JP6637627B1 - Composition for inhibiting GABA-degrading enzyme - Google Patents
Composition for inhibiting GABA-degrading enzyme Download PDFInfo
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- JP6637627B1 JP6637627B1 JP2019043675A JP2019043675A JP6637627B1 JP 6637627 B1 JP6637627 B1 JP 6637627B1 JP 2019043675 A JP2019043675 A JP 2019043675A JP 2019043675 A JP2019043675 A JP 2019043675A JP 6637627 B1 JP6637627 B1 JP 6637627B1
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
【課題】ＧＡＢＡを豊富に含むネギ属植物を原料とした、生体内におけるＧＡＢＡの有効利用率を向上させることのできる組成物や食品の提供。【解決手段】ＧＡＢＡ（γ−ａｍｉｎｏｂｕｔｙｒｉｃ ａｃｉｄ）分解酵素阻害用ノルメコニン（Ｎｏｒｍｅｃｏｎｉｎ ６，７−Ｄｉｈｙｄｏｒｏｘｙ−４−ｍｅｔｈｙｌ−１（３Ｈ）−ｉｓｏｂｅｎｚｏｆｕｒａｎｏｎｅ）を有する組成物。原料となるネギ属植物は、＜Ａ＞、＜Ｂ＞に列挙されたもののいずれかであるＧＡＢＡ分解酵素阻害用ノルメコニン。＜Ａ＞タマネギ、エシャロット、ニンニク、ラッキョウ、ツリーオニオン、アサツキ、ネギ（ナガネギ、青ネギ）、ワケギ、チャイブ、ニラ、ヤグラネギ、白ネギ、リーキ、ノビル、ギョウジャニンニク。＜Ｂ＞＜Ａ＞に列挙した植物の加工物。【選択図】図１The present invention provides a composition or food that can improve the effective utilization rate of GABA in a living body, using a raw material of an Allium genus plant that is rich in GABA. A composition having normeconin 6,7-dihydroxy-4-methyl-1 (3H) -isobenzofuranone for inhibiting GABA (γ-aminobutyric acid) degrading enzyme. Normeconin for inhibiting GABA-degrading enzyme, wherein the allium plant as a raw material is any one of those listed in <A> and <B>. <A> Onion, shallot, garlic, rakkyo, tree onion, asatsuki, leek (leek onion, green onion), scallion, chives, leek, yagura onion, white leek, leek, nobile, ginger garlic. <B> Processed products of the plants listed in <A>. [Selection diagram] Fig. 1
Description
本発明は、ニンニクなどのネギ属植物を原料とするＧＡＢＡ分解酵素阻害用組成物に関するものである。
TECHNICAL FIELD The present invention relates to a composition for inhibiting a GABA-degrading enzyme, which is derived from a plant of the genus Allium such as garlic.
ニンニク（Ａｌｌｉｕｍ ｓａｔｉｖｕｍ）は、古来より滋養強壮に用いられる食品としても親しまれているだけではなく、殺菌作用１）、抗炎症作用２）、抗腫瘍作用３）、抗糖尿病作用４）、抗酸化活性５）など多くの生理活性も有していることが報告されている。ニンニクを湿度６０〜９０％、温度４０〜９０℃の環境下で段階的に熟成させる発酵処理や、メイラード反応（アミノカルボキシル反応）処理して製造されるのが黒ニンニク（琥珀ニンニク等と言われることもあるが、以下、単に「黒ニンニク」と言う）である。なかでも、青森県産黒ニンニクは、品質、機能性ともに注目されている機能性食品のうちのひとつである。
〔参照文献〕
1) Idris AR, Afegbua SL., Trans. R．Soc．Trop．Med．Hyg., 111, 478 (2017)．
2) Baluchnejadmojarad T, Kiasalari Z, Afshin-Majd S, Ghasemi Z, Roghani M., Ｅｕｒ．J．Pharmacol., 5, 69-76 (2017)．
3) Block E, Bechand B, Gundala S, Vattekkatte A, Wang K, Mousa SS, Godugu K, Yalcin M, Mousa SA., Molecules., 22(12). pii: E2081. doi: 10.3390/molecules22122081.
4) Oboh G, Ademiluyi AO, Agunloye OM, Ademosun AO, Ogunsakin BG., J Diet Suppl. 16, 105-118(2019).
5) Zhang H, Wang P, Xue Y, Liu L, Li Z, Liu Y., Tissue Cell. 50, 89-95(2018).
Garlic (Allium sativum) is not only popular since ancient times as a nutritious and tonic food, but also has a bactericidal action 1) , anti-inflammatory action 2) , anti-tumor action 3) , anti-diabetic action 4) , antioxidant It has been reported that it also has many physiological activities such as activity 5) . Black garlic (amber garlic, etc.) is produced by fermentation in which garlic is matured stepwise in an environment at a humidity of 60 to 90% and a temperature of 40 to 90 ° C, or a Maillard reaction (aminocarboxyl reaction). In some cases, it is simply referred to as "black garlic". Above all, black garlic from Aomori Prefecture is one of the functional foods that has attracted attention in both quality and functionality.
(References)
1) Idris AR, Afegbua SL., Trans. Soc. Trop. Med. Hyg., 111, 478 (2017).
2) Baluchnejadmojarad T, Kiasalari Z, Afshin-Majd S, Ghasemi Z, Roghani M., Eur. J. Pharmacol., 5, 69-76 (2017).
3) Block E, Bechand B, Gundala S, Vattekkatte A, Wang K, Mousa SS, Godugu K, Yalcin M, Mousa SA., Molecules., 22 (12). Pii: E2081. Doi: 10.3390 / molecules22122081.
4) Oboh G, Ademiluyi AO, Agunloye OM, Ademosun AO, Ogunsakin BG., J Diet Suppl. 16, 105-118 (2019).
5) Zhang H, Wang P, Xue Y, Liu L, Li Z, Liu Y., Tissue Cell. 50, 89-95 (2018).
黒ニンニクについては従来、さまざまな技術的提案もなされている。たとえば後掲特許文献１には、ニンニク原料の湿度管理等を要しない簡便な手段で高生理活性物質（Ｓ−アリルシステイン、シクロアリイン等）が保持された黒ニンニクエキスを製造する技術として、ニンニク原料（乾燥ニンニク、生ニンニク等）を水抽出して得られる抽出物を、９０℃以上の温度で加熱熟成させる方法が開示されている。また特許文献２には、生ニンニクが本来有していないミネラル成分を含有した黒ニンニクを、食味を損うことなく製造する技術として、生ニンニクを６０℃の温泉水に６時間以内、より好ましくは略３時間浸漬させて温泉水処理生ニンニクとし、これを発酵させて黒ニンニクを得る方法が開示されている。 Conventionally, various technical proposals have been made on black garlic. For example, Patent Literature 1 listed below discloses a technique for producing a black garlic extract in which a highly bioactive substance (S-allyl cysteine, cycloallyin, etc.) is retained by a simple means that does not require humidity control of garlic raw materials. A method is disclosed in which an extract obtained by extracting raw materials (such as dried garlic and raw garlic) with water is heated and aged at a temperature of 90 ° C. or higher. Patent Literature 2 discloses a technique for producing black garlic containing a mineral component not originally possessed by raw garlic without impairing the taste. A method is disclosed in which garlic is immersed in hot spring water for about 3 hours to obtain raw garlic, which is fermented to obtain black garlic.
さて、青森県産のものを初めとして黒ニンニクでは、熟成過程においてＳ−ａｌｌｙｌ ｃｙｓｔｅｉｎｅ、 Ｃｙｃｌｏ−ａｌｌｉｉｎ、Ｐｙｒｏｇｌｕｔａｍｉｃ ａｃｉｄなどの多くの機能性成分が増加することが報告されており、とりわけＧＡＢＡ（γ−ａｍｉｎｏｂｕｔｙｒｉｃ ａｃｉｄ）においては８倍程度もの増加が認められている。既に、ＧＡＢＡを効率良く摂取することは血圧低下６）、中性脂肪の抑制７）、精神の安定８）に寄与することが知られているが、特に近年は、腸管ＧＡＢＡ受容体への刺激が中枢神経系を介して脳に作用する腸脳相関が注目されている。
〔参照文献〕
6）Vemulapalli S, Barletta M., Arch. Int. Pharmacodyn. Ther., 267, 46-58(1984).
7）Ho XL, Tsen SY, Ng MY, Lee WN, Low A, Loke WM., J. Med. Food., Published Online:1 Oct 2016https://doi.org/10.1089/jmf.2016.3693（2016）.
8）堀江健二, 東口伸二, 横越英彦，Food Style 21,7, 64-68(2003).
By the way, in black garlic including those from Aomori Prefecture, it is reported that many functional components such as S-allyl cysteine, Cyclo-alliin, Pyroglutamic acid, etc. increase in the ripening process, and in particular, GABA (γ- In aminobutyric acid), an increase of about 8 times has been observed. It is already known that the efficient intake of GABA contributes to blood pressure lowering 6) , suppression of triglycerides 7) , and mental stability 8) . In particular, in recent years, stimulation of intestinal GABA receptors has been reported. Intestinal-brain correlation, which acts on the brain via the central nervous system, has attracted attention.
(References)
6) Vemulapalli S, Barletta M., Arch. Int. Pharmacodyn. Ther., 267, 46-58 (1984).
7) Ho XL, Tsen SY, Ng MY, Lee WN, Low A, Loke WM., J. Med. Food., Published Online: 1 Oct 2016 https://doi.org/10.1089/jmf.2016.3693 (2016).
8) Kenji Horie, Shinji Higashiguchi, Hidehiko Yokogoshi, Food Style 21, 7, 64-68 (2003).
しかし、ＧＡＢＡは生体内では極めて分解されやすく、経口的に摂取したＧＡＢＡのほとんどはＧＡＢＡ−Ｔ（ＧＡＢＡ ｔｒａｎｓａｍｉｎａｓｅ）やＳＳＡＤＨ（ｓｕｃｃｉｎｉｃ ｓｅｍｉａｌｄｅｈｙｄｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ）などのＧＡＢＡ代謝系の酵素群により分解されてしまう。だが、翻せばこのことは、これらの酵素群に対して阻害作用を有する化合物さえあれば、それによって生体内におけるＧＡＢＡの有効利用率を向上させられることを示すものである。これは、多くの疾患予防の観点から注目すべきことである。 However, GABA is extremely easily decomposed in vivo, and most of GABA taken orally is decomposed by a group of GABA metabolic enzymes such as GABA-T (GABA transamine) and SSADH (succinic semidehydrodehydrogenase). However, in turn, this indicates that any compound having an inhibitory effect on these enzyme groups can improve the effective utilization rate of GABA in a living body. This is remarkable from the viewpoint of prevention of many diseases.
したがって、ＧＡＢＡを豊富に含むとされる機能性食品である、青森県産のものを初めとする黒ニンニクの、ＧＡＢＡ代謝酵素系の酵素であるＧＡＢＡ−Ｔ、およびＳＳＡＤＨの混合酵素ＧＡＢａｓｅに対する阻害作用についての検討は大いに有意義であるが、この問題をターゲットとした黒ニンニクの研究は未だなされていない。もし、かかる研究において一定の成果が得られた場合には、それは黒ニンニクのさらなる普及拡大に資するのみならず、広くニンニク以外のネギ属植物にも敷衍できる可能性がある。 Therefore, the inhibitory effect of black garlic, such as that produced from Aomori Prefecture, which is a functional food which is considered to be rich in GABA, on GABA-T, which is a GABA-metabolizing enzyme, and a mixed enzyme GABase of SSADH, Although research on garlic is very significant, no research has been done on black garlic targeting this problem. If a certain result is obtained in such research, it may contribute not only to the further spread of black garlic, but also to other allium plants other than garlic.
そこで本発明が解決しようとする課題は、このような従来技術の状況を踏まえ、ＧＡＢＡを豊富に含むネギ属植物をターゲットとし、生体内におけるＧＡＢＡの有効利用率向上を可能ならしめる成分の存否を研究によって明らかにし、それに基づいて、生体内におけるＧＡＢＡの有効利用率を向上させることのできる組成物や食品等を提供することである。 Therefore, the problem to be solved by the present invention is based on the state of the prior art described above, and it is intended to target allium genus plants containing GABA abundantly and to determine the presence or absence of a component capable of improving the effective utilization rate of GABA in vivo. An object of the present invention is to provide a composition, a food, or the like that can be clarified by research and that can improve the effective utilization rate of GABA in a living body based on the result.
本願発明者は上記課題について検討を行った。その結果、ＧＡＢＡを豊富に含むとされる黒ニンニクには、ＧＡＢＡ代謝酵素系の酵素であるＧＡＢＡ−ＴおよびＳＳＡＤＨの混合酵素ＧＡＢａｓｅに対する阻害作用があることを見出し、さらに、その活性成分をノルメコニン（Ｎｏｒｍｅｃｏｎｉｎ ６，７−Ｄｉｈｙｄｒｏｘｙ−１（３Ｈ）−ｉｓｏｂｅｎｚｏｆｕｒａｎｏｎｅ）と同定することができた。そして、これらを基に本願発明は完成された。すなわち、上記課題を解決するための手段として本願で特許請求される発明、もしくは少なくとも開示される発明は、以下の通りである。
The inventor of the present application has studied the above problem. As a result, it was found that black garlic, which is considered to be rich in GABA, had an inhibitory effect on GABase, a mixed enzyme of GABA-T and SSADH, which is a GABA-metabolizing enzyme, and furthermore, its active ingredient was normeconin ( Normeconin 6,7-Dihydroxy-1 (3H) -isobenzofuranone ). And based on these, the present invention was completed. That is, the invention claimed in the present application as a means for solving the above-mentioned problem, or at least the disclosed invention is as follows.
〔１〕 ノルメコニン（Ｎｏｒｍｅｃｏｎｉｎ ６，７−Ｄｉｈｙｄｒｏｘｙ−１（３Ｈ）−ｉｓｏｂｅｎｚｏｆｕｒａｎｏｎｅ）を有効成分とすることを特徴とする、ＧＡＢＡ（γ−ａｍｉｎｏｂｕｔｙｒｉｃ ａｃｉｄ）分解酵素阻害用組成物（「組成物」には「固体状または液体状のエキス（抽出物）」も含まれる。以下同じ）。 [1] A composition for inhibiting GABA (γ-aminobutyric acid) -degrading enzyme (“composition”), comprising normeconin (Normeconin 6,7-Dihydroxy-1 (3H) -isobenzofuranone) as an active ingredient. "Solid or liquid extract (extract)" is also included.
〔２〕 「ＧＡＢＡ分解酵素を阻害する」旨の表示が付されている、下記＜Ｆ＞列記のいずれかであることを特徴とする、〔１〕に記載のＧＡＢＡ分解酵素阻害用組成物。 [2] The composition for inhibiting a GABA-degrading enzyme according to [1], wherein the composition is any one of the following <F>, which is labeled "inhibits GABA-degrading enzyme".
＜Ｆ＞ 固体状のエキス、液体状のエキス、食品<F> Solid extract, liquid extract, food
〔３〕 黒ニンニク由来であることを特徴とする、〔１〕、〔２〕のいずれかに記載のＧＡＢＡ分解酵素阻害用組成物。[3] The composition for inhibiting a GABA-degrading enzyme according to any one of [1] and [2], which is derived from black garlic.
〔４〕 前記黒ニンニクは発酵処理、加熱処理、またはメイラード反応処理の少なくともいずれかの処理済みの加工物であることを特徴とする、〔３〕に記載のＧＡＢＡ分解酵素阻害用組成物。[4] The composition for inhibiting a GABA-degrading enzyme according to [3], wherein the black garlic is a processed product that has been subjected to at least one of a fermentation treatment, a heat treatment, and a Maillard reaction treatment.
