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國立屏東科技大學熱帶農業暨國際合作系 Department of Tropical Agriculture and International Cooperation National Pingtung University of Science and Technology 博士學位論文 Ph.D Dissertation 草藥 (龍牙草、鱧腸、仙草、地耳草、甜珠草及曲序香茅)之栽 培及生物活性研究 Study in Cultivation and Bioactivities of Herbs Agrimonia pilosa, Eclipta alba, Mesona procumbens, Hypericum japonicum, Scoparia dulcis, and Cymbopogon flexuosus 指導教授 Advisors: 吳明昌博士(Ming-Chang Wu, Ph.D.) 賴宏亮博士(Horng-Liang Lay, Ph.D.) 研究生 Student: 黎光鷹 (Le Quang Ung) 中華民國 108 年 月 15 日 June 15, 2019 摘要 學號：P10422023 論文題目: 草藥 (龍牙草、鱧腸、仙草、地耳草、甜珠草及曲序香茅)之栽 培及生物活性研究 總頁數：138 頁 學校名稱：國立屏東科技大學 系（所）別：熱帶農業暨國際合作系 畢業時間及摘要別：107 學年度第 學期博士學位論文摘要 研究生: 黎光鷹 指導教授: 吳明昌 博士 賴宏亮 博士 論文摘要內容： 本次研究中選擇六種受關注的傳統中草藥應用於天然產品。該研究 選用五項體外實驗進行研究。第一項目的為探討蚯蚓糞(VC)和豬糞肥對 龍牙草（Agrimonia pilosa Ledeb.）生長、產量和化學成分含量的影響。 其他四項研究目的為研究龍牙草 (Agrimonia pilosa Ledeb.)提取物的抗氧 化活性以及對 HepG2（人類肝癌細胞）和 A549（人類肺癌細胞）的生 長抑製效果。鱧腸（ Eclipta alba Hassk.）、仙草(Mesona procumbens Hemsl.) 、 地 耳 草 （ Hypericum japonicum Thunb ） 、 甜 珠 草 (Scoparia dulcis L.)和曲序香茅(Cymbopogon flexuosus (Nees ex Steud.) W Watson) 以乙醇萃取後進行實驗。實驗項目為總酚和類黃酮含量以及包含總抗氧 化能力(ABTS)、清除自由基能力(DPPH)以及羥自由基清除能力等抗氧 化實驗。在某些實驗中還評估了培養基中的乳酸脫氫酶含量。並利用高 效液相色譜（HPLC）方法用於分析龍牙草（Agrimonia pilosa Ledeb.）、 鱧腸（Eclipta alba Hassk.）、仙草(Mesona procumbens Hemsl.) 、 地耳 草（Hypericum japonicum Thunb.）及 甜珠草 (Scoparia dulcis L.) 中的酚 類 化 合 物 。 另 外 透 過 質 譜 儀 (MS) 和 核 磁 共 振 (NMR) 建 立 曲 序 香 茅 (Cymbopogon flexuosus (Nees ex Steud.) W Watson) 的化學結構和地上部 I 分的外部特徵。並透過西方墨點法分析凋亡因子 bcl-2、bax、bad、 caspase-3、caspase-9 和 p53 蛋白。 結果表明、對於第一項研究（1）、施用有機肥料增強了龍牙草 （Agrimonia pilosa Ledeb.）的生長、產量和化學成分含量。 pH 值在 6.9 和 7.4 之間、導電率(EC)從 0.5-0.6mS cm-1 為此種草藥生長的最佳條件。 蚯蚓糞(VC)和豬糞的最佳比例為 12.5 + 16.875 ton ha-1 的比例製成。這一 結果表明、使用有機肥料可以提升其他草藥的生長、產量和化學成分含 量 、 並進 行 進一步 的 詳細 調 查。在 第 二項 研 究（ 2） 中 、在 龍 牙草 （Agrimonia pilosa Ledeb.）萃取物中鑑定出由 4-羥基苯甲酸和對香豆酸 組成的兩種單獨的酚類化合物。與地上部分萃取物相比、根部萃取物有 更高的抗氧化力及 A549 細胞生長抑制能力。在第三項研究（3）中、鱧 腸（Eclipta alba Hassk.）萃取物經鑑定過後得知含有 4-羥基苯甲酸、咖 啡酸、對香豆酸和迷迭香酸等化合物。仙草(Mesona procumbens Hemsl.) 萃取物經鑑定過後得知含有 7-羥基香豆素、阿魏酸和芸香苷是新化合物 和兩種已知化合物。從鱧腸（Eclipta alba Hassk.）萃取物中檢測到較高 清除自由基能力(DPPH)和總抗氧化能力(ABTS)及抗癌活性。在第四項 研 究 （ ） 中 、 綠 原 酸 和 迷 迭 香 酸 是 地 耳 草 （ Hypericum japonicum Thunb.）萃取物經鑑定後所得知的化合物。芸香苷和迷迭香酸是從甜珠 草(Scoparia dulcis L.)萃取物中新測得的化合物。地耳草（Hypericum japonicum Thunb.）萃取物有更高水平的抗氧化和抗癌能力。最後的研究 （5）、1,3-O-二-E-咖啡酰甘油（SA3）和 1-O-p-香豆酰-3-O-咖啡酰甘 油（SA4）兩者為從曲序香茅(Cymbopogon flexuosus (Nees ex Steud.) W Watson) 萃取物中分離出來化合物。 SA3 化合物擁有最高的抗氧化力和 抗癌能力。 根據本次實驗結果顯示有些中草藥可能有助於為癌症疾病患者開發 新型天然藥物、還可以進一步了解草藥的功能和分子機制。製作龍牙草 （ Agrimonia pilosa Ledeb ） 、 鱧 腸 （ Eclipta alba Hassk ） 、 地 耳 草 （Hypericum japonicum Thunb.）、曲序香茅(Cymbopogon flexuosus (Nees ex Steud.) W Watson) 的機能性食品或營養補充品的膠囊。 關 鍵 字 ： 抗 癌 、 龍 牙 草 、 曲 序 香 茅 、 鱧 腸 、 地 耳 草 II Abstract Student ID: P10422023 Title of Dissertation: Study in Cultivation and Bioactivities of Herbs Agrimonia pilosa, Eclipta alba, Mesona procumbens, Hypericum japonicum, Scoparia dulcis, and Cymbopogon flexuosus Total Page: 138 pages Name of Institute: Department of Tropical Agricultural and International Cooperation, National Pingtung University of Science and Technology Graduate Date: June15, 2019 Name of Student: Le Quang Ung Degree Conferred: Doctoral Degree Advisors: Ming-Chang Wu, Ph.D Horng-Liang Lay, Ph.D