phytochemistry and bioassay for natural weed control

THESIS 

PHYTOCHEMISTRY AND BIOASSAY FOR NATURAL WEED 

CONTROL COMPOUNDS FROM Ageratum conyzoides L. 

BUAKHAO HONGSACHUM 

GRADUATE SCHOOL, KASETSART UNIVERSITY 

2008

THESIS 

PHYTOCHEMISTRY AND BIOASSAY FOR NATURAL WEED CONTROL 

COMPOUNDS FROM Ageratum conyzoides L. 

BUAKHAO HONGSACHUM 

A Thesis Submitted in Partial Fulfillment of 

the Requirements for the Degree of 

Master of Science (Botany) 

Graduate School, Kasetsart University 

2008

ACKNOWLEDGEMENTS 

I wish to express my grateful appreciation and deeply indebted to my chairman, 

Associate Professor Srunya Vajrodaya, for her valuable advice, encouragement and 

stimulating and helpful suggestion throughout the course of my graduate study at 

Department of Botany, Faculty of Science, Kasetsart University and for completely 

writing of this thesis. I would like to sincerely grateful to Associate Professor 

Sureeya Tantiwiwat, my major advisor, for her kindly encouragement, valuable 

suggestion and helpful assistance. I gratefully thank Associate Professor Poontariga 

Harinasut, my minor advisor from Department of Biochemistry, Faculty of Science, 

Kasessart University, for her valuable suggestion and also Associate Professor Decha 

Wiwatwitaya, the Graduate School representative from Department of Forest Biology, 

Faculty of Forestry, Kasetsart University for his valuable comments. 

I would like to deeply indebted to ledturers at Department of Botany, Faculty of 

Science, Kasetsart University for their encouragement and helpful suggestion. I am heartfelt 

thank to my friends at Department of Botany, Faculty of Science, Kasetsart University, for 

their assistance and encouragement. I would like to especially thank to Miss Somnuk 

Promdang, my pen sister at the laboratory, for her helpful suggestion and encouragement 

throughout this thesis. My appreciation is also extended to the Department of Chemistry, 

Faculty of Science, Kasetsart University for supporting of some instrumentations 

Grateful acknowledgement is made to Development and Promotion of Science 

and Technology Talented Project (DPST Project) for dissertation supported fund 

throughout my study and research. 

Finally, I am especially appreciated my parents, Mr. Roemma and Mrs. Kampon 

Hongsachum and my sisters, Miss Kwandao, Miss Pawadee and Miss Kesinee 

Hongsachum, for their love and encouragements. 

Buakhao Hongsachum 

April 2008

TABLE OF CONTENTS 

Page 

TABLE OF CONTENTS i 

LIST OF TABLES ii 

LIST OF FIGURES iv 

LIST OF ABBREVIATIONS vii 

INTRODUCTION 1 

OBJECTIVES 3 

LITERATURE REVIEW 4 

MATERIALS AND METHODS 38 

Materials 38 

Methods 43 

RESULTS AND DISCUSSION 50 

CONCLUSION AND RECOMMENDATION 107 

Conclusion 107 

Recommendation 108 

LITERATURE CITED 109 

APPENDIX 127 

i

LIST OF TABLES 

Table Page 

1 Selected ethno pharmacological applications of A. conyzoides L. 7 

2 Pharmacological activities of A. conyzoides L. 12 

3 Biological activities of A. conyzoides L. on insects 14 

4 Biological activities of A. conyzoides L. on microbial 16 

5 Allelopathy of A. conyzoides L. 18 

6 Mono- and sequiterpenes from A. conyzoides L. 20 

7 Chromenes, benzofurans and coumarins from A. conyzoides L. 22 

8 Flavonoids from A. conyzoides L. 27 

9 Alkaloids from A. conyzoides L. 33 

10 Triterpenes and sterols from A. conyzoides L. 35 

11 Phytochemical screening of POP1a, POP2a, POP3a, POP1b, 

POP2b, POP3b and callus 63 

12 Secondary metabolite screening on TLC plates of POP1a, POP2a, 

POP3a, POP1b, POP2b, POP3b and callus 69 

13 Secondary metabolites survey in lipophilic crude extracts of A. 

conyzoides L. 71 

14 Rf values of the compound detected from A. conyzoides L. 

lipophilic crude extracts detected under long wave length UV light 74 

15 Column chromatography information of lipophilic extract of 

POP1b 87 

16 Germination percentage of cultivated plants at 3,5 and 7 days 92 

17 Seedling growth of cultivated plants at the 7 th date 93 

18 Germination percentage of weeds at 3,5 and 7 days 99 

19 Seedling growth of weeds at the 7 th date 100 

20 Effect of the lipophilic crude extract from A. conyzoides L. on 

seed germination and seedling growth of tested seed 104 

ii

Appendix Table 

LIST OF TABLES (Continued) 

1 Retention time (Rt) and UV spectrum of peak detected from 

HPLC chromatogram 131 

2 Analysis of Variance (ANOVA) of germination percentage, shoot 

and root length of Oryza sativa L. cultivar Hom Mali 105 at 95% 

significant level 133 


and root length of Brassica chinensis L. var. chinensis at 95% 



and root length of Ipomoea aquatica Forssk. at 95% significant 

level 135 


and root length of Tridax procumbens L. at 95% significant level 136 


and root length of Mimosa pigra L. at 95% significant level 137 


and root length of Cenchrus echinatus L. at 95% significant level 138 


and root length of Echinochloa colona (L.) Link at 95% 


iii

LIST OF FIGURES 

Figure Page 

1 Ageratum conyzoides L. herbarium specimen 6 

2 Phytochemical method 48 

3 Alkaloidal test by using Dragendorff’s reagent 51 

4 Coumarins test by using 10% NaOH 54 

5 Unsaturated lactone ring test by using Kedde’s reagent 56 

6 Unsaturated lactone ring test by using Raymond’s reagent 57 

7 Steroid and triterpenoid test by using Libermann-Burchard test 59 

8 Flavonoid test by using Cyanidin test 61 

9 Flavonoid test by using FeCl3 solution 62 

10 Alkaloid detection from POP1a (1a), POP2a (2a), POP3a (3a), 

POP1b (1b), POP2b (2b), POP3b (3b) and callus 66 

11 Terpenoid detection from POP1a (1a), POP2a (2a), POP3a (3a), 


12 Coumarin detection from POP1a (1a), POP2a (2a), POP3a (3a), 


13 Unsaturated lactone ring detection from POP1a (1a), POP2a (2a), 

POP3a (3a), POP1b (1b), POP2b (2b), POP3b (3b) and callus 70 

14 TLC pattern of POP1a (1a), POP2a (2a), POP3a (3a), POP1b (1b), 

POP2b (2b), POP3b (3b) and callus under long wavelength 

(365 nm) UV light 72 

15 TLC pattern of POP1a (1a), POP2a (2a), POP3a (3a), POP1b (1b), 

POP2b (2b), POP3b (3b) and callus after treated with iodine crystal 73 

16 Chemical profiles and UV spectra of POP1a and POP1b 77 



19 Chemical profiles and UV spectra of POP1a, POP2a and POP3a 81 

20 Chemical profiles and UV spectra of POP1b, POP2b and POP3b 82 

iv

LIST OF FIGURES (Continued) 

Figure Page 

21 Chemical profiles and UV spectra of callus, POP1a and POP1b 84 

22 Chromatogram comparison of callus and POP1a 85 

23 Chromatogram comparison of callus and POP1b 86 

24 Drusses crystal isolated and purified from fraction C 88 

25 HPLC chromatogram and UV spectrogram of the crystal 89 

26 Oryza sativa L. cultivar Hom Mali 105 seedling treated with 

A. conyzoides extracts and herbicides at the 7 th date 94 

27 Brassica chinensis L. var. chinensis seedling treated with 

A. conyzoides extracts and herbicides at the 7 th date 95 

28 Ipomoea aquatica Forssk. seedling treated with A. conyzoides 

extracts and herbicides at the 7 th date 96 

29 Tridax procumbens L. seedling treated with A. conyzoides 

extracts and herbicides at the 7 th date 101 

30 Mimosa pigra L. seedling treated with A. conyzoides extracts 

and herbicides at the 7 th date 102 

31 Cenchrus echinatus L. seedling treated with A. conyzoides 

extracts and herbicides after the 7 th date 103 

Appendix Figure 

1 Three populations of A. conyzoides L. 140 

2 MPLC Instrumentation 141 

3 Agilent Technologies Instrumentation 142 

4 TLC information (under 254 nm UV light) of eighteen fractions 

collected from Column Chromatography 143 

5 TLC information (under 365 nm UV light) of eighteen fractions 

collected from Column Chromatography 144 

v

LIST OF FIGURES (Continued) 

Appendix Figure Page 

6 Dragendorff’s reagent detection of fraction D combined from 

Column Chromatography 145 

7 TLC chromatogram of crystal from fraction C developing in 

dichloromethane:ethyl acetate:methanol (75:20:5) solvent system 146 


dichromethane solvent system 147 


benzene:chloroform (7:3) solvent system 148 


chloroform:acetone (9:1) solvent system 149 

vi

LIST OF ABBREVIATIONS 

A. = Ageratum 

No = number 

BKF = Bangkok Forestry Herbarium 

TLC = Thin layer chromarography 

mm = millimeter 

cm = centimeter 

cm 2 = centimeter square 

nm = nanometer 

µ = micron/micrometer 

˚C = degree Celsius 

UV = ultraviolet 

Rf = retardation factor 

g = gram 

ml = milliter 

MPLC = medium pressure liquid chromatography 

HPLC = high performance liquid chromatography 

min. = minute 

M = molar 

N = normal 

AR = analytical reagent 

MeOH = methanol 

Ø = diameter 

MΩ = mega ohm 

POP = population 

cc = column chromatography 

IR = infrared 

NMR = nuclear magnetic resonance 

MS = mass spectrometry 

conc. = concentrated 

vii

LIST OF ABBREVIATIONS (Continued) 

g/l = gram per liter 

% = percent 

CRD = complete randomized design 

ANOVA = analysis of variance 

CV = coefficient of variance 

Rt = retention time 

λmax = lambda max 

sh = shoulder 

DMRT = Duncan’s multiple range test 

GERM = germination 

viii

PHYTOCHEMISTRY AND BIOASSAY FOR NATURAL WEED 

CONTROL COMPOUNDS FROM Ageratum conyzoides L. 

INTRODUCTION 

Careless use of synthetic chemical is the main cause of environmental problem 

in Thailand. In 2002, Department of Agriculture reported that Thailand imported 

large commercial chemicals up to 50,331 tons (11,341 million Baht) and herbicides 

are the largest amount (31,879 tons, 6,101 million Baht). 

Herbicides are contaminated in the groundwater and soil in many agricultural 

regions over Thailand. Water pollution is the major problem in the country. Soil 

pollution and soil erosion is a concern in many regions. Weed resistance to herbicides 

continues to grow, and the problem of herbicides residues in food has yet to be 

resolved. The use of herbicides implies a risk of accumulation of residues in 

conventionally cultivated plant foods. It was concluded that far from all conventionally 

grown fruits and vegetables contain herbicide residues and if they contain herbicides, 

the maximum residue limits are far from exceeded 

Because of these concerns, some farmers have begun to adopt sustainable 

farming practices with the goals of reducing input coats, preserving the resource base, 

and protecting human health. The sustainable systems are more deliberately integrate 

and take advantage of naturally occurring beneficial interactions between organisms. 

The practice farming method attempts to use organic system, such as apply the 

advantage of plant competition. The study in term of ‘allelopathy’ become interesting 

for the researchers that now realize the important of allelopathy in the world’s 

agricultural and forestry supplies. 

Allelopathy is the reaction of plants producing secondary metabolites called 

allelochemical that affect to other plants neighboring them. The effect can be both 

inhibited and promoted the growth of plants. In Thailand, many of the plant species 

have been analyzed and identified as containing biologically active secondary 

1

compounds derived from polyketide pathway and mevalonic acid pathway. These 

secondary compounds are such as sesquiterpene lactone, phenolic compound, terpene, 

alkaloid, etc. And those plants species may be common plant in the country, and even 

weeds that seem useless but may play an important role to reduce other disadvantage 

weeds and become natural weed control in the future. 

2

OBJECTIVES 

1. To investigate the secondary metabolites from Ageratum conyzoides L. 

2. To investigate biological activities of the secondary metabolites from 

Ageratum conyzoides L 

3

LITERATURE REVIEWS 

Ageratum was derived from the Greek words ‘a geras’, meaning non-aging, 

referring to the longevity of the whole plant. Conyzoides one on the other hand was 

derived from ‘konyz’ the Greek name of Inula helenium which the plant resembles. 

(Kissmann and Groth, 1993) 

Ageratum conyzodes L. belongs to the family Asteraceae tribe Eupatoriae. 

This family is well marked in their characteristics and can not be confused with any 

other. A large majority of the plants in this family are herbaceous while trees and 

shrubs were comparatively rare. The genus Ageratum consists of approximately thirty 

species but only a few species have been phytochemically investigated (Burkill, 

1985). 

The synonyms of A. conyzoides include A. album Stend, A. caeruleum Hort. 

ex Poir., A. coeruleum Desf., A. cordifolium Roxb., A. hirsutum Lam., A. humile 

Salisb., A. latifolium Car., A. maritimum H.B.K., A. mexicanum Sims., A. obtusifolium 

Lam., A. odoratum Vilm. and Cacalia mentrasto Vell. (Jaccoud, 1961). 

In Thailand, local names of A. conyzoides L. were listed as follow: Tapsuea 

lek (Sing Buri), Thiam mae haang (Loei), Saap raeng saap kaa (Chiang Mai), Yaa 

saap haeng (Chiang Mai) and Yaa saap raeng (Ratchaburi) (Smitinand, 1980). 

Johnson (1971) classified A. conyzoides into two subspecies, latifolium and 

conyzoides. Subsp. latifolium is found in the entire USA continental and subsp. 

conyzoides has a pantropical distribution. The basic chromosome number is 2n = 20 

but natural tetraploids are found. A. conyzoides subsp. latifolium is diploid while A. 

conyzoides subsp. conyzoides is tetraploid. 

Backer and van den Brink (1968) described the genus Ageratum L. are erect 

herb in the smelling of cumarine Lower leaves arrangement are opposite, petioled, 

serrate-crenate above the entire base, penninerved or subtrinerved, higher ones 

4

alternate, petiolate, ovate, dentate or serate. Heads corymbose or loosely peniculate, 

homogamous, discoid, many flowered. Involucre campanulate, imbricate, bracts 2-3 

seriates, linear, acute to acuminate, sub equal; receptacle flat or nearly so, naked or 

with caduceus scales. Corollas all tubular, equal, regular, the limb 5-fid. Anthers with 

and apical appendage, the base entire, obtuse. Style-arms long, slender, obtuse and 

pubescent at the apex. Achenes oblong, 5-angular. Pappus uniseriate, of 5 short free 

or connate scales, or of 10-20 narrow acuminate unequal scales. 

Ageratum conyzodes L. is an annual branching erect herb with grows to 

approximately 1 m. in height. The stems and leaves are covered with fine white hairs, 

the leaves are petiolate, ovate up to 7.5 cm long, the apex acute, the base truncate to 

rounded, rarely cordate, the margins crenate. The inflorescence is purple to white 

head, less than 6 mm across and arrange in close terminal corymbs of 8-15 heads. 

