JP6800733B2 - Glutathione-S-transferase expression promoter and food composition for expression promotion - Google Patents
Glutathione-S-transferase expression promoter and food composition for expression promotion Download PDFInfo
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Description
本発明は、１−ケストースを有効成分とする、グルタチオン−Ｓ−転移酵素の発現促進剤および発現促進用食品組成物に関する。 The present invention relates to an expression-promoting agent for glutathione-S-transferase and a food composition for promoting expression, which comprises 1-kestose as an active ingredient.
グルタチオン−Ｓ−転移酵素（グルタチオン−Ｓ−トランスフェラーゼ、ＧＳＴ）は、ほとんどの真核生物に広く存在する酵素である。哺乳動物のＧＳＴは、特に肝臓に多く存在するが、その他脾臓、腎臓、脳、骨格筋、睾丸、小腸、胎盤、赤血球などの器官にも存在する。ＧＳＴは、グルタチオンとリガンドとの結合（グルタチオン抱合）を触媒する。リガンドとしては、例えば、活性酸素や毒素・薬物・代謝産物などの生体にとって不要な物質が挙げられる。グルタチオン抱合のリガンドが活性酸素であれば還元されて消去され、不要物質であれば、そのグルタチオン抱合体が更なる代謝を受け、最終的に体外へ排出される。すなわち、ＧＳＴは、抗酸化や解毒など、生体にとって重要な役割を果たす酵素であることが知られている。 Glutathione-S-transferase (glutathione-S-transferase, GST) is an enzyme that is widely present in most eukaryotes. Mammalian GST is particularly abundant in the liver, but is also present in other organs such as the spleen, kidneys, brain, skeletal muscle, testes, small intestine, placenta, and erythrocytes. GST catalyzes the binding of glutathione to a ligand (glutathione conjugation). Examples of the ligand include substances unnecessary for the living body such as active oxygen, toxins, drugs, and metabolites. If the ligand for glutathione conjugation is active oxygen, it is reduced and eliminated, and if it is an unnecessary substance, the glutathione conjugate undergoes further metabolism and is finally excreted from the body. That is, GST is known to be an enzyme that plays an important role in living organisms such as antioxidant and detoxification.
一方、近年、活性酸素は、その強い酸化力により細胞内のＤＮＡを傷つけるため、癌や心筋梗塞、脳血管障害、アルツハイマー症等の疾患の発症ないし重症化に関与することが報告されている。このことから、ＧＳＴの発現を促進することにより活性酸素の消去を促すことができれば、上記疾患の予防や治療に寄与することが期待される。 On the other hand, in recent years, it has been reported that active oxygen is involved in the onset or aggravation of diseases such as cancer, myocardial infarction, cerebrovascular accident, and Alzheimer's disease because it damages intracellular DNA due to its strong oxidizing power. From this, if the elimination of active oxygen can be promoted by promoting the expression of GST, it is expected to contribute to the prevention and treatment of the above-mentioned diseases.
また、上述のとおりＧＳＴは、抗酸化機能の他にも、解毒機能を有するため、ＧＳＴの発現を促進することにより発ガン性物質などの生体外への排出を促すことができれば、癌の予防や健康の維持・増進に寄与することが期待される。 Further, as described above, GST has a detoxification function in addition to an antioxidant function. Therefore, if the expression of GST can be promoted to promote the excretion of carcinogenic substances to the outside of the living body, cancer can be prevented. It is expected to contribute to the maintenance and promotion of health and health.
そこで、ＧＳＴの発現を促進する物質が研究開発されており、例えば、特許文献１にはイチョウ葉抽出物を有効成分とするＧＳＴ活性化剤が、特許文献２には生体内でのＧＳＴ生成作用を有するセレン含有組成物が、特許文献３にはウコギ科人参抽出物を有効成分とするＧＳＴ発現促進剤が、それぞれ開示されている。 Therefore, substances that promote the expression of GST have been researched and developed. For example, Patent Document 1 describes a GST activator containing an ginkgo biloba extract as an active ingredient, and Patent Document 2 describes a GST-producing action in vivo. Patent Document 3 discloses a selenium-containing composition having the above, and a GST expression promoter containing an extract of Araliaceae ginseng as an active ingredient.
しかしながら、特許文献１に記載のイチョウ葉抽出物、特許文献２に記載のセレン含有組成物、特許文献３に記載のウコギ科人参抽出物は、いずれも特有の風味を有すると考えられるため、様々な食品や医薬品等に容易に配合することないし日常的に簡便に摂取することができるものとはいえない。すなわち、上記特許文献を鑑みても、ＧＳＴの発現を効果的に促進することができ、かつ、安全性が高く、様々な食品や医薬品等に容易に配合することないし日常的に簡便に摂取することができる物質は未だ十分に提供されておらず、そのような物質の開発が求められていた。 However, the ginkgo biloba extract described in Patent Document 1, the selenium-containing composition described in Patent Document 2, and the Araliaceae ginseng extract described in Patent Document 3 are all considered to have a peculiar flavor, and therefore vary. It cannot be said that it can be easily mixed with various foods and pharmaceuticals and can be easily taken on a daily basis. That is, even in view of the above patent documents, the expression of GST can be effectively promoted, the safety is high, the substance is not easily blended in various foods and pharmaceuticals, and the drug is easily ingested on a daily basis. The substances that can be produced have not yet been sufficiently provided, and the development of such substances has been sought.
本発明は、このような課題を解決するためになされたものであって、ＧＳＴの発現を効果的に促進することができ、かつ、安全性が高く、様々な食品や医薬品等に容易に配合することないし日常的に簡便に摂取することができるＧＳＴの発現促進剤および発現促進用食品組成物を提供することを目的とする。 The present invention has been made to solve such a problem, can effectively promote the expression of GST, has high safety, and can be easily blended in various foods, pharmaceuticals, etc. An object of the present invention is to provide an expression-promoting agent for GST and a food composition for promoting expression, which can be easily taken on a daily basis.
