CHAPTER ONE

INTRODUCTION
1.1 Background of the study
Traditional societies in Africa have historically devised comprehensive methods of
providing essential healthcare to individuals within the community through accessible means by
applying indigenous resources such as plants, animals, and minerals (Chhabra et al., 1987).
Herbal medicine, deeply rooted in the socio-cultural background of every country, remains an
integral part of primary healthcare, particularly in developing nations where access to modern
pharmaceuticals may be limited by cost or availability. Of late, despite the significant emphasis
placed on the research and development of synthetic drugs, a renewed and robust interest in
medicinal plants has emerged globally. This resurgence is partly due to the fact that many
synthetic drugs are potentially toxic, not free of side effects on the host, and that the
effectiveness of many herbal medicines is now an accepted scientific fact (Thomson, 1978;
Geddes, 1985). Further, herbal preparations constitute a valuable natural resource from which
chemicals of great potential interest for medicine, agriculture, and industry can be identified and
isolated. Such valuable drugs as atropine, reserpine, quinine, and morphine were discovered from
traditional herbal remedies, validating the ethnomedicinal approach to drug discovery (Sneader,
1985).

Figure 1.1: Chemical structures of Atropine, Reserpine, Quinine, and Morphine

It is estimated that about 25% of the drugs prescribed worldwide are derived from plants,
and 121 such active compounds are in current use (Sahoo et al., 2010). Between 2005 and 2007,
13 drugs derived from natural products were approved in the United States. More than 100
natural product-based drugs are in clinical studies (Li and Vederas, 2009), and out of the total
252 drugs in the World Health Organization's (WHO) essential medicine list, 11% are
exclusively of plant origin (Sahoo et al., 2010). This has necessitated microbiologists all over the
world to search for the formulation of new antimicrobial agents and evaluate the efficacy of
natural plant products as a substitute for chemical antimicrobial agents (Pandian et al., 2006).
Many microorganisms and plants produce compounds that are not related to the basic
metabolism of the producing organism, called secondary metabolites, as their defense