The content of abstract in this dissertation: Six herbs chosen in this work have received considerable attention as natural products for their applications in traditional medicine The study consists of five studies being conducted under in vitro conditions The first study aims to investigate effects of vermicompost and hog manure on growth, yield, chemical composition of Agrimonia pilosa Ledeb Four other studies aim to investigate antioxidant activities, HepG2 (human liver cancer cell line) and A549 (adenocarcinomic human alveolar basal epithelial cell line) growth inhibitory effect from extracts of Agrimonia pilosa Ledeb Eclipta alba Hassk Mesona procumbens Hemsl Hypericum japonicum Thunb Scoparia dulcis L.and Cymbopogon flexuosus (Nees ex Steud.) W Watson Extracts of ethanol were chosen for the assays Total phenolic and flavonoid content and the antioxidant activities comprising ABTS+, DPPH, hydroxyl radical III scavenging systems were determined Lactate dehydrogenase release in medium was also evaluated in some experiments High performance liquid chromatography (HPLC) method was used to analysize phenolic compounds from Agrimonia pilosa Ledeb Eclipta alba Hassk Mesona procumbens Hemsl Hypericum japonicum Thunb Scoparia dulcis L Chemical structural characterization from the aerial parts of Cymbopogon flexuosus was established by MS and NMR spectra techniques.The apoptotic factors bcl-2, bax, bad, caspase-3 and caspase-9, p53 were analyzed by western blotting assays The results showed that, for the first study (1), application of ogarnic fertilizers enhanced the morphological growth, yield, chemical composition of Agrimonia pilosa Ledeb The pH between 6.9 and 7.4, EC from 0.5-0.6 mS cm-1 are optimal for this herbal growth A mixture of the Vermicompost and Hog manure made with the rate of 12.5 + 16.875 ton -1 produced best parameters This result suggests that using of organic fertilizers could completely enhance the morphological growth, yield, chemical composition of other herbs and futher detailed investigations are in progress In the second study (2), two individual phenolic compounds consisting of 4-hydroxybenzoic acid and p-coumaric acid were firstly identified from Agrimonia pilosa Ledeb The root extract exhibited higher antioxidant and A549 inhibitory capacity compared to the aerial part extract In the third study (3), the 4hydroxybenzoic acid, caffeic acid, p-coumaric acid and rosmarinic acid were the identified compounds in Eclipta alba Hassk The 7-hydroxycoumarin, ferulic acid and rutin being new compounds and two known compounds were identidied in the Mesona procumbens Hemsl The higher levels of DPPH and ABTS+ radical scavenging and anticancer activities were detected from Eclipta alba Hassk In the fourth study (4), the chlorogenic acid and rosmarinic acid were the identified compounds in the Hypericum japonicum Thunb The rutin and rosmarinic acid were newly identified compound from the Scoparia dulcis L The higher levels of antioxidant and anticancer IV capacity were detected from Hypericum japonicum Thunb The last study (5), 1,3-O-di-E-caffeoylglycerol (SA3) and 1-O-p-coumaroyl-3-O- caffeoylglycerol (SA4) were firstly isolated from Cymbopogon flexuosus (Nees ex Steud.) W