JP7493852B2 - Composition for promoting tissue differentiation, composition for improving liver function - Google Patents
Composition for promoting tissue differentiation, composition for improving liver function Download PDFInfo
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Description
テクトリゲニンやテクトリゲニンの配糖体及び誘導体（テクトリゲニン類）はアヤメ科やマメ科の植物に存在するフラボノイドの一つであり、サーチュインを活性化すること（特許文献１参照）、Ｂｍａｌ１遺伝子の発現を活性化することなどが知られている（特許文献２参照）。
テクトリゲニン類は植物に含まれていることから、安全性が比較的に高く日常的に摂取しやすいと考えられるが、その活性や機能については未だ不明な点も多い。
Tectorigenin and its glycosides and derivatives (tectorigenins) are flavonoids present in plants of the Iridaceae and Fabaceae families, and are known to activate sirtuins (see Patent Document 1) and to activate the expression of the Bmal1 gene (see Patent Document 2).
Since tectorigenins are found in plants, they are thought to be relatively safe and easy to ingest on a daily basis, but there are still many unknowns about their activity and functions.
そこで、本発明においては、テクトリゲニン類の新たな用途及び機能を見い出すことを目的に、種々の検討を行った。 Therefore, in the present invention, various studies were conducted with the aim of finding new uses and functions for tectorigenins.
本発明者らは、テクトリゲニン類の作用効果について鋭意研究したところ、テクトリゲニン類において、アグリコンとしてのテクトリゲニンとテクトリゲニンの配糖体とを特定の比率で有する組成物が、驚くべきことに、組織分化促進や肝機能改善作用等、テクトリゲニンとテクトリゲニンの配糖体が元来有する機能が飛躍的に向上することを見い出した。また、特定比率でテクトリゲニンとテクトリゲニンの配糖体を有する組成物は、肌質改善や脂肪分解促進にとっても有利な効果を有することを見い出した。 The inventors of the present invention have conducted extensive research into the effects of tectorigenins, and have surprisingly found that a composition containing tectorigenin as an aglycone and tectorigenin glycosides in a specific ratio dramatically improves the functions that tectorigenin and tectorigenin glycosides originally possess, such as promoting tissue differentiation and improving liver function. They have also found that a composition containing tectorigenin and tectorigenin glycosides in a specific ratio has advantageous effects on improving skin quality and promoting fat decomposition.
すなわち、本発明は、アグリコンとしてのテクトリゲニンとテクトリゲニン配糖体とを、質量比で１：１０以上の量で含む、組織分化促進用組成物に関する。 That is, the present invention relates to a composition for promoting tissue differentiation that contains tectorigenin as an aglycone and tectorigenin glycoside in a mass ratio of 1:10 or more.
また、本発明は、テクトリゲニン及びテクトリゲニン配糖体を、質量比で１：１０以上の量で含む、脂肪分解促進用組成物に関する。 The present invention also relates to a composition for promoting lipolysis, which contains tectorigenin and tectorigenin glycoside in a mass ratio of 1:10 or more.
また本発明は、テクトリゲニンとテクトリゲニン配糖体とを、質量比で１：１０以上の量で含む、肝機能改善用組成物に関する。 The present invention also relates to a composition for improving liver function, which contains tectorigenin and tectorigenin glycoside in a mass ratio of 1:10 or more.
また、テクトリゲニン及びテクトリゲニン配糖体を、質量比で１：１０以上の量で含み、角層水分量向上、皮膚弾力向上又は皮膚水分蒸散抑制に用いられる組成物に関する。 The present invention also relates to a composition that contains tectorigenin and tectorigenin glycoside in a mass ratio of 1:10 or more and is used to improve the moisture content of the stratum corneum, improve skin elasticity, or inhibit moisture loss from the skin.
また、本発明は、テクトリゲニン及びテクトリゲニン配糖体を、質量比で１：１０以上の量で含む組成物に係るものである。 The present invention also relates to a composition containing tectorigenin and tectorigenin glycosides in a mass ratio of 1:10 or more.
本発明によれば、骨や筋肉等の組織について優れた分化促進作用を有する組織分化促進用組成物を提供することができる。
本発明によれば、優れた脂肪分解促進作用を有する脂肪分解促進用組成物を提供することができる。
本発明によれば、優れた肝機能改善作用を有する肝機能改善剤及び肝機能改善用組成物を提供することができる。
本発明によれば、角層水分量向上、皮膚弾力向上又は皮膚水分蒸散抑制について優れた作用を有し、これら角層水分量向上、皮膚弾力向上又は皮膚水分蒸散抑制に用いられる組成物を提供することができる。
本発明によれば、上記の各作用に優れた組成物を提供することができる。
According to the present invention, a composition for promoting tissue differentiation having an excellent differentiation promoting effect on tissues such as bone and muscle can be provided.
According to the present invention, it is possible to provide a composition for promoting lipolysis having an excellent effect of promoting lipolysis.
According to the present invention, it is possible to provide a liver function improving agent and a composition for improving liver function that have an excellent liver function improving effect.
According to the present invention, it is possible to provide a composition which has an excellent effect of improving the moisture content of the stratum corneum, improving skin elasticity, or inhibiting moisture evaporation from the skin, and which can be used for improving the moisture content of the stratum corneum, improving skin elasticity, or inhibiting moisture evaporation from the skin.
According to the present invention, a composition excellent in each of the above-mentioned functions can be provided.
以下、本発明の実施の形態を挙げて本発明を更に詳細に説明するが、本発明はこれらに限定されるものではない。以下、本発明の組織分化促進用組成物、脂肪分解促進用組成物、肝機能改善用組成物、角層水分量向上、皮膚弾力向上又は皮膚水分蒸散抑制に用いられる組成物及び組成物をまとめて「本発明の組成物」と記載する。 The present invention will be described in more detail below with reference to embodiments of the present invention, but the present invention is not limited thereto. Hereinafter, the compositions for promoting tissue differentiation, the compositions for promoting lipolysis, the compositions for improving liver function, and the compositions used to increase the moisture content of the stratum corneum, increase skin elasticity, or inhibit moisture loss from the skin and compositions of the present invention will be collectively referred to as the "compositions of the present invention."
（テクトリゲニン）
本発明で使用するテクトリゲニンは分子式Ｃ16Ｈ12Ｏ6で表され、5,7-Dihydroxy-3-(4-hydroxyphenyl)-6-methoxy-4H-1-benzopyran-4-one又は6-Methoxy-5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-oneと呼ばれる場合もある。本明細書中で単にテクトリゲニンという場合、このようにアグリコンの形態のものをいう。以下テクトリゲニンをＴＧＥとも記載する。テクトリゲニンの化学式は下記の通りである。
(tectorigenin)
The tectorigenin used in the present invention is represented by the molecular formula C16H12O6, and may be called 5,7-Dihydroxy-3-(4-hydroxyphenyl)-6-methoxy-4H-1-benzopyran-4-one or 6-Methoxy-5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one. When simply referring to tectorigenin in this specification, this refers to the aglycone form. Hereinafter, tectorigenin will also be referred to as TGE. The chemical formula of tectorigenin is as follows:
（テクトリゲニン配糖体）
テクトリゲニン配糖体（以下ＴＧＥ配糖体ともいう）としては、上記のＴＧＥに、グルコース、キシロース、ソルボース、ガラクトース、アピオース、ラムノースから選ばれる１種又は２種以上の糖を結合させたものが挙げられる。組織分化促進、脂肪分解促進、肝機能改善、肌質改善といった本発明の効果を高める観点から、ＴＧＥに、グルコース、キシロースから選ばれる１種又は２種以上の糖を結合させたものが好ましい。配糖体においてこれらの糖は、通常ＴＧＥの４位及び/又は７位のヒドロキシル基と結合しているが、前記の効果がより確実に得られる点や配糖体の入手しやすさの点から、７位のヒドロキシル基と結合しているものが好ましい。ＴＧＥ配糖体中の糖の数（糖の結合数ともいう）は、例えば１個以上が挙げられる。前記の効果を高める観点から、糖の結合数は好ましくは１個又は２個であるが、３個以上であってもよい。ここでいう糖の数とは、単糖単位の数をいう。ＴＧＥ配糖体としては、１種のみを用いてもよいが、２種以上を組み合わせることが好ましい。ＴＧＥ及びＴＧＥ配糖体は、本発明の組成物において、有機合成品であってもよく、植物等から抽出したものであってもよい。ＴＧＥ及びＴＧＥ配糖体は、粉末状等の固形状であってもよく、水や有機溶媒に溶解した状態であってもよい。ＴＧＥ及びＴＧＥ配糖体は、それらの混合物のみで存在していてもよく、その他の物質に溶解、分散ないし混合した状態であってよい。
(tectorigenin glycoside)
Examples of tectorigenin glycosides (hereinafter also referred to as TGE glycosides) include those obtained by binding one or more sugars selected from glucose, xylose, sorbose, galactose, apiose, and rhamnose to the above-mentioned TGE. From the viewpoint of enhancing the effects of the present invention, such as promoting tissue differentiation, promoting lipolysis, improving liver function, and improving skin quality, it is preferable to bind one or more sugars selected from glucose and xylose to TGE. In glycosides, these sugars are usually bound to the hydroxyl groups at the 4th and/or 7th positions of TGE, but from the viewpoint of more reliably obtaining the above-mentioned effects and ease of obtaining glycosides, it is preferable to bind to the hydroxyl group at the 7th position. The number of sugars in the TGE glycoside (also referred to as the number of sugar bonds) can be, for example, one or more. From the viewpoint of enhancing the above-mentioned effects, the number of sugar bonds is preferably one or two, but may be three or more. The number of sugars referred to here refers to the number of monosaccharide units. As the TGE glycoside, only one kind may be used, but it is preferable to use two or more kinds in combination. In the composition of the present invention, the TGE and TGE glycoside may be an organic synthesis product or may be extracted from a plant or the like. The TGE and TGE glycoside may be in a solid form such as a powder, or may be dissolved in water or an organic solvent. The TGE and TGE glycoside may exist only as a mixture thereof, or may be dissolved, dispersed or mixed in other substances.
