1. health and fitness
2. disease

Abstract
This study was conducted for the purpose of extracting Trigonella
foenum-graecium plant and Acacia nilotica plant by methanol and
water at the following concentrations (30%, 20%, and 10%) of infectious
bacteria and pathogens E. coli and salmonella bacteria.
So was the impact of the Acacia nilotica plant on the bacteria
methanol extraction Trigonella foenum-graecium higher than the ring
on the bacteria and gave the following results: the E. coli bacteria
between 12-15 As a result of the Acacia nilotica plant extraction with
water on Salmonella between 15-8 and when extraction plant with
water on loan bacteria E. coli was the result of a weak effect. And also
the result was weak when Trigonella foenum-graecium extract with
water and methanol to bacteria E. coli and salmonella bacteria.
‫ملــخــص البـــحــــــث‬
‫أجريت هذه الدراسة بغرض إستخالص نبات الحلبة ونبات القرض بواسطة الميثانول والماء‬
‫عند التراكيز اآلتية (‪ )10%,20%,30 %‬على البكتيريا المعدية والممرضة االشيرشيا كوالي‬
‫وبكتيريا السالمونيال‪.‬‬
‫بحيث كان تأثير نبات القرض على البكتريا بإستخالص الميثانول اعلى من تأثيرنبات الحلبة‬
‫على البكتريا وأعطى النتائج اآلتية‪ :‬على بكتيريا االشيرشيا كوالي مابين‬
‫‪ 12 -15‬أما نتيجة‬
‫إستخالص نبات القرض بالماء على بكتريا السالمونيال مابين ‪ 8 –15‬وعند إستخالص نبات‬
‫القرض بالماء على بكتيريا االشيرشيا كوالي كانت النتيجة ذات تأثير ضعيف‪.‬‬
‫وايضا كانت النتيجة ضعيفة عند إستخالص نبات الحلبة بواسطة الماء والميثانول على بكتيريا‬
‫االشيرشيا كوالي وبكتيريا السالمونيال‪.‬‬
CHAPTER ONE
INTRODUCTION
1.1. Introduction
Sudan is endowed with a wealth of medicinal plants. These plants are so
important in the traditional healers’ practice. In spite of the rich heritage of
knowledge on the use of plants as drugs, little attention had been paid to grow
them as field crops in the country until the latter part of the nineteenth
century. During the past eight decades, there has been a rapid extension of
the all opathic system of medical treatment in Sudan. It generated a
commercial demand for pharmacopoeia drugs and products in the country
(Elghazali et al., 1994)
In the last two decades, the pharmaceutical industry has made massive
investments in pharmacological ,clinical and chemical research all over the
world in an effort to discover more effective drugs from plants (Tabuti et al.,
2003).
Sudan is a large tropical Sub-Saharan country that has been considered as
a treasure house of valuable aromatic and medicinal plants species due to its
characteristic geographical position
This unique position of Sudan is reflected in its diverse habitats ranging
from desert and semi-desert in the north, Acacia-wooded grassland in the
Sahel zone of the central part of the country (El Tohami et al.,1997).
In addition, there are areas with a high-naturalized value, rich in official
botanical species, which grow wild and are potential candidates for crop
development and commercialization (Elamin et al., 1990).
1.2. Justification
Sudan’s economy is highly dependent on agriculture, which contributes
about 43.6 of the Gross Domestic Product,
In recent years, numerous food
poisoning outbreaks, involving various pathogens in food products, have
spurred growing awareness of the importance of food safety. Intensive use of
antibiotics is often followed by the development of resistant strains, because
this drug resistance, the search for new antibiotics continues unabated. The
interest in the study of medicinal plants as source of pharmacologically active
compounds has increased worldwide. In Sudan, plants are the main medicinal
source to treat infectious diseases.
1.3.Objective
The objective of study we investigate in vitro antibacterial activity of
different concentrations of the methanolic extract and aqueous extract from
tthe pods of Acacia nilotica and Trigonella foenum-graecium against two
standard bacterial strains (Escherichia coli and Salmonella), which are common
cause of wound infections.
1.4. Specific Objectives
1- To evaluate the antimicrobial effect of Acacia nilotica and Trigonella
foenum-graecium extract against Escherichia coli and Salmonella bacterial
that causes the infections.
2-To compare the effect of Acacia nilotica and Trigonella foenumgraecium extract against Escherichia coli and Salmonella bacterial that
causes the infections that causes the infections.
3. To investigate the antibacterial activity of different doses extracts from
Acacia nilotica and Trigonella foenum-graecium against Escherichia coli
and Salmonella bacterial that causes the infections.
CAPTER TWO
2. REVIEW OF LITERATURE
2.1. Medicinal Plants
Herbal materials continue to be used as the primary source of medicines
(Chitme et al., 2003). About 80% of the people in developing countries use
traditional medicines for their primary health care (Kim, 2005). Antibiotics have
undesirable side effects while the emergence of previously uncommon
infections is also a serious medical problem (Marchese and Shito, 2001). Over
75 % of the antibacterial in clinical use are of natural origin and most of them
are obtained from fungal sources (Newman et al., 2003).
