Dissertation 國國 國國國國國國國國國國國國國國國國國國國國國國國國 國國國國國國國國 Study in Cultivation and Bioactivities of Herbs Agrimonia pilosa, Eclipta alba, Mesona procumbens, Hypericum japonicum, Scoparia dulcis,
Trang 1國國國國國國國國國國國國國國國國國國 Department of Tropical Agriculture and International Cooperation
National Pingtung University of Science and Technology
國國國國國國 Ph.D Dissertation
國國 (國國國國國國國國國國國國國國國國國國國國國國)國國 國國國國國國國國
Study in Cultivation and Bioactivities of Herbs Agrimonia pilosa, Eclipta alba, Mesona procumbens, Hypericum japonicum, Scoparia
dulcis, and Cymbopogon flexuosus
國國國國 Advisors: 國國國國國(Ming-Chang Wu, Ph.D.)
國國國國國(Horng-Liang Lay, Ph.D.)
國國國 Student: 國國國 (Le Quang Ung)
國國國國 108 國 6 國 15 國 June 15, 2019
Trang 2國國國國國國 HepG2國國國國國國國國國 A549國國國國國國國國國國 國國國國國國國國國 Eclipta
國國國國國國
國國國(ABTS)國國國國國國國國(DPPH)國國國國國國國國國國國國國 國國國國國國國國國國國國國國國國國
國國國國國國國國國國國國國國 國國國國國國HPLC國國國國國國國國國國國Agrimonia pilosa
Ledeb.國國 國國國Eclipta alba Hassk.國國國國(Mesona procumbens Hemsl.) 國 國國 國
國Hypericum japonicum Thunb.國國 國國國 (Scoparia dulcis L.) 國國國
國 國 國 國 國 國 國 國 國 國 國 國 (MS) 國 國 國 國 國 (NMR) 國 國 國 國 國 國
(Cymbopogon flexuosus (Nees ex Steud.) W Watson) 國國國國國國國國國
Trang 3國5國國1,3-O-國-E-國國國國國國SA3國國 1-O-p-國國國-3-O-國國國國 國國SA4國國國國國國國國
國(Cymbopogon flexuosus (Nees ex Steud.) W Watson) 國國國國國國國國國國國國
國國國國國國國國國國國國國國國國國國國國國國國國國國國國國
國國國國國國國國國國國國國國國國國國國國國國國國國國國國國國國
國 Agrimonia pilosa Ledeb 國 國 國 國 國 Eclipta alba Hassk 國 國 國 國 國
國 Hypericum japonicum Thunb 國 國 國 國 國 國 (Cymbopogon flexuosus (Nees ex
Steud.) W Watson) 國國國國國國國國國國國國國國國國
國 國 國 國 國 國 國 國 國 國 國 國 國 國 國 國 國 國 國 國 國 國
Trang 4Abstract
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 Scienceand Technology
Graduate Date: June15, 2019 Degree Conferred: Doctoral DegreeName 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
Trang 5scavenging systems were determined Lactate dehydrogenase release inmedium was also evaluated in some experiments High performance liquidchromatography (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 blottingassays
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
Trang 6capacity were detected from Hypericum japonicum Thunb The last study (5),
1-O-p-coumaroyl-3-O-caffeoylglycerol (SA4) were firstly isolated from Cymbopogon flexuosus
(Nees ex Steud.) W Watson The SA3 compound showed the highestantioxidant and anticancer potent
The information obtained from this work will likely contribute to thedevelopment of novel natural medicine for cancer diseasers It will alsoprovide new insights for the futher understanding of the functions andmolecular mechanism of herbs An insightful investigation to establish andmanufacture 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
Trang 7Acknowledgements
I would like to express sincere thanks to my two advisors, Professor Dr.Ming-Chang Wu and Professor Dr Horng-Liang Lay, for their invaluableguidance 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 valuablecomments and constructive suggestions for the successful completion of thisresearch work
I am grateful to Taiwan Scholarship (Ministry of Education) for budgetsupport 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 TropicalAgriculture and International Cooperation; Office of International Affair,Chinese Herbal Medicine and FP201 Laboratory for their help during thisstudy program
Lastly, I would like to thank my parents for their confidence and love;
my colleague, my friends for their encouragement and affection andespecially my wife who was always there with wisdom, inspiration and cheer
Trang 8Table of Contents
國 國 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
Trang 92.1.3.2 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
Trang 102.5 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
Trang 113.5 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
Trang 124.2 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
Trang 134.4.5 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
Trang 14List 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
Trang 16List 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) 18Figure 3 Generic structure of a flavonoid molecule 19Figure 4 Structure of hydrolyzable (a) and condensed tannins (b) (Luthar,1992) 19Figure 5 Antioxidant behavior (Source :h t tp : / / m ulti mm u n it y co m / w p -
