JP2018100235A - Agent and food composition for promoting expression of glutathione-s-transferase - Google Patents
Agent and food composition for promoting expression of glutathione-s-transferase Download PDFInfo
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- JP2018100235A JP2018100235A JP2016246699A JP2016246699A JP2018100235A JP 2018100235 A JP2018100235 A JP 2018100235A JP 2016246699 A JP2016246699 A JP 2016246699A JP 2016246699 A JP2016246699 A JP 2016246699A JP 2018100235 A JP2018100235 A JP 2018100235A
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Abstract
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 promoter for glutathione-S-transferase and a food composition for promoting expression containing 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 spleen, kidney, brain, skeletal muscle, testis, small intestine, placenta, and red blood cells. 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 glutathione-conjugated ligand 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 for the living body, 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, active oxygen has recently been reported to be involved in the onset or aggravation of diseases such as cancer, myocardial infarction, cerebrovascular disorder, and Alzheimer's disease because active oxygen damages intracellular DNA by its strong oxidizing power. Therefore, 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 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 GST expression can be promoted to promote excretion of a carcinogen or the like in vitro, cancer prevention can be achieved. It is expected to contribute to the maintenance and promotion of 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。 Accordingly, substances that promote the expression of GST have been researched and developed. For example, Patent Document 1 discloses a GST activator containing ginkgo biloba extract as an active ingredient, and Patent Document 2 discloses GST generation action in vivo. A selenium-containing composition having a GST expression promoter is disclosed in Patent Document 3 as an active ingredient of the ginseng extract.
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, since the ginkgo biloba extract described in Patent Document 1, the selenium-containing composition described in Patent Document 2, and the arginaceae ginseng extract described in Patent Document 3 are considered to have unique flavors, various It cannot be said that it can be easily blended into simple foods and medicines and can be easily ingested on a daily basis. That is, even in view of the above-mentioned patent documents, the expression of GST can be effectively promoted, and it is highly safe and can be easily blended into various foods and pharmaceuticals, or can be easily taken on a daily basis. Substances that can be used have not been sufficiently provided, and 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 problems, can effectively promote the expression of GST, has high safety, and is easily incorporated into various foods and pharmaceuticals. An object of the present invention is to provide an expression promoter for GST and a food composition for promoting expression that 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 intensive studies, the present inventors have found that GST expression can be effectively promoted by eating 1-kestose. Accordingly, the following inventions have been completed based on this finding.
（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 promoter 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 expression promoter for glutathione-S-transferase 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 expression promoter for glutathione-S-transferase according to the present invention can be used as a discharge promoter for 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 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 expression of glutathione-S-transferase according to the present invention can be used for antioxidant purposes.
（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 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 as an active ingredient of the GST expression promoter and 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 with a long history of eating. In addition, 1-kestose has not been toxic in any of mutagenicity tests, acute toxicity tests, subchronic toxicity tests, and chronic toxicity tests. From these facts, it can be said that the safety of 1-kestose is extremely high (Food and Development, Vol. 49, No. 12, page 9, 2014).
In addition, 1-kestose is highly water-soluble and has a good sweetness similar to sugar. Therefore, 1-kestose can be easily ingested as it is or as a sweetener on a daily basis. Can be easily blended.
Therefore, according to the present invention, the expression of GST can be effectively promoted, and the safety is high, so that it can be easily blended into various foods and pharmaceuticals, or can be easily ingested on a daily basis. A GST expression promoter and a food composition for promoting expression can be obtained.
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 expression promoter for GST and the food composition for promoting expression according to the present invention will be described in detail.
The GST expression promoter and food composition for promoting expression 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 composed of one molecule of glucose and two molecules of fructose. 1-kestose can be obtained by performing an enzyme 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 allowed to stand at 37 ° C. to 50 ° C. for about 20 hours to perform an enzyme 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, tetrasaccharides or more) Oligosaccharide) is separated and purified to obtain a high purity 1-kestose solution. Subsequently, 1-kestose can be obtained as crystals by concentrating the high-purity 1-kestose solution and then crystallizing it by a crystallization method as disclosed in Japanese Patent Publication No. 6-70075.
