ABSTRACT
The antimicrobial activity and phytochemical analysis on the leaves of Carica papaya, Citrus sinensis, Citrus paradisi and Vernonia amygdalina was carried out. The four plant species were found to contain alkaloid, saponin, tannins, flavonoid and phenols in varying proportions. The concentration of the phytochemicals are as follows: alkaloid of paw-paw (1.11 ± 0.02%), saponins (0.53 ± 0.04%) tannin (0.43 ± 0.4%), flavonoid (0.67 ± 0.01%) and phenols (0.91 ± 0.03%), for grape alkaloid content was (0.31 ± 0.05%), saponins (0.19 ± 0.01%), tannins (0.25 ± 0.01%), flavonoid (0.36 ± 0.02%), phenol (0.52 ± 0.04%) In orange, alkaloid was (0.23 ± 0.01%), saponin (0.23 ± 0.01%), tannin (0.15 ± 0.01%), flavonoid (0.33 ± 0.01), phenol (0.59 ± 0.07%) For bitter leaf, alkaloid (1.31 ± 0.03%), saponin (1.15 ± 0.01%), flavonoid (0.71 ± 0.02%), tannin (0.43 ± 0.01%), phenol (1.15 ± 0.01%). The plant contain more alkaloid than other phytochemical. The anti-microbial activity of the ethanolic and aqueous extracts of leaves of the plants were tested against four microorganism. Klebsiella pneumonia, Salmonella pullorum, Enterobacter aerogenes and Proteus vulgaris. The susceptibility of the microorganisms was determined using the disk diffusion method. The leaf extract of these plants inhibited the growth of these pathogens. The minimum inhibitory concentration was analyzed using agar well diffusion method with values ranging between 50 mg/ml and 200 mg/ml against all the clinical isolates. There were variations in the lowest concentrations of the different plant extracts that caused inhibitions against the various microorganisms.
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgement v
Table of Contents vi
List of Tables viii
List of Figures ix
Abstract x
CHAPTER ONE
1.0 INTRODUCTION
1.1. Aim and Objectives 3
1.2 Justification 3
1.3 Botany of Carica papaya (Paw-Paw) 3
1.4 Botany of Citrus sinensis (Orange) 4
1.5 Botany of Vernonia amygdalina (Bitter leaf) 5
1.6. Botany of Citrus Paradisi (Grape) 6
CHAPTER TWO
2.0 LITERATURE
REVIEW
2.1 Phytochemical
Screening of Plants 7
2.2 Antimicrobial
Activity of Plants 8
CHAPTER TWO
3.0 MATERIALS
AND METHOD 9
3.1 Collection of Plant Materials and
Identification 9
3.1.1 Preparation
of the Sample for Analysis 9
3.2 Phytochemical Analysis 10
3.2.1 Qualitative Analysis on Leaf Extract 10
3.2.1.1
Test for the Presence of Tannins 10
3.2.1.2
Test for the Presence of Saponins 10
3.2.1.3
Test for the presence of Flavonoids 10
3.2.1.4 Test
for the presence of alkaloids 10
3.2.1.5 Test for
the Presence of phenols 11
3.3 Quantitative
Analysis of Phytochemicals 11
3.3.1 Determination
of Tannins 11
3.3.2
Determination of Alkaloid 12
3.3.3 Determination of Saponin 12
3.3.4 Determination of Phenols By
Spectrophotometer Methods 13
3.3.5 Determination of flavonoid 14
3.4 Determination
of anti- microbial activity 14
3.4.1 Preparation of the plant extracts 14
3.4.2
Sources
of pathogen 15
3.4.3 Anti-microbial Test 15
3.4.4 Determination of minimum inhibitory
concentration 16
3.4.5 Statistical
Analysis 16
CHAPTER
FOUR
4.0 RESULT 21
CHAPTER
FIVE
DISCUSSION,
CONCLUSION AND RECOMMENDATION
5.1 Discussion 30
5.2 Conclusion
and Recommendation 31
References 32
LIST
OF TABLES
Table
1: Phytochemical
Sample of Test Plants 21
Table
2: Phytochemical
Content of Test Plants 22
Table 3: Antimicrobial
Activity of Aqueous and Ethanolic Extracts of
leaves
Carica papaya, Citrus paradisi, Citrus
sinensis and
Vernonia amygdalina 23
Table
4: MIC
of extracts of Carica papaya, Citrus
paradisi, Citrus sinensis
and Vernonia
amygdalina concentrations (mg/ml) on Salmonella
pullorum
24
Table 5. MIC
