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

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 dulc

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

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。

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.)

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

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

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

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

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

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

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

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

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

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 HepG 2 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

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

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

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

List of Figures

Figure 1 The herbs used in study Agrimonia pilosa Ledeb (a); Eclipta alba Hassk (b); Mesona procumbens Hemsl (c); Hypericum japonicum Thunb (d); Scoparia dulcis L (e); and Cymbopogon flexuosus (Nees ex Steud.) W Watson.(f) 15

Figure 2 Examples of hydroxybenzoic acid (a) and hydroxycinnamic acid (b) 18

Figure 3 Generic structure of a flavonoid molecule 19

Figure 4 Structure of hydrolyzable (a) and condensed tannins (b) (Luthar, 1992) 19

Figure 5 Antioxidant behavior content/uploads/2013/11/antioxidant-c60.jpg) 19

(Source:http://multimmunity.com/wp-Figure 6 Summary of the extrinsic and intrinsic pathway in apoptosis 31

Figure 7 The schematic diagram of SA3 and SA4 compounds isolation from ethyl acetate fraction 37

Figure 8 Dry material yield per ha after 120 days planting of Agrimonia

pilosa Ledeb 60

Figure 9 ABTS+ radical scavenging activity in the presence of the standard

(BHT) and in samples of Agrimonia pilosa Ledeb 66

Figure 10 DPPH radical scavenging activity in the presence of the standard

(BHT) and in samples of Agrimonia pilosa Ledeb.a-bvalues (mean ± SD) with

different small letters were significantly different at P less than 0.01 (n=3) at

the same time and concentration 66

Figure 11 HPLC chromatograms of phenolic components of AL (A) standard phenolic, (B) peaks of aerial parts, (C) peaks of root: (1) 4-hydroxybenzoic

acid; (2), chlorogenic acid; (3), p-coumaric acid 69

Figure 12 LDH release in cell culture supernatant of different concentration from various extracts of Agrimonia pilosa on A549 after 24 hr treatment a-cvalues (mean±SD) with different small letter being significantly different at the same concentration 71

Figure 13 The RE activated apoptosis-related proteins (caspase-3, caspase-9, bcl-2, bax and bad) A549 cells after 24hr 71

Figure 14 DPPH free radical scavenging activities of EH and MH 78

Figure 15 HPLC chromatograms of phenolic components of EH 78

Figure 16 HPLC chromatograms of phenolic components of MH 81

Figure 17 LDH released in cell culture supernatant of different concentration ofEH and MH compared to BHA 81

Figure 18 EH and MH altered the expression of apoptosis –related proteins in HepG2 cells after 24 hr 82

Figure 19 HepG2 cells morphology after injured by different concentrations of the EH after 24 hr treatment (A) without sample, (B) 62.5 µg/mL, (C) 500 µg/mL, (D) 750 µg/mL 82

Figure 20 (a) HPLC chromatograms of phenolic components of the HT; A- Standard phenolic peaks: (1), 4-hydroxybenzoic acid; (2), chlorogenic acid; (3), benzoic acid; (4), p-coumaric acid; (5), caffeic acid; (6), ferulic acid; (7), rosmarinic acid; B- Sample phenolic peaks 86

Figure 21 ABTS+ radical scavenging capacity of the HT, SL, and BHT 88

Figure 22 DPPH radical scavenging capacity of the HT, SL, and BHT 88

Figure 23 Hydroxyl radical scavenging capacity of the HT, SL, and BHT 88

Figure 24 The HT and SL extracts induced growth inhibition in HepG2 after

24 hr (A) and after 48 hr (B); in A549 after 24 hr (C) and after 48 hr (D) 90

Figure 25 The HT altered the expression of apoptosis-related protein (caspase-3, caspase-9, bcl-2 and bad) in HepG2 after 24 hr 91

Figure 26 The HT altered the expression of apoptosis-related protein (caspase-3, bad, bax, p53) in A549 cells after 24 hr 91

Figure 27 Mass spectrum of compounds SA3 and SA4 96

Figure 28 Chemical structure of compounds SA3 (a) and SA4 (b) 96

Figure 29 ABTS+ radical scavenging capacity of the E50, SA3, SA4, and BHT a-bvalues (mean ± SD) with different small letters were significantly

different at P less than 0.01 (n=3) at the same time and concentration 99

Figure 30 DPPH radical scavenging capacity of the E50, SA3, SA4, and BHT 99

Figure 31 SA3 altered the expression of apoptosis- related proteins caspase-3, bax, bcl-2 in HepG2 (A) and caspase-3, caspase-9, p53 in A549 cells (B) after

24 hr 100

List of Abbreviation

AL Agrimonia pilosa Ledeb.

