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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 孫璐西 | |
dc.contributor.author | Yi-Shiuan Ho | en |
dc.contributor.author | 何怡萱 | zh_TW |
dc.date.accessioned | 2021-06-08T04:42:26Z | - |
dc.date.copyright | 2009-08-14 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-08-07 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23110 | - |
dc.description.abstract | 老化是生物體的一種自然生理變化,然而不同個體老化的速率卻不盡相同,影響老化速率的因素有很多,其中一個主要的因素是所攝入的食物。故本研究以秀麗隱桿線蟲 (Caenorhabditis elegans) 為實驗模式,並以 EUK-8 及 resveratrol 為正控制組,探討巴西洋菇 (Agaricus blazei Murill (Ab))、蓮子心(embryo of Nelumbo nucifera Gaertn. seed (Nn))、(-)-epicatechin 及 proanthocyanidin A2 (PA2) 這些食材及所含純品在於延緩老化的功效。
首先,利用線蟲耐高溫存活試驗去篩選具有延緩老化活性的物質,結果顯示正控制組 EUK-8 及 resveratrol 都可增加線蟲在高溫環境下的存活率。而樣品中 (-)-epicatechin、PA2、巴西洋菇水萃物 (Ab-W)、蓮子心 70% 甲醇萃物 (Nn-M)、蓮子心甲醇萃物之正丁醇區分層 (Nn-M-B),及其第五區分物 (Nn-M-B-V),也都具有增加線蟲耐熱的活性。 為得知具有增加線蟲耐熱性的樣品對延緩老化方面的影響,故進行壽命實驗。結果顯示,正控制組 EUK-8 (50 μM) 及 resveratrol (100 μM) 均可延長線蟲壽命分別達 10.7% 及 3.0%。在樣品方面,(-)-epicatechin (50 μM)、PA2 (100 μM)、Ab-W (100 ppm)、Nn-M (1000 ppm)、Nn-M-B (100 ppm) 及 Nn-M-B-V (25 ppm) 也都可延長線蟲的壽命,延長程度分別為 12.7%、8.9%、9.3%、12.0%、12.5% 及 8.2%。由此結果發現,這些樣品確實具有延緩老化的活性。 在過氧化氫誘導的氧化壓力實驗中,EUK-8 (50 μM)、(-)-epicatechin (50 μM)及 PA2 (100 μM) 分別可增加線蟲存活率達 63.3%、71.1% 及 78.5%,在統計上三者的效果是沒有差異的,而resveratrol (100 μM) 則僅增加 15.6% 的存活率。已知 EUK-8 主要是透過抗氧化活性來延長壽命,而 (-)-epicatechin 及 PA2 也具有很強的抗氧化活性,推測這兩個純物質可能是透過抗氧化活性,來延長線蟲壽命。雖然 Ab-W (100 ppm) 及 Nn-M-B (100 ppm) 延長線蟲壽命的能力與 EUK-8 相似,但在 H2O2 誘導的氧化壓力實驗中,卻僅能增加20.3% 與 44.5% 的存活率,推測這兩個混合物可能不完全是透過抗氧化活性,來達到延長壽命的效果。 在咽喉收縮率試驗部分,發現 (-)-epicatechin、PA2、Ab-W 及 Nn-M-B 均有延緩咽喉收縮率下降的情況,顯示樣品不會降低線蟲的攝食量,故可排除樣品延長壽命的作用機制是透過熱量限制 (caloric restriction)。 為瞭解具有延緩線蟲老化活性的樣品其抗氧化的能力,故進行體外 DPPH自由基清除能力分析及總酚含量測定,發現(-)-epicatechin 及 PA2 具有最好的清除力,次佳者為正控制組 trolox 及α-tocopherol,Nn-M-B 再次之,而 Ab-W 及巴西洋菇乙醇萃物 (Ab-E) 為最差。 總酚含量測定結果,Nn-M-B 含有 279.5 ± 5.7 (mg catechin/g of dry weight),Ab-E 含有 37.6 ± 2.5 (mg catechin/g of dry weight),Ab-W 含量最少為 22.6 ± 0.8 (mg catechin/g of dry weight)。 綜合以上結果,推測 (-)-epicatechin 與 PA2 主要是透過抗氧化活性增加線蟲壽命,而巴西洋菇及蓮子心延長線蟲的壽命,並非透過抗氧化活性,可能還有其他作用機制。 | zh_TW |
dc.description.abstract | Aging is one of the natural physiological changes in living organisms. However, the aging rate is different for each individual. Many factors influence aging and one major factor is food intake. Therefore, the aim of this study is to evaluate the potential of some edible materials, including Agaricus blazei Murill, embryo of Nelumbo nucifera Gaertn. seed, (-)-epicatechin, and proanthocyanidin A2 to delay aging in C. elegans model and we used EUK-8 and resveratrol as the positive controls.
