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Digitally Signed by: Content manager’s Name
DN : CN = Webmaster’s name
O = University of Nigeria, Nsukka
OU = Innovation Centre
Ugboaku, Edith J.
FACULTY OF BIOLOGICAL SCIENCES
DEPARTMENT OF DEPARTMENT OF DEPARTMENT OF DEPARTMENT OF MICROBIOLOMICROBIOLOMICROBIOLOMICROBIOLOGYGYGYGY
Antibacterial Activity of Piper guineense, Xylopia aethiopica
and Allium cepa against Bacteria Isolated from Spoilt Soup
Preparations
MARTIN, HANNAH CHINENYE
PG/M.Sc/10/57261
ii
Antibacterial Activity of Piper guineense, Xylopia aethiopica
and Allium cepa against Bacteria Isolated from Spoilt Soup
Preparations
BY
MARTIN, HANNAH CHINENYE
PG/M.Sc/10/57261
Department of Microbiology
UNIVERSITY OF NIGERIA, NSUKKA
DECEMBER, 2014
i
TITLE PAGE
Antibacterial Activity of Piper guineense, Xylopia aethiopica
and Allium cepa against Bacteria Isolated from Spoilt Soup
Preparations
BY
MARTIN HANNAH CHINENYE
PG/M.Sc/10/57261
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF MASTER
OF SCIENCE (M.Sc.) DEGREE IN MEDICAL MICROBIOLOGY
SUPERVISOR: PROF C.U. IROEGBU.
DECEMBER, 2014
ii
CERTIFICATION
Miss Martin Hannah Chinenye, a post graduate student in the department of Microbiology,
majoring in medical microbiology has satisfactorily completed the requirements for course work
and research for the degree of masters in science (M.Sc) in Microbiology. The work embodied in
this project is original and has not been submitted in part or full for either diploma or degree of
this university or any other university.
______________________ _______________________
Prof A. N. Moneke Prof. C. U. Iroegbu
Head, Supervisor
Department of Microbiology, Department of Microbiology,
University of Nigeria, Nsukka. University of Nigeria, Nsukka.
iii
DEDICATION
This work is dedicated to God almighty whose love and grace saw me through this program.
iv
ACKNOWLEDGEMENT
My heartfelt gratitude goes to my supervisor Prof. C. U. Iroegbu for his fatherly
disposition, attention, guidance and patience in the course of this research project.
My thanks also goes to Mr. A. A. Ngene and Dr. A. C. Ike for their support during this
research and also Prof K. F. Chah who provided me with some test organisms used in this
research
I lack both words and space to appreciate all my friends in this department and my
friends outside the department for their help in several ways, especially Chinazor Araonu, Obudu
Uche, Joy, Patricia Kalu, Akudo Osuji, Iyke Ibe. God bless all of you.
Finally I wish to thank my parents Mr and Mrs Martin Ibekwe, and my lovely husband
Mr. Chrys Duru for their great love and care.
v
TABLE OF CONTENTS
Title page - - - - - - - - - - i
Certification - - - - - - - - - - ii
Dedication - - - - - - - - - - iii
Acknowledgement - - - - - - - - - iv
Table of contents - - - - - - - - - v
List of tables - - - - - - - - - - vi
List of appendices - - - - - - - - - vii
Abstract - - - - - - - - - viii
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW- - - 1
1.2: LITERATURE REVIEW- - - - - - - - 4
1.2.1 General Characteristics of Piper guineense - - - - - 7
1.2.2 General Characteristics of Xylopia aethiopica - - - - - 8
1.2.3 General Characteristics of Allium cepa - - - - - - 9
1.2.4 Review of Some Medicinal Spices- - - - - - 11
1.2.5 Some Spices reported to Possess Antibacterial Properties - - - 13
1.2.6 Some Major Groups of Antimicrobial Phytochemicals from Plants- - - 15
vi
1.2.7 Description of Test Organisms- - - - - - - - 19
CHAPTER TWO: MATERIALS AND METHOD- - - - - - 24
2.1 Collection and Identification of Plant Materials - - - - - 24
2.2 Isolation and identification of Microorganisms - - - - - 24
2.3 Test Microorganism- - - - - - - - 24
2.4 Sample Preparation and Extraction Procedures - - - - - 25
2.5 Media Preparation - - - - - - - - - 25
2.6 Preparation of Crude Plant Extracts - - - - - - 26
2.7 Determination of Antimicrobial Activity of Extracts- - - - - 26
2.8 Determination of Minimum Inhibitory Concentration (MIC) and
Minimum Bactericidal Concentration (MBC) of Crude Extracts- - - 27
2.9 Phytochemical Screening of the Plant Extract- - - - - - 27
2.9.1 Test for Alkaloids - - - - - - - - 28
2.9.2 Test for Flavonoids- - - -- - - - - - 28
2.9.3 Test for Glycosides- - - - - - - - - 28
2.9.4 Test for Saponins - - - - - - - - - 29
2.9.5 Test for Tannins.- - - - - - - - - 29
2.9.5 Test for Fats and Oil - - - - - - - - 29
2.10 Screening of ground Spices for Inhibitory Activity against Test Organisms- 29
vii
CHAPTER THREE RESULT- - - - - - - - 31
3.1 Isolation and Characterisation of Test Organisms- - - - 31
3.2 Yield from Aqueous and Ethanol Extractions- - - - - - 31
3.3 Chemical Constituents in Ethanol, Cold and Hot water of the Extracts- - - 34
3.4 Antimicrobial Activity of the Extracts- - - - - - - 34
3.5 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal
Concentration (MBC) of the Extracts- - - - - - - 43
3.6 Screening of Spices for Inhibitory Activity against Test Organisms - - 43
CHAPTER FOUR: DISCUSSION - - - - - - - 51
REFRENCES 55
APPENDICES 60
viii
List of Tables
Table Title Page
1 Characterization of theTest Organisms- - - - - 32
2 Yields from the Ethanol and Aqueous Extractions - - - 33
3 Chemical Constituents in Ethanol, Hot and Cold Water Extracts of
Piper guineense- - - - - - - - 36
4 Chemical Constituents in Ethanol, Hot and Cold Water Extracts of
Xylopia aethiopica- - - - - - - - 37
5 Chemical Constituents in Ethanol, Hot and Cold Water Extracts of
Allium cepa- - - - - - - - - 38
6 Inhibition of Microrganisms by Hot Water Extract of Piper guineense 39
7 Inhibition of Microrganisms by Hot Water Extract of Xylopia aethiopica 40
8 Inhibition of Microrganisms by Cold Water Extract of Allium cepa 41
9 Inhibition of Microrganisms by Cold Water Extract of Xylopia aethiopica 42
10 Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
of Ethanol Extracts of X.aethiopicaI, P.guineense and A.cepa 44
11 Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
of Cold Water Extracts of X.aethiopicaI, P.guineense and A.cepa 45
12 Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
of Hot Water Extracts of X.aethiopicaI, P.guineense and A.cepa - 46
ix
13 Effect of Spice Combination (X. aethiopica: P. guineense ) on Test Organisms 47
14 Effect of Spice Combination ( A. cepa: X. aethiopica) on Test Organisms 48
15 Effect of Spice Combination (P. guineense : A.cepa) on Test Organisms 49
16 Effect of Spice Combination (X. aethiopica: P. guineense: A.cepa ) on
Test Organisms - - - - - - - 50
x
Appendices
Appendix Title Page
1 Univariate Analysis of Variance- - - 60
2 Post Hoc Tests- - - - - 72
3 Laboratory Media and Reagents - - - 95
xi
ABSTRACT
The antibacterial activity of ethanol, hot water and cold water extracts of Allium cepa, Xylopia
aethiopica and Piper guineense were determined against bacteria isolated from spoilt egusi soup
and veterinary unit of the university. Antimicrobial testing was by both broth –dilution and agar
diffusion methods. The spices tested, Allium cepa, Xylopia aethiopica and Piper guineense were
extracted with cold, hot water and with ethanol. The ethanol extract of Piper guineense gave the
highest yield of extract 4.8g (32%). The highest IZD (14.7±0.3mm) was achieved with hot water
extract at concentration of 400mg/ml against Escherichia coli. The hot water extract also had
activity against Bacillus sp (IZD= 12.7±0.6mm) isolated from spoilt egusi-soup. The Bacillus
strain isolated from spoilt pepper-soup was not susceptible to the hot water extract and indeed
any other extract even at the highest concentration. The most susceptible test organism was
E.coli with IZD ranges of 12.0± 0.1mm (obtained from cold water extract of X. aethiopica at
400mg/ml) to 14.7±0.2mm (from hot water extract of Piper guineense at 400mg/ml). The least
susceptible were Enterobacter sp and Proteus sp. which were only susceptible to cold water
extract of A. cepa (IZD= 10.0± 0.2mm and 12.0±0.2mm both at 400mg/ml, respectively).
Ethanol extracts showed no activity against any test organisms. Result show that the spices have
potentials for use as food preservatives while still acting as food condiments.
1
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.1. Introduction
Food borne illness caused by consumption of foods contaminated with pathogenic
bacteria or their toxins has been of great public health concern. In recent times, consumers are
even more concerned of the processed foods they eat not only because of the high risk of
contamination but also because of the added synthetic preservatives which may be hazardous to
health. Food additives such as monosodium glutamate, aspartame, saccharin, sodium cyclamate,
sulfites, nitrates, nitrites and antibiotics have all been reported to cause clinical conditions
manifesting variously as headache, nausea, weakness, mental retardation, seizures, cancer and
anorexia (Rangan and Barceloux, 2009; Wroblewska, 2009). The increasing demand for food
with longer shelf life, food with little or no chemical preservatives coupled with the concern
about toxic effects of some preservatives has resulted in increased pressure to find alternatives
for better healthcare. Therefore, there is a considerable interest to stop the disease outbreaks
caused by pathogenic and/or spoilage food microorganisms among food processors, food safety
researchers and regulatory agencies (Marija et al., 2009). Antimicrobial agents of plant origin
have been documented and spices are among those perceived to have great potentials for use as
antimicrobial agents (Arora and Kaur, 1999; Okeke et al., 2001).
Spices are defined by Corn (1999) as dried seeds, fruits, roots, barks, leaves or vegetables
used in nutritionally insignificant quantities as food additives for the purpose of flavour, colour
or as preservative that kill harmful bacteria or suppress their growth. Spices, which include plant
materials of medicinal importance, have been used for the treatment of human ailments way back
2
in the history of man. In Nigeria, some spices are used for the preparation of special types of
soup. These include soup for newly delivered mothers to accelerate blood flow leading to the
elimination of blood clots from her womb and blood system. Some have been recommended for
fast relief of ailments such as cholera, diarrhea, dysentery and wound sepsis (Inyang, 2003,
Olumsimbo et al., 2011).
It is now recognized that spices and herbs may fulfill more than one function in foods to
which they are added. These include imparting flavour, prolonging the storage life of foods by
their bacterostatic or bacterocidal activity, in addition to being nutrients. These appeal to
consumers who tend to question the safety of synthetic food additives (Eruteya and Odunfa,
2009). The medicinal and preservative values of spices have been attributed to the presence of
bioactive antimicrobial compounds (Lai and Roy, 2004).
Piper guineense (Igbo: Uziza) is a flowering vine in the family Piperaceae, cultivated for
its fruit which is usually dried and used as spice for seasoning. In the dried form the fruit is often
referred to as peppercorn or simply pepper. Pepper gets its spicy heat mostly from the piperine
compound which has been reported to exhibit antimicrobial properties detectable both in the
outer fruit and in the seed (Oladosun et al., 2012).
Xylopia aethiopica (Igbo: Uda) is an evergreen, aromatic tree of the Annonaceae family
that can grow up to 20m high. It is a plant used both as a spice and as a herb. It has been reported
in folklore that X. aethiopica is very potent in curing several ailments including cough,
rheumatism and nerve pains as well as in elimination of blood clots when used to prepare
peppersoup for newly delivered mothers (Ekpo et al., 2012).
Allium cepa (common onion) is a biennial garden plant, it is usually thought of as a
vegetable, and it also has a long medicinal use history. Principally, the fleshy bulb that grows
3
below the ground is used as medicine and as food; but other parts of the plant have also been
used in traditional medicine for the treatment of various ailments (Azu et al., 2007).
The fruits of the guinea pepper (Piper guineense, Uziza in Igbo) and seeds of the African
pepper (Xylopia aethiopica, Uda in Igbo) are common spices and condiments included in a
variety of indigenous Nigerian recipes particularly among the Igbos of southern Nigeria (Okeke
et al., 2001). In a recent survey, respondents in the region indicated that the two spices act as
stimulants and laxatives, used to smoothen the skin and cure fever, cough and stomach disorders.
They are also used as abortificients to treat amenoria and cleanse the womb after childbirth
(Okeke, 1998).
Studies in the past decades confirm that the growth of both Gram-negative and Gram-
positive food borne bacteria, yeasts and mold can be inhibited by spices (Eruteya and Odunfa,
2009). Monodora myristica, Piper guineense and Xylopia aethiopica were screened for fungi-
toxic activity of their essential oils against mycelial growth of 3 food contaminants, Aspergillus
fumigatus, Aspergillus nidulans and Mucor hiemalis. The essential oils from all the spices were
fungi-toxic to varying degrees (Nwaiwu and Imo, 1999). Johnson and Vaugh (1969) reported the
inhibitory activity of reconstituted onion and garlic preparations against Salmonella typhimurium
and Escherichia coli. According to Shelef (1983) garlic inhibited Salmonella typhymurium,
Escherichia coli, Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, mycotoxigenic
Aspergillus and Candida albicans.
1.1.1. Statement of Problem
Although a number of spices have been reported in research or folklore to exhibit
antimicrobial activity against different types of microorganisms, this varies widely depending on
4
the type of spices or herbs, test medium and microorganism. Besides, some of these claims still
need to be authenticated through scientific testing. Food preparations are known to be spoiled by
microbial contaminants that cause them to ferment and sour; some of these contaminants are
potent pathogens causing food poisoning or food infection when they multiply to high doses.
Thus many local foods need preservation without synthetic preservatives and need to be free
from pathogens. Thus, this study was undertaken to evaluate the antimicrobial and, by
implication, the potential preservative effects of these natural spices used in making soup on
some food spoilage isolates.
1.1.2. Aim
The aim of this research is to determine the antimicrobial effect of three spices used in
preparing pepper-soup on some food contaminating organisms and to determine the quantity of
the spices needed to inhibit the growth of the organisms.
1.1.3. Objectives
- To isolate and characterize microorganisms associated with spoilage of egusi- and
pepper-soups.
- To determine the antimicrobial activity of the spices against a variety of test
organisms including those isolated from spoilt egusi soup and peppersoup
- To determine the minimal concentrations of the spices needed to inhibit the
growth of the organisms.
1.2. Literature Review
Plants and their products have been used by humans in diverse ways, and the most
common uses are as food, spices and medicines. The uses of spice are not limited to flavouring
5
agents. They possess potent medicinal properties such as antimicrobial activity, antioxidants,
anticancer, anorexia, bronchitis and rheumatic complaints and as a post-operative antiemetic. The
medicinal property under review is the antimicrobial activity. The antimicrobial property can be
merely inhibitory or cytostatic or total killing or cytocidal. The main advantage of using the
herbal antimicrobial drug is that there are little or no side effects. Such side effects as depletion of
the normal intestinal flora, bone marrow depression, dysentery, local inflammation, damage to the
liver and kidney are largely overcome by using herbal preparations either as drug or as spices
(Saha Rajekhar et al., 2012).
Spoilage is a metabolic process that causes food to be undesirable or unacceptable for
human consumption due to changes in sensory and nutritional characteristics (Doyle, 2007).