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
Trang 17Figure 11 HPLC chromatograms of phenolic components of AL (A) standardphenolic, (B) peaks of aerial parts, (C) peaks of root: (1) 4-hydroxybenzoicacid; (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 atthe 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 concentrationofEH and MH compared to BHA 81
Figure 18 EH and MH altered the expression of apoptosis –related proteins inHepG2 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) 500µg/mL, (D) 750 µg/mL 82Figure 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),
Trang 19Figure 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 91Figure 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, andBHT 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, andBHT 99Figure 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
Trang 20List 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
Trang 21Chapter 1 Introduction1.1 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, hydroxylradicals, 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 butylatedhydroxytoluene (BHT) and butylated hydroxyanisole (BHA) were created due
to demand However, there has been concern regarding the toxicity andcarcinogenic effects of synthetic antioxidants The identification of new safeand 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 areexcellent sources of therapeutic compounds It is particularly evident incancer 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 majorcauses of death in the world The factor of cancer disease included people’slifestyle, environmental factors, and the most important are dietary factors
Trang 22(Takaski et al., 1999) Liver cancer is also more common because liver is the
key organ of metabolism and excretion and is continously and variedlyexposed to xenobiotics because of its strategic placement in the body Thetoxins absorbed from the intestinal tract gain access first to the liver resulting
in a variety of liver ailments Therefore many reactive intermediates couldform in the liver during the detoxifying mechanisms The incidence ofprimary liver cancer is increasing in several developed countries, includingthe 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 than75% of lung cancer is non-small cell lung cancer and chemotherapy is thestandard 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 liverdisorders The pharmacological activity of these herbs may be attributedprimarily to phenolic compounds in them Although the recent studies aboutthese herbs mainly focused on analyzing components, bioactivities andanticancer capacity, the researches about HepG2 and A549 cells inhibitorcapacity of their extract and bioactive compounds were limited Thus, thepresent study was based on the ethnomedical data described above anddesigned to evaluate the anti-proliferative and molecular mechanisms of theirethanol extracts on human HepG2, A549 cells
Trang 231.2 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
Trang 24Literature Review2.1 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 undergroundbuds Stems have sparsely pilose and pubescent, or densely rigidly hairy(rarely sparsely hirsute) in lower part Stipules green, falcate, rarely ovate orovate-lanceolate, herbaceous, margin sharply serrate or lobed, rarely entire,apex acute or acuminate; petiole sparsely pilose or pubescent; leaf bladeinterrupted imparipinnate with (2 or) 3 or 4 pairs of leaflets, reduced to 3leaflets on upper leaves; leaflets sessile or shortly petiolulate, obovate,obovate-elliptic, or obovate-lanceolate, 1.5–5 × 1–2.5 cm, abaxially appressedpilose 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 withsegments linear; bracteoles in 1 pair, ovate, margin entire or lobed Sepals 5,triangular-ovate Petals are yellow and oblong Stamens (5–) 8–15 Stylefiliform; stigma capitate Fruiting hypanthium obovoid-conic, 7–8 × 3–4 mmincluding prickles, abaxially 10-ribbed, pilose, with a multiseriate crown of
Trang 25prickles; 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).