Involucres are campanulate, the bracts are 2-3 seriate, linear, sub equal, acute, 

sparsely pilose outside; corollas are all tubular, 1-1.5 mm long, the limb 5-cleft. The 

fruits are linear-oblong black achene with 5-angled and are easily dispersed while the 

seed are photoblastic and often lost within 12 months; pappus are 5 short scales, the 

scales are often serrate below ending in a long awn (Backer and van den Brink, 1968). 

A. conyzoides L. are native to tropical America. It is now found in all warm 

and subtropical areas of the world that is very common in West Africa and some parts 

of Asia and South America. It is usually found in waste places, rice fields, gardens, 

old cultivations, low secondary growth forests, forest-edges, roadsides, water courses 

etc., where there is ample exposure to sunlight (Dung et al., 1996). It has a particular 

odor likened in Australia to that of a male goat and hence its name ‘goat weed’ or 

billy goat weed’ (Okunade, 2002). 

A. conyzoides L. was beneficial in conventional medicine all cover the tropical 

areas. The biological activities of the plant have been undertaken. Some of the 

significant ethno pharmacological applications of A. conyzoides L. were shown in Table 

1. 

5

Figure 1 Ageratum conyzoides L. herbarium specimen 

6

Table 1 Selected ethno pharmacological applications of A. conyzoides L. 

Geological area Part used Indication References 

Africa, Asia and 

South America 

whole 

plants 

Colombia Whole 

plants 

Folk remedies, purgative, 

febrifuge, Opthalmia, 

Colic, Ulcer, Wound 

dressing 

Insecticidal repellent or 

antifeedant properties 

India Root Possess anthelmintic and 

antidysenteric properties 

Senagal - Anti-enteralgic 

Antipyretic 

Kenya whole 

plants 

Thailand Whole 

plant 

Central Africa whole 

plants 

Antiasthmatic 

Antispasmodic 

Haemostatic effects 

Juice from plant taken 

orally as carminative 

property 

Githens, 1948 

Pérez, 1953 

Chopra et al., 1956 

Kerhoro and Adam, 

1974 

Kokwaro, 1976 

Boonyarattanakornkit 

and Supawita, 1977 

Burned wound Durodola, 1977 

India - Treat pneumonia 

Cure wounds and burns 

Nigeria Leaves Skin diseases 

Wound healing 

Diarrhea 

Relieve pain 

Brazil Teas from 

plants 

Anti-inflammatory 

Analgesic, Antidiarrhoeic 

Durodola, 1977 

Okunade, 1981 

Corea, 1984 

7

Table 1 (Continued) 

Geological 

area 

Part used Indication References 

Cameroon Leaves Treat conjunctivitis, 

ophthalmia, headache and 

otitis 

India - Bacteriocide, 

antidysenteric, antilithic 

Africa Whole plants Mental diseases 

Headaches 

Dyspnea 

Northeastern 

India 

Central 

Nigeria 

Leaves Applied on cuts and 

injuries to stop bleeding, 

as an antidote for 

snakebite 

Fresh leaves For body infection, neck 

pain, 

Kenya - Dermatological remedy 

(wound) 

Nepal Leaves Applied to cuts and 

wounds for 

antihemorragic and 

antiseptic properties 

Vietnam - Treatment of 

gynecological disease 

India Whole plants Treat leprosy and as oil 

lotion for purulent 

opthalmia 

Brazil - Anti-inflammatory and 

analgesic 

Sofowora, 1984 

Borthakur and 

Baruah, 1987 

Adjonohoun et al., 

1988 

Neogi et al., 1989 

Bhat et al., 1990 

Johns et al., 1990 

Bhattarai, 1991 

Dung and Loi, 1991 

Kasturi et al., 1973; 

Kirtikar and Badu, 

1991 

Elisabetsky and 

Wannmacher, 1993 

8



Madagascar Aerial parts Febrifuge Rasaonaivo et al., 

1992 

The island of 

Mauritius 

Mauritiua and 

Rodrigues 

India Aqueous 

extract of 

whole plants 

Leaves Release gas in stomach, cure 

diarrhea, skin infection 

Leaves Used as a diuretic in urinary 

diseases 

Treat colic, cold, fever, 

diarrhea, rheumatism, 

spasms, as a tonic 

Gurib-Fakim et 

al., 1993 

Gurib-Fakim et 

al., 1997 

Oliveira et al., 

1993 

Nepal Leaves Juice applied on fresh cut Shrestha and 

Joshi, 1993 

Gabon Leaves Eaten with cola fruit and salt 

to treat pain 

Tamil Nadu Leaves Antiinflammatory, diuretic, 

haemostatic 

India Leaves Chewed and applied over 

fresh cuts to stop bleeding 

and prevent infection 

Vietnam Whole 

plants 

Vietnam Whole 

plants 

Gynecological diseases 


Anti-allergic 


Anti-allergic 

Cure allergic rhibitis and 

simisitic,post partum uterine 

haemorrhage 

Madagascar Leaves Juice as a coagulant, 

Tea for diarrhea 

Akendengue and 

Louis, 1994 

Suresh et al., 1994 

Bhandary et al., 

1995 

Sharma and 

Sharma, 1995 

Dung et al., 1996 

Novy, 1997 

9



Brazil Leaves Treatment of malaria and 

yellow fever 

Ming, 1999 

India Fresh leaves Anthelmintic properties Perumal Samy et 

al., 1999 

Nepal Leaves, 

aerial parts 

The tribals of 

Bangangte 

(Western 

Cameroon) 

Northwest 

Argentina 

Juice of 

whole plants 

Whole 

plants 

Treat cuts and wounds, for 

stomach-ache 

Joshi and Joshi, 

2000 

Use for peptic ulcer Noumi, 2004 

Against cough by drunk as 

syrup during sickness 

Hilgert, 2001 

India Leaves Used for haemostat Kshirsagar and 

Singh, 2001 

Brunei 

Darussalam 

Decoction 

of whole 

plants 

Brazil Aerial parts Tonic, stimulant, 

emmenagogue 

Kenya Leaves, 

roots 

India Whole 

plants 

Taken for cough and fever Holdsworth et al., 

2001 

de Melo Junior et 

al., 2002 

Stomach ache Geissler et al., 

2002 

Applied on tumour and 

swelling, as an antidote to 

snakebite and stings 

Singh et al., 2002 

Vietnam Aerial parts Applied inflammation Ueda et al., 2002 

Ivory Coast Whole 

plants 

Against abdominal pain Diehl et al., 2004 

Cameroon Leaves Emetic effect Noumi, 2004 

10



Mexico Aerial parts As hypoglycemic effect for 

treat diabetes 

India Leaves Juice with leaves of 

Cocculus hirsutus taken to 

cure diarrhoea 

Andrade-Cetto and 

Heinrich, 2005 

Ayyanar and 

Ignacimuthu, 2005 

India Leaves As coagulant Jain et al., 2005 

Tanzania Seeds Treatment of epilepsy Moshi et al., 2005 

A. conyzoides received much attention from phytochemical viewpoint because 

of their plentiful of aromatic compounds. Many kinds of A. conyzoides essential oils 

possess wide varieties of biological activities. The biological activities in pharmacology 

were listed in Table 2. The activities affected on insects and microbial were listed in 

Table 3 and 4 respectively 

11

Table 2 Pharmacological activities of A. conyzoides L. 

Plant parts Solvents Biological activity References 

Leaves Aqueous extract Prevent coagulation of the 

whole blood 

Whole plants Aqueous extract Analgesic effect and 

improvement in 

articulation mobility 

Whole plants Aqueous extract Analgesic activity and antiinflammatory 

action 

Akah, 1988 

Marques-Neto et 

al., 1988 

Silva and Vale, 

1991 

Leaves Aqueous extract Analgesic activity Abena et al., 1993 

Leaves Aqueous extract Analgesic action in rat Bioka et al., 1993 

Whole plants Aqueous extract Muscle relaxing activities Achola et al., 1994 

Leaves Acetone/ 

Methanol 

Aerial parts, 

roots 

Antiinflammatory; paw 

edema in mice 

Essential oil Anti-inflammatory, 

analgesic and antipyretic in 

mice and rats 

Methanol Neuromuscular blocking 

activity 

Suresh et al., 

1994 

Abena et al., 1996 

Achola and 

Munenge, 1997 

Whole plants Aqueous extract Myorelaxing activity Magalhães et al., 

1997 

Leaves Water soluble 

fraction 

Aerial parts Petroleum ether, 

Dichloromethane, 

Ethyl acetate 

Analgesic and antiinflammatory 

activity in 

rats 

Magalhaes et al., 

1997 

Antimalarial activity Madureira et al., 

2002 

Whole plants Ethanol Exhibited DPPH 

scavenging and nitric oxide 

generation 

Shirwaikar et al., 

2003 

12



Aerial parts Ethanol Anti-inflammatory in 

Wistar albino rats 

Fresh leaves Methanol Demonstrated wound 

healing properties 

Moura et al., 2005 

Chah et al., 2006 

13

Table 3 Biological activities of A. conyzoides L. on insects 


Fresh 

leaves 

Whole 

plant 

Whole 

plants 

Precocenes I 

and II 

Precocene I 

and II 

Accelerate larval 

metamorphosis, resulted in 

juvenile form or weak and small 

adults of Musca domestica 

Antijuvenile hormone of 

Dysdercus flavidus 

Antijuvenile hormone of 

Sitophilus oryzae, Thlaspida 

japonica, Leptocarsia chinesis 

methanol Produce deficiency of juvenile 

hormone of sorghum pests 

(Chilo partellus) 

Petroleum 

ether 

Flowers Petroleum 

ether 

Whole 

plants 

Whole 

plants 

Induce morphogenetic 

abnormalities in the formation of 

mosquitoes larvae (Culex 

quinquefasciatus, Aedes aegypt 

and Anopheles stephensi 

Vyas and 

Mulchandani, 

1986 

Fagoonee and 

Umrit, 1981 

Lu, 1982 

Raja et al., 1987 

Sujatha et al., 

1988 

Hexane Against Musca domestica larvae Gonzales et al., 

1991 

Aqueous 

extract 

Against mosquitoes (Anopheles 

stephensi) 

Reduce larvae emergence of 

Meloidogyne incognita 

Methanol Suppress population of 

Anopheless stephensi at 

preimaginal stage 

Kamal and 

Mehra, 1991 

Shabana et al., 

1991 

Saxena and 

Saxena, 1992 

14



Whole 

plants 

Aqueous 

extract 

Essential oil 

(precocenes) 

Essential oil 

(precocenes) 

Leaves Distilled 

water 

Sheltered predators of spidermite 

(Panonychus citri, 

Phyllocoptruta oleivora, 

Brevipalpus phoenicis) 

Caused nymphal mortality of 

Schistocerca gregaria 

Toxicity on adults of cowpea 

weevil (Callosobruchus 

maculates) 

Cause mortality of the maize 

grain weevil (Sitophilus 

zeamais) 

Pu et al., 1990; 

Gravena et al., 

1993; Liang et al., 

1994 

Pari et al., 1998 

Gbolade et al., 

1999 

Bouda et al., 2001 

15

Table 4 Biological activities of A. conyzoides L. on microbial 


Whole 

plants 

Whole 

plants 

Whole 

plants 

Fresh 

leaves 

Whole 

plant 

Whole 

plant 

Ether and 

chloroform 

Against in vitro development 

of Staphylococus aureus 

Methanol Inhibit growth and 

development of S. aureus, 

Bacillus subtilis, Escherichia 

coli and Pseudomonas 

aeruginosa 

Durodola, 1977 

Almagboul et al., 

1985 

Aqueous extract Reduction of leprosy virus Gravena et al., 

1993 

Essential oil Inhibit the growth of Candida 

albicans SP-14, Cryptococcus 

neoformas SP-16, Sclerotium 

rolfsii SP-5, Trichophyton 

mentagrophytes SP-12 

Essential oil Against fungi; Penicillium 

chrysogenum and P. javanicum 

Essential oil 100% inhibition of the 

mycelial growth and 

germination of spores of 

Didymella bryoniae 

Distilled water Controlled the growth of 

Alkaligens viscolactis, 

Klebsiella aerogenas, Bacillus 

cerues and 

Dichloromethane/ 

Methanol 

Inhibit Plasmodium 

falciparum 

Pattnaik et al., 

1996 

Ekundayo et al., 

1988 

Fiori et al., 2000 

Perumal Samy et 

al., 1999 

Clarkson et al., 

2004 

Methanol Against Tychophyton spp. Moody et al., 

2004 

16




water 

Against Epidermophyton 

floccosum and Trichophyton 

mentengrophytes 

Leaves Ethanol Inhibit E. coli, Microsporum 

canis, Trichophyton 

mentagrophytes 

Whole 

plants 

Leaves, 

stems, roots 

Ethanol Inhibit Streptococcus pyogenous 

and Neisseria gonorrhoea 

Cold/hot 

water, 

Methanol/ 

Hexane 

Susceptibility of Staphylococcus 

aureus, Yersinia enterocolitica, 

Salmonella gallinarum and 

Escherichia coli 

Leaves Acetone Against the plant pathogenic 

fungi, Aspergillus niger 

Mishra et al., 

1991 

Vlietinck et al., 

1995 

Geyid et al., 2005 

Okwori et al., 

2007 

Widodo et al., 

2008 

In natural ecosystem, many higher plants may hold stronger allelopathic 

potential and may exhibit a higher weed reduction. A. conyzoides L. was assessed to 

have a strong invasion capacity in plant communities, significantly reducing natural 

growth of weeds in its vicinity. The allelopathic effects of A. conyzoides L.were 

listed in Table 5. 

17

Table 5 Allelopathy of A. conyzoides L. 


Whole plants Volatile oil, 

aqueous 

extract 

Leaves, stem, 

roots 

Distilled 

water 


water 


water 


water 

Aerial parts Distilled 

water 

Aerial parts Distilled 

water 

Inhibit seedling growth of radish 

(Raphanus sativus L.), tomato ( ) 

and ryegrass ( ) 

Leaf extract inhibited radish 

germination and shoot and root 

length 

Leaf extract reduced dry weight of 

radish 

Inhibited germination and growth 

of Monochoria vaginalis, 

Echinochloa crus-galli and 

Aeschynomene indica 

Promoted rice (Oryza sativa L. 

var. indica) growth: plant height, 

tiller number, panicle number, 

grain number and yield 

Controlled emergence of weeds in 

paddy field; Echinochloa 

oryzicola, Eleochalis acicularis, 

Linderna pyxidaria, Monochoria 

vaginalis and Rotala indica 

Inhibited germination of wheat 

(Triticum aestivum L.) and rice 

seeds 

Caused lower reduction of peanut 

seed (Arachis hypogaea L.) 

Kong et al., 

1999 

Xuan et al., 

2004 

Xuan et al., 

2004 

Xuan et al., 

2004 

Xuan et al., 

2004 

Jha and Dhakal, 

1990 

Parasad and 

Srivastava, 

1991 

18



Whole plants Distilled 

water 

Whole plants Distilled 

water 

Controlled emergence of 

Monochoria vaginalis, Rotala 

indica, Marsilea quadrifolia, 

Leptochloa chinensis, Cyperus 

difformis, Sphenochlea 

zeylanica, Commelina diffusa, 

Dactyloctenium aegyptium and 

Brachiaria mutica 

Promoted emergence of 

Fimbristylis miliacea, 

Murdannia keisak and Jussiaea 

decurrens 

Whole plants polyethylene Reduced growth of chickpea 

(Cicer arietinum) 

Hong et al., 

2004 

Hong et al., 

2004 

Batish et al., 

2006 

Beside this, A. conyzoides L. were noted for being a profuse source of many 

compounds of phytochemical interest. Many interesting compounds isolated from the 

plant can categorize including mono- and sesquiterpenes (Table 6), chromene, 

benzofuran and coumarin (Table 7), flavonoids (Table 8), alkaloids (Table 9) and 

Triterpenes and sterols (Table 10) 

19

Table 6 Mono- and sequiterpenes from A. conyzoides L. 