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。 Moreover, since 1-kestose is contained in commercially available fructooligosaccharides, it may be used as it is or after separating and purifying 1-kestose from fructooligosaccharides by the method described above. That is, a 1-kestose-containing composition such as an oligosaccharide containing 1-kestose may be used as the 1-kestose of the present invention. When the 1-kestose-containing composition is used, the purity of 1-kestose is preferably 80% or more, more preferably 85% or more, and further preferably 90% or more. In the present invention, the “purity” of 1-kestose refers to 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 pharmaceuticals, food additives, supplements and the like by ingestion by humans or animals. In addition, 1-kestose may be added to enteral nutrients, and this may be used by a method of administering by enteral nutrition 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 pharmaceutical or a supplement, the dosage form is, for example, powder, tablet, sugar coating, capsule, granule, dry syrup, liquid, syrup, drop, drink, etc. Or a 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。 The pharmaceuticals and supplements of the above dosage forms can be produced by methods known to those skilled in the art. For example, in the case of a powder, 800 g of 1-kestose and 200 g of lactose are mixed well, and then 300 mL of 90% ethanol is added and moistened. Subsequently, after the wet powder is granulated, it is dried by ventilation at 60 ° C. for 16 hours, and then sized to obtain 1000 g of powder with an appropriate fineness (1-kestose content of 800 mg / 1 g). In the case of a tablet, 300 g of 1-kestose, 380 g of powdered reduced starch syrup, 180 g of rice starch and 100 g of dextrin are mixed well, and then 300 mL of 90% ethanol is added and moistened. Subsequently, the wet powder is extruded and granulated, and then air-dried at 60 ° C. for 16 hours to obtain granules. Thereafter, this granule is sized using a sieve of 850 μm, and then 50 g of sucrose fatty acid ester is added to and mixed with 470 g of the granule, followed by punching using a rotary tableting machine (6B-2, manufactured by Kikusui Seisakusho). It is possible to obtain 5000 tablets (1-kestose content 60 mg / 1 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。 Moreover, the food composition for promoting expression 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 also be added and used in the normal manufacturing process of various foods and drinks. Since the sweetness of 1-kestose is 30 and its taste, physical properties and processability are close to sucrose, in the production process of various foods and drinks, part or all of the sugar is replaced with 1-kestose, Various foods and drinks can be produced in the same manner 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 animal feed.
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。 Examples of the intake amount (dosage) of the active ingredient when the expression promoter or expression promoting food composition of the present invention is used for humans or animals include 0.04 g / kg body weight or more per day. Can do. 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 having two domains, homozygous or heterozygous having a very similar structure. Is a soluble protein present in GST is classified into many classes such as α, μ, π, σ, θ, δ, ε, Ω, ζ, λ, and T 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 notation of GST is, for example, “GST A 1-1”, and the class is expressed in Greek letters or Roman letters such that the subunit 1 first found in the α (A) class is composed of homo. In capital letters, the constituent subunits (in the order in which they are found) are written in Arabic numerals. Before GST, species (h for human, r for rat, m for mouse, etc.) may be written in lower case letters (Toru Miyamoto, “Structure and function of glutathione S-transferase”, Tokyo, Japan). Agricultural University Journal of Agriculture, 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 be composed of any subunit, and may be derived from any organism such as animals, plants, fungi, and bacteria, but preferably includes GST derived from animals. Can do.
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 GST protein, or GST protein in any cell or tissue / organ of the living body. 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 GST expression is promoted can be confirmed by methods 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.) conventionally known to contain GST is collected as a sample, and the expression promoter or food composition for promoting expression of the present invention is ingested. If not, the content of messenger RNA of GST in the sample is compared. If the former has a larger messenger RNA content, it can be determined that the expression promoter or the food composition for promoting expression 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 GST expression is promoted, for example, the amount of GST protein in the above sample is confirmed by Western blotting as described in paragraph [0024] of Patent Document 2. The method of doing can be mentioned.
That is, after separating the protein in the sample according to the peptide chain length by electrophoresis using sodium dodecyl sulfate and polyacrylamide gel (SDS-PAGE), the membrane is brought into close contact with the gel, and the separated protein is subjected to voltage application. Transfer from gel to membrane (eg nitrocellulose membrane). A GST-specific antibody (primary antibody) is bound to this membrane, washed, and then reacted with a secondary antibody labeled with an enzyme such as alkaline farifatase. Then, it detects by the chemiluminescent method or coloring method using the activity of a labeling enzyme. Since the detection intensity (luminescence intensity or color intensity) is proportional to the amount of GST protein, if the former sample has a higher detection intensity, the expression promoter or the food composition for promoting expression of the present invention can express GST. Can be determined 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。
In addition, as another method for confirming whether or not the expression of GST is promoted, for example, as described in paragraphs [0022] to [0023] of Patent Document 2, the activity value of GST protein can be determined in the above sample. The method of confirming can be mentioned.
That is, the sample is disrupted in 0.2 M sucrose / 10 mM phosphate buffer to obtain a supernatant. A reaction solution containing this supernatant and 100 mM phosphate buffer (pH 6.5), 1 mM 1-chrolo-2,4-dinitrobenzene (CDNB) and 1 mM reduced glutathione (GSH) was prepared. Absorbance at 340 nm at 0 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 expression of GST was promoted by the expression promoter or food composition for promoting expression of the present invention. 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 described 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 University of Agriculture) Shukan, 2013, 57, 4, p.247-260; Akira Hiratsuka, “Recent Advances in Liver Drug Metabolism 5. Glutathione S-transferase”, Liver, 42, 6, p. 302-308). That is, since the oxidative stress in the living body can be reduced by promoting the expression of GST, the expression promoter for GST and the food composition for promoting expression can be used for the purpose of preventing or reducing the oxidation of the living body. Therefore, the expression promoter or food composition for promoting expression of the present invention can be used as an antioxidant or a food composition for antioxidant.
