ABSTRACT
The ethanoic and aqueous extract of seeds of Alligator pepper, Avocado pear, Bitter kola and Paw-paw were subjected to phytochemical screening and antimicrobial activity study. The study indicates that the extract of the seeds of Bitter kola (ethanoic) was active against all the test organisms at 100mg/ml zone of inhibition ranging from 8.33mm in Staphylococcus aureus and Pseudomonas aureginosa to 11mm in Salmonella typhi. The ethanoic extract of Paw-paw inhibited Pseudomonas aureginosa the most at 100mg/ml at the diameter of 10.33mm. The aqueous extract of Avocado pear seed best inhibits Pseudomonas aureginosa at 150mg/ml at 8.33mm, Alligator pepper aqueous extract inhibits two test organisms at 150mg/ml ranging from 9mm to 9.67mm in Staphylococcus aureus and Bacillus substilis respectively. The result reveals the presence of compounds in the seed extract and were found to be a rich source of phytochemical. The studies on Bitter kola, Paw-paw, Avocado pear and Alligator pepper extract exhibited their antimicrobial potential which could be exploited for pharmaceutical purposes and therapeutic potentials. The minimum inhibitory concentration (MIC) of extracts were determined for the various organism. Staphylococcus aureus has a remarkable inhibition with the aqueous extract of Carica papaya at the diameter 8.33 1.53 as with ethanoic extract of Aframomum melegueta that inhibits staphylococcus aureus at 17.667 1.53, Salmonella typhi was best inhibited with the aqueous extract of Persea Americana at the diameter of 8.663 1.53 against Garcinia kola that inhibits Salmonella typhi at the diameter of 20.333 .Bacillus substilis was inhibited at a good diameter of11.667 1.15 with aqueous extract of Carica papaya against ethanoic extract of Garcinia kola at the diameter of 21.000 2.00 and Pseudomonas aeroginosa was inhibited with the aqueous extract of Aframomum melegueta against a high diameter of 19.667 1.15. The standard antibiotic used showed the highest diameter of inhibition constituent against the four test seed sample.
TABLE OF CONTENT
TITLE
PAGE
........................................................................................................................I
CERTIFICATION
..................................................................................................................II
DEDICATION
....................................................................................................................III
ACKNOWLEDGEMENT
........................................................................................................IV
TABLE
OF CONTENT
............................................................................................................V
LIST
OF TABLES
....................................................................................................................VII
ABSTRACT
..............................................................................................................................VIII
CHAPTER
ONE
1.0 INTRODUCTION
...................................................................................................................1
1.1
BOTANY OF AFRAMOMUM MELEGUETA
........................................................................3
1.2
BOTANY OF GARCINIA KOLA .............................................................................................4
1.3
BOTANY OF AMERICANA PERSEA.......................................................................................5
1.4
BOTANY OF CARICA PAPAYA ..............................................................................................6
1.5
OBJECTIVES OF THE RESEARCH .......................................................................................7
CHAPTER
TWO
2.0
LITERATURE REVIEW
..........................................................................................................8
CHAPTER
THREE
3.0
MATERIAL AND METHOD .................................................................................................10
3.1
COLLECTION AND IDENTIFICATION OF SAMPLE
......................................................10
3.1.1.
SAMPLE PREPARATION .................................................................................................10
3.1.2.
PHYTOCHEMICAL SCREENING
...................................................................................10
3.2.0.
QUALITATIVE ANALYSIS OF PHYTOCHEMICAL
....................................................10
3.2.1
TEST FOR TANNIN
...........................................................................................................10
3.2.2.
TEST FOR SAPONIN
........................................................................................................11
3.2.3.
TEST FOR
FLAVOINOID..................................................................................................11
3.2.4.
TEST FOR ALKALOID
.....................................................................................................11
3.2.5.
TEST FOR PHENOL
..........................................................................................................12
3.3.0.
QUANTITATIVE DETERMINATION OF PHYTOCHEMICALS
..................................12
3.3.1. DETERMINATION OF ALKALOIDS
..............................................................................12
3.3.2.
DETERMINATION OF FLAVONOID
.............................................................................13
3.3.3.
DETERMINATION OF PHYTATE ...................................................................................13
3.3.4.
DETERMINATION OF TANNIN
.....................................................................................14
3.3.5.
DETERMINATION OF SAPONIN ...................................................................................15
3.3.6.
DETERMINATION OF PHENOL
.....................................................................................16
3.3.7.
DETERMINATION OF CYANOGENIC GLYCOSIDE
...................................................17
3.3.8.
DETERMINATION OF OXALATE
..................................................................................17
3.3.8.1.
DIGESTION......................................................................................................................18
3.3.8.2.
