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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