201219044 六、發明說明： 【發明所屬之技術領域】 本發明相關於豬肺萃取物作為基質金屬蛋白酶（Matrix Metalloproteinase，MMP)抑制劑之新用途，特別是用於抑 制 MMP-1、MMP-2 或 MMP-9。 【先前技術】 金屬蛋白酶為蛋白酶（酵素）之超族群，其數目於近年來 已急驟地增加。基於結構與功能性考量，此等酶已被分類 成數族群與亞族群。金屬蛋白酶之實例，包括基質金屬蛋 白酶（MMP)，譬如膠原酶（MMP-1，MMP-8,MMP-13)、明膠 酶（MMP-2，MMP-9)、基質溶素（MMP-3，MMP10，MMP-ll)、 間質溶素（MMP-7)、金屬彈性蛋白酶（MMP-12)、釉質溶素 (MMP-19)、MT-MMP(MMP-14，MMP-15,MMP-16，MMP-17) :生殖溶素或齒轴質溶素或MDC族群，其包括分泌酶與流 出酶，譬如TNF轉化酶（ADAM10與TACE):蝦紅素族群， 其包括一些酶，譬如原膠原處理蛋白酶（PCP);及其他金 屬蛋白酶，譬如聚集原酶，内皮肽轉化酶族群及血管收縮 素轉化酶族群。其中已知MMP與多種疾病相關，如MMP 於癌組織中血管新生或癌轉移時，其表現量會升高或者酵 素會產生活性化，另外，對於潰瘍形成、慢性關節風濕病 、骨質疏鬆症、牙周病等各種病症中細胞外基質之分解亦 具有作用。再者，皮膚因紫外線等外部刺激使活性亢進之 MMP將維持皮膚構造上重要成分分解的現象，在最近，因 其為由於紫外線而活化之老化促進因子而特別受到重視， 149806.doc 201219044 尤以MMP-l、MMP-2及MMP-9特別受到重視，其原因有兩 個，其一是這些MMP的底物為皮膚的重要結構成分，其二 是皮膚長期暴露在會刺激這些MMP形成病理狀態的因素下 ，例如炎症反應、氧化性應激及紫外線。因此，目前認為 選擇性抑制MMP-l、MMP-2及MMP-9比抑制所有金屬蛋白 酶在減緩皮膚老化上更有益處及有效。 MMP-1是通稱的膠原蛋白分解酶的一種，為調節光老化 重要因子，纖維母細胞受陽光之紫外線曝曬會引起基質金 屬蛋白酶大量表現，進而降解細胞外基質（ECM)，也就是 說MMP-1負責真皮組織的膠原蛋白降解。 MMP-2(解膠酵素72_kDa)的作用在皮膚生理上的重要性 也已經得到證實。雖然MMp_2在第1型膠原的分解上是否 扮演直接的角色尚未定案，但第j型膠原是其酵解物之_ 則已獲得證實，另外，第IV型膠原（基底膜）、第VII型膠原 (真皮-表皮定錨微纖維）、明膠（變質的第〗型膠原）、彈性蛋 白和纖維蛋白冑，也都是MMP-2的酵解物。隨著年齡之日 長，MMP-2在皮膚裡的酵素活動量會增加，因此， 在胞外基質的萎縮上，亦扮演重要的角色。 ΜΜΡ_9(明膠酶B ; 92kDa類型IV膠原酶；92kDa明膠酶） 為=種分泌之蛋白質，其係在1989年首先經純化，然後無 '、及疋序MMp_9之表現於正常情況下係被限制於少 數、、田胞類型’包括滋胚層、破骨細胞、嗜中性白血球及巨 紀仁是，其表現可在此等相同細胞中，及在其他細 胞類型中，藉數種介體誘發，包括此等細胞曝露至生長因 149806.doc 201219044 子或細胞活素。其係為經常與引發炎性回應有關聯之相同 介體。與其他經分泌之MM卜樣，MMp_9係以不活性酶原 釋出，其係隨後分裂而形成具酵素活性之酶。於活體内供 此活化作用所需要之蛋白酶，係為未知。活㈣刚對不 活性酶之平衡’係& 一步於活體内經由與一帛天然生成之 蛋白質ΤΙΜΡ·1(金屬蛋白酶」之組織抑制劑)之交互作用作 調節。TIMP-1係結合至!^河!>_9之^末端區域，導致1^河15_ φ 9催化功能部位之抑制。Pr〇MMP-9經誘發表現之平衡， ΡΓ〇ΜΜΡ-9之分裂成活性MMP9，及ΤΙΜρ ι之存在，係合 併以決定存在於局部位置之具催化活性mmp_9之量。具蛋 白分解活性之MMP-9會攻擊受質，其包括明膠、彈性蛋白 及天然類型iv與類型v膠原；其對於天然類膠原、蛋白 多醣或昆布胺酸未具有活性。已有成長中之資料體與 MMP-9在不同生理學與病理學過程中之角色有關聯。生理 學角色包括在胚胎移植之早期階段中，胚胎滋胚層經過子 φ 呂上皮之侵入；在骨骼生長與發展中之某種角色；及炎性 細胞從血管分佈潛移至組織中。使用酶免疫分析度量之 MMP-9釋出，在流體中，及在來自未經治療之氣喘病患者 之AM上層清液中，與來自其他個體群者比較，係顯著地 提高。增加之MMP-9表現亦已被發現於某些其他病理學症 狀中’於是使MMP-9與疾病過程產生關聯，譬如copd、 關節炎、腫瘤轉移、阿耳滋海默氏病、多發性硬化，及在 動脈粥瘤硬化中之斑破裂，導致急性冠狀症狀，譬如心肌 梗塞。 149806.doc 201219044 由於天然產物的安全性佳，現今對於天然來源產物的開 發盈發重視，仍有需要開發抑制MMp的天然產物。 【發明内容】 在本發明之前，並沒有任何報告或先前技術指出或推測 豬肺萃取物具有抑制基質金屬蛋白酶之功效，而在本發明 中，出人意料地發現豬肺萃取物可有效抑制基質金屬蛋白 酶活性。藉由該抑制活性，特別是抑制ΜΜΙΜ、μμρ·Μ /及ΜΜΡ-9的活性，減少彈性蛋白之分解，因而可改善/減 緩/防止皮膚老化。 本發明之一目的是提供一種豬肺萃取物的用途，其係用 於製備抑制基質金屬蛋白酶之組合物。 除非本文中另外定義，否則結合本發明使用的科技術語 應具有所屬領域的技術人員冑常理冑的含義。術語的含義 及範疇應為明確的；然而，若存在任何潛在含糊性，則本 文所提供的定義優先於任何詞典或外部來源之定義。而除 非上下文另外需要，否則單數術語應包括複數且複數術語 應包括單數。 術語”豬肺萃取物"表示將豬肺經萃取後的萃取物，其中 該豬肺不限於新鮮或冷;東之豬肺。於一較佳實施例中，、本 發明之豬料取物萃取方式係（_肺絞碎後加人緩衝液調 整PH值約6.0_9.〇 ;⑻加入酵素於步驟⑷中保溫酶解，酶 解後升溫使酵素失活；⑷加人酸液於步驟（b)中進行酸沉 澱後，過濾收集濾液，且（d)加入有機溶劑於濾液中，收集 沉澱物’得到豬肺萃取物。 149806.doc 201219044 —較佳實例中，該㈣係去除氣管部分後再絞碎。於 —較佳實例中’本發明步驟（a)中之pH值為約6.〇·8.〇、65_ 8·〇、6.0-7.5、或約7·〇。於一較佳實例中，本發明步驟⑷ 中所使用之緩衝液包括但不限於檸檬酸鈉。於一較佳實例 中，本發明步驟（b)中所使用之酵素包括但 或蛋白咖或其組合。於一較佳實例中，本發明步鳳： 所使用之酸液為強酸，其包括但不限於鹽酸、過氣酸及三 氟醋酸。於-較佳實射，本發明㈣⑷巾所使用之有機 溶液包括但不限於甲醇、乙醇或異丙醇’更佳係乙醇，最 佳係食品級乙醇。 於本發明之一較佳實施例中，本發明之豬肺萃取物係可 抑制MMP活性，所述之MMP較佳為MMIM、MMp_2或 MMP-9。於另一較佳實施例中，本發明之豬肺萃取物係可 同時抑制MMP-1、MMP-2及MMP-9之活性。 於另一方面，本發明之豬肺萃取物係可經由抑制MMp之 活性而具有改善、減緩或防止皮膚老化，特別是防止皮膚 老化。 術語"防止"表示預防或阻止症狀產生，或改善或回復已 產生的症狀。 術語”皮膚老化”一詞包括此種老化跡象包括但不限於皮 膚脆性，膠原或彈性蛋白之喪失；皮膚中之雌激素平衡缺 失，皮膚萎縮，線條或皺紋之出現或深度，包括微細線條 ;皮膚變色，包括黑眼圈；皮膚鬆弛；皮膚疲勞或壓力， 例如由於環境壓力譬如污染或溫度變化所致之皮膚突出； 149806.doc 201219044 皮膚乾燥性；皮膚片狀剝落；細胞老化；皮膚張力、彈性 或光澤之喪失；皮膚堅牢性之喪失；粗皮質地；皮膚彈性 或回彈性之喪失。 對皮膚美觀之利益與改良，可以任何下述証明：孔隙大 小之降低；皮膚張力、光彩'透明性或拉緊性之改良；抗 氧化劑活性之促進；皮膚堅牢性、膨脹性、柔曲性或柔軟 度之改良’原膠原或膠原生產上之改良；皮膚質地上之改 良或再變形之促進；皮膚障壁修復或功能上之改良：皮膚 輪廊外觀上之改良；皮膚光澤或亮度之修復；於皮膚中因 老化或斷經所減少之必須營養物或組份之補充；皮膚細胞 中連通之改良；細胞增生或繁殖上之增加；因老化或斷經 而降低之皮膚細胞新陳代謝作用上之增加；皮膚潤濕作用 上之改良，細胞轉換之促進或加速；皮膚厚度之加強；皮 膚彈性或回彈性上之增加；及脫落之加強。 於另一較佳實例中，本發明之豬肺萃取物，其中之組合 物另包含構成該組合物所需的載劑。 術語"載劑"或"化妝品中可接受的載劑"是指稀釋劑、賦 形劑或類似物，其為各該行業所屬技術領域的技術人員所 熟知。 步包含可用於防止皮膚老化的 、維生素A醇、熊果素、玻尿 本發明的組合物還可進一 活性成分’例如維生素A酸 酸、表皮生長因數及其它天然美白成分等。