Prevention of pathogenic and spoilage microorganisms in food is usually achieved by using
chemical preservatives but they have been associated with many carcinogenic and teratogenic side
effects as well as residual toxicity. There is, also, growing concern about microbial resistance
towards conventional preservatives; consumers tend to be suspicious of chemical additives,
hence, the exploration and exploitation of naturally occurring antimicrobial herbal preparations
are receiving increasing attention in food preservation (George et al., 2010). There has been an
increasing consumer demand for foods free of or low in, added synthetic preservatives because
synthetic preservatives could be toxic to humans (Bedin, et al., 1999). Concomitantly, consumers
have also demanded for wholesome and safe food with long shelf lives. These requirements are
often contradictory and have put pressure on the food industry for progressive removal of
chemical preservatives and adoption of natural alternatives to control food borne pathogens and
spoilage microorganisms (Brull and Coote, 1999). Many plant derived products such as spices,
fruit preparations, vegetable preparations or extracts have been used for centuries for the
6
preservation and extension of the shelf life of foods (Chattopadhyay and Bhattacharyya, 2007).
Numerous naturally occurring antimicrobials are present plant tissues and many studies have
evaluated the antimicrobial activities of several plant extracts, including Sesamum radiatum
(Shittu et al., 2007), Allium cepa (Agatemor, 2009), Olives, Chardonnay grapes, black
raspberries and orange essential oils (George et al.,2010).
Spices are the most common plant materials with potential antimicrobial properties that
are used in foods; and they have been used traditionally for thousands of years by many cultures
for preserving foods and as food additives to enhance aroma and flavour (Souza et al., 2005).
Spices may be indigenous or exotic, aromatic or with strong taste, but used in all cultures to
enhance the taste of foods. Spices may come in form of rhizomes, bulbs, barks, flower buds,
stigmas, fruit, seeds and leaves. They are categorized into tiny wild fruits, nuts, herbs, spices and
leafy vegetables. Some of them are not only used for food, but also in folklore medicine for
treatment of minor ailments. Spice ingredients produced from roots, barks, probably evolved as
part of the defense mechanisms of leaves, bulbs, stems flowers and seeds of certain plants
against microbial invasion. Each spice has a unique aroma and flavor, which is derived from
chemical constituents of the plant, generally designated “secondary metabolites” and so called
because they are secondary to the plant’s basic metabolism. Most spices contain dozens of
secondary metabolites or compounds. These are the plant’s recipes for survival-legacies of their
co-evolutionary races against biotic enemies. Some of the secondary metabolites that are active
against microorganisms fall into groups of compounds generically known as alkaloids,
flavonoids, glycosides etc. Spices include leaves (bay, mint, rosemary, coriander, laurel,
oregano), flowers (clove), bulbs (garlic, onion), fruits (cumin, red chilli, black pepper), stems
(coriander, cinnamon), rhizomes (ginger) and other plant parts (Shelef, 1983).
7
1.2.1 General Characteristics of Piper guineense
Piper guineense also known as African black Pepper, Ashanti Pepper, Benin Pepper,
False Cubeb, Guinea Cubeb, Uziza Pepper is very similar to Piper nigrum which is the true
pepper of commerce from which black and white peppers are processed (Isawumi, 1984). It has
more than 700 species widely distributed throughout the tropical and subtropical regions of the
world. It is known with different vernacular names in Nigeria: Igbo (Uziza), Yoruba (Iyere) and
Ibibios of Akwa Ibom (Odusa). P. guineense has culinary, medicinal, cosmetic and insecticidal
uses (Dalziel, 1955).
The plants that provide Ashanti pepper are climbing vines that can grow up to 20m in
length. These are native to tropical regions of central and Western African and are Semi-
cultivated in countries such as Nigeria where the leaves are used as a flavoring in soups recipes.
It is used in West African cuisine where it imparts "heat" (piquantness) and a spicy, pungent
aroma to classic West African "soups" (stews).
Ethnomedicinal uses of Piper guineense
Piper guineense has culinary, medicinal, cosmetic and insecticidal uses (Dalziel, 1955).
P. guineense insecticidal activity against Zonocerus variegatus is attributable to the piperine-
amide constituent of the plant. The leaves are considered aperitive, carminative and eupeptic.
They are also used for the treatment of cough, bronchitis, intestinal diseases and rheumatism
(Essiett and Ibanga, 2012). In Chinese folk medicine; black pepper is used to treat epilepsy.
Piperine, the active component of black pepper blocks convulsions induced by ‘kainte’ but not
by glutamate. It is also used in Chinese medicine for the treatment of rheumatism, toothache and
stomach ache. It is crushed and eaten by pregnant women in Caspian Littoral of Iran where
8
esophageal cancer rate is high. Black pepper has been prepared in the form of pills as a remedy
for cholera and syphilis. It has also been used in tooth powder for toothache and for sore throat
and hoarseness. It could be chewed to reduce throat inflammation. Other applications of black
pepper include treatment of boils, hair loss and skin diseases. It alleviates itching and paralysis.
A mixture of black pepper and honey serves as a remedy for night blindness. Black pepper is
also useful in hepatitis as well as urinary and reproductive disorders.
1.2.2 General Characteristics of Xylopia aethiopica
Xylopia aethiopica is known by different names in different languages – English (negro
peppr), Yoruba (eru), Igbo (Uda), Hausa (kimbara) and Kugbo (alilaar). It is widely cultivated
in West Africa, Central and Southern Africa. Xylopia aethiopica is an angiosperm of the family
Annonaceae. It grows into a tall tree of about 20m high and 75cm stem girth. The fruits are
rather small and look like twisted bean-pods in clusters of up to 40 green or red monocarps when
fresh but turn dark brown when dry (Burkill, 1985). It has been reported, that there are between
100 and 150 species of Xylopia distributed throughout the tropical regions of the world,
particularly Africa, among which are, X. aethiopica, X. brasiliensis, X. frutenscens, X.
grandiflora, X. aromatic. It is used as a spice and possesses great nutritional and medicinal
values in folklore (Oluwatosin et al, 2010). The various extracts from Xylopia spp. have been
shown to possess antiseptic and analgesic properties, and insecticidal activity against adult
mosquitoes, several leaf-eating insects and houseflies. Various parts of the plant have been
traditionally employed in different therapeutic preparations (Konning et al., 2004).
Phytochemichal evaluation shows that X. aethiopica is rich in alkaloids, tannins, flavonoids,
steroids, oligosaccharides and has tolerable levels of cyanogenic glycosides (Ijeh et al., 2004).
9
Ethnomedicinal uses of Xylopia aethiopica
X. aethiopica has an attractive aroma and has been applied in ethnomedicine in the
treatment of cough, bronchitis and dysentery (Iwu, 1986). In Ivory Coas,t the fruit extract is used
as tonic to encourage female fertility, for ease of childbirth and as a woman remedy after child
birth for relief of pains in the ribs, chest, lumbargo, neuralgia and in treatment of boils and skin
eruptions (Acquaya et al., 2002); for increasing menstrual flow and was accordingly deemed to
have abortifacient properties (Burkill, 1985; Nwafor and Gwotmut, 2006)). Some of its
investigated uses include termite anti-feedant activity (Murray, 1995) and antiseptic properties
(Iwu, 1995). Xylopia aethiopica has a wide variety of applications; the very odorous roots of the
plant are employed in West Africa in tinctures, administered orally to expel worms and other
parasitic animals from the intestines, or in teeth-rinsing and mouth-wash extracts against
toothaches. Crushed powdered fruits can also be mixed with shea butter fat and coconut oil and
used as creams, cosmetic products, and perfumes (Burkill 1985), and the dried fruits are also
used as spices in the preparation of two special local soups named “obe ata” (Yoruba) and “isi-
ewu” (Igbo) taken widely in the southwest and southeastern parts of Nigeria.
1.2.3 General Characteristics of Allium cepa
Allium cepa (Onion) which belongs to the family Alliaceae, is also known as ‘garden
onion’or ‘bulb’onion. It is one of the oldest cultivated vegetables in history. Onion is a biennial
plant, growing from a subterranean bulb. It can grow up to 70 cm in height. It has an erect stem
and an umbel of soft, white to pink flowers on its top. Its underground bulb carries small,
shallow roots. Above ground, the onion shows only a single vertical shoot; the bulb grows
underground, and is used for energy storage, leading to the possibility of confusion with a tuber
10
which it is not. The leaves are bluish-green and hollow, the bulbs are large, fleshy and firm.
Three main varieties of onion are available viz: red, white and purple skinned. Onions are easily
propagated, transported and stored. Onions are effective against common cold, heart disease,
diabetes, osteoporosis, coughs and sore throat. They also act as bacteristatic. Certain chemical
compounds believed to have anti-inflammatory, anti-cholesterol, anticancer and antioxidant
properties including quercetin are present in onions. They are high in flavonoids which is
concentrated on the outer layer of the flesh. Onions are also rich in Calcium, iron, phosphorus,
vitamin C, riboflavin, niacin, thiamine, carotene and polphenols than other allium vegetables.
Ethnomedicinal uses of Allium cepa
Onion has a great variety of medicinal uses. It is considered to have antihelmintic,
antioxidant, antiseptic, carminative, diuretic, expectorant, febrifuge and vulnerary properties.
Onion is said to help in cases ranging from the common cold to heart disease and diabetes. In
traditional medicine, Onion had been used for colds, coughs, flu and bronchitis. During winter
times, onion juice sweetened with honey can be used for prevention of common cold. It is also
said that chewing of fresh Onion can kill germs in mouth and soothe toothache. Recent studies
are showing its beneficial effect in the treatment of high blood pressure and high blood
cholesterol (Azu et al., 2007). It can be a good medicine for prevention of cardiovascular
disease, and even certain head and neck tumors. Some studies suggest that high consumption of
Onion, along with Garlic lowers the possibility of appearance of stomach cancers by 40% (Indu
et al., 2006). It can also be used for prevention of osteoporosis and in treatment of blisters, boils
and topical scars.
11
1.2.4. A Review of other Medicinal Spices
Medicinal plants are of great importance to the health of individuals and communities.
The medicinal value of plants lies in some chemical substances that produce a definite
physiological action in the human body. Many of these indigenous medicinal plants are used as
spices and food plant. They are sometimes added to foods meant for pregnant and nursing
mothers for medicinal purposes (Edeoga et al., 2005).
Ginger, the rhizome of Zingiber officinale, is one of the most widely used species of the
ginger family Zingiberaceae and is a common condiment for various foods and beverages.
Ginger is a creeping perennial on a thick tuberous rhizome which spreads underground. The
odour and taste are characteristically aromatic and pungent. The plant is indigenous to southern
Asia and is cultivated in a number of countries including India. The medicinal part of the herb is
the dried roots. It is now recongnised as a drug of choice for nausea and vomiting. It has been
found useful in pregnancy-related morning sickness. In rheumatoid arthritis and Osteoarthritis, it
is used as natural pain reliever and an inflammatory agent. It is also used in curing ulcer and
preventing heart attack and stroke (Samir and Amrit, 2003). Raw ginger is chewed to stimulate
the flow of saliva and to relax congested nostrils. Ginger tea is prescribed for cough, colds and
influenza (Gill, 1992). The juice of the rhizome served with honey is a very efficacious remedy
for cough and asthma (Okanla et al., 1990). It is recommended for ailments of the digestive
system, rheumatism and piles.
Allium sativum, commonly known as garlic is a specie in the onion family
Amaryllidaceae. It is known that Allium sativum possesses antimicrobial, antiprotozoal,
antimutagenic, antiplatelet and antihyperlipidemic properties. Allium sativum has been used in
world cuisines as well as in herbal medicine for thousands of years. It is used to prevent heart
12
diseases (including arteriosclerosis and high blood pressure) and cancer including stomach and
colon cancer. Allium sativum has been found to reduce platelet aggregation and hyperlipidemia
(Kojuri et al., 2007).
Studies have shown other relevant spices such as Monodora myristica seed used as
condiment in West Africa, and a decoction of the seed used to treat guinea worm infection. The
seeds are used as a remedy for constipation when mixed with palm oil. Roasted and powdered
seeds of the plant are very effective in curing stomach ache. The seeds are rubbed on the
forehead to cure headache (Gill, 1992). Another spice, cinnamon or Cinnamomum zeylanicum,
found in the inner bark of Cinnamomun trees, is commonly used in cooking for its aroma, flavor,
and taste. Historically, cinnamon has been used by the Egyptians for embalming, presumably
based on its antimicrobial properties. Eugenol and cinnamaldehyde are the two major chemical
components in cinnamon that are responsible for its health benefits. Eugenol, a phenol
compound, inhibits mold and adds flavor and aroma to bakery items. It also contains antiviral
properties in vitro. Additionally, eugenol and cinnamaldehyde inhibit Helicobacter pylori growth
at a low pH, showing their efficacy in eliminating the bacteria present in the human stomach (Ali
et al., 2005). The electronegative cinnamaldehyde also inhibits amino acid decarboxylase.
Cinnamaldehyde interferes with electron transfers and reacts with nitrogen-containing
compounds, resulting in impeded growth of microorganisms.
Clove (Syzygium aromaticum) has been proven to be active against many types of
bacteria. Cloves are the dried immature flower buds of a tropical tree of the Myrtaceae family.
These trees are native to Indonesia but also cultivated in other tropical regions. Cloves have been
used for centuries as natural medicine against illnesses such as diarrhea, ringworm and nausea
and have been shown to be effective in reducing toothache. They also have a strong inhibitory
13
effect against microbes and are able to kill species of bacteria and fungi such as Staphylococcus
aureus, Listeria monocytogenes, and Aspergillus. This property is attributed to the presence of
eugenol, the phenol compound also found in cinnamon. Eugenol makes up the majority of clove
bud oil. Other spices such as Curcuma longa (Turmeric), Piper nigrum (Black pepper) etc have
been found to have varied medicinal values (Shelef, 1983).
1.2.5 Some spices Reported to Possess Antibacterial Properties.
The antimicrobial properties of substances are desirable tools in control of infections and
food spoilage or food preservation. Antimicrobial activity depends on the type of spice or herb,
type of food and microorganism, as well as on the chemical composition and constituents of
extracts and essential oils. The antimicrobial activity of some plant extracts on some organisms
associated with fish spoilage was studied by George et al. (2010). They observed that
marceration in hot water was the best extraction method followed by ethanol and cold water
extraction methods. Among the plant materials they evaluated, Citrus paradise had the best
antimicrobial activity followed by Piper guineense and Carica papaya.
Aqueous and ethanol extracts of Occimum gratissum and P. guineense leaves were
screened for antibacterial activity against Escherichia coli and Staphylococcus aureus by Nwinyi
et al. (2009). Both extracts were found to exhibit selective inhibition against the isolates. Ethanol
extracts showed more inhibitory effect compared to the aqueous extracts.
Antimicrobial activity of some extracts of Allium sativum (garlic), Myristica fragrans
(nutmeg), Zingiber officinale (ginger), Allium cepa (onion) and Piper nigrum (pepper) was
evaluated against 20 different strains of Escherichia coli, 8 serotypes of Salmonella, Listeria
mononcytogenes and Aeromonas hydrophila by Indu et al. (2006). The result showed that garlic,
14
ginger and nutmeg showed some inhibitory activity on different test organisms while extracts of
Onion and Pepper did not show any activity against the test organisms.
Olusimbo et al. (2011) studied the aqueous and ethanol extracts of four spices (Monodora
myristica, Piper.guineense, Xylopia aethiopica and Tetrapleura tetraptera) on some pathogens.
The aqueous extracts had antimicrobial activity on all test organisms used while the ethanol
extracts were less active. The antimicrobial activity of essential oils of Xylopia aethiopica was
evaluated on some Gram-positive and Gram-negative pathogens by Fleischer et al. (2008), the
fresh and dried fruits, leaf, stem bark and root bark essential oils showed varied activity on the
test organisms except on Escherichia coli.
The effect of Eugenia aromatic (clove), Allium sativum (garlic) and Piper guineense
(brown pepper), three spices commonly used in the preparation of suya against Bacillus spp,
Enterobacter spp Aspergillus niger and Rhizopus stolonifer were studied by Eruteya and Odunfa
(2009). The sensitivity of the organism revealed that Clove is outstanding compared to the much
studied garlic and that Gram positive bacteria showed higher susceptibility to spices than Gram
negative organisms. R. stolonifer showed higher sensitivity to brown pepper than it did to garlic.