ＴＧＥに糖が１個結合した配糖体としては、例えば、テクトリジン、テクトリジン４′‐グルコシドが挙げられる。また、ＴＧＥに糖が２個結合した配糖体としては、テクトリゲニン－７－Ｏ－キシロシルグルコシドが挙げられる。ここで、ＴＧＥにｎ個糖が結合しているとは、ｎ個の糖の連結体が結合しているのであってもよく、テクトリジンの別の箇所に結合している糖の合計数がｎ個なのであってもよい。テクトリジンとは、ＴＧＥの７位にグルコースが１個結合した配糖体であり、例えば、テクトリゲニン7-O-β-D-グルコピラノシド又は7-(β-D-Glucopyranosyloxy)-4',5-dihydroxy-6-methoxyisoflavone等とも表される。テクトリジン４′‐グルコシドとは、ＴＧＥの４位にグルコースが１個結合した配糖体であり、例えば、テクトリゲニン4'-O-β-D-グルコピラノシド 又は3-[4-(β-D-Glucopyranosyloxy)phenyl]-5,7-dihydroxy-6-methoxy-4H-1-benzopyran-4-one等とも称される。テクトリゲニン－７－Ｏ－キシロシルグルコシドはＴＧＥの７位に、グルコースが１つ結合し、このグルコースにキシロースが更に結合した配糖体であり、例えば6"-O-Xylosyltectoridinとも称される。例えば、テクトリジン（以下ＴＤともいう）は以下の化学式の構造を有する。 Examples of glycosides with one sugar bound to TGE include tectoridin and tectoridin 4'-glucoside. Examples of glycosides with two sugars bound to TGE include tectorigenin-7-O-xylosylglucoside. Here, "n sugars bound to TGE" may mean that a linker of n sugars is bound, or the total number of sugars bound to different positions on tectoridin may be n. Tectoridin is a glycoside with one glucose bound to the 7th position of TGE, and is also represented by, for example, tectorigenin 7-O-β-D-glucopyranoside or 7-(β-D-Glucopyranosyloxy)-4',5-dihydroxy-6-methoxyisoflavone. Tectoridin 4'-glucoside is a glycoside in which one glucose is bound to the 4th position of TGE, and is also called, for example, tectorigenin 4'-O-β-D-glucopyranoside or 3-[4-(β-D-Glucopyranosyloxy)phenyl]-5,7-dihydroxy-6-methoxy-4H-1-benzopyran-4-one. Tectorigenin-7-O-xylosylglucoside is a glycoside in which one glucose is bound to the 7th position of TGE, and xylose is further bound to this glucose, and is also called, for example, 6"-O-Xylosyltectoridin. For example, tectoridin (hereinafter also referred to as TD) has the following chemical structure:
また例えば、テクトリゲニン－７－Ｏ－キシロシルグルコシド（以下ＴＧＸＧともいう）は以下の化学式の構造を有する。式中、Ｇｌｕはグルコース基であり、Ｘｙｌはキシロース基である。 For example, tectorigenin-7-O-xylosylglucoside (hereinafter also referred to as TGXG) has the following chemical structure: In the formula, Glu is a glucose group, and Xyl is a xylose group.
本発明においては、ＴＧＥ配糖体が、ＴＧＥに糖が１個結合した配糖体とＴＧＥに糖が２個結合した配合体とを含むことが、前記の効果が優れていることから好ましい。
ＴＧＥ配糖体が、ＴＧＥに糖が１個結合した配糖体とＴＧＥに糖が２個結合した配合体とを含む場合、ＴＧＥに糖が１個結合した配糖体とＴＧＥに糖が２個結合した配糖体との質量比は、前者：後者が１：０．０１以上７．５以下であることが好ましい。この質量比が１：０．０１以上であることには本発明の高い作用を得ながらＴＤ等のＴＧＥに糖が１個結合した配糖体の量を一定量に制限できる利点がある。またこの質量比が、１：７．５以下であることで、前記の効果を更に一層高めることができる。これらの観点からＴＧＥに糖が１個結合した配糖体とＴＧＥに糖が２個結合した配糖体との量比は、前者：後者が１：０．０１以上５以下であることが好ましく、０．１以上２．５以下であることが特に好ましい。
In the present invention, it is preferable that the TGE glycoside contains a glycoside having one sugar bonded to TGE and a combination having two sugars bonded to TGE, since the above-mentioned effects are excellent.
When the TGE glycoside contains a glycoside having one sugar bound to TGE and a combination having two sugars bound to TGE, the mass ratio of the glycoside having one sugar bound to TGE and the glycoside having two sugars bound to TGE is preferably 1:0.01 or more and 7.5 or less. This mass ratio of 1:0.01 or more has the advantage that the amount of the glycoside having one sugar bound to TGE such as TD can be limited to a certain amount while obtaining the high effect of the present invention. Furthermore, this mass ratio of 1:7.5 or less can further enhance the above-mentioned effect. From these points of view, the mass ratio of the glycoside having one sugar bound to TGE and the glycoside having two sugars bound to TGE is preferably 1:0.01 or more and 5 or less, and particularly preferably 0.1 or more and 2.5 or less.
また、ＴＧＥ配糖体としては、前記の効果をより確実に高める観点、及び、配糖体の入手しやすさ等の観点から、ＴＧＥに糖が１個結合した配糖体としてＴＤを含むともに、ＴＧＥに糖が２個結合した配合体としてＴＧＸＧを含み、ＴＤとＴＧＸＧとの配合比が上記の好ましい比率１：０．０１以上７．５以下であることも好ましい。特にＴＤとＴＧＸＧとの配合比が、１：０．０１以上５以下であることが好ましく、０．１以上２．５以下であることが特に好ましい。 In addition, from the viewpoint of more reliably enhancing the above-mentioned effects and from the viewpoint of ease of obtaining the glycoside, it is also preferable that the TGE glycoside contains TD as a glycoside in which one sugar is bound to TGE, and contains TGXG as a compound in which two sugars are bound to TGE, and the compounding ratio of TD to TGXG is the above-mentioned preferred ratio of 1:0.01 or more and 7.5 or less. In particular, the compounding ratio of TD to TGXG is preferably 1:0.01 or more and 5 or less, and particularly preferably 0.1 or more and 2.5 or less.
本発明の特徴の一つは、ＴＧＥとＴＧＥ配糖体との量比にある。
本発明者らは、ＴＧＥ又は／及びＴＧＥ配糖体等のＴＧＥ類における作用について鋭意検討した。その結果、ＴＧＥとＴＧＥ配糖体とを特定の比率で組み合わせた場合に、質量比が１：１の組み合わせと比べて、飛躍的に脂肪分解促進作用が高まり、肝機能改善作用も高まることを見出した。具体的には、本発明の組成物において、ＴＧＥとＴＧＥ配糖体とは、前者：後者の質量比が１：１０以上である必要がある。更に、本発明者らは、脂肪分解促進作用や肝機能改善効果をとりわけ優れたものとする観点から、ＴＧＥとＴＧＥ配糖体の質量比は、１：１３以上であることがより好ましく、とりわけ、１：１８以上であることが好ましいことを見出した。ＴＧＥとＴＧＥ配糖体との質量比の下限値が前記の値である本発明の組成物は、組織分化促進、角層水分量向上、皮膚弾力向上又は皮膚水分蒸散抑制といった効果においても優れていることが判った。ＴＧＥとＴＧＥ配糖体との質量比の下限値が前記の値である本発明の組成物は、アヤメ科のヒオウギ（Ｂｅｌａｍｃａｎｄａ ｃｈｉｎｅｎｓｉｓ）やマメ科のクズ（Ｐｕｅｒａｒｉａ ｔｈｏｍｓｏｎｉｉ、Ｐｕｅｒａｒｉａ ｌｏｂａｔａ、Ｐｕｅｒａｒｉａ ｔｈｕｎｂｅｒｇｉａｎａ等）の花部などの植物の抽出物であってもよく、植物の抽出物に有機合成品を添加して調製してもよく、植物からの抽出物を組み合わせて調製してもよく、有機合成品を組み合わせて調製してもよい。
One of the features of the present invention is the ratio of TGE to TGE glycoside.
