山藥儲藏性蛋白質水解胜肽的抗氧化和延緩老化活性評估

先前研究指出台農一號山藥 (Tainong No.1)中的主要儲藏性蛋白dioscorin，和胃蛋白酶水解產物具有清除自由基活性。本研究從cDNA轉譯之dioscorin A和dioscorin B蛋白質序列中，以電腦模擬經過胃蛋白酶作用，挑選5段含有Cys和一段含有Trp序列的胜肽 (diotide 1-6)，以這六段山藥胜肽序列合成胜肽 (diotides)，進行抗氧化試驗研究。結果發現，在ESR做清除氫氧自由基活性中發現，100μM的diotide 6 (序列為NW)活性最好 (88.80%)。在保護紅血球延緩AAPH攻擊發生溶血實驗活性中，100μM的diotide 6活性最好 (83.39%)。在ORAC試驗中，山藥合成胜肽相同濃度 (5μM)的diotide 6活性最好 (84.8μM TE)。以胎牛血清蛋白(BSA)分別和5mM methylglyoxal (MGO)反應三天，或0.4M的D-半乳糖 (BSA/gal)或0.4 M的D-葡萄糖反應 (BSA/glu)10天，生成glycation end product (AGE)的蛋白質醣基化模式系統中發現，diotide 6 (50μM和100μM)抑制BSA的醣基化 (BSA/gal)效果最佳。挑選dioscorin和NW (diotide 6，山藥合成胜肽)進行動物實驗相關研究。在dioscorin動物實驗研究，以D-半乳糖誘導老化模式小鼠四週後，用管餵方式餵食dioscorin (20和80 mg/kg b. w. /day，分別為20 dio組和80 dio組) 連續六週，並同時以D-半乳糖進行皮下注射。結果顯示，餵食20 dio組和80 dio組，血漿中MDA大幅下降 (p<0.001，和對照組比較)。以水迷宮評估記憶學習試驗中，高劑量組 (80 dio組)明顯改善學習記憶能力，在找到平台時間及水池中游泳錯誤距離皆明顯改善，並與對照組呈現明顯差異 (p<0.01)。犧牲後，在80 dio組的血漿中GSH含量及ORAC活性明顯提高，MDA含量明顯下降 (p<0.001)。在腦和肝臟組織中GSH含量和ORAC活性明顯提高，MDA含量明顯下降(p<0.01)。從腦和血漿中蛋白質醣基化程度，由抗argpyrimidine和抗CML免疫染色結果，發現20 dio組和80 dio組的血漿和腦組織，有抑制蛋白質醣基化現象。再以NW (diotide 6，合成胜肽)進行抗老化動物實驗研究，同樣以D-半乳糖誘導老化模式小鼠四周後，用管餵方式餵食NW (10 和40 mg/kg b. w. /day，分別為10NW組和40NW組)連續四週，並同時皮下注射D-半乳糖。結果顯示，以水迷宮評估記憶學習試驗中，40NW組，在找到平台時間及水池中游泳錯誤距離皆明顯改善，並與對照組呈現明顯差異 (p<0.01)。被動迴避實驗評估記憶學習試驗中，40NW組活性和空白組有相當的學習活性，並與對照組呈現明顯差異 (p<0.01)。犧牲後，在40NW組的腦、肝臟組織和血漿中GSH含量及ORAC活性明顯提高，MDA含量明顯下降 (p<0.001)。從腦和血漿中醣基化程度，由抗argpyrimidine和抗CML免疫染色結果，發現10NW組和40NW組，也有抑制蛋白質醣基化現象。以上的結果，我們推測山藥儲藏性蛋白質dioscorin延緩半乳糖模式誘導老化模式，可能來自於水解胜肽抗氧化能力，而相關機制須再進一步研究。

關鍵字

山藥, 儲藏性蛋白質, 胜肽, 抗氧化, 延緩老化活性

並列摘要

Yam (Dioscorea alata. L. cv. Tainong NO.1) storage proteins and its peptic hydrolysates have been reported to have radical scavenging activities; however, the active peptides are still under investigations. Dioscorin A and dioscorin B, the deduced proteins from our reported cDNA sequences, were used to hydrolyzed by computer-aided pepsin simulative hydrolysis (pH <2). In this study, five thiol-contained peptides (diotide1~5) and one Trp-contained peptides (diotide 6) were selected and synthesized for series of antioxidant and anti-radical activity assays, including hydroxyl radical scavenging activities by ESR, anti-AAPH mediated hemolytic activities and ORAC activities. Among tested peptides which, the diotide 6 (NW) exhibited the best inhibitory activity in these assays. For anti-advanced glycation endproducts (AGE) formation experiment, BSA/methylglyoxal (MGO), BSA/gal, BSA/glu were used as models without or with diotide additions. It was found that diotide 6 was the most potent inhibitors against AGE formation. Then, dioscorin and diotide 6 were further tested in D-galactose-induced aging mouse model mice. In this study, D-galactose (Gal) was subcutaneously injected into the dorsal necks of BALB/c mice daily for 10 weeks (Gal group) to induce oxidative stresses. By the 5th week, 20 or 80 mg dioscorin/ kg b. w. (20 dio group and 80 dio group) were orally administered daily together with daily Gal injection until the end of the study. The plasma malondialdehyde (MDA) level and advanced glycation endproducts obtained after dioscorin oral administrations were lower than those in the Gal group (p<0.001). The latency and swimming distance for 80 dio group were significantly better than those for the Gal group in the Morris water maze (p<0.01). Dioscorin administration resulted in higher GSH levels and oxygen radical antioxidant capacity (ORAC) and lower MDA in the brain and/or liver than those for the mice in the Gal group. Then, NW were used for D-galactose-induced aging model mice for 8 weeks, 10 or 40 mg diotide 6 (NW)/ kg b. w. / day (10NW group and 40NW group) were orally administered daily together with daily Gal injection for 4 weeks in the study. The plasma MDA level and advanced glycation endproducts obtained after NW oral administrations were lower than those in the Gal group (p<0.001). The latency and swimming distance for 40NW group were significantly better than those for the Gal group in the Morris water maze (p<0.01). In the experiments of passive avoidance, the 40NW group and the blank group have considerable memory learning activity, better than those for the Gal group (P <0.01). 40NW group resulted in higher GSH levels and oxygen radical antioxidant capacity (ORAC) and lower MDA in the brain and/or liver than those for the mice in the Gal group. These results suggest that peptic hydrolysates derived from dioscorin exhibit antioxidant and anti-aging activities, and need further investigation to confirm these results.