The growth of A. niger was not completely inhibited by brown pepper or a combination of both.
The percentage composition of these three spices affected their inhibitory effects on
microorganisms in suya condiment.
Azu et al. (2007) investigated the antimicrobial activity of raw and aqueous extract of
Allium cepa and Zingiber officinale against Staphlococcus aureus and Pseudomonas aeruginosa
that are common cause of nosocomial infection and urinary tract infection using cup–plate
diffusion technique. The result showed that ethanol extract of ginger gave the widest zone of
inhibition against the two test organisms at the concentration of 0.8gml-1
. However,
15
Pseudomonas aeruginosa was more sensitive to the extract of onion bulbs compared to
Staphylococcus aureus. It was also observed that the solvent of extraction and its varying
concentrations affected the sensitivity of the two organisms to the plant materials. The minimum
inhibitory concentration (MIC) of ginger extracts on the test organisms ranged from 0.1gml-1
-
0.2gml-1
, showing that ginger was more effective and produced marked inhibitory effect on the
two test organisms compared to the onion extracts. This investigation indicates that, though both
plants had antibacterial activity on the two test organisms, ginger had more inhibitory effect thus
confirming their use in folk medicine.
Marija and Nevena (2009) reviewed the antimicrobial activity of essential oils of widely
used spices and herbs, such as garlic, mustard, cinnamon, cumin, clove, bay, thyme, basil,
oregano, pepper, ginger, sage, rosemary etc., against most common bacteria and fungi that
contaminate food (Listeria spp., Staphylococcus spp., Salmonella spp., Escherichia spp.,
Pseudomonas spp., Aspergillus spp., Cladosporium spp. and many others). The result shows that
cinnamon, cloves and mustard have very strong antimicrobial potential, cumin, oregano, sage,
thyme and rosemary show medium inhibitory effect, and spices such as pepper and ginger have
weak inhibitory effect.
1.2.6 Some Major Groups of Antimicrobial Phytochemicals from Plants.
Phytochemicals are non nutritive compounds found in plant and which may have
protective or disease preventive properties. These phytochemicals are mostly secondary
metabolites of which over 10,000 have been isolated. In many cases, these substances serve as
plant defense mechanism against predation by microorganism, insects and herbivores. However,
it has been demonstrated that these chemical substances can also protect human against diseases.
They include the following;
16
Alkaloids
They are natural plant compounds with a basic character and usually contain one or more
nitrogen atom in a heterocyclic ring. They are usually colourless, crystalline, non volatile solids
which are insoluble in water but soluble in ethanol, ether, chloroform and other organic solvents.
Only very few liquids are soluble in water. Most alkaloids have a bitter taste and are optically
active. Most alkaloids are physiologically active while some are extremely poisonous. The first
medically useful example of an alkaloid was morphine isolated in 1805 fom the opium Papaver
somniferum. Many alkaloids are commonly found to have antimicrobial properties. The
mechanism of action of highly aromatic planar quaternary alkaloids such as berberine and har
ane is attributed to their ability to intercalate with DNA.
Flavonoids
Flavonoids are a class of water soluble plant pigments. They are a group of polyphenolic
compounds possessing 15 carbon atoms; two benzene rings joined by a linear three carbon chain.
Since the flavonoids are known to be synthesized by plants in response to microbial infection, it
should not be surprising that they have been found in vitro to be effective antimicrobial
substances against a wide array of microorganisms. Their activity is probably due to their ability
to complex with extracellular and soluble proteins and to complex with bacterial cell wall (Ajali,
2004). More lipophilic flavonoids may also disrupt microbial membranes. Human studies
suggest that flavonoids may reduce the risk of cardiovascular disease and stroke (Knek et al.,
1997).
Saponins
Saponins are glycosides with distinctive foaming characteristics. They are natural
detergents found in certain plants. They are found in many plants especially certain desert plants.
17
They got their name from the soapwort plant (Saponaria) the root of which was used historically
as a soap. Saponins have detergent or surfactant properties because they contain both water
soluble and fat soluble component. Saponins are amphipathic compounds, possessing both
hydrophilic and lipophilic portions. They are, therefore, surface active and can be used as
emulsifiers. Molecular weight is of the order 180-2000 Daltons. At concentrations below 200-
500 ppm saponins exist as monomers; above 200-500ppm, they aggregate as micelles with a
molecular weight of approximately 100,000 Dalton. Some saponins are sweet while others are
bitter.
The antifungal and antibacterial properties of saponins are important in cosmetic
application in addition to their emollient effects. Saponins have both current and potential
applications in animal and human nutrition, in pig and poultry raising facilities and in dog and
cat foods. Saponins have ammonia binding activity when added to the diet,can bind to ammonia
and certain other odoriferous components in the excreta and prevent them from being released
into the air. It is however interesting that human do not suffer severe poisoning from saponins.
Tannins
Tannins is a general descriptive name for a group of polymeric phenolic substances
capable of tanning leather or precipitating gelatin from solution, a property known as
astringency. Their molecular weight range from 500 to 3000 kD and are found in almost every
plant part: bark, wood, leaves, fruits and roots. Tannins are divided into two groups,
hydrolysable and condensed tannins. Hydrolysable tannins are based on Gallic acid, usually as
multiple esters with D-glucose; while the more numerous condensed tannins often called
proanthocyanidins are derived from flavonoid monomers. Tannins may be formed by
18
condensation of flavan derivatives which have been transported to woody tissues of plants.
Alternatively; tannins may be formed by polymerization of quinone units.
This group of compounds has received a great deal of attention in recent years, since it
was suggested that the consumption of tannin-containing beverages, especially green teas and
red wines, can cure or prevent a variety of illness (Herbert, 1989). Many human physiological
activities, such as stimulation of phagocytic cells, host mediated tumor activity, and a wide range
of anti-infective activities have been assigned to tannins. One of their molecular actions is to
complex with proteins through so-called nonspecific forces such as hydrogen bonding and
hydrophobic effects, as well as by covalent bond formation. Thus, their mode of antimicrobial
action may be related to their ability to inactivate microbial adhesions, enzymes, cell envelope
transport proteins etc.
Phenolics and Polyphenols
Some of the simplest bioactive phytochemicals consists of a single phenolic ring.
Cinnamic and caffeic acids are common representatives of a wide group of phenylpropane-
derived compounds which are in the highest oxidation state. The common herbs, Tarragon and
Thyme, both contain caffeic acid, which is effective against viruses (Wild, 1994), bacteria
(Bratner and Grein, 1994) and fungi (Duke, 1985).
Catochol and pyrogallol both are hydroxylated phenols shown to be toxic to
microorganisms. Catochol has two –OH groups, and pyrogallol has three. The site(s) and number
of hydroxyl groups on the phenol group are thought to be related to their relative toxicity to
microorganisms, with evidence that increased hydroxylation results in increased toxicity
(Geissman, 1963). In addition, some authors have found that more highly oxidized phenols are
inhibitors. The mechanisms thought to be responsible for phenolic toxicity to microorganisms
19
include enzyme inhibition by the oxidized compounds, possibly through more non specific
interactions with the proteins (Mason and Wesserman, 1987). Phenolic compounds possessing a
C3 side chain at lower level of oxidation and containing no oxygen are classified as essential oils
and often cited as antimicrobial as well. Eugenol is a well characterized representative found in
clove oil. Eugenol is considered bacteriostatic against both fungi and bacteria (Duke, 1985).
1.2.7 Description of Test Organisms
Food borne pathogens are widely distributed in the environment and may be a significant
cause of mortality and morbidity in the population (Indu et al., 2006). Escherichia coli is a
significant food borne hazard in many countries around the world. Infection often causes
haemorrhagic diarrhoea, and occasionally to kidney failure and death. Salmonella is another
bacteria that is the cause of food borne illness mainly from foods of animal origin throughout the
world. Staphylococcus aureus and Bacillus cereus cause foodborne illness due to their ability to
form heat stable toxins (WHO, 2007).
Salmonella spp
Salmonella species are gram negative, aerobic, rod-shaped, zoonotic bacteria that can
infect humans, birds, reptiles, and other animals. Salmonella spp. are a group of bacteria which
reside in the intestinal tract of human beings and warm blooded animals and are capable of
causing disease. They are members of the Enterobacteriaceae group. The genus Salmonella
contains 2 species: Salmonella enterica and Salmonella bongori. Salmonella enterica is an
important agent of food borne illness.
Salmonella spp. are not particularly heat resistant and most serotypes are killed by normal
cooking conditions, i.e. cooking to a core temperature of 75ºC instantaneously or an equivalent
20
time temperature combination, e.g. 70ºC for 2 minutes. However, a few highly heat resistant
serotypes have been reported, e.g. S. senftenberg 775W and S. irumu. Heat resistance is
influenced by water activity (aw), nature of the solutes and pH of the suspending medium.
Greater heat resistance is observed for cells in sucrose compared with NaCl at the same aw
values. The incidence of various Salmonella species seems to vary with geographic location and
the types of food consumed. Imported birds and animals may serve to introduce different
Salmonella species to the local area that can cause new and devastating outbreaks. They are the
causative agents of typhoid fever, enteric fever, gastroenteritis and septicemia.
Escherichia coli
Escherichia coli is a facultative, anaerobic, motile, gram negative rods that ferment
sugars to produce acid and gas. It belongs to the family Enterobacteriaceae which are bacteria
that normally live in the intestines of animals; including humans.There are approximately 100
strains of E. coli most of which are beneficial as normal flora. Although E. coli inhabit the
intestinal tract as beneficial microorganisms, there are also strains of E. coli that are known to
produce toxins. E.coli strains that contain enterotoxins and other virulence factors including
invasiveness and colonization factors cause diarrheal disease. Four such strains have been
identified. The National Center for Infectious Diseases in the United States , Centers for Disease
Control (CDC), particularly warns of the dangers posed by the rare strain E. coli O157:H7, a
pathogenic strain isolated from manure from cattle, sheep, pigs, deer and poultry. This strain can
cause severe diarrhea and kidney damage. Young children, the elderly, and those with weakened
immune systems are the most vulnerable. It is this particular strain that has been highly
publicized. E.coli is also a major cause of urinary tract infections and noscomial infections
including septicemia and meningitis.
21
Staphylococcus spp
They are gram positive cocci and occur most commonly as irregular cluster of spherical cells.
They are mesophilic non spore formers; however they are generally highly resistant to drying,
especially when sequestered in organic matter such as blood, pus and tissue fluids. They are
capable of surviving outside the body for extended period of time, even up to several months.
The genus Staphylococcus comprises both of pathogenic and non-pathogenic organisms. Most
Staphylococci are indigenous to skin surfaces and mucus membranes of the upper respiratory
tract. Breaks in the skin and mucus lining may serve as portals of entry to the underlying tissue.
With the possibility of infection by virulent strains, the three major species include S. aureus, S.
saprophyticus and S. epidermidis. Strains of the last two species are generally avirulent,
however, under special circumstances where a suitable portal of entry is provided. S. epidermidis
may be the aetiological agent for skin lesion and endocarditis and S. saprophyticus has been
implicated in some urinary tract infections. S.aureus is mainly associated with the skin and
mucous membrane of warm blooded vertebrates but is often isolated from food products, dust
and water. Some species are opportunistic pathogens of human and animals or produce
extracellular toxins.
Enterobacter spp
Enterobacter spp are in the family Enterobacteriaceae. Enterobacter spp are facultatively
anaerobic gram negative bacilli, motile by means of peritrichous flagella and have class 1
fimbriae. They produce acid upon glucose fermentation are methyl red negative and Voges-
Proskauer positive, with an optimal growth temperature of 30oC, about 80% are encapsulated
(Hart, 2006). They are widely distributed in nature occurring in fresh water, soil sewage plants,
vegetable and animals and human feces. Several strains of this bacterial organism are pathogenic
22
and cause opportunistic infection in immunocompromised hosts and in those who are on
mechanical ventilation. The urinary and respiratory tracts are the most common site of infection.
The genus Enterobacter is a member of the coliform group of bacteria. It does not belong
to the fecal coliform group of bacteria as does E.coli because it is incapable of growth at 44.5oC
in the presence of bile salts. Two clinically important species from this genus are E. aerogenes
and E.cloacae (Cabral, 2010).
Proteus spp
Proteus is a genus of gram negative proteobacteria, which includes pathogens responsible
for many urinary tract infections. Proteus exhibit characteristic swarming and they are part of the
normal flora of the gastrointestinal tract. It occurs in intestine of humans and a wide variety of
animal; also occur in manure, soil and polluted water. Three of the Proteus species, P. vulgaris,
P. mirabilis and P. penneri, are pathogenic to humans causing chronic urinary tract infections,
bacteremia, pneumonia and focal lesions. These species only become pathogenic if present
outside the gastro intestinal tract. Proteus also hydrolyzes urea, which alters the pH of urine and
may lead to the formation of kidney stones. Some Proteus species are motile, and all are oxidase
negative, urease positive, aerobic, rod shaped bacilli that do not ferment lactose.
Bacillus spp
Bacillus is a genus of gram positive rod shaped bacteria and a member of the phylum
Firmicutes. Bacillus species can be obligate aerobes or facultative anaerobes, and test positive
for the enzyme catalase. Bacillus includes both free living and pathogenic species. Under
stressful conditions, the cell produces oval endospores that can dormant for extended periods
(Madigan, 2005). They are found in a wide range of habitats, a few species are pathogenic to
vertebrates and invertebrates.
23
Two Bacillus species are considered medically significant: B. anthracis which causes
anthrax and B. cereus which causes a food borne illness similar to that of Staphylococcus. The
type species is B. subtilis an important model organism. It is also a food spoiler, causing ropiness
in bread and related food. Some environmental and commercial strains B. coagulans may play a
role in food spoilage of highly acidic tomato based products (Ryan and Ray, 2004).
24
CHAPTER TWO
MATERIALS AND METHOD
2.1 Collection and Identification of Plant Material
The plant material of the spices Piper guineeense (guinea pepper), Xylopia aethiopica
(African pepper) and Allium cepa (common onion) were purchased locally from Ogige market in
Nsukka. The plant seeds were identified by Mr Ugwuozor of Plant Science and Biotechnology
Department, University of Nigeria, Nsukka.
2.2 Isolation and identification of Microrganisms
Egusi soup which was observed to have spoilt after 24 h storage without reheating and
pepper-soup perceived to spoil after 48 h were obtained from a restaurant and cultured to isolate
contaminant and presumed spoilage organisms using standard bacteriological techniques. A
loopful of each spoilt soup sample was inoculated on sterile nutrient agar plates and incubated at
37oC for 24h. Discrete colonies obtained on the plate were isolated and purified by streaking and
re-isolation three successive times in nutrient agar plates. The pure cultures were subsequently
characterized and tentatively identified on the basis of their cultural, morphological and
biochemical properties with reference to Bergey’s Manual of Determinative Bacteriology, 8th
edition (Bergey and Breed, 1957)
2.3 Test Microorganism
The test organisms were a strain each of Escherichia coli, Enterobacter sp. and
Proteus sp. isolated from egusi soupand two strains of Bacillus spp. – one isolated from egusi-
soup and the other from pepper soup. Other test bacterial strains, namely, Staphylococcus sciuri,
25
Staphylococcus. aureus and Salmonella guineum, were obtained from Professor Char of the
Department of Veterinary Microbiology, University of Nigeria, Nsukka.
2.4 Sample Preparation and Extraction Procedures
The seeds of Piper guineense and X. aethiopica were spread and dried in the sun for 3
to 4 days and then pulverised with a mechanical grinder. The bulbs of Allium cepa were washed
and then air dried at room temperature for one week and pulverised into fine powder using an
electric milling machines.
A 15 g sample of each powdered plant tissue was soaked for 24 h in 100 ml of
absolute ethanol. After 24 h, the extracts were filtered using a clean muslin cloth and then
filtered with Whatman No.1 filter paper. The filtrates were evaporated under forced air current
and the extract obtained. The same procedure was repeated for hot water and cold water in
extraction of the various powdered plant materials. All extracts were stored dry in sterile
containers and refrigerated until used for phytochemical analyses and antimicrobial testing.
