ABSTRACT
Methanol, Chloroform and N-Hexane fractions of Newbouldia leavis root bark and leaf extract were analyzed in-vitro for their antibacterial, antioxidant and phytochemical properties against resistant bacteria isolate from infected wound. The antibacterial activity of the plant extracts were evaluated against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli using agar well diffusion method. It was observed that methanol extract of the plant leaf had the highest activity against E. coli with the mean zone of inhibition diameter of 17.67±0.67mm. While the least was N-Hexane and chloroform leaf extracts of the plant against Staph. aureus and E. coli respectively with a mean zone of inhibition diameter of 12.00 ± 0.00 mm each. Broth dilution method was used to determine the Minimum Inhibitory Concentration (MIC) in which methanol leaf extract against E. coli and Pseudomonas aeruginosa, N-hexane root extract against E. coli and chloroform root extract against Staph. aureus were observed at 25mg/ml while others were in 50 and 100 mg/ml. Minimum Bactericidal Concentration (MBC) of the extracts against the three test organisms were almost at the double concentration of the MIC. Antioxidant screening was done employing 2.2’- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method and Ferric reducing antioxidant power method- at the range of 25 – 400 µg/mL concentration in triplicate. The antioxidant activity shows positive results in most of the extracts. Their activities increased as the concentration increased with methanol leaf extract showing the highest activity, almost the same with the standard (ascorbic acid). The GC-MS analysis showed 17,32,18,16 and 22 compounds from methanol leaf extract, N-Hexane leaf extract, Chloroform leaf extract, Methanol root extract and Chloroform root extract of N. leavis respectively. The high bioactive activity of the methanol leaf extract is majorly due to the presence of Carbamodithioic acid, 5-Chlorovaleric acid, 1-Methylverbenol, Methylverbenol ether, arachidonic acid semicarbamide, N-Desmethyl Tapentadol, benzonitril, cyclopentene butylperoxy- phtalan present only in this extract. The study shows that the plant had broad spectrum in activity which justified the traditional use of this plant in treatment of wound and other bacterial infections.
TABLE
OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgment v
Table of Contents vi
List of Tables vii
List of Figures viii
Abstract xi
CHAPTER 1: INTRODUCTION 1
1.2 Background
Information
1.3 Statement of Problem 4
1.4 Aim and
Objectives 4
1.5 Scope of Study 5
1.6 Justification 5
CHAPTER
2:
LITERATURE REVIEW
2.1 Common
Names and Folkloric Uses of Newbouldia leavis 6
2.2 Morphology 7
2.3 Health Benefits of Newbouldia laevis Plant 8
2.4
Other Uses of Newbouldia laevis
Plant 10
2.5.1 Phytochemistry of Newbouldia
leavis plant 10
2.5.2 Geographic and genetic variation effect on
yield and composition of
plant secondary metabolites 10
2.5.3 Biological and pharmacological
activities of the phytochemical
constituents 13
2.5.4 Factors affecting the antimicrobial activities of the plant
extract 21
2.5.5
Pathogenicity of some bacteria
associated with wound infection 22
2.6
Methods Used in Evaluation of
Antimicrobial Properties of the
Plant
Extracts 25
2.6.1 Agar well diffusion test 25
2.6.2
Dilution
test 26
CHAPTER 3: MATERIALS AND METHODS
3.1 Source,
Sample Collection and Identification 27
3.1.1 Confirmation of the test bacteria 28
3.1.2 Molecular identification 31
3.2 Processing of the Leaf and the Root Bark
Samples 33
3.3 Extraction Process 33
3.4 Preparation
of Stock Solution of the Extracts 34
3.5 Phytochemical Screening of the Leaf and
Root Bark Sample 34
3.5.1
Qualitative analysis of
the phytochemical properties of the plant samples 34
3.5.2
Quantitative analysis
of the phytochemical properties of the plant samples 37
3.5.3
Gas chromatography/mass spectrometry analysis 41
3.5.4 Antioxidant
test 42
3.5.5 Preparation of 0.5 McFarland standard 43
3.5.6 Preparation
of inoculum suspension 43
3.6 Antibacterial Bioassay 43
3.6.1 Determination
of the zone of inhibition diameter of the leaf and root
bark extract of N. leavis 44
3.6.2 Determination
of minimum inhibitory concentration (MIC) 44
3.6.3 Determination of minimum bactericidal concentration (MBC) 45
3.6.4 Statistical Analysis 45
CHAPTER
4: RESULTS AND DISCUSSION 46
4.1 Results 46
4.2 Discussion 74
CHAPTER
5: CONCLUSION AND RECOMMENDATION
5.1 Conclusion 78
5.2
Recommendations 78
References
Appendices
LIST OF
TABLES
4.1: The
mean zone of inhibition diameter of the leaf and root
bark extract of N. leavis (mm) 49
4.2: Minimum inhibitory concentration 52
4.3: Minimum bactericidal concentration 53
4.4: The FRAP values of the extracts 57
4.5: The
