ABSTRACT
Aqueous (cold and hot water)
and ethanol extracts of Piper guineense
seeds were screened for antibacterial activity against four microbes, Escherichia coli, Salmonella typhi, Pseudomonas
aeruginosa and Staphylococcus aureus.
The aqueous extract of this plant showed minimal level of inhibition on E.coli and S.aureus at the highest concentration (2000µg/ml),with no effect at
all on P.aeruginosa and S. typhi. The ethanol extract exhibited
more inhibitory effect on Staphylococcus
aureus, E. coli, and Salmonella typhi with no effect on Pseudomonas aeruginosa. The minimum inhibitory concentration of the
aqueous extract (cold and hot water extract) was at 500µg/ml, while that of
ethanol was at 62.5µg/ml. The qualitative determination of chemical constituent
of the plant confirmed the presence of certain metabolites such as Alkoloids,
Saponins, Tannins, Oleoresin and Glycosides. The results obtained show that the
extract of Piper guineense possess
some level of antibacterial activity against some common pathogens.
TABLE OF CONTENTS
Cover page i
Certification ii
Dedication iii
Table of contents
Acknowledgements iv
Abstract v
CHAPTER
ONE
1.0 INTRODUCTION 1
1.1 OBJECTIVE
OF THE WORK 3
1.2 LITERATURE REVIEW 4
1.2.1 Morphology and
Description 4
1.3 ECOLOGY AND METHOD
OF CULTIVATION 5
1.3.1 Vegetative
propagation: 5
1.3.2 Diseases Of Plant 7
1.4 USES OF PIPER GUINEENSE (UZIZA) 9
1.4.1 Medicinal Uses 9
1.4.2 Culinary Uses 9
1.4.3 Pharmacological,
toxicological and clinical applications: 10
1.4.4 Bioavailability
enhancement: 10
1.4.5 As Antioxidant: 10
1.5 CHEMICAL
CONSTITUENTS OF PIPER GUINEENSE 11
1.5.1 Vitamin Content 13
1.5.2 Mineral Content 13
1.6 ROLES OF TEST
ORGANISMS IN RELATION TO DISEASES 13
1.6.1 Staphylococcus aureus 13
1.6.2 Pseudomonas aeruginosa 13
1.6.3 Escheriachia coli 14
1.6.4 Salmonella typhi 15
CHAPTER
TWO
2.0 MATERIALS AND
METHODS 16
2.1 COLLECTION OF PLANT
MATERIAL 16
2.2 PREPARATION OF PLANT
MATERIAL 16
2.3 ETHANOL EXTRACT PREPARATION 16
2.4 AQUEOUS EXTRACT
PREPARATION 16
2.5 CULTURE MEDIA USED 17
2.6 SOURCE AND
MAINTENANCE OF TEST ORGANISMS 17
2.6.1 Gram Staining 17
2.6.2 Catalase Test 18
2.6.3 Coagulase Test 18
2.6.4 Oxidase Test 19
2.6.5 Indole Test 19
2.7 ANTIBACTERIAL
TESTING 19
2.8 DISC DIFFUSION
METHOD 20
2.9 DETERMINATION OF
MINIMUM INHIBITORY
CONCENTRATION (MIC) 20
2.10 DETERMINATION OF
MINIMUM BACTERIAL
CONCENTRATION (MBC). 21
2.11 QUALITATIVE
DETERMINATION OF THE CHEMICAL
CONSTITUENTS 21
2.11.1 Test for Tannins 21
2.11.2 Test for Saponnin 21
2.11.3 Test for flavonoids 22
2.11.4 Test for Alkaloid 22
2.11.5 Test for glycoside 22
CHAPTER
THREE
3.0 RESULTS 23
3.1 Qualitative
Phytochemical Screening 29
CHPTER
FOUR
4.0 DISCUSSION 30
4.1 CONCLUSION 31
4.2 RECOMMENDATION 31
REFERENCES
APPENDICES
LIST OF TABLES
Title page
Table 1: Antibacterial activity of cold water extract of Piper
guineense 24
Table 2: Antibacterial
activity of hot water extract of Piper
guineense 25
Table 3: Antibacterial activity of ethanol extract of Piper guineense 26
Table 4:
shows the minimum inhibitory concentration 27
Table 5 :
shows the minimum bactericidal concentration 28
Table 6: Qualitative Phytochemical Screening 29
CHAPTER ONE
1.1 INTRODUCTION
There is a growing interest in exploiting plants for
medicinal purposes especially in Africa. This stems from the fact that
micro-organisms are developing resistance to many drugs and as such created
situation where some of the common and less expensive anti-microbial agents are
losing effectiveness (Montefore et al., 1989). Herbal medicine which uses medicinal
plants primarily presents as an alternative to such situation (Sofowora, 1993).