Trang 262.1.2 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, usuallybranched 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; stipitateglobose-globose, involucres bracts green, herbaceous, 5-6 rowed, 2-layered,oblong or oblong-shaped needle shape, outer layer slightly shorter than innerlayer, abaxially and marginally white with short hairs; peripheral femaleflowers 2 layers, tongue-like, 2-3 mm long, tongues short, distally 2-lobed orentire, central bisexual most flowers, corolla tubular, white, ca 1.5 mm,apically 4-dentate; style branches blunt, papillate; receptacles convexlanceolate or linear brackets Above the middle of the truncated piece is micropilose; achene dark brown, 2.8 mm long, achenes triangular to female flowers,amphipod flowers oblate, prismatic, apical truncate, 1-3-toothed, base slightlynarrowed 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 andditches in marshes and on the dikes of rice paddies Howevers, it is alsocommon 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 driersites in plantation crops This herb is a native of Asia, but has a general
Trang 27distribution over the world It is on all continents in tropical, subtropical andwarm 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 andNarasimhan, 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 hairgrowth, 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 bristlyglabrescent Petiole 2-15 mm; leaf blade narrowly ovate to subcircular, 2-5 x0.8-2.8 cm, finely bristly, villous, or glabrescent or abaxially hairy alongveins, base cuneate to sometimes rounded, margin serrate, apex acute toobtuse.Verticillasters in pedunculate, terminal, erect or upwardly oblique
Trang 28panicles 2-10(-13) cm; bracts circular to rhombic-ovate, sublanceolate, apexcaudate, 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 orreddish, ca 3 mm, puberulent, throat very dilated; upper lip 4-lobed, lateral 2lobes distinct, longer than middle lobes, or upper lip subentire Anteriorstamens 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 alsoused as a herbal remedy in the Traditional Vietnamese Medicine to treat heat-stock, diabetes, liver and colon diseases It is reported that the herb containedhypoglycemic 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).
Trang 292.1.4 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 orbrached dichotomously, rooting at basal nodes, 4 lined, glabrous or sparselyglandular Leaves simple, opposite, oblanceolate-elliptic to ovate, about 3-10
x 1-5 mm across, base cordate or amplexicaul, margins entire, apex obtuse orrounded, leaf lamina contain obscurely pellucid punctuate grands, especiallynear the margins beneath, chartaceous, green glabrous above and palerglaucous beneath, midrib impressed above and prominent beneath, midribimpressed above and prominent beneath, lateral veins about 2-6 on either side
of the midrib, petiole sessile, exstipulate Inflorescence 1-25 flowered axillarymonochasial 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, veinedprominently, glandular punctuate with pellucid black glands, president, about3-4.5 x 1-2.5 mm across, petals 5, oblanceolate-oblong, yellow or brightyellow, 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 Ovarysuperior, 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).
Trang 302.1.4.2 Chemical composition in HT
Different classes of chemicals such as flavonoids, xanthonoids,chromone glycosides, phloroglucinol derivatives and lactones have beenfound in this plant Among these isolated components, some single flavonoidcompounds 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 forrelieving internal heat or fever, hemostasis and detimescence In addition, ithas also been used as a medicinal herb in Asian countries, such as Japan,South Korea, Thailan, Nepal, India, Vietnam and Philippines The HT hasbeen studied due to its high value in traditional medicine The results ofmodern pharmacological studies have shown that the HT could be used forthe treatment of bacterial diseases, infectious hepatitis, acute and chronichepatitis, 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 arewhite or brown, fibrous The stem is polygonal and full It is often woody atthe base and hairless The leaves are opposite or verticillate in three They aresimple and sessile The lamina is oblanceolate, 2.5 to 5 cm long and 1.5 cmwide The base is attenuated by acute corner, forming a pseudo-petiole Thetop corner is wide Both sides are smooth and riddled with green glands,
Trang 31mm 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 andSrivastava, 2005) It also have been reported that this herb containsscopadulcic acid B (SA-B), a novel diterpenoid, is the main ingredient of thebiologically 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
Trang 322.1.6 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 Spikeletsbinate, one sessile, other pedicelled Sessile spikelets 4-5 mm long, elliptic-lanceolate; lower glume c 3 x 1 mm, elliptic-lanceolate, keels wingedtowards apex; upper glume ovate-lanceolate, margins ciliate; lower floretempty; 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 mmlong, 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; lowerfloret 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 chemicalcomposition 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)
Trang 332.1.6.3 Pharmacological activities and the use of CF in traditional medicine
Lemongrass essential oil has been traditionally used as a remedy for avariety of health conditions Recent scientific studies have provided evidencesupporting 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 stressand 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 withenvironment protection and attention to social and economic relationships, isimportant Also, there is a perception that organic farming will contribute toalleviate problems associated with food safety, environmental quality andimpact, market concentration, and the improvement of quality of life for ruralcommunities Research in these areas is invaluable for both organic andconventional growers, especially because soil science is a relatively new field
of study (Carla and Antonio, 2005) Moreover, materials used in organicfarming are considered one of the main pillars of soil fertility because theyhave beneficial effects on the physical, chemical, and biological properties ofsoils and on soil fertility too The using of organic meterials helps farmerssave inputs and energy Organic farming has applications in various sciencessuch as agriculture, ecology, and environmental sciences; and it has generatedincreasing interest among farmers, researchers, officials, and policy makers
(Tahami et al., 2011).
