數種花果萃出物及 stilbenoids 化合物之抗氧化活性與線蟲模式下之抗老化功效

Abstract

老化是一種生物體漸進式喪失生理功能、活動能力與認知能力的現象。過去的許多研究曾指出，氧化壓力是造成老化發生的重要因素，因此本研究選取了一些在先前研究中被證實具有良好抗氧化能力的樣品，包括龍眼花水萃物 (L-W)、桑椹水萃物 (M-W)、洛神葵水萃物 (R-W)、蓮子心乙醇萃物的水區分層 (N-E-W)、何首烏 stilbene glycoside (stilbene glycoside from Polygonum multiflorum Thunb., PM-SG) 及 pterostilbene，於秀麗隱桿線蟲 (Caenorhabditis elegans) 模式下進行抗老化活性的探討，並於化學模式與細胞模式中探討其抗氧化活性。 論文工作的第一部份為化學性的體外抗氧化實驗，選取四種抗氧化活性評估方法包括 DPPH 自由基清除能力試驗、總抗氧化能力 (TEAC) 試驗、氧自由基吸收能力 (ORAC) 試驗及還原能力測定，另外也進行總酚含量的分析。實驗結果顯示，L-W、PM-SG 與 pterostilbene 有較優異的體外抗氧化活性，而 N-E-W、M-W 與 R-W 則依序為抗氧化能力最差的三者，而總酚含量的分析結果也顯示 L-W 的總酚含量最高，依序為 N-E-W、M-W 與 R-W，與抗氧化能力一致。 論文工作的第二部份為細胞模式的抗氧化實驗，根據各樣品對 Hep G2 細胞存活率的影響結果，決定各樣品之最高實驗濃度。實驗結果顯示 L-W 具有最好的細胞抗氧化活性，其 EC50 值為 25.51 ± 2.17 μg/mL，其次依序為 PM-SG (EC50 = 211.35 ± 34.11 μg/mL)、N-E-W (EC50 = 457.83 ± 34.33 μg/mL) 與 M-W (EC50 = 776.66 ± 28.20 μg/mL)；而 R-W 達 500 μg/mL 尚未展現顯著之活性，pterostilbene 則是受限於細胞毒性作用而無法展現其抗氧化活性。與化學性抗氧化實驗結果相比，PM-SG 與 pterostilbene 的活性表現有較大差異，推測為兩者化學結構之差異影響在細胞模式中表現的抗氧化能力。 論文工作的第三部份為線蟲模式的抗老化實驗，首先於壽命期實驗中進行各樣品的延緩老化能力測定，結果發現只有 PM-SG、L-W 與 N-E-W 有顯著的活性表現，其延長線蟲壽命之最大程度分別為 15.8%、17.1% 與 15.5%，與體外抗氧化實驗的結果相比對，發現只有在細胞模式中有表現出抗氧化活性的樣品，才能在線蟲模式中表現出延緩老化的活性。進一步測定 PM-SG、L-W 與 N-E-W 對線蟲老化過程中生理表徵變化的影響，發現三者皆不影響線蟲的自體受孕生殖期，但皆能延緩咽喉收縮率的降低，顯示樣品延緩老化的效果應該不是經由影響其生殖能力所致。咽喉收縮率是線蟲模式的老化研究中已被證實的一項老化指標，因此可藉此佐證樣品延緩老化的活性。此外也測定 PM-SG、L-W 與 N-E-W 對改善線蟲的環境壓力抗性是否有幫助，發現三者皆可有效提高線蟲在高溫環境下的存活率，且最大程度分別達 46.8%、53.6% 與 32.3%，但僅 PM-SG 與 L-W 可有效提高線蟲在高氧化壓力環境下的存活率，其最大程度分別達 81.7% 與 113.7%，推測此實驗系統之氧化壓力過高，以致抗氧化能力較低的 N-E-W 無法與之抗衡而表現出其抗氧化活性。 本研究結果顯示，在化學性抗氧化實驗與細胞抗氧化實驗中皆表現出抗氧化能力的前三者，即 L-W、PM-SG 與 N-E-W，也同時是線蟲模式抗老化實驗結果中具有活性者，顯示抗氧化能力與延緩老化之功效具有相關性。

