200914061 九、發明說明： 【發明所屬之技術領域】 本案係關於一種抗皮膚細胞外因性老化的方法以及組成物。在本案中 所指的皮膚外因性老化係起因於紫外線過度照射者，而本案以綠藻萃取物 對抗因务、外線而致生之角負層粗化、纹理粗輪、敵紋、鬆弛等皮膚老化問 題。 【先前技術】200914061 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a method and composition for anti-aging of skin cells. The external skin aging referred to in this case is caused by ultraviolet radiation. In this case, the green algae extract is used to counteract the cause of the negative angle of the horn, rough texture, enemy pattern, slack and other skin. Aging problem. [Prior Art]
Fisher等人於2000年提出，紫外線照射會傷害人類皮膚’並引起一 些早發性的皮膚問題，例如角質層粗化、紋理粗糙、皱紋以及色素沈著斑 點。Fisher et al. (2000) proposed that ultraviolet radiation can damage human skin' and cause some early-onset skin problems such as coarsening of the stratum corneum, rough texture, wrinkles, and pigmentation spots.
Jenkins於2002年提出’過度曝露於紫外線_A(UVA)及紫外線_B(UVB) 會造成皮膚損害。Jenkins proposed in 2002 that 'excessive exposure to UV-A (UVA) and UV-B (UVB) can cause skin damage.
Debelle等人於1999年提出’膠原蛋白及彈力蛋白是兩種主要的細胞 表面間質(ECM，Extracellular m—的蛋白質。足量的彈力蛋白使皮膚顯 得年輕。膠原蛋白則維持皮膚的亮澤光彩。Debelle et al. proposed in 1999 that 'collagen and elastin are two major cell surface interstitial (ECM, Extracellular m-proteins. A sufficient amount of elastin makes the skin look younger. Collagen maintains the radiance of the skin. .
Fisher等人於2002年提出’紫外線會活化皮膚纖維母細胞(Fjbr〇b丨郎” 中的金屬蛋白分解_MP，Meta|_teinase)，皮膚献過度會使金屬蛋 白刀解酶(MMP)被過度活化’破壞正常纖維母細胞㈣灿㈣)内的膠原蛋 白及彈性蛋白。In 2002, Fisher et al. proposed that 'ultraviolet light activates metalloproteinases in skin fibroblasts (Fjbr〇b丨lang)_MP, Meta|_teinase), and excessive skin overgrowth causes metalloproteinase (MMP) to be overexposed. Activates 'destroy collagen and elastin in normal fibroblasts (4) Can (4).
Fisherl及Watanabe等人分別於1996及2〇〇4年提出，紫外線隱、 UVB在真皮層的纖維母細胞中，引起蛋白激素酶(pKc，p「輸⑷赚〇 去活化金屬蛋白分解酶(MMp)。 由上述之子理基礎’吾等可知皮膚受到紫外線過度侵害時，纖維母細 胞__ast)内會職不完全氧化現象，造成自由基鱗，聽離的自由 200914061 基會活化金屬蛋白分解酶_p)，被過度活化的金屬蛋白分解酶_?)會 破壞正常纖維母細胞(Fibroblast)内的膠原蛋白、彈性蛋白、纖維母細胞、 細胞膜、細胞核，引起皮膚細胞外基f萎縮，真皮層厚度逐漸減少，角質 層粗化、紋理粗糙、皺紋、鬆弛等老化問題。 目刖經皮吸收用以改善皮膚老化問題的外用製劑，包括果酸、維他命 A酸、維他命A、左旋維他命C等。果酸可使老舊角質脫落，表面性的改 善皮膚角化現象。果酸之使用濃度有所區分及限制，2〇%以下的低濃度果 , 酸可以作為日常保養，而20%〜70%的高濃度，則是由皮膚科醫師進行果 酸換膚時使用。維他命A酸制及功效與概触，但對皮膚之刺激性更 南，使財甚至會出現脫皮、紅腫等副侧，因此__定要在醫師指示下使 用，且使用期間嚴格防曬。左旋維他命C具有抗氧化功能，可以保護細胞 不受紫外線縣以及巾和游離自域，左雜他命c村祕合成膠原蛋 白，可以改善皮膚皺紋及鬆弛現象。 【發明内容】 本發明之主要目的，係在提出一種經科學驗證的抗皮膚細胞外因性老 _ί, 化的原料，並研製經皮吸收的外用製劑。 上述抗皮膚細胞外因性老化的原料係綠藻萃取物，本發明主要方法係 以一有效I之綠藻萃取物抑制皮膚纖維母細胞(F jbr〇blast)中的金屬蛋白分 解酶(MMP)之活性，以及促進皮膚纖維母細胞(Fibr〇b丨ast)中的彈性蛋白 (Elastin)及膠原蛋白(Collagen)增生。該綠藻萃取物可混合於一經皮吸收之 化粧品組成物中，使該組成物具有抗外因性皮膚老化之作用。 本發明抗皮膚細胞外因性老化的方法’係以綠藻萃取物抑制皮膚纖維 母細胞(Fibroblast)中的金屬蛋白分解酶(MMp)。 200914061 本發明包含以綠藻萃取物抑制皮膚纖維母細胞(Fibroblast)中的金屬蛋 白分解酶(MMP)而達到促進彈性蛋白(Elastin)及膠原蛋白(Collagen)增生的 效果。 本發明較佳方案係於可使用於人類皮膚之化粧品組成物中加強併入有 效量之綠藻萃取物’以增加該組成物抗皮膚細胞外因性老化之功效。 已知於紫外線過度照射皮膚時，皮膚纖維母細胞(Fibroblast)中的金屬 蛋白分解酶(MMP)將被過度活化，因此破壞正常纖維母細胞(Fibr〇b|ast)内 的彈性蛋白(Elastin)及膠原彈白(C0||agen)。因此，本發明係基於綠藻萃取 物對金屬蛋白分解酶(MMP)的活性抑制，使併入本發明綠藻萃取物之化粧 品組成物特別適用於保護皮膚，減少紫外線對皮膚侵襲而引起的老化現 象，包括角質層粗化、紋理粗糙、皺紋、鬆弛等皮膚問題。 本發明所使用之綠藻萃取物的量無法絕對地規定，因其依製備方式不 同而將有所改變。 本發明所使用之綠藻萃取物以及併入該綠藻萃取物製成經皮吸收之組 成物可為各種形式’例如水液狀、凝雜、乳液狀、霜狀、膏狀、粉狀、 油液狀。其中該組成物全量中包括約_至約5〇重量％之綠藻萃取物。 【實施方式】 依本案發_容之所述，—種抗皮膚細胞外因性老化之方法，係提供 彔冰萃取物做為抑制皮膚纖維母細胞(Fjb「〇b|ast)中的金屬蛋白分解酶 (MMP) ’以及促進彈性蛋白(曰astjn)及前勝原蛋白(p_丨㈣卽)基因增生之 主要成份。該、賴萃取物可贿何適合的方式製造，主要以在淡水中生長 的單、田御取成水錄萃取物。驗外線光譜分析(第八圖至第十圖， 、為00 cm_1〜40〇 cm-1)得知該綠藻萃取物係有機化合物，碳、氫、 7 200914061 氮為其主要元素’元素含量：氮(Ν)7·06。/。〜7.66。/。、氫(h)5.9%〜6 51%、石炭 (C)35.67%〜38.1%。 為示範本發明綠藻萃取物之抗皮膚細胞外因性老化之功效，茲以下列 所述之實驗結果說明。實驗係在兩種條件下進行，其中之一係以蛋白激素 誘導劑(PMA’ phorbol. 12-myristate 13-acetate)誘導皮膚細胞產生金屬蛋 白分解酶(MMP)為實驗條件，測試本發明綠藻萃取物對金屬蛋白分解酶 (MMP)的活性抑制效用。另一係以紫外線照射誘導皮膚細胞產生金屬蛋白 分解酶(MMP)為實驗條件，測試本發明綠藻萃取物對金屬蛋白分解酶(MMp) 的活性抑制效用。 【以蛋白激素誘導劑(PMA)誘導皮膚纖維母細胞(Fibroblast)產生金屬蛋白 分解酶(MMP)】 【實驗1】 第一圖之實驗例’係在正常皮膚纖維母細胞(Fibr〇b丨ast)以蛋白激素誘 導劑(PMA)誘導產生金屬蛋白分解酶(MMP)，測試本發明綠藻萃取物對金 屬蛋白分解酶(MMP)生成之抑制效用，並以量化之柱狀圖表表示。熟悉此 一領域者可知’蛋白激素誘導劑(PMA)為真皮層之纖維母細胞(Rbr〇b丨ast) 中蛋白激素轉(PKC, protein kinase C)之活化劑，而蛋白激素酶(pkc)為金 屬蛋白分解酶(MMP)之活化劑，而過度被活化的金屬蛋白分解酶(MMp)會 破壞正常皮膚纖維母細胞(Fibroblast〉的膠原蛋白（Collagen)及彈性蛋白 (Elastin)而致皮膚老化。第一圖之實驗例，編號為控制組，編號(2〉為對 8 200914061 照^編號⑶(4)(5)⑹為實驗組，實驗所需之培養時間為5小時。 ,(制、、且，表示正常纖維母細胞(F丨經5小時之實驗期間 所釋放的金屬蛋白分解酶_P)的含量大約為25 ng/mL。 '域(2)對&、組’表示加人蛋自激錢___議，金屬蛋白分 解酶(MMP)平均含量升高為7〇叩社。 —編號(3)實驗組，表示加人蛋白激«導劑(P_Q_以及本發明綠 為卞取物2Qnag/m| ’結果齡金輕白分麟(_卩)平均含量降低至約％ ng/mL。 —編礙4)貫驗組，表示加人蛋白激素誘導劑(pMA)她Μ以及本發明綠 漂萃取物15mg/m|，結果顯示金屬蛋白分解酶_〇平均含量降低至約筘 ng/mL。 編號(5)實驗組，表示加人蛋白激素誘導劑___以及習知的金 屬蛋白刀解酶抑制劑(GMXMnM，結果顯示金屬蛋白分解酶(MMp)平均含 量約為50 ng/mL。 編號⑹貫驗組，表示加人蛋自激讀導劑(pMA)iQ_以及維生素c 〇·15_，結果顯示金屬蛋白分解酶_P)平均含量約為60 ng/mL。 實驗1之結果顯示，綠藻萃取物可以抑制金屬蛋白分解酶_p)之活 性’且以較高的有效量可得到較佳的抑制效果。且由編號⑶及編號⑷實驗 數據可知，本發明綠藻萃取物對金屬蛋白分解酶卿p)的活性抑制功效係 顯然地優於其他實驗組。 【實驗2】 9 200914061 第二圖之貫驗例’係以蛋白激素誘導劑(PMA)誘導產生金屬蛋白分解酶 (MMP)，綱本發鴨藻萃取婦金屬蛋白分解酶(MMp)之活性抑制效用 之西方墨點法結果。第一列影像係表示金屬蛋白分解酶(Μ Μ p)的活性反 應。弟*一列影像係為一對照組。 編號(1)表示對正常皮膚纖維母細胞(Fjbr〇biast)以蛋白激素誘導劑 (PMA)100mM誘導產生金屬蛋白分解酶(MMp)，結果顯示金屬蛋白分解酶 (MMP)的活性反應增加。 編號(2)可視為一基礎樣態’表示正常皮膚纖維母細胞(nbr〇b丨ast)在正 常情況下之金屬蛋白分解酶(MMP)的活性反應。 編號(3)表示對正常皮膚纖維母細胞(Fibroblast)加入蛋白激素誘導劑 (PMA)100mM，以及0.2⑽蛋白激素酶(PKC)抑制劑，與編號⑴結果比較， 蛋白激素酶(PKC)抑制劑些微地抑制了金屬蛋白分解酶(|^|^卩)的活性反應。 編號(4)表示對正常皮膚纖維母細胞(Fibr〇b丨ast)加入蛋白激素誘導劑 (PMA)100mM ’以及0·4ηΜ的已知金屬蛋白分解酶抑制劑(GM)，與編號⑴ 結果比較，已知金屬蛋白分解酶抑制劑(GM)些微地抑制了金屬蛋白分解酶 (MMP)的活性反應。 編號(5)表示對正常皮膚纖維母細胞(Fibroblast)加入蛋白激素誘導劑 (PMA)100mM，以及20mg/ml綠藻萃取物，與編號⑴(2)(3)(4)結果比較， 本發明綠藻萃取物顯然地抑制了金屬蛋白分解酶(MMP)的活性反麻。 實驗2之結果顯示’本發明綠藻萃取物可以抑制金屬蛋白分解酶(MMp) 之活性’且抑制成效齡優於習知的蛋白激麵(PKC)抑制劑和已知金屬蛋 白分解酶抑制劑(GM)。 10 200914061 【實驗3】 第三圖之實關，係金騎白分__p)被蛋白激讀導劑(pMA) 活化之條件下，以西方墨點法絲本發縣鮮取物具有使皮膚彈性蛋白 (Ela_增生之效用。第-列影像絲示皮赫性蛋白邮伽)增生反應。 第二列影像係為一對照組。 編號⑴可視為-基礎樣態，絲正f皮雜維母細胞㈣灿㈣在正 常情況下之彈性蛋白(Elastin)含量。 編號(2)表示對正常皮膚纖維母細胞(Fjbr〇b丨娜添加蛋白激素誘導巧 (ΡΜΑ__ ’結果顯示彈性蛋白_增量顯著減少。其原因係蛋白激 素誘導劑(PMA)活化金屬蛋白分解酶(MMp)，被過度活化的金屬蛋白分解酶 (MMP)破壞正常纖維母細胞(Fjb「〇b|ast)内的膠原蛋白及彈性蛋白。 編號(3)表示對正常皮膚纖維母細胞(Fibroblast)添加蛋白激素誘導劑 (PMA)100mM，以及10mg/m丨之本發明綠藻萃取物。與編號⑺比較彈性 蛋白(Elastin)顯著增加。 編號(4)表示對正常皮膚纖維母細胞(Fibroblast)添加蛋白激素誘導劑 (PMA)1〇OmM ’以及2〇mg/m丨之本發明綠藻萃取物。與編號⑺⑼比較， 彈性蛋白(Elastin)更顯著增加。 Λ 實驗3之結果顯示，綠藻萃取物採用一有效量具有增生彈性蛋白之咬 用。且以較高的有效量可得到較佳的彈性蛋白(Elastin)增生結果。 【實驗4】 11 200914061 . 第四®之實驗例’細蛋白激素料劑(PMA)料金屬蛋白分解酶 (_P)生成的條件下，本發縣鮮取物對金屬蛋自分解酶(_卩)的基因 表見抑制>文用之RT-PCR結果。圖中的細胞内抑制蛋白(τίΜρι及丁丨Mp3) 係存在於纖維母細胞(Fibr〇blast)中用以抗衡金属蛋白分解酶(_^、 MMP3)的物質。 ’’扁號(1)表示對正常皮膚纖維母細胞(Fjbr〇blast)以蛋白激素誘導劑 (PMA)IGQmM ’結果顯示細胞崎制蛋白(了哪彳及T|Mp3)減少致使金 ， 屬蛋白分解酶(MMP1、MMP3)基因表現增加。 編號(2)可視為一基礎樣態，表示正常皮膚纖維母細胞((rjbr〇b丨郎丨)在正 常情況下之金屬蛋白分解酶卿…、_p3)以及細胞内抑制蛋白(T|Mpi 及TIMP3)之含量。 編號⑶表示對正常皮膚纖維母細胞(Fibroblast)加入蛋白激素誘導劑 (P_ 1GQmM，以及α2υΜ蛋白激域PKC)抑侧，與編號⑴結果比較， 蛋白激素酶(pkc)抑制劑並未使細胞内抑制蛋白(TIMP1及T|Mp3)基因表 現顯著增加’以致金屬蛋白分解酶(MMP1、MMP3)基因表現並未顯著減少。 編號(4)表示對正常皮膚纖維母細胞(Fibroblast)加入蛋白激素誘導劑 (PMA)100mM，以及〇.捕的已知金屬蛋白分解酶抑制劑(GM〉，與編號⑴ 結果比較，已知金屬蛋白分解酶抑制劑(GM)並未使細胞内抑制蛋白(丁丨Μρι 及TIMP3)基因表現顯著增加，以致金屬蛋白分解_(_ρι、MMp3)基 因表現並未顯著減少。 編號(5)表示對正常皮膚纖維母細胞(Fjbr〇biast)加入蛋白激素誘導劑 (PMA) 100mM，以及20mg/m丨綠藻萃取物，與編號⑴(2)(3)⑷結果比較， 本發明綠澡萃取物雖然沒有使細胞内抑制蛋白(TIMPi及timp3)基因表現 200914061 顯著增加，然而金屬蛋白分解__P1、_P3)基因表現卻有顯著減少。 實驗4之料顯示，本發明綠藻萃取物可以抑制金屬蛋白分解酶卿p) 之基因表現，且抑制成效顯然優於已知的蛋白激素酶(pKc)抑制劑和已知金 屬蛋白分解酶抑制劑(GM)。 【以i外線射誘導皮膚纖維母細胞(Fjb_a啦生金屬蛋白分解酶 (MMP)】 【實驗1】 第圖之實驗例，係採正常皮膚纖維母細胞(Fibroblast)以紫外線 (UVB)騎15小時，以量化圖絲示金屬蛋白分麟(關p)的活性， 以及採用本發明綠藻萃取物—有效量後金屬蛋白分解酶_卩)的活性抑制 效用。 編號⑴控她’表示在無料線照射之下皮膚纖維母細胞(nbfOb_ 所釋放的金屬蛋白分解酶_P)的含量大約為3·5 ng/mL。 扁號(2)對知組，表示正常皮膚纖維母細胞(F丨纪)以紫外線照射1 $ 小時，金屬蛋白分解酶(MMP)平均含量升高為約6 〇 ng/mL。 、’扁號(3)員驗組’表示正常皮膚纖維母細胞(Fjb「〇b|ast)以紫外線照射1.5 小時，於照射紫外線前加入本發明綠藻萃取物1〇mg/m卜結果顯示金屬蛋 白分解酶(MMP)平均含量降低至約3 5ng/mL。 編唬(4)實驗組，表示正常皮膚纖維母細胞(Rbr〇b丨as〇以紫外線照射1 5 小呀，於照射紫外線前加入本發明綠藻萃取物 20mg/ml，結果顯示金屬蛋 13 200914061 白分解酶_卩)平均含量降低至約1.5 ng/mL。 編號(5)實驗組，表示正常皮膚纖維母細胞(Fibr〇b|ast)以紫外線照射"5 I寸於^射i Hil加人本發鴨鮮取物3C)mg/m卜結果顯示金屬蛋 白分解酶(MMP)平均含量降低至彳〇 ng/mL以下。 、’扁號(6)貫驗組’表示正常皮膚纖維母細胞(Fibr〇b|ast)以紫外線照射1 5 小’於照射紫外線前加入維生素Cai5_，結果顯示金屬蛋白分解酶(_鬥 平均含量反而增昇高達約9.0 ng/mi_。 編號⑺實驗組，表示正常皮膚纖維母細胞(Fibr〇b丨ast)以紫外線照射】5 小，於照射紫外線前加入維生素E 25 uM，結果顯示金屬蛋白分解酶(_門 平均含量維持不變，即約6.〇 ng/mL。 貫驗1之結果顯示，綠藻萃取物可以抑制金屬蛋白分解酶(_p)之活 !·生且以較尚的有效置可得到較佳的抑制效果。且由編號⑶⑷⑼實驗數據 可知’本發縣鮮取物對金屬蛋白分解酶(_p)的活性抑制功效係顯然 地優於其他實驗組。 【實驗2】 第，、圖之實驗例’係以紫外線照射正常皮膚纖維母細胞(Fjbr〇b㈣3〇 分鐘’金屬蛋白分解酶(MMP)及彈性蛋白(曰astin)的變化，以及本發明綠藻 萃取物具有使皮膚彈性蛋白(Elastin)增生之效用之西方墨點法結果。 編號(2)控制組’标在簡紫外線之前皮麵料細胞(nb「Qb丨糾的 金屬蛋白分解酶(MMP)以及彈性蛋白(Elastin)含量。 編號(1)對照組，表示皮膚纖維母細胞(Fibrob丨ast)以紫外線照射3〇分 14 200914061 鐘’金屬蛋白分»_p)平均含量顯著升高 ，而彈性蛋白(Elastin)含量顯 著降低。 、° ()貝驗組’表示皮膚纖維母細胞(Fibroblast)以紫外線照射30分 !里於如、射紫外線前加入本發明綠藻萃取物鳩_丨，與編號⑴對照組比 幸又、°果顯不金屬蛋白分解酶(MMP)平均含量大幅降低，雜蛋白(日astjn) 含量顯著提升。 、、Ό貝驗組’表示皮膚纖維母細胞(「化恤丨如)以紫外線照射3〇分 ;’、射兔外線月；j加人本發明綠讓萃取物，〇mg/m卜與編號⑴對照組比 較’結果顯示金屬奸分解酶_卩)平均含量大幅降低，彈性蛋白(曰astin) 含量顯著提升。 貝驗2之結果顯不，綠藻萃取物具有抑制金屬蛋白分解酶(剛門之活 性以及使彈性蛋白(日峨)增生之功效。紹較高的有效量對金屬蛋白分解 酶(MMP)的活性有較佳的抑制效果，同時使彈性蛋自(&as㈣增生。 