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研究生: 黎光鷹
Quang Ung Le
論文名稱: 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: 吳明昌
Ming-Chang Wu
賴宏亮
Horng-Liang Lay
學位類別: 博士
Doctor
系所名稱: 國際學院 - 熱帶農業暨國際合作系
Department of Tropical Agriculture and International Cooperation
畢業學年度: 107
語文別: 英文
論文頁數: 138
中文關鍵詞: 抗癌龍牙草曲序香茅鱧腸地耳草
外文關鍵詞: anticancer, Agrimonia pilosa Ledeb, Cymbopogon flexuosus (Nees ex Steud.) W. Watson, Eclipta alba Hassk, Hypericum japonicum Thunb
DOI URL: http://doi.org/10.6346/DIS.NPUST.TAIC.003.2019.D04
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  • 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(4)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。

    Six herbs chosen in this work have received considerable attention as natural products for their applications in traditional medicine. The study consists of fivestudies 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 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 ha-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 4-hydroxybenzoic acid, caffeic acid, p-coumaric acid and rosmarinic acid were the identified compounds inEclipta 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 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.

    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 1 Introduction ................................................................................... 1
    1.1 Research background ............................................................................... 1
    1.2 Research objectives ................................................................................... 3
    Chapter 2 Literature Review ......................................................................... 4
    2.1 Herbs used in the study ............................................................................. 4
    2.1.1 Agrimonia pilosa Ledeb. (AL) ............................................................... 4
    2.1.1.1 Botany ............................................................................................. 4
    2.1.1.2 Chemical composition in AL .......................................................... 5
    2.1.1.3 Pharmacological activities and the use of AL in traditional medicine ...................................................................................................... 5
    2.1.2 Eclipta alba Hassk (EH) ......................................................................... 6
    2.1.2.1 Botany ............................................................................................. 6
    2.1.2.2 Chemical composition in EH ......................................................... 7
    2.1.2.3 Pharmacological activities and the use of EH in traditional medicine ...................................................................................................... 7
    2.1.3 Mesona procumbens Hemsl (MH) ......................................................... 7
    2.1.3.1Botany .............................................................................................. 7



    VIII
    2.1.3.2 Chemical composition in MH ......................................................... 8
    2.1.3.3 Pharmacological activities and the use of MH in traditional medicine ...................................................................................................... 8
    2.1.4 Hypericum japonicum Thunb (HT) ....................................................... 9
    2.1.4.1 Botany ............................................................................................. 9
    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



    IX
    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 3 Research Methodology ............................................................... 32
    3.1 The study setup ........................................................................................ 32
    3.1.1 Study 1 ................................................................................................... 32
    3.1.2 Study 2 ................................................................................................... 32
    3.1.3 Study 3 ................................................................................................... 32
    3.1.4 Study 4 ................................................................................................... 32
    3.1.5 Study 5 ................................................................................................... 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



    X
    3.5 Component ananalysis by high performance liquid chromatography (HPLC) ........................................................................................................... 38
    3.5.1 The following gradients for study 2 .................................................... 38
    3.5.2 The following gradients for study 3 .................................................... 39
    3.5.3 The following gradients for study 4 .................................................... 39
    3.5.4 Calibration curve method .................................................................... 40
    3.6 Bioactivitie assays .................................................................................... 40
    3.6.1 Antioxidant activities ........................................................................... 40
    3.6.2 Anticancer activities ............................................................................. 42
    3.6.2.1 Cell line resouce ........................................................................... 42
    3.6.2.2 Cell culture and treatments .......................................................... 43
    3.6.2.3 Analysis of cell morphology change ......................................... 46
    3.6.2.4 Cell viability assay (MTT assay) ............................................... 47
    3.6.2.5 Lactate dehydrogenase (LDH) released determination ............ 48
    3.6.2.6 Western blotting assay .............................................................. 49
    3.7 Statistical analysis ................................................................................ 53
    Chapter 4 Results and Discussion ................................................................ 54
    4.1 Study 1: Effects of vermicompost and hog manure on growth, yield, chemical composition of Agrimonia pilosa Ledeb. ..................................... 54
    4.1.1 Chemical and physical characterization of media ............................ 54
    4.1.2 Morphological parameters of Agrimonia pilosa Ledeb. ................ 55
    4.1.3 Effects of fertilizer on total phenolic and flavonoid content and antioxidant capacity of Agrimonia pilosa Ledeb ................................. 61
    4.1.4 Conclusion ............................................................................................. 62



    XI
    4.2. Study 2: Antioxidant activities and A549 cells growth inhibitory capacity from 50% ethanol extracts of various parts of Agrimonia pilosa Ledeb. 64
    4.2.1 Total phenolic and flavonoid content ................................................. 65
    4.2.2 Antioxidant properties ......................................................................... 65
    4.2.3 High performance liquid chromatography (HPLC) analysis .......... 68
    4.2.4 Extracts affecting growth in A549 cells .............................................. 68
    4.2.5 Conclusion ......................................................................................... 72
    4.3 Study 3: Antioxidant activities and HepG2 cells growth inhibitory capacity of whole plant 50% ethanol extracts (Eclipta alba Hassk and Mesona procumbens Hemsl) ......................................................................... 73
    4.3.1 Total phenolic and flavonoid content ................................................. 74
    4.3.2 Antioxidant properties ......................................................................... 74
    4.3.3 Phenolic identification by high performance liquid chromatography
    ................................................................................................................... 76
    4.3.4 EH and MH extracts affecting growth in HepG2 cells ...................... 77
    4.3.5 Conclusion ............................................................................................. 83
    4.4 Study 4: Evaluation of antioxidant and HepG2 and A549 cells growth inhibitory capacity of whole plant 50 % ethanol extracts (Hypericum japonicum Thunb and Scoparia dulcis L) .................................................... 84
    4.4.1 Total phenolic (TPC) and flavonoid content (TFC).......................... 84
    4.4.2 Phenolic identification by high performance liquid chromatography
    ................................................................................................................... 85
    4.4.3 Antioxidant properties ..................................................................... 85
    4.4.4 The HT and SL extracts affecting growth in HepG2 and A549 cells
    ................................................................................................................... 89



    XII
    4.4.5 The HT altered the expression of apoptosis-related proteins in HepG2 and A549 cells ............................................................................ 89
    4.4.6 Conclusion ............................................................................................. 92
    4.5 Study 5: The isolation, structural characterization and anti-cancer activity from the aerial parts of Cymbopogon flexuosus (Nees ex Steud.) W. Watson. ..................................................................................................... 93
    4.5.1 Total phenolic and flavonoids content ............................................... 93
    4.5.2 Antioxidant properties ......................................................................... 98
    4.5.3 SA3, SA4 and CF extract affecting growth in HepG2 and A549 cells ......................................................................................................................... 98
    4.5.4 Conclusion ........................................................................................... 101
    Chapter 5 Conclusions and Recommendations ........................................ 103
    5.1 Conclusions ............................................................................................ 103
    5.1.1 Application of organic fertilizer in Agrimona pilosa Ledeb cultivation .............................................................................................. 103
    5.1.2 Chemical compounds isolated from the studied herbs ................... 103
    5.1.3 Antioxidant potential of the studied herbs ................................... 104
    5.1.4 Anticancer potential of the studied herbs ........................................ 104
    5.2 Limitations ............................................................................................. 104
    5.3 Future study ........................................................................................... 105
    References .................................................................................................... 107
    Appendices ................................................................................................... 135
    Bio-Sketch of Author .................................................................................. 136

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