AE Aerial parts extract

ABTS+ 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid

CF Cymbopogon flexuosus (Nees ex Steud.) W Watson

DAT Days after transplanting

DNA Deoxyribonucleic acid

I.D Internal Diameter

iNOS Inducible nitric oxide

Chapter 1 Introduction 1.1 Research background

Free radicals can be generated in biological systems in the form of

reactive oxygen species (ROS) (Halliwell et al., 1995) They comprise

various forms of activated oxygen including superoxide radicals, hydroxyl radicals, hydrogen peroxide, and nitric oxide, which often are generated as

products of biologic reactions or from exogenous factors (Pan et al., 2008) High free radical levels or oxygen species create oxidative stress (Ordonez et

al., 2006) The ROS had been implicated in the etiology of various chronic

diseases such as Alzheimer’s and Parkinson’s diseases (Uttara et al., 2009); cancer, diabetes and obesity (Ames et al., 1993) In order to protect human

beings against oxidative damage, synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) were created due

to demand However, there has been concern regarding the toxicity and carcinogenic effects of synthetic antioxidants The identification of new safe and inexpensive antioxidant sources being of natural origin is necessary Therefore, in the recent decades many herbs have been commodiously studied

for their antioxidant activity and radical scavenging activity (De las heras et

al., 1998; Schinella et al., 2002; Vanderjagt et al., 2002), and much attention

has been focused on exploring therapeutic compounds Natural products are excellent sources of therapeutic compounds It is particularly evident in cancer therapy where more than 60% of approved drugs are of natural origin

(Newman et al., 2003) Therefore, a new natural source of anti-cancer

compounds would be a valuable tool in cancer therapy

Cancer currently remains a very serious disease and is one of the major causes of death in the world The factor of cancer disease included people’s lifestyle, environmental factors, and the most important are dietary factors

(Takaski et al., 1999) Liver cancer is also more common because liver is the

key organ of metabolism and excretion and is continously and variedly exposed to xenobiotics because of its strategic placement in the body The toxins absorbed from the intestinal tract gain access first to the liver resulting

in a variety of liver ailments Therefore many reactive intermediates could form in the liver during the detoxifying mechanisms The incidence of primary liver cancer is increasing in several developed countries, including the United States and the increasing will likely continue for some decades

Lung cancer accounts for more solid tumor deaths than colorectal, breast, prostate and pancreatic cancers in Taiwan Over the past decade, the mortality

of lung cancer has risen dramatically for both men and women More than 75% of lung cancer is non-small cell lung cancer and chemotherapy is the standard treatment for patients who have a comparatively good performance

status (Chou et al., 2003) The high prevalence and high death rate require

novel strategies for the prevention and treatment of hepatic and lung cancer

Agrimonia pilosa Ledeb Eclipta alba Hassk Mesona procumbens

Hemsl Hypericum japonicum Thunb Scoparia dulcis L and Cymbopogon

flexuosus (Nees ex Steud.) W Watson are herbs used in traditional medicine

with diverse properties including analgesic, anti-infammatory, and liver disorders The pharmacological activity of these herbs may be attributed primarily to phenolic compounds in them Although the recent studies about these herbs mainly focused on analyzing components, bioactivities and anticancer capacity, the researches about HepG2 and A549 cells inhibitor capacity of their extract and bioactive compounds were limited Thus, the present study was based on the ethnomedical data described above and designed to evaluate the anti-proliferative and molecular mechanisms of their ethanol extracts on human HepG2, A549 cells

1.2 Research objectives

The specific objectives included:

1 To evaluate effects of application of organic fertilizer in

Agrimonia pilosa Ledeb cultivation

2 To identify chemical compounds isolated from studied herbs

3 To evaluate antioxidant potential of studied herbs

4 To evaluate anticancer potential of studied herbs

Chapter 2 Literature Review 2.1 Herbs used in the study

2.1.1 Agrimonia pilosa Ledeb (AL)

2.1.1.1 Botany

The plant is scientifically classified as belonging to the Kingdom Plantae

It is of the order Rosids, of the family Rosaceae, genus Agrimonia, and species Agrimonia pilosa The full binomial name is Agrimonia pilosa

Ledeb (Figure 1a)