The present inventors have intensively studied the action of TGEs such as TGE and/or TGE glycoside. As a result, they have found that when TGE and TGE glycoside are combined at a specific ratio, the fat decomposition promoting effect is dramatically increased and the liver function improving effect is also increased, compared with a combination with a mass ratio of 1:1. Specifically, in the composition of the present invention, the mass ratio of TGE to TGE glycoside must be 1:10 or more. Furthermore, the present inventors have found that, from the viewpoint of particularly excellent fat decomposition promoting effect and liver function improving effect, the mass ratio of TGE to TGE glycoside is more preferably 1:13 or more, and particularly preferably 1:18 or more. It has been found that the composition of the present invention, in which the lower limit of the mass ratio of TGE to TGE glycoside is the above-mentioned value, is also excellent in the effects of promoting tissue differentiation, improving the stratum corneum moisture content, improving skin elasticity, and suppressing skin moisture evaporation. The composition of the present invention, in which the lower limit of the mass ratio of TGE to TGE glycoside is the above-mentioned value, may be an extract of a plant such as the flower parts of Belamcanda chinensis of the Iridaceae family or Pueraria thomsonii, Pueraria lobata, Pueraria thunbergiana, etc. of the Leguminosae family, or may be prepared by adding an organic synthetic product to a plant extract, or may be prepared by combining extracts from plants, or may be prepared by combining organic synthetic products.
ＴＧＥとＴＧＥ配糖体の質量比は、ＴＧＥ配糖体の量を一定以下とすることがＴＧＥ配糖体による皮膚炎、嘔吐、下痢、胃腸炎などの副作用を防ぐ観点から好ましい。この観点からＴＧＥとＴＧＥ配糖体の質量比は、好ましくは１：５００以下、より好ましくは１：２５０以下、さらに好ましくは１：１００以下、特に好ましくは１：５０以下とすることが好ましい。 It is preferable that the mass ratio of TGE to TGE glycoside is set to a certain amount or less in order to prevent side effects such as dermatitis, vomiting, diarrhea, and gastroenteritis caused by TGE glycoside. From this viewpoint, the mass ratio of TGE to TGE glycoside is preferably set to 1:500 or less, more preferably 1:250 or less, even more preferably 1:100 or less, and particularly preferably 1:50 or less.
本発明の組成物におけるＴＧＥ及びＴＧＥ配糖体の量の測定は、ＨＰＬＣ法にて、定量的または定性的に確認することができる。
ＨＰＬＣに供するための試料は、適宜不純物の除去や濃度調整等、公知の方法により調製される。
The amount of TGE and TGE glycoside in the composition of the present invention can be quantitatively or qualitatively confirmed by HPLC.
A sample to be subjected to HPLC is prepared by known methods, such as removing impurities and adjusting the concentration, as appropriate.
本発明の組成物は、上記ＴＧＥ及びＴＧＥ配糖体以外に、通常使用される他の成分を、本発明の効果を損なわない範囲で含有してもよい。このような成分としては、種々の賦形剤、結合剤、光沢剤、滑沢剤、安定剤、希釈剤、増量剤、増粘剤、乳化剤、酸化防止剤、ｐＨ調整剤、着色料、香料、添加剤などを挙げることができる。その他の成分の含有量は、本発明の組成物の形態等に応じて適宜選択することができる。 In addition to the above TGE and TGE glycoside, the composition of the present invention may contain other commonly used ingredients to the extent that the effects of the present invention are not impaired. Examples of such ingredients include various excipients, binders, gloss agents, lubricants, stabilizers, diluents, bulking agents, thickeners, emulsifiers, antioxidants, pH adjusters, colorants, fragrances, additives, etc. The content of the other ingredients can be appropriately selected depending on the form of the composition of the present invention, etc.
本発明の組成物は、外用又は経口用として使用することができる。外用剤としては、皮膚、頭皮等に塗布して用いるものであれば、特に制限はなく、その形態としては、軟膏剤、クリーム剤、ジェル剤、ローション剤、乳液剤、パック剤、湿布剤等の皮膚外用剤や、注射剤等の形態を挙げることができる。 The composition of the present invention can be used externally or orally. There are no particular limitations on the external preparation, so long as it is applied to the skin, scalp, etc., and the form of the external preparation can be, for example, an ointment, cream, gel, lotion, emulsion, pack, poultice, or injection.
また、本発明の組成物を経口剤として用いる場合、その形態としては、例えば、錠剤、カプセル剤、粉末剤、顆粒剤、液剤、粒状剤、棒状剤、板状剤、ブロック状剤、固形状剤、丸状剤、ペースト状剤、クリーム状剤、カプレット状剤、ゲル状剤、チュアブル状剤、スティック状剤等を挙げることができる。これらの中でも、錠剤、カプセル剤、粉末剤、顆粒剤、液剤の形態が特に好ましい。具体的には、サプリメント、食品添加剤、ペットボトル、缶、瓶等に充填された容器詰飲料、水（湯）、牛乳、果汁等に溶解して飲むためのインスタント粉末（顆粒）飲料等を例示することができる。これらは食事の際などに手軽に飲食しやすく、また嗜好性を高めることができるという点で好ましい。 When the composition of the present invention is used as an oral preparation, its form may be, for example, a tablet, capsule, powder, granule, liquid, granular, bar, plate, block, solid, pill, paste, cream, caplet, gel, chewable, or stick. Among these, tablets, capsules, powder, granule, and liquid forms are particularly preferred. Specific examples include supplements, food additives, packaged beverages filled in PET bottles, cans, bottles, and the like, and instant powder (granule) beverages to be dissolved in water (hot water), milk, fruit juice, and the like before drinking. These are preferred because they are easy to eat and drink during meals and can increase palatability.
一般的には、本発明の組成物が、錠剤，カプセル剤である場合には、本発明による肝機能改善効果をより高める観点から、有効成分がその固形分中、ＴＧＥを固形分全体の２．０～０．００１質量％含むことが好ましく、１．０～０．０１質量％含むことがより好ましく、０．５～０．０２５質量％含むことが更に好ましい。またＴＧＥ及びＴＧＥ配糖体の合計量は、本発明の組成物の固形分中、例えば０．５質量％以上、特に１質量％以上であると、肝機能の改善に十分な効果を発揮できるため好ましい。錠剤又はカプセル剤である本発明の組成物としては、例えば医薬品やサプリメント等が挙げられる。 In general, when the composition of the present invention is in the form of a tablet or capsule, from the viewpoint of further enhancing the effect of improving liver function according to the present invention, the active ingredient preferably contains TGE in an amount of 2.0 to 0.001% by mass of the total solid content, more preferably 1.0 to 0.01% by mass, and even more preferably 0.5 to 0.025% by mass. Furthermore, the total amount of TGE and TGE glycoside is, for example, 0.5% by mass or more, particularly 1% by mass or more, in the solid content of the composition of the present invention, which is preferable since it can exert a sufficient effect in improving liver function. Examples of the composition of the present invention in the form of a tablet or capsule include medicines and supplements.
本発明の組成物が液剤である場合には、本発明による前述した各効果をより高める観点から、有効成分がその液剤中、ＴＧＥを全体の０．００００１～０．５質量％含むことが好ましく、０．００００５～０．１質量％含むことがより好ましく、０．０００１～０．０５質量％含むことが更に好ましい。またＴＧＥ及びＴＧＥ配糖体の合計量は、本発明の組成物の固形分中、例えば０．０００１質量％以上、特に０．１質量％以上であると、前述した各効果が一層優れるため好ましい。液剤である本発明の組成物としては容器詰飲料等が挙げられる。 When the composition of the present invention is a liquid formulation, from the viewpoint of further enhancing each of the above-mentioned effects of the present invention, the active ingredient preferably contains 0.00001 to 0.5 mass% of TGE in the liquid formulation as a whole, more preferably 0.00005 to 0.1 mass%, and even more preferably 0.0001 to 0.05 mass%. Furthermore, the total amount of TGE and TGE glycoside is, for example, 0.0001 mass% or more, particularly 0.1 mass% or more, based on the solid content of the composition of the present invention, which is preferable since the above-mentioned effects are further improved. Examples of the composition of the present invention that is a liquid formulation include packaged beverages, etc.
また、本発明の組成物が粉末剤又は顆粒剤である場合には、本発明による前述した各効果をより高める観点から、有効成分がその固形分中、ＴＧＥを固形分全体の０．０００１～２．５質量％含むことが好ましく、０．００１～２．０質量％含むことがより好ましく、０．００５～１．５質量％含むことが更に好ましい。またＴＧＥ及びＴＧＥ配糖体の合計量は、本発明の組成物の固形分中、例えば０．００１質量％以上、特に０．０１質量％以上であると、前述した各効果が一層優れるため好ましい。粉末剤又は顆粒剤である本発明の組成物としてはインスタント粉末飲料、インスタント顆粒飲料等が挙げられる。 When the composition of the present invention is in the form of a powder or granules, from the viewpoint of further enhancing the above-mentioned effects of the present invention, the active ingredient preferably contains TGE in an amount of 0.0001 to 2.5% by mass, more preferably 0.001 to 2.0% by mass, and even more preferably 0.005 to 1.5% by mass, of the total solid content. Furthermore, the total amount of TGE and TGE glycoside is, for example, 0.001% by mass or more, particularly 0.01% by mass or more, of the solid content of the composition of the present invention, which is preferable since the above-mentioned effects are further improved. Examples of the composition of the present invention in the form of a powder or granules include instant powdered beverages and instant granulated beverages.
