JP2021001132A - Blood vessel aging inhibitory composition - Google Patents
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Abstract
Description
本開示は、血管老化抑制用組成物等に関する。詳細には、キサントフモールを含有する血管老化抑制用組成物等に関する。 The present disclosure relates to a composition for suppressing vascular aging and the like. More specifically, the present invention relates to a composition for suppressing vascular aging containing xanthohumol and the like.
血管内皮は血管の最内層に存在しており、血液成分などの血管透過性、血管の収縮や弛緩反応、凝固線溶系に関与することによる血液凝固性の調節、血小板機能の制御等、様々な機能を有している。これらの機能は、血管内皮細胞が様々な生理活性因子を産生することと深く関与している。 The vascular endothelium exists in the innermost layer of blood vessels, and has various functions such as vascular permeability of blood components, contraction and relaxation reactions of blood vessels, regulation of blood coagulation by participating in the coagulation / fibrinolytic system, and control of platelet function. It has a function. These functions are deeply involved in the production of various bioactive factors by vascular endothelial cells.
サーチュインは、カロリー制限による細胞老化の抑制、寿命の延長などに関与することが知られており、サーチュインファミリーの中でも最も研究の進んでいるＳｉｒｔ１は、細胞死抑制、ストレス耐性、代謝調節、抗炎症、がんや細胞分化、細胞移動、うつ病、神経変性疾患、糖尿病など、多岐にわたる生命現象に関与していることが知られている。 Sirtuins are known to be involved in suppressing cell senescence and prolonging lifespan by calorie restriction, and the most studied Sirtuin in the sirtuin family is cell death suppression, stress tolerance, metabolic regulation, and anti-inflammatory. , Cancer, cell differentiation, cell migration, depression, neurodegenerative diseases, diabetes, etc., are known to be involved in a wide range of life phenomena.
血管内皮細胞において、Ｓｉｒｔ１を阻害すると細胞老化が誘導され、過剰発現すると酸化ストレスによる細胞老化誘導を回避できることが知られている（非特許文献１）。また、Ｓｉｒｔ1は、血管内皮型一酸化窒素産生酵素（ｅＮＯＳ）と直接結合し、その活性を上昇させ、ＮＯ産生の増加をもたらし、血管を拡張することも知られている。 In vascular endothelial cells, it is known that inhibition of Sirtu1 induces cellular senescence, and overexpression can avoid induction of cellular senescence due to oxidative stress (Non-Patent Document 1). It is also known that Sirtuin directly binds to vascular endothelial nitric oxide-producing enzyme (eNOS), increases its activity, increases NO production, and dilates blood vessels.
また、Ｓｉｒｔ６も細胞老化において重要な役割を担っており、ヒト由来の若い細胞のＳｉｒｔ６の発現を抑制することにより細胞老化が誘導されることが報告されている（非特許文献２）。 In addition, Sirt6 also plays an important role in cell senescence, and it has been reported that cell senescence is induced by suppressing the expression of Sirt6 in young human-derived cells (Non-Patent Document 2).
新規な血管老化抑制用組成物を提供することを目的とする。 An object of the present invention is to provide a novel composition for suppressing vascular aging.
本発明者らは、キサントフモールがサーチュインの発現を亢進する作用を有することを見出し、さらに改良を重ねた。 The present inventors have found that xanthohumol has an action of enhancing the expression of sirtuin, and further improved it.
本開示は、例えば以下の項に記載の主題を包含する。
項１．
キサントフモールを含有する、血管老化抑制用組成物。
項２．
キサントフモールを含有する、サーチュイン活性化用組成物。
項３．
前記サーチュインが、Ｓｉｒｔ１、Ｓｉｒｔ６、及びＳｉｒｔ３からなる群より選択される１種以上である、項２に記載の組成物。
項４．
経口組成物である、項１〜３のいずれか１項に記載の組成物。
項５．
食品組成物又は医薬組成物である、項１〜４のいずれか１項に記載の組成物。
The present disclosure includes, for example, the subjects described in the following sections.
Item 1.
A composition for suppressing vascular aging containing xanthohumol.
Item 2.
A sirtuin activation composition containing xanthohumol.
Item 3.
Item 2. The composition according to Item 2, wherein the sirtuin is at least one selected from the group consisting of Sirt1, Sirt6, and Sirtu3.
Item 4.
Item 2. The composition according to any one of Items 1 to 3, which is an oral composition.
Item 5.
Item 2. The composition according to any one of Items 1 to 4, which is a food composition or a pharmaceutical composition.
本開示によれば、血管老化抑制用組成物を得ることができる。また本開示によれば、一実施形態において、サーチュイン活性化用組成物を得ることができる。 According to the present disclosure, a composition for suppressing vascular aging can be obtained. Further, according to the present disclosure, in one embodiment, a composition for activating sirtuin can be obtained.
以下、本開示に包含される各実施形態について、さらに詳細に説明する。 Hereinafter, each embodiment included in the present disclosure will be described in more detail.
本開示は、キサントフモールを含有する組成物を包含する。以下、当該組成物を、「本開示の組成物」と表記することがある。本開示の組成物は、血管老化抑制用、又はサーチュイン活性化用に好ましく用いることができる。 The present disclosure includes compositions containing xanthohumol. Hereinafter, the composition may be referred to as "the composition of the present disclosure". The compositions of the present disclosure can be preferably used for suppressing vascular aging or for activating sirtuins.
キサントフモールは、天然物であっても合成物であってもよく、天然物から精製したものや商業的に入手可能なものなど、特に限定されない。キサントフモールを含む天然物としては、例えばホップなどが挙げられる。また、キサントフモールとしては、キサントフモールを含有する限り、例えばホップ抽出物などを用いることができる。 Xanthohumol may be a natural product or a synthetic product, and is not particularly limited to a product refined from a natural product or a commercially available product. Examples of natural products containing xanthohumol include hops and the like. As the xanthohumol, for example, a hop extract or the like can be used as long as it contains xanthohumol.
