ABSTRACT
This research work was carried out to identify the bioactive constituents of the leaves and stem barks of Ageratum conyzoides Linn, a medicinally important plant of the Asteraceae family. This plant was selected on the basis of its widespread use in traditional herbal medicine.The present study deals with extraction, isolation and characterization of the bioactive compounds from the ethanol extract of leaves and stem barks of Ageratum conyzoides Linn. The extraction was done by macerating the air dried powdered samples into the solvent followed by filtration and concentration. Separation and purification of the various constituents of the crude extracts were done using chromatographic techniques.TLC and Column chromatography were carried out using different mobile phases and the solvent blends used were petroleum ether .ethyl acetate, chloroform and Methanol. Qualitative thin layer chromatography was used to monitor the column fractions and also to ascertain the purity of the isolates. The chemical constituents by GC-MS analysis of whole plant extract of Ageratum conyzoides Linn revealed seventy one phytochemicals ranging from high and low molecular weight chemical entities with varying quantities present. The chemical investigation resulted in the isolation of four compounds. Compound [1] 3-hydroxylup-20(29)-ene was isolated at Rf 0.69 from the stem bark The MS spectrum showed a molecular ion peak at m/z 426, which corresponded to a molecular formula of C30H50O. Compound [2] (β-Amyrin acetate) was obtained from the leaf extract of Ageratum conyzoides Linn with Rf 0.63. The MS spectrum showed a molecular ion peak at m/z 467, which corresponded to a molecular formula of C32H52O2. Compound [3] 3,3-dimethyl, (1- hydroxy) cyclohexene propanenitrile, 4-benzoate was obtained from the leaf extract. The MS spectrum showed a molecular ion peak at m/z 297, which corresponded to a molecular formula of C18H19O3N FTIR revealed the presence of alcohol (3298 cm-1), nitrite (2250 cm-1), carbonyl (1726 cm-1), aromatic (1451 cm-1}. Compound [4] ] 3-keto lupane was obtained at Rf of 0.78 from the leaf extract. Mass spectrum gave a molecular ion peak at m/z 424, which was consistent with a molecular formula of C30H48O.The FTIR spectrum showed a carbonyl stretch at 1707 cm-1 and CH stretches at 2922 and 2858 cm-1. The structural elucidation of the compounds were deduced using MS, IR, 1D NMR (1H and 13C) along with 2D NMR Spectra ( DEPT ,COSY, HMBC and HSQC). . The antibacterial activity of crude extracts from parts of the plant were tested against four bacteria species; two gram positive pathogens namely (Salmonella typhi,, and Staphylococcus aureus ) and two gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa), using disc diffusion technique. All the crude plant extracts showed inhibitory activity against the gram positive bacteria, and gram negative bacteria. At concentrations of 12.5-100 mg/ml, the extracts possesses antibacterial activity (from being partially active to very active)
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Acknowledgement iv
Table of contents v
List of tables vi
List of plate vii
List of figures
viii
Abstract ix
CHAPTER 1: INTRODUCTION
1.1
Background of the Study 1
1.2 Justification of Research Work 4
1.3
Objectives of the Research 4
CHAPTER 2: LITERATURE REVIEW
2.1 Origin
of Geographical Distribution of the Ageratum
conyzoides 5
2.1.1 Description
of the plant 5
2.1.2 Ageratum conyzoides species 8
2.1.3 Pharmacognosy
of Ageratum conyzoides 8
2.1.4 Scientific
classification of A. conyzoides 9
2.1.5 Ethnomedical
uses of A.conyzoides 9
2.1.6 Chemical
constituents of A. conyzoides 11
2.1.7 Pharmacological
uses of Ageratum conyzoides 19
2.1.8 Agricultural
and environmental application 21
2.1.9 Phytochemicals
with medicinal activity 22
2.2. Alkaloids
24
2.2.1 Saponins
25
2.2.2 Major
actions of saponins in the body 26
2.2.3 Flavonoids
27
2.2.4 Carotenoids
30
2.2.5
Anthocyanins 30
2.2.6
Reservatol 31
2.2.7
Tanins 32
2.2.8
Phenolic compounds as antioxidants 33
2.2.9
Overview of pants in Asteraceae family 33
2.3
Haemorrage Plant (Aspilia Africana
) 34
2.3.1
Emilia coccinea –Asteraceae 35
2.3.2
The botany of ethnobotanical history of Emilia coccinea 37
2.4.
