ABSTRACT
Oral
and Skin infections are among the leading causes of death worldwide. Pathogenic
bacteria are becoming resistance to the existing antibiotics and many of the
commercially available antimicrobial drugs. Therefore, there is need to explore
medicinal plants as rich reservoir for the discovery of a safe complementary
and alternative drugs that can be harnessed for human benefit. The present work
aimed at investigating the total phenolic, total flavonoid, antibacterial,
antioxidant and TLC profiling of some medicinal plants used traditionally for
the treatment of oral and skin infections in Ohafia, Abia State. Such plants
are: Ipomoea involucrata, Acalypha
hispida, Breynia nivosa, Jatropha curas, Chromolaena odorata, Macrolobium
macrophyllum, Eleusine indica, Baphia nitida, Burkea africana, and Cassia alata. The antioxidant potential,
antibacterial, total phenolic and flavonoid of methanolic extract were
investigated using in vitro standard
bioassays methods. DPPH, ABTS, and FRAP were used to evaluate the antioxidant
ability of the extract. Disc diffusion agar and Minimum Inhibitory Concentration
(MIC) were used to evaluate the antibacterial activities. TLC profiling using
silica gel coated on a glass with a solvent ratios of chloroform and methanol
(6:4) and toluene, ethyl acetate and acetic acid (5:4:1) were used to separate
polar and mid-polar compounds respectively. The plant extracts demonstrated a
robust antioxidative potential and a potent antibacterial activity against the
clinically isolated bacteria. The result of this study partly lend credence to
the ethno-medicinal use of these plant by the traditional healers. Further work
is suggested to isolate, characterize, and identify active principles in these
botanicals via bio-guided assays and their toxicological
studies for drug discovery.
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Fig.1
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Picture
of Chromolaena odorata
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Fig.2
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Picture
of Breynia nivosa
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Fig.3
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Picture
of Jatropha curcas
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Fig.4
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Picture
of Acalypha hispida
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Fig.5
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Picture
of Cassia alata
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Fig.6
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Picture
of Ipomoea involucrata
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Fig.7
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Picture
of Baphia nitida
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Fig.8
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Picture
of Burkea africana
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Fig.9
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Picture
of Eleusine indica
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Fig.10
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Picture
of Macrolobium macrophyllum
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Fig.11
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Map
satellite locating the place of collection of plant samples
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Fig.16
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IC50
of DPPH on a bar chart
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Fig.17
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IC50
of ABTS on a bar chart
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Fig18
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TFC
on a bar chart
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Fig.19
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TPC
on a bar chart
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Fig.20
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TLC
result
List of plates
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Plate
1a
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Zone
of inhibition of test extract against E.coli
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Plate
1b
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Zone
of inhibition of test extract against E.coli
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Plate
2
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Zone
of inhibition of test extract against Proteus
mirabilis
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Plate
3
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Zone
of inhibition of test extract against Staphylococcus
aureus
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Plate
4
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Zone
of inhibition of test extract against Klebsiella pneumonia
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Plate
5
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Zone
of inhibition of test extract against Klebsiella
pneumonia
List
of tables
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Table
1
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Medicinal plants used for the management
of skin infection in Ohafia, Abia state, Nigeria.
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Table
2
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Medicinal plants used for the management
of oral infection in Ohafia, Abia state, Nigeria.
