ABSTRACT
This research is designed to determine the antibacterial and antibiofilm activity of Zapotecca portoricensis roots, Artemisia annua leaves and Moringa oleifera seed oil. To verify the uses, of root, leaf extracts and seed oil of these plants for medicinal purpose. They were screened for antibiofilm, antibacterial and antioxidant effects against some isolates which includes; Salmonella typhi, Staphylococcus aureus and Escherichia coli. Biofilm quantification and tissue culture method was used to determine the percentage biofilm inhibition. Agar well diffusion method was used to check the antibacterial activity of the various plant extracts, The lowest minimum inhibitory concentration (MIC) was done using the 2,3,5-triphenyl tetrazolium chloride (TTC). Gas chromatography-mass spectroscopy (GC-MS) was used to identify bioactive compounds in these extract which include. 15.63 mg/ml was shown by Z. portoricensis and A. annua against E.coli. Extracts of A. annua leaves gave large inhibition zone diameter of 14mm against S aureus at 500 mg/ml. These extract gave high inhibition zone diameter of 12mm, 12.27mm and 14mm for M. oleifera, Z. portoricensis and A. annual respectively with % DPPH antioxidant activity of 32.50%, 45.24% and 73.25%. Maximum FRAP antioxidant activity and antibiofilm inhibition of extracts of M. oleifera, Z. portoricensis and A. annua observed were 1.02µm, 6.72µm and 0.54 µm for FRAP and 56.06%, 66.45% and 70.84% for % antibiofilm inhibition respectively. Results from this study indicate that extracts of M. oleifera, Z. portoricensis and A. annua contain bioactive compounds with possible potential as antibacterial, antioxidant and antibiofilm agents.
TABLE OF CONTENTS
Title
i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table
of Contents vi
List
of Tables viii
List
of Appendices ix
List
of Figures x
Abstract xi
CHAPTER 1: INTRODUCTION
1.1 Medicinal Plants as Antimicrobial Agents 1
1.2 Microbial
Biofilms 2
1.3 Statement
of Problem 3
1.4 Justification
of the Research 5
1.5 Significance
of the Study 5
1.6 Aim
of the Study 5
1.7 Objectives
of the Study 6
CHAPTER 2: LITERATURE
REVIEW
2.1
The Impact of Antimicrobial Resistance and Medicinal
Plant 8
2.2
Mechanism of Action of
Botanicals 10
2.3
Bacterial biofilms 13
2.4
Biofilms and its Role in Resistance 16
2.4.1 Physiological State 16
2.4.2 Extracellular matrix (ECM) 16
2.5 Quorum Sensing (QS) 16
2.6
Zapoteca
portoricensis 16
2.7 Artemisia
annua 16
CHAPTER 3: MATERIALS AND
METHODS
3.1
Collection of Plant Materials 21
3.2
Preparation of the Plant Extracts 21
3.3 Gas Chromatograph-Mass
Spectrometry (GC-MS) of Plant Extracts 22
3.4 Identification and Confirmation of the Test
Organisms 22
3.5
Biochemical Tests 26
3.5.1
Gram staining 27
3.5.3 Oxidase test 27
3.5.4 Motility test 27
3.5.5 Coagulase test 28
3.5.6 Methyl-red test 28
3.5.7 Indole test 29
3.6
Determination of Minimum Inhibitory Concentration (MIC) 30
3.7 Biofilm Formation and Inhibition Analysis 31
3.7.1 Tissue culture plate method 31
3.7.2 Biofilm
inhibition assay 31
3.7.3
Antioxidant test 31
3.7.3.1 2, 2-Diphenyl-1-Picrylhydrazyl (DPPH) photometric assay procedure 31
3.7.3.2 Ferric reducing antioxidant power
procedure 31
3.8 Data
Analyses and Confirmation of Values 31
CHAPTER
4: RESULTS AND DISCUSSION
4.1
Presentation of Results 32
4.2
Discussion 42
CHAPTER 5: CONCLUSION AND
RECOMMENDATION
5.1Conclusion 42
5.2
Recommendation 42
REFERENCES 43
APPENDIX 49
FIGURES 49
CHAPTER 1
INTRODUCTION
Plants have
been in use for the management and treatment of diseases and it started long
ago with life (Sarita, et al., 2019).