These medicinal plants have immensely contributed to the development of human
health and welfare. Concomitantly, there is an increase in data and huge patronage
to herbal products round the world (Elsenberg et al., 1990, Omoseyindemi, 2003).
Studies on the use of plant extracts for control of
diseases have shown the importance of natural chemicals as possible source of
non-phytotoxic and easily biodegradable alternative fungicides and antibiotics
(Akueshi et al., 2002)and the
advantages of these natural plant products include local availability, little
or no toxicity to humans and simple preparation procedures and plant herb
biological control generally favoured as a method of disease management because
it does not have the disadvantages of chemicals and tends to be more durable in
its effect.
Piper guineense
(uziza) common referred to as African black pepper or Ashanti pepper is very
similar to Piper nigrum which is the
true pepper of commerce from which black and white peppers are processed
(Isawumi, 1984). P. guineense belongs
to the family piperaceae. It has more
than 700 species throughout the tropical and subtropical regions of the world.
It is known with different vernacular names in Nigeria, Igbo (Uziza), and
Yoruba (Iyere). P. guineense has
culinary, medicinal, cosmetic and insecticides uses (Dalziel, 1995, Okwute, 1992).
P. guineense insecticidal activity against Zonocerus variegatus is attributed to the piperine-amide composed
by the plant. It is widely consumed in some part of West Africa especially Nigeria
and Ghana on account of its nutritional and medicinal properties. (Negbenebor et al., 1999). P. guineense berries, like other types of pepper, yield an aromatic
essential oil on steam distillation. Nothing is known about the chemical composition of the volatile oil of this plant
in Nigeria until in 1988 when Ekundayo et
al., (1988) first reported chemical composition of this plant. It was
reported that phenyl propanoid-rich essential oil was comprised of myristicin,
Sarison, Safron and elemicin as a dominant component and fifty-one mono and
sesquiterpenoids as a minor component, also preliminary toxicity of the
volatile oil was also evaluated.
In tradition herbal medicine, the seed of Piper guineense are put into variety of
uses for instance, in some parts of Nigeria, the seeds are consumed by women
after childbirth to enhance uterine contraction for the expulsion of placenta
and other remains from the womb (Udoh et
al., 1999). Extracts of P. guineense
has been reported to stimulate digestion of food by stimulating secretion of
digestive enzymes, pancreatic amylase, trypsin and chymotrypsin and is thus used
for the treatment of digestive disorders (Platel and Srinivasan, 2000).
Piper guineense
can also be used as a food preservative. Food poisoning is still a concern for
both consumers and the food industry despite the use of various preservation
methods. Food processors, food safety researchers and regulations agencies are
continuously concerned with the high and growing number of illness out breaks
caused by some pathogenic and spoilage microorganisms in food. (Meng et al., 1998). Consequently there is
considerable interest in ways to stop this upward trend and reduce the
incidence of food poisoning.
Spices and herbs have been added to food since ancient
times, not only as flavoring agents, but also as folk medicine and food
preservatives (Nakatani, 1994).