results of the qualitative phytochemical tests of the leaf and root
bark
of Newbouldia laevis. 59
4.6: The results of the quantitative
phytochemical analysis of the leaf
and root bark of N. leavis. (Mean
± SD). 61
4.7: GC-MS analysis of the methanol fraction of
N. leavis root extract 63
4.8: GC-MS analysis of the methanol fraction of
N. leavis leaf extract 65
4.9: GC-MS analysis of the N-hexane fraction of
N. leavis leaf extract 67
4.10: GC-MS analysis of the chloroform fraction of
N. leavis leaf extract 70
4.11: GC-MS analysis of the chloroform fraction of
N. leavis root extract 72
LIST
OF FIGURES
2.1 Newbouldia laevis in its natural
environment 7
2.2 Newbouldia laevis plant with flower 8
4.1 Phylogenetic
tree showing the evolutionary distance between
the bacterial isolates 47
4.2 Agarose gel electrophoresis lanes B1, B2,
B3 represent the
16SrRNA 48
4.3 DPPH radical inhibitory effects of the
extracts 55
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND
INFORMATION
The traditional medicinal practices is employed for
the treatment of various ailments in many societies especially the African
society. This practice continues to exist in the developing nations including
Nigeria. It is on this basis that researchers keep on working on medicinal
plants in order to produce the new medicines for therapeutic uses (Yadav and
Munin, 2011).
The leaves, flowers, stems, roots, seed, fruit, or
bark of these plants can be constituents of these herbal remedies (Criagg and David,
2001; Yadav and Munin, 2011). Since it is now clear that modern pharmaceuticals
cannot treat every condition effectively and also for the fact that some drugs
have unwanted side effects, it is necessary that scientist take objectives
approach in investigating the effectiveness of these herbal medicines or in
isolation of the active agents and utilizing it in development of safe drugs
with standardized dosages. This is imperative since studies have shown that
herbal medicine is relatively less toxic, widely available and cheaper than
synthetic drugs (Jahan et al., 2007).
Nowadays, the use of phytochemical constituents for
pharmaceutical purposes has gradually increased in many countries. The World
Health Organization (WHO, 2001) estimates that 80% of the world population
depends on traditional medicine, predominantly originated from plants for their
primary healthcare (WHO, 1996).
The medicinal values of plant lie in the presence of
some endogenous substances that produce definite physiological or
pharmacological actions on the human body. The most important of these
bioactive constituents include alkaloids, tannins and flavonoids. Others are
steroids, saponins, phenols, terpenoids and glycosides. In recent years, a
number of studies have been conducted in various countries to prove such
efficiency (Gill, 1992).
Phytochemicals, are produced by plants as protective
agents against external stress and pathogenic attack, hence source of plant’s
defense and survival ((Ullah
et al. 2012). According
to Ullah et al. (2012), medicinal plants produce secondary metabolites
that are responsible for their therapeutic properties but the presence of these
molecules and conversely, their activities are affected by environmental
factors like geographical locations, fertility of cultivars, parts used, season
and time of collection. Nikolic and Zlatkovic (2010) noted that chemical
profiles of plants and the accumulation levels of special metabolites in plant
tissues can be influenced by factors such as temperature, light quality and
light intensity. Thus, the yield and composition of secondary metabolites
within species vary between and within plants from different geographical
locations and may be influenced by environmental and genetic differences
(White, 2006).
Wound have a potential for severe bacterial infections,
including gas gangrene and tetanus, and these in turn may lead to long term
disabilities, chronic wound or bone infection and death. Wound infection is
particularly of concern when multi-drug resistance organisms are involved in
the infection. In recent times, there
has been increase in bacterial resistant strains of clinical importance which have
resulted in the emergence of new multi-drug resistant bacterial strains (WHO,
2001). The non-availability and high cost of synthetic drugs with limited efficacy
has led to increased morbidity and mortality (Williams, 2000). This has led to
the search for new, safe and effective antibacterial agent of
plant origin with the aim of discovering potentially useful active ingredient
that can serve as source and template for the synthesis of new anti-bacterial drugs which can
be used to treat bacterial wound infection and other infectious diseases (Mamah et al., 2014; Pretorious et al., 2003).
