ABSTRACT
Medicinal plants have been found to play a significant role in the treatment of human ailments due to the resistance nature of synthetic drugs aside been very expensive. One of the leading medicinal plant found to have therapeutic power is Amaranthus spinosus Linn. This present study is aimed at investigating the antioxidant and antimicrobial activities of ethanolic root extract of Amaranthus spinosus Linn. The antioxidant activity of root extract of Amaranthus spinosus was accessed by in- vitro experimental parameters such as DPPH (2,2-Diphenly -1-picrylhydrazyl) free radical scavenging activity and Nitric oxide radical assay and Anti-lipid peroxidation. In this study, DPPH was found to be concentration-dependent. The result showed that Amaranthus spinosus ethanolic root extract have showed potent in vitro antioxidant activity that may be attributed to the phenolic compounds present. The ethanolic root extract of Amaranthus spinosus was evaluated for antimicrobial activity against 6 bacteria’s such as (Proteus mirabillis, Streptococcus pneumoniae, Proteus aureginosa, Lactococcus bacillus, Klebsiella pnueumoniae, and Staphylococcus aureus) the in -vitro antimicrobial activity was performed by Agar Disc Diffusion method with Potato Dextrose Agar (PDA). The extract of 1000mg showed the highest inhibitory zone (33.00mm±1.00mm) on Staphylococcus aureus, while the extract of 62.5mg showed the least inhibitory zone (1.66mm±5.77mm) on Proteus mirabillis. All the organisms were susceptible to the plant extracts used for the antimicrobial test. This study strongly recommends the use of Amaranthus spinosus ethanolic root extracts as an Antimicrobial agent and can be used to cure many ailments caused by these microorganisms.
TABLE OF CONTENTS
Title Page i
Certification ii
Declaration iii
Dedication iv
Acknowledgment v
Table of content vii
List of table’s viii
List of Graphs ix
List of Plates x
Abstract xi
CHAPTER
ONE
1.1.1 Introduction 1
1.2
Review of Antioxidant
Activity 5
1.2.1 Pathophysiology
Of Oxidative Stress 5
1.2.2 Antioxidants
and Its Role In Biological System 5
1.2.3 DPPH as an antioxidant assay 6
1.2.4 Nitric
oxide as an antioxidant assay 7
1.2.5
Lipid Peroxidation as an antioxidant assay 7
1.3 Justification 8
1.4 Aims and Objectives 8
CHAPTER
TWO
2.0 Literature
review 9
2.1 Antimicrobial
activity of plant extract 9
2.2
Botanical Description of Amaranthus spinosus 12
2.2.1 Taxanomy of Amaranthus
spinosus 13
2.3 Description of Amaranthus spinousus 13
2.3.1
Soil Requirement of Amaranthus spinosus 14
2.3.2
Propagation 14
2.3.3
The Etymology of Amaranthus spinosus 15
2.4
Uses of Amaranthus spinosus 15
2.5
Chemical Composition of Amaranthus spinosus 16
2.6
Pharmacological Properties of Amaranthus spinosus 16
CHAPTER THREE
3.0
Materials and method 19
3.1
Background 19
3.2
Collection and identification of plant sample 19
3.3
Extraction carried out on the plant 19
3.4
Preparation of inocula 20
3.5
Antimicrobial activity test 20
3.6
Minimum inhibitory concentration (mic) 20
3.7
Preparation of antibiotics stock solution 21
3.8 Antioxidant activity 22
3.9 Statistical analysis 24
CHAPTER
FOUR
4.1Result 25
4.2
Antioxidant activity result 28
CHAPTER FIVE
5.0
Discussion and Conclusion 34
5.1
Discussion 34
5.2
Conclusion 35
REFERENCES
LIST OF TABLES
Table
1.Antimicrobial Activity of Root extract
of Amaranthus spinosus.
Table
2. Result of Minimum inhibitory concentration (MIC) of the plant extract.
Table
3. Result of Anti-lipid peroxidation.
Table
4. Result of 2, 2 Diphenyl -1- Picrylhydrazyl
(DPPH).