本發明的組合 物另可包含習用化妝品活性劑，譬如其他習用低色素沉著 劑，譬如對苯二齡、抗壞血酸或甘草精萃液；#炎劑；抗 I49806.doc 201219044 粉刺劑，譬如柳酸；剝落劑，譬如α_羥基酸、卜羥基酸、 酮基酸、氧酸或氧二酸；抗壞血酸基_磷醯基_膽固醇；防 晒劑，譬如氧基苯酮、曱氧基桂皮酸辛酯、柳酸辛酯、八 可烯、二氧化鈦、氧化鋅、丁基曱氧基二苯甲醯曱烷、亞 甲基雙-苯并三唑基四曱基丁基酚（ΜΒΒΤ);或抗老化劑； 或其任何組合。 於另一個較佳實施例中，本發明的組合物包含最終濃度 約50//g/mL至500 yg/mL的豬肺萃取物。較佳地，本發明 的組合物包含約200 yg/mL至400 yg/mL的豬肺萃取物。最 佳係包含約300 yg/mL的豬肺萃取物。 本發明之豬肺萃取物可以所萃取溶液原狀使用，或依需 要進行浪辦、稀釋、過遽等處理及以活性碳等進行脫色、 脫臭處理後使用。又，將萃取後溶液進行濃縮乾固、喷霧 乾燥、冷康乾燥等處理，以乾燥物之形式使用亦可。 本發明之豬肺萃取物可應用於對皮膚老化之預防、抑制 或症狀改善之醫藥品、準醫藥品、化粧品或食品中，並可 使用上述萃取物之原樣，或在不損害萃取物範圍内，與普 通醫藥品、準醫藥品、化粧品或食品中使用之成分：賦形 劑、安定劑、保存劑、結合劑、崩散劑、烴類、脂肪酸類 、醇類、酯類、界面活性劑、金屬肥皂、pH調節劑、防腐 劑、香料、保濕劑、粉體、紫外線吸收劑、增黏劑、色素 、抗氧化劑、美白劑、螯合劑、油脂類或蠟類等成分配合 使用。 本發明之豬肺萃取物可呈現之劑型例如有散劑、丸劑、 149806.doc •9- 201219044 键劑、注射劑、检劑、乳劑、膠囊、顆粒劑、液劑（包括 酊劑、流體酊劑、酒精劑、懸濁劑、檸檬水劑等）、化粧 水、乳膏、乳液、凝膠劑、喷霧劑”谓膚膏、洗淨劑、浴 用劑、基底、打粉、口紅、軟膏、溫濕布、糊狀劑、膏藥 、精油、糖果鍵或飲料等。 以下實施例不應視為過度地限制本發明。本發明所屬技 術領域中具有通常知識者可在不背離本發明之精神或範疇 的情況下對本文所討論之實施例進行修改及變化，而仍屬 於本發明之範圍。 【實施方式】 實施例一、豬肺萃取物萃取方式 冷凍豬肺解凍後以清水洗淨後去除氣管部分，先切割成 約10公分大小塊狀，以絞肉機絞碎至無大塊狀組織，加入 豬肺2倍重量之萃取液（〇.〇2 Μ檸檬酸鈉pH 7.0 ± 0.2)，以 授拌機充分咸合均勻’秤取絞碎緒肺臟重量之5 鳳梨酵素（2000 GDU/g，ST BIO)與〇·ι %(w/w)之蛋白酶M 酵素（5500 untis/g，Aman〇) ’以攪拌機充分攪拌均勻，於 5〇°C水浴反應3小時，反應完成後再以水浴9〇它i 〇分鐘讓 酵素失活，靜置於冰水中降溫至25。(：，加入1.5 %(W/W) 6N鹽酸攪拌均勻後靜置室溫下1小時，進行酸沈澱反應 。上述樣品以1 μηι孔徑紙板直接進行板式過遽，收集過滤 液。以3倍體積之95%乙醇（食品級）沈澱，放入4它冷房i 2 小時。以8000 (xg)，3〇分鐘離心收集沈澱物，沈澱物再以 〇·〇2 Μ檸檬酸鈉pH 7.0回溶。 149806.doc •10· 201219044 實施例二、豬肺萃取物對纖維母細胞之毒性及抑制 MMP-1之功效 MMP-1同功酵素圖譜分析 1.細胞培養和毒性分析 (1) 細胞培養： 以含10°/。FBS的a-MEM培養基，種植總體積u mL的 lxlO4 WS1(BCRC 60300)細胞於96孔盤’隔夜去除上清液 ’取已知濃度之樣品，以含有血清的培養基配製成 0,0.125，0.25，〇_5，1 mg/mL 5個分析劑量，各3重複。再以 0·2 μπι的過渡膜過渡’取〇. 1 mL加入細胞中，處理24 hr後 去除上清液’再以無血清但含有10 ng/mL TNF-的a_MEM 培養基配製成0,0.125，0.25，0.5，1 mg/mL 5個分析劑量各3 重複’以0.2 μπι的過濾膜過濾，取〇.1 mL加入細胞中，再 處理24小時後收集上清液’保存在-8〇。〇，作為後續 MMP-1同功酵素圖譜分析樣品。 (2) 毒性分析： 上述仍留在96孔盤之細胞，加入含有血清的培養基與 MTS(100 mL : 20 mL)之混合液，37。(：反應3小時，在〇D. 490/630 nm分析細胞存活率。取存活率高於8〇%(定義為無 毒性）之最高濃度者，分析MMP-1同功酵素圖譜。 2 ·同功酵素圖譜分析 (1)配製酵素電泳膠及電泳分析201219044 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a novel use of pig lung extract as a matrix metalloproteinase (MMP) inhibitor, particularly for inhibiting MMP-1, MMP-2 or MMP-9. [Prior Art] Metalloproteinases are supergroups of proteases (enzymes), and their numbers have increased sharply in recent years. Based on structural and functional considerations, these enzymes have been classified into several ethnic groups and sub-populations. Examples of metalloproteinases include matrix metalloproteinases (MMPs) such as collagenase (MMP-1, MMP-8, MMP-13), gelatinase (MMP-2, MMP-9), and matrix lysin (MMP-3, MMP10, MMP-ll), interstitial lysin (MMP-7), metallo-elastase (MMP-12), enamel lysin (MMP-19), MT-MMP (MMP-14, MMP-15, MMP-16) , MMP-17): a group of germin or lysin or MDC, including secretase and efflux enzymes, such as TNF-converting enzymes (ADAM10 and TACE): a group of astaxanthin, including some enzymes, such as procollagen Processing proteases (PCP); and other metalloproteinases, such as aggregating proenzymes, endothelin converting enzyme populations and angiotensin converting enzyme populations. Among them, MMP is known to be associated with various diseases. For example, when MMP is in angiogenesis or cancer metastasis in cancer tissues, its expression will increase or enzymes will be activated. In addition, for ulcer formation, chronic articular rheumatism, osteoporosis, Decomposition of the extracellular matrix in various conditions such as periodontal disease also plays a role. Furthermore, MMP, which is activated by external stimuli such as ultraviolet rays, will maintain the decomposition of important components of the skin structure. Recently, it has been particularly valued because it is an aging