本発明の組成物を経口的に摂取する場合、その経口投与量は、上記ＴＧＥ及びＴＧＥ配糖体の乾燥物合計量で、成人１日当りおよそ５ｍｇ以上２００ｍｇ以下であることが好ましく、１０ｍｇ以上１００ｍｇ以下であることがより好ましい。また本発明の組成物の１回の摂取量は、好ましくは、上記ＴＧＥ及びＴＧＥ配糖体の乾燥物合計量で、成人１日当りおよそ１５ｍｇ以上５０ｍｇ以下であることが好ましい。本発明の組成物は、1日の摂取量が前記摂取量となるように、１つの容器に、又は例えば２～３の複数の容器に分けて、１日分として収容することができる。 When the composition of the present invention is orally ingested, the oral dosage is preferably from 5 mg to 200 mg, and more preferably from 10 mg to 100 mg, per day for an adult, in terms of the total amount of the dry matter of the TGE and TGE glycoside. The amount of the composition of the present invention ingested at one time is preferably from 15 mg to 50 mg, in terms of the total amount of the dry matter of the TGE and TGE glycoside, per day for an adult. The composition of the present invention can be stored as a daily dose in a single container, or divided into, for example, 2 to 3 multiple containers, so that the daily intake is the above-mentioned amount.
本発明の組成物の利用形態としては、具体的には、医薬品（医薬部外品を含む）、化粧品、一般食品、栄養機能食品、所定機関により効能の表示が認められた特定保健用食品、機能性表示食品等のいわゆる健康食品等を挙げることができる。 Specific examples of applications of the composition of the present invention include medicines (including quasi-drugs), cosmetics, general foods, functional foods, foods for specified health uses whose efficacy has been approved by a designated organization, and foods with functional claims, so-called health foods.
本発明の組成物の利用形態のうち、食品としては、例えば、炭酸飲料、栄養飲料、果実飲料、乳酸飲料、スムージー、青汁等の飲料；アイスクリーム、アイスシャーベット、かき氷等の冷菓；そば、うどん、はるさめ、中華麺、即席麺等の麺類；飴、キャンディー、ガム、チョコレート、錠菓、スナック菓子、ビスケット、ゼリー、ジャム、クリーム、焼き菓子、パン等の菓子類；かまぼこ、ハム、ソーセージ等の水産・畜産加工食品；加工乳、発酵乳、ヨーグルト等の乳製品；サラダ油、てんぷら油、マーガリン、マヨネーズ、ショートニング、ホイップクリーム、ドレッシング等の油脂及びその加工食品；ソース、醤油等の調味料；カレー、シチュー、親子丼、お粥、雑炊、中華丼、かつ丼、天丼、牛丼、ハヤシライス、オムライス、おでん、マーボドーフ、餃子、シューマイ、ハンバーグ、ミートボール、各種ソース、各種スープ等のレトルトパウチ食品などを挙げることができる。 Among the uses of the composition of the present invention, food products include, for example, beverages such as carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, smoothies, and green juice; frozen desserts such as ice cream, ice sherbet, and shaved ice; noodles such as soba, udon, harusame, Chinese noodles, and instant noodles; sweets such as candy, candy, gum, chocolate, tablet sweets, snacks, biscuits, jellies, jams, creams, baked goods, and bread; processed seafood and livestock foods such as kamaboko, ham, and sausages; processed milk, Examples include dairy products such as fermented milk and yogurt; fats and oils such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing, and processed foods; seasonings such as sauces and soy sauce; retort pouch foods such as curry, stew, oyakodon, rice porridge, porridge, Chinese rice bowl, katsudon, tempura bowl, beef bowl, hayashi rice, omelet rice, oden, mapo dolphin, gyoza, shumai, hamburger steak, meatballs, various sauces, and various soups.
本発明の組成物は、安価で安全に経口摂取できるのみならず、後述する実施例の記載から明らかな通り、ＴＧＥ及びＴＧＥ配糖体を特定比率で含有することによりこれを摂取することで、横紋筋等の筋芽細胞から筋肉分化を促進することができるほか、前骨芽細胞から骨芽細胞への分化を促進でき、骨分化を促進することができる。
また本発明の組成物はＴＧＥ及びＴＧＥ配糖体を特定比率で含有することによりこれを摂取することで、優れた脂肪分解作用を得ることができる。
例えば、後述する実施例の記載から明らかな通り、特定比率のＴＧＥ及びＴＧＥ配糖体は、これを摂取することにより、体脂肪を分解する酵素の遺伝子の発現を促進して、体脂肪分解を促進することができる。
The composition of the present invention can be orally ingested at low cost and safely. As will be apparent from the description of the Examples below, the composition contains TGE and TGE glycoside in a specific ratio. By ingesting the composition, it is possible to promote muscle differentiation from myoblasts such as striated muscles, as well as promote differentiation from pre-osteoblasts to osteoblasts, thereby promoting bone differentiation.
Furthermore, the composition of the present invention contains TGE and TGE glycoside in a specific ratio, and by ingesting this composition, an excellent lipolytic effect can be obtained.
For example, as will be apparent from the description of the examples below, ingesting a specific ratio of TGE and TGE glycoside can promote the expression of genes encoding enzymes that break down body fat, thereby promoting the breakdown of body fat.
更に本発明において、特定比率のＴＧＥ及びＴＧＥ配糖体は、肝機能改善作用を効果的に高める。具体的には、後述する実施例の記載から明らかな通り、特定比率のＴＧＥ及びＴＧＥ配糖体は、さまざまなストレスが誘因となって細胞死滅が生じやすい等の問題点がある無血清培地で培養した肝細胞の活性（酵素活性）を高めることができる。すなわち特定比率のＴＧＥ及びＴＧＥ配糖体が肝細胞の活性低下を抑制する、つまり保護している。このように、特定比率のＴＧＥ及びＴＧＥ配糖体は肝細胞を保護できるため、肝細胞の傷害を防ぎ、肝細胞の活性を維持又は増進し、肝機能を改善する。特に、さまざまなストレスから肝細胞を保護し、肝細胞の障害を防ぐことで、肝細胞中のアスパラギン酸アミノトランスフェラーゼ（ＡＳＴ）、アラニンアミノ基転移酵素（Alanine transaminase、ＡＬＴ）、γ－グルタミルトランスペプチダーゼ（γ－glutamyltransferase、γ－ＧＴＰ）の血中への逸脱が抑制されることが期待される。 Furthermore, in the present invention, TGE and TGE glycoside at a specific ratio effectively enhance the liver function improving effect. Specifically, as is clear from the description of the examples described later, TGE and TGE glycoside at a specific ratio can enhance the activity (enzyme activity) of liver cells cultured in a serum-free medium, which has problems such as cell death being easily induced by various stresses. In other words, TGE and TGE glycoside at a specific ratio suppress the decrease in activity of liver cells, that is, they protect them. In this way, TGE and TGE glycoside at a specific ratio can protect liver cells, so that they prevent damage to liver cells, maintain or enhance the activity of liver cells, and improve liver function. In particular, by protecting liver cells from various stresses and preventing damage to liver cells, it is expected that the escape of aspartate aminotransferase (AST), alanine transaminase (ALT), and gamma-glutamyltransferase (gamma-GTP) in liver cells into the blood will be suppressed.
また、特定比率のＴＧＥ及びＴＧＥ配糖体は、これを摂取することにより、皮膚における角層水分量向上作用、皮膚弾力向上作用、皮膚水分蒸散抑制作用を得ることができる。これらの肌質改善作用は継続的に本発明の組成物を摂取することにより顕著に示され、例えば、経口摂取開始から４週間以内に現れ、更に８週間の継続的摂取、特に１２週間の継続的摂取により顕著に顕れる。ここでいう継続的な摂取とは、例えば１～３日間に少なくとも１回の摂取する状態を継続することをいう。 Furthermore, by ingesting a specific ratio of TGE and TGE glycoside, it is possible to obtain the effects of increasing the moisture content of the stratum corneum of the skin, improving skin elasticity, and inhibiting skin moisture evaporation. These effects of improving skin quality are notable when the composition of the present invention is ingested continuously; for example, they appear within four weeks of starting oral intake, and are more notable when the composition is ingested continuously for eight weeks, and particularly for twelve weeks. "Continuous intake" here means, for example, continuing to ingest the composition at least once every one to three days.
このような本発明の組成物は、筋肉分化促進用組成物、筋肉形成促進用組成物、骨分化促進用組成物、骨形成促進用組成物、脂肪分解促進用組成物、脂質代謝促進用組成物、脂肪低減用組成物、抗肥満用組成物、ダイエット用組成物、美容組成物、皮膚水分量増加用組成物、水分蒸散抑制用組成物、肌弾力向上用組成物、肌質改善組成物などとして使用できるほか、肝細胞の逸脱酵素流出予防又は抑制作用、肝細胞障害予防又は抑制作用、肝細胞保護作用、肝機能保護又は改善作用による、肝機能改善剤又は肝機能改善組成物として使用でき、ウイルス性肝炎、薬物性肝障害、自己免疫性肝炎、原発性胆汁性肝硬変、Budd-Chiari症候群等の肝機能障害の治療又は予防に使用できる。このように、本発明の組成物は、二日酔い防止等の飲酒と関連した疾病以外の障害に適用することが可能である。 The composition of the present invention can be used as a composition for promoting muscle differentiation, a composition for promoting muscle formation, a composition for promoting bone differentiation, a composition for promoting bone formation, a composition for promoting fat decomposition, a composition for promoting lipid metabolism, a composition for reducing fat, an anti-obesity composition, a diet composition, a beauty composition, a composition for increasing skin moisture content, a composition for suppressing moisture evaporation, a composition for improving skin elasticity, a composition for improving skin quality, etc. In addition, it can be used as a liver function improving agent or liver function improving composition by preventing or suppressing the outflow of enzymes from liver cells, preventing or suppressing liver cell damage, protecting liver cells, and protecting or improving liver function, and can be used to treat or prevent liver dysfunction such as viral hepatitis, drug-induced liver damage, autoimmune hepatitis, primary biliary cirrhosis, and Budd-Chiari syndrome. In this way, the composition of the present invention can be applied to disorders other than those related to drinking, such as preventing hangovers.
