Table of Content
Title ………………………………………………………………i
Certification ……………………………………………………………………………ii
Dedication ……………………………………………………….iii
Acknowledgement …………………………………………………iv
Table of content ………………………………………………….vi
List of tables ……………………………………………………….ix
List of figures …………………………………………….…………x
Appendix …………………………………………………………..xi
Abstract ………..………………………………………………….xii
Chapter one
Introduction …………………………………………………1
1.1 Aims and Objectives ……………………………………….….6
8
Chapter two
2.0 Literature review ……………………………………………….7
2.1 Plants extract review…………….…………………………….7
2.1.1 Brief history of medicinal plant …………………………..….9
2.1.2 Phytochemical components with antimicrobial activity ….…16
2.1.3 Characteristics of phytomedicine ……………………………18
2.1.4 Why the Demand for Phytomedicine ……………………….19
2.1.5 Challenges and Development in the use of phytomedicine……20
2.1.6 Possible Solutions ………………………………………………22
2.2 A review of Hibiscus sabdariffa ………………………..……..24
2.2.1 Scientific classification ……………………………….….……24
2.2.2 Description ……………………………………….…..……….25
2.2.3 Origin and distribution ……………………………………….26
2.2.4 Various names of H. sabdariffa ……………………………..28
2.2.5 Climate …………….………………………………………..30
2.2.6 Cultivation and harvesting …………………….………….. 30
2.2.7 Pests and diseases ………………………………………… 32
2.2.8 General uses OF H. sabdariffa ……………………………….. 33.
9
2.2.9 Phytomedicine ………………………………………….….38
2.2.3.1 Various research on H. Sabdariffa ……………..….…….38
2.3 Description of Salmonella typhi………………………………..40
2.3.1 Scientific classification OF Salmonella typhi ………….…41
2.3.2 History of typhoid fever ………….……………………….41
2.3.3 Causative Agent ………………………….……………….43
2.3.4 Mode of transmission ……………………………………..44
2.3.5 Signs and symptoms………………………………………46
2.3.6 Epidemiology of typhoid fever ………………………….48
2.3.7 Pathogenesis of Typhoid Fever …………………………..49
2.3.8 Prevention and control ……………………………………50
2.3.9 Diagnosis ………………………..………………………..51
2.34.1Risk factors ……………………………………………….52
2.3.4.2Treatment …………………………………………………52
2.3.4.3Other pathogens …………………………………….…….53
10
Chapter three
Materials………………………………………………….…..57
3.1.1 Hard Equipments …………………..……………………57
3.1.2 Soft wares …………………………….…………………57
3.1.3 Media used……………………………….………………57
3.2. Collection and confirmatory of test organisms……..….58
3.2.1 Gram staining……………………………………………58
3.2.2 Biochemical Tests ………………………………..…….59
3.3 Collection and identification of plant material….…..….65
3.4 Extraction of plant material……………………………..65
3.5 Preparation of the Mcfarland’s Standard………….……66
3.6 Preparation of the cell suspension (inocular)……….…….67
3.7 Serial dilutions of the extract ……………………………67
3.8 Antibiotics Susceptibility testing ………………………..68
3.9 Susceptibility testing using H. sabdariffa…………..……..70.
11
Chapter four
4.1
Results…………………………………………………………..71
Chapter five
5.1 Discussions of results……………………………………….77
5.2 Conclusion……..……………………………………………81
5.3
Recommendation……………………………………………………………….…..82
References…………………………………………….…………….83
12
List of tables
Table 1: Some African medicinal plants……………………..…….15
Table 2: Constituents of H. sabdariffa…………………………………37.
