201105356 六、發明說明： c發明所屬之技術領域3 相關申請案的交互參照 本申請案係以已先行提申之日本專利申請案特願 2009-148772號（申請日：2009年6月23日）為基礎，並主張其 優先權之利益，且藉由交互參照而將其整體揭露内容納入 於此。。 技術領域 本發明係有關於一種以燕窩之酵素分解物作為有效成 分之真皮纖維母細胞之膠原蛋白產生低落之抑制劑。更詳 而言之，本發明係有關於一種可抑制因紫外線照射而導致 皮膚真皮纖維母細胞中發生之I型膠原蛋白之產生低落之 膠原蛋白產生低落抑制劑。進而，本發明係有關於一種包 含前述有效成分之燕窩之酵素分解物之皮膚外用組成物及 化粧品。 L先前技術3 背景技術 肌膚乾燥、皺紋、肌膚鬆弛、彈性不佳等肌膚問題， 尤其受到女性重視與關心。該等肌膚問題通常被認為係由 環境因素、老化等增齡之因素、甚至壓力等心理因素等所 導致。尤其，近年紫外線增加及空調之泛用等所伴隨之生 活環境之乾燥化等所導致對肌膚造成不良影響之環境因素 正逐漸增加。 皮膚組織係由表皮、真皮、皮下組織所構成。真皮含 201105356 有多量膠原蛋白及坡尿酸，而與皮膚之保水性及彈力息息 相關。膠原蛋白及坡尿酸已知係由存在真皮中之真皮纖維 母細胞所產生。 據稱膠原蛋白約占人體内存在之蛋白質之30%，其中 70%存在於真皮中。存在真皮中之膠原蛋白雖包含丨型膠原 蛋白及111型膠原蛋白，但主要則為I型膠原蛋白。 以往’為提昇肌膚彈性及光澤、皺紋之預防改善之目 的’多採用以外用方式使用添加有膠原蛋白之化粧品，而 對皮膚直接補充膠原蛋白之方法。然而，以外用方式補充 膠原蛋白時’不易使皮膚真皮之膠原蛋白增加，故難謂絕 對充分可使肌膚之彈性及光澤提昇、預防改善皺紋。 皮膚若曝曝於包含紫外線之光線下，已知占真皮之細 胞外基質之大半之膠原蛋白將顯著減少，並使其纖維束排 列不規則。因此’真皮之膠原蛋白量增加有助於皺紋之改 善及預防等（非文獻1「用於機能性化粧品材料之開發之實 驗法」（芋皮玄爾監修），2007年發行，CMC出版，第45〜47 頁）。又，其中亦揭露中波紫外線(UVB)之照射將使真皮纖 維母細胞之膠原蛋白合成大幅減少。其中，！型膠原蛋白合 成促進劑係例示為維他命C誘導體、乳清成分等。 另’文獻（Akiko Kojima-Yuasa，et al·, Fragrance Journal, April’ 2009’ No’ 346(V〇1.37, No. 4)，pp. 103-)中則揭露了膠 原蛋白增生能力之降低之抑制有助於q〇L之提昇改善，皮 膚之膠原蛋白量之降低則為皺紋及鬆弛等之成因。 因此’抑制或改善業經紫外線照射之真皮纖維母細胞 4 201105356 之膠原蛋白合成之減少現象，就曝曬紫外祕之皮膚可能 毛生之膠原蛋白減少所伴隨之肌膚之彈性及光澤之降低之 預防或改善或者皺紋之預防或改善之觀點而言，可謂甚為 重要。 然而’设今，雖已有促進真皮_母細麟產生之段 膠原蛋白之增生之多種嘗試(諸如特開細8_ 1G5984號公報 及腦爾_9)，但就本發明人所知，關於藉由直接抑 制或改善業經紫外線照射之真皮纖維母細胞之膠原蛋白合 成之減少現象本身’而抑制真皮之膠原蛋白之變化現象， 尚未受到檢討。 燕窩係金絲燕以自己的唾液構成絲狀而築成之巢，已 知其成分包含多量之酶蛋白f，而幾乎不含脂質。燕高迄 今使用於中菜作為食材’亦為巾藥之原料等。近年，亦已 開發以燕“原料之錢食品# ,至於外㈣及化粧品等 外用用途之應用例則尚少為人知。 特開綱3-95961號公報中已揭露使用燕离之酵素分解 物之口服用之美肌促進劑。然而，复 ” T僅揭露口服用途， 而未就外用用途之利用可能性作任何揭露及暗二 效果係肌膚保濕感之提昇及肌理細緻肌膚之^又，其 可提昇肌㈣性及料纽善缺以料衫白^ = 促進能力作任何揭露，且，亦未就業經紫外線 纖維母細胞之膠原蛋白合成之減少 "、、射之真皮 - 之抑制作任何揭露或暗 7Κ 。 WO99/022709 中， 已揭露包含燕窝 之含水溶劑萃取 5 201105356 物’特別是熱水萃取物之化挺品。該化粧品可使用於皮膚 :養用途，係制於外㈣途者。又，其中亦揭露燕寫之 3水卒取物具有皮膚細胞之膠原蛋白合成促進效果'然 而，其使^萃取祕就—加叫料㈣擔固態部 分而成者，與减窩整料㈣核㈣加以直接使用之 燕高酵素分解物㈣不同。X，關於業經紫外線照射之真 皮纖維母細胞之膠原蛋白合成之減少之抑制，其中亦無任 何揭露或暗示。 【發明内容】 發明概要 本發明人此次意外發現了燕离之酵素分解物具有可有 效抑制業經紫外線照射之皮膚真皮纖維母細胞中可見之1 型膠原蛋白合成之減少現象之效果。本發明即以前述之發 現為依據。 另，本發明人亦同時發現了燕窩之酵素分解物具有皮 膚真皮纖維母細胞中之丨型膠原蛋白之優良增生促進效果。 因此，本發明之目的在提供可有效率地生產且安全性 較高之真皮纖維母細胞中之膠原蛋白產生低私優良抑制 劑及包含前者之外用劑與化铍品。 本發明之膠原蛋白產生低落抑制劑係—種以燕寫之酵 素分解物作為有效齡，可抑職皮纖料細胞因紫外線 照射而導致膠原蛋白產生低落之抑制劑。 依據本發明之-較佳態樣，前述酵素分解物係蛋白酶 之分解物。 6 201105356 依據本發明之-更佳態樣，前述酵素分解物之分子量 為 70〜200,000。 依據本發明之另-更佳態樣，前述酵素分解物之平均 分子量為1000〜50,〇〇〇。 依據本發明之較佳態樣，本發明之膠原蛋白產生低落 抑制劑可抑個紫外線照射而導致皮膚真皮纖維母細胞中 發生之I型膠原蛋白之產生低落。 依據本發明之其它態樣，可提供—種皮膚外用組成 物，其包含本發明之膠原蛋白產生低落抑·。該組成物 宜使用於錢料躲料錢之皮膚料，偏於預防 ，改善業經紫外線照射之皮膚所發生之肌膚彈性及光澤低 落以及預防或改善皺紋則更佳。 依據本發明之其它更佳態樣’本發明之皮膚外用 物係化粧品。 依據本發明之其它態樣，可提供一種燕寫之酵素分解 物之用途，其㈣製造涵括，仙 、=母細胞受紫外線照射而導致膠原蛋白產生低落之抑2 Μ。依據本發明之另一態樣， ,, 杈供種燕窩之酵素分解 物之用途，其用以製造接受紫 線關4後之皮膚保養 用之皮膚外用組成物。 依據本發明之再一態樣，可 以供種方法，包含：將 有攻量之本發明有效成分之酵 :之哺乳動物之皮膚上，抑: 線照射而導致膠原蛋白之產生 胞因备、外 氐洛。依據本發明之另一態 201105356 樣，則可提供—種皮膚保養方法，包含 明有效成分應用於涵括人類在内之=有效量之本發 照射前或後之皮膚上。同樣地物在接雙紫外線 將有效量之本發日椒成分㈣，包含： 物之皮膚·’而可改善業經紫外線昭 在内之哺乳動 彈性及光澤之降低，以及_或改善㈣ 之肌膚 可有==:=:料解物作為有—， =生低落。尤其對於業經紫外線照射之皮膚真 =:胞中可見之1型膠原蛋白之產生低落現象，可獲 致有效抑制或改善之效果❶ 之膠原蛋Q纽於上述真纽維母細胞 士 “田 抑制效果’而_素分解物使用於 或化桩品用途，則可改善纖維母細胞之膠原 蛋 低洛所伴隨之肌膚之彈性（彈力）及肌膚光澤之降 低，進而預防或改善皺紋。 尤二上述效果可改善曰曬後肌膚在膠原蛋 白合成減 /狀下之肌膚。如此’依據本發明，可實和肖費者之q〇l 之七昇X所使用之酵素分解物係使用亦公知為食材之 热窩’故安全性較高’消費者接受度亦較高 。進而，酵素 分解物可直接就燕窩進行酵素處理而取得，故可有效率進 行製造，且降低製造成本。 圖式簡單說明 第1圖係顯示燕窩樣本中之延酉曼量之測定結果之圖表。 第2圖係顯不燕高樣本中之蛋白質量之測定結果之圖 8 201105356 表。 第3圖係顯示採GPC-HPLC之燕离酵素分解物丨之測定 結果之圖表。 第4圖係顯示採GPC-HPLC之燕高酵素分解物2之測定 結果之圖表。 第5圖係顯示膠原蛋白之產生促進能力之測定結果之 圖表。 本發明之具體說明 有效成分 本發明之膠原蛋白產生低落抑制劑一如前述，係以燕 窩之酵素分解物作為有效成分。 酵素分解物之原料燕窩係金絲燕將自己的唾液構成絲 狀而築成之巢，其成分包含多量之醣蛋白質，幾乎不含有 脂質。在此，金絲燕係指雨燕科（Apodidae)之金絲燕屬 (Collocalia)下之燕類，可例舉諸如小灰腰金絲燕（Ap〇didae201105356 VI. OBJECTS OF THE INVENTION: C TECHNICAL FIELD OF THE INVENTION The present invention is based on the Japanese Patent Application No. 2009-148772 (Application Date: June 23, 2009), which is hereby incorporated by reference. Based on and claiming the benefits of its priority, and including its overall disclosure by cross-reference. . BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a collagen-producing inhibitor of dermal fibroblasts which utilizes an enzyme decomposition product of bird's nest as an effective component. More specifically, the present invention relates to a collagen-lowering inhibitor which inhibits the production of type I collagen which occurs in skin dermal fibroblasts by ultraviolet irradiation. Further, the present invention relates to a skin external composition and a cosmetic of an enzyme decomposition product of bird's nest containing the above-mentioned active ingredient. L Prior Art 3 Background Art Skin problems such as dry skin, wrinkles, sagging skin, and poor elasticity are particularly valued and cared for by women. These skin problems are usually thought to be caused by environmental factors, ageing factors such as ageing, and even psychological factors such as stress. In particular, environmental factors that have adversely affected the skin caused by the increase in ultraviolet rays and the drying of the living environment in recent years have been increasing. The skin tissue consists of epidermis, dermis, and subcutaneous tissue. The dermis contains 201105356 with a large amount of collagen and uric acid, which is closely related to the water retention and elasticity of the skin. Collagen and uric acid are known to be produced by dermal fibroblasts present in the dermis. Collagen is said to account for about 30% of the protein in the body, 70% of which is found in the dermis. Collagen in the dermis contains sputum type collagen and type 111 collagen, but mainly type I collagen. In the past, in order to improve the skin's elasticity, luster, and wrinkles, the use of a collagen-added cosmetic and a method of