Plants have not only provided mankind with food, clothing, flavors,
cosmetic, ornamental, fumigants, insect deterrents and fragrance, but have
also served humanity in the treatment of ailments. According to World Health
Organization (WHO) report in 1993, about three-quarters of the world’s
population which live in developing countries rely on plants for the treatment
of many illnesses (Akerele, 1993).
Plants are always surrounded by an enormous number of potential
enemies such as bacteria, viruses, fungi, wild fires and sometimes flood. Plants
protect themselves through a chemical defence system (Van Wyk and Gericke,
2000). Therefore, it is expected that biological active compounds are produced
by plants as chemical defense measures against their enemies. The search for
biologically active agents is part of a wider renaissance of scientific significance
to bring into being new chemotherapeutics (Moundipa et al., 2001). Plants
synthesize very complex molecules with specific stereochemistry and can show
biological activity with new modes of action (Houghton, 1996). Several useful
drugs have been developed from medicinal plants used in traditional medicine
in the treatment of a variety of illnesses.
According to Gilani and Atta-ur- Rahaman (2005), the use of plant extracts
or plant-derived pure chemicals to treat diseases is a therapeutic modality
which has stood the test of time. Many studies indicated that medicinal plants
contain substances like peptide, unsaturated long chain fatty acids, aldehydes,
alkaloids, essential oils, phenols and water or ethanol soluble compounds.
Most of the clinical drugs that are currently in use were derived from plants
and developed because of their use in the traditional medicine. The plants that
possess therapeutic properties or exert beneficial pharmacological effects on
the animal body are generally designated as (Medicinal Plants).
2.1.1. Fenugreek (Trigonella foenum-graecium)
2.1.1.1. Taxonomy of Trigonella foenum-graecium
Trees
Seeds
Fig (1) Trigonella foenum-graecium (Helba)
Latin Name: Trigonella foenum-graecium. Fenugreek Family: Fabaceae
2.1.1.2. Part used: Seeds
Trigonella foenum -graecium Plant is a native of Southern Europe and Asia
and has been grown in India and parts of North Africa for centuries and used as
a flavoring material. Considerable quantities are exported from India and
Morocco but the plant is mainly used locally for many purposes (Leung et al.,
1996).
2.1.1.3. Market Potential
Trigonella foenum -graecium is an important cash crop in India and
Pakistan as well as in Iran, Egypt and Sudan. The seeds are potential antihyper
glycimic, antihyperlipidimic, antimicrobial and more effective antiulcer than
omeprazole (Harbi et al., 2007).
2.1.1.4. Uses of Trigonella foenum-graecium
Decoction of whole plant is used as seat bath for uterus affection. Seeds
are tonic, aphrodisiac, galactogogue, laxative, used for gastro-intestinal
troubles and borncho-pulmonary infections, analeptic, stimulant emollients
febrifuge stimulate childbirth, curative of tonsillitis and itch, cataplasm
obtained by boiling the flour of seeds with vinegar and saltpeter used for
swelling of spleen (Fourmen et al., 1941).
Seeds are also used internally to treat anorexia, dyspepsia and topical
for frunculosis, myalgia, lymphadenitis, gout wounds and leg ulcers. They are
also indicated for use externally as an emollient for treating furuncles, boils
and eczema (Harbi et al., 2007).
2.1.1.5. Botanical Description of Fenugreek seed
Trigonella foenum-graecium is a member of the Papilionaceae family.
The Latin name of the genus Trigonella means little triangle, referring to the
shape of the leaflet of this herb from its corolla, the species name foenum
graecium means (Greek hay) (Fourmen et al., 1941).
2.1.1.6. Geographical Distribution
Trigonella foenum-graecium Plant is wildly grown today in the
Mediterranean countries, Argentina, France, India,
North Africa, Austria,
Belgium, Chile, Egypt, Spain, Sudan, Turkey and U.S.A (Algeria et al., 1978).
2.1.1.7 Climatic Requirement
The reported zone life of Fenugreek seed is 8 to 27°C with an annual
Precipitation of 0.4 to 1.5 meters and soil pH of 5.3 to 8.2. The plant thrives in
full sun rich, well-drained soils, growth is slow and weak in cold temperature
and wet soil. Fenugreek seed is a fairly salt tolerant Plant. It grows well in
strong clay soil which is hard and grows fairly on deep loamy, well-drained soil,
also it grows fairly well on gravelly or sandy soil (although it prefers natural
soil) As a leguminous plant, fenugreek seed needs a little if any nitrogen
fertilizer, and the plant can enrich soil with nitrogen. There is a considerable
commercial interest in breeding and growing fenugreek cultivars. The seeds
are highly aromatic and contain about 23% protein, 9% oil, 10% carbohydrate
and a considerable quantity of resinous material.
There are many interesting active constituents in fenugreek, they include,
dioscin, diosgenin, gitogenin, tigogenins, yamogenin (all of which are steroidal
saponins), also it contain trigenllie, arginine, glutamic acid,gama-amino butyric
acids, glycine, histidine, methionine, phenyl alanine, serine, tryptophan,
tyrosine (all of which are amino acid). In addition to minerals such as
zinc,chromium(Zn,Cr) and niacine (vitamin) (Newall et al., 1996).