本開示の組成物におけるキサントフモールの含有量は、例えば、０．００１〜９５質量％が好ましく、０．０１〜９０質量％がより好ましく、０．１〜８０質量％がさらに好ましい。 The content of xanthohumol in the compositions of the present disclosure is, for example, preferably 0.001 to 95% by mass, more preferably 0.01 to 90% by mass, still more preferably 0.1 to 80% by mass.
また、本開示の組成物による、キサントフモールの摂取量としては、キサントフモールが成人１日あたり１〜５００ｍｇ、好ましくは１０〜４００ｍｇ、更に好ましくは３０〜３００ｍｇとなる量が例示できる。 Further, as the intake amount of xanthohumol according to the composition of the present disclosure, an amount of xanthohumol in an amount of 1 to 500 mg, preferably 10 to 400 mg, more preferably 30 to 300 mg per day for an adult can be exemplified.
本開示の組成物は、経口組成物として利用することができる。経口組成物としては、例えば医薬組成物、食品組成物（飲料組成物及び食品添加物組成物を包含する）などが挙げられる。 The compositions of the present disclosure can be used as oral compositions. Examples of the oral composition include pharmaceutical compositions, food compositions (including beverage compositions and food additive compositions) and the like.
本開示の組成物は、上記成分を含み、さらに他の成分を含むことができる。当該他の成分は、当該組成物を用いる分野に応じて適宜選択することができる。例えば、薬学的又は食品衛生学的に許容される担体を用いることができる。 The composition of the present disclosure contains the above-mentioned components, and may further contain other components. The other component can be appropriately selected depending on the field in which the composition is used. For example, pharmaceutically or food hygiene-acceptable carriers can be used.
医薬組成物として用いる場合、他の成分としては、薬学的に許容される基剤、担体、及び／又は添加剤（例えば溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤等）等が例示できる。また、医薬組成物の形態も特に制限されず、錠剤、丸剤、散剤、液剤、懸濁剤、乳剤、顆粒剤、カプセル剤、クリーム剤、パップ剤等が例示できる。これらの形態の医薬組成物は、必要に応じて当該他の成分とキサントフモールを組み合わせて常法により調製することができる。 When used as a pharmaceutical composition, other ingredients include pharmaceutically acceptable bases, carriers, and / or additives (eg, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders). , Disintegrant, solvent, etc.) can be exemplified. The form of the pharmaceutical composition is not particularly limited, and examples thereof include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, creams, and poultices. The pharmaceutical compositions in these forms can be prepared by a conventional method in combination with the other component and xanthohumol, if necessary.
食品組成物として用いる場合、他の成分としては、食品衛生学上許容される基剤、担体、添加剤や、その他食品として利用され得る成分・材料などが例示できる。また、食品組成物の形態も特に制限されず、例えば加工食品、健康食品（栄養補助食品、栄養機能食品、病者用食品、特定保健用食品、機能性表示商品等）、サプリメント、病者向け食品（病院食、病人食又は介護食等）等が例示できる。これらは常法により調製することができる。特に、健康食品（栄養補助食品、栄養機能食品、病者用食品、特定保健用食品、機能性表示商品等）、又はサプリメントとして、食品組成物を調製する場合は、継続的な摂取が行いやすいように、例えば顆粒、カプセル、錠剤（チュアブル剤等を含む）、飲料（飲料パウダー、ドリンク剤、スムージー等）等の形態で調製することが好ましく、なかでもカプセル、タブレット、錠剤、飲料パウダー、ドリンク剤、ゼリー、グミの形態が摂取の簡便さの点からは好ましいが、特にこれらに限定されるものではない。なお、食品組成物の中でも食品添加物組成物として用いる場合には、その形態として、例えば液状、粉末状、フレーク状、顆粒状、ペースト状のものが挙げられる。より具体的には、調味料（醤油、ソース、ケチャップ、ドレッシング等）、フレーク（ふりかけ）、焼き肉のたれ、スパイス、ルーペースト（カレールーペースト等）等が例示できる。 When used as a food composition, examples of other ingredients include bases, carriers, and additives that are acceptable in terms of food hygiene, and other ingredients and materials that can be used as foods. In addition, the form of the food composition is not particularly limited, and for example, processed foods, health foods (nutrition supplements, nutritionally functional foods, foods for the sick, foods for specified health use, functionally labeled products, etc.), supplements, for the sick. Foods (hospital food, sick food, nursing food, etc.) can be exemplified. These can be prepared by a conventional method. In particular, when preparing food compositions as health foods (food supplements, foods with functional nutrition, foods for the sick, foods for specified health use, foods with functional claims, etc.) or supplements, continuous intake is easy. As described above, for example, it is preferable to prepare in the form of granules, capsules, tablets (including chewable agents), beverages (beverage powders, drinks, smoothies, etc.), among which capsules, tablets, tablets, beverage powders, drinks, etc. The forms of the agent, jelly, and gummy are preferable from the viewpoint of ease of ingestion, but are not particularly limited thereto. When used as a food additive composition among food compositions, examples thereof include liquid, powder, flakes, granules, and pastes. More specifically, seasonings (soy sauce, sauce, ketchup, dressing, etc.), flakes (sprinkle), roasted meat sauce, spices, roux paste (curry roux paste, etc.) and the like can be exemplified.