directly supplementing the skin with collagen was used. However, when collagen is added by external application, it is difficult to increase the collagen of the skin dermis, so it is difficult to increase the elasticity and gloss of the skin and prevent wrinkles. If the skin is exposed to light containing ultraviolet light, it is known that most of the collagen in the extracellular matrix of the dermis will be significantly reduced and the fiber bundles will be irregularly arranged. Therefore, the increase in the amount of collagen in the dermis contributes to the improvement and prevention of wrinkles (Non-document 1 "Experimental method for the development of functional cosmetic materials" (Supervisor of the skin), issued in 2007, CMC Publishing, 45~47 pages). In addition, it is also revealed that ultraviolet light (UVB) irradiation will greatly reduce the collagen synthesis of dermal fibroblasts. among them,! The type collagen synthesis promoter is exemplified by a vitamin C inducer, a whey component, and the like. Another 'document (Akiko Kojima-Yuasa, et al., Fragrance Journal, April' 2009' No' 346 (V〇 1.37, No. 4), pp. 103-) reveals the inhibition of the decrease in collagen proliferative capacity. It helps to improve the improvement of q〇L, and the decrease in the amount of collagen in the skin is the cause of wrinkles and sagging. Therefore, 'reducing or improving the reduction of collagen synthesis in the ultraviolet-irradiated dermal fibroblasts 4 201105356, the prevention or improvement of the decrease in the elasticity and gloss of the skin associated with the reduction of the collagen which may be caused by the ultraviolet-visible skin. Or the viewpoint of prevention or improvement of wrinkles is very important. However, there are various attempts to promote the proliferation of collagen in the segment of the dermis-female syllabus (such as the special publication 8_1G5984 and the brain _9), but as far as the inventors are aware, The phenomenon of inhibiting the collagen change of the dermis by directly inhibiting or improving the reduction of collagen synthesis by the ultraviolet-irradiated dermal fibroblasts has not been reviewed. The bird's nest swiftlet is made up of a silky form of its own saliva. It is known that its composition contains a large amount of enzyme protein f, and contains almost no lipid. Yangao has been used in Chinese cuisine as a food ingredient, and it is also a raw material for towel medicine. In recent years, it has not been known to develop an application example of Yan's “Materials for Raw Materials, For Foreign (4) and Cosmetics, etc. The use of Yancheng's enzyme decomposition products has been disclosed in Japanese Patent Laid-Open No. 3-95961. A skin-enhancing agent for oral administration. However, the complex T only exposes the oral use, and does not expose the use of the external use for any exposure and darkness effect, and the skin moisturizing effect and the texture of the skin are improved. Muscle (4) sex and material New good lack of shirt white ^ = promote the ability to make any exposure, and, also, the employment of UV fiber fibroblasts reduced collagen synthesis, ", the dermal leather - inhibition of any exposure or Dark 7Κ. In WO 99/022709, an aqueous solvent extraction comprising a bird's nest 5 201105356 has been disclosed, in particular a hot water extract. The cosmetic can be used for the skin: for the purpose of use, and for the external (four) way. In addition, it also reveals that the 3 water-stained materials written by Yan have the effect of promoting the synthesis of collagen in skin cells. However, it makes the extraction secrets--adding materials (4) to the solid part, and the litter (4) core. (4) The Yangao enzyme decomposition products (4) used directly are different. X, Regarding the inhibition of the reduction of collagen synthesis by UV-irradiated real-skinned fibroblasts, there is no disclosure or suggestion. SUMMARY OF THE INVENTION The present inventors have unexpectedly discovered that the enzyme-decomposing product of Yanyan has an effect of effectively suppressing the decrease in the synthesis of type 1 collagen which is visible in the dermal fibroblasts which are irradiated with ultraviolet rays. The present invention is based on the foregoing findings. Further, the inventors have also found that the enzyme decomposition product of bird's nest has an excellent proliferation promoting effect of sputum type collagen in skin dermal fibroblasts. Accordingly, it is an object of the present invention to provide a collagen-producing low-good-good inhibitor in dermal fibroblasts which can be efficiently produced and which is highly safe, and to contain the former external preparations and cosmetics. The collagen-producing low-lowering inhibitor of the present invention is an enzyme-decomposing substance of the genus, which is an effective inhibitor of the collagen, which inhibits the production of collagen by ultraviolet rays. According to a preferred embodiment of the present invention, the aforementioned enzyme decomposition product is a decomposition product of a protease. 