JP2013018715A - Mfap-4 production promoter - Google Patents
Mfap-4 production promoter Download PDFInfo
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- JP2013018715A JP2013018715A JP2011151224A JP2011151224A JP2013018715A JP 2013018715 A JP2013018715 A JP 2013018715A JP 2011151224 A JP2011151224 A JP 2011151224A JP 2011151224 A JP2011151224 A JP 2011151224A JP 2013018715 A JP2013018715 A JP 2013018715A
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- mfap
- skin
- sesame seeds
- extracts
- extract
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Abstract
Description
本発明は、ＭＦＡＰ−４産生促進、ならびに皮膚の光損傷／光老化を予防又は改善することができる物質に関する。 The present invention relates to a substance capable of promoting MFAP-4 production and preventing or improving photo-damage / photoaging of the skin.
光損傷や光老化とは、慢性的な日光照射、特に紫外線（ＵＶ）への曝露によって皮膚の内的な老化プロセスが加速されて起こる皮膚の変化である。皮膚の光損傷／光老化は、真皮におけるマトリックスメタロプロテアーゼ（ＭＭＰ）の発現上昇や、コラーゲンやエラスチンなどの細胞外マトリックス（ＥＣＭ）の発現減少・分解、又は表皮におけるケラチン６及び１６の発現上昇等の、種々の生理学的変化を伴うことが知られている。また光老化した皮膚においては、ＭＭＰ−１２やエラスターゼなどの弾性線維分解酵素の活性が亢進しており、分解された弾性線維やコラーゲン線維が真皮網状層に蓄積しているのがよく観察される（日光性弾性線維変性）。しかし、皮膚の光損傷／光老化の発症やそれに伴って見られる上記の様々な生理学的変化が、どのようなメカニズムで発生しているのかは未だ十分には明らかにされていない。 Photodamage and photoaging are skin changes caused by acceleration of the skin's internal aging process by exposure to chronic sunlight, particularly ultraviolet light (UV). Skin photodamage / photoaging is caused by increased expression of matrix metalloproteinase (MMP) in the dermis, decreased expression / degradation of extracellular matrix (ECM) such as collagen and elastin, or increased expression of keratins 6 and 16 in the epidermis. It is known to be accompanied by various physiological changes. In photoaged skin, the activity of elastic fiber degrading enzymes such as MMP-12 and elastase is enhanced, and it is often observed that the decomposed elastic fibers and collagen fibers are accumulated in the dermal reticulate layer. (Sunlight elastic fiber degeneration). However, it has not yet been fully clarified by what mechanism the occurrence of the photodamage / photoaging of the skin and the various physiological changes seen therewith are occurring.
真皮の弾性の維持には、細胞外マトリックス（ＥＣＭ）に存在する弾性線維やコラーゲン線維が重要な役割を担っている。弾性線維は、ミクロフィブリルにトロポエラスチン分子の凝集体が重合して構築されている。弾性線維の土台となるミクロフィブリルは、３５０ｋＤａの糖タンパク質であるフィブリリン−１から主に構成されており、直列及び並列に重合したフィブリリン−１に種々のタンパク質が作用することで構築される。 Elastic fibers and collagen fibers existing in the extracellular matrix (ECM) play an important role in maintaining the elasticity of the dermis. Elastic fibers are constructed by polymerizing aggregates of tropoelastin molecules on microfibrils. Microfibrils that form the basis of elastic fibers are mainly composed of fibrillin-1, which is a 350 kDa glycoprotein, and are constructed by the action of various proteins on fibrillin-1 polymerized in series and in parallel.
ｍｉｃｒｏｆｉｂｒｉｌｌａｒ−ａｓｓｏｃｉａｔｅｄ ｐｒｏｔｅｉｎ ４（ＭＦＡＰ−４）は、Ａｒｇ−Ｇｌｙ−Ａｓｐ（ＲＧＤ）配列とフィブリノーゲン様ドメインを含む２５５アミノ酸からなるタンパク質として最初に同定された。ＭＦＡＰ−４は、顔形状に異変（四角の顔、深くくぼんだ目、膨らんだ頬、低く突き出た顎など）が認められるＳｍｉｔｈ−Ｍａｇｅｎｉｓ ｓｙｎｄｒｏｍｅの原因遺伝子として知られている（非特許文献１）ほか、光老化した真皮ではその発現が消失しており、エラスチンに関係のある疾患ｐｓｅｕｄｏｘａｎｔｈｏｍａ ｅｌａｓｔｉｃｕｍにおいては、分解した弾性線維の蓄積部位に所見が認められることが報告されている（非特許文献２）。しかし、ＭＦＡＰ−４の機能や作用機序については未だ十分明らかにされていなかった。 microfibrillar-associated protein 4 (MFAP-4) was first identified as a 255 amino acid protein containing an Arg-Gly-Asp (RGD) sequence and a fibrinogen-like domain. MFAP-4 is known as a causative gene of Smith-Magenis syndrome in which abnormal facial shape (square face, deeply indented eyes, swollen cheeks, low protruding jaw, etc.) is recognized (Non-patent Document 1). In addition, the expression has disappeared in photoaged dermis, and in the disease related to elastin, pseudoxanthoma elasticum, it has been reported that findings are observed in the accumulation site of decomposed elastic fibers (Non-patent Document 2). . However, the function and mechanism of action of MFAP-4 have not yet been fully clarified.
ＭＦＡＰ−４が皮膚の光損傷／光老化に何らかの役割を果たしている可能性がある。さらに、ＭＦＡＰ−４の発現を制御することで皮膚の光損傷／光老化を改善できると期待される。 MFAP-4 may play a role in skin photodamage / photoaging. Furthermore, it is expected that skin photodamage / photoaging can be improved by controlling the expression of MFAP-4.
ゴマノハグサは、ゲンジン（玄參）とも称され、ゴマノハグサ科ゴマノハグサ属のScrophularia buergerianaを指す。これは従来、消炎、強心、利胆、利尿、傷薬等のために利用されてきた植物である。ゴマ（胡麻）は、クロゴマ（黒芝麻）、クロシマ（黒脂麻）とも称され、英明はsesameであり、ゴマ科ゴマ属のSesamum orientale又はSesamum indicumを指す。これは従来、虚弱体質、病後、便秘、消炎等のために利用されてきた植物である。ウメノキゴケ（梅樹苔）は、セキカ（石花）とも称され、ウメノキゴケ科ウメノキゴケ属のParmelia tinctorumを指す。これは従来、止血、消腫、利尿等のため、又はリトマス色素の原料として利用されてきた植物である。キンバイザサ（金梅笹）は、センボウ（仙茅）とも称され、英名Curculigo Gaertnであり、キンバイザサ科クルグリゴ属のCurculigo orchioidesを指す。これは従来、強壮薬、散湿薬として利用されてきた植物である。チョウカシ（澄茄子）は、ヒッチョウカとも称され、クスノキ科ハマビワ属のLitsea mollifoliaを指す。これは従来、食欲不振、消化不良、下痢、嘔吐等のために利用されてきた植物である。エゾミクリ（蝦夷実栗）は、サンリョウ（三稜）とも称され、ミクリ科ミクリ属のSparganium simplex又はSparganium emersumを指す。これは従来、痛経、生理不順、産後の腹痛、貧血、利尿等のために利用されてきた植物である。ショウロシランは、ロウロ（漏蘆）、テンセイソウ（天生草）とも称され、ユリ科のDiuranthera minorを指す。これは従来、清熱、解毒等のために利用されてきた植物である。ヨロイグサ（鎧草）は、ビャクシ、カラビャクシ、ウドモドキとも称され、英名Angelicaであり、セリ科シシウド属のAngelica dahuricaを指す。これは従来、消炎、鎮痛、排膿、肉芽形成、痒み防止のために利用されてきた植物である。ウチワドコロ（団扇野老）は、センザンリュウ（穿山竜）、コウモリドコロ（蝙蝠野老）とも称され、ヤマノイモ科ヤマノイモ属のDioscorea nipponicaを指す。これは従来、止痛、去痰、止咳のために利用されてきた植物である。ビャクダン（白檀）は、サンダルウッド（Sandalwood）又はサンダル（Sandal）とも称され、ビャクダン科ビャクダン属のSantalum albumを指す。これは従来香木として利用されてきた植物である。 It is also called Genzin (genus) and refers to Scrophularia buergeriana belonging to the genus Ganoderma. This is a plant that has been used for anti-inflammation, cardiotonia, bile, diuresis, wound medicine and the like. Sesame (sesame) is also called black whale and black sesame, and Eisei is sesame, which refers to Sesamum orientale or Sesamum indicum in the sesame family. This is a plant conventionally used for frail constitution, post-disease, constipation, anti-inflammation and the like. Plum tree moss (Plum tree moss), also called Sekika (stone flower), refers to Parmelia tinctorum of the genus Plumaceae. This is a plant conventionally used for hemostasis, antitumor, diuresis, etc., or as a raw material for litmus pigment. Kinbaisasa (also known as senbo) is the English name Curculigo Gaertn, and refers to Curculigo orchioides belonging to the genus Curgrigo . This is a plant that has been used as a tonic and a moisturizer. Butterflies (also known as Hitsuka ) refers to Litsea mollifolia of the genus Aceraceae . This is a plant conventionally used for anorexia, indigestion, diarrhea, vomiting and the like. Ezomikuri (Satsumi Chestnut) is also called Sanryo (Sanryo) and refers to Sparganium simplex or Sparganium emersum of the Mikuri family Mikuri genus. This is a plant conventionally used for pain, irregular menstruation, postpartum abdominal pain, anemia, diuresis and the like. Shorosilane is also called Loro (leakage) and Tenseiso (natural grass), and refers to the Diuranthera minor of the lily family. This is a plant that has been used for heat purification, detoxification and the like. Yoroigusa (Armorweed) is also known as “Bikaku”, “Karabakushi” or “Udomodoki”, and its English name is Angelica, which refers to Angelica dahurica belonging to the genus Ceramidae. This is a plant that has been used for anti-inflammatory, analgesic, drainage, granulation, and itching prevention. Uchiwadokoro (Dan Ogiro) is also called Senzanryu (Kukiyama Ryu) and bats (Kanoyamaro), and refers to Dioscorea nipponica in the genus Dioscorea . This is a plant that has been conventionally used for pain relief, expectoration and cough. Sandalwood (sandalwood), also called sandalwood or sandal, refers to the Santalum album of the sandalwood family sandalwood genus. This is a plant that has been conventionally used as an incense tree.