〔５〕 「黒」、「琥珀」、「発酵」、「熟成」、またはその他の発酵処理・加熱処理・メイラード反応処理の少なくともいずれかの処理済み黒ニンニク由来である旨を示す表示が付されている、下記＜Ｆ＞列記のいずれかであることを特徴とする、〔４〕に記載のＧＡＢＡ分解酵素阻害用組成物。 [5] A label indicating that the product is derived from black garlic that has been subjected to at least one of “black”, “amber”, “fermentation”, “aging”, or other fermentation treatment, heating treatment, or Maillard reaction treatment The composition for inhibiting a GABA-degrading enzyme according to [4], which is any one of the following <F>.
＜Ｆ＞ 固体状のエキス、液体状のエキス、食品<F> Solid extract, liquid extract, food
本発明のＧＡＢＡ分解酵素阻害用組成物は上述のように構成されるため、これらによれば、ＧＡＢＡを豊富に含むネギ属植物を原料として、生体内におけるＧＡＢＡの有効利用率向上を可能ならしめる成分であるところのノルメコニンを得ることができ、またそれを含む組成物や食品を得ることができ、生体内におけるＧＡＢＡの有効利用率を向上させることができる。すなわち、ＧＡＢＡを豊富に含有し、なおかつＧＡＢａｓｅ阻害作用を示すノルメコニンも有する黒ニンニクを摂取することは、ＧＡＢＡを効率的に摂取できることに繋がり、多くの疾患の予防策への寄与が期待できる。また、本発明は、黒ニンニク以外のネギ属植物またはその加工物における応用も期待できる。
Since the composition for inhibiting a GABA-degrading enzyme of the present invention is constituted as described above, according to these, it is possible to improve the effective utilization rate of GABA in a living body by using an Allium plant rich in GABA as a raw material. Normeconin as a component can be obtained, and a composition or food containing the same can be obtained, and the effective utilization rate of GABA in a living body can be improved. In other words, ingesting black garlic that is rich in GABA and also has normeconin that exhibits GABase inhibitory action leads to efficient intake of GABA, and is expected to contribute to preventive measures for many diseases. The present invention is also expected to be applied to allium plants other than black garlic or processed products thereof.
また、ノルメコニンがかかるＧＡＢＡ分解酵素阻害作用を有することは、本発明において初めて明らかにされた。上述の通り、ＧＡＢＡおよびその分解酵素阻害作用のあるノルメコニンをいずれも含有することにより、黒ニンニク自体や黒ニンニクからの抽出物・組成物、あるいはそれらを用いた加工物は、従来にはない有効なＧＡＢＡ摂取手段（方法）となる。しかし他方、由来の如何に関わらず、あるいはまた化学合成されたものであれ、ノルメコニン自体をＧＡＢＡ分解酵素阻害用として用いることもできる。 The present invention has also revealed for the first time that normeconin has such a GABA-degrading enzyme inhibitory action. As described above, by containing both GABA and normeconin, which has a degrading enzyme inhibitory effect, black garlic itself, extracts and compositions from black garlic, or processed products using them have an unprecedented effectiveness. GABA intake means (method). On the other hand, however, normeconin itself, regardless of its origin or chemically synthesized, can also be used for GABAase inhibition.
なお、出願人は本願に先立って、ニンニクなどのネギ属植物を原料とする組成物等において、従来未知の機能・作用を研究によって明らかにし、それに基づいて、高機能の組成物、およびその製造方法を提供するという課題に取り組んだ。その結果、ＡＧＥｓ（ａｄｖａｎｃｅｄ ｇｌｙｃａｔｉｏｎ ｅｎｄ−ｐｒｏｄｕｃｔｓ、終末糖化産物）の生成を阻害する作用、ＡＧＥｓの中間体の生成を阻害する作用、またはＡＧＥｓのＲＡＧＥ（ｒｅｃｅｐｔｏｒ ｆｏｒ ＡＧＥｓ、ＡＧＥｓ受容体）への接着を阻害する作用、といったＡＧＥｓ等阻害性のうち、少なくともいずれかを有するＡＧＥｓ等阻害性ネギ属植物製組成物を発明するに至り、これを開示した（特願２０１８−１５２１４７「ＡＧＥｓ等阻害性ネギ属植物製組成物、その製造方法、飲食品、およびＡＧＥｓ阻害用Ｎｏｒｍｅｃｏｎｉｎ」 本願出願時に未公開）。 Prior to the present application, the applicant clarified previously unknown functions and effects in compositions such as garlic and other allium genus plants by research, and based on them, based on them, highly functional compositions and their production Addressed the challenge of providing a method. As a result, the action of inhibiting the production of AGEs (advanced glycation end-products, advanced glycation end products), the action of inhibiting the production of intermediates of AGEs, or the adhesion of AGEs to RAGE (receptor for AGEs, AGEs receptor). The present invention has led to the invention of a composition made of an allium-inhibiting plant of the genus Allium having at least one of the inhibitory effects of AGEs, such as AGEs, and disclosed the same (Japanese Patent Application No. 2018-152147, "Inhibitory Allium of AGEs etc." Botanical Composition, Production Method, Food and Beverage, and Normeconin for Inhibiting AGEs ”Unpublished at the time of filing the present application).
このＡＧＥｓ等阻害性ネギ属植物製組成物等発明では、ＡＧＥｓの中間体の生成を阻害する作用は、３−ＤＧ（３−デオキシグルコソン）産生阻害作用とすることができ、また、組成物はエキスまたは飲食品の形態をとることができる。この発明によれば、黒ニンニクがカルボニルストレスを抑制できること、それによりＡＧＥｓ関連疾患の予防・治療に効果が期待でき、したがってＡＧＥｓ関連疾患の効果的な予防・治療を実現できるとともに、黒ニンニクの付加価値向上、普及拡大に資することができる。またこれらの効果は、広くニンニクなどのネギ属植物を原料とする組成物等に敷衍できると考えられ、ネギ属植物の付加価値向上、普及拡大に資することも期待できる。そしてこの発明においては特に、黒ニンニク含有成分の一つがノルメコニンであることを明らかにし、そのＡＧＥ反応性ＡＧＥｓ阻害活性を初めて確認でき、黒ニンニク等の付加価値等拡大のみならず、ＡＧＥｓ阻害というノルメコニンの新たな用途をした。 In the invention such as a composition made of an inhibitory Allium plant such as AGEs, the action of inhibiting the production of an intermediate of AGEs can be a 3-DG (3-deoxyglucosone) production inhibiting action. Can be in the form of an extract or food or drink. ADVANTAGE OF THE INVENTION According to this invention, black garlic can suppress carbonyl stress, it can be expected to be effective in the prevention and treatment of AGEs-related diseases, and therefore can realize effective prevention and treatment of AGEs-related diseases, and can add black garlic. It can contribute to value enhancement and spread. In addition, it is considered that these effects can be widely extended to compositions and the like using leeks such as garlic as a raw material, and it is expected that the effects of the welsh onions can be improved and their use can be increased. In the present invention, in particular, it was revealed that one of the black garlic-containing components is normeconin, and its AGE-reactive AGEs inhibitory activity can be confirmed for the first time. With new uses.
今回、本願においてさらに、ノルメコニンの新たな用途すなわちＧＡＢＡ分解酵素阻害用途を開示することにより、ノルメコニンはＡＧＥｓ阻害作用およびＧＡＢＡ分解酵素阻害作用（生体内におけるＧＡＢＡの有効利用率向上作用）の両方を併せ持つことが明らかとなった。また、ノルメコニンとＧＡＢＡをともに含有する、黒ニンニクに代表されるネギ属植物加工物は、ＡＧＥｓ阻害作用およびＧＡＢＡ分解酵素阻害作用（生体内におけるＧＡＢＡの有効利用率向上作用）の両方を併せ持つ、極めて有用性の高い素材であることが明らかとなった。 In this application, by further disclosing a new use of normeconin, namely, a use for inhibiting GABA-degrading enzyme, normeconin has both an AGEs-inhibiting effect and a GABA-degrading enzyme-inhibiting effect (an effect of improving the effective utilization rate of GABA in a living body). It became clear. Further, a processed product of the genus Allium, represented by black garlic, containing both normeconin and GABA, has both an AGEs inhibitory activity and a GABA-degrading enzyme inhibitory activity (an effect of improving the effective utilization rate of GABA in a living body). It became clear that the material was highly useful.
以下は実施例（第１部）に係る図である。
以下は実施例（第２部）に係る図である。
以下、本発明を図面も用いつつより詳細に説明する。
上述の通り本願が最も基本とする発明は、ＧＡＢＡ（γ−ａｍｉｎｏｂｕｔｙｒｉｃ ａｃｉｄ）分解酵素阻害用ノルメコニン（Ｎｏｒｍｅｃｏｎｉｎ ６，７−Ｄｉｈｙｄｒｏｘｙ−１（３Ｈ）−ｉｓｏｂｅｎｚｏｆｕｒａｎｏｎｅ）である。そして本分解酵素阻害用ノルメコニンは、ネギ属植物由来、すなわちネギ属植物を原料として得られるものとすることができる。この場合、用いるネギ属植物は、下記＜Ａ＞、＜Ｂ＞に列挙されたものの少なくともいずれかとすることができる。
＜Ａ＞ タマネギ、エシャロット、ニンニク、ラッキョウ、ツリーオニオン、アサツキ、ネギ（ナガネギ、青ネギ）、ワケギ、チャイブ、ニラ、ヤグラネギ、白ネギ、リーキ、ノビル、ギョウジャニンニク
＜Ｂ＞ ＜Ａ＞に列挙した各植物の加工物 例．黒ニンニク
Hereinafter, the present invention will be described in more detail with reference to the drawings.
As described above, the most basic invention of the present application is normeconin 6,7-Dihydroxy-1 (3H) -isobenzofuranone for inhibiting GABA (γ-aminobutyric acid) degrading enzyme. The degrading enzyme-inhibiting normeconin can be derived from a plant of the genus Allium, that is, obtained from a plant of the genus Allium. In this case, the Allium plant to be used may be at least one of the following <A> and <B>.
<A> Onion, shallot, garlic, rakkyo, tree onion, asatsuki, green onion (leek onion, green onion), scallion, chives, leek, yagura onion, white leek, leek, nobile, ginger garlic
<B> Processed products of each plant listed in <A> Example. Black garlic
原料として用いるネギ属植物としては特に、発酵処理、加熱処理、またはメイラード反応処理の少なくともいずれかの処理済みの加工物、たとえば黒ニンニクを好適に用いることができる。実施例（第１部）に後述するように、黒ニンニクの場合、発酵処理やメイラード反応処理を含む黒ニンニク製造過程を経たものにおいてノルメコニン生成が確認された。したがってノルメコニン生成機作としては、発酵処理またはメイラード反応処理による効果が挙げられる。これに加え、グルコースやフルクトースの加熱処理によるノルメコニン生成の機作も大いに可能性が示唆されるため、ここに開示するものである。 As the Allium plant used as a raw material, particularly, a processed product, for example, black garlic, which has been subjected to at least one of fermentation treatment, heat treatment, and Maillard reaction treatment can be suitably used. As described later in Examples (Part 1), in the case of black garlic, production of normeconin was confirmed in a black garlic production process including a fermentation treatment and a Maillard reaction treatment. Accordingly, the effect of the fermentation treatment or the Maillard reaction treatment is mentioned as the normeconin production mechanism. In addition, the mechanism of the production of normeconin by heat treatment of glucose or fructose suggests a great possibility, and is disclosed here.
なお、「黒」、「琥珀」、「発酵」、「熟成」のような黒ニンニク等のネギ属加工物製品に見られる表示、あるいはその他、発酵処理・加熱処理・メイラード反応処理の少なくともいずれかの処理済みネギ属植物由来である旨が示されていると認められる表示が付されているＧＡＢＡ分解酵素阻害用ノルメコニンも、本発明の範囲内である。また、「ＧＡＢＡ分解酵素を阻害する」旨の表示が付されているＧＡＢＡ分解酵素阻害用ノルメコニンも、同様である。 In addition, the label found on processed products of Allium genus such as black garlic, such as "black", "amber", "fermented", "aged", or at least one of fermentation treatment, heat treatment, Maillard reaction treatment Normeconin for inhibiting GABA-degrading enzyme, which is indicated to be derived from the treated Allium plant, is also within the scope of the present invention. The same applies to normeconin for inhibiting GABA-degrading enzyme, which is labeled "inhibits GABA-degrading enzyme".
本願発明者は、ノルメコニンにＧＡＢＡ分解酵素阻害機能があることを明らかにした。かかる作用を有するノルメコニンを用いて行うＧＡＢＡ分解酵素阻害方法もまた、本発明の範囲内である。そして、このノルメコニンの製造原料として上記＜Ａ＞、＜Ｂ＞記載のネギ属植物を用いることができる。すなわち、ネギ属植物を原料としたノルメコニンを用いて行うＧＡＢＡ分解酵素阻害方法もまた、本発明の範囲内である。 The present inventors have clarified that normeconin has a GABA-degrading enzyme inhibitory function. A method for inhibiting GABA-degrading enzyme using normeconin having such an action is also within the scope of the present invention. As the raw material for producing normeconin, the allium plants described in the above <A> and <B> can be used. That is, a method for inhibiting a GABA-degrading enzyme carried out using normeconin obtained from a plant of the genus Allium is also within the scope of the present invention.
図１は、本発明のＧＡＢＡ摂取用組成物の基本構成を示す概念図である。図示するように本ＧＡＢＡ分解酵素阻害用組成物３は、ノルメコニン１を有効成分とすることを最も基本的な構成とする。なお、本願において「組成物」には固体状または液体状のエキス（抽出物）も、食品も含まれる。すなわち組成物とは、複数の成分や構成要素により組成されているものである。また、「食品」は飲料を含む。なお、組成物の一類型である食品については、以降の説明において特に取り上げて述べることがある。 FIG. 1 is a conceptual diagram showing the basic constitution of the composition for ingesting GABA of the present invention. As shown in the figure, the composition 3 for inhibiting GABA-degrading enzyme has the most basic configuration containing normeconin 1 as an active ingredient. In the present application, the “composition” includes solid or liquid extracts (extracts) and foods. That is, the composition is composed of a plurality of components and constituent elements. “Food” includes beverages. In addition, the food which is a type of the composition may be particularly taken up and described in the following description.
図示するように本ＧＡＢＡ分解酵素阻害用組成物３には表示部２が付されていて、その表示部２が「ＧＡＢＡ分解酵素を阻害する」旨の表示であるものとすることができる。また、ネギ属植物由来、すなわち原料としてネギ属植物を用いるものとすることができ、たとえば下記の少なくともいずれかを使用できる点は、上記ノルメコニン発明と同様である。
＜Ａ＞ タマネギ、エシャロット、ニンニク、ラッキョウ、ツリーオニオン、アサツキ、ネギ（ナガネギ、青ネギ）、ワケギ、チャイブ、ニラ、ヤグラネギ、白ネギ、リーキ、ノビル、ギョウジャニンニク
＜Ｂ＞ ＜Ａ＞に列挙した各植物の加工物
As shown in the figure, the present GABA-degrading enzyme inhibiting composition 3 is provided with a display portion 2, and the display portion 2 can be a display indicating “inhibits GABA-degrading enzyme”. In addition, it is possible to use allium plants as a raw material, that is, to use at least one of the following, as in the case of the normeconin invention.