6 201105356 According to a preferred aspect of the present invention, the aforementioned enzyme decomposition product has a molecular weight of 70 to 200,000. According to another preferred aspect of the invention, the enzyme decomposition product has an average molecular weight of from 1000 to 50, 〇〇〇. According to a preferred embodiment of the present invention, the collagen-lowering inhibitor of the present invention inhibits ultraviolet light irradiation and causes the production of type I collagen in the skin dermal fibroblast to be low. According to another aspect of the present invention, a skin external composition comprising the collagen of the present invention can be provided. The composition should be used for skin materials that are used for money, and it is better to prevent and improve the elasticity and luster of the skin caused by ultraviolet rays and to prevent or improve wrinkles. According to still other preferred aspects of the present invention, the skin external cosmetic of the present invention. According to another aspect of the present invention, there is provided a use of an enzyme decomposition product of the Swallow, wherein (4) manufacturing includes, and the mother cells are exposed to ultraviolet rays to cause a decrease in collagen production. According to another aspect of the present invention, the use of an enzyme decomposition product for the supply of bird's nest is for the manufacture of a skin external composition for skin care after receiving the purple line. According to still another aspect of the present invention, there may be provided a method comprising: a yeast having an attacking amount of the active ingredient of the present invention: on the skin of a mammal, wherein: the irradiation of the line causes the production of collagen. Luo Luo. According to another aspect of the present invention, 201105356, a skin care method can be provided which comprises applying the active ingredient to the skin before or after the irradiation of an effective amount of human hair. In the same way, in the case of the double-ultraviolet light, the effective amount of the pepper component (4), including: the skin of the object, can improve the elasticity and luster of the lactation, and the skin of the skin can be improved. There is ==:=: the material is dissolved as -, = is low. Especially for the skin that has been exposed to ultraviolet light =: the type 1 collagen that is visible in the cell is low, and the effect of effectively inhibiting or improving the effect can be obtained. The collagen egg Q is added to the above-mentioned true New Zealand cell line "Field inhibition effect" When the _-decomposed substance is used in or used for the pile, it can improve the elasticity (elasticity) and skin gloss of the skin associated with the collagen of the fibroblast, thereby preventing or improving wrinkles. It improves the skin of the skin under collagen synthesis after drying. Thus, according to the present invention, the use of the enzyme decomposition product used in the seven liters of X of the shovel is also known as the heat of the food. The safety of the nest is high, and the consumer acceptance is also high. Further, the enzyme decomposition product can be directly obtained by the enzyme treatment of the bird's nest, so that the manufacturing can be efficiently performed and the manufacturing cost can be reduced. The figure shows the results of the measurement of the amount of delayed mangium in the bird's nest sample. Figure 2 shows the measurement result of the amount of protein in the sample of the non-yan high. Figure 8 201105356 Table. A graph showing the results of measurement of the enzyme-decomposing substance of Ginc-HPLC by GPC-HPLC. Fig. 4 is a graph showing the results of measurement of Yangao Enzyme Decomposition 2 by GPC-HPLC. Figure 5 shows the production of collagen. A graph showing the results of the measurement of the ability. DETAILED DESCRIPTION OF THE INVENTION Active ingredient The collagen-producing inhibitor of the present invention, as described above, is an enzyme decomposition product of bird's nest as an active ingredient. The raw material of the enzyme decomposition product is swiftlet swiftself. The saliva constitutes a filamentous and nested nest, the composition of which contains a large amount of glycoprotein and contains almost no lipids. Here, the swiftlet refers to the swallows of the genus Collocalia of Apodidae. Can be exemplified by a small gray waist swiftlet (Ap〇didae
Collocalia francica)、苔巢金絲燕（Apodidae Collocalia salanga)、爪哇金絲燕（Apodidae Collocalia fuciphaga)、印度 食用巢金絲燕(Apodidae Collocalia inexpectat)、褐腰金絲燕 (Apodidae Collocalia vestita)、白腹金絲燕（Apodidae Collocalia esculent)、大金絲燕（Apodidae Collocalia maximus)等。 本發明中，燕窩可使用市售品。一般市售之燕窩從僅 去除燕毛及糞便等污垢而經清洗者，至收集燕窩之碎屑而 201105356 反覆進竹漂白與清洗而成形者，涵括多種各類，但本發明 所使用之燕窩則宜使用未在前置處理時經過度清洗及漂白 等之燕窩。 热窩之酵素分解物係指就燕窩進行酵素反應加以分解 所得者，可使用蛋白酶或含有蛋白酶之複合酵素等分解燕 窩或其處理物而製得。在此，分解處理所使用之酵素宜為 蛋白酶，使用胰酶則更佳。 燕窩之酵素分解物通常可先經加熱殺菌處理，然後於 水中進行酵素處理。殺菌處理之條件可適當參考慣用之加 熱殺菌條件而料’酵素處理之條件亦可對應所使用之酵 素之種類及燕窩之狀態而適當加以設定。 更具體之例，則可依以下程序調製燕窩之酵素分解物。 舉例言之，對已磨碎之燕窩加入其質量之1〇〜5〇倍之水 而使其膨脹後，在60〜130°C下進行加熱殺菌處理5秒至3〇 分鐘。然後，直接添加適量之酵素，而在酵素之最佳pH、 最佳溫度下進行酵素反應〇·5〜48小時。酵素反應結束後， 藉加熱處理較料不活性化，並過歧缝而去除不溶 物，而可製得燕窩之酵素分解物。又，上述酵素分解物亦 可視需要而乾燥成粉狀。另，上述各程序中，亦可適當進 行pH調整、脫色、脫臭等操作。 他例則在就热窩之處理物加以酵素分解時，就對粒徑 2mm以下、150"m以下更佳之已磨碎之燕窩加入其質量之 H)〜H)0倍之水’並在丨〜6(TC下予以靜置或攪拌〇 5〜48小時 而使其膨服後’在60〜13(TC下進行加熱殺菌處理5秒〜扣分 201105356 鐘，並視需要進行過濾而製得溶液，再與上述同樣施以酵 素處理，而可製得燕窩之酵素分解物。 酵素分解物之調製法係酵素反應之時間愈長，蛋白質 之分解程度愈高，而可獲致包含更低分子之蛋白質之酵素 分解物。本發明中，酵素反應時間為0.5〜48小時，而3〜36 小時更佳，約24小時程度最佳，並宜使用酵素反應較充分 進行後之酵素分解物。 如此製得之燕窩之酵素分解物之分子量宜為 70〜200,000，70〜180,000則更佳。分子量為70〜150,000則極 佳，70〜120,000則最佳。 又，燕窩之酵素分解物之平均分子量（重量平均分子量） 宜為300〜100,000，而以500〜70,000為更佳。平均分子量 700〜60,000則甚佳，1,000〜50,000則最佳。 另，視需要而就燕窩酵素分解物適當變更上述操作條 件，或應用慣用之精製或區分方法，可進而將前述平均分 子量之下限值設為5,000以上，而以10,000以上為佳，15,000 以上更佳，20,000以上則最佳。 皮膚外用組成物 本發明之膠原蛋白產生低落抑制劑雖可單獨直接使 用，但亦可内含於化粧品、醫藥品、準藥物等各種皮膚外 用組成物中作為添加劑，而製得具有真皮纖維母細胞之膠 原蛋白產生低落之抑制效果之組成物。