【實驗3】 第七圖之實驗例’係本發明綠藻萃取物於紫外線照射皮膚纖維母細胞 (Fibroblast) 30分鐘金屬蛋白分解酶_?)及使前膠原蛋⑽隱响卽) 基因變化的RT.PCR結果。圖中的ΤΙΜΡι及T|Mp3係存在於纖維母細胞 (Fibroblast)中用以抗衡金屬蛋白分解酶(MMP1、MMp3)的物質。 編號⑴為控制組，表示正常皮膚纖維母細胞(Rb_ast)在未照射紫外 線之前金屬蛋白分解酶_P1、MMP3)、抑制劑(T|MP1、T|Mp3)以及前 膠原蛋白(Procollagen)之基因表現。 200914061 編號(2)為對如、組，表示對正常皮膚纖維母細胞(円br〇b|ast)照射紫外線 30分鐘’結果顯示金屬蛋白分解酶(mmp1、_P3)明顯增加，抑制劑谓η 及ΤΙΜΡ3基因表現無太大差異，但前膠原蛋白(ρ「。。。丨丨印印)基因表現減少。 編號⑶為實驗組，表轉正常植齡母細胞(Rb_ast)照射紫外線 30分鐘’於照射紫外線前加入1〇mg/m|綠鮮取物，與編號⑴控制組及 、扁號(2)對,’、、、、：£比較，本發明綠藻萃取物使金屬蛋白分解酶(_^1、 基因表現減少、抑制劑(τ丨MP1、谓p3)基因表現無差異而前膠原蛋白 (Procollagen)顯著增加。 、、扁號（）為實驗組’表示對正常皮膚纖維母細胞(F_b丨㈣照射紫外線 30分鐘，於照射紫外線前加入2〇m_丨綠藻萃取物，與編號⑴控制組及 編號(2)»触較，本發縣鮮取物使金屬蛋白分觸(Μ·、_p3) 基因表現減少、抑制劑(TIMP1、馨3) _表現無差異而前膠原蛋白 (Procollagen)顯著增加。 (ProcoNagen)增生 實驗3之結果顯示，綠藻萃取物可以抑制金屬蛋白分解酶剛p)基因 表現’以及使前膠原蛋白(P「QC。丨丨a_增加之功效。且啸高的有效量對 金屬蛋白分解酶(MMP)絲因表現有較佳的抑制效果，同時使前膠原蛋白 而非限制本發明之 以上所述之實驗例係為示範及證明本發明之效用， 實施範圍。 【圖式簡單說明】 第-圖為柱狀圖，係表示於蛋白激素活化劑(pMA)誘導纖維母細胞 (__中產蝴蛋白分解_Mp)之實驗财，本發賴萃取物 對金屬蛋白分解酶(MMP)具抑制效用。 16 200914061 的 量 第三圖，表示金屬蛋白分解酶_P)被蛋白激素活蝴PMA)活化之實驗例 中，本發明綠藻萃取物對彈性蛋白具增生效用之西方墨點法結果。 第四圖，表示以蛋白激素活倾PMA)鱗產生金屬蛋白分解酶(瞻)基因 表現之貫驗射，本發縣鮮取物對金屬蛋自分鱗(MMP)的活性抑制 效用之RT-PCR結果。 第五圖為柱狀圖，表示皮膚纖維母細胞以紫外線照射彳·5小時後釋放金屬蛋 白分解酶(ΜΜΡ)的情形，以及制本發縣鮮取物—有效量對金屬蛋白 分解酶(ΜΜΡ)的活性具抑制效用。 第六圖係在紫外線照射皮膚纖維母細胞30分鐘，金屬蛋白分解酶(ΜΜρ) 及彈性蛋白的變化，以及本發明綠藻萃取物對金屬蛋白分解酶(ΜΜρ)的活 性抑制以及彈性蛋白(Elastin)增生之效用的西方墨點法結果。 第七圖係本發明綠藻萃取物於紫外線照射皮膚纖維母細胞時使金屬蛋白分 解酶(MMP)的基因表現受到抑制及使前膠原蛋白(p「〇C〇||agen)基因表現增 加的RT-PCR結果。 第八圖為本發明綠藻萃取物之紅外線光譜分析(丨R)圖。 第九圖為本發明綠藻萃取物之紅外線光譜分析(丨R)圖。 第十圖為本發明綠藻萃取物之紅外線光譜分析(丨R)圖。 【主要元件符號說明】 無Fisherl and Watanabe et al. proposed in 1996 and 2004, respectively, UV-induced, UVB in the dermal layer of fibroblasts, causing protein hormone enzymes (pKc, p "transport (4) earned deactivated metalloproteinase (MMp) According to the above-mentioned sub-theory "we can know that the skin is over-infested by ultraviolet rays, the fibroblasts __ast" in-service incomplete oxidation phenomenon, causing free radical scales, the freedom of hearing off 200914061 base will activate metalloproteinase _ p), the over-activated metalloproteinase _?) destroys collagen, elastin, fibroblasts, cell membranes, and nucleus in normal fibroblasts, causing skin exogenous f-atrophy, dermal thickness Gradually reduce the aging of the stratum corneum roughening, rough texture, wrinkles, sagging, etc. See the percutaneous absorption of external preparations to improve skin aging, including fruit acid, vitamin A acid, vitamin A, L-vitamin C, etc. Acid can make the old keratin fall off, and the surface can improve the keratinization of the skin. The concentration of fruit acid is differentiated and limited, and the low concentration of fruit below 2〇%, acid can be For daily maintenance, the high concentration of 20%~70% is used by dermatologists for skin acid rejuvenation. Vitamin A acid system and efficacy and touch, but the skin is more irritating, making money There may even be a side effect such as peeling and redness, so __ must be used under the direction of the doctor, and strict sun protection during use. L-Vitamin C has antioxidant function, can protect cells from UV county and towel and free self-domain, left The main purpose of the present invention is to provide a scientifically proven anti-skin cell anti-existing raw material, and to improve the skin wrinkles and relaxation phenomenon. Development of a percutaneous absorption external preparation. The above-mentioned anti-skin cell extracellular aging raw material is a green algae extract, and the main method of the present invention is to inhibit skin fibroblasts (F jbr〇blast) by an effective I green algae extract. The activity of metalloproteinase (MMP) and the promotion of elastin and collagen in skin fibroblasts (Fibr〇b丨ast). The green algae extract can be mixed. In a cosmetic composition for percutaneous absorption, the composition has an anti-exogenous skin aging effect. The method for anti-aging of skin cells of the present invention is based on the inhibition of skin fibroblasts by green algae extract. Metalloproteinolytic enzyme (MMp). 