Organisms such as Escherichia
Coli and Staphylococcus aureus
are associated with the gastro intestinal tract of man and animals. They occur
in other environments. S. aureus is
among the invasive gram positive known as pyogenic coci implicated in several disease
of human. It has been found to be normal flora of upper respiratory tract and
vagina.
S. aureus
has been known to produce heat stable toxins which are implicated in illness
and stomach upset (Mosset et al., 1990).
From literatures, S. aureus has shown
to be very resistant to a wide variety of antibiotics (Nwinyi et al., 2008). E. Coli is a facultative
anaerobe of wide distribution in the environment, it has been implicated in the
cause of diarrhea, Urinary tract infection, meningitis, sepsis,
wound-infections, nosocomial pneumonia and arthritis. Salmonella typhi is of intestinal origin and infection is spread by
consumption of food contaminated by faeces. The clinical syndrome is
gastroenteritis.
Many researchers have reported the plant to possess
antimicrobial activity therefore, this work reports on the study of the
anti-bacterial activity of extracts of Piper
guineense against some common human pathogen.
1.1 Objective of the work
·
To determine the antibacterial activity of
aqueous (cold and hot water) and ethanol extract of Piper guineense against some common pathogens
·
To identify some of the chemical
constituents present in the plant.
1.2 LITERATURE REVIEW
1.2.1 Morphology and Description
Piper guineense is
a spices that belong to the piperaceae family. It is a climbing perennial
forest liana plant with gnarled branch lets spiraling on shrubs to about 10m
(Iwu, 1993). Piper guineense is a
perennial plant that is characterized by heart-shaped leaves and oval
petiolate, alternate 12cm long. The inflorescence is a pedicelled flower spike
between 3 and 6cm long, flowers are greenish yellow and arranged in a spiral
along the spine (Ayodele, 2012). The fruits
from P. guineese have some common and
native names in which it is identified in different areas as locations.
The common names include; West African black pepper,
Uziza, Ashanti pepper, Pepper Benin, False cubeb, Guinea cubeb, Guinea peper,
Dooje. The fruits of piper guineense are a drup mesocarp or fleshy
oval 3-5mm in diameter (Iwu, 1993).
Uziza is highly medicinal. It is found to have a
stimulating property like pepper and excellent flavouring properties. The seeds
are used in preparing pepper soup, especially for lactating mothers. It is
traditionally believed that uziza functions as an ointment for amounting
corpses. It cannot be eaten or consumed raw, rather the goodness in uziza and
the worldwide demand for it is derived from certain of its attributes, which
have been found to be very useful as additives blending of food and pharmaceutical
industries. The attributes are the aroma, the extraction of which is the sole
object of processing uziza either into oleoresin, essential oil, uziza powder
or similar concentrates which can later be used in the manufacture of
confectionery, beverages and pharmaceutical products (oletunji,1980).
1.3
ECOLOGY AND METHOD OF CULTIVATION
Piper guineense
plant has a pan tropical distribution and it is most commonly found in the low
land tropical rainforests, but can also occur in higher elevation life zones
such as cold forests. It is native to tropical regions of central and western
Africa and are semi—cultivated in countries such as Nigeria where the leave is
used as a flavoring for stews (Ayodele, 2012). The trees are widely distributed
in humid forest zones of West Africa. It thrives well in humus rich virgin
soil, fertile, well drake loamy soil. Soil pH should be between 5.5 to 7.0. It
is susceptible to water logging and hence is planted by preparing heap of soil
on field. High humidity helps for luxuriant crop growth and better yield. Very
high or low temperature is not conducive for growth. Temperature ranges between
10-40c. The crop is pollinated through rain drops and hence frequent showers.