本開示の組成物は、キサントフモールを含有することにより、サーチュイン（好ましくは、Ｓｉｒｔ１、Ｓｉｒｔ６、又はＳｉｒｔ３）の発現を亢進することができる。また、一実施形態において、本開示の組成物は、キサントフモールを含有するため、酸化ストレスを抑制することができる。また、一実施形態において、本開示の組成物は、キサントフモールを含有するため、炎症を抑制することができる。また、一実施形態において、本開示の組成物は、キサントフモールを含有するため、活性酸素を抑制することができる。また、一実施形態において、ヒストン３の脱アセチル化を介してテロメアクロマチンを安定化することができる、すなわち、本開示の組成物は、キサントフモールを含有するため、血管老化を抑制することができる。 The composition of the present disclosure can enhance the expression of sirtuin (preferably Sirtu1, Sirt6, or Sirt3) by containing xanthohumol. Moreover, in one embodiment, since the composition of the present disclosure contains xanthohumol, oxidative stress can be suppressed. Moreover, in one embodiment, since the composition of the present disclosure contains xanthohumol, inflammation can be suppressed. Further, in one embodiment, since the composition of the present disclosure contains xanthohumol, active oxygen can be suppressed. Also, in one embodiment, telomere chromatin can be stabilized through deacetylation of histone 3, that is, the compositions of the present disclosure contain xanthohumol and thus can suppress vascular aging. it can.
また、好適な一実施形態において、本開示の組成物は、ヘムオキシゲナーゼ−１（ＨＯ−１）の発現を亢進することができる。ＨＯ−１は、ヘム代謝の律速酵素であり、血管内皮機能を改善する効果を有することが知られている。すなわち、本開示の組成物は、ＨＯ−１の発現を亢進することにより、血管内皮機能を増強することができる。 Also, in a preferred embodiment, the compositions of the present disclosure can enhance the expression of heme oxygenase-1 (HO-1). HO-1 is a rate-determining enzyme of heme metabolism and is known to have an effect of improving vascular endothelial function. That is, the composition of the present disclosure can enhance the vascular endothelial function by enhancing the expression of HO-1.
本開示の組成物は、サーチュインを活性化（サーチュインの発現を亢進）させるために用いることができる。特に、本開示の組成物は、Ｓｉｒｔ１、Ｓｉｒｔ６、又はＳｉｒｔ３を活性化（Ｓｉｒｔ１、Ｓｉｒｔ６、又はＳｉｒｔ３の発現を亢進）させるために用いることができる。また、一実施形態において、本開示の組成物は、血管老化を抑制させるために用いることができる。また、一実施形態において、本開示の組成物は、酸化ストレスを抑制させるために用いることができる。また、一実施形態において、本開示の組成物は、炎症を抑制させるために用いることができる。また、一実施形態において、本開示の組成物は、活性酸素を抑制させるために用いることができる。また、一実施形態において、本開示の組成物は、Ｓｉｒｔ１の発現を亢進することができるため、抗細胞老化作用、抗動脈硬化作用、血管拡張作用、糖尿病合併症予防などの作用を発揮することができ、本開示の組成物はこのような作用を目的とした用途に好ましく用いることができる。例えば、本開示の組成物は、抗老化用、抗動脈硬化用、血管拡張用、糖尿病合併症予防などの用途に用いることができる。また、一実施形態において、本開示の組成物は、Ｓｉｒｔ６の発現を亢進することができるため、抗細胞老化作用、抗炎症作用、歯周病予防などの作用を発揮することができ、本開示の組成物はこれらの作用を目的とした用途に好ましく用いることができる。例えば、本開示の組成物は、抗細胞老化用、抗炎症用、歯周病予防などの用途に用いることができる。また、一実施形態において、本開示の組成物は、Ｓｉｒｔ３の発現を亢進することができるため、抗酸化作用などの作用を発揮することができ、本開示の組成物はこれらの作用を目的とした用途に好ましく用いることができる。例えば、本開示の組成物は、抗酸化用などの用途に用いることができる。また、本開示の組成物は、酸化ストレスを抑制することができるため、酸化ストレスの増加に起因する疾患（例えば、癌；高血圧症；動脈硬化症；脂質異常症；上昇高脂血症；血栓症；狭心症、心筋梗塞などの虚血性心疾患；糖尿病；糖尿病合併症；歯周病など）の予防及び／又は治療に好ましく用いることができる。また、本開示の組成物は、炎症を抑制することができるため、血管の炎症に起因する疾患（例えば、高血圧症、動脈硬化症、脂質異常症、上昇高脂血症、血栓症、糖尿病、糖尿病合併症、歯周病など）の予防及び／又は治療に好ましく用いることができる。また、本開示の組成物は、活性酸素を抑制することができるため、活性酸素の増加に起因する疾患（例えば、癌、高血圧症、動脈硬化症、脂質異常症、上昇高脂血症、血栓症、虚血性心疾患、糖尿病、糖尿病合併症、歯周病など）の予防及び／又は治療に好ましく用いることができる。また、本開示の組成物は、血管の老化を抑制することができるため、血管老化に起因する疾患（例えば、動脈硬化症；狭心症、心筋梗塞などの虚血性心疾患；脳出血、脳梗塞などの脳卒中；歯周病など）の予防及び／又は治療に好ましく用いることができる。 The compositions of the present disclosure can be used to activate sirtuins (enhance sirtuin expression). In particular, the compositions of the present disclosure can be used to activate Sirtuin 1, Sirtuin 6, or Sirtuin 3 (enhance the expression of Sirtuin 1, Sirtuin 6, or Sirtuin 3). Also, in one embodiment, the compositions of the present disclosure can be used to suppress vascular aging. Also, in one embodiment, the compositions of the present disclosure can be used to suppress oxidative stress. Also, in one embodiment, the compositions of the present disclosure can be used to suppress inflammation. Moreover, in one embodiment, the composition of the present disclosure can be used to suppress active oxygen. Further, in one embodiment, since the composition of the present disclosure can enhance the expression of Sirtu1, it exerts actions such as anti-cell senescence action, anti-arteriosclerosis action, vasodilatory action, and prevention of diabetic complications. The compositions of the present disclosure can be preferably used for applications aimed at such an action. For example, the compositions of the present disclosure can be used for anti-aging, anti-arteriosclerosis, vasodilation, prevention of diabetic complications, and the like. Further, in one embodiment, since the composition of the present disclosure can enhance the expression of Sirtu6, it can exert actions such as anti-cell senescence action, anti-inflammatory action, and prevention of periodontal disease. The composition of can be preferably used for the purpose of these actions. For example, the compositions of the present disclosure can be used for anti-cell senescence, anti-inflammatory, periodontal disease prevention and the like. Further, in one embodiment, since the composition of the present disclosure can enhance the expression of Sirt3, it is possible to exert an action such as an antioxidant action, and the composition of the present disclosure aims at these actions. It can be preferably used for the above-mentioned applications. For example, the compositions of the present disclosure can be used for applications such as antioxidants. In addition, since the composition of the present disclosure can suppress oxidative stress, diseases caused by an increase in oxidative stress (for example, cancer; hypertension; arteriosclerosis; dyslipidemia; elevated hyperlipidemia; thrombosis) It can be preferably