本発明者らは、鋭意研究の結果、１−ケストースを摂食することにより、ＧＳＴの発現を効果的に促進できることを見出した。そこで、この知見に基づいて下記の各発明を完成した。 As a result of diligent research, the present inventors have found that the expression of GST can be effectively promoted by feeding 1-kestose. Therefore, based on this finding, each of the following inventions was completed.
（１）本発明に係るグルタチオン−Ｓ−転移酵素の発現促進剤は、１−ケストースを有効成分とする。 (1) The expression-promoting agent for glutathione-S-transferase according to the present invention contains 1-kestose as an active ingredient.
（２）本発明に係るグルタチオン−Ｓ−転移酵素の発現促進剤は、抗酸化剤として用いることができる。 (2) The glutathione-S-transferase expression promoter according to the present invention can be used as an antioxidant.
（３）本発明に係るグルタチオン−Ｓ−転移酵素の発現促進剤は、不要物質の排出促進剤として用いることができる。 (3) The glutathione-S-transferase expression promoter according to the present invention can be used as an emission promoter of unnecessary substances.
（４）本発明に係るグルタチオン−Ｓ−転移酵素の発現促進用食品組成物は、１−ケストースを有効成分とする。 (4) The food composition for promoting the expression of glutathione-S-transferase according to the present invention contains 1-kestose as an active ingredient.
（５）本発明に係るグルタチオン−Ｓ−転移酵素の発現促進用食品組成物は、抗酸化用に用いることができる。 (5) The food composition for promoting the expression of glutathione-S-transferase according to the present invention can be used for antioxidants.
（６）本発明に係るグルタチオン−Ｓ−転移酵素の発現促進用食品組成物は、不要物質の排出促進用に用いることができる。 (6) The food composition for promoting the expression of glutathione-S-transferase according to the present invention can be used for promoting the discharge of unnecessary substances.
本発明に係るＧＳＴの発現促進剤および発現促進用食品組成物が有効成分とする１−ケストースは、オリゴ糖の一種であり、タマネギやニンニク、大麦、ライ麦などの野菜や穀物にも含まれていて、古来より食経験を有する物質である。また、１−ケストースは、変異原性試験、急性毒性試験、亜慢性毒性試験および慢性毒性試験のいずれにおいても毒性が認められていない。これらのことから、１−ケストースの安全性は極めて高いといえる（食品と開発、Ｖｏｌ．４９、Ｎｏ．１２、第９頁、２０１４年）。
また、１−ケストースは、水溶性が高く、砂糖に似た良好な甘味質を有するため、そのまま、あるいは甘味料等として、日常的に簡便に摂取することができるほか、様々な食品や医薬品等に容易に配合することができる。
したがって、本発明によれば、ＧＳＴの発現を効果的に促進することができ、かつ、安全性も高く、様々な食品や医薬品等に容易に配合することないし日常的に簡便に摂取することができるＧＳＴの発現促進剤および発現促進用食品組成物を得ることができる。
1-Kestose, which is an active ingredient of the GST expression-promoting agent and the expression-promoting food composition according to the present invention, is a kind of oligosaccharide and is also contained in vegetables and grains such as onion, garlic, barley and rye. It is a substance that has been eaten since ancient times. In addition, 1-kestose has not been found to be toxic in any of the mutagenicity test, acute toxicity test, subchronic toxicity test and chronic toxicity test. From these facts, it can be said that the safety of 1-kestose is extremely high (Food and Development, Vol. 49, No. 12, p. 9, 2014).
In addition, 1-kestose is highly water-soluble and has a good sweetness similar to sugar, so that it can be easily ingested as it is or as a sweetener on a daily basis, as well as various foods and pharmaceuticals. Can be easily blended into.
Therefore, according to the present invention, the expression of GST can be effectively promoted, the safety is high, the mixture is not easily blended in various foods and pharmaceuticals, and it can be easily ingested on a daily basis. It is possible to obtain a GST expression-promoting agent and a food composition for promoting expression.
以下、本発明に係るＧＳＴの発現促進剤および発現促進用食品組成物について、詳細に説明する。
本発明に係るＧＳＴの発現促進剤および発現促進用食品組成物は、１−ケストースを有効成分とする。
Hereinafter, the GST expression-promoting agent and the expression-promoting food composition according to the present invention will be described in detail.
The GST expression-promoting agent and the expression-promoting food composition according to the present invention contain 1-kestose as an active ingredient.
１−ケストースは、１分子のグルコースと２分子のフルクトースからなる三糖類のオリゴ糖である。１−ケストースは、スクロースを基質として、特開昭５８−２０１９８０号公報に開示されているような酵素による酵素反応を行うことにより得ることができる。具体的には、まず、β−フルクトフラノシダーゼをスクロース溶液に添加し、３７℃〜５０℃で２０時間程度静置することにより酵素反応を行って、１−ケストース含有反応液を得る。この１−ケストース含有反応液を、特開２０００−２３２８７８号公報で開示されているようなクロマト分離法に供することよって、１−ケストースと他の糖（ブドウ糖、果糖、ショ糖、４糖以上のオリゴ糖）とを分離して精製し、高純度１−ケストース溶液を得る。続いて、この高純度１−ケストース溶液を濃縮した後、特公平６−７００７５号公報に開示されているような結晶化法で結晶化することにより、１−ケストースを結晶として得ることができる。 1-Kestose is a trisaccharide oligosaccharide consisting of one molecule of glucose and two molecules of fructose. 1-Kestose can be obtained by performing an enzymatic reaction with an enzyme as disclosed in JP-A-58-201980 using sucrose as a substrate. Specifically, first, β-fructofuranosidase is added to a sucrose solution, and the mixture is allowed to stand at 37 ° C. to 50 ° C. for about 20 hours to carry out an enzymatic reaction to obtain a 1-kestose-containing reaction solution. By subjecting this 1-kestose-containing reaction solution to a chromatographic separation method as disclosed in JP-A-2000-232878, 1-kestose and other sugars (glucose, fructose, sucrose, tetrasaccharide or more) can be used. (Oligosaccharide) is separated and purified to obtain a high-purity 1-kestose solution. Subsequently, this high-purity 1-kestose solution is concentrated and then crystallized by a crystallization method as disclosed in Japanese Patent Publication No. 6-70075, whereby 1-kestose can be obtained as crystals.