2.5 Media Preparation
All media used were prepared according to the manufacturer’s directives. Nutrient
Agar (Fluka) was prepared by suspending 23grams of the medium in one liter of distilled water
and sterilized by autoclaving at 121oC for 15 mins and checked for sterility at 37
oC for 24h. The
Muller- Hinton Agar (Biotec) was prepared by suspending 38 grams of the medium in one liter
of distilled water. It was mixed well and boiled for about one minute and sterilized in an
autoclave at 121oC for 15 mins and checked for sterility at 37
oC for 24h. The Nutrient Agar was
used for plating out the organisms as well as storing them in slants while the Muller- Hinton
Agar was used for sensitivity test.
26
2.6 Preparation of Crude Plant Extracts
A 2 g amount of extract was weighed out, using a Mettler balance, and then dissolved in
5 ml of dimethyl sulphoxide (ethanol extract) or 5 ml of water (water extracts). Subsequently,
each solution was serially diluted two-fold to obtain 400, 200, 100, 50, 25, 12.5, 6.25 and 3.125
mg/ml concentrations.
2.7 Determination of Antimicrobial Activity of Extracts
The antimicrobial activity was evaluated using the agar well diffusion method as
described by Okeke et al. (2001). The dried extracts were reconstituted as described above. Prior
to use, the stock cultures of the test organisms were sub-cultured on nutrient broth and incubated
at 37oC for 12 h. The concentration of the 12 h culture was adjusted to 0.5 McFarland Standard (
i.e. about 105 cfu/ml). A 0.1 ml volume of the standard suspension (10
5 cfu/ml) of each test
bacterial strain was spread evenly on Muller Hinton agar plates using sterile glass rod spreader
and the plates were allowed to dry at room temperature. Subsequently, 6 mm-diameter wells
were bored on the agar and100 µl of each reconstituted plant extract was pipetted into triplicate
wells. After holding the plates at room temperature for 1 hour to allow diffusion of extract into
the agar, they were incubated at 37oC for 24 h and the inhibition zone diameter (IZD) was
measured to the nearest mm. Antimicrobial activities were expressed as the IZD (mm) produced
by the plant extracts.
27
2.8 Determination of Minimum Inhibitory Concentration (MIC) and Minimum
Bactericidal Concentration (MBC) of Crude Extracts
The MIC and MBC of the extracts for each susceptible test organism were determined
by a modification of the broth macro tube dilution method described by Okeke et al. (2001) and
Okoli et al. (2002). Two-fold serial dilutions of the reconstituted extract 400 mg/ml were made
in nutrient broth to achieve a concentration range of approximately 3.125- 400 mg/ml. A 0.1 ml
suspension of the test organism was inoculated into 1 ml of each concentration of the extract in
duplicates. A tube containing nutrient broth only was seeded with the test organism as described
above to serve as control. All culture tubes were incubated at 37oC for 24 h. Growth was scored
visually by the turbidity of the culture. The least concentration showing no growth was taken to
be the MIC.
To determine the MBC, 0.1 ml inoculum was taken from each of the last three
consecutive tubes in which there was no growth and sub cultured on Muller-Hinton Agar plates.
After incubation at 37oC for 24 h, the plates were observed for bacterial growth. The least
concentration showing no growth was taken as the MBC.
2.9 Phytochemical Screening of the Plant Extract
The phytochemical screening was carried out using the methods described by
Farnsworth (1996), Harbone (1998) and Sofowara (1993).
28
2.9.1 Test for Alkaloids
A 0.2 g weight of each plant extracts was boiled with 5 ml of 2% hydrochloric acid in
a water bath for 10 min. The mixture was filtered and 1 portion of each extract treated with 2
drops of the following reagents.
1) Drangendroff’s Reagent: A red precipitate indicates the presence of alkaloids
2) Mayer’s Reagent: A red precipitate indicates the presence of alkaloids
3) Picric Acid (1%). A yellow precipitate indicates the presence of alkaloids.
2.9.2 Test for Flavonoids
A 0.2 g weight of the extracts was heated with 10 ml of ethyl acetate in a boiling water
bath for 3 minutes. The mixture was cooled and filtered. The filtrate was used for the following
test.
1. Ammonium test. Approximately 4 ml of the filtrate was shaken with 1 ml of dilute
ammonia (1%). The layers were allowed to separate and a yellow colour in the ammonia layer
indicates the presence of flavonoid.
2. Aluminum Chloride test. About 4 ml of the filtrate was shaken with 1 ml of dilute 1%
aluminum chloride solution and observed for yellow coloration in the aluminum chloride layer.
2.9.3 Test for Glycosides
This was performed by weighing 2 g of the extract and adding to 30 ml of distilled
water. The mixture was heated for 5 min in a boiling water bath, allowed to cool and filtered.
Fehlings solution A and B were added to the filtrate until it turned alkaline when tested with red
litmus paper. The alkaline mixture was heated in a boiling water bath for 2 minutes. A brick red
precipitate indicates the presence of glycosides.
29
2.9.4 Test for Saponins
To conduct this test, 0.1 g of the extract was boiled with 5 ml of distilled water in a boiling water
bath for 2 min. The mixture was filtered while still hot and allowed to cool. The filtrate was used
for the following tests.
Frothing test: Exactly 1ml of the filtrate was diluted with 4ml of distilled water. The mixture
was vigorously shaken and then observed on standing for a stable froth.
Emulsion Test: To conduct this test, 2 drops of olive oil was added to 1ml of the filtrate. The
mixture was shaken and observed for the formation of emulsion.
2.10.5 Test for Tannins.
To perform this test, 1 g of the extract was boiled with 5 ml of 45% ethanol (45ml of
ethanol in 100ml of distilled water) for 5 min, the solution was filtered and the filtrate treated
with ferric chloride solution. Using a pipette, 2 drops of ferric chloride was added to 1 ml of the
filtrate. A greenish black precipitate indicates the presence of tannins.
2.10.6 Test for Fats and Oil.
About 0.2 g of the extract was pressed between filter paper and the paper observed. A
control was also prepared by placing 2 drops of olive oil in filter paper. Translucency of the filter
paper indicates the presence of fats and oil.
2.10 Screening of ground Spices for Inhibitory Activity against Test Organisms
This was carried out using the method described by Eruteya and Odunfa (2009).
Different concentrations(0.5%, 1.5%, and 3.0%) of grounded powder of Piper guineense,
Xylopia aethiopica and Alium cepa were added to nutrient agar before autoclaving at 1210C for
30
15 min. Using pour plate method 0.1 ml of 18-24 h of the different test organisms was added to
the sterilized nutrient agar containing the different spice concentrations respectively. Plates
without spice but with organisms served as control while plates with spice but no organism
served as standard. The plates were incubated at 370Cfor 24 h. After 24 h the plates were
checked for the concentration that inhibited the growth of the organisms. The inhibitory activity
was recorded by reduction in colony count on agar.
31
CHAPTER THREE
RESULT
3.1 Isolation and Characterization of Test Organisms
A total of seven organisms were used in this study; four, namely, E. coli, Proteus sp.,
Enterobacter sp.and Bacillus strain isolated from spoilt egusi; and three – Staph sciuri, Staph
aureus and Salmonella guineum obtained from Veterinary Microbiology Department. The
Bacillus strain isolated from pepper soup was not susceptible to the spices preparations at the
preliminary screening and, therefore, was not used further. The three strains obtained from
Veterinary Microbiology Department were re-characterised to confirm their identity. All test
organisms were tentatively identified or had their identity confirmed using standard
bacteriological techniques as shown in Table 1.
3.2 Yield from Aqueous and Ethanol Extractions
The spices were extracted with cold water, hot water and ethanol, respectively. The
highest yield was achieved with ethanol P. guineense 4.8g (32%), followed by cold water P.
guineense, 3.7g (24.7%). The least were cold water A. cepa and hot water X. aethiopica. Table 2.
32
Table 1: Characterization of the Test Organisms
Test strain/
(Code)
Cell morphology
and Gram reaction
Biochemical Characteristics
Tentative
organism Catalase Lactose Mannose Glucose Sucrose Citrate Motility Sporulation
ES-1 Gram – rod + - + + + + + - Enterobacter
sp
ES-2 Gram – rod + - + + + - + - Escherichia
coli
ES-3 Gram – rod - - -
-
+ + - + - Proteus sp
ES-4 Gram + rod - + - - - + + Bacillus sp
VS-1 Gram + Cocci + - + `ND + ND ND ND Staphylococc
us sciuri
VS-2 Gram + cocci + + + ND + ND ND ND Staphylococc
us aureus
VS-3 Gram - rod + ND ND + ND + + ND Salmonella
guineum
Key: Key: ES - Egusi Isolate; VS – Vet Isolate; ND – Not Determined
33
Table 2: Yield from the Ethanol and Aqueous Extractions
Extract Weight of
spice(g)
Weight of
Extract(g)
Percentage
Yield
Ethanol X. aethiopica 15 1.9 12.7%
Cold-Water X. aethiopica 15 0.6 4%
Hot-Water X. aethiopica 15 1.1 7.3%
Ethanol P. guineense 15 4.8 32%
Hot-Water P. guineense 15 3.7 10.7%
Cold-Water P. guineense 15 1.6 24.7%
Ethanol A. cepa 15 0.8 5.3%
Hot-Water A. cepa 15 0.7 4.7%
Cold-Water A. cepa 15 0.6 4%
34
3.3 Chemical Constituents in Ethanol, Cold and Hot water of the Extracts.
The secondary metabolites tested were alkaloids, flavonoids, glycosides, Saponins,
Tannins, Fats and oil. Alkaloids were detected in low or moderate amounts from extracts of all
the spices except ethanol extract of P. guineense and A. cepa and Cold water extracts of X.
aethiopica and A. cepa. Flavonoid was not detected in any of the extracts. Glycosides were
detected in high amounts in cold water extracts of P. guineense, in moderate amount in hot
water P. guineense and in low amount in ethanol extract of A. cepa and not at all in the other
extracts. Saponins were detected in moderate amount in ethanol P. guineense, low amount in
cold and hot water extracts of P. guineense and ethanol extract of X. aethiopica but not in
others.
Cold water extracts of P. guineense and X. aethiopica yielded tannins in high amount
while hot water extracts of P. guineense and ethanol extracts of A. cepa yielded moderate
amount. Cold water extracts of X. aethiopica and A. cepa had no detectable tannin. With the
exception of hot water extract of A. cepa and ethanol extract of A. cepa in which fats and oil
occurred in high amounts, it was moderately present in the other extracts. (Table 3-5).
3.4 Anti-bacterial Activity of the Extracts
Tables 6-9 show the inhibition zone diameter (IZD) measurement obtained for
antibacterial activity of the extracts. The highest inhibition zone diameter was achieved with
hot water extract of P. guineense on E.coli 14±0.2mm at a concentration of 400mg/ml
followed by the activity of P. guineense against Bacillus spp 12.7±0.2mm at a concentration of
400mg/ml (Table 6).
35
Table 7 shows the activity of X. aethiopica on E.coli, Bacillus spp, Staph sciuri and
Salmonella guineum. The extract exhibited activity at 400 and 200mg/ml with E. coli showing
the highest IZD 13.1±0.1mm at 400mg/ml. Proteus, Staph aureus and Enterobacter spp
showed no susceptibility at any concentration. The hot water extracts of all the three spices
showed no activity against Proteus and Enterobacter spp. Similarly, the hot water extract of A.
cepa did not show activity against any of the test organisms.
A 400mg/ml concentration of cold extract of A. cepa showed activity of 10±0.2mm
against Enterobacter spp and 12±0.1mm against other test organisms except Bacillus spp
which showed no susceptibility to any of the cold water extracts (Table 8).
As shown in table 9, cold water extract of X. aethiopica was not active against most test
organisms even at 400mg/ml concentration except E.coli, which showed susceptibility to 400,
200 and 100mg/ml, and slightly against Staph aureus IZD 7.8±0.1mm. Finally, cold water
extract of P. guineense and ethanol extract of all the spices showed no activity on any of the
test organisms.
36
Table 3: Chemical Constituents in Ethanol, Hot and Cold Water Extracts of Piper
guineense
Metabolites Ethanol Hot water Cold water
Alkaloids _ ++ ++
Flavonoids _ _ _
Glycosides _ ++ +++
Saponins ++ + +
Tannins + ++ +++
Fats and Oil ++ ++ ++
KEY
_ Absent
+ Low
++ Moderate
+++ High
37
Table 4: Chemical Constituents in Ethanol, Hot and Cold Water Extracts of Xylopia
aethiopica
Metabolites Ethanol Hot water Cold water
Alkaloids + + _
Flavonoids _ _ _
Glycosides _ _ _
Saponins + _ _
Tannins ND ++ +++
Fats and oil ++ ++ ++
KEY
_ Absent
+ Low
++ Moderate
+++ High
ND Not determined
38
Table 5: Chemical Constituents in Ethanol, Hot and Cold Water Extracts of Allium
cepa
Metabolites Ethanol Hot water Cold water
Alkaloids _ ++ _
Flavonoids _ _ _
Glycosides + _ ND
Saponins _ _ ND
Tannins ++ + ND
Fats and oil +++ +++ ++
KEY
- Absent
+ Low
++ Moderate
+++ High
ND Not determined
39
Table 6: Inhibition of Microorganisms by Hot Water Extract of Piper guineense
Test organisms
Inhibition Zone Diameter (IZD mm) achieved at each extract
concentration
400 200 100 50 25 12.5 6.25 3.125
Escherichia coli 14.7±0.3 9.9±0.2 7.9±0.1 0.0 0.0 0.0 0.0 0.0
Salmonella guineum 8.2±0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Proteus 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Bacillus spp
Staphylococcus aureus
12.7±0.6
8.0±0.1
10.8±0.3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Staphylococcus sciuri 9.1±0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Enterobacter spp
0.0 0.0 0.0 0.0 0.0 0.0 0.0
0.0
KEY
0.0 = No Activity
40
Table 7: Inhibition of Microorganisms by Hot water Extract of Xylopia aethiopica
Inhibition Zone Diameter (IZD mm) achieved at each extract
concentration
Test Organisms 400 200 100 50 25 12.5 6.25 3.125
Escherichia coli 13.1±0.2 11±0.2 10.1±0.1 0.0 0.0 0.0 0.0 0.0
Salmonella guineum 9.9±0.1 7.7±0.3 0.0 0.0 0.0 0.0 0.0 0.0
Proteus 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Bacillus spp 9.1±0.1 7.3±0.3 0.0 0.0 0.0 0.0 0.0 0.0
Staphylococcus aureus 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Staphylococcus sciuri 10.1±0.1 7.4±0.1 0.0 0.0 0.0 0.0 0.0 0.0
Enterobacter spp. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
KEY
0.0 = No Activity
41
Table 8: Inhibition of Microorganisms by Cold Water Extract of Allium cepa
Inhibition Zone Diameter (IZD mm) achieved at each extract
concentration
Test organism 400 200 100 50 25 12.5 6.25 3.125
Escherichia coli 12.0±0.2 10.2±0.2 9.8±0.2 0.0 0.0 0.0 0.0 0.0
Salmonella guineum 12.0±0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Proteus 12±0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Bacillus spp 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Staphylococcus aureus 12±0.2 10±0.2 10±0.1 0.0 0.0 0.0 0.0 0.0
Staphylococcus sciuri 12±0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Enterobacter spp 10±0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0
KEY
0.0 = No Activity
42
Table 9: Inhibition of Microorganism by Cold Water Extract of Xylopia aethiopica
Inhibition Zone Diameter (IZD mm) achieved at each extract
concentration
Test Organisms 400 200 100 50 25 12.5 6.25 3.125
Escherichia coli 12±0.1 10±0.1 7.2±0.1 0.0 0.0 0.0 0.0 0.0
Salmonella guineum 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Proteus 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Bacillus spp 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Staphylococcus aureus 7.8±0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Staphylococcus sciuri 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Enterobacter spp. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
KEY
0.0 = No Activity
43
3.5 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration
(MBC) of the Extracts
The result revealed that the MIC and MBC of hot water extracts were generally lower
than those of cold water and ethanol extracts. The least activity was recorded for the ethanol
extracts of all the spices (Table 10).
