200908989 九、發明說明： 【發明所屬之技術領域】 本發明是有關於一種多醣體組成物，且特別是有關於一 種褐藻多醣體組成物。 【先前技術】 皮膚的老化除了年齡增加的自然老化外，亦起因於外在 環境因素：生活飲食習慣、藥物以及光線，其中又以陽光為 皮膚老化的主要因素。 目前的研究顯示，可以活化基質金屬蛋白酶（MatHxBACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a polysaccharide composition, and more particularly to a brown algae polysaccharide composition. [Prior Art] In addition to the increased ageing of the skin, aging of the skin is also caused by external environmental factors: living habits, drugs, and light, and sunlight is the main cause of skin aging. Current research shows that matrix metalloproteinases can be activated (MatHx
Metall〇proteinases; MMPs) ’並且使皮膚真皮層中的膠原蛋白 產生降解’導致老化現象。 根據已知的文獻記載，多醣體（P〇lysacchaddes)具有抗氧 化' 抗發炎、增進皮膚免疫力、促進纖維母細胞增生以及保 濕效果。而由所產生的含有岩藻糖（Fuc〇se)的募糖 (Oligosaccharides)或多醣體，具有抑制基質金屬蛋白酶 MMP-2和MMP-9的活性，並促進纖維母細胞與真皮層大分 子蛋白質增生等防止皮膚老化的功效。 至於將多聽體應用於化妝品方面的研究，目前僅有採用 由裂褶菌<Sc/h’z叩comm⑽e發酵所產生的多酶體β葡 聚糖（P-U-glucan)來製作化妝品，其主要的功效在於促進纖 維母細胞增生與膠原蛋白合成，可降低皮膚曬傷所引起的紅 斑’並且具有優良的保濕與抗發炎效果。 因此本發明提供一種富含岩藻糖的褐藻多醣體組成 200908989 物’其抑制基質金屬蛋白酶活化的能力，並應用於抗老化化 妝品。 【發明内容】 本發明的目的在提供—種抗老化褐蒸多醣體组成物，其 係t木用0.1N氣化氫水溶液的萃取製程，再以重量百分濃度實 質介於別至75的酒精溶液沉殺萃取褐藻多醣體所形成，其 中此褐51體抗老化組成物的分子量實質介於2,咖，〇⑼Metall〇 proteinases; MMPs) and [degradation of collagen in the dermis layer of the skin] cause aging. According to the known literature, the polysaccharide (P〇lysacchaddes) has anti-oxidation, anti-inflammatory, skin immunity, fibroblast proliferation and moisturizing effect. The resulting fucose-containing oligosaccharides or polysaccharides have the activity of inhibiting matrix metalloproteinases MMP-2 and MMP-9 and promoting macromolecular proteins in fibroblasts and dermis. Proliferation and other effects to prevent skin aging. As for the application of multi-audio to cosmetics, only the multi-enzyme beta-glucan (PU-glucan) produced by the fermentation of Schizophyllum sp. < Sc/h'z叩comm(10)e is used to make cosmetics. The main effect is to promote fibroblast proliferation and collagen synthesis, reduce erythema caused by sunburn and have excellent moisturizing and anti-inflammatory effects. Accordingly, the present invention provides a fucose-rich fucoidan composition 200908989 which inhibits the activation of matrix metalloproteinases and is applied to anti-aging cosmetics. SUMMARY OF THE INVENTION The object of the present invention is to provide an anti-aging brown steamed polysaccharide composition, which is an extraction process of t-wood with 0.1N hydrogenated hydrogen aqueous solution, and then is substantially equal to 75 alcohol in weight percent concentration. The solution kills and extracts the brown algae polysaccharide, wherein the brown 51 anti-aging composition has a molecular weight of substantially 2, coffee, 〇 (9)
Da 至 40,〇〇〇,〇〇〇 Da 之間。 可抑制基質金屬蛋白轉活性的褐藻多醣體組成物，其係 採用0.1N氯化氫水溶液的萃取製程，再以重量百分濃度實質 介於50至75的酒精溶液沉殺萃取褐藻多畴體所形成二其中 褐蕩多醣體抗老化組成物的分子量實質介於2,8〇〇,〇〇〇以 至 40,0〇〇,〇〇〇 Da 之間。 本發明的又-目的在提供—種化妝品组成物，包括有一 種褐藻多醣體組成物’此一褐❹醣體組成物係採用〇·1Ν 氯化風水溶液的萃取製程，再以重量百分濃度實質介於咒 至75的酒精溶液㈣萃取褐藻多釀體所形成，其中此 多醣體抗老化組成物的分子量實質介於至 40,000,000 Da 之間。 ，叫 Da 至 根據以上所述之實施例，本發明的技術特、 氯化氯水溶液的萃取製程，再以重量百分濃度實質介用：1: 至75的酒精溶液沉澱萃取褐藻多醣體，以得 、 膽體組成物’其中此-具褐藻多醣體組成物的分子量= 200908989 於2,800,000 Da至40,〇〇〇,〇〇〇 Da之間，可抑制基質金屬蛋 白酶的活化，進而延緩膠原蛋白的降解，達到皮膚抗老化的 功能。可添加於化妝品之中作為抗皮膚老化的成分。 由於上述實施例所提供的褐藻多醣體組成物具有製程 簡單，可兼顧抗老化效果與成本優勢的優點，因此可以解決 習知技術投入成本較大’萃取程序較為繁雜的缺點。 【實施方式】 本發明係提供一種製程簡單，可兼顧抗老化效果與成本 優勢之含有褐藻多醣體的褐藻多醣體組成物。 有鑑於台灣北部、東北部、恆春半島及澎湖等地盛產褐 藻，而褐藻富含褐藻酸（Alginate)、褐藻澱粉（Laminadn卜甘 露醇（Mannitol)、岩藻多醣體（Fuc〇idan)及精油。經檢測，褐 藻中的岩藻多醣體（Fucoidan)是一種硫酸多醣體，主要包括 硫酯化的L-岩藻糖，以及微量的半乳糖（Galact〇se)、果糖 (Fructose)、木糖（Xyi〇se)和葡萄糖醛酸。因此可藉由直接對 褐藻進行純化，以得到具有抑制基質金屬蛋白酶分泌，以抵 抗皮膚老化的褐藻多醣體組成物。並可將此褐藻多醣體組成 物添加於化妝品之中作為化妝品抗老化的有效成分。 為了讓本發明的目的、特徵、優點與實施例能更明顯易 懂，特舉出一種馬尾藻科（Sargassaceae)的重緣葉馬尾蕩 (Sargassum duplicatum)作為較佳實施例來進行純化，並藉 由-系列定量或定性的試驗，來實證並說明本發明的技術特 徵，任何在不脫離本發日月之精神和範_所進行的材料與褐 200908989 藻種類之代換，皆包含與本發明的專利申請範圍 本發明的較佳實施例係採用0.1N氯化氫水二:萃 5〇-75%酒精沉㈣製程，來萃取重緣葉馬尾藻中的褐 體組成物，並進-步證實本案所提供之褐藻多_植成物， 確實具有可抑制基質金屬蛋白酶的功能。 刀此碣藻多醣體組成物 的卒取方法以及體外試驗的相關材料與分析方法詳述如 萃取方法 'Da to 40, 〇〇〇, 〇〇〇 Da. A fucoidan composition capable of inhibiting the transcription of matrix metalloproteins by using an extraction process of 0.1 N aqueous hydrogen chloride solution, and then extracting a multi-domain of brown algae by weighting the alcohol solution in a concentration of 50 to 75 The molecular weight of the anti-aging composition of the browning polysaccharide is substantially between 2, 8 〇〇, 〇〇〇 to 40, 0 〇〇, 〇〇〇 Da. A further object of the present invention is to provide a cosmetic composition comprising a brown algae polysaccharide composition, wherein the brown sugar composition is an extraction process using a chlorinated aqueous solution of chlorinated air, and then in a concentration by weight. The alcohol solution of the curse to 75 (4) is formed by extracting the brown algae multi-breast, wherein the molecular weight of the anti-aging composition of the polysaccharide is substantially between 40,000,000 Da. According to the above-mentioned embodiments, the extraction process of the technical special chlorine chloride aqueous solution of the present invention is further applied in a weight percent concentration: 1:75 to 75 alcohol solution is precipitated to extract the brown algae polysaccharide, The molecular weight of the composition of the biliary body, which has a brown algae polysaccharide composition = 200908989, between 2,800,000 Da and 40, between 〇〇〇 and 〇〇〇D, inhibits the activation of matrix metalloproteinases, thereby delaying collagen Degradation, to achieve the anti-aging function of the skin. It can be added to cosmetics as an ingredient against skin aging. Since the fucoidan polysaccharide composition provided by the above embodiment has a simple