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mechanism. Many of these products play important roles as therapeutics, stimulants, and feed
additives among others (Hans, 1993). Medicinal plants are well-known natural sources for the
treatment of various diseases since antiquity. About 20,000 plant species used for medicinal
purposes are reported by the World Health Organization (WHO) (Gullece et al., 2006; Maregesi
et al., 2008). Furthermore, natural products, either as pure compounds or as standardized plant
extracts, provide unlimited opportunities for new drug leads because of the unmatched
availability of chemical diversity (Cos et al., 2006). In the past, the wide range of antimicrobial
agents from lower organisms and synthetic drugs sufficed in the treatment or control of
infectious diseases. However, currently, there is a problem of microbial drug resistance, and there
is an increase of opportunistic infections, especially with acquired immune deficiency syndrome
(AIDS) patients and individuals on immunosuppressive chemotherapy. Many antifungal and
antiviral drugs are of limited use due to toxicity, while other viral diseases have not yet found a
cure. These problems pose a need for searching for more new drug substances. Specifically,
Vernonia amygdalina, commonly known as bitter leaf, has been widely used in traditional
medicine across Africa for the treatment of various ailments including fever, malaria, and
gastrointestinal disorders which are often associated with bacterial infections such as those
caused by Escherichia coli (E. coli). The resurgence of interest in this plant is driven by the need
to validate these traditional claims scientifically and to explore its potential as a source of novel
antibacterial agents in the face of rising antibiotic resistance.
1.2 Phytomedicine
People all over the world have used plants as medicine from time immemorial. It is
estimated by WHO that 80% of the population, the majority of this in developing countries, still
rely on plant-based medicine for primary healthcare (Evans, 1997; Farnsworth, 1998). WHO
further estimated that over 80% of the population residing in developing countries depends
directly on plants for their primary medical requirements (Czygan, 1993; WHO, 2008). This is
attributed to the fact that plant-derived medicines can be easily accessed and are also cheap
(Amin and Mousa, 2007; Ramawat and Goyal, 2008; WHO, 2008). The use of some crude
extracts whose specific evaluations have not been done could lead to serious complications,
overdose, and intake of toxic substances. Ineffective herbs could also be used as a matter of
belief or tradition (Baker et al., 1995). In most countries, the use of herbal medicine continues to
coexist with modern pharmacology (Ernest, 2005). The worldwide upsurge in the use of herbal
preparations and active ingredients isolated from medicinal plants in healthcare (Jassim and Naji,
2003) is due to increased side effects, lack of curative treatment for several chronic diseases,
high cost of new drugs, microbial resistance, and emerging diseases (Humber, 2002). This is
indicated by the growing popularity of Traditional Chinese Medicine (TCM) and Ayurvedic
medicine of India (Jiang et al., 2000; Dubey et al., 2004). In Africa, the use of traditional
medicine is very high as indicated by the fact that between 70 and 80% of the population depend
on herbal preparations for primary healthcare with little or no scientific information on efficacy
and side effects (Kokwaro, 1996). Furthermore, even the people of the developed world are also
dependent directly or indirectly on plants for their health care. In the United States, 25% of the
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prescriptions given from community pharmacies consisted of plant extracts or active ingredients
of plant origin (Cragg and Newman, 2005). In Dar es Salaam, Tanzania, 21% of patients who
visited public hospitals had consulted a traditional healer before they came to hospital (de Boer
et al., 2005). Plant-derived medicines are offered/taken in the form of tinctures, teas, poultices,
and powders, depending on the knowledge of the use and application method of a particular plant
for a given ailment (Fennell et al., 2004; Balunas and Kinghorn, 2005). Vernonia amygdalina is
typically consumed as a vegetable soup, decoction, or crushed leaf extract, reflecting the diverse
methods of administration prevalent in African traditional medicine. In Nigeria, debate on the
incorporation of traditional medicine into primary health care has been ongoing for several
decades. There have been efforts to regulate the practice of traditional medicine through
legislation. The Traditional Health Practitioners Bill, 2014, was published in February 2014 and
tabled before the National Assembly by the then Chairperson of the Senate Committee on
Health, Senator Omoba Lanre Tejuoso. The Bill underwent its first reading on 3rd June 2014,
and the Committee submitted its report on the deliberations regarding the Bill on 25th April
2015. Since then, however, there has been no further progress. If passed into law, the Bill seeks
to provide for the training, regulation, and licensing of traditional practitioners, thereby ensuring
proper oversight of their practice. It also proposes the establishment of a Traditional Health
Practitioners Council to regulate the profession, as well as a Disciplinary Committee to enforce
compliance, set offenses, and determine penalties under the law.
Many of the plants traditionally used in Nigeria for the treatment of various ailments have
not been scientifically studied for their efficacy or potential side effects (Kokwaro, 1993).
Ethnobotanical knowledge is gradually disappearing, as older generations holding this
information are passing away, and there is insufficient transmission of knowledge to younger
Nigerians. Some plant species also face imminent loss due to population pressures and over-
exploitation in the absence of proper conservation policies. Improper preparation, handling, and
storage of crude plant extracts can lead to decomposition or transformation into ineffective or
even harmful products (Baker et al., 1995). This highlights the urgent need to evaluate and
establish scientific protocols for the use of Nigerian medicinal plants through chemical,
pharmacological, toxicological, and microbial studies. Nigerian scientists should therefore
investigate the antimicrobial properties of these plants, with particular focus on widely used
species such as Vernonia amygdalina. Available data indicate that medicinal plants in Nigeria
could also serve as a source of foreign exchange through export to international markets (Busia,
2005). The pharmaceutical potential of many tropical medicinal plants could be leveraged to
generate revenue and create employment opportunities across the country. This potential,
however, cannot be realized without rigorous biological studies that provide evidence of efficacy
and safety, which this study partially addresses. Vernonia amygdalina, in particular, is promising
due to its widespread availability in Nigeria and well-documented ethnobotanical profile, making
it a prime candidate for commercial development if its antibacterial properties against pathogens
like E. coli are scientifically validated.
1.3 Antibiotics.
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 With the discovery of microorganisms as the causative agents of infectious diseases,
many substances including those of plant origin became recognized as "antiseptics". These are
substances having the ability to inhibit the growth of microorganisms. Later on, this term was
changed to "antibiotics" from the term "antibiosis", which describes antagonism between
different species and even between members of the same species in nature. An antibiotic is a
biochemical drug, derived from one or more kinds of microorganisms. It may have the ability to
inhibit the growth of (bacteriostatic agent) or to kill (bactericidal agent) a number of other
microorganisms. This makes antibiotics be of immense value in the treatment of a number of
diseases that result from microbial infection (Amit and Shailendra, 2005). Antibiotics, also
known as antimicrobial drugs, are therefore drugs that fight infections caused by bacteria, either
by inhibiting the growth of or by destroying these microbes. Their therapeutic success relies on
their pharmacokinetic behavior and the contribution that the host's own defenses are able to make
towards clearance of the offending microorganism (Amit and Shailendra, 2005). Escherichia coli
is a Gram-negative bacterium that is part of the normal flora of the human intestine but certain
strains can cause serious infections including urinary tract infections, respiratory illness, and
bloodstream infections. The treatment of E. coli infections has become increasingly challenging
due to the emergence of multidrug-resistant strains, necessitating the search for alternative
therapeutic agents such as those found in Vernonia amygdalina.
1.3.1 Bacteriostatic agents
Bacteriostatic agents are substances that inhibit the growth of other microorganisms by
interfering with bacterial protein production, bacterial DNA production, and bacterial cellular
metabolism. Bacteriostatic antibiotics inhibit growth and reproduction of bacteria without killing
them, and killing is done by the bactericidal agents. Examples of synthetic bacteriostatic agents
include; sulphonamides like furosemide, trimethoprim, tetracycline, chloramphenicol, and
erythromycin and their derivatives among others (Sritharan and Sritharan, 2004).

