細葉山茶樹的有益健康效應及其調控機制

The Beneficial and Healthy Effects of Camellia tenuifolia and the Underlying Mechanisms

魏嘉徵(Chia-Cheng Wei)

指導教授 ： 廖秀娟

國立臺灣大學/生物資源暨農學院/生物環境系統工程學研究所/博士(2017年)

https://doi.org/10.6342/NTU201700294

摘要

在亞洲地區，山茶屬(Camellia)的植物常被視為製作苦茶油的重要原料。雖然苦茶油為高品質食用油，但其對人類健康效益之相關科學數據卻很有限，因此本研究論文利用秀麗隱桿線蟲(Caenorhabditis elegans)作為活體模式生物來探討其保健功效及了解其背後的分子調控機制。本研究論文的具體研究目標有三項：(1) 評估生物廢棄物細葉山茶(Camellia tenuifolia)苦茶粕之潛在健康效益。(2) 鑑定細葉山茶苦茶油中主要及次要成分，並分析其在線蟲體內的抗氧化活性及基因調控機制。(3) 探討生物活性化合物β-amyrin在線蟲模式中的抗帕金森氏症效應及其分子調控機制。
針對研究目標 (1) 的部分，本研究探討生物廢棄物細葉山茶苦茶粕之保健功效。利用DPPH自由基清除及總酚化合物含量定量等試驗，評估苦茶粕抽出物其生物體外活性，並利用線蟲做為活體模式生物，研究苦茶粕抽出物於生物體內如抗氧化、抗老化及降低amyloid-β毒性等保健效應。苦茶粕抽出物之各分離部中，甲醇分離部的生物體內抗氧化活性最好，接著利用Diaion HP-20 管柱將其分成六個次分離部，其中，編號3次分離部的總酚化合物含量不僅最高，且生物體內及體外抗氧化活性最好。進一步研究發現，編號3次分離部 (1 and 10 micro;g/ml)能顯著地提升線蟲體內的抗氧化活性、降低線蟲體內活性氧物質的累積、延長線蟲壽命並延緩線蟲因amyloid-β之累積所造成的癱瘓。經分析鑑定後發現，此次分離部中主要有兩個kaempferol衍生物，且皆能在線蟲體內產生良好的抗氧化活性。總結來說，細葉山茶苦茶粕之甲醇抽出物能在線蟲體內產生多種保健效應，非常值得開發為保健食品；此外，農業生物廢棄物再利用之概念也能符合環保上之考量。
針對研究目標 (2) 的部分，本研究目標分析鑑定細葉山茶苦茶油中主要及次要之成分，並探討其在線蟲體內之抗氧化活性及調控機制。結果顯示，細葉山茶苦茶油中最主要的成分為不飽和脂肪酸(~78.4 %)，而另外兩個次成分則為 β‐amyrin 與β-sitosterol，其抗氧化活性也一併探討。研究結果顯示苦茶油(0.01%)及油酸(oleic acid, 0.066 mg/ml)皆能顯著提升線蟲的抗氧化能力，且苦茶油顯著提升線蟲抗氧化能力主要來自於油酸。更進一步的研究結果顯示，油酸與苦茶油對線蟲產生之抗氧化活性主要是經由線蟲體內的轉錄因子DAF-16/FOXO蛋白所調控。
針對研究目標 (3) 的部分，本研究目標探討苦茶油之生物活性物質β-amyrin 在線蟲模式中抗帕金森氏症之功效及其調控機制。研究結果顯示β-amyrin 能顯著提升線蟲在氧化壓力下之存活率，並且能保護線蟲抵抗因6-hydroxydopamine (6-OHDA) 所誘導之多巴胺神經細胞的損傷。此外，β-amyrin能減少線蟲體內α-synuclein的累積。更進一步研究結果顯示，β-amyrin 能提升線蟲體內LGG-1蛋白其mRNA表達量，並且能增加線蟲體內LGG-1蛋白轉移聚集的數量，表示β-amyrin可能是藉由提升線蟲體內和LGG-1相關的自噬作用(autophagy)以達到抗帕金森氏症之效應。這些研究結果顯示β-amyrin在醫學應用與營養補充上，擁有對帕金森氏症之治療與減緩病症之潛力。
總結而言，本研究結果顯示生物廢棄物細葉山茶苦茶粕具有值得進一步開發成營養保健產品之可能性，同時此農業副產物苦茶粕的再利用也符合環境保護的概念；又本研究論文之結果也提供食用細葉山茶苦油在藥理應用上的基礎知識，並提出細葉山茶苦茶油或其所含成分具有開發成對抗氧化壓力及神經退化性疾病的營養補給品、功能性食品，及其在醫療應用上的潛力。除此之外，本研究成果也能推動苦茶樹相關產業之發展，使苦茶樹成為高經濟價值之作物，並增進農民栽種本土種苦茶樹之意願，進而促進本土農業之發展。