OXALATE PRECIPITATION
........................................................................................18
3.3.8.3.
PERMANGANATE TITRATION
...................................................................................18
3.3.9.
ANTIMICROBIAL ACTIVITY
.........................................................................................19
3.4.0.
STATISTICAL ANALYSIS
...............................................................................................19
CHAPTER
FOUR
4.0.
RESULT
.................................................................................................................................20
4.1.
PHYTOCHEMICAL COMPOSITION
.................................................................................20
4.2.
ANTIMICROBIAL ACTIVITY
............................................................................................21
4.3.
ZONE OF INHIBITION
........................................................................................................23
CHAPTER
FIVE
5.0.
DISCUSSION
........................................................................................................................25
5.1. CONCLUSION
......................................................................................................................27
5.2. RECOMMENDATION
..........................................................................................................27
REFERENCES
......................................................................................................................28
APPENDIX
...........................................................................................................................33
LIST OF TABLES
TABLE
1: The qualitative
analysis of the phytochemical presence in the seeds of Carica Papaya, Garcinia Kola, Aframomum melegueta, and Americana Persea
TABLE
2: The percentage of
alkaloid, tannin, saponin, flavonoid, phenol, oxalate, cyanogenic glycoside in
the seeds of Carica Papaya, Garcinia
Kola, Aframomum melegueta, and Persea americana
TABLE
3: Antimicrobial
activity of the seeds of Carica Papaya,
Garcinia Kola, Aframomum melegueta, and Persea americana
TABLE
4: Zone of inhibition of
Salmonella typhi using the four seed
extract
TABLE
5: Zone of inhibition of
Staphylococcus aureus using the four
test seeds samples
TABLE
6: Zone of inhibition of
Pseudomonas aeruginosa using the four
test seed samples
TABLE
7: Zone of inhibition of
Bacillus substilis using the four
test seed samples
CHAPTER ONE
1.0
INTRODUCTION
The use of plants and plant products for
treatment, control and management of diseases is as old as mankind. Plants are
able to synthesize and accumulate active substances as naturally occurring chemical
compounds and some of them are able to provide health benefits to human.
The aims of using plants as source of
therapeutic agents are to isolate its bioactive compounds which are directly
used as drug e.g. atropine, digoxin, digitoxin, morphine and taxol and also in
the production of pharmacological tools e.g. lysergic and diethylamide. Also
the use of some plant parts or whole plant parts as herbal remedy was reported
by Fabricants and Farnsworth, (2001)
In recent years, drug resistance to human
pathogenic bacteria has been commonly reported from all over the world (Piddock
et al., 1989; Singh et al., 1984; Mulligen et al., 1993, Davis, 1994; Robin et al., 1998).
However, the situation is commonly
alarming in developing as well as developed countries due to indiscriminate use
of antibiotics. Therefore, alternative antimicrobial strategies are urgently
needed, and thus this situation has led to a re-evaluation of the therapeutic
use of remedies from plant. (Levy and Marshall, 2004; Basualdo et al., 2007).
According to World Health Organization
(2001), medicinal plants would be best source to obtain variety of drugs.
Medicinal plants are the richest bio-source of drugs, modern medicine, food
supplements, folk’s medicine, pharmaceutical intermediates and chemical
entities (Ncube et al., 2008).
The medicinal value of these plants lie on
some chemical active substances found in the plants known as their
phytochemical content that produce definite physiological action on the human
body. The most important phytochemical content of the plants are alkaloid,
tannins, saponins, steroids, phenolic compounds and cyanogenic glycoside
(Edeoga et al., 2005).
Natural products of some typical plants
may possess a new source of antimicrobial agents with possible novel mechanism
of action (Ahmed and Aquil, 2007). They
are effective in treatment of infectious diseases, while simultaneously
mitigating many of the side effects that are often associated with the
synthetic antimicrobials (Iwu et al, 1999).
Many studies have been undertaken with the aim of determining the different
antimicrobial and phytochemical constituents of medicinal plants and using them
for the treatment of both tropical and systematic microbial infection and also
as possible alternative to synthetic drugs to which many infectious
microorganisms have become resistant to (Akinpelu et al, 2006; Chopra, 1992).
Alkaloids has been reported as vital constituents
in drug production and are used in the production of steroidal drugs (Maxwell et al, 1995). Saponins are used as an
emulsifying agent in medicine, Tannin have been found to possess oxidative
inhibitory property (Ihekoronye et al,
1985). Also proximate content of some Nigeria plants have been studied to be
good source of carbohydrate, protein, ash, food energy, crude fibre, mineral
and vitamins (Aletor and Adeogun, 1995).