process and can take advantage of both the anti-aging effect and the cost advantage, it can solve the disadvantage that the conventional technology has a large input cost and the extraction process is complicated. [Embodiment] The present invention provides a fucoidan composition containing a brown algae polysaccharide which is simple in process and can take into consideration both anti-aging effects and cost advantages. In view of the fact that northern China, northeastern part, Hengchun Peninsula and Wuhu are rich in brown algae, brown algae are rich in alginate, brown algae (Mannitol), fucoidan (Fuc〇idan) and essential oils. Fucoidan in brown algae is a sulfated polysaccharide, mainly including thioesterified L-fucose, and trace amounts of galactose, fructose, and xylose. (Xyi〇se) and glucuronic acid. Therefore, the brown algae can be directly purified to obtain a fucoidan composition having inhibition of matrix metalloproteinase secretion to resist skin aging, and the fucoidan composition can be added. In the cosmetics, as an active ingredient for anti-aging of cosmetics. In order to make the objects, features, advantages and embodiments of the present invention more apparent, the Sargassum duplicatum of the Sargassaceae family (Sargassum duplicatum) is specifically mentioned. Purification is carried out as a preferred embodiment, and the technical features of the present invention are demonstrated and illustrated by a series of quantitative or qualitative tests, without departing from the present invention. The material of the sun and the moon and the material and the substitution of the brown variety 200908989 are included in the preferred embodiment of the invention. The preferred embodiment of the invention uses 0.1N hydrogen chloride water: extract 5〇-75% alcohol The Shen (4) process was used to extract the brown body composition in the sphaerotheca fuliginea, and further confirmed that the brown algae _ plant formed in the present case did have the function of inhibiting the matrix metalloproteinase. The method of stroke and the related materials and analytical methods of the in vitro test, such as the extraction method
。首先將重緣葉馬尾藻以自來水洗淨，置於烘箱以邮至 5〇t的溫度加以乾燥後’㈣碎機打成粉末。接著將乾燥粉 碎後的㈣進行粗萃取製程，以！比15的重量比率與〇 的氣化氫（HC1)混合，以每分鐘155轉的轉速進行授摔^個 小時。再以兩層紗布過濾，取過濾液以每分鐘8〇〇〇轉4艽的 環境中離心20分鐘，分離出粗萃取液（上清液）。並且將粗萃 取液保存於-20°C的環境之中。 ⑴接著，將粗萃取液與濃度（重量百分濃度以下以％表 示之)〜99.5%的酒精以1:3的重量比例進行混合，形成最終 漠度實質為75%的溶液，並在_2〇t的環境下靜置隔夜（8至 12小時）’再以每分鐘8〇〇〇轉，在4t的環境中離心分鐘。 在倒除離讀的上清液可得到第—褐藻多㈣組成物，並以 去離子水回溶沉澱物，以形成第-褐藻多II體組成物溶液。 (2)將第一褐藻多醣體組成物溶液與濃度99.5%的酒精 。以1比1的重量比例混合，使酒精溶液的最終濃度實質為5〇 %。之後在-20。(：的環境下靜置隔夜（8至12小時），再將酒精 溶液以每分鐘8000轉的轉速，在4t的環境中離心2〇分鐘。 200908989 在倒除上清液之後得到第二褐蕩多醣體組成物，再以去離子 水回溶沉澱物，以形成第二褐藻多醣體組成物溶液。 (3)再將所倒除的上清液與濃度99·5%的酒精以1比1的 重量比例混合，使酒精溶液的最終濃度實質為5〇%至75% 。 之後在-2(TC的環境下靜置隔夜（8至12小時），再將酒精溶液 以每分鐘8000轉的轉速，在4°c的環境中離心2〇分鐘。在 倒除上清液之後以得到第三褐藻多醣體組成物，再以去離子 水回溶沉澱物，以形成第三褐藻多醣體組成物溶液。 揭藻多醣體的澧度分折： 採用硫酸本紛法（Phenol-Sulfuric Acid Assay)，針對上述 萃取方法所得到的粗萃取液、第一褐藻多醣體組成物溶液、 第二褐藻多醣體組成物溶液和第三褐藻多醣體組成物溶液 進行分析。 在本發明的實施例之中，經由硫酸笨酚法分析，1公克 的重緣葉馬尾藻粉末所調製成的粗萃取液，約可萃取出29.86 mg的總糖（Total Sugar)。以最終濃度範圍實質為75%的酒精 沉殺萃取分離所得的褐藻多醣體組成物，具有17.32 mg的有 效褐藻多醣體，約佔總醣的58.0%。以最終濃度範圍實質為 50%的酒精溶液沉澱萃取分離所得的第二褐藻多醣體組成 物’可分離出12.19 mg的褐藻多醣體，約佔總糖的40.8%， 約佔有效褐藻多醣的70.4%。以最終濃度範圍實質為5〇%至 75%的酒精溶液沉澱萃取分離所得的第三褐藻多聽體組成 物’可分離出5.13 mg的褐藻多醣體，約佔總糖的17.2% ; 200908989 約佔有效褐藻多醣的29.6%。 接著再對第一褐藻多醣體組成物溶液及第三褐藻多_ 體組成物溶液進行褐藻多醣體的分子量分析測定及單醋< 份分析。 褐蕩多醣體分子量的分佈測定 取第一褐藻多醣體組成物溶液和第三褐藻多醣體組& 物溶液，用0.45μπι過濾膜過濾至樣品瓶後，使用膠體過濟、 層析（Gel Filtration Chromatography, GFC)做分析。在本發明 的實施例中，係使用TSKgel GMPWXL column管枝對褐蕩多 醣體組成物溶液進行膠體過濾層析。利用分子篩層析以蒸發 光散射 4貞測器（Evaporative Light Scattering Detectors, ELSD) 與分子量標準品 Polyethylene oxide Aqueous Calibration Kit (Mw = 5,900 ' 11,800 ' 22,800 ' 47,300 ' 112,000 ' 212,000 ' 404,000、788,000 Da)比對，以求出樣品的分子量。請參照表 1和第1A圖。表1為所使用的多醣體標準品之分子量、滯 留時間及標準品分子量的log值，其分子量的範圍是由5, 900至788,000Da。第1A圖則是以多醣體標準品之滯留時間 (Retention Time)及標準品分子量的log值所作之標準曲線。 表1 : 滯留時間（分鐘） 標準品分子量的log值 多醣體標準品之分子量 29.48 3.770852012 5900 28.83 4.071882007 11800 200908989 28.16 4.357934847 22800 27.19 4.674861141 47300 25.91 5.049218023 112000 25.03 5.326335861 212000 23.79 5.606381365 404000 22.84 5.896526217 788000 褐藻多醣體分子量的分佈測定的結果請參照第1B圖。 第1B圖係繪示褐藻多醣體組成物溶液所含之褐藻多醣體的 分子量分佈圖。 由第1B圖可得知褐藻多醣體組成物溶液所含之褐藻多 醣體的分子量約40,000,000 Da，遠大於標準品分子量最大值 的788,000 Da，褐藻多醣體分子量是相當龐大。 再根據第1C圖，其中褐藻多醣體的波峰滯留時間有延 後的現象，顯示第三褐藻多醣體組成物溶液所含之褐藻多醣 體的分子量較小，換算分子量約2,800,000 Da。 椹荡$醣體單醣成份分妍 根據 Chen, S. C·, Lu, Μ. K.，Cheng, J· J·，Wang, D. L.等 人於2005年發表於期刊『FEMS Microbiology Letters.』第 249 期 247-254 頁：「Antiangiogenic activities of polysaccharides isolated from medicinal fungi.」以及 Cheng, J. J., Huang, N. K., Chang, T. T., Wang, D. L., Lu, Μ. K.等人 200908989 於2005年發表於期刊『Life Sciences』第76期3029-3042 頁：「Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial cells」戶斤提 供的方法進行分析。 請參照第2A圖，第2A圖係繪示使用HPLC分析褐藻多 醣體組成物溶液的多醣體的單醣組成份分析圖。 由2A圖可發現褐藻多醣體組成物溶液只有出現兩個較 明顯的波峰，顯示褐藻多醣體組成物溶液中岩藻糖與半乳糖 含量較多，而其他糖類則屬少數。 經換算得知第三褐藻多醣體組成物溶液的岩藻糖與半 乳糖組成百分比分別為39.50%、36.21%，而第一褐藻多醣體 組成物溶液的岩藻糖與半乳糖組成百分比分別為17.89%、 19.77%(請參見表2)。 表2 單醣組成份（重 量百分比％ ) 第一褐藻多醣體組成物 溶液 第三褐藻多醣體組 成物溶液 岩藻糖 17.89% 39.5% 半乳糖 19.77% 36.21% 由表2的實驗資料，第三褐藻多醣體組成物溶液中含有 較多的岩藻糖與半乳糖。 再經由體外動物細胞毒性評估，採用MTT Assay分析 12 200908989 來決定出褐藻多醣體對動物細胞的較佳濃度添加量’而不至 於傷害動物細胞，導致後續的基質金屬蛋白酶（MMPs)活性分 析產生誤差。 體外動物細胞喜性評估 (1)細胞培養 採用購自食品工業研究所生物資源保存及研究中心’編 號 BCRC60071，美國菌種中心（American Type Culture Collection; ATTC)，編號CCL-92的3T3老鼠胚胎纖維母細胞。 將3T3老鼠胚胎纖維母細胞培養於25T培養瓶中，使用 内含有 3ml DMEM (DULBECCO’S MODIFIED MEM)、重量 百分濃度10FBS、重量百分濃度1NEA以及重量百分濃度1 抗生素的培養基中，並放置於37°C二氧化碳濃度5%的環境 中衡溫培養三天。之後移除瓶中培養基，並加入lml磷酸緩 衝液（Phosphate Buffer Saline; PBS)進行清洗，再移除填酸緩 衝液並加入lml的胰蛋白酶（Trypsin)，搖晃瓶身使一面均勻 浸泡，將培養瓶靜置1置5分鐘。當3T3老鼠胚胎纖維母細 胞漂浮於液面時，以微量低管沖洗的方式使細胞分散。再加 入2ml DMEM培養基，待混合均勻之後再取出2ml的細胞 液，並加入2ml DMEM培養基培養三天之後再進行繼代培養。 (2) MTT assay: MTT[3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazo liumbromide ] assay是一種常用於檢測細胞生存率的方法。 首先以濃度範圍分別為 0.1mg/ml、0.2mg/ml、0.3 13 200908989 mg/ml、0.4 mg/ml、0.5mg/ml、0.75mg/ml 和 1 mg/ml 的第一 褐藻多醣體組成物溶液和第三褐藻多醣體組成物溶液來處 理3T3老鼠胚胎纖維母細胞，並分別培養24、48、72小時 後，進行MTT細胞毒性測試分析，實驗結果顯示，經過以 下之第一褐藻多醣體組成物溶液和第三褐藻多醣體組成物 溶液處理後的3T3老鼠胚胎纖維母細胞，在培養24小時以 後，均略有刺激細胞增生的現象，且在培養48小時之後細 胞的存活率仍對照組相似。另外劑量範圍0.4 mg/ml的實驗 組中，培養72小時後的細胞的存活率仍有60%。因此第一 褐藻多醣體組成物和第三褐藻多醣體組成物溶液的安全有 效劑量實質介於0.1 mg/ml至0.5 mg/ml之間。 接著，進行基質金屬蛋白酶（MMPs)的活性分析，以證實 本發明的較佳實施力所提供的褐藻多醣體組成物具有抑制 基質金屬蛋白酶（MMPs)的活性，並抑制細胞分泌基質金屬蛋 白酶MMP-2、MMP-9的功效。有關基質金屬蛋白酶（MMPs) 活性分析的詳細操作步驟，請參考Riley SC,Thomassen R,Bae SE,等人發表於 Anim Reprod Sci,2004,81:329-339 中的 Matrix metalloproteinase-2 and -9 secretion by the equine ovary during follicular growth and prior to ovulation ° 基皙金屬蛋白醢（MMPs)的活性分折 基質金屬蛋白酶MMP-2、MMP-9活性抑制分析之樣品 處理： (1)將3T3老鼠胚胎纖維母細胞培養於含DMEM(内含 14 200908989 10% FBS)的培養瓶中’培養三天，並 A質今屬細胞培養液(内含 基質金屬蛋㈣的活性），分別取2 ml加到二個培養皿中， :中；個：入劑量實質為。.一的第一褐藻多黯體組成 物浴液，另-個加入同體積的無菌水作為對照組。在 句後放置培養箱（37 t：，5%⑽)培養，並依不同的㈣點 (〇、2、4'7、12、24、48、72小時）分別吸取相同體積的細 胞培養液缝量離4，並置於_2() t冷Μ存. First, the S. cerevisiae was washed with tap water, placed in an oven and dried by post to a temperature of 5 〇t, and then the (4) crusher was powdered. Then, the dry pulverized (four) is subjected to a crude extraction process to! The weight ratio of 15 is mixed with hydrazine vaporized hydrogen (HC1), and it is thrown at 155 rpm for one hour. The mixture was filtered through two layers of gauze, and the filtrate was centrifuged for 20 minutes at 8 rpm for 4 minutes to separate the crude extract (supernatant). The crude extract was stored in an environment at -20 °C. (1) Next, the crude extract is mixed with the concentration (expressed as % by weight or less) to 99.5% of the alcohol in a weight ratio of 1:3 to form a solution having a final resolution of substantially 75%, and at _2 Allow to stand overnight (8 to 12 hours) in a 〇t environment and then centrifuge at 8 rpm for 8 minutes in a 4t environment. The supernatant of the first-brown algae (four) is obtained by decanting the supernatant, and the precipitate is re-dissolved in deionized water to form a solution of the first-halved algal complex composition. (2) The first fucoidan composition solution and the alcohol having a concentration of 99.5%. The mixture was mixed at a weight ratio of 1 to 1, so that the final concentration of the alcohol solution was substantially 5 %. After that at -20. (In the environment of :: overnight (8 to 12 hours), then centrifuge the alcohol solution at 8000 rpm for 2 〇 minutes in a 4 t environment. 200908989 After the supernatant is removed, the second browning is obtained. The polysaccharide composition is re-dissolved in deionized water to form a second fucoidan composition solution. (3) The supernatant after decanting is further 1:1 with the concentration of 99.5% alcohol. The weight ratio is mixed so that the final concentration of the alcohol solution is substantially 5〇% to 75%. After that, it is left overnight (8 to 12 hours) in the environment of -2, and then the alcohol solution is 8000 rpm. Centrifuge in a 4 ° C environment for 2 minutes. After the supernatant is removed to obtain a third fucoidan composition, the precipitate is re-dissolved in deionized water to form a third fucoidan composition solution. The twisting degree of the polysaccharides of the algae-removing polysaccharide: using the Phenol-Sulfuric Acid Assay, the crude extract obtained by the above extraction method, the first fucoidan composition solution, and the second fucoidan composition Solution and third fucoidan composition solution In the embodiment of the present invention, a crude extract prepared by 1 gram of S. cerevisiae powder can be extracted by extracting 29.86 mg of total sugar (Total Sugar). The final concentration range is 75% of the alcohol-killing extract of the fucoidan composition, with 17.32 mg of effective fucoidan, accounting for 58.0% of the total sugar. Precipitated with an alcohol solution with a final concentration range of 50%. The second fucoidan composition obtained by extraction and separation can separate 12.19 mg of fucoidan polysaccharide, which accounts for 40.8% of the total sugar, accounting for 70.4% of the effective fucoidan. The final concentration range is substantially 5〇% to 75. The third brown algae multi-audio composition obtained by the precipitation and extraction of the alcohol solution can separate 5.13 mg of fucoidan, accounting for 17.2% of the total sugar; 200908989 accounts for 29.6% of the effective fucoidan. A brown algae polysaccharide composition solution and a third brown algae multi-body composition solution were used for molecular weight analysis of the brown algae polysaccharide and a single vinegar < part analysis. The distribution of the molecular weight distribution of the brown polysaccharide was determined by the first brown algae. The saccharide composition solution and the third fucoidan group & solution were filtered through a 0.45 μm filter membrane to a vial, and analyzed by gel permeation and chromatography (Gel Filtration Chromatography, GFC). In the example, the TSHgel GMPWXL column tube is used for colloidal filtration chromatography of the brownish polysaccharide composition solution. The molecular sieve is used to evaporate the light scattering electron detector (ELSD) and the molecular weight standard Polyethylene oxide. Aqueous Calibration Kit (Mw = 5,900 ' 11,800 ' 22,800 ' 47,300 ' 112,000 ' 212,000 ' 404,000, 788,000 Da) was compared to determine the molecular weight of the sample. Please refer to Table 1 and Figure 1A. Table 1 