しかし、これらの植物が皮膚の光損傷／光老化に関与していることはこれまで知られていなかった。 However, it has not been known so far that these plants are involved in photo-damage / photoaging of the skin.
本発明は、ＭＦＡＰ−４産生促進、ならびに皮膚の光損傷／光老化に伴う症状を予防又は改善することができる天然由来素材の提供に関する。 The present invention relates to the provision of a naturally-derived material that can promote or prevent MFAP-4 production and symptoms associated with photodamage / photoaging of the skin.
近年、光照射により真皮におけるＭＭＰの発現上昇やコラーゲンやエラスチンなどのＥＣＭの発現減少・分解や、表皮でのケラチン６と１６の発現上昇を伴うとともに、皮膚の表面粗さが増加し、且つ弾性が低下するヒト皮膚モデルが開発された（Hachiya et al, 2009, Am J Pathol 174:401-413）。さらに、この皮膚モデルにレンチウイルスを用いて目的の因子を過剰発現させるモデルも開発された（Hachiya et al, 2007, Gene Ther 14:648-656）。これらのモデルは、皮膚の光損傷／光老化に関与する因子を調べるためのモデルとして有望である。 In recent years, exposure to light has led to increased expression of MMP in the dermis, decreased expression and degradation of ECM such as collagen and elastin, increased expression of keratins 6 and 16 in the epidermis, increased skin surface roughness, and elasticity A human skin model has been developed (Hachiya et al, 2009, Am J Pathol 174: 401-413). In addition, a model has also been developed in which lentivirus is overexpressed in this skin model (Hachiya et al, 2007, Gene Ther 14: 648-656). These models are promising as models for investigating factors involved in photodamage / photoaging of the skin.
最近、一部に本発明者らを含む研究グループによって、上記モデル皮膚を用いた皮膚の光損傷／老化に関与する因子の探索とその機能検証が行われた。その結果、ＭＦＡＰ−４が光損傷／光老化皮膚や、加齢皮膚において顕著に発現減少していることが観察された（図３）。またＭＦＡＰ−４を過剰発現させたモデルでは、ＵＶＢ照射による皮膚表面粗さの増加や弾性の低下（すなわち、皮膚光老化）が有意に抑制されていること（図４）、及びＵＶＢ照射によるＥＣＭの減少が抑制されていることが示された（図５）。また、同モデルでＭＭＰ−１２の活性化が抑制されること（図６）、及び真皮線維芽細胞におけるＭＦＡＰ−４がＭＭＰの発現に関わっていることが示された（図７）。さらに、ＭＦＡＰ−４が、トロポエラスチン及びフィブリリン−１からなる重合体の形成に寄与していること（図８）、及び皮膚弾性線維の土台となるミクロフィブリルを構成するフィブリリン−１と直接相互作用していることが明らかになった（図９）。これらの研究により、ＭＦＡＰ−４が酵素ＭＭＰの活性化抑制等を介してＵＶＢ曝露によるコラーゲンの減少を抑制するとともに、ＥＣＭ内でフィブリリン−１からミクロフィブリルへの重合化を促進することで弾性線維の形成を促し（図１０）、結果としてＥＣＭの構造を維持する働きがあることが示された。 Recently, a research group including the present inventors partially searched for a factor involved in photodamage / aging of the skin using the model skin and verified its function. As a result, it was observed that the expression of MFAP-4 was significantly reduced in photodamaged / photoaged skin and aging skin (FIG. 3). In the model in which MFAP-4 is overexpressed, increase in skin surface roughness and decrease in elasticity (ie, skin photoaging) due to UVB irradiation are significantly suppressed (FIG. 4), and ECM due to UVB irradiation. It was shown that the reduction | decrease of is suppressed (FIG. 5). Moreover, it was shown that the activation of MMP-12 was suppressed in the same model (FIG. 6), and that MFAP-4 in dermal fibroblasts was involved in the expression of MMP (FIG. 7). Furthermore, MFAP-4 contributes to the formation of a polymer composed of tropoelastin and fibrillin-1 (FIG. 8), and directly interacts with fibrillin-1 constituting the microfibril that forms the basis of skin elastic fibers. It became clear that it was acting (FIG. 9). As a result of these studies, MFAP-4 suppresses the decrease in collagen due to UVB exposure through inhibition of the activation of the enzyme MMP, etc., and promotes the polymerization from fibrillin-1 to microfibrils in the ECM, thereby allowing elastic fibers (Fig. 10), and as a result, it was shown that it has the function of maintaining the structure of ECM.
従って、ＭＦＡＰ−４の発現を促進することができれば、真皮におけるＥＣＭ及び弾性線維の構築を促すとともにその分解を抑えて、その弾性構造を維持し、結果として、皮膚の光損傷／光老化による症状、例えば皮膚のしわ、たるみ、はりの低下等の予防及び／又は改善を図ることができる。 Therefore, if the expression of MFAP-4 can be promoted, it promotes the construction of ECM and elastic fibers in the dermis and suppresses the degradation thereof to maintain its elastic structure. As a result, symptoms caused by photodamage / photoaging of the skin For example, it is possible to prevent and / or improve skin wrinkles, sagging, and reduction of a beam.
上記の研究結果に基づいて、本発明者らは、ＭＦＡＰ−４の発現を促進する天然由来の物質を探索した結果、ある種の植物又はそれらの抽出物に、細胞のＭＦＡＰ−４の発現を促進する作用があり、且つ真皮におけるＥＣＭ及び弾性線維の形成促進及び分解抑制、皮膚のしわ、たるみ、はりの低下等の皮膚の光損傷／光老化による症状の予防及び／又は改善のための素材として有用であることを見出した。 Based on the above research results, the present inventors searched for naturally-occurring substances that promote the expression of MFAP-4. As a result, the expression of cellular MFAP-4 in certain plants or their extracts was determined. Material that promotes and promotes the formation and suppression of ECM and elastic fiber formation in the dermis, skin wrinkles, sagging, and reduction of skin, etc. As found useful.
すなわち、本発明は、ゴマノハグサ、ゴマ、ウメノキゴケ、キンバイザサ、チョウカシ、エゾミクリ、ショウロシラン、ヨロイグサ、ウチワドコロ及びビャクダン、ならびにそれらの抽出物から選択される少なくとも１つを有効成分とするＭＦＡＰ−４産生促進剤を提供する。
また本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とするミクロフィブリル形成促進剤を提供する。
また本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とする弾性線維形成促進剤を提供する。
また本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とする弾性線維分解抑制剤を提供する。
また本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とするコラーゲン分解抑制剤を提供する。
また本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とする皮膚光老化予防及び／又は改善剤を提供する。
また本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とする皮膚しわ予防及び／又は改善剤を提供する。
また本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とする皮膚たるみ予防及び／又は改善剤を提供する。
That is, the present invention relates to a MFAP-4 production promoter comprising, as an active ingredient, at least one selected from sesame seeds, sesame seeds, umenosokeki, kingweeds, butterflies, ezomikuri, shorosilane, armor rush, prickly pear and sandalwood, and extracts thereof. I will provide a.
Moreover, this invention provides the microfibril formation promoter which uses at least 1 selected from the said plant and those extracts as an active ingredient.
Moreover, this invention provides the elastic fiber formation promoter which uses as an active ingredient at least 1 selected from the said plant and those extracts.
Moreover, this invention provides the elastic fiber degradation inhibitor which uses as an active ingredient at least 1 selected from the said plant and those extracts.
Moreover, this invention provides the collagen degradation inhibitor which uses as an active ingredient at least 1 selected from the said plant and those extracts.
Moreover, this invention provides the skin photoaging prevention and / or improvement agent which uses as an active ingredient at least 1 selected from the said plant and those extracts.
Moreover, this invention provides the skin wrinkle prevention and / or improvement agent which uses at least 1 selected from the said plant and those extracts as an active ingredient.
Moreover, this invention provides the skin sagging prevention and / or improvement agent which uses at least 1 selected from the said plant and those extracts as an active ingredient.
本発明によれば、細胞のＭＦＡＰ−４産生を促進することができる。また本発明によれば、当該ＭＦＡＰ−４産生促進を介して、真皮や結合組織におけるコラーゲンや弾性線維の構造を維持することができ、結果として皮膚のしわ、たるみ、はりの低下等の皮膚の光損傷／光老化による症状の予防及び／又は改善をもたらすことができる。 According to the present invention, MFAP-4 production of cells can be promoted. Further, according to the present invention, the structure of collagen and elastic fibers in the dermis and connective tissue can be maintained through the promotion of MFAP-4 production, and as a result, skin wrinkles, sagging, reduction of the skin, etc. Prevention and / or amelioration of symptoms due to photodamage / photoaging.
本明細書において、「非治療的」とは、医療行為、すなわち治療による人体への処置行為を含まない概念である。 In the present specification, “non-therapeutic” is a concept that does not include a medical act, that is, a treatment act on the human body by therapy.
本明細書において、「改善」とは、疾患又は症状の好転、疾患又は症状の悪化の防止又は遅延、あるいは疾患又は症状の進行の逆転、防止又は遅延をいう。 In the present specification, “improvement” refers to improvement of a disease or symptom, prevention or delay of worsening of the disease or symptom, or reversal, prevention or delay of progression of the disease or symptom.
本明細書において、「予防」とは、個体における疾患若しくは症状の発症の防止又は遅延、あるいは個体の疾患若しくは症状の発症の危険性を低下させることをいう。 As used herein, “prevention” refers to preventing or delaying the onset of a disease or symptom in an individual, or reducing the risk of developing an individual's disease or symptom.
本明細書において、皮膚の「光損傷」と「光老化」とは同義に解される。本明細書において、皮膚の「光損傷」又は「光老化」とは、光、特に紫外線（ＵＶ）への慢性的な曝露によって皮膚に生じるしわ、たるみ、はりの低下等の皮膚弾性の低下に伴う種々の症状をいう。光老化は、通常に使用される意味での「老化」、すなわち加齢に伴う老化とは区別され得る。すなわち、光老化によって生じるしわやたるみは、加齢に伴う代謝の低下によって生じるしわ、たるみや、皮膚の乾燥に伴うしわ（小じわ）とは区別される。 In the present specification, “photodamage” and “photoaging” of the skin are understood to be synonymous. As used herein, “photodamage” or “photoaging” of skin refers to a decrease in skin elasticity such as wrinkles, sagging, and a decrease in beam caused by chronic exposure to light, particularly ultraviolet (UV) light. It refers to various symptoms that accompany it. Photoaging can be distinguished from “aging” in the commonly used sense, ie aging associated with aging. That is, wrinkles and sagging caused by photoaging are distinguished from wrinkles caused by a decrease in metabolism accompanying aging, sagging, and wrinkles (fine wrinkles) caused by skin dryness.