Table
5. Result of Nitric oxidescavenging activity.
LIST OF GRAPH
Fig 1. Bar chart
showing the Antimicrobial activity of Amaranthus
spinosus root extract at 1000mg, and antimicrobial activity of
ciprofloxacin at 500mg.
Fig 2. Bar chart
showing the Antioxidant activity of DPPH scavenging activity, Anti-lipid
peroxidation and Nitric oxide activity.
LIST OF PLATES
Plate
1: Amaranthus spinosus.
Plate
2: Prepared disc in different Concentrations and the control (Ciprofloxacin).
Plate 3: Zone of inhibition of
ethanolic extract of Root of Amaranthus spinosus
on Klebsiella pneumonia,
Staphylococcus aureus, Streptococcus pneumonia, Poteus mirabillis, Lactococcus
bacillus, Pseudomonas aeruginosa.
CHAPTER ONE
1.1
INTRODUCTION
Medicinal
plants are part and parcel of human society to combat diseases, from the dawn
of civilization, medicinal plants can be an important source of previous
unknown chemical substances with potential therapeutic effects (Vander et al., 2013).The use of natural
products with therapeutic properties has been a major sources of drugs, the
traditional healers and modern medicine have employed over the decade. (Ishrat,
et al., 2011) in spite of modern
development of sophisticated pharmaceutical chemicals to treat illness (Kavitha
et al., 2010). India is one of the
most medico-culturing diverse countries in the world where the main traditional
systems and exploiting their potential based on different health care systems,
the evaluation of the rich heritage of traditional medicine is essential(
Sharma, et al.,2013). Medicinal
plants contain number of medicinal properties because of the various
metabolites found in them like phenols, flavonoids, tannis, oils and resins,
saponins etc. and one of such plants is Amaranthus
spinosus Linn.
Infectious
diseases are one of the leading cause of premature death. Infective diseases
account for approximately one-half of all the death in tropics (Miwu et al., 1999). Resistance to antibiotics
drugs is a major global public health problem (Ibezim, 2005), partly due to
indiscriminate use of antibiotics (Ahmed et
al., 2011). Despite seeming progress made in the development of
antimicrobial agents, occurrence of drug resistance microorganisms and the
emergence of unknown disease causing microbes, pose enormous public health
concerns (Ibezim, 2005). Staphylococici
and Streptococci are among the
organism that cause respiratory infections. Candida
albicans is the common commensals of the gastro-instestinal and urogenital
tracts in human. (Cheesbrough, M, 2000). As well as Candidiasis in women. Pseudomonas species are particularly
noted for causing urinary tract infections and sepsis which are currently
resistance to virtually all the older Antibiotics (Harold, 1992). Many
infectious diseases are known to be treated with herbal medicines throughout
the human civilization. Even till today plant materials continue to play major
role in primary health care and higher plants have been shown to be potential
sources for new antimicrobial agents (Mitscher et al., 1988). Indigenous plants are reservoirs of
variousmetabolites and provide a limitless source of important chemicals that
have diverse biological properties( Cowan, 1999) many of modern day drugs have
their origin in traditional plant medicine(Farnsworth et al.,1985) in the area of Anti-infective, about 70% are naturally
derived( Cragg et al.,2005). The
screening of plant extracts for Antimicrobial activity has shown that higher
plants represents a potential source of novel Antibiotics Chemotherapies.
The
therapeutic efficacies of many indigenous plants for treating ailments have
been described by practitioners of traditional herbal medicines (Mann,1998,
Mann. et al.,2007) .The practice of
herbal medicines which employs plants as its major components is an integral
part of traditional and culture of Africans( Mann et al.,2003) . Most therapeutic attributes of medicinal plants are
traced to the plant constituents and the medicinal actions of those
constituents are unique to particular species or family. The antimicrobial
activity of plant extracts has formed the basis of many applications including
traditional medicine, pharmaceuticals natural and orthodox therapies.