200914061 The present invention comprises the use of a green algae extract to inhibit metalloproteinolytic enzyme (MMP) in skin fibroblasts to promote the proliferation of elastin and collagen (Collagen). Effect of the Invention A preferred embodiment of the present invention is to enhance the incorporation of an effective amount of green algae extract in a cosmetic composition for human skin to increase the effect of the composition against skin cell extracellular aging. It is known that when ultraviolet rays are excessively irradiated to the skin, the metalloproteinolytic enzyme (MMP) in the fibroblast will be excessively activated, thereby destroying elastin in normal fibroblasts (Fibr〇b|ast). And collagen bomb white (C0||agen). Therefore, the present invention is based on the inhibition of the activity of metalloproteinase (MMP) by the green algae extract, so that the cosmetic composition incorporating the green algae extract of the present invention is particularly suitable for protecting the skin and reducing the aging caused by ultraviolet rays on the skin. Phenomena, including skin problems such as thickening of the stratum corneum, rough texture, wrinkles, and sagging. The amount of the green algae extract used in the present invention cannot be absolutely specified, and it will vary depending on the manner of preparation. The green algae extract used in the present invention and the composition obtained by incorporating the green algae extract into percutaneous absorption may be in various forms such as a liquid liquid, a condensed, an emulsion, a cream, a paste, a powder, Oily. Wherein the total amount of the composition comprises from about _ to about 5% by weight of the green algae extract. [Embodiment] According to the method of the present invention, the method for anti-skin aging of the skin is to provide an ice-ice extract as a metalloprotein in the skin fibroblast (Fjb "〇b|ast"). Decomposing enzyme (MMP) 'and the main component that promotes the proliferation of elastin (曰astjn) and pre-regeneration protein (p_丨(四)卽). This extract can be made in a suitable way, mainly in fresh water. The single and Tian Yu were taken into the water extract. The spectral analysis of the external line (Fig. 8 to Fig. 10, 00 cm_1~40〇cm-1) revealed that the green algae extract is an organic compound, carbon and hydrogen. , 7 200914061 Nitrogen as its main element 'element content: nitrogen (Ν)7·06./.~7.66./, hydrogen (h) 5.9%~6 51%, charcoal (C) 35.67%~38.1%. The efficacy of the green algae extract of the present invention against skin cell extracellular aging is demonstrated by the following experimental results. The experiment is carried out under two conditions, one of which is a protein hormone inducer (PMA' phorbol. 12-myristate 13-acetate) induces the production of metalloproteinase (MMP) in skin cells as experimental conditions. The green algae extract of the present invention inhibits the activity of metalloproteinolytic enzyme (MMP). The other is to induce the production of metalloproteinase (MMP) by skin cells under ultraviolet irradiation as experimental conditions, and test the green algae extract of the present invention against metalloprotein Inhibition of the activity of the enzyme (MMp) [Molecular proteolytic enzyme (MMP) induced by protein hormone inducer (PMA)] [Experiment 1] The experimental example of the first figure is in normal Skin fibroblasts (Fibr〇b丨ast) induce the production of metalloproteinase (MMP) by protein hormone inducer (PMA), and test the inhibitory effect of the green algae extract of the present invention on metalloproteinolytic enzyme (MMP) production, and It is represented by a quantified bar graph. It is known to those skilled in the art that 'protein hormone inducer (PMA) is an activator of protein kinase C in fibroblasts (Rbr〇b丨ast) of the dermis layer. And protein hormone enzyme (pkc) is an activator of metalloproteinolytic enzyme (MMP), while over-activated metalloproteinolytic enzyme (MMp) destroys normal skin fibroblasts (Fibroblast) collagen (Collagen) Elastin causes skin aging. The experimental example in the first figure is numbered as control group, number (2> is for 8 200914061 photo ^ number (3) (4) (5) (6) for the experimental group, the culture required for the experiment The time was 5 