ABSTRACT
This research work was carried out to identify the bioactive constituents of the leaves, stems and roots of Lactuca (Launaea) taraxacifolia, a medicinally important plant of the Asteraceae family. The plant was selected for this study due to its widespread use in traditional medicine and on the basis that the plant is almost going into extinct in Nigeria. The present study deals with the extraction, isolation and characterization of the bioactive compounds of the chloroform extracts of the leaves, stems and roots of L. taraxacifolia. The extraction was done by subjecting the air dried pulverized samples into the chloroform solvent for a period of some days, followed by filtration and concentration. Separation and purification of the various constituents of the crude extracts were carried out using thin layer chromatography and column chromatography. The TLC and column chromatography were carried out using different mobile phases and the solvent blends used were: normal hexane, chloroform, ethyl acetate and methanol. Qualitative TLC on precoated aluminium silicate strips was used to monitor the column fractions to ascertain the degree of purity of the eluents. The structural elucidation of the isolated compounds was deduced using MS, FTIR, NMR (1H and 13C) and 2D COSY NMR Spectra. This chemical investigation resulted in the isolation of two novel compounds: SB2 (Octadecahydro-4,4-dimethoxy-12b, 14b-dimethyl-10-(napthalen-2-yl) Picen-3-(4H, 6bH, 14bH)-one and SB3 (Octadecahydro-10-(1, 2, 3, 4-tetrahydronaphtalen-3-yl)-4,4-dimethoxy-6b, 12b, 14b-trimethyl Picen-3-(4H, 6bH, 14bH)-one. Compound SB2 was isolated using 90% chloroform and 10% ethyl acetate at an Rf value of 0.821, while compound SB3 was isolated using 50% hexane and 50% chloroform at an Rf value of 0.692. The MS spectrum of compound SB2 gave a molecular ion peak at m/z 527.75, which corresponds to a molecular formular of C36H47O3.The MS spectrum of compound SB3 gave a molecular ion peak at m/z 545.81, which corresponds to a molecular formular of C37H53O3. These terpenoid derivatives characterized by the presence of carbonyl and methoxy groups accounted for the antimicrobial, anti-tumor and anti-oxidant properties of the plant.The antimicrobial activities of the crude extracts were carried out using the disc diffusion technique. Fifteen micro-organisms (five-Gram positive, five Gram-negative and five fungi) were selected for the antimicrobial screening of the solvent extracts of the leaves, stems and roots of L. taraxacifolia. The zone diameters of inhibition of the extracts ranged from 10.1 mm to 17.2 mm. In all cases, the phytochemicals in the chloroform extracts had greater broad spectrum effects when compared to other extracts used in this work, thus inhibiting all the test bacteria used in this work, though with varying diameters of inhibition, while the phytochemicals in methanol extract had the least effect, inhibiting six out of the ten bacteria used in the study. Generally, the extracts had negligible inhibition effects on the selected fungi. Phytochemicals in n-hexane and ethyl acetate were only effective on Candida albicans at zone diameters of inhibition of 17.2 mm and 15.2 mm respectively. A major set-back associated with the intake of herbal medicine is the problem of overdose, which may lead to the destruction of tissues and cells. Hence the minimum inhibitory, bactericidal and fungicidal concentrations of the extracts were carried out. The results showed that the minimum inhibitory concentrations (MIC) of the extracts ranged from 200 mg/ml to greater than 250 mg/ml. Some of the extracts had minimum bactericidal concentration (MBC) of 250 mg/ml, which is the concentration of most conventional drugs.