It has been noted and accepted that many plants do have medicinal value and
extracts from these plants have been used to formulate modern drugs.
Phytochemical screening evaluates the presence of biologically active,
non-nutritive compounds that adds to the flavour, colour and other
characteristics of plant parts. These compounds (eg alkaloids, tannins, cardiac
glycoside, terpenoids, saponins, anthraquinones, flavonoids etc) are the main
basis of pharmacological activities of medicinal plants (Artur, et al., 2020). Morphine alkaloids are
reliable pain relievers and narcotics (Lee et
al., 2019) while saponins are natural antibiotics that helps in fighting
infections and microbial invasion (Turmagambetova
et al., 2017). Tannins have been demonstrated to inhibit
multiplication of HIV and herpes simplex virus (Ueda et al., 1994). Presently, the use of phytochemicals for pharmaceutical use has
gradually increased in many countries. The ideal source for obtaining a variety
of medications would be medicinal plants (WHO, 2016). The WHO (2016) estimates
that almost 80% of the populace in the world depends on traditional medicine,
mostly originated from plants for their primary healthcare. From the time immemorial,
medicinal plants were the major source of treatment for disease conditions.
1.1
MEDICINAL PLANTS AS ANTIMICROBIAL AGENTS
Today, medicinal plants are used not only in
developing countries, but also in developed countries where modern medicine are
commonly used (Junaid, et al 2008). Plants most especially the higher plants
contain varieties of substances which are used as food additives and medicine
in treatment of various diseases of man plus animal. (Chattopadhyay et al 2018). The usage of herbal medicine in the cure of diseases is gradually
gaining acceptance and approval in the developed and developing countries. The
potency of plants used for treatment of diseases is due to the active
ingredients produced in the course of their metabolism (Kiranmayee et al., 2010). 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; however, environmental factors such as geographical
locations, cultivar fertility, parts used, season, and time of collection
determine the existence of these molecules plus their actions (Nikolic and
Zlatkovic, 2010). Thus, the yield plus constituents of secondary metabolites
within species vary between and within plants from diverse geographic locations
and may be predisposed by environmental and genetic differences. Many researchers have reported the activity
of plants against pathogens.
Since it is now certain that modern drugs cannot
treat every condition effectively and also for the fact that some drugs have
unwanted side effects, it is necessary that scientist take objective approach
in investigating the activities of these herbal medicines or in isolation of
the active agents and utilizing it in development of safe drugs with standardized
dosages. Thus, its imperative scientific researches have shown that herbal
medicine is relatively less toxic, widely available and less expensive than
synthetic drugs (Jahan et al., 2018).
In this current study, screening of crude extracts of Zapoteca portoricensis
roots, Moringa oleifera seed
oil and Artemisia annua leaves for antibiofilm and antibacterial activity might confirm that higher
plants represent source of novel anti-bacterial prototypes. There has been an
increase in bacterial resistant strains of clinically significence in recent
years, resulting in an urgent need for new multi-resistant bacterium strains
(WHO, 2006). The non-availability and high cost of synthetic drugs with limited
efficacy have led to the increase in morbidity and mortality (Williams, 2000)
which resulted in looking for substances with proven antibiofilm and antibacterial activities. This has triggered
the search for new, safe and effective bactericidal agent among materials of
plant origin with the aim of discovering potentially useful active ingredient
that can serve as source and template for the creation of new drugs (Pretorious
et al., 2003) which could be used to
fight bacteria involved in enteric infection and other infectious diseases
(Mamah et al., 2014).