<A> Onions, shallots, garlic, rakkyo, tree onions, asatsuki, green onions (leeks, green onions), scallions, chives, leek, yagura onions, white onions, leek, nobile, ginger garlic <B> Each plant listed in <A> Workpiece of
本ＧＡＢＡ分解酵素阻害用組成物３の原料とするネギ属植物は、発酵処理、加熱処理、またはメイラード反応処理の少なくともいずれかの処理済みの加工物とすることができる。なお、表示部２として、「黒」、「琥珀」、「発酵」、「熟成」、またはその他の発酵処理・加熱処理・メイラード反応処理の少なくともいずれかの処理済みネギ属植物由来である旨が、上述「ＧＡＢＡ分解酵素を阻害する」旨の表示と併せて、あるいは単独で、示されていてもよい。 The allium plant used as a raw material of the composition 3 for inhibiting GABA-degrading enzyme can be a processed product that has been subjected to at least one of fermentation treatment, heat treatment, and Maillard reaction treatment. In addition, as the display part 2, "Black", "Amber", "Fermentation", "Aging", or other fermentation treatment, heat treatment, and Maillard reaction treatment at least one of the fact that it is derived from allium plants , Or together with the above-mentioned "inhibiting GABAase" or alone.
図２は、本発明のＧＡＢＡ摂取用組成物の基本構成を示す概念図である。本ＧＡＢＡ摂取用組成物６は、発酵処理、加熱処理、またはメイラード反応処理の少なくともいずれかの処理済みのネギ属植物由来であり、すなわち、かかる処理済みのネギ属植物を原料として組成されているものである。そして、図示するように本ＧＡＢＡ摂取用組成物６はＧＡＢＡ４を含有しており、さらにノルメコニン１を一つの有効成分として含有する構成である。 FIG. 2 is a conceptual diagram showing the basic constitution of the composition for ingesting GABA of the present invention. The present composition 6 for GABA ingestion is derived from a processed Allium plant that has been subjected to at least one of fermentation treatment, heat treatment, and Maillard reaction treatment, that is, is composed of such a treated Allium plant as a raw material. Things. As shown in the figure, the composition 6 for GABA ingestion contains GABA4, and further contains normeconin 1 as one active ingredient.
すなわち本発明ＧＡＢＡ摂取用組成物６には、ＧＡＢＡ４が含まれているともに、これが生体内に摂取された際におけるＧＡＢＡ分解酵素による分解を阻害する作用を有するノルメコニン１もが含まれている。したがって本ＧＡＢＡ摂取用組成物６を体内に摂取した場合、摂取されたＧＡＢＡ４のＧＡＢＡ分解酵素による分解がノルメコニン１によって阻害されるため、生体内におけるＧＡＢＡの有効利用率を向上させることができる。 That is, the composition 6 for GABA ingestion of the present invention contains GABA4, and also contains normeconin 1, which has an action of inhibiting degradation by GABA-degrading enzymes when it is ingested into a living body. Therefore, when the present composition 6 for ingesting GABA is ingested into the body, the degradation of the ingested GABA4 by GABA-degrading enzyme is inhibited by normeconin 1, so that the effective utilization rate of GABA in the living body can be improved.
図示するように本発明ＧＡＢＡ摂取用組成物６には、「ＧＡＢＡ分解酵素を阻害する」旨、または「ＧＡＢＡを摂取できる」旨の少なくともいずれかの表示が付された表示部５が設けられている構成とすることができる。表示部５にはまた、かかる表示に併せて、または単独で、「黒」、「琥珀」、「発酵」、「熟成」、またはその他の発酵処理・加熱処理・メイラード反応処理の少なくともいずれかの処理済みネギ属植物由来である旨を示す表示が付されているものとすることもできる。 As shown in the figure, the composition for ingesting GABA 6 of the present invention is provided with a display section 5 to which at least one of a message indicating "inhibiting GABA-degrading enzyme" and a message "can ingest GABA" is provided. Configuration. The display unit 5 may also display at least one of “black”, “amber”, “fermentation”, “aging”, or other fermentation treatment, heating treatment, or Maillard reaction treatment in conjunction with such an indication or alone. A sign indicating that it is derived from a treated Allium plant may be added.
上述の通り、本願発明での「組成物」には食品も含まれる。したがって、ネギ属植物を原料とするＧＡＢＡ摂取用食品、そのネギ属植物が上記＜Ａ＞、＜Ｂ＞であるＧＡＢＡ摂取用食品、ネギ属植物としての発酵処理・加熱処理・またはメイラード反応処理の少なくともいずれかの処理済みの加工物の使用、さらにはノルメコニンを一有効成分とするＧＡＢＡ摂取用食品もまた、本発明の範囲内である。また、「ＧＡＢＡ分解酵素を阻害する」旨、または「ＧＡＢＡを摂取できる」旨の少なくともいずれかの表示が付されているＧＡＢＡ摂取用食品も、同様である。 As described above, the “composition” in the present invention includes food. Therefore, a food for GABA ingestion using a leek plant as a raw material, a food for GABA ingestion in which the leek plant is the above <A> or <B>, a fermentation treatment, a heating treatment, or a Maillard reaction treatment as the leek plant Use of at least one of the processed products, and foods for ingesting GABA containing normeconin as an active ingredient are also within the scope of the present invention. The same applies to foods for ingesting GABA, which are labeled with "inhibiting GABA-degrading enzyme" or "ingesting GABA".
なお、食品は組成物であり、すなわち原料ネギ属植物が生のままではなく何らかの加工処理をなされてなる加工物である。しかしながら、その加工の程度や処理過程の数は問われない。たとえば生の原料に対して所定の発酵処理がなされたのみの物でも、有効成分を含む抽出物、その抽出物を添加した物、分離された有効成分を添加した物など、全て食品に該当する。また、固形・半固形・粉末状・液状など食品としての形態も限定されない。 The food is a composition, that is, a processed product obtained by subjecting a raw material of the genus Allium to raw processing, rather than raw processing. However, the degree of processing and the number of processing steps are not limited. For example, even a product obtained by subjecting a raw material to a predetermined fermentation treatment, an extract containing an active ingredient, a product to which the extract is added, a product to which a separated active ingredient is added, etc., all fall under food. . Further, the form of the food, such as solid, semi-solid, powder, and liquid, is not limited.
図３は、本発明のＧＡＢＡ摂取用組成物製造方法の基本構成を示すフロー図である。図示するように本ＧＡＢＡ摂取用組成物等製造方法は、以上説明したいずれかのＧＡＢＡ分解酵素阻害用組成物１１０ａ、ＧＡＢＡ摂取用組成物１１０ｂ、またはＧＡＢＡ摂取用食品１１０ｃ（以上をまとめて、ＧＡＢＡ摂取用組成物等１１０ｘ）のいずれかを製造する方法であって、原料ネギ属植物１１に対する発酵処理Ｐ０、加熱処理Ｐ１、またはメイラード反応処理Ｐ２の少なくともいずれかの処理過程、およびその後の濃縮処理過程Ｐ３を含んで行われることを、主たる構成とする。 FIG. 3 is a flowchart showing the basic configuration of the method for producing a composition for ingesting GABA of the present invention. As shown in the figure, the method for producing a composition for ingesting GABA, etc., uses any one of the composition 110a for inhibiting GABA-degrading enzyme, the composition 110b for ingesting GABA, or the food 110c for ingesting GABA as described above. 110x), which comprises at least any one of a fermentation treatment P0, a heat treatment P1, and a Maillard reaction treatment P2 with respect to the raw material of the genus Allium plant 11, and a subsequent concentration treatment What is performed including the process P3 is a main configuration.
かかる構成により本ＧＡＢＡ摂取用組成物製造方法によれば、原料ネギ属植物１１が発酵処理Ｐ０、加熱処理Ｐ１、またはメイラード反応処理Ｐ２の少なくともいずれかの処理過程に供されて発酵処理、加熱処理、またはメイラード反応処理され、得られた処理物はついで濃縮処理過程Ｐ３によって濃縮され、最終的にＧＡＢＡ分解酵素阻害用組成物１１０ａ、ＧＡＢＡ摂取用組成物１１０ｂ、またはＧＡＢＡ摂取用食品１１０ｃ（ＧＡＢＡ摂取用組成物等１１０ｘ）のいずれかが得られる。 According to the method for producing a composition for GABA ingestion having such a configuration, the raw material of Allium sp. 11 is subjected to at least one of the fermentation treatment P0, the heat treatment P1, and the Maillard reaction treatment P2 to perform the fermentation treatment and the heat treatment. Or the Maillard reaction treatment, and the resulting processed product is then concentrated by the concentration process P3, and finally the composition 110a for GABA-degrading enzyme inhibition, the composition 110b for GABA ingestion, or the food 110c for GABA ingestion (GABA ingestion) 110x) is obtained.
図３−２は、本発明のＧＡＢＡ摂取用組成物製造方法の別の構成を示すフロー図である。図示するように本製造方法は、図３に示したフローに加えて、濃縮処理過程Ｐ３の後に乾燥処理過程Ｐ４を設けたフローとすることができる。かかるフローによれば、原料ネギ属植物１１は濃縮処理過程Ｐ３までの各過程による処理を経た後、乾燥処理過程Ｐ４にて乾燥処理され、最終的にＧＡＢＡ分解酵素阻害用組成物１１４ａ、ＧＡＢＡ摂取用組成物１１４ｂ、またはＧＡＢＡ摂取用食品１１４ｃ（ＧＡＢＡ摂取用組成物等１１４ｘ）のいずれかが得られる。 FIG. 3-2 is a flowchart showing another configuration of the method for producing a composition for ingesting GABA of the present invention. As shown in the drawing, the present manufacturing method can be a flow in which a drying process P4 is provided after the concentration process P3 in addition to the flow shown in FIG. According to this flow, the raw material of Allium sp. 11 is subjected to the treatments in each step up to the concentration treatment step P3, and then dried in the drying treatment step P4. Finally, the GABA-degrading enzyme inhibiting composition 114a, GABA intake Of the composition 114b for food or the food 114c for GABA intake (composition 114x for GABA intake, etc.).
乾燥処理過程Ｐ４における乾燥処理としては、温風乾燥、熱風乾燥、フリーズドライ（凍結乾燥処理）など任意の乾燥方法を用いることができる。なお図３、図３−２により説明したＧＡＢＡ摂取用組成物製造方法は、後述するＡＧＥｓ等阻害用兼ＧＡＢＡ摂取用ネギ属植物製組成物製造方法にも用いることができるが、その場合は、スプレードライ（噴霧乾燥処理）を除外するものとすることができる。スプレードライではシクロデキストリン等のホスト分子を用いてゲスト分子であるＡＧＥｓ等阻害性ネギ属植物製組成物を包摂する必要があり、その分純度が低くなってしまう。そこで、この乾燥法を用いないことで、純度の高い、あるいは固形分として原料ネギ属植物以外を含まない組成物を得ることができるからである。 As the drying treatment in the drying treatment process P4, any drying method such as hot air drying, hot air drying, freeze drying (freeze drying treatment) can be used. Note that the method for producing a composition for ingesting GABA described with reference to FIGS. 3 and 3-2 can also be used for a method for producing a composition made of an Allium plant for inhibiting AGEs and the like and for ingesting GABA described below. Spray drying (spray drying treatment) can be excluded. In spray drying, it is necessary to use a host molecule such as cyclodextrin to cover a composition made of an inhibitory allium plant such as AGEs, which is a guest molecule, and the purity is reduced accordingly. Thus, by not using this drying method, it is possible to obtain a composition having high purity or containing no solid matter other than the raw materials of the genus Allium.
図４は、本発明のＡＧＥｓ等阻害用兼ＧＡＢＡ摂取用ネギ属植物製組成物の基本構成を示す概念図である。図示するように本ＡＧＥｓ等阻害用兼ＧＡＢＡ摂取用ネギ属植物製組成物２２０は、ＡＧＥｓ（ａｄｖａｎｃｅｄ ｇｌｙｃａｔｉｏｎ ｅｎｄ−ｐｒｏｄｕｃｔｓ、終末糖化産物）の生成（産生）を阻害する作用（ＡＧＥｓ産生阻害作用）Ｆ１、ＡＧＥｓの中間体の生成（産生）を阻害する作用（ＡＧＥｓ中間体産生阻害作用）Ｆ２、またはＡＧＥｓのＲＡＧＥ（ｒｅｃｅｐｔｏｒ ｆｏｒ ＡＧＥｓ、ＡＧＥｓ受容体）への接着を阻害する作用（ＡＧＥ−ＲＡＧＥ接着阻害作用）Ｆ３の少なくともいずれかを有し、かつ、およびＧＡＢＡ分解酵素阻害作用Ｆ５をも有することを、主たる構成とする。なお、ＡＧＥｓ産生阻害作用Ｆ１、ＡＧＥｓ中間体産生阻害作用Ｆ２、ＡＧＥ−ＲＡＧＥ接着阻害作用Ｆ３をまとめて、ＡＧＥｓ等阻害作用Ｆ４とも言う。 FIG. 4 is a conceptual diagram showing the basic constitution of the composition of the genus Allium plant for inhibiting AGEs and the like and for ingesting GABA of the present invention. As shown in the figure, the composition of the Allium genus for inhibiting AGEs and the like and for ingesting GABA 220 has an action of inhibiting the generation (production) of AGEs (advanced glycation end-products, advanced glycation end products) (ages production inhibitory action) F1. , An action of inhibiting the generation (production) of intermediates of AGEs (AGEs intermediate production inhibiting action) F2, or an action of inhibiting the adhesion of AGEs to RAGE (receptor for AGEs, AGEs receptor) (AGE-RAGE adhesion inhibition) Action) The main configuration is to have at least one of F3 and also have a GABAase inhibitory action F5. The AGEs production inhibitory action F1, the AGEs intermediate production inhibitory action F2, and the AGE-RAGE adhesion inhibitory action F3 are collectively referred to as AGEs or other inhibitory action F4.
すなわち本ＡＧＥｓ等阻害用兼ＧＡＢＡ摂取用ネギ属植物製組成物２２０は、これらの少なくともいずれかのＡＧＥｓ等阻害作用Ｆ４とＧＡＢＡ分解酵素阻害作用Ｆ５とを兼ね備えた極めて有用な組成物である。なおＡＧＥｓ等阻害作用に関しては追って、その概要および実施例にて補足する。 That is, the composition of the genus Allium plant 220 for inhibiting AGEs and the like and ingesting GABA is an extremely useful composition having at least one of the inhibitory action F4 for AGEs and the like and the inhibitory action F5 for GABAase. The inhibitory action of AGEs and the like will be supplemented later with an outline and examples.
本ＡＧＥｓ等阻害用兼ＧＡＢＡ摂取用ネギ属植物製組成物２２０の原料ネギ属植物は、発酵処理、加熱処理、またはメイラード反応処理の少なくともいずれかの処理済みの加工物とすることができる。また、下記＜Ｄ１＞〜＜Ｄ４＞の中から少なくともいずれかと、および下記＜Ｅ１＞〜＜Ｅ４＞の中から少なくともいずれかの表示が併せて付された表示部２２５が設けられた構成としてもよい。
＜Ｄ１＞ ＡＧＥｓ生成阻害用である旨
＜Ｄ２＞ ＡＧＥｓの中間体の生成阻害用である旨
＜Ｄ３＞ ＡＧＥｓのＲＡＧＥへの接着阻害用である旨
＜Ｄ４＞ 固形分として原料とするネギ属植物以外のものが含まれていない旨
＜Ｅ１＞ＧＡＢＡ分解酵素阻害用である旨
＜Ｅ２＞ＧＡＢＡ摂取可能である旨
The allium plant of the allium plant composition 220 for inhibiting AGEs and the like and for ingesting GABA can be a processed product that has been subjected to at least one of fermentation treatment, heat treatment, and Maillard reaction treatment. Further, the display unit 225 may be provided with at least one of the following <D1> to <D4> and at least one of the following <E1> to <E4>. Good.
