ABSTRACT
This work was carried out to determine the physicochemical analysis of honey, phytochemical analysis of cinnamon bark powder and to examine their antibacterial activity against isolated bacteria concerned with respiratory tract infections (RTI), which are; Staphylococcusaureus, Escherichiacoli and Pseudomonasaeruginosa, isolated from sputum and throat swab specimens. The agar well diffusion method was employed for both honey and the two cinnamon bark powder extracts using nutrient agar and the inhibition zones around the six wells containing different concentrations of the samples (ranging from 3.125%-100%) were measured. Significant antibacterial activity of honey was observed against Staphylococcus aureus with a least zone of inhibition of 20mm at 6.25% concentration and a maximum inhibition zone of 45mm at 100% concentration. Also against Pseudomonas aeruginosa with a least inhibition zone of 15mm at 12.5% concentration and maximum inhibition zone of 24mm at 100% concentration and lastly against Escherichia coli with a least inhibition zone of 15mm at 3.125% concentration and maximum inhibition zone of 35mm at 100% concentration. The Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the honey sample was further determined, giving values of 12.5% concentration (MIC) and 25% (MBC) for Staphylococcus aureus, 25% (MIC) and 25% (MBC) for Escherichia coli and 25% (MIC) and 50% (MBC) for Pseudomonas aeruginosa. Significant antibacterial activity of ethanol extract of cinnamon bark powder was observed against Staphylococcusaureus with least inhibition zone of 10mm at 3.125% concentration and maximum inhibition zone of 22mm at 100% concentration, against Escherichiacoli with least inhibition zone of 13mm at 12.5% concentration and maximum inhibition zone of 20mm at 100% concentration but Pseudomonasaeruginosa was completely resistant. The test organisms were all resistant to aqueous extraction except that Staphylococcusaureus was susceptible by 8mm at 100% concentration only. Synergistic test using the double disk diffusion method and agar well diffusion technique was carried out. There was no synergistic effect of honey and cinnamon bark powder extracts in this study, but antagonistic effect. More effective research work is recommended for the synergy, to checkmate reviews encouraging the intake of honey and cinnamon powder together against respiratory problems.
TABLE OF CONTENTS
TITLE
PAGE
i
CERTIFICATION
PAGE
ii
DEDICATION
iii
ACKNOWLEDGEMENTS
iv
TABLE
OF CONTENTS
v-ix
LIST
OF TABLES
x
ABSTRACT
xi
CHAPTER ONE
1.0 Introduction
1
1.1 Background of the Study
2
1.2 Statement of Research Problem
3
1.3 Aim of the Study 3
1.4 Specific Objectives
3
CHAPTER TWO
2.0 Literature Review
5
2.1 Honey
5
2.1.1 Ancient History of Honey
5
2.1.2 Properties of Honey
6
2.1.2.1 Granulation Tendency
6
2.1.2.2 Tendency to Absorb Moisture
7
2.1.2.3 Viscosity
7
2.1.2.4 Food Value 7
2.1.3 Antimicrobial Properties
8
2.1.3.1 Osmolarity
8
2.1.3.2 Low Water Activity and pH
8
2.1.4 Composition of Honey
9
2.1.4.1 Water Content
9
2.1.4.2 Sugars
9
2.1.4.3 Organic Acids
10
2.1.4.4 Proteins, Enzymes and Amino Acids
10
2.1.4.5 Minerals
11
2.1.4.6 Vitamins
11
2.1.4.7 Essential Oils
11
2.1.5 Nutritional Content of Honey 11
2.1.6 Medicinal Uses of Honey
13
2.1.6.1 Other Uses of Honey 14
2.2 Cinnamon
14
2.2.1 History and Origin of Cinnamon 14
2.2.2 Varieties of Cinnamon
15
2.2.3 Plant Hierarchy 16
2.2.4 Macroscopic Characteristics
16
2.2.5 Constituents of Cinnamon 17
2.2.6 Nutritional Contents and Antibacterial
Properties of Cinnamon 19
2.2.7 Health Benefits and Uses of
Cinnamon 19
2.3 The Human Respiratory Tract
21
2.4 Description of the Organisms 23
2.4.1 Escherichia
coli23
2.4.2 Pseudomonas aeruginosa24