We first investigated the thermotolerance effects of different samples. Results showed that the positive controls, EUK-8 and resveratrol, could increase thermotolerance in C. elegans. In the experimental samples, (-)-epicatechin, proanthocyanidin A2 (PA2), water extract of Agaricus blazei Murill (Ab-W), 70% methanol extract of embryo of N. nucifera Gaertn. seed (Nn-M), n-butanol fraction of Nn-M (Nn-M-B) and the fifth sub-fraction obtained after column chromatography of Nn-M-B (Nn-M-B-V) all displayed thermotolerance activities. To further examine the anti-aging effect, the lifespan assay was carried out for the samples possessing thermotolerance activities. Both EUK-8 (50 μM) and resveratrol (100 μM) could increase lifespan of C. elegans by 10.5% and 3.0%, and the other test samples including (-)-epicatechin (50 μM), PA2 (100 μM), Ab-W (100 ppm), Nn-M (1000 ppm), Nn-M-B (100 ppm), and Nn-M-B-V (25 ppm) could also increase lifespan of C. elegans by 12.7%, 8.9%, 9.3%, 12.0%, 12.5%, and 8.2%, respectively. The results suggested that test samples had good anti-aging activities. In the assay of oxidative-stress induced by H2O2, EUK-8 (50 μM), (-)-epicatechin (50 μM), and PA2 (100 μM) raised the survival rates by 63.3%, 71.1%, and 78.5%, respectively, statistically all three of them possessed the same level of activity. It is known that EUK-8 extends the lifespan of C. elegans by acting as an antioxidant, so (-)-epicatechin and PA2 may also prolong lifespan by the strong antioxidant activities. Ab-W (100 ppm) and Nn-M-B (100 ppm) only increased the survival rates by 20.3% and 44.5%. Although the ability of EUK-8, Ab-W and Nn-M-B in extending the lifespan were similar, their effects in increasing the survival rate under oxidative stress were quite different. It is, therefore, suggested that Ab-W and Nn-M-B prolong the lifespan not only by antioxidant property but also by other mechanisms. In pharyngeal pumping rate test, we found that (-)-epicatechin, PA2, Ab-W, and Nn-M-B could delay the age-related declines of pharyngeal pumping. The results also indicated that those samples did not decrease the food intake of C. elegans, so the extended lifespan by caloric restriction could be excluded. To understand the antioxidant activity of the samples with anti-aging activities, we conducted in vitro antioxidative experiments, including DPPH free radical scavenging and total polyphenols assays. In DPPH free radical scavenging assay, (-)-epicatechin and PA2 showed better activity than the positive controls trolox and α-tocopherol followed by Nn-M-B. Howerver, Ab-W and ethanol extract of Agaricus blazei Murill (Ab-E) displayed the least activity. In the total polyphenols assay, Nn-M-B had higher total polyphenols content than Ab-E and Ab-W. In this research, we found that (-)-epicatechin and PA2 extended the lifespan of C. elegans mainly by antioxidant activity. However, Nn-M-B and Ab-W may increase the lifespan of C. elegans by other mechanisms than antioxidant activity. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T04:42:26Z (GMT). No. of bitstreams: 1 ntu-98-R96641001-1.pdf: 2263214 bytes, checksum: d9df9e3e7e232c75f3649d78105277f4 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 摘要................................................................................................................................ I
Abstract.......................................................................................................................III 目錄…………………………………………………………………………………...V 表次...............................................................................................................................X 圖次..............................................................................................................................XI 壹、前言........................................................................................................................1 貳、文獻整理................................................................................................................2 一、老化....................................................................................................................2 二、老化的相關理論................................................................................................4 (一) 演化理論 (Evolutionary theories)................................................................4 (二) 基因調節理論 (Gene Regulation Theory)...................................................4 (三) 細胞衰老理論 (Cell senescence theory) .....................................................5 (四) 神經內分泌理論 (Neuroendocrine theory) .................................................6 (五) 神經內分泌免疫理論 (Neuroendocrine-immuno theory)...........................6 (六) 自由基理論 (Free radical theory) -氧化壓力理論 (Oxidative stress theory) ...............................................................................................................................6 三、秀麗隱桿線蟲 (Caenorhabditis elegans).........................................................9 四、線蟲與老化研究..............................................................................................13 (一) 線蟲在學術研究上之歷史與應用.............................................................13 (二) 延長線蟲壽命相關的基因.........................................................................15 (三) 線蟲老化相關的生理變化.........................................................................16 1、咽喉收縮率 (Pharyngeal pumping rate)...................................................17 2、身體的移動性 (Body movement) ............................................................17 3、自體授精繁殖能力 (Self-fertile reproduction) ........................................18 4、知覺 (Sensory perception) ........................................................................18 5、排便 (Defecation) .....................................................................................18 6、螢光物質的累積 (Lipofuscin)..................................................................18 7、壓力抗性的降低........................................................................................19 8、基因對於線蟲生長階段的影響................................................................21 (四) 延長線蟲壽命相關研究介紹.....................................................................21 五、材料介紹..........................................................................................................28 (一) 蓮子心 (Embryo of N. nucifera Gaertn. seed) ..........................................28 1. 蓮之發現與由來.........................................................................................28 2. 蓮之分類及型態特徵.................................................................................28 3. 蓮子心之生理活性.....................................................................................29 (二) 巴西洋菇 (Agaricus blazei Murill)............................................................31 1. 巴西洋菇之發現與由來.............................................................................31 2. 巴西洋菇之分類及型態特徵.....................................................................31 3. 巴西洋菇之組成.........................................................................................31 4. 巴西洋菇之生理活性及其活性成分.........................................................31 (三) (-)-Epicatechin...........................................................................................33 (四) Proanthocyanidin A2.................................................................................34 (五) 白黎蘆醇 (resveratrol) (正控制組) ...........................................................36 (六) EUK-8 (正控制組)....................................................................................36 參、研究目的與實驗架構..........................................................................................37 一、研究目的..........................................................................................................37 二、實驗架構..........................................................................................................38 (一) 巴西洋菇 (Agaricus blazei Murill)............................................................38 (二) 蓮子心 (Embryo of N. nucifera Gaertn. seed)...........................................39 (三) (-)-Epicatechin 及 Proanthocyanidin A2.................................................41 肆、材料與方法..........................................................................................................42 VII 一、實驗材料..........................................................................................................42 (一) 巴西洋菇 (Agaricus blazei Murill)............................................................42 (二) 蓮子心 (Embryo of N. nucifera Gaertn. seed)...........................................42 (三) (-)-Epicatechin...........................................................................................42 (四) Proanthocyanidin A2.................................................................................43 (五) Resveratrol ………………………………………………………………44 二、實驗線蟲與菌種..............................................................................................43 (一) 線蟲品系 (Caenorhabditis elegans strains)...............................................43 (二) 菌種.............................................................................................................43 三、試劑與試藥......................................................................................................43 (一) 化學藥品.....................................................................................................43 (二) 溶劑.............................................................................................................45 四、線蟲實驗之溶液與培養基配方......................................................................45 (一) NGM agar.....................................................................................................45 (二) M9 buffer......................................................................................................46 (三) S medium .....................................................................................................46 (四) 含 0.16 mM H2O2 之 S medium 配製....................................................47 (五) Escherichia coli OP50 之培養基................................................................47 五、儀器設備..........................................................................................................48 (一) 樣品製備及化學抗氧化能力分析相關儀器設備.....................................48 (二) 線蟲模式實驗相關儀器設備.....................................................................49 六、實驗方法..........................................................................................................50 (一) 樣品製備.....................................................................................................50 1. 