used for the prevention and / or treatment of ischemic heart disease such as angina pectoris and myocardial infarction; diabetes; diabetic complications; periodontal disease, etc.). In addition, since the compositions of the present disclosure can suppress inflammation, diseases caused by inflammation of blood vessels (for example, hypertension, arteriosclerosis, dyslipidemia, hyperlipidemia, thrombosis, diabetes, etc.) It can be preferably used for the prevention and / or treatment of (diabetic complications, periodontal disease, etc.). In addition, since the composition of the present disclosure can suppress active oxygen, diseases caused by an increase in active oxygen (for example, cancer, hypertension, arteriosclerosis, dyslipidemia, elevated hyperlipidemia, thrombosis) It can be preferably used for the prevention and / or treatment of diseases, ischemic heart disease, diabetes, diabetic complications, periodontal disease, etc.). In addition, since the composition of the present disclosure can suppress aging of blood vessels, diseases caused by vascular aging (for example, arteriosclerosis; ischemic heart diseases such as angina and myocardial infarction; cerebral hemorrhage, cerebral infarction) It can be preferably used for the prevention and / or treatment of stroke (such as periodontal disease).
本開示の組成物は、その用途、効能、効果、機能、有効成分の種類、機能性成分の種類、摂取方法などに関する記載を表示したものであってもよい。「表示」とは、医薬組成物、健康食品（栄養補助食品、栄養機能食品、病者用食品、特定保健用食品、機能性表示商品等）、サプリメント、病者向け食品（病院食、病人食又は介護食等）等のそれぞれに適した表示を意味する。また、「表示」には、消費者に対して本開示の組成物の用途、効能、効果、機能等を知らせるための全ての表示が含まれる。この表示は、前段落の内容を想起・類推させ得るような表示であればよく、表示の目的、表示の内容、表示する対象物・媒体などに関わらず全ての表示を含み得る。例えば、製品の包装・容器での表示、製品に関する広告・価格表もしくは取引書類への表示およびこれらを展示もしくは頒布すること、またはこれらを電磁気的（インターネットなど）方法により提供すること等が挙げられる。 The composition of the present disclosure may display a description regarding its use, efficacy, effect, function, type of active ingredient, type of functional ingredient, ingestion method, and the like. "Indication" means pharmaceutical compositions, health foods (nutritional supplements, nutritionally functional foods, foods for the sick, foods for specified health use, foods with functional claims, etc.), supplements, foods for the sick (hospital foods, sick foods, etc.) Or, it means a display suitable for each of (nursing food, etc.). In addition, "labeling" includes all labeling for informing consumers of the use, efficacy, effect, function, etc. of the composition of the present disclosure. This display may be any display that can recall or infer the contents of the previous paragraph, and may include all displays regardless of the purpose of the display, the contents of the display, the object / medium to be displayed, and the like. For example, labeling of products on packaging / containers, advertising / price lists of products or labeling on transaction documents, displaying or distributing them, or providing them by electromagnetic (Internet, etc.) methods. ..
本開示の組成物が機能性表示食品である場合、例えば、「血管をしなやかに保つのを助ける（サポートする）」、「血管の柔らかさを保つのを助ける（サポートする）」、「血管の柔軟性を保つのを助ける（サポートする）」、「血管の抗酸化力を保つのを助ける（サポートする）」、「血管を若々しく保つのを助ける（サポートする）」、「血管を強くするのを助ける（サポートする）」、「血管力を保つのを助ける（サポートする）」、「血管のしなやかさが気になる方に」、「血管の柔らかさが気になる方に」、「血管の柔軟性が気になる方に」、「血管の抗酸化力が気になる方に」、「血管の若々しさが気になる方に」、「血管力が気になる方に」、「血管年齢が気になる方に」、「血管の老化防止に」、「血のめぐりをスムーズに維持する」、「血のめぐりをスムーズにするのを助ける（サポートする）」、「血流をスムーズにするのを助ける（サポートする）」、「血管本来の力を維持するために」「血管本来の力を引きだすために」、「血管の若返りのために」、「血管機能の維持を助ける（サポートする）」「血管機能の改善を助ける（サポートする）」、「血管内環境を良好にする」「血管内環境の維持を助ける（サポートする）」、「血管内環境の改善を助ける（サポートする）」、「血管の調子を整える」、「血管の調子を維持する」、「血管の調子を助ける（サポートする）」等の表示を付すことができる。なお、上述の例に限定されず、そのような意味と同義である文言を使用することができる。 When the composition of the present disclosure is a food with a functional claim, for example, "helping (supporting) keeping blood vessels supple", "helping (supporting) keeping blood vessels soft", "helping to keep blood vessels soft", "vascular Help keeps flexibility (support), "helps keep blood vessels antioxidative (supports)", "helps keep blood vessels youthful (supports)", "strengthens blood vessels" "Help (support)", "Help (support) to maintain blood vessel strength", "For those who are concerned about the suppleness of blood vessels", "For those who are concerned about the softness of blood vessels", "For those who are concerned about the flexibility of blood vessels", "For those who are concerned about the antioxidant power of blood vessels", "For those who are concerned about the youthfulness of blood vessels", "For those who are concerned about blood vessel strength" "For those who are concerned about blood vessel age", "For prevention of blood vessel aging", "Keep blood circulation smooth", "Help (support) smooth blood circulation", " "Helping (support) smoothing blood flow", "To maintain the original power of blood vessels", "To draw out the original power of blood vessels", "For rejuvenation of blood vessels", "For vascular function "Help (support) maintenance" "Help (support) improve vascular function" "Improve intravascular environment" "Help (support) maintain intravascular environment", "Improve intravascular environment" It is possible to add indications such as "help (support)", "adjust the condition of blood vessels", "maintain the condition of blood vessels", and "help (support) the condition of blood vessels". In addition, the wording is not limited to the above-mentioned example, and the wording synonymous with such a meaning can be used.