2.1.1.8. Chemical Composition of the seed
- Moisture: The moisture content of Indian fenugreek seeds is in the range of
5.77% to 0.30% determined a moisture content of 9% in fenugreek seed (Abdel
Aal et al., 1985).
Found that the Egyptian fenugreek seed powder contains about 5.05%
moisture while its flour contains about 3.4% moisture (El-Hassan et al., 1991).
- Lipids: Fenugreek seed contains about 7% oil which is used as galactogog. A
value of 8.4% crude oil in the seeds was also determined Fenugreek seed
contains small amount of volatile oil (0.2%) who also found that the seed
contains about 7% fixed oil( Sabryaid et al., 1959) .
The seed oil has marked drying properties, the dried oil being of golden
yellow color and insoluble in ether and has got disagreeable odor and bitter
taste Egyptian fenugreek seed
7.6%crude oil (Latham et al., 1965).
was reported to contain about 7% to
The oil had a strong celery odour, ether extract(fat%) range from 6.0 to
10.3% the fatty acid content include linoleic, oleic, linolenic and Palmitic
(Gupta et al., 1996), .
- Crude Protein (CP): In as early studies of fenugreek seed Protein by a value of
27% CP and also he separated the different protein fractions found in
fenugreek seeds as globulin (6.8%) albumin (5.4%) and nucleoproteins (14%)
(Wunschendroff et al., 1919).
However, indicated that the seeds contain 26.67%cp of which 25.2% as
true protein. The
content of raw and roasted fenugreek seed of 24 Indian
samples ranges from 27.68% to 38.56% (Mohammedain et al., 2000).
In Sudan that promising cultivars of fenugreek seed selected according to
their protein content (Berber4, Dongla 7 and Kassala 9) their protein was rich
in lysine, Lucien valine, and phenyl alanine but poor in the sulphur – containing
amino acids which are considered limiting ones (Abdel-Nabey et al., 1990).
- Carbohydrates (CHO): A value of 53.19% total(CHO) in addition to that
determined the total (CHO)content soluble in 70% methanol and reported a
value of 4.22% soluble(CHO) for ungerminated seeds, and a value of 14.26%
for germinated seeds (Latham et al., 1985). Reported a value of 60.0% total
(CHO) in fenugreek seed powder and 76.97% in fenugreek flour, where as
Reported a value of 40.6% (CHO).Amongst Sudanese cultivars; Kassala cultivar
contained the highest level of (CHO) fat, and minerals (Abdel Aal et al., 1986).
2.1.2. Acacia nilotica
2.1.2.1. Taxonomy of Acacia nilotica
Trees
Fruits
Plate (2) Acacia nilotica (Garad)
Latin
Name:
Acacia
nilotica.
Fenugreek
Family:
Mimosaceae
Synonyms: Mimosa nilotica or Acacia nilotica , Local name: Sonoot (tree) Garad
(fruit)
2.1.2.2. Distribution
Acacia nilotica is native to Africa including Egypt, Mozambique, Sudan,
Natal, Zambia and Botswana Ali et al., 1973).
The sub species tomentosa occurs in Senegal, Mali, Ivory Coast, Ghana, Niger,
Nigeria, the Sudan and Ethiopia (Monod et al., 1974).
2.1.2.3. Distribution in Sudan
It is widely distributed along the Nile bank and its tributaries, especially in
northern and central Sudan (El Atta et al., 1993).
2.1.2.4. Botanical descriptions
Acacia nilotica is a small tree about (2.5-14 meters) tall and it has
branches full of long sharp thorns. It can grow up to 20 meters but this
attenuated by site. It is usually no more than 10 meters high and in very
unfavorable location is only a shrub. It has a flattish or umbrella shaped crown
and it easily identified by its bright yellow sweet scented flower heads (El Amin
et al., 1973).
- Leaves: The Leaves are often with( 1-2 ) petiolar glands and other glands
between all only the upper most pinnate, (2-11) pairs, leaflets (7-52) pairs (0.51.5) mm wide, glabrous or pubescent apex obtuse.Peduncles clustered at
nodes of leafy and leafless branches (Duke et al., 1981) .
- Flowers: The Flowers are bright or golden yellow sweetly scented, numerous,
with bisexual and male flowers on the same globosely inflorescence. Flowering
is prolific, can occur a number of times in a season on current seasons growth,
but often only about 0.1% of flowers set pods (Brenan et al., 1959).
- Fruits: Pods are grey, thick, softly tomentose, and straightly curved, 10-15 cm
long on a pedicle, 0.5-1.2 cm long, with constrictions between the seeds, giving
a necklace appearance. Fleshy when young, becoming black and hard when
maturity (ALI et al., 1980).
- Seeds: Seeds are deep blackish-brown, smooth, sub circular, compressed
areole about (6-7) mm wide (Clemens et al., 1977).
2.1.2.4. Maturity age
Maturation takes 8 months (ideal) to 12 years (harsh condition). (5-7)
years are typical (Kriticos et al., 1999).