It is either propagated vegetatively or by seed (Oletunji et al., 1980), propagated by seed
Propagation by seed is carried out by soaking fully
ripened barriers in water for 2-3days them removing the flesh and drying them
in the shade afterwards, they are planted out in moist, shaded beds filled with
a mixture of humus and lots of sand, at a distance of one hand-width to
one-another. They will begin to germinate after 30 days and can be transplanted
to their final sites after further 6 months-when they have produced 4 leaves
(Udo et al, 2001).
1.3.1 Vegetative propagation:
The most widely spread form of propagation is with the
use of cuttings. They should be selected from the area of the main shoot of a
strong, healthy and highly productive parent plant. Before the shoot is cut
from the main shoot, the vegetative apex, as well as the leaves and lateral
shoots from the 3rd to the 7th knots, should be removed.
As soon as the plants apex has regenerated, the shoot underneath the 7th
knot is removed and planted in a seedling bed. The seedling should be set in
the soil at an angle of 450 with 3-4 knots. The shoots bed must be
kept moist and shaded. The shoots will have taken root after 2 months, whereby
only a 30% rate of success should be expected. They can then be planted at
their final sites (Udo et al., 2001).
1.3.2 Diseases of Piper Guineense
|
Germ
|
Symptoms
|
|
Phytophtera
palmivora
|
Leaf
wilt, yellow discoloring with lot of leaves, shoots and finally entire
plants.
|
|
Fusariu
Solani
|
Leaf
Wilt
|
|
Ganoderma
lacidum
|
Leaf
Wilt
|
|
Colletotriclum,
Rhizoctonia
|
Leaf
Flecks
|
|
Pseudomonas
(Bacteria)
|
Leaf
Flecks
|
PEST
Nematode infestation by meloidogyne Spp. Causes the main problem on conventional pepper
cultivation greater damage, especially in Indonesia are caused by various bugs,
green flies, beetles as well as butterfly, caterpillars.
1.4 USES
OF PIPER GUINEENSE (UZIZA)
The use of Uziza is very rampant though little is
known of its importance in our daily cure of sickness and prevention of allergy
and running nose. Based on the usefulness and importance, among all the spices,
black pepper is commonly referred as “The king of spices”. It is spicy, aromatic
and carminative. It is a natural antioxidant. It acts as anti-inflammatory,
anti-cancer, anti-periodic and anti-pyretic. It is rubefacient in nature and
can help to lower body cholesterol levels. It contains mainly vitamins A, C, E,
K, niacin and B-Carotene and traces of minerals such as iron, calcium,
phosphorus. The amino acids contained by black pepper works for enhancing the bioavailability
of nutrients (Deluxe et al., 2013).
These acid constituents can be used in anti-microbial, anti-bacterial
applications.
1.4.1 Medicinal Uses
Seeds of Piper
guineense have been used internally as medicinal agents for the treatment
of worm infestation, tonsillitis, bronchitis and gastrointestinal diseases.
In traditional herbal medicine, the seeds are put into
variety of uses for instance, in some parts of Nigeria, the seeds are consumed
by women after child birth to enhance uterine contraction for expulsion of
placenta and other remains from the womb (Okigbo et al., 2005). Also the powder from the dried fruits mixed with
honey acts as carminative and relieves stomach aches.
1.4.2 Culinary Uses
In some parts of Nigeria it is used for preparing
pepper soups and different kinds of traditional soups. The leaves are used as
flavouring for stew. This is as a result of spicy and aromatic properties.
1.4.3 Pharmacological, toxicological and clinical
applications:
Black pepper is described as a drug which increases
digestive power, improves appetite, cures cold, cough, diseases of the throat,
intermittent fever, Colic, dysentery system. It possesses a broad spectrum
antimicrobial activity. (Srimivasan, 2005).
1.4.4 Bioavailability enhancement:
Black pepper and its volatile oil are used in food and
food items to aid digestion, relieve gas, Treat food poisoning, Stomach chills,
cholera, Dysentary. Piperine increases
the bioavailability of valuable phytochemicals present in food items and can
boost the activity of biochemically active compounds contained in it. (Agbor et al., 2006).