本明細書において、「ＭＦＡＰ−４」（ｍｉｃｒｏｆｉｂｒｉｌｌａｒ−ａｓｓｏｃｉａｔｅｄ ｐｒｏｔｅｉｎ ４）とは、Ｇｅｎｅ ＩＤ：４２３９、ＭＩＭ ＩＤ：６００５９６として知られているタンパク質である。
本発明における「ＭＦＡＰ−４産生促進」は、ＭＦＡＰ−４タンパク質の量の増加、ＭＦＡＰ−４タンパク質の発現レベルの亢進、ＭＦＡＰ−４タンパク質をコードするｍＲＮＡの発現レベルの亢進、ＭＦＡＰ−４タンパク質をコードする遺伝子の転写制御領域の活性化レベルの亢進等の任意のパラメーターに基づいて測定することができる。
In the present specification, “MFAP-4” (microfibrillar-associated protein 4) is a protein known as Gene ID: 4239 and MIM ID: 6000059.
In the present invention, “MFAP-4 production promotion” refers to an increase in the amount of MFAP-4 protein, an increase in the expression level of MFAP-4 protein, an increase in the expression level of mRNA encoding MFAP-4 protein, It can be measured based on an arbitrary parameter such as an increase in the activation level of the transcription control region of the gene to be encoded.
本明細書において、「ゴマノハグサ」とは、ゴマノハグサ科ゴマノハグサ属のScrophularia buergerianaを指し、「ゴマ」とは、ゴマ科ゴマ属のSesamum orientale又はSesamum indicumを指し、「ウメノキゴケ」とは、ウメノキゴケ科ウメノキゴケ属のParmelia tinctorumを指し、「キンバイザサ」とは、キンバイザサ科クルグリゴ属のCurculigo orchioidesを指し、「チョウカシ」とは、クスノキ科ハマビワ属のLitsea mollifoliaを指し、「エゾミクリ」とは、ミクリ科ミクリ属のSparganium simplex又はSparganium emersumを指し、「ショウロシラン」とは、ユリ科のDiuranthera minorを指し、「ヨロイグサ」とは、セリ科シシウド属のAngelica dahuricaを指し、「ウチワドコロ」とは、ヤマノイモ科ヤマノイモ属のDioscorea nipponicaを指し、「ビャクダン」とは、ビャクダン科ビャクダン属のSantalum albumを指す。 In the present specification, “Gomanohagusa” refers to Scrophularia buergeriana belonging to the genus Lomaceae , “Sesame” refers to Sesamum orientale or Sesamum indicum , and “Umenookeoke” refers to the genus Pleurotus genus refers to the Parmelia tinctorum, and "Kinbaizasa" refers to the Curculigo orchioides of hypoxidaceae Kurugurigo genus, the "Choukashi", refers to the Litsea mollifolia of Lauraceae Hamabiwa genus, the "sparganium emersum", Sparganium of Mikuri family sparganiaceae Simplex or Sparganium emersum , `` Shorosilane '' refers to Diuranthera minor of the lily family, ` ` Yoroigusa '' refers to Angelica dahurica of the genus Aceraceae, `` Uchiwadokoro '' refers to Dioscorea of the genus Yamanoimo nipponica refers to sandalwood, a santalum album belonging to the sandalwood family Point to.
本発明において、上記に挙げた植物は、特に限定されない限り、当該植物の任意の部位、例えば全草、全木、葉、茎、芽、花、蕾、樹木、木質部、樹皮、根、根茎、仮球茎、塊根、地衣体、葉状体、種子、果実、若しくは樹脂等、又はそれらの組み合わせを利用すればよい。好ましい部位は、ゴマノハグサの場合は根茎、ゴマの場合は種子、ウメノキゴケの場合は葉状体、キンバイザサの場合は根茎、チョウカシの場合は果実、エゾミクリの場合は根茎、ショウロシランの場合は根、ヨロイグサの場合は根、ウチワドコロの場合は根茎、及びビャクダンの場合は樹木である。 In the present invention, the plant listed above is not limited to any part of the plant, for example, whole plant, whole tree, leaf, stem, bud, flower, bud, tree, wood part, bark, root, rhizome, A temporary corm, tuberous root, lichen, frond, seed, fruit, resin, or the like, or a combination thereof may be used. Preferred sites are rhizomes for sesame seeds, seeds for sesame seeds, fronds for umemos, rhizomes for yellow moss, fruits for butterflies, fruits for rhizomes, roots for shrimp, roots for yorogusa In the case of roots, rhizomes in the case of prickly pears, and trees in the case of sandalwood.
上記に挙げた植物の抽出物は、上述した当該植物の任意の部位又はそれらの組み合わせからの抽出物であればよいが、上述した当該植物の好ましい部位からの抽出物であるのが好ましい。これらの植物の部位は、そのまま抽出工程に付されてもよく、又は粉砕、切断若しくは乾燥された後に抽出工程に付されてもよい。 Although the extract of the plant mentioned above should just be the extract from the arbitrary site | parts or the combination of the said plant mentioned above, it is preferable that it is the extract from the preferable site | part of the said plant mentioned above. These plant parts may be subjected to the extraction process as they are, or may be subjected to the extraction process after being pulverized, cut or dried.
上記抽出物としては、市販されているものを利用してもよく、又は常法により得られる各種溶媒抽出物、又はその希釈液、その濃縮液、その乾燥末、ペースト若しくはその活性炭処理したものであってもよい。一例として、本発明における抽出物は、上記植物の部位を室温若しくは加温下にて抽出するか、又はソックスレー抽出器等の抽出器具を用いて抽出することにより得ることができる。 As the extract, commercially available products may be used, or various solvent extracts obtained by a conventional method, or diluted solutions thereof, concentrated solutions thereof, dried powders thereof, pastes or activated carbon treatments thereof. There may be. As an example, the extract in the present invention can be obtained by extracting the plant part at room temperature or under heating, or by using an extraction device such as a Soxhlet extractor.
抽出のための溶媒には、極性溶媒、非極性溶媒のいずれをも使用することができる。溶媒の具体例としては、例えば、水；メタノール、エタノール、プロパノール、ブタノール等のアルコール類；プロピレングリコール、ブチレングリコール等の多価アルコール類；アセトン、メチルエチルケトン等のケトン類；酢酸メチル、酢酸エチル等のエステル類；テトラヒドロフラン、ジエチルエーテル等の鎖状及び環状エーテル類；ポリエチレングリコール等のポリエーテル類；スクワラン、ヘキサン、シクロヘキサン、石油エーテル等の炭化水素類；トルエン等の芳香族炭化水素類；ジクロロメタン、クロロホルム、ジクロロエタン等のハロゲン化炭化水素類；及び超臨界二酸化炭素；ピリジン類；油脂、ワックス等その他オイル類等の有機溶媒；ならびにこれらの混合物が挙げられる。好適には、水、アルコール類及びその水溶液が挙げられ、アルコール類としてはエタノールが好ましい。より好ましい溶媒は、水及びエタノール水溶液である。 As the solvent for extraction, either a polar solvent or a nonpolar solvent can be used. Specific examples of the solvent include water; alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; methyl acetate, ethyl acetate, and the like. Esters; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; hydrocarbons such as squalane, hexane, cyclohexane and petroleum ether; aromatic hydrocarbons such as toluene; dichloromethane and chloroform And halogenated hydrocarbons such as dichloroethane; and supercritical carbon dioxide; pyridines; organic solvents such as fats and waxes and other oils; and mixtures thereof. Preferable examples include water, alcohols and aqueous solutions thereof, and ethanol is preferable as the alcohols. More preferred solvents are water and an aqueous ethanol solution.
アルコール類と水との配合割合（容量比）としては、９９．９９９〜０：０．００１〜１００が好ましく、９５〜５：５〜９５がより好ましく、８０〜２０：２０〜８０がさらに好ましく、７０〜３０：３０〜７０がさらにより好ましく、６０〜４０：４０〜６０がなお好ましい。
溶媒の使用量としては、上記植物（乾燥質量換算）１ｇに対して１〜１００ｍＬが好ましく、抽出時間としては、１分間〜３ヶ月間が好ましく、１０分間〜２ヶ月間がより好ましい。このときの抽出温度は、０℃〜溶媒沸点、より好ましくは１０〜６０℃、さらに好ましくは１５〜４０℃である。
The blending ratio (volume ratio) of alcohols and water is preferably 99.999-0: 0.001-100, more preferably 95-5: 5-95, and still more preferably 80-20: 20-80. 70-30: 30-70 are still more preferable, and 60-40: 40-60 are still more preferable.
As a usage-amount of a solvent, 1-100 mL is preferable with respect to 1 g of said plants (dry mass conversion), As extraction time, 1 minute-3 months are preferable, and 10 minutes-2 months are more preferable. The extraction temperature at this time is 0 ° C. to the solvent boiling point, more preferably 10 to 60 ° C., and further preferably 15 to 40 ° C.
抽出物を得る抽出手段は、具体的には、固液抽出、液液抽出、浸漬、煎出、浸出、還流抽出、超音波抽出、マイクロ波抽出、攪拌等の手段を用いることができる。例えば、浸漬の好適な一例として、０℃〜溶媒沸点（好ましくは１５〜４０℃）で、１時間〜２ヶ月間の浸漬が挙げられる。また、抽出時間を短縮する場合には、攪拌を伴う固液抽出が望ましい。抽出物の酸化を防止するため、煮沸脱気や窒素ガス等の不活性ガスを通気して溶存酸素を除去しつつ、いわゆる非酸化的雰囲気下で抽出する手段を併用してもよい。 Specific examples of the extraction means for obtaining the extract include solid-liquid extraction, liquid-liquid extraction, immersion, decoction, leaching, reflux extraction, ultrasonic extraction, microwave extraction, and stirring. For example, as a suitable example of immersion, immersion for 1 hour to 2 months at 0 ° C. to a solvent boiling point (preferably 15 to 40 ° C.) can be mentioned. Moreover, when shortening extraction time, solid-liquid extraction with stirring is desirable. In order to prevent the oxidation of the extract, a means for extracting under a so-called non-oxidizing atmosphere while removing dissolved oxygen by bubbling degassing or inert gas such as nitrogen gas may be used in combination.