また、１−ケストースは市販のフラクトオリゴ糖に含まれているため、これをそのまま、あるいは、フラクトオリゴ糖から上述の方法により１−ケストースを分離精製して用いてもよい。すなわち、本発明の１−ケストースとして、１−ケストースを含有するオリゴ糖などの１−ケストース含有組成物を用いてもよい。１−ケストース含有組成物を用いる場合、１−ケストースの純度は８０％以上であることが好ましく、８５％以上であることがより好ましく、９０％以上であることがさらに好ましい。なお、本発明において、１−ケストースの「純度」とは、糖の総質量を１００とした場合の、１−ケストースの質量％をいう。 Further, since 1-kestose is contained in a commercially available fructooligosaccharide, 1-kestose may be used as it is, or 1-kestose may be separated and purified from the fructooligosaccharide by the above-mentioned method. That is, as the 1-kestose of the present invention, a 1-kestose-containing composition such as an oligosaccharide containing 1-kestose may be used. When a 1-kestose-containing composition is used, the purity of 1-kestose is preferably 80% or more, more preferably 85% or more, and even more preferably 90% or more. In the present invention, the "purity" of 1-kestose means the mass% of 1-kestose when the total mass of sugar is 100.
本発明の発現促進剤は、そのまま、あるいは医薬品や食品添加剤、サプリメントなどの形態で、ヒトまたは動物に経口摂取させることにより使用することができる。その他、１−ケストースを経腸栄養剤に添加して、これを、胃や小腸などの消化管に挿入したチューブを経由して経腸栄養法により投与する方法により使用してもよい。 The expression-promoting agent of the present invention can be used as it is, or in the form of a drug, a food additive, a supplement, or the like, by being orally ingested by a human or an animal. Alternatively, 1-kestose may be added to an enteral nutritional supplement and used by the enteral nutrition method via a tube inserted into the digestive tract such as the stomach or small intestine.
本発明の発現促進剤を医薬品やサプリメントの形態で用いる場合、その剤型は、例えば、散剤、錠剤、糖衣剤、カプセル剤、顆粒剤、ドライシロップ剤、液剤、シロップ剤、ドロップ剤、ドリンク剤等の固形または液状の剤型にすることができる。 When the expression-promoting agent of the present invention is used in the form of a drug or supplement, the dosage form is, for example, powder, tablet, sugar coating, capsule, granule, dry syrup, liquid, syrup, drop, drink, etc. Can be in solid or liquid dosage form.
上記各剤型の医薬品やサプリメントは、当業者に公知の方法で製造することができる。例えば、散剤であれば、１−ケストース８００ｇおよび乳糖２００ｇをよく混合した後、９０％エタノール３００ｍＬを添加して湿潤させる。続いて、湿潤粉末を造粒した後、６０℃で１６時間通風乾燥し、その後、整粒して、適当な細かさの散剤１０００ｇ（１−ケストース含有量８００ｍｇ／１ｇ）を得ることができる。また、錠剤であれば、１−ケストース３００ｇ、粉末還元水飴３８０ｇ、コメデンプン１８０ｇおよびデキストリン１００ｇをよく混合した後、９０％エタノール３００ｍＬを添加して湿潤させる。続いて、湿潤粉末を押し出し造粒した後、６０℃で１６時間通風乾燥して顆粒を得る。その後、この顆粒を８５０μｍの篩を用いて整粒し、続いて顆粒４７０ｇにショ糖脂肪酸エステル５０ｇを添加して混合した後、ロータリー打錠機（６Ｂ−２、菊水製作所製）を用いて打錠し、直径８ｍｍ、重量２００ｍｇの錠剤５０００錠（１−ケストース含有量６０ｍｇ／１錠）を得ることができる。 Each of the above dosage forms of drugs and supplements can be produced by a method known to those skilled in the art. For example, in the case of powder, 800 g of 1-kestose and 200 g of lactose are mixed well, and then 300 mL of 90% ethanol is added to moisten the powder. Subsequently, the wet powder is granulated, air-dried at 60 ° C. for 16 hours, and then granulated to obtain 1000 g (1-kestose content 800 mg / 1 g) of a powder having an appropriate fineness. In the case of tablets, 300 g of 1-kestose, 380 g of powdered reduced starch syrup, 180 g of rice starch and 100 g of dextrin are well mixed, and then 300 mL of 90% ethanol is added to moisten the tablets. Subsequently, the wet powder is extruded and granulated, and then air-dried at 60 ° C. for 16 hours to obtain granules. Then, the granules were sized using a 850 μm sieve, then 50 g of sucrose fatty acid ester was added to 470 g of the granules and mixed, and then beaten using a rotary tableting machine (6B-2, manufactured by Kikusui Seisakusho). It is possible to obtain 5000 tablets (1-kestose content 60 mg / tablet) having a diameter of 8 mm and a weight of 200 mg.