<D1> Inhibition of AGEs generation <D2> Inhibition of AGEs intermediate formation <D3> Inhibition of AGEs adhesion to RAGE <D4> Allium genus plant as solid material <E1> Indicates that it is for GABAase inhibition <E2> Indicates that GABA can be consumed
以上説明したいずれかのＡＧＥｓ等阻害用兼ＧＡＢＡ摂取用ネギ属植物製組成物を製造する方法であって、原料ネギ属植物に対する発酵処理、加熱処理、またはメイラード反応処理の少なくともいずれかの処理過程、およびその後の濃縮処理過程を含んで行われるＡＧＥｓ等阻害用兼ＧＡＢＡ摂取用ネギ属植物製組成物製造方法もまた、本発明の範囲内である（前出図３、図３−２参照）。同じく、本発明ＡＧＥｓ等阻害用兼ＧＡＢＡ摂取用ネギ属植物製組成物、または上記製造方法により得られるＡＧＥｓ等阻害用兼ＧＡＢＡ摂取用ネギ属植物製組成物を用いてなる食品もまた、本発明の範囲内である。 A method for producing any one of the above-described compositions for inhibiting AGEs or the like and for ingesting GABA, the composition comprising at least one of a fermentation treatment, a heating treatment, and a Maillard reaction treatment for a raw material of allium. Also, a method for producing a composition made of Allium plants for inhibiting AGEs or the like and ingesting GABA, which is carried out including a concentration treatment step thereafter, is also within the scope of the present invention (see FIG. 3 and FIG. 3-2 described above). . Similarly, a food composition using the composition of the genus Allium plant for inhibiting AGEs and the like and ingesting GABA or the composition of the genus Allium plant for inhibiting AGEs and the like and ingesting GABA obtained by the above-mentioned production method is also the present invention. Is within the range.
さらに、本項の冒頭に説明した本発明ノルメコニンについて補足する。ＡＧＥｓ等およびＧＡＢＡ分解酵素の阻害用ノルメコニンも本発明の範囲内である。また、ネギ属植物由来である、すなわちネギ属植物を原料としてなるＡＧＥｓ等およびＧＡＢＡ分解酵素の阻害用ノルメコニンも、本発明の範囲内である。 Furthermore, the normeconin of the present invention described at the beginning of this section is supplemented. Normeconin for inhibiting AGEs and the like and GABA-degrading enzymes are also within the scope of the present invention. Further, AGEs and the like derived from Allium plants, that is, normeconins for inhibiting GABA-degrading enzymes, which are obtained from Allium plants are also included in the scope of the present invention.
上述のＡＧＥｓ等阻害用兼ＧＡＢＡ摂取用ネギ属植物製組成物発明の補足説明として、出願人による「ＡＧＥｓ等阻害性ネギ属植物製組成物、その製造方法、飲食品、およびＡＧＥｓ阻害用Ｎｏｒｍｅｃｏｎｉｎ」出願（特願２０１８−１５２１４７ 本願出願時に未公開）に記載された発明の概要を、若干の追記や表記変更を含めて下記の通り転記する。 As a supplementary explanation of the above-mentioned composition of the genus Allium plant for inhibiting AGEs and the like and ingesting GABA, "Applicant's composition for inhibiting allium genus such as AGEs, its production method, food and drink, and Normeconin for inhibiting AGEs" was filed by the applicant. The summary of the invention described in the application (Japanese Patent Application No. 2018-152147, unpublished at the time of filing the present application) is transcribed as follows, with some additions and notational changes.
〔１８−１〕 ＡＧＥｓ（ａｄｖａｎｃｅｄ ｇｌｙｃａｔｉｏｎ ｅｎｄ−ｐｒｏｄｕｃｔｓ、終末糖化産物）もしくはその中間体の生成を阻害する作用、またはＡＧＥｓのＲＡＧＥ（ｒｅｃｅｐｔｏｒ ｆｏｒ ＡＧＥｓ、ＡＧＥｓ受容体）への接着を阻害する作用（以下、まとめて「ＡＧＥｓ等阻害性」という。）の少なくともいずれかを有することを特徴とする、ＡＧＥｓ等阻害性ネギ属植物製組成物。
〔１８−２〕 前記ＡＧＥｓ等阻害性の少なくとも一つが、ＡＧＥｓ産生阻害作用に係ることを特徴とする、〔１８−１〕に記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
〔１８−３〕 前記ＡＧＥｓ等阻害性の少なくとも一つが、３−ＤＧ（３−デオキシグルコソン）産生阻害作用に係ることを特徴とする、〔１８−１〕または〔１８−２〕に記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
〔１８−４〕 前記ＡＧＥｓ等阻害性の少なくとも一つが、ＡＧＥ−ＲＡＧＥ接着阻害作用に係ることを特徴とする、〔１８−１〕ないし〔１８−３〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
〔１８−５〕 飲食品であることを特徴とする、〔１８−１〕ないし〔１８−４〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
[18-1] An action of inhibiting the production of AGEs (advanced glycation end-products, advanced glycation end products) or an intermediate thereof, or an action of inhibiting adhesion of AGEs to RAGE (receptor for AGEs, AGEs receptor) (hereinafter, referred to as AGEs) AGEs and the like, collectively referred to as “AGEs and other inhibitory properties”).
[18-2] The composition made of an inhibitory Allium plant such as AGEs according to [18-1], wherein at least one of the AGEs or the like has an inhibitory effect on AGEs production.
[18-3] The method according to [18-1] or [18-2], wherein at least one of the inhibitors such as AGEs is related to an inhibitory action on 3-DG (3-deoxyglucosone) production. A composition made of an inhibitory allium plant such as AGEs.
[18-4] At least one of the AGEs and the like has a AGE-RAGE adhesion inhibitory action, wherein the AGEs or the like as described in any one of [18-1] to [18-3]. Allium genus composition.
[18-5] The composition made of an inhibitory Allium plant such as AGEs according to any of [18-1] to [18-4], which is a food or drink.
〔１８−６〕 固体状または液体状のエキス（抽出物）であることを特徴とする、〔１８−１〕ないし〔１８−５〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
〔１８−７〕 前記ネギ属植物は下記＜１８−Ａ＞のいずれかであることを特徴とする、〔１８−１〕ないし〔１８−６〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
＜１８−Ａ＞タマネギ、エシャロット、ニンニク、ラッキョウ、ツリーオニオン、アサツキ、ネギ（ナガネギ、青ネギ）、ワケギ、チャイブ、ニラ、ヤグラネギ、白ネギ、リーキ、ノビル、ギョウジャニンニク
〔１８−８〕 前記ネギ属植物は発酵処理、加熱処理（追記）またはメイラード反応処理の少なくともいずれかの処理済みネギ属植物であることを特徴とする、〔１８−１〕ないし〔１８−７〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
〔１８−９〕 前記ネギ属植物は発酵処理またはメイラード反応処理の少なくともいずれかの処理済みニンニクであり、「黒」、「琥珀」、「発酵」または「熟成」の少なくともいずれかの語を「ニンニク」の語に付した表示が付されていることを特徴とする、〔１８−１〕ないし〔１８−７〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
〔１８−１０〕 固形分として原料ネギ属植物以外のものが含まれていないことを特徴とする、〔１８−１〕ないし〔１８−９〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
[18-6] The composition made of an inhibitory Allium plant such as AGEs according to any of [18-1] to [18-5], which is a solid or liquid extract (extract). object.
[18-7] The allium plant is any one of the following <18-A>, and the inhibitory allium genus such as AGEs according to any of [18-1] to [18-6] is provided. Botanical composition.
<18-A> Onion, shallot, garlic, rakkyo, tree onion, asatsuki, leek (leek onion, green onion), scallion, chives, chive, leek, leek, white onion, leek, nobile, ginger garlic [18-8] The above Leek genus plant AGEs or the like according to any one of [18-1] to [18-7], which is a processed Allium plant that has been subjected to at least one of fermentation treatment, heat treatment (additional treatment) and Maillard reaction treatment. An inhibitory leek composition.
[18-9] The Allium plant is a treated garlic of at least one of a fermentation treatment and a Maillard reaction treatment, and at least one of the words “black”, “amber”, “fermentation” or “aged” is “ The composition according to any one of [18-1] to [18-7], wherein the composition is an inhibitory welsh onion plant according to any one of [18-1] to [18-7].
[18-10] An inhibitory allium plant such as AGEs according to any one of [18-1] to [18-9], wherein the solid content does not include anything other than the raw material of the allium plant. Composition.
〔１８−１１〕 有効成分としてノルメコニンが含有されていることを特徴とする、〔１８−１〕ないし〔１８−１０〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
〔１８−１２〕 有効成分として５−Ｈｙｄｒｏｘｙｍｅｔｈｙｌ ｆｕｒｆｕｒａｌ（５−ＨＭＦ）が含有されていることを特徴とする、〔１８−１〕ないし〔１８−１１〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
〔１８−１３〕 ＡＧＥｓ生成阻害用であることを特徴とする、〔１８−１〕ないし〔１８−１２〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
〔１８−１４〕 ＡＧＥｓの中間体の生成阻害用であることを特徴とする、〔１８−１〕ないし〔１８−１３〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
〔１８−１５〕 ＡＧＥｓのＲＡＧＥへの接着阻害用であることを特徴とする、〔１８−１〕ないし〔１８−１４〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
[18-11] The composition according to any of [18-1] to [18-10], which comprises normeconin as an active ingredient.
[18-12] An inhibitory leek such as AGEs according to any one of [18-1] to [18-11], characterized by containing 5-hydroxymethyl furfural (5-HMF) as an active ingredient. A genus plant composition.
[18-13] The composition made of an inhibitory Allium plant such as AGEs according to any one of [18-1] to [18-12], which is for inhibiting AGEs production.
[18-14] The composition according to any one of [18-1] to [18-13], which is for inhibiting the production of an intermediate of AGEs.
[18-15] The composition made of an inhibitory Allium plant such as AGEs according to any one of [18-1] to [18-14], which is used for inhibiting adhesion of AGEs to RAGE.
〔１８−１６〕 飲食品用組成物であることを特徴とする、〔１８−１〕ないし〔１８−１５〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物。
〔１８−１７〕 〔１８−１〕ないし〔１８−１６〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物、または請求項１１ないし１３のいずれかに記載の方法により得られるＡＧＥｓ等阻害性ネギ属植物製組成物であって、下記＜１８−Ｄ１＞〜＜１８−Ｄ４＞の少なくともいずれかの旨の表示が付されていることを特徴とする、ＡＧＥｓ等阻害性ネギ属植物製組成物。
＜１８−Ｄ１＞ ＡＧＥｓ生成阻害用である旨
＜１８−Ｄ２＞ ＡＧＥｓの中間体の生成阻害用である旨
＜１８−Ｄ３＞ ＡＧＥｓのＲＡＧＥへの接着阻害用である旨
＜１８−Ｄ４＞ 固形分として原料とするネギ属植物以外のものが含まれていない旨
〔１８−１８〕 〔１８−１〕ないし〔１８−１７〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物を製造する方法であって、原料ネギ属植物を発酵処理、加熱処理（追記）またはメイラード反応処理の少なくともいずれかの処理をし、その後濃縮処理を行なうことを特徴とする、ＡＧＥｓ等阻害性ネギ属植物製組成物製造方法。
〔１８−１９〕 前記濃縮処理後、乾燥処理を行なうことを特徴とする、〔１８−１８〕に記載のＡＧＥｓ等阻害性ネギ属植物製組成物製造方法。
〔１８−２０〕 前記乾燥処理は任意の乾燥方法（ただし、スプレードライを除く）により行なうことを特徴とする、〔１８−１９〕に記載のＡＧＥｓ等阻害性ネギ属植物製組成物製造方法。
[18-16] The composition made of an inhibitory Allium plant such as AGEs according to any of [18-1] to [18-15], which is a composition for food or drink.
[18-17] The composition made of an inhibitory Allium plant such as AGEs according to any one of [18-1] to [18-16], or AGEs obtained by the method according to any one of claims 11 to 13. A composition made of an equi-inhibiting Allium plant, wherein the composition is provided with at least one of the following <18-D1> to <18-D4>. Botanical composition.
<18-D1> AGEs production inhibition <18-D2> AGEs intermediate production inhibition <18-D3> AGEs adhesion to RAGE inhibition <18-D4> solid [18-18] A composition made of an inhibitory Allium plant such as AGEs according to any of [18-1] to [18-17]. A method for producing, comprising: performing a fermentation treatment, a heating treatment (additional treatment) or a Maillard reaction treatment on a raw material of an allium genus plant, and then performing a concentration treatment; A method for producing a plant-made composition.
[18-19] The method for producing a composition made of an inhibitory Allium plant such as AGEs according to [18-18], wherein a drying treatment is performed after the concentration treatment.
[18-20] The method for producing a composition made of an inhibitory Allium plant such as AGEs according to [18-19], wherein the drying treatment is performed by any drying method (except for spray drying).
〔１８−２１〕 〔１８−１〕ないし〔１８−１７〕のいずれかに記載のＡＧＥｓ等阻害性ネギ属植物製組成物、または〔１８〕ないし〔２０〕のいずれかに記載の方法により得られるＡＧＥｓ等阻害性ネギ属植物製組成物を用いた、飲食品。
〔１８−２２〕 〔１８−２１〕に記載の飲食品であって、下記＜１８−Ｄ１＞〜＜１８−Ｄ４＞の少なくともいずれかの内容の表示が付されていることを特徴とする、飲食品。
＜１８−Ｄ１＞ ＡＧＥｓ生成阻害用である旨
＜１８−Ｄ２＞ ＡＧＥｓの中間体の生成阻害用である旨
＜１８−Ｄ３＞ ＡＧＥｓのＲＡＧＥへの接着阻害用である旨
＜１８−Ｄ４＞ 固形分として原料とするネギ属植物以外のものが含まれていない旨
〔１８−２３〕 下記＜１８−Ｎ１＞〜＜１８−Ｎ３＞の少なくともいずれかに用いられることを特徴とする、ＡＧＥｓ阻害用ノルメコニン。
＜１８−Ｎ１＞ ＡＧＥｓ生成阻害用
＜１８−Ｎ２＞ ＡＧＥｓの中間体の生成阻害用
＜１８−Ｎ３＞ ＡＧＥｓのＲＡＧＥへの接着阻害用
〔１８−２４〕 ネギ属植物由来であることを特徴とする、〔１８−２３〕に記載のＡＧＥｓ阻害用ノルメコニン。
[18-21] The composition according to any one of [18-1] to [20], wherein the composition is an inhibitory allium plant such as AGEs according to any of [18-1] to [18-17]. Foods and drinks using a composition made of an inhibitory allium plant such as AGEs.
[18-22] The food or beverage according to [18-21], wherein the content is marked with at least one of the following <18-D1> to <18-D4>, Food and drink.
<18-D1> AGEs production inhibition <18-D2> AGEs intermediate production inhibition <18-D3> AGEs adhesion to RAGE inhibition <18-D4> solid [18-23] for use in at least any one of the following <18-N1> to <18-N3> for inhibiting AGEs Normeconin.
<18-N1> For inhibiting AGEs production <18-N2> For inhibiting the production of intermediates of AGEs <18-N3> For inhibiting adhesion of AGEs to RAGE [18-24] It is characterized by being derived from Allium genus plants Normeconin for inhibiting AGEs according to [18-23].
