxide and iron oxide.
Examples of the flavoring/flavoring agent include sucrose, orange peel, citric acid, and tartaric acid.

The oral liquid preparation can be produced, for example, by adding additives such as flavoring agents, buffering agents, stabilizers, etc. to the active ingredient and by a conventional method.

Examples of the flavoring/flavoring agent include sucrose, orange peel, citric acid, and tartaric acid.
Examples of the buffer include sodium citrate and the like.
Examples of the stabilizer include tragacanth, gum arabic, gelatin and the like.

For the injection, for example, the active ingredient is blended with a pH adjusting agent, a buffering agent, a stabilizer, a tonicity agent, a local anesthetic, etc., and is administered subcutaneously, intramuscularly, or intravenously by a conventional method. etc. can be manufactured.
Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of the isotonizing agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.

Animals to which the agent of the present invention is administered include, but are not limited to, humans; monkeys; horses; domestic animals such as cows, pigs, goats and chickens; pets such as cats and dogs; ; and the like.

The dosage of the agent of the present invention is not particularly limited, and can be appropriately selected according to the age and body weight of the subject to be administered, the degree of desired effect, etc. For example, the daily dosage for adults is , The amount of the water-soluble viscous fraction of natto is preferably 1 mg to 30 g, more preferably 10 mg to 10 g, and particularly preferably 100 mg to 3 g.
The preferred dosage of the sugar chain component (enzyme-treated polysaccharide fraction), or when it is contained in an oral solid preparation or oral liquid preparation, the above preferred dosage is applied in terms of the amount of the water-soluble viscous fraction of natto. be done.

EXAMPLES The present invention will be described in more detail below based on examples and study examples, but the present invention is not limited to the specific scope of the following examples and the like.

Evaluation example 1
<Types of silkworms, rearing conditions, and method of collecting bodily fluids>
Silkworms were reared as previously reported (Kaito C, Akimitsu N, Watanabe H, Sekimizu K. Silkworm larvae as an animal model of bacterial infection pathogenic to humans. Microbial pathogenesis. 2002; 32:183-190. Kurokawa K, Kaito C, Sekimizu K. Two-component signaling in the virulence of Staphylococcus aureus: a silkworm larvae-pathogenic agent infection model of virulence. Methods Enzymol. 2007; 422:233-244.).

Silkworm fertilized eggs (hybrid fu-yo x tsukuba-ne) were purchased from Ehime Sericulture Co., Ltd.
The hatched larvae were fed artificial feed Silkmate 2S (Nippon Nosan Kogyo Co., Ltd.) at room temperature and raised to the 5th instar larvae. As the rearing container, square No. 2 petri dishes (Eiken Kizai) were used for eggs to second instar larvae, and disposable plastic food packs (Food Pack FD Obuka, Chuo Kagaku Co., Ltd.) were used for the rearing containers. The breeding temperature was 27°C. Unless otherwise specified, larvae on the 1st day of the 5th instar that had been fasted after the 4th instar sleep were used in the experiments.

Sucrose was mixed with artificial feed so that the sucrose content was 10% by mass, and a sample (specimen) to be evaluated was further mixed. After feeding the 5th instar silkworm with a sample (sample) mixed food or unmixed food for 1 hour, the silkworm's legs were cut with scissors to collect body fluid, and the glucose concentration was measured with a glucometer ( Accu-Chek, Roche).

Example 1
<Proof that the water-soluble viscous fraction of natto has the effect of suppressing the increase in glucose concentration in the blood after sucrose intake and the effect of suppressing the blood sugar spike after sucrose intake (1)>
Water was added to 4 types of commercially available natto (#1, #2, #3, #4), and the mixture was stirred well, after which soybeans were removed to obtain a water extract fraction. Twice as much ethanol was added thereto, and the filamentous precipitates formed were collected by centrifugation and designated as "water-soluble viscous fractions #1, #2, #3 and #4".