The
Amaranthus genus is one of about 160
genera in the flowering plant family Amarnthaceae. Members of this family have
simple leaves that are opposite or alternate, with margins entire or coarsely
toothed, and without stipules. The flowers are solitary or aggregated in cymes,
spikes or panicles and typically perfect (bisexual) and actinomorphic. There
are 1 to 5 stamen, the hypogenous ovary has 3 to 5 joined sepals. AmaranthusspinosusL.is an annual or
perennial monoecious herb, native to tropical American and found throughout
India as a weed in cultivated as well as fallow land (Anonymous, 1988). Whole
plant is used as a traditional medicine to treat diabetics. The plant is
Astringent, diaphoretic, diuretic, emollient, febrifuge and galactogugue (Bown,
1995; Manaadhar, 2002; Usher, 1974) it is also used in treatment of snake
bites. The plant is used to treat ulcerated mouths, virginal discharge, nose
bleeds and wounds (Bown, 1995). Amaranthus
spinosus (Linn) is reported for its Anti-inflammatory properties and immunomodulatory
activity (Murgan et al.,
1993;Olumayokumet al., 2004). The
Antioxidant capacity of Amaranthus spinosus was studied in roadside plants
which were postulated to be continuously exposed to the high levels of nitrogen
oxides and sulphur oxides from automobile emissions (Singh and Dahiya, 2002).
Different extracts from traditional medicinal plants have been tested to
identify the source of therapeutic effects. Amaranthus
spinosus (Linn)plants contain a lot of substances having medicinal value,
which are yet to be explored.
Plate 1: Amaranthus
spinosus
1.2 REVIEW OF ANTIOXIDANT
ACTIVITY
1.2.1
Pathophysiology
Of Oxidative Stress
It
has been established that the oxidative stress is among the major causative
factors in induction of many chronic and degenerative diseases including
atherosclerosis, ischemic heart diseases, ageing diabetes mellitus, cancer,
immune suppression, neurodegenerative diseases and others (Young and Woodside,
2001). A great number of aromatic, medicinal, spice and other plants contain
chemical compounds exhibiting antioxidant properties. Oxidative process is of
the most important routes for producing free radicals in foods, drug and even
in living systems (Halliwell, 1994). The most effective path to eliminate and
diminish the action of free radicals which cause the oxidative stress is Anti
-oxidative defense mechanisms. Antioxidants are those substances which
possesses free radical chain reaction breaking properties.Recently there has
been an upsurge of interest in the therapeutic potential of medicinal plants as
antioxidants in re-antioxidant in reducing oxidative stress induced tissue
injury (Pourmoradet al.,2006) . among
the numerous naturally occurring Antioxidants, ascorbic acid, carotenoids and
phenolic compounds are more effective (Duh et
al.,1999).the study is done on medicinal plants and vegetables strongly
supports the idea that plant constituents with antioxidant activity are capable
of exerting protective effects against oxidative stress in biological systems (
Cao et al.,1996).
1.2.2
Antioxidants
and Its Role In Biological System
Antioxidants
is a molecule that inhibits the oxidation of other molecules, Antioxidants are
also phytochemicals, vitamins and other nutrients that protect the cells from
damage caused by free radicals (Baille et
al.,2009). Antioxidants protects
cells against the damaging effects of reactive oxygen, superoxide, nitric
oxide, Peroxyl radicals and so on which results in oxidative stress leading to
cellular damage (Mattson and Cheng, 2006). Natural oxidation play a key role in
health maintenance and prevention of chronic and degenerative diseases such as
atherosclerosis, cardiac, and cerebral ischemia, rheumatic disorder, DNA damage
and aging ( Uddin et al.,2008) .
Antioxidants exert their activity by scavenging free radicals are unstable
chemical species which tends to trap electrons from surrounding. These
radicals, if not scavenged effectively in time, they may damage crucial
biomolecules. Plants also contain a wide variety of free radicals scavenging molecules
like phenols, flavonoids, vitamins, terpenoids, tannis and other phytochemicals
that are rich in antioxidant activity (Cai et
al., 2005).