以下、本発明の実施例を説明するが、本発明がかかる実施例に限定されるものではない。なお、本発明完成に至る研究過程の一部を説明することによって、実施例に替える。また、本説明は第１部と第２部に分かれるが、両部を通しての章立てとする。
＊＊＊＊＊＊＊＊＊＊＊＊
研究テーマ：黒ニンニクに含まれる有用成分の探索
第１部 黒ニンニクに含まれるＲＡＧＥ阻害活性成分について
第１章 黒ニンニクエキスのＡＧＥｓ産生阻害活性の検討
第１節 黒ニンニクエキスのＡＧＥｓ産生阻害活性の検討
（１） 黒ニンニクエキスの抽出と結果
青森県産黒ニンニク２００ｇにエタノール５００ｍＬを加え、黒ニンニクが浸る状態で２４時間冷浸を行った後、ろ過および濃縮した。この操作を３回繰り返した後、ろ過、濃縮し乾燥させたものを黒ニンニクエキスとした。この時黒ニンニクエキスは６２．４６ｇ得られ、収率は６２．４６ｇ／２００ｇ×１００＝３１．２％であった。
（２） 実験方法
Ｄ−グルコース（Ｇｌｕ、和光純薬株式会社）および牛血清アルブミン（ＢＳＡ、Ｓｉｇｍａ−Ａｌｄｒｉｃｈ Ｃｏ．，ＬＴＤ．）を各々１０％、１％となるように１ｘＰＢＳ（リン酸緩衝生理食塩水 ｐＨ７．０）に溶解したものをＢＳＡ−Ｇｌｕ溶液とした。また、１０％ＤＭＳＯ（ジメチルスルホキシド）含有１ｘＰＢＳにて４８３μｇ／μＬ、４８．３μｇ／μＬ、４．８３μｇ／μＬ、０．４８３μｇ／μＬに調製した黒ニンニクエキスを試料とした。無菌操作下において、ＢＳＡ−Ｇｌｕ溶液９００μＬとサンプル溶液１００μＬを混合し、６０℃、湿度１００％にて４週間インキュベーションした。
Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples. It should be noted that some of the research processes leading to the completion of the present invention will be described in order to replace the embodiments. In addition, the present description is divided into a first part and a second part.
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Research theme: Search for useful ingredients contained in black garlic
Part 1. About RAGE inhibitory active ingredient contained in black garlic
Chapter 1 Investigation of AGEs production inhibitory activity of black garlic extract Section 1 Investigation of AGEs production inhibitory activity of black garlic extract (1) Extraction and results of black garlic extract To 200 g of Aomori black garlic, add 500 mL of ethanol and add black garlic Was subjected to cold soaking for 24 hours, then filtered and concentrated. After repeating this operation three times, the mixture was filtered, concentrated and dried to obtain a black garlic extract. At this time, 62.46 g of black garlic extract was obtained, and the yield was 62.46 g / 200 g × 100 = 31.2%.
(2) Experimental method D-glucose (Glu, Wako Pure Chemical Industries, Ltd.) and bovine serum albumin (BSA, Sigma-Aldrich Co., LTD.) Were adjusted to 1% PBS (phosphate buffered physiology) to 10% and 1%, respectively. The solution dissolved in saline (pH 7.0) was used as a BSA-Glu solution. In addition, a black garlic extract prepared with 1 × PBS containing 10% DMSO (dimethyl sulfoxide) to 483 μg / μL, 48.3 μg / μL, 4.83 μg / μL, and 0.483 μg / μL was used as a sample. Under aseptic operation, 900 μL of the BSA-Glu solution and 100 μL of the sample solution were mixed and incubated at 60 ° C. and 100% humidity for 4 weeks.
産生されたＡＧＥｓ量は蛍光強度法（励起波長：３７５ｎｍ、吸収波長：４４０ｎｍ）にて、１０倍希釈した混合溶液を１週間ごとに測定し、下記の計算式により阻害率を算出した。
阻害率（％）＝［１−（Ｆsample−Ｆsample Blank）／（Ｆcontrol−Ｆnormal）］ｘ１００
Ｆcontrol：ＰＢＳ
Ｆsample：試料溶液を加えたｇｌｕｃｏｓｅ−ＢＳＡ溶液
Ｆsample Blank：試料溶液（インキュベーション無し）
Ｆnormal：Ｇｌｕｃｏｓｅ−ＢＳＡ溶液（インキュベーション無し）
The amount of AGEs produced was measured every week for a 10-fold diluted mixed solution by a fluorescence intensity method (excitation wavelength: 375 nm, absorption wavelength: 440 nm), and the inhibition rate was calculated by the following formula.
Inhibition rate (%) = [1− (Fsample−Fsample Blank) / (Fcontrol−Fnormal)] × 100
Fcontrol: PBS
Fsample: glucose-BSA solution with sample solution added Fsample Blank: sample solution (no incubation)
Fnormal: Glucose-BSA solution (no incubation)
（３）結果・考察
グルコースおよび牛血清アルブミンにおけるメイラード反応において産生するＡＧＥｓに対する黒ニンニクエキスの阻害作用について検討した結果を図５に示す。これは、黒ニンニクエキスのＡＧＥｓ産生阻害活性の阻害率推移を吸光度により示したグラフである。図示するように、陽性対照薬として用いた３００μＭケルセチンの抑制作用は、反応開始時から添加していた場合、１週間後では４．２％、２週間後では１３．９％、３週間後では１２．４％、４週間後では１３．４％の阻害作用を認めた。一方、かかる対照群のＡＧＥｓ産生量に比較して、黒ニンニクエキス４８３μｇ／μＬを反応開始時から添加していた場合には、反応開始の１週間後で８３％、２週間後からは１００％の極めて強いＡＧＥｓ産生阻害作用が認められた。
(3) Results and Discussion FIG. 5 shows the results of an investigation on the inhibitory effect of black garlic extract on AGEs produced in the Maillard reaction with glucose and bovine serum albumin. This is a graph showing the change in the inhibition rate of the AGEs production inhibitory activity of black garlic extract by absorbance. As shown in the figure, the inhibitory effect of 300 μM quercetin used as a positive control drug was 4.2% after 1 week, 13.9% after 2 weeks, and 33.9% after 3 weeks when added from the beginning of the reaction. At 12.4% and 4 weeks later, an inhibitory effect of 13.4% was observed. On the other hand, when 483 μg / μL of black garlic extract was added from the start of the reaction, 83% after one week from the start of the reaction, and 100% after 2 weeks compared to the AGEs production amount of the control group. Has an extremely strong AGEs production inhibitory effect.
この阻害作用は用量依存的であり、４８．３μｇ／μＬでは反応開始１週間後で３７％、２週間後からは５０％程度の阻害作用を認めたが、４．８３μｇ／μＬでは反応開始１週間後では抑制は認められなかった。そして反応開始２週間後で１６％の阻害作用が認められたが、反応開始３週間後からは阻害作用はほとんど認められなかった。また、０．４８３μｇ／μＬではいずれの測定時間においても阻害は認められなかった。以上の通り、黒ニンニクエキスは用量依存的にＡＧＥｓ産生に対する強い阻害活性があることが認められた。 This inhibitory effect was dose-dependent. At 48.3 μg / μL, an inhibitory effect of 37% was observed one week after the start of the reaction and about 50% after 2 weeks, but at 4.83 μg / μL, the inhibitory effect was 1%. After a week, no suppression was observed. Two weeks after the start of the reaction, an inhibitory effect of 16% was observed, but almost three weeks after the start of the reaction, almost no inhibitory effect was observed. At 0.483 μg / μL, no inhibition was observed at any measurement time. As described above, it was confirmed that black garlic extract had a strong inhibitory activity on AGEs production in a dose-dependent manner.
第２節 黒ニンニクエキスの３−デオキシグルコソン産生阻害活性の検討＿ＨＰＬＣによる中間生成物３−デオキシグルコソンの定量
（１） 試料調製方法
上記第１節と同様の反応条件において、各濃度に調整した黒ニンニクエキス試料１００μおよびＰＢＳ９００μＬに内標準物質として０．００５％ ２，３−ｂｕｔａｎｅｄｉｏｎｅ（ジアセチル）５μＬを加えた後、３−デオキシグルコソン（３−ＤＧ）を誘導体化するために、０．１％ジアミノナフタレン（ＤＡＮ）１０μＬを加え、４℃で１４時間反応させた。これに酢酸エチル４ｍＬを加えて、１５分間振とう、静置させて反応生成物を抽出、上清３．５ｍＬを蒸発乾固した後、メタノール１５０μＬにて再溶解したものをＨＰＬＣ（高速液体クロマトグラフィー）サンプルとした。
Section 2 Investigation of the activity of black garlic extract for inhibiting 3-deoxyglucosone production_Quantification of intermediate product 3-deoxyglucosone by HPLC (1) Sample preparation method Adjusted to each concentration under the same reaction conditions as in Section 1 above After adding 5 μL of 0.005% 2,3-butanedione (diacetyl) as an internal standard to 100 μL of the thus-obtained black garlic extract sample and 900 μL of PBS, 0.1% was added to derivatize 3-deoxyglucosone (3-DG). 10 μL of 1% diaminonaphthalene (DAN) was added and reacted at 4 ° C. for 14 hours. To this was added 4 mL of ethyl acetate, shaken for 15 minutes, allowed to stand to extract the reaction product, 3.5 mL of the supernatant was evaporated to dryness, then redissolved in 150 μL of methanol and subjected to HPLC (high performance liquid chromatography). Graphy) as a sample.
（２）装置および分析条件
検出器； ＳＰＤ−２０ＡＤ（ＳＨＩＭＡＤＺＵ）
送液ポンプ； ＬＣ−２０ＡＤ（ＳＨＩＭＡＤＺＵ）
脱気ユニット； ＤＧＵ−２０Ａ３Ｒ（ＳＨＩＭＡＤＺＵ）
カラム； ＣＯＳＵＭＯＳＩＬ（登録商標）５ＰＥ−ＭＳ ４．６×２５０ｍｍ（Ｎａｋａｌａｉ Ｔｅｓｑｕｅ，Ｋｙｏｔｏ，Ｊａｐａｎ）
移動相； アセトニトリル：メタノール：５０ｍＭリン酸水溶液＝２０：２０：６０
流速； １．０ｍＬ／ｍｉｎ
検出波長； ２６８ｎｍ
(2) Apparatus and analytical condition detector; SPD-20AD (SHIMADZU)
Liquid sending pump; LC-20AD (SHIMADZU)
Degassing unit; DGU-20A3R (SHIMADZU)
Column; COSUMOSIL (registered trademark) 5PE-MS 4.6 x 250 mm (Nakalai Tesque, Kyoto, Japan)
Mobile phase; acetonitrile: methanol: 50 mM phosphoric acid aqueous solution = 20: 20: 60
Flow rate: 1.0 mL / min
Detection wavelength: 268 nm
（３）結果・考察
グルコースおよび牛血清アルブミンにおけるメイラード反応において産生するＡＧＥｓ産生経路における中間体である３−ＤＧに対する黒ニンニクエキスの阻害作用について検討した結果を図６に示す。これは、３−ＤＧ産生阻害活性を、３−ＤＧ産生量の推移により示したグラフである。図示するように、黒ニンニクエキス無添加の対照群では反応開始１週間後に１２７μＭの３−ＤＧの生成が認められたのに対し、黒ニンニクエキス４８３μｇ／μＬを反応開始時から添加していた場合には、反応開始の１週間後で０．０８μＭ、２週間後からは１．０μＭ以下の極めて強いＡＧＥｓ産生阻害作用が認められた。
(3) Results and consideration
The results of examining the inhibitory effect of black garlic extract on 3-DG, which is an intermediate in the AGEs production pathway produced in the Maillard reaction with glucose and bovine serum albumin, are shown in FIG. This is a graph showing the 3-DG production inhibitory activity by the transition of the amount of 3-DG production. As shown in the figure, in the control group without the addition of black garlic extract, the production of 127 μM 3-DG was observed one week after the start of the reaction, whereas the case where 483 μg / μL of the black garlic extract was added from the start of the reaction. Showed an extremely strong AGEs production inhibitory effect of 0.08 μM one week after the start of the reaction and 1.0 μM or less after two weeks.
この阻害作用は用量依存的であり、４８．３μｇ／μＬでは反応開始１週間後で２６．５μＭ、２週間後からは１６μＭ程度の阻害作用を認めた。また、４．８３μｇ／μＬおよび０．４８３μｇ／μＬでは反応開始１週間後で約８０μＭ程度の産生量であった。しかし、反応開始４週間後には対照群での３−ＤＧ産生量は低下してきたため、用量依存的な阻害は認められなかった。これは、３−ＤＧは反応開始１週間後をピークとし産生されるが、その後、反応中間体として消費されてＡＧＥｓに変換されたためと考えられる。本結果は、第１節で得られた阻害率の結果とも相関性が認められたため、黒ニンニクエキスには３−ＤＧの産生を抑制することでＡＧＥｓの生成を阻害する作用があると結論付けられた。 This inhibitory effect was dose-dependent. At 48.3 μg / μL, an inhibitory effect of about 26.5 μM was observed one week after the start of the reaction and about 16 μM after two weeks. At 4.83 μg / μL and 0.483 μg / μL, the production amount was about 80 μM one week after the start of the reaction. However, 4 weeks after the start of the reaction, the amount of 3-DG produced in the control group had decreased, and no dose-dependent inhibition was observed. This is considered to be because 3-DG was produced with a peak one week after the start of the reaction, but was subsequently consumed as a reaction intermediate and converted to AGEs. Since this result was also correlated with the result of the inhibition rate obtained in Section 1, it was concluded that black garlic extract had an effect of inhibiting the production of AGEs by suppressing the production of 3-DG. Was done.
第３節 黒ニンニクエキスのＲＡＧＥ反応性ＡＧＥｓ阻害活性＿ＥＬＩＳＡ法によるＲＡＧＥ反応性ＡＧＥｓ阻害活性の検討
ＲＡＧＥ反応性ＡＧＥｓ阻害活性を測定するにあたり、コスモバイオ社（ＣＯＳＭＯ ＢＩＯ Ｃｏ．，ＬＴＤ．）製の「Ａｇｉｎｇ／Ｇｌｙｃａｔｉｏｎ Ａｓｓａｙ Ｋｉｔ Ｓｅｒｉｅｓ，ＲＡＧＥ Ｒｅａｃｔｉｖｅ ＡＧＥｓ Ａｓｓａｙ Ｋｉｔ，Ｇｌｙｃｅｒａｌｄｅｈｙｄｅ」を使用した。
Section 3 RAGE-Reactive AGEs Inhibitory Activity of Black Garlic Extract_Investigation of RAGE-Reactive AGEs Inhibitory Activity by ELISA Method In measuring the RAGE-reactive AGEs inhibitory activity, "COSMO BIO Co., LTD." Aging / Glycation Assay Kit Series, RAGE Reactive AGEs Assay Kit, Glyceraldehyde "was used.
（１） 実験方法
（ｉ） 試料の調製
付属の検体希釈液を用いて試料を溶解し、０．４５μｍメンブランフィルター（Ｎａｃａｌａｉ ｔｅｓｑｕｅ．Ｉｎｃ．，Ｋｙｏｔｏ，Ｊａｐａｎ）を用いてろ過した後、検体希釈液で黒ニンニクエキスは４８３μｇ／μＬ、４８．３μｇ／μＬ、４．８３μｇ／μＬ、０．４８３μｇ／μＬに、陽性対照薬であるアミノグアニジンは２０ｍＭに調製した。
(1) Experimental method (i) Preparation of sample The sample was dissolved using an attached sample diluent, and filtered using a 0.45 μm membrane filter (Nacalai tesque. Inc., Kyoto, Japan). The black garlic extract was adjusted to 483 μg / μL, 48.3 μg / μL, 4.83 μg / μL and 0.483 μg / μL, and the aminoguanidine as a positive control drug was adjusted to 20 mM.