2.1.2.5. Habitat
In Africa, sub species nilotica and tomentosa are restricted to riverine
habitats and seasonally flooded areas on clay alluvial soils (Fagg et al., 1990).
2.1.2.6. Ecology
Acacia nilotica is a relatively fast growing, nitrogen-fixing tree on arid
sites. It is a moderate- sized tree that can withstand extreme environment in
semi-arid areas. Can survive very arid sites, thrives under irrigation and endes
flood. It is an important riverine tree in Egypt, India, Sudan and Senegal
(Kayanja et al., 1990).
- Temperature: Trees can withstand extremes of temperatures, but are frost
tender when Young. Average annual temperatures commonly from (15 to
28°C). Acacia nilotica prefers dry conditions, with an annual rainfall of from
250 to 1500 mm; although under irrigation some varieties will grow in areas
with less than 100mm (Duke et al., 1983).
- PH range: pH range from 2.5 to 8.0 (Duke et al., 1983).
2.1.2.7. Uses of Acacia nilotica
- Firewood-Charcoal: Excellent firewood and charcoal, Used to fuel
Locomotives, river steamers and boilers in some small industries (Firewood
Crops, 1980).
- Timber-wood: Hard and tough wood, resistant to termites, tool handles,
carts, oars, posts and buildings.Attractive wood good for carving, turnery and
boat building (Van Wyk et al., 2000).
- Pulp and paper: Wood is good for paper and pulp making (Dwivedi et al.,
1993).
- Forage-Fodder: Leaves tender pods and shoots used as forage for goats,
sheep and camels. Seeds are also a valuable cattle food .Pods contain 12- 15%
crude protein.Rich in minerals (Firewood Crops, 1980).
- Tannins: Bark has high levels of tannin (12-20%) for sub species indicia. Used
for tanning and dyeing leathers. Pods of sub species nilotica used for tanning
(Fagg et al., 1990).
2.1.2.8. Chemical constituents
Acacia nilotica has reported to contain galantine, galactose, Nacetyldjenkolicacid, sulphoxides pentosan, saponin and tannin (Pande et al.,
1981).
Seeds contain crude protein, fiber, nitrogen- free extract, ash and silica,
epoxy and hydroxyl fatty acid.The bark and sap contain quartering and
saponins (Watt et al., 1962).
Various flavonoids and Phenolic compounds have been isolated from the
flowers whereas tannins, gallic acid and m-digallic acids were isolated from the
pods, epoxy and hydroxyl fatty acid from the bark and dip respectively.
Catechins-s-galloyl esters were isolated from the bark (Khalid et al., 1989).
2.1.2.9. Uses of Acacia nilotica in folk medicinal
The Zulu of South Africa take the bark for cough. An astringent from the
bark used for diarrhea, dysentery and leprosy. Bruised leaves poultice were
used for treatment of ulcers. According to Hart well, the gum or bark were
used for cancer and tumors of ear, eye, or testicles and indurations of liver and
spleen, Candylomas and excess flesh In Lebanon the resin is mixed with an
orange-flower, use as infusion for typhoid convalesces. Egyptions Nubians
believe than diabetics may eat unlimited carbohydrates without any
consequences as long as they consume the pods in powder form (Duke et al.,
1983).
Acacia nilotica is traditionally used to treat sore throat, cold, bronchitis,
pneumonia, diarrhea, dysentery, leprosy, venereal disease and hemorrhage
because of it is tonic, astringent and stimulant properties (Duke et al., 1983).
Aqueous extract of fruits showed activity against candida albicans, gram
positive and gram negative bacteria it was found that an extract from the fruits
of Acacia nilotica inhibited the growth of many bacteria. This activity was not
related to the low pH of the extract. Different bacteria showed different
degrees of sensitivity to the extract (Suhair et al., 1999).
2.1.2.10. Anti-microbial activity for Acacia nilotica
Aqueous extracts of fruits (collected from central Sudan) showed activity
against Candida albicans and gram positive and gram negative bacteria
(Hussein et al., 1999).
- Antimicrobial Agents: Any chemical Substance inhibiting the growth or
causing the death of a microorganism is known as antimicrobial agent
(Kenaway et al., 2007).
Antimicrobial agent are generally described as bacteriostatic when, at
usual dosages, they prevent the active multiplication of bacterial, for example
,chloramphenicol,tetracycline, and erythromycin and described as bactericidal
when, at usual dosages, they kill bacterial, for example, the penicillin
cephalosporins,polymyxin,
and
amino
glycosides.
Some
bacteriostatic
antimicrobial become bactericidal when used at higher concentration, for
example, erythromycin (Ward et al., 2006).
Not all antimicrobials at the concentration required to be effective are
completely non-toxic to human cells, most, however, show sufficient selective
toxicity to be of value in the treatment to microbial diseases (Maggi et al.,
1965) .
Antimicrobial drugs are effective in the treatment of infection because of
their selective toxicity. In most instances, the selective toxicity is relative,
rather than absolute, requiring that the concentration of the drug be carefully
controlled to attack the microorganism while still being tolerated by the host
(Harvey et al., 2001).