TABLE
OF CONTENTS
Title
Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgement v
Table
of Contents vi
List
of Tables xii
List
of Figures xiii
List
of Plates xiv
Abstract xv
CHAPTER 1: INTRODUCTION
1.1
Background of the Study 1
1.2
Significance of the Study 3
1.3
Justification of the
Study 4
1.4
Statement of the Problem 5
1.5
Aims and Objectives of
the Study 5
1.6
Scope of the Study 6
CHAPTER
2: LITERATURE REVIEW
2.1 Historical
and Common Applications of Wild Lettuce 7
2.1.1
Common names of wild lettuce 8
2.1.2
Taxonomy of Lactuca taraxacifolia 8
2.1.3
Biological diversity and divergency of the
genius 11
2.2 Taxonomy of the Genus Lactuca Species 11
2.2.1
Lactuca 12
2.2.2
Phaenixopus 12
2.2.3
Mulgedium 13
2.2.4
Lactucopsis
13
2.2.5
Tuberosae,
Micranthae and Sororiae 13
2.2.6
African group 14
2.2.7
North American group 14
2.2.8
Geographical distribution and ecology of
genus Lactuca species 16
2.2.9
Karyological status, biochemical and
molecular markers 17
2.3
Gene Pool of Lactuca Sativa and Crossability 17
2.3.1
Regeneration of accessions of wild Lactuca species 19
2.3.2
Chemical composition of wild lettuce 19
2.3.3
Pharmacology of wild lettuce 20
2.3.4
Demulcent and prebiotic effects of wild
lettuce 20
2.3.5
Digestion, inflammation and angiogenesis
of wild lettuce 20
2.3.6
Hypoglycemic activities of wild lettuce 21
2.3.7
Immunity 21
2.3.8
Varieties of preclinical studies of wild
lettuce 22
2.3.9
Hepatobiliary effects of wild lettuce 22
2.4 Inhibitory Effects of Wild Lettuce 22
2.4.1
Clinical applications, western studies 23
2.4.2
Clinical applications, Chinese studies 24
2.4.3 Dosage and sustainability of wild lettuce 25
2.4.4 Safety and drug
interaction of wild lettuce 26
2.4.5
Medical indications of wild lettuce 26
2.4.6
Liver Gall Bladder Statsis 27
2.4.7
Edema 27
2.4.8
Colitis 27
2.4.9
Side effects and toxicity of wild lettuce 27
2.5
Bioactive Compounds Found in Asteraceae 28
2.5.1
Nutritional
factors 29
2.5.2
Structures of sesquiterpenes 30
2.5.3
Role
of the µ -
methylene and – Y – lactone groups 33
2.5.4
Supression
of NF – KB 34
2.5.5
Role of sesquiterpene lactones in people 36
2.5.6
Tumor inhibition by sesquiterpene lactones 37
2.5.7
Alternative mechanism of anti-inflammatory
activities 39
2.5.8
The function of parthenolide in anti
cancer therapy 40
2.5.9
Sesquiterpenoids in herbal medicine 41
2.6
Antioxidant Function of Sesquiterpene
Lactone 42
2.6.1
Bitter properties of sesquiterpene lactone 43
2.6.2
Importance of sesquiterpenes in plants 45
2.6.3
Anti-herbivory activities of sesquiterpene lactones 46
2.6.4
Antimicrobial roles of sesquiterpene lactones 47
2.6.5
The use of sesquiterpene lactones to protect
ozone layer 48
2.6.6
Allelophathy 49
2.6.7
Implications for crop production 50
2.7
Active Chemical Constituents 51
2.7.1
Cycloartenol 51
2.7.2
Beta – sitosterol 53
2.7.3
Vitamin A 54
2.7.4
Ascorbic acid (vitamin C) 56
2.7.5
Tannins 58
2.7.6
Alkaloids 59
2.7.7
Cardiac glycosides 62
2.8 The
Biochemistry of Cardiac Glycosides 63
2.8.1 Medical analysis of the activities of
cardiac glycosides 66
2.8.2 Anticancer characteristics and the
mechanisms of cardiac glycosides 66
2.9 Apigenin (Flavonoids) 69
CHAPTER 3:
MATERIALS AND METHODS
3.1 Preparation of Plant Materials for
Column Chromatography 71
3.1.1 Packing
of the large column 71
3.1.2 Packing of micro column 72
3.1.3 Thin
layer chromatography 72
3.2 Phytochemical
Screening of the Extracts 72
3.2.1 Determination of
saponins 72
3.2.2 Determination
of phenols 73
3.2.3 Determination of alkaloids 73
3.2.4 Determination of tannins 74
3.2.5 Determination of cardiac glycosides 75
3.2.6 Determination of flavonoids
75
3.2.7 Determination of Steroids 76
3.2.8 Determination of terpenoids 76
3.3 Proximate Composition 77
3.3.1 Determination of ash content 77
3.3.2 Determination of crude fat 77
3.3.3 Determination of crude fibre 77
3.3.4 Determination of crude protein by Kjedahl
method 78
3.3.5 Determination of moisture 79
3.3.6 Determination of available carbohydrate 79
3.3.7 Determination of energy value 80
3.4 Preparation and
Collection of Plant Materials for Antimicrobial
Susceptibility Test. 80
3.4.1 Extraction of plant materials 80
3.4.2 Preparation
of test organisms 80
3.4.3 Antimicrobial
susceptibility test of the L.