1.2.3
DPPH
as an antioxidant assay
DPPH
is a common abbreviation for an organic chemical compound 2,2-diphenly-1-picrylhydrazyl.
It is a dark coloured crystalline powder composed of stable free radical
molecules. DPPH is applicable in laboratory research to monitor chemical
reactions involving radicals most notably in an antioxidant assay (Sharma and
Tej, 2009). DPPH is a well-known radical and a trap for other radical
therefore, rate reduction of a chemical reaction upon additionof DPPH, is used
as an indicator of the radical nature of that colour in solution and it becomes
colourless or pale yellow when neutralized as a result of strong absorption
band centered at about 520nm. This property allows visual monitoring of the
reaction and the numberof initial radicals can be counted from the damage in
optical absorption at 520nm (Mar, 1997) since DPPH is an efficient radical
trap, it is also a strong inhibitor of radical mediated polymerization (Cowie
and Arrighi, 2008).
1.2.4
Nitric
oxide as an antioxidant assay
Nitric
oxide (NO) is a colourless gas and a binary molecule acting as a free radical
which has been an important intermediate and biological regulator component in
the fields of neuroscience, physiology and immunology. Nitric oxide has been
produced by numerous cells which are involved in the immune response, in
particular cytokine-activated macrophages have been noted to produce high
levels of NO which is involved in the killing of targeted cells as tumors and
bacteria ( Shirawasa et al.,2000) moreover , Nitric oxide
acts as a mediator in inflammatory process by which Nitric oxide acts as a
mediator in inflammatory process by which Nitric oxide enhanced the
cyclooxygenases(COX) enzymes effect ultimately leading in increased production
of Pro-inflammatory eiconsids ( Kousai et
al.,2004). Moreover, Nitric oxide has the capability to react with
metelloxo and metalloxo complexes. Further, the presence of NO has been noted
to prevent peroxide mediated tissue damage by scavenging metal Oxo spices
(Liebman et al.,2011). It inhibits lipid oxygenase activity by
reacting with non heme iron at the active site. No plays modulatory roles in
fighting cardiovascular diseases (Watanabe, 2011).
1.2.5
Lipid
Peroxidation as an antioxidant assay
Currently,
lipid peroxidation is considered as the main molecular mechanism involved in
the oxidative damage to cell structures and in the toxicity process that lead
to cell death. Researchers considered that lipid peroxidation was the
consequence of toxic metabolites that produced highly reactive species,
disruption of the intercellular membrane and cellular damage (Dianzani and
Barrera, 2008). Lipid peroxidation is a complex process known to occur in both
plants and animals. It involves the formation and propagation of lipid
radicals, uptake of oxygen, rearrangement of the double bonds in unsaturated
lipids and eventual destruction of membrane lipidswith the production of
variety of breakdown products. The mechanisms of biological damage and toxicity
on biological system are explained by the sequential stages of reversible
oxidative stress and irreversible oxidative damage( Boveris et al.,2008). Lipid peroxidation has a
role in pathogenesis in several pathologies as neuro degradative, inflammatory
and infectious diseases (Reptto et al.,
2010).
1.3 JUSTIFICATION
Antimicrobial
diseases still remain one of the major causes of human morbidity and mortality
in developing countries, especially in rural areas, where there is inadequate
production of pharmaceuticaldrugs, the possibility of use of plants that are
inexpensive and easily available such as Amaranthus
spinosus Linn. The use of Amaranthus spinosus
Linn. Has been revealed by many herbal practitioners to cure STDs diseases,
but there was no scientific prove or backup. Base on this background, the
present study was intended to muster or gather information about the
antioxidants and antimicrobial activities of Amaranthus spinosus root Linn.
1.4 AIMS AND OBJECTIVES
Ø To
determine the Antimicrobial Activity of
Ethanolic extract of root of Amaranthus spinosus
on selected clinical isolates.
Ø To
Evaluate the Antioxidant Activity of root extract of Amaranthus spinosus
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