また、本発明の組成物は、筋肉分化促進、筋肉形成促進、骨分化促進、骨形成促進、脂肪分解促進、抗肥満、ダイエット、美容、肌質改善、肝機能改善の用途に用いられる点において、製品として他の製品と区別できるものであればよく、例えば、本発明の肝機能改善剤に係る製品の本体、包装、説明書、宣伝物のいずれかに肝機能改善の機能がある旨を表示したものを挙げることができる。例えば、肝機能改善機能がある旨の表示とは、肝機能が気になる方に、肝臓の健康が気になる方に、などのように肝臓の働きを気にする対象者に訴えかける表示や、肝臓の健康を維持する、肝臓の働きをサポートする、健康な肝臓を維持する、健康な肝臓の機能を維持する、肝機能酵素に対して健常域で高めの数値を低下するなどのように肝臓の健康の維持増進に役立つことを表示するものをいう。また、脂肪分解促進の機能がある旨の表示とは、肥満が気になる方に、お腹まわりが気になる方に、体重が気になる方に、お腹の脂肪（内臓脂肪と皮下脂肪など）が気になる方に、などのように体脂肪を気にする対象者に訴えかける表示や、体重を減らすのを助ける、お腹の脂肪（内臓脂肪と皮下脂肪など）を減らすのを助ける、ウエスト周囲径を減らすのを助ける、肥満解消をサポートする、ダイエットをサポートするなどのように体の脂肪を低減するのに役立つことを表示するものをいう。 In addition, the composition of the present invention may be any product that can be distinguished from other products in terms of its use for promoting muscle differentiation, muscle formation, bone differentiation, bone formation, lipolysis, anti-obesity, dieting, beauty, skin quality improvement, and liver function improvement. For example, the product of the liver function improver of the present invention may be indicated on the product itself, packaging, instructions, or advertising materials to indicate that it has a function of improving liver function. For example, an indication of liver function improvement function refers to an indication that appeals to subjects who are concerned about liver function, such as those who are concerned about liver function or those who are concerned about liver health, or an indication that it is useful for maintaining and promoting liver health, such as maintaining liver health, supporting liver function, maintaining a healthy liver, maintaining healthy liver function, and reducing high values of liver function enzymes in the healthy range. In addition, a display indicating that a product has the function of promoting fat decomposition refers to a display that appeals to subjects who are concerned about body fat, such as those who are concerned about obesity, those who are concerned about their stomach area, those who are concerned about their weight, and those who are concerned about abdominal fat (visceral fat, subcutaneous fat, etc.), as well as a display indicating that a product is useful for reducing body fat, such as helping to lose weight, helping to reduce abdominal fat (visceral fat, subcutaneous fat, etc.), helping to reduce waist circumference, supporting obesity relief, and supporting dieting.
本発明の組成物は安全であり、長期間（例えば３か月間以上）投与（例えば１日に上記の投与量で投与した場合）を継続しても差し支えない。本発明の組成物は、このように継続的に使用することが好ましいものである。また本発明の組成物の投与対象としては、その肝細胞保護作用を生かして、ＡＳＴ、ＡＬＴ、γ－ＧＴＰなどの逸脱酵素の血中濃度が高い傾向のある対象者が好適に挙げられる。このような対象者が本発明の組成物を摂取することで、肝機能の低下防止効果をより一層高めることができる。 The composition of the present invention is safe and can be administered continuously for a long period of time (e.g., for three months or more) (e.g., when administered at the above-mentioned dosage per day). It is preferable to use the composition of the present invention continuously in this manner. In addition, subjects for whom the composition of the present invention is administered are preferably those who tend to have high blood concentrations of deviation enzymes such as AST, ALT, and γ-GTP, taking advantage of its hepatocyte protective effect. By ingesting the composition of the present invention by such subjects, the effect of preventing the decline of liver function can be further enhanced.
以下、実施例を挙げて本発明を更に詳細に説明する。しかし本発明の範囲はかかる実施例に限定されない。以下、特に断らない場合「％」は質量％、「部」は質量部を表す。 The present invention will be described in more detail below with reference to examples. However, the scope of the present invention is not limited to these examples. In the following, unless otherwise specified, "%" means "mass %" and "parts" means "mass parts".
〔実施例１〕
実施例１の被験物質として、質量比が、テクトリゲニン（ＴＧＥ）：テクトリゲニン配糖体＝１：２１（配糖体の内訳は質量比でＴＤ：ＴＧＸＧ＝１：１．９）の粉末状組成物を用いた。
これを下記の（１）～（３）の手順の筋肉分化試験、及び、下記（ア）～（オ）の手順の骨分化試験にそれぞれ供した。
Example 1
As the test substance in Example 1, a powdered composition having a mass ratio of tectorigenin (TGE):tectorigenin glycoside=1:21 (the breakdown of glycosides was TD:TGXG=1:1.9 in mass ratio) was used.
These were subjected to a muscle differentiation test according to the following procedures (1) to (3), and a bone differentiation test according to the following procedures (a) to (e).
（筋肉分化試験）
（１）マウス横紋筋由来筋芽細胞Ｃ２Ｃ１２（理化学研究所より入手）を、１０％ＦＢＳ含有ＤＭＥＭで所定の数になるまで、５容量％ＣＯ2インキュベーターにて、３７℃、湿潤条件で培養した。
次いで、培地を取り除き、ＤＰＢＳで３度洗浄したのち、Ｔｒｙｐｓｉｎ－ＥＤＴＡで細胞を剥離した。
新鮮な１０％ＦＢＳ含有ＤＭＥＭを加えてトリプシン反応を停止したのち、細胞をチューブへ集め、遠心機で８００ｒｐｍ、３分間遠心して細胞を沈殿させた。
２×１０4 ｃｅｌｌｓ／ｍＬになるように新鮮な１０％ＦＢＳ含有ＤＭＥＭに細胞を懸濁し、９６ｗｅｌｌ ｐｌａｔｅに１００μＬ／ｗｅｌｌで播種して、２日間、５容量％ＣＯ2インキュベーターにて、３７℃、湿潤条件で前培養した。
(Muscle differentiation test)
(1) Mouse striated muscle-derived myoblasts C2C12 (obtained from RIKEN) were cultured in 10% FBS-containing DMEM in a 5% by volume CO 2 incubator at 37° C. under humid conditions until they reached a predetermined number.
Then, the medium was removed, the cells were washed three times with DPBS, and the cells were detached with Trypsin-EDTA.
Fresh DMEM containing 10% FBS was added to stop the trypsin reaction, and the cells were then collected into a tube and centrifuged at 800 rpm for 3 minutes to precipitate the cells.
The cells were suspended in fresh 10% FBS-containing DMEM to a concentration of 2 x 104 cells/mL, seeded at 100 µL/well in a 96-well plate, and precultured for 2 days in a 5% by volume CO2 incubator at 37°C under humid conditions.
（２） 実施例１の被験物質をＴＧＥ及び配糖体の総量の濃度が４０ｍＭになるようにＤＭＳＯに溶解し、これを２％ウマ血清（ＨＳ）含有ＤＭＥＭで２００倍に希釈し、これを更に所定の２倍の濃度になるように０．５％ＤＭＳＯを含む２％ＨＳ含有ＤＭＥＭで希釈した。ここでいう所定濃度とは、ＴＧＥ及び配糖体の総量の濃度として２５μＭである。 (2) The test substance of Example 1 was dissolved in DMSO so that the total concentration of TGE and glycosides was 40 mM, and this was diluted 200-fold with DMEM containing 2% horse serum (HS), and this was further diluted with DMEM containing 2% HS containing 0.5% DMSO so that the concentration became twice as high as the specified concentration. The specified concentration here is 25 μM as the total concentration of TGE and glycosides.
（３）（１）で前培養をした細胞の培地を除き、分化誘導培地である２％ＨＳ含有ＤＭＥＭを５０μＬ、及び、前記（２）で調製した２％ＨＳ含有ＤＭＥＭを５０μＬ添加して、更に５％容量ＣＯ2インキュベーターにて、３７℃、湿潤条件で２４時間培養を続けた。一方、コントロールとして分化誘導培地である２％ＨＳ含有ＤＭＥＭを１００μＬ添加したものを同様に培養した。
２４時間後、培地を除き、細胞からRneasy mini(Qiagen社製)を用いてＲＮＡを精製した。
精製したＲＮＡよりReverTra Ace(登録商標) qPCR RT Master Mix with gDNA Remover(東洋紡社製)でｃＤＮＡを合成した。
内部標準としてGAPDH(Mm_Gapdh_3_SG QuantiTect Primer Assay、Qiagen社製)、測定遺伝子としてMyogenin(Mm_Myog_1_SG QuantiTect primer assay、Qiagen社製)のプライマー、QuantiNOVA SYBR GREEN(Qiagen社製)を用いて、Rotor-Gene Q(Qiagen社製)でＰＣＲを
おこなった。
ＰＣＲの結果はRotor Gene Q Pure Detection(Qiagen社製)を用いて解析した。
解析により得られた遺伝子発現量について、コントロール（比較例１）を１００％とした相対値を算出した。結果を図１に示す。
(3) The medium of the cells precultured in (1) was removed, and 50 μL of 2% HS-containing DMEM, which is a differentiation-inducing medium, and 50 μL of the 2% HS-containing DMEM prepared in (2) were added, followed by further culturing for 24 hours in a 5% CO2 incubator at 37° C. under humid conditions. On the other hand, as a control, a medium to which 100 μL of 2% HS-containing DMEM, which is a differentiation-inducing medium, was added was similarly cultured.