Compounds Plant parts References 

sabinene Whole plants Ekundayo et al., 1988 

camphene Aerial parts Dung et al., 1996 

cubebene Aerial parts Dung et al., 1996 

elemene Aerial parts Dung et al., 1996 

farnesol Aerial parts Dung et al., 1996 

β-farnesene Aerial parts Dung et al., 1996 

β-myrcene Aerial parts Dung et al., 1996 

β -pinene Whole plants Ekundayo et al., 1988; 

Dung et al., 1996 

α-pinene Whole plants Rao and Nigam, 1973; 

Dung et al., 1996; 

Ekundayo et al., 1988 

β -phelandrene Whole plants Ekundayo et al., 1988 

β-selinene Aerial parts Dung et al., 1996 

1,8-cineole Whole plants Ekundayo et al., 1988 

limonene Whole plants Ekundayo et al., 1988 

terpinen-4-ol Whole plants Ekundayo et al., 1988 

α-terpineol Whole plants Ekundayo et al., 1988 

α-terpinene Aerial parts Dung et al., 1996 

ocimene Whole plants Rao and Nigam, 1973 

eugenol Whole plants Rao and Nigam, 1973 

methyleugenol Whole plants Rao and Nigam, 1973 

20



β-caryophyllene Whole plants Ekundayo et al., 1988; 

Dung et al., 1996; 

Chalchat et al., 1997; 

Riaz et al., 1995; Nébié 

et al., 2004 

δ-cadinene Whole plants Rao and Nigam, 1973 

sesquiphellandrene Whole plants Ekundayo et al., 1988 

O 

H 

Caryophyllene epoxide 

H 

Whole plants Ekundayo et al., 1988; 

21

Table 7 Chromenes, benzofurans and coumarins from A. conyzoides L. 

MeO 

H 


7-methoxyageratochromene (Precocene I) 

MeO 

MeO 

O 

O 

Me 

Me 

Me 

Me 

Ageratochromene (Precocene II) 

MeO 

O 

MeO 

Encecalin 

6-vinyl-7-methoxy-2,2-dimethylchromene 

O 

O 

Me 

Me 

Me 

Me 

Whole plants Dung et al., 1996; 

Wandji et al., 

1996; Pham et al., 

1976; Nébié et 

al., 2004; Quijano 

et al., 1982; Pari 

et al., 1998 

Whole plant Dung et al., 1996; 

Nébié et al., 

2004; Pham et al., 

1976; Quijano et 

al., 1982; Pari et 

al., 1998 

Whole plant 

Ekundayo et al., 

1988; Gonzalez et 

al., 1991a 

Whole plant Ekundayo et al., 


al., 1991a 

22


∆ 3 

∆ 3 

∆ 3 

∆ 3 

MeO 

O 

H 

O 


O 

Dihydroencecalin 

O 

Me 

Me 

Me 

Me 

Dihydrodemethoxyencecalin 

,H 

O 

,HO 

O 

Demethoxyencecalin 

Demethylencecalin 

O 

O 

Me 

Me 

Me 

Me 


1988 


1988 


1988 


1988 

23


HO 

HO 

MeO 

MeO 


O 

Me 

C 

O 

i-Pr 

2-(1´-oxo-2´-methylpropyl)-2methyl-6,7-dimethoxychromene 

HO 

O 

OH 

O 

2,2-dimethylchromene-7-O-βglucopyranoside 

MeO 

MeO 

O 

O 

Me 

Me 

6-(1-methoxyethyl)-7-methoxy-2,2dimethylchromene 

MeO 

O 

H 

O 

Me 

Me 

6-(1-hydroxyethyl)-7-methoxy-2,2dimethylchromene 

Me 

Me 

Whole plant Pari et al., 1998 

Whole plant Ahmad et al., 

1999 

Aerial part Gonzalez et al., 

1991a 


1991a 

24


Me 

Me 

MeO 

Et 

O 


O 

Me 

Me 

6-(1-ethoxyethyl)-7-methoxy-2,2dimethylchromene 

O 

O 1' 

2' 

4' 

H 

3' 

MeO 

5' 

6-angeloyloxy-7-methoxy-2,2dimethylchromene 

O OMe 

MeO 

MeO 

O 

MeO 

Encecanescin 

O 

O 

i-Pr 

2-(2´-methylethyl)-5,6-dimethoxybenzofuran 

Me 

O 

Me 

Me 

Me 


1991a 


1991a 


1991a 


25


Me 

O 

HO 


O 

OH 

14-hydroxy-2Hβ,3-dihydroeuparine 

MeO 

OMe 

O 

O 

Me 

i-Bu 

3-(2´-methylpropyl)-2-methyl-6,8dimethoxychrom-4-one 

MeO 

MeO 

O 

O 

Me 

CH=CMe 2 

2-(2´-methoxyprop-2´-enyl)-2-methyl-6,7dimethoxychroman-4-one 

Whole plant Ahmed et al., 

1999 



26

Table 8 Flavonoids from A. conyzoides L. 

MeO 

MeO 

MeO 

MeO 

MeO 

MeO 

OMe 


O 

OMe O 

OMe 

nobiletin 

O 

OMe O 

OMe 

OMe 

5´-methoxynobiletin 

O 

OMe O 

Ageconyflavone A 

O 

OMe 

H 

OMe 

OMe 

O 

H 

Whole plant Vyas and 

Mulchandani. 


al., 1991b 



1986; Adesogan 

and Okunade, 


al., 1991b 



1986 

27


MeO 

MeO 

MeO 

MeO 

MeO 

MeO 


O 

OMe O 

Ageconyflavone B 

O 

OMe O 

Ageconyflavone C 

OMe 

O 

OMe O 

Linderoflavone B 

OMe 

OMe 

O 

OH 

H 

OH 

OMe 

O 

H 



1986 



1986 




al., 1991b 

28


MeO 

MeO 

H 


O 

OMe O 

O 

O 

OMe 

5,6,7,5´-tetramethoxy-3´,4´-methylene 

dioxyflavone 

MeO 

MeO 

MeO 

MeO 

O 

OMe O 

H 

Sinensetin 

O 

OMe O 

OMe 

OMe 

OMe 

H 

OMe 

OMe 

5,6,7,3´,4´,5´-hexamethoxyflavone 

whole plant Gonzalez et al., 

1991b 




al., 1991b 

Whole plant, 

aerial part 

Gonzalez et al., 

1991b; Vyas and 



al., 1984 

29


H 

MeO 

OMe 


O 

OMe O 

OMe 

5,6,8,3´,4´,5´-hexamethoxyflavone 

MeO 

MeO 

HO 

OMe 

O 

OMe O 

Eupalestin 

O 

OH O 

Quercetin 

OH 

O 

OH 

O 

OMe 

OMe 

OMe 

OH 

aerial part Gonzalez et al., 

1991b 

aerial part Gonzalez et al., 

1991b; Gonzalez 

et al., 1984; 

Adesogan and 

Okunade, 1979 

Whole plant Gill et al., 1978 

30


HO 

HO 

HO 


O 

OH O 

O 

OH 

Quercetin-3-rhamnopyranoside 

O 

OH O 

Kaempferol 

O 

OH O 

OH 

H 

OH 

rhamnopyranosyl 

Kaempferol-3-rhamnopyranoside 

O 

H 

OH 

OH 

rhamnopyranosyl 




31


lysonarypoculg 


O 

OH O 

O 

H 

OH 

glucopyranosyl 

Kaempferol-3,7-diglucopyranoside 

HO 

(H 3C) 2C=HC-H 2C 

O 

O O 

HO 

Rha-( 1-5)-Rha 5,7,2´,4´-tetrahydroxy-6,3´-di- 

(3,3-dimethylallyl)-isoflavone 5-Οα-L-rhamnopyranosyl-(1→4)α-L-rhamnopyranoside 

OH 

CH 2-CH=C(CH 3) 2 

Whole plant Gill et al., 1978; 

Nair et al., 1977 

Stems Yadara and 

Kumar, 1999 

32

Table 9 Alkaloids from A. conyzoides L. 

OH O 

N 


H 3C CH 3 

O 

HO 

OH 

CH 3 

Lycopsamine (pyrrolizidine alkaloids) 

HO O 

O 

O 

N 

Whole plant Wiedenfeld 

and Roder, 

1991; Horie et 

al., 1993 

1,2-desilropirrolizidinic whole plant Horie et al., 

1993 

H 3C CH 3 

O 

HO 

OH 

CH 3 

Echinatine (pyrrolizidine alkaloids) 

O 

H 

H 

H 

H 

O 

(+)-sesamin 

H 

O 

O 

Whole plant Wiedenfeld 

and Roder, 

1991 

Whole plant Gonzalez et 

al., 1991b 

33


HO 

H 3C 

HO 

CH 3 

AcO 


Caffeic acid 

H 

N 

O 

HN 

O 

COOH 

Aurantiomide acetate 

CH 3 

Phytol 

CH 3 

CH 3 

OH 

Whole plant Nair et al., 

1977 

Whole plant Sur et al., 

1997 

Whole plant Vera, 1993 

34

Table 10 Triterpenes and sterols from A. conyzoides L. 

HO 

HO 

O 


H H H 

Friedelin 

β -Sitosterol 

∆ 22 Stigmatosterol 

Whole plant Dubey et al., 1989; 

Horng et al., 1976; 

Hui and Lee, 1971 







35


HO 

HO 

HO 


∆ 22 Brassicasterol 

Dihydrobrassicasterol 

∆ 22 Spinasterol 










36


HO 


Dihydrospinasterol 




37

1. Plant Materials 

METERIALS AND METHODS 

Materials 

1.1 Plant materials for phytochemical method 

Whole plants of A. conyzoides utilized in this study were collected from 

Trat Agroforestry Research Station, Kasertsart University Research and Development 

Institute, Trat province in the eastern part of Thailand in December 2004 and October 

2005. Botanical identification was achieved through comparison with specimens (No 

074019, 075554, 075556, 07179, 3159, 076326, 55755, 075552, 45867, 37342, 

075001, 075563 and 124878) deposited in the Bangkok Forestry Herbarium (BKF). 

Callus utilized in this experiment was obtained from less hairy-blue head A. 

conyzoides collected from Chantaburi Province in December 2003. 

1.2 Plant materials for biological activity test 

The seed of plants utilized in this study were both cultivated plants and 

weeds. The cultivated plants were Ipomoea aquatica Forssk., Brassica chinensis L. 

var. chinensis and Oryza sativa L. cultivar Hom Mali 105. The weeds were Mimosa 

pigra L., Tridax procumbens L., Echinochloa colona (L.) Link and Cenchrus 

echinatus L. 

2. Instrumentations 

2.1 Labatory Instruments 

The instruments utilized in this study were rotary evaporator (Buchi 

Rotavapor R-114, R-205), UV cabinet (CN-6.T) with Ultraviolet radiation obligatory 

eye protection 254 nm and 365 nm (Vilber Lourmal serial number V01 5636), 

38

analyical balance (Mettler Toledo AG 204), oven (National EH 5741), deep freeze 

(Sanyo -85 ˚C), blender, desicator, TCL tank, filter paper (110 mm and 185 mm Ø 

Whatman No. 1) and glasswares such as burettes, pipettes, Erlenmeyer flask, 

Buchner funnel, separatory funnel, 6´´ diameter Petri dish, volumetric flask, beaker. 

2.2 Chromatographic Techniques 

2.2.1 Thin layer chromatography (TLC) 

Technique : one way, ascending 

Absorbent : silica gel 60 F254 (0.2 mm thickness, 20x20 

cm 2 Merck) supported on mirror plate 

Plate size : 10 cm x 20 cm and 20 cm x 20 cm 

Layer thickness : 250 µ 

Solvent system : dichloromethane : ethyl acetate : methanol 

(75:20:5) 

Distance : 15 cm 

Temperature : 25-30 ºC 

Detection : 254 nm and 365 nm UV light (Ultraviolet 

Radiation Obligatory eye protection: Vilber 

Lourmal serial No V01 5636) 

The position of a substance zone (spot) in a thin layer chromatogram 

can be described as Retardation Factor (Rf). 

Rf = distance of the substance zone from the starting line (cm) 

distance of the solvent front from the starting line(cm) 

(Hahn-Deinstrop, 1997) 

39

2.2.2 Dry column chromatography 

Column size : glass column 80 cm long, 1.7 cm wide 

(diameter inside the column) 

Absorbent : 60 g of silica gel 60 (0.2-0.5 mm, 35-70 mesh: 

Merck) 

Sample : 1 g of lipophilic extract 

Mobile phase : hexane, diethyl ether and methanol from 

95:5:0 to 0:0:100 

Fraction volume : 50 ml in Erlenmeyer flask 

Examination : TLC monitoring 

2.2.3 Medium pressure liquid chromatography (MPLC) 

MPLC technology : ISCO Type 9 optical unit 

Pump : The FMI lab pump model RP-D Fluid 

Column : glass column (400 x 40 mm) 

Absorbent : Lichroprep silica gel 60 (25-40 µm, 132 TA 

145390) 

Mobile phase : mixture of 5%, 10%, 15%, 30%, 50% and 

70% ethyl acetate in hexane 

Flow rate : 30 ml/min 

Sample : 1 g 

Detector : absorbance/ fluorescence detector with wave 

length 254 nm: ISCO UA-5 

Fraction : collect the fraction eluted from column by 

chromatogram signal 

40

2.2.4 High Performance Liquid Chromatography (HPLC) 

2.3 Spectroscopy 

HPLC technology : Agilent 1100 series 

Detector : UV photodiode array detector 230 nm wave 

length 

Column : reverse phase ChromSepher 5 C18 column 

(250 x 4.6 mm; part number CP 29358) 

Sample : 10 mg/ml of lipophilic extract and 1 mg/ml of 

pure compound filtered with 13 mm x 0.45 µm 

Nylon filter (Iso-discTM N-13-4) 

Injection : 20 µl 

Flow rate : 1.0 ml/min 

Time : 30 mins 

Solvent system : methanol gradient 60%-100% (HPLC grade 

Merck) in aqueous buffer (0.015 M tetrabutyl 

ammonium hydroxide (C16H37NO, AR grade 

Fluka) and 0.015 M ortho-phosphoric acid 

(AR grade Merck), pH 3) 

Mobile phase : Time (Min) MeOH Buffer 

0.10 60 40 

17.00 90 10 

22.00 100 0 

28.00 100 0 

29.00 60 40 

2.3.1 Ultraviolet Spectroscopy 

Ultraviolet (UV) spectra were determined on Agilent 1100 series 

UV photodiode array detector 230 nm wave length at Scientific Instrumentation 

Center, Faculty of Science, Kasetsart University, Bangkok, Thailand. 

41

3. Chemicals 

The organic solvents utilized in extraction were methanol (CH3OH: AR grade 

Merck), chloroform (CHCl3: AR grade Merck) and distilled water (H2O: 16 MΩ /cm, 

Millipore). 

The chemicals for chromatography were dichloromethane (CH3Cl2: AR grade 

Merck, Fisher), ethyl acetate (CH3COOC2H5: AR grade Merck, Fisher), hexane 

(CH3(CH2)4CH3: AR grade Merck, Labscan) and diethyl ether ((C2H5)2O: AR grade 

Merck). 

