JP7563674B2 - An agent for improving or preventing the decline in function of connective tissues whose main component is collagen by suppressing the deterioration of collagen structure caused by hypoxic conditions - Google Patents
An agent for improving or preventing the decline in function of connective tissues whose main component is collagen by suppressing the deterioration of collagen structure caused by hypoxic conditions Download PDFInfo
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- JP7563674B2 JP7563674B2 JP2023078383A JP2023078383A JP7563674B2 JP 7563674 B2 JP7563674 B2 JP 7563674B2 JP 2023078383 A JP2023078383 A JP 2023078383A JP 2023078383 A JP2023078383 A JP 2023078383A JP 7563674 B2 JP7563674 B2 JP 7563674B2
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- hypoxic conditions
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Description
本発明は、低酸素条件によるコラーゲン構造の悪化を抑制することによる、コラーゲンを主成分とする結合組織の機能低下の改善又は予防剤に関する。 The present invention relates to an agent for improving or preventing the decline in the function of connective tissues whose main component is collagen by suppressing the deterioration of collagen structure caused by hypoxic conditions.
近年、加齢に伴う人体の各組織の機能低下、すなわち老化現象を抑制することを目的とするアンチエイジングに関する研究が盛んに行われている。特に美容、化粧料の技術分野においては加齢に伴う肌のしわ、たるみ、しみなどの老化現象を抑制する有効成分の探索がされている。 In recent years, there has been active research into anti-aging, which aims to suppress the decline in function of various tissues in the human body that occurs with age, i.e., the aging phenomenon. In particular, in the fields of beauty and cosmetics, there has been a search for active ingredients that suppress aging phenomena such as wrinkles, sagging skin, and age spots that come with age.
例えば、特許文献１には、真皮の構造形成の制御に関わる遺伝子の発現を指標とすることで、皮膚のシワ、たるみ、ハリの低下を改善する素材をスクリーニングする方法が開示されている。
特許文献２には、細胞の核膜異常状態を指標として、シワの予防又は改善効果を有する成分をスクリーニングする方法が開示されている。
For example, Patent Document 1 discloses a method for screening materials that improve wrinkles, sagging, and loss of elasticity of the skin by using the expression of genes involved in the control of the formation of the dermis structure as an indicator.
Patent Document 2 discloses a method for screening components having wrinkle-preventing or improving effects, using an abnormal state of the nuclear membrane of a cell as an indicator.
ところで、血液ガス分析による解析により、動脈血のｐＯ２（酸素分圧）が低下することが知られている（非特許文献１）。これは加齢に伴い人体の各組織が低酸素の状態に置かれることを示している。
このような知見により、低酸素状態と老化現象の関連が示唆されているが、低酸素状況下でどのような生理学的変化が起こることで老化が起こるのか、そのメカニズムの全容は明らかとなっていない。
Meanwhile, it is known that the pO2 (oxygen partial pressure) of arterial blood decreases according to blood gas analysis (Non-Patent Document 1), which indicates that the tissues of the human body are placed in a state of hypoxia with aging.
These findings suggest a link between hypoxia and the aging phenomenon; however, the full mechanism by which physiological changes occur under hypoxic conditions that result in aging remains unclear.
一方、加齢に伴い結合組織が次第に柔軟性、弾力性を失い硬くなっていくことが知られている。これは、結合組織の主要成分であるコラーゲン線維が加齢とともに架橋し、結束してしまうことに起因していると考えられている（非特許文献２）。
しかし、加齢に伴うコラーゲン線維の結束がどのようなメカニズムで生じるのかについては明らかとなっていない。
On the other hand, it is known that connective tissues gradually lose flexibility and elasticity and become hard with age. This is thought to be due to the fact that collagen fibers, the main component of connective tissues, cross-link and bind with age (Non-Patent Document 2).
However, the mechanism by which collagen fiber bundling occurs with aging remains unclear.
生体より採取した組織片は、新規の素材を探索する際のツールとして用いられることがある。生体におけるコラーゲン構造に影響を与える成分の探索に当たっても、コラーゲン構造を含む真皮組織等の組織片を生体より採取し、これをモデルとして用いることは理論上可能である。
しかし、組織片は生体より採取されたものであるが故、その品質の均一性が担保されておらず、再現性に悖るという問題点がある。
Tissue fragments collected from living organisms can be used as tools for searching for new materials. In searching for components that affect collagen structures in living organisms, it is theoretically possible to collect tissue fragments such as dermal tissue that contains collagen structures from living organisms and use them as models.
However, because the tissue slices are collected from a living body, the uniformity of their quality cannot be guaranteed, which poses the problem of lack of reproducibility.
このような問題に鑑み、本発明の解決しようとする課題は、生体におけるコラーゲン構造の悪化を抑制する効果のある成分をスクリーニングするための新たな技術を提供することにある。 In view of these problems, the objective of the present invention is to provide a new technology for screening components that are effective in suppressing the deterioration of collagen structure in the body.
細胞を分散させたコラーゲン含有組成物を低酸素条件下でインキュベーションしたところ、驚くべきことにコラーゲン線維の結束度が上昇することが、発明者の鋭意研究努力により判明した。
この結果は、加齢に伴い人体の各組織が低酸素条件にさらされること（非特許文献１）、並びに、加齢に伴い結合組織におけるコラーゲン線維の結束が昂進すること（非特許文献２）、という２つの知見と合致するものである。
つまり、本発明者が構築した上記試験系は、人体の結合組織におけるコラーゲン線維の加齢に伴う結束度の向上を再現できる良好なモデルとして活用できることが明らかとなった。
Through the diligent research efforts of the inventors, it was surprisingly discovered that when a collagen-containing composition in which cells are dispersed is incubated under hypoxic conditions, the degree of collagen fiber cohesion increases.
This result is consistent with two findings: that with age, each tissue in the human body is exposed to hypoxic conditions (Non-Patent Document 1), and that with age, the bundling of collagen fibers in connective tissues increases (Non-Patent Document 2).
In other words, it has become clear that the above-mentioned test system constructed by the present inventors can be utilized as a good model capable of reproducing the increase in cohesion of collagen fibers in the connective tissue of the human body that occurs with aging.
かかる知見に基づき、本発明者は本発明を完成させた。
すなわち、上記課題を解決する本発明は、細胞を分散させたコラーゲン含有組成物に、被検成分を添加して、低酸素条件でインキュベーションしたときの、コラーゲン線維の結束度の上昇低減効果を指標とすることを特徴とする、低酸素条件及び／又は加齢による、コラーゲン構造の悪化を抑制する成分のスクリーニング方法である。
本発明によれば、低酸素条件や加齢に伴うコラーゲン構造の悪化を抑制する成分を簡便にスクリーニングすることができる。
Based on these findings, the present inventors have completed the present invention.
That is, the present invention, which solves the above-mentioned problems, is a method for screening components that suppress the deterioration of collagen structure due to hypoxic conditions and/or aging, characterized by using as an indicator the effect of reducing the increase in the degree of collagen fiber cohesion when a test component is added to a collagen-containing composition in which cells are dispersed and incubated under hypoxic conditions.
According to the present invention, it is possible to simply screen for components that suppress the deterioration of collagen structure that occurs under hypoxic conditions or with aging.
本発明の好ましい形態では、前記被検成分の存在下及び非存在下で、前記コラーゲン含有組成物を低酸素条件下でインキュベーションし、
被検成分の存在下でインキュベーションした後のコラーゲン線維の結束度が、被検成分の非存在下でインキュベーションした後のコラーゲン線維の結束度よりも低い場合に、
前記被検成分が、低酸素条件及び／又は加齢による、コラーゲン構造の悪化を抑制する成分であると判定することを特徴とする。
このように対照試験を実施する形態とすることにより、より正確なスクリーニングが可能となる。
In a preferred embodiment of the present invention, the collagen-containing composition is incubated under hypoxic conditions in the presence and absence of the test component,
If the degree of integrity of collagen fibers after incubation in the presence of the test component is lower than the degree of integrity of collagen fibers after incubation in the absence of the test component,
The test component is characterized in that it is determined to be a component that suppresses the deterioration of collagen structure due to hypoxic conditions and/or aging.
By carrying out a control test in this manner, more accurate screening is possible.
本発明は、低酸素条件や加齢に伴う結合組織のコラーゲン構造の悪化を抑制する成分のスクリーニングに応用することができる。 The present invention can be applied to screening for components that suppress the deterioration of collagen structure in connective tissues that occurs under hypoxic conditions or with aging.
本発明の好ましい形態では、前記細胞が結合組織細胞である。結合組織を構成する結合組織細胞を用いることで、結合組織におけるコラーゲン構造の悪化抑制成分をより精度よくスクリーニングすることができる。 In a preferred embodiment of the present invention, the cells are connective tissue cells. By using connective tissue cells that constitute connective tissue, it is possible to more accurately screen for components that inhibit the deterioration of collagen structure in connective tissue.