また、本発明の発現促進用食品組成物は、そのまま、飲食物としてヒトまたは動物に摂取させることにより使用することができる。その他、各種の飲食物の通常の製造過程で、添加して使用することもできる。１−ケストースの甘味度は３０で、その味質・物性・加工性はショ糖に近いことから、各種飲食物の製造過程において、砂糖の一部または全部を１−ケストースに置き換えるなどして、砂糖と同様に扱って各種飲食物を製造することができる。また、本発明の発現促進用食品組成物を動物に摂取させる場合は、動物飼料に添加して用いてもよい。 In addition, the expression-promoting food composition of the present invention can be used as it is by ingesting it as a food or drink to humans or animals. In addition, it can be added and used in the normal manufacturing process of various foods and drinks. The sweetness of 1-kestose is 30, and its taste, physical properties, and processability are close to those of sucrose. Therefore, in the manufacturing process of various foods and drinks, part or all of sugar is replaced with 1-kestose. Various foods and drinks can be produced by treating it in the same way as sugar. In addition, when the food composition for promoting expression of the present invention is ingested by an animal, it may be added to an animal feed and used.
本発明の発現促進剤や発現促進用食品組成物をヒトや動物に対して用いる場合の有効成分の摂取量（投与量）としては、例えば、１日あたり０．０４ｇ／ｋｇ体重以上を挙げることができる。係る摂取量は、１日１回に限らず、複数回に分割して摂取してもよい。 When the expression-promoting agent or the expression-promoting food composition of the present invention is used for humans or animals, the intake (dose) of the active ingredient is, for example, 0.04 g / kg body weight or more per day. Can be done. The amount of such intake is not limited to once a day, and may be divided into a plurality of times.
グルタチオン−Ｓ−転移酵素（グルタチオン−Ｓ−トランスフェラーゼ、ＧＳＴ）は、２つのドメインをもつサブユニットを、ホモ、あるいは極めて構造がよく似たヘテロで構成された２量体であり、生体内では細胞質に存在する可溶性タンパク質である。ＧＳＴは、一次構造と基質特異性を基にα、μ、π、σ、θ、δ、ε、Ω、ζ、λ、Ｔ等といった多くのクラスに分類される。例えば、ラットでは少なくとも１８、ヒトでは少なくとも１６のサブユニットが報告されている（平塚明、「肝薬物代謝の最近の進歩 ５．グルタチオン Ｓ−トランスフェラーゼ」、肝臓、第４２巻、第６号、ｐ．３０３−３０４）。 Glutathione-S-transferase (glutathione-S-transferase, GST) is a dimer composed of subunits with two domains, homozygous or heterozygous for very similar structures, and is cytoplasmic in vivo. It is a soluble protein present in. GST is classified into many classes such as α, μ, π, σ, θ, δ, ε, Ω, ζ, λ, T, etc. based on the primary structure and substrate specificity. For example, at least 18 subunits have been reported in rats and at least 16 subunits in humans (Akira Hiratsuka, "Recent Advances in Liver Drug Metabolism 5. Glutathione S-Transferase", Liver, Vol. 42, No. 6, p. .303-304).
ＧＳＴの表記は、例えば、「ＧＳＴ Ａ １−１」であれば、α（Ａ）クラスで最初に発見されたサブユニット１がホモで構成されたものというように、クラスをギリシャ文字またはローマ字の大文字で、構成サブユニット（発見された順）をアラビア数字で、それぞれ表記する。ＧＳＴの前には、生物種（ヒトはｈ、ラットはｒ、マウスはｍなど）がローマ字の小文字で記載される場合もある（宮本徹、「グルタチオンＳ−転移酵素の構造と機能」、東京農大農学集報、２０１３年、第５７巻、第４号、ｐ．２４７−２６０）。 The GST notation is, for example, "GST A 1-1", where the subunit 1 first discovered in the α (A) class is composed of homo, and the class is in Greek or Roman letters. Uppercase letters indicate the constituent subunits (in order of discovery) in Arabic numerals. Prior to GST, species (h for humans, r for rats, m for mice, etc.) may be listed in lowercase Roman letters (Toru Miyamoto, "Structure and Function of Glutathione S-Transferase", Tokyo. Agricultural University Agricultural Bulletin, 2013, Vol. 57, No. 4, p. 247-260).
本発明が対象とするＧＳＴは、いずれのサブユニットからなるものであってもよく、動物、植物、菌類、細菌等いずれの生物に由来するものでもよいが、好ましくは動物由来のＧＳＴを挙げることができる。 The GST targeted by the present invention may consist of any subunit, and may be derived from any organism such as an animal, a plant, a fungus, or a bacterium, but a GST derived from an animal is preferable. Can be done.
本発明において、「ＧＳＴの発現を促進する」とは、生体のいずれかの細胞ないし組織・器官において、ＧＳＴ遺伝子の転写量を大きくすること、ＧＳＴタンパク質の量を大きくすること、または、ＧＳＴタンパク質の活性を大きくすることをいう。 In the present invention, "promoting the expression of GST" means increasing the transcription amount of the GST gene, increasing the amount of the GST protein, or the GST protein in any cell, tissue, or organ of the living body. It means to increase the activity of.
本発明において、ＧＳＴの発現が促進されたか否かは、当業者に公知の方法により確認することができる。具体的には、例えば、後述する実施例に示すように、リアルタイムＰＣＲ法によりＧＳＴ遺伝子の転写量を確認する方法を挙げることができる。
すなわち、従来、ＧＳＴが存在していることが知られている組織（血液、肝臓など）の一部を試料として採取し、本発明の発現促進剤または発現促進用食品組成物を摂取した場合としていない場合とで、当該試料におけるＧＳＴのメッセンジャーＲＮＡの含有量を比較する。前者の方がメッセンジャーＲＮＡの含有量が大きければ、本発明の発現促進剤または発現促進用食品組成物により、ＧＳＴの発現が促進されたと判断することができる。
In the present invention, whether or not the expression of GST is promoted can be confirmed by a method known to those skilled in the art. Specifically, for example, as shown in Examples described later, a method of confirming the transcription amount of the GST gene by a real-time PCR method can be mentioned.