本開示の組成物は、１回又は複数回（好ましくは２〜３回）に分けて摂取することができる。また、摂取対象としてはヒトが好ましいが、ヒト以外の非ヒト哺乳動物であってもよい。摂取対象としては、例えば、サーチュインの発現量が低いヒト、Ｓｉｒｔ１の発現量が低いヒト、Ｓｉｒｔ６の発現量が低いヒト、Ｓｉｒｔ３の発現量が低いヒト、血液中の活性酸素量が多いヒト、血管における炎症の抑制を希望するヒト、血管の酸化ストレスの抑制を希望するヒト、酸化ストレスの増加に起因する疾患（例えば、癌；高血圧症；動脈硬化症；脂質異常症；上昇高脂血症；血栓症；狭心症、心筋梗塞などの虚血性心疾患；糖尿病；糖尿病合併症；歯周病など）を有する又は有する疑いがあるヒト、血管の炎症に起因する疾患（例えば、高血圧症、動脈硬化症、脂質異常症、上昇高脂血症、血栓症、糖尿病、糖尿病合併症、歯周病など）を有する又は有する疑いがあるヒト、活性酸素の増加に起因する疾患（例えば、癌、高血圧症、動脈硬化症、脂質異常症、上昇高脂血症、血栓症、虚血性心疾患、糖尿病、糖尿病合併症、歯周病など）を有する又は有する疑いがあるヒト、血管老化に起因する疾患（例えば、動脈硬化症；狭心症、心筋梗塞などの虚血性心疾患；脳出血、脳梗塞などの脳卒中；歯周病など）を有する又は有する疑いがあるヒト、血管老化の抑制を希望するヒトなどが挙げられる。 The compositions of the present disclosure can be ingested once or in multiple doses (preferably 2-3 doses). In addition, although humans are preferable as the ingestion target, non-human mammals other than humans may be used. Ingestion targets include, for example, humans with low sirtuin expression, low Sirt1 expression, low Sirt6 expression, low Sirt3 expression, high blood active oxygen, and blood vessels. People who want to suppress inflammation in blood vessels, people who want to suppress oxidative stress in blood vessels, diseases caused by increased oxidative stress (eg, cancer; hypertension; arteriosclerosis; dyslipidemia; elevated hyperlipidemia; Ischemic heart disease such as thrombosis; angina, myocardial infarction; diabetes; diabetic complications; periodontal disease, etc. in humans, diseases caused by inflammation of blood vessels (eg, hypertension, arteries) People with or suspected of having sclerosis, dyslipidemia, elevated hyperlipidemia, thrombosis, diabetes, diabetic complications, periodontal disease, etc., diseases caused by increased active oxygen (eg, cancer, hypertension) Diseases, arteriosclerosis, dyslipidemia, elevated hyperlipidemia, thrombosis, ischemic heart disease, diabetes, diabetic complications, periodontal disease, etc.) in humans, diseases caused by vascular aging People who have or are suspected of having (eg, arteriosclerosis; ischemic heart disease such as angina, myocardial infarction; stroke such as cerebral hemorrhage, cerebral infarction; periodontal disease, etc.) And so on.
なお、本明細書において「含む」とは、「本質的にからなる」と、「からなる」をも包含する（The term "comprising" includes "consisting essentially of” and "consisting of."）。また、本開示は、本明細書に説明した構成要件を任意の組み合わせを全て包含する。 In addition, in this specification, "including" also includes "consisting essentially" and "consisting of" (The term "comprising" includes "consisting essentially of" and "consisting of."). The present disclosure also includes all combinations of the constituent requirements described herein.
また、上述した本開示の各実施形態について説明した各種特性（性質、構造、機能等）は、本開示に包含される主題を特定するにあたり、どのように組み合わせられてもよい。すなわち、本開示には、本明細書に記載される組み合わせ可能な各特性のあらゆる組み合わせからなる主題が全て包含される。 In addition, the various properties (property, structure, function, etc.) described for each embodiment of the present disclosure described above may be combined in any way in specifying the subject matter included in the present disclosure. That is, the present disclosure includes all subjects consisting of any combination of each of the combinable properties described herein.
本開示の内容を以下の実施例を用いて具体的に説明する。しかし、本開示はこれらに何ら限定されるものではない。下記において、特に言及する場合を除いて、実験は大気圧及び常温条件下で行っている。また特に言及する場合を除いて、「％」は「質量％」を意味する。 The contents of the present disclosure will be specifically described with reference to the following examples. However, this disclosure is not limited to these. In the following, the experiments are carried out under atmospheric pressure and normal temperature conditions, unless otherwise specified. Also, unless otherwise specified, "%" means "mass%".