所製得之組成物可 有效使用於皮膚保養，更宜使用於接受紫外線照射之前或 後之皮膚保養，使用於業經紫外線照射之皮膚所發生之肌 11 201105356 膚彈性與光澤之降低之改善及皺紋之預防或改善則更佳。 本發明之有效成分之燕窩之酵素分解物具有可有效抑 制業經紫外線照射之人類皮膚真皮纖維母細胞中可見之】 型膠原蛋白之產生低落之活性（後述實施例之測試結果）。 因此，本發明中’所謂膠原蛋白之產生低落之抑制活 性意指抑制或改善人類皮膚真皮之纖維母細胞中可能發生 之膠原蛋白之產生能力之降低之作用，意在藉此可抑制或 改善人類皮膚真皮中之膠原蛋白量之現象。抑制或改善真 皮之朦原蛋白量之降低，即可獲致皺紋改善及預防效果， 並可預防或改善膠原蛋白產生低落所導致肌膚彈性（彈力） 及肌膚光澤之降低，本業之技術人員尤已周知，亦可輕易 理解(諸如前述之文獻(「用於機能性化粧品材料之開發之實 驗法」（芋皮玄爾監修），2007年發行’CMC出版，第45〜47 頁（非專利文獻 1))及文獻（Akiko Kojima-Yuasa，et al， Fragrance Journal, April，2009, No. 346(Vol.37, No. 4)，pp 103_(非專利文獻2))等中均已明示此點）。 尤其’上述效果可謂可改善日曬後之肌膚中膠原蛋白 合成減少之狀態之肌膚。因此，本發明之有效成分已確認 具有真皮之纖維母細胞之膠原蛋白產生低落之抑制活性， 故本發明之有效成分明顯有助於皮膚保養，尤其，本發明 之有效成分明顯有助於接受紫外線照射之前或後之皮膚保 養’更適用於業經紫外線照射之皮膚所發生之肌膚之彈性 及光澤之降低之改善，以及皺紋之預防或改善。進而，本 發明之有效成分亦有助於老化所導致之肌膚之彈力降低之 12 201105356 改善、皺紋之預防等。 力，尽說明書中，所謂肌膚 ^ ^ ，'丄六疋净之降低、皺 二膚或皮膚之狀態之「預防或改善」，包含其狀態之調 二1 減輕、發作預防、復發預防、抑制等在 故而 成八有太/ ，依據本發明之其它態樣，可提供構 成3有本♦明之膠原蛋自產生低落抑_ 物。皮膚外用組成物宜提供作為醫藥品、準藥物或化粧品成 而更宜提供作為化粧品。 •本發明之皮膚外用組成物（尤其化粧品）中，有效成分之 燕窩之酵素分解物之含量(乾物換算)並無特限制，依組 成物形態而不同，但一般作為固態部分而為0 001〜20重量 百分比，而以0·005〜5重量百分比之範圍為佳，可對應組成 物之形態而適當加以變更。 或’本發明之皮膚外用組成物之燕窩酵素分解物之含 量（乾物換算）宜為可實現每曰於每平方公分之皮膚上使用 O.Olmg以上，而以o oimg〜2mg為佳之量。 在本發明之皮膚外用組成物包含有效成分，且無損真 皮纖維母細胞之膠原蛋白之產生低落之抑制效果之限度 内’本發明之皮膚外用組成物可進而包含其它任意成分。 上述之任意成分可為諸如皮膚外用劑、化粧品慣用之各種 成分’可例舉諸如保濕劑、低級醇、多價醇、醣類、界面 活性劑、緩衝劑、乳化劑、安定劑' 增黏劑、抗氧化劑、 防腐抗卤劑、螫合劑、pH調整劑、香料、色素、紫外線吸 13 201105356 收劑、紫外線散射劑、維他命類、胺基酸類、抗炎劑、水、 胜肽、糖醇類、酵素類、植物萃取類、抗氧化物質類、滑 石粉、黏土、花粉、珍珠粉等。 在此，保濕劑可例舉諸如聚乙二醇、聚丙二醇、丙三 醇、丙二醇、山梨糖醇、玻尿酸及硫酸軟骨素等酸性黏多 醣類、胺基酸、膠原蛋白、彈力蛋白等。 低級醇可例舉諸如乙醇、丙醇、異丙醇等。 多價醇可例舉諸如丙三醇、異戊四醇、雙異戊四醇、 乙二醇、丙二醇、聚丙二醇、1，3-丁二醇等。 醣類則可例舉諸如葡萄糖、麥芽糖、乳糖、D-葡萄醣 醛酸、蔗糖、D-山梨醣醇、山梨醇酐、纖維素、澱粉等醣 類、寡糖類、多醣類及其等之誘導體。 本發明之皮膚外用組成物可採用公知之方法適當調配 包含燕窩酵素分解物之膠原蛋白產生低落抑制劑及上述之 任意成分，而構成洗劑、乳液、乳霜、保濕液、敷面劑、 化粧水、洗面乳、身體乳、身體霜等各種化粧品之慣用產 品形態。本發明之皮膚外用組成物亦可進而提供作為粉底 類、脣膏、眼影、腮紅等彩粧化粧品及防曬用產品、防臭 化粧品等藥用化粧品、洗髮精及潤絲精、造型品等髮粧品、 皮膚清潔劑及入浴劑之浴用化桩品等之使用用途。 依據本發明之其它態樣，一如前述，可提供燕窩之酵 素分解物之使用，目的在製造涵括人類在内之哺乳動物之 真皮纖維母細胞因紫外線照射而導致之膠原蛋白產生低落 之抑制劑。在此，真皮纖維母細胞因紫外線照射而導致之 14 201105356 膠原蛋白產生低落之抑制宜為紫外線照射所致皮膚真皮纖 維母細胞中發生之I型膠原蛋白產生低落之抑制。 依據本發明之另一態樣，一如前述，可提供燕窩之酵 素分解物之使用，目的在製造接受紫外線照射之前或後之 皮膚保養用之皮膚外用組成物。在此，皮膚外用組成物宜 使用於業經紫外線照射之皮膚所發生之肌膚彈性與光澤之 降低以及皺紋之預防或改善。又，皮膚外用組成物宜為化 粧品。 依據本發明之另一態樣，一如前述，可提供一種方法， 包含對涵括人類在内之哺乳動物之皮膚使用本發明之有效 成分之酵素分解物之有效量之程序，而可抑制真皮纖維母 細胞因紫外線照射所致之膠原蛋白之產生低落。另，在此 所謂「有效量」係指因使用而可於體内之預定領域内發揮 膠原蛋白之產生低落之抑制活性之充足量。又，此之所謂 真皮纖維母細胞因紫外線照射所致之膠原蛋白之產生低落 之抑制宜為紫外線照射所致之皮膚真皮纖維母細胞中發生 之I型膠原蛋白產生低落之抑制。 進而，依據本發明之另一其它態樣，可提供一種皮膚 保養方法，包含對涵括人類在内之哺乳動物之接受紫外線 照射之前或後之皮膚使用本發明之有效成分之有效量之程 序。同樣地，亦可提供包含對涵括人類在内之哺乳動物之 皮膚使用本發明之有效成分之有效量之程序，而可改善肌 膚之彈性及光澤之降低，並預防或改善皺紋之方法。另， 此之所謂「有效量」係指獲致所欲之效果所需之充足量。 15 201105356 另，本說明書中，使用「約」及「程度」之用語之值， 包含在達成設定其值之目的之前提下，本業之技術人員所 可容許之值之變動之意義。舉例言之，意指可容許預定值 或範圍之20°/。以内之變動，而以10%以内為佳，5%以内為 更佳。 實施例 參照以下之例具體說明本發明，但本發明並不受限於 以下之例。 (1)燕窩樣本之調製 (1-1)燕窩酵素分解物1 藉碾磨機磨碎市售之未漂白之燕窩，而製得100篩目大 小（粒徑150//m以下）之燕窩粉末，再對該燕窩粉末加入約 50倍量（質量）之水而在5°C下使其膨脹20小時後，在121°C下 進行加熱殺菌處理15分鐘。 在製得之處理液冷卻後，調整pH，並對燕窩粉末添加2 質量百分比之量之含蛋白酶酵素（商品名「Pancreatin F」， 天野製藥株式會社出品），而在45°C下進行反應3小時。 將上述酵素反應液調整成pH7.0後，在90°C下加熱5分 鐘而使酵素不活性化，再加以過濾、並回收遽、液。濃縮上述 濾液後，予以凍結乾燥而製得「燕窩酵素分解物1」 (1-2)燕窩酵素分解物 除酵素反應之時間改為24小時以外，製程均與前述（1-1) 之酵素分解物1相同，而製得「燕窩酵素分解物2」。 (1-3)燕窩熱水萃取物（比較例） 16 201105356 藉碾磨機磨碎市售之未漂白之燕寫(乾燥物），並將其磨 碎物加人l_ml之錢水巾，再藉加熱迴流進行2小時之萃 取處理。加熱迴流後’再予靜置，然後分取上澄液加以過 渡:而回收渡液。殘渣則進而與前述相同，予以加入麵W 之蒸顧水巾’藉加熱㈣而進行再萃取處理後，過渡固態 物殘’查而力σ以去除，並回收據液^所得之滤液(萃取液)則一 同在減壓_境下加以濃縮’所得之絲物料以康結乾 燥，而製得黃白色之「燕窩熱水萃取物」（比較例）。 (2)燕窩樣本之分析 製得之各燕窩樣本(_酵素分_丨、絲酵素分解 物2及燕寫熱水萃取物）中之—量與蛋白質量已測定如 下。 涎酸之量係在各試料之酸性水解後藉高速液體層析法 而測定游離Ν-乙醯神經氨酸所得。 蛋白質之量則使用基於Brad ford法之Bio Rad公司之 Protein Assay Kit而加以測定。 結果則如第1及第2圖所示。 其次，為測定燕窩酵素分解物丨及燕窩酵素分解物2所 含之蛋白質之分子量及其分布，而在以下條件下，進行了 GPC之分析。 [HPLC之測定條件]Collocalia francica), Apodidae Collocalia salanga, Apodidae Collocalia fuciphaga, Apodidae Collocalia inexpectat, Apodidae Collocalia vestita, white belly Apodidae Collocalia esculent, Apodidae Collocalia maximus, etc. In the present invention, commercially available products can be used for the bird's nest. Generally, the bird's nest, which is commercially available, is formed by removing only the dirt such as swallows and feces, and cleaning the debris of the bird's nest, and 201105356 is repeatedly formed into bamboo bleaching and washing, and includes various types, but the bird's nest used in the present invention. It is advisable to use bird's nest that has not been cleaned and bleached prior to pretreatment. The enzyme decomposition product of the heat nest is obtained by decomposing an enzyme reaction in bird's nest, and can be obtained by decomposing a bird's nest or a treatment thereof using a protease or a compound enzyme containing a protease. Here, the enzyme used in the decomposition treatment is preferably a protease, and trypsin is more preferable. The enzyme decomposition products of bird's nest can usually be heat-sterilized and then treated with enzymes in water. The conditions of the sterilization treatment can be appropriately referred to the conventional heating and sterilization conditions, and the conditions of the enzyme treatment can be appropriately set depending on the type of the enzyme to be used and the state of the bird's nest. More specifically, the enzyme decomposition product of bird's nest can be prepared according to the following procedure. For example, after the pulverized bird's nest is added with 1 〇 to 5 〇 times its mass and expanded, it is subjected to heat sterilization at 60 to 130 ° C for 5 seconds to 3 minutes. Then, directly add the right amount of enzyme, and carry out the enzyme reaction at the optimum pH and optimal temperature of the enzyme for 5 to 48 hours. After the completion of the enzyme reaction, the heat treatment is inactivated, and the insoluble matter is removed by the