Vermicompost (VC) and hog manure are environmental friendlymaterials VC has many favorable physicochemical characteristics, making itsuitable 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
Trang 34accelerated 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, andwater-holding capacity (Ewards and Burrows, 1988) Compared toconventional compost, which passes a thermophilic stage, vermicompostusually has a much finer structure and larger surface area, providing strongabsorbability and retention of nutrients (Shi-wei and Fu-zhen, 1991) Theycontain nutrients in a soluble form that is readily taken up by plants, such asnitrates, soluble potassium, exchangeable phosphorus, and magnesim, andcalcium (Krishnamoorthy and Vajranabhaiah, 1986; Grappelli, 1985) andmay contain biologically active substances such as plant growth regulators.Moreover, vermicompost could release nutrients slowly and steadily into thesystem and enables the plants to absorb these nutrients over time (Sharma,2003) Based on all these characteristic, earthworm-processed organic wastewould have great commercial potential in the herbal industry as containermedia for growing bedding and herb plants Vermicomposts have asignificant effect on the growth and productivity of plants (Edwards, 1998)
Trang 36Organic fertilizers are obtained from animal manure or plant sourceslike 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) Inaddition, organic fertilizer resulted in significant increase in soil carbon,nitrogen, pH, cation exchange capacity and exchageable Ca, Mg and K whichinvariably enhance crop yield and productivity (Ayoola and Makinde, 2008).The biosynthesis of secondary metabolites in herbal plants is stronglyinfluenced by environmental factors (Stutte, 2006) These conditions causevariations in the fresh and dry weight, as well as active components In thiscontext, the application of organic and chemical fertilizers can increase theessential 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 phenoliccontent, antioxidant activity in this plant
2.3 The chemistry of phenolic compounds
Structurally, phenolic compounds comprise an aromatic ring, bearingone or more hydroxyl substituents, and range from simple phenolic molecules
to highly polymerised compounds (Bravo, 1998) Despite this structuraldiversity, the group of compounds is often referred to as “polyphenols” Mostnaturally occurring phenolic compounds are present as conjugates with mono-and polysaccharides, linked to one or more of the phenolic groups, and mayalso 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)
Trang 372.3.1 Phenolic acid
Phenolic acids consist of two subgroups, i.e., the hydroxybenzoic andhydroxycinnamic acids (Figure 2) Hydroxybenzoic acids include gallic, p-hydroxybenzoic, protocatechuic, vanillic and syringic acids, which incommon 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, accountingfor 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 a3-carbon bridge, usually in the form of a heterocyclic ring, C (Figure 3) Thearomatic ring A is derived from the acetate/malonate pathway, while ring B isderived from phenylalanine through the shikimate pathway (Merken andBeecher, 2000) Variations in substitution patterns to ring C result in themajor flavonoid classses, i.e., flavonols, flavones, flavanones, flavanols (orcatechins), 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)
Trang 38Table 1.Classes of phenolic compounds in plants
Class StructureSimple phenolics, benzoquinones C6
Hydroxybenzoic acids C6-C1Acethophenones, phenylacetic acids C6-C2
Trang 39Hydroxycinamic acids, phenylpropanoids
(courmarins, isocoumarins, chromones, chromenes) C6-C2
p-coumaric acid Caffeic acid
Ferulic acid Sinapic acidFigure 2 Examples of hydroxybenzoic acid (a) and hydroxycinnamic acid (b)
Trang 40Figure 3 Generic structure of a flavonoid molecule