Agrimonia pilosa Ledeb is herb medicine with 30–120 cm tall Rhizome

short, usually tuberous, with many lateral roots and 1 to several underground buds Stems have sparsely pilose and pubescent, or densely rigidly hairy (rarely sparsely hirsute) in lower part Stipules green, falcate, rarely ovate or ovate-lanceolate, herbaceous, margin sharply serrate or lobed, rarely entire, apex acute or acuminate; petiole sparsely pilose or pubescent; leaf blade interrupted imparipinnate with (2 or) 3 or 4 pairs of leaflets, reduced to 3 leaflets on upper leaves; leaflets sessile or shortly petiolulate, obovate, obovate-elliptic, or obovate-lanceolate, 1.5–5 × 1–2.5 cm, abaxially appressed pilose on veins, or densely pubescent or tomentose-pubescent between veins, rarely glabrescent, markedly or sparsely glandular punctate, adaxially pilose,

or hirsute or hirtellous on veins, rarely glabrescent, base cuneate to broadly so, margin acutely to obtusely serrate, apex rounded to acute, rarely acuminate Inflorescence terminal, spicate-racemose, branched or not; rachis pilose Flowers 6–9 mm in diam; pedicel 1–5 mm, pilose; bract usually 3-parted with segments linear; bracteoles in 1 pair, ovate, margin entire or lobed Sepals 5, triangular-ovate Petals are yellow and oblong Stamens (5–) 8–15 Style filiform; stigma capitate Fruiting hypanthium obovoid-conic, 7–8 × 3–4 mm including prickles, abaxially 10-ribbed, pilose, with a multiseriate crown of

prickles; prickles erect when young, connivent at maturity Flower and fruit

are from May to December Agrimonia pilosa Ledeb is found in thinned

forests, forest margins, thickets, meadows, stream banks, roadsides; 100-3800

m This herb distributes throughout China Bhutan, Northern India, Japan, Northern Laos, Korea, Mongolia, Myanmar, Nepal, Russia, Sikkim, Northern

Thailand, Northern Vietnam and East Europe (Le et al., 2018a)

2.1.1.2 Chemical composition in AL

Chemical studies have shown that its extracts contain flavonoids (Xu et

al., 2005; Jung and Park, 2007), terpenoids (An et al., 2005; Xie et al., 2013)

Its overground part contains phenol compounds: flavonoids and their

glycosides: apigenin, kaempferol, quercetin, rutin, and luteolin (Kimura et al.,

1968); hydroxy-cinnamonic acids: caffeic acid and chlorogenic acid (Khanina

et al., 2011).The 54 compounds were also systematically reported (Le et al.,

2018a)

2.1.1.3 Pharmacological activities and the use of AL in traditional medicine Agrimonia pilosa Ledeb has been used traditionally for treatment of

abdominal pain, sore throat, headaches, bloody discharge, parasitic infections

and eczema (Kato et al., 2010) Pharmacologically, the AL had been reported

to prossess tumor (Koshiura et al., 1985; Miyamoto et al., 1987), viral (Li et al., 2004; Shin et al., 2010), anti-oxidant (Zhu et al., 2009), anti- microbial (Yamaki et al., 1989) and anti-hyperglycemic activities (Jung et al., 2006), anti-bacterial and hepatoprotective activities (Park et al., 2004; Kwon

anti-et al., 2005) Reports also indicated that the AL extract inhibited the

inflammatory process by suppression of iNOS, ROS and inflammatory

cytokine production in microglial cells (Jung et al., 2010)

2.1.2 Eclipta alba Hassk (EH)

2.1.2.1 Botany

The plant is scientifically classified as belonging to the Kingdom Plantae

It is of the order Asterales, of the family Asteraceae, genus Eclipta, and species Eclipta alba The full binomial name is Eclipta alba (L.) Hassk

(Figure 1b)

Stems erect, obliquely ascending or supine, up to 60 cm, usually branched from base, adnate to hairs Leaves long-lanceolate or lanceolate, sessile or shortly stalked, 3-10 cm long, 0.5-2.5 cm wide, apical or acuminate, margin serrulate or sometimes undulate, bilaterally dense hard rough hairs

The capitulum 6-8 mm in diam, with peduncles 2-4 cm long; stipitate globose-globose, involucres bracts green, herbaceous, 5-6 rowed, 2-layered, oblong or oblong-shaped needle shape, outer layer slightly shorter than inner layer, abaxially and marginally white with short hairs; peripheral female flowers 2 layers, tongue-like, 2-3 mm long, tongues short, distally 2-lobed or entire, central bisexual most flowers, corolla tubular, white, ca 1.5 mm, apically 4-dentate; style branches blunt, papillate; receptacles convex lanceolate or linear brackets Above the middle of the truncated piece is micro pilose; achene dark brown, 2.8 mm long, achenes triangular to female flowers, amphipod flowers oblate, prismatic, apical truncate, 1-3-toothed, base slightly narrowed margins with the white ribs have small nodutes and are hairless Fowering is from June to September