That is, as a case where a part of a tissue (blood, liver, etc.) in which GST is known to be present is collected as a sample and the expression-promoting agent or the expression-promoting food composition of the present invention is ingested. The content of GST messenger RNA in the sample is compared with and without. If the former has a higher content of messenger RNA, it can be determined that the expression-promoting agent or the expression-promoting food composition of the present invention promoted the expression of GST.
また、ＧＳＴの発現が促進されたか否かを確認する他の方法としては、例えば、上記試料において、特許文献２の段落[0024]に記載のようなウエスタンブロット法により、ＧＳＴタンパク質の量を確認する方法を挙げることができる。
すなわち、Sodium dodecyl sulfateとポリアクリルアミドゲルを利用した電気泳動（SDS-PAGE）により試料中のタンパク質をペプチド鎖長に応じて分離した後、ゲルにメンブレンを密着させ、分離したタンパク質を電圧をかけてゲルからメンブレン（ニトロセルロース膜など）に移す。このメンブレンに、ＧＳＴ特異的抗体（一次抗体）を結合させ、洗浄した後、アルカリファリフォスファターゼなどの酵素で標識した二次抗体を反応させる。その後、標識酵素の活性を利用した化学発光法もしくは発色法により検出する。検出強度（発光強度もしくは発色強度）は、ＧＳＴタンパク質の量に比例するため、前者の試料の方が検出強度が大きければ、本発明の発現促進剤または発現促進用食品組成物により、ＧＳＴの発現が促進されたと判断することができる。
As another method for confirming whether or not the expression of GST is promoted, for example, in the above sample, the amount of GST protein is confirmed by Western blotting as described in paragraph [0024] of Patent Document 2. There are ways to do this.
That is, proteins in a sample are separated according to the peptide chain length by electrophoresis (SDS-PAGE) using Sodium dodecyl sulfate and polyacrylamide gel, a membrane is adhered to the gel, and the separated proteins are applied with a voltage. Transfer from gel to membrane (such as nitrocellulose membrane). A GST-specific antibody (primary antibody) is bound to this membrane, washed, and then reacted with an enzyme-labeled secondary antibody such as alkaline fariphosphatase. Then, it is detected by a chemiluminescence method or a color development method utilizing the activity of the labeling enzyme. Since the detection intensity (emission intensity or color development intensity) is proportional to the amount of GST protein, if the detection intensity of the former sample is higher, the expression of GST is expressed by the expression promoter of the present invention or the expression-promoting food composition. Can be judged to have been promoted.
また、ＧＳＴの発現が促進されたか否かを確認する他の方法としては、例えば、上記試料において、特許文献２の段落[0022]-[0023]に記載のように、ＧＳＴタンパク質の活性値を確認する方法を挙げることができる。
すなわち、上記試料を０．２Ｍスクロース／１０ｍＭリン酸緩衝液中で破砕して上清を得る。この上清と、１００ｍＭのリン酸緩衝液（ｐＨ６．５)、１ｍＭの1-chrolo-2,4-dinitrobenzene（ＣＤＮＢ）および１ｍＭの還元型グルタチオン（ＧＳＨ）とを含む反応溶液を調製し、３０℃における３４０ｎｍの吸光度を１５秒毎に３分間測定する。
ＣＤＮＢ は、ＧＳＴの基質でありＧＳＨと反応する。この反応によって生成するグルタチオン抱合体は ３４０ｎｍの吸収をもつため、前者の試料の方が吸光度が大きければ、本発明の発現促進剤または発現促進用食品組成物により、ＧＳＴの発現が促進されたと判断することができる。
Further, as another method for confirming whether or not the expression of GST is promoted, for example, in the above sample, as described in paragraphs [0022]-[0023] of Patent Document 2, the activity value of the GST protein is determined. The method of confirmation can be mentioned.
That is, the sample is crushed in 0.2 M sucrose / 10 mM phosphate buffer to obtain a supernatant. A reaction solution containing 100 mM phosphate buffer (pH 6.5), 1 mM 1-chrolo-2,4-dinitrobenzene (CDNB) and 1 mM reduced glutathione (GSH) was prepared from this supernatant, and 30 The absorbance at 340 nm at ° C. is measured every 15 seconds for 3 minutes.
CDNB is a substrate for GST and reacts with GSH. Since the glutathione conjugate produced by this reaction has an absorption of 340 nm, if the former sample has a higher absorbance, it is judged that the expression promoter of the present invention or the expression-promoting food composition promoted the expression of GST. can do.
上述のとおり、ＧＳＴは、強力な酸化力をもつ活性酸素や過酸化物を、グルタチオン抱合して還元することにより消去する（宮本徹、「グルタチオンＳ−転移酵素の構造と機能」、東京農大農学集報、２０１３年、第５７巻、第４号、ｐ．２４７−２６０；平塚明、「肝薬物代謝の最近の進歩 ５．グルタチオン Ｓ−トランスフェラーゼ」、肝臓、第４２巻、第６号、ｐ．３０２−３０８）。すなわち、ＧＳＴの発現を促進することにより、生体内の酸化ストレスを軽減できることから、ＧＳＴの発現促進剤や発現促進用食品組成物は、生体の酸化を予防ないし軽減する用途に用いることができる。従って、本発明の発現促進剤や発現促進用食品組成物は、抗酸化剤や抗酸化用食品組成物として用いることができる。 As mentioned above, GST eliminates active oxygen and peroxides with strong oxidizing power by conjugating and reducing glutathione (Toru Miyamoto, "Structure and function of glutathione S-transferase", Tokyo Agricultural University Agriculture. Shuho, 2013, Vol. 57, No. 4, p.247-260; Akira Hiratsuka, "Recent Advances in Liver Drug Metabolism 5. Glutathione S-Transferase," Liver, Vol. 42, No. 6, p. .302-308). That is, since oxidative stress in the living body can be reduced by promoting the expression of GST, the GST expression promoter and the food composition for promoting the expression can be used for preventing or reducing the oxidation of the living body. Therefore, the expression-promoting agent and the expression-promoting food composition of the present invention can be used as an antioxidant or an antioxidant food composition.