Aging is a progressive phenomenon of losing physiological function, behavior and cognitive ability over time. Previous studies have pointed out that oxidative stress is an important factor that causes aging. Therefore, some previously shown antioxidative materials were chosen, including water extract of Longan flower (Dimocarpus longan Lour.) (L-W), water extract of mulberry (Morus alba Linn.) (M-W), water extract of roselle (Hibiscus sabdariffa Linn.) (R-W), water fraction from ethanol extract of embryo of Nelumbo nucufera Gaertn.(N-E-W), stilbene glycoside from Polygonum multiflorum Thunb.(PM-SG) and pterostilbene. The objective is to investigate the anti-aging ability of these materials in the Caenorhabditis elegans model. The first part of this study was the chemical in vitro antioxidative experiments, including DPPH free radicals scavenging assay, trolox equivalent antioxidant capacity assay, oxygen radical absorbance capacity assay, reducing power assay and total polyphenols analysis. Results showed that L-W, PM-SG and pterostilbene had excellent activities followed by N-E-W, M-W and R-W. The second part of this study was the cellular antioxidant activity assay. The concentrations of each sample used in the assay were determined according to the cytotoxicity of each sample on Hep G2 cell. Results showed that L-W had the best cellular antioxidant activity with EC50 value 25.51 ± 2.17 μg/mL, followed by PM-SG (EC50 = 211.35 ± 34.11 μg/mL), N-E-W (EC50 = 457.83 ± 34.33 μg/mL) and M-W (EC50 = 776.66 ± 28.20 μg/mL). At 500 μg/mL R-W still did not have apparent activity while the cytotoxicity of pterostilbene was too high to express its antioxidant activity. Compared with the results of chemical antioxidant experiments, there were large differences in the activities of PM-SG and pterostilbene, indicating that the cellular antioxidant activity of these two compounds were influencesd by the structure. The third part of this study was the anti-aging tests in C. elegans model. We first investigated the lifespan prolonging effects of different samples. Results showed that only PM-SG、L-W and N-E-W had apparent anti-aging activities, with greatest degree of lifespan extention of 15.8%, 17.1% and 15.5%, respectively. Compared with the results of chemical antioxidant experiments, only samples exhibiting cellular antioxidant activities demonstrated anti-aging effects. Further examination of the effects of PM-SG、L-W and N-E-W on age-related changes of physiological processes, the results showed that PM-SG, L-W and N-E-W did not influence the self-fertile reproductive span, but did delay the decrease of pharyngeal pumping rate. This finding indicated that the lifespan prolonging effects of the three samples were not via influencing reproduction ability. On the other hand, since pharyngeal pumping rate had already been proved as one of the aging index, the finding of pharyngeal pumping rate test can be another evidence of the anti-aging ability of these samples. In addition, experiments to test if PM-SG, L-W and N-E-W are helpful to worms to resist the environmental pressure were also conducted, and the results showed that PM-SG, L-W and N-E-W can improve the survival rate of worms under thermal-stress, with greatest degree of lifespan extention of 46.8%, 53.6% and 32.3%, respectively. Only PM-SG and L-W, however, are helpful to worms to resist the oxidative-stress from H2O2, with greatest degree of lifespan extention of 81.7% and 113.7%, respectively. We speculated that the oxidative-stress was too high in this system, so that N-E-W with less antioxidant activity, can not express its antioxidant ability. In summery, samples, which showed better in vitro antioxidant activity, seemed to be the more effective ones in anti-aging tests in C. elegans model as well. This indicated that antioxidant activities correlate closely to anti-aging effects.