hours. (Production, and, indicating that the content of normal fibroblasts (metalloproteinase_P released during the 5-hour experiment) was about 25 ng/mL. The 'domain (2) pair & group ' indicates that the egg is self-exciting ___, and the average content of metalloproteinase (MMP) is increased to 7 〇叩. - No. (3) Experimental group, indicating that the addition of human protein stimulating agent (P_Q_ and the green of the invention is 2Qnag/m| of the extract], the average content of the golden white lining (_卩) is reduced to about % ng /mL - Intervention 4) The test group indicates that the human protein hormone inducer (pMA) and the green bleach extract of the present invention are 15 mg/m|, and the average content of the metalloproteinase_〇 is reduced to about 筘. Ng/mL. No. (5) The experimental group indicated that the human protein hormone inducer ___ and the known metalloproteinase inhibitor (GMXMnM) showed an average content of metalloproteinolytic enzyme (MMp) of about 50 ng/mL. (6) The test group indicated that the self-excited reading agent (pMA) iQ_ and vitamin c 〇·15_ were added, and the results showed that the average content of metalloproteinase _P) was about 60 ng/mL. As a result of Experiment 1, it was revealed that the green algae extract can inhibit the activity of the metalloproteinase_p) and a preferable inhibitory effect can be obtained with a higher effective amount. Further, from the experimental data of No. (3) and No. (4), it was found that the activity of the green algae extract of the present invention against the metalloproteinolytic enzyme p) was clearly superior to that of the other experimental groups. [Experiment 2] 9 200914061 The second example of the experiment is based on the induction of metalloproteinase (MMP) by protein hormone inducer (PMA), and the activity inhibition of the metalloproteinase (MMp) extracted from duck algae Western blotting results for utility. The first column of images represents the active reaction of the metalloproteinase (Μ Μ p). The younger brother* is a control group. The number (1) indicates that metalloproteinase (MMp) was induced to 100 mM protein hormone inducer (PMA) against normal skin fibroblasts (Fjbr〇biast), and the activity of metalloproteinolytic enzyme (MMP) was increased. The number (2) can be regarded as a basic state 'representing the active reaction of normal skin fibroblasts (nbr〇b丨ast) under normal conditions of metalloproteinolytic enzyme (MMP). No. (3) indicates the addition of protein hormone inducer (PMA) 100 mM to normal skin fibroblasts (PMA) and 0.2 (10) protein hormone enzyme (PKC) inhibitors, compared with the number (1) results, protein hormone enzyme (PKC) inhibitors. The activity of the metalloproteinolytic enzyme (|^|^卩) was slightly suppressed. No. (4) indicates the addition of a protein metalloproteinase (PMA) 100 mM ' and a known metalloproteinase inhibitor (GM) to normal skin fibroblasts (Fibr〇b丨ast), compared with the number (1). It is known that metalloproteinase inhibitors (GM) slightly inhibit the activity of metalloproteinolytic enzymes (MMPs). No. (5) indicates that a protein hormone inducer (PMA) 100 mM, and a 20 mg/ml green algae extract were added to normal skin fibroblasts, compared with the results of numbers (1), (2), (3), (4), and the present invention. The green algae extract apparently inhibited the activity of metalloproteinolytic enzyme (MMP). The results of Experiment 2 show that 'the green algae extract of the present invention can inhibit the activity of metalloproteinolytic enzyme (MMp)' and inhibit the effect of age superior to conventional protein kim surface (PKC) inhibitors and known metalloproteinase inhibitors. (GM). 