蓮子心與芝麻粕之抗氧化活性及其延長線蟲壽命功效之研究

Abstract

老化是一種漸進性的退化過程，包含許多分子、細胞、組織及系統方面的改變。關於老化的原因，迄今所提出的學說不勝枚舉，其中較受公認的為自由基老化理論。經由體內各種過程產生之活性氧物質 (ROS) 會造成氧化傷害。當體內之氧化傷害無法修復時，則造成組織器官功能喪失或老化相關疾病之發生。而現代研究指出飲食中補充抗氧化劑可有效減少老化相關疾病發生而達平均壽命延長之效果。本研究室先前研究，以「Aβ 聚集導致 PC-12 細胞死亡之抑制」為模式，發現蓮子心與芝麻粕具有抗阿滋海默症之能力。基於阿滋海默症等神經性退化疾病與老化之發生皆與體內氧化傷害相關，本研究以蓮子心與芝麻粕為材料，探討兩食材之體外抗氧化能力及體內抗老化活性。 實驗方面，首先以水及四種有機溶劑萃取蓮子心與芝麻粕，其萃取方式為沸點下熱水迴流萃取及室溫下以 95% 乙醇、丙酮、乙酸乙酯和正己烷四種溶劑之攪拌萃取。再以兩種評估方法檢測抗氧化活性，包括清除 DPPH 自由基和 TEAC (Trolox equivalent antioxidant capacity)，另外以「線蟲壽命延長」為模式，評估抗老化活性，並以 EUK-8 作為抗老化研究模式之正對照組。結果顯示，兩食材不同溶劑粗萃物中以蓮子心乙醇粗萃物於抗氧化實驗中展現較高之抗氧化力，其清除 DPPH 自由基之 EC50 為 47.32±1.20 μg/mL，TEAC 值為 0.31 mM Trolox equiv.；而 DPPH 試驗之正對照組為 α-tocopherol (EC50 為 8.40±0.13 μg/mL)，TEAC 試驗之正對照組為 ascorbic acid (TEAC值為 1.16 mM Trolox equivalent)。整體而言抗氧化活性以蓮子心乙醇粗萃物最佳，其次為水、丙酮、乙酸乙酯和正己烷粗萃物；而芝麻粕各溶劑粗萃物之抗氧化活性皆不佳。以線蟲壽命延長為模式之抗老化實驗結果顯示，兩食材各溶劑粗萃物中以蓮子心乙醇粗萃物具有最佳延長線蟲壽命之效果。蓮子心乙醇粗萃物 1000 μg/mL 處理下線蟲平均壽命 23.4±5.7 天（較未處理組增加 25.8% (p＜ 0.001) ），最大壽命期 35 天，80% 線蟲死亡時間 28.2 天，其效果與 100 μM (35.5 μg/mL) EUK-8 （平均壽命增加 24.1% (p＜ 0.001)，最大壽命期 35 天，80% 線蟲死亡時間 27.0 天 ）十分接近。 進一步將蓮子心乙醇粗萃物以正己烷、乙醚、乙酸乙酯及水四種溶劑進行分配萃取，其中以水區分層之抗氧化效果最佳。其對於清除 DPPH 自由基之 EC50 為 25.17±0.19 μg/mL，TEAC 值為 0.41 mM Trolox equiv.。四種區分物於抗老化模式實驗中，亦以蓮子心乙醇粗萃物之水區分物效果最佳，100 μg/mL 的水區分物可延長線蟲平均壽命（16.3% (p=0.0036)），最大壽命期增加 17.2%，80% 線蟲死亡時間延後 12.5%，且於較高濃度處理下具有濃度效應。水區分物 100 μg/mL 之效果與正對照組 25 μM (8.9 μg/mL) EUK-8 相近（增加了 16.3% 的壽命期 (p=0.0044)，且延長最長存活時間（增加 13.8%）與 80% 線蟲死亡時間（增加 14.3%）。 本研究結果顯示蓮子心乙醇粗萃物之水區分物具有相當良好的抗氧化能力與延緩老化使線蟲壽命延長之功效，有潛力發展為保健食品。