It is usually found on poorly drained, wet areas; along streams and ditches in marshes and on the dikes of rice paddies Howevers, it is also common in lawns and in upland conditions where rainfall is about 1200 mm

or more It can grow under wet, saline conditions but is often a weed of drier sites in plantation crops This herb is a native of Asia, but has a general

distribution over the world It is on all continents in tropical, subtropical and warm temperate regions (''Botanical Description of Eclipta Prostrata'', 2018)

2.1.2.2 Chemical composition in EH

Eclipta alba (L.) Hassk (EH) is an annual herbaceous plant belonging

to the Asteraceae family It has high therapeutic and medicinal value due to

its chemical composition The whole plant contains nicotine (Pal and Narasimhan, 1943), ecliptine and coumarin (Nguyen and Doan, 1989),

wedelolactone (Wagner et al., 1986), Dimethylwedelolactone-7-glucoside (Zhang and Guo, 2001), stigmasterol (Han et al., 1998), ecliptal (Das and Chakravarty, 1991), α-formyl terthienyl (Zhang et al., 1997), triterpine glycosides and eclalbasaponins (Yahara et al., 1997)

2.1.2.3 Pharmacological activities and the use of EH in traditional medicine

This herb shows versatile pharmacological effects that include hair growth, antioxidant, antimicrobial, anti-inflammatory and antihepatotoxic

(Jadhav et al., 2009; Chuahan et al., 2012); hepatoprotective activity (Ma-Ma

et al., 1978) and anticancer activities (Chaudhary et al., 2011)

2.1.3 Mesona procumbens Hemsl (MH)

2.1.3.1Botany

The plant is scientifically classified as belonging to the Kingdom Plantae

It is of the order Lamiales, of the family Lamiaceae, genus Platostoma The full binomial name is Mesona procumbens Hemsl (Figure 1c)

Stems are 15-100 cm; branches and stems have pilose or finely bristly glabrescent Petiole 2-15 mm; leaf blade narrowly ovate to subcircular, 2-5 x 0.8-2.8 cm, finely bristly, villous, or glabrescent or abaxially hairy along veins, base cuneate to sometimes rounded, margin serrate, apex acute to obtuse.Verticillasters in pedunculate, terminal, erect or upwardly oblique

panicles 2-10(-13) cm; bracts circular to rhombic-ovate, sublanceolate, apex caudate, mucronate, shorter to slightly longer than flowers, bright colored Pedicel 3-4(-5) mm, short hairy Calyx 2-2.5 mm, densely white pilose, middle lobe of upper lip acute to obtuse, lower lip sometimes emarginated; fruiting calyx 3-5 mm, subglabrous or hairy on veins Corolla white or reddish, ca 3 mm, puberulent, throat very dilated; upper lip 4-lobed, lateral 2 lobes distinct, longer than middle lobes, or upper lip subentire Anterior stamens longer than basally hirsute posterior pair Nutlets black, oblong

Flowering and fruiting are from July to October (''Mesona chinensis in Flora

of China @ efloras.org'', 2018)

2.1.3.2 Chemical composition in MH

The herb Mesona procumbens Hemsl.(MH), called “suong sao” in

Vietnam, is consumed as both herbal drink and jelly-type dessert, and also used as a herbal remedy in the Traditional Vietnamese Medicine to treat heat-stock, diabetes, liver and colon diseases It is reported that the herb contained hypoglycemic and hypertension substances, including oleanolic acid, β-sitosterol, α and β - amyrin, maslinin acid, stigmasterol and β-sitosterol

glycoside (Yen et al., 2000); kaempferol, apigenin, caffeic acid,

protocatechuic acid, syringic acid, vanillic acid and p-hydrobenzoic acid (Yen

et al., 2003)

2.1.3.3 Pharmacological activities and the use of MH in traditional medicine

This herb shows versatile pharmacological effects including antioxidant

(Hung and Yen, 2001); lowering blood pressure (Lai et al., 2000), liver protection (Shyu et al., 2008), antimicrobial (Chiu and Lai, 2010), and antimutagenic (Yen et al., 2001)

2.1.4 Hypericum japonicum Thunb (HT)

2.1.4.1 Botany

The plant is scientifically classified as belonging to the Kingdom Plantae

It is of the order Malpighiales, of the family Hypericaceae, genus Hypericum, and species Hypericum japonicum Thunb The full binomial name

is Hypericum japonicum Thunb (Figure 1d)