- Antibiotics: Antibiotics are antimicrobial substances produced by living
microorganisms. They include the culture extracts and filtrates of fungi such as
Penicillium and Cephalosporium, and bacteria such as Streptomyces and
Bacillus species. Many antibiotics in common use are synthetic derivatives of
microbial (Strobel et al., 2003).
- Antibiotic Sensitivity: Antibiotic Sensitivity is unpredictable, because
enterobacteria readily acquire resistance-coding plasmids, which can spread to
other strain (Heddle et al., 2002).
The main antibiotics used against enterobacteria are:
Ampicillin/amoxicillin - Amino glycoside – Trimethoprim Chloramphenicol Ciprofloxacin
- Resistance of bacteria to antibiotics: The bacteria overcome the action of
antimicrobial agents through the following by:
1- Genetic mutation which changes the proteins and other components of
bacterial cell that antimicrobials use as binding sites. Changes of genetic origin
are associated with resistance to amino glycosides, lincomycin, and
erythromycin (Barnard et al., 2000).
2- Producing enzymes that destroy or inactivate antimicrobials, examples of
enzyme beta-lactamase which destroies .The beta-lactam rings that forms part
of the structure of Penicillin and cephalosporin (Cheesbrough et al., 1984).
3- changing to other metabolic pathway not affected by the antimicrobial
being used. This mechanism of resistance is found in some sulphonamideresistant bacteria (Anderson et al., 2005).
4- Altering the permeability of their cell membrane, making it difficult for
antimicrobials to enter. This type of resistance is found in bacteria resistant to
poly myxins and tetracyclines (Cheesbrough et al., 1984).
2.2. Anti-microbial activity
2.2.1 Antimicrobial Sensitivity Test
In the treatment and control of infectious diseases specially when caused
by pathogens that are often drug resistant, sensitivity test must be used to
select effective antimicrobial drugs (Jelager et al., 1998).
Sensitivity test must never be performed on commensally organisms or
contaminants because this would mislead the clinician and could result in the
patient receiving ineffective and unnecessary antimicrobial therapy, causing
possible side effects and resistance to other potentially pathogenic organisms
(Barry et al., 1989).
1.3. Food Spoilage and Pathogenic Bacteria
Although it is extremely difficult to pinpoint the precise beginnings of
human awareness of the presence and role of microorganisms in foods, the
available evidence indicates that this knowledge preceded the establishment
of bacteriology and microbiology as a science. The era prior to the
establishment of bacteriology as a science may be designated the pre scientific
era (James, 2000).
Food quality and safety is an increasingly important public health issue.
Nowadays, the topics “food quality” and “food safety” are very close and two
important issues in the food sector, due to the globalization of the food supply
and the increased complexity of the food chain. The consumers need to
purchase safe products that do not involve any kind of risk for health (FAO,
2003).
The contamination of food products with microorganisms presents a
problem of global concern, since the growth and metabolism of
microorganisms can cause serious foodborne intoxications and a rapid spoilage
of the food products. Thus, the acceptance and safety of a food product for the
consumers depends in great part on the presence and nature of
microorganisms. Besides molds and yeasts, bacteria are the principle
responsible for various types of food spoilage and foodborne intoxications
(Blackburn, 2006).
1.3.1. Spoilage bacteria
Spoilage bacteria are microorganisms that cause the deterioration of food
and develop odors, tastes, and textures. A spoiled food has lost the original
nutritional value, Spoilage bacteria normally do not cause illness; however,
when consumed in high concentration, they can cause gastrointestinal
disturbance (Blackburn, 2006). In general, bacteria can spoil different foods
depending on the physical-chemical preservation profile (Gram et al., 2002).
1.3.2. Pathogenic bacteria
Foodborne diseases are caused by agents that enter the body through the
ingestion of food. Food can transmit disease from person to person, as well as
serve as a growth medium for bacteria that can cause food poisoning.
A great proportion of these cases can be attributed to the consumption of
contaminated food and water. The percentage of the population suffering
from foodborne diseases each year has been reported to be up to 30% (WHO,
2007).
Pathogenic bacteria often do not change the color, odor, taste or texture
of a food product, being hard to recognize if the product is contaminated.
Food-borne infection is caused by bacteria in food. If bacteria become
numerous and the food is eaten, bacteria may continue to grow in intestines
and cause illness. Food intoxication results from consumption of toxins (or
poisons) produced in food as a by-product of bacterial growth and
multiplication in food. In this case the toxins and not bacteria cause illness.
Toxins may not alter the appearance, odor or flavor of food. Common bacteria
that produce toxins include Staphylococcus aureus and Clostridium botulinum.
In some cases, such as Clostridium perfringens, illness is caused by toxins
released in the gut, when large numbers of vegetative cells are eaten. Bacterial
food poisoning is commonly caused by bacterial pathogenic species such as
Escherichia coli, Salmonella spp., Listeria monocytogenes, S. aureus, Bacillus
cereus, C. perfringens, Campylobacter spp., Shigella spp., Streptococcus spp.,
Vibrio cholerae, including O1 and non-O1, Vibrio parahaemolyticus, Vibrio
vulnificus, Yersinia enterocolitica and Yersinia pseudotuberculosis. Emerging
foodborne pathogens may refer to new pathogens, pathogens that emerge
due to changing ecology or changing technology that connects a potential
pathogen with the food chain or emerge de novo by transfer of mobile
virulence factors (Tauxe, 2002). Emerging foodborne pathogens include E. coli
O157:H7, Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria,
Mycobacterium
Helicobacter
spp.,
spp.,
vancomycin-resistant
Enterobacter
sakazakii,
enterococci,
non-jejuni/coli
non-gastric
species
of
Campylobacter, and non-O157 Shiga toxin-producing E. coli.