2.4.3 Staphylococcus aureus25
2.5 Phytochemicals
27
2.5.1 Classification of Phytochemicals
27
CHAPTER THREE
3.0 Materials and Methods 28
3.1 Study Area
28
3.2 Collection of Materials 28
3.2.1 Preliminary Test for the Confirmation of
the Purity of Honey 28
3.2.1.1 Dissolution Test 28
3.2.1.2 Crystallization Test
28
3.2.1.3 Flame Test 28
3.3 Processing Of Herbal Sample
29
3.4 Physicochemical and Phytochemical
Screening of Honey and Cinnamon Bark
Powder Extracts
29
3.4.1 Physico-Chemical Screening Of Honey 29
3.4.1.1 Moisture Content
29
3.4.1.2 Ash Content 30
3.4.1.3 Water Activity
30
3.4.1.4 Color 30
3.4.1.5 pH
30
3.4.2 Phytochemical Screening of Cinnamon Bark
Powder Extract 30
3.4.2.1 Test for Flavonoids
30
3.4.2.2 Test for Alkaloids
30
3.4.2.3 Test for Tannins
30
3.4.2.4 Test for Phenols
30
3.4.2.5 Test for steroids
31
3.5 Culture Media Preparation
31
3.6 Isolation of Organisms
31
3.7 Identification and Confirmation of
Isolated Bacteria from Respiratory
Tract Infections
31
3.7.1 Biochemical Tests
31
3.7.1.1 Catalase Test
31
3.7.1.2 Citrate Utilization Test
32
3.7.1.3 Coagulase Test
32
3.7.1.4 Methyl Red (MR) Test
32
3.7.1.5 Oxidase (Cytochrome Oxidase) Test 32
3.7.1.6 Indole Test
33
3.7.1.7 Voges-Proskauer (VP) Test 33
3.8 Isolation and Purification of
Isolates
33
3.9 Antibacterial Activity Testing of
Honey 33
3.9.1 Preparation of Dilution
33
3.9.2 Agar Well Diffusion Technique 34
3.9.3 Determination of Minimum Inhibitory
Concentration (MIC) and Minimum
Bactericidal Concentration (MBC)
of Honey.
34
3.91 Antibacterial Activity Testing of
Cinnamon
35
3.91.1 Processing of Herbal Sample
35
3.91.2 Preparation of Dilution
35
3.91.3 Agar Well Diffusion Technique
35
3.92 Synergistic Testing of Cinnamon Bark
Powder and Honey 36
3.92.1 Agar Well Diffusion Technique
36
3.92.3 Wet Disk Diffusion or Double Disk
Diffusion Technique 36
CHAPTER FOUR
RESULTS
38-50
CHAPTER FIVE
5.0
DISCUSSION, CONCLUSION AND RECOMMENDATION 51
5.1
DISUSSION
51
5.2
CONCLUSION 53
5.3
RECOMMENDATION
54
REFERENCES 55-60
APPENDIX
LIST OF TABLES
Table
2.1 Nutritional Content of Honey
Table
2.2 Constituents of Cinnamon
Table
1 Physico-chemical Analysis of the
Analyzed Honey Sample
Table
2 Phytochemical Analysis of Ethanol
and Aqueous Extracts of Cinnamon Bark Powder
Table
3 Colonial Morphology of the
Isolates
Table
4 Biochemical Analysis of the
Isolates
Table
5 Zones of Inhibition Produced by
the Honey against the Isolates
Table
6 MIC and MBC of Honey against the
Isolates
Table
7 Zones of Inhibition Produced by
Ethanol Extract of Cinnamon Bark Powder against the Isolates
Table
8 Zones of Inhibition Produced by
Aqueous Extract of Cinnamon Bark Powder against the Isolates
Table
9 Zones of Inhibition Produced by
Synergistic Test Using Agar well Diffusion Technique against the Isolates
Table
10 Zones of Inhibition Produced by
Synergistic Test Using Wet Disk Diffusion Technique against the
Isolates
CHAPTERONE
1.0
INTRODUCTION
Honey
is a sweet food made by bees using nectar from flowers. The variety produced by
honey bees (the genus Apis) is the one most commonly referred to and is the
type of honey collected by beekeepers and consumed by humans. The various species
of Apis include; Apisandreniformis, Apisflorea, Apisdorsata,Apiscerana,
Apiskoschevnikovi, Apismellifera,Apisnigrocinta (Silveira etal., 2002).
Honey
gets its sweetness from the monosaccharides: fructose and glucose, and has
about the same relative sweetness as granulated sugar (National honey Board,
2012).It has attractive chemical properties for baking and a distinctive flavor
that leads some people to prefer it over sugar and other sweeteners. Most
microorganisms do not grow in honey because of its low water activity of 0.6
(Bata, 2009).