Medicinal Plants as Important Source of Natural and Drug Lead Molecules 38
2.4.1
Significance of medicinal plants in drug discovery 39
2.4.2
Bronchodilator 40
CHAPTER 3: MATERIAL AND METHODS
3.1
Research Design 42
3.2
Sample Collection 43
3.3
Sample Preparation 43
3.4
Phytochemical Determination 44
3.4.1
Determination of alkaloid 44
3.4.2
Determination of saponin 44
3.4.3
Determination of tannin by Folin, Dennis colorimeter method 45
3.4.4
Determination of flavonoid 45
3.4.5
Determination of phenol by A.O.A.C method 46
3.5
Extraction of the Plant Material 46
3.6 Isolation and Purification of the
Compounds 50
3.7
Thin layer Chromatography 50
3.7.1
Preparing plates for thin layer chromatography 51
3.7.2
Spotting, developing and visualization 51
3.8
Experimental Procedure for Gas Chromatography – Mass Spectrometry
(GC –MS) of whole Plant of Ageratum
conyzoides living 52
3.8.1 Components identification 53
3.8.2 NMR
characterization technique 53
3.8.3 NMR
structure elucidation 53
3.8.4 1HNMR
spectroscopy 54
3.8.5 13C
NMR 54
3.8.6 Correlation
spectroscopy COSY 54
3.8.7 Heteronuclear
multiple bond coherence (HMBC) 55
3.9 Antimicrobial
Evaluation of Ageratum conyzoides Plant
Parts Leaves and Stem Bark
55
3.9.1 Test
organisms 55
3.9.2 Media
preparation 55
3.9.3 Sub-culturing
of text organisms 55
3.9.4 Antibacterial assay of the plant leaf and stem
extracts 56
CHAPTER 4: RESULTS AND
DISCUSSIONS
4.1 Percentage
Yield of Extracts (leaves & stem bark) 57
4.2 Phytochemical Composition of Leaves
and Stem Bark of Ageratum conyzoides 57
4.3 Antimicrobial
Activity of Organic Crude Plant Extract of
A. conyzoides leaves
and stem bark 59
4.3.1 Antibacterial
activity of crude plant extract of Ageratum
conyzoides leaf extract and
Stem barks 60
4.4 Isolation and
Structural Elucidation of Compound [1]
(3-hydroxylup-20(29)-ene 63
4.5 Structural
Characterization of Compound [1]
3-hydroxylup-20(29)-ene 66
4.6 Isolation
and Elucidation of Compound [2] β-amyrin acetate 71
4.6.1 Structural Characterization of Compound [2] β-amyrin
acetate 74
4.7 Isolation
and Structural Elucidation of Compound [3]
4.7.1 Structural Characterization of Compound
[3] 3,3 dimethyl,(1 hydroxy) cyclohexene
propanenitrile, 4-benzoate C18H19O3 80
4.8 Isolation and Structural Elucidation of
Compound [4] 3- keto lupine 84
4.9 Structural Characterization
of Compound [4] 3- keto lupine 87
4.9.3 G.C-MS
of whole pant chloroform extract of Ageratum
conyzoides 107
CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusion
5.2 Recommendations
References
Appendices
LIST OF TABLE
3.1
TLC identification text
3.2 Microbe names and type used
4.1 Phytochemical
composition of Leaves and stem bark of Ageratum
conyzoides
4.2 Minimum
inhibitory concentration of plant extracts
4.3 13C NMR and 1H NMR
of compound [1]
4.4 FTIR Analysis of compound [1]
4.5 The m/z and the molecular formula of
compound [1]
4.6 FTIR Analysis of compound [2]
4.6.1 13C NMR and 1H NMR
of compound [2]
4.6.2 The m/z and the molecular formula of
compound [2]
4.7 Infra-Red Analysis of the compound [3]
4.8 13C
NMR and 1H NMR of compound [3]
4.8.1 The m/z and the molecular formula of
compound [3]
4.9 FTIR Analysis of compound [4]
4.9.1 13Carbon and 1H NMR
data for compound [4] lupenone
4.9.2 The m/z and the molecular formula of
compound [4]
4.9.3 GC-MS analysis of Ageratum conyzoides showing molecular formula, molecular weight, percentage
content, retention time
LIST OF PLATE
2.1 Ageratum conyzoides plant, stem, leaves and flowers
3.1 Beakers
of extracted samples (leaves and stem extracts) of A conyzoides
3.2 Picture of the student carrying out column
chromatographic in the night
4.1
TLC Chromatogram showing different spots
4. 2
TLC Chromatogram showing different spots on different solvent bend
LIST OF FIGURES
3.1 Flow
chart of research design for the thesis
3.2 Milled
plant parts of Ageratum conyzoides
(a) Whole plant for GC-MS (b) Leaves (c) Stem
3.3 Extraction
procedure of the leaves and stem barks of Ageratum
conyzoides
4.1
Structural characterization of Compound [1]
3-hydroxylup-20(29)-ene, m/z