Table 3: Results of identification test…………………………..….71
Table 4: Inhibitions zone diameters (IZDs) of antibiotics…………72
Table 5: IZDs of H. sabdariffa on the test organisms..………………75
13
List of figures
Fig 1: Hibiscus sabdariffa calyces………………………………..26
Fig 2: serial dilution of plant extract………………………………68
Fig 3: Agar plate showing the effect of antibiotics on S. typhi…….73
Fig4: Agar plates showing effect of antibiotics on other test
organisms…………………………………………………………………………….73
Fig 5: Agar plate showing the effect of H. sabdariffa extract on
S.typhi……………………………………………………………….75
Fig 6: Agar plate showing effect of H. sabdariffa extract on S.
aureus………………………………………………………………………..75
Fig 7: Agar plate showing the effect of H. sabdariffa on E. coli……76
Fig 8: Agar plate showing Effect of H. sabdariffa on klebsiella spp.76
14
APPENDIX
Appendix 1: Media preparation…………………………………95
Appendix 2: Reagents used……………………………………..97
Appendix 3: interpretative chart for antibiotic for testing………98
15
ABSTRACT
The susceptibilities of Salmonella typhi and other pathogens to
antibiotics and hot aqueous extract of Hibiscus sabdariffa were
investigated using agar diffusion and agar well diffusion methods
respectively. Salmonella typhi was sensitive to ampicillin, cetriaxone,
ciprofloxacin, gentamycin of ofloxacin and perfloxacin.
Nitrofurantoin, ampicillin, clarithomycin and augumentin are
resistant. Escherichia coli, Klebsiella spp, and Staphylococcus aureus
were sensitive to 50%, 70% and 60% of the antibiotics respectively.
Pseudomonas aeruginosa was resistant to all antibiotics. Hibiscus
sabdariffa extract (0.6g in 6ml of sterile distilled water) was active
against S. typhi at concentrations of 100mg/ml, 50mg/ml and 25mg/ml
(inhibitions zone diameter IZDs = 23mm, 20mm and 16mm
respectively). Staphylococcus aureus was susceptible to 100mg/ml,
50mg/ml, 25mg/ml and 12.5mg/ml of the extract with IZDs of 29mm,
18mm, 17mm and 14mm respectively. Klebsiella spp was susceptible
to concentrations of 25mg/ml and 12.5mg/ml of the extract with IZDs
of 15mm and 10mm respectively. Escherichia coli and Pseudomonas
aeruginosa were resistant to all the concentrations of H. sabdariffa
extract. It is therefore imperative to note that the use of medicinal
plants is recommended to the Government and Industry.
16
CHAPTER ONE
INTRODUCTION
Long before mankind discovered the existence of microbes, the idea
that plants have some healing potentials, i.e. that they contain what we
will currently characterize as antimicrobial principle was well
accepted (Doughari, mahmood & Tyoyina, 2011). In whatever
manner early man gained his knowledge of the curative powers of
plants, one must assume that the was able thereafter to recognize the
plant, since the detailed flora available today, were not in existence
then (Sofowora, 2008). The use of higher plants and their extracts to
treat infectious diseases is an age old practice in traditional African
medicine (Onyeagba, ugbogu, Okeke & Iroakasi, 2004). Traditional
medicine practice has been known for centuries in many parts of the
world (Sofowora, 1984). It is however observed that these practices
vary from one country to another (Onyeagba, ugbogu, Okeke &
Iroakasi 2004). Nature has been a source of medicinal agents for
thousands of years. The use of herbs is the most ancient approach to
healing known (Apata, 1979).World Health Organization (WHO) in
17
1978 defined traditional medicine as the sum total of knowledge or
practices whether explicable or inexplicable used in diagnosing and
preventing a physical, mental or social disease which may rely
exclusively on past experience or observation handed down from
generation to generation, verbally or in writings. Since antiquity, man
has used plants to treat common infectious diseases and some of these
traditional medicines are still included as part of habitual treatment of
various maladies (Doughari et al). Numerous plants and herbs are
used all over Nigeria as phytomedicine by traditional medicine
practioners. Plant extracts are given singly or as concoction for
various ailments. The medicine could be either in the form of
powders, liquids, liniments and inclusion accoding to Apata. More
than 70% of people living in Nigeria depend on these various forms of
concoctions and herbal decocotions for the treatment of some diseases
(Kimbi-Beyioku, 1996). Many investigators have demonstated the
antimicrobial activity of the constituents of some higher plants
(Akobundu & Agykara, 1987; Rocio and Rion, 1982; Almagboul et al
1988; Misra et al, 1992; Hablemariam et al; 1993) and quite a number
18
of chemical compounds of plant origin have been shown to possess
antimicrobial activity (Corthout, Piefers & Cleays, 1992). One of
such plants is Hibiscus sabdariffa.