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 described above, GST urges substances that are unnecessary for the living body such as toxins, drugs, and metabolites to be conjugated with glutathione, and finally excreted from the body through further metabolism in the kidney (Toru Miyamoto). "Structure and function of glutathione S-transferase", Tokyo University of Agriculture, Journal of Agriculture, Vol. 57, No. 4, p.247-260; Akira Hiratsuka, "Recent Advances in Hepatic Drug Metabolism 5. Glutathione" S-transferase ", Liver, Vol. 42, No. 6, p. 302-308). That is, by promoting the expression of GST, detoxification or discharge of substances unnecessary for the living body can be promoted. Therefore, the expression promoter for GST and the food composition for promoting expression are used for the purpose of promoting the discharge of the unnecessary substances. be able to. Therefore, the expression promoter and food composition for promoting expression of the present invention can be used as an unnecessary substance discharge promoter or 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 expression promoting agent and the food composition for promoting expression of GST 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 by 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”. Further, in this example, a significant difference between the average values of each group was tested by T test, and a risk rate of less than 5% was considered 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 upon ingestion of 1-kestose (1) Breeding of mice ingested 1-kestose orally In the formulation of a mouse standard purified feed “AIN-93G” (CLEA Japan), corn starch By substituting a portion of this with 1-kestose (purity 99%; product food science company), a feed containing 1-kestose at 5% by mass was commissioned to Claire Japan, A kestose-containing feed was used. The formulation of AIN-93G is shown below.
<< Composition of AIN-93G (unit: mass%) >> corn starch 39.7486%, milk casein 20%, pregelatinized corn starch 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 Science Materials Co., Ltd.) were divided into two groups of 10 mice, one being the kestose diet group and the other being the control group. AIN-93G was fed to the control group, and the kestose diet group was reared for 10 weeks while freely taking the kestose-containing feed. 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, body weight was measured every week, and a 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 breeding period, each group of mice was dissected to remove the liver, the weight was measured, and an average value and a standard deviation were obtained for each group, and a significant difference test was performed. The result is shown in FIG.
As shown in FIGS. 1 and 2, there was no significant difference in body weight and liver weight 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 liver of Example 1 (1), individuals with average food intake and weight gain were selected for each group for 5 individuals. Total RNA was extracted from these 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 reverse transcriptase ArrayScript and T7 Oligo dT Primer. Next, double-stranded cDNA was synthesized using dNTP mix, DNA ligase, DNA Polymerase and Rnase H (Ambion). The resulting double-stranded cDNA is purified using the Min Elute PCR Purification Kit (QIAGEN), biotin-labeled using the Massage Amp-Biotin Kit (Ambion), and purified using the Rneasy MinElute Cleanup Kit (QIAGEN). Biotin-labeled cDNA was obtained. Subsequently, a 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. 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。 The fragmented biotin-labeled cDNA was hybridized to an oxidative stress anti-aging chip “Genopal (registered trademark) ROSM-JX (mouse version; equipped with 219 gene)” (Mitsubishi Rayon Co., Ltd.). This reaction was carried out in a hybridization buffer at 65 ° C. for 16 hours. Then, the fluorescence signal was detected using Biochip Reader MB-M3C (Yokogawa Electric Corp.). Genes having a fluorescence signal measurement value of 100 or more were extracted as positive, and the ratio of “measurement value of kestose diet group” to “measurement value of control group” (expression ratio: measurement value of kestose diet group / The measured value of the control group) was determined. Genes with an expression ratio of 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, 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 were used as candidate genes. Two genes (Glutathione S-transferase alpha 4; Gsta4) (expression ratio: 1.50 times) were extracted. Therefore, the expression level of these genes was quantitatively analyzed by real-time PCR.
（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 (Toyobo)” 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. For the relative expression level, a mean value and a standard deviation were calculated for each group, a significant difference test was performed, and the result was expressed in a bar graph. 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; CGGCAAATATGTCACCCTCATCTA (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 and 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, but about 4.3 in the kestose diet group, and there was a significant difference between the two groups. That is, it was revealed that the expression level of the Gstp1 gene was about 2.5 times and the expression level of the Gsta4 gene was about 4 times larger in the kestose diet group than in the control group.
From this result, it became clear that the expression of GST is promoted by feeding 1-kestose.
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