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Table
3
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FRAP
result
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Table
4
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Standard
drugs used in the analysis
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Table
5
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TLC
results
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Table
6
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Result
of zone of inhibition
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Table
7
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Result
of MIC
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Table
8
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Result
of DPPH % inhibition
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Table
9
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Result
of ABTS % inhibition
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Table
10
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Result
of TFC
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Table
11
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Result
of TPC
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TABLE OF CONTENT
Cover
page………………………………………………………………………………...0
Title
page………………………………………………………………………………..…i
Declaration………………………………………………………………………………....ii
Certification…………………………………………………………………………….…iii
Dedication………………………………………………………………………………....iv
Acknowledgement……………………………………………………………………….....v
Abstract…………………………………………………………………………………….vi
Table
of figures, plate and tables…………………………………………………………..vii
Table
of content……………………………………………………………………………viii
CHAPTER ONE
1.0 Introduction……………………………………………………………………………1
1.1 Aim…………………………………………………………………………………….3
1.2 Objectives………………………………………………………………………….......3
1.3 Justification……………………………………………………………………….……3
CHAPTER TWO
2.0 Literature
Review……………………………………………………………..………..4
2.1.1 Chromolaena odorata……………………………………………………...................5
2.1.2 Breynia nivosa …………………………………………………………………..……7
2.1.3 Jatropha curcas…………………………………………………………………….....9
2.1.4 Acalypha hispida……………………………………………………………………...11
2.1.5 Cassia alata…………………………………………………………………………...13
2.1.6 Ipomoea involucrata………………………………………………………………….....14
2.1.7 Baphia nitida…………………………………………………………………………..16
2.1.8 Burkea africana………………………………………………………………………..18
2.1.9 Eleusine indica…………………………………………………………………………20
2.1.10 Macrolobium macrophyllum……………………………………………………….…21
2.2.0 Antioxidant
activities……………………………………………………….………….22
2.2.1 DPPH
Radical Scavenging activities…………………………………………….……22
2.2.2 ABTS
Radical Scavenging activities…………………………………………….…....22
2.2.3 Ferric
Reducing Antioxidant Power…………………………………………..………22
2.3.0 Antibacterial
activities…………………………………………………………............23
2.3.1 Disc
diffusion assay…………………………………………………………….……...23
2.3.2 Minimum
Inhibitory concentration (MIC)……………………………………………..23
2.4.0 Thin
Layer Chromatographic Profiling………………………………………………...23
CHAPTER THREE
3.0 Materials
and Method…………………………………………………………………..25
3.1.0 Plant Sample collection and
Identification……………………………………………..25
3.1.1 Extraction……………………………………………………………………………….26
3.1.2 Chemicals
and Reagents………………………………………………………….……..26
3.1.3 Bacterial
Strain collection………………………………………………………....…….26
3.2.0 Quantification
of Phenolic compounds………………………………………….…........27
3.2.1 Determination
of total flavonoid content………………………………………….…….27
3.2.2 Determination
of total Phenolic acid content……………………………………….…...27
3.3.0 Quantification
of Antioxidant activities…………………………………………………28
3.3.1 DPPH
(1, 1-Diphenyl-2-picrylhydrazyl) Scavenging assay…………………………......28
3.3.2 ABTS
(2, 2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) Scavenging assay….....29
3.3.3 FRAP
(Ferric Reducing Antioxidant Power) assay………………………………….…..30
3.4.0 Antibacterial
activity…………………………………………………………………......31
3.4.1 Media
Preparation…………………………………………………………………….….31
3.4.2 Disc
diffusion method……………………………………………………………….…..32
3.4.3 Minimum
inhibition concentration (MIC)…………………………………………..…..32
3.5.0 Thin
Layer Chromatography (TLC) Profiling………………………………………...…32
CHAPTER FOUR
4.0 Result………………………………………………………………………...…………...34
4.1 Description
and Biological activities of medicinal plants during the ethno-botanical survey.34
4.2.0 Antioxidant
activities………………………………………………………….………….36
4.2.1 DPPH
Assay………………………………………………………….……….…………..36
4.2.2 ABTS
Assay………………………………………………………………...………….…37
4.2.3 FRAP
Assay……………………………………………………………...……………....38
4.3.0 Determination of polyphenol content…………………………….…………………….....40
4.3.1 Total Flavonoid Content……………………………………………………………..…...40
4.3.2 Total Phenolic Content…………………………………………………….……….….…41
4.4.0
Thin Layer Chromatographic
Profiling…………………………………...……...….......42
4.5.0 Antibacterial Activities……………………………………………………….……….….44
4.5.1 Disc diffusion assay……………………………………………………………………....44
4.5.2 Minimum Inhibitory Concentration
(MIC)…………………………………………….…47
CHAPTER FIVE
5.1.0 Discussion………………………………………………………………………….…….49
5.2.0 Conclusion………………………………………………………………………………..52
5.3.0 References…………………………………………………………………………….......53
5.4.0 Appendix………
CHAPTER ONE
1.0 INTRODUCTION
The
discovery and development of antibiotics took a leap over 60 years ago, with
credit to Alexander Fleming in 1928 for the discovery of the β-lactam
penicillin, a natural product which came into clinical use in 1944 (Saga and
Yamaguchi 2009). Drugs from medicinal plants are important sources of
therapeutic remedies of various ailments using the crude extract and dry powder
samples from medicinal plants extensively for the development and preparation
of alternative traditional medicine (Karou et
al., 2005). Scientific experiments on the antimicrobial properties of plant
component from different part of medicinal plants used in Nigeria are been
researched recently.