本発明の好ましい形態では、前記インキュベーション後のコラーゲン線維の顕微鏡撮影画像に基づき、前記コラーゲン線維の結束度を評価することを特徴とする。
顕微鏡撮影画像に基づく形態とすることで、コラーゲン線維の結束度の評価を容易に行うことができる。
In a preferred embodiment of the present invention, the degree of bundling of the collagen fibers is evaluated based on a microscopic image of the collagen fibers after the incubation.
By using a morphology based on a microscopic image, the degree of cohesion of collagen fibers can be easily evaluated.
本発明の好ましい形態では、前記顕微鏡撮影画像に対して画像解析処理を施し、前記コラーゲン線維の結束度を定量化した画像解析処理結果に基づき、前記コラーゲン線維の結束度を評価することを特徴とする。
このように定量的に評価する形態とすることにより、より精度の高いスクリーニングが実現できる。
In a preferred embodiment of the present invention, the microscopic image is subjected to image analysis processing, and the degree of cohesion of the collagen fibers is evaluated based on the image analysis processing results that quantify the degree of cohesion of the collagen fibers.
By adopting such a quantitative evaluation format, screening with higher accuracy can be achieved.
本発明の好ましい形態では、前記画像解析処理において、前記顕微鏡撮影画像に対してフーリエ変換処理を施して２次元空間周波数パワースペクトルを表すフーリエ変換画像を取得し、
該フーリエ変換画像の少なくとも原点を通過する直線を設定し、該直線の長さ方向について、該直線上における該フーリエ変換画像のパワーをプロットして得られる波形、又は、
該フーリエ変換画像から、少なくともその原点を含む略矩形領域画像を切り出し、切り出された略矩形領域画像の短径方向のパワーの平均値を、該略矩形領域画像の長径方向についてプロットして得られる波形、
を得ることを特徴とする。
フーリエ変換を用いることにより、複雑な顕微鏡撮影画像の定量的な評価が容易となる。
In a preferred embodiment of the present invention, the image analysis process includes performing a Fourier transform process on the microscopic image to obtain a Fourier transform image representing a two-dimensional spatial frequency power spectrum,
A waveform obtained by setting a straight line passing through at least the origin of the Fourier transform image and plotting the power of the Fourier transform image on the straight line in the length direction of the straight line, or
a waveform obtained by cutting out an approximately rectangular region image including at least the origin from the Fourier transform image, and plotting an average value of power in the short diameter direction of the cut out approximately rectangular region image in the long diameter direction of the approximately rectangular region image;
The present invention is characterized in that:
The use of Fourier transformation facilitates quantitative evaluation of complex microscopic images.
本発明の好ましい形態では、前記波形の傾斜部分の少なくとも一部を切り出し、前記傾斜部分の近似直線を作成し、該近似直線に対する前記波形を構成するデータのばらつきの程度が小さいほど、前記コラーゲン構造の悪化を抑制する効果に優れるものと判断することを特徴とする。
データのばらつきの程度という一次元的な尺度に基づきコラーゲン構造の結束度を評価することで、より簡便なスクリーニングが可能である。
In a preferred embodiment of the present invention, at least a portion of the sloping portion of the waveform is cut out, an approximation line of the sloping portion is created, and it is determined that the smaller the degree of variation of the data constituting the waveform with respect to the approximation line, the more excellent the effect of suppressing deterioration of the collagen structure.
By evaluating the degree of integrity of the collagen structure based on a one-dimensional measure, that is, the degree of data variability, screening can be performed more simply.
本発明の好ましい形態では、前記ばらつきの程度を標準偏差により評価することを特徴とする。
標準偏差により評価することにより、統計学的観点から精度の高い評価が可能となる。
In a preferred embodiment of the present invention, the degree of the variation is evaluated by a standard deviation.
Evaluation based on the standard deviation enables highly accurate evaluation from a statistical point of view.
本発明の好ましい形態では、前記低酸素条件が、細胞培養雰囲気中の酸素濃度が５％以下の条件であることを特徴とする。
このような条件下でインキュベーションを行うことにより、より効果的にスクリーニングを行うことができる。
In a preferred embodiment of the present invention, the hypoxic conditions are characterized in that the oxygen concentration in the cell culture atmosphere is 5% or less.
By carrying out the incubation under such conditions, screening can be carried out more effectively.
本発明は抗老化成分のスクリーニング方法に応用することができる。 The present invention can be applied to screening methods for anti-aging ingredients.
また本発明は、加齢に伴うシワ、たるみ又はハリの低下の改善又は予防成分のスクリーニング方法に応用することができる。 The present invention can also be applied to screening methods for ingredients that improve or prevent wrinkles, sagging, or loss of firmness associated with aging.
また、本発明は、アオイ科ゼニアオイ属（Ｍａｌｖａｃｅａｅ Ｍａｌｖａ）に属する植物の抽出物を有効成分として含む、低酸素条件及び／又は加齢による、コラーゲン構造の悪化の抑制剤にも関する。 The present invention also relates to an agent for inhibiting deterioration of collagen structure due to hypoxic conditions and/or aging, comprising an extract of a plant belonging to the genus Malvaceae Malva of the family Malvaceae as an active ingredient.
本発明によれば、低酸素条件及び／又は加齢によるコラーゲン構造の悪化を抑制する成分を容易にスクリーニングすることができる。 The present invention makes it easy to screen for components that suppress the deterioration of collagen structure due to hypoxic conditions and/or aging.
＜１＞スクリーニング方法
本発明は低酸素条件において進行するコラーゲン線維の架橋化による、コラーゲン構造の悪化を抑制する成分をスクリーニングする方法である。また、加齢に伴い組織が低酸素条件に置かれることから（非特許文献１）、本発明は、加齢によるコラーゲン構造の悪化を抑制する成分をもスクリーニングの対象とすることができる。
<1> Screening method The present invention is a method for screening components that suppress the deterioration of collagen structure caused by crosslinking of collagen fibers that progresses under hypoxic conditions. In addition, since tissues are placed under hypoxic conditions with aging (Non-Patent Document 1), the present invention can also be used to screen components that suppress the deterioration of collagen structure due to aging.
なお、骨、歯、軟骨、脂肪、腱、靱帯、真皮、皮下組織などの結合組織の主成分はコラーゲンである。よって、本発明は結合組織のコラーゲン構造の悪化を抑制する成分のスクリーニング方法に応用することができる。 The main component of connective tissues such as bones, teeth, cartilage, fat, tendons, ligaments, dermis, and subcutaneous tissue is collagen. Therefore, the present invention can be applied to a screening method for components that suppress the deterioration of the collagen structure of connective tissues.
特に、真皮組織のコラーゲン線維の架橋が進み、コラーゲン構造が悪化すると、皮膚のシワ、たるみ又はハリの低下に代表される老化が進む。
したがって、本発明は、抗老化成分、より具体的には、加齢に伴うシワ、たるみ又はハリの低下の改善又は予防成分のスクリーニング方法に応用することが好ましい。
以下、本発明の構成について詳述する。
In particular, when cross-linking of collagen fibers in the dermis tissue progresses and the collagen structure deteriorates, aging, typified by wrinkles, sagging, and loss of elasticity of the skin, progresses.
Therefore, the present invention is preferably applied to a method for screening anti-aging ingredients, more specifically, ingredients that improve or prevent wrinkles, sagging, or loss of firmness associated with aging.
The configuration of the present invention will be described in detail below.
（１）コラーゲン含有組成物
本発明においては、細胞を分散させたコラーゲン含有組成物を用いる。コラーゲン含有組成物は、コラーゲンを含有する組成物であり、細胞培養を阻害するような組成のものでなければ、その具体的構成は限定されない。
(1) Collagen-containing composition In the present invention, a collagen-containing composition having cells dispersed therein is used. The collagen-containing composition is a composition containing collagen, and the specific composition is not limited as long as it is not a composition that inhibits cell culture.