Annual herbs, about 5-35 cm tall, stem erect or decumbent, simple or brached dichotomously, rooting at basal nodes, 4 lined, glabrous or sparsely glandular Leaves simple, opposite, oblanceolate-elliptic to ovate, about 3-10

x 1-5 mm across, base cordate or amplexicaul, margins entire, apex obtuse or rounded, leaf lamina contain obscurely pellucid punctuate grands, especially near the margins beneath, chartaceous, green glabrous above and paler glaucous beneath, midrib impressed above and prominent beneath, midrib impressed above and prominent beneath, lateral veins about 2-6 on either side

of the midrib, petiole sessile, exstipulate Inflorescence 1-25 flowered axillary monochasial or dichasial cymes Flowers bisexual, actinomorphic, about 8-10

mm across, 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, veined prominently, glandular punctuate with pellucid black glands, president, about 3-4.5 x 1-2.5 mm across, petals 5, oblanceolate-oblong, yellow or bright yellow, veined prominently, persistent, as long as sepels or about 2-4 x 2-3

mm across Stamens 5-30, inbudles or fascicles 3-5, free, filaments linear, diliform, glabrous, of unequal length and anthers dorsifixed, yellow Ovary superior, ovoid-globose, unilocular, about 2-3 mm long, ovules many, styles 3, free from the base, erect, about 1.2 mm long Fruit capsule septicidal, ovoid-subglobose, about 4-4.5 x 2.5-3 mm long, apex obtuse, longitudinally vittate, with persistent style Seeds numerous, oblong, ribbed, testa transverse-

scalariform densely, brown (''Hypericum Japonicum Thunb.'', 2018)

2.1.4.2 Chemical composition in HT

Different classes of chemicals such as flavonoids, xanthonoids, chromone glycosides, phloroglucinol derivatives and lactones have been found in this plant Among these isolated components, some single flavonoid compounds such as quercitrin, isoquercitrin and quercetin-7-O-α-L-rhamnose

are show to have a variety of bioactivities in vivo or in vitro, and thereby are thought as the bioactive components of the HT (Liu et al., 2014)

2.1.4.3 Pharmacological activities and the use of HT in traditional medicine

It has been used in Traditional Chinese Medicine for a long time for relieving internal heat or fever, hemostasis and detimescence In addition, it has also been used as a medicinal herb in Asian countries, such as Japan, South Korea, Thailan, Nepal, India, Vietnam and Philippines The HT has been studied due to its high value in traditional medicine The results of modern pharmacological studies have shown that the HT could be used for the treatment of bacterial diseases, infectious hepatitis, acute and chronic hepatitis, gastrointestinal disorders, internal hemoorhages and tumors, which

generally matches its traditional uses (Lin et al., 2007) So far, no studies on the toxicity of the HT have been reported (Liu et al., 2014)

2.1.5 Scoparia dulcis L (SL)

2.1.5.1 Botany

Scoparia dulcis L (SL) being a member of Plantaginaceae family

(Figure 1e) It is terrestrial, annual, erect herb, up to 75 cm tall Roots are white or brown, fibrous The stem is polygonal and full It is often woody at the base and hairless The leaves are opposite or verticillate in three They are simple and sessile The lamina is oblanceolate, 2.5 to 5 cm long and 1.5 cm wide The base is attenuated by acute corner, forming a pseudo-petiole The top corner is wide Both sides are smooth and riddled with green glands,

brilliant The margin is entire in the lower half of the leaf blade and toothed in the upper half

Flowers are a bluish color They are carried by a peduncle 6 to 8 mm long The calyx consists of 5 sepals almost free to the base They are elliptical

in shape, tapering at the top They are finely hairy The corolla consists of 4 petals, rarely 5, free nearly to the base They are oval, apiculate at the top The entire calyx and corolla is 3 to 4 mm long The four stamens have anthers 2-celled equal The ovary is surmounted by a filiform style not exceeding the corolla The fruit is a dehiscent capsule, ovoid, surmounted by the style It is 4

mm long and contains numerous seeds When ripe, it opens in two valves The seeds are extremely small, they measure 0.1 mm long They are

obconical shape (''Plantaginaceae-Scoparia dulcis L.'', 2018)

2.1.5.2 Chemical composition in SL

The SL is a rich source of flavones, terpenes and steroids (Jain and Srivastava, 2005) It also have been reported that this herb contains scopadulcic acid B (SA-B), a novel diterpenoid, is the main ingredient of the biologically active compounds and its debenzoyl derivative, diacety scopadol