promoting factor activated by ultraviolet rays, 149806.doc 201219044 MMP-1, MMP-2 and MMP-9 are particularly valued for two reasons. One is that the substrates of these MMPs are important structural components of the skin, and the second is that long-term skin exposure can stimulate the pathological state of these MMPs. Factors such as inflammatory response, oxidative stress and ultraviolet light. Therefore, it is currently believed that selective inhibition of MMP-1, MMP-2, and MMP-9 is more beneficial and effective than inhibiting all metalloproteinases in slowing skin aging. MMP-1 is a commonly known collagen degrading enzyme. In order to regulate important factors of photoaging, the ultraviolet exposure of fibroblasts to sunlight causes a large amount of matrix metalloproteinases to be expressed, thereby degrading the extracellular matrix (ECM), that is, MMP- 1 is responsible for collagen degradation of dermal tissue. The physiological importance of the action of MMP-2 (debonding enzyme 72_kDa) has also been confirmed. Although MMp_2 has not been finalized in the decomposition of type 1 collagen, the j-type collagen is the lysate of it, and it has been confirmed. In addition, type IV collagen (basement membrane) and type VII collagen (dermal-skinned anchor microfibers), gelatin (deteriorated collagen), elastin and fibrin, are also the yeasts of MMP-2. As the age increases, the amount of enzyme activity in the skin of MMP-2 increases, and therefore plays an important role in the shrinkage of the extracellular matrix. ΜΜΡ_9 (gelatinase B; 92kDa type IV collagenase; 92kDa gelatinase) is a protein secreted by the species, which was first purified in 1989, and then the absence of ', and the order of MMp_9 is normally limited to A few, field type 'including dermal layer, osteoclast, neutrophil and giant genomics, its performance can be induced in these same cells, and in other cell types, by several mediators, including this The cells are exposed to growth factors 149806.doc 201219044 or cytokines. It is the same mediator that is often associated with an inflammatory response. Like other secreted MMs, MMp_9 is released as an inactive zymogen, which then divides to form an enzyme-active enzyme. The protease required for this activation in vivo is unknown. The activity (4) just balances the inactive enzymes's in one step in vivo through the interaction with a naturally occurring protein ΤΙΜΡ·1 (metalloproteinase) tissue inhibitor. The TIMP-1 line binds to the end region of the ^^河!>_9, resulting in inhibition of the catalytic function of 1^河15_φ9. The equilibrium of the induced expression of Pr〇MMP-9, the division of ΡΓ〇ΜΜΡ-9 into the active MMP9, and the presence of ΤΙΜρ ι, is combined to determine the amount of catalytic activity mmp_9 present at the local position. MMP-9 with proteolytic activity attacks the host, including gelatin, elastin, and native type iv and type v collagen; it is not active against native collagen, proteoglycan or laminbumin. The growing body of data is associated with the role of MMP-9 in different physiological and pathological processes. Physiological roles include the invasiveness of the embryonic dermal layer through the sub- φ ly epithelium in the early stages of embryo transfer; a role in bone growth and development; and the inflammatory cells that migrate from the vascular distribution to the tissue. MMP-9 release measured by enzyme immunoassay was significantly improved in fluids, and in AM supernatants from untreated asthma patients, as compared to those from other individual populations. Increased MMP-9 performance has also been found in certain other pathological conditions', thus linking MMP-9 to disease processes such as copd, arthritis, tumor metastasis, Alzheimer's disease, multiple sclerosis And plaque rupture in atherosclerosis, leading to acute coronary symptoms, such as myocardial infarction. 