1.3.2.1. Escherichia coli
This bacterium is shortly written as E .coli ;was named after Escherichia
who was the first one to describe the colon bacillus Escherichia coli .It lives in
intestine of human and animal as part of the normal microbial flora (Evans et
al., 2007).
- Characteristics: E. coli is a member of the enterobacteriacea. It is a nonspore forming gram-negative bacillus. Many strains are capsulated .E .coli is
about 2 micrometres (μm) long and 0.5 μm in diameter with a cell volume
between 0.6 and 0.7 μm3. It is motile by peritrichous flagella ,though some
strains may be non-motile. Many strains have a combination of characters. This
is because of conjugation and transductions between bacterial strains
Pathogenic strains of E. coli are mainly responsible for three types of infections
in humans: urinary tract infections (UTI), neonatal meningitis, and intestinal
diseases (gastroenteritis( )Todar et al., 2007).
- Culture requirements: E .coli is a facultative anaerobe and it grows in a
temperature ranging from 10 - 46 °C with an optimum of 37 °C. It grows in
MacConkey agar forming large lactose fermenting colonies (Fotadar et al.,
2005).
1.3.2.2. Salmonella
This bacterium is a genus of rod-shaped ,Gram-negative bacteria there
are only two species of Salmonella, Salmonella bongori and Salmonella
enterica, of which there are around six subspecies and innumerable serovar.
The genus belongs to the same family as Escherichia ,which includes the
species E.coli (Ryan et al., 2004).
- Characteristics: Salmonella are non-spore-forming ,predominantly motile
enterobacteria with diameters around 0.7 to 1.5 µm, lengths from 2 to 5 µm,
and peritrichous flagella ( flagella that are all around the cell body). They are
chemoorganotrophs, obtaining their energy from oxidation and reduction
reactions using organic sources, and are facultative anaerobes (Fabrega et al.,
2013) .
- Culture requirement :Most subspecies of Salmonella produce hydrogen
sulfide which can readily be detected by growing them on media containing
ferrous sulfate ,such as is used in the triple sugar iron test (TSI) . Most isolates
exist in two phases: a motile phase I and a no motile phase II (Clark et al.,
1987).
CHAPTER FOUR
4. RESULTS & DISCUSSION
The pods of Acacia nilotica have been popularly used in traditional
medicine for a long time which cannot be backdated to a specific era. Its fruit
is used as a herbal medicine for treatment of different health problems .It is
used to relieve nasal congestion and sore throat in influenza. Moreover it is
used topically as treatment for human and animal wounds and a deodorant to
get rid of the offensive smell of the infected wounds.The long experience in
use of the plants may prove its safety (Satish, 2008( .The seeds of Trigonella
foenum-greacium that known in the Sudan as Helba and is commonly
cultivated since the ancient time (Chevallier, 2001).
The pods and seeds have been used in food and traditional medicine,
especially in India and the Nile Valley (Joy et al., 1998).
It was a medical plant used by the ancient Arabs as medicinal diet in the
treatment of infections of the stomach and intestines (Balch et al., 2000) .
The seeds of the helba increase the milk yield and urine and help in the
process of digestion. It is also used as a topical treatment in cases of abscesses
and wounds (Chevallier, 2001).
Traditionally the plant is used as crude extract and treatment does not
aim at using the pure isolate. It is not known which of its components has the
antibacterial activity. The crude concentrated extract in high concentration
may be harmful. This point necessitates further studies to find the safe
effective concentration and dose. This herb may be quite suitable as
antimicrobial in form of an antiseptic and a drug if used topically (Pak, 2012).
In this study, the methanolic extract of the pod of Acacia nilotica
showed an in vitro antibacterial activity in the three concentrations (10%, 20%
and 30%). The inhibition zones of E .coli and Salmonella by the disc diffusion
method ranged between 8 mm and 15 mm. a known bactericidal drug against
E .coli gave an inhibition zone of 15 mm. This result was comparable to report
most of the bacterial species showed a high degree of sensitivity to the
methanolic extracts of Acacia nilotica ssp nilotica pods (Z = range between 2137 mm( )Hatil et al., 2009) .
The methanolic extract of the seeds of Trigonella foenum showed an in
vitro antibacterial resistant in the three concentrations (10%, 20% and 30%).
The inhibition zones of E. coli by the disc diffusion technique ranged between
(0 mm and 8 mm) for the E. coli. This study was in accordance with the study,
which was done in the University of Kushtia, Bangladesh (It reported an
inhibition zone of 8mm and 7mm against E.coli (Life Sciences and Medicine
Research, 2011).