There
is a widespread of interest in evaluating drugs derived from plant source. This
interest primarily stems from the belief that green medicine is safe and
dependable, when compared to the costly synthesized drugs which are invariably associated
with adverse effect (Ramarathnam et al
1995). Natural antimicrobial have been derived from plants, animal tissues and
micro-organisms. The adverse effect of drugs available today necessitate the
discovery of new pharmacological agent from medicinal plants.
Medicinal
value of plant lie in their phytochemical component, otherwise known as
secondary metabolites which are synthesized during the secondary metabolism of
plant (Hill, 1952). This phytochemicals are bioactive compounds found in plant
that work with nutrient and dietary fibers to protect against disease.
Phytochemicals such as alkaloid, tannins, flavonoid and other phenolic
compounds was found to give definite physiological action on human body,
especially in the treatment of oral and skin infections (Hostettmann and
maston, 2002). Hence a systemic research
for useful bioactivities from medicinal plant is now considered to be a
rational approach in drug research.
The
pathogenic micro-organisms are becoming resistance to many of the commercially
available antimicrobial drugs. Efforts by scientist in the area of chemotherapy
have been increased to a great extent in the last two decades especially in natural
products considered as a good source of obtaining highly safe, potent and low
cost antibacterial drugs.
Reactive
oxygen species (free radicals) ROS are the metabolic by-product produced in
human body. These free radicals are very harmful to our body and can damage the
cell membrane and nucleic acid of our body if produced in excess quantity. In
order to reduce the oxidative stress caused by various endogenous and exogenous
free radicals, antioxidants are useful in combating the effects of the free
radicals (Rajesh et al., 2008). These
antioxidant compound react with the free radicals and neutralize them. Some
synthetic antioxidant such as Butylated hydroxyl anisole (BHA) and Butylated
hydroxyl toluene (BHT) are powerful. However, they are proved to be toxic to
human health. Therefore it is urgent that research should go on to find out
natural antioxidant.
The
present study investigates the natural antioxidant, antibacterial, total
phenolics, total flavonoid, and the thin-layer chromatographical profile of
some medicinal plants like: Baphia nitida,
Cassia alata, Ipomoea involucrata, Acalypha hispida, Macrolobium macrophyllum,
Eleusine indica, Jatropha curas, Burkea
africana, Chromolaena odorata and Breynia nivosa, on their biological
activities on clinically isolated bacteria implicated in oral and skin
infections.
1.1 AIM
The
aim of the present study is to investigate the total phenolic and flavonoid
content, and to determine the antibacterial, thin layer chromatography (TLC)
profiling, and antioxidant activities of the aforementioned ten medicinal
plants used locally in oral and skin infection treatment.
1.2 OBJECTIVE
1. To
evaluate the antibacterial properties of the test medicinal plants
2. To
determine the antioxidant properties of the test medicinal plants
3. To
determine the phytochemical profile of the plants.
1.3 JUSTIFICATION
This
study is to provide scientific evidence to the acclaimed medicinal activities
of these plants used in traditional herbal medicine practice in Ebem Ohafia
L.G.A, Abia state, Nigeria. The analysis of their antioxidant, antibacterial
and phytochemical constituent aid to reveal the presence and activities of the
phytochemicals which could be responsible individually or mostly in synergistic
manner for treatments used locally in oral and skin infections.
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