コラーゲン含有組成物は、細胞培養のための各種成分を含んでいることが好ましい。例えば、ＭＥＭ（Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ）、ＢＭＥ（Ｂａｓａｌ Ｍｅｄｉｕｍ Ｅａｇｌｅ）、ＤＭＥＭ（Ｄｕｌｂｅｃｃｏ´ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ）、ＥＭＥＭ（Ｅａｇｌｅ´ｓ ｍｉｎｉｍａｌ ｅｓｓｅｎｔｉａｌ ｍｅｄｉｕｍ）、ＩＭＤＭ（Ｉｓｃｏｖｅ´ｓ Ｍｏｄｉｆｉｅｄ Ｄｕｌｂｅｃｃｏ´ｓ Ｍｅｄｉｕｍ）、ＧＭＥＭ（Ｇｌａｓ－ ｇｏｗ´ｓ ＭＥＭ）、Ｆ１２（Ｈａｍ´ｓ Ｆ１２ Ｍｅｄｉｕｍ）、ＤＭＥＭ／Ｆ１２、ＲＰＭＩ１６４０、ＢＭＯＣ－３（Ｂｒｉｎｓｔｅｒ´ｓ ＢＭＯＣ－３ Ｍｅｄｉｕｍ）、ＣＭＲＬ－１０６６、Ｌ－１５培地（Ｌｅｉｂｏｖｉｔｚ´ｓ Ｌ－１５ ｍｅｄｉｕｍ）、ＭｃＣｏｙ’ｓ ５Ａ、Ｍｅｄｉａ １９９、ＭＥＭ αＭｅｄｉａ、ＭＣＤＢ１０５、ＭＣＤＢ１３１、ＭＣＤＢ１５３、ＭＣＤＢ２０１、Ｗｉｌｌｉａｍｓ’ ｍｅｄｉｕｍ Ｅなど細胞培養に常用される基本培地；ＦＢＳなどの血清；ＫＳＲなどの血清代替品；ＮａＨＣＯ３、ＨＥＰＥＳなどの緩衝剤；ｐＨ調製の目的でのアルカリや酸；ＦＧＦなどの各種成長因子などを例示できる。
コラーゲン含有組成物に分散させる細胞の種類により、上記任意成分の種類及びその濃度を適宜選択することができる。
The collagen-containing composition preferably contains various components for cell culture, such as MEM (Minimum Essential Medium), BME (Basal Medium Eagle), DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's minimal essential medium), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glas-gow's MEM), F12 (Ham's F12 Medium), DMEM/F12, RPMI1640, BMOC-3 (Brinster's BMOC-3), etc. Examples of such media include basal media commonly used in cell culture, such as CMRL-1066, L-15 medium (Leibovitz's L-15 medium), McCoy's 5A, Media 199, MEM αMedia, MCDB105, MCDB131, MCDB153, MCDB201, and Williams' medium E; serum, such as FBS; serum substitutes, such as KSR; buffers, such as NaHCO 3 and HEPES; alkalis or acids for the purpose of adjusting pH; and various growth factors, such as FGF.
The type and concentration of the optional components can be appropriately selected depending on the type of cells to be dispersed in the collagen-containing composition.
コラーゲン含有組成物は、液体状であってもゲル状であってもよい。生体内におけるコラーゲンの存在態様、より具体的には結合組織の態様に近づけるという観点から、コラーゲン含有組成物はコラーゲンゲルの形態とすることが好ましい。 The collagen-containing composition may be in the form of a liquid or a gel. From the viewpoint of approximating the state of collagen in the living body, more specifically, the state of connective tissue, it is preferable that the collagen-containing composition be in the form of a collagen gel.
コラーゲン含有組成物に含まれるコラーゲンの種類は特に限定されないが、Ｉ型～ＩＩＩ型、Ｖ型、ＸＩ型などの線維性コラーゲンが好適に例示できる。コラーゲンは、動物の皮膚などの結合組織より抽出することにより得ることができる。 The type of collagen contained in the collagen-containing composition is not particularly limited, but suitable examples include fibrous collagens such as types I to III, V, and XI. Collagen can be obtained by extraction from connective tissues such as animal skin.
コラーゲンとしては、市販品を特に制限なく用いることができる。例えば、Ａｔｅｌｏｃｏｌｌａｇｅｎ／Ｎａｔｉｖｅ Ｃｏｌｌａｇｅｎ Ａｃｉｄｉｃ Ｓｏｌｕｔｉｏｎｓ（株式会社高研社製）、Ｃｅｌｌｍａｔｒｉｘ Ｔｙｐｅ Ｉ－Ａ（新田ゼラチン株式会社製）、コラーゲンタイプＩＩＩウシ真皮由来（株式会社ニッピ製）などが挙げられる。 As collagen, commercially available products can be used without particular restrictions. Examples include Atelocollagen/Native Collagen Acidic Solutions (manufactured by Kokensha Co., Ltd.), Cellmatrix Type I-A (manufactured by Nitta Gelatin Co., Ltd.), and collagen type III derived from bovine dermis (manufactured by Nippi Co., Ltd.).
コラーゲン含有組成物におけるコラーゲンの濃度は特に限定されないが、好ましくは０．０１～１０質量％、より好ましくは０．０５～８質量％、さらに好ましくは０．１～５質量％、さらに好ましくは０．３～２質量％、さらに好ましくは０．５～１．５質量％である。 The concentration of collagen in the collagen-containing composition is not particularly limited, but is preferably 0.01 to 10% by mass, more preferably 0.05 to 8% by mass, even more preferably 0.1 to 5% by mass, even more preferably 0.3 to 2% by mass, and even more preferably 0.5 to 1.5% by mass.
（２）細胞
コラーゲン含有組成物に分散させる細胞の種類は特に限定されず、生体から採取した初代培養細胞や、株化された培養細胞を用いることができる。
なお、コラーゲンは結合組織の主要成分である。よって、生体における結合組織を再現したモデルとしての精度を向上させる観点からは、線維芽細胞、細網細胞、組織球、形質細胞、リンパ球、脂肪細胞、肥満細胞、顆粒白血球、色素細胞などの結合組織細胞を用いることが好ましく、中でも線維芽細胞を用いることが特に好ましい。
これら細胞については、初代培養細胞や株化細胞が市販されており、これを本発明のために制限無く使用することができる。
(2) Cells The type of cells to be dispersed in the collagen-containing composition is not particularly limited, and primary cultured cells collected from a living body or established cultured cell lines can be used.
Collagen is a major component of connective tissue, and therefore, from the viewpoint of improving the accuracy of a model that reproduces connective tissue in a living body, it is preferable to use connective tissue cells such as fibroblasts, reticular cells, histiocytes, plasma cells, lymphocytes, adipocytes, mast cells, granular leukocytes, and pigment cells, and among these, it is particularly preferable to use fibroblasts.
Regarding these cells, primary cultured cells and established cell lines are commercially available, and these can be used without restriction for the present invention.
コラーゲン含有組成物に分散させる細胞の数は特に限定されない。コラーゲン含有組成物の１ｍＬ当たりの細胞数としては、好ましくは１×１０２個～１×１０６個、より好ましくは１×１０３個～１×１０５個、さらに好ましくは５×１０３個～５×１０４個を目安とすることができる。 The number of cells dispersed in the collagen-containing composition is not particularly limited. The number of cells per mL of the collagen-containing composition is preferably 1× 10 to 1×10, more preferably 1× 10 to 1 × 10 , and even more preferably 5× 10 to 5× 10 .
コラーゲン含有組成物の調製方法は特に限定されない。
まず、コラーゲン含有組成物を構成するコラーゲン溶液を調製し、これに別途用意した細胞の懸濁液を加えて混合することでコラーゲン含有組成物を調製することが好ましい。
The method for preparing the collagen-containing composition is not particularly limited.
It is preferable to first prepare a collagen solution that constitutes the collagen-containing composition, and then add a separately prepared cell suspension to the collagen solution and mix the solution to prepare the collagen-containing composition.
（３）被検成分
被検成分をコラーゲン含有組成物に添加する際の態様は限定されない。好ましくは被検成分を含む溶液をコラーゲン含有組成物に添加する形態とする。被検成分を含む溶液は、コラーゲン含有組成物の調製に使用したものと同じ基本培地や血清などの培地成分を含むことが好ましい。
(3) Test Component The mode of adding the test component to the collagen-containing composition is not limited. Preferably, a solution containing the test component is added to the collagen-containing composition. The solution containing the test component preferably contains medium components such as the same basal medium and serum as those used in the preparation of the collagen-containing composition.
コラーゲン含有組成物をコラーゲンゲルの形態として本発明を実施する場合には、コラーゲン含有組成物がゲル化する前の溶液を培養容器に充填し、ＣＯ２インキュベーター内で数時間静置し、完全にゲル化した状態となってから、被検成分を添加することが好ましい。
また、被検成分の添加前に、ゲル化したコラーゲン含有組成物を培養容器の内壁から剥離させてから被検成分を添加することが好ましい。
When carrying out the present invention with the collagen-containing composition in the form of a collagen gel, it is preferable to fill a culture vessel with a solution of the collagen-containing composition before it gels, leave it in a CO2 incubator for several hours, and add the test component after it has completely gelled.
In addition, it is preferable to detach the gelled collagen-containing composition from the inner wall of the culture vessel before adding the test component.
コラーゲン含有組成物に対する、被検成分の溶液の添加量も特に限定されない。コラーゲン含有組成物に対する被検成分の溶液の体積比は、好ましくは０．１～１０、より好ましくは０．２～５、さらに好ましくは０．３～２を目安とすることができる。 There are no particular limitations on the amount of the solution of the test component added to the collagen-containing composition. The volume ratio of the solution of the test component to the collagen-containing composition is preferably 0.1 to 10, more preferably 0.2 to 5, and even more preferably 0.3 to 2.