（ｉｉ） 洗浄液の調製
付属の洗浄液を超純水にて１０倍希釈して使用した。
（ｉｉｉ） 発色液の調製
発色用タブレット１個を発色液調製用緩衝液５ｍＬに入れ、完全に溶解させた。なお、この操作は発色液添加直前に調製を行った。
(Ii) Preparation of washing solution The attached washing solution was diluted 10 times with ultrapure water and used.
(Iii) Preparation of color-developing solution One tablet for color-developing was placed in 5 mL of a buffer for color-developing solution preparation and completely dissolved. This operation was prepared immediately before the addition of the coloring solution.
（ｉｖ） ＲＡＧＥ反応性ＡＧＥｓの測定
１）２０ｍＭアミノグアニジン溶液、および試験試料液（ｃｏｎｔｒｏｌは検体希釈液）を５０μＬ／ｗｅｌｌ用いた。
２）１００ｍＭグリセルアルデヒド（ＧＡ）溶液を５０μＬ／ｗｅｌｌ添加し、ＢＳＡ固層化プレートにシールし密封した。
３）湿潤状態下の３７℃インキュベーターにて２４時間静置し、固相化ＢＳＡ を糖化させた。
４）反応溶液を捨て、洗浄液２００μＬ／ｗｅｌｌで３回洗浄した。
５）ブロッキング液１００μＬ／ｗｅｌｌを添加して室温にて１時間静置した。
６）ブロッキング液を捨て、洗浄液２００μＬ／ｗｅｌｌで３回洗浄した。
(Iv) Measurement of RAGE-reactive AGEs 1) A 20 mM aminoguanidine solution and a test sample solution (control is a sample diluent) were used at 50 μL / well.
2) A 100 mM glyceraldehyde (GA) solution was added at 50 μL / well, and the plate was sealed and sealed in a BSA-solidified plate.
3) The mixture was allowed to stand for 24 hours in a humidified 37 ° C. incubator to saccharify the immobilized BSA.
4) The reaction solution was discarded, and the well was washed three times with 200 μL / well of a washing solution.
5) 100 μL / well of a blocking solution was added, and the mixture was allowed to stand at room temperature for 1 hour.
6) The blocking solution was discarded, and the plate was washed three times with 200 μL / well of a washing solution.
７）Ｒｅｃｏｍｂｉｎａｎｔ Ｒｅｃｅｐｔｏｒ ｏｆ ＡＧＥｓ Ｆｃ（ＲＡＧＥ−ＦＣ）溶液５０μＬ／ｗｅｌｌを添加して室温にて１時間静置した。
８）ＲＡＧＥ−ＦＣ溶液を捨て、洗浄液２００μＬ／ｗｅｌｌで３回洗浄した。
９）ＡＬＰ標識プロテインＡ／Ｇを５０μＬ／ｗｅｌｌを添加して室温にて１時間静置した。
１０）ＡＬＰ標識プロテインＡ／Ｇを捨て、洗浄液２００μＬ／ｗｅｌｌで３回洗浄した。
１１）．発色液を１００μＬ／ｗｅｌｌを添加して３０分間室温にて静置後、波長４０５ｎｍにて吸光度を測定（Ｉｍｍｕｎｏ Ｍｉｎｉ ＮＪ−２３００， ＢＩＯ ＴＥＫ，ＬＴＤ．）した。
（ｖ） 統計処理
有意差の検定は、Ｓｉｇｍａ Ｓｔａｔ ｓｔａｔｉｓｔｉｃａｌ ｓｏｆｔｗａｒｅ ｖｅｒ．２．０３（ＳＰＳＳ Ｉｎｃ．，ＩＬ，Ｕ．Ｓ．Ａ）を用いて行った。測定値は平均値±標準誤差で示し、Ｋｒｕｓｋａｌ−Ｗａｌｌｉｓ ＡＮＯＶＡ Ｒａｎｋ Ｔｅｓｔ（ｐ＜０．０５）により有意差を検討した。
7) Recombinant Receptor of AGEs Fc (RAGE-FC) solution (50 μL / well) was added, and the mixture was allowed to stand at room temperature for 1 hour.
8) The RAGE-FC solution was discarded and washed three times with 200 μL / well of a washing solution.
9) ALP-labeled protein A / G was added at 50 μL / well and left at room temperature for 1 hour.
10) The ALP-labeled protein A / G was discarded, and the well was washed three times with 200 μL / well of a washing solution.
11). After 100 µL / well of the coloring solution was added and left at room temperature for 30 minutes, the absorbance was measured at a wavelength of 405 nm (Immuno Mini NJ-2300, BIO TEK, LTD.).
(V) Statistical processing The test for a significant difference was performed according to Sigma Statistical software version. 2.03 (SPSS Inc., IL, USA). The measured values were shown as an average value ± standard error, and significant differences were examined using the Kruskal-Wallis ANOVA Rank Test (p <0.05).
（２）結果・考察
黒ニンニクエキスのＲＡＧＥ反応性ＡＧＥｓ阻害活性に対する阻害作用について検討した結果を、図７に示す。これは、ＲＡＧＥ反応性ＡＧＥｓ産生量を吸光度により示したグラフである（＊Ｐ＝０．００２）。図示するように、対照群でのＲＡＧＥ反応性ＡＧＥｓ産生量は０．５５４であったが、陽性対照薬のアミノグアニジンではその産生量は０．４４２であり、約２０％の阻害を認めた。一方、黒ニンニクエキス４８３μｇ／μＬでは、その産生量は０．３３５であり、約４０％の強い阻害が認められ、陽性対照薬のアミノグアニジンに比較して２倍もの強い阻害活性が認められた。
(2) Results and Discussion FIG. 7 shows the results of examining the inhibitory effect of black garlic extract on the RAGE-reactive AGEs inhibitory activity. This is a graph showing the amount of RAGE-reactive AGEs produced by absorbance ( * P = 0.002). As shown in the figure, the production of RAGE-reactive AGEs in the control group was 0.554, whereas the production of aminoguanidine as a positive control drug was 0.442, indicating about 20% inhibition. On the other hand, in the case of 483 μg / μL of black garlic extract, the production amount was 0.335, and a strong inhibition of about 40% was recognized. As a result, an inhibitory activity twice as strong as that of the positive control drug aminoguanidine was recognized. .
この阻害作用は用量依存的であり、４８．３μｇ／μＬではＲＡＧＥ反応性ＡＧＥｓの産生量は０．５０９５であって阻害は約１０％と減弱し、４．８３μｇ／μＬでも同様の結果であった。以上の結果より、黒ニンニクエキスはＡＧＥｓとＲＡＧＥの結合を阻害することを介して、もしくはグリセルアルデヒドがタンパク質中のアミノ酸残基との結合を阻害することを介して、ＡＧＥｓ産生を抑制し、結合を抑制するという機序が考えられた。 This inhibitory effect was dose-dependent. At 48.3 μg / μL, the amount of RAGE-reactive AGEs produced was 0.5095, the inhibition was attenuated to about 10%, and the same result was obtained at 4.83 μg / μL. Was. From the above results, black garlic extract suppresses AGEs production through inhibiting the bond between AGEs and RAGE, or through inhibiting glyceraldehyde from binding to amino acid residues in proteins, A mechanism for suppressing binding was considered.
第２章 黒ニンニクエキスの分画と精製
（１） 実験方法
第１章第１節で述べた黒ニンニクエキスの調製方法にて得られた黒ニンニクエキス５９．２３ｇを精製水２００ｍＬに溶解した。この溶液を分液ロートに入れ、ジエチルエーテル２００ｍＬを加えた後、１５分間振とうし、得られたエーテル溶液を黒ニンニクエーテル層分画（収率：３．９９％）とした。この操作を順番に酢酸エチル、ブタノールでも繰り返し行い、各々得られた分画を黒ニンニク酢酸エチル層分画（収率：１．９０％）、黒ニンニクブタノール層分画（収率：２．３６％）とし、最終的に残った水溶液を黒ニンニク水層分画（収率：９１．７３％）とした。図８に分液ロートによる分画操作後の黒ニンニクエキス分画図を示す。得られた各サンプルは、濃縮し乾燥させた。また、４層に分画したものを各移動相で順次ＨＰＬＣを用いて分離・精製（パックドカラム５Ｃ１８−ＡＲ−II：コスモシール株式会社，Ｍｉｇｈｔｙｓｉｌ（登録商標）ＧＰ２５０−１０：関東化学株式会社，Ｃ３０−ＵＧ：野村化学株式会社，ＴＯＳＯＨ（登録商標）−ＯＤＳ１２０−Ｔ：東ソー株式会社）した。
Chapter 2 Fractionation and Purification of Black Garlic Extract (1) Experimental Method 59.23 g of black garlic extract obtained by the method for preparing black garlic extract described in Chapter 1, Section 1 was dissolved in 200 mL of purified water. This solution was put into a separating funnel, 200 mL of diethyl ether was added, and the mixture was shaken for 15 minutes, and the obtained ether solution was used as a black garlic ether layer fraction (yield: 3.99%). This operation was repeated in sequence with ethyl acetate and butanol, and the obtained fractions were subjected to black garlic ethyl acetate layer fractionation (yield: 1.90%) and black garlic butanol layer fractionation (yield: 2.36). %), And the final remaining aqueous solution was used as a black garlic aqueous layer fraction (yield: 91.73%). FIG. 8 shows a black garlic extract fractionation diagram after the fractionation operation using a separating funnel. Each of the obtained samples was concentrated and dried. Separation and purification of the four-layer fraction using each mobile phase sequentially using HPLC (packed column 5C18-AR-II: Cosmoseal Co., Ltd., Mightysil (registered trademark) GP250-10: Kanto Chemical Co., Ltd.) C30-UG: Nomura Chemical Co., Ltd., TOSOH (registered trademark) -ODS120-T: Tosoh Corporation.
（２）結果・考察
ＡＧＥｓ阻害活性成分の探索を目的として、黒ニンニク原末から図８に示したエーテル層、酢酸エチル層、ブタノール層および水層を分離・精製し、１９３の分画画分を得た。
(2) Results and Discussion In order to search for AGEs-inhibiting active ingredients, the ether layer, ethyl acetate layer, butanol layer and aqueous layer shown in FIG. 8 were separated and purified from the raw powder of black garlic, and 193 fractions were obtained. I got
第３章 黒ニンニク含有成分の同定
（１）実験方法
各種分析方法、すなわち、１Ｈ−ＮＭＲ（プロトン各磁気共鳴）、１３Ｃ−ＮＭＲ（炭素１３各磁気共鳴）、ＥＩ−ＭＳ（電子イオン化質量分析）、ＮＯＥ（オーバーハウザー効果差スペクトル）を用いて、各種サンプルの重さを測定後調製し、計測した（重メタノール、重クロロホルム等を使用した）。
Chapter 3 Identification of Black Garlic-Containing Components (1) Experimental Methods Various analytical methods, namely 1 H-NMR (proton magnetic resonance), 13 C-NMR (carbon 13 magnetic resonance), EI-MS (electron ionization mass) Analysis) and the weight of each sample was measured and measured using NOE (Overhauser effect difference spectrum) and measured (using heavy methanol, heavy chloroform, etc.).
（２）結果・考察
第２章（２）の結果・考察に示す化合物群を得た酢酸エチル層における分画・精製図を図９に示す。今回の分画により得た化合物群について、ＡＧＥｓ産生阻害活性によりスクリーニングを行い、活性が認められた画分を単離・精製し、構造決定を行った。このうち、同定できた化合物は下記の３つであった。
５−ＨＭＦ（５−Ｈｙｄｒｏｘｙｍｅｔｈｙｌ ｆｕｒｆｕｒａｌ） （図１０）
５−ＨＭＦＡ（５−Ｈｙｄｒｏｘｙｍｅｔｈｙ−ｌ−２−ｆｕｒａｎｏｉｃ ａｃｉｄ） （図１１）
Ｎｏｒｍｅｃｏｎｉｎ（６，７−Ｄｉｈｙｄｒｏｘｙ−１（３Ｈ）−ｉｓｏｂｅｎｚｏｆｕｒａｎｏｎｅ） （図１２）
以下、順に構造決定方法について考察した。
(2) Results and consideration
FIG. 9 shows a fractionation / purification diagram in the ethyl acetate layer from which the compounds shown in the results and discussion in Chapter 2 (2) were obtained. The compounds obtained by this fractionation were screened for their AGEs production inhibitory activity, and fractions showing the activity were isolated and purified, and the structure was determined. Among these, the following three compounds could be identified.
5-HMF (5-Hydroxymethyl furfural) (FIG. 10)
5-HMFA (5-hydroxymethy1-2-furanoic acid) (FIG. 11)
Normeconin ( 6,7-Dihydroxy-1 (3H) -isobenzofuranone ) (FIG. 12)
Hereinafter, the structure determination method was considered in order.
初めに、１Ｈ−ＮＭＲデータを参考に比較することで、化学構造の基本構造について推定した。図１３は、５−ＨＭＦに関して１Ｈ−ＮＭＲから得られたスペクトルを示すグラフである（２７０．０５ＭＨｚ，ＣＤ３ＯＤ）。図示するように、９．５２ｐｐｍからアルデヒド基を同定、δ７．３７３から７．３６０ｐｐｍ、およびδ６．５７５から６．５６１ｐｐｍにかけて見られる１Ｈ分のｓｉｎｇｌｅｔシグナルが２つ観測されていることからフラン環を同定、４．５９８ｐｐｍからメチレン基を同定し、５−ＨＭＦに代表的な構造が同定された。これらの結果とマススペクトルによる分子量等の結果とを合わせて、下記２文献９）、１０）における文献値を比較し、５−ＨＭＦであると決定した。
文献：
9) Liang T., Wei F., Lu Y., Kodani Y., Nakada M., Miyakawa T., Tanokura M., J. Agric. Food. Chem., 63, 683-691 (2015).
10) Matsui T., Kudo A., Tokuda S., Matsumoto K., Hosoyama H., J. Agric. Food. Chem. 58, 10876-10879 (2010).
First , the basic structure of the chemical structure was estimated by comparing with reference to 1 H-NMR data. FIG. 13 is a graph showing a spectrum of 5-HMF obtained from 1 H-NMR (270.05 MHz, CD3OD). As shown in the figure, the aldehyde group was identified from 9.52 ppm, and the furan ring was identified from two singlet signals for 1H observed from δ7.373 to 7.360 ppm and δ6.575 to 6.561 ppm. Identification A methylene group was identified from 4.598 ppm, and a typical structure of 5-HMF was identified. By combining these results with the results of the molecular weight and the like based on the mass spectrum, the literature values in the following two literatures 9) and 10) were compared and determined to be 5-HMF.
Literature:
9) Liang T., Wei F., Lu Y., Kodani Y., Nakada M., Miyakawa T., Tanokura M., J. Agric. Food. Chem., 63, 683-691 (2015).
10) Matsui T., Kudo A., Tokuda S., Matsumoto K., Hosoyama H., J. Agric. Food. Chem. 58, 10876-10879 (2010).
次に、図１４は、５−ＨＭＦＡ（５−Ｈｙｄｒｏｘｙｍｅｔｈｙｌ−２−ｆｕｒａｎｏｉｃ ａｃｉｄ）に関して１Ｈ−ＮＭＲから得られたスペクトルを示すグラフである（３９９．６５ＭＨｚ，ＣＤ３ＯＤ）。図示する構造と下記文献１１）の文献値を比較し、これが５−ＨＭＦに類似の構造であり、図１３に示したグラフと比較して９．５２ｐｐｍのアルデヒド基特有のピークがなくなったことから、５−ＨＭＦが酸化されたことにより生成した５−ＨＭＦＡ（５−Ｈｙｄｒｏｘｙｍｅｔｈｙｌ−２−ｆｕｒａｎｏｉｃ ａｃｉｄ）であると決定した。
文献：
11) Murai N., Yonaga M., Tanaka K., Org. Lett., 14, 1278-1281 (2014).