426 molecular
formula of C30H50O
4.2
Fragmentation pattern of Compound [1] 3-hydroxylup-20(29)-ene
4.3 Structural characterisation of Compound [2] β- Amyrin
acetate
4.3.1 Fragmentation
Pattern of Compound [2]
4.4 Structural
characterisation of Compound [3] 3, 3-dimethyl, (1 hydroxy) cyclohexene propanenitrile, 4-benzoate C18H19O3 M/Z =
297
4.5 Fragmentation
pattern of the Nitrile [3]
4.6 Structural
characterization of Compound [4 ] 3-Keto lupine
4.7 Fragmentation
pattern of compound [4]
4.8 GC-MS
chromatogram of Ageratum conyzoides
whole plant extract
4.9 GC- MS of Mass Spectra Ageratum conyzoides
whole plant extract
CHAPTER 1
INTRODUCTION
1.1
BACKGROUND OF THE STUDY
Plant-based remedies
enjoy a reputable position today, particularly in the developing countries,
where basic health care facilities are inadequate. Herbal medications which are
more effective, safe and cheap are gaining ground in both urban and rural
communities (Shariat and Noor, 2018). Information from ethnic groups or
traditional medicine practitioners has played an important role in the
development of novel bioactive medicinal products from herb as therapeutic
agents (Katewa et al., 2004)
Medicinal plants are in high demand
in the developed as well as developing countries for basic healthcare need,
because of their wide biological and medicinal properties, higher safety
margins and lesser costs. Over the years, the WHO suggested that countries
should get involved in the use of herbal medicine with the aim of exploiting the
secondary metabolites that provide effective and safe solutions for diseases of
both non-microbial and microbial origins (Fatema., 2013). Medicinal plants have
been revealed to have valid utility and over 80% people living in rural
communities rely on its efficacy for their primary health care. The efficacy of
herbal medicines against ill health is possible due to presence of so many
bioactive constituents such as nutrients, phytochemicals, etc, which have
pharmacological activities in the body of living organisms (Amadi et
al., 2012).
Ageratum conyzoides Linn is
among the annual herb with a long history of traditional medicinal applications
in both sub-tropical and tropical zones of the world. Ageratum is coiled from the Greek words ‘a geras’ meaning non-aging
which refers to long life-time of plant. It is generally refered to as billy
goat weeds. Asteraceae family comprises over 35 species with Ageratum
conyzoides as one of the genus in the family (Okunade., 2002). Among the
species is the Ageratum conyzoides L., which is generally refered to
as “billy goat weed”, “mentrasto” and “catinga-de-bode” (Asicumpo., 2005).
It can survive in both tropical and
subtropical areas where it thrives as ornamental during summer (Greuter.,
2016). The species is now common in Europe,
Africa, North America, Central America, the Caribbean, South America, and
Oceania. A.conyzoides is
generally used in folk medicine in many nations of the world as analgesic,
febrifuge, anti-inflammatory agent and purgative (Okunade., 2002).The plant when mature is used
for its antispasmodic haemostatic, anti-inflammatory, antiasthmatic,properties
for bacterial infections and the treatment of wounds (Gonzalez et
al., 1991; Adetutu
et al., 2012).
Ageratum conyzoides is used for treatment of infective
hepatitis, constipation, intestinal worms, filariasis and cuts. The whole plant
possesses antiallergic, antinematocidal, anticoagulant, smooth muscle relaxant,
haemostatic, analgesic, antifungal, antibacterial and hypothermic activities
(Nogueira et al., 2010; Adetutu et al., 2012).The
essential oil found in it can inhibit the growth and production of toxigenic
strain of Aspergillu sparasiticus (Patil et al., 2010). A. conyzoides has growth inhibitory actions and
larvicidal activities in the second and fourth instar larvae of the Anopheles
stephensi (Neetu et al., 2011). Phytochemical
examination of A. conyzoides indicated presence of alkaloids, resins,
saponins, tannins, glycosides and flavonoids (Kamboj and Saluja., 2008).