(DAS), has been shown to inhibit gastric H+, K(+)-ATPase (Asano et al., 1990) and antiviral effect (Hayashi et al., 1988)

2.1.5.3 Pharmacological activities and the use of SL in traditional medicine

Scoparia dulcis L (SL) is widely used in Indian folk medicine for the

treatment of diabetes mellitus The whole plant is used for ailments like

diarrhea, stomach-ache, kidney stones, kidney problems, and fever (Patra et

al., 2014) It also used against stomach aches, injuries, wounds, bronchitis,

coughs, diarrhoea, eye infection, fever, and kidney failure and liver diseases

2.1.6 Cymbopogon flexuosus (Nees ex Steud.)W Watson (CF)

2.1.6.1 Botany

The plant is scientifically classified as belonging to the Kingdom Plantae

It is of the order poales of the family poaceae, genus Cymbopogon, and species Cymbopogon flexuosus The full binomial name is Cymbopogon

flexuosus (Nees ex Steud.)W Watson.(Figure 1f)

Perennials, Culms 80-240 cm high, tufted; nodes glabrous Leaves 20-80

x 1-2 cm, linear, rounded at base, flat; sheaths glabrous; ligule ovate, membranous Inflorescence a large complex grayish panicle; branches slender, flexuous, often drooping Racemes paired, with a spatheole beneath Spikelets binate, one sessile, other pedicelled Sessile spikelets 4-5 mm long, elliptic-lanceolate; lower glume c 3 x 1 mm, elliptic-lanceolate, keels winged towards apex; upper glume ovate-lanceolate, margins ciliate; lower floret empty; upper floret bisexual; first lemma 2.5-3 x 1 mm, lanceolate, purplish, epaleate; second lemma c 2 x 0.4 mm, deeply 2- fid, awned, hyaline, awn 6-

10 mm; stamens 3, anthers 2 mm long; ovary c 1 mm, oblong, stigmas 1.5 mm long; caryopsis c 2 mm long, elliptic Pedicelled spiketets 3-4 mm long, lanceolate; pedicels c 2 mm long, densely villious; lower glume c 3 x 1

1-mm, ovate-lanceolate; upper glume c 3 x 1 1-mm, elliptic-lanceolate; lower floret male; upper floret wanting; firt lemma c 3 x 1 mm, elliptic-lanceolate;

stamens 3, anthers 1.5-2 mm long (''Cymbopogon flexuosus'', 2018)

2.1.6.2 Chemical composition in CF

There are no studies on chemical composition CF extracts The chemical composition of the CF oil has been reported The various constituents (%) present in the oil from lemon grass variety of the CF such as geranial (20.08), geranyl acetate (12.2), α–bisabolol (8.42) and isointermedeol (24.97),

limonene (3.5%) and borneol (1.9) (Sharma et al., 2009)

2.1.6.3 Pharmacological activities and the use of CF in traditional medicine

Lemongrass essential oil has been traditionally used as a remedy for a variety of health conditions Recent scientific studies have provided evidence supporting its antimicrobial, antioxidant, antifungal and anti-inflammatory

properties in several disease models (Tsai et al., 2011) Jiang et al., (2016)

found that its oil protected against benzo-α-pyrene-induced oxidative stress and DNA demage in human embryonic lung fibroblast cells

2.2 Application of organic materials in plant cultivation

Organic farming is a farming method that enjoys a special position today

In this method, sustainable production of food crops, in combination with environment protection and attention to social and economic relationships, is important Also, there is a perception that organic farming will contribute to alleviate problems associated with food safety, environmental quality and impact, market concentration, and the improvement of quality of life for rural communities Research in these areas is invaluable for both organic and conventional growers, especially because soil science is a relatively new field

of study (Carla and Antonio, 2005) Moreover, materials used in organic farming are considered one of the main pillars of soil fertility because they have beneficial effects on the physical, chemical, and biological properties of soils and on soil fertility too The using of organic meterials helps farmers save inputs and energy Organic farming has applications in various sciences such as agriculture, ecology, and environmental sciences; and it has generated increasing interest among farmers, researchers, officials, and policy makers

(Tahami et al., 2011)

