JP2020180075A - Prophylactic and therapeutic agents for diabetes, blood glucose level elevation inhibitor, blood glucose level spike inhibitor, as well as glucose uptake inhibitor - Google Patents
Prophylactic and therapeutic agents for diabetes, blood glucose level elevation inhibitor, blood glucose level spike inhibitor, as well as glucose uptake inhibitor Download PDFInfo
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- JP2020180075A JP2020180075A JP2019084096A JP2019084096A JP2020180075A JP 2020180075 A JP2020180075 A JP 2020180075A JP 2019084096 A JP2019084096 A JP 2019084096A JP 2019084096 A JP2019084096 A JP 2019084096A JP 2020180075 A JP2020180075 A JP 2020180075A
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Abstract
Description
本発明は、納豆由来の、糖尿病予防治療剤、血糖値上昇抑制剤、血糖値スパイク抑制剤及びグルコース取り込み阻害剤に関する。 The present invention relates to a diabetes preventive and therapeutic agent, a blood glucose elevation inhibitor, a blood glucose spike inhibitor and a glucose uptake inhibitor derived from natto.
現在、過剰なカロリー摂取による肥満や、その後に発症する糖尿病等の生活習慣病の発症が問題となっている。日頃から血糖値が上昇しないように注意することは、生活習慣病の発症を抑制する上で重要であるとされている。生活習慣病の予防のためには、食事療法や運動療法が効果的とされており、食事療法においては、主に血糖値の上昇が起こらないように摂取カロリーが制限される。
しかしながら、そのような食事療法を継続することは心理的に困難の場合があり、血糖値の上昇を抑制する物質（成分、剤等）や、該物質を含む飲食品（健康食品を含む）の開発が、継続的な療法を実施する上で有効であると考えられている。
At present, obesity due to excessive calorie intake and the subsequent onset of lifestyle-related diseases such as diabetes have become problems. It is said that it is important to be careful not to raise the blood sugar level on a daily basis in order to suppress the onset of lifestyle-related diseases. Diet therapy and exercise therapy are considered to be effective for the prevention of lifestyle-related diseases, and in diet therapy, calorie intake is mainly restricted so that the blood sugar level does not rise.
However, it may be psychologically difficult to continue such a diet, and substances (ingredients, agents, etc.) that suppress the rise in blood sugar level and foods and drinks (including health foods) containing the substances Development is believed to be effective in implementing continuous therapy.
納豆は、大豆を納豆菌（Buccilus subtilis var natto）で発酵させて製造する日本で伝統的な食品である。納豆菌が大豆の表面で増殖すると、様々な多糖が産生され粘性を示すようになる。 Natto is a traditional food in Japan produced by fermenting soybeans with Bacillus subtilis (Buccilus subtilis var natto). When Bacillus natto grows on the surface of soybeans, various polysaccharides are produced and become viscous.
納豆菌が関連している技術として、ナットウキナーゼ（nattokinase）が糖尿病治療薬として有用であることが知られている（特許文献１）。しかしながら、ナットウキナーゼは、納豆に含有される酵素タンパク質の一種であって、そもそも水溶性の糖（鎖成分）ではないことに加え、特許文献１は、インスリン量を増加させることによる糖尿病治療薬であった。
As a technique related to Bacillus natto, nattokinase is known to be useful as a therapeutic agent for diabetes (Patent Document 1). However, nattokinase is a kind of enzyme protein contained in natto and is not a water-soluble sugar (chain component) in the first place. In addition,
大豆タンパク質及び米糠を納豆菌で発酵させた発酵物を、更にプロテアーゼで処理してなる組成物を含有する抗糖尿病組成物が知られている（特許文献２）。
また、大豆摩砕物の固形画分を発酵させた大豆発酵組成物を含有する抗糖尿病組成物が知られている（特許文献３）
しかしながら、これらは、全てが水溶性でなかったり、インスリン量の増加に関係した糖尿病治療薬であったりした。
An antidiabetic composition containing a composition obtained by further treating a fermented product obtained by fermenting soybean protein and rice bran with Bacillus natto with a protease is known (Patent Document 2).
Further, an antidiabetic composition containing a fermented soybean composition obtained by fermenting a solid fraction of pyroclastic soybean is known (Patent Document 3).
However, these were not all water-soluble or were antidiabetic agents associated with increased insulin levels.
納豆に含有される糖に関しては、現時点で以下のような状況にある。
すなわち、納豆には、ポリグルタミン酸とレバンという多糖が含まれており、ポリグルタミン酸（poly-γ-glutamic acid）は、インスリン等の医薬品の送達システム（Drug delivery system）の基材として利用されている。しかしながら、ポリグルタミン酸にショ糖摂取後の血糖値の上昇を抑制する活性があるという報告はない。
At present, the sugar contained in natto is as follows.
That is, natto contains polysaccharides called polyglutamic acid and levan, and poly-γ-glutamic acid is used as a base material for a drug delivery system for drugs such as insulin. .. However, there is no report that polyglutamic acid has an activity of suppressing an increase in blood glucose level after ingestion of sucrose.
また、レバンには、保湿性等の様々な機能があり、食品添加物、化粧品、医薬品開発に利用されている。しかしながら、納豆由来のレバンを投与しても、通常ラットやストレプトゾトシン処理した糖尿病ラットに対して血糖値を下げる効果は見られず、糖尿病症状の改善はないことが報告されている（非特許文献１）。 Levan has various functions such as moisturizing properties, and is used for the development of food additives, cosmetics, and pharmaceuticals. However, it has been reported that administration of levan derived from natto does not have an effect of lowering blood glucose level in normal rats and diabetic rats treated with streptozotocin, and does not improve diabetic symptoms (Non-Patent Document 1). ).
すなわち、納豆由来の水溶性粘性成分（画分）にショ糖摂取後の血糖値の上昇を抑制する効果があることを示した報告はない。 That is, there is no report showing that the water-soluble viscous component (fraction) derived from natto has an effect of suppressing an increase in blood glucose level after ingestion of sucrose.
一方、従来、食後高血糖の抑制や糖尿病の予防・治療剤の評価には、マウスやラット等の哺乳動物が用いられてきた。しかし、多数の哺乳動物を実験に供することに対しては、コストばかりでなく動物愛護の観点からも多くの問題が指摘されている。
本発明者らは、カイコを用いた評価系により、ヒトの血糖値上昇抑制剤や、ヒトの食後高血糖を抑制する活性物質の探索法（スクリーニング法）を確立し、食後高血糖の抑制剤や糖尿病の予防・治療剤を新たに見出すことに成功している（特許文献４、５、非特許文献２、３、４等）。
On the other hand, conventionally, mammals such as mice and rats have been used for suppressing postprandial hyperglycemia and evaluating preventive / therapeutic agents for diabetes. However, many problems have been pointed out not only from the viewpoint of cost but also from the viewpoint of animal protection for using a large number of mammals for experiments.
The present inventors have established a search method (screening method) for a human blood glucose level increase inhibitor and an active substance that suppresses human postprandial hyperglycemia by an evaluation system using silkworms, and an agent for suppressing postprandial hyperglycemia. We have succeeded in finding new preventive / therapeutic agents for diabetes and diabetes (
また、ショ糖摂取による血糖値の一過的な上昇は、血糖値スパイクと呼ばれ、糖尿病を引き起こす要因の１つである。この血糖値スパイクの抑制は、糖尿病発症の予防に役立つと考えられ、そのような効果を有する物質（成分）、薬剤、健康食品、飲食品等の開発が望まれている。 In addition, a transient increase in blood glucose level due to sucrose intake is called a blood glucose level spike, and is one of the factors that cause diabetes. Suppression of this blood glucose spike is considered to be useful for prevention of the onset of diabetes, and development of substances (ingredients), drugs, health foods, foods and drinks having such effects is desired.