Hibiscus sabdariffa (called Roselle in English or Zobo in the northern
part of Nigeria) which belongs to mallow family (malvaceae) is native
to West Africa. Hibiscus sadariffa is cultivated in loamy, well drained
soil mainly in tropical climates and requires rainfall averaging about
10 inches (15 cm) each month throughout the growth season. Hibiscus
sabdrriffa is of several use, it is considered to have anti-hypertensive
properties. In some places the plant has been used in folk medicine as
diuretic, mild laxative and treatment for cardiac and nerve diseases
and cancer to mention but a few.
However, the plant (Hibiscus sabdariffa) is rich in anthocyanin. The
dried calyces contained flavonoids gossypetin, hibiscentine and
sabdaretine. The major flavonoid formerly reported as hibiscin, has
been identified as daphniphylline. Small amounts of myrillin
(delphinidin 3- monoglucoside), chrysanthenin (cyanide 3-
monoglucoside) and delphinidin are also present (Mohammed,
19
Fernandez, Pineda & Aguilar, 2007). All these gave it the qualities to
be regarded as a chemotherapeutic agent against some
microorganisms especially some pathogens of man.
Salmonella typhi is an enteric bacterium responsible for causing
typhoid fever which has affected mankind since human population
became large enough to contaminate the supply of its water. It is a
food borne disease contracted by ingestion of bacteria in contaminated
food or water (jerry, 2007). The sources of infection could be through
infected food, poor kitchen hygiene, and excretions from either sick
people or infected but apparently clinically healthy people and
animals, polluted surface water standing water and so on. The signs
and symptoms of the disease has 4 phases, first week involves the
slow rise in temperature, headache, cough, malaise and abdominal
pain. In the second week of the infection, high fever in plateau around
40oC (104o
F) and bradycardiac (Sphygmothermic dissociation) and
delirium is frequent. The patient may be calm but sometimes agitated,
thus it gave typhoid fever the nickname called “Nervous fever”. In
the third week, intestinal hemorrhage occurs, Encephalitis,
20
Neuropsychiatric symptoms, metastatic abscesses and endocarditis is
seen. The final week (fourth), the patient enters into the typhoid state.
The incidence of Salmonella infection may not be perfectly known.
This is because the majority of patients are treated as outpatients and
therefore hospital based studies will underestimate the true incidence
(W.H.O, 2006). However, the incidence of typhoid fever in
developing countries is higher compared to other developed countries.
The pathogenesis of typhoid fever is a complex process which
proceeds through several stages with an asymptomatic incubation
period of 7-14days inversely related to the size of the infecting dose
during which bacteria invade macrophages spread throughout the
reticuloendothelial system. After passing through the pathological
stages, necrosis may occur. Salmonella typhi infection can be best
prevented by sanitation and hygiene. It can be controlled using a wide
variety of methods such as the use of vaccines e.g live oral Ty2la
vaccine (sold at Vivotif Berna). Diagnosis is by bone marrow or stool
culture and with a widal test i.e (demonstration of Salmonella
antibodies against O and H antigens). Treatment is by the use of
21
antibiotics such as ampicillin, chloramphenicol, trimethoprim,
sulfomethriazole and ciprofloxacin. Since some plants have been
shown to have antimicrobial effect against some pathogenic bacteria,
especially antibiotic resistant pathogens, this study is aimed at testing
the effect of H. sabdariffa on S. typhi and other pathogens such as
Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa,
and Klebsiella pneumonia. The aim of the study is to determine the
invitro effect of H. sabdariffa on a clinical isolate of S. typhi and other
pathogens. Below are the objectives of this study.
1.1. Objectives
Collection and identification of H. sabdariffa flower
Antibiotic sensitivity testing of the test organism
Antimicrobial screening of H. sabdariffa extract against the test
organisms.
Investigation of the susceptibility of S. typhi and other
pathogens to hot water extract of H. sabdariffa flower.
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