Cold water extract of P. guineense showed no bacterial activity while cold water extract
of X. aethiopica and A. cepa had MBC values ranging from 50mg/ml to 100mg/ml against all
test organisms except Proteus (Table 11). The highest activity was recorded for hot water P.
guineense with MIC and MBC values of 3.125mg/ml and 25 mg/ml, respectively, for both
Salmonella guineum and Staph. sciuri followed by hot water extracts of A. cepa with MIC and
MBC values of 3.125mg/ml and 50mg/ml, respectively, for both E. coli and Staph. sciuri.
None of the extract showed bactericidal activity against Bacillus spp (Table 12).
3.6 Screening of Spices for Inhibitory Activity against Test Organisms
Tables 13-16 show the effects of combinations (ratio of 1:1 or 1:1:1) of ground spices used
against each test organisms.The combination of X. aethiopica and P. guineense had
inhibitory effect on S.guineum, Staph aureus and Enterobacter spp at concentrations of 3%
(Table 13). The combination of A. cepa : X. aethiopica at concentration of 3% had inhibitory
effect on all test organisms except Staph aureus and E.coli (Table 14). The combination of P.
guineense and A .cepa and X. aethiopica, P. guineense and A.cepa had an inhibitory effect at
concentration of 3.0% on all the test organisms (Table15 & 16).
44
Table 10: Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
of Ethanol Extract of X. aethiopica, P. guineense and A.cepa
Test organism Piper guineense
(mg/ml)
Xylopia aethiopica
(mg/ml)
Alium cepa
(mg/ml)
MIC MBC MIC MBC MIC MBC
Escherichia coli 50 _ 100 _ 100 _
Salmonella guineum 25 _ 50 _ 50 50
Proteus 50 _ 100 _ 50 _
Bacillus spp 100 _ 100 _ 50 _
Staphylococcus sciuri 50 _ 50 _ _ _
Enterobacter spp 50 _ 25 _ _ _
Staphylococcus aureus 100 _ 50 _ _ _
KEY
- = No Activity
45
Table 11: Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
of Cold Water Extract of X. aethiopica, P. guineense and A. cepa
Test organism Piper guineense
(mg/ml)
Xylopia aethiopica
(mg/ml)
Allium cepa
(mg/ml)
MIC MBC MIC MBC MIC MBC
Escherichia coli 100 _ 100 400 50 50
Salmonella guineum 100 _ 100 _ 25 50
Proteus _ _ 100 _ _ _
Bacillus spp 100 _ 100 _ 50 100
Staphylococcus sciuri 50 _ 50 100 100 100
Enterobacter spp _ _ 100 _ 100 50
Staphylococcus aureus _ _ 50 400 50 100
KEY
- = No Activity
46
Table 12: Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
of Hot Water Extract of X. aethiopica, P. guineense and A. cepa
Test organism Piper guineense
(mg/ml)
Xylopia aethiopica
(mg/ml)
Allium cepa
(mg/ml)
MIC MBC MIC MBC MIC
MBC
Escherichia coli 25 100 3.125 100 3.125 50
Salmonella guineum 3.125 25 50 200 12.5 50
Proteus 25 25 3.125 _ 12.5 100
Bacillus spp 3.125 _ 3.125 _ 12.5 _
Staphylococcus sciuri 3.125 25 3.125 400 3.125 50
Enterobacter spp 25 100 50 _ 6.25 100
Staphylococcus aureus 3.125 50 3.125 _ 3.125 100
KEY
- = No Activity
47
Table 13: Effect of Spice Combination (X. aethiopica: P. guineense ) on Test Organisms
Test organisms No Spice 0.5% 1.5% 3.0%
Escherichia coli +++ +++ +++ +
Salmonella guineum +++ +++ ++ _
Proteus +++ ++ ++ +
Bacillus spp +++ +++ ++ +
Staphylococcus aureus +++ +++ +++ ++
Staphylococcus sciuri +++ +++ +++ _
Enterobacter spp +++ +++ ++ _
KEY
+++ = Abundant growth
++ = Growth (numerous separate colonies)
+ = limited growth
- = No growth
48
Table 14: Effect of Spice Combination (P. guineense: A. cepa) on Test Organisms
Test organisms No Spice 0.5% 1.5% 3.0%
Escherichia coli +++ ++ ++ +
Salmonella guineum +++ +++ _ _
Proteus +++ +++ ++ _
Bacillus spp +++ +++ ++ _
Staphylococcus aureus +++ +++ +++ ++
Staphylococcus sciuri +++ +++ + _
Enterobacter spp +++ +++ ++ _
KEY
+++ = Abundant growth
++ = Growth (numerous separate colonies)
+ = limited growth
- = No growth
49
Table 15: Effect of Spice Combination (Alium cepa: X.aethiopica) on Test Organisms
Test Organisms No Spice 0.5% 1.5% 3.0%
Escherichia coli +++ ++ + _
Salmonella guineum +++ +++ _ _
Proteus +++ +++ + _
Bacillus spp +++ +++ +++ _
Staphylococcus aureus +++ +++ +++ _
Staphylococcus sciuri +++ + _ _
Enterobacter spp +++ +++ + _
KEY
+++ = Abundant growth
++ = Growth (numerous separate colonies)
+ = limited growth
- = No growth
50
Table 16: Effect Spice Combination (X. aethiopica : P. guineense : A. cepa ) on Test
Organisms
Test organisms No Spice 0.5% 1.5% 3.0%
Escherichia coli +++ ++ + _
Salmonella guineum +++ ++ _ _
Proteus +++ +++ ++ _
Bacillus spp +++ +++ +++ _
Staphylococcus aureus +++ ++ + _
Staphylococcus sciuri +++ ++ ++ _
Enterobacter spp +++ + + _
KEY
+++ = Abundant growth
++ = Growth (numerous separate colonies)
+ = limited growth
- = No growth
51
CHAPTER FOUR
DISCUSSION
Additives preserve foods by inhibiting microbial growth or inhibiting enzyme activity
in case of fruits and forestalling spoilage. It is on this premise that spices are being tested for
antibacterial properties; and presumably those with antibacterial activity could be further
studied for use as natural preservatives for recipes where they are used for spicing or as
condiments. This trend is being promoted because of the safety concerns surrounding the use
of chemical additives for food preservation. In this study, the preservative effect of three
Nigerian spices Piper guineense, Xylopia aethiopica and Allium cepa used in the preparation of
pepper-soup was evaluated.
The phytochemical screening of the plant crude extracts revealed the presence of
Alkaloids, Glycosides, Saponnins, Tannins, Fats and oil in varying proportions and the absence
of Flavonoids. These compounds have variously been reported to possess antimicrobial activity
(Okeke et al., 2001, Mahajan et al., 2008). The absence of flavonoids was reported in all the
extracts and flavonoids have been found in–vitro to be effective antimicrobial substance
against a wide array of microorganisms (Azu et al., 2007; Ijeh et al, 2004; Ekpo et al; 2012).
The results show that there are differences in phytochemical constituents extractable by the
different solvents (cold water, hot water and ethanol) used. These differences seem to be
reflected in the difference in spectrum and degree of antibacterial activity of extracts on the test
organisms. Almost all the metabolites detected have been suspected to contribute to
antimicrobial activity of extracts in other reports (Okeke et al., 2001, Mahajan et al., 2008).
For example, tannins have been found to form irreversible complexes with proline–rich
proteins resulting in the inhibition of the cell protein synthesis; besides, herbs that contain
52
tannin are astringent in nature and are used for treating intestinal disorders such as diarrhea and
dysentery but this use has not been specifically found to be due to their antimicrobial activities
(Shimada, 2006 and Dharmanda, 2003). There was no experiment in this work designed to
trace the specific active compounds in the extracts.
From this investigation, the aqueous extract of the spices have more potential as an
antimicrobial agent than its ethanolic extracts. This result is in agreement with Ijeh et al.
(2005), who reported the higher susceptibility of test organisms to aqueous extract of O.
grattisimum and X. aethiopica, Olusimbo et al. (2011) who reported the antimicrobial potential
of the aqueous extract of Piper guineense and Azu et al. (2007) who reported the antibacterial
properties of the water soluble extracts of onion. The high level of activity observed in the
aqueous extracts against the bacterial pathogens showed that the active components were
soluble in water. This property is very desirable as these spices are used as condiments in food
preparation (Olusimbo et al., 2011). This also supports the extensive inclusion of these spices
in folklore medicinal preparations in various cultures in Africa. For example it is believed that
Piper guineense stimulates the production of hydrochloric acid in the stomach and promotes
the health of the digestive tract (Olusimbo et al., 2011).
It was observed that the hot water extracts of Piper guineense and Xylopia aethiopica
showed activity against all the test organisms except Proteus spp and Enterobacter spp. (Table
6 & 7). This may mean that heating of these spices during cooking would not inactivate the
ingredients that are active against the suscepetible bacterial strains. In other words the spices in
the cooked food samples may already be active against some contaminants, thereby providing
a measure of food preservation. It was also observed that hot water extract of Allium cepa did
not show any activity. This may be explained by the fact that the antimicrobial substance in the
53
onion extracts, which are mainly phenolic compounds are heat labile and might have been
denatured during the extraction process (Azu et al., 2007).
The cold water extract of Allium cepa exhibited activity at (400mg/ml) on Salmonella
guineum, Proteus, Staphylococcus scuiri, Enterobacter spp, Escherichia coli and Staph. aureus
and inactivity on Bacillus spp. This would imply that for A. cepa to be applied in food
preservation, its cold water extract should not be subjected to heating or cooking. This leads to
the speculation that chewing of raw onions may reduce presence or number/type of pathogens
in the oral cavity, thus helping to maintain oral hygiene. The non-susceptibility of Bacillus spp
may be explained by the physiology of the strain, since the organism has the ability to form
resting spores under adverse environmental conditions, it may also form resting spores when
exposed to theeffect of the extracts. Specific investigation needs to be carried out on the
differential reactions of the vegetative cells and spores of Bacillus sp to exposure to different
concentrations of cold extracts of A. cepa. Also, the cold water extract of Xylopia aethiopica
had activity only on E. coli at 400mg/ml while cold water Piper guineense was not active
against the test organism, suggesting that cold water as a solvent could dissolve the active
constituents of Allium cepa better compared to X. aethiopica and P. guineense extract (Azu et
al, 2007). It is not known to what extent the activity of cold water extract of X. aethiopica
could affect the intestinal E. coli in vivo and whether this could partly explain the diarrhea that
results in some individuals after consumption of much pepper.
It was also observed that there were differences in susceptibility of the test
microorganisms used in the research to each extract. Escherichia coli was the most inhibited
organism by P. guineense, A. cepa and X. aethiopica with a mean inhibition zone diameter
(IZD) of 3.99mm while the least inhibited was Enterobacter spp with a mean inhibition zone
54
diameter of 0.314mm by cold water A. cepa. Escherichia. coli was best inhibited by hot water
extract of Piper guineense with an average IZD of 14.7±0.3mm. Varied susceptibility of each
test organisms to the extracts usually reflect the differences in physiology of individual
bacterial species as stated by Garret et al., (2000) or differences in the quantity and quality of
the active ingredients (Cowan, 1999; Adetiyi et al., 2004), extraction methods employed, the
dosage of extract applied and the diffusion properties of these extracts in the agar. (Ekwenye
and Elegalam, 2005).
A 50:50 combination of the extracts of A.cepa and X.aethiopica had the same activity
(IZD) as the ground spice material applied directly in 1:1 proportions also. The later was not
estimated by IZD measurement but by reduction in colony count in agar. All materials used in
this assay were heat- treated. In the first place the activity shown by both after heat treatment
can only come from heat-stable components present in both the extract and in the unextracted
spice preparation. Both materials having relatively equivalent activity may indicate that it is the
same components that show activity in them. A residual activity after heat treatment is
desirable since almost all recipes in which the spices are applied are cooked at high heat. This
means that the heat stable antimicrobial constituent confers a measure of preservation on the
food for a period.
CONCLUSION
This research shows that the spices have a measure of antimicrobial actvivity and also
potentials for use as preservative. More studies need to be carried out to determine how these
spices can be used as preservatives- in what form, what quantity and how long.
55
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60
APPENDIX 1
UNIANOVA Clearance BY Organisms Conc
/METHOD=SSTYPE(3)
/INTERCEPT=INCLUDE
/EMMEANS=TABLES(Organisms)
/EMMEANS=TABLES(Conc)
/EMMEANS=TABLES(Organisms*Conc)
/PRINT=DESCRIPTIVE
/CRITERIA=ALPHA(.05)
/DESIGN=Organisms Conc Organisms*Conc.
UNIANOVA Clearance BY Organisms Conc Groups
/METHOD=SSTYPE(3)
/INTERCEPT=INCLUDE
/POSTHOC=Organisms Conc Groups(LSD)
/EMMEANS=TABLES(Organisms)
/EMMEANS=TABLES(Conc)
/EMMEANS=TABLES(Groups)
/EMMEANS=TABLES(Organisms*Conc)
/EMMEANS=TABLES(Organisms*Groups)
/EMMEANS=TABLES(Organisms*Conc*Groups)
/PRINT=DESCRIPTIVE
/CRITERIA=ALPHA(.05)
/DESIGN=Organisms Conc Groups Organisms*Conc Organisms*Groups Conc*Groups Organisms*Conc*Groups.