（４）インキュベーション
被検成分の添加の後、コラーゲン含有組成物を低酸素条件下でインキュベーションする。
通常の細胞培養におけるＣＯ２インキュベーターにおいて酸素濃度は１８～１９％程度であるので、これよりも低い酸素濃度でインキュベーションする。具体的には、好ましくは１５％以下、より好ましくは１０％以下、より好ましくは７％以下、さらに好ましくは５％以下、さらに好ましくは３％以下の酸素濃度でインキュベーションする。
酸素濃度の下限は、コラーゲン含有組成物に分散された細胞が死滅しなければ特に制限されないが、好ましくは０．１％以上、より好ましくは０．５％以上の酸素濃度である。
(4) Incubation After the addition of the test component, the collagen-containing composition is incubated under hypoxic conditions.
In a CO2 incubator for normal cell culture, the oxygen concentration is about 18 to 19%, so incubation is performed at an oxygen concentration lower than this. Specifically, incubation is performed at an oxygen concentration of preferably 15% or less, more preferably 10% or less, more preferably 7% or less, even more preferably 5% or less, and even more preferably 3% or less.
The lower limit of the oxygen concentration is not particularly limited as long as the cells dispersed in the collagen-containing composition are not killed, but is preferably 0.1% or more, more preferably 0.5% or more.
低酸素条件でのインキュベーションにおいては、炭酸ガスの他に窒素ガスや混合ガスなどのボンベを併設した低酸素濃度培養用ＣＯ２インキュベーター（例えば、池本理化工業社製）を用いてもよいし、ガス濃度調節剤を備えるガスバリア性パウチからなる低酸素培養器具（例えば、スギヤマ技研製）を用いてもよい。 For incubation under low-oxygen conditions, a CO2 incubator for low-oxygen culture (e.g., manufactured by Ikemoto Rika Kogyo Co., Ltd.) equipped with a cylinder of nitrogen gas or mixed gas in addition to carbon dioxide gas may be used, or a low-oxygen culture device (e.g., manufactured by Sugiyama Giken Co., Ltd.) consisting of a gas barrier pouch equipped with a gas concentration regulator may be used.
インキュベーション期間は特に制限されず、コラーゲン含有組成物に分散した細胞の種類や数、これに基づき予想されるコンフルエントに達する時間等を考慮して適宜設計することができる。具体的には、好ましくは１２時間～１０日、より好ましくは１日～９日、さらに好ましくは３日～８日を目安とすることができる。
このインキュベーションの期間中、被検成分を含む培地を用いて培地交換をしてもよい。
The incubation period is not particularly limited, and can be appropriately designed taking into consideration the type and number of cells dispersed in the collagen-containing composition, the time to reach confluence predicted based on the type and number of cells, etc. Specifically, the incubation period can be preferably set to 12 hours to 10 days, more preferably 1 day to 9 days, and even more preferably 3 days to 8 days.
During this incubation period, the medium may be exchanged with medium containing the component to be tested.
なお、対照として、被検成分を添加せずにコラーゲン含有組成物をインキュベーションすることが好ましい。対照との比較によって、後述するコラーゲン線維の結束度の上昇低減効果の確認が容易となるからである。 As a control, it is preferable to incubate the collagen-containing composition without adding the test component. This is because comparison with the control makes it easier to confirm the effect of reducing the increase in the degree of collagen fiber cohesion, which will be described later.
（５）コラーゲン線維の結束度の観察
生体内では加齢に伴い結合組織のコラーゲンの架橋が進み、コラーゲン線維の結束度が向上する。細胞が分散されたコラーゲン含有組成物を低酸素条件下でインキュベーションしたときにも、この生体内での事象と同じく、コラーゲン含有組成物中のコラーゲン線維の結束度が上昇する。
本発明においては、被検成分を添加したときのコラーゲン含有組成物におけるコラーゲン線維の結束度の上昇低減効果を指標とする。
(5) Observation of the degree of collagen fiber cohesion In vivo, the degree of collagen fiber cohesion increases with age due to the progression of collagen cross-linking in connective tissues. When a collagen-containing composition in which cells are dispersed is incubated under hypoxic conditions, the degree of collagen fiber cohesion in the collagen-containing composition increases, similar to the phenomenon observed in vivo.
In the present invention, the index is the effect of reducing the increase in the degree of collagen fiber cohesion in a collagen-containing composition when a test component is added.
インキュベーション後のコラーゲン含有組成物におけるコラーゲン線維の結束度の確認方法は特に限定されないが、顕微鏡、特に電子顕微鏡による観察を好適に例示することができる。
電子顕微鏡としては走査型電子顕微鏡（ＳＥＭ）、透過型電子顕微鏡（ＴＥＭ）、及び走査型透過電子顕微鏡（ＳＴＥＭ）の何れを用いても構わない。
The method for confirming the degree of collagen fiber bundling in the collagen-containing composition after incubation is not particularly limited, but a suitable example is observation with a microscope, particularly an electron microscope.
As the electron microscope, any of a scanning electron microscope (SEM), a transmission electron microscope (TEM), and a scanning transmission electron microscope (STEM) may be used.
本発明においては顕微鏡撮影画像に基づき、コラーゲン含有組成物におけるコラーゲン線維の結束度を評価する形態とすることが好ましい。
顕微鏡で観察したとき、コラーゲン線維が束になって凝集しているように見える箇所が、コラーゲン線維の結束部分である。この結束部分の数や大きさなどを観察し、その程度が大きい場合に、結束度が高いと評価することができる。
In the present invention, it is preferable to evaluate the degree of bundling of collagen fibers in a collagen-containing composition based on a microscopic image.
When observed under a microscope, the collagen fibers are bundled and appear to be aggregated. The number and size of these bundles are observed, and when the degree of bundling is large, it can be evaluated as high.
顕微鏡撮影画像に基づく評価の実施の形態は特に制限されない。
例えば、予め用意しておいた基準写真を基に官能的に結束度を評価する形態としてもよい。
また、顕微鏡撮影画像に対して画像解析処理を施し、コラーゲン線維の結束度を定量化した画像解析処理結果に基づいて、結束度を評価する形態としてもよい。
The embodiment of the evaluation based on the microscopic image is not particularly limited.
For example, the degree of cohesion may be evaluated sensually based on a reference photograph prepared in advance.
Alternatively, an image analysis process may be performed on the microscopic image, and the degree of cohesion of collagen fibers may be evaluated based on the results of the image analysis process that quantifies the degree of cohesion.
ここでいう画像解析処理の手法は特に限定されない。顕微鏡撮影画像において結束部分は高強度（高光度、高明度、高輝度など）で表示される傾向がある。そのため、画像解析処理によって、強度（光度、明度、輝度など）のパラメータを計算処理することで、結束度を定量的に評価することができる。 The image analysis method referred to here is not particularly limited. In microscopic images, the bound portions tend to be displayed with high intensity (high luminosity, high brightness, high luminance, etc.). Therefore, the degree of bounds can be quantitatively evaluated by calculating the parameters of intensity (luminosity, brightness, luminance, etc.) through image analysis processing.
以下、画像解析処理としてフーリエ変換処理による手法を具体的に説明する。
フーリエ変換は周期性の評価手法である。２次元の画像に対してフーリエ変換を行った場合、パワーが濃淡で表された２次元空間周波数パワースペクトルを表すフーリエ変換画像が得られる。このフーリエ変換画像の中心は波数０の原点であり、原点より離れた位置ほど高波数を表す。
A method using Fourier transform processing as the image analysis processing will be specifically described below.
The Fourier transform is a method for evaluating periodicity. When a two-dimensional image is subjected to a Fourier transform, a Fourier transform image is obtained that represents a two-dimensional spatial frequency power spectrum in which power is represented by shading. The center of this Fourier transform image is the origin of wave number 0, and positions farther away from the origin represent higher wave numbers.
フーリエ変換の手法としては、離散フーリエ変換（ＤＦＴ）や、演算量を減らした高速フーリエ変換（ＦＦＴ）などが挙げられる。
なお、フーリエ変換で扱えるのは通常グレースケールの画像なので、顕微鏡撮影画像をグレースケールに変換してからフーリエ変換することが好ましい。
Examples of Fourier transform techniques include discrete Fourier transform (DFT) and fast Fourier transform (FFT), which requires a reduced amount of calculation.
Since the Fourier transform can usually handle grayscale images, it is preferable to convert the microscopic image to grayscale before performing the Fourier transform.
次に、フーリエ変換画像に基づき、パワーの波形に関するデータを得る。
パワーの波形は、以下の何れかの方法により算出することができる。
（ｉ）フーリエ変換画像の少なくとも原点を通過する直線を設定し、該直線の長さ方向について、該直線上における該フーリエ変換画像のパワーをプロットして得る（図１に概略図を示す）。
（ｉｉ）フーリエ変換画像から、少なくともその原点を含む略矩形領域画像を切り出し、切り出された略矩形領域画像の短径方向のパワーの平均値を、該略矩形領域画像の長径方向についてプロットして得る（図２に概略図を示す）。
Next, data regarding the power waveform is obtained based on the Fourier transform image.
The power waveform can be calculated by any of the following methods.
(i) A straight line is set that passes through at least the origin of the Fourier transform image, and the power of the Fourier transform image on the straight line is plotted in the length direction of the line (a schematic diagram is shown in FIG. 1).