taraxacifolia extracts 81
3.4.4 Test for minimum inhibitory concentration
(MIC) of the
L. taraxacifolia extracts
82
3.4.5 Test for minimum bactericidal (MBC) and
fungicidal
concentration
(MFC) of the extracts 82
CHAPTER
4: RESULTS AND DISCUSSION
4.1 Chromatographic Separation of L. taraxacifolia Extracts 83
4.2 Isolation of compound SB2 87
4.2.1 13CNMR Spectrum of compound
SB2 88
4.2.2 1HNMR Spectrum of compound SB2 90
4.2.3 COSY – 2D Spectrum of compound SB2 93
4.2.4 Infra-red spectrum of compound SB2 95
4.2.5 Characterization of compound SB2 98
4.2.6 Mass spectral analysis of compound SB2 99
4.3 Isolation of Compound SB3 102
4.3.1 13CNMR spectrum of compound
SB3 103
4.3.2 1HNMR spectrum of compound SB3 105
4.3.3 COSY – 2D spectrum of compound SB3 108
4.3.4 Infra-red spectrum of compound SB3 110
4.3.5 Characterization of compound SB3 113
4.3.6 Mass spectral analysis of compound SB3 114
4.4. Phytochemical Composition of L. taraxacifolia 117
4.5 Proximate Analysis of L. taraxacifolia Leaves. 122
4.6 Antimicrobial Screening of the
Extracts of L. taraxacifolia 125
4.7 Minimum
Inhibitory/Bactericidal/Fungicidal Concentration of
Extracts
of L. taraxacifolia 132
CHAPTER
5: CONCLUSION AND RECOMMENDATIONS
5.1
Conclusion 134
5.2
Contributions to
Knowledge 135
5.3
Recommendations 136
References
137
LIST
OF TABLES
2.0
The
taxonomy of Lactuca Species, genetic
resources
andtheir
categorization to sections, subsections and groups 15
2.1 Survey of wild Lactuca and related genera
germplasm
maintained in genebank collections 18
2.2 Antiproliferrative
activities of cardiac glycosides 68
4.1 Large column and
thin layer chromatography of
L. taraxacifolia extracts
84
4.1.1 Micro
column and thin layer chromatography
of
L. taraxacifolia extracts 86
4.2 1HNMR
and 13CNMR chemical shift of compound SB2 92
4.3 Infra-red
spectrum of compound SB2 97
4.4 1HNMR
and 13CNMR chemical shift of compound SB3 107
4.5 Infra-red
spectrum of compound SB3 112
4.6 Qualitative
screening of photochemicals in L.
taraxacifolia 120
4.7 Quantitative
screening of phytochemicals in L.
taraxacifolia 121
4.8 Proximate
composition and energy content of the
Leaves
of L. taraxacifolia 124
4.9 Antimicrobial
activities of the extracts of Lactuca
taraxacifolia 127
4.9.1 MIC,
MBC & MFC of the extracts of Lactuca
taraxacifolia 133
LIST OF FIGURES
2.1 Sesquiterpene lactones 32
2.2 Structural characteristics of
cardiac glycosides 65
4.1
13CNMR
spectrum of compound SB2 89
4.1.1
1HNMR
spectrum of compound SB2 91
4.1.2
COSY – 2D spectrum of
compound SB2 94
4.1.3
Infra-red spectrum of
compound SB2 96
4.1.4
Mass spectral analysis of
compound SB2 100
4.1.5
Fragmentation scheme of
compound SB2 101
4.2
13CNMR
spectrum of compound SB3 104
4.2.1
1HNMR
spectrum of compound SB3 106
4.2.2
COSY – 2D spectrum of compound SB3 109
4.2.3
Infra-red spectrum of
compound SB3 111
4.2.4
Mass spectral analysis of
compound SB3 115
4.2.5
Fragmentation scheme of compound
SB3 116
4.3 Proximate composition of the leaves
of L. taraxacifolia 123
4.4 Zones of inhibition of chloroform
extract on different bacterial species 128
4.5 Zones of inhibition of hexane
extract on different bacterial species
and on Candida
albicans 129
4.6 Zone of inhibition of methanol
extract on different bacterial species 130
4.7 Zones of inhibition of ethyl
acetate extract on different bacterial
Species and on Candida albicans 131
LIST OF PLATES
2.0 Lactuca
taraxacifolia 10
CHAPTER 1
INTRODUCTION
1.1: BACKGROUND OF THE STUDY
Data obtained from recent researches
showed that a range of 65% to 90% of people
in most developing countries rely on traditional medical practices in addition to herbal
medicines in the treatment of ailments and to manage their basic health-care requirements (WHO, 2002
– 2005). The application of traditional herbal medication is also essential in certain
developed nations. Between 68% and 85% of the populace consume traditional
medicines under the titles: “Complementary”, “alternative”, or
“nonconventional” medications has been reported in developed nations like
Canada, France, and Germany (WHO, 2005; Barnes et al., 2007 Traditional Medicine, 2008).
