ABSTRACT
The knowledge of traditional medicine, medicinal plants and the study of their scientific chemical principles may lead to the discovery of newer and cheaper drugs. Azadirachta indica is a medicinal plant of Indian origin, commonly known as ‘Neem’ or ‘Dogoyaro’. It belongs to the family Melaceae and subfamily Melioidea. This plant has diverse and potential medicinal uses in traditional medicine for treating malaria, stomach and intestinal ulcer, pain, fever, skin diseases, diabetes, urinary tract disorder, leprosy, bloody nose and other diseases. This study was undertaken to investigate the antibacterial activity of Azadirachta indica on common pathogenic organism; Staphylococcus aureus, E. coli and Pseudomonas aeruginosa. Agar diffusion assay was employed to determine the antibacterial activity of the ethanol and aqueous extracts of the neem leaf at five concentration levels 200 mg/l, 100 mg/l, 50 mg/l, 25 mg/l and 12.5 mg/l by measuring the diameter of zones of inhibition around the tested organisms. The results obtained showed that ethanol extract was more effective in inhibiting the growth of S. aureus than aqueous extract as inhibition was observed from 25 mg/l to 200 mg/ml concentrations. While aqueous extract was found to inhibit S. aureus from 100 mg/l to 200 mg/ml. E. coli strain responded in similar way to both ethanol and aqueous leaf extracts, as both extracts were found to inhibit growth at 200 mg/ml and 100 mg/ml concentrations. Pseudomonas strain also responded in similar way to both ethanol and aqueous leaf extracts. Both ethanol and aqueous leaf extracts were found to inhibit growth at 200 mg/ml and 25 mg/ml concentrations. This study has shown that ethanol and aqueous extracts of Dogoyaro leaves possess high antibacterial activities against pathogenic bacteria (S. aureus, E. coli and Pseudomonas). The more effective concentration was between 100 mg/l to 200 mg/ml extracts and zone of inhibition was increased with the increase in concentration of extracts.
TABLE OF CONTENTS
Title page i
Certification ii
Dedication iii
Acknowledgements iv
Table of contents v
List of tables viii
Abstract i
CHAPTER ONE
1.1 Introduction
1 - 4
1.5 Aim of the study 5
1.6 Objectives of the study
5
CHAPTER TWO
2.0 Literature Review 6
2.1 Historical Use of Plants as Antimicrobials
6 - 7
2.1.1 Major
groups of antimicrobial compounds from plants 7 -
8
2.1.1.1 Phenolics
and Polyphenols, simple phenols and phenolic acids 8
2.1.1.2 Quinones
8
- 9
2.1.1.3 Flavones,
flavonoids, and flavonols
9 - 10
2.1.1.4 Tannins
10
- 11
2.1.1.5 Lectins
and Polypeptides
11
2.2 Dogoyaro
Plant (Neem) leaves 12
2.2.1 Neem
as a medicinal plant
12 - 13
2.3 Neem
leaves
14
2.3.1 Antiviral
activity of Neem leaves
14 - 15
2.3.2 Antifungal
properties of Neem leaves
15
2.3.3 Antibacterial
properties of Neem leaves
16
CHAPTER THREE
3.0 Materials and Methods
17
3.1 Materials
17
3.2 Methods
17
3.2.1 Sample collection
17
3.2.2 Sample Preparation
17
3.2.3 Preparation
of the Extracts for Antibacterial Assay
17 - 18
3.3 Isolation and
Characterization of Microbes
18
3.3.1 Gram
staining
18
3.3.2. Cultural Characteristics
18
3.3.3. Morphological Characteristics
19
3.4
Biochemical characteristics
19
3.4.1
Motility test
19
3.4.2
Catalase test
19
3.4.3
Oxidase test
20
3.4.4
Indole production
20
3.4.5
Methyl red reaction
20
3.4.6
Voges Proskaur (VP) reaction
20
3.4.7
Citrate utilization
21
3.5
Antibacterial Activity Testing
21
3.5.1 Preparation of stock solution
21
3.5.2 Preparation of Muller-Hinton agar
22
CHAPTER
FOUR
4.0 Results
23 - 31
CHAPTER
FIVE
5.0 Discussion, Conclusion and
Recommendations
32
5.1 Discussion of results
32 - 33
5.2 Conclusion
33
5.3 Recommendations
33 - 34
REFERENCES
35 - 40
LIST OF TABLES
Table
4.1 Antibacterial effect of ethanoic
and aqueous extracts of Dogoyaro leaves on S.