(ii) An approximately rectangular area image including at least the origin is cut out from the Fourier transform image, and the average value of the power in the short diameter direction of the cut out approximately rectangular area image is plotted in the long diameter direction of the approximately rectangular area image (a schematic diagram is shown in FIG. 2).
図１及び図２の下段において、縦軸はパワーであるが、フーリエ変換画像においてパワーは濃淡により表現される。したがって、パワーについてはフーリエ変換画像の光度や輝度、明度に基づき算出することができる。 In the lower part of Figures 1 and 2, the vertical axis is power, but in the Fourier transform image, power is expressed by shading. Therefore, power can be calculated based on the luminance, brightness, and luminosity of the Fourier transform image.
また、図１及び図２の下段において、横軸は周波数であるが、フーリエ変換画像はデジタル画像としてコンピュータ上で処理されるため、画素（ｐｉｘｅｌ）に基づく位置情報として取得される。 In addition, in the lower part of Figures 1 and 2, the horizontal axis represents frequency, but since the Fourier transform image is processed on a computer as a digital image, it is obtained as position information based on pixels.
元データである顕微鏡撮影画像においては、上述した通り、コラーゲン構造の結束部分は高光度、高明度又は高輝度で表示される傾向がある。すなわち、コラーゲン構造の結束度が高い場合には、顕微鏡撮影画像において高光度、高明度又は高輝度の領域が高頻度に出現することとなる。そのため、これをフーリエ変換した２次元空間周波数パワースペクトルにおいては、周波数ごとのパワーにばらつきが生じやすい傾向となる。
したがって、波形で表されるパワーのばらつきの程度が高いほど、元データである顕微鏡撮影画像に表されたコラーゲン線維の結束度は高いと評価することができる。
In the original data, that is, the microscopic image, the bound portion of the collagen structure tends to be displayed with high luminance, high brightness, or high luminance, as described above. In other words, when the degree of binding of the collagen structure is high, areas of high luminance, high brightness, or high luminance appear frequently in the microscopic image. Therefore, in the two-dimensional spatial frequency power spectrum obtained by Fourier transforming this, the power for each frequency tends to vary.
Therefore, it can be evaluated that the greater the degree of variation in power represented by the waveform, the higher the degree of cohesion of the collagen fibers represented in the microscopic image, which is the original data.
このばらつきの程度は、波形の傾斜部分における近似直線（図３）に対する、波形を構成するデータのばらつきにより評価可能である。
例えば、近似直線と波形を構成するデータとのパワーの差分（Δパワー）を算出し、Δパワーのデータ集合についての標準偏差によって、波形を構成するデータのばらつきを客観的に評価することができる。
The degree of this variation can be evaluated from the variation of the data constituting the waveform with respect to the approximation line (FIG. 3) in the slope portion of the waveform.
For example, the power difference (Δpower) between the approximation line and the data constituting the waveform is calculated, and the variability of the data constituting the waveform can be objectively evaluated based on the standard deviation of the data set of Δpower.
＜２＞コラーゲン構造の悪化の抑制剤
アオイ科ゼニアオイ属（Ｍａｌｖａｃｅａｅ Ｍａｌｖａ）に属する植物の抽出物には、低酸素条件や加齢による、コラーゲン構造の悪化を抑制する効果がある。
つまり、アオイ科ゼニアオイ属に属する植物の抽出物は、コラーゲンを主成分とする組織である、骨、歯、軟骨、脂肪、腱、靱帯、真皮、皮下組織などの結合組織の機能低下の改善又は予防に効果を奏する。
<2> Inhibitor of Deterioration of Collagen Structure Extracts from plants belonging to the genus Malvaceae Malva of the family Malvaceae have the effect of inhibiting the deterioration of collagen structure caused by hypoxic conditions and aging.
In other words, extracts of plants belonging to the genus Malvaceae of the family Malvaceae are effective in improving or preventing functional decline in connective tissues, such as bones, teeth, cartilage, fat, tendons, ligaments, dermis, and subcutaneous tissue, which are tissues whose main component is collagen.
また、真皮組織のコラーゲン構造が悪化すると、皮膚のシワ、たるみ又はハリの低下に代表される老化が進む。
したがって、アオイ科ゼニアオイ属に属する植物の抽出物には、コラーゲン構造の悪化を抑制する効果に付随して、抗老化作用、より具体的には、加齢に伴う皮膚のシワ、たるみ又はハリの低下の改善又は予防効果があると言える。
Furthermore, deterioration of the collagen structure of the dermis tissue accelerates aging, typified by wrinkles, sagging, and loss of firmness of the skin.
Therefore, it can be said that extracts of plants belonging to the genus Malvaceae in the family Malvaceae have an anti-aging effect, more specifically, an effect of improving or preventing wrinkles, sagging, or loss of firmness in the skin that accompanies aging, in addition to the effect of suppressing the deterioration of collagen structure.
本発明の有効成分であるアオイ科ゼニアオイ属に属する植物としては、Ｍａｌｖａ ａｅｇｙｐｔｉａ Ｌ．、Ｍａｌｖａ ａｌｃｅａ Ｌ．、Ｍａｌｖａ ａｌｃｅａ ｖａｒ． ｆａｓｔｉｇｉａｔａ （Ｃａｖ．） Ｋ． Ｋｏｃｈ、Ｍａｌｖａ ａｒｂｏｒｅａ （Ｌ．） Ｗｅｂｂ ＆ Ｂｅｒｔｈｅｌ．、Ｍａｌｖａ ａｓｓｕｒｇｅｎｔｉｆｌｏｒａ （Ｋｅｌｌｏｇｇ） Ｍ．Ｆ．Ｒａｙ、Ｍａｌｖａ ｂｏｒｅａｌｉｓ Ｗａｌｌｍａｎ、Ｍａｌｖａ ｃａｎａｒｉｅｎｓｉｓ Ｍ．Ｆ．Ｒａｙ、Ｍａｌｖａ ｃａｔｈａｙｅｎｓｉｓ Ｍ．Ｇ． Ｇｉｌｂｅｒｔ， Ｙ． Ｔａｎｇ ＆ Ｄｏｒｒ、Ｍａｌｖａ ｃｒｅｔｉｃａ Ｃａｖ．、Ｍａｌｖａ ｃｒｅｔｉｃａ ｓｕｂｓｐ． ａｌｔｈａｅｏｉｄｅｓ （Ｃａｖ．） Ｄａｌｂｙ、Ｍａｌｖａ ｄｕｒｉｅｕｉ Ｓｐａｃｈ、Ｍａｌｖａ ｅｒｅｃｔａ Ｊ． Ｐｒｅｓｌ ＆ Ｃ． Ｐｒｅｓｌ、Ｍａｌｖａ ｈｉｓｐａｎｉｃａ Ｌ．、Ｍａｌｖａ ｉｌｊｉｎｉｉ Ｒｉｅｄｌ、Ｍａｌｖａ ｌｉｎｄｓａｙｉ （Ｍｏｒａｎ） Ｍ．Ｆ．Ｒａｙ、Ｍａｌｖａ ｍｏｓｃｈａｔａ Ｌ．（ジャコウアオイ）、Ｍａｌｖａ ｍｕｌｔｉｆｌｏｒａ （Ｃａｖ．） Ｓｏｌｄａｎｏ， Ｂａｎｆｉ ＆ Ｇａｌａｓｓｏ、Ｍａｌｖａ ｎｅｇｌｅｃｔａ Ｗａｌｌｒ．、Ｍａｌｖａ ｎｉｃａｅｅｎｓｉｓ Ａｌｌ．、Ｍａｌｖａ ｏｃｃｉｄｅｎｔａｌｉｓ （Ｓ．Ｗａｔｓｏｎ） Ｍ．Ｆ．Ｒａｙ、Ｍａｌｖａ ｐａｃｉｆｉｃａ Ｍ．Ｆ．Ｒａｙ、Ｍａｌｖａ ｐａｒｖｉｆｌｏｒａ Ｌ．（ウサギアオイ）、Ｍａｌｖａ ｐｒｅｉｓｓｉａｎａ Ｍｉｑ．、Ｍａｌｖａ ｐｓｅｕｄｏｌａｖａｔｅｒａ Ｗｅｂｂ ＆ Ｂｅｒｔｈｅｌ．、Ｍａｌｖａ ｐｕｓｉｌｌａ Ｓｍ．、Ｍａｌｖａ ｓｔｉｐｕｌａｃｅａ Ｃａｖ．、Ｍａｌｖａ ｓｕｂｏｖａｔａ （ＤＣ．） Ｍｏｌｅｒｏ ＆ Ｊ．Ｍ．Ｍｏｎｔｓ．、Ｍａｌｖａ ｓｙｌｖｅｓｔｒｉｓ Ｌ．（ウスベニアオイ）、Ｍａｌｖａ ｓｙｌｖｅｓｔｒｉｓ ｖａｒ． ｍａｕｒｉｔｉａｎａ （Ｌ．） Ｂｏｉｓｓ．（ゼニアオイ）、Ｍａｌｖａ ｔｏｕｒｎｅｆｏｒｔｉａｎａ Ｌ．、Ｍａｌｖａ ｖｅｒｔｉｃｉｌｌａｔａ Ｌ．（フユアオイ）、Ｍａｌｖａ ｖｉｄａｌｉｉ （Ｐａｕ） Ｍｏｌｅｒｏ ＆ Ｊ．Ｍ．Ｍｏｎｔｓ．などが挙げられる。
特に好ましくはゼニアオイ（Ｍａｌｖａ ｓｙｌｖｅｓｔｒｉｓ ｖａｒ． ｍａｕｒｉｔｉａｎａ （Ｌ．） Ｂｏｉｓｓ．）を例示できる。
Examples of plants belonging to the genus Malvaceae, which are the active ingredient of the present invention, include Malva aegyptia L., Malva alcea L., Malva alcea var. fastigiata (Cav.) K. Koch, Malva arborea (L.) Webb & Berthel., Malva assurgentiflora (Kellogg) M. F. Ray, Malva borealis Wallman, Malva canariensis M. F. Ray, Malva cathayensis M. G. Gilbert, Y. Tang & Dorr, Malva cretica Cav. , Malva cretica subsp. althaeoides (Cav.) Dalby, Malva durieui Spach, Malva erecta J. Presl & C. Presl, Malva hispanica L. , Malva iljinii Riedl, Malva lindsayi (Moran) M. F. Ray, Malva moschata L. (Musk mallow), Malva multiflora (Cav.) Soldano, Banfi & Galasso, Malva neglecta Wallr., Malva nicaensis All., Malva occidentalis (S. Watson) M. F. Ray, Malva pacifica M. F. Ray, Malva parviflora L. (Rabbit mallow), Malva preissiana Miq., Malva pseudolavatera Webb & Berthel., Malva pusilla Sm. , Malva stilacea Cav., Malva subovata (DC.) Molero & J.M. Monts., Malva sylvestris L. (mallow), Malva sylvestris var. mauritian (L.) Boiss. (mallow), Malva tournefortiana L., Malva verticillata L. (winter mallow), Malva vidali (Pau) Molero & J.M. Monts. and the like.