本発明の課題は、安心・安全な天然由来の成分を含有し、インスリン分泌促進とは異なる作用機序で血糖値の上昇を抑制して糖尿病を予防・治療する剤を提供することにある。 An object of the present invention is to provide an agent that contains a safe and secure naturally-derived component and suppresses an increase in blood glucose level by a mechanism of action different from that of promoting insulin secretion to prevent or treat diabetes.
本発明者は、上記の課題を解決すべく鋭意検討を重ねた結果、納豆の水溶性粘性画分が、カイコにおけるショ糖やグルコースの摂取による血糖値の上昇に対して抑制効果を示すことを見出した。
また、納豆の水溶性粘性画分の、酵素処理による多糖画分、すなわち糖鎖成分も、ショ糖摂取後の血糖値の上昇に対する抑制活性を有することを見出して本発明に至った。
なお、カイコを用いた評価系を用いて、前記した通り、ヒトの血糖値上昇抑制や、ヒトの食後高血糖の抑制を評価できることが確かめられており、カイコを用いた評価系を用いて、ヒトに対する上記効果を有する物質の探索法（スクリーニング法）も確立されている（特許文献４、５、非特許文献２、３、４等）。
本発明では、カイコを用いる糖に関する上記評価系を、単に「カイコ評価系」と略記する。
As a result of diligent studies to solve the above problems, the present inventor has found that the water-soluble viscous fraction of natto has an inhibitory effect on the increase in blood glucose level due to the intake of sucrose and glucose in silkworms. I found it.
Further, they have found that the polysaccharide fraction of natto by enzyme treatment, that is, the sugar chain component, also has an inhibitory activity against an increase in blood glucose level after ingestion of sucrose, and has reached the present invention.
As described above, it has been confirmed that the suppression of human blood glucose elevation and the suppression of human postprandial hyperglycemia can be evaluated using the evaluation system using silkworms, and the evaluation system using silkworms can be used. A method for searching for a substance having the above-mentioned effect on humans (screening method) has also been established (
In the present invention, the above evaluation system for sugars using silkworms is simply abbreviated as "silkworm evaluation system".
更に、本発明者は、納豆の酵素処理多糖画分（糖鎖成分）が、ヒトの腸管細胞におけるグルコースの取り込みを阻害することを、ヒトの腸管由来のＣａｃｏ−２細胞を用いて立証することによって本発明に至った。 Furthermore, the present inventor uses Caco-2 cells derived from the human intestine to prove that the enzyme-treated polysaccharide fraction (sugar chain component) of natto inhibits glucose uptake in human intestinal cells. This led to the present invention.
すなわち、本発明は、納豆の水溶性粘性画分、又は、納豆の水溶性粘性画分に含まれる糖鎖成分を含有することを特徴とする糖尿病予防治療剤を提供するものである。 That is, the present invention provides a diabetes preventive and therapeutic agent, which comprises a water-soluble viscous fraction of natto or a sugar chain component contained in the water-soluble viscous fraction of natto.
また、本発明は、納豆の水溶性粘性画分、又は、納豆の水溶性粘性画分に含まれる糖鎖成分を含有することを特徴とする血糖値上昇抑制剤を提供するものである。 The present invention also provides an agent for suppressing an increase in blood glucose level, which comprises a water-soluble viscous fraction of natto or a sugar chain component contained in the water-soluble viscous fraction of natto.
また、本発明は、納豆の水溶性粘性画分、又は、納豆の水溶性粘性画分に含まれる糖鎖成分を含有することを特徴とする腸管からのグルコース取り込み阻害剤を提供するものである。 The present invention also provides an inhibitor of glucose uptake from the intestinal tract, which comprises a water-soluble viscous fraction of natto or a sugar chain component contained in the water-soluble viscous fraction of natto. ..
また、本発明は、納豆の水溶性粘性画分、又は、納豆の水溶性粘性画分に含まれる糖鎖成分を含有することを特徴とする血糖値スパイク抑制剤を提供するものである。 The present invention also provides a blood glucose spike inhibitor characterized by containing a water-soluble viscous fraction of natto or a sugar chain component contained in the water-soluble viscous fraction of natto.
また、本発明は、カイコ評価系を利用して納豆の種類をスクリーニングする工程を有することを特徴とする、糖尿病予防治療剤、血糖値上昇抑制剤、グルコース取り込み阻害剤、又は、血糖値スパイク抑制剤の、製造方法若しくはスクリーニング方法を提供するものである。 Further, the present invention is characterized by having a step of screening the type of natto using a silkworm evaluation system, which comprises a diabetes preventive treatment agent, a blood glucose level rise inhibitor, a glucose uptake inhibitor, or a blood glucose level spike suppressant. It provides a method for producing or screening an agent.
本発明によれば、食品として馴染みのある納豆を原料として用いて、そこから血糖値上昇を抑制することができる剤を得ることができる。すなわち、本発明によれば、安全であり安心感を与える天然由来（一般食品由来）の血糖値上昇抑制剤、グルコース取り込み（輸送）阻害剤等を提供することができる。 According to the present invention, it is possible to obtain an agent capable of suppressing an increase in blood glucose level by using natto, which is familiar as a food, as a raw material. That is, according to the present invention, it is possible to provide a naturally-derived (general food-derived) blood glucose level increase inhibitor, glucose uptake (transport) inhibitor, and the like that are safe and give a sense of security.
また、本発明の剤によれば、腸管からのグルコース取り込みを阻害することができるので、グルコースの血中濃度を抑制することができる。 In addition, according to the agent of the present invention, glucose uptake from the intestinal tract can be inhibited, so that the blood glucose concentration can be suppressed.
上記の本発明の効果は、何れも、ショ糖等の糖の摂取による血中グルコース濃度の抑制に係るものであり、インスリンに依存した効果は確認されていないので、糖を含有する食事をした直後に血糖値が急激に上昇する血糖値スパイクの抑制に極めて効果があるものである。 All of the above-mentioned effects of the present invention relate to suppression of blood glucose concentration by ingestion of sugar such as sucrose, and no insulin-dependent effect has been confirmed. Therefore, a diet containing sugar was eaten. It is extremely effective in suppressing blood glucose spikes in which the blood glucose level rises sharply immediately afterwards.
糖尿病を治療する剤は、インスリン分泌を促進（分泌不全を改善）するもの、インスリン抵抗性を排除（改善）するもの、糖吸収を抑制するもの等があるが、本発明は「糖吸収を抑制するもの」である。
糖の吸収・輸送には、糖を腸管の細胞に取り込むこと、及び、腸管の細胞から糖を血液中に放出することを含むが、本発明によれば、実施例５に示したように、特に前者を抑制すること、すなわち、グルコースの腸の細胞への取り込みを抑制することができる。
Agents for treating diabetes include those that promote insulin secretion (improve secretory deficiency), those that eliminate (improve) insulin resistance, those that suppress glucose absorption, etc., but the present invention "suppresses glucose absorption". What to do ".
Absorption and transport of sugar include taking sugar into intestinal cells and releasing sugar from the intestinal cells into blood. According to the present invention, as shown in Example 5, the sugar is absorbed and transported. In particular, the former can be suppressed, that is, the uptake of glucose into intestinal cells can be suppressed.
上記した血糖値スパイクは、健常人でも起こるものである。本発明は、糖尿病患者に適用できるだけではなく、糖尿病を予防したい人、糖尿病を軽減したい人、健常人等にも好適に適用することができる。 The above-mentioned blood glucose spikes also occur in healthy people. 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.