後記実施例に示すように、上記植物の抽出物は、細胞におけるＭＦＡＰ−４の発現を有意に促進する作用を有する。従って、上記植物及び／又はそれらの抽出物は、ＭＦＡＰ−４産生促進のために使用することができる。また、上記植物及び／又はそれらの抽出物は、当該ＭＦＡＰ−４産生促進作用を介して、真皮における弾性線維の形成促進及び分解抑制、又はコラーゲンの分解抑制、皮膚の光損傷／光老化の予防及び／又は改善、あるいは皮膚のしわ、たるみ等の予防及び／又は改善のために使用することができる。当該使用は、ヒト若しくは非ヒト動物、又はそれらに由来する検体における使用であり得、また治療的使用であっても非治療的使用であってもよい。 As shown in Examples below, the plant extract has an effect of significantly promoting the expression of MFAP-4 in cells. Therefore, the said plant and / or those extracts can be used for MFAP-4 production promotion. Further, the plant and / or an extract thereof promotes the formation and suppression of elastic fiber formation in the dermis, or suppresses the degradation of collagen, and prevents photodamage / photoaging of the skin through the MFAP-4 production promoting action. And / or improvement, or prevention and / or improvement of skin wrinkles and sagging. The use can be in humans or non-human animals, or specimens derived therefrom, and can be therapeutic or non-therapeutic.
従って、一態様として、本発明は、ゴマノハグサ、ゴマ、ウメノキゴケ、キンバイザサ、チョウカシ、エゾミクリ、ショウロシラン、ヨロイグサ、ウチワドコロ及びビャクダン、ならびにそれらの抽出物から選択される少なくとも１つを有効成分とするＭＦＡＰ−４産生促進剤を提供する。
別の態様として、本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とするミクロフィブリル形成促進剤を提供する。
別の態様として、本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とする弾性線維形成促進剤を提供する。
別の態様として、本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とする弾性線維分解抑制剤を提供する。
別の態様として、本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とするコラーゲン分解抑制剤を提供する。
別の態様として、本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とする皮膚光老化予防及び／又は改善剤を提供する。
別の態様として、本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とする皮膚しわ予防及び／又は改善剤を提供する。
別の態様として、本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを有効成分とする皮膚たるみ予防及び／又は改善剤を提供する。
本発明において、上記剤は、本質的にゴマノハグサ、ゴマ、ウメノキゴケ、キンバイザサ、チョウカシ、エゾミクリ、ショウロシラン、ヨロイグサ、ウチワドコロ及びビャクダン、ならびにそれらの抽出物から選択される少なくとも１つから構成される。
本発明において、これらの植物及び抽出物は、単独で使用されてもよく、いずれか２以上の組み合わせで使用されてもよい。
Therefore, as one aspect, the present invention provides an MFAP- containing, as an active ingredient, at least one selected from sesame seeds, sesame seeds, apricot moss, golden moss, butterfly, butterfly, sprout, shorosilane, armor rush, prickly pear and sandalwood, and extracts thereof. 4. Provide production promoter.
As another aspect, the present invention provides a microfibril formation promoter containing at least one selected from the above plants and their extracts as an active ingredient.
As another aspect, the present invention provides an elastic fiber formation promoter comprising as an active ingredient at least one selected from the above-mentioned plants and extracts thereof.
As another aspect, the present invention provides an elastic fiber degradation inhibitor comprising as an active ingredient at least one selected from the above plants and extracts thereof.
As another aspect, the present invention provides a collagen degradation inhibitor comprising as an active ingredient at least one selected from the above plants and extracts thereof.
As another aspect, the present invention provides a skin photoaging preventive and / or ameliorating agent comprising at least one selected from the above plants and their extracts as an active ingredient.
As another aspect, the present invention provides an agent for preventing and / or improving skin wrinkles comprising at least one selected from the above-mentioned plants and extracts thereof as an active ingredient.
As another aspect, the present invention provides an agent for preventing and / or improving skin sagging, comprising at least one selected from the above plants and their extracts as an active ingredient.
In the present invention, the agent is essentially composed of at least one selected from sesame seeds, sesame seeds, apricot moss, yellow foxtails, butterflies, ezomikuri, shorosilane, armor rushes, prickly pears and sandalwood, and extracts thereof.
In the present invention, these plants and extracts may be used alone or in any combination of two or more.
上記植物及び／又はそれらの抽出物は、ＭＦＡＰ−４産生促進、真皮における弾性線維の形成促進及び分解抑制、又はコラーゲンの分解抑制、皮膚の光損傷／光老化の予防及び／又は改善、あるいは皮膚のしわ、たるみ、はりの低下等の予防及び／又は改善のための組成物、医薬、医薬部外品、化粧料等の製造のために使用することができる。当該組成物、医薬、医薬部外品、化粧料等もまた、本発明の範囲内である。 The above plants and / or their extracts are MFAP-4 production promotion, elastic fiber formation promotion and degradation inhibition in the dermis, or collagen degradation inhibition, skin photodamage / photoaging prevention and / or improvement, or skin It can be used for the production of compositions, medicaments, quasi-drugs, cosmetics and the like for the prevention and / or improvement of wrinkles, sagging, and reduction of beam. Such compositions, pharmaceuticals, quasi drugs, cosmetics and the like are also within the scope of the present invention.
上記組成物、医薬、医薬部外品、化粧料等は、ヒト又は非ヒト動物用として製造され、又は使用され得る。上記植物及び／又はそれらの抽出物は、当該組成物、医薬、医薬部外品、化粧料等に素材として配合され、ＭＦＡＰ−４産生促進、真皮における弾性線維の形成促進及び分解抑制、又はコラーゲンの分解抑制、皮膚の光損傷／光老化の予防及び／又は改善、あるいは皮膚のしわ、たるみ、はりの低下等の予防及び／又は改善のための有効成分であり得る。 The above composition, medicine, quasi-drug, cosmetic and the like can be produced or used for human or non-human animals. The plant and / or extract thereof is blended as a raw material in the composition, medicine, quasi-drug, cosmetics, etc., and promotes MFAP-4 production, formation of elastic fibers in the dermis and inhibition of degradation, or collagen It may be an active ingredient for preventing and / or improving skin degradation, prevention and / or improvement of photodamage / photoaging of the skin, or reduction of wrinkles, sagging, and beam.
上記医薬又は医薬部外品は、上記植物及び／又はそれらの抽出物を有効成分として含有する。当該医薬又は医薬部外品は、任意の投与形態で投与され得る。投与形態は、経口投与でも外用剤等の非経口投与でもよい。経口投与のための剤型としては、例えば、錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤のような固形投薬形態、ならびにエリキシロール、シロップおよび懸濁液のような液体投薬形態が挙げられ、非経口投与のための剤型としては、注射、輸液、局所、外用剤、経皮、経粘膜、経鼻、経腸、吸入、坐剤、ボーラス、貼布剤等が挙げられる。
一例として、当該医薬又は医薬部外品は、ローション、クリーム、ジェル、軟膏、貼布剤等の形態の皮膚外用剤、又は皮膚用注射剤であり得る。
The said pharmaceutical or quasi-drug contains the said plant and / or those extracts as an active ingredient. The pharmaceutical or quasi drug can be administered in any dosage form. The administration form may be oral administration or parenteral administration such as external preparation. Dosage forms for oral administration include, for example, solid dosage forms such as tablets, coated tablets, granules, powders, capsules, and liquid dosage forms such as elixirol, syrups and suspensions, Examples of the dosage form for parenteral administration include injection, infusion, topical, external preparation, transdermal, transmucosal, nasal, enteral, inhalation, suppository, bolus, patch and the like.
As an example, the medicine or quasi drug may be a skin external preparation in the form of a lotion, cream, gel, ointment, patch or the like, or an injection for skin.
上記医薬や医薬部外品は、上記植物又はそれらの抽出物を単独で含有していてもよく、又は薬学的に許容される担体と組み合わせて含有していてもよい。斯かる担体としては、例えば、賦形剤、被膜剤、結合剤、増量剤、崩壊剤、界面活性剤、滑沢剤、希釈剤、分散剤、緩衝剤、浸透圧調整剤、ｐＨ調整剤、分散剤、乳化剤、防腐剤、安定剤、酸化防止剤、着色剤、紫外線吸収剤、保湿剤、増粘剤、活性増強剤、抗炎症剤、殺菌剤、香料、矯味剤、矯臭剤等が挙げられる。また、当該医薬や医薬部外品は、上記植物又はそれらの抽出物のＭＦＡＰ−４産生促進作用が失われない限り、他の有効成分や薬理成分を含有していてもよい。 The said pharmaceutical and quasi-drug may contain the said plant or those extracts alone, or may contain it in combination with a pharmaceutically acceptable carrier. Examples of such carriers include excipients, coating agents, binders, extenders, disintegrants, surfactants, lubricants, diluents, dispersants, buffers, osmotic pressure adjusting agents, pH adjusting agents, Dispersants, emulsifiers, preservatives, stabilizers, antioxidants, colorants, UV absorbers, moisturizers, thickeners, activity enhancers, anti-inflammatory agents, bactericides, fragrances, flavoring agents, flavoring agents, etc. It is done. Moreover, the said pharmaceutical and quasi-drug may contain the other active ingredient and pharmacological component, unless the MFAP-4 production promotion effect of the said plant or those extracts is lost.
上記医薬又は医薬部外品は、上記植物及び／又はそれらの抽出物から、あるいは必要に応じて上記担体及び／又は他の有効成分や薬理成分を組みあわせて、常法により製造することができる。当該医薬又は医薬部外品における上記植物及び／又はそれらの抽出物の含有量は、例えば、当該抽出物の乾燥重量に換算して、通常０．００１〜９９．９９９質量％であり、０．０１〜２０質量％とするのが好ましい。 The above-mentioned medicine or quasi-drug can be produced from the above-mentioned plant and / or an extract thereof or, if necessary, by combining the above-mentioned carrier and / or other active ingredient or pharmacological ingredient by a conventional method. . Content of the said plant and / or those extracts in the said medicine or quasi-drug is 0.001-99.999 mass% normally in conversion of the dry weight of the said extract, for example. It is preferable to set it as 01-20 mass%.