The reagent for phytochemical screening were Dragendorff’s reagent (bismuth 

sub nitrate (BiO(NO3).H2O: AR grade Merck), glacial acetic acid (CH3COOH: AR 

grade Merck), distilled water (H2O: 16 MΩ/cm Millipore) and potassium iodide (KI: 

AR grade Merck)), 10% Sodium hydroxide (NaOH: AR grade Mallinckrodt), Kedde’s 

reagent (3,5-dinitrobenzoic acid (C7H4N2O6 : AR grade Fluka), potassium hydroxide 

(KOH: AR grade Univar) and ethanol (C2H5OH: AR grade Merck)), Raymond’s 

reagent (1,3-dinitrobenzene (C6H4N2O4 : AR grade Fluka)), acetic anhydride, 97% 

sulfuric acid (H2SO4 : AR grade Fisher Scientific), hydrochloric acid (HCl: AR grade 

Merck), ferric chloride (FeCl3), anisaldehyde (C8H8O2 : AR grade Fluka), iodine 

crystals (May & Baker LTD Dagenham England) 

The commercial herbicides utilized for seed germination and seedling growth 

tests were paraquat dichloride (glumoxone syngenta) and glyphosate 

isopropylammonium (glyphosate 48) 

42

Extractions 

Methods 

Three populations of A. conyzoides L.; less hairy-blue head (POP1), more 

hairy-blue head (POP2) and more hairy-white head (POP3) plants and calli from in 

vitro induction at Department of Botany, Faculty of Science, Kasetsart University 

were utilized in the experiment. The samples were collected twice, December 2004 

and October 2005, the populated were designated as: 

Collection 1:- 

less hairy-blue head : POP1a 

more hairy-blue head : POP2a 

more hairy-white head : POP3a 

Collection 2:- 

less hairy-blue head : POP1b 

more hairy-blue head : POP2b 

more hairy-white head : POP3b 

Air-dried whole plants of POP1a (236.67 g), POP2a (236.32 g), POP3a (30.33 

g), POP1b (372.70 g), POP2b (412.43 g) and POP3b (261.30 g) were cut into 

approximately 1 cm pieces and then crushed into powder. The plant powder and 

callus were macerated with methanol for 7 days in the dark at room temperature, then 

the extracts were filtered through Whatman no. 1 filter paper and subsequently 

concentrated by using rotary evaporator at 37 °C afforded dark green semi-solid crude 

extract. The concentrated crude extract was successively partitioned into two parts: 

hydrophilic extract and lipophilic crude extract with distilled water and chloroform 

respectively. The lipophilic crude extract was then evaporated into dryness for further 

experiments. 

43

Lipophilic crude extract of POP1a (2.121 g), POP2a (3.487 g), POP3a (0.26 g), 

POP1b (8.086 g), POP2b (6.248 g) and POP3b (4.302 g) and callus (0.058 g) were 

screened by preliminary test and analyzed by using Thin Layer Chromatography (TLC) 

and High Performance Liquid Chromatography (HPLC), only POP1b lipophilic crude 

extract was utilized in bioassay test, separated and subsequently purified by using 

column chromatography and Medium Pressure Liquid Chromatography (MPLC). 

Phytochemical Screening (Preliminary Test) 

Portion of the lipophilic crude extract subjected for the biological screening 

was used for the identification of the major secondary metabolites employing the 

methodology outlined by Farnsworth (1966) as following; 

1. Screening for alkaloids 

The alkaloids nucleus could be detected by Dragendorff’s reagent. The 

lipophilic extract was dropped into a porcelain basin, dried by blower and added with 

a few drops of Dragendorff’s reagent. A change occurred with red-brown 

precipitations within several minutes indicated the presence of alkaloid nucleus. 

Anyway, these might be false positive because of Dragendorff’s reagent 

2. Screening for coumarins 

The sample was dissolved with methanol in a flask. The flask was first 

covered with a piece of filter paper moistened with 10% sodium hydroxide, finally the 

filter paper was dried by blower and then examined under the UV light (365 nm). The 

appearance of yellow-green fluorescence after a short time indicated the presence 

of coumarin. In this study, the method was applied by dropping of the extract onto 

filter paper. When the extract dried, dropped 10% sodium hydroxide and then 

examined under the UV light (365 nm). 

44

3. Screening for unsaturated lactone ring 

For confirmation of unsaturated lactone ring, the test must be done 

paralelly, both test with Kedde’s reagent and the test with Raymond’s reagent. When 

the sample was dropped into porcelain basin, dried by blower and added with a few 

drops of Kedde’s and Raymond’s reagent. Positive test after dropping Kedde’s 

reagent, violet-pink color could be detected. Positive test after dropping Raymond’s 

reagent, violet-blue color could be detected. 

4. Screening for steroids and triterpenoids 

Libermann-Burchard (L-B) test: This method was used for testing of 

steroidal and triterpenoidal nucleus. The sample was dropped into a porcelain basin, 

dried by blower and added with a few drops of acetic anhydride followed by one drop 

of conc. sulfuric acid (conc. H2SO4). A change in color to blue or blue-green within a 

minute indicated the presence of steroidal nucleus whereas triterpenoidal nucleus gave 

the purple, pink or red color. 

5. Screening for flavonoids 

Cyanidin test: This method was used for testing of γ-benzopyrone nucleus. Put 

a small piece of magnesium ribbon into methanolic extract of sample and added few 

drops of conc. hydrochloric acid (conc. HCl). The presence of bubble color 

ranging from orange to red which indicated the presence of flovone, red to crimson 

indicated the presence of flavonol, crimson to magenta indicated the presence of 

flavanone and occasionally green or blue was a positive reaction for either aglycone 

or heteroside. Xanthone had also given the positive cyaniding reaction whereas 

chalcone and aurone would not give the positive testing. 

Another method used to test for flavonoids was ferric chloride (FeCl3). 

The sample was dropped into a porcelain basin, dried by blower and added with a few 

drops of ferric chloride. A change in color to blue-green within several minutes 

indicated the presence of flavonoids. 

45

All results of preliminary tests were compared with the results from TLC 

spraying technique 

Qualitative analyses 

1. TLC screening 

The TLC plates were subsequently sprayed with detecting reagents so as to 

screen major secondary compounds. This method allowed not only for detection of 

the compounds, but also for an estimate of the number present. These reagents were 

almost similar to those used for phytochemical sceening as following; 

For alkaloid detection, sprayed TLC plate by Dragendorff’s reagent. 

Positive test after spraying the reagent, red-orange color could be detected 

(Farnsworth, 1966). 

For terpenoid detection, sprayed TLC plate by Anisaldehyde-sulfuric acid 

and following by heating in oven at 100-105 ˚C for 5-10 miniutes. Positive test after 

spraying this reagent, colorful spot will occure varies on the compound; red (terpene), 

green (steroid), blue (phenol) and grey (sugar) (Merck, 1980). 

For unsaturated lactone ring detection, the test must be done pararelly by 

sprayed TLC plate both with Kedde’s reagent and with Raymond’s reagent. Positive 

test after spraying Kedde’s reagent, violet-pink color could be detected. Positive test 

after spraying Raymond’s reagent, violet-blue color could be detected (Farnsworth, 

1966). 

For coumarin detection, sprayed TLC plate by 10% NaOH. When the plate 

dried, exposed the plate under UV light at wavelength 365 nm. If coumarin was 

present, a yellow-green fluorescence appeared (Applied from Farnsworth, 1966). 

For general detection, put the TLC plate in iodine vapor chamber and 

watched the spots appearing more clearly (Merck, 1980) 

46

2. Comparative analysis 

2.1 TLC 

A small spot of solution containing the sample was applied to a plate, 

about 1 cm from the base. The plate was then dipped in to solvent system and placed 

in a sealed container. Different compounds in the sample mixture traveled at different 

rates due to differences in solubility in the solvent, and due to differences in their 

attraction to the stationary phase 

2.2 HPLC 

Prepared 10 mg of samples in methanol (HPLC grade). The HPLC 

analysis was undertaken on Agilent 1100 series at Scientific Instrument Center, 

Faculty of Science, Kasetsart University. 

Separation and purification 

Lipophilic crude extract of POP1b were separately fractionated using dry 

column chromatography over silica gel eluting with hexane, hexane-ethyl acetate, 

ethyl acetate-methanol and methanol gradient. Combined the collected fractions from 

each extracts on the basis of their TLC patterns. Interesting fractions were further 

purified by MPLC technique subsequently recrystallized to give pure compound with 

diethyl ether. 

The structure of pure compound was elucidated by spectroscopy technique and 

analyzed melting point by Melting point Apparatus (Fisher John apparatus serial 

number 4017) 

The flowchart of phytochemical method was showed in Figure 2 

47

H2O fraction 

(hydrophilic crude extract) 

Extraction 

Plant samples 

Figure 2 Phytochemical method 

Ground 

Macerated in MeOH for 7 days 

Filtered and evaporated 

Crude extract 

Partition between H2O and CHCl3 

TLC and HPLC analyses 


Roughly separation by dry CC 

MPLC 

CHCl3 fraction 

(lipophilic crude extract) 

Recrystallization 

Pure compound 

Structure elucidation and identification by melting 

point measure, UV, IR, NMR and MS 

Biotest 

Phytochemical screening 

48

Biological activities test 

To examine the inhibitory effect of the extract on seed germination and 

seedling growth, lipophilic extract of less hairy-blue head (POP2b) was diluted to be 

0.25, 0.50, 1.0 and 2.0 g/l by methanol. The test was compared with chemical 

herbicide (paraquat dichloride and glyphosate). 

Cultivated plant and weed seeds were rinsed with distilled water. Oryza sativa 

L. cultivar Hom Mali 105, Cenchrus echinatus L. and Echinochloa colona (L.) Link 

were soaked in distilled water at 8 °C for 1 day. Ipomoea aquatica Forssk., Brassica 

chinensis L. var. chinensis and Tridax procumbens L. were soaked in distilled 

water at room temperature for 1 day and Mimosa pigra L. were soaked in distilled 

water at 100 °C and allowed to cool for 1 day. Twenty seeds were seeded on three 

germination blotter papers in each Petri dish and all treatments were replicated 

four times. The treatments were incubated at room temperature. Amount of 

germinated seeds in each Petri dish were counted at every 3, 5 and 7 days during 

incubation. Seedling growth was also recorded in shoot and root length at the last day 

of the experiment. Methanol without extract was used as a control. Standard error 

was calculated based on four measurements of replicate dishes. 

Germination rate was expressed as percent of germinated seeds over total 

seeds seeded. The inhibition percentage of the study was calculated as follows: 

Germination percentage = Amount of germinated seeds x 100 

Amount of total seeds 

The experiment was designed as Complete Randomized Design (CRD). Data 

are presented as means and subjected to one-way analysis of variance (one-way ANOVA) 

followed by Duncan’s multiple range rest. All the statistical analysis was performed 

using SPSS software version 13.0 

49

Extraction 

RESULT AND DISCUSSION 

In this experiment, the three-population dried plants of Ageratum conyzoides L. 

from two collections (Collection 1: POP1a, POP2a and POP3a; Collection 2: POP1b, 

POP2b and POP3b) and callus were ground and extracted with methanol for 7 days in 

total darkness at ambient temperature. The filtered extract was concentrated to be 

semi-solid residue giving the crude extract which was partitioned by chloroform and 

water. Combined chloroform fractions were evaporated to dryness called lipophilic 

crude extract, dissolved in methanol, and stored at -80 ˚C until proceeding further. 

From crude methanolic extract of POP1a, POP2a, POP3a, POP1b, POP2b, 

POP3b and callus, the amount of 2.121 g, 3.487 g, 0.26 g, 8.086 g, 6.248 g, 4.302 g 

and 0.058 g of lipophilic crude extract was obtained respectively. These lipophilic 

crude extracts were analyzed the major secondary metabolites by phytochemical 

screening technique. 

Phytochemical screening 

1. Screening for alkaloids 

For detection of alkaloids in phytochemical screening, Dragendorff’s 

reagent was utilized as alkaloidal precipitants commonly used for the detection of 

general alkaloids. The positive reaction was that, heavy metal containing in the 

reagent (Bismuth (III)) would react to nitrogenous base in alkaloidal extracts and form 

insoluble complex heavy metal salt as red-brown precipitation. The results of 

alkaloids screening were shown as Figure 3 and Table 11 

50

POP1a POP2a POP3a POP1b POP2b POP3b 

Before test before test before test before test before test before test 


after test after test after test after test after test after test 

(positive test) (negative test) (negative test) (positive test) (negative test) (negative test) 

Figure 3 Alkaloidal test by using Dragendorff’s reagent 

51 

51

From the result, only less hairy-blue head Ageratum conyzoides (both 

POP1a and POP1b) was detected as red-orange precipitation, meaning that this 

population might contain of alkaloid, while the other two populations, more hairyblue 

head (POP2a and POP2b) and more hairy-white head (POP3a and POP3b), 

alkaloid were absent. 

Variability of results in alkaloid testing of plant material could be induced 

by a number of factors such as age, climate, habitat, plant part tested, season, 

chemical race of plants, etc. All of three A. conyzoides populations were collected 

together in the same habitat, however, there were some different characteristics; 

complexion (more and less hair), inflorescence color, even age. A few examples 

regarding these factors should serve to point out their importance. For example, 

Geijera salicifolia gave consistently better alkaloid tests as the broad leaf form than 

the narrow leaf form, even plants with the two characters were growing side by side in 

the field (Webb, 1949). In certain groups of plants, i.e. Compositae, alkaloids are 

often found only in or near the flower tops and in the Apocynaceae, alkaloids 

generally trend to concentrate in the root or bark (Raffauf and Flagler, 1960). 

Furthermore, other substances could give false-positive to Dragendorff’s 

reagent. These substances reported in the literature as false-positive alkaloid reactions 

are certain glycosides, carbohydrate, betalain, choline, purines, methylated amines, 

tannins and ammonium salts (Rosenthaler, 1930; Raffauf, 1962; Webb, 1949). To 

confirm the result, different types of alkaloidal precipitating reagent, i.e., 

Bouchardat’s reagent, Hager’s reagent, Mayer’s reagent or Wagner’s reagent or 

different way to analyze should be found out, such as chromatography. Many 

investigators utilized 4 or 5 reagents in their screening of plant extracts, and only 

samples yielding precipitate with all reagents were considered to contain alkaloids 

(Farnsworth, 1966) 

However, this was similar to the finding of Okwori et al. (2007), who 

documented alkaloids containing in A. conyzoides L. by phytochemical screening. 

There were the studies finding alkaloids, especially pyrrolizidine alkaloids in A. 

conyzoides L. (Trigo et al., 1988; Wiedenfeld and Roder, 1991; Horie et al., 1993). 

52

2. Screening for coumarins 

Coumarins, which are benz-α-pyrone derivatives, is a group appearing 

most interesting because of their coagulation, estrogenic, thermal photosensizing, 

antibacterial, molluscacidal, anthelmintic, sedative and hypnotic, analgesic and 

hypothermal effects (Bose, 1958; Soine, 1964). According to these interesting 

properties, coumarin was one of compounds should be analyzed before point out the 

biological activity of A. conyzoides lipophilic crude extract. 

Coumarin itself could be easily detected in the extract simply by 10% 

sodium hydroxide solution. Positive test is yellow-green fluorescence which could be 

detected after exposure to UV light at wavelength 365 nm within few minutes. The 

result of coumarin detection was shown in Figure 4 and Table 11. 