aureus
Table
4.2 Antibacterial effect of ethanoic
and aqueous extracts of Dogoyaro leaves on E.
coli
Table
4.3 Antibacterial effect of ethanoic and aqueous extracts of Dogoyaro leaves on
pseudomonas species
Table
4.4 Zone of inhibition (mm) of varying concentrations of extracts (mg/ml) on S. aureus
Table
4.5 Zone of inhibition (mm) of varying concentrations of extracts (mg/ml) on E. coli
Table
4.6 Zone of inhibition (mm) of varying concentrations of extracts (mg/ml) on pseudomonas
CHAPTER 1
1.1 INTRODUCTION
Medicinal
plants have a long history of use and their use is widespread in both
developing and developed countries. According to reports of the World Health
Organization (WHO), 80% of the world’s population relies mainly on traditional
therapies which involve the use of plant extracts or their active substances
(WHO, 2003). Microorganisms have developed resistance against many antibiotics
due to the indiscriminate use of antimicrobial drugs (Ahmad et al., 2008).
Furthermore,
antibiotics are sometimes associated with side effects (Cunha, 2001), whereas
there are some advantages of using antimicrobial compounds of medicinal plants,
such as fewer side effects, better patient tolerance, relatively less
expensive, acceptance due to long history of use and being renewable in nature
(Vermani and Garg, 2002). It is known that more than 400, 000 species of
tropical flowering plants have medicinal properties and this has made
traditional medicine cheaper than modern medicine (Odugbemi, 2006). Some plant
decoctions are of great value in the treatment of diarrhoea or gastrointestinal
disorder, urinary tract infections, skin infections, infertility, wound and
cutaneous abscesses (Ergene et al.,
2006).
The
tree, Azadirachta indica of the
family Maliaceae, popularly known as neem tree or dogoyaro (Hausa) is an
evergreen tree, native to the Southeast Asia and found in most tropical
countries. It has been in use since ancient times, to treat a number of human
ailments and also as household pesticide (Chattopadhyay et al., 2003; Chattopadhyay and Bandyopadhyay, 2005). Extracts from
the bark, leaves, fruits and roots have been used to control leprosy,
intestinal helminthosis and respiratory disorders (Ketkar and Ketkar, 2005).
Every part of the neem tree has been used as traditional medicine for house-hold
remedy against various human ailments from antiquity. The tree is still
regarded as ‘Village dispensary’. It is a plant known over 2000 years as one of
the most versatile medicinal plants having a wide spectrum of activity (Larkshmanan
and Subramanian, 2006).
1.1.1 PATHOGENIC ORGANISMS
Pathogenic
organisms can contaminate fresh fruits and vegetable through a wide range of
mechanisms. Contamination can occur in the fields or orchards while growing,
during harvesting, postharvest handling, processing or distribution. The
contamination may come from people/workers, animals, faeces, sewage, water, or
soil (Beuchat, 2006). Of particular concern are: (a) Listeria due to the
ubiquitous nature of the bacteria in the environment, and the severe effects of
its growth on a fetus,( b) pathogenic E. coli with the severity of illness
associated with infections, and (c) Salmonella, which is the second greatest cause
of bacterial-induced diarrhoea in the United States (Beuchat, 2006).
Enterobacteriaceae,
the enteric bacteria are facultative anaerobic Gram-negative rods that live in
the intestinal tract of animals in health and diseases. The Enterobacteriaceae
are among the most important bacteria medically. A number of genera within the
family are human and animal intestinal pathogens (for example, Salmonella, Shigella and Yersinia).
Several others are normal colonist of the human gastrointestinal tract (for
example, Escherichia coli, Enterobacter,
Klebsiella), but these bacteria, as well, may occasionally be associated
with diseases in humans and animals (Todar, 2008).