After 24 hours, the medium was removed, and RNA was purified from the cells using Rneasy mini (Qiagen).
cDNA was synthesized from the purified RNA using ReverTra Ace (registered trademark) qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd.).
PCR was performed using GAPDH (Mm_Gapdh_3_SG QuantiTect Primer Assay, Qiagen) as an internal standard, primers for myogenin (Mm_Myog_1_SG QuantiTect primer assay, Qiagen) as the measurement gene, and QuantiNOVA SYBR GREEN (Qiagen) using Rotor-Gene Q (Qiagen).
The PCR results were analyzed using Rotor Gene Q Pure Detection (Qiagen).
The gene expression levels obtained by the analysis were calculated as relative values with the control (Comparative Example 1) set at 100%. The results are shown in FIG.
一般に筋芽細胞において、Ｍｙｏｇｅｎｉｎ遺伝子の発現は、筋肉組織への分化誘導マーカーとして広く用いられている。図１に示すように、特定のＴＧＥ：ＴＧＥ配糖体量比を有する実施例１の組成物を分化誘導培地中の筋芽細胞に添加することで、Ｍｙｏｇｅｎｉｎ遺伝子発現量が向上している。このことは、実施例１の組成物が添加された筋芽細胞において、筋肉組織への分化が促進されたことを示している。 In general, in myoblasts, expression of the Myogenin gene is widely used as a marker for differentiation into muscle tissue. As shown in Figure 1, the amount of Myogenin gene expression is improved by adding the composition of Example 1 having a specific TGE:TGE glycoside ratio to myoblasts in a differentiation induction medium. This indicates that differentiation into muscle tissue is promoted in myoblasts to which the composition of Example 1 has been added.
（骨分化試験）
（ア）：マウス頭蓋冠由来前骨芽細胞ＭＣ３Ｔ３－Ｅ１細胞（ＤＳファーマバイオメディカル社より入手）を１０％ＦＢＳ含有ＭＥＭα培地（アスコルビン酸不含）で所定の数になるまで５容量％ＣＯ2インキュベーターにて、３７℃、湿潤条件で培養した。培地を取り除き、ＤＰＢＳで２度洗浄したのち、Ｔｒｙｐｓｉｎ－ＥＤＴＡで細胞を剥離した。新鮮な１０％ＦＢＳ含有ＭＥＭα培地を加えてトリプシン反応を停止したのち、細胞をチューブへ集め、遠心機で８００ｒｐｍ、３分遠心して細胞を沈殿させた。４×１０5 ｃｅｌｌｓ／ｍＬになるように新鮮な１０％ＦＢＳ含有ＭＥＭα培地に細胞を懸濁し、９６ｗｅｌｌ ｐｌａｔｅに１００μＬ／ｗｅｌｌで播種して１日間、５容量％ＣＯ2、３７℃、湿潤条件で前培養した。
(Bone differentiation test)
(A): Mouse calvaria-derived preosteoblast MC3T3-E1 cells (obtained from DS Pharma Biomedical) were cultured in 10% FBS-containing MEMα medium (ascorbic acid-free) in a 5% CO2 incubator at 37°C under humid conditions until a predetermined number of cells were obtained. The medium was removed, the cells were washed twice with DPBS, and then the cells were detached with trypsin-EDTA. After adding fresh 10% FBS-containing MEMα medium to stop the trypsin reaction, the cells were collected in a tube and centrifuged at 800 rpm for 3 minutes in a centrifuge to precipitate the cells. The cells were suspended in fresh 10% FBS-containing MEMα medium to a concentration of 4 x 105 cells/mL, seeded at 100 μL/well in a 96-well plate, and precultured for 1 day under 5% CO2 , 37°C, and humid conditions.
（イ）：アスコルビン酸ナトリウムとβグリセロリン酸二ナトリウムを１０％ＦＢＳ含有ＭＥＭα培地にそれぞれ５０μｇ／ｍＬ、１０ｍＭになるように溶解し、骨組織への分化を誘導する分化誘導培地とした。実施例１の被験物質を、ＴＧＥ及び配糖体の総量の濃度として２０ｍＭになるようにＤＭＳＯに溶解し、これを分化誘導培地で２００倍に希釈し、これをさらに所定の２倍の濃度になるように０．５％ＤＭＳＯを含む１０％ＦＢＳ含有ＭＥＭα培地で希釈し、被験物質含有分化誘導培地とした。ここでいう所定濃度とは、ＴＧＥ及び配糖体の総量の濃度として１００μＭである。 (a): Sodium ascorbate and disodium β-glycerophosphate were dissolved in 10% FBS-containing MEMα medium to 50 μg/mL and 10 mM, respectively, to prepare a differentiation-inducing medium for inducing differentiation into bone tissue. The test substance of Example 1 was dissolved in DMSO to a total concentration of TGE and glycosides of 20 mM, which was then diluted 200-fold with differentiation-inducing medium, and this was further diluted with 10% FBS-containing MEMα medium containing 0.5% DMSO to a predetermined concentration of twice the amount, to prepare a differentiation-inducing medium containing the test substance. The predetermined concentration here refers to a total concentration of TGE and glycosides of 100 μM.
（ウ）：（ア）で前培養をした細胞の培地を除き、（イ）で調製した被験物質含有分化誘導培地を１００μＬ／ｗｅｌｌ添加し、２日又は３日に一度、培地を交換しながら、１４日間５容量％ＣＯ2、３７℃、湿潤条件で培養を続けた。一方、コントロールとして、被験物質非含有分化誘導培地を別のｗｅｌｌに同量添加以外は、同様に培養した。 (C): The medium of the cells precultured in (A) was removed, and 100 μL/well of the differentiation-inducing medium containing the test substance prepared in (B) was added, and the culture was continued for 14 days under humid conditions of 5% by volume CO2 and 37°C, with the medium being replaced once every 2 or 3 days. On the other hand, as a control, the same amount of differentiation-inducing medium not containing the test substance was added to another well, and the cells were cultured in the same manner.
（エ）上記（ウ）の培養後、培地を除き、無血清ＤＭＥＭで１度洗浄したのち、無血清ＤＭＥＭ培地で３０倍に希釈したCell Counting-Kit 8（同仁化学社製）を１５０μＬ添加して３７℃で適当な発色まで保温した。４５０ｎｍの吸光度（Ａ１）をプレートリーダー（ThermoScientific社製）で測定した。
（オ）（エ）の測定後の培地からCell Counting-Kit 8を除き、ＴＲＡＰ／ＡＬＰ染色キット（和光純薬社製）の説明書に従い、アルカリホスファターゼ（ＡＬＰ）活性測定用の染色を行った。
次いで、グリシン、無水塩化マグネシウム、塩化亜鉛をそれぞれ０．１Ｍ、１ｍＭ、１ｍＭになるように超純水に溶解し、水酸化ナトリウムでｐＨ１０．４に調整し、グリシン緩衝液とした。得られたグリシン緩衝液５ｍｌに４－ニトロフェニルリン酸二ナトリウム塩六水和物 ５ｍｇタブレット（Ｓｉｇｍａ Ａｌｄｒｉｃｈ社製）を一粒溶解した。得られた液を、ＡＬＰ染色後の各ウェルに１５０μＬ加えた。
３７℃で３０分加温した後、上清１００μＬをアッセイプレート（ＡＧＣテクノグラス社製）に移し４０５ｎｍの吸光度（Ａ２）をプレートリーダーで測定した。
上記で測定した吸光度Ａ１及びＡ２を用いて、ＡＬＰ活性を下記式により求めた。
ＡＬＰ活性＝Ａ２／Ａ１
得られたＡＬＰ活性の値について、コントロール（比較例１）を１００％として相対値を算出した。結果を図２に示す。
(D) After the above culture (C), the medium was removed, the cells were washed once with serum-free DMEM, and 150 μL of Cell Counting-Kit 8 (Dojindo Chemical Industries, Ltd.) diluted 30-fold with serum-free DMEM medium was added and incubated at 37° C. until appropriate color development occurred. The absorbance (A1) at 450 nm was measured using a plate reader (ThermoScientific Corporation).
(E) and (D) were subjected to staining for measuring alkaline phosphatase (ALP) activity according to the instructions of the TRAP/ALP staining kit (Wako Pure Chemical Industries, Ltd.), after removing Cell Counting Kit 8 from the medium after the measurements.
Next, glycine, anhydrous magnesium chloride, and zinc chloride were dissolved in ultrapure water to 0.1 M, 1 mM, and 1 mM, respectively, and the pH was adjusted to 10.4 with sodium hydroxide to prepare a glycine buffer solution. One 5 mg tablet of 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma Aldrich) was dissolved in 5 ml of the obtained glycine buffer solution. 150 μL of the obtained solution was added to each well after ALP staining.