crevice, and the enzyme decomposition product of the bird's nest can be obtained. Further, the above-mentioned enzyme decomposition product may be dried into a powder form as needed. Further, in the above respective procedures, operations such as pH adjustment, decolorization, and deodorization may be appropriately performed. In his case, when the enzyme is decomposed in the heat treatment, the powdered bird's nest with a particle size of 2 mm or less and 150 or less is added to the mass of H)~H) 0 times of water' ~6 (Still it under TC for 5~48 hours and let it swell after '60' at 60~13 (heat sterilization treatment for TC for 5 seconds~ deducting 201105356 minutes, and filter if necessary) Then, the same enzyme treatment as described above can be used to obtain the enzyme decomposition product of the bird's nest. The longer the reaction time of the enzyme decomposition process, the higher the decomposition degree of the protein, and the protein containing the lower molecule can be obtained. The enzyme decomposition product. In the present invention, the enzyme reaction time is 0.5 to 48 hours, and 3 to 36 hours is better, about 24 hours is the best, and it is preferable to use an enzyme decomposition product after the enzyme reaction is sufficiently performed. The molecular weight of the enzyme decomposition product of the bird's nest is preferably 70 to 200,000, more preferably 70 to 180,000. The molecular weight is 70 to 150,000, and the optimum is 70 to 120,000. The average molecular weight of the enzyme decomposition product of the bird's nest (weight average) Molecular weight) 300 to 100,000, and more preferably 500 to 70,000. The average molecular weight of 700 to 60,000 is very good, and 1,000 to 50,000 is optimal. Further, if necessary, the above operating conditions are appropriately changed for the bird's nest enzyme decomposition product, or the conventional purification is applied. Alternatively, the lower limit of the average molecular weight may be 5,000 or more, more preferably 10,000 or more, more preferably 15,000 or more, and most preferably 20,000 or more. Skin external composition The collagen of the present invention produces a low inhibitor Although it can be used as it is, it can also be used as an additive in various skin external compositions, such as a cosmetics, a pharmaceutical, and a quasi-drug, and it is set as the composition which has the fall of the collagen of the dermal fibroblasts. The obtained composition can be effectively used for skin care, and is preferably used for skin care before or after exposure to ultraviolet rays, and is used for muscles which are produced by ultraviolet light-irradiated skin. 11 201105356 Improvement of skin elasticity and gloss and wrinkles It is more preferable to prevent or improve. The enzyme decomposition product of the bird's nest of the active ingredient of the present invention can be effectively inhibited. In the human skin dermal fibroblasts which are exposed to ultraviolet light, the activity of producing collagen is low (the test results of the examples described later). Therefore, in the present invention, the so-called collagen production inhibitory activity means inhibition. Or the effect of reducing the ability of collagen production in fibroblasts of human skin dermis, which is intended to inhibit or improve the amount of collagen in the dermis of human skin. Inhibition or improvement of prion protein in the dermis When the amount is reduced, wrinkle improvement and prevention effects can be obtained, and the decrease in collagen production (elasticity) and skin gloss can be prevented or improved, which is well known to those skilled in the art and can be easily understood (such as the foregoing ("Experimental method for the development of functional cosmetic materials" (Supervisor of the skin), issued in 2007, "CMC Publishing, pages 45 to 47 (Non-Patent Document 1)) and documents (Akiko Kojima-Yuasa) , et al, Fragrance Journal, April, 2009, No. 346 (Vol. 37, No. 4), pp 103_ (Non-Patent Document 2), etc. Express this point). In particular, the above effects can improve the skin in which the collagen synthesis is reduced in the skin after sun exposure. Therefore, the active ingredient of the present invention has confirmed that the collagen having the dermal fibroblasts has a low inhibitory activity, so that the active ingredient of the present invention contributes significantly to skin care, and in particular, the active ingredient of the present invention is significantly useful for receiving ultraviolet rays. Skin care before or after exposure is more suitable for the improvement of the elasticity and gloss of the skin caused by UV-irradiated skin, as well as the prevention or improvement of wrinkles. Further, the active ingredient of the present invention contributes to the improvement of the elasticity of the skin caused by aging, and the prevention of wrinkles. In the manual, the so-called skin ^ ^ , 'the prevention or improvement of the sputum of the sputum, the wrinkle skin or the state of the skin, including the adjustment of its state 2 mitigation, seizure prevention, recurrence prevention, inhibition, etc. In the other case, according to other aspects of the present invention, it is possible to provide a composition 3 with a collagen egg self-generating. The external composition for skin is preferably provided as a pharmaceutical, quasi-drug or cosmetic, and is preferably provided as a cosmetic. In the skin external composition (especially cosmetic) of the present invention, the content of the enzyme decomposition product (dry matter conversion) of the bird's nest of the active ingredient is not particularly limited, and varies depending on the form of the composition, but is generally 0 001 as a solid portion. It is 20% by weight, and preferably in the range of 0. 