1.1.1.1 Escherichia
coli
Studies
of Escherichia coli have been ongoing
since 1885 when it was isolated from children’s faeces. Since then, the
scientific attention given to E. coli
has probably made it the best understood free living organism (Adams and Moss,
2005). This organism is of the Enterobacteriaceae family. E. coli is almost universally found within the gut of humans and
warm blooded animals (Friedman et al.,
2002). It is a predominant facultative anaerobe, but is only a small component
of the total microflora. Generally, most strains of this bacterium are harmless
to the carrier, but can be an opportunistic pathogen and cause human disease. E. coli is a gram negative,
facultative anaerobe. It is capable of growing in a minimal water activity
range of 93-95, a pH range as low as 3.6 and a maximum pH of 9.5. E. coli can grow in environments of up
to 8% salt by weight, with a minimum growth temperature ranging from 33-37oF
(0.6-3) and a maximum temperature of 113oF (45oC) (Corlett,
2008).
The
pathogenic strains of E. coli are
divided according to clinical symptoms and mechanisms of pathogenesis. These
groups classically have been enterpathogenic E. coli (EPEC), enterotoxigenic E.
coli (ETEC), enteroinvasive E. coli
(EIEC), and enterohemorrhagic E. coli
(EHEC) (Balows, 2001). Recently, new groups have been added: the
enteroaggregative (EaggEC) (Jay, 2006), and diffusely adherent (DAEC) groups (Forsythe,
2002). Strain O157:H7 of E. coli is an EHEC (Forsythe, 2002), but one of the several
organisms that produces Shiga-like toxins (STEC), also called verotoxins. Other
STEC serogroups include; O26, O111, O103, O128, O91, O113, O2, O9, O145, OX3,
O1, O8, O22, 121, O146, O5, O18, O117, and O118. These groups have been
isolated from bovine stools, sheep at slaughter in Germany, goats at slaughter
in the Czech Republic, pigs, cats and dogs, but not from poultry. STEC
serogroups have also been isolated from ground beef, raw milk in Austria, and
from milk filters in Italy (Kaper et al.,
2008). E. coli O157:H7 is the most recognized serotype of E. coli in the United
States due to outbreaks in the last 20 years and is the only serotype that can
be easily isolated and identified in the clinical microbiology laboratory
(Balows, 2001). E. coli O157:H7 has been shown to be a food borne pathogen and
has been associated with ground beef, venison jerky, raw vegetables
(particularly lettuce and sprouts), apple juice or cider, and in water
(swimming and drinking water). Studies have shown ground beef to be
contaminated with E. coli O157:H7 with rates as high as 23 to 25% (Samadpour et al., 2004). Vegetables and fruits can
also become contaminated with E. coli O157:H7 while growing in the field or
orchard. Contamination can also occur during harvesting, post harvest handling,
processing and distribution (Beuchat, 2006).
1.1.1.2 Staphylococcus
aureus
Since
its discovery by Dr Alexander Ogston in 1880, Staphylococcus aureus has been recognized as a versatile micro-organism
worldwide (Friedman et al., 2002). S. aureus may colonize the human body as
a part of the normal flora. Approximately 30 % of healthy people are inhabited
by S. aureus, mostly in the anterior
nares. S. aureus is also a leading
cause of hospital-associated (HA) and community-associated(CA) bacterial
infections in humans, associating with numerous mild skin and soft tissue
infections, as well as life-threatening pneumonia, bacteraemia, osteomyelitis, endocarditis,
sepsis and toxic shock syndrome. The increasing prevalence of
meticillin-resistant S. aureus (MRSA)
and its ability to resist multiple drugs has posed a serious challenge for
infection control. HA-MRSA often infects individuals with health care risk
factors, such as surgery or residence in a long-term care facility. By
contrast, many CA-MRSA and meticillin-susceptible S. aureus (MSSA) often infect healthy persons who do not have such
risk factors (Samadpour et al., 2004).
1.2 AIM OF
THE STUDY
The
aim of the present study is to determine the antibacterial activity of Aqueous
and Ethanol extracts of dogoyaro leaf on pathogenic organisms.
1.3 OBJECTIVES OF THE STUDY
Specific
Objectives include;
1. To
evaluate the antimicrobial activities of aqueous and ethanol extracts of
dogoyaro leaf on E. coli, pseudomonas species and Staphylococcus
species
2. To
determine the minimum inhibitory Concentration (MIC) of the aqueous and ethanol
extracts of dogoyaro leaf.
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