また、上述のとおり、ＧＳＴは、毒素・薬物・代謝産物などの生体にとって不要な物質をグルタチオン抱合し、腎臓での更なる代謝等を経て最終的に体外へ排出されることを促す（宮本徹、「グルタチオンＳ−転移酵素の構造と機能」、東京農大農学集報、２０１３年、第５７巻、第４号、ｐ．２４７−２６０；平塚明、「肝薬物代謝の最近の進歩 ５．グルタチオン Ｓ−トランスフェラーゼ」、肝臓、第４２巻、第６号、ｐ．３０２−３０８）。すなわち、ＧＳＴの発現を促進することにより、生体にとって不要な物質の解毒ないし排出を促進できることから、ＧＳＴの発現促進剤や発現促進用食品組成物は、当該不要物質の排出を促進する用途に用いることができる。従って、本発明の発現促進剤や発現促進用食品組成物は、不要物質の排出促進剤や排出促進用食品組成物として用いることができる。 In addition, as mentioned above, GST conjugates glutathione-unnecessary substances such as toxins, drugs, and metabolites, and promotes them to be finally excreted from the body through further metabolism in the kidney (Toru Miyamoto). , "Structure and Function of Glutathione S-Transfer Enzyme", Tokyo Agricultural University Agricultural Bulletin, 2013, Vol. 57, No. 4, p.247-260; Akira Hiratsuka, "Recent Advances in Liver Drug Metabolism 5. Glutathione "S-Transferase", Liver, Vol. 42, No. 6, p. 302-308). That is, since the detoxification or excretion of substances unnecessary for the living body can be promoted by promoting the expression of GST, GST expression promoters and food compositions for promoting expression are used for promoting the excretion of the unnecessary substances. be able to. Therefore, the expression-promoting agent and the expression-promoting food composition of the present invention can be used as an emission-promoting agent for unnecessary substances and a food composition for promoting discharge.
以下、本発明に係るＧＳＴの発現促進剤および発現促進用食品組成物について、各実施例に基づいて説明する。なお、本発明の技術的範囲は、これらの実施例によって示される特徴に限定されない。 Hereinafter, the GST expression-promoting agent and the expression-promoting food composition according to the present invention will be described based on each example. The technical scope of the present invention is not limited to the features shown in these examples.
本実施例においては、所定の純度で１−ケストースを含有する組成物を、「１−ケストース」という。また、本実施例において、各群の平均値の間の有意差はＴ検定により検定し、危険率５％未満を有意とみなした。 In this example, a composition containing 1-kestose with a predetermined purity is referred to as "1-kestose". In addition, in this example, the significant difference between the mean values of each group was tested by T-test, and a risk rate of less than 5% was regarded as significant.
＜実施例１＞１−ケストース摂取時の遺伝子発現量の検討
（１）１−ケストースを経口摂取させたマウスの飼育
マウスの標準精製飼料「ＡＩＮ−９３Ｇ」（日本クレア社）の配合において、コーンスターチの一部を１−ケストース（純度９９％；物産フードサイエンス社）に置換することにより、１−ケストースを５質量％となるように配合した飼料を日本クレア社に委託して調製し、これをケストース配合飼料とした。ＡＩＮ−９３Ｇの配合を以下に示す。
《ＡＩＮ−９３Ｇの配合（単位は質量％）》コーンスターチ ３９．７４８６％、ミルクカゼイン ２０％、アルファ化コーンスターチ １３．２％、グラニュー糖 １０％、精製大豆油 ７％、セルロースパウダー ５％、ミネラルミックス ３．５％、ビタミンミックス １％、Ｌ−シスチン ０．３％、重酒石酸コリン ０．２５％、第３ブチルヒドロキノン ０．００１４％。
<Example 1> Examination of gene expression level at the time of ingestion of 1-kestose (1) Breeding of mice ingested 1-kestose Orally ingested cornstarch in the formulation of the standard purified feed "AIN-93G" (Claire Japan) of mice. By substituting 1-kestose (purity 99%; Bussan Food Science Co., Ltd.) for a part of 1-kestose, a feed containing 1-kestose in an amount of 5% by mass was outsourced to Japan Claire Co., Ltd. It was used as a kestose-blended feed. The formulation of AIN-93G is shown below.
<< Combination of AIN-93G (unit: mass%) >> Cornstarch 39.7486%, milk casein 20%, pregelatinized cornstarch 13.2%, granulated sugar 10%, refined soybean oil 7%, cellulose powder 5%, mineral mix 3.5%, vitamin mix 1%, L-cystine 0.3%, choline bitartrate 0.25%, tertiary butylhydroquinone 0.0014%.