Descriptive Statistics
Dependent Variable:Clearance
Organisms Conc Groups Mean Std. Deviation N
E.coli 400 mg/ml Hot water Piper guineense 14.733 .2517 3
Hot water Xylopia aethiopica 13.100 .1732 3
Cold water Allium cepa 12.000 .2000 3
Cold water Xylopia aethiopica 11.967 .1528 3
Total 12.950 1.1882 12
200 mg/ml Hot water Piper guineense 9.867 .2309 3
Hot water Xylopia aethiopica 11.000 .2000 3
Cold water Allium cepa 10.200 .2000 3
Cold water Xylopia aethiopica 10.000 .1000 3
Total 10.267 .4868 12
100 mg/ml Hot water Piper guineense 7.867 .1155 3
61
Hot water Xylopia aethiopica 10.133 .1155 3
Cold water Allium cepa 9.833 .2082 3
Cold water Xylopia aethiopica 7.233 .0577 3
Total 8.767 1.3020 12
50 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
25 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
12.5 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
6.25 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
3.125 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
Total Hot water Piper guineense 4.058 5.6488 24
62
Hot water Xylopia aethiopica 4.279 5.6974 24
Cold water Allium cepa 4.004 5.3147 24
Cold water Xylopia aethiopica 3.650 4.9647 24
Total 3.998 5.3330 96
Salmonella spp 400 mg/ml Hot water Piper guineense 8.167 .2887 3
Hot water Xylopia aethiopica 9.867 .1155 3
Cold water Allium cepa 12.000 .2000 3
Cold water Xylopia aethiopica .000 .0000 3
Total 7.508 4.7473 12
200 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica 7.867 .2309 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total 1.967 3.5592 12
100 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
50 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
25 mg/ml Hot water Piper guineens .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
12.5 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
63
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
6.25 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
3.125 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
Total Hot water Piper guineense 1.021 2.7603 24
Hot water Xylopia aethiopica 2.217 3.9558 24
Cold water Allium cepa 1.500 4.0544 24
Cold water Xylopia aethiopica .000 .0000 24
Total 1.184 3.2044 96
Proteus 400 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa 12.100 .2000 3
Cold water Xylopia aethiopica .000 .0000 3
Total 3.025 5.4731 12
200 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
100 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
64
50 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
25 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
12.5 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
6.25 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
3.125 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
Total Hot water Piper guineense .000 .0000 24
Hot water Xylopia aethiopica .000 .0000 24
Cold water Allium cepa 1.513 4.0882 24
Cold water Xylopia aethiopica .000 .0000 24
Total .378 2.1166 96
Bacillus spp 400 mg/ml Hot water Piper guineense 12.667 .5774 3
Hot water Xylopia aethiopica 9.067 .1155 3
65
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total 5.433 5.8340 12
200 mg/ml Hot water Piper guineense 10.800 .3464 3
Hot water Xylopia aethiopica 7.333 .3055 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total 4.533 4.9089 12
100 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
50 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
25 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
12.5 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
6.25 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
66
Total .000 .0000 12
3.125 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
Total Hot water Piper guineense 2.933 5.2156 24
Hot water Xylopia aethiopica 2.050 3.6553 24
Cold water Allium cepa .000 .0000 24
Cold water Xylopia aethiopica .000 .0000 24
Total 1.246 3.3893 96
Staphylococcus aureus 400 mg/ml Hot water Piper guineense 8.000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa 11.933 .2082 3
Cold water Xylopia aethiopica 7.833 .0577 3
Total 6.942 4.5242 12
200 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa 10.000 .2000 3
Cold water Xylopia aethiopica .000 .0000 3
Total 2.500 4.5235 12
100 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa 10.033 .0577 3
Cold water Xylopia aethiopica .000 .0000 3
Total 2.508 4.5378 12
50 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
25 mg/ml Hot water Piper guineense .000 .0000 3
67
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
12.5 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
6.25 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
3.125 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
Total Hot water Piper guineense 1.000 2.7027 24
Hot water Xylopia aethiopica .000 .0000 24
Cold water Allium cepa 3.996 5.3005 24
Cold water Xylopia aethiopica .979 2.6464 24
Total 1.494 3.5412 96
Staphylococcus sciuri 400 mg/ml Hot water Piper guineense 9.067 .1155 3
Hot water Xylopia aethiopica 10.133 .1155 3
Cold water Allium cepa 11.967 .2517 3
Cold water Xylopia aethiopica .000 .0000 3
Total 7.792 4.8235 12
200 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica 7.433 .1155 3
Cold water Allium cepa .000 .0000 3
68
Cold water Xylopia aethiopica .000 .0000 3
Total 1.858 3.3622 12
100 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
50 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
25 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
12.5 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
6.25 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
3.125 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
69
Total Hot water Piper guineense 1.133 3.0632 24
Hot water Xylopia aethiopica 2.196 3.9461 24
Cold water Allium cepa 1.496 4.0434 24
Cold water Xylopia aethiopica .000 .0000 24
Total 1.206 3.2615 96
Enterobacter spp 400 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa 10.033 .2517 3
Cold water Xylopia aethiopica .000 .0000 3
Total 2.508 4.5390 12
200 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
100 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
50 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
25 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
12.5 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
70
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
6.25 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
3.125 mg/ml Hot water Piper guineense .000 .0000 3
Hot water Xylopia aethiopica .000 .0000 3
Cold water Allium cepa .000 .0000 3
Cold water Xylopia aethiopica .000 .0000 3
Total .000 .0000 12
Total Hot water Piper guineense .000 .0000 24
Hot water Xylopia aethiopica .000 .0000 24
Cold water Allium cepa 1.254 3.3904 24
Cold water Xylopia aethiopica .000 .0000 24
Total .314 1.7553 96
Total 400 mg/ml Hot water Piper guineense 7.519 5.4082 21
Hot water Xylopia aethiopica 6.024 5.4758 21
Cold water Allium cepa 10.005 4.2463 21
Cold water Xylopia aethiopica 2.829 4.7208 21
Total 6.594 5.5513 84
200 mg/ml Hot water Piper guineense 2.952 4.7920 21
Hot water Xylopia aethiopica 4.805 4.4235 21
Cold water Allium cepa 2.886 4.6766 21
Cold water Xylopia aethiopica 1.429 3.5858 21
Total 3.018 4.4799 84
100 mg/ml Hot water Piper guineense 1.124 2.8210 21
Hot water Xylopia aethiopica 1.448 3.6337 21
Cold water Allium cepa 2.838 4.5991 21
Cold water Xylopia aethiopica 1.033 2.5937 21
71
Total 1.611 3.5141 84
50 mg/ml Hot water Piper guineense .000 .0000 21
Hot water Xylopia aethiopica .000 .0000 21
Cold water Allium cepa .000 .0000 21
Cold water Xylopia aethiopica .000 .0000 21
Total .000 .0000 84
25 mg/ml Hot water Piper guineense .000 .0000 21
Hot water Xylopia aethiopica .000 .0000 21
Cold water Allium cepa .000 .0000 21
Cold water Xylopia aethiopica .000 .0000 21
Total .000 .0000 84
12.5 mg/ml Hot water Piper guineense .000 .0000 21
Hot water Xylopia aethiopica .000 .0000 21
Cold water Allium cepa .000 .0000 21
Cold water Xylopia aethiopica .000 .0000 21
Total .000 .0000 84
6.25 mg/ml Hot water Piper guineense .000 .0000 21
Hot water Xylopia aethiopica .000 .0000 21
Cold water Allium cepa .000 .0000 21
Cold water Xylopia aethiopica .000 .0000 21
Total .000 .0000 84
3.125 mg/ml Hot water Piper guineense .000 .0000 21
Hot water Xylopia aethiopica .000 .0000 21
Cold water Allium cepa .000 .0000 21
Cold water Xylopia aethiopica .000 .0000 21
Total .000 .0000 84
Total Hot water Piper guineense 1.449 3.6684 168
Hot water Xylopia aethiopica 1.535 3.5883 168
Cold water Allium cepa 1.966 4.2509 168
Cold water Xylopia aethiopica .661 2.4437 168
Total 1.403 3.5720 672
72
Post Hoc Tests
Groups
Multiple Comparisons
Clearance
LSD
(I) Groups (J) Groups
Mean Difference
(I-J) Std. Error Sig.
95% Confidence Interval
Lower Bound
Upper
Bound
Hot water Piper guineense Hot water Xylopia aethiopica -.085* .0087 .000 -.102 -.068
Cold water Allium cepa -.517* .0087 .000 -.534 -.500
Cold water Xylopia aethiopica .788* .0087 .000 .771 .805
Hot water Xylopia aethiopica Hot water Piper guineense .085* .0087 .000 .068 .102
Cold water Allium cepa -.432* .0087 .000 -.449 -.414
Cold water Xylopia aethiopica .873* .0087 .000 .856 .890
Cold water Allium cepa Hot water Piper guineense .517* .0087 .000 .500 .534
Hot water Xylopia aethiopica .432* .0087 .000 .414 .449
Cold water Xylopia aethiopica 1.305* .0087 .000 1.288 1.322
Cold water Xylopia aethiopica Hot water Piper guineense -.788* .0087 .000 -.805 -.771
Hot water Xylopia aethiopica -.873* .0087 .000 -.890 -.856
Cold water Allium cepa -1.305* .0087 .000 -1.322 -1.288
Based on observed means.
The error term is Mean Square(Error) = .006.
*. The mean difference is significant at the .05 level.
Conc
Multiple Comparisons
Clearance
LSD
(I) Conc (J) Conc
Mean Difference
(I-J) Std. Error Sig.
95% Confidence Interval
Lower Bound Upper Bound
73
400 mg/ml 200 mg/ml 3.576* .0123 .000 3.552 3.600
100 mg/ml 4.983* .0123 .000 4.959 5.007
50 mg/ml 6.594* .0123 .000 6.570 6.618
25 mg/ml 6.594* .0123 .000 6.570 6.618
12.5 mg/ml 6.594* .0123 .000 6.570 6.618
6.25 mg/ml 6.594* .0123 .000 6.570 6.618
3.125 mg/ml 6.594* .0123 .000 6.570 6.618
200 mg/ml 400 mg/ml -3.576* .0123 .000 -3.600 -3.552
100 mg/ml 1.407* .0123 .000 1.383 1.431
50 mg/ml 3.018* .0123 .000 2.994 3.042
25 mg/ml 3.018* .0123 .000 2.994 3.042
12.5 mg/ml 3.018* .0123 .000 2.994 3.042
6.25 mg/ml 3.018* .0123 .000 2.994 3.042
3.125 mg/ml 3.018* .0123 .000 2.994 3.042
100 mg/ml 400 mg/ml -4.983* .0123 .000 -5.007 -4.959
200 mg/ml -1.407* .0123 .000 -1.431 -1.383
50 mg/ml 1.611* .0123 .000 1.587 1.635
25 mg/ml 1.611* .0123 .000 1.587 1.635
12.5 mg/ml 1.611* .0123 .000 1.587 1.635
6.25 mg/ml 1.611* .0123 .000 1.587 1.635
3.125 mg/ml 1.611* .0123 .000 1.587 1.635
50 mg/ml 400 mg/ml -6.594* .0123 .000 -6.618 -6.570
200 mg/ml -3.018* .0123 .000 -3.042 -2.994
100 mg/ml -1.611* .0123 .000 -1.635 -1.587
25 mg/ml .000 .0123 1.000 -.024 .024
12.5 mg/ml .000 .0123 1.000 -.024 .024
6.25 mg/ml .000 .0123 1.000 -.024 .024
3.125 mg/ml .000 .0123 1.000 -.024 .024
25 mg/ml 400 mg/ml -6.594* .0123 .000 -6.618 -6.570
200 mg/ml -3.018* .0123 .000 -3.042 -2.994
100 mg/ml -1.611* .0123 .000 -1.635 -1.587
50 mg/ml .000 .0123 1.000 -.024 .024
74
12.5 mg/ml .000 .0123 1.000 -.024 .024
6.25 mg/ml .000 .0123 1.000 -.024 .024
3.125 mg/ml .000 .0123 1.000 -.024 .024
12.5 mg/ml 400 mg/ml -6.594* .0123 .000 -6.618 -6.570
200 mg/ml -3.018* .0123 .000 -3.042 -2.994
100 mg/ml -1.611* .0123 .000 -1.635 -1.587
50 mg/ml .000 .0123 1.000 -.024 .024
25 mg/ml .000 .0123 1.000 -.024 .024
6.25 mg/ml .000 .0123 1.000 -.024 .024
3.125 mg/ml .000 .0123 1.000 -.024 .024
6.25 mg/ml 400 mg/ml -6.594* .0123 .000 -6.618 -6.570
200 mg/ml -3.018* .0123 .000 -3.042 -2.994
100 mg/ml -1.611* .0123 .000 -1.635 -1.587
50 mg/ml .000 .0123 1.000 -.024 .024
25 mg/ml .000 .0123 1.000 -.024 .024
12.5 mg/ml .000 .0123 1.000 -.024 .024
3.125 mg/ml .000 .0123 1.000 -.024 .024
3.125 mg/ml 400 mg/ml -6.594* .0123 .000 -6.618 -6.570
200 mg/ml -3.018* .0123 .000 -3.042 -2.994
100 mg/ml -1.611* .0123 .000 -1.635 -1.587
50 mg/ml .000 .0123 1.000 -.024 .024
25 mg/ml .000 .0123 1.000 -.024 .024
12.5 mg/ml .000 .0123 1.000 -.024 .024
6.25 mg/ml .000 .0123 1.000 -.024 .024
Based on observed means.
The error term is Mean Square(Error) = .006.
*. The mean difference is significant at the .05 level.
75
Homogeneous Subsets
Organisms
Multiple Comparisons
Clearance
LSD
(I) Organisms (J) Organisms
Mean Difference
(I-J) Std. Error Sig.
95% Confidence Interval
Lower Bound Upper Bound
E.coli Salmonella spp 2.814* .0115 .000 2.791 2.836
Proteus 3.620* .0115 .000 3.597 3.642
Bacillus spp 2.752* .0115 .000 2.729 2.775
Staphylococcus aureus 2.504* .0115 .000 2.482 2.527
Staphylococcus sciuri 2.792* .0115 .000 2.769 2.814
Enterobacter spp 3.684* .0115 .000 3.662 3.707
Salmonella spp E.coli -2.814* .0115 .000 -2.836 -2.791
Proteus .806* .0115 .000 .784 .829
Bacillus spp -.061* .0115 .000 -.084 -.039
Staphylococcus aureus -.309* .0115 .000 -.332 -.287
Staphylococcus sciuri -.022 .0115 .058 -.044 .001
Enterobacter spp .871* .0115 .000 .848 .893
Proteus E.coli -3.620* .0115 .000 -3.642 -3.597
Salmonella spp -.806* .0115 .000 -.829 -.784
Bacillus spp -.868* .0115 .000 -.890 -.845
Staphylococcus aureus -1.116* .0115 .000 -1.138 -1.093
Staphylococcus sciuri -.828* .0115 .000 -.851 -.806
Enterobacter spp .065* .0115 .000 .042 .087
Bacillus spp E.coli -2.752* .0115 .000 -2.775 -2.729
Salmonella spp .061* .0115 .000 .039 .084
Proteus .868* .0115 .000 .845 .890
Staphylococcus aureus -.248* .0115 .000 -.271 -.22
Staphylococcus sciuri .040* .0115 .001 .017 .062
76
Enterobacter spp .932* .0115 .000 .910 .955
Staphylococcus aureus E.coli -2.504* .0115 .000 -2.527 -2.482
Salmonella spp .309* .0115 .000 .287 .332
Proteus 1.116* .0115 .000 1.093 1.138
Bacillus spp .248* .0115 .000 .225 .271
Staphylococcus sciuri .288* .0115 .000 .265 .310
Enterobacter spp 1.180* .0115 .000 1.158 1.203
Staphylococcus sciuri E.coli -2.792* .0115 .000 -2.814 -2.769
Salmonella spp .022 .0115 .058 .000 .044
Proteus .828* .0115 .000 .806 .851
Bacillus spp -.040* .0115 .001 -.062 -.017
Staphylococcus aureus -.288* .0115 .000 -.310 -.265
Enterobacter spp .893* .0115 .000 .870 .915
Enterobacter spp E.coli -3.684* .0115 .000 -3.707 -3.662
Salmonella spp -.871* .0115 .000 -.893 -.848
Proteus -.065* .0115 .000 -.087 -.042
Bacillus spp -.932* .0115 .000 -.955 -.910
Staphylococcus aureus -1.180* .0115 .000 -1.203 -1.158
Staphylococcus sciuri -.893* .0115 .000 -.915 -.870
Based on observed means.
The error term is Mean Square(Error) = .006.
*. The mean difference is significant at the .05 level.