Aging is an inherently complex process that is manifested within an organism at molecular, cellular, organ, and system levels. There are many theories that have been put forward to explain the process of aging, and one mechanism involving free radical damage seems to emerge in a quite consensual manner. Reactive oxygen species (ROS), produced by a variety of processes, can cause extensive oxidative damages, which would be responsible for age-associated loss of functional capacity. Dietary interventions with antioxidants seem to demonstrate some potential benefits on the average life expectancy, probably by reducing the onset of age-related diseases. In the previous study conducted in our laboratory, using “inhibition of aggregated Aβ-induced PC-12 cell death” as the model for Alzheimer’s disease, has shown that embryo of Nelumbo nucifera Gaertn. seed and defatted Sesamum indicum L. have good anti-Alzheimer’s disease capacity. Since both aging and neurodegenerative disease such as Alzheimer’s disease are associated with in vivo oxidative damage, we are therefore interested in investigating the in vitro antioxidation and in vivo anti-aging effects of embryo of Nelumbo nucifera Gaertn. seed and defatted Sesamum indicum L. The crude extracts from different solvents were prepared by extracting these two raw materials with boiling water and four solvents (95% ethanol, acetone, ethyl acetate and n-hexane) at room temperature. Then the different solvent extracts of embryo of Nelumbo nucifera Gaertn. seed and defatted Sesamum indicum L. were tested for two antioxidative assays, including DPPH free radical scavenging effect and Trolox equivalent antioxidant capacity (TEAC) assay. They were also examined for anti-aging activity using the “prolongation of C. elegans lifespan” as the model, and EUK-8 as the positive control. The results of antioxidant assays revealed that the best effects was exhibited by the 95% ethanol extract of the embryo of Nelumbo nucifera Gaertn. seed, followed by its water, acetone, ethyl acetate and n-hexane extract. The EC50 value of embryo of Nelumbo nucifera Gaertn. seed 95% ethanol extract in scavenging DPPH free radicals was 47.32±1.20 μg/mL, while its TEAC value was 0.31 mM Trolox equivalent. The EC50 of the positive control (α-tocopherol) is 8.40±0.13 μg/mL for DPPH scavenging ability and ascorbic acid for TEAC assay (TEAC value was 1.16 mM Trolox equivalent). Different solvents extracts of the defatted Sesamum indicum L. did not show good antioxidant activity from both antioxidant assays. Results of anti-aging model experiment showed that treatment of worms with embryo of Nelumbo nucifera Gaertn. seed 95% ethanol extract had the best effect on extension the lifespan of C. elegans. Treatment of worms with 1000 μg/mL embryo of Nelumbo nucifera Gaertn. seed 95% ethanol extract resulted in an increase in mean lifespan of 25.8% (p＜ 0.001) and also extended maximum lifespan (35 days) and 80% death time (28.2 days). The effect was similar to that of 100 μM (35.5 μg/mL) EUK-8 which can increase mean lifespan by 24.1% (p＜0.001) and can also extend maximum lifespan (35 days) and 80% death time (27.0 days). After liquid-liquid partition of the 95% ethanol extract of the embryo of Nelumbo nucifera Gaertn. seed with n-hexane, ethyl ether, ethyl acetate and water consecutively, the water fraction showed the best antioxidant activity. The EC50 value of the water fraction in scavenging DPPH free radical was 25.17±0.19 μg/mL, and the result of TEAC assay (0.41 mM Trolox equivalent) was also the highest. Among the four fractions, water fraction of the embryo of Nelumbo nucifera Gaertn. seed 95% ethanol extract had the best effect on the lifespan of C. elegans. Treatment of worms with 100 μg/mL water fraction increased their mean (16.3% (p=0.0036)) and maximum (17.2%) lifespan in addition to 80% death time (12.5%). Extension of the lifespan of C. elegans also exhibited dose response was observed. The effect of 100 μg/mL water fraction had similar effect as the 25 μM (8.9 μg/mL) EUK-8 which increased mean lifespan by 16.3% (p=0.0044) and also extended maximum lifespan (13.8%) and 80% death time (14.3%). This study showed that the water fractions of the 95% ethanol extract of the embryo of Nelumbo nucifera Gaertn. seed contained components with antioxidant activity and could also prolong the lifespan of C. elegans. It thus has good potential to be developed as a functional food for anti-aging.