Next, FIG. 14 is a graph showing a spectrum obtained from 1 H-NMR for 5-HMFA (5-hydroxymethyl-2-furanoic acid) (399.65 MHz, CD3OD). The structure shown in the figure and the literature value of the following literature 11) were compared, and this was a structure similar to 5-HMF, and the peak specific to the aldehyde group at 9.52 ppm disappeared as compared with the graph shown in FIG. , 5-HMF (5-Hydroxymethyl-2-furanoic acid) generated by oxidation of 5-HMF.
Literature:
11) Murai N., Yonaga M., Tanaka K., Org. Lett., 14, 1278-1281 (2014).
最後に、図１５は、ノルメコニン（６，７−Ｄｉｈｙｄｒｏｘｙ−１（３Ｈ）−ｉｓｏｂｅｎｚｏｆｕｒａｎｏｎｅ）に関して１３Ｃ−ＮＭＲから得られたスペクトルを示すグラフである（６７．８０ＭＨｚ，ＣＤ３ＯＤ）。図示するように、ノルメコニンについてはＭＳスペクトルを用いて解析を行なった結果、ピークがｍ／ｚ１６６に観測された。このことから、推定した化合物の分子量は１６６であり，ＭＳスペクトルの結果と合致した。
Finally, FIG. 15 is a graph showing the spectrum obtained from 13 C-NMR for normeconin ( 6,7-Dihydroxy-1 (3H) -isobenzofuranone ) (67.80 MHz, CD3OD). As shown in the figure, the analysis of normeconin using the MS spectrum showed that a peak was observed at m / z 166. From this, the estimated molecular weight of the compound was 166, which was consistent with the result of the MS spectrum.
また、図１６は、ノルメコニンに関してＥＩ−ＭＳによる解析結果を示すグラフである（Ｌｏｗ，ＭｅＯＨ）。図示するように、炭素数は８個であり、１７２．９ｐｐｍのピークからエステル基、また、７０．７ｐｐｍのピークからメチレン基の構造があることがわかり、フタリド骨格を有する化合物であることが推定された。 FIG. 16 is a graph showing the analysis results of normeconin by EI-MS (Low, MeOH). As shown in the figure, the carbon number is 8, and the peak at 172.9 ppm indicates the structure of an ester group, and the peak at 70.7 ppm indicates the structure of a methylene group, indicating that the compound has a phthalide skeleton. Was done.
次に、図１７は、ノルメコニンに関して１Ｈ−ＮＭＲから得られたスペクトルを示すグラフである（２７０．０５ＭＨｚ，ＣＤ３ＯＤ）。図示するように、芳香族領域に認められるδ７．１４２から７．１１３、δ６．８３７から６．８０８、の２Ｈ分のｄｏｕｂｌｅｔシグナルが８Ｈｚでカップリングしたことから、これらがオルト位の関係にあることがわかった。 Next, FIG. 17 is a graph showing a spectrum of normeconin obtained from 1 H-NMR (270.05 MHz, CD3OD). As shown in the figure, the double signals of 2H of δ7.142 to 7.113 and δ6.837 to 6.808 observed in the aromatic region were coupled at 8 Hz, and these signals are in the ortho position. I understand.
最後に、図１８は、ノルメコニンに関してＮＯＥ−１Ｈ−ＮＭＲから得られたスペクトルを示すグラフである（２７０．０５ＭＨｚ，ＣＤ３ＯＤ）。図示するように、５．２ｐｐｍ付近に照射して測定したところ、６位にプロトンが付くことがわかった。以上より、下記各文献１２）、１３）の文献値と比較して、ノルメコニン（６，７−Ｄｉｈｙｄｒｏｘｙ−１（３Ｈ）−ｉｓｏｂｅｎｚｏｆｕｒａｎｏｎｅ）であると決定した。
文献：
12) Andrew M., Norma T., Mark F., Scott C., David M., J. Am. Chem. Soc., 119, 6084-6094 (1997).
13) Thomas P., Olof T . Acta. Chem. Scand., B30, 397-402 (1976)
Finally, Figure 18 is a graph showing a spectrum obtained from NOE- 1 H-NMR with regard Norumekonin (270.05MHz, CD3OD). As shown in the figure, measurement was performed by irradiating around 5.2 ppm, and it was found that a proton was attached to the 6-position. From the above, it was determined that it was normeconin ( 6,7-Dihydroxy-1 (3H) -isobenzofuranone ) as compared with the literature values of the following literatures 12) and 13).
Literature:
12) Andrew M., Norma T., Mark F., Scott C., David M., J. Am. Chem. Soc., 119, 6084-6094 (1997).
13) Thomas P., Olof T. Acta. Chem. Scand., B30, 397-402 (1976)
第４章 黒ニンニク含有成分のＲＡＧＥ反応性ＡＧＥｓ阻害活性の検討
（１）実験方法
同定された黒ニンニク含有成分について、第１章第３節（ＥＬＩＳＡ法によるＲＡＧＥ反応性ＡＧＥｓ阻害活性の検討）における「（ｉｖ）ＲＡＧＥ反応性ＡＧＥｓの測定」に示した方法の通りに検討した。
Chapter 4 Investigation of RAGE-Reactive AGEs Inhibitory Activity of Black Garlic-Containing Components (1) Experimental Method For the identified black garlic-containing components, see Chapter 1, Section 3 (Study of RAGE-reactive AGEs inhibitory activity by ELISA). Investigation was performed according to the method shown in “(iv) Measurement of RAGE-reactive AGEs”.
（２）結果・考察
ノルメコニンのＲＡＧＥ反応性ＡＧＥｓ阻害活性に対する阻害作用について検討した。図１９は、ノルメコニンのＲＡＧＥ反応性ＡＧＥｓ阻害活性に対する阻害作用を示すグラフである（＊Ｐ＝０．０４３）。同一濃度にて陽性対照薬の１０ｍＭアミノグアニジンとノルメコニンとを比較した。その結果、図示するように対照群での吸光度は０．３９であった。それに対して陽性対照薬１０ｍＭアミノグアニジンでは０．３１となり、対照群よりも強い阻害作用、対照群と比較して２０％の阻害作用が確認された。
(2) Results and Discussion The inhibitory effect of normeconin on RAGE-reactive AGEs inhibitory activity was examined. FIG. 19 is a graph showing the inhibitory effect of normeconin on RAGE-reactive AGEs inhibitory activity ( * P = 0.043). At the same concentration, 10 mM aminoguanidine as a positive control drug and normeconin were compared. As a result, as shown in the figure, the absorbance in the control group was 0.39. On the other hand, the positive control drug, 10 mM aminoguanidine, was 0.31, indicating a stronger inhibitory effect than the control group and a 20% inhibitory effect as compared with the control group.
一方、１０ｍＭ ノルメコニンでは０．２７であり、陽性対照薬の１０ｍＭアミノグアニジンよりも強い阻害作用、対照群と比較して３０％の阻害作用が認められた。すなわちノルメコニンの阻害作用は、１０ｍＭアミノグアニジンの１．５倍であった。以上より、黒ニンニクエキスがＲＡＧＥ反応性ＡＧＥｓ阻害活性を示す活性本体のうちの一つが、ノルメコニンであることが明らかとなった。 On the other hand, the value was 0.27 for 10 mM normeconin, indicating a stronger inhibitory effect than 10 mM aminoguanidine as a positive control drug, and a 30% inhibitory effect as compared with the control group. That is, the inhibitory effect of normeconin was 1.5 times that of 10 mM aminoguanidine. From the above, it was clarified that one of the active bodies of the black garlic extract showing the RAGE-reactive AGEs inhibitory activity is normeconin.
ノルメコニンのＲＡＧＥ反応性ＡＧＥｓ阻害活性を示した機序について考察する。本化合物は、フタリド骨格により非常に安定な構造であることに加え、分子内に窒素原子を含んでいないことから、グリセルアルデヒドとの直接的な求核反応等によりクロスリンカー的役割を果たし、それによって活性を示す、という機序は考えにくい。しかし、カテコール骨格を部分構造に含んでいることから、一般的にフェノール類よりも酵素やたんぱく質との吸着、結合性が強い傾向にある。このため、ＲＡＧＥのリガンド結合部位やサブユニット中の疎水性ポケット等と相互作用することで、受容体のコンフォメーション変化を起こし、そのことがＡＧＥｓとの結合を阻害する機序となっている可能性がある。また、タンパク質中のアミノ酸残基とノルメコニンの構造に含まれるヒドロキシ基や酸素原子との相互作用により、グリセルアルデヒドとの結合が円滑に進行しない環境を作り出すことで、反応を抑制するという可能性も考えられる。 The mechanism by which normeconin exhibited RAGE-reactive AGEs inhibitory activity will be discussed. This compound has a very stable structure due to the phthalide skeleton and, since it does not contain a nitrogen atom in the molecule, plays a crosslinker role by direct nucleophilic reaction with glyceraldehyde, etc. It is difficult to imagine the mechanism of activity. However, since it contains a catechol skeleton in its partial structure, it generally tends to have stronger adsorption and binding to enzymes and proteins than phenols. Therefore, by interacting with the ligand binding site of RAGE and the hydrophobic pocket in the subunit, the conformational change of the receptor is caused, which may be the mechanism that inhibits the binding to AGEs. There is. In addition, the interaction between amino acid residues in proteins and hydroxy groups and oxygen atoms contained in the structure of normeconin may create an environment in which the bond with glyceraldehyde does not proceed smoothly, thereby suppressing the reaction. Is also conceivable.
第５章 総括
蛍光強度法によるＡＧＥｓ産生阻害活性、および３−ＤＧの定量の結果から、黒ニンニクエキスは、３−ＤＧ産生を用量依存的に産生阻害することでＡＧＥｓ産生を抑制すると考えられた。加えて、ＥＬＩＳＡ法によるＲＡＧＥ反応性ＡＧＥｓ阻害活性の結果より、黒ニンニクエキスは用量依存的な阻害作用を示し、陽性対象薬のアミノグアニジンよりも強い阻害作用を示したことから、ＲＡＧＥとの結合を阻害できた、もしくはＡＧＥｓとグリセルアルデヒドのアミノ酸残基との結合を阻害した可能性もある。
Chapter 5 Summary From the results of the AGEs production inhibitory activity by the fluorescence intensity method and the results of quantification of 3-DG, it was considered that black garlic extract suppresses AGEs production by inhibiting the production of 3-DG in a dose-dependent manner. . In addition, from the results of the RAGE-reactive AGEs inhibitory activity by the ELISA method, black garlic extract showed a dose-dependent inhibitory effect, and showed a stronger inhibitory effect than the positive target drug, aminoguanidine. May have been inhibited, or the binding of AGEs to the amino acid residue of glyceraldehyde may have been inhibited.
また、黒ニンニク含有成分の分画・精製を行い、構造決定に至った３つの化合物のうち、ノルメコニンにおいてＲＡＧＥ反応性ＡＧＥｓ阻害活性が認められた。このことから、本化合物は黒ニンニクエキスに含まれるＲＡＧＥ反応性ＡＧＥｓ阻害活性を示す活性本体の１つであることが分かった。その他、５−ＨＭＦについては、ＰＩ３−Ａｃｔ系（ＰＩ３Ｋ ／ Ａｋｔ Ｓｉｇｎａｌｉｎｇ、ＰＩ３Ｋ ／ Ａｋｔ シグナル伝達系）の活性化によるｅＮＯＳ（内皮型一酸化窒素合成酵素）の産生に続くＮＯ（一酸化窒素）産生に起因する血管拡張作用も報告されている（下記文献１４））が、これは、黒ニンニクに期待される冷え、肩こり、疲労回復効果を示す活性物質の一つであると考えられる。
文献：
14) O'Donnell G., Poeschl R., Zimhony O., Gunaratnam M., Moreira JB., Neidle S., Evangelopoulos D., Bhakta S., Malkinson JP., Boshoff HI., Lenaerts A., Gibbons S., J . Nat. Prod., 72, 360-365 (2009).
In addition, among the three compounds that were subjected to fractionation and purification of the black garlic-containing component and the structure was determined, normeconin exhibited RAGE-reactive AGEs inhibitory activity. From this result, it was found that the present compound is one of the active components that exhibit RAGE-reactive AGEs inhibitory activity contained in black garlic extract. In addition, regarding 5-HMF, production of eNOS (endothelial nitric oxide synthase) following activation of a PI3-Act system (PI3K / Akt Signaling, PI3K / Akt signaling system) followed by NO (nitric oxide) production A vasodilator effect due to garlic has also been reported (Reference 14 below)), which is considered to be one of the active substances that exhibit the effects of cooling, shoulder stiffness and fatigue recovery expected from black garlic.
Literature:
14) O'Donnell G., Poeschl R., Zimhony O., Gunaratnam M., Moreira JB., Neidle S., Evangelopoulos D., Bhakta S., Malkinson JP., Boshoff HI., Lenaerts A., Gibbons S. ., J. Nat. Prod., 72, 360-365 (2009).
第２部 黒ニンニクに含まれるＧＡＢＡおよび関連成分について
第６章 黒ニンニク抽出エキスおよびその含有成分のＧＡＢＡ代謝酵素阻害作用
第１節 黒ニンニク抽出エキスおよびその含有成分のＧＡＢＡ代謝酵素阻害作用
（１） 研究の目的
ＧＡＢＡを豊富に含むとされる機能性食品である黒ニンニクの、ＧＡＢＡ代謝酵素系の酵素であるＧＡＢＡ−ＴおよびＳＳＡＤＨの混合酵素ＧＡＢａｓｅに対する阻害作用について検討した。
Part 2 About GABA and related components contained in black garlic
Chapter 6 Inhibitory Effect of Black Garlic Extract and Its Constituents on GABA-Metabolizing Enzyme Section 1 Inhibitory Effect of Black Garlic Extract and Its Constituents on GABA-metabolizing Enzyme (1) Purpose of Research Functionality that is considered to be rich in GABA The inhibitory effect of black garlic, a food, on the mixed enzyme GABase of GABA-T and SSADH, which are GABA-metabolizing enzyme enzymes, was examined.
（２） 実験方法
青森県産黒ニンニク２ｋｇを粉砕し、アセトン：水（４：１）で加温抽出を行った。この操作を２回繰り返し、減圧濃縮し、黒ニンニク抽出エキスとした。その収量は４００．５５ｇであった。抽出エキスは水：ジエチルエーテル（２：１）に溶解後、イオン交換樹脂（ダイヤイオン（商標）ＨＰ２０（三菱ケミカル）に水層を吸着させた。その後、水：ＭｅＯＨ（０：１００〜１００：０）で段階的に溶出し、表１に示す各画分（１Ａ〜１Ｆ）を得た。
(2) Experimental method 2 kg of black garlic produced in Aomori prefecture was pulverized and subjected to warm extraction with acetone: water (4: 1). This operation was repeated twice and concentrated under reduced pressure to obtain a black garlic extract. The yield was 400.55 g. After the extract was dissolved in water: diethyl ether (2: 1), the aqueous layer was adsorbed on an ion exchange resin (Diaion (trademark) HP20 (Mitsubishi Chemical). Then, water: MeOH (0: 100 to 100: The fractions were eluted stepwise in 0) to obtain the fractions (1A to 1F) shown in Table 1.
（３）ＧＡＢＡｓｅ阻害活性成分の特定
得られた画分は、（４）に後述するＧＡＢＡｓｅ阻害活性測定法によりその阻害活性を比較した結果、１Ｃ画分に最も強い阻害を認めた。このため、以下の条件でその活性成分の検索を行った。その結果、その活性成分として化合物Ｘを得た。
(3) Identification of GABAase inhibitory activity component The obtained fractions were compared for their inhibitory activity by the GABAase inhibitory activity measurement method described later in (4). As a result, the 1C fraction showed the strongest inhibition. Therefore, the active ingredient was searched under the following conditions. As a result, compound X was obtained as the active ingredient.