Particularly preferred is mallow (Malva sylvestris var. mauritian (L.) Boiss.).
アオイ科ゼニアオイ属に属する植物の抽出物を得る際の抽出部位は、特に限定されず、植物の花、葉、茎、根、種から選ばれる１種又は２種以上を用いて、抽出物を得ることができる。このなかでも、特に花から得られる抽出物が好ましい。 When obtaining an extract from a plant belonging to the genus Malvaceae in the family Malvaceae, there is no particular limitation on the part of the plant to be extracted, and the extract can be obtained using one or more parts selected from the flowers, leaves, stems, roots, and seeds of the plant. Among these, an extract obtained from the flowers is particularly preferred.
本発明における前記の植物の抽出物は、日本において自生又は生育された植物、漢方生薬原料などとして販売される日本産のものを用い抽出物を作製することもできるし、丸善株式会社などの植物抽出物を扱う会社より販売されている市販の抽出物を購入し、使用することもできる。 The plant extracts of the present invention can be prepared from plants that grow wild or are cultivated in Japan, or from plants that are sold in Japan as raw materials for traditional Chinese medicines, or commercially available extracts sold by companies that deal in plant extracts, such as Maruzen Co., Ltd., can be purchased and used.
抽出に際し、植物は予め、粉砕或いは細切して抽出効率を向上させるように加工することが好ましい。抽出物は、植物またはその乾燥物１質量に対して、溶媒を１～３０質量部加え、室温であれば数日間、沸点付近の温度であれば数時間浸漬する。浸漬後は、室温まで冷却し、所望により不溶物を除去した後、溶媒を減圧濃縮するなどにより除去することができる。その後、シリカゲルやイオン交換樹脂を充填したカラムクロマトグラフィ－などで分画精製し、所望の抽出物を得ることができる。 When extracting, it is preferable to process the plant in advance by crushing or cutting it into small pieces to improve extraction efficiency. To prepare the extract, 1 to 30 parts by mass of a solvent is added to 1 mass of the plant or its dried product, and the plant is soaked for several days at room temperature, or for several hours at a temperature near the boiling point. After soaking, the plant is cooled to room temperature, and insoluble matter is removed as desired, after which the solvent can be removed by concentrating under reduced pressure, for example. The desired extract can then be obtained by fractionation and purification using column chromatography filled with silica gel or ion exchange resin.
抽出溶媒としては、極性溶媒が好ましく、水、エタノ－ル、イソプロピルアルコ－ル、ブタノ－ルなどのアルコ－ル類、１，３－ブタンジオ－ル、ポリプロピレングリコ－ルなどの多価アルコ－ル類、アセトン、メチルエチルケトンなどのケトン類、ジエチルエ－テル、テトラヒドロフランなどのエ－テル類から選択される１種乃至は２種以上が好適に例示できる。 The extraction solvent is preferably a polar solvent, and suitable examples include one or more selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol, polyhydric alcohols such as 1,3-butanediol and polypropylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran.
本発明は外用剤又は経口剤の形態とすることが好ましい。
外用剤としては化粧料、医薬部外品、医薬品などが好適に例示でき、本発明の効果を損ねない限度において、通常使用される任意成分を含有することもできる。
The present invention is preferably in the form of an external preparation or an oral preparation.
Suitable examples of external preparations include cosmetics, quasi-drugs, and pharmaceuticals, and they may contain any optional ingredients that are commonly used to the extent that they do not impair the effects of the present invention.
このような任意成分としては、例えば、マカデミアナッツ油、アボカド油、トウモロコシ油、オリーブ油、ナタネ油、ゴマ油、ヒマシ油、サフラワー油、綿実油、ホホバ油、ヤシ油、パーム油、液状ラノリン、硬化ヤシ油、硬化油、モクロウ、硬化ヒマシ油、ミツロウ、キャンデリラロウ、カルナウバロウ、イボタロウ、ラノリン、還元ラノリン、硬質ラノリン、ホホバロウ等のオイル、ワックス類；流動パラフィン、スクワラン、プリスタン、オゾケライト、パラフィン、セレシン、ワセリン、マイクロクリスタリンワックス等の炭化水素類；オレイン酸、イソステアリン酸、ラウリン酸、ミリスチン酸、パルミチン酸、ステアリン酸、ベヘン酸、ウンデシレン酸等の高級脂肪酸類；セチルアルコール、ステアリルアルコール、イソステアリルアルコール、ベヘニルアルコール、オクチルドデカノール、ミリスチルアルコール、セトステアリルアルコール等の高級アルコール等；イソオクタン酸セチル、ミリスチン酸イソプロピル、イソステアリン酸ヘキシルデシル、アジピン酸ジイソプロピル、セバチン酸ジ－２－エチルヘキシル、乳酸セチル、リンゴ酸ジイソステアリル、ジ－２－エチルヘキサン酸エチレングリコール、ジカプリン酸ネオペンチルグリコール、ジ－２－ヘプチルウンデカン酸グリセリン、トリ－２－エチルヘキサン酸グリセリン、トリ－２－エチルヘキサン酸トリメチロールプロパン、トリイソステアリン酸トリメチロールプロパン、テトラ－２－エチルヘキサン酸ペンタンエリトリット等の合成エステル油類等の油剤類；脂肪酸セッケン（ラウリン酸ナトリウム、パルミチン酸ナトリウム等）、ラウリル硫酸カリウム、アルキル硫酸トリエタノールアミンエーテル等のアニオン界面活性剤類；塩化ステアリルトリメチルアンモニウム、塩化ベンザルコニウム、ラウリルアミンオキサイド等のカチオン界面活性剤類；イミダゾリン系両性界面活性剤（２－ココイル－２－イミダゾリニウムヒドロキサイド－１－カルボキシエチロキシ２ナトリウム塩等）、ベタイン系界面活性剤（アルキルベタイン、アミドベタイン、スルホベタイン等）、アシルメチルタウリン等の両性界面活性剤類；ソルビタン脂肪酸エステル類（ソルビタンモノステアレート、セスキオレイン酸ソルビタン等）、グリセリン脂肪酸類（モノステアリン酸グリセリン等）、プロピレングリコール脂肪酸エステル類（モノステアリン酸プロピレングリコール等）、硬化ヒマシ油誘導体、グリセリンアルキルエーテル、ＰＯＥソルビタン脂肪酸エステル類（ＰＯＥソルビタンモノオレエート、モノステアリン酸ポリオキエチレンソルビタン等）、ＰＯＥソルビット脂肪酸エステル類（ＰＯＥ－ソルビットモノラウレート等）、ＰＯＥグリセリン脂肪酸エステル類（ＰＯＥ－グリセリンモノイソステアレート等）、ＰＯＥ脂肪酸エステル類（ポリエチレングリコールモノオレート、ＰＯＥジステアレート等）、ＰＯＥアルキルエーテル類（ＰＯＥ２－オクチルドデシルエーテル等）、ＰＯＥアルキルフェニルエーテル類（ＰＯＥノニルフェニルエーテル等）、プルロニック型類、ＰＯＥ・ＰＯＰアルキルエーテル類（ＰＯＥ・ＰＯＰ２－デシルテトラデシルエーテル等）、テトロニック類、ＰＯＥヒマシ油・硬化ヒマシ油誘導体（ＰＯＥヒマシ油、ＰＯＥ硬化ヒマシ油等）、ショ糖脂肪酸エステル、アルキルグルコシド等の非イオン界面活性剤類；ポリエチレングリコール、グリセリン、エリスリトール、ソルビトール、キシリトール、マルチトール、プロピレングリコール、２，４－ヘキサンジオール等の多価アルコール類；ピロリドンカルボン酸ナトリウム、乳酸、乳酸ナトリウム等の保湿成分類；パラアミノ安息香酸系紫外線吸収剤；アントラニル酸系紫外線吸収剤；サリチル酸系紫外線吸収剤；桂皮酸系紫外線吸収剤；ベンゾフェノン系紫外線吸収剤；糖系紫外線吸収剤；２－（２'－ヒドロキシ－５'－ｔ－オクチルフェニル)ベンゾトリアゾール、４－メトキシ－４'－ｔ－ブチルジベンゾイルメタン等の紫外線吸収剤類；エタノール、イソプロパノール等の低級アルコール類フェノキシエタノール等の抗菌剤などが好ましく例示できる。 Such optional ingredients include, for example, oils and waxes such as macadamia nut oil, avocado oil, corn oil, olive oil, rapeseed oil, sesame oil, castor oil, safflower oil, cottonseed oil, jojoba oil, coconut oil, palm oil, liquid lanolin, hydrogenated coconut oil, hydrogenated oil, Japan wax, hydrogenated castor oil, beeswax, candelilla wax, carnauba wax, Ibota wax, lanolin, reduced lanolin, hard lanolin, and jojoba wax; liquid paraffin, squalane, pristane, ozokerite, paraffin, and ceramide. hydrocarbons such as oleic acid, isostearic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, undecylenic acid, and other higher fatty acids; higher alcohols such as cetyl alcohol, stearyl alcohol, isostearyl alcohol, behenyl alcohol, octyldodecanol, myristyl alcohol, and cetostearyl alcohol; cetyl isooctanoate, isopropyl