靈芝酪胺酸酶抑制劑之特性

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Title:	靈芝酪胺酸酶抑制劑之特性 Characteristics of tyrosinase Inhibitor from Ganoderma lucidum
Authors:	陳彥瑾 Yen-Chin Chen
Contributors:	朱惠鈴郭建民嘉南藥理科技大學：生物科技研究所
Keywords:	酪胺酸酶 tyrosinase
Date:	2003
Issue Date:	2008-10-08 15:45:19 (UTC+8)
Abstract:	本研究的目的為萃取靈芝酪胺酸酶抑制劑，探討其最適萃取、純化、分離條件以及抑制劑之生化特性。酪胺酸酶為黑色素生合成關鍵酵素，酪胺酸酶抑制劑則可調控黑色素生合成，具有減少皮膚或食品不良反應之作用。以不同溶液進行靈芝酪胺酸酶抑制劑萃取分析，結果以25mM 磷酸鈉緩衝溶液pH6.8萃取率為最佳（80％），其次為甲醇、正丁醇、正己烷和乙酸乙酯。靈芝酪胺酸酶抑制劑在-18℃及4℃儲存180天後，其相對殘留抑制活性分別為98％和88％。該抑制劑粗抽出液繼續利用Amberlite XAD-7、Sephadex G-25和Superdex peptide管柱層析進行純化，其回收率分別為83％、50％和15％，而半抑制濃度（IC50）分別為5.8、3.19和0.15 mg/ml。經部分純化的靈芝酪胺酸酶抑制劑以L-DOPA當基質，進行生化性質之探討，抑制劑和酪胺酸酶最適反應時間會隨著時間延長而抑制作用越強，在0分鐘時為47％，在反應18小時後，抑制活性最高可達成100％。該抑制劑最適作用溫度為45℃-55℃，最適作用酸鹼值為pH 5.0，而從pH4.0到pH9.0都很穩定。在動力學方面，分別以L-DOPA及L-tyrosine當基質時，靈芝酪胺酸酶抑制劑均屬於mix-type抑制作用，抑制常數（Ki）分別為1.1 mg/ml及0.3 mg/ml；而以Superdex peptide HR管柱層析測量此抑制劑分子量大約為0.3kDa。該抑制劑與Fehling 試劑和ninhydrin呈現反應，推測其結構中可能含有醣基和胺基。 The purpose of this study is to isolate and purify the tyrosinase inhibitor from Ganoderma lucidum. Tyrosinase is a key enzyme in melanogenesis, therefore tyrosinase inhibitor can regulate the enzyme activity of tyrosinase and prevent the undesirable effect of skin as well as browning reaction of food. The tyrosinase inhibitor from Ganoderma lucidum was extracted by various extraction media, and that extracted by 25mM sodium phosphate buffer pH6.8 showed the highest extraction efficiency, followed by methanol n-butanol n-hexane and ethyl acetate . The inhibitor was then stored at -18℃ and 4℃, the relative inhibition activity of tyrosinase were 98% and 88% after 6 month storage, respectively. The tyrosinase inhibitor from Ganoderma lucidum was further purified with Amberlite XAD-7 Sephadex G-25 and Superdex Peptide column chromatography, and the recovery was 83% 50% and 15%, respectively; the inhibitory concentration caused 50% inhibition (IC50) was 5.8 3.19 and 0.15mg/ml, respectively. The inhibition activity of tyrosinase was 47% when preincubated with the inhibitor for 0 minute while 100% for 18 hour, indicating a slow binding behavior. The optimal temperature of the inhibition activity of tyrosinase treated with the inhibitor was 45℃-55℃; optimal pH 5.0. The inhibitor was stable at pH 4.0-9.0. In Kinetic study, the inhibitor showed mixed types and the inhibitory constant (Ki) 1.1mg/ml using L-DOPA as substrate, while mixed types and Ki 0.3mg/ml using L-tyrosine as substrate. Moreover, molecular mass of the partially purified tyrosinase inhibitor from Ganoderma lucidum was 0.3kDa examined by Superdex Peptide column gel filtration. The tyrosinase inhibitor from Ganoderma lucidum may contain sugar residue or amino group, according to the positive reaction with Fehling reagent and ninhydrin.
Relation:	校內外均一年後公開
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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