Newbouldia
leavis commonly called ‘Aduruku’
in Hausa, ‘Ogirisi’ in Igbo and ‘Akoko’ in Yoruba languages (Hutchinson and
Dalziel, 1963) used for this research is a medium sized angiosperm which
belongs to the Bignoniaceae family. It grows to a height of about 7-8(up to 15)
meters, usually a shrub of 2-3 meters, many-stemmed forming clumps of gnarled
branches. Newbouldia leavis is native
to tropical Africa and grows from Guinea Savannahs to dense forests (Arbonnier,
2004).
It is one of the plants with magical effect (Idu et al., 2003). Scientifically it has been reported to have
medicinal value ranging from anti-inflammatory, anti malarial, antioxidant,
antibacterial, antifungal, analgesic and wound healing properties (Aladesanmi et al., 1998; chukwujekwu et al., 2005, Kuete et al., 2007; Usman and Osuji, 2007; Akere et al., 2011; Omokpo et al.,
2012). Specifically, the leaves have been used in the south-Eastern and Western
part of Mid-Western Nigeria, for the treatment of septic wounds and eye
problems (Akere et al., 2011; Egba et al., 2014).
In Nigeria, the bark is used in the treatment of
stomach pains, diarrhea and toothache (Lewis and Manony, 1977). The plant has
been found to be effective in the treatment of elephantiasis, dysentery,
rheumatic swellings, constipation, pile and as vermifuge to round worms (Iwu,
2000). It has also been used for the treatment of earache, sore feet, chest
pain, epilepsy and children’s convulsion (Akunyili, 2000). Some parts of the
plant have been used for febrifuge; wound dressing and stomach ache (Iwu,
2000).
1.2 STATEMENT OF PROBLEM
Newbouldia
leavis is one of the valuable medicinal trees found in
Africa. The species are facing threat of destruction due to over-exploitation
by traditional medicinal practitioners based on its wide application in
treatment and management of various diseases. Previous studies have shown
significant geographical and genetic differentiation within its populations (Muchugi et
al., 2008; Muchugi et al., 2012). It is not known if such
differentiation also exists in its antimicrobial activity and phytochemical properties.
Since there are high chances
of pathogen specificity, microbial resistances of clinically important pathogens have led to
emergence of new multi-resistance strains of microorganisms (WHO, 2001). However,
the synthetic antimicrobial drugs are expensive and not readily available (WHO,
2001) with many side effects (Williams, 2000). The antibacterial activities of the plant extracts
are influenced by the solvent used in the extraction (Nikolic and Zlatkovic,
2010).
1.3
AIM
AND OBJECTIVES
1.3.1 Aim
To evaluate the antibacterial, antioxidant
and bioactive properties of the leaf and root bark extract of Newbouldia leavis.
1.3.2 Specific
objectives
v
To access the antibacterial activities of leaf and
root bark extracts of Newbouldia leavis on some selected bacteria from infected
wound.
v
To evaluate the bioactive constituents of the leaf
and root bark extracts of Newbouldia leavis.
v
To investigate the antioxidant activities of N-hexane,
chloroform and methanol extracts of leaf and root bark of Newbouldia leavis.
1.4 SCOPE OF STUDY
To evaluate the antibacterial, antioxidant
and bioactive properties of the leaf and root bark extract of Newbouldia leavis.
1.5 JUSTIFICATION
Ø
The
knowledge on antibacterial, antioxidant and bioactive properties of the extracts
of Newbouldia leavis will
help to identify plants with high bioactivity with regard to the percentage
relative abundance of the active compounds expressed in the plant species. This
will assist in designing strategies to identify suitable genotypes and parts of
the plant species with most
effective antibacterial and antioxidant properties. Some plant parts can be more sustainably harvested
than others. Therefore, it is important to assess levels of expression of these
active compounds in different parts of Newbouldia leavis.
Ø
This
study would provide the solvent with most significant effect on the extraction
yield and antioxidant properties. This could be due to the differences in the
polarity of the compound which were selectively more soluble in different
solvents.
Ø
The study will also provide information on the antibacterial sensitivity
of the extracts against some selected bacteria from infected wound. The plant
extracts showed remarkable inhibitory effects on the entire test bacteria used
for this work. Both root and leaf extracts of Newbouldia leavis inhibited the growth of both Gram negative and
Gram positive bacteria. Findings
from this study will be valuable in providing data that will support the
existing information on the use of Newbouldia leavis in traditional medicine therapy.
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