149806.doc 201219044 Due to the safety of natural products, there is still a need to develop natural products that inhibit MMp. SUMMARY OF THE INVENTION Prior to the present invention, there was no report or prior art to indicate or speculate that pig lung extract has the effect of inhibiting matrix metalloproteinases, whereas in the present invention, it has been surprisingly found that pig lung extract can effectively inhibit matrix metalloproteinases. active. By this inhibitory activity, in particular, the activity of ΜΜΙΜ, μμρ·Μ / and ΜΜΡ-9 is suppressed, and the decomposition of elastin is reduced, so that skin aging can be improved/reduced/prevented. It is an object of the present invention to provide a use of a pig lung extract for the preparation of a composition for inhibiting matrix metalloproteinases. Unless otherwise defined herein, the technical terms used in connection with the present invention should have the meaning of ordinary skill in the art. The meaning and scope of the term should be clear; however, if there is any potential ambiguity, the definition provided in this paper takes precedence over the definition of any dictionary or external source. Unless the context requires otherwise, the singular terms shall include the plural and the plural terms shall include the singular. The term "porcine lung extract" means an extract obtained by extracting pig lungs, wherein the pig lung is not limited to fresh or cold; Dong pig lung. In a preferred embodiment, the pig feed of the present invention The extraction method is (_the lung is smashed and added to the buffer to adjust the pH value of about 6.0_9. 〇; (8) the enzyme is added to the enzyme in step (4), and the enzyme is inactivated after enzymatic hydrolysis; (4) adding acid solution in the step ( After acid precipitation in b), the filtrate is collected by filtration, and (d) an organic solvent is added to the filtrate to collect the precipitate to obtain a pig lung extract. 149806.doc 201219044 - In a preferred embodiment, the (4) is after removal of the tracheal portion Further, in the preferred embodiment, the pH in the step (a) of the present invention is about 6. 〇·8.〇, 65_8·〇, 6.0-7.5, or about 7·〇. In an embodiment, the buffer used in the step (4) of the present invention includes, but is not limited to, sodium citrate. In a preferred embodiment, the enzyme used in the step (b) of the present invention includes but or a protein coffee or a combination thereof. In a preferred embodiment, the phoenix of the present invention: the acid used is a strong acid, including but not limited to hydrochloric acid, Acid and trifluoroacetic acid. Preferably, the organic solution used in the (4) (4) towel of the present invention includes, but is not limited to, methanol, ethanol or isopropanol, more preferably ethanol, preferably food grade ethanol. In a preferred embodiment, the pig lung extract of the present invention inhibits MMP activity, and the MMP is preferably MMIM, MMp_2 or MMP-9. In another preferred embodiment, the pig lung extract of the present invention The system can simultaneously inhibit the activities of MMP-1, MMP-2 and MMP-9. In another aspect, the pig lung extract of the present invention can improve, slow down or prevent skin aging by inhibiting the activity of MMp, in particular Prevents skin aging. The term "prevent" means preventing or preventing the onset of symptoms, or improving