After heating at 37° C. for 30 minutes, 100 μL of the supernatant was transferred to an assay plate (AGC Technoglass) and the absorbance (A2) at 405 nm was measured using a plate reader.
Using the absorbances A1 and A2 measured above, the ALP activity was calculated according to the following formula.
ALP activity = A2/A1
The obtained ALP activity values were calculated as relative values, with the control (Comparative Example 1) set at 100%. The results are shown in FIG.
一般に、ＡＬＰ活性は、ＭＣ３Ｔ３－Ｅ１細胞に係る骨芽細胞への分化誘導マーカーとして広く用いられている。図２に示すように、特定のＴＧＥ：ＴＧＥ配糖体量比を有する実施例１の組成物を分化誘導培地中のＭＣ３Ｔ３－Ｅ１細胞に添加することで、ＡＬＰ活性が向上している。このことは、実施例１の組成物が添加されたＭＣ３Ｔ３－Ｅ１細胞において、骨芽分化が促進されたことを示している。 In general, ALP activity is widely used as a marker for inducing differentiation of MC3T3-E1 cells into osteoblasts. As shown in Figure 2, ALP activity is improved by adding the composition of Example 1 having a specific TGE:TGE glycoside ratio to MC3T3-E1 cells in a differentiation induction medium. This indicates that osteoblast differentiation is promoted in MC3T3-E1 cells to which the composition of Example 1 has been added.
〔実施例２－１～２－６、比較例２－１～２－４〕
被験物質として、ＴＤ、ＴＧＸＧ及びＴＧＥを以下の表１に記載で配合したものを用いた。表１中の配糖体とはＴＤ及びＴＧＸＧの合計量を指す。これら実施例及び比較例の被験物質を、下記の（ｉ）～（viii)の手順の（脂肪分解酵素発現試験）に供した。
[Examples 2-1 to 2-6, Comparative Examples 2-1 to 2-4]
The test substances used were TD, TGXG and TGE blended as shown in Table 1 below. The glycoside in Table 1 refers to the total amount of TD and TGXG. These test substances of the examples and comparative examples were subjected to the following procedures (i) to (viii) (lipolytic enzyme expression test).
（脂肪分解酵素発現試験）
ホルモン感受性リパーゼ（ＨＳＬ）の遺伝子発現を下記方法にて測定した。
（ｉ）３７℃、５容量％ＣＯ2インキュベーター内で、７５ｃｍ2フラスコを用いて脂肪細胞に分化するマウス線維芽細胞(３Ｔ３－Ｌ１)を前培養培地で培養した。前培養培地としては、１０％ＦＢＳ－ＤＭＥＭを用いた。
（ii)トリプシン処理により浮遊させた細胞を１０％ＦＢＳ－ＤＭＥＭに懸濁し、９６ｗｅｌｌ ｐｌａｔｅの各ｗｅｌｌに２×１０3ｃｅｌｌｓ／ｗｅｌｌの細胞密度で播種した。播種後の細胞を、３７℃、５容量％ＣＯ2インキュベーター内で２日間前培養した。
（iii)培地を除き、あらかじめ、デキサメタゾン、3-Isobutyl-1-methylxanthine、インスリンをそれぞれ１μＭ、１ｍＭ、２０μｇ／ｍＬ含むように調製しておいた１０％ＦＢＳ－ＤＭＥＭ（脂肪細胞への分化誘導培地）を１００μＬ／ｗｅｌｌ添加し、２日間３７℃、５容量％ＣＯ2インキュベーター内で培養した。
（iv)各ｗｅｌｌから培地を半分除き、あらかじめインスリンを２０μｇ／ｍＬ含むように調製しておいた１０％ＦＢＳ－ＤＭＥＭ（脂肪細胞分化維持培地）に交換し、さらに５日間３７℃、５容量％ＣＯ2インキュベーター内で培養した。この間、２日おきに培地の半分をインスリンを含む１０％ＦＢＳ－ＤＭＥＭに交換した。
（v) 各ｗｅｌｌから培地を完全に除き、あらかじめ被験物質が１００μｇ／ｍＬになるように調製しておいた１０％ＦＢＳ－ＤＭＥＭを１００μＬ／ｗｅｌｌ（ＴＧＥ及び配糖体の総量の濃度が１００μＭ）添加して、さらに４８時間３７℃、５容量％ＣＯ2インキュベーター内で培養を続けた。
（vi)培地を完全に除き、氷冷したＤＰＢＳで２回洗浄後、アイソジェン（ニッポンジーン社製）を１００μＬ／ｗｅｌｌ加え、取扱説明書の通り、総ＲＮＡを精製した。
（vii) （vi)で得られた総ＲＮＡから、ReverTra AceR qPCR RT Master Mix with gDNA Removerで取扱説明書に従い、ｃＤＮＡを合成した。
（viii)QuantiNova SYBR Green PCR Kitを用いて、（vii)で得られたｃＤＮＡを鋳型としてＲＴ－ＰＣＲをおこなった。プライマーとしてはＨＳＬ遺伝子発現用としてMm_Lipe_1_SG QuantiTect Primer Assayを用いた。内部標準にはActbをMm_Actb_1_SG QuantiTect Primer Assayを用いて行った。比較例１（コントロール）の遺伝子発現量を１とした時の相対値を図３に示す。
(Lipogenic enzyme expression test)
The expression of the hormone-sensitive lipase (HSL) gene was measured by the following method.
(i) Mouse fibroblasts (3T3-L1) that differentiate into adipocytes were cultured in a preculture medium using a 75 cm flask at 37° C. in a 5% CO incubator. 10% FBS- DMEM was used as the preculture medium.
(ii) The cells were suspended in 10% FBS-DMEM and seeded in each well of a 96-well plate at a cell density of 2 x 10 cells/well. The seeded cells were pre-cultured for 2 days in a 5% CO incubator at 37°C.
(iii) The medium was removed, and 100 μL/well of 10% FBS-DMEM (adipocyte differentiation-inducing medium) that had been previously prepared to contain 1 μM, 1 mM, and 20 μg/mL of dexamethasone, 3-isobutyl-1-methylxanthine, and insulin, respectively, was added, and the cells were cultured for 2 days in a 37° C., 5 % by volume CO incubator.
(iv) Half of the medium was removed from each well and replaced with 10% FBS-DMEM (adipocyte differentiation maintenance medium) that had been prepared in advance to contain 20 μg/ mL insulin, and the cells were further cultured for 5 days in a 5% by volume CO incubator at 37° C. During this period, half of the medium was replaced with 10% FBS-DMEM containing insulin every 2 days.
(v) The medium was completely removed from each well, and 100 μL/well of 10% FBS-DMEM (total concentration of TGE and glycosides: 100 μM) that had been previously prepared so that the test substance was 100 μg/mL was added, and the culture was continued for a further 48 hours at 37° C. in a 5% by volume CO incubator .
(vi) The medium was completely removed, and the plate was washed twice with ice-cold DPBS. Isogen (Nippon Gene) was added at 100 μL/well, and total RNA was purified according to the instruction manual.
(vii) cDNA was synthesized from the total RNA obtained in (vi) using ReverTra AceR qPCR RT Master Mix with gDNA Remover in accordance with the manufacturer's instructions.
(viii) RT-PCR was performed using the cDNA obtained in (vii) as a template with QuantiNova SYBR Green PCR Kit. As a primer for HSL gene expression, Mm_Lipe_1_SG QuantiTect Primer Assay was used. As an internal standard, Actb was used with Mm_Actb_1_SG QuantiTect Primer Assay. The relative values when the gene expression level in Comparative Example 1 (control) was set to 1 are shown in Figure 3.
図３に示すように、各実施例の被験物質を用いることにより、ホルモン感受性リパーゼ（ＨＳＬ）の遺伝子発現量が、各比較例を大幅に上回った。ホルモン感受性リパーゼ（ＨＳＬ）は中性脂肪分解酵素であり、脂肪細胞内に存在し、トリグリセリドを遊離脂肪酸とグリセロールとに分解し、遊離脂肪酸を血液中に放出させることが知られている。従って、本発明の組成物が、ホルモン感受性リパーゼ（ＨＳＬ）遺伝子発現の促進を通じて、脂肪細胞における脂肪分解を効果的に促進することが判る。 As shown in Figure 3, by using the test substances of each Example, the gene expression level of hormone-sensitive lipase (HSL) was significantly higher than that of each Comparative Example. Hormone-sensitive lipase (HSL) is a neutral fat-decomposing enzyme that is present in fat cells and is known to decompose triglycerides into free fatty acids and glycerol, releasing the free fatty acids into the blood. Therefore, it can be seen that the composition of the present invention effectively promotes fat decomposition in fat cells by promoting the expression of the hormone-sensitive lipase (HSL) gene.