Aqucous extract of Acacia nilotica ssp nilotica pods was highly active
against all tested Gram-positive and Gram-negative bacteria (1z=range
betwen21-43mm) (Hatil et al., 2009).
Aqucous extract of Trigonella foenum-greacum the all bacterial species
were found to be resistant except Pseudomonas aeruginosa (Hatil et al.,
2009).
Hatil et al (2009) reported that the different extracts of Trigonella
foenum-greacum seeds did not show any activity against all tested Grampositive and Gram-negative bacteria.
The E.coli count presented in table (1) Fig (1) Plate (3 and 4) was highest in
concentration of Aqucous extract of Acacia nilotica.)36.00 ( %30 And the
lowest count obtained by Acacia nilotica extraction by methanol in 10%
concentration (12.20) .
Kavitha et al (2013) reported that the antimicrobial activity of crude
methanolic extracts of acacia nilotica ,was performed by the method
described earlier and then and then analyzed for phytocompounds present in
the antibacterial activity in terms of zone of inhibition .The methanolic extract
of Acacia nilotica showed highest zone of inhibition against a E.coli exhibits
moderate zone of inhibition 18 mm, 17 mm and 19 mm respectively .
The separated bioactive compounds of methanolic extract of acacia
nilotica the presence of Alkaloids ,Flavonoids, saponins, Tanins, Terpenoids,
Anthraquinone. The antibacterial activity of each bioactive compounds revels
that the Flavonoids exhibit a very good antibacterial activity showed the
highest 21 mm of zone against E.coli .The other bioactive compoundstannins,
terpinoids and anthraquinonehave not showed their existence.
Table1. Activity of methanol and aqueous extract of Acacia nilotica
(Pods) on E. coli
Con. (%)
10
20
30
Methanol
Aqueous
12.20e
26.00c
±1.04
±1.00
12.47e
30.00b
±0.58
±1.00
15.60d
36.00a
±0.53
±3.00
2.588*
Lsd0.05
a, b, c, d & e: Means having different superscripts differ significantly (P≤0.05).
40
zone (mm)
35
30
25
20
15
10
5
0
Con10% Con20% Con30% Con10% Con20% Con30%
Methanolic
Aqueous
Fig (1): Activity of methanol and aqueous extract of
Acacia nilotica (Pods)
on E. coli
Plate 3: Inhibition Zone of Acacia nilotica Methanol extracts
(10% 20% and 30%) E.coli.
Plate 4: Inhibition Zone of Acacia nilotica aqueous extracts
(10% 20% and 30%) E.coli.
The Salmonella typhi count presented in table (2) Fig (2) Plate (5 and 6)
was highest in concentration of Aqucous extract of Acacia nilotica 30 )15.00(.
And the lowest count obtained by Acacia nilotica extraction by methanol in
%10concentration (10.33).
Kavitha et al (2013) reported that the antimicrobial activity of crude
methanolic extracts of acacia nilotica ,was performed by the method
described earlier and then and then analyzed for phytocompounds present in
the antibacterial activity in terms of zone of inhibition .The methanolic extract
of Acacia nilotica showed highest zone of inhibition against a gram + vecocci,
S. Aries 20 mm and least 16 mm respectively .The separated bioactive
compounds of methanolic extract of acacia nilotica indicate the presence of
Alkaloids, Flavonoids, saponins, Tanins, Terpenoids, Anthraquinone .The
antibacterial activity of each bioactive compounds revels that the alkaloids
showed 06, 08, 05, 04 and 01 mm zone of inhibition against S.aureus. The
other bioactive compounds tannins, terpinoids and anthraquinonehave not
showed their existence.
The E.coli count presented in table (3) Fig (3) Plate (7 and 8) was
highest in concentration of Aqucous extract of Trigonella foenum graecium
.)14.67 ( %30And the lowest count obtained by Trigonella foenum
graecium extraction by methanol in 10% concentration (0. 0).
Hatil et al (2009) reported that the antibacterial properties of the
ethanol, petroleum ether, ethyl acetate, methanol and water extracts of some
Sudanese medicinal plants Trigonella foenum-greacum seeds) at concentration
100 mg/ml.The all bacterial species were found to be resistant against
Trigonella foenum-greacum seeds ,except Pseudomonas aeruginosa.
Table2. Activity of methanol and aqueous extract of Acacia nilotica (Pods) on
Salmonella typhi.
Con. (%)
Methanol
Aqueous
10.33b
8.67b
±0.58
±1.53
12.67ab
12.67ab
±1.15
±1.15
12.33ab
15.00a
±4.73
±1.00
10
20
30
3.889*
Lsd0.05
Methanol
Con30%
Con20%
Con10%
Con30%
Con20%
16
14
12
10
8
6
4
2
0
Con10%
zone (mm)
a &b: Means having different superscripts differ significantly (P≤0.05).