From Figure 4, under UV light wavelength 365 nm, the yellow-green 

fluorescence appeared in the lipophilic crude extracts of more hairy A. conyzoides. 

The detections were not different between blue head (POP2a and POP2b) and white 

head (POP3a and POP3b). On the other hand, the extract of less hairy plants (POP1a 

and POP1b) and even their callus did not show yellow-green fluorescence under UV 

light. It could be concluded that coumarin contain in A. conyzoides but only in more 

hairy populations. It has also been reported that A. conyzoides contained coumarins 

(Ladeira et al., 1987; Ming, 1999) from the phytochemical screening. Similarly, 

Widodo et al. (2008) have found coumarin from leaves of this plant species. 

This procedure, however, was applicable only to coumarin and related 

volatile compounds. Most methods that appear useful for coumarins detection were 

followed by chromatography of the extract and revelation of the coumarins with spray 

reagent such as phenylboric acid, β-aminoethyl ester (Stahl and Schorn, 1961), KOH 

and diazotized sulfanilic acid (Sundt and Saccardi, 1962). 

53

POP1a POP2a POP3a POP1b POP2b POP3b Callus 

Before test under UV light at wavelength 365 nm 


(negative test) (positive test) (positive test) (negative test) (positive test) (positive test) (positive test) 

After test under UV light at wavelength 365 nm 

Figure 4 Coumarins test by using 10% NaOH 

54 

54

3. Screening for unsaturated lactone ring 

Kedde’s reagent and Raymond’s reagent were used for unsaturated lactone 

ring detection in this study. These reagents react with active methylene groups of 

unsaturated lactone (Farnsworth, 1966). These reagents give violet-pink and violetblue 

colors, respectively. The result of unsaturated lactone ring screening was shown 

in Figure 5-6 and Table 11. 

After testing with Kedde’s reagent, the color of all lipophilic crude extracts 

changed to be violet-pink, but disappeared within few minutes. By contrast, no color 

changes could be observed when tested with Raymond’s reagent. So, it could not be 

confirmed for unsaturated lactone ring contained in this extract. Nevertheless, there 

was no report for unsaturated lactone ring containing in A. conyzoides L. 

To confirm the result, different reagent such as Baljet’s reagent (Baljet, 

1918) and Legal’s reagent (Legal, 1948) should be tested paralelly. 

55





(positive test) (positive test) (positive test) (positive test) (positive test) (positive test) 

Figure 5 Unsaturated lactone ring test by using Kedde’s reagent 56 

56





(negative test) (negative test) (negative test) (negative test) (negative test) (negative test) 

Figure 6 Unsaturated lactone ring test by using Raymond’s reagent 57 

57

4. Screening for steroid and triterpenoid 

For steroid and triterpenoid, the Libermann-Burchard (L-B) test was used 

to detect these classes of compounds. According to Farnsworth (1966), blue or bluegreen 

colors are formed in the L-B test with steroid and red, pink or purple colors 

with triterpenoids. 

From this experiment, the changing in blue-green color occured with all 

lipophilic crude extracts of A. conyzoides. So it could be interpreted that steroid 

contained in all populations of A. conyzoides (Figure 7) agreed with literature review 

mentioned in Table 10 as well. 

However, many investigators noted that there was variation in the colors 

produced, depending on the manner in which the test was conducted. For example, 

Simes et al. (1959) stated that triterpenoid gave blue-green color in solution, but if the 

test is applied directly to solid material, the colors were only purple or violet. 

Whereas Wall et al. (1954) used chloroform extracts of plant material, pointed out 

those interfering substances such as carotene and xanthophylls produce immediate 

color change in the L-B test, as also do the saturated sterols. 

58





(positive test) (positive test) (positive test) (positive test) (positive test) (positive test) 

Figure 7 Steroid and triterpenoid test by using Libermann-Burchard test 

59 

59

5. Screening for flavonoid 

A number of specific color reactions for various types of flavonoids have 

been reported that could be adapted to screening large numbers of plant samples, but 

specificity of a sort was usually not desirable for the initial testing. One of the most 

useful general tests was called cyanidin reaction utilized in this screening. The 

Cyanidin test would detect compounds having the γ-benzopyrone nucleus 

(Farnsworth, 1966). Lipophilic crude extract was added a small piece of magnesium 

ribbon, followed by the dropwise addition of conc. HCl. Bubble color ranging from 

orange to red (flavones), red to crimson (flavonols), crimson to magenta (flavanones) 

and green to blue (aglycone or heteroside). However, subject to variation in intensity 

depending on the concentration of flavonoid present in the sample (Geissman, 1955) 

From the result (Table 11 and Figure 8) showed positive test of less hairy-blue 

head A. conyzoides (POP1a and POP1b) as orange bubble whereas there was not 

present in more hair populations both blue and white head indicated that the lipophilic 

crude extract of less hairy-blue head population contained flavone. The flavone 

bearing of A. conyzoides was according to Table 8 in review literature enumerated 

many types of flavone such as Ageconyflavone A, B and C (Vyas and Mulchandani. 

1986). Previous studied of this plant in screening test also showed positive result for 

flavonoids (Vajrodaya, 1986; Okwori et al., 2007). 

Flavonoid detection by using ferric chloride showed negative results with all 

lipophilic crude extracts (Figure 9). However, this screening method was used for 

free OH group detection. So it could be interpreted that there was no free OH group 

in the lipophilic crude extracts of A. conyzoides L. 

60




(positive test) (negative test) (negative test) (positive test) (negative test) (negative test) 

Figure 8 Flavonoid test by using Cyanidin test 

61 

61





(negative test) (negative test) (negative test) (negative test) (negative test) (negative test) 

Figure 9 Flavonoid test by using FeCl3 solution 

62 

62

Table 11 Phytochemical screening of POP1a, POP2a, POP3a, POP1b, POP2b, 

POP3b and callus 

lipophilic 

crude extract 

Alkaloid Coumarin Unsaturated 

lactone ring 

Dragendorff’s 

reagent 

NaOH Kedde’s Raymond’s 

reagent reagent 

Triterpenoid 

and steroid 

Libermann- 

Burchard test 

Cyanidin 

test 

Flavonoid 

POP1a +++ - + - + +++ - 

POP2a - +++ + - + - - 

POP3a - +++ + - + - - 

POP1b +++ - + - + + - 

POP2b - +++ + - + - - 

POP3b - +++ + - + - - 

FeCl3 

Callus NT + NT NT NT NT NT 

Remarks - no detection (negative test) 

+ detected 

+++ dominantly detected 

NT No test (too small amount to test) 

63

For general considerations, a method for use in phytochemical screening was 

simple, rapid, designed for a minimum of equipment, reasonably selective for the 

class of compounds under study and give additional information as the presence or 

absence of specific members of the group being evaluated. Hence, with the selection 

of a specific plant for phytochemical investigation, either on the basis of one or more 

approaches, or through some other avenue, phytochemical screening techniques could 

be a valuable aid. Because an initial selection of investigational plants should be 

made, not on evidence that extracts elicit a particular and interesting biological 

activity, but rather on the basis that certain chemicals are present in the plant, 

relatively of which can usually be associated with biological activity. Ultimately, the 

goal in surveying plants for biologically active or medicinally useful compounds 

should be isolated for a particular activity. 

64

Qualitative analyses 

1. TLC screening 

In addition to phytochemical screening, specific color reagents were 

sprayed onto the TLC plates for an estimate of the number of secondary metabolites 

present; alkaloids, terpenoids, coumarins and unsaturated lactone ring detections. The 

results of separated compound detection by color reagent were shown in Table 12. 

1.1 Screening for alkaloid 

After spraying Dragendorff’s reagent onto the TLC plate of A. 

conyzoides extracts, the result showed that POP1a and POP1b gave positive test as 

orange spots at Rf value 0.65 and 0.78, while it could not be detected in the other 

lipophilic extracts (Fig 10). 

From the result, only less hairy-blue head A. conyzoides (both POP1a 

and POP1b) was detected as orange color at Rf values 0.65 and 0.78 for POP1a and 

only 0.65 for POP1b, respectively; meaning that there might be two types of alkaloid 

containing POP1a and one in POP1b. While no alkaloid containing in the other two 

populations, more hairy-blue head (POP2a and POP2b) and more hairy-white head 

(POP3a and POP3b). This result was not different from alkaloid precipitating reagent 

test in phytochemical screening. So, it could be confirmed that only less hairy-blue 

head A. conyzoides contain alkaloid. However, the other alkaloid detecting reagent 

mentioned in the screening test should be sprayed in order to confirm this result. 

In this experiment, callus which was obtained from tissue culturing of 

less hairy-blue head plant, did not showed positive test for alkaloid. The result was 

different because of their quite different concentration. The callus had so less 

concentration that it could not be detected by the reagent. Moreover, the less hairyblue 

head plants used to in vitro culture were collected from different time and place 

as POP1a and POP1b. To confirm this result, original plant of tissue culture should 

be analyzed 

65

Rf 0.78 

Rf 0.65 

15 cm 

1a 2a 3a 1b 2b 3b callus 

Figure 10 Alkaloid detection from POP1a (1a), POP2a (2a), POP3a (3a), 

POP1b (1b), POP2b (2b), POP3b (3b) and callus 

66

1.2 Screening for terpenoid 

When sprayed the TLC plate by Anisaldehyde-sulfuric acid reagent for 

terpenoidsdetection. After heating TLC plate at the tempersture 110 ˚C until maximal 

visualization of the spots. Color ranged from violet (terpenes), blue (sugar), red 

(steroids) and grey-green (phenol) (Merck, 1980). The result showed that all 

lipophilic crude extracts gave a change color to violet at Rf values 0.13, 0.20, 0.25, 

0.42, 0.61 and 0.85. (Figure 11) indicated the lipophilic crude extracts of less hairyblue 

head, more hairy-blue head and more hairy-white head A. conyzoides and callus 

contained terpene at those Rf values. 

15 cm 


Rf 0.85 

Rf 0.61 

Rf 0.42 

Rf 0.25 

Rf 0.20 

Rf 0.13 

Figure 11 Terpenoid detection from POP1a (1a), POP2a (2a), POP3a (3a), POP1b 

(1b), POP2b (2b), POP3b (3b) and callus 

67

1.3 Screening for coumarin 

For revelation of coumarin, the TLC plate was sprayed with 10% 

NaOH and exposed with UV light at long wavelength (365 nm). The result showed 

that POP2a, POP2b, POP3a and POP3b gave yellow-green fluorescence. The reagent 

detected coumarin at Rf values 0.40 and 0.90 (Fig 12). The result indicated that more 

hairy-blue head (POP2a and POP2b) and more hairy-white head (POP3a and POP3b) 

plants contain coumarin. This result was similar to phytochemical screening. So it 

could be concluded that A. conyzoides with more hair populations even blue or white 

head contained coumarin. 

Other reagent useful for detecting coumarin in chromatography such as 

phenylboric acid, β-aminoethyl ester (Stahl and Schorn, 1961), KOH and diazotized 

sulfuric acid (Sundt and Saccardi, 1962) should be used to support and make the result 

more reasonable. 

15 cm 


Rf 0.90 

Rf 0.40 

Figure 12 Coumarin detection from POP1a (1a), POP2a (2a), POP3a (3a), POP1b 

(1b), POP2b (2b), POP3b (3b) and callus 

68

1.4 Screening for unsaturated lactone ring 

There was no presence of purple or blue color when spray TLC plate 

with Kedde’s reagent and Raymond’s reagent (Figure 13). This test gave different 

results from phytochemical screening test appearing positive Kedde reaction. 

Actually, TLC technique is more sensitive than phytochemical screening. Thus from 

this reason the chromatography should give positive test. However, the appearance of 

color changing in screening test might be false-positive interpretation. 

Table 12 Secondary metabolite screening on TLC plates of POP1a, POP2a, POP3a, 

POP1b, POP2b, POP3b and callus 

lipophilic 

crude 

Alkaloid Terpenoid Coumarin 

Unsaturated 

lactone ring 

extract Dragendorff’s 

reagent 

Anisaldehydesulfuric 

acid 

10% NaOH Kedde’s 

reagent 

Raymond’s 

reagent 

POP1a +++ +++ + - - 

POP2a - +++ +++ - - 

POP3a - +++ +++ - - 

POP1b +++ +++ + - - 

POP2b - +++ +++ - - 

POP3b - +++ +++ - - 

Callus - + - - - 

Remarks - no detection 

+ detected 

+++ dominant detection 

69

Kedde’s 

reagent 

Raymond’s 

reagent 

15 cm 

15 cm 



Figure 13 Unsaturated lactone ring detection from POP1a (1a), POP2a (2a), POP3a 

(3a), POP1b (1b), POP2b (2b), POP3b (3b) and callus 

70

From both phytochemical screening and TLC screening of A. conyzoides L. 

lipophilic crude extract, major secondary metabolites detected in the extracts were 

listed in Table 13 

Table 13 Secondary metabolites survey in lipophilic crude extracts of A. conyzoides L. 

Lipophilic 

crude 

extract 

Alkaloid Coumarin Terpenoid Steroid Unsaturated 

lactone ring 

Flavonoids 

POP1a √ √ √ √ 

POP2a √ √ √ 

POP3a √ √ √ 

POP1b √ √ √ √ 

POP2b √ √ √ 

POP3b √ √ √ 

Callus √ 

71

2. Comparative analysis 

2.1 TLC 

All lipophilic crude extracts were analyzed on the basis of their TLC 

pattern. The chemical constituents were detected by visualization under UV light 

both at long wavelength (365 nm). Positions of separated zones on thin-layer 

chromatograms were described as the Rf values of each substances. The Rf values of 

all lipophilic extracts were listed as Figure 14 and Table 14. 

Rf 0.65 

Rf 0.51 

Rf 0.47 

Rf 0.42 

15 cm 


Rf 0.88 

Rf 0.84 

Rf 0.77 

Rf 0.65 

Rf 0.50 

Rf 0.39 

Rf 0.25 

Rf 0.13 

Rf 0.09 

Figure 14 TLC pattern of POP1a (1a), POP2a (2a), POP3a (3a), POP1b (1b), POP2b 

(2b), POP3b (3b) and callus under long wavelength (365 nm) UV light 

72

Another method existed to visualize the spots was iodine vaporing. 

Iodine vapor chamber was made from a TLC jar by adding iodine crystals. The 

stained TLC plate was shown as Figure 15 that all Rf values mentioned above could 

be detected by iodine vapor. This method worked on variety compounds but does not 

often very sensitive. 

Rf 0.65 

15 cm 


Figure 15 TLC pattern of POP1a (1a), POP2a (2a), POP3a (3a), POP1b (1b), POP2b 

(2b), POP3b (3b) and callus after treated with iodine crystal 

73

Table 14 Rf values of the compound detected from A. conyzoides L. lipophilic crude 

extracts detected under long wave length UV light 


0.88 0.88 0.88 0.88 0.88 0.88 - 

0.84 0.84 0.83 0.83 0.85 0.85 - 

0.77 - 0.77 0.77 - 0.77 - 

0.65 0.65 0.65 0.65 - 0.65 - 

0.51 - - 0.51 - - - 

- 0.50 0.50 - 0.50 0.50 - 

0.47 - - 0.47 - - - 

0.42 - - 0.42 - - - 

- 0.39 0.39 - 0.39 0.39 - 

0.25 0.25 0.25 0.25 0.25 0.25 - 

0.13 0.13 0.13 0.11 0.13 0.13 0.13 

- - 0.09 - - 0.09 - 

From Figure 14 and Table 14, mobilities of all compounds separated 

from the three populations of A. conyzoides extracts; less hairy-blue head (POP1a and 

POP1b), more hairy-blue head (POP2a and POP2b) and more hairy-white head 

(POP3a and POP3b) and callus were compared. Aloisi et al., (1990) described that a 

match in Rf values, color, size and shape of zones detection in 365 nm UV light 

among samples was evidence for the identity of the samples. 