Figure 1.2 : Chemical structures of Sulphonamides, Trimethoprim, Tetracycline,

Chloramphenicol, and Erythromycin

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 These agents are crucial in managing infections where the immune system is capable of
eliminating the inhibited bacteria. In the context of *E. coli*, bacteriostatic agents prevent the
proliferation of the bacteria, allowing the host's immune response to clear the infection.
However, resistance to these agents has been documented, highlighting the need for new sources
of bacteriostatic compounds from plants.
1.3.2 Bactericidal agents
These are substances that kill microorganisms and, preferably, nothing else. Bactericides are
disinfectants, antiseptics, or antibiotics. Members in this class include; benzylpenicillin,
cephalosporins like cefalexin, and amino-glycosides like gentamicin (Sritharan and Sritharan,
2004).

Figure 1.3: Chemical structures of Benzylpenicillin, Cefalexin, and Gentamicin

Effective chemotherapy in the use of antibiotics depends upon selective toxicity, defined
as the ability of the drug to inhibit microorganisms at concentrations tolerated by the host (Amit
and Shailendra, 2005). For E. coli infections, bactericidal agents are often preferred in severe
cases such as sepsis or meningitis. However, the overuse of these agents has led to the selection
of resistant strains. Plant extracts from Vernonia amygdalina may offer compounds with
bactericidal properties that operate through different mechanisms than conventional antibiotics,
potentially overcoming existing resistance patterns.
1.4 Active compounds in plant extracts
Plants produce biologically active secondary metabolites often with highly complex
chemical structures. These compounds are concerned with the survival of the plants and are
known to have antimicrobial activity (Waterman, 1992). The compounds vary from plant to plant
and are found to be in different concentrations in different parts of the same plant (Lawrence,
2000). In Vernonia amygdalina, these active compounds are primarily found in the leaves, which
are the most commonly used part in traditional medicine. The major classes of phytochemicals
identified in Vernonia amygdalina include flavonoids, alkaloids, saponins, tannins, and
terpenoids. Flavonoids are known for their antioxidant and antimicrobial properties, often acting