以下、本発明について説明するが、本発明は、以下の具体的態様に限定されるものではなく、技術的思想の範囲内で任意に変形することができる。 Hereinafter, the present invention will be described, but the present invention is not limited to the following specific aspects, and can be arbitrarily modified within the scope of the technical idea.
本発明は、納豆の水溶性粘性画分、又は、納豆の水溶性粘性画分に含まれる糖鎖成分を含有することを特徴とする糖尿病予防治療剤である。
また、本発明は、納豆の水溶性粘性画分、又は、納豆の水溶性粘性画分に含まれる糖鎖成分を含有することを特徴とする血糖値上昇抑制剤でもある。
The present invention is a diabetes preventive and therapeutic agent, which comprises a water-soluble viscous fraction of natto or a sugar chain component contained in the water-soluble viscous fraction of natto.
The present invention is also an agent for suppressing an increase in blood glucose level, which is characterized by containing a water-soluble viscous fraction of natto or a sugar chain component contained in the water-soluble viscous fraction of natto.
本発明は、限定はされないが、インスリン分泌促進用ではない糖尿病予防治療剤又はインスリン分泌促進用ではない血糖値上昇抑制剤でもある。すなわち、作用機序的に若干のインスリン分泌が付随的に予期せず関与してしまう場合まで本発明から排除されるものではないが、本発明は、非インスリン分泌促進用糖尿病予防治療剤又は非インスリン分泌促進用血糖値上昇抑制剤でもある。
また、言い換えると、本発明は、インスリン非依存性の糖尿病予防治療剤又はインスリン非依存性の血糖値上昇抑制剤であり、インスリン分泌促進用でもなくインスリン抵抗性改善（若しくは抵抗性軽減）用でもない、糖尿病予防治療剤又は血糖値上昇抑制剤でもある。
The present invention is also, but not limited to, a diabetes preventive and therapeutic agent that is not for promoting insulin secretion or an agent that suppresses an increase in blood glucose level that is not for promoting insulin secretion. That is, although it is not excluded from the present invention until a case where some insulin secretion is incidentally and unexpectedly involved in the mechanism of action, the present invention is a non-insulin secretagogue prophylactic therapeutic agent for diabetes or non-insulin It is also an inhibitor of blood glucose elevation for promoting insulin secretion.
In other words, the present invention is an insulin-independent preventive treatment for diabetes or an insulin-independent inhibitor of blood glucose elevation, which is not for promoting insulin secretion but for improving (or reducing resistance) insulin resistance. It is also a preventive treatment for diabetes or an inhibitor for increasing blood glucose level.
本発明によって、納豆の水溶性粘性画分、又は、納豆の水溶性粘性画分に含まれる糖鎖成分が、血糖値スパイクを抑制すること、腸管からのグルコース取り込みを阻害することが見出された。
上記した効果は、何れも、インスリン分泌とは関係がないので、本発明は、インスリン分泌促進用ではない、糖尿病予防治療剤又は血糖値上昇抑制剤である。
According to the present invention, it has been 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 glucose spikes and inhibits glucose uptake from the intestinal tract. It was.
Since none of the above effects is related to insulin secretion, the present invention is a diabetes preventive therapeutic agent or a blood glucose level increase inhibitor, which is not for promoting insulin secretion.
β細胞刺激によるインスリン分泌促進は、β細胞の疲弊をまねいたり、膵臓に副作用を与えたりするおそれもあるため、上記したような効果を有し、上記した効果を奏する用途に用いられる本発明の糖尿病予防治療剤又は血糖値上昇抑制剤は極めて有用である。 Promotion of insulin secretion by β-cell stimulation may lead to exhaustion of β-cells and may cause side effects on the pancreas. Therefore, the present invention has the above-mentioned effects and is used for applications that exert the above-mentioned effects. Diabetes preventive and therapeutic agents or blood glucose elevation inhibitors are extremely useful.
本願の実施例に示した通り、腸管からのグルコース取り込み阻害は、糖尿病予防用、血糖値上昇抑制用、血糖値スパイク抑制用等として有用であることから、本願発明の糖尿病予防治療剤又は血糖値上昇抑制剤は、２型糖尿病の患者のみならず、それらの予備軍や、１型糖尿病の患者や、それらを予防したい健常人等にも好適に適用できる。
As shown in the examples of the present application, inhibition of glucose uptake from the intestinal tract is useful for preventing diabetes, suppressing an increase in blood glucose level, suppressing spikes in blood glucose level, and the like. Therefore, the antidiabetic therapeutic agent or blood glucose level of the present invention. The ascending inhibitor can be suitably applied not only to patients with
「納豆の水溶性粘性画分」や「納豆の水溶性粘性画分に含まれる糖鎖成分」の原料となる納豆の種類は、本発明の前記した効果を発揮するものであれば特に限定はない。実施例１に示したように、評価した納豆＃１、＃２、＃３及び＃４の中には、ショ糖摂取後の血中グルコース濃度の上昇を大きく抑制したものがあった（図１）。
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 particularly limited as long as it exhibits the above-mentioned effects of the present invention. Absent. As shown in Example 1, some of the evaluated
ただし、実施例１で評価したもののうち納豆＃４が最も効果があった（納豆の種類の間で差が見られた）ことから、複数の納豆を評価して、効果の高い納豆を採用することが好ましい。「納豆」は、原料となる豆と納豆菌を含んでいる。従って、本発明で「納豆の種類」とは、少なくとも、大豆等の種類と納豆菌の種類のことを言う。
納豆の種類ごとにカイコにおけるショ糖摂取後の血糖値の上昇に対する抑制効果は異なっていた。従って、カイコ評価系を用いて、より活性の高い納豆の種類の探索ができると考えられる。
従って、本発明は、カイコ評価系を利用して、前記効果又は下記剤の効果を特に奏する納豆をスクリーニングする工程を有することを特徴とする、糖尿病予防治療剤、血糖値上昇抑制剤、グルコース取り込み阻害剤又は血糖値スパイク抑制剤の、製造方法若しくはスクリーニング方法でもある。
However, since
The effect of suppressing the increase in blood glucose level after ingestion of sucrose in silkworm was different for each type of natto. Therefore, it is considered that the silkworm evaluation system can be used to search for more active types of natto.
Therefore, the present invention is characterized by having a step of screening natto that particularly exerts the above-mentioned effect or the effect of the following agent by using a silkworm evaluation system, which comprises a diabetes preventive treatment agent, a blood glucose level increase inhibitor, and glucose uptake. It is also a method for producing or screening an inhibitor or a blood glucose spike inhibitor.
「納豆の水溶性粘性画分」とは、納豆に水を該納豆の２〜１０質量倍量加えて、撹拌機で、２０℃で撹拌し、その後、残存している大豆を除いて濾過し、濾液である水溶液を得て、該水溶液に２容量倍量のエタノールを加えたときの沈殿物のことを言う。
ただし、上記のようにして得られるようなものならば、本発明における「納豆の水溶性粘性画分」であり、それは実際に上記具体的獲得方法で得られたものには限定されない。
The "water-soluble viscous fraction of natto" means that water is added to natto in an amount of 2 to 10 times by mass of the natto, stirred with a stirrer at 20 ° C., and then filtered by removing the remaining soybeans. , A precipitate obtained when an aqueous solution as a filtrate is obtained and 2 volumes of ethanol is added to the aqueous solution.
However, if it can be obtained as described above, it is the "water-soluble viscous fraction of natto" in the present invention, and it is not limited to that actually obtained by the above-mentioned specific acquisition method.