Watson The SA3 compound showed the highest antioxidant and anticancer potent The information obtained from this work will likely contribute to the development of novel natural medicine for cancer diseasers It will also provide new insights for the futher understanding of the functions and molecular mechanism of herbs An insightful investigation to establish and manufacture capsules used as function and supplemental food from Agrimonia pilosa Ledeb Eclipta alba Hassk Hepericum japonicum Thunb Cymbopogon flexuosus (Nees ex Steud.) W Watson was recommended Keywords: anticancer, Agrimonia pilosa Ledeb, Cymbopogon flexuosus (Nees ex Steud.) W Watson, Eclipta alba Hassk, Hypericum japonicum Thunb V Acknowledgements I would like to express sincere thanks to my two advisors, Professor Dr Ming-Chang Wu and Professor Dr Horng-Liang Lay, for their invaluable guidance on my research I greatly appreciate their intellectual instructions, kindness, and supports during my Ph.D research program I would like to thank the advisory committee members for their valuable comments and constructive suggestions for the successful completion of this research work I am grateful to Taiwan Scholarship (Ministry of Education) for budget support for my Ph.D program in Taiwan I would also like to thank NPUST, DTAIC for offering me this good opportunity to pursue my doctoral study I would like to thank the personnel of the Department of Tropical Agriculture and International Cooperation; Office of International Affair, Chinese Herbal Medicine and FP201 Laboratory for their help during this study program Lastly, I would like to thank my parents for their confidence and love; my colleague, my friends for their encouragement and affection and especially my wife who was always there with wisdom, inspiration and cheer VI Table of Contents 摘要 I Abstract III Acknowledgements VI Table of Contents VII List of Tables XIII List of Figures XV List of Abbreviation XVIII Chapter Introduction 1.1 Research background 1.2 Research objectives Chapter Literature Review 2.1 Herbs used in the study 2.1.1 Agrimonia pilosa Ledeb (AL) 2.1.1.1 Botany 2.1.1.2 Chemical composition in AL 2.1.1.3 Pharmacological activities and the use of AL in traditional medicine 2.1.2 Eclipta alba Hassk (EH) 2.1.2.1 Botany 2.1.2.2 Chemical composition in EH 2.1.2.3 Pharmacological activities and the use of EH in traditional medicine 2.1.3 Mesona procumbens Hemsl (MH) 2.1.3.1Botany VII 2.1.3.2 Chemical composition in MH 2.1.3.3 Pharmacological activities and the use of MH in traditional medicine 2.1.4 Hypericum japonicum Thunb (HT) 2.1.4.1 Botany 2.1.4.2 Chemical composition in HT 10 2.1.4.3 Pharmacological activities and the use of HT in traditional medicine 10 2.1.5 Scoparia dulcis L (SL) 10 2.1.5.1 Botany 10 2.1.5.2 Chemical composition in SL 11 2.1.5.3 Pharmacological activities and the use of SL in traditional medicine 11 2.1.6 Cymbopogon flexuosus (Nees ex Steud.)W Watson (CF) 12 2.1.6.1 Botany 12 2.1.6.2 Chemical composition in CF 12 2.1.6.3 Pharmacological activities and the use of CF in traditional medicine 13 2.2 Application of organic materials in plant cultivation 13 2.3 The chemistry of phenolic compounds 16 2.3.1 Phenolic acid 17 2.3.2 Flavonoids 17 2.3.3 Tannins 20 2.4 Quantification of phenolics 20 2.4.1 Spectrophotometric assays 20 2.4.2 High performance liquid chromatogaraphy (HPLC) 21 VIII 2.5 Antioxidants 22 2.5.1 What is an antioxidant? 