is a log of the molecular weight, residence time, and standard molecular weight of the polysaccharide standard used, and the molecular weight ranges from 5,900 to 788,000 Da. Figure 1A is a standard curve based on the retention time of the polysaccharide standard and the log value of the molecular weight of the standard. Table 1: Retention time (minutes) The logarithm of the molecular weight of the standard product The molecular weight of the polysaccharide standard 29.48 3.770852012 5900 28.83 4.071882007 11800 200908989 28.16 4.357934847 22800 27.19 4.674861141 47300 25.91 5.049218023 112000 25.03 5.326335861 212000 23.79 5.606381365 404000 22.84 5.896526217 788000 Fucoidan molecular weight Please refer to Figure 1B for the results of the distribution measurement. Fig. 1B is a graph showing the molecular weight distribution of the brown algae polysaccharide contained in the solution of the brown algae polysaccharide composition. It can be seen from Fig. 1B that the molecular weight of the brown algae polysaccharide contained in the solution of the brown algae polysaccharide composition is about 40,000,000 Da, which is much larger than the maximum molecular weight of 788,000 Da of the standard product, and the molecular weight of the brown algae polysaccharide is quite large. Further, according to Fig. 1C, the peak residence time of the fucoidan polysaccharide was delayed, and it was revealed that the brown algae polysaccharide contained in the third fucoidan polysaccharide composition solution had a small molecular weight and a molecular weight of about 2,800,000 Da. According to Chen, S. C., Lu, Μ. K., Cheng, J. J., Wang, DL et al., published in the journal "FEMS Microbiology Letters." 249, pp. 247-254, "Antiangiogenic activities of polysaccharides isolated from medicinal fungi." and Cheng, JJ, Huang, NK, Chang, TT, Wang, DL, Lu, Μ. K. et al. 200908989, published in the journal in 2005. "Life Sciences" 76th 3029-3042: "Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial cells". Referring to Fig. 2A, Fig. 2A is a graph showing the analysis of the monosaccharide composition of the polysaccharide of the solution of the brown algae polysaccharide composition by HPLC. It can be seen from Fig. 2A that only two distinct peaks appear in the solution of the brown algae polysaccharide composition, indicating that the fucoidan and galactose content in the solution of the brown algae polysaccharide composition are more, while other sugars are a minority. The percentages of fucose and galactose of the solution of the third fucoidan polysaccharide composition were 39.50% and 36.21%, respectively, and the percentage of fucose and galactose of the first fucoidan composition solution was 17.89, respectively. %, 19.77% (see Table 2). Table 2 Monosaccharide component (% by weight) First brown algae polysaccharide composition solution Third brown algae polysaccharide composition solution fucose 17.89% 39.5% Galactose 19.77% 36.21% From the experimental data of Table 2, the third brown algae The polysaccharide composition solution contains more fucose and galactose. Based on the in vitro animal cytotoxicity assessment, MTT Assay analysis 12 200908989 was used to determine the optimal concentration of fucoidan polysaccharides on animal cells, without harming animal cells, leading to errors in subsequent matrix metalloproteinase (MMPs) activity analysis. . In vitro animal cell benign evaluation (1) Cell culture was performed using 3T3 mouse embryonic fiber purchased from the Center for Bioresource Conservation and Research of the Food Industry Research Institute, number BCRC60071, American Type Culture Collection (ATTC), number CCL-92. Mother cell. 3T3 mouse embryonic fibroblasts were cultured in a 25T culture flask, and used in a medium containing 3 ml of DMEM (DULBECCO'S MODIFIED MEM), a weight percent concentration of 10 FBS, a weight percent concentration of 1 NEA, and a weight percent concentration of 1 antibiotic. Incubate at 37 ° C in a 5% carbon dioxide atmosphere for three days. Then remove the medium in the bottle and add 1 ml of phosphate buffer (Phosphate Buffer Saline; PBS) for washing, then remove the acid buffer and add 1 ml of trypsin (Trypsin), shake the bottle to evenly soak, and culture The bottle is allowed to stand for 1 minute for 5 minutes. When the 3T3 mouse embryonic fibroblasts floated on the liquid surface, the cells were dispersed by a small amount of low-tube washing. Further, 2 ml of DMEM medium was added, and after mixing well, 2 ml of the cell solution was taken out, and cultured in 2 ml of DMEM medium for 3 days, followed by subculture. (2) MTT assay: MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazo liumbromide ] assay is a commonly used method for detecting cell viability. First, the first fucoidan composition having a concentration range of 0.1 mg/ml, 0.2 mg/ml, 0.3 13 200908989 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.75 mg/ml, and 1 mg/ml, respectively. The solution and the third brown algae polysaccharide composition solution were used to treat 3T3 mouse embryonic fibroblasts, and cultured for 24, 48, and 72 hours, respectively, and subjected to MTT cytotoxicity test analysis, and the experimental results showed that the following first fucoidan composition was formed. The 3T3 mouse embryonic fibroblasts treated with the solution of the solution and the third fucoidan composition solution slightly stimulated cell proliferation after 24 hours of culture, and the cell survival rate remained similar after the culture for 48 hours. . In the experimental group with a dose range of 0.4 mg/ml, the survival rate of the cells after 72 hours of culture was still 60%. Therefore, the safe and effective dose of the first fucoidan composition and the third fucoidan composition solution is substantially between 0.1 mg/ml and 0.5 mg/ml. Next, the activity analysis of matrix metalloproteinases (MMPs) was carried out to confirm that the fucoidan composition provided by the preferred embodiment of the present invention has the activity of inhibiting matrix metalloproteinases (MMPs) and inhibiting the secretion of matrix metalloproteinase MMP by cells. 