「納豆の水溶性粘性画分に含まれる糖鎖成分」とは、「酵素処理多糖画分」と同義であり、上記した納豆の水溶性粘性画分を、実施例３に示したように酵素で処理した後、エタノールを加えて生じた沈殿物のことを言う。
ただし、上記のようにして得られるようなものならば、本発明における「納豆の水溶性粘性画分に含まれる糖鎖成分」「酵素処理多糖画分」であり、それは上記具体的獲得方法で実際に得られたものには限定されない。
The "sugar chain component contained in the water-soluble viscous fraction of natto" is synonymous with the "enzyme-treated polysaccharide fraction", and the above-mentioned water-soluble viscous fraction of natto is an enzyme as shown in Example 3. Refers to the precipitate formed by adding ethanol after treatment with.
However, if it can be 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 the above-mentioned specific acquisition method. It is not limited to what is actually obtained.
本発明は、納豆の水溶性粘性画分、又は、納豆の水溶性粘性画分に含まれる糖鎖成分を含有することを特徴とする、腸管からのグルコース取り込み阻害剤であり、血糖値スパイク抑制剤でもある。
「納豆の水溶性粘性画分に含まれる糖鎖成分（酵素処理多糖画分）」が、腸管からのグルコースの取り込み（又は輸送）を阻害することは、実施例５で立証された。従って、それを含む「納豆の水溶性粘性画分」もグルコース取り込み阻害剤となり得る。
The present invention is an inhibitor of glucose uptake from the intestinal tract, which comprises a water-soluble viscous fraction of natto or a sugar chain component contained in the water-soluble viscous fraction of natto, and suppresses blood glucose spikes. It is also an agent.
It was proved in Example 5 that the "sugar chain component (enzyme-treated polysaccharide fraction) contained in the water-soluble viscous fraction of natto" inhibits the uptake (or transport) of glucose from the intestinal tract. Therefore, the "water-soluble viscous fraction of natto" containing it can also be a glucose uptake inhibitor.
更に、「納豆の水溶性粘性画分」が、「腸管からのグルコース取り込み阻害剤」となり得ることは、実施例４において、ショ糖のみならずグルコースを摂取させた場合でも血糖値の上昇を抑制したことからも立証された。 Furthermore, the fact that the "water-soluble viscous fraction of natto" can be a "glucose uptake inhibitor from the intestinal tract" suppresses an increase in blood glucose level even when glucose is ingested as well as sucrose in Example 4. It was also proved by what I did.
また、実施例１〜４は全て、食事（給餌）から１時間後の血中グルコース濃度を測定し、グルコース濃度の上昇を抑制する効果を確認していることから、納豆の水溶性粘性画分も、納豆の水溶性粘性画分に含まれる糖鎖成分（酵素処理多糖画分）も、血糖値スパイク抑制剤となり得ることが立証された。納豆の水溶性粘性画分は、腸管細胞のグルコース取り込みを阻害することにより、食後高血糖を抑制するので、食後高血糖抑制剤として有用である。
In addition, since all of Examples 1 to 4 measured the
また、少なくともグルコースの取り込みが阻害されれば、血糖値上昇抑制剤、糖尿病予防治療剤となり得ることは明らかである。 In addition, it is clear that if at least glucose uptake is inhibited, it can be an inhibitor of blood glucose elevation and a preventive and therapeutic agent for diabetes.
本発明の剤は、一般飲食品、健康飲食品、薬剤等に含有させて使用できる。本発明の剤は、水等を含んで湿潤状態になっているものでもよく、乾燥して粉末状態になったものでもよい。 The agent of the present invention can be used by being contained in general foods and drinks, healthy foods and drinks, drugs and the like. The agent of the present invention may be moistened with water or the like, or may be dried and powdered.
一般飲食品に含有させて使用するときは、本発明の剤自体を飲食してもよく、水等の液体又は可食性の固体で希釈して使用することも好ましい。また、一般飲食品に含有させるときは、料理の際に配合させてもよいし、ふりかけ・調味料等として使用することも好ましい。 When used by containing it in general foods and drinks, the agent of the present invention itself may be eaten or eaten, and it is also preferable to dilute it with a liquid such as water or an edible solid. Further, when it is contained in general foods and drinks, it may be blended at the time of cooking, and it is also preferable to use it as a sprinkle, a seasoning or the like.
上記一般飲食品の種類としては、特に限定はないが、例えば、ゼリー、キャンディー、チョコレート、ビスケット、グミ等の菓子類；緑茶、紅茶、コーヒー、清涼飲料等の嗜好飲料；醗酵乳、ヨーグルト、アイスクリーム、ラクトアイス等の乳製品；野菜飲料、果実飲料、ジャム類等の野菜・果実加工品；スープ等の液体食品；御飯類、パン類、麺類等の穀物（加工）品；ふりかけ等の各種調味料；肉、魚、野菜等の副食類；等が挙げられる。 The types of general foods and drinks are not particularly limited, but for example, confectioneries such as jelly, candy, chocolate, biscuits, and gummy; favorite beverages such as green tea, tea, coffee, and soft drinks; fermented milk, yogurt, and ice cream. Dairy products such as cream and lacto ice; processed vegetables and fruits such as vegetable drinks, fruit drinks and jams; liquid foods such as soups; grain (processed) products such as rice, breads and noodles; various seasonings such as sprinkles Foods; side dishes such as meat, fish and vegetables; etc.
健康飲食品、薬剤等に含有させて使用するときの剤型としては、特に限定はないが、具体的には、例えば、経口固形剤（錠剤、被覆錠剤、顆粒剤、散剤、ハードカプセル剤、ソフトカプセル剤等）、経口液剤（内服液剤、シロップ剤、エリキシル剤等）、注射剤（溶剤、懸濁剤等）等が挙げられる。
中でも、経口固形剤又は経口液剤が、簡便で前記効果を発揮し易い点から好ましい。
The dosage form when used in healthy foods and drinks, drugs, etc. is not particularly limited, but specifically, for example, oral solid preparations (tablets, coated tablets, granules, powders, hard capsules, soft capsules). Agents, etc.), oral liquids (oral liquids, syrups, elixirs, etc.), injections (solvents, suspensions, etc.) and the like.
Of these, an oral solid preparation or an oral liquid preparation is preferable because it is simple and easily exerts the above-mentioned effect.
上記経口固形剤としては、本発明の「納豆の水溶性粘性画分又は納豆の水溶性粘性画分に含まれる糖鎖成分」に、例えば、賦形剤、結合剤、崩壊剤、滑沢剤、着色剤、矯味・矯臭剤、保存安定剤、腸溶剤、崩壊遅延剤等の添加剤を加え、常法により製造することができる。 Examples of the oral solid preparation include excipients, binders, disintegrants, and lubricants in the "sugar chain component contained in the water-soluble viscous fraction of natto or the water-soluble viscous fraction of natto" of the present invention. , Coloring agent, flavoring / odorant, storage stabilizer, enteric solvent, disintegration retarder and other additives can be added and produced by a conventional method.
該賦形剤としては、例えば、乳糖、白糖、塩化ナトリウム、ブドウ糖、デンプン、炭酸カルシウム、カオリン、微結晶セルロース、珪酸等が挙げられる。
該結合剤としては、例えば、水、エタノール、プロパノール、単シロップ、ブドウ糖液、デンプン液、ゼラチン液、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルスターチ、メチルセルロース、エチルセルロース、シェラック、リン酸カルシウム、ポリビニルピロリドン等が挙げられる。
該崩壊剤としては、例えば、乾燥デンプン、アルギン酸ナトリウム、カンテン末、炭酸水素ナトリウム、炭酸カルシウム、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド、乳糖等が挙げられる。
該滑沢剤としては、例えば、精製タルク、ステアリン酸塩、ホウ砂、ポリエチレングリコール等が挙げられる。
該着色剤としては、例えば、酸化チタン、酸化鉄等が挙げられる。
前記矯味・矯臭剤としては、例えば、白糖、橙皮、クエン酸、酒石酸等が挙げられる。
Examples of the excipient include lactose, sucrose, 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, shelac, calcium phosphate, polyvinylpyrrolidone and the like. Be done.