myristate, and hexyl isostearate. Decyl, diisopropyl adipate, di-2-ethylhexyl sebacate, cetyl lactate, diisostearyl malate, ethylene glycol di-2-ethylhexanoate, neopentyl glycol dicaprate, di-2-heptylundecanoate, glycerin tri-2-ethylhexanoate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, pentane erythritol tetra-2-ethylhexanoate and other synthetic ester oils; anionic surfactants such as fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, and alkyl triethanolamine ether sulfate; cationic surfactants such as stearyl trimethylammonium chloride, benzalkonium chloride, and laurylamine oxide; imidazoline amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, etc.), betaine surfactants (alkyl betaine amphoteric surfactants such as acylmethyltaurine; sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acids (glycerin monostearate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hydrogenated castor oil derivatives, glycerin alkyl ethers, POE sorbitan fatty acid esters (POE sorbitan monooleate, poly(ethylene glycol) monostearate, etc.), POE sorbitan fatty acid esters (POE-sorbitan monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE 2-octyldodecyl ether, etc.), POE alkyl phenyl ethers (POE nonylphenyl ether, etc.), Pluronic types, POE/POP Nonionic surfactants such as alkyl ethers (POE/POP 2-decyltetradecyl ether, etc.), tetronics, POE castor oil/hydrogenated castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), sucrose fatty acid esters, and alkyl glucosides; polyhydric alcohols such as polyethylene glycol, glycerin, erythritol, sorbitol, xylitol, maltitol, propylene glycol, and 2,4-hexanediol; sodium pyrrolidone carboxylate, lactic acid, and lactic acid Preferred examples include moisturizing ingredients such as sodium; para-aminobenzoic acid-based UV absorbers; anthranilic acid-based UV absorbers; salicylic acid-based UV absorbers; cinnamic acid-based UV absorbers; benzophenone-based UV absorbers; sugar-based UV absorbers; UV absorbers such as 2-(2'-hydroxy-5'-t-octylphenyl)benzotriazole and 4-methoxy-4'-t-butyldibenzoylmethane; lower alcohols such as ethanol and isopropanol; and antibacterial agents such as phenoxyethanol.
経口剤としては、例えば、菓子やパン、麺などの一般食品、ドリンク製剤、カプセル剤や錠剤の形態をとる健康増進の目的を有する食品群（例えば、特定保健用食品等）、顆粒剤、粉末剤、カプセル剤や、錠剤の形態をとる経口投与医薬品等が例示できる。 Examples of oral preparations include general foods such as sweets, bread, and noodles, drink preparations, food groups intended to promote health in the form of capsules or tablets (e.g., foods for specified health uses, etc.), and orally administered pharmaceuticals in the form of granules, powders, capsules, or tablets.
経口剤の形態とする場合においては、許容される任意成分を含有することができる。この様な任意成分としては、食品であれば、塩、砂糖、グルタミン酸ナトリウム、イノシン酸ナトリウム、酢等の調味成分、着色成分、フレーバー等の矯臭成分、増粘剤、乳化・分散剤、保存料、安定剤、各種ビタミン類等が好適に例示でき、健康増進の目的を有する食品群や医薬品であれば、結晶セルロース、乳糖等の賦形剤、アラビヤガムやヒドロキシプロピルセルロース等の結合剤、クロスカルメロースナトリウム、デンプン等の崩壊剤、ステアリン酸マグネシウム等の滑沢剤、矯味、矯臭剤、着色剤、各種ビタミン類等が好ましく例示できる。これらを常法に従って処理することにより、本発明の経口投与組成物を製造することができる。 When the composition is in the form of an oral preparation, it may contain any acceptable optional ingredient. Suitable examples of such optional ingredients for foods include seasoning ingredients such as salt, sugar, monosodium glutamate, sodium inosinate, and vinegar, coloring ingredients, odor-correcting ingredients such as flavors, thickeners, emulsifiers/dispersants, preservatives, stabilizers, and various vitamins. Suitable examples of such optional ingredients for foods and pharmaceuticals that are intended to promote health include excipients such as crystalline cellulose and lactose, binders such as gum arabic and hydroxypropyl cellulose, disintegrants such as croscarmellose sodium and starch, lubricants such as magnesium stearate, flavorings, odorants, colorants, and various vitamins. The oral administration composition of the present invention can be produced by processing these ingredients according to conventional methods.
経口剤における前記植物の抽出物の総含有量は、固形分として、０．０５～１００質量％、より好ましくは３０～８０質量％とすることができる。
また、固形分として前記植物の抽出物を１日あたり１０～１０００ｍｇを１回又は数回に分けて飲用する形態とすることが好ましい。
The total content of the plant extract in the oral preparation can be 0.05 to 100% by mass, more preferably 30 to 80% by mass, in terms of solid content.
It is also preferable that the extract of the plant is taken in a form in which 10 to 1000 mg of solid content is taken once or in divided doses per day.
＜１＞コラーゲン含有組成物の調製
表１上段に示した成分を同表に示した質量比で氷冷しながら混合し、コラーゲン溶液Ａを調製した。コラーゲン溶液Ａと水を表１中段に示す質量比で混合し、コラーゲン溶液Ｂを調製した。２４ｗｅｌｌプレートにコラーゲン溶液Ｂを２５０μＬ／ｗｅｌｌ添加し、ＣＯ２インキュベーター内で１５分静置した。
一方、予め培養していた細胞を回収し、１×１０５ｃｅｌｌｓ／ｍＬに調製した細胞懸濁液と、コラーゲン溶液Ａとを表１下段に記載の割合で混合し、細胞を含むコラーゲン溶液Ｃを得た。
コラーゲン溶液Ｃを上で説明した２５０μＬ／ｗｅｌｌのコラーゲン溶液Ｂが入ったｗｅｌｌに１ｍＬ／ｗｅｌｌ添加し、ＣＯ２インキュベーター内で４時間静置することで、細胞が分散されたゲル状のコラーゲン含有組成物を調製した。
<1> Preparation of collagen-containing composition The components shown in the upper part of Table 1 were mixed in the mass ratio shown in the same table while cooling on ice to prepare collagen solution A. Collagen solution A and water were mixed in the mass ratio shown in the middle part of Table 1 to prepare collagen solution B. Collagen solution B was added to a 24-well plate at 250 μL/well and allowed to stand in a CO2 incubator for 15 minutes.
Meanwhile, cells that had been cultured in advance were collected, and a cell suspension adjusted to 1 x 10 5 cells/mL was mixed with collagen solution A in the ratio shown in the lower part of Table 1 to obtain collagen solution C containing cells.