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by disrupting bacterial cell walls or inhibiting enzyme function. Alkaloids, such as vernodalin
and vernolide, have shown significant biological activity including antibacterial effects. Saponins
possess detergent-like properties that can disrupt microbial membranes. Tannins are known to
bind to proteins and inhibit microbial growth. Terpenoids, including sesquiterpene lactones, are
characteristic of the Vernonia genus and contribute to the plant's bitter taste and medicinal
properties. The synergistic effect of these compounds in crude extracts may provide a broader
spectrum of activity compared to isolated compounds, which is a significant advantage in
treating infections caused by resilient bacteria like *E. coli*. Understanding the specific
contribution of each class of compounds is essential for standardizing herbal preparations and
developing new drugs.
1.5 Statement of the Problem
Indigenous Nigerian communities have long incorporated the use of traditional
medicines, primarily derived from plants, to treat or alleviate the effects of common ailments.
Across Nigeria, numerous plants used in folk medicine have been identified, and the application
of their crude extracts has been documented (Kokwaro, 1993). A wide variety of medicinal plants
are employed to manage diseases such as diarrhoea and infections, which remain major public
health concerns in the country. Vernonia amygdalina is one such plant, widely used for
gastrointestinal issues that are often caused by bacterial pathogens like E. coli. Despite its
widespread use, limited information exists on the phytochemistry of the active ingredients in
these plants, particularly regarding their efficacy against E. coli isolates. The combination of
health-related challenges—including rapid population growth, the emergence of antimicrobial-
resistant pathogenic strains, frequent side effects from conventional drugs, the failure of modern
medicine to provide effective treatment, high costs of new drugs, and emerging diseases—has
intensified the need for research into natural products with antimicrobial activity. Establishing a
scientific basis for the use of medicinal plants through biological screening is therefore crucial.
Systematic pharmacological studies of the genus Vernonia have been conducted to some extent.
However, several species, including Vernonia amygdalina, require further investigation to
identify the active principles, determine their efficacy, and elucidate their mechanisms of action
against specific isolates like E. coli. Furthermore, little information is available on the
phytochemistry of Vernonia amygdalina leaves in relation to specific antibacterial activity
against E. coli within the Nigerian context. This study seeks to determine the phytochemical
constituents and evaluate the antibacterial activity of crude extracts from Vernonia amygdalina
against E. coli isolates. The absence of standardized dosages and scientific validation poses a
potential public health risk, as users may rely on ineffective preparations or suffer from toxicity.
By investigating the specific activity against E. coli, this study aims to bridge the gap between
traditional usage and modern scientific evidence in Nigeria.
1.6 Hypotheses

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 i. Extracts of Vernonia amygdalina contain compounds that exhibit antibacterial activity
against E. coli.
ii. These compounds are stable and can be extracted and detected through phytochemical
screening.
iii. The extracts remain biologically active once extracted from the plant material.
These hypotheses are grounded in the ethnobotanical evidence of Vernonia amygdalina usage
and the known presence of bioactive secondary metabolites in the plant. Testing these hypotheses
will provide concrete data on the plant's potential as an antimicrobial agent.
1.7 Objectives
1.7.1 General objective
The study was aimed at investigating the phytochemical constituents and antibacterial
components from the leaves of Vernonia amygdalina.
1.7.2 Specific objectives
The specific objectives of the study were:
i. To determine the phytochemical constituents present in Vernonia amygdalina leaf extracts.
ii. To determine antibacterial activity of leaf crude extracts of Vernonia amygdalina against E.
coli isolate by in vitro assays.
iii. To determine the Minimum Inhibitory Concentration (MIC) of the active extracts against E.
coli.
iv. To determine the Minimum Bactericidal Concentration (MBC) of the active extracts against
E. coli.
These objectives are designed to systematically evaluate the plant's potential, starting
from chemical identification to quantitative assessment of antibacterial potency. The inclusion of
MIC and MBC determination will provide critical information on the concentration required for
therapeutic effect.
1.8 Justification and Significance of the Study
More than 80% of the population in developing countries, including Nigeria, depends directly on
herbal drugs and traditional medicines for primary healthcare (WHO, 2008). Many of the drugs
in use today were originally discovered through their ethnobotanical roots (Kareru et al., 2007).
Additionally, pathogen resistance to existing drugs, increased side effects, the lack of curative
treatment for several chronic diseases, the high cost of drugs, and the emergence of new diseases
remain major challenges in modern healthcare (Humber, 2002). This underscores the need for
research on medicinal plants to identify potent pharmacological agents, as natural products and

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their derivatives have historically been an invaluable source of therapeutic compounds (Koehn
and Carter, 2005). Bacterial infections remain a significant public health problem in Nigeria,
particularly in rural areas where traditional medicines are commonly used as part of primary
healthcare. Vernonia amygdalina, widely employed by Nigerian communities for traditional
therapy of infections, has not been fully investigated phytochemically for its antimicrobial
activity against specific isolates such as E. coli. E. coli is a leading cause of diarrheal diseases
and urinary tract infections, which contribute significantly to morbidity and mortality, especially
among children and immunocompromised individuals. The findings of this study are expected to
provide detailed information on the phytochemistry of the main bioactive chemical constituents
in the leaves of Vernonia amygdalina, which could be evaluated for the treatment of bacterial
infections and serve as templates for the development of new drugs. Furthermore, the study will
scientifically validate the traditional use of the plant against bacterial pathogens. If successful,
this research could lead to the development of affordable, locally sourced antibacterial agents,
reducing reliance on expensive imported antibiotics and mitigating the impact of antibiotic
resistance. In addition, it contributes to the conservation of indigenous knowledge by
documenting the scientific basis of traditional medicinal practices in Nigeria.