Examples of the disintegrant include dried starch, sodium alginate, canten powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, lactose and the like.
Examples of the lubricant include purified talc, stearate, borax, polyethylene glycol and the like.
Examples of the colorant include titanium oxide, iron oxide and the like.
Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.
前記経口液剤としては、例えば、前記有効成分に、矯味・矯臭剤、緩衝剤、安定化剤等の添加剤を加え、常法により製造することができる。 The oral solution can be produced by a conventional method, for example, by adding additives such as a flavoring / odorant, a buffering agent, and a stabilizer to the active ingredient.
該矯味・矯臭剤としては、例えば、白糖、橙皮、クエン酸、酒石酸等が挙げられる。
該緩衝剤としては、例えば、クエン酸ナトリウム等が挙げられる。
該安定化剤としては、例えば、トラガント、アラビアゴム、ゼラチン等が挙げられる。
Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.
Examples of the buffering agent include sodium citrate and the like.
Examples of the stabilizer include tragant, gum arabic, gelatin and the like.
前記注射剤としては、例えば、前記有効成分に、ｐＨ調節剤、緩衝剤、安定化剤、等張化剤、局所麻酔剤等を配合し、常法により皮下用、筋肉内用、静脈内用等の注射剤を製造することができる。
該ｐＨ調節剤及び該緩衝剤としては、例えば、クエン酸ナトリウム、酢酸ナトリウム、リン酸ナトリウム等が挙げられる。前記安定化剤としては、例えば、ピロ亜硫酸ナトリウム、ＥＤＴＡ、チオグリコール酸、チオ乳酸等が挙げられる。前記等張化剤としては、例えば、塩化ナトリウム、ブドウ糖等が挙げられる。前記局所麻酔剤としては、例えば、塩酸プロカイン、塩酸リドカイン等が挙げられる。
As the injection, for example, a pH adjuster, a buffer, a stabilizer, an isotonic agent, a local anesthetic, etc. are blended with the active ingredient, and for subcutaneous, intramuscular, or intravenous use by a conventional method. Etc. can be produced.
Examples of the pH regulator and the buffering agent include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium metabisulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of the tonicity agent include sodium chloride, glucose and the like. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride and the like.
本発明の剤の投与対象動物としては、特に限定はないが、例えば、ヒト；サル；ウマ；ウシ、ブタ、ヤギ、ニワトリ等の家畜；ネコ、イヌ等のペット；マウス、ラット等の実験動物；等が挙げられる。 The animals to which the agent of the present invention is administered are not particularly limited, but for example, humans; monkeys; horses; domestic animals such as cows, pigs, goats, and chickens; pets such as cats and dogs; experimental animals such as mice and rats. ; Etc. can be mentioned.
本発明の剤の投与量としては、特に限定はなく、投与対象の年齢、体重、所望の効果の程度等に応じて適宜選択することができるが、例えば、成人への１日の投与量は、納豆の水溶性粘性画分の量として、１ｍｇ〜３０ｇが好ましく、１０ｍｇ〜１０ｇがより好ましく、１００ｍｇ〜３ｇが特に好ましい。
糖鎖成分（酵素処理多糖画分）の好ましい投与量や、経口固形剤又は経口液剤に含有されているときは、納豆の水溶性粘性画分の量に換算して、上記好適投与量が適用される。
The dose of the agent of the present invention is not particularly limited and may be appropriately selected depending on the age, body weight, degree of desired effect, etc. of the subject to be administered. For example, the daily dose to an adult may be determined. As the amount of the water-soluble viscous fraction of natto, 1 mg to 30 g is preferable, 10 mg to 10 g is more preferable, and 100 mg to 3 g is particularly preferable.
When it is contained in a preferable dose of a sugar chain component (enzyme-treated polysaccharide fraction) or an oral solid preparation or an oral liquid preparation, the above-mentioned suitable dose is applied in terms of the amount of the water-soluble viscous fraction of natto. Will be done.
以下、実施例及び検討例に基づき本発明を更に詳細に説明するが、本発明は以下の実施例等の具体的範囲に限定されるものではない。 Hereinafter, the present invention will be described in more detail based on Examples and Study Examples, but the present invention is not limited to the specific scope of the following Examples and the like.
評価例１
＜カイコの種類、飼育条件及び体液採取方法＞
カイコは、以前報告した方法で飼育した（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.、及び、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.）。
Evaluation example 1
<Types of silkworms, breeding conditions and body fluid collection methods>
Silkworms were bred 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.).
カイコの受精卵（交雑種ふ・よう×つくば・ね）は、愛媛養蚕株式会社から購入した。
孵化した幼虫は、室温で人工飼料シルクメイト２Ｓ（日本農産工業株式会社）を与えて５齢幼虫まで育てた。飼育容器は、卵から２齢幼虫までを角型２号シャーレ（栄研器材）、それ以降をディスポーザブルのプラスチック製フードパック（フードパックＦＤ 大深、中央化学株式会社）を用いた。飼育温度は２７℃とした。特に記載しない限り、実験には４齢眠以後絶食させた５齢１日目の幼虫を用いた。
Fertilized silkworm eggs (hybrid fu, yo x Tsukuba, ne) were purchased from Ehime Sericulture Co., Ltd.
The hatched larvae were fed with artificial feed Silkmate 2S (Nosan Corporation) at room temperature and raised to the 5th instar larvae. As the breeding container, a square No. 2 petri dish (Eiken equipment) was used for eggs to the second instar larvae, and a disposable plastic food pack (Food Pack FD Ofuka, Chuo Kagaku Co., Ltd.) was used for the rest. The breeding temperature was 27 ° C. Unless otherwise stated, larvae of the 5th instar and 1st day fasted after the 4th instar sleep were used in the experiment.
ショ糖を人口餌に、ショ糖１０質量％となるように混合し、更に評価するサンプル（試料）を混合した。５齢カイコに、サンプル（試料）を混合した餌、又は、混合していない餌を１時間与えた後、カイコの足をハサミで切って体液を採取し、グルコース濃度をグルコメーターで測定した（Accu-Chek,Roche）。 Sucrose was mixed with artificial diet so as to have 10% by mass of sucrose, and a sample to be further evaluated was mixed. After feeding the 5th instar silkworm with a mixed diet or a non-mixed diet for 1 hour, the silkworm's legs were cut with scissors to collect body fluid, and the glucose concentration was measured with a glucometer (glucose meter). Accu-Chek, Roche).
実施例１
＜納豆の水溶性粘性画分には、ショ糖摂取後の血液中のグルコース濃度の上昇を抑制する効果や、ショ糖摂取後の血糖値スパイクを抑制する効果があることの証明（１）＞
市販の納豆４種（＃１、＃２、＃３、＃４）に、それぞれ水を加えてよく撹拌し、その後、大豆を除き、水抽出画分を得た。これに２倍量のエタノールを加え、生じた糸状の沈殿を遠心により集め、「水溶性粘性画分(Crude fraction)＃１、＃２、＃３、＃４」とした。
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 ingestion of sucrose and the effect of suppressing the spike in blood glucose level after ingestion of sucrose (1)>
Water was added to each of the four commercially available natto (# 1, # 2, # 3, and # 4) and stirred well, and then soybeans were removed to obtain a water-extracted fraction. Twice the amount of ethanol was added thereto, and the resulting filamentous precipitate was collected by centrifugation to obtain "water-soluble viscous fractions (Crude fractions) # 1, # 2, # 3, # 4".