１．タンパク発現に対する作用
キサントフモール（ＸＨ）、８−プレニルナリンゲニン（８−ＰＮ）、又はイソキサントフモール（ＩＸＨ）、並びに１容量％ＦＢＳ及び２ｍＭ Ｌ‐グルタミンを含有するＭＣＤＢ１３１培地で培養したヒト臍帯静脈内皮細胞（ＨＵＶＥＣｓ、三光純薬社製）を冷ＰＢＳ（−）で２回洗浄し、氷上でプロテアーゼ阻害剤（Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ社製)、及びホスファターゼ阻害剤（Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ社製）を添加したＲＩＰＡ緩衝液、又はＭ−ＰＥＲ緩衝液（Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ社製）により細胞を回収した。回収した細胞を１０秒間超音波処理した後、４℃で遠心分離（１５，０００g、２０分）を行い、上清を採取した。得られたタンパク抽出液の総タンパク質濃度をＢＣＡアッセイ（Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ社製）により測定した。それらのタンパク質濃度を一定に揃えた後、Ｌａｅｍｍｌｉ Ｓａｍｐｌｅ Ｂｕｆｆｅｒ（ＢＩＯ−ＲＡＤ社製）を添加、５分間煮沸することによってタンパク質を変性させた。その後、ＳＤＳ−ＰＡＧＥによってタンパク質の電気泳動を行い、分離したタンパク質をＰＶＤＦ膜に転写した。タンパク質を転写した膜を、５％スキムミルク、および０．０５容量％ Ｔｗｅｅｎ−２０を含有するＰＢＳにてブロッキングし、次いで、１次抗体、２次抗体と反応させた後、基質としてＳｕｐｅｒＳｉｇｎａｌ Ｗｅｓｔ Ｄｕｒａ Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｔ Ｓｕｂｓｔｒａｔｅ、又はＳｕｐｅｒＳｉｇｎａｌ Ｗｅｓｔ Ｐｉｃｏ Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｔ Ｓｕｂｓｔｒａｔｅ（Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ社製）を用いて膜を化学発光させ、ＬＡＳ４０００ｍｉｎｉ（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製）システムにより発光強度を検出した。バンド強度の定量は、Ｉｍａｇｅ Ｊを用いて行った。Ｓｉｒｔ１、Ｓｉｒｔ６、Ｓｉｒｔ３の１次抗体、および２次抗体にはＣｅｌｌ Ｓｉｇｎａｌｉｎｇ社製の抗体を使用した。ＨＯ−１の１次抗体には、Ｅｎｚｏ Ｌｉｆｅ Ｓｃｉｅｎｃｅｓ社の抗体を使用した。なお、対照として、キサントフモール（ＸＨ）、８−プレニルナリンゲニン（８−ＰＮ）、およびイソキサントフモール（ＩＸＨ）のいずれも含有しない培地で培養した細胞を用いて同様に分析を行った。対照におけるβ−アクチンの発現量に対するＳＩＲＴ１、ＳＩＲＴ６、又はＳＩＲＴ３の発現量を１としたときの、相対値を図１〜図３に示す。なお、キサントフモール（ＸＨ）、８−プレニルナリンゲニン（８−ＰＮ）、およびイソキサントフモール（ＩＸＨ）の構造式を、式（１）、（２）、及び（３）に示す。また、サントフモール（ＸＨ）、８−プレニルナリンゲニン（８−ＰＮ）、およびイソキサントフモール（ＩＸＨ）の含有量は、各図中に最終濃度として示す。
1. 1. Effect on protein expression Human umbilical vein cultured in MCDB131 medium containing xanthofumol (XH), 8-prenylnaringenin (8-PN), or isoxanthohumol (IXH), and 1 volume% FBS and 2 mM L-glutamine. RIPA in which venous endothelial cells (HUVECs, manufactured by Sanko Junyaku Co., Ltd.) were washed twice with cold PBS (-), and a protease inhibitor (manufactured by Thermo Scientific) and a phosphatase inhibitor (manufactured by Thermo Scientific) were added on ice. Cells were harvested with buffer or M-PER buffer (manufactured by Thermo Scientific). The collected cells were sonicated for 10 seconds and then centrifuged (15,000 g, 20 minutes) at 4 ° C. to collect the supernatant. The total protein concentration of the obtained protein extract was measured by BCA assay (manufactured by Thermo Scientific). After adjusting the protein concentrations to a constant level, Laemmli Sample Buffer (manufactured by BIO-RAD) was added and the proteins were denatured by boiling for 5 minutes. Then, the protein was electrophoresed by SDS-PAGE, and the separated protein was transferred to the PVDF membrane. The protein-transferred membrane is blocked with PBS containing 5% skim milk and 0.05% by volume Tween-20, then reacted with the primary and secondary antibodies and then SuperSignal West Dura Chemiluminescence as a substrate. The membrane was chemiluminescent using Substrate or SuperSignal West Pico Chemiluminescent Substrate (manufactured by Thermo Scientific), and the luminescence intensity was detected by the LAS4000mini (manufactured by GE Healthcare) system. The band strength was quantified using Image J. Antibodies manufactured by Cell Signaling Co., Ltd. were used as the primary antibody of Sirt1, Sirt6, and Sirt3, and the secondary antibody. As the primary antibody of HO-1, an antibody manufactured by Enzo Life Sciences was used. As a control, the same analysis was performed using cells cultured in a medium containing no xanthohumol (XH), 8-prenylnaringenin (8-PN), or isoxanthohumol (IXH). The relative values when the expression level of SIRT1, SIRT6, or SIRT3 is 1 with respect to the expression level of β-actin in the control are shown in FIGS. 1 to 3. The structural formulas of xanthohumol (XH), 8-prenylnaringenin (8-PN), and isoxanthohumol (IXH) are shown in the formulas (1), (2), and (3). The contents of santohumol (XH), 8-prenylnaringenin (8-PN), and isoxanthohumol (IXH) are shown as final concentrations in each figure.