1.4.5 As Antioxidant:
An antioxidant is defined as any substance which is
capable of delaying, retarding or preventing the development in food of
rancidity or either flavour deterioration due to oxidation. Antioxidant
compounds in food items play import roles as health protecting factors.
Antioxidants are widely used as additives in fats, oils and in food processing
to prevent or delay spoilage of foods. Black pepper is a centre of attraction
as source of effective antioxidants. It contains several powerful antioxidants
and it thus one of the most spices for preventing and curtailing oxidative
stress. In addition to their direct antioxidant properties, several of these
compounds work indirectly by enhancing the action of other antioxidants.
Saturated fats in food are one of the main causes of oxidative stress and black
pepper minimizes it.
1.5 CHEMICAL CONSTITUENTS OF PIPER GUINEENSE
1.
PHENYLPROPANDIDS
It was reported that piper guineense contains phenylpropanoid, rich essential oil which
was comprised of myristicin, sarisan, safrone and elemicin as a dominant
component and fifty-one mono and sesquiterpenoids as a minor component
(Ekundayo et al., 1998).
2.
ALKALOIDS
A chemical substance of plant origin composed of
carbon, hydrogen, nitrogen and (usually) oxygen. The alkaloids are organic
basis similar to the alkalies (Inorganic basis); the name means alkali like. Most
alkaloids have pronounced effects on the nervous system of humans and other
animals. Many are used as drugs. Some similar alkaloids are caffeine, nicottine,
quinine, cocaine and morphine. Alkaloids occur mostly in various genera of seed
plants.
3.
FLAVONOIDS
Flavonoids are naturally occurring biochemical
substances found throughout plants. They are often referred to as
bioflavonoids. There are thousands of different plant flavonoid. Generally
speaking they are all chemically related because they have polyphenol
structures in laboratory research tests, flavonoids tend to prevent other
substances from undergoing oxidation reaction. People often refer to flavonoids
as antioxidants for this reason, and there is no shortage of people expounding
the benefits of flavonoid supplements.
4.
SAPONINS
Saponins are glycosides with foaming characteristics.
Saponins consists of a polycyclic aglycones attached to one or more sugar side
chains. The aglycone part, which is also called sapogenin, is either steroid or
a triterpene. The foaming ability of saponins is caused by the combination of a
hydrophobic (fat-soluble) sapogenin and hydrophilic (water-soluble) sugar part.
Saponins have a bitter taste. Some saponins are toxic and are known as sapotoxin.
5.
TANNINS
A tannin is an astringent, bitter plant polyphenolic compounds
binds to and precipitates proteins and various other organic compounds
including amino acids and alkaloids. The term tannins refers to the use of wood
tannins from oak in tanning animal hide into leather; hence the words “Tan” and
tannin for the treatment of leather; however, the term tannins by extension is
widely applied to any large polyphenolic compound containing sufficient
hydroxyls and other suitable groups (such as carboxyls) to form strong
complexes with various macromolecules. The tannin compounds are widely distributed
in many species of plants. Where they
play a role in protection from predation, and perhaps also as pesticides, and
in plant growth regulation.
6.
OLEORESIN
Piper guneense
also contains Oleoresin which is a mixture of a resin (volatile) and essential oil
(Oletunji, 1999). Oleoresins are obtained by solvent extraction of the spice.
Oleoresins consist of the volatile essential oil and the non-volatile resinous
fraction comprising of taste components, fixatives, anti-oxidants, pigments and
fixed oils that are naturally present in the spice, in a highly concentrated
form. Oleoresins, the true essence of the spice represents the over all flavor
profile of the spice. They serve as convenient and hygienic substitutes for
spice powders in food products without impairing any flavor characteristics.
1.5.1 Vitamin Content
Choline, folic acid, Niacin, Pyridoxine, Ruboflavim,
Thiamin, Vitamin A and Vitamin C. Vitamin E, Vitamin K is the major Vitamin found
in black pepper .