The obtained water-soluble viscous fractions (Crude fraction) #1-4, respectively, 25 mg / g diet), and voglibose (40 mg / g diet), which is an α-glycosidase inhibitor, were added to 10% by mass of sucrose. was mixed into a bait containing
The silkworms were fed with the bait mixed with each sample (water-soluble viscous fraction #1-4, voglibose) or with the unmixed bait (Control), and after 1 hour the glucose concentration in the bodily fluid of the silkworm was measured. .

The results are shown in FIG.
n=4-5/group, statistical significance test was performed using Student's t-test. Bars in FIG. 1 indicate the average value of each sample.
Data marked with "*" are P<0.05.

<<Results>>
From FIG. 1, it was suggested that the water-soluble viscous fraction obtained from natto has the effect of suppressing the increase in blood sugar level after ingestion of sucrose in silkworms. Compared with the feed (Control) that did not contain the water-soluble viscous fraction of natto, some of the silkworm body fluids showed suppression of the increase in glucose concentration. In particular, among the four types of natto evaluated, the water-soluble viscous fraction obtained from one type of natto (#4) was found to greatly suppress the increase in the blood sugar level of silkworms.

As described above, the suppression of blood sugar level elevation in silkworms by the administered test substance is correlated with humans (because it has been confirmed that similar results are obtained in humans). It was suggested that the water-soluble viscous fraction has an effect of suppressing elevation of blood sugar level, and that it can be used as an agent for suppressing elevation of blood sugar level.
Moreover, since the above results were obtained one hour after feeding, it was suggested that there is also a use as a blood sugar level spike inhibitor.

Example 2
<Proof that the water-soluble viscous fraction of natto has the effect of suppressing the increase in glucose concentration in the blood after sucrose intake and the effect of suppressing the blood sugar spike after sucrose intake (2)>
<<Dose dependence of suppression (inhibition) by the water-soluble viscous fraction of natto on the increase in blood glucose level>>
The dose dependence of the water-soluble viscous fraction #4 of natto was measured. A water-soluble viscous fraction of natto #4 (Natto #4) was mixed with 10% by mass sucrose-containing feed at concentrations of 13, 25, and 50 mg/g diet. As a positive control, voglibose (40 mg/g diet) was mixed with 10% by mass sucrose-containing feed.

Silkworms were fed with the above-described sample-mixed feed or unmixed feed (Control), and one hour later, the glucose concentration in the body fluid was measured.

The results are shown in Figure 2A.
n=5-10/group, statistical significance test was performed using Student's t-test. Bars in FIG. 2A indicate mean values for each sample.
Data marked with "*" are P<0.05.

<<Dose dependence of suppression (inhibition) by indigestible dextrin against elevation of blood glucose level>>
Indigestible dextrin was mixed with 10% by mass sucrose-containing feed at concentrations of 50, 100 and 200 mg/g diet. As a positive control, acarbose (40 mg/g diet) was mixed with 10% by mass sucrose-containing feed.

Silkworms were allowed to eat the food mixed with each sample or the food (Control) not mixed with each sample, and the glucose concentration in the body fluid was measured after 1 hour.

The results are shown in Figure 2B.
n=5-10/group, statistical significance test was performed using Student's t-test. Bars in FIG. 2B indicate mean values for each sample.
Data marked with "*" are P<0.05.

<<Results>>
Water-soluble viscous fraction #4 obtained from natto dose-dependently inhibited the increase in blood sugar level of silkworms due to sucrose intake (Fig. 2A).

On the other hand, indigestible dextrin is a representative water-soluble dietary fiber, its solution exhibits viscosity, and it has been reported that it has the effect of lowering postprandial hyperglycemia in rats (Wakabayashi S, Kishimoto Y, Matsuoka A Effects of indigestible dextrin on glucose tolerance in rats. J Endocrinol. 1995; 144:533-538.).
However, even when this indigestible dextrin was added to silkworm feed at 200 mg/g diet, it did not suppress the increase in blood glucose level after ingestion of sucrose (Fig. 2B).