In the United States of America the application
of complementary and combined medical methods in health care delivery is
gaining grounds. A certain research shows that 43% of Americans used alternative
therapies in 1997; 41% for treatment of acute ailments and 59% for disease control.
In 2007, the most popularly applied complementary and traditional medical therapy
include: non vitamins, non minerals and natural products constituting 17.7%
(Barnes et al., 2007). Inspite of
this rapid rise in the application of herbal medicines, limited evidence exists
for their potency and toxic nature. More researches need to be carried out to
develop the evidence base for herbals, botanicals and dietary supplements
(Turner, 1965).
Conversely, natural products have been
very essential for effective treatment and amelioration of disease burdens of humans
and animals (Perry, 1980). Thus it is very important to evaluate natural
products for their potency in the diagnosis of ailments. Most plants manufacture
substances that are essential for the sustenance of health in humans and other
animals. Such organic compounds include: aromatics like phenols, tannins and
secondary metabolites of which not more than 12% have been isolated. In most
situations, molecules such as alkaloids protect the plant against predators by
micro-organisms, insects and herbivores. Most of the herbs and spices applied by
humans to season their food give important medicinal phytochemicals (Lai and
Roy, 2004; Tapsell et al., 2006). Analogous
to the recommended drugs, a range of herbs are thought to have undesirable
activities (Talalay, 2001). Additionally, adulteration, wrong combination or
lack of knowledge of plants and mechanisms of drug interactions has caused
negative effects that are mostly risky to life (Elvin-Lewis, 2001).The application
of herbs to mitigate ailments is almost common with non-industrialized communities
(Edgar et al., 2002). Most of the drugs
currently accessible to medical practitioners have a connection to herbs as a major
component as compared to other natural resources because of the belief that
they have no side effects (Veale et al.,
1992; Ram et al., 1997). The application
of such and dietary supplement obtained from plants has greatly increased in
recent years. Microbiologists, Pharmacologists, Botanists and Natural Product
Chemists are carrying out research into phytochemicals for solutions that could
be developed for the diagnosis of different ailments. According to World Health
Organization, as much as 26% of the modern drugs obtained from plant products
are used in the United States. Over the past one hundred years, about one
hundred and twenty one pharmaceutical products have been found as a result of
the information obtained from traditional healers (Anesini and Pereez, 1993).
In certain Asian and African nations, 82% of the recommendations are to evaluate
the safety and efficacy of each plant before they are prescribed for medical
use (Vickers, 2007). Invariably, most consumers are of the thought that herbal medicine
and synthetic drugs may act in synergy causing harm to the patient. It may also
be severely contaminated and without proven potency may ignorantly be used to
substitute drugs that do have similar potency (Ernst, 2007).
The use of Lactuca (Launea) taraxacifolia as a health-care remedy is scarcely
documented. Despite the fact that not much studies have been done
scientifically on the plant, claims from the public and herbalists recommended that
the plant is very beneficial in the management of diabetes and as a mild
laxative. The plant is also thought to provide a cure to high blood pressure,
heart attack, stroke and other cardiovascular diseases (Leung and Foster, 1996).
L. taraxacifolia leaves are consumed
by lactating cows in Northern part of
Nigeria to enhance milk production; it
is also used to induce multiple birth rates in sheep and goats (Adinortey et al., 2012).Medicinally, the leaves
are rubbed on legs to make children in Nigeria and Ghana walk; their leaves are
combined with ashes to cure yaws (Ayensu,
1978).
Lactuca
(Launaea) taraxacifolia (Wild) is an annual West
African tropical herb popularly known as Wild Lettuce or Dandelion. The plant
is known in French as langue de vache
which means “tongue of the cow”. There are some common names of the plant; Ga; agbloke, Ewe (anlo); anoto, Tivi; dadeou, Akan Akuapem, nne noa (boil today), Hausa: namijin dayii (applied loosely), Yoruba:
efo yanrin, Sierra Leone: Kissi; bekuhoa-pomboe. The plant has leaves
which are in arranged basal rosette form of 3-5, pinnately lobed with ultimate
margins dentate. The stem is upright towering to about 1-3 meters high from a
woody rhizome which is solitary branched and borne with 25-30 floret flowers
with yellow corollas in a convex receptacle at the apex slightly narrowed,
which gives rise to a white 7-8 mm long pappus air-borne seeds (Burkill,
1985).