005 to 5 weight percent, and can be appropriately changed depending on the form of the composition. Or the content of the bird's nest enzyme decomposition product (dry matter conversion) of the external composition for skin of the present invention is preferably such that it can be used in an amount of O.Omg or more per square centimeter of the skin, and preferably in an amount of o oimg to 2 mg. In the skin external composition of the present invention, the active ingredient is contained, and the skin-inhibiting effect of the collagen of the present invention is not limited. The skin external composition of the present invention may further contain other optional components. Any of the above-mentioned components may be various ingredients such as external preparations for skin and cosmetics, and may be exemplified by humectants, lower alcohols, polyvalent alcohols, saccharides, surfactants, buffers, emulsifiers, stabilizers, and tackifiers. , antioxidants, anti-corrosion and anti-halogen agents, chelating agents, pH adjusters, perfumes, pigments, UV absorbers 13 201105356 Collecting agents, UV scattering agents, vitamins, amino acids, anti-inflammatory agents, water, peptides, sugar alcohols , enzymes, plant extracts, antioxidants, talcum powder, clay, pollen, pearl powder, etc. Here, the humectant may, for example, be an acidic mucopolysaccharide such as polyethylene glycol, polypropylene glycol, glycerin, propylene glycol, sorbitol, hyaluronic acid or chondroitin sulfate, amino acid, collagen, elastin or the like. The lower alcohol may, for example, be ethanol, propanol, isopropanol or the like. The polyvalent alcohol may, for example, be glycerol, isovaerythritol, diisoamyltetraol, ethylene glycol, propylene glycol, polypropylene glycol, 1,3-butylene glycol or the like. The sugar may, for example, be an inducement of sugars such as glucose, maltose, lactose, D-glucuronic acid, sucrose, D-sorbitol, sorbitol anhydride, cellulose, starch, oligosaccharides, polysaccharides, and the like. body. The composition for external use on skin of the present invention can be appropriately formulated into a collagen-producing inhibitor containing a bird's nest enzyme decomposition product and any of the above-mentioned components by a known method to form a lotion, lotion, cream, moisturizer, face lotion, makeup Forms of conventional products such as water, facial cleanser, body lotion, body cream, etc. The skin external composition of the present invention can further provide hair cosmetic products such as foundations, lipsticks, eye shadows, and blushes, sunscreen products, deodorant cosmetics, and other cosmetic products such as shampoos, moisturizers, and styling products. , use of skin cleansers and bathing baths for bathing agents, etc. According to another aspect of the present invention, as described above, the use of an enzyme decomposition product of bird's nest can be provided for the purpose of producing collagen fibroblasts of mammals including humans, which are caused by ultraviolet light irradiation and inhibiting collagen production. Agent. Here, the dermal fibroblasts are caused by ultraviolet irradiation. 14 201105356 The inhibition of collagen production is preferably suppressed by the low-level type I collagen production in the skin dermal fibroblasts caused by ultraviolet irradiation. According to another aspect of the present invention, as described above, the use of an enzyme decomposition product of bird's nest can be provided for the purpose of producing a skin external composition for skin care before or after exposure to ultraviolet rays. Here, the external composition for skin is preferably used for the reduction of skin elasticity and gloss which occurs in the skin which is irradiated with ultraviolet rays, and the prevention or improvement of wrinkles. Further, the composition for external use of the skin is preferably a cosmetic. According to another aspect of the present invention, as described above, there can be provided a method comprising the step of using an effective amount of an enzyme decomposition product of the active ingredient of the present invention on the skin of a mammal including a human, thereby inhibiting the dermis The production of collagen due to ultraviolet irradiation of fibroblasts is low. Here, the "effective amount" means a sufficient amount of inhibitory activity which exerts a low level of collagen production in a predetermined field in the body by use. Further, it is preferable that the so-called dermal fibroblasts are inhibited by the low-level production of collagen due to ultraviolet irradiation, and it is preferable that the type I collagen produced in the dermal fibroblasts due to ultraviolet irradiation is suppressed. Further, according to still another aspect of the present invention, there is provided a skin care method comprising the step of using an effective amount of the active ingredient of the present invention on a skin before or after exposure to ultraviolet rays to a mammal including a human. Similarly, a method of using an effective amount of the active ingredient of the present invention for the skin of a mammal including a human, and improving the elasticity and gloss of the skin and preventing or improving wrinkles can be provided. In addition, the so-called "effective amount" refers to the sufficient amount required to achieve the desired effect. 