（肝機能改善試験）
〔実施例３－１～３－１１並びに比較例３－１～３－４〕
（１）細胞培養
３７℃、５容量％ＣＯ2インキュベーター内で、７５ｃｍ2フラスコを用いて、ヒト肝癌由来細胞（ＨｅｐＧ２、ＡＴＣＣ社製）を１０％ウシ胎児血清（ＦＢＳ）含有培地により培養した。次いでトリプシン処理により浮遊させた細胞を、７５ｃｍ2フラスコから９６ｗｅｌｌ ｐｌａｔｅの各ｗｅｌｌに４．０×１０4ｃｅｌｌｓ／ｗｅｌｌの細胞密度で播種した。その後、３７℃、５容量％ＣＯ2インキュベーター内で２４時間前培養した。
各ｗｅｌｌより培地を除去後、所定濃度に調製した被験物質含有培地を１００μＬ／ｗｅｌｌ添加し、ＣＯ2インキュベーター内で２４時間培養した。被験物質含有培地における培地としては無血清ＤＭＥＭを使用した。被験物質としては、ＴＤ、ＴＧＸＧ及びＴＧＥを以下の表２に記載で配合したものを用いた。表２中の配糖体とはＴＤ及びＴＧＸＧの合計量を指す。上記所定濃度としては、ＴＤ、ＴＧＸＧ及びＴＧＥの総量の濃度として４００μｇ／ｍｌとした。比較例３－１はｃｏｎｔｒｏｌであり、被験物質含有培地の代わりに０．５％ＤＭＳＯ含有無血清ＤＭＥＭを用いた。
(Liver function improvement test)
[Examples 3-1 to 3-11 and Comparative Examples 3-1 to 3-4]
(1) Cell culture Human hepatoma-derived cells (HepG2, manufactured by ATCC) were cultured in a 75 cm2 flask in a 5% CO2 incubator at 37°C in a medium containing 10% fetal bovine serum (FBS). The cells were then suspended by trypsinization and seeded from the 75 cm2 flask into each well of a 96-well plate at a cell density of 4.0 x 104 cells/well. The cells were then precultured for 24 hours in a 5% CO2 incubator at 37°C.
After removing the medium from each well, 100 μL/well of medium containing the test substance prepared to a predetermined concentration was added, and the cells were cultured in a CO2 incubator for 24 hours. Serum-free DMEM was used as the medium for the test substance-containing medium. As the test substance, a mixture of TD, TGXG, and TGE as shown in Table 2 below was used. The glycoside in Table 2 refers to the total amount of TD and TGXG. The above-mentioned predetermined concentration was 400 μg/ml as the total concentration of TD, TGXG, and TGE. Comparative Example 3-1 was a control, and serum-free DMEM containing 0.5% DMSO was used instead of the test substance-containing medium.
＜細胞活性測定＞
上記の２４時間培養後、培地を除去し、無血清ＤＭＥＭで３０容量倍に希釈したCell Counting Kit-8溶液（同仁化学製）を各ｗｅｌｌに１５０μｌずつ添加した。３７℃、５容量％ＣＯ2インキュベーター内に静置し適度に発色させた後、４５０ｎｍにおける吸光度を測定した。
得られたデータを元に% of controlを算出した。
% of control = (Data sample－Data blank) ／（Data control－Data blank）×100
得られた結果を図４として示す。図４には、３連の平均値及び標準偏差を記載した。
<Cell activity measurement>
After 24 hours of culture, the medium was removed, and 150 μl of Cell Counting Kit-8 solution (Dojindo Chemical Industries, Ltd.) diluted 30 times by volume with serum-free DMEM was added to each well. The plate was placed in a 5% CO2 incubator at 37°C to allow adequate color development, and the absorbance at 450 nm was measured.
The % of control was calculated based on the obtained data.
% of control = (Data sample - Data blank) / (Data control - Data blank) x 100
The results are shown in Figure 4. In Figure 4, the average value and standard deviation of triplicate results are shown.
図４に示す結果から明らかな通り、ＴＧＥ及びその配糖体の量比が特定であるテクトリゲニン類は、無血清ＤＭＥＭで培養された肝細胞の活性を保護する優れた作用を有することが判る。従って、この質量比が特定であるテクトリゲニン類を用いることにより、肝細胞を保護し、肝機能を改善することが出来る。 As is clear from the results shown in Figure 4, tectorigenins with a specific mass ratio of TGE and its glycosides have an excellent effect of protecting the activity of liver cells cultured in serum-free DMEM. Therefore, by using tectorigenins with this specific mass ratio, liver cells can be protected and liver function can be improved.
〔実施例４〕
実施例４の被験物質として、質量比が、ＴＧＥ：配糖体＝１：２２．１９（ＴＤ：ＴＧＸＧ＝１：１．８１）の粉末状組成物４３．１４ｍｇ（ＴＧＥ及び配糖体の総量）を用いた。これを、下記美容試験に供した。
Example 4
As the test substance of Example 4, 43.14 mg (total amount of TGE and glycoside) of a powder composition having a mass ratio of TGE:glycoside=1:22.19 (TD:TGXG=1:1.81) was used. This was subjected to the following cosmetic test.
（美容試験）
健常な女性２人（平均年齢１９．７歳）を被験者とした。
被験者に水に溶解した実施例４の被験物質を毎朝摂取させた。これを１２週間継続させた。摂取開始前、摂取開始後（４，８，１２週間後）に左頬部（目尻と小鼻を直線で結んだときの中央付近）における角層水分量、水分蒸散量、弾力をそれぞれ下記の測定装置で測定した。角層水分量の平均値を図５に、水分蒸散量の平均値を図６に、弾力の平均値を図７に、それぞれ示す。
(Cosmetic Exam)
Two healthy women (mean age 19.7 years) participated in the study.
The test substance of Example 4 dissolved in water was administered to each subject every morning. This was continued for 12 weeks. Before and after the start of administration (4, 8, and 12 weeks later), the stratum corneum moisture content, moisture evaporation rate, and elasticity were measured on the left cheek (near the center of a straight line drawn between the outer corner of the eye and the nostril) using the following measuring device. The average values of the stratum corneum moisture content, moisture evaporation rate, and elasticity are shown in FIG. 5, FIG. 6, and FIG. 7, respectively.
・角層水分量：皮表角層水分量測定装置SKICON-200EX（アイ・ビイ・エス社製）で測定した。この皮表角層水分量測定装置は、皮膚のコンダクタンス（電気伝導度、単位：μＳ）を角層の水分量として評価したものである。
・水分蒸散量：Tewameter TM300（Courage+Khazaka社製）で測定した。
・弾力：皮膚粘弾力測定装置Cutometer MPA580（Courage+Khazaka社製）で測定した。弾力としては、測定器であるキュートメーターで得ることのできる皮膚粘弾性値の測定パラメータとして、測定部の皮膚を一定時間陰圧で吸引し、引き込まれた高さと吸引部頂点から戻った分の皮膚の変位を解析して求められる戻り弾性比率を用いた。戻り弾性比率は、１．００に近いほど、皮膚の弾力性は高いと言える。
これらの測定は、機器に付属の説明書に記載される標準的な方法で実施した。
- Stratum corneum moisture content: Measured using a skin epidermal stratum corneum moisture content measuring device SKICON-200EX (manufactured by IBS Co., Ltd.) This skin epidermal stratum corneum moisture content measuring device evaluates the skin's conductance (electrical conductivity, unit: μS) as the moisture content of the stratum corneum.
Water evaporation rate: Measured using a Tewameter TM300 (Courage+Khazaka).
Elasticity: Measured using a skin viscoelasticity measuring device Cutometer MPA580 (manufactured by Courage+Khazaka). Elasticity was measured using a skin viscoelasticity measuring device, Cutometer MPA580. The skin at the measurement point was sucked with negative pressure for a certain period of time, and the return elasticity ratio was calculated by analyzing the height of the skin that was sucked in and the amount of skin that returned from the apex of the suction point. The closer the return elasticity ratio is to 1.00, the higher the elasticity of the skin is.
These measurements were performed according to standard procedures described in the instructions provided with the instrument.
図５～図７の結果から、特定のＴＧＥ：配糖体量比を有する本発明の組成物摂取時期が長くなるにつれて、肌の角層水分量が向上し、水分蒸散量が抑制され、弾力が向上したことが判る。 The results in Figures 5 to 7 show that as the period of ingestion of the composition of the present invention having a specific TGE:glycoside ratio increases, the moisture content of the stratum corneum of the skin increases, the amount of water evaporation is suppressed, and elasticity improves.
Claims (1)
テクトリゲニン：（テクトリジン及びテクトリゲニン－７－Ｏ－キシロシルグルコシドの合計）の質量比が１：１８以上であり、
テクトリジン：テクトリゲニン－７－Ｏ－キシロシルグルコシドの質量比が１：０．１以上１．８１以下の量である、
角層水分量向上、皮膚弾力向上又は皮膚水分蒸散抑制に用いられる組成物。 Contains tectorigenin, tectoridin and tectorigenin-7-O-xylosylglucoside;
The mass ratio of tectorigenin: (the sum of tectoridin and tectorigenin-7-O-xylosylglucoside) is 1:18 or more,
The mass ratio of tectoridin to tectorigenin-7-O-xylosylglucoside is 1:0.1 or more and 1:81 or less;
A composition used for improving the moisture content of the stratum corneum, improving skin elasticity, or suppressing moisture evaporation from the skin.
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| JP2023108048A (en) | 2023-08-03 |
| JP2022113789A (en) | 2022-08-04 |
| JP2021102658A (en) | 2021-07-15 |
| JP6873467B2 (en) | 2021-05-19 |
| JP7304655B2 (en) | 2023-07-07 |
| JP2017171654A (en) | 2017-09-28 |
| JP7094049B2 (en) | 2022-07-01 |
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