2.1.1 Comparison between two collection 

From Table 14, Rf values of detected compounds comparing 

between POP1a and POP1b were almost similar. However, Figure 15 showed that the 

size of zone detection in long wave length was different, even in same color and 

shape. This might result from different concentration. For POP2a and POP2b, 

chemical profiles of extracts were almost similar, only at Rf values 0.65 of POP2a 

which appeared different between two collections. While the chemical profiles were 

74

completely similar in POP3a and POP3b. Hence, it could be assumed that less hairyblue 

head, more hairy-blue head and more hairy white head A. conyzoides collected 

different times showed similar profiles. 

In this experiment, the two collections were collected during the 

same season even defferent year and A. conyzoides L. is annual plant. So, plant age 

might not affect to chemical accumulation. 

2.1.2 Comparisom among three populations 

Among three populations were identified as termed “systematic 

analysis”. From the result shown in Figure 14, Rf values, color, size and shape of 

detected spots that appeared the same characteristic in all of the three populations 

were 0.13, 0.25, 0.84 and 0.88. On the other hand, the spots which specific for only 

less hairy-blue head (POP1a and POP1b) were light blue fluorescence spots at Rf 

values 0.42, 0.47, 0.52 and 0.65, especially Rf values 0.65 presented as major 

compound (Figure 15). For more hairy-blue head (POP2a and POP2b) and white 

head (POP3a and POP3b), spots separated in different Rf values from less hairy-blue 

head were yellow-green fluorescence spot at Rf values 0.39, red fluorescence spot at 

Rf values 0.50 and blue fluorescence spot at Rf values 0.65. Rf values 0.65 was the 

same position as spot detected from less hairy-blue head. 

It could be assumed that three populations of A. conyzoides L. 

gave two chemical profiles; less hair and more hair profile. Whereas chemical 

profiles from different inflorescence color in more hair populations were similar. 

2.1.3 Comparison between less hairy-blue head and callus 

Because the lipophilic extract of callus had so small amount that 

it might not be compared clearly. However, callus extract appeared red fluorescence 

spot at Rf values 0.13 which similar to less hairy-blue head extract. It could be 

assumed that one compound from callus extract could be comparable to compound 

75

detected from the extract of less hairy-blue head. Nevertheless, these results could be 

compared with the results from HPLC. 

From this experiment, TLC pattern was detected by using 

dichloromethane, ethyl acetate and methanol in rate 70:20:5, respectively. Chemical 

patterns in TLC plate i.e. alkaloid, coumarin, terpenoid, steroid and flavonoid 

detection showed that two collections of less hairy-blue head were the same but 

different from the collections of more hairy-blue and white head. The two collections 

of more hairy A. conyzoides L., both blue and white were similar. 

For more information of systematic analysis, chromatographic spectra 

or profiles of compounds should be plotted as Rf values in a group of different solvent 

systems and those solvent systems should be judiciously chosen so as to separate the 

chemical compound clearly. 

2.2 HPLC 

The HPLC analysis showed out two types of data images. One is called 

a chemical profile or chromatogram, and the other is called spectrum (UV spectrum).. 

In this experiment, both chromatogram and UV spectra were used to determine a 

probability of identifying and comparing a chemical in each sample. 

2.2.1 Comparison between two collections 

It was shown that chrmical profiles and UV spectrum of 

lipophilic extracts from two collections of less hairy-blue head (POP1a-POP1b) and 

more hairy-blue head (POP2a-POP2b) almost the same (Fifure 16 and 17, Appendix 

Table 1). While more hairy-white head (POP3a-POP3b), showed more dominant 

peaks from collection two and the UV spectrum of peak J and U were different 

(Figure 18 and Appendix Table 1). To clarify this question, more number of each 

population should be collected. However TLC analysis showed the same TLC pattern 

between two collections. Thus, in this experiment, it could be assumed that A. 

conyzoides from different collections were the same species. 

76

Absorbtion 

Absorbtion 

POP1a D UV of D 

B 

C UV of E 

A E 

POP1b O 

Retention time (min) 

M 

N 

P 

L Q 


Figure 16 Chemical profiles and UV spectra of POP1a and POP1b 

F G 

UV of O 

UV of P 

77

Absorbtion 

Absorbtion 

POP2a H UV of H 

POP2b 



I 

UV of I 

R UV of R 

S UV of S 


78

Absorbtion 

Absorbtion 

POP3a J UV of J 

K 



UV of K 

POP3b U UV of U 

T 


V 

UV of V 

79

2.2.2 Comparison among three populations 

In plant collection 1, chemical profiles of less hairy-blue head 

(POP1a) gave obviously more peaks while the other two populations, more hairy-blue 

head (POP2a) and more hairy-white head (POP3a) gave only two dominant peaks 

(Figure 19). However there was one dominant peak from all three chromatograms 

that presented at the same retention time, around 14.3 minutes labeled peak number E, 

H and J for POP1a, POP2a and POP3a respectively. But when compared these peak 

numbers as UV spectra, UV spectrum of E looked quite different from H and J, which 

showed the same UV spectrum (Appendix Table 1). 

The extract of less hairy-blue head plant collection 2 (POP1b) 

presented six dominant peaks, while more hairy-blue head (POP2b) presented two 

peaks and three peaks for more hairy-white head (POP3b) (Figure 20). And also, 

three chromatograms had the same peak around 14.4 minutes labeled as peak P, R and 

U for POP1b, POP2b and POP3b, respectively. But the UV spectra looked different 

though, especially at peak U of more hairy-white head (Figure 20 and Appendix Table 

1). 

From two collections, comparison of the extracts among three 

populations of A. conyzoides by HPLC technique showed that less hairy-blue head 

extract presented more peaks than the two more hairy extracts; blue and white head.. 

From this result, it might be assumed that inflorescence color could not be used for 

identification, but the amount of hair should be in consideration. As well as the other 

factors such as plant age, plant part, environment, number of population, etc. 

80

Absorbtion 

Absorbtion 

Absorbtion 


B 

C 

UV of E 

A E 




F G 

POP2a H UV of H 

I 

UV of I 

POP3a J UV of J 

Figure 19 Chemical profiles and UV spectra of POP1a, POP2a and POP3a 

K 

UV of K 

81

Absorbtion 

Absorbtion 

Absorbtion 

POP1b 

POP2b 

M 

O UV of O 

N UV of P 

P 

L Q 


R UV of R 



S UV of S 

POP3b U UV of U 

T 

Figure 20 Chemical profiles and UV spectra of POP1b, POP2b and POP3b 

V 

UV of V 

82

2.2.3 Comparison between less hairy-blue head and callus 

From Figure 21, it was shown that chromatogram of callus 

extract had fewer peaks than chromatogram of two collections of less haory-blue head 

(POP1a and POP1b). However, one peak of callus and less hairy-blue head extract 

presented at the same retention time about 14.3 minutes labeled as peak X, E and P 

for chromatogram of callus, POP1a and POP1b, respectively, and its UV spectrum 

were the same (Figure 21 and Appendix Table 1). It could be assumed that callus 

from less hairy-blue head produced one compound as same as the plant from natural 

habitat. 

83

Absorbtion 

Absorbtion 

Absorbtion 

Callus UV of W UV of X 

W 

X Y UV of Y 



B 

C UV of E 

A E 

POP1b O 


M 

N 

P 

L Q 


F G 

Figure 21 Chemical profiles and UV spectra of callus, POP1a and POP1b 

UV of O 

UV of P 

84

Absorbtion 


Figure 22 Chromatogram comparison of callus and POP1a 

Absorbtion 


Figure 23 Chromatogram comparison of callus and POP1b 

POP1a 

callus 

POP1b 

callus 

85

From comparative phytochemical analysis using both TLC and HPLC, 

it was shown that there were differences in chemical patterns among populations of 

A. conyzoides L. In less hair populations, more chemical compounds could be 

detected than the more hair populations. The chemical patterns of more hair 

populations, blue and white inflorescence are similar eventhough the color are 

different. So, inflorescence color should not be used as morphological character for 

identification. But from this study the difference between less and more hair 

characteristic might be evidence for identification of Ageratum into taxa under 

species. According to Johnson (1971) had splitted A. conyzoides L. into two 

subspecies by using chromosome number, so the phytochemical evidence may be 

used to support the identification of Ageratum conyzoides as well. 

The evidences from TLC analysis could support the phytochemical screening 

of lipophilic crude extracts from three A. conyzoides L. populations (less hairy-blue 

head, more hairy-blue head and more hairy-white head). However, the evidences 

from TLC showed that the chemical patterns from more hairy-blue head and more 

hairy-white head were not definitely the same. Moreover, chemical profiles from 

HPLC analysis showed more differences between these two populations. Hence, 

further evidence such as cytology, molecular evidence etc.were need in order to 

clarify this problem. 

86


From lipophilic extract of POP1b (8.086 g), eighteen fractions were collected 

by a column of silica gel eluting with hexane, diethyl ether and methanol. Five 

fractions were obtained in accordance with the information from TLC pattern (Table 

15 and Appendix Figure 4-6). 

Table 15 Column chromatography information of lipophilic extract of POP1b 

Solvent system 

hexane:diethyl ether:methanol 

Combined 

fraction 

Remark 

100:0:0 – 50:50:0 A Non fluorescence 

50:50:0 B 

25:75:0 – 0:100:0 C 

Pink fluorescence under 365 

nm UV light (Appendix 

Figure 5) 

Red fluorescence under 365 

nm UV light 

(Appendix Figure 5) 

0:100:0 – 0:75:25 D - Blue fluorescence under 

254 and 365 nm UV light 

(Appendix Figure 4 and 5) 

- Positive to Dragendorff’s 

reagent (Appendix Figure 6) 

0:50:50 – 0:0:100 E Blue and red fluorescence 

under 365 nm UV light 


Combined fraction C was evaporated to dryness under reduced pressure. 

Subsequently, fraction C was isolated by MPLC technique .The drusses crystal (4.8 mg) 

was isolated from fraction C which was recrystallized by diethyl ether (Figure 24). 

87

Figure 24 Drusses crystal isolated and purified from fraction C 

Characterization of crystal 

Characteristic : Colourless, coumarine like odor, drusses crystal 

Solubility : Soluble in methanol, ethanol, ethyl acetate, chloroform, and 

dichloromethane 

Rf values : The Rf values were detected by using four different solvent 

system as following: 

1) 0.83 in dichloromethane:ethyl acetate:methanol (75:20:5) 


2) 0.28 in dichromethane (Appendix Figure 9) 

3) 0.34 in benzene:chloroform (7:3) (Appendix Figure 10) 

4) 0.77 in chloroform:acetone (9:1) (Appendix Figure 11) 

Melting point : 156-158 ˚C 

UV spectra : peak 1 : UV λmax MeOH (nm) 219.5, 277, 302.5 and 315 (sh) 

peak 2 : UV λmax MeOH (nm) 222.5, 272 (sh), 286, 307, 317.5 (sh) 

88

HPLC chromatogram 

Absorbtion 

1 (13.438) 

2 (14.370) 


Figure 25 HPLC chromatogram and UV spectrogram of the crystal 

UV of 1 

UV of 2 

TLC chromatogram of the crystal showed the presence of one spot in all four 

solvent systems. This could indicate the purification of the crystal. To confirm this 

result, the crystal was examined by HPLC technique. 

The HPLC chromatogram showed 2 dominant peaks (Figure 26) indicated 

that the crystal was not pure. Peak number (1) presented at retention time 13.438 

minute and peak number (2) at 14.370 minute. When compared the crystal’s 

chromatogram to POP1b’s, there was no peak presented around the retention time of 

13 in POP1b chromatogram. So, this peak could be artifact. While peak (2) was 

comparable to peak P in lipophilic extract of POP1b (Appendix Table 1). 

Because of small amount of this crystal (4.8 mg), so it was not enough for 

purification . 

89

Biological activities test 

When treated cultivated and weed seeds with 4 diluted concentrations of less 

hairy-blue head (POP1b) lipophilic crude extract and herbicides, the percentage of 

germination was calculated at the 3 rd , 5 th and 7 th day. While shoot and root length of 

tested seeds were measured at the last date of the experiment. 

1. Cultivated plants 

1.1 Oryza sativa L. cultivar Hom Mali 105 

Germination rate of Oryza sativa L. cultivar Hom Mali 105 were 

negatively correlated with the concentrations of the extracts after 5 and 7 days of 

experiment. All concentrations tended to stimulate germination the seeds. However, 

those rates were not significantly different from the control (Table 16). The seed 

germinated only radical when treated with glyphosate, but no germination occurred in 

paraquat (Table 16 and 17, Figure 27) 

There was significant inhibition on shoot elongation of the seedling for 

the 0.50 g/l concentration and root elongation was significantly reduced by 0.50 g/l 

and 2.0 g/l concentration. Yet, the other concentrations were not significantly 

different from the control (Table 17). 

1.2 Brassica chinensis L. var. chinensis 

It seemed that at 0.25, 0.5 and 1.0 g/l concentration inhibited seed 

germination of Brassica chinensis L. var. chinensis, but in statistic, it was not 

different from the control (Table 16). The seeds germinated only radical when treated 

with glyphosate, but no germination occurred in paraquat (Table 16 and 17, Figure 

28) 

In term of seedling growth, both shoot and root lengths of Brassica 

chinensis L. var. chinensis were inhibited. The extracts reduced growth of the 

90

seedlings when applied higher concentration and the data showed significantly 

different as compared with control (Table 17). Nevertheless, at low concentration 

(0.25 g/l), there was non-significant reduction in shoot growth. 

1.3 Ipomoea aquatica Forssk. 

Data in Table 16 showed that all concentrations of lipophilic extracts 

of A. conyzoides promoted seed germination of Morning glory since 3 to 7 day of 

experiment. At concentrations 0.5, 1.0 and 2.0 g/l, seed germination was significant 

stimulated compared with control. The seed germination also occurred in glyphosate, 

but only short radical. While it was not occurred in paraquat (Table 16 and 17, Figure 

29) 

Like seed germination, the extracts tended to promoted growth of 

seedlings, not only shoot length but also root length was increased. However, it was 

not different from control. Even if at dose 2.0 (4.908 cm.), it seemed that the root 

growth was inhibited, but it was not different from control as well (Table 17) 

91

Table 16 Germination percentage of cultivated plants at 3,5 and 7 days 

Concentration 

(g/l) 

Percentage of germination (%) 1/ 

Oryza sativa L. cultivar Hom Mali 105 Brassica chinensis L. var. chinensis Ipomoea aquatica Forssk. 