上記化粧料は、上記植物及び／又はそれらの抽出物を有効成分として含有する。上記化粧料は、上記植物又はそれらの抽出物を単独で含有していてもよく、又は化粧料として許容される担体と組み合わせて含有していてもよい。
斯かる担体としては、例えば、賦形剤、被膜剤、結合剤、増量剤、崩壊剤、界面活性剤、滑沢剤、希釈剤、分散剤、緩衝剤、浸透圧調整剤、ｐＨ調整剤、分散剤、乳化剤、防腐剤、安定剤、酸化防止剤、着色剤、紫外線吸収剤、保湿剤、増粘剤、活性増強剤、抗炎症剤、殺菌剤、香料、矯味剤、矯臭剤等が挙げられる。また、当該化粧料は、上記植物又はそれらの抽出物のＭＦＡＰ−４産生促進作用が失われない限り、他の有効成分や化粧成分、例えば、保湿剤、美白剤、紫外線保護剤、細胞賦活剤、洗浄剤、角質溶解剤、メークアップ成分（例えば、化粧下地、ファンデーション、おしろい、パウダー、チーク、口紅、アイメーク、アイブロウ、マスカラ、その他）等を含有していてもよい。
化粧料の形態としては、クリーム、乳液、ローション、懸濁液、フォーム、ジェル、パウダー、パック、シート、パッチ、スティック、ケーキ等、化粧料に使用され得る任意の形態が挙げられる。
一例として、上記化粧品は、しわ、たるみ、はり改善用のローション、乳液、クリーム、フォーム、ジェル、パック、シート等の形態であり得る。
The said cosmetics contain the said plant and / or those extracts as an active ingredient. The cosmetic may contain the plant or an extract thereof alone or in combination with a carrier acceptable as a cosmetic.
Examples of such carriers include excipients, coating agents, binders, extenders, disintegrants, surfactants, lubricants, diluents, dispersants, buffers, osmotic pressure adjusting agents, pH adjusting agents, Dispersants, emulsifiers, preservatives, stabilizers, antioxidants, colorants, UV absorbers, moisturizers, thickeners, activity enhancers, anti-inflammatory agents, bactericides, fragrances, flavoring agents, flavoring agents, etc. It is done. In addition, as long as the MFAP-4 production promoting action of the above plants or their extracts is not lost, the cosmetics are other active ingredients and cosmetic ingredients such as moisturizers, whitening agents, UV protection agents, cell activators. , Cleaning agents, keratolytic agents, make-up ingredients (for example, makeup bases, foundations, funny, powder, teak, lipstick, eye makeup, eyebrow, mascara, etc.) and the like.
Examples of cosmetic forms include creams, emulsions, lotions, suspensions, foams, gels, powders, packs, sheets, patches, sticks, cakes, and any other form that can be used for cosmetics.
As an example, the cosmetic product may be in the form of wrinkles, sagging, a lotion for improving a beam, emulsion, cream, foam, gel, pack, sheet, and the like.
上記化粧料は、上記植物及び／又はそれらの抽出物から、あるいは必要に応じて上記担体及び／又は他の有効成分や化粧成分を組みあわせて、常法により製造することができる。当該化粧料における上記植物及び／又はそれらの抽出物の含有量は、当該抽出物の乾燥重量に換算して、通常０．０００１〜９９．９９９質量％であり、０．００１〜１０質量％とするのが好ましい。 The cosmetic can be produced by a conventional method from the plant and / or an extract thereof, or in combination with the carrier and / or other active ingredients and cosmetic ingredients as necessary. Content of the said plant and / or those extracts in the said cosmetics is 0.0001-99.999 mass% normally in conversion of the dry weight of the said extract, and 0.001-10 mass% It is preferable to do this.
また本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを投与することを特徴とするＭＦＡＰ−４産生促進方法を提供する。当該方法において、上記植物及びそれらの抽出物から選択される少なくとも１つは、細胞のＭＦＡＰ−４産生促進のために、それを必要とする対象に有効量で投与される。
また本発明は、上記植物及びそれらの抽出物から選択される少なくとも１つを投与することを特徴とする、真皮における弾性線維の形成促進及び分解抑制、又はコラーゲンの分解抑制方法、皮膚の光損傷／光老化の予防及び／又は改善方法、あるいは皮膚のしわ、たるみ、はりの低下等の予防及び／又は改善方法を提供する。当該方法において、上記植物及びそれらの抽出物から選択される少なくとも１つは、真皮における弾性線維の形成促進及び分解抑制、又はコラーゲンの分解抑制、皮膚の光損傷／光老化の予防及び／又は改善、あるいは皮膚のしわ、たるみ、はりの低下等の予防及び／又は改善のために、それらを必要とする対象に有効量で投与される。
Moreover, this invention provides the MFAP-4 production promotion method characterized by administering at least 1 selected from the said plant and those extracts. In the method, at least one selected from the plants and extracts thereof is administered in an effective amount to a subject in need thereof to promote MFAP-4 production of cells.
Further, the present invention provides at least one selected from the above plants and their extracts, a method for promoting the formation and suppression of the degradation of elastic fibers in the dermis, the method for inhibiting the degradation of collagen, and the photodamage of the skin Provided is a method for preventing and / or improving photoaging, or a method for preventing and / or improving skin wrinkles, sagging, and reduction of a beam. In the method, at least one selected from the above-mentioned plants and their extracts is the formation promotion and inhibition of elastic fibers in the dermis, or the inhibition of collagen degradation, the prevention and / or improvement of skin photodamage / photoaging. Alternatively, it is administered in an effective amount to a subject in need thereof for the prevention and / or improvement of skin wrinkles, sagging, reduction of a beam and the like.
投与する対象としては、動物、好ましくはヒト又は非ヒト哺乳動物、より好ましくはヒトが挙げられる。好ましくは、当該投与は、美容又は健康維持を目的として、非治療的に投与され得る。あるいは、投与する対象は、動物由来の組織、器官、細胞、又はそれらの分画物であり得る。当該組織、器官、細胞、又はそれらの分画物は、好ましくは、ＭＦＡＰ−４発現能を有する天然由来又は生物学的若しくは生物工学的に改変された組織、器官、細胞、又はそれらの分画物であり、より好ましくは皮膚組織、皮膚組織由来の細胞、又はそれらの培養物である。 Subjects to be administered include animals, preferably humans or non-human mammals, more preferably humans. Preferably, the administration can be administered non-therapeutically for the purpose of cosmetics or health maintenance. Alternatively, the subject to be administered can be an animal-derived tissue, organ, cell, or fraction thereof. The tissue, organ, cell, or fraction thereof is preferably a naturally-derived or biologically or bioengineered tissue, organ, cell, or fraction thereof having the ability to express MFAP-4. More preferably, it is skin tissue, cells derived from skin tissue, or cultures thereof.
好ましい投与量は、対象の種、体重、性別、年齢、状態又はその他の要因に従って変動し得る。投与の用量、経路、間隔、及び摂取の量や間隔は、当業者によって適宜決定され得る。例えば、上記植物の抽出物の乾燥物換算で、成人１人当たり、０．０１〜１０００ｍｇ／日とすることが好ましく、０．１〜１００ｍｇ／日がより好ましい。 The preferred dosage may vary according to the subject's species, weight, sex, age, condition or other factors. The dose, route, interval, and amount and interval of intake can be determined appropriately by those skilled in the art. For example, it is preferably 0.01 to 1000 mg / day, more preferably 0.1 to 100 mg / day per adult in terms of dry matter of the plant extract.
以下、実施例を示し、本発明をより具体的に説明する。 EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.
製造例 植物抽出物の調製
製造例１ ゴマノハグサ抽出物の調製
ゴマノハグサ根茎（新和物産）４０ｇに５０％エタノール水溶液４００ｍＬを加え、室温で２５日間浸漬した。これをろ過し、ゴマノハグサ抽出液を得た。この抽出液を濃縮したところ、その固形分は１３．５４ｇであった。この抽出物を５０％エタノールにて希釈し、１％（ｗ／ｖ）の溶液とした。
Production Example Preparation of plant extract Production Example 1 Preparation of sesame seed extract Extract 40 mL of 50% ethanol aqueous solution was added to 40 g of sesame rhododendron rhizome (produced by Shinwa), and immersed at room temperature for 25 days. This was filtered to obtain a sesame extract. When this extract was concentrated, the solid content was 13.54 g. This extract was diluted with 50% ethanol to give a 1% (w / v) solution.
製造例２ ゴマ抽出物の調製
ゴマ種子（新和物産）４０ｇに５０％エタノール水溶液４００ｍＬを加え、室温で２７日間浸漬した。これをろ過し、ゴマ抽出液を得た。この抽出液を濃縮したところ、その固形分は１．５２ｇであった。この抽出物を５０％エタノールにて希釈し、１％（ｗ／ｖ）の溶液とした。
Production Example 2 Preparation of Sesame Extract 400 g of 50% ethanol aqueous solution was added to 40 g of sesame seeds (Shinwa product) and immersed for 27 days at room temperature. This was filtered to obtain a sesame extract. When this extract was concentrated, the solid content was 1.52 g. This extract was diluted with 50% ethanol to give a 1% (w / v) solution.
製造例３ ウメノキゴケ抽出物の調製
ウメノキゴケ葉状体（新和物産）４０ｇに５０％エタノール水溶液４００ｍＬを加え、室温で２７日間浸漬した。これをろ過し、ウメノキゴケ抽出液を得た。この抽出液を濃縮したところ、その固形分は０．８０ｇであった。この抽出物を５０％エタノールにて希釈し、１％（ｗ／ｖ）の溶液とした。
Production Example 3 Preparation of Apricot Mushroom Extract To 40 g of apricot foliage (Shinwa product) was added 400 mL of a 50% aqueous ethanol solution and immersed for 27 days at room temperature. This was filtered to obtain an Apricot extract. When this extract was concentrated, the solid content was 0.80 g. This extract was diluted with 50% ethanol to give a 1% (w / v) solution.