of extracts of Carica papaya, Citrus
paradisi, Citrus sinensis
and
Vernonia amygdalina concentrations
(mg/ml) on Klebsiella
pneumonia 25
Table 6. MIC
of extracts of Carica papaya, Citrus
paradisi, Citrus sinensis and Veronica
amygdalina concentrations (mg/ml) on Enterobacter
aerogenes 26
Table 7. MIC
of extracts of Carica papaya, Citrus
paradisi, Citrus sinensis
and
Veronica amygdalina concentrations
(mg/ml) on Proteus vulgaris 27
LIST
OF PLATES
Plate
1. The
leaves of Carica papaya 17
Plate 2 The
leaves of Vernonia amygdalina 17
Plate 3 The
leaves of Citrus sinensis 18
Plate 4 The
leaves of Citrus paradise 18
Plate 5: Zone
of inhibition for aqueous and ethanol leaf extract of Vernonia
qamygdalina
on Salmonella purthorum 19
Plate 6: Solidified
Muller Hinton agar in sterile petri dishes 20
CHAPTER ONE
1.0 INTRODUCTION
An
anti-microbial is an agent that kills micro-organism or stop their growth It is
derived from Greek word. Anti (against) micros (little) and bio (life) and
refers to all agent that act against microbial organism or substance of natural
semi-synthetic origin that kill or inhibits the growth of the host. (Setzer et al, 2000). Anti-microbial medicine
can be grouped according to the micro-organisms they act primarily against for
example antibiotics are used against bacteria and antifungals are used against
fungi. They can also be classified according to their functions: agents that
kill microbes are called microbial while those that inhibit the growth are
called biostatics.
A special
features of higher plants is their capacity to produce large number of organic
chemicals called the secondary metabolites (Agatemar et al, 2009). Plants are rich in a wide variety of second
metabolite with anti-microbial properties such as flavonoid, tannins, phenols
and alkaloids (Almomam, 2007). The study of most research recently is to
evaluate the anti-microbial activity of some local natural plants which have
potential of treating infectious diseases and with lessen side effects compared
to the synthetic drug agents. Plant based drugs have survived through ages and
it is still catering for health needs of millions all over the world (Kapor,
2011). The use of plant extracts and phytochemical, both with known
antimicrobial properties can be great significance in the treatment of
diseases. Many plants have been used because of their anti-microbial properties
of traits. This is mostly due to synthesis of secondary metabolism by the plants
(Prusti, 2008). Discovery of new antimicrobial compounds with diverse chemical
structures and novel mechanism of action becomes of urgent attention.
Currently, the development of resistant strains of bacteria has increased the
need for new antibiotics (Eloft, 1998). The antimicrobial compounds produced by
plants are active against plants and human pathogenic microorganism (Sarac and
Ugwu, 2007).
Isolation of
bacteria less susceptible to regular antibiotics and recovery of resistant
isolates during antibacterial therapy is now a global problem (Muhammed and
Muhammed, 2005). In the developing world, the situation is even worse because
of poor sanitation and ignorance of good hygiene practice thus exposing a large
number of people to infectious agents.
Phytochemicals
are chemical compounds that occur naturally in plants (phyto means “plants” in
Greeek). Some are responsible for color and other organoleptic properties, such
as the deep purple of blueberries and the smell of ginger phytochemical has
bio-active constitutes such as alkaloids, tannis, flavonoids, saponins and
phenolic compounds (Okwu, 2001). They are also found in vegetables and fruits.
