Nghiên cứu canh tác và các hoạt động sinh học của các thảo dược: long nha thảo; hạn liên thảo, sương sáo, điền cơ hoàng, cam thảo đất và sả dịu

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 6 月 15 日

June 15, 2019

I

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 2 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

II

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。

關鍵字 ： 抗 癌 、 龍 牙 草 、 曲 序 香 茅 、 鱧 腸 、 地 耳 草

III

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 Degree Conferred: Doctoral Degree

Name of Student: Le Quang Ung 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

IV

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 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

V

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

VI

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.

VII

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

XIII

List of Tables

Table 1.Classes of phenolic compounds in plants ..........................................18

Table 2. Composition of transfer buffer (pH 8.4-9.2, 1L)..............................52

Table 3. Component of TBST-1L...................................................................52

Table 4. Component of sperating gel (2 gels).................................................52

Table 5. Component of stacking gel (2 gels) ..................................................52

Table 6. Chemical and physical characteristics of different vermicompost rate

mixture with hog manure ................................................................................57

Table 7. Effects of different fertilizer rates on the morphological growth of

Agrimonia pilosa Ledeb. after 120 days.........................................................58

Table 8. Effects of different fertilizer rates on the morphological growth of

Agrimonia pilosa Ledeb. after 120 days.........................................................58

Table 9. Effects of different fertilizer rates on total phenolic content and

antioxidant activity of Agrimonia pilosa Ledeb. ............................................59

Table 10. Pearson correlation between macro- and micro nutrients and herbal

quality of Agrimonia pilosa Ledeb .................................................................59

Table 11. Various extracts induced growth inhibition in A549 cells .............69

Table 12. DPPH free radical scavenging activities of EH and MH................75

Table 13. The contents of phenolic components of EH and MH....................79

Table 14. The EH and MH extracts induced growth inhibition in HepG2.....79

XIV

Table 15. Phenolic contents, flavonoid contents and antioxidant activities of

the HT and SL extracts....................................................................................87

Table 16. Total phenolic and flavonoid contents obtained by different solvents

.........................................................................................................................95

Table 17. 1H (500 MHz) and 13C-NMR (125 MHz) data for compounds SA3

and SA4 ...........................................................................................................95

Table 18. The antioxidant effects and cytotoxicity of compounds SA3, SA4

and ethanol 50% extract against two cancer cell lines..................................100