1.2: SIGNIFICANCE OF THE STUDY
Due to the increasing use of herbal medicine in the
treatment of disease, it is pertinent to evaluate the extraction and
bioactivity of endangered medicinal plants such as L. taraxacifolia in the treatment of wide range of diseases and in
the production of conventional drugs.
1.3: JUSTIFICATION OF THE STUDY
The worldwide use for medicinal plants is
fast advancing (Yadav et al., 2010). The
use of traditional medicine to aid orthodox medicine in the treatment of
ailments in developing nations is increasing (Salahdeen and Yemitan, 2006). Conversely,
these traditional medicines do not have enough proof for their potency or
safety (WHO, 2000; Patwardhan, 2005; Mosihuzzaman and Choudhary, 2008).
Plant extracts and phytochemicals are very
essential in therapeutic medicine (Varalakshmi et al., 2011). Safety evaluations of these
plants for medicines are very important since plants have many phytochemicals
which may lead to toxicity either on their own or by interacting with other
chemicals (Teixera et al., 2003).
Chaya leaves (Cnidoscolous acontifolius) are toxic as they contain cyanogenic
glycosides that can cause electrolyte imbalance particularly hypokalemia
(Westendorf, 1993). It was discovered that a high risk of lung cancer occurred
in individuals consuming a mixture of betacarotene and vitamin E supplements
(Goodman et al., 2004). Plants of the
genus Euphorbia synthesize caustic lattices,
which poses a health problem to humans and livestocks (Adedapo et al., 2004).
Launaea (Lactuca)
taraxacifolia is a plant used in traditional herbal medicine for the treatment
of skin and eye diseases – conjunctivitis, measles, diabetes mellitus and also
rubbed on the legs of toddlers to
enhance walking (Ayensu, 1978; Adebisi,
2004; Obi et al.,2006), abdominal
disorders, heart burn, dyslipidaemia, liver diseases and also as food
(Adinortey, 2012). Unfortunately, there is scarcity of evidence to support
these uses and to determine the bioactive compounds in this plant. The World
Health Organization supports research into the agents for diagnosis, management
and control of ailments by the application of traditional medical practices
(Atta and Mouneir, 2004).
Here, the plant Launaea taraxacifolia is evaluated for its phytochemical
composition, antimicrobial activities, minimum inhibitory concentration (MIC),
minimum bactericidal and fungicidal concentrations (MBC & MFC) of the
extracts. In addition, advanced chromatographic techniques followed by spectral
analysis of the extracts were used to elucidate the structures of the bioactive
compounds of the plant. It is expected that the findings from this work may add
to the overall value of the medicinal potential of the Launaea taraxacifolia.
1.4: STATEMENT OF THE PROBLEM
There has been few documented work on L. taraxacifolia. These reports focused
on the extraction of active components of L.
taraxacifolia using a specific solvent and its antimicrobial activities on
few microbes. In view of this, this current research evaluates the
effectiveness of four different solvent extracts of the plant on microbes.
There is few documented work on the structures of the bioactive compounds in L. taraxacifolia, hence there is need to
carry out spectral analysis on the isolated components. A problem or
disadvantage of herbal medicine is the consumption of overdose leading to the
destruction of tissues and cells.
Hence, this research work further evaluates
the Minimum Inhibitory Concentration and Minimum Bactericidal Concentration of
the results of antimicrobial screening.
1.5: AIM AND OBJECTIVES OF THE STUDY
The aim of this present
study is to isolate and characterize the bioactive compounds of the plant Launaea (Lactuca) taraxacifolia.
The aim will be achieved
using the following objectives:
ü
Effective utilization of
chromatographic techniques and other extractive processes to isolate the
components of L. taraxacifolia.
ü
Determination of the
phytochemicals of L. taraxacifolia using
different conventional methods
ü
Determination of the
antimicrobial potency of solvent extracts of L. taraxacifolia
ü
Determination of the
minimum inhibitory concentration, minimum bacterial concentration and minimum
fungicidal concentration of the solvent extracts.
1.6: SCOPE OF THE STUDY
This research work involved the separation of the
components of air-dried plant materials of L.
taraxacifolia by means of column chromatography. Four solvents namely:
hexane, chloroform, ethyl acetate and methanol were employed in the
chromatographic separation. The extracts obtained were then subjected to Thin
Layer Chromatography (TLC) using pre-coated aluminium-silicate strips, in order
to isolate pure samples for spectral analysis. The biological properties
(antimicrobial, phytochemical content, minimum bactericidal concentration and
minimum fungicidal concentration) of the extracts from the four solvents were
also evaluated. Fifteen micro-organisms which include: five gram positive bacteria,
five gram negative bacteria and five fungi were used in the antimicrobial
susceptibility tests.
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