Aqueous
Fig (2): Activity of methanol and aqueous
extract of Acacia nilotica (Pods) on Salmonella…
Plate 5: Inhibition Zone of Acacia nilotica Methanol extracts
(10% 20% and 30%) Salmonella
Plate 6: Inhibition Zone of Acacia nilotica aqueous extracts
(10% 20% and 30%) Salmonella
Table3. Activity of methanol and aqueous extract of Trigonella foenumgraecium (Seeds) on E. coli
Con. (%)
10
20
30
Methanol
Aqueous
0.0d
6.00c
±0.0
±2.00
0.0d
9.00b
±0.0
±3.00
5.33c
14.67a
±0.58
±1.15
2.781*
Lsd0.05
a, b, c & d: Means having different superscripts differ significantly (P≤0.05).
zone (mm)
12
10
8
6
4
2
0
Con10%
Con20%
Methanol
Con30%
Con10%
Con20%
Con30%
Aqueous
Fi (3): Activity of methanol and aqueous extract of
Trigonella foenum-graecium (Seeds) on E. coli
Plate 7: Inhibition Zone of Trigonella foenum graecum Methanol extracts
(10% 20% and 30%) E.coli.
Plate 8: Inhibition Zone of Trigonella foenum graecum aqueous extracts (10%
20% and 30%) E.coli.
The Salmonella typhi count presented in table (4) Fig (4) Plate (9 and
10) was highest in concentration of Aqucous extract of Trigonella foenum
graecium% 30 )11.00(. And the lowest count obtained by Trigonella foenum
graecium extraction by methanol in %10 concentration (0.0).
All bacterial species were found to be resistant against extract of Cordia
sinensis stem bark and Trigonella foenum-greacum seed methanol and water
extracts of Acacia methanol and water extracts of Acacia nilotica ssp. nilotica
it showed exceptionally stronger activity against E. coli than other plant
extracts yet having moderate /poor activity on Gram-positive bacteria, a trend
not observed for other species of plants. The antibacterial activity Acacia
nilotica ssp. nilotica Lawsonia inermis and Azadirachta indica extracts was
more pronounced on the Gram-positive bacteria( Staphylococcus aureus )than
the Gram-negative bacteria a trend not observed for other species of plants.
The antibacterial activity Acacia nilotica ssp. nilotica , Lawsonia inermis
and Azadirachta indica extracts was more pronounced on the Gram-positive
bacteria( Staphylococcus aureus )than the Gram-negative bacteria( Klebsiella
pneumoniae and Pseudomonas aeruginosa
The reason for the difference in sensitivity between Gram positive and
Gram-negative bacteria might be ascribed to the differences in morphological
constitutions between these microorganisms, Gram-negative bacteria having
an outer phospholipidic membrane carrying the structur lipopolysaccharide
components. This makes the cell wall impermeable to antimicrobial chemical
substances. The Gram-positive bacteria on the other hand are more
susceptible having only an outer.
Table4. Activity of methanol and aqueous extract of Trigonella foenumgraecium (Seeds) on Salmonella typhi.
Con. (%)
Methanol
Aqueous
0.0d
4.00c
±0.0
±1.00
0.0d
7.33b
±0.0
±1.53
4.33c
11.00a
±2.52
±2.65
10
20
30
2.965*
Lsd0.05
Methanol
Con30%
Con20%
Con10%
Con30%
Con20%
12
10
8
6
4
2
0
Con10%
zone (mm)
a, b, c, d e, f, g, h &i: Means having different superscripts differ significantly (P≤0.05).
Aqueous
Fig (4): Activity of methanol and aqueous extract of
Trigonella foenum-graecium (Seeds) on Salmonella
typhi.
Plate 9: Inhibition Zone of Trigonella foenum graecum Methanol extracts
(10% 20% and 30%) Salmonella typhi
Plate 10: Inhibition Zone of Trigonella foenum graecum aqueous extracts
(10% 20% and 30%) Salmonella typhi
peptidoglycan layer which is not an effective permeability barrier. Therefore,
the cell wall of Gram-negative organisms which are more complex than the
Gram-positive ones act as a diffusional barrier and making them less
susceptible to the antimicrobial agents than are Gram-positive In spite of this
permeability differences ,however, some of the extracts have still exerted
some degree of inhibition against Gram-negative organisms as well (Hodges et
al., 2002).
CHAPTER FIVE
5. CONCLUSIONS & RECOMMENDATIONS
The study has clearly shown the in vitro antibacterial activity of the crude
methanolic and aqueous extract of A. nilotica on E. coli and its possible use in
treatment of its infections. There is also a need to establish standard dosages
for A. nilotica preparations and to investigate its toxicity in medical use.
Moreover, testing the antimicrobial effect of Acacia nilotica against other
bacteria, fungi and some viruses can be considered in further research.
Although the methanol and aqueous extract of Trigonella foenumgreacium did not show any antimicrobial activity against E. coli, the
methanolic and aqueous extracts of Acacia nilotica is inhibitory effect against
the tested micro-organisms E. coli Similar to the effectiveness of
ciprofloxacin. Alkaloids by methanolic extract of Acacia nilotica showed zone of
inhibition against hospital isolates. Flavonoids are promising bioactive
compounds against S.aureus gram negative and E.coli.
These results act as pointers to authenticity of traditional medicinal
knowledge.
Recommendations:
- Sudan is full of plants that can be used as an alternative to antibiotics.
- Development of methods of extraction of medicinal plants.
- The use of the Acacia nilotica in the manufacture of some kinds of medicines
and drugs.
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