The above results suggest that the water-soluble viscous fraction (ingredient) of natto has the activity of suppressing the elevation of blood sugar level after ingestion of sucrose, which is higher than that of indigestible dextrin.
It is suggested that the water-soluble viscous fraction obtained from natto can be a blood sugar level increase inhibitor, and the above results are the results of 1 hour after feeding, so it can be used as a blood sugar level spike inhibitor. It was also suggested that

Example 3
<Proof that the sugar chain component of natto has the effect of suppressing the increase in blood glucose concentration after ingestion of sucrose and the effect of suppressing the spike in blood sugar level after ingestion of sucrose>
The water-soluble viscous fraction obtained from natto as in Example 1 was subjected to DNase, RNase, proteinse K treatment, and phenol treatment to obtain an enzyme-treated polysaccharide fraction, that is, a sugar chain component in natto. Ta.
Specifically, DNaseI (1000 unit/mL; Promega) and RNaseA (10 µg/mL; NIPPON GENE CO., LTD.) were added to the ethanol precipitated fraction, incubated at 37°C for 24 hours, and further , Proteinase K was added, and after incubation at 37°C for 24 hours, an equal volume of phenol/chloroform/isoamyl alcohol (50:49:1) was added. was added, and the resulting precipitate was collected by centrifugation and dried to obtain an enzyme-treated polysaccharide fraction.

The amount of sugar, DNA, RNA, and protein contained per 1 g of dry weight of this enzyme-treated polysaccharide fraction (sugar chain component in natto) was quantified, and the results were as shown in Table 1. The sugar content was 32. The amounts of DNA, RNA and protein were all 1% by mass or less (see Table 1).

Water-soluble viscous fraction of natto (Non-treated Natto # 4, 50 mg / g diet), DNase, RNase, proteinse K treatment, and enzyme-treated polysaccharide fraction (sugar chain component in natto) ( Enzyme-treated Natto #4, 50 mg/g diet) and acarbose (40 mg/g diet) were each mixed with 10% by weight sucrose-containing diet.

Silkworms were allowed to eat the food mixed with each sample or the food (Control) not mixed with each sample, and the glucose concentration in the body fluid was measured after 1 hour.

The results are shown in FIG.
n=5-10/group, statistical significance test was performed using Student's t-test. Bars in FIG. 3 indicate the average value of each sample.
Data marked with "*" are P<0.05.

<<Results>>
When the enzyme-treated polysaccharide fraction (sugar chain component in natto) was added to the sucrose-containing feed, similar to the results of the water-soluble viscous fraction obtained in Examples 1 and 2, silkworm The increase in blood sugar level was suppressed (Fig. 3).
The above results suggest that the polysaccharide component of natto has the activity of suppressing the increase in blood sugar level after ingestion of sucrose.
It is suggested that the polysaccharide component contained in natto can be a blood sugar level increase inhibitor, and since the above results are the results of 1 hour after feeding, it is also suggested that there is a use as a blood sugar level spike inhibitor. was done.

Example 4
<Proof that the water-soluble viscous fraction of natto has the activity of inhibiting glucose uptake (transport)>
Natto water-soluble viscous fraction #4 (50 mg/g diet) was mixed with 10% by mass sucrose-containing feed or 10% by mass glucose-containing feed.
The silkworms were fed with the sample-mixed feed or unmixed feed (Control), and one hour later, the glucose concentration in the body fluid was measured.

The results are shown in FIG.
n=6-7/group, statistical significance test was performed using Student's t-test. Bars in FIG. 4 indicate the average value of each sample.
Data marked with "*" are P<0.05.

Sucrose in the intestinal tract is degraded into glucose and fructose by α-glycosidase and taken up by intestinal cells (Matsumoto Y, Ishii M, Sekimizu K. An in vivo invertebrate evaluation system for identifying substances that suppress sucrose-induced postprandial hyperglycemia). Scientific reports. 2016; 6:26354.).
Acarbose and voglibose, which are α-glycosidase inhibitors in humans, are known to inhibit the elevation of blood glucose levels after ingestion of sucrose in silkworms as well, but do not suppress the elevation of blood glucose levels due to glucose ingestion (ibid.). ).