2, the efficacy of MMP-9. For detailed procedures for the analysis of matrix metalloproteinase (MMPs) activity, please refer to Riley SC, Thomassen R, Bae SE, et al., Matrix metalloproteinase-2 and -9 secretion, published in Anim Reprod Sci, 2004, 81: 329-339. By the equine ovary during follicular growth and prior to ovulation ° The activity of matrix metalloproteinases (MMPs) is divided into matrix metalloproteinases MMP-2, MMP-9 activity inhibition analysis of sample processing: (1) 3T3 mouse embryonic fibrils The cells were cultured in a culture flask containing DMEM (containing 14 200908989 10% FBS) for three days, and A cell culture medium (containing matrix metal egg (IV) activity), respectively, 2 ml was added to two In the culture dish, : middle; one: the dose is substantial. One of the first brown algae polysteroid composition baths, and the other one added the same volume of sterile water as a control group. Place the incubator (37 t:, 5% (10)) after the sentence, and draw the same volume of cell culture solution according to different (four) points (〇, 2, 4'7, 12, 24, 48, 72 hours). Measured by 4 and placed in _2() t cold storage
(2) 將3Τ3老鼠胚月台纖維母細胞培養於二個培養皿中，盆 :-個加人劑量實質為G.5mg/ml㈣—褐藻多醣體組成物 溶液，另一個加入同體積的無菌水作為對照組。在混合均勻 後放置培養箱（37 I，5%⑽)培養三天，依不同的時間點 (〇 2、4、7、12、24、48、72小時）分別吸取相同體積的細 胞培養液到微量離心'管，並置於_2G t冷隸存作為樣本。 (3) 將3T3老鼠胚胎纖維母細胞培養於培養亚中，分別再 不同培養皿中加入不同劑量，例如〇mg/ml 、〇 lmg/mh〇 15 mg/ml、0.2 mg/m卜 0.25 mg/m卜 0.3 mg/m卜 0.35 mg/m卜 0.4 mg/m卜0.45 mg/ml及0.5 mg/ra卜的第一褐藻多醣體，在混 &均勻後放置培養箱（37 C , 5。/。C02)培養7.5小時之後分別 吸取相同體積的細胞培養液到微量離心管，並置於_2〇它冷 )東保存作為樣本。 (4)將3T3老鼠胚胎纖維母細胞培養於含DMEM(内含 10 FBS)的培養瓶中，培養三天，並抽取細胞培養液（内含 基質金屬蛋白酶的活性），分別取2 ml加到二個培養皿中， 其中一個加入劑量實質為〇.5mg/ml的第三褐藻多醣體組成 15 200908989 物溶液，另—個加入同體積的無菌水作為對照組。在混合均 勻後放置培養箱（37 5% c〇2)培養，並依不同的時間點 (0' 2、4、6、10、24、48小時）分別吸取相同體積的細胞培 養液到微量離心管，並置於_20 t冷凍保存作為樣本。 (5) 將3T3老鼠胚胎纖維母細胞培養於二個培養皿中，其 中一個加入第三褐藻多醣體组成物溶液，另一個加入同體積 的無菌水作為對照組。在混合均勻後放置培養箱（37。匸， C02)培養三天，依不同的時間點（0、2、4、7、12、24、48、 72小時）分別吸取相同體積的細胞培養液到微量離心管，並 置於-20 °C冷凍保存作為樣本。 (6) 將3T3老鼠胚胎纖維母細胞培養於培養皿中，分別 再不同培養皿中加入不同劑量，例如〇 mg/ml 、〇」mg/ml、 0.15 mg/ml ^ 0.2 mg/ml ^ 0.25 mg/ml > 0.3 mg/ml > 0.35 mg/ml ^ 0.4 mg/nU、0.45 mg/ml及0.5 mg/m卜的第三褐藻多醣體， 在混合均勻後放置培養箱（37 t，5% C02)培養7.5小時之後 分別吸取相同體積的細胞培養液到微量離心管，並置於_2〇 C冷束保存作為樣本。 接著以基質金屬蛋白酶的酵素活性電泳分析法 (Gelatin-based Zymography)分析上述樣本。 基質金屬蛋白酶（MMPs)的活性抑制分析結果請參照第 3A圖至第3C圖。第3A圖係根據本發明的較佳實施例，以 第一褐藻多醣體組成物溶液來處理移除3T3老鼠胚胎纖維母 細胞後的細胞培養液，經〇至72小時反應後，再進行基質 金屬蛋白酶活性分析所繪示的酵素活性電泳膠片影像。第3Β 16 200908989 ^係根據本發明的較佳實施例，將3T3老鼠胚胎纖維母細胞 養於s有第一褐藻多醣體組成物溶液的細胞培養液中，經 Q 至.f-j ry . 口* 小時培養後’再進行基質金屬蛋白酶活性分析所繪示 的酵素活性電泳膠片影像。第3C圖係'根據本發明的較佳實 施例，以第一褐藻多醣體組成物溶液處理3T3老鼠胚胎纖維 母細胞’經7.5小時培養後，再進行基質金屬蛋自酶活性分 析後所繪示的酵素活性電泳"影像。圖中c代表未加入多 膽體的對照組’培養第〇小時後的電泳膠片影像;C72代表未 加入多醣體的對照組，培養第72小時後的電泳膠片影像。 由第3A圖的實驗結果顯示，以第一褐藻多醣體組成物 於〇.5 mg/ml劑量處理細胞培養液72小時後，發現培養液内 所含基質金屬蛋白酶MMP_2的活性產生了抑制作用。 第圖的實驗結果也顯示’以含有〇_5 mg/ml第一褐藻 多醣體組成物的細胞培養液培養3T3老鼠胚胎纖維母細胞， 在培養0至4個小時後即對3Τ3老鼠胚胎纖維母細胞產生了 抑制基質金屬蛋白酶ΜΜΡ-2分泌的作用。 此外，由第3C圖更可發現3Τ3老鼠胚胎纖維母細胞培 養液内所含基質金屬蛋白酶ΜΜΡ-2的活性，會隨著第一褐 藻多醣體組成物溶液的處理劑量增加而減少，並具有劑量依 隨（Dose-Dependent)的趨勢。 請參照第4A圖至第4C圖。第4A圖係根據本發明的較 佳實施例，以第三褐藻多醣體組成物溶液來處理移除3丁3老 鼠胚胎纖維母細胞後的細胞培養液，經〇至7 2小時反庳後， 再進行基質金屬蛋白酶活性分析所繪示的酵素活性電泳膠 17 200908989 "像第4B圖係根據本發明的較佳實施例，肖仍老鼠 胚胎纖維母細胞培養於含有第三褐藻多賴組成物溶液的 ，σ養液中’、⑳G至72 時培養後，再進行基質金屬蛋 白酶活性分析所緣示的酵素活性電泳勝片影像。第牝圖係 根據本發明的較佳實施例 筮__ 平又狂1她例以第二褐溱多醣體組成物溶液處 理3T3老鼠胚胎纖維母細胞，經7 5小時培養後，再進行基 質金屬蛋白酶活性分析後輯示的酵素活性電泳膠片影像: 其中C代表未加入多醣體的對照組，培養第〇小時後的 電泳膠片影像；c2代表未加人多酿體的對照組，培養第2小 時後的電泳膠片影像;C4代表未加人多骑體的對照組，培養第 4小時後的電泳膠片影像；^代表未加人多㈣的對照組， 培養第6小時後的電泳膠片影像；Ci。代表未加人多醣雜的對 照組’培養第10小時後的電泳膠片影像；CM代表未加入多醣 體的對照組，培養第72小時後的電泳膠片影像。 由第4A圖的實驗結果顯示’以第三褐藻多酷體組成物 於0.5 mg/ml劑量處理細胞培養液72小時後，發現培養液内 所含MMP-2的活性產生了抑制作用。 第4B圖的實驗結果也顯示’以含有〇.5 mg/w第三褐藻 多醣體組成物的細胞培養液直接培養3T3老鼠胚胎纖維母細 胞，在培養〇、2、4、6及10個小時後分別與控制組c、C2、 C4、C6和CIO的作比較，細胞培養液内所含基質金屬蛋白 酶MMP-2的活性皆較實驗組來得高。因此可以推知，含有 劑置範圍實質為0.5 mg/ml的第三褐藻多醣體组成物溶液會 抑制3T3老鼠胚胎纖維母細胞的基質金屬蛋白酶ΜΜρ·2酵 18 200908989 素的活性。 而且由第4B圖更可.發現在處理3T3老鼠胚胎纖維母細 胞培養液10小時之内，細胞培養液内所含基質金屬蛋白酶 MMP-2的分泌量並沒有緩慢増加的現象產生，因此更可證明 第三褐藤多醣體組成物溶液具有抑制3T3老鼠胚胎纖維母細 胞分泌的新的基質金屬蛋白酶MMp_2的功能。更可以此推 論第三褐藻多_體組成物溶液具有抑制打3老鼠胚胎纖維母 細胞分泌基質金屬蛋白酶MMP_2之蛋白質前驅物pr〇_ MMP-2的功能。 此一推論可由第4C圖得到證實，此外由第4(：圖更可發 現3T3老鼠胚胎纖維母細胞培養液内所含基質金屬蛋白酶 MMP-2的活性，會隨著第三褐藻多醣體組成物溶液的處理劑 量增加而減少，並具有劑量依隨的趨勢。 由以上述實驗資料，已可證實使用劑量實質為〇.5mg/ml 的第三褐藻多醣體組成物溶液可直接抑制細胞培養液中由 3T3老鼠胚胎纖維母細胞所分泌之基質金屬蛋白酶mmp-2 的活性°而且藉由劑量實質為0.5 mg/ml的第三褐藻多醣體 組成物溶液直接處理3T3老鼠胚胎纖維母細胞，可以在7.5 小時之内抑制3T3老鼠胚胎纖維母細胞分泌基質金屬蛋白酶 MMP·2和蛋白質pro-MMP-2。而上述功效，可採用Sircol Collagen Assay kit分析，藉由測試細胞培養基中膠原蛋白的 含量疋否因基質金屬蛋白酶的活性抑制或分泌抑制而使得 膠原蛋白降解（Degradation)速率減緩，使膠原蛋白含量隨時 間增加而出現累積的效果來進一步加以證實。 