15 201105356 In addition, the values of the terms "approximately" and "degree" are used in this manual to include the meaning of the changes that can be tolerated by the skilled person in the industry before the purpose of setting the value. By way of example, it is intended to allow a predetermined value or range of 20°/. Within the change of 10%, it is better, and within 5% is better. EXAMPLES The present invention will be specifically described with reference to the following examples, but the present invention is not limited by the following examples. (1) Preparation of bird's nest sample (1-1) Bird's nest enzyme decomposition product 1 The unwashed bird's nest was ground by a mill to obtain a bird's nest powder of 100 mesh size (particle size 150//m or less). Then, about 50 times (mass) of water was added to the bird's nest powder, and it was expanded at 5 ° C for 20 hours, and then heat-sterilized at 121 ° C for 15 minutes. After the obtained treatment liquid was cooled, the pH was adjusted, and 2% by mass of the protease-containing enzyme (trade name "Pancreatin F", manufactured by Amano Pharmaceutical Co., Ltd.) was added to the bird's nest powder, and the reaction was carried out at 45 ° C. hour. After the above enzyme reaction solution was adjusted to pH 7.0, the enzyme was inactivated by heating at 90 ° C for 5 minutes, and then filtered and recovered. After concentrating the filtrate, it is freeze-dried to obtain "bird's nest enzyme decomposition product 1" (1-2) The decomposition of the enzyme decomposition reaction of the bird's nest enzyme is changed to 24 hours, and the process is decomposed with the enzyme of the above (1-1). The substance 1 is the same, and the "bird's nest enzyme decomposition product 2" is obtained. (1-3) Hot water extract of bird's nest (Comparative example) 16 201105356 Grinding the commercially available unbleached swallow (dry matter) by a mill, and adding the grind to the l_ml money towel, and then The extraction treatment was carried out by heating under reflux for 2 hours. After heating and refluxing, the mixture was allowed to stand still, and then the supernatant liquid was taken for the transition: the liquid was recovered. The residue is further the same as described above, and the steamed towel which is added to the surface W is subjected to re-extraction treatment by heating (4), and then the transition solid matter is removed to check the force σ to be removed, and the filtrate obtained by the liquid is recovered (extract solution) The silk material obtained by concentrating under reduced pressure was dried by Kangkang to obtain a yellow-white "swallow's nest hot water extract" (Comparative Example). (2) Analysis of bird's nest samples The amount and amount of protein in each bird's nest sample (_enzyme fraction _ 丨, silk fibroin decomposition product 2 and Swallow written hot water extract) were determined as follows. The amount of citric acid was determined by high-speed liquid chromatography after the acidic hydrolysis of each sample to determine the free oxime-acetamidine. The amount of protein was determined using Bio Rad's Protein Assay Kit based on the Brad ford method. The results are shown in Figures 1 and 2. Next, in order to determine the molecular weight and distribution of the protein contained in the bird's nest enzyme decomposition product and the bird's nest enzyme decomposition product 2, GPC analysis was carried out under the following conditions. [HPLC measurement conditions]
.管柱：ShodexAsahipakGS320HQ( 0 7.6x300mm) •管柱溫度：35°C •移動相：50mMCH3COONH3pH6.7 17 201105356 .流速：0.4ml/min •試料注入量：1〇#1 •偵測器：紫外分光偵測器（UV22〇nm之測定） [使用之分子量標示器] • BSAMW66,000 •甘胺醯甘胺酸(Gly-gly)MW132.1 •三胜肽（Gly-gly-gly)MW189.17 .L-天冬胺酸(L-Asparaginic acid)MW133.1 .DL-天冬胺酸(DL-Asparaginic acid)MW150.1 .N-乙醯神經氨酸(N-Acetylneuraminic acid)MW309.3 測定結果則如第3及第4圖所示。 進而，已就燕寓酵素分解物所含之蛋白質之分子量， 使用西方墨點法而加以評價。具體而言，係先依循前述之 測定法而確認燕窩酵素分解物中之蛋白質之量。就燕寓酵 素分解物來源之蛋白質（100 " g)使用SDS-PAGE而藉 10〜15°/〇之聚丙醯胺加以分離。將已分散於膠體中之蛋白質 轉印至lmmunoblot PVDF膜（聚偏二氟乙烯（p〇iyviny丨idene difluoride) ’取自Bio-Lad Lab公司）。再於包含3%之脫脂乳 與0.1%之Tween-20之PBS中對PVDF膜進行阻隔1小時，再 加以進行視覺上之判定。 另’評價蛋白質之分子量時，亦已參考公知文獻(Guo, C. T., Suzuki Y., et al., Antiviral Res. 70, pp.140-146(2006)) 所揭露之資料。 由上述結果可知，燕窩酵素分解物中所含之蛋白質分 18 201105356 子里存在約70〜120,〇〇〇程度（另，視評價條件之不同，上限 值可達20〇,〇〇〇程度）之範圍内。 進而，由所得結果可知，燕窩酵素分解物丨之蛋白質之 平均刀子里約為55，GGG °又’可知燕窩酵素分解物2之蛋白 負之平均分子量約為33,000。 (3)1外線照射所導致真皮纖維母細胞之膠原蛋自之產生低 落之抑制效果 已使用含有0.5%幼牛血清(FBS)之DMEM培養基（取自 SIGMA)於96孔板上按2X1 〇4ceus之細胞密度種植正常人類 皮膚真皮纖維母細胞(KF_4l〇9，取自KURABO公司）。種植 24小時後，更換為内含含有預定濃度之各燕窩樣本之 〇.5%FBS之DMEM培養基，再進行培養24小時。 又’樣本濃度如下所示： (0酵素分解物之濃度(〇、78.1、312.5、1250、5000"g/ml) (ii)熱水萃取物之濃度(0、1.56、6·25、25、i〇0/CZg/ml) 培養後，回收培養基上澄液之一部分，並藉ELISA法 測定上澄液中之I型膠原蛋白量。 由測定結果可知’燕窩之酵素分解物可積極使人類皮 膚真皮纖維母細胞之I型膠原蛋白增生。其與比較例之熱水 萃取物相較，差異甚為顯著。 培養後’進而，於含有預定濃度之各燕窩樣本之Hank，s 緩衝液中，在照射UVB(中波紫外線)後(使用紫外線照射器 (PHILIPS公司出品之TL20W/12RS)，UV短波長截止濾波器 (截通波長300nm))，更換成含〇.5%FBS之DMEM培養基，再 201105356 進行培養24小時。 培養後，回收培養基上澄液，並藉ELISA法測定上澄 液中之I型膠原蛋白量。 以0.1%之Triton X-100溶液溶解細胞後，就蛋白質量進 行了定量。 ELISA 法係使用 Anti-Human Collagen Type 1 抗體 (Rabit)作為一次抗體，並使用Histofine PO(Rabit)作為二次 抗體’進而發色劑則使用2,2^連氮基-雙-(3-乙基苯並二氫噻 唑啉-6-項酸)(ABTS)，而藉微量盤判讀儀測定了 4〇5nm之吸 光度。 蛋白質量之定量則藉BCA法（二辛可寧酸法使已與胜 肽鍵反應之銅離子與BCA(二辛可寧酸）反應，而測定波長 562nm之吸收之方法）而進行。 培養基中之I型膠原蛋白量已藉培養基中膠原蛋 白量除以全部細胞之蛋白質量而算出各單位蛋白質量之工 型膠原蛋白產生量。 結果則如第3圖所示。 由結果已輕’燕窩之料分解物可_抑制謂昭 射所導致真皮纖維母細胞之!型膠原蛋白產生低落。又，熱 水萃取物（比較例）則未出現此種抑制效果。 … I：圖式簡單說明】 第1圖係顯示燕窩樣本中之誕酸量之測定結果之圖表。 