製造例４ キンバイザサ抽出物の調製
キンバイザサ根茎（新和物産）４０ｇに５０％エタノール水溶液４００ｍＬを加え、室温で２２日間浸漬した。これをろ過し、キンバイザサ抽出液を得た。この抽出液を濃縮したところ、その固形分は３．８６ｇであった。この抽出物を５０％エタノールにて希釈し、１％（ｗ／ｖ）の溶液とした。
Production Example 4 Preparation of Kinbizasa Extract 40 ml of 50% ethanol aqueous solution was added to 40 g of Kinbizasa rhizome (produced by Shinwa), and immersed for 22 days at room temperature. This was filtered to obtain an extract of Kinbisazasa. When this extract was concentrated, the solid content was 3.86 g. This extract was diluted with 50% ethanol to give a 1% (w / v) solution.
製造例５ チョウカシ抽出物の調製
チョウカシ果実（新和物産）４０ｇに５０％エタノール水溶液４００ｍＬを加え、室温で２５日間浸漬した。これをろ過し、チョウカシ抽出液を得た。この抽出液を濃縮したところ、その固形分は１．６８ｇであった。この抽出物を５０％エタノールにて希釈し、１％（ｗ／ｖ）の溶液とした。
Production Example 5 Preparation of butterfly extract To 400 g of butterfly fruit (Shinwa product), 400 mL of a 50% aqueous ethanol solution was added and immersed for 25 days at room temperature. This was filtered to obtain a butterfly extract. When this extract was concentrated, the solid content was 1.68 g. This extract was diluted with 50% ethanol to give a 1% (w / v) solution.
製造例６ エゾミクリ抽出物の調製
エゾミクリ根茎（新和物産）ｇに５０％エタノール水溶液４００ｍＬを加え、室温で４６日間浸漬した。これをろ過し、エゾミクリ抽出液を得た。この抽出液を濃縮したところ、その固形分は２．３１ｇであった。この抽出物を５０％エタノールにて希釈し、１％（ｗ／ｖ）の溶液とした。
Production Example 6 Preparation of Ezo Mikuri Extract 400 mL of 50% ethanol aqueous solution was added to g of Ezo Mikuri rhizome (produced by Shinwa) and immersed for 46 days at room temperature. This was filtered to obtain an ezomikuri extract. When this extract was concentrated, the solid content was 2.31 g. This extract was diluted with 50% ethanol to give a 1% (w / v) solution.
製造例７ ショウロシラン抽出物の調製
ショウロシラン根（新和物産）４０ｇに５０％エタノール水溶液４００ｍＬを加え、室温で４１日間浸漬した。これをろ過し、ショウロシラン抽出液を得た。この抽出液を濃縮したところ、その固形分は７．８１ｇであった。この抽出物を５０％エタノールにて希釈し、１％（ｗ／ｖ）の溶液とした。
Production Example 7 Preparation of Shorosilane Extract To 40 g of shorosilane root (produced by Shinwa Bussan), 400 mL of 50% aqueous ethanol solution was added and immersed for 41 days at room temperature. This was filtered to obtain a shorosilane extract. When this extract was concentrated, the solid content was 7.81 g. This extract was diluted with 50% ethanol to give a 1% (w / v) solution.
製造例８ ヨロイグサ抽出物の調製
ヨロイグサ根（新和物産）４０ｇに５０％エタノール水溶液４００ｍＬを加え、室温で２５日間浸漬した。これをろ過し、ヨロイグサ抽出液を得た。この抽出液を濃縮したところ、その固形分は５．２４ｇであった。この抽出物を５０％エタノールにて希釈し、１％（ｗ／ｖ）の溶液とした。
Production Example 8 Preparation of Euglena extract 400 g of 50% aqueous ethanol solution was added to 40 g of euglena root (Shinwa product) and immersed at room temperature for 25 days. This was filtered to obtain an extract of Euglena. When this extract was concentrated, the solid content was 5.24 g. This extract was diluted with 50% ethanol to give a 1% (w / v) solution.
製造例９ ウチワドコロ抽出物の調製
ウチワドコロ根茎（新和物産）４０ｇに５０％エタノール水溶液４００ｍＬを加え、室温で２８日間浸漬した。これをろ過し、ウチワドコロ抽出液を得た。この抽出液を濃縮したところ、その固形分は４．８８ｇであった。この抽出物を５０％エタノールにて希釈し、１％（ｗ／ｖ）の溶液とした。
Production Example 9 Preparation of Prickly Pendulum Extract 400 ml of 50% ethanol aqueous solution was added to 40 g of prickly pear rhizome (Shinwa product) and immersed for 28 days at room temperature. This was filtered to obtain a prickly extract. When this extract was concentrated, the solid content was 4.88 g. This extract was diluted with 50% ethanol to give a 1% (w / v) solution.
製造例１０ ビャクダン抽出物の調製
ビャクダン樹木（新和物産）４０ｇに５０％エタノール水溶液４００ｍＬを加え、室温で１５日間浸漬した。これをろ過し、ビャクダン抽出液を得た。この抽出液を濃縮したところ、その固形分は０．６４ｇであった。この抽出物を５０％エタノールにて希釈し、１％（ｗ／ｖ）の溶液とした。
Production Example 10 Preparation of Sandalwood Extract 400 ml of 50% ethanol aqueous solution was added to 40 g of sandalwood tree (Shinwa Bussan) and immersed at room temperature for 15 days. This was filtered to obtain a sandalwood extract. When this extract was concentrated, the solid content was 0.64 g. This extract was diluted with 50% ethanol to give a 1% (w / v) solution.
実施例１ 植物抽出物によるＭＦＡＰ−４産生促進
正常ヒト真皮線維芽細胞（倉敷紡績株式会社）を、３．０×１０5細胞/ウェルで２４ウェルプレートに添加し、２４時間培養した。培地を交換し、次いで各ウェルに、試験物質として製造例１〜１０で調製した植物抽出物のいずれか（最終濃度０．１％（ｖ／ｖ）；エキス分として０．００１％（ｗ／ｖ））、又は対照として同量の５０％エタノールを添加した。３日後、培養上清を回収し、それをサンプルとしてウェスタンブロティング法によりＭＦＡＰ−４のバンドを検出してＭＦＡＰ−４タンパク質の発現量を定量し、対照に対する相対発現量を求めた。
結果を表１に示す。表１記載の植物抽出物は、いずれもＭＦＡＰ−４発現を促進した。
Example 1 Promotion of MFAP-4 Production by Plant Extract Normal human dermal fibroblasts (Kurashikibo Co., Ltd.) were added to a 24-well plate at 3.0 × 10 5 cells / well and cultured for 24 hours. The medium was changed, and then each well was extracted with any of the plant extracts prepared in Production Examples 1 to 10 as a test substance (final concentration: 0.1% (v / v); 0.001% (w / v)), or the same amount of 50% ethanol was added as a control. Three days later, the culture supernatant was collected, and a MFAP-4 protein band was detected by Western blotting using the culture supernatant as a sample to quantify the expression level of MFAP-4 protein, and the relative expression level relative to the control was determined.
The results are shown in Table 1. All plant extracts listed in Table 1 promoted MFAP-4 expression.
実施例２ 植物抽出物による弾性線維形成促進
実施例１と同様の手順で、但し細胞を１．０×１０5細胞/ウェルでプレートに添加し、２４時間培養した後、培地を交換し、ウェルに試験物質としてゴマノハグサ抽出物（最終濃度０．２％（ｖ／ｖ）；エキス分として０．００２％ｗ／ｖ％）、又は対照として同量の５０％エタノールを添加した。７日後、メタノールで細胞を固定し、トロポエラスチン抗体及びフィブリリン−１抗体を用いて免疫染色を、ＤＡＰＩを用いて核染色を行い、蛍光顕微鏡下で弾性線維の形成を観察した。
結果を図１に示す。ゴマノハグサ抽出物によりトロポエラスチン及びフィブリリン−１の密度が高まっており、弾性線維形成が促進されているのが観察された。
Example 2 Promotion of elastic fiber formation by plant extract In the same procedure as in Example 1, except that cells were added to the plate at 1.0 × 10 5 cells / well and cultured for 24 hours. In addition, sesame extract was added as a test substance (final concentration 0.2% (v / v); 0.002% w / v% as an extract), or the same amount of 50% ethanol as a control. Seven days later, the cells were fixed with methanol, immunostained with tropoelastin antibody and fibrillin-1 antibody, and nuclear stained with DAPI, and the formation of elastic fibers was observed under a fluorescence microscope.
The results are shown in FIG. It was observed that the density of tropoelastin and fibrillin-1 was increased by the licorice extract, and the formation of elastic fibers was promoted.
実施例３ 植物抽出物によるＭＭＰ−１発現抑制
実施例１と同様の手順で、但し細胞を１．０×１０5細胞/ウェルでプレートに添加し、２４時間培養した後、培地を交換し、細胞を４群に分けて８日間培養した。２群は、１０ｎＭの組換えＭＦＡＰ−４（ｒＭＦＡＰ−４）の存在下又は非存在下で培養し、培養中に２回、ＭＦＡＰ−４に対する特異性がない対照ｓｉＲＮＡ配列（２０μＭで３．７５μｌ）を培地に添加することにより、ｓｉＲＮＡを細胞に導入した（ａ群およびｃ群）。残りの２群は、ｒＭＦＡＰ−４非存在下で、ＭＦＡＰ−４ ｓｉＲＮＡ配列（２０μＭで３．７５μｌ）を培養中に２回培地に添加して細胞に導入したか（ｂ群）、又はゴマノハグサ抽出物（最終濃度０．２％（ｖ／ｖ）；エキス分として０．００２％ｗ／ｖ％）存在下で培養した（ｄ群）。培養後、培養上清を回収し、それをサンプルとしてウェスタンブロティング法によりＭＭＰ−１のバンドを検出した。
結果を図２に示す。コラーゲン分解酵素であるＭＭＰ−１は、ｓｉＲＮＡにより細胞のＭＦＡＰ−４発現を抑制した場合に高発現したが（ｂ）、ゴマノハグサ抽出物により発現が抑制された（ｄ）。ゴマノハグサ抽出物によるＭＭＰ−１発現抑制効果は、ｒＭＦＡＰ−４による発現抑制効果（ｃ）とほぼ同程度であった。ゴマノハグサ抽出物により皮膚組織におけるコラーゲン分解を抑制することができる。
Example 3 Suppression of MMP-1 expression by plant extract In the same procedure as in Example 1, except that cells were added to the plate at 1.0 × 10 5 cells / well and cultured for 24 hours, then the medium was changed, The cells were divided into 4 groups and cultured for 8 days. The two groups were cultured in the presence or absence of 10 nM recombinant MFAP-4 (rMFAP-4) and twice in culture, a control siRNA sequence with no specificity for MFAP-4 (3.75 μl at 20 μM). ) Was added to the medium to introduce siRNA into the cells (groups a and c). In the other two groups, in the absence of rMFAP-4, the MFAP-4 siRNA sequence (3.75 μl at 20 μM) was added to the medium twice during the culture and introduced into the cells (group b), or sesame leaf extract In the presence of the product (final concentration 0.2% (v / v); 0.002% w / v% as extract) (group d). After culturing, the culture supernatant was recovered, and a MMP-1 band was detected by Western blotting using it as a sample.