Homogeneous Subsets
Estimated Marginal Means
1. Organisms
Dependent Variable:Clearance
Organisms Mean Std. Error
95% Confidence Interval
Lower Bound Upper Bound
E.coli 3.998 .008 3.982 4.014
77
Salmonella spp 1.184 .008 1.168 1.200
Proteus .378 .008 .362 .394
Bacillus spp 1.246 .008 1.230 1.262
Staphylococcus aureus 1.494 .008 1.478 1.510
Staphylococcus sciuri 1.206 .008 1.190 1.222
Enterobacter spp .314 .008 .298 .330
2. Conc
Dependent Variable:Clearance
Conc Mean Std. Error
95% Confidence Interval
Lower Bound Upper Bound
400 mg/ml 6.594 .009 6.577 6.611
200 mg/ml 3.018 .009 3.001 3.035
100 mg/ml 1.611 .009 1.594 1.628
50 mg/ml 7.344E-17 .009 -.017 .017
25 mg/ml 7.344E-17 .009 -.017 .017
12.5 mg/ml 7.344E-17 .009 -.017 .017
6.25 mg/ml 7.344E-17 .009 -.017 .017
3.125 mg/ml 4.837E-16 .009 -.017 .017
3. Groups
Dependent Variable:Clearance
Groups Mean Std. Error
95% Confidence Interval
Lower Bound Upper Bound
Hot water Piper guineense 1.449 .006 1.437 1.461
Hot water Xylopia aethiopica 1.535 .006 1.522 1.547
Cold water Allium cepa 1.966 .006 1.954 1.978
Cold water Xylopia aethiopica .661 .006 .649 .673
78
4. Organisms * Conc
Dependent Variable:Clearance
Organisms Conc Mean Std. Error
95% Confidence Interval
Lower Bound Upper Bound
E.coli 400 mg/ml 12.950 .023 12.905 12.995
200 mg/ml 10.267 .023 10.221 10.312
100 mg/ml 8.767 .023 8.721 8.812
50 mg/ml 2.288E-16 .023 -.045 .045
25 mg/ml 2.288E-16 .023 -.045 .045
12.5 mg/ml 2.288E-16 .023 -.045 .045
6.25 mg/ml 3.891E-16 .023 -.045 .045
3.125 mg/ml -7.118E-15 .023 -.045 .045
Salmonella spp 400 mg/ml 7.508 .023 7.463 7.554
200 mg/ml 1.967 .023 1.921 2.012
100 mg/ml -1.099E-15 .023 -.045 .045
50 mg/ml -4.341E-16 .023 -.045 .045
25 mg/ml -4.341E-16 .023 -.045 .045
12.5 mg/ml -4.712E-16 .023 -.045 .045
6.25 mg/ml -4.036E-16 .023 -.045 .045
3.125 mg/ml -2.641E-15 .023 -.045 .045
Proteus 400 mg/ml 3.025 .023 2.980 3.070
200 mg/ml -1.339E-16 .023 -.045 .045
100 mg/ml 5.240E-16 .023 -.045 .045
50 mg/ml 2.003E-16 .023 -.045 .045
25 mg/ml 2.743E-16 .023 -.045 .045
12.5 mg/ml 1.492E-16 .023 -.045 .045
6.25 mg/ml -2.168E-17 .023 -.045 .045
3.125 mg/ml -6.482E-16 .023 -.045 .045
Bacillus spp 400 mg/ml 5.433 .023 5.388 5.479
200 mg/ml 4.533 .023 4.488 4.579
100 mg/ml 3.090E-17 .023 -.045 .045
79
50 mg/ml -3.613E-17 .023 -.045 .045
25 mg/ml -7.784E-17 .023 -.045 .045
12.5 mg/ml -1.373E-16 .023 -.045 .045
6.25 mg/ml -4.328E-16 .023 -.045 .045
3.125 mg/ml 8.762E-16 .023 -.045 .045
Staphylococcus aureus 400 mg/ml 6.942 .023 6.896 6.987
200 mg/ml 2.500 .023 2.455 2.545
100 mg/ml 2.508 .023 2.463 2.554
50 mg/ml -1.910E-16 .023 -.045 .045
25 mg/ml -2.017E-16 .023 -.045 .045
12.5 mg/ml -3.184E-16 .023 -.045 .045
6.25 mg/ml -8.340E-16 .023 -.045 .045
3.125 mg/ml 7.693E-16 .023 -.045 .045
Staphylococcus sciuri 400 mg/ml 7.792 .023 7.746 7.837
200 mg/ml 1.858 .023 1.813 1.904
100 mg/ml 9.828E-16 .023 -.045 .045
50 mg/ml 6.636E-16 .023 -.045 .045
25 mg/ml 5.711E-16 .023 -.045 .045
12.5 mg/ml 3.349E-16 .023 -.045 .045
6.25 mg/ml 7.713E-17 .023 -.045 .045
3.125 mg/ml 4.728E-16 .023 -.045 .045
Enterobacter spp 400 mg/ml 2.508 .023 2.463 2.554
200 mg/ml -1.593E-15 .023 -.045 .045
100 mg/ml -2.192E-15 .023 -.045 .045
50 mg/ml 8.256E-17 .023 -.045 .045
25 mg/ml 1.535E-16 .023 -.045 .045
12.5 mg/ml 7.280E-16 .023 -.045 .045
6.25 mg/ml 1.740E-15 .023 -.045 .045
3.125 mg/ml 1.167E-14 .023 -.045 .045
5. Organisms * Groups
80
Dependent Variable:Clearance
Organisms Groups Mean Std. Error
95% Confidence Interval
Lower Bound Upper Bound
E.coli Hot water Piper guineense 4.058 .016 4.026 4.090
Hot water Xylopia aethiopica 4.279 .016 4.247 4.311
Cold water Allium cepa 4.004 .016 3.972 4.036
Cold water Xylopia aethiopica 3.650 .016 3.618 3.682
Salmonella spp Hot water Piper guineense 1.021 .016 .989 1.053
Hot water Xylopia aethiopica 2.217 .016 2.185 2.249
Cold water Allium cepa 1.500 .016 1.468 1.532
Cold water Xylopia aethiopica -8.341E-16 .016 -.032 .032
Proteus Hot water Piper guineense 5.464E-16 .016 -.032 .032
Hot water Xylopia aethiopica 5.763E-16 .016 -.032 .032
Cold water Allium cepa 1.512 .016 1.481 1.544
Cold water Xylopia aethiopica -3.664E-16 .016 -.032 .032
Bacillus spp Hot water Piper guineense 2.933 .016 2.901 2.965
Hot water Xylopia aethiopica 2.050 .016 2.018 2.082
Cold water Allium cepa -9.836E-16 .016 -.032 .032
Cold water Xylopia aethiopica 5.312E-16 .016 -.032 .032
Staphylococcus aureus Hot water Piper guineense 1.000 .016 .968 1.032
Hot water Xylopia aethiopica -1.527E-15 .016 -.032 .032
Cold water Allium cepa 3.996 .016 3.964 4.028
Cold water Xylopia aethiopica .979 .016 .947 1.011
Staphylococcus sciuri Hot water Piper guineense 1.133 .016 1.101 1.165
Hot water Xylopia aethiopica 2.196 .016 2.164 2.228
Cold water Allium cepa 1.496 .016 1.464 1.528
Cold water Xylopia aethiopica 2.269E-15 .016 -.032 .032
Enterobacter spp Hot water Piper guineense -1.739E-15 .016 -.032 .032
Hot water Xylopia aethiopica 5.388E-16 .016 -.032 .032
Cold water Allium cepa 1.254 .016 1.222 1.286
Cold water Xylopia aethiopica -1.014E-15 .016 -.032 .032
81
6. Organisms * Conc * Groups
Dependent Variable:Clearance
Organisms Conc Groups Mean Std. Error
95% Confidence Interval
Lower Bound Upper Bound
E.coli 400 mg/ml Hot water Piper guineense 14.733 .046 14.643 14.824
Hot water Xylopia aethiopica 13.100 .046 13.010 13.190
Cold water Allium cepa 12.000 .046 11.910 12.090
Cold water Xylopia aethiopica 11.967 .046 11.876 12.057
200 mg/ml Hot water Piper guineense 9.867 .046 9.776 9.957
Hot water Xylopia aethiopica 11.000 .046 10.910 11.090
Cold water Allium cepa 10.200 .046 10.110 10.290
Cold water Xylopia aethiopica 10.000 .046 9.910 10.090
100 mg/ml Hot water Piper guineense 7.867 .046 7.776 7.957
Hot water Xylopia aethiopica 10.133 .046 10.043 10.224
Cold water Allium cepa 9.833 .046 9.743 9.924
Cold water Xylopia aethiopica 7.233 .046 7.143 7.324
50 mg/ml Hot water Piper guineense 3.216E-16 .046 -.090 .090
Hot water Xylopia aethiopica -3.993E-18 .046 -.090 .090
Cold water Allium cepa -3.223E-16 .046 -.090 .090
Cold water Xylopia aethiopica 9.200E-16 .046 -.090 .090
25 mg/ml Hot water Piper guineense 2.741E-16 .046 -.090 .090
Hot water Xylopia aethiopica -1.849E-17 .046 -.090 .090
Cold water Allium cepa -3.469E-16 .046 -.090 .090
Cold water Xylopia aethiopica 1.007E-15 .046 -.090 .090
12.5 mg/ml Hot water Piper guineense 2.472E-16 .046 -.090 .090
Hot water Xylopia aethiopica 8.702E-17 .046 -.090 .090
Cold water Allium cepa -2.888E-16 .046 -.090 .090
Cold water Xylopia aethiopica 8.699E-16 .046 -.090 .090
6.25 mg/ml Hot water Piper guineense 3.533E-16 .046 -.090 .090
Hot water Xylopia aethiopica 1.489E-16 .046 -.090 .090
82
Cold water Allium cepa 2.441E-16 .046 -.090 .090
Cold water Xylopia aethiopica 8.099E-16 .046 -.090 .090
3.125 mg/ml Hot water Piper guineense -2.800E-15 .046 -.090 .090
Hot water Xylopia aethiopica -2.402E-15 .046 -.090 .090
Cold water Allium cepa 2.428E-15 .046 -.090 .090
Cold water Xylopia aethiopica -2.570E-14 .046 -.090 .090
Salmonella spp 400 mg/ml Hot water Piper guineense 8.167 .046 8.076 8.257
Hot water Xylopia aethiopica 9.867 .046 9.776 9.957
Cold water Allium cepa 12.000 .046 11.910 12.090
Cold water Xylopia aethiopica -6.327E-16 .046 -.090 .090
200 mg/ml Hot water Piper guineense -1.173E-15 .046 -.090 .090
Hot water Xylopia aethiopica 7.867 .046 7.776 7.957
Cold water Allium cepa -4.406E-16 .046 -.090 .090
Cold water Xylopia aethiopica 1.391E-15 .046 -.090 .090
100 mg/ml Hot water Piper guineense -9.605E-16 .046 -.090 .090
Hot water Xylopia aethiopica -2.758E-16 .046 -.090 .090
Cold water Allium cepa -3.445E-15 .046 -.090 .090
Cold water Xylopia aethiopica 2.870E-16 .046 -.090 .090
50 mg/ml Hot water Piper guineense -3.104E-16 .046 -.090 .090
Hot water Xylopia aethiopica -5.624E-16 .046 -.090 .090
Cold water Allium cepa -1.312E-15 .046 -.090 .090
Cold water Xylopia aethiopica 4.481E-16 .046 -.090 .090
25 mg/ml Hot water Piper guineense -3.375E-16 .046 -.090 .090
Hot water Xylopia aethiopica -7.641E-16 .046 -.090 .090
Cold water Allium cepa -1.310E-15 .046 -.090 .090
Cold water Xylopia aethiopica 6.755E-16 .046 -.090 .090
12.5 mg/ml Hot water Piper guineense -3.645E-16 .046 -.090 .090
Hot water Xylopia aethiopica -6.082E-16 .046 -.090 .090
Cold water Allium cepa -1.228E-15 .046 -.090 .090
Cold water Xylopia aethiopica 3.158E-16 .046 -.090 .090
6.25 mg/ml Hot water Piper guineense -3.018E-16 .046 -.090 .090
Hot water Xylopia aethiopica -7.466E-16 .046 -.090 .090
83
Cold water Allium cepa -7.626E-16 .046 -.090 .090
Cold water Xylopia aethiopica 1.966E-16 .046 -.090 .090
3.125 mg/ml Hot water Piper guineense -1.527E-15 .046 -.090 .090
Hot water Xylopia aethiopica 1.282E-15 .046 -.090 .090
Cold water Allium cepa -9.652E-16 .046 -.090 .090
Cold water Xylopia aethiopica -9.354E-15 .046 -.090 .090
Proteus 400 mg/ml Hot water Piper guineense -2.293E-15 .046 -.090 .090
Hot water Xylopia aethiopica 2.863E-15 .046 -.090 .090
Cold water Allium cepa 12.100 .046 12.010 12.190
Cold water Xylopia aethiopica 7.325E-16 .046 -.090 .090
200 mg/ml Hot water Piper guineense -3.449E-16 .046 -.090 .090
Hot water Xylopia aethiopica -2.144E-15 .046 -.090 .090
Cold water Allium cepa 1.008E-15 .046 -.090 .090
Cold water Xylopia aethiopica 9.451E-16 .046 -.090 .090
100 mg/ml Hot water Piper guineense 1.156E-15 .046 -.090 .090
Hot water Xylopia aethiopica 1.026E-15 .046 -.090 .090
Cold water Allium cepa -5.673E-16 .046 -.090 .090
Cold water Xylopia aethiopica 4.811E-16 .046 -.090 .090
50 mg/ml Hot water Piper guineense 1.209E-15 .046 -.090 .090
Hot water Xylopia aethiopica 3.422E-16 .046 -.090 .090
Cold water Allium cepa -2.964E-16 .046 -.090 .090
Cold water Xylopia aethiopica -4.538E-16 .046 -.090 .090
25 mg/ml Hot water Piper guineense 1.308E-15 .046 -.090 .090
Hot water Xylopia aethiopica 5.501E-16 .046 -.090 .090
Cold water Allium cepa -1.929E-16 .046 -.090 .090
Cold water Xylopia aethiopica -5.685E-16 .046 -.090 .090
12.5 mg/ml Hot water Piper guineense 9.732E-16 .046 -.090 .090
Hot water Xylopia aethiopica -8.851E-17 .046 -.090 .090
Cold water Allium cepa -4.995E-16 .046 -.090 .090
Cold water Xylopia aethiopica 2.115E-16 .046 -.090 .090
6.25 mg/ml Hot water Piper guineense 9.319E-16 .046 -.090 .090
Hot water Xylopia aethiopica 2.244E-16 .046 -.090 .090
84
Cold water Allium cepa -4.438E-16 .046 -.090 .090
Cold water Xylopia aethiopica -7.992E-16 .046 -.090 .090
3.125 mg/ml Hot water Piper guineense 1.431E-15 .046 -.090 .090
Hot water Xylopia aethiopica 1.837E-15 .046 -.090 .090
Cold water Allium cepa -2.381E-15 .046 -.090 .090
Cold water Xylopia aethiopica -3.480E-15 .046 -.090 .090
Bacillus spp 400 mg/ml Hot water Piper guineense 12.667 .046 12.576 12.757
Hot water Xylopia aethiopica 9.067 .046 8.976 9.157
Cold water Allium cepa 9.602E-16 .046 -.090 .090
Cold water Xylopia aethiopica -2.460E-15 .046 -.090 .090
200 mg/ml Hot water Piper guineense 10.800 .046 10.710 10.890
Hot water Xylopia aethiopica 7.333 .046 7.243 7.424
Cold water Allium cepa -2.499E-15 .046 -.090 .090
Cold water Xylopia aethiopica 4.775E-15 .046 -.090 .090
100 mg/ml Hot water Piper guineense 2.126E-16 .046 -.090 .090
Hot water Xylopia aethiopica 7.325E-16 .046 -.090 .090
Cold water Allium cepa -8.656E-16 .046 -.090 .090
Cold water Xylopia aethiopica 4.411E-17 .046 -.090 .090
50 mg/ml Hot water Piper guineense -4.817E-16 .046 -.090 .090
Hot water Xylopia aethiopica -2.419E-16 .046 -.090 .090
Cold water Allium cepa -1.805E-15 .046 -.090 .090
Cold water Xylopia aethiopica 2.384E-15 .046 -.090 .090
25 mg/ml Hot water Piper guineense -5.639E-16 .046 -.090 .090