Hydrogen
peroxide (H202), methylglyoxal (MGO) bee defensin, pH and
Osmotic effect of honey are known to be responsible for the antimicrobial
effects (Mandal, 2011).
Cinnamon
comes from the bark of trees native to china, India and Southeast Asia. It is a
popular cooking spice in many cultures for centuries and also has a long
history of use as a traditional medicine (National institutes of Healthetal.,2012).
In
ancient Indian philosophy of medicine, cinnamon is used as a remedy fordiabetes,indigestion
and colds. In traditional Chinese medicine, Cassia cinnamon is used for colds,
flatulence, nausea, diarrhea and painful menstrual periods. It’s also believed
to improve energy, vitality, and circulation. It is particularly useful for
people who tend to feel hot in their upper body but have cold feet (paradise
India, 2014). Cinnamon bark is used to make powders, capsules, teas and liquid
extracts (National institutes of health etal.,
2012)
Based
on the place grown there are hundreds of varieties of cinnamon, which can be
broadly classified into two varieties: Ceylon cinnamon andcassia cinnamon.
Eugenol and cinnamaldehyde are the two major chemical components in cinnamon
that are responsible for its health benefits (Agabalogun etal., 2013).
1.1 BACKGROUND OF THE
STUDY
The
primary function of the respiratory tract is to supply the body with oxygen and
remove carbon dioxide. During inspiration, outside air (Oxygen) is inspired
into the body which travels from either the nose or mouth down to the lungs.
During exhalation,carbon dioxide is expelled into the outside air (Tuetal., 2013). Anatomically, the
respiratory tract can be separated into the upper respiratory tract comprising
the nasal cavity, pharynx and larynx, and the lower respiratory tract
comprising the trachea, bronchi and lungs. The respiratory tract is also connected
to the ears via the Eustachian tubes and thus conditions that affect the
respiratory tract can also produce symptoms in the ear (Addison, 2011).The
upper respiratory tract is generally considered to be the air way above the
glottis or vocal cords (Sabyasachi, 2008).
Microbes
that infect the respiratory tract include: Bacteria; Streptococcuspneumoniae, Staphylococcusaureus,Corynebacteriumdiphtheria, Klebsiellapneumoniae, Pseudomonasaeruginosa, Haemophilusinfluenza, Legionellapneumophila, Mycoplasmapneumoniae, Chlamydia spp., Streptococcuspyogenes, Mycobacteriumtuberculosis,
Fungi: Aspergillusniger, Viruses;
Rhinoviruses, Coronaviruses, Coxsackieviruses, Adenoviruses, Influenza virus,
Parainfluenza virus (Udda, 2007).
Infectious
diseases of the respiratory tract include; pharyngitis, common cold, influenza,
otitis media, otitis externa, acute sinusitis, laryngitis bronchitis, pneumonia
(which is dividedinto acute community-acquired, acute hospital-acquired and
chronic in AIDS), pulmonary tuberculosis, Empyema, croup, epiglottis,
bronchitis (Udda, 2007).
1.2 STATEMENT OF RESEARCH
PROBLEM
Respiratory
tract infection (RTI) occurs commonly in both children and adults and is a
major cause of mild morbidity. It has a high cost to society, being responsible
for absenteeism from school and work and unnecessary medical care and is
occasionally associated with serious sequelae. It also has a direct influence
on changing environment like industrialization and air pollution. Solution has
been found for many infectious illness, but common cold and other RTI has been and
is still a problem to man. The use of honey and cinnamon by mankind as food and
medicine has a long history down to the primitive men. However, their knowledge
of the efficacy of these natural products is not vast, most especially that of
cinnamon bark powder.
1.3 AIM OF THE STUDY
To
determine active components in honey and cinnamon bark powder and evaluate the
antibacterial activities of honey and cinnamon on some isolated bacteria
species identified as responsible for respiratory tract infections.
1.4 SPECIFIC OBJECTIVES
Ø To
isolate and identify some bacteria associated with respiratory tract
infections.
Ø To
determine and know the active components in honey and cinnamon bark powder
responsible for their antibacterial effects by physicochemical and
phytochemical analysis.
Ø To
evaluate the antibacterial activities of honey and cinnamon at varied
concentrations against the bacteria isolates from the respiratory tract, both
synergistically and separately, by agar well diffusion, disc diffusion method
and tube dilution assays.
Ø To
determine the minimum inhibitory concentration andminimumbactericidal concentration
of honey against the bacteria isolates.
Ø To
determine the susceptibilities of the respiratory tract organisms to honey and
cinnamon, both as synergy and separately.
Ø To
know for effective prescribing at individual patient level.
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