1.2 MICROBIAL BIOFILMS
Microbial biofilms are communities of
bacteria, embedded in a self-producing matrix, forming on living and nonliving
solid surfaces (Kim and 2004). Biofilm-associated cells have the capability to
adhere irreversibly on several varieties of surfaces, both living tissues and
indwelling medical devices as catheters, valves, prosthesis, and others
(Stewart et al 2003). They are
considered an important virulence factor that is accountable for persistent
chronic and recurrent infections; they are strongly resistant to antibiotics
and host immune defenses. Antibiotic resistance is many times higher in
bacteria protected by biofilm exopolysaccharides than in planktonic
(free-floating) cells, which has important implications for therapy and
complicates treatment alternatives. Biofilms are protected by an extracellular
matrix in approximately 75% of bacterial infections (Kim, 2004). Biofilm
resistance is occasioned by several reasons, like restricted dispersal of
antibiotics into biofilm matrix, expression of multidrug efflux pushes, type
IVsecretion systems, decreased permeability, and the action of
antibiotic-modifying enzymes. (Jahan et al., 2008). Biofilm resistance to standard treatments is
increasing, necessitating the development of new control measures. Biofilm
inhibition is a popular medical target for the treatment of a variety of
bacterial and fungal infection, and the pharmacological development of these
therapies is now being researched extensively. Many green nonlethal biofilm
management tactics have been established in recent years, making the mode of
action of these novel antibiofilm agents far less prone to the establishment of
resistance. (Trautner et al, 2004).
One interesting alternative is to look for naturally occurring plant-derived
chemicals that can prevent biofilm formation. Historically, plant extracts and
their biologically active compounds have been reliable sources of natural
products, which have perform a central role in the deterrence and cure of
diseases, helping to maintain human health. They are also widely accepted since
they are perceived to be harmless and have a lengthy history of use in folk
medicine to cure diseases and illnesses since ancient times (Stewart et al., 2003).
1.3 STATEMENT OF THE PROBLEM
The synthetic
antibacterials are expensive and rarely available (WHO, 2006) and also due to
the upsurge of resistance, there is need for new drugs or medicines of plant
source. Synthetic drugs have side effects (Williams, 2000) and high price of
the solvent for extraction, proximity of the extraction site to the research
laboratory is very far, some of the used media were scarce, the site of sample
collection was also far from the laboratory, there was poor power supply during
the research that warranted the use of generator for preservation and further
research.
1.4 JUSTIFICATION OF STUDY
Challenges associated with synthetic drugs
motivated researchers to look for substances with proven antibiofilm and
antimicrobial actions among plant origin. This has triggered the search for
new, safe and effective antibacterial agent among materials of plant origin.
However, based on the properties of Zapoteca portoricensis root, Moringa
oleifera seed oil and Artemisia annua leaves, this work was designed to investigate the biological
activities of Zapoteca portoricensis
root, Artemisia annua leaves and Moringa oleifera seed oil extract on
some isolates.
1.5 AIM OF THE STUDY
To determine the antibacterial and antibiofilm
activities of some plant extracts on selected microorganisms.
1.6 OBJECTIVES
OF THE STUDY
1. To
determine the phytochemical contents of Zapoteca
portoricensis root, Artemisia annua
leaves and Moringa oleifera seed oil extracts.
2. To
determine the antioxidant activity of Zapotecca
portoricensis root, Artemisia annua
leaves and Moringa oleifera seed oil extracts
using 2, 2- Pipheny1-1- Picrylhydrazyl (DPPH)
and ferric reducing antioxidant power (FRAP) methods.
3. To
determine the antibacterial activities of Zapotecca
portoricensis root, Artemisia and
Moringa oleifera seed oil extracts using
antibacterial susceptibility test (AST).
4. To determine the percentage antibiofilm inhibition
of Zapotecca portoricensis root, Artemisia annua leaves and Moringa oleifera seed oil extracts using tissue
culture plate method.
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