得られた、水溶性粘性画分(Crude fraction)＃１−４を、それぞれ、２５ｍｇ／ｇ ｄｉｅｔ）、及び、α−グリコシダーゼ阻害剤であるボグリボース（４０ｍｇ／ｇ ｄｉｅｔ）を、１０質量％ショ糖を含有する餌に混合した。
各試料（水溶性粘性画分＃１−４、ボグリボース）を混合した餌、又は、混合しなかった餌（Ｃｏｎｔｒｏｌ）をカイコに食べさせ、１時間後のカイコの体液中のグルコース濃度を測定した。
The obtained water-soluble viscous fraction (Crude fraction) # 1-4 was added to 25 mg / g diet, respectively, and voglibose (40 mg / g diet), which is an α-glycosidase inhibitor, was added to 10% by mass of sucrose. Was mixed with a diet containing.
The silkworms were fed a diet in which each sample (water-soluble viscous fraction # 1-4, voglibose) was mixed or not mixed (Control), and the glucose concentration in the body fluid of the silkworm was measured after 1 hour. ..
結果を図１に示す。
ｎ＝４−５／ｇｒｏｕｐ、統計学的有意差検定は、Student’s t-testを用いて行った。図１中の棒線は、各試料の平均値を示す。
「＊」を付したデータは、Ｐ＜０．０５である。
The results are shown in FIG.
n = 4-5 / group, statistical significance test was performed using Student's t-test. The bar in FIG. 1 shows the average value of each sample.
The data with "*" is P <0.05.
＜＜結果＞＞
図１より、納豆から得られた水溶性粘性画分には、カイコにおけるショ糖摂取後の血糖値の上昇を抑制する効果があることが示唆された。納豆の水溶性粘性画分を混合しなかった餌（Ｃｏｎｔｒｏｌ）に比べ、カイコの体液中のグルコース濃度の上昇抑制が見られたものがあった。特に、評価した納豆４種のうち、１種の納豆（＃４）から得た水溶性粘性画分は、カイコの血糖値の上昇を大きく抑制することが分かった。
<< Result >>
From FIG. 1, it was suggested that the water-soluble viscous fraction obtained from natto has an effect of suppressing an increase in blood glucose level after ingestion of sucrose in silkworm. Compared with the diet (Control) that did not mix the water-soluble viscous fraction of natto, there were some that showed suppression of the increase in glucose concentration in the body fluid of silkworms. In particular, it was found that the water-soluble viscous fraction obtained from one of the four types of natto evaluated (# 4) greatly suppressed the increase in the blood glucose level of silkworms.
前記した通り、投与した被験物質によるカイコでの血糖値の上昇抑制は、ヒトと相関がとれていることから（ヒトでも同様な結果が出ることが確かめられていることから）、納豆から得られた水溶性粘性画分には、血糖値上昇抑制効果があることが示唆され、血糖値上昇抑制剤となり得ることが示唆された。
また、上記結果は、給餌後１時間の結果であることから、血糖値スパイク抑制剤としての用途があることも示唆された。
As described above, the suppression of the increase in blood glucose level in silkworms by the administered test substance is obtained from natto because it is correlated with humans (since it has been confirmed that similar results are obtained in humans). It was suggested that the water-soluble viscous fraction had an effect of suppressing the increase in blood glucose level, and that it could be an agent for suppressing the increase in blood glucose level.
In addition, since the above results were obtained 1 hour after feeding, it was suggested that the results could be used as a blood glucose spike inhibitor.
実施例２
＜納豆の水溶性粘性画分には、ショ糖摂取後の血液中のグルコース濃度の上昇を抑制する効果や、ショ糖摂取後の血糖値スパイクを抑制する効果があることの証明（２）＞
＜＜血糖値上昇に対する納豆の水溶性粘性画分による抑制（阻害）の用量依存性＞＞
納豆の水溶性粘性画分＃４の用量依存性を測定した。納豆＃４の水溶性粘性画分（Ｎａｔｔｏ＃４）を、１３、２５、５０ｍｇ／ｇ ｄｉｅｔとなるように、１０質量％ショ糖含有餌に混合した。また、ポジティブコントロールとして、ボグリボース（４０ｍｇ／ｇ ｄｉｅｔ）を１０質量％ショ糖含有餌に混合した。
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 ingestion of sucrose and the effect of suppressing the spike in blood glucose level after ingestion of sucrose (2)>
<< Dose dependence of suppression (inhibition) of natto by water-soluble viscous fraction on blood glucose elevation >>
The dose dependence of the water-soluble
上記試料を混合した餌、又は、混合しなかった餌（Ｃｏｎｔｒｏｌ）をカイコに食べさせ、１時間後の体液中のグルコース濃度を測定した。 The silkworms were fed a diet mixed with the above samples or a diet not mixed (Control), and the glucose concentration in the body fluid after 1 hour was measured.
結果を図２Ａに示す。
ｎ＝５−１０／ｇｒｏｕｐ、統計学的有意差検定は、Student’s t-testを用いて行った。図２Ａ中の棒線は、各試料の平均値を示す。
「＊」を付したデータは、Ｐ＜０．０５である。
The results are shown in FIG. 2A.
n = 5-10 / group, statistical significance test was performed using Student's t-test. The bar in FIG. 2A shows the average value of each sample.
The data with "*" is P <0.05.
＜＜血糖値上昇に対する難消化性デキストリンによる抑制（阻害）の用量依存性＞＞
難消化性デキストリン（Indigestible dextrin）を、５０、１００、２００ｍｇ／ｇ ｄｉｅｔとなるように、１０質量％ショ糖含有餌に混合した。また、ポジティブコントロールとして、アカルボース（４０ｍｇ／ｇ ｄｉｅｔ）を、１０質量％ショ糖含有餌に混合した。
<< Dose dependence of suppression (inhibition) by indigestible dextrin on blood glucose elevation >>
Indigestible dextrin was mixed with a 10 mass% sucrose-containing diet to 50, 100, 200 mg / g diet. In addition, as a positive control, acarbose (40 mg / g diet) was mixed with a diet containing 10% by mass of sucrose.
各試料を混合した餌、又は、混合しなかった餌（Ｃｏｎｔｒｏｌ）をカイコに食べさせ、１時間後の体液中のグルコース濃度を測定した。 The silkworms were fed a diet in which each sample was mixed or a diet in which they were not mixed (Control), and the glucose concentration in the body fluid after 1 hour was measured.
結果を図２Ｂに示す。
ｎ＝５−１０／ｇｒｏｕｐ、統計学的有意差検定は、Student’s t-testを用いて行った。図２Ｂ中の棒線は、各試料の平均値を示す。
「＊」を付したデータは、Ｐ＜０．０５である。
The results are shown in FIG. 2B.
n = 5-10 / group, statistical significance test was performed using Student's t-test. The bar in FIG. 2B shows the average value of each sample.
The data with "*" is P <0.05.
＜＜結果＞＞
納豆から得られた水溶性粘性画分＃４は、用量依存的にショ糖摂取によるカイコの血糖値の上昇を抑制した（図２Ａ）。
<< Result >>
The water-soluble
一方、難消化性デキストリンは、代表的な水溶性食物繊維であり、その溶液は粘性を示し、ラットにおける食後高血糖を下げる効果があることが報告されている（Wakabayashi S, Kishimoto Y, Matsuoka A. Effects of indigestible dextrin on glucose tolerance in rats. J Endocrinol. 1995; 144:533-538.）。
しかしながら、この難消化性デキストリンは、２００ｍｇ／ｇ ｄｉｅｔとなるようにカイコの餌に加えても、ショ糖摂取後の血糖値の上昇は抑制されなかった（図２Ｂ）。
On the other hand, indigestible dextrin is a typical water-soluble dietary fiber, and its solution is viscous and has been reported to have an 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 if this indigestible dextrin was added to the silkworm diet so as to be 200 mg / g diet, the increase in blood glucose level after ingestion of sucrose was not suppressed (Fig. 2B).