Hot water Xylopia aethiopica -8.819E-16 .046 -.090 .090
Cold water Allium cepa -1.019E-15 .046 -.090 .090
Cold water Xylopia aethiopica 2.153E-15 .046 -.090 .090
12.5 mg/ml Hot water Piper guineense -8.943E-16 .046 -.090 .090
Hot water Xylopia aethiopica -7.140E-16 .046 -.090 .090
Cold water Allium cepa -5.069E-16 .046 -.090 .090
Cold water Xylopia aethiopica 1.566E-15 .046 -.090 .090
6.25 mg/ml Hot water Piper guineense -1.260E-15 .046 -.090 .090
Hot water Xylopia aethiopica -5.651E-16 .046 -.090 .090
85
Cold water Allium cepa -6.233E-16 .046 -.090 .090
Cold water Xylopia aethiopica 7.174E-16 .046 -.090 .090
3.125 mg/ml Hot water Piper guineense 6.253E-15 .046 -.090 .090
Hot water Xylopia aethiopica 3.691E-15 .046 -.090 .090
Cold water Allium cepa -1.510E-15 .046 -.090 .090
Cold water Xylopia aethiopica -4.930E-15 .046 -.090 .090
Staphylococcus aureus 400 mg/ml Hot water Piper guineense 8.000 .046 7.910 8.090
Hot water Xylopia aethiopica -4.116E-15 .046 -.090 .090
Cold water Allium cepa 11.933 .046 11.843 12.024
Cold water Xylopia aethiopica 7.833 .046 7.743 7.924
200 mg/ml Hot water Piper guineense -2.898E-15 .046 -.090 .090
Hot water Xylopia aethiopica -5.634E-15 .046 -.090 .090
Cold water Allium cepa 10.000 .046 9.910 10.090
Cold water Xylopia aethiopica 7.581E-15 .046 -.090 .090
100 mg/ml Hot water Piper guineense -1.747E-15 .046 -.090 .090
Hot water Xylopia aethiopica -2.127E-16 .046 -.090 .090
Cold water Allium cepa 10.033 .046 9.943 10.124
Cold water Xylopia aethiopica -4.653E-15 .046 -.090 .090
50 mg/ml Hot water Piper guineense 3.396E-16 .046 -.090 .090
Hot water Xylopia aethiopica -1.277E-15 .046 -.090 .090
Cold water Allium cepa -1.677E-15 .046 -.090 .090
Cold water Xylopia aethiopica 1.850E-15 .046 -.090 .090
25 mg/ml Hot water Piper guineense 1.266E-16 .046 -.090 .090
Hot water Xylopia aethiopica -1.857E-15 .046 -.090 .090
Cold water Allium cepa -2.006E-15 .046 -.090 .090
Cold water Xylopia aethiopica 2.929E-15 .046 -.090 .090
12.5 mg/ml Hot water Piper guineense -5.572E-17 .046 -.090 .090
Hot water Xylopia aethiopica -1.514E-15 .046 -.090 .090
Cold water Allium cepa -1.927E-15 .046 -.090 .090
Cold water Xylopia aethiopica 2.222E-15 .046 -.090 .090
6.25 mg/ml Hot water Piper guineense -5.170E-16 .046 -.090 .090
Hot water Xylopia aethiopica -5.751E-16 .046 -.090 .090
86
Cold water Allium cepa -1.484E-15 .046 -.090 .090
Cold water Xylopia aethiopica -7.596E-16 .046 -.090 .090
3.125 mg/ml Hot water Piper guineense 6.958E-15 .046 -.090 .090
Hot water Xylopia aethiopica 2.965E-15 .046 -.090 .090
Cold water Allium cepa -2.184E-15 .046 -.090 .090
Cold water Xylopia aethiopica -4.661E-15 .046 -.090 .090
Staphylococcus sciuri 400 mg/ml Hot water Piper guineense 9.067 .046 8.976 9.157
Hot water Xylopia aethiopica 10.133 .046 10.043 10.224
Cold water Allium cepa 11.967 .046 11.876 12.057
Cold water Xylopia aethiopica 1.173E-14 .046 -.090 .090
200 mg/ml Hot water Piper guineense -7.776E-16 .046 -.090 .090
Hot water Xylopia aethiopica 7.433 .046 7.343 7.524
Cold water Allium cepa 5.857E-16 .046 -.090 .090
Cold water Xylopia aethiopica 2.865E-15 .046 -.090 .090
100 mg/ml Hot water Piper guineense 1.760E-15 .046 -.090 .090
Hot water Xylopia aethiopica 4.886E-16 .046 -.090 .090
Cold water Allium cepa 2.830E-16 .046 -.090 .090
Cold water Xylopia aethiopica 1.400E-15 .046 -.090 .090
50 mg/ml Hot water Piper guineense 1.048E-15 .046 -.090 .090
Hot water Xylopia aethiopica 2.052E-16 .046 -.090 .090
Cold water Allium cepa -3.187E-16 .046 -.090 .090
Cold water Xylopia aethiopica 1.720E-15 .046 -.090 .090
25 mg/ml Hot water Piper guineense 1.012E-15 .046 -.090 .090
Hot water Xylopia aethiopica -3.425E-16 .046 -.090 .090
Cold water Allium cepa 3.365E-16 .046 -.090 .090
Cold water Xylopia aethiopica 1.278E-15 .046 -.090 .090
12.5 mg/ml Hot water Piper guineense 3.469E-16 .046 -.090 .090
Hot water Xylopia aethiopica -8.722E-16 .046 -.090 .090
Cold water Allium cepa 2.715E-17 .046 -.090 .090
Cold water Xylopia aethiopica 1.838E-15 .046 -.090 .090
6.25 mg/ml Hot water Piper guineense 2.732E-16 .046 -.090 .090
Hot water Xylopia aethiopica -4.229E-16 .046 -.090 .090
87
Cold water Allium cepa -1.082E-15 .046 -.090 .090
Cold water Xylopia aethiopica 1.540E-15 .046 -.090 .090
3.125 mg/ml Hot water Piper guineense 3.792E-15 .046 -.090 .090
Hot water Xylopia aethiopica 1.629E-15 .046 -.090 .090
Cold water Allium cepa 6.901E-16 .046 -.090 .090
Cold water Xylopia aethiopica -4.220E-15 .046 -.090 .090
Enterobacter spp 400 mg/ml Hot water Piper guineense -1.988E-14 .046 -.090 .090
Hot water Xylopia aethiopica 1.382E-15 .046 -.090 .090
Cold water Allium cepa 10.033 .046 9.943 10.124
Cold water Xylopia aethiopica -3.654E-14 .046 -.090 .090
200 mg/ml Hot water Piper guineense 7.384E-15 .046 -.090 .090
Hot water Xylopia aethiopica -9.027E-16 .046 -.090 .090
Cold water Allium cepa 7.567E-15 .046 -.090 .090
Cold water Xylopia aethiopica -2.042E-14 .046 -.090 .090
100 mg/ml Hot water Piper guineense 6.567E-16 .046 -.090 .090
Hot water Xylopia aethiopica 2.798E-15 .046 -.090 .090
Cold water Allium cepa -1.093E-14 .046 -.090 .090
Cold water Xylopia aethiopica -1.289E-15 .046 -.090 .090
50 mg/ml Hot water Piper guineense 1.300E-15 .046 -.090 .090
Hot water Xylopia aethiopica 3.332E-15 .046 -.090 .090
Cold water Allium cepa 4.379E-15 .046 -.090 .090
Cold water Xylopia aethiopica -8.680E-15 .046 -.090 .090
25 mg/ml Hot water Piper guineense 1.606E-15 .046 -.090 .090
Hot water Xylopia aethiopica 5.108E-15 .046 -.090 .090
Cold water Allium cepa 3.187E-15 .046 -.090 .090
Cold water Xylopia aethiopica -9.286E-15 .046 -.090 .090
12.5 mg/ml Hot water Piper guineense 3.174E-15 .046 -.090 .090
Hot water Xylopia aethiopica 5.504E-15 .046 -.090 .090
Cold water Allium cepa 3.070E-15 .046 -.090 .090
Cold water Xylopia aethiopica -8.835E-15 .046 -.090 .090
6.25 mg/ml Hot water Piper guineense 3.744E-15 .046 -.090 .090
Hot water Xylopia aethiopica 3.497E-15 .046 -.090 .090
88
Cold water Allium cepa 1.202E-15 .046 -.090 .090
Cold water Xylopia aethiopica -1.484E-15 .046 -.090 .090
3.125 mg/ml Hot water Piper guineense -1.190E-14 .046 -.090 .090
Hot water Xylopia aethiopica -1.641E-14 .046 -.090 .090
Cold water Allium cepa -3.427E-15 .046 -.090 .090
Cold water Xylopia aethiopica 7.843E-14 .046 -.090 .090
Descriptive Statistics
Dependent Variable:Clearance
Organisms Conc Mean Std. Deviation N
E.coli 400 mg/ml 14.733 .2517 3
200 mg/ml 9.867 .2309 3
100 mg/ml 7.867 .1155 3
50 mg/ml .000 .0000 3
25 mg/ml .000 .0000 3
12.5 mg/ml .000 .0000 3
6.25 mg/ml .000 .0000 3
3.125 mg/ml .000 .0000 3
Total 4.058 5.6488 24
Salmonella spp 400 mg/ml 8.167 .2887 3
200 mg/ml .000 .0000 3
100 mg/ml .000 .0000 3
50 mg/ml .000 .0000 3
25 mg/ml .000 .0000 3
12.5 mg/ml .000 .0000 3
6.25 mg/ml .000 .0000 3
3.125 mg/ml .000 .0000 3
Total 1.021 2.7603 24
Proteus 400 mg/ml .000 .0000 3
200 mg/ml .000 .0000 3
89
100 mg/ml .000 .0000 3
50 mg/ml .000 .0000 3
25 mg/ml .000 .0000 3
12.5 mg/ml .000 .0000 3
6.25 mg/ml .000 .0000 3
3.125 mg/ml .000 .0000 3
Total .000 .0000 24
Bacillus spp 400 mg/ml 12.667 .5774 3
200 mg/ml 10.800 .3464 3
100 mg/ml .000 .0000 3
50 mg/ml .000 .0000 3
25 mg/ml .000 .0000 3
12.5 mg/ml .000 .0000 3
6.25 mg/ml .000 .0000 3
3.125 mg/ml .000 .0000 3
Total 2.933 5.2156 24
Staphylococcus aureus 400 mg/ml 8.000 .0000 3
200 mg/ml .000 .0000 3
100 mg/ml .000 .0000 3
50 mg/ml .000 .0000 3
25 mg/ml .000 .0000 3
12.5 mg/ml .000 .0000 3
6.25 mg/ml .000 .0000 3
3.125 mg/ml .000 .0000 3
Total 1.000 2.7027 24
Staphylococcus sciuri 400 mg/ml 9.067 .1155 3
200 mg/ml .000 .0000 3
100 mg/ml .000 .0000 3
50 mg/ml .000 .0000 3
25 mg/ml .000 .0000 3
12.5 mg/ml .000 .0000 3
6.25 mg/ml .000 .0000 3
90
3.125 mg/ml .000 .0000 3
Total 1.133 3.0632 24
Enterobacter spp 400 mg/ml .000 .0000 3
200 mg/ml .000 .0000 3
100 mg/ml .000 .0000 3
50 mg/ml .000 .0000 3
25 mg/ml .000 .0000 3
12.5 mg/ml .000 .0000 3
6.25 mg/ml .000 .0000 3
3.125 mg/ml .000 .0000 3
Total .000 .0000 24
Total 400 mg/ml 7.519 5.4082 21
200 mg/ml 2.952 4.7920 21
100 mg/ml 1.124 2.8210 21
50 mg/ml .000 .0000 21
25 mg/ml .000 .0000 21
12.5 mg/ml .000 .0000 21
6.25 mg/ml .000 .0000 21
3.125 mg/ml .000 .0000 21
Total 1.449 3.6684 168
Dependent Variable:Clearance
Source
Type III Sum of
Squares df Mean Square F Sig.
Corrected Model 2245.940a 55 40.835 3.363E3 .000
91
Intercept 352.930 1 352.930 2.906E4 .000
Organisms 328.695 6 54.783 4.512E3 .000
Conc 1043.897 7 149.128 1.228E4 .000
Organisms * Conc 873.348 42 20.794 1.712E3 .000
Error 1.360 112 .012
Total 2600.230 168
Corrected Total 2247.300 167
a. R Squared = .999 (Adjusted R Squared = .999)
Estimated Marginal Means
1. Organisms
Dependent Variable:Clearance
Organisms Mean Std. Error
95% Confidence Interval
Lower Bound Upper Bound
E.coli 4.058 .022 4.014 4.103
Salmonella spp 1.021 .022 .976 1.065
Proteus 1.654E-16 .022 -.045 .045
Bacillus spp 2.933 .022 2.889 2.978
Staphylococcus aureus 1.000 .022 .955 1.045
Staphylococcus sciuri 1.133 .022 1.089 1.178
Enterobacter spp -4.777E-16 .022 -.045 .045
2. Conc
Dependent Variable:Clearance
Conc Mean Std. Error
95% Confidence Interval
Lower Bound Upper Bound
400 mg/ml 7.519 .024 7.471 7.567
200 mg/ml 2.952 .024 2.905 3.000
100 mg/ml 1.124 .024 1.076 1.171
50 mg/ml -9.640E-17 .024 -.048 .048
92
25 mg/ml -9.640E-17 .024 -.048 .048
12.5 mg/ml -9.640E-17 .024 -.048 .048
6.25 mg/ml -9.640E-17 .024 -.048 .048
3.125 mg/ml 6.498E-16 .024 -.048 .048
3. Organisms * Conc
Dependent Variable:Clearance
Organisms Conc Mean Std. Error
95% Confidence Interval
Lower Bound Upper Bound
E.coli 400 mg/ml 14.733 .064 14.607 14.859
200 mg/ml 9.867 .064 9.741 9.993
100 mg/ml 7.867 .064 7.741 7.993
50 mg/ml 1.082E-15 .064 -.126 .126
25 mg/ml 1.082E-15 .064 -.126 .126
12.5 mg/ml 1.378E-15 .064 -.126 .126
6.25 mg/ml 1.516E-15 .064 -.126 .126
3.125 mg/ml -1.688E-15 .064 -.126 .126
Salmonella spp 400 mg/ml 8.167 .064 8.041 8.293
200 mg/ml -1.168E-15 .064 -.126 .126
100 mg/ml -1.124E-16 .064 -.126 .126
50 mg/ml -5.481E-16 .064 -.126 .126
25 mg/ml -4.741E-16 .064 -.126 .126
12.5 mg/ml -5.605E-16 .064 -.126 .126
6.25 mg/ml -4.458E-16 .064 -.126 .126
3.125 mg/ml 1.367E-16 .064 -.126 .126
Proteus 400 mg/ml 1.808E-15 .064 -.126 .126
200 mg/ml -2.610E-16 .064 -.126 .126
100 mg/ml 1.806E-16 .064 -.126 .126
50 mg/ml 1.148E-16 .064 -.126 .126
25 mg/ml 2.481E-16 .064 -.126 .126
12.5 mg/ml 3.646E-16 .064 -.126 .126
93
6.25 mg/ml 1.804E-16 .064 -.126 .126
3.125 mg/ml -1.312E-15 .064 -.126 .126
Bacillus spp 400 mg/ml 12.667 .064 12.541 12.793
200 mg/ml 10.800 .064 10.674 10.926
100 mg/ml -1.284E-15 .064 -.126 .126
50 mg/ml -9.801E-16 .064 -.126 .126
25 mg/ml -1.032E-15 .064 -.126 .126
12.5 mg/ml -3.042E-16 .064 -.126 .126
6.25 mg/ml -9.927E-16 .064 -.126 .126
3.125 mg/ml 4.614E-16 .064 -.126 .126
Staphylococcus aureus 400 mg/ml 8.000 .064 7.874 8.126
200 mg/ml -7.353E-16 .064 -.126 .126
100 mg/ml -2.870E-16 .064 -.126 .126
50 mg/ml -5.007E-16 .064 -.126 .126
25 mg/ml -4.415E-16 .064 -.126 .126
12.5 mg/ml -9.236E-16 .064 -.126 .126
6.25 mg/ml -1.254E-15 .064 -.126 .126
3.125 mg/ml 9.624E-16 .064 -.126 .126
Staphylococcus sciuri 400 mg/ml 9.067 .064 8.941 9.193
200 mg/ml -1.022E-15 .064 -.126 .126
100 mg/ml -2.222E-16 .064 -.126 .126
50 mg/ml -4.730E-16 .064 -.126 .126
25 mg/ml -4.322E-16 .064 -.126 .126
12.5 mg/ml -7.885E-16 .064 -.126 .126
6.25 mg/ml -1.233E-15 .064 -.126 .126
3.125 mg/ml 2.935E-16 .064 -.126 .126
Enterobacter spp 400 mg/ml -7.867E-15 .064 -.126 .126
200 mg/ml -3.620E-15 .064 -.126 .126
100 mg/ml -7.477E-16 .064 -.126 .126
50 mg/ml 6.299E-16 .064 -.126 .126
25 mg/ml 3.745E-16 .064 -.126 .126
12.5 mg/ml 1.589E-16 .064 -.126 .126