10 200914061 [Experiment 3] The third figure is the real thing, the golden riding white __p) is activated by the protein-activated reading agent (pMA), and the Western blotting method is used to make the skin fresh. Elastin (Ela_proliferation effect. The first-column image shows the pilgrin protein). The second column of images is a control group. The number (1) can be regarded as - the basic state, the elastin content of the silk fibroblasts (4) can (4) under normal conditions. No. (2) indicates that the normal skin fibroblasts (Fjbr〇b丨na added protein hormone induces cleverness (ΡΜΑ__ ' results show a significant decrease in elastin_ increment. The reason is that protein hormone inducer (PMA) activates metalloproteinolytic enzyme (MMp), over-activated metalloproteinase (MMP) destroys collagen and elastin in normal fibroblasts (Fjb "〇b|ast". Number (3) indicates normal fibroblasts (Fibroblast) Protein hormone inducer (PMA) 100 mM, and 10 mg/m 本 of the green algae extract of the present invention. Compared with the number (7), elastin was significantly increased. No. (4) indicates addition to normal skin fibroblasts (Fibroblast). Protein hormone inducer (PMA) 1 〇OmM ' and 2 〇mg/m 丨 of the green algae extract of the present invention. Compared with the number (7) (9), elastin (Elastin) is more significantly increased. Λ The results of Experiment 3 show that green algae extraction The substance uses an effective amount of proliferating elastin biting, and a higher effective amount can obtain a better elastin proliferation result. [Experiment 4] 11 200914061 . The fourth example of the experiment 'fine protein stimulation Under the conditions of the production of the metalloproteinase (_P) of the material (PMA), the results of the RT-PCR results of the gene expression of the metal egg self-decomposing enzyme (_卩) in the county. The intracellular inhibitory protein (τίΜρι and 丨Mp3) is a substance that is present in fibroblasts to counter metalloproteinases (_^, MMP3). ''Broad number (1) means Normal skin fibroblasts (Fjbr〇blast) show that the expression of cytosine protein (M. sinensis and T|Mp3) is reduced by the protein hormone inducer (PMA) IGQmM' results, resulting in the expression of gold, genus proteolytic enzymes (MMP1, MMP3). The number (2) can be regarded as a basic state, indicating normal skin fibroblasts ((rjbr〇b丨lang) under normal conditions, metalloproteinases..., _p3) and intracellular inhibitory proteins (T| The content of Mpi and TIMP3). The number (3) indicates that the protein hormone inducer (P_ 1GQmM, and α2υΜ protein domain PKC) is added to the normal skin fibroblast (Fibroblast), and compared with the number (1), the protein hormone enzyme (pkc) Inhibitors did not show intracellular inhibitory proteins (TIMP1 and T|Mp3) genes The increase in 'metalloproteinase (MMP1, MMP3) gene performance was not significantly reduced. Number (4) indicates that protein stimulating agent (PMA) was added to normal skin fibroblasts (Fibroblast) 100 mM, and 捕. Metalloproteinase inhibitor (GM), compared with the number (1) results, it is known that metalloproteinase inhibitors (GM) do not significantly increase the expression of intracellular inhibitory proteins (Ding 丨Μι and TIMP3), resulting in metalloprotein decomposition _(_ρι, MMp3) gene performance was not significantly reduced. No. (5) indicates that protein stimulating agent (PMA) 100 mM and 20 mg/m chlorella extract were added to normal skin fibroblasts (Fjbr〇biast), compared with the results of numbers (1)(2)(3)(4). Although the invention of the green bath extract did not significantly increase the intracellular inhibitory protein (TIMPi and timp3) gene expression 200914061, the metalloproteinase __P1, _P3) gene performance was significantly reduced. The experiment 4 shows that the green algae extract of the present invention can inhibit the gene expression of the metalloproteinase enzyme p), and the inhibition effect is obviously superior to the known protein hormone enzyme (pKc) inhibitor and the known metalloproteinase inhibitor. Agent (GM). [Inducing skin fibroblasts by i-line injection (Fjb_a Lasheng metalloproteinase (MMP)] [Experiment 1] The experimental example of the figure is a normal skin fibroblast (Fibroblast) riding with ultraviolet rays (UVB) for 15 hours. In order to quantify the activity of the metalloprotein fragment (the p), and the activity inhibition effect of the green algae extract of the present invention - an effective amount of the metalloproteinolytic enzyme _ 卩. The number (1) controls her to indicate that the content of skin fibroblasts (nbfOb_ released metalloproteinase_P) is about 3.5 ng/mL under no-line irradiation. The squamous (2) pair of knowledge groups indicated that normal skin fibroblasts (F丨) were irradiated with ultraviolet light for 1 hour, and the average content of metalloproteinolytic enzyme (MMP) was increased to about 6 ng ng/mL. 