巴西洋菇.....................................................................................................50 a 熱水萃出物..............................................................................................50 b 乙醇、丙酮、乙酸乙酯及正己烷萃出物..............................................50 2. 蓮子心.........................................................................................................50 a. 70 %甲醇粗萃物製備.............................................................................50 b. 70 %甲醇萃物之溶劑區分萃取 (liquid-liquid partition) ......................50 c. Sephadex LH-20 管柱層析......................................................................50 (二) 樣品萃出率之測定.....................................................................................53 (三) 以線蟲為模式之延緩老化活性測定.........................................................53 1. E. coli OP50 之培養...................................................................................53 2. 含有 E. coli OP50 之 NGM agar plates之製備......................................53 3. 線蟲之單一寄生物培養 (Monoxenic culture) ....................................54 4. 線蟲之蟲卵分離與同步化 (Synchronize)................................................54 5. 線蟲保存與解凍.........................................................................................54 6. 線蟲壽命期測定.........................................................................................55 7. 線蟲耐高溫試驗.........................................................................................56 8. 線蟲耐高氧化壓力試驗.............................................................................57 9. 線蟲咽喉收縮率.........................................................................................57 (四) 抗氧化活性測定.........................................................................................58 1. DPPH 自由基清除能力之測定.................................................................58 2. 總酚含量測定.............................................................................................58 (五) 統計分析.....................................................................................................59 伍、 結果與討論........................................................................................................60 一、不同溶劑萃取巴西洋菇發酵產物及蓮子心之萃出率..................................60 (一) 巴西洋菇 (Agaricus blazei Murill)............................................................60 (二) 蓮子心 (Embryo of N. nucifera Gaertn. seed)...........................................61 二、以線蟲為模式評估延緩老化之活性..............................................................63 (一) 線蟲耐高溫試驗.........................................................................................63 (二) 線蟲壽命期測試.........................................................................................78 (三) 線蟲耐高氧化壓力試驗.............................................................................91 (四) 線蟲咽喉收縮率測定.................................................................................93 三、體外抗氧化活性測定......................................................................................96 (一) DPPH 自由基清除能力..............................................................................96 (二) 總酚含量測定.............................................................................................99 四、討論................................................................................................................100 陸、結論....................................................................................................................104 柒、後續研究方向建議............................................................................................105 捌、參考文獻............................................................................................................106 玖、附錄....................................................................................................................119 | |
dc.language.iso | zh-TW | |
dc.title | 蓮子心、巴西洋菇、(-)-epicatechin及proanthocyanidin A2
之延緩線蟲老化功效 | zh_TW |
dc.title | Effects of embryo of Nelumbo nucifera Gaertn. seed, Agaricus blazei Murill, (-)-epicatechin and proanthocyanidin A2 on anti-aging of Caenorhabditis elegans | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蕭明熙,羅時成,周成功,何其儻 | |
dc.subject.keyword | 線蟲,老,化,蓮,子心,巴西洋菇,(-)-epicatechin,proanthocyanidin A2, | zh_TW |
dc.subject.keyword | Caenorhabditis elanegs,aging,Nelumbo nucifera Gaertn.,Agaricusblazei Murill,(-)-epicatechin,proanthocyanidin A2, | en |
dc.relation.page | 124 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2009-08-07 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 食品科技研究所 | zh_TW |
顯示於系所單位: | 食品科技研究所 |
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