19 200908989(2) The 3Τ3 mouse embryonic fibroblasts were cultured in two culture dishes, the pots: one plus human dose was G.5 mg/ml (four)-fucoid polysaccharide composition solution, and the other added the same volume of sterile water. As a control group. After mixing well, place the incubator (37 I, 5% (10)) for three days, and draw the same volume of cell culture solution at different time points (〇2, 4, 7, 12, 24, 48, 72 hours). The tube was microcentrifuged and placed in a _2 G t cold stock as a sample. (3) 3T3 mouse embryonic fibroblasts were cultured in culture medium, and different doses were added to different culture dishes, such as 〇mg/ml, 〇1mg/mh〇15 mg/ml, 0.2 mg/m 0.25 mg/ m Bu 0.3 mg/m b 0.35 mg/m b 0.4 mg/m b 0.45 mg/ml and 0.5 mg/ra b of the first fucoidan, placed in an incubator after mixing & even (37 C, 5 ./ C02) After 7.5 hours of incubation, the same volume of cell culture medium was aspirated into a microcentrifuge tube and placed in _2 〇 it cold) and stored as a sample. (4) 3T3 mouse embryonic fibroblasts were cultured in a DMEM containing 10 FBS, cultured for three days, and the cell culture medium (containing matrix metalloproteinase activity) was taken and added to 2 ml respectively. One of the two culture dishes was added with a third fucoidan composition having a dose of substantially 0.5 mg/ml, and a solution of 15 200908989, and the same volume of sterile water was added as a control group. After mixing well, place the incubator (37 5% c〇2) and incubate the same volume of cell culture medium to microcentrifugation at different time points (0' 2, 4, 6, 10, 24, 48 hours). Tube and place in _20 t for cryopreservation as a sample. (5) 3T3 mouse embryonic fibroblasts were cultured in two culture dishes, one of which was added with a third fucoidan composition solution, and the other was added with the same volume of sterile water as a control group. After mixing well, place the incubator (37.匸, C02) for three days, and draw the same volume of cell culture solution at different time points (0, 2, 4, 7, 12, 24, 48, 72 hours). Microcentrifuge tubes were placed and stored frozen at -20 °C as a sample. (6) 3T3 mouse embryonic fibroblasts were cultured in culture dishes, and different doses were added to different culture dishes, such as 〇mg/ml, 〇"mg/ml, 0.15 mg/ml ^ 0.2 mg/ml ^ 0.25 mg /ml > 0.3 mg/ml > 0.35 mg/ml ^ 0.4 mg/nU, 0.45 mg/ml and 0.5 mg/m b of the third fucoidan, placed in an incubator after mixing evenly (37 t, 5% C02) After culturing for 7.5 hours, the same volume of cell culture medium was aspirated into a microcentrifuge tube and placed in a _2〇C cold bundle for storage as a sample. The above samples were then analyzed by matrix metalloproteinase enzyme electrophoresis (Gelatin-based Zymography). For the results of inhibition assay of matrix metalloproteinases (MMPs), please refer to Figures 3A to 3C. 3A is a view of a preferred embodiment of the present invention, wherein the cell culture medium after removing the 3T3 mouse embryonic fibroblast is treated with the first fucoidan composition solution, and the substrate metal is further reacted after 72 hours of reaction. Protease activity analysis shows the enzyme active electrophoresis film image. 3rd Β 16 200908989 ^ According to a preferred embodiment of the present invention, 3T3 mouse embryonic fibroblasts are cultured in a cell culture solution having a solution of the first fucoidan composition, via Q to .fj ry. After the culture, the enzyme activity electrophoresis film image shown by the matrix metalloproteinase activity analysis was performed. 3C is a diagram showing the treatment of 3T3 mouse embryonic fibroblasts with the first fucoidan composition solution after 7.5 hours of cultivation according to a preferred embodiment of the present invention, and then analyzing the self-enzymatic activity of the matrix metal eggs. Enzyme Active Electrophoresis &Image; In the figure, c represents an electrophoretic film image of the control group which was not added to the biliary body after the culture for the second hour; C72 represents the electrophoresis film image after the 72 hours of the culture control group which was not added with the polysaccharide. From the experimental results in Fig. 3A, it was found that the activity of the matrix metalloproteinase MMP_2 contained in the culture solution was inhibited by treating the cell culture medium with the first fucoidan composition at a dose of 5 mg/ml for 72 hours. The experimental results in the figure also show that '3T3 mouse embryonic fibroblasts are cultured in a cell culture medium containing 〇_5 mg/ml of the first fucoidan composition, and 3 to 3 mouse embryonic fibrils are cultured after 0 to 4 hours of culture. The cells produce a role in inhibiting the secretion of matrix metalloproteinase-2. In addition, it can be found from Fig. 3C that the activity of matrix metalloproteinase-2 contained in the culture medium of 3Τ3 mouse embryonic fibroblasts decreases with the increase of the treatment dose of the first fucoidan composition solution, and has a dose. Follow the trend of (Dose-Dependent). Please refer to Figures 4A to 4C. 4A is a view of a preferred embodiment of the present invention, wherein the cell culture medium after removing the 3D3 mouse embryonic fibroblasts is treated with a solution of the third fucoidan polysaccharide composition, and after sputuming for 72 hours, Further, the enzyme active electrophoresis gel represented by the matrix metalloproteinase activity analysis is shown in Fig. 4B. According to a preferred embodiment of the present invention, the mouse embryonic fibroblast is cultured in a composition containing the third brown algae. In the solution, the sputum culture solution was cultured at 20G to 72°C, and then the enzyme activity electrophoresis was recorded on the substrate metalloproteinase activity analysis. The first diagram is based on the preferred embodiment of the present invention. __ Ping and mad 1 her case treated 3T3 mouse embryonic fibroblasts with a solution of the second brown peony polysaccharide composition, and cultured for 75 hours, followed by matrix metal Enzyme activity electrophoresis film image after protease activity analysis: C represents the control group without polysaccharide added, and the electrophoresis film image after