第2圖係顯示燕窩樣本中之蛋白質量之测定結果之圖 20 201105356 第3圖係顯示採GPC-HPLC之燕窩酵素分解物1之測定 結果之圖表。 第4圖係顯示採GPC-HPLC之燕窩酵素分解物2之測定 結果之圖表。 第5圖係顯示膠原蛋白之產生促進能力之測定結果之 圖表。 【主要元件符號說明】 (無） 21Column: ShodexAsahipakGS320HQ (0 7.6x300mm) • Column temperature: 35°C • Mobile phase: 50mMCH3COONH3pH6.7 17 201105356 . Flow rate: 0.4ml/min • Sample injection amount: 1〇#1 • Detector: UV spectrometry Detector (measurement of UV22〇nm) [molecular weight marker used] • BSAMW66,000 • Gly-gly MW132.1 • Gly-gly-gly MW189.17 .L-Asparaginic acid MW133.1 .DL-Asparaginic acid MW150.1 .N-Acetylneuraminic acid MW309.3 Determination The results are shown in Figures 3 and 4. Further, the molecular weight of the protein contained in the enzyme decomposition product of Yanxiang has been evaluated using the Western blot method. Specifically, the amount of protein in the decomposition of the bird's nest enzyme is confirmed by the above-described measurement method. The protein (100 " g) derived from the enzyme decomposition product of Yanxiang was separated by 10 to 15 °/〇 polyacrylamide using SDS-PAGE. The protein dispersed in the colloid was transferred to a lmmunoblot PVDF membrane (polyvinylidene fluoride (p〇iyviny丨idene difluoride) was taken from Bio-Lad Lab). The PVDF membrane was blocked for 1 hour in PBS containing 3% skim milk and 0.1% Tween-20, and visually judged. Further, when evaluating the molecular weight of a protein, reference has been made to the information disclosed in the well-known literature (Guo, C. T., Suzuki Y., et al., Antiviral Res. 70, pp. 140-146 (2006)). From the above results, it can be seen that the protein contained in the bird's nest enzyme decomposition product has a degree of about 70 to 120 in the 201105356, and the degree of sputum is different (in addition, depending on the evaluation conditions, the upper limit can reach 20 〇, the degree of 〇〇〇 Within the scope of). Further, from the results obtained, it is found that the average knives of the protein of the bird's nest enzyme decomposition product are about 55, and the GGG ° 'is known that the protein of the bird's nest enzyme decomposition product 2 has an average molecular weight of about 33,000. (3) The inhibitory effect of collagen injection on dermal fibroblasts caused by external irradiation has been reduced to 2X1 〇 4ceus in 96-well plates using DMEM medium (taken from SIGMA) containing 0.5% young bovine serum (FBS). The cell density was implanted in normal human skin dermal fibroblasts (KF_4l〇9, taken from KURABO). After 24 hours of planting, the cells were replaced with DMEM medium containing 5% 5% FBS containing a predetermined concentration of each bird's nest sample, and cultured for another 24 hours. 'The sample concentration is as follows: (0 concentration of enzyme decomposition product (〇, 78.1, 312.5, 1250, 5000 " g / ml) (ii) concentration of hot water extract (0, 1.56, 6 · 25, 25, I〇0/CZg/ml) After culture, one part of the liquid on the culture medium was recovered, and the amount of type I collagen in the supernatant liquid was determined by ELISA. From the measurement results, it was found that the enzyme decomposition product of bird's nest can actively make human skin. The type I collagen of dermal fibroblasts is proliferated. Compared with the hot water extract of the comparative example, the difference is very significant. After the cultivation, 'in the Hank, s buffer containing the predetermined concentration of each bird's nest sample, After UVB (medium-wave ultraviolet ray) was irradiated (using UV illuminator (TL20W/12RS from PHILIPS), UV short-wavelength cut-off filter (cut-off wavelength 300 nm), and replaced with DMEM medium containing 5% FBS. 201105356 was cultured for 24 hours. After the culture, the medium was recovered and the amount of type I collagen in the supernatant was determined by ELISA. After dissolving the cells in 0.1% Triton X-100 solution, the amount of protein was quantified. ELISA uses Anti-Human Collagen Typ The e 1 antibody (Rabit) is used as a primary antibody, and Histofine PO (Rabit) is used as a secondary antibody. Further, a coloring agent is used, and 2,2^azino-bis-(3-ethylbenzodihydrothiazoline- 6-item acid (ABTS), and the absorbance of 4〇5nm was determined by a microplate reader. The protein quality was quantified by the BCA method (the di-cinchonine method to make the copper ion and BCA which have reacted with the peptide bond). (dioctocinic acid) reaction, and measuring the absorption at a wavelength of 562 nm). The amount of type I collagen in the medium has been calculated by dividing the amount of collagen in the medium by the amount of protein in all cells to calculate the amount of protein per unit. The amount of collagen produced is as shown in Fig. 3. The result is lighter, 'the decomposition of the bird's nest material can be suppressed, and the dermal fibroblasts are caused by the low-grade collagen. The extract (comparative example) did not show such an inhibitory effect. ... I: A brief description of the pattern] Fig. 1 is a graph showing the measurement results of the amount of acid in the bird's nest sample. Fig. 2 shows the protein in the bird's nest sample. Figure 20 of the measurement results of the quantity 2011-05356 A graph showing the measurement results of the bird's nest enzyme decomposition product 1 by GPC-HPLC. Fig. 4 is a graph showing the measurement results of the bird's nest enzyme decomposition product 2 by GPC-HPLC. Fig. 5 is a graph showing the production promoting ability of collagen. Chart of measurement results [Explanation of main component symbols] (None) 21