A number of flavones have been
identified including 8-hydroxy-5, 6, 7, 3, 4, 5- hexamethoxy flavones (Singh et
al., 2013). Humans do not eat the plant except for medicinal
purposes; however, the leaves are used to feed fish, domestic guinea pigs,
cattle and horses. It has also been reported that the plant might contain
allele chemicals when the acetone extract residue of the plant inhibits the
growth and germination of the shoots and roots of other plants (Kato-Naguchim.,
2001). Thus,it has been discovered that crude extracts from A. conyzoides offer
the possibility of biological control of plant pathogenic fungi (Iqbal et al., 2004). The plant leaves have
been reported to exhibit
anti-inflammatory properties, with no apparent hepatotoxicity (Moura et al., 2005). A tea made from Ocimum tenuifolium mixed with flower
heads is used to treat colds, catarrh and coughs. The juice from the flower
heads is applied externally to treat scabies, and then a paste made from the
flower head is used in treatment of rheumatism. The juice of the plant is also
used to treat bruises, cuts and wounds. An extract of the dried plant or the
fresh juice of the plant is used in the treatment of allergic rhinitis and
sinusitis and also in treating post-partum uterine haemorrhage. Ageratum conyzoides
is the only plant Igede people in Nigeria use in the treatment of HIV/AIDS (Igoli et al., 2005).
The plant as a whole produces strong
smelled volatile oil which has several biological activities. It is used for dressing
wounds, curing various skin diseases, ophthalmic, colic, ulcers treatment, as purgative
and febrifuge. The infusion or decoction of the herb is taken in stomach
ailments such as intestinal colic, diarrhoea, dysentery, flatulence, (Chopra et al., 2002), Many other traditional
remedies include mouthwash for toothache, anti-itch, sleeping sickness, antitusive,
vermifuge, tonic and killing lice (Burkill, 1985; Kapur, 1993).A freshly made decoction of the plant
is used as a hair wash, leaving the hair soft, fragrant and dandruff free.
The plant leaves are reported to have hematopoietic activity which could
possibly cure anemia (Burkill., 1985). It was further reported to have
gastroprotective potentials (Shirwaikar et
al., 2003). The WSF of the plant extract produces peripheral analgesic
activity and an anti-inflammatory action, which seems to occur in
leucocyte-dependent inflammatory events. They are implicated in the mediation
of acute pain in the central nervous system of mammals (Sampson et al., 2000). Aqueous extract of the
plant was tested against three Gram-positive bacteria and seven Gram-negative
bacteria and evaluated by the filter paper disc diffusion method. Results
showed a significant control of the growth of Alcaligenes viscolactis,
Klebsiella aerogenes, Bacillus cereus and Streptococcus pyogenes (Moody
et al., 2004). N-hexane extracts of A.
conyzoides with LC50 values 1925.60 and 267.90 ppm respectively
was found to have inhibitory activity against the mosquito, Culex quinque fasciatus
larvae (Preeti et al., 2009). The
most important considerable biological activity of Ageratum conyzoides is in fact, its insecticidal activity which may
play a valuable role in agriculture economically as well as effectively. Both
the oil as well as its extracts have insecticidal activity (Liu
and Liu, 2014) It can exhibit insecticidal property
against cowpea weevil, Callosobruchus maculatus F. (Gbolade et al., 1990) and high nymphal
mortality, that is, 90 % to the nymphs of Schistocerca gregaria (Pari et al., 1998). N-hexane extract was
active against the larvae of mosquito, C.quinquefasciatus (Preeti et al., 2009). The crude plant extract
also showed insecticidal and pesticidal activities against various types of
insects and pests.
1.2
JUSTIFICATION OF RESEARCH WORK
Ageratum
conyzoides Linn has been described to possess
various biological activities. It is used in folk medicine as a purgative,
febrifuge, anti-inflammatory, analgesic, anesthetic and in treatment of ulcers.
The mature plant is used for its haemostatic, anti-spasmodic, anti-asthmatic,
properties for the treatment of wounds and in bacterial infections. The plant
has played a valuable role in agriculture economically as the extract has
showed insecticidal and pesticidal activities against various types of insects
and pests. Ageratum conyzoides has
been reported to have displayed considerable larvicidal activity and
inhibited emergence of adult mosquitoes, thus improved mosquito control and
reduced malaria transmission rates. The
bioactive constituents of the leaves and stem bark of Ageratum conyzoides
Linn have not been fully documented.
1.3 OBJECTIVES
OF THE RESEARCH
Ageratum conyzoides Linn plant is quite popular in Nigeria. The objectives
of this research are as follows:
To identify
the chemical constituents of the leaves and stem bark of Ageratum
conyzoides Linn
Extraction,
fractionation and isolation of pure compounds from the plant using
chromatographic techniques, TLC and Column Chromatography.
Structural elucidation of pure compounds
by spectroscopic (1D NMR and 2D NMR, IR) and spectrometric (MS) method
Identify the phytochemicals present using
Gas Chromatography-Mass Spectrometry analyses.
To examine the
antimicrobial activity of the crude
extracts against Pseudomonas
aeruginosa ,Escherichia
coli, Staphylococus aureus and Samonella typhi.
To ascertain the
usefulness of the plant in the treatment of various diseases as claimed by
herbalist.
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