the second hour of culture; c2 represents the control group without added multi-breast, the second hour of culture After the electrophoresis film image; C4 represents the control group without human body riding, the electrophoresis film image after the 4th hour of culture; ^ represents the control group without the addition of more people (4), the electrophoresis film image after the 6th hour of culture; Ci . The electrophoresis film image after the 10th hour of the control group was cultured without the addition of the polysaccharide; CM represents the control group without the addition of the polysaccharide, and the electrophoretic film image after the 72nd hour of culture was cultured. From the experimental results in Fig. 4A, it was revealed that the activity of MMP-2 contained in the culture solution was inhibited by treating the cell culture medium at a dose of 0.5 mg/ml for 72 hours with the third brown algae composition. The experimental results in Fig. 4B also show that '3T3 mouse embryonic fibroblasts were directly cultured in a cell culture medium containing 〇.5 mg/w of the third fucoidan composition, and cultured for 2, 4, 6 and 10 hours. After comparison with control groups c, C2, C4, C6 and CIO, the activity of matrix metalloproteinase MMP-2 contained in the cell culture medium was higher than that of the experimental group. Therefore, it can be inferred that the solution of the third fucoidan composition containing the agent in the range of substantially 0.5 mg/ml inhibits the activity of the matrix metalloproteinase 3ρ·2 fermentation of 3T3 mouse embryonic fibroblasts. Furthermore, it can be found from Fig. 4B that within 10 hours of treatment of 3T3 mouse embryonic fibroblast culture medium, the secretion of matrix metalloproteinase MMP-2 contained in the cell culture solution is not slowly increased, so it is more It was confirmed that the third brown vine polysaccharide composition solution has a function of inhibiting the secretion of the new matrix metalloproteinase MMp_2 by 3T3 mouse embryonic fibroblasts. It can be further inferred that the third brown algae multi-body composition solution has the function of inhibiting the protein precursor pr〇_MMP-2 which secretes the matrix metalloproteinase MMP_2 of the mouse embryonic fibroblast. This inference can be confirmed by Figure 4C. In addition, the activity of matrix metalloproteinase MMP-2 contained in the culture medium of 3T3 mouse embryonic fibroblasts can be found along with the third fucoidan composition. The treatment dose of the solution is increased and decreased, and has a tendency of dose dependency. From the above experimental data, it has been confirmed that the solution of the third fucoidan polysaccharide composition having a dose of substantially 〇5 mg/ml can directly inhibit the cell culture solution. The activity of matrix metalloproteinase mmp-2 secreted by 3T3 mouse embryonic fibroblasts and direct treatment of 3T3 mouse embryonic fibroblasts by a solution of a third fucoidan composition with a dose of 0.5 mg/ml, which can be at 7.5 Within 3 hours, 3T3 mouse embryonic fibroblasts are inhibited from secreting matrix metalloproteinase MMP·2 and protein pro-MMP-2. The above effects can be analyzed by Sircol Collagen Assay kit, by testing the content of collagen in cell culture medium. Matrix metalloproteinase activity inhibition or secretion inhibition causes the rate of collagen degradation to slow down, allowing collagen content to be readily available The cumulative effect and the cumulative effect are further confirmed. 19 200908989
Sircol Collagen Assay kit 分折 本實施例係以劑量實質為0.5 mg/ml的第三褐藻多醣體 組成物溶液處理移除3T3老鼠胚胎纖維母細胞後的細胞培養 液，分別於反應0、24、48、72小時後，檢測細胞培養液内 第一型（Type I)膠原蛋白含量的變化情形。（操作步驟根據 Sircol™ soluble collagen assay kit) 實驗結果請參照第5A圖，由第5A圖中顯示，第三褐藻 多醣體組成物溶液處理後的細胞培養液内，其第一型膠原蛋 白含量有維持一定量的趨勢，而未以第三褐藻多醣體組成物 溶液處理的細胞培養液内第一型膠原蛋白含量在24小時後 已破壞超過50% (由68.70 g/ml降至33.48 g/ml)。 由以上的實驗結果’可證實第三褐藻多醣體組成物溶液 具有抑制3T3老鼠胚胎纖維母細胞分泌基質金屬蛋白酶 MMP-2酵素及抑制基質金屬蛋白酶MMP-2酵素活性的功 效’因此可減緩細胞培養液内膠原蛋白的破壞，而達到膠原 蛋白含量累積的現象。 根據以上所述之實施例，本發明的技術特徵係採用酒精 萃取製程，以重量百分濃度實質介於50至75的酒精溶液萃 取褐藻，以得到一種褐藻多醣體組成物，其中此一褐藻多醣 體組成物的分子量實質介於2,800,000 Da至40,〇〇〇,〇〇〇 Da 之間’具有可抑制基質金屬蛋白酶活化的功能，可添加於化 妝品之中作為以抗皮膚老化的成分。 藉由基質金屬蛋白酶（MMPs)的活性分析可證實，上述實 20 200908989 施例所提供的褐藻多冑體組成物確實具有抑制基質金屬蛋 白酶活性並抑制動物細胞分泌基質金屬蛋白酶的功能。進而 可延缓膠原蛋白的降解達到皮膚抗老化的功能。 因此上述實施例所提供的褐藻多醣體組成物，具有製裎 簡單可兼顧抗老化效果與成本優勢的優點，可以解決習知技 術投入成本較大’萃取程序較為繁雜的缺點。 雖然本發明已以較佳實施例揭露如上，然其並非用以限 又本發月，任何相關技術領域具有通常知識者，在不脫離本 發明之精神和範圍内，當可作各種之更動與潤飾，因此本發 明之保護範圍當視後附之申請專利範圍所界定者為準。 【圖式簡單說明】 為讓本發明之上述和其他目的、特徵、優點與實施例能 更明顯易懂，所附圖式之詳細說明如下： 第1A圖則是以多醣體標準品之滯留時間及標準品分子 量的log值所作之標準曲線。 第1B圖係繪示第一褐藻多醣體組成物溶液所含之褐藤 多塘體的分子量分佈圖。 第1 c圖係繪示第三褐藻多醣體組成物溶液所含之褐藻 >聰體的分子量分佈圖。 ,第2A圖係繪示使用HPLC分析第一褐藻多醣體組成物 溶液的多醣體的單醣組成份分析圖。 第2B圖係繪示使用HPLC分析第三褐藻多醣體組成物 21 200908989 '合液的多醣體單醣組成份分析圖。 第3A圖係根據本發明的較佳實施例，以第一褐藻多醣 體組成物⑷液來處理移除3T3老鼠胚胎纖維母細胞後的細胞 培養液m至72小時反應後，再進行基質金屬蛋白酶活 性分析所緣示的酵素活性電泳膠片影像。 第3B圖係根據本發明的較佳實施例，將3丁3老鼠胚胎 纖維母細胞培養於含有第一帛藻多醣體組成物溶液的細胞 培養液中’經〇至72小時培養後，再進行基f金屬蛋白酶 活!·生刀析所繪不的酵素活性電泳膠片影像。 第3C圖係根據本發明的較佳實施例以第一褐藻多醣 體組成物#液處理3T3老I胚胎纖維母細胞’經7 5小時培 養後，再進行基質金屬蛋白酶活性分析後所繪示的酵素活性 電泳膠片影像。 第4A圖係根據本發明的較佳實施例，以第三褐藻多醣 體組成物洛液來處理移除3T3老鼠胚胎纖維母細胞後的細胞 培養液，經〇至72小時反應後，再進行基質金屬蛋白酶活 性分析所繪示的酵素活性電泳膠片影像。 第4Β圖係根據本發明的較佳實施例，將3Τ3老鼠胚胎 纖維母細胞培養於含有第三褐藻多醣體組成物溶液的細胞 培養液中，經〇至72小時培養後，再進行基質金屬蛋白酶 活性分析所繪示的酵素活性電泳膠片影像。 第4C圖係根據本發明的較佳實施例，以第三褐藻多醣 體組成物溶液處理3Τ3老鼠胚胎纖維母細胞，經7.5小時培 養後S進行基質金屬蛋自酶活性分析後所繪示的酵素活性 22 200908989 電泳膠片影像。 