'Broad number (3) member test group' indicates that normal skin fibroblasts (Fjb "〇b|ast" are irradiated with ultraviolet rays for 1.5 hours, and the green algae extract of the present invention is added 1 〇 mg/m before the irradiation of ultraviolet rays. The average content of metalloproteinase (MMP) was reduced to about 35 ng/mL. Codification (4) Experimental group, indicating normal skin fibroblasts (Rbr〇b丨as〇 irradiated with ultraviolet light for 15 hours, before ultraviolet irradiation) When the green algae extract of the present invention was added at 20 mg/ml, the average content of the metal egg 13 200914061 white degrading enzyme _ 卩 was reduced to about 1.5 ng/mL. No. (5) Experimental group, indicating normal skin fibroblasts (Fibr〇b) |ast) UV irradiation "5 I inch in ^I iil plus human hair duck fresh extract 3C) mg / m results show that the average content of metalloproteinase (MMP) decreased below 彳〇 ng / mL. The 'flat number (6) test group' indicates that normal skin fibroblasts (Fibr〇b|ast) are irradiated with ultraviolet rays 1 5 small ' before adding ultraviolet light to the vitamin Cai5_, and the results show that the metalloproteinase (the average content of the bucket) Instead, it increased by about 9.0 ng/mi_. No. (7) Experimental group, indicating normal skin fiber The cells (Fibr〇b丨ast) were irradiated with ultraviolet light for 5 hours, and vitamin E 25 uM was added before the irradiation of ultraviolet rays. The results showed that the metalloproteinase (the average content of the _ gate remained unchanged, that is, about 6. ng/mL. The results of the test 1 show that the green algae extract can inhibit the activity of the metalloproteinase (_p)! It is better to obtain a better inhibitory effect by the effective setting. The experimental data of No. (3)(4)(9) can be seen that 'Benfa County The activity of the fresh extract against the metalloproteinase (_p) was clearly superior to that of the other experimental groups. [Experiment 2] The experimental example of the figure was irradiated with ultraviolet rays to normal skin fibroblasts (Fjbr〇b (4) 3〇 Minutes of changes in metalloproteinases (MMP) and elastin (曰astin), and the results of Western blotting methods for the effect of the green algae extract of the present invention on the proliferation of elastin. No. (2) Control group 'The skin fabric cells (nb "Qb 丨 的 金属 金属 金属 金属 金属 金属 金属 金属 金属 n n n n n n n n n n n n n n n n n n n n n 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线Irradiation 3 points 14 200914061 The 'metal protein score»_p) increased significantly, while the content of elastin was significantly reduced. ° () Pei test group ' indicates that Fibroblast is irradiated with ultraviolet light for 30 minutes! Before the ultraviolet irradiation, the green algae extract 鸠_丨 of the present invention was added, and the number of the control group (1) was significantly lower than that of the control group, and the average content of the metalloproteinase (MMP) was significantly decreased, and the content of the heteroprotein (day astjn) was significantly increased. ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, (1) Compared with the control group, the results showed that the average content of metalloproteinase _卩 was significantly reduced, and the content of elastin (曰astin) was significantly increased. The results of Bayesi 2 were not observed, and the green algae extract had inhibitory metalloproteinase (Gangmen) The activity and the effect of elastin (coronal) hyperplasia. The higher effective amount has a better inhibitory effect on the activity of metalloproteinase (MMP), and at the same time makes the elastic egg self (&as (4) hyperplasia. 