1.5.2 Mineral Content
Calcium, copper, iron, magnesium, manganese,
phosphorus, zinc are the main mineral found in the black pepper.
1.6 ROLES OF TEST ORGANISMS IN RELATION TO
DISEASES
1.6.1 Staphylococcus aureus
Staphylococcus
aureus is a gram positive coccus that produces a wide
variety of toxins. It is a facultative anaerobe which appears as grape like
cluster when viewed through a microscope and has large round, golden-yellow
colonies, often with haemolysis when grown in blood agar plate (Ryan and Ray, 2004).
Each year about 500,000 patients are affected by staphylococcus infection in
American hospitals. Staphylococcus aureus
may occur as a commensal on human skin sometimes it infects other tissue when
normal barrier have been reached, this leads to furuncle (boils) and carbuncles
(a collection of furuncles). In infants staphylococcus infection can cause a
severe disease staphylococcus scaled skin syndrome. (Curran and Al-Salihi, 1980).
It also causes some diseases like atopic dermatitis, Toxic shock syndrome, and
mastitis in cow.
1.6.2 Pseudomonas aeruginosa
Pseudomonas
aeruginosa is a member of the Gamma proteobacteria class of
bacteria. It is a Gram-negative, aerobic rod belonging to the bacterial family Pseudomonadaceae.
Pseudomonas is a Gram-negative rod
measuring 0.5 to 0.8µm by 1.5 to 3.0µm. Almost all strains are motile by means
of a single polar flagellum. Pseudomonas
aeruginosa is an opportunistic pathogen. It causes urinary tract infection,
respiratory system infections, dermatitis, soft tissue infections, bacteremia,
bone and joint infections, dermatitis, soft tissue infection, bacteremia bone
and joint infections, gastrointestinal infections and a variety of systematic
infections, particularly in patients with severe burns and in cancer and AID patients
who are immune suppressed. Pseudomonas
aeruginosa infections is a serious problem in patients hospitalized with
cancer, Cystic fibrosis, and burns.
A significant number of pseudomonas can produce
exopolysaccharides that are known as slime layers. Secretion of exopolysaccharide
makes it difficult for pseudomonads to be phagocytosed by mammalian white blood
cells (Ryan and Rey, 2004).
1.6.3 Escheriachia coli
E.Coli
is a gram negative, facultative aneaerobic, rod-shaped and non-sporulating
organism. The cells are about 2µ long and 0.5µ in diameter with a cell volume
of 0.6 to 0.7µm3. The optimal growth of E.Coli occurs at 370 C but some laboratory stains can
multiply at temperature of up to 490C (Fotader et al., 2005). Some strains of E.
Coli possess flagella with
peritrichus arrangement (Darnton, 2007).
Some virulent strains of E.Coli causes
Gastroenteritis, Urinary tract infections, and neonatal meningitis, in rare
cases, virulent strains are also responsible for Hemolytic-uremic syndrome
(HUS), peritonitis, mastitis, septicemia and Gram negative pneumonia (Toder, 2007).
Bacteria infections are usually treated with antibiotic,
Antibiotic resistance is a growing problem, some of this is due to overuse of
antibiotics in humans but some of it is probably due to the use of antibiotics
as growth promoters in food of animals (Johnson et al., 2006).
1.6.4 Salmonella typhi
This gram-negative enteric bacillus belongs to the
family enterobaceriaceae. It is a motile, facultative anaerobe that is
susceptible to various antibiotics. Currently, 107 strains of this organism
have been isolated, many containing varying metabolic characteristics, levels
of virulence, and multi-drug resistance is prevalent. Diagnostic identification
can be attained by growth on MacConkey and the bacteria is trickly non-lactose
fermenting. It also produces no gas when grown in TSI media which is sued to
differentiate it from other enterobacteriaceae (Dennis et al., 2003). Infection of S.typhi
lead to the development of typhoid or enteric fever.
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