3 5 7 3 5 7 3 5 7 

0.00 62.50 a 2/ 

78.75 a 78.75 a 62.50 a 68.75 a 66.25 a 68.75 a 70.00 a 70.00 a 

0.25 58.75 a 87.50 a 87.50 a 60.00 a 75.00 a 60.00 a 75.00 ab 76.25 ab 77.50 ab 

0.50 71.25 a 85.00 a 85.00 a 58.75 a 85.00 a 61.25 a 85.00 b 86.25 b 90.00 b 

1.00 73.75 a 82.50 a 82.50 a 56.25 a 85.00 a 61.25 a 85.00 b 87.50 b 88.75 b 

2.00 75.00 a 83.75 a 85.00 a 63.75 a 86.25 a 66.25 a 86.25 b 88.75 b 92.50 b 

Glyphosate 62.50 a 78.75 a 80.00 a 66.25 a 82.50 a 66.25 a 82.50 ab 85.00 ab 85.00 ab 

Paraquat 0.00 b 0.00b 0.00 b 0.00 b 0.00 b 0.00 b 0.00 c 0.00 c 0.00 c 

F-test ** ** ** ** ** ** ** ** ** 

CV (%) 19.86 12.34 11.94 19.72 16.66 16.49 14.20 14.11 15.37 

1/ Average of 4 replications 

2/ Values in the column with same letter are not significantly different at 95% significant level applying Duncan’s multiple range test 

(DMRT) method 

** significantly different at 95% significant level 

92 

92

Table 17 Seedling growth of cultivated plants at the 7 th date 

Concentration 

(g/l) 

Length of seedlings (cm.) 1/ 

Oryza sativa L. cultivar Hom Mali 105 Brassica chinensis L. var. chinensis Ipomoea aquatica Forssk. 

Shoot Root Shoot Root Shoot Root 

0.00 3.095 a 2/ 5.517 a 3.273 a 3.553 a 4.803 a 5.135 a 

0.25 3.017 a 5.215 a 3.094 a 2.645 b 5.045 a 5.588 a 

0.50 1.890 b 2.605 b 2.443 b 2.123 c 5.183 a 5.575 a 

1.00 2.626 a 3.664 ab 2.243 bc 1.658 d 5.438 a 5.773 a 

2.00 2.535 ab 3.155 b 1.905 c 1.318 d 5.073 a 4.908 a 

Glyphosate 0.000 c 0.342 c 1.078 d 0.180 e 2.600 b 0.655 b 

Paraquat 0.000 c 0.000 c 0.000 e 0.000 e 0.000 c 0.000 b 

F-test ** ** ** ** ** ** 

CV (%) 24.02 41.13 16.47 15.96 18.96 17.26 



(DMRT) method 


93 

93

Control 0.25 g/l 0.50 g/l 1.00 g/l 

2.00 g/l Glyphosate Paraquat 

Figure 26 Oryza sativa L. cultivar Hom Mali 105 seedling treated with A. conyzoides extracts and herbicides at the 7 th date 

94 

94



Figure 27 Brassica chinensis L. var. chinensis seedling treated with A. conyzoides extracts and herbicides at the 7 th date 

95 

95



Figure 28 Ipomoea aquatica Forssk. seedling treated with A. conyzoides extracts and herbicides at the 7 th date 

96 

96

2. Weeds 

2.1 Tridax procumbens L. 

From Table 18, it was shown that A. conyzoides significant inhibited 

seed germination the 3 rd date of experiment at concentration 0.25, 0.5, 1.0 and 2.0 g/l. 

But when the germination took longer, the inhibition was reduce, that was the extracts 

inhibited tridax seed germination at concentration 0.5, 1.0 and 2.0 g/l at the 5 th date 

and 1.0 and 2.0 g/l at 7 th date of experiment. The seed germination also occurred in 

glyphosate, but only short radical. While it was not occurred in paraquat (Table 18 

and 19 and Figure 30) 

In term of seedling growth, shoot length of tridax seedling was 

inhibited at the highest dose, while the others seemed to promote shoot growth. For 

root length, the result was not different to the control (Table 19). 

2.2 Mimosa pigra L. 

Data in Table 25 showed that at doses 0.5 g/l of lipophilic extracts of 

A. conyzoides suppressed seed germination of mimosa glory since 3 to 7 day of 

experiment, however, it was not different to the control. The seed germination also 

occurred in glyphosate and paraquat, but only short shoot and radical. (Table 18 and 

19, Figure 31) 

The extracts seemed not have effect to seedling growth of mimosa. 

Because some dose promoted and some inhibited, but not significant in term of (Table 

19) 

97

2.3 Cenchrus echinatus L. 

Germination rate of cenchrus were negatively correlated with the 

concentrations of the extracts at 3 days of experiment. That was most concentrations 

stimulated germination of seeds. However, those were not significantly different from the 

control (Table 18). At the end of experiment, the inhibition occurred at concentration 0.25 

g/l significantly. In herbicides treated seed, the germination also occurred, but for short 

shoot and radical only (Table 18 and 19, Figure 32) 

There was significant inhibition on shoot and root elongation of 

cenchrus for the 0.50, 1.0 and 2.0 g/l doses (Table 19). 

2.4 Echinochloa colona (L.) Link 

Data in Table 18 showed that all doses of lipophilic extracts of A. 

conyzoides promoted seed germination of Morning glory since 5 to 7 day of 

experiment. At concentrations 0.5, 1.0 and 2.0 g/l, seed germination was significant 

stimulated when the seeds were treated 5 days. The seed germination also occurred in 

glyphosate, but only short radical. While it was not occurred in paraquat (Table 18 

and 19) 

The extracts seemed not have effect to seedling growth of mimosa. 

Because some dose promoted and some inhibited, but not significant in term of 

statistic (Table 19) 

98

Table 18 Germination percentage of weeds at 3,5 and 7 days 

Contration 

(g/l) 

Percentage of germination (%) 1/ 

Tridax procumbens L. Mimosa pigra L. Cenchrus echinatus L. Echinochloa colona (L.) Link 

3 5 7 3 5 7 3 5 7 3 5 7 

0.00 86.25 a 2/ 90.00 a 87.50 a 100.0 a 100.0 a 100.0 a 50.00 a 60.00 a 65.00ab 42.50 a 42.50 a 77.50 a 

0.25 67.50 b 83.75 a 90.00 a 100.0 a 100.0 a 100.0 a 50.00 a 56.25 a 56.25 b 40.00 a 46.25 a 83.75 a 

0.50 42.50 c 61.25 bc 81.25 a 95.00 ab 97.50 ab 97.50 a 57.50 a 68.75 a 71.25 a 47.50 a 62.50 b 82.50 a 

1.00 31.25 c 48.75 c 61.25 b 97.50 a 100.0 a 100.0 a 52.50 a 62.50 a 62.50ab 46.25 a 55.00ab 82.50 a 

2.00 17.50 d 17.50 d 27.50 c 100.0 a 100.0 a 100.0 a 57.50 a 58.75 a 58.75ab 41.25 a 50.00ab 77.50 a 

Glyphosate 57.50 b 75.00 ab 80.00 a 95.00 ab 95.00 bc 97.50 a 35.00 b 37.50 b 37.50 c 25.00 b 28.75 c 50.00 b 

paraquat 0.00 d 0.00 e 0.00 d 90.00 b 91.25 c 87.50 b 17.50 c 23.73 c 23.75 d 0.00 c 0.00 d 0.00 c 

F-test ** ** ** ** ** ** ** ** ** ** ** ** 

CV (%) 19.45 19.39 17.64 3.83 2.68 1.94 20.25 18.24 16.42 25.88 22.66 14.94 



(DMRT) method 


99 

99

Table 19 Seedling growth of weeds at the 7 th date 

Concentration 

(g/l) 

Length of seedlings (cm.) 1/ 

Tridax procumbens L. Mimosa pigra L. Cenchrus echinatus L. Echinochloa colona (L.) Link 

Shoot Root Shoot Root Shoot Root Shoot Root 

0.00 1.132 a 2/ 1.174 a 5.893 a 1.818 a 3.526 a 1.778 a 2.132 a 0.558 ab 

0.25 1.879 c 1.790 a 6.034 a 2.188 a 3.129 a 2.355 a 1.646 ab 0.502 ab 

0.50 1.785 ac 1.226 a 5.665 ab 1.995 a 1.820 b 1.289 abc 2.198 a 1.150 c 

1.00 1.519 ac 1.459 a 6.369 ab 1.590 ab 0.890 c 0.819 cd 1.417 ab 0.486 ab 

2.00 0.542 bd 1.294 a 5.733 ab 2.118 a 0.558 cd 0.578 cd 1.795 a 0.769 ac 

Glyphosate 0.798 ab 0.560 b 0.136 c 0.510 c 0.428 cd 0.397 d 0.277 bc 0.155 b 

Paraquat 0.000 d 0.000 b 0.170 c 0.528 c 0.075 d 0.075 d 0.000 c 0.000 b 

F-test ** ** ** ** ** ** ** ** 

CV (%) 38.92 36.46 8.27 16.60 32.03 52.74 66.56 72.32 



(DMRT) method 


100 

100


101 


Figure 29 Tridax procumbens L. seedling treated with A. conyzoides extracts and herbicides at the 7 th date 

101


102 


Figure 30 Mimosa pigra L. seedling treated with A. conyzoides extracts and herbicides at the 7 th date 

102


103 


Figure 31 Cenchrus echinatus L. seedling treated with A. conyzoides extracts and herbicides after the 7 th date 

103

Table 20 Effect of the lipophilic crude extract from A. conyzoides L. on seed germination and seedling growth of tested seed 

Tested plant 

Cultivated plants: 

Oryza sativa L. cultivar Hom 

Mali 105 

Brassica chinensis L. var. 

chinensis 

Seed germination 

104 

Concentration of the extract affecting on tested plant (g/l) 

Seedling growth 

Shoot Root 

Inhibition Promotion Inhibition Promotion Inhibition Promotion 

- 

- 

0.5 

- 

0.5 

2.0 

- - 0.5, 1.0, 2.0 - 0.25, 0.5, 

1.0, 2.0 

Ipomoea aquatica Forssk. - 0.5, 1.0, 2.0 - - - - 

Weeds: 

Tridax procumbens L. 

0.25, 0.5, 

1.0, 2.0 

- 

Mimosa pigra L. - - - - - - 

Cenchrus echinatus L. 0.25 - 0.5, 1.0, 2.0 - 0.5, 1.0, 2.0 - 

Echinochloa colona (L.) Link. - 0.5 - - 0.5 - 

- 

0.25 

- 

- 

- 

- 

104

From the result, the lipophilic crude extract from A. conyzoides L. could 

inhibit seed germination and seedling growth of many tested plants. Tongma et al. 

(1997) reported inhibitory effect of secendary metabolites from Asteraceae plants on 

other plants. Okwori et al. (2007) documented active phytochemicals of the plant A. 

conyzoides L. must be more lipid soluble or non-polar compound. However, there 

were studies found aqoueous extract from A. conyzoides L. inhibit seed germination 

of weeds Monochoria vaginalis, Echinochloa crus-galli, Aeschynomene indica (Xuan 

et al., 2004). Hong et al. (2004) have shown that aqueous extract of whole plants 

controlled emergence of weeds Monochoria vaginalis, Rotala indica, Marsilea 

quadrifolia, Leptochloa chinensis, Cyperus difformis, Sphenochlea zeylanica, 

Commelina diffusa, Dactyloctenium aegyptium and Brachiaria mutica. Similarly, 

Xuan et al. (2004) found that leaves aqueous extract of A. conyzoides L. controlled 

emergence of weeds in paddy field such as Echinochloa oryzicola, Eleochalis 

acicularis, Linderna pyxidaria, Monocharia vaginalis and Rotala indica. 

In this study, non polar extract (lipophilic crude extract) also showed 

inhibitory effect on seed germination of weeds; Tridax procumbens L. and Cenchrus 

echinatus L. In cultivated plants, the extracts inhibited growth of Oryza sativa L. 

cultivar Hom Mali 105 and Brassica chinensis L. var. chinensis. However, the 

inhibitory effects had low potential when compared with commercial herbicides 

which could strongly inhibited the germination and growth of tested seed especially 

paraquat. Hoagland and Williams, (2004) described that the concentrations of the 

allelopathic compounds are generally low in a crude extract and nonactive compounds 

in the crude extracts may physically or chemically bind or mask the action of an 

allelochemical. Nevertheless, A. conyzoides L. extract might can be used initially and 

followed with commercial herbicides that can reduce the use of herbicide. 

Molisch (1937) pointed out that allelopathic interactions could be either 

inhibitory or stimulatory to growth. There was promotion effect found in this 

experiment also. The extract promoted seed germination of Ipomoea aquatica Forssk. 

and shoot length of Tridax procumbens L., but only in low concentration. This result 

agree with Xuan et al. (2004), who documented leaves aqueous extract of A. 

conyzoides L. promoted rice Oryza sativa L. var. indica growth. 

105

This experiment used methanol as a solvent for initial extraction, and 

chloroform for partitioned then. Secendary metabolites extracted by alcohol might be 

the compound from Shikimic acid pathway (Vickery and Vickery, 1981). The most 

common phenolic compound derived from Shikimic acid pathway are the derivatived 

of cinnamic acid, benzoic acid, coumarins, tannins, flavonoids and other polyphenolic 

complexes. The phenolic acid, coumarins and tanins appear to have quite similar 

mechanisms of action, inhibiting plant growth through cytotoxiccity. The initial 

actions are on celmembranes, resulting in nonspecific permeability changes that alter 

ion fluxes and hydraulic conductivity of roots. Membrane perturbations are followed 

by a cascade of physiological effects that include alterations in ion balance, plantwater 

relationships, stomatal functions, and rates of photosynthesis and respiration. 

Allelopathic flavonoids are potent inhibitors of energy metabolism, blocking 

mitochondrial and chloroplast functions (Einhellig, 2004). Alkaloids could be utilized 

as intra- or interspecific competition of plant species (Blum, 2004). 

Problems with using crude extracts might result from osmotic potential. 

When testing extracted plant material, care should be taken to ensure that seed 

germination is not delayed by the osmotic potential of the extract solution. Sorbitol or 

manitol could be used for osmotic potential control. Moreover, insensitive bioassays 

with crude extracts might be occurred. 

106

CONCLUSION AND RECOMMENDATION 

Conclusion 

1. For phytochemical screening and TLC screening, the lipophilic crude 

extract of three populations of Ageratum conyzoides L., it was shown that: 

Less hairy-blue head contained alkaloids, terpenoids, steroids and 

triterpenoids and flavonoids. More hairy-blue head contained coumarins, terpenoid 

and steroids and triterpenoids. More hairy-white head contained coumarins, terpenoid 

and steroids and triterpenoids. Callus contained terpenoid 

2. For TLC and HPLC analyses, the result were: 

TLC and HPLC chromatograms of all three populations of A. conyzoides 

L. from two collections showed similar pattern. Among three populations, TLC and 

HPLC profiles of more hairy-blue head and more hairy-white head were similar. 

More secondary metabolites could be detected from the profile of less hairy-blue 

head. HPLC analysis showed that callus extract contained three compounds and the 

main compound was comparable to one compound found in the extract from less 

hairy-blue head. 

3. The purification and structure elucidation of the crystal isolated from A. 

conyzoides L. with less hairy-blue head could not be carried on. It might be the result 

from artifact together with scantly amount of crystal. 

4. The biological activity of lipophilic crude extract from less hairy-blue head 

A. conyzoides can be concluded as follow: 

In cultivated plants: The lipophilic crude extract at concentrations more 

than 0.5 g/l could inhibit the growth of Oryza sativa L. cultivar Hom Mali 105 

seedling, all concentrations could inhibit the growth of Brassica chinensis L. var. 

107

chinensis seedling, and at concentrations more than 0.5 g/l could promote seed 

germination of Ipomoea aquatica Forssk. 