The results are shown in FIG. MMP-1, which is a collagenolytic enzyme, was highly expressed when siRNA suppressed the expression of MFAP-4 in the cells (b), but its expression was suppressed by the sesame extract (d). The MMP-1 expression inhibitory effect of the sesame extract was almost the same as the expression inhibitory effect (c) of rMFAP-4. Collagen degradation in the skin tissue can be suppressed by the sesame extract.
参考例１ 光老化皮膚及び加齢皮膚におけるＭＦＡＰ−４発現減少
Ｈａｃｈｉｙａら（Ａｍ．Ｊ．Ｐａｔｈｏｌ．１７４：４０１−４１３，２００９）に従ってＳＣＩＤマウスにヒト皮膚を移植し、移植ヒト皮膚モデル（ｘｅｎｏｇｒａｆｔｅｄ ｈｕｍａｎ ｓｋｉｎ）を作製した。移植の創傷が治癒した約１０週間後、移植皮膚にＢ波紫外線（ＵＶＢ）照射を行い光老化させた。照射は３０２ｎｍ付近にピークを持つフィルターを装着したＵＶＢランプを用い、１〜２ＭＥＤで週に５回、６週間行った。照射後、移植皮膚を採取し、トータルＲＮＡを抽出して、ＴａｑＭａｎ（登録商標）ｇｅｎｅ ｅｘｐｒｅｓｓｉｏｎ ａｓｓａｙｓ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いてＲＴ−ＰＣＲを行い、ＭＦＡＰ−４発現を測定し、内部標準（ＲＰＬＰ０）に対する相対値として定量化した。対照として、ＵＶＢ非照射皮膚で同様にＭＦＡＰ−４に発現を定量した。
さらに、２０代（Ｙｏｕｎｇ）及び５０代（Ｏｌｄ）のヒトから皮膚検体を採取し、上記と同様の手順でＭＦＡＰ−４発現を定量した。
結果を図３に示す。ＵＶＢ照射された光老化皮膚では、非照射皮膚と比べてＭＦＡＰ−４発現が有意に低下した（Ａ）。ＭＦＡＰ−４発現の低下は加齢皮膚でも見られた（Ｂ）。光老化や加齢に伴う老化により、皮膚におけるＭＦＡＰ−４発現は減少する。
Reference Example 1 Reduction of MFAP-4 Expression in Photoaged Skin and Aged Skin According to Hachiya et al. (Am. J. Pathol. 174: 401-413, 2009), human skin was transplanted into SCID mice, and a transplanted human skin model (xenografted human) skin). About 10 weeks after the transplant wound was healed, the transplanted skin was irradiated with B-wave ultraviolet light (UVB) and photoaged. Irradiation was performed 5 times a week for 6 weeks at 1-2 MED using a UVB lamp equipped with a filter having a peak near 302 nm. After irradiation, the transplanted skin was collected, total RNA was extracted, RT-PCR was performed using TaqMan (registered trademark) gene expression assays (Applied Biosystems), MFAP-4 expression was measured, and internal standard (RPLP0) Quantified as a relative value to. As a control, expression in MFAP-4 was similarly quantified in non-UVB irradiated skin.
Furthermore, skin samples were collected from humans in their 20s (Young) and 50s (Old), and MFAP-4 expression was quantified by the same procedure as described above.
The results are shown in FIG. In photoaged skin irradiated with UVB, MFAP-4 expression was significantly reduced compared to non-irradiated skin (A). A decrease in MFAP-4 expression was also seen in aging skin (B). Due to photoaging and aging accompanying aging, MFAP-4 expression in the skin decreases.
参考例２ 皮膚光老化に対するＭＦＡＰ−４過剰発現の影響
Ｈａｃｈｉｙａら（Ｇｅｎｅ Ｔｈｅｒ．１４：６４８−６５６，２００７）に従って、参考例１で作製した移植ヒト皮膚に、ＶＳＶ−Ｇエンベロープを有するシュードタイプレンチウイルスベクター（ＶＳＶ−Ｇ ｐｓｅｕｄｏｔｙｐｅｄ ｌｅｎｔｉｖｉｒａｌ ｖｅｃｔｏｒ）を用いてＭＦＡＰ−４を過剰発現させた。１週間後、この皮膚に１〜２ＭＥＤで週に５回、８週間ＵＶＢ照射を行った。対照として同様の手順でレポーター遺伝子を発現する移植皮膚を作製し、ＵＶＢ照射及び非照射皮膚を作製した。
次いで、これらの皮膚のレプリカを作製し、ＰＲＩＭＯＳ Ｃｏｍｐａｃｔ（ＧＦ Ｍｅｓｓｔｅｃｈｎｉｋ ＧｍｂＨ）を用いて、領域（４〜５ｍｍ×７〜９ｍｍ）の３次元解析をおこない、Ｓａ値（解析領域の凹部及び凸部の高さの絶対値の平均）、及びＳｚ（凸部のピークから凹部の底までの高低差の平均）を求め、皮膚の表面粗さを評価した。
結果を図４に示す。ＵＶＢ照射によって起こる皮膚表面粗さの亢進が、ＭＦＡＰ−４過剰発現によって抑制された。またＵＶＢ照射により皮膚のしわが増え弾性が低下するが、これらもＭＦＡＰ−４過剰発現によって抑制された（データ示さず）。
Reference Example 2 Effect of MFAP-4 overexpression on skin photoaging According to Hachiya et al. (Gene Ther. 14: 648-656, 2007), the transplanted human skin prepared in Reference Example 1 has a pseudo-type wrench having a VSV-G envelope. MFAP-4 was overexpressed using a viral vector (VSV-G pseudotyped longitudinal vector). One week later, the skin was irradiated with UVB at 1-2 MED 5 times a week for 8 weeks. As a control, transplanted skin expressing the reporter gene was prepared by the same procedure, and UVB irradiated and non-irradiated skin were prepared.
Next, replicas of these skins were prepared, and a three-dimensional analysis of the region (4-5 mm × 7-9 mm) was performed using PRIMOS Compact (GF Messtechnik GmbH), and the Sa value (the concave and convex portions of the analysis region was measured). The average of the absolute value of the height) and Sz (average of the height difference from the peak of the convex part to the bottom of the concave part) were determined, and the surface roughness of the skin was evaluated.
The results are shown in FIG. Increased skin surface roughness caused by UVB irradiation was suppressed by MFAP-4 overexpression. UVB irradiation increased skin wrinkles and decreased elasticity, but these were also suppressed by MFAP-4 overexpression (data not shown).
参考例３ ＭＦＡＰ−４発現抑制が細胞外マトリックス（ＥＣＭ）に与える影響
参考例２と同様の手順でＭＦＡＰ−４過剰発現ＵＶＢ照射皮膚、レポーター遺伝子発現ＵＶＢ照射皮膚、及びレポーター遺伝子発現ＵＶＢ非照射皮膚を作製した。移植皮膚をホルマリン固定して皮膚切片を作製し、コラーゲン抗体を用いた免疫染色を行った。対照として正常ウサギＩｇＧ抗体による非特異的な免疫染色を行った。
結果を図５に示す。ＵＶＢ照射によって起こる真皮コラーゲンの減少は、ＭＦＡＰ−４過剰発現によって抑制された。
Reference Example 3 Effect of inhibition of MFAP-4 expression on extracellular matrix (ECM) In the same procedure as in Reference Example 2, MFAP-4 overexpressing UVB-irradiated skin, reporter gene-expressing UVB-irradiated skin, and reporter gene-expressing UVB-irradiated skin Was made. The transplanted skin was fixed with formalin to prepare a skin section, and immunostaining was performed using a collagen antibody. As a control, non-specific immunostaining with normal rabbit IgG antibody was performed.
The results are shown in FIG. The decrease in dermal collagen caused by UVB irradiation was suppressed by MFAP-4 overexpression.
参考例４ ＭＭＰ活性に対するＭＦＡＰ−４の影響
参考例２と同様の手順でＭＦＡＰ−４過剰発現ＵＶＢ照射皮膚、レポーター遺伝子発現ＵＶＢ照射皮膚、及びレポーター遺伝子発現ＵＶＢ非照射皮膚を作製した。
移植皮膚を採取し、バッファー（２００ｍＭ ＮａＣｌ、５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）、５ｍＭ ＣａＣｌ2、２０μＭ ＺｎＳＯ4、０．０５％ Ｂｒｉｊ３５）に溶解させて組織抽出物を得、これをサンプルとして、ＳｅｎｓｏＬｙｔｅTM５２０ ＭＭＰ−１２ Ｆｌｕｏｒｉｍｅｔｒｉｃ Ａｓｓａｙ Ｋｉｔ（ＡｎａＳｐｅｃ Ｃｏｒｐｏｒａｔｅ）を用いて、蛍光標識ＭＭＰ−１２基質に対する酵素活性を蛍光的に測定した。
さらに、参考例３と同様の手順で遺伝子導入線維芽細胞を調製した。細胞からトータルＲＮＡを抽出して、ＴａｑＭａｎ（登録商標）ｇｅｎｅ ｅｘｐｒｅｓｓｉｏｎ ａｓｓａｙｓ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いたＲＴ−ＰＣＲによりＭＭＰ−１２発現及びＭＭＰ−１発現を測定し、内部標準（ＧＡＰＤＨ）に対する相対値として定量化した。
結果を図６及び７に示す。ＵＶＢ照射によって起こるＭＭＰ−１２活性の亢進が、ＭＦＡＰ−４過剰発現によって抑制された。また、ＭＦＡＰ−４発現を抑えた場合、ＭＭＰ−１２やＭＭＰ−１の発現は増加した。ＭＭＰ−１２やＭＭＰ−１はＥＣＭ分解に関わる酵素であることから、ＭＦＡＰ−４はコラーゲン線維や弾性線維などのＥＣＭの分解に関わることが示された。
Reference Example 4 Effect of MFAP-4 on MMP Activity MFAP-4 overexpressing UVB-irradiated skin, reporter gene-expressing UVB irradiated skin, and reporter gene-expressing UVB non-irradiated skin were prepared in the same manner as in Reference Example 2.