Vermicompost (VC) and hog manure are environmental friendly materials VC has many favorable physicochemical characteristics, making it suitable for mixture in substrates, including high porosity and good aeration, drainage, and water-holding capacity (Edwards and Burrows, 1988) Vermicompost, in contrast to conventional compost, is the product of an

accelerated bio-oxidation of organic matter by the use of high densities of

earthworm populations without passing a thermophilic stage (Domínguez et

al., 1997; Subler et al., 1998) The earthworm-processed organic wastes are

finely divided peat-like materials with high porosity, aeration, drainage, and water-holding capacity (Ewards and Burrows, 1988) Compared to conventional compost, which passes a thermophilic stage, vermicompost usually has a much finer structure and larger surface area, providing strong absorbability and retention of nutrients (Shi-wei and Fu-zhen, 1991) They contain nutrients in a soluble form that is readily taken up by plants, such as nitrates, soluble potassium, exchangeable phosphorus, and magnesim, and calcium (Krishnamoorthy and Vajranabhaiah, 1986; Grappelli, 1985) and may contain biologically active substances such as plant growth regulators Moreover, vermicompost could release nutrients slowly and steadily into the system and enables the plants to absorb these nutrients over time (Sharma, 2003) Based on all these characteristic, earthworm-processed organic waste would have great commercial potential in the herbal industry as container media for growing bedding and herb plants Vermicomposts have a significant effect on the growth and productivity of plants (Edwards, 1998)

Figure 1 The herbs used in study Agrimonia pilosa Ledeb (a); Eclipta alba Hassk (b); Mesona procumbens Hemsl (c); Hypericum japonicum Thunb (d); Scoparia dulcis L (e); and Cymbopogon flexuosus (Nees ex Steud.) W Watson.(f)

Organic fertilizers are obtained from animal manure or plant sources like green manure, which can serve as alternative to mineral fertilizers (Dauda

et al., 2008) and microbial biomass (Suresh et al., 2004) Organic manures

have beneficial impacts in soil properties and produce safe plants with good, neat source of better availability of nutrienties (O’Brien and Barker, 1996) In addition, organic fertilizer resulted in significant increase in soil carbon, nitrogen, pH, cation exchange capacity and exchageable Ca, Mg and K which invariably enhance crop yield and productivity (Ayoola and Makinde, 2008) The biosynthesis of secondary metabolites in herbal plants is strongly influenced by environmental factors (Stutte, 2006) These conditions cause variations in the fresh and dry weight, as well as active components In this context, the application of organic and chemical fertilizers can increase the essential oil yield and main components of medicinal plants (Economakis,

2005; Khalid et al., 2006; Naghdibadi et al., 2004; Shalby and Razin, 1992)

In this study, we have cultured Agrimonia pilosa Ledeb in net house condition

and examined the effects of vermicompost on growth, yield, total phenolic content, antioxidant activity in this plant

2.3 The chemistry of phenolic compounds

Structurally, phenolic compounds comprise an aromatic ring, bearing one or more hydroxyl substituents, and range from simple phenolic molecules

to highly polymerised compounds (Bravo, 1998) Despite this structural diversity, the group of compounds is often referred to as “polyphenols” Most naturally occurring phenolic compounds are present as conjugates with mono-and polysaccharides, linked to one or more of the phenolic groups, and may also occur as fuctional derivatives such as esters and methyl esters (Harborne

et al., 1999) Though such structural diversity results in the wide range of

phenolic compounds that occur in nature, phenolic compounds can basically

be categorised into several classes as shown in Table 1 (Harborne, 1989;

Harborne et al., 1999) Of these, phenolic acids, flavonoids and tannins are

regarded as the main dietary phenolic compounds (King and Young, 1999)

2.3.1 Phenolic acid

Phenolic acids consist of two subgroups, i.e., the hydroxybenzoic and hydroxycinnamic acids (Figure 2) Hydroxybenzoic acids include gallic, p-hydroxybenzoic, protocatechuic, vanillic and syringic acids, which in common have the C6-C1 structure Hydroxycinnamic acids, on the other hand, are aromatic compounds with a three-carbon side chain (C6-C3), with caffeic, ferulic, p-coumaric and sinapic acids being the most common (Bravo, 1998)

2.3.2 Flavonoids

Flavonoids constitute the largest group of plant phenolics, accounting for over half the eight thounsand naturally occurring phenolic compounds

(Harborne et al., 1999) Flavonoids are low molecular weight compounds,

consisting of fifteen carbon atoms, arranged in a C6-C3-C6 configuration Essentially the structure consists of two aromatic rings A and B, joined by a 3-carbon bridge, usually in the form of a heterocyclic ring, C (Figure 3) The aromatic ring A is derived from the acetate/malonate pathway, while ring B is derived from phenylalanine through the shikimate pathway (Merken and Beecher, 2000) Variations in substitution patterns to ring C result in the major flavonoid classses, i.e., flavonols, flavones, flavanones, flavanols (or catechins), isoflavones, flavanonols, and anthocyanidins (Hollman and Katan, 1999), of which flavones and flavonols are the most widely occurring and

structurally diverse (Harborne et al., 1999) Substitutions to rings A and B

give rise to the different compounds within each class of flavonoids (Pietta, 2000) These substitutions may include oxygenation, alkylation, glycosylation, acylation, and sulfation (Balasundram, 2006; Hollman and Katan, 1999)