or reverting to the symptoms that have already occurred. The term "skin aging" includes such signs of aging including but not limited to skin fragility, collagen or elastin. Loss; loss of estrogen balance in the skin, skin atrophy, appearance or depth of lines or wrinkles, including fine lines; skin discoloration, including dark circles; skin relaxation; skin fatigue or stress, for example due to Environmental stress such as skin damage caused by pollution or temperature changes; 149806.doc 201219044 Skin dryness; skin exfoliation; cell aging; loss of skin tone, elasticity or luster; loss of skin fastness; rough cortical; skin elasticity Loss of resilience. Benefits and improvements in skin aesthetics can be demonstrated by any reduction in pore size; improvement in skin tension, radiance 'transparency or tension; promotion of antioxidant activity; skin fastness, swelling Improvement of sex, flexibility or softness 'improvement of procollagen or collagen production; promotion of skin texture or re-deformation; skin barrier repair or functional improvement: skin appearance improvement; skin gloss Or the repair of brightness; the supplement of essential nutrients or components that are reduced in the skin due to aging or menstruation; the improvement of connectivity in skin cells; the increase in cell proliferation or reproduction; the reduction of skin cells due to aging or menopause Increased metabolism; improved skin moisturization, promotion or acceleration of cell turnover; skin thickness Strengthen; skin elasticity increase on the back or elastic; and shedding of strengthening. In another preferred embodiment, the pig lung extract of the present invention, wherein the composition further comprises a carrier which is required to form the composition. The term "carrier" or "cosmetic acceptable carrier" refers to a diluent, excipient or the like, which is well known to those skilled in the art. The step comprises vitamin A alcohol, arbutin, and hyaluronic which are useful for preventing skin aging. The composition of the present invention may further comprise an active ingredient such as vitamin A acid, epidermal growth factor and other natural whitening ingredients. The composition of the present invention may further comprise a conventional cosmetic active agent, such as other conventional low pigmentation agents, such as benzoate, ascorbic acid or licorice extract; #炎剂;抗I49806.doc 201219044 acne, such as salicylic acid; Exfoliating agent, such as α-hydroxy acid, hydroxy acid, keto acid, oxyacid or oxyacid; ascorbyl phosphinyl-cholesterol; sunscreen, such as oxybenzophenone, octyl octyl cinnamate, willow Octyl octanoate, octadecene, titanium dioxide, zinc oxide, butyl decyl benzophenone, methylene bis-benzotriazolyl tetradecyl butyl phenol (ΜΒΒΤ); or an anti-aging agent; or Any combination of them. In another preferred embodiment, the compositions of the present invention comprise a pig lung extract having a final concentration of from about 50//g/mL to 500 yg/mL. Preferably, the compositions of the present invention comprise from about 200 yg/mL to 400 yg/mL of porcine lung extract. The best system contains about 300 yg/mL of pig lung extract. The pig lung extract of the present invention can be used as it is in the form of the extracted solution, or can be subjected to treatment such as wave treatment, dilution, and sputum treatment as needed, and decolorization and deodorization treatment with activated carbon or the like. Further, the extracted solution is subjected to a treatment such as concentration drying, spray drying, cold drying, and the like, and may be used in the form of a dried