以上の結果は、納豆の水溶性粘性画分（成分）には、ショ糖摂取後の血糖値の上昇を抑制する活性があり、それは、難消化性デキストリンより高いことを示唆している。
納豆から得られた水溶性粘性画分は、血糖値上昇抑制剤となり得ることが示唆され、また、上記結果は、給餌後１時間の結果であることから、血糖値スパイク抑制剤としての用途があることも示唆された。
The above results suggest that the water-soluble viscous fraction (ingredient) of natto has an activity of suppressing an increase in blood glucose 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 an inhibitor of blood glucose elevation, and since the above result is the result of 1 hour after feeding, it can be used as a blood glucose spike inhibitor. It was also suggested that there was.
実施例３
＜納豆の糖鎖成分には、ショ糖摂取後の血液中のグルコース濃度の上昇を抑制する効果や、ショ糖摂取後の血糖値スパイクを抑制する効果があることの証明＞
実施例１のようにして納豆から得た水溶性粘性画分に対して、DNase、RNase、proteinse K処理、及び、フェノール処理を行い、酵素処理多糖画分、すなわち納豆中の糖鎖成分を得た。
具体的には、エタノール沈殿画分に、ＤＮａｓｅＩ（１０００ｕｎｉｔ／ｍＬ；Ｐｒｏｍｅｇａ）、及び、ＲＮａｓｅＡ（１０μｇ／ｍＬ；ＮＩＰＰＯＮ ＧＥＮＥ ＣＯ．，ＬＴＤ．）を添加して、３７℃で２４時間インキュベーションし、更に、Ｐｒｏｔｅｉｎａｓｅ Ｋを加え、３７℃で２４時間インキュベーションした後、等体積のフェノール・クロロホルム・イソアミルアルコール（５０：４９：１）を加え、懸濁後、遠心し、上層画分に２倍量のエタノールを加え、生じた沈殿を遠心により集め乾燥させ、酵素処理多糖画分とした。
Example 3
<Proof that the sugar chain component of natto has the effect of suppressing the increase in glucose concentration in the blood after ingestion of sucrose and the effect of suppressing the spike in blood glucose level after ingestion of sucrose>
The water-soluble viscous fraction obtained from natto as in Example 1 was treated with DNase, RNase, proteins K, and phenol to obtain an enzyme-treated polysaccharide fraction, that is, a sugar chain component in natto. It was.
Specifically, DNaseI (1000 unit / mL; Promega) and RNaseA (10 μg / mL; NIPPON GENE CO., LTD.) Were added to the ethanol precipitation fraction, and the mixture was incubated at 37 ° C. for 24 hours, and further. , Proteinase K, incubate at 37 ° C. for 24 hours, add equal volumes of phenol, chloroform, isoamyl alcohol (50:49: 1), suspend, centrifuge, and double the amount of ethanol in the upper fraction. Was added, and the resulting precipitate was collected by centrifugation and dried to obtain an enzyme-treated polysaccharide fraction.
この酵素処理多糖画分（納豆中の糖鎖成分）の乾燥重量１ｇ当たりに含まれる、糖、ＤＮＡ、ＲＮＡ及びタンパク質の量を定量した結果、表１の通りであり、糖の含有量は３２質量％であるのに対し、ＤＮＡ、ＲＮＡ及びタンパク質の量は、何れも１質量％以下であった（表１参照）。 Table 1 shows the results of quantifying the amounts of sugar, DNA, RNA and protein contained in 1 g of dry weight of this enzyme-treated polysaccharide fraction (sugar chain component in natto), and the sugar content is 32. The amount of DNA, RNA and protein was 1% by mass or less, whereas the amount was 1% by mass (see Table 1).
納豆の水溶性粘性画分（Non-treated Natto ＃４、５０ｍｇ／ｇ ｄｉｅｔ）、DNase、RNase、proteinse K処理、及び、フェノール処理を行った酵素処理多糖画分（納豆中の糖鎖成分）（Enzyme-treated Natto ＃４、５０ｍｇ／ｇ ｄｉｅｔ）、及び、アカルボース（４０ｍｇ／ｇ ｄｉｅｔ）を、それぞれ１０質量％ショ糖含有餌に混合した。
Water-soluble viscous fraction of natto (
各試料を混合した餌、又は、混合しなかった餌（Ｃｏｎｔｒｏｌ）をカイコに食べさせ、１時間後の体液中のグルコース濃度を測定した。 The silkworms were fed a diet in which each sample was mixed or a diet in which they were not mixed (Control), and the glucose concentration in the body fluid after 1 hour was measured.
結果を図３に示す。
ｎ＝５−１０／ｇｒｏｕｐ、統計学的有意差検定は、Student’s t-testを用いて行った。図３中の棒線は、各試料の平均値を示す。
「＊」を付したデータは、Ｐ＜０．０５である。
The results are shown in FIG.
n = 5-10 / group, statistical significance test was performed using Student's t-test. The bar in FIG. 3 shows the average value of each sample.
The data with "*" is P <0.05.
＜＜結果＞＞
酵素処理多糖画分（納豆中の糖鎖成分）を、ショ糖を含有する餌に加えると、実施例１及び実施例２で得られた、水溶性粘性画分の結果と同様に、カイコの血糖値の上昇が抑制された（図３）。
以上の結果は、納豆の多糖成分が、ショ糖摂取後の血糖値の上昇を抑制する活性を有することを示唆している。
納豆に含まれる多糖成分は、血糖値上昇抑制剤となり得ることが示唆され、また、上記結果は、給餌後１時間の結果であることから、血糖値スパイク抑制剤としての用途があることも示唆された。
<< Result >>
When the enzyme-treated polysaccharide fraction (sugar chain component in natto) was added to the diet containing sucrose, the silkworm's results were similar to the results of the water-soluble viscous fractions obtained in Examples 1 and 2. The rise in blood glucose level was suppressed (Fig. 3).
The above results suggest that the polysaccharide component of natto has an activity of suppressing an increase in blood glucose level after ingestion of sucrose.
It is suggested that the polysaccharide component contained in natto can be an inhibitor of blood glucose elevation, and since the above result is the result of 1 hour after feeding, it is also suggested that it may be used as a blood glucose spike inhibitor. Was done.
実施例４
＜納豆の水溶性粘性画分に、グルコースの取り込み（輸送）を阻害する活性があることの証明＞
納豆の水溶性粘性画分＃４（５０ｍｇ／ｇ ｄｉｅｔ）を、１０質量％ショ糖含有餌、又は、１０質量％グルコース含有餌に混合した。
試料を混合した餌、又は、混合しなかった餌（Ｃｏｎｔｒｏｌ）をカイコに食べさせ、１時間後の体液中のグルコース濃度を測定した。
Example 4
<Proof that the water-soluble viscous fraction of natto has an activity to inhibit glucose uptake (transportation)>
The water-soluble viscous fraction # 4 (50 mg / g diet) of natto was mixed with a 10% by mass sucrose-containing diet or a 10 mass% glucose-containing diet.
The silkworms were fed a diet mixed with the sample or a diet not mixed (Control), and the glucose concentration in the body fluid after 1 hour was measured.
結果を図４に示す。
ｎ＝６−７／ｇｒｏｕｐ、統計学的有意差検定は、Student’s t-testを用いて行った。図４中の棒線は、各試料の平均値を示す。
「＊」を付したデータは、Ｐ＜０．０５である。
The results are shown in FIG.
n = 6-7 / group, statistical significance test was performed using Student's t-test. The bar in FIG. 4 shows the average value of each sample.
The data with "*" is P <0.05.
