Isolation of proteoglycan from sea cucumber and investigation on its antioxidant, antimutagenic and hypolipidemic activities

Abstract

本研究以市售海參黃光參（Metriatyla scabra）為材料，分離與鑑定其內含機能性成分蛋白多醣（glycosaminoglycan, GAG），藉由腸道細胞（intestine 407）模式系統與添加致突變劑（4NQO與MNNG）探討其抗氧化與抗致突變之能力。此外，應用動物試驗分析，配合高膽固醇飲食與管餵GAG，評估海參體GAG對降膽固醇之功用與可能之作用機制。試驗結果如下： 1.乾海參之基本組成以重量百分率計，水分3%、蛋白質88%、碳水化合物 4%、脂質 2%、灰分 3%；復水後依次為 95%、4%、0.2%、0.1%、0.1%。依水、鹼、酸、熱處理之順序分畫海參體壁中之膠原蛋白，其含量分別為42%、6%、48%、2%，其中以水溶性(佔 42%)及酸溶性 (佔 48%)者居多，合佔總量之 90%。且以Type I與II collagen為主。 2.市售黃光參經由papain分解後所得之crude PG經離子交換管柱與膠體過濾層析，可分劃出2個peak （peak-1與peak-2），其分子量分別為200~500, 40~200 kDa，進一步分析內在成分發現，peak-1（GAG）含hexuronic acid、hexosamine為主，而peak-2（free glycan） 內含量以fucan為主。 3.單一細胞膠體電泳分析顯示，海參體蛋白多醣包括crude PG, GAG, free glycan等均可降低細胞DNA受傷指數；分析其抗氧化能力發現因具有清除DPPH自由基、超氧陰離子、清除過氧化氫以及螯合鐵與銅金屬離子的能力等抗氧化能力，因此可清除致突變劑所生成自由基與活性氧，進而保護細胞DNA。硝酸鹽還原測試，結果發現海參體蛋白多醣對亞硝基化合物不具有還原能力，可證明蛋白多醣對致突變劑4NQO與MNNG具有抑制與抗突變之效果。 4.動物試驗分析結果得知，海參體蛋白多醣GAG可降低大鼠血漿中總膽固醇、LDL-膽固醇濃度、肝臟與血漿過氧化程度及血清中抗氧化酵素活性SOD, GSH-Px活性。此影響與GAG之管餵量有關，在管餵量高於20mg/kg b.w.，隨著GAG濃度增加而有下降趨勢。 5.經由血清GSH與GSSG分析，餵食1%膽固醇之大鼠血清中GSH有顯著下降情況，但隨著餵食GAG含量增加而有上升趨勢，然而血清中GSSG其變化趨勢與GSH相同，由GSSG/GSH之比值可知管餵海參體GAG對於降低生物體內氧化壓力之途徑與GSH系統的相關性並不明顯。此外，餵食1％膽固醇與海參體GAG大鼠其糞便中膽酸排出並無顯著影響(p>0.05)，因此推斷，GAG可能藉由其中影響脂質代謝或合成的途徑，達到降低血清與肝臟中脂質濃度。

Glycosaminoglycan (GAG), recognized as a functional component with antioxidant, antimutagenic and hypolipidemic activities, was isolated and identified from body wall of sea cucumber Metriatyla scabra. Antioxidative and antimutagenic activities were investigated by using intestine 407 cell model system and additing of 4NQO (4-nitroquinoline-1-oxide) and MNNG (N-methyl-N-nitroso-guanidine)as mutagenic agents. Hypolipidemic effects and mechanism of the GAG were also evaluated through cholesterol supplemented diet with oral administration of GAG to rats. The results are: 1. Compositions of dried sea cucumber were: moisture 3%, protein 88%, carbohydrate 4%, lipid 2%, ash 3%; after rehydration they were 95%, 4%, 0.2%, 0.1%, 0.1%, respectively. Extraction in the order of water, alkali solution, acidic solution, and hot water indicates that major collagen contains in sea cucumber body wall were water-soluble (42%) and acid-soluble (48%), i.e., Type I and Type II collagens. 2. Two peaks (peak-1 and peak-2) were observed for the sea cucumber hydrolyzed with papain and followed by ion-exchange and gel-filtration chromatography. Peak-1 M.W. 200-500kDa, contained mainly GAG as hexuronic acid and hexosamine, while peak-2 M.W. 40-200kDa, contained mostly free glycan as fucose with little hexuronic acid or hexosamine. The peak-1 fraction was used to evaluate hypolipidemic effects. 3. Single cell gel electrophoresis (comet assay) shows that crude proteoglycan (crude PG), GAG (peak-1) and free glycan exhibited DNA damage, which may result from good antimutagenic effects, inducted by 4NQO and MNNG. GAG exhibited antioxidant capacity including scavenging of DPPH free radical, H2O2, superoxide anion and chelating Fe++ and Cu++. No ability for reduction of nitrate further verified the inhibition and antimutagenic activity. 4. Oral administration of GAG to male Wister rats shows that plasma levels of total cholesterol, LDL-cholesterol and atherogenic index, SOD, GSH-Px activity in plasma were significantly decreased, while HDL-cholesterol was significantly increased, and plasma and hepatric tissue MDA concentraction, although these effects of the GAG were only dose-dependent at dose higher than 20mg/kg b.w. Similarly, the GAG significantly prevented the increase (p<0.05) in hepatic contents of triglyceride, cholesterol and phospholipid. 5. GAG increased serum GSH levels. Howerever, the relationship between antioxidant activities and GSH levels was not obvious. Furthermore, the GAG did not significantly prevent the increase of bile acid in the fecal. Sea cucumber GAG probably was identified to attain the reduction of plasma and hepatic cholesterol.