product. The pig lung extract of the present invention can be applied to medicines, quasi-drugs, cosmetics or foods for preventing, inhibiting or improving symptoms of skin aging, and can use the above-mentioned extracts as they are, or without damaging the extracts. , ingredients used in common pharmaceuticals, quasi-drugs, cosmetics or foods: excipients, stabilizers, preservatives, binders, disintegrating agents, hydrocarbons, fatty acids, alcohols, esters, surfactants, Metal soap, pH adjuster, preservative, perfume, moisturizer, powder, UV absorber, tackifier, pigment, antioxidant, whitening agent, chelating agent, grease or wax. The pig lung extract of the present invention can be formulated into a dosage form such as a powder, a pill, a 149806.doc •9-201219044 bond, an injection, a test, an emulsion, a capsule, a granule, a liquid (including an expectorant, a fluid tincture, an alcohol). , suspending agent, lemonade, etc.), lotion, cream, lotion, gel, spray "pretend skin cream, detergent, bath, base, powder, lipstick, ointment, warm and damp cloth, Pastes, plasters, essential oils, candy keys or beverages, etc. The following examples are not to be construed as unduly limiting the invention, and those of ordinary skill in the art to which the present invention pertains, without departing from the spirit or scope of the invention Modifications and variations of the embodiments discussed herein remain within the scope of the invention. [Embodiment] Example 1 Extraction method of pig lung extract Frozen lung lungs are thawed, washed with water, and then the trachea portion is removed, first cut It is about 10 cm in size and is ground in a meat grinder to a non-bulk structure. Add 2 times the weight of the pig lung extract (〇.〇2 Μ sodium citrate pH 7.0 ± 0.2) to the salt mixer. Combined Uniform 'weighing the weight of the lungs 5 pineapple enzyme (2000 GDU / g, ST BIO) and 〇 ·ι % (w / w) protease M enzyme (5500 untis / g, Aman 〇) 'mixed thoroughly with a blender Uniform, react in a water bath at 5 ° C for 3 hours. After the reaction is completed, let the enzyme be inactivated in a water bath for 9 minutes. Allow to cool in ice water to 25. (:, add 1.5% (W/W) 6N After the hydrochloric acid was stirred uniformly, the mixture was allowed to stand at room temperature for 1 hour to carry out an acid precipitation reaction. The above sample was directly plated with a 1 μm aperture paperboard, and the filtrate was collected, precipitated in 3 volumes of 95% ethanol (food grade), and placed. 4 It was cold room i for 2 hours. The precipitate was collected by centrifugation at 8000 (xg) for 3 minutes, and the precipitate was reconstituted with 〇·〇2 Μ sodium citrate pH 7.0. 149806.doc •10· 201219044 Example 2, Pig Toxicity of lung extract to fibroblasts and inhibition of MMP-1 MMP-1 isozyme map analysis 1. Cell culture and toxicity analysis (1) Cell culture: A-MEM medium containing 10 ° /. FBS, Plant a total volume of u mL of lxlO4 WS1 (BCRC 60300) cells in a 96-well plate 'over the supernatant overnight' to a known concentration The product was prepared into serum containing medium, 0, 0.125, 0.25, 〇_5, 1 mg/mL, 5 analytical doses, 3 replicates each. Then the transition membrane of 0·2 μπι was used to 'take 〇. 1 mL In the cells, the supernatant was removed after 24 hr of treatment and then prepared in a_MEM medium without serum but containing 10 ng/mL TNF-, 0, 0.125, 0.25, 0.5, 1 mg/mL 5 analytical doses each 3 replicates' The membrane was filtered through a 0.2 μm filter membrane, and 1 mL was added to the cells, and after 24 hours, the supernatant was collected and stored at -8 Torr. 〇, as a follow-up sample of MMP-1 isozyme analysis. (2) Toxicity analysis: The above cells remained in the 96-well plate, and a mixture of serum-containing medium and MTS (100 mL: 20 mL) was added, 37. (: The reaction was analyzed for 3 hours, and the cell viability was analyzed at 〇D. 490/630 nm. The highest concentration of the survival rate was higher than 8〇% (defined as non-toxicity), and the MMP-1 isozyme map was analyzed. Analysis of the enzyme enzyme map (1) preparation of enzyme electrophoresis gel and electrophoresis analysis