腸管内のショ糖は、α−グリコシダーゼにより、グルコースとフルクトースに分解され、腸管細胞に取り込まれる（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.）。
ヒトにおけるα−グリコシダーゼ阻害剤であるアカルボースやボグリボースでは、カイコにおいてもショ糖摂取後の血糖値の上昇を阻害するが、グルコース摂取による血糖値の上昇は抑制しないことが知られている（同上論文）。
Sucrose in the intestinal tract is decomposed 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.).
It is known that acarbose and voglibose, which are α-glycosidase inhibitors in humans, also inhibit the increase in blood glucose level after ingestion of sucrose in silkworm, but do not suppress the increase in blood glucose level due to glucose intake (ibid. ).
一方、図４から分かるように、納豆から得た水溶性粘性画分の餌への添加は、ショ糖及びグルコース何れの摂取後も、カイコの血糖値の上昇を抑制した。この結果は、納豆の水溶性粘性画分は、グルコースの輸送を阻害する活性を有することを示唆している。
納豆の水溶性粘性画分は、血糖値上昇抑制剤となり得ること、また、グルコースの取り込み（輸送）阻害剤となり得ることが示唆された。
On the other hand, as can be seen from FIG. 4, the addition of the water-soluble viscous fraction obtained from natto to the diet suppressed the increase in the blood glucose level of silkworms after ingestion of both sucrose and glucose. This result suggests that the water-soluble viscous fraction of natto has an activity of inhibiting glucose transport.
It was suggested that the water-soluble viscous fraction of natto could be an inhibitor of blood glucose elevation and an inhibitor of glucose uptake (transportation).
実施例５
＜納豆の糖鎖成分に、グルコース取り込み阻害活性があることの証明＞
既に、本願発明の発明者は、カイコにおけるグルコース摂取後の高血糖を抑制する乳酸菌、Enterococcus faecalis ＹＭ０８３１株が、ヒトの腸管由来であるＣａｃｏ−２細胞のグルコース取り込みを阻害することを確認している。
Ｃａｃｏ−２細胞における２−ＮＢＤＧの取り込み系において、納豆の酵素処理多糖画分（納豆中の糖鎖成分）（Enzyme-treated Natto ＃４）を加えて、Ｃａｃｏ−２細胞におけるグルコースの取り込みを蛍光測定によって定量した。
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 ingestion in silkworm, inhibits glucose uptake by Caco-2 cells derived from the human intestinal tract. ..
In the 2-NBDG uptake system in Caco-2 cells, the enzyme-treated polysaccharide fraction of natto (sugar chain component in natto) (Enzyme-treated Natto # 4) is added to fluoresce glucose uptake in Caco-2 cells. Quantified by measurement.
具体的には、Ｃａｃｏ−２細胞を用いたグルコース取り込み実験は、以下に示す方法に従って行った。
Ｃａｃｏ−２細胞は、American Type Cell Collection (ATCC, Manassas, VA, USA)から分与された。Ｃａｃｏ−２細胞は、１０％ＦＢＳ (Gibco, NY, USA)、１％ penicillin/streptomycin (Gibco, NY, USA)を加えたＤＭＥＭ (Gibco, NY, USA)培地中で、３７℃、５％ＣＯ２条件下にて培養した。
Ｃａｃｏ−２細胞を、９６ウェルプレート(Tissue culture testplate 96F: TPP, Switzerland)で単層になるまで培養し、更に血清を含まないＤＭＥＭ培地中で２４時間培養した。
Specifically, the glucose uptake experiment using Caco-2 cells was performed according to the method shown below.
Caco-2 cells were donated from the American Type Cell Collection (ATCC, Manassas, VA, USA). Caco-2 cells were prepared at 37 ° C., 5% CO in DMEM (Gibco, NY, USA) medium supplemented with 10% FBS (Gibco, NY, USA) and 1% penicillin / streptomycin (Gibco, NY, USA). It was cultured under two conditions.
Caco-2 cells were cultured in a 96-well plate (Tissue culture testplate 96F: TPP, Switzerland) until they became monolayer, and further cultured in DMEM medium containing no serum for 24 hours.
その後、培養液をNa buffer [10 mM HEPES (pH 7.4), 140 mM NaCl, 20 mg/ml BSA]に置換し、１５分間培養した。更に、Na bufferに最終濃度５０μＭ 2-deoxy-2-[(7-nitro-2, 1, 3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG: Cayman Chemical, MI, USA)を加え、１０分間インキュベーションした。その後、細胞を冷やしたＰＢＳで２回洗浄し、細胞を蛍光顕微鏡で観察した。細胞の蛍光強度をImage J ver. 1.43u (National Institutes of Health, USA)で定量した。 Then, the culture solution was replaced with Na buffer [10 mM HEPES (pH 7.4), 140 mM NaCl, 20 mg / ml BSA] and cultured for 15 minutes. In addition, the final concentration of Na buffer is 50 μM 2-deoxy-2-[(7-nitro-2, 1, 3-benzoxadiazol-4-yl) amino] -D-glucose (2-NBDG: Cayman Chemical, MI, USA) Was added and incubated for 10 minutes. The cells were then washed twice with chilled PBS and the cells were observed under a fluorescence microscope. The fluorescence intensity of cells was quantified by Image J ver. 1.43u (National Institutes of Health, USA).
結果を図５に示す。
ｎ＝３／ｇｒｏｕｐ、統計学的有意差検定は、Student’s t-testを用いて行った。図４中の棒線は、各試料の平均値を示す。
「＊」を付したデータは、Ｐ＜０．０５ vs control、エラーバーは、標準誤差（ＳＥＭ）を示す。
The results are shown in FIG.
The n = 3 / group, statistical significance test was performed using Student's t-test. The bar in FIG. 4 shows the average value of each sample.
Data with "*" indicates P <0.05 vs control, and error bars indicate standard error (SEM).
図５に示すように、納豆の酵素処理多糖画分（納豆中の糖鎖成分）の添加により、ヒト腸管細胞であるＣａｃｏ−２細胞のグルコース取り込みが阻害されることを見出した。
一方、難消化性デキストリンを添加しても、Ｃａｃｏ−２細胞のグルコース取り込みは阻害されなかった（図５）。
以上の結果から、納豆の酵素処理多糖画分（納豆中の糖鎖成分）は、腸管細胞のグルコース取り込みを阻害する高い活性を有すると考えられる。
納豆の酵素処理多糖画分（納豆中の糖鎖成分）は、グルコース取り込み阻害剤となり得ることが示唆された。
As shown in FIG. 5, it was found that the addition of the enzyme-treated polysaccharide fraction of natto (sugar chain component in natto) inhibits glucose uptake by Caco-2 cells, which are human intestinal cells.
On the other hand, the addition of indigestible dextrin did not inhibit glucose uptake by 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.
本発明の剤は、腸管からのグルコースの取り込みを阻害するので、血糖値スパイク抑制剤、食後高血糖抑制剤等の他、血糖値上昇抑制剤、糖尿病予防治療剤としても有用なので、健康食品分野、医薬品分野、食品業界等で広く利用可能である。
Since the agent of the present invention inhibits the uptake of glucose from the intestinal tract, it is useful as a blood glucose spike inhibitor, postprandial hyperglycemia inhibitor, blood glucose elevation inhibitor, diabetes preventive treatment agent, etc. , Pharmaceutical field, food industry, etc.
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| JP2002275027A (en) * | 2001-03-22 | 2002-09-25 | Nikko Seiyaku Kk | Cosmetic and cosmetic impregnated in nonwoven fabric |
| JP2009058500A (en) * | 2007-08-06 | 2009-03-19 | Genome Soyaku Kenkyusho:Kk | Evaluating method, screening method, and manufacturing method of matter for lowering blood sugar level |
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