關鍵字

細葉山茶 ； 苦茶油 ； 苦茶粕 ； 秀麗隱桿線蟲 ； 抗氧化 ； 抗帕金森氏症 ； 自噬作用 ； 油酸 ； β?amyrin ； β-sitosterol ； DAF-16轉錄因子 ； kaempferol ； 油酸 ； 氧化壓力 ； 壽命 ； amyloid-β

並列摘要

In Asia, the genus Camellia is important for tea seed oil production. Although tea seed oil is a high quality edible oil, scientific data to support the beneficial and healthy effects of tea seed oil to human beings are limited. Herein, this study investigated the beneficial and healthy effects and the underlying mechanisms, using an in vivo model organism Caenorhabditis elegans. The specific aims of this study are: (1) to evaluate the potential beneficial health effects from the biowaste Camellia tenuifolia tea seed pomace, (2) to identify major and minor components in C. tenuifolia seed oil, and to examine the antioxidant activity and its regulating mechanisms in C. elegans, and (3) to examine the bioactive compound β-amyrin on the anti-Parkinsonian effects in C. elegans disease models and its underlying mechanisms.
For specific aim1, this study evaluated the potential beneficial health effects from the biowaste tea seed pomace. DPPH radical scavenging and total phenolic content assays were performed to investigate the in vitro activities of the extracts. C. elegans was applied as in vivo model to evaluate the beneficial health effects, including antioxidant activity, delayed aging, and reduced amyloid-β toxicity. Among all soluble fractions obtained from the extracts of tea seed pomace from C. tenuifolia, the methanol (MeOH)-soluble fraction has the best in vivo antioxidant activities. The MeOH-soluble extraction was further divided into six fractions by chromatography with a Diaion HP-20 column, and fraction 3 not only had the highest phenolic contents but also showed the best in vitro and in vivo antioxidant activities. Further analysis in C. elegans showed that the MeOH extract (fraction 3) (1 and 10 micro;g/ml) of tea seed pomace significantly increased antioxidative activity, decreased intracellular reactive oxygen species level, prolonged lifespan, and delayed amyloid-β-induced paralysis in C. elegans. Moreover, bioactivity-guided fractionation yielded two kaempferol derivatives, and both compounds exhibited excellent in vivo antioxidant activity. In summary, MeOH extracts of tea seed pomace from C. tenuifolia have multiple beneficial health effects, suggesting that the reuse of agricultural biowaste tea seed pomace might be valuable to be explored for further development as nutraceutical products and also fulfills the environmental perspective.
For specific aim 2, this aim identified major and minor components in C. tenuifolia seed oil, and examined the antioxidant activity and its underlying mechanisms in C. elegans. The results showed that the major constitutes in C. tenuifolia seed oil were unsaturated fatty acids (~78.4 %). Moreover, two minor compounds, β‐amyrin and β‐sitosterol, were identified and their antioxidant activity was examined. Both C. tenuifolia seed oil (0.01 %) and oleic acid (0.066 mg/ml) significantly enhanced antioxidative activities in C. elegans. Oleic acid was found as the main constitute in C. tenuifolia seed oil and contributed to the antioxidant activity of C. tenuifolia seed oil in C. elegans. Further evidences revealed that the transcription factor DAF-16/FOXO was involved in both oleic acid- and C. tenuifolia seed oil-mediated oxidative stress resistance in C. elegans.
For specific aim 3, this aim examined the anti-Parkinsonian effects of β-amyrin, the bioactive compound in C. tenuifolia seed oil, in C. elegans disease models and its underlying mechanisms. The results showed that β-amyrin significantly increased the survival of C. elegans under oxidative stress. β-Amyrin also showed a protective effect on dopaminergic neurons against 6-hydroxydopamine (6-OHDA) induced cell damage. In addition, β-amyrin reduced the α-synuclein aggregation in C. elegans. Moreover, β-amyrin up-regulated LGG-1 mRNA expression and increased the number of localized LGG-1 puncta in C. elegans. This suggests that anti-Parkinsonian effects of β-amyrin might be regulated via LGG-1 involved autophagy pathway in C. elegans. These results suggest the potential of β-amyrin on the therapeutic applications or supplements to treat or slow the progression of PD.
In summary, this dissertation suggests that the biowaste tea seed pomace might be valuable to be explored for further development as nutraceutical products. Moreover, the reuse of agricultural byproduct tea seed pomace also fulfills the environmental perspective. The findings of this study provide fundamental knowledge of pharmacological application of C. tenuifolia seed oil diet and suggest the potential of C. tenuifolia seed oil or the contained bioactive compounds as nutritional supplements, functional foods or medical applications for oxidative stress and neurodegenerative related diseases. Furthermore, it can also promote the implementation of Camellia tree related industries and make the Camellia tree with high economic value, which can improve the farmers’ willingness to plant theses native species and promote the local agricultural development.

並列關鍵字

Camellia tenuifolia ； tea seed oil ； tea seed pomace ； Caenorhabditis elegans ； antioxidant ； autophagy ； oleic acid ； β?amyrin ； β?sitosterol ； DAF-16 ； kaempferol ； oleic acid ； oxidative stress ； lifespan ； amyloid-β

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