以含有0.2%酪蛋白（casein)受質溶液，配製成1〇% SDS_ PAGE電泳膠。取4 μί的樣品染料（sampie dye)(2〇〇 mM 149806.doc _ιι 201219044The solution was prepared to contain 1% SDS_PAGE electrophoresis gel containing 0.2% casein. Take 4 μί of sample dye (sampie dye) (2〇〇 mM 149806.doc _ιι 201219044
Tris-HCl，pH值 6.8，8% SDS，0.4°/。溴紛藍，40%甘油）和 20 μί的樣品混合均勻（樣品染料：樣品=1 : 5)，置於室溫 1 0分鐘’注入（loading)到上述配製之電泳膠進行電泳分析 ’以固定80伏特至電泳結束。 (2) 同功酵素圖譜反應 取完成膠片浸泡在清洗緩衝液I (Wash buffer I)，於室溫 下搖晃洗務60分鐘。再換浸泡在清洗緩衝液n (Wash buffer II)於室溫下搖晃洗滌4〇分鐘。最後於反應緩衝液 (Reaction buffer)，37。(：作用 20小時。 (3) 染色、退染及條帶分析 反應完成膠片放置於染色溶液（〇 15%考馬斯藍R25〇)染 色60分鐘。再置於去染溶液（4〇%甲醇，1〇%醋酸）去色約 30分鐘，搖晃至透明帶出現。最後浸泡在R〇水中震盪⑺分 鐘。以Bio-Rad影像處理系統定量透明條帶的光密度值。 由圖1A之豬肺萃取物對纖維母細胞之毒性試驗圖表可知 ，即便豬肺萃取物濃度達4〇〇 gg/mL，也不會對細胞產生 毒性而使細胞死亡。由圖1B可知，對於對照組，豬肺萃取 物濃度越高，確越可抑制ΜΜΡ·卜圖1C則將不同濃度之豬 肺萃取物以及對照組·ΜΜΙΜ之抑制率做成圖表，顯示 越高濃度的豬肺萃取物具有抑制MMpq的功效，而對照組 則未有相同的抑制作用。 實施例三、豬肺萃取物對纖維母細胞之毒性及抑制 ΜΜΡ-2/-9之功效 ΜΜΡ-2/-9同功酵素圖譜分析 I49806.doc 201219044 1 ·細胞培養和毒性分析 (1) 細胞培養 以含10% FBS的DMEM培養基’種植總體積〇1 mL的 lxlO4 NIH/3T3(BCRC 60008)細胞於96孔盤，隔夜去除上 清液’取已知濃度的樣品’以含有血清的培養基配製成 0,0.125, 0.25，0.5，1 mg/mL 5個分析劑量，各3重複，再以 0.2μηι的過濾、膜過渡，取〇· 1 mL加入細胞中，處理24 hr後 去除上清液’再以未含有血清但含有TNF-的DMEM培養基 配製成〇,〇.125，0.25，0.5，11^/111[5個分析劑量各3重複， 以0.2 μιη的過濾膜過濾，取〇·ι mL加入細胞中，再處理 24小時後收集上清液，保存在’作為後續mmp-2/-9 同功酵素圖譜分析樣品。 (2) 細胞毒性分析：同實施例二之細胞毒性分析試驗步 驟。 2.同功酵素圖譜分析 (1) 配製酵素電泳膠及電泳分析 以含有動物膠/酪蛋白受質溶液配製成1〇% SDS-PAGE電 泳膠。取4 μί的樣品染料（2〇〇 mM Tris-HCl，pH值6.8， 8% SDS，0_4%溴酚藍，40%甘油）和20 的樣品混合均勻 (樣品染料：樣品=1 : 5)，置於室溫10分鐘，注入到上述 配製之電泳膠進行電泳分析，以固定8〇伏特至電泳結束。 (2) 同功酵素圖譜反應 步驟同實施例二之功酵素圖譜反應所述 (3) 染色、退染及條帶分析 H9806.doc 201219044 步驟同實施例二之染色、退染及條帶分析所述。 由圖2 A為豬肺萃取物對纖維母細胞之毒性試驗圖表可之 ，豬肺萃取物濃度達3〇〇 pg/mL，也不會對細胞產生毒性Tris-HCl, pH 6.8, 8% SDS, 0.4°/. Blend blue, 40% glycerol) and 20 μί sample are evenly mixed (sample dye: sample = 1: 5), placed at room temperature for 10 minutes 'loading' to the above prepared electrophoresis gel for electrophoresis analysis' to fix 80 volts to the end of the electrophoresis. (2) Isozyme mapping reaction The finished film was immersed in Wash buffer I and shaken at room temperature for 60 minutes. Then change to soak in Wash buffer II (Wash buffer II) and shake for 4 minutes at room temperature. Finally, in Reaction buffer, 37. (: 20 hours of action. (3) Dyeing, de-staining and strip analysis reaction The film was placed in a staining solution (〇15% Coomassie Blue R25〇) for 60 minutes, and then placed in a de-staining solution (4% methanol). , 1〇% acetic acid) Decoloring for about 30 minutes, shaking to the zona pellucida. Finally immersed in R 〇 water for shaking (7) minutes. Quantify the optical density value of the transparent strip by Bio-Rad image processing system. The toxicity test chart of the extract on the fibroblasts shows that even if the concentration of the pig lung extract reaches 4 〇〇 gg/mL, the cells will not be toxic and the cells will die. As shown in Fig. 1B, for the control group, pig lung extraction The higher the concentration of the substance, the more it can be inhibited. Figure 1C shows the inhibition rates of different concentrations of pig lung extract and control group, showing that the higher concentration of pig lung extract has the effect of inhibiting MMpq. The control group