These compounds that are antimicrobial
agents can also be used to inhibit the growth of microorganism depending on
their phytochemical properties such microorganisms include Staphylococcus aureus, Salmonella typhi, Pseudomonas aeroginosa, Bacillus
subtilis. Staphylococcus aureus
is a gram positive bacteria which can be inhibited by antibacterial agent from
plant extract Against these background, these project was designed to study the
antimicrobial activity of the seeds of four plants against some bacterial
pathogens with a view to ascertain their relative potencies. The extract for
the inhibition of the microorganism were gotten from the seeds of Carica papaya L, Garcinia kola Heckel, Aframomum
melegueta K. Schum and Persea americana
Mill
1.1.2. BOTANY
OF AFRAMOMUM MELEGUETA K.Schum (Grain
of Paradise)
Aframomum
melegueta is a species in the ginger family, Zingiberaceae.
These species commonly known as Ossame, Grains of paradise, Melegueta pepper, Alligator
pepper, Black pepper, Guinea grain, Guinea pepper is obtained from the ground
seed.
It impart a pungent, peppery flavour with
hint of citrus. Although it is native to West Africa, it is also an important
cash crop in the Basketo district (Basket special woreda) of Southern Ethopia
(SNNPR, 2005).
A.
melegueta is a herbaceous perennial plant native to swampy
habitat along the West African coast. Its trumpet-shaped purple flower develop
into 5 – 7cm long pods containing numerous small, reddish – brown seeds. The
pungent peppery taste of the seed is caused by aromatic ketones e.g. 6-paradol
(systematic name: 1-4-hydroxyl – 3 – methoxylphenyl-decan-3-one).
Essential oils, which are the dominating
flavour component in the closely related cardamom occur only in traces(Paul
1995).The stem at time can be short and usually show signs of scars and fallen
leaves. The average leaves are usually 35cm in length and 15cm wide, with a well-structured
vascular system. The flower of the herbaceous plant are described as “handsome”,
aromatic with an orange – coloured lip and rich pinkish orange upper part. The
fruit contain numerous small, golden brown seeds.
Aframomum
melegueta is used in the cuisines of West and North Africa, where
it has been traditionally imported by Caravan route through Sahara desert and
whence they are distributed to the rest of Italy.
They were rename “grain of paradise” a
substitute for the formerly called black
pepper in Europe in the 14th and 15th centuries (Daniel
and Austin, 2004).
It is used to improve the “stale smell” of
wine, it is also used in cardiovascular treatment of lowland gorillas in the
zoo. (Dybas et al., 2007). It is used
in religious (voodoo) rite in
Caribbean and Latin America. (Moret, 2013). It is used as addictive to impact
heat and pungent aroma to food.
Sofowara (1993), reported the use of Aframomum melegueta in traditional
medicinal formula for the treatment of stomach ache, tuberculosis, diarrhoea
and remedy for snake and scorpion bite and stings.
1.1.3. BOTANY
OF GARCINIA KOLA
Garcinia
kola (Bitter kola, a name sometimes also used for G. afzelii) is a species of flowering
plant in the Clusiaceae family. It is
found in Benin, Cameroun, Democratic Republic of Congo, Ivory Coast, Gabon,
Ghana, Liberia, Nigeria, Senegal and Sierra leone.
Its natural habitat is subtropical or
tropical moist lowland forests. Garcinia
kola or bitter kola is a tree that grows in rain forest of West Africa. The
fruit, seeds, nuts and bark of the tree of the plant have been used for
centuries in folk medicine to treat ailments from cough to fever.
According to a report from the centre of
International Forestry Research, Garcinia
kola trade is still important to the tribes and villages in Nigeria.
As with any herb, never consume Garcinia kola in large quantity without
first discussing it’s use and benefit with your physician especially if you are
currently being treated for other medical condition.
Traditionally, Garcinia kola is used by African herbal practitioners who believe
that it has purgative, antiparasitic and antimicrobial properties. The seeds
are used for bronchitis, throat infection, colic, head and chest colds and
cough. It is also used for liver disorder and as chewing stick
1.1.4. BOTANY
OF PERSEAAMERICANA
Avocado pear is a tree that is native to
South Central Mexico classified as a member of the flowering plant family Lauraceae (Morton, 1987). Avocado pear (also
alligator pear) is botanically a large berry containing a single seed (Storey,
1973). They have a green-skinned, fleshly body that may be pear shaped, egg
shaped, or spherical and fleshy body that may ripen after harvesting. Avocado
tree are partially self-pollinated and often are propagated by grafting to
maintain a predictable quality and quantity of the fruit.
Persea
American is believed to originate in the state of Puebla, Mexico
(Galindo – Jovar et al., 2007).