Plants are
composed entirely of chemicals of various kinds (Breslin, Andrew, 2007).
Phytochemicals are chemical produced by plants through primary or secondary
metabolism (Harbone, 1999). They generally have biological activity on
predadors and help in plant growth.
The medicinal
values of some plants lie in chemical substances that produce definite physiological
actions in the human body. The most important of these bioactive constituents
are alkaloids, tannins, flavonoids and phenolic compounds. Many of these
indigenous medicinal plants are used as spices and food plants (Bratner, 1994).
Plant serves as rich resources of natural drugs for research and development
(Kong et al., 2008). The beneficial
medicinal effects of plant materials typically results from the combinations of
secondary products present in plants. The medicinal actions of plants are unique
to particular plant species or groups as the combination of secondary products
in a plant are often taxonomically distinct (Ali et al., 2008).
Phytochemicals
generally are regarded as research compounds rather than essential nutrients
because proof of their possible health effect has not been established yet
(Oregoh, 2017). Phytochemical are usually extracted, isolation of compounds
from plants followed by defining their structure or testing in laboratory model
systems.
1.1. Aim
and Objectives
The aim and
objectives of this work are to :
i) determine the phytochemicals of Citris paradisi (grape), Citrus
sinensis C. (orange), Carica papaya
(paw-paw) and Vernonia amygdalina (bitter
leaf).
ii) determine antimicrobial activities of the aqueous and
ethanolic leaf extracts of Citris
paradisi, Citrus sinensis Carica papaya and Vernonia amygdalina, on Salmonella
pullorum, Klebsiella pneumonia, Enterobacter aerogenes and Proteus vulgaris
1.2 Justification
The use of plant
products in the treatment of diseases has been controversial, some people are
of the view that it is not good for the health, others said that it could lead
to more deadly diseases while others are of the view that it does not have a
prescribed dosage. The result of this study will therefore
(i)
demonstrate
the constituents of the plant extracts that enable it to attack the pathogens
responsible for bacterial infection.
(ii)
the
concentrations of plant extract that could be effective in treating microbial
infection.
1.3 Botany of Carica papaya (Paw-Paw)
The genus Carica belongs to the family Caricaceae which is usually a
single-stemmed, semi-woody giant herb with fast, indeterminate growth (3m
during the first year). Some branding may occur if apical dominance is lost due
to tip damage and in tall plants (Morton, 1987). Carica papaya is a sole species of the genus Carica. Caricaeceae, a family well represented in the neotropics that
includes six genera with at least 35 species (Fisher 1980; Ming et al., 2008; Carvalho and Rennel,
2013). It is spread to many tropical and subtropical regions around the world
(Kim et al., 2002). The plant
produces large palmate leaves (0.6m2) with five to none pinnate
lobes of various widths between 40 to 60cm (Ming et al., 2008). The papaya plant develops very fast taking 3-8
months from seed germination to flowering and 9-15 months for harvest (Paterson
et al., 2008). The leaves are used in
herbal medicine to remove intestinal worms. The stem grows from 5 to 10m (16 to
33ft) tall, with spirally arranged leaves confined to the top of the trunk. The
lower trunk is conspicuously scarred when the leaves and fruit were borne. The
leaves are large, 50-70cm (20-28m) in diameter, deeply palmately lobed, with
seven lobes, all parts of the plant contain latex in articulated laticifers (Hemood
et al 2007). The ripe fruit of papaya
is usually eaten raw without skin or seeds. The unripe green fruit can be eaten
cooked, usually in curries, salads and stew. (Netsawan et al 2013).