図２２は、化合物Ｘに関して１Ｈ−ＮＭＲから得られたスペクトルを示すグラフである。また、
図２３は化合物Ｘに関して１３Ｃ−ＮＭＲから得られたスペクトルを示すグラフ、図２４は化合物Ｘに関してＥＩ−ＭＳによる解析結果を示すグラフ、図２５は化合物Ｘに関してに関してＮＯＥ−１Ｈ−ＮＭＲから得られたスペクトルを示すグラフである。本化合物Ｘは、各図に示した各種スペクトル分析値を文献値１５）、１６）と比較した結果、ノルメコニン（Ｎｏｒｍｅｃｏｎｉｎ ６，７−Ｄｉｈｙｄｒｏｘｙ−１（３Ｈ）−ｉｓｏｂｅｎｚｏｆｕｒａｎｏｎｅ）と同定された。また、（５）に後述するＨＰＬＣによる測定の結果、黒ニンニク原末中にノルメコニンは０．００８％含有されている結果が得られた。
文献：
15) Andrew M., Norma T., Mark F., Scott C., David M., J. Am. Chem. Soc., 119, 6084-6094 (1997).
16) Thomas P., Olof T., Acta. Chem. Scand., B30, 397-402 (1976).
FIG. 22 is a graph showing a spectrum of Compound X obtained from 1 H-NMR. Also,
Figure 23 is derived from NOE- 1 H-NMR with respect to 13 graph illustrating a spectrum obtained from C-NMR, the graph Figure 24 showing the analysis result by EI-MS for compound X, FIG. 25 for compounds X for compounds X 9 is a graph showing the obtained spectrum. This compound X was identified as normeconin ( Normeconin 6,7-Dihydroxy-1 (3H) -isobenzofuranone ) as a result of comparing the various spectral analysis values shown in each figure with literature values 15) and 16). Further, as a result of the measurement by HPLC described later in (5), a result was obtained in which the raw powder of black garlic contained normeconin in 0.008%.
Literature:
15) Andrew M., Norma T., Mark F., Scott C., David M., J. Am. Chem. Soc., 119, 6084-6094 (1997).
16) Thomas P., Olof T., Acta. Chem. Scand., B30, 397-402 (1976).
（４）ＧＡＢＡｓｅ阻害活性の検討方法（文献１７））
得られたノルメコニンのＧＡＢＡｓｅ阻害活性検討は次のように行った。
ノルメコニンを、１．４ｍＭ ＮＡＤＰ＋（和光純薬工業）、７５０ｍＭ Ｎａ２ＳＯ４、２．０ｍＭ α−ｋｅｔｏｇｌｕｔａｒｉｃ ａｃｉｄ（和光純薬工業）を含む８０ｍＭトリス塩酸緩衝液（ｐＨ９．０）に溶解したものを反応液とした。反応液６０μＬ／ｗｅｌｌ、被験画分の１０％メタノール溶液１０μＬ／ｗｅｌｌ、ＷＳＴ−８（テトラゾリウム塩：ナカライテスク）１０μＬ／ｗｅｌｌ、ＧＡＢａｓｅ（４−Ａｍｉｎｏｂｕｔｙｒｉｃ Ａｃｉｄ：東京化成工業）０．０１Ｕ／１０μＬ（ｗｅｌｌ）、ＧＡＢＡ（４−Ａｍｉｎｏｂｕｔｙｒｉｃ Ａｃｉｄ：東京化成工業）溶液（５００μＭ、２００μＭ、１００μＭ、６０μＭ、５０μＭ）１０μＬ／ｗｅｌｌを順に混合し、全量１００μＬ／ｗｅｌｌとした。
(4) Method for examining GABAase inhibitory activity (Reference 17))
The GABAase inhibitory activity of the obtained normeconin was examined as follows.
Normeconin dissolved in 80 mM Tris-HCl buffer (pH 9.0) containing 1.4 mM NADP + (Wako Pure Chemical Industries), 750 mM Na 2 SO 4 , and 2.0 mM α-ketoglutaric acid (Wako Pure Chemical Industries) Was used as a reaction solution. Reaction solution 60 μL / well, 10% methanol solution of test fraction 10 μL / well, WST-8 (tetrazolium salt: Nacalai Tesque) 10 μL / well, GABase (4-Aminobutylic Acid: Tokyo Chemical Industry) 0.01 U / 10 μL (well) ), 10 μL / well of GABA (4-Aminobutyric Acid: Tokyo Chemical Industry) solution (500 μM, 200 μM, 100 μM, 60 μM, 50 μM) were mixed in order to make a total volume of 100 μL / well.
その後、３７℃でインキュベートしながら、１０分間隔で吸光度を測定（測定波長：４５０ｎｍ）した。なお、対照にはエタノール１μＬ／ｗｅｌｌ、トリス塩酸緩衝液（ｐＨ９．０）９μＬ／ｗｅｌｌを添加した。その後、Ｅｎｚｙｍｅ Ｋｉｎｅｔｉｃｓ Ｍｏｄｕｌｅ１．１（Ｖｅｒ．６．１，ＳＰＳＳ Ｉｎｃ．，２００２）を用いて反応速度の解析を行った。
文献：
17) Tsukatani T, Higuchi T, Matsumoto K., Analytica Chimica Acta, 540, 293-297(2005).
Thereafter, the absorbance was measured at 10 minute intervals (measurement wavelength: 450 nm) while incubating at 37 ° C. As a control, ethanol 1 μL / well and Tris-HCl buffer (pH 9.0) 9 μL / well were added. Thereafter, the reaction rate was analyzed using Enzyme Kinetics Module 1.1 (Ver. 6.1, SPSS Inc., 2002).
Literature:
17) Tsukatani T, Higuchi T, Matsumoto K., Analytica Chimica Acta, 540, 293-297 (2005).
（５）ノルメコニンの精製方法
ノルメコニンは、高速液体クロマトグラフィー（ＨＰＬＣ）を繰り返すことで精製した。その装置条件は、カラム；ＵＬＴＲＡ ＰＡＫ ＯＤＳ−ＳＭ−５０Ｃ−Ｍ（山善株式会社）、Ｃａｐｃｅｌ Ｐａｋ Ｃ８（資生堂）、ＴＯＳＯＨ ＴＳＫｇｅｌ（登録商標） １２０Ｔ−ＯＤＳ（東ソー株式会社）、Ｃｏｓｍｏｓｉｌ（登録商標）ＡＲ−ＩＩ（ナカライテスク株式会社）。移動相；１０％ＣＨ３ＣＮ＋ＴＦＡ 、２０％ＭｅＯＨ、２０％ＣＨ３ＣＮ。測定波長（検出波長）；２１０ｎｍである。
(5) Normeconin Purification Method Normeconin was purified by repeating high performance liquid chromatography (HPLC). The apparatus conditions are as follows: Column: ULTRA PAK ODS-SM-50CM (Yamazen Corporation), Capcel Pak C8 (Shiseido), TOSOH TSKgel (registered trademark) 120T-ODS (Tosoh Corporation), Cosmosil (registered trademark) AR -II (Nacalai Tesque, Inc.). Mobile phase; 10% CH 3 CN + TFA , 20% MeOH, 20% CH 3 CN. Measurement wavelength (detection wavelength): 210 nm.
（６）実験結果
ノルメコニンのＧＡＢＡｓｅ阻害活性を測定した結果、濃度依存性のＧＡＢＡｓｅ阻害活性が認められた。図２６は、測定されたノルメコニンの阻害様式の速度論的な解析結果を示すグラフである。解析結果から、ノルメコニンのＧＡＢＡｓｅ阻害様式は非競合性であり、そのパラメータ値（μＭ）はそれぞれＶｍａｘ：８０．２，Ｋｍ：１６３．７，Ｋｉ：１７０．７であった。ＧＡＢＡｓｅ阻害薬として知られているバルプロ酸は、本来ＳＳＡＤＨに対し競合的阻害を示し、ＧＡＢＡ−Ｔに対してはアロステリックな阻害を示すことが報告されている（文献１８））。
(6) Experimental Results As a result of measuring the GABAse inhibitory activity of normeconin, a concentration-dependent GABAase inhibitory activity was observed. FIG. 26 is a graph showing the results of kinetic analysis of the measured inhibition pattern of normeconin. According to the analysis results, the GABAase inhibition mode of normeconin was non-competitive, and the parameter values (μM) were Vmax: 80.2, Km: 163.7, and Ki: 170.7, respectively. It has been reported that valproic acid, which is known as a GABAase inhibitor, originally shows competitive inhibition on SSADH and shows allosteric inhibition on GABA-T (Reference 18).
本研究結果より、ノルメコニンのＧＡＢＡｓｅ阻害様式も同様にＧＡＢＡ−Ｔへの影響が大きいため、アロステリックな阻害様式が強くモニタリングされ、非競合的阻害が確認できたと考えられる。
18） Piplani S, Verma PK, Kumar A., Biomed Pharmacother., 81, 402-410, (2016).
From the results of this study, it is considered that the GABAase inhibition mode of normeconin also has a large effect on GABA-T, so that the allosteric inhibition mode was strongly monitored and non-competitive inhibition could be confirmed.
18) Piplani S, Verma PK, Kumar A., Biomed Pharmacother., 81, 402-410, (2016).
（７）結論
ＧＡＢＡを豊富に含有し、なおかつＧＡＢａｓｅ阻害作用を示すノルメコニンも有する黒ニンニクを摂取することは、ＧＡＢＡを効率的に摂取できることに繋がり、したがって多くの疾患の予防に寄与することが期待される。
＊＊＊＊＊＊＊＊＊＊＊＊
(7) Conclusion Ingestion of black garlic that is rich in GABA and also has normeconin that exhibits GABase inhibitory action leads to efficient intake of GABA, and is therefore expected to contribute to the prevention of many diseases. Is done.
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本発明のＧＡＢＡ分解酵素阻害用組成物によれば、ＧＡＢＡを豊富に含むネギ属植物を原料として、生体内におけるＧＡＢＡの有効利用率向上を可能ならしめる成分であるところのノルメコニンを得ることができ、またそれを含む組成物や食品を得ることができ、生体内におけるＧＡＢＡの有効利用率を向上させることができる。 According to the composition for inhibiting a GABA-degrading enzyme of the present invention, it is possible to obtain normeconin, which is a component capable of improving the effective utilization rate of GABA in a living body, from a raw material of the genus Allium rich in GABA. In addition, it is possible to obtain a composition or food containing the same, and it is possible to improve the effective utilization rate of GABA in a living body.
また、ノルメコニンがかかるＧＡＢＡ分解酵素阻害作用を有することは本発明において初めて明らかにされた。先に出願人による別出願にて開示されたＡＧＥｓ等阻害用（本願出願時未公開）とも相俟って、ノルメコニンの利用価値が新たに開拓された。 The present invention has also revealed for the first time that normeconin has such a GABA-degrading enzyme inhibitory action. The utility value of normeconin has been newly pioneered in conjunction with the inhibition of AGEs and the like (not disclosed at the time of filing of this application) previously disclosed in a separate application filed by the applicant.
本願発明では特に、黒ニンニクにおける生体内摂取ＧＡＢＡ利用率向上効果、カルボニルストレスの抑制効果とそれによるＡＧＥｓ関連疾患の予防・治療に効果が大いに期待できることが示された。したがって本発明は、機能性の高い素材としての黒ニンニクの付加価値向上、普及拡大に資することができるものでもある。また以上の効果は、広くニンニクなどのネギ属植物を原料とする組成物等に敷衍できると考えられ、ネギ属植物の付加価値向上、普及拡大に資することも期待される。したがって本発明は、農産加工分野、食品製造分野、健康産業分野、その他関連する全分野において、産業上利用性が高い発明である。 In particular, the present invention has shown that black garlic is expected to be highly effective in improving the in vivo GABA utilization rate, inhibiting carbonyl stress, and thereby preventing and treating AGEs-related diseases. Therefore, the present invention can contribute to the enhancement of added value and the spread of black garlic as a highly functional material. Further, it is considered that the above effects can be broadly applied to compositions and the like using leeks such as garlic as a raw material, and are expected to contribute to the enhancement of added value and the spread of leeks. Therefore, the present invention has high industrial applicability in the agricultural processing field, the food manufacturing field, the health industry field, and all other related fields.
１…ノルメコニン
２、５…表示部
３…ＧＡＢＡ分解酵素阻害用組成物
４…ＧＡＢＡ
６…ＧＡＢＡ摂取用組成物
１１…原料ネギ属植物
１１０ａ、１１４ａ…ＧＡＢＡ分解酵素阻害用組成物
１１０ｂ、１１４ｂ…ＧＡＢＡ摂取用組成物
１１０ｃ、１１４ｃ…ＧＡＢＡ摂取用食品
１１０ｘ、１１４ｘ…ＧＡＢＡ摂取用組成物等
２２０…ＡＧＥｓ等阻害用兼ＧＡＢＡ摂取用ネギ属植物製組成物
２２５…表示部
DESCRIPTION OF SYMBOLS 1 ... Normeconin 2, 5 ... Display part 3 ... Composition for inhibiting GABAase 4 ... GABA
6 ... composition for GABA ingestion 11 ... raw material Allium plants 110a, 114a ... composition for GABA degrading enzyme inhibition 110b, 114b ... composition for GABA ingestion 110c, 114c ... food for GABA ingestion 110x, 114x ... composition for GABA ingestion Etc. 220 ... Composition made of Allium plant for inhibiting AGEs etc. and ingesting GABA 225 ... Display
Ｆ１…ＡＧＥｓ産生阻害作用
Ｆ２…ＡＧＥｓ中間体産生阻害作用
Ｆ３…ＡＧＥ−ＲＡＧＥ接着阻害作用
Ｆ４…ＡＧＥｓ等阻害作用
Ｆ５…ＧＡＢＡ分解酵素阻害作用
Ｐ０…発酵処理
Ｐ１…加熱処理
Ｐ２…メイラード反応処理
Ｐ３…濃縮処理過程
Ｐ４…乾燥処理過程
F1 AGEs production inhibitory action F2 AGEs intermediate production inhibitory action F3 AGE-RAGE adhesion inhibitory action F4 AGEs etc. inhibitory action F5 GABA degrading enzyme inhibitory action P0 Fermentation treatment P1 Heat treatment P2 Maillard reaction treatment P3 Concentration process P4 ... Dry process
Claims (5)
＜Ｆ＞ 固体状のエキス、液体状のエキス、食品 The composition for inhibiting a GABA-degrading enzyme according to claim 1, wherein the composition is any one of the following <F>, which is labeled "inhibits GABA-degrading enzyme".
<F> Solid extract, liquid extract, food
＜Ｆ＞ 固体状のエキス、液体状のエキス、食品
`` Black '', `` amber '', `` fermented '', `` aged '', or other fermentation treatment, heat treatment, Maillard reaction treatment at least one of the treated black garlic is attached to indicate that it is derived from, The composition for inhibiting a GABA-degrading enzyme according to claim 4, wherein the composition is any one of the following <F>.
<F> Solid extract, liquid extract, food
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| JP4907477B2 (en) * | 2007-09-14 | 2012-03-28 | 株式会社スパン・ライフ | Black garlic manufacturing method and food containing black garlic |
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| JP6845537B2 (en) * | 2017-09-05 | 2021-03-17 | 株式会社スパン・ライフ | Inhibitive black garlic extract such as AGEs, its production method, and food and drink |
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