Ｃ５７ＢＬ／６マウス（中部科学資材社）を１０匹ずつ２つの群に分け、一方をケストース食群、他方を対照群とした。対照群にはＡＩＮ−９３Ｇを、ケストース食群にはケストース配合飼料をそれぞれ自由摂取させながら、１０週間飼育した。飼育条件は温度２３±１℃、明期１２時間（８：００〜２０：００）および暗期１２時間（２０：００〜８：００）とした。 C57BL / 6 mice (Chubu Scientific Materials Co., Ltd.) were divided into two groups of 10 mice each, one of which was a kestose diet group and the other of which was a control group. The control group was bred with AIN-93G, and the kestose diet group was bred with a kestose-blended diet for 10 weeks. The breeding conditions were a temperature of 23 ± 1 ° C., a light period of 12 hours (8:00 to 20:00) and a dark period of 12 hours (20:00 to 8:00).
飼育期間中は、一週間経過毎に体重を測定し、各群毎に平均値および標準偏差を求めて有意差検定を行った。その結果を図１に示す。また、飼育期間終了後に各群のマウスを解剖して肝臓を摘出し、その重量を測定して、各群毎に平均値および標準偏差を求めて有意差検定を行った。その結果を図２に示す。
図１および図２に示すように、ケストース食群と対照群とで、体重および肝臓重量に有意な差は見られなかった。
During the breeding period, the body weight was measured every week, and the mean value and standard deviation were obtained for each group and a significant difference test was performed. The result is shown in FIG. In addition, after the end of the breeding period, the mice in each group were dissected, the liver was removed, the weight was measured, and the mean value and standard deviation were obtained for each group to perform a significance test. The result is shown in FIG.
As shown in FIGS. 1 and 2, no significant difference in body weight and liver weight was observed between the kestose diet group and the control group.
（２）ＤＮＡマイクロアレイによる候補遺伝子の選定
本実施例１（１）のマウス肝臓から、摂食量および体重増加量が平均的であった個体のものを、各群毎に５個体分ずつ選んだ。それらの肝臓組織からNucleo Spin RNAキット（タカラバイオ社）を用いて総ＲＮＡを抽出して、各群毎に、等量ずつ混合した。この総ＲＮＡを鋳型として、逆転写酵素ArrayScriptおよびT7 Oligo dT Primerを用いてｃＤＮＡを合成した。次に、dNTP mix、DNA ligase、DNA PolymeraseおよびRnase Hを用いて２本鎖ｃＤＮＡを合成した（以上、Ambion社）。得られた２本鎖ｃＤＮＡをMin Elute PCR Purification Kit（QIAGEN社）を用いて精製した後、Massage Amp-Biotin Kit（Ambion社）によりビオチン標識し、Rneasy MinElute Cleanup Kit（QIAGEN社）で精製してビオチン標識ｃＤＮＡを得た。続いて、３０ｍＭの酢酸マグネシウムおよび１００ｍＭの酢酸カリウムを含む４０ｍＭのトリス−酢酸緩衝液（ｐＨ８．１）中で、９４℃で３５分間加熱することにより断片化反応を行い、断片化ビオチン標識ｃＤＮＡを得た。
(2) Selection of Candidate Genes by DNA Microarray From the mouse livers of Example 1 (1), individuals with average food intake and weight gain were selected for 5 individuals in each group. Total RNA was extracted from those liver tissues using the Nucleo Spin RNA Kit (Takara Bio Inc.) and mixed in equal amounts for each group. Using this total RNA as a template, cDNA was synthesized using the reverse transcriptase ArrayScript and T7 Oligo dT Primer. Next, double-stranded cDNA was synthesized using dNTP mix, DNA ligase, DNA Polymerase and Rnase H (above, Ambion). The obtained double-stranded cDNA was purified using the Min Elute PCR Purification Kit (QIAGEN), biotin-labeled with the Massage Amp-Biotin Kit (Ambion), and purified with the Rneasy MinElute Cleanup Kit (QIAGEN). Biotin-labeled cDNA was obtained. Subsequently, the fragmentation reaction was carried out by heating at 94 ° C. for 35 minutes in 40 mM Tris-acetate buffer (pH 8.1) containing 30 mM magnesium acetate and 100 mM potassium acetate to obtain the fragmented biotin-labeled cDNA. Obtained.
断片化ビオチン標識ｃＤＮＡを、酸化ストレス・アンチエイジングチップ 「ジェノパール（登録商標） ＲＯＳＭ−ＪＸ（マウス版；２１９遺伝子搭載）」（三菱レイヨン社）へハイブリダイズさせた。この反応は、ハイブリダイゼーション用バッファー中で６５℃にて１６時間行った。その後、Biochip Reader MB-M3C（横河電機社）を用いて蛍光シグナルの検出を行った。蛍光シグナルの測定値が１００以上を示した遺伝子を陽性として抽出し、それらについて、「対照群の測定値」に対する「ケストース食群の測定値」の比（発現比：ケストース食群の測定値／対照群の測定値）を求めた。発現比が１．５倍以上であった遺伝子を、１−ケストースの摂食により発現が促進される候補遺伝子として抽出した。 Fragmented biotin-labeled cDNA was hybridized to the oxidative stress anti-aging chip "Genopal (registered trademark) ROSM-JX (mouse version; with 219 genes)" (Mitsubishi Rayon Co., Ltd.). This reaction was carried out at 65 ° C. for 16 hours in hybridization buffer. After that, the fluorescence signal was detected using Biochip Reader MB-M3C (Yokogawa Electric Co., Ltd.). Genes showing a fluorescence signal measurement value of 100 or more were extracted as positive, and the ratio of the "measured value of the Kestose diet group" to the "measured value of the control group" (expression ratio: measured value of the Kestose diet group /). The measured value of the control group) was determined. Genes whose expression ratio was 1.5 times or more were extracted as candidate genes whose expression is promoted by feeding 1-kestose.