図１に示す通り、キサントフモール（ＸＨ）は、ＳＩＲＴ１の発現を亢進することが分かった。一方、キサントフモールと構造が類似する、８−プレニルナリンゲニン（８−ＰＮ）およびイソキサントフモール（ＩＸＨ）は、いずれもＳＩＲＴ１の発現を亢進しないことが分かった。また、図２に示す通り、キサントフモール（ＸＨ）は濃度依存的にＳＩＲＴ６の発現を亢進するのに対して、８−プレニルナリンゲニン（８−ＰＮ）およびイソキサントフモール（ＩＸＨ）は、いずれもＳＩＲＴ６の発現を亢進しないことが分かった。さらに、図３に示す通り、キサントフモール（ＸＨ）は、濃度依存的にＳＩＲＴ３の発現を亢進することが分かった。また図４に示す通り、キサントフモールは、濃度依存的にＨＯ−１の発現を亢進することが分かった。この結果から、キサントフモールは、Ｓｉｒｔ１、Ｓｉｒｔ６、Ｓｉｒｔ３、ＨＯ−１の発現を亢進することによって、優れた血管老化抑制作用、血管内皮機能増強作用などを奏することが考察される。 As shown in FIG. 1, xanthohumol (XH) was found to enhance the expression of SIRT1. On the other hand, neither 8-prenylnaringenin (8-PN) nor isoxanthohumol (IXH), which are similar in structure to xanthohumol, were found to enhance SIRT1 expression. Further, as shown in FIG. 2, xanthohumol (XH) enhances the expression of SIRT6 in a concentration-dependent manner, whereas 8-prenylnaringenin (8-PN) and isoxanthohumol (IXH) eventually increase. Was also found not to enhance the expression of SIRT6. Furthermore, as shown in FIG. 3, xanthohumol (XH) was found to enhance SIRT3 expression in a concentration-dependent manner. Further, as shown in FIG. 4, it was found that xanthohumol enhances the expression of HO-1 in a concentration-dependent manner. From this result, it is considered that xanthohumol exerts an excellent vascular aging inhibitory action, vascular endothelial function enhancing action, and the like by enhancing the expression of Sirt1, Sirt6, Sirt3, and HO-1.
２．活性酸素（ＲＯＳ）の観察
１０μＭのキサントフモール、１容量％ＦＢＳ、及び２ｍＭ Ｌ‐グルタミンを含有するＭＣＤＢ１３１培地で２４時間培養したヒト臍帯静脈内皮細胞を、ＰＢＳ（−）で洗浄し、５００μＭの過酸化水素を含有する無血清培地で１時間刺激を行った。刺激後、ＰＢＳ（−）で洗浄し、ＣｅｌｌＲＯＸ Ｇｒｅｅｎ（Ｉｎｖｉｔｒｏｇｅｎ社製）、Ｈｏｅｃｈｓｔ ３３３４２（Ｉｎｖｉｔｒｏｇｅｎ社製）を添加し、３７℃で３０分間インキュベートすることによってＲＯＳ、および核を蛍光染色した。染色後、４重量％パラホルムアルデヒドで細胞を固定化した後、蛍光顕微鏡ＢＺ‐ＩＩ（キーエンス社製）にて蛍光観察を行った。なお、キサントフモール又は過酸化水素で処理しない細胞を対照とした。図５にキサントフモール及び過酸化水素で処理していない細胞（左上）、過酸化水素のみを処理した細胞（左下）、キサントフモールのみを処理した細胞（右上）、並びにキサントフモール及び過酸化水素で処理した細胞（右下）の観察結果を示す。
2. 2. Observation of Reactive Oxygen Species (ROS) Human umbilical vein endothelial cells cultured for 24 hours in MCDB131 medium containing 10 μM xanthophumol, 1 volume% FBS, and 2 mM L-glutamine were washed with PBS (-) and 500 μM. Stimulation was performed for 1 hour in serum-free medium containing hydrogen peroxide. After stimulation, the cells were washed with PBS (−), CellROX Green (manufactured by Invitrogen), Hoechst 33342 (manufactured by Invitrogen) were added, and ROS and nuclei were fluorescently stained by incubating at 37 ° C. for 30 minutes. After staining, cells were immobilized with 4 wt% paraformaldehyde, and then fluorescence was observed with a fluorescence microscope BZ-II (manufactured by KEYENCE CORPORATION). Cells not treated with xanthohumol or hydrogen peroxide were used as controls. In FIG. 5, cells not treated with xanthohumol and hydrogen peroxide (upper left), cells treated with hydrogen peroxide only (lower left), cells treated with only xanthohumol (upper right), and xanthohumol and excess. The observation results of cells treated with hydrogen peroxide (lower right) are shown.
キサントフモールは、過酸化水素によって誘導されるＲＯＳの産生を抑制することが分かった。この結果から、キサントフモールは、優れた活性酸素の抑制作用などを奏することが分かった。 Xanthohumol was found to suppress hydrogen peroxide-induced production of ROS. From this result, it was found that xanthohumol exerts an excellent inhibitory effect on active oxygen.
３．細胞老化の観察
老化細胞では、Ｓｅｎｅｓｃｅｎｃｅ β‐Ｇａｌａｃｔｏｓｉｄａｓｅが過剰発現することが知られている。１０μＭのキサントフモール、１容量％ＦＢＳ、及び２ｍＭ Ｌ‐グルタミンを含有するＭＣＤＢ１３１培地で２４時間培養したヒト臍帯静脈内皮細胞を、ＰＢＳ（−）で洗浄し、５００μＭの過酸化水素、１容量％ＦＢＳ、及び２ｍＭ Ｌ‐グルタミンを含有するＭＣＤＢ１３１培地で２４時間刺激を行った。刺激後、ＰＢＳ（−）で洗浄し、Ｓｅｎｅｓｃｅｎｃｅ β‐Ｇａｌａｃｔｏｓｉｄａｓｅ Ｓｔａｉｎｉｎｇ Ｋｉｔ（Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ社製）を使用してβ‐Ｇａｌａｃｔｏｓｉｄａｓｅ染色を行い、顕微鏡観察を行った。なお、キサントフモール又は過酸化水素で処理しない細胞を対照とした。図６にキサントフモール及び過酸化水素で処理していない細胞（左上）、過酸化水素のみを処理した細胞（左下）、キサントフモールのみを処理した細胞（右上）、並びにキサントフモール及び過酸化水素で処理した細胞（右下）の観察結果を示す。
3. 3. Observation of Cellular Senescence It is known that Senescence β-galactosidase is overexpressed in senescent cells. Human umbilical vein endothelial cells cultured for 24 hours in MCDB131 medium containing 10 μM xanthohumol, 1 volume% FBS, and 2 mM L-glutamine were washed with PBS (-) and 500 μM hydrogen peroxide, 1 volume%. Stimulation was performed for 24 hours with MCDB131 medium containing FBS and 2 mM L-glutamine. After stimulation, the cells were washed with PBS (-), stained with β-Galactosidese using a Sensoring β-Galactose Staining Kit (manufactured by Cell Signaling), and observed under a microscope. Cells not treated with xanthohumol or hydrogen peroxide were used as controls. FIG. 6 shows cells not treated with xanthohumol and hydrogen peroxide (upper left), cells treated with hydrogen peroxide only (lower left), cells treated with only xanthohumol (upper right), and xanthohumol and excess. The observation results of cells treated with hydrogen peroxide (lower right) are shown.