The transplanted skin was collected and dissolved in a buffer (200 mM NaCl, 50 mM Tris-HCl (pH 7.6), 5 mM CaCl 2 , 20 μM ZnSO 4 , 0.05% Brij 35) to obtain a tissue extract, which was used as a sample. Enzyme activity against fluorescently labeled MMP-12 substrate was measured fluorescently using SensoLite ™ 520 MMP-12 Fluorometric Assay Kit (AnaSpec Corporation).
Furthermore, gene-introduced fibroblasts were prepared by the same procedure as in Reference Example 3. Total RNA was extracted from the cells, MMP-12 expression and MMP-1 expression were measured by RT-PCR using TaqMan (registered trademark) gene expression assays (Applied Biosystems), and relative values to the internal standard (GAPDH) Quantified.
The results are shown in FIGS. Enhancement of MMP-12 activity caused by UVB irradiation was suppressed by MFAP-4 overexpression. Moreover, when MFAP-4 expression was suppressed, the expression of MMP-12 and MMP-1 increased. Since MMP-12 and MMP-1 are enzymes involved in ECM degradation, MFAP-4 was shown to be involved in the degradation of ECM such as collagen fibers and elastic fibers.
参考例５ ＭＦＡＰ−４発現抑制がフィブリリン−１の重合化に与える影響
実施例１と同様の手順で、正常ヒト真皮線維芽細胞を１．０×１０5細胞/ウェルで２４ウェルプレートに添加し、２４時間培養した。培地を交換し、細胞を３群に分けて８日間培養した。２つの群は、１０ｎＭの組換えＭＦＡＰ−４（ｒＭＦＡＰ−４）の存在下又は非存在下で培養し、培養中に２回、ＭＦＡＰ−４に対する特異性がない対照ｓｉＲＮＡ配列（２０μＭで３．７５μｌ）を培地に添加することにより、ｓｉＲＮＡを細胞に導入した。残りの１群はｒＭＦＡＰ−４非存在下で培養し、培養中に２回、ＭＦＡＰ−４ ｓｉＲＮＡ配列（２０μＭで３．７５μｌ）を培地に添加して細胞に導入した。
培養後、メタノールで細胞を固定し、トロポエラスチン抗体及びフィブリリン−１抗体を用いて免疫染色を、ＤＡＰＩを用いて核染色を行い、蛍光顕微鏡下で弾性線維の形成を観察した。
結果を図８に示す。ｓｉＲＮＡでＭＦＡＰ−４発現を抑えた場合、トロポエラスチン及びフィブリリン−１からなる重合体の形成が損なわれたことから、ＭＦＡＰ−４がフィブリリン−１の重合化に寄与していることが示された。
Reference Example 5 Effect of inhibition of MFAP-4 expression on polymerization of fibrillin-1 In the same procedure as in Example 1, normal human dermal fibroblasts were added to a 24-well plate at 1.0 × 10 5 cells / well. And cultured for 24 hours. The medium was changed and the cells were divided into 3 groups and cultured for 8 days. The two groups were cultured in the presence or absence of 10 nM recombinant MFAP-4 (rMFAP-4) and twice in culture, a control siRNA sequence with no specificity for MFAP-4 (3. The siRNA was introduced into the cells by adding 75 μl) to the medium. The remaining one group was cultured in the absence of rMFAP-4, and twice during the culture, the MFAP-4 siRNA sequence (3.75 μl at 20 μM) was added to the medium and introduced into the cells.
After culturing, the cells were fixed with methanol, immunostained with tropoelastin antibody and fibrillin-1 antibody, and nuclear stained with DAPI, and the formation of elastic fibers was observed under a fluorescence microscope.
The results are shown in FIG. When MFAP-4 expression was suppressed with siRNA, the formation of a polymer composed of tropoelastin and fibrillin-1 was impaired, indicating that MFAP-4 contributes to the polymerization of fibrillin-1. It was.
参考例６ ＭＦＡＰ−４とフィブリリン−１との相互作用
実施例１と同様の手順で、正常ヒト真皮線維芽細胞を１．０×１０5細胞／ウェルで２４ウェルプレートに添加し、２４時間培養した。培地を交換し、細胞を８日間培養した。培養上清を回収し、０．４５μｍメンブレン（Ｍｉｌｌｉｐｏｒｅ）で濾過した後３ｋＤａカットオフメンブレン（Ｍｉｌｌｉｐｏｒｅ）を用いて１０倍に濃縮し、濃縮液５００μＬを１０μｇのＭＦＡＰ−４抗体又は正常ウサギＩｇＧとともにインキュベートした。これに２０μＬのＰｒｏｔｅｉｎ Ｇセファロースビーズ（ＧＥ Ｈｅａｌｔｈｃａｒｅ）を添加してインキュベートした後、遠心によりビーズを回収した。回収したビーズからタンパク質を集め、１×ＳＤＳサンプルバッファー（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ ｓｃｉｅｎｔｉｆｉｃ）に懸濁し、フィブリリン−１抗体を用いたウェスタンブロティング法に供した。
結果を図９に示す。ＭＦＡＰ−４抗体により回収した画分にフィブリリン−１が検出されたことから、ＥＣＭ内でＭＦＡＰ−４とフィブリリン−１とが共存していることが示唆された。この結果を参考例４で示されたＭＦＡＰ−４のフィブリリン−１の重合化に対する影響とあわせて考慮すると、ＭＦＡＰ−４は、ＥＣＭ内でフィブリリン−１と相互作用して、フィブリリン−１からミクロフィブリルへの重合化を促進し、弾性線維形成に関与していることが示唆される（図１０）。
Reference Example 6 Interaction between MFAP-4 and fibrillin-1 In the same procedure as in Example 1, normal human dermal fibroblasts were added to a 24-well plate at 1.0 × 10 5 cells / well and cultured for 24 hours. did. The medium was changed and the cells were cultured for 8 days. The culture supernatant is collected, filtered through a 0.45 μm membrane (Millipore), concentrated 10-fold using a 3 kDa cut-off membrane (Millipore), and 500 μL of the concentrated solution is incubated with 10 μg of MFAP-4 antibody or normal rabbit IgG. did. 20 μL of Protein G Sepharose beads (GE Healthcare) was added thereto and incubated, and then the beads were collected by centrifugation. Proteins were collected from the collected beads, suspended in 1 × SDS sample buffer (Thermo Fisher Scientific), and subjected to Western blotting using fibrillin-1 antibody.
The results are shown in FIG. Since fibrillin-1 was detected in the fraction collected by the MFAP-4 antibody, it was suggested that MFAP-4 and fibrillin-1 coexist in the ECM. Considering this result together with the influence of MFAP-4 on the polymerization of fibrillin-1 shown in Reference Example 4, MFAP-4 interacts with fibrillin-1 in the ECM, and from fibrillin-1 to microarray. It is suggested that it promotes polymerization to fibrils and is involved in elastic fiber formation (FIG. 10).
参考例まとめ
参考例１〜２に示されるように、ＭＦＡＰ−４は、光老化による皮膚弾性の低下や、しわ、滑らかさの低下を防ぐ働きがある一方で、その発現は皮膚光老化や加齢によって減少する。参考例３〜５に示すように、ＭＦＡＰ−４は、酵素ＭＭＰの活性化抑制等を介してＵＶＢ曝露によるコラーゲンの減少を抑制するとともに、ＥＣＭ内でフィブリリン−１と相互作用してフィブリリン−１からミクロフィブリルへの重合化を促進することで弾性線維の形成を促し、結果としてＥＣＭの構造を維持するように機能する。
従って、ＭＦＡＰ−４発現を促進することによって、上記ＭＦＡＰ−４の機能を介して皮膚の弾性を向上させることができ、ひいては皮膚光老化を改善することができる。
Summary of Reference Examples As shown in Reference Examples 1 and 2, MFAP-4 has a function of preventing the skin elasticity, wrinkles, and smoothness from being reduced by photoaging, while its expression is influenced by skin photoaging and aging. Decreases with age. As shown in Reference Examples 3 to 5, MFAP-4 suppresses the decrease in collagen due to UVB exposure through inhibition of the activation of the enzyme MMP and the like, and also interacts with fibrillin-1 in the ECM. It promotes the polymerization of fibrils to microfibrils to promote the formation of elastic fibers and consequently functions to maintain the structure of the ECM.
Therefore, by promoting the expression of MFAP-4, the elasticity of the skin can be improved through the function of the MFAP-4, and the skin photoaging can be improved.
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| JP2013035807A (en) * | 2011-08-10 | 2013-02-21 | Rohto Pharmaceutical Co Ltd | Elastic-fiber formation promotor |
| CN104721706A (en) * | 2015-04-16 | 2015-06-24 | 王景明 | Traditional Chinese medicine for treating globus hysteriocus |
| JP2017218429A (en) * | 2016-06-08 | 2017-12-14 | ポーラ化成工業株式会社 | Skin ligament-improving agent |
| JP2020103198A (en) * | 2018-12-28 | 2020-07-09 | ポーラ化成工業株式会社 | Anti-skin aging agent screening method |
| JP2021513360A (en) * | 2017-11-01 | 2021-05-27 | スイダンスク ウニバーシティトＳｙｄｄａｎｓｋ Ｕｎｉｖｅｒｓｉｔｅｔ | Antibodies to MFAP4 |
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| JP2013035807A (en) * | 2011-08-10 | 2013-02-21 | Rohto Pharmaceutical Co Ltd | Elastic-fiber formation promotor |
| CN104721706A (en) * | 2015-04-16 | 2015-06-24 | 王景明 | Traditional Chinese medicine for treating globus hysteriocus |
| JP2017218429A (en) * | 2016-06-08 | 2017-12-14 | ポーラ化成工業株式会社 | Skin ligament-improving agent |
| JP2021100977A (en) * | 2016-06-08 | 2021-07-08 | ポーラ化成工業株式会社 | Skin ligament-improving agent |
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