22 2.5.2 Basics of antioxidant characterization 23 2.5.3 Reactive oxygen species 23 2.5.4 Antioxidant assay 24 2.5.5 Synthetic phenolic and natural antioxidants 24 2.6 Introduction of cancers 25 2.6.1 Cancer 25 2.6.2 Introduction of human tumor cell lines 26 2.7 Introduction of apoptosis 27 2.7.1 What is apoptosis? 27 2.7.2 Bcl-2 family protein in apoptosis 28 2.7.3 Caspases in apoptosis 29 2.7.4 Effects of antioxidants on cancer 29 Chapter Research Methodology 32 3.1 The study setup 32 3.1.1 Study 32 3.1.2 Study 32 3.1.3 Study 32 3.1.4 Study 32 3.1.5 Study 32 3.2 Plant cultivation of Agrimonia pilosa Ledeb 33 3.3 Preparation of samples 35 3.4 Determination of total phenolic and flavonoid contents 36 IX Nguyen, V.D., andDoan, T.N (1989) Medicinal Plants in Vietnam, World Health Organization, 101 Nho, K.J., Chun, J.M., and Kim, H.K (2011) Agrimonia pilosa ethanol extract induces apoptotic cell death in HepG2 cells Journal of Ethnopharmacology 138: 358-363 O’ Brien, T.A., and Barker, A.V (1996) Growth of peppermint in compost Journal of Herbs,Spices& Medicinal Plants 4: 19-27 Ola, M.S., Nawaz, M., and Ahsan, H (2011) Role of Bcl-2 family proteins and caspases in the regulation of apoptosis Molecular and Cellular Biochemistry 351: 41-58 Orsi, F., and Varano, G (2015) Minimal invasive treatments for liver malignancies Ultrasonic Sonochemistry 27: 659-667 doi: 10.1016/j.ultsonch.2015.05.030 Ordonez, A.L., Gomez, J.D., Vattuone, M.A., and Isla, M.I (2006) Antioxidant activities of Sechium edule (Jacq.) 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Hassk Acta Pharmaceutica Sinica 32: 633-634 Zhang, J.S., and Guo, Q.M (2001) Studies on the chemical constituents of Eclipta prostrate (L) Acta Pharmaceutica Sinica 36: 34-37 Zhu, L., Tan, J., Wang, B., He, R., Liu, Y., and Zheng, C (2009) Antioxidant activities of aqueous extract from Agrimonia pilosa Ledeb and its fractions Chemistry and Biodiversity 6: 1716-1726 134 Appendices Appendix Mass spectrum of compounds SA3 and SA4 Inten.(x1,000,000) 259.15 2.5 215.10 2.0 1.5 245.15 383.05 303.50 1.0 338.45 0.5 0.0 200 289.10 250 300 527.45 455.45 417.30 361.20 350 400 622.85 543.95 450 500 671.60 550 600 650 m/z SA3-ESI+ Inten.(x10,000,000) 1.00 0.75 464 401 0.50 383 423 0.25 237 0.00 200 250 282 311 300 350 527 543 450 338 400 SA4-ESI+ 135 450 500 550 600 594 m/z Bio-Sketch of Author Full name: Le Quang Ung Gender: Male Nationality: Vietnamese Education background Major Education Ph.D Herbal medicine and natural products Master Crop Sciences Bachelor Crop Sciences Date Institution 2015 – National Pingtung University of 2019 Science and Technology, Taiwan 2011– 2013 2006– 2011 Thai Nguyen University, Vietnam Thai Nguyen University, Vietnam Published Papers Le, Q.U., Lay, H.L., and Wu, M.C (2017) Antioxidant activities and HepG2 cells growth inhibitory capacity of whole plant ethanol extracts (Eclipta alba Hassk and Mesona procumbens Hemsl) Journal of Food Biochemistry, Doi: 10.1111/jfbc.12454 42(2) (SCI paper) Le, Q.U., Lay, H.L., and Wu, M.C (2018) The