キサントフモールは、過酸化水素によって誘導される老化マーカーの発現を抑制することが分かった。この結果から、キサントフモールは、優れた血管老化抑制作用などを奏することが分かった。 Xanthohumol was found to suppress the expression of hydrogen peroxide-induced aging markers. From this result, it was found that xanthohumol exerts an excellent vascular aging inhibitory effect.
４．遺伝子発現に対する作用
１０μＭのキサントフモール、１容量％ＦＢＳ、及び２ｍＭ Ｌ‐グルタミンを含有するＭＣＤＢ１３１培地で２４時間培養したヒト臍帯静脈内皮細胞を、ＰＢＳ（−）で洗浄し、１００ｎｇ／ｍＬのＥ．ｃｏｌｉ ＬＰＳ、１容量％ＦＢＳ、及び２ｍＭ Ｌ‐グルタミンを含有するＭＣＤＢ１３１培地でさらに６時間培養した。細胞をＰＢＳ（−）で洗浄後、ＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ（Ｑｉａｇｅｎ社製）を使用して総ＲＮＡを抽出した。１本鎖のｃＤＮＡは０．５μgの総ＲＮＡよりＰｒｉｍｅＳｃｒｉｐｔ ＲＴ ｒｅａｇｅｎｔ Ｋｉｔ（タカラバイオ社製）を使用して合成した。ＣＣＬ２およびＩＣＡＭ−１のｍＲＮＡの定量分析はリアルタイムＰＣＲシステム（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）を使用して実施した。Ｐｒｅｍｉｘ Ｅｘ Ｔａｑ（タカラバイオ社製）、Ａｓｓａｙ−ｏｎ−Ｄｅｍａｎｄ，Ｇｅｎｅ Ｅｘｐｒｅｓｓｉｏｎ Ｐｒｏｄｕｃｔｓ（Ｈｓ００２３４１４０＿ｍ１ ｆｏｒ ＣＣＬ２、ＨＳ００１６４９３２＿ｍ１ ｆｏｒ ＩＣＡＭ−１：Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）を定量的リアルタイムＰＣＲ分析に使用した。定量データはそれぞれ、Ｒｉｂｏｓｏｍａｌ ｐｒｏｔｅｉｎ Ｓ１８（ＲＰＳ１８）の発現レベルで補正した。なお、対照として、キサントフモール又はＥ．ｃｏｌｉ ＬＰＳを含有しない培地で培養した細胞を用いて同様に分析を行った。キサントフモール及びＥ．ｃｏｌｉ ＬＰＳのいずれも含有しない培地で培養した細胞から抽出したＲＰＳ１８の発現量に対するＣＣＬ２、又はＩＣＡＭ−１の発現量を１としたときの、相対値を図７および図８に示す。
4. Effect on gene expression Human umbilical vein endothelial cells cultured for 24 hours in MCDB131 medium containing 10 μM xanthophumol, 1 volume% FBS, and 2 mM L-glutamine were washed with PBS (-) and 100 ng / mL E. .. The cells were cultured in MCDB131 medium containing coli LPS, 1 volume% FBS, and 2 mM L-glutamine for an additional 6 hours. After washing the cells with PBS (−), total RNA was extracted using RNeasy Mini Kit (manufactured by Qiagen). The single-stranded cDNA was synthesized from 0.5 μg of total RNA using PrimeScript RT reagent Kit (manufactured by Takara Bio Inc.). Quantitative analysis of CCL2 and ICAM-1 mRNA was performed using a real-time PCR system (Applied Biosystems). Premix Ex Taq (manufactured by Takara Bio Inc.), Assay-on-Demand, Gene Expression Products (Hs00234140_m1 for CCL2, HS00164932_m1 for ICAM-1: Applied Biosystems) Quantitative analysis using real-time analysis by Applied Biosystems. Quantitative data were each corrected for expression levels of ribosome protein S18 (RPS18). As a control, xanthohumol or E.I. The same analysis was performed using cells cultured in a medium containing no coli LPS. Xanthohumol and E. The relative values when the expression level of CCL2 or ICAM-1 is 1 with respect to the expression level of RPS18 extracted from cells cultured in a medium containing neither E. coli LPS are shown in FIGS. 7 and 8.
Ｅ．ｃｏｌｉ ＬＰＳは炎症性遺伝子である、ＣＣＬ２やＩＣＡＭ−１の発現を誘導することを確認した。これに対して、キサントフモールは、Ｅ．ｃｏｌｉ ＬＰＳによって誘導されるＣＣＬ２やＩＣＡＭ−１の遺伝子発現を顕著に抑制することが分かった。この結果から、キサントフモールは、優れた抗炎症作用などを奏することが分かった。 E. It was confirmed that E. coli LPS induces the expression of inflammatory genes such as CCL2 and ICAM-1. On the other hand, xanthohumol is E. coli. It was found that the gene expression of CCL2 and ICAM-1 induced by E. coli LPS was remarkably suppressed. From this result, it was found that xanthohumol exerts an excellent anti-inflammatory effect and the like.
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