did not have the same inhibitory effect. Example 3, toxicity of porcine lung extract on fibroblasts and inhibition of ΜΜΡ-2/-9 ΜΜΡ-2/-9 isozyme map analysis I49806.doc 201219044 1 · Cell culture and toxicity analysis (1) Cell culture In a DMEM medium containing 10% FBS, 'plant a total volume of 1 mL of lxlO4 NIH/3T3 (BCRC 60008) cells in a 96-well plate, and remove the supernatant 'take a sample of known concentration' overnight to contain The serum medium was formulated into 5, 0.125, 0.25, 0.5, 1 mg/mL 5 analytical doses, 3 replicates each, and then filtered with 0.2 μηι, membrane transition, and 1 mL was added to the cells for 24 hrs. The supernatant was removed and then formulated in DMEM medium containing no serum but containing TNF-, 〇.125, 0.25, 0.5, 11^/111 [5 analytical doses each for 3 replicates, filtered through a 0.2 μιη filter membrane 〇·ι mL was added to the cells, and the supernatant was collected after 24 hours of treatment, and stored in 'as a follow-up mmp-2/-9 isozyme pattern analysis sample. (2) Cytotoxicity analysis: same as Example 2 Cytotoxicity analysis test procedure 2. Isozyme analysis (1) Preparation of enzyme electrophoresis gel and electrophoresis analysis to prepare 1〇% SDS-PAGE electrophoresis gel containing animal glue/casein receptor solution. Take 4 μί sample Dye (2 mM Tris-HCl, pH 6.8, 8% SDS, 0_4% bromophenol blue, 40% glycerol) and 20 The sample was uniformly mixed (sample dye: sample = 1: 5), placed at room temperature for 10 minutes, and injected into the above-mentioned prepared electrophoresis gel for electrophoresis analysis to fix 8 volts to the end of electrophoresis. (2) Isozyme reaction step (3) Dyeing, de-staining and band analysis H9806.doc 201219044 The procedure is the same as in the dyeing, de-staining and strip analysis of Example 2. Figure 2A shows the toxicity test of pig lung extract on fibroblasts. The concentration of pig lung extract is up to 3〇〇 pg/mL, which is not toxic to cells.
而使細胞死亡’證實豬肺萃取物對細胞是安全的。由圖2B 可知’對於對照組，豬肺萃取物濃度越高，確越可抑制 MMP-2以及MMP-9。圖2C則將不同濃度之豬肺萃取物以及 對照組對於MMP-2之抑制率做成圖表，顯示越高濃度的豬 肺萃取物具有抑制MMP_2的功效，而對照組則未有相同的 抑制作用。圖2D則將不同濃度之豬肺萃取物以及對照組對 於MMP-9之抑制率做成圖表，顯示越高濃度的豬肺萃取 物具有抑制MMP-9的功效，而對照組則未有相同的抑制 作用。 【圖式簡單說明】 圖1A係豬肺萃取物對纖維母細胞之毒性試驗圖表；圖 1B及C係各種濃度之豬肺萃取物抑制mmp_ 1活性之膠體電 泳圖及圖表。 圖2A係豬肺萃取物對纖維母細胞之毒性試驗圖表；圖 2丑及C係各種濃度之豬肺萃取物抑制MMp_2及 mmP-9活性 之膠體電泳圖及圖表。 149806.docAnd let the cells die 'to confirm that the pig lung extract is safe for the cells. As can be seen from Fig. 2B, for the control group, the higher the concentration of the pig lung extract, the more the MMP-2 and MMP-9 were inhibited. Figure 2C shows the inhibition rates of MMP-2 in different concentrations of pig lung extract and control group, showing that the higher concentration of pig lung extract has the effect of inhibiting MMP_2, while the control group has no similar inhibition. . Figure 2D is a graph showing the inhibition rates of MMP-9 in different concentrations of pig lung extract and control group, showing that the higher concentration of pig lung extract has the effect of inhibiting MMP-9, while the control group does not have the same Inhibition. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A is a graph showing the toxicity test of pig lung extract on fibroblasts; Fig. 1B and Fig. C are colloidal electrophoresis charts and graphs of various concentrations of pig lung extracts for inhibiting mmp-1 activity. Fig. 2A is a graph showing the toxicity test of pig lung extract on fibroblasts; Fig. 2 is a colloidal electrophoresis pattern and graph showing the inhibition of MMp_2 and mmP-9 activities by porcine lung extracts of various concentrations of ugly and C strains. 149806.doc