3] The experimental example of the seventh figure is the RT of the present invention. The green algae extract of the present invention is irradiated with ultraviolet light to the skin fibroblast (Fibroblast) for 30 minutes (metalloproteinase _?) and the procollagen egg (10) is occluded. PCR results: ΤΙΜΡι and T|Mp3 in the figure are found in fibroblasts to counter metalloproteinases (MMP1, MMp3). Number (1) is the control group, indicating normal skin fibroblasts (Rb_ast) ) gold before it is irradiated Gene expression of proteolytic enzymes _P1, MMP3), inhibitors (T|MP1, T|Mp3), and procollagen (Procollagen) 200914061 No. (2) for pairs of groups, indicating normal skin fibroblasts (円br〇b|ast) irradiated with ultraviolet light for 30 minutes' results showed that metalloproteinase (mmp1, _P3) increased significantly, and the inhibitors showed that η and ΤΙΜΡ3 genes did not differ much, but procollagen (ρ".丨丨Printing) The gene expression was reduced. No. (3) for the experimental group, the table was transferred to normal planting mother cells (Rb_ast) for 30 minutes of ultraviolet light. Add 1 〇mg/m|green fresh extract before irradiation with ultraviolet light, and control with number (1) Group and squad (2) pairs, ',,,,: £ comparison, the green algae extract of the present invention enables metalloproteinase (_^1, gene expression reduction, inhibitor (τ丨MP1, pre-p3) gene There was no difference in expression and the procollagen (Procollagen) was significantly increased. The squaring () is the experimental group' indicates that the normal skin fibroblasts (F_b丨(4) are irradiated with ultraviolet rays for 30 minutes, and 2〇m_丨 green is added before the ultraviolet rays are irradiated. Algae extract, compared with number (1) control group and number (2)» The fresh extracts from the county reduced the expression of metalloproteins (Μ·, _p3), and the inhibitors (TIMP1, Xin 3) _ showed no difference and the procollagen (Procollagen) increased significantly. (ProcoNagen) results of proliferation experiment 3 showed , Chlorella extract can inhibit the metalloproteinolytic enzyme just p) gene expression 'and make procollagen (P "QC. 丨丨a_ increase the effect. And the effective amount of Xiao Gao on metalloproteinolytic enzyme (MMP) silk The performance of the present invention is demonstrated by demonstrating that the procollagen is not limited to the above-described experimental examples of the present invention. [Simplified description of the diagram] The first-graph is a histogram, which is expressed in the protein hormone activator (pMA)-induced fibroblasts (__中中-producing protein decomposition_Mp) Decomposing enzymes (MMPs) have inhibitory effects. 16 The amount of 200914061 The third figure shows an example in which the metalloproteinase _P) is activated by the protein hormone PMA). The green algae extract of the present invention has an effect of Western blotting on the effect of elastin. The fourth figure shows the RT-PCR of the inhibitory effect of the extract of the metalloproteinase (MMP) from the freshly harvested protein in the county. result. The fifth picture is a histogram showing the release of metalloproteinase (ΜΜΡ) from skin fibroblasts after exposure to ultraviolet light for 5 hours, and the extraction of the metalloproteinase from the hair of the county. The active agent inhibits the utility. Figure 6 is a graph showing changes in metalloproteinolytic enzymes (ΜΜρ) and elastin in UV-irradiated skin fibroblasts for 30 minutes, and inhibition of metalloproteinolytic enzymes (ΜΜρ) by the green algae extract of the present invention and elastin (Elastin) The result of the Western blot method of the effect of hyperplasia. The seventh figure shows that the green algae extract of the present invention inhibits the gene expression of metalloproteinase (MMP) and increases the expression of procollagen (p"〇C〇||agen) gene when ultraviolet rays are irradiated to skin fibroblasts. RT-PCR results. The eighth figure is the infrared spectrum analysis (丨R) diagram of the green algae extract of the present invention. The ninth figure is the infrared spectrum analysis (丨R) diagram of the green algae extract of the present invention. Infrared spectrum analysis (丨R) diagram of the invention green algae extract. [Main component symbol description]