1.4 Botany of Citrus sinensis (Orange)
The genus citrus
belong to the family Rutaceae. The
orange tree is evergreen, flowering tree, with an average height of 9 to 10m
(30 to 33ft), although some very old specimens can reach 15m (49ft) The leaves
are oval, alternatively arranged 4 to 10cm and have crenulate margins. Sweet
orange grow in range of different sizes, and shapes varying from spherical to
oblong. When unripe, the fruit is green, the irregular rind of the ripe fruit
can range from bright orange to yellow-orange. Sweet orange were mentioned in
Chinese literature in 314BC. As of 1987 orange trees were found to be the most cultivated
fruit tree in the world (Willard, 1989). It is widely grown in tropical and
sub-tropical climates for their sweet fruit, the fruit can be eaten fresh, or
processed for its juice or fragrant peel. As of 2012, sweet orange accounted
for approximately 70% of citrus production. In 2014, 70.9million tonnes of orange
were grown worldwide, with Brazil producing 24% of the world total followed by
China and India. The orange peel is used as a dried seasoning, orange are
effective in the management of asthma, arthritis, cholera etc. Citrus sinensis (orange) peel extracts contain bioflavonoids including
poly methoxylated flavours (pmfs), which have anti-inflaammatory and
hypolipodemic effects (Julius et al.,
2009).
1.5 Botany of Vernonia amygdalina (Bitter leaf)
Vernonia amygdalina, a member of the Asteraceae family, is a shrub or small tree of 2-5m with petiolate
leaf of about 6mm diameter and elliptic shape. The leaves are green with a
characteristics odour and a bitter taste. No seeds are produced and the tree
has therefore to be distributed through cutting. It is known in Nigeria local
languages as etidot (Efila), uzi (Ebira), onughu (Igbo) and chusar duku
(Hausa). Elsewhere in Africa it is called ndole (Cameroon), and tuntirano
(Tanzania). (Mbang et al, 2008). It
grows under a range of ecological zones in Africa with 200 species of Verona,
the leaves are used for human consumption and washed before eating to get rid
of the bitter taste. They are used as vegetable and stimulate the digestive
system, as well as they reduce fever.
They are used as
local medicine against leech. Free living chimpanzees eat the leaves, if they
have attacked by parasite. Vernonia
amygdalina is also used, instead of hops to make beer in Nigeria It is used
in medicine as anti-malarial, anti-microbial laxative, antihelmitic, antithrombotic
and both hypoglycemic and hypohpidaemic effect in diabetic – hyperlipidamic
also in breast cancer treatment. Vernonia
amygdalina can also be used alone or in combination with known drugs. Vernonia amygdalina extracts may help
suppress, delay kill cancerous cell in many ways, such as induction of
apoptosis as determined in cell culture and animal studies (Song et al (2005). Other medicinal importance
include, it helps on digestive system and reduce fever, it is also herb against
parasites. (Atanwho et al, 2010, Ho et al., 2012). Vernonia
amygalina also enhances chemotherapy sensitivity, extract may render
cancerous cells to be more sensitive to chemotherapy (Sweemy, 2005).
1.6. Botany of Citrus Paradisi
(Grape)
Citrus paradise (grape) belong to the family Rutaceae, order Sapindales and species Citrus paradise, it is a subtropical citrus tree known for its sour
to semi-sweet somewhat bitter fruit. (Fraseret
al., 2003), The evergreen grape fruit trees usually grow to around 5-6
meters (16-20ft) tall, though they can reach 13-15m (43-49ft), the leaves are
glossy dark green, long (up to 15 centimeters (5.9inches) and thin. It produces
5cm (2inches) while four-petaled flowers. The fruit is yellow-orange skinned
and generally an oblate spheroid in shape, it ranges in diameter from 10-15cm
(3.9 – 5.9inches). The flesh is segmented an acidic varying in color depending
on the cultivars which include, white, pink and red pulps of varying sweetness.
Grape has been investigated in cancer medicine pharmacodynamics and also good
source of vitamin C (Fraseret al.,
2003, Duarte et al., 2010). The genetic origin of the grape is a hybrid mix (Xiaomeng
et al 2017).
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