In weeds: The lipophilic crude extract at all concentrations could inhibit 

seed germination of Tridax procumbens L., but at the concentration 0.25 g/l the 

growth of seedling could be promoted. At all concentrations could neither affect to 

seed germination nor seedling growth of Mimosa pigra L. At the concentration 0.25 

g/l could inhibit seed germination of Cenchrus echinatus L. while higher 

concentrations could inhibit seedling growth. The concentrations 0.25 and 0.5 g/l 

could promote seedling growth of Echinochloa colona (L.) Link. 

Recommendation 

1. There are many groups of chemical compounds in the lipophilic crude 

extract of A. conyzoides L. Thus, this plant species might have economic value and 

could be used as a natural source for those compounds. However, it was rough 

investigation, so the other methods should be used for confirmation. 

2. The callus of less-hairy blue head Ageratum conyzoides L.contained 

interesting chemical compound eventhough the amount of callus was so small. Thus, 

it was recommended to increase amount of callus to clarify this compound. 

3. Different character of A. conyzoides gave different profile of the extract. 

So whenever the researchers collected the plants from any where, TLC or HPLC 

analyses should be done firstly. 

4. The artifact could be occurred during either the long process of experiment 

or from organic solvents. Thus, in the process of separation and purification, it was 

strongly recommended to carry on as soon as possible. Then, the fractions in each 

steps should be compared with the original crude extract. 

5. The biological activity tests were done only in laboratory condition. Field 

study should be found out to clarify the effect of extract on tested seeds. 

108

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55(3): 225-276. 

Widodo, G.P., E.Y. Sukandar and J.K. Adyana. 2008. A coumarin from Ageratum 

leaves (Ageratum conyzoides L.). International Journal of Pharmacology 

4(1): 56-59. 

Wiedenfeld, H. and E. Roder. 1991. Pyrrolizidine alkaloids from Ageratum 

conyzoides. Planta Med 57: 578-579. 

Xuan, T.D., T. Shinkichi, N.H. Hong, T.D. Khanh and C.I. Min. 2004. Assessment 

of phytotoxic action of Ageratum conyzoides L. (billy goat weed) on weeds. 

Crop Protection 23: 915-922. 

Yadara, R.N. and S. Kumar. 1999. A novel isoflavone from the stems of Ageratum 

conyzoides . Fitoterapia 70: 475-477. 

126

APPENDIX 

127

Preparation of reagents for phytochemical screening 

1. Dragendorff’s reagent 

Solution A: 

Bismuth sub nitrate (BiO(NO3).H2O): AR grade Merck 0.85 g 

Glacial acetic acid (CH3COOH): AR grade Merck 10.0 ml 

Distilled water (H2O): 16 MΩ/cm Millipore 40.0 ml 

Solution B: 

Potassium iodide (KI): AR grade Univar 8.0 g 


Solution C: 

Glacial acetic acid (CH3COOH): AR grade Merck 20.0 ml 


Before use, 5 ml of solution A was mixed with 5 ml of solution B and 100 

ml of solution C. Amines and basic heterocycles like pyridine produce brown-orange 

spots at retention time. Phosphines and crown ethers are also detected (Farnsworth, 

1966). 

2. Anisaldehyde-sulfuric acid 

The reagent was freshly prepared before use by mixture of 0.5 ml 

anisaldehyde (C8H8O2 : AR grade Fluka) in 50 ml Glacial acetic acid and 1 ml 97% 

sulfuric acid (H2SO4 : AR grade Fisher Scientific) following by heating on oven 

(Sanyo OMT) at 100-105 °C until maximal visualization of the spots. 

Colorful spots will occur varies on the compound: terpene (red), sugar 

(grey), steroid (green), phenol (blue) and lichen (violet). Good for all things with 

128

active methylene, and for distinguishing closely-spaced spots on TLC by their color 

difference (Merck, 1980). 

3. Kedde’s reagent 

The reagent was freshly prepared by mixture of Kedde I, 5 ml of 3% 

ethanolic-3,5-dinitrobenzoic acid (C7H4N2O6 : AR grade Fluka) and Kedde II, 5 ml of 

2 M ethanolic potassium hydroxide (KOH: AR grade Univar) and evaluated in the air. 

The reagent was useful for detection of C17 unsaturated lactone ring like cardiac 

glycoside. The present of purple color was assessed as positive result (Farnsworth, 1966). 

4. Raymond’s reagent 

The reagent was freshly prepared by Raymond I, 5 ml of 2% ethanolic- 

1,3-dinitrobenzene (C6H4N2O4 : AR grade Fluka) mixed with Raymond II, 5 ml of 5% 

ethanolic potassium hydroxide and evaluated in the air. 

The reagent was useful for detection of C17 unsaturated lactone ring like 

cardiac glycoside. The present of blue color was assessed as positive result 

(Farnsworth, 1966). 

5. 10% Sodium hydroxide 

Dissolved 8 g of pellets sodium hydroxide (NaOH: AR grade Mallinckrodt) 

in 100 ml ethanol. After spraying, evaluate in the air. The chemical constituent was 

detected as yellow-green fluorescence by visualization under UV light at wavelength 

365 nm (Farnsworth, 1966). 

129

6. Iodine 

Iodine vapor chamber is made from a TLC jar by adding iodine crystals 

(May & Baker LTD Dagenham England). Put dry TLC in the chamber and watch the 

spots to going. This technique works on variety compounds but often not very 

sensitive. Iodine stained TLC can be developed subsequently with other stains 

(Merck, 1980) 

130

Appendix Table 1 Retention time (Rt) and UV spectrum of peak detected from 

Peak Rt 

(min) 

A 

B 

C 

D 

E 

F 

G 

H 

I 

J 

K 

L 

M 

HPLC chromatogram 

9.122 

10.595 

10.991 

12.364 

14.357 

21.842 

25.133 

14.389 

15.622 

14.346 

15.583 

9.178 

10.647 

UV spectrum 

λmax MeOH (nm) 202, 202.5(sh), 210(sh), 242, 267, 

336 

λmax MeOH (nm) 197(sh), 200, 214, 265, 320 

λmax MeOH (nm) 197, 200(sh), 225, 227(sh), 284 

λmax MeOH (nm) 202(sh), 207, 212, 264, 266(sh), 

327 

λmax MeOH (nm) 198, 217.5, 273, 282.5(sh), 302, 

314(sh) 

λmax MeOH (nm) 198, 200(sh), 233(sh) 

λmax MeOH (nm) 200(sh), 202 

λmax MeOH (nm) 200(sh), 212, 230(sh), 270(sh), 

282(sh), 302, 314(sh) 

λmax MeOH (nm) 198, 222, 274(sh), 285(sh), 305, 

317.5(sh) 

λmax MeOH (nm) 209, 230(sh), 270(sh), 282(sh), 302, 

314(sh) 

λmax MeOH (nm) 198, 202.5(sh), 222, 275(sh), 

286(sh), 306, 317(sh) 

λmax MeOH (nm) 198(sh), 200, 207(sh), 242, 265, 

335 

λmax MeOH (nm) 198(sh), 200, 214, 216(sh), 265, 

320 

131

Appendix Table 1 (Continued) 

Peak Rt 

(min) 

N 

O 

P 

Q 

R 

S 

T 

U 

V 

W 

X 

Y 

11.041 

12.436 

14.402 

20.136 

14.345 

15.601 

10.214 

14.365 

15.583 

8.305 

14.372 

21.183 

UV spectrum 

λmax MeOH (nm) 198, 200(sh), 212(sh), 225, 227(sh), 

283 

λmax MeOH (nm) 206, 212(sh), 218(sh), 268, 323 

λmax MeOH (nm) 198, 218, 272, 282(sh), 302, 

314(sh) 

λmax MeOH (nm) 198, 200(sh), 226, 233(sh) 

λmax MeOH (nm) 208, 232.5(sh), 272(sh), 282(sh), 

302, 314(sh) 

λmax MeOH (nm) 198, 223, 274(sh), 286(sh), 305, 

315(sh) 

λmax MeOH (nm) 197(sh), 199, 220, 228(sh), 275, 

285(sh), 320 

λmax MeOH (nm) 208(sh), 212(sh), 222(sh), 237(sh), 

268(sh), 272(sh), 285, 314, 

λmax MeOH (nm) 196(sh), 198, 215, 227(sh), 275(sh), 

285(sh), 305, 316(sh) 

λmax MeOH (nm) 197, 199, 223, 225(sh), 270(sh), 

280, 292(sh) 

λmax MeOH (nm) 198, 217, 272, 282(sh), 302, 

314(sh) 

λmax MeOH (nm) 197.5, 200, 232(sh) 

132

Appendix Table 2 Analysis of Variance (ANOVA) of germination percentage, shoot 

and root length of Oryza sativa L. cultivar Hom Mali 105 at 95% 

significant level 

GERM 

3 DAY 

GERM 

5 DAY 

GERM 

7 DAY 

SHOOT 

ROOT 

df Sum of Square Mean Square F 

Between groups 6 16467.857 2744.643 20.912** 

Within groups 21 2756.250 131.250 

Total 27 19224.107 


Within groups 21 1606.250 76.488 

Total 27 25302.679 

Between groups 6 23912.500 3985.417 55.107 ** 

Within groups 21 1518.750 72.321 

Total 27 25431.250 


Within groups 21 4.284 0.204 

Total 27 47.581 



Total 27 142.004 

CV (GERM 3 DAY) = 19.86 % 

CV (GERM 5 DAY) = 12.34 % 

CV (GERM 7 DAY) = 11.94 % 

CV (SHOOT) = 24.02 % 

CV (ROOT) = 41.13 % 


133


and root length of Brassica chinensis L. var. chinensis at 95% 


GERM 

3 DAY 

GERM 

5 DAY 

GERM 

7 DAY 

SHOOT 

ROOT 



Within groups 21 2250.000 107.143 

Total 27 15375.000 


Within groups 21 1706.250 81.250 

Total 27 15602.679 


Within groups 21 1693.750 80.655 

Total 27 15716.964 



Total 27 34.010 



Total 27 40.742 

CV (GERM 3 DAY) = 19.72 % 

CV (GERM 5 DAY) = 16.66 % 

CV (GERM 7 DAY) = 16.49 % 

CV (SHOOT) = 16.47 % 

CV (ROOT) = 15.96 % 


134


and root length of Ipomoea aquatica Forssk. at 95% significant 

level 

GERM 

3 DAY 

GERM 

5 DAY 

GERM 

7 DAY 

SHOOT 

ROOT 



Within groups 21 2012.500 95.833 

Total 27 25167.857 


Within groups 21 2081.250 99.107 

Total 27 26416.964 


Within groups 21 2568.750 122.321 

Total 27 28216.964 



Total 27 109.432 



Total 27 159.443 

CV (GERM 3 DAY) = 14.20 % 

CV (GERM 5 DAY) = 14.11 % 

CV (GERM 7 DAY) = 15.37 % 

CV (SHOOT) = 18.96 % 

CV (ROOT) = 17.26 % 


135


and root length of Tridax procumbens L. at 95% significant level 

GERM 

3 DAY 

GERM 

5 DAY 

GERM 

7 DAY 

SHOOT 

ROOT 



Within groups 21 1387.500 66.071 

Total 27 25060.714 


Within groups 21 2281.250 108.631 

Total 27 30081.250 


Within groups 21 2437.50 116.071 

Total 27 31067.857 



Total 27 15.267 



Total 27 11.846 

CV (GERM 3 DAY) = 19.45 % 

CV (GERM 5 DAY) = 19.36 % 

CV (GERM 7 DAY) = 17.64 % 

CV (SHOOT) = 38.92 % 

CV (ROOT) = 36.46 % 


136


and root length of Mimosa pigra L. at 95% significant level 

GERM 

3 DAY 

GERM 

5 DAY 

GERM 

7 DAY 

SHOOT 

ROOT 



Within groups 21 225.000 10.714 

Total 27 560.714 


Within groups 21 143.750 6.845 

Total 27 424.107 



Total 27 575.000 



Total 27 181.782 



Total 27 13.861 

CV (GERM 3 DAY) = 3.83 % 

CV (GERM 5 DAY) = 2.68 % 

CV (GERM 7 DAY) = 1.94 % 

CV (SHOOT) = 8.27 % 

CV (ROOT) = 16.60 % 


137


and root length of Cenchrus echinatus L. at 95% significant level 

GERM 

3 DAY 

GERM 

5 DAY 

GERM 

7 DAY 

SHOOT 

ROOT 



Within groups 21 1800.000 85.714 

Total 27 6885.714 


Within groups 21 1925.000 91.667 

Total 27 8025.000 


Within groups 21 1625.000 77.381 

Total 27 8442.857 



Total 27 49.976 



Total 27 22.107 

CV (GERM 3 DAY) = 20.25 % 

CV (GERM 5 DAY) = 18.24 % 

CV (GERM 7 DAY) = 16.42 % 

CV (SHOOT) = 32.03 % 

CV (ROOT) = 52.74 % 


138


and root length of Echinochloa colona (L.) Link at 95% 


GERM 

3 DAY 

GERM 

5 DAY 

GERM 

7 DAY 

SHOOT 

ROOT 



Within groups 21 1687.500 80.357 

Total 27 8596.429 


Within groups 21 1787.500 85.119 

Total 27 12185.714 


Within groups 21 1968.750 93.750 

Total 27 24874.107 



Total 27 35.388 



Total 27 6.401 

CV (GERM 3 DAY) = 25.88 % 

CV (GERM 5 DAY) = 22.66 % 

CV (GERM 7 DAY) = 14.94 % 

CV (SHOOT) = 66.56 % 

CV (ROOT) = 72.32 % 


139

Less hairy-blue head more hairy-blue head more hairy-white head 

Appendix figure 1 Three populations of A. conyzoides L. 

140

pump 

Mobile phase 

reservoir 

Monometer 

sample 

254 nm UV 

detector 

Appendix Figure 2 MPLC Instrumentation 

Reference cell 

column 

fractions 

141

pump 

Mixer 

Solvent 

(mobile phase) 

Injector 

Column 

Waste 

Appendix Figure 3 Agilent Technologies Instrumentation 

Chromatogram 

Detector 

UV/DAD 

142

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 

A B C D E 

Appendix Figure 4 TLC information (under 254 nm UV light) of eighteen fractions 

collected from Column Chromatography 

143

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 

A B C D E 

Appendix Figure 5 TLC information (under 365 nm UV light) of eighteen fractions 

collected from Column Chromatography 

144

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 

A B C D E 

Appendix Figure 6 Dragendorff’s reagent detection of fraction D combined from 

Column Chromatography 

145

15 cm 

Rf value 0.83 

Appendix Figure 7 TLC chromatogram of crystal from fraction C developing in 

dichloromethane:ethyl acetate:methanol (75:20:5) solvent system 

146

15 cm 

Rf value 0.28 


dichromethane solvent system 

147

15 cm 


benzene:chloroform (7:3) solvent system 

Rf value 0.34 

148

15 cm 


chloroform:acetone (9:1) solvent system 

Rf value 0.77 

149

CURRICULUM VITAE 

NAME : Ms. Buakhao Hongsachum 

BIRTH DATE : February 01, 1981 

BIRTH PLACE : Nakhon Phanom, Thailand 

EDUCATION : YEAR INSTITUTE DEGREE/DIPLOMA 

2004 Kasetsart Univ. B.Sc. (Biology) 

2008 Kasetsart Univ. M.S. (Botany) 

POSITION/TITLE : 

WORK PLACE : 

SCHOLARSHIP/AWARD : Development and Promotion of Science and 

Technology Talented Project (DPST Project)

phytochemistry and bioassay for natural weed control

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