Table 1.Classes of phenolic compounds in plants

Hydroxycinamic acids, phenylpropanoids

(courmarins, isocoumarins, chromones, chromenes)

Condensed tannins (proanthocyanidins or flavolans) (C6-C3-C6)n

Figure 2 Examples of hydroxybenzoic acid (a) and hydroxycinnamic acid (b)b)

a)

Figure 3 Generic structure of a flavonoid molecule

Figure 4 Structure of hydrolyzable (a) and condensed tannins (b) (Luthar, 1992)

Figure 5 Antioxidant behavior

(Source:http://multimmunity.com/wp-content/uploads/2013/11/antioxidant-c60.jpg)

2.3.3 Tannins

The properties of tannins are based on their chemical structures having two or three phenolic hydroxyl groups on a phenyl ring, in a molecule of moderately large size Tannins were once classified into two groups: hydrolyzable tannins (HTs) and condensed tannins (CTs) densed tannins (CTs)

(Romani et al., 2012) In the HTs a carbohydrate (Figure 4a), usually

D-glucose, is partially or totally esterified with phenolic groups such as gallic acid (gallotannins, GTs) or ellagic acid (allagitannins, Ets) and several others workers studied the biosynthesis of gallic acid Depending on the polyphenolic acids that are obtained as products of hydrolysis, these are again sub-divided into: gallotannins and ellagitannins Gallotannins yield gallic acid and glucose on hydrolysis Ellagitannins are different from gallotannins in that they deposit on standing (hydrolysis) ellagic acid in addition to gallic acid and glucose from their tannin influsions They show the characteristic phenomenon of the formation of sludge or bloom on leathers

Condensed tannins (CTs) (Figure 4b) are chemicall oligomers of hydroxyflanvan-3-ol (commomly catechin or epicatechin) and polyhydroxyflavan-3,4-diols (leucoantho-cyanidin) or oligomers of a combination of those two compounds The basis flavonoid structure is condensed tannins in flavan Structurally related to flavonoids, these tannins are distributed widely in nature and constitute a heterogeneous group The C15

skeleton of the flavonoids is made up of two distinct units, A ring (consisting

of a C6 unit) and B ring (made up of C6-C3 unit) (Luthar, 1992)

2.4 Quantification of phenolics

2.4.1 Spectrophotometric assays

Spectrophotometry is one of the relatively simple techniques for quantification of plant phenolics The Folin-Denis and Folin-Ciocalteu methods were the two widely used spectrophotometric assays to measure total

phenolics in plant materials for many years (Lapornik et al., 2005) Both

methods are based on a chemical reduction involving reagents containing tungsten and molybdenum (Stalikas, 2007) The products of this reduction in the presence of phenolic compounds have a blue color with a broad ligh absorption spectrum around 760 nm The reagents for both methods do not react specifically with only phenols but also with other substances like ascorbic acid, aromatic amines and sugars (Box, 1983)

Total phenolic quantification, total flavonoids, proanthocyanidin (condensed tannin) and hydrolysable tannin can also be estimate by colorimetric methods Methanolic or ethanolic extracts of plant phenols mixed with AlCl3 allow measurement of total flavonoids in the range 410-423

nm (Huang et al., 2009)

2.4.2 High performance liquid chromatogaraphy (HPLC)

HPLC is the preferred technique for both separation and quantification

of phenolic compounds Various factors affect HPLC analysis of phenolics, including sample purification, mobile phase, column types and detectors (Stalikas, 2007) In general, purified phenolics are applied to an HPLC instrument utilizing a reversed phase C18 column (RP-C18), photo diode

array detector (PDA) and polar acidified organic solvents (Ignat et al., 2011)

Several reviews are available on the application of HPLC and the quantification of phenolics (Robbins, 2003; Kalili and De Villiers, 2011; Merken and Beecher, 2000) Normally, HPLC sensitivity and detection is based on purification of phenolics and pre-concentration from complex matrics of crude plant extracts

The purification stage includes removing the interfering compounds from the crude extract with partitionable solvents and using open column chromatography or an adsorption-desorption process Sephadex LH-20, polyamide, Amberlite, solid phase extraction (SPE) cartridges and styrene-divinylbenzene (XAD 4, XAD16, EXA-90, EXA 118, SP70), acrylic resins