On the other hand, as can be seen from FIG. 4, the addition of the water-soluble viscous fraction obtained from natto to the feed suppressed the increase in the blood sugar level of silkworms after ingestion of both sucrose and glucose. This result suggests that the water-soluble viscous fraction of natto has activity to inhibit glucose transport.
It was suggested that the water-soluble viscous fraction of natto can be a blood glucose level suppressor and a glucose uptake (transport) inhibitor.

Example 5
<Proof that the sugar chain component of natto has glucose uptake inhibitory activity>
The inventor of the present invention has already confirmed that Enterococcus faecalis YM0831 strain, a lactic acid bacterium that suppresses hyperglycemia after glucose intake in silkworms, inhibits glucose uptake of Caco-2 cells derived from the human intestinal tract. .
In the 2-NBDG uptake system in Caco-2 cells, an enzyme-treated polysaccharide fraction of natto (sugar chain component in natto) (Enzyme-treated Natto #4) was added, and glucose uptake in Caco-2 cells was measured by fluorescence. Quantified by measurement.

Specifically, glucose uptake experiments using Caco-2 cells were performed according to the method shown below.
Caco-2 cells were a gift from the American Type Cell Collection (ATCC, Manassas, VA, USA). Caco-2 cells were grown in DMEM (Gibco, NY, USA) medium supplemented with 10% FBS (Gibco, NY, USA), 1% penicillin/streptomycin (Gibco, NY, USA) at 37°C, 5% CO It was cultured under _two conditions.
Caco-2 cells were cultured to monolayer in 96-well plates (Tissue culture testplate 96F: TPP, Switzerland) and further cultured in serum-free DMEM medium for 24 hours.

Thereafter, the culture medium was replaced with Na buffer [10 mM HEPES (pH 7.4), 140 mM NaCl, 20 mg/ml BSA] and cultured for 15 minutes. Furthermore, 2-deoxy-2-[(7-nitro-2, 1, 3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG: Cayman Chemical, MI, USA) was added to a final concentration of 50 μM in Na buffer. was added and incubated for 10 minutes. Cells were then washed twice with cold PBS and observed under a fluorescence microscope. Fluorescence intensity of cells was quantified with Image J ver. 1.43u (National Institutes of Health, USA).

The results are shown in FIG.
n=3/group, statistical significance test was performed using Student's t-test. Bars in FIG. 4 indicate the average value of each sample.
Data with "*" are P<0.05 vs control, and error bars indicate standard error (SEM).

As shown in FIG. 5, it was found that addition of enzyme-treated polysaccharide fractions of natto (sugar chain components in natto) inhibits glucose uptake of Caco-2 cells, which are human intestinal cells.
On the other hand, addition of indigestible dextrin did not inhibit glucose uptake of Caco-2 cells (Fig. 5).
From the above results, it is considered that the enzyme-treated polysaccharide fraction of natto (sugar chain component in natto) has a high activity of inhibiting glucose uptake by intestinal cells.
It was suggested that the enzyme-treated polysaccharide fraction of natto (sugar chain component in natto) could be a glucose uptake inhibitor.

Since the agent of the present invention inhibits glucose uptake from the intestinal tract, it is also useful as a blood sugar level spike inhibitor, a postprandial hyperglycemia inhibitor, etc., as well as a blood glucose level elevation inhibitor, and a diabetes prevention and treatment agent. , the pharmaceutical field, the food industry, etc.

Claims (4)

Add water to natto in an amount of 2 to 10 times the weight of the natto, stir with a stirrer at 20 ° C., then remove the remaining soybeans and filter to obtain an aqueous solution that is a filtrate, and add it to the aqueous solution. A water-soluble viscous fraction of natto , which is a precipitate when two volumes of ethanol are added , or a sugar chain component contained in the water-soluble viscous fraction of natto . Dependent intestinal glucose uptake inhibitor. 2. A preventive and therapeutic agent for diabetes , which is the insulin-independent intestinal glucose uptake inhibitor according to claim 1 . 2. A blood glucose elevation suppressor, which is the insulin-independent intestinal glucose uptake inhibitor according to claim 1 . 2. A blood sugar level spike inhibitor, which is the insulin-independent intestinal glucose uptake inhibitor according to claim 1 .

Images