【主要元件符號說明】Sircol Collagen Assay kit The cell culture medium after removal of 3T3 mouse embryonic fibroblasts was treated with a solution of the third fucoidan polysaccharide composition at a dose of 0.5 mg/ml, respectively, in reactions 0, 24, 48 After 72 hours, the change in the type I collagen content in the cell culture medium was examined. (According to the SircolTM soluble collagen assay kit), please refer to Figure 5A. Figure 5A shows that the first type of collagen in the cell culture solution after the treatment of the third fucoidan solution solution has Maintaining a certain amount of trend, while the type 1 collagen content in the cell culture medium not treated with the third fucoidan composition solution has destroyed more than 50% after 24 hours (from 68.70 g/ml to 33.48 g/ml) ). From the above experimental results, it can be confirmed that the third fucoidan polysaccharide composition solution has the effect of inhibiting the secretion of matrix metalloproteinase MMP-2 enzyme and inhibiting the activity of matrix metalloproteinase MMP-2 enzyme by 3T3 mouse embryonic fibroblasts, thereby slowing down cell culture. The destruction of collagen in the liquid, and the accumulation of collagen content is reached. According to the embodiments described above, the technical feature of the present invention is to extract a brown algae by using an alcohol extraction process in an alcohol solution having a weight percentage of substantially 50 to 75 to obtain a fucoidan polysaccharide composition, wherein the fucoidan polysaccharide The molecular weight of the bulk composition is substantially between 2,800,000 Da and 40, and the relationship between 〇〇〇 and 〇〇〇D' has the function of inhibiting the activation of matrix metalloproteinase, and can be added to cosmetics as an ingredient against skin aging. It was confirmed by the activity analysis of matrix metalloproteinases (MMPs) that the brown algae polysteroid composition provided by the above example 20 200908989 did have a function of inhibiting matrix metalloproteinase activity and inhibiting secretion of matrix metalloproteinase by animal cells. In turn, the degradation of collagen can be delayed to achieve the anti-aging function of the skin. Therefore, the brown algae polysaccharide composition provided by the above embodiments has the advantages of simple anti-aging effect and cost advantage, and can solve the disadvantages that the conventional technology has a large input cost, and the extraction procedure is complicated. Although the present invention has been disclosed in the above preferred embodiments, it is not intended to limit the scope of the present invention, and it is intended that various modifications may be made without departing from the spirit and scope of the invention. The scope of protection of the present invention is therefore defined by the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS In order to make the above and other objects, features, advantages and embodiments of the present invention more obvious, the detailed description of the drawings is as follows: Figure 1A is the retention time of the polysaccharide standard. And the standard curve of the log value of the standard molecular weight. Fig. 1B is a graph showing the molecular weight distribution of the brown vines contained in the solution of the first fucoidan polysaccharide composition. Fig. 1c is a graph showing the molecular weight distribution of the brown algae > genus contained in the solution of the third fucoidan polysaccharide composition. Fig. 2A is a graph showing the analysis of the monosaccharide composition of the polysaccharide of the first fucoidan polysaccharide solution solution by HPLC. Fig. 2B is a graph showing the analysis of the composition of the polysaccharide monosaccharide of the liquid mixture using the HPLC analysis of the third fucoidan composition 21 200908989 '. 3A is a method for treating a cell culture medium in which 3T3 mouse embryonic fibroblasts are removed by treatment with a first fucoidan composition (4) solution for m to 72 hours, followed by matrix metalloproteinases according to a preferred embodiment of the present invention. The enzyme activity electrophoresis film image indicated by the activity analysis. Fig. 3B is a view showing a preferred embodiment of the present invention, wherein the 3D3 mouse embryonic fibroblasts are cultured in a cell culture medium containing a solution of the first polysaccharide composition, 'after culturing for 72 hours, and then Base f metalloproteinase live! · Biofilm electrophoresis film image. 3C is a diagram showing the activity of matrix metalloproteinase activity after the culture of 3T3 old I embryonic fibroblasts of the first fucoidan composition #liquid treatment 3T3 old I embryonic fibroblasts according to a preferred embodiment of the present invention. Enzyme active electrophoresis film image. 4A is a diagram of a cell culture medium in which 3T3 mouse embryonic fibroblasts are removed by treatment with a third fucoidan composition, according to a preferred embodiment of the present invention, and then subjected to a reaction for 72 hours. The enzyme activity electrophoresis film image shown by the metalloproteinase activity analysis. According to a preferred embodiment of the present invention, 3Τ3 mouse embryonic fibroblasts are cultured in a cell culture medium containing a solution of a third fucoidan composition, and cultured for 30 hours, followed by matrix metalloproteinase Activity analysis of the enzyme-active electrophoresis film image. According to a preferred embodiment of the present invention, the 3D3 mouse embryonic fibroblasts are treated with the third fucoidan polysaccharide composition solution, and the enzymes are analyzed after the 7.5-hour culture of the matrix metal egg self-enzymatic activity analysis. Activity 22 200908989 Electrophoresis film image. [Main component symbol description]