Through Fossil evidence suggest similar species were much more widespread millions
of years ago occurring as far Northern California This took place when the
climate of that region was more hospitable to them (Morton, 1987).
The tree grows to 20m (66ft) with
alternately arranged leaves of 12 – 25cm (4.7 – 9.8in) long. The flower are
inconspicuous greenish yellow, 5 – 10mm 90.2 – 0.4in) wide. The pear – shaped
fruit is 7 – 20cm (2.8 – 7.9in) long, weighing between 100 and 1,000g 93.5 and
35.5oɛ), and has a large central seed, 5 – 6.4cm (2.0 – 2.5in) long.
It can also be propagated by seed, taking
roughly four to six years to bear fruit. Although in some case seedling can
take 10 years to bear fruit. (Boning, 2006; Morton, 1987).
Avocado tree is vulnerable to bacterial,
viral, fungal and malnutrition disease especially when there is excess and
deficiency of key nutrient materials.
Disease can affect all parts of the plant
causing spotting, rotting, cankers, pitting and discoloration (Ohr et al., 2003).
1.1.5. BOTANY
OF CARICA PAPAYA
Paw-paw is the fruit of the plant Carica papaya, and is one of the 22
accepted species in the genus Carica
of the plant family Caricaceae.
It is native to the tropic of the
Americans, perhaps from the southern Mexico and neighbouring Central America.
(Morton, 1987). It was first cultivated in Mexico several centuries before the
emergence of the Mesoamerican classical civilization. The papaya is the large,
tree-like plant, with a single stem growing from 5 – 10cm (16 – 33ft) tall with
a spirally arranged leaves confined to the top of the trunk. The lower trunk is
conspicuously scarred where leaves and fruits were borne. The leaves are large,
50 – 70cm (20 – 28in) in diameter, deeply palmately lobed with seven lobes
usually for such large plant. The tree is dioecious, usually unbranched, unless
loped. The flowers are large with fruits 15 – 45cm (5.39 – 17.7in) long and 10
– 30cm (3.9 – 11.8in) in diameter. The fruit is a type of berry (Rivra-Pistrana et al,
2010).
It is ripe when it feels soft (as soft as a ripe avocado or a bit softer) and
its skin has attained an amber to orange colour.
Carica
papaya was the first transgenic fruit tree to have it genome
sequenced. Papaya plant grows in
three sexes namely male, female, and hermaphrodite. The male produces only
pollen never fruits. The female will produce small, inedible fruit unless
pollinated. The hermaphrodite can self-pollinate since its flower contain both
male and female ovaries.
Almost all commercial Papaya orchard contain only hermaphrodite Papaya prefer sandy, well-drained soil as standing water will kill
the plant within 24 hours
Papaya
is affected by disease (bacteria, fungi and viral disease) which Papaya ring spot virus is one of the well-known
viruses (Morton, 1987). The first sign
of the virus are yellowing and vein clearing of younger leaves, as well as
mottling yellow leaves. Infected leaves may have blade sticking upward from the
middle of the leaves.
As of 2010, the only way to protect Papaya from these virus is by genetic
modification (Gonsalves et al.,
2010).
Papaya
mosaic virus destroys the plant until only a small part of leaves are left. The
ripen fruit of the Papaya is usually
eaten raw, without skin or seeds. The unripe green fruit can be eaten cooked,
usually in curries, salad and stews. Green Papaya
is used in Southern Asian cooking (Natty, 2013). Papaya flower are used in vegetable dish preparation, the black seeds
of the Papaya are edible and have a
sharp, spicy taste (Morton, 1987). They are sometimes grounded and used as a
substitute for black pepper or alligator pepper. Papaya skin, pulp and seed contain a variety of phytochemicals
including carotenoids and polyphenol (Rivera-pastrana et al., 2010). Papaya also contain cyanogenic
substance, “Prunasin” In some part of the world, Papaya leaves are made into tea as a treatment for malaria, but the
mechanism is not understood and no treatment method based on these result has
been scientifically proven. (Titanji et al., 2008).
1.1.6. OBJECTIVES
OF THE RESEARCH
The general objectives of the work is to
determine the antimicrobial effects of the seeds of the test plants, Carica papaya L,Garcinia kola HECKEL, Aframomum
meleguata K. Schum Persea americana Mill extract on specific
bacteria pathogen including
staphylococcus aureus, Bacillus Subtilis, Psuedomonas aeroginosa and Salmonella
typhi.
The specific objectives include the
following:
1.
To determine the phytochemical
constituents of the test plant seeds.
2.
To determine the activity of the extract
against bacteria.
3.
To assess the potency of the extract
relative to standard antibiotic
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