isolation, structural characterization and anti-cancer activity from the aerial parts of Cymbopogon flexuosus Journal of Food Biochemistry Doi: 10.1111/jfbc.12718 (SCI paper) Le, Q.U., Lay, H.L., Wu, M.C., Nguyen, D.L., and Nguyen, T.H.H (2018) Phytoconstituents and Biological Activities of Panax vietnamensis (Vietnamese Ginseng): A Precious Ginseng and Call for Further Research-A systematic review Natural product communications 13(10), 1381-1384 (SCIE paper) 136 Le, Q.U., Lay, H.L., and Wu, M.C (2018) Comparative study of Anti-oxidant and alpha amylase Inhinitory Activity of Six herbs used in traditional medicine International Journal of Scientific and Engineering Research, 9(3), 510-529 Le, Q.U., Lay, H.L., and Wu, M.C (2018) Effects of Vermicompost and Hog Manure on Growth, Yield and Antioxidant Activities of Agrimonia Pilosa Ledeb as a medicine plant Biological forum- An International Journal, 10(1), 56-63 (Web of Science Platform) Le, Q.U., Joshi, R.K., Lay, H.L., and Wu, M.C (2018) Agrimonia pilosa Ledeb: Phytochemistry, Ethnopharmacology, Pharmacology of an important traditional herbal medicine Journal of Pharmacognosy and Phytochemistry, 7(4), 3202-3211 Le, Q.U., Lay, H.L., and Wu, M.C (2018) Antioxidant activities and A549 growth inhibitory capacity from various extracts of Agrimonia pilosa Ledeb Journal of Ayurvedic and herbal medicine.4(3), 114-119 Le, Q.U., Lay, H.L., Wu, M.C., and Nguyen, T.H.H (2018) Natural plant colorants widely used widely in Vietnam traditional food culture Journal of Food, nutrition and Agriculture 1(1), 40-46 Le, Q.U., Lay, H.L., Wu, M.C., Nguyen, D.L., and Nguyen, T.H.H (2018) Hovenia dulcis Thunb revisited-A mini critical review and call for further research to insightfully elucidate Journal of Pharmacognosy and Phytochemistry 7(6), 1137-1141 10 Le, Q.U., Lay, H.L., and Wu, M.C (2019) Antioxidant potential and HepG2 and A549 cell growth inhibitory capacity of Hypericum japonicum Thunb and Scoparia dulcis L Journal of medicinal plants studies.Accepted 137 11 Le, Q.U., Lay, H.L., and Wu, M.C (2019) Medicinal botanicals in the osteoarthritis treatment and management in traditional Vietnamese medicine International Journal of Botany Studies 4(3), 59-62 12 Le, Q.U., Lay, H.L., and Wu, M.C (2018) Phytoconstituents and pharmacological activities of Silybum marianum (Milk Thistle): A critical review American Journal of Essential Oils and Natural Products 6(4):41-47 13 Le, Q.U., Lay, H.L., and Wu, M.C (2019) Herbs for the Management of Diabetes Mellitus in Traditional Vietnamese Medicine Journal of Applied Biopharmaceutics and Pharmacokinetics 7, 1-6 138 ... stamens 3, anthers 1-2 mm long; ovary c mm, oblong, stigmas 11.5 mm long; caryopsis c mm long, elliptic Pedicelled spiketets 3-4 mm long, lanceolate; pedicels c mm long, densely villious; lower... unilocular, about 2-3 mm long, ovules many, styles 3, free from the base, erect, about 1.2 mm long Fruit capsule septicidal, ovoidsubglobose, about 4-4.5 x 2.5-3 mm long, apex obtuse, longitudinally vittate,... pedicel about 4-7 mm long, bracts linear lanceolate, foliaceous, about 2-2.5 mm long, caduceus, sepals 5, unequal, quincuncial, outer 2, lanceolate-ovate, inner 3, oblanceolate-oblong, chartaceous,