その結果、候補遺伝子として、グルタチオン−Ｓ−トランスフェラーゼのクラスπのサブユニット１（Glutathione S-transferase pi 1；Ｇｓｔｐ１）（発現比２．１３倍）およびグルタチオン−Ｓ−トランスフェラーゼのクラスαのサブユニット４（Glutathione S-transferase alpha 4；Ｇｓｔａ４）（発現比１．５０倍）の２遺伝子が抽出された。そこで、これらの遺伝子について、リアルタイムＰＣＲ法により発現量の定量的解析を行うこととした。 As a result, as candidate genes, Glutathione S-transferase class π subunit 1 (Glutathione S-transferase pi 1; Gstp1) (expression ratio 2.13 times) and glutathione-S-transferase class α subunit 4 Two genes (Glutathione S-transferase alpha 4; Gsta4) (expression ratio 1.50 times) were extracted. Therefore, we decided to quantitatively analyze the expression levels of these genes by the real-time PCR method.
（３）リアルタイムＰＣＲ法による遺伝子発現量の定量的解析
本実施例１（１）のマウス肝臓から、Nucleo Spin RNAキット（タカラバイオ社）を用いて総ＲＮＡを抽出した。この総ＲＮＡから、ReverTra Ace qPCRRT Master Mix（東洋紡社）を用いて逆転写反応を行い、ｃＤＮＡを作成した。このｃＤＮＡを鋳型として、下記の遺伝子特異的プライマー、リアルタイムＰＣＲ試薬「THUNDERBIED SYBR qPCR Mix（東洋紡社）」およびリアルタイムＰＣＲ装置「Thermal Cycler Dice Real Time System（タカラバイオ社）」を用いてリアルタイムＰＣＲを行った。内部標準遺伝子としてβ−Ａｃｔｉｎ遺伝子の発現量を同様に測定し、Ｇｓｔｐ１遺伝子およびＧｓｔａ４遺伝子の発現量は、β−Ａｃｔｉｎ遺伝子の発現量に対する比率（相対発現量）として算出した。相対発現量は、各群ごとに平均値および標準偏差を算出して有意差検定を行い、棒グラフに表した。その結果を図３に示す。
(3) Quantitative analysis of gene expression level by real-time PCR method Total RNA was extracted from the mouse liver of Example 1 (1) using the Nucleo Spin RNA Kit (Takara Bio Inc.). From this total RNA, reverse transcription reaction was performed using ReverTra Ace qPCRRT Master Mix (Toyobo Co., Ltd.) to prepare cDNA. Using this cDNA as a template, real-time PCR is performed using the following gene-specific primers, real-time PCR reagent "THUNDERBIED SYBR qPCR Mix (Toyo Boseki)" and real-time PCR device "Thermal Cycler Dice Real Time System (Takara Bio)". It was. The expression level of the β-Actin gene as an internal standard gene was measured in the same manner, and the expression levels of the Gstp1 gene and the Gsta4 gene were calculated as a ratio (relative expression level) to the expression level of the β-Actin gene. The relative expression level was shown in a bar graph by calculating the mean value and standard deviation for each group and performing a significant difference test. The result is shown in FIG.
なお、遺伝子特異的プライマーの配列を以下に示す。
《Ｇｓｔｐ１遺伝子》
フォワードプライマー；CGGCAAATATGTCACCCTCATCTA（配列番号１）
リバースプライマー；TCTGGGACAGCAGGGTCTCA（配列番号２）
《Ｇｓｔａ４遺伝子》
フォワードプライマー；TGACACAGACCAGGGCCATC（配列番号３）
リバースプライマー；ATCAGGTCCTGGGTGCCATC（配列番号４）
《β−Ａｃｔｉｎ遺伝子》
フォワードプライマー；CATCCGTAAAGACCTCTATGCCAAC（配列番号５）
リバースプライマー；ATGGAGCCACCGATCCACA（配列番号６）
The sequence of the gene-specific primer is shown below.
<< Gstp1 gene >>
Forward primer; CGGCAAAATATGTCACCCTCATCTA (SEQ ID NO: 1)
Reverse primer; TCTGGGACAGCAGGGTCTCA (SEQ ID NO: 2)
<< Gsta4 gene >>
Forward primer; TGACACAGACCAGGGCCATC (SEQ ID NO: 3)
Reverse primer; ATCAGGTCCTGGGTGCCATC (SEQ ID NO: 4)
<< β-Actin gene >>
Forward primer; CATCCGTAAAGACCTCTATGCCAAC (SEQ ID NO: 5)
Reverse primer; ATGGAGCCACCGATCCACA (SEQ ID NO: 6)
図３に示すように、Ｇｓｔｐ１遺伝子の相対発現量は、対照群では約３であったのに対して、ケストース食群では約７．５であり、両群間には有意差があった。また、Ｇｓｔａ４遺伝子の相対発現量は、対照群では約１であったのに対して、ケストース食群では約４．３であり、両群間には有意差があった。すなわち、ケストース食群では、対照群と比較して、Ｇｓｔｐ１遺伝子の発現量が約２．５倍、Ｇｓｔａ４遺伝子の発現量が約４倍、大きいことが明らかになった。
この結果から、１−ケストースの摂食により、ＧＳＴの発現が促進されることが明らかになった。
As shown in FIG. 3, the relative expression level of the Gstp1 gene was about 3 in the control group, whereas it was about 7.5 in the Kestose diet group, and there was a significant difference between the two groups. The relative expression level of the Gsta4 gene was about 1 in the control group, whereas it was about 4.3 in the Kestose diet group, and there was a significant difference between the two groups. That is, it was clarified that the expression level of the Gstp1 gene was about 2.5 times higher and the expression level of the Gsta4 gene was about 4 times higher in the Kestose diet group than in the control group.
From this result, it was clarified that the expression of GST was promoted by feeding 1-kestose.
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