Collagen solution C was added at 1 mL/well to wells containing 250 μL/well of collagen solution B described above, and the mixture was allowed to stand in a CO2 incubator for 4 hours to prepare a gel-like collagen-containing composition in which cells were dispersed.
マイクロスパーテルでコラーゲン含有組成物をプレートの内壁から剥離した。ここへ被検成分を含有する溶液（１０％ＦＢＳ ＤＭＥＭ）を７５０μＬ／ｗｅｌｌ添加した。
脱酸素剤を備えた低酸素培養器具（ＢＩＯＮＩＸ、スギヤマ技研製）に、この２４ｗｅｌｌプレートを封入し、酸素濃度１％に調整した状態で、ＣＯ２インキュベーター内でのインキュベーション（低酸素状態）を開始した。
インキュベーション開始から７２時間後、ｗｅｌｌ内の培地を再び被検成分を含有する溶液（１０％ＦＢＳ ＤＭＥＭ）７５０μＬ／ｗｅｌｌで交換した。その後、再びプレートを低酸素培養器具に封入し、９６時間インキュベーション（低酸素状態）した。
The collagen-containing composition was peeled off from the inner wall of the plate with a microspatula, and 750 μL/well of a solution containing a test component (10% FBS DMEM) was added thereto.
The 24-well plate was sealed in a hypoxic culture device (BIONIX, manufactured by Sugiyama Giken) equipped with an oxygen scavenger, and incubation (hypoxic condition) was started in a CO 2 incubator with the oxygen concentration adjusted to 1%.
After 72 hours from the start of incubation, the medium in the wells was replaced with 750 μL/well of a solution containing the test component (10% FBS DMEM).Then, the plate was again sealed in the hypoxic culture device and incubated (hypoxic condition) for 96 hours.
なお、本試験例においては被検成分としてゼニアオイ花エキスを用いた。
また、対照試験として、被検成分溶液に代えて培地（１０％ＦＢＳ ＤＭＥＭ）を添加したコラーゲン含有組成物を、低酸素培養器具へ封入（低酸素状態）又は未封入（通常酸素状態）の状態で同様にインキュベーションした。
In this test example, mallow flower extract was used as the test ingredient.
As a control test, a collagen-containing composition to which culture medium (10% FBS DMEM) was added instead of the test component solution was similarly incubated in a hypoxic culture device (hypoxic condition) or without being enclosed (normoxic condition).
＜２＞顕微鏡観察
インキュベーション後のゲル状のコラーゲン含有組成物を回収し、表２に示すフローで固定、脱水、真空凍結乾燥を行い、走査型電子顕微鏡（ＳＥＭ）によりコラーゲン構造を観察した。電子顕微鏡撮影画像を図４に示す。
<2> Microscopic Observation After incubation, the gel-like collagen-containing composition was collected, and then fixed, dehydrated, and vacuum freeze-dried according to the flow shown in Table 2. The collagen structure was observed with a scanning electron microscope (SEM). The electron microscope image is shown in FIG.
図４に示すように、通常酸素状態でインキュベーションしたものと比較し、低酸素状態でインキュベーションしたコラーゲン含有組成物においては、コラーゲン線維が結束し凝集した部分（図４の矢印で示す部分）が顕著に観察された。
一方、ゼニアオイ花エキスを添加したコラーゲン含有組成物は、同エキス非添加のものに比べてコラーゲン線維の顕著な結束が見られなかった。
As shown in FIG. 4, compared to that incubated under normal oxygen conditions, in the collagen-containing composition incubated under hypoxic conditions, areas in which collagen fibers were bundled and aggregated (areas indicated by arrows in FIG. 4) were prominently observed.
On the other hand, the collagen-containing composition to which mallow flower extract was added did not show significant bundling of collagen fibers compared to the composition to which the extract was not added.
この結果は、細胞を分散させたコラーゲン含有組成物に、被検成分を添加して、低酸素条件でインキュベーションしたときの、コラーゲン線維の結束度の上昇低減効果を指標とすることで、低酸素条件によるコラーゲン構造の悪化を抑制する成分をスクリーニングできることを示している。 These results indicate that by adding a test component to a collagen-containing composition containing dispersed cells and incubating it under hypoxic conditions, and using the effect of reducing the increase in collagen fiber cohesion as an indicator, it is possible to screen for components that suppress the deterioration of collagen structure due to hypoxic conditions.
また、加齢に伴い組織が低酸素条件に置かれることから（非特許文献１）、上記試験系は、加齢によるコラーゲン構造の悪化を抑制する成分のスクリーニングにも応用できることを示している。 In addition, because tissues are placed under hypoxic conditions as we age (Non-Patent Document 1), the above test system has been shown to be applicable to screening for components that suppress the deterioration of collagen structure due to aging.
また、本試験例の結果は、アオイ科ゼニアオイ属に属する植物の抽出物には、低酸素条件におけるコラーゲン構造の悪化を抑制する効果があることを示している。 The results of this test example also show that extracts from plants belonging to the genus Malvaceae in the family Malvaceae have the effect of suppressing the deterioration of collagen structure under hypoxic conditions.
＜３＞画像解析
図４に示す顕微鏡撮影画像について画像解析ソフト（IｍａｇｅＪ）を用いて高速フーリエ変換（ＦＦＴ）を施し、２次元空間周波数パワースペクトルを表す、フーリエ変換画像（ＦＦＴ画像）を取得した（図５）。
ＦＦＴ画像の中心（すなわち２次元空間周波数パワースペクトルの波数０の原点）を中心とした矩形領域を選択した（図５）。
選択した領域の縦方向の強度（つまり２次元空間周波数パワースペクトルのパワーに相当）を平均した数値を、同領域の横方向についてプロットした波形データを抽出した（図６）。
<3> Image Analysis The microscopic image shown in FIG. 4 was subjected to a fast Fourier transform (FFT) using image analysis software (ImageJ) to obtain a Fourier transform image (FFT image) representing a two-dimensional spatial frequency power spectrum (FIG. 5).
A rectangular region centered on the center of the FFT image (i.e., the origin of wavenumber 0 of the two-dimensional spatial frequency power spectrum) was selected (Figure 5).
The vertical intensity of the selected region (corresponding to the power of the two-dimensional spatial frequency power spectrum) was averaged and plotted against the horizontal intensity of the same region to extract waveform data (Figure 6).
この波形データにおける傾斜部分の一部（図６における２５４ｐｉｘｅｌ～３８１ｐｉｘｅｌの領域）を切り出し、この傾斜部分の波形を構成するデータについて近似直線を作成した（図７）。 A portion of the slope of this waveform data (the area from 254 pixels to 381 pixels in Figure 6) was extracted, and an approximation line was created for the data that constitutes the waveform of this slope portion (Figure 7).
この近似直線と波形を構成するデータとのパワーの差分（Δパワー、Δｐｏｗｅｒ）を算出し（図８）、標準偏差を計算した（表３）。 The power difference (Δpower) between this approximation line and the data that constitutes the waveform was calculated (Figure 8), and the standard deviation was calculated (Table 3).
表３に示す通り、低酸素状態でインキュベーションしたコラーゲン含有組成物の電子顕微鏡撮影画像の解析により算出された標準偏差は、通常酸素状態でインキュベーションしたものと比較し、顕著に大きかった。
一方、ゼニアオイ花エキスを添加したコラーゲン含有組成物においては、同エキス非添加のものに比べて、顕著に標準偏差が小さかった。
As shown in Table 3, the standard deviations calculated by analysis of electron microscopy images of collagen-containing compositions incubated under hypoxic conditions were significantly larger than those incubated under normal oxygen conditions.
On the other hand, the collagen-containing composition containing the mallow flower extract had a significantly smaller standard deviation than that containing no mallow flower extract.
この結果は、顕微鏡撮影画像より得られたフーリエ変換画像に表された、２次元空間周波数パワースペクトルのパワーの値のばらつきに基づき、被検成分のコラーゲン線維の結束度の向上低減効果を評価できることを示している。 These results indicate that the effect of the test component in improving or reducing the degree of collagen fiber cohesion can be evaluated based on the variation in the power values of the two-dimensional spatial frequency power spectrum shown in the Fourier transform image obtained from the microscopic image.
本発明はアンチエイジングに関する有効成分の探索に応用することができる。
The present invention can be applied to the search for effective ingredients for anti-aging.
Claims (5)
前記低酸素条件によるコラーゲン構造の悪化により、皮膚のシワ、たるみ又はハリが低下することを改善又は予防する、請求項１又は２に記載のコラーゲンを主成分とする結合組織の機能低下の改善又は予防剤。 The connective tissue mainly composed of collagen is the dermis,
3. An agent for improving or preventing functional decline of connective tissue whose main component is collagen, as described in claim 1 or 2 , which improves or prevents wrinkles, sagging or loss of firmness of the skin caused by deterioration of collagen structure due to the hypoxic conditions.
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